Donald G. Rainnie - US grants

DESCRIPTION (Adapted from applicant's abstract): Schizophrenia is a debilatating disease that affects nearly 1% of the world population, some of cardinal signs of which are fear, paranoia, inappropriate emotional responses, and social withdrawal. One of the more consistent findings from the brains of schizophrenia patients has been a decreased grey matter volume in the temporal lobe, especially in the region of the amygdala. The amygdala has long been associated with emotional processing, and is thought to be a critical locus where cognitive events acquire their emotional significance. Lesions of the amygdala induce a permanent disruption of APDs, such as clozapine, are particularly efficacious in treating negative symptoms of schizophrenia. In addition to their being dopamine receptor antagonists, the atypical APDs, are also potent 5-HT receptor antagonists. It is possible, that the efficacy of atypical APDs in treating negative symptoms lies in their ability to disrupt 5-HT transmission in areas such as the amygdala. Unfortunately, little is known about signal processing in the amygdala at the cellular level. The long-term objectives of this application are to use whole-cell patch clamp recording from identified amygdala neurones in the in vitro slice preparation to determine how information is processed within the amygdala, how it may be regulated by neurotransmitters such as 5-HT, and how alterations in transmission/modulation may contribute to pathological states such as schizophrenia. The hypothesis to be tested is that: one of the primary models of action of chronic atypical APDs application is an alteration of 5-HTergic modulation of neurotransmission in the amygdala, and that this is related to their clinical efficacy in alleviating negative symptoms of schizophrenia. The following specific aims will address this hypothesis: (1) Characterization of intrinsic membrane properties of neurones within the BLA and ACe nuclei of the amygdala, and determination of the neurotransmitters involved in signal transduction in these same nuclei. (2) Examination of the effects of 5-HTergic modulation of intrinsic membrane properties and synaptic transmission in the BLA and ACe, and its interaction with acute administration of the atypical APDs, clozapine and risperidone. Application of 5-HT receptor subtype specific agonists, and antagonists, will be used to determine the nature of the 5-HT receptor/s involved in reach response. (3) Examination of the effects of chronic APDs administration on intrinsic membrane properties, synaptic transmission, and 5-HTergic modulation of BLA and ACe neurones.

neurophysiology; neuropeptide Y; interneurons; amygdala; Primates; animal colony;

neurons; classification; amygdala; Primates; animal colony;

neuroregulation; amygdala; Primates; animal colony; synapses;

neurons; limbic system; biophysics; Primates; animal colony;

neuroregulation; dopamine; neurophysiology; amygdala; Primates; animal colony;

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. In 2010 we concluded the studies for our parent R01 (Functional Neuroanatomy of the Basolateral Amygdala) and using preliminary data generated from the parent grant we successfully applied to NIH for continuing R01 funding (Fear and Dopamine in the Basolateral Amygdala). Based on our previous studies, we knew that a subpopulation of BLA interneurons, the parvalbumin containing cells, were key regulators of synchronized neural activity in the BLA. The results of this study were submitted to the Journal of Physiology [Lond.] and are currently being revised (Jasnow et al., in revision). The results of our previous studies had also suggested that the excitability of the parvalbumin interneurons was tightly controlled by expression of the transient outward potassium current, IA. These channels form macromolecular complexes with chaperone molecules, including the potassium channel interacting proteins (KChIPs). We have examined the relative distribution of KChIPs (KChIP1 [unreadable]KChIP4) in the BLA and noted that KChIP1 was selectively expressed in a subpopulation of neurons. We have extended this study to show that KChiP1 is almost exclusively expressed in the population of parvalbumin expressing interneurons. In addition, we completed our study into the dopamine-dependence of LTP induction in principal neurons of the BLA. Moreover, we extended these studies to show that a synaergistic interaction between dopamine, brain derived nerve growth factor, and synaptically released zinc, are a prerequisite for LTP induction, and by extrapolation fear memory formation.

DESCRIPTION (provided by applicant): The amygdala is critically involved in human psychiatric diseases such as alcohol addiction, depression, bipolar illness, and anxiety disorders. A better understanding of the functional microcircuitry within the primate amygdala is required for insight into the pathophysiology of these human disorders. The principle goal of this project is to optimize methods that will allow differential visualization and electrophysiological recording from specific populations of interneurons based on differential cell-type specific promoter usage. The tools that will be created and optimized here will be applicable to a large range of neuroscience questions across most species. The specific experiments proposed in this project are aimed toward understanding the rat and primate amygdala. The amygdala offers a particularly rich substrate for research because it is among the best understood brain regions in terms of complex mammalian behavior. However, what remains to be determined is how the complex heterogeneity of neurons within the amygdala encodes and modulates information flow, and how activity in this neural circuit relates to behavior. The primary limitation to progress in this area is a lack of tools for examining neuronal functioning in defined neural populations. A second limitation is that there are sufficient dissimilarities in receptor expression and functional modulation between the rodent amygdala and that of higher primates to make translational research problematic. We intend to consolidate molecular biology, electrophysiology, and optical techniques into a united research approach to overcome these limitations. This R21 developmental grant hypothesizes that fluorescence-based reporters can be transiently expressed by cell-type specific promoters in neurons within organotypic slice cultures of the rodent and primate amygdala. Interneurons of basolateral amygdala can be divided into distinct subgroups based on the expression pattern of four marker genes: Parvalbumin, Cholecystokinin, Somatostatin, and Vasoactive Intestinal Peptide. These interneuron subgroups are thought to play distinct roles in modulating the input/output properties of excitatory pyramidal neurons within the amygdala. The defined promoters for these genes will be cloned upstream of a red fluorescent reporter (DsRed). Following the demonstration of efficient cell-type specific expression, visually-guided whole-cell patch clamp recordings of fluorescent interneurons wjll be obtained, and electrophysiological properties determined. Post-hoc dual immunofluorescent labeling will be used to verify the phenotype of each recorded neuron. Finally, defined promoters will be expressed in a lentivirus to determine how recordings from subacute slice cultures compares to recordings from acute brain slices following in vivo infection and expression of cell-type specific reporters. These experiments will advance our understanding of the primate amygdala, a necessary step for understanding the pathophysiology of and development of novel treatments for disorders of alcohol addiction and other psychiatric disorders.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The goal of this collaboration was to use electrophysiological and immunohistochemical techniques to identify characteristic physiological properties of interneurons of the basolateral amygdala based on their expression of cell-specific calcium binding proteins. In 2009 we finalized our examinations of the effects of serotonin receptor activation on the excitability of interneurons in the BLA, as well as our studies to examine the relative distribution of interneuron subtypes in the non-human primate BLA. The final stage of this project was devoted to analyzing and publishing data.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. In 2010 we concluded the studies outlined in our original R01 submission (Stress Allostasis: CRF, Serotonin and the BNST) and successfully applied to NIH for continuation R01 (Stress-Induced Gene Regulation: BNST CRF Neurons and the Physiology of Anxiety). We have continued to work with the repeated unpredictable shock stress (USS) protocol to examine its effects on the gene expression of serotonin receptor subtypes as well as ion channel subunit expression in BNST neurons. As a foundation for these studies we have conducted a study designed to look at the cell-specific expression of four key ion channel subunits, namely those of Ih, IT, IA, and IAR. The results of these studies were recently published in Molecular and Cellular Neuroscience (Hazra et al., 2011, In Press). We have also published the results of our studies into the physiological properties of CRF-containing neurons that were made possible through the production of a CRF-GFP transgenic mouse (Martin et al., 2010). Another manuscript is in preparation describing the physiological and genetic properties of CRF-containing neurons in the BNST. We have also used this transgenic mouse to examine the relationship between BNST CRF neurons and oxytocin-containing neurons in the hypothalamus. Our studies have revealed a reciprocal relationship between these two neuropeptide systems, such that CRF neurons innervate the oxytocin neurons, and vice versa. The results of this study were submitted and are current under review in Neuropsychoendocrinology (Dabrowska et al., submitted).

Amygdala; Amygdaloid Body; Amygdaloid Nucleus; Amygdaloid structure; Animals; CCK; CRISP; Cholecystokinin; Common Rat Strains; Computer Retrieval of Information on Scientific Projects Database; Connector Neuron; Cyclic Somatostatin; Funding; GFP; Grant; Green Fluorescent Proteins; Growth Hormone Inhibiting Factors; Growth Hormone-Inhibiting Hormone; Institution; Intercalary Neuron; Intercalated Neurons; Interneurons; Internuncial Cell; Internuncial Neuron; Investigators; Lentiviral Vector; Lentivirus Vector; Light; Mammals, Primates; Mammals, Rats; Mammals, Rodents; Maps; NIH; National Institutes of Health; National Institutes of Health (U.S.); Nerve Cells; Nerve Unit; Neural Cell; Neurocyte; Neurons; Pancreozymin; Parvalbumins; Photoradiation; Physiologic; Physiological; Primates; Promoter; Promoters (Genetics); Promotor; Promotor (Genetics); Property; Property, LOINC Axis 2; Proteins; Rat; Rattus; Reporter; Reporting; Research; Research Personnel; Research Resources; Researchers; Resources; Rhodopsin; Rodent; Rodentia; Rodentias; SRIH; SRIH-14; Slice; Somatostatin; Somatostatin-14; Somatotropin Release Inhibiting Factors; Somatotropin Release-Inhibiting Hormone; Source; Spinal Column; Spine; Technology; United States National Institutes of Health; Uropancreozymin; Vertebral column; Visual Purple; Work; amygdaloid nuclear complex; awake; backbone; base; calbindin 2; calretinin; cell type; gene product; growth hormone release inhibiting factor; in vivo; neuronal

Activation of the basolateral amygdala (BLA) plays a critical role in the normal adaptive response to negative emotional stimuli. Abnormal activity of BLA output neurons has been implicated in the etiology of several mood disorders. For example, depressed and anxious individuals show exaggerated amygdala activation in response to negative emotional stimuli, which is now recognized as a trait marker for mood disorders. Hyperactivation is also a predictor of positive treatment outcome as it normalizes with the onset of therapeutic action of drug treatment, suggesting that the amygdala is a key component of a mood-regulatory system that is dysregulated in anxiety and depression. Selective serotonin reuptake inhibitors (SSRIs) are a first-line treatment for many mood disorders, and the amygdala has a high density of SSRI binding sites. Significantly, negative emotional stimuli trigger serotonin (5HT) release into the BLA where it acts to decrease the excitability of BLA output neurons. Moreover, 5HT levels in the BLA are finely regulated by the activity of 5HT transporter proteins, suggesting that SSRIs may exert their therapeutic effects by raising BLA 5HT levels and thus normalizing the activity of its output neurons. However, the slow onset of action of SSRIs and their unwanted side effects are driving the search for faster acting, and more targeted treatments for anxiety and depression. Recently, a novel antidepressant agent has been identified, GSK-1, which is a mixed SHTwiB/iD receptor antagonist that has a rapid onset of action. Multiple serotonin receptor subtypes are expressed in the BLA. Hence, drugs acting at one or more 5HT receptors could have a profound impact on the excitability of BLA output neurons, and hence mood disorders. However, little is known about how individual serotonin receptor activation may modulate the activity of BLA output neurons, let alone how mixed 5HT receptor antagonists may affect these neurons. In this study, we will use patch clamp recording in an in vitro slice preparation to compare the response of BLA neurons to administration of a classic SSRI, citalopram, with that of GSK-1 before, during, and after a challenge with exogenous 5HT. The hypothesis to be tested is that: acute administration of GSK-1 will mimic the net effect of chronic administration of SSRIs on the activity of BLA output neurons. Three specific aims will test this hypothesis: Aim 1: Compare and contrast the effects of acute in vitro administration of GSK-1 on serotonin receptor-mediated activity in BLA projection neurons and interneurons. Aim 2: Compare and contrast the effects of in vivo administration of GSK-1 on serotonin receptor-mediated activity in BLA projection neurons and interneurons. Aim 3: Compare and contrast the effects of GSK-1 and citalopram on serotonin receptor-mediated activity in BLA projection neurons and interneurons following sustained fear conditioning.

DESCRIPTION (provided by applicant): Activation of neurons in the basolateral amygdala (BLA) plays an essential role in the cellular processes that underlie the normal, adaptive, behavioral response to threatening, as well as rewarding, environmental stimuli. Importantly, release of the neurotransmitter dopamine has also been shown to play a central role in the response to threatening or rewarding stimuli. Moreover, several neuropsychiatric disorders such as schizophrenia, which are commonly associated with emotional disturbances, are thought to result, at least in part, from abnormal dopamine transmission. Compelling evidence now suggests that region-specific release of dopamine into the BLA is an absolute requirement for the formation of fearful memories. Hence, dopamine depletion prevents the formation of fear memories, an effect that can be rescued by allowing dopamine release to occur only within the BLA. More specifically, pharmacological agents that selectively modulate the activity of D1 family dopamine receptors (D1R, including D1 and D5) in the BLA can also modulate fear memory formation and consolidation. Significantly, gene knockout mice with a global deletion of D1 receptors show an impairment of fear memory formation. In other brain regions, D1 receptors are believed to activate the protein kinase-A (PKA) cascade. Importantly, inactivation of the PKA pathway impairs fear memory formation. Together, these data suggest that activation of the D1 - PKA cascade in the BLA may play a critical role in the formation of fear memories. Similarly, recent studies have indicated that synchronized neural activity, both within the BLA and between the BLA and target structures such as the medial prefrontal cortex (mPFC), play a major role in memory formation and recall. Dopamine has long been known to play a critical role in synchronizing neural activity. However, no study has systematically examined the molecular, cellular, and network-level mechanisms by which D1 receptor activation may facilitate fear memory formation. The studies outlined in this proposal are designed to address this significant knowledge gap. We have strong preliminary data to support our hypothesis that: D1 receptor activation in BLA principal neurons acts to facilitate synaptic plasticity by a PKA-dependent enhancement of intrinsic membrane oscillations and spike timing precision. The resulting highly synchronized firing of principal neurons in distinct frequency ranges, and subsequent phase locking of synchronized activity between the BLA and mPFC, facilitates fear memory formation. The specific aims of this proposal have been designed to answer three specific questions relating to this hypothesis: SA#1. Does activation of the D1 - PKA cascade facilitate intrinsic oscillatory activity in the BLA? SA#2. Is activation of the D1 - PKA cascade necessary for synaptic plasticity in BLA afferent inputs? SA#3. Can activation of the D1 - PKA cascade facilitate coherent oscillations between the BLA and mPFC during fear learning? PUBLIC HEALTH RELEVANCE: Compelling evidence suggests that dopamine release in the amygdala is a prerequisite for the formation and expression of fear memory, and long-term changes in dopaminergic signaling are thought to underlie a number of psychiatric disorders, including schizophrenia, that are often associated with disturbances of emotion. However, the cellular and subcellular mechanisms that underlie the dopamine effect on fear learning are poorly understood. This proposal will use electrophysiological, molecular-biological, and behavioral techniques to examine the effect of site-specific modulation of the dopamine system in the basolateral amygdala, with the goal of identifying novel points for clinical intervention in several psychiatric disorders.

DESCRIPTION (provided by applicant): Activity of neurons in the bed nucleus of the stria terminalis (BNST) plays a central role in the normal adaptive response to stress. However, chronic release of stress hormones into the BNST also plays a critical role in several central and peripheral pathologies, including anxiety disorders, posttraumatic stress disorder (PTSD), stress-induced drug abuse, cardiovascular disease, as well as gastrointestinal disorders. To date the cellular mechanisms underlying the switch from a normal adaptive response to a psychopathological state remain unknown. The long-term objectives of this proposal are to delineate the cellular mechanisms contributing to the pathological switch in BNST function, with the hope of identifying novel targets for clinical intervention. The selective serotonin (5-HT) reuptake inhibitors (SSRIs) are the first line drugs of choice in treating many stress-related disorders suggesting that abnormal 5-HT function in key areas, such as the BNST may play an important role in the development of these disorders. We have shown that 5-HT inhibits the majority of BNST neurons in vitro, and evokes an anxiolytic response in vivo. Moreover, acute release of the stress hormone corticotrophin releasing factor (CRF) facilitates the inhibitory response of BNST neurons to 5-HT, suggesting that an interaction between these two systems contributes to the normal adaptive response to stress. Our data suggest that repeated restraint stress (RRS) results in a long lasting enhancement of anxiety-like behavior that is associated with a significant reduction in the mRNA expression of inhibitory 5-HT1A receptor subunits, and an increase in excitatory 5-HT2C/7 receptor subunits in BNST neurons. These data suggest that RRS switches the 5-HT response of BNST neurons from inhibition to excitation. In addition, RRS selectively attenuates the expression of mRNA for the Kv4.2 subunit of the inhibitory transient outward potassium current (IA). Significantly, pilot data suggests that the response to RRS can be blocked by prior administration of a CRF1 receptor antagonist, or a histone-deacetylase inhibitor, which alters gene transcription. Our hypothesis is that in RRS, repeated CRF1 receptor activation initiates a cascade of events that disrupts transcriptional regulation of gene expression resulting in an increase in the excitability of BNST neurons, and particularly CRF-containing neurons, and shifting their response to 5-HT in favor of excitation. We propose that similar shifts in BNST excitability may contribute to the etiology of anxiety disorders and PTSD. Here, we will use patch clamp electrophysiology, molecular biology, and behavioral studies in rats and in a novel transgenic mouse in which a green fluorescent protein (GFP) is expressed in CRF-neurons to test our hypotheses. Two specific aims are proposed. Specific Aim #1 will characterize the effects of repeated restraint stress on gene expression and physiological properties of neurons in the anterolateral BNST of the rat. Specific Aim #2 will characterize the effects of repeated restraint stress on gene expression and physiological properties of CRF-containing neurons of the mouse anterolateral BNST. PUBLIC HEALTH RELEVANCE: Trauma or chronic stress is a major precipitating factor in the development and expression of many anxiety disorders, including post-traumatic stress disorder. Similarly, disruption of normal BNST function is thought to contribute to the development of many anxiety disorders. This proposal will use electrophysiological, molecular biological, and behavioral techniques to examine the effect of repeated restraint stress on the physiological properties and genetic profile of BNST neurons with the goal of identifying novel points for clinical intervention in anxiety disorders and PTSD.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. In our initial experiments we focused on increasing the sample size of our pilot data for the control postsynaptic response of BLA neurons to serotonin receptor activation, as well as the modification of synaptic transmission by presynaptic serotonin receptor activation. For these experiments, we examined the postsynaptic response of BLA projection neurons and interneurons to exogenous application of 5HT (Aim 1). The results of these studies show that 5-HT application causes a significant reduction of presynaptic glutamate release (54% of baseline) onto BLA principal neurons, which is mediated by activation of 5-HT1B receptors. Significantly, this action was fully blocked with prior application of the test compound, GSK1. As predicted by earlier studies, 5-HT also caused an indirect inhibition of principal neurons by exciting local circuit interneurons thereby increasing tonic GABA release. In a few cells, 5-HT also caused a direct 5-HT1A receptor-mediated inhibition in principal neurons, which appeared to be insensitive to GSK1. In association with these studies we also conducted a single cell RT-PCR study on an additional 12 BLA projection neurons to confirm our preliminary observations on 5HT receptor mRNA expression in this cell population.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. In 2010, we continued work on Aim 1: Characterize spontaneous activity in the BLA and PFC, and evaluate the presence of synchronous oscillations. We have collected data from six rats undergoing fear conditioning and extinction training, and have begun work on a manuscript describing our findings. We have demonstrated that delta (1.5-4Hz) and gamma (30-80Hz) frequency bands in the local field potentials of the BLA and mPFC show unique patterns of increased coherence during fear learning and expression, and that these patterns are not seen in rats that do not learn the fear association, indicating a functional relationship with the emotional state of the animal. We have now begun experiments related to Aim 2: Evaluate neural and behavioral response to PFC stimulation. We have applied electrical stimulation modeled after human DBS parameters to the mPFC region thought to be homologous to Cg25, while recording behavior and neural responses in the BLA. By stimulating during the fear extinction training protocol, we hope to demonstrate a suppression of negative emotion and an acceleration of extinction learning. The rats being used in this experiment have just arrived and are being weighed daily in preparation for food restriction, the first step in the protocol.

PROJECT SUMMARY (See instructions): Behavioral evidence across species suggests that oxytocin plays a general role in many aspects of social cognition, yet the neurobiological substrates through which it acts at the neural circuit level are not fully understood. An intriguing but untested idea is that centrally released oxytocin acting on limbic brain regions allows for the neural processing of social cues to gate activity in areas involved in seeking reward, thus facilitating the motivation to socially interact and the reinforcement of conspecific cues. Our long-term goal is to elucidate how oxytocin modulates the oxytocin receptor rich regions underlying social information processing and reward to enhance social cognition. The objective here is to record from chronic electrode implants within these regions during social behavioral paradigms in rodents. Our central hypothesis is that the motivation to interact socially is determined by a balance between positive and negative valence cues, and that oxytocin acts to enhance how positive valence cues and/or suppress how negative valence cues modulate the functional neural connections between cue and reward processing areas, helping to reinforce their encoding. The rationale for our proposal is that, once we know how oxytocin affects functional connectivity between these areas in natural social contexts, our improved knowledge about oxytocin's sites of action will enable direct manipulation of these circuits to enhance prosocial behavior. Two complementary specific aims in two different rodent models will be pursued, each chosen to maximize our ability to deduce the electrophysiological effects of either oxytocin loss of function (Aim 1) or gain of function (Aim 2) during social interactions. Our proposal's significance lies in the fact that it will implicate a specific central limbic circuit in mediating oxytocin's role in facilitating social motivation and socially reinforced learning. The combination of in vivo electrophysiology with oxytocin manipulation in freely moving, socially interacting rodents is an innovation that will enable key questions to be addressed about how real-time neural activity within limbic circuits is dynamically modulated by oxytocin in natural social interactions.

? DESCRIPTION (provided by applicant): Anxiety disorders are the most prevalent psychiatric disorders and affect over 40 million American adults over the age of 18 every year. Significantly, ~ 20% of individuals who seek treatment for independent anxiety disorders also have a current drug use disorder. The extended amygdala is thought to play a critical role in adaptive motivational behavior, and has been implicated in the pathophysiology of maladaptive fear, anxiety, and addiction. Two key elements of the extended amygdala are the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST). Notably, evidence from clinical and preclinical studies suggests that differences in BNST activity determine individual differences in trait anxiety levels and also anticipatory anxiety (AA). Activity of corticotropin release factor (CRF) containing neurons in the BNST plays a central role in the normal adaptive response to stress. However, chronic release of CRF also plays a critical role in several psychopathologies, including anxiety disorders, posttraumatic stress disorder (PTSD), stress-induced drug recidivism, all of which have excessive AA as a core symptom. To date the cellular mechanisms underlying the switch from a normal adaptive response to a psychopathological state remain unknown. However, evidence from imaging studies suggests that activation of a circuit comprising the insular cortex (IC), amygdala, BNST, and ventrolateral periaqueductal grey (vlPAG) plays a key role in regulating the expression of AA. Using a transgenic CRF-Cre mouse line, we now have exciting pilot data showing that cell type-selective inhibition of CRF neurons in the BNST blocks the development of AA. The proposed work will use a multidisciplinary approach to define a functional circuit by testing the hypothesis that BNSTov CRF neurons act to integrate viscerosensory and emotional information from the IC and amygdala and relay this information to the vlPAG to regulate the expression of AA. The long-term objectives of this proposal are to delineate the cellular mechanisms contributing to the pathological switch in BNST function, with the hope of identifying novel targets for clinical intervention. Three Specific Aims will test the hypothesis: Aim 1 will examine the necessity / sufficiency of BNSTov CRF neuron activation in the expression of AA. Aim 2 will examine the role of the insular cortex (IC) and posterior basolateral amygdala (BLAp) in activating BNSTov CRF neurons during AA and their downstream connection with the vlPAG, and Aim 3 will examine the effects of chronic stress on the functioning of the proposed pathway.

? DESCRIPTION (provided by applicant): Major depressive disorder (MDD) is one of the most common mental disorders in the United States, with a lifetime prevalence of ~17%. Significantly, the risk of MDD is more than doubled in patients with type 2 diabetes mellitus (T2DM), which is also associated with a 10 fold greater risk for suicide. With the incidence of T2DM growing annually, the need for more rapidly effective treatment strategies for MDD is also growing. We hypothesize that metabolic stress-induced dysfunction of cellular energy-sensing homeostatic mechanisms contributes to the etiology of MDD, and drugs targeting neuronal energy sensors may provide novel, rapid-acting treatments for depression. Imaging studies revealed that hyperactive metabolism of the basolateral amygdala (BLA) correlates with MDD symptom severity and normalizes with successful pharmacotherapy. We have strong preliminary data showing that BLA manipulations of the ubiquitous cellular energy sensor, AMP-activated protein kinase (AMPK), can significantly disrupt affective behavior and dramatically alter electrophysiological properties of principal neurons. Thus, we further hypothesize that AMPK signaling plays a major role in regulating homeostatic plasticity in BLA principal neurons, and dysfunction of this signaling cascade could contribute to the etiology of depression in T2DM. In T2DM, insulin-resistance prevents insulin-induced glucose uptake into cells, thereby disturbing cellular energy balance. Metformin, the drug of choice for treating T2DM, works by activating AMPK. Activated AMPK functions to restore cellular energy balance by inhibiting energy utilizing and enhancing energy producing processes, such as increasing cellular glucose uptake. Significantly, the BLA expresses a high density of insulin receptors and insulin-regulated glucose transporters, suggesting that insulin-dependent regulation of glucose uptake is a key modulator of BLA metabolic function. Importantly, insulin-resistance occurs in both CNS and periphery. However, as metformin does not easily cross the blood brain barrier the central effects of metabolic disruption in T2DM largely remain unchecked. Recent metabolomics studies have shown a significant overlap in biomarkers involved in metabolic- and mood disorders. Indeed, diet-induced obesity in rodents results in insulin-resistance, depression-like behavior, enhanced BLA activation to emotional stimuli, and a reduced threshold for LTP induction. Notably, the insulin- resistance and depression-like behavior could be alleviated by administration of a selective AMPK activator, AICAR, suggesting that targeting the AMPK signaling cascade may be a novel avenue of research for treating depression. However, surprisingly little is known about the role of the AMPK signaling cascade in regulating neuronal function in areas outside the hypothalamus, or how it is affected by metabolic stress. This proposal seeks to address this critical knowledge gap.

Project Summary (Project 3, Rainnie) The ability to recognize the identity and intentions of others and to react accordingly is an evolutionarily adaptive process with relevance to psychiatric disorders. However, the cellular and neurotransmitter systems that regulate this process remain largely unknown. One region consistently shown to play a pivotal role in regulating the behavioral response to both appetitive and aversive sensory and/or social stimuli is the basolateral amygdala (BLA). The activity of neurons in the BLA has been shown to signal preference in a social recognition task, and in the previous Conte Center funding period we showed that during social interaction between same sex rats neural activity in the BLA and a key component of reward circuitry, the nucleus accumbens (NAc), became highly synchronized. Synchronization was associated with markedly enhanced ?-? cross-frequency-coupling (?-? CFC) similar to that seen in non-human primates (NHP) during performance of a social preference task (see Project 4) and between the mPFC and NAc during pair bonding in voles (Project 2). Together, these data suggest that ?-? CFC may represent a canonical mechanism for integrating executive, emotion, and reward circuits to drive appropriate behavioral responses during social interaction. We have successfully developed a novel rat social recognition task, which mirrors the task being utilized in the NHP studies of Project 4, and with which we can directly examine the role of two neurotransmitters, acetylcholine (ACh) and oxytocin (OT) in the modulation of social recognition as well as ?-? CFC in the pathway from the BLA to the NAc. These two neurotransmitters have been shown to play key roles in regulating cue discrimination, ?-? CFC, and social interaction in rodents and NHPs. However, ACh and OT are usually studied independently of one another. It is our contention that ACh and OT act synergistically in the BLA to facilitate social recognition in conspecifics. Here, we will test the hypothesis that OT release in the BLA acts to facilitate social recognition by enhancing ACh release in the BLA and promoting ?-? CFC in the pathway from the BLA to NAc. To challenge this hypothesis we will use state-of- the-art gene transfer and gene deletion techniques in conjunction with pathway specific viral vector manipulations to selectively target specific neural circuits that are thought to regulate BLA neural activity during social recognition and discrimination. The PI of Project 3, is an internationally recognized expert in the field of amygdala anatomy and physiology and has a track record of using state-of-the-art viral vector manipulations to examine the fine structure of neural circuits that regulate affective behavior. In addition, the research team for Project 3 have all of the necessary expertise to successfully complete the proposed studies. We anticipate that at the end of Project 3 we will have markedly increased our understanding of the interaction between two critical neurotransmitter systems that are known to play a major role in social discrimination. By better understanding the systems and circuits that guide prosocial behavior we will be able to develop more targeted therapeutic approaches for disorders that share a common pathology of deficits in social behavior.