Study on the plasticity of ion-channel in the bladder afferent and efferent neurons associated with bladder hypesrefl

(1)在雌性成年大鼠的膀胱壁注射荧光染料（固蓝）逆行轴突运输标记的背根神经节（DRG）的初级传入神经元和主要盆神经节支配膀胱的传出神经元。然后使用包括胰蛋白酶的酶溶液分离膀胱传入和传出神经元，并通过荧光显微镜进行鉴定。(2)采用全细胞膜片钳技术研究了调节下尿路功能的神经递质一氧化氮（NO）对离体膀胱传入和传出神经元的作用。应用一氧化氮供体SNAP可抑制膀胱传入神经元钙通道电流30%。通过应用8-溴-环-GMP、膜渗透性环-GMP来模拟这些抑制作用，然后通过预孵育ODQ（一种NO依赖性鸟苷酸环化酶抑制剂）来拮抗NO介导的钙通道电流抑制。这些结果表明，NO通过环GMP依赖的机制对膀胱传入通路具有抑制作用（这些数据已在美国神经科学学会第27届年会上发表）。与NO相反，应用神经生长因子增加钙通道电流的30%，在膀胱传入神经元。NGF对钙通道电流的易化作用主要表现在河豚毒素（TTX）抗性钠电流的C纤维膀胱传入神经元。这些结果表明，NGF可以调节膀胱传入神经元的兴奋性，因此可能有助于膀胱反射亢进的出现。

(1) Primary afferent neurons in dorsal root ganglia (DRG) and efferent neurons in major pelvic ganglia innervating the urinary bladder were labeled by retrograde axonal transport of a fluorescent dye (Fast Blue) injected into the bladder wall in female adult rats. Bladder afferent and efferent neurons were then dissociated using enzymatic solution including trypsin and identified by a fluorescent microscope.(2) Electrophysiological studies using whole-cell patch clamp recording techniques were performed to examine the effects of nitric oxide (NO), which is one of neurotransmitters regulating lower urinary tract function on dissociated bladder afferent and efferent neurons. An application of SNAP,an NO donor, inhibited calcium channel currents by 30% in bladder afferent neurons. These inhibitory effects were mimicked by an application of 8-bromo-cyclic-GMP,membrane permeable cyclic-GMP,and the NO-mediated inhibition of calcium channel currents was then antagonized by an preincubation of ODQ,a NO-dependent guanylate cyclase inhibitor. These results indicate that NO has an inhibitory effect on bladder afferent pathway via cyclic-GMP dependent mechanisms (these data have been presented at 27th Annual meeting of Society for Neuroscience, USA)Effects of nerve growth factor (NGF) on bladder afferent and efferent neurons were investigated using whole-cell patch clamp recording techniques. In contrast to NO,an application of NGF increased calcium channel currents by 30% in bladder afferent neurons. These facilitatory effects of NGF on calcium channel currents were mainly observed in C-fiber bladder afferent neurons which exhibited tetrodotoxin (TTX)-resistant sodium currents. These results indicate that NGF can modulate excitability of bladder afferent neurons, and therefore might contribute to an emergence of bladder hyperreflexia.