Bestrophin1 Channels are Insensitive to Ethanol and Do not Mediate Tonic GABAergic Currents in Cerebellar Granule Cells

The granule cell layer of the cerebellum functions in spatio-temporal encoding of information. Granule cells (GCs) are tonically inhibited by spillover of GABA released from Golgi cells and this tonic inhibition is facilitated by acute ethanol. Recently, it was demonstrated that a specialized Ca2+-activated anion-channel, bestrophin1 (Best1), found on glial cells, can release GABA that contributes up to 50–75% of the tonic GABAergic current. However, it is unknown if ethanol has any actions on Best1 function. Using whole-cell electrophysiology, we found that recombinant Best1 channels expressed in HEK-293 cells were insensitive to 40 and 80 mM ethanol. We attempted to measure the Best1-mediated component of the tonic current in slices using 5-nitro-2-(3-phenylpropylamino)benzoic acid (NPPB). We confirmed that this agent blocks recombinant Best1 channels. Unexpectedly, we found that NPPB significantly potentiated the tonic current and the area and decay of GABAA-mediated spontaneous inhibitory post-synaptic currents (IPSCs) in GCs in rodent slices under two different recording conditions. To better isolate the Best1-dependent tonic current component, we blocked the Golgi cell component of the tonic current with tetrodotoxin and found that NPPB similarly and significantly potentiated the tonic current amplitude and decay time of miniature IPSCs. Two other Cl−-channel blockers were also tested: 4′-diisothiocyanatostilbene-2,2′-disulfonic acid disodium salt hydrate (DIDS) showed no effect on GABAergic transmission, while niflumic acid (NFA) significantly suppressed the tonic current noise, as well as the mIPSC frequency, amplitude, and area. These data suggest that acute ethanol exposure does not modulate Best1 channels and these findings serve to challenge recent data indicating that these channels participate in the generation of tonic GABAergic currents in cerebellar GCs

The endocannabinoid system controls food intake via olfactory processes

Comment in Sensory systems: the hungry sense. [Nat Rev Neurosci. 2014] Inhaling: endocannabinoids and food intake. [Nat Neurosci. 2014]; International audience; Hunger arouses sensory perception, eventually leading to an increase in food intake, but the underlying mechanisms remain poorly understood. We found that cannabinoid type-1 (CB1) receptors promote food intake in fasted mice by increasing odor detection. CB1 receptors were abundantly expressed on axon terminals of centrifugal cortical glutamatergic neurons that project to inhibitory granule cells of the main olfactory bulb (MOB). Local pharmacological and genetic manipulations revealed that endocannabinoids and exogenous cannabinoids increased odor detection and food intake in fasted mice by decreasing excitatory drive from olfactory cortex areas to the MOB. Consistently, cannabinoid agonists dampened in vivo optogenetically stimulated excitatory transmission in the same circuit. Our data indicate that cortical feedback projections to the MOB crucially regulate food intake via CB1 receptor signaling, linking the feeling of hunger to stronger odor processing. Thus, CB1 receptor-dependent control of cortical feedback projections in olfactory circuits couples internal states to perception and behavior

Na+/K+-ATPase inhibition partially mimics the ethanol-induced increase of the Golgi cell-dependent component of the tonic GABAergic current in rat cerebellar granule cells.

Cerebellar granule cells (CGNs) are one of many neurons that express phasic and tonic GABAergic conductances. Although it is well established that Golgi cells (GoCs) mediate phasic GABAergic currents in CGNs, their role in mediating tonic currents in CGNs (CGN-I(tonic)) is controversial. Earlier studies suggested that GoCs mediate a component of CGN-I(tonic) that is present only in preparations from immature rodents. However, more recent studies have detected a GoC-dependent component of CGN-I(tonic) in preparations of mature rodents. In addition, acute exposure to ethanol was shown to potentiate the GoC component of CGN-I(tonic) and to induce a parallel increase in spontaneous inhibitory postsynaptic current frequency at CGNs. Here, we tested the hypothesis that these effects of ethanol on GABAergic transmission in CGNs are mediated by inhibition of the Na(+)/K(+)-ATPase. We used whole-cell patch-clamp electrophysiology techniques in cerebellar slices of male rats (postnatal day 23-30). Under these conditions, we reliably detected a GoC-dependent component of CGN-I(tonic) that could be blocked with tetrodotoxin. Further analysis revealed a positive correlation between basal sIPSC frequency and the magnitude of the GoC-dependent component of CGN-I(tonic). Inhibition of the Na(+)/K(+)-ATPase with a submaximal concentration of ouabain partially mimicked the ethanol-induced potentiation of both phasic and tonic GABAergic currents in CGNs. Modeling studies suggest that selective inhibition of the Na(+)/K(+)-ATPase in GoCs can, in part, explain these effects of ethanol. These findings establish a novel mechanism of action of ethanol on GABAergic transmission in the central nervous system

TTX-induced decreases of CGN-I_tonic and sIPSCs are correlated.

<p>Representative traces of the effect of TTX and subsequent gabazine application on (A) CGN-I_tonic and (B) sIPSC frequency and amplitude. Time courses of the effect of TTX on (C) normalized amplitude of the CGN-I_tonic and (D) sIPSC frequency. Plots of the (E) CGN-I_tonic and (F) sIPSC frequencies of individual cells before and after TTX; * <i>p</i><0.05, paired t-test. (G) Correlation between basal sIPSC frequency (Hz) and TTX-induced change in tonic current amplitude (pA); slope significantly different from zero, <i>p</i><0.05, r² = 0.63.</p

Effect of ouabain on the frequency and amplitude of simulated CGN IPSPs (phasic component only).

<p>IPSP traces for (A1) control condition and (A2) in the presence of ouabain with phasic component only. Effect of ouabain on (B1) inter-event interval and (B2) amplitude in the presence of the phasic component only. Traces represent recorded inter-event intervals and amplitudes of 50 CGNs.</p

Effect of ouabain on the frequency and amplitude of simulated CGN IPSPs (phasic and perisynaptic components).

<p>IPSP traces for (A1) control condition and (A2) in the presence of ouabain including both phasic and perisynaptic “spillover” components. The effect of ouabain on (B1) inter-event interval and (B2) amplitude with both phasic and perisynaptic “spillover” components present. Traces represent recorded inter-event intervals and amplitudes of 50 CGNs.</p

Structure of the network computer model.

<p>(A) The model consists of three populations. A two dimensional layer represents each population. The GoC population is indicated by black color, CGN in blue, mossy fibres (MFs) in green, and the parallel fibers (PFs) emanating from CGNs are shown in red. (B) Postsynaptic receptors of a modeled GoC and neurotransmitters activating them. Each modeled GoC has AMPA receptors activated by glutamate either from MFs or PFs. The Na⁺/K⁺-ATPase has been incorporated in the soma of the model. (C) CGNs have NMDA and AMPA receptors activated by glutamate from MFs and GABA receptors activated by GABA neurotransmission from GoCs. CGNs also have two types of GABA_A receptors on them: GABA_AR1 (phasic) that has rapid rise (0.31 ms) and decay time (8.8 ms) and GABA_AR2 (perisynaptic) that has slow rise (6.8 ms) and decay time (232.5 ms). D) Sample traces illustrating the events mediated by synaptic and perisynaptic receptors.</p

The magnitude of tonic current amplitudes and sIPSC frequencies are not correlated.

<p>Exemplar traces of two cells with similar tonic current amplitudes (A1 – 21.6 pA and B1 – 20.5 pA), but different sIPSC frequencies (A2 – 0.24 Hz and B2 – 0.73 Hz). Dashed lines and arrows illustrate that the CGN-I_tonic amplitude during baseline and in the presence of gabazine is nearly identical in both exemplar traces. (C) Plot of basal sIPSC frequency (Hz) versus basal current amplitude (pA) shows no correlation (slope not significantly different from zero, <i>p</i>>0.05, r² = 0.012).</p

Ouabain partially mimics the EtOH-induced, action potential-dependent potentiation of CGN-I_tonic.

<p>Exemplar traces of the effect of (A1) 80 mM EtOH, (B1), 0.1 µM ouabain, and (C1) 0.1 µM ouabain in the presence of TTX on the CGN-I_tonic. Summary of the effect of (A2) EtOH, (B2) ouabain, and (C2) TTX+ouabain on the amplitude of CGN-I_tonic. * <i>p</i><0.05, paired t-test.</p