No abstract

Mashour et al. review more than two decades of research on the global neuronal workspace theory of conscious processing, examine recent data related to unconscious states, and present a synthesis that links conscious access, attention, and working memory.

Nicotinic acetylcholine receptors (nAChRs) expressed by dopaminergic (DA) neurons have long been considered as potential therapeutic targets for the treatment of several neuropsychiatric diseases, including nicotine and cocaine addiction or Parkinson's disease. However, DA neurons express mRNAs coding for most, if not all, neuronal nAChR subunits, and the subunit composition of functional nAChRs has been difficult to establish. Immunoprecipitation experiments performed on mouse striatal extracts allowed us to identify three main types of heteromeric nAChRs (alpha4beta2*, alpha6beta2*, and alpha4alpha6beta2*) in DA terminal fields. The functional relevance of these subtypes was then examined by studying nicotine-induced DA release in striatal synaptosomes and recording ACh-elicited currents in DA neurons fromalpha4, alpha6, alpha4alpha6, and beta2 knock-out mice. Our results establish that alpha6beta2* nAChRs are functional and sensitive to alpha-conotoxin MII inhibition. These receptors are mainly located on DA terminals and consistently do not contribute to DA release induced by systemic nicotine administration, as evidenced by in vivo microdialysis. In contrast, (nonalpha6)alpha4beta2* nAChRs represent the majority of functional heteromeric nAChRs on DA neuronal soma. Thus, whereas a combination of alpha6beta2* and alpha4beta2* nAChRs may mediate the endogenous cholinergic modulation of DA release at the terminal level, somato-dendritic (nonalpha6)alpha4beta2* nAChRs most likely contribute to nicotine reinforcement.

A variety of ligand-gated ion channels undergo a fast activation process after the rapid application of agonist and also a slower transition towards desensitized or inactivated closed channel states when exposure to agonist is prolonged. Desensitization involves at least two distinct closed states in the acetylcholine receptor, each with an affinity for agonists higher than those of the resting or active conformations. Here we investigate how structural elements could be involved in the desensitization of the acetylcholine-gated ion channel from the chick brain alpha-bungarotoxin sensitive homo-oligomeric alpha 7 receptor, using site-directed mutagenesis and expression in Xenopus oocytes. Mutations of the highly conserved leucine 247 residue from the uncharged MII segment of alpha 7 suppress inhibition by the open-channel blocker QX-222, indicating that this residue, like others from MII, faces the lumen of the channel. But, unexpectedly, the same mutations decrease the rate of desensitization of the response, increase the apparent affinity for acetylcholine and abolish current rectification. Moreover, unlike wild-type alpha 7, which has channels with a single conductance level, the leucine-to-threonine mutant has an additional conducting state active at low acetylcholine concentrations. It is possible that mutation of Leu 247 renders conductive one of the high-affinity desensitized states of the receptor.

Allosteric regulation plays an important role in many biological processes, such as signal transduction, transcriptional regulation, and metabolism. Allostery is rooted in the fundamental physical properties of macromolecular systems, but its underlying mechanisms are still poorly understood. A collection of contributions to a recent interdisciplinary CECAM (Center Européen de Calcul Atomique et Moléculaire) workshop is used hereto provide an overview of the progress and remaining limitations in the understanding of the mechanistic foundations of allostery gained from computational and experimental analyses of real protein systems and model systems. The main conceptual frameworks instrumental in driving the field are discussed. We illustrate the role of these frameworks in illuminating molecular mechanisms and explaining cellular processes, and describe some of their promising practical applications in engineering molecular sensors and informing drug design efforts.

Although the neuronal nicotinic receptor alpha 6 subunit was cloned several years ago, its functional significance remains to be investigated. Here we describe an in situ hybridization study of the mRNA for this subunit in the adult rat central nervous system using oligonucleotide probes. Specific alpha 6 mRNA labelling was restricted to a few nuclei throughout the brain; it was particularly high in several catecholaminergic nuclei [the locus coeruleus (A6), the ventral tegmental area (A10) and the substantia nigra (A9)] at levels significantly higher than those found for any other known nicotinic receptor subunit mRNA. Labelling for alpha 6 mRNA was also detected at lower levels in the reticular thalamic nucleus, the supramammillary nucleus and the mesencephalic V nucleus. Some cells of the medial habenula (medioventral part) and of the interpeduncular nucleus (central and lateral parts) were also labelled. The distribution of alpha 6 mRNA was compared with the distribution of the other known nicotinic acetylcholine receptor subunit mRNAs. In several nuclei, the expression of alpha 6 was complementary to those of other alpha subunits. Moreover, some of the cell groups (such as the substantia nigra, the ventral tegmental area and the locus coeruleus) previously thought to contain mainly alpha 3 mRNA in fact were found to contain high levels of alpha 6 mRNA. Finally, we found extensive colocalization of alpha 6 and beta 3, indicating the possible existence of nicotinic receptor hetero-oligomers containing both subunits. The present results show that alpha 6 is the major nicotinic acetylcholine receptor alpha subunit expressed in dopaminergic cell groups of the mesencephalon and noradrenergic cells of the locus coeruleus. This suggests the involvement of the alpha 6 subunit in some of the major functions of central nicotinic circuits, including the modulation of locomotor behaviour and reward.

The relative permeability for sodium, potassium, and calcium of chicken a7 neuronal nicotinic receptor was investigated by mutagenesis of the channel domain M2.Mutations in the "intermediate ring" of negatively charged residues, located at the cytoplasmic end of M2 (site 1), reduce calcium permeability without significantly modifying other functional properties (activation and desensitization) of the receptor; a similar change of ion selectivity is also noticed when mutations at site 1 are done in the context of a receptor mutant that conducts ions in a desensitized state. Moreover, mutations of two adjacent rings of leucines at the synaptic end of M2 (site 2) have multiple effects. They abolish calcium permeability, increase the apparent affinity for acetylcholine by 10-to 100-fold, augment Hill numbers (up to 4.6-5.0) of acetylcholine dose-response relationships, slow rates of ionic response onset, and lower the extent of desensitization. Mutations at these two topographically distinct sites within M2 selectively alter calcium transport without affecting the relative permeabilities for sodium and potassium.Influx of calcium (Ca2+) through peripheral and brain nicotinic acetylcholine receptors (nAChRs) has been described in several preparations including muscle (1, 2) (23,27,28). In another study, the simultaneous introduction of three mutations in the M2 segment of the a7 receptor converted its ionic selectivity from cationic to anionic (14). For one of the mutationsThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.(E237A), indirect evidence suggested significant alterations of Ca2+ permeability (14).In this study, we further investigate the monovalent vs. divalent cation selectivity of the a7 receptor. We show that a mutation at the cytoplasmic end of M2 (E237A) abolishes Ca2+ permeability without significantly affecting other properties of the pharmacological and physiological responses to ACh. We further identify another site of two adjacent amino acids, close to the extracellular end of M2 (Leu-254 and Leu-255), where mutations reduce Ca2+ permeability and affect other physiological properties of the response such as its apparent affinity for ACh and the rate of currents onset and desensitization.MATERIALS AND METHODS Mutagenesis. Mutants were prepared as described (23,27). Their coding sequence was checked.Electrophysiology. Oocytes were prepared, injected, and recorded as described (29). For voltage-clamp measurements, cells were incubated in OR2 medium (solution B, Table 1) and challenged by ACh application. Data from one oocyte are given in the figures and values (mean ± SEM) determined from 5 to 10 oocytes from more than one donor are given in Table 2.Current-voltage (I-V) curves were obtained by subtracting passive membrane currents from currents evoked in the presence of ACh. ACh was applied at a concentration close to its EC50 value ...

In the mammalian visual system the formation of eye-specific layers at the thalamic level depends on retinal waves of spontaneous activity, which rely on nicotinic acetylcholine receptor activation. We found that in mutant mice lacking the ␤2 subunit of the neuronal nicotinic receptor, but not in mice lacking the ␣4 subunit, retinofugal projections do not segregate into eye-specific areas, both in the dorso-lateral geniculate nucleus and in the superior colliculus. Moreover, ␤2؊͞؊ mice show an expansion of the binocular subfield of the primary visual cortex and a decrease in visual acuity at the cortical level but not in the retina. We conclude that the ␤2 subunit of the nicotinic acetylcholine receptor is necessary for the anatomical and functional development of the visual system. I n the visual system of mammals, the projections of retinal ganglion cells (RGCs) from each eye are initially intermixed in their thalamic target, the dorso-lateral geniculate nucleus (dLGN), and subsequently segregate into eye-specific layers through an activity-dependent process (1-4). RGCs spontaneously fire periodic bursts of action potentials that sweep across the immature retina in a wave-like manner from embryonic age until eye opening, at the time retinogeniculate projections segregate (5-11). The mechanism of wave propagation is not fully understood. Yet, synaptic transmission mediated by nicotinic acetylcholine receptors (nAChRs) on RGCs appears to be necessary for the genesis of these waves and for the segregation process (12, 13).Neuronal nAChRs are pentameric ligand-gated ion channels encoded by a large multigene family consisting of at least eight ␣ (␣2-9) and three ␤ (␤2-4) subunit genes (14-16). These subunits associate into functional homopentamers (␣7, ␣8, or ␣9) or heteropentamers, most likely comprised of two ␣ and three ␤ subunits.In recent years, the availability of mutant mice lacking single nAChR subunits has circumvented the lack of subtype-specific agonists and antagonists and given new impetus to the study of nAChR physiology (17). These transgenic animals represent unique tools to study the role of nAChRs in complex circuits and the related functions or behaviors.In particular, in the case of retinal development, it has been shown that mice lacking the ␤2 subunit exhibit no correlated spontaneous activity in the retina during the first week of postnatal life, indicating that ␤2 subunit-containing nAChRs are critical in establishing this phenotype (18). The aim of the present paper is to exploit the facilities offered by the animal model of ␤2Ϫ͞Ϫ mice (19) to investigate the role of nAChRs in the development of the connectivity and related function of the visual system. In particular, we evaluate the hypothesis that the lack of spontaneous retinal activity in ␤2Ϫ͞Ϫ mice causes abnormalities in the development of retinogeniculate connections. As the distribution of ␤2 and ␣4 nAChR subunits largely overlaps in the brain and these subunits are thought to combine to form the predominant nAChR isoform in centra...