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Scopolamine inhibition of lordosis in naturally cycling female rats
Physiology& Behavior,Vol. 45, pp. 819-823. © PergamonPress plc., 1989. Printed in the U.S.A.
0031-9384/89 $3.00 + .00
Scopolamine Inhibition of Lordosis in Naturally Cycling Female Rats I C H E R Y L S. M E N A R D A N D G A R Y P. D O H A N I C H 2
Department o f Psychology and Neuroscience Program, Tulane University, New Orleans, LA R e c e i v e d 6 S e p t e m b e r 1988
MENARD, C. S. AND G. P. DOHANICH. Scopolamine inhibition of lordosis in naturally cyclingfemale rats. PHYSIOL BEHAV 45(4) 819-823, 1989.--Cholinergic antagonists, such as scopolamine, atropine, and hemicholinium-3, have been found previously to inhibit lordosis in ovariectomized rats primed with estrogen and progesterone. The present study further examined this effect using intact cycling female rats. Cycling was determined by daily monitoring of sexual behavior and vaginal cytology. In the first experiment, intraventricular administration of the muscarinic receptor blocker, scopolamine, was found to significantly inhibit lordosis behavior during natural estrus (10 or 20 Ixg bilaterally). In the second experiment, systemic administration of scopolamine was also found to significantly inhibit lordosis behavior during natural estrus (4 mg/kg, IP). Administration of the cholinergic antagonist did not significantly interrupt cyclicity patterns. These results indicate that central cholinergic muscarinic systems contribute to the regulation of lordosis during natural behavioral estrus in intact female rats. Scopolamine
Muscarinic
Cholinergic
Acetylcholine
Lordosis
Female sexual behavior
More recently, intraventricular infusion of scopolamine, a cholinergic muscarinic antagonist, has been shown to significantly inhibit lordosis behavior in ovariectomized, hormonally primed rats (2,8). In addition, systemic administration of scopolamine was also found to inhibit lordosis in ovariectomized, hormonally treated rats (8,17). The present study examined the inhibitory effects of scopolamine on the lordotic response in naturally cycling female rats. Extending this analysis to naturally cycling animals verifies the importance of cholinergic regulation of lordosis during natural receptivity, as well as validates the use of ovariectomized, hormonally primed rats in the examination of these effects. Since similar effects were previously reported using systemic and intracerebral administrations of scopolamine in ovariectomized, hormonally treated animals, both routes of administrations were used in this study in order to fully compare the inhibitory effects of scopolamine in intact and ovariectomized females. Based on previous results reported by this laboratory, it was expected that the inhibition of lordosis produced by the intracerebral and systemic administration of scopolamine in intact rats would be similar to the inhibition previously seen with scopolamine administration in ovariectomized females.
FEMALE rats exhibit 4- to 5-day estrous cycles, with the greatest occurrence of sexual receptivity displayed for 8-10 hours during the proestrous-estrous stage of the cycle. Sexual behavior in female rats is characterized by soliciting behaviors, such as hopping and darting, in the presence of a male rat and lordosis when mounted by the male rat. Lordosis, the ventral arching of the spine with the elevation of the perineum, is believed to facilitate vaginal penetration by the male during mating. The occurrence of this lordotic response in females is activated and maintained by estrogen and progesterone released by the ovaries (1). Previous evidence indicates that these hormones modify brain functioning, and consequently sexual behavior, by regulating central cholinergic activity (4). Intracerebral administration of cholinergic agonists, such as carbachol, bethanechol, and oxotremorine, have been shown to induce lordosis in ovariectomized rats primed with low hormonal levels (2, 6, 7, 12). These facilitative effects were reversed by the administration of the cholinergic muscarinic receptor blockers, atropine and scopolamine (2, 6, 7, 12). In addition, it has been found that intracerebral infusion of eserine, an acetylcholinesterase inhibitor, also facilitates lordosis in ovariectomized rats primed with low levels of hormone (2). Again, this facilitative effect was reversed by the administration of atropine (2). Furthermore, intracerebral infusions of hemicholinium-3, an acetylcholine synthesis inhibitor, have been found to inhibit hormonally primed sexual receptivity in ovariectomized female rats (3,5). This inhibitory effect was reversed by the administration of carbachol (5) and choline (3).
GENERAL METHOD
Animals Subjects were 24 Sherman female rats, 175-200 g in weight,
ISupported by U.S.P.H.S. Grant No. HD22235-01AI to Gary P. Dohanich. 2Requests for reprints should be addressed to Gary P. Dohanich, Ph.D., Department of Psychology, Tulane University, New Orleans, LA 70118.
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purchased from Camm Research Co., Wayne, NJ. The animals were individually housed and maintained on a 10/14 light/dark cycle (lights off at 0700 hr) in a temperature controlled vivarium throughout the experiments. Six Long-Evans males, used in behavior testing, were also individually housed in the same vivarium as the females. To avoid interruptions in the estrous cycle due to irregularities in the light/dark cycle, all manipulations and testing, with the exception of stereotaxic surgery, were done within the vivarium. Prior to surgery, which was performed during the dark phase of the light cycle, animals were visually masked to reduce exposure to light.
Cycling Cycling of female rats was determined by the daily examination of vaginal cytology and sexual behavior. Vaginal smears were collected twice daily, at the beginning and end of each dark cycle. Smears were then stained with toluidine blue and examined microscopically. Sexual behavior was observed once a day during the afternoon of the dark cycle. Animals were considered to be successfully cycling when vaginal cytology and sexual behavior, corresponding to normal 4- or 5-day cycles, occurred for at least 2 cycles. This criterion was maintained throughout both experiments.
Stereotaxic Surgery On the first day of diestrus after successfully cycling twice, animals were anesthetized with Ketaset (100 mg/kg: Bristol Laboratories; Syracuse, NY) and Rompun (7.4 mg/kg: Miles Laboratories; Shawnee, KA). Double-barrel cannulae were placed bilaterally in the lateral ventricles of each female rat in preparation for the infusion to be received in Experiment 1. Each cannula consisted of a guide constructed from 23 gauge stainless steel tubing, fitted with an insert constructed from 28-gauge tubing. The guides were anchored to the skull with machine screws and dental acrylic. Inserts extended into the ventricles 1 mm beyond the guide tips and were removed only during infusion.
Behavior Testing Behavior testing was conducted in the vivarium in a semicircular metal arena with a glass front (30 cm radius, 25 cm height). During daily behavior testing used to monitor cycling, females were introduced to an arena occupied by a male Long-Evans rat and allowed 3 mounts. If lordotic responses were observed for at least 2 of the 3 mounts, then the animal was considered to be receptive. Females failing to receive 3 mounts within 10 min were transferred to another arena and testing was completed with a different male. During behavior testing used to determine drug effects, females received 10 mounts. Each response by a female was recorded as either 0 for no lordosis or 1 for lordosis. The lordosis quotient [(number of lordoses/number of mounts) x 100] was computed in this study. Again, females failing to receive the 10 mounts within 10 min were transferred to another arena for the completion of the test. During all sexual behavior testing, females were vaginally masked with masking tape to avoid pregnancy. EXPERIMENT 1 In the first experiment, the incidence of lordosis in intact cycling female rats during natural estrus was examined following infusion of scopolamine into the lateral ventricles. This compound, when infused into this area, has been shown to inhibit the
incidence of lordosis in ovariectomized rats that have been primed with estrogen and progesterone (8). METHOD
After surgery, the vaginal cytology and sexual behavior of each rat were monitored daily. Each animal was allowed to cycle twice before drug testing. Drug testing occurred at the first indication of sexual receptivity following this period. Prior to drug treatment, the female rat was introduced into the behavior testing arena where she received 10 mounts by a male. The female was then randomly assigned to receive one of three bilateral drug administrations: saline vehicle. 10 txg scopolamine/ cannula, or 20 Ixg scopolamine/cannula. Prior to infusion, ( - )-scopolamine hydrobromide (Sigma Chemical Co., St. Louis, MO) was dissolved in saline vehicle solution. A Hamilton microsyringe, connected to a 28-gauge infusion insert by PE 20 polyethylene tubing, was used to deliver 0.5 Ixl/cannula of the infusion solutions. The solution was infused into the lateral ventricle over a 30 sec period, then allowed to diffuse for 30 sec before the infusion insert was removed from the guide and replaced with the temporary insert. This procedure was repeated contralaterally. After infusions were completed bilaterally, the animal was placed in her home cage for 15 min. She was then returned to the testing arena where she received 10 mounts by a male. Daily monitoring of cyclic activity continued for 2 cycles following drug testing to determine the effects of scopolamine infusions on cyclicity. RESULTS
Cycling before and after surgery was compared to determine if surgery or anesthesia administered prior to surgery disrupted cycling. A cycling percentage (No. of cycles/number of days) was used to estimate cycling efficiency. A cycling percentage of 25% would, therefore, be characteristic of a 4-day cycling animal, while a cycling percentage of 20% would be characteristic of a 5-day cycling animal. Comparison of cycling percentages before surgery (mean= 19.4%, S D = 3 . 9 % ) and after surgery (mean= 16.5%, SD = 5.9%) indicated that surgery performed under anesthetic administrations of Ketaset/Rompun did not significantly interrupt normal cycling activity in female Sherman rats. Cycling before and after infusion were also compared to determine if infusion disrupted cycling. Comparison of cycling percentages before infusion (mean = 16.5%, SD = 5.9%) and after infusion (mean= 16.5%, S D = 8 . 5 % ) indicated that infusion of either saline or scopolamine did not interrupt normal cycling activity. Furthermore, comparisons of cycling percentages between groups indicated that there was no significant difference in cycling activity between groups receiving infusions of saline (mean = 20.9%, S D = 3.2%), 10 Ixg scopolamine (mean = 17.2%, SD = 9.6%), or 20 Ixg scopolamine (mean = 9.6%, SD = 9.3%). Although no significant differences in cycling among groups were found, large variabilities in cycling among Sherman female rats were evident. More recently in this laboratory, it has been found that intracerebral infusion of the acetylacholinesterase inhibitor, eserine, in Long-Evans Hooded rats maintained on a 12/12 light/dark cycle similarly does not affect cycling. There was also found less variability in cyclicity using Long-Evans Hooded rats on this light/dark cycle. Therefore, much of cycling variability evident in this study may have been avoided by using a different strain of rat or a 12/12 light/dark cycle. The large variability in cycling among Sherman females in this study and the small number of cycles monitored limits any conclusions about the lack of effects of surgery, anesthesia, and/or drug treatments on cyclicity.
INHIBITION OF NATURAL RECEPTIVITY
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Nineteen of the 24 female rats were found to be cycling normally at the time of drug testing in Experiment I. Six animals were assigned to each of the two drug testing conditions, while seven were assigned to the saline group. For the purpose of analysis, lordosis quotients (LQ) were converted into Percents of Control (POC). POC for each animal was calculated as follows (See Fig. 11:
POC =
LQ after infusion × I00 LQ before infusion
Intraventricular infusions of both high (mean POC=30.00%, S D = 3 0 . 9 8 % ) and low (mean POC=23.33%, S D = 2 2 . 5 1 % ) doses of scopolamine were found to significantly inhibit lordosis behavior in female rats (p<0.0001), whereas infusions of saline (mean POC=98.21%, S D = 4 . 7 2 ) was not. These results are consistent with those reported using ovariectomized female rats primed with estrogen and progesterone (8). Post hoc comparison of POC for high and low dose groups further indicated that there was no significant dose-dependent difference. Although no dose-dependent difference was detected at these drug levels, this does not suggest that there would not be a dose-dependent relationship at other dose levels as reported with ovariectomized, hormone-primed females (8). EXPERIMENT 2 In Experiment 2, the incidence of lordosis in intact cycling female rats during natural estrus was examined following intraperitoneal injection of scopolamine hydrobromide. This compound has been shown to inhibit the incidence of lordosis when injected systemically in ovariectomized rats that have been primed with estrogen and progesterone (8,17). METHOD
Cycling was again monitored daily by examination of vaginal
cytology and sexual behavior. At first sign of sexual receptivity following two successful cycles, drug testing occurred. Prior to drug administration, (-)-scopolamine hydrobromide was dissolved in saline solution and prepared for injection. Again, the female rat was introduced into the behavior testing arena where she received 10 mounts by a male. Animals which received saline infusions in Experiment 1, received a 0.2 ml intraperitoneal injection of saline solution, while animals which received scopolamine infusions in Experiment 1, received a 0.2 ml intraperitoneal injection of I m g scopolamine (4 mg/kg). This dose level of scopolamine has been found previously to inhibit lordosis in ovariectomized female rats primed with estrogen and progesterone (8,17). Immediately after injection, the female was placed in her home cage for 15 rain before returning to the testing arena to receive 10 mounts by a male. Daily monitoring of cyclic activity continued for 2 cycles past drug testing to determine the effects of scopolamine infusions on cycling behavior. RESULTS
Cycling before and after injections were compared to determine if systemic injection disrupted cycling. Cycling percentages were again calculated as in Experiment 1. Comparison of cycling percentages before injection (mean = 21.4%, SD = 4.0%) and after injection (mean = 17,4%, SD = 9.7%) indicated that injections in general did not interrupt normal cycling in female rats. Further comparison of cycling percentages for the group receiving scopolamine (mean = 18.5c~, SD = 9.29/) and the group receiving saline (mean = 15.7c~. SD = 10.9c~ I indicated no significant difference, suggesting that intraperitoneal administration of scopolamine at this dose level did not significantly interrupt normal cycling activity in this experiment. As in Experiment 1, this interpretation of cycling activity is limited due to the large variability in cycling anaong animals and the small number of cycles monitored. Fourteen of the females were found to be normally cycling at the time of drug testing during this experinaent. Therefore 7 were assigned to each group. As in Experiment 1, LQs were converted into POCs for the purpose of analysis. The mean POC for the
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[] P~+m-
& .,+
'm
,40
20
Saline
II
10 ug/cannula
20 ug/cannuia
InlravenlricularTreatment FIG. 1. Inhibition of lordosis during natural behavioral estrus in intact female rats 15 rain after intraventricular infusion of low and high doses of scopolamine or saline vehicle (17<0.0001 ).
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100
.=o 60
40
20
Saline
4 mglkg
SystemicTreatment FIG. 2. Inhibition of lordosis during natural behavioral estrus in intact female rats 15 min after systemic injection of scopolamine or saline vehicle (p<0.0001 ).
scopolamine group (24.29%, S D = 3 0 . 4 8 % ) was significantly lower (p<0.0001) than the mean POC of the saline group (98.57%, SD = 3.78%) suggesting that intraperitoneal administration of scopolamine inhibits lordosis behavior in female rats (See Fig. 2). These results are consistent with those reported in Experiment 1. They are also consistent with those results reported using ovariectomized female rats primed with estrogen and progesterone (8,17). GENERAL DISCUSSION Intraventricular infusions and systemic injections of scopolamine, a cholinergic muscarinic blocker, significantly inhibited naturally occurring lordosis behavior in intact cycling female rats.
These results are consistent with previous findings that scopolamine inhibits sexual receptivity in ovariectomized, hormonally primed female rats (2, 8, 17). These findings provide further support for the hypothesis that the cholinergic system regulates female sexual behavior. Although scopolamine was found to have a significant effect on sexual receptivity, it was not found to have a significant effect on cyclicity as measured in this study. Since interpretation of cyclicity data are limited due to sample size and variability, the concept of independent mechanisms in the regulation of sexual receptivity and the regulation of cyclicity requires more rigorous analysis. While it appears that specific cyclic events, such as sexual receptivity, are regulated by cholinergic mechanisms, the general regulation of the estrous cycle may not be under cholinergic control. Cholinergic regulation of other cyclic events, including the release of gonadotropins and prolactin during proestrus, have been reported (13,14). Furthermore, administrations of pilocarpine, a muscarinic agonist, has been shown to advance ovulation during normal 5-day cycling (18). This evidence does not, however, suggest an interruption or resetting of the estrous cycle. Instead, cyclicity appears to be regulated by circadian clock mechanisms (21). After the administration of scopolamine in the present study, it was also observed during behavior testing that sexual proceptivity, although somewhat diminished in a proportion of females, was still prevalent in a number of the animals. Although proceptivity was not measured systematically in this experiment, this observation suggests that proceptivity and receptivity are independently regulated. In previous studies, cholinergic agonists have activated lordosis but not proceptivity in ovariectomized, estrogen primed females (5-7). Further investigation of the cholinergic regulation of sexual proceptivity in intact females is planned. Although the present study offers support for the cholinergic regulation of sexual behavior, it does not identify the anatomical location of these effects in the brain. Estrogen has been shown to alter cholinergic receptor binding (9-11, 19, 20) and related enzymatic activity (15,16) in areas of the brain implicated in the regulation of sexual behavior, including the medial preoptic area and the ventromedial hypothalamus. However, conclusive localization of the cholinergic regulation of sexual behavior in the brain has yet to be documented.
REFERENCES 1. Boling, J. L.; Blandau, R. J. The estrogen-progesterone induction of mating responses in the spayed female rat. Endocrinology 25:359364; 1939. 2. Clemens, L. G.; Barr, P. J.; Dohanich, G. P. Cholinergic regulation of female sexual behavior in rats demonstrated by manipulation of endogenous acetylcholine. Physiol. Behav. 45(2): 437442; 1989. 3. Clemens, L. G.; Dohanich, G. P. Inhibition of lordotic behavior in female rats following intracerebral infusion of anticholinergic agents. Pharmacol. Biochem. Behav. 13:89-95; 1980. 4. Clemens, L. G.; Dohanich, G. P.; Barr, P. J. Cholinergic regulation of feminine sexual behavior in laboratory rats. In: Hormones and behavior in higher vertebrates. New York: Springer-Verlag; 1983: 56--68. 5. Clemens, L. G.; Donhanich, G. P.; Witcher, J. A. Cholinergic influences of estrogen-independent sexual behavior in female rats. J. Comp. Physiol. Psychol. 95:763-770; 1981. 6. Clemens, L. G.; Humphrys, R. R.; Donhanich, G. P. Cholinergic brain mechanisms and the hormonal regulation of female sexual behavior in the rat. Pharmacol. Biochem. Behav. 13:81-88; 1980. 7. Dohanich, G. P.; Barr, J.; Witcher, J. A.; Clemens, L. G. Pharmacological and anatomical aspects of cholinergic activation of female sexual behavior. Physiol. Behav. 32:1021-1026; 1984. 8. Dohanich, G. P.; Holland, R.; Menard, C.; McMullan, M.; Cada, D. Effects of cholinergic muscarinic antagonists on female sexual behav-
ior. Conf. Reprod. Behav. Prog. Abstr. 20:20; 1988. 9. Dohanich, G. P.; Witcber, J. A.; Weaver, D. R.; Clemens, L. G. Alteration of muscarinic binding in specific brain areas following estrogen treatment. Brain Res. 241:347-350; 1982. 10. Egozi, Y.; Avissar, S.; Sokolovsky, M. Muscarinic mechanisms and sex hormone secretions in rat adenohypophysis and preoptic area. Neuroendocrinology 35:93-97; 1982. 11. Egozi, Y.; Kloog, Y. Muscarinic receptors in the preoptic area are sensitive to 17[3-Estradiol during the critical period. Neuroendocrinology 40:385-392; 1985. 12. Kaufman, L. S.; McEwen, B. S.; Pfaff, D. W. Cholinergic mechanisms of lordotic behavior in rats. Physiol. Behav. 43:507-514; 1988. 13. Libertun, C.; McCann, S. M. Blockade of the release of gonadotrophins and prolactin by subcutaneous or intraventricular injection of atropine in male and female rats. Endocrinology 92:1714-1724; 1973. 14. Libertun, C.; McCann, S. M. Further evidence for cholinergic control of gonadotrophin and prolactin secretion. Proc. Soc. Exp. Biol. Med. 147:498-504; 1974. 15. Luine, V. N.; Khylchevskaya, R. 1.; McEwen, B. S. Effects of gonadal steroids on activities of monoamine oxidase and choline acetylase in rat brain. Brain Res. 86:293-306; 1975. 16. Luine, V.; Park, D.; Tong, J.; Reis, D.; McEwen, B. S. Immunochemical demonstration of increased choline acetyltransferase concentration in rat preoptic areas after estradiol administration. Brain Res.
INHIBITION OF NATURAL RECEPTIVITY
191:273-277; 1980. 17. Meyers, T. C.; Clemens, L. G. Scopolamine inhibition of lordosis at high levels of estrogen priming. Conf. Reprod. Behav. Prog. Abstr. 18:72; 1986. 18. Meyerson, B. J.; Palis, A. Advancement of the time for ovulation in the 5-day cyclic rat by pilocarpine. Acta Pharmacol. Toxicol. (Suppl.) 28:68; 1970. 19. Olsen, K. L.; Edwards, E.; Schechter, N.; Whalen, R. E. Muscarinic
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receptors in preoptic area and hypothalamus: effects of cyclicity, sex, and estrogen treatment. Brain Res. 448:223-229; 1988. 20. Rainbow, T. C.; DeGroff, V.; Luine, V. N.; McEwen, B. S. Estradiol 17-13 increases the number of muscarinic receptors in hypothalamic nuclei. Brain Res. 198:239-243; 1980. 21. Schwartz, N. B. A model for the regulation of ovulation in the rat. Recent Prog. Horm. Res. 25:1-55; 1969.
0031-9384/89 $3.00 + .00
Scopolamine Inhibition of Lordosis in Naturally Cycling Female Rats I C H E R Y L S. M E N A R D A N D G A R Y P. D O H A N I C H 2
Department o f Psychology and Neuroscience Program, Tulane University, New Orleans, LA R e c e i v e d 6 S e p t e m b e r 1988
MENARD, C. S. AND G. P. DOHANICH. Scopolamine inhibition of lordosis in naturally cyclingfemale rats. PHYSIOL BEHAV 45(4) 819-823, 1989.--Cholinergic antagonists, such as scopolamine, atropine, and hemicholinium-3, have been found previously to inhibit lordosis in ovariectomized rats primed with estrogen and progesterone. The present study further examined this effect using intact cycling female rats. Cycling was determined by daily monitoring of sexual behavior and vaginal cytology. In the first experiment, intraventricular administration of the muscarinic receptor blocker, scopolamine, was found to significantly inhibit lordosis behavior during natural estrus (10 or 20 Ixg bilaterally). In the second experiment, systemic administration of scopolamine was also found to significantly inhibit lordosis behavior during natural estrus (4 mg/kg, IP). Administration of the cholinergic antagonist did not significantly interrupt cyclicity patterns. These results indicate that central cholinergic muscarinic systems contribute to the regulation of lordosis during natural behavioral estrus in intact female rats. Scopolamine
Muscarinic
Cholinergic
Acetylcholine
Lordosis
Female sexual behavior
More recently, intraventricular infusion of scopolamine, a cholinergic muscarinic antagonist, has been shown to significantly inhibit lordosis behavior in ovariectomized, hormonally primed rats (2,8). In addition, systemic administration of scopolamine was also found to inhibit lordosis in ovariectomized, hormonally treated rats (8,17). The present study examined the inhibitory effects of scopolamine on the lordotic response in naturally cycling female rats. Extending this analysis to naturally cycling animals verifies the importance of cholinergic regulation of lordosis during natural receptivity, as well as validates the use of ovariectomized, hormonally primed rats in the examination of these effects. Since similar effects were previously reported using systemic and intracerebral administrations of scopolamine in ovariectomized, hormonally treated animals, both routes of administrations were used in this study in order to fully compare the inhibitory effects of scopolamine in intact and ovariectomized females. Based on previous results reported by this laboratory, it was expected that the inhibition of lordosis produced by the intracerebral and systemic administration of scopolamine in intact rats would be similar to the inhibition previously seen with scopolamine administration in ovariectomized females.
FEMALE rats exhibit 4- to 5-day estrous cycles, with the greatest occurrence of sexual receptivity displayed for 8-10 hours during the proestrous-estrous stage of the cycle. Sexual behavior in female rats is characterized by soliciting behaviors, such as hopping and darting, in the presence of a male rat and lordosis when mounted by the male rat. Lordosis, the ventral arching of the spine with the elevation of the perineum, is believed to facilitate vaginal penetration by the male during mating. The occurrence of this lordotic response in females is activated and maintained by estrogen and progesterone released by the ovaries (1). Previous evidence indicates that these hormones modify brain functioning, and consequently sexual behavior, by regulating central cholinergic activity (4). Intracerebral administration of cholinergic agonists, such as carbachol, bethanechol, and oxotremorine, have been shown to induce lordosis in ovariectomized rats primed with low hormonal levels (2, 6, 7, 12). These facilitative effects were reversed by the administration of the cholinergic muscarinic receptor blockers, atropine and scopolamine (2, 6, 7, 12). In addition, it has been found that intracerebral infusion of eserine, an acetylcholinesterase inhibitor, also facilitates lordosis in ovariectomized rats primed with low levels of hormone (2). Again, this facilitative effect was reversed by the administration of atropine (2). Furthermore, intracerebral infusions of hemicholinium-3, an acetylcholine synthesis inhibitor, have been found to inhibit hormonally primed sexual receptivity in ovariectomized female rats (3,5). This inhibitory effect was reversed by the administration of carbachol (5) and choline (3).
GENERAL METHOD
Animals Subjects were 24 Sherman female rats, 175-200 g in weight,
ISupported by U.S.P.H.S. Grant No. HD22235-01AI to Gary P. Dohanich. 2Requests for reprints should be addressed to Gary P. Dohanich, Ph.D., Department of Psychology, Tulane University, New Orleans, LA 70118.
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purchased from Camm Research Co., Wayne, NJ. The animals were individually housed and maintained on a 10/14 light/dark cycle (lights off at 0700 hr) in a temperature controlled vivarium throughout the experiments. Six Long-Evans males, used in behavior testing, were also individually housed in the same vivarium as the females. To avoid interruptions in the estrous cycle due to irregularities in the light/dark cycle, all manipulations and testing, with the exception of stereotaxic surgery, were done within the vivarium. Prior to surgery, which was performed during the dark phase of the light cycle, animals were visually masked to reduce exposure to light.
Cycling Cycling of female rats was determined by the daily examination of vaginal cytology and sexual behavior. Vaginal smears were collected twice daily, at the beginning and end of each dark cycle. Smears were then stained with toluidine blue and examined microscopically. Sexual behavior was observed once a day during the afternoon of the dark cycle. Animals were considered to be successfully cycling when vaginal cytology and sexual behavior, corresponding to normal 4- or 5-day cycles, occurred for at least 2 cycles. This criterion was maintained throughout both experiments.
Stereotaxic Surgery On the first day of diestrus after successfully cycling twice, animals were anesthetized with Ketaset (100 mg/kg: Bristol Laboratories; Syracuse, NY) and Rompun (7.4 mg/kg: Miles Laboratories; Shawnee, KA). Double-barrel cannulae were placed bilaterally in the lateral ventricles of each female rat in preparation for the infusion to be received in Experiment 1. Each cannula consisted of a guide constructed from 23 gauge stainless steel tubing, fitted with an insert constructed from 28-gauge tubing. The guides were anchored to the skull with machine screws and dental acrylic. Inserts extended into the ventricles 1 mm beyond the guide tips and were removed only during infusion.
Behavior Testing Behavior testing was conducted in the vivarium in a semicircular metal arena with a glass front (30 cm radius, 25 cm height). During daily behavior testing used to monitor cycling, females were introduced to an arena occupied by a male Long-Evans rat and allowed 3 mounts. If lordotic responses were observed for at least 2 of the 3 mounts, then the animal was considered to be receptive. Females failing to receive 3 mounts within 10 min were transferred to another arena and testing was completed with a different male. During behavior testing used to determine drug effects, females received 10 mounts. Each response by a female was recorded as either 0 for no lordosis or 1 for lordosis. The lordosis quotient [(number of lordoses/number of mounts) x 100] was computed in this study. Again, females failing to receive the 10 mounts within 10 min were transferred to another arena for the completion of the test. During all sexual behavior testing, females were vaginally masked with masking tape to avoid pregnancy. EXPERIMENT 1 In the first experiment, the incidence of lordosis in intact cycling female rats during natural estrus was examined following infusion of scopolamine into the lateral ventricles. This compound, when infused into this area, has been shown to inhibit the
incidence of lordosis in ovariectomized rats that have been primed with estrogen and progesterone (8). METHOD
After surgery, the vaginal cytology and sexual behavior of each rat were monitored daily. Each animal was allowed to cycle twice before drug testing. Drug testing occurred at the first indication of sexual receptivity following this period. Prior to drug treatment, the female rat was introduced into the behavior testing arena where she received 10 mounts by a male. The female was then randomly assigned to receive one of three bilateral drug administrations: saline vehicle. 10 txg scopolamine/ cannula, or 20 Ixg scopolamine/cannula. Prior to infusion, ( - )-scopolamine hydrobromide (Sigma Chemical Co., St. Louis, MO) was dissolved in saline vehicle solution. A Hamilton microsyringe, connected to a 28-gauge infusion insert by PE 20 polyethylene tubing, was used to deliver 0.5 Ixl/cannula of the infusion solutions. The solution was infused into the lateral ventricle over a 30 sec period, then allowed to diffuse for 30 sec before the infusion insert was removed from the guide and replaced with the temporary insert. This procedure was repeated contralaterally. After infusions were completed bilaterally, the animal was placed in her home cage for 15 min. She was then returned to the testing arena where she received 10 mounts by a male. Daily monitoring of cyclic activity continued for 2 cycles following drug testing to determine the effects of scopolamine infusions on cyclicity. RESULTS
Cycling before and after surgery was compared to determine if surgery or anesthesia administered prior to surgery disrupted cycling. A cycling percentage (No. of cycles/number of days) was used to estimate cycling efficiency. A cycling percentage of 25% would, therefore, be characteristic of a 4-day cycling animal, while a cycling percentage of 20% would be characteristic of a 5-day cycling animal. Comparison of cycling percentages before surgery (mean= 19.4%, S D = 3 . 9 % ) and after surgery (mean= 16.5%, SD = 5.9%) indicated that surgery performed under anesthetic administrations of Ketaset/Rompun did not significantly interrupt normal cycling activity in female Sherman rats. Cycling before and after infusion were also compared to determine if infusion disrupted cycling. Comparison of cycling percentages before infusion (mean = 16.5%, SD = 5.9%) and after infusion (mean= 16.5%, S D = 8 . 5 % ) indicated that infusion of either saline or scopolamine did not interrupt normal cycling activity. Furthermore, comparisons of cycling percentages between groups indicated that there was no significant difference in cycling activity between groups receiving infusions of saline (mean = 20.9%, S D = 3.2%), 10 Ixg scopolamine (mean = 17.2%, SD = 9.6%), or 20 Ixg scopolamine (mean = 9.6%, SD = 9.3%). Although no significant differences in cycling among groups were found, large variabilities in cycling among Sherman female rats were evident. More recently in this laboratory, it has been found that intracerebral infusion of the acetylacholinesterase inhibitor, eserine, in Long-Evans Hooded rats maintained on a 12/12 light/dark cycle similarly does not affect cycling. There was also found less variability in cyclicity using Long-Evans Hooded rats on this light/dark cycle. Therefore, much of cycling variability evident in this study may have been avoided by using a different strain of rat or a 12/12 light/dark cycle. The large variability in cycling among Sherman females in this study and the small number of cycles monitored limits any conclusions about the lack of effects of surgery, anesthesia, and/or drug treatments on cyclicity.
INHIBITION OF NATURAL RECEPTIVITY
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Nineteen of the 24 female rats were found to be cycling normally at the time of drug testing in Experiment I. Six animals were assigned to each of the two drug testing conditions, while seven were assigned to the saline group. For the purpose of analysis, lordosis quotients (LQ) were converted into Percents of Control (POC). POC for each animal was calculated as follows (See Fig. 11:
POC =
LQ after infusion × I00 LQ before infusion
Intraventricular infusions of both high (mean POC=30.00%, S D = 3 0 . 9 8 % ) and low (mean POC=23.33%, S D = 2 2 . 5 1 % ) doses of scopolamine were found to significantly inhibit lordosis behavior in female rats (p<0.0001), whereas infusions of saline (mean POC=98.21%, S D = 4 . 7 2 ) was not. These results are consistent with those reported using ovariectomized female rats primed with estrogen and progesterone (8). Post hoc comparison of POC for high and low dose groups further indicated that there was no significant dose-dependent difference. Although no dose-dependent difference was detected at these drug levels, this does not suggest that there would not be a dose-dependent relationship at other dose levels as reported with ovariectomized, hormone-primed females (8). EXPERIMENT 2 In Experiment 2, the incidence of lordosis in intact cycling female rats during natural estrus was examined following intraperitoneal injection of scopolamine hydrobromide. This compound has been shown to inhibit the incidence of lordosis when injected systemically in ovariectomized rats that have been primed with estrogen and progesterone (8,17). METHOD
Cycling was again monitored daily by examination of vaginal
cytology and sexual behavior. At first sign of sexual receptivity following two successful cycles, drug testing occurred. Prior to drug administration, (-)-scopolamine hydrobromide was dissolved in saline solution and prepared for injection. Again, the female rat was introduced into the behavior testing arena where she received 10 mounts by a male. Animals which received saline infusions in Experiment 1, received a 0.2 ml intraperitoneal injection of saline solution, while animals which received scopolamine infusions in Experiment 1, received a 0.2 ml intraperitoneal injection of I m g scopolamine (4 mg/kg). This dose level of scopolamine has been found previously to inhibit lordosis in ovariectomized female rats primed with estrogen and progesterone (8,17). Immediately after injection, the female was placed in her home cage for 15 rain before returning to the testing arena to receive 10 mounts by a male. Daily monitoring of cyclic activity continued for 2 cycles past drug testing to determine the effects of scopolamine infusions on cycling behavior. RESULTS
Cycling before and after injections were compared to determine if systemic injection disrupted cycling. Cycling percentages were again calculated as in Experiment 1. Comparison of cycling percentages before injection (mean = 21.4%, SD = 4.0%) and after injection (mean = 17,4%, SD = 9.7%) indicated that injections in general did not interrupt normal cycling in female rats. Further comparison of cycling percentages for the group receiving scopolamine (mean = 18.5c~, SD = 9.29/) and the group receiving saline (mean = 15.7c~. SD = 10.9c~ I indicated no significant difference, suggesting that intraperitoneal administration of scopolamine at this dose level did not significantly interrupt normal cycling activity in this experiment. As in Experiment 1, this interpretation of cycling activity is limited due to the large variability in cycling anaong animals and the small number of cycles monitored. Fourteen of the females were found to be normally cycling at the time of drug testing during this experinaent. Therefore 7 were assigned to each group. As in Experiment 1, LQs were converted into POCs for the purpose of analysis. The mean POC for the
IUO
[] P~+m-
& .,+
'm
,40
20
Saline
II
10 ug/cannula
20 ug/cannuia
InlravenlricularTreatment FIG. 1. Inhibition of lordosis during natural behavioral estrus in intact female rats 15 rain after intraventricular infusion of low and high doses of scopolamine or saline vehicle (17<0.0001 ).
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MENARD AND DOHANICH
100
.=o 60
40
20
Saline
4 mglkg
SystemicTreatment FIG. 2. Inhibition of lordosis during natural behavioral estrus in intact female rats 15 min after systemic injection of scopolamine or saline vehicle (p<0.0001 ).
scopolamine group (24.29%, S D = 3 0 . 4 8 % ) was significantly lower (p<0.0001) than the mean POC of the saline group (98.57%, SD = 3.78%) suggesting that intraperitoneal administration of scopolamine inhibits lordosis behavior in female rats (See Fig. 2). These results are consistent with those reported in Experiment 1. They are also consistent with those results reported using ovariectomized female rats primed with estrogen and progesterone (8,17). GENERAL DISCUSSION Intraventricular infusions and systemic injections of scopolamine, a cholinergic muscarinic blocker, significantly inhibited naturally occurring lordosis behavior in intact cycling female rats.
These results are consistent with previous findings that scopolamine inhibits sexual receptivity in ovariectomized, hormonally primed female rats (2, 8, 17). These findings provide further support for the hypothesis that the cholinergic system regulates female sexual behavior. Although scopolamine was found to have a significant effect on sexual receptivity, it was not found to have a significant effect on cyclicity as measured in this study. Since interpretation of cyclicity data are limited due to sample size and variability, the concept of independent mechanisms in the regulation of sexual receptivity and the regulation of cyclicity requires more rigorous analysis. While it appears that specific cyclic events, such as sexual receptivity, are regulated by cholinergic mechanisms, the general regulation of the estrous cycle may not be under cholinergic control. Cholinergic regulation of other cyclic events, including the release of gonadotropins and prolactin during proestrus, have been reported (13,14). Furthermore, administrations of pilocarpine, a muscarinic agonist, has been shown to advance ovulation during normal 5-day cycling (18). This evidence does not, however, suggest an interruption or resetting of the estrous cycle. Instead, cyclicity appears to be regulated by circadian clock mechanisms (21). After the administration of scopolamine in the present study, it was also observed during behavior testing that sexual proceptivity, although somewhat diminished in a proportion of females, was still prevalent in a number of the animals. Although proceptivity was not measured systematically in this experiment, this observation suggests that proceptivity and receptivity are independently regulated. In previous studies, cholinergic agonists have activated lordosis but not proceptivity in ovariectomized, estrogen primed females (5-7). Further investigation of the cholinergic regulation of sexual proceptivity in intact females is planned. Although the present study offers support for the cholinergic regulation of sexual behavior, it does not identify the anatomical location of these effects in the brain. Estrogen has been shown to alter cholinergic receptor binding (9-11, 19, 20) and related enzymatic activity (15,16) in areas of the brain implicated in the regulation of sexual behavior, including the medial preoptic area and the ventromedial hypothalamus. However, conclusive localization of the cholinergic regulation of sexual behavior in the brain has yet to be documented.
REFERENCES 1. Boling, J. L.; Blandau, R. J. The estrogen-progesterone induction of mating responses in the spayed female rat. Endocrinology 25:359364; 1939. 2. Clemens, L. G.; Barr, P. J.; Dohanich, G. P. Cholinergic regulation of female sexual behavior in rats demonstrated by manipulation of endogenous acetylcholine. Physiol. Behav. 45(2): 437442; 1989. 3. Clemens, L. G.; Dohanich, G. P. Inhibition of lordotic behavior in female rats following intracerebral infusion of anticholinergic agents. Pharmacol. Biochem. Behav. 13:89-95; 1980. 4. Clemens, L. G.; Dohanich, G. P.; Barr, P. J. Cholinergic regulation of feminine sexual behavior in laboratory rats. In: Hormones and behavior in higher vertebrates. New York: Springer-Verlag; 1983: 56--68. 5. Clemens, L. G.; Donhanich, G. P.; Witcher, J. A. Cholinergic influences of estrogen-independent sexual behavior in female rats. J. Comp. Physiol. Psychol. 95:763-770; 1981. 6. Clemens, L. G.; Humphrys, R. R.; Donhanich, G. P. Cholinergic brain mechanisms and the hormonal regulation of female sexual behavior in the rat. Pharmacol. Biochem. Behav. 13:81-88; 1980. 7. Dohanich, G. P.; Barr, J.; Witcher, J. A.; Clemens, L. G. Pharmacological and anatomical aspects of cholinergic activation of female sexual behavior. Physiol. Behav. 32:1021-1026; 1984. 8. Dohanich, G. P.; Holland, R.; Menard, C.; McMullan, M.; Cada, D. Effects of cholinergic muscarinic antagonists on female sexual behav-
ior. Conf. Reprod. Behav. Prog. Abstr. 20:20; 1988. 9. Dohanich, G. P.; Witcber, J. A.; Weaver, D. R.; Clemens, L. G. Alteration of muscarinic binding in specific brain areas following estrogen treatment. Brain Res. 241:347-350; 1982. 10. Egozi, Y.; Avissar, S.; Sokolovsky, M. Muscarinic mechanisms and sex hormone secretions in rat adenohypophysis and preoptic area. Neuroendocrinology 35:93-97; 1982. 11. Egozi, Y.; Kloog, Y. Muscarinic receptors in the preoptic area are sensitive to 17[3-Estradiol during the critical period. Neuroendocrinology 40:385-392; 1985. 12. Kaufman, L. S.; McEwen, B. S.; Pfaff, D. W. Cholinergic mechanisms of lordotic behavior in rats. Physiol. Behav. 43:507-514; 1988. 13. Libertun, C.; McCann, S. M. Blockade of the release of gonadotrophins and prolactin by subcutaneous or intraventricular injection of atropine in male and female rats. Endocrinology 92:1714-1724; 1973. 14. Libertun, C.; McCann, S. M. Further evidence for cholinergic control of gonadotrophin and prolactin secretion. Proc. Soc. Exp. Biol. Med. 147:498-504; 1974. 15. Luine, V. N.; Khylchevskaya, R. 1.; McEwen, B. S. Effects of gonadal steroids on activities of monoamine oxidase and choline acetylase in rat brain. Brain Res. 86:293-306; 1975. 16. Luine, V.; Park, D.; Tong, J.; Reis, D.; McEwen, B. S. Immunochemical demonstration of increased choline acetyltransferase concentration in rat preoptic areas after estradiol administration. Brain Res.
INHIBITION OF NATURAL RECEPTIVITY
191:273-277; 1980. 17. Meyers, T. C.; Clemens, L. G. Scopolamine inhibition of lordosis at high levels of estrogen priming. Conf. Reprod. Behav. Prog. Abstr. 18:72; 1986. 18. Meyerson, B. J.; Palis, A. Advancement of the time for ovulation in the 5-day cyclic rat by pilocarpine. Acta Pharmacol. Toxicol. (Suppl.) 28:68; 1970. 19. Olsen, K. L.; Edwards, E.; Schechter, N.; Whalen, R. E. Muscarinic
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receptors in preoptic area and hypothalamus: effects of cyclicity, sex, and estrogen treatment. Brain Res. 448:223-229; 1988. 20. Rainbow, T. C.; DeGroff, V.; Luine, V. N.; McEwen, B. S. Estradiol 17-13 increases the number of muscarinic receptors in hypothalamic nuclei. Brain Res. 198:239-243; 1980. 21. Schwartz, N. B. A model for the regulation of ovulation in the rat. Recent Prog. Horm. Res. 25:1-55; 1969.