Taschenberger H, Grantyn R
Developmental Neurobiology Group, Max Planck Institute for Psychiatry, Martinsried, Germany.
J Neurosci. 1995 Mar;15(3 Pt 2):2240-54. doi: 10.1523/JNEUROSCI.15-03-02240.1995.
A dissociated cell culture from the postnatal rat retina was established to characterize the synapses formed by retinal ganglion neurons (RGNs) in vitro. An antibody against Thy-1.1 was used to preselect putative RGNs for pair patch-clamp recording with the principal aim of identifying the released transmitter(s) and estimating the role of different types of voltage-activated Ca2+ channels in evoked transmitter release. The population of Thy-1+ neurons was heterogeneous. Staining patterns, soma-dendritic geometries and axon length displayed variations that could be related to basic electrophysiological properties, such as amplitudes of voltage-activated Na+ currents (INa(V)), action potential size and capacity for repetitive discharge. Out of 73 coupled connections, 33 pairs were glutamatergic. With no exception, these connections were formed by the axons of strongly labeled Thy-1+ neurons with large INa(V) (typically > 2 nA) and repetitive firing over a broad current range. Such neurons were classified as RGNs. Forty out of 73 coupled pairs were GABAergic. These connections were always formed by weakly stained Thy-1+ neurons with small INa(V) (typically < 2 nA) and very limited capacity for repetitive discharge. Such neurons were tentatively classified as displaced amacrine cells. Evoked EPSCs in response to RGN activation were completely blocked by low concentrations of Cd2+ or Gd3+. omega-CgTx-GVIA (5 microM) reduced EPSCs to 67 +/- 29%, omega-AgaTx-IVA (200 nM) had no effect, and nifedipine (15 microM) enhanced the evoked EPSCs. Our experiments indicate that (1) the transmitter released by RGNs is glutamate and (2) the major part of synaptic glutamate release is governed by a novel toxin-resistant Ca2+ channel. The results further suggest that the characteristic phenotype of RGNs is well maintained in dissociated cell culture. In conjunction with electrophysiological tests Thy-1+ labeling can be used for RGN identification.
建立了出生后大鼠视网膜的解离细胞培养体系，以在体外表征视网膜神经节神经元（RGNs）形成的突触。使用抗Thy-1.1抗体预先选择假定的RGNs用于配对膜片钳记录，主要目的是鉴定释放的递质，并评估不同类型的电压激活Ca2+通道在诱发递质释放中的作用。Thy-1+神经元群体是异质的。染色模式、胞体-树突几何形状和轴突长度表现出的变化可能与基本电生理特性有关，如电压激活Na+电流（INa(V)）的幅度、动作电位大小和重复放电能力。在73对耦合连接中，33对是谷氨酸能的。无一例外，这些连接由强标记的Thy-1+神经元的轴突形成，这些神经元具有大的INa(V)（通常>2 nA），并在宽电流范围内重复放电。这类神经元被归类为RGNs。73对耦合对中有40对是GABA能的。这些连接总是由弱染色的Thy-1+神经元形成，这些神经元具有小的INa(V)（通常<2 nA），重复放电能力非常有限。这类神经元暂被归类为移位无长突细胞。对RGN激活的反应中诱发的兴奋性突触后电流（EPSCs）被低浓度的Cd2+或Gd3+完全阻断。ω-芋螺毒素GVIA（5 μM）将EPSCs降低到67±29%，ω-阿加毒素IVA（200 nM）没有作用，硝苯地平（15 μM）增强了诱发的EPSCs。我们的实验表明：（1）RGNs释放的递质是谷氨酸；（2）突触谷氨酸释放的主要部分由一种新型的抗毒素Ca2+通道控制。结果进一步表明，RGNs的特征表型在解离细胞培养中得到了很好的维持。结合电生理测试，Thy-1+标记可用于RGNs的鉴定。
