Jean-Pierre Changeux

Crucial Role of α4 and α6 Nicotinic Acetylcholine Receptor Subunits from Ventral Tegmental Area in Systemic Nicotine Self-Administration

The identification of the molecular mechanisms involved in nicotine addiction and its cognitive consequences is a worldwide priority for public health. Novel in vivo paradigms were developed to match this aim. Although the β2 subunit of the neuronal nicotinic acetylcholine receptor (nAChR) has been shown to play a crucial role in mediating the reinforcement properties of nicotine, little is known about the contribution of the different α subunit partners of β2 (i.e., α4 and α6), the homo-pentameric α7, and the brain areas other than the ventral tegmental area (VTA) involved in nicotine reinforcement. In this study, nicotine (8.7–52.6 μg free base/kg/inf) self-administration was investigated with drug-naive mice deleted (KO) for the β2, α4, α6 and α7 subunit genes, their wild-type (WT) controls, and KO mice in which the corresponding nAChR subunit was selectively re-expressed using a lentiviral vector (VEC mice). We show that WT mice, β2-VEC mice with the β2 subunit re-expressed exclusively in the VTA, α4-VEC mice with selective α4 re-expression in the VTA, α6-VEC mice with selective α6 re-expression in the VTA, and α7-KO mice promptly self-administer nicotine intravenously, whereas β2-KO, β2-VEC in the substantia nigra, α4-KO and α6-KO mice do not respond to nicotine. We thus define the necessary and sufficient role of α4β2- and α6β2-subunit containing nicotinic receptors (α4β2*- and α6β2*-nAChRs), but not α7*-nAChRs, present in cell bodies of the VTA, and their axons, for systemic nicotine reinforcement in drug-naive mice.

Evaluating the suitability of nicotinic acetylcholine receptor antibodies for standard immunodetection procedures.

Nicotinic acetylcholine receptors play important roles in numerous cognitive processes as well as in several debilitating central nervous system (CNS) disorders. In order to fully elucidate the diverse roles of nicotinic acetylcholine receptors in CNS function and dysfunction, a detailed knowledge of their cellular and subcellular localizations is essential. To date, methods to precisely localize nicotinic acetylcholine receptors in the CNS have predominantly relied on the use of anti‐receptor subunit antibodies. Although data obtained by immunohistology and immunoblotting are generally in accordance with ligand binding studies, some discrepancies remain, in particular with electrophysiological findings. In this context, nicotinic acetylcholine receptor subunit‐deficient mice should be ideal tools for testing the specificity of subunit‐directed antibodies. Here, we used standard protocols for immunohistochemistry and western blotting to examine the antibodies raised against the α3‐, α4‐, α7‐, β2‐, and β4‐nicotinic acetylcholine receptor subunits on brain tissues of the respective knock‐out mice. Unexpectedly, for each of the antibodies tested, immunoreactivity was the same in wild‐type and knock‐out mice. These data imply that, under commonly used conditions, these antibodies are not suited for immunolocalization. Thus, particular caution should be exerted with regards to the experimental approach used to visualize nicotinic acetylcholine receptors in the brain.

Functional architecture of the nicotinic acetylcholine receptor: A prototype of ligand-gated ion channels

The nicotinic acetylcholine receptor (nAChR) ~ is involved in chemo-electrical transduction at the neuromuscular junction and at cholinergic synapses of the central nervous system. At the motor endplate, invasion of the motor nerve ending by an action potential causes the release in the synaptic cleft of a brief pulse of acetylcholine (ACh), whose local concentration reaches 0. I to 1 mM (Katz & Miledi, 1977) for less than 1 msec (see also Clements et al., 1992 in the case of GluR). ACh diffuses through the cleft and binds to the nAChR present in the postsynaptic membrane, where it triggers the all-ornone opening of cation-selective ion channels through which Na+/K + ions flow passively. When depolarization reaches a threshold, muscle contraction occurs. In the cleft, ACh concentration rapidly declines to background levels (10 .9 M) as a consequence of diffusion and degradation by ACh esterase (Kuffler & Yoshikami, 1975; Katz & Miledi, 1977).

Functional significance of aromatic amino acids from three peptide loops of the α7 neuronal nicotinic receptor site investigated by site-directed mutagenesis

Three aromatic amino acids, Tyr92, Trp148 and Tyr187 belonging to three separate domains of the α7‐subunit of neuronal nicotinic acetylcholine receptor were mutated to phenylalanine, and the electrophysiological response of the resulting mutant receptors analyzed in the Xenopus oocyte expression system. All mutations significantly decreased the apparent affinities for acetylcholine and nicotine, and to a lesser extent, those for the competitive antagonists dihydro‐β‐erythroidine and α‐bungarotoxin. Other properties investigated, such as the voltage dependency of the ion response as well as its sensitivity to the open channel blocker QX222, were not significantly changed, indicating that the mutations affected selectively the recognition of cholinergic ligands by the receptor protein. The maximal rates for the rapid desensitization process were slightly modified, suggesting that the contribution of Tyr92, Trp148 and Tyr187 to the binding area might differ in the various conformations of the nicotinic receptor. Other mutations at nearby positions (S94N, W153F, G151D and G82E) did not affect the properties of the electrophysiological response. These data point to the functional significance of Tyr92, Trp148 and Tyr187 in the binding of cholinergic ligands and ion channel activation of the nicotinic receptor, thus supporting a multiple loop model [(1990) J. Biol. Chem. 265, 10430–10437] for the ligand binding area.

Nicotinic agonists stimulate acetylcholine release from mouse interpeduncular nucleus: a function mediated by a different nAChR than dopamine release from striatum

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 [3H]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. α‐Conotoxin MII did not inhibit this release, while α‐conotoxin AuIB inhibited 50% of agonist‐stimulated release. Comparison of this process with [3H]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 β2 subunit null mutation. The deletion of the β2 subunit had no effect on agonist‐stimulated acetylcholine release, but abolished agonist‐stimulated release of dopamine from striatal synaptosomes. Mice heterozygous for the β2 subunit null mutation showed decreased dopamine release evoked by l‐nicotine with no apparent change in EC50 value, as well as similar decreases in both transient and persistent phases of release with no changes in desensitization rates.

Mutational Analysis of the Charge Selectivity Filter of the α7 Nicotinic Acetylcholine Receptor

In the alpha7 nicotinic acetylcholine receptors, we analyze the contribution of mutations E237A and V251T, together with the proline insertion P236, in the conversion of the charge selectivity from cationic to anionic. We show that the triple mutant exhibits spontaneous openings displaying anionic selectivity. Furthermore, at position 251, hydrophilic or even negatively charged residues are compatible with an anionic channel. In contrast, the additional proline yields an anionic channel only when inserted between positions 234 and 237; insertion before 234 yields a cationic channel and after 238 alters the receptor surface expression. The coiled 234-238 loop thus directly contributes to the charge selectivity filter of the alpha7 channel.

Structure and pharmacology of pentameric receptor channels: from bacteria to brain.

Orthologs of the pentameric receptor channels that mediate fast synaptic transmission in the central and peripheral nervous systems have been found in several bacterial species and in a single archaea genus. Recent X-ray structures of bacterial and invertebrate pentameric receptors point to a striking conservation of the structural features within the whole family, even between distant prokaryotic and eukaryotic members. These structural data reveal general principles of molecular organization that allow allosteric membrane proteins to mediate chemoelectric transduction. Notably, several conformations have been solved, including open and closed channels with distinct global tertiary and quaternary structure. The data reveal features of the ion channel architecture and of diverse categories of binding sites, such as those that bind orthosteric ligands, including neurotransmitters, and those that bind allosteric modulators, such as general anesthetics, ivermectin, or lipids. In this review, we summarize the most recent data, discuss insights into the mechanism of action in these systems, and elaborate on newly opened avenues for drug design.

Distinct contributions of nicotinic acetylcholine receptor subunit α4 and subunit α6 to the reinforcing effects of nicotine

Nicotine is the primary psychoactive component of tobacco. Its reinforcing and addictive properties depend on nicotinic acetylcholine receptors (nAChRs) located within the mesolimbic axis originating in the ventral tegmental area (VTA). The roles and oligomeric assembly of subunit α4- and subunit α6-containing nAChRs in dopaminergic (DAergic) neurons are much debated. Using subunit-specific knockout mice and targeted lentiviral re-expression, we have determined the subunit dependence of intracranial nicotine self-administration (ICSA) into the VTA and the effects of nicotine on dopamine (DA) neuron excitability in the VTA and on DA transmission in the nucleus accumbens (NAc). We show that the α4 subunit, but not the α6 subunit, is necessary for ICSA and nicotine-induced bursting of VTA DAergic neurons, whereas subunits α4 and α6 together regulate the activity dependence of DA transmission in the NAc. These data suggest that α4-dominated enhancement of burst firing in DA neurons, relayed by DA transmission in NAc that is gated by nAChRs containing α4 and α6 subunits, underlies nicotine self-administration and its long-term maintenance.

Nicotine and serotonin in immune regulation and inflammatory processes: a perspective

Nicotine and serotonin modulate the innate and adaptive immune responses and the inflammatory states. Several nicotinic cholinergic and serotonergic receptor subtypes have been characterized in B and T lymphocytes, monocytes, macrophages, and dendritic cells. The use of knockout mice has allowed a better characterization of nicotinic receptors and their role in anti‐inflammatory processes in these cells. Cytokines play a crucial role in controlling inflammatory reactions. Nicotine and serotonin have been reported to regulate cytokine release. Cholinergic mechanisms also play an important role in inflammation through endogenous acetylcholine. Nicotine mimics this effect by activating the cholinergic anti‐inflammatory pathways. New concepts of reciprocal interactions between nicotine and serotonin are emerging. The role of nicotine as an anti‐inflammatory agent has been established, whereas that of serotonin remains more controversial.

Tonic nicotinic modulation of serotoninergic transmission in the spinal cord

The spinal serotoninergic projection from the raphe magnus has been shown to modulate nociceptive inputs, and activation of this projection mediates nicotine-elicited analgesia. Here, we investigate the interactions between cholinergic and serotoninergic systems in the spinal cord, by conducting serotonin [5-hydroxytryptamine (5-HT)] efflux experiments on mouse spinal slices. At least three spinal populations of nicotinic receptors are distinguished that affect 5-HT release. The first could be directly located on serotoninergic terminals, is insensitive to nanomolar concentrations of methyllicaconitine (MLA), and may be subjected to a basal (not maximal) cholinergic tone. The second is tonically and maximally activated by endogenous acetylcholine, insensitive to nanomolar concentrations of MLA, and present on inhibitory neurons. The last is also present on inhibitory neurons but is sensitive to nanomolar concentrations of MLA and not tonically activated by acetylcholine. Multiple nicotinic acetylcholine receptor populations thus differentially exert tonic or not tonic control on 5-HT transmission in the spinal cord. These receptors may be major targets for nicotine effects on antinociception. In addition, the presence of a tonic nicotinic modulation of 5-HT release indicates that endogenous acetylcholine plays a role in the physiological regulation of descending 5-HT pathways to the spinal cord.