Flank Marking Research Articles

Brief, high-frequency stimulation of the corticomedial amygdala induces a delayed and prolonged increase of aggressiveness in male Syrian golden hamsters.

Brief 200-Hz stimulation of the corticomedial amygdala (CMA) increases the aggressiveness of male Syrian golden hamsters for about 30 min; the effect peaks 10-15 min after stimulation. This effect is sensitive to stimulation amplitude and frequency. Stimulation at the parameters that reduce attack latency increases flank marking but does not affect copulation latency or general activity. Immunocytochemical analysis suggests that stimulation effects may be coupled to c-fos expression and that unilateral stimulation has bilateral effects. CMA stimulation effects appear to mimic part of the time course of behaviorally induced attack priming. The temporal persistence of aggression may result from long-term potentiation-like changes within CMA-related neural circuitry.

Ovarian hormones alter the behavioral response of the medial preoptic anterior hypothalamus to arginine-vasopressin.

In Syrian hamsters (Mesocricetus auratus) arginine-vasopressin (AVP) within the medial preoptic-anterior hypothalamus (MPOA-AH) plays a critical role in the control of a hormone-dependent behavior called flank marking. The present study investigated whether ovarian hormones influence flank marking by altering the response of the MPOA-AH to AVP. The amount of flank marking stimulated by microinjection of AVP (9 microM in 200 nl saline) into the MPOA-AH varied significantly over the 4 days of the estrous cycle with the lowest levels of flank marking observed on estrus. A second experiment demonstrated that administration of progesterone significantly reduced AVP-stimulated flank marking in estradiol-treated ovariectomized hamsters. These data support the hypothesis that the changing levels of estradiol and progesterone during the estrous cycle influence flank marking by altering the sensitivity or response of the MPOA-AH to AVP.

Brief, high-frequency stimulation of the corticomedial amygdala induces a delayed and prolonged increase of aggressiveness in male Syrian golden hamsters.

Brief 200-Hz stimulation of the corticomedial amygdala (CMA) increases the aggressiveness of male Syrian golden hamsters for about 30 min; the effect peaks 10-15 min after stimulation. This effect is sensitive to stimulation amplitude and frequency. Stimulation at the parameters that reduce attack latency increases flank marking but does not affect copulation latency or general activity. Immunocytochemical analysis suggests that stimulation effects may be coupled to c-fos expression and that unilateral stimulation has bilateral effects. CMA stimulation effects appear to mimic part of the time course of behaviorally induced attack priming. The temporal persistence of aggression may result from long-term potentiation-like changes within CMA-related neural circuitry.

Effects of testosterone on the behavioral response to arginine vasopressin microinjected into the central gray and septum

Arginine vasopressin (AVP) plays an important role in the control of a gonadal hormone-dependent communicative behavior in the Syrian hamster ( Mesocricetus auratus) called flank marking. Previous studies have shown that gonadal hormones alter the amount of flank marking stimulated by the microinjection of AVP into the medial preoptic area-anterior hypothalamus (MPOA-AH). The purpose of the present study was to determine if testicular hormones alter the amount of flank marking stimulated by the microinjection of AVP into two other sites involved in the control of flank marking, the lateral septum-bed nucleus of the stria terminalis (LS-BNST) and the central gray. The data of the present study indicate that testicular hormones may influence the amount of AVP-stimulated marking in the central gray and LS-BNST; however, these effects are subtle and appear to occur primarily at high concentrations of AVP. When taken together with previous studies, these data indicate that gonadal hormones have greater effects on AVP-stimulated marking in the MPOA-AH than in the LS-BNST or central gray.

Vasopressin and sex differences in hamster flank marking

Vasopressin (AVP) within the medial preoptic-anterior hypothalamic continuum (MPOA-AH) plays an essential role in the control of flank marking in Syrian hamsters. Sex differences are found in the scent marking of many mammalian species, including hamsters. The first two experiments tested the hypothesis that sex differences in flank marking are the result of sex differences in the availability of AVP for release in several CNS sites. No support for this hypothesis was provided because neither immunohistochemical analysis nor radioimmunoassay of tissue punches revealed sex differences in AVP immunoreactivity in the MPOA-AH or other sites likely to be involved in flank marking. The third experiment, which tested the hypothesis that sex differences in flank marking are the result of sex differences in the sensitivity or response of the MPOA-AH to AVP, found no sex differences in the amount of flank marking stimulated by microinjection of AVP in the MPOA-AH. These data provide no support for the hypothesis that sex differences in vasopressinergic activity are responsible for sex differences in flank marking.

Septo-hypothalamic organization of a stereotyped behavior controlled by vasopressin in golden hamsters

In golden hamsters, microinjections of arginine vasopressin (AVP) within the anterior hypothalamus (AH) and lateral septum (LS) elicit the display of a stereotyped behavior: flank marking. As these areas are reciprocally connected, we tested whether AVP-sensitive sites constitute an organized network. Flank marking was recorded in animals with ibotenic acid lesions within the AH or LS after AVP injections within the LS or AH. While AVP injections in the AH and LS induced high flank-marking scores, certain lesions blocked the behavior. Lesions of the LS failed to affect flank marking induced by injections within the AH. In contrast, unilateral AH lesions blocked flank marking induced either by LS injections or AH injections in the contralateral side. These results suggest that the bilateral integrity of the AH is critical for the activation of flank-marking behavior by AVP. Together, these data suggest that the AH is an important relay of the neural network controlling flank-marking behavior.

Vasopressin and serotonin interactions in the control of agonistic behavior

In hamsters, dominant/subordinate relationships are initially determined by overt aggression, but subsequently communicated by flank marking, an arginine vasopressin (AVP)-dependent behavior. Once a relationship is established, dominant males will flank mark at a higher frequency than their subordinate partners. Flank marking displayed during social encounters can be turned “on or off” by microinjection of AVP or AVP-receptor antagonist within the anterior hypothalamus (AH). For instance, microinjecting dominant hamsters with AVP-receptor antagonist blocks their flank marking and provokes an immediate induction of flank marking by subordinate animals. The central effects of AVP have been extended to include a role in offensive aggression. Microinjection of AVP-receptor antagonist into the AH inhibits the aggression of a resident hamster toward an intruder and diminishes aggression between hamsters placed into a neutral arena. Microinjection of AVP into the ventrolateral hypothalamus (VLH) facilitates offensive aggression of a resident toward an intruder. As AVP receptors in the VLH are testosterone-dependent, it is possible that the reduction of aggression observed in castrated hamsters is due to a loss of AVP responsiveness in the VLH. Recent work has focused on the notion that serotonin (5-HT) antagonizes AVP activity in the CNS. The AH and VLH have a high density of 5-HT terminals and binding sites. Indeed, there appear to be 5-HT synapses on AVP neurons in the AH. Microinjection of 5-HT into the AH inhibits AVP-induced flank marking while IP injection of fluoxetine a serotonin reuptake inhibitor inhibits AVP-induced offensive aggression in the VLH. It is possible that serotonin interacts with AVP to modulate offensive aggression.

An iodinated vasopressin (V 1) antagonist blocks flank marking and selectively labels neural binding sites in golden hamsters

An arginine-vasopressin (AVP) derivative, [d(CH 2) 5,Sar 7]AVP (SAVP), has been characterized as an antagonist to vasopressin V 1 receptors. Using AVP-dependent flank-marking behavior as a bioassay, it was possible to verify that iodinated SAVP (I-SAVP) retains biological activity within the central nervous system, as the antagonist blocked the behavior. Furthermore, 125I-SAVP was used to localize specific V 1 binding sites in the brain. The resulting binding was localized to discrete anatomical sites, and highly specific to V 1-like receptors. While we confirmed previous findings using 3H-AVP in golden hamsters, we also identified binding in many areas previously unreported (e.g., arcuate and paraventricular nuclei of the hypothalamus, tenia tecta, posteromedial cortical nucleus of the amygdala, and zona incerta), suggesting that 125I-SAVP provides a greater level of resolution. In addition, specific binding was observed in the lateral septum, anterior hypothalamus, and midbrain central gray, areas that have previously been shown to trigger flank marking in response to AVP microinjection. The presence of AVP binding sites in limbic and mesencephalic areas involved in the regulation of flank marking suggests that this neuropeptide may play an important role as a neurotransmitter at multiple levels in the neural circuits controlling this behavior.

Estradiol increases the behavioral response to arginine vasopressin (AVP) in the medial preoptic-anterior hypothalamus

Flank marking, a form of hamster scent marking controlled by arginine vasopressin (AVP) in the medial preoptic-anterior hypothalamus (MPOA-AH), is altered by circulating levels of gonadal hormones. We hypothesized that gonadal hormones influence flank marking either by altering the availability of AVP for release in the MPOA-AH or by altering the sensitivity or responsiveness of MPOA-AH neurons to AVP. We examined the levels of AVP immunoreactivity (AVP-IR) over the estrous cycle in the MPOA-AH and other areas. Arginine vasopressin immunoreactivity did not vary in the areas examined as a function of the stage of the estrous cycle. In Experiment 2 we measured flank marking after MPOA-AH microinjection of AVP in ovariectomized hamsters receiving estradiol or empty Silastic capsules. Hamsters implanted with estradiol capsules marked significantly more in response to AVP than did hamsters receiving no hormone replacement. These results support the hypothesis that estradiol influences flank marking by altering the sensitivity or responsiveness of the MPOA-AH or its efferents to AVP. Additionally, we observed an unexpected effect of AVP in estradiol-treated hamsters. After microinjection with 90 μ M AVP, lordosis occurred spontaneously in 60% of the hamsters even though no male was present. We suggest that female hamsters may be a useful model to further investigate the role of AVP and AVP-like peptides in female sexual behavior.

Sex Differences and the Role of Aromatization in the Control of Sexually Dimorphic Behavior and Morphology in Gray Short-Tailed Opossums ( Monodelphis domestica)

Sex differences in the activating effects of an aromatizable (testosterone, T) and a nonaromatizable (dihydrotesterone, DHT) androgen on sexually dimorphic scent marking and aggressive behavior were examined in gonadectomized gray short-tailed opossums. When compared with males, females showed less chest, head, flank, and hip marking and more fighting behavior in tests with stimulus females and threat behavior in tests with stimulus males following receipt of subcutaneous T, DHT, or blank (B) implants. Testosterone but not DHT activated hip and head marking while both T and DHT activated flank marking. In tests with stimulus females, only males showed clicking vocalizations and T but not DHT or B stimulated clicking. Animals that received T had significantly larger suprasternal scent glands than those that received DHT or B. The significance of these findings is discussed with respect to the development of sex differences in behavior in eutherian mammals.

Norepinephrine Inhibits Vasopressin-Stimulated Flank Marking in the Syrian Hamster by Acting Within the Medial Preoptic-Anterior Hypothalamus

Syrian hamsters exhibit a form of scent marking behavior called flank marking. Flank marking, which is elicited during social contact with other hamsters and by the odors of other hamsters, communicates socially important information such as mate choice and dominance status. Vasopressinergic activity within the medial preoptic-anterior hypothalamic continuum (MPOA-AH) is essential for the expression of flank marking. In female hamsters, an inverse relationship exists between the expression of flank marking and sexual receptivity during the 4-day estrous cycle. Since norepinephrine (NE) appears to facilitate sexual receptivity, the present study investigated whether NE might have an inhibitory effect on flank marking by acting on Vasopressinergic activity within the MPOA-AH. Microinjection of 9.0 μM arginine vasopressin (AVP) into the MPOA-AH stimulated high levels of flank marking. Microinjection of 9.0 μM AVP combined with NE in concentrations of 4.0, 0.4 or 0.2 nM, drastically reduced or eliminated flank marking. In contrast, AVP in combination with 0.09, 0.04 or 0.004 nM NE produced no significant reductions in flank marking. In addition, microinjection of 9.0 μM AVP, in combination with epinephrine (4.0 nM), but not dopamine (4.0 nM), serotonin (4.0 nM) or neuropeptide Y (900 μM), significantly reduced AVP-induced flank marking. In male hamsters, microinjection of NE (4.0 nM) combined with AVP (9.0 μM) into the MPOA-AH was not found to inhibit AVP-stimulated flank marking. These results suggest that NE is involved in regulating the expression of flank marking during the estrous cycle by acting on Vasopressinergic activity within the MPOA-AH.

Sex differences in odor-stimulated flank marking in the golden hamster ( Mesocricetus auratus)

To determine whether sex differences exist in the frequency of odor-stimulated flank marking, intact male and female hamsters were exposed to the recently vacated home cages of male stimulus hamsters for a 10-min test on 4 consecutive days. Females were found to mark at significantly higher levels than males. To investigate the role of gonadal hormones in the sex differences in flank marking, gonadectomized male and female hamsters were implanted with Silastic capsules containing estradiol or testosterone. Females exhibited twofold higher levels of odor-stimulated flank marking than males, and the amount of flank marking was significantly higher when the hamsters were administered testosterone than when they were administered estradiol. These data demonstrate that sex differences exist in the frequency of flank marking stimulated by the odors of male hamsters, and that these sex differences do not appear to result from the typical sexspecific patterns of circulating levels of estradiol and testosterone.

Afferent projections of the hamster periaqueductal gray. A neural site where vasopressin can stimulate flank marking.

Afferent projections of the hamster periaqueductal gray. A neural site where vasopressin can stimulate flank marking.

Functionally and Anatomically Distinct Populations of Vasopressinergic Magnocellular Neurons in the Female Golden Hamster

The present study was done to determine whether the vasopressinergic neurons in the hypothalamus controlling flank marking behavior are distinct from the magnocellular neurons comprising the hypothalamo-neurohypophysial system. Animals were either hypophysectomized or injected with a suicide transport lectin, volkensin, into the neurohypophysis. Both procedures resulted in a pronounced loss of vasopressin-immunoreactive perikarya throughout the hypothalamus concomitant with increases in water intake and urine output and decreases in circulating levels of vasopressin. The loss of the hypothalamo-neurohypophysial system was most pronounced in volkensin-treated animals that presented with frank diabetes insipidus and exceedingly low levels of plasma vasopressin. However, the vasopressinergic fibers and magnocellular neurons in and around the anterior hypothalamus implicated in the control of flank marking survived the volkensin treatment. Volkensin-treated animals exhibited levels of flank marking typical of untreated animals. These data suggest the presence of anatomically and functionally distinct populations of vasopressinergic magnocellular neurons in the hypothalamus of the golden hamster.

Vomeronasal and/or olfactory mediation of ultrasonic calling and scent marking by female golden hamsters

The role of the olfactory and vomeronasal systems in mediating odor-stimulated ultrasonic calling, flank marking, and vaginal marking by female hamsters was investigated by selective lesions of either system. Removal of the vomeronasal organ resulted in reduced frequencies of ultrasonic calling by estrous and nonestrous females in response to conspecific odors but it had no influence on either scent marking behavior during the same tests. When tested immediately after separation from a male, ultrasonic calling was not reduced by vomeronasal removal, indicating that such surgery does not cause deficits in calling ability and that the vomeronasal organ specifically mediates odor-stimulated calling. Zinc sulfate treatment of the olfactory mucosa led to reduction in the frequency of ultrasonic calling, flank marking, and vaginal marking in response to conspecific odors. Females' ability to discover buried food was also impaired by this treatment. Thus, the stimulation of both scent marking behaviors due to the perception of conspecific odors appears to be mediated primarily by the olfactory system, whereas stimulation of ultrasonic calling is mediated by both olfactory and vomeronasal systems.

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

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 n 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.

Steroid-specific regulation of agonistic responding in the anterior hypothalamus of male hamsters

The agonistic behaviors of adult male golden hamsters ( N = 108 dyads) were examined 5 min after stereotaxic microinjection of adrenal and gonadal steroids into the anterior hypothalamus. Flank marks, attacks, bites, and retreats were scored over a 15 min test period during which steroid-injected animals were paired in a neutral arena with vehicle-injected conspecifics. Animals microinjected with either 10 −6 M cortisol or 10 −6 M β-estradiol displayed significantly ( p<0.05) higher levels of flank marking than other steroid-treated animals. Animals microinjected with 10 −6 M cortisol displayed significantly higher levels of aggression than their opponents. In contrast, the behavior of the vehicle-injected animals paired with 10 −6 M cortisol-treated opponents was characterized by submissive responding. This profile of the 10 −6 M cortisol treatment, i.e., promoting aggression in a steroid-treated animal while eliciting submission from its vehicle-treated opponent, was not observed in pairs in which steroid-injected animals were treated with equimolar concentrations of testosterone, dihydrotestosterone, progesterone, β-estradiol, or deoxycorticosterone. These findings suggest steroids exert immediate effects on agonistic responding in the anterior hypothalamus of male hamsters. The immediate action(s) of cortisol appear to include facilitating aggression and flank marking, while the immediate action(s) of β-estradiol appears to be confined to the communicative aspect of agonistic responding in this species.

Vasopressin in the septal area of the golden hamster controls scent marking and grooming

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 V1-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 V1-receptor antagonist into these sites was able to temporarily block flank marking triggered by odors from conspecifics.

Kainic Acid Lesion of Vasopressinergic Neurons in the Hypothalamus Disrupts Flank Marking Behavior in Golden Hamsters

Abstract It is well established that flank marking behavior in the Golden hamster is controlled by vasopressin-sensitive neurons localized to the anterior hypothalamus; however, the source(s) of vasopressinergic innervation to this area is unknown. Previous analysis by immunocytochemistry showed distinct populations of vasopressinergic magnocellular neurons localized to the supraoptic nucleus, paraventricular nucleus and the nucleus circularis that did not project to the neurohypophysis. In the present study, these same hypothalamic nuclei were lesioned by microinjection of kainic acid to determine which, if any, of these populations of vasopressin neurons are involved in the control of flank marking. Unilateral lesions in the areas of the nucleus circularis and supraoptic nucleus at the rostro-caudal plane of the anterior hypothalamus abolished odor-induced flank marking behavior. Lesions in the paraventricular nucleus at the level of the anterior hypothalamus did not consistently inhibit flank marking, while lesions of magnocellular neurons rostral or caudal to the anterior hypothalamus were ineffective. The microinjection of vasopressin into the anterior hypothalamus following lesion of the paraventricular and supraoptic nuclei stimulated flank marking, evidence that treatment with kainic acid did not damage the efferent component of this behavior. However, animals with lesions in the nucleus circularis did not respond to the microinjection of vasopressin; hence, it is uncertain whether lesions in this area disrupt vasopressinergic innervation to the anterior hypothalamus or simply destroy the motor neurons controlling flank marking. In summary, the data clearly demonstrate that vasopressin neurons localized primarily to the medial aspect of the supraoptic nucleus are necessary for sensory integration of odor-induced flank marking, and as such, may be one possible source of neurotransmitter controlling this behavior.

Computer‐Aided Mapping of Vasopressin Neurons in the Hypothalamus of the Male Golden Hamster: Evidence of Magnocellular Neurons that do not Project to the Neurohypophysis

Abstract Vasopressin-sensitive neurons in the region of the anterior hypothalamus are necessary for the mediation of flank marking behavior in the Golden hamster. The precise nature of the vasopressinergic innervation to the anterior hypothalamus is unknown. In this study we seek to examine the potential sources of this innervation by mapping and counting the vasopressin-immunoreactive neurons that contribute to the hypothalamo-neurohypophysial system, and those that do not. Vasopressin-immunoreactive neurons in the hypothalamus were visualized by immunocytochemistry. Sections were mapped with a computer-aided microscope system, and labeled neurons counted. Two-dimensional maps were stacked into a three-dimensional wireframe model which could be manipulated for further examination. The average number of vasopressin neurons was 3,135, with over 60% of all perikarya localized to the lateral supraoptic nucleus. In a double-labeling study, neurons contributing to the hypothalamo-neurohypophysial system were retrogradely labeled by the injection of horseradish peroxidase into the neurohypophysis. The enzyme reaction product was visualized by treatment with tetramethylbenzidine followed by nickel-conjugated diaminobenzidine. Sections were subsequently stained for vasopressin by immunocytochemistry. Single- and double-stained neurons from serial sections were mapped and counted. Wireframe and contoured three-dimensional representations were generated. The average number of neurons projecting to the neurohypophysis was 5,619. However, an average of 981 neurons was immunoreactive to vasopressin but devoid of horseradish peroxidase. The greatest number of these non-projecting perikarya were found in and around the anterior hypothalamus, localized primarily in the lateral and medial aspect of the supraoptic nuclei, the ventral area of the paraventricular nucleus, and the nucleus circularis. By comparing the number of non-projecting neurons found by double-staining to the total cell count of the entire vasopressin system, it was estimated that approximately 30% of all vasopressin neurons in and around the anterior hypothalamus did not project to the neurohypophysis. Based on the distribution and localization of the non-projecting perikarya, it is speculated that these neurons may provide neurotransmitter for vasopressin-dependent flank marking in the male Golden hamster.