Joseph F. Margiotta, PhD - US grants

DESCRIPTION: (adapted from Applicant's Abstract) Studies of the chick ciliary ganglion using electrophysiological, molecular, and microscopic techniques are proposed to test two hypotheses. One hypothesis is that extrasynaptic AChRs influence the extent of neuronal survival and maturation during development. A second hypothesis is that presynaptic AChRs, implicated in ACh release, are influenced by neuropeptide-generated signals thereby providing a means of modulating release from nerve terminals. Alpha7 AChRs have been found in ciliary ganglion neurons in addition to synaptic a3 AChRs on the same neurons. Dr. Margiotta proposes to investigate the role of a7 subunits in development by measuring a7 transcripts during the course of development, testing the effects of AChR blockade on neuron survival, maturation, and synapse formation. Presynaptic AChRs will be sought on terminals of cultured and freshly isolated neurons and tested for their effects on ACh release. To examine if synaptic and presynaptic AChRs are targeted by different intracellular signals, the subtype-specific effects of activating separate signaling pathways by neuropeptides will then be elucidated.

IBN: 95154560 PI: Margiotta Nerve cells, or neurons, communicate with each other primarily at functional contacts called chemical synapses. Each synapse is highly specialized for the release of neurotransmitter molecules from the presynaptic cell terminal, and for molecular reception of the particular neurotransmitter molecules by the membrane on the postsynaptic cell. The receptor structures are made of membrane proteins, and these receptors often are clustered right near the sites of presynaptic release. It remains unknown how those receptor molecules become clustered in the postsynaptic membranes. This project utilizes molecular and biochemical approaches to isolate and identify the genetic sequences, expression, and functional role of a potentially important protein that is associated with clustering of receptors for a common neurotransmitter, acetylcholine. Results will be important for neuroscience in general because synapses are crucial for neuronal signaling. Identification of the molecules important for receptor clustering will be central to achieving a better understanding of how synapses form and how they function.

[unreadable] DESCRIPTION (provided by applicant): Tobacco consumption, driven by addiction to nicotine, is the leading cause of preventable death in the United States. In the period from 1995-1999, the CDC reported 440,00 deaths/year from illnesses attributable to smoking (cancer, respiratory, and cardiovascular diseases) and $157 billion/year in related economic losses. Neuronal nicotinic acetylcholine receptors (nAChRs) initiate and reinforce the process of nicotine addiction. These receptor molecules aggregate at pre- and postsynaptic sites in the brain and autonomic nervous system, allowing them to both modulate and directly mediate synaptic transmission once activated by acetylcholine (ACh) released from cholinergic nerve terminals. Recent studies demonstrate that chronic exposure to levels of nicotine seen in smokers' blood, causes sustained changes in both pre- and postsynaptic nAChRs. It is widely presumed that such changes alter synaptic circuits underlying addictive behaviors associated with nicotine, and possibly other commonly abused drugs. Unfortunately, these circuits are embedded in brain regions where experimental control of pre- and postsynaptic elements is difficult or impossible to achieve, preventing discovery of precise mechanisms relevant to nicotine-induced changes in synaptic function. To address this problem, our laboratory proposes to combine state of the art single-cell gene transfection and high-resolution synaptic stimulation/recording methods in a model culture system where nAChRs and nicotinic synapses are abundant and accessible. One goal is to manipulate gene expression in pre- and postsynaptic neurons that can subsequently be examined in detailed electrophysiological experiments (Aim 1). Realizing this goal will provide a cutting-edge advance relevant to many studies of synapse formation, function and regulation. A second goal is to test the utility of our approach by probing for alterations in synaptic function expected to accompany sustained changes in nicotinic receptor activation (Aim 2). It is anticipated that these latter experiments will provide new answers concerning how alterations of nicotinic synaptic components modify circuit function to initiate and reinforce addiction to nicotine and other abused drugs. [unreadable] [unreadable] [unreadable]

Synapses are specialized cell junctions that link neurons into functional networks, thereby providing the basis of all integrative neural signaling. Synaptic transmission involves release of neurotransmitter from presynaptic neuron terminals and capture by specific postsynaptic receptors that transit ions to alter the target cell's excitability. Nicotinic acetylcholine (ACh) receptors (nAChRs) bind ACh to mediate excitatory synaptic transmission in autonomic ganglia controlling homeostasis and modulating the output of brain circuits that control coordinated movement and behaviors underlying pleasure and reward. Despite their broad relevance, the molecular mechanisms regulating function and development of nicotinic synapses remain poorly understood. This project will explore changes triggered by a neuropeptide (PACAP) released from the pituitary at nicotinic synapses on autonomic ciliary ganglion (CG) neurons. Recent findings indicate that PACAP rapidly regulates synapses on CG neurons in culture. Electrophysiological, imaging, and biochemical approaches will be used to identify altered synaptic properties and determine the protein-protein interactions that mediate this regulation. Since PACAP levels are developmentally regulated, and exogenous PACAP enhances neuronal survival in an activity dependent fashion, the possibility that PACAP persistently influences synapse formation and function will also be explored. It is expected that PACAP-triggered signaling will be critical both for building nicotinic synapses and controlling their function. Lastly, outreach programs administered through the University of Toledo will be used to train visiting undergraduates, high school students, and high school teachers. Thus, this project will further elucidate molecular mechanisms relevant to homeostatic regulation, behavior, and development while simultaneously fostering the scientific literacy and training of underrepresented groups.