Department of Anesthesiology, University of Pittsburgh, Pittsburgh, PA, USA.
Neuroscience. 2012 Apr 19;208:133-42. doi: 10.1016/j.neuroscience.2012.01.050. Epub 2012 Feb 8.
Despite evidence that high-affinity GABA(A) 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 GABA(A) currents in DRG neurons. To test this hypothesis, we studied dissociated DRG neurons 2 h (acute) and 24 h (cultured) after plating with whole-cell patch-clamp techniques, Western blot, and semiquantitative reverse transcriptase polymerase chain reaction (sqRT-PCR) analysis. GABA(A) 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 ρ3-subunit mRNA in cultured DRG neurons. Our results suggest that high-affinity δ-subunit-containing GABA(A) 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.
尽管有证据表明高亲和力 GABA(A) 受体亚基 mRNA 和蛋白存在于背根神经节 (DRG) 中，但在体外急性分离的 DRG 神经元中检测到的低亲和力电流占主导地位。这一观察结果提出了一种可能性，即高亲和力受体通常被运出 DRG 到外周和中枢末端。因此，我们假设在培养过程中，DRG 神经元中的高亲和力 GABA(A) 电流会增加。为了验证这一假设，我们使用全细胞膜片钳技术、Western blot 和半定量逆转录聚合酶链反应 (sqRT-PCR) 分析研究了培养 2 小时 (急性) 和 24 小时 (培养) 后的分离 DRG 神经元。随着培养时间的延长，GABA(A) 电流密度显著增加，同时出现两种持续电流，其 EC50 分别为 0.25±0.01 μM 和 3.2±0.02 μM，用于 GABA 激活。在亚群神经元中，随着培养时间的延长，瞬时电流对 GABA 的激活效力也从 EC50 的 78.16±10.1 μM 增加到 9.56±1.3 μM。一部分高亲和力电流被 δ 亚基激动剂 4,5,6,7-四氢异恶唑[5,4-c]吡啶-3-醇 (THIP) 增强。δ 亚基免疫反应性在急性 DRG 神经元中主要局限于细胞质部分，但在培养的 DRG 神经元中局限于膜部分，δ 亚基 mRNA 没有检测到变化。然而，在培养的神经元亚群中也检测到一种被 THIP 阻断且对 BIC 不敏感的高亲和力电流，同时在培养的 DRG 神经元中 ρ2-和 ρ3-亚基 mRNA 增加。我们的结果表明，正常情况下，高亲和力 δ 亚基 GABA(A) 受体被运出 DRG，然后被靶向到外周和中枢过程。它们还强调，从培养的 DRG 神经元中获得的数据的解释应该谨慎进行。
