Jessica A. Cardin - US grants

DESCRIPTION (provided by applicant): One of the central goals of neuroscience research is to understand how the brain learns. One model system that has contributed to our understanding of learning is vocal learning in songbirds. Song learning and production in passerine birds is governed by the song system, a set of discrete brain nuclei. Because the song system is both experimentally accessible and dedicated to the production of a learned behavior, researchers have generated a great deal of information about how the avian brain learns and produces song. Both song learning and song maintenance require constant auditory feedback to the song system. In fact, there is a population of neurons in the song system which respond selectively to the bird?s own song. These song-selective auditory responses are modulated by the behavioral state of the animal. This observation, in combination with the extensive existing knowledge of the anatomy, development, and function of the song system, makes the avian song system a prime candidate for studying the modulation of complex sensory processing. Therefore, the proposal outlined below focuses on determining how thalamic input to the song system modulates auditory responses by song system neurons. An interdisciplinary approach will be used, including neuroanatomical techniques and intra and extracellular electrophysiology. The main hypothesis guiding this work is that modulation of activity in a thalamic song system nucleus mediates state-dependent regulation of auditory inputs to the main song system pathways.

spaceprovided) Neurons in sensory areas of the brain receive continuously varying inputs in response to stimuli in the surrounding environment. In addition, they also receive spontaneous, intrinsically generated background input from the local network of cells. Despite numerous advances in neuroscience that have begun to elucidate sensory system function at the cellular level, we know surprisingly little about how individual brain cells integrate and process these two kinds of input. The experiments proposed here will examine the basic cellular rules governing the integration of visually driven synaptic inputs by single cortical neurons and explore how changes in network activity modulate synaptic input integration and cortical cells' input-output gain. The cellular mechanisms of input integration will be examined by recording intracellularly from primary visual cortex neurons in intact animals and analyzing the synaptic potentials and spike output evoked by visual stimulation. This work will provide insights into the fundamental cellular mechanisms underlying visual processing and elucidate basic principles of sensory cortical function.

The long-term objective ofthis project is to contribute to our understanding ofthe mechanisms of visual representation by cortical networks. Neuronal gain, measured as the continuous slope ofthe relationship between stimulus input and cellular outpiut, is a measure of neuronal sensitivity to the stimulus and a defining element ofthe contribution of single neurons to network operations. Previous work has identified a role for network-driven synaptic activity in modulating the input-output gain of cortical neurons. In addition, synchroiQ' between local network mputs may determine the magnitude ofthe impact of network activity on neuronal gain. Previous studies have also suggested that gain modulation and population synchrony play roles in mediating visual perception. However, even at the earliest stages of cortical visual processing, die relationship between individual neurons and the surrounding local network is poorly understood. The main goals ofthe work proposed here are therefore 1) to determine the relationship betweoi cellular mechanisms of contrast gain control and networic synchrony and 2) to examine tiie interacti(H) between population synchrony and visual perception. To tiiat end, the first Aim focused on many simultaneous recordings of cortical neurons during presentation of stimuli with varying prqierties. The second Aim will use a behavioral task in which awake bdiaving animals discriminate between visual stimuli of vaiying contrast In one series of expraiments, tiiis task will be combined witii population recordings of neimms that make up local netwoiks in primary visual cortex. The results of these eiqieriments are expected to provide novel insights into the relation^p between tiie temporal dynamics of population activity and cellular mechanisms of gain control. In addition, they will graierate a better understanding of tiie role of synchronous cortical networic activity in visual perception.

DESCRIPTION (provided by applicant): GABAergic inhibitory interneurons are thought to play a powerful role in regulating the ongoing pattern of activity in the cortex. Interneurons can be divided into many classes based on their intrinsic properties, synaptic targets, and molecular markers. The two largest groups are the parvalbumin-expressing interneurons that target the soma and the somatostatin-expressing interneurons that target the dendrites. Identifying the mechanisms by which these two sources of synaptic inhibition regulate sensory processing is a critical step towards understanding the complex cellular interactions underlying active network function in the brain. However, little is known about the activity pattern or impact of these cells during wakefulness. Using the primary visual system as a model system, we will record the activity of many excitatory and inhibitory neurons in awake, moving animals. Using dense extracellular recordings of identified neurons, we will examine the temporal pattern of interneuron recruitment by sensory stimuli and the contrast-dependence of those activity patterns. We will use a combination of intracellular recordings and cell type-specific optogenetic manipulations to test the impact of parvalbumin and somatostatin interneurons on input integration and spike generation by their postsynaptic target excitatory neurons. Inhibition is thought to play a major role in facilitating the functional flexibility of cortical networks and allowing adaptive scaling of neuronal output to match the range of inputs present in the surrounding sensory environment. To understand the dynamic role that inhibitory interneurons play in regulating the input-output relationship of local cortical networks, we will test the impac of parvalbumin and somatostatin interneurons, as well as excitatory neurons, in modulating the sensitivity, or gain, of cortical responses to visual stimuli. We will further test the behavioral tate dependence of inhibitory gain modulation. These studies will reveal fundamental mechanisms of visual processing in the awake brain and lead to a more complete understanding of cortical network function. Results from our experiments will answer fundamental questions about key interneuron populations that have historically not been possible to target in vivo. Because input integration and gain control are global elements of neural function, our results will be applicable to systems throughout the brain and will elucidate the function and dysfunction of cortical circuits critical for information encoding, perception, and behavior.

DESCRIPTION (provided by applicant): Genetic factors appear to play a major role in the etiology of schizophrenia, a devastating mental illness that affects up to 1% of the worldwide population. Recent evidence suggests disruption of GABAergic inhibitory interneurons in the brain as a strong candidate underlying mechanism. Our long-term goal is therefore to understand the role of inhibitory dysfunction in the pathophysiology of this psychiatric disease. Identifying the mechanisms by which inhibitory dysregulation during development leads to perturbation of cortical function will give insight into the neurodevelopmental cellular mechanisms underlying schizophrenia and may provide new cellular targets for therapeutic strategies. Human screening studies have identified Neuregulin-1 (Nrg-1), an extracellular signaling factor, and its membrane-bound receptor ERBB4 (ErbB4), as strong candidate genes for schizophrenia. Most evidence indicates that ErbB4 protein is expressed predominantly in GABAergic cells throughout the brain. Global disruptions of ErbB4 result in decreased GABA release in the cerebral cortex, and mice lacking Nrg-1 or ErbB4 exhibit key behavioral deficits associated with schizophrenia. The ErbB4 model of schizophrenia is thus an ideal system in which to examine the cell type-specific role of inhibitory interneuron dysregulation in cortical network perturbation in this complex disorder. A major issue in using genetic mouse models to study psychiatric disease has been that many studies focus on single cells or synapses in vitro. In contrast, the cognitive and perceptual processes disrupted in schizophrenia rely on large-scale neural network interactions in the intact brain. We will therefore examine neural function in vivo in primary visual cortex circuits in the ErbB4 deletion model of schizophrenia. Synaptic and circuit function in the healthy visual cortex and the contribution of these circuits to visual processing are well characterized, providing a critical framework in which to interpret disease-related alterations in cellular and network function. Furthermore, schizophrenic patients exhibit specific deficits in basic visual processing and perception that rely on primary visual cortex function. We will use a combination of intra- and extracellular recordings and cell type-specific optogenetic manipulations to test the impact of ErbB4 deletion on the function of inhibitory interneurons in cortical networks in vivo in awake behaving animals. Using measurements of key aspects of cortical circuit function at the synaptic and circuit levels, we will assess the rol of ErbB4 signaling in interactions between inhibitory and excitatory neurons. We will further use targeted genetic approaches to examine the developmental role of this signaling pathway in the survival and maturation of specific populations of GABAergic cells. These studies will reveal fundamental mechanisms underlying circuit dysfunction in this genetic model of schizophrenia and lead to a more complete understanding of the cell type-specific role of GABAergic dysfunction in psychiatric disease. Because the cellular and circuit interactions identified in thi work are core elements of neural function, our results will be applicable to systems throughout the brain.

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.

PROJECT SUMMARY To understand brain function, it is essential to identify how information is represented in neuronal population activity and how it is transformed by individual neurons as it flows through microcircuits. ?Two-photon (2P) microscopy is a core tool for this because it enables neuronal activity to be monitored at high spatial resolution deep within brain tissue in behaving animals?. ?However, ?t?he temporal resolution of conventional galvanometer-based 2P microscopy severely limits measurements of fast signaling in 3D neuronal circuits. Acousto-optic lens (AOL) microscopy, which enables fast focussing and selective imaging of regions of interest distributed within the imaging volume, has substantially improved the temporal resolution of 3D 2P microscopy. But current AOL microscopes, which rely on ?linear acoustic drive waveforms, suffer from limitations that make them ine?fficient to monitor signaling in structures that project in the Z dimension. ?Each change in the focus requires a 24 ?µ?s ?dead time? to refill the AOL aperture and continuous line scanning is restricted to the selected X-Y focal plane, limiting imaging rates for 3D dendritic trees to a few Hz, rather than the 100-1000 Hz required for monitoring neurotransmitter reporters and voltage indicators. ?The main aim of this project is to optimize and disseminate ?nonlinear ?AOL 3D microscopy, a technology we have invented to overcome these limitations by enabling ultra-fast line scanning (up to 40 kHz) in any arbitrary direction in X, Y and Z. By developing a prototype ?nonlinear AOL 2P microscope with real time correction of brain movement, we have demonstrated the performance of this technology for high-speed multiscale 3D imaging of neural circuits in awake behaving animals. We will build on these results by optimizing ?nonlinear AOL microscopy for imaging entire 3D dendritic trees and the surrounding neuronal population at unprecedented speeds. We will develop variants of this dendritic ?arboreal imaging? approach to provide low spatial resolution, ultra-high-speed 3D imaging (up to 1 kHz) by combining the fast scanning and adaptive optics properties of ?nonlinear ?AOLs. We will also extend the real time FPGA analysis used in our closed loop 3D movement correction to enable ?attentional imaging? where active regions of a dendritic tree, or circuit, are rapidly detected and imaged at higher spatio-temporal resolution. These applications ?will provide the temporal resolution required for monitoring voltage across the entire 3D dendritic tree of pyramidal cells in awake animals for the first time. Moreover, attentional imaging will enable neurotransmitter release to be mapped at high spatiotemporal resolution. Low cost dissemination of this powerful new technology will be achieved by providing US labs and an imaging facility with compact ?nonlinear AOL modules that will be added to their existing conventional 2P microscopes. By extending our open source microscope GUI software, standardizing data formats with NWB2 and refining automated analysis pipelines, we will also deliver reliable user-friendly microscope control and a semiautomated data analysis framework for the collaborators to carry out experiments on a range of different neural circuits.

PROJECT SUMMARY The goal of this proposal is to provide highly focused and advanced training in the development, function and dysfunction of the mammalian cerebral cortex at Yale University. The philosophy of this program is to preserve and foster integrative approaches to neurobiology that will interface across genetics, molecular biology, cell biology, systems neuroscience and clinical medicine with respect to the development, organization, function and plasticity of the mammalian cortex. Twenty-seven (27) faculty from eleven (11) basic and clinical departments at both Yale School of Medicine and Yale College are participants in this multidisciplinary program. The program offers both depth and breadth. The depth derives from its unique substantive focus on cortical circuits of the rodent, primate and human brain. The breadth of the program derives from the diversity of approaches, spanning genetic, molecular, developmental, systems, theory and cognitive neuroscience. Faculty interests span cortical morphogenesis and axon guidance mechanisms in embryos to memory decline and stroke in elderly humans. Methodologies include deep sequencing; cloning; cell culture; immunocytochemistry; in situ hybridization; electron and two photon microscopy; voltage clamp and whole cell recording; calcium and other forms of optical imaging; biochemistry and molecular analyses; psycho-pharmacology; rodent, monkey and human behavior; in vivo extracellular recording in behaving animals; and fMRI and PET imaging in human subjects. Four predoctoral positions are requested. Trainees are selected by the Executive Committee from an outstanding pool of advanced Neuroscience graduate students at Yale, which are amongst the best in the country and come from a variety of backgrounds in biological and physical sciences. Trainees are selected on the basis of their potential for excellence and leadership in research focused on the mammalian neocortex. Mentors are Ph.D.s and M.D.s. with substantial NIH support and NINDS related research foci on the neurobiology of cortical systems, particularly the development, function and dysfunction, including Alzheimer?s, Schizophrenia and Autism. Training includes focused coursework, instruction in experimental design and statistical methods, aggressive mentorship through advisory committees, intensive research apprenticeship, structured seminar programs, career skills development, extensive oral and written feedback, and numerous opportunities for written and oral presentation of research progress and analysis.

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.