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Background and purpose:Smoking cessation trials with three high-affinity partial agonists of a4b2 neuronal nicotinic acetylcholine receptors (nAChRs) have demonstrated differences in their clinical efficacy. This work examines the origin of the differences by taking into account brain exposure and pharmacological effects at human a4b2 nAChRs. Experimental approach: Rat plasma and brain pharmacokinetics were characterized and used to predict human steady-state plasma and brain concentrations following recommended doses of each of the three compounds. The pharmacological characterization included in vitro affinities at different nAChR subtypes, functional efficacies and potencies at the human a4b2 nAChR, as well as in vivo effects on rat mesolimbic dopamine turn-over. Key results: A comparison of predicted human brain concentrations following therapeutic doses demonstrated that varenicline and nicotine, but not dianicline and cytisine, can extensively desensitize and, to a lesser extent, activate a4b2 nAChRs. The limited clinical efficacy of dianicline may be accounted for by a combination of weak functional potency at a4b2 nAChRs and moderate brain penetration, while recommended doses of cytisine, despite its high in vitro potency, are predicted to result in brain concentrations that are insufficient to affect a4b2 nAChRs. Conclusions and implications:The data provide a plausible explanation for the higher abstinence rate in smoking cessation trials following treatment with varenicline than with the two other a4b2 nAChR partial agonists. In addition, this retrospective analysis demonstrates the usefulness of combining in vitro and in vivo parameters with estimated therapeutic human brain concentrations for translation to clinical efficacy.

GABA A receptors form ClϪ permeable channels that mediate the majority of fast synaptic inhibition in the brain. The K ϩ /Cl Ϫ cotransporter KCC2 is the main mechanism by which neurons establish low intracellular Cl Ϫ levels, which is thought to enable GABAergic inhibitory control of neuronal activity. However, the widely used KCC2 inhibitor furosemide is nonselective with antiseizure efficacy in slices and in vivo, leading to a conflicting scheme of how KCC2 influences GABAergic control of neuronal synchronization. Here we used the selective KCC2 inhibitor VU0463271 [N-cyclopropyl-N-(4-methyl-2-thiazolyl)-2-[(6-phenyl-3-pyridazinyl)thio]acetamide] to investigate the influence of KCC2 function. Application of VU0463271 caused a reversible depolarizing shift in E GABA values and increased spiking of cultured hippocampal neurons. Application of VU0463271 to mouse hippocampal slices under low-Mg 2ϩ conditions induced unremitting recurrent epileptiform discharges. Finally, microinfusion of VU0463271 alone directly into the mouse dorsal hippocampus rapidly caused epileptiform discharges. Our findings indicated that KCC2 function was a critical inhibitory factor ex vivo and in vivo.

Genome-wide association studies (GWASs) have identified associations between the CHRNA5–CHRNA3–CHRNB4 gene cluster and smoking heaviness and nicotine dependence. Studies in rodents have described the anatomical localisation and function of the nicotinic acetylcholine receptors (nAChRs) formed by the subunits encoded by this gene cluster. Further investigations that complemented these studies highlighted the variability of individuals’ smoking behaviours and their ability to adjust nicotine intake. GWASs of smoking-related health outcomes have also identified this signal in the CHRNA5–CHRNA3–CHRNB4 gene cluster. This insight underpins approaches to strengthen causal inference in observational data. Combining genetic and mechanistic studies of nicotine dependence and smoking heaviness may reveal novel targets for medication development. Validated targets can inform genetic therapeutic interventions for smoking cessation and tobacco-related diseases.

Stem cell biology offers advantages to investigators seeking to identify new therapeutic molecules. Specifically, stem cells are genetically stable, scalable for molecular screening, and function in cellular assays for drug efficacy and safety. A key hurdle for drug discoverers of central nervous system disease is a lack of high quality neuronal cells. In the central nervous system, ␣-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) subtype glutamate receptors mediate the vast majority of excitatory neurotransmissions. Embryonic stem (ES) cell protocols were developed to differentiate into neuronal subtypes that express AMPA receptors and were pharmacologically responsive to standard compounds for AMPA potentiation. Therefore, we hypothesized that stem cell-derived neurons should be predictive in high-throughput screens (HTSs). Here, we describe a murine ES cell-based HTS of a 2.4 ؋ 10 6 compound library, the identification of novel chemical "hits" for AMPA potentiation, structure function relationship of compounds and receptors, and validation of chemical leads in secondary assays using human ES cell-derived neurons. This reporting of murine ES cell derivatives being formatted to deliver HTS of greater than 10 6 compounds for a specific drug target conclusively demonstrates a new application for stem cells in drug discovery. In the future new molecular entities may be screened directly in human ES or induced pluripotent stem cell derivatives.Cognitive impairment is a fundamental trademark of many CNS 2 diseases such as Alzheimer disease, schizophrenia, and attention deficit hyperactivity disorder. Unfortunately few pharmaceutical agents have proven to be efficacious in treating cognitive impairment. As such, the medical need for new therapeutic approaches for improving cognitive deficits has emerged as an important frontier in treating central nervous system diseases. Glutamate mediates the vast majority of excitatory neurotransmission in the CNS with post-synaptic signaling regulated by either ligand-gated cation channels (ionotropic) or G-protein-coupled receptors (metabotropic). Ionotropic ␣-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors are tetrameric aggregates composed of combinations of one or more of four subunits (GluR1-4) that can each bind a glutamate neurotransmitter molecule. Upon binding of glutamate, the associated ion channel pore opens, allowing cations to cross the membrane and depolarize the postsynaptic cell. Termination of the postsynaptic signal is driven primarily by the removal of glutamate from the synaptic cleft, reducing the occupancy of the glutamate binding site and subsequent channel closure (deactivation). Additionally, the conduction pathway will close in the continued presence of bound agonist as a result of conformational changes secondary to glutamate binding (desensitization).AMPA receptor pharmacology has identified agonists, competitive and non-competitive antagonists, and positive allosteric modulators (3). This latter class of pharmacologic agen...

1 UK-78,282, a novel piperidine blocker of the T lymphocyte voltage-gated K + channel, Kv1.3, was discovered by screening a large compound ®le using a high-throughput 86 Rb e ux assay. This compound blocks Kv1.3 with a IC 50 of *200 nM and 1 : 1 stoichiometry. A closely related compound, 325, containing a benzyl moiety in place of the benzhydryl in UK-78,282, is signi®cantly less potent. 2 Three lines of evidence indicate that UK-78,282 inhibits Kv1.3 in a use-dependent manner by preferentially blocking and binding to the C-type inactivated state of the channel. Increasing the fraction of inactivated channels by holding the membrane potential at 750 mV enhances the channel's sensitivity to 282. Decreasing the number of inactivated channels by exposure to *160 mM external K + decreases the sensitivity to UK-78,282. Mutations that alter the rate of Ctype inactivation also change the channel's sensitivity to UK-78,282 and there is a direct correlation between t h and IC 50 values.3 Competition experiments suggest that UK-78,282 binds to residues at the inner surface of the channel overlapping the site of action of verapamil. Internal tetraethylammonium and external charybdotoxin do not compete UK-78,282's action on the channel. 4 UK-78,282 displays marked selectivity for Kv1.3 over several other closely related K + channels, the only exception being the rapidly inactivating voltage-gated K + channel, Kv1.4. 5 UK-78,282 e ectively suppresses human T-lymphocyte activation.

The neuronal K+-Cl− co-transporter KCC2 (also known as solute carrier family 12 member 5, or SLC12A5) critically maintains the neuronal Cl− gradient to establish hyperpolarizing signaling by GABA A receptors, the primary mediators of fast synaptic inhibition in the brain. These receptors are also the principal targets of benzo-diazepines, neurosteroids, and intravenous general anesthetics 1 . The downregulation of KCC2 in neuropathic pain models

The behavioral and anatomical deficits seen in fragile X syndrome (FXS) are widely believed to result from imbalances in the relative strengths of excitatory and inhibitory neurotransmission. Although modified neuronal excitability is thought to be of significance, the contribution that alterations in GABAergic inhibition play in the pathophysiology of FXS are ill defined. Slow sustained neuronal inhibition is mediated by γ-aminobutyric acid type B (GABA) receptors, which are heterodimeric G-protein-coupled receptors constructed from R1a and R2 or R1b and R2 subunits. Via the activation of G, they limit cAMP accumulation, diminish neurotransmitter release, and induce neuronal hyperpolarization. Here we reveal that selective deficits in R1a subunit expression are seen in Fmr1 knock-out mice (KO) mice, a widely used animal model of FXS, but the levels of the respective mRNAs were unaffected. Similar trends of R1a expression were seen in a subset of FXS patients. GABA receptors (GABARs) exert powerful pre- and postsynaptic inhibitory effects on neurotransmission. R1a-containing GABARs are believed to mediate presynaptic inhibition in principal neurons. In accordance with this result, deficits in the ability of GABARs to suppress glutamate release were seen in Fmr1-KO mice. In contrast, the ability of GABARs to suppress GABA release and induce postsynaptic hyperpolarization was unaffected. Significantly, this deficit contributes to the pathophysiology of FXS as the GABAR agonist ()-baclofen rescued the imbalances between excitatory and inhibitory neurotransmission evident in Fmr1-KO mice. Collectively, our results provided evidence that selective deficits in the activity of presynaptic GABARs contribute to the pathophysiology of FXS.