Yves Pichon

Electrophysiological analysis of the peripheral response to odours in wild type and smell-deficient olf C mutant of Drosophila melanogaster

Abstract A few smell deficient mutants of Drosophila have been characterized recently. In a series of experiments, we have compared the slow electrical response of the antenna (EAG) to calibrated concentrations of butanol and aliphatic n-acetates of olf C mutants with those of wild flies. This mutation was found to affect the dose-response curves specifically. Our data can be explained by assuming that this mutation affects the peripheral olfactory membrane at several levels.

Modifications of the ionic content of the haemolymph and of the activity of Periplaneta americana in relation to diet

Abstract The concentration of K + , Na + , and Ca 2+ ions in the haemolymph of Periplaneta americana is compared for insects fed on different diets. The Na + /K + ratio is the same when the insect is fed on milk powder or wheat flour; it is lower on a diet of lettuce, the sodium ion concentration decreasing and potassium increasing. The activity of insects fed on milk powder is greater than that of the insects fed on lettuce. This result can be interpreted as being due to modifications in the potassium ion concentration of the haemolymph.

Some aspects of the physiological role of ion channels in the nervous system

Recent analyses of the genomes of several animal species, including man, have revealed that a large number of ion channels are present in the nervous system. Our understanding of the physiological role of these channels in the nervous system has followed the evolution of biophysical techniques during the last century. The observation and the quantification of the electrical events associated with the operation of the ionic channels has been, and still is, one of the best tools to analyse the various aspects of their contribution to nerve function. For this reason, we have chosen to use electrophysiological recordings to illustrate some of the main functions of these channels. The properties and the roles of Na+ and K+ channels in neuronal resting and action potentials are illustrated in the case of the giant axons of the squid and the cockroach. The nature and role of the calcium currents in the bursting behaviour of the neurons are illustrated for Aplysia giant neurons. The relationship between presynaptic calcium currents and synaptic transmission is shown for the squid giant synapse. The involvement of calcium channels in survival and neurite outgrowth of cultured neurons is exemplified using embryonic cockroach brain neurons. This same neuronal preparation is used to illustrate ion channel noise and single-channel events associated with the binding of agonists to nicotinic receptors. Some features of the synaptic activity in the central nervous system are shown, with examples from the cercal nerve giant-axon preparation of the cockroach. The interplay of different ion conductances involved in the oscillatory behaviour of the Xenopus spinal motoneurons is illustrated and discussed. The last part of this review deals with ionic homeostasis in the brain and the function of glial cells, with examples from Necturus and squids.

Effects of nicotinic and muscarinic ligands on embryonic neurones of Periplaneta americana in primary culture: a whole cell clamp study

The pharmacological properties of acetylcholine (ACh) receptors of cultured neurones from embryonic cockroach brains were studied using the whole-cell configuration of the patch-clamp technique. More than 90% of the studied neurones responded to ACh by a monophasic inward current, the intensity of which varied from cell to cell. The sequence of potency of the five tested agonists was ACh > nicotine=carbamylcholine > suberyldicholine=oxotremorine. The dose-response relationship was complex, suggesting the existence of two populations of receptors: high-affinity receptors (extrapolated K(d) around 10(-7) M) and low-affinity receptors (extrapolated K(d) around 5x10(-5) M). The current-voltage relationship of the induced current was linear between -80 and -40 mV and the extrapolated reversal potential was not significantly different from 0 mV. The sequence of decreasing potency of the antagonists of the ACh response was: methyllycaconitine > alpha-bungarotoxin > mecamylamine > curare > strychnine > bicuculline > atropine > picrotoxin. These results show: (1) that, in embryonic brain neurones, the response to ACh corresponds to the opening of non-selective cationic channels; and (2) that the pharmacology of the ACh receptors is mainly but not solely nicotinic. The nature of the single events which underlie this response, as well as the structure of the channels (homo or hetero-oligomeric) remain to be investigated.

Properties and development of calcium currents in embryonic cockroach neurons.

In freshly dissociated neurons from embryonic cockroach (Periplaneta americana L.) brains, voltage-dependent calcium currents appear early in development (E14). Their intensity increases progressively during embryonic life until eclosion (E35). Their time course and voltage dependency are characteristic of high voltage activated (HVA) currents although a 10 mV shift of the I/V curve towards more negative potentials was observed between E18 and E23. Their sensitivity to omega-AgaTx-IVA and omega-CgTx-GVIA and insensitivity to both amiloride and isradipine indicate that the corresponding channels are of the P/Q and N types. These channels, as well as a small proportion of toxin-resistant (R) channels (about 20%), are blocked by mibefradil and verapamil. The physiological significance of these currents and their modifications during embryonic life is discussed.

Mutually exclusive blockage of sodium channels of myelinated frog nerve fibres by benzocaine and the indole alkaloid ervatamine

Abstract In frog nerve fibres the indole alkaloid ervatamine blocks sodium channels in a frequency-dependent fashion as studied in voltage clamp experiments. In the presence of ervatamine (3 μM), adding or taking away benzocaine (0.5 mM) leads to a non-monotonic further increase or relief of block. This strong interaction is interpreted as competition of the two drugs for the same binding site, the receptor of classical local anaesthetics.

In vitro development of P- and R-like calcium currents in insect (Periplaneta americana) embryonic brain neurons

Voltage-gated calcium currents are important for the survival and growth of embryonic cockroach brain neurons in primary culture. In the present experiments, we have studied, using the patch-clamp technique, the evolution with time in culture of the voltage-dependency and of the pharmacological properties of the calcium conductance of these neurons during the formation of a network. We have observed a progressive increase of the high-voltage-activated calcium conductance and a 10mV shift of the voltage-dependency of activation towards more negative potentials. The proportion of the R-like calcium current component increased during network formation. At the same time, the highly omega-AgaTxIVA-sensitive P-like component of the current is progressively replaced by a component which is less sensitive to the toxin. The origin and functional implications of these modifications are discussed.

Role of ligand-gated ion channels in the swimming behaviour of Xenopus tadpoles: experimental data and modelling experiments.

The swimming behaviour of lower vertebrates has been used as a model to study the function of simple neuronal circuits. Good examples are the lamprey and the Xenopus tadpole. In these two cases, glutamate-activated NMDA receptors are involved, and the relative importance of the NMDA and non-NMDA receptors as well as the involvement of other ion channels has been studied using a combination of electrophysiological recordings and modelling experiments, but little attention had been paid to their evolution during development. In the present experiments, which have been performed on Xenopus embryos from stages 31 to 42, we have probed the relative importance of the two categories of receptors using selective blockers [respectively dl-2-amino-5-phosphonovaleric acid (APV) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX)]. The sensitivity of the swimming behaviour to APV was found to increase during development and that to CNQX to decrease. Furthermore, it has been observed that the spike activity recorded from the ventral roots is more complex in late embryonic stages that in early embryos. These modifications are associated with changes of the neuronal circuit, some of which correspond to a lengthening of the axon and an increased complexity of the dendritic tree of the motoneurons. We have incorporated these modifications in a simplified model of the central pattern generator built with Neuron software. The results indicate that at least part of the observed changes can be associated with changes in the length of the dendrites and axons.

Ion channels: from biophysics to disorders

Biological membranes have been known for a long time to be permeable to ions and small molecules. This permeability reflects the function of specialized membrane proteins within the hydrophobic lipid bilayer. Some of these proteins, the ion channels, have received special attention, being responsible for the ionic currents underlying nerve and muscle excitability as first demonstrated by Hodgkin and Huxley (1952) for the squid giant axon. Since then, the nature of these ion channels and their mode of operation have been the object of intense research, and half of the 2003 Nobel Prize in chemistry was awarded to Roderick MacKinnon who unveiled, using X-ray crystallography, the 3D structure of a bacterial voltage-dependent potassium channel (Jiang et al, 2003). At the same time, an increasing number of reports have indicated that these same ion channels are involved in a variety of diseases called channelopathies. Furthermore, molecular biology has shown that the number of genes coding for ion channels is much higher than initially expected from electrophysiological recordings of the ionic currents, and the precise functions of the proteins encoded by these genes often remain to be discovered. Whereas it is still possible to discuss these different aspects at specialized meetings, the need for a broader view of this constantly moving field was becoming apparent. In this context, we organized, with the help of the British and French Biophysical Societies, an informal meeting entitled: ‘‘Ion Channels: from Biophysics to Disorders’’, which was held in Rennes (France) in May 2003 . This special issue of the European Biophysics Journal assembles the contributions of the participants who volunteered to take part in this follow-up of the meeting. Special care has been taken to present the results in a way that would be comprehensible by non-specialists. The first set of papers deals with model channels. In his contribution on artificial porous systems, C.L. Bashford summarizes the results of his investigations on the role of surface charges as a basic determinant of the conductance, selectivity, and fluctuation of the conducting states of ion channels. L. Whitmore and B.A. Wallace illustrate how one can extract from the peptaibol database the salient features of these peptide molecules related to their propensity to form ion channels. The third paper of this set is by H. Duclohier and illustrates how the bending of the transmembrane helices formed by three peptaibol molecules (alamethicin, trichotoxin, and antiamoebin) is correlated with different patterns of activity as recorded electrophysiologically using planar lipid bilayers. In the last contribution of this set, P. Llanos, M. Henriquez, J. Minic, K. Elmorjani, D. Marion, G. Riquelme, J. Molgo, and E. Benoit indicate that wheat endosperm proteins form ion pores when incorporated into giant liposomes. The second set of papers concerns emerging studies on the essential structural features of ion channels. In the first paper, D. Doyle summarizes some of the lessons or common structural themes that can be drawn after 5 years of crystallographic studies of ion channels, in particular from the inwardly rectifying bacterial potassium channel KirBac1.1. The second contribution, by J. Holyoake, C. Domene, J.N. Bright and M.S.P. Sansom, compares, using modelling and simulation studies, the H. Duclohier (&) Æ Y. Pichon Interactions Cellulaires et Moleculaires, UMR 6026 CNRS-Universite de Rennes I, Batiment 13, Campus de Beaulieu, 35042 Rennes, France E-mail: herve.duclohier@univ-rennes1.fr

Calcium currents in developing neurones from cockroach brains in primary culture

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