Investigating role of ASIC2 in synaptic and behavioral responses to drugs of abuse.

Drugs of abuse produce rearrangements at glutamatergic synapses thought to contribute to drug-reinforced behaviors. Acid-Sensing Ion Channels (ASICs) have been suggested to oppose these effects, largely due to observations in mice lacking the ASIC1A subunit. However, the ASIC2A and ASIC2B subunits are known to interact with ASIC1A, and their potential roles in drugs of abuse have not yet been investigated. Therefore, we tested the effects of disrupting ASIC2 subunits in mice exposed to drugs of abuse. We found conditioned place preference (CPP) to both cocaine and morphine were increased in Asic2 -/- mice, which is similar to what was observed in Asic1a -/- mice. Because nucleus accumbens core (NAcc) is an important site of ASIC1A action, we examined expression of ASIC2 subunits there. By western blot ASIC2A was readily detected in wild-type mice, while ASIC2B was not, suggesting ASIC2A is the predominant subunit in nucleus accumbens core. An adeno-associated virus vector (AAV) was used to drive recombinant ASIC2A expression in nucleus accumbens core of Asic2 -/- mice, resulting in near normal protein levels. Moreover, recombinant ASIC2A integrated with endogenous ASIC1A subunits to form functional channels in medium spiny neurons (MSNs). However, unlike ASIC1A, region-restricted restoration of ASIC2A in nucleus accumbens core was not sufficient to affect cocaine or morphine conditioned place preference, suggesting effects of ASIC2 differ from those of ASIC1A. Supporting this contrast, we found that AMPA receptor subunit composition and the ratio of AMPA receptor-mediated current to NMDA receptor-mediated current (AMPAR/NMDAR) were normal in Asic2 -/- mice and responded to cocaine withdrawal similarly to wild-type animals. However, disruption of ASIC2 significantly altered dendritic spine morphology, and these effects differed from those reported previously in mice lacking ASIC1A. We conclude that ASIC2 plays an important role in drug-reinforced behavior, and that its mechanisms of action may differ from ASIC1A.

Preclinical Models of Chronic Stress: Adaptation or Pathology?

The experience of prolonged stress changes how individuals interact with their environment and process interoceptive cues, with the end goal of optimizing survival and well-being in the face of a now-hostile world. The chronic stress response includes numerous changes consistent with limiting further damage to the organism, including development of passive or active behavioral strategies and metabolic adjustments to alter energy mobilization. These changes are consistent with symptoms of pathology in humans, and as a result, chronic stress has been used as a translational model for diseases such as depression. While it is of heuristic value to understand symptoms of pathology, we argue that the chronic stress response represents a defense mechanism that is, at its core, adaptive in nature. Transition to pathology occurs only after the adaptive capacity of an organism is exhausted. We offer this perspective as a means of framing interpretations of chronic stress studies in animal models and how these data relate to adaptation as opposed to pathology.

A Unique Role for Protocadherin γC3 in Promoting Dendrite Arborization through an Axin1-Dependent Mechanism.

The establishment of a functional cerebral cortex depends on the proper execution of multiple developmental steps, culminating in dendritic and axonal outgrowth and the formation and maturation of synaptic connections. Dysregulation of these processes can result in improper neuronal connectivity, including that associated with various neurodevelopmental disorders. The γ-Protocadherins (γ-Pcdhs), a family of 22 distinct cell adhesion molecules that share a C-terminal cytoplasmic domain, are involved in multiple aspects of neurodevelopment including neuronal survival, dendrite arborization, and synapse development. The extent to which individual γ-Pcdh family members play unique versus common roles remains unclear. We demonstrated previously that the γ-Pcdh-C3 isoform (γC3), via its unique "variable" cytoplasmic domain (VCD), interacts in cultured cells with Axin1, a Wnt-pathway scaffold protein that regulates the differentiation and morphology of neurons. Here, we confirm that γC3 and Axin1 interact in the cortex in vivo and show that both male and female mice specifically lacking γC3 exhibit disrupted Axin1 localization to synaptic fractions, without obvious changes in dendritic spine density or morphology. However, both male and female γC3 knock-out mice exhibit severely decreased dendritic complexity of cortical pyramidal neurons that is not observed in mouse lines lacking several other γ-Pcdh isoforms. Combining knock-out with rescue constructs in cultured cortical neurons pooled from both male and female mice, we show that γC3 promotes dendritic arborization through an Axin1-dependent mechanism mediated through its VCD. Together, these data identify a novel mechanism through which γC3 uniquely regulates the formation of cortical circuitry.SIGNIFICANCE STATEMENT The complexity of a neuron's dendritic arbor is critical for its function. We showed previously that the γ-Protocadherin (γ-Pcdh) family of 22 cell adhesion molecules promotes arborization during development; it remained unclear whether individual family members played unique roles. Here, we show that one γ-Pcdh isoform, γC3, interacts in the brain with Axin1, a scaffolding protein known to influence dendrite development. A CRISPR/Cas9-generated mutant mouse line lacking γC3 (but not lines lacking other γ-Pcdhs) exhibits severely reduced dendritic complexity of cerebral cortex neurons. Using cultured γC3 knock-out neurons and a variety of rescue constructs, we confirm that the γC3 cytoplasmic domain promotes arborization through an Axin1-dependent mechanism. Thus, γ-Pcdh isoforms are not interchangeable, but rather can play unique neurodevelopmental roles.

Activity in a prefrontal-periaqueductal gray circuit overcomes behavioral and endocrine features of the passive coping stress response.

The question of how the brain links behavioral and biological features of defensive responses has remained elusive. The importance of this problem is underscored by the observation that behavioral passivity in stress coping is associated with elevations in glucocorticoid hormones, and each may carry risks for susceptibility to a host of stress-related diseases. Past work implicates the medial prefrontal cortex (mPFC) in the top-down regulation of stress-related behaviors; however, it is unknown whether such changes have the capacity to buffer against the longer-lasting biological consequences associated with aversive experiences. Using the shock probe defensive burying test in rats to naturalistically measure behavioral and endocrine features of coping, we observed that the active behavioral component of stress coping is associated with increases in activity along a circuit involving the caudal mPFC and midbrain dorsolateral periaqueductal gray (PAG). Optogenetic manipulations of the caudal mPFC-to-dorsolateral PAG pathway bidirectionally modulated active (escape and defensive burying) behaviors, distinct from a rostral mPFC-ventrolateral PAG circuit that instead limited passive (immobility) behavior. Strikingly, under conditions that biased rats toward a passive coping response set, including exaggerated stress hormonal output and increased immobility, excitation of the caudal mPFC-dorsolateral PAG projection significantly attenuated each of these features. These results lend insight into how the brain coordinates response features to overcome passive coping and may be of importance for understanding how activated neural systems promote stress resilience.

Environmental certainty influences the neural systems regulating responses to threat and stress.

Flexible calibration of threat responding in accordance with the environment is an adaptive process that allows an animal to avoid harm while also maintaining engagement of other goal-directed actions. This calibration process, referred to as threat response regulation, requires an animal to calculate the probability that a given encounter will result in a threat so they can respond accordingly. Here we review the neural correlates of two highly studied forms of threat response suppression: extinction and safety conditioning. We focus on how relative levels of certainty or uncertainty in the surrounding environment alter the acquisition and application of these processes. We also discuss evidence indicating altered threat response regulation following stress exposure, including enhanced fear conditioning, and disrupted extinction and safety conditioning. To conclude, we discuss research using an animal model of coping that examines the impact of stressor controllability on threat responding, highlighting the potential for previous experiences with control, or other forms of coping, to protect against the effects of future adversity.

Comparison of murine behavioural and physiological responses after forced exercise by electrical shock versus manual prodding.

NEW FINDINGS: What is the central question of this study? Forced treadmill exercise using electrical shock is the most common technique in rodent exercise studies. Here, we examined how the use of electrical shock during forced treadmill exercise affects behavioural and physiological responses in comparison to a novel non-electrical shock technique. What is the main finding and its importance? In comparison to mice that underwent traditional treadmill running induced by electrical shock, mice that underwent forced running using a novel technique involving gentle prodding to induce running showed: (i) higher locomotor activity; (ii) less anxiety-like behaviour; and (iii) altered exercise-induced muscle pain immediately after exercise. ABSTRACT: Animal models of exercise have been useful to understand underlying cellular and molecular mechanisms. Many studies have used methods of exercise that are unduly stressful (e.g., electrical shock to force running), potentially skewing results. Here, we compared physiological and behavioural responses of mice after exercise induced using a prodding technique that avoids electrical shock versus a traditional protocol using electrical shock. We found that exercise performance was similar for both techniques; however, the shock group demonstrated significantly lower locomotor activity and higher anxiety-like behaviour. We also observed divergent effects on muscle pain; the prodding group showed hyperalgesia immediately after exercise, whereas the shock group showed hypoalgesia. Corticosterone concentrations were elevated to a similar extent for both groups. In conclusion, mice that were exercised without shock generated similar maximal exercise performance, but postexercise these mice showed an increase in locomotor activity, less anxiety-like behaviour and altered muscle pain in comparison to mice that exercised with shock. Our data suggest that running of mice without the use of electrical shock is potentially less stressful and might be a better technique to study the physiological and behavioural responses to exercise.

Bed nuclei of the stria terminalis modulate memory consolidation via glucocorticoid-dependent and -independent circuits.

There is extensive evidence that glucocorticoid hormones enhance memory consolidation, helping to ensure that emotionally significant events are well remembered. Prior findings suggest that the anteroventral region of bed nuclei of the stria terminalis (avBST) regulates glucocorticoid release, suggesting the potential for avBST activity to influence memory consolidation following an emotionally arousing learning event. To investigate this issue, male Sprague-Dawley rats underwent inhibitory avoidance training and repeated measurement of stress hormones, immediately followed by optogenetic manipulations of either the avBST or its projections to downstream regions, and 48 h later were tested for retention. The results indicate that avBST inhibition augmented posttraining pituitary-adrenal output and enhanced the memory for inhibitory avoidance training. Pretreatment with a glucocorticoid synthesis inhibitor blocked the memory enhancement as well as the potentiated corticosterone response, indicating the dependence of the memory enhancement on glucocorticoid release during the immediate posttraining period. In contrast, posttraining avBST stimulation decreased retention yet had no effect on stress hormonal output. Subsequent experiments revealed that inhibition of avBST input to the paraventricular hypothalamus enhanced stress hormonal output and subsequent retention, whereas stimulation did not affect either. Conversely, stimulation-but not inhibition-of avBST input to the ventrolateral periaqueductal gray impaired consolidation, whereas neither manipulation affected glucocorticoid secretion. These findings indicate that divergent pathways from the avBST are responsible for the mnemonic effects of avBST inhibition versus stimulation and do so via glucocorticoid-dependent and -independent mechanisms, respectively.

Evidence for Similar Prefrontal Structural and Functional Alterations in Male and Female Rats Following Chronic Stress or Glucocorticoid Exposure.

Previous work of ours and others has documented regressive changes in neuronal architecture and function in the medial prefrontal cortex (mPFC) of male rats following chronic stress. As recent focus has shifted toward understanding whether chronic stress effects on mPFC are sexually dimorphic, here we undertake a comprehensive analysis to address this issue. First, we show that chronic variable stress (14-day daily exposure to different challenges) resulted in a comparable degree of adrenocortical hyperactivity, working memory impairment, and dendritic spine loss in mPFC pyramidal neurons in both sexes. Next, exposure of female rats to 21-day regimen of corticosterone resulted in a similar pattern of mPFC dendritic spine attrition and increase in spine volume. Finally, we examined the effects of another widely used regimen, chronic restraint stress (CRS, 21-day of daily 6-h restraint), on dendritic spine changes in mPFC in both sexes. CRS resulted in response decrements in adrenocortical output (habituation), and induced a pattern of consistent, but less widespread, dendritic spine loss similar to the foregoing challenges. Our data suggest that chronic stress or glucocorticoid exposure induces a relatively undifferentiated pattern of structural and functional alterations in mPFC in both males and females.

Prefrontal-Bed Nucleus Circuit Modulation of a Passive Coping Response Set.

One of the challenges facing neuroscience entails localization of circuits and mechanisms accounting for how multiple features of stress responses are organized to promote survival during adverse experiences. The rodent medial prefrontal cortex (mPFC) is generally regarded as a key site for cognitive and affective information processing, and the anteroventral bed nuclei of the stria terminalis (avBST) integrates homeostatic information from a variety of sources, including the mPFC. Thus, we proposed that the mPFC is capable of generating multiple features (endocrine, behavioral) of adaptive responses via its influence over the avBST. To address this possibility, we first optogenetically inhibited input to avBST from the rostral prelimbic cortical region of mPFC and observed concurrent increases in immobility and hypothalamo-pituitary-adrenal (HPA) output in male rats during tail suspension, whereas photostimulation of this pathway decreased immobility during the same challenge. Anatomical tracing experiments confirmed projections from the rostral prelimbic subfield to separate populations of avBST neurons, and from these to HPA effector neurons in the paraventricular hypothalamic nucleus, and to aspects of the midbrain periaqueductal gray that coordinate passive defensive behaviors. Finally, stimulation and inhibition of the prelimbic-avBST pathway, respectively, decreased and increased passive coping in the shock-probe defensive burying test, without having any direct effect on active coping (burying) behavior. These results define a new neural substrate in the coordination of a response set that involves the gating of passive, rather than active, coping behaviors while restraining neuroendocrine activation to optimize adaptation during threat exposure.SIGNIFICANCE STATEMENT The circuits and mechanisms accounting for how multiple features of responses are organized to promote adaptation have yet to be elucidated. Our report identifies a prefrontal-bed nucleus pathway that organizes a response set capable of gating passive coping behaviors while concurrently restraining neuroendocrine activation during exposure to inescapable stressors. These data provide insight into the central organization of how multiple features of responses are integrated to promote adaptation during adverse experiences, and how disruption in one neural pathway may underlie a broad array of maladaptive responses in stress-related psychiatric disorders.

Amygdala central nucleus modulation of cerebellar learning with a visual conditioned stimulus.

Previous studies found that reversible inactivation of the central amygdala (CeA) severely impairs acquisition and retention of cerebellum-dependent eye-blink conditioning (EBC) with an auditory conditioned stimulus (CS). A monosynaptic pathway between the CeA and basilar pontine nuclei (BPN) may be capable of facilitating cerebellar learning. However, given that the CeA projects to the medial auditory thalamus, a critical part of the auditory CS pathway in EBC, the CeA influence on cerebellar learning could be specific to auditory stimuli. Here we examined the generality of CeA facilitation of EBC acquisition and retention in rats using a visual CS. As in our previous studies using an auditory CS, inactivation of the CeA with muscimol severely impaired acquisition and retention of EBC with a visual CS. Extending training to 15 100-trial sessions resulted in acquisition of EBC, indicating that the CeA plays a modulatory role in cerebellar learning and is not part of the necessary neural circuitry for EBC. Tract-tracing experiments verified that axons from the CeA reach both the BPN and medial auditory thalamus (part of the necessary auditory CS pathway), but were not found in the ventral lateral geniculate (part of the necessary visual CS pathway). The neuroanatomical results suggest that the CeA most likely modulates cerebellar learning through its projection to the BPN. The findings of the current study are consistent with the hypothesis that the CeA modulates cerebellar learning by increasing CS-related sensory input to the cerebellar cortex and interpositus nucleus via the BPN. This increase in CS-related input is thought to constitute an increase in attention to the CS during EBC.

Anteroventral bed nuclei of the stria terminalis neurocircuitry: Towards an integration of HPA axis modulation with coping behaviors - Curt Richter Award Paper 2017.

A network of interconnected cell groups in the limbic forebrain regulates hypothalamic-pituitary-adrenal (HPA) axis activation and behavioral responses to emotionally stressful experiences, and chronic disruption of these systems chronically is implicated in the pathogenesis of psychiatric illnesses. A significant challenge has been to unravel the circuitry and mechanisms providing for regulation of HPA activity, as these limbic forebrain regions do not provide any direct innervation of HPA effector cell groups in the paraventricular hypothalamus (PVH). Moreover, information regarding how endocrine and behavioral responses are integrated has remained obscure. Here we summarize work from our laboratory showing that anteroventral (av) bed nuclei of the stria terminalis (BST) acts as a point of convergence between the limbic forebrain and PVH, receiving and coordinating upstream influences, and restraining HPA axis output in response to inescapable stressors. Recent studies highlight a more expansive modulatory role for avBST as one that coordinates HPA-inhibitory influences while concurrently suppressing passive behavioral responses via divergent pathways. avBST is uniquely positioned to convey endocrine and behavioral alterations resulting from chronic stress exposure, such as HPA axis hyperactivity and increased passive coping strategies, that may result from synaptic reorganization in upstream limbic cortical regions. We discuss how these studies give new insights into understanding the systems-level organization of stress response circuitry, the neurobiology of coping styles, and BST circuit dysfunction in stress-related psychiatric disorders.

Circuit and synaptic mechanisms of repeated stress: Perspectives from differing contexts, duration, and development.

The current review is meant to synthesize research presented as part of a symposium at the 2016 Neurobiology of Stress workshop in Irvine California. The focus of the symposium was "Stress and the Synapse: New Concepts and Methods" and featured the work of several junior investigators. The presentations focused on the impact of various forms of stress (altered maternal care, binge alcohol drinking, chronic social defeat, and chronic unpredictable stress) on synaptic function, neurodevelopment, and behavioral outcomes. One of the goals of the symposium was to highlight the mechanisms accounting for how the nervous system responds to stress and their impact on outcome measures with converging effects on the development of pathological behavior. Dr. Kevin Bath's presentation focused on the impact of disruptions in early maternal care and its impact on the timing of hippocampus maturation in mice, finding that this form of stress drove accelerated synaptic and behavioral maturation, and contributed to the later emergence of risk for cognitive and emotional disturbance. Dr. Scott Russo highlighted the impact of chronic social defeat stress in adolescent mice on the development and plasticity of reward circuity, with a focus on glutamatergic development in the nucleus accumbens and mesolimbic dopamine system, and the implications of these changes for disruptions in social and hedonic response, key processes disturbed in depressive pathology. Dr. Kristen Pleil described synaptic changes in the bed nuclei of the stria terminalis that underlie the behavioral consequences of allostatic load produced by repeated cycles of alcohol binge drinking and withdrawal. Dr. Eric Wohleb and Dr. Ron Duman provided new data associating decreased mammalian target of rapamycin (mTOR) signaling and neurobiological changes in the synapses in response to chronic unpredictable stress, and highlighted the potential for the novel antidepressant ketamine to rescue synaptic and behavioral effects. In aggregate, these presentations showcased how divergent perspectives provide new insights into the ways in which stress impacts circuit development and function, with implications for understanding emergence of affective pathology.

γ-Protocadherins Interact with Neuroligin-1 and Negatively Regulate Dendritic Spine Morphogenesis.

The 22 γ-Protocadherin (γ-Pcdh) cell adhesion molecules are critical for the elaboration of complex dendritic arbors in the cerebral cortex. Here, we provide evidence that the γ-Pcdhs negatively regulate synapse development by inhibiting the postsynaptic cell adhesion molecule, neuroligin-1 (Nlg1). Mice lacking all γ-Pcdhs in the forebrain exhibit significantly increased dendritic spine density in vivo, while spine density is significantly decreased in mice overexpressing one of the 22 γ-Pcdh isoforms. Co-expression of γ-Pcdhs inhibits the ability of Nlg1 to increase spine density and to induce presynaptic differentiation in hippocampal neurons in vitro. The γ-Pcdhs physically interact in cis with Nlg1 both in vitro and in vivo, and we present evidence that this disrupts Nlg1 binding to its presynaptic partner neurexin1ß. Together with prior work, these data identify a mechanism through which γ-Pcdhs could coordinate dendrite arbor growth and complexity with spine maturation in the developing brain.

A Basal Forebrain Site Coordinates the Modulation of Endocrine and Behavioral Stress Responses via Divergent Neural Pathways.

UNLABELLED: The bed nuclei of the stria terminalis (BST) are critically important for integrating stress-related signals between the limbic forebrain and hypothalamo-pituitary-adrenal (HPA) effector neurons in the paraventricular hypothalamus (PVH). Nevertheless, the circuitry underlying BST control over the stress axis and its role in depression-related behaviors has remained obscure. Utilizing optogenetic approaches in rats, we have identified a novel role for the anteroventral subdivision of BST in the coordinated inhibition of both HPA output and passive coping behaviors during acute inescapable (tail suspension, TS) stress. Follow-up experiments probed axonal pathways emanating from the anteroventral BST which accounted for separable endocrine and behavioral functions subserved by this cell group. The PVH and ventrolateral periaqueductal gray were recipients of GABAergic outputs from the anteroventral BST that were necessary to restrain stress-induced HPA activation and passive coping behavior, respectively, during TS and forced swim tests. In contrast to other BST subdivisions implicated in anxiety-like responses, these results direct attention to the anteroventral BST as a nodal point in a stress-modulatory network for coordinating neuroendocrine and behavioral coping responses, wherein impairment could account for core features of stress-related mood disorders. SIGNIFICANCE STATEMENT: Dysregulation of the neural pathways modulating stress-adaptive behaviors is implicated in stress-related psychiatric illness. While aversive situations activate a network of limbic forebrain regions thought to mediate such changes, little is known about how this information is integrated to orchestrate complex stress responses. Here we identify novel roles for the anteroventral bed nuclei of the stria terminalis in inhibiting both stress hormone output and passive coping behavior via divergent projections to regions of the hypothalamus and midbrain. Inhibition of these projections produced features observed with rodent models of depression, namely stress hormone hypersecretion and increased passive coping behavior, suggesting that dysfunction in these networks may contribute to expression of pathological changes in stress-related disorders.

Loss of estrogen-related receptor alpha disrupts ventral-striatal synaptic function in female mice.

Eating disorders (EDs), including anorexia nervosa, bulimia nervosa and binge-ED, are mental illnesses characterized by high morbidity and mortality. While several studies have identified neural deficits in patients with EDs, the cellular and molecular basis of the underlying dysfunction has remained poorly understood. We previously identified a rare missense mutation in the transcription factor estrogen-related receptor alpha (ESRRA) associated with development of EDs. Because ventral-striatal signaling is related to the reward and motivation circuitry thought to underlie EDs, we performed functional and structural analysis of ventral-striatal synapses in Esrra-null mice. Esrra-null female, but not male, mice exhibit altered miniature excitatory postsynaptic currents on medium spiny neurons (MSNs) in the ventral striatum, including increased frequency, increased amplitude, and decreased paired pulse ratio. These electrophysiological measures are associated with structural and molecular changes in synapses of MSNs in the ventral striatum, including fewer pre-synaptic glutamatergic vesicles and enhanced GluR1 function. Neuronal Esrra is thus required for maintaining normal synaptic function in the ventral striatum, which may offer mechanistic insights into the behavioral deficits observed in Esrra-null mice.

Prolonged corticosterone exposure induces dendritic spine remodeling and attrition in the rat medial prefrontal cortex.

The stress-responsive hypothalamo-pituitary-adrenal (HPA) axis plays a central role in promoting adaptations acutely, whereas adverse effects on physiology and behavior following chronic challenges may result from overactivity of this system. Elevations in glucocorticoids, the end-products of HPA activation, play roles in adaptive and maladaptive processes by targeting cognate receptors throughout neurons in limbic cortical networks to alter synaptic functioning. Because previous work has shown that chronic stress leads to functionally relevant regressive alterations in dendritic spine shape and number in pyramidal neurons in the medial prefrontal cortex (mPFC), this study examines the capacity of sustained increases in circulating corticosterone (B) alone to alter dendritic spine morphology and density in this region. Subcutaneous B pellets were implanted in rats to provide continuous exposure to levels approximating the circadian mean or peak of the steroid for 1, 2, or 3 weeks. Pyramidal neurons in the prelimbic area of the mPFC were selected for intracellular fluorescent dye filling, followed by high-resolution three-dimensional imaging and analysis of dendritic arborization and spine morphometry. Two or more weeks of B exposure decreased dendritic spine volume in the mPFC, whereas higher dose exposure of the steroid resulted in apical dendritic retraction and spine loss in the same cell population, with thin spine subtypes showing the greatest degree of attrition. Finally, these structural alterations were noted to persist following a 3-week washout period and corresponding restoration of circadian HPA rhythmicity. These studies suggest that prolonged disruptions in adrenocortical functioning may be sufficient to induce enduring regressive structural and functional alterations in the mPFC. J. Comp. Neurol. 524:3729-3746, 2016. © 2016 Wiley Periodicals, Inc.

Amygdala Modulation of Cerebellar Learning.

Previous studies showed that amygdala lesions or inactivation slow the acquisition rate of cerebellum-dependent eyeblink conditioning, a type of associative motor learning. The current study was designed to determine the behavioral nature of amygdala-cerebellum interactions, to identify the neural pathways underlying amygdala-cerebellum interactions, and to examine how the amygdala influences cerebellar learning mechanisms in rats. Pharmacological inactivation of the central amygdala (CeA) severely impaired acquisition and retention of eyeblink conditioning, indicating that the amygdala continues to interact with the cerebellum after conditioning is consolidated (Experiment 1). CeA inactivation also substantially reduced stimulus-evoked and learning-related neuronal activity in the cerebellar anterior interpositus nucleus during acquisition and retention of eyeblink conditioning (Experiment 2). A very small proportion of cerebellar neurons responded to the conditioned stimulus (CS) during CeA inactivation. Finally, retrograde and anterograde tracing experiments identified the basilar pontine nucleus at the confluence of outputs from CeA that may support amygdala modulation of CS input to the cerebellum (Experiment 3). Together, these results highlight a role for the CeA in the gating of CS-related input to the cerebellum during motor learning that is maintained even after the conditioned response is well learned. SIGNIFICANCE STATEMENT: The current study is the first to demonstrate that the amygdala modulates sensory-evoked and learning-related neuronal activity within the cerebellum during acquisition and retention of associative learning. The findings suggest a model of amygdala-cerebellum interactions in which the amygdala gates conditioned stimulus inputs to the cerebellum through a direct projection from the medial central nucleus to the basilar pontine nucleus. Amygdala gating of sensory input to the cerebellum may be an attention-like mechanism that facilitates cerebellar learning. In contrast to previous theories of amygdala-cerebellum interactions, the sensory gating hypothesis posits that the gating mechanism continues to be necessary for retrieval of cerebellar memory after learning is well established.

The Contingency of Cocaine Administration Accounts for Structural and Functional Medial Prefrontal Deficits and Increased Adrenocortical Activation.

The prelimbic region (PL) of the medial prefrontal cortex (mPFC) is implicated in the relapse of drug-seeking behavior. Optimal mPFC functioning relies on synaptic connections involving dendritic spines in pyramidal neurons, whereas prefrontal dysfunction resulting from elevated glucocorticoids, stress, aging, and mental illness are each linked to decreased apical dendritic branching and spine density in pyramidal neurons in these cortical fields. The fact that cocaine use induces activation of the stress-responsive hypothalamo-pituitary-adrenal axis raises the possibility that cocaine-related impairments in mPFC functioning may be manifested by similar changes in neuronal architecture in mPFC. Nevertheless, previous studies have generally identified increases, rather than decreases, in structural plasticity in mPFC after cocaine self-administration. Here, we use 3D imaging and analysis of dendritic spine morphometry to show that chronic cocaine self-administration leads to mild decreases of apical dendritic branching, prominent dendritic spine attrition in PL pyramidal neurons, and working memory deficits. Importantly, these impairments were largely accounted for in groups of rats that self-administered cocaine compared with yoked-cocaine- and saline-matched counterparts. Follow-up experiments failed to demonstrate any effects of either experimenter-administered cocaine or food self-administration on structural alterations in PL neurons. Finally, we verified that the cocaine self-administration group was distinguished by more protracted increases in adrenocortical activity compared with yoked-cocaine- and saline-matched controls. These studies suggest a mechanism whereby increased adrenocortical activity resulting from chronic cocaine self-administration may contribute to regressive prefrontal structural and functional plasticity. SIGNIFICANCE STATEMENT: Stress, aging, and mental illness are each linked to decreased prefrontal plasticity. Here, we show that chronic cocaine self-administration in rats leads to decrements in medial prefrontal structural and functional plasticity. Notably, these impairments were largely accounted for in rats that self-administered cocaine compared with yoked counterparts. Moreover, we verified previous reports showing that adrenocortical output is augmented by cocaine administration and is more protracted in rats that were permitted to receive the drug contingently instead of passively. These studies suggest that increased adrenocortical activity resulting from cocaine self-administration may contribute to regressive prefrontal structural and functional plasticity.

Evidence for involvement of a limbic paraventricular hypothalamic inhibitory network in hypothalamic-pituitary-adrenal axis adaptations to repeated stress.

Emotional stressors activate a stereotyped set of limbic forebrain cell groups implicated in constraining stress-induced hypothalamic-pituitary-adrenal (HPA) axis activation by inhibiting hypophysiotropic neurons in the paraventricular hypothalamic nucleus (PVH). We previously identified a circumscribed, anterior part of the bed nuclei of the stria terminalis (aBST) that houses stress-sensitive, PVH-projecting, γ-aminobutyric acid (GABA)-ergic neurons as representing a site of convergence of stress-inhibitory influences originating from medial prefrontal and hippocampal cortices. Here we investigate whether exaggerated HPA axis responses associated with chronic variable stress (CVS; daily exposure to different stressors at unpredictable times over 14 days, followed by restraint stress on day 15) and diminished HPA output seen following repeated (14 days) restraint-stress exposure are associated with differential engagement of the limbic modulatory network. Relative to acutely restrained rats, animals subjected to CVS showed the expected increase (sensitization) in HPA responses and diminished levels of activation (Fos) of GABAergic neurons and glutamic acid decarboxylase (GAD) mRNA expression in the aBST. By contrast, repeated restraint stress produced habituation in HPA responses, maintained levels of activation of GABAergic neurons, and increased GAD expression in the aBST. aBST-projecting neurons in limbic sites implicated in HPA axis inhibition tended to show diminished activational responses in both repeated-stress paradigms, with the exception of the paraventricular thalamic nucleus, in which responsiveness was maintained in repeatedly restrained animals. The results are consistent with the view that differential engagement of HPA inhibitory mechanisms in the aBST may contribute to alterations in HPA axis responses to emotional stress in sensitization and habituation paradigms.

Behavioral disturbances in estrogen-related receptor alpha-null mice.

Eating disorders, such as anorexia nervosa and bulimia nervosa, are common and severe mental illnesses of unknown etiology. Recently, we identified a rare missense mutation in the transcription factor estrogen-related receptor alpha (ESRRA) that is associated with the development of eating disorders. However, little is known about ESRRA function in the brain. Here, we report that Esrra is expressed in the mouse brain and demonstrate that Esrra levels are regulated by energy reserves. Esrra-null female mice display a reduced operant response to a high-fat diet, compulsivity/behavioral rigidity, and social deficits. Selective Esrra knockdown in the prefrontal and orbitofrontal cortices of adult female mice recapitulates reduced operant response and increased compulsivity, respectively. These results indicate that Esrra deficiency in the mouse brain impairs behavioral responses in multiple functional domains.