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Estrogen-progesterone and 8-OH-DPAT attenuate the lordosis-inhibiting effects of the 5-HT1A agonist in the VMN
BRAIN RESEARCH ELSEVIER
Brain Research 637 (1994) 173-180
Research Report
Estrogen-progesterone and 8-OH-DPAT attenuate the lordosis-inhibiting effects of the 5-HTIA agonist in the VMN Lynda Uphouse *, Marjay Caldarola-Pastuszka, Sharmin Maswood, Martha Andrade, Nekayla Moore Department of Btology, Texas Woman's Unwerstty, Denton, TX 76204, USA (Accepted 5 October 1993)
Abstract
Ovariectomized rats were treated for 2 consecutive weeks with 25 Izg estradiol benzoate followed 48 h later with 500 ~zg progesterone. Bilateral infusions of 200 ng 8-hydroxy-2-(dl-n-propylamino)tetralin (8-OH-DPAT) into the ventromedial nucleus of the hypothalamus (VMN) inhibited female sexual behavior on the first but not the second week of hormone priming. Such attenuation on the second week of priming did not appear to result from an enhanced receptivity of the female rats since there were no differences in the L / M ratios prior to drug infusion; nor was the attenuation a consequence of infusion-induced VMN damage since neither saline nor 8-OH-DPAT preinfusions prevented the later inhibitory effects of 8-OH-DPAT on lordosis behavior. However, preinfusion with 8-OH-DPAT may have reduced the duration of the inhibition. Hormone priming (without any VMN infusion) also partially attenuated the effect of 8-OH-DPAT. Both hormone priming and treatment with 8-OH-DPAT were required to eliminate the effects of the second 8-OH-DPAT treatment. Thus, the present results suggest that gonadal hormones, alone, slightly attenuate the effects of agonlst activation of 5-HT~A receptors involved in the inhibition of lordosis behavior; that agonist activation of 5-HT1A receptors also produces a slight attenuation; but that both treatments together have a robust protective action against the inhibitory effect of a 5-HT~A agonist on female lordosis behavior.
Key words: SerotoninlA receptor; Ovariectomized rat; Intact rat; Desensitization; Female sexual behavior
1. Introduction
In recent years, it has been recognized that serotonin's (5-HT) role in regulating female sexual behavior includes both facilitatory and inhibitory mechanisms. Facilitatory effects are thought to result from 5-HT's interaction with m e m b e r s of the 5-HT 2 receptor family [15,17]. Inhibitory effects are mediated by agonist activation of the 5-HT1A receptor [1,16]. Work in our laboratory has demonstrated that 5-HTIA receptors in the ventromedial nucleus of the hypothalamus (VMN) are partially responsible for the inhibitory effects of 5-HTIA agonists on female rodent sexual behaviors [24]. Since inhibition occurs when V M N presynaptic terminals are lesioned by 5,7-DHT 2, postsynaptic 5-HTIA receptors a p p e a r to be responsible for the suppression of lordosis behavior. It is therefore reason-
* Corresponding author 0006-8993/94/$07.00 © 1994 Elsevier Soence B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) E 1 3 7 8 - G
able to expect that on the night of proestrus (when sexual behavior is present in the intact, regularly cycling female rat), such 5-HTtA activation must be suppressed. Although it is not known how such suppression might occur, Lakoski [12] has suggested that somatodendritic 5-HTIA autoreceptors of the dorsal raphe neurons ( D R N ) are reduced by estrogen treatment. This suggestion is consistent with observations that the 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), induces less hyperphagia in proestrous than in diestrous rats [26] and that estrogen contributes significantly to the reduced sensitivity of the proestrous females [20]. Although effects of estrogen on the postsynaptically mediated events of select 5-HT receptors has received less attention, the 'serotonin syndrome', which is believed to result from stimulation of postsynaptic receptors in the brainstem [21], can be elicited more easily in female than in male rats [8]. Moreover, 3H-5-HT binding in several brain areas is decreased after estradiol benzoate treatment [4,5,27]. The studies
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L Uphouseet al /Brain Research 637 (1994) 173-180
described below suggest that the 5-HTIA receptors which contribute to 8 - O H - D P A T ' s inhibition of lordosts behavior are modulated by exposure to gonadal hormones and by prior exposure to 8-OH-DPAT.
2. Materials 8-Hydroxy-2-(dl-n-propylammo)tetrahn(8-OH-DPAT) was purchased from Research Blochemlcals (Nauck, MA) Estradiol benzoate and progesterone were purchased from Sigma Chemical (St. Lores, MO) Intracranial cannulae were purchased from Plastic Products (Roanoke, VA) and dental acryhc was purchased from Rehance Dental Mfg. (Worth, IL). All other supplies came from Fisher Scientific (Houston, TX)
3. General methods 3 1. Housmg and treatment of ammals Adult, female rats (CDF-344) were bred in the laboratory from stock obtained from Sasco Laboratories (Omaha, Nebraska). Rats were weaned into polycarbonate shoebox cages at 25 days of age and were housed three or four per cage with like-sex littermates. The colony room was maintained at 22°C and 55% humidity on a 12/12 h l i g h t / d a r k cycle with lights off at 12 noon (CST). Food and water were available ad lib. When ovariectomized rats were used, bilateral ovariectomy was p e r f o r m e d with methoxyflurane (Metofane) anesthesia. For hormone priming, ovariectomized rats were injected s.c. with 25 /~g estradiol followed 48 h later with 500 /zg progesterone (s.c.). When intact females were used, behavioral testing took place on the afternoon of proestrus. Vaginal smears with nucleated cells or primarily nucleated with a few cornified cells, but an absence of leucocytes, were judged as proestrous smears. All females were virgin at the initiation of the experiment.
3.2 Intracrantal tmplantation and testing procedures Rats were anesthetized with Metofane and implanted bilaterally with 22 gauge stainless steel guide cannulae advanced stereotactically into the ventromedial nucleus of the hypothalamus (VMN) [atlas coordinates from Konig and Klippel [11], AP 4.38; D V 7.8; ML + 0.4] as previously described [23]. On the day that females were to receive intracranial infusions, females received a brief pretest to confirm their sexual receptivity prior to initiation of the experiment. Only highly receptive females ( L / M ratio >_ 0.90) were included in the studies (it was necessary to exclude only one rat on the basis of this criterion). Sexually receptive females had their dummy cannulae replaced with 28 gauge
stamless steel mternal cannulae (terminating 0.5 m m below the guide cannulae), attached by tubing (ID = 0.58 mm; O D = 0.96 mm) to a BAS ( C M A / 1 0 0 ) microinjector. Sexual behawor of the females was tested within a C M A / 1 2 0 containment system (BAS). This system consists of a clear, round-bottom, plcx~glass chamber (13 1 / 2 inch high, 15 3 / 8 inch diameter at the top, 12 inch diameter at the level of the rat) especially designed for infusion of awake animals. The system is equipped w~th an overhanging 'arm' and liquid swivel for attachment of tubing from the animal's cannulae to the infusion pump. Testing was inittated within the first 1-3 h after lights off. The female was allowed to adjust to the chamber for 5-10 min. The male (previously adapted to the containment system and the infusion apparatus) was then placed with the female. The female's behavior was recorded continuously for 5 - 1 0 mounts prior to infusion, during the infusion and for 30 min after the infusion. Infusions were administered at 0.24 to 0.26 / z l / m i n to a final infusion volume per cannula site of 0.5/~1.
3.3. Testing for sexual behactor Sexual receptivity was monitored as previously described [25] and was quantified as the lordosis to mount ( L / M ) ratio (e.g., number of lordosis responses by the female divided by the number of mounts by the male).
3.4. Histological procedures Females, deeply anesthetized with Metofane, were perfused intracardially with 0.9% saline, followed by 10% buffered formalin. The brain was excised and placed in 10% buffered formalin for a minimum of 24 h before sectioning (100/zm) with a Lancer vibratome. Tissue sections were stained with cresyl violet and cannulae locations were verified according to Konig and Klippel [11]. The location of each cannula was determined by an individual without knowledge of the experimental treatment or behavioral results. For each cannula, the most ventral location of the tract was determined and the cannula location was taken as the first section in which the most ventral location occurred. Only rats with V M N implants were included in the studies.
4. Statistical procedures The data were analyzed by repeated measures A N O V A . When data within an individual treatment were compared to their pretreatment data, individual means were compared by Dunnett's test. When data between treatments were compared within a testing
L Uphouse et al ~Brain Research 637 (1994) 173-180
interval, individual means were compared by the Tukey test. The statistical reference was Zar [29] and an a-level of 0.05 was required for rejection of the null hypothesis.
5. Specific methods In the first experiment, 2 weeks after ovariectomy, eight rats were implanted with guide cannulae in the VMN. Two weeks later (4 weeks after ovariectomy), rats were injected s.c. with 25 ~g estradiol benzoate (50 pl/rat, s.c.) followed 48 h later by 500 pg progesterone (100/xl/rat, s.c.). Four to 6 h later, rats were screened for sexual receptivity. After a 5-10 mount pretest, sexually receptive females were infused into the VMN with 200 ng 8-OH-DPAT. Sexual behavior was recorded during the pretest, the 2 min infusion and for six consecutive 5-min intervals after the infusion. After testing, the female was returned to her home cage. The next week, the procedures were repeated. The effect of 8-OH-DPAT during the first and second week of hormone priming was evaluated by 2-way repeated measures ANOVA with week of priming as the independent factor and time after infusion as the repeated factor. In the second experiment, 21 ovariectomized rats were implanted with guide cannulae in the VMN. Intracranial surgery was performed 2 weeks after ovariectomy and all behavioral studies were performed on the second week after implantation (4 weeks after ovariectomy). One week before behavioral testing, 12 rats received an infusion with either 0.5 /xl of 0.9% saline (S-EPD, n = 8) or 200 ng 8-OH-DPAT (D-EPD, n = 4). Five rats (EPS-EPD) received estradiol benzoate (25/xg) and progesterone (500 ~g) priming plus a saline infusion (0.5/xl) 1 week before testing; four rats received only the hormone priming (EP-EPD). The effect of the saline infusion in rats that were hormonally primed and infused with saline (EPS-EPD) was evaluated on the first week of hormone priming. On the week of behavioral testing, all rats were treated s.c. with 25/zg estradiol benzoate and 500/~g progesterone followed 4-6 h later with 200 ng 8-OH-DPAT. Data were evaluated by 2-way repeated measures ANOVA with type of pretreatment and time after infusion as the repeated factor. In the third experiment, intact, regularly cycling female rats received VMN implants. Seven rats received a preinfusion with 200 ng 8-OH-DPAT one estrous cycle prior to the behavioral testing. Rats were preinfused on the day of proestrus (determined by the vaginal smear) with 0.5 /xl saline containing 200 ng 8-OH-DPAT and returned to their home cage. When the female again had a vaginal smear indicative of proestrus, the effects of 8-OH-DPAT on lordosis be-
175
havior were examined. Eleven additional intact rats received no preinfusion but were infused with 8-OHDPAT on the day of behavioral testing. L / M ratios were evaluated by 2-way repeated measures ANOVA with type of pretreatment as the independent factor and time after infusion as the repeated factor. In the final experiment, female rats were first implanted with VMN cannulae. Two weeks later, rats were ovariectomized. Eight rats (no deprivation) were injected with 25 /xg estradiol benzoate immediately after ovariectomy and were injected with 500/zg progesterone 40-48 h later. The effect of 200 ng 8-OHDPAT infusions on lordosis behavior was examined 4-6 h later. Seven days after ovariectomy, the hormone priming was repeated and the effect of 8-OH-DPAT was again examined. Six rats (deprivation) were injected with sesame oil immediately after ovariectomy. Seven days later they were primed with estrogen and progesterone and subjected to behavioral testing. Data were compared by repeated measures ANOVA.
6. Results In the first experiment, eight female rats that had been ovariectomized for 2 weeks were implanted bilaterally with cannulae in the VMN. Two weeks later (4 weeks after ovariectomy), the females received their first treatment with estrogen. Forty-eight h later, rats received 500 pg progesterone and the effects of 8OH-DPAT were examined that afternoon. Five days after testing, rats were again primed with estrogen followed 48 h later with progesterone and the effects of 8-OH-DPAT were examined 4-6 h later. As is evident from Fig. 1, the effects of 8-OH-DPAT were dependent on the week of testing. Infusion with 200 ng 8-OHDPAT produced a significant decline in the L / M ratio on the first, but not the second, week of hormone priming. As a result, there were significant effects of the number of weeks of hormone treatment (F1,14 = 45.63, P < 0.0001), time after infusion (F7,98 = 9.96, P < 0.0001) and the time-week of treatment interaction (F7,98 = 5.82, P < 0.0001). On the first week of treatment, there was a significant decline in the L / M ratio (different from the pretest interval by 5 min after infusion and at every interval thereafter (all q98,8 >~2.39; P _<0.05; Dunnett's test)). In contrast, on the second week of infusion, the L / M ratio was never significantly different from the pretest. Consequently, by 5 min after infusion, the L / M ratio on the second week of hormonal priming and 8-OH-DPAT infusion was significantly greater than that on the first week (Tukey test; all q98,2 >-- 2.83, P < 0.05). L / M ratios during the pretest and the infusion period were not different on the 2 weeks (Tukey, P > 0.05). Thus, females appeared to differ in their response to 8-OH-DPAT on the 2
L Uphou,w et al /Bram Research 637 (1994) 173-180
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Fig 1. Effects of 2 consecutwe weeks of 8 - O H - D P A T infusions into the VMN on lordosis behavior. This shows the mean_+ S.E.M L / M ratios for eight hormonally primed, ovarlectomlzed rats infused with 200 ng 8 - O H - D P A T during the first and second weeks of hormone priming The figure shows the average L / M ratio during the preinfusion period, the infusion interval and six consecutive 5-mln intervals after infusion. Asterisks indicate the intervals during which the L / M rauo was significantly lower on the first relative to the second week of hormonal priming At these same intervals, the L / M ratio in animals gwen their first infusion with 8 - O H - D P A T was significantly different from their premfuslon interval
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weeks of treatment and not in their overall level of receptivity. These results suggested that on the second week of hormone priming, the lordosis-inhibiting effects of 8OH-DPAT had been attenuated. Such an outcome could occur if the first infusion had damaged tissue at the cannulae tips, thus reducing the effectiveness of the second infusion. Alternatively, such an outcome could occur if prior infusion with 8-OH-DPAT reduced the effectiveness of a second treatment. Finally, attenuation might result from the female's prior experience in the testing cylinder. That none of these explanations, alone, can account for the reduced sensitivity to 8-OH-DPAT after prior hormone priming and 8-OHDPAT infusion is clear from Fig. 2. Fig. 2A shows the effects of an 8-OH-DPAT infusion into the VMN of female rats primed for the first time with estrogen and progesterone but preinfused 7 days earlier with either 0.5 /xl saline or 0.5 /~1 saline containing 200 ng 8-OH-DPAT (respectively S-EPD and D-EPD). A decline in the L / M ratio was present in both treatment conditions (ANOVA F7,70---4.85, P < 0.0002) by 10 min after infusion. Animals preinfused with saline were significantly different from their pretest interval by 10 min after infusion and at every interval thereafter (Dunnett's q > 2.69, P < 0.05). Females preinfused with 8-OH-DPAT had reduced L / M ratios at 10, 15 and 20 min after infusion. Neither the treatment nor the time-treatment interaction were significant (P > 0.05), but females preinfused with 8-OHDPAT appeared to recover from the inhibitory effects
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Fig 2 Various pretreatments and the effects of 8 - O H - D P A T infusions into the V M N on lordosis behavior Fig. 2A shows the mean +_ S.E M L / M ratios for ovanectomlzed rats infused with either sahne (S-EPD, n = 8) or 200 ng 8 - O H - D P A T (D-EPD, n = 4) 1 week prior to behavioral testing. On the week of behavioral testing, all rats received 25 txg estradlol benzoate followed 48 h later with 500 ~ g progesterone, Behavioral testing before and after infusion with 200 ng 8 - O H - D P A T occurred 4 - 6 h after progesterone. Asterisks indicate those time intervals during which the L / M ratio was s]gnificantly different from the pretest interval. Fig. 2B shows the m e a n + S.E.M L / M ratios for ovanectomlzed rats treated 1 week before behavioral testing w~th estradiol benzoate (25 tzg) and progesterone (500 /zg). Fwe of these rats (EPS-EPD) were tested for sexual behavior before and after recewing an infusion with 0.5 /~1 saline (4-6 h after progesterone) (data not shown in the figure), four rats (EP-EPD) received no infusion. The next week, all rats were primed with estradiol benzoate and progesterone and infused with 8-OHD P A T 4 - 6 h later The data mdicate the L / M ratios before, during and after 8 - O H - D P A T infusion. The asterisks indicate those intervals during which the L / M ratio was significantly different from the pretest interval
L Uphouse et al ~Brain Research 637 (1994) 173-180
in the L / M ratio after 8-OH-DPAT t r e a t m e n t (F7,49 = 3.93, P < 0.002) but not until 20 min after infusion (Dunnett's q >_ 2.02, P _ 0.05). Both groups continued to exhibit reduced L / M ratios 25 min after infusion but by 30 min, the EP-EPD group was no longer different from its pretest interval. Neither the treatment nor the time-treatment interaction were significant (P > 0.05). Although none of these pretreatments prevented the inhibitory effects of 8-OH-DPAT on lordosis behavior, prior treatment with gonadal hormones appeared to delay the onset and reduce the duration of inhibition; preinfusion with 8-OH-DPAT appeared to reduce the duration but not the onset of the inhibition. It is important to note that, in each case, inhibition was less than that observed in Experiment 1 in rats treated for the first time with estrogen, progesterone and 8OH-DPAT and greater than that observed in rats treated for the second time with estrogen, progesterone and 8-OH-DPAT (Fig. 1). To see if the intact female would also show some protection from prior infusion with 8-OH-DPAT, we infused seven intact, regularly cycling female rats on the day of proestrus with 0.5/.~l saline containing 200 ng 8-OH-DPAT. No behavior testing was performed after this first infusion. When females again showed a proestrous smear, they were again infused with 200 ng 8-OH-DPAT and behavior was monitored for 30 min after infusion (Fig. 3). Although a decline in the L / M ratio was present (F7,112 = 9.6, P _<0.0001), it was less robust in females previously infused with 8-OH-DPAT than in those previously infused for the first time with 8-OH-DPAT (ANOVA for time after infusion by pretreatment interaction F7,112 ---2.11, P_< 0.05). Rats in-
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Fig. 3. Effects of premfusion with 8-OH-DPAT on a subsequent 8-OH-DPAT infusion into the VMN of regularly cychng, intact female rats. This shows the mean+S.E.M. L / M ratio for seven regularly cycling, proestrous rats (preinf) infused bilaterally into the VMN with 200 ng 8-OH-DPAT after a prior infusion with 200 ng 8-OH-DPAT. Also shown are the mean± S.E.M. L / M ratios for 11 proestrous rats (no preinf) that were infused for the first time with 8-OH-DPAT. Asterisks indicate those intervals which were significantly different from the pretest interval.
177
fused for the first time with 8-OH-DPAT showed a rapid onset of inhibition (significantly different from their pretest interval by 5 min after infusion and at every interval thereafter; Dunnett's q112,8 ~- 2.65, P _< 0.05). Rats preinfused with 8-OH-DPAT were significantly different from their pretest interval between 10 and 20 min after infusion but not at 25 or 30 min (P > 0.05). Thus, the previous findings suggest that both preinfusion (particularly with 8-OH-DPAT) and prior hormone priming each offer some protection against the inhibitory effects of VMN infusions with 8-OH-DPAT on lordosis behavior. Although arguable, the hormone pretreatment may be more effective than the 8-OHDPAT pretreatment. The possibility that gonadal hormones may attenuate the effects of VMN infusions with 8-OH-DPAT on female lordosis behavior was unexpected since a similar dose of 8-OH-DPAT is effective in decreasing L / M ratios in intact proestrous rats [25,26]. However, it may explain why the magnitude of inhibition was more robust in ovariectomized rats primed for the first time with estrogen and progesterone than it was in the intact, regularly cycling female rats. Nevertheless, regardless of the relative effectiveness of either pretreatment, alone, it is clear that preinfusion with 8-OH-DPAT as well as pretreatment with estrogen essentially eliminates the effects of 8-OH-DPAT in inhibiting lordosis behavior. These results are consistent with the observations that the effects of estrogen treatment in ovariectomized rats extend beyond the 24-48 h required for elicitation of lordosis behavior [7]. We would like to suggest that a reduced sensitivity to 5-HT~A agonists (or alternatively a facilitation of events antagonistic to 5-HT1A activation) are included in these longer term effects of estrogen. However, rats used in these experiments were ovariectomized 2 weeks prior to VMN implantation and then allowed 2 weeks recovery from surgery before their use in the study. It might, therefore, be argued that the robust difference observed between the first and second treatment with estrogen, progesterone and 8-OH-DPAT reflects an enhanced sensitivity during the first week of treatment and not an attenuation during the second week of treatment (though either explanation would suggest an important interaction between the hormone priming and the 5HT1A agonist). Since lordosis behavior of intact, proestrous rats is inhibited by 8-OH-DPAT, it is unlikely that sensitivity to 8-OH-DPAT requires deprivation from estrogen. Nevertheless, to distinguish between these two possibilities, female rats received bilateral VMN implants before ovariectomy and were injected immediately after ovariectomy with either sesame oil or 25/zg estradiol benzoate. The oil-injected rats (deprivation group) received no other treatment until the next week. The estrogen-treated rats were injected
L Uphouse et al /Bram Research 637 (1994) 173-180
178
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Fig 4 Effects of Immediate hormone priming m ovar]ectom,zed rats on the effects of VMN mfus,ons of 8-OH-DPAT, showing the mean_+ S E.M. L / M ratio for eight ovarlectomlzed rats (no deprivation-first), rejected s c w,th 25 /zg estradlol benzoate immediately after ovariectomy. Forty-eight h later, rats were injected s c with 500 /zg progesterone, followed 4-6 h later by behaworal testing before and after VMN infusions with 200 ng 8-OH-DPAT One week after ovanectomy, the hormone priming was repeated and the effects of 200 ng 8-OH-DPAT were examined a second t,me (no deprivationsecond). The figure also shows the mean + S E M L / M ratios for six rats (deprwatzon) injected with sesame od immediately after ovariectomy. primed 1 week later with estradiol benzoate and progesterone and refused with 200 ng 8-OH-DPAT. Asterisks indicate those intervals m which the L / M rat,o was slgmficantly less than the pretest interval
40-48 h later with 500 ~g progesterone and the effects of 8-OH-DPAT were examined 4-6 h thereafter (no deprivation group). Seven days after ovariectomy, all rats were injected with 25 /~g estradiol benzoate, followed 48 h later with 500/.~g progesterone and behavioral testing that afternoon. The results, shown in Fig. 4, clearly demonstrate that the different response to 8-OH-DPAT on the first and second week of estrogen, progesterone and 8-OH-DPAT treatment does not result from ovariectomy-induced hormone deprivation. A significant effect of treatment (ANOVA F2,19= 5.55, P < 0.02) and the significant time-treatment interaction ( F 7 , 1 3 3 = 3.40, P < 0.0001) reflected the attenuated response to 8-OH-DPAT in the no deprivation animals tested for the second time with 8-OH-DPAT. Females injected for the first time with estrogen on the day of ovariectomy (no deprivation-First) and those injected for the first time with estrogen 1 week after ovariectomy (deprivation) showed comparable inhibition of the L / M ratio after infusion with 8-OH-DPAT (decrease in L / M between 15 and 30 min; Dunnett's q133,8 ~ 2.65, P < 0.05) while rats infused for the second time were never significantly different from their pretest interval ( P > 0.05). Thus, ovariectomy-induced deprivation from gonadal hormones cannot be responsible for the difference between a first and second week of treatment with estrogen, progesterone and 8-OH-DPAT.
The present results demonstrate that prior treatment with estrogen plus progesterone and 8-OH-DPAT in ovariectomized rats can reduce the effectiveness of a second 8-OH-DPAT treatment in decreasing lordosis behavior. Both hormone priming and pretreatment with 8-OH-DPAT appear to contribute to this attenuation. Whether both estrogen and progesterone are required is currently unknown. Both hormones have been reported to modulate the functioning of the 5-HT system [5,13,18,19,28] but studies of the effects of gonadal hormones on select 5-HT receptors is still limited. Lakoski [12] provided the initial evidence that the 5-HT1A autoreceptors, located on soma and dendrites of 5-HT neurons, were modulated by estrogen and work in our laboratory has been consistent with her findings [26]. However, the current findings most likely reflect an altered sensitivity of postsynaptic 5-HT1A receptors in the VMN. Further studies with a wider range of 8-OH-DPAT doses and additional 5-HTlA agonlsts will be required to substantiate this suggestion. It should, however, be noted that these experiments cannot rule out the possibility of potential confounding due to a ceiling effect for the measurement of lordosis behavior. Although the ovariectomized females showed a high level of lordosis behavior (e.g., L / M ratios between 0.9 and 1.0) in both the first and second weeks of hormone treatment, we can never completely eliminate the possibility that females primed for 2 consecutive weeks with estrogen and progesterone were actually more receptive than those in the first week of priming. However, preliminary work in our laboratory suggests that the effect of 8-OH-DPAT on behaviors other than the lordosis reflex are also influenced by hormone priming (unpublished observations). Hypothalamic release, reuptake and metabolism of 5-HT are altered by estrogen [13]. Altered sensitivity (or endogenous activation) of postsynaptic 5-HT1A sites could result from any or all of these mechanisms. It. is also of interest that hormonal priming has also been reported to reduce the inhibitory effect of the muscarinic blocker, scopolamine, on lordosis behavior [14]. It is, therefore, possible that the effects of estrogenprogesterone priming include an attenuation of multiple neurotransmitter events that are inhibitory to lordosis behavior. Estrogen and progesterone priming, alone, however, were considerably less effective in attenuating the effects of 8-OH-DPAT than was the combination of hormone priming and prior agonist activation. Although the somatodendritic 5-HTIA autoreceptors have been reported to rapidly desensitize in response to agonist activation [3,10], the ease with which postsynaptic 5-HT1A receptors can be desensitized by agonist
L. Uphouse et al. / Brain Research 637 (1994) 173-180
activation has been questioned [10]. Nevertheless, in the current studies, a single VMN infusion with 8-OHDPAT partially attenuated the effects of a second 8-OH-DPAT infusion into the VMN and gonadal hormones appeared to amplify this effect. A potential regulation of 5-HT receptors by estrogen is not without precedent. The binding of 3H-5-HT (reflecting primarily 5-HT 1 receptors rather than 5-HT 2 receptors) to cortical and hypothalamic membranes was reportedly decreased by estrogen treatment of ovariectomized rats [5,27]. Interestingly, 2 weeks of treatment with estrogen was also reported to increase 3H-spiroperidol binding with mianserin as a competitor (presumably reflecting 5-HT 2 rather than 5-HT 1 binding sites). Since the 5-HT 2 family of receptors is thought to facilitate female lordosis behavior, these findings raise the interesting possibility that estrogen might enhance the effectiveness of 5-HT-mediated facilitatory events that modulate female lordosis behavior. Since the endogenous ligand, 5-HT, would be expected to activate both facilitatory and inhibitory events, an inverse modulation of these events by the gonadal hormones that facilitate lordosis behavior would be an efficient mechanism for controlling the timing of female sexual receptivity. Thus, the present results suggest that estrogen and/or progesterone reduces the inhibitory effect on lordosis behavior of agonist activation of the 5-HT1A site. Whether or not a comparable enhancement of 5-HT's facilitation of lordosis behavior occurs after hormonal treatment is presently unknown. Acknowledgments The authors thank Mr. T~m Lair for carmg for animals within the ammal research facility. The research was supported in part by NIH GM08256 and by NIH RO1 HD28419 to LU.
8. References [1] Ahlenms, S.N., Fernandez-Guastl, A., Hjorth, S. and Larsson, D. Suppression of lordosis behavior by the putative 5-HT receptor agomst, 8-OH-DPAT, m the rat, Eur. J. Pharmacol, 124 (1986) 361-363. [2] Alello-Zaldivar, M., Luine, V. and Frankfurt, M. 5,7-DHT facilitated lordosis: effects of 5-HT agonists, NeuroReport, 3 (1992) 542-544. [3] Beer, M., Kennett, G.A. and Curzon, G. A single dose of 8-OH-DPAT reduces raphe binding of 3H-OH-DPAT and increases the effect of raphe stimulation on 5-HT metabolism, Eur. J Pharmacol., 178 (1990) 179-187. [4] Blegon, A., Bercovltz, H and Samuel, D. Serotonm receptor concentrations during the estrous cycle of the rat, Brain Research, 187 (1980) 221-225. [5] Biegon, A., Reches, A., Snyder, L. and McEwen, B.S. Serotonergic and noradrenergic receptors in the rat brain: modulation by chronic exposure to ovarian hormones, Life Sct., 32 (1983) 2015-2021. [6] Brown, H., Stewart, G., Salamanca, S. and Uphouse, L. Chlordecone on the night of proestrus inhibits female sexual
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behavior m the cychng Fischer-344 rat, Toxtcol. Appl Pharmacol., 110 (1991) 97-106. [7] Cohen, R.S. and Pfaff, D.W. Ventrome&al hypothalamic neurons in the mediation of long-lasting effects of estrogen on lordosis behavior, Progr. NeurobtoL, 38 (1992) 423-453. [8] Fischette, C.T., Blegon, A. and McEwen, B.S. Sex steroid modulation of the serotonm behavioral syndrome, Ltfe Sct., 35 (1984) 1197-1206. [9] Hardy, D.F. and DeBold, J.F Effect of coital stimulation upon behavior of the female rat, J Comp. Phystol Psychol, 78 (1972) 400-408 [10] Kennett, G A., Chaouloff, F., Marcou, M and Curzon, G. Female rats are more vulnerable than males in an animal model of depression, the possible role of serotonm, Brain Res, 382 (1986) 416-421 [11] Komg, J. and Khppel, R, The Rat Bram A Stereotaxw Atlas of the Forebram and Lower Parts of the Brain Stem, Wilhams and Wilkms, Baltimore, 1963 [12] Lakoskl, J.M, Cellular electrophystologlcal approaches to the central regulation of female reproductwe aging. In J.M. Lakoskl, J R. Perez-Polo and D.K. Rassin (Eds), Neurology and Neurobzology. Vol 50 Neural Control of Reproductwe Functton, Alan R. Liss, New York, 1989, pp. 209-220. [13] Lulne, V.N and Paden, C.M., Effects of monoamme oxidase inhibition on female sexual behawor, serotonin levels and type A and B monoamine oxldase activity, Neuroendocrmology, 34 (1982) 245-251. [14] Menard, C.S., Hebert, T J., Ross, S.M. and Dohanlch, G.P., The effect of estrogen treatment on scopolamine inhibition of lordosis, Horm Behau, 26 (1992) 364-374. [15] Mendelson, S D and Gorzalka, B.B., A facihtatory role for serotonm in the sexual behavior of the female rat, Pharmacol. Btochem Behav., 22 (1985) 1025-1033 [16] Mendelson, S D. and Gorzalka, B.B 5-HTIA Receptors. differential involvement in female and male sexual behavior in the rat, Phystol Behav, 37 (1986) 345-351. [17] Mendelson, S.D. and Gorzalka, B.B. Sex differences in the effects of 1-(m-trlfluoromethylphenyl) piperazine and l-(m-chlorophenyl) piperazme on copulatory behavior m the rat. Neuropharmacology, 29 (1990) 783-786. [18] Meyerson, B.J, Malmnas, C.O. and Everitt, B J Neuropharmacology, neurotransmitters and sexual behavior in mammals. In N. Adler, D Pfaff and R.W Goy (Eds.), Handbook of Be. havzoural Neurobtology, Plenum Press, New York, 1985, pp 495-536. [19] Rehavi, M., Sepcutl, H and Weizman, A., Upregulation of ~mipramine binding and serotonm uptake by estra&ol in female rat brain, Brain Res, 410 (1987) 135-139. [20] Salamanca, S. and Uphouse, L Estradiol modulation of the hyperphagm induced by the 5-HT~A agonist, 8-OH-DPAT. Pharmacol Btochem. Behav., 43 (1992) 953-955. [21] Tncklebank, M.D., Mlddlemlss, D N. and Nedl, J. Pharmacological analysis of the behavioural and therrnoregulatory effects of the putatwe 5-HT~A receptor agomst, RU 24969, m the rat, Neuropharmacology, 25 (1986) 877-886. [22] Uphouse, L. and Caldarola-Pastuszka, M. Female sexual behavior following intracerebral infusions of the 5-HTIA agonist, 8-OH-DPAT, Bram Research, 601 (1993) 203-208 [23] Uphouse, L., Caldarola-Pastuszka, M. and Droge, M. 8-OHDPAT m the mldbraln central gray inhibits lordosis behawor. Pharmacol. Btochem Behav , 43 (1992) 833-838. [24] Uphouse, L., Caldarola-Pastuszka, M. and Montanez, S. Intracerebral actions of the 5-HTIA agomsts, 8-OH-DPAT and buspirone and of the 5-HTIA partml agonist/antagomst, NAN-190, on female sexual behawor. Neuropharmacology, 31 (1992) 969981 [25] Uphouse, L., Montanez, S, Rlchards-Hlll, R , Caldarola-
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Pastuszka, M. and Droge, M., Effects of 8-OH-DPAT on sexual behaviors of the proestrous rat, Pharmacol Btochem BehaL,, 39 (1991) 635-640 [26] Uphouse, L, Salamanca, S and Caldarola-Pastuszka, M Gender and estrous cycle differences in the response to the 5-HTIA agomst 8-OH-DPAT, Pharmacol Btochem Behat', 40 (1991) 901-906 [27] Wllhams, J and Uphouse, L Serotonm binding sites during
proestrus and following estradlol treatment, Pharma¢ol Btochem Behat,, 33 (1989) 615-620 [28] Wdson, C A and Hunter, A J Progesterone stimulates sexual behavior m female rats by increasing 5-HT actwlty on 5-HT, receptors, Brain Research, 333 (1985) 223-229 [29] Zar, J H, Btostatlsttcal Anal wts, 2nd edn, Prentice-Hall, Englewood Cliffs, NJ, 1984
Brain Research 637 (1994) 173-180
Research Report
Estrogen-progesterone and 8-OH-DPAT attenuate the lordosis-inhibiting effects of the 5-HTIA agonist in the VMN Lynda Uphouse *, Marjay Caldarola-Pastuszka, Sharmin Maswood, Martha Andrade, Nekayla Moore Department of Btology, Texas Woman's Unwerstty, Denton, TX 76204, USA (Accepted 5 October 1993)
Abstract
Ovariectomized rats were treated for 2 consecutive weeks with 25 Izg estradiol benzoate followed 48 h later with 500 ~zg progesterone. Bilateral infusions of 200 ng 8-hydroxy-2-(dl-n-propylamino)tetralin (8-OH-DPAT) into the ventromedial nucleus of the hypothalamus (VMN) inhibited female sexual behavior on the first but not the second week of hormone priming. Such attenuation on the second week of priming did not appear to result from an enhanced receptivity of the female rats since there were no differences in the L / M ratios prior to drug infusion; nor was the attenuation a consequence of infusion-induced VMN damage since neither saline nor 8-OH-DPAT preinfusions prevented the later inhibitory effects of 8-OH-DPAT on lordosis behavior. However, preinfusion with 8-OH-DPAT may have reduced the duration of the inhibition. Hormone priming (without any VMN infusion) also partially attenuated the effect of 8-OH-DPAT. Both hormone priming and treatment with 8-OH-DPAT were required to eliminate the effects of the second 8-OH-DPAT treatment. Thus, the present results suggest that gonadal hormones, alone, slightly attenuate the effects of agonlst activation of 5-HT~A receptors involved in the inhibition of lordosis behavior; that agonist activation of 5-HT1A receptors also produces a slight attenuation; but that both treatments together have a robust protective action against the inhibitory effect of a 5-HT~A agonist on female lordosis behavior.
Key words: SerotoninlA receptor; Ovariectomized rat; Intact rat; Desensitization; Female sexual behavior
1. Introduction
In recent years, it has been recognized that serotonin's (5-HT) role in regulating female sexual behavior includes both facilitatory and inhibitory mechanisms. Facilitatory effects are thought to result from 5-HT's interaction with m e m b e r s of the 5-HT 2 receptor family [15,17]. Inhibitory effects are mediated by agonist activation of the 5-HT1A receptor [1,16]. Work in our laboratory has demonstrated that 5-HTIA receptors in the ventromedial nucleus of the hypothalamus (VMN) are partially responsible for the inhibitory effects of 5-HTIA agonists on female rodent sexual behaviors [24]. Since inhibition occurs when V M N presynaptic terminals are lesioned by 5,7-DHT 2, postsynaptic 5-HTIA receptors a p p e a r to be responsible for the suppression of lordosis behavior. It is therefore reason-
* Corresponding author 0006-8993/94/$07.00 © 1994 Elsevier Soence B.V. All rights reserved SSDI 0 0 0 6 - 8 9 9 3 ( 9 3 ) E 1 3 7 8 - G
able to expect that on the night of proestrus (when sexual behavior is present in the intact, regularly cycling female rat), such 5-HTtA activation must be suppressed. Although it is not known how such suppression might occur, Lakoski [12] has suggested that somatodendritic 5-HTIA autoreceptors of the dorsal raphe neurons ( D R N ) are reduced by estrogen treatment. This suggestion is consistent with observations that the 5-HT1A agonist, 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), induces less hyperphagia in proestrous than in diestrous rats [26] and that estrogen contributes significantly to the reduced sensitivity of the proestrous females [20]. Although effects of estrogen on the postsynaptically mediated events of select 5-HT receptors has received less attention, the 'serotonin syndrome', which is believed to result from stimulation of postsynaptic receptors in the brainstem [21], can be elicited more easily in female than in male rats [8]. Moreover, 3H-5-HT binding in several brain areas is decreased after estradiol benzoate treatment [4,5,27]. The studies
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L Uphouseet al /Brain Research 637 (1994) 173-180
described below suggest that the 5-HTIA receptors which contribute to 8 - O H - D P A T ' s inhibition of lordosts behavior are modulated by exposure to gonadal hormones and by prior exposure to 8-OH-DPAT.
2. Materials 8-Hydroxy-2-(dl-n-propylammo)tetrahn(8-OH-DPAT) was purchased from Research Blochemlcals (Nauck, MA) Estradiol benzoate and progesterone were purchased from Sigma Chemical (St. Lores, MO) Intracranial cannulae were purchased from Plastic Products (Roanoke, VA) and dental acryhc was purchased from Rehance Dental Mfg. (Worth, IL). All other supplies came from Fisher Scientific (Houston, TX)
3. General methods 3 1. Housmg and treatment of ammals Adult, female rats (CDF-344) were bred in the laboratory from stock obtained from Sasco Laboratories (Omaha, Nebraska). Rats were weaned into polycarbonate shoebox cages at 25 days of age and were housed three or four per cage with like-sex littermates. The colony room was maintained at 22°C and 55% humidity on a 12/12 h l i g h t / d a r k cycle with lights off at 12 noon (CST). Food and water were available ad lib. When ovariectomized rats were used, bilateral ovariectomy was p e r f o r m e d with methoxyflurane (Metofane) anesthesia. For hormone priming, ovariectomized rats were injected s.c. with 25 /~g estradiol followed 48 h later with 500 /zg progesterone (s.c.). When intact females were used, behavioral testing took place on the afternoon of proestrus. Vaginal smears with nucleated cells or primarily nucleated with a few cornified cells, but an absence of leucocytes, were judged as proestrous smears. All females were virgin at the initiation of the experiment.
3.2 Intracrantal tmplantation and testing procedures Rats were anesthetized with Metofane and implanted bilaterally with 22 gauge stainless steel guide cannulae advanced stereotactically into the ventromedial nucleus of the hypothalamus (VMN) [atlas coordinates from Konig and Klippel [11], AP 4.38; D V 7.8; ML + 0.4] as previously described [23]. On the day that females were to receive intracranial infusions, females received a brief pretest to confirm their sexual receptivity prior to initiation of the experiment. Only highly receptive females ( L / M ratio >_ 0.90) were included in the studies (it was necessary to exclude only one rat on the basis of this criterion). Sexually receptive females had their dummy cannulae replaced with 28 gauge
stamless steel mternal cannulae (terminating 0.5 m m below the guide cannulae), attached by tubing (ID = 0.58 mm; O D = 0.96 mm) to a BAS ( C M A / 1 0 0 ) microinjector. Sexual behawor of the females was tested within a C M A / 1 2 0 containment system (BAS). This system consists of a clear, round-bottom, plcx~glass chamber (13 1 / 2 inch high, 15 3 / 8 inch diameter at the top, 12 inch diameter at the level of the rat) especially designed for infusion of awake animals. The system is equipped w~th an overhanging 'arm' and liquid swivel for attachment of tubing from the animal's cannulae to the infusion pump. Testing was inittated within the first 1-3 h after lights off. The female was allowed to adjust to the chamber for 5-10 min. The male (previously adapted to the containment system and the infusion apparatus) was then placed with the female. The female's behavior was recorded continuously for 5 - 1 0 mounts prior to infusion, during the infusion and for 30 min after the infusion. Infusions were administered at 0.24 to 0.26 / z l / m i n to a final infusion volume per cannula site of 0.5/~1.
3.3. Testing for sexual behactor Sexual receptivity was monitored as previously described [25] and was quantified as the lordosis to mount ( L / M ) ratio (e.g., number of lordosis responses by the female divided by the number of mounts by the male).
3.4. Histological procedures Females, deeply anesthetized with Metofane, were perfused intracardially with 0.9% saline, followed by 10% buffered formalin. The brain was excised and placed in 10% buffered formalin for a minimum of 24 h before sectioning (100/zm) with a Lancer vibratome. Tissue sections were stained with cresyl violet and cannulae locations were verified according to Konig and Klippel [11]. The location of each cannula was determined by an individual without knowledge of the experimental treatment or behavioral results. For each cannula, the most ventral location of the tract was determined and the cannula location was taken as the first section in which the most ventral location occurred. Only rats with V M N implants were included in the studies.
4. Statistical procedures The data were analyzed by repeated measures A N O V A . When data within an individual treatment were compared to their pretreatment data, individual means were compared by Dunnett's test. When data between treatments were compared within a testing
L Uphouse et al ~Brain Research 637 (1994) 173-180
interval, individual means were compared by the Tukey test. The statistical reference was Zar [29] and an a-level of 0.05 was required for rejection of the null hypothesis.
5. Specific methods In the first experiment, 2 weeks after ovariectomy, eight rats were implanted with guide cannulae in the VMN. Two weeks later (4 weeks after ovariectomy), rats were injected s.c. with 25 ~g estradiol benzoate (50 pl/rat, s.c.) followed 48 h later by 500 pg progesterone (100/xl/rat, s.c.). Four to 6 h later, rats were screened for sexual receptivity. After a 5-10 mount pretest, sexually receptive females were infused into the VMN with 200 ng 8-OH-DPAT. Sexual behavior was recorded during the pretest, the 2 min infusion and for six consecutive 5-min intervals after the infusion. After testing, the female was returned to her home cage. The next week, the procedures were repeated. The effect of 8-OH-DPAT during the first and second week of hormone priming was evaluated by 2-way repeated measures ANOVA with week of priming as the independent factor and time after infusion as the repeated factor. In the second experiment, 21 ovariectomized rats were implanted with guide cannulae in the VMN. Intracranial surgery was performed 2 weeks after ovariectomy and all behavioral studies were performed on the second week after implantation (4 weeks after ovariectomy). One week before behavioral testing, 12 rats received an infusion with either 0.5 /xl of 0.9% saline (S-EPD, n = 8) or 200 ng 8-OH-DPAT (D-EPD, n = 4). Five rats (EPS-EPD) received estradiol benzoate (25/xg) and progesterone (500 ~g) priming plus a saline infusion (0.5/xl) 1 week before testing; four rats received only the hormone priming (EP-EPD). The effect of the saline infusion in rats that were hormonally primed and infused with saline (EPS-EPD) was evaluated on the first week of hormone priming. On the week of behavioral testing, all rats were treated s.c. with 25/zg estradiol benzoate and 500/~g progesterone followed 4-6 h later with 200 ng 8-OH-DPAT. Data were evaluated by 2-way repeated measures ANOVA with type of pretreatment and time after infusion as the repeated factor. In the third experiment, intact, regularly cycling female rats received VMN implants. Seven rats received a preinfusion with 200 ng 8-OH-DPAT one estrous cycle prior to the behavioral testing. Rats were preinfused on the day of proestrus (determined by the vaginal smear) with 0.5 /xl saline containing 200 ng 8-OH-DPAT and returned to their home cage. When the female again had a vaginal smear indicative of proestrus, the effects of 8-OH-DPAT on lordosis be-
175
havior were examined. Eleven additional intact rats received no preinfusion but were infused with 8-OHDPAT on the day of behavioral testing. L / M ratios were evaluated by 2-way repeated measures ANOVA with type of pretreatment as the independent factor and time after infusion as the repeated factor. In the final experiment, female rats were first implanted with VMN cannulae. Two weeks later, rats were ovariectomized. Eight rats (no deprivation) were injected with 25 /xg estradiol benzoate immediately after ovariectomy and were injected with 500/zg progesterone 40-48 h later. The effect of 200 ng 8-OHDPAT infusions on lordosis behavior was examined 4-6 h later. Seven days after ovariectomy, the hormone priming was repeated and the effect of 8-OH-DPAT was again examined. Six rats (deprivation) were injected with sesame oil immediately after ovariectomy. Seven days later they were primed with estrogen and progesterone and subjected to behavioral testing. Data were compared by repeated measures ANOVA.
6. Results In the first experiment, eight female rats that had been ovariectomized for 2 weeks were implanted bilaterally with cannulae in the VMN. Two weeks later (4 weeks after ovariectomy), the females received their first treatment with estrogen. Forty-eight h later, rats received 500 pg progesterone and the effects of 8OH-DPAT were examined that afternoon. Five days after testing, rats were again primed with estrogen followed 48 h later with progesterone and the effects of 8-OH-DPAT were examined 4-6 h later. As is evident from Fig. 1, the effects of 8-OH-DPAT were dependent on the week of testing. Infusion with 200 ng 8-OHDPAT produced a significant decline in the L / M ratio on the first, but not the second, week of hormone priming. As a result, there were significant effects of the number of weeks of hormone treatment (F1,14 = 45.63, P < 0.0001), time after infusion (F7,98 = 9.96, P < 0.0001) and the time-week of treatment interaction (F7,98 = 5.82, P < 0.0001). On the first week of treatment, there was a significant decline in the L / M ratio (different from the pretest interval by 5 min after infusion and at every interval thereafter (all q98,8 >~2.39; P _<0.05; Dunnett's test)). In contrast, on the second week of infusion, the L / M ratio was never significantly different from the pretest. Consequently, by 5 min after infusion, the L / M ratio on the second week of hormonal priming and 8-OH-DPAT infusion was significantly greater than that on the first week (Tukey test; all q98,2 >-- 2.83, P < 0.05). L / M ratios during the pretest and the infusion period were not different on the 2 weeks (Tukey, P > 0.05). Thus, females appeared to differ in their response to 8-OH-DPAT on the 2
L Uphou,w et al /Bram Research 637 (1994) 173-180
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weeks of treatment and not in their overall level of receptivity. These results suggested that on the second week of hormone priming, the lordosis-inhibiting effects of 8OH-DPAT had been attenuated. Such an outcome could occur if the first infusion had damaged tissue at the cannulae tips, thus reducing the effectiveness of the second infusion. Alternatively, such an outcome could occur if prior infusion with 8-OH-DPAT reduced the effectiveness of a second treatment. Finally, attenuation might result from the female's prior experience in the testing cylinder. That none of these explanations, alone, can account for the reduced sensitivity to 8-OH-DPAT after prior hormone priming and 8-OHDPAT infusion is clear from Fig. 2. Fig. 2A shows the effects of an 8-OH-DPAT infusion into the VMN of female rats primed for the first time with estrogen and progesterone but preinfused 7 days earlier with either 0.5 /xl saline or 0.5 /~1 saline containing 200 ng 8-OH-DPAT (respectively S-EPD and D-EPD). A decline in the L / M ratio was present in both treatment conditions (ANOVA F7,70---4.85, P < 0.0002) by 10 min after infusion. Animals preinfused with saline were significantly different from their pretest interval by 10 min after infusion and at every interval thereafter (Dunnett's q > 2.69, P < 0.05). Females preinfused with 8-OH-DPAT had reduced L / M ratios at 10, 15 and 20 min after infusion. Neither the treatment nor the time-treatment interaction were significant (P > 0.05), but females preinfused with 8-OHDPAT appeared to recover from the inhibitory effects
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Fig 2 Various pretreatments and the effects of 8 - O H - D P A T infusions into the V M N on lordosis behavior Fig. 2A shows the mean +_ S.E M L / M ratios for ovanectomlzed rats infused with either sahne (S-EPD, n = 8) or 200 ng 8 - O H - D P A T (D-EPD, n = 4) 1 week prior to behavioral testing. On the week of behavioral testing, all rats received 25 txg estradlol benzoate followed 48 h later with 500 ~ g progesterone, Behavioral testing before and after infusion with 200 ng 8 - O H - D P A T occurred 4 - 6 h after progesterone. Asterisks indicate those time intervals during which the L / M ratio was s]gnificantly different from the pretest interval. Fig. 2B shows the m e a n + S.E.M L / M ratios for ovanectomlzed rats treated 1 week before behavioral testing w~th estradiol benzoate (25 tzg) and progesterone (500 /zg). Fwe of these rats (EPS-EPD) were tested for sexual behavior before and after recewing an infusion with 0.5 /~1 saline (4-6 h after progesterone) (data not shown in the figure), four rats (EP-EPD) received no infusion. The next week, all rats were primed with estradiol benzoate and progesterone and infused with 8-OHD P A T 4 - 6 h later The data mdicate the L / M ratios before, during and after 8 - O H - D P A T infusion. The asterisks indicate those intervals during which the L / M ratio was significantly different from the pretest interval
L Uphouse et al ~Brain Research 637 (1994) 173-180
in the L / M ratio after 8-OH-DPAT t r e a t m e n t (F7,49 = 3.93, P < 0.002) but not until 20 min after infusion (Dunnett's q >_ 2.02, P _ 0.05). Both groups continued to exhibit reduced L / M ratios 25 min after infusion but by 30 min, the EP-EPD group was no longer different from its pretest interval. Neither the treatment nor the time-treatment interaction were significant (P > 0.05). Although none of these pretreatments prevented the inhibitory effects of 8-OH-DPAT on lordosis behavior, prior treatment with gonadal hormones appeared to delay the onset and reduce the duration of inhibition; preinfusion with 8-OH-DPAT appeared to reduce the duration but not the onset of the inhibition. It is important to note that, in each case, inhibition was less than that observed in Experiment 1 in rats treated for the first time with estrogen, progesterone and 8OH-DPAT and greater than that observed in rats treated for the second time with estrogen, progesterone and 8-OH-DPAT (Fig. 1). To see if the intact female would also show some protection from prior infusion with 8-OH-DPAT, we infused seven intact, regularly cycling female rats on the day of proestrus with 0.5/.~l saline containing 200 ng 8-OH-DPAT. No behavior testing was performed after this first infusion. When females again showed a proestrous smear, they were again infused with 200 ng 8-OH-DPAT and behavior was monitored for 30 min after infusion (Fig. 3). Although a decline in the L / M ratio was present (F7,112 = 9.6, P _<0.0001), it was less robust in females previously infused with 8-OH-DPAT than in those previously infused for the first time with 8-OH-DPAT (ANOVA for time after infusion by pretreatment interaction F7,112 ---2.11, P_< 0.05). Rats in-
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Fig. 3. Effects of premfusion with 8-OH-DPAT on a subsequent 8-OH-DPAT infusion into the VMN of regularly cychng, intact female rats. This shows the mean+S.E.M. L / M ratio for seven regularly cycling, proestrous rats (preinf) infused bilaterally into the VMN with 200 ng 8-OH-DPAT after a prior infusion with 200 ng 8-OH-DPAT. Also shown are the mean± S.E.M. L / M ratios for 11 proestrous rats (no preinf) that were infused for the first time with 8-OH-DPAT. Asterisks indicate those intervals which were significantly different from the pretest interval.
177
fused for the first time with 8-OH-DPAT showed a rapid onset of inhibition (significantly different from their pretest interval by 5 min after infusion and at every interval thereafter; Dunnett's q112,8 ~- 2.65, P _< 0.05). Rats preinfused with 8-OH-DPAT were significantly different from their pretest interval between 10 and 20 min after infusion but not at 25 or 30 min (P > 0.05). Thus, the previous findings suggest that both preinfusion (particularly with 8-OH-DPAT) and prior hormone priming each offer some protection against the inhibitory effects of VMN infusions with 8-OH-DPAT on lordosis behavior. Although arguable, the hormone pretreatment may be more effective than the 8-OHDPAT pretreatment. The possibility that gonadal hormones may attenuate the effects of VMN infusions with 8-OH-DPAT on female lordosis behavior was unexpected since a similar dose of 8-OH-DPAT is effective in decreasing L / M ratios in intact proestrous rats [25,26]. However, it may explain why the magnitude of inhibition was more robust in ovariectomized rats primed for the first time with estrogen and progesterone than it was in the intact, regularly cycling female rats. Nevertheless, regardless of the relative effectiveness of either pretreatment, alone, it is clear that preinfusion with 8-OH-DPAT as well as pretreatment with estrogen essentially eliminates the effects of 8-OH-DPAT in inhibiting lordosis behavior. These results are consistent with the observations that the effects of estrogen treatment in ovariectomized rats extend beyond the 24-48 h required for elicitation of lordosis behavior [7]. We would like to suggest that a reduced sensitivity to 5-HT~A agonists (or alternatively a facilitation of events antagonistic to 5-HT1A activation) are included in these longer term effects of estrogen. However, rats used in these experiments were ovariectomized 2 weeks prior to VMN implantation and then allowed 2 weeks recovery from surgery before their use in the study. It might, therefore, be argued that the robust difference observed between the first and second treatment with estrogen, progesterone and 8-OH-DPAT reflects an enhanced sensitivity during the first week of treatment and not an attenuation during the second week of treatment (though either explanation would suggest an important interaction between the hormone priming and the 5HT1A agonist). Since lordosis behavior of intact, proestrous rats is inhibited by 8-OH-DPAT, it is unlikely that sensitivity to 8-OH-DPAT requires deprivation from estrogen. Nevertheless, to distinguish between these two possibilities, female rats received bilateral VMN implants before ovariectomy and were injected immediately after ovariectomy with either sesame oil or 25/zg estradiol benzoate. The oil-injected rats (deprivation group) received no other treatment until the next week. The estrogen-treated rats were injected
L Uphouse et al /Bram Research 637 (1994) 173-180
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7. Discussion 0
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Fig 4 Effects of Immediate hormone priming m ovar]ectom,zed rats on the effects of VMN mfus,ons of 8-OH-DPAT, showing the mean_+ S E.M. L / M ratio for eight ovarlectomlzed rats (no deprivation-first), rejected s c w,th 25 /zg estradlol benzoate immediately after ovariectomy. Forty-eight h later, rats were injected s c with 500 /zg progesterone, followed 4-6 h later by behaworal testing before and after VMN infusions with 200 ng 8-OH-DPAT One week after ovanectomy, the hormone priming was repeated and the effects of 200 ng 8-OH-DPAT were examined a second t,me (no deprivationsecond). The figure also shows the mean + S E M L / M ratios for six rats (deprwatzon) injected with sesame od immediately after ovariectomy. primed 1 week later with estradiol benzoate and progesterone and refused with 200 ng 8-OH-DPAT. Asterisks indicate those intervals m which the L / M rat,o was slgmficantly less than the pretest interval
40-48 h later with 500 ~g progesterone and the effects of 8-OH-DPAT were examined 4-6 h thereafter (no deprivation group). Seven days after ovariectomy, all rats were injected with 25 /~g estradiol benzoate, followed 48 h later with 500/.~g progesterone and behavioral testing that afternoon. The results, shown in Fig. 4, clearly demonstrate that the different response to 8-OH-DPAT on the first and second week of estrogen, progesterone and 8-OH-DPAT treatment does not result from ovariectomy-induced hormone deprivation. A significant effect of treatment (ANOVA F2,19= 5.55, P < 0.02) and the significant time-treatment interaction ( F 7 , 1 3 3 = 3.40, P < 0.0001) reflected the attenuated response to 8-OH-DPAT in the no deprivation animals tested for the second time with 8-OH-DPAT. Females injected for the first time with estrogen on the day of ovariectomy (no deprivation-First) and those injected for the first time with estrogen 1 week after ovariectomy (deprivation) showed comparable inhibition of the L / M ratio after infusion with 8-OH-DPAT (decrease in L / M between 15 and 30 min; Dunnett's q133,8 ~ 2.65, P < 0.05) while rats infused for the second time were never significantly different from their pretest interval ( P > 0.05). Thus, ovariectomy-induced deprivation from gonadal hormones cannot be responsible for the difference between a first and second week of treatment with estrogen, progesterone and 8-OH-DPAT.
The present results demonstrate that prior treatment with estrogen plus progesterone and 8-OH-DPAT in ovariectomized rats can reduce the effectiveness of a second 8-OH-DPAT treatment in decreasing lordosis behavior. Both hormone priming and pretreatment with 8-OH-DPAT appear to contribute to this attenuation. Whether both estrogen and progesterone are required is currently unknown. Both hormones have been reported to modulate the functioning of the 5-HT system [5,13,18,19,28] but studies of the effects of gonadal hormones on select 5-HT receptors is still limited. Lakoski [12] provided the initial evidence that the 5-HT1A autoreceptors, located on soma and dendrites of 5-HT neurons, were modulated by estrogen and work in our laboratory has been consistent with her findings [26]. However, the current findings most likely reflect an altered sensitivity of postsynaptic 5-HT1A receptors in the VMN. Further studies with a wider range of 8-OH-DPAT doses and additional 5-HTlA agonlsts will be required to substantiate this suggestion. It should, however, be noted that these experiments cannot rule out the possibility of potential confounding due to a ceiling effect for the measurement of lordosis behavior. Although the ovariectomized females showed a high level of lordosis behavior (e.g., L / M ratios between 0.9 and 1.0) in both the first and second weeks of hormone treatment, we can never completely eliminate the possibility that females primed for 2 consecutive weeks with estrogen and progesterone were actually more receptive than those in the first week of priming. However, preliminary work in our laboratory suggests that the effect of 8-OH-DPAT on behaviors other than the lordosis reflex are also influenced by hormone priming (unpublished observations). Hypothalamic release, reuptake and metabolism of 5-HT are altered by estrogen [13]. Altered sensitivity (or endogenous activation) of postsynaptic 5-HT1A sites could result from any or all of these mechanisms. It. is also of interest that hormonal priming has also been reported to reduce the inhibitory effect of the muscarinic blocker, scopolamine, on lordosis behavior [14]. It is, therefore, possible that the effects of estrogenprogesterone priming include an attenuation of multiple neurotransmitter events that are inhibitory to lordosis behavior. Estrogen and progesterone priming, alone, however, were considerably less effective in attenuating the effects of 8-OH-DPAT than was the combination of hormone priming and prior agonist activation. Although the somatodendritic 5-HTIA autoreceptors have been reported to rapidly desensitize in response to agonist activation [3,10], the ease with which postsynaptic 5-HT1A receptors can be desensitized by agonist
L. Uphouse et al. / Brain Research 637 (1994) 173-180
activation has been questioned [10]. Nevertheless, in the current studies, a single VMN infusion with 8-OHDPAT partially attenuated the effects of a second 8-OH-DPAT infusion into the VMN and gonadal hormones appeared to amplify this effect. A potential regulation of 5-HT receptors by estrogen is not without precedent. The binding of 3H-5-HT (reflecting primarily 5-HT 1 receptors rather than 5-HT 2 receptors) to cortical and hypothalamic membranes was reportedly decreased by estrogen treatment of ovariectomized rats [5,27]. Interestingly, 2 weeks of treatment with estrogen was also reported to increase 3H-spiroperidol binding with mianserin as a competitor (presumably reflecting 5-HT 2 rather than 5-HT 1 binding sites). Since the 5-HT 2 family of receptors is thought to facilitate female lordosis behavior, these findings raise the interesting possibility that estrogen might enhance the effectiveness of 5-HT-mediated facilitatory events that modulate female lordosis behavior. Since the endogenous ligand, 5-HT, would be expected to activate both facilitatory and inhibitory events, an inverse modulation of these events by the gonadal hormones that facilitate lordosis behavior would be an efficient mechanism for controlling the timing of female sexual receptivity. Thus, the present results suggest that estrogen and/or progesterone reduces the inhibitory effect on lordosis behavior of agonist activation of the 5-HT1A site. Whether or not a comparable enhancement of 5-HT's facilitation of lordosis behavior occurs after hormonal treatment is presently unknown. Acknowledgments The authors thank Mr. T~m Lair for carmg for animals within the ammal research facility. The research was supported in part by NIH GM08256 and by NIH RO1 HD28419 to LU.
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