Roger A. Nicoll - US grants

The strength of synapses in many areas of the nervous system is not fixed. When activated repetitively their strength can be altered for prolonged periods of time. The use of dependent plasticity, referred to as long-term potentiation (LTP) is thought to play a role in certain forms of learning and memory. There are clearly at least two distinct forms of LTP; an N-methyl-D-aspartate (NMDA)-dependent form as expressed in the CA1 region of the hippocampus and an NMDA-independent form of LTP as expressed at mossy fiber synapses in the CA3 region. The studies proposed here will focus on aspects of the induction and expression in both forms of LTP. NMDA-dependent LTP. We will carry out experiments to determine if Ca++ entry via the NMDA receptor is necessary for LTP. We will use whole- cell voltage clamp to determine if a suppression potential can be found for LTP. Ca++ imaging will be performed with Dr. John Connor to measure directly Ca++ transients at various positive holding potentials. We will also address whether a rise in Ca++ is sufficient to generate stable potentiation. The Ca=== rise will be generated by activating voltage-dependent Ca++ channels which ca, under certain conditions, cause a transient potentiation. We will maximize conditions for Ca++ entry (e.g., raised extracellular Ca++) to see if Ca++ alone can evoke stable potentiation. If not, voltage-dependent Ca++ entry will be paired with synaptic stimulation in the presence of an NMDA antagonist in an attempt to reconstitute stable potentiation. If successful we will identify the necessary component provided by synaptic stimulation. Three types of experiment will address the expression of LTP. 1.) Whole-cell recording to reexamine the relative effect of LTP on the NMDA and non-NMDA component of the evoked and miniature EPSC. 2.) The sensitivity of the postsynaptic membrane to the glutamate agonist AMPA will be tested before and after NMDA application. 3.) We will use outside-out membrane patches from the soma to monitor the synaptic release of glutamate as detected by NMDA channel activity in the patch. Mossy fiber LTP. We will examine the action of a number of drugs which affect transmitter release and/or various signaling pathways in an attempt to localize the site of induction and expression of mossy fiber LTP. The manipulations will include changing Ca++/Mg++ ratio, phorbol esters, forskolin, kinase inhibitors, 4 amino-pyridine, CNQX and 2- amino-4-phosphonobutyrate (AP-4). A second series of experiments will examine mechanism underlying the modulation of mossy fiber LTP by various neurotransmitters known to alter this form of LTP, e.g., opioid peptides, norepinephrine and acetylcholine. The proposed studies will greatly advance our understanding of the cellular and molecular events underlying synaptic plasticity in the CNS. It can be anticipated that this knowledge will lead to the design of drugs that will have therapeutic value in such devastating conditions as Alzheimer's Disease.

Neurons in the central nervous system can be broadly classified into two strikingly different systems: 1) highly organized, hierarchical systems and 2) diffuse, non-specific systems. The norepinephrine-locus coeruleus (NE-LC) system best typified the second category. In the past neurophysiologists have focused primarily on the first category. Recent work on the NE-LC system indicates that there are fundamental differences in the synaptic mechanisms involved in this system. For instance it has been proposed that NE can simultaneously inhibit spontaneous activity and yet enhance the response to excitatory synaptic inputs thus increasing the signal-to-noise ratio of neurons. A satisfactory explanation for these seemingly paradoxical effects is not available. Our research will focus on the action of NE. We have preliminary evidence in the hippocampal slice that NE exerts a direct action on relay neurons to increase the signal to noise ratio, in agreement with previous proposals, and an indirect action involving a blockade of inhibitory pathways. The direct action appears to involve a cyclic AMP modulation of a Ca++ activated K+ conductance (GK(Ca)), while the site of action on inhibitory pathways is unclear. Both of these actions stand in marked contrast to the traditional view that NE is an inhibitory transmitter. The specific goals of this proposal, which will rely on intracellular recording are 1) to elucidate the mechanism and receptor type underlying the direct and indirect action of NE in the hippocampal slice, 2) to determine if similar results can be obtained from other cell types in slice preparations, 3) to determine if electrical stimulation of the NE-LC pathway in vivo mimics the effects of bath applied NE. The NE-containing locus coeruleus neurons are thought to be involved in such global brain functions as arousal and transitions between behavioral states as well as in numerous psychiatric disorders. The proposed experiments will not only provide deeper insight in the neurophysiological and neuropharmacological organization of NE in cortical regions and how NE is involved processing information, but also may well provide important clues about the role of NE in normal and abnormal behavior.

Serotonin has long been recognized as a neurotransmitter in the mammalian central nervous system, and as such has been implicated in a variety of behavioral functions. While substantial knowledge has accumulated regarding the biochemical processes involved in its synthesis, release and reuptake, its localization in the central nervous system and its binding sites, relatively little is still known about its postsynaptic actions, their pharmacology and their ionic and molecular bases. This application proposes, therefore, to examine the effects of serotonin in two regions of the mammalian brain; the hippocampus and prefrontal cortex. Based upon binding studies it has been proposed that these two areas are particularly enriched in the two best known subtypes of serotonin binding sites and therefore are potentially ideal locations to study serotonergic receptors and their mechanisms of actions. Four issues will be addressed in these areas: 1) the electrophysiological actions of serotonin; 2) the pharmacology of the serotonin receptor involved in these actions; 3) the final effector mechanisms responsible for the observed effects; and 4) the transmembrane signalling mechanism used by these receptors. These studies will be conducted using intracellular recordings in in vitro rat brain slices and will involve the use of current and voltage clamp methods. This study should elucidate some of the mechanisms involved in the actions of serotonin in the central nervous system and therefore should ccontribute significantly to our present understanding of serotonergic functions in the central nervous system as well as its associated pathological states.

Serotonin has long been recognized as a neurotransmitter in the mammalian central nervous system, and as such has been implicated in a variety of behavioral functions. While substantial knowledge has accumulated regarding the biochemical processes involved in its synthesis, release and reuptake, its localization in the central nervous system and its binding sites, relatively little is still known about its postsynaptic actions, their pharmacology and their ionic and molecular bases. This application proposes, therefore, to examine the effects of serotonin in two regions of the mammalian brain; the hippocampus and prefrontal cortex. Based upon binding studies it has been proposed that these two areas are particularly enriched in the two best known subtypes of serotonin binding sites and therefore are potentially ideal locations to study serotonergic receptors and their mechanisms of actions. Four issues will be addressed in these areas: 1) the electrophysiological actions of serotonin; 2) the pharmacology of the serotonin receptor involved in these actions; 3) the final effector mechanisms responsible for the observed effects; and 4) the transmembrane signalling mechanism used by these receptors. These studies will be conducted using intracellular recordings in in vitro rat brain slices and will involve the use of current and voltage clamp methods. This study should elucidate some of the mechanisms involved in the actions of serotonin in the central nervous system and therefore should ccontribute significantly to our present understanding of serotonergic functions in the central nervous system as well as its associated pathological states.

Serotonin has long been recognized as a neurotransmitter in the mammalian central nervous system, and as such has been implicated in a variety of behavioral functions. While substantial knowledge has accumulated regarding the biochemical processes involved in its synthesis, release and reuptake, its localization in the central nervous system and its binding sites, relatively little is still known about its postsynaptic actions, their pharmacology and their ionic and molecular bases. This application proposes, therefore, to examine the effects of serotonin in two regions of the mammalian brain; the hippocampus and prefrontal cortex. Based upon binding studies it has been proposed that these two areas are particularly enriched in the two best known subtypes of serotonin binding sites and therefore are potentially ideal locations to study serotonergic receptors and their mechanisms of actions. Four issues will be addressed in these areas: 1) the electrophysiological actions of serotonin; 2) the pharmacology of the serotonin receptor involved in these actions; 3) the final effector mechanisms responsible for the observed effects; and 4) the transmembrane signalling mechanism used by these receptors. These studies will be conducted using intracellular recordings in in vitro rat brain slices and will involve the use of current and voltage clamp methods. This study should elucidate some of the mechanisms involved in the actions of serotonin in the central nervous system and therefore should ccontribute significantly to our present understanding of serotonergic functions in the central nervous system as well as its associated pathological states.

The strength of synapses in many areas of the nervous system is not fixed. When activated repetitively their strength can be altered for prolonged periods of time. The use of dependent plasticity, referred to as long-term potentiation (LTP) is thought to play a role in certain forms of learning and memory. There are clearly at least two distinct forms of LTP; an N-methyl-D-aspartate (NMDA)-dependent form as expressed in the CA1 region of the hippocampus and an NMDA-independent form of LTP as expressed at mossy fiber synapses in the CA3 region. The studies proposed here will focus on aspects of the induction and expression in both forms of LTP. NMDA-dependent LTP. We will carry out experiments to determine if Ca++ entry via the NMDA receptor is necessary for LTP. We will use whole- cell voltage clamp to determine if a suppression potential can be found for LTP. Ca++ imaging will be performed with Dr. John Connor to measure directly Ca++ transients at various positive holding potentials. We will also address whether a rise in Ca++ is sufficient to generate stable potentiation. The Ca=== rise will be generated by activating voltage-dependent Ca++ channels which ca, under certain conditions, cause a transient potentiation. We will maximize conditions for Ca++ entry (e.g., raised extracellular Ca++) to see if Ca++ alone can evoke stable potentiation. If not, voltage-dependent Ca++ entry will be paired with synaptic stimulation in the presence of an NMDA antagonist in an attempt to reconstitute stable potentiation. If successful we will identify the necessary component provided by synaptic stimulation. Three types of experiment will address the expression of LTP. 1.) Whole-cell recording to reexamine the relative effect of LTP on the NMDA and non-NMDA component of the evoked and miniature EPSC. 2.) The sensitivity of the postsynaptic membrane to the glutamate agonist AMPA will be tested before and after NMDA application. 3.) We will use outside-out membrane patches from the soma to monitor the synaptic release of glutamate as detected by NMDA channel activity in the patch. Mossy fiber LTP. We will examine the action of a number of drugs which affect transmitter release and/or various signaling pathways in an attempt to localize the site of induction and expression of mossy fiber LTP. The manipulations will include changing Ca++/Mg++ ratio, phorbol esters, forskolin, kinase inhibitors, 4 amino-pyridine, CNQX and 2- amino-4-phosphonobutyrate (AP-4). A second series of experiments will examine mechanism underlying the modulation of mossy fiber LTP by various neurotransmitters known to alter this form of LTP, e.g., opioid peptides, norepinephrine and acetylcholine. The proposed studies will greatly advance our understanding of the cellular and molecular events underlying synaptic plasticity in the CNS. It can be anticipated that this knowledge will lead to the design of drugs that will have therapeutic value in such devastating conditions as Alzheimer's Disease.

cGMP dependent protein kinase; laboratory mouse; long term potentiation; voltage /patch clamp

Long-term potentiation (LTP) is a phenomenon in which brief repetitive synaptic stimulation results in the persistent enhancement in synaptic strength. Most of our understanding of synaptic plasticity comes from studies in the CAI region of the hippocampus. At these synapses, LTP is induced by the activation of postsynaptic NMDA receptors, while the site of expression remains controversial. A less-studied form of synaptic plasticity, which has been the topic of this grant, is found at mossy fiber synapses in the CA3 region of the hippocampus. We have accomplished most of the goals laid out int he previous grant. This form of LTP is independent of NMDA receptors, and we have provided evidence that it involves entirely presynaptic mechanisms. It requires the entry of Ca into the terminal, which activates a Ca/calmodulin-sensitive adenylyl cyclase (ACI). Activation of PKA then results in the persistent enhancement of transmitter release. The goals of the present grant are seven-fold: (1) We have evidence that mossy fiber synapses activate NMDA receptors. What role might these receptors have; is there a small NMDA-sensitive component to mossy fiber LTP: Might low frequency stimulation reveal an NMDA-dependent LTD at these synapses? (2) We would predict that the presence of ACI in presynaptic terminals may be critical for the expression of mossy fiber-type LTP. We will determine if an LTP with the same properties as mossy fiber LTP can be found in the CNS by examining synapses that express ACI. Specifically, we will examine the cerebellar parallel synapses, whose cell bodies express ACI, for a mossy fiber-type, (3) We will determine the role that zinc, which is contained and released from mossy fibers, plays at these synapses. Might it normally prevent the expression of nMDA-dependent forms of plasticity by blocking these receptors: (4) If our model of LTP is correct, it should be possible to demonstrate this form of LTP at autapses in single-neuron cultures. Since all the synapses will express LTP, this reduced system will greatly facilitate studies on this form of lTP. For instance, we can determine if changes occur int he frequency or amplitude of miniature EPSCs; (5) We will apply PKA inhibitors on synapses expressing LTP to determine if the maintenance requires kinase activity; (6) We wish to develop a method to induce LTP at all mossy fiber synapses in the slice, which will permit biochemical studies on molecular mechanisms. We have preliminary evidence that high K can selectively and persistently enhance mossy fiber responses; (7) Using mouse genetics, we will determine the effects of deleting or over-expressing proteins that are candidates for involvement in mossy fiber LTP. A comparison of the results on mossy fiber LTP to those on NMDA dependent LTP will markedly advance our understanding of the basic rules underlying synaptic plasticity in the brain. Such an understanding will be indispensable in designing strategies for preventing and/or correcting the cognitive defects associated with diseases such a Alzheimer's.

DESCRIPTION: (Applicant's Abstract) The physiological role of neuropeptides in the brain has received much attention, but we still have a rudimentary understanding of their synaptic roles. Recently we have found that the opioid peptide dynorphin is released during high-frequency stimulation from mossy fibers (mfs), and causes a presynaptic inhibition of neighboring mf synapses by acting on kappa 1 opioid receptors. It also raises the threshold for the induction of mf long-term potentiation (LTP). This grant proposes to examine 7 questions: (1) how does dynorphin inhibit transmitter release from mfs? Experiments will examine the effects of selective Ca channel blockers on the action of dynorphin as well as the effect of dynorphin on the frequency of miniature excitatory postsynaptic currents; (2) what are the properties controlling the release of dynorphin from mf synapses? We will apply AM esters of slow Ca buffers, such as EGTA, to selectively block peptide release; (3) what factors are responsible for the remarkably slow time course of the synaptically-released dynorphin? We will apply a brief puff of dynorphin and terminate the action of dynorphin by rapidly applying opioid antagonists. Application of a cocktail of peptidase inhibitors will determine if the time course is governed by enzymatic degradation; (4) are kappa 1 receptors actually present in the CA3 region? We will design experiments to localize the action of dynorphin, (5) what is the potency of the various opioid peptides that are contained in mfs? This information will help identify the most likely candidate for the synaptic effects; (6) does activation of kappa 2 receptors inhibit mf fiber evoked N-methyl-D-aspartate receptor mediated responses, and if so, what is the mechanism? (7) are there similarities between our results with dynorphin at mf synapses and the possible role of enkephalins at the lateral perforant path in the dentate gyrus? We will test the hypothesis that enkephalin controls the induction of LTP at this synapse by causing disinhibition during the tetanus. These studies will involve characterizing opioid mediated synaptic effects on identified interneurons. While neuropeptides have received much attention, particularly opioid peptides, the role of this class of signaling molecule remains very unclear. The present study will help define the physiological role of neuropeptides in the CNS at the cellular level which is absolutely essential for understanding the role of these peptidergic synapses in affect, cognition, substance abuse, and the management of pain.

DESCRIPTION OF OVERALL CENTER (Provided by Applicant): The Silvio Conte Center for Neuroscience Research at UCSF will explore the central hypothesis that receptors and ion channels serve as organizing centers and membrane attachment points for large, multi-protein complexes that control neuronal excitability. The Center will explore how the various proteins are trafficked and targeted to specific sites on the surface of the cell and the functional consequences of these multi-protein complexes. We have assembled a group of investigators who have common interests and goals, but who bring unique neuropharmacological approaches to the analysis of a common biological problem. We will 1) employ organisms amenable to genetics for the identification of novel genes important for receptor and ion channels aggregation, 2) use cellular, molecular and pharmacological approaches to understand the mechanisms involved in the delivery of these membrane proteins to the surface and their targeting to specific sites on the cell?s surface, and 3) use structural analysis to elucidate how the various proteins within the multi-protein complex interact with each other. Results from these studies will be of fundamental importance for understanding normal and abnormal brain function. For instance, most psychiatric and neurological diseases are believed to result from alterations in synaptic transmission and neuronal excitability. Furthermore, accumulating evidence indicates that synaptic plasticity involves the rapid and long lasting modification of the receptor composition of the postsynaptic membrane. Thus, an understanding of the principles involved in the formation and maintenance of multi-protein signaling complexes will be invaluable in the development of therapeutic drugs for conditions such as depression, schizophrenia, Alzheimer's disease and Parkinson's disease.

Most excitatory synapses in the brain release glutamate. These synapses show a remarkable degree of plasticity, in which brief repetitive activation results in a long term potentiation or LTP. At most synapses LTP requires the activation of postsynaptic NMDA receptors. However, hippocampal mossy fiber synapses, exhibit a form of LTP that is independent of NMDA receptor activation. Our experiments during the past decade strongly suggest that mossy fiber LTP (mfLTP) is an entirely presynaptic form of plasticity. This grant will explore three separate, but overlapping, issues concerning the mechanisms underlying mfLTP. 1) The role of kainate receptors (KARs) in mfLTP will be explored, using KAR knockout mice and a novel KAR antagonist. Preliminary results suggest that KARs can control the threshold for the induction of mfLTP. 2) We have recently found that the hyperpolarization-activated nonselective cation current l-h mediates the expression of mfLTP. In our model we propose that the cAMP-dependent enhancement of l-h causes a depolarization of the mossy fibers which enhances transmitter release secondary to spike broadening. We will test this model with a number of experiments, a) If mfLTP is associated with a depolarization of the terminals, then the enhancement of synaptic transmission by K+ should occlude with mfLTP, b) We will record from mossy fiber boutons to directly determine if depolarization causes spike broadening and whether mfLTP is associated with a depolarization, c) Our model predicts that all synapses formed by dentate granule cells should show an LTP similar to that found at the mossy fiber to pyramidal cell synapse. However, others have reported that the synapses made by mossy fibers onto interneurons in s. lucidum do not show LTP. Thus we will analyze the properties of this synapse to understand why it fails to generate LTP. 3) We have preliminary results indicating that ambient extracellular adenosine exerts a profound tonic inhibition of mossy fiber transmitter release and, indeed, is necessary for mossy fibers to express both short-term and long-term plasticity. We will analyze the role that this tonic action has on the properties of mossy fiber synapses and why adenosine has such a pronounced and selective action on mossy fibers. These experiments will help elucidate the mechanisms involved in controlling transmitter release and more specifically how this control is involved in short term and long term synaptic plasticity. Our results should provide insight into the cellular mechanisms underlying learning and memory.

DESCRIPTION (provided by applicant): Transmission at excitatory synapses in mammalian brain is mediated primarily by glutamate acting on AMPA receptors and NMDA receptors, two classes of ligand gated ion channels. Whereas NMDA receptors are stable components of the postsynaptic density (PSD), AMPA receptors cycle on and off the synaptic membrane in a manner that is tightly controlled by neuronal activity. This regulated insertion and removal of AMPA receptors at the synapse provides a mechanism for altering synaptic efficacy and for storing information in brain. Our preliminary data show that functional expression of AMPA receptors in cerebellar granule cells requires stargazin, a member of a large family of four-pass transmembrane proteins. And, we have defined a family of transmembrane AMPA receptor regulatory proteins (TARPs), which comprise stargazin, gamma3., gamma-4 and gamma-8 - but not related proteins - that mediate surface expression of AMPA receptors. TARPs mediate synaptic trafficking of AMPA receptors by interacting with the postsynaptic density protein, PSD-95. Whether stargazin-like proteins control AMPA receptor turnover and synaptic plasticity in forebrain regions such as hippocampus remains uncertain. However, we found that one of the TARPs, gamma-8, is uniquely enriched in hippocampus, where it interacts with AMPA receptors. We now propose to determine whether gamma-8 regulates the activity-dependent AMPA receptor trafficking that underlies aspects of synaptic plasticity. Because phosphorylation plays a major role in activity-dependent AMPA receptor trafficking, we will assess functional roles for phosphorylation of gamma-8 in hippocampus. We will also characterize functional domains and protein interactions with the unique C-terminal tail of gamma-8. We will also take genetic approaches and determine how overexpression or targeted disruption of gamma-8 modulates AMPA receptor targeting and turnover at synapses. These studies will provide fundamental insight into mechanisms for postsynaptic development and function. Understanding mechanisms that control synaptic targeting of glutamate receptors will help clarify the role that this plasticity plays in learning and memory.

DESCRIPTION (provided by applicant): Synaptic development and function require assembly of protein complexes at synaptic sites. A key breakthrough in understanding molecular mechanisms for synapse development was the discovery that PDZ proteins play central roles in scaffolding receptors and signaling elements at pre- and postsynaptic sites. Mutations in lin-2, lin-7 or lin-10, genes encoding PDZ proteins in C. elegans, disrupt vulval differentiation. Elegant biochemical and genetic analyses indicate that LIN-2/-7/-10 form a protein complex that mediates subcellular localization of an EGF receptor essential for vulval development in worms. The LIN-2/-7/-10 complex also occurs prominently in neurons. Disruption of lin-10 in C. elegans prevents proper postsynaptic sorting of a glutamate receptor. In mammals, close homologues of LIN-2 (CASK), LIN-7 (MALS/Veli) and LIN-10 (Mint/X11) have all been identified and the LIN-2/-7/-10 complex occurs at highest levels at pre- and postsynaptic junctions in mammalian brain. We found that the PDZ domains of MALS bind the C-termini of NMDA receptor subunits, and biochemical studies implicate the CASK/MALS/Mint complex in microtubule-dependent trafficking of NMDA receptors. Based on these data, we hypothesize that MALS regulate receptor trafficking at mammalian cell junctions, especially synapses. To address this hypothesis, we are generating mutant mice that lack the three MALS isoforms (MALS-1/- 2/-3). We previously reported that mice lacking MALS-1 and MALS-2 are without any discernable phenotype. In preliminary unpublished studies, we now find that MALS-3 knockouts (KOs) have developmental defects. Whereas mice that lack any pair of MALS genes are viable for at least one week of life, triple mutant mice die within 1 hour of birth. Furthermore, hypomorphic mutants that lack MALS-1/-3 or MALS-2/-3 have prominent neurological phenotypes. We now plan to study these mutant mice to determine the essential roles for MALS in regulating ion channels and growth factor receptors at mammalian cell junctions, especially synapses. Because MALS proteins regulate growth factor receptors and interact with NMDA type glutamate receptors at synapses, this work will be relevant to both tissue development and synaptic plasticity.

DESCRIPTION (provided by applicant): Excitatory synapses in the CNS release glutamate, which acts on two types of ionotropic receptors: AMPA receptors (AMPARs) and NMDA receptors (NMDARs). Evidence indicates that AMPARs, in contrast to NMDARs, are highly mobile and that activity can rapidly change the number of receptors at the synapse. To begin to understand the molecular basis underlying the regulation of synaptic AMPARs, we have focused on the ataxic and epileptic mouse stargazer. Cerebellar granule cells in this mouse lack functional AMPARs, although NMDARs are normal and excitatory synapses release normal amounts of glutamate. During the current Conte grant we have carried out a series of studies on the role of stargazin (v-2), the mutated gene in the stargazer mouse. We have found that stargazin is an auxiliary subunit of AMPARs, not only controlling their trafficking to the cell surface and to the synapse, but also controlling their biophysical properties. We have identified a total of three additional proteins (v-3, y-4, v-8) that are expressed throughout the CNS and can rescue the AMPAR defect in cerebellar granule cells. We refer to these proteins as transmembrane AMPAR regulatory proteins (TARPs). We have succeeded in deleting the gene for each of the TARPs in mice. These mutant mice will form the basis for many of the proposed experiments in this RO1 grant, which is a continuation of the work carried out on the Conte Grant. There are 4 Specific Aims. (1) Determine the role of v-3 in the mouse brain, (2) determine the role of y-4 in the mouse brain, (3) determine the functional differences among TARPs, and (4) determine whether TAPRs may have AMPAR-independent roles in the CNS. The role of TARPs in AMPAR trafficking, synaptic transmission and plasticity will be studied primarily in the hippocampus. Preliminary studies indicate that y-8 plays an important role in AMPAR trafficking in the hippocampus, but substantial AMPAR transmission remains. We will also compare the ability of various TARPs to modify the deactivation of AMPARs and the kinetics of synaptic transmission. We have evidence that each of the roles TARPs play (i.e., surface delivery, synaptic targeting and receptor gating) all vary for each of the TARPs. We have found that the Y-2/Y-3A/-4, triple KO is lethal and the newborn pups are completely immobile. We will determine why the spinal cord is nonfunctional. These studies may uncover novel roles for TARPs in the nervous system. Given the critical role that receptor trafficking plays in synaptic plasticity and, by implication in certain aspects of learning and memory, it is anticipated that findings from these studies will have direct clinical impact. Indeed, clinically promising AMPAkines exert their effect, in part, by controlling the kinetics of AMPAR gating similar to TARPs. In addition, TARPs modify the pharmacological properties of AMPAkines and, thus, represent a novel target for drug design.

DESCRIPTION (provided by applicant): Central to the focus of this grant is the AMPA receptor (AMPAR), which drives most fast synaptic transmission in the brain. Remarkably the AMPAR is the final common path for at least three mechanistically distinct forms of plasticity. This grant will compare and contrast these forms of plasticity. To accomplish these goals we use a combination of techniques. Central to our studies is electrophysiology, since this is the most critical and quantitative way to measure the functional consequences of our molecular manipulations. Most of the experiments involve a molecular replacement strategy that we recently developed, in which endogenous receptor subunits are genetically deleted and replaced with mutated receptors. The three forms of activity- dependent trafficking of AMPARs that will be studied are: 1) Long-term potentiation (LTP). Using a molecular replacement strategy we have recently found that LTP can be normally elicited without the C-tails of GluA1 or GluA2 and is also normal when exogenous kainate receptors replace AMPARs. This raises many issues. Two types of experiments are planned. First, we will determine if there are shared features of the non-NMDARs, such as the N-Terminal Domain (NTD), that are required. Second, we will test whether in more intact conditions TARPs/NETOs do play a role. 2) Long term depression (LTD). A substantial literature has argued that the C-tails of both GluA1 and GluA2 are required for LTD. We will use the same molecular replacement strategy that was used to study LTP to determine the minimal requirements for LTD. It has also been reported that, although LTD requires glutamate binding to the NMDAR, it does not requires calcium entry, a finding that we have confirmed. Using a molecular replacement strategy for NMDARs we will determine the critical domain(s) of the NMDAR that is/are required for this metabotropic action. 3) Homeostatic synaptic scaling/distance-dependent dendritic scaling. Homeostasis involves the global change in synaptic strength following prolonged changes in activity, whereas distance-dependent dendritic scaling is a phenomenon in which distant synapses have twice as many AMPARs as proximal synapses. We have found that the GluA2 subunit is absolutely required for both homeostasis and for distance-dependent dendritic scaling, raising the possibility that these two forms of plasticity use similar mechanisms. We will define which domains of GluA2 are required for both forms of plasticity. Given the central role that AMPARs plays in synaptic plasticity it is anticipated that findings from these studies will have direct clinical impact.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. This project will examine the role of activity in controlling the excitability and morphology of neurons. We have succeeded in making conditional mice in which we can, in a cell autonomous manner, silence all excitatory input to that neuron, by deleting all AMPA and NMDA receptors. These neurons can then be studied over various time intervals after silencing to examine the effects on neuronal excitability and morphology.

DESCRIPTION (provided by applicant): The overall goal of my research program is to elucidate the underlying molecular principles that govern the assembly of the postsynaptic component of a synapse. There are three main questions we wish to address. First, what are the sequences of events that occur during synapse formation? Second, how does a synapse maintain a stable anatomical identity? Finally, what is the mechanism whereby activity can induce a change in synapse function? Central to the understanding of synaptic transmission are the glutamate receptors embedded in the postsynaptic density (PSD). To tackle these ambitious goals we use a combination of a number of techniques. The most central to our studies is electrophysiology, since this is the most critical way to measure the functional consequences of our molecular manipulations. This grant is focused on a variety of proteins that act as glutamate receptor auxiliary subunits. While voltage gated ion channels have long been know to be decorated with auxiliary subunits, which control all aspects of trafficking and function, the notio that ligand gated ion channels also associate with auxiliary subunits is quite new. The most studied family of auxiliary subunits is the TARPs, which selectively control the trafficking and function of the AMPAR subtype of glutamate receptor. However, recent studies indicate that other structurally unrelated proteins, such as CNIH2, CKAMP44, and SynDIG1 also serve as AMPAR auxiliary subunits. In addition, NETO-1/2 has been shown to serve a similar role for kainate receptors. In this renewal we will characterize the role of CNIH2 in the brain with the use of conditional knockout mice. Initial results suggest widespread effects of deleting CNIH2. We will also determine the physiological role of TARP ¿-7, an unusual TARP, which sets it apart from the other well characterize TARPs. Understanding the role of SynDIG1 forms the third Aim of this grant. Both overexpression and RNAi in slice culture will be used for these experiments. Finally we will use the CA1 synapse, which normally lacks kainate receptors, as a null to determine the role of NETO-1/2 in trafficking and gating of kainate receptors. It is hoped that these studies will uncover novel roles for glutamate auxiliary proteins in the nervous system. Given the critical role that receptor trafficking plays in synaptic plasticity it is anticipated tht findings from these studies will have direct clinical impact. Indeed, clinically promising AMPAkines exert their effect, in part, by controlling the kinetics of AMPAR gating similar to TARPs.

DESCRIPTION (provided by applicant): This proposal requests R13 support for a longstanding, well-attended, and well-received Gordon Research Conference (GRC) on Excitatory Synapses and Brain Function. The synapse is central to our understanding of circuit function and behavior. In the central nervous system, excitatory synapses represent the primary means of information processing by local circuits and communication between brain regions. Synapses serve as the site of action for many commonly prescribed medications and their disruption contributes to many neurological and psychiatric disorders. These include schizophrenia, autism, depression, substance abuse and addiction, Parkinson's disease, Alzheimer's disease, traumatic brain injury, stroke and epilepsy. In some cases, synaptic dysfunction is causal in disease, whereas in other cases it represents the downstream sequelae of one or more underlying molecular defects. In either case, a fundamental understanding of the formation, structure, molecular organization, signaling function, and plasticity of synapses is essential to progress in lessening the burden of human neurological disease and for predicting and improving mental health. This conference is unique in its focus on the excitatory synapse, and in its multidisciplinary group of participants including structural biologists, molecular and developmental biologists, cell biologists, biochemists, cell/molecular imagers, biophysicists and neurophysiologists. The conference is intended to relate fundamental insights in excitatory synaptic function to the impairments in synaptic function that occur in disease, as well as the maladaptive plasticity that occurs in substance abuse. The goal of the conference is to identify and highlight fundamental new insights into synaptic function and dysfunction from a thematic approach. The program has been designed to also highlight cutting edge approaches and to stimulate new concepts, methods and technologies within a sound biological framework of fundamental neuroscience. The conference will bring together expert scientists worldwide in an environment that is conducive to discussion and exchange of ideas. The exchange of ideas at this conference has been a driving force for the field. We expect the 2011 GRC on Excitatory Synapses and Brain Function will shape future scientific directions, and provide critical support for the mission of multiple institutes at NIH including NIMH, NINDS, NIDA and NIA.

DESCRIPTION (provided by applicant): This is an application for renewal of support for a successful training program for students admitted to the UCSF graduate program in Neuroscience. The goal of this training program will continue to be to provide the best possible education and training of students in concepts and methods of modern neuroscience in order to give these students the skills, knowledge and enthusiasm needed for them to make creative contributions to Neuroscience during their entire careers. The Neuroscience Program currently has 72 faculty members in 18 different basic science and clinical departments and affiliated institutes. Program membership is restricted to faculty who contribute to program activities and faculty memberships are periodically reviewed. Virtually all areas of neuroscience are encompassed by the research interests of our faculty who have received numerous honors, including Nobel and other highly prestigious prizes. Our faculty includes many members of the National Academy of Sciences, the Institute of Medicine and the American Academy of Arts and Sciences. Over the past 3 decades this program has recruited many of the most able and imaginative students interested in Neuroscience. Many of these have now initiated or are well established in careers at leading American or international universities and elsewhere. Several of our older graduates already have careers of distinction recognized by prestigious awards, such as MacArthur and McKnight faculty awards. Our more recent graduates have received also prestigious awards, including Helen Hay Whitney postdoctoral scholarships, and Harold Weintraub and Donald B. Lindsley Awards for outstanding Ph.D. theses. During their first year, trainees will receive a strong foundation in modern neuroscience as well as scientific writing and oral presentation skills through an introductory 2-quarter-long core course, intensive mini- courses, a grant-writing workshop, and oral defense of a written minor proposal. The minicourses are sponsored in collaboration with other biomedical science graduate programs and are intended to stimulate creative theses at the interface of disciplines as well as provide training in how to efficiently identify the major obstacles to progress and opportunities in a new research area. The trainees will attend a weekly seminar series and weekly student-faculty journal club. They will also complete at least three laboratory rotations in more than one area of Neuroscience before choosing a thesis laboratory. The program introduces students to our many laboratories through faculty and advanced student/postdoc presentations at our annual retreat and a series of dinners for first year students with small groups of faculty. During their second year, students will complete a Scientific Ethics course, begin advanced literature-based special topics courses for 2nd year and more senior students, and continue attendance at seminars, journal clubs, and the annual retreat. They will also write and orally defend a thesis proposal in order to advance to candidacy. All trainees will receive comprehensive research training in the laboratory of their Ph.D. supervisor and this will be their major commitment after advancement to candidacy although they are expected to continue participating in program intellectual activities. Students meet no less than annually with their thesis committees and at least every six months with their thesis committee chair, who is not their research supervisor. Student progress is also supervised by a Student Progress Oversight Committee that reviews thesis committee reports. At every stage of their training, students have access to activities sponsored by the UCSF Graduate Education in Medical Science Program (GEMS) which is an HHMI and School of Medicine-funded program that sponsors courses and activities aimed at increasing the medical literacy and knowledge of graduate students. The UCSF Office of Career and Professional Development sponsors many workshops for our students to help them develop their leadership, time management, oral and written presentation and other skills. This office also sponsors many seminars and information sessions aimed at increasing the knowledge of our students about their future career options. This training grant has been the single most important pillar underlying our program's success over the past 30 years. This training grant is used to support students during their first and second years of study before they have advanced to candidacy and initiated full-time Ph.D. thesis research. Continued support is essential for us to continue to provide graduate students with the education they will need to become leaders in their field in the future.

All ionotropic glutamate receptors share the same domain structure with two extracellular domains; the amino terminal domain (ATD) and ligand binding domain (LBD), a transmembrane domain (TM) and a cytoplasmic carboxy terminal domain (CTD). The synaptic trafficking of glutamate receptors is of fundamental importance for synapse development and plasticity. Virtually all the work on this topic has focused on the CTD of the various subclasses of glutamate receptor. These studies, while contributing importantly to our understanding, left many unanswered question. On the other hand virtually nothing is known about the role of the ATDs of these receptors, which account for approximately 50% of the protein. Our recent results have established a critical role of the ATD for the subunit specific synaptic trafficking, not only for AMPA receptors, but also for kainate receptors and for the Delta1 glutamate receptor. The overall goals of this project is to 1) determine the functional similarities and differences of the ATDs of subunits within a class of glutamate receptor as well as between different classes of receptor and 2) identify synaptic cleft proteins that specifically interact with the extracellular domains of the various glutamate receptors. To accomplish these objectives the first approach will be to carry out a series of deletions of the ATD to determine what regions are necessary for their function. Based on the regions we identify, we will carry out a series of domain swapping experiments to determine if the regions we identify are sufficient for their function. The second approach will focus on identifying synaptic cleft proteins that interact with the extracellular domains of the glutamate receptors. This will rely on both a candidate approach and an unbiased proteomic approach. Specifically, we will 1) determine the role of the ATD in subtype- and subunit-specific ionotropic glutamate receptors in constitutive and plasticity-dependent synaptic targeting; 2) determine the role of GluD1 in excitatory synaptic transmission 3) characterize MDGA Proteins as novel ATD binding proteins These results will provide basic information about the rules and proteins involved in the extracellular control of basal and activity-dependent synaptic glutamate receptor trafficking.