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

本研究旨在探讨甘氨酸受体是否参与大鼠排尿反射的中枢机制。大鼠（S.D.，250- 300 g），用乌拉坦（0.96mg/kg，i. p.）用于所有体内实验。从9-14天的Wistar大鼠急性解剖脑的单个神经元。[结果] 1）右美沙芬（DM，5-10 mg/kg，i. v.）抑制膀胱和尿道压力2)将甘氨酸前药Z-甘氨酰胺（50-100 mg/kg，i. p.）以剂量依赖性方式诱导节律性膀胱收缩。40 min内，Z-甘氨酰胺治疗组的平均收缩次数为22.2±2.6次，显著高于对照组的7.5±2.3次。3)在膀胱测压图中，100μg而非9μg甘氨酸i. c. v.给药显著降低膀胱容量。4)将甘氨酸3μg直接注入导水管周围灰质（PAG）（-8300-8800 UNFm，来自前囟）的尾侧区，可促进排尿反射，表现为最大膀胱压增加和排尿反射阈值降低。但在PAG的喙部注射相同剂量的甘氨酸对反射影响不大。5)在PAG的整个区域中，证实了甘氨酸受体α亚基mRNA的表达。6)用DiI示踪神经元回路的研究表明，PAG尾侧的神经元投射到中脑的Me 5区和延髓的最后区。7)膜片钳研究表明，甘氨酸引起的士的宁敏感电流的单个神经元从整个区域的巴林顿核。8)膀胱灌注醋酸溶液可使排尿中枢及相关核团表达Fos蛋白。DM可明显抑制Barington核和排尿反射相关区域的Fos蛋白表达。提示甘氨酸受体可能部分参与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.