Several lines of evidence suggest a link between the ␣7 neuronal nicotinic acetylcholine receptor (nAChR) and brain disorders including schizophrenia, Alzheimer's disease, and traumatic brain injury. The present work describes a novel molecule, 1-(5-chloro-2,4-dimethoxy-phenyl)-3-(5-methyl-isoxazol-3-yl)-urea (PNU-120596), which acts as a powerful positive allosteric modulator of the ␣7 nAChR. Discovered in a high-throughput screen, PNU-120596 increased agonist-evoked calcium flux mediated by an engineered variant of the human ␣7 nAChR. Electrophysiology studies confirmed that PNU-120596 increased peak agonist-evoked currents mediated by wild-type receptors and also demonstrated a pronounced prolongation of the evoked response in the continued presence of agonist. In contrast, PNU-120596 produced no detectable change in currents mediated by ␣4␤2, ␣3␤4, and ␣9␣10 nAChRs. PNU-120596 increased the channel mean open time of ␣7 nAChRs but had no effect on ion selectivity and relatively little, if any, effect on unitary conductance. When applied to acute hippocampal slices, PNU-120596 increased the frequency of ACh-evoked GABAergic postsynaptic currents measured in pyramidal neurons; this effect was suppressed by TTX, suggesting that PNU-120596 modulated the function of ␣7 nAChRs located on the somatodendritic membrane of hippocampal interneurons. Accordingly, PNU-120596 greatly enhanced the ACh-evoked inward currents in these interneurons. Systemic administration of PNU-120596 to rats improved the auditory gating deficit caused by amphetamine, a model proposed to reflect a circuit level disturbance associated with schizophrenia. Together, these results suggest that PNU-120596 represents a new class of molecule that enhances ␣7 nAChR function and thus has the potential to treat psychiatric and neurological disorders.

Nicotine, a component of tobacco, is highly addictive but possesses beneficial properties such as cognitive improvements and memory maintenance. Involved in these processes is the neuronal nicotinic acetylcholine receptor (nAChR) alpha7, whose activation triggers depolarization, intracellular signaling cascades, and synaptic plasticity underlying addiction and cognition. It is therefore important to investigate intracellular mechanisms by which a cell regulates alpha7 nAChR activity. We have examined the role of phosphorylation by combining molecular biology, biochemistry, and electrophysiology in SH-SY5Y neuroblastoma cells, Xenopus oocytes, rat hippocampal interneurons, and neurons from the supraoptic nucleus, and we found tyrosine phosphorylation of alpha7 nAChRs. Tyrosine kinase inhibition by genistein decreased alpha7 nAChR phosphorylation but strongly increased acetylcholine-evoked currents, whereas tyrosine phosphatase inhibition by pervanadate produced opposite effects. Src-family kinases (SFKs) directly interacted with the cytoplasmic loop of alpha7 nAChRs and phosphorylated the receptors at the plasma membrane. SFK inhibition by PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] or SU6656 (2,3-dihydro-N,N-dimethyl-2-oxo-3-[(4,5,6,7-tetrahydro-1H-indol-2-yl)methylene]-1H-indole-5-sulfonamide) increased alpha7 nAChR-mediated responses, whereas expression of active Src reduced alpha7 nAChR activity. Mutant alpha7 nAChRs lacking cytoplasmic loop tyrosine residues because of alanine replacement of Tyr-386 and Tyr-442 were more active than wild-type receptors and insensitive to kinase or phosphatase inhibition. Because the amount of surface alpha7 receptors was not affected by kinase or phosphatase inhibitors, these data show that functional properties of alpha7 nAChRs depend on the tyrosine phosphorylation status of the receptor and are the result of a balance between SFKs and tyrosine phosphatases. These findings reveal novel regulatory mechanisms that may help to understand nicotinic receptor-dependent plasticity, addiction, and pathology.

The objective of the present work was double. (i) Light microscopic autoradiography was used to determine the distribution of vasopressin and oxytocin binding sites in the spinal cord of rats. (ii) Whole-cell recordings were performed in lumbar spinal cord slices in order to assess whether these receptors are functional, whether they are located pre- or postsynaptically and whether they are present in motoneurons. In newborns, vasopressin binding sites of the V1a type were present in all laminae of the central gray at all segmental levels, whereas oxytocin binding sites were found only in the superficial layers of the dorsal horn. In adults, binding sites for both neuropeptides were also present, but were less dense. The dissociation constants for vasopressin were similar in newborns and adults. Whole-cell recordings showed that in identified motoneurons vasopressin exerted a direct effect, by inducing a membrane depolarization or by generating a sustained inward current, and an indirect effect, by enhancing glycinergic and GABAergic inhibitory transmission. Vasopressin-induced facilitation of inhibitory transmission could also be demonstrated in unidentified ventral horn neurons. All these effects were mediated by V1a but not V1b receptors. In some neurons, glycinergic transmission was also facilitated by a selective oxytocin receptor agonist. Our data, together with data obtained previously in brainstem motor nuclei, suggest that vasopressin of hypothalamic origin could play a role in motricity. The neuropeptide could act as a neuromodulator, because it would not directly activate motoneurons, but rather render them more responsive to incoming excitatory inputs. Vasopressin may thus act as a regulator of muscular force.

The pudendal motor system is constituted by striated muscles of the pelvic floor and the spinal motoneurons that innervate them. It plays a role in eliminative functions of the bladder and intestine and in sexual function. Pudendal motoneurons are located in the ventral horn of the caudal lumbar spinal cord and send their axon into the pudendal nerve. In the rat, binding sites for vasopressin and tachykinin are present in the dorsomedial and dorsolateral pudendal nuclei, suggesting that these neuropeptides may affect pudendal motoneurons. The aim of the present study was to investigate possible effects of vasopressin and tachykinins on these motoneurons. Recordings were performed in spinal cord slices of young male rats using the whole-cell patch-clamp technique. Before recording, motoneurons were identified by 1,1Ј-dilinoleyl-3,3,3Ј,3Ј-tetramethylindocarbocyanine, 4-chlorobenzenesulfonate retrograde labeling. The identification was confirmed, a posteriori, by choline acetyltransferase immunocytochemistry. Vasopressin and tachykinins caused a powerful excitation of pudendal motoneurons. The peptide-evoked depolarization, or the peptide-evoked inward current, persisted in the presence of tetrodotoxin, indicating that these effects were mainly postsynaptic. By using selective receptor agonists and antagonist, we determined that vasopressin acted via vasopressin 1a (V1a), but not V1b, V2, or oxytocin receptors, whereas tachykinins acted via neurokinin 1 (NK1), but not NK2 or NK3, receptors. Vasopressin acted by enhancing a nonselective cationic conductance; in some motoneurons, it also probably suppressed a resting K ϩ conductance. Our data show that vasopressin and tachykinins can excite pudendal motoneurons and thus influence the force of striated perineal muscles involved in eliminative and sexual functions.

Substance P and other neuropeptides of the tachykinin family can powerfully excite CA1 hippocampal interneurons present in the CA1 region. In the present work we show that, by exciting hippocampal interneurons, tachykinins can indirectly inhibit pyramidal neurons. We found that tachykinins caused a decrease in the inhibitory synaptic current interval and an increase in the inhibitory synaptic current amplitude in almost all pyramidal neurons tested. This effect was tetrodotoxin sensitive. Tachykinins did not alter the frequency or amplitude of miniature inhibitory synaptic currents and were without effect on evoked inhibitory synaptic currents. Thus, these neuropeptides acted at the somatodendritic membrane of GABAergic interneurons, rather than at their axon terminals. The effect of substance P on spontaneous inhibitory synaptic currents could be mimicked by a selective agonist of NK1 receptors, but not by selective agonists of NK2 and NK3 receptors. It was suppressed by an NK1 receptor antagonist. In CA1 interneurons located in stratum radiatum, substance P generated a sustained tetrodotoxin-insensitive inward current or induced membrane depolarization and action potential firing. This direct excitatory action was mediated by NK1 receptors. Current-voltage relationships indicate that the net tachykinin-evoked current reversed in polarity at or near the K+ equilibrium potential, suggesting that a suppression of a resting K+ conductance was involved. By increasing the excitability of CA1 GABAergic interneurons, tachykinins can powerfully facilitate the inhibitory synaptic input to pyramidal neurons. This indirect inhibition could play a role in regulating short-term and/or long-term synaptic plasticity, promoting neuronal circuit synchronization or, in some physiopathological situations, influencing epileptogenesis.