James E. Vaughn, Ph.D. - US grants

The proposed research focuses on the role that neuronal migration may have in the development of neuronal diversity. Specifically, we propose to explore the hypothesis that the phenotypic differences between two closely related subsets of spinal cord cells, somatic motor neurons (SMNs) and autonomic motor neurons (AMNs) arise as a result of signals these cells encounter along their respective migration pathways. Initially, we plan to test hypotheses concerned with mechanisms of AMN migration. By necessity, these hypotheses will be examined in cultured spinal slice preparations that support normal AMN migration. We will seek to determine whether trophic and cell surface interactions play roles in AMN migration. Questions that arise in this regard include: Is AMN migration dependent upon axonal connections with peripheral targets? Do molecules on the surfaces of early developing circumferential axons guide the nonradial migration of AMNs? Do extracellular matrix molecules play a role in AMN movements? Furthermore, we propose to conduct experiments that explore the roles of receptors and intracellular ion levels in AMN movements. For example, questions important to this part of our proposed studies are: Do glutamate receptors gate intracellular changes important to AMN migration? Is this migration dependent on Ca2+ levels? Can intracellular levels of nitric oxide modulate AMN migration? Second in addition to the studies of migratory mechanisms, we will examine the subsequences that AMN migration has for the molecular differentiation of these cells. For instance: Is the characteristic expression of NADPH diaphorase in AMNs triggered by their dorsal migration? Is the motor neuronal, subset-specific expression of neuropeptides related to the migrational differences between SMNs and AMNs? Questions such as these muse be answered because they are important to understanding both normal brain function and degenerative neurological disorders, such as amyotrophic lateral sclerosis (ALS), where differential vulnerability of neuronal populations is a hallmark of the disease process.

Objectives for the next year of support are: Project 1. We intend to complete immunocytochemical studies of cholinergic neurons and synapses in rat neostriatum, and finish quantitative analyses of the temporal decrease of GABAergic terminals that is manifest in a monkey model of focal epilepsy. Moreover, we will initiate studies where both cholinergic and GABAergic markers are simultaneously localized by immunocytochemistry within the cerebral cortex, neostriatum, thalamus, and basal forebrain. Project 2. Our plans include studies to: a) purify rat brain ChAT in sufficient quantities for amino acid sequence determination; b) generate new monoclonal antibodies to the enzyme surface and/or amino acid sequence. We intend to use the antibodies and amino acid sequence information for molecular cloning of the rat ChAT gene, using either the synthetic oligonucleotide approach or immunological screening of rat cDNA expression vector libraries. Project 3. An inhibitory mechanism of neurotransmission will be studied for synapses of the thick accessory neuron that terminate on crayfish stretch receptor neurons. Morphological evidence suggests that these synapses may exert a powerful influence on stretch receptor neurons, but this has never been analyzed physiologically. Inhibitory function will be studied both by stimulating the thick accessory neuron and by iontophoretic application of GABA. In addition, the relationships between the formation of synaptic vesicles and availability of transmitter will be investigated in the temperature-sensitive ChAT mutant of Drosophila, where the amount of acetylcholine in terminals can be regulated experimentally. Project 4. A search will be made among the several substances that are potent competitive inhibitors of GABA transport in the P3 fraction to obtain a substance which could serve as a covalently-linked ligand for the transporter. Upon labeling such a substance, we should be able to develop a method for the isolation of the transporter in a purified form, and to begin to study its molecular properties. We also plan to obtain monoclonal antibodies to the P3 fraction, select antibodies that inhibit the GABA transporter specifically, and employ these antibodies in affinity columns for purification of the transporter.

Phenotypic diversity of its constituent cells is a hallmark of the nervous system. This characteristic is the fabric underlying the complexities of normal nervous system function as well as a variety of neurological disorders. The three projects comprising our proposed program share the goal of understanding how this remarkable degree of diversity is generated during neurodevelopment. The main thrust of this revised renewal program project grant application is an integrated, multidisciplinary assault on developmental gene neurobiological problems. We will conduct studies of neuronal determination, regulation of developmental gene expression, and epigenetic influences on neuronal differentiation. Dr. Paul Salvaterra is interested in the molecular logic that regulates neuronal determination. He will study the regulation of a neuron specific gene in the developing nervous system of Drosophila. Dr. Linda Iverson is interested in the regulation of gene expression during the differentiation of voltage-gated ion channels. She will examine the expression of the Drosophila gene Shaker that encodes functionally distinct potassium channels. Dr. James Vaughn is interested in the role of cellular interactions in the differentiation of neuronal phenotypes. He will study the migration of four subsets of cholinergic neurons in rat spinal cord to see whether these neurons develop their diverse characteristics in response to epigenetic cues encountered on different migration pathways. While the main focus of this program- project is on normal development, we anticipate that it will contribute information that is fundamental to understanding neurological and neuromuscular disorders.