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.

Arginine-vasopressin and the regulation of aggression in female Syrian hamsters (Mesocricetus auratus).

Arginine-vasopressin (AVP) is critical for the expression of a variety of social behaviors in many species. Previous studies have demonstrated that AVP regulates behaviors such as social communication and aggression in Syrian hamsters through the V1a receptor subtype. In male hamsters, AVP injected into the anterior hypothalamus (AH) stimulates aggression, while injection of a V1a receptor antagonist inhibits the behavior. The purpose of the present studies was to determine whether AVP influences aggression by its action in the AH in female hamsters. In the first experiment, we were surprised to find that injection of the V1a receptor antagonist, Manning compound, into the AH of intact female hamsters increased aggression. The second experiment confirmed the ability of the V1a receptor antagonist to increase aggression and found that the largest effects of the antagonist occurred at intermediate concentrations of the compound. The next experiment found that injection of AVP into the AH significantly reduced the latency to attack and the duration of aggression. Finally, we examined whether the effects of AVP and the V1a receptor antagonist on aggression differed in hamsters exposed to long 'summer-like' photoperiods or short 'winter-like' photoperiods, and found that their effects on aggression were not photoperiod dependent. In summary, contrary to what is observed in males, these data suggest that AVP in the AH may play an inhibitory role on aggression in female Syrian hamsters.

Avian pancreatic polypeptide phase shifts hamster circadian rhythms when microinjected into the suprachiasmatic region.

The suprachiasmatic nucleus has been identified tentatively as a circadian pacemaker. To examine the functional role of peptides found within suprachiasmatic neurons, avian pancreatic polypeptide and vasopressin were microinjected into the suprachiasmatic region. Avian pancreatic polypeptide, but not vasopressin, shifted the phase of the wheelrunning rhythm as a function of the time of its injection within the circadian cycle. Avian pancreatic polypeptide or a similar peptide may be one component of the neurochemical processes underlying entrainment to the light-dark cycle.

Vasopressin injected into the hypothalamus triggers a stereotypic behavior in golden hamsters.

Microinjection of arginine vasopressin into the medial preoptic area of the hypothalamus of male and female golden hamsters triggered a complex, stereotypic behavior--flank marking--a type of scent marking used in olfactory communication. The flank marking was not elicited by saline, oxytocin, neurotensin, or angiotensin II. Vasopressin was ineffective when injected into other areas of the hypothalamus or into the lateral cerebroventricle.

Role for the NR2B subunit of the N-methyl-D-aspartate receptor in mediating light input to the circadian system.

Light information reaches the suprachiasmatic nucleus (SCN) through a subpopulation of retinal ganglion cells that utilize glutamate as a neurotransmitter. A variety of evidence suggests that the release of glutamate then activates N-methyl-D-aspartate (NMDA) receptors within the SCN and triggers a signaling cascade that ultimately leads to phase shifts in the circadian system. In this study, we first sought to explore the role of the NR2B subunit in mediating the effects of light on the circadian system of hamsters and mice. We found that localized microinjection of the NR2B subunit antagonist ifenprodil into the SCN region reduces the magnitude of light-induced phase shifts of the circadian rhythm in wheel-running activity. Next, we found that the NR2B message and levels of phospho-NR2B vary with time of day in SCN tissue using semiquantitative real-time polymerase chain reaction and western blot analysis, respectively. Functionally, we found that blocking the NR2B subunit with ifenprodil significantly reduced the magnitude of NMDA currents recorded in SCN neurons. Ifenprodil also significantly reduced the magnitude of NMDA-induced Ca2+ changes in SCN cells. Together, these results demonstrate that the NR2B subunit is an important component of NMDA receptor-mediated responses within SCN neurons and that this subunit contributes to light-induced phase shifts of the mammalian circadian system.

Activation of NMDA receptors in the suprachiasmatic nucleus produces light-like phase shifts of the circadian clock in vivo.

Although there is substantial evidence that glutamate mimics the effects of light on the mammalian circadian clock in vitro, it has been reported that microinjection of glutamate into the suprachiasmatic nucleus of the hypothalamus (SCN) region in vivo does not result in a pattern of phase shifts that mimic those caused by light pulses. The present study was designed to test the hypothesis that microinjection of NMDA into the SCN would induce light-like phase shifts of the circadian clock through activation of the NMDA receptor. Hamsters housed in constant darkness received microinjections of NMDA through guide cannulas aimed at the SCN region at various times throughout the circadian cycle. Wheel running was monitored as a measure of circadian phase. Microinjection of NMDA resulted in circadian phase shifts, the size and direction of which were dependent on the time of injection. The resulting phase-response curve closely resembled that of light. The circadian response showed a clear dose-dependence at circadian time (CT) 13.5 but not at CT19. Both phase delays and advances induced by NMDA were blocked by coinjection of the NMDA antagonist 2-amino-5-phosphopentanoic acid but were slightly attenuated by the non-NMDA antagonist 6-nitro-7-sulfamoylbenzo[f]quinoxaline-2,3-dione disodium. The ability of NMDA to induce phase shifts was not altered by coinjection with tetrodotoxin. These data are consistent with the hypothesis that activation of NMDA receptors is a critical step in the transmission of photic information to the SCN.

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.

Cold exposure elevates cellular levels of messenger ribonucleic acid encoding thyrotropin-releasing hormone in paraventricular nucleus despite elevated levels of thyroid hormones.

Cold exposure increases blood levels of TSH and thyroid hormones by stimulating the secretion of TRH from the median eminence. Thyroid hormones reduce TRH release and cellular levels of TRH mRNA. Using quantitative in situ hybridization to measure changes in cellular levels of various neuropeptide mRNAs, the present studies demonstrate that cold exposure also increases cellular levels of TRH mRNA in neurons of the paraventricular nucleus (PVN), supporting the concept that TRH mRNA levels are reflective of TRH secretion in these neurons. The effect of cold appeared to be specific for TRH expression in the PVN, because cold exposure did not influence cellular levels of TRH in the reticular thalamic nucleus or beta-actin and oxytocin mRNAs in the PVN. Cellular levels of mRNA encoding CRH were elevated by cold exposure. This latter observation is predictable based on the cold-induced activation of the hypothalamic-pituitary-adrenal axis. There was a 24-h rhythm and a time of day difference in the effect of cold on TRH mRNA levels in the PVN. Time of day differences in the effect of cold on CRH mRNA levels were not apparent. Cold exposure appeared to elevate TRH mRNA levels in all neurons of the PVN, indicating that the neurally mediated effect of cold on TRH expression can override the inhibitory effects of circulating T3 within the same neuronal population.

Neurotensin levels in the hepatic-portal circulation are inversely related to the circadian feeding cycle in rats.

To investigate whether the circulating level of neurotensin (NT) in the rat is related to either the 24-h pattern in food consumption or environmental lighting conditions, the plasma level of NT was determined every 4 h in the hepatic-portal vein and the abdominal aorta over the course of 24 h. At each time interval, pooled plasma samples from groups of 4 rats were extracted, lyophilized, reconstituted, and subjected to HPLC. Column fractions were radioimmunoassayed with both N- and C-terminal directed antisera. Animals housed in a 12-h light, 12-h dark cycle and given food and water ad libitum had a significant (P less than 0.05) 24-h variation in the level of chromatographically and immunochemically identified NT in the portal circulation while the level of NT in the systemic circulation remained unchanged. The level of NT in portal blood ranged from 12-38 fmol/ml and was highest in the afternoon, 12-16 h after peak feeding. The level of NT in aortic blood never exceeded 7 fmol/ml. Similar results were obtained from animals exposed to constant illumination for 13-32 h with free access to food and water. The release of NT during the fasting phase of the feeding cycle was dependent upon the prior intake of food, since the level of NT in the hepatic-portal circulation of rats housed in 12-h light, 12-h dark cycle and fasted for 20-24 h was about 2-fold less than that observed in animals allowed free access to food. In summary, these data show that the release and circulation of NT are tightly linked to the circadian pattern of food intake and that the greatest release of NT into the hepatic-portal circulation occurs 5-10 h after the cessation of eating during the fasting phase of the feeding cycle.

Neural circuitry controlling vasopressin-stimulated scent marking in Syrian hamsters (Mesocricetus auratus).

In Syrian hamsters, arginine vasopressin (AVP) plays a critical role in the control of a form of scent marking called flank marking. Microinjection of AVP into the medial preoptic-anterior hypothalamus (MPOA-AH), lateral septal nucleus (LS), bed nucleus of the stria terminalis (BNST), and the periaqueductal gray (PAG) stimulates high levels of flank marking. Microinjection of an antagonist of the V1a-AVP receptor into sites such as the MPOA-AH inhibits expression of flank marking. The purpose of the present study was to investigate the neural circuit controlling flank marking by localizing the induction of Fos protein in response to the microinjection of AVP, a V1a-AVP antagonist (AVPA) or saline into the MPOA-AH. Immediately after microinjection, hamsters were placed in a clean cage and their behavior was videotaped for 10 minutes. Ninety minutes after the behavioral experiment hamsters were perfused and their brains removed for subsequent immunocytochemical localization of Fos protein. The number of Fos-positive neurons was significantly greater in the BNST, PAG, and central amygdala (Ce) following the microinjection of AVP than following the microinjection of either AVPA or saline. In AVP-injected animals, the number of Fos-labeled cells in the Ce, PVN, and PAG increased with increased frequency of either flank marking or flank gland grooming. These data support the hypothesis that neurons within the MPOA-AH, BNST, and PAG play an important role in the control of flank marking and suggest that the Ce may be a previously unrecognized part of this neural circuit.

Three-dimensional structure of the mammalian suprachiasmatic nuclei: a comparative study of five species.

The suprachiasmatic nuclei from five mammalian species (rat, hamster, cat, rhesus, and squirrel monkey) were reconstructed in three-dimensions by use of computer graphics and conventional histological techniques. The gross nuclear structures of the suprachiasmatic nuclei have complex three-dimensional geometries in every plane of orientation, and between the five species there are marked differences in the three-dimensional morphology of the suprachiasmatic nuclei. These dimensionally accurate reconstructions are discussed in relation to previous data suggesting morphological and/or functional specialization within specific regions of the suprachiasmatic nuclei.

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.

Disruption of human circadian and cognitive regulation following a discrete hypothalamic lesion: a case study.

We report a patient with rostral hypothalamic damage that significantly disrupted temporal patterning of the sleep-wake cycle, body temperature, and cognitive and behavioral functioning. The findings suggest that the suprachiasmatic region of the hypothalamus is important for the circadian control of human behavior, and that circadian organization may be essential for normal cognitive functioning.

Disruption of short-duration timing associated with damage to the suprachiasmatic region of the hypothalamus.

The neural bases of circadian rhythmicity have been demonstrated in a variety of animal species, including primates. Yet, the brain mechanisms underlying time experience and the timing of behaviors of shorter duration are still not well understood. In the present study, we demonstrate disruption of short-duration timing capacity in AH, a patient with damage to the suprachiasmatic (SCN) region of the hypothalamus. AH exhibited extreme inconsistency in her rate of tapping production on a motor continuation paradigm. Her inter-response intervals (IRIs) were extremely large compared with normal control subjects and were similar to those previously reported in patients with cerebellar dysfunction. Increased variability of both central timing and motor implementation processes was evident compared with both age-matched and elderly normal control subjects. Severe impairment of time perception was also evident on duration discrimination, whereas auditory loudness discrimination was intact. These findings suggest that a hierarchic relationship between long-duration (circadian) and short-duration timing exists, and that in addition to the cerebellum, intact hypothalamic functioning is necessary for short-duration timing.

Neuropeptide Y and N-methyl-D-aspartic acid interact within the suprachiasmatic nuclei to alter circadian phase.

Circadian rhythms are reset by exposure to photic stimuli and nonphotic stimuli. Glutamate appears to be the primary neurotransmitter that communicates photic stimuli to the circadian clock located in the suprachiasmatic nucleus. There is also substantial evidence that neuropeptide Y (NPY) mediates the effects of at least some nonphotic stimuli on the circadian clock. The purpose of this study was to investigate how NPY and glutamate receptor activation interact to reset the phase of the circadian clock. Microinjection of the glutamate agonist N-methyl-D-aspartic acid (NMDA) during the subjective day significantly decreased NPY-induced phase advances. During the late subjective night, NMDA induced light-like phase advances, which were significantly reduced by microinjection of NPY. Microinjection of NPY inhibited NMDA-induced phase advances during the late subjective night, even when sodium-dependent action potentials were inhibited by tetrodotoxin. These data support the hypothesis that, during the subjective night, NPY and NMDA act on the same clock cells or on cells that communicate with clock cells by mechanisms not requiring action potentials. Although NPY and NMDA appear to be mutually inhibitory during both the day and the night, the mechanisms of this inhibition appear to be different during the day versus the night.

Transduction of light in the suprachiasmatic nucleus: evidence for two different neurochemical cascades regulating the levels of Per1 mRNA and pineal melatonin.

The suprachiasmatic nucleus (SCN) contains a circadian clock and regulates melatonin synthesis in the pineal gland. Light exposure during the subjective night acutely increases the mRNA levels of the Period (Per)1 gene in the SCN and acutely suppresses melatonin levels in the pineal gland. Activation of N-methyl-D-aspartate (NMDA) receptors in the SCN has been demonstrated to phase-shift the circadian clock in a manner similar to light. We tested the hypothesis that activation of excitatory amino acid (EAA) receptors in the SCN mediates the acute effects of light on Per1 mRNA levels and pineal melatonin. NMDA, injected into the SCN of Syrian hamsters during the night, acutely suppressed melatonin levels in the pineal gland. Both the NMDA receptor antagonist 2-amino-5-phosphonopentanoic acid (AP5) and the alpha-amino-3-hydroxy-5-methylisoxazoleproprionic acid (AMPA)/kainate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione (DNQX) inhibited the light-induced increase of Per1 mRNA levels in the SCN. In the same animals, however, these antagonists had no effect on the ability of light to suppress pineal melatonin. These results support the hypothesis that EAA receptor activation in the SCN is necessary for the acute effects of light on Per1 mRNA levels. They also indicate that NMDA receptor activation in the SCN is sufficient but may not be necessary for the acute effects of light on pineal melatonin. These data suggest that there may be at least two different neurochemical cascades that transduce the effects of light in the SCN

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.