Lipton S A
Department of Neurology, Children's Hospital, Boston, Massachusetts 02115.
J Neurosci. 1988 Oct;8(10):3857-68. doi: 10.1523/JNEUROSCI.08-10-03857.1988.
Nictonic cholinergic responses have been previously documented in mammalian retinal ganglion cells. In the present study, dissociated retinal cells were densely plated and grown in a specific batch of rat serum. When held at negative potentials during whole-cell recording with a patch electrode, the retinal ganglion cells located close to presumptive cholinergic amacrine cells in these cultures were found to respond to acetylcholine (ACh; 20-200 microM) with an apparent outward current in the presence of physiological salines on both sides of the membrane. Other nicotinic agonists (nicotine, carbachol) produced the same effect. Conductance measurements revealed that this apparent outward current was actually a decrease in a tonic inward current. Nicotinic antagonists such as d-turbocurarine (10 microM) and dihydro-beta-erythroidine (20 microM), when applied in dishes that had never been exposed to exogenous ACh, produced a similar decrease in a tonic inward current. The reversal potential of the tonic current suppressed by ACh was similar to the nicotinic current previously studied in these central neurons. Furthermore, purified acetylcholinesterase was capable of modulating the tonic inward current of the retinal ganglion cells. Lowering an excised patch of muscle into dense areas of the retinal cultures activated nicotinic channels in the muscle membrane, indicating the presence of endogenous ACh in the culture fluid. Divalent cations such as Co2+ blocked the tonic inward current of retinal ganglion cells in these cultures. Finally, direct biochemical measurements indicated that low levels of endogenous ACh (on the order of 0.5 microM near putative cholinergic amacrine cells) were present in the retinal cultures. Taken together, these results show that ACh was being spontaneously released into these cultures, resembling at least to some degree the tonic leakage of ACh found in the intact retina. This concentration of ACh was capable of tonically depolarizing the membrane potential of retinal ganglion cells exposed to this dosage. This culture system allows the study of trophic effects of ACh on a central mammalian neuron in a precisely controlled extracellular environment.
此前已有文献记载，在哺乳动物视网膜神经节细胞中存在烟碱型胆碱能反应。在本研究中，将解离的视网膜细胞密集接种，并在特定批次的大鼠血清中培养。在用膜片电极进行全细胞记录时，当保持在负电位时，发现在这些培养物中靠近假定的胆碱能无长突细胞的视网膜神经节细胞，在膜两侧存在生理盐溶液的情况下，对乙酰胆碱（ACh；20 - 200微摩尔）产生明显的外向电流反应。其他烟碱型激动剂（尼古丁、卡巴胆碱）也产生相同的效果。电导测量表明，这种明显的外向电流实际上是持续性内向电流的减少。烟碱型拮抗剂，如d -筒箭毒碱（10微摩尔）和二氢β -刺桐碱（20微摩尔），当应用于从未接触过外源性ACh的培养皿中时，也会使持续性内向电流产生类似的减少。被ACh抑制的持续性电流的反转电位与先前在这些中枢神经元中研究的烟碱型电流相似。此外，纯化的乙酰胆碱酯酶能够调节视网膜神经节细胞的持续性内向电流。将一块切除的肌肉片放入视网膜培养物的密集区域会激活肌肉膜中的烟碱型通道，表明培养液中存在内源性ACh。二价阳离子如Co2 +会阻断这些培养物中视网膜神经节细胞的持续性内向电流。最后，直接生化测量表明，视网膜培养物中存在低水平的内源性ACh（在假定的胆碱能无长突细胞附近约为0.5微摩尔）。综上所述，这些结果表明ACh正在自发释放到这些培养物中，至少在某种程度上类似于在完整视网膜中发现的ACh的持续性泄漏。这种ACh浓度能够使暴露于该剂量的视网膜神经节细胞的膜电位发生持续性去极化。这种培养系统允许在精确控制的细胞外环境中研究ACh对中枢哺乳动物神经元的营养作用。
