Jt,,z, .U? - Sezione di Farmacologia

Transcription

UNIVERSITÀ DEGLI STUDI DI BRESCIA
DOTTORATO DI RICERCA IN
NEUROSCIENZE
Settore Scientifico Disciplinare Preminente
BI0/14 Farmacologia
XXVI Ciclo
A. A. 2013 - 2014
Tesi di
MARCO BORTOLATO
Role of neurosteroids in the modulation of
behavioral responses elicited by dopaminergic agonists:
relevance to Tourette Syndrome
Il coordinatore
~·
/Jt,,z,· .U?_g_
Il relatore
..,
i /]fuA--e.z
Il dottorando
A papà e mamma
i
ABSTRACT – VERSIONE ITALIANA
La sindrome di Tourette è un disturbo neuropsichiatrico dello sviluppo ad alta prevalenza nel
sesso maschile, caratterizzato dalla presenza cronica e ricorrente di tic motori e fonici. Queste
manifestazioni, tipicamente semi-volontarie ed esplosive, sono aggravate in presenza di
condizioni ambientali stressanti, e rispondono generalmente alla somministrazione di farmaci
antipsicotici, che sopprimono l’espressione di tic per via del blocco dei recettori D2 per la
dopamina. Tali farmaci, nonostante siano efficaci nel ridurre i tic, inducono spesso una serie di
onerosi effetti collaterali, che spesso comportano una significativa riduzione della compliance
terapeutica. Queste premesse sottolineano la necessità di sviluppare nuovi approcci terapeutici
per la sindrome di Tourette. Recenti studi neuropsicologici hanno dimostrato che, al di là della
loro natura di manifestazione patologica motoria, i tic sono espressioni epifenomeniche di
urgenze psicologiche incoercibili, tipicamente avvertite prima dell’esecuzione dei tic stessi, e
direttamente collegate ad alterazioni della sfera sensoriale. In particolare, lo studio della
fenomenologia di queste pulsioni ha rivelato che i pazienti affetti da sindrome di Tourette
presentano caratteristicamente un deficit del sensorimotor gating, ovvero la capacità di filtrare
adeguatamente informazioni provenienti dall’ambiente. Il quadro risultante comporta un
sovraccarico percettivo che faciliterebbe l’insorgenza dei tic. Evidenze precliniche suggeriscono
che il sensorimotor gating sia regolato dagli stessi processi imputati come responsabili per la
manifestazione dei tic, ovvero alterazioni del segnale dopaminergico nel contesto
dei circuiti cortico-striatali. Ciononostante, i meccanismi neurobiologici che comportano lo
scatenamento di tic in presenza di stress, nonché le cause fisiopatologiche della predominanza
maschile della sindrome rimangono essenzialmente ignoti. A questo proposito, recenti studi
hanno evidenziato la possibilità che tali fenomeni riflettano meccanismi modulatori della
neurotransmissione dopaminergica operati da steroidi neuroattivi. Nella fattispecie, il nostro
gruppo di lavoro ha dimostrato che la 5α- riduttasi, uno degli enzimi chiave nella sintesi di
steroidi neuroattivi e metaboliti androgenici del testosterone, è direttamente coinvolta nella
fisiopatologia della sindrome di Tourette. In uno studio clinico preliminare in aperto, la
finasteride, l’inibitore prototipico della 5α- riduttasi approvato in clinica da oltre vent’anni, ha
infatti significamente ridotto l’intensità dei tic in pazienti resistenti agli antipsicotici, senza
ii
indurre alcuno degli effetti collaterali extrapiramidali tipicamente associati a questi farmaci.
Inoltre, in modelli animali, la finasteride ha prevenuto i deficit di sensorimotor gating ed altri
fenomeni correlati ai tic (quali le stereotipie), secondari alla stimolazione non selettiva di
recettori dopaminergici. La ricerca presentata in questa tesi riguarda una serie di esperimenti,
condotti su modelli animali, finalizzati alla delucidazione delle basi neuroanatomiche
e neurochimiche degli effetti della finasteride e del coinvolgimento della 5α-riduttasi ed altri
enzimi steroidogenici rispetto alla modulazione degli effetti comportamentali derivati
dall’attivazione dei recettori dopaminergici. In particolare, tali studi sono stati condotti con una
particolare attenzione rivolta verso il protocollo della prepulse inhibition del riflesso di startle
acustica, il parametro di elezione per la misurazione del sensorimotor gating. I risultati di questi
studi dimostrano un coinvolgimento diretto dei neurosteroidi rispetto ai meccanismi di segnale
del recettore dopaminergico D1 nel contesto della corteccia prefrontale e del nucleo accumbens,
aree critiche per la fisiopatologia della sindrome di Tourette. Nell’insieme, questi risultati
potrebbero fornire importanti indicazioni sui meccanismi tramite i quali lo stress e i fattori
ormonali maschili possano influenzare il decorso della sindrome di Tourette. Inoltre, i nostri dati
mettono in luce come i pathway di sintesi steroidea possano rappresentare una fonte importante
per lo sviluppo di nuovi farmaci per la sindrome di Tourette, con un migliore profilo di sicurezza
e tollerabilità.
iii
ABSTRACT – ENGLISH VERSION
Tourette syndrome (TS) is a neurodevelopmental disorder, with a marked male predominance,
characterized by chronic, recurring motor and phonic tics. These semi-voluntary and explosive
manifestations, are typically exacerbated by stressful environmental contigencies, and
suppressed by antipsychotic drugs, through the antagonism of dopamine D2 receptors. These
drugs, albeit generally effective in reducing tic severity, are often associated to burdensome
extrapyramidal side effects, which often result in poor therapeutic compliance. This background
highlights the need for the development of novel approaches for TS treatment. Recent
neuropsychological studies have shown that tics are the epiphenomena of premonitory urges,
underpinned by sensory alterations. In particular, the phenomenological analysis of these
endophenotypes has revealed that TS patients exhibit deficits in sensorimotor gating, i.e. the
ability to filter out irrelevant environmental information. This defect leads to perceptual overload
and may facilitate tic ontogeny. Notably, preclinical evidence suggests that sensorimotor gating
is regulated by the same processes responsible for tics, namely dopaminergic signaling within
cortico-striatal circuits. Although the involvement of dopamine in TS has been confirmed by
ample experimental evidence, the neurobiogical bases of its male predominance and stress
sensitivity remain unknown. Recent studies have evidenced that these phenomena are likely
reflective of the modulation of dopamine neurotransmission by neuroactive steroids. Specifically,
our group has documented that the key neurosteroidogenic enzyme 5α-reductase (5αR) is
implicated in TS pathophysiology. In a preliminary open-label clinical trial, the 5αR inhibitor
finasteride - which was approved for clinical use more than twenty years ago - significantly
reduced tic severity without inducing any of the extrapyramidal effects typically associated with
benchmark antipsychotic drugs used in TS treatment. Furthermore, in animal models, finasteride
potently counters the gating deficits and other behavioral effects (such as stereotypies) induced
by non-selective dopamine agonists. The research presented in this thesis focuses on a series of
experiments on rodent models, aimed at elucidating the neuroanatomical and neurochemical
bases of the effects of finasteride, as well as the implication of 5αR and other neurosteroidogenic
enzymes in the modulation of dopamine signal. In particular, the main theme of this research is
the implication of neuroactive steroids in sensorimotor gating, as examined by the prepulse
inhibition of the acoustic startle reflex. Our results indicate that neurosteroids are directly
iv
involved in the modulation of the behavioral effects induced by dopamine D1 receptor activation,
and that these processes are likely mediated by key brain dopaminoceptive regions, such as the
prefrontal cortex and nucleus accumbens. Taken together, these results may shed light on the
mechanisms whereby stress and male sex can predispose to TS, and point to novel molecular
targets for the development of novel treatments with better safety and tolerability profiles.
v
ACKNOWLEDGMENTS
I would like to thank my supervisor, Prof. M. Cristina Missale, for the help and support she has
provided over the years, as well as my colleagues at the Universities of Southern California and
Kansas, and my collaborators at the Universities of Cagliari and Milan, without whom this work
would not have been possible. My sincere gratitude should be particularly addressed to Prof.
GianLuigi Gessa, PierFranco Spano, Daniele Piomelli, Francesco Marrosu and Jean C. Shih,
who mentored me during my scientific and professional career in its early steps.
The financial and infrastructural support for this work was partially provided by several funding
bodies, including the Tourette Syndrome Association, Italian Ministry of Health and
Government of Sardinia.
A special and heartfelt thanks goes out to my parents, who have been always and consistently
dedicated to foster my curiosity and have given me unflinching support through any rough period
of my life. I also wish to thank my friends, and in particular Matt and Sean, whose support has
been tremendously important for my life away from my family.
This work is also dedicated to the loving memory of Joel, Emanuele and Priscilla. You are not
forgotten.
vi
TABLE OF CONTENTS
ABSTRACT – ITALIAN VERSION
ii
ABSTRACT – ENGLISH VERSION
iv
ACKNOWLEDGMENTS
vi
TABLE OF CONTENTS
vii
LIST OF ABBREVIATIONS
xi
CHAPTER 1 – GENERAL INTRODUCTION
1
1.1 TIC DISORDERS AND TOURETTE SYNDROME (TS)
2
1.1.1. Clinical definition and phenomenology of tics
2
1.1.2. Prevalence and clinical features of tic disorders and TS
3
1.1.3. Pathophysiology and neurobiology of TS
3
1.1.4. Etiology of TS: genetic, environmental and sex factors
5
1.1.5. Pharmacological therapy of TS
8
1.1.6. Animal models of tic disorders and TS
9
1.1.7. TS-related neurobehavioral phenotypes in animal models
11
1.1.8. Models of TS based on pharmacological stimulation of dopamine receptors
15
1.1.9. Models of TS based on environmental manipulations
16
1.2. ROLE OF NEUROACTIVE STEROIDS IN TS
17
1.2.1. Pathways of synthesis and metabolism of neuroactive steroids
17
1.2.2. Steroid 5α-reductase (5αR)
20
1.2.3. 5αR inhibitors
22
1.2.4. Implication of neuroactive steroids in TS
25
vii
1.2.5. Application of 5αR inhibitors in TS therapy
1.3. AIMS OF THE THESIS
26
30
CHAPTER 2 – ROLE OF STEROIDOGENIC ENZYMES IN THE MODULATION OF
SENSORIMOTOR GATING
31
2.1. RATIONALE OF THE STUDIES
32
2.2. MATERIALS AND METHODS
32
2.3. RESULTS
36
2.3.1. Effects of abiraterone and finasteride on startle and PPI parameters
36
2.3.2. Effects of indomethacine on startle and PPI parameters
36
2.3.3. Effects of trilostane on startle and PPI parameters
39
2.3.4. Effects of androgen-receptor ligands on startle and PPI parameters
39
2.4. DISCUSSION
43
CHAPTER 3 – ANATOMICAL SUBSTRATES OF THE EFFECTS OF 5αR INHIBITORS
ON SENSORIMOTOR GATING
48
49
49
3.3. RESULTS
53
3.3.1. Effects of FIN on behavioral performances of castrated rats
54
3.3.2. Effects of intracerebroventricular FIN on APO-induced PPI disruption
57
viii
3.3.3. Effects of local FIN injections on APO-induced PPI disruption
57
3.3.4. Effects of systemic and local FIN on dopamine extracellular levels
61
3.4. DISCUSSION
64
CHAPTER 4 – ROLE OF 5αR AND NEUROSTEROIDS IN THE GATING DEFICITS
SECONDARY TO ISOLATION REARING
70
71
71
4.3. RESULTS
74
4.3.1. Effects of FIN and HAL on IR-induced changes in startle and PPI
74
4.3.2. Effects of FIN on IR-induced changes in NS profile in the NAc
76
4.4. DISCUSSION
78
CHAPTER 5 – IDENTIFICATION OF THE DOPAMINE RECEPTORS MEDIATING
THE ANTIPSYCHOTIC-LIKE EFFECTS OF 5αR INHIBITORS: EVIDENCE IN MICE
83
84
84
5.3. RESULTS
87
5.3.1. Effects of FIN and dopaminergic agonists on startle and PPI
87
5.3.2. Effects of FIN and DAergic agonists on open-field behaviors
90
ix
5.3.3. Effects of FIN on APO-induced stereotypies and catalepsy
5.4. DISCUSSION
93
94
CHAPTER 6 – IDENTIFICATION OF THE DOPAMINE RECEPTORS MEDIATING
THE ANTIPSYCHOTIC-LIKE EFFECTS OF 5αR INHIBITORS: EVIDENCE IN RATS
100
101
101
6.3. RESULTS
104
6.3.1. Effects of FIN versus the non-selective DAergic agonist APO on ASR and PPI in
SD and LE rats
104
6.3.2. Effects of FIN versus D1 agonists on ASR and PPI in SD and LE rats
105
6.3.3. Effects of of FIN versus D2 agonists on ASR and PPI in SD and LE rats
107
6.3.4. Effects of FIN versus D3 agonist on ASR and PPI in SD rats
109
6.4. DISCUSSION
110
CHAPTER 7 – GENERAL DISCUSSION
114
REFERENCES
120
LIST OF PUBLICATIONS
152
x
LIST OF ABBREVIATIONS
%PPI: percent prepulse inhibition
17β-HSD: 17β-hydroxysteroidodehydrogenase
3α-diol: 5α-androstane, 3α-diol
3α-HSOR: 3α- hydroxysteroid oxidoreductase
3β-diol: 5α-androstane,3β diol
3β-HSD: 3β-hydroxysteroid dehydrogenase
3β-HSOR: 3β-hydroxysteroid oxidoreductase
5-HT2: Serotonin 2 receptor
5-HT3: Serotonin 3 receptors
5αR: 5α-reductase
5αR1: 5α-reductase type 1
5αR2: 5α-reductase type 2
ABI: abiraterone
ADHD: attention-deficit hyperactivity disorder
AI: activity index
AMPA: α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid
AMPH: amphetamine
ANOVA: analysis of variance
AP: allopregnanolone (3α,5α-tetrahydroprogesterone)
AP: anterior-posterior
APO: apomorphine
AR: androgen receptor
ASD: autism-spectrum disorder
ASR: acoustic startle reflex
BPH: benign prostatic hyperplasia
CDX/R: hydroxypropyl-β-cyclodextrin/Ringer solution
CNS: central nervous system
CREB: cAMP response element-binding protein
CSTC: cortico-striatal-thalamo-cortical
D1: dopamine 1 receptors
DA: dopamine
xi
DARPP-32: dopamine- and cAMP-regulated neuronal phosphoprotein, 32 KDa
DHEA: dehydroepiandrosterone
DHP: dihydroprogesterone
DHT: dihydrotestosterone
DMSO: Dimethyl sulfoxide
DOPAC: 3,4-Dihydroxyphenylacetic acid
DSM-5: Diagnostic and Statistical Manual of Mental Disorders- 5th Edition
DV: dorsal-ventral
ERα: Estrogen receptor α
ERβ: Estrogen receptor β
FIN: finasteride
FLU: flutamide
GABA: γ-amino butyric acid
GABA-A: γ-amino butyric acid A receptor
GABA-B: γ-amino butyric acid B receptor
GPER1: G-protein-coupled estrogen receptor 1
GWAS: Genome-wide association study
GxExS: gene x environment x sex
HAL: haloperidol
HPA: hypothalamic-pituitary-adrenal
HPLC: High-performance liquid chromatography
ICDs: impulse control disorders
ICV: intracerebroventricular
INDO: indomethacine
IP: intraperitoneal
IR: Isolation rearing
Ki : Inhibition constant
KO: knock-out
KO: Long-Evans
LH: luteinizing hormone
ML: medial-lateral
mPFC: medial prefrontal cortex
mPR: membrane progesterone receptor
MS: mass spectrometry
NAc: Nucleus Accumbens
NADP: Nicotinamide adenine dinucleotide phosphate, oxidated form
xii
NADPH: Nicotinamide adenine dinucleotide phosphate, reduced form
NMDA: N-methyl-D-aspartate
NS: neurosteroids
OCD: obsessive-compulsive disorder
ORX: orchidectomized
PFC: prefrontal cortex
PGRMC1: progesterone membrane receptor component 1
PKA: protein kinase A
PPI: prepulse inhibition
PR: cytoplasmic progesterone receptor
PREG: pregnenolone
PROG: progesterone
pXR: pregnane X receptor
QUIN: quinpirole
RIA: radioimmunoassay
SAL: saline
SC: subcutaneous
SD: Sprague-Dawley
SHBG: Sex hormone binding globulin
SNP single-nucleotide polymorphism
SR: Social rearing
SUM: sumanirole
T: testosterone
T1/2 : half-life
THDOC: 3α,5α-tetrahydrodeoxycorticosterone
TRI: trilostane
TS: Tourette syndrome
TSA: Tourette Syndrome Association
TSAICG: Tourette Syndrome Association International Consortium for Genetics
VEH: vehicle
∆PPI: differential prepulse inhibition
σ1 receptors: sigma-1 receptors
xiii
CHAPTER 1
GENERAL INTRODUCTION
1
1.1 TIC DISORDERS AND TOURETTE SYNDROME (TS)
1.1.1. Clinical definition and phenomenology of tics
Tics are repetitive, semi-voluntary, sudden movements and/or vocalizations, which typically
mimic normal and purposeful motor or vocal activities (such as eye blinking, touching objects,
hopping, uttering meaningful words) in a contextually incongruous fashion (Leckman et al.,
2006).
Tics are manifested across highly heterogeneous modalities and localizations, with variable
degrees of frequency, duration and complexity. Motor tics are non-rhythmic movements,
frequently confined to the head, neck, face and mouth muscles, but also observed in the trunk
and limbs (Jankovic, 1997). Phonic tics are rapid vocalizations due to rapid air movements
through the upper respiratory tract, which can sometimes be associated with copro-, echo- or
palilalia (Jankovic, 2001).
Tics can also be classified as simple or complex, based on the degree of involvement of different
muscles. Simple tics are brief and repetitive actions, such as eye blinking, facial grimacing, head
jerking, sniffing or grunting sounds; conversely, complex tics engage multiple muscle groups in
coordinated and stereotyped patterns akin to purposeful activities, including touching objects or
people, hopping and jumping as well as uttering words or phrases (Jankovic, 1992).
Another pathognomonic characteristic of tics, which is commonly used as a key criterion for
differential diagnosis from other similar manifestations (such as twitches and myoclonus), lies in
their association with premonitory urges. These sensations, which precede tic execution, are
typically perceived as increasingly intrusive and uncomfortable feelings, sometimes
accompanied by a sense of somatic tension (Kwak et al., 2003; Belluscio et al., 2011; Cohen et
al., 2013). Although the nature of premonitory urges has not yet been fully clarified, these
manifestations have been proposed to reflect an interoceptive “hyperattentional state”,
characterized by psychological fixation and excessive awareness of stimuli from specific body
parts (Kane, 1994). The intensity of the urges is typically exacerbated by stress and by any
attempt to suppress tics, while it is alleviated by tic execution; nevertheless, the severities of tics
2
and urges are not correlated (Steinberg et al., 2010; Ganos et al., 2012), and urges appear to be
directly linked to obsessive-compulsive and depressive symptoms (Steinberg et al., 2010).
1.1.2. Prevalence and clinical features of tic disorders and TS
Although tics can occasionally occur in every individual, their persistent manifestation is
regarded as pathological (in view of potentially serious repercussions on psychosocial and
professional functioning of the affected subjects) and classified as a tic disorder.
Tic disorders are neurodevelopmental conditions affecting nearly 3% of the population (Knight
et al., 2012). The most severe tic disorder, Tourette syndrome (TS), is a familial, childhood-onset
neurobehavioral disorder characterized by multiple motor tics and at least one phonic tic, with a
duration greater than one year (APA, 2013). The prevalence of TS has been recently estimated at
0.4-1% of the population (Robertson, 2008). In addition to tics, approximately 90% of patients
are affected by comorbid psychiatric conditions, including attention-deficit hyperactivity
disorder (ADHD), obsessive-compulsive disorder (OCD) and impulse-control disorders (ICDs)
(Ghanizadeh and Mosallaei, 2009; Frank et al., 2011).
The typical onset of TS occurs at 6-7 years of age and is characterized by the appearance of
simple, recurrent motor tics, followed by the manifestation of phonic tics after several months
(Robertson, 2011). In most children, TS symptoms undergo a progressive exacerbation, which
reaches its zenith at the beginning of puberty (11-12 years of age), and is then followed by a
gradual remission in the majority of patients (Leckman et al., 1998); conversely, 30-40% of TSaffected children retain their symptoms in adulthood (Singer and Walkup, 1991). In addition to
these temporal changes, tic severity exhibits numerous fluctuations throughout life and is
typically increased during periods of high mental and physical stress (Leckman, 2002).
1.1.3. Pathophysiology and neurobiology of TS
Tics are commonly regarded as isolated or concatenated segments of automatic behavioral
sequences, which are dissociated from their original valence and thereby executed in a misplaced,
maladaptive and often reverberative fashion. Although the neurobiological bases of tics remain
3
partially unclear, converging lines of evidence have shown that the disorder is underpinned by
functional and/or morphological impairments of the cortico-striatal-thalamo-cortical (CSTC)
pathway, and in particular, of the basal ganglia (Peterson et al., 1996; Albin and Mink, 2006;
Gilbert et al., 2006; Yoon et al., 2007; Gomis et al., 2008; Wong et al., 2008; Leckman et al.,
2010; Felling and Singer, 2011; Ranjan et al., 2011; Eichele and Plessen, 2013). These structures
are directly implicated in the generation of extrapyramidal motor patterns and modulation of
automated motor performances.
Tics appear to result from imbalances of the inhibitory and excitatory inputs in these regions,
either related to insufficient inhibition from select families of corticostriatal interneurons
(Kalanithi et al., 2005; Kataoka et al., 2010), or excessive stimulation of specific neuronal
clusters (Albin and Mink, 2006). Irrespective of the source, the common outcome of these
impairments is likely an inadequate “center-on surround-off” interaction in these structures,
which leads to the activation of ectopic foci and a partial inability to suppress competing or
unwanted motor sequences (for a comprehensive review of this issue, see Albin and Mink, 2006
and Singer, 2005).
It should be noted that most tics tend to involve the activation of facial and neck muscles,
possibly suggesting a preferential localization of ectopic foci within the neurons of the
ventromedial in the striatum (which represent these regions, based on the somatotopic
organization of this brain area; see Maillard et al., 2000; and Nambu, 2011) and their
downstream projections.
While multiple lines of evidence have underscored the role of basal ganglia in TS ontogenesis,
several findings indicate that the prefrontal cortex (PFC) may have an equally important role in
tics. Indeed, the ability to suppress tics temporarily is likely to reflect the ability of this region to
control the automatic functions of the basal ganglia (Peterson et al., 1998a). Cortical
disinhibition of striatal circuits has been documented in TS (Heise et al., 2010). Furthermore,
impairments in cortical activation have also been evidenced in the execution of “go- no go” tasks
in TS patients (Ganos et al., 2014; Thomalla et al., 2014), which likely underpin their slower
performance in these tests (Eichele et al., 2010).
Although the alterations in CSTC circuits underlying tics involve multiple neurotransmitter
systems, a number of neuroimaging studies and postmortem analyses have clearly documented
4
that tics are likely supported by excess activation of dopamine (DA) receptors or other alterations
of its system (Minzer et al., 2004; Gilbert et al., 2006; Steeves et al., 2010). Accordingly, DA
receptor antagonists, such as haloperidol (HAL) and pimozide, are highly effective in reducing
tic severity (Bloch et al., 2011; Roessner et al., 2013), while DAergic agonists have been
associated with an exacerbation of tics (Shale et al., 1986).
Recent studies indicate that one of the main impairments in the involvement of DAergic
neurotransmission in tics may lie in the dysregulation of (reduced) tonic and (overactive) phasic
DA levels in the basal ganglia (Wong et al., 2008; Buse et al., 2013). Tics may be underpinned
by rapid variations in synaptic DA content, leading to a prominent activation of postsynaptic D1
receptors in the striatum. These receptors govern the activation of the “direct pathway”
projections to globus pallidus and substantia nigra pars reticulata, and may therefore lead to the
stimulation of ectopic foci. Accordingly, the results of a recent clinical trial sponsored by the
Tourette Syndrome Association (TSA) on the D1 receptor antagonist ecopipam suggest that this
drug may be highly effective as a therapeutic option for TS (Gilbert et al., 2014).
It is worth noting that the implication of the DAergic system in the pathophysiology of TS may
also involve the key role of this neurotransmitter in the ventral striatum with respect to the
orchestration of critical behavioral functions, such as habit formation, incentive motivation,
configuration of salience maps and sensorimotor gating (Berns and Sejnowski, 1998; Suri and
Schultz, 1998; Hikosaka et al., 2002; Seymour et al., 2004). Indeed, TS patients feature
alterations in all these behavioral domains (Castellanos et al., 1996; Swerdlow and Young, 2001;
Marsh et al., 2004; Palminteri et al., 2011).
In addition to DA, other neurotransmitters are implicated in tic disorders, such as γ-aminobutyric acid (GABA). The contribution of serotonin and norepinephrine in tics is more
controversial and may be related to specific subgroups of TS and comorbid manifestations, rather
than to the whole spectrum of tic disorders (Steeves and Fox, 2008; Udvardi et al., 2013).
1.1.4. Etiology of TS: genetic, environmental and sex factors
Over the past decade, research into the etiology of TS has afforded fundamental contributions to
our current understanding of the biological bases of this disorder. In particular, a large body of
5
evidence has indicated that, similarly to other neuropsychiatric conditions, TS is a multifactorial
disorder governed by multiple genetic, environmental and sex-related factors (Deng et al., 2012;
Paschou, 2013; Hoekstra et al., 2013).
Genetic factors. The genetic basis of TS was originally postulated by several groups in the late
1970’s, based on clinical observations on the high familiality of the syndrome (Kidd et al., 1980;
Pauls et al., 1981). These findings spurred a great number of analyses on genetic variants in TS.
Specifically, some of the first genetic studies on TS focused on genes directly implicated in DA
and serotonin regulation. Recently, several candidate genes have been discovered based on
sporadic and familial mutations associated with TS. Among these genes, particular interest has
been recently raised by SLITRK1 (Abelson et al., 2005; Miranda et al., 2009; Karagiannidis et al.,
2012), which encodes for a molecule involved in the organization of neurite growth.
In addition to specific studies on select genes, recent whole-genome analyses have been
conducted to identify potential single-nucleotide polymorphism (SNP) variants associated with
TS. Recently, the TSA International Consortium for Genetics (TSAICG) reported the results of
the first genome-wide association study (GWAS) on TS, based on the analysis of 484,000 SNPs
in the DNA of 1496 TS patients and 5249 controls (Scharf et al., 2013). Although the data
revealed the possible association of TS with several genes, none of the identified SNPs reached
the threshold of genome-wide significance, further confirming the complex genetic architecture
of TS inheritance.
An alternative approach to study inheritance patterns in TS is afforded by genetic linkage studies
across families with high TS prevalence. The largest linkage study for TS and tic disorder to date,
also conducted by the TSAICG (TSAICG, 2007) on 238 nuclear families and 18 large
multigenerational families totaling 2040 individuals, identified regions of high linkage to the
disorder in the chromosome 2p23 (TSAICG, 2007; O’Rourke et al., 2009).
Environmental factors: TS pathogenesis is influenced by the exposure to several environmental
variables (Swain et al., 2007). The severity of tics and other behavioral symptoms in TS is
typically exacerbated by the exposure to environmental and psychosocial stress (Bornstein et al.,
1990; Lombroso et al., 1991; Nelson and Pribor, 1993; Silva et al., 1995; Conelea and Woods,
2008). For example, stressful contingencies lead to a reduced ability in suppressing tics (Conelea
et al., 2011). The relation between TS and stress appears to be bidirectional, insofar as patients
6
have higher stress perception than non-affected controls, and short-term future tic severity is
predicted by current levels of psychosocial stress (Lin et al., 2007). These findings underscore a
critical involvement of stress-response mechanisms in TS pathogenesis.
In addition to the emotional impact of current contingencies, TS has been associated with the
occurrence of several adverse events during pre- and perinatal stages (Saccomani et al., 2005;
Motlagh et al., 2010; Bos-Veneman et al., 2011), including maternal psychosocial stress
(Leckman et al., 1990) as well as severe nausea and vomiting during the first gestational
trimester (Leckman et al., 1990). Maternal smoking and consumption of medications are also
significant risk factors for the disorder (Mathews et al., 2006). Finally, exposure to infections
(particularly from β-hemolytic streptococcus) has been associated with higher incidence of TS
and associated syndromes (Swedo et al., 1998). In general, the involvement of
neuroinflammatory events in TS is suggested by numerous findings (Mell et al., 2005; Leslie et
al., 2008), and may also reflect the involvement of autoimmune processes (Martino et al., 2009).
Notably, it is possible that the exposure to prenatal complications (including infections) may
indirectly influence the clinical course of TS by altering stress reactivity (Lin et al., 2010; Zohar
and Weinstock, 2011).
Sex factors and gender differences in TS. One of the most striking epidemiological aspects of TS
lies in its marked gender differences. Similarly to other neurodevelopmental conditions, such as
ADHD and autism-spectrum disorder (ASD), male gender is a major risk factor for TS (with a
male: female prevalence ratio estimated at ~4:1) (Jankovic and Kurlan, 2011). Although the
biological mechanisms underlying the higher TS vulnerability in boys remain elusive, genetic
studies have clearly ruled out that this phenomenon may reflect the involvement of X-linked
heritability patterns.
The implication of sex factors in TS is also indirectly indicated by the observation that temporal
variations of tic severity are characteristically time-locked with all the major phases of sex
maturation. For example, the typical age of onset coincides with adrenarche (6-7 years old);
symptoms increase in severity until the beginning of puberty (12 years old) and then undergo a
spontaneous amelioration, which becomes apparent with the end of puberty (at 18-19 years of
age).
7
In males, TS onset is characterized by anger-related manifestations and simple tics; conversely,
females exhibit complex tics more often than males. TS is diagnosed later in females than males,
with different age distributions (Santangelo et al., 1994); furthermore, recent data indicate that,
while male gender increases vulnerability for tics in childhood, female gender may predict
greater tic severity in adulthood (Lichter, 2008). Interestingly, male TS patients exhibit
significant deficits in cortical and callosal thickness, which are not observed in females
(Baumgardner et al., 1996; Mostofsky et al., 1999; Fahim et al., 2010).
1.1.5. Pharmacological therapy of TS
The current armamentarium for the therapy of tic disorders is based on drugs that target the key
neurotransmitter systems implicated in TS, such as DA receptor antagonists (antipsychotics) and
the α2 receptor agonist clonidine. While antipsychotics are often effective in reducing the severity
and frequency of tics, their numerous side effects, including extrapyramidal symptoms, sedation,
cognitive impairments and anhedonia, often result in poor therapeutic compliance.
In addition to standard therapies, GABA-A receptor positive allosteric modulators (such as
clonazepam) (Jimenez-Jimenez and Garcia-Ruiz, 2001; Reid, 2004), GABA-B receptor agonists
(baclofen) (Singer et al., 2001) and blockers of 5-HT2 receptors (such as atypical antipsychotics)
(Bruun and Budman, 1996; Budman et al., 2001) have shown some therapeutic efficacy in TS.
The lack of diagnostic biomarkers for TS dictates that the bases for the therapeutic management
of this disorder are empirical, often resulting in suboptimal clinical outcomes. In this perspective,
the recent advances in our understanding of the endophenotypic architecture and multifactorial
nature of TS, may facilitate the development of novel therapeutic agents tailored for specific
“neurobiological subtypes” of tic disorders, with greater tolerability and fewer side effects.
Animal models capturing a distinct set of deficits related to specific genetic and environmental
vulnerability factors promise to afford highly valuable experimental platforms for the
development of more selective and effective therapeutic strategies for tic disorders.
8
1.1.6. Animal models of tic disorders and TS
As in the case of other neuropsychiatric disorders, animal models provide a powerful tool to test
hypotheses on the biological substrates of TS and other tic disorders in a controlled experimental
setting. Given the high complexity of tics and related behavioral phenomena, animal modeling of
these condition is generally based on mammalian species, and, in particular, rodents, given their
high cost-effectiveness and acceptable degree of neurobiological similarity with humans.
The validation of an animal model of TS is essentially based on three major criteria (Willner,
1986):
1. Face validity, which refers to the analogy between the behavioral performance of the animal
models and the signs and symptoms in tic disorders;
2. Construct validity (encompassing also etiological validity), which evaluates the congruence
between the etiological and pathophysiological processes in tic disorders and the
neurobiological basis of the behavioral manifestations in the animal models;
3. Predictive validity, which qualifies the responsiveness of the animal model to treatments
validated for tic disorders (such as antipsychotic agents and clonidine).
The application of each of these criteria to animal models of tic disorders poses a number of
challenges. For example, testing an animal model for face validity implies the presentation of ticlike behaviors; nevertheless, given that the behavioral repertoire of rodents is distinctly different
from that of humans, and tics reproduce purposeful behaviors in a repetitive and maladaptive
fashion, it is to be expected that tic-like behaviors in these species may markedly diverge from
those observed in TS patients. For example, it is difficult to predict whether these manifestations
should involve vocalizations, in consideration of the different development of laryngeal motor
apparatus in humans, as compared to rats and mice. Thus, it is clear that the substantiation of a
potential model of TS based on face validity bears significant risks of anthropomorphic bias, and
can lead to numerous confounds. In relation to this issue, Swerdlow and Sutherland (2006)
indicated several instances of animal models (such as the stargazer rats and mice) in which
spontaneous motor jerks in animals have been sometimes framed as “tic-like” manifestations,
9
even though their neurobiological underpinnings are strikingly different from the substrates of
TS. These examples show that, while face validity remains a key criterion in the analysis of
animal models of tic disorders, a superficial assessment based uniquely on this parameter has
very limited translational value and should not be deemed sufficient for drug development
studies. The low reliability of face validity in the evaluation of tic-like manifestations is further
underscored by recent studies, showing marked variations in the expression of tic-like behaviors
induced pharmacologically across different murine strains (Proietti-Onori et al., 2014). Finally,
the nature of tic-like behaviors cannot be fully validated due to the impossibility to ascertain the
existence of premonitory urges or internal experiences in animals.
While predictive validity can be an effective complement to face validity, excessive reliance
upon this criterion should also be avoided, in consideration of the fact that several TS patients do
not respond to any of the available treatments. Furthermore, the excessive refinement of animal
models and behavioral paradigms aimed at enhancing their responsiveness to well-validated
therapies can constrain their translational potential, by reduce their ability to capture the effects
of novel treatments based on divergent mechanisms of action.
The most problematic aspect of the verification of construct validity in animal models of tic
disorders lies in our limited knowledge of the pathophysiological bases of these conditions. This
problem is compounded by the limitations of our current diagnostic classification of tic disorders,
which are based on symptomatic parameters, but not on quantitative, measurable indices. Indeed,
the diagnostic guidelines of the DSM-5 (APA, 2013) differentiate subtypes of tic disorders based
on the severity and pervasiveness of tics, but not on their neurobiological bases. This
classification is likely to mix in the same category a number of potentially heterogeneous
conditions that share similar symptomatic aspects, but depend on different pathophysiological
mechanisms and also respond to different therapies. This reliance on signs and symptoms as
central diagnostic criteria may also be applied to several other types of neuropsychiatric
conditions.
In order to overcome these limitations, researchers have begun dissecting complex
neuropsychiatric conditions, such as TS into more elementary “building blocks”. In this respect,
a very important innovation in our conceptual framework for animal models of tic disorders is a
new focus on intermediate phenotypes, (Leboyer et al., 1998) which are defined as measurable
10
indices posited to reflect a more elementary set of neuroanatomic, functional or psychological
deficits, than the whole array of deficits associated with TS. The best-known example of
intermediate phenotypes is afforded by endophenotypes, defined as heritable features
corresponding to elements of vulnerability to a given disorder (Gottesman and Shields, 1973).
Endophenotypes may encompass behavioral, neuroanatomical, biochemical, neurophysiological,
neuropsychological, or cognitive traits related to specific genetic factors (Gould and Gottesman,
2006; Arts et al., 2008; Viswanath et al., 2009). Endophenotypes are not inherently pathological,
but should be regarded as vulnerability elements, which can facilitate the development of a
disorder in the presence of other critical abnormalities (derived from other genetic factors or
environmental variables).
It is worth noting that the “atomistic” approach afforded by intermediate phenotypes is more
amenable to the implementation of effective translational strategies, particularly when referring
to cross-species parameters (which can be dependably measured in both humans and animal
models) (Rutter, 2008; Bearden et al., 2009; Markou et al., 2009).
This background highlights that research on animal models of tic disorders and TS refers to two
main objectives: 1) the development of animal models based on either genetic mutations or
environmental interventions aimed at replicating the key factors associated with the etiology of
these disorders; and 2) the confirmation of endophenotypes in TS models across the criteria of
face, construct and predictive validity. While it is beyond the scope of this section to illustrate
the multiple models based on genetic manipulations (for a general review on the topic, the
interested reader can refer to Godar et al., 2014), we will mostly focus on key phenotypes and
paradigms that enable the analysis of the DAergic functions in TS, as well as the impact of stress
as an environmental trigger.
1.1.7. TS-related neurobehavioral phenotypes in animal models
Neurobiological deficits in the CSTC circuits. As stated above, several findings have elucidated
that tics are underpinned by alterations of the basal ganglia. In particular, a host of studies have
documented that TS is accompanied by low caudate volume, thinning of sensorimotor cortices
and high corticostriatal activity during tic execution (Hyde et al., 1995; Peterson et al., 2003;
Bloch et al., 2005; Makki et al., 2009; Plessen et al., 2009; Fahim et al., 2010).
11
The reduction in caudate volume has been highlighted as a potential predictive measure of tic
severity in adulthood (Bloch et al., 2005). Based on the well-documented striatal disinhibition in
TS (Baym et al., 2008; Mazzone et al., 2010; Wang et al., 2011; Bronfeld et al., 2013) and the
finding of a selective loss in parvalbumin-positive interneurons in the striatum of TS patients
(Kalanithi et al., 2005; Kataoka et al., 2010), a number of studies have begun addressing direct
neurobiological hypotheses by producing selective lesions and/or pharmacological treatments in
the basal ganglia. Remarkably, selective ablation of D1-receptor expressing striatal neurons has
recently been shown to exhibit tic-like movements sensitive to treatment with the DAergic
blocker HAL, as well as other locomotor impairments and striatal atrophy (Kim et al., 2014).
The thinning of the somatosensory cortex in TS (Fahim et al., 2010) has also appeared to serve
as a potential index for symptom severity (Sowell et al., 2008; Fahim et al., 2010), and may
underpin the perceptual alterations associated with tics. Interestingly, Steiner and Kitai (2000)
found that DAergic stimulation activates barrel fields (the rodent equivalent of somatosensory
cortex, which regulate the sensory input and activity from vibrissae) through D1-like receptors in
the striatum, suggesting that the connectivity between these two regions may play a key role in
the execution of tics.
Stereotyped behaviors. Stereotypies are defined as motor and behavioral sequences that are
repeated purposelessly (Ridley, 1994). These behaviors are typically exhibited by most captive
animals kept in spatial restriction (which interferes with the expression of behavioral needs) and
are interpreted as a spontaneous manifestation of environmental discomfort, likely linked to the
expression of functional responses (such as foraging) in the absence of adequate sensory
feedback (Dantzer, 1991).
The relevance of animal stereotypies to TS is defined by all three validity criteria; like tics,
stereotypies are repetitive, habit-forming motor patterns, which typically mimic purposeful
behaviors. Oro-facial and head-bobbing stereotypies can be induced in experimental rodents by
several pharmacological agents that impinge on neurochemical substrates related to tics,
including agonists for DA and serotonin 5-HT2 receptors. Studies have shown that, like tics,
animal stereotypies (either spontaneous or pharmacologically induced) reflect alterations of the
basal ganglia (Garner and Mason, 2002); more specifically, the injection of DAergic agonists in
the dorsal striatum has been shown to evoke stereotyped behaviors in rodents, with a mechanism
12
that involves both D1-like and D2-like DA receptors. Finally, the predictive validity of
stereotyped behaviors with respect to tics, is confirmed by the ability of antipsychotic agents to
fully suppress these manifestations (Arnt, 1985; Arnt et al., 1988; Conti et al., 1997).
With respect to the analogy between tics and stereotypies in humans, it should be noted that,
while tics used to be regarded as stereotyped behaviors, the DSM-5 has clearly defined a
separation between these two phenomena. According to the current diagnostic classifications,
stereotypies are defined as more severe and pervasive than tics, and typically associated with
intellectual disabilities (such as in fronto-temporal dementia, ASD and some subtypes of
schizophrenia). Furthermore, they are characterized by greater rhythmicity, fewer temporal
fluctuations, and no relation to premonitory antecedents. However, it is worth noting that this
distinction has not yet led to a reorganization of the corresponding nomenclature in animal
ethology, and thus it is likely that animal stereotypies may correspond to a broader set of
phenomena than the human homonyms.
Prepulse inhibition (PPI) of the startle reflex. PPI is the reduction in startle response elicited by a
strong sensory stimulus that occurs when the latter is preceded by a weaker signal (Fig.1.1)
(Hoffman and Ison, 1980; Braff et al., 1992; Braff et al., 2001a). This index is considered a
highly dependable measure of sensorimotor gating, the cognitive function that enables the
formation of salience maps by filtering out non-relevant information. PPI deficits have been
documented in TS (Castellanos et al., 1996; Zebardast et al., 2013). Although the severity of tics
and OCD manifestations is not correlated with PPI impairments, this index is regarded as a key
intermediate phenotype for tic disorders. Of note, PPI disruptions are found in several other
neuropsychiatric disorders, thus studies that examine the effects of pharmacological
manipulations on PPI should be interpreted in this context. The phenomenological connection
between sensorimotor gating deficits and PPI is posited to reflect sensory alterations in TS
patients, which may also underpin the enhanced sensory feedback and somatic sensitivity in this
disorder (Bliss, 1980; Kane, 1994; Biermann-Ruben et al., 2012; Cohen et al., 2013). In addition,
the high construct validity of PPI for tic disorders is supported by the overlapping
neurobiological and neurochemical substrates of sensorimotor gating with the brain regions
involved in TS pathophysiology; indeed, PPI deficits can be induced in rodent models by lesions
of the CSTC loops as well as stimulation of DA and 5-HT2 receptors (Geyer et al., 2001;
13
Swerdlow et al., 2001a). In parallel, PPI deficits in TS patients have been shown to be associated
with altered patterns of caudate activation (Zebardast et al., 2013). Finally, PPI deficits in
rodents can be reversed by TS medications (Geyer et al., 2001). Nevertheless, it should be noted
that the efficacy of antipsychotic agents in reversing PPI alterations has not been validated in TS
patients and has produced equivocal results in mental patients with gating disturbances (Kumari
et al., 2000; Graham et al., 2001; Kumari and Sharma, 2002; Kumari et al., 2002; Xue et al.,
2012). Of relevance to the work presented in this thesis, it should be noted that, although the
focus of our sensorimotor gating and PPI research remains on TS, this paradigm is used also in
relation to multiple other neuropsychiatric disorders, such as schizophrenia, OCD and mania. For
a more detailed description of the importance of PPI in schizophrenia research, see Braff et al.,
2001a and Geyer et al, 2001).
Fig. 1.1 Prepulse inhibition (PPI) of the acoustic startle reflex. (A) Photograph of acoustic
startle apparatus (Med Associates, St. Albans, VT). (B) Image of a mouse placed into the
testing cage, mounted across a speaker and on a piezoelectric platform for signal
transduction. (C and D) Examples of pulse-alone and prepulse-pulse waves during PPI
testing.
14
1.1.8. Models of TS based on pharmacological stimulation of dopamine receptors
In line with the multifactorial nature of tic disorders, multiple neurotransmitters have been
implicated in the pathophysiology of TS by brain-imaging studies, postmortem analyses, and,
above all, the effectiveness of specific pharmacological agents in reducing tic severity and
frequency. In addition to the well-known implication of DA in tics, deficits in GABAergic
neurotransmission are a likely concurring factor of TS pathogenesis, as signified by the reduction
in GABAergic interneurons documented in this disorder (Kalanithi et al., 2005; Kataoka et al.,
2010). While the implications of other neurotransmitters in TS are less well-documented, it is
likely that both serotonin (at least through 5-HT2A receptor activation) and norepinephrine may
exert a modulatory function on the alterations of the CSTC circuitry observed in tic disorders.
Based on these notions, systemic or intracerebral treatment with drugs that can reproduce some
of the neurochemical alterations observed in TS can afford a crude, yet rapid, cost-effective way
to investigate the pathophysiology of tic disorders and screen for novel potential therapies.
The stimulation of DA receptors through non-selective indirect or direct agonists is known to
produce stereotypies as well as PPI deficits (Randrup et al., 1963; Ridley et al., 1982; Mansbach
et al., 1988; Geyer et al., 2001; Ralph et al., 2001; Lind et al., 2004). In addition, both D1-like
and D2-like receptor activation plays a role in these two phenomena (Doherty et al., 2008; Frau
et al., 2013). The impact of D1-like and D2-like receptor agonists on PPI varies depending on the
species and strain of experimental animals (Ralph and Caine, 2005; Ralph and Caine, 2007);
however, it appears that, in most cases, these phenotypes are contributed by both families of DA
receptors. The mechanisms supporting the cooperation between D1 and D2 receptors in the
striatum remain partially unclear, but are likely to reflect their confinement on striatonigral
neurons of the direct pathway and striatopallidal neurons of the indirect pathway, respectively
(Gerfen et al., 1990; Robertson et al., 1992; Planert et al., 2013). In this perspective, it is worth
noting that the stereotypies induced by stimulant drugs, such as amphetamine (AMPH) and
cocaine, have been shown to reflect the disequilibrium of activation in the striosomes (which
display abundant D1 receptors) with respect to the matrix (which features high levels of D2
receptors) (Canales and Graybiel, 2000). Conversely, PPI deficits in response to DAergic
agonists have been shown to reflect the activation of D1 and D2 receptors in the nucleus
15
accumbens (Wan et al., 1995; Wan and Swerdlow, 1996; Swerdlow et al., 2007). As expected,
the behavioral changes produced by DAergic stimulation are sensitive to antipsychotic agents,
signifying high predictive validity for these preparations (Arnt, 1995; Geyer et al., 2001; Gilbert
et al., 2014).
1.1.9. Models of TS based on environmental manipulations
Pharmacologically induced states, albeit extremely useful to represent specific aspects of TS,
afford a non-naturalistic approach that is poorly suited for the study of neurochemical and
functional imbalances in TS. This limitation is particularly problematic when addressing the
relation between tics and their environmental triggers, and particularly stress. An experimental
model that more closely capture the neurobiological relationship between stress and TS is the
isolation-rearing (IR) paradigm (for a general review on this mdoel, see Fone and Porkess, 2008).
IR consists of subjecting rats to the deprivation of social interactions for 6–9 weeks after
weaning, a critical phase of rodent life span characterized by the development of social play
(Einon and Morgan, 1977). As a result of this manipulation, IR rats exhibit a spectrum of
persistent behavioral alterations in adulthood, including hyperactivity, increased responsiveness
to psychostimulants, perseverative behaviors, cognitive deficits, and, particularly, PPI disruption
(Weiss and Feldon, 2001). These abnormalities are paralleled by multiple neurochemical
changes, such as aberrant DA levels in PFC and NAc (Fulford and Marsden, 1998; Brenes et al,
2008), and altered mesocortical transmission (Peters and O’Donnell, 2005).
Although IR has not been generally studied as a model of schizophrenia, rather than TS, the PPI
deficits induced by this manipulation are particularly relevant to the relation between dopamine,
stress and the pathophysiology of TS. Accordingly, these impairments are countered by typical
antipsychotics. Furthermore, IR facilitates the ontogeny of stereotypies induced by dopaminergic
agonists and leads to CTSC deficits (see Heidbreder et al., 2000).
16
1.2 ROLE OF NEUROACTIVE STEROIDS IN TS
The epidemiological evidence outlined in the previous section suggests that the neural
underpinnings of TS may result from complex gene-environment-sex (GxExS) interactions.
Although the molecular bases of these putative interactions remain unknown, one of the best
candidate systems that may contribute to these mechanisms is based on neuroactive steroids,
which play key roles in both stress response regulation and sex differences. In this section, we
will briefly outline the pathways of steroidogenesis related to these molecules, and then focus
more specifically on 5α-reductase, the enzyme catalyzing one of the main rate-limiting steps in
this process. We will then review the available evidence on the involvement of neuroactive
steroids in TS, and highlight the major knowledge gaps in this emerging field of research.
1.2.1. Pathways of synthesis and metabolism of neuroactive steroids
While the influence of stress on the brain has been long known, converging lines of research
have elucidated that the CNS itself can synthesize multiple classes of endogenous steroids
(named neurosteroids; NSs), which then act directly in the modulation of their major
neurotransmitter systems. These compounds act in coordination with the abundant endocrine
input received from adrenal and gonadal steroids to regulate a broad set of neurobehavioral
functions, including stress response and gender-related characteristics. Interestingly, various
studies indicated that neuroactive steroids mediate pivotal processes to be considered with
specific attention in the pathophysiology of multiple neural disorders (Mensah-Nyagan et al.,
2001; Mensah-Nyagan et al., 2008). Although not all the details of steroidogenic reactions have
been fully elucidated, it has become clear that both the biosynthesis and metabolism of all major
sex steroids can occur in the brain, through a number of tightly interwoven reactions (Fig.1.2).
The precursor of all steroids is cholesterol, which is converted into pregnenolone (PREG) by
cytochrome P450 side-chain cleavage (CYP450scc). PREG is then further transformed into
progesterone (PROG) by the action of the enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD)
(Melcangi et al., 2008). PROG undergoes multiple metabolic processes, mediated by different
enzymes expressed in neurons, astrocytes, oligodendrocytes and Schwann cells (Pelletier, 2010).
One of the most predominant and best-characterized enzymatic reactions of PROG metabolism is
17
its transformation into dihydroPROG (DHP) by the enzyme 5αR; in turn, DHP is further
converted into either 3α,5α-tetrahydroPROG (commonly named AP; AP) or 3α,5βtetrahydroPROG (isopregnanolone) by 3α-hydroxysteroid oxidoreductase (3α-HSOR) and 3βhydroxysteroid oxidoreductase (3β-HSOR), respectively (Melcangi et al., 2008). Alternatively to
this metabolic pathway, both PREG and PROG can be substrates for 17α-hydroxylase/C17,20
Fig. 1.2 Schematization of the main pathways of neurosteroidogenesis. Enzymes: CYP450scc:
Cytochrome P450 side chain cleavage; CYP450-17A1: 17α-hydroxylase/17,20 lyase 3β-HSD: 3βhydroxysteroid dehydrogenase; 5α-R; 5α-reductase; 3α-HSOR; 3α-hydroxysteroid oxidoreductase; 3β HSOR; 3β-hydroxysteroid oxidoreductase 17β-HSD: 17β-hydroxysteroid dehydrogenase; CYP45019A1: aromatase. Receptors: PGRMC1: Progesterone receptor membrane component 1; αER: estrogen
receptor α; βER: estrogen receptor β; GPER1: G-protein estrogen receptor 1; mPRs: membrane
progesterone receptors; PR: cytoplasmic progesterone receptor; AR: androgen receptor; PXR:
Pregnane X receptor; GABA-A: γ-amino butyric acid A receptor
18
lyase (cytochrome P450 17A1; CYP17A1) which converts them into their 17-hydroxylated
metabolites and then into dehydroepiandrosterone (DHEA) and 4-androstenedione (Melcangi et
al., 2008). These neuroactive steroids are further converted into T and other androgens by 17βhydroxysteroidodehydrogenase (17β-HSD).
Similarly to PROG, in neurons and glial cells T is then converted by 5αR into dihydroT (DHT)
(Lephart et al., 2001) and subsequently by the action of 3α-HSOR or 3β-HSOR into 5αandrostane, 3α-diol (3α-diol) or 5α-androstane,3β diol (3β-diol) respectively (Jin and Penning,
2001). It is worth noting that, in neurons and glia, 4-androstenedione and T can also be
respectively converted into estrone and 17β-estradiol by aromatase (Garcia-Segura et al., 2003).
While estrone mediates its effects through activation of the estrogen receptor α (ERα), estradiol
also activates estrogen receptor β (ERβ) and the membrane receptor GPER1.
The metabolism of PROG and T into their numerous derivatives has a deep impact on the
mechanism of action of these neuroactive steroids, through the involvement of multiple receptors
and signaling pathways.
For example, while PROG and DHP interact with the classical PROG receptor (PR), AP is a
potent ligand of a non-classical steroid receptor, such as GABA-A receptor (Lambert et al., 2003;
Belelli and Lambert, 2005). Conversely, 3β-THP, albeit unable to bind directly to GABA-A, can
antagonize the effect of AP on this receptor (Melcangi et al., 2008). In addition to these wellknown mechanisms, emerging lines of evidence are revealing a number of novel receptors that
may contribute to the effects of PROG and AP. For example, PROG has been shown to bind to a
number of membrane receptors, including the family of membrane PROG receptors (mPRs) and
the PROG membrane receptor component 1 (PGRMC1), which are abundantly distributed in
several brain regions (Intlekofer and Petersen, 2011; Thomas and Pang, 2012; Cooke et al.,
2013). Furthermore, several effects of AP appear to be mediated by mPRs and by the pregnane X
receptor (PXR) (Cooke et al., 2013), as well as the modulation of the glutamate N-methyl-Daspartate (NMDA) receptor.
A similar variety of receptor-mediated mechanisms has been shown for T and its 5α-reduced
metabolites; whereas DHT and T activate ARs (ARs), 3α-diol and 3β-diol act as a positive
allosteric modulator of GABA-A receptor and an a potent ERβ agonist, respectively (Melcangi et
al., 2008; Handa et al., 2008). Although several authors posited the involvement of membrane19
bound receptors to account for several rapid actions of T, the molecular identity of these targets
remains currently elusive (Foradori et al, 2008).
From this perspective, it is apparent that 5αR plays a key role in modulating the functions and
signaling mechanisms of NSs in the brain, by enabling the conversion of PROG and T into
numerous neuroactive metabolites that can modulate brain functions and behavior through the
action of a broad array of receptors. In the next sections, we will review the current background
on this family of enzymes, and highlight our previous work on it, which will serve as
fundamental background for the hypotheses and experimental work presented in this dissertation.
1.2.2 Steroid 5α-reductase (5αR)
The family of 5αRs catalyzes the saturation of the 4,5 double bond of the A ring of several ∆4-3ketosteroid substrates, including PROG, deoxycorticosterone, corticosterone, aldosterone,
androstenedione and T (Fig.1.3).
This process, which requires nicotinamide adenine dinucleotide phosphate (NADPH) as a cofactor, consists in the formation of an enolate intermediate on the C3 position of the substrate
ketosteroid, which consequently results in the protonation of C4 and direct transfer of a hydride
ion from NADPH to C5 (Berseus, 1967; Bjorkem, 1969; Wilton, 1976). The reaction catalyzed
by 5αR increases the susceptibility of the carbonyl group in C3 to be reduced by 3α-HSOR or
3β-HSOR, or conjugated with hydrophilic moieties, such as sulfate and glucuronyl groups, in
order to facilitate their elimination. In addition to its role in the regulation of steroid catabolism,
5α reduction is the rate-limiting step in the metabolism of non-aromatic steroids, in view of its
irreversibility under physiological conditions.
Several 5αR products play a pivotal role in a large number of physiological processes. For
example, DHT is the most potent androgen hormone in vivo, and orchestrates the development
of male external genitalia and secondary sex traits, as well as the trophism of the prostate (Liao,
1969; Gomez and Frost, 1972; Mooradian et al., 1987).
The role of 5αR in male hormone metabolism also encompasses the conversion of the adrenal
androgen androstenedione into its 5α-reduced metabolite androstanedione, which is further
converted into androsterone by 3α-HSOR (Weisser et al., 1996). The functions of 5αRs are not
20
Fig. 1.3. Scheme of the reaction catalyzed by steroid 5α-reductase (5αR). NADP+/NADPH:
nicotinamide adenine dinucleotide phosphate (in reduced and oxidized form). The atomic
numbering (C1-C17) and ring nomenclature (A-D) refer to the cyclopentanoperhydrophenantrene ring system.
Fig. 1.4 Structure of the FDA-approved 5αR inhibitors finasteride and dutasteride
21
limited to endocrine regulation. Several 5α-reduced steroids play central roles in the modulation
of key physiological functions across different organs and systems. In the eye, the metabolite of
cortisol, 5α-dihydrocortisol, plays a role in the production of aqueous humor (Weinstein et al.,
1991).
In the kidney, 5α-dihydroaldosterone exerts a potent natriuretic function (Morris et al., 1989). In
the liver, the 5αR-mediated conversion of cholestenone into cholestanone is a fundamental step
for the synthesis of cholestanol (Bjorkhem and Karlmar, 1974); moreover, the glucocorticoids
5α-dihydrocorticosterone and 3α,5α-tetrahydrocorticosterone are posited to regulate glucose
metabolism (McInnes et al., 2004). Finally, in the brain, the 5α-reduced metabolites of PROG
and deoxycorticosterone, DHP and 5α-dihydrodeoxycorticosterone, are respectively converted
by 3α-HSD in AP and 3α,5α-tetrahydrodeoxycorticosterone (THDOC), which modulate the
behavioral reaction to environmental stress by activating the GABA-A receptor (Barbaccia et al.,
2001; Girdler and Klatzkin, 2007).
Of the five types of 5αR enzymes characterized to date, the first two (termed 5αR1 and 5αR2)
play major roles in steroidogenesis and mediate overlapping reactions (for a thorough
presentation on the characteristics of both isoenzymes and their regulation, see Paba et al., 2011);
however, they differ by patterns of localization and expression. 5αR1 is highly expressed in the
epidermal cells, neurons and adrenal glands; conversely, 5αR2 is predominantly expressed in the
male urogenital tract, as well as genital skin, hair follicles and liver. While 5αR2 is not as
abundant as 5αR1 in the brain, it can be found across most structures, and particularly in the
cortex and cerebellum (Castelli et al., 2013).
1.2.3. 5αR inhibitors
The prototypical 5αR inhibitor, finasteride (FIN) [17β-(N-tert-butylcarbamoyl)-4-aza-5αandrost-1-en-3-one] (Fig.1.4) is an unsaturated derivative of 4-MA, with relatively high
selectivity for 5αR2 over 5αR1 (Rasmusson et al., 1986). The inhibition of 5αR mediated by FIN
is based on the transfer of a hydride group from NADPH to the ∆1-double bond of the drug. This
process results in the formation of a covalent NADP-dihydroFIN adduct, which potently binds to
the free 5αR enzyme, competing with its endogenous substrates (Bull et al., 1996). The
extremely low dissociation constant of this compound for 5αR2 (Ki = 3–5 nM), however, results
22
in a very slow turnover of the enzyme-adduct complex (T1/2 ≈ 30 days), rendering the inhibition
virtually irreversible.
In the 1990s, the quest for novel 5αR inhibitors led to the development of dutasteride ((5α,17β)N-{2,5-bis(trifluoromethyl)-phenyl}-3-oxo-4-azaandrost-1-ene-17-carboxamide) (Fig. 1.4), a 4azasteroid with much higher potency than FIN in inhibiting both 5αR1 and 5αR2 (100 and 3
times greater, respectively).
A large body of data from randomized clinical trials has demonstrated the efficacy of 5αR
inhibitors in the treatment of benign prostatic hyperplasia (BPH). Several clinical trials have
shown the long-term efficacy of FIN and dutasteride in alleviating symptoms, improving urinary
flow rates, reducing prostate volume and decreasing the risk of acute urinary retention and the
need for surgical intervention (Nickel et al., 1996).
Lower doses of FIN are also indicated for the treatment of androgenetic alopecia, both orally (1
mg/day) or locally (by gel). The drug significantly diminishes the progression of the baldness
and often stimulates new growth. The mechanism of action is based on the reduction of DHT
levels, which play a key role in male-pattern hair loss (Price et al., 2000). Both FIN and
dutasteride are reported to elicit very limited side effects in volunteers and patients (Paba et al.,
2011). The most common untoward effects induced by 5αR inhibitors include decreased libido,
ejaculatory disorders and erectile dysfunction, and are ascribed to the reduction in DHT levels as
well as the increased estrogen synthesis (due to enhanced availability of T and androstenedione
for aromatization) (Amory et al., 2007). The reported incidence of these adverse effects is
variable across different studies, but is generally low (around 5%) and mainly reported during
the initial stages of treatment (Ishikawa et al., 2006). None of these side effects has been found
to significantly interfere with therapeutic compliance, as indicated by comparable rates of
treatment withdrawal between FIN- and placebo-treated groups (Makridakis and Reichardt,
2005).
In addition to its endocrine and metabolic effects, inhibition of 5αR results in a broad array of
effects in emotional and cognitive regulation in rodents, which stem from the ensuing alterations
in the profile of neuroactive steroids within cortical and limbic brain regions. While only few
clinical studies have analyzed the psychotropic effects of 5αR inhibitors, numerous
investigations in experimental animals have focused on the behavioral implication of FIN
treatment.
23
The best-characterized and most frequently investigated behavioral effect of pharmacological
5αR blockade in rodents is the suppression of the synthesis of AP and THDOC, which leads to
perturbed modulation of GABA-A receptor function and altered responsiveness to environmental
stimuli and stress (Mostallino et al., 2009; Molina-Hernandez et al., 2009; de Sousa et al., 2010).
Indeed, both AP and THDOC have been shown to elicit anxiolytic-like actions in different
animal models (Crawley et al., 1986; Zimmerberg et al., 1994). Furthermore, AP has been
shown to regulate aggression and fear responses in male mice (Agis-Balboa et al., 2007; Pibiri et
al., 2008).
Systemic administration of FIN has been shown to increase indices of anxiety-like behaviors in
the open field and elevated plus maze paradigms and depression-like behavior in the forced swim
test (Frye and Walf, 2002; Mann, 2006). Several brain regions, including hippocampus and
amygdala, have been shown to play a role in these effects (Frye and Walf, 2002; Walf et al.,
2006; Martin-Garcia et al., 2008).
FIN may exert its effects on anxiety and mood by decreasing the levels of other 5α-reduced
neuroactive steroids, such as androsterone and 3α-diol, which have been shown to exert
anxiolytic and antidepressant properties (Reddy and Rogawski, 2002; Reddy, 2003; Frye et al.,
2008).
The translational value of the preclinical investigations on FIN’s behavioral effects is partially
limited by the fact that, in rats, FIN has a high affinity for both 5αR1 and 5αR2 (Azzolina et al.,
1997). This characteristic is strikingly at variance with the relative selectivity of FIN for 5αR2 in
humans, suggesting possible differences in the neuropsychological outcomes of this drug.
Accordingly, several studies have even reported potential beneficial effects of FIN on anxiety
and mood in BPH patients, but this outcome is arguably influenced by their enhancement in
quality of life due to the reduction of urinary symptoms (Girman et al., 1996).
Few studies have actually shown an association between FIN administration and increased
anxiety and depression in patients with BPH and androgenetic alopecia (Altomare and Capella,
2002; Rahimi-Ardabili et al., 2006). In line with this concept, several studies have reported an
inverse relationship between AP levels and anxiety and depression symptoms in patients
(Uzunova et al., 1998). The role of NSs in mediating antidepressant effects, however, has been
recently challenged (Rupprecht, 2003; Eser et al., 2006).
24
1.2.4. Implication of neuroactive steroids in TS
The first studies on endocrine changes in TS were published in the late 1980’s, and suggested
that this disorder may feature functional alterations in the secretion of luteinizing hormone (LH),
the main regulator of gonadal androgen synthesis (Sandyk and Bamford, 1988a; Sandyk et al.,
1988). In the following years, a number of clinical observations showed that tics in TS patients
could be exacerbated by anabolic androgens (Leckman and Scahill, 1990). In addition, TS
patients were found to exhibit behavioral features typically associated with androgens, including
aggressiveness, precocious sex drive and pervasive erotic urges (Comings and Comings, 1987;
Budman et al., 1998). Furthermore, studies on the behavioral characteristics of TS-affected
children have also assessed that tic severity correlates with their preference for masculine play,
irrespective of gender (Alexander and Peterson, 2004).
One of the most intriguing aspects of the postulated involvement of male sex hormones in TS is
the possibility that steroidogenic enzymes and ARs may serve as putative therapeutic targets for
this disorder. The AR antagonists flutamide and cyproterone were tested in adult TS patients
with positive results (Peterson et al., 1994; Peterson et al., 1998b; Izmir and Dursun, 1999). In
particular, flutamide (750 mg/day) was tested in a cross-over, double-blind, placebo-controlled
trial with 10 men and 3 women affected by TS. Treatment was evaluated for 21 days, and
resulted in a very modest (7%), yet significant amelioration of motor tic severity; conversely, no
significant effects were observed on phonic tics and OCD symptoms (Peterson et al., 1998b).
Overall, the limited and short-lived efficacy of the drug undermined its therapeutic suitability,
also in consideration of its potential severe hepatic side effects (Brahm et al., 2011).
The temporal trajectory of TS in females diverges from that in males, in that tic severity is
typically increased after puberty. Interestingly, preliminary surveys appeared to indicate that, in
women, tic severity can be conditioned by variations in hormonal profile during the menstrual
cycle. In particular, 26% of females were found to experience exacerbation of tics in the
estrogenic phase of the menstrual cycle, and this phenomenon was found to be correlated with
increased tic severity at menarche (Schwabe and Konkol, 1992). While these data clearly support
direct implication of estrogens in TS pathogenesis, evidence in this respect remains limited and
controversial. Indeed, in a different study, no significant correlation was found between
25
fluctuation in tic severity and frequency and variations in estradiol or PROG through the
menstrual cycle in female TS patients (Kompoliti et al., 2001).
Although the neurobiology of sex steroid involvement in TS is not clear, numerous animal
studies have shown that sex hormones (including T, estradiol and PROG) yield multiple
modulatory effects on DAergic responses in the striatum and nucleus accumbens (Di Paolo, 1994;
Petitclerc et al., 1995; Tomas-Camardiel et al., 2002; De Souza et al., 2009; Tye et al., 2009;
Sanchez et al., 2010).
The involvement of neuroactive steroids in TS is also postulated in view of the increased stress
sensitivity of TS patients (Leckman et al., 1995; Chappell et al., 1996), which has been linked to
alterations of the hypothalamic-pituitary-adrenal (HPA) axis and glucocorticoid receptor
function. Indeed, abnormal functional enhancements of HPA axis were found in TS patients in
response to stressful medical procedures (Chappell et al., 1994; Corbett et al., 2008), as well as
injection of the opioid antagonist naloxone (Sandyk and Bamford, 1988b). Furthermore,
synthetic glucocorticoid treatment increased tics in two cases of tic disorders (Dietl et al., 1998).
Several rodent studies suggest that glucocorticoids modulate brain DA levels (Imperato et al.,
1989; Piazza et al., 1996), underscoring the possibility that this neurotransmitter may be
involved in the link between these neuroactive steroids and TS. It should be noted, however, that
cortisol does not appear to play a prominent role in the variations in stress responsiveness in TS;
indeed, Corbett and coworkers (2008) did not identify significant differences in the natural
cortisol circadian variations between TS-affected children and healthy probands. Further studies
are required to ascertain the degree of implication of each major glucocorticoid hormone in the
modulation of TS symptoms.
1.2.5. Application of 5αR inhibitors in TS therapy
As stated above, both 5αR1 and 5αR2 are present in the brain. In particular, the expression of
5αR2 in the brain appears to be related to the surges in T levels in this organ. In fact, the
expression of this enzyme has been mainly documented in early developmental stages. In adults,
the expression of the enzyme is posited to depend on androgens. Although the reactions
mediated by 5αR1 and 5αR2 are largely overlapping, it is interesting to notice that 5αR2 has a
26
higher affinity for PROG and T, possibly suggesting that these two isoenzymes may be
differentially activated in the brain, in relation to different concentrations of substrates.
Furthermore, unlike 5αR1, 5αR2 is distinctly absent from glial cells (Castelli et al., 2013), likely
signifying a topographical segregation of their functional roles.
Both 5αRs have been shown to play a role in stress response. Specifically, short-term stress
increases the expression of these enzymes, thereby allowing the synthesis of neuroactive steroids.
In the brain, the increased 5αR activity leads to higher synthesis of AP, THDOC and other NSs,
which appear to modulate stress response through multiple mechanisms, including the direct
regulation of HPA axis (Sarkar et al., 2011). The best characterized of such mechanisms is the
positive modulation of GABA-A receptor by AP; however, emerging data indicate that other 5αreduced NSs may play a key role in the responses to stress. In addition, it should be noted that,
while stress-induced increases in 5αR activity have been well characterized in brain regions,
recent data support that this phenomenon may also occur in peripheral organs, such as the
prostate (Sanchez et al., 2013).
In view of these premises, our group worked on the evaluation of FIN and other 5αR inhibitors
as potential therapeutic tools for TS. Our studies began with the preclinical evaluation of FIN
and dutasteride in animal models. In particular, we observed that these agents could lead to a
dramatic reduction of the PPI deficits and a significant decrease in stereotypies induced by the
non-selective DAergic agonists AMPH and apomorphine (APO) (Bortolato et al., 2008)
(Fig.1.5). Although FIN exhibited anti-DAergic mechanisms similar to those elicited by HAL
across several behavioral tasks, it strikingly failed to induce catalepsy (Bortolato et al., 2008).
Prompted by these preclinical results, we studied the therapeutic potential of FIN in adult male
TS patients. The first patient who gave informed consent for experimental treatment with FIN as
an adjunctive therapy was a severe case of TS with explosive vocalizations, stereotyped
coprolalic utterances, self-injuring motor tics and excessive sex drive. Previous therapeutic
attempts with typical antipsychotics had resulted in transient improvements, but the high rate of
extrapyramidal and cognitive side effects had led him to repeated withdrawals (Bortolato et al,
2007). FIN (5 mg/day) led to a gradual improvement of his motor and vocal tics, as assessed by
the Yale Tic Severity Scale with no reported side effects. The discontinuation of the regimen
after 18 weeks, however, resulted in an abrupt, dramatic exacerbation of the symptoms, which
was countered by reinstatement of the 5αR inhibitor.
27
The therapeutic effects of FIN as an adjunctive treatment in TS have been confirmed in a first
open-label study with adult male patients, who exhibited a significant reduction of the severity of
tics and associated compulsive (but not obsessive) manifestations by the sixth week of therapy
(Muroni et al, 2011). At the time of this writing, the open trial of FIN has been extended to 16
TS male adult patients.
As shown in Fig. 5, our patients consistently showed a fully significant reduction in tic severity
by the 6th week of treatment, and reached a plateau in therapeutic effects by the 12th week of FIN
administration. Notably, three patients have shown that FIN discontinuation led to a sudden
exacerbation of their symptoms. Interestingly, in contrast with antipsychotics, FIN does not elicit
extrapyramidal side effects in patients (Paba et al., 2011; Muroni et al., 2011).
Our preliminary surveys on the psychological mechanisms of FIN in tic suppression revealed
that, in most patients, this drug confers an attenuation of the premonitory urges and a greater
ability to control the execution of tics and other impulses, resulting in lower interference and
higher functioning. In addition, our results seem to suggest that FIN is more efficacious in
reducing simple, rather than complex tics. Following these results, our group has begun a
double-blind, placebo-controlled clinical trial at the TS Center of the University of Cagliari, Italy.
The idea that 5αR inhibitors may reduce tic severity by improving impulse control is indirectly
supported by our recent clinical observations on the effects of FIN in ICDs. Indeed, we found
that, in males, FIN reduced pathological gambling induced by DA receptor agonists + levodopa
in Parkinson’s disease patients (Bortolato et al, 2012).
Although our current data do not allow for a full characterization of the role of this enzyme in TS,
a number of interesting observations raise the possibility that both 5αR isoenzymes may be
directly involved in the genetic bases of the disorder. Specifically, the largest linkage study for
TS and tic disorder has identified a region of high linkage in the chromosome 2p23 (TSAICG,
2007; O’ Rourke et al, 2009), in a position directly proximal to (or partially coinciding with) the
gene SRD5A2, which encodes for 5αR2 (Morissette et al, 1996). Furthermore, two previous TS
genome scan studies pointed to chromosomal regions proximal to the gene SRD5A1 (encoding
for 5αR1) on chromosome 5p15 (Barr et al, 1999; Curtis et al., 2004).
While FIN is not likely to be an optimal therapy for TS, given its potentially serious side effects
on masculinization in boys, ongoing studies in our laboratory are currently analyzing the
contribution of different androgen steroids in the anti-tic mechanisms of FIN, using animal
28
models. These investigations may yield important contributions to our understanding of the
neuroendocrine alterations underpinning tic disorders. Another corollary of our study has led to
the analysis of association of TS and SRD5A2, the gene encoding 5αR type 2 (the primary target
of FIN in humans). Indeed, the location of this gene on chromosome 2p23 (Morissette et al.,
1996) is directly proximal to one of the regions with high linkage for TS, as recognized by the
largest linkage study for tic disorders to date (TSAICG, 2007).
Fig.1.5 Effects of finasteride (FIN) on TS male adult patients (n=16). Readapted from
Bortolato et al, 2013. See text for more details
29
1.3. AIMS OF THE THESIS
As shown in the previous section, our preliminary clinical and preclinical work has documented
that the pharmacological inhibition of 5αR reduces the impact of DA receptor agonist in a
number of rodent models related to TS and other tic disorders. The purpose of the studies
presented in this thesis is to further the understanding of the neural mechanisms whereby
neuroactive steroids influence DAergic signals in sensorimotor gating and other behavioral
manifestations related to TS. In particular, three main issues will be explored. First, these studies
will analyze the mechanisms of steroid involvement in sensorimotor gating, by verifying the
implication of neurosteroidogenic enzymes other than 5αR in the regulation of DAergic
modulation of gating, and by verifying the role of this enzyme in the PPI deficits induced by IR.
Second, we will present evidence on the neuroanatomical substrates accounting for the effects of
finasteride, as injected in different brain regions. Third, this thesis will determine the specific
role of DA receptors in the effects of FIN using both mouse and rat models of PPI deficits, in
consideration of the differences between these two species with respect to PPI sensitivity to D1
or D2 receptor agonists.
The studies presented in Chapter 2 will examine what neurosteroidogenic enzymes other than
5αR contribute to the modulation of dopaminergic responses in PPI, through the employment of
selective inhibitors. This perspective will be particularly useful to chart the specific involvement
of specific subsets of neuroactive steroids in the regulation of DAergic responses.
In chapter 3, we will present the results of studies aimed at the characterization of the anatomical
substrates of the PPI-ameliorating effects of FIN, by investigating the contribution of gonads and
different brain regions to the antiDAergic effect of this 5αR inhibitor.
Chapter 4 will focus on the impact of FIN on the spontaneous PPI deficits and alterations in
brain-regional neurosteroid profiles induced by IR, an experimental manipulation that may help
capture the relation between psychosocial stress and DAergic neurotransmission, particularly vis
a vis the regulation of sensorimotor gating.
Finally, Chapters 5 and 6 will present the results of studies aimed at identifying the mechanisms
of the DAergic receptors responsible that mediate the effects of FIN in mouse and rat models,
respectively. These results wil help understand why FIN elicits antiDAergic and PPIameliorative effects without inducing extrapyramidal effects.
30
CHAPTER 2
ROLE OF STEROIDOGENIC ENZYMES IN THE
MODULATION OF SENSORIMOTOR GATING
31
2.1. RATIONALE OF THE EXPERIMENTAL STUDIES
Our previous studies showed that the pharmacological inhibition of 5αR, the key rate-limiting
enzyme in NS and androgen metabolism (Martini et al., 1993; Martini et al., 1996; Paba et al.,
2011), counters several behavioral effects of non-selective DAergic receptor agonists in SpragueDawley rats, such as the deficits in sensorimotor gating (Bortolato et al., 2008). In addition, we
have documented that the anti-DAergic actions of the prototypical 5αR inhibitor FIN, albeit
strikingly akin to those induced by classical antipsychotic agents, are not accompanied by
catalepsy (Bortolato et al., 2008). These results have been supported by preliminary clinical
observations documenting the efficacy and high tolerability of FIN in adult male, treatmentrefractory patients affected by TS (Bortolato et al., 2007; Muroni et al., 2011) and chronic
schizophrenia (Koethe et al., 2008).
These premises highlight that the enzymatic machinery for synthesis, metabolism and signaling
of androgenic NSs may be a very promising source of novel therapeutic options for
schizophrenia and TS. To fully explore this issue, in the present study we analyzed whether the
PPI disruption induced by the DAergic receptor agonist APO may be affected by the blockade of
three critical enzymes in neurosteroidogenesis and androgen synthesis, CYP17A1, 3αHSOR/HSD, 3β- HSOR/HSD, as well as ARs (see Fig.2.1).
Animals. A total of 327 male Sprague—Dawley (Harlan, Italy) rats weighing 250—300 g were
used for this study. Animals were group-housed in cages (n=4) with ad libitum access to food
and water. The room was maintained at 22±0.2ºC on a 12/12-h dark/light cycle (with lights off at
07:00 PM). Each animal was used only once throughout the study and all efforts were made to
minimize animal suffering throughout this study. All experimental procedures were executed in
compliance with the National Institute of Health guidelines and approved by the Animal Use
Committees at the University of Cagliari and University of Kansas.
Drugs. The following drugs were used: abiraterone (ABI), FIN, T, DHT, FLU, trilostane (TRI),
indomethacin (INDO) and (R)-(-)-APO hydrochloride. For systemic injections,
32
Fig. 2.1. Schematic view of main steroid pathways of androgen synthesis and metabolism. The
numerical codes represent the enzymes described in the table. Listed are all enzyme inhibitors
used in the present study.
33
ABI, FIN, T, DHT, FLU, TRI, INDO were suspended in a VEH solution containing 5% Tween
80 and 95% SAL (0.9% NaCl). For intracerebral infusions, ABI was dissolved in hydroxypropylβ-cyclodextrin/Ringer solution (CDX/R). APO was dissolved in 0.9% SAL containing 0.1% (v/v)
of ascorbic acid to prevent oxidation. All solutions were freshly prepared on the day of testing
and administered SC and IP in an of injection volume of 1 and 2 ml/kg body weight, respectively.
The doses of each compound were selected based on preliminary data or on previous reports
indicating their biological and behavioral efficacy (Nayebi and Rezazadeh, 2004; Ugale et al.,
2004; Ahboucha et al., 2008).
Stereotaxic surgery and intracerebroventricular (ICV) Infusion procedures. Rats were
anaesthetized with Equithesin and placed in a stereotaxic apparatus (Kopf, Tujunga, CA), with
blunt ear bars to avoid damage of the tympanic membranes. Under aseptic conditions, rats were
shaved and their scalp was retracted. Bilateral craniotomies were performed above the target
sites, and stainless steel 22-G guide-cannulae (Plastics One, Roanoke, VA) were lowered slowly
into place and implanted using dental cement and two skull screws. The lengths of the cannulae
were selected so as to end 1 mm above the targeted areas with the corresponding injector
projecting 1 mm beyond guide tip. Cannulae were plugged with wire stylets, and wounds were
closed with surgical staples. According to the stereotaxic brain atlas (Paxinos and Watson, 1998),
the stereotaxic coordinates for lateral ventricles were: AP = -1 mm, ML = ±1 mm; DV = -3 mm,
referring to bregma. Rats were given antibiotic therapy for 5 days (enrofloxacin, Bayer
HealthCare, Shawnee Mission, KS) and allowed to recover in their home cages for 7-10 days
prior to testing. The day of the test, rats were subjected to ICV administrations through 33-gauge
internal cannulae (Plastics One) connected to a 50 µl syringe (Hamilton, Reno, NV, USA) by PE
tubing (Intramedic, New York, NY, USA). The rate of infusion (0.5 µl/min) was controlled by
microinjection pumps (CMA Microdialysis, Stockholm, Sweden). Injections were confirmed by
monitoring movement of liquid in the tubing via a small air bubble. The injectors were left in
place for 2 min after infusion, to fully allow diffusion of fluid. PPI testing took place
immediately after completion of infusion. On completion of testing, the location of cannula tips
was verified by injecting 5 µl of methylene blue dye into the cannula. Rats were euthanized after
5 min and their brain was removed and sliced at the point of cannula entry. Trained operators
assessed the diffusion of the dye through the target ventricle into both hemispheres.
34
Startle reflex and PPI. Startle and PPI testing were performed as previously described (Bortolato
et al., 2004), between 10 AM and 4 PM. Rats were placed in the PPI chamber for a 5-min
acclimatization period consisting of 70 dB background white noise, which continued for the
remainder of the entire session. The PPI protocol included three consecutive blocks of pulse, prepulse+pulse and “no-stimulus” trials. Specifically, unlike the first and the third block, during
which rats received only five pulse-alone trials of 115 dB, throughout the second block rats were
exposed to a pseudorandom sequence of 50 trials, consisting of 12 pulse-alone trials, 30 trials of
pulse preceded by 74, 78 or 82 dB pre-pulses intensities (ten for each level of prepulse loudness)
and eight “no stimulus” trials, where the only background noise was delivered. Intertrial intervals
were selected randomly between 10 and 15 s. Sound levels were assessed using an A Scale
setting. Percent PPI was calculated with the following formula:
with
and
representing the mean startle amplitudes for all pre-pulse+pulse trials and pulse
alone trials, respectively. The first 5 pulse-alone bursts were excluded from the calculation.
Except for the ICV microinjection experiment, all rats received two systemic injections: a
pretreatment IP 40 min and a treatment SC immediately before starting PPI session.
Data analysis. Normality and homoscedasticity of data distribution were verified by using the
Kolmogorov-Smirnov and Bartlett’s tests. Analyses were performed by multiple-way ANOVAs,
as appropriate, followed by Tukey’s test (with Spjøtvoll-Stoline correction for unequal N
whenever required) for post-hoc comparisons of the means. For % PPI analyses, main effects for
prepulse levels were consistently found throughout all the analyses, showing loudness-dependent
effects. Since no interactions between prepulse levels and other factors were found, however,
data relative to different prepulse levels were collapsed. Significance threshold was set at 0.05.
35
2.3. RESULTS
2.3.1.Effects of abiraterone and finasteride on startle and PPI parameters.
The first experiment was aimed at investigating the impact of the selective CYP17A1 inhibitor
ABI (10-50 mg/kg, IP), on the effects on startle and PPI mediated by APO (0.25 mg/kg, SC), in
comparison with the 5αR inhibitor FIN (100 mg/kg, IP). Neither ABI pretreatment [F(3,67)=1.20,
NS] nor APO treatment [F(1,67)=0.16, NS] had significant main effects on startle amplitude.
Furthermore, ANOVA did not reveal any significant ABI x APO interactions [F(3, 67)=0.61, NS]
(Fig. 2.2A). In contrast with ABI, FIN significantly reduced baseline startle amplitude [main
effect of pretreatment: F(1,35)=32.85, P<0.001]. The combination of FIN and APO, however,
did not produce significant effects on startle parameters [pretreatment x treatment interaction:
F(1,35)=1.03, NS]. The analysis of PPI revealed a significant pre-treatment x treatment
interaction, indicating that the dose of 25 mg/kg of ABI significantly reduced the PPI-disruptive
properties of APO {[F(3,67)=3.16, P<0.05]; P<0.05 for comparison VEH+APO vs ABI 25+APO;
Tukey's}, albeit only partially (Ps<0.01 for all comparisons between SAL- and APO-treated rats)
(Fig. 2.2B). In line with our previous results, FIN significantly countered APO-mediated PPI
disruption [F(1,35)=4.33, P<0.05] (Fig. 2.2B).
To verify the involvement of brain structures in the PPI-ameliorating properties of systemic ABI,
we studied the effects of ICV injections of this compound (1 µg/1 µl) on startle reflex and PPI
parameters. Startle analyses did not disclose significant main effects of ABI [F(1, 30)=3.80, NS]
or APO [F(1,30)=0.46, NS]. The interaction of the two treatments also failed to affect startle
amplitude [F(1,30)=0.41, NS] (Fig. 2.3A). In addition, we found a significant pre-treatment x
treatment interaction with respect to PPI values, indicating that ICV ABI infusions significantly
ameliorated APO-mediated PPI deficits [F(1, 30)=3.63, P<0.05] (Fig. 2.3B).
2.3.2.Effects of indomethacine on startle and PPI parameters.
In the third set of experiments, we tested the effects of the 3α-HSD inhibitor INDO (5 mg/kg, IP),
on the PPI disruption mediated by APO (0.25 mg/kg, SC). The analysis of startle amplitude
revealed a significant interaction between pretreatment and treatment [F(1, 31)=5.47, P<0.05]
36
Fig. 2.2. Effects of systemic finasteride (FIN, 100mg/kg, IP) and abiraterone (ABI, 1050mg/kg, IP) on startle reflex (A) and %PPI (B) in relation to the gating deficits induced by
systemic apomorphine (APO, 0.25mg/kg, SC). FIN and ABI doses are indicated in mg/kg.
VEH, vehicle of FIN and ABI; SAL, saline. Values are expressed as mean ± S.E.M. N= 814/group. **, P<0.01; ***, P<0.001 vs rats treated with the same pre-treatment and SAL
(pretreatment x treatment interaction); #, P<0.05 vs rats treated with VEH and APO
(pretreatment x treatment interaction). Main effects are not indicated; for further details, see
text.
37
Fig.2.3. Effects of intracerebroventricular abiraterone (ABI, 1µg/µl) on startle reflex (A)
and %PPI (B) in relation to the gating deficits induced by systemic apomorphine (APO,
0.25mg/kg, SC). ABI doses are indicated in µg (in 1µl of solution). VEH, vehicle of ABI
(Hydroxypropyl β-Cyclodextrin/Ringer solution, 20% w/v). Values are expressed as mean ±
S.E.M, N= 8-9/group. *, P<0.05 vs rats treated with VEH and saline (pretreatment x
treatment interaction); #, P <0.05 vs rats treated with VEH and APO. For further details, see
text.
38
(Fig. 2.4A). Post-hoc comparisons disclosed that this effect reflected a statistical trend for a
difference between INDO+SAL vs INDO+APO (P<0.10; Tukey’s test).
PPI analyses disclosed a significant main effect for APO [F(1, 31)=37.52, P<0.001], but not for
INDO [F(1, 31)=0.53, NS] treatment. Finally, no significant pretreatment x treatment
interactions were found [F(1, 31)=0.31, NS], indicating that INDO was not able to counter the
PPI disruption mediated by APO (Fig. 2.4B).
2.3.3. Effects of trilostane on startle and PPI parameters.
To investigate the role of 3β-HSD in the DAergic regulation of PPI, we then measured the
impact of different doses of TRI (15-60 mg/kg, IP), the selective inhibitor of this enzyme, in
association with APO (0.25 mg/kg, SC). The analysis of startle amplitude detected a significant
pretreatment x treatment interaction [F(3,72)=4,56, P<0.01] (Fig. 2.4C). Post-hoc analyses
evidenced that the combination of APO and the higher dose of TRI significantly decreased startle
amplitude in comparison with the association of VEH and SAL (P<0.05, Tukey’s). PPI analyses
did not reveal a significant main effect for pretreatment [F(3, 72)=0.19, NS]. As expected,
however, APO produced a marked PPI deficit [main treatment effect F(1, 72)=109.2, P<0.001],
which was not reversed by TRI pretreatment [pretreatment x treatment interaction: F(3,72)=0.64,
NS] (Fig. 2.4D).
2.3.4. Effects of Androgen-receptor ligands on startle and PPI parameters.
For the evaluation of the role of AR on startle reflex and gating functions, we studied the effect
of its natural ligands (T and DHT; 100 mg/kg, IP) and antagonist (FLU, 10 mg/kg, IP). As shown
in Fig. 2.5A, startle amplitude was not modified by either T pretreatment [F(1,22)=0.81, NS] or
APO treatment [F(1,22)=0.04, NS]. A statistical trend was identified for pretreatment x treatment
interaction [F(1,22)=3.34; P=0.07]. PPI analyses (Fig. 15B) revealed that T did not modify PPI,
while APO significantly reduced gating [F(1,22)=26.08, P<0.001], even though this effect was
not prevented by T pretreatment [F(1, 22)=0.01, NS].
The systemic effects of DHT (Fig.2.5C-D) were similar to those observed after T injection, with
no overall effects on startle amplitude [F(1,27)=0.05, NS]. In addition, statistical trends were
39
Fig. 2.4. Effects of systemic indomethacin (INDO, 5mg/kg, IP) and trilostane (TRI, 1560mg/kg, IP) on startle reflex (A-C) and %PPI (B-D) in relation to the gating deficits induced by
systemic apomorphine (APO, 0.25mg/kg, SC). VEH, vehicle of TRI and INDO; SAL, saline.
Values are expressed as mean ± S.E.M., N= 7-17/group. #, p<0.05 vs rats treated with VEH
and APO; °, p<0.05, °°, p<0.01 vs rats treated with TRI 15 and APO (pretreatment x treatment
interaction). Main effects are not indicated. For further details, see text.
found for the main effect of APO [F(1,27)=3.38, P=0.07] and for pretreatment x treatment
interactions [F(1,27)=3.87, P=0.06]. Furthermore, DHT did not significantly affect PPI [F(1, 27)
= 0.05, NS], while APO significantly reduced this parameter [F(1, 27)=36.33, P<0.001]. Finally,
the administration of DHT was not able to reverse APO-mediated PPI impairments [F(1,
27)=2,46, NS].
40
Fig. 2.5. Effects of systemic testosterone (T, 100mg/kg, IP) and dihydrotestosterone (DHT,
100mg/kg, IP) on startle reflex (A-C) and %PPI (B-D) in relation to the gating deficits induced
by systemic apomorphine (APO, 0.25mg/kg, SC). VEH, vehicle of T and DHT; SAL, saline.
Values are expressed as mean ± S.E.M. N= 6-10/group. Main effects are not indicated. For
further details, see text.
The last series of experiments investigated the effects of AR blockade by systemic injections of
FLU, on startle and PPI parameters. ANOVA detected a significant pretreatment x treatment
interaction on startle amplitude [F(1, 28)=30.82, p<0.001] (Fig. 2.6A). Post-hoc analyses
conducted with Tukey’s test revealed significant differences between VEH+SAL and FLU+SAL
(P<0.001), VEH+APO and FLU+SAL, (p<0.01), VEH+APO and FLU+APO (P<0.05),
FLU+SAL and FLU+APO (p<0.001). Finally, PPI analysis detected a significant main effect for
APO treatment [F(1, 28)=31.30, p<0.001], but not for FLU pretreatment [F(1, 28)=0.20, NS]. No
pretreatment x treatment interactions were found [F(1, 28)=0.72, NS] (Fig. 2.6B).
41
Fig. 2.6. Effects of systemic flutamide (FLU, 10mg/kg, IP) on startle reflex (A) and %PPI
(B) in relation to the gating deficits induced by systemic apomorphine (APO, 0.25mg/kg,
SC). VEH, vehicle of FLU; SAL, saline. Values are expressed as mean ± S.E.M. N=
8/group. #, p<0.05 vs rats treated with VEH and APO. Main effects are not indicated.
For further details, see text.
42
2.4. DISCUSSION
The major result of this study is that ABI, the selective CYP17A1 inhibitor, led to a dosedependent reversal of the PPI deficits induced by the direct DA receptor agonist APO. Notably,
these effects were observed after both systemic and ICV injections, and were not paralleled by
changes in startle amplitude. CYP17A1 is a member of the cytochrome P450 enzyme family,
responsible for the biosynthesis of androgens from pregnane derivatives (such as PROG and
PREG). The enzyme catalyzes two separate reactions, consisting in the hydroxylation of the 17α
terminal (17α-hydroxylase) and the cleavage of the bond between carbons 17 and 20 (C17,20
lyase), respectively (for a review on the enzyme, see Porubek, 2013). In particular, the latter
component is responsible for the synthesis of androgenic steroids; accordingly, males with a
selective C17,20 lyase deficiency exhibit reduced sex hormone levels and severe
undervirilization (Miller, 2012). These premises, together with our previous evidence on the
antipsychotic-like actions of 5αR inhibitors (Bortolato et al., 2008; Devoto et al., 2012), point to
a potential involvement of androgen synthesis for the modulation of DAergic control of
information processing, and may help account for the male predominance of schizophrenia, TS
and other neuropsychiatric disorders featuring gating deficits and DAergic perturbations.
The effects of ICV infusion of ABI were comparable with those ensuing systemic treatment,
suggesting the involvement of brain CYP17A1 in mediating the antipsychotic-like effects of ABI.
Although CYP17A1 expression is most abundant in the adrenal cortex, this enzyme has also
been documented in several brain regions by immunoreactivity, such as hippocampus,
hypothalamus, brainstem and cerebellum (Stromstedt and Waterman, 1995; Yamada et al., 1997;
Kohchi et al., 1998; Hojo et al., 2004). In addition, in situ hybridization studies have
documented the presence of CYP17A1 mRNA across the whole brain (Allen Atlas;
http://mouse.brain-map.org/gene/show/12855), including the cortex and the striatum, key regions
for the DAergic regulation of PPI (Swerdlow et al., 2001a). While our experiments do not allow
for a characterization of the specific brain regions involved in the anti-DAergic effects of ABI, it
is worth noting that APO-induced PPI deficits are supported by multiple areas of the CSTC
circuitry, including the NAc core, medial PFC and ventral subiculum (Hart et al., 1998;
Swerdlow et al., 2001a).
43
Our results showed that ABI and FIN elicited analogous effects on PPI regulation, although the
effects of the latter drug were more marked. It is possible that the antipsychotic-like properties of
these agents may be partially supported by similar substrates. With respect to this hypothesis, it
is worth noting that the systemic effects of FIN were reproduced by local injections into the shell
and core of the nucleus accumbens, but not other brain regions (Devoto et al., 2012). These
findings suggest that the nucleus accumbens may be responsible for the contribution of NSs in
sensorimotor gating modulation, and point to the implication of this region in the antipsychoticlike properties of ABI.
The possibility that ABI and FIN elicit similar effects is particularly interesting, in view of the
translational potential of our results. Indeed, our preclinical findings on the antipsychotic-like
properties of FIN (Bortolato et al., 2008) were matched by therapeutic effects of this agent in
patients affected by TS (Bortolato et al., 2007; Muroni et al., 2011) as well as schizophrenia
(Koethe et al., 2008) and other disorders (Paba et al., 2011). Notably, ABI is already approved
for clinical use as a chemotherapeutic agent for the treatment of castration-resistant prostate
cancer (Attard et al., 2009; de Bono et al., 2011).
Inhibitions of 5αR and CYP17A1 share a number of similar outcomes with respect to
neurosteroidogenesis. Indeed, both enzymes lead to a significant reduction of 5α-reduced
androstane derivatives, such as androsterone and 5α-androstanedione. It is worth noting that this
latter compound, in particular, may be implicated in the antiDAergic effects of FIN and ABI, as
it is postulated to result from the combined metabolic action of 5αR and CYP17A1. Nevertheless,
the lack of neurochemical analyses in the present study does not allow us to fully exclude that
the anti-DAergic effects of these antiandrogenic drugs may be supported by differential
underpinnings; indeed, only FIN produced a significant reduction of startle amplitude, likely
reflecting partially divergent mechanisms of the two drugs on behavioral reactivity.
It is worth noting that the greater impact of FIN on PPI regulation in comparison with ABI may
suggest the participation of non-androgenic NSs (not directly affected by ABI), such as AP.
Recent studies have actually suggested the implication of this neurosteroid in the regulation of
PPI (Darbra and Pallares, 2010; Darbra et al., 2012; Darbra et al., 2013).
We found that the inhibition of 3α-HSD and 3β-HSD by INDO and TRI, respectively, did not
elicit significant effects on sensorimotor gating. Both enzymes have been described in the brain
of humans and rodents (Dupont et al., 1994; Khanna et al., 1995a; Khanna et al., 1995b; Yu et
44
al., 2002), where they are posited to play key roles in neurosteroid biosynthesis, by catalyzing
the conversion of ∆5-hydroxysteroids into ∆4-ketosteroids. Nevertheless, it should be noted that
the failure to elicit effects may signify that INDO and TRI are competitive inhibitors and their
activity may therefore be limited by substrate accumulation. Thus, a conclusive assessment of
the role of 3α-HSD and 3β-HSD in PPI regulation awaits the development of novel, highly
selective non-competitive inhibitors for these enzymes.
The lack of effects of FLU suggests that the effects of androgens in PPI might not be mediated
by AR. The existence of membrane-bound receptors has been postulated by several authors,
based on the ability of androgens to rapidly modulate the activity of ion channels and
intracellular calcium levels (Heinlein and Chang, 2002). Non-genomic effects are likely
mediated through membrane ARs or act through SHBG or the c-Src kinase-AR complex.
Interestingly, a human membrane receptor for PROG has been cloned (Gerdes et al., 1998;
Bernauer et al., 2001). Furthermore, new membrane receptors for other steroid hormones have
been recently identified (Fernández-Pérez et al., 2008).
Notably, FLU has been used as a potential therapy for TS (Peterson et al., 1998), yielding very
limited and short-lived effects. Thus, while both FIN and FLU are generally labeled as “antiandrogenic” drugs, the contrast in effects between these two agents may reveal a greater
implication of 5αR in TS pathophysiology. Future double-blind, placebo-controlled clinical trials
on FLU will help settle this interesting issue.
Systemic administrations of both T and DHT did not exert any intrinsic PPI-disrupting properties,
in further support that these potent activators of AR may not be directly responsible for the
effects of FIN and ABI on DAergic responses. Interestingly, our data complement previous
results by Van den Buuse and Eikelis (2001), who documented that, in female rats, T and
estradiol, but not DHT, enhanced PPI. These divergent effects suggest the existence of sexspecific differences in the metabolic fate of T, which may result in different roles of this steroid
with respect to the regulation of PPI. In addition, our results do not exclude the possibility that,
in males, T may still play a facilitatory role for DAergic impairments. Acute T has been reported
to increase DA levels in the neostriatum and in the nucleus accumbens (de Souza Silva et al.,
2009), while chronic T may increase DA metabolism and turnover, but not content (Thiblin et al.,
1999). In addition, recent findings indicate that T and DHT increase the expression of
biosynthetic and metabolic DA enzymes in the substantia nigra of male rats (Purves-Tyson et al.,
45
2012). Future studies are warranted to analyze whether the combination of these steroids with
sub-threshold doses of DA releasers (such as d-AMPH) may result in PPI impairments.
Although the inability of T, DHT and FLU to affect APO-induced PPI deficits may seemingly
contradict our findings on the anti-DAergic properties of ABI and FIN, it should be noted that,
unlike T and DHT, most 5α-reduced androgenic NSs do not exert their neuroactive properties
through ARs. For example, 3β-diol has been shown to be a potent agonist of β estrogen receptors
(Kuiper et al., 1997), while 3α,5α-androstanediol acts as a positive modulator of GABA-A
receptors (Reddy and Jian, 2011). Furthermore, androsterone has been shown to activate
farnesoid receptors (Wang et al., 2006). The implication of these receptors in information
processing awaits further investigations.
Several limitations in the present study should be acknowledged. First, our analyses were
restricted to males, thereby limiting our ability to predict whether the observed antipsychotic-like
effects of CYP17A1 inhibition may be applicable to females. Second, another important caveat
of our study lies in the lack of available selective 3α- and 3β-HSD inhibitors. Indeed, both INDO
and TRI are known to elicit their actions also through other mechanisms, namely the inhibition
of prostaglandin synthesis and the activation of β-estrogen receptors, respectively (Vane and
Botting, 1998; Espallergues et al., 2011). Third, the conclusions that INDO and FLU did not
protect from the APO-induced PPI deficits are limited by the lack of dose-response curves;
although the doses for both compounds were selected based on a careful literature search, we
cannot completely rule out that higher concentrations (or chronic treatments) may have elicited
ameliorative effects in sensorimotor gating. Fourth, our interpretation of the reported effects of
neurosteroidogenic inhibitors on startle and PPI is restricted by the lack of accompanying
measurements of their effects on steroid profile; nevertheless, it should be clarified that this
limitation may not be easily overcome at present, since the levels of most NSs (particularly
among 3α,5α- and 3β,5α-androstane derivatives) cannot be accurately detected in rat brain
regions, due to their relatively low content.
Irrespective of the specific mechanisms, the present set of results point to CYP17A1 as a
potential target for certain behavioral disorders characterized by gating disturbances, such as
schizophrenia and TS. In particular, following our discovery of 5αR inhibitors as antipsychoticlike agents using the rat model of APO-induced PPI disruption, we have pursued the possibility
that 5αR inhibitors may have therapeutic efficacy for these conditions (Bortolato et al., 2007;
46
Koethe et al., 2008; Muroni et al., 2011). Following this perspective, it is possible that
CYP17A1 inhibitors may be novel therapeutic avenues for mental disorders featuring alterations
of sensorimotor gating. In particular, this possibility is highlighted by the recent development of
drugs that inhibit more specifically C17,20 lyase and spare 17α-hydroxylase, such as orteronel
(TAK-700; Hara et al., 2013). These next-generation drugs, which induce fewer side effects due
to the lack of effects on glucocorticoid synthesis, may have important implications in the therapy
of psychiatric disorders.
47
CHAPTER 3
ANATOMICAL SUBSTRATES OF THE EFFECTS OF 5αR
INHIBITORS ON SENSORIMOTOR GATING
48
The background section presented in Chapter 1 showed that extensive evidence suggests a role of
NSs in the pathophysiology of TS. Our previous findings showed that 5αR blockade elicits
antipsychotic-like effects (Bortolato et al, 2008). Based on this evidence, we sought to identify
the anatomical substrates of FIN-mediated antipsychotic-like effects, using the rat model of PPI
deficits induced by DA receptor agonists. Furthermore, we explored whether the effects of FIN
may be paralleled by brain-regional changes in DA efflux, as measured by microdialysis.
Animals. A total of 448 male Sprague–Dawley albino rats (Harlan, San Pietro al Natisone, Italy)
weighing 225–300 g were kept on a 12/12-h reverse light/dark cycle (with lights off from 7 AM
to 7 PM), with food and water available ad libitum. All experimental protocols were carried out
in strict accordance with the Italian Ministry of Health regulation for the care and use of
laboratory animals (DL 11692). Each animal was used only once throughout the study, with the
exception of 8 gonadectomized and 12 sham-operated rats, which were treated with vehicle
(VEH) + SAL in the first PPI experiment and subsequently (7 days after) injected with either
VEH + SAL or VEH + AMPH for the second PPI experiment.
Drugs. For systemic injections, FIN (Sigma Aldrich, St Louis, MO) was suspended in a VEH
(VEH) solution of Tween 80 in distilled water (1:9 vol:vol). For intracerebroventriculal infusions,
FIN was dissolved in DMSO-Ringer solution (final concentration, 1:1 vol:vol). Intracerebral
regional infusions were performed with a solution of FIN in cyclodextrine/Ringer solution (final
concentration, 1:5 vol:vol). APO (Sigma Aldrich) was dissolved in a solution containing 0. 9%
SAL with 0.1 mg/ml ascorbic acid to prevent oxidization. AMPH (Sigma-Aldrich) was dissolved
in SAL. All systemic administrations were performed in an injection volume of 1 (subcutaneous,
SC) or 2 ml/kg body weight (intraperitoneal, IP). Doses of FIN (both systemic and intracerebral),
APO and AMPH were based on preliminary studies.
49
Orchidectomy. Gonadectomy and sham surgeries were performed under aseptic conditions using
Equithesin (0.97 g pentobarbital, 2.1 g MgSO4, 4.25 g chloral hydrate, 42.8 ml propylene glycol,
11.5 ml 90% ethanol, distilled water up to 100 ml, 5 ml/kg, IP) for anesthesia. For both
operations, the sac of the scrotum and underlying tunica were incised; orchidectomy was
performed by bilateral ligation of the vas deferens and removal of the testes. Incisions were
closed using sterile surgical staples.
Stereotaxic surgery. Rats were anaesthetised with Equithesin and placed in a stereotaxic
apparatus (Kopf, Tujunga, CA), with blunt ear bars to avoid damage of the tympanic membranes.
Under aseptic conditions, rats were shaved and their scalp was retracted. Bilateral craniotomies
were performed above the target sites, and stainless steel 22-G guide-cannulae (Plastics One,
Roanoke, VA) were lowered slowly into place and implanted using dental cement and two skull
screws. The lengths of the cannulae were selected so as to end 1 mm above the targeted areas
with the corresponding injector projecting 1mm beyond guide tip. Cannulae were plugged with
wire stylets, and wounds were closed with surgical staples. The target locations for cannulation
from bregma were: lateral ventricles (monolaterally; AP= -1 mm, ML= ± 1 mm; DV= -3 mm);
medial PFC (mPFC) (AP= +3.0 mm, ML= ± 0.5 mm; DV= -3 mm from the skull surface);
nucleus accumbens (NAc) core (AP= + 1.2 mm, ML= ± 2 mm; DV= -7 mm from the skull
surface); NAc shell (AP= +1.7 mm, ML= ± 0.8 mm; DV= -7.4 mm from the skull surface),
dorsal caudate nucleus (AP= +0.5 mm, L= ± 3.0 mm; DV= -4 mm from the skull surface),
basolateral amygdala (AP= -2.6 mm, L= ± 4.8 mm; DV= -7 mm from the skull surface), and
ventral hippocampus (AP= -5.0 mm, L= ± 5.0 mm; DV= -6 mm from the skull surface). These
locations were selected based on their well-characterized role in the regulation of sensorimotor
gating (Swerdlow et al., 2001a).
Coordinates were taken from bregma, according to the stereotaxic brain atlas (Paxinos and
Watson, 1998). Bilateral guides were used for mPFC and NAc shell, with center-to-center
distances between the stainless steel tubing of 1 and 1.6 mm, respectively. Rats were given
antibiotic therapy for 5 days (enrofloxacin, Bayer HealthCare, Shawnee Mission, KS) and
allowed to recover in their home cages for 10-15 days before testing.
For microdialysis, animals were implanted with a vertical microdialysis probe (membrane AN
69-HF, Hospal-Dasco, Bologna, Italy; cut-off 40,000 Dalton) as previously described (Devoto et
50
al., 2008). For intracerebral administration, microdialysis probes were coupled to a 26-G
injection cannula, whose tip terminated just above the dialyzing portion of the probe and 1 mm
lateral. These modified probes were placed in the mPFC (AP= +3.0 mm, L= ±0.6 mm, V= -6.5
mm from bregma), or in the NAc shell (AP= +1.9 mm, L= ±0.7 mm, V= -8.3 mm from bregma),
according to the rat stereotaxic atlas by Paxinos and Watson (1998). The probes were oriented to
set dialysis and injection cannulae along the antero-posterior axis. The dialyzing membrane
length was 3 mm in mPFC and 2 mm in NAc shell.
On completion of testing, rats were sacrificed and the locations of cannula tips and dialysis
probes were histologically verified by trained operators blind to behavioral results. Rats with
errant locations of either device or damage of the targeted areas were excluded from analysis.
et al., 2008). All animals used in these studies were tested between 10 AM and 3 PM. Each
session was performed with a 70-dB white noise background and consisted of a 5-min
acclimatization, followed by five 115-dB pulse-alone trials and a pseudo-random sequence of
trials, including: 17 pulse-alone trials; 20 prepulse+pulse trials, in which the same acoustic bursts
were preceded by 74, 78 or 82 dB prestimuli; 8 no-stimulus trials (with only background noise).
Sound levels were assessed using an A Scale setting. All experiments were performed with a
between-subject design.
The first series of experiments was performed on orchidectomized (ORX) rats or their shamoperated (sham) controls (N=109), to verify whether gonadectomy may either reproduce or limit
FIN-induced behavioral effects by reducing plasma levels of T and its 5α-reduced metabolite
DHT. Fourteen days after castration, rats were injected with FIN (50-100 mg/kg, IP) or its VEH.
Forty min later, each group received either APO (0.25 mg/kg, SC) or SAL. After 5 min, all
animals were placed in the testing cages. In a second experiment (N=64), we injected ORX and
sham rats with FIN (100 mg/kg, IP) followed by AMPH (2.5 mg/kg, SC). The time interval
between AMPH administration and testing lasted 10 min.
The second group of experiments (N=53) was aimed at the evaluation of the ICV effects of FIN
(1-10 µg/1 µl) or its VEH (DMSO/Ringer solution, 1:1, v:v) in relation to the PPI deficits
induced by subcutaneous APO (0.25 mg/kg) or SAL. Immediately after APO injection, rats
were subjected to administration of FIN or DMSO/Ringer solution through 33-gauge internal
51
cannulae (Plastics One) connected to a 10-µl syringe (Hamilton, Reno, NV, USA) by PE tubing
(Intramedic, New York, NY, USA). The rate of infusion (0.5 µl/ min) was controlled by
microinjection pumps (CMA Microdialysis, Stockholm, Sweden). Injections were confirmed by
monitoring movement of liquid in the tubing via a small air bubble. The injectors were left in
place for 2 min after infusion, to allow diffusion of fluid. PPI testing took place immediately
after completion of infusion.
The third set of experiments mirrored the previous one, but targeted six brain areas (mPFC, NAc
core and shell, dorsal caudate, basolateral amygdala and ventral hippocampus) in bilaterally
cannulated rats (N=216: 8-10 rats/treatment group/region). Following APO (0.25 mg/kg, SC) or
SAL, rats immediately received either intracerebral FIN (0.5 µg/0.5 µl/side) or VEH
(cyclodextrine/Ringer solution, 1:5, v:v) with the aforementioned infusion conditions, and were
then tested for startle and PPI.
Microdialysis. Experiments were performed as previously described (Devoto et al., 2008). The
day after probe implantation, an artificial cerebrospinal fluid (147 mM NaCl, 4 mM KCl, 1.5
mM CaCl2, pH 6-6.5) was pumped through the dialysis probes at a constant rate of 2.2 µl/min
via a CMA/100 microinjection pump (CMA Microdialysis, Stockholm, Sweden). Samples were
collected every 20 min, and DA and DOPAC simultaneously evaluated in real time by HPLC
with electrochemical detection (ESA Coulochem II detectors, Chelmford, MA, USA).
In the first experiment (N=15), we tested the effects of FIN (100 mg/kg, IP) on extracellular DA
and DOPAC values. When a stable baseline was obtained, FIN was injected and changes in DA
and DOPAC levels were calculated as percent of mean basal value obtained from three
consecutive samples with a variance not exceeding 15%.
In the second series of experiments (N=27), we tested the effects of intracerebral FIN injections
(0.5 µg/0.5 µl for each side) in either mPFC or NAc shell on the local DA and DOPAC
concentrations. When a stable baseline was obtained (with variations ≤15% over three
consecutive time points), 33-G injection cannulae connected to pump-operated Hamilton
syringes were inserted into the guide cannulae, and either FIN (0.5 µg/0.5 µl for each side) or its
VEH (DMSO-Ringer solution) were bilaterally infused. Then, two further samples were
collected and analyzed.
52
The third series of experiments (N=28) was aimed at ascertaining whether the combined action
of intracerebral FIN and systemic APO (with the same regimen and treatment schedule used to
test PPI effects) may affect DA extracellular concentrations in the areas of infusion (mPFC and
NAc shell). Testing was performed with the same procedure as in the previous one, but
intracerebral infusions of FIN were administered 5 min following systemic injection of APO
(0.25 mg/kg, SC).
Kolmogorov-Smirnov and Bartlett’s tests. For PPI analysis, data relative to different prepulse
levels were collapsed, as no interaction between prepulse and any other factor was found in any
experiment. Percent PPI was calculated according to the formula:
with
and
,
indicating the mean startle amplitudes for all pre-pulse+pulse and pulse-alone
trials, respectively. The first 5 pulse-alone trials were excluded from the calculation. In
consideration of FIN’s ability to reduce startle magnitude (Bortolato et al., 2008), we envisioned
the possibility of drug-induced artifacts in %PPI calculation due to “floor” effects; to avoid this
occurrence, the results of %PPI analyses were always confirmed using ∆PPI values, calculated
with the formula: ∆PPI =
defined as the ratio of
(Bortolato et al., 2004). Furthermore, an activity index (AI),
and mean activity during no-stimulus trials (
was calculated and
analyzed for each animal.
Analyses were performed by multiple-way ANOVAs (with repeated measures for microdialysis
data), as appropriate, followed by Tukey’s test (with Spjøtvoll-Stoline correction for unequal N
whenever required) for post-hoc comparisons of the means. Significance threshold was set at
0.05.
3.3. RESULTS
Throughout all PPI experiments, AIs were always between the values of 8 and 12. Furthermore,
this parameter was never affected significantly by any treatment combination; thus, its analysis
will not be presented here.
53
3.3.1. Effects of FIN on behavioral performances of castrated rats.
The effects of FIN and APO on the magnitude of startle reflex in ORX rats were compared with
those observed in sham-operated controls (Fig. 3.1A). Values were analyzed by a 3-way
ANOVA, with post-surgical conditions (ORX vs sham), pre-treatment (FIN doses vs VEH) and
treatment (APO vs SAL) as factors. Startle amplitude was not affected by orchidectomy
[F(1,97)=1.29, NS], but was reduced by FIN [F(2,97)=26.85, P<0.001; P<0.01 VEH vs FIN 100,
Tukey’s] and APO [F(1,97)=6.45, P<0.05]. No specific interactions among factors were detected
[F(2,97)=0.49, NS], indicating that orchidectomy did not significantly affect the decrement in
startle produced by FIN or APO. The %PPI analysis, performed with the same design as the one
employed for startle amplitude values (Fig. 3.1B), detected that ORX rats did not exhibit any
change in PPI parameters [F(1,97)=1.29, NS]. Conversely, main effects were found for both
pretreatment [F(2,97)=6.04, P<0.01] and treatment [F(1,97)=4.80, P<0.05]. A highly significant
pre-treatment x treatment interaction was found [F(2,97)=11.38, P<0.001]. Post-hoc comparisons
revealed that APO significantly disrupted PPI (VEH+APO vs VEH+SAL, P<0.001, Tukey’s)
and that this effect was prevented by FIN (FIN 50+SAL vs FIN 50+APO, NS; FIN 100+SAL vs
FIN 100+APO, NS) specifically, while only a statistical trend was found for the 50 mg/kg dose
of FIN (VEH+APO vs FIN 50+APO, P<0.10, Tukey’s), the 100 mg/kg dose fully countered the
gating deficits induced by APO (VEH+APO vs FIN 100+APO, P<0.001, Tukey’s). FIN did not
affect the baseline %PPI in comparison to VEH-treated animals. The effects of FIN and APO on
%PPI were not influenced by surgical castration, as indicated by the lack of interactions among
the three factors [F(2,97)=0.52, NS].
The analysis of ∆PPI values (Fig.3.1C) did not confirm any main effect for pre-treatment
[F(2,97)=0.41, NS], but revealed significant main effects for treatment [F(1,97)=13.60, P<0.001].
Orchidectomy did not appear to affect ∆PPI [F(1,97)=0.57, NS]. In parallel to the previous set of
results, ANOVA identified a significant pre-treatment x treatment interaction [F(2,97)= 11.68,
P<0.001]. Tukey’s test revealed significant ∆PPI differences between VEH+APO and
VEH+SAL (P<0.001) and between VEH+APO and FIN 100-APO (P<0.05).
In a second set of experiments, we tested the effect of FIN (100 mg/kg, IP) on the PPI deficits
induced by AMPH (2.5 mg/kg, SC) (Fig.3.2A) in ORX and sham-operated rats. ANOVA
revealed that, while orchidectomy did not induce changes in startle amplitude [F(1,56)=0.79, NS],
54
this parameter was reduced by FIN [F(1,56)=17.72, P<0.001] and enhanced by AMPH
[F(1,56)=5.84, P<0.05]. No interactions among factors were found.
In line with the results obtained on APO, we found that AMPH induced a deficit in %PPI
[F(1,56)=9.76, P<0.01], while neither orchidectomy [F(1,56)=1.95, NS] nor FIN pretreatment
[F(1,56)=3.32 NS] had any significant effect on this index (Fig. 7B). A significant pretreatment x
Fig. 3.1 Effects of systemic finasteride (FIN, 50-100 mg/kg, IP) and apomorphine (APO,
0.25 mg/kg, SC) on startle reflex (A), %PPI (B) and ∆PPI (C) in sham-operated (SHAM) and
orchidectomized (ORX) rats. FIN doses are indicated in mg/kg (IP). VEH, VEH of FIN; SAL,
SAL. Values are expressed as mean ± S.E.M. N=8-12/group. ***, P<0.001 vs rats treated
with VEH and SAL (pre-treatment x treatment interaction); ###, P<0.05; ###, P<0.001 vs rats
treated with VEH and APO. Main statistical effects are not indicated. For further details, see
text.
55
treatment interaction was found [F(1,56)=10.69, P<0.01].
Post-hoc analyses revealed that the latter effect depended on the %PPI-disrupting effects of
AMPH (VEH+SAL vs VEH+AMPH, P<0.01, Tukey’s), which were reversed by FIN
(VEH+AMPH vs FIN+AMPH, P<0.001, Tukey’s). The evaluation of ∆PPI did not reveal any
main effect {orchidectomy: [F(1,56)=0.91, NS]; pre-treatment [F(1,56)=0.18, NS]; treatment
[F(1,56)=0.30, NS]}, but confirmed a significant pre-treatment x treatment interaction
[F(1,56)=7.60, P<0.01]. In contrast with the results on %PPI, Tukey’s test identified no
significant difference among treatment groups, although a statistical trend (P<0.10) was found
for the comparison between VEH+SAL and VEH+AMPH.
Fig. 3.2 Effects of systemic finasteride (FIN, 50-100 mg/kg, IP) and d-amphetamine (AMPH,
2.5 mg/kg, SC) on startle reflex (A), %PPI (B) and ∆PPI (C) in sham-operated (SHAM) and
orchidectomized (ORX) rats. VEH, VEH of FIN; SAL, SAL. Values are expressed as mean ±
S.E.M. N=8/group. *,P<0.05; **,P<0.01 vs rats treated with VEH and SAL (pre-treatment x
treatment interaction); ###, P<0.001 vs rats treated with VEH and AMPH. Statistical trends
(P<0.10) for comparisons with rats treated with VEH and SAL (both SHAM and ORX) are
indicated on the respective columns. Main statistical effects are not indicated. For further
details, see text.
56
3.3.2. Effects of intracerebroventricular FIN on APO-induced PPI disruption.
To verify whether the effects of FIN reflect central mechanisms, we next measured the effects of
ICV injections of FIN (1,10 µg/1 µl) in countering the PPI disruption mediated by subcutaneous
APO (0.25 mg/kg). While no significant change was observed in startle amplitude (Fig. 3.3A),
the analysis of %PPI revealed a main effect of APO [F(1,47)=32.77; P<0.001], and a significant
interaction of APO and FIN [F(2,47)=4.74; P<0.05]. Post-hoc analyses revealed that rats treated
with DMSO/Ringer solution + APO and FIN (1 µg) + APO manifested a significant %PPI deficit
in comparison with their SAL-treated counterparts (P<0.001 and P<0.05, respectively).
Conversely, no significant difference was found between animals treated with FIN (10 µg) +
APO and FIN (10 µg) + SAL. Moreover, the highest ICV FIN dose was found to significantly
prevent APO-mediated PPI disruption, as revealed by the significant enhancement of PPI in the
FIN (10 µg) + APO group as compared to the rats treated with DMSO/Ringer solution and APO
(P<0.05) (Fig. 3.3B). These results were further borne out by the examination of ∆PPI values
[FIN x APO interaction: F(2,47)= 4.92, P<0.05] (Fig. 3.3C).
3.3.3. Effects of local FIN injections on APO-induced PPI disruption.
The next series of experiments was aimed at the identification of the brain structures involved in
the antipsychotic-like effects of FIN. Thus, we tested the effects of FIN injections (0.5 µg/0.5
µl/side) in different forebrain regions, in combination with systemic APO (0.25 mg/kg, SC), on
startle and PPI parameters.
While APO significantly reduced startle amplitude in all experiments [Main effects for APO:
mPFC: F(1,36)=19.91, P<0.001; NAc shell: F(1,36)=17.66, P<0.001; NAc core: F(1,36)=5.20,
P<0.05; dorsal caudate: F(1,28)=30.59, P<0.001; basolateral amygdala: F(1,28)=40.79, P<0.001;
ventral hippocampus: F(1,28)=61.00, P<0.001], locally administered FIN failed to elicit any
significant alterations in startle amplitude or to elicit significant interactions with APO on this
parameter, irrespective of the region of infusion (Table 3.1).
The analysis of %PPI parameters in mPFC (Fig.3.4A-B) revealed a significant main effect for
APO [F(1,36)=66.50, P<0.001], but not for FIN [F(1,36)=1.40, NS]. The interaction between the
two treatments was also significant [F(1,36)=4.70, P<0.05]; post-hoc calculations revealed that
57
this effect was due to significant differences between the VEH+SAL and the VEH+APO groups
(P<0.001) and between the FIN+SAL and the FIN+APO groups (P<0.001); furthermore, the
difference between VEH+APO and FIN+APO, although not significant, was found to be
associated to a statistical trend (P<0.10, Tukey’s test; Fig.9B).
Fig. 3.3 Effects of intracerebroventricular finasteride (FIN) on startle reflex (A), %PPI
(B) and ∆PPI (C) in relation to the gating deficits induced by systemic apomorphine
(APO, 0.25 mg/kg, SC). FIN doses are indicated in µg (in 1 µL of solution). VEH, VEH
of FIN (DMSO-Ringer solution, v:v=1:1). Values are expressed as mean ± S.E.M. N=614/group. *, P<0.05; **, P<0.01; ***, P<0.001 vs rats treated with DMSO-Ringer
solution (or FIN1) and SAL (pre-treatment x treatment interaction); #, P<0.05 vs rats
treated with DMSO-Ringer Solution and APO. For further details, see text.
58
PFC
NAcS
NAcC
DC
BLA
VHip
INTRACEREBRAL
TREATMENT
SYSTEMIC
TREATMENT
STARTLE
AMPLITUDE
S.E.M.
VEH
SAL
689.11
±
FIN
SAL
653.56
±
40.82
32.92
VEH
APO
436.82
±
31.33
FIN
APO
495.97
±
21.67
VEH
SAL
691.20
±
FIN
SAL
711.77
±
43.12
38.15
VEH
APO
413.20
±
26.61
FIN
APO
555.48
±
28.92
VEH
SAL
680.26
±
FIN
SAL
632.42
±
65.84
34.46
VEH
APO
443.62
±
29.69
FIN
APO
616.66
±
44.63
VEH
SAL
715.57
±
FIN
SAL
720.74
±
18.53
30.15
VEH
APO
362.21
±
28.04
FIN
APO
505.20
±
40.21
VEH
SAL
730.52
±
FIN
SAL
714.96
±
37.50
47.82
VEH
APO
423.50
±
30.55
FIN
APO
427.25
±
31.41
VEH
SAL
727.16
±
FIN
SAL
720.86
±
43.96
35.12
VEH
APO
346.82
±
25.64
FIN
APO
360.83
±
12.18
}***
}***
}*
}***
}***
}***
Table 3.1 Effects of intracerebral finasteride (FIN; 0.5 µg/0.5 µl/side, bilaterally) on startle reflex
across medial prefrontal cortex (mPFC), nucleus accumbens shell (NAcS), nucleus accumbens
59
core (NAcS), dorsal caudate (DCau), basolateral amygdala (BLA) and ventral hippocampus
(VHip). APO, apomorphine (0.25 mg/kg, SC). SAL, SAL; VEH, VEH of FIN (CyclodextrineRinger solution). Braces indicate main effect for APO vs SAL. Values are expressed in arbitrary
units, as mean ± S.E.M. N=8-10/group.*, P<0.05; ***, P<0.001 vs SAL. For further details, see
text.
60
Fig. 3.4 Topographical schematizations of cannulae placements and effects of local FIN
(FIN, 0.5 µg/0.5 µl/side, bilaterally) on %PPI and ∆PPI across medial prefrontal cortex
(mPFC; A-C), nucleus accumbens shell (NAcS, D-F), nucleus accumbens core (NAcC, G-I),
dorsal caudate (CPu, J-L), basolateral amygdala (BLA, M-O) and ventral hippocampus
(VHip, P-R). APO, apomorphine (0.25 mg/kg, SC). The black lines represent the injectors
placements while the signs + and – refer to the presence of a drug or its VEH (Ringer
solution-cyclodextrine for FIN, SAL for APO). Values are expressed as mean ± S.E.M. N=810/group **, P<0.01; ***, P<0.001 vs rats treated with Ringer solution-cyclodextrine and SAL
(pre-treatment x treatment interaction); #, P<0.05; ##, P<0.01 vs rats treated with VEH and
APO. Statistical trends (P<0.10) for comparisons with rats treated with VEH and APO are
indicated on the respective columns. Significant main effects are not indicated. For further
details, see text.
61
In the analysis of %PPI values related to the experiments in the NAc core (Fig. 3.4G-I), ANOVA
disclosed significant main effects for both FIN [F(1,36)=7.10, P<0.05] and APO [F(1,36)=21.20,
P<0.001], as well as a significant interaction between the treatments [F(1,36)=6.13, P<0.05].
Post-hoc scrutiny of this effect revealed that FIN reversed the PPI decrement produced by
subcutaneous APO (Fig. 3.4H) (VEH+SAL vs VEH+APO, P<0.001; VEH+APO vs FIN+APO,
P<0.01, Tukey’s].
∆PPI analysis (Fig. 3.4I) revealed a significant main effect for APO
[F(1,36)=15.68, P<0.001], but not FIN [F(1,36)=0.21, NS]. A significant FIN x APO interaction
was also found [F(1,36)=10.35, P<0.01], which was found to depend on significant differences
between VEH+SAL and VEH+APO (P<0.001; Tukey’s test). The comparison between
VEH+APO and FIN+APO, albeit not significant, was associated with a statistical trend (P<0.10).
Administration of FIN in dorsal caudate (Fig. 3.4J-L), basolateral amygdala (Fig. 3.4M-O) and
ventral hippocampus (Fig. 3.4P-R) did not elicit any significant effect on either startle or PPI,
and failed to reverse the %PPI deficit caused by systemic APO administration [Main effects for
APO: dorsal caudate: F(1,28)=26.95, P<0.001; basolateral amygdala: F(1,28)=34.20, P<0.001;
ventral hippocampus: F(1,28)=65.49, P<0.001]. Analyses of ∆PPI values confirmed these results
[Main effects for APO: dorsal caudate: F(1,28)=64.77, P<0.001; basolateral amygdala:
F(1,28)=63.97, P<0.001; ventral hippocampus: F(1,28)=117.80, P<0.001].
3.3.4. Effects of systemic and local FIN injections on dopamine extracellular levels.
We then tested whether the effects of systemic FIN (100 mg/kg, IP) in PPI may be accompanied
by changes in DA levels in the two regions that mediated its antipsychotic-like effects, mPFC
and NAc shell. In the mPFC (mean baseline values ± S.E.M: DA= 1.2 ± 0.1 pg/40 µl dialysate;
DOPAC= 162.4±30.2 pg/40 µl dialysate), FIN induced a significant increase in DA extracellular
concentrations [F(11,83)=4.33, P<0.001], which started at 40 min after FIN injection and lasted
for the whole duration (3 h) of the experiment (Fig. 3.5A). Likewise, DOPAC concentrations
were significantly enhanced [F(11,83)=6.53, P<0.001] in a time-dependent fashion, from 100
min after FIN injection onwards (Fig. 3.5A). In the NAc shell (mean baseline values ± S.E.M:
DA = 6.0 ± 1.6 pg/40 µl dialysate; DOPAC = 3046.7 ± 681.9 pg/40 µl dialysate). FIN also
induced a significant enhancement in DA [F(11,95)=5.66, P<0.001] and DOPAC [F(11,95)=4.05,
P<0.001], starting by 80 and 180 min after FIN injection, respectively (Fig. 3.5B).
62
Fig. 3.5. Time-related effects of systemic FIN (FIN, 100 mg/kg, IP) on extracellular concentrations of
DA(DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) in medial prefrontal cortex (mPFC) (A) and
nucleus accumbens shell (NAcS) (B). Arrows represent injection time of FIN or its VEH. The period
corresponding to PPI testing is indicated by a bar alongside the time axis. Values are expressed as
mean percent of the baseline (average values of the three first samples) ± S.E.M. N=7-8/group. *,
In contrast with these results, intracerebral FIN injections failed to induce alterations of local
levels of DA (Fig.3.6) and DOPAC (data not shown) in either mPFC [F(2,22)=0.11, NS] (Fig.
3.6A) or NAc shell [F(2,28)=1.57, NS] (Fig. 3.6B). As expected (Shilliam and Heidbreder, 2003;
Devoto and Flore, 2006), APO time-dependently decreased extracellular DA levels in the Nac
shell [2-way, repeated measure ANOVA: Main effect for time: F(2,20) = 23.59, p<0.001; APO
0’ vs APO 40’, P<0.01, Tukey’s test], but not in the mPFC [F(2,28) = 4.09, NS]; notably, no
significant treatment x time interaction was found, indicating that these effects were equivalent
for FIN- and VEH-treated animals (Fig.3.6 C-D).
63
Fig.3.6 Time-related effects of local infusions of FIN (FIN, 0.5 µg/0.5 µl/side) in the medial prefrontal
cortex (mPFC; left column) and nucleus accumbens shell (NAcS; right column) on local DA(DA)
concentrations, by itself (A-B) or in combination with apomorphine (APO, 0.25 mg/kg, SC) (B-C). VEH,
VEH of FIN. Arrows represent the injection time for FIN/VEH (A-B) or APO+FIN/APO+VEH (with APO
injection immediately preceding FIN/VEH administration) (C-D). The period corresponding to PPI
testing is indicated by a bar alongside the time axis. Each time point represents the content of DA in
the sample relative to the baseline. N=6-7/group. **, P<0.05 vs baseline (Main time effect; APO 0’ vs
APO 40’). Values are expressed as mean ± S.E.M. For further details, see text.
64
3.4. DISCUSSION
The major finding of the present study is that PPI deficits induced by DAergic stimulation were
dose-dependently reversed by systemic and intracerebral administration of the 5αR inhibitor FIN.
This phenomenon was mainly observed in the NAc (particularly in its shell subdivision), the key
terminal of the DAergic mesolimbic pathway; additionally, our data indicated a statistical trend
for the involvement of the mPFC in FIN’s role on PPI regulation. Conversely, we found that the
same dose of FIN did not affect DAergic modulation of PPI when infused in other forebrain
regions, such as the dorsal caudate, basolateral amygdala and ventral hippocampus. In addition,
orchidectomy did not modify the effects of FIN on PPI, suggesting that the mechanisms
mediating these effects are not directly dependent on plasma levels of androgens.
These results extend and complement our previous reports on the antipsychotic-like profile of
5αR inhibitors in rats (Bortolato et al., 2008) and in patients affected by schizophrenia (Koethe et
al., 2008), TS (Bortolato et al., 2007; Muroni et al., 2011) and levodopa-induced pathological
gambling (Bortolato et al., 2011). In addition, these findings delineate a preliminary
topographical assessment of the neural underpinnings of 5αR’s role in the regulation of
sensorimotor gating and DAergic signaling. The implication of NAc in the 5αR-mediated
modulation of DA signaling is supported by recent studies from our group and others,
documenting the expression of 5αR isoforms and their products in this region (Saalmann et al.,
2007; Bortolato et al., 2011). Furthermore, both the core and the shell of this area have been
implicated in the behavioral actions of FIN and 5αR products (Rhodes and Frye, 2001; Frye et
al., 2002; Molina-Hernandez et al., 2005; Engin and Treit, 2007) and are pivotal substrates for
the DAergic regulation of PPI (reviewed in Swerdlow et al., 2001a). In particular, the activation
of accumbal DA receptors induces a remarkable PPI disruption in rats (Swerdlow et al., 1992;
Wan et al., 1994; Wan et al., 1996; Kretschmer and Koch, 1998). Notably, Hart and colleagues
(1998) showed that the PPI deficits induced by systemic APO administration was significantly
prevented by infusions of the typical antipsychotic HAL in mPFC, NAc core, ventral tegmental
area and ventral subiculum, but not in NAc shell.
Although the role of both compartments of NAc, core and shell, was tested in our study, it should
be noted that, in consideration of the lipid nature of FIN and the extremely rapid rate of diffusion
of steroids throughout biological membranes (Oren et al., 2004), it is extremely likely that the
65
effect of the 5αR inhibitor in each subdivision of NAc may have been affected by the
contribution of the other area or other adjacent structures. Future studies with non-lipophilic 5αR
inhibitors (with lower levels of diffusion) will be required to ultimately define the potential
contribution of NAc core and shell on the control of DAergic modulation of sensorimotor gating.
Notably, FIN injection in other brain regions, such as amygdala, hippocampus and caudate,
failed to counter APO-induced PPI deficits, suggesting that the involvement of these areas in the
5αR-mediated gating regulation may be less critical than NAc (and possibly mPFC).
Nevertheless, the participation of those structures in the systemic effects of FIN in PPI cannot be
excluded; in fact, each experiment was performed following intracerebral administration of only
one dose of the 5αR inhibitor in one individual region, raising the possibility that either higher
doses of the compound or the concomitant engagement of different regions (with multiple
simultaneous stereotaxic injections) could still attenuate the PPI deficits induced by DA receptor
activation.
In agreement with previous data, we showed that the reduction of plasma androgen levels in
ORX rats did not elicit any significant effect on startle and PPI responses at 2 weeks after
surgery (Gogos and van den Buuse, 2003; Turvin et al., 2007); additionally, gonadectomy failed
to change the effects of DAergic agonists (Wong et al., 2002) and FIN on PPI responses. These
results strongly suggest that peripheral sex hormones may play only a negligible role on gating
functions. This possibility is also confirmed by our observation that FIN exerts significant
antipsychotic-like effects also in female rats, irrespective of the phase of estrous cycle
(unpublished data).
Similarly to our previous results (Bortolato et al., 2008), FIN IP administration induced a
significant reduction in startle amplitude, irrespective of gonadectomy; in contrast, intracerebral
FIN injections that reduced the PPI-disruptive action of APO in NAc and mPFC did not
significantly affect startle magnitude, indicating a dissociation between the outcomes of FIN on
startle reflex and gating regulation. Notably, the reduction in startle amplitude elicited by
systemic administration of the 5αR inhibitor was accompanied by hypolocomotion (Bortolato et
al., 2007), as well as overt muscle relaxation (unpublished data); this last outcome was also
observed in orchidectomized rats, but not in animals subjected to intracerebral FIN infusion.
These phenomena could be due to non-androgenic 5αR substrates, such as PROG; the blockade
of 5αR mediated by FIN is likely to enhance the concentrations of this hormone by inhibiting its
66
conversion into its 5α-reduced metabolites, 5α-DHP and AP. Interestingly, PROG has been
found to reduce locomotor activity and startle response in rats (Rupprecht et al., 1999); the
apparent lack of involvement of forebrain regions may indicate that the hormone may induce
myorelaxation via the brainstem nuclei (which also regulate startle reflex) or by reducing the
respiratory function of skeletal muscles (Gras et al., 2007).
Throughout the study, startle amplitude was significantly reduced by APO and increased by
AMPH. Previous evidence showed that, while indirect DAergic activators are generally
conducive to a potentiation of startle reactivity (Kehne and Sorenson, 1978; Davis, 1980;
Johansson et al., 1995), the effects of APO on this parameter vary in relation to the dose, with
low dosages typically exerting a depressing influence on startle amplitude (likely due to the
preferential activation of presynaptic autoreceptors) and high doses increasing it (Davis and
Aghajanian, 1976). In addition, other factors have been shown to govern the effect of APO on
startle responsiveness in rats, including: genetic background (Swerdlow et al., 2000a; Swerdlow
et al., 2000b; Conti et al., 2006); duration of the time interval between treatment and testing
(Davis and Aghajanian, 1976; Young et al., 1991); functional integrity of specific brain areas,
such as the hippocampus and PFC (Swerdlow et al., 1995; Swerdlow et al., 2000c). Based on
these premises, the opposite effects of APO and AMPH on startle amplitude may reflect
differential degrees of activation of DAergic receptors, with respect to their synaptic and brainregional location.
Irrespective of this issue, the finding that both systemic FIN and DAergic activators induced
significant changes in
introduces potential confounds in the interpretation of %PPI data
(Mansbach et al., 1988; Davis et al., 1990; Swerdlow and Geyer, 1993; Kinney et al., 1999).
Because %PPI is defined as a function of
and
, its alterations can be unequivocally
assessed when the reduction of the startle reflex induced by pre-pulses are not accompanied by
significant changes in overall startle reactivity. A major caveat in %PPI computation is that
increases or reductions in startle magnitude can respectively lead to artefactual changes of this
index, due to “ceiling” or “floor” effects (Davis et al., 1990; Swerdlow et al., 2000a). Floor
effects consequent to marked reductions of
variations of
are particularly problematic, since even small
or general activity may lead to substantial changes in %PPI. Thus, in the event
of significant modifications of
, alternative strategies are required to rule out false-positive
results due to computational issues. In our study, two complementary approaches were adopted
67
to ascertain this eventuality. First, we verified that AI (the activity index, defined as the
/
ratio) was always comprised between 8 and 12 for all animals; in fact, preliminary tests
conducted in our laboratory found that AI values in this range robustly predict against floor and
ceiling effects (unpublished observations); furthermore, statistical analyses showed that AI was
not significantly affected by any treatment combination (data not shown). Second, we controlled
for computational false positives by counter-analyzing the results of each experiment using ∆PPI
values; this double assessment is a stringent and dependable method to exclude potential floor
effects in %PPI calculations due to overall reductions in startle magnitude (Bortolato et al.,
2004). The majority of significant effects in the analysis of %PPI were matched by similar
results in the statistical computation of ∆PPI, albeit with generally lower Ps or, in the case of FIN
infusion in NAc core, only a statistical trend.
Collectively, these convergent results indicate that, across most experiments, the impact of FIN
and/or DAergic agonists on PPI did not reflect substantial modifications of startle amplitude.
This conclusion was also confirmed by separate analyses on
unlike
values, which showed that,
, this parameter was not diminished by VEH+APO treatment in comparison with
VEH+SAL controls; conversely, the combination of systemic FIN with either SAL or APO
produced consensual reductions of both indices (data not shown).
The main discrepancy between %PPI and cognate ∆PPI values was found in the post-hoc
analysis of the significant interactions between FIN and AMPH. The possibility that the effects
on PPI of the two drugs may be spurious is challenged by several observations. Both APO and
AMPH are posited to disrupt PPI by activating postsynaptic DAergic receptors in NAc
(Swerdlow et al., 1990), in a fashion unrelated to variations in startle amplitude (Kinney et al.,
1999); thus, it appears unlikely that FIN’s ability to counter the PPI impairment caused by APO
is not matched by analogous properties in the presence of AMPH. Additionally, we previously
reported that FIN significantly reversed the %PPI deficits induced by 5 mg/kg (s.c.) of AMPH
(Bortolato et al., 2008) - a result that was further corroborated by ∆PPI analyses (unpublished
data). Notably, the effects of direct DA agonists in PPI are consistently more pronounced than
those elicited by AMPH, due to the latter’s dependence on endogenous mesolimbic DA levels to
exert its effects on sensorimotor gating (Geyer et al., 2001; Swerdlow et al., 2001b). Given the
statistical strictness of ∆PPI analysis, this parameter appears only sensitive to profound gating
68
impairments, and higher doses of AMPH may therefore be needed to elicit significant reductions
of ∆PPI and capture FIN’s antipsychotic-like effects with this computational approach.
In keeping with previous evidence, we found that the antipsychotic-like effects of systemic FIN
treatment were paralleled by a time-dependent increase in extracellular DA and DOPAC levels
in both NAc and mPFC (Dazzi et al., 2002). These neurochemical effects, however, were
dissociated from the antipsychotic-like actions of FIN and did not reflect the blockade of 5αR in
either region, as shown by the lack of effects on DA concentrations following intracerebral FIN
treatment under the same conditions that elicited PPI-restorative effects.
The divergence between the neurochemical effects of IP and intracerebral injections suggests
that the increase in accumbal and medial prefrontal DA levels induced by systemic FIN may
reflect the modulation of its release due to a direct action of 5αR on the somata of DA neurons in
the ventral tegmental area or on other regions, rather than alterations in DA reuptake in the
presynaptic boutons. Notably, the administration of 5αR substrate PROG has been shown to
induce DA release in striatum (Petitclerc et al., 1995), while its metabolite AP has been reported
to induce a reduction of DA release in NAc (Motzo et al., 1996; but see Rouge-Pont et al., 2002
for contrasting evidence). T and its metabolite β-estradiol have also been shown to affect DA
release (Thompson and Moss, 1994; Putnam et al., 2003).
Although the subcutaneous doses of APO used in this study are arguably consistent with
activation of DA autoreceptors and post-synaptic receptors, the observed effects of intracerebral
FIN injections suggest that the antipsychotic-like actions of this drug are likely supported by
post-synaptic processes, as they were not accompanied by significant alterations in DA
concentrations. Furthermore, previous studies suggest that the gating impairments induced by
DA direct agonists is mainly mediated by post-synaptic mechanisms (Swerdlow et al., 2001b).
Interestingly, both prefrontal pyramidal cells and accumbal medium spiny neurons, which
receive most DAergic projections in mPFC and NAc respectively (Meredith et al., 1992; Santana
et al., 2009), feature high levels of 5αR (Agis-Balboa et al., 2006). In these neurons, 5αR
substrates and products are known to modulate the signaling of several targets, such as GABAA,
AMPA, NMDA, 5-HT3 and σ1 receptors (Rupprecht and Holsboer, 1999), which may in turn
interact with - and modify - the downstream cascade of DA receptors. In line with this
possibility, preliminary studies have provided functional and anatomical evidence on a
modulatory role of NSs on cortical and striatal DAergic functions (Engel et al., 1979; Menniti
69
and Baum, 1981; Savageau and Beatty, 1981; Beatty et al., 1982; Dluzen et al., 1986; Bitar et al.,
1991; Hernandez et al., 1994; Fabre-Nys, 1998). Since 5αR isoforms catalyze an irreversible
reaction on the metabolism of several ketosteroids, including progestagens, androgens and
glucocorticoids, its blockade is likely to induce profound modifications of the steroidal profile in
the targeted neurons.
Several limitations of the present study need to be acknowledged. First, we did not analyze the
specific impact of FIN on steroid concentrations in each brain region; nevertheless, it should be
noted that the content of several NSs in rat mPFC and NAc may be below the detection limit of
currently available methods, such as radioimmunoassay or HPLC/MS (Donatella Caruso,
personal communication). Second, our study did not include the analyses of full intracerebral
dose-response curves for FIN and omitted several important brain structures implicated in gating
regulation, such as the ventral tegmental area, dorsal subiculum and globus pallidus. Third, the
high affinity of FIN for both 5αR1 and 5αR2 isozymes in rats (Thigpen and Russell, 1992) does
not allow us to elucidate the specific contribution of each isoenzyme to the antipsychotic-like
effects of 5αR inhibition. Fourth, our behavioral analyses were mainly focused on startle reflex
and PPI; however, the reduction of this index is currently regarded as one of the endophenotypes
with highest relevance to schizophrenia-associated perturbations, in view of its high degree of
isomorphism with the alterations featured in psychotic disorders (Braff et al., 2001b; Cadenhead
et al., 1993) and its sensitivity to antipsychotic agents (Braff et al., 2001a; Leumann et al., 2002;
Kumari et al., 2007). Whereas further research is needed to address these limitations, our
findings highlight the critical role of 5αR in the pathophysiology of gating deficits and psychosis,
and point to both NAc subdivisions as the key substrates implicated in the link between NSs and
a number of disorders featuring gating deficits and alterations of DAergic signaling. In particular,
given the growing evidence collected by our group in support of a potential therapeutic efficacy
of FIN in TS (Bortolato et al., 2007; Muroni et al., 2011), the current results may help elucidate
the neurobiological underpinnings of the well-characterized male predominance of this condition
(Tanner and Goldman, 1997) as well as the mechanism of action of 5αR inhibitors in this and
other related neuropsychiatric disorders (see Paba et al., 2011 for an overview of the issue).
70
CHAPTER 4
ROLE OF 5αR AND NEUROSTEROIDS IN THE GATING
DEFICITS SECONDARY TO ISOLATION REARING
71
4.1.RATIONALE OF THE STUDIES
Isolation rearing (IR) is an experimental manipulation consisting in subjecting rodents to
prolonged social deprivation from weaning through adulthood. This manipulation results in PPI
deficits. Recent findings have shown that these impairments are underpinned by alterations in
DA (Jones et al., 1992; Hall et al., 1998; Roncada et al., 2009), one of the key neurotransmitters
implicated in the pathophysiology of psychotic disorders. In particular, IR has been shown to
lead to PPI disruption through DAergic imbalances in the NAc (Powell et al., 2003), the
terminal of the mesolimbic DA system.
We recently showed that IR results in a profound reduction in 5αR expression in the NAc
(Bortolato et al., 2011). This finding, however, did not qualify whether this down-regulation is
etiologically relevant with respect to the PPI deficits observed in IR-subjected rats, or rather
represent a compensatory adaptive response aimed at balancing other molecular changes
associated with those impairments. To address this question, in the present study we examined
the functional role of 5αR in IR-induced PPI deficits by testing the impact of FIN on the PPI
deficits and NS profile in the NAc of IR-subjected rats, as compared with socially reared (SR)
counterparts.
Animals and isolation procedure. Sprague–Dawley (SD) dams (Harlan Italy, S. Pietro al
Natisone, Italy) were mated with sires and single-housed for the whole duration of their
pregnancy. Following delivery, litters were culled to 6 pups. At postnatal day 22, rats were
weaned and males were randomly assigned to either IR or SR groups. To avoid litter effects, no
more than two SR littermates were placed together in the same cage. IR-subjected rats were
reared individually in plastic cages, while SR rats were housed four per cage. Animals were
disturbed only for cleaning purposes, which consisted of changing the cage (once a week for IRsubjected rats and twice a week for SR controls). Both groups were housed in the same room so
that IR rats maintained visual, auditory, and olfactory contact with the other animals. The room
72
was kept under standard conditions of temperature and humidity, and food and water was
available ad libitum. Artificial light was on from 8 PM to 8 AM. Experiments were conducted
during the light-off phase of the day.
Drugs. FIN and HAL were used in this study. FIN (Polichimica, Bologna, Italy) was suspended
in Tween 80 and diluted with 0.9% SAL solution (1% Tween 80/SAL; 1:9 vol:vol). HAL
(Sigma Aldrich, Milan, Italy) was dissolved in a single drop of 1 M HCl and diluted with SAL.
Both drugs were administered IP in an injection volume of 2 ml/kg, 40 min before testing. All
experimental procedures were approved by the local ethics committee and carried out in strict
accordance with the guidelines for experimental animals care (EEC Council 86/609; Italian D.L.
27/01/92, No. 116).
Startle reflex and PPI. Startle testing was performed as described in Frau et al. (2007). Briefly,
the apparatus used for detection of startle reflexes (Med Associates, St Albans, VT, USA)
consisted of four standard cages placed in sound-attenuated chambers with fan ventilation. Each
cage consisted of a Plexiglas cylinder of 9 cm diameter, mounted on a piezoelectric
accelerometric platform connected to an analog-digital converter. Two separate speakers
conveyed background noise and acoustic bursts, each one properly placed so as to produce a
variation of sound within 1 dB across the startle cage. Both speakers and startle cages were
connected to a main PC, which detected and analyzed all chamber variables with specific
software. Before each testing session, acoustic stimuli and mechanical responses were calibrated
via specific devices supplied by Med Associates. After 8 weeks of IR manipulation, IR- and SRsubjected rats were injected with FIN (25-100 mg/kg, i.p.), HAL (0.1 mg/kg, i.p., as positive
control) or their VEHs, and placed 35 min later in the testing cages, for a 5-min acclimatization
period with a 70 dB white noise background that continued for the remainder of the session.
Each session consisted of three consecutive sequences of trials (periods). Unlike the first and the
third period, during which rats were presented with only five pulse-alone trials of 115 dB, the
second period consisted of a pseudorandom sequence of 50 trials, including 12 pulse-alone trials,
30 trials of pulse preceded by 74, 78, or 82 dB pre-pulses (10 for each level of pre-pulse
loudness), and eight no stimulus trials, where only the background noise was delivered. Intertrial intervals were selected randomly between 10 and 15 s. Percent PPI was calculated using the
73
following formula: 100-[(mean startle amplitude for pre-pulse pulse trials/mean startle
amplitude for pulse alone trials) x 100]. As no significant interactions between prepulse levels
and treatment were found in the statistical analysis, the % PPI values were collapsed to represent
average PPI.
Liquid chromatography-tandem mass spectrometry analyses. A separate set of IR-subjected rats
and SR controls were sacrificed. NAc samples were harvested according to the indication of the
atlas of Paxinos and Watson (1998). Given the low size of this region, analyses were performed
on a broader area of the brain, also including the ventral and mediodorsal striatum, in order to
achieve a critical content of NSs that may be detected by LC-MS/MS analysis. Sample
extraction and purification were performed as previously described (Caruso et al., 2008). Briefly,
samples were added with internals standards, homogenized in 2 ml of MeOH/acetic acid (99:1,
v/v) using a tissue lyser (Qiagen, Italy). After an overnight extraction at 4 °C, samples were
centrifuged at 12,000 rpm for 5 min and the pellet was extracted twice with 1 ml of
MeOH/acetic acid (99:1, v/v). The organic phases were combined and dried with a gentle stream
of nitrogen in a 40 C water bath. The samples were resuspended with 3 ml of MeOH/H2O (10:90,
v/v) and passed through a SPE cartridges, previously activated with MeOH (5 ml) and
MeOH:H2O 1:9 (v/v) (5 ml), the steroids were eluted in MeOH, concentrated and transferred in
autosampler vials before the LC–MS/MS analysis.
Positive atmospheric pressure chemical ionization (APCI+) experiments were performed with a
linear ion trap-mass spectrometer (LTQ, ThermoFisher Co., San Jose, CA, USA) using nitrogen
as sheath, auxiliary and sweep gas. The instrument was equipped with a Surveyor liquid
chromatography (LC) Pump Plus and a Surveyor Autosampler Plus (ThermoFisher Co., San Jose,
CA, USA). The mass spectrometer was employed in MS/MS mode using helium as collision gas.
The LC mobile phases were (A) H2O/0.1% formic acid and (B) methanol (MeOH)/0.1% formic
acid. The gradient (flow rate 0.5 ml/min) was as follows: T0 70%A, T1.5 70%A, T2 55%A, T3
55%A, T35 36%A, T40 25%A, T41 1%A, T45 1%A, T45.2 70%A, T55 70%A. The split valve was
set at 0–6.99 min to waste, 6.99–43.93 min to source and 43.93–55 to waste. The Hypersil Gold
column (100 mm × 3 mm, 3 µm; ThermoFisher Co., San Jose, CA, USA) was maintained at
40 C. The injection volume was 25µl and the injector needle was washed with MeOH/water 1/1
(v/v). Peaks of the LC–MS/MS were evaluated using a Dell workstation by means of the
74
software Excalibur® release 2.0 SR2 (ThermoFisher Co., San Jose, CA, USA). Quantitative
analyses were performed on the basis of calibration curves prepared and analyzed using internal
standards. Analyses targeted the following NSs: PREG, PROG, DHP, AP, isopregnanolone,
DHEA, T, DHT, 3α-diol and 17β-estradiol. Calibration curves were extracted and analyzed as
described above for samples. Limits of quantification, precision, and accuracy have been
previously reported (Caruso et al., 2008).
Statistical analyses. Normality and homoscedasticity of data distribution were verified by using
the Kolmogorov-Smirnov and Bartlett’s test. Statistical analyses were performed with one-way
or two-way ANOVAs, as appropriate. Post-hoc comparisons were performed by Tukey’s test for
factorial designs and Dunnett’s test for repeated measures. Significance threshold was set at 0.05.
4.3. RESULTS
4.3.1. Effects of FIN and HAL on IR-induced changes in startle and PPI.
The effects of FIN on startle magnitude were analyzed by a two-way ANOVA design, with
treatment and rearing condition as factors (Fig. 4.1A). While no effect of rearing conditions was
identified [F(1,63)=2.38, NS], ANOVA detected a significant main effect for FIN treatment
[F(3,63)=2.79, P<0.05], which was found to reflect a significant difference between the dose of
100 mg/kg (i.p.) and the VEH (P<0.05, Tukey’s test). Furthermore, a significant interaction
between rearing conditions and treatment was found [F(1,63)=3.69, P<0.05]. Post-hoc
comparisons assessed that this effect reflected a significant difference between SR rats treated
with the dose of 100 mg/kg and VEH (P<0.01).
Analyses of %PPI values, performed with the same statistical design, (Fig. 4.1A), disclosed that
IR-subjected rats displayed a significant reduction in this index in comparison with SR controls
[F(1,63)=4.62, P<0.05]. While no main effects were found for the treatment [F(1,63)=2.20, NS],
a significant rearing condition x treatment interaction was detected [F(3,63)=4.06, P<0.05].
Tukey’s test revealed that IR-induced PPI disruption (P<0.05 for IR-VEH vs SR-VEH
comparison) was reversed by both 50 mg/kg (P<0.05) and 100 mg/kg (P<0.01) FIN doses.
75
Fig. 4.1 Effects of finasteride (F) and haloperidol (HAL) on startle magnitude (A) and
%PPI (B) in isolation-reared (IR) and socially-reared (SR) rats. Values represent mean ±
SEM for each experimental group. Treatments are indicated below the horizontal axis.
Data related to animals treated with HAL’s vehicle are not represented. All doses are
given in mg/kg (IP). VEH, vehicle of finasteride. * P<0.05, ** P<0.01 in comparison to SRVEH group; # P<0.05, ## P<0.01, in comparison to IR-VEH group. For further details, see
results section.
76
It should be noted that, in a separate set of observations, none of the doses of FIN induced
catalepsy or other extrapyramidal manifestations in either IR or SR rats (data not shown).
In a parallel set of studies, we verified the effects of HAL on IR-induced changes in startle
amplitude and PPI (Fig. 4.1B). HAL reduced startle amplitude [Main treatment effect:
F(1,33)=7.18, P<0.05]. Conversely, no significant main effects for rearing condition or
significant interactions between factors were found. PPI analyses detected significant
differences between SR and IR-subjected rats [F(1,33)=26.69, P<0.001]. Furthermore, ANOVA
found a main effect for treatment [F(1,33)=22.56, P<0.001], as well as a significant rearing
condition x treatment interaction [F(1,33)=5.89, P<0.05]. Post-hoc analyses revealed that HAL
significantly reversed the PPI deficit induced by IR condition (P<0.001).
4.3.2 .Effects of FIN on IR-induced changes in NS profile in the NAc.
We then evaluated the effect of FIN (100 mg/kg, i.p.) on the NS profile in the NAc and striatum
of IR-subjected rats and their controls. As shown in Fig. 4.2A, IR resulted in a profound
reduction in the concentration of PREG. [Main effect for condition: F(1,36)=11.55, P<0.05].
Furthermore, FIN led to a marked enhancement in the concentration of this NS [Main effect for
treatment: F(1,36)=25.10; P<0.0001]. No interactions between condition and treatment were
found [F(1,36)=7.63, NS]. Analyses of PROG levels (Fig. 4.2B) found that this NS was not
influenced by IR [[Main effect for condition: F(1,36)=1.42, NS], FIN treatment [Main effect for
treatment: F(1,36)=0.45, NS] or their interaction [F(1,36)=1.00, NS]. In line with these results,
the analysis of PROG/PREG ratio disclosed a significant enhancement in IR-subjected rats
(Main effect: IR vs SR: P<0.05) and a dramatic reduction caused by FIN treatment (Main effect:
FIN vs VEH: P<0.0001) (Fig. 4.2H). As shown in Fig. 4.2C, we found that DHP levels were
significantly reduced by IR [Main effect for condition: F(1,38)=4.25, P<0.05], but not by FIN
[Main effect for treatment: F(1,38)=0.98, NS] or by condition x treatment interactions
[F(1,38)=1.49, P<0.05]. In addition, the DHP/PROG ratio (Fig. 4.2I) was found to be reduced
by IR (Main effect for rearing condition: P<0.05). Analysis of AP levels revealed no main
effects for either rearing condition [F(1,36)=0.11, NS] or treatment [F(1,36)=0.34, NS]; however,
a significant interactions of these factors was found [F(1,36)=7.86, P<0.01]. Post-hoc analyses
77
Fig. 4.2. Effects of finasteride (FIN; 100 mg/kg, i.p.) and its vehicle (VEH) on the levels of
neuroactive steroids in the Nucleus Accumbens and striatum of isolation-reared (IR) and
socially-reared (SR) rats. The administration of FIN inverted these imbalances increasing
the AP levels in the same brain region. Values are expressed as pg/mg ± SEM for each
experimental group. Treatments are indicated below the horizontal axis. **, P<0.01; ***,
P<0.001 in comparison with vehicle-treated rats (main treatment effect); °, P<0.05; °° ,
P<0.01 in comparison with SR rats (main rearing condition effect); ^ P<0.05, ^^ p<0.01 in
comparison to VEH-SR group (rearing condition x treatment interaction); $$ p<0.01 in
comparison to VEH-IR group (rearing condition x treatment interaction). VEH, vehicle; FIN,
finasteride. For further details, see results section.
78
revealed that, while VEH-treated IR-subjected rats exhibited a significant reduction in AP levels
in comparison with their SR counterparts (P<0.05), FIN treatment induced a paradoxical
enhancement in AP levels in IR rats (P<0.05) (Fig. 4.2D). These results were paralleled by the
changes in AP/DHP ratio, which revealed a positive rearing x treatment interaction
[F(1,36)=9.14; P<0.01]. Indeed, SR VEH-treated rats were found to display higher ratios than
either IR VEH-treated (P<0.05) or SR FIN-treated (P<0.01) rats. Furthermore, in IR rats FIN
enhanced this ratio (P<0.01) in comparison with VEH-treated counterparts (Fig. 4.2J).
While DHEA levels were not affected by IR [Main effect for condition: F(1,39)=0.77, P<0.05],
we found a statistical trend for a reduction induced by FIN
[Main effect for treatment:
F(1,39)=3.43, P=0.07], but no condition x treatment interactions (Fig. 4.2E). The DHEA/PREG
ratio was found to be increased by IR and reduced by FIN (Main effects: Ps<0.01) (Fig. 4.2K).
While IR failed to affect T levels (Fig. 4.2F), it caused a reduction in estradiol levels [Main
effect for condition: F(1,38)=5.51, P<0.05] (Fig. 4.2G); conversely, FIN failed to affect the
levels of either steroid (Figs. 3F-G). No significant differences were found in the analysis of the
estradiol/T ratio (Fig. 4.2L). Levels of isopregnanolone, DHT, and 3α-diol could not be
compared reliably, as their concentrations remained under detection limits in more than 70% of
samples.
4.4. DISCUSSION
The main finding of the present study is that the potent 5αR inhibitor FIN dose-dependently
countered the deficits in PPI caused by IR, a well-characterized neurodevelopmental model of
juvenile psychosocial stress leading to schizophrenia-related alterations. Similarly to previous
experiments (Bortolato et al., 2008), the effects of FIN were generally similar to those of the
benchmark antipsychotic HAL; in spite of this analogy, the 5αR blocker failed to induce
catalepsy or other overt extrapyramidal manifestations. This result confirms and extends our
previous observations on the ameliorative effects of FIN and other 5αR inhibitors on psychosisrelated alterations elicited by direct and indirect DAergic agonists in rodents (Bortolato et al.,
2008; Devoto et al., 2012; Frau et al., 2013). Furthermore, these data support preliminary
clinical evidence on the possible therapeutic potential of FIN in schizophrenia (Koethe et al.,
2008), as well as other disorders featuring DAergic alterations, including TS (Bortolato et al.,
79
Fig. 4.3 Synoptic schematization of the observed effects of isolation rearing (IR) and
finasteride (FIN) on neurosteroid (NS) levels in the nucleus accumbens (NAcc). Black and
white arrows represent increased and decreased enzyme activities, respectively. For
further details, see text.
2007; Muroni et al., 2011) and impulsive behaviors induced by DAergic agonists in susceptible
patients (Bortolato et al., 2012).
In agreement with our previous findings (Bortolato et al., 2008; Devoto et al., 2012), startle
reflex was significantly reduced by FIN in SR animals. This phenomenon, however, was not
observed in IR-exposed animals, suggesting that IR may reduce sensitivity to the effects of 5αR
inhibitors with respect to startle reactivity. Notably, the lack of a selective effects of FIN on
startle amplitude in IR animals also rules out possible confounds in PPI analysis due to
alterations in this parameter (Swerdlow et al., 2000a).
We previously showed that the effects of FIN on the regulation of PPI are likely supported by
changes in the signaling of the postsynaptic DA receptors in the NAc (Devoto et al, 2009).
80
Furthermore, several studies have shown that the gating deficits induced by IR are primarily
mediated by this region (Powell et al., 2003; Leng et al., 2004). Our results showed that IR leads
to significant reductions in PREG, DHP, AP and estradiol in the NAcc. These data are in
substantial agreement with previous findings from our group and others, indicating that IR leads
to an overall reduction in NS levels in the cortex (Serra et al., 2000; Bortolato et al., 2011). We
also documented no significant changes in PROG, DHEA and T levels, suggesting that the
generalized reduction in NS biosynthetic pathways may be partially offset by the downregulation of catabolic enzymes and/or the activation of alternative anabolic processes. The
significant changes in steroid ratios are in keeping with the previously documented downregulation of 5αR in IR-subjected rats (as indicated by the reduction in DHP/PROG ratio);
furthermore, our data suggest that this manipulation may lead to functional alterations of other
neurosteroidogenic enzymes, such as a potential enhancement of the activities of 3β-HSD and
CYP17A1, which catalyze the conversion of PREG into PROG and DHEA, respectively (see
Fig. 4.3).
The behavioral effects of FIN were paralleled by a marked increase in the concentrations of
PREG and a statistical trend for an enhancement of DHEA levels in the NAc and striatum. The
increased content of these 3β-hydroxy-∆5-steroids may signify their accumulation in response to
the inhibition of 5αR, given that they are the precursors of the two main 5αR substrates, i.e.
PROG and T. The possibility that the antipsychotic-like actions of FIN may be contributed by
the enhancement of PREG (and, possibly, DHEA) levels is in keeping with emerging evidence
supporting the therapeutic potential of these NSs for cognitive and negative symptoms in
schizophrenia (Strous et al., 2003; Marx et al., 2009; Marx et al., 2011; Ritsner et al., 2014).
Furthermore, PREG was recently shown to rescue the spontaneous PPI deficits in DA
transporter knockout mice, which are also reflective of DA receptor hyperactivation in the NAc
(Wong et al., 2012). Although the biological actions of PREG and DHEA are not completely
understood, they act as potent agonists of σ1 receptors (Maurice et al., 2001), which have been
shown to be essential for the modulation of DA D1 receptor signaling (Navarro et al., 2010).
Notably, we recently showed that the PPI-ameliorating properties of FIN are due to the
suppression of D1 receptor signaling (Frau et al., 2013). In addition, PREG and DHEA have
been shown to exert neuroprotective properties against apoptosis and oxidative damage, which
may play an important role in cognitive deficits in schizophrenia. Future studies will be needed
81
to elucidate whether the effects of FIN in IR-subjected rats are mediated by PREG and DHEA,
and substantiate the role of σ1 receptors on the regulation of PPI and other schizophrenia-related
endophenotypes.
We found that, while FIN reduced AP levels and AP/DHP ratio in SR rats, this drug had
surprisingly opposite effects in IR-subjected rats. This paradoxical phenomenon may have been
caused by functional changes in 3α-HSOR, the reversible enzyme involved in the interconversion of AP and DHP. Indeed, this enzyme has been shown to serve both the synthesis and
degradation of AP, depending on the prevalence of its cytosolic or membrane-bound isoform
(Mellon and Vaudry, 2001). Depending on the changes induced by IR on the expression and
activity of this enzyme, the acute 5αR inhibition may have altered the inter-regulatory cross-talk
between these two enzymes, leading to a reduction of the conversion of AP into DHP.
Alternatively, the increase in AP may reflect an altered mechanism of action of FIN, due to
alterations in 5αR structure and intracellular localization in IR-subjected rats. In order to inhibit
5αR, FIN needs to be accepted as a substrate of this enzyme and reduced to dihydroFIN; in turn,
this metabolite forms an adduct with NADP, which is then covalently bound to the enzyme
(Bull et al., 1996). Thus, it is possible that the down-regulation of 5αR may limit its
transformation into dihydroFIN and unmask other secondary mechanisms of this drug, such as
the inhibition of 5β-reductase (Drury et al., 2009), which may paradoxically enhance the
synthesis of AP and other 5α-reduced NSs.
The enhancement in AP in IR rats may play a key contributory role in the effects of FIN. AP is
known to activate GABA-A receptors, whose action may indeed interact with potential changes
in DA receptor signaling in DAergic regions. Interestingly, AP has been shown to counter the
enhancement of aggression and fear-related memory in isolated mice (Pinna et al., 2008). Future
studies will be needed to test the potential role of AP in the modulation of PPI by FIN in IRsubjected rats.
Although our results may provide critical element of insight into the role of 5αR in the
regulation of PPI, several limitations need to be acknowledged. Our neuroactive steroid analyses
could not be limited to NAcc, in view of the small size of this region, and incorporated also the
striatum; however, it is possible that the two regions, albeit organized similarly, may different
82
significantly with respect to NS profiles. In addition, our studies failed to test whether the
changes in NSs may be directly conducive to changes in PPI. Future studies with systemic and
intra-accumbal infusion of PREG and AP are warranted to further confirm the potential
involvement of these NSs in the therapeutic actions of FIN.
Another important limitation of our study lies in our lack of data on the relevance of the two
major 5αR types, 1 and 2, in the anti-psychotic-like effects of FIN in IR-subjected rats. Both
isoforms are expressed in the NAc and down-regulated by IR (Bortolato et al., 2011; Castelli et
al., 2013). In particular, the involvement of 5αR2 in the observed effects of FIN may be
particularly meaningful from a translational perspective, given that FIN has a higher affinity for
this isoenzyme in humans (Paba et al., 2011). Furthermore, given the importance of 5αR2 in the
synthesis of androgens, its implication in the antiDAergic mechanisms of FIN may account for
recent findings from our group on PPI-ameliorating properties of abiraterone, the main
androgen-synthetic enzyme, in rats (Frau et al., 2014). The involvement of androgens in the
effects of FIN may also provide a mechanistic frame to account for the well-documented sex
differences in schizophrenia and their relation to DAergic signaling (Godar and Bortolato, 2014).
Due to the relatively small size of our biological samples, we were unfortunately unable to
obtain a reliable measurement of 5α-reduced androgens, such as DHT, 3α-diol and 3β-diol.
Future studies with different detection systems, such as RIA, will be necessary to verify the
potential involvement of androgens in the sequelae of IR and antipsychotic-like effects of FIN.
Irrespective of these limitations, our findings have confirmed FIN’s antipsychotic-like properties,
by documenting its ability to correct the gating alterations associated with IR, a
neurodevelopmental model of schizophrenia with high face, construct and predictive validity.
The actions of FIN are accompanied by changes in NS levels, such as an increase in PREG and
AP, which countered the effects of IR. These data further confirm previous evidence pointing to
5αR as a promising target for the development of novel treatments for schizophrenia and other
neuropsychiatric disorders featuring stress-related gating impairments, such as TS and OCD.
83
CHAPTER 5
IDENTIFICATION OF THE DOPAMINE RECEPTORS
MEDIATING THE ANTIPSYCHOTIC-LIKE EFFECTS OF 5αR
INHIBITORS: EVIDENCE IN MICE
84
As highlighted in the background, our research has shown that the pharmacological inhibition of
5αR, counters several behavioral effects of non-selective DAergic receptor agonists in SpragueDawley rats, including hyperlocomotion, stereotypies and sensorimotor gating deficits (Bortolato
et al., 2008; Devoto et al., 2012). In addition, we have documented that the antiDAergic actions
of the prototypical 5αR inhibitor FIN, albeit strikingly akin to those induced by classical
antipsychotic agents, are not accompanied by overt extrapyramidal effects (Bortolato et al.,
2008). Although these promising translational findings highlight the potential for 5αR inhibitors
as a valuable therapeutic target for TS, the biological bases of the neuropsychiatric effects of
5αR inhibitors, however, remain elusive. In particular, a crucial yet unresolved issue concerns
the specific involvement of the two main DA receptor families, D1- and D2 –like, in the
antipsychotic-like profile of 5αR inhibitors. To address this problem, in the present study we
analyzed the impact of FIN on the behavioral responses to DA receptor agonists in C57BL/6
mice, including the disruption of PPI. Previous studies on the pharmacological modulation of
PPI have clearly indicated marked interspecies and interstrain differences in the response of rats
and mice to different DAergic agonists (Ralph and Caine, 2005; Ralph and Caine, 2007). For
example, previous research has shown that, in sheer contrast with rats, the activation of D1-like,
but not D2 –like receptors induces hyperlocomotion and PPI deficits in C57BL/6 mice (GeterDouglass et al., 1997; Halberda et al., 1997; Ralph and Caine, 2005; Ralph-Williams et al.,
2003a), the most common inbred murine strain used in research and the only one whose genome
has been entirely sequenced to date (Waterston et al., 2002). This background prompted us to
test the differential impact of FIN on PPI and other behavioral responses of C57BL/6 male mice
to D1-and D2-like, as well as non-selective agonists. The rationale for these experiments was
further informed by our need to broaden the translational value of our previous findings with an
additional preclinical platform that may allow for future investigations on transgenic mice.
Animals. A total of 242 male adult C57BL/6 mice (25-35 g) (Charles River; Como, Italy and
Wilmington, MA, USA) were used. Animals were group-housed in cages (n=4) with ad libitum
85
access to food and water. The room was maintained at 22±0.2ºC on a 12/12-h dark/light cycle
(with lights off at 07:00 PM). All experimental procedures were executed in compliance with the
National Institute of Health guidelines.
Drugs. For systemic injections, FIN (Sigma Aldrich, St Louis, MO, USA) was suspended in a
VEH (VEH) of 1% Tween 80 in 0.9% SAL. APO (Sigma Aldrich) was dissolved in SAL with
0.1 mg/ml ascorbic acid to prevent oxidization. The full D1-like agonist SKF82958 and D2-like
agonist quinpirole (QUIN) (Sigma-Aldrich) were dissolved in SAL. Systemic administration
volume was 10 ml/kg body weight (IP).
et al., 2007), between 10 AM and 3 PM. Animals were injected with either FIN (25-50 mg/kg, IP)
or VEH, followed, 30 min later, by a DAergic agonist [SKF82958 (0.3 mg/kg, IP), QUIN (0.5
mg/kg, IP), APO (0.5 mg/kg, IP)] or SAL. Behavioral testing began 10 min after the last
injection; each session lasted 28-30 min and was performed with a 70-dB white-noise
background. Following a 5-min acclimatization period, mice were exposed to five consecutive
115-dB pulse-alone bursts; subsequently, the speakers delivered a pseudo-random sequence of
trials, including: 1) pulse-alone 115-dB trials (n=17); 2) pre-pulse+pulse trials, in which the same
pulse were preceded by 74, 78 or 82 dB pre-pulses (n=60; 20 for each pre-pulse level); 3) nostimulus trials, in which only background noise was delivered (n=8). Sound levels were assessed
using an A Scale setting. Percent PPI was calculated with the following formula:
with
and
representing the mean startle amplitudes for all
pre-pulse+pulse trials and pulse alone trials, respectively. The first 5 pulse-alone bursts were
excluded from the calculation.
Open-field locomotor behaviour. Locomotor activity was measured in a novel open field. The
apparatus was a Plexiglas square grey arena (40 x 40 cm) surrounded by 4 black walls (40 cm
high). On the floor, two concentric zones of equivalent areas were defined: a central square
quadrant and a peripheral frame directly adjacent to the walls. Background light and noise were
maintained at 10 lux and 70 dB, respectively. Each experimental session lasted 120 min. Mice
86
were initially placed in the center of the arena; after 30 min, they were briefly removed and
treated with either FIN (50 mg/kg, IP) or VEH, and quickly repositioned in the center of the open
field. Thirty min later, mice were treated with SKF82958 (0.3 mg/kg, IP), QUIN (0.5 mg/kg, IP)
or SAL, and monitored for the remainder of the session (60 min). Analyses of locomotor activity
were performed using Ethovision pathway tracking software (Noldus Instruments, Wageningen,
The Netherlands). Behavioral measures included the distance travelled, time spent in the central
zone, number of rearing episodes and meandering (defined as the ratio between turn-angle
degrees and total distance) (Kalueff et al., 2007).
Stereotyped behavior. Mice were injected with either FIN (50 mg/kg, IP) or its VEH; 30 min
later, they received either APO (3 mg/kg, IP) or SAL. Stereotyped behaviors were then evaluated
throughout the following 30-min period, by two separate trained observers blinded to the
treatments. Behaviors were monitored for 30 s, at intervals of 2 min, for a total of 15
observations. At the end of each period, behaviors were assigned numerical scores based on a
modified version of the rating scale used by Benus et al. (1991) for the evaluation of APOinduced stereotyped behaviors (for a detailed description of the scale, please see the legend of
Fig. 6). Stereotypy scores were calculated as mean values of the scores of the two observers.
Inter-observer consistency was evaluated by Cronbach’s alpha coefficient (Cronbach, 1951),
which was consistently higher than 0.85.
Catalepsy. Catalepsy, defined as the reduction in the ability to initiate movement and a failure to
achieve correct posture, was assessed via the bar test. Thirty minutes following treatment with
FIN (25-200 mg/kg, IP), the forepaws of the mice were placed on a bar positioned 4-cm above
the bench and the length of time during which the animal retained this position was recorded by
an observer unaware of the treatment.
Kolmogorov-Smirnov and Bartlett’s tests. Analyses were performed by multiple-way ANOVAs
(with repeated measures for the analyses of the time-related effects on locomotor behaviors in
the open field and stereotypies), as appropriate, followed by Tukey’s test (with Spjøtvoll-Stoline
correction for unequal N whenever required) for post-hoc comparisons of the means. For %PPI
87
analyses, data relative to different prepulse levels were collapsed, since no interactions were
found between prepulse levels and other factors throughout the study. Significance threshold was
set at 0.05.
5.3. RESULTS
5.3.1. Effects of FIN and DAergic agonists on startle and PPI.
The first experiment (n = 63; 9—14/group) was aimed at the evaluation of the effects of FIN
(25—50 mg/kg, IP) or VEH on the PPI disruption induced by the D1-receptor agonist SKF82958 (0.3 mg/kg, IP) (Fig. 5.1). The effects of FIN on startle amplitude were studied with a
two-way ANOVA, with pre-treatment (FIN vs VEH) and treatment (SKF-82958 vs SAL) as
independent factors (Fig. 5.1A). ANOVA revealed that SKF- 82958 significantly decreased
baseline startle magnitude [main effect: F(1,57) = 4.54, P < 0.05]. Conversely, FIN did not
reduce startle amplitude [main effect: F(2,57) = 1.50, NS]; furthermore, no significant
pretreatment x treatment interactions were detected [F(2,57) = 0.05, NS]. The same design was
Fig.5.1. Effects of systemic finasteride (FIN, 25—50 mg/kg, IP) and SKF-82958 (SKF, 0.3
mg/kg, IP) on startle reflex (A) and %PPI (B) in C57BL/6 mice. FIN doses are indicated in
mg/kg. VEH, vehicle of FIN; SAL, saline. Values are expressed as mean ± S.E.M., N = 9—14/
group. *P < 0.05, ***P < 0.001 vs rats treated with SAL (pre-treatment x treatment interaction);
ºººP < 0.001 vs rats treated with VEH and SKF (pre-treatment x treatment interaction).
88
used to run %PPI analyses (Fig. 5.1B). Significant main effects were found for both pretreatment
[F(2,57) = 8.30, P < 0.001] and treatment [F(1,57) = 28.53, P < 0.001]. Furthermore, pretreatment x treatment interactions were also statistically significant [F(2,57) = 5.16, P < 0.01].
Post hoc comparisons revealed that SKF-82958 produced a PPI impairment (P < 0.001 for VEH
+ SAL vs VEH + SKF-82958 comparisons) which was completely reversed by the highest FIN
dose (P < 0.001 for VEH + SKF- 82958 vs FIN 50 + SKF-82958; Tukey’s test).
In the second experiment (n = 57; 8—10/group), we studied the combined impact of FIN (25—
50 mg/kg, IP) and QUIN (0.5 mg/kg, IP) on the startle magnitude and PPI (Fig. 5.2).
Analyses of startle amplitude (Fig. 5.2A) revealed significant effects for the pre-treatment (FIN
vs VEH) [main effect: F(2,51) = 4.10, P < 0.05], treatment (QUIN vs SAL) [main effect: F(1,51)
= 46.91, P < 0.001], and their interaction [F(2,51) = 7.19, P < 0.01]. This latter effect was found
to reflect a significant reduction induced by the combination of the highest FIN dose with QUIN
as compared with their counterparts treated with FIN and SAL (P < 0.001 for comparison FIN 50
+ QUIN vs FIN 50 + SAL).
The analysis of %PPI revealed significant main effects for pre-treatment [F(2,51) = 4.76, P <
0.05], treatment [F(1,51) = 37.62, P < 0.001] and their interaction [F(2,51) = 3.80, P < 0.05].
Fig. 5.2 Effects of systemic finasteride (FIN, 25—50 mg/kg, IP) and quinpirole (QUIN, 0.5
mg/kg, IP) on startle reflex (A) and %PPI (B) in C57BL/6 mice. FIN doses are indicated in
mg/kg. VEH, vehicle of FIN; SAL, saline. Values are expressed as mean ± S.E.M., N = 8-10/
group. **P < 0.01, ***P < 0.001 vs rats treated with SAL (pre-treatment x treatment
interaction); ººP < 0.01 vs rats treated with VEH and QUIN (pre-treatment x treatment
interaction).
89
Post-hoc analyses revealed that, while neither FIN nor QUIN significantly affected PPI, this
parameter was markedly reduced by their combination (P < 0.001 for FIN 50 + QUIN vs FIN 50
+ SAL; Tukey’s test) (Fig. 5.2B). These results were confirmed by ∆PPI analyses {main effect
for pre-treatment: [F(2,51) = 3.78, P < 0.05]; main effect for treatment: [F(1,51) = 42.91, P <
0.001]; pre-treatment x treatment interaction: [F(2,51) = 4.42, P < 0.05]; post hoc comparisons: P
< 0.001 for FIN 50 + QUIN vs FIN 50 + SAL} (data not shown).
The third experiment (n = 32; 8/group) (Fig. 5.3) was aimed at the assessment of the combined
effects of FIN and APO (0.5 mg/kg, IP). With respect to startle, ANOVA detected a main effect
only for the treatment [F(1,30) = 28.61, P < 0.001], but not for either pre-treatment [F(1,30) =
2.44, NS] or pre-treatment x treatment interactions [F(1,30) = 3.38, NS] (Fig. 5.3A). Similarly,
%PPI analyses highlighted a main effect for treatment [F(1,30) = 46.21, P < 0.001], but not for
either pre-treatment [F(1,30) = 0.07, NS] or pre-treatment x treatment interactions [F(1,30) =
0.01, NS] (Fig. 5.3B). The evaluation of ∆PPI also revealed a significant effect for treatment
[F(1,30) = 38.59, P < 0.001], but not pre-treatment [F(1,30) = 1.67, NS] or their interactions
[F(1,30) = 1.64, NS] (data not shown).
Fig.5.3.Effects of systemic finasteride (FIN, 50 mg/kg, IP) and apomorphine (APO, 0.5
mg/kg, IP) on startle reflex (A) and %PPI (B) in C57BL/6 mice. VEH, vehicle of FIN; SAL,
saline. Values are expressed as mean ± S.E.M., N = 8/group.
90
5.3.2. Effects of FIN and DAergic agonists on open-field behaviors.
We then studied the combined impact of FIN and DAergic agonists (SKF-82958 and QUIN) on
locomotor activity and other open-field behaviors. After 30 min in the arena, FIN (50 mg/kg, IP)
produced a significant reduction in locomotor activity [main effect for treatment: F(1,43) =
60.67,P < 0.001] at 15-20 min after administration [time x treatment interaction: F(6,258) =
17.05, P < 0.001; P < 0.05 for comparisons with baseline values, Tukey’s].
Subsequently, mice were injected with either SAL or DAergic agonists, and their behavior was
studied for further 60 min. The analysis of the effects of FIN and SKF-82958 on locomotor
parameters was run with 3-way ANOVA designs, factors being pre-treatment (FIN vs VEH),
treatment (SKF-82958 vs SAL) and time (repeated measures, using the last measure before SKF82958/SAL injection as baseline). FIN significantly reduced the locomotor activity {main pretreatment effect: [F(1,26) = 24.85, P < 0.001]}, while SKF-82958 increased it {main treatment
effect: [F(1,26) = 29.33, P < 0.001]}. A significant interaction between these two drugs was also
found [F(1,26) = 9.89, P < 0.01], which was shown to reflect the ability of SKF-82958 to
enhance locomotor activity in both VEH- and FIN-pretreated animals, as compared to SALtreated counterparts (P < 0.001 for both comparisons, Tukey’s). A significant effect for time
[F(12,312) = 6.22, P < 0.01] was found. Dunnett’s test revealed that the hyperlocomotive effect
of SKF-82958 was significant after 150 from its injection (Fig. 5.4A). Meandering was
significantly increased by FIN {main pretreatment effect: [F(1,24) = 18.35, P < 0.001]}. Notably,
ANOVA detected a significant pre-treatment x treatment x time interaction [F(12, 288) = 3.23, P
< 0.001]. Post hoc comparisons revealed that SKF-82958 significantly reduced meandering in
VEH-, but not FIN-pretreated mice for the whole duration of the experiment, with the greatest
effects measured at 25—30 min after treatment (Fig. 5.4B). Rearing behavior was significantly
reduced by FIN [F(1,24) = 8.70, P < 0.01] and increased by SKF-82958 [F(1,24) = 11.80, P <
0.01]. Significant pre-treatment x - treatment [F(1,24) = 5.05, P < 0.05] and pre-treatment x treatment x time [F(6,144) = 2.25, P < 0.05] interactions were found. Post hoc scrutiny of this
effect indicated that SKF-82958 increased rearing in VEH-pretreated mice, at 20— 40 min after
treatment. Of note, this effect was significantly antagonized by FIN pre-treatment (Fig. 5.4C).
91
Fig. 5.4 Effects of systemic finasteride (FIN, 50 mg/kg, IP) and SKF-82958 (SKF, 0.3 mg/kg, IP)
on locomotor and exploratory behavior in an open field. (A) Distance traveled, (B) meandering,
(C) rearing behaviors, and (D) Time spent in the central compartment. VEH, vehicle of FIN; SAL,
saline. Arrows correspond to the times of injection. Values are expressed as mean ± S.E.M., N =
8—12/group. *P < 0.05, **P < 0.01, ***P < 0.001 for comparisons with baseline values at 60 min;
8P < 0.05, ººP < 0.01 for comparisons between VEH + SKF and FIN + SKF (pre-treatment x
treatment x time interaction).
92
No statistically significant differences were detected with respect to the effect of SKF-82958 on
the duration of the time spent in the central compartment (Fig. 5.4D).
The analysis of the effects of QUIN and FIN co-treatment on locomotor activity was conducted
with the same design as described for the experiment with SKF-82958. ANOVA revealed main
effects for pre-treatment [F(1,26) = 39.69, P < 0.001], treatment [F(1,26) = 4.51, P < 0.05] and
time [F(12,312) = 4.41, P < 0.001], but not for their interactions (Fig. 5.5A). No significant
differences were observed on meandering, rearing or time spent in center (Figs. 5B-D).
Fig.5.5. Effects of systemic finasteride (FIN, 50 mg/kg, IP) and quinpirole (QUIN, 0.5
mg/kg, IP) on locomotor and exploratory behavior in an open field. (A) Distance traveled,
(B) meandering, (C) rearing behaviors, and (D) time spent in the central compartment.
VEH, vehicle of FIN; SAL, saline. Arrows correspond to the times of injection. Values are
expressed as mean ± S.E.M., N = 8-12/group.
93
5.3.3. Effect of FIN on APO-induced stereotypies and catalepsy.
As shown in Fig. 5.6A, APO caused a significant increase in stereotyped behavior in a timerelated fashion [F(6,84) = 96.36, P < 0.001], which was not affected by FIN (50 mg/k g, IP) at
any time [pre-treatment x time interaction: F(6,84) = 1.31, NS]. In line with our previous results
in rats (Bortolato et al., 2008), FIN did not induce catalepsy at any tested dose [F(4,20) = 0.76,
NS]; conversely, the antipsychotic agent HAL induced a robust cataleptic effect [F(1,8) = 114.06,
P < 0.001] (Fig. 5.5B).
Fig. 5.6. (A) Effects of systemic finasteride (FIN, 50 mg/kg, IP) on the stereotyped responses
induced by apomorphine (APO, 3 mg/ kg, IP) The average stereotypy score was based on a
modification of the rating scale of Benus et al. (1991), as follows: 0: nonstereotyped behaviors
(locomotion, grooming, rearing, sniffing, digging, sitting); 1: hypolocomotion and behavioral
freezing; 2: hypolocomotion with bouts of stereotyped sniffing and gnawing the sawdust; 3:
stereotyped behavior in a particular pattern; 4: compulsive sniffing and gnawing the sawdust;
front paws occasionally on cage wall; 5: slow climbing or stretched-out, near-vertical position;
6: continuous gnawing and/or licking the walls and bars of the cage. (B) Effects of FIN and
haloperidol (HAL) on catalepsy. FIN and HAL doses are indicated in mg/kg. VEH, vehicle of
FIN. Values are expressed as mean ± S.E.M., N = 8/group. ***P < 0.001 for comparisons with
baseline values at 30 min.
94
5.4. DISCUSSION
We found that, in C57BL/6 mice, the prototypical 5αR inhibitor FIN produced a dramatic
reduction in locomotor activity, but failed to modify startle reflex, impair PPI, or induce
catalepsy at any tested dose. Furthermore, this agent exerted divergent modulatory actions on the
effects of D1- and D2–like receptor activation, insofar as it reduced the PPI deficits and rearing
responses induced by the full D1 receptor agonist SKF82958, but surprisingly synergized with the
D2-like activator QUIN to produce marked gating impairments. Taken together, the present
findings extend and complement previous evidence on the anti-DAergic properties of 5αR
blockers (Bortolato et al., 2008; Devoto et al., 2012) and suggest that, at least in C57BL/6 mice,
the role of 5αR in behavioral organization may vary, in relation to the differential engagement of
distinct DAergic receptor classes.
In agreement with previous findings, the full D1 receptor agonist SKF82958 significantly reduced
startle and PPI (Ralph-Williams et al., 2002; Ralph-Williams et al., 2003), increased locomotor
activity and enhanced rearing responses. FIN potently prevented the gating and rearing
alterations induced by SKF-82958, but did not significantly affect the startle deficits elicited by
this agent. Furthermore, although FIN countered the overall motor activation in response to D1
receptor stimulation, this phenomenon did not reflect a specific effect, but rather FIN’s intrinsic
hypolocomotive properties, as revealed by the lack of significant statistical interactions between
the two treatments. These results may signify that FIN is able to significantly modulate only
select effects of D1 receptor activation, such as its role in the regulation of sensorimotor gating
and exploratory behavior, rather than motor organization. Accordingly, we have recently
identified that, in rats, the anti-DAergic effects of systemic FIN administration are reproduced by
local infusions of this agent in the NAc, but not in dorsal striatum (Devoto et al., 2012); within
this conceptual framework, it is worth noting that, in contrast with the well-documented role of
the dorsal striatum in motor orchestration (Voorn et al., 2004), the NAc has been shown to
mediate and organize motor activity mainly in response to motivational inputs and limbic
activation (Groenewegen and Trimble, 2002). Irrespective of these considerations, the ability of
SKF82958 to produce a significant increase in locomotion in FIN-treated animals, together with
previous binding data (see Bortolato et al., 2008) clearly indicates that FIN is not a D1–like
receptor antagonist; nonetheless, it is likely that this compound may still affect open-field
95
behaviors through the negative modulation of D1 downstream signaling cascade. In further
support of this possibility, multiple studies have shown that the negative modulation of D1
receptors does not produce intrinsic changes in PPI (Doherty et al., 2008; Ralph et al., 2001;
Ralph-Williams et al., 2003), but markedly reduces locomotor activation (Centonze et al., 2003;
Kelly et al., 2008) and increases meandering (unpublished observations).
In conformity with other reports, the D2 receptor agonist QUIN did not elicit any significant
change in PPI in C57BL/6 mice (Ralph-Williams et al., 2003). Nevertheless, its combination
with FIN surprisingly reduced this parameter in a fashion dependent on the dose of the 5αR
blocker. Furthermore, in striking contrast with our previous findings in Sprague-Dawley rats
(Bortolato et al., 2008), FIN failed to affect the PPI deficits and stereotyped behaviors induced
by the mixed D1-D2 agonist APO. The divergent effects mediated by FIN on the behavioral
outcomes of the two families of DA receptors may suggest that the signaling of these targets may
be distinctively modulated by different balances in NSs, at least with respect to their roles on the
regulation of PPI and other specific behavioral responses.
Ample evidence has documented that, while both D1-like and D2-like receptors participate in the
regulation of sensorimotor gating and information processing, the relative contribution of these
two subfamilies is highly variable, depending on the species and the genetic background (Kinney
et al., 1999; Ralph and Caine, 2005; Ralph and Caine, 2007; Swerdlow et al., 2000a; Swerdlow
et al., 2000b). While most D1-like agonists have been shown to have no intrinsic PPI-disrupting
effects in rats, they robustly impair this index in C57BL/6 and other commonly used inbred and
outbred strains of mice (Holmes et al., 2001; Ralph and Caine, 2005); vice versa, activation of
D2-like receptors appears to be a fundamental prerequisite for the PPI deficits induced by
DAergic compounds in rats (Geyer et al., 2001), but not in C57BL/6 mice (Ralph-Williams et al.,
2003; Ralph and Caine, 2005). Irrespective of these differences, the DAergic regulation of PPI,
locomotor activity and other behaviors has been shown to be contributed by a complex set of
synergistic and antagonistic interactions between D1- and D2-like receptors (Eagle et al., 2011;
Ralph and Caine, 2005; Swerdlow et al., 2005; Wan et al., 1996). The finding that the same
doses of FIN that silenced the PPI-disrupting effects of SKF82958 precipitated the gating
impairments induced by QUIN suggests that different conditions of baseline NS concentrations
may partially contribute to the interstrain and interspecies differences in DAergic regulation. In
96
this respect, it is interesting to note that previous studies documented very low levels of PROG,
one of the key brain substrates for 5αR, in C57BL/6 mice (Phan et al., 2002).
The signaling of D1-like receptors is largely mediated by Gαs and Gαolf proteins, which stimulate
adenylyl cyclase and trigger the activation of cyclic-AMP-dependent protein kinase (PKA) and
its downstream targets, including several ion channels, CREB and DARPP-32 (Romanelli et al.,
2010). Conversely, D2-like receptors exert opposite intracellular effects through the inhibition of
adenylyl cyclase mediated by Gi and Go proteins. Given that the action of FIN on PPI regulation
are likely to be postsynaptic (Devoto et al., 2012), our results may signify that the variations in
NS levels may interfere with the common signaling pathways of D1 and D2 receptor signaling.
The two best-characterized isoforms of 5αR (1 and 2) catalyze the irreversible saturation of the
4,5 double bond of the A ring of ∆4-3 ketosteroids, such as PROG, deoxycorticosterone,
androstenedione and T (Paba et al., 2011). The neuroactive metabolites of these precursors are
involved in the organization of several key brain functions. For example, the 5α-reduced product
of PROG is converted into the potent NS AP, which plays an important role in stress
responsiveness and other brain functions by acting as a positive allosteric modulator of the γamino-butyric acid (GABA)A ionotropic receptor (Purdy et al., 1991; Morrow et al., 1995;
Barbaccia et al., 2001). The T derivative dihydroT (DHT), on the other hand, is a potent
activator of ARs in the brain and contributes to a number of critical behavioral functions
(Zuloaga et al., 2009).
In separate experiments, we found that the NS concentrations in mouse brain regions were
generally below detectability levels for HPLC/MS analyses; thus, we are currently unable to
fully elucidate the specific molecular mechanisms underlying the antiDAergic properties of FIN.
Nevertheless, given that 5αRs catalyze the rate-limiting step of neurosteroidogenesis (Paba et al.,
2011), it is likely that their inhibition results in profound imbalances in NS concentrations, due to
the decreased synthesis of AP and DHT and accumulation (or metabolic shift towards alternative
pathways) of ketosteroid precursors, such as PROG and T. Notably, previous studies have
documented that PROG and other NSs interact with the signaling and functions of D1 receptors
(Apostolakis et al., 1996; Petralia and Frye, 2006; Dong et al., 2007). Interestingly, DARPP-32
has been shown to mediate some of the actions of PROG on D1-like receptors (Mani et al., 2000;
97
Frye and Walf, 2010). Another interesting possibility to partially account for the observed effects
may reflect the action of PROG and other NSs on σ1 receptors; indeed, recent studies show that
these targets amplify DA D1 receptor signaling (Fu et al., 2010) and form heteromers with D1
receptors in the brain (Navarro et al., 2010).
In line with previous results in rats (Bortolato et al., 2008), FIN significantly reduced open-field
locomotion in C57BL/6 mice. This phenomenon was paralleled by a marked increase in
meandering and a decrease in rearing responses and time of permanence in the center.
Collectively, these findings indicate that FIN shifted the locomotor patterns of mice from broad
exploratory movements across the arena to a predominantly local activity in the periphery. This
variation was not induced by intrinsic alterations in motor competence, as indicated by the
absence of catalepsy in the present study and/or impairments in the rotorod test (unpublished
results). The decrement in the time spent in the center quadrant of the open field may signify an
increase in anxiety-like behavior; this interpretation is consistent with the notion that 5αR
inhibition results in the block of the synthesis of AP, which exerts an anxiolytic action by
positive modulation of GABA-A receptors (Dubrosky et al., 2006; Eser et al., 2008).
Nevertheless, this possibility is challenged by several ideas: first, we previously showed that the
same doses of FIN used in the present study reduced, instead of increasing, marble burying in
mice (Bini et al., 2009); second, in our experimental setting FIN was injected after 30 min of
acclimation to the open field; evaluation of thigmotaxis are generally considered meaningful of
anxiety during the first 5-10 min of introduction of a rodent in an open arena; third, alterations in
locomotor activity greatly limit the interpretation of variations in locomotor trajectory as
dependent on anxiety-like responses. This caveat is particularly relevant in the interpretation of
our data, in consideration of the highly significant correlation between locomotor activity and
time spent in center in FIN-treated animals.
In sheer contrast with our previous data in Sprague-Dawley rats, FIN did not prevent the PPI
deficits or the stereotyped responses mediated by the mixed D1/D2 agonist APO (Bortolato et al.,
2008). Although most of the behavioral effects of APO have been shown to depend on the
synergism of D1 and D2 receptors (Braun and Chase, 1986; Bordi and Meller, 1989; Plaznik et
al., 1989; LaHoste and Marshall, 1996), previous research documented that, in C57BL/6 mice,
the PPI deficits induced by this drug depend on the activation of D1, rather than D2 receptors
98
(Ralph-Williams et al., 2003a; Ralph-Williams et al., 2003b). In this perspective, it is somewhat
surprising that the same dose that fully antagonized the PPI deficits induced by SKF82958 did
not attenuate the behavioral outcomes of APO on gating; nevertheless, the PPI-disrupting
potential of FIN in the presence of D2 receptor activation may offset and counterbalanced its
PPI-ameliorating properties related to the outcomes of D1 receptor. More in general, the
divergent effects of FIN on APO-mediated effects in Sprague-Dawley rats and C57BL/6 mice
are likely to depend on interspecies and interstrain differences in the role of DA receptors on the
regulation of sensorimotor gating. For example, unlike C57BL/6 mice, Sprague-Dawley rats do
not generally exhibit PPI deficits following activation of D1 receptors (Bortolato et al., 2005;
Wan et al., 1996; Wan and Swerdlow, 1993), although these targets afford a major contribution
to the PPI-disrupting properties of APO (Hoffman and Donovan, 1994; Wan et al., 1996). On the
other hand, QUIN produces a significant reduction of PPI in rats, but not in C57BL/6 mice
(Ralph and Caine, 2005). These divergences parallel other differential outcomes of QUIN on
locomotor activity: while this drug yields biphasic effects on locomotor activity in rats, it lowers
locomotor activity in C57BL/6 mice irrespective of the dose (Halberda et al., 1997), possibly
reflecting different patterns of D2 and D3 receptor activation across different species and strains.
Further studies on the effects of FIN on the PPI-disrupting effects of DAergic agonists in
Sprague-Dawley rats are needed to better qualify potential interspecies differences in the role of
NSs in gating regulation.
Throughout our experiments, startle amplitude was significantly reduced by all DA receptor
agonists, in line with previous results (Byrnes et al., 2007; Zhang et al., 2000). These results
suggest that activation of both D1- and D2-like receptors may exert a startle-attenuating effect in
mice. Indeed, previous studies showed that the reduction in startle amplitude induced by APO
remains unaffected in D1 or D2 knockout mice (Ralph-Williams et al., 2002), potentially
suggesting a contribution of both DA receptor subfamilies in this phenomenon. In contrast with
our results in rats, FIN did not inherently affect startle amplitude in C57BL/6 mice, potentially
suggesting a different role of NSs in the modulation of startle responses. While future studies are
warranted to explore the neurobiological bases of these differences, the fact that the marked
reduction in locomotor activity induced by FIN was not paralleled by changes in startle
amplitude confirms the dissociation between these two phenomena, in line with previous results
(Davis et al., 1986).
99
Several limitations of the present study should be acknowledged, including the lack of
experiments that may better differentiate the relative contribution of subtypes within D1-like and
D2-like receptors. Furthermore, the high affinity of FIN for both 5αR1 and 5αR2 isozymes in
rodents (Thigpen and Russell, 1992) does not allow us to elucidate the specific contribution of
each isoenzyme to the antipsychotic-like effects of 5αR inhibition. Whereas further research is
needed to address these limitations, our findings highlight the critical role of 5αR in the
pathophysiology of gating deficits and psychosis, and point to an important functional link
between NSs and the signaling cascade of D1 receptors, which may be involved in the
pathophysiology of a number of neuropsychiatric disorders, including TS and schizophrenia.
100
CHAPTER 6
IDENTIFICATION OF THE DOPAMINE RECEPTORS
MEDIATING THE ANTIPSYCHOTIC-LIKE EFFECTS OF 5αR
INHIBITORS: EVIDENCE IN RATS
101
In general, most studies have shown that the role of DA in PPI is mediated by both D1, D2 and D3
receptors. Nevertheless, the specific contribution of these receptors to these phenomena has been
shown to vary across different rodent models. In SD albino rats, PPI deficits are elicited by
D2 and D3 DAreceptor agonists, but not by D1 receptor agonists (Peng et al., 1990; Bristow et al.,
1996; Varty and Higgins, 1998); nevertheless, D1 receptor activation has been shown to be
directly involved in the PPI deficits induced by non-specific DAergic agonists, such as APO
(Peng et al., 1990). Conversely, we recently found that the selective activation of D1 and D2, but
not D3 receptors produces a stable PPI deficits in the hooded Long-Evans (LE) strain (Mosher et
al., submitted).
In previous studies we documented that, in SD rats, the PPI deficits induced by non-selective
DAergic agonists are enabled by 5αR (Bortolato et al., 2008), the enzyme catalyzing the ratelimiting step in the synthesis of NSs and androgenic metabolites of T (Martini et al., 1993;
Martini et al., 1996; Paba et al., 2011). Accordingly, the selective 5αR inhibitor FIN attenuates
the PPI deficits induced by non-selective DAergic agonists in SD rats (Bortolato et al., 2008;
Devoto et al., 2012). In parallel with these preclinical results, our group found that FIN elicited
therapeutic properties in patients affected by chronic schizophrenia (Koethe et al., 2008) and TS
(Bortolato et al., 2007; Muroni et al., 2011). Notably, the anti-DAergic effects of FIN and other
5αR blockers were not accompanied by extrapyramidal side effects. While these premises point
to these agents as promising options for the therapy of these neuropsychiatric conditions, the
specific involvement of DA receptors in the PPI-enhancing effects of FIN remain elusive. Based
on this background, in the present study we investigated the specific contributions of different
DA receptor subtypes on sensorimotor gating using SD and Long Evans (LE) rats.
Subjects. A total of 240 male SD (Harlan, Italy) and 186 LE rats (Harlan, U.S.) weighing
between 275 and 320 g were used for these experiments. Animals were group-housed in cages
(n=4) with ad libitum access to food and water. The room was maintained at 22±0.2ºC on
reversed 12-hr light/dark cycle (with lights off at 07:00 PM). Each animal was used only once
throughout the study and all efforts were made to minimize animal suffering throughout this
102
study. PPI and microdialysis studies occurred between 11:00 AM and 5:00 PM. All experimental
procedures were executed in compliance with the National Institute of Health guidelines and
approved by the Animal Use Committees at the University of Cagliari and University of Kansas.
Drugs and solutions. The following drugs were used in the present study: FIN, (R)-(-)-APO
hydrochloride, SKF 38,393 hydrobromide, SKF 82,958 hydrobromide, SKF-83959, Sumanirole
(SUM) maleate, (-)-QUIN hydrochloride, (+)-PD 128907 hydrochloride and GR-103691. FIN
was suspended in a VEH solution containing 5% Tween 80 and 95% sterile SAL, while the other
drugs were dissolved in SAL solution. Drug doses are based on mg/kg of salts. All solutions
were freshly prepared on the day of testing and administered SC and IP in injection volumes of 1
and 2 ml/kg body weight, respectively. The doses and the latency time of the drug used in these
experiments were determined by our previous studies and in accordance with those commonly
used in PPI studies on rats (Bortolato et al., 2008; Wan et al., 1996; Zhang et al., 2007; for a
review, see Geyer et al., 2001).
Startle Reflex and PPI. Startle and PPI testing were performed as previously described in Frau et
al. (2013). Rats were placed in the PPI chamber for a 5-min acclimatization period consisting of
70 dB background white noise, which continued for the remainder of the entire session. The PPI
protocol consists of three consecutive blocks with pulse, pre-pulse+pulse and “no-stimulus” trials.
Specifically, during the first and the third block rats received only five pulse-alone trials of 115
dB, whereas throughout the second block they were exposed to a pseudorandom sequence of 50
trials, including 12 pulse-alone trials, 30 trials of pulse preceded by 74, 78 or 86 dB pre-pulses
intensities (ten for each level of prepulse levels) and eight “no stimulus” trials, in which the only
background noise was delivered. Intertrial intervals were randomly selected between 10 and 15 s.
The sound levels were assessed using an A Scale setting and the sensitivity of the stabilimeter
was calibrated among the four startle chambers each day prior to start the PPI sessions. Percent
PPI was calculated with the following formula:
103
with
and
representing the mean startle amplitudes for all pre-pulse+pulse trials
and pulse alone trials, respectively. The first 5 pulse-alone bursts were excluded from the
calculation.
Experimental Design. The present study was composed of five distinct experiments:
In the first experiment, we tested the impact of FIN on APO-induced PPI deficits in SD and LE
rats. Briefly, rats (n=8–12 per group) were treated with FIN (100 mg/kg, IP) or VEH. Sixty
minutes later, rats were treated with either APO (0.5 mg/kg, SC) or SAL (control), and
immediately subjected to behavioral testing.
The second experiment was aimed at evaluating the combined effects of FIN and the selective D1
agonists on startle and PPI values of SD and LE rats. Both strain of rats (n=8–12 per group) then
received FIN or VEH followed by SKF-38,393 (5 mg/kg, SC), SKF-82,958 (1 mg/kg, SC), SKF83,959 (10 mg/kg, SC) or SAL.
The goal of the third and fourth experiments was to study the ability of FIN to counter the PPI
impairments induced by D2 agonists, quinpirole (QUIN) and sumanirole (SUM) (n=8-10 animals
per group). Animals were treated with FIN or VEH (100 mg/kg, IP) QUIN and SUM (or SAL
control) were injected 60 min after. 10 min after the last treatment, rats were underwent PPI
sessions.
In the fifth experiment, FIN (100 mg/kg, IP) or VEH (control) was administered 50 min
before PD (0.1 mg/kg, IP) or SAL (control), in SD and LE rats. Furthermore, in a separate
cohort of SD rats, we also tested the efficacy of the selective D3 antagonist GR in attenuating the
PPI disruptive effects of PD. Thus, animals were injected with GR or SAL, and 10 min after,
with PD. Then, 10 min after the latter treatment, animals were placed in the startle chamber and
tested (n=8–11 per group).
Kolmogorov-Smirnov and Bartlett’s tests. Analyses were performed by multiple-way ANOVAs,
as appropriate, followed by Tukey’s test (with Spjøtvoll-Stoline correction for unequal N
whenever required) for post-hoc comparisons of the means. For % PPI analyses, main effects for
104
prepulse levels were consistently found throughout all the analyses, showing loudness-dependent
effects. Since no interactions between prepulse levels and other factors were found, however,
data relative to different prepulse levels were collapsed. Significance threshold was set at 0.05.
6.3. RESULTS
6.3.1. Effects of FIN versus the non-selective DAergic agonist APO on ASR and PPI in SD
and LE rats.
We first investigated the effects of FIN and APO on sensorimotor gating in LE and SD rat strains.
In line with previous studies from our group (Bortolato et al., 2008), FIN significantly reduced
baseline startle magnitude [main effect of pretreatment: F(1, 35)=32.85, P<0.001], while APO
significantly increased startle amplitude in SD rats [main effect of treatment: F(1, 35)=6.91,
P<0.05]. However, no significant interactions between pretreatment and treatment were found
[F(1, 35)=1.03, NS]. In contrast to SD rats, APO induced a significant reduction in startle
amplitude in LE rats [(main effect of treatment: F(1, 29)=4.63, P<0.05], however, FIN did not
show any effect on startle [F(1, 29)=0.18, NS]. No significant interactions between pretreatment
Fig. 6.1. Effects of finasteride (FIN, 100 mg/kg, IP) and apomorphine (APO, 0.25 mg/kg, SC)
on Startle Reflex (A) and PPI (B) in Sprague Dawley (SD) and Long Evans (LE) rats. Results
are expressed as mean ± SEM. Veh, vehicle of FIN; Sal, saline. N=8-12/group. ***p<0.001
vs rats treated with Veh and Sal, °°p <0.01 vs rats treated with Veh and APO.
105
and treatment were found in LE rats [F(1, 29)=0.79, NS] (Fig. 6.1A).
Examination of sensorimotor gating in SD rats revealed main effects for both pretreatment
[F(1, 35)=10.93, P<0.01] and treatment [F(1, 35)=15.62, P<0.001]. Furthermore, PPI analysis
also showed a significant pretreatment X treatment interaction [F(1, 35)=4.33, P<0.05]. In
keeping with previous findings by our group (Bortolato et al., 2008; Devoto et al., 2012), FIN
prevented APO-induced PPI disruptions in this strain (P<0.05). Conversely, PPI analyses
conducted on LE rats showed robust main effects of APO [main effects of treatment:
F(1, 29)=15.62, P<0.001], but not of FIN [main effects of pretreatment: F(1, 29)=0.09, NS].
Interestingly, no APO X FIN interaction was detected, suggesting that FIN was not able to
counteract the APO-induced PPI deficits in this strain [pretreatment X treatment: F(1, 29)=0.08,
NS] (Fig. 6.1B).
6.3.2. Effects of FIN versus D1 agonists on ASR and PPI in SD and LE rats.
Our results in the first experiment suggest that the PPI-disruptive effects of APO may be due to
the activation of different DA receptors. Thus, we investigated the impact of D1 receptors in
sensorimotor gating using the selective D1 agonists. In comparison with SAL-treated controls,
SKF-38,393 and SKF-82,958 significantly modified startle parameters of SD rats, by increasing
and decreasing their values, respectively [P<0.05]. SKF-83,959, however, did not induce any
alterations in startle amplitude. Conversely, neither SKF-82,958 nor FIN affected startle
parameters [main effect of pretreatment: F(1, 28)=0.03, NS; main effect of treatment:
F(1, 28)=1.07, NS; pretreatment
treatment interaction: F(1, 28)=0.99, NS] (Fig. 6.2A).
In SD rats, D1 receptor activation did not affect sensorimotor gating [SKF-38, 393: t(19)=2,11
NS; SKF-82,958: t(18)=0.09, NS; SKF-83,959, t(23)=1.65, NS]. Two-way ANOVA analyses of
treatment and pretreatment in LE rats revealed a significant main effects [treatment:
F(1, 28)=52.01, P<0.001; pretreatment: F(1, 28)=8.25, P<0.01]. Moreover, we found a
significant pretreatment X treatment interaction [F(1, 28)=15.39, P<0.001]. Post-hoc
comparisons showed that D1 activation significantly impaired PPI of LE rats (P<0.001), an
effect that was countered by FIN (P<0.001) (Fig. 6.2B).
106
Fig. 6.2. Effects of finasteride (FIN, 100 mg/kg, IP), SKF 38,393 (5 mg/kg, SC), SKF 82, 958
(APB; 1 mg/kg, SC), SKF 83,959 (10 mg/kg, SC) on startle reflex (A) and PPI (B) in Sprague
Dawley (SD) and Long Evans (LE) rats. Results are expressed as mean ± SEM. Veh, VEH of
FIN; Sal, Saline. N=8-10/group. ***p<0.001vs rats treated with Veh and Sal, °°°p <0.001 vs
rats treated with Veh and APB.
Fig. 6.3. Effects of finasteride (FIN, 100 mg/kg, IP), sumanirole (SUM, 3 mg/kg, SC) on Startle
Reflex (A) and PPI (B) in Sprague Dawley rats. Results are expressed as mean ± SEM. Veh,
vehicle of FIN; Sal, SAL. N=8-10/group.
107
6.3.3. Effects of FIN versus D2 agonists on ASR and PPI in SD and LE rats.
Next, we evaluated the role of the D2 receptor in sensorimotor gating. In SD rats, SUM did not
affect startle magnitude (Fig. 6.3A) [F(1,31)=1.16; NS]. Although FIN significantly reduced
acoustic startle response [F(1,31)=20.50; P<0.001], no significant effect interaction between
SUM and FIN was observed [F(1,31)=0.81; NS]. Analyses of sensorimotor gating showed that
SUM disrupted PPI [F(1,31)=8.64; P<0.001], however, this effect was not affected by FIN
pretreatment (Fig. 6.3B) [F(1,31)=0.09; NS].
Treatment with QUIN did not elicit any alterations in startle amplitude in SD rats (Fig. 6.4A)
[F(1,37)=2.80; NS], however, this parameter was reduced by FIN [F(1,37)=13.50;
P<0.001]. Two-way ANOVA revealed a marked difference in the interaction between FIN and
QUIN [F(1,37)=46.68; P<0.001]. In particular, both FIN and QUIN reduced startle responses
compared to VEH and SAL treatments (Ps<0.001), but the combination of QUIN-FIN
significantly increased startle magnitude compared to animals treated with SAL and FIN
(P<0.001). Although startle values were not affected by FIN pretreatment in LE rats
[F(1,33)=1.71; NS], QUIN induced a marked reduction of this index [F(1,33)=11.27; P<0.01].
No differences in the FIN X QUIN interaction were found [F(1,33)=0.01; NS].
Examination of the D2 receptor effects on PPI in SD rats showed that while QUIN did reduced
sensorimotor gating (Fig. 6.4B) [F(1,37)=16.76; P<0.001], FIN increased this parameter
[F(1,37)=8.64; P<0.001]; however, no significant pretreatment X treatment interaction was
detected between FIN and QUIN [F(1,37)=0.80; NS]. In marked contrast to SD animals, the PPI
disruptive effect of QUIN [F(1,33)=48.47; P<0.001] was not affected by FIN [Main effect of
FIN: F(1,33)=0.34; NS; pretreatment X treatment interaction: F(1,33)=0.90; NS].
108
Fig. 6.4. Effects of finasteride (FIN, 100 mg/kg, IP) and quinpirole (QUIN, 0.6 mg/kg, SC) on
Startle Reflex (A) and PPI (B) in Sprague Dawley (SD) and Long Evans (LE) rats. Veh, VEH
of FIN; Sal, SAL. Results are expressed as mean ± SEM. N=8-10/group. **p<0.01,
***p<0.001 vs rats treated with Veh and Sal. °°°p <0.001 vs rats treated with Veh and QUIN.
Fig. 6.5. Effects of finasteride (FIN, 100 mg/kg, SC) and PD 128907 (PD, 0.1 mg/kg, IP) on
Startle Reflex (A) and PPI (B) in Sprague Dawley rats. Veh, VEH of FIN; Sal, SAL. Results are
expressed as mean ± SEM. N=8-12/group. *p<0.05 vs rats treated with Veh and Sal, °°p <0.01
vs rats treated with Veh and PD.
109
6.3.4. Effects of FIN versus D3 agonist on ASR and PPI parameters in SD rats.
To investigate the contribution of D3 receptors in sensorimotor gating, SD rats were treated with
the D3 agonist PD 128907 (PD). FIN [F(1,43)=25.39; P<0.001] and PD [F(1,43)=6.10; P<0.05]
altered acoustic startle amplitude, but not the combination of both agents [F(1,43)=1.36; NS] (Fig.
6.5A). Two-way ANOVA analyses of PPI parameters showed a significant main effect of
pretreatment [F(1,43)=10.56; P<0.01] and a significant pretreatment x treatment interaction (Fig.
6.5B) [F(1,43)=6.71; P<0.05], but not treatment [F(1,43)=1.03; NS]. In particular, PD induced
PPI deficits (P<0.05) that were prevented by FIN (P<0.01).
To reveal whether the PPI-disruptive effects of PD were mediated by D3 receptor subtypes, and
not by the contributions of other DAergic receptors (Zhang et al., 2007), we investigated the
effects of the selective D3 antagonist GR-103691 on the PPI impairments produced by PD in SD
rats. Startle amplitude values were evaluated with a two-way ANOVA, with pretreatment (GRor VEH) and treatment (PD or SAL) as factors. Although PD reduced startle parameters (Fig.
6.6A) [F(1,28)=4.41, P<0.05], GR showed no effect [F(1,28)=0.75, NS]. Similarly, we detected
an interaction between PD X GR [F(1,28)=3.99, P<0.06], however, this effect did not reach
statistical significance. Two-way ANOVA showed that both compounds altered PPI values (Fig.
6.6B) [main effect of pretreatment: F(1, 28)=21.07, P<0.001; main effect of treatment:
F(1, 28)=14.00, P<0.001; pretreatment x treatment interaction: F(1, 28)=6.97, P<0.05]. Posthoc comparisons revealed that GR significantly attenuated PD-mediated PPI disruption
(Ps<0.001).
110
Fig. 6.6. Effects of GR-103691 (GR, 0.2 mg/kg, SC) and PD 128907 (PD, 0.1 mg/kg, IP) on
Startle Reflex (A) and PPI (B) in Sprague Dawley rats. Results are expressed as mean ±
SEM. Veh, VEH of GR; Sal, SAL. N=8-11/group. ***p<0.001 vs rats treated with Veh and Sal,
°°°p <0.001 vs rats treated with Veh and PD.
6.4. DISCUSSION
The major result of the present study is that the prototypical 5αR inhibitor FIN prevented the PPI
deficits induced by the activation of D1 and D3, but not D2 receptors across different rat strains.
Specifically, in LE rats - which exhibit PPI deficits following activation of D1 and D2, but not D3
receptor agonists (Mosher et al., submitted) - FIN significantly prevented the PPI deficits
mediated by the potent D1 receptor agonist SKF82958, but not by the D2 receptor agonist
quinpirole or by the non-selective, D1-D2 receptor agonist APO. The involvement of D1, but not
D2 receptors in FIN-mediated effects was supported by our findings on SD rats (a strain that
exhibits PPI impairments following treatment with D2 and D3, but not D1 receptor agonists)
(Bortolato et al., 2005; Mosher et al., submitted). While FIN treatment was confirmed to reduce
APO-mediated PPI deficits in SD rats (Bortolato et al., 2008), this mechanism was likely not
supported by an action on D2 receptors, as confirmed by the inability of the 5αR inhibitor to
affect the behavioral changes induced by the D2 receptor agonists quinpirole and sumanirole.
Indeed, previous studies showed that D1 receptor antagonists potently attenuate the PPI deficits
induced by APO in SD rats (Bortolato et al., 2005; Wan et al., 1996).
111
Taken together, these findings extend and complement previous evidence on the involvement of
D1 receptors in the antiDAergic properties of FIN in C57BL/6 mice (Frau et al., 2013). In
particular, our data strongly suggest that, irrespective of interspecies and interstrain differences,
the regulation of sensorimotor gating is contributed by a direct interaction between D1 receptors
and 5αR, plausibly through the mediation of 5α-reduced NSs or neuroactive androgens. Indeed,
we previously showed that the PPI-ameliorative effects of FIN do not depend on gonadal
androgens, but rather its effect on neurosteroid synthesis in the NAc, which are likely related to
the modulation of the responses of DA receptors. From this perspective, it is particularly
interesting to observe that the changes mediated by FIN are related to both D1 and D3 receptors,
which are abundantly distributed in extrasynaptic locations in the striosomes of the striatum and
NAc shell (Gonon et al., 1997; Diaz et al., 2000). This configuration may be particularly
important for volume transmission and phasic activation (Fuxe et al., 2012). Thus, it is possible
to speculate that the importance of NSs and 5αR in the regulation of the role of DA in
sensorimotor gating may be reflective of a specific dynamic activity of this neurotransmitter,
which may be particularly relevant in the context of extrasynaptic locations.
The signaling of D1 receptors is largely mediated by Gαs and Gαolf proteins, which stimulate
adenylyl cyclase and trigger the activation of cyclic-AMP-dependent protein kinase A (PKA) and
its downstream targets, including several ion channels, CREB and DARPP-32 (Romanelli et al.,
2010). Given that the action of FIN on PPI regulation are likely to be postsynaptic (Devoto et al.,
2012), our results may signify that the variations in neurosteroid levels may interfere with the
common signaling pathways of D1 receptor signaling. In line with this evidence, previous studies
have shown that key NSs, such as pregnanolone, potently modulate the phenotypes associated
with D1 receptor activation (for a review, see Paba et al., 2011).
In line with previous studies, we found that APO induced marked PPI deficits in both SD and LE
rats (Swerdlow et al., 2011; Breier et al., 2010; Qu et al., 2009). While FIN was confirmed to
counter this deficit in SD rats, it did not in LE counterparts, possibly due to a greater contribution
of D2 receptor in APO-mediated effects in the latter strain. Indeed, SD and LE rats have been
shown to exhibit different sensitivity to the PPI-disruptive effects of APO and quinpirole
(Swerdlow et al., 2001b). This divergence is likely to parallel other documented variations in the
behavioral (Qu et al., 2009), genetic (Swerdlow et al., 2004b), and neurochemical (Swerdlow et
al, 2004a) profiles of these two strains. Further comparative studies will be needed to compare
112
the relative contribution of D1 and D2 receptors in the regulation of sensorimotor gating across
SD and LE rats.
Irrespective of the potential elements of difference between LE and SD rats, the lack of
involvement of D2 receptors in the effects of FIN may help explain the lack of extrapyramidal
effects associated with this drug, which sets it apart from other antiDAergic drugs currently used
in the therapy of schizophrenia and TS. This aspect may be particularly important in
consideration of preliminary data from our group indicating the therapeutic potential of FIN in
the treatment of these disorders. Furthermore, these data hint at the possibility that antiDAergic
drugs devoid of D2 receptor antagonist properties may still exert significant antipsychotic and/or
anti-tic properties; this intriguing concept may have profound implications in the design and
development of novel drugs with better tolerability and compliance than most of the currently
available therapeutic agents.
In line with previous findings (Zhang et al., 2007), PD exerted PPI deficits in SD rats in a D3dependent fashion, as shown by the reversal of this effect mediated by the selective inhibition of
this receptor. Interestingly, the effects of APO on PPI appear to be exacerbated, instead of
attenuated, by D3 receptor antagonists, likely highlighting a clear distinction between the two
antiDAergic mechanisms of FIN. D3 receptors have been widely implicated in schizophrenia and
other neuropsychiatric disorders associated with DA and gating dysfunctions, such as TS and
ICDs, has been recently proposed (Lieberman et al., 1997; Richtand et al., 2001). Our data
strongly suggest that NSs may play a prominent role in the modulation of the behavioral
outcomes of D3 receptor activation, which may be particularly relevant for those disorders.
The two best-characterized isoforms of 5αR (1 and 2) catalyze the irreversible saturation of the
4,5 double bond of the A ring of ∆4-3 ketosteroids, such as PROG, deoxycorticosterone,
androstenedione and T (Paba et al., 2011). The neuroactive metabolites of these precursors are
involved in the organization of several key brain functions. For example, the 5α-reduced product
of PROG is converted into the potent NS AP, which plays an important role in stress
responsiveness and other brain functions by acting as a positive allosteric modulator of the γamino-butyric acid (GABA)A ionotropic receptor (Barbaccia et al., 2001; Morrow et al., 1995;
Purdy et al., 1991). The T derivative DHT, on the other hand, is a potent activator of ARs in the
brain and contributes to a number of critical behavioral functions (Zuloaga et al., 2009).
113
Nevertheless, given that 5αRs catalyze the rate-limiting step of neurosteroidogenesis (Paba et al.,
2011), it is likely that their inhibition results in profound imbalances in NS concentrations, due to
the decreased synthesis of AP and DHT and accumulation (or metabolic shift towards alternative
pathways) of ketosteroid precursors, such as PROG and T. Notably, previous studies have
documented that PROG and other NSs interact with the signaling and functions of D1 receptors
(Apostolakis et al., 1996; Dong et al., 2007; Petralia and Frye, 2006). Interestingly, DARPP-32
has been shown to mediate some of the actions of PROG on D1-like receptors (Mani et al., 2000;
Frye and Walf, 2010). Another interesting possibility to partially account for the observed effects
may reflect the action of PROG and other NSs on σ1 receptors; indeed, recent studies show that
these targets amplify DAD1 receptor signaling (Fu et al., 2010) and form heteromers with
D1 receptors in the brain (Navarro et al., 2010).
Several limitations of the present study should be acknowledged. For example, the high affinity
of FIN for both 5αR1 and 5αR2 isozymes in rodents (Thigpen and Russell, 1992) does not allow
us to elucidate the specific contribution of each isoenzyme to the antipsychotic-like effects of
5αR inhibition. In addition, most experiments were not performed with a full dose-response
curve, thereby limiting a comprehensive assessment of the interstrain differences in the DAergic
regulation of rat PPI, as well as a closer understanding of the relation between NSs and DA
receptors.
Whereas further research is needed to address these limitations, our findings highlight the critical
role of 5αR in the pathophysiology of gating deficits and psychosis, and point to an important
functional link between NSs and the signaling cascade of D1 and D3 receptors, which may be
involved in the pathophysiology of a number of neuropsychiatric disorders, including TS and
schizophrenia.
114
CHAPTER 7
GENERAL DISCUSSION
115
The studies described in the previous chapters explored the relation between the key
neurosteroidogenic enzyme 5αR, its steroid products and the modulation of PPI and other
endophenotypes related to TS, using a variety of animal models and complementary approaches.
The results of the studies presented in Chapter 2 elucidated that, in addition to 5αR, the enzyme
CYP17A1, which is essential for the synthesis of androgen steroids, is also likely to participate
to the contro of DAergic modulation of PPI. These findings shed further light into the
involvement of androgens in TS and DAergic neurotransmission, and may help provide
important elements of insight into the male predominance of the syndrome. In contrast with these
findings, however, we found that neither T nor DHT are directly able to reduce PPI, and that
FLU, the prototypical AR antagonist, is equally ineffective to counter PPI deficits induced by
APO. These data suggest that other androgenic steroids, which enact their physiological effects
through different receptors, may be ultimately responsible for the mechanisms of FIN and ABI.
Several potential candidates can be considered, such as 3α-diol, 3β-diol and androsterone. As
discussed in Chapter 1, these steroids mediate their neuroactive effects through different
receptors, such as GABA-A, ERβ and PXR. Unfortunately, current analytical methodologies do
not allow for the measurement of these NSs in small brain regions, such as the NAc; however,
future studies will need to investigate whether exogenous administration of these molecules
(both systemic and intracerebral) may result in alterations of DAergic signaling and/or
modifications of sensorimotor gating.
The possibility that 3α-diol, 3β-diol and androsterone, rather than T and DHT, may be implicated
in the pathophysiology of TS is particularly intriguing, as it may provide a solution for one of the
most puzzling aspects of the male predominance in TS. Indeed, the pubertal surge in T and other
gonadal androgens typically coincides with an amelioration, rather than an exacerbation, of the
severity of tics and other TS symptoms. As noted above, the median age of TS onset coincides
essentially with adrenarche, the first stage of sexual maturation characterized by the development
of the inner zona reticularis in the adrenal cortex. The biochemical hallmark of adrenarche is the
acquisition of 17,20 lyase activity by CYP17A1 (Mapes et al., 1999), which is promoted by
cytochrome b5 and the phosphorylation of serine residues (Katagiri et al., 1995; Zhang et al.,
1995; Auchus et al, 1998; Geller et al., 1999; Pattison et al, 2007). The result of this process is
the increased synthesis of DHEA and androstenedione, which leads to the growth of axillary and
116
pubic hair as well as enhancement in the oiliness of the skin (Auchus and Rainey, 2004). Recent
studies have documented the existence of an alternative “backdoor pathway” for the synthesis of
androgens, which appears to be predominant before adrenarche. In this series of reactions, 17OH PREG is converted into 17-OH AP, and then androsterone (Kamrath et al., 2012). Recent
findings show that the shift from the backdoor pathway to the main pathway of androgen
synthesis in puberty (∆5 pathway) is based on the functional antagonism between 5αR and
CYP17A1. The prevailing activity of 5αR allows the predominance of the backdoor pathway, by
facilitating the synthesis of 17-OH-AP. This premise suggests that 5αR hyperactivation in the
periphery may lead to the persistence of the backdoor pathway even after adrenarche; upon these
conditions, it is possible that the imbalance in androgens would lead to a persistent increase in
3α-diol, 3β-diol and androsterone. Alternatively, it is even possible that the persistence of 5αR2
throughout childhood may facilitate the conversion of androstenedione (which has high affinity
for this isoenzyme) into androstanedione, which would then be re-converted into androsterone by
3α-HSOR.
The results of Chapter 3 indicate that, in line with the observed effects of FIN, its mechanisms
are likely based on key dopaminoceptive brain areas, such as the NAc and, possibly, the PFC.
The finding that the effects of FIN are not associated with changes in the concentrations of DA
in either region is particularly significant, as it appears to indicate that the mechanisms are likely
mediated by post-synaptic, rather than presynaptic effects of NSs. Indeed, this possibility is in
alignment with the general interpretation of the PPI-disrupting properties of APO, which are
assumed to reflect the activation of postsynaptic DA receptors. As mentioned in Chapter 1, both
major 5αR isoforms have been found in the dopaminoceptive neurons of each regions, i.e. the
pyramidal cells of the PFC and the medium-spiny neurons of the NAc. The lack of available
isoform-selective inhibitors for 5αR in rats leaves the question open as to whether these effects
are primarily mediated by 5αR1 or 5αR2. In humans, FIN is considered to be relatively selective
for 5αR2, suggesting that this enzyme may be directly implicated in the pathophysiology of TS.
This possibility would reveal an important, as-yet unknown function of 5αR2 in the regulation of
behavioral responses and information processing. Indeed, although both isoforms serve similar
enzymatic functions, they are likely to play a significantly different functional role. To address
this issue, ongoing studies in our lab are analyzing the differential effects of FIN and D1 receptor
117
agonists in 5αR1 and 5αR2 knockout (KO) mice. Our preliminary results indicate that, while
5αR1 may be primarily implicated in the regulation of gating functions, 5αR2 may be important
in the regulation of the role of NSs in stress response. In fact, 5αR1 KO mice failed to exhibit
any PPI deficits in response to D1 receptor stimulation, while 5αR2 KO did not respond to other
behavioral effects of FIN, including the reduction in locomotor activity and stress-coping
responses. If confirmed, these data may indicate that both isoenzymes may play a key role
(possibly through multiple brain-region-specific contributions) in the pathophysiology of TS and
other neuropsychiatric disorders. An alternative approach that may be particularly helpful in
providing meaningful answers about the region-specific implication of each isoenzyme in
sensorimotor gating regulation may lie in the injection of antisense oligonucleotides for both
genes within the NAc and PFC.
The findings of Chapter 4 revealed that the PPI deficits induced by IR, a manipulation aimed at
reproducing the impact of psychosocial stress throughout adolescence, are reversed by FIN
injection, through alterations of the NS profile in the NAc/striatum. In particular, it was found
that FIN increased the concentrations of PREG in IR rats. This result sheds light on another
important mechanism that may account for the effects of FIN, consisting in the accumulation of
NS precursors. Taken together with the evidence outlined in Chapter 3, these data suggest that
the involvement of 5αR in the regulation of gating is likely to be mediated by multiple,
potentially synergistic mechanisms, which may be mediated in turn by a constellation of
different receptors. Given that TS is primarily a childhood disorder, FIN is not a viable therapy
for the majority of patients, given its potential interference with sexual development. Our hope is
to map the NS pathways involved by the actions of FIN, so as to identify novel therapeutic
targets that may reduce tic severity without inducing extrapyramidal symptoms. If PREG plays a
role in the therapeutic effects of FIN, this would indicate that its attending receptor, σ1, may
have an important role in TS pathophysiology. Indeed, this receptor has been implicated in the
modulation of D1 receptor agonists, through the formation of homo- and heterodimers (Navarro
et al., 2001). PREG has been successfully used in recent clinical trials for schizophrenia,
suggesting that this NS may be also available for TS therapy. Ongoing studies in our lab are
testing whether PREG and synthetic σ receptor ligands can modify the behavioral effects of
118
DAergic agonists (and alter the properties of FIN in co-treatment with these drugs) to verify
whether this receptor may be an interesting target for TS.
The studies reported in Chapters 5 and 6 focused on the specific role of D1 and D2 receptors
with respect to the antipsychotic-like effects of FIN. This issue was addressed in both mice and
rats, given that evidence on these species has shown some elements of divergence with respect to
their sensitivity to DAergic agonists in the modulation of PPI. Indeed, C57/BL6 and other major
murine strains have been shown to be highly sensitive to D1, but not D2 receptor agonists; in
contrast, D1 receptor agonists do not induce PPI deficits in albino rats (SD and Wistar), but
potentiate the effects of D2 receptor stimulation in this paradigm. Our collective results
unequivocally indicate that, in both species, the effects of FIN were based on the silencing of D1,
but not D2, receptor signal. Assuming that the therapeutic effects of FIN in TS rely on the same
mechanisms, these results highlight the potential importance of D1 receptors in TS
pathophysiology, and explain why FIN does not induce extrapyramidal symptoms, which are
primarily driven by D2 antagonism. Interestingly, our experiments on mouse models
documented that FIN treatment dose-dependently sensitized C57BL/6 mice to D2-dependent PPI
deficits; these results possibly indicate that the changes in NS profile secondary to 5αR inhibition
may play an important role in the responsiveness to different classes of DA receptors with
respect to sensorimotor gating. Previous studies have shown that DARPP-32, a key gatekeeper of
DA receptor signaling, is targeted by PROG, possibly through the activation of mPRs (see
Chapter 1). These data point to DARPP-32 as a key candidate mediator for the effects of FIN.
While the involvement of DARPP-32 in TS remains unknown, future studies are warranted to
explore the potential of this enzyme as a therapeutic target for TS.
In addition to these results, our experiments on SD rats showed that FIN also interferes with the
effects on PPI of D3 receptor agonists. While these effects are not likely implicated in the ability
of FIN to counter the outcomes of AMPH and APO in PPI, these results highlight an implication
of NSs in the regulation of these receptors. In line with this concept, we recently showed that
FIN treatment reduced the severity of pathological gambling in Parkinson’s disease patients
treated with the D3 receptor agonist pramipexole. Ongoing studies in our lab have replicated
these results in animal models of probability discounting, which recapitulate key neuroeconomic
functions related to pathological gambling.
119
One of the major limitations of our studies lies in the fact that our key readout criterion, PPI, is
not specific for TS, but is also compromised in a broad number of mental disorders, such as
schizophrenia and mania. A more direct approach to study the mechanisms of FIN (and,
potentially, ABI) in TS may be based on the utilization of specific models that feature
spontaneous tic-like responses, such as the D1CT-7 mice (Campbell et al., 1999). In these
transgenic animals, the promoter region for the D1 receptor was fused with the enzymatic portion
of cholera toxin subunit α1 gene, which leads to a persistent activation of Gs proteins. D1CT-7
mice display explosive jerking movements of the head, trunk and limbs, which are highly
reminiscent of tics (Nordstrom and Burton, 2002). In conformity with the gender discrepancies
in TS patients, D1CT-7 male mice exhibit more tic flurries than females. Furthermore, the onset
of twitching occurs at postnatal day 16 (Nordstrom and Burton, 2002); interestingly, recent
studies have found that the changes in steroidal profile at day 16 in rodents are similar to those
featured in adrenarche in primates (Pignatelli et al., 2006). Initial studies in our lab recently
showed that D1CT-7 mice exhibit a significant reduction in tic severity and stress-dependent PPI
deficits following FIN and ABI treatment, further supporting the potential relevance of these
mechanisms in TS.
In summary, the results of the studies presented in this thesis point to a highly complex set of
processes and mechanisms related to the enzymatic functions of 5αR, and reveal this enzyme as
a core mediator of important behavioral processes, ranging from motor control to information
processing and emotional/motivational functions. Irrespective of mechanistic issues, which await
further studies, the scientific progress outlined in this thesis underscores the importance of NSs
in the pathophysiology of TS and other psychopathological states related to DAergic imbalances.
In particular, these results may be highly important for the definition of the neurochemical bases
of the male predominance and stress-sensitivity of TS; furthermore, the knowledge provided by
these studies prove essential for the identification of novel, brain-specific therapeutic targets for
TS as well as its comorbid issues, such as OCD and ADHD, with limited endocrinological
untoward effects.
120
REFERENCES
Abelson JF, Kwan KY, O'Roak BJ, Baek DY, Stillman AA, Morgan TM, Mathews CA, Pauls
DL, Rasin MR, Gunel M, Davis NR, Ercan-Sencicek AG, Guez DH, Spertus JA, Leckman JF,
Dure L, Kurlan R, Singer HS, Gilbert DL, Farhi A, Louvi A, Lifton RP, Sestan N, State MW.
Sequence variants in SLITRK1 are associated with Tourette's syndrome. Science. 2005; 310:
317-20
Agis-Balboa RC, Pinna G, Pibiri F, Kadriu B, Costa E, Guidotti A. Down-regulation of
neurosteroid biosynthesis in corticolimbic circuits mediates social isolation-induced behavior
in mice. Proc Natl Acad Sci USA. 2007; 104: 18736-41
Agís-Balboa RC, Pinna G, Zhubi A, Maloku E, Veldic M, Costa E, Guidotti A. Characterization
of brain neurons that express enzymes mediating neurosteroid biosynthesis. Proc Natl Acad
Sci USA. 2006; 103: 14602-07
Ahboucha S, Jiang W, Chatauret N, Mamer O, Baker GB, Butterworth RF. Indomethacin
improves locomotor deficit and reduces brain concentrations of neuroinhibitory steroids in
rats following portacaval anastomosis. Neurogastroenterol Motil. 2008; 20: 949-57
Albin RL, Mink JW. Recent advances in Tourette syndrome research. Trends Neurosci. 2006;
29: 175-82.
Alexander GM, Peterson BS. Testing the prenatal hormone hypothesis of tic-related disorders:
gender identity and gender role behavior. Dev Psychopathol. 2004; 16: 407-20.
Altomare G, Capella GL. Depression circumstantially related to the administration of finasteride
for androgenetic alopecia. J Dermatol. 2002; 29: 665-9.
American Psychiatric Association (APA). The Diagnostic and Statistical Manual of Mental
Disorders: DSM 5. Bookpoint, US, 2013
Amory JK, Wang C, Swerdloff RS, Anawalt BD, Matsumoto AM, Bremner WJ, Walker SE,
Haberer LJ, Clark RV. The effect of 5alpha-reductase inhibition with dutasteride and
finasteride on semen parameters and serum hormones in healthy men. J Clin Endocrinol
Metab. 2007; 92: 1659-65.
Apostolakis EM, Garai J, Fox C, Smith CL, Watson SJ, Clark JH, O'Malley BW. Dopaminergic
regulation of progesterone receptors: brain D5 dopamine receptors mediate induction of
lordosis by D1-like agonists in rats. J Neurosci. 1996; 16: 4823-34.
Arnt J, Hyttel J, Meier E. Inactivation of dopamine D-1 or D-2 receptors differentially inhibits
stereotypies induced by dopamine agonists in rats. Eur J Pharmacol. 1988; 155: 37-47
Arnt J. Antistereotypic effects of dopamine D-1 and D-2 antagonists after intrastriatal injection
in rats: Pharmacological and regional specificity. Naunyn Schmiedebergs Arch Pharmacol.
1985; 330: 97-104
Arnt J. Differential effects of classical and newer antipsychotics on the hypermotility induced by
two dose levels of D-amphetamine. Eur J Pharmacol. 1995; 283: 55-62
121
Arts B, Jabben N, Krabbendam L, van Os J. Meta-analyses of cognitive functioning in euthymic
bipolar patients and their first-degree relatives. Psychol Med. 2008; 38: 771-85
Attard G, Reid AH, Olmos D, de Bono JS. Antitumor activity with CYP17 blockade indicates
that castration-resistant prostate cancer frequently remains hormone driven. Cancer Res. 2009;
69: 4937-40
Auchus RJ, Lee TC, Miller WL. Cytochrome b5 augments the 17,20-lyase activity of human
P450c17 without direct electron transfer. J Biol Chem. 1998; 273: 3158-65
Azzolina B, Ellsworth K, Andersson S, Geissler W, Bull HG, Harris GS. Inhibition of rat alphareductases by finasteride: evidence for isozyme differences in the mechanism of inhibition. J
Steroid Biochem Mol Biol. 1997; 61: 55-64
Barbaccia ML, Serra M, Purdy RH, Biggio G. Stress and neuroactive steroids. Int Rev Neurobiol.
2001; 46: 243-72
Barr CL, Wigg KG, Pakstis AJ, Kurlan R, Pauls D, Kidd KK, Tsui LC, Sandor P. Genome scan
for linkage to Gilles de la Tourette syndrome. Am J Med Gen. 1999; 88: 437-45
Baumgardner TL, Singer HS, Denckla MB, Rubin MA, Abrams MT, Colli MJ, Reiss AL.
Corpus callosum morphology in children with Tourette syndrome and attention-deficit
hyperactivity disorder. Neurology. 1996; 47: 477-82
Baym CL, Corbett BA, Wright SB, Bunge SA. Neural correlates of tic severity and cognitive
control in children with Tourette syndrome. Brain. 2008; 131:165-79
Bearden CE, Jasinska AJ, Freimer NB. Methodological issues in molecular genetic studies of
mental disorders. Annu Rev Clin Psychol. 2009; 5:49-69
Beatty WW, Dodge AM, Traylor KL. Stereotyped behavior elicited by amphetamine in the rat:
influences of the testes. Pharmacol Biochem Behav. 1982; 16: 565-8
Belelli D, Lambert JJ. Neurosteroids: endogenous regulators of the GABA(A) receptor. Nat Rev
Neurosci. 2005; 6: 565-75.
Belluscio BA, Jin L, Watters V, Lee TH, Hallett M. Sensory sensitivity to external stimuli in
Tourette syndrome patients. Mov Disord. 2011; 26:2538-43
Benus RF, Bohus B, Koolhaas JM, van Oortmerssen GA. Behavioural differences between
artificially selected aggressive and non-aggressive mice: response to apomorphine. Behav
Brain Res. 1991; 43: 203-8
Bernauer S, Wehling M, Gerdes D, Falkenstein E. The human membrane progesterone receptor
gene: genomic structure and promoter analysis DNA sequence. J DNA Seq Map. 2001; 12:
13-25
Berns GS, Sejnowski TJ. A computational model of how the basal ganglia produce sequences. J
Cog Neurosci. 1998; 10: 108-21
122
Berseus O. Conversion of cholesterol to bile acids in rat: purification and properties of a delta-43-ketosteroid 5-beta-reductase and a 3-alpha-hydroxysteroid dehydrogenase. Eur J Biochem.
1967; 2: 493-502
Biermann-Ruben K, Miller A, Franzkowiak S, Finis J, Pollok B, Wach C et al. Increased sensory
feedback in Tourette syndrome. Neuroimage. 2012; 63:119-25
Bini V, Frau R, Collu M, Paba S, Devoto P, Marrosu F, Bortolato M. Blockade of 5-alpha
reductase reduces compulsive behaviors in mice. 22nd ECNP Congress, 12-16 September
2009, Istanbul, Turkey.
Bitar MS, Ota M, Linnoila M, Shapiro BH. Modification of gonadectomy-induced increases in
brain monoamine metabolism by steroid hormones in male and female rats.
Psychoneuroendocrinology. 1991; 16: 547-57
Bjorkhem I, Karlmar KE. Biosynthesis of cholestanol: conversion of cholesterol into 4cholesten-3-one by rat liver microsomes. Biochem Biophys Acta. 1974; 337: 129-31
Bjorkhem I. Mechamism and stereochemistry of the enzymatic conversion of a delta 4-3oxosteroid into a 3-oxo-5alpha-steroid. Eur J Biochem. 8: 345-51
Bliss J. Sensory experiences of Gilles de la Tourette syndrome. Arch Gen Psychiatry. 1980; 37:
1343-7
Bloch M, State M, Pittenger C. Recent advances in Tourette syndrome. Curr Opin Neurol. 2011;
24: 119-25
Bloch MH, Leckman JF, Zhu H, Peterson BS. Caudate volumes in childhood predict symptom
severity in adults with Tourette syndrome. Neurology. 2005; 65:1253-8
Bordi F, Meller E. Enhanced behavioral stereotypies elicited by intrastriatal injection D1 and D2
dopamine agonists in intact rats. Brain Res. 1989; 504: 276-283
Bornstein RA, Stefl ME, Hammond L. A survey of Tourette syndrome patients and their
families: the 1987 Ohio Tourette Survey. J Neuropsych Clin Neurosci. 1990; 2: 275-81
Bortolato M, Aru GN, Fà M, Frau R, Orrù M, Salis P, Casti A, Luckey GC, Mereu G, Gessa GL.
Activation of D1 but not D2 receptors potentiates dizocilpine-mediated disruption of prepulse
inhibition of the startle. Neuropsychopharmacology. 2005; 30: 561-74
Bortolato M, Cannas A, Solla P, Bini V, Puligheddu M, Marrosu F. Finasteride attenuates
pathological gambling in patients with Parkinson disease. J Clin Psychopharmacol. 2012; 32:
424-5
Bortolato M, Devoto P, Roncada P, Frau R, Flore G, Saba P, Pistritto G, Soggiu A, Pisanu S,
Zappalà A, Ristaldi MS, Tattoli M, Cuomo V, Marrosu F, Barbaccia ML. Isolation rearinginduced reduction of brain 5α-reductase expression: relevance to dopaminergic impairments.
Neuropharmacology. 2011; 60: 1301-8
123
Bortolato M, Frau R, Aru GN, Orru M, Gessa GL. Baclofen reverses the reduction in prepulse
inhibition of the acoustic startle response induced by dizocilpine but not by apomorphine.
Psychopharmacology. 2004; 171: 322-30
Bortolato M, Frau R, Godar SC, Mosher LJ, Paba S, Marrosu F et al. The implication of
neuroactive steroids in tourette's syndrome pathogenesis: a role for 5alpha-reductase? J
Neuroendocrinol. 2013; 25:1196-208
Bortolato M, Frau R, Orru M, Bourov Y, Marrosu F, Mereu G, Devoto P, Gessa GL.
Antipsychotic-like properties of 5-alpha-reductase inhibitors. Neuropsychopharmacology.
2008; 33: 3146-56
Bortolato M, Muroni A, Marrosu F. Treatment of Tourette's syndrome with finasteride. Am J
Psychiatry. 2007; 164: 1914-5
Bos-Veneman NG, Olieman R, Tobiasova Z, Hoekstra PJ, Katsovich L, Bothwell AL et al.
Altered immunoglobulin profiles in children with Tourette syndrome. Brain Behav Immun
.2011; 25:532-8
Braff DL, Geyer MA, Light GA, Sprock J, Perry W, Cadenhead KS, Swerdlow NR. Impact of
prepulse characteristics on the detection of sensorimotor gating deficits in schizophrenia.
Schizophr Res. 2001; 49: 171-8
Braff DL, Geyer MA, Swerdlow NR. Human studies of prepulse inhibition of startle: normal
subjects patient groups and pharmacological studies. Psychopharmacology. 2001; 156: 234–
58
Braff DL, Grillon C, Geyer MA. Gating and habituation of the startle reflex in schizophrenic
patients. Arch Gen Psychiatry. 1992; 49: 206-15
Brahm J, Brahm M, Segovia R, Latorre R, Zapata R, Poniachik J, Buckel E, Contreras L. Acute
and fulminant hepatitis induced by flutamide: case series report and review of the literature.
Annals of hepatology. 2011; 10: 93-8
Braun AR, Chase TN. Obligatory D1/D2 receptor interaction in the generation of dopamine
agonist related behaviors. Eur J Pharmacol. 1986; 131: 301-6
Breier MR, Lewis B, Shoemaker JM, Light GA, Swerdlow NR. Sensory and sensorimotor
gating-disruptive effects of apomorphine in Sprague Dawley and Long Evans rats. Behav
Brain Res. 2010; 208: 560–5
Brenes JC, Rodríguez O, Fornaguera J. Differential effect of environment enrichment and social
isolation on depressive-like behavior, spontaneous activity and serotonin and norepinephrine
concentration in prefrontal cortex and ventral striatum. Pharmacol Biochem Behav. 2008; 89:
85-93
Bristow LJ, Cook GP, Gay JC, Kulagowski JJ, Landon L, Murray F, Saywell KL, Young L,
Hutson PH. The behavioural and neurochemical profile of the putative dopamine D3 receptor
agonist (+)-PD 128907 in the rat. Neuropharmacology. 1996; 35: 285-94
124
Bronfeld M, Yael D, Belelovsky K, Bar-Gad I. Motor tics evoked by striatal disinhibition in the
rat Front Syst Neurosci. 2013; 7:50
Bruun RD, Budman CL. Risperidone as a treatment for Tourette's syndrome. J Clin Psychiatry.
1996; 57:29-31
Budman CL, Bruun RD, Park KS, Olson ME. Rage attacks in children and adolescents with
Tourette's disorder: a pilot study. J Clin Psychiatry. 1998; 59: 576-80
Budman CL, Gayer A, Lesser M, Shi Q, Bruun RD. An open-label strudy of the treatment
efficacy of olanzapine for Tourette’s disorder. J Clin Psychiatry 2001; 62: 290-4
Budman CL. Treatment of aggression in Tourette syndrome. Adv Neurol. 2006; 99: 222-6
Bull HG, Garcia-Calvo M, Andersson S, Baginsky WF, Chan HK, Ellsworth DE, Miller RR,
Stearns RA, Bakshi RK, Rasmusson GH, Tolman RL, Myers RW, Kozarich JW, Harris GS.
Mechanism-Based Inhibition of Human Steroid 5α-Reductase by Finasteride: EnzymeCatalyzed Formation of NADP−Dihydrofinasteride, a Potent Bisubstrate Analog Inhibitor. J
Am Chem Soc. 1996; 118: 2359-65
Buse J, Schoenefeld K, Munchau A, Roessner V. Neuromodulation in Tourette syndrome:
Dopamine and beyond. Neurosci Biobehav Rev. 2013; 37:1069-84
Byrnes EM, Bridges RS, Scanlan VF, Babb JA, Byrnes JJ. Sensorimotor gating and dopamine
function in postpartum rats. Neuropsychopharmacology. 2007; 32: 1021-31
Cadenhead KS, Geyer MA, Braff DL. Impaired startle prepulse inhibition and habituation in
patients with schizotypal personality disorder Am. J Psychiatry. 1993; 150: 1862-67
Campbell KM, de Lecea L, Severynse DM, Caron MG, McGrath MJ, Sparber SB, Sun LY,
Burton FH. OCD-Like behaviors caused by a neuropotentiating transgene targeted to cortical
and limbic D1+ neurons. J Neurosci 1999; 19: 5044-53
Canales JJ, Graybiel AM. A measure of striatal function predicts motor stereotypy. Nat
Neurosci. 2000; 3: 377-83
Caruso D, Scurati S, Maschi O, De Angelis L, Roglio I, Giatti S, Garcia-Segura LM, Melcangi
RC. Evaluation of neuroactive steroid levels by liquid chromatography-tandem mass
spectrometry in central and peripheral nervous system: effect of diabetes. Neurochem Int.
2008; 52: 560-8
Castellanos FX, Fine EJ, Kaysen D, Marsh WL, Rapoport JL, Hallett M. Sensorimotor gating in
boys with Tourette's syndrome and ADHD: preliminary results. Biol Psychiatry. 1996;3 9:3341
Castelli MP, Casti A, Casu A, Frau R, Bortolato M, Spiga S, Ennas MG. Regional distribution of
5alpha-reductase type 2 in the adult rat brain: an immunohistochemical
analysis .Psychoneuroendocrinology. 2013; 38: 281-93
125
Centonze D, Grande C, Saulle E, Martin AB, Gubellini P, Pavón N, Pisani A, Bernardi G,
Moratalla R, Calabresi P. Distinct roles of D1 and D5 dopamine receptors in motor activity
and striatal synaptic plasticity. J Neurosci. 2003; 23: 8506-12
Chappell P, Leckman J, Goodman W, Bissette G, Pauls D, Anderson G, Riddle M, Scahill L,
McDougle C, Cohen D. Elevated cerebrospinal fluid corticotropin-releasing factor in
Tourette's syndrome: comparison to obsessive-compulsive disorder and normal controls. Biol
Psychiatry. 1996; 39: 776-83
Chappell P, Riddle M, Anderson G, Scahill L, Hardin M, Walker D, Cohen D, Leckman J.
Enhanced stress responsivity of Tourette syndrome patients undergoing lumbar puncture. Biol
Psychiatry. 1994; 36: 35-43
Cohen SC, Leckman JF, Bloch MH. Clinical assessment of Tourette syndrome and tic disorders.
Neurosci Biobehav Rev. 2013; 37: 997-1007
Comings DE, Comings BG. A controlled study of Tourette syndrome II Conduct. Am J Hum
Genetics. 1987; 41: 742-60
Conelea CA, Woods DW, Brandt BC. The impact of a stress induction task on tic frequencies in
youth with Tourette Syndrome. Behav Res Ther. 2011; 49: 492-7
Conelea CA, Woods DW. The influence of contextual factors on tic expression in Tourette's
syndrome: a review. J Psychosom Res. 2008; 65: 487-96
Conti LH, Berridge CW, Tayler JE. Both corticotropin-releasing factor and apomorphine reduce
prepulse inhibition following repeated central infusion of corticotropin-releasing factor.
Pharmacol Biochem Behav. 2006; 85: 261-72
Conti LH, Segal DS, Kuczenski R. Maintenance of amphetamine-induced stereotypy and
locomotion requires ongoing dopamine receptor activation. Psychopharmacology. 1997;
130:183-8
Cooke PS, Nanjappa MK, Yang Z, Wang KK. Therapeutic effects of progesterone and its
metabolites in traumatic brain injury may involve non-classical signaling mechanisms. Front
Neurosci. 2013; 7:108.
Corbett BA, Mendoza SP, Baym CL, Bunge SA, Levine S. Examining cortisol rhythmicity and
responsivity to stress in children with Tourette syndrome. Psychoneuroendocrinology. 2008;
33: 810-20
Crawley JN, Glowa JR, Majewska MD, Paul SM. Anxiolytic activity of an endogenous adrenal
steroid. Brain Res. 1986; 398: 382-85
Cronbach LJ. Coefficient alpha and the internal structure of tests. Psychometrika. 1951; 16: 297334
Curtis D, Brett P, Dearlove AM, McQuillin A, Kalsi G, Robertson MM, Gurling HM. Genome
scan of Tourette syndrome in a single large pedigree shows some support for linkage to
regions of chromosomes 5 10 and 13. Psych Gen. 2004; 14: 83-7
126
Dantzer R. Stress stereotypies and welfare Behav Processes 1991; 25:95-102
Darbra S, Mòdol L, Pallarès M. Allopregnanolone infused into the dorsal (CA1) hippocampus
increases prepulse inhibition of startle response in Wistar rats. Psychoneuroendocrinology.
2012; 37: 581-85
Darbra S, Mòdol L, Vallée M, Pallarès M. Neonatal neurosteroid levels are determinant in
shaping adult prepulse inhibition response to hippocampal allopregnanolone in rats.
Psychoneuroendocrinology. 2013; 38:1397-406
Darbra S, Pallarès M. Alterations in neonatal neurosteroids affect exploration during adolescence
and prepulse inhibition in adulthood. Psychoneuroendocrinology. 2010; 35: 525-35
Davis M, Aghajanian GK. Effects of apomorphine and haloperidol on the acoustic startle
response in rats. Psychopharmacology. 1976; 47: 217-23
Davis M, Commissaris RL, Cassella JV, Yang S, Dember L, Harty TP. Differential effects of
dopamine agonists on acoustically and electrically elicited startle responses: comparison to
effects of strychnine. Brain Res. 1986; 371: 58-69
Davis M, Mansbach RS, Swerdlow NR, Braff DL, Geyer MA. Apomorphine disrupts the
inhibition of acoustic startle induced by weak prepulses in rats. Psychopharmacology. 1990;
102: 1–4
Davis M. Neurochemical modulation of sensory-motor reactivity: Acoustic and tactile startle
reflexes. Neurosci Biobehav Rev. 1980; 4: 241-263
Dazzi L, Serra M, Seu E, Cherchi G, Pisu MG, Purdy RH, Biggio G. Progesterone enhances
ethanol-induced modulation of mesocortical dopaminergic neurons: antagonism by finasteride.
J Neurochem. 2002; 83: 1103-9
de Bono JS, Logothetis CJ, Molina A, Fizazi K, North S, Chu L, Chi KN, Jones RJ, Goodman
OB, Saad F, Staffurth JN, Mainwaring P, Harland S, Flaig TW, Hutson TE, Cheng T,
Patterson H, Hainsworth JD, Ryan CJ ,Sternberg CN, Ellard SL, Fléchon A, Saleh M, Scholz
M, Efstathiou E, Zivi A, Bianchini D, Loriot Y, Chieffo N, Kheoh T, Haqq CM, Scher HI.
Abiraterone and increased survival in metastatic prostate cancer. N Engl J Med. 2011; 364:
1995-2005
de Sousa FL, Lazzari V, de Azevedo MS, de Almeida S, Sanvitto G,L Lucion AB, Giovenardi M.
Progesterone and maternal aggressive behavior in rats. Behav Brain Res. 2010; 212: 84-9
De Souza MM, Pereira MA, Ardenghi JV, Mora TC, Bresciani LF, Yunes RA, Delle Monache F,
Cechinel-Filho V. Filicene obtained from Adiantum cuneatum interacts with the cholinergic
dopaminergic glutamatergic GABAergic and tachykinergic systems to exert antinociceptive
effect in mice. Pharmacol Biochem Behav. 2009; 93: 40-6
de Souza Silva MA, Mattern C, Topic B, Buddenberg TE, Huston JP. Dopaminergic and
serotonergic activity in neostriatum and nucleus accumbens enhanced by intranasal
administration of testosterone. Eur Neuropsychopharmacology. 2009; 19: 53-63
127
Deng H, Gao K, Jankovic J. The genetics of Tourette syndrome. Nat Rev Neurology. 2012; 8:
203-13
Devoto P, Flore G, Saba P, Castelli MP, Piras AP, Luesu W, Viaggi MC, Ennas MG, Gessa GL.
6-Hydroxydopamine lesion in the ventral tegmental area fails to reduce extracellular
dopamine in the cerebral cortex. J Neurosci Res. 2008; 86: 1647-58
Devoto P, Flore G. On the Origin of Cortical dopamine: Is it a Co-Transmitter in Noradrenergic
Neurons? Curr Neuropharmacol. 2006; 41:15-125
Devoto P, Frau R, Bini V, Pillolla G, Saba P, Flore G, Corona M, Marrosu F, Bortolato M.
Inhibition of 5alpha-reductase in the nucleus accumbens counters sensorimotor gating deficits
induced by dopaminergic activation. Psychoneuroendocrinology. 2012; 37: 1630-45
Di Paolo T. Modulation of brain dopamine transmission by sex steroids. Rev Neurosci. 1994; 5:
27-41
Diaz J, Pilon C, Le Foll B, Gros C, Triller A, Schwartz JC, Sokoloff P. Dopamine D3 receptors
expressed by all mesencephalic dopamine neurons. J Neurosci, 2000; 20: 8677-84
Dietl T, Kumpfel T, Hinze-Selch D, Trenkwalder C, Lechner C. Exacerbation of tics by
prednisolone. Nervenarzt. 1998; 69: 1111-4
Dluzen DE, Green MA, Ramirez VD. The effect of hormonal condition on dose-dependent
amphetamine-stimulated behaviors in the male rat. Horm Behav. 1986; 20: 1-6
Doherty JM, Masten VL, Powell SB, Ralph RJ, Klamer D, Low MJ et al. Contributions of
dopamine D1 D2 and D3 receptor subtypes to the disruptive effects of cocaine on prepulse
inhibition in mice. Neuropsychopharmacology. 2008; 33: 2648-56
Dong LY, Cheng ZX, Fu YM, Wang ZM, Zhu YH, Sun JL, Dong Y, Zheng P. Neurosteroid
dehydroepiandrosterone sulfate enhances spontaneous glutamate release in rat prelimbic
cortex through activation of Dopamine D1 and sigma-1 receptor. Neuropharmacology. 2007;
52: 966-74
Drury JE, Di Costanzo L, Penning TM, Christianson DW. Inhibition of human steroid 5betareductase (AKR1D1) by finasteride and structure of the enzyme inhibitor complex. J Biol
Chem. 2009; 24: 284 19786-90
Dubrovsky B. Neurosteroids neuroactive steroids and symptoms of affective disorders.
Pharmacol Biochem Behav. 2006; 84: 644-55
Dupont E, Simard J, Luu-The V, Labrie F, Pelletier G. Localization of 3 beta-hydroxysteroid
dehydrogenase in rat brain as studied by in situ hybridization. Mol Cell Neurosci. 1994; 5:
119–23
Eagle DM, Wong JC, Allan ME, Mar AC, Theobald DE, Robbins TW. Contrasting roles for
dopamine D1 and D2 receptor subtypes in the dorsomedial striatum but not the nucleus
accumbens core during behavioral inhibition in the stop-signal task in rats. J Neurosci. 2011;
31: 7349-56
128
Eichele H, Eichele T, Hammar A, Freyberger HJ, Hugdahl K, Plessen KJ. Go/NoGo
performance in boys with Tourette syndrome. Child Neuropsychol. 2010; 16:162-8
Eichele H, Plessen KJ. Neural plasticity in functional and anatomical MRI studies of children
with Tourette syndrome. Behav Neurol. 2013; 27: 33-45
Einon DF, Morgan MJ. A critical period for social isolation in the rat. Dev Psychobiol. 1977;
10: 123-32
Engel J, Ahlenius S, Almgren O, Carlsson A, Larsson K, Södersten P. Effects of gonadectomy
and hormone replacement on brain monoamine synthesis in male rats. Pharmacol Biochem
Behav. 1979; 10: 149-54
Engin E, Treit D. The anxiolytic-like effects of allopregnanolone vary as a function of
intracerebral microinfusion site: the amygdala medial prefrontal cortex or hippocampus.
Behav Pharmacol. 2007; 18: 461-70
Eser D, Baghai TC, Schüle C, Nothdurfter C, Rupprecht R. Neuroactive steroids as endogenous
modulators of anxiety. Curr Pharm Des. 2008; 14: 3525-33
Eser D, Romeo E, Baghai TC, di Michele F, Schule C, Pasini A, Zwanzger P, Padberg F,
Rupprecht R. Neuroactive steroids as modulators of depression and anxiety. Neuroscience.
2006; 138: 1041-8
Espallergues J, Temsamani J, Laruelle C, Urani A, Maurice T. The antidepressant-like effect of
the 3β-hydroxysteroid dehydrogenase inhibitor trilostane involves a regulation of β-type
estrogen receptors. Psychopharmacology. 2011; 214: 455-63
Fabre-Nys C. Steroid control of monoamines in relation to sexual behaviour. Rev Reprod. 1998;
3: 31-41
Fahim C, Yoon U, Das S, Lyttelton O, Chen J, Arnaoutelis R et a.l Somatosensory-motor bodily
representation cortical thinning in Tourette: effects of tic severity age and gender. Cortex.
2010; 46:750-60
Felling RJ, Singer HS. Neurobiology of tourette syndrome: current status and need for further
investigation. J Neurosci. 2011;31:12387-95
Fernández-Pérez L, Flores-Morales A, Chirino-Godoy R, Díaz-Chico JC, Díaz-Chico BN.
Steroid binding sites in liver membranes: interplay between glucocorticoids sex steroids and
pituitary hormones. J Steroid Biochem Mol Biol. 2008; 109: 336-43
Fone KCF, Porkess MV. Behavioural and neurochemical effects of post-weaning social isolation
in rodents-Relevance to developmental neuropsychiatric disorders. Neurosci Biobehav. 2008;
32: 1087-102
Foradori CD, Weiser MJ, Handa RJ. Non-genomic actions of androgens. Front Neuroendocrinol.
2008;29: 169-81.
Frank MC, Piedad J, Rickards H, Cavanna AE. The role of impulse-control disorders in Tourette
syndrome: an exploratory study. J Neurol Sci. 2011; 310:276-8
129
Frau R, Bini V, Pes R, Pillolla G, Saba P, Devoto P, Bortolato M. Inhibition of 17alphahydroxylase/C1720 lyase reduces gating deficits consequent to dopaminergic activation.
Psychoneuroendocrinology. 2014; 39:204-13
Frau R, Orrù M, Fà M, Casti A, Manunta M, Fais N, Mereu G, Gessa G, Bortolato M. Effects of
topiramate on the prepulse inhibition of the acoustic startle in rats.
Neuropsychopharmacology. 2007; 32: 320-31
Frau R, Pillola G, Bini V, Tambaro S, Devoto P, Bortolato M. Inhibition of 5α-reductase
attenuates behavioral effects of D1- but not D2-like receptor agonists in C57BL/6 mice.
Psychoneuroendocrinology. 2013; 38: 542–51
Frye CA, Koonce CJ, Edinger KL, Osborne DM, Walf AA. Androgens with activity at estrogen
receptor beta have anxiolytic and cognitive-enhancing effects in male rats and mice. Horm
Behav. 2008; 54: 726-34
Frye CA, Rhodes ME, Rosellini R, Svare B. The nucleus accumbens as a site of action for
rewarding properties of testosterone and its 5alpha-reduced metabolites. Pharmacol Biochem
Behav. 2002; 74: 119-27
Frye CA, Walf AA. Changes in progesterone metabolites in the hippocampus can modulate open
field and forced swim test behavior of proestrous rats. Horm Behav. 2002; 41: 306-15
Frye CA, Walf AA. Infusions of anti-sense oligonucleotides for DARPP-32 to the ventral
tegmental area reduce effects of progesterone- and a dopamine type 1-like receptor agonist to
facilitate lordosis. Behav Brain Res. 2010; 206: 286-92
Fu Y, Zhao Y, Luan W, Dong LY, Dong Y, Lai B, Zhu Y, Zheng P. Sigma-1 receptors amplify
dopamine D1 receptor signaling at presynaptic sites in the prelimbic cortex. Biochim Biophys
Acta. 2010; 1803: 1396-408
Fulford AJ, Marsden CA Effect of isolation-rearing on conditioned dopamine release in vivo in
the nucleus accumbens of the rat. J Neurochem. 1998; 70: 384-90
Fuxe K, Borroto-Escuela DO, Romero-Fernandez W, Diaz-Cabiale Z, Rivera A, Ferraro L,
Tanganelli S, Tarakanov AO, Garriga P, Narváez JA, Ciruela F, Guescini M, Agnati LF.
Extrasynaptic neurotransmission in the modulation of brain function. Focus on the striatal
neuronal-glial networks. Front Physiol. 2012; 3:136
Ganos C, Kahl U, Schunke O, Kuhn S, Haggard P, Gerloff C et al. Are premonitory urges a
prerequisite of tic inhibition in Gilles de la Tourette syndrome? J Neurol Neurosurg
Psychiatry. 2012; 83:975-8
Ganos C, Kuhn S, Kahl U, Schunke O, Brandt V, Baumer T et al. Prefrontal cortex volume
reductions and tic inhibition are unrelated in uncomplicated GTS adults. J Psychosom Res.
2014; 76:84-7
Garcia-Segura LM, Veiga S, Sierra A, Melcangi RC, Azcoitia I. Aromatase: a neuroprotective
enzyme. Prog Neurobiol. 2003; 71: 31-41.
130
Garner JP, Mason GJ. Evidence for a relationship between cage stereotypies and behavioural
disinhibition in laboratory rodents. Behav Brain Res. 2002;136:83-92
Geller DH, Auchus RJ, Miller WL. P450c17 mutations R347H and R358Q selectively disrupt
17,20-lyase activity by disrupting interactions with P450 oxidoreductase and cytochrome b5.
Mol Endocrinol. 1999; 13: 167-75
Gerdes D, Wehling M, Leube B, Falkenstein E. Cloning and tissue expression of two putative
steroid membrane receptors. Biol Chem. 1998; 379: 907-11
Gerfen CR, Engber TM, Mahan LC, Susel Z, Chase TN, Monsma Jr FJ, Sibley DR. D1 and D2
dopamine receptor-regulated gene expression of striatonigral and striatopallidal neurons.
Science. 1990; 250:1429-32
Geter-Douglass B, Katz JL, Alling K, Acri JB, Witkin JM. Characterization of unconditioned
behavioral effects of dopamine D3/D2 receptor agonists. J Pharmacol Exp Ther. 1997; 283: 715.
Geyer MA, Krebs-Thomson K, Braff DL, Swerdlow NR. Pharmacological studies of prepulse
inhibition models of sensorimotor gating deficits in schizophrenia: a decade in review
Psychopharmacology 2001;156:117-54
Ghanizadeh A, Mosallaei S. Psychiatric disorders and behavioral problems in children and
adolescents with Tourette syndrome. Brain Dev. 2009; 31:15-9
Gilbert DL, Budman CL, Singer HS, Kurlan R, Chipkin RE. A D1 receptor antagonist,
ecopipam, for treatment of tics in Tourette syndrome. Clin Neuropharmacol. 2014; 37:26-30
Gilbert DL, Christian BT, Gelfand MJ, Shi B Mantil J, Sallee FR. Altered mesolimbocortical and
thalamic dopamine in Tourette syndrome. Neurology. 2006; 67: 1695-7
Girdler SS, Klatzkin R. Neurosteroids in the context of stress: implications for depressive
disorders. Pharmacol Ther. 2007; 116: 125-39
Girman CJ, Kolman C, Liss CL, Bolognese JA, Binkowitz BS, Stoner E. Effects of finasteride
on health-related quality of life in men with symptomatic benign prostatic hyperplasia.
Finasteride Study Group. Prostate. 1996; 29: 83-90
Godar SC, Bortolato M. Gene-sex interactions in schizophrenia: focus on dopamine
neurotransmission. Front Behav Neurosci. 2014; 8: 71
Gogos A, van den Buuse M. Castration reduces the effect of serotonin-1A receptor stimulation
on prepulse inhibition in rats. Behav Neurosci. 2003; 117: 1407-1415
Gomez EC, Frost P. Testosterone metabolism and androgenic effect. Adv Biol Skin. 1972; 12:
367-379
Gomis M, Puente V, Pont-Sunyer C, Oliveras C, Roquer J. Adult onset simple phonic tic after
caudate stroke. Mov Disord. 2008; 23:765-6
Gonon F. Prolonged and extrasynaptic excitatory action of dopamine mediated by D1 receptors
in the rat striatum in vivo. J Neurosci. 1997; 17:5972-8
131
Gottesman II, Shields J. Genetic theorizing and schizophrenia. Br J Psychiatry. 1973;122:15-30
Gould TD, Gottesman II. Psychiatric endophenotypes and the development of valid animal
models. Genes Brain Behav. 2006; 5:113-9
Graham SJ, Langley RW, Bradshaw CM, Szabadi E. Effects of haloperidol and clozapine on
prepulse inhibition of the acoustic startle response and the N1/P2 auditory evoked potential in
man. J Psychopharmacol. 2001;15:243-50
Gras F, Brunmair B, Quarré L, Szöcs Z, Waldhäusl W, Fürnsinn C. Progesterone impairs cell
respiration and suppresses a compensatory increase in glucose transport in isolated rat skeletal
muscle: a non-genomic mechanism contributing to metabolic adaptation to late pregnancy?
Diabetologia. 2007; 50: 2544-52
Groenewegen HJ, Trimble M. The ventral striatum as an interface between the limbic and motor
systems. CNS Spectr. 2007; 12: 887-92
Halberda JP, Middaugh LD, Gard BE, Jackson BP. Dopamine D1- and dopamine D2-like agonist
effects on motor activity of C57 mice: differences compared to rats. Synapse. 1997; 26: 81-92
Hall FS, Wilkinson LS, Humby T, Inglis W, Kendall DA, Marsden CA, Robbins TW. Isolation
rearing in rats: pre- and postsynaptic changes in striatal dopaminergic systems. Pharmacol
Biochem Behav. 1998; 59: 859-72
Handa RJ, Pak TR, Kudwa AE, Lund TD, Hinds L. An alternate pathway for androgen
regulation of brain function: activation of estrogen receptor beta by the metabolite of
dihydrotestosterone, 5alpha-androstane-3beta,17beta-diol. Horm Behav. 2008;53: 741-52.
Hara T, Kouno J, Kaku T, Takeuchi T, Kusaka M, Tasaka A, Yamaoka M. Effect of a novel
1720-lyase inhibitor orteronel (TAK-700) on androgen synthesis in male rats. J Steroid
Biochem Mol Biol. 2013; 134: 80-91
Hart S, Zreik M, Carper R, Swerdlow NR. Localizing haloperidol effects on sensorimotor gating
in a predictive model of antipsychotic potency. Pharmacol Biochem Behav. 1998; 61: 113-9
Heidbreder CA, Weiss IC, Domeney AM, Pryce C, Homberg J, Hedou G, Feldon J, Moran MC,
Nelson P. Behavioral, neurochemical and endocrinological characterization of the early social
isolation syndrome. Neuroscience. 2000; 100: 749-68
Heinlein CA, Chang C. The roles of androgen receptors and androgen-binding proteins in
nongenomic androgen actions. Mol Endocrinol. 2002; 16: 2181-7
Heise KF, Steven B, Liuzzi G, Thomalla G, Jonas M, Muller-Vahl K et al. Altered modulation of
intracortical excitability during movement preparation in Gilles de la Tourette syndrome.
Brain. 2010;133:580-90
Hernandez L, Gonzalez L, Murzi E, Paez X, Gottberg E, Baptista T. Testosterone modulates
mesolimbic dopaminergic activity in male rats. Neurosci Lett. 1994; 71: 172-4
Hikosaka O, Nakamura K, Sakai K, Nakahara H. Central mechanisms of motor skill learning.
Curr Opin Neurobiol. 2002; 12: 217-22
132
Hoekstra PJ, Dietrich A, Edwards MJ, Elamin I, Martino D. Environmental factors in Tourette
syndrome. Neurosci Biobehav Rev. 2013; 37:1040-9.
Hoenig K, Hochrein A, Quednow BB, Maier W, Wagner M. Impaired prepulse inhibition of
acoustic startle in obsessive-compulsive disorder Biol Psychiatry 2005; 57: 1153-8
Hoffman DC, Donovan H. D1 and D2 dopamine receptor antagonists reverse prepulse inhibition
deficits in an animal model of schizophrenia. Psychopharmacology. 1994; 115: 447-53
Hoffman HS, Ison JR. Reflex modification in the domain of startle: I. Some empirical findings
and their implications for how the nervous system processes sensory input. Psychol Rev.
1980; 87:175-89
Hojo Y, Hattori TA, Enami T, Furukawa A, Suzuki K, Ishii HT, Mukai H, Morrison JH, Janssen
WG, Kominami S, Harada N, Kimoto T, Kawato S. Adult male rat hippocampus synthesizes
estradiol from pregnenolone by cytochromes P45017alpha and P450 aromatase localized in
neurons. Proc Natl Acad Sci USA. 2004; 101: 865-70
Holmes A, Hollon TR, Gleason TC, Liu Z, Dreiling J, Sibley DR, Crawley JN. Behavioral
characterization of dopamine D5 receptor null mutant mice. Behav Neurosci. 2001; 115: 112944
Hyde TM, Stacey ME, Coppola R, Handel SF, Rickler KC, Weinberger DR. Cerebral
morphometric abnormalities in Tourette's syndrome: a quantitative MRI study of monozygotic
twins. Neurology. 1995; 45:1176-82
Imperato A, Puglisi-Allegra S, Casolini P, Zocchi A, Angelucci L. Stress-induced enhancement
of dopamine and acetylcholine release in limbic structures: role of corticosterone. Eur J
Pharmacol. 1989; 165: 337-8
Intlekofer KA, Petersen SL. Distribution of mRNAs encoding classical progestin receptor,
progesterone membrane components 1 and 2, serpine mRNA binding protein 1, and progestin
and ADIPOQ receptor family members 7 and 8 in rat forebrain. Neuroscience. 2011; 172: 5565.
Ishikawa T, Glidewell-Kenney C, Jameson JL. Aromatase-independent testosterone conversion
into estrogenic steroids is inhibited by a 5 alpha-reductase inhibitor. J Steroid Biochem Mol
Biol. 2006; 98: 133-38
Izmir M, Dursun SM. Cyproterone acetate treatment of Tourette's syndrome. Can J Psychiatry.
1999; 44: 710-1
Jankovic J, Kurlan R. Tourette syndrome: evolving concepts. Mov disorders. 2011; 26: 1149-56
Jankovic J. Diagnosis and classification of tics and Tourette syndrome. Adv Neurol. 1992; 58:714
Jankovic J. Tourette syndrome Phenomenology and classification of tics. Neurol clin. 1997; 15:
267-75
Jankovic J. Tourette's syndrome. New Engl J Med. 2001; 345: 1184-92
133
Jimenez-Jimenez FJ, Garcia-Ruiz PJ. Pharmacological options for the treatment of Tourette’s
disorder. Drugs. 2001; 61:2207-20
Jin Y, Penning TM. Steroid 5alpha-reductases and 3alpha-hydroxysteroid dehydrogenases: key
enzymes in androgen metabolism. Best Pract Res Clin Endocrinol Metab. 2001; 15: 79-94
Johansson C, Jackson DM, Zhang J, Svensson L. Prepulse inhibition of acoustic startle a
measure of sensorimotor gating: Effects of antipsychotics and other agents in rats. Pharmacol
Biochem Behav. 1995; 52: 649-54
Jones GH, Hernandez TD, Kendall D A, Marsden CA, Robbins TW. Dopaminergic and
serotonergic function following isolation rearing in rats: study of behavioural responses and
postmortem and in vivo neurochemistry. Pharmacol Biochem Behav. 1992; 43: 17-35
Kalanithi PS, Zheng W, Kataoka Y, DiFiglia M, Grantz H, Saper CB et al. Altered parvalbuminpositive neuron distribution in basal ganglia of individuals with Tourette syndrome. Proc Natl
Acad Sci U S A. 2005; 102: 13307-12
Kalueff AV, Jensen CL, Murphy DL. Locomotory patterns spatiotemporal organization of
exploration and spatial memory in serotonin transporter knockout mice. Brain Res. 2007;
1169: 87-97
Kamrath C, Hartmann MF, Remer T, Wudy SA. The activities of 5alpha-reductase and 17,20lyase determine the direction through androgen synthesis pathways in patients with 21hydroxylase deficiency. Steroids. 2012; 77: 1391-7
Kane MJ. Premonitory urges as "attentional tics" in Tourette's syndrome. J Am Acad Child
Adolesc Psychiatry. 1994; 33:805-8
Karagiannidis I, Rizzo R, Tarnok Z, Wolanczyk T, Hebebrand J, Nothen MM, Lehmkuhl G,
Farkas L, Nagy P, Barta C, Szymanska U, Panteloglou G, Miranda DM, Feng Y, Sandor P,
Barr C ,TsgeneSee, Paschou P. Replication of association between a SLITRK1 haplotype and
Tourette Syndrome in a large sample of families. Mol Psychiatry. 2012; 17:665-8
Katagiri M, Kagawa N, Waterman MR. The role of cytochrome b5 in the biosynthesis of
androgens by human P450c17. Arch Biochem Biophys. 1995; 317: 343-7
Kataoka Y, Kalanithi PS, Grantz H, Schwartz ML, Saper C, Leckman JF et al. Decreased
number of parvalbumin and cholinergic interneurons in the striatum of individuals with
Tourette syndrome. J Comp Neurol. 2010; 518:277-91
Kehne JH, Sorenson CA. The effects of pimozide and phenoxybenzamine pretreatments on
amphetamine and apomorphine potentiation of the acoustic startle response in rats.
Psychopharmacology. 1978; 58137-144
Kelly MA, Low MJ, Rubinstein M, Phillips TJ. Role of dopamine D1-like receptors in
methamphetamine locomotor responses of D2 receptor knockout mice. Genes Brain Behav.
2008; 7: 568-77
134
Khanna M, Qin KN, Cheng KC. Distribution of 3 alpha-hydroxysteroid dehydrogenase in rat
brain and molecular cloning of multiple cDNAs encoding structurally related proteins in
humans. J Steroid Biochem Mol Biol. 1995a; 53: 41-46
Khanna M, Qin KN, Wang RW, Cheng KC. Substrate specificity gene structure and tissuespecific distribution of multiple human 3 alpha-hydroxysteroid dehydrogenases. J Biol Chem,
1995b; 270: 20162-8
Kidd KK, Prusoff BA, Cohen DJ. Familial pattern of Gilles de la Tourette syndrome. Arch Gen
Psych. 1980; 37: 1336-9
Kim HA, Jiang L, Madsen H, Parish CL, Massalas J, Smardencas A et al. Resolving
pathobiological mechanisms relating to Huntington disease: gait balance and involuntary
movements in mice with targeted ablation of striatal D1 dopamine receptor cells. Neurobiol
Dis. 2014; 62: 323-37
Kinney GG, Wilkinson LO, Saywell KL, Tricklebank MD. Rat strain differences in the ability to
disrupt sensorimotor gating are limited to the dopaminergic system specific to prepulse
inhibition and unrelated to changes in startle amplitude or nucleus accumbens dopamine
receptor sensitivity. J Neurosci. 1999; 19: 5644-53
Knight T, Steeves T, Day L, Lowerison M, Jette N, Pringsheim T. Prevalence of tic disorders: a
systematic review and meta-analysis. Pediatr Neuro.l 2012; 47:77-90
Koethe D, Bortolato M, Piomelli D, Leweke FM. Improvement of general symptoms in a
chronic psychotic patient treated with finasteride: case report. Pharmacopsychiatry. 2008; 41:
115-6
Kohchi C, Ukena K, Tsutsui K. Age- and region-specific expressions of the messenger RNAs
encoding for steroidogenic enzymes p450scc, P450c17, and 3beta-HSD in the postnatal rat
brain. Brain Res. 1998; 801: 233-8
Kompoliti K, Goetz CG, Leurgans S, Raman R, Comella CL. Estrogen progesterone and tic
severity in women with Gilles de la Tourette syndrome. Neurology. 2001; 57: 1519
Kretschmer BD, Koch M. The ventral pallidum mediates disruption of prepulse inhibition of the
acoustic startle response induced by dopamine agonists but not by NMDA antagonists. Brain
Res. 1998; 798: 204-10
Kuiper GG, Carlsson B, Grandien K, Enmark E, Häggblad J, Nilsson S, Gustafsson JA.
Comparison of the ligand binding specificity and transcript tissue distribution of estrogen
receptors alpha and beta. Endocrinology. 1997a; 138: 863-70
Kumari V, Antonova E, Geyer MA, Ffytche D, Williams SC, Sharma T. A fMRI investigation of
startle gating deficits in schizophrenia patients treated with typical or atypical antipsychotics.
Int J Neuropsychopharmacol. 2007; 10: 463-77
Kumari V, Sharma T. Effects of typical and atypical antipsychotics on prepulse inhibition in
schizophrenia: a critical evaluation of current evidence and directions for future research.
Psychopharmacology. 2002; 162:97-101
135
Kumari V, Soni W, Mathew VM, Sharma T. Prepulse inhibition of the startle response in men
with schizophrenia: effects of age of onset of illness symptoms and medication. Arch Gen
Psych. 2000; 57:609-14
Kumari V, Soni W, Sharma T. Prepulse inhibition of the startle response in risperidone-treated
patients: comparison with typical antipsychotics. Schizophr Res. 2002; 55:139-46
Kwak C, Dat Vuong K, Jankovic J. Premonitory sensory phenomenon in Tourette's syndrome.
Mov Disord. 2003; 18:1530-3
LaHoste GJ, Ruskin DN, Marshall JF. Cerebrocortical Fos expression following dopaminergic
stimulation: D1/D2 synergism and its breakdown. Brain Res. 1996; 728: 97-104
Lambert JJ, Belelli D, Peden DR, Vardy AW, Peters JA. Neurosteroid modulation of GABAA
receptors. Prog Neurobiol. 2003; 71: 67-80.
Leboyer M, Bellivier F, Nosten-Bertrand M, Jouvent R, Pauls D, Mallet J. Psychiatric genetics:
search for phenotypes. Trends Neurosci. 1998; 21:102-5
Leckman JF, Bloch MH, King RA, Scahill L. Phenomenonlogy of tics and natural history of tic
disorders. Adv Neurol. 2006; 99:1-16
Leckman JF, Bloch MH, Smith ME, Larabi D, Hampson M. Neurobiological substrates of
Tourette's disorder. J Child Adolesc Psychopharmacol. 2010; 20: 237-47
Leckman JF, Dolnansky ES, Hardin MT, Clubb M, Walkup JT, Stevenson J et al .Perinatal
factors in the expression of Tourette's syndrome: an exploratory study. J Am Acad Child
Adolesc Psychiatry. 1990; 29:220-6
Leckman JF, Goodman WK, Anderson GM, Riddle MA, Chappell PB, McSwiggan-Hardin MT,
McDougle CJ, Scahill LD, Ort SI, Pauls DL et al. Cerebrospinal fluid biogenic amines in
obsessive-compulsive
disorder,
Tourette's
syndrome
and
healthy
controls.
Neuropsychopharmacology. 1995; 12: 73-86
Leckman JF, Scahill L. Possible exacerbation of tics by androgenic steroids. N Engl J Med. 1990;
322: 1674
Leckman JF, Zhang H, Vitale A, Lahnin F, Lynch K, Bondi C, Kim YS, Peterson BS. Course of
tic severity in Tourette syndrome: the first two decades. Pediatrics. 1998; 102: 14-9
Leckman JF. Tourette's syndrome. Lancet. 2002 360:1577-86.
Leng A, Feldon J, Ferger B. Long-term social isolation and medial prefrontal cortex:
dopaminergic and cholinergic neurotransmission. Pharmacol Biochem Behav. 2004; 77: 3719
Lephart ED, Lund TD, Horvath TL. Brain androgen and progesterone metabolizing enzymes:
biosynthesis, distribution and function. Brain Res Brain Res Rev. 2001; 37: 25-37.
Leslie DL, Kozma L, Martin A, Landeros A, Katsovich L, King RA et al. Neuropsychiatric
disorders associated with streptococcal infection: a case-control study among privately
insured children. J Am Acad Child Adolesc Psychiatry. 2008; 47:1166-72
136
Leumann L, Feldon J, Vollenweider FX, Ludewig K. Effects of typical and atypical
antipsychotics on prepulse inhibition and latent inhibition in chronic schizophrenia. Biol
Psychiatry. 2002; 52729-739
Liao S, Fang S. Receptor-proteims for androgens and the mode of action of androgens on gene
transcription in ventral prostate. Vitam Horm. 1969; 27: 17-90
Lichter DG. Female gender is an independent predictor of tic severity in adult Tourette syndrome.
Mov Disord. 2008; 23S:146-S7
Lieberman JA, Sheitman BB, Kinon BJ. Neurochemical sensitization in the pathophysiology of
schizophrenia: deficits and dysfunction in neuronal regulation and plasticity
.Neuropsychopharmacology. 1997; 17: 205–29
Lin H, Katsovich L, Ghebremichael M, Findley DB, Grantz H, Lombroso PJ, King RA, Zhang H,
Leckman JF. Psychosocial stress predicts future symptom severities in children and
adolescents with Tourette syndrome and/or obsessive-compulsive disorder. J Child Psych
Psychiatry All Discip. 2007; 48: 157-66
Lin H, Williams KA, Katsovich L, Findley DB, Grantz H, Lombroso PJ, King RA, Bessen DE
Johnson D, Kaplan EL, Landeros-Weisenberger A, Zhang H, Leckman JF. Streptococcal
upper respiratory tract infections and psychosocial stress predict future tic and obsessivecompulsive symptom severity in children and adolescents with Tourette syndrome and
obsessive-compulsive disorder. Biol Psychiatry. 2010; 67: 684-91
Lind NM, Arnfred SM, Hemmingsen RP, Hansen AK. Prepulse inhibition of the acoustic startle
reflex in pigs and its disruption by d-amphetamine. Behav Brain Res. 2004; 155:217-22
Lombroso PJ, Mack G, Scahill L, King RA, Leckman J. Exacerbation of Gilles de la Tourette's
syndrome associated with thermal stress: a family study. Neurology. 1991; 41: 1984-7
Maillard L, Ishii K, Bushara K ,Waldvogel D, Schulman AE,. Hallett M. Mapping the basal
ganglia: fMRI evidence for somatotopic representation of face hand and foot. Neurology.
2000; 55:377-83
Makki MI, Govindan RM, Wilson BJ, Behen ME, Chugani HT. Altered fronto-striato-thalamic
connectivity in children with Tourette syndrome assessed with diffusion tensor MRI and
probabilistic fiber tracking. J Child Neurol. 2009; 24:669-78
Makridakis N, Reichardt JK. Pharmacogenetic analysis of human steroid 5 alpha reductase type
II: comparison of finasteride and dutasteride. J Mol Endocrinol. 2005; 34: 617-23
Mani SK, Fienberg AA, O'Callaghan JP, Snyder GL, Allen PB, Dash PK, Moore AN, Mitchell
AJ, Bibb J, Greengard P, O'Malley BW. Requirement for DARPP-32 in progesteronefacilitated sexual receptivity in female rats and mice. Science. 2000; 287: 1053-6
Mann PE. Finasteride delays the onset of maternal behavior in primigravid rats. Physiol Behav.
2006; 88: 333-338
Mansbach RS, Geyer MA, Braff DL. Dopaminergic stimulation disrupts sensorimotor gating in
the rat. Psychopharmacology. 1988; 94:507-14
137
Mapes S, Corbin CJ, Tarantal A, Conley A. The primate adrenal zona reticularis is defined by
expression of cytochrome b5, 17alpha-hydroxylase/17,20-lyase cytochrome P450 (P450c17)
and NADPH-cytochrome P450 reductase (reductase) but not 3beta-hydroxysteroid
dehydrogenase/delta5-4 isomerase (3beta-HSD). J Clin Endocrinol Metabol. 1999; 84: 33825
Markou A, Chiamulera C, Geyer MA, Tricklebank M, Steckler T. Removing obstacles in
neuroscience drug discovery: the future path for animal models. Neuropsychopharmacology.
2009; 34:74-89
Marsh R, Alexander GM, Packard MG, Zhu H ,Wingard JC, Quackenbush G, Peterson BS.
Habit learning in Tourette syndrome: a translational neuroscience approach to a
developmental psychopathology. Arch Gen Psychiatry. 2004; 61: 1259-68
Martin-Garcia E, Darbra S, Pallares M. Neonatal finasteride induces anxiogenic-like profile and
deteriorates passive avoidance in adulthood after intrahippocampal neurosteroid
administration. Neuroscience. 2008; 154: 1497-505
Martini L, Celotti F, Melcangi RC. Testosterone and progesterone metabolism in the central
nervous system: cellular localization and mechanism of control of the enzymes involved. Cell
Mol Neurobiol. 1996; 16: 271–82
Martini L, Melcangi RC, Maggi R. Androgen and progesterone metabolism in the central and
peripheral nervous system. J Steroid Biochem Mol Biol. 1993; 47195-205
Martino D, Dale RC, Gilbert DL, Giovannoni G, Leckman JF. Immunopathogenic mechanisms
in tourette syndrome: A critical review. Mov Disord. 2009; 24: 1267-79
Marx CE, Bradford DW, Hamer RM, Naylor JC, Allen TB, Lieberman JA, Strauss JL, Kilts JD.
Pregnenolone as a novel therapeutic candidate in schizophrenia: emerging preclinical and
clinical evidence. Neuroscience. 2011; 191: 78-90
Marx CE, Keefe RS, Buchanan RW, Hamer RM, Kilts JD, Bradford DW, Strauss JL, Naylor JC,
Payne VM, Lieberman JA, Savitz AJ, Leimone LA Dunn L, Porcu P, Morrow AL, Shampine
LJ. Proof-of-concept trial with the neuroster,oid pregnenolone targeting cognitive and
negative symptoms in schizophrenia. Neuropsychopharmacology. 2009; 34: 1885-903
Mathews CA, Bimson B, Lowe TL, Herrera LD, Budman CL, Erenberg G, Naarden A, Bruun
RD, Freimer NB, Reus VI. Association between maternal smoking and increased symptom
severity in Tourette's syndrome. Am J Psychiatry. 2006; 163: 1066-73
Maurice T, Urani A, Phan VL, Romieu P. The interaction between neuroactive steroids and the
sigma1 receptor function: behavioral consequences and therapeutic opportunities. Brain Res
Rev. 2001; 37: 116-32
Mazzone L, Yu S, Blair C, Gunter BC, Wang Z, Marsh R et al. An FMRI study of frontostriatal
circuits during the inhibition of eye blinking in persons with Tourette syndrome. Am J
Psychiatry. 2010; 167:341-9
138
McGrath MJ, Campbell KM, Parks CR, Burton FH. Glutamatergic drugs exacerbate
symptomatic behavior in a transgenic model of comorbid Tourette's syndrome and obsessivecompulsive disorder. Brain Res. 2000; 877: 23-30
McInnes KJ, Kenyon CJ, Chapman KE, Livingstone DE, Macdonald LJ, Walker BR, Andrew R.
5alpha-reduced glucocorticoids novel endogenous activators of the glucocorticoid receptor. J
Biol Chem. 2004; 279: 22908-12
Melcangi RC, Garcia-Segura LM, Mensah-Nyagan AG. Neuroactive steroids: state of the art and
new perspectives. Cell Mol Life Sci. 2008; 65:777-97.
Mell LK, Davis RL, Owens D. Association between streptococcal infection and obsessivecompulsive disorder Tourette's syndrome and tic disorder. Pediatrics. 2005;116:56-60
Mellon SH, Vaudry H. Biosynthesis of neurosteroids and regulation of their synthesis. Int Rev
Neurobiol. 2001; 46: 33-78
Menniti FS ,Baum MJ. Differential effects of estrogen and androgen on locomotor activity
induced in castrated male rats by amphetamine a novel environment or apomorphine. Brain
Res. 1981; 216: 89-107
Mensah-Nyagan AG, Do-Régo JL, Beaujean D, Luu-The V, Pelletier G, Vaudry H. Regulation
of neurosteroid biosynthesis in the frog diencephalon by GABA and endozepines. Horm
Behav. 2001; 40: 218-25
Mensah-Nyagan AG, Kibaly C, Schaeffer V, Venard C, Meyer L, Patte-Mensah C. Endogenous
steroid production in the spinal cord and potential involvement in neuropathic pain
modulation. J Steroid Biochem Mol Biol. 2008; 109: 286-93
Meredith GE, Agolia R, Arts MP, Groenewegen HJ, Zahm DS. Morphological differences
between projection neurons of the core and shell in the nucleus accumbens of the rat.
Neuroscience. 1992; 50: 149-62
Miller WL. P450 oxidoreductase deficiency: a disorder of steroidogenesis with multiple clinical
manifestations. Sci Signal. 2012; 5: 11
Minzer K, Lee O, Hong JJ, Singer HS. Increased prefrontal D2 protein in Tourette syndrome: a
postmortem analysis of frontal cortex and striatum. J Neurol Sci. 2004;219:55-61
Miranda DM, Wigg K, Kabia EM, Feng Y, Sandor P, Barr CL. Association of SLITRK1 to
Gilles de la Tourette Syndrome. Am J Med Genet B Neuropsychiatr Genet. 2009; 150B:483-6
Molina-Hernández M, Tellez-Alcántara NP, García JP, Lopez JI, Jaramillo MT. Antidepressantlike actions of intra-accumbens infusions of allopregnanolone in ovariectomized Wistar
rats .Pharmacol Biochem Behav. 2005 ; 80: 401-409
Molina-Hernandez M, Tellez-Alcantara NP, Olivera-Lopez JI, Jaramillo MT. Olanzapine plus
17-beta estradiol produce antidepressant-like actions in rats forced to swim. Pharmacol
Biochem Behav. 2009; 93: 491-7
139
Mooradian AD, Morley JE, Korenman SG. Biological actions of androgens. Endocr Rev. 1987; 8:
1-28
Morissette J, Durocher F, Leblanc JF, Normand T, Labrie F, Simard J. Genetic linkage mapping
of the human steroid 5 alpha-reductase type 2 gene (SRD5A2) close to D2S352 on
chromosome region 2p23-->p22. Cytogen Cell Genet. 1996; 73: 304-7
Morris DJ, Souness GW, Saccoccio NA, Harnik M. The effects of infusions of ring-A-reduced
derivatives of aldosterone on the antinatriuretic and kaliuretic actions of aldosterone. Steroids.
1989; 53: 21-36
Morrow AL, Devaud LL, Purdy RH, Paul SM. Neuroactive steroid modulators of the stress
response. Ann N Y Acad Sci. 1995; 771: 257-72
Mostallino MC, Sanna E, Concas A, Biggio G, Follesa P. Plasticity and function of extrasynaptic
GABA(A) receptors during pregnancy and after delivery. Psychoneuroendocrinology. 2009;
34: S74-83
Mostofsky SH, Wendlandt J, Cutting L ,Denckla MB, Singer HS. Corpus callosum
measurements in girls with Tourette syndrome. Neurology. 1999; 53: 1345-7
Motlagh MG, Katsovich L ,Thompson N, Lin H, Kim YS, Scahill L, Lombroso PJ, King RA,
Peterson BS, Leckman JF. Severe psychosocial stress and heavy cigarette smoking during
pregnancy: an examination of the pre- and perinatal risk factors associated with ADHD and
Tourette syndrome. European child & adolescent psychiatry. 2010; 19: 755-64
Motzo C, Porceddu ML, Maira G, Flore G, Concas A, Dazzi L, Biggio G. Inhibition of basal and
stress-induced dopamine release in the cerebral cortex and nucleus accumbens of freely
moving rats by the neurosteroid allopregnanolone. J Psychopharmacol. 1996; 10: 266-72
Muroni A, Paba S, Puligheddu M, Marrosu F, Bortolato M. A preliminary study of finasteride in
Tourette syndrome. Mov Disord. 2011; 26: 2146–7
Nambu A. Somatotopic organization of the primate Basal Ganglia. Front Neuroanat. 2011; 5:26
Navarro G, Moreno E, Aymerich M, Marcellino D, McCormick PJ, Mallol J, Cortés A, Casadó V,
Canela EI, Ortiz J, Fuxe K, Lluís C, Ferré S, Franco R. Direct involvement of sigma-1
receptors in the dopamine D1 receptor-mediated effects of cocaine. Proc Natl Acad Sci. 2010;
107: 18676-81
Nayebi AR, Rezazadeh H. Involvement of serotoninergic mechanism in analgesia by castration
and flutamide a testosterone antagonist in the rat formalin test. Pharmacol Biochem Behav.
2004; 77: 9-14
Nelson EC, Pribor EF. A calendar savant with autism and Tourette syndrome Response to
treatment and thoughts on the interrelationships of these conditions. Ann Clin
Psychiatry.1993; 5: 135-40
Nickel JC, Fradet Y, Boake RC, Pommerville PJ, Perreault JP, Afridi SK, Elhilali MM. Efficacy
and safety of finasteride therapy for benign prostatic hyperplasia: results of a 2-year
140
randomized controlled trial (the PROSPECT study). PROscar Safety Plus Efficacy Canadian
Two year Study. CMAJ. 1996; 155: 1251-9
Nordstrom EJ, Burton FH. A transgenic model of comorbid Tourette's syndrome and obsessivecompulsive disorder circuitry. Mol Psychiatry. 2002; 7: 617-25
Oren I, Fleishman SJ, Kessel A, Ben-Tal N. Free diffusion of steroid hormones across
biomembranes: a simplex search with implicit solvent model calculations. Biophys. 2004; J
87: 768-79
O'Rourke JA, Scharf JM, Yu D, Pauls DL. The genetics of Tourette syndrome: a review. J
Psychosom Res. 2009; 67: 533-45
Paba S, Frau R, Godar SC, Devoto P, Marrosu F, Bortolato M. Steroid 5α-reductase as a novel
therapeutic target for schizophrenia and other neuropsychiatric disorders. Curr Pharm Des.
2011; 17: 151–67
Palminteri S, Lebreton M, Worbe Y, Hartmann A, Lehericy S, Vidailhet M, Grabli D,
Pessiglione M. Dopamine-dependent reinforcement of motor skill learning: evidence from
Gilles de la Tourette syndrome. Brain. 2011; 134(Pt 8): 2287-301
Paschou P. The genetic basis of Gilles de la Tourette Syndrome. Neurosci Biobehav Rev. 2013;
37:1026-39
Pattison JC, Saltzman W, Abbott DH, Hogan BK, Nguyen AD, Husen B, Einspanier A, Conley
AJ, Bird IM. Gender and gonadal status differences in zona reticularis expression in
marmoset monkey adrenals: Cytochrome b5 localization with respect to cytochrome P450
17,20-lyase activity. Mol Cell Endocrinol. 2007; 265-266: 93-101
Pauls DL, Cohen DJ, Heimbuch R, Detlor J, Kidd KK. Familial pattern and transmission of
Gilles de la Tourette syndrome and multiple tics. Arch Gen Psychiatry. 1981; 38: 1091-3
Paxinos G, Watson C. The rat brain in stereotaxic coordinates. 3rd Edition Academic Press San
Diego California. 1998
Pelletier G. Steroidogenic enzymes in the brain: morphological aspects. Prog Brain Res. 2010;
181: 193-207.
Peng RY, Mansbach RS, Braff DL, Geyer MA. A D2 dopamine receptor agonist disrupts
sensorimotor gating in rats Implications for dopaminergic abnormalities in schizophrenia.
Neuropsychopharmacology. 1990; 3: 211–8
Peters YM, O'Donnell P. Social isolation rearing affects prefrontal cortical response to ventral
tegmental area stimulation. Biol Psychiatry. 2005; 57: 1205-8
Peterson BS, Bronen RA, Duncan CC. Three cases of symptom change in Tourette's syndrome
and obsessive-compulsive disorder associated with paediatric cerebral malignancies. J Neurol
Neurosurg Psychiatry. 1996; 61:497-505
141
Peterson BS, Leckman JF, Scahill L, Naftolin F, Keefe D, Charest NJ, King RA, Hardin MT,
Cohen DJ. Steroid hormones and Tourette's syndrome: early experience with antiandrogen
therapy. J Clin Psychopharmacol. 1994; 14: 131-5
Peterson BS, Skudlarski P, Anderson AW, Zhang H, Gatenby JC, Lacadie CM et al. A
functional magnetic resonance imaging study of tic suppression in Tourette syndrome. Arch
Gen Psychiatry. 1998a; 55:326-33
Peterson BS, Thomas P, Kane MJ, Scahill L, Zhang H, Bronen R et al. Basal Ganglia volumes
in patients with Gilles de la Tourette syndrome. Arch Gen Psychiatry. 2003; 60:415-24
Peterson BS, Zhang H, Anderson GM, Leckman JF. A double-blind placebo-controlled
crossover trial of an antiandrogen in the treatment of Tourette's syndrome. J Clin
Psychopharmacol. 1998b; 18: 324-31
Petitclerc M, Bedard PJ, Di Paolo T. Progesterone releases dopamine in male and female rat
striatum: a behavioral and microdialysis study. Prog Neuro-psychopharmacol Biol Psychiatry.
1995; 19: 491-7
Petralia SM, Frye CA. In the ventral tegmental area G-proteins mediate progesterone's actions at
dopamine type 1 receptors for lordosis of rats and hamsters. Psychopharmacology. 2006; 186:
133-42
Phan VL, Urani A, Romieu P, Maurice T. Strain differences in sigma(1) receptor-mediated
behaviours are related to neurosteroid levels. Eur J Neurosci. 2002; 15: 1523-34
Piazza PV, Rougé-Pont F, Deroche V, Maccari S, Simon H, Le Moal M .Glucocorticoids have
state-dependent stimulant effects on the mesencephalic dopaminergic transmission. Proc Natl
Acad Sci USA. 1996; 93: 8716-20
Pibiri F, Nelson M, Guidotti A, Costa E, Pinna G. Decreased corticolimbic allopregnanolone
expression during social isolation enhances contextual fear: A model relevant for
posttraumatic stress disorder. Proc Natl Acad Sci USA. 2008; 105: 5567-72
Pignatelli D, Xiao F, Gouveia AM, Ferreira JG, Vinson GP. Adrenarche in the rat. J
Endocrinology. 2006; 191: 301-8
Pinna G, Agis-Balboa RC, Pibiri F, Nelson M, Guidotti A, Costa E. Neurosteroid biosynthesis
regulates sexually dimorphic fear and aggressive behavior in mice. Neurochem Res. 2008; 33:
1990-2007
Planert H, Berger TK, Silberberg G. Membrane properties of striatal direct and indirect pathway
neurons in mouse and rat slices and their modulation by dopamine. PLoS One. 2013;
8:e57054
Plaznik A, Stefanski R, Kostowski W. Interaction between accumbens D1 and D2 receptors
regulating rat locomotor activity. Psychopharmacology. 1989; 99: 558-62
Plessen KJ, Bansal R, Peterson BS. Imaging evidence for anatomical disturbances and
neuroplastic compensation in persons with Tourette syndrome. J Psychosom Res. 2009;
67:559-73
142
Porubek D. CYP17A1: A Biochemistry Chemistry and Clinical Review. Curr Top Med Chem.
2013; 13: 1364-84.
Powell SB, Geyer MA, Preece MA, Pitcher LK, Reynolds GP, Swerdlow NR. Dopamine
depletion of the nucleus accumbens reverses isolation-induced deficits in prepulse inhibition
in rats .Neuroscience. 2003; 119: 233-40
Price VH, Roberts JL, Hordinsky M, Olsen EA, Savin R, Bergfeld W, Fiedler V, Lucky A,
Whiting DA, Pappas F, Culbertson J,Kotey P, Meehan A, Waldstreicher J. Lack of efficacy of
finasteride in postmenopausal women with androgenetic alopecia. J Am Acad Dermatol. 2000;
43: 768-76
Proietti Onori M, Ceci C, Laviola G, Macri S. A behavioural test battery to investigate tic-like
symptoms stereotypies attentional capabilities and spontaneous locomotion in different mouse
strains. Behav Brain Res. 2014; 267:95-105
Purdy RH, Morrow AL, Moore PH, Paul SM. Stress-induced elevations of gamma-aminobutyric
acid type A receptor-active steroids in the rat brain. Proc Natl Acad Sci USA. 1991; 88: 45537
Purves-Tyson TD, Handelsman DJ, Double KL, Owens SJ, Bustamante S, Weickert CS. 2012
Testosterone regulation of sex steroid-related mRNAs and dopamine-related mRNAs in
adolescent male rat substantia nigra BMC Neurosci. 2012; 6: 95.
Putnam SK, Sato S, Hull EM. Effects of testosterone metabolites on copulation and medial
preoptic dopamine release in castrated male rats. Horm Behav. 2003; 44: 419-26
Qu Y, Saint Marie RL, Breier MR, Ko D, Stouffer D, Pearsons LH et al. Neural basis for a
heritable phenotype: differences in the effects of apomorphine on startle gating and ventral
pallidal GABA efflux in male Sprague-Dawley and Long-Evans rats. Psychopharmacology.
2009; 207: 271–80
Rahimi-Ardabili B, Pourandarjani R, Habibollahi P, Mualeki A. Finasteride-induced depression:
a prospective study. BMC Clin Pharmacol. 2006; 6: 7
Ralph RJ, Caine SB. Dopamine 1 and D2 agonist effects on prepulse inhibition and locomotion:
comparison of Sprague-Dawley rats to Swiss-Webster 129X1/SvJ C57BL/6J and DBA/2J
mice. J Pharmacol Exp Ther. 2005; 312:733-41
Ralph RJ, Caine SB. Effects of selective dopamine D1-like and D2-like agonists on prepulse
inhibition of startle in inbred C3H/HeJ SPRET/EiJ and CAST/EiJ mice. Psychopharmacology.
2007; 191: 731-39
Ralph RJ, Paulus MP, Fumagalli F, Caron MG, Geyer MA. Prepulse inhibition deficits and
perseverative motor patterns in dopamine transporter knock-out mice: differential effects of
D1 and D2 receptor antagonists. J Neurosci. 2001; 21:305-13
Ralph-Williams RJ, Lehmann-Masten V, Geyer MA. 2003a Dopamine D1 rather than D2
receptor agonists disrupt prepulse inhibition of startle in mice Neuropsychopharmacology. 28
108-18
143
Ralph-Williams RJ, Lehmann-Masten V, Otero-Corchon V, Low MJ, Geyer MA. Differential
effects of direct and indirect dopamine agonists on prepulse inhibition: a study in D1 and D2
receptor knock-out mice. J Neurosci. 2002; 22: 9604-11
Ralph-Williams RJ, Paulus MP, Zhuang X, Hen R ,Geyer MA. Valproate attenuates hyperactive
and perseverative behaviors in mutant mice with a dysregulated dopamine system. Biol
Psychiatry .2003; 53:352-9
Randrup A, Munkvad I, Udsen P. Adrenergic Mechanisms and Amphetamine Induced Abnormal
Behaviour. Acta Pharmacol Toxicol. 1963; 20:145-57
Ranjan N, Nair KP, Romanoski C, Singh R, Venketswara G. Tics after traumatic brain injury
.Brain Inj. 2011; 25:629-33
Rasmusson GH, Reynolds GF, Steinberg NG, Walton E, Patel GF, Liang T, Cascieri MA,
Cheung AH, Brooks JR, Berman C. Azasteroids: structure-activity relationships for inhibition
of 5 alpha-reductase and of androgen receptor binding. J Med Chem. 1986; 29: 2298-315
Reddy DS, Jian K. The testosterone-derived neurosteroid androstanediol is a positive allosteric
modulator of GABAA receptors. J Pharmacol Exp Ther. 2011; 334: 1031-41
Reddy DS, Rogawski MA. Stress-induced deoxycorticosterone-derived neurosteroids modulate
GABA(A) receptor function and seizure susceptibility. J Neurosci. 2002; 22: 3795-805
Reddy DS. Pharmacology of endogenous neuroactive steroids. Crit Rev Neurobiol. 2003; 15:
197-234
Reid SD. Neuroleptic-induced Tardive Tourette treated with clonazepam: a case report and
literature review. Clin Neuropharmacol. 2004; 27:101-4
Rhodes ME, Frye CA. Inhibiting progesterone metabolism in the hippocampus of rats in
behavioral estrus decreases anxiolytic behaviors and enhances exploratory and antinociceptive
behaviors. Cogn Affect Behav Neurosci. 2001; 1: 287-96
Ridley RM, Baker HF, Owen F, Cross AJ, Crow TJ. Behavioural and biochemical effects of
chronic amphetamine treatment in the vervet monkey. Psychopharmacology. 1982; 78:245-51
Ridley RM. The psychology of perserverative and stereotyped behaviour. Prog Neurobiol. 1994;
44: 221-31
Ritchtand NM, Woods SC, Berger SP, Strakowski SM. D3 dopamine receptor behavioral
sensitization and psychosis. Neurosci Biobehav Rev. 2001; 25: 427–43
Ritsner MS, Bawakny H, Kreinin A. Pregnenolone treatment reduces severity of negative
symptoms in recent-onset schizophrenia: an 8-week double-blind randomized add-on twocenter trial. Psychiatry Clin Neurosci. 2014; 68: 432-40
Robertson GS, Vincent SR, Fibiger HC. D1 and D2 dopamine receptors differentially regulate
c-fos expression in striatonigral and striatopallidal neurons. Neuroscience. 1992; 49: 285-96
Robertson MM. Gilles de la Tourette syndrome: the complexities of phenotype and treatment. Br
J Hosp Med. 2011; 72:100-7
144
Robertson MM. The prevalence and epidemiology of Gilles de la Tourette syndrome Part 1: the
epidemiological and prevalence studies. J Psychosom Res. 2008; 65: 461-72
Roessner V, Schoenefeld K, Buse J, Bender S, Ehrlich S, Munchau A. Pharmacological
treatment of tic disorders and Tourette Syndrome. Neuropharmacology. 2013; 68:143-9
Romanelli RJ, Williams JT, Neve KA. Dopamine receptor signaling: Intracellular pathways to
behavior. In: Neve KA (ed) The Dopamine Receptors 2nd edn. Humana Totawa. 2010; 137–
173
Roncada P, Bortolato M, Frau R, Saba P, Flore G, Soggiu A, Pisanu S, Amoresano A, Carpentieri
A, Devoto P. Gating deficits in isolation-reared rats are correlated with alterations in protein
expression in nucleus accumbens. J Neurochem. 2009; 108: 611-620
Rougé-Pont F, Mayo W, Marinelli M, Gingras M, Le Moal M, Piazza PV. The neurosteroid
allopregnanolone increases dopamine release and dopaminergic response to morphine in the
rat nucleus accumbens Eur. J Neurosci. 2002; 16: 169-173
Rupprecht R, Holsboer F. Neuroactive steroids: mechanisms of action
neuropsychopharmacological perspectives Trends. Neurosci. 1999; 22: 410-416
and
Rupprecht R, Koch M, Montkowski A, Lancel M, Faulhaber J, Harting J, Spanagel R.
Assessment of neuroleptic-like properties of progesterone. Psychopharmacology. 1999; 143:
29-38
Rupprecht R. Neuroactive steroids: mechanisms of action and neuropsychopharmacological
properties. Psychoneuroendocrinology. 2003; 28: 139-68
Rutter M. Biological implications of gene-environment interaction. J Abnorm Child Psychol.
2008; 36:969-75
Saalmann YB, Kirkcaldie MT, Waldron S, Calford MB. Cellular distribution of the GABAA
receptor-modulating 3alpha-hydroxy 5alpha-reduced pregnane steroids in the adult rat brain. J
Neuroendocrinol. 2007; 19: 272–84
Saccomani L, Fabiana V, Manuela B, Giambattista R. Tourette syndrome and chronic tics in a
sample of children and adolescents. Brain Develop. 2005; 27: 349-52
Sanchez MG, Bourque M, Morissette M, Di Paolo T. Steroids-dopamine interactions in the
pathophysiology and treatment of CNS disorders. CNS Neurosci Therapeutics. 2010; 16: e4371
Sanchez P, Torres JM, Olmo A, O'Valle F,. Ortega E. Effects of environmental stress on mRNA
and protein expression levels of steroid 5alpha-Reductase isozymes in adult rat brain. Horm
Behav. 2009; 56: 348-353
Sandyk R ,Bamford CR. Heightened cortisol response to administration of naloxone in Tourette's
syndrome. Intl J Neurosci. 1988b; 39: 225-7
Sandyk R, Bamford CR ,Binkiewicz A, Finley PR. Gonadotropin deficiency in Tourette's
syndrome: a preliminary communication. Intl J Neurosci. 1988; 42: 121-5
145
Sandyk R, Bamford CR. Deregulation of hypothalamic opioid and luteinizing hormone releasing
factor (LHRF) activity and its relevance to Tourette's syndrome. Intl J Neurosci. 1988a; 39:
223-4
Santana N, Mengod G, Artigas F. Quantitative analysis of the expression of dopamine D1 and
D2 receptors in pyramidal and GABAergic neurons of the rat prefrontal cortex. Cereb Cortex.
2009; 19: 849-860
Santangelo SL, Pauls DL, Goldstein JM, Faraone SV, Tsuang MT, Leckman JF. Tourette's
syndrome: what are the influences of gender and comorbid obsessive-compulsive disorder? J
Am Acad Child Adolesc Psychiatry. 1994; 33: 795-804
Sarkar J, Wakefield S, MacKenzie G, Moss SJ, Maguire J. Neurosteroidogenesis is required for
the physiological response to stress: role of neurosteroid-sensitive GABAA receptors. J
Neurosci. 2011; 31: 18198-210
Savageau MM, Beatty WW. Gonadectomy and sex differences in the behavioral responses to
amphetamine and apomorphine of rats. Pharmacol Biochem Behav. 1981; 14: 17-21
Scharf JM, Yu D, Mathews CA, Neale BM, Stewart SE, Fagerness JA et al. Genome-wide
association study of Tourette's syndrome. Mol Psychiatry. 2013; 18:721-8
Schwabe MJ, Konkol RJ. Menstrual cycle-related fluctuations of tics in Tourette syndrome.
Pediatric neurology. 1992; 8: 43-6
Serra M, Pisu MG, Littera M, Papi G, Sanna E ,Tuveri F. Social isolation-induced decreases in
both the abundance of neuroactive steroids and GABA(A) receptor function in rat brain. J
Neurochem. 2000; 75: 732-40
Seymour B, O'Doherty JP, Dayan P, Koltzenburg M, Jones AK, Dolan RJ, Friston
KJ ,Frackowiak RS. Temporal difference models describe higher-order learning in humans.
Nature. 2004; 429: 664-7
Shale H, Fahn S, Mayeux R. Tics in a patient with Parkinson's disease. Mov disord. 1986; 1: 7983
Shilliam CS, Heidbreder. Gradient of dopamine responsiveness to dopamine receptor agonists in
subregions of the rat nucleus accumbens. Eur J Pharmacol. 2003; 47:7113-122
Silva RR, Munoz DM, Barickman J, Friedhoff AJ. Environmental factors and related fluctuation
of symptoms in children and adolescents with Tourette's disorder. J Child Psychol Psychiatry.
1995; 36: 305-12
Singer HS ,Wendlandt J, Krieger M, Giuliano J. Baclofen treatment in Tourette syndrome: a
double-blind placebo-controlled crossover trial. Neurology. 2001; 56:599-604
Singer HS, Walkup JT. Tourette syndrome and other tic disorders. Diagn Pathophysiol Treat
Med. 1991; 70:15-32
Singer HS. Tourette's syndrome: from behaviour to biology. Lancet Neurology. 2005; 4: 149-59
146
Sowell ER, Kan E, Yoshii J, Thompson PM, Bansal R, Xu D et al. Thinning of sensorimotor
cortices in children with Tourette syndrome. Nat Neurosci. 2008; 11:637-9
Steeves TD, Fox SH. Neurobiological basis of serotonin-dopamine antagonists in the treatment
of Gilles de la Tourette syndrome In: Di Giovanni G, Di Matteo V, Esposito E (Eds).
Serotonin-Dopamine Interaction: Experimental and Therapeutic Evidence. Elsevier,
Netherlands; 2008; 495-513
Steeves TD, Ko JH, Kideckel DM, Rusjan P, Houle S, Sandor P, Lang AE, Strafella AP.
Extrastriatal dopaminergic dysfunction in tourette syndrome. Ann Neurol. 2010; 67:170-81
Steinberg T, Shmuel Baruch S ,Harush A, Dar R,Woods D ,Piacentini J et al. Tic disorders and
the premonitory urge. J Neural Transm. 2010; 117: 277-84
Steiner H, Kitai ST. Regulation of rat cortex function by D1 dopamine receptors in the striatum.
J Neurosci. 2000; 20: 5449-60
Strömstedt M, Waterman MR. Messenger RNAs encoding steroidogenic enzymes are expressed
in rodent brain. Brain Res Mol Brain Res. 1995; 34: 75-88
Strous RD, Maayan R, Lapidus R ,Stryjer R, Lustig M, Kotler M, Weizman A.
Dehydroepiandrosterone augmentation in the management of negative depressive and anxiety
symptoms in schizophrenia. Arch Gen Psychiatry. 2003; 60: 133-41
Suri RE, Schultz W. Learning of sequential movements by neural network model with
dopamine-like reinforcement signal. Exp Brain Res. 1998; 121: 350-4
Swain JE, Scahill L, Lombroso PJ, King RA, Leckman JF .Tourette syndrome and tic disorders:
a decade of progress. J Am Acad Child Adol Psychiatry. 2007; 46: 947-68
Swedo SE, Leonard HL, Garvey M, Mittleman B, Allen AJ, Perlmutter S et al. Pediatric
autoimmune neuropsychiatric disorders associated with streptococcal infections: clinical
description of the first 50 cases. Am J Psychiatry. 1998; 155: 264-71
Swerdlow NR, Braff DL, Geyer MA. Animal models of deficient sensorimotor gating: what we
know what we think we know and what we hope to know soon. Behav Pharmacol. 2000a; 11:
185-204
Swerdlow NR, Braff DL, Lehmann-Masten V, Geyer MA. Schizophrenic-like sensorimotor
gating abnormalities in rats following dopamine infusion into the nucleus accumbens.
Psychopharmacology. 1990; 101: 414-420
Swerdlow NR, Caine SB, Geyer MA. Regionally selective effects of intracerebral dopamine
infusion on sensorimotor gating of the startle reflex in rats. Psychopharmacology. 1992; 108:
189-195
Swerdlow NR, Geyer MA, Braff DL. Neural circuit regulation of prepulse inhibition of startle in
the rat: current knowledge and future challenges. Psychopharmacology. 2001a; 156:194-215
Swerdlow NR, Geyer MA. Clozapine and haloperidol in an animal model of sensorimotor gating
deficits in schizophrenia. Pharmacol Biochem Behav. 1993; 44: 741-4
147
Swerdlow NR, Kuczenski R, Goins JC, Ma LT, Bongiovanni MJ, Shoemaker JM. Neurochemical
analysis of rat strain differences in the startle gating-disruptive effects of dopamine agonists.
Pharmacol Biochem Behav. 2004a; 80: 203–11
Swerdlow NR, Martinez ZA, Hanlon FM, Platten A, Farid M, Auerbach P, Braff DL, Geyer MA.
Toward understanding the biology of a complex phenotype: rat strain and substrain
differences in the sensorimotor gating-disruptive effects of dopamine agonists. J Neurosci.
2000b; 20: 4325-36
Swerdlow NR, Platten A, Kim YK, Gaudet I, Shoemaker J,. Pitcher L et al. Sensitivity to the
dopaminergic regulation of prepulse inhibition in rats: evidence for genetic but not
environmental determinants. Pharmacol Biochem Behav. 2001b; 70: 219–26
Swerdlow NR, Shoemaker JM, Bongiovanni MJ, Neary AC, Tochen LS, Saint Marie RL. Strain
differences in the disruption of prepulse inhibition of startle after systemic and intraaccumbens amphetamine administration. Pharmacol Biochem Behav. 2007; 87:1-10
Swerdlow NR, Shoemaker JM, Bongiovanni MJ, Neary AC, Tochen LS, Saint Marie RL.
Reduced startle gating after D1 blockade: effects of concurrent D2 blockade. Pharmacol
Biochem Behav. 2005; 82: 293-9
Swerdlow NR, Shoemaker JM, Crain S, Goins J ,Onozuka K, Auerbach PP. Sensitivity to drug
effects on prepulse inhibition in inbred and outbred rat strains. Pharmacol Biochem Behav.
2004b; 77: 291–302
Swerdlow NR, Sutherland AN. Preclinical models relevant to Tourette syndrome. Adv Neurol.
2006; 99:69-88
Swerdlow NR, Taaid N, Halim N, Randolph E, Kim YK, Auerbach P. Hippocampal lesions
enhance startle gating-disruptive effects of apomorphine in rats: a parametric assessment.
Neuroscience. 2000c; 96: 523-36
Swerdlow NR, Talledo J, Sutherland AN, Nagy D, Shoemaker JM. Antipsychotic effects on
prepulse inhibition in normal 'low gating' humans and rats. Neuropsychopharmacology. 2011;
31: 2011–21
Swerdlow NR, Young AB. Neuropathology in Tourette syndrome: an update. Advances in
neurology. 2001; 8:51-61
Swerdlow NW, Lipska BK, Jaskin GE, Geyer MA. Increased sensitivity to the sensorimotor
gating- disruptive effects of apomorphine after lesions of medial prefrontal cortex or ventral
hippocampus in adult rats. Psychopharmacology. 1995; 122: 27-34
Tanner CM, Goldman SM. Epidemiology of Tourette syndrome. Neurol Clin. 1997; 15: 395-402
Thiblin I, Finn A, Ross SB, Stenfors C. Increased dopaminergic and 5-hydroxytryptaminergic
activities in male rat brain following long-term treatment with anabolic androgenic steroids.
Br J Pharmacol. 1999; 126: 1301-6
Thigpen AE, Russell DW. Four-amino acid segment in steroid 5 alpha-reductase 1 confers
sensitivity to finasteride, a competitive inhibitor. J Biol Chem. 1992; 267: 8577–83
148
Thomalla G, Jonas M, Baumer T, Siebner HR, Biermann-Ruben K, Ganos C et al. Costs of
control: decreased motor cortex engagement during a Go/NoGo task in Tourette's syndrome.
Brain. 2014; 137:122-36
Thomas P, Pang Y. Membrane progesterone receptors: evidence for neuroprotective,
neurosteroid signaling and neuroendocrine functions in neuronal cells. Neuroendocrinology.
2012; 96:162-71.
Thompson TL, Moss RL. Estrogen regulation of dopamine release in the nucleus accumbens:
genomic- and nongenomic-mediated effects. J Neurochem. 1994; 62: 1750-6
Tomas-Camardiel M, Sanchez-Hidalgo MC, Sanchez del Pino MJ, Navarro A, Machado A,
Cano J. Comparative study of the neuroprotective effect of dehydroepiandrosterone and
17beta-estradiol against 1-methyl-4-phenylpyridium toxicity on rat striatum. Neuroscience.
2002; 109: 569-84
Tourette Syndrome Association International Consortium for Genetics. Genome scan for
Tourette disorder in affected-sibling-pair and multigenerational families. Am J Hum Genet.
2007; 80: 265-72
Turvin JC, Messer WS Jr, Kritzer MF. On again off again effects of gonadectomy on the
acoustic startle reflex in adult male rats. Physiol Behav. 2007; 90: 473-82
Tye SJ, Miller AD, Blaha CD. Differential corticosteroid receptor regulation of mesoaccumbens
dopamine efflux during the peak and nadir of the circadian rhythm: a molecular equilibrium in
the midbrain? Synapse. 2009; 63: 982-90
Udvardi PT, Nespoli E, Rizzo F, Hengerer B, Ludolph AG.
Nondopaminergic
neurotransmission in the pathophysiology of Tourette syndrome. Int Rev Neurobiol. 2013;
112:95-130
Ugale RR, Hirani K, Morelli M, Chopde CT. Role of neuroactive steroid allopregnanolone in
antipsychotic-like action of olanzapine in rodents. Neuropsychopharmacology. 2004; 29:
1597-1609
Uzunova V, Sheline Y, Davis JM, Rasmusson A, Uzunov DP, Costa E, Guidotti A. Increase in
the cerebrospinal fluid content of neurosteroids in patients with unipolar major depression
who are receiving fluoxetine or fluvoxamine. Proc Natl Acad Sci USA. 1998; 95: 3239-44
Van den Buuse M, Eikelis N. Estrogen increases prepulse inhibition of acoustic startle in rats.
Eur J Pharmacol. 2001; 425: 33-41
Vane JR, Botting RM. Mechanism of action of nonsteroidal anti-inflammatory drugs. Am J Med.
1998; 104: 2-8
Varty GB, Higgins GA. Dopamine agonist-induced hypothermia and disruption of preulse
inhibition: evidence for a role of D3 receptors? Behav Pharmacol. 1998; 9: 445-55
Viswanath B, Janardhan Reddy YC, Kumar KJ, Kandavel T, Chandrashekar CR. Cognitive
endophenotypes in OCD: a study of unaffected siblings of probands with familial OCD. Prog
Neuropsychopharmacol Biol Psychiatry. 2009; 33:610-5
149
Voorn P Vanderschuren LJ, Groenewegen HJ, Robbins TW, Pennartz CM. Putting a spin on the
dorsal-ventral divide of the striatum. Trends Neurosci. 2004; 8: 468-74
Walf AA, Sumida K, Frye CA. Inhibiting 5alpha-reductase in the amygdala attenuates
antianxiety and antidepressive behavior of naturally receptive and hormone-primed
ovariectomized rats. Psychopharmacology. 2006; 186: 302-11
Wan FJ, Geyer MA, Swerdlow NR. Accumbens D2 modulation of sensorimotor gating in rats:
assessing anatomical localization. Pharmacol Biochem Behav. 1994; 49: 155-63
Wan FJ, Geyer MA, Swerdlow NR. Presynaptic Dopamine-glutamate interactions in the
nucleus accumbens regulate sensorimotor gating. Psychopharmacology. 1995; 120:433-41
Wan FJ, Swerdlow NR. Intra-accumbens infusion of quinpirole impairs sensorimotor gating of
acoustic startle in rats. Psychopharmacology. 1993; 113: 103-9
Wan FJ, Swerdlow NR. Sensorimotor gating in rats is regulated by different dopamineglutamate interactions in the nucleus accumbens core and shell subregions. Brain Res. 1996;
722:168-76
Wan FJ, Taaid N, Swerdlow NR. Do D1/D2 interactions regulate prepulse inhibition in rats?
Neuropsychopharmacology. 1996; 14: 265–74
Wang S,. Lai K, Moy FJ, Bhat A, Hartman HB, Evans MJ. The nuclear hormone receptor
farnesoid X receptor (FXR) is activated by androsterone. Endocrinology. 2006; 147: 4025-33
Wang Z, Maia TV, Marsh R, Colibazzi T, Gerber A, Peterson BS. The neural circuits that
generate tics in Tourette's syndrome. Am J Psychiatry. 2011; 168:1326-37
Waterston RH, Lindblad-Toh K, Birney E, Rogers J, Abril JF et al. Initial sequencing and
comparative analysis of the mouse genome. Nature. 2002; 420: 520–562
Weinstein BI, Kandalaft N, Ritch R, Camras CB, Morris DJ, Latif SA, Vecsei P, Vittek J,Gordon
GG, Southren AL. 5 alpha-dihydrocortisol in human aqueous humor and metabolism of
cortisol by human lenses in vitro. Invest Ophthalmol Vis Sci. 1991; 32: 2130-35
Weiss IC, Feldon J. Environmental animal models for sensorimotor gating deficiencies in
schizophrenia: a review. Psychopharmacology. 2001; 156: 305-26
Weisser H, Tunn S, Behnke B, Krieg M. Effects of the sabal serrulata extract IDS 89 and its
subfractions on 5 alpha-reductase activity in human benign prostatic hyperplasia. Prostate.
1996; 28: 300-306
Willner P. Validation criteria for animal models of human mental disorders: learned helplessness
as a paradigm case. Prog Neuropsychopharmacol Biol Psychiatry. 1986; 10: 677-90
Wilton DC. Is a Schiff base involved in the mechanism of the delta4-3-oxo steroid 5alpha- or
5beta-reductases from mammalian liver? Biochem J. 1976; 155: 487-91
Wong DF, Brasic JR, Singer HS, Schretlen DJ, Kuwabara H, Zhou Y et al. Mechanisms of
dopaminergic and serotonergic neurotransmission in Tourette syndrome: clues from an in vivo
neurochemistry study with PET. Neuropsychopharmacology. 2008; 33:1239-51
150
Wong DF, Brasic JR, Singer HS, Schretlen DJ, Kuwabara H, Zhou Y, Nandi A, Maris MA,
Alexander M, Ye W, Rousset O, Kumar A, Szabo Z, Gjedde A, Grace AA. Mechanisms of
dopaminergic and serotonergic neurotransmission in Tourette syndrome: clues from an in vivo
neurochemistry study with PET. Neuropsychopharmacology. 2008; 33: 1239-51
Wong J, Shoemaker JM, Platten A, Martinez ZA, Pitcher L, Hakimzada F, Auerbach P,
Swerdlow NR. Atypical antipsychotic effects on startle gating in adult rats: lack of sex
difference and impact of prepubertal castration. Abstr Biol Psychiatry. 2002; 51120S
Wong P, Chang CC, Marx CE, Caron MG, Wetsel WC, Zhang X. Pregnenolone rescues
schizophrenia-like behavior in dopamine transporter knockout mice. PLoS One. 2012; 7:
51455
Xue YY, Wang HN, Xue F, Tan QR. Atypical antipsychotics do not reverse prepulse inhibition
deficits in acutely psychotic schizophrenia. J Int Med Res. 2012; 40:1467-75
Yamada H, Kominami S, Takemori S, Kitawaki J, Kataoka Y. Immunohistochemical
localization of cytochrome P450 enzymes in the rat brain considering the steroid-synthesis in
the neurons .Acta Histochem Cytochem. 1997; 30: 609–16
Yoon DY, Gause CD, Leckman JF, Singer HS. Frontal dopaminergic abnormality in Tourette
syndrome: a postmortem analysis. J Neurol Sci. 2007; 255:50-6
Young KA, Randall PK, Wilcox RE. Dose and time response analysis of apomorphine's effect on
prepulse inhibition of acoustic startle. Behav Brain Res. 1991; 42: 43-8
Yu L, Romero DG, Gomez-Sanchez CE, Gomez-Sanchez EP. Steroidogenic enzyme gene
expression in the human brain. Mol Cell Endocrinol. 2002; 190: 9-17
Zebardast N, Crowley MJ, Bloch MH, Mayes LC ,Wyk BV, Leckman JF et al .Brain
mechanisms for prepulse inhibition in adults with Tourette syndrome: initial findings.
Psychiatry Res. 2013; 214: 33-41
Zhang J, Forkstam C, Engel JA, Svensson L. Role of dopamine in prepulse inhibition of the
acoustic startle. Psychopharmacology. 2000; 149: 181-8
Zhang LH, Rodriguez H, Ohno S, Miller WL. Serine phosphorylation of human P450c17
increases 17,20-lyase activity: implications for adrenarche and the polycystic ovary
syndrome. Proc Natl Acad Sci USA. 1995; 92: 10619-23
Zhang M, Ballard ME, Unger LV, Haupt A, Gross G, Decker MW et al. Effects of antipsychotics
and selective D3 antagonists on PPI deficits induced by PD 128907 and apomorphine Behav
.Brain Res. 2007; 182: 1–11
Zimmerberg B, Brunelli SA, Hofer MA. Reduction of rat pup ultrasonic vocalizations by the
neuroactive steroid allopregnanolone. Pharmacol Biochem Behav. 1994; 47: 735-8
Zohar I, Weinstock M. Differential effect of prenatal stress on the expression of corticotrophinreleasing hormone and its receptors in the hypothalamus and amygdala in male and female
rats. J Neuroendocrinol. 2011; 23: 320-8
151
Zuloaga DG, McGivern RF, Handa RJ. Organizational influence of the postnatal testosterone
surge on the circadian rhythm of core body temperature of adult male rats. Brain Res. 2009;
1268: 68-75
LIST OF PUBLICATIONS (in reverse chronological order)
1.
Moskovitz J, Walss-Bass C, Cruz DA, Thompson PM, Hairston J, Bortolato M. The
enzymatic activities of brain COMT and methionine sulfoxide reductase are correlated in a
COMT Val/Met allele-dependent fashion. Neuropath. Appl. Neurobiol, in press
152
2.
Solla P*, Bortolato M*, Cannas A, Mulas CS, Marrosu F. Paraphilias and paraphilic
disorders in Parkinson’s disease: a systematic review of the literature, Mov Disord, in press
3.
Godar SC, Mosher LJ, Di Giovanni G, Bortolato M. Animal models of Tourette Syndrome:
a translational perspective. J Neurosci Methods, 2014 Dec 30;238C:54-69.
4.
Fite PJ, Brown S, Gabrielli J, DiPierro M, Pederson C, Blossom JB, Cooley JL, Bortolato
M. The Role of Negative Life Events in Comorbid Reactive Aggression and Marijuana Use
Initiation among Latino Adolescents. J Aggress Maltreat Trauma, in press
5.
Stetler DA, Davis C, Leavitt K, Schriger I, Benson K, Bhakta S, Wang LC, Oben C, Watters
M, Haghnegahdar T, Bortolato M. Association of low-activity MAOA allelic variants with
violent crime in incarcerated offenders. J Psychiatr Res. 2014 Nov;58:69-75
6.
Fraschini M, Demuru M, Puligheddu M, Floridia S, Polizzi L, Maleci A, Bortolato M,
Hillebrand A, Marrosu F. The re-organization of functional brain networks in
pharmacoresistant epileptic patients who respond to VNS. Neurosci Lett, 2014 Sep
19;580:153-7.
7.
Godar SC, Bortolato M, Castelli MP, Casti A, Casu A, Chen K, Ennas MG, Tambaro S,
Shih JC. The aggression and behavioral abnormalities associated with monoamine oxidase A
deficiency are rescued by acute inhibition of serotonin reuptake. J Psychiatr Res. 2014
Sep;56:1-9.
8.
Moskovitz J, Walss-Bass C, Cruz DA, Thompson PM, Bortolato M. Methionine sulfoxide
reductase
regulates
brain
catechol-O-methyl
transferase
activity.
Int
J
Neuropsychopharmacol. 2014 Oct;17(10):1707-13.
9.
Frau R, Bini V, Pillolla G, Malherbe P, Pardu A, Thomas AW, Devoto P, Bortolato M.
Positive Allosteric Modulation of GABA(B) Receptors Ameliorates Sensorimotor Gating in
Rodent Models. CNS Neurosci Ther. 2014 Jul;20(7):679-84.
10.
Godar SC, Bortolato M. Gene-sex interactions in schizophrenia: focus on dopamine
neurotransmission. Front Behav Neurosci. 2014 Mar 6;8:71.
11.
Bortolato M, Bini V, Frau R, Devoto P, Pardu A, Fan Y, Solbrig MV. Juvenile cannabinoid
treatment induces frontostriatal gliogenesis in Lewis rats. Eur Neuropsychopharmacol. 2014
Jun;24(6):974-85.
12.
Frau R, Bini V, Pes R, Pillolla G, Saba P, Devoto P, Bortolato M. Inhibition of 17αhydroxylase/C17,20 lyase reduces gating deficits consequent to dopaminergic activation.
Psychoneuroendocrinology. 2014 Jan;39:204-13.
13.
Bortolato M, Godar SC, Tambaro S, Li FG, Devoto P, Coba MP, Chen K, Shih JC. Early
postnatal inhibition of serotonin synthesis results in long-term reductions of perseverative
behaviors, but not aggression, in MAO A-deficient mice. Neuropharmacology. 2013
Dec;75:223-32.
14.
Singh C, Bortolato M, Bali N, Godar SC, Scott AL, Chen K, Thompson RF, Shih JC.
Cognitive abnormalities and hippocampal alterations in monoamine oxidase A and B
knockout mice. Proc Natl Acad Sci U S A. 2013 Jul 30;110(31):12816-21.
153
15.
Bortolato M, Frau R, Godar SC, Mosher LJ, Paba S, Marrosu F, Devoto P. The implication
of neuroactive steroids in tourette's syndrome pathogenesis: a role for 5α-reductase? J
Neuroendocrinol. 2013 Nov;25(11):1196-208.
16.
Davis DA*, Bortolato M*, Godar SC, Sander TK, Iwata N, Pakbin P, Shih JC, Berhane K,
McConnell R, Sioutas C, Finch CE, Morgan TE. Prenatal exposure to urban air
nanoparticulate in mice causes neuronal differentiation and depression-like responses. PLoS
One. 2013 May 29;8(5):e64128.
17.
Wyatt LR, Godar SC, Khoja S, Jakowec MW, Alkana RL, Bortolato M, Davies DL. Sociocommunicative and sensorimotor impairments in male P2X4-deficient mice.
Neuropsychopharmacology. 2013 Sep;38(10):1993-2002.
18.
Tambaro S, Tomasi ML, Bortolato M. Long-term CB1 receptor blockade enhances
vulnerability to anxiogenic-like effects of cannabinoids. Neuropharmacology. 2013
Jul;70:268-77.
19.
Bortolato M, Pivac N, Muck Seler D, Nikolac Perkovic M, Pessia M, Di Giovanni G. The
role of the serotonergic system at the interface of aggression and suicide. Neuroscience 2013
Apr 16;236: 160-85.
20.
Fraschini M, Puligheddu M, Demuru M, Polizzi L, Maleci A, Tamburini G, Congia S,
Bortolato M, Marrosu F. VNS induced desynchronization in gamma bands correlates with
positive clinical outcome in temporal lobe pharmacoresistant epilepsy. Neurosci Lett. 2013
Mar 1;536: 14-8.
21.
Bortolato M, Yardley MM, Khoja S, Godar SC, Asatryan L, Finn DA, Alkana RL, Louie
SG, Davies DL. Pharmacological insights into the role of P2X4 receptors in behavioural
regulation: lessons from ivermectin. Int J Neuropsychopharmacol. 2013 Jun;16(5):1059-70.
22.
Stancampiano R, Frau R, Bini V, Collu M, Carta M, Fadda F, Bortolato M. Chronic
tryptophan deprivation attenuates gating deficits induced by 5-HT(1A), but not 5-HT(2)
receptor activation. Eur Neuropsychopharmacol. 2013 Oct;23(10):1329-35.
23.
Alzghoul L, Bortolato M, Delis F, Thanos PK, Darling RD, Godar SC, Zhang J, Grant S,
Wang GJ, Simpson KL, Chen K, Volkow ND, Lin RC, Shih JC. Altered cerebellar
organization and function in monoamine oxidase A hypomorphic mice. Neuropharmacology.
2012 Dec;63(7):1208-17.
24.
Frau R, Pillolla G, Bini V, Tambaro S, Devoto P, Bortolato M. Inhibition of 5α-reductase
attenuates behavioral effects of D(1)-, but not D(2)-like receptor agonists in C57BL/6 mice.
Psychoneuroendocrinology. 2013 Apr;38(4):542-51.
25.
Bortolato M, Godar SC, Alzghoul L, Zhang J, Darling RD, Simpson KL, Bini V, Chen K,
Wellman CL, Lin RC, Shih JC. Monoamine oxidase A and A/B knockout mice display
autistic-like features. Int J Neuropsychopharmacol. 2013 May;16(4):869-88.
26.
Castelli MP, Casti A, Casu A, Frau R, Bortolato M, Spiga S, Ennas MG. Regional
distribution of 5α-reductase type 2 in the adult rat brain: An immunohistochemical analysis.
Psychoneuroendocrinology. 2013 Feb;38(2):281-93.
154
27.
Bortolato M, Godar SC, Melis M, Soggiu A, Roncada P, Casu A, Flore G, Chen K, Frau R,
Urbani A, Castelli MP, Devoto P, Shih JC. NMDARs mediate the role of monoamine
oxidase A in pathological aggression. J Neurosci. 2012 Jun 20;32(25):8574-82.
28.
Davies DL, Bortolato M, Finn DA, Ramaker MJ, Barak S, Ron D, Liang J, Olsen RW.
Recent advances in the discovery and preclinical testing of novel compounds for the
prevention and/or treatment of alcohol use disorders. Alcohol Clin Exp Res. 2013
Jan;37(1):8-15.
29.
Bortolato M, Cannas A, Solla P, Bini V, Puligheddu M, Marrosu F. Finasteride attenuates
pathological gambling in patients with Parkinson disease. J Clin Psychopharmacol. 2012
Jun;32(3):424-5.
30.
Yardley MM, Wyatt L, Khoja S, Asatryan L, Ramaker MJ, Finn DA, Alkana RL, Huynh N,
Louie SG, Petasis NA, Bortolato M, Davies DL. Ivermectin reduces alcohol intake and
preference in mice. Neuropharmacology. 2012 Aug;63(2):190-201.
31.
Tambaro S, Bortolato M. Cannabinoid-related agents in the treatment of anxiety disorders:
current knowledge and future perspectives. Recent Pat CNS Drug Discov. 2012 Apr
1;7(1):25-40.
32.
Devoto P, Frau R, Bini V, Pillolla G, Saba P, Flore G, Corona M, Marrosu F, Bortolato M.
Inhibition of 5α-reductase in the nucleus accumbens counters sensorimotor gating deficits
induced by dopaminergic activation. Psychoneuroendocrinology. 2012 Oct;37(10):1630-45.
33.
Bortolato M, Shih JC. Behavioral outcomes of monoamine oxidase deficiency: preclinical
and clinical evidence. Int Rev Neurobiol. 2011;100:13-42.
34.
Bortolato M, Chen K, Godar SC, Chen G, Wu W, Rebrin I, Farrell MR, Scott AL,
Wellman CL, Shih JC. Social deficits and perseverative behaviors, but not overt aggression,
in MAO-A hypomorphic mice. Neuropsychopharmacology. 2011 Dec;36(13):2674-88.
35.
Muroni A, Paba S, Puligheddu M, Marrosu F, Bortolato M. A preliminary study of
finasteride in Tourette syndrome. Mov Disord. 2011 Sep;26(11):2146-7.
36.
Paba S, Frau R, Godar SC, Devoto P, Marrosu F, Bortolato M. Steroid 5α-reductase as a
novel therapeutic target for schizophrenia and other neuropsychiatric disorders. Curr Pharm
Des. 2011;17(2):151-67.
37.
Bortolato M, Devoto P, Roncada P, Frau R, Flore G, Saba P, Pistritto G, Soggiu A, Pisanu
S, Zappala A, Ristaldi MS, Tattoli M, Cuomo V, Marrosu F, Barbaccia ML. Isolation
rearing-induced reduction of brain 5α-reductase expression: relevance to dopaminergic
impairments. Neuropharmacology. 2011 Jun;60(7-8):1301-8.
38.
Godar SC*, Bortolato M*, Frau R, et al. Maladaptive defensive behaviors in monoamine
oxidase A-deficient mice. Int J Neuropsychopharmacol. 2011 Oct;14(9):1195-207.
155
DICHIARAZIONE DI CONFORMITÀ DELLE TESI
PER IL CONSEGUIMENTO DEL TITOLO DI DOTTORE DI RICERCA
(DICHIARAZIONE SOSTITUTIVA DI ATTO NOTORIO E DI CERTIFICAZIONE
(artt. 46-47 del D.P.R. 445 del 28.12.00 e relative modifiche)
Il/La sottoscritto/a…MARCO BORTOLATO………… Nato/a il…9/1/1976…………………….
a …CAGLIARI……………….………………. Provincia/Stato …CA……………………………….
Dottorato di ricerca in …NEUROSCIENZE…………………………………………………………….
………………………………………………………………..……………………………………………
a conoscenza del fatto che in caso di dichiarazioni mendaci, oltre alle sanzioni previste dal Codice
Penale e dalle Leggi speciali per l’ipotesi di falsità in atti ed uso di atti falsi, decade dai benefici
conseguenti al provvedimento emanato sulla base di tali dichiarazioni,
DICHIARA
sotto la propria responsabilità, ai fini dell’ammissione all’esame finale per il conseguimento del titolo di
Dottore di ricerca,
di essere a conoscenza che,
in conformità al Regolamento dell’Università degli Studi di Brescia per l'ammissione all'esame finale ed il
rilascio del titolo per il conseguimento del titolo di Dottore di Ricerca, è tenuto a depositare all’U.O.C.
Dottorati e Scuole di Specializzazione:
- n. 1 copia in formato cartaceo della propria tesi di dottorato e l’esposizione riassuntiva (abstract) in
italiano, se la redazione della tesi è stata autorizzata in lingua straniera;
- n. 2 copie della tesi su DVD o CD-ROM per il deposito presso le Biblioteche Nazionali di Roma e di
Firenze;
DICHIARA inoltre
- che il contenuto e l’organizzazione della tesi sono opera originale e non compromettono in alcun modo i
diritti di terzi,
- che sarà consultabile immediatamente dopo il conseguimento del titolo di Dottore di ricerca, in quanto non
è il risultato di attività rientranti nella normativa sulla proprietà industriale, non è stata prodotta nell’ambito
di progetti finanziati da soggetti pubblici o privati con vincoli alla divulgazione dei risultati, e non è oggetto
di eventuali registrazioni di tipo brevettale o di tutela.
- che l’Università è in ogni caso esente da qualsiasi responsabilità di qualsivoglia natura, civile,
amministrativa o penale e sarà tenuta indenne da qualsiasi richiesta o rivendicazione da parte di terzi;
- che la tesi in formato elettronico (DVD o CD-ROM ) è completa in ogni sua parte ed è del tutto identica a
quella depositata in formato cartaceo all’U.O.C. Dottorati e Scuole di Specializzazione dell’Università degli
Studi di Brescia e inviata ai Commissari. Di conseguenza va esclusa qualsiasi responsabilità dell’Ateneo per
quanto riguarda eventuali errori, imprecisioni od omissioni nei contenuti della tesi.
Dichiara inoltre di essere consapevole che saranno effettuati dei controlli a campione. Eventuali discordanze
od omissioni potranno comportare l’esclusione dal dottorato di ricerca;
Luogo e data Cagliari, 20/1/2015..
Firma del dichiarante ……………………….
155
MODULO DI EMBARGO DELLA TESI
(da compilare solo se si richiede un periodo di segretazione della tesi)
Il/La sottoscritto/a…MARCO BORTOLATO…………… Nato/a il…9/1/1976……………………………….
a (indicare anche l’eventuale paese estero)……CAGLIARI……………………………………..
provincia di (ovvero sigla del paese estero)……CAGLIARI…………………………………….
dottorato di ricerca in ……NEUROSCIENZE........………………………………………….
………………………………………………………………..……………………………………………
DICHIARA
- che il contenuto della tesi non può essere immediatamente consultabile per il seguente motivo:
PARTE DELLA RICERCA NON È ANCORA STATA PUBBLICATA SU RIVISTE SCIENTIFICHE
---------------------------------------------------------------------------------------------------------------------La motivazione deve essere dettagliata e controfirmata obbligatoriamente dal Tutor e/o Coordinatore
(Brevetto, segreto industriale, motivi di priorità nella ricerca, motivi editoriali, altro)
- che il testo completo della tesi potrà essere reso consultabile dopo:
X 12 mesi dalla data di conseguimento titolo
24 mesi dalla data di conseguimento titolo
altro periodo
_______________
- che sarà comunque consultabile immediatamente l’abstract della tesi, che viene consegnato alla U.O.C.
Dottorati e Scuole di Specializzazione
Luogo e Data
Firma del Dichiarante
Cagliari, 20/1/2015
_____________________________________
Controfirma del Tutor e/o Relatore e/o Coordinatore del
Dottorato per la motivazione di embargo e il periodo.
______________________________________
156

Jt,,z, .U? - Sezione di Farmacologia

Transcription

Similar documents

DarkStar instructions.cdr

Catálogo Brises Hunter Douglas

alpha omega minilight vehicule light vehicule

On PPI - Gismad

Surftech Australia

Scottish Equitable PIMCO Select Global Bond

milgard vinyl 9-20-04.qxd (Page 1)

THE FirsT AMEriCAN rECord oF Aspidopleurus

descriptions of a new species of serranid fish from western australia