The principal inhibitory neurotransmitter in the mammalian brain, ␥-aminobutyric acid (GABA), is thought to regulate memory processes by activating transient inhibitory postsynaptic currents. Here we describe a nonsynaptic, tonic form of inhibition in mouse CA1 pyramidal neurons that is generated by a distinct subpopulation of GABA type A receptors (GABA ARs). This tonic inhibitory conductance is predominantly mediated by ␣5 subunit-containing GABAARs (␣5GABAARs) that have different pharmacological and kinetic properties compared to postsynaptic receptors. GABAARs that mediate the tonic conductance are well suited to detect low, persistent, ambient concentrations of GABA in the extracellular space because they are highly sensitive to GABA and desensitize slowly. Moreover, the tonic current is highly sensitive to enhancement by amnestic drugs. Given the restricted expression of ␣5GABAARs to the hippocampus and the association between reduced ␣5GABAAR function and improved memory performance in behavioral studies, our results suggest that tonic inhibition mediated by ␣5GABAARs in hippocampal pyramidal neurons plays a key role in cognitive processes. The ␥-aminobutyric acid (GABA) subtype A receptor (GABA A R) is a pentameric anion-selective ion channel that assembles from different classes of subunits (␣1-6, ␤1-3, ␥1-3, ␦, , , and ) (1). The combination of various GABA A R subunits confers different biophysical and pharmacological properties and regulates regional and subcellular patterns of distribution (2, 3). The subunit composition critically determines agonist affinity, receptor kinetics, and sensitivity to a variety of clinically important drugs, including benzodiazepines and general anesthetics.Studies of gene-targeted mice have implicated specific GABA A R subunit isoforms in critical aspects of information processing in the brain. Notably, ␣5-null mutant mice (␣5Ϫ͞Ϫ) exhibit improved performance in the water maze model of spatial learning, a hippocampus-dependent learning task (4). Further, mice carrying a point mutation at position 105 of the ␣5 subunit (H105R) experience an unexpected selective reduction of ␣5GABA A Rs in hippocampal pyramidal neurons and improved performance for learning tasks (5). Pharmacological studies further support the involvement of ␣5GABA A Rs in learning processes; for example, ␣5 subunit-selective inverse agonists such as L-655,708 enhance learning performance in rats in the Morris water maze test (6, 7). Moreover, ␣5 subunitselective inverse agonists exhibit desirable nootropic effects without causing adverse convulsant activities associated with nonselective GABA A R inverse agonists. Thus, inhibition of ␣5GABA A Rs presents an attractive strategy for developing memory-enhancing drugs.The neuronal substrates underlying improved cognitive performance associated with reduced ␣5GABA A R function remain unknown. The ␣5 subunit has a unique and limited pattern of distribution in the mammalian brain. Although ␣5-containing receptors constitute Ͻ5% of the total GABA A R population...

We present a simple tool that uses PS alone to prognosticate in advanced cancer, and has similar discriminatory ability to more complex models.

Volatile (inhaled) anesthetics cause amnesia at concentrations well below those that cause loss of consciousness and immobility; however, the underlying neuronal mechanisms are unknown. Although many anesthetics increase inhibitory GABAergic synaptic transmission, this effect occurs only at high concentrations (Ͼ100 M). Molecular targets for low concentrations of inhaled anesthetics have not been identified. Here, we report that a tonic inhibitory conductance in hippocampal pyramidal neurons generated by ␣5 subunitcontaining GABA A receptors is highly sensitive to low concentrations of the volatile anesthetic isoflurane (ISO) (25 and 83.3 M). The ␣5 subunit is necessary for enhancement of the tonic current by these low concentrations of isoflurane because potentiation is absent in neurons from ␣5 Ϫ/Ϫ mice. Furthermore, ISO (25 M) potentiated recombinant human ␣5␤3␥2L GABA A receptors, whereas this effect was not seen with ␣1␤3␥2L GABA A receptors. These studies suggest that an increased tonic inhibition in the hippocampus may contribute to amnestic properties of volatile anesthetics.

Glycine receptors (GlyRs) are transmitter‐gated channels that mediate fast inhibitory neurotransmission in the spinal cord and brain. The GlyR β subunit contains a putative tyrosine phosphorylation site whose functional role has not been determined. To examine if protein tyrosine kinases (PTKs) regulate the function of GlyRs, we analysed whole‐cell currents activated by applications of glycine to CA1 hippocampal neurons and spinal neurons. The role of a putative site for tyrosine phosphorylation at position 413 of the β subunit was examined using site‐directed mutagenesis and expression of recombinant (α1βY413F) receptors in human embryonic kidney (HEK 293) cells. Lavendustin A, an inhibitor of PTKs, depressed glycine‐evoked currents (IGly) in CA1 neurons and spinal neurons by 31 % and 40 %, respectively. In contrast, the intracellular application of the exogenous tyrosine kinase, cSrc, enhanced IGly in CA1 neurons by 56 %. cSrc also accelerated GlyR desensitization and increased the potency of glycine 2‐fold (control EC50= 143 μm; cSrc EC50= 74 μm). Exogenous cSrc, applied intracellularly, upregulated heteromeric α1β receptors but not homomeric α1 receptors. Substitution mutation of the tyrosine to phenylalanine at position β‐413 prevented this enhancement. Furthermore, a selective inhibitor of the Src family kinases, PP2, down‐regulated wild‐type α1β but not α1βY413F receptors. Together, these findings indicate that GlyR function is upregulated by PTKs and this modulation is dependent on the tyrosine‐413 residue of the β subunit.

The mechanisms by which insulin modulates neuronal plasticity and pain processes remain poorly understood. Here we report that insulin rapidly increases the function of glycine receptors in murine spinal neurons and recombinant human glycine receptors expressed in human embryonic kidney cells. Whole-cell patch-clamp recordings showed that insulin reversibly enhanced current evoked by exogenous glycine and increased the amplitude of spontaneous glycinergic miniature inhibitory postsynaptic currents recorded in cultured spinal neurons. Insulin (1 M) also shifted the glycine concentration-response plot to the left and reduced the glycine EC 50 value from 52 to 31 M. Currents evoked by a submaximal concentration of glycine were increased to approximately 140% of control. The glycine receptor ␣ subunit was sufficient for the enhancement by insulin because currents from recombinant homomeric ␣ 1 receptors and heteromeric ␣ 1 ␤ receptors were both increased. Insulin acted at the insulin receptor via pathways dependent on tyrosine kinase and phosphatidylinositol 3 kinase because the insulin effect was eliminated by the insulin receptor antagonist, hydroxy-2-naphthalenylmethylphosphonic acid trisacetoxymethyl ester, the tyrosine kinase inhibitor lavendustin A, and the phosphatidylinositol 3 kinase antagonist wortmannin. Together, these results show that insulin has a novel regulatory action on the potency of glycine for ionotropic glycine receptors.

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