Jacques I. Wadiche - US grants

DESCRIPTION (provided by applicant): Brain function relies on information transfer between neurons at structurally and functionally defined sites called synapses. Considerable effort is directed at understanding the detailed mechanisms underlying synaptic transmission because it is increasingly clear that many diseases of the brain, including neurodevelopmental disorders and the earliest stages of neurodegenerative diseases, are associated with dysregulation of synaptic function. Synaptic transmission is composed of three components including the presynaptic release of neurotransmitter containing vesicles, diffusion of neurotransmitter through the space between neurons, and the activation of receptors that generate a response in the postsynaptic neuron. These events take place on a millisecond time scale, and the timing of each component is tightly controlled to ensure precise and efficient information transfer. Yet transmission is also highly dynamic, showing many types of plasticity in response to natural stimulus patterns. The overall goal of this project is to determine how activity controls the synchrony of neurotransmitter release from the presynaptic terminal, and how this activity-dependent plasticity contributes to the timing of information transfer through the synapse. We will first establish the conditions and mechanisms that control the synchronicity of transmitter release. We will subsequently test physiological consequences of activity dependent asynchronous transmitter release in terms of neural output and postsynaptic Ca2+ signaling. These studies will be conducted in brain slices from mice using voltage and current clamp recordings as well as calcium imaging and pharmacological manipulations. We will use a cerebellar synapse where it is well established that precise timing of synaptic transmission is critical for behaviors such as motor control and motor learning. The results from these studies will provide insight into an important presynaptic mechanism for regulating synaptic transmission that likely plays a role in neural timing throughout the CNS.

AMPA receptors (AMPARs) mediate the majority of excitatory glutamatergic synaptic transmission in the central nervous system. Most AMPARs, once bound to glutamate, allow Na+ and K+ flux across the cell membrane, causing neurons to depolarize. However, AMPARs that lack the GluR2 subunit are also permeable to Ca2+. These Ca2-permeable (CP) AMPARs are highly expressed during development when they are essential for activity-dependent plasticity, and this function persists at some synapses throughout adulthood. A biophysical characteristic known as rectification is commonly used to differentiate CP-AMPARs from Ca2+-impermeable (CI) AMPARs. Whereas CP-AMPARs exhibit strong inward rectification, CI-AMPA receptors display linear current-voltage relationships. Inward rectification of CP-AMPARs results from intracellular polyamines that act as open channel blockers to prevent outward current flux. Thus, inward rectification and sensitivity to antagonists that bind at the polyamine site provide biophysical signatures of AMPAR subunit composition and hence Ca2+ permeability, and these characteristics have been widely used to establish rules of AMPAR subunit plasticity. Molecular layer interneurons of the cerebellum provide a well-established model system for understanding AMPAR localization and trafficking because repetitive synaptic stimulation or a single experience of fear triggers a form of plasticity called subunit-switching wherein CP-AMPARs at synapses are replaced by CI-AMPARs from a pool of extrasynaptic AMPARs. Although rectification index and sensitivity to polyamine site toxins are widely used to distinguish between GluR2-containing and -lacking AMPARs, there are many examples from the literature that show these biophysical properties do not exclusively reflect subunit composition. A separate literature has converged on gating models of AMPARs that include multiple conductance states, but the functional implications are unclear. Now, our preliminary data show that CP-AMPAR rectification and pharmacology are sensitive to factors that regulate AMPAR conductance states, potentially complicating the interpretation of results using these biophysical properties as sole proxies of subunit composition. We propose to understand how the multiple sub-conductance states of AMPARs contribute to the hallmark biophysical properties CP-AMPARs. We will use high resolution Ca2+ imaging, heterologous expression systems and genetic manipulation to understand regulation of CP-AMPAR biophysical properties and use that understanding to critically evaluate CP-AMPAR localization and plasticity in cerebellar molecular layer interneurons. AMPAR subunit composition has important functional consequences, ranging from regulating the ability of postsynaptic cells to precisely follow high-frequency synaptic activity and mediating Ca2+ influx that can trigger plasticity or pathology. Successful completion of the proposed studies will reveal novel properties of AMPARs that are essential for understanding their function within synapses and intact circuits in the normal and diseased brain.

Abstract Identification of the mechanism(s) responsible for drug reinforcement is a key step in understanding the mechanism of reinforcement learning, but has so far proven elusive. The dopamine neurons of the ventral tegmental area and substantia nigra pars compacta, located within the ventral mesencephalon, are a central locus for drug reinforcement. Even a single exposure to cocaine is sufficient to alter synaptic transmission to dopamine neurons, with attention focused on postsynaptic mechanisms of plasticity mediated by AMPA receptors (AMPARs). Most AMPARs are impermeable to Ca2+ (CI-AMPAR) whereas receptors that lack the GluR2 subunit are permeable to Ca2+ (CP-AMPAR). A biophysical characteristic known as rectification is commonly used to differentiate CP-AMPARs from the more common CI-AMPARs . It is commonly accepted that cocaine exposure alters rectification of AMPAR synaptic currents on dopamine neurons without affecting measures of release probability, pointing to postsynaptic mechanisms of synaptic plasticity. However, our new data challenges the assumptions that rectification is sufficient to infer AMPAR subunit composition and that release probability is sufficient to assess presynaptic efficacy. Rather, our data shows that changes in the readily-releasable pool of vesicles can robustly alter presynaptic efficacy without a change in the release probability and that presynaptic mechanisms can affect rectification properties of AMPAR synaptic currents. Based on our data, we hypothesize that presynaptic mechanisms contribute to synaptic changes in dopamine neurons following cocaine exposure. We will first test AMPAR properties in dopamine neurons from naïve and cocaine-treated mice under conditions that isolate postsynaptic mechanisms. We will then follow up to test whether presynaptic changes contribute to synaptic plasticity induced by cocaine exposure. Presynaptic efficacy and AMPAR subunit composition have important functional consequences ranging from regulating the ability of postsynaptic cells to precisely follow high-frequency synaptic activity and mediating Ca2+ influx that can trigger plasticity or pathology like addiction. Successful completion of the proposed studies has potential to reveal novel mechanisms underlying synaptic plasticity at synapses onto dopamine neurons following exposure to drugs of abuse.