Lucas Pozzo-Miller, Ph.D. - US grants

BDNF has emerged as a potent modulator of activity-dependent synaptic development and plasticity. Dysfunctions in its trafficking and release, as well as in signaling through its receptor TrkB, have been implicated in the etiology of numerous developmental and neurodegenerative brain disorders. The prevailing notion is that Ca2+ release from IPs-sensitive stores is the only mechanism for BDNF to modulate Ca2+ homeostasis. However, direct evidence identifying the specific signaling and the sources of Ca2+ ions mediating those actions is limited and contradictory. In addition, nothing is known about the actions of native BDNF released during neuronal activity, despite the extensive evidence of the effects of exogenously applied BDNF. The long-term goal of this project is to identify the mechanisms by which TrkB activation sets in motion the wide range of BDNF effects on hippocampal neurons and synapses. In this competing renewal we will focus on our observation that BDNF elicits slow and sustained Ca2+ signals associated with membrane currents, reminiscent of capacitative Ca2+ entry and non-selective cationic currents mediated by TRPC channels, respectively. The specific hypothesis is that BDNF triggers TrkB-dependent PLCy activation followed by Ca2+ mobilization from IP- sensitive stores in CA1 pyramidal neurons, leading to the activation of capacitative Co2* entry and a sustained inward current mediated by TRPC channels. The first two Aims will identify the elementary actions of exogenously applied BDNF on membrane currents and intracellular Ca2+ levels, while the third Aim will use this knowledge to identify similar responses evoked by native BDNF released during afferent stimulation. Simultaneous Ca2+ imaging and electrophysiological recording, combined with pharmacological inhibitors, function-blocking antibodies, and siRNA- mediated knockdown will be used to identify the components of the signaling pathway. BDNF scavengers will allow determining whether BDNF released by afferent activity evokes similar Ca2+ signals and inward currents. We expect the proposed studies to provide the most comprehensiveunderstatingto date of the immediateactions of BDNF on membrane currents and intracellular Ca2+ homeostasis, leading to enduring changes in synaptic function, structure, and plasticity.

The objective of the Developmental Neurobiology Imaging Core is to provide state-of-the-art equipment and technical support for experimental projects on the assembly and modulation of synaptic structure and function in the cerebral cortex. By sharing technical expertise, equipment, facilities, and professional staff, this Core will facilitate cross-project collaborations among the different projects of the proposed Program Project Grant The Imaging Core will perform cytological and histological processing of experimental tissues, and perform image analysis in a regular reliable fashion for the development neurobiology projects outlined in the Program Project. New projects initiated through the shared use of the Core by the Program Project P.I.s will also be supported by the Developmental Neurobiology Imaging Core. In particular, the Developmental Neurobiology confocal, and electron microscopic levels for all five projects compromising the Program Project. In each of these projects, experimental nervous system or cells from fetal and neonatal animals will be routinely processed in substantial volumes for microscopy, and quantitative image analysis. In addition, luminometric analysis of samples from cultured neurons, slices, and synaptosomal preparations will allow for rapid, highly efficient quantitative analysis of glutamate release.

DESCRIPTION: The Developmental Neurobiology Imaging and Tissue Processing Core is an extension of the Imaging Core for a pending program project. This Core provides the equipment for basic histology, immunocytochemistry, and light, confocal, laser scanning confocal, as well as electron microscopy. The Core will also provide the technical support to train staff and MRRC investigators the histological and cytological processing of experimental tissues or cells and quantitative image analysis. The Core represents a continuation of services offered and equipment purchased through the previous P50 center grant that has been supported for five years. This Core would be used potentially by 15 of the MRRC investigators for their research, but primarily by the seven MRRC investigators who are principal investigators or co-investigators on individual projects in the program project application. The Core Director indicates that if the program project is not funded, the request for this Core will be withdrawn.

DESCRIPTION: The Simultaneous Laser Scanning Imaging Core will allow investigators to perform simultaneous electrophysiological recordings and single-cell imaging of intracellular ion concentrations, presynaptic vesicle recycling, or cellular morphology of cells transfected with GFP in neurons and glial cells from brain slices. The major core facility will consist of one dedicated laser scanning microscope for confocal microscopy and electrophysiological setup for patch-clamping. The Core requests funding for a conventional confocal light microscope in Year -01, an entire new electrophysiology setup, including a patch-clamp microscope in Year -02, upgrading the conventional confocal light microscope to a two-photon laser scanning microscope in Year -03, and addition of UV flash unit and an additional postdoctoral fellow in Year -04. This Core will most likely be used by ten investigators whose research projects demand simultaneous imaging during electrophysiological recording. It is predicted that the Core will be utilized by one investigator for 10 days to two weeks at a time.

[unreadable] DESCRIPTION (provided by applicant): The neuropathology of mental retardation is thought to be associated with deficits in synaptic structure and function. Our goal is to make contributions into understanding the formation and maintenance of dendritic spines and of postsynaptic Ca2+ homeostasis in neurons expressing Rett Syndrome-associated mutations in MECP2. Rett syndrome (RTT) is an X- linked developmental disorder and the leading cause of mental retardation in females. Mutations in the transcriptional represser MECP2 have been identified in >90% of RTT cases. One of the target genes of MeCP2 is bdnf, brain-derived neurotrophic factor. Considering that BDNF has recently emerged as a potent modulator of activity-dependent synaptic development and plasticity in the postnatal brain, including fundamental neuronal properties such as dendritic spine density and form and neuronal Ca2+ signaling, we hypothesize that a deregulation of BDNF signaling may underlie the dendritic pathologies observed in RTT. The specific hypothesis to be tested is twofold: 1) RTT-associated MECP2 mutations cause dendritic spine loss leading to Impaired dendritic Ca2+ signaling in hippocampal pyramidal neurons through reduced BDNF signaling; 2) impaired dendritic structure in MECP2 mutant neurons can be reverted by BDNF treatment. The consequences of mutant MECP2 expression will be evaluated in neurons maintained in organotypic slice cultures and transfected by particle-mediated gene-transfer. The biolistic gene-transfer approach provides a more flexible way to introduce different mutant forms of MECP2 compared to the generation of transgenic or knockout mice, in addition to allow the co-transfection of other cDNAs or knockdown siRNA constructs of interest. Thus, it represents a novel cellular model of RTT. Transfected neurons will be studied by laser-scanning confocal and time-lapse multiphoton microscopy, as well as by simultaneous Ca2+ imaging and whole-cell intracellular recordings. This combination of state-of-the-art approaches has never been used to investigate MECP2 function, or applied to animal models of RTT. We expect the proposed studies to provide novel insights into the consequences of mutant MECP2 expression in hippocampal neurons in a cellular model of RTT. [unreadable] [unreadable] [unreadable]

Core C The Developmental Neurobiology Imaging and Tissue Processing Core (Core C) provides state-of-the-art equipment and technical support for experimental projects on the assembly and modulation of synaptic, neuronal, and glial structure and function. By sharing technical expertise, equipment, facilities, and professional staff, the Core facilitates cost-effective, cross-project collaborations. The Developmental Neurobiology Imaging and Tissue Processing Core performs cytological and histological processing of experimental tissues, and performs image analysis in a regular and reliable fashion for the developmental synaptic neurobiology, neuroimmunoendocrinology and infectious disease, and molecular biological and genetic studies projects outlined in the MRRC. In particular, the Developmental Neurobiology Imaging and Tissue Processing Core provides a facility for cell and tissue processing and quantitative image analysis at the light, confocal, and electron microscopic levels for many projects comprising the MRRC portfolio. In this regard, the Imaging Core contributes to an interrelationship and synergism among the component projects, resulting in greater scientific productivity and improved cost effectiveness than individual projects could achieve separately. The currently re-configured Neurobiology Tissue Processing and Imaging Core is the result of a merger of two previously existing cores (developmental neurobiology and tissue processing AND combined LSCM imaging and electrophysiology) that had two clearly delineated objectives from the original P30. For many investigators, these two objectives are sequential and therefore have led to a sequential use of two non-overlapping facilities. Now, these tasks are integrated allowing an investigator to accomplish cell or tissue processing and various imaging modalities including: time-lapse live imaging for stable, long-term high-resolution morphological studies;simultaneous real time two photon laser scanning confocal microscopy imaging and recording of plasma membrane potential by the whole-cell patch-clamp technique;monitoring of presynaptic release probability of individual synapses by the de-staining rate of the FM fluorescent dyes;high resolution electron microscopy including serial section analysis of reconstructed cells;and combined immunohistochemical staining and electron microscopy for subcellular localization of epitopes a single unit. These techniques require constantly evolving skills on the side of the investigators and a significant investment in instrumentation.

DESCRIPTION (provided by applicant): Rett syndrome (RTT), an autism spectrum disorder, is a devastating childhood disorder due to its impact on individuals (1:10,000-15,000 births worldwide), their families and society. RTT is caused by loss-of- function mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2), a transcriptional regulator that binds to methylated CpG sites in promoter regions of DNA. An imbalance of excitatory and inhibitory synaptic function in the hippocampus has been implicated in neurodevelopmental disorders associated with cognitive impairments and mental retardation. Mouse cortical neurons lacking Mecp2 show low levels of neuronal activity caused by an excitation/inhibition imbalance that favors synaptic inhibition, and Mecp2 expression levels modulate excitatory synapse formation between hippocampal neurons. One of the target genes of Mecp2 transcriptional control is Brain-derived neurotrophic factor (Bdnf), a potent modulator of activity-dependent synaptic development, function and plasticity. Considering that BDNF is critical for the maturation of inhibitory GABAergic synapses, and based on our Preliminary Results, our general hypothesis is that impaired development of inhibitory GABAergic synapses due to reduced activity- dependent BDNF release from Mecp2-deficient neurons causes an imbalance of excitatory and inhibitory synaptic function in the hippocampus. We propose the following four Specific Aims: (1) test if the hyperexcitable hippocampal network of neuronal Mecp2 null mice is caused by impaired GABAergic synapse function in area CA3; (2) test whether activity-dependent BDNF release from mossy fibers, the axons of dentate gyrus granule cells, is reduced in neuronal Mecp2 null mice; (3) generate a novel RTT model - dentate granule cell-specific Mecp2 knockout mice - and test whether hippocampal hyperexcitability is associated with impaired activity-dependent BDNF release from granule cell mossy fibers; (4) test if enhancing BDNF expression or mimicking BDNF/TrkB signaling prevents hippocampal hyperexcitability in Mecp2 null mice and dentate granule cell-specific Mecp2 knockout mice. We anticipate that the proposed experiments will yield novel information regarding the consequences of Mecp2 deletion for the excitation/inhibition balance in the hippocampus, uncovering fundamental brain mechanisms involved in the neuropathology of RTT and Autism Spectrum Disorders, and testing an experimental rationale to relieve cognitive impairments and mental retardation in children with associated neurodevelopmental disorders. PUBLIC HEALTH RELEVANCE: Rett syndrome (RTT) is an X-linked neurodevelopmental disorder associated with autism and mental retardation, which is caused by mutations in MECP2, a DNA-binding protein that regulates target genes, including Bdnf. We will test whether impaired development of hippocampal inhibitory synapses due to reduced BDNF release contributes to the excitatory/inhibitory imbalance of synaptic function implicated in cognitive impairments and autism in RTT.

DESCRIPTION (provided by applicant): Rett syndrome (RTT), an autism spectrum disorder, is a devastating childhood disorder due to its impact on individuals (1:10,000-15,000 births worldwide), their families and society. RTT is caused by loss-of- function mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2), a transcriptional regulator that binds to methylated CpG sites in promoter regions of DNA. An imbalance of excitatory and inhibitory synaptic function in the hippocampus has been implicated in neurodevelopmental disorders associated with cognitive impairments and mental retardation. Mouse cortical neurons lacking Mecp2 show low levels of neuronal activity caused by an excitation/inhibition imbalance that favors synaptic inhibition, and Mecp2 expression levels modulate excitatory synapse formation between hippocampal neurons. One of the target genes of Mecp2 transcriptional control is Brain-derived neurotrophic factor (Bdnf), a potent modulator of activity-dependent synaptic development, function and plasticity. Considering that BDNF is critical for the maturation of inhibitory GABAergic synapses, and based on our Preliminary Results, our general hypothesis is that impaired development of inhibitory GABAergic synapses due to reduced activity- dependent BDNF release from Mecp2-deficient neurons causes an imbalance of excitatory and inhibitory synaptic function in the hippocampus. We propose the following four Specific Aims: (1) test if the hyperexcitable hippocampal network of neuronal Mecp2 null mice is caused by impaired GABAergic synapse function in area CA3; (2) test whether activity-dependent BDNF release from mossy fibers, the axons of dentate gyrus granule cells, is reduced in neuronal Mecp2 null mice; (3) generate a novel RTT model - dentate granule cell-specific Mecp2 knockout mice - and test whether hippocampal hyperexcitability is associated with impaired activity-dependent BDNF release from granule cell mossy fibers; (4) test if enhancing BDNF expression or mimicking BDNF/TrkB signaling prevents hippocampal hyperexcitability in Mecp2 null mice and dentate granule cell-specific Mecp2 knockout mice. We anticipate that the proposed experiments will yield novel information regarding the consequences of Mecp2 deletion for the excitation/inhibition balance in the hippocampus, uncovering fundamental brain mechanisms involved in the neuropathology of RTT and Autism Spectrum Disorders, and testing an experimental rationale to relieve cognitive impairments and mental retardation in children with associated neurodevelopmental disorders.

DESCRIPTION (provided by applicant): Understanding the pathophysiological mechanisms of Rett syndrome (RTT) at the cellular and molecular levels, and establishing successful bioassays for evaluation of potential therapeutic strategies take priority in the path of research on this neurodevelopmental disorder. RTT, an autism spectrum disorder, is a devastating childhood disability due to its impact on individuals (1:10,000 births worldwide), their families and society RTT is caused by loss-of-function mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2), a transcriptional regulator that binds to methylated CpG sites in promoter regions of several genes, including the neurotrophin Bdnf, and the Ca2+-permeable non-selective cationic channel subunits Trpc3 and Trpc6. The availability of endogenously expressed BDNF for its activity-dependent release can be monitored with membrane currents and dendritic Ca2+ signals mediated by TRPC channels. Preliminary Results demonstrate that TRPC currents and Ca2+ signals evoked in CA3 pyramidal neurons by stimulation of presynaptic mossy fiber (MF) are smaller in symptomatic Mecp2 mutant mice. Responses evoked by either recombinant BDNF or a non-hydrolyzable DAG analog (to activate TRPC channels) are also impaired in Mecp2 mutant neurons. Consistently, mRNA and protein levels of both BDNF and TRPC3 are lower in Mecp2 mutant hippocampus. Preliminary Results show that the TRPC6 channel activator hyperforin evokes membrane currents and Ca2+ signals, and promotes dendritic spine maturation in CA3 pyramidal neurons, resembling well-known actions of BDNF. Based on these Preliminary Results and since TRPC3 and TRPC6 form heteromultimers, our hypothesis is that impaired BDNF signaling through TRPC3/6 channels in Mecp2 mutant mice can be overcome by treatment with the selective TRPC6 activator hyperforin to reverse two RTT-like phenotypes: hippocampal network hyperactivity and immature dendritic spines. We propose two Specific Aims: 1. Test whether membrane currents and Ca2+ signals evoked by BDNF and mediated by TRPC channels in CA3 pyramidal neurons and GABAergic interneurons are impaired in Mecp2 mutant mice; and 2. Test whether treatment with hyperforin reverses hippocampal phenotypes in Mecp2 mutant mice, i.e. hippocampal network hyperactivity and immature dendritic spines. Identifying TRPC3/6 channels as novel targets for pharmacological intervention is necessary for pre-clinical trials leading to rational treatments for RTT and other neurodevelopmental disorders associated with MECP2 mutations and impaired BDNF signaling.

PROJECT SUMMARY/ABSTRACT Rett syndrome (RTT), an X-linked autism spectrum disorder, is a devastating childhood disability and has a tremendous impact on individuals (1:10,000 births), their families and society. The majority of RTT cases are caused by loss-of-function mutations in the gene encoding methyl-CpG-binding protein 2 (MeCP2). RTT girls are born without any obvious problems but abruptly develop a host of neurological deficits, including irregular breathing, loss of purposeful hand movement and speech, seizures, and intellectual disability. Among these symptoms, motor dysfunction and deficits in attention-related behavioral shifting are the most profound, bearing resemblance to several brain disorders of basal ganglia origin. Disease-modifying therapies that are designed for the treatment of RTT require a clear understanding of the underlying pathophysiology at the molecular, cellular, and network levels. Dysfunction of the striatum, where most inputs enter the basal ganglia, may play a significant role in RTT pathogenesis. The striatum receives glutamatergic excitatory projections from the thalamus, which integrate and modulate cortical inputs for proper striatal output. Our preliminary data demonstrate altered function and plasticity of thalamo-striatal synapses in Mecp2 knockout (KO) mice, which may contribute to dysfunction of cortico-striatal synaptic. Our hypothesis is that altered thalamo-striatal synaptic function in Mecp2 KO mice disintegrates the striatal role in integrating cortical inputs. We propose two Specific Aims: (1) characterize thalamic neurons and their synaptic projections to the dorsal striatum of Mecp2 KO mice; (2) test whether the impact of thalamic inputs on cortico-striatal system in dorsal striatum is impaired in Mecp2 KO mice. We anticipate that these experiments will yield novel information regarding the consequences of MeCP2 loss on thalamo-striatal synaptic transmission and plasticity, as well as on its integration and control of cortico-striatal connections. These findings will uncover fundamental brain mechanisms involved in RTT neuropathology, and aid to develop and test novel therapeutic approaches. Our studies will also have deep implications for the understanding and treatment of other neurological disorders with common symptoms and neural substrates, including Huntington and Parkinson diseases, as well as Tourette and Angelman syndromes.

This REU site award to the University of Alabama at Birmingham (UAB), in Birmingham, AL, will support 10 students for 10 weeks during the summers of 2017-2019. The theme of the Summer Program in Neuroscience (SPIN) REU program, hosted by the Department of Neurobiology, is the development and plasticity of the nervous system. The program will allow students to conduct fundamental neuroscience research across a range of research questions and methodological approaches. Students will participate in activities and seminars to develop their graduate school application and prepare for graduate school interview. This REU site is devoted to supporting students seriously considering post-baccalaureate research careers following completion of their undergraduate degree program. While research experience is not a pre-requisite, students should be able to demonstrate a commitment to fundamental discovery science. Applications are accepted online through the program website (below). Students will be selected by the SPIN admissions committee based on their cumulative GPA in a STEM major, evidence of upper level coursework to prepare for research in a neuroscience lab, response to questions in the application, and recommendation letters.

It is anticipated that a total of 30 students primarily from schools with limited local research opportunity will be trained through the REU program. Students from under-represented groups are strongly encouraged to apply. Students will learn how research is conducted, will integrate into an active research team, will participate in professional development activities, and prepare for success in graduate level education. Students will build their technical skills, participate in journal clubs, present their research results, and attend the annual Neural Conference at UAB.

A common web-based assessment tool used by all REU Site programs funded by the Division of Biological Infrastructure will be used to determine the effectiveness of the training program. Students will be tracked after the program concludes to determine their career paths. Students will be asked to respond to an automatic email sent via the NSF reporting system. More information about the program is available by visiting http://www.uab.edu/medicine/neurobiology/education/undergraduate-summer-research or by contacting the program directors: PI, Dr. Lucas Pozzo-Miller (lucaspm@uab.edu) or co-PI, Dr. Gwendalyn King (gdking@uab.edu).

PROJECT SUMMARY / ABSTRACT The hippocampus is critical for the formation and consolidation of spatial memories and, through its efferent projections, also contributes to other cognitive tasks. The goal of this project is to determine whether altered neuronal network activity in the hippocampus of a mouse model of autism propagates to, and alters distal cortical regions involved in social behaviors. A deficit in social interaction is one of the core symptoms of autism spectrum disorders, which includes Rett syndrome (RTT), a neurodevelopmental disorder caused by loss-of-function mutations in the transcriptional regulator MECP2. Altering the excitation and inhibition (E/I) balance in the medial prefrontal cortex (mPFC) of mice causes social behavior impairments reminiscent of autism spectrum disorders. The ventral hippocampus (vHIP) of Mecp2 knockout mice (KO) mice is hyperactive due to an E/I imbalance driven by impaired inhibition and enhanced excitation, which results in saturated synaptic plasticity at excitatory synapses. Intriguingly, CA1 pyramidal neurons of the vHIP project their axons to the mPFC, making direct monosynaptic connections with excitatory pyramidal neurons and inhibitory interneurons. We propose to characterize the influence of the vHIP on the activity of the mPFC through this long-range monosynaptic glutamatergic projection, testing whether altered vHIP network activity is causal to mPFC dysfunction and social interaction deficits. We hypothesize that hyperactive hippocampal afferents alter network activity in the mPFC of Mecp2 KO mice by affecting the E/I balance, which contributes to social interaction deficits. To test this hypothesis, we will identify the cellular targets of direct vHIP afferents in the mPFC of RTT mice and characterize their function and role in social behaviors using a combination of optogenetics, chemogenetics, ex vivo and in vivo electrophysiology and Ca2+ imaging, anterograde and retrograde tract tracing, and behavioral assessments. We propose the following Specific Aims: (1) characterize the cellular targets of the monosynaptic projection from the vHIP to the mPFC in Mecp2 KO mice; (2) characterize synaptic function and long-term synaptic plasticity of the monosynaptic projection from the vHIP to the mPFC in Mecp2 KO mice; (3) test if chemogenetic modulation of vHIP activity restores mPFC network activity and social behaviors in Mecp2 KO mice. In addition to defining novel pathophysiological mechanisms of RTT, these studies will provide fundamental information regarding the functional and structural properties of the long-range monosynaptic connection between the vHIP and mPFC in typically developing brains. Thus, the impact of this work will extend beyond RTT to other neuropsychiatric disorders in which propagation of network dysfunction from the hippocampus to the mPFC is thought to contribute to cognitive deficits.

ABSTRACT Astrocytes are a key element of brain connectivity because they regulate neuronal communication at synapses. Bergmann glial cells (BGCs), a major type of astrocytes in the cerebellum, are intimately associated with excitatory glutamatergic synapses between Purkinje cell (PC) spines and parallel fibers or climbing fibers, playing a significant role in synaptic stability and long-term plasticity. BGCs express a high density of Ca2+- permeable GluA2-lacking AMPA receptors (CP-AMPARs), which are required for the maintenance of the BGC processes that ensheath PC spine synapses. Genetic deletion of CP-AMPARs results in impaired associative motor learning. Our preliminary data indicate that CP-AMPARs in BGCs are subject to dopaminergic (DAergic) modulation. Pharmacological stimulation of D1 receptors enhances CP-AMPARs-mediated currents in BGCs, and phosphorylates the GluA1 subunit at Ser845. Using transgenic mice with fluorescently-tagged D1 receptors and anterograde axonal tracing, we determined that D1 receptors are exclusively expressed in BGCs, which receive DAergic afferents from the ventral tegmental area and substantia nigra pars compacta in the midbrain. Intriguingly, D1 receptor activation also increases PC firing and locomotor activity. Based on these preliminary findings, we hypothesize that activation of D1 receptors induces insertion of CP- AMPARs in BGCs, which in turn modulates PC synaptic function. To test this hypothesis, we will use a combination of experimental approaches, including whole-cell recordings with optogenetic stimulation, immunocytochemistry, molecular biology, and pharmacology. We propose two Specific Aims: (1) characterize membrane insertion of GluA1 in Bergmann glial cells induced by DA; and (2) define the role of DAergic modulation of Bergmann glial cells in PC activity. We expect to generate conceptually novel knowledge regarding DA modulation of cerebellar circuitry function by defining the contribution of CP-AMPARs in BGCs to synaptic function in PCs. These new findings will not only deepen our understanding of DAergic function in the healthy brain, but also provide additional avenues for the development of disease-modifying therapies for motor-related neurological disorders.

PROJECT SUMMARY / ABSTRACT The hippocampus is critical for the formation and consolidation of spatial memories and contributes to other cognitive tasks through its efferent projections. The goal of this project is to determine whether altered neuronal network activity in the hippocampus of an autism mouse model propagates to, and alters distal cortical regions involved in social behaviors. One of the core symptoms of autism spectrum disorders, such as Rett syndrome (RTT), is a deficit in social interaction. In mice, similar social behavior impairments can be modeled by altering the excitation and inhibition (E/I) balance in the medial prefrontal cortex (mPFC). For RTT, knocking out the gene that is mutated in humans, Mecp2, causes hyperactivity in the ventral hippocampus (vHIP) due to an E/I imbalance driven by impaired inhibition and enhanced excitation, resulting in saturated synaptic plasticity at excitatory synapses. Intriguingly, CA1 pyramidal neurons of the vHIP project their axons to the mPFC, making direct monosynaptic connections with excitatory pyramidal neurons and inhibitory interneurons. We propose to characterize how the vHIP influences mPFC activity through this long-range monosynaptic glutamatergic projection. Importantly, we will test whether altered vHIP network activity is causal to mPFC dysfunction and deficits in social interaction. We hypothesize that atypically strong vHIP afferents alter network activity in the mPFC of Mecp2 KO mice by affecting the E/I balance and synaptic plasticity, which in turn contributes to social interaction deficits. To test this hypothesis, we will identify the cellular targets of direct vHIP afferents in the mPFC of RTT mice and characterize their function in social behaviors using a combination of optogenetics, chemogenetics, ex vivo and in vivo electrophysiology and Ca2+ imaging, anterograde and retrograde tract tracing, and unbiased machine-learning behavioral assessments. We propose the following Specific Aims: 1) identify and characterize the cellular targets of the monosynaptic projection from the vHIP to the mPFC in Mecp2 KO mice; 2) characterize long-term synaptic plasticity of the vHIP-mPFC projection in Mecp2 KO mice; and 3) determine if chemogenetic modulation of the activity of mPFC-projecting vHIP neurons alters mPFC function and social behaviors. These studies will provide fundamental information on the functional and structural properties of the long- range connection between the vHIP and mPFC in the developing brain. The impact of this work extends beyond RTT to other neuropsychiatric disorders in which propagation of network dysfunction from the hippocampus to the mPFC is believed to contribute to cognitive deficits.