Daniel Kerschensteiner - US grants

DESCRIPTION (provided by applicant): Neurons receive information through synaptic connections on their dendrites. Dendrites span long distances and branch intricately to contact the appropriate synaptic partners. The size and complex patterns of dendrites pose unique cell biological challenges. Thus, cellular organelles need to traffic far from the soma to maintain dendritic structure and support synaptic function. Mitochondria - the cell's power plants - are found throughout neuronal dendrites. In addition to producing ATP, mitochondria take up Ca2+ and participate in neuronal signaling. The special importance of mitochondria to neurons is highlighted by the fact, that, while all cells contain mitochondria, mutations that affect their trafficking and function manifest specifically as diseases of the nervous system. In particular, these diseases frequently affect retinal ganglion cells (RGCs), the output neurons of the eye, and cause vision loss. Despite their importance, we know little about how dendritic mitochondria move through neurons, how they target specific regions within dendrites, and how their local function shapes and supports neural development and function. This proposal uses a multidisciplinary approach to address these questions in RGCs in the intact retina. A combination of genetic strategies will be used to simultaneously label RGC dendrites, synapses and mitochondria. Using static high resolution imaging and time-lapse microscopy, the distribution, development and dynamic interaction of these structures will be analyzed. Next, the hypothesis that synaptic activity guides the movements of mitochondria during development and locally controls their function will be tested in transgenic mice in which synaptic input to RGCs is modified in vivo. Bimolecular sensors have been developed and tested to monitor mitochondrial function dynamically in their natural environment using optical approaches. Finally, the specific contribution of mitochondria to dendritic and synaptic development and function will be tested using genetic techniques to selectively interfere with mitochondrial localization or sensitize them to laser-ablation. The consequences of these manipulations for RGC development will be assessed using live imaging and their impact on visual function will be evaluated using patch-clamp electrophysiology. Together, the proposed experiments will not only advance our understanding of the fundamental cell biology underlying retinal circuit development and function, but also provide insight into the mechanisms of a growing number of nervous system disorders - involving eye and brain - that are caused by mutations in mitochondrial genes (e.g. dominant optic atrophy) and/or associated with mitochondrial dysfunction (e.g. Parkinson's and Alzheimer's disease).

DESCRIPTION (provided by applicant): Developing neural circuits undergo critical periods of refinement to establish precise connectivity. During these critical periods, neuronal activity and programmed cell death (PCD) shape the anatomy and function of neurons (i.e. neuronal plasticity). Dysregulation of plasticity has been identified as a common step in the etiology of neurodevelopmental disorders such as autism and schizophrenia. Neuronal plasticity encompasses axon and dendrite remodeling, synapse formation and elimination, changes in the molecular architecture of pre- and postsynaptic specializations, and adjustments to intrinsic excitability. While much is known about the regulation and action of individual plasticity mechanisms, how different plasticity mechanisms cooperate during neural development is not well understood. Recent evidence indicates that crosstalk between these mechanisms governs their function , and suggests that the interplay of plasticity mechanism depends on neuron type and in vivo circuit context. Here, we propose to study how diverse plasticity mechanisms cooperate across different levels of in vivo neural organization (synapse, neuron and circuit), in different cellular compartments (dendrite and axon), and in response to different triggers (neuronal activity and PCD) to shape the development and function of retinal bipolar cells (BCs), glutamatergic second order neurons of the visual system. Towards this end, we have generated transgenic mouse lines that selectively interfere with synaptic input to or output from BCs, or in which BCs can be removed in a graded manner concurrent with their naturally occurring PCD. To analyze structural and functional plasticity, we have established optical approaches from superresolution microscopy, to confocal reconstructions and 2-photon live imaging and optimized methods for targeted patch-clamp and anatomically aligned multielectrode array (MEA) recordings. Thus, we aim to provide an integrated view how diverse activity- and cell- density-dependent plasticity mechanisms cooperate to guide the development of a specific class of neurons and their integration into precise circuits in vivo.

In many parts of the nervous system, interneurons, which mediate local interactions within a circuit, are more diverse than projection neurons, which transmit information between subsequent circuits in a pathway. Due in part to this diversity, the functions of many interneurons are unknown and general operating principles of interneuron circuits remain to be identified. The diversity of interneurons may be greatest in the retina, where approximately 40 distinct types of amacrine cells (ACs) form specific patterns of connections with bipolar cells, which transmit photoreceptor signals from the outer to the inner retina, and retinal ganglion cells, which transmit retinal information to the brain. Most AC types release GABA or glycine, and many release excitatory neurotransmitters or neuromodulators as well (i.e. dual transmitter neurons), further enhancing the diversity of their signals. Here, we will analyze the contributions of specific AC types to motion processing in the retina and to characteristic behaviors elicited by different forms of visual motion. In doing so, we will test a set of general principles (i.e. functional modularity), which we hypothesize govern the operation of AC circuits. We recently identified VGluT3-expressing ACs (VG3-ACs) as local motion detectors in the retina, and showed that VG3-ACs provide excitatory input to object motion sensitive ganglion cells. The selectivity of this circuit relies on fast inhibitory inputs that cancel responses to global motion stimuli. Which AC type(s) provide this input is currently unknown. Preliminary results show that two genetically identified wide-field AC types form inhibitory connections with object motion sensitive ganglion cells. In Aim 1, we will test whether either or both AC types inhibit additional tiers of the excitatory axis of this circuit (i.e. bipolar cells, VG3-ACs). We will then use mice in which these ACs are transiently or stably silenced, or are removed from mature retinas, to probe the functional contribution of their input to motion processing in the object motion sensitive circuit. In addition, we will assess their influence on orienting responses of mice to local motion stimuli. Optogenetic experiments suggest that VG3-ACs provide excitatory input to additional ganglion cell types, with distinct motion preferences. Whether this input occurs during vision, and how VG3-ACs contribute to motion processing in these circuits and influence characteristic behaviors elicited by different forms of visual motion is unclear. In Aim 2, we will test the functional significance and anatomical basis of excitatory input from VG3-ACs to different motion sensitive ganglion cells and assess changes in behavioral responses to visual motion in mice in which VG3-ACs are transiently or stably silenced, or are removed from mature retinas. Intriguingly, preliminary results indicate that VG3-ACs provide selective inhibitory input to a ganglion cell that is suppressed by motion. We will analyze the patterns and function of these connections and test the contribution of this target-specific use of dual transmitters to suppressive responses of these ganglion cells.

The morphology of axons and dendrites shapes the connectivity and function of neuronal circuits; and dysmorphic axons and dendrites are a common feature of neurodevelopmental disorders. To establish cell-type-specific morphologies, developing neurites need to (1) grow towards and branch in the right places (i.e. neurite targeting), (2) elaborate arbors with distinct branching patterns and geometries (i.e. neurite shape), and (3) occupy appropriate territories (i.e. neurite size). How axons and dendrites grow to an exact size, how arbor size regulates connectivity, and how it influences specific circuit computations is not well understood. In preliminary studies, we identified four cell adhesion molecules (CAMs; Amigo1, Amigo2, netrin-G1, and NGL1) that regulate dendrite and axon size of neurons in two circuits of the retina: the direction selective (DS) circuit, which extracts motion information in the inner retina, and the rod bipolar pathway, which transmits dim-light-signals from the outer to the inner retina. Starburst cells have radially symmetric arbors that overlap extensively among neighbors and express Amigo2. The central two thirds of each arbor receive input and the peripheral third provides output. Inhibitory input from starburst cells is critical for DS responses of ganglion cells. Neurite size of starburst cells is increased in Amigo2 knockout (Amigo2-/-) mice, while functional compartmentalization is maintained. In Aim 1, we will analyze the molecular mechanisms of Amigo2?s actions, test its influence on neurite morphology and connectivity, DS circuit function, and image stabilizing head and eye movements. At the first stage of the rod bipolar pathway, horizontal cell axons mediate lateral inhibition among rods, which provide input to rod bipolar dendrites. Horizontal cells express Amigo1. In Amigo1-/- mice, horizontal cell axons and rod bipolar dendrites are both reduced in size. In Aim 2, we will characterize the signaling mechanism of Amigo1, explore territory matching between synaptic partners, analyze effects on connectivity and measure light sensitivity along the rod bipolar pathway, and in behavioral responses. At the second stage of the rod bipolar pathway, netrin-G1-expressing rod bipolar axons synapse onto NGL1-expressing AII cells. Rod bipolar axon size is reduced in netrin- G1-/- and NGL1-/- mice, suggesting that retrograde signals of trans-synaptic netrin-G1/NGL1 complexes regulates axon growth. In Aim 3, we will explore whether forward signals control AII arbor size. We will determine how netrin-G1/NGL1 complexes affect the number, ultrastructure and function of synapses between rod bipolar and AII cells, and assess their influences on light responses along the rod bipolar pathway and on the ability of mice to detect dim light flashes. Together these studies will provide insights into the molecular mechanisms that control axon and dendrite size in the retina, reveal how neurite size regulates connectivity, and how it shapes specific circuit computations and influences visually guided behaviors.

SUMMARY Neuronal plasticity is high during development and low at maturity. High plasticity enables developing circuits to refine their connections and attain specific functions, whereas low plasticity restricts rewiring in mature circuits, limiting functional recovery from neurodegeneration and injury. Our proposal focuses on three questions: (1) How do cell-type-specific plasticity mechanisms support the development of specific circuits? (2) What controls the maturational switch from high to low plasticity? (3) How can we enhance plasticity of mature neurons to promote functional recovery from neurodegeneration and injury? We will address these questions in retinal bipolar cells. Bipolar cells are second-order neurons of the visual system and relay photoreceptor signals from the outer retina to amacrine and ganglion cells in the inner retina. Bipolar cells lose input when photoreceptors die in retinal degeneration, the most common heritable cause of visual impairment (i.e., > 1:2000 people worldwide). Retinal degeneration, a heterogeneous group of diseases, often progresses slowly, leaving a window of opportunity, in which rewiring of bipolar cells with remaining photoreceptors could rescue vision. In Aim 1 of our proposal, we will characterize the developmental plasticity of three bipolar cell types, which participate in two retinal circuits that support specific visually guided behaviors. Thus, we will link cell-type-specific plasticity mechanisms to the development of specific circuits and the behaviors they support. In Aim 2, we will characterize molecules and mechanisms that contribute to the maturational switch from high to low plasticity in the same bipolar cells. We will translate insights into these molecules and mechanisms into viral tools to restore developmental plasticity to mature neurons. In Aim 3, we will test the ability of these tools to rescue connectivity and function in two mouse models of retinal degeneration. Throughout this proposal, we will use adeno- associated viruses to manipulate plasticity. We will analyze bipolar cell morphology and connectivity by confocal and superresolution imaging. We will monitor circuit function by two-photon Ca2+ imaging and patch clamp electrophysiology, and we will test optokinetic responses and perceptual contrast sensitivity to assess visual function of mice. Together, these studies will provide insights into the expression and control mechanisms of plasticity and translate these insights into strategies to enhance plasticity of mature bipolar cells to rescue vision during retinal degeneration.

PROJECT SUMMARY Many computations in the nervous system occur at the level of individual neurites within large extensively branched arbors (i.e., subcellular processing). Most neurites operate in dense neuropils, in which processes of diverse cell types are tightly packed and abundantly interconnected. To understand subcellular processing, we need to measure neurite responses to physiological stimuli and relate them to local patterns of synaptic inputs. To delineate the functional architecture of neuropils and reveal the logic of their connectivity, we need to characterize neurite responses and synapse patterns at high density. Neurite responses can be observed by two-photon imaging, and synaptic inputs can be reconstructed in serial-section electron microscopy (ssEM). A number of technical obstacles have precluded the combination of these techniques (i.e., functional connectomics) to study subcellular processing in dense neuropils. Here, we develop new tools and approaches to overcome these obstacles. In Aim 1, we develop genetic, viral, and computational tools for multispectral two-photon calcium imaging and signal demixing to enable dense functional characterization of neuropils. In Aim 2, we devise a novel strategy for combining two-photon imaging and ssEM (i.e., multimodal imaging), and establish a high-throughput ssEM method for analyzing local synaptic connectivity patterns in the context of larger-scale circuit wiring (i.e., multiresolution imaging). We use our advances to study amacrine cells (ACs), a diverse class of retinal interneurons. The neurites of more than 50 AC types extract salient visual information in a dense neuropil the inner retina. We will acquire a complete functional connectomic dataset of ACs. This dataset, which will be made publicly available, will form the basis of a future R01 application to study the mechanisms of subcellular processing in ACs, the functional architecture of the AC neuropil, and the logic of its connectivity.

Project Summary All animals need to detect threats in their environment to survive. Objects on a collision course cast expanding shadows on the retina (i.e., looming) that elicit innate defensive responses from insects to humans. During the previous award of this grant, we discovered that a retinal interneuron, the VGLUT3-expressing amacrine cell (VG3-AC), detects looming and drives innate defensive responses in mice. Here, we follow up on this discovery to understand how dendritic processing gives rise to feature-selective responses of VG3-ACs (Aim 1), and how VG3-ACs use dual transmitters (glutamate and glycine) to generate divergent feature representations downstream and guide behavior (Aim 2). Dendritic processing and dual transmission are features of subcellular modularity, which we propose as an organizing principle of interneurons. To explore subcellular modularity, we developed methods to combine two-photon calcium imaging and serial-section electron microscopy in the same tissue (i.e., functional connectomics). In Aim 1, we will combine functional connectomics with computational modeling and cell-type-specific genetic manipulations to test the hypotheses that synaptic inhibition and arbor morphology compartmentalize VG3-AC dendrites and that dendritic compartmentalization generates looming- selective responses. In Aim 2, we combine functional connectomics, optogenetics, and cell-type-specific genetic manipulations, to test the hypotheses that VG3-ACs use glutamate and glycine to communicate their responses with opposite sign to two categories of ganglion cells and that this target-specific use of dual transmitters generates divergent representations of looming in the retinal output. We know little about how retinal processing relates to visual processing in the brain and behavior. To fill this gap in our knowledge, we have established projection-specific large-scale recordings from retinal ganglion cells, large-scale recordings from subcortical ganglion cell targets, and behavioral assays. This allows us to track how looming signals of VG3-AC dendrites are transformed across subsequent stages of processing to guide behavior. In Aim 1, we will test the hypotheses that downstream neurons lose their feature selectivity and that innate defensive responses generalize to non- threatening stimuli when dendritic processing of VG3-ACs is disrupted (i.e., when local processing becomes global). In Aim 2, we will test the hypothesis that VG3-ACs use glutamate and glycine to generate impressed- and suppressed-by-looming responses in two categories of ganglion cells and that these ganglion cells converge in the superior colliculus to drive defensive behaviors and control the contrast gain of these responses, respectively. Together, our studies will provide insights into the specifics and general principles of interneuron organization, mechanisms, and functions and bridge that gap in our understanding from retinal processing to visual processing in the brain and behavior for a conserved retinal interneuron and its downstream pathways performing a conserved visual computation that drives a survival behavior.