Wade G. Regehr - US grants

DESCRIPTION: (Applicant's Abstract) At fast chemical synapses, neurotransmitter release is dynamically modified according to the pattern and frequency of presynaptic spikes. Normally, neuronal firing is variable and can reach frequencies of over 100 Hz. Depending upon the type and state of the synapse, neurotransmitter release can either be enhanced or reduced more than ten-fold compared to release evoked by an isolated stimulus. This has a profound impact on the ability of a neuron to influence the firing of its targets. Our primary goal is to determine how synaptic strength is controlled when firing rates are in the physiological range. These studies have important implications for signal processing and drug actions under realistic conditions. We will first study individual mechanisms and then examine how they interact during trains. We will build upon our previous work to answer unresolved questions regarding three prominent forms of short-term synaptic plasticity: facilitation, delayed release and depression. Because presynaptic calcium participates in all of these forms of synaptic plasticity, optical measurements of presynaptic calcium will be an important part of our experimental approach. First, we will study facilitation, an enhancement of the probability of release that lasts for hundreds of milliseconds. We will determine how presynaptic calcium and the initial release probability control the amplitude and time course of facilitation. Second, we will study delayed release, a long-lived (hundreds of milliseconds) component of release that follows the large, brief phasic release of neurotransmitter. We will determine if delayed release becomes increasingly important during trains, which elevate presynaptic calcium. Third, we will study depression, a use-dependent reduction in synaptic strength, by quantifying the calcium-dependence of recovery from depression. Finally, the resulting insights into these three forms of synaptic plasticity will form the basis of our studies of the behavior of synapses during spike trains, when these mechanisms interact to dictate synaptic strength, Experiments will be conducted in rodent cerebellar brain slices. Two classes of synapses will be studied: facilitating synapses between granule cells and either stellate or Purkinje cells, and depressing synapses between climbing fibers and Purkinje cells. By understanding these two very different types of synapses we hope to develop a general framework for understanding use-dependent changes in synaptic strength.

Clarifying the function of acetylcholine (ACh) receptors in the brain promises to have important implications for understanding Alzheimer's disease, addiction to nicotine, learning and memory. The overall goal of this proposal is to determine how ACh affects synapses and to ascertain the consequences of changes in synaptic strength. Initial experiments will be conducted in the hippocampus, a brain region involved in learning and memory. ACh is known to play an important modulatory role in the hippocampus by activating different types of receptors. There are, however, many unanswered questions regarding the mechanisms of the modulation and their physiological consequences. One major goal of the proposal is to clarify the types of synaptic connections that are regulated by ACh to determine the mechanisms of the modulation. Another goal is to determine the ultimate effects of such changes in synaptic strength for conditions that would be encountered in different behavioral states in the brain.

DESCRIPTION (provided by applicant): This a proposal to continue a successful and strong training program at Harvard Medical School that is a cohesive postdoctoral training program in fundamental neuroscience. The essence of the program is to train exceptional postdoctoral fellows in the most advanced methods and concepts of modern neurobiology to prepare them for the transition to independent careers in biomedical sciences. The training supervisors include 32 faculties located primarily on or adjacent to the Harvard Medical School campus. Together this faculty represents the following research areas: integrative neuroscience, developmental neuroscience, molecular and cellular neuroscience and neurological disease mechanisms. The training program has four major components: (1) background coursework in neuroscience and collateral areas, (2) regular attendance at two scientific neuroscience seminar series, (3) supervised research in the laboratory of a program faculty member, and (4) regular meetings with a trainee-specific advisory committee. We plan to support eight postdoctoral trainees at levels 1-3 for two years each. The primary facilities for the training include the laboratories of the program faculty at the Harvard Medical School and adjacent institutions.

DESCRIPTION (provided by applicant): Cannabinoids, such as d9-THC, affect the brain by activating G-protein coupled CB1 receptors that can inhibit adenylate cyclase, modulate a variety of ion channels, and inhibit synaptic transmission. These receptors are expressed widely throughout the brain, with particularly high levels of expression found in the cerebellum, the cortex, the hippocampus and the striatum. Recent studies provide new insight into the physiological role of the cannabinoid system and suggest that cannabinoids can act as retrograde messengers. Elevations of calcium in the dendrites of some types of neurons result in the cleavage of phospholipids leading to the formation and liberation of endogenous cannabinoids, which bind to presynaptic CB1 receptors to inhibit synaptic transmission. This retrograde inhibition lasts for tens of seconds. Our primary goal is to clarify the properties and mechanisms of this retrograde inhibition and to determine its physiological role. Studies will be performed in rodent cerebellar brain slice on excitatory and inhibitory synapses that are known to be retrogradely inhibited by cannabinoids released from Purkinje cell dendrites. These synapses are well suited to these studies because cells are readily identified, whole cell voltage clamp is straight forward, and both presynaptic and postsynaptic calcium levels can be monitored optically. Mechanistic studies will concentrate on the calcium dependence of cannabinoid release, identification of the presynaptic targets of that modulation, and determination of the factors governing its time course. Factors governing the spread of retrograde inhibition to synapses onto neighboring cells and its spatial extent will also be determined. In addition, we will determine the physiological role of retrograde inhibition in controlling synaptic strength and determine if it is a mechanism that can provide synapse-specific regulation or if it provides a homeostatic mechanism for a cell to regulate all of the synaptic inputs it receives. These basic mechanistic studies promise to help cannabinoids realize their great therapeuptic potential as an appetite stimulant, as an anticonvulsant, and in the treatment of Huntington's disease and Parkinson's disease.

DESCRIPTION (provided by applicant): On the time scale of milliseconds to minutes synapses are dynamically regulated in ways that are vital to brain function. Such short-term synaptic plasticity has many potential functional roles including rapid computation, coincidence detection, improving temporal precision, dynamic gain control, frequency-dependent filtering, sensory adaptation, and increasing information transfer. However, many aspects of synaptic modification under physiological conditions are poorly understood because synaptic strength during realistic activity patterns reflects the complex interaction of multiple processes. The overall goals of this project are to understand individual mechanisms of synaptic modulation, to determine how these processes combine to control synaptic strength under physiological conditions, and to determine the functional significance of short-term synaptic plasticity. Initially, individual mechanisms of use-dependent plasticity will be studied, such as presynaptic depression of release, facilitation, desensitization of postsynaptic receptors and postsynaptic receptor saturation. In addition, modulation via chemical messenger activation of presynaptic ionotropic and metabotropic receptors will be examined. The manner in which these forms of plasticity interact to control release during physiological patterns of activity will then be determined. Finally, we will determine the functional consequences of short-term synaptic plasticity and evaluate the manner in which plasticity at different types of synapses is tailored to particular roles. Experiments will be conducted in brain slices from rats and mice. Studies of individual mechanisms will use whole-cell voltage clamp recordings of synaptic strength and mEPSC frequency, optical measurement of pre-and post-synaptic calcium and presynaptic waveforms, and serial electron microscopy. Functional consequences of short-term synaptic plasticity will be determined using activation patterns and experimental conditions that approximate physiological conditions. Responses will be measured in current clamp and further characterization will be made in dynamic clamp. All of the techniques required in this study are routinely used in the laboratory, making it likely that the proposed experiments will be completed in the allocated time. These studies will lead to a deeper understanding of the factors controlling the strength of central synapses, and they are relevant for understanding complex conditions including epilepsy, schizophrenia and depression.

DESCRIPTION (provided by applicant): We request funds to purchase a fluorescence microscope equipped with a sensitive CCD and a motorized stage for array tomography to determine with high resolution the location of multiple proteins in brain tissue. Array tomography is a new approach that overcomes two major limitations of conventional immunohistochemical approaches: the poor resolution in the z direction and the inability to image large numbers of proteins in a single sample. The poor z resolution is overcome by embedding tissue in a polymer that is then sectioned in a series of slices of about 100 nm in thickness. The small slice thickness results in a z resolution that is much better than can be achieved with conventional fluorescence imaging or confocal imaging. The second limitation is resolved by the fact that array tomography allows a large number of epitopes to be examined per section because antibodies can be efficiently stripped from the preparation and new antibodies can be applied. We propose to use array tomography to characterize cells, presynaptic boutons, postsynaptic densities and spines, and the distribution of proteins in the cell body. Preliminary experiments have established our ability to prepare tissue, cut arrays of thin sections, and image individual immunostained slices. However, we lack an appropriate microscope that will allow automated collection of a large number of images from serial sections. There is currently no suitable microscope at Harvard Medical School that is available for the long blocks of time necessary to image slice arrays at high-resolution. We are therefore requesting funds to purchase a microscope suitable for array tomography for the use of the major users on this proposal.

The major goal of this project is to provide a core that allows array tomography to be used to analyze brain tissue. This is a powerful new approach that allows high resolution imaging in the x, y and z planes, and is particularly well suited to using immunohistochemical approaches to determine the distribution of many different proteins in the same sample. We have the trained personnel and equipment to prepare and image the samples and to analyze the resulting images.

? DESCRIPTION (provided by applicant): The cerebellum is involved in behaviors ranging from motor learning to social behaviors. It is therefore important to understand the cerebellar circuit and how it's dysfunction can lead to neurological disorders, such as ataxia and autism. To accomplish this it is necessary to clarify the function and connectivity of Purkinje cells (PCs), te sole output cells of the cerebellar cortex. Much is known about the intrinsic firing properties, synaptic integration and plasticity of PC inputs. However, it is unclear how PC collaterals allow PCs to influence cells within the cerebellar cortex. Our primary goal is to elucidate the propertie and functional roles of PC collaterals in juveniles and adults by answering several fundamental questions. First, what are the properties of PC collaterals in juveniles and adults and what are their targets? It is known that in newborn mice PC collaterals are prominent. They provide the primary source of inhibition to PCs, and they can mediate widespread travelling waves of activity. This is not the case in older animals where it is not clear if PC collaterals are extensie and what their targets are. Our initial studies indicate that all PCs have extensive collaterals confined to a parasagittal plane. A variety of light and electron microscopy techniques will be used in combination with electrophysiological investigations to identify cellular targets. Second, do PC collaterals allow PCs to regulate the excitability of the input layer of the cerebellum? Optogenetic approaches will be combined with slice and in vivo electrophysiology to determine the targets of PC collaterals within the granular layer and to test the hypothesis that a primary function of the collateral is to allow the output of the cerebellar cortex to feedback and regulate the input layer. Third, do PC collaterals promote synchronous PC firing and do synchronously firing PCs converge on cells in the deep cerebellar nuclei (DCN) to control their spiking? It has been proposed that synchrony allows PCs to control the spiking of cells in the DCN: If PCs fire asynchronously they suppress DCN neuron firing, and if they fire synchronously they promote phase-locked DCN firing. Our initial slice studies suggest that PC collaterals inhibit neighboring PCs and can promote synchronous activity. We will test the hypothesis that the spatial extent of PC collaterals determines the range of synchronous firing in vivo. We will also test the hypothesis that synaptically-connected PCs converge onto the same DCN neuron and regulate its firing. These studies will extend our understanding of cerebellar processing and will provide important insights into neurological disorders that arise from cerebellar dysfunction.

? DESCRIPTION (provided by applicant): Viral expression vectors are widely used to either promote or knockdown the expression of specific genes. In optogenetic studies, viruses can express channelrhodopsin-2 or halorhodopsin to allow optical regulation of neuronal activity. Viruses can also express genetically encoded calcium and voltage indicators to allow optical monitoring of neuronal activity. But current methods have limitations. Here we develop methods for silk-based delivery of adeno-associated virus (AAV) in order to improve the localization of expression, to reduce immunogenic responses and to improve transduction efficiency. Silk is a biocompatible material that when implanted into tissue can dissolve to release viruses. We have found that silk/AAV can be used to express proteins at the tip of an optrode. This leads to alignment with the area of expression and obviates the need of a second surgery to inject AAV. This is simpler than existing methods and promises to increase throughput, lead to more reliable experimental results, and greatly reduce the number of animals and number of experiments required. We will adjust processing conditions to vary the properties of silk films to control the rates of release in order to obtain reliable localized expression and eliminate unwanted expression. We will also determine if silk/AAV reduces inflammatory responses. Injecting AAVs can lead to reactive gliosis, which has been implicated in perturbing synaptic properties. Silk may shield viruses from host immune responses, preventing degradation and improving transduction efficiency. We will compare reactive gliosis and synaptic properties for silk/AAV mixtures and conventional injections of AAV. We will also evaluate the performance of silk/AAV-dependent expression in in vivo imaging (using silk/AAV-coated endoscopes) and optogenetic (using silk/AAV-coated optrodes) studies. Another major goal is to obtain efficient transduction of a large fraction of cells over large regions. We will determine if thin sheets of silk/AAV can b patterned and placed on the surface of the brain to obtaining widespread expression in a defined cortical region. We also propose to use silk/AAV particles to obtain widespread expression. Our preliminary experiments suggest that some formulations of silk/AAV produce stronger and more widespread expression than that produced by injection of virus alone. We will develop approaches to obtain similar expression patterns by implanting small prefabricated silk/AAV particles in the brain. We will assess the utility of silk/AAV in rescue experiments that require widespread expression in a large fraction of a population of cells. We will also determine the utility of silk to express GCaMP in cortical neurons for imaging. If silk can be used to obtain either localized or widespread viral expression it will represent a major technical advance that will make an important contribution to the application of optogenetic approaches and more generally to viral delivery.

Abstract In this Institutional Center Core Grant to Support Neuroscience Research, we propose to continue to develop an innovative Imaging Center that serves NINDS-funded Harvard Medical School (HMS) and Boston Children's Hospital (BCH) investigators. This state-of-the-art core facility provides important resources to the HMS and BCH neuroscience community, and will perform essential services that are difficult and impractical for individual laboratories to provide on their own. The experimental opportunities and innovative services provided by the Imaging Center will give area neuroscientists access to unique equipment and training in several new cutting-edge methodologies, greatly benefiting the research programs of NINDS-funded investigators at these institutions. Moreover, the Center will function as the centerpiece of a concerted effort to strengthen ties between the neuroscience communities at HMS and BCH. Through the continued development of this Center, we hope to shift from a complete reliance on individual laboratory-centered research to a more cost-effective and productive use of extraordinary cores while further deepening existing ties between these two vibrant neuroscience communities.

Project Summary The ultimate goal of this grant is to determine how synapses and circuits function in vivo to control behaviors. One major focus is to clarify the mechanisms of short-term plasticity, and to understand the functional and behavioral role of short term plasticity at different synapses. Although synaptic plasticity plays a crucial role throughout the brain, and dysfunction has been implicated in numerous neurological disorders, the many interacting forms of plasticity remain poorly understood. We will focus on the hypothesis that specialized calcium sensors respond to presynaptic calcium signals to enhance neurotransmitter release. Our findings suggest that facilitation and posttetanic potentiation (PTP) use 2 different types of calcium sensors to enhance transmission on different time scales. Facilitation is a form of synaptic enhancement that lasts for hundreds of milliseconds. We have found that facilitation is mediated by synaptotagmin 7 (syt7), which is a calcium- sensitive isoform with slow kinetics. In preliminary studies we find that in syt7 knockout mice, facilitation is eliminated even though the initial probability of release and presynaptic calcium signals are unaltered. Viral expression of syt7 restores facilitation in syt7 knockout animals. These studies indicate that we have identified the long sought after calcium sensor for facilitation. Future studies will clarify the role of syt7 in facilitation in contributing to different behaviors. PTP is a form of synaptic enhancement lasting for tens of seconds following a period of high-frequency firing of presynaptic neurons. We have recently shown that PKC? is a calcium sensor for PTP at the calyx of Held. We will continue to clarify the mechanism of PTP and ultimately plan to use molecular genetics to selectively eliminate PTP from specific synapses to determine the role of PTP in different behaviors. A second major focus is to clarify cerebellar circuitry and understand how different circuit elements contribute to cerebellar function, which regulates motor learning, sensorimotor integration and social behaviors. We have recently shown that all Purkinje cells (PCs) have collaterals that target many types of cells within the cerebellar cortex, even in adults. This indicates it is necessary to consider feedback from the output of PCs to the cerebellar cortex. We will determine if PC?PC synapses promote synchronous activity also test the hypothesis that synaptically-connected PCs converge onto the same DCN neuron and regulate its firing. These studies will extend our understanding of cerebellar processing and will provide important insights into neurological disorders that arise from cerebellar dysfunction. We will also determine how specific regions of the cerebellar cortex regulate specific behaviors.

Abstract/Summary In addition to its well-established role in motor function and motor learning, the cerebellum is implicated in a myriad of non-motor behaviors. In humans, cerebellar damage can impair abstract reasoning and working memory, and result in PTSD. In animal models, the cerebellum regulates heart rate, breathing, aggression, appetite, fear conditioning and many other behaviors. In general, the role of the cerebellum in regulating these behaviors is not well understood. An important step in understanding these nonmotor behaviors is to determine the output pathway and downstream targets that allow the cerebellum to regulate these behaviors. It is known that Purkinje cells (PCs) relay signals from the cerebellar cortex to the deep cerebellar nuclei (DCN), which in turn activate the motor thalamus. It was assumed that DCN outputs are also responsible for nonmotor behaviors. Here, we describe powerful and direct inhibitory connections between PCs in regions of the cerebellum implicated in nonmotor behaviors, and neurons in the parabrachial nucleus (PBN). This is intriguing, because the PBN contributes to many of the same nonmotor behaviors influenced by the cerebellum. Based on our preliminary findings, we hypothesize that the PBN is a specialized cerebellar output that allows the cerebellum to regulate nonmotor behaviors. The first step in testing this hypothesis will be to characterize the connections between PCs and the PBN. The studies will determine the strength and prevalence of the direct PC to PBN synapse and determine the extent to which PCs regulate the activity of neurons in the PBN. The second major step is to identify the regions targeted by the PC?PBN pathway and to determine the contribution of this pathway to various behaviors. Preliminary studies suggest that this pathway projects to the hypothalamus, the amygdala, and the basal forebrain, and thus has the appropriate connectivity to allow the cerebellum to regulate diverse behaviors ranging from aggression to fear extinction. This promises to lead to a new appreciation of the roles of the cerebellum in numerous behaviors, it will provide insight into the circuits involved in these behaviors and it has important implications for many neurological disorders.