Richard J. Weinberg - US grants

Recent receptive field mapping studies of primary somatosensory cortex (SI) in cat and monkey have demonstrated that the body surface is represented as a mosaic of discrete placed-defined cortical columns, "segregates", approximately 0.3-0.6 mm in diameter. The neuroanatomy of these cortical modules will be studied in cats and rats: Small injections of retrograde tracers, including gold-labeled wheat germ agglutinin conjugated to apo- horseradish peroxidase (WGA-apoHRP-Au) and various fluorescent tracers, will be made in individual physiologically-identified SI segregates to establish their thalamic input. Similar experiments will be performed using neurotropic viral tracers to permit transsynaptic retrograde labeling. In this way, the disynaptic pathways to the cortex from the dorsal column nuclei and the spinal dorsal horn will be studied. The organization of inhibitory intracortical connections of electrophysiologically-defined segregates will be studied using the transmitter-selective retrograde tracer [3H]GABA, and by combining GABA immunocytochemistry with WGA-apoHRP-Au pathway tracing. Retrograde transport of the transmitter-selective tracer [3H]D-aspartate will be used to study the organization of excitatory intracortical connections; immunocytochemistry with antibodies for glutamate and aspartate will be used to further define the transmitter used by these neurons. This analysis of the modular connectivity of SI directly addresses fundamental principles of cortical organization.

DESCRIPTION (Adapted from the Investigator's Abstract): Glutamate is the major excitatory neurotransmitter in cerebral cortex. Electrophysiology and pharmacology have distinguished several types of glutamate receptors, but only with the advent of modern molecular techniques has their full diversity been recognized. Given the central role of glutamate in excitatory neurotransmission, understanding its receptors is of fundamental significance to neuroscience. It may also have practical implications: Drugs acting on glutamergic systems have not yet had a major impact on clinical practice, presumably because the transmitter is so ubiquitous that the selectivity of these agents is limited. Rational design based on a better understanding of receptor subtypes may yield a new generation of drugs with greater specificity of action. Novel research on the synaptic localization of glutamate receptor subunits in dentate gyrus and somatic sensory cortex is proposed, utilizing a new postembedding gold immunocytochemical technique for electron microscopic localization. This will allow us to investigate the subcellular distribution of glutamate receptors and their relationship to the plasma membrane and the active zone, as well as to assess how faithfully light microscopy reflects the underlying distribution of receptors. To explore how the arrangement of glutamate receptors may influence calcium entry into neurons, we will study the pattern of colocalization of AMPA subunits, and the distribution of AMPA and NMDA receptors on spines and dendritic shafts. The information thus gathered will allow study of the dynamics of glutamate receptors. The third part of the proposed research will test whether there are changes in distribution, concentration, or expression of these receptors associated with altered synaptic efficacy in two models of cortical plasticity: responsiveness in the barrel field of somatic sensory cortex after modification of its input, and long-term potentiation in the perforant pathway from entorhinal cortex to dentate gyrus. A deeper knowledge of mechanisms that may underlie cortical plasticity is of potential significance in understanding the reorganization associated with recovery from brain injury.

DESCRIPTION: Glutamate, the major excitatory transmitter in cortex, is also the transmitter used in the main afferent pathway from thalamus to cortex. The proposed study of glutamate receptors in thalamocortical transmission is designed further to elucidate mechanisms underlying initial steps of sensory processing in neocortex. Glutamate receptors in S1 postsynaptic to terminals from the ventrobasal thalamus will be characterized by combining pathway tracing with immunogold electron microscopy. The glutamate receptors in dendrites of excitatory neurons that receive thalamic information will be compared with those in dendrites of inhibitory neurons. The glutamate receptors that receive thalamic information will be compared with those used by the same neurons to receive information from cortex. Besides contributing to our understanding of thalamocortical mechanisms, the research relates to the basic issues in development, cell biology, and synaptic physiology.

DESCRIPTION (Adapted from applicant's abstract): Excitatory neurotransmission in the brain is mediated mainly via ionotropic glutamate receptors. These receptors are anchored at the postsynaptic membrane by cytoplasmic proteins concentrated in the postsynaptic density (PSD). PSD proteins may help to shape the postsynaptic response to glutamate, but even after the full range of possible biochemical and electrophysiological effects of these molecules has been documented in vitro, their actions in vivo will depend on how they are physically arranged and organized at the synapse. Modern biochemical techniques permit construction of a topological diagram of protein-protein interactions within the macromolecular complex of the PSD, but provide no direct information on the positioning of the elements of this complex, the "supramolecular architecture" of these molecules. The present research will harness new tools for ultrastructural localization of proteins to investigate the supramolecular architecture of the PSD, as a step toward elucidation of the structure and function of the excitatory synapse.

Calcium is a key intracellular messenger, but excessive levels of Ca 2+ are toxic. This work addresses Ca 2+ signaling in glutamatergic axospinous synapses in the CA1 region of the rat hippocampus. Specifically: 1. To provide structural evidence on the extent of interactions among Ca 2+ sources in dendritic spines, the spatial organization of NMDA receptors and voltage-dependent calcium channels will be determined in individual dendritic spines. 2. Since the enzymatic activation of CaMKII is likely to be sensitive to its location in relationship to Ca 2+ microdomains, the organization of CaMKII within the spine will be studied. 3. Restoration of [Ca2+]i to resting levels is important not only to maintain the fidelity of Ca 2+ signals, but also to avoid the potential cytotoxicity associated with excessive calcium loads. Therefore, the organization of Ca 2+ pumps likely to be concentrated in the plasma membrane and organelles of the spine will be studied, as will calcium-binding proteins that may contribute to shaping Ca 2+ transients. Quantitative anatomical data generated by this work will be used to refine computer simulations running under the MCell program, allowing better insight into the functional significance of dendritic spines.

DESCRIPTION (provided by applicant): Long-term modifications in synaptic efficacy are implicated in learning and memory; dysfunction of this plasticity is implicated in a variety of neurodevelopmental and neuropsychiatric disorders. The long-term goal of this project is to elucidate the supramolecular organization of the postsynaptic density (PSD), which plays a central role in synaptic signal processing. This information may ultimately prove very useful in designing novel approaches to the prevention or treatment of brain disorders. The proposal for this funding cycle includes three specific aims: Aim 1 is to develop new electron microscopic tools to study the ultrastructure of synapses, and to use these tools to study abnormalities of the PSD in mouse models of autism and schizophrenia. Aim 2 is to examine how actin filaments attach to the PSD, to study the organization of two isoforms of a protein that links actin to the PSD, and to study two PSD-associated enzymes that control Rho-family proteins (molecular switches that modulate actin remodeling). Aim 3 is to study the alignment of presynaptic release sites with postsynaptic receptors, and to explore possible disruptions in synaptic structure and receptor expression in mice that have mutations in synaptic adhesion molecules.

Calcium is a key intracellular messenger, but excessive levels of Ca 2+ are toxic. This work addresses Ca 2+ signaling in glutamatergic axospinous synapses in the CA1 region of the rat hippocampus. Specifically: 1. To provide structural evidence on the extent of interactions among Ca 2+ sources in dendritic spines, the spatial organization of NMDA receptors and voltage-dependent calcium channels will be determined in individual dendritic spines. 2. Since the enzymatic activation of CaMKII is likely to be sensitive to its location in relationship to Ca 2+ microdomains, the organization of CaMKII within the spine will be studied. 3. Restoration of [Ca2+]i to resting levels is important not only to maintain the fidelity of Ca 2+ signals, but also to avoid the potential cytotoxicity associated with excessive calcium loads. Therefore, the organization of Ca 2+ pumps likely to be concentrated in the plasma membrane and organelles of the spine will be studied, as will calcium-binding proteins that may contribute to shaping Ca 2+ transients. Quantitative anatomical data generated by this work will be used to refine computer simulations running under the MCell program, allowing better insight into the functional significance of dendritic spines.

DESCRIPTION (provided by applicant): LTP involves the reorganization of AMPA receptors (AMPARs) in the postsynaptic membrane. Most current work on neural plasticity in mammals involves systems more tractable than intact brain, but it remains unclear how suitable these are as models for brain synapses. Therefore, Specific Aim 1 asks whether synapses in brain resemble synapses in slice-culture or cultured neurons. Excitatory synapses in hippocampus express AMPARs comprising GluR1, GluR2, and GluR3 subunits;GluR2 and GluR3 are trafficked and anchored at the synapse by the same mechanisms, distinct from the mechanisms that handle GluR1. Specific Aim 2 uses immunogold electron microscopy to gain new insight into the organization of AMPA receptors at the synapse, to determine the organization of AMPAR subunits in a synapse, how their organization changes after LTP and LTD, and where new AMPARs are inserted in the synapse. Spine volume is correlated with the number of AMPA receptors in the synaptic membrane, implying that AMPAR expression must be coupled to spine size. While the causal relationship is not clear, this implies a linkage between the signaling pathways that mediate synaptic plasticity, and those that mediate changes in the actin cytoskeleton. Specific Aim 3 examines how LIM kinase and Slingshot, two major proteins involved in the control of actin remodeling, are organized in a spine, whether their organization differs between large spines and small spines, and how their organization and level of activation may change after LTP/LTD. These studies will help to elucidate basic mechanisms underlying learning and memory. Their successful completion may help to gain new insight into the biological basis for memory disorders and mental retardation.

This project addresses the architecture of neuropil in the human cerebral cortex. Aim 1 uses material from surgically-resected temporal lobe to perform high-resolution quantitative electron microscopic studies of the structure of excitatory synapses, including synapses onto identified classes of interneurons. Using the same biological material, Aim 2 performs quantitative immunogold electron microscopy to define the architecture of a number of different molecules abundant in the postsynaptic density, focusing on proteins whose mutations are implicated in neuropsychiatric and neurodevelopmental disease, especially schizophrenia. Aim 3 performs work similar to that specified for Aims 1 and 2, but focusing on cortical organoids cultured in vitro. These results will make it possible to compare the structure and molecular organization of synapses in culture with synapses in intact brain.