Diaz Marvin R, Wadleigh Aya, Hughes Benjamin A, Woodward John J, Valenzuela C Fernando
Department of Neurosciences, University of New Mexico Health Sciences Center Albuquerque, NM, USA.
Front Neurosci. 2012 Jan 11;5:148. doi: 10.3389/fnins.2011.00148. eCollection 2011.
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 Ca(2+)-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 GABA(A)-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.
小脑颗粒细胞层在信息的时空编码中发挥作用。颗粒细胞（GCs）受到从高尔基细胞释放的GABA溢出的持续性抑制，而急性乙醇会促进这种持续性抑制。最近，研究表明在胶质细胞上发现的一种特殊的钙激活阴离子通道——最佳rophin1（Best1），可以释放GABA，其对持续性GABA能电流的贡献高达50 - 75%。然而，乙醇是否对Best1功能有任何作用尚不清楚。使用全细胞膜片钳电生理学技术，我们发现HEK - 293细胞中表达的重组Best1通道对40和80 mM乙醇不敏感。我们试图使用5 - 硝基 - 2 -（3 - 苯丙基氨基）苯甲酸（NPPB）来测量切片中持续性电流的Best1介导成分。我们证实该试剂可阻断重组Best1通道。出乎意料的是，我们发现在两种不同的记录条件下，NPPB显著增强了啮齿动物切片中GCs的持续性电流以及GABA(A)介导的自发性抑制性突触后电流（IPSCs）的面积和衰减。为了更好地分离Best1依赖的持续性电流成分，我们用河豚毒素阻断了持续性电流的高尔基细胞成分，发现NPPB同样显著增强了微小IPSCs的持续性电流幅度和衰减时间。还测试了另外两种Cl(-)通道阻滞剂：4'-二异硫氰酸芪 - 2,2'-二磺酸二钠盐（DIDS）对GABA能传递没有影响，而氟灭酸（NFA）显著抑制了持续性电流噪声以及微小IPSC的频率、幅度和面积。这些数据表明急性乙醇暴露不会调节Best1通道，这些发现对最近表明这些通道参与小脑GCs中持续性GABA能电流产生的数据提出了挑战。
