Muriel Amar

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.

The marine phycotoxin gymnodimine targets muscular and neuronal nicotinic acetylcholine receptor subtypes with high affinity

Gymnodimines (GYMs) are phycotoxins exhibiting unusual structural features including a spirocyclic imine ring system and a trisubstituted tetrahydrofuran embedded within a 16‐membered macrocycle. The toxic potential and the mechanism of action of GYM‐A, highly purified from contaminated clams, have been assessed. GYM‐A in isolated mouse phrenic hemidiaphragm preparations produced a concentration‐ and time‐dependent block of twitch responses evoked by nerve stimulation, without affecting directly elicited muscle twitches, suggesting that it may block the muscle nicotinic acetylcholine (ACh) receptor (nAChR). This was confirmed by the blockade of miniature endplate potentials and the recording of subthreshold endplate potentials in GYM‐A paralyzed frog and mouse isolated neuromuscular preparations. Patch‐clamp recordings in Xenopus skeletal myocytes revealed that nicotinic currents evoked by constant iontophoretical ACh pulses were blocked by GYM‐A in a reversible manner. GYM‐A also blocked, in a voltage‐independent manner, homomeric human α7 nAChR expressed in Xenopus oocytes. Competition‐binding assays confirmed that GYM‐A is a powerful ligand interacting with muscle‐type nAChR, heteropentameric α3β2, α4β2, and chimeric α7‐5HT3 neuronal nAChRs. Our data show for the first time that GYM‐A broadly targets nAChRs with high affinity explaining the basis of its neurotoxicity, and also pave the way for designing specific tests for accurate GYM‐A detection in shellfish samples.

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.

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.

Homeostatic control of the excitation-inhibition balance in cortical layer 5 pyramidal neurons

Homeostatic regulation in the brain is thought to be achieved through a control of the synaptic strength by close interactions between excitation and inhibition in cortical circuits. We recorded in a layer 5 pyramidal neuron of rat cortex the composite response to an electrical stimulation of various layers (2–3, 4 or 6). Decomposition of the global conductance change in its excitatory and inhibitory components permits a direct measurement of excitation–inhibition (E‐I) balance. Whatever the stimulated layer was, afferent inputs led to a conductance change consisting of 20% excitation and 80% inhibition. Changing synaptic strengths in cortical networks by using a high‐frequency of stimulation (HFS) protocol or a low‐frequency of stimulation (LFS) protocol (classically used to induce long‐term potentiation or long‐term depression at the synaptic level) were checked in order to disrupt this balance. Application of HFS protocols in layers 2–3, 4 or 6, or of LFS protocols in layer 4 induced, respectively, long‐term paralleled increases or long‐term paralleled decreases in E and I which did not change the E‐I balance. LFS protocols in layers 2–3 or 6 decreased E but not I and disrupted the balance. It is proposed that regulatory mechanisms might be mainly sustained by recurrent connectivity between excitatory and inhibitory neuronal circuits and by modulation of shunting GABAA inhibition in the layer 5 pyramidal neuron.

Involvement of Nicotinic and Muscarinic Receptors in the Endogenous Cholinergic Modulation of the Balance between Excitation and Inhibition in the Young Rat Visual Cortex

This study aims to clarify how endogenous release of cortical acetylcholine (ACh) modulates the balance between excitation and inhibition evoked in visual cortex. We show that electrical stimulation in layer 1 produced a significant release of ACh measured intracortically by chemoluminescence and evoked a composite synaptic response recorded intracellularly in layer 5 pyramidal neurons of rat visual cortex. The pharmacological specificity of the ACh neuromodulation was determined from the continuous whole-cell voltage clamp measurement of stimulation-locked changes of the input conductance during the application of cholinergic agonists and antagonists. Blockade of glutamatergic and gamma-aminobutyric acid (GABAergic) receptors suppressed the evoked response, indicating that stimulation-induced release of ACh does not directly activate a cholinergic synaptic conductance in recorded neurons. Comparison of cytisine and mecamylamine effects on nicotinic receptors showed that excitation is enhanced by endogenous evoked release of ACh through the presynaptic activation of alpha(*)beta4 receptors located on glutamatergic fibers. DHbetaE, the selective alpha4beta2 nicotinic receptor antagonist, induced a depression of inhibition. Endogenous ACh could also enhance inhibition by acting directly on GABAergic interneurons, presynaptic to the recorded cell. We conclude that endogenous-released ACh amplifies the dominance of the inhibitory drive and thus decreases the excitability and sensory responsiveness of layer 5 pyramidal neurons.

Alternative Splicing Controls Neuronal Expression of v-ATPase Subunit a1 and Sorting to Nerve Terminals

Vacuolar proton ATPase accumulates protons inside various intracellular organelles such as synaptic vesicles; its membrane domain V0 could also be involved in membrane fusion. These different functions could require vacuolar proton ATPases possessing different V0 subunit a isoforms. In vertebrates, four genes encode isoforms a1–a4, and a1 variants are also generated by alternative splicing. We identified a novel a1 splice variant a1-IV and showed that the two a1 variants containing exon C are specifically expressed in neurons. Single neurons coexpress a2, a1-I, and a1-IV, and these subunit a isoforms are targeted to different membrane compartments. Recombinant a2 was accumulated in the trans-Golgi network, and a1-I was concentrated in axonal varicosities, whereas a1-IV was sorted to both distal dendrites and axons. Our results indicate that alternative splicing of exon N controls differential sorting of a1 variants to nerve terminals or distal dendrites, whereas exon C regulates their neuronal expression.

Blockade of different muscarinic receptor subtypes changes the equilibrium between excitation and inhibition in rat visual cortex

We have shown that cortical acetylcholine modulates the balance between excitation and inhibition evoked in layer 5 pyramidal neurons of rat visual cortex [Lucas-Meunier E, Monier C, Amar M, Baux G, Frégnac Y, Fossier P (2009) Cereb Cortex 19:2411-2427]. Our aim is now to establish a functional basis for the role of the different types of muscarinic receptors (MRs) on glutamate fibers and on GABAergic interneurons and to analyse their contribution to the modulation of excitation-inhibition balance in the rat visual cortex. To ascertain that there was a basis for our functional study, we first checked for the presence of the various MR subtypes by single cell RT-PCR and immunolabeling experiments. Then, recording the composite responses in layer 5 pyramidal neurons to layer 1-2 stimulation (which also recruits cholinergic fibers) in the presence of specific antagonists of the different types of MR allowed us to determine their modulatory role. We show that the specific blockade of the widely distributed M1R (with the mamba toxin, MT7) induced a significant increase in the excitatory conductance without modifying the inhibitory conductance, pointing to a localization of M1R on glutamatergic neurons where their activation would decrease the release of glutamate. From our functional results, M2/M4Rs appear to be located on glutamatergic neurons afferent to the recorded layer 5 pyramidal neuron and they decrease glutamate release. The extended distribution of M4Rs in the cortex compared to the restricted distribution of M2R (layers 3-5) is in favour of a major role as a modulator of M4R. The selective antagonist of M3Rs, 4-DAMP, decreased the inhibitory conductance, showing that activated M3Rs increase the release of GABA and thus are located on GABAergic interneurons. The activation of the different types of MRs located either on glutamatergic neurons or on GABAergic interneurons converges to reinforce the dominance of inhibitory inputs thus decreasing the excitability of layer 5 pyramidal neurons.

Impaired GABAergic transmission disrupts normal homeostatic plasticity in rat cortical networks

In the cortex, homeostatic plasticity appears to be a key process for maintaining neuronal network activity in a functional range. This phenomenon depends on close interactions between excitatory and inhibitory circuits. We previously showed that application of a high frequency of stimulation (HFS) protocol in layer 2/3 induces parallel potentiation of excitatory and inhibitory inputs on layer 5 pyramidal neurons, leading to an unchanged excitation/inhibition (E/I) balance. These coordinated long‐term potentiations of excitation and inhibition correspond to homeostatic plasticity of the neuronal networks. We showed here, on the rat visual cortex, that blockade (with gabazine) or overactivation (with 4,5,6,7‐tetrahydroisoxazolo[5,4‐c]pyridin‐3‐ol) of GABAA receptors enhanced the E/I balance and prevented the potentiation of excitatory and inhibitory inputs after an HFS protocol. These impairements of the GABAergic transmission led to a long‐term depression‐like effect after an HFS protocol. We also observed that the blockade of inhibition reduced excitation (by 60%), and conversely, the blockade of excitation decreased inhibition (by 90%). These results support the idea that inhibitory interneurons are critical for recurrent interactions underlying homeostatic plasticity in cortical networks.

Involvement of NR2A-or NR2B-containing N-methyl-D-aspartate receptors in the potentiation of cortical layer 5 pyramidal neurone inputs depends on the developmental stage

In the cortex, N‐methyl‐d‐aspartate receptors (NMDARs) play a critical role in the control of synaptic plasticity processes. We have previously shown in rat visual cortex that the application of a high‐frequency stimulation (HFS) protocol used to induce long‐term potentiation in layer 2/3 leads to a parallel potentiation of excitatory and inhibitory inputs received by cortical layer 5 pyramidal neurones without changing the excitation/inhibition balance of the pyramidal neurone, indicating a homeostatic control of this parameter. We show here that the blockade of NMDARs of the neuronal network prevents the potentiation of excitatory and inhibitory inputs, and this result leaves open to question the role of the NMDAR isoform involved in the induction of long‐term potentiation, which is actually being strongly debated. In postnatal day (P)18–23 rat cortical slices, the blockade of synaptic NR2B‐containing NMDARs prevents the induction of the potentiation induced by the HFS protocol, whereas the blockade of NR2A‐containing NMDARs reduced the potentiation itself. In P29–P32 cortical slices, the specific activation of NR2A‐containing receptors fully ensures the potentiation of excitatory and inhibitory inputs. These results constitute the first report of a functional shift in subunit composition of NMDARs during the critical period (P12–P36), which explains the relative contribution of both NR2B‐ and NR2A‐containing NMDARs in synaptic plasticity processes. These effects of the HFS protocol are mediated by the activation of synaptic NMDARs but our results also indicate that the homeostatic control of the excitation/inhibition balance is independent of NMDAR activation and is due to specialized recurrent interactions between excitatory and inhibitory networks.