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.我们采用单细胞逆转录聚合酶链反应（RT-PCR）技术来检测大鼠脊髓切片浅表背角的小神经元。在超过一半的研究浅表背角神经元中观察到了奈硫宁-脑啡肽序列特异性 PCR 产物。这让人想起之前的免疫组织化学报告，即浅表背角中存在高密度的脑啡肽神经元。在电流钳条件下，去极化电流注入刺激背角神经元产生尖峰。典型地，脑啡肽能神经元通常表现出明显的尖峰调节，并且尖峰间隔逐渐延长。此外，脑啡肽能神经元在超极化电流注入后很少表现出初始下垂，表明脑啡肽能神经元缺乏时间依赖性内向整流通道。3．在电压钳条件下，研究了由电压指令引起的离子电流。在脑啡肽能神经元中观察到以下钾电流：瞬时 IA 电流（在研究的 46 个脑啡肽能神经元中，有 39 个存在）；内向整流器（39 中的 18）； Ca2+ 依赖性钾电流（31 例中有 14 例）；和延迟整流（在所有研究的脑啡肽能神经元中）。4。巴比妥类药物使脑啡肽能神经元超极化并抑制去极化电流注射后尖峰的产生。5．研究还发现，约 40% 的脑啡肽神经元中检测到了 5-HT3 受体的 mRNA。强烈建议脑啡肽能抑制性神经元介导血清素诱导的脊髓镇痛，至少部分归因于脑啡肽能神经元中表达的 5-HT3 受体的激活。

项目成果

Shinpei Hanazawa: "Enhancement of formalin-induced licking response in mutant mice lacking D-amino-acid oxidase." Pain Research. 12・3. 151-151 (1997)

Shinpei Hanazawa：“缺乏 D-氨基酸氧化酶的突变小鼠中福尔马林诱导的舔舐反应的增强”12・3（1997）。

Yuuichi Hori: "Bell-Magendie's controversy (written in Japanese)"Brain Science. vol.21. 873-876 (1999)

堀雄一：《贝尔-马让迪的争议（日文写）》《脑科学》。

Megumi Shimoyama: "Gabapentin affects synaptically-evoked NMDA current in rat dorsal horn neuons"Pain. (発表予定、in press). (1999)

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。

Masaru Tsuchiya,et al.: "Enkephalinergic neurons express 5-HT3 receptors in the sponal cord dorsal horn: Single cell RT-PCR analysis"NeruoReport. vol.10. 2749-2753 (1999)

Masaru Tsuchiya 等人：“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”NeruoReport。