Grant: Discovery Grants Program - Individual

Chemical transmission is one of the most fundamental processes of the nervous system, which depends on the release, diffusion and clearance of neuroactive substances detected by various receptors. Particularly, neuron-to-neuron communication mediated by glutamate, the primary excitatory neurotransmitter in the CNS, is critical for information flow within neuronal networks. In addition, recent research has established that astrocytes can play multiple roles in glutamatergic transmission. Astrocytes sense, uptake and release glutamate and hence regulate the extracellular glutamate levels, which can underlie basal or tonic excitability in some neurons. The distinct time course of neuronal and astrocytic glutamatergic transmission suggests different mechanisms, regulatory processes and functional roles. However, these mechanisms remain obscure. Neuropeptide release from peptidergic neurons typically require prolonged firing activity, thus sustained elevation in excitability supported by astrocytes may have significant functional consequences. Melanin-concentrating hormone (MCH) neurons are peptidergic neurons in the hypothalamus critical for the control of energy balance, sleep-wake cycle and motivated behaviors. Our preliminary study indicates that MCH neurons represent a unique model to study neuronal and non-neuronal chemical transmission as they display several glutamatergic currents with distinct time courses: fast EPSC, slow EPSC and tonic inward current. Fast and slow EPSCs are triggered by synaptic stimulation while tonic currents are induced by glutamate transport inhibitors and are due to ambient glutamate that appear to be astrocytic in origin (gliotransmission). Furthermore, these currents are modulated/mediated by different glutamate transporters and receptors. Based on these results, we will test the hypothesis that depending on the source (presynaptic neuron or astrocyte), extracellular glutamate is regulated by distinct glutamate transporters and detected by different pools of receptors on MCH neurons. SHORT-TERM AIMS 1)Determine the mechanism of fast and slow EPSCs in MCH neurons 2)Identify the source of ambient glutamate and its mechanism of release 3)Determine the role of EAAC1 (neuronal glutamate transporter) and NMDA receptors in ambient glutamate action EPSCs and tonic currents will be recorded in mice brain slices using whole cell patch clamp, while extracellular glutamate levels will be assessed by genetically encoded glutamate sensors iGluSnFR expressed on MCH neurons. Subcellular localization of receptors and transporters will be investigated by electrophysiology and immunofluorescence microscopy combined with super-resolution radial fluctuation analysis. Proposed studies will be significant steps towards my long-term goal to establish a better understanding of neuronal and non-neuronal chemical transmission in the central nervous system.