Assessment of Methods for the Intracellular Blockade of GABAA Receptors.
Assessment of Methods for the Intracellular Blockade of GABAA Receptors.
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DOI：
10.1371/journal.pone.0160900
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发表时间：
2016
期刊：
影响因子：
3.7
通讯作者：
Mellor JR
中科院分区：
文献类型：
--
作者：
Atherton LA;Burnell ES;Mellor JR
选择性阻断抑制性突触传递到特定神经元是解剖正在进行的网络活动的兴奋性和抑制性突触成分的有用工具。为了实现这一目标，使用能够阻断GABAA受体的膜片溶液进行细胞内记录比其他操作（如GABAA能拮抗剂的药理学应用或对中间神经元群体的光遗传抑制）具有优势，因为大多数抑制传递不受影响，因此保留了剩余的网络活性。在这里，我们评估了先前描述的三种阻断抑制的方法：细胞内应用分子微毒素，4,4 ' -二硝基二苯乙烯-2,2 ' -二磺酸（DNDS）和4,4 ' -二异硫氰基二苯乙烯-2,2 ' -二磺酸（DIDS）。研究发现，DNDS和微毒素对阻断小鼠CA1锥体细胞上的单突触抑制突触后电流（IPSCs）均无效。含有DIDS和氟化铯的细胞内溶液，但缺乏核苷酸ATP和GTP，可有效降低IPSCs的振幅。然而，发现这种作用与DIDS和细胞内核苷酸的缺乏无关，而是由于细胞内溶液中氟离子的存在，氟离子也阻断了海马锐波期间自发产生的IPSCs。关键的是，细胞内氟离子也引起自发和诱发兴奋性突触电流的减少，并阻止核苷酸在细胞内溶液中的包含。因此，在所测试的方法中，只有氟离子对IPSCs的细胞内阻断有效，但这种方法具有额外的细胞效应，降低了其选择性和实用性。
Selective blockade of inhibitory synaptic transmission onto specific neurons is a useful tool for dissecting the excitatory and inhibitory synaptic components of ongoing network activity. To achieve this, intracellular recording with a patch solution capable of blocking GABAA receptors has advantages over other manipulations, such as pharmacological application of GABAergic antagonists or optogenetic inhibition of populations of interneurones, in that the majority of inhibitory transmission is unaffected and hence the remaining network activity preserved. Here, we assess three previously described methods to block inhibition: intracellular application of the molecules picrotoxin, 4,4’-dinitro-stilbene-2,2’-disulphonic acid (DNDS) and 4,4’-diisothiocyanostilbene-2,2’-disulphonic acid (DIDS). DNDS and picrotoxin were both found to be ineffective at blocking evoked, monosynaptic inhibitory postsynaptic currents (IPSCs) onto mouse CA1 pyramidal cells. An intracellular solution containing DIDS and caesium fluoride, but lacking nucleotides ATP and GTP, was effective at decreasing the amplitude of IPSCs. However, this effect was found to be independent of DIDS, and the absence of intracellular nucleotides, and was instead due to the presence of fluoride ions in this intracellular solution, which also blocked spontaneously occurring IPSCs during hippocampal sharp waves. Critically, intracellular fluoride ions also caused a decrease in both spontaneous and evoked excitatory synaptic currents and precluded the inclusion of nucleotides in the intracellular solution. Therefore, of the methods tested, only fluoride ions were effective for intracellular blockade of IPSCs but this approach has additional cellular effects reducing its selectivity and utility.