Sex-dependent effects of social status on the regulation of arginine-vasopressin (AVP) V1a, oxytocin (OT), and serotonin (5-HT) 1A receptor binding and aggression in Syrian hamsters (Mesocricetus auratus).

Dominance status in hamsters is driven by interactions between arginine-vasopressin V1a, oxytocin (OT), and serotonin 1A (5-HT1A) receptors. Activation of V1a and OT receptors in the anterior hypothalamus (AH) increases aggression in males, while decreasing aggression in females. In contrast, activation of 5-HT1A receptors in the AH decreases aggression in males and increases aggression in females. The mechanism underlying these differences is not known. The purpose of this study was to determine if dominance status and sex interact to regulate V1a, OT, and 5-HT1A receptor binding. Same-sex hamsters (N = 47) were paired 12 times across six days in five min sessions. Brains from paired and unpaired (non-social control) hamsters were collected immediately after the last interaction and processed for receptor binding using autoradiography. Differences in V1a, OT, and 5-HT1A receptor binding densities were observed in several brain regions as a function of social status and sex. For example, in the AH, there was an interaction between sex and social status, such that V1a binding in subordinate males was lower than in subordinate females and V1a receptor density in dominant males was higher than in dominant females. There was also an interaction in 5-HT1A receptor binding, such that social pairing increased 5-HT1A binding in the AH of males but decreased 5-HT1A binding in females compared with unpaired controls. These results indicate that dominance status and sex play important roles in shaping the binding profiles of key receptor subtypes across the neural circuitry that regulates social behavior.

Distinct subcircuits within the mesolimbic dopamine system encode the salience and valence of social stimuli.

The mesolimbic dopamine (DA) system (MDS) is the canonical "reward" pathway that has been studied extensively in the context of the rewarding properties of sex, food, and drugs of abuse. In contrast, very little is known about the role of the MDS in the processing of the rewarding and aversive properties of social stimuli. Social interactions can be characterized by their salience (i.e., importance) and their rewarding or aversive properties (i.e., valence). Here, we test the novel hypothesis that projections from the medial ventral tegmental area (VTA) to the nucleus accumbens (NAc) core codes for the salience of social stimuli through the phasic release of DA in response to both rewarding and aversive social stimuli. In contrast, we hypothesize that projections from the lateral VTA to the NAc shell codes for the rewarding properties of social stimuli by increasing the tonic release of DA and the aversive properties of social stimuli by reducing the tonic release of DA. Using DA amperometry, which monitors DA signaling with a high degree of temporal and anatomical resolution, we measured DA signaling in the NAc core or shell while rewarding and aversive social interactions were taking place. These findings, as well as additional anatomical and functional studies, provide strong support for the proposed neural circuitry underlying the response of the MDS to social stimuli. Together, these data provide a novel conceptualization of how the functional and anatomical heterogeneity within the MDS detect and distinguish between social salience, social reward, and social aversion. Significance Statement: Social interactions of both positive and negative valence are highly salient stimuli that profoundly impact social behavior and social relationships. Although DA projections from the VTA to the NAc are involved in reward and aversion little is known about their role in the saliency and valence of social stimuli. Here, we report that DA projections from the mVTA to the NAc core signal the salience of social stimuli, whereas projections from the lVTA to the NAc shell signal valence of social stimuli. This work extends our current understanding of the role of DA in the MDS by characterizing its subcircuit connectivity and associated function in the processing of rewarding and aversive social stimuli.

Rhythms of glutamic acid decarboxylase mRNA in the suprachiasmatic nucleus.

The purpose of the present study was to determine whether there is a rhythm in glutamic acid decarboxylase (GAD) message in the suprachiasmatic nucleus (SCN) of rats housed in a light:dark cycle. The mRNAs encoding two isoforms of GAD (i.e., GAD65 and GAD67) were examined using in situ hybridization histochemistry. Computerized image analysis of film autoradiographs revealed that GAD65 mRNA was significantly higher in the light than it was in the dark. GAD67 mRNA levels were lower overall and did not decrease significantly in the dark. Following emulsion autoradiography, silver grain counts over individual SCN cells indicated that GAD65 mRNA was highest in the dorsomedial hypothalamus during the light. These data suggest that GAD mRNA varies rhythmically in the SCN and that mRNA levels are regulated differently within SCN subdivisions during the light:dark cycle.

Bicuculline increases and muscimol reduces the phase-delaying effects of light and VIP/PHI/GRP in the suprachiasmatic region.

The present study investigated the effects of gamma-amino butyric acid (GABA)A-active drugs on the ability of light or coadministration of vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI), and gastrin-releasing peptide (GRP) to phase delay hamster activity rhythms. Microinjection of the GABAA agonist, muscimol, significantly (p < .01) reduced the phase-delaying effect of light administered at circadian time (CT) 13.5. By contrast, microinjection of the GABAA antagonist, bicuculline, significantly (p < .01) increased the phase-delaying effect of light administered at CT 13.5. Microinjection of muscimol or bicuculline into the suprachiasmatic nucleus (SCN) produced little or no effect on circadian phase when no light pulses were provided. Coadministration of muscimol with VIP/PHI/GRP significantly (p < .01) reduced the phase-delaying effect of VIP/PHI/GRP, whereas administration of bicuculline with VIP/PHI/GRP significantly (p < .05) increased the phase-delaying effect of these peptides. These data indicate that changes in GABAA activity within the SCN can modulate the phase-delaying effects of light and VIP/PHI/GRP during the early portion of subjective night.

Dopamine in the nucleus accumbens modulates the memory of social defeat in Syrian hamsters (Mesocricetus auratus).

Conditioned defeat (CD) is a behavioral response that occurs in Syrian hamsters after they experience social defeat. Subsequently, defeated hamsters no longer produce territorial aggression but instead exhibit heightened levels of avoidance and submission, even when confronted with a smaller, non-aggressive intruder. Dopamine in the nucleus accumbens is hypothesized to act as a signal of salience for both rewarding and aversive stimuli to promote memory formation and appropriate behavioral responses to significant events. The purpose of the present study was to test the hypothesis that dopamine in the nucleus accumbens modulates the acquisition and expression of behavioral responses to social defeat. In Experiment 1, bilateral infusion of the non-specific D1/D2 receptor antagonist cis(z)flupenthixol (3.75 µg/150 nl saline) into the nucleus accumbens 5 min prior to defeat training significantly reduced submissive and defensive behavior expressed 24h later in response to a non-aggressive intruder. In Experiment 2, infusion of 3.75 µg cis-(Z)-flupenthixol 5 min before conditioned defeat testing with a non-aggressive intruder significantly increased aggressive behavior in drug-infused subjects. In Experiment 3, we found that the effect of cis-(Z)-flupenthixol on aggression was specific to defeated animals as infusion of drug into the nucleus accumbens of non-defeated animals did not significantly alter their behavior in response to a non-aggressive intruder. These data demonstrate that dopamine in the nucleus accumbens modulates both acquisition and expression of social stress-induced behavioral changes and suggest that the nucleus accumbens plays an important role in the suppression of aggression that is observed after social defeat.

GABAergic regulation of light-induced c-Fos immunoreactivity within the suprachiasmatic nucleus.

Analysis of the photic induction of c-Fos immunoreactivity (-ir) within the suprachiasmatic nucleus (SCN) has proven to be a powerful tool with which to study the neurochemical mechanisms involved in phase shifting the circadian clock. Some systemically administered GABAergic drugs inhibit light-induced phase shifts and c-Fos-ir, whereas others inhibit light-induced phase shifts without affecting c-Fos-ir. More recently, we have found that injection of GABAergic drugs directly into the SCN region can have dramatically different effects on light-induced phase shifts than following their systemic administration. The present study investigated the effects of GABA(A) and GABA(B) agonists and antagonists injected into the SCN region on c-Fos-ir within the SCN. Microinjection of either a GABA(A) agonist, muscimol, or a GABA(B) agonist, baclofen, into the SCN region significantly reduced light-induced c-Fos-ir within the SCN when administered before light exposure at circadian time (CT) 13.5 or CT 19. In contrast, microinjection of a GABA(A) antagonist, bicuculline, but not a GABA(B) antagonist, CGP-35348, into the SCN region increased light-induced c-Fos-ir within the SCN when administered before light exposure at CT 13.5 or CT 19. These data indicate that GABAergic agonists and antagonists injected directly into the SCN region alter light-induced Fos-ir in a manner similar to their effects on light-induced phase shifts. Comparison of these data with previous studies examining the effects of systemically administered GABAergic drugs suggests that GABA(B)-active drugs have similar effects whether given systemically or within the SCN, but that GABA(A)-active drugs have more complex effects on c-fos induction and have multiple sites of action.

N-methyl-D-aspartate receptors in the amygdala are necessary for the acquisition and expression of conditioned defeat.

Here, we describe a biologically relevant model called conditioned defeat that is used to examine behavioral responses to social defeat in Syrian hamsters. In this model experimental animals that are normally aggressive experience social defeat and consequently display high levels of submissive/defensive behavior even in response to non-threatening conspecifics. N-methyl-D-aspartate (NMDA) receptors within the amygdala play an important role in conditioned fear; therefore, the purpose of this study was to examine whether NMDA receptors within the amygdala are necessary for the acquisition and expression of conditioned defeat. Specifically, the present study examined whether bilateral infusions of the NMDA receptor antagonist DL-2-amino-5-phosphonopentanoic acid (AP5; 0.625, 1.25, 2.5, 5.0, 10.0 microg) into the amygdala would block the acquisition of conditioned defeat. Subsequently, we examined whether bilateral infusions of AP5 (0.625, 1.25, 2.5, 5.0 microg) into the amygdala prior to testing would block the expression of conditioned defeat. Infusions of AP5 into the amygdala immediately before the initial social defeat significantly reduced submissive/defensive behavior when hamsters were tested the following day with a non-aggressive intruder. Similarly, infusions of AP5 into the amygdala immediately before exposure to a non-aggressive intruder significantly attenuated the display of submissive/defensive behavior. These data demonstrate that NMDA receptors are necessary for both the acquisition and expression of conditioned defeat. We believe that conditioned defeat is a unique and valuable animal model with which to investigate the neurobiology of fear-related changes in social behavior.

Serotonergic regulation of circadian rhythms in Syrian hamsters.

This study investigated the effects of (+/-)-2-dipropylamino-8-hydroxy-1,2,3,4-tetrahydronaphthaline hydrobromide (8-OH-DPAT) on circadian rhythms in Syrian hamsters. Systemic administration of 8-OH-DPAT (0.75 mg in 150 microl saline) at circadian time 7 produced phase advances in the circadian activity rhythm. These 8-OH-DPAT-induced phase advances were blocked by microinjection of bicuculline (166 ng, 200 nl) into the suprachiasmatic nucleus, suggesting that GABAergic activity in the suprachiasmatic nucleus mediates the phase shifts produced by systemic injections of 8-OH-DPAT. Microinjection of 8-OH-DPAT (1 microg, 200 nl) or serotonin (0.7 microg, 200 nl) directly into the suprachiasmatic nucleus did not induce phase shifts at circadian time 7, suggesting that the phase shifting effects of systemic injection of 8-OH-DPAT are mediated outside the suprachiasmatic nucleus. To examine possible sites of action of 8-OH-DPAT, 8-OH-DPAT (0.5 microg (100 nl) or 1.0 microg (200 nl)) was microinjected into the intergeniculate leaflet, dorsal raphe nuclei, and the median raphe nucleus at circadian time 7. Significant phase advances were observed after microinjection into the dorsal raphe and median raphe but not the intergeniculate leaflet. These results support the hypothesis that systemic injection of serotonergic agonists can alter circadian rhythms via action in the midbrain raphe nucleus, and that the phase shifts induced by microinjection of 8-OH-DPAT into the raphe nuclei are mediated by a neurotransmitter other than serotonin within the suprachiasmatic nucleus.

Social experience and social context alter the behavioral response to centrally administered oxytocin in female Syrian hamsters.

The type of social behavior displayed by an individual is profoundly influenced by its immediate social environment or context and its prior social experience. Although oxytocin is important in the expression of social behavior in several species, it is not known if social factors alter the ability of oxytocin to influence behavior. The purpose of the present study was to test the hypothesis that social experience and social context alter the ability of oxytocin to regulate flank marking (a form of scent marking) in female Syrian hamsters. Oxytocin was microinjected into the medial preoptic anterior hypothalamic continuum (MPOA-AH) of socially experienced, dominant female hamsters which were then tested with either a subordinate partner, with a novel partner, or alone. Oxytocin induced flank marking in a dose-dependent manner but only when the experienced dominant hamsters were tested with their familiar, subordinate partners. Oxytocin did not induce flank marking when injected into socially naive female hamsters that were tested with an opponent or alone. In males, by contrast, oxytocin induced flank marking in dominant hamsters when they were tested with their subordinate partner or alone. These data support the hypothesis that social experience and social context interact to regulate the ability of oxytocin to stimulate flank marking by its actions in the MPOA-AH in female hamsters.

GABA interacts with photic signaling in the suprachiasmatic nucleus to regulate circadian phase shifts.

Circadian rhythms of physiology and behavior in mammals are driven by a circadian pacemaker located in the suprachiasmatic nucleus of the hypothalamus. The majority of neurons in the suprachiasmatic nucleus are GABAergic, and activation of GABA receptors in the suprachiasmatic nucleus can induce phase shifts of the circadian pacemaker both in vivo and in vitro. GABA also modulates the phase shifts induced by light in vivo, and photic information is thought to be conveyed to the suprachiasmatic nucleus by glutamate. In the present study, we examined the interactions between GABA receptor agonists, glutamate agonists, and light in hamsters in vivo. The GABA(A) receptor agonist muscimol and the GABA(B) receptor agonist baclofen were microinjected into the suprachiasmatic nucleus at circadian time 13.5 (early subjective night), followed immediately by a microinjection of N-methyl-D-aspartate (NMDA). Both muscimol and baclofen significantly reduced the phase shifting effects of NMDA. Further, coadministration of tetrodotoxin with baclofen did not alter the inhibition of NMDA by baclofen, suggesting a postsynaptic mechanism for the inhibition of NMDA-induced phase shifts by baclofen. Finally, the phase shifting effects of microinjection of muscimol into the suprachiasmatic nucleus during the subjective day were blocked by a subsequent light pulse. These data suggest that GABA regulates the phase of the circadian clock through both pre- and postsynaptic mechanisms.

Plasma cyclic AMP increases in hamsters following exposure to a graded footshock stressor.

To assess the utility of plasma cyclic AMP (cAMP) as a sensitive physiological index of an animal's arousal level, we exposed male hamsters to various intensities (0.0-2.0 mA) of a footshock stressor. The plasma cAMP response was directly related to stimulus intensity. Ratings of behavioral arousal were positively correlated with plasma cAMP values. We conclude that plasma cAMP may be a useful index of arousal.

Oxytocin inhibits aggression in female Syrian hamsters.

Dominant subordinate relationships are formed as the result of social conflict and are maintained at least in part by communication. At this time, little is known about the neural mechanisms that are responsible for coordinating the social behaviours (e.g. aggression) that occur in association with the formation and maintenance of these relationships. The purpose of the present study was to investigate the role of oxytocin (OXT) within the medial preoptic anterior hypothalamic continuum (MPOA-AH) in the control of aggression in female hamsters. OXT injected into the MPOA-AH immediately before testing significantly reduced the duration of aggression in a dose-dependent manner. Injection of an OXT antagonist 30 min before testing significantly increased the duration of aggression. In contrast, the duration of aggression was not altered when hamsters were tested either 30 min after injection of OXT or immediately following injection of an OXT-antagonist. These data support the hypothesis that OXT release within the MPOA-AH regulates social behaviours important in the formation and maintenance of dominant subordinate relationships in female hamsters.

Neuropeptide Y phase shifts circadian rhythms in vivo via a Y2 receptor.

Neuropeptide Y (NPY) injected into the suprachiasmatic region 6 h before the onset of locomotor activity produces phase advances in circadian rhythms. The present study investigated whether the phase advances produced by NPY in Syrian hamsters are mediated by a Y1- or Y2-like NPY receptor by comparing the phase advancing effects of the Y1 agonist, [Leu31, Pro34]NPY, and the Y2 agonist, NPY(3-36), following their injection into the suprachiasmatic region. Microinjection of the Y2 agonist produced phase advances that were significantly greater than those produced by the microinjection of the Y1 agonist. These data support the hypothesis that the phase advancing effects of NPY in the suprachiasmatic region are mediated by a Y2-like NPY receptor, similar to results found in vitro.

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 microM 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.

Neuropeptide Y microinjected into the suprachiasmatic region phase shifts circadian rhythms in constant darkness.

The geniculohypothalamic tract (GHT) is a projection from the intergeniculate leaflet to the suprachiasmatic nucleus (SCN). The GHT exhibits neuropeptide Y (NPY) immunoreactivity and appears to communicate photic information to the SCN. Microinjection of NPY into the SCN has been found to phase shift circadian rhythms of hamsters housed in constant light in a manner similar to the phase shifts produced by pulses of darkness or triazolam injections. In the present study, NPY was injected into the SCN of Syrian hamsters housed in constant darkness and was found to produce phase shifts similar to those seen in hamsters housed in constant light. Microinjections were not followed by wheel running during the subjective day (the time when NPY microinjections are followed by significant phase advances). These data suggest that NPY produces phase shifts by some mechanism other than by inducing wheel running or by inhibiting the response of SCN neurons to light and supports a role for NPY in nonphotic shifting of the circadian clock.

Activation of GABA(A) receptors in the amygdala blocks the acquisition and expression of conditioned defeat in Syrian hamsters.

Social defeat is a powerful experience that often leads to drastic physiological and behavioral changes in many animal species. An example of such a change is conditioned defeat in Syrian hamsters. The neurophysiological mechanisms that underlie such changes are not yet fully understood, however, there is evidence that the amygdala plays an essential role in behavioral and emotional responses to a variety of stressors. The goal of the present study was to determine whether GABAergic neurotransmission in the amygdala is a critical component of conditioned defeat in male Syrian hamsters. Experiment 1 examined whether infusion of the GABA(A) receptor agonist, muscimol (0.0, 4.4, 8.8 nmol), into the amygdala would block the acquisition of conditioned defeat. Experiment 2 examined whether infusion of muscimol into the amygdala prior to testing would block expression of conditioned defeat. Submissive behavior during testing was significantly reduced in animals receiving infusions of muscimol immediately prior to initial defeat training. Animals that received infusions of muscimol immediately prior to being tested with a non-aggressive intruder also displayed significantly less submissive behavior than did animals receiving vehicle control. These data indicate that infusion of muscimol into the amygdala can block the acquisition and expression of conditioned defeat, a finding that indicates that GABAergic neurotransmission within the amygdala is involved in the acquisition and expression of fear or stress-induced behavioral changes. This is the first evidence indicating that the neural circuits involved in Pavlovian fear conditioning are also involved in more ethologically-relevant models examining stress-related behavioral plasticity.

Bicuculline blocks neuropeptide Y-induced phase advances when microinjected in the suprachiasmatic nucleus of Syrian hamsters.

Microinjection of neuropeptide Y (NPY) into the suprachiasmatic nucleus of the hypothalamus (SCN) during the middle of the subjective day (i.e. circadian time 6) causes large phase advances in circadian rhythms. The present study demonstrates that microinjection of the gamma-aminobutyric acid (GABA) antagonist, bicuculline, completely blocks NPY-induced phase advances. These data indicate that GABAA activity within the SCN may mediate the phase shifting effects of some stimuli on the circadian pacemaker.

GABA(A) and GABA(B) agonists and antagonists alter the phase-shifting effects of light when microinjected into the suprachiasmatic region.

GABAergic drugs have profound effects on the regulation of circadian rhythms. The present study evaluated the effects of microinjections of GABAergic drugs into the suprachiasmatic region in hamsters on phase shifts induced by light and by microinjection of a cocktail containing vasoactive intestinal peptide (VIP), peptide histidine isoleucine (PHI) and gastrin-releasing peptide (GRP). The phase-advancing effects of light at circadian time (CT) 19 were significantly reduced by microinjection of GABA(A) or GABA(B) agonists into the SCN, but were not altered by microinjection of GABA(A) or GABA(B) antagonists. Microinjection of a GABA(B) agonist also reduced the phase-delaying effects of light at CT 13.5-14 while a GABA(B) antagonist increased the phase delays caused by light. Neither GABA(B) drug altered the phase delays produced by microinjection of a peptide cocktail containing VIP, PHI, GRP. These data indicate that changes in GABA(A) or GABA(B) activity within the SCN can alter the phase-shifting effects of light on circadian rhythms and support a role for GABA in gating photic input to the circadian clock.

Tetrodotoxin blocks NPY-induced but not muscimol-induced phase advances of wheel-running activity in Syrian hamsters.

During the middle of the subjective day, circadian activity rhythms in Syrian hamsters can be phase advanced by a variety of stimuli including microinjection of neuropeptide Y (NPY) or muscimol into the suprachiasmatic nucleus (SCN). It is not known, however, if these treatments shift activity rhythms by acting directly on pacemaker cells within the SCN. In the present study NPY and muscimol were microinjected with either tetrodotoxin or saline in order to determine whether classical synaptic transmission within the SCN is necessary for the phase advances produced by NPY or muscimol. Blockade of sodium-dependent action potentials within the SCN prevented NPY- but not muscimol-induced phase advances. These data, along with our previous finding that bicuculline blocks NPY-induced phase advances, suggest that NPY requires sodium-dependent action potentials within GABAergic neurons in order to phase-shift the circadian pacemaker.

Differential effects of two corticotropin-releasing factor antagonists on conditioned defeat in male Syrian hamsters (Mesocricetus auratus).

The purpose of the present study was to determine whether corticotropin-releasing factor (CRF) is involved in mediating the expression of conditioned defeat in male Syrian hamsters. The present study examined the effects of two different competitive CRF receptor antagonists on the expression of conditioned defeat. Specifically, Experiment 1 examined whether peripheral administration of CP-154,526, a specific non-peptide CRF1 receptor antagonist, would reduce the expression of conditioned defeat. Experiment 2 examined whether D-Phe CRF(12-41), a nonspecific CRF1/CRF2 receptor antagonist, infused directly into the brain, would reduce the expression of conditioned defeat. The results revealed that i.p. injections of CP-154,526 did not reduce the expression of conditioned defeat, whereas i.c.v. injections of D-Phe CRF(12-41) successfully reduced the expression of conditioned defeat. The duration of submissive/defensive behaviors in hamsters that received the high dose of D-Phe CRF(12-41) was significantly less than that exhibited by animals that received a vehicle control. The present data suggest that central CRF may be involved in mediating the expression of conditioned defeat and other behavioral responses to stressful stimuli.