Guenther E, Rothe T, Taschenberger H, Grantyn R
Department of Neurophysiology, Max Planck Institute for Psychiatry, Martinsried, Germany.
Brain Res. 1994 Jan 7;633(1-2):223-35. doi: 10.1016/0006-8993(94)91543-1.
A culture system of the postnatal rat retina was established to investigate Ca2+ currents and synaptic transmission in identified neurons. Methods are described that allowed us to select retinal ganglion neurons (RGNs) in short term cultures (up to 48 h in vitro) and in long-term cultures (3 to 21 days in vitro). The specific aim of the present study was to identify channel specific components in whole-cell Ca2+ currents of RGNs and to clarify the potential use of the lanthanide Gd3+ as a selective Ca2+ channel blocker. About one third of freshly dissociated RGNs generated both low voltage activated Ca2+ currents (ICa(LVA)) and high voltage activated Ca2+ currents (ICa(HVA)). The remaining 2/3 or RGNs in short term culture and most RGNs in long-term culture displayed only ICa(HVA). The latter comprised at least three different components that were functionally rather similar, but could be separated pharmacologically. A significant portion (about 40%) of ICa(HVA) was irreversibly blocked by the N channel antagonist omega-CgTx (5 microM). The L channel antagonist nifedipine (10 microM) eliminated about 25% of ICa(HVA). Thus, about 1/3 of the HVA Ca2+ or Ba2+ current remained unaffected by either omega-CgTx or nifedipine. omega-AgaTx (200 nM) completely failed to block HVA Ca2+ or Ba2+ currents in RGNs. Gd3+ exerted contrasting actions on LVA and HVA Ca2+ currents. While ICa(LVA) consistently increased in the presence of Gd3+ (0.32-3.2 microM), ICa(HVA) always decreased, especially when using higher concentrations of Gd3+ (10-32 microM). The blocking action of Gd3+ was not restricted to the omega-CgTx-sensitive HVA current component, but also concerned omega-CgTx- and nifedipine-resistant components. The decay of Ca2+ currents was accelerated in the presence of Gd3+. Even in RGNs lacking ICa(LVA), application of 3.2 microM Gd3+ significantly reduced the time constant of decay from an average of 64 ms to 36 ms (voltage steps from -90 to 0 mV; 10 mM [Ca2+]o; 26 degrees C). This is in contrast to what had to be expected if an N-type HVA current component was selectively suppressed by Gd3+.Gd3+ diminished glutamatergic spontaneous synaptic activity in retinal cultures tested during the 3rd week in vitro. Both frequency and amplitude were reduced. Occasionally, the application was followed by a rebound increase of EPSC frequency. A stimulatory effect during application of Gd3+ has never been observed. These experiments indicate that RGNs express at least 4 different types of Ca2+ currents, that resemble in some aspects T, N and L channel currents.(ABSTRACT TRUNCATED AT 400 WORDS)
建立了产后大鼠视网膜培养系统，以研究特定神经元中的Ca2+电流和突触传递。本文描述了在短期培养（体外培养长达48小时）和长期培养（体外培养3至21天）中选择视网膜神经节神经元（RGNs）的方法。本研究的具体目的是确定RGNs全细胞Ca2+电流中的通道特异性成分，并阐明镧系元素Gd3+作为选择性Ca2+通道阻滞剂的潜在用途。约三分之一的新鲜分离RGNs产生低电压激活Ca2+电流（ICa(LVA)）和高电压激活Ca2+电流（ICa(HVA)）。短期培养中其余的2/3 RGNs和长期培养中的大多数RGNs仅显示ICa(HVA)。后者至少包含三个功能上相当相似但可通过药理学分离的不同成分。ICa(HVA)的很大一部分（约40%）被N通道拮抗剂ω-CgTx（5 microM）不可逆地阻断。L通道拮抗剂硝苯地平（10 microM）消除了约25%的ICa(HVA)。因此，约1/3的HVA Ca2+或Ba2+电流不受ω-CgTx或硝苯地平的影响。ω-AgaTx（200 nM）完全不能阻断RGNs中的HVA Ca2+或Ba2+电流。Gd3+对LVA和HVA Ca2+电流有相反的作用。在Gd3+（0.32 - 3.2 microM）存在的情况下，ICa(LVA)持续增加，而ICa(HVA)总是减少，尤其是使用较高浓度的Gd3+（10 - 32 microM）时。Gd3+的阻断作用不仅限于ω-CgTx敏感的HVA电流成分，还涉及ω-CgTx和硝苯地平耐药的成分。在Gd3+存在的情况下，Ca2+电流的衰减加速。即使在缺乏ICa(LVA)的RGNs中，应用3.2 microM Gd3+也显著将衰减时间常数从平均64毫秒降低到36毫秒（电压从 - 90步进到0 mV；10 mM [Ca2+]o；26℃）。这与如果N型HVA电流成分被Gd3+选择性抑制所预期的情况相反。Gd3+减少了体外培养第3周测试的视网膜培养物中的谷氨酸能自发突触活动。频率和幅度均降低。偶尔，应用后会出现EPSC频率的反弹增加。从未观察到Gd3+应用期间的刺激作用。这些实验表明，RGNs表达至少4种不同类型的Ca2+电流，在某些方面类似于T、N和L通道电流。（摘要截断于400字）
