Vincent A. Chiappinelli, Ph.D. - US grants

Nicotinic acetylcholine receptors of neurons are poorly characterized in comparison with the nicotinic receptors of skeletal muscle. A major reason for this is that snake venom alpha-neurotoxins, which have been valuable probes for muscle nicotinic receptors, are often completely ineffective at neuronal nicotinic receptors. Recently, a related family of snake neurotoxins has been described. The kappa-neurotoxins show considerable sequence homology (approximately-50%) with the alpha- neurotoxins, but have an opposite selectivity for nicotinic receptors. Kappa-Neurotoxins are potent antagonists of neuronal nicotinic receptors in avian and murine autonomic ganglia, but bind with much lower affinity to nicotinic receptors of muscle. Radiolabeled kappa-neurotoxins have been used to distinguish physiologically-defined nicotinic receptors from ganglionic alpha- neurotoxin sites, which at present have no known function. In this proposal, two kappa-neurotoxins (kappa-bungarotoxin and kappa-flavitoxin) will be used in electrophysiological, biochemical and morphological studies designed to characterize neuronal nicotinic receptors. For the first time, the effects of kappa- neurotoxins on central vertebrate neurons will be determined. Intracellular recordings will examine the electrophysiology of neuronal nicotinic receptors in slices of chick optic lobe and rat locus coeruleus. Binding and localization studies will be done in chick optic lobe, where three different nicotinic sites have been detected. Kappa-Bungarotoxin binds to at least one of these sites with high affinity, but it is presently not known which of these nicotinic sites is involved in physiological nicotinic responses. Autoradiographic studies using radiolabeled kappa-bungarotoxin, alpha-bungarotoxin, nicotine and acetylcholine will localize these sites in the optic lobe. Binding experiments will compare the properties of the biochemically-defined sites with those of physiologically-defined nicotinic receptors. The biochemistry of kappa-neurotoxins will be further defined to gain information about the active sites of neuronal nicotinic receptors. Synthetic peptides based on the known amino acid sequences of kappa-neurotoxins will be tested for activity at neuronal receptors, with the goal of defining the critical structural differences between kappa-neurotoxins and alpha- neurotoxins which mirror the structural differences between neuronal and muscle nicotinic receptors. Several disease states involve alterations in neuronal cholinergic function. A marked decrease in cholinergic transmission has been observed in patients suffering from Alzheimer's disease and Huntington's disease. By further characterizing cholinergic neurotransmission, the experiments proposed in this study may provide a better understanding of these diseases.

The parasympathetic component of the oculomotor pathway is responsible for a number of critical non-retinal ocular processes, including control of the light aperture, accommodation and regulation of blood flow t the retina. The cell bodies of autonomic ganglion cells in this pathway are located in the ciliary ganglion. All presynaptic terminals in the ciliary ganglion originate in the midbrain in the Edinger-Westphal nucleus. Acetylcholine, contained within these terminals, is the only proven neurotransmitter in the ganglion, acting at nicotinic receptors on postsynaptic ganglion cells. Substance P-like and leucine-enkephalin-like immunoreactivities have been localized within dense core vesicles in a majority of the cholinergic terminals in the avian ciliary ganglion, suggesting that these neuroactive peptides are released along with acetylcholine following nerve stimulation. The role of these peptides in neurotransmission in the oculomotor system is not known and will be examined in this proposal. Intact ciliary ganglia obtained from chick embryos and hatched chickens will be studied in an in vitro electrophysiological chamber. Intracellular and extracellular recording methods will be used to characterize non-nicotinic transmission in the ganglion. The effects of substance P and leucine-enkephalin will be examined by application of these and related peptides. Specific opioid antagonists will be used to reveal any endogenous opioid actions. The avian ciliary ganglion contains two distinct types of postsynaptic cells, the ciliary and choroid neurons. In addition, presynaptic terminals which form calyciform endings on the ciliary neurons can be impaled in this ganglion. The ability to record from presynaptic and postsynaptic neurons in the ganglion is of critical importance to the project, since we have shown that the neuroactive peptides present have effects on both sides of the ganglionic synapse. Using intracellular recording in a brain slice preparation, the effects of tachykinins and opioid peptides will be examined in the Edinger-Westphal nucleus, which is known to receive a substance P-positive innervation from the contralateral suprachiasmatic nucleus. Of particular interest will be a comparison of the peptide responses of cell bodies in the nucleus to those of their terminals within the ciliary ganglion. This proposal should provide important information on the role of endogenous neuroactive peptides in synaptic transmission in the oculomotor pathway. A number of clinical diseases of the eye involve defects in this system. These include some defects in accommodation, internal ophthalmoplegia and tonic pupil. In addition, the condition of the pupil is an important diagnostic sign in numerous ocular syndromes and in traumatic injuries to the head.

nicotinic receptors; neural transmission; synapses; acetylcholine; central nervous system; nimodipine; neurotransmitter transport; bungarotoxins; nifedipine; neuropharmacology; gamma aminobutyrate; lateral geniculate body; carbachol; glutamates; evoked potentials; developmental neurobiology; dopamine; voltage gated channel; nicotine; confocal scanning microscopy; immunocytochemistry; chickens; chick embryo; voltage /patch clamp; tissue /cell culture;

The Cys-loop receptor family includes the nicotinic acetylcholine receptor (nAChR) and the serotonin type 3 receptor (5-HT3R). Cys-loop receptors are neurotransmitter-activated ion channels participating in neurotransmission in the nervous system. They are targets of drugs for treating disorders from nausea to anxiety. Mutations in Cys-loop receptors can lead to diseases including epilepsy. Neurotransmitter binding opens the ion channel by initiating the movement of the second transmembrane (TM2) domain of each of the channel's five subunits. In the conventional view of the channel an outer vestibule narrows to the rate-limiting region within the membrane where the gate is located; the pore then opens into the cytoplasm. Thus, in this scheme, determinants of ion conduction reside exclusively in TM2. However, recent evidence renders this textbook view of Cys-loop receptors obsolete: First, cryoelectron microscopy revealed that the nAChR has narrow "portals" at the cytoplasmic interfaces of each subunit; Second, mutagenesis of these regions in the 5-HT3R influences ion conduction. These findings necessitate revision of the model for the ion permeation pathway. Thus this project addresses a fundamental question in neuroscience: does conduction through ion channels involve cytoplasmic elements? Data from this laboratory demonstrate that intracellular regions of 5-HT¬3Rs affect all aspects of ion conduction: conductance, voltage-dependence and ionic selectivity. This study will identify the mechanisms involved, creating a new model for Cys-loop receptor function. A key objective of the study is to continue to develop a Cys-loop receptor database accessible to physicians, researchers and educators. Thus this project will enhance the infrastructure for research and education at The George Washington University and beyond. Undergraduate students spearhead the development of the Cys-loop receptor database.