Philippe Fossier

Cholinergic modulation of the cortical neuronal network.

Acetylcholine (ACh) is an important neurotransmitter of the CNS that binds both nicotinic and muscarinic receptors to exert its action. However, the mechanisms underlying the effects of cholinergic receptors have still not been completely elucidated. Central cholinergic neurons, mainly located in basal forebrain, send their projections to different structures including the cortex. The cortical innervation is diffuse and roughly topographic, which has prompted some authors to suspect a modulating role of ACh on the activity of the cortical network rather than a direct synaptic role. The cholinergic system is implicated in functional, behavioural and pathological states including cognitive function, nicotine addiction, Alzheimers disease, Tourettes syndrome, epilepsies and schizophrenia. As these processes depend on the activation of glutamatergic and GABAergic systems, the cholinergic terminals must exert their effects via the modulation of excitatory and/or inhibitory neurotransmission. However, the understanding of cholinergic modulation is complex because it is the result of a mixture of positive and negative modulation, implying that there are various types, or even subtypes, of cholinergic receptors. In this review, we summarize the current knowledge on central cholinergic systems (projections and receptors) and then aim to focus on the implications for ACh in the modulation of cortical neuronal activity.

Cyclic ADP‐ribose and calcium‐induced calcium release regulate neurotransmitter release at a cholinergic synapse of Aplysia

1 Presynaptic injection of cyclic ADP‐ribose (cADPR), a modulator of the ryanodine receptor, increased the postsynaptic response evoked by a presynaptic spike at an identified cholinergic synapse in the buccal ganglion of Aplysia californica. 2 The statistical analysis of long duration postsynaptic responses evoked by square depolarizations of the voltage‐clamped presynaptic neurone showed that the number of evoked acetylcholine (ACh) quanta released was increased following cADPR injection. 3 Overloading the presynaptic neurone with cADPR led to a transient increase of ACh release followed by a depression. 4 cADPR injections did not modify the presynaptic Ca2+ current triggering ACh release. 5 Ca2+ imaging with the fluorescent dye rhod‐2 showed that cADPR injection rapidly increased the free intracellular Ca2+ concentration indicating that the effects of cADPR on ACh release might be related to Ca2+ release from intracellular stores. 6 Ryanodine and 8‐amino‐cADPR, a specific antagonist of cADPR, decreased ACh release. 7 ADP‐ribosyl cyclase, which cyclizes NAD+ into cADPR, was present in the presynaptic neurone as shown by reverse transcriptase‐polymerase chain reaction experiments. 8 Application of NAD+, the substrate of ADP‐ribosyl cyclase, increased ACh release and this effect was prevented by both ryanodine and 8‐amino‐cADPR. 9 These results support the view that Ca2+‐induced Ca2+ release might be involved in the build‐up of the Ca2+ concentration which triggers ACh release, and thus that cADPR might have a role in transmitter release modulation.

Nitric oxide transforms serotonin into an inactive form and this affects neuromodulation.

Nitric oxide is a highly reactive molecule, diffusible and therefore ubiquitous in the central nervous system. Consequently, nitric oxide or nitric oxide-derived nitrogen oxides must enter into contact with neuromodulators and they can modify these molecules, especially monoamines, and thus change their regulatory action on synaptic transmission. We tested this possibility on a well-known, identified cholinergic synapse of Aplysia buccal ganglion, in which we have found that evoked acetylcholine release was decreased by extracellularly applied serotonin. We show that this modulatory effect of serotonin was largely reduced not only in the presence of 3-morpholinosydnonimine, a nitric oxide donor, but also when endogenous nitric oxide synthase was activated. We have shown that this decrease in the serotonin effect is due to the formation of chemical derivatives of serotonin, mainly a symmetric serotonin dimer, 4-nitroso-serotonin and 4-nitro-serotonin, which are ineffective in reproducing the modulatory effect of serotonin. Serotonin is involved in the regulation of several central functions, such as sleep-wake activity or mood. The consequences of chemical modifications of serotonin by nitric oxide must be taken into account in physiological as well as pathological situations. In addition, our results highlight the importance of the physiological implications of interactions between free radicals and neuromediators in the nervous system.

Regulatory roles of complexins in neurotransmitter release from mature presynaptic nerve terminals

Complexins are presynaptic proteins whose functional roles in synaptic transmission are still unclear. In cultured rat hippocampal neurons, complexins are distributed throughout the cell bodies, dendrites and axons, whereas synaptotagmin I and synaptobrevin/VAMP‐2, essential proteins for neurotransmitter release, accumulated in the synaptic‐releasing sites as early as 1 week in culture. With a maturation of synapses in vitro, complexins also accumulated in the synaptic release sites and co‐localized with synaptotagmin I and synaptobrevin/VAMP‐2 after 3–4 weeks in culture. Complexins I and II were expressed in more than 90 and 70% of the cultured neurons, respectively; however, they were largely distributed in different populations of synaptic terminals. In the developing rat brain, complexins were distributed in neuronal cell bodies in the early stage of postnatal development, but gradually accumulated in the synapse‐enriched regions with development. In mature presynaptic neurons of Aplysia buccal ganglia, injection of anticomplexin II antibody caused a stimulation of neurotransmitter release. Injection of recombinant complexin II and αSNAP caused depression and facilitation of neurotransmitter release from nerve terminals, respectively. The effect of complexin was reversed by a subsequent injection of recombinant αSNAP, and vice versa. These results suggest that complexins are not essential but have some regulatory roles in neurotransmitter release from presynaptic terminals of mature neurons.

Nitric Oxide and l-Arginine Cause an Accumulation of Utrophin at the Sarcolemma: A Possible Compensation for Dystrophin Loss in Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD), a severe X-linked recessive disorder which results in progressive muscle degeneration, is due to a lack of dystrophin, a membrane cytoskeletal protein. An approach to treatment is to compensate for dystrophin loss with utrophin, another cytoskeletal protein with over 80% homology with dystrophin. Utrophin is expressed, at the neuromuscular junction, in normal and DMD muscles and there is evidence that it may perform the same cellular functions as dystrophin. So, the identification of molecules or drugs that could up-regulate utrophin is a very important goal for therapy. We show that in adult normal and mdx mice (an animal model of Duchenne myopathy) treated with l-arginine, the substrate of nitric oxide synthase (NOS), a pool of utrophin localized at the membrane appeared and increased, respectively. In normal and mdx myotubes in culture, l-arginine, nitric oxide (NO), or hydroxyurea increased utrophin levels and enhanced its membrane localization. This effect did not occur with d-arginine, showing the involvement of NOS in this process. The NO-induced increase in utrophin was prevented by oxadiazolo-quinoxalin-1-one, an inhibitor of a soluble guanylate cyclase implicated in NO effects. These results open the way to a potential treatment for Duchenne and Becker dystrophies.

Serotoninergic Fine-Tuning of the Excitation–Inhibition Balance in Rat Visual Cortical Networks

Fundamental brain functions depend on a balance between excitation (E) and inhibition (I) that is highly adjusted to a 20-80% set point in layer 5 pyramidal neurons (L5PNs) of rat visual cortex. Dysregulations of both the E-I balance and the serotonergic system in neocortical networks lead to serious neuronal diseases including depression, schizophrenia, and epilepsy. However, no link between the activation of neuronal 5-hydroxytryptamine receptors (5-HTRs) and the cortical E-I balance has yet been reported. Here we used a combination of patch-clamp recordings of composite stimulus-locked responses in L5PN following local electrical stimulations in either layer 2/3 or 6, simultaneous measurement of excitatory and inhibitory conductance dynamics, together with selective pharmacological targeting and single-cell reverse transcriptase-polymerase chain reaction. We show that cortical serotonin shifts the E-I balance in favor of more E and we reveal fine and differential modulations of the E-I balance between 5-HTR subtypes, in relation to whether layer 2/3 or 6 was stimulated and in concordance with the specific expression pattern of these subtypes in pyramidal cells and deep interneurons. This first evidence for the functional segregation of 5-HTR subtypes sheds new light on their coherent functioning in polysynaptic sensory circuits.

Ryanodine-, IP3- and NAADP-dependent calcium stores control acetylcholine release

Injections of inositol trisphosphate (IP3) or nicotinamide adenine dinucleotide phosphate (NAADP) into the presynaptic neurone of an identified cholinergic synapse in the buccal ganglion of Aplysiacalifornica increased the amplitude of the inhibitory postsynaptic current evoked by a presynaptic action potential. This suggests that Ca2+ release from various Ca2+ stores can modulate acetylcholine (ACh) release. Specific blockade of the calcium-induced calcium release (CICR) mechanism with ryanodine, or of IP3-induced calcium release with heparin, abolished the effects of IP3, but not the effects of NAADP, suggesting the presence of an intracellular Ca2+ pool independent of those containing ryanodine receptors (RyR) or IP3 receptors. To reinforce electrophysiological observations, intracellular [Ca2+]i changes were measured using the fluorescent dye rhod-2. Injections of cyclic ADP-ribose (an activator of RyR), IP3 or NAADP into the presynaptic neurone induced transient increases in the free intracellular Ca2+ concentration. RyR- and IP3-induced increases were prevented by application of respective selective antagonists but not NAADP-induced increases. Our results show that RyR-dependent, IP3-dependent, and NAADP-dependent Ca2+ stores are present in the same presynaptic terminal but are differently involved in the regulation of the presynaptic Ca2+ concentration that triggers transmitter release.

The V-ATPase membrane domain is a sensor of granular pH that controls the exocytotic machinery

The V0 membrane domain of the V-ATPase reversibly dissociates from V1 at acidic intragranular pH and is necessary for normal exocytosis and synaptic transmission.

Calcium transients and neurotransmitter release at an identified synapse

It is widely accepted that the modulation of the presynaptic Ca2+ influx is one of the main mechanisms by which neurotransmitter release can be controlled. The well-identified cholinergic synapse in the buccal ganglion of Aplysia has been used to study the modulations that affect presynaptic Ca2+ transients and to relate this to quantal evoked neurotransmitter release. Three types of Ca2+ channel (L, N and P) are present in the presynaptic neurone at this synapse. Influxes of Ca2+ through N- and P-type channels trigger the release of ACh with only N-type Ca2+ channels being regulated by presynaptic neuromodulator receptors. In addition, presynaptic Ca2+ stores, via complex mechanisms of Ca2+ uptake and Ca2+ release, control the Ca2+ concentration that triggers this evoked ACh release.

A nitric oxide synthase activity is involved in the modulation of acetylcholine release inAplysia ganglion neurons: A histological, voltammetric and electrophysiological study

Abstract The role of nitric oxide or related molecules as neuromodulators was investigated in the buccal and the abdominal ganglia of the mollusc Aplysia californica . In a first step we showed that reduced nicotinamide adenine dinucleotide phosphate-diaphorase histochemistry and specific nitric oxide synthase immunohistochemistry labelled the same neurons and fibres in both ganglia, pointing to the presence of a neuronal nitric oxide synthase. In a second step, we performed voltammetric detection of nitric oxide-related molecules using a microcarbon electrode in a reduction mode. A peak identified as N -nitroso- l -arginine was detected at −1.66 V in both ganglia. The identification of this compound as a product of endogenous nitric oxide synthase activity was reinforced by the fact that its peak amplitude was decreased in the presence of N G -monomethyl- l -arginine, an inhibitor of nitric oxide synthase, and increased with its substrate, l -arginine. An additional proof of a nitric oxide synthase activity was the detection of nitrites and nitrates in high concentrations (millimolar range) by capillary electrophoresis. We also showed that these nitric oxide-related molecules modulated acetylcholine release at two identified synapses in these ganglia. l -Arginine decreased acetylcholine release it the inhibitory synapse (buccal ganglion), whereas it increased acetylcholine release at the excitatory synapse (abdominal ganglion). The nitric oxide synthase inhibitors, N ω -nitro- l -arginine and N G -monomethyl- l -arginine, had opposite effects. Moreover, the exogenous nitric oxide donor, 3-morpholinosydnonimine hydrochloride mimicked the effects of l -arginine on both inhibitory and excitatory cholinergic synapses. The identification of two cholinergic synapses where nitric oxide affects acetylcholine release in opposite ways provides a useful tool to study the cellular mechanisms through which nitric oxide-related molecules modulate transmitter release.