BAB receptors by baclofen appears to have a specific inhibitory effect on sexual behavior, primarily af- fecting precopulatory behaviors (Paredes & /kgmo,. 1989) ...
BEHAVIORALAND NEURALBIOLOGY60, 118--122 (1993)
Electromyographic Activity of Rat Ischiocavernosus Muscles during Copulation after Treatment with a GABA-Transaminase Inhibitor RAf~L G. PAREDES,* GREGORY M. HOLMES,t BENJAMIN D.
SACHS,tAND ANDERS /~GMO*'1
*Escuela de Psicologia, Universidad Andhuac, Mdxico D.F. 11000; and tDepartment of Psychology, University of Connecticut, Storrs, Connecticut 06268
proate (VAL) and T-acetylen-GABA, consistently reduce the intromission ratio, i.e., the proportion of mounts that result in intromissions. Mounting behavior is not inhibited by GABA-TIs, suggesting t h a t the sexual potency is reduced while sexual motivation is not affected. Furthermore, the dose of the GABA-TIs required to obtain an effect on intromission ratio is within the range that produces motor incoordination, as evaluated by a treadmill test (/kgmo et al., 1987). It was proposed t h a t elevated brain GABA levels indirectly inhibit sexual behavior, the primary effect being on motor execution. In an effort to analyze the mechanism that might be responsible for the effects of GABA-TIs on sexual behavior, copulatory thrusting patterns were recorded with the accelerometric technique (/~gmo & Contreras, 1990). It was assumed that inadequate pelvic thrusting could account for the deficiencies in intromissive behavior. However, when a drugtreated animal displayed a mount with pelvic thrusting, the pattern was well coordinated (Agmo & Contreras, 1990). It appears, then, that alterations in copulatory thrusting patterns are not the cause of the reduction of intromissive behavior seen after administration of GABA-TIs. It was therefore suggested that some effects on penile activities could be responsible for this effect. The ischiocavernosus (IC) muscles of rats promote penile insertion by directing the penile body anteriorly and increasing its rigidity. This straightening aligns the penis, which is normally directed posteriorly, with the vagina. The bulbospongiosus (BS) muscles pump blood from the penile bulb, thereby augmenting the engorgement of the corpus spongiosum and consequently of the glans penis (for a discussion, see Sachs & Meisel, 1988). Electromyographic (EMG) analysis of these muscles during
The administration of GABA--transaminase inhibitors (GABA-TIs) to male rats reduces the proportion of mounts that result in intromissions. Copulatory pelvic thrusting remains normal, despite the fact that animals treated with GABA-TIs show gross deficiencies in other motor acts. In order to determine whether altered sexual behavior produced by GABA-TI could be due to deficiencies in activity of striated penile muscles, we recorded the electromyographic (EMG) activity of the ischiocavernosus (IC) muscle during copulation in male rats treated with sodium valproate. The duration of IC EMG bursts was reduced by sodium valproate in separate tests that allowed or prevented intromission. There was no effect on EMG amplitude or frequency. It is suggested that insufficient activity of the IC muscles reduces the likelihood of vaginal penetration. The actions of GABA may be localized to hypothalamic or brain stem nuclei with GABAergic projections to the spinal motoneurons controlling the IC muscles, or GABA may act directly on these neurons. © 1993 Academic Press, Inc. The systemic administration of GABA receptor agonists produces a strong inhibition of sexual behavior in male rats (/~gmo & Paredes, 1985;/~gmo, Fernandez, & Paredes, 1987) and rabbits (/~gmo, Contreras, & Paredes, 1991). The stimulation of GABAB receptors by baclofen appears to have a specific inhibitory effect on sexual behavior, primarily affecting precopulatory behaviors (Paredes & /kgmo, 1989). No functional deficits in erectile or ejaculatory mechanisms are produced by baclofen (McQuade and Sachs, 1989). In contrast, the GABAtransaminase inhibitors (GABA-TIs), sodium val1 This project was supported by the National Council for Science and Technology (CONACYT, Project 890219-SA-7) and by research grants to B.D.S from NICHD (HDO8933) and the University of Connecticut Research Foundation. Address correspondence and reprint requests to Ra~l G. Paredes, Escuela de Psicologla, Universidad An~ihuac, Apdo. Postal 10-844, M~xico D.F. 11000. Fax: (525)589 9796. 118
0163-1047/93 $5.00 Copyright © 1993 by Academic Press, Inc. All rights of reproduction in any form reserved.
GABA AND EMG IN PENILE MUSCLES copulation has demonstrated that they are activated differently. Prior to the deep intromissive thrust, there is relatively high activity in the IC and relatively low activity in the proximal BS. Intromission with or without ejaculation is characterized by a transition to high activity in the proximal BS (Holmes, Chapple, Leipheimer, & Sachs, 1991). These observations clearly indicate that activity in the IC is associated with events preceding intromission, whereas BS activity is mainly postinsertive. Thus, a reduced intromission ratio would be expected to be more closely related to disturbance of activity in the IC than in the BS muscles. In the present study, the EMG activity of the IC muscles was recorded during sexual behavior in male rats treated with VAL in order to evaluate if the inhibitory effects of this drug upon sexual behavior is related to alterations of IC muscle activity. To further analyze the effects of the drug on EMG activity, the same animals were observed with a receptive female whose vagina was covered by masking tape. If VAL-induced changes in EMG activity during copulation were due to reduced sensory feedback, then no differences should be seen in subjects paired with a female with occluded vagina. METHOD
Subjects Sexually experienced male rats ( H a r l a n - B l u e Spruce hooded, N = 11) were housed singly with free access to commercial pelleted rodent chow and tap water. They were maintained on a LD 12:12 cycle, with lights o f f a t 1100. Stimulus females were brought into estrus by injection of estradiol benzoate (50/xg/female) 48 h before testing and progesterone (300 /xg/female) 4 h before testing.
Surgery Under deep sodium pentobarbital anesthesia (55 m g / k g intraperitoneally, ip), the animals were implanted in the IC muscles with a bipolar hook electrode (detailed description can be found in Holmes et al., 1991). Briefly, a midline incision was made posterior to the preputial sheath exposing the IC muscles. Through a midline incision made along the back of the skull, PE-90 tubing attached to a thin metal hook was passed until it was just rostral to the IC muscles. The electrodes were bilaterally implanted in the muscle with the aid of a 20-gauge hypodermic needle, and the free ends of the wire were passed through the tubing and attached to a female amphenol (220-SO2) connector. The con-
119
hectors were snapped into a plastic Amphenol conector strip, and the assembly was placed between two screws in the skull, one of which served as a ground. The assembly was fixed to the skull with dental acrylic. All wounds were sutured closed and swabbed with antibiotic ointment. Males were given a l-week recovery period, which also allowed the electrodes to stabilize within the muscle.
Drugs The GABA-transaminase inhibitor sodium valproate (VAL; Ciba-Geigy Mexicana) was dissolved in physiological saline and injected ip 15 min before behavioral observation. A dose of 200 m g / k g was administered in a volume of 5 ml/kg. This dose was used since it reliably reduces the intromission ratio (/~gmo et al., 1987; ~ g m o & Contreras, 1990). Vehicle and drug injections were counterbalanced.
Apparatus During copulation, the male's upper torso was fitted with a cloth harness (Stoelting 50556, Chicago, IL) and a 4-mm diameter tether was attached to the harness. The animal's headpiece was connected to a commutator (Stoelting 50506) mounted above the testing arena by means of a cable constructed of double-strand, shielded wire (Belden Corp. 8429, Chicago IL) with a matching plug consisting of gold-plated, subminiature male connectors (Amphenol 220-PO2). The commutator, in turn, was connected to a Grass Model 7D polygraph by a similar cable. The raw signal was low-pass, half-amplitude, filtered at 75 Hz, and low-frequency motion artifact was removed using a high-pass, fourth-order Bessel filter with a 30-Hz cutoff. The data were digitized at 500 Hz through a Dash-16 analog/digital board (Keithley Metrabyte, Taunton, MA) in an IBM-AT computer.
Testing Procedure The test animal was connected to the polygraph and placed in a test chamber, into which a receptive female was placed 15 min later. Mount, intromission, and ejaculation latency (each timed from introduction of female) were recorded, as were the number of mounts with and without intromission. The intromission ratio was calculated by dividing the number of intromissions by the number of mounts with and without intromission. The test was ended after ejaculation or if no sexual behavior was displayed within 30 min. In a second series of tests a few days later, masking tape was applied to cover the anogenital region of stimulus females before
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P A R E D E S E T AL.
TABLE 1 S e x u a l B e h a v i o r in Male Rats T r e a t e d with S o d i u m V a l p r o a t e , 200 m g / k g , a n d T e s t e d w i t h an U n t a p e d F e m a l e (N -- 11) Vehicle N u m b e r of m o u n t s N u m b e r of i n t r o m i s s i o n s Intromission ratio I n t r o m i s s i o n latency" E j a c u l a t i o n latency"
4.55 8.45 76.95 5.51 5.33
± ± ± ± ±
VAL
2.44 1.67 5.13 1.38 2.05
i0.00 6.72 45.74 9.47 4.70
± -+ ± _-: ±
2.17"* 0.33 4.61"* 2.47 0.72
Note. V a l u e s a r e m e a n s _+ SEM. a In min. ** D i f f e r e n t f r o m vehicle, p < .01.
each test. Seven of the previously used males were allowed to make five mounts before the female was removed from the chamber. The remaining four males, whose electrodes had become unreliable, were excluded from this phase of the study.
Data Analysis The following EMG variables were analyzed by computer (Asystant +, Keithley Asyst, Taunton, MA, and "in-house" software): peak amplitude, burst duration (measured as 25 x the number of 40 ms bins with activity above resting baseline), frequency (spikes/second), and average mean amplitude of the burst. For each animal in each condition, three mounts without intromission were selected for analysis on the basis of signal clarity. The means of the means were compared by paired t tests, using SYSTAT (Mcmillan Software, New York, NY). The EMG data from one animal in the first experiment could not be used because unreliable recordings. Only nonintromissive mounts were analyzed because the GABA-TIs reliably reduces intromission
ratio. As mentioned above, this effect m a y be attributed to difficulties in achieving penile insertion. Such a difficulty would be most evident when a mount ends without intromission. All sex behavior data were analyzed by paired t tests. All probabilities are two-tailed. RESULTS All subjects achieved ejaculation both after treatment with saline and VAL. As can be seen in Table 1, VAL significantly increased the number of mounts preceding ejaculation (tl0 = 3.70, p = .004) and significantly reduced the intromission ratio (tl0 = 6.04, p < .001). In tests with a female with closed vagina, all subjects completed five mounts. The duration of EMG bursting associated with mounts was significantly reduced after VAL, both in tests with an untaped female (t9 = 3.437, p = .007) and when the animals were tested with a taped female (t6 = 2.654, p = .038). No effect on EMG amplitude or spike frequency was observed (amplitude: untaped female, t9 = .619, NS; taped female, t6 = 2.166, NS. Frequency: untaped female t9 = .959, NS: taped female, t6 = 2.006, NS). Data are shown in Table 2. DISCUSSION In this experiment the burst duration of the IC muscles was reduced after VAL in animals observed with either an untaped or a taped female. In this context it is important to know that even during mounts without intromission the penis makes occasional contacts with the vaginal orifice (Morali & Beyer, 1992). The data obtained in the tests with taped females imply that the sensory stimulation
TABLE 2 E l e c t r o m y o g r a p h i c Activity o f the I s c h i o c a v e r n o s u s M u s c l e s in A n i m a l s T r e a t e d w i t h S o d i u m V a l p r o a t e , 200 m g / k g , (N = 10) Untaped female
Duration
Taped female
Vehicle
VAL
Vehicle
VAL
613.8 -+ 52.5
422.3 -+ 39.35**
477.3 -+ 70.0
396.3 -+ 61.75"
15 +- 2
13 -+ 2
25 ± 2
22 ± 2
(ms) Amplitude
(mV) Frequency
243.2 +- 18.3
372.4 -+ 31.3
(Hz) Note. V a l u e s a r e m e a n s -+ S E M ( u n t a p e d f e m a l e , N = 10; t a p e d f e m a l e , N = 7). * D i f f e r e n t f r o m vehicle, p < .05; **p < .01.
257.3 -+ 23.0
220.3 -+ 21.5
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GABA AND EMG IN PENILE MUSCLES
provided by contacts between penis and vagina are not important for the actions of VAL. Thus, it could be proposed that the effects of the GABA-TI are not due to a deficit in peripheral sensory input. The pudendal nerve, which innervates the striated perineal muscles and carries afferents from the penis, has its motoneurons located in the ventral L5 and L6 segments and its sensory neurons in the dorsal L5-S1 segments (McKenna & Nadelhaft, 1986). It has been shown that a substantial part of the descending projections in the rat from the ventromedial lower brain stem to lumbar motoneural cell groups contain GABA as a transmitter (Holstege, 1991). It is possible, then, that some of the neurons involved in the regulation of penile actions are under inhibitory GABAergic influence. In fact, it has been shown that injections of baclofen, a GABAB receptor agonist, into the subarachnoid space of the lumbosacral spinal cord (L5-S1) inhibits penile reflexes (Bitran, Miller, McQuade, Leipheimer, & Sachs, 1989). In contrast, injection of baclofen at thoracic segments (T8-T10) does not affect these reflexes. The GABAA agonist THIP had only a weak effect on penile reflexes at very high doses. These results were interpreted as indicating that stimulation of GABAB receptors in the lumbosacral spinal cord inhibits erectile mechanisms ex copula. Hypothalamic centers can also influence the function of penile muscles. The paraventricular and lateral hypothalamic nuclei, as well as the dorsal area of the hypothalamus, project to lower lumbar regions of the spinal cord, specifically to the L5-L6 segments (Wagner & Clemens, 1991). Two motor nuclei in this region, the spinal nucleus of the bulbospongiosus and the dorsolateral nucleus, innervate the BS and the IC, respectively (McKenna & Nadelhaft, 1986; Schroder, 1980). The effects of systemically administered GABATIs on penile EMG activity may therefore be localized within the spinal motoneurons at the L5L6 segments, in the brain stem (ventral part of the medial reticular formation, between the caudal part of the facial nucleus and the rostral half of the inferior olive) from where GABAergic neurons project (Holstege, 1991), or in the hypothalamus. Present data suggest that GABAergic systems in the brain and/or the spinal cord participate in the control of penile muscle activity. Because VAL treatment significantly reduced the duration of EMG bursts in the IC muscles without reliable effects on burst amplitude and frequency, we infer that GABA does not control the intensity of muscular activity, but rather participates in the timing
of that activity. We cannot say whether VAL acted to delay the onset of IC contraction, to hasten the end of contraction, or both. Furthermore, although the IC muscle is critical for intromission (Sachs & Meisel, 1988), we cannot be sure that the shortened duration of IC activity is reliably related to the reduced likelihood of gaining insertion. Indeed, Holmes et al. (1991) did not observe a reliable difference in the duration of IC activity between mounts with and without intromission. They did report, however, that failures of intromission were sometimes associated with EMG activity in the IC starting together with, rather than before, activity in the BS muscles. It remains to be determined whether the reduced intromission ratio of VALtreated males is due only to the effects on burst duration in the IC muscles or whether this behavioral effect may be symptomatic of other problems of penile muscle coordination. REFERENCES ~,gmo, A., & Contreras, J. L. (1990). Copulatory thrusting pattern in the male rat after acute treatment with GABA transaminase inhibitors. Physiology and Behavior, 47, 311-314. /~gmo, A., & Paredes, R. (1985). GABAergic drugs and sexual behavior in the male rat. European Journal of Pharmacology, 112, 371-378. /kgmo, A., Contreras, J. L., & Paredes, R. (1991). Sexual behavior and thrusting patterns in male rabbits treated with GABA transaminase inhibitors. Physiology and Behavior, 49, 7378. /~gmo, A., Fernandez, H., & Paredes, R. (1987). Differential effects of GABA transaminase inhibitors on sexual behavior, locomotor activity, and motor execution in the male rat. Pharmacology Biochemistry and Behavior, 29, 47-52. Bitran, A. P., Miller, S. A., McQuade, D. B., Leipheimer, R. E., & Sachs, B. D. (1989). Inhibition of sexual reflexes by lumbosacral injection of a GABAs agonist in the male rat. Pharmacology Biochemistry and Behavior, 31, 657-666. Holmes, G. M., Chapple, W. D., Leipheimer, R. L., & Sachs, B. D. (1991). Electromyographic analysis of male rat perineal muscles during copulation and reflexive erections. Physiology and Behavior, 49, 1235-1246. Holstege, J. C. (1991). Ultrastructural evidence for GABAergic brain stem projections to spinal motoneurons in the rat. Journal of Neuroscience, 11, 159-167. McKenna, K. E., & Nadelhaft, I. (1986). The organization of the pudendal nerve in the male and female rat. Journal of Comparative Neurology, 248, 532-549. McQuade, D., & Sachs, B. D. (1989). Context determines effects of a GABAs agonist on erection, copulatory behavior and fertility in male rats. 21st Conference on Reproductive Behavior, Saratoga Springs, NY. MoraH, G. & Beyer, C. (1992). Motor aspects of masculine sexual behavior in rats and rabbits. Advances in the Study of Behavior, 21, 201-238.
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Paredes, R., &/~gmo, A. (1989). Stereospecific actions of baclofen on sociosexual behavior, locomotor activity and motor execution. Psychopharmacology, 97, 358-364.
Schroder, H. D. (1980). Organization of the motoneurons innervating the pelvic muscles of the male rat. Journal of Comparative Neurology, 192, 567-587.
Sachs, B. D., & Meisel, R. L. (1988). The physiology of male sexual behavior. In E. Knobil & J. Neill (Eds.), The physiology of reproduction (pp. 1393-1485). New York: Raven Press.
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