John P. Horn - US grants

Sympathetic ganglia convey commands that implement reactions to bodily stress. The long-term goals of this project are to understand how three slow synaptic potentials contribute to the normal integrative function of bullfrog sympathetic ganglia and to identify factors that regulate the expression of muscarinic synapses in ganglia. The working hypotheses for this proposal are that 1) ganglia contain physiologically specialized and antomically separate functional channels that modulate different classes of peripheral end organs, 2) slow potentials enable ganglion cells to generate characteristic patterns of activity to drive optimally their specific targets and 3) the synaptic connections of ganglion cells regulate their expression of muscarinic synapses. Isolated preparations containing the ninth and tenth paravertebral ganglia will be used to study the muscarinic excitatory postsynaptic potential (epsp), the muscarinic inhibitory post synaptic potential (ipsp) and a peptidergic epsp mediated by leuteinizing hormone releasing hormone (LHRH). Three physiologically identifiable types of neurons (fast B, slow B, C) within these ganglia innvervate viscera in the lower abdomen (e.g. bladder) and other targets in the hindlimbs (e.g. exocrine glands, vasculature, sensory receptor). However, the relation between these cell types and the hypothesized functional channels is uncertain. A combination of electrophysiological and anatomical methods will be used to provide a detailed description of ganglionic synapses, the morphology of identified sympathetic neurons and the projections of identified cells into cutaneous, motor and visceral branches or peripheral nerves. Next, reports that muscarinic epsps are variable in their voltage-sensitivity will be pursued to determine how heterogeneity in muscarinic excitation is related to the subclasses of B cells and how muscarinic epsps differ in their effects upon repetitive firing. Then muscarinic inhibition of repetitive firing will be analyzed by voltage clamping C cells. After the full range of muscarinic modulation of repetitive firing has been characterized, the influence of specific connections between the ganglia, cord and periphery upon the differential expression of muscarinic responses in ganglion cell types will be studied during axotomy, denervation and re-innervation.

The objectives of this application are to intensify ongoing research on the physiology of synaptic cotransmission in sympathetic ganglia and to learn molecular and biological methods that will be applied in new studies of neuronal differentiation. The P.I. is an assistant professor of Physiology and is in the 05 year of independent research with RO1 support. An excellent collegial environment and adequate support services exist within the local University community. The propose plan will insure continued development of the P.I.'s research career. It will limit growth of non-research related activities and provide resources to pursue new methodologies, experiments and collaborations. The experiments will utilize a combination of electrophysiological, anatomical and biochemical methods in isolated preparations and in primary cell cultures. The new methods to be learned include nucleic acid isolation,Northern analysis and in situ hybridization. The experimental goals of the proposal are to determine the integrative function and the developmental origins of synapses that utilize slowly acting peptidergic co-transmitters in addition to the classical transmitters acetylcholine (ACH) and epinephrine (EPI). The roles of 4 neuropeptides, luteinizing hormone releasing hormone (LHRH), neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP) and substance P (SP), will be analyzed in the sympathetic system of bullfrog. Although these peptides are co-expressed with other transmitters in many regions of the peripheral and central nervous system, the functional dynamics and ontogeny of synaptic co-transmission is largely unknown. Bullfrog sympathetic ganglia express a diversity of synaptic mechanisms and yet they are accessible to analysis at the cellular and molecular levels. In the lumbar ganglia, there exist 3 subclasses of sympathetic neurons that can be distinguished by their functions, electrophysiological properties, connectivity with the spinal cord and with peripheral targets, and by their expression of muscarinic cholinergic receptors and neuropeptides. In the vasomotor sympathetic C system, LHRH and ACH are co-released by preganglionic neurons and elicit 3 postsynaptic potentials in a subset of ganglion cells that co-express EPI and NPY-like immunoreactivity. The first specific aim of this proposal is to test the hypothesis that co-transmission at ganglionic and end-organ synapses in this circuit enhances the dynamic range of vascular contractions that can be elicited by different patterns of preganglionic stimulation. The second specific aim is to test the hypothesis that CGRP is co-released with ACH by preganglionic B neurons and produces trophic effects and an excitatory postsynaptic potential in B neurons. The third goal is to establish the timetable during development in tadpoles for cell-specific expression of neuropeptide genes and their products. Using tissue culture, the final goal of the project will be to test the hypothesis that expression of NPY is controlled by molecules in the extracellular environment and/or by cellular interactions, and that such factors also control the electrophysiological differentiation of vasomotor neurons.

The long-term goal of this project is to elucidate the integrative function of muscarinic and peptidergic synapses in bullfrog sympathetic ganglia. The bullfrog was chosen for study because its anatomical features are optimal for electrophysiological experiments that can be performed at the cellular level and directly related to the control of vascular tone and glandular secretion. The proposed experiments exploit the fact that paravertebral sympathetic neurons in the bullfrog are organized into 2 major subsystems, B and C, that can be identified by their axonal conduction velocities, and selectively activated due to the anatomical separation of their preganglionic inputs. These cell types are further distinguished by their expression of muscarinic synapses and neuropeptides, and by their functional roles. The experiments will employ electrophysiological methods to study isolated preparations containing either ganglia and their end-organs, ganglia alone, or end-organs alone, and to study dissociated neurons in primary cell culture. The proposal has 3 specific aims: 1) To test the hypothesis that co-transmitters endow the vasomotor C system with variable synaptic gain that is regulated by temporal patterns of preganglionic activity. The hypothetical model postulates that synapses in the ganglia and at end-organs function as 2 variable gain stages in series. In ganglia, acetylcholine (ACH) and luteinizing hormone releasing hormone (LHRH) are co-released to produce a nicotinic epsp, a slow muscarinic ipsp, and a slow peptidergic epsp. Temporal interactions between the 2 slow synaptic potentials are hypothesized to control ganglionic gain. In the periphery, it is postulated that the release of the co-transmitters epinephrine (EPI) and neuropeptide Y (NPY) is differentially regulated by activity. When NPY is released, it enhances end-organ gain by potentiating vascular responses to EPI. 2) To test the hypotheses that the B system innervates cutaneous mucous glands and that calcitonin gene-related peptide (CGRP) is both a ganglionic co-transmitter that mediates a slow epsp and a trophic factor that regulates the expression of nicotinic ACH receptors. 3) To test the hypothesis that LHRH released by preganglionic C fibers acts heterosynaptically to increase the gain of synapses between pre- and postganglionic B neurons. Results of the proposed studies will provide a conceptual framework for understanding the integrative function of synaptic co-transmission in more complex circuits including mammalian autonomic ganglia and the brain.

The objectives of this application are to intensify ongoing research on the physiology of synaptic cotransmission in sympathetic ganglia and to learn molecular and biological methods that will be applied in new studies of neuronal differentiation. The P.I. is an assistant professor of Physiology and is in the 05 year of independent research with RO1 support. An excellent collegial environment and adequate support services exist within the local University community. The propose plan will insure continued development of the P.I.'s research career. It will limit growth of non-research related activities and provide resources to pursue new methodologies, experiments and collaborations. The experiments will utilize a combination of electrophysiological, anatomical and biochemical methods in isolated preparations and in primary cell cultures. The new methods to be learned include nucleic acid isolation,Northern analysis and in situ hybridization. The experimental goals of the proposal are to determine the integrative function and the developmental origins of synapses that utilize slowly acting peptidergic co-transmitters in addition to the classical transmitters acetylcholine (ACH) and epinephrine (EPI). The roles of 4 neuropeptides, luteinizing hormone releasing hormone (LHRH), neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP) and substance P (SP), will be analyzed in the sympathetic system of bullfrog. Although these peptides are co-expressed with other transmitters in many regions of the peripheral and central nervous system, the functional dynamics and ontogeny of synaptic co-transmission is largely unknown. Bullfrog sympathetic ganglia express a diversity of synaptic mechanisms and yet they are accessible to analysis at the cellular and molecular levels. In the lumbar ganglia, there exist 3 subclasses of sympathetic neurons that can be distinguished by their functions, electrophysiological properties, connectivity with the spinal cord and with peripheral targets, and by their expression of muscarinic cholinergic receptors and neuropeptides. In the vasomotor sympathetic C system, LHRH and ACH are co-released by preganglionic neurons and elicit 3 postsynaptic potentials in a subset of ganglion cells that co-express EPI and NPY-like immunoreactivity. The first specific aim of this proposal is to test the hypothesis that co-transmission at ganglionic and end-organ synapses in this circuit enhances the dynamic range of vascular contractions that can be elicited by different patterns of preganglionic stimulation. The second specific aim is to test the hypothesis that CGRP is co-released with ACH by preganglionic B neurons and produces trophic effects and an excitatory postsynaptic potential in B neurons. The third goal is to establish the timetable during development in tadpoles for cell-specific expression of neuropeptide genes and their products. Using tissue culture, the final goal of the project will be to test the hypothesis that expression of NPY is controlled by molecules in the extracellular environment and/or by cellular interactions, and that such factors also control the electrophysiological differentiation of vasomotor neurons.

The long-term goal of this project is to determine how diversity in the cellular expression of neuronal nicotinic receptors contributes to synaptic function in mammalian sympathetic ganglia. This research will be done in Kiev, Ukraine as an extension of NIH grant #R01 NS21065. The project's central hypothesis states that neurons serving different effector modalities express distinct combinations of nicotinic receptor subunits, as a means for regulating the duration of fast excitatory postsynaptic potentials and thereby controlling the synaptic amplification of preganglionic activity. The proposed experiments will use electrophysiology to assess nicotinic receptors in rat sympathetic and parasympathetic neurons. Specific aim 1 is to identify kinetic features that distinguish nicotinic receptors on functional subsets of sympathetic and parasympathetic neurons. The integrative consequences of these data will then be evaluated using computational models that simulate synaptic activity in autonomic ganglia. Specific aim 2 is to extend the pharmacological profile of synaptic receptors on vasomotor sympathetic neurons by systematic characterization of novel open-channel blockers and antisera against alpha-subunits of nicotinic receptors. The goal is to develop a rational basis for designing new compounds that would be highly selective for vasomotor sympathetic neurons and other autonomic modalities. Such compounds could be used to further dissect the roles of different autonomic cell groups and may have clinical applications. Specific aim 3 is to determine whether alpha-bungarotoxin-sensitive nicotinic receptors contribute to fast synaptic transmission in sympathetic ganglia. This will test whether recent work on avian autonomic ganglia also holds true in mammalian neurons that are known to express the alpha-7 nicotinic subunit. Resolving the issues posed by each specific aim would have fundamental implications for our understanding of ganglionic integration. Nicotinic synapses in autonomic ganglia are important for public health because they are essential for autonomic behaviors that are disrupted by aging and neurological disease (e.g. cardiovascular adaptation to exercise, reproduction, thermoregulation and defensive adaptation to stress).

DESCRIPTION (provided by applicant): The goal of this exploratory proposal is to discover genes whose expression defines the specialization of vasomotor sympathetic neurons. Such genes represent new tools for manipulating sympathetic neurons and blood pressure control in experimental studies and for developing new therapeutic strategies. The approach will exploit a transgenic reporter mouse in which NPY-promoter sequences drive expression of green fluorescent protein. Messenger RNA will be purified from manually sorted identified neurons, then amplified and analyzed with microarrays to detect gene expression. The proposal is predicated on the novel concept that half of the entire sympathetic outflow of spike activity originates in the ganglia, not the central nervous system, and upon the hypothesis that synaptic amplification in functional subsets of vasomotor sympathetic neurons is tuned to the needs of different vascular beds. Experiments will address one specific aim, which is to discover genes that distinguish vasomotor from non-vasomotor neurons and that identify subsets of sympathetic neurons controlling different aspects of cardiovascular function. Gene expression will be analyzed in four different ganglia that control brain circulation, the heart, the kidney and limb muscle vasculature. The expression analysis will be further refined by using a retrograde tracer to back label neurons that control circulation in muscles, the skin and the kidney and through analysis of gene expression in single cells. Validation studies will employ quantitative PCR, immunocytochemistry and electrophysiology. This project has potential for high-impact. Strong evidence now indicates that hyperactivity in vasomotor postganglionic sympathetic neurons presages 50% of human hypertension and further contributes to problems associated with heart failure and renal failure. These conditions impose a tremendous public health burden, yet existing therapies for clinical management of hypertension remain inadequate. Finding tools for selectively manipulating postganglionic sympathetic activity in cell types that regulate specific vascular beds will advance fundamental scientific and medical understanding of integrated autonomic physiology.