Stefano Vicini - US grants

This work will be complementary to patch-clamp recordings of amino acid- activated single channel currents in outside-out patches excised from neurons in brain slices in comparison to amino acid-activated single channel currents in outside-out patches excised from cell in culture transiently transfected with specific subunits of excitatory amino acid receptors, thereby expressing only special recombinant receptor. I also propose to study long-term changes of excitatory synapses during normal and experimentally modified development in neurons of rat primary visual cortex. Finally, in light of experimental evidence for a role of neurotrophic factors underlying the plasticity of synaptic connections during development of the visual system, I will also study the effects of endogenously provided neurotrophins on AA-mediated synapses during normal development and during visual deprivation. Data obtained from these specific aims will permit the relative contributions of variations in the molecular forms of excitatory amino acid receptors to be distinguished as determinants of synaptic functions that underlie behavior and that are relevant in various CNS disorders.

Gamma-aminobutyric/A (GABA/A) receptors form part of a major neurotransmitter system in the brain that contributes to the regulation of anxiety, sleep and the pathogenesis of many neurological disorders. Recent developments in molecular cloning of GABA/A receptors have revealed an impressive heterogeneity of molecular forms of constituent subunits. There is growing evidence that an aspect of this large heterogeneity could be a functional heterogeneity of inhibitory synapses. Furthermore, the selectivity of drug action for distinct molecular forms of GABA/A receptors raise hopes for better treatment of disorders related to inhibitory GABAergic pathways. We have evidence that in cerebellum two functionally and pharmacologically distinct populations of inhibitory synapses can be found in relation to specific cell types. We propose to test the hypotheses that the GABA/A receptor subunits composing postsynaptic receptor-channel complexes play a major role at these synapses as determinants of synaptic strength. GABA-mediated synapses in visually identified neurons in the rat cerebellum, will be analyzed using whole-cell voltage-clamp recordings of synaptic currents. We will attempt to correlate functional differences of cerebellar synapses with the presence of the mRNA for specific GABA/A receptor subunits in these neurons determined with single-cell polymerase chain reaction and with the study of the expression of these subunits revealed by immunocytochemical techniques. This work will be complementary to patch-clamp recordings of GABA-activated channel currents in outside- out patches excised from neurons in brain slices and from cells transiently transfected with specific subunits of GABA/A receptors. We will also test the hypothesis that the presence of structurally distinct GABA/A receptor subtypes in the cerebellum, are the basis of specific pharmacological properties of agonists, antagonists as well as partial agonists of the benzodiazepine receptor. Lastly, we propose to provide the correlation between long-term changes in rat cerebellar inhibitory synapses and pharmacological treatments with clinically relevant compounds such as benzodiazepines, with acute and chronic ethanol treatments and with these experimental conditions combined. Data obtained from these specific aims will permit us to distinguish the contribution of the various molecular forms of GABA/A receptors as determinants of synaptic functions that underlie behavior and that are relevant in various CNS disorders.

DESCRIPTION: N-Methyl-D-Aspartic acid (NMDA) receptors contribute to synapse formation, learning and memory and the pathogenesis of neurological disorders. Recent developments in molecular cloning of NMDA receptors have revealed a substantial heterogeneity of molecular forms of these receptors. It is proposed that the heterogeneity of molecular forms of NMDA receptors at excitatory synapses and their posttranslational modifications, underlie the effectiveness of the NMDA component of EM mediated synaptic transmission and in turn synaptic plasticity. Specific physiological manipulations that result in differential subunit expression and posttranslational modifications leading to alteration of NMDA receptor-mediated synaptic function will be studied. The functional diversity of synaptic and extrasynaptic NMDA in distinct neuronal populations in brain slices and primary culture will be investigated with electrophysiological, anatomical and pharmacological techniques. The result of this study will reveal how the relative contribution of the NMDA subunit NR1 and NR2B determines the kinetics of NMDA-mediated synaptic currents in developing rat neocortical and cerebellar neurons. The main hypothesis is that the relative proportion of NR1/NR2A heteromers are the crucial component that controls the efficacy of synaptic NMDA receptors. Recombinant receptors will be used to compare and contrast functional differences related to those observed in neurons and to understand mechanisms underlying the kinetic differences observed between different molecular forms of NMDA receptors. Preliminary results with recombinant receptors suggest that subunit composition is not the only kinetic determinant of synaptic NMDA receptors. Therefore, the role for posttranslational modifications in producing distinct kinetics of synaptic NMDA currents in cortical neurons will be determined. Lastly, experimental strategies will be used to regulate expression of distinct NMDA receptor subtypes in neocortical and cerebellar neurons resulting in the understanding of how neuronal activity, protein kinases and neurotrophins control the expression of NMDA receptor subtypes. Data obtained from these specific aims will permit the understanding of the molecular determinant responsible for synaptic plasticity in CNS.

DESCRIPTION (provided by applicant): GABAa receptors play a critical role in anxiety, sleep and the pathogenesis of many neurological disorders. Molecular cloning of gamma-aminobutyric A (GABA)a receptors has revealed a considerable heterogeneity of constituent subunits. Different GABAa receptor isoforms in cells transfected with specific combinations of subunits have unique rates of channel transition between distinct conformational states that determines a "molecular kinetic fingerprint" of GABA responses. Using mice with specific deletion of subunit genes we will prove that this molecular fingerprint set the time course of inhibitory synaptic currents (IPSCs) a crucial factor in determining the strength of the inhibitory synapse and a target of many commonly prescribed drugs. We focus on cerebellar granular and stellate neurons where a progressive developmental decrease in IPSCs duration parallels changes in GABAa receptor subunit expression. Supported by our preliminary finding that the deletion of the alpha1 subunit of GABAa receptor prevents developmental changes in IPSCs' kinetics, our hypothesis is that distinct GABAa receptor subtypes, anatomically restricted and developmentally regulated lead to the specific functional properties of inhibitory activity that underlie behavior and neurological disorders. The outcome of our study will allow linking the heterogeneity of molecular structures to the functional heterogeneity of inhibitory synapses. Whole-cell recordings of synaptic and extrasynaptic GABA currents in neurons of the mouse cerebellum in slices and primary neuronal cultures will be complementary to recordings of GABA-activated channel currents in outside-out patches excised from these neurons. The biophysical study of native GABA channel will be integrated by studying those recorded from cells transiently transfected with specific subunits of GABAa receptors and from transgenic mice missing specific subunits. The goal is to identify native subunit combinations and ultimately link these different receptor combinations to their particular role in GABA-mediated inhibition in cerebellar neurons.

DESCRIPTION (provided by applicant): The striatum controls the execution of learned motor behaviors as well as the suppression of unwanted movements. The main neuronal type in the striatum, the medium spiny neuron (MSN), receives converging glutamatergic innervation from the cortex and thalamus and dopaminergic innervation from the substantia nigra. In addition a small population of GABAergic interneurons controls the excitability of a vast population of MSNs to maintain the proper balance of information outflow for smooth control of motor output. This control is exerted via both phasic and tonic GABAa receptor activation. This project aims to compare GABA receptor-mediated currents and the receptor subtypes amongst MSNs originating the striatopallidal and the striatonigral pathways. We will utilize corticostriatal slices made from strains of mice which selectively express green or red fluorescent protein in D1 or D2 dopamine receptor expressing cells to identify unique properties of GABAa receptors in these cells. Single cell electrophysiology in corticostriatal slices will be used to test the hypothesis that that a portion of striatal GABAa receptors have unique properties and are unequally distributed between the two major subtypes of striatal projection neurons. Based on preliminary data showing that striatopallidal MSN express a tonic conductance mediated by a subtype of GABAa channel, the first specific aim will strive to understand the role of alpha and beta subunits of GABAa receptors in producing this tonic activation in MSNs. This will be done with subunit selective drugs, mice lacking specific subunits and recombinant GABA channels relevant to striatal MSNs. The second aims will test the hypothesis that protein kinase phosphorylation regulates the functional properties of tonically active GABA channels. The last aim will investigate if the presence of the specific and distinct dopamine receptors in MSN subtypes plays a role in setting the properties and regulating the expression of GABA receptor subtypes that can be target for pharmacological therapy of disorders associated with striatal dysfunction.

? DESCRIPTION (provided by applicant): This proposal focus on the cellular properties of neurons of Barrington's nucleus (BRN) and their responses to afferent or pharmacological stimulation. BRN neurons are positioned to coordinate micturition with arousal and voiding behaviors as they innervate the preganglionic neurons that regulate detrusor contraction as well as the locus coeruleus (LC), the major brain norepinephrine nucleus. Dysfunctions in this circuit may also underlie bladder and voiding disorders, particularly Underactive Bladder (UAB). We will take advantage of technical advances combining genetics, optical stimulation and imaging to elucidate how this nucleus functions and how it can be manipulated to treat disorders such as UAB. As the majority of BRN neurons express the stress related neuropeptide, corticotropin-releasing factor (CRF), they can be easily identified and studied in brain slices from mice with CRF-Cre dependent expression of fluorescence reporter proteins. The close proximity of the LC and BRN in these slices we will allow us to directly quantify the relationship between BRN and LC neurons and to better understand this central branch of the micturition reflex. In aim one we will characterize with patch clamp recordings the cellular properties of neurons in the BRN-LC circuit and how they change with aging. This will be paired with immunocytochemical (IHC) determination of neurotransmitter phenotype. We hypothesize that the majority of BRN neurons co-localize CRF and glutamate. Then we will use CRF- Channelrhodopsin 2 mice to control CRF expressing BRN neurons with different frequencies of optical stimulation and quantify their synaptic action in the LC. In aim two using CRF-GCaMP6f mice that express a Ca2+ indicator in CRF neurons we will be able to investigate the functional BRN circuitry as it modifies with age and thus identify cellular changes in the pontine micturition center that may be causal to age-related UAB. Our exploratory proposal will provide us with the conceptual framework and essential preliminary data to develop a mechanistic knowledge of the functional and pharmacological regulation of the BRN circuitry relevant for the diagnosis, evaluation, and treatment of UAB. The goal of the application is to set the stage for a combined in vitro and in vivo approach to the understanding of the role of the pontine micturition center in UAB.

The goal of this proposal is to determine the role of two specific GABAergic interneurons, somatostatin (Sst) and neuropeptide Y (Npy) expressing neurons, in the regulation of Brainstem neural circuitry controlling gastric function. Our studies, focusing on the major brain nuclei of this circuit that form the dorsal vagal complex (DVC) in the hindbrain, namely the medial nucleus tractus solitarius (mNTS) and dorsal motor nucleus of the vagus (DMV), indicate that local GABA signaling in these nuclei is critical for modulation of vagal output to the stomach. However, the identity of the GABA neurons to which this inhibitory signaling can be attributed is lacking. Recent advances in transgenic mouse models, virus injection and optogenetic techniques have made it possible to isolate and selectively stimulate specific cell types. Using these technologies, we have begun to acquire nascent data on the identity and role of the neurons involved in GABA signaling in the DVC. Furthermore, these techniques have been instrumental in obtaining information on the likely mechanisms by which a regulator of energy homeostasis, namely an endogenous agonist of the melanocortin receptor system (McR), produces its differential effects on gastric function (e.g., tone and motility) in the NTS (inhibitory) and in the DMV (excitatory). The focus of the present proposal is on the genetically and virally defined Sst and Npy neurons in the DVC. We will investigate the functional role of these neurons in DVC circuitry by determining their electrophysiological and morphological characteristics in relation to other neurons that comprise the neural circuitry controlling gastric function via trans-synaptic labeling. Our previously published data and preliminary findings show that optogenetic stimulation of Sst/Npy neurons in the NTS or DMV strongly influence the activity of neurons along the Brain-Gut axis. Based on these results, we will pursue our overall hypothesis that Sst and Npy neurons in the DVC serve as key complementary components that regulate the vago-vagal gastric circuitry. Finally, we will assess the role of Sst and Npy neurons in an in vivo model of gastric function. In this model, we will test the hypothesis that stimulation of Sst and Npy GABA neurons in the mNTS and DMV will selectively affect gastric tone and motility. The knowledge gained from our proposed studies will contribute greatly to understanding vago-vagal circuitry controlling gastric function, with the potential of providing an important template for studying homeostasis in other autonomic reflex systems.