Effects of mianserin and ketanserin on lordosis behavior after systemic treatment or infusion into the ventromedial nucleus of the hypothalamus

BRAIN RESEARCH ELSEVIER

Brain Research 718 (1996) 46-52

Research report

Effects of mianserin and ketanserin on lordosis behavior after systemic treatment or infusion into the ventromedial nucleus of the hypothalamus Lynda Uphouse *, Lucy Colon, Amy Cox, Marjay Caldarola-Pastuszka,Amy Wolf Department of Biology, Texas Woman's University, Denton, 7X 76204, USA

Accepted 20 December 1995

Abstract The effects of the 5-HT2A/2 C antagonists, mianserin and ketanserin, were investigated in proestrous rats and in ovariectomized rats hormonally primed with estradiol benzoate and progesterone. Drugs were administered systemically or by infusion into the vicinity of the ventromedial nucleus of the hypothalamus (VMN). By both routes of administration, inhibition of lordosis behavior was seen following treatment with the 5-HT2A/2 C antagonists, but the magnitude of the decline was smaller in proestrous than in hormone-primed, ovariectomized rats. The results of these studies are discussed in terms of the dual role of serotonin in the control of female sexual behavior. Keywords: 5-HT2A/2c antagonist;Intact proestrous rat; Ovariectomizedrat; Lordosisbehavior

1. Introduction Serotonin (5-HT) is believed both to inhibit and facilitate female sexual behavior by acting on 5-HT1A and 5-HT2A/2c receptors, respectively [8]. Data leading to the assumption of 5-HT's dual role in the regulation of lordosis behavior have been based primarily on the use of 5-HT]A receptor agonists and 5-HT2A/2 C receptor antagonists to inhibit lordosis behavior of ovariectomized, hormone-primed female rats. Less emphasis has been placed on the effects of the compounds in the intact female. However, the inhibitory effects of 5-HT m receptor agonists have been readily confirmed in the intact, regularly cycling rat and the ventromedial nucleus of the hypothalamus (VMN) has been identified as an effective site for this inhibition [1,15]. The neural location of the putative lordosis-facilitating 5-HT2A/2 C receptors has not been determined. However, there is reason to suspect that the VMN may also be involved in 5-HT2A/2c receptor facilitation of lordosis behavior. The VMN is critical for gonadal hormone facilitation of the lordosis reflex [12]; hormone-induced changes in the VMN are required for execution of the reflex in response to the male's mount [13,14]; and 5-HT produces biphasic effects on neuronal firing in many brain areas, including

* Corresponding author. Fax: (1) (817) 898-2382. 0006-8993/96/$15.00 © 1996Elsevier ScienceB.V. All rights reserved PII S0006- 8993(96)00049-2

the VMN [6,10]. In hypothalamic slices including the VMN, the 5-HT m receptor agonist, (_)-8-hydroxy-2-(din-propylamino)tetralin (8-OH-DPAT), mimics the inhibitory effect and the 5-HT2A/2c receptor agonist, ( _ ) (2,5-dimethoxy-4-iodophenyl)-2-aminopropane HC1 (DOI), mimics the facilitatory effect of 5-HT on neuronal firing [6]. Furthermore, 5-HT's inhibition of neuronal firing was attenuated by DOI. Thus, Kow et al. [6] suggested that both the inhibitory and facilitatory effects of 5-HT on lordosis could be exerted in the VMN. Recent studies in our laboratory, in which the lordosis-inhibiting effect of VMN infusions with 8-OH-DPAT were blocked by co-infusion of DOI, are consistent with this suggestion [16]. The present studies were designed to determine if the reported inhibitory effects of 5-HT2A/2c antagonists in hormoneprimed ovariectomized rats also occurred in intact, regularly cycling proestrous rats and to determine if the VMN was an effective site for this inhibition.

2. Materials and methods 2.1. Materials

Ketanserin and mianserin were purchased from Sigma Chemical Co. (St. Louis, MO). Ketanserin tartrate was obtained from Research Biochemicals (Natick, MA). In-

L. Uphouseet al. / Brain Research 718 (1996) 46-52 tracranial (i.c.) cannulae were purchased from Plastics One (Roanoke, VA) and dental acrylic was purchased from Reliance Dental Mfg. Co. (Worth, IL). Methoxyflurane (Metofane) was purchased fi:om Pitman Moore (Mundelein, IL). All other supplies came from Fisher Scientific (Houston, TX). 2.2. Methods 2.2.1. Animals and housing conditions Adult, female rats (CDF-344), bred in our laboratory from stock obtained from Sasco Laboratories (Omaha, NE), were used in the experiments. After weaning, rats were housed 3 or 4 per cage with like-sex littermates in a colony room maintained at 22°C and 55% humidity on a 12:12 h light/dark cycle with lights off at 12 noon. Food and water were available ad libitum. When proestrous rats were used, rats were selected on the basis of their vaginal smear history. When i.c. infusions were performed with hormone-primed rats with VMN implants, rats were ovariectomized 2 weeks after implantation of VMN cannulae and hormone priming began 1 week after ovariectomy. 2.2.2. Surgical procedures Rats were anesthetized with Metofane and implanted bilaterally with 22 gauge stainless steel guide cannulae advanced stereotactically into the VMN (atlas coordinates from Konig and Klippel [5]: AP 4.38; DV 7.8; ML 0.4) as previously described [15-17]. Guide cannulae were secured with dental acrylic aJad anchored to the skull with 3 stainless steel screws. St~finless steel dummy cannulae, terminating approximately 0.5 mm below the guide, were placed in the guide cannulae at the time of surgery to prevent clogging. Bilateral ovariectomy was performed under Metofane anesthesia. Postsurgical monitoring was performed to assure the ~tnimal's comfort after surgical procedures. All procedures were approved by the IACUC. 2.2.3. Vaginal smearing procedures Vaginal smears were monitored as previously described [15]. Proestrous females were selected on the basis of their past vaginal smear history, the presence of nucleated cells and the absence of leucocytes, and the presence of sexual receptivity during the pretesting procedures. 2.2.4. Behavioral testing procedures Sexual receptivity was tested as previously described [15-17]. In all studies, sexual behavior was tested during the dark phase of the light/dark cycle. For intracranial procedures, rats had their dummy cannulae replaced with 28-gauge stainless steel internal cannulae (terminating 0.5 mm below the guide cannulae), attached by tubing to a BAS (CMA/100) microinjector. The sexual behavior of the females was tested within a C M A / 1 2 0 containment system (BAS). The female was allowed to adjust to the chamber for 5 - 1 0 min before the male (previously adapted

47

to the containment system and the infusion apparatus) was placed with the female. For each mount by the male, the presence or absence of a lordosis reflex by the female was recorded. Sexual receptivity 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). The female's behavior was recorded for 5 - 1 0 mounts prior to infusion with the appropriate agent, during the infusion, and continuously for 30 min (or 20 min in the study with mianserin) after the infusion. Infusions were delivered at 0.24 to 0.26 /xl/min to a final infusion volume per bilateral site of 0.5 ~1. For i.p. treatments, rats were pretested in the home cage of a sexually experienced male. The female was removed and injected with the test agent and placed back into the male's cage where sexual behavior was monitored continuously for 30 min after injection. For both i.p. and i.c. studies, males that failed to actively mount the female were replaced so that females were always tested with sexually active males. By this procedure, females received at least three mounts during each 5 min period. Any animal that failed to receive a sufficient number of mounts were excluded from the data analysis. 2.2.5. Drug treatments Mianserin was dissolved in 0.9% saline; ketanserin was dissolved in 0.01 N HC1; and ketanserin tartrate was dissolved in distilled/deionized water. For intracranial treatments, drugs were prepared so that the appropriate concentration per bilateral VMN site was present in 0.5 /xl of solution. Mianserin doses examined ranged from 250 ng to 4000 ng per 0.5/~1. Ketanserin doses ranged from 50 ng to 2500 ng per 0.5 /xl. Doses of ketanserin tartrate ranged from 500 ng to 3000 ng per 0.5 /zl. For i.p. treatments, drugs were prepared so that the appropriate concentration was delivered in a volume of 0.1 ml per 100 g body weight. Mianserin was used at doses from 1 m g / k g to 8 m g / k g ; doses of ketanserin varied from 1 m g / k g to 5 mg/kg. In the first three experiments, respectively, 42 proestrous rats (i.c. mianserin), 63 proestrous rats (i.c. ketanserin), and 30 proestrous rats (i.c. ketanserin tartrate) were used. In the fourth experiment, 27 ovariectomized rats, hormonally primed with 0.5 ~ g estradiol benzoate and 500/xg progesterone, and 13 proestrous rats were used to compare the effects of VMN infusion with ketanserin tartrate in ovariectomized and intact rats. Estradiol benzoate was dissolved in sesame seed oil and was administered subcutaneously (s.c.) at a volume of 0.1 ml per rat. 48 h after treatment with estradiol benzoate, rats were injected with 500 ~ g progesterone dissolved either in sesame seed oil or in propylene glycol so that each rat received a s.c. injection of 0.1 ml. Behavioral testing occurred 4 - 6 h after the progesterone injection. Thirty-six and 29 proestrous rats were used, respectively, for studying the effects of i.p. treatment with mi-

48

L. Uphouse et al./ Brain Research 718 (1996) 46-52

anserin and ketanserin. Eleven ovariectomized rats, hormortally primed with 25/xg estradiol benzoate and 500/zg progesterone, and 8 proestrous rats were used to compare the systemic effects of ketanserin in ovariectomized and intact rats.

2.2.6. Histological procedures Females were anesthetized with Metofane and 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 vibratome sectioning (100 /zm). Tissue sections were stained with cresyl violet and cannulae locations were verified according to Konig and Klippel [5]. The location of each cannula was determined by an individual without knowledge of the experimental treatment or behavioral results. Only rats with cannulae located in or near the VMN were used in the data analysis. 2.2.7. Statistical methods In all cases, the statistical reference was Zar [20] and an alpha level of 0.05 was required for rejection of the null hypothesis. Data were organized into pretest period, infusion period (when appropriate) and consecutive 5 min or 10 min intervals after infusion (or injection). The data were analyzed by repeated-measures ANOVA with time after infusion (injection) as the repeated measure and treatment or treatments as the independent factors. Timedependent effects within treatment were compared to the pretest interval with Dunnett's tests. Differences between groups were compared by Tukey's test.

3. Results All females were actively mounted by the experienced males and there were no differences in the number of mounts received by females in the different treatment conditions (ANOVA, P > 0.05). The effects of bilateral VMN infusion with mianserin, ketanserin, or ketanserin tartrate on L / M ratios of proestrous rats are shown in Figs. 1-3. Each drug produced statistically significant decrements in the L / M ratio. For mianserin, there was a significant effect of dose (F4,37 = 3.57, P < 0.02), time after infusion (F5,185 = 16.06, P < 0.0001), and the dose × time interaction (F20,]85 = 2.48, P < 0.0001). Relative to the pretest interval, infusion with either 2000 or 4000 ng mianserin significantly reduced the L / M ratio by the first 10 min interval after infusion and at every interval thereafter (Dunnett's, all q185,6 >--2.26, P < 0.05). Rats infused with 250 ng mianserin showed a modest but significant decline in the L / M ratio at 15 min and at 20 min after infusion. Never were rats infused with 1000 ng significantly different from the pretest. For ketanserin, the overall effect of drug dose on the L / M ratio was not significant (F6,56 = 1.99, P < 0.09) but

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Fig. 1. Intracranial effects of mianserin on lordosis behavior in proestrous rats. Data are the mean+S.E.M. L / M ratios for regularly cycling, proestrous rats infused bilaterally into the vicinity of the VMN with saline (n = 6) or with 250 ng (n = 7), 1000 ng (n = 9), 2000 ng (n = 9) or 4000 ng (n = 11) of mianserin immediately following a pretest for sexual receptivity. Data were collected during the pretest (PRE), infusion interval (INF), and during each 5 min interval for 20 min following infusion. Asterisks indicate significant differences (Dunnett's, P < 0.05) from the pretest prior to drug infusion.

the time after infusion (F7.392 = 15.54, P < 0.0001) and the time × dose interaction (F42,392 = 1.79, P < 0.003) were significant. Following infusion with 50 ng, there was a decline in the L / M by 10 min after infusion and recovery to pretest levels was not evident until 30 min after the treatment (Dunnett's, all q39L8 -> 2.65, P < 0.05). A similar pattern was seen for 100 ng ketanserin with a significant decline in the L / M ratio occurring 10-20 min after infusion (Dunnett's, all q392,8 -> 2.65, P < 0.05). In contrast, the onset of inhibition following infusion with higher doses did not occur until at least 15-20 min after treatment. Infusion with 2000 ng or 2500 ng ketanserin did not reduce the L / M ratio until 15 min after infusion (for

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Fig. 2. Intracranial effects of ketanserin on lordosis behavior in proestrous rats. Data are the mean L / M ratios for regularly cycling, proestrous rats infused bilaterally into the vicinity of the VMN with 0.01 N HCI (acid control) (n = 14) or with 50 ng (n = 10), 100 ng (n = 6), 250 ng (n = 7), 1000 ng (n = 8), 2000 ng (n = 8) or 2500 ng (n = 10) ketanserin immediately following a pretest for sexual receptivity. S.E.M., omitted from the figure for ease of viewing, averaged less than 0.1. Data were computed during the pretest (PRE), infusion interval (INF), and during each 5 min interval for 30 min following infusion. Asterisks indicate significant differences (Dunnett's, P _< 0.05) from the pretest prior to drug infusion.

L. Uphouse et al. / Brain Research 718 (1996) 46-52

2000 ng, inhibition was present at 15, 20, and 25 min; for 2500 ng, inhibition was present at 15 and 20 min after infusion). For 1000 ng, the inhibition did not occur until 20 min after infusion but continued for the remainder of the testing period. The intermediate dose of 250 ng showed only a transient reduction of lordosis behavior at 25 min after infusion. Following infusion with ketanserin tartrate (Fig. 3), there was some evidence for a dose-dependent decline in the L / M ratio (ANOVA for dose, F3,26 = 4.29, P < 0.02). The time after i n f u s i o n (177,182 = 13.86, P < 0.0001) and the time × dose interaction (F21,182 = 2.32, P < 0.02) were also significant. Relative to the pretest interval, the earliest onset of inhibition of the L / M ratio was seen with 1000 and 3000 ng 15 min after infusion (Dunnett's, all q182.8 > 2.65, P < 0.05) and animals infused with both doses continued to show inhibition throughout the remainder of the 30 min testing period. With the 500 ng dose, inhibition was present between 20 and 30 min after infusion. Although VMN infusion with mianserin, ketanserin, or ketanserin tartrate did inhibit lordosis behavior, such inhibition was less consistent than expected if an action of 5-HT on 5-HT2A/2 c receptors in the VMN were essential for the occurrence of the lordosis reflex. Even at 4000 ng mianserin, 5 of the 11 rats showed little decline in the L / M ratio (e.g. L / M ratio was greater than 0.75 after treatment). Similarly, 9 of the 18 rats infused with 2000 ng or 2500 ng ketanserin had L / M ratios greater than or equal to 0.75 and 4 of the 9 rats infused with 3000 ng ketanserin tartrate continued to show L / M ratios at least as high as 0.75 throughout the majority of the testing period. Thus, even at the highest doses of the drugs examined, lordosis behavior of approximately 50% of the proestrous rats were inhibited by the compounds while the

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Fig. 4. Comparison of the VMN effects of ketanserin tartrate in hormone-treated, ovariectomized and intact, proestrous rats. Intact, proestrous rats or ovariectomized rats, hormonally primed with 0.5 /xg estradiol benzoate and 500/xg progesterone, were bilaterally infused with ketanserin tartrate into the VMN. Data are the mean _ S.E.M. L / M ratios for regularly cycling, proestrous rats infused with 500 ng (n = 4), 1000 ng (n = 3), or 3000 ng (n = 6) ketanserin tartrate and for hormone-primed ovariectomized rats infused with 500 ng (n = 9), 1000 ng (n = 9) or 3000 ng (n = 9) ketanserin tartrate. Data are presented for the pretest (PRE) and for each 5 min interval for 30 min following infusion. Single asterisks indicate significant differences (Dunnett's, P < 0.05) from the pretest interval. Double asterisks indicate significant differences (Tukey's, P < 0.05) between intact and hormone-primed, ovariectomized rats for the same dose treatment within time intervals.

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TIME AFTER INFUSION(mln) Fig. 3. Intracranial effects of ketanserin tartrate on lordosis behavior of proestrous rats. Data are the mean+ S.E.M. L / M ratios for regularly cycling, proestrous rats infused bilaterally into the vicinity of the VMN with water (n = 6) or with 500 ng (n = 6), 1000 ng (n = 9), or 3000 ng ( n = 9) ketanserin tartrate immediately following a pretest for sexual receptivity. Data were computed during the pretest (PRE), infusion interval (INF), and during each 5 min interval for 30 min following infusion. Asterisks indicate significant differences (Dunnett's, P < 0.05) from the pretest prior to drug intiasion.

remaining rats appeared to be less sensitive to the lordosis-inhibiting actions of the 5-HT2A/: c receptor antagonists. To see if this variability in response was unique to proestrous rats, we compared the effects of ketanserin tartrate in intact and hormone-primed, ovariectomized rats (Fig. 4). Although in both groups, ketanserin tartrate significantly reduced lordosis behavior in a dose-dependent manner (ANOVA for drug dose, F2,34 = 6.35, P < 0.005), the effects were greater in hormone-primed ovariectomized rats than in the intact rats (F1.34 = 8.92, P < 0.005). As a result, both time after infusion (FT,z38 = 12.62, P < 0.0001) and the type of animal × time after infusion interaction (F7.238 = 2.70, P < 0.01) were significant. Following infusion with 500 ng, the L / M ratio of intact rats was significantly greater than that of hormone-primed, ovariec-

L. Uphouse et al. / Brain Research 718 (1996) 46-52

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Fig. 5. Effects of i.p. treatment with mianserin on lordosis behavior of proestrous rats. Intact proestrous rats were pretested for sexual receptivity for approximately 10 mounts. The females were immediately injected i.p. with mianserin (1, 3, 5, or 8 m g / k g ) or the saline vehicle and were tested for sexual behavior for 30 min after injection. Data are grouped into the pretest (PRE) interval and 5 consecutive 5 min intervals after injection. The figure shows the mean+S.E.M. L / M ratio for 8 saline-treated females, 6 females given 1 m g / k g , 7 females treated with 3 m g / k g , 8 females treated with 5 m g / k g and 7 females treated with 8 m g / k g mianserin. Asterisks indicate significant differences (Dunnett's, P < 0.05) from the pretest interval.

tomized rats at 15 and 30 min; for 1000 ng, the two groups were significantly different from 15 through 30 min after infusion; and for 3000 ng, differences were present at 10, 20 and 30 min following infusion (Tukey's, all q238, 2 > 2.8, P < 0.05). For the 3000 ng dose of ketanserin tartrate, 9 of the 9 ovariectomized rats showed a decline in the L / M ratio while only 4 of the 6 intact rats showed a consistent decline in the L / M ratio. Thus, the variability in response to the 5-HT2A/2c receptor antagonist that was evident in the proestrous rat was not present in the hormone-primed, ovariectomized rat. Since prior studies in which the putative involvement of the 5-HT2A/2c receptor in lordosis behavior have relied upon the results of systemic treatment with 5-HT2A/2c antagonists, the effects of i.p. treatment with mianserin and ketanserin were evaluated in proestrous rats. The results are shown in Figs. 5 and 6. For mianserin there was a significant effect of dose (F4,31 = 10.91, P < 0.0001), time after treatment (F6:86 = 18.58, P < 0.00021) and dose × time i n t e r a c t i o n (F24,186 = 2.30, P < 0.0001). For the doses of 5 m g / k g and 8 m g / k g , there was a significant decrease in the L / M ratio by 5 min after injection and at every interval thereafter (Dunnett's, all q186,7 ~ 2.32, P < 0.05). Thirteen of these 15 rats showed a decline in the L / M ratio. Animals injected with 3 m g / k g mianserin did not decrease the L / M ratio until 10 min after injection, but thereafter, the L / M ratio remained lower than the pretest interval during the 30 min testing session (Dunnett's, all q186,7 >- 2.26, P < 0.05). Treatment with 1 m g / k g mianserin produced a smaller decline in the L / M ratio. Following i.p. treatment with ketanserin, there was a

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Fig. 6. Effects of i.p. treatment with ketanserin on lordosis behavior of proestrous rats. Intact proestrous rats were pretested for sexual receptivity for approximately 10 mounts. The females were immediately injected i.p. with ketanserin (1, 3, or 5 m g / k g ) or with the 0.01 N HC1 vehicle (acid control) and were tested continuously for sexual behavior for 30 min after injection. Data are grouped for the period before injection (PRE) and for each 10 min interval after injection. The figure shows the mean + S.E.M. L / M ratio for 8 vehicle-treated females, 7 females given 1 m g / k g ketanserin, 7 females treated with 3 m g / k g ketanserin, and 7 females treated with 5 m g / k g ketanserin. Asterisks indicate significant differences (Dunnett's, P < 0.05) from the pretest interval.

dose-dependent decline in the L / M ratio (ANOVA for dose, F3,25 = 9.29, P < 0.0005) and there was a significant effect of time after treatment (F5.~25 = 11.8, P < 0.0001) and a significant time × treatment interaction (F~5.~25 = 2.42, P < 0.004). Doses of 3 m g / k g and 5 m g / k g reduced the L / M ratio by 10 min after injection (Dunnett's, all q125,6 ~ 2.55, P < 0.05). In a final experiment, hormone-primed ovariectomized rats and proestrous rats were compared in their response to 3 m g / k g ketanserin given i.p. (Fig. 7). Although both groups were inhibited after t r e a t m e n t (F2,34 = 38.02, P < 1.0'

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Fig. 7. Comparison of the i.p. effects of ketanserin in hormone-primed, ovariectomized and in intact rats. Proestrous rats or ovariectomized rats, hormonally primed with 25 txg estradiol benzoate and 500 ~ g progesterone, were injected i.p. with 3 m g / k g ketanserin. Lordosis behavior was monitored for 10 min before injection and 30 min after the injection. Prior to injection, all rats showed L / M ratios > 0.9. The figure shows the mean + S.E.M. L / M for intact (n = 8) and hormone-primed, ovariectomized (OVX) ( n = 11) rats before (PRE) and 30 min after the ketanserin injection. The asterisk indicates a significant difference between the ovariectomized and the intact rats (Tukey's, P < 0.05).

L Uphouse et al./Brain Research 718 (1996) 46-52

0.0001), there was a significant effect of the type of animal (ovariectomized versus intact, Fl.17 = 10.7, P < 0.005); the type of animal × time after treatment interaction was also significant (F2,34 = 4.81, P < 0.02) with the L / M ratio of intact females significantly higher than that of the ovariectomized rats following treatment with ketanserin (Tukey's, all q34,2 -----4.38, P < 0.05).

4. Discussion This is the first report in which the lordosis-inhibiting effects of 5-HTEA/2 C antagonists in intact, regularly cycling females have been examined. Furthermore, it is the first report in which the i ntracranial effects of the compounds on lordosis behavior have been examined. In general, these studies support previous arguments for a role of 5-HT2A/2 C receptors in the control of female lordosis behavior [8,9,19]. Furthermore, the data are consistent with previous suggestions that the VMN contains 5-HTxA/2 C receptors important in the: control of the lordosis reflex [6,8]. However, relative to hormone-primed ovariectomized rats, proestrous rats may be less likely to show inhibition of lordosis behavior following treatment with 5-HTEA/2c antagonists. Even at the highest doses examined, only 50-60% of proestrous rats showed a substantial decline ( < 0.75) in the L / M ratio after VMN infusion, and there was a wide range of individual responsivity to the drug. Within any tre~ttment condition, some animals responded to the infusion with a robust decline in the L / M ratio while other animals failed to respond to the infusion, but there was no obvious difference between animals or their cannula placements in the two groups of rats. In contrast, 100% of the suboptimally hormone-primed rats were inhibited after VMN infusion of ketanserin tartrate. These findings lead to the suggestion that the degree of hormone priming may be an important factor in determining the female's response to the 5-HT2A/2 c antagonists. Exactly why proestrous ral:s and hormone-primed, ovariectomized rats differ in their sensitivity to the 5-HT2A/2 c antagonists remains to be determined. One possible explanation is that gonadal hormones modulate the number a n d / o r functioning of 5-HT2A/2 c receptors. Or gonadal hormones may alter other neurotransmitters or intracellular events (e.g. phosphoinositide hydrolysis) which in turn modify the response to 5-HT2-active drugs. Alternatively, reproductively relevant events such as the release of gonadotropin releasing factor, luteinizing hormone, or prolactin may differ between proestrous rats and those primed exogenously with estrogen and progesterone. The idea that 5-HT2A/2 c receptor activation facilitates lordosis behavior was pioneered by observations that significant decrements in le,rdosis behavior occurred when hormone-primed ovariectomized rats were injected with several 5-HT2A/2 C antagonists, including mianserin and ketanserin [9,19]. In the present study, systemic treatment

51

with either mianserin or ketanserin inhibited lordosis behavior, but consistent with the intracranial treatment, hormone-primed ovariectomized rats appeared to be more sensitive than the proestrous rats. Although it was possible to obtain a more uniform inhibition of lordosis behavior after i.p. treatment of proestrous rats with the 5-HT2A/2 C antagonists, with the exception of the highest i.p. doses of mianserin and ketanserin (where flattened posture a n d / o r the appearance of lethargy were present), i.p. and i.c. administration of the compounds produced comparable findings. If the dual hypothesis regarding 5-HT's role in the control of lordosis behavior does have merit, and given that estrous cyclicity is characterized by an increased probability of sexual receptivity as the female moves toward the stage of proestrus, proestrous rats must experience a reduction in endogenous activation of the inhibitory 5-HT1A receptors a n d / o r an elevation in the activation of facilitatory 5-HT2A/2 C receptors. In previous studies, we have shown that 5-HTtA agonists inhibit (and can even eliminate) lordosis behavior of both proestrous and hormone-primed, ovariectomized rats [15,17]. In comparison to these 5-HT1A receptor agonists, 5-HT2A/2 c receptor antagonists were relatively ineffective in inhibiting lordosis behavior of proestrous rats. Since both mianserin and ketanserin affect receptors in addition to 5-HT2A/2 C receptors, it is possible that nonserotonergic actions of the drugs attenuated a lordosis-inhibiting action of 5-HT2A/2 c receptor antagonism. Both mianserin and ketanserin are antagonists at c~-adrenergic receptors [2,7,11] while ketanserin antagonizes histamine receptors [18]. We do not, however, believe these nonserotonergic actions can account for the modest effect of the drugs in the proestrous rats. Over the time course examined in the present experiment, both c~and /3-noradrenergic antagonists are more likely to inhibit than to facilitate lordosis behavior [3,4]. Thus, while activation of 5-HTIA receptors in the VMN is clearly incompatible with lordosis behavior, activation of 5-HT2A/2 C receptors in the VMN may not be required for occurrence of the reflex in the endogenously primed, proestrous rat. In contrast to the present findings, in previous studies, we have shown that 5-HT2A/2 ¢ agonists are very efficient in protecting against the lordosis-inhibiting effects of 5HT~A agonists following infusion into the VMN [16]. 5-HT2A/2 C receptors in the VMN may, therefore, provide a type of 'fail-safe' mechanism with the potential to override inhibitory effects of 5-HT in the event that a sudden or inappropriate increase in the release of 5-HT occurs on the day of proestrus. In summary, the 5-HT2A/2 c receptor antagonists, mianserin and ketanserin, were shown to inhibit lordosis behavior in both proestrous rats and in ovariectomized rats, primed with estradiol benzoate and progesterone. Furthermore, 5-HT2A/2 c receptors in the VMN appear to be an effective site for the inhibitory effects of the receptor antagonists. However, for both routes of treatment, hor-

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L. Uphouse et al./ Brain Research 718 (1996) 46-52

mone-primed, ovariectomized rats appeared to be more sensitive to the disrupting effects of the 5-HT2A/2 c antagonists. In combination with previous studies, these findings are consistent with arguments that the VMN contains both 5-HTIA and 5-HT2A/2 c receptors and that a functional balance between these receptors types is important in 5-HT's modulation of lordosis behavior.

Acknowledgements Special appreciation is given to Dr. Sharmin Maswood for her encouragement during these studies and for reading prior versions of the manuscript. The animal care provided by Mr. Tim Lair is acknowledged. The research was supported in part by NIH GM08256, by NIH RO1 HD28419 and by a TWU Research Enhancement Grant to L.U.

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