Elucidation of central mechanisms of micturition reflex for developing novel medicine of micturition disorder, especially a reinforcement drug of micturition reflex

在这个项目中，我们研究了甘氨酸受体是否参与了大鼠排尿反射的中枢机制。大鼠(S.D.，250-300g)用乌拉坦(0.96 mg/kg，ip)麻醉。在所有活体实验中都使用过。取出生9~14天的Wistar大鼠，急性解剖脑内单个神经元。[结果]1)右美沙芬(DM，5~10 mg/kg，静脉注射)抑制膀胱和尿路压力。2)甘氨酸前药z-甘氨酰胺(50-100 mg/kg，i.p.)以剂量依赖的方式诱导节律性膀胱收缩。Z-甘氨酸胺组平均收缩次数为22.2±2.6次，持续40min。该值显著高于对照组(7.5±2.3)。3)膀胱测定术中，i.c.v。甘氨酸剂量为100μg，但不是9μg时，可显著降低膀胱容量。4)将甘氨酸-3-μ-g直接注入中脑导水管周围灰质尾侧(-8300~-8800μm，来自前庭)可促进排尿反射，表现为最大膀胱压力升高，排尿反射阈值降低。但PAG吻区注射相同剂量的甘氨酸对反射的影响不大。5)甘氨酸受体α亚单位m RNA在大脑中隔核全区均有表达。6)DiI对神经元回路的追踪研究显示，PAG尾侧投射到中脑的Me5区和延髓的最后区。7)膜片钳研究表明，甘氨酸可引起分离的巴灵顿核全区单个神经元的士的宁敏感电流。8)膀胱内注入醋酸溶液后，在排尿中心及相关核团表达Fos蛋白。DM对巴林顿核和多个参与排尿反射的区域的Fos蛋白表达有明显抑制作用。这些结果提示甘氨酸受体可能部分参与了排尿反射的中枢机制。

In this project, we investigated whether glycine receptors are involved in central mechanisms of micturition reflex in rats. Rats (S.D., 250-300g) under anesthesia with urethane (0.96 mg/kg, i.p.) were used in all in vivo experiments. Single neurons of the brain were acutely dissected from 9-14 days of Wistar rat. 【Results】 1) Dextromethorphan (DM, 5-10 mg/kg, i.v.) suppressed the bladder and urethral pressure. 2) A glycine prodrug, z-glycinamide (50-100 mg/kg, i.p.) induced rhythmic bladder contractions in a dose-dependent manner. The mean number of contractions in z-glycinamide treated group was 22.2±2.6 for 40 min. this value was significantly higher than that in control group (7.5±2.3). 3) In cystometrogram, i.c.v. administration of glycine at 100μg but not 9μg significantly reduced the capacity of the bladder. 4) Direct administration of glycine 3μg into the caudal region of periaqueductal gray matter (PAG) (-8300〜-8800μm, from bregma) facilitated micturition reflex as indicated by an increase in maximal bladder pressure and a decrease in a threshold of micturition reflex. But injection of the same dose of glycine into the rostal region of the PAG had little influence on the reflex. 5) In the whole region of the PAG, expression of glycine receptor α-subunit mRNA was confirmed. 6) Tracing study of neuronal circuits using DiI showed neurons in the caudal PAG projected to the region Me5 in the midbrain and the area postrema in the medulla oblongata. 7) Patch-clamp study showed that glycine caused strychnine-sensitive current in single neurons dissected from the whole region of the Barington's nucleus. 8) Infusion of acetic acid solution into the bladder expressed Fos protein in the micturition center and related nuclei. DM strongly inhibited the Fos protein expression in the Barington's nucleus and several regions involved in micturition reflex. These results suggest that glycine receptors may be involved partly in central mechanisms of micturition reflex.