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Effects of estradiol replacement in ovariectomized rats on conditioned avoidance responses and other behaviors
Physiology & Behavior, Vol. 50, pp. 61-65. ©Pergamon Press pie, 1991. Printed in the U.S.A.
0031-9384/91 $3.00 + .00
Effects of Estradiol Replacement in Ovariectomized Rats on Conditioned Avoidance Responses and Other Behaviors I GABRIELA DIAZ-VELIZ, FABIO URRESTA, NELSON DUSSAUBAT AND SERGIO MORA 2
Departamento Preclinicas, Facultad de Medicina, Division Ciencias Medicas Oriente, Universidad de Chile P.O. Box 16038, Santiago 9, Chile R e c e i v e d 3 July 1990 DIAZ-VELIZ, G., F. URRESTA, N. DUSSAUBAT AND S. MORA. Effects of estradiol replacement in ovariectomized rats on conditioned avoidance responses and other behaviors. PHYSIOL BEHAV 50(1) 61-65, 1991.--The acquisition of conditioned avoidance responses along with spontaneous behaviors were studied in ovarieetomized rats. Fourteen days after ovariectomy, they were injected subcutaneously with one of the following doses of estradiol benzoate: 0.2, 2 or 20 p,g/rat. Behavioral tests were applied 3, 24, 48 or 72 hours after estradiol treatment. Although estradiol 2 p,g/rat induced a decrease in acquisition of conditioned avoidance responses at all times tested, this effect was maximum at 48 h. Estradiol 0.2 and 20 p,g/kg decreased and stimulated, respectively, the acquisition performance, as tested 3 h after injection. All doses increased global motility and rearing behavior. This hypermotility disappeared at 24 h, but it was observed again at 48 and 72 h after estradiol 0.2 and 20 p.g/rat. The hormone also induced an increase in head shaking and a decrease in grooming. Although the behavioral changes are more significant in presence of very low serum levels of estradiol, they seem to be triggered by the previous increase in the estradiol levels. The possible sites and mechanisms of action of estradiol on behavior are discussed. Ovariectomy
Estradiol
Conditioned avoidance responses
SEVERAL nonsexual behaviors such as locomotion (21), running wheel activity (4, 19, 34), rotational behavior (5), food intake (35), reactivity to footshock (3,12) and intracranial selfstimulation (34) have been found to vary with the estrous state of the rat. There is evidence suggesting that the acquisition of conditioned avoidance responses is also influenced by the hormonal changes that occur during the estrous cycle. This influence seems to be a facilitation of the response at diestrus and a deterioration at estrus and metestrus (11). It has been suggested that these changes in the avoidance performance across the estrous cycle could be associated with endogenous estradiol level fluctuations (11,32). Although the effect of ovarian hormones upon acquisition of conditioned avoidance behavior has been investigated (2), the role of estradiol remains unclear. Considering that estrus is associated with lower serum estradiol and reduction in avoidance response, some authors have postulated a facilitatory effect of this hormone (32). Recently we have informed that ovariectomy induces a facilitation of the avoidance performance, which is reversed with dally administration of low doses of estradiol, suggesting an inhibitory rather than facilitatory effect of estradiol upon the response (11). The present study was designed to provide more information about the influence of estradiol upon avoidance conditioning and spontaneous motor responses. We investigated the effect of a single injection of the hormone in ovariectomized rats, which
Motor activity
Grooming
Head shaking
were behaviorally tested 3, 24, 48 and 72 hours later. METHOD
Animals Female Sprague-Dawley rats, weighing 180-200 g, were maintained housed in groups of six per cage under a 12:12 light/ dark cycle (lights on from 0800 to 2000 h) with free access to food and water. Vaginal smears were taken dally for determination of different stages of estrous cycle. Only rats exhibiting consistent 4-day estrous cycles were used in this study. All the animals were bilaterally ovariectomized under light ether anesthesia. Ten days after surgical removal of the ovaries, they were assigned to four groups that received either corn oil vehicle (0.2 ml) or estradiol benzoate (0.2, 2 or 20 i~g/rat), injected SC in the dorsal region of the neck. The behavioral experiments were carded out 3, 24, 48 or 72 hours after corn oil or estradiol treatment. Since each animal was tested only once, there was a total of 12 experimental groups treated with estradiol (7 to 10 rats per group). Although separate control groups were run at each time point, they were considered as a homogeneous group (N = 16) because statistical analysis of the behavioral data revealed no significant differences between them.
1This work was supported by grants 1084-89 from FONDECYT and B-2707 from DTI, Universidad de Chile, Chile. 2Requests for reprints should be addressed to Dr. Sergio Mora.
61
62
DIAZ-VELIZ, URRESTA, DUSSAUBAT AND MORA
The behavioral tests were applied in the morning, immediately after the analysis of the vaginal smears. Before estradiol administration, the vaginal smears were invariably found to be diestrus, confirming the completeness of ovariectomy. All doses of estradiol induced vaginal smears similar to that observed at proestrus and estrus, with latencies of 48 and 72 hours, respectively.
Spontaneous Motor Activity The animals were individually placed into a Plexiglas cage (30 × 30 × 30 cm) contained in a sound-attenuated room. The floor of the cage was an activity platform (Lafayette Instrument Co.) connected to an electromechanical counter. Spontaneous motor activity was monitored automatically for 30 min, and simultaneously the following responses were recorded: number of times each animal reared, number of head shakes and the time (seconds) spent in grooming behavior. Each animal was observed continuously for the 30-min observation period, via a Sony video camera (Model AVC-1420) connected to a Panasonic VHS tape recorder (Model PV-4000). Scores were made from the live observations, and the video sequences were used for a subsequent reanalysis.
Active Avoidance Conditioning The conditioning experiments were carried out with a twoway shuttle box (Lafayette Instrument Co.) composed of two stainless steel modular testing units, which were equipped with an 18-bar insulated shock grid floor, two 28-V I)(2 lights, and a tone generator (Mallory Sonalert 2800 Hz). Electric shocks were delivered through the grid floor by a Master shock supply (Lafayette Instrument Co.). Immediately after the spontaneous motility test, the rats were placed into the shuttle box and trained over 50 trials. Each trial consisted of the presentation of a tone which, after 5 s, was overlapped with a 0.20-mA foot-shock until the animal escaped to the opposite chamber. A conditioned avoidance response was defmed as a crossing within the first 5 s (tone). Each rat was tested only once, since the purpose of this study was to investigate whether the hormonal status might influence the avoidance behavior.
Statistics Two-way analysis of variance (ANOVA) followed by Newman-Keuls and Dunnett multiple comparison procedures were applied to evaluate the statistical significance of the results. In all cases, differences were considered to be significant when p was equal to or less than 0.05.
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FIG. 1. Effects of estradiol replacement in ovariectomized rats (ovex) on the acquisition of conditioned avoidance responses expressed as the percentage out of 50 trials. Each point of the curves represents the mean + S.E.M. of the observations derived from separate groups of 8 to 10 animals. Hatched area represents the mean_+S.E.M, of the ovex control group (N= 16). Comparisons were made by using two-way ANOVA followed by Dunnetrs test. Levels of significance for comparisons with the ovex controls: *p<0.05; **p<0.005. decrease in the acquisition of conditioned avoidance responses only when the animals were tested 3 h after the injection (p<0.05). On the contrary, the higher dose (20 ~g/rat) significantly improved the performance at the same time (p<0.01). Estradiol 2 I~g/rat, which is near the minimum amount needed to induce sexual receptivity in ovariectomized rats (10), significantly decreased the acquisition of the response at every time after the injection. A marked impairment in the performance was evident 48 h after treatment. In fact, Newman-Keuls analysis showed that the response at that time was significantly lower than that observed at all other times.
Spontaneous Motor Activity The effects of estradiol on the spontaneous motility vs. time are presented in Fig. 2. Two-way ANOVA revealed that the hormone induced significant effects, F(3,124) = 16.6794, p<0.01, which varies with the time, F(3,124)=21.0388, p<0.01. Dunnetrs test showed that the three doses of estradiol induced a significant increase in motility 3 h after treatment. This stimulating effect was not significant at 24 h, but a rebound of the hypermotility was observed 48 and 72 h following injection in the animals treated with estradiol 0.2 and 20 ~g/rat.
Rearing Behavior RESULTS
Active Avoidance Conditioning The effects of the three doses of estradiol benzoate on the acquisition of conditioned avoidance responses vs. time are presented in Fig. 1. A two-way ANOVA was performed to assess main effects due to treatment and time of treatment, and the interaction between treatment and time. The data revealed that estradiol induced significant effects on acquisition, F(3,125)= 22.9005, p<0.01, and that this effect changed across the time, F(3,125)=3.4621, p<0.05. The interaction between these two factors was also significant, F(9,125)=3.0366, p<0.01. Dunnett's test for comparisons with the controls (ovariectomized plus corn oil) indicated that estradiol 0.2 p,g/rat induced a significant
The influence of estradiol on the number of rears is shown in Fig. 3. Two-way ANOVA indicated significant effects of estradiol upon this behavior, F(3,124)=29.6136, p<0.01. These effects changed with the time, F(3,124)=5.7613, p<0.01, and there is an interaction between treatment and time, F(9,124)= 5.7662, p<0.01. Dunnett's test showed that the three doses of estradiol significantly increased the number of rearings during the observation period. This effect decays at 24 h, with a rebound at 48 and 72 h in the animals treated with estradiol 0.2 and 20 0,g/rat.
Head Shaking Behavior The influence of estradiol on the number of head shakes is shown in Fig. 4. Two-way ANOVA revealed significant effects
OVARIECTOMY, ESTRADIOL AND NONSEXUAL BEHAVIORS >~-
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FIG. 2. Effects of estradiol replacement in ovariectomized rats (ovex) on spontaneous motor activity expressed as the number of counts in 30 min. Each point of the curves represents the mean-S.E.M, of the observations derived from separate groups of 8 to 10 animals. Hatched area represents the mean+--S.E.M, of the ovex control group (N= 16). Comparisons were made by using two-way ANOVA followed by Dunnett's test. Levels of significance for comparisons with the ovex controls: *p<0.05; **p<0.005. of the ovarian hormone upon this behavior, F(3,124)= 16.9445, p < 0 . 0 1 , which were not modified across the time. Durmett's test indicated that estradiol 0.2 and 20 p,g/rat increased the number of head shakes tested at every time after injection, whereas estradiol 2 p,g/rat induced a significant increase in this behavior at 24 and 48 h.
Grooming Behavior The influence of estradiol on the time spent in grooming behavior is presented in Fig. 5. Two-way ANOVA revealed sig-
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FIG. 3. Effects of estradiol replacement in ovariectomized rats (ovex) on rearing behavior, expressed as the number of rears performed in 30 rain. Each point of the curves represents the mean+--S.E.M, of the observations derived from separate groups of 8 to 10 animals. Hatched area represents the mean+S.E.M, of the ovex control group (N= 16). Comparisons were made by using two-way ANOVA followed by Dunnett's test. Levels of significance for comparisons with the ovex controls: *,o<0.05; **p<0.005.
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FIG. 4. Effects of estradiol replacement in ovariectomized rats (ovex) on head-shaking behavior, expressed as the number of head shakes in 30 min. Each point of the curves represents the mean+-S.E.M, of the observations derived from separate groups of 8 to 10 animals. Hatched area represents the mean_S.E.M, of the ovex control group (N= 16). Comparisons were made by using two-way ANOVA followed by Dunnett's test. Levels of significance for comparisons with the ovex controis: *p<0.05; **p<0.005.
nificant main effects of estradiol, F(3,124)= 18.9430, p<0.01, which vary with the time, F(3,124)=8.2641, p<0,O1, and an interaction between treatment and time, F(9,124)=2.1605, p<0.05. Dunnett's test showed that all doses of estradiol induced a significant decrease in grooming behavior at 24 h after treatment. This decrease is also observed at 48 and 72 h after treatment with estradiol 2 and 20 p,g/rat. DISCUSSION The present study demonstrates that systemic administration of a single dose of estradiol benzoate to ovariectomized rats induces immediate and delayed effects on behavior. Recently, we have reported that ovariectomy, which reduces estradiol to very low endogenous levels, facilitates the acquisition of a conditioned avoidance response and increases the time spent in grooming behavior (11), suggesting an inhibitory control of estradiol on these behaviors. The present results contribute to support this view. The administration of estradiol to ovariectomized rats induces biphasic acute effects on acquisition of conditioned avoidance responses, which are dose-dependent and time-dependent. Whereas small doses (0.2 and 2 p~g/rat) lead the acquisition of the conditioned response to levels similar to that observed in the intact rat at diestrus (11), high doses (20 p,g/rat) stimulate the performance. These effects are evident at least 3 h after the injection. Besides, a marked disruption of the response, similar to that observed in the intact rat at estrus (11), occurs 48 hours after estradiol 2 I~g/rat. This dose is near the minimum amount sufficient to reverse the typical changes induced by ovariectomy in endocrine target organs and to elicit sexual receptivity, and it could be considered within the general physiological range (10). In addition, it has been established that it produces serum levels approximately equal to those observed at proestrus, which decrease after 12 hours and become undetectable by 48 hours (9). One important point brought out by these results is that changes in acquisition of conditioned avoidance responses observed across the estrous cycle can be reproduced by a single
64
DIAZ-VELIZ, URRESTA, DUSSAUBAT AND MORA
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FIG. 5. Effects of estradiol replacement in ovariectomized rats (ovex) on grooming behavior, expressed as the time in s spent in this behavior. Each point of the curves represents the mean---S.E.M, of the observations derived from separate groups of 8 to 10 animals. Hatched area represents the mean-S.E.M, of the ovex control group (N= 16). Comparisons were made by using two-way ANOVA followed by Dunnett's test. Levels of significance for comparisons with the ovex controls: *p<0.05; **p<0.005.
subcutaneous injection of estradiol, in absence of other ovarian hormones, indicating that avoidance conditioning could be considered as another estrogen-dependent behavior. The most severe impairment in the conditioned performance is evident when the serum estradiol levels are almost undetectable, that is 48 hours after estradiol replacement in the ovariectomized rat (9) or during the estrus stage of the cycle (8). Nevertheless, a previous increase in the serum estradiol that could be able to trigger delayed behavioral effects seems to be necessary. It is known that the high levels of estradiol during the proestrus are able to trigger a sequence of neuroendocrine events in the female rat that include secretion of LHRH and LH and ovulation, as well as lordosis reflex (29). These events occur during the night of the estrus, that is in the presence of the lowest serum levels of estradiol (8). The ovarian steroids, estradiol and progesterone, induce feedback actions on pulsatile LHRH and LH secretion which are dose- and time-dependent (24). We do not have evidence about putative effects of LH upon conditioning, but we have demonstrated in our laboratory that LHRH, injected either subcutaneously (26,27) or intracerebrally in the stfiatum (28), induces an impairment in the acquisition of avoidance responses in male rats. The role of this hypothalamic hormone in the behavioral changes across the estrous cycle is under study. There are estrous cycle-dependent variations in other nonsexual behaviors, such as locomotor activity (19,35), exploratory behavior (35), nmning wheel activity (4,35) and sensorimotor performance (6). All these behaviors increase after estrogen treatment. Estradiol, in our experimental conditions, appears to stimulate general motor activity and rearing behavior in ovariectomized rats. In fact, all doses increased motility and rearing 3
h after treatment. This effect disappeared at 24 h but increases in these behaviors were observed again 48 and 72 h after injection of estradiol 0.2 and 20 ~g/rat. These results agree with studies indicating a locomotor-stimulating effect of estradiol in ovariectomized rats (35). The striatum is one brain region that has been postulated as a possible site of the estrogenic effect on behavior. Moreover, behavioral, neurochemical and pharmacological data have indicated a possible interaction between estrogen and dopamine (DA) at striatal level. In fact, behaviors induced by injections of DA and amphetamine into the dorsal striatum are suppressed with low doses of estradiol, whereas locomotor activity elicited by ventral striatal injections of amphetamine appears to be facilitated by estrogen (23), leading to the suggestion that the behaviors mediated by the striatum could be differently affected by this hormone (6). It has been suggested that estradiol exerts acute effects on the sensitivity of the nigrostriatal DA system, while it produces chronic changes in the mesolimbic DA system (7). Several antidopaminergic-like effects of estradiol at striatal level have been described (7, 13, 22). For instance, estrogen potentiates DA turnover and increases the number of striatal DA receptors (20). Besides, estrogens can inhibit the supersensitivity induced by neuroleptics (15). Gordon et al. (16) have demonstrated that ovariectomized rats treated acutely (3 days) with estradiol show a reduced response to apomorphine in terms of development of stereotyped behavior. They postulate a possible decrease in either the number or the affinity of DA receptors. Pharmacological doses of estrogen induce a biphasic response in striatal DA sensitivity: there is a suppression of apomorphineinduced stereotypy at 24 h after the last injection of estradiol, which is followed by an increased sensitivity to apomorphine at 48 h (17,18). Active avoidance conditioning and grooming behavior have been related to the activity of central DA systems, and the effects of estradiol on these behaviors could be, in part, the consequence of its interaction with DA systems. Inhibition of conditioned behavior, without affecting escape responding, is generally considered as a characteristic action of almost all drugs which block central DA transmission (31), and it has been classically used for detection of potential antipsychotic actions in man (1). A disruption of striatal DA function is thought to underlie this behavioral effect (25,33). The effects of estradiol on motor activity and rearing behavior could also be explained through an interaction with DA systems, but in other areas of the brain, such as the mesolimbic DA system, for instance. On the other hand, estradiol also enhances head-shaking behavior. Although the neurochemical mechanism involved in the production of head shakes remains unclear, there is evidence that it is a 5-HT-dependent behavior (14,30). Thus the possibility of interactions between estradiol and other neurotransmitter systems cannot be overlooked. In conclusion, although the exact mechanism of action of estradiol on behavioral patterns is unclear, this study gives further support to the view that the increase in estradiol levels could trigger a delayed inhibition of avoidance behavior and induce other behavioral changes, either by stimulating the release of other neuroendocrine agents, such as LHRH, or by changing neurotransmitter receptors' sensitivity.
REFERENCES
1. Amt, J. Pharmacological especificity of conditioned avoidance response inhibition in rats: Inhibition by neuroleptics and correlation to dopamine receptor blockade. Acta Pharmacol. Toxicol. 51:321329; 1982. 2. Banerjee, U. Influence of pseudopregnancy and sex hormones on
conditioned behavior in rats. Neuroendocrinology 7:278-290; 1971. 3. Beatty, W. W.; Beatty, P. A. Hormonal determinants of sex differences in avoidance behavior and reactivity to electric shock in the rat. J. Comp. Physiol. Psychol. 73:446--455; 1970. 4. Beatty, W. W. Gonadal hormones and sex differences in nonrepro-
OVARIECTOMY, ESTRADIOL AND NONSEXUAL BEHAVIORS
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ductive behaviors in rodents: Organizational and activational influences. Horm. Behav. 12:112-163; 1979. Becket, J. B.; Robinson, T. E.; Lorenz, K. A. Sex differences and estrous cycle variations in amphetamine-elicited rotational behavior. Eur. J. Pharmacol. 80:65-72; 1982. Becker, J. B.; Snyder, P. J.; Miller, M. M.; Westgate, S. A.; Jenuwine, J. The influence of estrous cycle and intrastriatal estradiol on sensory motor performance in the female rat. Pharmacol. Biocbem. Behav. 27:53-57; 1987. Bedard, P.; Langelier, P.; Dankova, J.; Villeneuve, A.; Di Paolo, T.; Barren, N.; Labile, F.; Boissier, J. R.; Euvrad, C. Estrogens, progesterone and the extrapyramidal system. In: PoLder, L. J.; Sourkes, T. L.; Bedard, P., eds. Extra-pyramidal system and its disorders. Advances in neurology series. New York: Raven Press; 1979:411-422. Butcher, R. L.; Collins, W. E.; Fugo, N. W. Plasma concentration of LH, FSH, prolactin, progesterone and estradiol-17B throughout the 4-day estrous cycle of the rat. Endocrinology 94:1704-1708; 1974. Cheng, H. C.; Johnson, D. C. Temporal changes in serum estradiol and the prolactin in immature female rats given a single injection of estradiol benzoate. Steroids 24:657-664; 1974. Davidson, J. M.; Smith, E. R.; Rodgers, C. H.; Bloch, G. J. Relative thresholds of behavioral and somatic responses to estrogen. Physiol. Behav. 3:227-229; 1968. Diaz-Veliz, G.; Soto, V.; Dussaubat, N.; Mora, S. Influence of the estrous cycle, ovafiectomy and estradiol replacement upon the acquisition of conditioned avoidance responses in rats. Physiol. Behay. 46:397-401; 1989. Drury, R. A.; Gold, R. M. Differential effects of ovarian hormones on reactivity to electric footshock in the rat. Physiol. Behav. 20: 187-191; 1978. Euvrard, C.; Oberlander, C.; Boissier, J. R. Antidopaminergic effect of estrogens at the sLdatal level. J. Pharmacol. Exp. Ther. 14: 179-185; 1980. Fernando, J. C. R.; Curson, G. Behavioral responses to drugs releasing 5-hydroxytryptamine and catecholamines: Effects of treatments altering precursor concentrations in brain. Neuropharmacology 20:115-122; 1981. Fields, J. Z.; Gordon, J. H. Estrogen inhibits the dopaminergic supersensitivity induced by neuroleptics. Life Sci. 30:229-234; 1982. Gordon, J. H. Modulation of apomorphine-induced stereotypy by estrogen: Time course and dose response. Brain Res. Bull. 5:679682; 1980. Gordon, J. H.; Gorski, R. A.; Borison, R. L.; Diamond, B. L. Postsynaptic efficacy of dopamine: Possible suppression by estrogen. Pharmacol. Biochem. Behav. 12:515-518; 1980. Gordon, J. H.; Perry, K. O. Pie- and postsynaptic neurochemical alterations following estrogen-induced striatal hypo- and hypersensitivity. Brain Res. Bull. 10:425-428; 1983. Hitchcock, F. A. The comparative activity of male and female al-
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bino rats. Am. J. Physiol. 75:205-21; 1925. 20. Hruska, R. E.; Silbergeld, E. K. Estrogen treatment enhances dopamine receptor sensitivity in the rat sLdatum. Eur. J. Pharmacol. 61: 397--400; 1980. 21. Hyde, J. F.; Jerussi, T. P. Sexual dimorphism in rats with respect to locomotor activity and circling behavior. Pharmacol. Biochem. Behav. 18:725-729; 1983. 22. Joyce, J. N.; Van Hartesveldt, C. Behaviors induced by intrasLdatal dopamine vary independently across the estrous cycle. Pharmacol. Biochem. Behav. 20:551-557; 1984. 23. Joyce, J. N.; Montero, E.; Van Hartesveldt, C. Dopamine-mediated behaviors: Characteristics of modulation by estrogen. Pharmacol. Biocbem. Behav. 21:791-800; 1980 24. Karsh, F. J. Central actions of ovarian steroids in the feedback regulation of pulsatile secretion of luteinizing hormone. Annu. Rev. Physiol. 49:365-82; 1987. 25. Kelly, P. H.; Seviour, P. W.; Iversen, S. D. Amphetamine and apomorphine responses in the rat after 6-OHDA lesions of the nucleus accumbens septi and corpus sLdatum. Brain Res. 94:507; 1975. 26. Mora, S.; Nasello, A. G.; Mandelli-Lopes, M.; Diaz-Veliz, G. LHRH and rat avoidance behavior: Influence of castration and testosterone. Physiol. Behav. 30:19-22; 1983. 27. Mora, S.; Caro, F.; Cardenas, G.; Espinoza, M.; Diaz-Veliz, G. Dose-dependent and time-dependent effects of luteinizing hormone releasing hormone on active avoidance behaviour in rats. IRCS Meal. Sci. 11:1108-1109; 1983. 28. Mora, S.; Diaz-Veliz, G.; Belmar, J. Is LHRH a modulator of brain dopaminergic activity in the rat? Arch. Biol. Med. Exp. (Santiago) 20:R161; 1987. 29. Moss, R. L.; Riskind, P.; Dudley, C. A. Effects of LHRH on sexual activities in animal and man. In: Collu, R.; Barbeau, A.; Duchanne, J. R.; Rochefort, J. G., eds. Central nervous system effects of hypothalamic hormones and other peptides. New York: Raven Press; 1979. 30. Nakamura, M.; Carney, J. M. Separation of clonazepam induced head twitched and muscle relaxation in mice. Pharmacol. Biocbem. Behav. 19:549-552; 1983. 31. Niemegeers, C. J. E.; Verbruggen, F. J.; Janssen, P. A. J. The influence of various neuroleptic drugs on shock avoidance responding in rats. Psychopharmacologia 16:161-174; 1969. 32. Sfikakis, A.; Spyraki, C.; Sitaras, N.; Varonos, D. Implications of the estrous cycle on conditioned avoidance behavior in the rat. Physiol. Behav. 21:441-446; 1978. 33. Sherman, A. D.; Petty, F. Locus of action of antipsychotic drugs. Soc. Neurosci. Abstr. 7:203; 1981. 34. Steiner, M.; Katz, R. J.; Caroll, B. J. Behavioral effects of dopamine agonists across the estrous cycle in rats. Psychopharmacology (Berlin) 71:147-151; 1980. 35. Wade, G. N. Gonadal hormones and behavioral regulation of body weight. Physiol. Behav. 8:523-534; 1972.
0031-9384/91 $3.00 + .00
Effects of Estradiol Replacement in Ovariectomized Rats on Conditioned Avoidance Responses and Other Behaviors I GABRIELA DIAZ-VELIZ, FABIO URRESTA, NELSON DUSSAUBAT AND SERGIO MORA 2
Departamento Preclinicas, Facultad de Medicina, Division Ciencias Medicas Oriente, Universidad de Chile P.O. Box 16038, Santiago 9, Chile R e c e i v e d 3 July 1990 DIAZ-VELIZ, G., F. URRESTA, N. DUSSAUBAT AND S. MORA. Effects of estradiol replacement in ovariectomized rats on conditioned avoidance responses and other behaviors. PHYSIOL BEHAV 50(1) 61-65, 1991.--The acquisition of conditioned avoidance responses along with spontaneous behaviors were studied in ovarieetomized rats. Fourteen days after ovariectomy, they were injected subcutaneously with one of the following doses of estradiol benzoate: 0.2, 2 or 20 p,g/rat. Behavioral tests were applied 3, 24, 48 or 72 hours after estradiol treatment. Although estradiol 2 p,g/rat induced a decrease in acquisition of conditioned avoidance responses at all times tested, this effect was maximum at 48 h. Estradiol 0.2 and 20 p,g/kg decreased and stimulated, respectively, the acquisition performance, as tested 3 h after injection. All doses increased global motility and rearing behavior. This hypermotility disappeared at 24 h, but it was observed again at 48 and 72 h after estradiol 0.2 and 20 p.g/rat. The hormone also induced an increase in head shaking and a decrease in grooming. Although the behavioral changes are more significant in presence of very low serum levels of estradiol, they seem to be triggered by the previous increase in the estradiol levels. The possible sites and mechanisms of action of estradiol on behavior are discussed. Ovariectomy
Estradiol
Conditioned avoidance responses
SEVERAL nonsexual behaviors such as locomotion (21), running wheel activity (4, 19, 34), rotational behavior (5), food intake (35), reactivity to footshock (3,12) and intracranial selfstimulation (34) have been found to vary with the estrous state of the rat. There is evidence suggesting that the acquisition of conditioned avoidance responses is also influenced by the hormonal changes that occur during the estrous cycle. This influence seems to be a facilitation of the response at diestrus and a deterioration at estrus and metestrus (11). It has been suggested that these changes in the avoidance performance across the estrous cycle could be associated with endogenous estradiol level fluctuations (11,32). Although the effect of ovarian hormones upon acquisition of conditioned avoidance behavior has been investigated (2), the role of estradiol remains unclear. Considering that estrus is associated with lower serum estradiol and reduction in avoidance response, some authors have postulated a facilitatory effect of this hormone (32). Recently we have informed that ovariectomy induces a facilitation of the avoidance performance, which is reversed with dally administration of low doses of estradiol, suggesting an inhibitory rather than facilitatory effect of estradiol upon the response (11). The present study was designed to provide more information about the influence of estradiol upon avoidance conditioning and spontaneous motor responses. We investigated the effect of a single injection of the hormone in ovariectomized rats, which
Motor activity
Grooming
Head shaking
were behaviorally tested 3, 24, 48 and 72 hours later. METHOD
Animals Female Sprague-Dawley rats, weighing 180-200 g, were maintained housed in groups of six per cage under a 12:12 light/ dark cycle (lights on from 0800 to 2000 h) with free access to food and water. Vaginal smears were taken dally for determination of different stages of estrous cycle. Only rats exhibiting consistent 4-day estrous cycles were used in this study. All the animals were bilaterally ovariectomized under light ether anesthesia. Ten days after surgical removal of the ovaries, they were assigned to four groups that received either corn oil vehicle (0.2 ml) or estradiol benzoate (0.2, 2 or 20 i~g/rat), injected SC in the dorsal region of the neck. The behavioral experiments were carded out 3, 24, 48 or 72 hours after corn oil or estradiol treatment. Since each animal was tested only once, there was a total of 12 experimental groups treated with estradiol (7 to 10 rats per group). Although separate control groups were run at each time point, they were considered as a homogeneous group (N = 16) because statistical analysis of the behavioral data revealed no significant differences between them.
1This work was supported by grants 1084-89 from FONDECYT and B-2707 from DTI, Universidad de Chile, Chile. 2Requests for reprints should be addressed to Dr. Sergio Mora.
61
62
DIAZ-VELIZ, URRESTA, DUSSAUBAT AND MORA
The behavioral tests were applied in the morning, immediately after the analysis of the vaginal smears. Before estradiol administration, the vaginal smears were invariably found to be diestrus, confirming the completeness of ovariectomy. All doses of estradiol induced vaginal smears similar to that observed at proestrus and estrus, with latencies of 48 and 72 hours, respectively.
Spontaneous Motor Activity The animals were individually placed into a Plexiglas cage (30 × 30 × 30 cm) contained in a sound-attenuated room. The floor of the cage was an activity platform (Lafayette Instrument Co.) connected to an electromechanical counter. Spontaneous motor activity was monitored automatically for 30 min, and simultaneously the following responses were recorded: number of times each animal reared, number of head shakes and the time (seconds) spent in grooming behavior. Each animal was observed continuously for the 30-min observation period, via a Sony video camera (Model AVC-1420) connected to a Panasonic VHS tape recorder (Model PV-4000). Scores were made from the live observations, and the video sequences were used for a subsequent reanalysis.
Active Avoidance Conditioning The conditioning experiments were carried out with a twoway shuttle box (Lafayette Instrument Co.) composed of two stainless steel modular testing units, which were equipped with an 18-bar insulated shock grid floor, two 28-V I)(2 lights, and a tone generator (Mallory Sonalert 2800 Hz). Electric shocks were delivered through the grid floor by a Master shock supply (Lafayette Instrument Co.). Immediately after the spontaneous motility test, the rats were placed into the shuttle box and trained over 50 trials. Each trial consisted of the presentation of a tone which, after 5 s, was overlapped with a 0.20-mA foot-shock until the animal escaped to the opposite chamber. A conditioned avoidance response was defmed as a crossing within the first 5 s (tone). Each rat was tested only once, since the purpose of this study was to investigate whether the hormonal status might influence the avoidance behavior.
Statistics Two-way analysis of variance (ANOVA) followed by Newman-Keuls and Dunnett multiple comparison procedures were applied to evaluate the statistical significance of the results. In all cases, differences were considered to be significant when p was equal to or less than 0.05.
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FIG. 1. Effects of estradiol replacement in ovariectomized rats (ovex) on the acquisition of conditioned avoidance responses expressed as the percentage out of 50 trials. Each point of the curves represents the mean + S.E.M. of the observations derived from separate groups of 8 to 10 animals. Hatched area represents the mean_+S.E.M, of the ovex control group (N= 16). Comparisons were made by using two-way ANOVA followed by Dunnetrs test. Levels of significance for comparisons with the ovex controls: *p<0.05; **p<0.005. decrease in the acquisition of conditioned avoidance responses only when the animals were tested 3 h after the injection (p<0.05). On the contrary, the higher dose (20 ~g/rat) significantly improved the performance at the same time (p<0.01). Estradiol 2 I~g/rat, which is near the minimum amount needed to induce sexual receptivity in ovariectomized rats (10), significantly decreased the acquisition of the response at every time after the injection. A marked impairment in the performance was evident 48 h after treatment. In fact, Newman-Keuls analysis showed that the response at that time was significantly lower than that observed at all other times.
Spontaneous Motor Activity The effects of estradiol on the spontaneous motility vs. time are presented in Fig. 2. Two-way ANOVA revealed that the hormone induced significant effects, F(3,124) = 16.6794, p<0.01, which varies with the time, F(3,124)=21.0388, p<0.01. Dunnetrs test showed that the three doses of estradiol induced a significant increase in motility 3 h after treatment. This stimulating effect was not significant at 24 h, but a rebound of the hypermotility was observed 48 and 72 h following injection in the animals treated with estradiol 0.2 and 20 ~g/rat.
Rearing Behavior RESULTS
Active Avoidance Conditioning The effects of the three doses of estradiol benzoate on the acquisition of conditioned avoidance responses vs. time are presented in Fig. 1. A two-way ANOVA was performed to assess main effects due to treatment and time of treatment, and the interaction between treatment and time. The data revealed that estradiol induced significant effects on acquisition, F(3,125)= 22.9005, p<0.01, and that this effect changed across the time, F(3,125)=3.4621, p<0.05. The interaction between these two factors was also significant, F(9,125)=3.0366, p<0.01. Dunnett's test for comparisons with the controls (ovariectomized plus corn oil) indicated that estradiol 0.2 p,g/rat induced a significant
The influence of estradiol on the number of rears is shown in Fig. 3. Two-way ANOVA indicated significant effects of estradiol upon this behavior, F(3,124)=29.6136, p<0.01. These effects changed with the time, F(3,124)=5.7613, p<0.01, and there is an interaction between treatment and time, F(9,124)= 5.7662, p<0.01. Dunnett's test showed that the three doses of estradiol significantly increased the number of rearings during the observation period. This effect decays at 24 h, with a rebound at 48 and 72 h in the animals treated with estradiol 0.2 and 20 0,g/rat.
Head Shaking Behavior The influence of estradiol on the number of head shakes is shown in Fig. 4. Two-way ANOVA revealed significant effects
OVARIECTOMY, ESTRADIOL AND NONSEXUAL BEHAVIORS >~-
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FIG. 2. Effects of estradiol replacement in ovariectomized rats (ovex) on spontaneous motor activity expressed as the number of counts in 30 min. Each point of the curves represents the mean-S.E.M, of the observations derived from separate groups of 8 to 10 animals. Hatched area represents the mean+--S.E.M, of the ovex control group (N= 16). Comparisons were made by using two-way ANOVA followed by Dunnett's test. Levels of significance for comparisons with the ovex controls: *p<0.05; **p<0.005. of the ovarian hormone upon this behavior, F(3,124)= 16.9445, p < 0 . 0 1 , which were not modified across the time. Durmett's test indicated that estradiol 0.2 and 20 p,g/rat increased the number of head shakes tested at every time after injection, whereas estradiol 2 p,g/rat induced a significant increase in this behavior at 24 and 48 h.
Grooming Behavior The influence of estradiol on the time spent in grooming behavior is presented in Fig. 5. Two-way ANOVA revealed sig-
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FIG. 3. Effects of estradiol replacement in ovariectomized rats (ovex) on rearing behavior, expressed as the number of rears performed in 30 rain. Each point of the curves represents the mean+--S.E.M, of the observations derived from separate groups of 8 to 10 animals. Hatched area represents the mean+S.E.M, of the ovex control group (N= 16). Comparisons were made by using two-way ANOVA followed by Dunnett's test. Levels of significance for comparisons with the ovex controls: *,o<0.05; **p<0.005.
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FIG. 4. Effects of estradiol replacement in ovariectomized rats (ovex) on head-shaking behavior, expressed as the number of head shakes in 30 min. Each point of the curves represents the mean+-S.E.M, of the observations derived from separate groups of 8 to 10 animals. Hatched area represents the mean_S.E.M, of the ovex control group (N= 16). Comparisons were made by using two-way ANOVA followed by Dunnett's test. Levels of significance for comparisons with the ovex controis: *p<0.05; **p<0.005.
nificant main effects of estradiol, F(3,124)= 18.9430, p<0.01, which vary with the time, F(3,124)=8.2641, p<0,O1, and an interaction between treatment and time, F(9,124)=2.1605, p<0.05. Dunnett's test showed that all doses of estradiol induced a significant decrease in grooming behavior at 24 h after treatment. This decrease is also observed at 48 and 72 h after treatment with estradiol 2 and 20 p,g/rat. DISCUSSION The present study demonstrates that systemic administration of a single dose of estradiol benzoate to ovariectomized rats induces immediate and delayed effects on behavior. Recently, we have reported that ovariectomy, which reduces estradiol to very low endogenous levels, facilitates the acquisition of a conditioned avoidance response and increases the time spent in grooming behavior (11), suggesting an inhibitory control of estradiol on these behaviors. The present results contribute to support this view. The administration of estradiol to ovariectomized rats induces biphasic acute effects on acquisition of conditioned avoidance responses, which are dose-dependent and time-dependent. Whereas small doses (0.2 and 2 p~g/rat) lead the acquisition of the conditioned response to levels similar to that observed in the intact rat at diestrus (11), high doses (20 p,g/rat) stimulate the performance. These effects are evident at least 3 h after the injection. Besides, a marked disruption of the response, similar to that observed in the intact rat at estrus (11), occurs 48 hours after estradiol 2 I~g/rat. This dose is near the minimum amount sufficient to reverse the typical changes induced by ovariectomy in endocrine target organs and to elicit sexual receptivity, and it could be considered within the general physiological range (10). In addition, it has been established that it produces serum levels approximately equal to those observed at proestrus, which decrease after 12 hours and become undetectable by 48 hours (9). One important point brought out by these results is that changes in acquisition of conditioned avoidance responses observed across the estrous cycle can be reproduced by a single
64
DIAZ-VELIZ, URRESTA, DUSSAUBAT AND MORA
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FIG. 5. Effects of estradiol replacement in ovariectomized rats (ovex) on grooming behavior, expressed as the time in s spent in this behavior. Each point of the curves represents the mean---S.E.M, of the observations derived from separate groups of 8 to 10 animals. Hatched area represents the mean-S.E.M, of the ovex control group (N= 16). Comparisons were made by using two-way ANOVA followed by Dunnett's test. Levels of significance for comparisons with the ovex controls: *p<0.05; **p<0.005.
subcutaneous injection of estradiol, in absence of other ovarian hormones, indicating that avoidance conditioning could be considered as another estrogen-dependent behavior. The most severe impairment in the conditioned performance is evident when the serum estradiol levels are almost undetectable, that is 48 hours after estradiol replacement in the ovariectomized rat (9) or during the estrus stage of the cycle (8). Nevertheless, a previous increase in the serum estradiol that could be able to trigger delayed behavioral effects seems to be necessary. It is known that the high levels of estradiol during the proestrus are able to trigger a sequence of neuroendocrine events in the female rat that include secretion of LHRH and LH and ovulation, as well as lordosis reflex (29). These events occur during the night of the estrus, that is in the presence of the lowest serum levels of estradiol (8). The ovarian steroids, estradiol and progesterone, induce feedback actions on pulsatile LHRH and LH secretion which are dose- and time-dependent (24). We do not have evidence about putative effects of LH upon conditioning, but we have demonstrated in our laboratory that LHRH, injected either subcutaneously (26,27) or intracerebrally in the stfiatum (28), induces an impairment in the acquisition of avoidance responses in male rats. The role of this hypothalamic hormone in the behavioral changes across the estrous cycle is under study. There are estrous cycle-dependent variations in other nonsexual behaviors, such as locomotor activity (19,35), exploratory behavior (35), nmning wheel activity (4,35) and sensorimotor performance (6). All these behaviors increase after estrogen treatment. Estradiol, in our experimental conditions, appears to stimulate general motor activity and rearing behavior in ovariectomized rats. In fact, all doses increased motility and rearing 3
h after treatment. This effect disappeared at 24 h but increases in these behaviors were observed again 48 and 72 h after injection of estradiol 0.2 and 20 ~g/rat. These results agree with studies indicating a locomotor-stimulating effect of estradiol in ovariectomized rats (35). The striatum is one brain region that has been postulated as a possible site of the estrogenic effect on behavior. Moreover, behavioral, neurochemical and pharmacological data have indicated a possible interaction between estrogen and dopamine (DA) at striatal level. In fact, behaviors induced by injections of DA and amphetamine into the dorsal striatum are suppressed with low doses of estradiol, whereas locomotor activity elicited by ventral striatal injections of amphetamine appears to be facilitated by estrogen (23), leading to the suggestion that the behaviors mediated by the striatum could be differently affected by this hormone (6). It has been suggested that estradiol exerts acute effects on the sensitivity of the nigrostriatal DA system, while it produces chronic changes in the mesolimbic DA system (7). Several antidopaminergic-like effects of estradiol at striatal level have been described (7, 13, 22). For instance, estrogen potentiates DA turnover and increases the number of striatal DA receptors (20). Besides, estrogens can inhibit the supersensitivity induced by neuroleptics (15). Gordon et al. (16) have demonstrated that ovariectomized rats treated acutely (3 days) with estradiol show a reduced response to apomorphine in terms of development of stereotyped behavior. They postulate a possible decrease in either the number or the affinity of DA receptors. Pharmacological doses of estrogen induce a biphasic response in striatal DA sensitivity: there is a suppression of apomorphineinduced stereotypy at 24 h after the last injection of estradiol, which is followed by an increased sensitivity to apomorphine at 48 h (17,18). Active avoidance conditioning and grooming behavior have been related to the activity of central DA systems, and the effects of estradiol on these behaviors could be, in part, the consequence of its interaction with DA systems. Inhibition of conditioned behavior, without affecting escape responding, is generally considered as a characteristic action of almost all drugs which block central DA transmission (31), and it has been classically used for detection of potential antipsychotic actions in man (1). A disruption of striatal DA function is thought to underlie this behavioral effect (25,33). The effects of estradiol on motor activity and rearing behavior could also be explained through an interaction with DA systems, but in other areas of the brain, such as the mesolimbic DA system, for instance. On the other hand, estradiol also enhances head-shaking behavior. Although the neurochemical mechanism involved in the production of head shakes remains unclear, there is evidence that it is a 5-HT-dependent behavior (14,30). Thus the possibility of interactions between estradiol and other neurotransmitter systems cannot be overlooked. In conclusion, although the exact mechanism of action of estradiol on behavioral patterns is unclear, this study gives further support to the view that the increase in estradiol levels could trigger a delayed inhibition of avoidance behavior and induce other behavioral changes, either by stimulating the release of other neuroendocrine agents, such as LHRH, or by changing neurotransmitter receptors' sensitivity.
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