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

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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.