Herbert K. Proudfit - US grants

The aim of the proposed studies is to examine the influence of neurohypophyseal hormones and related peptides on the development and maintenance of CNS tolerance to ethanol. Previous work has implicated noradrenergic systems as necessary for development of ethanol tolerance, and the neurohypophyseal peptides have been reported to affect the metabolism of norepinephrine in brain. An animal model for chronic consumption of ethanol will be used in which the development and expression of tolerance and physical dependence can be monitored by behavioral and physiological means, and in which the effects of the peptides on these phenomena, both during normal and altered catecholaminergic function, can be assessed. In this context, ethanol tolerance may be considered as a model for CNS adaptive phenomena, some of which (i.e. learning or memory) are also influenced via centrally-mediated effects of neurohypophyseal peptides.

The proposed experiments have been designed to determine the anatomical and functional connections among specific neurons in the brainstem and spinal cord that are involved in modulating nociception. The neuronal cell groups to be examined include the nucleus raphe magnus, nucleus reticularis gigantocellularis pars a, periaqueductal gray, pedunculopontine tegmental nucleus, A5 and A7 catecholamine cell groups, and an enkephalin cell group in the dorsolateral pontine tegmentum that innervates the spinal cord dorsal horn. Neurons in these cell groups are neurochemically diverse and contain neurotransmitters such as serotonin, norepinephrine, acetylcholine, enkephalins, neurotensin, substance P, excitatory amino acids, and probably many other unidentified peptides and amino acids. These experiments will determine the neurotransmitters contained in these neurons, the anatomical connections among these immunochemically-identified neurons, including the axonal trajectories, and the location of synaptic contacts made by these neurons. Functional neuronal connections will be determined by activating selected neurons using electrical or chemical stimulation and determining the resulting effects on nociception and electrophysiological recordings of postsynaptic neurons. Pharmacological analysis using selective neurotransmitter agonists and antagonists will be used to provide evidence for the function of specific neurons in nociceptive modulation. These anatomical, electrophysiological, and pharmacological experiments are designed to provide converging evidence that will be used to identify and define specific neuronal circuits in the brainstem and spinal cord that modulate nociception. Understanding the neural circuits that regulate nociception could lead to new and effective treatments for the numerous pain states that are refractory to current therapeutic methods. In addition, understanding the pharmacology of pain modulatory circuits may provide insights into new drug treatments for pain states that are currently treated with opioid drugs. Thus, non-opioid drugs may be developed that lack the unfortunate and dangerous side effects of the opioid analgesic agents.

DESCRIPTION (Adapted From The Applicant's Abstract): The proposed experiments will determine the anatomical and functional connections among specific neurons in the brainstem and spinal cord that are involved in the modulation of nociception. The neuronal cell groups to be examined include substance P and met-enkephalin neurons in the ventromedial medulla (VMM) and the periaqueductal gray (PAG), the A7 catecholamine cell group, a group of enkephalin neurons in the dorsolateral pontine tegmentum (DLPT) that innervate the spinal cord dorsal horn, and GABA neurons in the DLPT. Anatomical connections among these identified neurons will be determined using immunocytochemical methods to characterize their neurotransmitters, projections, and the location of synapses formed by these neurons. Functional connections among these immunochemically-identified neurons will be established using pharmacological and electrophysiological approaches. Pharmacological studies will determine changes in nociception induced by electrical or chemical activation of selected, identified neurons and the effect of subsequent microinjection of receptor agonists or antagonists in the regions to which these neurons project. Electrophysiological studies will characterize the response properties of spinally-projecting enkephalin and noradrenergic A7 neurons, and determine the functional connections of substance P and enkephalin neurons in the VMM and the PAG, and GABA neurons in the DLPT, that project to these descending neurons. The importance of the proposed experiments is related to the contributions they will make toward understanding the mechanisms of endogenous pain modulation in the brainstem that also control nociceptive responses in the spinal cord. The ultimate goal of this research is to understand the pain modulatory circuits in sufficient detail to allow the derivation of a computational model of these neuronal circuits that could be used to develop more effective pain control methods. It is hoped that safer and more effective pain controls methods could be developed that do not depend on opioid drugs with their attendant serious side effects and addiction liability.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] We hypothesize that the formation of dendritic varicosities.in response to substance P (SubP) is a novel and as yet unappreciated morphological mechanism for the long-term regulation of neuronal excitability. The occurrence of dendritic varicosities (DVs) is well-documented and there is a sound theoretical basis for their ability to alter neuronal excitability, yet their functional significance remains unknown. We have preliminary evidence for their physiological relevance, and occurrence in brainstem neurons implicated in the regulation of nociception and reward mechanisms. The aim of this CEBRA application is to test the hypothesis that the formation of DVs is a morphological mechanism by which the excitability of neurons can be regulated longterm. Whole-cell voltage-clamp recordings from rostral ventromedial medullary (RVM) neurons in brainstem slices will be used to establish the functional relevance of DVs with respect to synaptic transmission. The first Specific Aim will use conventional and confocal light microscopic analyses to determine (1) the timecourse of DV formation in NK1-ir RVM GABA neurons, (2) the ability of microtubule stabilizing drugs, such as taxol, to prevent the formation of DVs, and (3) the ability of NK1 receptor antagonists to block the formation of DVs. Blockade of synaptic transmission by TTX will be used to determine whether DVs induced by SubP are mediated by a direct effect rather than the release of other transmitters. The results of these studies will form the anatomical basis for understanding the effect of DVs on the conduction of synaptic potentials in NK1-ir RVM GABA neurons. The second Specific Aim will use whole-cell voltage clamp recordings to (1) characterize how the formation of DVs alters the passive membrane properties of RVM neurons including cytosolic resistance and (2) determine whether the formation of DVs influences the conduction of synaptic potentials -in RVM neurons. These latter studies will determine whether the formation of DVs is associated with changes in (a) the resting membrane currents, (b) the conduction of synaptic currents, and (c) stimulus evoked input-output curves of RVM neurons. Collectively, the results of these studies will establish the formation of DVs as a means to regulate the conduction of synaptic potentials and excitability of brainstem neurons. The significance of this application lies in its characterization of a potentially important and novel mechanism that may control neuronal excitability. [unreadable] [unreadable] [unreadable] [unreadable]