2009 |
Higley, Michael James |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Dopamineergic Modulation of Synaptic Transmission in the Straitum
DESCRIPTION (provided by applicant): The striatum is a key component of the basal ganglia, a collection of forebrain nuceli whose function is critical for the proper execution of voluntary movements. Normal striatal activity is regulated by the neuromodulator dopamine, and loss of dopaminergic inputs to the striatum results in the profound neurological impairments seen in Parkinson's disease. Nevertheless, despite numerous advances in understanding the contribution of dopaminergic signaling to striatal function, the precise mechanisms of dopamine's actions remain elusive. Dopamine is thought to influence the response of striatal neurons to synaptic inputs that target dendritic spines, small ~T micron protrusions of the postsynaptic cell membrane not easily studied by conventional electrophysiological methods. However, recent developments using optical imaging have enabled the study of neuronal activity at this small spatial scale. The specific goal of this work is to obtain a detailed understanding of how dopamine regulates synaptic transmission and integration in the striatum. In particular, the experiments will address two key questions. First, how does dopamine modulate the response of striatal neurons to excitatory synaptic inputs at the level of single synapses? Second, how does dopamine alter the ability of neurons to integrate spatially and temporally distributed synaptic inputs? To address these questions, a combination of electrophysiology and 2-photon laser scanning microscopy will be used to stimulate and record synaptic activity within individual dendritic spines. More generally, this proposal aims to provide new insight into the relationship between dopaminergic modulation and normal striatal activity. By expanding our knowledge of the interactions between transmitter systems in the striatum, these studies will enhance our understanding of the role of the striatum in behavior and contribute to the development of better models of Parkinson's disease and other movement disorders.
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0.934 |
2012 — 2021 |
Higley, Michael James [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Cellular Mechanisms of Gabaergic Inhibition in Neocortical Dendrites
DESCRIPTION (provided by applicant): Cortical GABAergic interneurons (INs) play critical roles in controlling normal patterns of brain activity and are implicated in the pathophysiology of neuropsychiatric disease. While many INs target pyramidal neuron (PN) somata, where they regulate the magnitude and timing of spike output, the majority of GABAergic synapses are formed onto PN dendrites, where their role in cellular function is less well understood. Dendrite-targeting interneurons that express the peptide somatostatin (SOM-INs) are hypothesized to provide negative feedback to distal PN dendrites that scales with local network activity. However, technical limitations to selectively controlling the output of these neurons while simultaneously measuring dendritic activity with high spatial resolution have prevented a clear elaboration of SOM-IN function. Our long-term goal is to understand how distinct pools of GABAergic INs contribute to cellular and circuit regulation in the prefrontal cortex (PFC), a brain region associated with higher cognitive processes that may be disrupted in illnesses such as schizophrenia. In this proposal, our primary objective is to identify how SOM-INs regulate calcium (Ca) signaling in the dendrites of PFC PNs. We also focus on understanding how dendritic inhibition is shaped by the intrinsic voltage-gated properties of PN dendrites and the neuromodulator dopamine. Our central hypothesis is that GABAergic inhibition is both heterogeneous and compartmentalized in PN dendrites. We expect that this compartmentalization is dependent on many factors, including the spatiotemporal pattern of inhibitory synaptic activation and the electrical properties of dendritic structures such as spines Guided by strong preliminary data, we will examine this central hypothesis in three specific aims: 1) Determine the role of GABAergic inhibition in shaping dendritic Ca signaling. 2) Identify the voltage-gated dendritic conductances that contribute to inhibitory synaptic integration. 3) Determine the actions of dopamine on dendritic inhibition and Ca signaling. The data generated by these experiments will generate new insights into the contribution of GABAergic transmission to both neuronal cell biology and the function of cortical circuits. We expect our results will als highlight new avenues into the investigation of the pathophysiology underlying neuropsychiatric disorders resulting from perturbation of both GABAergic and dopaminergic signaling. PUBLIC HEALTH RELEVANCE: Inhibitory GABAergic neurons provide important control over the activity of neurons in the neocortex. This proposal will determine the role of GABAergic synapses in regulating calcium, a key biochemical signaling molecule, within the dendrites of cortical neurons. Results from these studies will help identify novel cellular mechanisms that may contribute to the pathophysiology of neuropsychiatric disorders and suggest new avenues for therapeutic intervention.
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1.009 |
2017 — 2021 |
Cardin, Jessica A [⬀] Higley, Michael James (co-PI) [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Role of Gabaergic Interneurons in Developmental Dysregulation of Cortical Function
Project Summary Neurodevelopmental disorders such as autism produce significant emotional, physical, and economic consequences for affected individuals and their families. Autism spectrum disorders (ASDs) affect approximately 1% of the worldwide population and are associated with cognitive deficits in perception, social interaction, and communication, all functions served by the cerebral cortex. While the cellular mechanisms underlying ASDs remain unclear, recent evidence suggests disruption of GABAergic inhibitory interneurons (INs) may contribute to abnormal development and function of cortical circuits. Genetic studies of ASD patients have identified several candidate genes including MeCP2, a gene strongly associated with Rett Syndrome (RTT), and IN-specific deletion of MeCP2 produces many ASD-like phenotypes. However, little is known about the specific cellular, synaptic, and circuit consequences of IN dysregulation. To address this question, we propose to use a mouse model in which MeCP2 is deleted in a distinct subpopulation of dendrite-targeting GABAergic INs, focusing on the mouse visual system. Altered sensory processing is a hallmark of ASDs, and the wealth of knowledge on the normal function of the visual cortex will provide critical context for interpreting the cellular mechanisms underlying observed circuit and behavioral abnormalities. Specifically, we will test the following three hypotheses: (1) MeCP2 expression in somatostatin-expressing (SOM) INs regulates cortical neuronal morphology and connectivity. (2) SOM-IN dysregulation contributes to cortical circuit dysfunction in the MeCP2 model. (3) SOM-IN-specific MeCP2 deletion impairs visual perception. We will combine electrophysiological and anatomical analyses ex vivo with high-density neuronal recordings and behavioral analyses in vivo. This approach will allow us to generate novel insights into the links between structural and synaptic dysregulation and dysfunction of neural circuits in an established model of neurodevelopmental disorders.
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1.009 |
2018 — 2021 |
Higley, Michael James (co-PI) [⬀] Koleske, Anthony J [⬀] |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Impact of Excitatory Synapse Maturation On Synaptic Plasticity and Stability
Immature excitatory synapses in the perinatal brain contain high release probability (Pr) presynaptic terminals coupled to postsynaptic specializations with GluN2B subunit-containing NMDA receptors (hi-Pr, hi-GluN2B synapses). For over two decades, we have known that these immature synapses mature in an activity- dependent manner to low-Pr, low-GluN2B synapses, but the mechanisms that coordinate this transition, why it occurs, and how it contributes to circuit plasticity and stability remain controversial and are fundamental unanswered questions. Addressing these issues will identify basic mechanisms that control synapse development and that may be disrupted in neurodevelopmental and psychiatric disorders. Disruption of the Arg/Abl2 kinase in mice yields a population of hi-Pr, hi-GluN2B synapses that persist into early adulthood. The persistence of these immature synapses drives a >40% net loss of hippocampal synapses between postnatal day (P) 21 and P42, and impairs synaptic plasticity and behavior. Building on these findings, we will identify new regulators of synapse maturation, and determine how they regulate synaptic plasticity and stability. In Aim 1, we will identify the cell surface receptors that activate Arg to coordinate the maturation from hi-Pr, hi- GluN2B synapses to low-Pr, low-GluN2B synapses. We provide preliminary data that integrin ?3?1 adhesion receptor and platelet-derived growth factor receptor ? (PDGFR?) act upstream of Arg to control synapse function and stability. We will use selective gene inactivation in the pre- and postsynaptic neurons along with genetic epistasis and rescue experiments to address how and where these receptors interact with Arg and each other to regulate Pr and postsynaptic GluN2B levels. In Aim 2, we will elucidate how Arg mediates GluN2B downregulation at the synapse. Arg-mediated signaling is critical to downregulate GluN2B during maturation. We identified the SHP2 tyrosine phosphatase and the NMDAR-associated protein BRAG1, both mutated in intellectual disability, as likely functional links between Arg and developmental GluN2B downregulation. We will use biochemical, cell-based, and genetic approaches to test how Arg interacts with SHP2 and BRAG1 to downregulate GluN2B function. In Aim 3, we will characterize how immature and mature synapses differentially contribute to plasticity and stability. We will use patterned glutamate uncaging at single synapses to test whether hi-Pr, hi-GluN2B and low-Pr, low-GluN2B synapses have altered ability to undergo long-term potentiation (LTP) and long-term depression (LTD) in arg?/? mice. We will use in vivo imaging to examine how enlarged dendritic spines at hi- Pr, hi-GluN2B cortical synapses in arg?/? mice differ from normal spines in their plasticity and stability. Our studies will elucidate the mechanisms by which receptors act through Arg and its downstream targets to control Pr and NMDAR composition during synapse maturation. Disruption of these mechanisms may underlie the defects in synapse development, plasticity, and stability in intellectual disability and other brain disorders.
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1.009 |
2020 — 2021 |
Higley, Michael James [⬀] |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Behavior-Dependent Classification of Neocortical Cell Types
PROJECT SUMMARY The classification of cell types in the cerebral cortex is a major goal in neuroscience, guided by the expectation that elaborating a complete neuronal census will provide important insights into both healthy brain function and the pathophysiology of disease. Traditional schemes for grouping cells have largely focused on morphological, electrophysiological, and hodological features derived from ex vivo preparations. However, the development of tools for labeling and recording neurons in vivo presents substantial opportunities to broaden our knowledge of what constitutes a class of cells. Here, we propose that in vivo activity is an additional axis on which to categorize neuronal types. Our overall goal is to develop a novel strategy for linking behaviorally relevant activity with a traditional characterization of cellular properties. To that end, we focus on the role of behavioral state in modulating the firing patterns of neurons in the mouse neocortex. Several recent studies have demonstrated that locomotion is associated with significant but heterogeneous alteration in activity, with different cells showing enhanced or suppressed output during periods of motor behavior. We will take advantage of a novel green fluorescent protein, called CaMPARI2, enabling us to label cortical neurons that are active during arousal by coupling light stimulation with real-time detection of locomotion. This approach is followed by ex vivo analyses comparing locomotion-sensitive (photo-converted, red) and -insensitive (green) cells side by side. In Aim 1, we characterize the regional and laminar differences in cells throughout the neocortex whose activity is modulated by locomotion. In Aim 2, we examine the electrophysiological, morphological, and transcriptional properties of these cells. Overall, these efforts to gain a complete picture of functional neuronal diversity will be essential for understanding healthy brain function and for driving new interventions aimed at the prevention and treatment of neuropsychiatric disorders.
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1.009 |
2021 |
Higley, Michael James [⬀] |
DP1Activity Code Description: To support individuals who have the potential to make extraordinary contributions to medical research. The NIH Director’s Pioneer Award is not renewable. |
Bridging Cellular and Systems Neuroscience: Synaptic Dynamics Underlying Behavior
SUMMARY A major challenge to our progress in understanding the functional organization of the nervous system is the practical schism between cellular/molecular and systems sub-fields within the broader neuroscience community. For example, synaptic transmission is the fundamental mechanism by which activity propagates between neurons. While we have a detailed understanding of the cellular and molecular mechanisms underlying this process, the dynamic range and operating regime of synapses in the intact, behaving animal is essentially unknown. Based on recent data from our laboratory, our overall goal in this proposal is to investigate the hypothesis that variations in behavior over multiple time scales are associated with fluctuations in the strength of synaptic transmission within neuronal networks of the mammalian neocortex. With a groundbreaking combination of conceptual and methodological innovations, we will specifically identify the modifications of synaptic function that correspond to changes in behavioral state and perceptual learning. Specifically, we propose to monitor variation in synaptic release probability, potency, and integration for targeted circuits within the mouse visual cortex, relating these properties to behavioral state transitions and enhanced perceptual ability associated with visuomotor learning. Overall, this ambitious paradigm will generate critical new insights into the relationships between synapses, circuits, and behavior and open up new avenues of exploration that unite diverse areas of the neuroscience community.
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1.009 |
2021 |
Cardin, Jessica A (co-PI) [⬀] Higley, Michael James (co-PI) [⬀] Koleske, Anthony J [⬀] |
R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
The Role of Trio Signaling in Neuronal Development, Synaptic Function, and Circuit Connectivity
Abstract Heterozygous loss-of-function (LOF) or damaging variants in the TRIO gene are associated with increased risk for schizophrenia and autism spectrum disorders. However, the functional role of TRIO in neuronal biology and circuit function are not well understood, which limits the advance of therapies for these disorders. TRIO acts downstream of cell surface receptors to control axon and dendrite pathfinding, synapse development, and synaptic transmission. Deletion of a single TRIO allele in mouse cortical excitatory neurons drives reductions in cortical neuropil and defects in dendrite and synapse development and function, yielding social and motor deficits and increased anxiety and compulsivity. However, the links between specific TRIO mutations and subsequent consequences for cortical function are unknown. Here, we will integrate a broad array of highly complementary, interdisciplinary approaches including genetics, biochemistry and proteomics, optogenetic analysis of synaptic function, and multimodal in vivo imaging of cortical network dynamics to address this question. Our first aim will identify the biochemical mechanisms by which TRIO regulates cortical neuron development. We identified several new candidate TRIO signaling partners (PDE4A5, L1CAM, and the LGI1/ADAM22/ADAM23 complex) and will elucidate how they interact with TRIO to regulate cortical neuron dendritic arbor, dendritic spine, and synapse development. We also generated CRISPR mice heterozygous for three disorder-related TRIO variants - K1431M (autism), K1918X (schizophrenia), M2145T (bipolar disorder) - that differentially impact TRIO?s biochemical activities and yield different anatomical and behavioral phenotypes. We will use mass spectrometry-based comparative proteomics to discover new signaling partners differentially impacted by these discrete TRIO alleles. Our second aim will determine how different TRIO variants impact neuronal connectivity and synaptic function. We will assess the consequences of our TRIO CRISPR variants for cortical neuron development by measuring how they impact axon, dendrite, and synapse development, synaptic transmission and plasticity. We will also use viral Cre-mediated sparse TRIO disruption and whole cell recordings to test which deficits reflect cell- autonomous versus network level effects. Our third aim will test how alterations in TRIO impact the functional organization of cortical networks in vivo, taking advantage of our recently developed strategies for combining single cell and mesoscopic imaging of GCaMP6-labeled neurons to measure circuit organization in awake, behaving mice. Our overall goal is to understand how altered TRIO function impacts neuronal function at the cellular, synaptic, and network levels, providing a broad framework for understanding how genetic dysregulation drives changes in behavior.
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1.009 |