Search

Vasopressin signaling has important effects on the regulation of social behaviors and stress responses, and is considered a promising pathway to target for new therapeutics of stress-induced psychiatric disorders. Although there is evidence for sex differences in the behavioral effects of arginine vasopressin (AVP), few data have directly compared the effects of stress on endogenous AVP signaling in males and females. We used California mice (Peromyscus californicus) to study the short and long term effects of social defeat stress on AVP immunoreactive cells in the paraventricular nucleus (PVN) and the posteromedial bed nucleus of the stria terminalis (BNSTmp). Acute exposure to defeat increased AVP/c-fos cells in the PVN and SON of both males and females. In contrast, there were sex differences in the long term effects of defeat. Males but not females exposed to defeat had less avp mRNA in the PVN, and in two experiments defeat reduced the number of AVP positive cells in the caudal PVN of males but not females. Interestingly, during relatively benign social encounters with a target mouse, there was a rapid decrease in AVP percent staining (including cell bodies and fibers) in the PVN of males but not females. Defeat reduced AVP percent staining in males, but did not block the socially induced decrease in percent staining. When mice were tested in resident-intruder tests, males exposed to defeat were no less aggressive than control males whereas aggression was abolished in females. However, bouts of aggression were positively correlated with the number of AVP neurons in the BNSTmp of control males but not stressed males, suggesting that different mechanisms mediate aggression in control and stressed males. These data show that while acute AVP responses to defeat are similar in males and females, the long term effects of defeat on AVP are stronger in males.

Alcohol use disorder (AUD) and anxiety disorders are frequently comorbid and share dysregulated neuroimmune-related pathways. Here, we used our established rat model of comorbid post-traumatic stress disorder (PTSD)/AUD to characterize the interleukin 18 (IL-18) system in the central amygdala (CeA). Male and female rats underwent novel (NOV) and familiar (FAM) shock stress, or no stress (unstressed controls; CTL) followed by voluntary alcohol drinking and PTSD-related behaviors, then all received renewed alcohol access prior to the experiments. In situ hybridization revealed that the number of CeA positive cells for Il18 mRNA increased, while for Il18bp decreased in both male and female FAM stressed rats versus CTL. No changes were observed in Il18r1 expression across groups. Ex vivo electrophysiology showed that IL-18 reduced GABAA-mediated miniature inhibitory postsynaptic currents (mIPSCs) frequencies in CTL, suggesting reduced CeA GABA release, regardless of sex. Notably, this presynaptic effect of IL-18 was lost in both NOV and FAM males, while it persisted in NOV and FAM females. IL-18 decreased mIPSC amplitude in CTL female rats, suggesting postsynaptic effects. Overall, our results suggest that stress in rats with alcohol access impacts CeA IL-18-system expression and, in sex-related fashion, IL-18's modulatory function at GABA synapses.

Neuroimmune pathways regulate brain function to influence complex behavior and play a role in several neuropsychiatric diseases, including alcohol use disorder (AUD). In particular, the interleukin-1 (IL-1) system has emerged as a key regulator of the brain's response to ethanol (alcohol). Here we investigated the mechanisms underlying ethanol-induced neuroadaptation of IL-1β signaling at GABAergic synapses in the prelimbic region of the medial prefrontal cortex (mPFC), an area responsible for integrating contextual information to mediate conflicting motivational drives. We exposed C57BL/6J male mice to the chronic intermittent ethanol vapor-2 bottle choice paradigm (CIE-2BC) to induce ethanol dependence, and conducted ex vivo electrophysiology and molecular analyses. We found that the IL-1 system regulates basal mPFC function through its actions at inhibitory synapses on prelimbic layer 2/3 pyramidal neurons. IL-1β can selectively recruit either neuroprotective (PI3K/Akt) or pro-inflammatory (MyD88/p38 MAPK) mechanisms to produce opposing synaptic effects. In ethanol naïve conditions, there was a strong PI3K/Akt bias leading to a disinhibition of pyramidal neurons. Ethanol dependence produced opposite IL-1 effects - enhanced local inhibition via a switch in IL-1β signaling to the canonical pro-inflammatory MyD88 pathway. Ethanol dependence also increased cellular IL-1β in the mPFC, while decreasing expression of downstream effectors (Akt, p38 MAPK). Thus, IL-1β may represent a key neural substrate in ethanol-induced cortical dysfunction. As the IL-1 receptor antagonist (kineret) is already FDA-approved for other diseases, this work underscores the high therapeutic potential of IL-1 signaling/neuroimmune-based treatments for AUD., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Alcohol use disorder (AUD) and affective disorders are frequently comorbid and share underlying mechanisms that could be targets for comprehensive treatment. Post-traumatic stress disorder (PTSD) has high comorbidity with AUD, but comprehensive models of this overlap are nascent. We recently characterized a model of comorbid AUD and PTSD-like symptoms, wherein stressed rats receive an inhibitory avoidance (IA)-related footshock on two occasions followed by two-bottle choice (2BC) voluntary alcohol drinking. Stressed rats received the second footshock in a familiar (FAM, same IA box as the first footshock) or novel context (NOV, single-chambered apparatus); the FAM paradigm more effectively increased alcohol drinking in males and the NOV paradigm in females. During abstinence, stressed males displayed avoidance-like PTSD symptoms, and females showed hyperarousal-like PTSD symptoms. Rats in the model had altered spontaneous action potential-independent GABAergic transmission in the central amygdala (CeA), a brain region key in alcohol dependence and stress-related signaling. However, PTSD sufferers may have alcohol experience prior to their trauma. Here, we therefore modified our AUD/PTSD comorbidity model to provide 3 weeks of intermittent extended alcohol access before footshock and then studied the effects of NOV and FAM stress on drinking and PTSD phenotypes. NOV stress suppressed the escalation of alcohol intake and preference seen in male controls, but no stress effects were seen on drinking in females. Additionally, NOV males had decreased action potential-independent presynaptic GABA release and delayed postsynaptic GABA A receptor kinetics in the CeA compared to control and FAM males. Despite these changes to alcohol intake and CeA GABA signaling, stressed rats showed broadly similar anxiogenic-like behaviors to our previous comorbid model, suggesting decoupling of the PTSD symptoms from the AUD vulnerability for some of these animals. The collective results show the importance of alcohol history and trauma context in vulnerability to comorbid AUD/PTSD-like symptoms., (© 2021 Wiley Periodicals LLC.)

Alcohol use disorder (AUD) and anxiety disorders are frequently comorbid and share mechanisms that could be therapeutic targets. To facilitate mechanistic studies, we adapted an inhibitory avoidance-based "2-hit" rat model of posttraumatic stress disorder (PTSD) and identified predictors and biomarkers of comorbid alcohol (ethanol)/PTSD-like symptoms in these animals. Stressed Wistar rats received a single footshock on two occasions. The first footshock occurred when rats crossed into the dark chamber of a shuttle box. Forty-eight hours later, rats received the second footshock in a familiar (FAM) or novel (NOV) context. Rats then received 4 weeks of two-bottle choice (2BC) ethanol access. During subsequent abstinence, PTSD-like behavior responses, GABAergic synaptic transmission in the central amygdala (CeA), and circulating cytokine levels were measured. FAM and NOV stress more effectively increased 2BC drinking in males and females, respectively. Stressed male rats, especially drinking-vulnerable individuals (≥0.8 g/kg average 2-h ethanol intake with >50% ethanol preference), showed higher fear overgeneralization in novel contexts, increased GABAergic transmission in the CeA, and a profile of increased G-CSF, GM-CSF, IL-13, IL-6, IL-17a, leptin, and IL-4 that discriminated between stress context (NOV > FAM > Control). However, drinking-resilient males showed the highest G-CSF, IL-13, and leptin levels. Stressed females showed increased acoustic startle and decreased sleep maintenance, indicative of hyperarousal, with increased CeA GABAergic transmission in NOV females. This paradigm promotes key features of PTSD, including hyperarousal, fear generalization, avoidance, and sleep disturbance, with comorbid ethanol intake, in a sex-specific fashion that approximates clinical comorbidities better than existing models, and identifies increased CeA GABAergic signaling and a distinct pro-hematopoietic, proinflammatory, and pro-atopic cytokine profile that may aid in treatment., (© 2020. The Author(s), under exclusive licence to Springer Nature Limited.)

Genetically-selected Marchigian Sardinian alcohol-preferring (msP) rats display comorbid symptoms of increased alcohol preference and elevated anxiety-like behavior. Heightened stress sensitivity in msPs is influenced by genetic polymorphisms of the corticotropin-releasing factor receptor in the central nucleus of the amygdala (CeA), as well as reduced influence of anti-stress mechanisms that normally constrain the stress response. Given this propensity for stress dysregulation, in this study, we expand on the possibility that msPs may display differences in neuroendocrine processes that normally terminate the stress response. We utilized behavioral, biochemical, and molecular assays to compare basal and restraint stress-induced changes in the hypothalamic-pituitary-adrenal (HPA) axis of male and female msPs relative to their nonselected Wistar counterparts. The results showed that msPs display deficits in marble-burying behavior influenced by environmental factors and procedures that modulate arousal states in a sex-dependent manner. Whereas male msPs display evidence of dysregulated neuroendocrine function (higher adrenocorticotropic hormone levels and subthreshold reductions in corticosterone), females display restraint-induced elevations in corticosterone levels that were persistently higher in msPs. A dexamethasone challenge reduced the circulation of these stress hormones, although the reduction in corticosterone was generally attenuated in msP versus Wistar rats. Finally, we found evidence of diminished stress-induced glucocorticoid receptor (GR) phosphorylation in the hypothalamic paraventricular nucleus of msPs, as well as innate increases in phosphorylated GR levels in the CeA of male msPs. Collectively, these findings suggest that negative feedback processes regulating HPA responsiveness are diminished in msP rats, possibly underlying differences in the expression of anxiety-like behaviors., (© 2020 Society for the Study of Addiction.)

Background: Microglia, the primary immune cells of the brain, are implicated in alcohol use disorder. However, it is not known if microglial activation contributes to the transition from alcohol use to alcohol use disorder or is a consequence of alcohol intake., Methods: We investigated the role of microglia in a mouse model of alcohol dependence using a colony stimulating factor 1 receptor inhibitor (PLX5622) to deplete microglia and a chronic intermittent ethanol vapor two-bottle choice drinking procedure. Additionally, we examined anxiety-like behavior during withdrawal. We then analyzed synaptic neuroadaptations in the central nucleus of the amygdala (CeA) and gene expression changes in the medial prefrontal cortex and CeA from the same animals used for behavioral studies., Results: PLX5622 prevented escalations in voluntary alcohol intake and decreased anxiety-like behavior associated with alcohol dependence. PLX5622 also reversed expression changes in inflammatory-related genes and glutamatergic and GABAergic (gamma-aminobutyric acidergic) genes in the medial prefrontal cortex and CeA. At the cellular level in these animals, microglia depletion reduced inhibitory GABA A and excitatory glutamate receptor-mediated synaptic transmission in the CeA, supporting the hypothesis that microglia regulate dependence-induced changes in neuronal function., Conclusions: Our multifaceted approach is the first to link microglia to the molecular, cellular, and behavioral changes associated with the development of alcohol dependence, suggesting that microglia may also be critical for the development and progression of alcohol use disorder., (Copyright © 2020 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

The pituitary adenylate cyclase-activating polypeptide (PACAP) system plays a central role in the brain's emotional response to psychological stress by activating cellular processes and circuits associated with threat exposure. The neuropeptide PACAP and its main receptor PAC1 are expressed in the rodent central amygdala (CeA), a brain region critical in negative emotional processing, and CeA PACAPergic signaling drives anxiogenic and stress coping behaviors. Despite this behavioral evidence, PACAP's effects on neuronal activity within the medial subdivision of the CeA (CeM, the major output nucleus for the entire amygdala complex) during basal conditions and after psychological stress remain unknown. Therefore, in the present study, male Wistar rats were subjected to either restraint stress or control conditions, and PACAPergic regulation of CeM cellular function was assessed using immunohistochemistry and whole-cell patch-clamp electrophysiology. Our results demonstrate that PACAP-38 potentiates GABA release in the CeM of naïve rats, via its actions at presynaptic PAC1. Basal PAC1 activity also enhances GABA release in an action potential-dependent manner. Notably, PACAP-38's facilitation of CeM GABA release was attenuated after a single restraint stress session, but after repeated sessions returned to the level observed in naïve animals. A single restraint session also significantly decreased PAC1 levels in the CeM, with repeated restraint sessions producing a slight recovery. Collectively our data reveal that PACAP/PAC1 signaling enhances inhibitory control of the CeM and that psychological stress can modulate this influence to potentially disinhibit downstream effector regions that mediate anxiety and stress-related behaviors. This article is part of the special issue on 'Neuropeptides'., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Glycogenolysis and lactate transport from astrocytes to neurons is required for long-term memory formation, but the role of this lactate is poorly understood. Here we show that the Krebs cycle substrates pyruvate and ketone body B3HB can functionally replace lactate in rescuing memory impairment caused by inhibition of glycogenolysis or expression knockdown of glia monocarboxylate transporters (MCTs) 1 and 4 in the dorsal hippocampus of rats. In contrast, either metabolite is unable to rescue memory impairment produced by expression knockdown of MCT2, which is selectively expressed by neurons, indicating that a critical role of astrocytic lactate is to provide energy for neuronal responses required for long-term memory. These responses include learning-induced mRNA translation in both excitatory and inhibitory neurons, as well as expression of Arc/Arg3.1. Thus, astrocytic lactate acts as an energy substrate to fuel learning-induced de novo neuronal translation critical for long-term memory., Competing Interests: Competing interestsThe authors declare no competing interests.

Oxytocin (OT) is widely known for promoting social interactions, but there is growing appreciation that it can sometimes induce avoidance of social contexts. The social salience hypothesis posed an innovative solution to these apparently opposing actions by proposing that OT enhances the salience of both positive and negative social interactions. The mesolimbic dopamine system was put forth as a likely system to evaluate social salience owing to its well-described role in motivation. Evidence from several sources supports the premise that OT acting within the nucleus accumbens and ventral tegmental area facilitates social reward and approach behavior. However, in aversive social contexts, additional pathways play critical roles in mediating the effects of OT. Recent data indicate that OT acts in the bed nucleus of the stria terminalis to induce avoidance of potentially dangerous social contexts. Here, we review evidence for neural circuits mediating the effects of OT in appetitive and aversive social contexts. Specifically, we propose that distinct but potentially overlapping circuits mediate OT-dependent social approach or social avoidance. We conclude that a broader and more inclusive consideration of neural circuits of social approach and avoidance is needed as the field continues to evaluate the potential of OT-based therapeutics., (Copyright © 2018 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

The interleukin-1 system (IL-1) is a prominent pro-inflammatory pathway responsible for the initiation and regulation of immune responses. Human genetic and preclinical studies suggest a critical role for IL-1β signaling in ethanol drinking and dependence, but little is known about the effects of chronic ethanol on the IL-1 system in addiction-related brain regions such as the central amygdala (CeA). In this study, we generated naïve, non-dependent (Non-Dep) and dependent (Dep) male mice using a paradigm of chronic-intermittent ethanol vapor exposure interspersed with two-bottle choice to examine 1) the expression of IL-1β, 2) the role of the IL-1 system on GABAergic transmission, and 3) the potential interaction with the acute effects of ethanol in the CeA. Immunohistochemistry with confocal microscopy was used to assess expression of IL-1β in microglia and neurons in the CeA, and whole-cell patch clamp recordings were obtained from CeA neurons to measure the effects of IL-1β (50 ng/ml) or the endogenous IL-1 receptor antagonist (IL-1ra; 100 ng/ml) on action potential-dependent spontaneous inhibitory postsynaptic currents (sIPSCs). Overall, we found that IL-1β expression is significantly increased in microglia and neurons of Dep compared to Non-Dep and naïve mice, IL-1β and IL-1ra bi-directionally modulate GABA transmission through both pre- and postsynaptic mechanisms in all three groups, and IL-1β and IL-1ra do not alter the facilitation of GABA release induced by acute ethanol. These data suggest that while ethanol dependence induces a neuroimmune response in the CeA, as indicated by increased IL-1β expression, this does not significantly alter the neuromodulatory role of IL-1β on synaptic transmission., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Aims: Stress induces neuroimmune responses via Toll-like receptor 4 (TLR4) activation. Here, we investigated the role of TLR4 in the effects of the stress peptide corticotropin-releasing factor (CRF) on GABAergic transmission in the central nucleus of the amygdala (CeA) following restraint stress., Methods: Tlr4 knock out (KO) and wild-type rats were exposed to no stress (naïve), a single restraint stress (1 h) or repeated restraint stress (1 h per day for 3 consecutive days). After 1 h recovery from the final stress session, whole-cell patch-clamp electrophysiology was used to investigate the effects of CRF (200 nM) on CeA GABAA-mediated spontaneous inhibitory postsynaptic currents (sIPSCs)., Results: TLR4 does not regulate baseline GABAergic transmission in the CeA of naive and stress-treated animals. However, CRF significantly increased the mean sIPSC frequencies (indicating enhanced GABA release) across all genotypes and stress treatments, except for the Tlr4 KO rats that experienced repeated restraint stress., Conclusions: Overall, our results suggest a limited role for TLR4 in CRF's modulation of CeA GABAergic synapses in naïve and single stress rats, though TLR4-deficient rats that experienced repeated psychological stress exhibit a blunted CRF cellular response., Short Summary: TLR4 has a limited role in CRF's activation of the CeA under basal conditions, but interacts with the CRF system to regulate GABAergic synapse function in animals that experience repeated psychological stress.

Repeated cycles of alcohol [ethanol (EtOH)] intoxication and withdrawal dysregulate excitatory glutamatergic systems in the brain and induce neuroadaptations in the medial prefrontal cortex (mPFC) that contribute to cognitive dysfunction. The mPFC is composed of subdivisions that are functionally distinct, with dorsal regions facilitating drug-cue associations and ventral regions modulating new learning in the absence of drug. A key modulator of glutamatergic activity is the holoenzyme calcium/calmodulin-dependent protein kinase II (CaMKII) that phosphorylates ionotropic glutamate receptors. Here, we examined the hypothesis that abstinence from chronic intermittent EtOH (CIE) exposure dysregulates CaMKII activity in the mPFC to impair cognitive flexibility. We used an operant model of strategy set shifting in male Long-Evans rats demonstrating reduced susceptibility to trial omissions during performance in a visual cue-guided task versus albino strains. Relative to naïve controls, rats experiencing approximately 10 days of abstinence from CIE vapor exposure demonstrated impaired performance during a procedural shift from visual cue to spatial location discrimination. Phosphorylation of CaMKII subtype α was upregulated in the dorsal, but not ventral mPFC of CIE-exposed rats, and was positively correlated with perseverative-like responding during the set shift. The findings suggest that abstinence from CIE exposure induces an undercurrent of kinase activity (e.g. CaMKII), which may promote aberrant glutamatergic responses in select regions of the mPFC. Given the role of the mPFC in modulating executive control of behavior, we propose that increased CaMKII subtype α activity reflects a dysregulated 'top-down' circuit that interferes with adaptive behavioral performance under changing environmental demands., (© 2017 Society for the Study of Addiction.)

Background: The neuropeptide oxytocin (OT) is a key regulator of social and emotional behaviors. The effects of OT are context dependent, and it has been proposed that OT increases the salience of both positive and negative social cues. Here we tested whether the bed nucleus of the stria terminalis (BNST) mediates anxiogenic effects of OT., Methods: First, we studied the effects of systemic administration of an OT receptor (OTR) antagonist L-368,899 on social behavior in male and female California mice exposed to social defeat. We examined the effect of L-368,899 on G protein activation and used early growth response factor 1 immunohistochemistry to identify potential sites of OTR action. Finally, we examined the effects of L-368,899 infused in the BNST on behavior., Results: A single dose of systemic L-368,899 increased social approach in stressed female mice and decreased social approach in male mice naïve to defeat. L-368,899 prevented OT activation of G proteins and did not activate G proteins in the absence of OT. Intranasal OT, which reduces social approach in female mice but not male mice, increased early growth response factor 1 immunoreactivity in the nucleus accumbens core and anteromedial BNST in female mice but not in male mice. Stressed female mice that received an infusion of L-368,899 into the anteromedial BNST but not the nucleus accumbens core increased social approach and decreased social vigilance responses., Conclusions: Our results suggest that OTR activation in anteromedial BNST induces a vigilance response in which individuals avoid, yet attend to, unfamiliar social contexts. Our results suggest that OTR antagonists may have unappreciated therapeutic potential for stress-induced psychiatric disorders., (Copyright © 2017 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Women are nearly twice as likely as men to be diagnosed with major depressive disorder, yet the use of female animal models in studying the biological basis of depression lags behind that of males. The social defeat model uses social stress to generate depression-like symptoms in order to study the neurobiological mechanisms. In general, social defeat is difficult to apply in female rodents. However, male and female California mice (Peromyscus californicus) are territorial. This allows defeat to be studied in both sexes. Males exposed to defeat tend to exhibit proactive coping mechanisms and demonstrate aggression and reduced cognitive flexibility. Females exposed to defeat engage more in reactive coping mechanisms which is highlighted by social avoidance and low aggression. Importantly, effects of defeat on social interaction behavior in females is independent of adult gonadal steroids. These behavioral phenotypes are associated with sex-specific changes in arginine vasopressin (AVP) and oxytocin (OT), closely related peptides that regulate social behavior and stress reactivity. In brain regions associated with stress responses and social behavior, defeat induced long term decreases in AVP activity and increases in OT activity in males and females respectively. Intranasal OT administration was shown to mimic the effects of defeat-induced increases in endogenous OT activity, causing social withdrawal in undefeated females. This suggests that inhibition of OT activity could reduce the impact of stress on behavior in females. These results highlight the value of maintaining diverse rodent models in the search for sex-specific pharmacological approaches to treating mood disorders., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Vasopressin V1a receptors (V1aR) are thought to contribute to the pathophysiology of psychiatric disorders such as anxiety and depression, sparking interest in V1aR as a therapeutic target. Although the global effects of V1aR have been documented, less is known about the specific neural circuits mediating these effects. Moreover, few studies have examined context-specific V1aR function in both males and females. By using the California mouse, we first studied the effects of sex and social defeat stress on V1aR binding in the forebrain. In females but not males, V1aR binding in the bed nucleus of the stria terminalis (BNST) was negatively correlated to social interaction behavior. In females stress also increased V1aR binding in the nucleus accumbens (NAc). Infusions of V1aR antagonist in to the medioventral BNST (BNSTmv) had anxiogenic effects only in animals naïve to defeat. For males, inhibition of V1aR in BNSTmv had anxiogenic effects in social and nonsocial contexts, but for females, anxiogenic effects were limited to social contexts. In stressed females, inhibition of V1aR in the NAc shell had no effect on social interaction behavior, but had an anxiogenic effect in an open field test. These data suggest that V1aR in BNSTmv have anxiolytic and prosocial effects in males, and that in females, prosocial and anxiolytic effects of V1aR appear to be mediated independently by receptors in the BNSTmv and NAc shell, respectively. These findings suggest that males have more overlap in neural circuits modulating anxiety in social and nonsocial contexts than females., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Background: Oxytocin (OT) is considered to be a stress-buffering hormone, dampening the physiologic effects of stress. However, OT can also be anxiogenic. We examined acute and long-lasting effects of social defeat on OT neurons in male and female California mice., Methods: We used immunohistochemistry for OT and c-fos cells to examine OT neuron activity immediately after defeat (n = 6-9) and 2 weeks (n = 6-9) and 10 weeks (n = 4-5) later. We quantified Oxt messenger RNA with quantitative polymerase chain reaction (n = 5-9). Intranasal OT was administered to naïve and stressed mice tested in social interaction and resident-intruder tests (n = 8-14)., Results: Acute exposure to a third episode of defeat increased OT/c-fos colocalizations in the paraventricular nucleus of both sexes. In the medioventral bed nucleus of the stria terminalis, defeat increased Oxt messenger RNA, total OT neurons, and OT/c-fos colocalizations in female mice but not male mice. Intranasal OT failed to reverse stress-induced social withdrawal in female mice and reduced social interaction behavior in female mice naïve to defeat. In contrast, intranasal OT increased social interaction in stressed male mice and reduced freezing in the resident-intruder test., Conclusions: Social defeat induces long-lasting increases in OT production and OT/c-fos cells in the medioventral bed nucleus of the stria terminalis of female mice but not male mice. Intranasal OT largely reversed the effects of stress on behavior in male mice, but effects were mixed in female mice. These results suggest that changes in OT-sensitive networks contribute to sex differences in behavioral responses to stress., (Copyright © 2015 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.)

Emotionally relevant experiences form strong and long-lasting memories by critically engaging the stress hormone/neurotransmitter noradrenaline, which mediates and modulates the consolidation of these memories. Noradrenaline acts through adrenergic receptors (ARs), of which β2-adrenergic receptors (βARs) are of particular importance. The differential anatomical and cellular distribution of βAR subtypes in the brain suggests that they play distinct roles in memory processing, although much about their specific contributions and mechanisms of action remains to be understood. Here we show that astrocytic rather than neuronal β2ARs in the hippocampus play a key role in the consolidation of a fear-based contextual memory. These hippocampal β2ARs, but not β1ARs, are coupled to the training-dependent release of lactate from astrocytes, which is necessary for long-term memory formation and for underlying molecular changes. This key metabolic role of astrocytic β2ARs may represent a novel target mechanism for stress-related psychopathologies and neurodegeneration.

Long-term memory formation, the ability to retain information over time about an experience, is a complex function that affects multiple behaviors, and is an integral part of an individual's identity. In the last 50 years many scientists have focused their work on understanding the biological mechanisms underlying memory formation and processing. Molecular studies over the last three decades have mostly investigated, or given attention to, neuronal mechanisms. However, the brain is composed of different cell types that, by concerted actions, cooperate to mediate brain functions. Here, we consider some new insights that emerged from recent studies implicating astrocytic glycogen and glucose metabolisms, and particularly their coupling to neuronal functions via lactate, as an essential mechanism for long-term memory formation.

Dopamine neurons in the ventral tegmental area (VTA) have important functions related to rewards but are also activated in aversive contexts. Electrophysiology studies suggest that the degree to which VTA dopamine neurons respond to noxious stimuli is topographically organized across the dorsal-ventral extent. We used c-fos immunohistochemistry to examine the responses of VTA dopamine neurons in contexts of social defeat and social approach. Studying monogamous California mice (Peromyscus californicus) allowed us to observe the effects of social defeat on both males and females. Females exposed to three episodes of defeat, but not a single episode, had more tyrosine hydroxylase (TH)/c-fos-positive cells in the ventral (but not dorsal) VTA compared with controls. This observation suggests that repeated exposure to aversive contexts is necessary to trigger activation of VTA dopamine neurons. Defeat did not affect TH/c-fos colocalizations in males. We also examined the long-term effects of defeat on c-fos expression in a social interaction test. As previously reported, defeat reduced social interaction in females but not males. Surprisingly, there were no effects of defeat stress on TH/c-fos colocalizations in any subregion of the VTA. However, females had more TH/c-fos-positive cells than males across the entire VTA, and also had greater c-fos-positive cell counts in posterior subregions of the nucleus accumbens shell. Our results show that dopamine neurons in the VTA are more responsive to social contexts in females and that the ventral VTA in particular is sensitive to aversive contexts., (© 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Behavioral flexibility is a component of executive functioning that allows individuals to adapt to changing environmental conditions. Independent lines of research indicate that the mu opioid receptor (MOR) is an important mediator of behavioral flexibility and responses to psychosocial stress. The current study bridges these two lines of research and tests the extent to which social defeat and MOR affect behavioral flexibility and whether sex moderates these effects in California mice (Peromyscus californicus). Males and females assigned to social defeat or control conditions were tested in a Barnes maze. In males, defeat impaired behavioral flexibility but not acquisition. Female performance was unaffected by defeat. MOR binding in defeated and control mice in the orbitofrontal cortex (OFC), striatum and hippocampus was examined via autoradiography. Stressed males had reduced MOR binding in the OFC whereas females were unaffected. The MOR antagonist beta-funaltrexamine (1 mg/kg) impaired performance in males naïve to defeat during the reversal phase but had no effect on females. Finally, we examined the effects of the MOR agonist morphine (2.5 and 5 mg/kg) on stressed mice. As expected, morphine improved behavioral flexibility in stressed males. The stress-induced deficits in behavioral flexibility in males are consistent with a proactive coping strategy, including previous observations that stressed male California mice exhibit strong social approach and aggression. Our pharmacological data suggest that a down-regulation of MOR signaling in males may contribute to sex differences in behavioral flexibility following stress. This is discussed in the framework of coping strategies for individuals with mood disorders., (© 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Vasopressin signaling has important effects on the regulation of social behaviors and stress responses, and is considered a promising pathway to target for new therapeutics of stress-induced psychiatric disorders. Although there is evidence for sex differences in the behavioral effects of arginine vasopressin (AVP), few data have directly compared the effects of stress on endogenous AVP signaling in males and females. We used California mice (Peromyscus californicus) to study the short and long term effects of social defeat stress on AVP immunoreactive cells in the paraventricular nucleus (PVN) and the posteromedial bed nucleus of the stria terminalis (BNSTmp). Acute exposure to defeat increased AVP/c-fos cells in the PVN and SON of both males and females. In contrast, there were sex differences in the long term effects of defeat. Males but not females exposed to defeat had less avp mRNA in the PVN, and in two experiments defeat reduced the number of AVP positive cells in the caudal PVN of males but not females. Interestingly, during relatively benign social encounters with a target mouse, there was a rapid decrease in AVP percent staining (including cell bodies and fibers) in the PVN of males but not females. Defeat reduced AVP percent staining in males, but did not block the socially induced decrease in percent staining. When mice were tested in resident-intruder tests, males exposed to defeat were no less aggressive than control males whereas aggression was abolished in females. However, bouts of aggression were positively correlated with the number of AVP neurons in the BNSTmp of control males but not stressed males, suggesting that different mechanisms mediate aggression in control and stressed males. These data show that while acute AVP responses to defeat are similar in males and females, the long term effects of defeat on AVP are stronger in males., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Glia regulate the hypothalamic-pituitary-gonadal (HPG) axis in birds and mammals. This is accomplished mechanically by ensheathing gonadotrophin-releasing hormone I (GnRH) nerve terminals thereby blocking access to the pituitary blood supply, or chemically in a paracrine manner. Such regulation requires appropriate spatial associations between glia and nerve terminals. Female turkeys (Meleagris gallopavo) use day length as a primary breeding cue. Long days activate the HPG-axis until the hen enters a photorefractory state when previously stimulatory day lengths no longer support HPG-axis activity. Hens must then be exposed to short days before reactivation of the reproductive axis occurs. As adult hens have discrete inactive reproductive states in addition to a fertile state, they are useful for examining the glial contribution to reproductive function. We immunostained tuberal hypothalami from short and long-day photosensitive hens, plus long-day photorefractory hens to examine expression of two intermediate filaments that affect glial morphology: glial fibrillary acidic protein (GFAP) and vimentin. GFAP expression was drastically reduced in the central median eminence of long day photosensitive hens, especially within the internal zone. Vimentin expression was similar among groups. However, vimentin-immunoreactive fibers abutting the portal vasculature were significantly negatively correlated with GFAP expression in the median eminence, which is consistent with our hypothesis for a reciprocal relationship between GFAP and vimentin expression. It appears that up-regulation of GFAP expression in the central median eminence of turkey hens is associated with periods of reproductive quiescence and that photofractoriness is associated with the lack of a glial cytoskeletal response to long days., (Copyright © 2013 Elsevier Inc. All rights reserved.)

In several vertebrate species, the effects of estrogens on male aggressive behavior can be modulated by environmental cues. In song sparrows and rodents, estrogens modulate aggression in the nonbreeding season or winter-like short days, respectively. The behavioral effects of estrogens are rapid, which generally is considered indicative of nongenomic processes. The current study further examined the hypothesis that estradiol acts nongenomically under short days by utilizing a protein synthesis inhibitor, cycloheximide (CX). Mice were housed in either short or long day photoperiods, and treated with an aromatase inhibitor. One hour before resident-intruder testing mice were injected with either CX or saline vehicle, and 30 min later were treated orally with either cyclodextrin conjugated estradiol or vehicle. Under short days, mice treated with estradiol showed a rapid decrease in aggressive behavior, independent of CX administration. CX alone had no effect on aggression. These results show that protein synthesis is not required for the rapid effects of estradiol on aggression, strongly suggesting that these effects are mediated by nongenomic processes. We also showed that estradiol suppressed c-fos immunoreactivity in the caudal bed nucleus of the stria terminalis under short days. No effects of estradiol on behavior or c-fos expression were observed in mice housed under long days. Previously we had also demonstrated that cage bedding influenced the directional effects of estrogens on aggression. Here, we show that the phenomenon of rapid action of estradiol on aggression under short days is a robust result that generalizes to different bedding conditions., (Copyright © 2013 Elsevier Inc. All rights reserved.)

One of the goals in neuroscience is to obtain tractable laboratory cultures that closely recapitulate in vivo systems while still providing ease of use in the lab. Because neurons can exist in the body over a lifetime, long-term culture systems are necessary so as to closely mimic the physiological conditions under laboratory culture conditions. Ideally, such a neuronal organoid culture would contain multiple cell types, be highly differentiated, and have a high density of interconnected cells. However, before these types of cultures can be created, certain problems associated with long-term neuronal culturing must be addressed. We sought to develop a new protocol which may further prolong the duration and integrity of E18 rat hippocampal cultures. We have developed a protocol that allows for culturing of E18 hippocampal neurons at high densities for more than 120 days. These cultured hippocampal neurons are (i) well differentiated with high numbers of synapses, (ii) anchored securely to their substrate, (iii) have high levels of functional connectivity, and (iv) form dense multi-layered cellular networks. We propose that our culture methodology is likely to be effective for multiple neuronal subtypes-particularly those that can be grown in Neurobasal/B27 media. This methodology presents new avenues for long-term functional studies in neurons.

There is compelling evidence for important sex differences in behavioral and hormonal responses to psychosocial stress. Here we examined the effects of gonadal hormones on behavioral responses to social defeat stress in monogamous California mice (Peromyscus californicus). Three episodes of social defeat induced social withdrawal in intact females but not males. Gonadectomy blocked corticosterone responses to defeat in females and sensitized male corticosterone responses. However, gonadectomy had no effects on social interaction behavior, suggesting that social withdrawal is not dependent on gonadal hormones in the adult California mouse. In contrast, defeat reduced exploratory behavior in the open field test for intact but not castrated males. We also examined the effects of social defeat on social interaction behavior when California mice were raised on corncob bedding, which has estrogenic properties. In this dataset of over 300 mice, we observed that social defeat did not induce social withdrawal when females were raised on corncob bedding. This finding suggests that the use of corncob in rodent studies could mask important sex differences in the effects of stress on brain and behavior. Although gonadal hormones do not affect social withdrawal behavior in adults, our data suggest that hormones may act earlier in development to induce a more resilient social phenotype., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Many temperate-zone animals use changes in photoperiod to time breeding. Shorter term cues, like food availability, are integrated with photoperiod to adjust reproductive timing under unexpected conditions. Many mice of the genus Peromyscus breed in the summer. California mice (Peromyscus californicus), however, can breed year round, but tend to begin breeding in the winter. Glial cells may be involved in transduction of environmental signals that regulate gonadotrophin releasing hormone I (GnRH) activity. We examined the effects of diet and photoperiod on reproduction in female California mice. Mice placed on either short days (8L:16D) or long days (16L:8D) were food restricted (80% of normal intake) or fed ad libitum. Short day-food restricted mice showed significant regression of the reproductive system. GnRH-immunoreactivity was increased in the tuberal hypothalamus of long day-food restricted mice. This may be associated with the sparing effect long days have when mice are food restricted. The number of GFAP-immunoreactive fibers in proximity to GnRH nerve terminals correlated negatively with uterine size in ad libitum but not food restricted mice, suggesting diet may alter glial regulation of the reproductive axis. There was a trend towards food restriction increasing uterine expression of c-fos mRNA, an estrogen dependent gene. Similar to other seasonally breeding rodents, short days render the reproductive system of female California mice more susceptible to effects of food restriction. This may be vestigial, or it may have evolved to mitigate consequences of unexpectedly poor winter food supplies., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Food restriction has been reported to have positive effects on cognition. This study examines how another environmental factor, daylength, can alter the impact of food restriction on the brain and behavior. Female California mice (Peromyscus californicus), housed on either long days (16 h of light and 8 h of darkness) or short days (8 h of light and 16 h of darkness), were restricted to 80% of their normal baseline food intake or provided with food ad libitum. Testing in a Barnes maze revealed that the effects of food restriction depended on photoperiod, and that these effects differed for acquisition vs. reversal learning. During acquisition testing, food restriction increased latency to finding the target hole in short-day mice but not in long-day mice. In reversal testing, food restriction decreased latency to finding the target hole in long-day mice but not in short-day mice. Latency to finding the hole was positively and independently correlated with both errors and time spent freezing, suggesting that changes in both spatial learning and anxiety-like behavior contributed to performance. Short days increased hippocampal expression of the synaptic protein, synapsin I, which was reversed by food restriction. Short days also reduced plasma corticosterone levels, but diet had no effect. There was no effect of diet or photoperiod on hippocampal expression of the glial marker, glial fibrillary acidic protein. The present findings suggest that, in female California mice, the differential effects of food restriction on acquisition and reversal learning are photoperiod-dependent. These results justify further testing of the relationship between food restriction and hippocampal synapsin I in the context of spatial learning., (© 2010 The Authors. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

The role of thyroid hormones in the expression of photosensitivity-photorefractoriness in female turkeys was investigated through the use of an antithyroidal agent, 6-n-propyl-2-thiouracil (PTU). In experiment 1, females held continuously from hatch on long day lengths (16L:8D; LD) and fed 0.1% PTU from 0 to 16 wk, began laying eggs at 26 wk of age, peaking at 75% hen-day egg production by 29 wk, whereas controls initiated lay 3 wk earlier but only achieved less than 50% hen-day egg production. In experiment 2, PTU treatment from 10 to 18 wk severely suppressed plasma triiodothyronine and thyroxine, as confirmed by RIA. Egg production of PTU and control hens held on LD from hatch began by 23 wk, with PTU hens reaching a substantially greater rate of lay than controls. Eggs were smaller initially in both treatments but exceeded 75 g by 28 wk. In experiment 3, recycled hens on short day lengths (8L:16D) received PTU for 2 wk before LD and 12 wk thereafter; a subset of these hens was killed after 48 h of LD for immunohistochemical analysis of fos-related antigen (FRA) expression in the tuberal hypothalamus as a marker of photoinduced neuronal activity. The PTU treatment completely forestalled egg production until its withdrawal; egg production then rose sharply to control levels before resuming, along with controls, a typical seasonal decline. The PTU treatment did not impair photoinduced FRA expression. Together, these results demonstrate the following: 1) that a period of pharmacological suppression of triiodothyronine and thyroxine can substitute for short day exposure in conferring photosensitivity on juvenile-aged turkeys (and is actually superior to short day exposure), 2) that reproductive development does not limit egg production of turkey hens photostimulated as young as approximately 20 wk of age, and 3) that effects of thyroid suppression on photostimulation lie downstream of photoinduced FRA expression. Taken together, these results suggest that there is ample physiological potential to substantially advance the age of photoinduced egg production in commercial flocks.

For many temperate-zone avian species, termination of breeding occurs when individuals no longer respond to previously stimulatory day lengths, a condition called photorefractoriness. Long day lengths induce significantly greater expression of c-fos and fos-related antigens (FRAs) in the tuberal hypothalamus of the photosensitive hen than that of the photorefractory hen. The tuber is also a site of photoinducible glial expression of type 2 iodothyronine deiodinase (Dio2), which converts thyroxine into its active form, triiodothyronine (T3). T3 induces withdrawal of glial processes from gonadotrophin-releasing hormone (GnRH) I nerve terminals, which is believed to permit the efficient release of GnRH I into the associated portal vasculature. Using a riboprobe, we tested whether long days induce Dio2 mRNA expression in the turkey tuber and, if so, whether this expression is reduced in photorefractory hens. Long days significantly induced rostral and caudal tuberal hypothalamic Dio2 expression in photosensitive hens. Photorefractory hens had reduced expression of Dio2 with most subjects expressing no detectable mRNA in the rostral tuber and variably attenuated amounts throughout the medial and caudal tuber. We also performed double-label immunohistochemistry to identify co-localisation between FRAs and glial fibrillary acidic protein, a glial marker. FRAs were present in the nuclei of a few astrocytes in the median eminence and infundibular nucleus of the tuber. The temporal and spatial coincidence between FRA and Dio2 expression, their mutual association with glia, and the attenuation of their response during photorefractoriness suggests that the two events are linked and that photorefractoriness involves a reduced capacity for photo-inducible gene expression within glia of the tuberal hypothalamus.