Thomas J. O'Dell - US grants

Within the hippocampus, a region of the brain important for some types of learning and memory, the strength of excitatory synapses onto CA1 pyramidal cells is persistently altered following certain patterns of synaptic activity. Long-term potentiation, a persistent enhancement of synaptic transmission that occurs at these synapses, is currently a leading candidate for a cellular mechanism involved in memory formation. On the basis of theoretical arguments it has been proposed that, in addition to long-term potentiation, the synaptic processes involved in memory may also include mechanisms that produce a long-term depression of synaptic transmission. Consistent with this prediction, two different types of homosynaptic LTD have been observed in the hippocampus. However, while LTD of basal synaptic transmission is a prominent phenomenon in the hippocampus of young animals,it is rarely seen at synapses in the CA1 region of the adult hippocampus. Thus these two forms of homosynaptic LTD may primarily be developmental forms of synaptic plasticity and are probably unlikely to contribute to the synaptic processes involved in memory in the adult hippocampus. Although not yet well understood, there is one form of long-term depression, known as depotentiation, that occurs at synapses in the adult hippocampus. Depotentiation refers to the persistent depression of synaptic transmission that occurs following low-frequency stimulation of synapses that have recently undergone LTP. Since depotentiation selectively occurs at synapses that have undergone LTP in the adult hippocampus, it may have an important role in hippocampal-dependent memory formation. Thus, the specific aim of the experiments described in this proposal is to identify the synaptic and molecular processes responsible for depotentiation. Electrophysiological techniques will be used to study synaptic transmission in the adult hippocampus and pharmacological manipulations will be used to probe the signal transduction pathways involved in depotentiation. Moreover, depotentiation will be studied in transgenic mice expressing a mutant form of a calcium/calmodulin-dependent kinase II, a protein kinase that may be an important modulatory role in hippocampal synaptic plasticity. The proposed experiments focus on depotentiation since to fully understand the cellular basis of memory in the adult hippocampus, it will be necessary to identify the synaptic and biochemical processes involved in forms of synaptic plasticity, such as depotentiation, that are present in the adult hippocampus. Moreover, understanding these processes will be important understanding, at the cellular level, how memory is impaired in pathological conditions such as Alzheimer's disease.

DESCRIPTION: (Applicant's Abstract) Activity-dependent processes that modify the strength of synaptic transmission are thought to play a crucial role in the formation of new memories during learning and in the refinement of synaptic connections that occurs during development. In the hippocampus, a region of the brain known to have a crucial role in memory formation, excitatory synapses express a number of different forms of synaptic plasticity. For instance, some patterns of synaptic activity induce a long-lasting enhancement of synaptic transmission known as long-term potentiation (LTP) while different patterns of activity can induce a long-lasting decrease in transmission known as long-term depression (LTD). The synaptic events leading to LTP and LTD induction are well characterized and key components of the signaling pathways responsible for these forms of plasticity have been identified. Little is known, however, about how the signaling pathways responsible for LTP and LTD interact to generate the "rules" that determine how synaptic strength is modified by different patterns of synaptic activity. In this project we will use synaptic stimulation protocols that mimic endogenous patterns of neural activity in the hippocampus to investigate the cellular and molecular mechanisms that regulate LTP induction at excitatory synapses onto hippocampal pyramidal cells. In particular, we will investigate the role of activity-dependent changes in NMDA-type glutamate receptor function, nitric oxide production, and cAMP signaling in the ability of certain patterns of synaptic stimulation to disrupt the induction of LTP. In addition, we will investigate synaptic transmission and plasticity in transgenic mice with a mutation in the postsynaptic density protein PSD-95, a protein thought to be an important organizer of NMDA receptor-dependent signaling pathways involved in synaptic plasticity. These experiments will provide insights into the cellular and molecular mechanisms controlling forms of synaptic plasticity thought to be involved in learning and memory and may provide insights into how changes in these processes might contribute to the impairment of memory formation that occurs in Alzheimer's disease and during normal aging.

DESCRIPTION (provided by applicant): This application is for a continuation of funding for a postdoctoral training program in Cellular Neurobiology. The goal of this program, which has been supported by NIH since 1968, is to provide postdoctoral fellows in their first 2 years after obtaining the Ph.D. with cutting-edge training in electrophysiological, optical, molecular, and/or structural techniques and their application to fundamental questions in neurobiology. Our program is currently training 33 pre-doctoral students and 49 postdoctoral fellows, of whom 4 are supported by this grant. Trainees, who typically spend a minimum of at least 2 years in the program, are selected for the program based on evidence indicating outstanding potential for a future career in research and are admitted based on prior research experience, publications, and letters of recommendation. Special efforts are in place to enhance the recruitment and retention of underrepresented minorities. In the program trainees are offered a rich selection of seminars, journal clubs, and technique workshops and are thus exposed to new techniques, findings and ideas. Outstanding research facilities and an exceptionally strong research environment are available to our trainees. Moreover, core facilities and shared resources offer our trainees access to state-or-the-art equipment in analytical biochemistry, molecular biology, electrophysiology, and microscopy. Our training faculty have strong research programs in their laboratories that are supported by both public and private funding agencies and are highly committed to both pre-doctoral and postdoctoral training.

[unreadable] DESCRIPTION (provided by applicant): Activity-dependent processes that persistently modify the strength of synaptic transmission are thought to have a crucial role in the formation of new memories during learning. In the hippocampus, a region of the brain known to have an important role in memory formation, excitatory synapses are capable of undergoing both long-term potentiation (LTP), a long-lasting increase in synaptic strength, as well as long-term depression (LTD), a persistent decrease in the strength of synaptic transmission. Although induced by very different patterns of synaptic activity, the induction of both LTP and LTD is dependent on activation of NMDA-type glutamate receptors. Importantly, NMDA receptors and many of the intracellular signaling pathways involved in plasticity are organized into multi-protein complexes by a family of scaffolding or adaptor proteins known as membrane-associated guanylate kinases (MAGUKs). Although this suggests that MAGUKs are responsible for the formation of NMDA receptor signaling complexes that enable rapid and selective activation of downstream signaling pathways underlying LTP and LTD, little is known about the specific roles these proteins have in LTP and LTD. In this project we will investigate synaptic transmission and plasticity in mice with mutations in MAGUKs that associate with NMDA receptors (SAP102, PSD-93, and PSD-95) to examine the role of these proteins in LTP and LTD. In addition, we will investigate the role of MAGUKs and other signaling molecules in NMDA receptor-dependent activation of the extracellular signal-regulated kinase pathway, a signaling pathway known to have a key role in LTP and learning. These experiments will provide fundamental insights into the molecular mechanisms underlying activity-dependent forms of synaptic plasticity. Moreover, mutations in the gene for the MAGUK SAP102 have recently been identified as a cause of nonsyndromic X-linked mental retardation. Thus, our studies of synaptic transmission and plasticity in SAP102 mutants will not only provide important insights into the roles of MAGUKs in activity-dependent forms of synaptic plasticity but will also identify potential changes in synaptic function that contribute to learning impairments in this form of metal retardation. [unreadable] [unreadable] [unreadable]

At many excitatory synapses in the brain the strength of synaptic transmission is not fixed but instead can be regulated by patterns of synaptic stimulation. These long lasting changes in synaptic strength, known as long-term potentiation (LTP) and long-term depression (LTD), have a crucial role in the storage of new information during memory formation and are a prominent feature of synaptic transmission in brain regions that have an important role in learning and memory, such as the hippocampus. Although it is not yet clear how physiological patterns of synaptic activity might induce LTP and LTD in vivo, a number of studies indicate that the induction of LTP and LTD is critically dependent on the precise timing and order of single pre- and postsynaptic action potentials, a phenomenon known as spike timing-dependent plasticity or STDP.

STDP not only represents a physiologically realistic basis for understanding how LTP and LTD can be induced by normal patterns of neuronal activity but also provides a computationally powerful formulation for how these forms of synaptic plasticity might be involved in complex computational tasks such as coincidence detection and sequence learning. Importantly, STDP has been primarily studied in the immature brain and recent studies suggest that the properties of STDP are very different in the adult hippocampus. Thus, the functional role of STDP in the adult hippocampus is unclear. In this project a combination of electrophysiological, pharmacological, and modeling approaches will be used to study three fundamental aspects of STDP at excitatory synapses in the adult hippocampus. First, studies of cells in the highly interconnected neuronal network found in the hippocampal CA3 region will be used to determine whether STDP has a crucial role in allowing interconnected cells to form networks that avoid runaway excitation or epileptic-like activity. Second, studies in the hippocampal CA1 region, where cells are not highly interconnected but instead form simple feed-forward networks, will be used to investigate whether STDP exists in a latent or hidden form at synapses in feed-forward networks. Finally, experiments will be done to examine whether modulatory neurotransmitters known to have an important role in learning and memory can modulate the induction of STDP in the hippocampus. Together, the results of these experiments will provide key information regarding how STDP may be involved in hippocampal information processing and hippocampal-dependent forms of learning in the adult brain. Moreover, the project will provide a unique opportunity for the multidisciplinary training of both undergraduate and graduate students in a highly interactive project using a combination of cellular neurophysiological and computational approaches.

DESCRIPTION (provided by applicant): Astrocytes are found throughout the brain and play well documented physiological roles. Emerging roles for astrocytes include signaling to and from neurons and regulation of local blood flow. Certain astrocyte functions are correlated with or regulated by cytosolic calcium transients, which are a physiological signal. During the previous grant cycle, we developed and used a membrane tethered genetically encoded calcium indicator called Lck-GCaMP3 with the aim of measuring astrocyte calcium transients. Using Lck-GCaMP3 we made the serendipitous discovery of a novel calcium signal in astrocytes due to transmembrane fluxes mediated by TRPA1 ion channels. Moreover, our ongoing experiments show that TRPA1 mediated calcium fluxes give rise to frequent and highly localized near membrane calcium signals that contribute significantly to the resting calcium levels of astrocytes not only within a single astrocyte, but also in a network of astrocytes and neurons in cell cultures, as well as in acute brain slices. Our preliminary data also show that pharmacological block or genetic deletion of TRPA1 channels reduced inhibitory synapse efficacy onto interneurons and long term synaptic potentiation of Schaffer collateral synapses onto pyramidal neurons. We have three specific aims with which we seek to further extend these findings, test novel hypotheses and evaluate the function of astrocyte TRPA1 mediated calcium signals. In Aim 1 we will study near membrane calcium signals in astrocytes within hippocampal slices. In Aim 2 we will employ a variety of methods to systematically evaluate why blocking astrocyte TRPA1 channels or buffering astrocyte calcium levels below rest reduces inhibitory synapse efficacy onto interneurons, but not pyramidal neurons in the stratum radiatum (s.r.) of the hippocampus. In Aim 3 we will study how long-term potentiation (LTP) is reduced by blocking TRPA1 channels. By completing these experiments we will provide new information on the function of a novel astrocyte calcium signal. This information will contribute significantly to our understanding of astrocytes in neuronal networks and allow us and others to test novel hypotheses on the roles of near membrane Ca2+ signals in astrocyte-neuron signaling, and lay the foundations for determining if TRPA1 channels are valid drug targets in brain disorders that involve astrocytes.

Project Summary/Abstract Homeostatic forms of plasticity are thought to have a crucial role in stabilizing neuronal activity in response to changes in excitatory synaptic drive that occur during development, following the induction of Hebbian forms of synaptic plasticity during learning, and in pathological conditions such as sensory impairment, stroke, and other types of neuronal damage. Although much has been learned from studies of homeostatic plasticity in dissociated neuronal cultures, we know relatively little about the mechanisms and properties of homeostatic plasticity at mature synapses in the adult brain. Moreover, computational studies indicate that known forms of homeostatic synaptic plasticity lack important properties needed to both efficiently stabilize neural networks over short time scales and preserve information encoded by differences in the strength of individual synapses. In recent experiments we discovered that decreases in Ca2+ influx via NMDA type glutamate receptors and/or voltage- gated Ca2+ channels induces a novel form of homeostatic potentiation at excitatory synapses onto CA1 pyramidal cells in adult mouse hippocampus. This form of homeostatic potentiation is exceptionally fast and is induced within minutes following a reduction in intracellular Ca2+ levels. Moreover, it may be mediated by the insertion of AMPA type glutamate receptors into silent synapses (i.e. synapses that only contain NMDARs). Thus, it may provide a mechanism that can homeostatically regulate overall excitatory synaptic strength without interfering with changes in synaptic weights induced by Hebbian forms of plasticity, such as long-term depression. However, many fundamental questions regarding the properties and mechanisms involved in this novel form of synaptic plasticity have not yet been investigated. Thus, in this project we propose to use a combination of electrophysiological, pharmacological and biochemical approaches to further characterize this unique form of homeostatic plasticity and begin to identify the underlying cellular and molecular mechanisms.