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Progesterone effects on the acquisition of conditioned avoidance responses and other motoric behaviors in intact and ovariectomized rats
Pergamon
Psychoneuroendocrinology, Vol. 19, No. 4, pp. 387-394, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0306-4530/94 $6.00 + .00
0306-4530(94)E0001-P PROGESTERONE EFFECTS ON THE ACQUISITION OF CONDITIONED AVOIDANCE RESPONSES AND OTHER MOTORIC BEHAVIORS IN INTACT AND OVARIECTOMIZED RATS GABRIELA DIAZ-VI~LIZ, FABIO URRESTA, NELSON DUSSAUBAT,
and SERGIO MORA Departamento Medicina Experimental, Facultad de Medicina, Divisi6n Ciencias M6dicas Oriente, Universidad de Chile, P.O. Box 16038, Santiago 9, Chile
(Receioed 9 July 1993; in final form 17 Nooember 1993)
SUMMARY This study demonstrates a significant impairment in the acquisition of conditioned avoidance responses in female rats during their estrus phase. Progesterone (PROG 5 mg) injected 6 h prior to the test, significantly enhanced the performance exhibited by rats at estrus, but not at diestrus. In ovariectomized rats, the acquisition of conditioned avoidance responses was similar to the exhibited during diestrus and this behavior was depressed by a single dose of estradiol benzoate (EB 2/~g) injected 48 h prior to the test. PROG antagonized the avoidance depression induced by EB, but it was not able to induce changes in the acquisition of conditioned avoidance response in ovariectomized rats without EB pretreatment. Estradiol appears to be the principal ovarian steroid modulating the acquisition of an avoidance task, whereas PROG seems to have a secondary role in this behavior, regulating the actions of estradiol on the brain. PROG failed to induce consistent changes in some spontaneous motor behaviors in intact and ovariectomized rats. KeywordsDRat; Ovariectomy; Progesterone; E stradiol; Conditioned avoidance responses; Motoric behaviors.
TIlE EFFECT OF gonadal hormones on reproductive behaviors have been studied in great detail and much is known about the neuroendocrine control of these activities (Lisk, 1967; Phoenix et al., 1967). The same hormones also have rather striking effects on several nonsexual behaviors such as open field ambulation (Birke et al., 1975), locomotion (Hyde & Jerussi, 1983), wheel running activity (Finger, 1969; Steiner et al., 1980), food intake (Wade, 1972), rotational behavior (Becket et al., 1982), reactivity to foot shock (Beatty & Beatty, 1970; Drury & Gold, 1978), sensorimotor performance (Becker et al., 1987), and avoidance conditioning (Banerjee, 1971; Beatty, 1979; Beatty & Beatty, 1970; Dfaz-V61iz et al., 1989; Sfikakis et al., 1978). All these behaviors have been found to vary with the sex and the estrous stage of the rat. Previously we found that female rats showed a significant impairment in the acquisition Address correspondence and reprint requests to: Gabriela Dfaz-V61iz. This work was supported by Grant 193-1071 from F O N D E C Y T , Chile. 387
388
G. DfAz-VI~LIZet al.
of a conditioned task at estrous and metestrous with minimal changes in spontaneous motor activity, while ovariectomy induced an enhancement of this response (Dfaz-V61iz et al., 1989). Systemic administration of a single dose of estradiol benzoate (EB 2 pg) to ovariectomized rats induced a decrease in acquisition of conditioned task tested at 3, 24, 48, and 72 h after injection (Dfaz-V61iz et al., 1991). Although progesterone (PROG) has been implicated in the control of sexual behavior (Edward et al., 1968; Rubin & Bartfield, 1983; Whalen, 1974) as well as reduction of anxiety in rodents (Rodriguez-Sierra et al., 1986), inhibition of aggressive behaviors in hamsters (Fraile et al., 1987), and inhibition of wheel-running activity in intact female rats (Rodier, 1971), it has not shown other nonsexual behavioral roles in female rats. The purpose of the present study was to investigate the influence of PROG administration on the acquisition of active avoidance responses and spontaneous motor activity in intact, ovariectomized and estrogen-primed rats. METHODS
Animals A total of 80 female Sprague-Dawley rats, weighing 180-200 g, were housed in groups of six per cage under a 12:12 light/dark cycle (ligths on from 0800 to 2000h) with free access to food and water. Intact female rats were submitted daily to vaginal smears for determination of different stages of estrous cycle. Only rats exhibiting three or more consistent 4-day cycles were utilized. On the basis of a previous report (Diaz-Vrliz et al., 1989) showing large differences in the acquisition of a conditioned avoidance response between diestrus and estrus females, only rats in these stages were included in this study. Forty cycling rats were assigned to two groups according to the phase of the cycle: diestrus and estrus. Animals of each group received either 5 mg PROG or corn oil vehicle (0.2 ml). The behavioral tests were performed 6 h after the injection. Another group of 40 rats was bilaterally ovariectomized under light ether anesthesia. Fourteen days after surgical removal of the ovaries, animals were randomly divided into two groups that received either 2/~g EB or corn oil vehicle (0.2 ml), 48 h prior to tests. Later, 6 h prior to behavioral tests, both groups received either 5 mg PROG or corn oil vehicle (0.2 ml). Both hormones were dissolved in corn oil and injected subcutaneously (SC) in the dorsal region of the neck. Each rat was tested only once between 1000 and 1400 h.
Spontaneous Motor Activity Each animal was individually placed in a Plexiglas cage (30 × 30 x 30 cm), located into 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 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 continously for the 30 min observation period, via a video camera connected to a VHS tape recorder. Scores were made from the live picture and the video sequences were used for a subsequent reanalysis.
Active Avoidance Conditioning The conditioning experiments were carried out with a two-way 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 DC lights, and a tone
PROGESTERONE AND CONDITIONED AVOIDANCE
389
PERCENT CARl
To
]
60 t
.................
6040-
30-
20-
10-
0
t
i
DI * OIL
DI * PROG
NORMALLY
E * 'OIL
CYCLING
E * PROG RATS
FIG. l: Effects of progesterone (PROG = 5 mg) on the acquisition of conditioned avoidance responses (CARs) in intact cycling female rats, at stages of diestrus (DI) and estrus (E). Each bar represent the mean -+ SEM of the percentage of CARs out 50 trials. Comparisons were made by using one-way ANOVA followed by Newman-Keuls test (*significantly different from all other groups, p < .01). The number of animals in each group was 10. For more details see text. generator (Mallory Sonalert 2800 Hz). Electric shocks were provided to the grid floor by a Master shock supply (Lafayette Instrument Co.). Immediately after the spontaneous motility test, the rats were placed in the shuttle box and trained over 50 trials. Each trial consisted of the presentation of a tone that, after 5 s, was overlapped with a 0.2-mA foot shock until the animal escaped to the opposite chamber; maximum shock duration was 10 s. A conditioned avoidance response (CAR) was defined as a crossing within the first 5 s (tone)•
Statistics One way analisis of variance (ANOVA) followed by Newman-Keuls procedure were applied to evaluate the statistical significance of the results. In all cases differences were considered to be significant when p -< .05. RESULTS
Active Avoidance Conditioning One-way analysis of variance (ANOVA) indicated significant modifications on acquisition of conditioned avoidance responses (CARs), F(3, 36) = 9.780, p < .01, after PROG injection to intact female rats according to the stage of the estrous cycle. Newman-Keuls analysis showed that the acquisition of CARs was significantly lower during estrus than during diestrus (p < .001). Figure 1 shows that PROG significantly increased the avoidance
390
G. DfAz-V~LIZet al.
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EBIOIL OILI'PROG EB*PROG OVARIECTOMIZED RATS FIG. 2: Effects of estradiol (EB = 2/zg) and progesterone (PROG = 5 mg) on the acquisition of conditioned avoidance responses (CARs) in ovariectomized rats. Each bar represent the mean _+ SEM of the percentage of CARs out 50 trials. Comparisons were made by using one-way ANOVA followed by Newman-Keuls test (*significantly different from all other groups, p < .001). The number of animals in each group was 10. For more details see text.
performance in rats at estrus (19 < .001), but the response of rats at diestrus was not affected by PROG administration. One-way ANOVA revealed significant effects due to hormonal treatment F(3, 36) = 28.103, p < .01, on acquisition of CARs in ovariectomized rats. Figure 2 shows that the administration of 2/~g of EB, drastically reduced the acquisition of CARs in ovariectomized rats (p < .001). This suppressive effect of EB on acquisition was not seen in animals receiving both EB and PROG. Spontaneous Motor Activity The effects of PROG on the spontaneous motor behaviors in intact female rats are presented in Table I. One-way ANOVA revealed that the hormone failed to induce significant changes on motor activity, number of rearing, number of head shakes, and time spent in grooming behavior, in both stages of the estrous cycle (Estrus and Diestrus). While in ovariectomized rats motor activity was not affected as a result of hormones administration, significant modifications were observed on the number of rearings, F(3, 36) = 3.088, p < .05, the number of head shakes, F(3, 36) = 5.125, p < .01, and grooming behavior, F(3, 36) = 4.4096, p < .01 (Table II). In fact, rearing and head shaking behaviors were enhanced after EB replacement (p < .025), but no significant effect of PROG on these behaviors was observed. Both EB and PROG significantly reduced the time spent
PROGESTERONE AND CONDITIONED AVOIDANCE
TABLE
I. PROGESTERONE
EFFECTS
ON SPONTANEOUS FEMALE
Treatment
Motor activity (counts)
DI + Oil DI + PROG (5 mg) E + Oil E + PROG (5 rag)
962.6 -+ 94.1 1134.5 +- 85.6 811.2 -+ 86.2 1003.3-+ 99.8
MOTOR
391
BEHAVIORS
IN INTACT
RATS
Rearing (number of times) 65.7 52.7 49.3 60.6
Head shaking (number of times)
-+ 6.2 - 5.1 - 5.5 + 5.5
11.8 12.2 5.5 8.3
--- 2.2 -+ 3.3 - 0.8 -+ 1.6
Grooming (seconds) 430.3 501.3 350.8 367.0
- 42.3 -+ 55.1 --- 41.6 + 24.1
Values are expressed as mean --- SEM of 10 intact female rats on each group. DI = Diestrus and E = Estrus. P R O G = progesterone injected SC 6 h prior to the test. Oil = Corn oil vehicle for PROG. Comparisons were made by using one-way A N O V A , no significant differences were detected.
in grooming behavior (p < .01). Comparing Table I and Table II, it can be observed that ovariectomy itself reduced motor activity and rearing behavior (p < .0025 in both cases). DISCUSSION As the plasma levels of estrogen and progesterone fluctuate (Butcher et al., 1974), the animal undergoes several modifications in its behavioral state (Beatty, 1979; B e c k e r et al., 1982, 1987; Birke & Archer, 1975; Diaz-V61iz et al., 1989; Drury & Gold, 1978; H y d e & Jerussi, 1983; Sfikakis et al., 1978; Wade, 1972). The present findings contribute to support the idea that the acquisition o f a conditioned avoidance response (CAR) and of some spontaneous motor behaviors of female rats are also influenced by the ovarian hormones. Previously we found a significant impairment of the conditioned avoidance performance during estrus compared with diestrus (Diaz-V61iz et al., 1989). This impairment has been associated with endogenous estradiol and/or progesterone fluctuations observed across the estrous cycle (Butcher et al., 1974). We observed that the impairment in avoidance conditioning begins during the morning of proestrus (Dfaz-V61iz et al., 1989), when the levels of estradiol are high, and the greatest impairment in acquiring a CAR
TABLE II.
PROGESTERONE
EFFECTS
ON SPONTANEOUS
OVARIECTOMIZED
Treatment Oil EB Oil EB
+ Oil (2/xg) + Oil + PROG (5 mg) + PROG
Motor activity (counts) 516.6 460.1 645.7 593.4
-+ 19.7 _+ 35.8 - 79.6 __+43.0
Rearing (number of times) 27.9 37.6 27.1 37.1
_ 2.9 _+ 3.7* _ 1.3 -+ 3.5*
MOTOR
BEHAVIORS
IN
RATS
Head shaking (number of times) 8.9 18.6 6.7 16.9
_-+ 1.3 _ 3.6* --- 0.9 _+ 2.3*
Grooming (seconds) 554.3 375.1 411.2 391.9
__+64.7 _+ 22.9* _ 24.1" -+ 33.2*
Values are expressed as mean -+ SEM of 10 ovariectomized rats on each group. EB = estradiol benzoate and PROG = progesterone injected 6 h prior to the test. Oil = Corn oil vehicle for EB and PROG. Comparisons were made by using oneway A N O V A followed by Newman-Keuls test. (*significantly different from the group receiving Oil + Oil, p < .025).
392
G. DIAz-VI~LIZet al.
was observed during estrus when the estrogen levels are low (Butcher et al., 1974), indicating that these events could be associated with estradiol withdrawal. This suggestion is further supported by our finding that the facilitation of avoidance performance induced by ovariectomy was reversed only 48 h after the administration of a physiologically relevant dose of estradiol (Dfaz-Vrliz et al., 1989, 1991). The dose of EB used in the present work (2 pg) is the minimum amount needed to induce sexual receptivity in ovariectomized rats (Davidson et al., 1968) and to reverse changes in endocrine target organs caused by ovariectomy, inducing vaginal smears similar to that observed at estrous, with a latency of 3 days (Davidson et al., 1968; DiazVrliz et al., 1991). It is important to consider that a minimal dose of estradiol is required to induce delayed effects on behavior when serum estradiol levels become undetectable (Cheng & Johnson, 1974). The dose of PROG used in this study (5 mg) has demonstrated to influence behavioral changes in intact and ovariectomized rats, which are not seen with lower doses (Drury & Gold, 1978; Rodier, 1971). The present study shows that PROG alone was not able to modify the acquisition of conditioned avoidance responses in intact rats at diestrus nor in ovariectomized rats not treated with EB. Nevertheless, PROG antagonized the impairment on the acquisition of CARs both in intact rats at estrus and in ovariectomized rats treated with EB. These observations indicate that the effect of PROG on the acquisition of an avoidance task may be dependent on the presence of the ovary or the previous treatment with EB. Then, PROG seems to have a secondary role in this behavior, probably modulating actions of estradiol on the brain. Our hormonal manipulations failed to induce consistent changes in spontaneous motor behaviors in intact female rats. EB treatment of ovariectomized rats resulted in an increase in the number of head shakes and the number or rears, but there was no significant evidence of an influence of PROG on these behaviors. The grooming behavior that increased after ovariectomy (Diaz-Vrliz et al., 1989, and this study), was inhibited by the administration of both ovarian hormones. In addition, ovariectomy caused a decrease in motor activity and rearing behavior, but hormonal treatments were not able to restore preovariectomized levels of these behaviors. Thus, it seems that active avoidance conditioning and spontaneous motor behaviors are responses which are independently modulated by ovarian hormones and that changes in one behavior are not consequence of changes in another. Besides, little is known about the neural pathways that are activated once these hormones reach the brain. There is several evidence for an interaction between estradiol and progesterone in a variety of functions. They interact in the secretion of gonadotrophins from the anterior pituitary (Karsch, 1987) and in the promotion of sexual and nonsexual behaviors. PROG can inhibit, as well as facilitate, estrogen's effects on lordosis (Edward et al., 1968). On the other hand, PROG injection decreases wheel running and increases body weight in intact female rats and in ovariectomized rats receiving EB replacement; however, PROG does not influence these behaviors in ovariectomized rats not treated with EB, suggesting that PROG effects requires the presence of endogenous or exogenous estrogen (Rodier, 1971). Ovarian steroids can play a modulatory role of dopaminergic activity in the rat central nervous system. For example, neurochemical data indicate that EB treatments can influence dopamine turnover (Di Paolo et al., 1985; Fern~indez-Ruiz et al., 1990), and DA receptor binding (Hruska et al., 1980). The effect of estradiol on the activity of dopaminergic neurons is not observed when estradiol-treated rats are injected with a
PROGESTERONE AND CONDITIONED AVOIDANCE
393
single dose of PROG (Fernfindez-Ruiz et al., 1990). In addition, estrogen had a stimulatory effect on stereotypy in nondrug-treated rats, while PROG had an inhibitory effect (Michanek & Meyerson, 1982). This suggests the possibility of a negative interaction between both steroids. In a previous paper we postulated that the effects of estradiol on active avoidance conditioning and other behaviors could be explained through an interaction with dopaminergic systems (Dfaz-Vrliz et al., 1991). According to the present results, PROG and EB could be acting in an opposite way on these systems. It has been postulated that sex hormones are capable to regulate neuronal functions by genomic and nongenomic mechanisms (McEwen, 1991). We observed that the steroid effects on avoidance behavior take many hours or days to appear and those of progesterone seem to be dependent on the stage of the estrous cycle or of prior estrogen priming; this implies a dependence of the actions of estradiol possibly via genomic mechanisms. The effects of PROG described in this report are very similar to that observed after the administration of a synthetic antagonist of LHRH. Recently (Mora et al., 1993) we demonstrated that a single injection of 10 /.~g of [N-acetyl-D-p-chloro-Phe~.2,D-TrP3 , DArg6,D-AIa~0]-LHRH stimulated the acquisition of CARs in intacts rats at estrus, and in ovariectomized treated with EB, but neither in rats at diestrus nor in ovariectomized animals without treatment with EB. In this context, the ability of PROG to block the estrogen-induced LH and FSH release has been established (Dierschke et al., 1973). To summarize, the results show that PROG treatment results in increased acquisition of CARs in intact rats at estrus and in estrogen-primed ovariectomized rats. In contrast, PROG does not influence this behavior in intact rats at diestrus nor in ovariectomized rats not treated with EB. Then, the PROG effect seems to require the presence of endogenous or exogenous estrogen. The nature of estradiol-progesterone interaction remains unclear, but it could explain the changes in the acquisition of CARs and other behaviors observed across the estrous cycle of the rat.
REFERENCES Banerjee U (1971) Influence of pseudopregnancy and sex hormones on conditioned behavior in rats. Neuroendocrinology 7:278-290. Beatty WW (1979) Gonadal hormones and sex differences in nonreproductive behaviors in rodents: Organizational and activational influences. Horm Behav 12:112-163. Beatty WW, Beatty PA (1970) Hormonal determinants of sex differences in avoidance behavior and reactivity to electric shock in the rat. J Comp Physiol Psychol 73:446-455. Becker JB, Robinson TE, Lorenz KA (1982) Sex differences and estrous cycle variations in amphetamine-elicited rotational behavior. Eur J Pharmacol 80:65-72. Becker JB, Snyder PJ, Miller MM, Westgate SA, Jenuwine J (1987) The influence of estrous cycle and intrastriatal estradiol on sensorimotor performance in the female rats. Pharmacol Biochem Behav 27:53-57. Birke LIA, Archer J (1975) Open-field behavior of oestrous and dioestrous rats: Evidence against an "emotionality" interpretation. Anim Behav 23:509-512. Butcher RL, Collins WE, Fugo NW (1974) Plasma concentration of LH, FSH, prolactin, progesterone and estradiol-17/3throughout the 4-day estrous cycle of the rat. Endocrinology 94:1704-1708. Cheng HC, Johnson DC (1974) Temporal changes in serum estradiol and prolactin in immature female rats given a single injection of estradiol benzoate. Steroids 24:657-664. Davidson JM, Smith ER, Rodgers CH, Bloch GJ (1968) Relative thresholds of behavioral and somatic responses to estrogen. Physiol Behav 3:227-229. Dfaz-Vrliz G, Soto V, Dussaubat N, Mora S (1989) Influence of the estrous cycle, ovariectomy and estradiol replacement upon the acquisition of conditioned avoidance responses in rats. Physiol Behav 46:397-401. Diaz-Vrliz G, Urresta F, Dussaubat N, Mora S (1991) Effects of estradiol replacement in ovariectomized rats on conditioned avoidance responses and other behaviors. Physiol Behav 50:61-65.
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al.
Dierschke DJ, Yamaji T, Karsch FJ, Weick RF, Weiss G, Knobil E (1973) Blockade by progesterone of estrogen-induced LH and FSH release in the rhesus monkey. Endocrinology 92:1496-1501. Di Paolo T, Rouillard C, B6dard PJ (1985) 17/3-Estradiol at a physiological dose acutely increases dopamine turnover in rat brain. Eur J Pharmacol 117:197-203. Drury RA, Gold RM (1978) Differential effects of ovarian hormones on reactivity to electric foot shock in the rat. Physiol Behav 20:187-191. Edward DA, Whalen RE, Nadler RD (1968) Induction of estrus: Estrogen-progesterone interactions. Physiol Behav 3:29-33. Fern,'indez-Ruiz JJ, de Miguel R, Hern,'indez ML, Ramos JA (1990) Time-course of the effects of ovarian steroids on the activity of limbic and striatal dopaminergic neurons in female rat brain. Pharmacol Biochem Behav 36:603-606. Finger FW (1969) Estrous and general activity in the rat. J Comp Physiol Psychol 68:461-466. Fraile I, McEwen BS, Pfaff DW (1987) Progesterone inhibition of aggresive behavior in hamster. Physiol Behav 39:225-229. Hruska RE, Ludmer LM, Silbergeld EK (1980) Characterization of the striatal dopamine receptor supersensitivity produced by estrogen treatment of male rats. Neuropharmacology 19:923-926. Hyde JF, Jerussi TP (1983) Sexual dimorphism in rats with respect to locomotor activity and circling behavior. Pharmacol Biochem Behav 18:725-729. Karsch FJ (1987) Central actions of ovarian steroids in the feedback regulation of pulsatile secretion of luteinizing hormone. Ann Rev Physiol 49:365-382. Lisk RD (1967) Sexual behavior: Hormonal control. In: Martini L, Ganong WF (Eds) Neuroendocrinology, Vol II. Academic Press, New York, pp 197-239. McEwen BS (1991) Nongenomic and genomic effects of steroids on neural activity. Trends Pharmacol Sci 12:141-147. Michanek A, Meyerson BJ (1982) Influence of estrogen and progesterone on behavioral effects of apomorphine and amphetamine. Pharmacol Biochem Behav 16:875-879. Mora S, Urresta F, Dussaubat N, Benavente F, Baeza R, Diaz-V61iz G (1993) Behavioral effects of a LHRH antagonist in intact and ovariectomized rats. Pharmacol Biochem Behav 46(3):673-677. Phoenix CH, Goy RW, Young WC (1967) Sexual behavior: General aspect. In: Martini L, Ganong WF (Eds) Neuroendocrinology, Vol II. Academic Press, New York, pp 163-196. Rodier WI (1971) Progesterone-estrogen interactions in the control of activity-wheel running in the female rat. J Comp Physiol Psychol 74:365-373. Rodriguez-Sierra JF, Hagley MT, Hendricks SE (1986) Anxiolytic effects of progesterone are sexually dimorphics. Life Sci 38:1841-1845. Rubin BS, Bartfield RJ (1983) Induction of estrus behavior in ovariectomized rats by sequential replacement of estrogen and progesterone to the ventromedial hypothalamus. Neuroendocrinology 37:218-224. Sfikakis A, Spyraki C, Sitaras N, Varonos D (1978) Implications of the estrous cycle on conditioned avoidance behavior in the rat. Physiol Behav 21:441-446. Steiner M, Katz RJ, Caroll BJ (1980) Behavioral effects of dopamine agonists across the estrous cycle in rats. Psychopharmacology (Berlin) 71:147-151. Wade GN (1972) Gonadal hormones and behavioral regulation of body weight. Physiol Behav 8:523-534. Whalen RE (1974) Estrogen-progesterone induction of mating in female rats. Horm Behav 5:157-162.
Psychoneuroendocrinology, Vol. 19, No. 4, pp. 387-394, 1994 Copyright © 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0306-4530/94 $6.00 + .00
0306-4530(94)E0001-P PROGESTERONE EFFECTS ON THE ACQUISITION OF CONDITIONED AVOIDANCE RESPONSES AND OTHER MOTORIC BEHAVIORS IN INTACT AND OVARIECTOMIZED RATS GABRIELA DIAZ-VI~LIZ, FABIO URRESTA, NELSON DUSSAUBAT,
and SERGIO MORA Departamento Medicina Experimental, Facultad de Medicina, Divisi6n Ciencias M6dicas Oriente, Universidad de Chile, P.O. Box 16038, Santiago 9, Chile
(Receioed 9 July 1993; in final form 17 Nooember 1993)
SUMMARY This study demonstrates a significant impairment in the acquisition of conditioned avoidance responses in female rats during their estrus phase. Progesterone (PROG 5 mg) injected 6 h prior to the test, significantly enhanced the performance exhibited by rats at estrus, but not at diestrus. In ovariectomized rats, the acquisition of conditioned avoidance responses was similar to the exhibited during diestrus and this behavior was depressed by a single dose of estradiol benzoate (EB 2/~g) injected 48 h prior to the test. PROG antagonized the avoidance depression induced by EB, but it was not able to induce changes in the acquisition of conditioned avoidance response in ovariectomized rats without EB pretreatment. Estradiol appears to be the principal ovarian steroid modulating the acquisition of an avoidance task, whereas PROG seems to have a secondary role in this behavior, regulating the actions of estradiol on the brain. PROG failed to induce consistent changes in some spontaneous motor behaviors in intact and ovariectomized rats. KeywordsDRat; Ovariectomy; Progesterone; E stradiol; Conditioned avoidance responses; Motoric behaviors.
TIlE EFFECT OF gonadal hormones on reproductive behaviors have been studied in great detail and much is known about the neuroendocrine control of these activities (Lisk, 1967; Phoenix et al., 1967). The same hormones also have rather striking effects on several nonsexual behaviors such as open field ambulation (Birke et al., 1975), locomotion (Hyde & Jerussi, 1983), wheel running activity (Finger, 1969; Steiner et al., 1980), food intake (Wade, 1972), rotational behavior (Becket et al., 1982), reactivity to foot shock (Beatty & Beatty, 1970; Drury & Gold, 1978), sensorimotor performance (Becker et al., 1987), and avoidance conditioning (Banerjee, 1971; Beatty, 1979; Beatty & Beatty, 1970; Dfaz-V61iz et al., 1989; Sfikakis et al., 1978). All these behaviors have been found to vary with the sex and the estrous stage of the rat. Previously we found that female rats showed a significant impairment in the acquisition Address correspondence and reprint requests to: Gabriela Dfaz-V61iz. This work was supported by Grant 193-1071 from F O N D E C Y T , Chile. 387
388
G. DfAz-VI~LIZet al.
of a conditioned task at estrous and metestrous with minimal changes in spontaneous motor activity, while ovariectomy induced an enhancement of this response (Dfaz-V61iz et al., 1989). Systemic administration of a single dose of estradiol benzoate (EB 2 pg) to ovariectomized rats induced a decrease in acquisition of conditioned task tested at 3, 24, 48, and 72 h after injection (Dfaz-V61iz et al., 1991). Although progesterone (PROG) has been implicated in the control of sexual behavior (Edward et al., 1968; Rubin & Bartfield, 1983; Whalen, 1974) as well as reduction of anxiety in rodents (Rodriguez-Sierra et al., 1986), inhibition of aggressive behaviors in hamsters (Fraile et al., 1987), and inhibition of wheel-running activity in intact female rats (Rodier, 1971), it has not shown other nonsexual behavioral roles in female rats. The purpose of the present study was to investigate the influence of PROG administration on the acquisition of active avoidance responses and spontaneous motor activity in intact, ovariectomized and estrogen-primed rats. METHODS
Animals A total of 80 female Sprague-Dawley rats, weighing 180-200 g, were housed in groups of six per cage under a 12:12 light/dark cycle (ligths on from 0800 to 2000h) with free access to food and water. Intact female rats were submitted daily to vaginal smears for determination of different stages of estrous cycle. Only rats exhibiting three or more consistent 4-day cycles were utilized. On the basis of a previous report (Diaz-Vrliz et al., 1989) showing large differences in the acquisition of a conditioned avoidance response between diestrus and estrus females, only rats in these stages were included in this study. Forty cycling rats were assigned to two groups according to the phase of the cycle: diestrus and estrus. Animals of each group received either 5 mg PROG or corn oil vehicle (0.2 ml). The behavioral tests were performed 6 h after the injection. Another group of 40 rats was bilaterally ovariectomized under light ether anesthesia. Fourteen days after surgical removal of the ovaries, animals were randomly divided into two groups that received either 2/~g EB or corn oil vehicle (0.2 ml), 48 h prior to tests. Later, 6 h prior to behavioral tests, both groups received either 5 mg PROG or corn oil vehicle (0.2 ml). Both hormones were dissolved in corn oil and injected subcutaneously (SC) in the dorsal region of the neck. Each rat was tested only once between 1000 and 1400 h.
Spontaneous Motor Activity Each animal was individually placed in a Plexiglas cage (30 × 30 x 30 cm), located into 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 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 continously for the 30 min observation period, via a video camera connected to a VHS tape recorder. Scores were made from the live picture and the video sequences were used for a subsequent reanalysis.
Active Avoidance Conditioning The conditioning experiments were carried out with a two-way 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 DC lights, and a tone
PROGESTERONE AND CONDITIONED AVOIDANCE
389
PERCENT CARl
To
]
60 t
.................
6040-
30-
20-
10-
0
t
i
DI * OIL
DI * PROG
NORMALLY
E * 'OIL
CYCLING
E * PROG RATS
FIG. l: Effects of progesterone (PROG = 5 mg) on the acquisition of conditioned avoidance responses (CARs) in intact cycling female rats, at stages of diestrus (DI) and estrus (E). Each bar represent the mean -+ SEM of the percentage of CARs out 50 trials. Comparisons were made by using one-way ANOVA followed by Newman-Keuls test (*significantly different from all other groups, p < .01). The number of animals in each group was 10. For more details see text. generator (Mallory Sonalert 2800 Hz). Electric shocks were provided to the grid floor by a Master shock supply (Lafayette Instrument Co.). Immediately after the spontaneous motility test, the rats were placed in the shuttle box and trained over 50 trials. Each trial consisted of the presentation of a tone that, after 5 s, was overlapped with a 0.2-mA foot shock until the animal escaped to the opposite chamber; maximum shock duration was 10 s. A conditioned avoidance response (CAR) was defined as a crossing within the first 5 s (tone)•
Statistics One way analisis of variance (ANOVA) followed by Newman-Keuls procedure were applied to evaluate the statistical significance of the results. In all cases differences were considered to be significant when p -< .05. RESULTS
Active Avoidance Conditioning One-way analysis of variance (ANOVA) indicated significant modifications on acquisition of conditioned avoidance responses (CARs), F(3, 36) = 9.780, p < .01, after PROG injection to intact female rats according to the stage of the estrous cycle. Newman-Keuls analysis showed that the acquisition of CARs was significantly lower during estrus than during diestrus (p < .001). Figure 1 shows that PROG significantly increased the avoidance
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EBIOIL OILI'PROG EB*PROG OVARIECTOMIZED RATS FIG. 2: Effects of estradiol (EB = 2/zg) and progesterone (PROG = 5 mg) on the acquisition of conditioned avoidance responses (CARs) in ovariectomized rats. Each bar represent the mean _+ SEM of the percentage of CARs out 50 trials. Comparisons were made by using one-way ANOVA followed by Newman-Keuls test (*significantly different from all other groups, p < .001). The number of animals in each group was 10. For more details see text.
performance in rats at estrus (19 < .001), but the response of rats at diestrus was not affected by PROG administration. One-way ANOVA revealed significant effects due to hormonal treatment F(3, 36) = 28.103, p < .01, on acquisition of CARs in ovariectomized rats. Figure 2 shows that the administration of 2/~g of EB, drastically reduced the acquisition of CARs in ovariectomized rats (p < .001). This suppressive effect of EB on acquisition was not seen in animals receiving both EB and PROG. Spontaneous Motor Activity The effects of PROG on the spontaneous motor behaviors in intact female rats are presented in Table I. One-way ANOVA revealed that the hormone failed to induce significant changes on motor activity, number of rearing, number of head shakes, and time spent in grooming behavior, in both stages of the estrous cycle (Estrus and Diestrus). While in ovariectomized rats motor activity was not affected as a result of hormones administration, significant modifications were observed on the number of rearings, F(3, 36) = 3.088, p < .05, the number of head shakes, F(3, 36) = 5.125, p < .01, and grooming behavior, F(3, 36) = 4.4096, p < .01 (Table II). In fact, rearing and head shaking behaviors were enhanced after EB replacement (p < .025), but no significant effect of PROG on these behaviors was observed. Both EB and PROG significantly reduced the time spent
PROGESTERONE AND CONDITIONED AVOIDANCE
TABLE
I. PROGESTERONE
EFFECTS
ON SPONTANEOUS FEMALE
Treatment
Motor activity (counts)
DI + Oil DI + PROG (5 mg) E + Oil E + PROG (5 rag)
962.6 -+ 94.1 1134.5 +- 85.6 811.2 -+ 86.2 1003.3-+ 99.8
MOTOR
391
BEHAVIORS
IN INTACT
RATS
Rearing (number of times) 65.7 52.7 49.3 60.6
Head shaking (number of times)
-+ 6.2 - 5.1 - 5.5 + 5.5
11.8 12.2 5.5 8.3
--- 2.2 -+ 3.3 - 0.8 -+ 1.6
Grooming (seconds) 430.3 501.3 350.8 367.0
- 42.3 -+ 55.1 --- 41.6 + 24.1
Values are expressed as mean --- SEM of 10 intact female rats on each group. DI = Diestrus and E = Estrus. P R O G = progesterone injected SC 6 h prior to the test. Oil = Corn oil vehicle for PROG. Comparisons were made by using one-way A N O V A , no significant differences were detected.
in grooming behavior (p < .01). Comparing Table I and Table II, it can be observed that ovariectomy itself reduced motor activity and rearing behavior (p < .0025 in both cases). DISCUSSION As the plasma levels of estrogen and progesterone fluctuate (Butcher et al., 1974), the animal undergoes several modifications in its behavioral state (Beatty, 1979; B e c k e r et al., 1982, 1987; Birke & Archer, 1975; Diaz-V61iz et al., 1989; Drury & Gold, 1978; H y d e & Jerussi, 1983; Sfikakis et al., 1978; Wade, 1972). The present findings contribute to support the idea that the acquisition o f a conditioned avoidance response (CAR) and of some spontaneous motor behaviors of female rats are also influenced by the ovarian hormones. Previously we found a significant impairment of the conditioned avoidance performance during estrus compared with diestrus (Diaz-V61iz et al., 1989). This impairment has been associated with endogenous estradiol and/or progesterone fluctuations observed across the estrous cycle (Butcher et al., 1974). We observed that the impairment in avoidance conditioning begins during the morning of proestrus (Dfaz-V61iz et al., 1989), when the levels of estradiol are high, and the greatest impairment in acquiring a CAR
TABLE II.
PROGESTERONE
EFFECTS
ON SPONTANEOUS
OVARIECTOMIZED
Treatment Oil EB Oil EB
+ Oil (2/xg) + Oil + PROG (5 mg) + PROG
Motor activity (counts) 516.6 460.1 645.7 593.4
-+ 19.7 _+ 35.8 - 79.6 __+43.0
Rearing (number of times) 27.9 37.6 27.1 37.1
_ 2.9 _+ 3.7* _ 1.3 -+ 3.5*
MOTOR
BEHAVIORS
IN
RATS
Head shaking (number of times) 8.9 18.6 6.7 16.9
_-+ 1.3 _ 3.6* --- 0.9 _+ 2.3*
Grooming (seconds) 554.3 375.1 411.2 391.9
__+64.7 _+ 22.9* _ 24.1" -+ 33.2*
Values are expressed as mean -+ SEM of 10 ovariectomized rats on each group. EB = estradiol benzoate and PROG = progesterone injected 6 h prior to the test. Oil = Corn oil vehicle for EB and PROG. Comparisons were made by using oneway A N O V A followed by Newman-Keuls test. (*significantly different from the group receiving Oil + Oil, p < .025).
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G. DIAz-VI~LIZet al.
was observed during estrus when the estrogen levels are low (Butcher et al., 1974), indicating that these events could be associated with estradiol withdrawal. This suggestion is further supported by our finding that the facilitation of avoidance performance induced by ovariectomy was reversed only 48 h after the administration of a physiologically relevant dose of estradiol (Dfaz-Vrliz et al., 1989, 1991). The dose of EB used in the present work (2 pg) is the minimum amount needed to induce sexual receptivity in ovariectomized rats (Davidson et al., 1968) and to reverse changes in endocrine target organs caused by ovariectomy, inducing vaginal smears similar to that observed at estrous, with a latency of 3 days (Davidson et al., 1968; DiazVrliz et al., 1991). It is important to consider that a minimal dose of estradiol is required to induce delayed effects on behavior when serum estradiol levels become undetectable (Cheng & Johnson, 1974). The dose of PROG used in this study (5 mg) has demonstrated to influence behavioral changes in intact and ovariectomized rats, which are not seen with lower doses (Drury & Gold, 1978; Rodier, 1971). The present study shows that PROG alone was not able to modify the acquisition of conditioned avoidance responses in intact rats at diestrus nor in ovariectomized rats not treated with EB. Nevertheless, PROG antagonized the impairment on the acquisition of CARs both in intact rats at estrus and in ovariectomized rats treated with EB. These observations indicate that the effect of PROG on the acquisition of an avoidance task may be dependent on the presence of the ovary or the previous treatment with EB. Then, PROG seems to have a secondary role in this behavior, probably modulating actions of estradiol on the brain. Our hormonal manipulations failed to induce consistent changes in spontaneous motor behaviors in intact female rats. EB treatment of ovariectomized rats resulted in an increase in the number of head shakes and the number or rears, but there was no significant evidence of an influence of PROG on these behaviors. The grooming behavior that increased after ovariectomy (Diaz-Vrliz et al., 1989, and this study), was inhibited by the administration of both ovarian hormones. In addition, ovariectomy caused a decrease in motor activity and rearing behavior, but hormonal treatments were not able to restore preovariectomized levels of these behaviors. Thus, it seems that active avoidance conditioning and spontaneous motor behaviors are responses which are independently modulated by ovarian hormones and that changes in one behavior are not consequence of changes in another. Besides, little is known about the neural pathways that are activated once these hormones reach the brain. There is several evidence for an interaction between estradiol and progesterone in a variety of functions. They interact in the secretion of gonadotrophins from the anterior pituitary (Karsch, 1987) and in the promotion of sexual and nonsexual behaviors. PROG can inhibit, as well as facilitate, estrogen's effects on lordosis (Edward et al., 1968). On the other hand, PROG injection decreases wheel running and increases body weight in intact female rats and in ovariectomized rats receiving EB replacement; however, PROG does not influence these behaviors in ovariectomized rats not treated with EB, suggesting that PROG effects requires the presence of endogenous or exogenous estrogen (Rodier, 1971). Ovarian steroids can play a modulatory role of dopaminergic activity in the rat central nervous system. For example, neurochemical data indicate that EB treatments can influence dopamine turnover (Di Paolo et al., 1985; Fern~indez-Ruiz et al., 1990), and DA receptor binding (Hruska et al., 1980). The effect of estradiol on the activity of dopaminergic neurons is not observed when estradiol-treated rats are injected with a
PROGESTERONE AND CONDITIONED AVOIDANCE
393
single dose of PROG (Fernfindez-Ruiz et al., 1990). In addition, estrogen had a stimulatory effect on stereotypy in nondrug-treated rats, while PROG had an inhibitory effect (Michanek & Meyerson, 1982). This suggests the possibility of a negative interaction between both steroids. In a previous paper we postulated that the effects of estradiol on active avoidance conditioning and other behaviors could be explained through an interaction with dopaminergic systems (Dfaz-Vrliz et al., 1991). According to the present results, PROG and EB could be acting in an opposite way on these systems. It has been postulated that sex hormones are capable to regulate neuronal functions by genomic and nongenomic mechanisms (McEwen, 1991). We observed that the steroid effects on avoidance behavior take many hours or days to appear and those of progesterone seem to be dependent on the stage of the estrous cycle or of prior estrogen priming; this implies a dependence of the actions of estradiol possibly via genomic mechanisms. The effects of PROG described in this report are very similar to that observed after the administration of a synthetic antagonist of LHRH. Recently (Mora et al., 1993) we demonstrated that a single injection of 10 /.~g of [N-acetyl-D-p-chloro-Phe~.2,D-TrP3 , DArg6,D-AIa~0]-LHRH stimulated the acquisition of CARs in intacts rats at estrus, and in ovariectomized treated with EB, but neither in rats at diestrus nor in ovariectomized animals without treatment with EB. In this context, the ability of PROG to block the estrogen-induced LH and FSH release has been established (Dierschke et al., 1973). To summarize, the results show that PROG treatment results in increased acquisition of CARs in intact rats at estrus and in estrogen-primed ovariectomized rats. In contrast, PROG does not influence this behavior in intact rats at diestrus nor in ovariectomized rats not treated with EB. Then, the PROG effect seems to require the presence of endogenous or exogenous estrogen. The nature of estradiol-progesterone interaction remains unclear, but it could explain the changes in the acquisition of CARs and other behaviors observed across the estrous cycle of the rat.
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