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

(1)对成年雌性大鼠膀胱壁注射荧光染料快蓝(Fast Blue)，逆行标记背根神经节(DRG)初级传入神经元和支配膀胱的盆大神经节传出神经元。用含胰酶的酶解液分离膀胱传入和传出神经元，并在荧光显微镜下进行鉴定。(2)采用全细胞膜片钳记录技术进行电生理研究，观察调节下尿路功能的神经递质之一一氧化氮(NO)对分离的膀胱传入和传出神经元的影响。应用NO供体SNAP可抑制膀胱传入神经元钙通道电流30%。这些抑制作用可被8-溴-环-GMP、膜通透性环-GMP所模拟，而NO介导的钙通道电流抑制可被NO依赖的鸟苷环化酶抑制剂ODQ所拮抗。这些结果表明，NO通过环GMP依赖机制抑制膀胱传入通路(这些数据已在美国神经科学学会第27届年会上发表)。应用全细胞膜片钳记录技术研究了神经生长因子(NGF)对膀胱传入和传出神经元的影响。与NO相反，应用NGF使膀胱传入神经元的钙通道电流增加30%。NGF对钙通道电流的易化作用主要见于具有河豚毒素(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，从而可能参与了膀胱反射亢进的发生。

(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.