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Brain Research, 512 (1990) 1-6 Elsevier BRES 15281 1 Research Reports Dopamine receptors in the ventral tegmental area modulate male sexual behavior in rats Elaine M. Hull, Terence J. Bazzett, Robert K. Warner, Robert C. Eaton and James T. Thompson Department of Psychology, State University of New York at Buffalo, Amherst, N Y 14260 (U.S.A.) (Accepted 8 August 1989) Key words: Sexual behavior; Dopamine; Apomorphine; c/s-Flupenthixol; Ventral tegmental area; Motivation; Depolarization block The mesocorticolimbic dopamine tract is considered to be a substrate for motivation and reward as well as for locomotor behavior. The present experiments assessed the role of dopamine cell bodies in the ventral tegmental area (VTA), the source of this tract, in the copulatory behavior of male rats. The dopamine agonist apomorphine or the dopamine antagonist c/s-flupenthixol were microinjected into the VTA immediately before sexual behavior tests with a receptive female. Apomorphine delayed the onset of copulation and slowed its rate, presumably by stimulating somatodendritic autoreceptors and thereby decreasing the firing rate of VTA neurons. Control injections of apomorphine into the substantia nigra were without effect, c/s-Flupenthixol, which would have blocked autoreceptors and thereby depolarized VTA neurons, shortened the latency to begin copulating in those animals that did copulate; however, fewer animals exhibited sexual behavior. One possible explanation for the apparently contradictory effects of c/s-flupenthixol may be that VTA neurons increased their rate of firing in some animals, leading to a faster onset of copulation, but that in other animals depolarization block in a substantial number of neurons resulted in a lack of copulation. These results are consistent with a contribution of the mesocorticolimbic dopamine tract to motivational and/or motor aspects of male copulatory behavior. INTRODUCTION T h e ventral tegmental a r e a ( V T A ) , the source of d o p a m i n e r g i c projections to various limbic and cortical structures, is thought to contribute to l o c o m o t o r and motivational aspects of behavior 5'14'37. Studies manipulating dopamine ( D A ) and/or opiate receptors in the V T A or the nucleus accumbens, one of its terminal fields, have suggested that this pathway may enhance feeding 9'16'27, locomotor activity1°,18,21,24, drug self administration 4,32 and electrical self stimulation of the brain 6'23'26'3°,33. The possible role of this system in sexual motivation has not been adequately assessed. Microinjection of the mu opiate agonist morphine 2, the D A agonist apomorphine 2° or the indirect catecholamine agonist amphetamine 12 into the nucleus accumbens resulted in dose related decreases in the latency to begin copulating; however, the apomorphine effect was of borderline statistical significance (P = 0.058). These findings suggest that sexual motivation, measured as the inverse of the latency to begin copulating, may be enhanced by D A and/or opiate activity in this terminal field of the mesocorticolimbic D A system. The present experiments investigated whether manipulation of impulse regulating D A autoreceptors, located on cell bodies and dendrites in the VTA, would affect copulation of male rats. D A agonists injected systemically or applied iontophoretically have been reported to decrease the firing rate of D A neurons in the V T A 1'36. D A antagonists, on the other hand, have generally increased the firing rates of these cells8'36. These results are assumed to result from stimulation or blocking, respectively, of D A autoreceptors in the VTA. We predicted that stimulation of these autoreceptors with apomorphine microinjections would increase the latency to begin copulating, by decreasing the firing rate of these neurons and therefore decreasing the release of D A in limbic terminal fields. O n the other hand, microinjections of the D A antagonist c/s-flupenthixol into the V T A were expected to decrease the latency to copulate. Control injections into the substantia nigra (SN) were expected to be ineffective. MATERIALS AND METHODS Animals Adult male Long-Evans rats were housed individually in large plastic cages with food and water available ad libitum. They were tested for sexual behavior 3 times before surgery; only those that copulated consistently were chosen. Ovariectomized females of the same strain were used as stimulus animals and were housed in a Correspondence: E.M. Hull, Department of Psychology, Park Hall, State University of New York at Buffalo, Amherst, NY 14260, U.S.A. separate room. Females were brought into behavioral estrus with a single s.c. injection of estradiol benzoate (20 ktg in oil) 48 h before behavioral testing. Surgery and cannulae The male rats were anesthetized with ketamine hydrochloride (50 mg/kg) and xylazine hydrochloride (4 mg/kg) i.m., and received bilateral stainless steel guide cannulae ending 1 mm above the VTA (AP: - 3.0 from bregma, ML: + 1.2, DV: - 8.2, incisor bar: + 5)2'~ or the SN (AP: - 3.0, ML: + 2.7, DV: 7.8, incisor bar: + 5) 2~. The guide cannulae were constructed of 23 gauge thin wall stainless steel tubing (o.d. = 0.6 mm; i.d. = 0.4 mm). An obturator constructed from 27 gauge stainless steel tubing (o.d. = 0.4 mm; i.d. = 0.2 mm) was cut flush with the end of the guide cannula and remained in the guide cannula except during microinjections. Injection cannulae were also constructed from 27 gauge tubing and extended 1 mm below the end of the guide cannula. Details of surgery and cannula construction are described in Hull et al. z~. Drugs The DA agonist apomorphine (Sigma Chemical) was dissolved in 0.5 #1 of 0.2% ascorbate. The DA antagonist cis-flupenthixol was a gift of H. Lundbeck A/S, and was dissolved in 1/zl sterile water. Procedures Two weeks following surgery, males were given a single postoperative baseline test for sexual behavior. Thereafter, behavioral tests were given at 1 week intervals. All animals received all doses in counterbalanced order. Drugs were administered using a Harvard infusion pump while the animal moved freely in his cage. The rate of injection was 1.0 #l/min, with the injection cannula being left in place for 30 s after the injection was completed. The male was then carried in his home cage to an adjacent testing room, where a stimulus female was introduced into his cage. Testing began immediately. Each test lasted for 30 min following the first intromission, or for a total of 30 min if the male failed to intromit. The occurrence and time of each mount, intromission, and ejaculation were recorded by a program written for the IBM XT microcomputer19. Measures derived from the data were: number of ejaculations per test, numbers of mounts and of intromissions preceding each ejaculation, latencies to the first mount and the first intromission, ejaculation latency (time from the first intromission of an ejaculatory series to the subsequent ejaculation), postejaculatory refractory period (time from the ejaculation to the next intromission), interintromission interval (the average time between intromissions), and intromission ratio (the number of intromissions divided by the number of mounts plus intromissions). Intromissions were distinguished behaviorally from mounts without intromission by the presence of a deep thrust followed by a rapid, springing dismount. Ejaculation patterns were characterized by longer, deeper thrusts, slow dismounts and a prolonged period of rest following ejaculation. Statistical analysis Data were analyzed by one way repeated measures analyses of variance, followed by Newman-Keuls comparisons among groups. Ordinal data were analyzed by Cochran's Q-tests, followed by paired comparisons using McNemar's test. All data are presented as means +_ S.E.M, ~" LU o~ 160+ T 140- >- o 120- ~- lO0- Z l.d ._J z / / / / / i / 80- i////// ///i/i/ 0 60- / i / 1 / 1 1 lll///, 0 ,,y ~Z 40- i 20. 0 VEH After all behavioral tests were completed, males were anesthetized and sacrificed by decapitation. Coronal sections of the brain were cut at 40 k~m, stained with cresylviolet, and examined with a projection magnifier. Data from animals with one or both cannulae located more than 0.5 mm from the VTA or SN were excluded from statistical analysis. Experiments In Expt. 1 apomorphine was microinjected bilaterally into the VTA of 18 animals with accurate cannula placements. Doses were 0.5 2.0 APOMORPHINE (p~g) Fig. 1. The effects of microinjections of apomorphine into the VTA on the latency (in sec) to the first intromission in sexual behavior tests. Values are means + S.E.M. The 2 #g dose significantly delayed the first intromission, compared to vehicle (*P < 0.05). 0 (vehicle), 0.1, 0.5 or 2.0/xg per cannula. In Expt. 2 apomorphine was microinjected bilaterally into the SN of 20 animals with accurate cannula placements. Doses were the same as in Expt. 1. In Expt. 3 cis-flupenthixol was microinjected bilaterally into the VTA of 14 animals with accurate placements. Doses were 0, 2, 5, and 10 #g per cannula. In Expt. 4 c/s-flupenthixol was microinjected bilaterally into the VTA of 14 animals with accurate placements. Doses were 0.0, 0.02, 0.2, and 2.0 #g per cannula. RESULTS Expt. 1: effects of apomorphine in the VTA As predicted, apomorphine microinjected into the VTA increased the latency to begin copulating (F3,sl = 3.14, P < 0.05; see Fig. 1). Apomorphine also reduced the number of ejaculations (F3,sl = 4.75, P < 0.01; see Fig. 2). This reduction reflected a slowed rate of t/3 Z O t-- 4 Ti ! 5 O < //////~ /////// laA laO L/L/L4 Y////// I.d CI:I Z Histology 0.1 ~/////,, O VEH 0.1 APOMORPHiNE Y////X 0.5 2.0 (~g) Fig. 2. The effects of microinjections of apomorphine into the VTA on the number of ejaculations during sexual behavior tests, which lasted for 30 min from the first intromission (30 min total, if no intromission occurred). The two highest doses (0.5 and 2.0 #g) significantly decreased the number of ejaculations, compared to vehicle (**P < 0.01). 4-- 11141 o lad '" 60. U3 Z 0 I--. .< --I 40. ---j W h 0 Z 7 Q 50. i 20. I,I DD o z . . . . . . . Z 10. I,l,I [-- VEH VEH O. 1 0.5 2.0 APOMORPHINE (p.g) I,Ll >- 4 0 0 0 Z W 300 ~ 200 ,,~ 1000 VEH 0.1 0.5 5.0 1 0.0 ClS-FLUPENTHIXOL (~zg) Fig. 4. The effects of microinjections of high doses of c/sflupenthixol into the VTA on the number of ejaculations during sexual behavior tests, which lasted 30 min from the first intromission (30 min total, if no intromission occurred). The highest dose (10/~g) significantly decreased the number of ejaculations, compared to vehicle (**P < 0.01). This decrease reflected a decrease in the number of animals copulating (see text). "-(.3 5 0 0 ~ 2.0 2.0 APOMORPHINE (p,g) Fig. 3. Top: the effects of microinjections of apomorphine on the mean interval (in sec, + S.E.) between intromissions in sexual behavior tests. The two highest doses (0.5 and 2.0/~g) significantly slowed the rate of intromitting, compared to vehicle (**P < 0.01). Bottom: the effects of microinjections of apomorphine into the VTA on the mean latency (in sec, + S.E.) from the first intromission to the first ejaculation. The two highest doses (0.5 and 2.0 /~g) significantly slowed the occurrence of the first ejaculation, compared to vehicle (**P < 0.01). copulation, rather than a decrease in the number of animals copulating. Specifically, the interval between intromissions was lengthened (F3,sl = 10.83, P < 0.001; see Fig. 3 top), resulting in an increased ejaculation latency (F3,51 = 6.22, P < 0.005; see Fig. 3 bottom). Measures that were not affected included intromission ratio, number of intromissions preceding ejaculation, postejaculatory latency to resume copulation, and number of animals copulating. the number of ejaculations during the test period (F3,39 = 6.61; P < 0.001; see Fig. 4). This decrease reflected a reduction in the number of animals copulating (Vehicle:14/14; 2 ag:14/14; 5/~g:12/14; 10/~g:8/14; Q(3) = 13.09; P < 0.005), since there was no drug related decline among animals that copulated consistently. There was no gross impairment of motor behavior by the doses used in this experiment; however, animals that did not copulate appeared generally less active. A higher dose (20/zg) in a pilot study did result in marked sedation. No other measures were significantly affected by cis-flupenthixol, although there was a nonsignificant trend towards decreased intromission latency among those animals that did copulate (0.05 < P < 0.1). This trend provided the rationale for testing lower doses in Expt. 4. ~" 600- >.. 500- IaJ 0 Z w p- 400- "5 z 0 bq o') 0 ,'," I-. No measure was affected by any dose of apomorphine in the SN. Expt. 3: effects of high doses of cis-flupenthixol in the VTA ct~-Flupenthixol produced a dose related decrease in "//N//~ //.'v//A "//,'Z/A :300200100- Z Expt. 2: effects of apomorphine in the SN i ;,//y//.~ 0 /// //i /// ii/ I/¢ /// //'/ /// i// VEH .02 .20 2.0 CIS-FLUPENTHIXOL (/zg) Fig. 5. Effects of microinjections of low doses of c/s-flupenthixol into the VTA on the mean latency (in sec, + S.E.) to the first intromission in sexual behavior tests. The highest dose (2 /~g) hastened the occurrence of the first intromission, compared to vehicle (*P < 0.05). Expt. 4: effects of low doses of cis-flupenthixol in the VTA The 2/*g dose of cis-flupenthixol decreased the number of animals copulating (Vehicle: 13/14; 0.02/~g: 14/14; 0.2 ~g:13/14; 2/~g:9/14; Q(3) = 8.6, P < 0.05). However, the decrease in number of animals copulating was offset by a nonsignificant increase in number of ejaculations in those animals that did copulate, resulting in no overall change in the number of ejaculations of the entire group. The 2 ~g dose also decreased the latency to intromit of those animals that did copulate (F3,36 = 3.02; P < 0.05; see Fig. 5). No other measures were affected. (The reason for the ineffectiveness of the 2/,g dose in Expt. 3 is not known. Failure to copulate after cis-flupenthixol did not appear to be correlated with cannula placement or with temporal order of testing.) DISCUSSION Stimulation of impulse regulating somatodendritic autoreceptors in the VTA with apomorphine increased the latency to begin copulating, suggesting that sexual motivation may have been impaired. This effect is consistent with the proposal that activation of the mesocorticolimbic D A tract enhances motivation and/or reward. However, other rate measures were lengthened as well, implying that motor behavior may have been generally slowed, although the animals did not appear to be sedated and all animals did copulate. Thus, the increased latency to begin copulating may have reflected a slight motor retardation, as well as, or instead of, a motivational reduction. A recently completed experiment designed to dissociate motor from motivational effects of these treatments suggests that locomotor slowing was the primary deficit 34. In that experiment apomorphine injections into the VTA slowed the rate of running to all arms of an X maze, but did not affect the percentage of trials on which the male chose the goal arm containing a receptive female. These effects of apomorphine appear to be relatively specific to the VTA. Previous studies have shown that apomorphine in the medial preoptic area increased the efficiency of copulation without affecting the latency to intromit 3,2°,2s, whereas when injected into the nucleus accumbens, it somewhat decreased the latency to the first intromission z~j. Apomorphine injections into several other structures (caudate nucleus, lateral septum, paraventricular nucleus) did not affect copulation (20, unpublished observations). In the present experiments apomorphine microinjected into the SN was likewise without effect. Cannulae in Expt. 2 were located in the lateral half of the SN, an area that probably projected to the caudate-putamen and/or amygdala 13. The coordinates were chosen to maximize the distance from the VTA, so as to decrease the probability of drug diffusion to that structure. We cannot exclude the possibility that injections through cannulae situated more medially in the SN may have affected copulation. However, this does not seem likely, since the drug probably diffused throughout a substantial volume of the SN, including both zona compacta and zona reticulata, and yet did not produce even a trend toward a behavioral effect. (We have previously found that similar microinjections of dye diffused in a volume no more than 1 mm ~ surrounding the tip of the cannula, and extending up the cannula track.) The DA antagonist cis-flupenthixol was expected to enhance sexual motivation and/or motor performance by blocking impulse regulating autoreceptors in the VTA and thereby increasing the firing of mesocorticolimbic DA neurons. In support of this hypothesis, cis-flupenthixol did decrease the latency to begin in those animals that did copulate, (this effect was statistically significant only in Expt. 4). However, the higher doses of cisflupenthixot (5 and 10/~g in Expt. 3, and 2/~g in Expt. 4) decreased the number of animals copulating, an unexpected effect. The apparent decrease in sexual motivation and/or motor performance in those animals that failed to copulate, coupled with an apparent increase among those that did copulate, appears paradoxical. One possible explanation for these results is a reduction in the number of spontaneously firing neurons, due to induction of depolarization block, in some VTA neurons and a simple excitation of others. Thus, removal of inhibition normally exerted by autoreceptors would be expected to result in a net depolarization of VTA neurons 36. In some neurons this would be reflected in an increased firing rate and a shift to a bursting pattern of firing that may result in a greater release of DA from the axon terminal 15. In other neurons the depolarization would be great enough to silence the firing as a result of depolarization block 817. According to this hypothesis, animals that began copulating with a shorter latency would be those whose VTA neurons primarily increased their firing rates; those that failed to copulate would be those whose VTA neurons were primarily blocked. No direct evidence for or against this explanation was obtained in these experiments. One factor mitigating against this explanation is the finding that acute treatment with neuroleptics is usually incapable of inducing depolarization block (reviewed in refs. 7,11). Typically, chronic treatment over a period of 2-3 weeks has been required ~35. However, these electrophysiological recording experiments have utilized anesthetized preparations. Decreased excitability of neurons resulting from the anesthesia may have reduced the incidence of depolarization block 22"25. In addition, the electrophysiological recording studies have generally employed either systemic drug injections or microiontophoresis. However, greater concentrations of drug at the active site may be produced by local microinjections than by systemic administration, and a larger portion of the neuron may be affected by microinjection than by microiontophoresis. Thus, our use of localized microinjections in unanesthetized animals may possibly have increased the likelihood of depolarization block in our animals. A recent report of depolarization block after acute neuroleptic treatment confirms that higher doses of neuroleptic drugs can sometimes induce such inactivation, even in anesthetized animals a7. Another behavioral example of apparent depolarization block was recently reported by Rompr6 and Wise 3a. Briefly, systemically administered pimozide elevated the threshold for electrical self stimulation of the brain, presumably by blocking postsynaptic D A receptors in limbic terminal areas. Low doses of morphine microinjected into the VTA reversed this desensitization, restoring self stimulation to normal. However, higher doses of morphine frequently resulted in a complete absence of self stimulation. Finally, the G A B A agonist muscimol, which normally inhibits neurons by hyperpolarization, was able paradoxically to restore self stimulation. Rompr6 and Wise suggested that the ability of high doses of morphine to block self stimulation resulted from depolarization block of VTA D A neurons, and that the hyperpolarizing influence of muscimol was able to restore the neurons to normal. While Rompr6 and Wise's regimen differs considerably from ours, their results are consistent with the ability of acute treatments to induce depolarization block in VTA neurons and with the detrimental effects of such inactivation on motivated behavior. An alternative explanation for the decrease in the number of animals copulating is that cis-flupenthixol may REFERENCES 1 Aghajanian, G.K. and Bunney, B.S., Dopamine 'autoreceptors'. Pharmacological characterization by microiontophoretic single cell recording studies, Naunyn Schmiedebergs Arch. Pharmacol., 297 (1977) 1-7. 2 Band, L.C. and Hull, E.M., Opioids microinjected into central dopaminergic pathways: effects on copulation in male rats, Soc. Neurosci. Abstr., 14 (1988) 293. 3 Bitran, D., Hull, E.M., Holmes, G.M. and Lookingland, K.J., Regulation of male rat copulatory behavior by preoptic incertohypothalamic dopamine neurons, Brain Res. Bull., 20 (1988) 323-331. 4 Bozarth, M.A., Opiate reward mechanisms mapped by intracranial self-administration. In J.E. Smith and J.D. Lane (Eds.), Neurobiology of Opiate Reward Processes, Elsevier, Amsterdam, 1983, pp. 331-359. 5 Bozarth, M.A., Ventral tegmental reward system. In L. Oreland and J. Engel (Eds.), Brain Reward Systems and Abuse, Raven Press, New York, 1987, pp. 1-17. 6 Broekkamp, C.L.E., Phillips, A.G. and Cools, A.R., Facilita- have resulted in maximal output of D A in those animals that failed to copulate, such that sexual behavior (or self stimulation in Rompr6 and Wise's experiment) could not further increase D A output. Thus, if one function of motivated behavior is to increase D A release in the nucleus accumbens, and if the behavior fails to increase D A output, then the behavior may not be performed. This explanation seems unlikely, however, since one would expect the animals to be hyperactive if D A output were high; instead, animals that failed to copulate seemed hypoactive, although not inactive. In summary, stimulation of D A autoreceptors in the VTA with microinjections of apomorphine delayed the onset and slowed the rate of copulation. Injections of apomorphine into the SN were ineffective. High doses of the D A antagonist cis-flupenthixol microinjected into the VTA decreased the number of animals copulating without noticeably sedating the animals. The 2/~g dose of cis-flupenthixol both decreased the number of animals copulating and decreased the latency to begin in those animals that did copulate. The latter result suggests that blockade of the D A autoreceptors in the VTA may result in simple excitation of some neurons, resulting in greater release of D A in their terminal fields. However, some neurons may be sufficiently depolarized to block their activity, resulting in a deficit in sexual motivation or motor performance. These results implicate the mesocorticolimbic D A tract in the motivational and/or motor aspects of male copulatory behavior. Acknowledgements. We wish to thank Mary Sue Weber, Vincent Markowski and Michael Swain for assistance in testing animals, and Drs. Francis White, Michael Bozarth, Roy Wise and Daniel Bitran for helpful comments. 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