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.

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, Alzheimer's disease, Tourette's 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.

Chimeric green fluorescent protein-aequorin as bioluminescent Ca2+ reporters at the single-cell level.

Monitoring calcium fluxes in real time could help to understand the development, the plasticity, and the functioning of the central nervous system. In jellyfish, the chemiluminescent calcium binding aequorin protein is associated with the green fluorescent protein and a green bioluminescent signal is emitted upon Ca(2+) stimulation. We decided to use this chemiluminescence resonance energy transfer between the two molecules. Calcium-sensitive bioluminescent reporter genes have been constructed by fusing green fluorescent protein and aequorin, resulting in much more light being emitted. Chemiluminescent and fluorescent activities of these fusion proteins have been assessed in mammalian cells. Cytosolic Ca(2+) increases were imaged at the single-cell level with a cooled intensified charge-coupled device camera. This bifunctional reporter gene should allow the investigation of calcium activities in neuronal networks and in specific subcellular compartments in transgenic animals.

A conotoxin from Conus textile with unusual posttranslational modifications reduces presynaptic Ca2+ influx.

Cone snails are gastropod mollusks of the genus Conus that live in tropical marine habitats. They are predators that paralyze their prey by injection of venom containing a plethora of small, conformationally constrained peptides (conotoxins). We report the identification, characterization, and structure of a gamma-carboxyglutamic acid-containing peptide, conotoxin epsilon-TxIX, isolated from the venom of the molluscivorous cone snail, Conus textile. The disulfide bonding pattern of the four cysteine residues, an unparalleled degree of posttranslational processing including bromination, hydroxylation, and glycosylation define a family of conotoxins that may target presynaptic Ca2+ channels or act on G protein-coupled presynaptic receptors via another mechanism. This conotoxin selectively reduces neurotransmitter release at an Aplysia cholinergic synapse by reducing the presynaptic influx of Ca2+ in a slow and reversible fashion. The three-dimensional structure, determined by two-dimensional 1H NMR spectroscopy, identifies an electronegative patch created by the side chains of two gamma-carboxyglutamic acid residues that extend outward from a cavernous cleft. The glycosylated threonine and hydroxylated proline enclose a localized hydrophobic region centered on the brominated tryptophan residue within the constrained intercysteine region.