A reinforcing circuit action of extrasynaptic GABAA receptor modulators on cerebellar granule cell inhibition.

GABAA receptors (GABARs) are the targets of a wide variety of modulatory drugs which enhance chloride flux through GABAR ion channels. Certain GABAR modulators appear to acutely enhance the function of δ subunit-containing GABAR subtypes responsible for tonic forms of inhibition. Here we identify a reinforcing circuit mechanism by which these drugs, in addition to directly enhancing GABAR function, also increase GABA release. Electrophysiological recordings in cerebellar slices from rats homozygous for the ethanol-hypersensitive (α6100Q) allele show that modulators and agonists selective for δ-containing GABARs such as THDOC, ethanol and THIP (gaboxadol) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in granule cells. Ethanol fails to augment granule cell sIPSC frequency in the presence of glutamate receptor antagonists, indicating that circuit mechanisms involving granule cell output contribute to ethanol-enhancement of synaptic inhibition. Additionally, GABAR antagonists decrease ethanol-induced enhancement of Golgi cell firing. Consistent with a role for glutamatergic inputs, THIP-induced increases in Golgi cell firing are abolished by glutamate receptor antagonists. Moreover, THIP enhances the frequency of spontaneous excitatory postsynaptic currents in Golgi cells. Analyses of knockout mice indicate that δ subunit-containing GABARs are required for enhancing GABA release in the presence of ethanol and THIP. The limited expression of the GABAR δ subunit protein within the cerebellar cortex suggests that an indirect, circuit mechanism is responsible for stimulating Golgi cell GABA release by drugs selective for extrasynaptic isoforms of GABARs. Such circuit effects reinforce direct actions of these positive modulators on tonic GABAergic inhibition and are likely to contribute to the potent effect of these compounds as nervous system depressants

Conformational Dynamics of hSGLT1 during Na+/Glucose Cotransport

This study examines the conformations of the Na+/glucose cotransporter (SGLT1) during sugar transport using charge and fluorescence measurements on the human SGLT1 mutant G507C expressed in Xenopus oocytes. The mutant exhibited similar steady-state and presteady-state kinetics as wild-type SGLT1, and labeling of Cys507 by tetramethylrhodamine-6-maleimide had no effect on kinetics. Our strategy was to record changes in charge and fluorescence in response to rapid jumps in membrane potential in the presence and absence of sugar or the competitive inhibitor phlorizin. In Na+ buffer, step jumps in membrane voltage elicited presteady-state currents (charge movements) that decay to the steady state with time constants τmed (3–20 ms, medium) and τslow (15–70 ms, slow). Concurrently, SGLT1 rhodamine fluorescence intensity increased with depolarizing and decreased with hyperpolarizing voltages (ΔF). The charge vs. voltage (Q-V) and fluorescence vs. voltage (ΔF-V) relations (for medium and slow components) obeyed Boltzmann relations with similar parameters: zδ (apparent valence of voltage sensor) ≈ 1; and V0.5 (midpoint voltage) between −15 and −40 mV. Sugar induced an inward current (Na+/glucose cotransport), and reduced maximal charge (Qmax) and fluorescence (ΔFmax) with half-maximal concentrations (K0.5) of 1 mM. Increasing [αMDG]o also shifted the V0.5 for Q and ΔF to more positive values, with K0.5's ≈ 1 mM. The major difference between Q and ΔF was that at saturating [αMDG]o, the presteady-state current (and Qmax) was totally abolished, whereas ΔFmax was only reduced 50%. Phlorizin reduced both Qmax and ΔFmax (Ki ≈ 0.4 μM), with no changes in V0.5's or relaxation time constants. Simulations using an eight-state kinetic model indicate that external sugar increases the occupancy probability of inward-facing conformations at the expense of outward-facing conformations. The simulations predict, and we have observed experimentally, that presteady-state currents are blocked by saturating sugar, but not the changes in fluorescence. Thus we have isolated an electroneutral conformational change that has not been previously described. This rate-limiting step at maximal inward Na+/sugar cotransport (saturating voltage and external Na+ and sugar concentrations) is the slow release of Na+ from the internal surface of SGLT1. The high affinity blocker phlorizin locks the cotransporter in an inactive conformation

A reinforcing circuit action of extrasynaptic GABAA receptor modulators on cerebellar granule cell inhibition.

GABAA receptors (GABARs) are the targets of a wide variety of modulatory drugs which enhance chloride flux through GABAR ion channels. Certain GABAR modulators appear to acutely enhance the function of δ subunit-containing GABAR subtypes responsible for tonic forms of inhibition. Here we identify a reinforcing circuit mechanism by which these drugs, in addition to directly enhancing GABAR function, also increase GABA release. Electrophysiological recordings in cerebellar slices from rats homozygous for the ethanol-hypersensitive (α6100Q) allele show that modulators and agonists selective for δ-containing GABARs such as THDOC, ethanol and THIP (gaboxadol) increased the frequency of spontaneous inhibitory postsynaptic currents (sIPSCs) in granule cells. Ethanol fails to augment granule cell sIPSC frequency in the presence of glutamate receptor antagonists, indicating that circuit mechanisms involving granule cell output contribute to ethanol-enhancement of synaptic inhibition. Additionally, GABAR antagonists decrease ethanol-induced enhancement of Golgi cell firing. Consistent with a role for glutamatergic inputs, THIP-induced increases in Golgi cell firing are abolished by glutamate receptor antagonists. Moreover, THIP enhances the frequency of spontaneous excitatory postsynaptic currents in Golgi cells. Analyses of knockout mice indicate that δ subunit-containing GABARs are required for enhancing GABA release in the presence of ethanol and THIP. The limited expression of the GABAR δ subunit protein within the cerebellar cortex suggests that an indirect, circuit mechanism is responsible for stimulating Golgi cell GABA release by drugs selective for extrasynaptic isoforms of GABARs. Such circuit effects reinforce direct actions of these positive modulators on tonic GABAergic inhibition and are likely to contribute to the potent effect of these compounds as nervous system depressants

Blocking glutamatergic transmission abolishes ethanol-effects on granule cell sIPSC frequency.

<p><i>A.</i> Representative granule cell current traces recorded in ACSF (CON, left panel) and during perfusion of 50 mM ethanol (EtOH, middle panel). Cumulative probability plots (right panel) compare the frequency of individual sIPSC in ACSF (CON) and after perfusion of 50 mM ethanol (same number of events from <i>n</i> = 5 cells; <i>p</i><0.05 by K-S test). <i>B</i>. Granule cell current traces recorded in the presence of the GluR antagonists 20 µM APV and 25 µM DNQX (CON, left panel) and after inclusion of 50 mM ethanol (EtOH, middle panel). Cumulative probability plots (right panel) show the frequency distribution of individual sIPSCs in APV/DNQX (CON) and after addition of 50 mM ethanol (identical number of events from <i>n</i> = 6 cells; <i>p</i><0.05 by K-S test). <i>C</i>. Summary data of the sIPSC frequency averaged over 30 second periods in control conditions before addition of ethanol (CON) and in the presence 50 mM ethanol. The frequency of sIPSCs recorded in ACSF is compared with the effect observed in DNQX/APV (# indicates p<0.05 by two-way repeated measures ANOVA followed by post-hoc analyses by Holm-Sidak method). <i>D</i>. Summary data of the sIPSC frequency averaged over 30 second periods in control conditions before addition of ethanol (CON) and in the presence 10 mM ethanol. Individual data points are represented by open circles connected by dotted lines. (** indicates p<0.05 by Wilcoxon Signed Rank test). <i>F</i>. Summary histogram of the sIPSC frequency in ethanol, normalized to frequency in the same cell prior to perfusion of ethanol (n.s. denotes p>0.05, * indicates p<0.05 by paired <i>t</i>-test and ## indicates p<0.05 by one-way ANOVA followed by post-hoc analyses by Holm-Sidak method).</p

Ethanol does not modulate sIPSC amplitude or decay.

<p><i>A.</i> Overlay of normalized granule cell sIPSC traces recorded in ACSF show that there was no difference in the IPSC kinetics under control conditions (black) and in 50mM ethanol (gray). <i>B.</i> Overlay of normalized granule cell sIPSC traces recorded in DNQX (25 µM) and APV (20 µM) show no difference in IPSC kinetics in the absence (black) and in 50mM ethanol (gray). <i>C</i>. Summary plots of sIPSC amplitude before and during ethanol perfusion illustrate the lack of ethanol-modulation both in ACSF and in GluR antagonists (DNQX/APV). <i>D</i>. Histogram of the weighted τ_decay of granule cell sIPSCs in ACSF in GluR antagonists (DNQX/APV) recorded before and during ethanol perfusion.</p

Blocking GABA or glutamate receptors reduces enhancement of Golgi cell firing by extrasynaptic GABAR agonists.

<p><i>A.</i> Example trace of loose-patch cell-attached recordings from a Golgi cell illustrates the spontaneous firing in ACSF (upper panel) and the increase in firing in the presence of 50 mM ethanol (lower panel). <i>B.</i> Golgi cell firing recorded in the presence of gabazine (10 µM) in loose-patch mode before (upper panel) and during application of 50 mM ethanol (lower panel). <i>C</i>. Summary plots of the frequency of Golgi cell firing in 50 mM ethanol, normalized to the firing frequency before ethanol perfusion in ACSF (white bar) and in gabazine (black bar). <i>D</i>. Summary plots of the frequency of Golgi cell firing in 1 µM THIP, normalized to the firing frequency before THIP perfusion in ACSF (white bar) and in 3 mM kynurenic acid and 20 µM APV (black bar). * indicates p<0.05 by paired <i>t</i>-test.</p

Schematic of the circuit hypothesis for ethanol-enhancement of synaptic GABA release from Golgi neurons.

<p><i>A.</i> Schematic of the connectivity between cerebellar granule cells and Golgi cells including the inhibitory connections between Golgi cells identified in recent studies <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072976#pone.0072976-Hull1" target="_blank">[33]</a>. Location of extrasynaptic GABARs in granule cell somata and parallel fiber axons is illustrated. Prediction (right panel), illustrates the proposed changes in neuronal activity in the circuit following increased activation of parallel fiber GABARs. <i>B</i>. Hypothesized effect of GluR antagonists on circuit activity following increase in activation of granule cell extrasynaptic GABARs. Red X indicates blocked glutamatergic synapses. <i>C</i>. Predicted effects of GABAR antagonists on granule and Golgi cell activity following increased activation of granule cell extrasynaptic GABARs. Red X indicates blocked GABA synapses and orange X denotes block of both somato-dendritic and axonal extrasynaptic GABARs.</p

THIP increases Golgi cell sEPSC frequency.

<p><i>A.</i> Representative Golgi cell current recordings obtained with a low chloride internal solution at a holding potential of –70 mV. Recordings in control ACSF (upper panel) and after perfusion of 1 µM THIP (lower panel) illustrate the increase in frequency of spontaneous excitatory synaptic events in THIP. <i>B</i>. Summary plot shows the effect of THIP on Golgi cell sEPSC frequency. Individual data points are represented by open circles connected by dotted lines. * indicates p<0.05 by paired <i>t</i>-test.</p

Ethanol enhances tonic GABA currents without increasing sIPSC frequency in the presence of glutamate receptor antagonists.

<p><i>A</i>. Voltage clamp recordings from a granule cell illustrates the effect of ethanol (50 mM) on synaptic and tonic GABA currents recorded in the presence of the GluR antagonists APV (20 µM) and kynurenic acid (KyA, 3 mM). To the right are histograms of all points in each segment. Tonic GABA currents were measured as the baseline current blocked by GABAR antagonist gabazine (10 µM). Gaussian fits to the positive half of the current trace under each condition are superimposed (right panel). The dashed lines indicate the mean current from these fits and difference currents are noted. <i>B</i>. Plot illustrates the time course of changes in baseline currents, averaged over 100 ms time intervals, during perfusion of ethanol (50 mM) and subsequent washout of ethanol. Baseline current averages during gabazine perfusion at the end of the recordings were used to measure tonic GABA current amplitude. Segments of representative current traces in A and baseline currents in B were obtained from the same recordings <i>C.</i> Summary plot of the effect of ethanol on granule cell sIPSC frequency in the presence of ACSF and in KyA/APV. In each case, sIPSC frequency was averaged over a 30 second recording periods. <i>C</i>. Histogram shows that the amplitude of tonic GABA currents in ethanol (50 mM) normalized to the tonic GABA current amplitude in the same cell prior to perfusion of ethanol, both in ACSF and in KyA/APV. * indicates p<0.05 by paired <i>t</i>-test.</p