R Alan North - US grants

The objectives of the proposed research are to understand the primary action of narcotic analgesics and the way in which these alter during the development of tolerance and physical dependence. Intracellular recordings will be made from neurons in the myenteric plexus of the guinea-pig ileum. Acute actions of opiates and opioid peptides will be studied on single membrane ion channels using intracellular voltage clamp (single electrode) combined with extracellular patch clamp recording. The effects of known concentrations of various opiate receptor subtype selective ligands will be applied. The role of intracellular and extracellular calcium ions will be assessed by applying calcium selectively to the inside of the plasma membrane using liposomes. Effects of opiates on transmitter release will be determined by recording synaptic potentials resulting from activity in single fibres. The longer term actions of opiates will be studied by repeating these experiments in neurons removed from guinea-pigs treated with morphine during several days. In addition, adult neurons will be maintained for several days or weeks as a primary explant culture, either with or without morphine. The results are expected to elucidate the primary mechanism of action of narcotic analgesics and opioid peptides on the membrane of excitable cells in both naive and drug dependent animals, and to provide insight into the way in which opiates exert their pharmacological effects and induce tolerance and dependence.

The nucleus locus coeruleus is a rather uniform collection of noradrenaline containing nerve cells. In the rat, there are about 1500 on each side, approximately half of the noradrenaline containing cells in the brain. These cells have been implicated in several of the acute actions of opioids, and their increased activity during withdrawal from opioids may contribute to several of the physical signs of withdrawal. Our previous work has determined that rat locus coeruleus neurones express Mu receptors; there is no evidence for delta or kappa receptors. Activation of the Mu receptors on these cells results in an increase in membrane potassium conductance. The objective of the proposed experiments is to determine the properties of this potassium conductance at the molecular level, to determine how this conductance is linked to the opioid receptor through a guanine nucleotide binding protein (G-protein), and to investigate the changes in this coupling which accompany, or cause, tolerance to the action of opioids. Electrophysiological methods will be used (whole cell recording with intracellular perfusion, and single channel recording) on cells dissociated from the locus coeruleus of rats and maintained in culture for periods of up to several days. The effects of full and partial agonists at the Mu receptor, with and without experimental manipulation of the coupling G protein will be studied. These experiments will be carried out on neurons removed from normal rats, neurons removed from morphine- treated rats, and on neurons removed from normal rats and maintained in primary culture for several days with and without morphine. The experiments form a test of the hypothesis that the Mu receptors couple directly through a G protein to a potassium channel, and impairment of this coupling is the primary defect in opioid tolerance.

Brain amines have become increasingly implicated in several types of mental illness, either the amine-containing cells themselves or discrete populations of target cells which bear receptors for amines. The proposed studies take advantage of two properties of these amine-containing cells to investigate the cellular consequences of activation of the various amine receptors in mammalian brain. First, the cell bodies of the neurones are densely clustered. Second, the cells themselves bear receptors for the amines which they release (autoreceptors). This enables a study to be made of the actions of the amine transmitters on the membrane of the same cells that produce them. Intracellular recordings will be made from single neurones in slices of rat midbrain superfused at 37 degrees C. Three series of experiments will be conducted - involving the 5-hydroxytryptamine (5-HT) cells of the dorsal raphe, the noradrenaline (NA) containing cells of the locus coeruleus and the dopamine (DA) cells of the substantia nigra. First, the ion channels affected by the natural agonists 5-HT, NA and DA will be determined by single-electrode voltage clamp experiments. In these experiments, stable analogues of the natural amines which are selective for particular receptor sub-types will be used. Second, receptors on the neurones will be characterized by pharmacological null methods at equilibrium (antagonist affinities by the Schild method and agonist affinities by the Furchgott technique). Third, the intracellular second messengers (linkage) which couple receptor activation to ion channel will be investigated. Fourth, transmitter release will be measured from the single tissue slice in vitro using tritium labeling techniques. The action of various agonists on transmitter release will be correlated with their actions on membrane conductances measured simultaneously. The proposed studies will significantly increase our understanding of mental illness because the receptors for a variety of psychomimetic and psychotherapeutic agents will be characterized on intact, single brain neurones and because the consequences of receptor activation or blockade will be determined at the level of membrane ion channel and neurotransmitter release.

The purpose of the proposed research is to understand more about the function of the enteric nervous system, and particularly the way in which it controls the motility and absorptive functions of gastrointestinal tract. The experimental approach will be electrophysiological, by making intracellular recordings from neurons lying in the myenteric and submucous plexuses of the guinea-pig small intestine. Three levels of approach are planned. The first is a study of the detailed properties of the individual neurons, particularly their membrane ion channels, their cell surface receptors (particularly for catecholamines and acetylcholine), and their intracellular second messengers which couple receptors to ion channels. The second is an investigation of the way in which the nerve cells are synaptically connected; this will be done by observing the effects of surgical lesions, and by recording from pairs of neurons at the same time. The third approach is to record from single cells in circumstances in which they are more or less intact in the wall of the intestine, and responsive to the physiological synaptic inputs during reflex activity. This may allow us to construct a model for the way in which the nerve cells control peristalsis. The health related significance of this work derives from the directness of its approach to the function of gastrointestinal nerves. The enteric nervous system is involved in primary disease states, and also contributes to the pathophysiology of a wide range of conditions in which it is not primarily involved. These range from peptic ulcer to irritable bowel syndrome, from dysphagia to diabetes. The long term objective of the work is to understand the role of the enteric nervous system and the way in which it contributes to these pathological conditions.

The overall aim of the experiments proposed is an increased understanding of the actions of opioids at the cellular and molecular level; this understanding is sought so that the full therapeutic potential of this class of drugs can be safely exploited, and so that the adverse effects of the drugs such as tolerance and dependence can be managed and even prevented. The work is predicated on prior knowledge that opioids affect nerve cells by acting on three or more distinct receptor types; the specific aim is to determine the consequences for individual nerve cells of activation of these receptors by endogenous and exogenous opioids and how these consequences change with long term exposure. The methods used will be primarily electrophysiological - intracellular recording of membrane ion currents using single electrode voltage clamp and patch clamp techniques. The currents of single cells will be separated by their voltage and time dependence, and by the effects of channel blockers. The cells are chosen for study (guinea-pig myenteric and submucous plexus neurons) because earlier work has shown them to express mu, delta, and kappa receptors. Agonists and antagonists will be applied to the cells in known concentrations under steady-state conditions, or instantaneously so as to measure the time course of opioid action. The nerve cells will be maintained in vitro, with experimental recording carried out during the first few hours after removal from the animal, or after up to three weeks in cell culture. Hypotheses to be tested are that (i) mu and delta receptors activate a potassium conductance by a direct mechanism not involving changes in intracellular calcium, (ii) that kappa agonists depress a single class of calcium conductance, (iii) that cell processes from which transmitter is released express opioid receptors, and that consequences of their activation can be assessed by Fura-two measurements of intracellular calcium, and that (iv) chronic treatment of guinea-pigs with morphine results in tolerance to actions at mu receptors, but not at delta or kappa receptors.

DESCRIPTION (Adapted from applicant's abstract): It is widely acknowledged that dopamine-containing neurons with cell bodies in the midbrain and health. The proposed work is an investigation of the properties and connections of these neurons in slices of brain tissue removed from rats and maintained in vitro. Electrical recordings will be made from neurons of the ventral tegmental area, the region of dopamine cell bodies, and the nucleus accumbens, an important limbic projection area. Intracellular whole-cell and single channel recording will be used as appropriate; currents through membrane ion channels will be isolated an measured, and excitatory and inhibitory synaptic inputs will be evoked. Particular emphasis will be accorded to investigating the actions of 5-HT and CCK since current theories of schizophrenia and anxiety hypothesize that the actions of these transmitters at 5-HT3 and CCK-B receptors may be involved. The primary objective of this project is to test nine hypotheses. Hypotheses about dopamine cells of the VTA" (1) The firing of dopamine cells is determined by the intrinsic conductance I-H; (2) Dopamine neurons receive strong GABA-A synaptic inputs from local interneurons; (3) 5-HT excites dopamine cells by inhibiting GABA release at GABA-B synapses; (4) 5-HT excites dopamine cells by activating 5-HT3 receptors; (5) CCK excites dopamine cells by acting at CCK-B receptors; and (6) CCK closes the same potassium channels that dopamine opens (at D2 receptors). Hypotheses regarding target cells of the nucleus accumbens: (7) 5-HT excites accumbens neurons by acting at 5-HT3 receptors; (8) CCK excites accumbens neurons by reducing membrane potassium conductance; (9) Dopamine acts at D3 receptors to depolarize some accumbens neurons.

The overall aim of the experiments proposed is an increased understanding of the actions of opioids at the cellular and molecular level; this understanding is sought so that the full therapeutic potential of this class of drugs can be safely exploited, and so that the adverse effects of the drugs such as tolerance and dependence can be managed and even prevented. The work is predicated on prior knowledge that opioids affect nerve cells by acting on three or more distinct receptor types; the specific aim is to determine the consequences for individual nerve cells of activation of these receptors by endogenous and exogenous opioids and how these consequences change with long term exposure. The methods used will be primarily electrophysiological - intracellular recording of membrane ion currents using single electrode voltage clamp and patch clamp techniques. The currents of single cells will be separated by their voltage and time dependence, and by the effects of channel blockers. The cells are chosen for study (guinea-pig myenteric and submucous plexus neurons) because earlier work has shown them to express mu, delta, and kappa receptors. Agonists and antagonists will be applied to the cells in known concentrations under steady-state conditions, or instantaneously so as to measure the time course of opioid action. The nerve cells will be maintained in vitro, with experimental recording carried out during the first few hours after removal from the animal, or after up to three weeks in cell culture. Hypotheses to be tested are that (i) mu and delta receptors activate a potassium conductance by a direct mechanism not involving changes in intracellular calcium, (ii) that kappa agonists depress a single class of calcium conductance, (iii) that cell processes from which transmitter is released express opioid receptors, and that consequences of their activation can be assessed by Fura-two measurements of intracellular calcium, and that (iv) chronic treatment of guinea-pigs with morphine results in tolerance to actions at mu receptors, but not at delta or kappa receptors.