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Coordination of hamster lordosis and flank marking behavior: Role of arginine vasopressin within the medial preoptic-anterior hypothalamus
Brain Research Bulletin, Vol. 23, pp. 105-109. IDPergamon Press plc, 1989. Printed in the U.S.A.
0361-9230/89 $3.00 + .OO
Coordination of Hamster Lordosis and Flank Marking Behavior: Role of Arginine Vasopressin Within the Medial Preoptic-Anterior Hypothalamus H. ELLIOTT ALBERS AND SUSAN RAWLS Laboratory of Neuroendocrinology and Behavior, Departments of Biology and Psychology, Georgia State University, Atlanta, GA 30303 Received 10 April 1989
ALBERS, H. E. AND S. RAWLS. Coordination of hamster lordosis andflank marking behavior: Role of arginine vasopressin within the medial preoptic-anterior hypothalamus. BRAIN RES BULL 23(1/2) 105-109, 1989.-The role of arginine vasopressin (AVP) within the medial preoptic area-anterior hypothalamus (MPOA-AH) in the neural coordination of lordosis and flank marking was investigated. AVP, but not saline, microinjected into the MPOA-AH of ovariectomized hamsters not given hormone replacement therapy stimulated high levels of flank marking when tested with a sexually experienced male, or when tested alone. In contrast, AVP microinjected into the MPOA-AH of ovariectomized hamsters given estradiol benzoate (EB) and progesterone did not stimulate flank marking or inhibit lordosis during tests with a sexually experienced male. However these same females exhibited high levels of flank marking in response to AVP when tested alone. A second experiment demonstrated that progesterone was not required for inhibition of AVP-induced flank marking in ovariectomized females given EB replacement and tested with sexually experienced males. The present study provides no evidence that AVP acts within the MF’OA-AH to inhibit hamster lordosis, but demonstrates that ovarian hormones and male social contact block the induction of flank marking by AVP microinjected into the MPOA-AH. These data suggest that one component in the neural coordination of lordosis and flank marking is inhibition of the response of the MPOA-AH to AVP. Peptides
Estrogen
Progesterone
Hypothalamus
Scent marking
increases. This inverse relationship results in the appropriate sequencing of the behaviors important for attraction and selection of a mate with the behaviors necessary for subsequent mating. The appropriate sequencing of scent marking and lordosis during the estrous cycle could be the result of the differential effect of ovarian hormones on these behaviors. In the hamster, estrogen is sufficient to stimulate lordosis, however estrogen combined with progesterone is more effective than estrogen alone (6,7). Estrogen increases the frequency of flank marking in response to male odors, while progesterone given in combination with estrogen reduces the frequency of flank marking (3). The stimulatory effects of estrogen and progesterone on lordosis, combined with their inhibitory effect on scent marking may account for the inverse relationship observed between these behaviors during the estrous cycle. In addition to ovarian hormones, stimulation of lordosis requires the appropriate social environment, specifically the presence of a male, or stimuli that can mimic the presence of a male (24). The social stimulation produced by the presence of the male, which is necessary for induction of lordosis, may also contribute- to the inhibition of flank marking on estrus. How the neural mechanisms that respond to ovarian hormones and social factors sequence the expression of lordosis and flank marking is not well understood. Two hypothalamic regions have been identified to be critical in the control of lordosis and flank
REPRODUCTION requires the coordination of social, behavioral and physiological factors. Potential mates must be attracted and the appropriate mate selected before the initiation of copulatory behavior. In spontaneous ovulators, the timing of these events must be closely synchronized with the ovulatory cycle to ensure that mating occurs when the female can conceive. Much of the coordination of reproductive behavior and physiology appears to result from how gonadal hormones simultaneously influence the expression of both behavior and physiology. For example, in hamsters where ovulation and receptive behavior have a precise 96-hr rhythm (15,38), the elevation of estrogen early in the estrous cycle and the surge of progesterone that occurs on the afternoon of proestrus are important for the stimulation of both ovulation and lordosis behavior (27,34). In addition to lordosis, many other behaviors contribute to successful reproduction. Prior to the opportunity for copulation on es&us, females engage in a number of behaviors important for attracting and choosing among potential mates (4, 5, 16). In hamsters, vaginal marking and flank gland marking are involved in mate attraction and choice (19-22, 32). Females housed with males show increasing amounts of vaginal and flank gland marking over the days preceding receptivity. As the time of ovulation approaches, the frequency of scent marking is significantly reduced as the potential for engaging in receptive behavior
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marking. The ventromedial hypothalamus (VMH) appears to be involved in the control of female receptive behavior in several mammalian species. In the hamster, the VMH contains estrogen concentrating cells (26) and local implants of estrogen into the VMH can restore lordosis in ovariectomized hamsters (9, 39, 40). Destruction of the VMH and inhibition of protein synthesis within the VMH reduces lordosis behavior (10,29). The medial preoptic area-anterior hypothalamus (MPOA-AH) plays a major role in scent marking behavior in a variety of mammalian species (17, 18, 42). In the hamster, arginine vasopressin (AVP) contained within neurons of the MPOA-AH is critical in the control of hamster flank marking. Microinjection of AVP, but no other peptides or neurotransmitters, within the MPOA-AH stimulates intense bouts of flank marking in a dosedependent manner (1, 11, 12, 14). AVP antagonists microinjected into the MPGA-AH inhibit flank marking stimulated by AVP, by odors of other hamsters and during encounters between male hamsters. The MPOA-AH may mediate the effects of gonadal hormones on flank marking since the MPOA-AH contains estrogen concentrating cells (25), and in male hamsters testosterone significantly increases the amount of flank marking in response to AVP microinjected into the MPOA-AH (2). Coordination of the expression of lordosis and flank marking during the estrous cycle could result from an inhibitory interaction between the VMH and MPGA-AH. Support of this view comes from studies which have shown that the VMH and MPGA-AH are reciprocally innervated in rats (8, 25, 35) and lesions of the MPOA-AH facilitate lordosis (33,41). More direct evidence has come from studies in which electrical stimulation of the MPOAAH suppressed hamster lordosis by influencing neurons within the VMH (31). These data combined with evidence that AVP has excitatory effects on MPGA-AH single units recorded in the hypothalamic slice preparation (Liou and Albers, unpublished data), and that intracerebroventricular injection of AVP inhibits rat receptive behavior (36), raise the possibility that AVP may be involved in the inhibition of lordosis by acting within the MPOAAH. According to this hypothesis, AVP may not only activate flank marking, but inhibit lordosis. It is also possible that the factors responsible for the induction of lordosis also inhibit flank marking by acting on the AVP neuronal system within the MPOA-AH. The present study investigated the role of AVP within the MPOA-AH in the neural coordination of flank marking and lordosis. The initial experiment examined whether AVP microinjetted into the MPOA-AH suppressed lordosis in estrogen and progesterone primed ovariectomized hamsters in tests conducted with a sexually experienced male, or whether mating induced under these conditions blocked or reduced the induction of flank marking in response to AVP microinjected into the MPOA-AH. Since AVP was found to have no suppressive effect on lordosis, and mating completely blocked the response of the MPOA-AH to AVP, subsequent experiments investigated the importance of and social (i.e., hormonal (i.e., estrogen and progesterone) presence of a sexually active male) factors in the inhibition of the AVP response within the MPGA-AH. More specifically, these studies examined whether: 1) estrogen and progesterone levels sufficient to stimulate lordosis are sufficient to block the induction of flank marking produced by AVP within the MPOA-AH, 2) the social stimulation produced by the presence of the male inhibits the response of the MPGA-AH to AVP, or whether 3) estrogen and progesterone must occur in combination with the social stimulation provided by the male to block the induction of flank marking by AVP within the MPGA-AH.
ALBERS AND RAWLS
METHOD
All hamsters were obtained from Harlan Sprague Dawley Inc. and housed under a light-dark cycle containing 14 hr of light (on at 0900) and 10 hr of darkness. Food and water were provided continuously throughout all experiments. All females were anesthetized with sodium pentobarbital and ovariectomized through incisions made on the dorsal flanks. Each hamster was implanted with a blank Silastic capsule (0.062 i.d., 0.125 o.d.; Dow Coming), or a capsule containing 5 mm of crystalline 17B estradiol-3-benzoate (EB). Silastic capsules of these dimensions packed with 5 mm of EB have been reported to produce blood levels of estrogen similiar to those observed during the proestrous phase of the hamster estrous cycle (23). Following ovariectomy each hamster was individually housed. Approximately 7 weeks later each hamster was anesthetized with sodium pentobarbital and stereotaxically implanted with a single, 4 mm 26-gauge guide cannula aimed at the MPOA-AH. The stereotaxic coordinates were - 7.3 mm ventral of dura and 1.1 mm anterior and 1.7 mm lateral of bregma. The guide cannula was implanted 10 degrees from perpendicular with the incisor bar set at 0. Behavioral testing was begun approximately two weeks later. Six hr before testing each female was subcutaneously injected with a control injection of 0.1 ml of corn oil or 0.5 mg progesterone dissolved in 0.1 ml corn oil. All testing was conducted during the dark phase of the light-dark cycle under dim red illumination. Immediately before testing each female was microinjected with 400 nl of saline, 0.9 PM arginine vasopressin (Experiment l), or 90 p.M arginine vasopressin (Experiment 2) dissolved in 400 nl of saline. Microinjections were made through a 33-gauge needle attached to a l-p1 Hamilton syringe with polyethylene tubing. In all experiments in which individual hamsters received injections of more than one substance, the order of the substances injected was counterbalanced among the subjects. Testing was conducted at 24-hr intervals in a clean 24 X 45 X 20 cm Plexiglas arena. A sexually experienced male hamster was placed in the testing arena prior to the female. During the IO-min test the amount of time spent in lordosis and the number of flank marks exhibited by the female were recorded. Statistical differences between conditions were analyzed with the one-way or Subjects/Groups x Trials classifications of the analysis of variance (37). Following the experiments each hamster was anesthetized with sodium pentobarbital and microinjected with 400 nl of dye to mark the site of injection. Each hamster was intracardially perfused with Perfix and the brain removed. The brain was dissected with the aid of a dissecting microscope and the site of injection was verified to be within the MPOA-AH. RESULTS
Experiment 1 Microinjection of AVP into the MPOA-AH of ovariectomized hamsters (N = 4) given estradiol benzoate (EB) and progesterone (P) did not induce flank marking, or reduce the duration of lordosis behavior (Fig. 1) during a 10-r& test with a sexually experienced male. No differences were observed in the duration of lordosis following microinjection with AVP or saline (SAL), i.e., AVP: 409.4 & 36.6 set and SAL: 394.8 k76.5. Although essentially no flank marking was observed in response to AVP microinjection during tests conducted with a male, when these hamsters were tested in the same protocol without a male in the arena, all hamsters exhibited high levels of flank marking behavior in response to microinjection of AVP, i.e., 36.025.5 flank marks/l0 min. To
107
LORDOSIS AND FLANK MARKING
BLANK+OIL
IO
P
5 5 4 20
a
1 AVP
FIG. 1. The amount of lordosis (white bars) and flank marking (black bars) (mean? SEM) following microinjection of arginine vasopressin (AVP; 0.9 p,M) and saline (SAL) into the medial preoptic-anterior hypothalamus of ovariectomized hamsters (N = 4) implanted with Silastic capsules containing estradiol benzoate and subcutaneously injected with progesterone 6 hr before testing. Flank marking was almost completely absent when a sexually experienced male was present throughout the IO-min test. However high levels of flank marking were observed in response to AVP microinjection when the females were tested alone (AVP posttest alone). The duration of lordosis was similar whether AVP or SAL was microinjetted into the MPOA-AH. All microinjections were given immediately prior to testing, with the order of administration of AVP and SAL across testing days counterbalanced among the subjects.
verify that each female remained capable of lordosis during this posttest, each hamster was placed with a male immediately following this lo-min test and was observed to exhibit lordosis shortly after it was placed with the male. A second group of ovariectomized hamsters (N =4) given blank Silastic capsules and injected with oil 6 hr prior to testing, flank marked at high levels following AVP microinjection in tests conducted in the presence of a sexually experienced male (Fig. 2). Despite the repeated attempts at copulation by the male, the females flank marked more than 20 times and displayed no lordosis behavior. When these same females received microinjections of saline prior to testing with the male neither flank marking nor lordosis behavior was observed. During the posttest, when each female was microinjected with AVP and tested without the male present, an average of more than 40 flank marks were observed during each lo-min observation period. The higher levels of flank marking observed in response to AVP when the females were tested alone, as compared to the test conducted in the presence of the male, appeared to result from the interference produced by the persistent copulatory advances of the male during the test period. Analysis of the flank marking data obtained from behavioral tests conducted in the presence of male hamsters revealed a statistically significant @
AVP
SAL
FIG. 2. The amount of lordosis (white bars) and flank marking (black bars) (mean k SEM) following microinjection of arginine vasopressin (AVP; 0.9 p.M) and saline (SAL) into the medial preoptic-anterior hypothalamus of ovariectomized hamsters (N=4) implanted with blank Silastic capsules and subcutaneously injected with oil 6 hr prior to testing. High levels of flank marking were observed in response to microinjection of AVP whether a sexually experienced male was present (AVP with male), or not (AVP posttest alone). During the AVP test with the male, flank marking appeared to be interrupted by persistent advances by the male. The order of administration of AVP and SAL microinjections across testing days was counterbalanced among the subjects. analysis of the frequency of lordosis behavior revealed only a statistically significant (~~~0.01) main effect of hormone treatment. Although the EB+P-treated group flank marked less than the OIL+BLANK group in response to AVP when individuals from these groups were tested alone, this difference was not statistically reliable (pBO.05). Experiment 2
A second experiment was conducted to determine whether the mating induced inhibition of AVP-stimulated flank marking persisted when a higher concentration of AVP (i.e., 90 pM versus 0.9 pM) was microinjected into the MPGA-AH, and whether the inhibition of AVP-induced flank marking required progesterone. Ovariectomized females (N = 7) implanted with EB-containing capsules were injected with P and OIL 6 hr prior to behavioral testing on consecutive days. The order of P and oil injection was counterbalanced among the subjects. As can be seen in Fig. 3 the induction of flank marking by AVP microinjected into the MPOAAH was inhibited when behavioral testing was conducted in the presence of a male regardless of whether the females had been treated with EB + P or EB + OIL. The duration of lordosis behavior during testing was significantly @<0.05) shorter when the hamsters were injected with oil than when injected with P 6 hr before testing. During the posttest when all females were tested without the male present, high levels of flank marking were observed in response to microinjection of AVP into the MPGA-AH. DISCUSSION The present study provides no evidence that AVP acts within the MPOA-AH to inhibit lordosis behavior in the hamster. Ovariectomized females given estrogen and progesterone exhib-
ALBERS AND RAWLS
FIG.
3. The amount of lordosis (white bars) and flank marking (black bars) (mean-C SEM) following microinjection of arginine vasopressin (AVP; 90 FM) into the medial preoptic anterior hypothalamus of ovariectomized hamsters (N=7) implanted with Silastic capsules containing estradiol benzoate (E) and subcutaneously injected with progesterone (PROG) and oil 6 hr prior to testing. Almost no flank marking was observed in response to AVP microinjection in the tests conducted with a sexually experienced male regardless of whether hamsters were given E+P or E+OIL. When tested alone all females exhibited high levels of flank marking in response to AVP microinjection. In contrast, the duration of lordosis was significantly @<0.05) shorter when the hamsters were given E+OIL than when given E+P. The order of administration of P and oil across testing days was counterbalanced among subjects.
ited the same duration of lordosis whether they were microinjected with AVP or SAL. Even microinjection of a high dose of AVP (i.e., 90 PM) within the MPOA-AH did not inhibit lordosis, whether the lordosis was stimulated by estrogen and progesterone or estrogen alone. It therefore seems unlikely that the inhibition of lordosis reported to result from electrical stimulation of the MPOA-AH (31) is mediated by AVP. In addition, these data suggest that the inhibition of receptivity produced by intraventricular administration of AVP in rats (36) may occur at sites other than the MPOA-AH. Plank marking behavior induced by AVP within the MPOAAH was completely blocked in ovariectomized hamsters adminis-
tered estrogen and progesterone, or estrogen alone and provided social contact with a male hamster. Inhibition of the behavioral effects of AVP within the MPOA-AH required both hormonal and social stimulation since neither ovarian hormones nor male social contact alone were sufficient to block the induction of flank marking. These data indicate that hormonal and social conditions sufficient to stimulate lordosis inhibit the expression of flank marking by rendering the MPOA-AH unresponsive to AVP. This inhibition could occur at the level of the neurons responding to AVP within the MPOA-AH, or on neural pathways controlling flank marking efferent to the MPOA-AH. These data further indicate that the inhibition of flank marking during copulation does not require inhibition of AVP release within the MPOA-AH. The neural mechanisms that inhibit the behavioral response of the MPOA-AH to AVP are not known. The inhibition of AVP within the MPOA-AH could be mediated by a number of MPOAAH afferents from CNS sites that are involved in the control of lordosis (35), such as projections from the VMH. It is also possible that this inhibition is controlled by a more diffuse system that serves to inhibit a number of behaviors, while allowing the expression of lordosis. While both the appropriate hormonal priming and the presence of the male were required for the inhibition of the AVP response, the precise signal necessary for activating this inhibitory system is not clear. This inhibition could be initiated when a hormonally primed female simply encounters a male, or this inhibition might only be initiated when the female has begun to engage in lordosis. While the present study did not attempt to resolve this question, it did not appear that the female had to be engaged in lordosis for AVP-induced flank marking to be blocked. Although these females spent the majority of their time in lordosis, almost no flank marking was observed during the remainder of the test period when they were engaged in other behaviors. In summary, inhibition of the responsiveness of the MPOA-AH to AVP appears to be one component in the neural circuitry that serves to coordinate the expression of flank marking and lordosis. The neural circuitry that coordinates lordosis with flank marking and other proceptive behaviors will most likely be complex given the variety of behaviors involved in successful copulation. The present data suggest that neural coordination of reproductive behaviors involves not only the activation of the behavior dictated by the hormonal and social stimuli present (e.g., lordosis), but also the simultaneous inhibition of other, potentially conflicting behaviors (flank marking).
ACKNOWLEDGEMENT
This work was supported by NSF Grant BNS-8711373.
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12. Ferris, C. F.; Pollock, P.; Albers, H. E.; Leeman, S. E. Inhibition of flank marking behavior in golden hamsters by microinjection of a vasopressin antagonist into the hypothalamus. Neurosci. Lett. 55: 163-168; 1985. 13. Ferris, C. F.; Meenan, D. M.; Albers, H. E. Microinjection of kainic acid into the hypothalamus of golden hamsters prevents vasopressindependent flank marking behavior. Neuroendocdnology 44: 112-l 16; 1986. 14. Ferris, C. F.; Meenan, D. M.; Axelson, J. F.; Albers, H. E. A vasopressin antagonist can reverse dominant/subordinate behavior in hamsters. Physiol. Behav. 38:135-138; 1986. 15. Fitzgerald, K. M.; Zucker, I. Circadian organization of the estrous cycle of the golden hamster. Proc. Natl. Acad. Sci. USA 73: 2923-2927; 1976. 16. Floody, 0. R. The hormonal control of ultrasonic communication in rodents. Am. Zool. 21:129-142; 1981. 17. Hart, B. L. Medial preoptic-anterior hypothalamic area and sociosexual behavior of male dogs: A comparative neuropsychological analysis. J. Comp. Physiol. Psychol. 86:328-349; 1974. 18. Hart, B. L.; Voith, V. L. Changes in urine spraying, feeding and sleep behavior of cats following medial preoptic-anterior hypothalamic lesions. Brain Res. 145:4Ow9; 1978. 19. Huck, U. W.; Lisk, R. D.; Gore, A. C. Scent marking and mate choice in the golden hamster. Physiol. Behav. 35:389-393; 1985. 20. Johnston, R. E. The causation of two scent marking behaviours in female golden hamsters. Anim. Behav. 25:317-327; 1977. 21. Johnston, R. E. Olfactory preferences, scent marking and proceptivity in female hamsters. Harm. Behav. 13:21-39; 1979. 22. Johnston, R. E. Communication. In: Siegel, H. I., ed. The hamster. New York: Plenum Press; 1985:121-155. 23. Jorgenson, K. L.; Schwartz, N. B. Effect of steroid treatment on tonic and surge secretion of LH and FSH in the female golden hamster: effect of photoperiod. Neuroendocrinology 39:549-554; 1984. 24. Kow, L.-M.; Malsbury, C. W.; Pfaff, D. W. Lordosis in the male golden hamster elicited by manual stimulation: Characteristics and hormonal sensitivity. J. Comp. Physiol. Psychol. 90:26-40; 1976. 25. Krieger, M. S.; Conrad, L. C. A.; Pfaff, D. W. An autoradiographic study of the efferent connections of the ventromedial nucleus of the hypothalamus. J. Comp. Neural. 183:785-816; 1979. 26. Krieger, M. S.; Morrell, J. I.; Pfaff, D. W. Autoradiographic localization of estradiol-concentrating cells in the female hamster brain. Neuroendocrinology 22: 193-205; 1976. 27. Lisk, R. D. The estrous cycle. In: Siegel, H. I., ed. The hamster. New York: Plenum Press; 1985:23-53. 28. Lisk, R. D.; Zeiss, J.; Ciaccio, L. A. The influence of olfaction on
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0361-9230/89 $3.00 + .OO
Coordination of Hamster Lordosis and Flank Marking Behavior: Role of Arginine Vasopressin Within the Medial Preoptic-Anterior Hypothalamus H. ELLIOTT ALBERS AND SUSAN RAWLS Laboratory of Neuroendocrinology and Behavior, Departments of Biology and Psychology, Georgia State University, Atlanta, GA 30303 Received 10 April 1989
ALBERS, H. E. AND S. RAWLS. Coordination of hamster lordosis andflank marking behavior: Role of arginine vasopressin within the medial preoptic-anterior hypothalamus. BRAIN RES BULL 23(1/2) 105-109, 1989.-The role of arginine vasopressin (AVP) within the medial preoptic area-anterior hypothalamus (MPOA-AH) in the neural coordination of lordosis and flank marking was investigated. AVP, but not saline, microinjected into the MPOA-AH of ovariectomized hamsters not given hormone replacement therapy stimulated high levels of flank marking when tested with a sexually experienced male, or when tested alone. In contrast, AVP microinjected into the MPOA-AH of ovariectomized hamsters given estradiol benzoate (EB) and progesterone did not stimulate flank marking or inhibit lordosis during tests with a sexually experienced male. However these same females exhibited high levels of flank marking in response to AVP when tested alone. A second experiment demonstrated that progesterone was not required for inhibition of AVP-induced flank marking in ovariectomized females given EB replacement and tested with sexually experienced males. The present study provides no evidence that AVP acts within the MF’OA-AH to inhibit hamster lordosis, but demonstrates that ovarian hormones and male social contact block the induction of flank marking by AVP microinjected into the MPOA-AH. These data suggest that one component in the neural coordination of lordosis and flank marking is inhibition of the response of the MPOA-AH to AVP. Peptides
Estrogen
Progesterone
Hypothalamus
Scent marking
increases. This inverse relationship results in the appropriate sequencing of the behaviors important for attraction and selection of a mate with the behaviors necessary for subsequent mating. The appropriate sequencing of scent marking and lordosis during the estrous cycle could be the result of the differential effect of ovarian hormones on these behaviors. In the hamster, estrogen is sufficient to stimulate lordosis, however estrogen combined with progesterone is more effective than estrogen alone (6,7). Estrogen increases the frequency of flank marking in response to male odors, while progesterone given in combination with estrogen reduces the frequency of flank marking (3). The stimulatory effects of estrogen and progesterone on lordosis, combined with their inhibitory effect on scent marking may account for the inverse relationship observed between these behaviors during the estrous cycle. In addition to ovarian hormones, stimulation of lordosis requires the appropriate social environment, specifically the presence of a male, or stimuli that can mimic the presence of a male (24). The social stimulation produced by the presence of the male, which is necessary for induction of lordosis, may also contribute- to the inhibition of flank marking on estrus. How the neural mechanisms that respond to ovarian hormones and social factors sequence the expression of lordosis and flank marking is not well understood. Two hypothalamic regions have been identified to be critical in the control of lordosis and flank
REPRODUCTION requires the coordination of social, behavioral and physiological factors. Potential mates must be attracted and the appropriate mate selected before the initiation of copulatory behavior. In spontaneous ovulators, the timing of these events must be closely synchronized with the ovulatory cycle to ensure that mating occurs when the female can conceive. Much of the coordination of reproductive behavior and physiology appears to result from how gonadal hormones simultaneously influence the expression of both behavior and physiology. For example, in hamsters where ovulation and receptive behavior have a precise 96-hr rhythm (15,38), the elevation of estrogen early in the estrous cycle and the surge of progesterone that occurs on the afternoon of proestrus are important for the stimulation of both ovulation and lordosis behavior (27,34). In addition to lordosis, many other behaviors contribute to successful reproduction. Prior to the opportunity for copulation on es&us, females engage in a number of behaviors important for attracting and choosing among potential mates (4, 5, 16). In hamsters, vaginal marking and flank gland marking are involved in mate attraction and choice (19-22, 32). Females housed with males show increasing amounts of vaginal and flank gland marking over the days preceding receptivity. As the time of ovulation approaches, the frequency of scent marking is significantly reduced as the potential for engaging in receptive behavior
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marking. The ventromedial hypothalamus (VMH) appears to be involved in the control of female receptive behavior in several mammalian species. In the hamster, the VMH contains estrogen concentrating cells (26) and local implants of estrogen into the VMH can restore lordosis in ovariectomized hamsters (9, 39, 40). Destruction of the VMH and inhibition of protein synthesis within the VMH reduces lordosis behavior (10,29). The medial preoptic area-anterior hypothalamus (MPOA-AH) plays a major role in scent marking behavior in a variety of mammalian species (17, 18, 42). In the hamster, arginine vasopressin (AVP) contained within neurons of the MPOA-AH is critical in the control of hamster flank marking. Microinjection of AVP, but no other peptides or neurotransmitters, within the MPOA-AH stimulates intense bouts of flank marking in a dosedependent manner (1, 11, 12, 14). AVP antagonists microinjected into the MPGA-AH inhibit flank marking stimulated by AVP, by odors of other hamsters and during encounters between male hamsters. The MPOA-AH may mediate the effects of gonadal hormones on flank marking since the MPOA-AH contains estrogen concentrating cells (25), and in male hamsters testosterone significantly increases the amount of flank marking in response to AVP microinjected into the MPOA-AH (2). Coordination of the expression of lordosis and flank marking during the estrous cycle could result from an inhibitory interaction between the VMH and MPGA-AH. Support of this view comes from studies which have shown that the VMH and MPGA-AH are reciprocally innervated in rats (8, 25, 35) and lesions of the MPOA-AH facilitate lordosis (33,41). More direct evidence has come from studies in which electrical stimulation of the MPOAAH suppressed hamster lordosis by influencing neurons within the VMH (31). These data combined with evidence that AVP has excitatory effects on MPGA-AH single units recorded in the hypothalamic slice preparation (Liou and Albers, unpublished data), and that intracerebroventricular injection of AVP inhibits rat receptive behavior (36), raise the possibility that AVP may be involved in the inhibition of lordosis by acting within the MPOAAH. According to this hypothesis, AVP may not only activate flank marking, but inhibit lordosis. It is also possible that the factors responsible for the induction of lordosis also inhibit flank marking by acting on the AVP neuronal system within the MPOA-AH. The present study investigated the role of AVP within the MPOA-AH in the neural coordination of flank marking and lordosis. The initial experiment examined whether AVP microinjetted into the MPOA-AH suppressed lordosis in estrogen and progesterone primed ovariectomized hamsters in tests conducted with a sexually experienced male, or whether mating induced under these conditions blocked or reduced the induction of flank marking in response to AVP microinjected into the MPOA-AH. Since AVP was found to have no suppressive effect on lordosis, and mating completely blocked the response of the MPOA-AH to AVP, subsequent experiments investigated the importance of and social (i.e., hormonal (i.e., estrogen and progesterone) presence of a sexually active male) factors in the inhibition of the AVP response within the MPGA-AH. More specifically, these studies examined whether: 1) estrogen and progesterone levels sufficient to stimulate lordosis are sufficient to block the induction of flank marking produced by AVP within the MPOA-AH, 2) the social stimulation produced by the presence of the male inhibits the response of the MPGA-AH to AVP, or whether 3) estrogen and progesterone must occur in combination with the social stimulation provided by the male to block the induction of flank marking by AVP within the MPGA-AH.
ALBERS AND RAWLS
METHOD
All hamsters were obtained from Harlan Sprague Dawley Inc. and housed under a light-dark cycle containing 14 hr of light (on at 0900) and 10 hr of darkness. Food and water were provided continuously throughout all experiments. All females were anesthetized with sodium pentobarbital and ovariectomized through incisions made on the dorsal flanks. Each hamster was implanted with a blank Silastic capsule (0.062 i.d., 0.125 o.d.; Dow Coming), or a capsule containing 5 mm of crystalline 17B estradiol-3-benzoate (EB). Silastic capsules of these dimensions packed with 5 mm of EB have been reported to produce blood levels of estrogen similiar to those observed during the proestrous phase of the hamster estrous cycle (23). Following ovariectomy each hamster was individually housed. Approximately 7 weeks later each hamster was anesthetized with sodium pentobarbital and stereotaxically implanted with a single, 4 mm 26-gauge guide cannula aimed at the MPOA-AH. The stereotaxic coordinates were - 7.3 mm ventral of dura and 1.1 mm anterior and 1.7 mm lateral of bregma. The guide cannula was implanted 10 degrees from perpendicular with the incisor bar set at 0. Behavioral testing was begun approximately two weeks later. Six hr before testing each female was subcutaneously injected with a control injection of 0.1 ml of corn oil or 0.5 mg progesterone dissolved in 0.1 ml corn oil. All testing was conducted during the dark phase of the light-dark cycle under dim red illumination. Immediately before testing each female was microinjected with 400 nl of saline, 0.9 PM arginine vasopressin (Experiment l), or 90 p.M arginine vasopressin (Experiment 2) dissolved in 400 nl of saline. Microinjections were made through a 33-gauge needle attached to a l-p1 Hamilton syringe with polyethylene tubing. In all experiments in which individual hamsters received injections of more than one substance, the order of the substances injected was counterbalanced among the subjects. Testing was conducted at 24-hr intervals in a clean 24 X 45 X 20 cm Plexiglas arena. A sexually experienced male hamster was placed in the testing arena prior to the female. During the IO-min test the amount of time spent in lordosis and the number of flank marks exhibited by the female were recorded. Statistical differences between conditions were analyzed with the one-way or Subjects/Groups x Trials classifications of the analysis of variance (37). Following the experiments each hamster was anesthetized with sodium pentobarbital and microinjected with 400 nl of dye to mark the site of injection. Each hamster was intracardially perfused with Perfix and the brain removed. The brain was dissected with the aid of a dissecting microscope and the site of injection was verified to be within the MPOA-AH. RESULTS
Experiment 1 Microinjection of AVP into the MPOA-AH of ovariectomized hamsters (N = 4) given estradiol benzoate (EB) and progesterone (P) did not induce flank marking, or reduce the duration of lordosis behavior (Fig. 1) during a 10-r& test with a sexually experienced male. No differences were observed in the duration of lordosis following microinjection with AVP or saline (SAL), i.e., AVP: 409.4 & 36.6 set and SAL: 394.8 k76.5. Although essentially no flank marking was observed in response to AVP microinjection during tests conducted with a male, when these hamsters were tested in the same protocol without a male in the arena, all hamsters exhibited high levels of flank marking behavior in response to microinjection of AVP, i.e., 36.025.5 flank marks/l0 min. To
107
LORDOSIS AND FLANK MARKING
BLANK+OIL
IO
P
5 5 4 20
a
1 AVP
FIG. 1. The amount of lordosis (white bars) and flank marking (black bars) (mean? SEM) following microinjection of arginine vasopressin (AVP; 0.9 p,M) and saline (SAL) into the medial preoptic-anterior hypothalamus of ovariectomized hamsters (N = 4) implanted with Silastic capsules containing estradiol benzoate and subcutaneously injected with progesterone 6 hr before testing. Flank marking was almost completely absent when a sexually experienced male was present throughout the IO-min test. However high levels of flank marking were observed in response to AVP microinjection when the females were tested alone (AVP posttest alone). The duration of lordosis was similar whether AVP or SAL was microinjetted into the MPOA-AH. All microinjections were given immediately prior to testing, with the order of administration of AVP and SAL across testing days counterbalanced among the subjects.
verify that each female remained capable of lordosis during this posttest, each hamster was placed with a male immediately following this lo-min test and was observed to exhibit lordosis shortly after it was placed with the male. A second group of ovariectomized hamsters (N =4) given blank Silastic capsules and injected with oil 6 hr prior to testing, flank marked at high levels following AVP microinjection in tests conducted in the presence of a sexually experienced male (Fig. 2). Despite the repeated attempts at copulation by the male, the females flank marked more than 20 times and displayed no lordosis behavior. When these same females received microinjections of saline prior to testing with the male neither flank marking nor lordosis behavior was observed. During the posttest, when each female was microinjected with AVP and tested without the male present, an average of more than 40 flank marks were observed during each lo-min observation period. The higher levels of flank marking observed in response to AVP when the females were tested alone, as compared to the test conducted in the presence of the male, appeared to result from the interference produced by the persistent copulatory advances of the male during the test period. Analysis of the flank marking data obtained from behavioral tests conducted in the presence of male hamsters revealed a statistically significant @
AVP
SAL
FIG. 2. The amount of lordosis (white bars) and flank marking (black bars) (mean k SEM) following microinjection of arginine vasopressin (AVP; 0.9 p.M) and saline (SAL) into the medial preoptic-anterior hypothalamus of ovariectomized hamsters (N=4) implanted with blank Silastic capsules and subcutaneously injected with oil 6 hr prior to testing. High levels of flank marking were observed in response to microinjection of AVP whether a sexually experienced male was present (AVP with male), or not (AVP posttest alone). During the AVP test with the male, flank marking appeared to be interrupted by persistent advances by the male. The order of administration of AVP and SAL microinjections across testing days was counterbalanced among the subjects. analysis of the frequency of lordosis behavior revealed only a statistically significant (~~~0.01) main effect of hormone treatment. Although the EB+P-treated group flank marked less than the OIL+BLANK group in response to AVP when individuals from these groups were tested alone, this difference was not statistically reliable (pBO.05). Experiment 2
A second experiment was conducted to determine whether the mating induced inhibition of AVP-stimulated flank marking persisted when a higher concentration of AVP (i.e., 90 pM versus 0.9 pM) was microinjected into the MPGA-AH, and whether the inhibition of AVP-induced flank marking required progesterone. Ovariectomized females (N = 7) implanted with EB-containing capsules were injected with P and OIL 6 hr prior to behavioral testing on consecutive days. The order of P and oil injection was counterbalanced among the subjects. As can be seen in Fig. 3 the induction of flank marking by AVP microinjected into the MPOAAH was inhibited when behavioral testing was conducted in the presence of a male regardless of whether the females had been treated with EB + P or EB + OIL. The duration of lordosis behavior during testing was significantly @<0.05) shorter when the hamsters were injected with oil than when injected with P 6 hr before testing. During the posttest when all females were tested without the male present, high levels of flank marking were observed in response to microinjection of AVP into the MPGA-AH. DISCUSSION The present study provides no evidence that AVP acts within the MPOA-AH to inhibit lordosis behavior in the hamster. Ovariectomized females given estrogen and progesterone exhib-
ALBERS AND RAWLS
FIG.
3. The amount of lordosis (white bars) and flank marking (black bars) (mean-C SEM) following microinjection of arginine vasopressin (AVP; 90 FM) into the medial preoptic anterior hypothalamus of ovariectomized hamsters (N=7) implanted with Silastic capsules containing estradiol benzoate (E) and subcutaneously injected with progesterone (PROG) and oil 6 hr prior to testing. Almost no flank marking was observed in response to AVP microinjection in the tests conducted with a sexually experienced male regardless of whether hamsters were given E+P or E+OIL. When tested alone all females exhibited high levels of flank marking in response to AVP microinjection. In contrast, the duration of lordosis was significantly @<0.05) shorter when the hamsters were given E+OIL than when given E+P. The order of administration of P and oil across testing days was counterbalanced among subjects.
ited the same duration of lordosis whether they were microinjected with AVP or SAL. Even microinjection of a high dose of AVP (i.e., 90 PM) within the MPOA-AH did not inhibit lordosis, whether the lordosis was stimulated by estrogen and progesterone or estrogen alone. It therefore seems unlikely that the inhibition of lordosis reported to result from electrical stimulation of the MPOA-AH (31) is mediated by AVP. In addition, these data suggest that the inhibition of receptivity produced by intraventricular administration of AVP in rats (36) may occur at sites other than the MPOA-AH. Plank marking behavior induced by AVP within the MPOAAH was completely blocked in ovariectomized hamsters adminis-
tered estrogen and progesterone, or estrogen alone and provided social contact with a male hamster. Inhibition of the behavioral effects of AVP within the MPOA-AH required both hormonal and social stimulation since neither ovarian hormones nor male social contact alone were sufficient to block the induction of flank marking. These data indicate that hormonal and social conditions sufficient to stimulate lordosis inhibit the expression of flank marking by rendering the MPOA-AH unresponsive to AVP. This inhibition could occur at the level of the neurons responding to AVP within the MPOA-AH, or on neural pathways controlling flank marking efferent to the MPOA-AH. These data further indicate that the inhibition of flank marking during copulation does not require inhibition of AVP release within the MPOA-AH. The neural mechanisms that inhibit the behavioral response of the MPOA-AH to AVP are not known. The inhibition of AVP within the MPOA-AH could be mediated by a number of MPOAAH afferents from CNS sites that are involved in the control of lordosis (35), such as projections from the VMH. It is also possible that this inhibition is controlled by a more diffuse system that serves to inhibit a number of behaviors, while allowing the expression of lordosis. While both the appropriate hormonal priming and the presence of the male were required for the inhibition of the AVP response, the precise signal necessary for activating this inhibitory system is not clear. This inhibition could be initiated when a hormonally primed female simply encounters a male, or this inhibition might only be initiated when the female has begun to engage in lordosis. While the present study did not attempt to resolve this question, it did not appear that the female had to be engaged in lordosis for AVP-induced flank marking to be blocked. Although these females spent the majority of their time in lordosis, almost no flank marking was observed during the remainder of the test period when they were engaged in other behaviors. In summary, inhibition of the responsiveness of the MPOA-AH to AVP appears to be one component in the neural circuitry that serves to coordinate the expression of flank marking and lordosis. The neural circuitry that coordinates lordosis with flank marking and other proceptive behaviors will most likely be complex given the variety of behaviors involved in successful copulation. The present data suggest that neural coordination of reproductive behaviors involves not only the activation of the behavior dictated by the hormonal and social stimuli present (e.g., lordosis), but also the simultaneous inhibition of other, potentially conflicting behaviors (flank marking).
ACKNOWLEDGEMENT
This work was supported by NSF Grant BNS-8711373.
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