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Studies are presented of the interaction in a physiological ionic environment of aromatic amine local anesthetics (prilocaine, lidocaine, and dimethisoquin) and Ca++ with receptor-rich membrane fragments isolated from Torpedo electric organ. The environmentaly sensitive fluorophore 1-(5-dimethylaminonaphthalene-1-sulfonamido)ethane 2-trimethylammonium iodide (DNS-chol)interacts with two classes of sites in the membrane fragments: the cholinergic receptor site and secondary sites characterized by probe emission properties (λmax) sensitive to the pharmacological nature (agonist or antagonist) of the cholinergic ligand bound to the receptor site. Fluorescence studies show that the local anesthetics cause an increase of affinity of the membrane-bound receptor for DNS-chol and for cholinergic ligands, both agonists and antagonists. The increase of affinity is not associated with a change of DNS-chol emission properties. At the same concentrations at which the anesthetics control receptor affinity, they also affect the fluorescence of DNS-chol bound to the secondary sites: the presence of local anesthetic causes a loss of the DNS-chol spectral properties characteristic of the binding of agonists to the receptor site. Local anesthetics also control the binding of [3H]acetylcholine to the membrane-bound receptor. In the absence of prilocaine the acetylcholine binding curve is slightly sigmoid (Hill coefficient, nH = 1.4, half-saturation at 10 nM free acetylcholine). In the presence of 3 mM prilocaine there is a decrease of cooperativity and an increase of affinity (nH = 1.0, half-saturation at 6 mM free acetylcholine). The concentrations at which the local anesthetics act on the membrane fragments are those at which they block the permeability response of Electrophorus electroplax upon addition to the bath of the agonist carbamylcholine. Fluorescence and radioactive ligand assays demonstrate that Ca++ also causes an increase of receptor affinity for cholinergic ligands, but in a manner significantly different from that observed with local anesthetics. Solubilization of membrane fragments by detergent leads to changes in the binding properties of the receptor protein. On the membrane fragments the binding data for each agonist can be analyzed in terms of a homogeneous population of sites, while after solubilization heterogeneity of the binding constants appears. Prilocaine or Ca+ no longer affects the binding of acetylcholine to the solubilized receptor protein. The observed effects of local anesthetics and Ca++ on the affinity of the cholinergic receptor are related to the phenomenon of receptor desensitization.

Recent molecular, immunologic, and physiological studies have revealed that wide variety of nicotinic acetylcholine receptors exist in the nervous system of vertebrates. Nicotinic systems in the brain appear to play significant roles in drug addiction and in cognitive functions, as well as in pathologies, such as Alzheimer's disease.

We have described a strategy to analyze the data available on brain genes expression, using the concept of coherent-gene groups controlled by transcription factors (TFs). A hierarchical model of gene-expression patterns during brain development was established that identified the genes assumed to behave as functionally coding. Analysis of the concerned signaling pathways and processes showed distinct temporal gene-expression patterns in relation with neurogenesis/synaptogenesis. We identified the hierarchical tree of TF networks that determined the patterns of genes expressed during brain development. Some 'master TFs' at the top level of the hierarchy regulated the expression of gene groups. Enhanced/decreased activity of a few master TFs may explain paradoxes raised by the genetic determination of autism-spectrum disorders and schizophrenia. Our analysis showed gene-TF networks, common or related, to these disorders that exhibited two maxima of expression, one in the prenatal and the other at early postnatal period of development, consistent with the view that these disorders originate in the prenatal period, develop in the postnatal period, and reach the ultimate neural and behavioral phenotype with different sets of genes regulating each of these periods. We proposed a strategy for drug design based upon the temporal patterns of expression of the concerned TFs. Ligands targeting specific TFs can be designed to specifically affect the pathological evolution of the mutated gene(s) in genetically predisposed patients when administered at relevant stages of brain development.

The nicotinic acetylcholine receptor is a transmembrane allosteric protein that mediates transduction of chemoelectric signals throughout the nervous system by opening an intrinsic ionic channel. This rapid pore opening enables flow of Na+, K+, and, in several instances, Ca2+ ions across the cell membrane. As a consequence, nicotinic acetylcholine receptors elicit fast changes in the membrane electric potential, but they also regulate transmission of electric signals by closing the pore through slower desensitization transitions. As such, nicotinic acetylcholine receptors play crucial physiological roles and, when altered, they cause pathologies in humans. This article discusses the functional organization of nicotinic acetylcholine receptors down to the atomic level.

We have examined several neurochemical and behavioural parameters related to the function of the mesolimbic dopamine (DA) pathway in animals treated with nicotine following three modes of drug administration, i.e. systemic intraperitoneal injection, intra-accumbens (Acb) infusion or intraventral tegmental area (intra-VTA) microinjection. The present modes of systemic, intra-Acb and intra-VTA nicotine administration elicited comparable acute increases in dialysate DA levels from the Acb. The increase in extracellular DA levels was paralleled by a significant enhancement of locomotion in a habituated environment in the case of systemic or intra-VTA nicotine administration, whereas unilateral or bilateral intra-Acb nicotine infusion was ineffective, showing that accumbal DA increase is not sufficient to elicit locomotion in this experimental paradigm. Intra-VTA, but not systemic or intra-Acb, nicotine administration caused a long-term (at least 24-h) increase in basal dialysate DA levels from the Acb. In addition, significant increases in tyrosine hydroxylase (TH) and GluR1 (but not dopamine transporter or NR1) mRNA levels in the VTA were detected 24 h after intra-VTA nicotine administration. Systemic nicotine injection caused only an increase in TH mRNA levels while intra-Acb infusion did not modify any of the mRNAs tested. The long-term increase in basal DA levels in the Acb and TH, and GluR1 mRNA levels in the VTA upon intra-VTA nicotine microinjection indicates that even a single nicotine injection can induce plastic changes of the mesolimbic DA pathway.

Nicotinic acetylcholine receptors (nAChR) composed of chick alpha7 subunits mutated to threonine at amino acid valine-251 in the putative channel-lining M2 domain were expressed heterologously in several neuron-like and non-neuronal mammalian cell lines. Expression of mutant alpha7-nAChR is toxic to neuron-like cells of the human neuroblastoma cell lines SH-SY5Y and IMR-32, but not to several other cell types. Growth in the presence of the alpha7-nAChR antagonist methyllycaconitine (MLA) protects against neurotoxicity, as does gradual downregulation of functional, mutant alpha7-nAChR in surviving transfected SH-SY5Y cells. Relative to wild-type alpha7-nAChR, functional alpha7-nAChR mutants show a higher affinity for agonists, slower rates of desensitization, and sensitivity to dihydro-beta-erythroidine (DHbetaE) as an agonist, but they retain sensitivity to MLA as a competitive antagonist. These findings demonstrate that expression of hyperfunctional, mutant forms of Ca2+-permeable alpha7-nAChR is toxic to neuron-like cells.

Acetylcholine release stimulated by nicotinic agonists was measured as radioactivity released from perfused synaptosomes prepared from mouse interpeduncular nucleus (IPN) that had been loaded with [(3)H]choline. Agonist-stimulated release was dependent upon external calcium and over 90% of released radioactivity was acetylcholine. The release process was characterized by dose response curves for 13 agonists and inhibition curves for six antagonists. alpha-Conotoxin MII did not inhibit this release, while alpha-conotoxin AuIB inhibited 50% of agonist-stimulated release. Comparison of this process with [(3)H]dopamine release from mouse striatal synaptosomes indicated that different forms of nicotinic acetylcholine receptors (nAChRs) may mediate these processes. This was confirmed by assays using mice homozygous for the beta 2 subunit null mutation. The deletion of the beta 2 subunit had no effect on agonist-stimulated acetylcholine release, but abolished agonist-stimulated release of dopamine from striatal synaptosomes. Mice heterozygous for the beta 2 subunit null mutation showed decreased dopamine release evoked by L-nicotine with no apparent change in EC(50) value, as well as similar decreases in both transient and persistent phases of release with no changes in desensitization rates.

Neuronal network models have been proposed for the organization of evaluation and decision processes in prefrontal circuitry and their putative neuronal and molecular bases. The models all include an implementation and simulation of an elementary reward mechanism. Their central hypothesis is that tentative rules of behavior, which are coded by clusters of active neurons in prefrontal cortex, are selected or rejected based on an evaluation by this reward signal, which may be conveyed, for instance, by the mesencephalic dopaminergic neurons with which the prefrontal cortex is densely interconnected. At the molecular level, the reward signal is postulated to be a neurotransmitter such as dopamine, which exerts a global modulatory action on prefrontal synaptic efficacies, either via volume transmission or via targeted synaptic triads. Negative reinforcement has the effect of destabilizing the currently active rule-coding clusters; subsequently, spontaneous activity varies again from one cluster to another, giving the organism the chance to discover and learn a new rule. Thus, reward signals function as effective selection signals that either maintain or suppress currently active prefrontal representations as a function of their current adequacy. Simulations of this variation-selection have successfully accounted for the main features of several major tasks that depend on prefrontal cortex integrity, such as the delayed-response test, the Wisconsin card sorting test, the Tower of London test and the Stroop test. For the more complex tasks, we have found it necessary to supplement the external reward input with a second mechanism that supplies an internal reward; it consists of an auto-evaluation loop which short-circuits the reward input from the exterior. This allows for an internal evaluation of covert motor intentions without actualizing them as behaviors, by simply testing them covertly by comparison with memorized former experiences. This element of architecture gives access to enhanced rates of learning via an elementary process of internal or covert mental simulation. We have recently applied these ideas to a new model, developed with M. Kerszberg, which hypothesizes that prefrontal cortex and its reward-related connections contribute crucially to conscious effortful tasks. This model distinguishes two main computational spaces within the human brain: a unique global workspace composed of distributed and heavily interconnected neurons with long-range axons, and a set of specialized and modular perceptual, motor, memory, evaluative and attentional processors. We postulate that workspace neurons are mobilized in effortful tasks for which the specialized processors do not suffice; they selectively mobilize or suppress, through descending connections, the contribution of specific processor neurons. In the course of task performance, workspace neurons become spontaneously co-activated, forming discrete though variable spatio-temporal patterns subject to modulation by vigilance signals and to selection by reward signals. A computer simulation of the Stroop task shows workspace activation to increase during acquisition of a novel task, effortful execution, and after errors. This model makes predictions concerning the spatio-temporal activation patterns during brain imaging of cognitive tasks, particularly concerning the conditions of activation of dorsolateral prefrontal cortex and anterior cingulate, their relation to reward mechanisms, and their specific reaction during error processing.

Nicotinic acetylcholine receptors (nAChRs) are expressed in the midbrain ascending dopaminergic system, a target of many addictive drugs. Here we assessed the intracellular Ca2+ level by imaging fura-2-loaded cells in substantia nigra pars compacta in mouse brain slices, and we examined the influence on this level of prolonged exposures to nicotine using mice lacking the nAChR beta2-subunit. In control cells, superfusion with nicotine (10-100 microM) caused a long-lasting rise of intracellular Ca2+ level which depended on extracellular Ca2+. This nicotinic response was almost completely absent in beta2-/- mutant mice, leaving a small residual response to a high concentration (100 microM) of nicotine which was inhibited by the alpha7-subunit-selective antagonist, methyllycaconitine. Conversely, the alpha7-subunit-selective agonist choline (10 mM) caused a methyllycaconitine-sensitive increase in intracellular Ca2+ level both in wild-type and beta2-/- mutant mice. Nicotine-elicited Ca2+ mobilization was reduced by the Na+ channel blocker tetrodotoxin (TTX) and by T-type Ca2+ channel blocking agents, whereas the choline-elicited Ca2+ increase was insensitive to TTX. Neither nicotine nor choline produced Ca2+ increase following inhibition of the release of Ca2+ from intracellular stores by dantrolene. These results demonstrate that in nigral dopaminergic neurons, nicotine can elicit Ca2+ mobilization via activation of two distinct nAChR subtypes: that of beta2-subunit-containing nAChR followed by activation of Na+ channel and T-type Ca2+ channels, and/or activation of alpha7-subunit-containing nAChR. The Ca2+ influx due to nAChR activation is subsequently amplified by the recruitment of intracellular Ca2+ stores. This Ca2+ mobilization may possibly contribute to the long-term effects of nicotine on the dopaminergic system.

Nicotinic acetylcholine receptors (nAChRs) are expressed in muscle, the central nervous system (CNS), and the peripheral nervous system (PNS). Nicotine, a specific agonist of these receptors, exerts diverse cellular and behavioural effects. Aside from its addictive properties, nicotine acts as a cognitive enhancer, an anxiolytic, an antinociceptive substance, and a seizure inducer. Pharmacological experiments with nicotinic agonists and antagonists have pharmacologically helped to elucidate the function of acetylcholine (ACh) and nAChRs in the CNS and in the periphery. However, selective ligands for the multiple isoforms of neuronal nAChRs are still scarce. A recent approach involves genetic manipulations in mice which result in “knockouts” with genomic null mutations. Specific genes encoding for receptors are deleted, thus, in essence, providing a highly selective, albeit irreversible, “antagonist”. Although there are some inherent problems in using this approach (developmental requirements or compensatory effects, for example), this genetic approach gives new insights into the pharmacology and functional role of neuronal receptors in complex neurobiological systems. In this chapter we will focus on knockout mice lacking a nAChR subunit that have allowed a molecular dissection of nAChR subtypes in the CNS and that have led to the identification of particular nAChR subunits involved in nicotine-elicited behaviours in addition to being used as models of several human pathologies.

We present here a systematic mapping of nAChR subunit mRNAs in Macaca mulatta brain. A fragment, from the transmembrane segments MIII to MIV of Macaca neuronal nAChR subunits was cloned, and shown to exhibit high identity (around 95%) to the corresponding human subunits. Then, specific oligodeoxynucleotides were synthesized for in situ hybridization experiments. Both alpha4 and beta2 mRNA signals were widely distributed in the brain, being stronger in the thalamus and in the dopaminergic cells of the mesencephalon. Most brain nuclei displayed both alpha4 and beta2 signals with the exception of some basal ganglia regions and the reticular thalamic nucleus which were devoid of alpha4 signal. alpha6 and beta3 mRNA signals were selectively concentrated in the substantia nigra and the medial habenula. The strongest signals for alpha3 or beta4 mRNAs were found in the epithalamus (medial habenula and pineal gland), whereas there were no specific alpha3 or beta4 signals in mesencephalic dopaminergic nuclei. alpha5 and alpha7 mRNA signals were found in several brain areas, including cerebral cortex, thalamus and substantia nigra, although at a lower level than alpha4 and beta2. The distribution of alpha3, alpha4, alpha5, alpha6, alpha7, beta2, beta3 and beta4 subunit mRNAs in the monkey is substantially similar to that observed in rodent brain. Surprisingly, alpha2 mRNA signal was largely distributed in the Macaca brain, at levels comparable with those of alpha4 and beta2. This observation represents the main difference between rodent and Macaca subunit mRNA distribution and suggests that, besides alpha4beta2*, alpha2beta2* nAChRs constitute a main nAChR isoform in primate brain.

Nicotinic acetylcholine receptors are homo- or heteropentameric proteins belonging to the superfamily of receptor channels including the glycine and GABA-A receptors. Affinity labelling and mutagenesis experiments indicated that the M2 transmembrane segment of each subunit lines the ion channel and is coiled into an alpha-helix. Comparison of the M2 sequence of the cation-selective alpha 7 nicotinic receptor to that of the anion-selective alpha 1 glycine receptor identified amino acids involved in charge selectivity. Mutations of the alpha 7 homo-oligomeric receptor within (or near) M2, namely E237A, V251T and a proline insertion P236' were shown to convert the ionic selectivity of alpha 7 from cationic to anionic. Systematic analysis of each of these three mutations supports the notion that the conversion of ionic selectivity results from a local structural reorganization of the 234-238 loop. The 234-238 coiled loop, previously shown to lie near the narrowest portion of the channel, is thus proposed to contribute directly to the charge selectivity filter. A possible functional analogy with the voltage-gated ion channels and related receptors is discussed.

Several recent electrophysiological studies have demonstrated that nicotinic agonists stimulate the release of gamma-aminobutyric acid (GABA) from rodent brain tissue. Our studies used a neurochemical approach to characterize nicotinic receptor-stimulated [3H]-GABA release from mouse brain synaptosomes. Nicotine increased [3H]-GABA release from synaptosomes preloaded with [3H]-GABA in a concentration-dependent manner. This release appeared rapidly, was Ca++ dependent, and was partially (about 50%) blocked by 100 nM tetrodotoxin and totally blocked by mecamylamine and dihydro-beta-erythroidine. alpha-Bungarotoxin had no effect. Twelve nicotinic agonists were compared for their effects on [3H]-GABA release. The agonists differed in potency (EC50) and efficacy (Emax). The EC50 and Emax values were significantly correlated (r = 0.95, P.001 for EC50; r = 0.93, P.01 for Emax) to values obtained for these same agonists when 86Rb+ efflux was determined. A significant correlation (r = 0.84, P.01) was found when the EC50 values for agonist-stimulated [3H]-GABA release and IC50 values for agonist inhibition of [3H]-L-nicotine binding were compared. Differences in [3H]-GABA release were detected in 12 brain regions and maximal release was significantly correlated with [3H]-nicotine binding. The pharmacological and regional comparisons suggest that the nAChR that stimulates [3H]-GABA release is the one that binds [3H]-nicotine with high affinity (alpha4beta2). Unequivocal evidence that the receptor that modulates nicotine-stimulated [3H]-GABA release contains a beta2 subunit was obtained in a study using wild-type, heterozygous and homozygous beta2 null mutant mice. [3H]-GABA release and [3H]-nicotine binding decreased along with the number of copies of the null mutant gene.

Neuronal nicotinic acetylcholine receptor (nAChR) subunit genes compose a family of genes. The major isoform of nAChR in the brain is made up of the alpha4 and beta2 subunits and possesses a high affinity for nicotine. To investigate the mechanisms of the regulation of the nAChR alpha4 gene expression in mouse, its genomic DNA was cloned and characterized. The transcription initiation site was mapped by primer extension and RNase protection experiments and localized at about 254 bp upstream of the translation initiation site. The 5' flanking region of this gene did not have typical TATA box but GC-rich sequences were found around the initiation site. Methylation analysis of this region revealed that genomic DNAs from liver and muscle are partially methylated, whereas little methylation was observed in genomic DNA from brain. To characterize the cis-acting elements driving cell-specific expression of the alpha4 subunit gene, we produced lines of transgenic mice which carry a series of fragments of the alpha4 gene fused with bacterial lacZ as a reporter gene. An 11.5-kb DNA fragment containing 9 kb of the region upstream of the transcription initiation site and the first intron was found to confer an expression pattern which coincides rather well with the endogenous gene expression pattern at early embryonic stages, suggesting that the elements necessary for the onset of alpha4 gene expression are located in this region. A DNA fragment containing the 1.8-kb upstream sequence and the first intron drove expression of lacZ in a limited subset of alpha4 expressing cells, whereas the 1.8-kb upstream sequence alone did not elicit any significant expression. These results show that both upstream and intronic sequences are important for cell-specific expression of the nAChR alpha4 gene.

Recent studies have suggested that alcohols can affect the function of neurotransmitter-gated ion channels by a direct interaction with the receptor protein. However, the molecular region of the receptor protein that mediates the alcohol action is not known. To address this question, we studied the effect of ethanol on the function of recombinant nicotinic acetylcholine type alpha 7 (nACh alpha 7) receptors, 5-hydroxytryptamine (serotonin) type 3 (5-HT3) receptors, and a chimeric receptor constructed from these two receptors. The receptors were expressed in Xenopus oocytes and their function was studied using the two-electrode voltage-clamp technique. Ethanol inhibited the response of nACh alpha 7 receptors in a concentration-dependent manner over the concentration range of 5-100 mM; the EC50 for this inhibition was 33 mM ethanol. Ethanol decreased the maximal amplitude (Emax) of the nACh alpha 7 receptor agonist concentration-response curve, without significantly affecting the EC50. In contrast, ethanol potentiated 5-HT3 receptor-mediated responses at low agonist concentrations. The potentiation was concentration-dependent over the concentration range of 10-100 mM; the EC50 for this potentiation was 57 mM ethanol. The magnitude of the ethanol potentiation of 5-HT3 receptor-mediated responses decreased with increasing agonist concentration. The chimeric receptor had the amino-terminal domain from the nACh alpha 7 receptor and the transmembrane and carboxyl-terminal domains from the 5-HT3 receptor. Ethanol was found to inhibit the function of this chimeric receptor in a manner similar to that of nACh alpha 7 receptors. Because the inhibition transfers with the amino-terminal domain of the receptor, the observations suggest that the amino-terminal domain of the receptor is involved in the inhibition.

The chick cerebellar kainate (KA) binding protein (KBP), a member of the family of ionotropic glutamate receptors, harbours a glycine-rich (GxGxxG) motif known to be involved in the binding of ATP and GTP to kinases and G proteins respectively. Here, we report that guanine, but not adenine, nucleotides interact with KBP by inhibiting [3H]KA binding in a competitive-like manner, displaying IC50 values in the micromolar range. To locate the GTP binding site, KBP was photoaffinity labelled with [alpha-32P]GTP. The reaction was blocked by KA, glutamate, 6-cyano-7-nitroquinoxaline-2,3-dione and antibodies raised against a peptide containing the glycine-rich motif. Site-directed mutagenesis of residues K72 and Y73 within the glycine-rich motif followed by the expression of the KBP mutants at the surface of HEK 293 cells showed a decrease in GTP binding affinity by factors of 10 and 100 respectively. The binding of [3H]KA to the K72A/T KBP mutants was not affected but binding to the Y73I KBP mutant was decreased by a factor of 10. Accordingly, we propose that the glycine-rich motif of KBP forms part of a guanine nucleotide binding site. We further suggest that the glycine-rich motif is the binding site at which guanine nucleotides inhibit the glutamate-mediated responses of various members of the subfamily of glutamate ionotropic receptors.

The neuronal nicotinic acetylcholine receptor gene family encodes proteins that form either homo­oligomeric or hetero-oligomeric ligand gated ion channels. Although the total number of combinations of possible receptors is large, only a few have been demonstrated to function in the oocyte expression system. However, those that have been expressed differ from each other in several important ways. Receptors expressed in oocytes differ with respect to the ligands that activate them, their single channel properties, their permselectivity, and their modulation by external calcium. Furthermore, some cells express several of the genes that encode these receptor subunits and assemble receptors that contain a subset of the expressed genes. There are, therefore, probably mechanisms responsible for the assembly and targeting of receptors containing specific combinations of subunits. We investigated this possibility initially focussing on the homo-oligomeric alpha7 receptor and identified a requirement for the activity of a peptidyl prolyl isomerase in the assembly of this receptor. This requirement is shared by the serotonin-gated ion channel. We have created a number of mutations in the alpha 7 protein to try to identify relevant proline residues and have examined the expression of these mutant receptors in the Xenopus oocyte expression system.

The effects of chlorpromazine (CPZ) on nicotinic acetylcholine receptor (nAChR) were re-investigated by patch-clamp recordings on a mouse muscle cell line: (1) CPZ decreased the channel-opening frequency and, thus, acted as a closed-channel blocker. This effect was independent of the membrane potential and was consistent with an enhanced desensitization of the nAChR. (2) In addition, CPZ decreased the mean channel open time of the nAChR in a concentration- and voltage-dependent manner and, thus, behaved as an open-channel blocker. The latter effect supports the notion that CPZ binds to a site within the nAChR ionic channel.

In the adult motor endplate the acetylcholine receptor protein (AChR) is strictly localized under the motor nerve ending, whereas in the noninnervated myotube it is distributed all over the surface of the cell. The genesis of this anisotropic distribution involves a differential regulation of AChR gene transcription. In situ hybridization with AChR subunit probes discloses high levels of unspliced and mature mRNA all over differentiating myotubes. After the entry of the exploratory motor axons, the mRNA clusters located outside the endplate decrease in number and become restricted to the subneural "fundamental" nuclei. Denervation causes a reappearance of unspliced and mature mRNA in extrajunctional areas. A compartmentalized expression of AChR genes take place during endplate formation. Chronic paralysis of the embryo interferes with the disappearance of extrajunctional AChR that, thus, represents an electrical activity-dependent repression of AChR genes. The entry of Ca2+ ions through the sarcolemmal membrane during electrical activity and the activation of protein kinase C plausibly contribute to this membrane-to-gene regulation. The maintenance and late increase in AChR number at the endplate requires the intervention of an anterograde signal or signals, of neural origin. Several factors have been suggested to play a role in this process, such as an acetylcholine receptor-inducing activity (ARIA), ascorbic acid, or calcitonin gene-related peptide (CGRP), a peptide known to coexist with acetylcholine in spinal cord motoneurons. In cultured chick muscle cells, CGRP increases the concentration of surface AChR and alpha-subunit unspliced and mature mRNA and stimulates membrane-bound adenylate cyclase, suggesting that distinct second messengers are involved in the regulation of AChR biosynthesis by electrical activity and by CGRP. The data are interpreted in terms of a model in which it is assumed that (i) in the adult muscle fiber, different stages of gene expression occur in the nuclei in subneural and extrajunctional areas, and (ii) different second messengers elicited by neural factors or electrical activity regulate the state of transcription of these nuclei via trans-acting allosteric proteins binding to cis-acting DNA regulatory elements. The upstream flanking regions of several of the AChR subunit genes reveal ubiquitous DNA elements such as TATA and CAAT boxes, Sp1 binding sites and SV40 core enhancer sites, and muscle-specific MyoD (CANNTG) elements. The contribution of some of these elements to the differential regulation of the multiple AChR subunits is discussed.

The chromosomal localization of four neuronal nicotinic acetylcholine receptor subunit genes was performed by following the mendelian segregation of their corresponding alleles in backcrosses involving the mouse species Mus spretus and the laboratory strains C57BL/6 or BALB/c. A similar analysis previously performed with muscle nicotinic acetylcholine receptor subunits revealed that the genes coding for the alpha and beta subunits are respectively located on chromosome 2 and 11, whereas the gamma and delta subunit coding genes are linked and located on mouse chromosome 1. In this study, we show that the genes coding for the neuronal nicotinic acetylcholine receptor alpha 2, alpha 3 and beta 2 subunits are dispersed on three different mouse chromosomes, viz. 14, 9 and 3 respectively. Moreover, the alpha 4 subunit gene is located on chromosome 2 but is not genetically linked to the alpha 1 subunit gene.

There is a strong association between perinatal tobacco (especially nicotine) exposure and poor infant outcome, but the mechanisms linking the two are not well understood. Here we determined how perinatal nicotine exposure compromises neonatal respiratory control by comparing the effects of nicotine in normal (wild-type, WT) and knockout (KO) mice lacking nicotinic acetylcoline receptors (nAChRs) containing the b2 subunit. These nAChRs are a major component of high-affinity nicotine binding sites in the nervous system. Pregnant WT and KO mice were implanted with micro-osmotic minipumps on embryonic day 14. Pumps delivered either water or nicotine (6.0 mg.kg-1 day-1). Breathing was measured using dual-chamber plethysmograhy. Control WT and KO pups had comparable breathing rhythms at rest. Stress, however, unmasked important differences between genotypes. KO pups responded briskly to hypoxia, but breathing became highly irregular, with frequent pauses (apnoeas) during recovery from hypoxia. Irregular breathing and apnoeas were never seen in WT pups post-hypoxia. Control KO and WT pups responded similarly to CO2, with no evidence of breathing irregularity or apnoea in either genotype during recovery from bouts of intermittent hypercapnia. Nicotine-exposure augmented the response of the WT pups to hypoxia. WT pups also developed an irregular breathing rhythm due to frequent apnoeas between bouts of hypoxia. Nicotine-exposed WT pups consequently resembled the KO. Nicotine-exposure did not alter the hypoxic breathing pattern of KO pups. Moreover, perinatal nicotine exposure did not alter responses of either WT or KO pups to intermittent hypercapnia. Conclusion: Our findings suggest that nicotine increases the risk of neonatal apnoea by disrupting nAChR-dependent mechanisms that fine-tune breathing rhythm. These mechanisms are particularly sensitive to sudden changes in reflex drive from O2rather than CO2-sensitive chemoreflexes.

Patch-clamp techniques were used to record acetylcholine- (ACh) activated single-channel currents in cell-attached membrane patches from myotubes of the mouse cell line, C2. The effects of the phenothiazine derivative chlorpromazine (CPZ) and of the hallucinogen phencyclidine (PCP) on ACh-activated single-channel properties were studied under conditions where both compounds are positively charged (pH 7.2). The single-channel conductance was unaffected by either CPZ or PCP at concentrations ranging from 10 to 500 nM. 10-200 nM-CPZ and PCP led to shortened mean burst times. CPZ and PCP effects on mean burst times were voltage independent and did not vary in a simple linear manner with concentration. 10-200 nM-CPZ and PCP did not reduce channel opening frequencies, suggesting that the fraction of non-conducting state (occupied, blocked or desensitized) favoured at equilibrium was not significant at these concentrations. On the other hand, concentrations of CPZ and PCP higher than 300 nM did lead to depressed channel opening frequencies. In addition, we observed that, at these concentrations, the shortened burst duration reverses to the longer values found at lower effector concentrations. The effects of CPZ and PCP on ACh-activated single-channel kinetics are interpreted in terms of current models of ACh-receptor structure and conformational transitions.

Tools from recombinant DNA technology are now available to analyse the mechanisms that regulate the expression of synaptic proteins during synapse formation and stabilization. This review focuses on the expression of the acetylcholine receptor, an essential and well-characterized component of the motor endplate.

Recently the purified alpha-subunit from Torpedo marmorata acetylcholine receptor was shown to bind alpha-bungarotoxin with a KD approximately 3 nM in the presence of sodium dodecyl sulfate (Tzartos, S.J., and Changeux, J.P. (1983) EMBO J. 2, 381-387). Here we describe a further significant step toward renaturation of the alpha-subunit as judged by toxin and monoclonal antibody binding. Purified T. marmorata receptor subunits were diluted with 1% lipids (asolectin) plus 0.5% Na+ cholate. An anion-exchange resin eliminated most of the detergents, leaving approximately 0.1% Na+ cholate and the lipids. After this treatment, about 20% of the alpha-subunit recovered (but not the beta-, gamma-, or delta-subunit) exhibited a high affinity for radioiodinated alpha-bungarotoxin with a KD approximately 0.5 nM. The 34,000- and 27,000-dalton proteolytic peptides of the alpha-subunit conserved this lipid-dependent toxin binding. Unlabeled alpha-toxins, hexamethonium, and carbamylcholine competed with alpha-bungarotoxin for the renatured alpha-subunit. Noncompetitive channel blockers doubled the lipid-dependent toxin-binding capacity of the alpha-subunit but had no effect on the 27,000-dalton peptide. The binding of several monoclonal antibodies to the main immunogenic region (which is particularly sensitive to denaturation) significantly increased. In particular, binding of antibody 16 changed from 1% to denatured to 100% to the lipid-renaturated alpha-subunit. The binding of these antibodies was lost with the lipid-renatured 34,000- and 27,000-dalton peptides.

The 5' end and promoter region of the alpha-subunit gene of chicken muscle acetylcholine receptor was mapped and sequenced. It includes a TATA and a CAAT box and a potential Sp1-binding site. When inserted in front of the chloramphenicol acetyltransferase gene, this promoter (including 850 base pairs of upstream sequence) directed high transient chloramphenicol acetyltransferase expression in transfected mouse C2.7 myotubes but not in C2.7 myoblasts or nonmyogenic 3T6 cells.

Treatment of isolated electroplax with physiological solutions supplemented with either 1 molar sodium chloride, 2 molar urea, or 2 molar sucrose renders the cell insensitive to carbamylcholine, phenyltrimethylammonium, or decamethonium even at high concentrations. The treated cells have a residual resting potential of -20 +/- 10 millivolts (negative inside) and are depolarized by acetylcholine at concentrations larger than 10(-3) mole per liter. This response is not affected by d-tubocurarine but is blocked by physostigmine, diisopropylphosphorofluoridate, or strong buffers and thus depends on the catalytic activity of the membrane-bound acetylcholinesterase.

3H chlorpromazine binds to acetylcholine receptor-rich membrane fragments prepared from Torpedo marmorata electric organ in three different manners: (1) at the level of high affinity sites from which it is displaced by perhydrohistrionicotoxin, (2) at the level of low affinity sites insensitive to this toxin, and (3) in a non saturable manner to a presumably lipidic phase. Binding of chlorpromazine to the first two categories of sites independently stabilizes the "desensitized" high affinity state of the receptor for cholinergic agonists. There exists one high affinity site per two snake alpha-toxin sites, thus per 250,000 daltons light form of the receptor. Under the conditions of 3H chlorpromazine high affinity binding, ultraviolet irradiation results in the covalent incorporation of this ligand to the four chains of the receptor.

The interaction of the cholinergic (nicotinic) receptor protein purified from Torpedo marmorata electric organ with lipidic monomolecular films is studied at constant film area or constant superficial pressure. In both experimental situations, it is incorporated more efficiently into cholesterol than into egg lecithin films without showing any selectivity for a particular phospholipid class.