Identification and characterization of enkephalinergic neurons in spinal cord dorsal horn: Single cell RT-PCR analysis

脊髓背角脑啡肽能神经元的鉴定和表征：单细胞 RT-PCR 分析

• 批准号: 09680818
• 负责人: HORI Yuuichi
• 金额: $ 0.64万
• 依托单位: Dokkyo University School of Medicine
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 1997
• 资助国家: 日本
• 起止时间: 1997 至 1999
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09680818/
• 关键词: Single cell RT-PCR; Path Clamp Methods; Superficial dorsal horn,; Enkephalin; Reverse Transcripcion; Polymerase Chain Reaction

1. We adapted the technique of single-cell reverse transcription- polymerase chain reaction (RT-PCR) for small neurons in the superficial dorsal horn of rat spinal cord slices. Nethionin-enkephalin sequence-specific PCR products were observed in more than half of superficial dorsal horn neurons studied. This is reminiscent of the previous immunohistochemical reports that a high density of enkephalin neurons are present in the superficial dorsal horn2. Under current clamp conditions, depolarizing current injection stimulated dorsal horn neurons to generate spikes. Characteristically, enkephalinergic neurons usually displayed marked spike accommodation with a progressive prolongation of interspike intervals. In addition, enkephalinergic neurons seldom showed an initial sag upon hyperpolarizing current injection, indicating enkephalinergic neurons lack a time-dependent inwardly rectifying channel.3. Under voltage clamp conditions, ionic currents evoked by voltage command was investigated. The following potassium currents were observed in enkephalinergic neurons: transient IA currents (in 39 of 46 enkephalinergic neurones studied); Inward rectifier ( in 18 of 39); Ca2+-dependent potassium currents (in 14 of 31); and delayed rectifier (in all enkephalinergic neurones studied).4. Barbiturates hyperpolarized enkephalinergic neurons and suppressed spike generation in response to depolarizing currents injection.5. It was also found that mRNA of 5-HT3 receptors was detected in some 40% of enkephalin-containing neurons. It is strongly suggested that enkephalinergic inhibitory neurons mediate Serotonin-induced spinal analgesia, owing at least partly to activation of 5-HT3 receptors expressed in enkephalinergic neurons.

1。我们适应了大鼠脊髓切片的浅表背角中的小神经元的单细胞逆转录聚合酶链反应（RT-PCR）的技术。在研究的一半以上的浅表背角神经元中，观察到nethionin-脑脑序列特异性PCR产物。这让人联想到以前的免疫组织化学报告，即浅表背角中存在高密度的Enkephalin神经元。在当前的夹具条件下，去极化电流注射刺激背角神经元产生尖峰。特征是，脑脑能神经元通常显示出明显的尖峰适应，并逐渐延长尖峰间隔。此外，脑脑能神经元很少在超极化电流注射时出现初始下垂，表明Enkephalinergic神经元缺乏时间依赖性的内部整流通道3。在电压夹条件下，研究了电压命令引起的离子电流。在脑脑能神经元中观察到以下钾电流：瞬态IA电流（在研究的46个enkephalinergic神经元中的39个中）；内向整流器（39中的18个）； Ca2+依赖性钾电流（31中的14个）；并延迟整流器（在所有研究的脑脑能神经元中）。4。巴比妥的超极化脑脑能神经元，并响应去极化电流注入而抑制尖峰产生。5。还发现，在含Enkephalin的40％的神经元中检测到5-HT3受体的mRNA。强烈建议，脑脑抑制性神经元介导5-羟色胺诱导的脊髓镇痛，至少部分是由于在邻脑能神经元中表达的5-HT3受体的激活。

项目成果

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Megumi Shimoyama：“加巴喷丁影响大鼠背角神经元中突触诱发的 NMDA 电流”疼痛（待公布，待出版）。

Masaru Tsuchiya: "Enkephalinergic neurons express 5-HT3 receptors in the spinal cord dorsal horn: Single cell RT-PCR analysis"NeuroReport. 10. 2749-2753 (1999)

Masaru Tsuchiya：“脑啡肽能神经元在脊髓背角表达 5-HT3 受体：单细胞 RT-PCR 分析”NeuroReport。

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Toshiaki Minami 等人：“初级传入 C 纤维参与前列腺素 E2 诱导的触摸诱发疼痛（异常性疼痛）”欧洲神经科学杂志。