Brahz Research, 587 (1992) 178-180 ~ 1q92 Elsevier Science Publishers B.V. All rights reserved U006-8993/92/$05.00
178
BRES 252~5
The role of the bulbocavernosus in penile reflex behavior in rats E d w a r d P. M o n a g h a n *, a n d S. M a r c B r e e d l o v e Dep,rtment of P~'t'holo,~,% Unh'ersio'of California. Berkeley. CA 94720 (USA) (Accepted 5 May 1992)
Key word.~': Bulbocavernosus; Penile reflex: Male sexual behavior; Sexual dimorphism
The sexuallydimorphic spinal nucleus of the bulbocavernosus (SNB) innervates the bulbocavernosus (BC) and levator ant (LA), striated perineal muscle,;. By tr;mseeting the pudendal nerve just proximal to the BC muscle, we removed spinal input to both mu~les while leaving them mechanically intact. Such denervation of the BC/LA muscles virtually eliminated the display of reflexive penile cups. while having nc significant effect an penile erections, or flips.
Sexual dimorphism of the central nervous system provides a model for studying neural development and hormonal influences upon behavior. The spinal nucleus of the bulbocavcrnosus (SNB) and its target muscles, the bulbocavernosus (BC) and levator ant (LA), eonstitutc such a sexually dimorphic system in the rat, Moton¢urons are significantly larger and more numerous in the SNB region of males than of females, and the target muscles arc present in adult male but are either absent ' or vestigial ~ in female rats. During the last decade, research has focused on understanding the role of hormones both in the development and maintenance of this neuromuscular system. The present investigation focuses on thu behavioral function of this system. When a rat is held in a supine position with the preputial sheath retracted to expose the glans penis, genital responses occur in either intact '~ males or in animals given a midthoracic spinal transection ~, These reflexes include erections (swelling of the penis), cups (intense swelling in which the distal portion of the penis flares to form a cup-like structure), and flips (dorsal deflection of the penis), Evidence implicating BC involvement in these reflexes includes electromyo, graphic recordings of activity in the BC during erections and cups r, and elimination of cups after surgical removal of the BC ~,x Although the reflexes do not correlate directly with traditional measures of copula-
tory behavior, removal of the BC significantly reduces the male's ability to deposit tight plugs and to dislodge previously deposited copulatory plugs ta thus influencing a male's ability to successfully impregnate a female. These excision and electromyographic studies provide evidence that the BC muscle contributes to the production of penile cups. However, removal of the muscle may have affected penile activity simply by eliminating structural support at the base of the penis or inducing local inflammatory responses to a rather drastic surgery, Although electromyographic recordings provide excellent correlational data, a muscle may be active during a behavior even if it is not contributing to the behavior, To further test SNB-BC involvement in penile reflexes we wanted to remove SNB input to the BC and LA while leaving the muscle, and hence the mechanical structures supporting the penis, intact, This was accomplished by measuring penile behavior before and after transecting the pudendal nerve just proximal to the BC, Male Sprague-Dawley rats (Charles River) weighing 300-450 g were housed in groups of 3 in opaque Plexiglas cages (44 x 24 x 20 cm) with ad lib access to Purina rat chow and water, The vivarium was mait~tained at a temperature of approximately 23°C and a 12:12 light:dark cycle with lights on at 07.00 h. Behavior tests were conducted between 08.30 and 13,30 h and consisted of holding a rat in a supine
Com',~'pmuh,m'e: E.P, Monaghan, PsychobiologyDepartment, University of California, Irvine, CA 92717,4550, USA. Fax: (I) (714) 725-2447. * Present address: PsychobiologyDepartment, University of California, lrvine, CA 92717-4550, USA,
179 position in a Plexiglas cylinder for a period of 25 min. After 5 rain of adaptation in the cylinder, the rat's penile sheath was rolled back with a wooden cottontipped applicator to expose the glans. The occurrence of erections, cups, and flips and the latency to the first erection were recorded on an event recorder for a period of 20 rain. Erections were scored when the penis became blood engorged and swollen in size, a cup involved the flaring out of the tip of the phallus to a circumference equal to or greater than the base of the glans, a flip involved a dorsal deflection of 30° or more from resting position. Animals received at least two behavior tests prior to denervation and one final test 2-3 days after surgery, before any noticeable amount of muscle atrophy occurred. To cut the pudendal nerve, eight rats were deeply anesthetized with a 1.0 ml/kg i.p. injection of a ketamine mixture (6% ketamine, 0.6% rompun, 0.1% acepromizine). Under aseptic conditions bilateral incisions were made just caudal to the thigh and dorsal to the scrotum allowing access to the pudendal nerve. The nerve was cut at the level of the bulbourethral gland just before it innervates the BC and LA muscles. Pilot work using H R P retrograde tracing demonstrated that this transection eliminated SNB innervation of the BC and LA, but did not interfere with ischiocavernosus (IC) innervation from the dorsal lateral motor nucleus. Five animals which "received a similar surgical procedure, with the exception that the pudendal nerve was exposed but not cut, served as sham.operated controls. Results were analyzed using a repeated measures t-test.
As can be seen in Fig. 1, denervation of the B C / L A virtually eliminated cups [t = 3.2 (dr = 7); P < 0.02], tended to reduce the number of erections [t = 2.3 (df = 7); P > 0.05], and had no effect on flips [t = 0.2 (df = 7); P > 0.05] or latency [t = 1.2 (df); P > 0.05]. Control animals showed no change in any measure of penile reflex activity [cups: t = 1.0 (df = 4), P > 0.05; erections: t = 1.7 (df = 4), P > 0.05; flips: t = 1.7 (df = 4), P > 0.05; late:~t~y: t = 2.4 (df = 4), P > 0.05]. Denervation of the B C / L A virtually eliminated the display of cups, tended to reduce the number of erections, and had no effect on flips or latency to the first response. These results indicate that these muscles must be present and under spinal control for an animal to display the full complement of penile responses, suggesting that it supplies more than a mere mechanical support for the penis. These findings support the earlier work of Hart & Melese-d'Hospital "and Sachs ~ who extirpated the BC. Although these results are consonant with earlier work, we felt that the extirpation studies left some room for doubt. The BC completely surrounds the base of the penis, and removal could result in unintentional mechanical and neural damage. Furthermore, such a large mass could provide a mechanical support which is necessary for the display of a behavior even if contraction of the muscle played no part in the reflexes. The present findings ameliorate thc~¢ doubts, and the similar effects produced by the two procedures, i.e., the slight reduction in expression of erections and virtual elimination of cups, provides further support for the
EFFECT OF BC DENERVATION ON PENILE REFLEXES
40'
i
• ] • []
30'
Pre.dencrvation Posbdenen~alion Pre.sharn surgery Post-sheen surgery
-I0
i
~20' 5
II0'
0
~
ERECTIONS
el
FLIPS
CUPS
LATENCY
Fig. 1. Penile reflex m e a s u r e s of rats before and after denervation of the bulbocavernosus muscle (BC) or s h a m surgery. Responses arc reported as m e a n s per animal per test + S.E.M. (n -- 8 for experi:,:ental animals and n = 5 for controls).
180 conclusion that the SNB-BC system is responsible for the expression of intense cup-like erections. The slight, but not significant, reduction in erections after denerration suggests that the SNB-BC may contribute to the production of erections, but it is clearly not necessary for their display and thus autonomic inner'cation of the penis may be primarily responsible for the erectile response. The finding that none of the responses were eliminated following removal of only the LA ~ proves that this muscle is not required for the production of cups. By elimination, we conclude that our dencrvation blocked the expression of cups by causing a loss of BC function. We intended "o look at the effect of IC dcnervation on penile reflexes. From the extirpation studies, we would expect such a procedure to eliminate the expression of penile flips but not cups. However, surgical exposure of the branch of the pudendal nerve proximal to the IC proved difficult. When taking a ventral approach the nerve could not be exposed without damaging either the pubis or ischium, and a dorsal approach required extensive cutting of overlying thigh musculature. We never felt justified in allowing such rats to recover from anesthesia because the extensive surgical damage would have made it difficult to distinguish effects of the surgery from the effects of IC denervalion per s¢. With the reasonable confidence that SNB input affects penile reflexes, we can now investigatg whether changes in SNB morphology correlate with changes in behavior, Like other male copulatory behaviors, penile reflexes are hormone sensitive. Castration significantly reduces the reflexes 4, SNB cell size ~, and dendritic arbor ,t,7 and results in atrophy of the BC/LA ~:, Sub. sequent androgen troatment restores these measures to prccastration levels, Indeed, spinal implants of testosterone, at concentrations small enough not to affect growth of accessory sex glands, have been reported to
maintain penile reflexes in castrates .s. We are presently looking at the effects of physiological levels of testosterone administered either intrathecally or systemically on penile reflex behavior and SNB morphology to detect whether a difference in motoneuronal size a n d / o r dendritic arbor contributes to differences in behavior. Supported by NIH Grant NS28421. I Breedlove. S,M. and Arnold. A.P.. Sexually dimorphic motor nucleus in the rat lumbar spinal cord: response to adult hormone manipulation, absence in androgen-insensitive rats. Brain Rt,s.. 225 ( 1981 ) 297-307. 2 Breedhwe, S.M. and Arnold, A,P,, Hormone accumulation in a sexm, lly dimorphic motor smcleus of the rat spinal ¢x)rd. Sciem'e. 210 (1980) 564-566, 3 Forger. N,G, and Br¢¢dlov¢, S.M. Seamnal variation in mammalian striated muscle mass and motoncuron morphology. J. Neurobiol,. 18 (1987) 155-165. 4 Hart. B,L,, Testosterone regulation of .sexual reflexes in spinal male rats, Sciem'e. 155 (1967) 1283-1284. 5 Itqrt. B,L. and ilaugen, C.M.. Activation of sexual reflexes in male rats by spinal implantation of tesloslcrone. Pto,siol. Belmv.. 3 (1968) 735-738, 6 Ilarl, B,L, and Melesc.d'ltospital. P.Y.. Penile mechanisms and the role of the striated penile muscles in penile reflexes. Phy,~ioL Ih'hm,, 31 (1083) 807-813, 7 Kurz, E.M., Sengelaub. D,R. and Arnold, A,P,, Androgens regulate the dendritic length of mammalian motoneurons in adulthood, Soft,nee, 232 (1986) 39~ =398, Sachs, B,D,, Roh: of the penile muscles in penile reflexes, copulation, and induction of pregnancy, J. Rffwml, Furl., 65 (1982) 4,13=443. ~) Sachs, B,I), and Oarin¢llo, L.I)., Spinal pacemaker ¢onlmllinll sexual rcllex~s ill male rats, ilm#r Ne~',, 171 (l*/?q) 1~2=15(i. I(I Sachs, It,I), and (iarlnell., L,D,, I lypolh¢lieul spinal pacemaker rcttulatint.t penile r¢llexes in rats: evldene¢ I'rom Irunsection of sptntd cord trod dorsM pc,lie nerves. J. ('OOllp. Phy.~iol. I~.vt'hol.. t)4 (IqH()) ,~3ql=33~, I I Tohin. C. ~md Joul~ert. Y,, T~Jslostelxnte-inducedduwlopmunt of the ral Ievator ant muscle. I.k.v. Bkd.. 14¢)(l~gl) 131=13tl. 12 Wainman. P. and Sltioounol'f. G.C. The ul't'uctstff castration and testosterone propionat¢ on the striated perineal musculature in the rat, Eml¢a'r#u#o.~,v, 2~ (1~41) 97~-978, 13 Wullach. SJ,R. and tlart, B.L,. The role of the slriatud l~nil¢ muscles of the male rat in ~minal plug dislodgement and depo.~ilion. Phy.~'iol. I/chin',, 31 (1~8.1)815-821,
178
BRES 252~5
The role of the bulbocavernosus in penile reflex behavior in rats E d w a r d P. M o n a g h a n *, a n d S. M a r c B r e e d l o v e Dep,rtment of P~'t'holo,~,% Unh'ersio'of California. Berkeley. CA 94720 (USA) (Accepted 5 May 1992)
Key word.~': Bulbocavernosus; Penile reflex: Male sexual behavior; Sexual dimorphism
The sexuallydimorphic spinal nucleus of the bulbocavernosus (SNB) innervates the bulbocavernosus (BC) and levator ant (LA), striated perineal muscle,;. By tr;mseeting the pudendal nerve just proximal to the BC muscle, we removed spinal input to both mu~les while leaving them mechanically intact. Such denervation of the BC/LA muscles virtually eliminated the display of reflexive penile cups. while having nc significant effect an penile erections, or flips.
Sexual dimorphism of the central nervous system provides a model for studying neural development and hormonal influences upon behavior. The spinal nucleus of the bulbocavcrnosus (SNB) and its target muscles, the bulbocavernosus (BC) and levator ant (LA), eonstitutc such a sexually dimorphic system in the rat, Moton¢urons are significantly larger and more numerous in the SNB region of males than of females, and the target muscles arc present in adult male but are either absent ' or vestigial ~ in female rats. During the last decade, research has focused on understanding the role of hormones both in the development and maintenance of this neuromuscular system. The present investigation focuses on thu behavioral function of this system. When a rat is held in a supine position with the preputial sheath retracted to expose the glans penis, genital responses occur in either intact '~ males or in animals given a midthoracic spinal transection ~, These reflexes include erections (swelling of the penis), cups (intense swelling in which the distal portion of the penis flares to form a cup-like structure), and flips (dorsal deflection of the penis), Evidence implicating BC involvement in these reflexes includes electromyo, graphic recordings of activity in the BC during erections and cups r, and elimination of cups after surgical removal of the BC ~,x Although the reflexes do not correlate directly with traditional measures of copula-
tory behavior, removal of the BC significantly reduces the male's ability to deposit tight plugs and to dislodge previously deposited copulatory plugs ta thus influencing a male's ability to successfully impregnate a female. These excision and electromyographic studies provide evidence that the BC muscle contributes to the production of penile cups. However, removal of the muscle may have affected penile activity simply by eliminating structural support at the base of the penis or inducing local inflammatory responses to a rather drastic surgery, Although electromyographic recordings provide excellent correlational data, a muscle may be active during a behavior even if it is not contributing to the behavior, To further test SNB-BC involvement in penile reflexes we wanted to remove SNB input to the BC and LA while leaving the muscle, and hence the mechanical structures supporting the penis, intact, This was accomplished by measuring penile behavior before and after transecting the pudendal nerve just proximal to the BC, Male Sprague-Dawley rats (Charles River) weighing 300-450 g were housed in groups of 3 in opaque Plexiglas cages (44 x 24 x 20 cm) with ad lib access to Purina rat chow and water, The vivarium was mait~tained at a temperature of approximately 23°C and a 12:12 light:dark cycle with lights on at 07.00 h. Behavior tests were conducted between 08.30 and 13,30 h and consisted of holding a rat in a supine
Com',~'pmuh,m'e: E.P, Monaghan, PsychobiologyDepartment, University of California, Irvine, CA 92717,4550, USA. Fax: (I) (714) 725-2447. * Present address: PsychobiologyDepartment, University of California, lrvine, CA 92717-4550, USA,
179 position in a Plexiglas cylinder for a period of 25 min. After 5 rain of adaptation in the cylinder, the rat's penile sheath was rolled back with a wooden cottontipped applicator to expose the glans. The occurrence of erections, cups, and flips and the latency to the first erection were recorded on an event recorder for a period of 20 rain. Erections were scored when the penis became blood engorged and swollen in size, a cup involved the flaring out of the tip of the phallus to a circumference equal to or greater than the base of the glans, a flip involved a dorsal deflection of 30° or more from resting position. Animals received at least two behavior tests prior to denervation and one final test 2-3 days after surgery, before any noticeable amount of muscle atrophy occurred. To cut the pudendal nerve, eight rats were deeply anesthetized with a 1.0 ml/kg i.p. injection of a ketamine mixture (6% ketamine, 0.6% rompun, 0.1% acepromizine). Under aseptic conditions bilateral incisions were made just caudal to the thigh and dorsal to the scrotum allowing access to the pudendal nerve. The nerve was cut at the level of the bulbourethral gland just before it innervates the BC and LA muscles. Pilot work using H R P retrograde tracing demonstrated that this transection eliminated SNB innervation of the BC and LA, but did not interfere with ischiocavernosus (IC) innervation from the dorsal lateral motor nucleus. Five animals which "received a similar surgical procedure, with the exception that the pudendal nerve was exposed but not cut, served as sham.operated controls. Results were analyzed using a repeated measures t-test.
As can be seen in Fig. 1, denervation of the B C / L A virtually eliminated cups [t = 3.2 (dr = 7); P < 0.02], tended to reduce the number of erections [t = 2.3 (df = 7); P > 0.05], and had no effect on flips [t = 0.2 (df = 7); P > 0.05] or latency [t = 1.2 (df); P > 0.05]. Control animals showed no change in any measure of penile reflex activity [cups: t = 1.0 (df = 4), P > 0.05; erections: t = 1.7 (df = 4), P > 0.05; flips: t = 1.7 (df = 4), P > 0.05; late:~t~y: t = 2.4 (df = 4), P > 0.05]. Denervation of the B C / L A virtually eliminated the display of cups, tended to reduce the number of erections, and had no effect on flips or latency to the first response. These results indicate that these muscles must be present and under spinal control for an animal to display the full complement of penile responses, suggesting that it supplies more than a mere mechanical support for the penis. These findings support the earlier work of Hart & Melese-d'Hospital "and Sachs ~ who extirpated the BC. Although these results are consonant with earlier work, we felt that the extirpation studies left some room for doubt. The BC completely surrounds the base of the penis, and removal could result in unintentional mechanical and neural damage. Furthermore, such a large mass could provide a mechanical support which is necessary for the display of a behavior even if contraction of the muscle played no part in the reflexes. The present findings ameliorate thc~¢ doubts, and the similar effects produced by the two procedures, i.e., the slight reduction in expression of erections and virtual elimination of cups, provides further support for the
EFFECT OF BC DENERVATION ON PENILE REFLEXES
40'
i
• ] • []
30'
Pre.dencrvation Posbdenen~alion Pre.sharn surgery Post-sheen surgery
-I0
i
~20' 5
II0'
0
~
ERECTIONS
el
FLIPS
CUPS
LATENCY
Fig. 1. Penile reflex m e a s u r e s of rats before and after denervation of the bulbocavernosus muscle (BC) or s h a m surgery. Responses arc reported as m e a n s per animal per test + S.E.M. (n -- 8 for experi:,:ental animals and n = 5 for controls).
180 conclusion that the SNB-BC system is responsible for the expression of intense cup-like erections. The slight, but not significant, reduction in erections after denerration suggests that the SNB-BC may contribute to the production of erections, but it is clearly not necessary for their display and thus autonomic inner'cation of the penis may be primarily responsible for the erectile response. The finding that none of the responses were eliminated following removal of only the LA ~ proves that this muscle is not required for the production of cups. By elimination, we conclude that our dencrvation blocked the expression of cups by causing a loss of BC function. We intended "o look at the effect of IC dcnervation on penile reflexes. From the extirpation studies, we would expect such a procedure to eliminate the expression of penile flips but not cups. However, surgical exposure of the branch of the pudendal nerve proximal to the IC proved difficult. When taking a ventral approach the nerve could not be exposed without damaging either the pubis or ischium, and a dorsal approach required extensive cutting of overlying thigh musculature. We never felt justified in allowing such rats to recover from anesthesia because the extensive surgical damage would have made it difficult to distinguish effects of the surgery from the effects of IC denervalion per s¢. With the reasonable confidence that SNB input affects penile reflexes, we can now investigatg whether changes in SNB morphology correlate with changes in behavior, Like other male copulatory behaviors, penile reflexes are hormone sensitive. Castration significantly reduces the reflexes 4, SNB cell size ~, and dendritic arbor ,t,7 and results in atrophy of the BC/LA ~:, Sub. sequent androgen troatment restores these measures to prccastration levels, Indeed, spinal implants of testosterone, at concentrations small enough not to affect growth of accessory sex glands, have been reported to
maintain penile reflexes in castrates .s. We are presently looking at the effects of physiological levels of testosterone administered either intrathecally or systemically on penile reflex behavior and SNB morphology to detect whether a difference in motoneuronal size a n d / o r dendritic arbor contributes to differences in behavior. Supported by NIH Grant NS28421. I Breedlove. S,M. and Arnold. A.P.. Sexually dimorphic motor nucleus in the rat lumbar spinal cord: response to adult hormone manipulation, absence in androgen-insensitive rats. Brain Rt,s.. 225 ( 1981 ) 297-307. 2 Breedhwe, S.M. and Arnold, A,P,, Hormone accumulation in a sexm, lly dimorphic motor smcleus of the rat spinal ¢x)rd. Sciem'e. 210 (1980) 564-566, 3 Forger. N,G, and Br¢¢dlov¢, S.M. Seamnal variation in mammalian striated muscle mass and motoncuron morphology. J. Neurobiol,. 18 (1987) 155-165. 4 Hart. B,L,, Testosterone regulation of .sexual reflexes in spinal male rats, Sciem'e. 155 (1967) 1283-1284. 5 Itqrt. B,L. and ilaugen, C.M.. Activation of sexual reflexes in male rats by spinal implantation of tesloslcrone. Pto,siol. Belmv.. 3 (1968) 735-738, 6 Ilarl, B,L, and Melesc.d'ltospital. P.Y.. Penile mechanisms and the role of the striated penile muscles in penile reflexes. Phy,~ioL Ih'hm,, 31 (1083) 807-813, 7 Kurz, E.M., Sengelaub. D,R. and Arnold, A,P,, Androgens regulate the dendritic length of mammalian motoneurons in adulthood, Soft,nee, 232 (1986) 39~ =398, Sachs, B,D,, Roh: of the penile muscles in penile reflexes, copulation, and induction of pregnancy, J. Rffwml, Furl., 65 (1982) 4,13=443. ~) Sachs, B,I), and Oarin¢llo, L.I)., Spinal pacemaker ¢onlmllinll sexual rcllex~s ill male rats, ilm#r Ne~',, 171 (l*/?q) 1~2=15(i. I(I Sachs, It,I), and (iarlnell., L,D,, I lypolh¢lieul spinal pacemaker rcttulatint.t penile r¢llexes in rats: evldene¢ I'rom Irunsection of sptntd cord trod dorsM pc,lie nerves. J. ('OOllp. Phy.~iol. I~.vt'hol.. t)4 (IqH()) ,~3ql=33~, I I Tohin. C. ~md Joul~ert. Y,, T~Jslostelxnte-inducedduwlopmunt of the ral Ievator ant muscle. I.k.v. Bkd.. 14¢)(l~gl) 131=13tl. 12 Wainman. P. and Sltioounol'f. G.C. The ul't'uctstff castration and testosterone propionat¢ on the striated perineal musculature in the rat, Eml¢a'r#u#o.~,v, 2~ (1~41) 97~-978, 13 Wullach. SJ,R. and tlart, B.L,. The role of the slriatud l~nil¢ muscles of the male rat in ~minal plug dislodgement and depo.~ilion. Phy.~'iol. I/chin',, 31 (1~8.1)815-821,
