Christophe Mulle

Calcium influx through nicotinic receptor in rat central neurons: Its relevance to cellular regulation

The Ca2+ permeability of a nicotinic acetylcholine receptor (nAChR) in the rat CNS was determined using both current and fluorescence measurements on medial habenula neurons. The elementary slope conductance of the nAChR channel was 11 pS in pure external Ca2+ (100 mM) and 42 pS in standard solution. Ca2+ influx through nAChRs resulted in the rise of cytosolic Ca2+ concentration ([Ca2+]i) to the micromolar range. This increase was maximal under voltage conditions (below -50 mV) in which Ca2+ influx through voltage-activated channels was minimal. Ca2+ influx through nAChRs directly activated a Ca(2+)-dependent Cl- conductance. In addition, it caused a decrease in the GABAA response that outlasted the rise in [Ca2+]i. These results underscore the physiological significance of Ca2+ influx through nAChR channel in the CNS.

Potentiation of nicotinic receptor response by external calcium in rat central neurons

Nicotinic acetylcholine receptor (nAChR) responses of rat medial habenular neurons are potentiated up to 3.5-fold by increasing the concentration of external Ca2+ in the millimolar range. This effect, independent of voltage, is probably due to the binding of Ca2+ to an external site. External Ca2+ decreases nAChR single-channel conductance at negative but not positive potentials, and it markedly enhances the frequency of opening of acetylcholine activated channels. The potentiating effect of Ca2+ is mimicked by Ba2+ and Sr2+, but barely by Mg2+. These data support the existence of positively acting allosteric sites for Ca2+, distinct from those involved in the decrease of single-channel conductance. A model in which external Ca2+ changes the properties of activation of the nAChR appears consistent with these data.

Existence of different subtypes of nicotinic acetylcholine receptors in the rat habenulo-interpeduncular system

Neuronal nicotinic ACh receptors (nAChRs) are present in the rat medial habenula (MHB) and interpeduncular nucleus (IPN), two brain regions connected through the fasciculus retroflexus (FR). The goal of the present study was to compare the electrophysiological and pharmacological characteristics of nAChRs located at pre- and postsynaptic sites within the MHB-IPN system. nAChRs located on the soma of IPN neurons were studied using patch-clamp techniques and a preparation of acutely isolated neurons. Whole-cell currents evoked by Ach and nicotine showed an intense rectification at positive membrane potentials. nAChR channels were relatively nonselective for cations, had a unitary conductance of 35 pS, and were activated by several nicotinic agonists with the following rank order: cytisine greater than ACh greater than nicotine greater than dimethylphenylpiperazinium (DMPP). They were blocked by mecamylamine, hexamethonium, curare, and dihydro-beta-erythroidine (DHBE), but were insensitive to alpha- bungarotoxin and neuronal bungarotoxin. In contrast, nAChRs recorded on the soma of MHB neurons under equivalent experimental conditions exhibited different characteristics for single-channel conductance and agonist and antagonist sensitivity. The pharmacological properties of presynaptic nAChRs in the IPN were analyzed in a rat brain slice preparation. Stimulation of the FR produced a presynaptic afferent volley recorded in the rostral subnucleus of the IPN. Nicotinic agonists decreased the amplitude of the afferent volley with different efficacies: nicotine greater than cytisine greater than ACh greater than DMPP. The action of nicotine was insensitive to alpha-bungarotoxin and to neuronal bungarotoxin, but was blocked by mecamylamine, hexamethonium, curare, and DHBE, with IC50 values different from those reported for IPN postsynaptic nAChRs. This study thus demonstrates the functional diversity of nAChRs in the rat CNS.

Peripheral maps and synapse elimination in the cerebellum of the rat.I. Representation of peripheral inputs through the climbing fiber pathway in the posterior vermis of the normal adult rat

The present study gives a detailed description of the functional characteristics and of the topographic distribution of responses, mediated through the climbing fiber pathway and elicited by electrical stimulation of several peripheral inputs, in a circumscribed region of the posterior vermis of the rat cerebellum. Experiments were carried out on normal adult rats under urethane anaesthesia. Purkinje cells (PCs) which responded to the electric stimulation of the contralateral snout, of the ipsilateral and contralateral hindlimb or forepad, and of the tail were recorded in an area extending 1000 microns laterally to the midline in the vermal part of lobules VII and VIII. Using precise micromapping techniques and computer analysis, we located the cells on the map of the unfolded PC layer. Taking into account the mean latency of the responses and the probability of discharge of the PCs, restricted areas of projection were found for the snout, the forepads and the tail. Zones of short-latency responses form compact patches of less than 1 mm2. There was some overlap of projection zones from tail and snout and from forepads and snout. In these zones, there was a convergence of several peripheral inputs on some of the PCs tested. No precise projection of the hindlimbs could be detected in the same lobules. These results fit well with the hypothesis already proposed that the representation of peripheral inputs through the climbing fiber pathway is fractured into a mosaic of patches, which are partly overlapping, and in which remote parts of the body are represented in adjacent areas.

The effects of QX314 on thalamic neurons

The effects of QX314 were studied in cat thalamic neurons recorded in vivo. Besides blocking Na+ spike electrogenesis, QX314 transformed spindle oscillations into a single long-lasting period of hyperpolarization that terminated with a rebound Ca2+ spike. Large sustained injections of the drug resulted in the occurrence of numerous fast prepotentials that had the characteristics of attenuated dendritic Ca2+ spikes. These effects are interpreted as resulting from the blockage by QX314 of a persistent Na+ current and a delayed rectifier K+ current which currents have already been disclosed in thalamic neurons recorded in vitro.

Electrophysiology of Neuronal Nicotinic Receptors in the CNS

We have analyzed the properties of nicotinic acetylcholine receptors (nAChRs) in several regions of rat and mouse central nervous system. We have demonstrated the existence of several functionally distinct subtypes of nAChRs. A direct role of nAChR in synaptic transmission could not be found under our experimental conditions and in these preparations. However, we have shown that nAChRs acting at a preterminal (tetrodotoxin-sensitive) or at a presynaptic (tetrodotoxin-insensitive) level can modulate GABAergic synaptic transmission in several brain regions.

Neuronal, trophic and endocrine effects of nicotine

A growing body of evidence indicates that nicotine, acting at fetal CNS nicotinic cholinergic receptors, produces neurobehavioral teratogenesis. By contrasting acute nicotine injections to pregnant rats with continuous infusions (implanted minipumps), we have been able to separate the contributing variables of hypoxia/ischemia, maternal nutrition and fetal growth retardation from the direct effects of nicotine exerted on developing neurons, Nicotine leads to premature arrest of neural cell replication, an effect mimicking the natural actions of acetylcholine as a neurotrophic factor controlling differentiation of cholinergic target cells. With exogenous nicotine administration, this trophic effect is elicited at an incorrect time, leading to aberrant macromolecule synthesis and lasting changes in activity of a variety of neurotransmitter pathways. Because nicotinic receptors also play similar roles in peripheral pathways, we have been able to demonstrate clear-cut functional deficits in cardiac-sympathetic activity in animals exposed to nicotine prenatally, characterized by deficiencies in sympathetic tone, in I3-adrenergic receptor binding sites and in heart rate responses to adrenergic agonists or electrical stimulation of sympathetic nerves. One important implication of neurotransmitter receptors as a target for trophic effects on the immature nervous system is that “classical” teratologic indices, such as growth retardation, may be inappropriate markers of developmental neurotoxicity, since receptor-mediated actions typically occur at drug doses orders of magnitude below those for nonselective toxic events. For nicotine, doses that do not interfere with maternal or fetal weight gain, and that do not evoke any signs of general toxicity, are still fully effective in perturbing CNS cell development and synaptic function.

Regulation of the Desensitization of the Acetylcholine Nicotinic Receptor

Among the several mechanisms which may potentially control synapse efficacy, several prominent ones have been assigned to the functional properties of receptors for neurotransmitters. Up to now, two types of receptors for neurotransmitters have been characterized: the ligand-gated channels are involved in fast synaptic transmission and the receptors coupled to a G-protein which indirectly regulate the opening ionic channels are involved in slow synaptic transmission.