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.

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.

Aggressive encounters alter the activation of serotonergic neurons and the expression of 5-HT1A mRNA in the hamster dorsal raphe nucleus.

Serotonergic (5-HT) neurons in the dorsal raphe nucleus (DRN) have been implicated in stress-induced changes in behavior. Previous research indicates that stressful stimuli activate 5-HT neurons in select subregions of the DRN. Uncontrollable stress is thought to sensitize 5-HT neurons in the DRN and allow for an exaggerated 5-HT response to future stimuli. In the current study, we tested the hypothesis that following aggressive encounters, losing male Syrian hamsters would exhibit increased c-Fos immunoreactivity in 5-HT DRN neurons compared to winners or controls. In addition, we tested the hypothesis that losers would have decreased 5-HT1A mRNA levels in the DRN compared to winners or controls. We found that a single 15-min aggressive encounter increased c-Fos expression in 5-HT and non-5-HT neurons in losers compared to winners and controls. The increased c-Fos expression in losers was restricted to ventral regions of the rostral DRN. We also found that four 5-min aggressive encounters reduced total 5-HT1A mRNA levels in the DRN in losers compared to winners and controls, and that differences in mRNA levels were not restricted to specific DRN subregions. These results suggest that social defeat activates neurons in select subregions of the DRN and reduces message for DRN 5-HT1A autoreceptors. Our results support the hypothesis that social stress can activate 5-HT neurons in the DRN, reduce 5-HT1A autoreceptor-mediated inhibition, and lead to hyperactivity of 5-HT neurons.

GABA(B) receptor activation in the suprachiasmatic nucleus of diurnal and nocturnal rodents.

Diurnal (day-active) and nocturnal (night-active) animals have very different daily activity patterns. We recently demonstrated that the suprachiasmatic nucleus (SCN) responds to GABAergic stimulation differently in diurnal and nocturnal animals. Specifically, GABAA receptor activation with muscimol during the subjective day causes phase delays in diurnal grass rats while producing phase advances in nocturnal hamsters. The aim of the following experiments was to determine if diurnal and nocturnal animals differ in their response to GABAB receptor activation in the SCN. Baclofen, a GABAB receptor agonist, was microinjected into the SCN region of grass rats or hamsters under free-running conditions and phase alterations were analyzed. Changes in phase were not detected after baclofen treatment during the subjective day in either grass rats or hamsters. During the night, however, GABAB receptor activation significantly decreased the ability of light to induce phase delays in grass rats. Taken together with previous data from our laboratory, these results demonstrate that, in both hamsters and grass rats, GABAB receptor activation in the SCN significantly affects circadian phase during the night, but not during the day.

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.

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.

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.

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.

The effects of endomorphin-1 on conditioned defeat in Syrian hamsters (Mesocricetus auratus).

The present study examined the effect of endomorphin-1 (EM1), an endogenous opioid with a high affinity for the mu opiate receptor, on conditioned defeat. Conditioned defeat is a phenomenon in which hamsters that have been defeated subsequently fail to exhibit normal territorial aggression and instead display submissive/defensive behaviors even when paired with a non-aggressive intruder. In experiment 1, animals were placed in the home cage of a larger resident for 15 min and were defeated. After 24 h, animals received a 3-microl injection of EM1 (0.0, 0.3, 3.0, or 10 nmol) into the left lateral cerebral ventricle 5 min before a smaller non-aggressive intruder was placed in the home cage of the experimental animal. In experiment 2, animals were infused with EM1 immediately after the initial defeat and were paired with a non-aggressive intruder 24 h later as in experiment 1. EM1 reduced the duration of submissive/defensive behavior in experiment 1 (P<0.05) but not in experiment 2 (P>0.05). These data support the hypothesis that the highly selective mu receptor agonist endomorphin-1 modulates the expression of conditioned defeat, but provides no support for the hypothesis that endomorphin-1 modulates the consolidation of conditioned defeat.

Acute and chronic social defeat suppresses humoral immunity of male Syrian hamsters (Mesocricetus auratus).

Stressors, both physical and psychological, can activate the hypothalamic-pituitary-adrenal (HPA) axis, leading to a wide range of physiological responses including increased glucocorticoid release and suppression of immune function. The majority of studies published to date have focused on the effects of physical stressors (e.g., cold exposure, electric shock) on immunity. The present study examined the role of a stressor, social defeat, on humoral immune function of Syrian hamsters (Mesocricetus auratus). Specifically, adult male Syrian hamsters experienced social defeat (i.e., exposure to a dominant animal in that animal's home cage) that was either acute (i.e., a single exposure) or chronic (i.e., daily exposures across 5 days). A control group of animals was placed in a resident's home cage without the resident animal present and did not experience defeat. After the last encounter, blood samples were drawn and animals were subsequently injected with keyhole limpet hemocyanin (KLH). Blood samples were again taken 5 and 10 days postimmunization and serum was analyzed to determine serum cortisol and anti-KLH immunoglobulin G (IgG) concentrations. Cortisol concentrations were elevated in both acutely and chronically defeated hamsters compared with control animals. In contrast, serum IgG concentrations were significantly reduced in both groups of defeated hamsters compared with control animals. Collectively, these results demonstrate that both acute social defeat and chronic social defeat lead to activation of the HPA axis and suppression of humoral immune function. These data suggest that social defeat is an important, ecologically relevant model with which to examine stress-induced immune suppression in rodents.

Short-day increases in aggression are inversely related to circulating testosterone concentrations in male Siberian hamsters (Phodopus sungorus).

Many nontropical rodent species display seasonal changes in both physiology and behavior that occur primarily in response to changes in photoperiod. Short-day reductions in reproduction are due, in part, to reductions in gonadal steroid hormones. In addition, gonadal steroids, primarily testosterone (T), have been implicated in aggression in many mammalian species. Some species, however, display increased aggression in short days despite basal circulating concentrations of T. The goal of the present studies was to test the effects of photoperiod on aggression in male Siberian hamsters (Phodopus sungorus) and to determine the role of T in mediating photoperiodic changes in aggression. In Experiment 1, hamsters were housed in long and short days for either 10 or 20 weeks and aggression was determined using a resident-intruder model. Hamsters housed in short days for 10 weeks underwent gonadal regression and displayed increased aggression compared to long-day-housed animals. Prolonged maintenance in short days (i.e., 20 weeks), however, led to gonadal recrudescence and reduced aggression. In Experiment 2, hamsters were housed in long and short days for 10 weeks. Half of the short-day-housed animals were implanted with capsules containing T whereas the remaining animals received empty capsules. In addition, half of the long-day-housed animals were castrated whereas the remaining animals received sham surgeries. Short-day control hamsters displayed increased aggression compared to either castrated or intact long-day-housed animals. Short-day-housed T treated hamsters, however, did not differ in aggression from long-day-housed animals. Collectively, these results confirm previous findings of increased aggression in short-day-housed hamsters and suggest that short-day-induced increases in aggression are inversely related to gonadal steroid hormones.

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.

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.

Glutamic acid decarboxylase mRNA in the suprachiasmatic nucleus of rats housed in constant darkness.

This study demonstrates that the levels of the mRNAs encoding the two isoforms of glutamic acid decarboxylase (GAD) (i.e., GAD65 and GAD67) do not differ over the circadian activity cycle in the suprachiasmatic nucleus (SCN) of rats housed in constant darkness. These data indicate that the rhythmic expression of GAD56 mRNA previously observed in animals housed in a light:dark cycle [K.L. Huhman, A.C. Hennessey, H.E. Albers, Rhythms of glutamic acid decarboxylase mRNA in the suprachiasmatic nucleus, J. Biol. Rhythms 11 (1996) 311-316.] is the result of the activity of retinal afferents.

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.

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.

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.

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.