NS-O15 - SC EPSCoR/IDeA

Galanin Expressing
Leptin Receptor Neurons
and Their Role in Anorexigenic Leptin Action
Yan Zhang1, Amanda Laque1, Miro Faouzi2, Gwendolyn Louis2, Rebecca Leshan2, Gina M.
Leininger2, Justin C. Jones2, Christopher J. Rhodes3, and Heike Münzberg1
1Pennington
Biomedical Research Center and Louisiana State University System
2Internal Medicine/MEND, University of Michigan,
3Medicine/Biological Science, University of Chicago
Leptin
Glucose homeostasis
Reproduction
Thermoregulation
Physical activity
Immune Function
Linear Growth …
Leptin target sites
express LepRb
Leptin
ob/ob
+/+
Satiety
Food intake
Adipose tissue
Montague CT et al, Nature 387, 903-908 1997
LepRb expression in the CNS
Behavior
Myers MG, Jr, Munzberg H, Leinninger GM, Leshan RL, Cell Metab 9:117-123, 2009
POMC neurons
AgRP neurons
3V
ARC
+
Leptin
POMC↑
CART
Activity (cFos)↑
NPY↓
AgRP↓
Activity (cFos)↓
Galanin
GalaninTgGFP mouse, IHC: GFP
Orexigenic neuropeptide (29 AA)
specific induction of fat intake
Orexigenic action proposed to be mainly mediated
via action in the PVN
DMH
LHA/
PFA
fx
VMH
ARC
Literature on interaction of leptin and galanin
system very controversial with regard to
LepRb/galanin co-localization.
Leptin induced pSTAT3 as a marker for LepRb
Saline
Leptin
LHA
Fasted wt mice
30’ treatment
DMH
VMH
3v
ARC
IHC: P-STAT3
E.
500
400
300
200
100
0
exPFA
Double colocalized cells
/pSTAT3 cells%
exPFA
Number of LepRb(Gal) neurons
LepRb Neurons in exPFA and NTS
Co-Express the neuropeptide Galanin
70
NTS
55%
60%
ExPFA
NTS
60
50
40
30
20
10
0
LepRb(Gal) Neurons are Distinct from Other Orexigenic
Neurons Known in the Perifornical Area
DMH
exPFA
orexin
GFP (Galanin)
pSTAT3 (LepRb)
fx
DMH
exPFA
MCH
GFP (Galanin)
pSTAT3 (LepRb)
fx
Unilateral cannulation of the exPFA
to measure anorexigenic leptin effects
GalaninTgGFP mouse
1μg
100ng
10ng
1μg
100ng
10ng
A.
PBS injection
leptin
***
-2
24h food intake (g)
6
5
4
3
2
1
0
-1
0
1
2
3
4 day
1.5
1.0
0.5
0.0
-0.5
-1.0
-1.5
-2.0
-2.5
-3.0
PBS injection
leptin
-2
-1
0
1
* ** **
2
3
Leptin injection into the exPFA reduces food intake
20ng leptin
1ng leptin
8
8
7
**
*
Body weight changes (g)
1.5
1.0
0.5
0.0
-0.5
-1.0
-1.5
-2.0
-2.5
-3.0
Leptin injection into the exPFA reduces body weight
1ng leptin
20ng leptin
B.
C.
**
24h food intake (g)
Body weight changes (g)
Leptin Action in the exPFA
Regulates Body Weight and Food Intake
7
D.
6
5
4
3
2
1
0
*
4 day
- Conditional Tracing: Viral cre/loxP System –
Do exPFA LepRb Neurons innervate the PVN?
PVN
stop
1.
LepRbCre
5’
PmeI
IRES
LepRb ex. 18b
Terminal fields
NheI
cre
LepRb
pA
neo
3’
other
stop
2. galCre
5’
gal
PmeI
IRES
NheI
cre
pA
neo
3’
Cre recombination at loxP sites
CMV
A. Ad-iZ/EGFPf
B. Ad-iN/Wed
DMH
C. Ad-iN/Ted
β-geo-pA
loxP
loxP
loxP
EGFPf
pA
3’
WGAEGFP DsRedf pA 3’
β-geo-pA
loxP
ARC
TTCEGFP DsRedf pA 3’
β-geo-pA
loxP
loxP
exPFA
Cre-expressing neurons
LepRbCre
LepRb specific:
EGFPf expression only in cre-expressing
neurons
Site specific (e.g. exPFA):
stereotaxic injections
Visible dendrites and axons:
Farnesylated EGFP
LepRb Neurons in the exPFA
vDMH LepRb neurons
project to the PVN
* IHC: GFP
PVN
*
DMH
fx
VMH
3V
ARC
ME
LepRbCre mouse
PFA LepRb neurons
* Ipsilateral
injection site
*
*
PVN
DMH
fx
3V
VMH
ARC
ME
LepRbCre mouse
Summary
Autonomic
Neuroendocrine
Behavior
Energy
homeostasis
1. LepRb(Gal) neurons describe a
confined neuronal population within
the exPFA, as well as the NTS.
2. LepRb(Gal) neurons in the exPFA
are distinct from well characterized
orexin and MCH neurons.
3. LepRb neurons in the exPFA
mediate anorexigenic leptin action.
PVN
exPFA
NTS
4. LepRb neurons in the vDMH and
PFA innervate the PVN and likely
regulate neuroendocrine, autonomic
and behavioral PVN outputs.
DMH
Future Directions:
fx
= LepRb(Galanin) neuron
exPFA
1. Conditional LepRbKO in galanin
neurons will identify the
physiological role of LepRb(Gal)
neurons in anorexigenic leptin
action.
2. Investigate if galanin (mRNA or
release) is regulated (inhibited?) by
leptin action.
Leptin
3. Discriminate between LepRb(Gal)
function in exPFA and brainstem.
Acknowledgement
PBRC
Yan Zhang
Amanda Laque
Hans-Rudolf Berthoud
Randy Mynatt
Jingying Zhang
Chris Morrison
Rick Rogers
Gerlinda Herman
COBRE
Tom Gettys
Internal Advisors:
Leslie Kozak
Jackie Stephens
External Advisors:
Barry Levin
Richard Hanson
University of Michigan
Martin Myers
and Lab Members
University of Chicago
Chris Rhodes
Columbia University
Streamson Chua
Mutant Mouse Regional Resource Ctr
(MMRRC)
Funding
AHA (Scientist Development Grant)
COBRE (1 P20 RR02195)
Conditional LepRb deletion in Galanin Neurons:
Generation of GalaninCre Mice
GalCre/+, RosaYFP/+
GalTgGFP
Classical retrograde tracing experiments:
Confirmation of DMH LepRb Projections
to the PVN and BST
IHC: pSTAT3/Fluorogold
Injection site with Fluorogold
PVN
DMH
3V
fx
PVN = paraventricular nucleus
3V = 3rd ventricle
DMH = dorsomedial hypothalamus
Location Might Encode Function
and Neuronal Population
Endocrine System (e.g. thyroid)
Autonomic System
Behavior
PVN
3V
Satiety
AP
NTS
DMH
3V
cc
LHA/PFA
fx
VMH
Mesolimbic Dopamine Sytem
Hedonic Food Intake/Addiction
Thermoregulation
Glycemic Control
ARC
ME
Reproduction