Upregulation of high-affinity GABA(A) receptors in cultured rat dorsal root ganglion neurons.
Upregulation of high-affinity GABA(A) receptors in cultured rat dorsal root ganglion neurons.
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DOI：
10.1016/j.neuroscience.2012.01.050
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发表时间：
2012-04-19
期刊：
影响因子：
3.3
通讯作者：
Gold, M. S.
中科院分区：
文献类型：
--
作者：
Lee, K. Y.;Charbonnet, M.;Gold, M. S.
尽管有证据表明高亲和力的GABAA受体亚单位mRNA和蛋白存在于背根神经节(DRG)中，但在体外急性分离的DRG神经元中，低亲和力电流占主导地位。这一观察结果提出了这样一种可能性，即高亲和力受体通常从背根节流向中枢和外周终末。因此，我们假设，随着培养时间的延长，DRG神经元的高亲和力GABAA电流将会增加。为了验证这一假设，我们用全细胞膜片钳技术、Western印迹和qRT-PCR分析研究了培养后2小时(急性)和24小时(培养)的分离的背根节神经元。随着培养时间的延长，GABA的电流密度急剧增加，并出现两种持续电流，激活GABA的EC50值分别为0.2 5±0.0 1μM和3.2±0.0 2μM。在神经元亚群中，随着培养时间的延长，瞬时电流激活GABA的效力也从78.16±10.1μM增加到9.56±1.3μM。部分高亲和力电流可被THIP亚基激动剂4，5，6，7-tetrahydroisoxazolo[5，4-c]pyridine-3-ol(δ)增强。急性期背根神经节细胞δ亚单位免疫反应主要局限于胞浆部分，而培养的背根神经节细胞膜部分δ亚单位表达无明显变化。然而，在培养的神经元亚群中发现了一种被THIP阻断的、对荷包牡丹碱不敏感的高亲和力电流，并与培养的背根节神经元中ρ2和3亚单位基因的表达增加有关。我们的结果表明，含有GABA受体的高亲和力δ亚单位通常被运输出背根神经节，在那里它们被靶向外周和中央突起。他们还强调，对从培养的背根节神经元获得的数据的解释应该谨慎。
Despite evidence that high-affinity GABAA receptor subunit mRNA and protein are present in dorsal root ganglia (DRG), low affinity currents dominate those detected in acutely dissociated DRG neurons in vitro. This observation raises the possibility that high affinity receptors are normally trafficked out of the DRG toward central and peripheral terminals. We therefore hypothesized that with time in culture, there would be an increase in high-affinity GABAA currents in DRG neurons. To test this hypothesis, we studied dissociated DRG neurons 2 hrs (acute) and 24 hrs (cultured) after plating with whole cell patch clamp techniques, western blot and qRT-PCR analysis. GABAA current density increases dramatically with time in culture in association with the emergence of two persistent currents with EC50’s of 0.25 ± 0.01 μM and 3.2 ± 0.02 μM for GABA activation. In a subpopulation of neurons, there was also an increase in the potency of GABA activation of the transient current from an EC50 of 78.16 ± 10.1 μM to 9.56 ± 1.3 μM with time in culture. A fraction of the high affinity current was potentiated by δ-subunit agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridine-3-ol (THIP). δ-subunit immunoreactivity was largely restricted to the cytosolic fraction in acute but the membrane fraction in cultured DRG neurons with no detectable change in δ-subunit mRNA. However, the emergence of a high affinity current blocked by THIP and insensitive to bicuculline was detected in a subpopulation of cultured neurons as well in association with an increase in ρ2 and 3-subunit mRNA in cultured DRG neurons. Our results suggest that high-affinity δ-subunit containing GABAA receptors are normally trafficked out of the DRG where they are targeted to peripheral and central processes. They also highlight that the interpretation of data obtained from cultured DRG neurons should be made with caution.
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影响因子：
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