Role of the extended amygdala in short-duration versus sustained fear: a tribute to Dr. Lennart Heimer

The concept of the "extended amygdala", developed and explored by Lennart Heimer, Jose de Olmos, George Alheid, and their collaborators, has had an enormous impact on the field of neuroscience and on our own work. Measuring fear-potentiated startle test using conditioned stimuli that vary in length we suggest that the central nucleus of the amygdala (CeA) and the lateral division of the bed nucleus of the stria terminalis (BNST(L)) are involved in short-term versus long-term fear responses we call phasic versus sustained fear, respectively. Outputs from the basolateral amygdala (BLA) activate the medial division of the CeA (CeA(M)) to very rapidly elicit phasic fear responses via CeA(M) projections to the hypothalamus and brainstem. The BLA also projects to the BNST(L), which together with other BNST(L) inputs from the lateral CeA (CeA(L)) initiate a slower developing, but sustained fear response, akin to anxiety. We hypothesize this occurs because the CeA(L) releases the peptide corticotropin releasing hormone (CRF) into the BNST(L) which facilitates the release of glutamate from BLA terminals. This activates the BNST(L) which projects to hypothalamic and brainstem areas similar to those innervated by the CeA(M) that mediate the specific signs of fear and anxiety. The generality of this idea is illustrated by selective studies looking at context conditioning, social defeat, drug withdrawal and stress induced reinstatement.     

Corticotropin-releasing factor type II (CRF-sub-2) receptors in the bed nucleus of the stria terminalis modulate conditioned defeat in Syrian hamsters (Mesocricetus auratus)

In Syrian hamsters (Mesocricetus auratus), social defeat produces a subsequent increase in submissive and defensive behavior and a loss of normal territorial aggression, which the authors have called conditioned defeat. In this study, the authors investigated the effect of blocking corticotropin-releasing factor (CRF) Type I and Type II receptors on conditioned defeat. Intracerebroventricular infusion of the CRF-sub-2 receptor antagonist antisauvagine-30 prior to testing significantly reduced conditioned defeat compared with vehicle controls, whereas the CRF-sub-1 receptor antagonist CP-154,526 had no effect. Also, infusion of antisauvagine-30 into the bed nucleus of the stria terminalis (BNST) 15 min, but not immediately, prior to testing reduced conditioned defeat in a dose-dependent manner. The authors' results provide evidence that CRF-sub-2 receptors in the BNST, but not CRF-sub-1 receptors, are an important component in the neural circuitry regulating conditioned defeat.     

Cocaine pre-exposure enhances CRF-induced expression of c-fos mRNA in the central nucleus of the amygdala: an effect that parallels the effects of cocaine pre-exposure on CRF-induced locomotor activity

There is evidence that cocaine pre-exposure produces changes in the responsivity of central corticotropin-releasing factor (CRF) systems and that these systems mediate some of the drug-related behavioural effects of acute stressors. The present experiment was conducted to assess the effects of repeated cocaine exposure on CRF-induced neuronal activation within two regions of the extended amygdala, the central nucleus of the amygdala (CeA) and lateral bed nucleus of the stria terminalis (BNST). In addition, CRF-induced neuronal activation was compared with CRF-induced locomotor activity. Rats were injected for 7 days with cocaine (days 1 and 7 in test chambers; days 2-6 in homecages) or saline. After 10 drug-free days, locomotor responsiveness to intracerebroventricular (i.c.v.) injections of CRF and Vehicle was assessed over 2-h test periods. Twenty-four to 48 h following testing for locomotor activity, animals were injected with either CRF or Vehicle, 30 min before being sacrificed. Subsequently, the brains were processed by in situ hybridization for c-fos mRNA, a widely used marker of neuronal activation, in the CeA and BNST. In CeA, i.c.v. CRF enhanced the expression of c-fos mRNA in cocaine, but not saline, pre-exposed animals; in the same animals, i.c.v. CRF resulted in enhanced locomotor activity in cocaine, but not saline, pre-exposed animals. The results demonstrate that repeated exposure to cocaine changes the neuronal response to CRF in the CeA; furthermore, they suggest that these changes in the CeA could potentially be of functional significance in the effects of repeated cocaine exposure on CRF-induced locomotor activity.     

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.     

Mu-opioid and corticotropin-releasing-factor receptors show largely postsynaptic co-expression,and separate presynaptic distributions,in the mouse central amygdala and bed nucleus of the stria terminalis

The anxiolytic effects of opiates active at the mu-opioid receptor (μ-OR) may be ascribed, in part, to suppression of neurons that are responsive to the stress-associated peptide, corticotropin releasing factor (CRF), in the central amygdala (CeA) and bed nucleus of the stria terminalis (BNST). The corticotropin releasing factor receptor (CRFr) and μ-OR are expressed in both the CeA and BNST, but their subcellular relationship to each other is not known in either region. To address this question, we used dual electron microscopic immunolabeling of μ-OR and CRFr in the mouse lateral CeA and anterolateral BNST. Immunolabeling for each receptor was detected in the same as well as in separate somatic, dendritic and axonal profiles of neurons in each region. CRFr had a plasmalemmal or cytoplasmic distribution in many dendrites, including those co-expressing μ-OR. The co-expression of CRFr and μ-OR also was seen near excitatory-type synapses on dendritic spines. In both the CeA and BNST, over 50% of the CRFr-labeled dendritic profiles (dendrites and spines) contained immunoreactivity for the μ-OR. However, less than 25% of the dendritic profiles containing the μ-OR were labeled for CRFr in either region, suggesting that opiate activation of the μ-OR affects many neurons in addition to those responsive to CRF. The dendritic profiles containing CRFr and/or μ-OR received asymmetric, excitatory-type synapses from unlabeled or CRFr-labeled axon terminals. In contrast, the μ-OR was identified in terminals forming symmetric, inhibitory-type synapses. Thus, in both the CeA and BNST, μ-OR and CRFr have strategic locations for mediation of CRF and opioid effects on the postsynaptic excitability of single neurons, and on the respective presynaptic release of excitatory and inhibitory neurotransmitters. The commonalities in the synaptic location of both receptors in the CeA and BNST suggest that this is a fundamental cellular association of relevance to both drug addiction and stress-induced disorders.     

The effect of escapable versus inescapable social defeat on conditioned defeat and social recognition in Syrian hamsters

Male Syrian hamsters are naturally aggressive animals that reliably defend their home territory against intruding conspecifics. Hamsters that lose agonistic encounters subsequently exhibit a striking change in their agonistic behavior, however, expressing no aggression and instead becoming highly submissive, a behavioral change that we have termed conditioned defeat. We have generally employed an inescapable defeat training protocol when studying conditioned defeat. The purpose of the present study was to determine if conditioned defeat is an epiphenomenon of the inescapable defeat experience by comparing the behavior of hamsters exposed to inescapable versus escapable defeat. In the conditioned defeat model, defeated hamsters subsequently generalize their submission and social avoidance to a novel, non-aggressive opponent, suggesting that hamsters subjected to inescapable defeat may not form a specific memory of their aggressive opponent. Thus, a secondary purpose of the present study was to determine whether hamsters subjected to our defeat protocol have the ability to recognize a familiar opponent following defeat. Our results provide evidence that conditioned defeat is not solely a by-product of inescapable defeat because all experimental animals, regardless of the type of defeat, expressed conditioned defeat during testing. We also found that animals experiencing an inescapable defeat avoided a familiar aggressor significantly more than they did an unfamiliar aggressor, demonstrating that these animals have the ability to recognize their previous attacker. Thus, we maintain that a variety of social defeat models, and conditioned defeat in particular, represent generalizable and ethologically valid models with which to study the effects of social stress on physiology and behavior.     

Differential brain-derived neurotrophic factor expression in limbic brain regions following social defeat or territorial aggression

Syrian hamsters readily form dominant-subordinate relationships under laboratory conditions. Winning or losing in agonistic encounters can have striking, long-term effects on social behavior, but the mechanisms underlying this experience-induced behavioral plasticity are unclear. The present study tested the hypothesis that changes in brain-derived neurotrophic factor (BDNF) may at least in part mediate this plasticity. Male hamsters were paired for 15-min using a resident-intruder model, and individuals were identified as winners or losers on the basis of their behavior. BDNF was examined with in situ hybridization 2 hr after treatment during the consolidation period of emotional learning. Losing animals had significantly more BDNF mRNA in the basolateral (BLA) and medial (MeA) nuclei of the amygdala when compared with winning animals as well as novel cage and home cage controls. Interestingly, winning animals had significantly more BDNF mRNA in the dentate gyrus of the dorsal hippocampus than did losing animals, novel, and home cage controls. No conflict-related changes in BDNF mRNA were observed in several other regions including the bed nucleus of the stria terminalis and central amygdala. Next, we demonstrated that K252a, a Trk receptor antagonist, significantly reduced the acquisition of conditioned defeat when administered within the BLA. These data support a model in which BDNF-mediated plasticity within the BLA supports learning of submission or subordinate social status in losing animals, whereas BDNF-mediated plasticity within the hippocampus may instantiate aspects of winning such as control of a territory in dominant animals.     

Rapid neuroadaptation in the nucleus accumbens and bed nucleus of the stria terminalis mediates suppression of operant responding during withdrawal from acute opioid dependence

Single injections of morphine induce a state of acute opioid dependence in humans and animals, measured as precipitated withdrawal when an antagonist is administered 4-24 h after morphine. Additional morphine exposure at daily or weekly intervals results in further increases in withdrawal severity, suggesting that acute opioid dependence reflects the early stages in the development of a chronic state of dependence. The current study evaluated the role of the nucleus accumbens (NAC), bed nucleus of stria terminalis (BNST), interstitial nucleus of posterior limb of the anterior commissure (IPAC), and central amygdala (CeA) in the expression of antagonist-precipitated suppression of operant responding for food as a measure of withdrawal from acute opioid dependence. Rats trained on a fixed-ratio 15 schedule received one or four daily injections of morphine, with the lipophobic opioid antagonist methylnaloxonium (16-2000 ng) infused into one of the brain regions or the lateral ventricle (i.c.v.) 4 h after the final morphine injection. After acute morphine methylnaloxonium was more potent upon infusion into the NAC (17.9-fold potency shift), BNST (6.8-fold) and CeA (5.5-fold) than it was upon i.c.v. administration. Following repeat morphine the NAC and BNST but not CeA continued to show greater sensitivity relative to i.c.v. infusion (12.9-, 8.7-, and 3.2-fold potency shifts, respectively). The IPAC was insensitive to methylnaloxonium after acute or repeat morphine at doses that reliably suppressed responding upon i.c.v. infusion (125-500 ng). Thus, among the components of extended amygdala examined in this study, rapid neuroadaptation within the nucleus accumbens and bed nucleus of the stria terminalis appear to play the most prominent role in antagonist-precipitated suppression of operant responding during the early stages in the development of opioid dependence.     

Glutamate and the aggression neural circuit in adolescent anabolic steroid-treated Syrian hamsters (Mesocricetus auratus)

Adolescent exposure to anabolic androgenic steroids (AAS) alters the development and activity of the glutamate neural system in the latero-anterior hypothalamus (LAH) in hamsters (Mesocricetus auratus); that is, an important neural component of the adolescent AAS-induced aggressive response. In this article, we used retrograde tracing to investigate glutamate-specific alterations in the connections between the LAH and several other nuclei implicated in adolescent AAS-induced aggression. Briefly, hamsters were treated with AAS or sesame-oil control during adolescence and then microinjected with retrograde tracer into the medial amygdala (MeA), lateral septum (LS), or bed nucleus of the stria terminalis (BNST). Brains were then processed for vesicular glutamate transporter 2 (VGLUT2) and examined for AAS-induced changes in the number VGLUT2 cells containing retrograde tracer (VGLUT2/tracer) within the LAH. It is interesting to note that while aggressive AAS-treated hamsters injected retrograde tracer into the MeA showed a significant reduction in the number of VGLUT2/tracer cells in the LAH, aggressive AAS-treated hamsters injected tracer into the BNST showed a significant increase in the number of VGLUT2/tracer cells in the LAH when compared with controls. Last, AAS hamsters injected with tracer into the LS had a comparable number of LAH-VGLUT2/tracer cells to controls. The current results indicate that glutamate likely functions as the major aggression output system from the LAH and that adolescent AAS treatment significantly alters the neural circuitry modulating aggression. Moreover, increases in the number of glutamate projections from the LAH to the BNST in AAS hamsters identify the BNST as an area particularly important for the regulation of AAS-induced aggression.     

Effects of neonatal oxytocin treatment on aggression and neural activities in mandarin voles

Neonatal manipulation of oxytocin (OT) has long-term effects on behavior and physiology. Here we test the hypothesis that neonatal OT treatment can affect the subsequent expression of intrasexual aggression partly by reprogramming the neural activities of relevant brain regions. To test this hypothesis, mandarin voles (Lasiopodomys mandarinus) received OT or isotonic saline treatment within 24 h of birth. At about 75 days of age, aggressive behaviors and Fos expression in different brain regions were tested. The results indicate that the (1) level of intrasexual aggression was higher and other social contact was lower in SAL-treated sexually na?ve males than in females and; (2) OT-treated females showed a greater increase in aggressive behaviors and Fos expression only after exposure to a male than SAL-treated females, but there were no significant changes in aggressive behaviors in males. These results demonstrate a sexual difference in aggression, and that neonatal exposure to OT may increase aggression in female mandarin voles. These effects may be based on changes in neural activities of relevant brain regions including the bed nucleus of the stria terminalis (BNST), lateral septal nucleus (LS), medial preoptic area (MPOA), the paraventricular nucleus of the hypothalamus (PVN), supraoptic nucleus (SON), mediodorsal thalamic nucleus (MD), ventromedial nucleus of hypothalamic (VMH), the medial amygdala (MeA) and central amygdala (CeA).     

Corticotropin-releasing factor-1 receptors in the basolateral amygdala mediate stress-induced anorexia

Corticotropin-releasing factor (CRF) receptor activation within the basolateral amygdala (BLA) has been relatively unexplored compared with the central nucleus of the amygdala (CeA), despite the fact that CRF receptors are more densely distributed in BLA than in CeA. The authors show that infusion of CRF into BLA, but not CeA, decreases feeding and increases grooming. These effects are mediated by CRF-sub-1 receptors, because they are blocked by intra-BLA treatment with NBI27914 (NBI), a CRF-sub-1 antagonist, but not Astressin 2B, a CRF-sub-2 antagonist. Exposure to a stressor results in behaviors identical to those seen after intra-BLA CRF infusion. These stress-induced changes are prevented by pre-stress treatment with NBI but not Astressin 2B. These data demonstrate that stimulation of intra-BLA CRF-sub-1 receptors is both necessary and sufficient for eliciting stress-induced anorexia and grooming.     

Blocking corticotropin-releasing factor-2 receptors, but not corticotropin-releasing factor-1 receptors or glucocorticoid feedback, disrupts the development of conditioned defeat

Several neuroendocrine signals of the hypothalamic-pituitary-adrenal (HPA) axis are released following exposure to stressful events. It has long been proposed that the signals in this cascade each act to modify ongoing and future behavior. In this study we investigated whether blocking glucocorticoid synthesis, corticotropin-releasing factor (CRF)-1 receptors, or CRF-2 receptors during social defeat would alter subsequent behavioral responses. We used a conditioned defeat model in Syrian hamsters in which social defeat results in a dramatic shift from territorial aggression to increased submissive and defensive behavior in future social encounters. We found that intracerebroventricular administration of anti-sauvagine-30, a CRF-2 receptor antagonist, prior to social defeat training reduced the acquisition of conditioned defeat. In contrast, the acquisition of conditioned defeat was not altered by the CRF-1 receptor antagonist CP-154,526 or the glucocorticoid synthesis inhibitor metyrapone. Our results suggest that CRF, and perhaps related neuropeptides such as urocortins, act at CRF-2 receptors to promote the development of defeat-induced changes in social behavior, whereas signaling at CRF-1 and glucocorticoid receptors plays a negligible role in this process.     

Effects of dominance status on conditioned defeat and expression of 5-HT1A and 5-HT2A receptors

Past experience can alter how individuals respond to stressful events. The brain serotonin system is a key factor modulating stress-related behavior and may contribute to individual variation in coping styles. In this study we investigated whether dominant and subordinate hamsters respond differently to social defeat and whether their behavioral responses are associated with changes in 5-HT1A and 5-HT2A receptor immunoreactivity in several limbic brain regions. We paired weight-matched hamsters in daily aggressive encounters for two weeks so that they formed a stable dominance relationship. We also included controls that were exposed to an empty cage each day for two weeks. Twenty-four hours after the final pairing or empty cage exposure, subjects were socially defeated in 3, 5-min encounters with a more aggressive hamster. Twenty-four hours after social defeat, animals were tested for conditioned defeat in a 5-min social interaction test with a non-aggressive intruder. We collected brains following conditioned defeat testing and performed immunohistochemistry for 5-HT1A and 5-HT2A receptors. We found that dominants showed less submissive and defensive behavior at conditioned defeat testing compared to both subordinates and controls. Additionally, both dominants and subordinates had an increased number of 5-HT1A immunopositive cells in the basolateral amygdala compared to controls. Subordinates also had more 5-HT1A immunopositive cells in the dorsal medial amygdala than did controls. Finally, dominants had fewer 5-HT1A immunopositive cells in the paraventricular nucleus of the hypothalamus compared to controls. Our results indicate that dominant social status results in a blunted conditioned defeat response and a distinct pattern of 5-HT1A receptor expression, which may contribute to resistance to conditioned defeat.     

Separate sets of neurons of the central nucleus of the amygdala project to the substantia innominata and the caudal pontine reticular nucleus in the rat

The central nucleus of the amygdala (CeA) is generally regarded as a control nucleus of subcortical target systems. Due to its widespread projections to different brain areas it is able to modulate emotional behavior of the organism. However, it is still not clear whether single neurons of the CeA project to different areas or to one target area. Injections of the retrograde tracers Fluorogold and True Blue into target regions of the central nucleus of the amygdala, i.e., the substantia innominata (SI) and the caudal pontine reticular nucleus (PNC), revealed overlapping but otherwise distinct neuronal populations within mainly the medial division of the CeA. From our study we conclude that SI and PNC receive input from different subsets of amygdala neurons.     

Effects of prenatal stress on defensive withdrawal behavior and corticotropin releasing factor systems in rat brain

Exposure of pregnant rats to stress results in offspring that exhibit abnormally fearful behavior and have elevated neuroendocrine responses to novelty and aversive stimuli. This study examined the effects of prenatal stress on plasma corticosterone, adrenal weight, defensive withdrawal behavior, and the density of receptors for corticotropin releasing factor (CRF) in the amygdala. Pregnant Sprague-Dawley rats were stressed by daily handling and saline injection (s.c., 0.9%, 0.1 mL) during the last week of gestation. Male offspring were studied at adulthood (60-120 days of age). Adrenal hypertrophy and increased plasma corticosterone were observed in the prenatally stressed offspring. Defensive withdrawal, an ethological measure of the conflict between exploratory behavior and retreat, was quantified in naive offspring, and in offspring exposed to restraint stress (2 h). Restraint stress increased defensive withdrawal in both control and prenatally stressed offspring. Both naive and restraint-stressed prenatally stressed offspring exhibited increased defensive withdrawal compared to control offspring. There was a significant interaction between prenatal stress and restraint stress, suggesting increased vulnerability of prenatally stressed offspring. The effects of restraint in the defensive withdrawal test were reduced by intracerebroventricular administration of the CRF antagonists, alpha-helical CRF9-41 (20 microg every hour) or D-phe(12), Nle(21, 38), C(alpha)-MeLeu(37)]-CRF((12-41)) (5 microg every hour) during the restraint period. The difference between control and prenatally stressed offspring was abolished by the CRF antagonists, suggesting that increased activation of CRF receptors may be a factor in the behavioral abnormalities of prenatally stressed rats. Measurement of CRF receptors in amygdala revealed a 2.5-fold increase in binding in prenatally stressed offspring. In light of previous work from this laboratory demonstrating increased content and release of CRF in amygdala from prenatally stressed offspring, the present study suggests that the increased fearfulness of prenatally stressed rats may be a consequence of increased activity of CRFergic systems in the amygdala.     

Long-term behavioral and neuronal cross-sensitization to amphetamine induced by repeated brief social defeat stress: Fos in the ventral tegmental area and amygdala

Repeated exposure to stress induces cross-sensitization to psychostimulants. The present study assessed functional neural activation during social defeat stress-induced sensitization to a subsequent amphetamine challenge. Social defeat stress was induced in intruder rats during short confrontations with an aggressive resident rat once every third day during the course of 10 days. Rats received d-amphetamine injections (1 mg/kg, i.p.) 17 or 70 days after the first social defeat stress exposure. Amphetamine administration induced a significantly higher frequency of locomotor activity in stressed animals than in handled control rats, which was still evident 2 months after the last social stress exposure. Immunohistochemistry for Fos-like proteins was used to detect activated neural profiles in the striatum, nucleus accumbens (NAc), prefrontal cortex, amygdala, and ventral tegmental area (VTA). Repeated social defeat stress significantly increased Fos-like immunoreactive (Fos-LI) labeling 17 days after the start of stress exposure in the prelimbic and infralimbic cortical regions, NAc shell and core, medial, central and basolateral amygdala, and VTA, which probably represented the expression of chronic Fos-related antigens. Amphetamine augmented stress-induced Fos-LI labeling 17 days after the first stress episode in the dorsal striatum, NAc core, and medial amygdala, reflecting a cross-sensitization of Fos response. Amphetamine challenge 70 days after social stress exposures revealed sensitized Fos-LI labeling in the VTA and the amygdala. These data suggest that episodes of repeated social stress induce a long-lasting neural change that leads to an augmented functional activation in the VTA and amygdala, which might represent a neurobiological substrate for long-lasting cross-sensitization of repeated social defeat stress with psychostimulant drugs.     

Corticotropin-releasing factor projections from limbic forebrain and paraventricular nucleus of the hypothalamus to the region of the ventral tegmental area

Corticotropin-releasing factor (CRF) is a peptide neurotransmitter with high numbers of cell bodies found in limbic regions of the rat brain including the oval nucleus of the bed nucleus of the stria terminalis (BNSTov) and central nucleus of the amygdala (CeA) as well as in the paraventricular nucleus of the hypothalamus (PVN). CRF systems are activated in response to acute stressors and mediate a wide variety of physiological and behavioral responses to acute stress including aversive responses and responses that support appetitive behaviors. CRF is released in the ventral tegmental area (VTA), the cell body region of the mesocorticolimbic dopaminergic neurons, in response to acute stress and plays a role in stress-activation of appetitive behavior [Wang B, Shaham Y, Zitzman D, Azari S, Wise RA, You ZB (2005) Cocaine experience establishes control of midbrain glutamate and dopamine by corticotropin-releasing factor: a role in stress-induced relapse to drug seeking. J Neurosci 25:5389-5396]. However, although it is known that the VTA region contains significant levels of CRF-immunoreactive fibers [Swanson LW, Sawchenko PE, Rivier J, Vale WW (1983) Organization of ovine corticotropin-releasing factor immunoreactive cells and fibers in the rat brain: an immunohistochemical study. Neuroendocrinology 36:165-186], the source of CRF input to the region has not been identified. We used infusions of a fluorescent retrograde tracer, fluorogold, into the VTA region, combined with fluorescent immunocytochemistry for CRF to identify sources of this input. Double-labeled cells were found in BNSTov, CeA and PVN. The percent of fluorogold-labeled cells in each region that were CRF-positive was 30.8, 28.0 and 16.7% respectively. These data point to diffusely distributed sources of CRF-containing fibers in the VTA.     

Subpallial and hypothalamic areas activated following sexual and agonistic encounters in male chickens

Male sexual and agonistic behaviors are controlled by the common social behavior network, involving subpallial and hypothalamic brain areas. In order to understand how this common network generates different behavioral outcomes, induction of FOS protein was used to examine the patterns of neuronal activation in adult male chickens following interaction with a female or a male. Males were subjected to one of the following treatments: handling control, non-contact interaction with a female, contact interaction with a live female, a taxidermy female model or another male. The number of FOS-immunoreactive (FOS-ir) cells, and the area and immunostaining density of individual cells were quantified in the medial preoptic nucleus (POM), medial extended amygdala (nucleus taeniae of the amygdala, TnA, and dorsolateral and ventromedial subdivisions of the medial portion of the bed nucleus of stria terminalis, BSTM1 and BSTM2, respectively), lateral septum (SL), hypothalamic paraventricular nucleus (PVN), bed nucleus of the pallial commissure (NCPa) and ventrolateral thalamic nucleus (VLT). An increase in FOS-ir cells following appetitive sexual behavior was found in BSTM2 and NCPa. Copulation augmented FOS-ir in POM, SL, VLT, and PVN. Intermale interactions increased FOS-ir in all examined brain regions except the TnA and BSTM. Within the SL, copulatory and agonistic behavior activated spatially segregated cell groups. In the PVN, different social behaviors induced significant changes in the distribution of FOS-ir cell sizes suggesting activation of heterogeneous subpopulations of cells. Collectively, behavioral outcomes of male-female and male-male interactions are associated with a combination of common and site-specific patterns of neural activation.     

Repeated social defeat increases reactive emotional coping behavior and alters functional responses in serotonergic neurons in the rat dorsal raphe nucleus

Chronic stress is a vulnerability factor for a number of psychiatric disorders, including anxiety and affective disorders. Social defeat in rats has proven to be a useful paradigm to investigate the neural mechanisms underlying physiologic and behavioral adaptation to acute and chronic stress. Previous studies suggest that serotonergic systems may contribute to the physiologic and behavioral adaptation to chronic stress, including social defeat in rodent models. In order to test the hypothesis that repeated social defeat alters the emotional behavior and the excitability of brainstem serotonergic systems implicated in control of emotional behavior, we exposed adult male rats either to home cage control conditions, acute social defeat, or social defeat followed 24 h later by a second social defeat encounter. We then assessed behavioral responses during social defeat as well as the excitability of serotonergic neurons within the dorsal raphe nucleus using immunohistochemical staining of tryptophan hydroxylase, a marker of serotonergic neurons, and the protein product of the immediate-early gene, c-fos. Repeated social defeat resulted in a shift away from proactive emotional coping behaviors, such as rearing (explorative escape behavior), and toward reactive emotional coping behaviors such as freezing. Both acute and repeated defeat led to widespread increases in c-Fos expression in serotonergic neurons in the dorsal raphe nucleus. Changes in behavior following a second exposure to social defeat, relative to acute defeat, were associated with decreased c-Fos expression in serotonergic neurons within the dorsal and ventral parts of the mid-rostrocaudal dorsal raphe nucleus, regions that have been implicated in 1) serotonergic modulation of fear- and anxiety-related behavior and 2) defensive behavior in conspecific aggressive encounters, respectively. These data support the hypothesis that serotonergic systems play a role in physiologic and behavioral responses to both acute and repeated social defeat.