GRAMICIDIN-PERFORATED PATCH RECORDING - GABA RESPONSE IN MAMMALIAN NEURONS WITH INTACT INTRACELLULAR CHLORIDE
GRAMICIDIN-PERFORATED PATCH RECORDING - GABA RESPONSE IN MAMMALIAN NEURONS WITH INTACT INTRACELLULAR CHLORIDE
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DOI：
10.1113/jphysiol.1995.sp020649
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发表时间：
1995-04-01
影响因子：
5.5
通讯作者：
AKAIKE, N
中科院分区：
文献类型：
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作者：
EBIHARA, S;SHIRATO, K;AKAIKE, N
1.通过建立一种新的短杆菌肽穿孔贴片法，在不干扰细胞内氯离子浓度的情况下，观察了GABA对大鼠黑质网状部（SNR）神经元的作用.使用含有短杆菌肽（100 μ g ml（-1））的贴片移液管溶液，在进行千兆欧姆密封后40分钟内，接入电阻下降到小于20 M欧姆。在电流钳条件下，GABA引起的超极化伴随着自发放电的封锁。在维持电位为-50 mV的电压钳下，GABA通过荷包牡丹碱和印防己毒素敏感的GABA（A）受体诱发外向电流.细胞外氯离子浓度的10倍变化导致GABA诱导的外向电流（E（GABA））的逆转电位偏移58 mV，表明膜在GABA存在下表现得像氯离子电极。使用细胞外氯离子活性和E（GABA）值用能斯特方程计算的细胞内氯离子活性（aCl（i））的范围为2.8至19.7 mM，平均值为9.5 mM。在短杆菌肽穿孔膜片钳技术记录脑片SNR神经元时，可记录到不同方向的抑制性和兴奋性突触后电流，前者可被荷包牡丹碱阻断.总之，短杆菌肽穿孔贴片法将揭示涉及Cl-的生物反应的以前未知的方面。
1. By the development of a new perforated patch method using gramicidin, the effects of GABA on neurones dissociated from the rat substantia nigra pars reticulata (SNR) were examined without disturbing the intracellular chloride concentration.2. Using the patch pipette solution containing gramicidin (100 mu g ml(-1)), the access resistance dropped to less than 20 M Ohm within 40 min after making the gigaohm seal.3. Under current-clamp conditions, GABA caused a hyperpolarization accompanied by a blockade of spontaneous firing. Under voltage clamp at a holding potential (V-h) of -50 mV, GABA evoked an outward current by way of bicuculline- and picrotoxin-sensitive GABA(A) receptors.4. A 10-fold change of extracellular chloride concentration resulted in a 58 mV shift of the reversal potential of GABA-induced outward current (E(GABA)), indicating that the membrane behaves like a chloride electrode in the presence of GABA.5. The intracellular chloride activities (aCl(i)), calculated with the Nernst equation using both extracellular chloride activity and E(GABA) values, ranged from 2.8 to 19.7 mM with a mean value of 9.5 mM. The aCl(i) was not affected either by different pipette solutions or by different holding potentials more hyperpolarized than -40 mV.6. In the recording from SNR neurones in brain slice using the gramicidin-perforated patch-clamp technique, the inhibitory and excitatory postsynaptic currents were recorded in different current directions and the former was blocked by bicuculline.7. In conclusion, the gramicidin-perforated patch method will disclose previously unknown aspects of biological responses involving Cl-.