The modulatory effects of glutamate, the main excitatory neurotransmitter in the central nervous system, are mediated by the activation of metabotropic glutamate receptors (mGluRs) [1]. Eight distinct mGluRs (mGluR1−8) have been classified into three groups (I−III) according to their sequence homology [2]. Group I (mGluRI; mGluR1 and mGluR5), group II (mGluRII: mGluR2 and mGluR3), and group III (mGluRIII: mGluR4, mGluR6, mGluR7, and mGluR8) mGluRs are widely distributed throughout the central nervous system [3]. However, the role of mGluRs in the regulation of neural mechanisms controlling the micturition reflex remains unknown. In the present study, we assessed whether RS-3,5-dihydroxyphenylglycine (RS-3,5-DHPG), a selective mGluRI agonist; 2R,4R-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC), a selective mGluRII agonist; and L-(+)-2-amino-4-phosphonobutyric acid (L-AP4), a selective mGluRIII agonist, affect the micturition reflex in urethane-anaesthetized rats.

Adult female Sprague-Dawley rats weighing 242 to 268 g were used in this study. The rats were anaesthetized with isoflurane followed by urethane (1.2 g/kg, administered subcutaneously). Thereafter, the abdomen was opened through a midline incision, and a PE-60 polyethylene catheter connected to a pressure transducer and amplifier was implanted into the bladder through the bladder dome. This catheter was used to fill the bladder by continuous infusion of saline and to record intravesical pressure during cystometry. Continuous cystometrograms (0.04 ml/min) were performed in two groups of urethane-anaesthetized rats. The first group of 54 rats was used for intracerebroventricular administration of 1−10 μg of RS-3,5-DHPG, 2R,4R-APDC, or L-AP4 via a catheter inserted into the lateral ventricle. For the experiment, saline was first continuously infused for 2 hours to evaluate bladder activity during a control period. Then, RS-3,5-DHPG, 2R,4R-APDC, or L-AP4 was administered intracerebroventricularly to evaluate changes in bladder activity. Using a stereotaxic micro-injector, a 30-gauge needle attached to a 10-μl Hamilton syringe was inserted into the lateral ventricle, and single doses of drugs were administered in a 2-μl volume over a 2-minute period. In the second group of 54 rats, 1−10 μg of RS-3,5-DHPG, 2R,4R-APDC, or L-AP4 was administered via a PE-10 intrathecal catheter that was implanted at Th11 via an incision in the dura under isoflurane anaesthesia 3 days prior to the experiments. For the experiment, saline was first continuously infused for 2 hours to evaluate bladder activity during a control period. Then, RS-3,5-DHPG, 2R,4R-APDC, or L-AP4 was administered intrathecally to evaluate changes in bladder activity. The catheter was directed caudally into the spinal subarachnoid space and positioned at the level of the L6-S1 spinal cord. The volume of fluid in the catheter was kept constant at 6 μl. Single doses of drugs were then administered in a 2-μl volume, followed by a 6-μl saline flush. Cystometric parameters were recorded and compared before and after drug administration. All data values are expressed as the mean±standard deviation. A one-way ANOVA followed by Dunnett’s multiple comparison test was used for the statistical analysis between the vehicle and drug-treated groups. Wilcoxon’s signed rank test was used to compare cystometric variables before and after treatment. For all statistical tests, p<0.05 was considered significant.

Intracerebroventricular administration of RS-3,5-DHPG at doses of 1, 3, and 10 μg (n=6 per dose) increased the intercontraction intervals in a dose-dependent manner to 111.8±9.4%, 121.8±8.7%, and 129.2±10.7% of the control value, respectively (p<0.01). Intracerebroventricular administration of L-AP4 at doses of 1, 3, and 10 μg (n=6 per dose) showed similar effects, with the intercontraction intervals increasing to 115.3±11.5%, 128.6±9.5%, and 132.2±13.6% of the control value, respectively (p<0.01). Both the intracerebroventricular and intrathecal administration of RS-3,5-DHPG or L-AP4 increased the threshold pressure in a dose-dependent manner. Intrathecal administration of RS-3,5-DHPG and L-AP4 at doses of 1, 3, and 10 μg (n=6 per dose) also increased the intercontraction intervals in a dose-dependent manner. However, intracerebroventricular or intrathecal administration of 2R,4R-APDC did not affect the intercontraction intervals, maximum pressure, and basal pressure at any tested dose.

In urethane-anaesthetized rats, intracerebroventricularly or intrathecally administered RS-3,5-DHPG or L-AP4 has an inhibitory effect on the micturition reflex, as shown by the observed increases in the intercontraction intervals and threshold pressure. The main function of RS-3,5-DHPG or L-AP4 seems to be mediated by the modulation of afferent activity, rather than efferent or smooth muscle activity, because RS-3,5-DHPG or L-AP4 induced increases in the intercontraction intervals and threshold pressure without affecting the maximum pressure or basal pressure. We postulate that the site of action may be the supraspinal and spinal sites.

The results of our study indicate that the activation of mGluRI and mGluRIII could inhibit the spinal and supraspinal micturition reflexes in urethane-anaesthetized rats. Thus, mGluRI and mGluRIII could be potential targets for the treatment of bladder dysfunction, such as overactive bladder.

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