Kimberly Huber - US grants

long term potentiation; glutamate receptor; neural plasticity; direct cortical response; developmental neurobiology; visual cortex; inositol phosphates; eye pharmacology; inhibitor /antagonist; electrophysiology; laboratory rat; laboratory mouse;

[unreadable] DESCRIPTION (provided by applicant): Activation of the Gq coupled metabotropic glutamate receptors (Group 1 mGluRs) induce long-term plasticity of neuronal and synaptic function which is mediated through direct regulation of new protein synthesis. Consequently, the group 1 mGluRs, mGluR1 and mGluR5 are implicated many long-term behavioral adaptions of brain function including postnatal cortical map formation, learning and memory, chronic pain and drug addiction. Elucidating the basic mechanisms of how mGluRs and other Gq coupled neurotransmitter receptors induce plasticity and how these new proteins alter synapse function is essential to understanding the neurobiological basis of these behaviors. The significance of mGluR- dependent plasticity to human cognitive function is highlighted by the recent findings of enhanced or unregulated mGluR-and protein synthesis dependent plasticity in the mouse model of fragile X syndrome (Fmr1 KO mice), the most common inherited form of mental retardation in humans. We have discovered and characterized a form of long-term synaptic depression in hippocampal area CA1 induced by group 1 mGluRs which relies on rapid (within minutes) protein synthesis in dendrites (mGluR-LTD). Furthermore, we have found that the mechanisms of mGluR-LTD are altered in Fmr1 KO mice. This proposal focuses on determining how mGluRs induce LTD and regulate protein synthesis machinery and how new proteins lead to persistent changes at synapses. Based on our new findings, we hypothesize and propose experiments to test if other Gq coupled neurotransmitter receptors induce LTD through similar mechanisms as group 1 mGluRs. From the knowledge gained from the study of the basic mechanisms of mGluR-LTD in normal rodents, we propose experiments to examine how and why mGluR-dependent plasticity is altered in Fmr1 KO mice. 1: Determine the role of Homer isoforms in mGluR-and protein synthesis dependent LTD mechanisms. 2: Determine mechanism for persistent decreases in AMPAR surface expression induced by mGluRs. 3: Explore the role of other Gq coupled receptors in protein synthesis dependent LTD .4: Role of Homer interactions and other Gq coupled receptors in LTD in Fmr1 KO mice. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Fragile X Syndrome (FXS) is the most common inherited form of mental retardation and is caused by loss of function mutations in the Fragile X Mental Retardation gene (FMR1). Patients with FXS as well as other forms of mental retardation have an excess of dendritic spines as well as longer spines, suggesting that abnormal postsynaptic function, development or plasticity contributes to the cognitive deficits of this disease. Fragile X Mental Retardation Protein (FMRP), the protein product of FMR1, is an RNA binding protein and is thought to regulate translation of proteins in dendrites and dendritic spines. Such local or synaptic protein synthesis regulates both synapse development and long-term plasticity in mature animals. Therefore, FMRP most likely mediates its neuronal effects through translational regulation of synaptic proteins. Consistent with this idea, we and others have discovered that FMRP regulates synapse pruning in adolescent neurons as well as long-term synaptic depression by metabotropic glutamate receptors (mGluRs). Exactly how FMRP regulates synapse development and plasticity is unknown. Whether these effects are due to translational regulation of dendritically synthesized proteins or the identity of such proteins is also unknown. Here we propose to examine the cellular mechanisms by which FMRP regulates synapse development and mGluR- dependent synaptic plasticity. We will also examine how FMRP regulates the synthesis of proteins in dendrites and test 2 candidate proteins for their role in FMRP mediated synaptic pruning and mGluR-induced synaptic depression. Developing and testing new therapeutic strategies for treatment of FXS and autism, such as mGluR antagonists, is a mission of the NIH. Our results are expected to provide knowledge of how FMRP, or its absence, regulates synapse maturation and mGluR-dependent synaptic plasticity in the adult. These results will help to determine the neurobiological basis of mental retardation and related disorders such as autism as well as test if mGluR antagonists are a suitable therapeutic strategy for FXS. The specific aims of the grant are: 1. Examine FMRP regulation of synapse development and elimination. 2 Determine the role of FMRP phosphorylation in regulation of synapse number and mGluR-induced dendritic protein synthesis, 3. Determine if FMRP is an acute regulator of LTD, 4. Test candidate proteins for their involvement in LTD and FMRP induced synapse elimination.Fragile X Syndrome (FXS) is the most common inherited form of mental retardation and a leading cause of autism. FXS is caused by loss of function mutations in the Fragile X Mental Retardation protein (FMRP). Our research will determine how FMRP normally works in the brain as well as how and why the brain functions differently without FMRP. This work is expected to provide important knowledge to development therapies for mental retardation and autism.

DESCRIPTION (provided by applicant): Fragile X Syndrome (FXS) is the most common, inherited form of intellectual disability and the leading genetic cause of autism 21, 160-164. Transcriptional silencing or loss of function mutations of a single gene, Fmr1, lead to FXS165. Fmr1 encodes a neuronal RNA binding protein, Fragile X Mental Retardation Protein (FMRP) 6. Accumulating evidence indicates that the Gq-coupled metabotropic glutamate receptor 5 (mGluR5) is dysfunctional and overactive in the mouse model of FXS, Fmr1 KO mice 26, 28, 29, 34, 56. Consequently, mGluR5 antagonists reverse many of the phenotypes in FXS animal models and are currently in clinical trials in FXS patients 30, 31, 33-38, 108. The cellular mechanisms of mGluR5 dysfunction in Fragile X have been elusive. A clue comes from the findings that mGluR5 is less associated with the synaptic scaffolding molecule Homer in Fmr1 KO mice 14. The N-terminal EVH1 domain of Homer binds to the C-terminus of mGluR5 as well as signaling effectors, ion channels, and other synaptic scaffolds. The C-terminal coiled-coil domain of Homer dimerizes with other Homer molecules to scaffold mGluR5 to its effectors and the postsynaptic density. A short variant of Homer, Homer1a, lacks the coiled-coil domain, disrupts long Homer scaffolds, which in turn, alters mGluR5 signaling, subcellular localization and causes agonist-independent activity of mGluR5 42, 43, 105, 137, 140, 166, 167. We find that the disrupted mGluR5-Homer interactions mediate much of the altered mGluR5 function as well as the neurophysiological and behavioral phenotypes of Fmr1 KO mice 13, 44. It is unknown if FXS results from a specific disruption in mGluR5-Homer interactions or a disruption of Homer scaffolds in general. To test these ideas, we will determine if Homer interactions with other proteins are affected in Fmr1 KO mice and if a transgenic mouse expressing a knockin mutation of mGluR5 (F1128R) that does not interact with Homer is sufficient to mimic the biochemical, neurophysiological and behavioral phenotypes of Fmr1 KO mice 45. It is unknown what leads to reduced mGluR5-Homer interactions in Fmr1 KO mice. Phosphorylation of mGluR5 20, 46, 47 and Homer at specific sites are known to regulate their interactions . In Aim 2, we will determine if mGluR5 and Homer phosphorylation are altered in Fmr1 KO mice and examine candidate mechanisms for this altered phosphorylation. FMRP functions to translationally suppress its mRNA targets, which leads to elevated levels of specific proteins in Fmr1 KO mice 6. In Aim 3, we will determine if elevated levels of a candidate protein in Fmr1 KO mice, known to interact with mGluR5, results in altered mGluR5 function and Homer scaffolds in Fmr1 KO neurons 15, 16. In Aim 4, we will examine another mouse model of autism to determine if altered mGluR5 function and disrupted mGluR5-Homer scaffolds constitute shared neurobiological mechanisms of autism and FXS 19, 51. Results of this work are expected to determine the core molecular deficits of FXS and autism and provide novel targets for pharmacological intervention. PUBLIC HEALTH RELEVANCE: Excessive mGluR5 activity has been proposed to cause mental retardation and autism associated with Fragile X Syndrome. Here we propose to determine the cellular mechanisms for altered mGluR5 function in Fragile X Syndrome and to determine if altered mGluR5 function is a common cause of autism.

DESCRIPTION (provided by applicant): Cortical structures (the hippocampus and neocortex) are critical for cognition and perception, and their improper function is implicated in intellectua disability and autism. The establishment of proper cortical circuits requires a complex interaction of neural activity and genetic programs that control the formation and elimination of specific synaptic connections. Studies of structural excitatory synapses, such as spines, find there is a rapid period of synaptogenesis early in postnatal development followed by a later period of elimination (or pruning) - in both humans and mice. Importantly, sensory experience and circuit activity drives the pruning of spines in vivo. Spine elimination is also triggered by learning in adults and may mediate the refinement of circuits that maintain memories. However, spines are an indirect measure of synaptic number and provide little information about how pruning regulates synaptic function and connectivity of specific cortical pathways. Furthermore, virtually nothing is known of the cellular and molecular mechanisms of activity and sensory experience-dependent synapse elimination in cortical neurons. Using assays of synaptic function in isolated cortical pathways, we have accumulated evidence indicating that activity-dependent synaptic pruning is regulated by the activation of the Myocyte-Enhancer Factor 2 (MEF2) family of transcription factors. We find that the RNA binding protein, Fragile X Mental Retardation Protein (FMRP) is required for MEF2- triggered synapse elimination by regulating the translation of MEF2-generated transcripts - including Protocadherin10 (Pcdh10) and Arc/Arg3.1. We find that Arc and Pcdh10 mediate elimination of synapses through distinct mechanisms. Importantly, loss of function mutations in MEF2C, FMRP and Pcdh10 are linked to intellectual disability (ID), autism and circuit hyperexcitability. Little is known of the physiological and in vivo conditions that lead to elimination of functional synaptic connections on cortical neurons and if or how this involves MEF2c, FMRP, Pcdh10, and Arc. In Aim 1 we will use optogenetics to induce physiological patterns of CA1 neuron firing and synapse elimination to determine the role of MEF2 isoforms, Fmr1 and Pcdh10 in physiological activity-dependent synapse elimination. In Aim 2 we will determine if endogenous MEF2 isoforms contribute to developmental pruning of functional excitatory synaptic connections onto cortical neurons in vivo. We also have new data suggesting that a novel experience activates MEF2-dependent Arc transcription which primes CA1 neurons for long-term synaptic depression upon activation of metabotropic glutamate receptors (mGluR-LTD). Novelty-priming of mGluR-LTD may be a precursor to synapse elimination and contribute to the formation of sparse cortical network representations of memories. In Aim 3 we propose to determine if MEF2 contributes to novelty-induced gene expression, priming of mGluR-LTD, and novelty habituation. In Aim 4 we will use optogenetics and electrical stimulation to establish an in vitro model of novelty-induced priming of LTD to reveal cellular mechanisms.

DESCRIPTION (provided by applicant): Sensory hypersensitivity is commonly seen in FXS patients and the FXS mouse model - the Fmr1 knockout (KO). Recent data suggests that this abnormality stems from hyperexcitability in sensory circuits. We have established that cortical microcircuits are hyperexcitable in the Fmr1 KO mouse model, and that sensory responses are enhanced in Fmr1 KO mice and FXS patients. Thus, investigation of sensory sensitivities is clinically relevant, but perhaps more important is the promise of sensory system studies to advance understanding of the mechanisms and consequences of hyperexcitability in neocortical circuitry that could represent a primary pathophysiological factor impacting the development of a wide range of perceptual, cognitive, and language skills in FXS. Further, we have identified biochemical signaling mechanisms that may underlie hyperexcitability involving processes that we and others have uncovered that can be examined in detail in KO mouse models and tested in FXS patients to develop a foundation for novel therapeutic development. The striking consistency of findings across levels of investigation and species offers an unprecedented opportunity to investigate mechanisms of brain dysfunction in a mouse disease model and translate it directly to patients - a multidisciplinary mission that is ideal for a Center environment. Our Center is organized to pursue precisely this aim with a tightly integrated and highly novel scientific program of translational research. Project 1 (Huber/Gibson; UTSW; co-investigators) will determine the cellular, molecular and synaptic mechanisms of auditory neocortical dysfunction using in vitro brain slices in FXS mouse models. Project 2 (Razak/Etheii/Binder; UCR; co-investigators) will study auditory sensory processing deficits in vivo in FXS mouse models, test mechanisms, and examine developmental and structural correlates of these deficits. Project 3 (Sweeney/Byerly, UTSW, co-investigators) will investigate auditory cortical processing deficits using novel neurophysiological strategies in individuals with FXS. All Projects will examine candidate mechanisms of sensory hyperexcitability with an acute pharmacological probe strategy to test mechanisms of interest in parallel studies of mice and patients.

Sensory hypersensitivity and abnormal sensory processing contribute significantly to behavioral problems associated with Fragile X Syndrome (FXS). Sensory hypersensitivity and audiogenic seizures in the FXS mouse model, Fmr1 knockout (KO) mice, suggest a hyperexcitability of sensory circuits. We have discovered a robust hyperexcitability, as well as changes in specific excitatory and inhibitory synaptic connections, in neocortical circuits in the Fmr1 KO mice. Our work has also revealed a novel molecular mechanism underlying hyperexcitability - impaired scaffolding among Homer proteins and resulting enhanced metabotropic glutamate receptor (mGluR5) signaling. We hypothesize that, in addition to enhancing mGluR5 function, impaired Homer scaffolding causes a displacement of the endocannabinoid (eCB) synaptic signaling resulting in differentially altered eCB-dependent plasticity of excitatory and inhibitory synapses in the Fmr1 KO. Evidence also indicates that ERK activity is involved in this altered plasticity. New data find that increased CamKlla phosphorylation of Homer causes the loss of scaffolding. As part of the FXS center, we propose to examine the role that these biochemical mechanisms and 3 synaptic pathways play in hyperexcitability of the auditory cortex in the Fmr1 KO mouse. Coordination of experiments are planned to link the alterations we find in the auditory neocortex with deficits in auditory sensory processing in Fmr1 KO mice (P2) and FXS patients (P3). In, Aim 1, we examine the developmental and circuit mechanisms underlying circuit hyperexcitability. Based on existing candidate targets for potential FXS treatment, we also examine the acute effects of clinically-approved, potential therapeutics on neocortical hyperexcitability. In Aim 2, we determine if mGluR5-eCB synaptic plasticity is dysregulated at 3 different neocortical synaptic pathways with known changes in the Fmr1 KO that could underlie hyperexcitability. In Aim 3, we determine if enhanced CaMKlla phosphorylation of Homer causes circuit hyperexcitibility and dysregulated mGluR5-eCB plasticity of specific synaptic circuits. In Aim 4, and in collaboration with P2, we examine the role of altered matrix-metalloproteinase (MMP9) signaling in hyperexcitability and disrupted Homer scaffolds

Sensory hypersensitivities are common and distressing feature of the Fragile X Syndrome. This clinical symptom is believed to be associated with neuronal hyperexcitability in neocortex. There are three primary goals of our highly novel and integrated Center program as pursued in Project 3. First, we aim to characterize the neural substrate of auditory sensory hypersensitivities in Fragile X patients using clinical neurophysiology. Second, we will establish mouse-human homologies via parallel 'in vivo' auditory neurophysiology studies in P2 and P3. Third, we will examine neurophysiological effects of single dose administration of minocycline, acamprosate, and lovastatin on resting EEG and auditory evoked responses in patients with FXS, using the same paradigms as in the Fmr1 mouse studies of P2. Auditory sensory responses, repeatedly shown to be highly abnormal in the Fmr1 mouse model and FXS patients, will be analyzed from a bottom-up, local circuit perspective by examining early sensory evoked response amplitudes and habituation to repeated tones. We will also analyze top-down corticocortical control of auditory processing using our recently established talk/listen paradigm. Last, we will perform a time-frequency decomposition of the EEG response to amplitude modulated (AM) chirp stimuli in order to examine local circuit-mediated neural oscillations. For the chirp paradigm, we will focus particularly on higher frequency gamma band activity, which we have found to be highly abnormal in Preliminary Studies in Fragile X as predicted by the neural circuitry model being tested in P1. Data will be examined for correlations between CGG repeat number and gene methylation, and with clinical ratings. Together with the mouse auditory circuit studies in PI & P2, pharmacological studies in P1 & P2, and 'in vivo' mouse auditory processing studies (P2), we will develop a mechanistic understanding of auditory hypersensitivity in Fragile X patients, and more broadly about illness mechanisms and translational strategies for evaluating neuronal hyperexcitability and its clinical impact.

Using a preclinical model system of Fragile X Syndrome (FXS), the proposed studies will identify new auditory processing based biomarkers that can be used as reliable outcome measures for therapeutics and provide new insights into neural mechanisms underlying auditory hypersensitivity in FXS. FXS is the most common inherited cause of mental impairment, and is a leading genetic cause of autism. Data from humans with FXS and the mouse model (Fmr1 KO) indicate auditory processing deficits. In vivo electrophysiological data from cortical neurons in adult Fmr1 KO mice suggest hyper-excitability in response to sounds and altered spectral and temporal responses. The proposed studies will determine the developmental time course of auditory cortical deficits in Fmr1 knock out (KO) mice, address mechanisms of these deficits and evaluate novel drugs in treating auditory functional deficits. Specific aim 1 will test the hypothesis that auditory processing deficits in the adult Fmr1 KO mice arise due to a deficit in developmental refinement of cortical processing. These studies will also compare auditory cortical responses between mice in which Fmr1 is deleted only in the excitatory neurons to address sources of deficits. In addition, recordings will be obtained from mice in which cortical Fmr1 is deleted only from 3 weeks postnatal to disambiguate developmental versus acute role for Fmr1 in establishing cortical responses in adult mice. Dendritic spine morphology of Al neurons will also be tracked during development to identify possible structural correlates of functional deficits. Specific aim 2 will determine baseline and sound evoked electroencephalogram (EEG) responses in awake, behaving mice. We will determine the effect of candidate drugs in reversing the auditory deficits at the single neuron and EEG/ERP levels. The EEG response will provide high throughput markers to test drugs. These studies will also identify new therapeutic strategies based on analysis of matrix metalloproteases in the auditory cortex in specific aim 3. An integrated approach based on in vivo electrophysiology, neuroanatomy and pharmacology will be used to address mechanisms of auditory processing deficits and drug effects in reversing these deficits.

? DESCRIPTION (provided by applicant): Activity dependent changes in synaptic efficacy, such as those which occur during long-term potentiation (LTP) and long-term depression (LTD), are believed to underlie learning and memory. A key event in both LTP and LTD is the induction of a set of immediate-early gene products, including the activity-regulated cytoskeletal- associated protein (arc, also known as arg 3.1). The best characterized function of Arc is enhancement of the endocytic internalization of AMPA receptors in dendritic spines, a process associated with LTD. Arc has also been implicated in the proteolytic processing of amyloid precursor protein (APP) on the surface of endosomes. To mediate these activities, Arc must associate with cellular membranes, but the mechanism of its binding to membranes is not understood. In addressing this question, we found that Arc undergoes palmitoylation in neurons, allowing it to insert directly into the lipid bilayer. Unlike other forms of protein lipidation, palmitoylation is reversible and, hence, may be subject to conditional regulation. In Aim 1 of this project we propose to define the mechanism and biochemical consequences of Arc palmitoylation. We will determine if the level of Arc palmitoylation is responsive to LTP- and LTD-inducing paradigms, and we will identify which of the 23 known human forms of palmitoyl acyltransferase is (are) responsible for modifying Arc. Using a combination of biochemical and imaging approaches, we will determine how palmitoylation influences the subcellular distribution of Arc, its ability to self-assemble on the membranes of living cells, and its susceptibility to undergo tyrosine phosphorylation, another Arc modification identified in our studies. In Aim 2 we will carry out electrophysiological investigations aimed at understanding how Arc palmitoylation affects synaptic plasticity. Our previous work implicated Arc in the weakening of synaptic strength and the elimination of excitatory synapses. Preliminary data indicate that these effects may be mediated by Arc palmitoylation. Changes in Arc expression have been linked to numerous cognitive disorders, including mental retardation, Alzheimer's disease, and substance abuse. Therefore, elucidation of novel mechanisms of Arc regulation has potential clinical significance.

A typical neocortical pyramidal neuron integrates information from thousands of excitatory inputs from both ?local? circuits, within a cortical region, and from many ?long-range? circuits from other cortical and non-cortical regions, both ipsi- and contralaterally. These long-range connections develop postnatally, are highly specified, and regulated by sensory experience. Remarkably, >50% of excitatory synapses a typical neocortical pyramidal neuron receives are from long-range connections, but virtually nothing is known of how functional long-range connections develop, are regulated by experience or if and how long-range and local inputs are balanced. In the last grant cycle, we discovered that the activity-dependent transcription factor, Myocyte-Enhancer Factor 2C (MEF2C) functions postnatally and cell autonomously to regulate the balance of local and long-range excitatory connectivity onto layer (L) 2/3 neocortical neurons in primary somatosensory cortex. Specifically, MEF2C promotes connectivity from multiple local excitatory circuits onto L2/3 neurons, while weakening callosal inputs from contralateral somatosensory cortex. Importantly, sensory experience is necessary for MEF2C regulation of both local and callosal circuits suggesting that MEF2C as a key player in experience-dependent, input-specific development of cortical circuits. Our findings have important implications for neurodevelopmental disorders. Brain mapping studies in humans with autism spectrum disorder (ASD) and schizophrenia (SCZ) reveal imbalances in local vs. long-range functional cortical connectivity. Furthermore, loss of function mutations in Mef2c are linked with intellectual disability (ID), ASD, epilepsy and SCZ in humans and mice. We hypothesize that sensory experience-driven neural activity regulates MEF2C-dependent transcriptional control of target genes to mediate input-specific development and plasticity of cortical circuits. Thus, a corollary of this hypothesis predicts that loss of function mutations in Mef2c or its effectors would result in imbalances of local and long-range connectivity, abnormal sensory-related behaviors and neuropsychiatric disease. To test these hypotheses: In Aim 1, we will use state-of-the-art optogenetic and photostimulation circuit mapping methods to determine if Mef2c deletion generally strengthens long-range inputs onto L2/3 neurons, bidirectionally regulates inputs based on their dendritic location and maintains the input-specificity of mature circuits. Aim 2: MEF2C functions both to repress transcription of target genes and stimulate their transcription in response to neural activity. Using MEF2C mutants, we will determine which of these functions mediates experience-dependent, input-specific regulation of cortical circuits. Aim 3: FMRP, mGluR5 and Arc are required for MEF2 regulation of synapses in culture neurons and associated with ASD, ID and SCZ. Here, we will test their role in MEF2C-mediated input-specific development of cortical circuits. Aim 4: To identify candidate genes that regulate cortical circuit input specificity, we will identify the postnatal MEF2C- and experience-dependent transcriptomes in L2/3 neocortical neurons using fluorescent-activated cell sorting (FACS) and RNA sequencing.

Summary This proposal requests R13 support for a longstanding, well-attended, and well-received Gordon Research Conference (GRC) on Excitatory Synapses and Brain Function on June 9-14, 2019 at University of New England, Biddeford ME. The GRC will be preceded by a Gordon Research Seminar (GRS) targeted towards graduate students and postdoctoral fellows on June 8, 9 2019 at the same location. The synapse is central to our understanding of brain function and behavior. In the central nervous system, excitatory synapses represent the primary means of information processing by local circuits and communication between brain regions and thus serve to mediate sensory processing, motor control, cognition and behavior. Importantly, synapses serve as the site of action for many commonly prescribed medications and synaptic dysfunction contributes to many neurological and psychiatric disorders. These include schizophrenia, autism, depression, drug addiction, Parkinson's disease, Alzheimer's disease, traumatic brain injury, stroke and epilepsy. In some cases, synaptic dysfunction is causal in disease, whereas in other cases it represents the downstream sequelae of one or more underlying molecular defects. Thus, therapeutic strategies for these diseases are targeted to modify, repair or maintain synaptic function. Therefore, a fundamental understanding of synapse development, structure, molecular organization, signaling function, and plasticity in both the healthy and diseased brain is essential to lessening the burden of human neurological disease and predicting and improving mental health. This conference is unique in its focus on excitatory synapses, the major synapse type in the brain, and in its multidisciplinary group of participants including structural biologists, molecular and developmental biologists, cell biologists, biochemists, biophysicists, neurophysiologists and systems neuroscientists. The conference is intended to relate fundamental insights in excitatory synaptic function to the impairments in synaptic function that occur in neurological disease, as well as the maladaptive plasticity that contributes to drug addiction. The goal of the conference is to identify and highlight fundamental new insights into synaptic function and dysfunction from a thematic approach. The program has been designed to also highlight cutting edge approaches and to stimulate new concepts, methods and technologies within a sound biological framework of fundamental neuroscience. The conference will bring together expert scientists worldwide in an environment that is conducive to discussion and exchange of ideas. The exchange of ideas at this conference has been a driving force for the field. We expect the 2019 GRC on Excitatory Synapses and Brain Function will shape future scientific directions and provide critical support for the mission of NINDS as well as other NIH institutes such as NIMH, NIDA and NIA.

Although ASD is more diagnosed in males, the behavioral manifestations of ASD, as well as the neuroanatomical changes and brain dysfunction associated with ASD, differ between males and females. There is little known of how ASD genes impact the female brain. Estrogen, acting through membrane bound estrogen receptor ? (ER?), interacts with and activates Gq-coupled Group 1 mGluRs, mGluR1 and mGluR5, selectively in female neurons in diverse brain regions to regulate signaling, neurophysiology, and behavior. Altered functioning of mGluRs, primarily mGluR5, is strongly implicated in the pathophysiology of ASD mouse models, most notably Fragile X Syndrome, but many others, and mGluR5 antagonists are in clinical trials of FXS children. We were the first to discover hyperactivity of mGluR5 function in the FXS mouse model, Fmr1 KO, and show this results from an abnormal mGluR5 complex; mGluR5 is dissociated from its postsynaptic scaffolding protein, Homer, which, in turn, leads to constitutive mGluR5 activity, abnormal signaling to downstream effectors and disease relevant phenotypes, such as circuit hyperexcitability. Despite the evidence for mGluR5 in ASD pathophysiology, the known sex-dependent regulation of mGluR1/5 by estrogen, little is known of how ASD-linked genes interact with estrogen to affect mGluR1/5 function and the consequences on ASD-relevant neurophysiology and behavior. We have discovered a sex-specific, mGluR5 -dependent dysfunction of sensory neocortical circuits in a mouse model of ASD that results from deletion of Pten (Phosphatase and tensin homolog deleted on chromosome 10), a suppressor of the PI3K/mTORC1 pathway. Specifically, we observe hyperexcitable neocortical circuit oscillations, termed UP states, in females of two distinct PTEN deletion models; an embryonic, knockout of Pten in hippocampus and layer 5 neocortical neurons, and a germline Pten heterozygous mouse (Pten-het), which is a genetically valid model for Pten-related ASD in humans. Hyperexcitable UP states in female Pten models are corrected by acute antagonism of mGluR5 or ER?, indicative of hyperactive mGluR5- ER? signaling. A candidate molecular substrate for enhanced mGluR5-ER? activity is the observed increase in mGluR5-ER?, and decreased mGluR5-Homer, complexes in female Pten-het cortex. We hypothesize that an imbalance in mGluR5 interactions with ERa and Homer in Pten deleted female neurons results in enhanced mGluR5-signaling in response to estrogen, acting on ER?, as well as constitutively active mGluR5 which leads to hyperexcitable cortical oscillations and ASD-relevant behaviors. We propose the following aims to test this hypothesis: Aim 1: Determine the sex-dependent interactions of Pten and ER? in cortical circuit dysfunction and mGluR5 complex regulation. Aim 2: Examine the sex-specific effects of mGluR5 and ER? on intrinsic and synaptic properties of Pten-deleted layer 5 neurons. Aim 3: Determine the sex-specific, mGluR5- and ER?-dependent in vivo cortical oscillations with EEG in Pten deletion models. Aim 4. Determine the role of female-specific, Pten-dependent cortical circuit hyperexcitability in ASD-relevant behaviors.