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Project 1  -  Brain function & Synaptic transmission

Background: Neurotransmitter release is a key process for intercellular communication in the brain. During the last decade, major molecular components important in synaptic release of neurotransmitter have been identified using model animals such as C. elegans, Drosophila and mice. Physiologically, regulated neurotransmitter release is important for short-term and long-term synaptic plasticity mediating learning and memory. A key challenge in modern neurobiology is to understand the essential molecular framework and its dynamics underlying regulated synaptic release of neurotransmitter in the central nervous system.

Project Description: The long-term goal of this project is to understand how neurotransmitter release is regulated at the molecular and cellular levels. We are particularly interested in elucidating genes/signaling pathways important in regulation of inhibitory GABA release in the central nervous system (CNS). g-Aminobutyric acid (GABA) is the main inhibitory transmitter in the CNS of both invertebrates and vertebrates. Given that inhibitory transmission has an equally important function in regulating the neuronal excitability, it is not surprising to find that GABA plays a critical role for higher brain functions such as learning/memory and coordinated behaviors. In this project, we investigate molecular and cellular mechanisms underlying (1) dopamine-mediated neuromodulation of inhibitory transmission, (2) Ca²⁺-dependent release of GABA, and (3) cAMP-dependent plasticity at the GABAergic synapse. These studies are conducted using Drosophila neuronal cultures in which spontaneously active, excitatory cholinergic and inhibitory GABAergic synapses form between primary neurons (Lee and O’Dowd, 1999; Lee et al, 2003). The project is organized into the following three specific aims:

Specific Aim 1: To characterize molecular and cellular mechanisms of dopamine modulation regulating GABA release at Drosophila central synapses.

      Dopamine neuromodulation and behavioral plasticity appear to involve similar molecular pathways in flies, mice, and human. This includes modulation through G protein coupled dopamine receptors. Indeed, genetic and pharmacological studies over last 20 years revealed that dopaminergic system in Drosophila play multiple roles in development, motor function and cognition. However, mechanisms underlying dopamine modulation in the Drosophila CNS remain to be explored. Our preliminary studies demonstrate that dopamine receptors are functionally expressed in Drosophila neuronal cultures, suggesting Drosophila neuronal culture can be an excellent model to study modulation of GABA release by dopamine receptors. In this aim, we are characterizing properties of dopamine D1 and/or D2 receptors in GABAergic neurons using specific pharmacological probes in conjunction with electrophysiology (e.g. whole-cell patch clamping). The subsequent studies will focus on G-protein signaling mechanisms which underlie dopamine modulation. The information generated by this specific aim will be important in understanding how dopamine regulates GABA release and what receptors are mediating dopamine signaling in Drosophila.

Specific Aim 2: To investigate functional and molecular properties underlying Ca²⁺-dependent inhibition of GABA release.

      Calcium triggers rapid neurotransmitter release (less than a millisecond), which is mediated by binding of fluxed calcium ions to a low-affinity binding protein such as synaptotagmin. In this view, Ca²⁺ plays a role as a positive regulator in neurotransmitter release. At the neuromuscular junction and central cholinergic synapses in Drosophila influx of Ca²⁺ into the presynapstic terminal enhances neurotransmitter release (Broadie and Bate, 1993; Lee and O’Dowd, 1999) as also observed in other chemical synapses. In contrast to ACh release, our recent finding suggested that GABA release is inversely regulated by extracellular Ca²⁺ concentrations. This observation is contrary to the central dogma of neurotransmitter release, which defines Ca²⁺ as an enhancer. Given that inhibitory transmission has an equally important function in regulating strength of excitatory synapses, suppression of GABA release may have a significant role in mediating certain forms of synaptic plasticity. However, inhibition of GABA release by Ca²⁺ has not been studies in Drosophila CNS. In this specific aim, we investigate molecular and cellular mechanisms by which Ca²⁺ inhibits release.

Specific Aim 3: To characterize mechanisms regulating cAMP-dependent plasticity at central GABAergic synapses.

      The cAMP-signaling pathway plays a central role in regulating plasticity at excitatory glutamatergic synapses underlying learning and memory in both mammals and Aplysia (Milner et al, 1998). Behavioral, biochemical, and molecular genetic studies indicate that this signaling cascade is also important in learning and memory. Considering that enhancement of the efficacy in excitatory transmission is mediating certain form of synaptic plasticity such as hippocampal long-term potentiation (LTP) and facilitation (LTF) in Aplysia (Milner et al, 1998), suppression of inhibitory transmission by a common stimulus such as cAMP, which enhances excitatory synaptic signaling at the same time, is an attractive and efficient way to increase the overall strength of synaptic transmission. However, it is not known how inhibitory GABAergic communication is altered by cAMP. This study aims to understand mechanisms underlying regulation of GABAergic transmission to mediate leaning and memory in Drosophila by using genetic and biochemical manipulations in addition to electrophysiological and immunostaining approaches.
 

Drosophila neuronal cultures: The ability to combine behavioral and biochemical approaches with sophisticated molecular genetics has made Drosophila an important model organism for identification of genes involved in synaptic vesicle cycling. Furthermore, the electrophysiological studies feasible at the larval and embryonic neuromuscular junction (NMJ) have made it possible to access the phenotypic consequences of mutating specific genes, providing important insights into the molecular mechanisms underlying Ca²⁺ regulated synaptic excytosis as well as endocytosis (Pennetta et al, 1999; Richmond and Broadie, 2002).
      Since synaptic exocytosis in central transmission is a pivotal process, modifications to exocytosis are likely to underlie certain forms of synaptic plasticity important in learning and memory formation. However, the molecular and functional properties of neurotransmitter release at central synapses in Drosophila have not been well described. This is largely due to difficulties in recording from single neurons in Drosophila CNS in vivo. In recent years, we have developed Drosophila neuronal cultures in which spontaneously active, excitatory cholinergic and inhibitory GABAergic synapses form (Lee and O’Dowd, 1999; Lee et al, 2003). Molecular, functional and pharmacological properties of synapses formed in cultured neurons so far examined (Lee & O’Dowd, 1999; Lee et al, 2003) are consistent with those observed in insect CNS (Restifo and White, 1990), strongly suggesting that they may serve as an in vitro model for central synapses in vivo. In conjunction with well developed genetics, this culture system will be instrumental in the study of mechanisms regulating Ca²⁺ dependent release of neurotransmitter at excitatory and inhibitory central synapses.