Microinjection of arginine-vasopressin into the periaqueductal gray stimulates flank marking in Syrian hamsters (Mesocricetus auratus)

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Brain Research, 569 (1992) 136-140 (~) 1992 Elsevier Science Publishers B.V. All rights reserved. 0006-8993/92/$05.00

BRES 24965

Short Communications

Microinjection of arginine-vasopressin into the periaqueductal gray stimulates flank marking in Syrian hamsters (Mesocricetus auratus) Ann C. Hennessey, D. Carol Whitman and H. Elliott Albers Laboratory of Neuroendocrinology and Behavior, Departments of Biology and Psychology, Georgia State University, Atlanta, GA 30303 (U.S.A.) (Accepted 10 September 1991)

Key words: Arginine-vasopressin; Scent marking; Periaqueductal gray; Proceptivity; Olfaction

Syrian hamsters can communicate using a distinctive form of scent marking called flank marking. Vasopressin-sensitive neurons within the medial preoptic-anterior hypothalamic continuum (MPOA-AH) play a critical role in the control of this form of olfactory communication. Extrahypothalamic regions may also mediate hamster flank marking. Since the MPOA-AH and the periaqueductal gray (PAG) are reciprocally connected, the present study investigated whether PAG neurons are involved in the control of flank marking. The first study found that microinjection of vasopressin, but not oxytocin or saline, into the PAG induced high levels of flank marking in male (n = 8) and female (n = 5) hamsters (P < 0.01). The second study demonstrated that microinjection of vasopressin into the PAG stimulated flank marking in a dose-dependent manner in both male (n = 7) and female (n = 11) hamsters (P < 0.01). These data suggest that vasopressin-responsive neurons within the periaqueductal gray participate in the control of hamster flank marking. Olfactory communication by scent marking is an important form of communication in many mammals 22'23'29'43'5°. Flank marking is one form of scent marking used by Syrian hamsters (Mesocricetus auratus). Hamsters flank mark by rubbing large sebaceous glands, one located on each dorsolateral flank, against vertical surfaces while moving in a forward direction 3°. Intense grooming of the flank glands often occurs immediately before and between bouts of flank marking 34. Flank gland grooming entails robust licking, chewing and scratching of the flank gland region and these actions can result in a notable wet area on and around the flank gland. Flank marking is naturally elicited by odors of conspecifics or can be triggered by social encounters with other hamsters 34. Flank marking appears to play a role in dominance interactions 15'34 and may contribute to mate attraction and selection 27'32. The function of flank gland grooming may be to increase dissemination of flank gland secretions and/or to stabilize body temperature 2L47. Microinjection of arginine-vasopressin (AVP), but not other peptides or neurotransmitters, into the medial preoptic-anterior hypothalamic continuum ( M P O A - A H ) can elicit intense bouts of flank marking in hamsters in a dose-dependent manner 3'14. Destruction of neuronal cell bodies within the M P O A - A H , or microinjection of AVP antagonists into this region, reduces or eliminates flank marking induced by AVP microinjection, or by exposure

to conspecific odors 17'18. Taken together these data indicate that flank marking requires the involvement of vasopressin-sensitive neurons within the M P O A - A H . More recent data suggest that AVP-sensitive neurons within the region of the lateral septum/bed nucleus of the stria terminalis (SEPT/BNST) may be involved in the control of flank marking 28. Flank marking is influenced by olfactory, social and hormonal factors 4'5'26'33'35'36. For flank marking to be expressed under the appropriate environmental and physiological conditions, there needs to be communication between brain regions essentially involved in coordinating these factors. The critical neurocircuitry that underlies this coordination most likely involves the M P O A - A H and other brain regions known to connect with this area. The periaqueductal gray (PAG) is a brain region known to be reciprocally connected with the M P O A - A H , SEPT and BNST 6'8'24'25'40'42'45'48. The P A G is anatomically well positioned to consolidate descending forebrain (olfactory), limbic (social) and hypothalamic (hormonal) input 1'6'8'9'13'24'49. In addition, the hamster P A G has vasopressin binding sites 12. Thus, from an anatomical perspective, the P A G is a viable candidate for involvement in the neural control of flank marking. The intent of the present study was to investigate if the P A G is involved in the neural control of hamster flank marking. The first experiment was designed to examine

Correspondence: A.C. Hennessey, Lab. of Neuroendocrinology & Behavior, Dept. of Biology, EO. Box 4010, Georgia State University, Atlanta, GA 30302-4010, U.S.A.

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Fig. 1. The frequency of flank marking (mean -+ S.E.M.) following mieroinjeetion of saline (SAL), oxytocin (OXY), or argininevasopressin (AVP) into the PAG of male (n = 8) and female (n = 5) Syrian hamsters.

whether AVP microinjected into the P A G could stimulate flank marking. The purpose of the second experiment was to investigate whether microinjection of AVP into the P A G stimulated flank marking in a dosedependent manner. Adult Syrian hamsters (Harlan Sprague-Dawley Inc.) were housed individually and maintained on a reversed light:dark cycle of 14:10 h. Lab chow and water were provided ad libitum. Hamsters were approximately 12 weeks of age and their weights ranged from 121 to 154 g at the time of stereotaxic surgery. Each hamster was deeply anesthetized with an intraperitoneal injection of sodium pentobarbital (90 mg/ kg) and was stereotaxically implanted with a single 4 mm, 26-gauge guide cannula aimed at the posterior PAG. Each guide cannula was positioned 5.0 mm posterior to bregma, 0.7 mm lateral to the midline suture and 2.8 mm below dura. Guide cannulae were implanted on the left side of the brain with the skull leveled be-

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Fig. 2. Dose-response curve for AVP-stimulated flank marking for male (n = 7) and female (n = 11) Syrian hamsters.

Fig. 3. Open circles indicate localized injection sites from representative hamsters (n = 12) in Experiment i and 2. The open circles are superimposed on schematic diagrams of coronal sections of the PAG arranged from anterior to posterior (top to bottom). The aqueduct is shown as the blackened area within the PAG. Brain region abbreviations: PAG, periaqueduetal gray; Pn, pontine nuclei; DR, dorsal raphe nuclei; IF, inferior colliculus.

tween bregma and lambda. Behavioral testing began 2 or 3 days after surgery. All testing was conducted under dim red illumination during the dark phase of the light/ dark cycle, the most active time of day for hamsters. In each experiment, individual hamsters received three (Experiment 1) or four (Experiment 2) microinjections such that the order of substances injected was counterbalanced among'subjects and across days. Each hamster was microinjected with 100 nl of saline, 9.0/~M oxytocin, and 9.0/~M arginine-vasopressin dissolved in 100 nl of saline (Experiment 1), or 0.9/tM, 9.0/tM, 90.0/~M, and 900.0 /~M arginine-vasopressin (Experiment 2) dissolved in 100 nl of saline. Microinjections were made with a 33-gauge needle (12.5 mm long such that the injection is ultimately administered 4.8 mm below dura) attached to a 1 ~1 Hamilton syringe. The 12.5 mm needle was inserted into the guide cannula and 100 nl of solution was injected within 1 s. The needle was removed 30 s after completion

138 of the injection. In Experiment 1, each hamster was placed in a clean 24 × 43 x 20 cm Plexiglas arena immediately after injection, and, various behaviors were scored by researchers cognizant of the solution microinjected. A flank mark was scored each time a hamster vigorously rubbed its flank gland region against the corners or sides of the cage while moving in a forward direction. Flank gland grooming was recorded whenever a hamster bit or licked its flank gland. Duration of flank gland grooming was determined using a cumulative stopwatch. Vaginal marking was scored each time a female placed her genital area on the floor of the cage and then moved in a forward direction in this position. In Experiment two, only flank marking was recorded. In both experiments, each behavioral test period lasted 10 min and testing was conducted at 24 h intervals. Estrous cycle day was determined by monitoring each female's vaginal discharge every day immediately after behavioral testing 41. Statistical differences between treatments were analyzed using a two-way analysis of variance with a repeated measures design 44. After completion of behavioral testing each hamster was administered a lethal dose of sodium pentobarbital. Prior to intracardial perfusion with neutral buffered formalin each hamster was microinjected with 100 nl of dye (India ink) into the PAG. After perfusion, each brain was removed and placed in a 30% sucrose 0.1 M phosphate buffered saline solution for a minimum of one week. Brains were sliced at 60 #m using a vibratome. Coronal brain sections were stained with 0.5% Neutral red. Injection sites were verified using a microscope. Each injection site was considered to be localized where the greatest extent of dye deposit was microscopically visualized. Microinjection of AVP, but not oxytocin or saline, into the periaqueductal gray induced intense bouts of flank marking in male and female hamsters (Fig. 1). There was a significant main effect of the solution microinjected into the P A G (F2,22 = 51.9, p < 0.01). No flank marking was observed in response to saline and only a small amount of flank marking was induced by oxytocin microinjection. In contrast, microinjection of AVP into the P A G stimulated high levels of flank marking in both male (66.3 -+ 13.1; ~ - S.E.M.) and female (79.0 -+ 13.5) hamsters. The amount of time spent flank gland grooming differed significantly across treatment groups (F2,22 = 4.2, P < 0.05). Male and female hamsters groomed the most after microinjection of AVP into the P A G (57.2 -+ 17.3 and 19.8 -+ 6.4, respectively). Flank gland grooming was also elicited, although to a lesser extent, by oxytocin in male and female hamsters (30.4 +- 13.8 and 7.6 -+ 4.3, respectively). Males rarely groomed flank glands and only one female groomed after microinjection of saline

(5.0 -+ 3.5 and 0.2 -+ 0.2, respectively). Irrespective of the solution microinjected, males groomed their flank glands significantly more than females (F1,11 = 5.6, P < 0.05). Female vaginal marking was not significantly influenced by the solution microinjected into the periaqueductal gray (P > 0.05). Females vaginal marked at about the same levels whether microinjected with saline (~ = 8.0 -+ 3.6), oxytocin (Y -- 6.8 -+ 3.0), or vasopressin (i = 11.6 - 7.0). Females never exhibited vaginal marking when they were tested during estrus, and, they marked an average of 10 times/10 min on non-estrous test days. In the second experiment, as is illustrated in Fig. 2, AVP microinjected into the P A G increased flank marking in a dose-dependent manner (F3,48 = 11.3, P < 0.01). Microinjection of AVP into the P A G stimulated increased amounts of flank marking as the AVP dose increased. The amount of flank marking leveled off after microinjection of 90.0 ktM AVP in males and 9.0 /~M AVP in females. Each injection site was verified histologically. Dye injections (100 nl) were found to primarily invade the left PAG, at the level of the pons. Representative injection sites (n = 12), depicted by open circles in Fig. 3, demonstrate the relative distribution of injection sites for these two experiments. The present study demonstrates that microinjection of AVP, but not oxytocin or saline, into the periaqueductal gray can induce intense bouts of flank marking in Syrian hamsters. Microinjection of AVP into the P A G stimulates flank marking in a dose-dependent manner. No sex difference in the effectiveness of AVP to stimulate flank marking was found after microinjection of AVP into the P A G despite the fact that odor-stimulated flank marking is greater in female hamsters than males (Albers and Prishkolnik, unpublished data). The results of the present study suggest that AVP-responsive neurons within the pontine PAG may contribute to the central control of hamster flank marking. The ability of AVP, but not oxytocin, to stimulate high levels of flank marking after microinjection into the P A G parallels the results obtained after microinjection of these peptides into the M P O A - A H T M and the SEPT/BNST 2s in hamsters. These findings are particularly interesting because the molecular structure of oxytocin differs from that of AVP in only 2 of 9 amino acid sequences. It has now been demonstrated that AVP may be involved in the central regulation of hamster flank marking within at least 3 neura ! regions, the MPOA-AH, SEPT/BNST and the PAG. The precise role AVP-sensitive neurons within each of these regions plays in the control of flank marking is not understood. Yet, since these neural sites are well con-

139 n e t t e d , it seems likely that neurons in these regions interact to control flank marking. In the Syrian hamster, the S E P T / B N S T and the M P O A - A H are reciprocally connected 16'25. In addition, recent anatomical w o r k in our l a b o r a t o r y indicates that the M P O A - A H and SEPT/ B N S T project to the AVP-sensitive site within the pontine P A G 24. It therefore seems plausible that these regions w o r k in concert to c o o r d i n a t e h o r m o n a l , limbic and olfactory inputs which influence flank marking. F l a n k gland grooming most often occurs in association with intense bouts of flank marking. B o t h A V P and oxytoein can induce flank gland grooming after microinjection into the M P O A - A H in male hamsters 2. T h e present study suggests that at the level of the pontine P A G , microinjection of AVP, and to a lesser extent oxytocin, can stimulate flank gland grooming in b o t h male and female hamsters. Intraventricular injection of A V P or oxytocin has b e e n r e p o r t e d to p r o d u c e grooming be-

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havior in other rodents t°'11'3s'39. Together these studies suggest that A V P and oxytocin play a role in r o d e n t grooming behavior. F l a n k marking, in the female hamster, can be conside r e d proceptive b e h a v i o r because it is involved in the attraction and selection of a m a t e 7'27'a2. Since the caudal P A G has b e e n implicated in another form of hamster proceptive behavior, namely ultrasound production 19'2°, the present study investigated whether A V P microinjected into the P A G influenced vaginal marking. In accord with its function as a proceptive behavior, female hamsters only exhibited vaginal marking prior to receptivity31'37'46. Microinjection of A V P or oxytocin into the P A G did not modify the frequency of vaginal marking. It appears that neither of these peptides m o d u l a t e s vaginal marking, at least within the pontine P A G . This work was supported by NSF BNS-8910863 to H.E.A.

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