Court Hull, PhD - US grants

DESCRIPTION (provided by applicant): Rapid and continuous chemical transmission between neurons requires that synaptic vesicles be recycled after fusing with the plasma membrane to release transmitter. Endocytosis, the process by which neurons reinternalize fused vesicular membrane, is the first essential step in this recycling process. Despite its clear necessity in maintaining an available pool of synaptic vesicles and balancing the size of a presynaptic terminal against membrane added during exocytosis, very little is known about endocytosis. This is especially true in neuronal systems. The proposed study seeks to characterize endocytosis from a physiological perspective using membrane capacitance measurements of single presynaptic bipolar cell terminals from the goldfish retina. The specific aims of this project are: 1) To characterize the effect of neurotransmitters on the kinetics of endocytosis, 2) To determine the effect of phosphatase inhibition on endocytosis in the presynaptic terminal, 3) To determine the quantitative relationship between membrane addition and retrieval during ongoing exocytosis and endocytosis. To measure endocytosis in the intact bipolar cells of a retinal slice. The results are expected to provide fundamental insight about presynaptic signal modulation in neurons and further elucidate the functional characteristics of retinal bipolar cells and their role in visual signal transduction.

[unreadable] DESCRIPTION (provided by applicant): PURPOSE: This proposal addresses the synaptic mechanisms by which thalamic afferents control the excitability of cortical neurons. It is focused on the thalamic projection to the rodent somatosensory "barrel" cortex, the cortical input of tactile information originating from the whiskers. Specifically, I will seek to determine how thalamocortical afferents produce differential excitation of interneurons and principal cells, and characterize the receptor subtypes that regulate transmission from the thalamus into the cortex. I will use the mouse thalamocortical slice as a model system, and electrophysiological, morphological and imaging techniques to assay synaptic function. BACKGROUND: Somatosensory information enters the cortex via afferent projections from the thalamus. In the primary somatosensory cortex of rodents, thalamocortical afferents carry information originating from the whiskers. Here, these afferents make synapses onto both inhibitory interneurons and excitatory principal cells. Thalamocortical fibers thus distribute information between two cortical cells types, setting the initial phase of cortical processing. The divergence of thalamocortical fibers onto interneurons and principal cells provides the basis for disynaptic feed-forward inhibition in the somatosensory cortex, which is critical for enforcing the temporal precision of cortical responses to whisker stimulation. Thalamocortical afferents support this vital disynaptic circuit by producing larger inputs onto interneurons than principal cells, thereby establishing a "fail-safe" to ensure feed-forward inhibition. It is not known, however, what mechanisms allow thalamocortical fibers to mediate differential input to interneurons and principal cells. AIM 1: Determine the mechanisms underlying the stronger input of thalamocortical afferents onto inhibitory interneurons. By recording from neurons in thalamocortical slices and stimulating the thalamus, I will determine what produces stronger inputs to inhibitory interneurons receiving input from the thalamus. AIM 2: Characterize the receptor subtypes responsible for synaptic transmission from the thalamus to the cortex, and determine their role in shaping the behavior of local feed-forward inhibitory circuits. PUBLIC HEALTH RELAVENCE: By determining the functional properties of transmission into the cortex using the thalamocortical system, I hope to gain insight into the mechanisms that control the balance between excitation and inhibition in thalamic recipient layers, and that contribute to the cortical discrimination of tactile stimuli. A deeper understanding of the factors that regulate cortical excitability may contribute to the development of therapies aimed at preventing epileptogenesis in cortical areas. [unreadable] [unreadable] [unreadable]

Sensorimotor integration in the cerebellum is essential for refining motor output. Much of this integration occurs at the initial stage of cerebellar processing, in the granule cell layer where mossy fibers carrying diverse sensory and motor information converge. While these computations have been thought to occur through rigid, anatomically defined circuits, recent evidence suggests that granule cell layer integration can be contextually modified. Neuromodulators represent a strong candidate for such regulation, and anatomical studies have revealed prominent cholinergic and serotonergic projections into the cerebellar granule cell layer. However, it is unknown how these neuromodulators act at the cellular and circuit level to control sensory and motor integration. Our preliminary data reveal that Golgi cells, interneurons that provide the sole source of inhibition to the granule cell layer, express receptors for both acetylcholine (ACh) and serotonin (5-HT). We find that these neuromodulators bi-directionally regulate the excitability of Golgi cells: ACh suppresses Golgi cell spiking while 5-HT elevates spiking. In addition, we find that granule cells are depolarized by ACh. This suggests that ACh may generally act to increase excitability in the granule cell layer. Using a combination of modern physiological, genetic and anatomical approaches in the mouse, we will test the following aims: In Aim 1 we will use an in vitro brain slice preparation to identify the sites of ACh and 5-HT receptor expression on the major cell classes of the granule cell layer: the granule cells, Golgi cells and mossy fibers. Using targeted application of neuromodulatory agonists and specific pharmacology, we will determine how these neuromodulators directly impact cellular excitability both acutely and after prolonged exposure. In Aim 2, we will use retrograde tracing to identify the sources of cerebellar cholinergic and serotonergic inputs. This will allow us to identify whether these neuromodulatory inputs are part of a larger, brain-wide system, and under what conditions they are active. Localizing the afferent neuromodulatory nuclei will also allow viral delivery of optogenetic proteins, and thus investigation of the effect of endogenously released neuromodulators on the intact granule cell circuit. In particular, we will test the hypothesis that ACh acts to increase excitability in the granule cell layer while 5-HT acts to decrease it. Then in Aim 3, we will test how these neuromodulatory effects on excitability regulate granule cell layer integration of sensory and motor input in vivo. We will use multi-unit electrophysiology and two-photon imaging to determine how activation of cholinergic and serotonergic inputs alter the activity of the population of granule cells. In particular, we will present sensory stimuli with graded intensity to test the hypothesis that ACh and 5-HT change the gain of the granule cell network. Together, these experiments will reveal the synaptic and circuit mechanisms that support context-dependent processing in the cerebellum. In the future, we hope to extend these studies to determine how these mechanisms support context-specific learning in behaving animals.

Abstract The cerebellum is critical for learning and executing coordinated, well-timed movements. The cerebellar cortex seems to have a particular role in learning to time movements. Since the 1960's and 70's, we have known the architecture of the cerebellar microcircuit, but most analyses of cerebellar function during behavior have focused on Purkinje cells. Here, we propose to investigate the cerebellar cortex at an entirely new level by asking how the full cerebellar microcircuit ? mossy fiber, granule cells, Golgi cells, molecular layer interneurons, and Purkinje cells ? performs neural computations during motor behavior and motor learning. We strive to ?crack? the circuit by identifying all elements, recording their electrical activity during movement and learning, and reconstructing a neural circuit model that reproduces the biological data. We will use three established learning systems that all can learn predictive timing: classical conditioning of the eyelid response (mice), predictive timing of forelimb movements (mice), and direction learning in smooth pursuit eye movements (monkeys). Our proposal has six key features. First, optogenetics (in mice) will link the discharge of different cerebellar interneurons during movement and learning to their molecular cell types. Second, a machine-learning clustering analysis (in mice and monkeys) will find analogies among the cell populations recorded in our three preparations and will classify neurons according to their putative cell types based on recordings of many parameters of non-Purkinje cells during movement and motor learning. Third, multi- contact electrodes will allow us to record simultaneously from multiple neighboring single neurons and compute spike-timing cross-correlograms (CCGs) to identify the sign of connections; we also will look for changes in CCGs that provide evidence of specific sites of plasticity during learning. Fourth, gCAMP imaging of the granule cell layer will reveal the temporal structure of inputs to the cerebellar microcircuit, and determine whether those inputs are modified in relation to motor learning. Fifth, a model neural network with realistic cerebellar architecture will reveal a single set of model parameters that will transform the measured inputs to the cerebellum in our three movement systems to the measured responses of all neurons in the cerebellar cortex. Sixth, the model will elucidate how mechanisms of synaptic and cellular plasticity at different sites in the cerebellar microcircuit work together to cause motor learning.

PROJECT SUMMARY The proposed research addresses a critically important question in autism spectrum disorder (ASD) research: how defects in cerebellar circuits contribute to ASD. In particular, it examines the role of the predominantly cerebellar gene ASTN2 in cerebellar circuit function and ASD-related behaviors. Copy number variations (CNVs) in ASTN2 have been identified as a significant risk factor for ASD (Lionel et al, 2014), suggesting that ASTN2 mutations such as those found in patients with ASTN2 CNVs, lead to altered cerebellar synaptic function. In addition, we recently reported a family with a paternally inherited intragenic ASTN2 duplication, which caused a heterozygous loss of function of ASTN2. The family manifested a range of neurodevelopmental disorders, including ASD, learning difficulties and speech and language delay (Behesti et al, 2018). Our cellular and molecular studies on mouse cerebellum show that ASTN2 binds to and regulates the trafficking of multiple synaptic proteins, including Neuroligins, which have been genetically linked to ASDs, and modulates cerebellar Purkinje cell (PC) synaptic activity (Behesti et al, 2018). To provide a genetic model to study cerebellar circuit function, we generated both a global loss of function Astn2 line and a floxed Astn2 line for conditional knockout experiments. New, preliminary evidence indicates that PCs in mice lacking Astn2 have a decrease in evoked excitation relative to inhibition in PCs and reduced PC dendritic spine density, suggesting specific cerebellar circuit defects. In addition, preliminary evidence shows mild motor deficits and defects in USVs and an open field assay, ASD-related behaviors. As other preliminary findings do not indicate major defects in cerebellar development, we hypothesize that the behavioral defects we observed relate to defects in the cerebellar circuitry with underlying changes in receptor trafficking. In the proposed research, we will 1) test how loss of Astn2 alters intrinsic excitability in PCs and the synaptic efficacy of their presynaptic inputs from GCs and molecular layer interneurons, 2) use proteomics to identify changes in the levels of synaptic proteins and live imaging to assess whether such changes relate to changes in the rate of endocytosis, 3) compare changes in PC dendritic branching as well as the regional distribution of PC spines in wild type and mutant animals to provide insight on whether there are changes in the organization of PC inputs during the establishment of the cerebellar circuitry, and 4) analyze changes in social behavior and ultrasonic vocalization in Astn2 wild type, heterozygous and mutant animals. Taken together, the proposed research will provide a new mouse model that allows us to link an ASD-related gene that is predominantly expressed in the cerebellum with specific cerebellar circuit function and molecular pathways.