Didier M, Berman S A, Lindstrom J, Bursztajn S
Department of Psychiatry, Harvard Medical School/McLean Hospital, Belmont, MA 02178, USA.
Brain Res Mol Brain Res. 1995 May;30(1):17-28. doi: 10.1016/0169-328x(94)00266-h.
Nicotinic acetylcholine receptors (nAChRs), like other calcium permeable channel receptors, may play a crucial role during neuronal development. We have characterized nAChRs in developing mouse cerebellar granule cells in primary culture. L-[3H]Nicotine, [3H]cytisine and [125I]alpha-bungarotoxin binding experiments revealed the presence of a single class of saturable and specific high affinity binding sites for each ligand. The expression of these nicotinic binding sites followed a developmental pattern reaching a maximum during the establishment of excitatory amino acid synaptic contacts. Immunolabeling with monoclonal antibodies to nAChR subunits revealed the presence of alpha 4 and beta 2 subunits in most neurons. Moreover, some neuronal cells displayed a somatic as well as a neuritic localization for the alpha 7 subunit as shown by [125I]alpha-bungarotoxin autoradiography. The reverse transcription-polymerase chain reaction (RT-PCR) detected the presence of mRNAs for alpha 3, alpha 4, alpha 5, alpha 7, beta 2 and beta 4 nAChR subunits. Non-neuronal cells did not express nAChRs, as shown by [3H]nicotine and [125I]alpha-bungarotoxin binding, immunocytochemistry and PCR. Maximum Ca2+ influx elicited by nicotine, and partly sensitive to alpha-bungarotoxin, was observed around 10-14 days after plating. This correlated with the time period at which the highest number of nicotine binding sites was detected. Sensitivity to several NMDA receptor antagonists as well as to removal of endogenous glutamate by pyruvate transaminase treatment revealed a glutamatergic component in the nicotine stimulated calcium influx. The time-dependent specific nAChR expression and the potential association between nAChRs and NMDA receptor activation suggest that nAChRs may regulate glutamatergic activity during synaptogenesis in cerebellar granule cells.
与其他钙通透性通道受体一样，烟碱型乙酰胆碱受体（nAChRs）可能在神经元发育过程中发挥关键作用。我们已对原代培养的发育中小鼠小脑颗粒细胞中的nAChRs进行了特性描述。L-[3H]尼古丁、[3H]金雀花碱和[125I]α-银环蛇毒素结合实验表明，每种配体均存在一类单一的可饱和且特异性的高亲和力结合位点。这些烟碱型结合位点的表达遵循一种发育模式，在兴奋性氨基酸突触联系建立期间达到最大值。用针对nAChR亚基的单克隆抗体进行免疫标记显示，大多数神经元中存在α4和β2亚基。此外，如[125I]α-银环蛇毒素放射自显影所示，一些神经元细胞的α7亚基在体细胞和神经突中均有定位。逆转录-聚合酶链反应（RT-PCR）检测到α3、α4、α5、α7、β2和β4 nAChR亚基的mRNA存在。如[3H]尼古丁和[125I]α-银环蛇毒素结合、免疫细胞化学和PCR所示，非神经元细胞不表达nAChRs。接种后约10 - 14天观察到尼古丁引发的最大Ca2+内流，且部分对α-银环蛇毒素敏感。这与检测到最高数量尼古丁结合位点的时间段相关。对几种NMDA受体拮抗剂的敏感性以及通过丙酮酸转氨酶处理去除内源性谷氨酸后显示，尼古丁刺激的钙内流中存在谷氨酸能成分。nAChR表达的时间依赖性特异性以及nAChRs与NMDA受体激活之间的潜在关联表明，nAChRs可能在小脑颗粒细胞突触形成过程中调节谷氨酸能活性。
