Characterization of bulbospongiosus muscle reflexes activated by

Transcription

Am J Physiol Regul Integr Comp Physiol 303: R737–R747, 2012.
First published August 8, 2012; doi:10.1152/ajpregu.00004.2012.
Characterization of bulbospongiosus muscle reflexes activated by urethral
distension in male rats
Masayuki Tanahashi,1 Venkateswarlu Karicheti,2 Karl B. Thor,2 and Lesley Marson2
1
Astellas Pharma Incorporated, Tsukuba, Japan; and 2Urogenix, Incorporated, Durham, North Carolina
Submitted 5 January 2012; accepted in final form 2 August 2012
urethrogenital reflex; ejaculation; neural regulation; pudendal nerve;
pelvic nerve
penile erection, emission,
and ejaculation of seminal fluid. These components are regulated by supraspinal and spinal mechanisms and require coordination of sympathetic, parasympathetic, and somatic nerves.
During the emission phase, autonomic nerves (pelvic and
hypogastric) regulate secretion of seminal fluids into the ure-
SEXUAL BEHAVIOR IN MALES COMPRISES
Address for reprint requests and other correspondence: L. Marson, 801
Capitola Drive Suite 12, Durham, NC 27713.
http://www.ajpregu.org
thra. Expulsion of the seminal fluids is thought to occur in
response to urethral afferent activation due to the presence of
seminal fluids in the urethra (via stretch or chemoreceptors)
and penile afferent activation due to mechanical stimulation of
the penis (via cutaneous mechanoreceptors) and is characterized by rhythmic contractions of striated perineal muscles,
primarily the bulbospongiosus muscle (BSM) (2, 14, 21, 42,
43, 56). In acutely spinalized (T8 –T10), urethane-anesthetized
rats, clamping the tip of the penis, while infusing fluid into the
urethra, produces repeated, phasic contractions of the BSM
(11, 30, 32). This pattern of BSM activity is similar to that
recorded during ejaculation in conscious, behaving rats and
was termed the urethrogenital reflex (UGR). The BSM rhythmic contractions recorded during the UGR in rats is also
similar to the BSM activity in men during ejaculation (4, 5,
43). In addition, the BSM activity in both men and rats,
involves a spinal reflex that can be triggered by stimulation of
penile afferent fibers of the dorsal nerve of the penis (18, 23,
46). This reflex is thought to mimic the expulsion phase of
ejaculation, since it is characterized by rhythmic contractions
of the perineal muscles, which is necessary to expel seminal
fluids (11, 18, 21, 24, 32, 40, 43, 44). Therefore, the UGR is
considered a surrogate for the peripheral and spinal components of somatic and autonomic reflexes that control ejaculation and has been used to determine the anatomical, physiological, and pharmacological characteristics of ejaculatory-like
reflexes (ELR).
Both penile and urethral stimulation (distension and/or pressure) are considered to provide ejaculatory sensory input in
humans and rats (9, 32, 41, 43, 44). The sensory branch of the
pudendal nerve (SbPN) carries both penile and urethral primary afferent fibers, while the pelvic nerve (PelN) and the
hypogastric nerve (HgN) carry urethral primary afferent fibers
(7, 13, 16, 17, 27). The role of the SbPN, PelN, and HgN in
afferent pathways of peripheral sexual stimuli evoking ELR
has been reported but are not fully understood (7, 12, 29, 45).
Penile clamping can activate various types of mechanoreceptors and nociceptors or even cause tissue damage depending on
how the “clamp” is applied. In preliminary experiments, we
attempted to minimize activation of glans penis mechanoreceptors by putting a thin ligature through the tip of the penis
and gently pulling the penis from the sheath. Fortunately, we
were able to occlude the urethra and cause urethral distension
that generated pressures equivalent to pressures produced by
penile clamping. Using this penile extension method to elevate
urethral pressure, we found significant differences in activation
of the UGR. The present study characterizes the two urethral
distension methods, as well as the role of the SbPN and PelN
in conveying sensory input to spinal ejaculation control centers
and establishes baseline data for further physiological charac-
0363-6119/12 Copyright © 2012 the American Physiological Society
R737
Downloaded from http://ajpregu.physiology.org/ by 10.220.33.4 on October 21, 2016
Tanahashi M, Karicheti V, Thor KB, Marson L. Characterization
of bulbospongiosus muscle reflexes activated by urethral distension in
male rats. Am J Physiol Regul Integr Comp Physiol 303: R737–R747, 2012.
First published August 8, 2012; doi:10.1152/ajpregu.00004.2012.—The urethrogenital reflex (UGR) is used as a surrogate model of the autonomic and somatic nerve and muscle activity that accompanies
ejaculation. The UGR is evoked by distension of the urethra and
activation of penile afferents. The current study compares two methods of elevating urethral intraluminal pressure in spinalized, anesthetized male Sprague-Dawley rats (n ⫽ 60). The first method, penile
extension UGR, involves extracting the penis from the foreskin, so
that urethral pressure rises due to a natural anatomical flexure in the
penis. The second method, penile clamping UGR, involves penile
extension UGR with the addition of clamping of the glans penis.
Groups of animals were prepared that either received no additional
treatment, surgical shams, or received bilateral nerve cuts (4 nerve cut
groups): either the pudendal sensory nerve branch (SbPN), the pelvic
nerves, the hypogastric nerves, or all three nerves. Penile clamping
UGR was characterized by multiple bursts, monitored by electromyography (EMG) of the bulbospongiosus muscle (BSM) accompanied by elevations in urethral pressure. The penile clamping UGR
activity declined across multiple trials and eventually resulted in only
a single BSM burst, indicating desensitization. In contrast, the penile
extension UGR, without penile clamping, evoked only a single BSM
EMG burst that showed no desensitization. Thus, the UGR is composed of two BSM patterns: an initial single burst, termed urethrobulbospongiosus (UBS) reflex and a subsequent multiple bursting
pattern (termed ejaculation-like response, ELR) that was only induced
with penile clamping urethral occlusion. Transection of the SbPN
eliminated the ELR in the penile clamping model, but the single UBS
reflex remained in both the clamping and extension models. Pelvic
nerve (PelN) transection increased the threshold for inducing BSM
activation with both methods of occlusion but actually unmasked an
ELR in the penile extension method. Hypogastric nerve (HgN) cuts
did not significantly alter any parameter. Transection of all three
nerves eliminated BSM activation completely. In conclusion, penile
clamping occlusion recruits penile and urethral primary afferent fibers
that are necessary for an ELR. Urethral distension without significant
penile afferent activation recruits urethral primary afferent fibers
carried in either the pelvic or pudendal nerve that are necessary for the
single-burst UBS reflex.
R738
CHARACTERIZATION OF UGR
terization of the “adequate stimuli” for urethral and glans penis
primary afferent fibers in triggering the UGR.
MATERIALS AND METHODS
Penis is inside the foreskin
Ligature through glans penis
Resting condition
Obstructive flexure
Dorsal penile vein
Extension UGR
Obstructive flexure
Rubber-tipped clamp
occlusion
Clamping UGR
Fig. 1. Photographs showing the procedures for extension and clamping
urethrogenital reflex (UGR). Top: under the resting conditions, the penis
remains within the foreskin, the tie through the penile glans is accessible for
extending the penis from its foreskin during the UGR procedure. Middle:
extension UGR shows the penis extended from the foreskin and the normal
kink of the penis (obstructive flexure) that is present, which allows urethral
pressure to rise during saline infusion. Bottom: clamping UGR shows the tip of
the penis clamped to provide a closed system during the clamping UGR.
immediately removed, allowing the penis to retract with release of
urethral fluid and pressure.
The penile extension and occlusion (with or without clamping)
were performed at least 30 min after the surgical procedures. Each
occlusion was repeated 5 times with 15-min intervals between each
occlusion in groups with intact peripheral nerves. The occlusion was
applied at least three times in the neurectomized rats.
Acute neurectomies. Seven groups of animals were prepared:
Group 1 had all nerves intact (n ⫽ 12). Group 2 had bilateral
transection of the SbPN (n ⫽ 12). The SbPN was isolated in the
ischiorectal fossa and dissected free from the internal pudendal vein
AJP-Regul Integr Comp Physiol • doi:10.1152/ajpregu.00004.2012 • www.ajpregu.org
Animals. Adult, male, Sprague-Dawley rats (250 – 400 g, n ⫽ 60;
Charles River Laboratories, Frederick, MD) were housed in cages
(three/cage) with free access to water and food. The colony room was
maintained on a 12:12-h light-dark cycle. All experiments were
performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were approved
by the Urogenix Institutional Animal Care and Use Committee. All
efforts were made to minimize animal stress and to reduce the number
of animals used.
Surgery and data collection. Rats were initially anesthetized with
3.0% isoflurane (VEDCO, Saint Joseph, MO) and subsequently injected subcutaneously with 1.2 g/kg urethane (Acros Organics, Geel,
Belgium). The skin and muscle over the middle thoracic vertebrae
were incised, and the spinal cord was exposed by a laminectomy and
transected at the T8 –T10 level. The spinal cord transection was
performed at least 90 min before the experiment started.
The abdomen was opened, and a saline-filled polyethylene (PE)
tubing with a cuff at the tip (PE-50; Becton Dickinson, Franklin
Lakes, NJ) was inserted through a small incision in the dome of the
bladder and passed caudally into the urethra just past the bladder neck.
The catheter was tied in place at the bladder neck. The other end of the
urethral catheter was connected to a T-tube, allowing continuous
urethral saline perfusion (5 ␮l/s) by an infusion pump (PHD2000
infusion; Harvard Apparatus, Holliston, MA) and measurement of
urethral pressure (CDXIII Transducer, Argon Medical Devices, Athens, TX). The signal was amplified using a bridge amplifier (Transbridge 4M, World Precision Instruments, Sarasota, FL). The seminal
vesicle ducts were tied bilaterally to prevent saline from flowing into
the seminal vesicle during urethral occlusion. A suture was passed
through the tip of the penis, and a loose loop was made that did not
constrict the penis (6 – 0 silk suture with a 26-mm ½ circle, cutting
needle). This suture remained loose and was used to gently extend the
penis from the foreskin, so that either the extension or clamping
method could be performed (Fig. 1).
After a perineal skin incision to expose the BSM, a pair of
Teflon-coated stainless-steel electrodes with tip exposed (0.05 mm in
diameter, MT GIKEN, Japan) was placed in the muscle for recording
BSM-electromyogram (BSM-EMG). The electrical signals were amplified (2K) and filtered (½ amp low pass at 10 Hz and ½ amp high
pass at 10 kHz) with a P511 AC amplifier (Grass Instruments
Division, Astro-Med, West Warwick, RI). Urethral pressure and
BSM-EMG signals were acquired continuously on a personal
computer using LabChart (version 7, ADInstruments, Colorado
Springs, CO).
Urethral occlusion. Two methods were used to evoke BSM-EMG
activity: penile extension and penile clamping (Fig. 1). For both
methods, the penis was gently pulled 1–1.5 cm from the foreskin
using the loose ligature through the tip of the penis (as described
above) until the dorsal penile vein was seen at the base of the penis.
This extension produces an anatomical occlusion of the urethra by
“kinking” the lumen and is responsible for the pressure increase
associated with urethral saline perfusion. The penis remained extended until a large burst of BSM-EMG activity and a rapid rise in
urethral pressure was noted (the threshold pressure), and the penile
extension was immediately released, allowing the penis to retract,
with subsequent expulsion of urethral fluid and rapid return of urethral
pressure to baseline (Fig. 2).
For penile clamping, immediately after extending the penis as
above, the glans penis was clamped with forceps (1.3 mm tip) whose
tips were rubber-coated (Fig. 1). The force of the clamp was estimated
to be 120 –300 g/mm2. The clamp remained on the extended penis
until the threshold pressure was reached, at which time, the clamp was
R739
A
UPP
(mmHg)
160
Occlusion
Release
5 sec
80
Threshold pressure
BSM-EMG
(mV)
0
2
Duration of bursting & Number of bursts
Basal
pressure
Latency to 1st burst of BSM-EMG
1
* *
0
2
2
3
4
Number of bursts
5
*
Urethrobulbospongiosus (UBS) Reflex
RMS of 1st BSM burst (1 sec)
Ejaculation-like response (ELR)
RMS of BSM bursting (30 sec)
1
BSM-EMG
(mV)
2
1
*
2
3
4
5
1 sec
*
0
-1
-2
C
BSM-EMG
(mV)
2
1
0
-1
-2
0.05 sec
Fig. 2. Example of penile clamping-induced urethral occlusion and resulting UGR in a control rat. A: urethral pressure (top) and bulbospongiosus
muscle-electromyography (BSM-EMG) (bottom). Start of urethral occlusion by penile clamping is indicated by the left, vertical dotted line, and the urethral
perfusion pressure (UPP) gradually increases. When the UPP reaches ⬃80 mmHg, a brief, large-amplitude, BSM-EMG burst is recorded that is accompanied
by a sharp increase in UPP. Immediately upon detecting the BSM-EMG burst, the clamp is released (noted by the right, vertical dotted line), fluid drains from
the urethra, and pressure drops. This pressure is recorded as the “threshold pressure”, and the time from start of occlusion to this first BSM-EMG burst is the
“latency to first BSM burst”. As UPP drops, additional bursts of brief, large-amplitude, BSM-EMG activity associated with sharp increases in UPP are recorded.
The duration of bursting activity and the number of bursts are measured. B: an expanded trace of the BSM-EMG activity. Because the first burst could be reliably
induced and exhibited consistent parameters under multiple conditions, it is termed the “urethrobulbospongiosus (UBS) reflex”. Its action potential duration and
RMS value (see Tables 1 and 2) were measured. Since the later bursting resembles BSM-EMG activity recorded during ejaculation, it is termed the
“ejaculation-like response” (ELR). B and C: an asterisk denotes BSM-EMG activity. C: further expansion of the BSM-EMG trace to show an example of the
first burst, UBS reflex. When urethral occlusion is induced by penile extension, only the UBS reflex (i.e., first BSM-EMG burst) is seen.
and cut. Group 3 had bilateral PelN transection (n ⫽ 12). The PelN
was exposed through a ventral abdominal incision and cut central to
the major pelvic ganglion at the point just distal to its separation from
the levator ani nerve (7). Group 4 had bilateral transection of the HgN
(n ⫽ 12). Hypogastric nerves were identified between the inferior
mesenteric ganglion and major pelvic ganglion and cut just distal to
the inferior mesenteric ganglion. Group 5 (n ⫽ 6) received bilateral
neurectomy of all three peripheral nerves (SbPN, PelN, and HgN).
These groups were randomly divided equally into penile extension or
penile clamping experiments. In addition, two sham groups were
examined: group 6 (n ⫽ 2) bilateral isolation of the SbPN as in group
2, without the nerves being cut; and group 7 (n ⫽ 4), in which the
PelN was isolated bilaterally as in group 3 but not cut. Both penile
extension and penile clamping were performed in the sham groups.
Measurements. The measurements taken to generate Tables 1 and
2 are diagrammed in Fig. 2. The BSM-EMG activity was measured as
the root mean square (RMS: the square root of the sum of the squared
amplitude values) over the designated time (1 or 30 s; see Fig. 2).
Statistical analysis. All data were presented as means ⫾ SE. All
statistical analysis was performed with Prism 5 for Windows (GraphPad Software, San Diego, CA). One-way and two-way ANOVA with
Dunnett’s multiple-comparison, or Bonferroni’s multiple-comparison
post hoc tests, respectively, were used to examine the effect of
repeated trials, the effect of various nerve cuts, and to compare
clamping versus extension methods. Individual tests are described in
each respective table legend. P ⬍ 0.05 was considered statistically
significant.
RESULTS
General characteristics. Baseline urethral pressure prior to
infusion ranged between 18 and 24 mmHg and urethral compliance ranged from 2.9 to 4.6 ␮l/mmHg across all trials in all
experiments, with no correlation to the method of occlusion
(i.e., penile clamping or extension), trial number, or neurectomy.
At variable times after initiation of filling (about 20 – 40 s), during
the linear portion of the volume-pressure curve, three to five
small BSM-EMG bursts might be recorded (see BSM-EMG
activity marked by an asterisk in Fig. 2). These small BSMEMG bursts were never accompanied with a sharp elevation in
urethral perfusion pressure (UPP, Fig. 2) and had smallamplitude, short-duration (usually ⬍1 s), and moderate frequency (about 70 –100 spikes/s). This activity was sporadic
and not reliably produced within or between individual animals
and is not considered a component of the UGR, so it will not
be discussed further.
B
R740
Table 1. UGR parameters measured repeatedly across five trials
Occlusion
1st
3rd
4th
5th
77.9 ⫾ 5.6
74.8 ⫾ 6.1
70.6 ⫾ 5.4
74.3 ⫾ 6.1
77.8 ⫾ 5.6
82.2 ⫾ 7.3
82.9 ⫾ 6.7
83.0 ⫾ 7.3
88.5 ⴞ 6.3#
80.1 ⫾ 6.5
37.0 ⫾ 3.2
39.1 ⫾ 3.1
27.3 ⫾ 2.0
35.4 ⫾ 2.0
33.3 ⫾ 4.2
41.8 ⫾ 4.0
34.6 ⫾ 3.5
38.1 ⫾ 3.9
36.1 ⫾ 4.4
39.3 ⫾ 3.4
15.6 ⴞ 2.3*
0.8 ⫾ 0.3
19.4 ⴞ 7.8*
2.1 ⫾ 0.5
17.7 ⴞ 6.4*
0.6 ⫾ 0.0
14.1 ⫾ 8.6
1.7 ⫾ 0.7
6.2 ⫾ 3.7
0.5 ⫾ 0.0
5.3 ⴞ 0.6**
1.2 ⫾ 0.2
43.4 ⴞ 2.9**
9.4 ⫾ 4.7
162.3⫾35.3
194.7 ⫾ 18.4
5.3 ⴞ 0.4**
1.7 ⫾ 0.2
39.2 ⴞ 5.8*
16.5 ⫾ 5.0
154.6 ⫾ 38.2
201.7 ⫾ 18.6
3.7 ⴞ 1.2**
1.0 ⫾ 0.0
2.8 ⴞ 0.7#
1.3 ⫾ 0.2
2.2 ⴞ 0.8##
1.0 ⫾ 0.0
26.2 ⴞ 8.5#*
5.5 ⫾ 1.2
22.9 ⴞ 7.0##
12.1 ⫾ 4.7
13.3 ⴞ 5.6##
5.0 ⫾ 1.3
165.2 ⫾ 43.1
182.1 ⫾ 29.8
155.4 ⫾ 40.8
189.9 ⫾ 23.1
160.1 ⫾ 42.0
186.0 ⫾ 26.5
Values are expressed as means ⫾ SE; n ⫽ 6/group. The name of each parameter corresponds to the description in Fig. 1. Statistically significant differences
are in bold type. Repeated-measures one-way ANOVA was done for each parameter to compare the trials. #P ⬍ 0.05; ##P ⬍ 0.01; compared with 1st occlusion
in respective group (paired Dunnett’s multiple-comparison test). Repeated-measures two-way ANOVA with Bonferroni’s multiple-comparison post hoc test was
done to compare each parameter between clamping and extension groups. UGR, urethrogenital reflex; UPP, urethral perfusion pressure; BSM, bulbospongiosus
muscle; RMS, root mean square. *P ⬍ 0.05; **P ⬍ 0.01 compared with extension group.
Penile clamping-evoked urethrogenital reflex (UGR). In this
study, the characteristics of the first trial of penile clampingevoked UGR were very similar to previous reports (9, 30, 32).
During occlusion and filling, urethral pressure increased in a
linear fashion until it reached ⬃80 ⫾ 10 mmHg (Figs. 2– 4,
Table 1). At that point (i.e., threshold pressure), a distinctive,
large-amplitude, high-frequency (492 ⫾ 48 spikes/s), shortduration (580 ⫾ 110 ms) burst of BSM-EMG activity was
recorded, which was accompanied by a sharp inflection in UPP
(Fig. 2). We have termed this response the urethrobulbospongiosus (UBS) reflex. At this point, the penile clamp was
released, fluid (and sometimes a seminal plug) was forcefully
expelled from the urethra, and UPP returned toward baseline.
While the UPP was falling and for a short time after its return
to baseline, additional bursts of BSM-EMG activity (Table 1)
were recorded with an interburst interval of ⬃1– 4 s; these
bursts were accompanied by transient elevations in intraurethral pressure. We have termed this response the ejaculatorylike response (ELR) (Fig. 2). Penile clamping alone (i.e.,
without infusion of the urethra to elevate urethral pressure) did
not produce this pattern of BSM-EMG bursting.
When penile clamping was repeated in each rat, there was
evidence of desensitization of the ELR (Fig. 3, Table 1). For
example, by the 4th or 5th occlusion, the number of bursts and
the RMS of BSM bursting (30 s) significantly decreased, and
the threshold UPP significantly increased. In addition, the
duration of the bursting appeared lower by the 5th trial. In
contrast to the waning of the ELR, the initial BSM-EMG burst
(the UBS reflex) remained consistent across multiple trials. In
summary, the multiple bursting BSM-EMG activity (i.e., ELR)
after release diminished over multiple trials. By the 5th trial,
the penile clamping-evoked BSM-EMG burst looked very
similar to the penile extension-evoked initial BSM-EMG burst,
i.e., the UBS reflex (see below).
Penile extension-evoked UGR. Penile extension produced a
linear increase in urethral pressure and small bursts of BSM-
EMG activity that were similar to the penile clamping-evoked
UGR (Table 1). Also, at a similar UPP threshold, a single,
large-amplitude, high-frequency (331 ⫾ 60 spikes/s), shortduration (580 ⫾ 40 ms) burst of BSM-EMG activity was
associated with a sharp increase in UPP with forceful expulsion
of the infused saline from the urethra (e.g., Figs. 3 and 4).
Therefore, penile extension UGR evoked the UBS reflex.
However, the ELR was not observed (Fig. 3 and Table 1). The
UBS reflex did not desensitize over multiple trails (Table 1).
Neurectomies. In sham groups 6 and 7, both the extension
and clamping UGR produced similar responses as the control
group, confirming that surgical manipulations did not alter the
evoked responses. Transection of the SbPN abolished the
multiple bursting BSM-EMG activity (i.e., the ELR) (Table 2,
Fig. 4B) in clamping UGR, but it did not affect the UBS reflex
in either clamping or extension UGR. A nonsignificant increase in latency and threshold UPP (Table 2, Fig. 4B) was also
observed in the clamping UGR.
Pelvic nerve transection resulted in a significant increase in
the latency and threshold UPP to the first BSM-EMG burst
during penile extension UGR, as well as a nonsignificant
increase in threshold UPP and latency in clamping UGR (Table
2 and Fig. 4C). Surprisingly, pelvic neurectomy “unmasked”
an ELR following release of penile extension (Table 2, Fig.
4C). This response was similar to the BSM-EMG activity seen
with the clamping UGR (Fig. 4C) in control animals.
Transection of the HgN did not change either the penile
clamping or penile extension-induced UGRs (Table 2, Fig.
4D). Transection of all three nerves completely eliminated both
the penile clamping- and penile extension-induced BSM-EMG,
i.e., both UBS and ELR were absent (data not shown).
DISCUSSION
The findings of the current study confirm and extend previous reports that have used urethral infusion with penile clamp-
Threshold UPP, mmHg
Clamping
Extension
Latency to 1st BSM burst, s
Clamping
Extension
Duration of BSM bursting, s
Clamping
Extension
BSM bursts (number)
Clamping
Extension
RMS of BSM bursting (30 s), ␮V
Clamping
Extension
RMS of 1st BSM burst (1 s) ␮V
Clamping
Extension
2nd
R741
UPP (mmHg)
B
1st occlusion
5th occlusion
100
100
100
80
80
80
60
60
60
40
40
40
20
20
20
1
1 2 3 4 5
12
2
1
1
00
0
-1
-1
-1-2
-2
C
D
1st occlusion
20sec
5th occlusion
UPP (mmHg)
100 00
100
80 80
80
60 60
60
40 40
40
20 20
BSM-EMG (mV)
Extension
120
1
2
20
1
1
2
1
1
0
0
0
-1
-1
-1 -2
-2
Fig. 3. Examples of UPP and BSM-EMG activity recorded during the 1st (A and C) and 5th trials (B and D) of urethral occlusion when induced with penile
clamping (A and B) or penile extension (C and D). The first trial with penile clamping-induced occlusion produces a robust ejaculation-like response composed
of multiple BSM-EMG bursts while the 5th trial results in only a single UBS reflex. In contrast, the first trial of penile extension-induced urethral occlusion
produces only a single burst (the UBS reflex), which remained consistent through the 5th trial. The black bar under the UPP indicates the time of urethral
occlusion.
ing to elevate urethral pressure and evoke the UGR (9, 29, 31).
By comparing extension- and clamping-evoked UGR activity,
we have been able to isolate two components, an initial
BSM-EMG burst that involves urethral afferent fibers carried
by the pelvic and pudendal nerves, which is termed the UBS
reflex. The second component of the UGR comprises multiple
bursts of BSM-EMG activity that follow release of penile
clamping (or release of penile extension in animals with a
pelvic neurectomy). These multiple bursts of EMG activity are
remarkably similar to BSM-EMG patterns recorded during
ejaculation in conscious rats and humans; thus, we have termed
this clustered pattern of BSM-EMG bursting an ELR (19, 22,
24, 28, 36, 39, 40).
The bursting BSM-EMG activity recorded during the UGR
is quite different from the bursting urethral rhabdosphincter
EMG activity and BSM EMG activity recorded during voiding
in rats (15, 49) (Karicheti V., unpublished observations). During voiding, the urethral rhabdosphincter (innervated by pudendal motor neurons like the BSM) shows a rhythmic EMG
bursting pattern, but the frequency of the bursting is 6 Hz,
much faster than the 0.25– 0.5 Hz bursting of the BSM-EMG
during the UGR (49). Furthermore, the 6-Hz bursting of the
urethral rhabdosphincter and pelvic floor muscles only occurs
at the peak of a micturition bladder contraction. Because the
bladder remained completely empty during the current study, it
is not reasonable to think that bladder contractions occurred at
any time during our experiments. In addition, the BSM-EMG
bursting was seen after bilateral transection of the pelvic
nerves, which eliminates afferent and efferent activity to and
from the bladder, respectively. Finally, high-frequency contractions of the urethral rhabdosphincter are only seen in rat,
while low-frequency rhythmic bursting of the BSM during
ejaculation is seen in multiple species (6, 10, 43) . Considering
the above evidence, it is reasonable to speculate that two
distinct “pattern generators” exist in the rat spinal cord, one
activated during micturition, and another activated during ejaculation. Future experiments recording EMG activity from both
the BSM and urethral rhabdosphincter simultaneously, while
inducing micturition and UGR activation, would be useful to
confirm this possibility.
Electrical stimulation of the dorsal nerve of the penis (a
branch of the SbPN) in multiple species, including humans and
rats, triggers rhythmic bursting of the BSM, which mimics
ejaculation, suggesting that penile activation alone might trig-
BSM-EMG (mV)
Clamping
A
R742
Table 2. UGR parameters after various selective neurectomies
Neurectomy
Control
PelN
HGN
77.9 ⫾ 5.6
74.8 ⫾ 6.1
90.5 ⫾ 9.1
88.5 ⫾ 4.5
104.1 ⫾ 20.6
138.8 ⴞ 15.7##
73.5 ⫾ 2.6
87.0 ⫾ 6.6
37.0 ⫾ 3.2
39.1 ⫾ 3.1
62.0 ⫾ 8.1
53.4 ⫾ 6.3
66.3 ⫾ 16.7
82.2 ⴞ 12.8##
31.8 ⫾ 2.5
50.4 ⫾ 5.3
15.6 ⴞ 2.3**
0.8 ⫾ 0.3
0.5 ⴞ 0.1##
0.5 ⫾ 0.1
7.8 ⴞ 1.7**
20.7 ⴞ 5.0##
19.5 ⴞ 3.8**
1.8 ⫾ 0.8
5.3 ⴞ 0.6**
1.2 ⫾ 0.2
1.0 ⴞ 0.0##
1.0 ⫾ 0.0
3.3 ⫾ 0.8
3.0 ⴞ 0.4##
5.8 ⴞ 0.9**
1.3 ⫾ 0.2
43.4 ⴞ 2.9**
9.4 ⫾ 4.7
5.5 ⴞ 0.7##
5.6 ⫾ 0.6
162.3 ⫾ 35.3
194.7 ⫾ 18.4
175.0 ⫾ 18.7
174.3 ⫾ 27.9
34.5 ⫾ 6.6
24.7 ⴞ 4.3#
112.2 ⫾ 16.1
107.3 ⴞ 16.7#
47.6 ⴞ 10.2**
8.0 ⫾ 2.9
125.0 ⫾ 27.4
188.1 ⫾ 14.9
Values are expressed as means ⫾ SE; n ⫽ 6 per group. The name of each parameter corresponds to the name of each parameter in Fig. 1. Statistically significant
differences are in bold type. One-way ANOVA with Dunnett’s multiple-comparison test was done for each parameter to compare the neurectomy models. SbPN,
pudendal sensory nerve branch; PelN, pelvic nerve; HGN, hypogastric nerve. #P ⬍ 0.05; ##P ⬍ 0.01, compared with control in respective parameter. Two-way
ANOVA with Bonferroni’s multiple-comparison post hoc test was done to compare each parameter between clamping and extension. **P ⬍ 0.01 compared with
extension group. Only the 1st UGR is presented in the table; subsequent UGRs showed similar responses.
ger ejaculation (38, 46, 48, 54, 55). However, studies conducted in humans have also shown that direct stimulation of the
bulbous and prostatic urethra can trigger reflex BSM-EMG
bursting, which is similar to ELRs (42, 59). In addition,
distension of the bulbous urethra produced rhythmic BSMEMG activity, which was reproducible and which disappeared
after anesthetization of the bulbous urethra, further suggesting
that urethral afferents may also play a role in ejaculation (42).
Little direct information is known about the nerves involved in
mediating this response. The BSM rhythmic contractions may
occur via more than one mechanism, which may depend on
other factors, such as the presence or absence of a full erection
or the presence of seminal fluids in the urethra. The hypothesis
that three factors are involved seems reasonable: seminal fluids
released during emission act on urethra afferents (chemosensitive and stretch receptors) to alter the sensitivity of penile
(DPN) afferents; penile erection alters the sensitivity of the
penile afferents; and that glans penis stimulation immediately
prior to ejaculation facilitates or triggers the strong muscle
contractions to eject the semen.
The ELR, observed in the present study is also similar to
BSM-EMG activity evoked by direct stimulation of the medial
region of the L3/L4 spinal cord, where the lumbar spinothalamic neurons (Lst), i.e., where the proposed spinal pattern
generator neurons for ejaculation (SPGE) are located (1, 6, 14,
50, 51). In behavioral copulation experiments, the SPGE was
shown to be essential for ejaculation, as lesions of the Lst
neurons resulted in animals that showed normal mounts and
intromissions, but these animals failed to ejaculate (1, 50). The
Lst neurons (SPGE) are activated with the UGR by both
urethral distension and stimulation of the dorsal penile nerve,
and contractions of the BSM muscle via dorsal penile nerve
stimulation can be prevented by inhibition of MAPK signaling
in Lst neurons (29, 46). Thus, it is proposed that the ELR
component of the UGR involves activation of the SPGE.
The current study demonstrated that the ELR evoked by
penile clamping desensitizes upon repeated activation in an
individual animal. However, the UBS remains and shows no
desensitization. This remaining single burst of BSM-EMG
activity is similar to the BSM-EMG burst produced by penile
extension-induced elevations in urethral pressure, as well as
the single burst produced by penile clamping-induced urethral
pressure elevation when the SbPN is transected. The phenomenon of desensitization of the UGR has been previously suggested, using a different experimental protocol and was termed
“exhaustion” of the response (9). Desensitization is not thought
to be due to activation of nociceptors or tissue damage as the
same phenomenon was observed when either forceps or a
plastic microvascular clip was used to clamp the urethral
meatus (Karicheti V., Marson L., unpublished observations). In
addition, desensitization of the ELR has been seen in our
laboratory’s preliminary experiments using electrical stimulation of the dorsal nerve of the penis or the SbPN (31), as well
as in behavioral studies of sexual exhaustion after prolonged
sexual activity (57, 58). The existence of the UBS that does not
desensitize suggests that desensitization may involve the pattern generator rather than the primary afferents or BSM motor
neurons.
The importance of penile afferent fibers in initiating the ELR
was also shown by transecting the SbPN, which carries afferent
fibers from the penis (dorsal penile nerve) and urethra to the
spinal cord (20, 37). This procedure resulted in an increase in
the UPP threshold and a decrease in burst duration and RMS
values of both the penile clamping-induced and penile extension-induced UGR. The UBS reflex (a single burst) was present after SbPN cuts, in both procedures, suggesting that this
response is mediated by urethral afferents that do not travel
through the SbPN (probably through the PelN; see below). The
importance of the SbPN in mediating the afferent input of the
ELR has previously been shown in the male and female rat, in
Threshold UPP, mmHg
Clamping
Extension
Latency to 1st BSM burst, s
Clamping
Extension
Duration of BSM bursting, s
Clamping
Extension
BSM bursts (number)
Clamping
Extension
RMS of BSM bursting (30 s), ␮V
Clamping
Extension
RMS of 1st BSM burst (1 s), ␮V
Clamping
Extension
SbPN
R743
Control
UPP (mmHg)
A
Clamping occlusion
Extension occlusion
100
100
100 00
80
80
80 80
60 60
60
60
40 40
40
40
20 20
20
20
1 2 3 4 5
BSM-EMG (mV)
1
1
0.5 1
0
00
0
-1
-0.5-1
1
-1-2
-2
SbPN
UPP (mmHg)
B
12
2
100
100
120
120
100
100
80
80
60
60
40
40
20
20
12
20sec
80
80
60
60
40
40
20
20
1
24
0.51
12
00
00
-0.5
-1
1
-1-2
-1
-2
-2-4
-3
Fig. 4. Effects of selective neurectomies on the UGR induced by penile clamping (left column) and penile extension (right column)-induced urethral occlusion
and UPP and BSM-EMG activity. A: control responses showing an ELR composed of multiple BSM-EMG bursts. B: following bilateral transection of the sensory
branch of the pudendal nerve (SbPN). Note the loss of the ELR from the penile clamping-induced UGR, but preservation of the UBS reflex in both the penile
clamping- and penile extension-induced UGR. C: following bilateral transection of the pelvic nerve (PelN). Note in the extension-induced UGR, there is an
increase in latency and threshold pressure, and the presence of an ELR (i.e., multiple bursts). D: following bilateral transection of the hypogastric nerves (HgN),
no changes were observed.
which bilateral transection of the SbPN abolished the UGR (7,
32). Moreover, electrical stimulation of the dorsal penile nerve
evokes the ELR, while bilateral transections of the dorsal
penile nerve prevent ejaculation in copulating rats and eliminates the ELR (38).
The role of pelvic afferent fibers was also examined in the
present study. Pelvic neurectomy produced increases in the
UPP threshold for both the penile clamping and the penile
extension-evoked UBS, suggesting that some urethral afferent
fibers mediating the UGR traverse the pelvic nerve. Surprisingly, pelvic neurectomy also “unmasked” a penile extensionevoked ELR, similar to that produced by the penile clampingevoked UGR. This finding is important because it suggests that
an ELR can be induced by urethral afferent fibers (presumably
traversing the pudendal nerve) without substantial input from
the glans penis afferent fibers. Because a significant increase in
the threshold pressure to evoke the UGR was seen after pelvic
neurectomy, we cannot rule out the possibility that this elevated pressure may be responsible for the ELR. An alternative
explanation is that there are afferent fibers in the pelvic nerve
that may inhibit the activation of the ELR. Thus, it may be that
the pelvic nerve contains separate populations of urethral
afferent fibers that can excite or inhibit BSM-EMG activation.
However, our experimental protocol does not allow us to rule
out involvement of pelvic efferent fibers that control urethral
smooth muscle tone, and this possibility should be explored in
future studies.
Transection of the HgNs did not alter either the penile
clamping or penile extension-evoked UGR. While a combination transection of the pelvic, pudendal, and hypogastric nerves
eliminated all BSM-EMG activity. These data suggest that the
HgN, under normal circumstances, does not contribute to the
UGR, while the SbPN and PelN carry all the urethral and
penile afferent fibers capable of activating the UGR. This
BSM-EMG (mV)
1
R744
Clamping occlusion
C
Extension occlusion
Pel N
120
100
100
80
80
60
60
40
40
20
20
150
150
UPP (mmHg)
120
100
100
50
50
0
12
D
HgN
UPP (mmHg)
2
3
0.5 1
00
-0.5-1
-1-2
100 00
100
80
80
60
60
40
40
20
20
100
BSM-EMG (mV)
1
1.0
0.25
0.5
0
0.0
-0.25
-0.5
-0.5
-1.0
-0.75
-1.5
80 80
60 60
40 40
20 20
1 2 345
6
0.5
1 2
1
0 0
-1 -2
Fig. 4—Continued.
agrees with previous studies examining the UGR in the female
rat (7).
Compiling all of the results from our studies, we propose a
model (Fig. 5) to describe the activation of BSM-EMG motor
neurons resulting from distension of the urethra following its
occlusion through clamping of the glans penis or through
extension of the penis without clamping. Our model proposes
that activation of urethral stretch receptors, whose primary
afferent axons are carried in both the SbPN and PelN, initiates
a single-burst discharge of BSM motor neurons, i.e., the UBS
reflex. When the urethral occlusion is released and stretch of
the urethra is absent, the driving force for BSM-EMG activity
is removed and the BSM motor neurons become quiet. When
penile afferent fibers are additionally stimulated by clamping
the glans penis to occlude the urethra, the combined input from
both penile mechanoreceptors and urethral stretch receptors via
thinly myelinated and unmyelinated fibers (A beta, A delta, and
C fibers), which mediate low-threshold slowly adapting afferent responses, as well as high-threshold noxious stimuli (25,
47), converge on the SPGE located in the L3/L4 spinal cord,
which subsequently produces a pattern of repeated activation
of BSM motor neurons and results in a cluster of multiple
BSM-EMG bursts that resemble ejaculation in behaving rats
(i.e., the ELR). Because neither urethral distension alone (i.e.,
penile extension with urethral infusion) nor penile stimulation
alone (i.e., penile clamping without urethral distension) activates the ELR when all innervation is intact, the model includes a convergence of urethral and penile afferent inputs onto
a common pathway. The convergence could occur at a number
of places in the spinal cord, but because penile and urethral
afferent fibers overlap in the L5/L6 dorsal horn and dorsal gray
commissure, we believe this is a reasonable proposal (3, 26, 27,
33–35, 52, 53).
For simplicity, the model shows pelvic and pudendal urethral afferent fibers converging on a single neuronal pathway
from the SPGE to subsequently produce a single burst of
activity from BSM motor neurons (i.e., the UBS reflex), but the
two groups of afferent fibers could have separate pathways.
BSM-EMG (mV)
12 3
12
0.5
0.51
00
-0.5
-0.5
-1
-1
-1
-2
-1.5
-3
1
R745
NTS
NTS
7N
NRM
LPGC
Primary afferent
neurons in the
L6 DRG
Medulla
I
py
Spinalization
Bladder
L3 spinal cord
A
Pelvic n.
I
A. UBS reflex
Urethra
Bulbospongiosus
muscle
Glans
Penis
B
+ B. ELR
Pudendal n.
Bulbospongiosus
motor neuron
L6 spinal
cord
Fig. 5. A proposed model for UGR pathways based on the results of the current study. Afferent terminals in the urethra are activated by urethral distension during
slow filling of the urethra and occlusion of the urethra through either penile clamping or penile extension. These urethral afferents, carried by both the pelvic
nerve (blue) and pudendal nerve (green), excite dorsal horn interneurons which, in turn, relay through the SPGE to produce a single burst of firing in
bulbospongiosus muscle (BSM) motor neurons and a single contraction of the BSM (pathway A and inset A in blue). Activation of urethral afferent fibers alone
is only sufficient to activate a single burst from BSM motor neurons (UBS reflex). Mechanoreceptive afferent terminals in the glans penis (brown) are activated
during penile clamping (but not during penile extension). These afferent fibers cannot activate a single burst from BSM motor neurons when stimulated by penile
clamping alone. However, when urethral afferent and glans penis afferent fibers are activated simultaneously by a combination of urethral distension and penile
clamping, this convergence of inputs relays through the SPGE to subsequently activate a multiple burst ELR from BSM motor neurons (pathway B and inset
B in red), which then produces multiple rhythmic contractions of the BSM and expulsion of urethral fluids (i.e., ejaculation). Since transection of the pelvic nerve
“unmasked” an ELR in the absence of penile clamping, an excitatory projection (dashed green line) from pudendal urethral afferent fibers to the ELR pathway
is proposed, along with an inhibitory pathway (dashed blue line) through an inhibitory interneuron (denoted by the small white circle with the black letter “I”)
from the pelvic nerve afferent fibers onto the ELR pathway. Spinal cord transection rostral to the L3 level is required to see this ELR because of descending
inhibition from supraspinal centers (e.g., the lateral paragigantocellularis nucleus of the medulla denoted by the large white circle with the black letter “I”), and
the model includes this supraspinal inhibitory input to the SPGE. LPGC, nucleus of the lateral paragigantocellularis; DRG, dorsal root ganglion; NTS, nucleus
tractus solitarius; NRM, nucleus raphe magnus; 7N, motor nucleus of the 7th cranial nerve; NRM, nucleus raphe magnus; py, pyramidal tract; UBS,
urethrobulbospongiosus; ELR, and ejaculation-like response; UBS, urethrobulbospongiosus; ELR, ejaculation-like response.
Furthermore, the model shows convergence of penile and
urethral fibers that are required to produce multiple bursting
(i.e., ELR), occurring in the SPGE, but the convergence could
occur at various points in the pathway. In addition, the model
shows a single pudendal urethral afferent providing input to
both the single-burst UBS reflex, and multiple-burst ELR
pathways and a single pelvic afferent fiber, providing both
excitatory input to the UBS reflex pathway and an inhibitory
input to the pudendal urethral activation of the ELR pathway.
However, it is likely that the excitatory and inhibitory inputs
arise from separate populations of neurons, and the inhibition
could occur at various points in the reflex pathway. Possibly
pelvic afferent inhibition of ELR could play a role in coordinating sexual and excretory functions of the urethra.
Perspectives and Significance
The UGR as originally described by McKenna and colleagues (11, 32) provided a starting point for the subsequent
study of various aspects of ejaculatory control, such as identification of L3/4 ejaculatory interneurons, supraspinal regulation, and pharmacological studies. It is expected that it will
continue to provide physiological and pharmacological information critical to understanding ejaculation in men. The current study provides critical baseline information regarding the
“adequate stimulus” to induce ejaculation and the relative
importance of primary afferent fibers carried in the sympathetic, parasympathetic, and somatic nerves innervating the
penis and urethra.
Spinal pattern
generator for
ejaculation
R746
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
DISCLOSURES
24.
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
25.
Author contributions: M.T., V.K., K.B.T., and L.M. conception and design
of research; M.T., V.K., and L.M. performed experiments; M.T., V.K., and
L.M. analyzed data; M.T., V.K., K.B.T., and L.M. interpreted results of
experiments; M.T., V.K., K.B.T., and L.M. prepared figures; M.T., V.K.,
K.B.T., and L.M. drafted manuscript; M.T., V.K., K.B.T., and L.M. approved
final version of manuscript; V.K., K.B.T., and L.M. edited and revised
manuscript.
26.
27.
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may provide a model applicable to study of the “refractory
period” observed during natural copulation. Importantly, the
initial BSM-EMG burst did not show desensitization, suggesting that the site of desensitization is the pattern generator as
opposed to primary afferent or bulbospongiosus motor neurons. Characterization of the adequate stimuli to trigger activation of the SPGE, the output of this pattern generator, and the
refractory nature of the pattern generator are likely to provide
useful insight regarding other spinal pattern generators that
control respiration and locomotion. The surprising finding that
pelvic nerve afferent fibers can provide an inhibitory signal to
ejaculation opens an avenue to future research aimed at understanding coordination of urethral function during micturition
and copulation. Obviously, it would be disruptive to micturition if urine flow into the urethra produced an ejaculation-like
response. It is hoped that our physiological characterization of
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Characterization of bulbospongiosus muscle reflexes activated by

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