Physiology& Behavior, Vol. 48, pp. 693-699. ©PergamonPress plc, 1990. Printedin the U.S.A.
0031-9384/90$3.00 + .00
Vasopressin in the Septal Area of the Golden Hamster Controls Scent Marking and Grooming R O B E R T W. I R V I N , P A T R I C I A S Z O T , * D A N I E L M. D O R S A , * M I C H A E L P O T E G A L t A N D C R A I G F. F E R R I S t
Department of Physiology, University of Massachusetts Medical Center 55 Lake Avenue North, Worcester, MA 01655 *Geriatric Research, Education, and Clinical Center Veterans Affairs Medical Center, 1660 S. Columbian Way, Seattle, WA 98108 "pDepartment of Developmental Psychobiology, New York State Psychiatric Institute, New York, N Y 10032 Received 7 June 1990
IRVIN, R. W., P. SZOT, D. M. DORSA, M. POTEGAL AND C. F. FERRIS. Vasopressin in the septal area of the golden hamster controls scent marking and grooming. PHYSIOL BEHAV 48(5) 693-699, 1990.--Microinjection of arginine vasopressin into the lateral septum and bed nucleus of the stria terminalis of male hamsters stimulates intense flank marking and flank gland grooming, while microinjections of vasopressin in sites immediately adjacent to these areas or in the lateral ventricle are ineffective. Microinjections of oxytocin, angiotensin II and the behaviorally active C-terminal fragment of vasopressin, metabolite neuropeptide, by comparison, do not stimulate flank marking. Effective sites for vasopressin injection are clearly superimposable upon autoradiographically defined sites of high Vl-receptor density. Furthermore, vasopressin-sensitive neurons in the lateral septum and bed nucleus of the stria terminalis are necessary for the expression of naturally elicited flank marking since the microinjection of a V1receptor antagonist into these sites was able to temporarily block flank marking triggered by odors from conspecifics. Flank marking
Autoradiography
Vl-receptor
Vasopressin receptor antagonist
THERE is substantial evidence that vasopressin functions as a neurotransmitter in the central nervous system of the rat, affecting memory consolidation and antipyresis (5, 16, 17, 19). One region of the brain that appears to be involved in the regulation of both functions is the septal area. The microinjection of arginine vasopressin (AVP) into the lateral septum can enhance social memory, i.e., recognition of conspeciflcs (3), while perfusion of AVP into the ventral septal area can lower pyrogen-induced fever (20). Conversely, microinjection of Vl-receptor antagonist into the same sites shortens social memory (3) and prolongs and exacerbates pyrogen-induced fever (2). The golden hamster (Mesocricetus auratus) has two vasopressinmediated behaviors, flank marking and flank gland grooming, that may be functionally similar to social memory and antipyresis in the rat. Flank marking is a stereotypic motor behavior used to disseminate odors throughout the environment (15). Following the establishment of dominant/subordinate relationships, hamsters flank mark to communicate their social status (10). Therefore, flank marking between conspecifics might be interpreted as a display of social memory. Prior to and during bouts of flank mark-
Thermoregulation
Oxytocin
ing, hamsters normally groom their flank glands by vigorously clawing and chewing a large area of the dorsolateral flanks. In many cases flank gland grooming results in a thorough soaking of the pelage with saliva (6), a condition associated with decreases in body temperature (14,22). Microinjections of AVP or AVP receptor antagonist into the anterior hypothalamus can, respectively, elicit and inhibit flank marking and the associated flank gland grooming under numerous experimental conditions (6, 7, 9). Other regions of the hypothalamus and medial preoptic area of the hamster are insensitive to the microinjection of AVP (6,8). It is unknown whether injection of AVP into extrahypothalamic sites can elicit flank marking and flank gland grooming. Therefore, the present study examines the potential role of AVP as a neurotransmitter in the septal area of the golden hamster controlling flank marking and flank gland grooming. METHOD
Stereotaxic Microinjections The microinjection studies were divided into three separate
~Requests for reprints should be addressed to Craig F. Ferris.
693
IRVIN ET AL.
694
experiments. In the first experiment, AVP was microinjected in different sites throughout the septum and around the anterior commissure for the purpose of mapping the AVP-sensitive area(s) that trigger flank marking and flank gland grooming. In the second, animals were microinjected into AVP-sensitive sites with different peptides in order to examine the specificity of AVPstimulated flank marking. In the third experiment, animals were microinjected into AVP-sensitive sites with an AVP-receptor antagonist, and subsequently tested for the flank marking behavior elicited by the odors of conspecifics. This study was designed to determine whether endogenous AVP in the septal area is necessary for odor-induced flank marking. In all studies male golden hamsters (110-120 g) obtained from Charles River Laboratories (Wilmington, MA) were housed individually in Plexiglas cages (24 × 22 x 20 cm). Animals were provided food and water ad lib and maintained for 2-3 weeks on a reverse light:dark cycle of 14:l0 (lights off at 0700 h). Hamsters anesthetized with sodium pentobarbital (50 mg/kg) were stereotaxically implanted with a 26-gauge guide cannula as previously described (8). All microinjections were given no less than three days after surgery, in unanesthetized, passively restrained animals. All test substances were prepared in 0.9% NaC1 and microinjected in a volume of 100 nl. Microinjections were delivered through a 33-gauge needle attached to a 1 ~l Hamilton syringe connected by PE 20 tubing. At the conclusion of each microinjection study, animals were anesthetized and sacrificed by decapitation. The brains were removed, fixed by immersion in Perfix (Fisher Scientific) and sectioned on a vibratome. The position of the tip of the needle for each microinjection site was determined on 100 i~m transverse sections stained with thionin.
Experiment I Hamsters (n =40) were microinjected in the septal area with 1 ng of AVP. The stereotaxic coordinates ranged between: 2.22.6 mm anterior to bregma, 0.1-0.8 mm lateral to the midsagittal suture and 4.0-5.0 mm below dura. The incisor bar was held level with the interaural line. To control for the possibility that AVP entered the cerebroventricular system and stimulated areas outside the microinjection sites, six animals were microinjected with AVP (1 ng/100 nl) directly into the lateral ventricle. Following microinjection, hamsters were placed in a clean Plexiglas cage (24 x 22 x 20 cm) and scored for flank marking and flank gland grooming over a 10-min test period. A flank mark was scored each time the animal rubbed its flank gland region against the corners or sides of the cage. Flank gland grooming was scored as the time spent grooming the flank gland region, but not the face or other areas of the body. During a bout of grooming animals would vigorously claw and chew the flank glands, often soaking the surrounding area with saliva. All behavioral tests were performed between 0900 and 1400 h under dim red illumination.
Experiment H Twenty-seven hamsters were prepared with septal cannulae in which pretests with AVP injection elicited flank marking. Afterward, animals received a single injection of angiotensin II (n = 7), oxytocin (n=7), [pGlu 4, cystine6]-AVP 4-8, metabolite neuropeptide (n = 6) or vehicle control of 0.9% NaC1 (n = 7). Each peptide was given in equimolar concentrations comparable to the concentration of AVP (ca. 9 txM). The stereotaxic coordinates for each of these microinjections were: 2.3 mm anterior to bregma; 0.8 mm lateral to the midsagittal suture; and 5.0 mm ventral to the dura mater. These coordinates marked the central area of the
AVP-sensitive site as determined in Experiment I. Immediately after microinjection the behavior of each animal was scored for a 10-min test period in a neutral arena as described above.
Experiment I11 Eight animals were tested for odor-induced flank marking by placing them into the recently vacated home cage of another male hamster for a 10-min period. On the following day these animals were microinjected with 10 ng of a Vl-receptor antagonist, [ (B-mercapto-B, B-cyclopentamethylene propionic acid), 2-(0methyl) tyrosine] arginine vasopressin (Peninsula Labs, Inc., CA), in a volume of 100 nl. The concentration of receptor antagonist employed (ca. 90 IxM) has been previously found to inhibit AVPstimulated flank marking in the hypothalamus for up to 24 h (11). The stereotaxic coordinates for each microinjection were the same as those used in Experiment II. Animals were tested for odor-induced flank marking 1 h and 48 h after the microinjection of antagonist. All tests were performed in the same stimulus cage as the preinjection test. To control for any nonspecific effects the microinjection procedure may have had on odor-induced flank marking, these procedures were also carried out in six animals microinjected with 0.9% NaCI, instead of AVP-receptor antagonist. These control animals were tested in the same order and time of day as experimental animals.
Autoradiographic Localization of 3H-AVP Binding Sites Adult male hamsters were decapitated and the brains frozen on COz ice and stored at - 8 0 ° C until sectioning. Coronal sections (16 txm) of forebrain were cut on a cryostat at - 14°C and thaw mounted on gelatin-coated slides. The slides containing tissue sections were stored at - 80°C for less than 2 weeks until the binding assay could be performed. Slides containing tissue were thawed to room temperature and placed flat on moist paper in a plastic tray. The tissue slices were covered with 200 txl of incubation medium of 100 mM Tris-HC1, pH. 8.0, 10 mM MgC12, 1% bovine serum albumin, 0.5 mg/ml bacitracin and 100 KIU/ml aprotinin containing 2.5 nM 3H-AVP. Nonspecific binding was determined in the presence of 1 txM AVP. The slides were incubated at room temperature for 30 min followed by two 2-minute successive rinses in ice cold 10 mM Tris-HC1, pH 8.0 containing 1 mM MgC1z and 0.1% bovine serum albumin and a 2-s ice cold water rinse. The sections were dried with a stream of cool, desiccated air and apposed to Hyperfilm-3H (Amersham Corp., Arlington Heights, IL) for 6 weeks. A complete description of the binding properties of 3H-AVP in the septum of the golden hamster appears elsewhere (21). RESULTS
Experiment I The site of the tip of the needle for each of 40 hamsters microinjected with AVP (1 ng/100 nl) is shown in Fig. 1. The distribution of flank marking scores was clearly bimodal. The solid circles (n= 20) denote sites where AVP stimulated 12-77 flank marks (median=27 flank marks/10 min). The stippled circles (n = 20) indicate sites where AVP elicited 6 or fewer flank marks (median=0 flank marks/10 min). Microinjection of AVP (1 ng/ 100 nl) directly into the lateral ventricle had no significant effect on flank marking or flank gland grooming. Figure 2 is a photomicrograph showing the precise location of a microinjection site that was effective in stimulating flank marking behavior. In all AVP-sensitive sites, the flank marking was accompanied by robust flank gland grooming (194± 14 s), and in 17 out
VASOPRESSIN AND SCENT MARKING
695
(Fig. 3) with the mapping of AVP-sensitive sites (Fig. 1) suggests a high degree of overlapping between the two.
Experiment H Injections of various peptides or saline were made into the septal area of hamsters from which flank marking had been previously elicited by AVP injection. Only four of the seven salineinjected animals flank marked and those only did so 1-3 times. Injections of peptides were similarly without effect: two of the seven animals injected with oxytocin marked 1--4 times, one of the seven angiotensin-II-injectedanimals marked once, and none of the six metabolite neuropeptide animals marked at all. There were no differences among these groups, F(3,23)= 1.8. Of the six animals with AVP injections into the ventricle, three marked 1-3 times each, the others did not mark. The scores of this latter group were not significantly different from 0, t(5)= 2.45, and were significantly less than the mean score obtained with AVP injection into effective septal sites in Experiment I, t(5)=61.7, p
0031-9384/90$3.00 + .00
Vasopressin in the Septal Area of the Golden Hamster Controls Scent Marking and Grooming R O B E R T W. I R V I N , P A T R I C I A S Z O T , * D A N I E L M. D O R S A , * M I C H A E L P O T E G A L t A N D C R A I G F. F E R R I S t
Department of Physiology, University of Massachusetts Medical Center 55 Lake Avenue North, Worcester, MA 01655 *Geriatric Research, Education, and Clinical Center Veterans Affairs Medical Center, 1660 S. Columbian Way, Seattle, WA 98108 "pDepartment of Developmental Psychobiology, New York State Psychiatric Institute, New York, N Y 10032 Received 7 June 1990
IRVIN, R. W., P. SZOT, D. M. DORSA, M. POTEGAL AND C. F. FERRIS. Vasopressin in the septal area of the golden hamster controls scent marking and grooming. PHYSIOL BEHAV 48(5) 693-699, 1990.--Microinjection of arginine vasopressin into the lateral septum and bed nucleus of the stria terminalis of male hamsters stimulates intense flank marking and flank gland grooming, while microinjections of vasopressin in sites immediately adjacent to these areas or in the lateral ventricle are ineffective. Microinjections of oxytocin, angiotensin II and the behaviorally active C-terminal fragment of vasopressin, metabolite neuropeptide, by comparison, do not stimulate flank marking. Effective sites for vasopressin injection are clearly superimposable upon autoradiographically defined sites of high Vl-receptor density. Furthermore, vasopressin-sensitive neurons in the lateral septum and bed nucleus of the stria terminalis are necessary for the expression of naturally elicited flank marking since the microinjection of a V1receptor antagonist into these sites was able to temporarily block flank marking triggered by odors from conspecifics. Flank marking
Autoradiography
Vl-receptor
Vasopressin receptor antagonist
THERE is substantial evidence that vasopressin functions as a neurotransmitter in the central nervous system of the rat, affecting memory consolidation and antipyresis (5, 16, 17, 19). One region of the brain that appears to be involved in the regulation of both functions is the septal area. The microinjection of arginine vasopressin (AVP) into the lateral septum can enhance social memory, i.e., recognition of conspeciflcs (3), while perfusion of AVP into the ventral septal area can lower pyrogen-induced fever (20). Conversely, microinjection of Vl-receptor antagonist into the same sites shortens social memory (3) and prolongs and exacerbates pyrogen-induced fever (2). The golden hamster (Mesocricetus auratus) has two vasopressinmediated behaviors, flank marking and flank gland grooming, that may be functionally similar to social memory and antipyresis in the rat. Flank marking is a stereotypic motor behavior used to disseminate odors throughout the environment (15). Following the establishment of dominant/subordinate relationships, hamsters flank mark to communicate their social status (10). Therefore, flank marking between conspecifics might be interpreted as a display of social memory. Prior to and during bouts of flank mark-
Thermoregulation
Oxytocin
ing, hamsters normally groom their flank glands by vigorously clawing and chewing a large area of the dorsolateral flanks. In many cases flank gland grooming results in a thorough soaking of the pelage with saliva (6), a condition associated with decreases in body temperature (14,22). Microinjections of AVP or AVP receptor antagonist into the anterior hypothalamus can, respectively, elicit and inhibit flank marking and the associated flank gland grooming under numerous experimental conditions (6, 7, 9). Other regions of the hypothalamus and medial preoptic area of the hamster are insensitive to the microinjection of AVP (6,8). It is unknown whether injection of AVP into extrahypothalamic sites can elicit flank marking and flank gland grooming. Therefore, the present study examines the potential role of AVP as a neurotransmitter in the septal area of the golden hamster controlling flank marking and flank gland grooming. METHOD
Stereotaxic Microinjections The microinjection studies were divided into three separate
~Requests for reprints should be addressed to Craig F. Ferris.
693
IRVIN ET AL.
694
experiments. In the first experiment, AVP was microinjected in different sites throughout the septum and around the anterior commissure for the purpose of mapping the AVP-sensitive area(s) that trigger flank marking and flank gland grooming. In the second, animals were microinjected into AVP-sensitive sites with different peptides in order to examine the specificity of AVPstimulated flank marking. In the third experiment, animals were microinjected into AVP-sensitive sites with an AVP-receptor antagonist, and subsequently tested for the flank marking behavior elicited by the odors of conspecifics. This study was designed to determine whether endogenous AVP in the septal area is necessary for odor-induced flank marking. In all studies male golden hamsters (110-120 g) obtained from Charles River Laboratories (Wilmington, MA) were housed individually in Plexiglas cages (24 × 22 x 20 cm). Animals were provided food and water ad lib and maintained for 2-3 weeks on a reverse light:dark cycle of 14:l0 (lights off at 0700 h). Hamsters anesthetized with sodium pentobarbital (50 mg/kg) were stereotaxically implanted with a 26-gauge guide cannula as previously described (8). All microinjections were given no less than three days after surgery, in unanesthetized, passively restrained animals. All test substances were prepared in 0.9% NaC1 and microinjected in a volume of 100 nl. Microinjections were delivered through a 33-gauge needle attached to a 1 ~l Hamilton syringe connected by PE 20 tubing. At the conclusion of each microinjection study, animals were anesthetized and sacrificed by decapitation. The brains were removed, fixed by immersion in Perfix (Fisher Scientific) and sectioned on a vibratome. The position of the tip of the needle for each microinjection site was determined on 100 i~m transverse sections stained with thionin.
Experiment I Hamsters (n =40) were microinjected in the septal area with 1 ng of AVP. The stereotaxic coordinates ranged between: 2.22.6 mm anterior to bregma, 0.1-0.8 mm lateral to the midsagittal suture and 4.0-5.0 mm below dura. The incisor bar was held level with the interaural line. To control for the possibility that AVP entered the cerebroventricular system and stimulated areas outside the microinjection sites, six animals were microinjected with AVP (1 ng/100 nl) directly into the lateral ventricle. Following microinjection, hamsters were placed in a clean Plexiglas cage (24 x 22 x 20 cm) and scored for flank marking and flank gland grooming over a 10-min test period. A flank mark was scored each time the animal rubbed its flank gland region against the corners or sides of the cage. Flank gland grooming was scored as the time spent grooming the flank gland region, but not the face or other areas of the body. During a bout of grooming animals would vigorously claw and chew the flank glands, often soaking the surrounding area with saliva. All behavioral tests were performed between 0900 and 1400 h under dim red illumination.
Experiment H Twenty-seven hamsters were prepared with septal cannulae in which pretests with AVP injection elicited flank marking. Afterward, animals received a single injection of angiotensin II (n = 7), oxytocin (n=7), [pGlu 4, cystine6]-AVP 4-8, metabolite neuropeptide (n = 6) or vehicle control of 0.9% NaC1 (n = 7). Each peptide was given in equimolar concentrations comparable to the concentration of AVP (ca. 9 txM). The stereotaxic coordinates for each of these microinjections were: 2.3 mm anterior to bregma; 0.8 mm lateral to the midsagittal suture; and 5.0 mm ventral to the dura mater. These coordinates marked the central area of the
AVP-sensitive site as determined in Experiment I. Immediately after microinjection the behavior of each animal was scored for a 10-min test period in a neutral arena as described above.
Experiment I11 Eight animals were tested for odor-induced flank marking by placing them into the recently vacated home cage of another male hamster for a 10-min period. On the following day these animals were microinjected with 10 ng of a Vl-receptor antagonist, [ (B-mercapto-B, B-cyclopentamethylene propionic acid), 2-(0methyl) tyrosine] arginine vasopressin (Peninsula Labs, Inc., CA), in a volume of 100 nl. The concentration of receptor antagonist employed (ca. 90 IxM) has been previously found to inhibit AVPstimulated flank marking in the hypothalamus for up to 24 h (11). The stereotaxic coordinates for each microinjection were the same as those used in Experiment II. Animals were tested for odor-induced flank marking 1 h and 48 h after the microinjection of antagonist. All tests were performed in the same stimulus cage as the preinjection test. To control for any nonspecific effects the microinjection procedure may have had on odor-induced flank marking, these procedures were also carried out in six animals microinjected with 0.9% NaCI, instead of AVP-receptor antagonist. These control animals were tested in the same order and time of day as experimental animals.
Autoradiographic Localization of 3H-AVP Binding Sites Adult male hamsters were decapitated and the brains frozen on COz ice and stored at - 8 0 ° C until sectioning. Coronal sections (16 txm) of forebrain were cut on a cryostat at - 14°C and thaw mounted on gelatin-coated slides. The slides containing tissue sections were stored at - 80°C for less than 2 weeks until the binding assay could be performed. Slides containing tissue were thawed to room temperature and placed flat on moist paper in a plastic tray. The tissue slices were covered with 200 txl of incubation medium of 100 mM Tris-HC1, pH. 8.0, 10 mM MgC12, 1% bovine serum albumin, 0.5 mg/ml bacitracin and 100 KIU/ml aprotinin containing 2.5 nM 3H-AVP. Nonspecific binding was determined in the presence of 1 txM AVP. The slides were incubated at room temperature for 30 min followed by two 2-minute successive rinses in ice cold 10 mM Tris-HC1, pH 8.0 containing 1 mM MgC1z and 0.1% bovine serum albumin and a 2-s ice cold water rinse. The sections were dried with a stream of cool, desiccated air and apposed to Hyperfilm-3H (Amersham Corp., Arlington Heights, IL) for 6 weeks. A complete description of the binding properties of 3H-AVP in the septum of the golden hamster appears elsewhere (21). RESULTS
Experiment I The site of the tip of the needle for each of 40 hamsters microinjected with AVP (1 ng/100 nl) is shown in Fig. 1. The distribution of flank marking scores was clearly bimodal. The solid circles (n= 20) denote sites where AVP stimulated 12-77 flank marks (median=27 flank marks/10 min). The stippled circles (n = 20) indicate sites where AVP elicited 6 or fewer flank marks (median=0 flank marks/10 min). Microinjection of AVP (1 ng/ 100 nl) directly into the lateral ventricle had no significant effect on flank marking or flank gland grooming. Figure 2 is a photomicrograph showing the precise location of a microinjection site that was effective in stimulating flank marking behavior. In all AVP-sensitive sites, the flank marking was accompanied by robust flank gland grooming (194± 14 s), and in 17 out
VASOPRESSIN AND SCENT MARKING
695
(Fig. 3) with the mapping of AVP-sensitive sites (Fig. 1) suggests a high degree of overlapping between the two.
Experiment H Injections of various peptides or saline were made into the septal area of hamsters from which flank marking had been previously elicited by AVP injection. Only four of the seven salineinjected animals flank marked and those only did so 1-3 times. Injections of peptides were similarly without effect: two of the seven animals injected with oxytocin marked 1--4 times, one of the seven angiotensin-II-injectedanimals marked once, and none of the six metabolite neuropeptide animals marked at all. There were no differences among these groups, F(3,23)= 1.8. Of the six animals with AVP injections into the ventricle, three marked 1-3 times each, the others did not mark. The scores of this latter group were not significantly different from 0, t(5)= 2.45, and were significantly less than the mean score obtained with AVP injection into effective septal sites in Experiment I, t(5)=61.7, p
