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

1. 我们采用单细胞逆转录聚合酶链反应（RT-PCR）技术对大鼠脊髓浅表背角小神经元切片进行检测。在研究的浅表背角神经元中，超过一半的神经元中观察到硫代肽-脑啡肽序列特异性PCR产物。这让人想起以前的免疫组织化学报道，高密度的脑啡肽神经元存在于浅表背角2。在电流箝位条件下，去极化电流注射刺激背角神经元产生尖峰。典型地，脑啡能神经元通常表现出明显的峰间调节，峰间间隔逐渐延长。此外，在注射超极化电流时，脑啡能神经元很少出现初始凹陷，这表明脑啡能神经元缺乏时间依赖性的内整流通道。在电压箝位条件下，研究了电压指令引起的离子电流。在脑啡能神经元中观察到以下钾电流：瞬态IA电流（在所研究的46个脑啡能神经元中的39个）；向内整流器（39个中的18个）；Ca2+依赖性钾电流（31人中有14人）；延迟整流（研究的所有脑啡能神经元）。巴比妥酸盐对脑啡能神经元的超极化和抑制去极化电流的产生。在约40%的含脑啡肽神经元中检测到5-HT3受体mRNA。这强烈表明，脑啡能抑制神经元介导5-羟色胺诱导的脊髓镇痛，至少部分原因是脑啡能神经元中表达的5-HT3受体的激活。

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.