Aaron P. Fox - US grants

Calcium channels play a fundamental role a in variety of neuronal processes including neurotransmitter release, neurite extension and electrical signalling. Calcium ions flowing through voltage- dependent calcium channels, operate as an intracellular second messenger by modulating various cellular constituents. In the hippocampus, calcium ions are also involved in the form of memory called long-term potentiation and may be involved in diseases such as epilepsy. The existence of multiple types of neuronal calcium channels is now widely accepted. Our work demonstrated that chick DRG neurons had three different types of calcium channels which we call "T", "N", and "L". These channels differed on the basis of pharmacology, single-channel conductance, and gating. The studies outlined in this grant would extend the Ca channel investigations into several different areas of the hippocampus, including the CA1, CA3 and dentate-gyrus regions. Patch-clamp electrophysiology and high-K+ or field electrode evoked release of radiolabelled neurotransmitters, will be the principal techniques used. Specifically, I propose to carry out the following studies. -1) Characterize the calcium present in hippocampal neurons. Both Ca channel getting and permeation will be investigated. -2) Study hippocampal calcium channel modulation by neurotransmitters, hormones and drugs. These experiments will focus mainly on single-channel mechanisms. Perfusion of the patch pipette while in the whole-cell recording configuration will allow us to introduce and study various intracellular second- messengers. -3) Try to correlate calcium types with cellular function. Included will be studies attempting to identify the type(s) of Ca channels involved in neurotransmitter release. -4) Prepare giant synaptosomes to directly investigate pre- synaptic calcium channels with electrophysiological techniques. One of the most interesting aspect of these studies will the comparisons made between the calcium channels found in the different hippocampal neurons.

DESCRIPTION (Adapted from Applicant's Abstract): Adrenaline and noradrenaline, hormones secreted by adrenal chromaffin cells directly into the circulatory system, have profound pharmacological effects on many different organ systems in the body including the heart, gut, central nervous system, immune system etc. Secretion is governed by Ca2+ entering chromaffin cells through voltage-dependent calcium channels. Acetylcholine, released by splanchnic nerve terminals in response to electrical activity, depolarizes the chromaffin cells thereby activating the calcium channels. Calcium channels play a dual role in chromaffin cells. First, they open in response to membrane depolarization and allow Ca2+ ions to flow down their electrochemical gradient; this influx carries depolarizing charge contributing to the cells electrical characteristics. Second, calcium channels act as transducers changing the signal encoded in the membrane potential into a chemical signal; changes in intracellular Ca2+ act as a second messenger that can modulate a wide variety of Ca2+-sensitive cellular processes. Our preliminary results show that there are three types of calcium channels present in chromaffin cells that are different than calcium channels found in other organs. What are the physiological roles of the various calcium channels of chromaffin cells? What are their electrical and pharmacological properties? Which calcium channel(s) governs the release of catecholamines? Can more than one channel play a role in secretion? Results also showed that there was not a good correlation between the Ca2+ ions entering the cells via calcium channels and the intracellular calcium-transients. Why don't calcium currents correspond to the calcium transients? What is the significance for catecholamine release? To answer some of these questions the applicant will perform the following studies: (1) Characterize both biophysically and pharmacologically the different bovine chromaffin cell's calcium channels. These results will be compared to feline chromaffin cells which may be dissimilar. (2) Study calcium channel modulation produced by adrenaline, noradrenaline and dopamine. The applicant will focus on single-channel mechanisms, effects of second messengers and GTP binding proteins. (3) Study the factors that control intracellular calcium homeostasis. (4) Determine which calcium channel(s) takes part in catecholamine secretion and the intracellular calcium buffering systems that may also regulate release.

This application focusses on the Ca channels and on mechanisms of exocytosis found in chromaffin cells. In the animal, chromaffin cells in the adrenal medulla secrete catecholamines and several neuropeptide hormones. Secretion is triggered by Ca2+ entering the cells through voltage-gated Ca channels. Chromaffin cells possess three different types of Ca channels. One type, the facilitation Ca channel, is not activated by single brief depolarizations. This channel is normally silent; it does not contribute to electrical activity or to Ca2+ influx. Facilitation Ca channels are activated by many depolarizations to physiological potentials, or by depolarizations to very positive potentials. Recruitment of these channels may involve a novel form of channel regulation, voltage- dependent phosphorylation. All three types of Ca channels found in chromaffin cells are capable of triggering secretion; facilitation Ca channels are most efficient. Secretion produced by facilitation Ca channels is larger, faster, and has a shorter latency when compared to release produced by the other channels types, even when the Ca current is smaller. By simultaneously measuring Ca currents, [Ca2+] and secretion (with capacitance), we hope to determine the optimal conditions for triggering release by each type of Ca channel. We will model secretion for each type of Ca channel present in chromaffin cells. Chromaffin cells store and secrete either epinephrine or norepinephrine (some cells may secrete both). In a few preliminary studies norepinephrine containing cells were found to have no facilitation Ca channels. Using voltammetric techniques to identify chromaffin cells as either epinephrine or norepinephrine secreting, we will verify this finding. Norepinephrine cells will be studied extensively to better understand catecholamine secretion from cells that presumably have no facilitation Ca channels. All the Ca channels in norepinephrine cells will be tested for efficacy in promoting secretion. These data will be compared to secretion data from epinephrine containing cells that have facilitation Ca channels. These studies may expose alterations in secretion resulting from differences in the Ca channels present. Ca currents recorded in chromaffin cells using the perforated patch whole- cell configuration are different than currents recorded with whole-cell patch clamp configuration with low EGTA in the pipette. This is an important point as almost all capacitance studies secretion have used whole-cell recording conditions with low EGTA. We will study secretion from chromaffin cells using perforated whole-cell recording conditions. Given our preliminary results I feel it's crucial to study secretion with the intracellular constituents intact. Finally, we will determine whether secretion can be modulated by neurotransmitters, while keeping Ca currents and [Ca2+]i constant. If modulation is found, we will try and elucidate the mechanism.

The objective of this proposal is to bring together a multidisciplinary group of investigators to study novel or poorly understood Ca2+ influx pathways, how these pathways are regulated and the physiological processes they control (i.e. synaptic transmission/ secretion). By combining a variety of experimental techniques including molecular biology, biochemistry, electrophysiology and [Ca2+]i measurements, we hope to aid our progress. We expect that bringing together a group of investigators with similar interests and goals but who use a variety of techniques in different preparations will aid each of the individual projects and provide a synergy not available when working alone. In project 1 - Dr. Miller will study novel Ca channels (type alpha1E) cloned in his lab (as well as others). Message for this channel is found in abundance throughout the nervous system, but functional channels have only been found in a couple of locations (i.e. cerebellum) and even there, they are rare. More in-depth studies of this Ca channel including localization and functional expression are planned. It is possible that these channels have not been identified using standard electrophysiological procedures because they are preferentially localized to synapses. In project 2 - Dr. Green will study neuronal ACh receptors in PC12 cells. Detailed studies of the pharmacological and physiological properties of these receptors are planned, with emphasis on their Ca2+ permeability. In addition, regulation and the subunit assembly of these receptors will be studied. Experiments to determine the subunit composition will continue. The goal is to gain a greater understanding of how neuronal ACh receptor diversity is achieved and the role played by these receptors in the nervous system. In project 3 - Dr. Fox will study the relationship between Ca2+ entry and secretion. Studies of membrane retrieval following stimulation are planned. The contribution made by activation of ACh receptors to secretion will be investigated. A thorough understanding of stimulus- secretion coupling and membrane recycling are essential for a complete understanding of the release process.

This application focusses on mechanisms of exocytosis and endocytosis in chromaffin cells. Secretion is triggered by Ca2+ entering the cells through voltage-gated Ca channels. Chromaffin cells possess three different types of Ca channels. One type, the facilitation Ca channel, is not activated by single brief depolarizations. Recruitment of these channels may involve a novel form of channel regulation, voltage- dependent phosphorylation. Ongoing studies will verify (or disprove) this hypothesis. In all of our experiments to date, catecholamine release is followed by rapid membrane retrieval (tau of seconds). We will investigate whether membrane retrieval rates are similar for large and small levels of secretion, whether membrane retrieval is Ca2+ dependent, and whether known kinases or phosphatases are important in this process. As we are one of the few labs that measuring retrieval rates in real-time, we believe that we have a contribution to make in understanding this important process. Acetylcholine, released from splanchnic nerve terminals within the adrenal medulla, depolarizes chromaffin cells by activating Ca2+- permeable nicotinic ACh receptors, leading to calcium entry. ACh also promotes calcium release from intracellular stores by activating muscarinic receptors. Three different Ca2+-signals summate to trigger release; these are calcium-entry through Ca2+ channels, calcium-entry through Ca2+-permeable nicotinic ACh receptors and calcium-release from intracellular stores following muscarinic ACh receptor activation. In collaboration with Dr. Green, we will investigate the relative importance of each of these signals, as well as how they work together. N-type Ca channels have been found in virtually every neuron. They are known to be involved in neurotransmitter release at a variety of synapses (as well as chromaffin cells), yet detailed biophysical information has been surprisingly difficult to obtain, in part because N-type Ca channels are found in cells with other types of channels. We have available cloned N-type Ca channels stably expressed in HEK cells. In collaboration with Dr. Richard Miller's lab we plan on carrying out extensive studies of N channel gating currents. This grant application may look diffuse but Specific Aims 3 and 4 are collaborative efforts that strengthen the interactions between my lab and those of Drs. Green and Miller and so should be judged in that context.

tissue /cell culture; biomedical facility; PC12 cells; chromaffin cells;

DESCRIPTION: (Applicant's Abstract) N-type calcium channels, widely distributed throughout the nervous system, have been unambiguously linked to a variety of important physiological processes including synaptic transmission. In secretory cells, like chromaffin cells, N-type Ca channels can trigger catecholamine secretion. The purpose of this grant is to characterize novel N-type Ca channels found in chromaffin cells. Typically, N-type Ca channels found in both peripheral and central neurons inactivate. Channel availability is greatly diminished at depolarized holding potentials and during prolonged depolarizations. Some chromaffin cells, in contrast, express N-type channels that do not inactivate. These channels do not exhibit decreased availability at depolarized holding potentials, nor do they show inactivation during long depolarizations. N-type Ca channels are thought to be composed of a pore forming alpha1B subunit and accessory subunits. In order to understand the molecular details of bovine chromaffin cell inactivation, we have cloned the bovine alpha1B and various accessory subunits. We have managed to recreate native non-inactivating properties in the Xenopus oocyte expression system by co-expressing alpha1B, beta2A and alpha2/delta. Channels made with other beta-subunits, beta1b, beta1c, beta2b and beta3a, show inactivation. We wish to identify regions in the beta-subunits that are critical in determining their inactivation properties. When beta2a is co-expressed with neuronal alpha1b subunits, inactivation although slowed, is still present. We wish to identify new regions in the alpha1b subunit that determine their inactivation properties. We will avail ourselves of the opportunity to extensively characterize single channel behavior of these non-inactivating N-type channels. In the past, this family of channels has been described as exhibiting modal behavior. Channels display periods of greatly diminished activity. We will determine whether these low Po periods are true channel gating states or whether they are induced by channel inactivation. G-protein betagamma subunits inhibit N-type Ca channels in a voltage-dependent manner; large depolarizations are thought to promote unbinding of the G-betagamma subunit from the channel. Unfortunately, it has been difficult to study the Ca channel/G-betagamma interactions, in part due to channel inactivation. Detailed studies of the interaction of these channels with G-betagamma subunits are planned. In some chromaffin cells N-type channels inactivate, while in others they do not. In cells which show inactivation, all the N-type channels inactivate. In cells which show no inactivation, no N-type Ca channels inactivate. Chromaffin cells will be segregated into two groups - those cells expressing inactivating and those cells expressing non-inactivating N-type Ca channels. N-type Ca currents can be observed in isolation in chromaffin cells, using appropriate antagonists. Using a variety of stimulation protocols we will determine what effect N-type Ca channel inactivation has on catecholamine release; these experiments should provide insights into neurotransmitter release at synapses regulated by either inactivating or non-inactivating Ca channels.

DESCRIPTION (provided by applicant): The addiction to nicotine results in a significantly shortened life span in millions of people and in billions of health care dollars spent every year in tobacco related illnesses. Although the mechanism of addiction is not understood there is a growing body of evidence pointing to the involvement of high-affinity neuronal nicotinic receptors (nAChR) in addiction. Our project will explore possible mechanisms that underlie nicotine addiction. Our studies will be carried out in adrenal chromaffin cells, which may be a primary peripheral target for nicotine. Chromaffin cells express neuronal nAChR similar to those in neurons; the subsequent catecholamine release triggered by nicotine may be involved in the addiction process. Release of catecholamine may affect blood pressure and heart rate and may be involved in some of the adverse physiological changes seen in smokers. In addition, we will study physiological release mechanisms mediated by ACh (or nicotine). We have helped develop a new model system that will allow us to directly test the efficacy of neuronal nicotinic receptors in initiating neurotransmitter release. Similar studies will be carried out in chromaffin cells. The specific aims of this grant are: Aim 1 (Chromaffin Cells) - Hypothesis: Activation of nicotinic receptors directly triggers or modulates neurotransmitter release. Nicotine levels in smokers are sufficient to produce significant desensitization of receptors, which alters release. Because nAChRs have a high permeability to Ca2+ our goal is to determine the importance of this Ca2+-influx pathway in triggering release using physiologically relevant applications of nicotine. These experiments will be repeated after both short- and long-term (20 mm. to 6 days) exposure to low levels of nicotine (100 -- 500 nM) similar to those observed in smokers. Because elevations of intracellular Ca2+ mobilize vesicles for release, we will try to determine whether low levels of nicotine (100 -- 500 nM) affect vesicular mobilization and the role of desensitization in this process. Aim 2 (mouse pheochromcytoma cells) - Hypothesis: Activation of a variety of different nAChR can directly trigger secretion. My lab (in collaboration with Dr. Art Tischler's lab) recently identified a mouse pheochromocytoma (MPC) cell line that is secretion competent but which expresses no nicotinic receptors and few or no endogenous Ca2+ channels. We will transfect these cells with different types of nicotinic receptors to determine whether they can trigger secretion or mobilize vesicles and the mechanisms involved. Subunits that will be tested include a7 (alone), a3 b2, a3 / b4 and a4/b2. For these experiments we will employ cells that express no endogenous Ca2+ currents (determined by briefly depolarizing every cell). Physiologically, activation of nAChRs depolarizes cells and activates Ca2+ channels. In other experiments we will co-transfect nAChRs and the a1b/a2d/b3a(N-type) Ca2+ channel subunits in order to study the synergy between these Ca2+-inf1ux pathways. This new aim is extremely exciting to us and is only possible because of the discovery of this remarkable cell line.

DESCRIPTION (provided by applicant): Despite a great deal of research, a complete understanding of the actions of general anesthetics is still not available. The objective of this research is to advance our understanding of isoflurane, with the hope of gaining insights into all anesthetics. Isoflurane, a halogenated volatile anesthetic, is thought to produce anesthesia by depressing central nervous system function. Many anesthetics, including isoflurane, are thought to modulate and/ or directly activate GABAA receptors. Isoflurane is also known to have effects on other channels and receptors. In a set of preliminary studies we observed that isoflurane, at clinically relevant concentrations, dramatically inhibited the neurotransmitter release machinery in PC12 cells. Etomidate and propofol also inhibited the release machinery in PC12 cells, suggesting that inhibition of neurotransmitter release may be an important general action of anesthetics. Because neurotransmitter release mechanisms are strongly conserved between PC12 and neurons we hypothesize that anesthetics interfere with the neuronal neurotransmitter release machinery and this may represent an important site of action for anesthesia in intact animals. The experiments outlined in this application will be carried out in cultured hippocampal neurons, PC12 cells and mutant mice. This proposal has three goals. First, we will determine whether isoflurane interferes with neurotransmitter release in hippocampal neurons at clinically relevant concentrations. Next, the anesthetic site of action will be identified. We start by examining syntaxin 1A and unc-13, but other sites including SNAP- 25, synaptobrevin and synaptotagmin 1, will be investigated as well. Details about why these sites were selected are provided in this application. Once neurotransmitter release machinery targets are identified they will be mutated in order to suppress the interaction between isoflurane and the target. This information will be used to design knock-in mice with the same mutations, which will permit us to determine, using a battery of physiological and behavioral tests, whether these animals still respond to isoflurane (or other anesthetics) or whether their responses to the anesthetics are altered.Project Narrative: Isoflurane is a widely used volatile anesthetic. Our goal is to find out how isoflurane produces anesthesia. In particular, we will explore the effect on the neurotransmitter release machinery, as our preliminary data suggests this may be a critical site of action for isoflurane and other anesthetics. This may allow for the design of new more beneficial drugs in the future.

? DESCRIPTION (provided by applicant): The objective of this research is to determine whether caffeine reverses anesthesia in humans. General anesthetics induce a coma-like state; recovery from anesthesia is passive and is due to the discontinuation of anesthetic. Problematically, recovery from anesthesia is somewhat random, dependent upon a variety of factors like age or genetics that are beyond the clinician's control. Although waking from anesthesia can be relatively rapid, cognitive abilities are depressed for hours. In addition, some patients wake very slowly. It would be extremely beneficial to be able to time recovery from anesthesia in a reproducible manner and to have that recovery be complete. Our preliminary results, carried out in cultured PC12 cells and hippocampal neurons, suggested that general anesthetics inhibit neurotransmitter release. We hypothesized that inhibition of neurotransmitter release plays a key role in how anesthetics produce anesthesia in animals and humans. Furthermore we hypothesized that drugs that reverse the inhibitory effects of anesthetics on the release machinery should reverse anesthesia. Historically, cAMP signaling has been shown to play a key role in synaptic function and plasticity. Elevating cAMP facilitates neurotransmitter release. We posited that elevating intracellular cAMP might alter anesthetic action by restoring neurotransmitter release. Three drugs that elevate [cAMP]i levels, were tested; these drugs were found to completely reverse the inhibitory effects of anesthetics on neurotransmitter release in in vitro studies. When tested, these same cAMP elevating drugs dramatically accelerated recovery from anesthesia in rats. The most effective drug tested was caffeine which dramatically accelerated recovery from anesthesia (isoflurane and propofol) at relatively modest concentrations. In addition to elevating [cAMP]i, caffeine also inhibits adenosine receptors. A2A receptors mediate caffeine's arousal effects since knocking out this receptor or blocking it pharmacologically suppresses caffeine mediated arousal. It is possible that caffeine's ability to inhibit adenosine receptors helps it to reverse anesthesia. The goals of this application are: 1) Blinded Behavioral Studies in Mice and Rats - a) Determine whether A2A receptors play a role in reversing anesthesia. b) Determine whether caffeine works for all anesthetics. c) Determine optimal caffeine timing for anesthesia reversal. 2) Blinded Studies in Human Volunteers - a) Determine whether caffeine accelerates recovery from anesthesia and whether it accelerates recovery of cognitive abilities. b) Determine the optimal caffeine concentration and timing for anesthesia reversal. c) Determine whether caffeine is effective for all anesthetics. If caffeine accelerates recovery from anesthesia and restore cognitive abilities, then it may have the potential to impact medicine in a positive manner and in a brief time frame.