In the lower urinary tract (LUT), nicotinic acetylcholine receptors (nAChRs) play an important role in regulation of urinary function through synaptic transmission in autonomic ganglia and neuromascular junction in urethral striated muscle.  The urinary function is also regluated by the brain in coordinating with the peripheral nervous system and the spinal cord.  Previously, intracerebroventricularly administered (±)-epibatidine (EP, a non-selective nAChR agonist) was reported to prolong intercontraction intervals (ICI) in anesthetized and conscious rats [1].  In addition, decreased bladder capacity in rats by cerebral infarction was improved by intracerebroventricularly administered donepezil (a cholinesterase inhibitor) [2], which is reported to activate nAChRs directly.  Therefore, brain nAChRs have a therapeutic potential for patients with combined neurogenic disorders and LUT dysfunctions (LUTDs).  However, central mechanisms for brain nAChR-mediated inhibitory regulation of the micturition reflex are still unknown.

In the brain, the abundant forms of nAChRs are heteropentameric α4β2 nAChRs and homopentameric α7 nAChRs [3].  These receptors are expressed at the synapse pre- and post-synaptically, and function to promote release of neurotransmitters such as GABA, glutamate, noradrenaline and ACh [3].  All of these transmitters have been implicated in control of the micturition reflex in the brain.  In this study, we investigated brain mechanisms for intracerebroventricularly administered EP-induced inhibition of the micturition reflex, focusing on brain nAChR subtyes and brain receptors of GABA, an inhibitory neurotransmitter, in rats.

Urethane anesthetized (0.8 g/kg, ip) male Wistar rats (300-450 g) were used.  A catheter was inserted into the bladder from the bladder dome to perform cystometry.  Two hours after the surgery, intravesical instillation of saline at 12 ml/h was started to evaluate ICI and maximal voiding pressure (MVP).  One hour after the start, EP (0.3 or 1 nmol/rat) or vehicle [2.5 µl N,N-dimethylformamide (DMF)/rat] was intracerebroventricularly administered.  Evaluations of ICI and MVP were continued 1 hour after the administration.  In some rats, through a catheter inserted into the femoral vein, EP was intravenously administered (1 nmol/rat) instead of intracerebroventricular administration.

We also investigated effects of intracerebroventricularly pretreated mecamylamine (MEC, a non-selective nAChR antagonist, 100 or 300 nmol in 5 µl saline/rat) methyllycaconitine (MLA, a selective α7 nAChR antagonist, 30 or 100 nmol in 5 µl saline/rat), dihydro-β-erythroidine (DHβE, a selective α4β2 nAChR antagonist, 100 or 300 nmol in 5 µl saline/rat), SR95531 (SR, a GABAA receptor antagonist, 0.03 or 0.1 nmol in 5 µl saline/rat) or SCH50911 (SCH, a GABAB receptor antagonist, 0.03 or 0.1 nmol in 5 µl saline/rat) on the intracerebroventricularly administered EP (1 nmol/rat)-induced responses.  Each pretreatment was performed 30 min before EP administration.  In addition, effects of intracerebroventricularly administered PHA568487 (PHA, a selective α7 nAChR agonist, 0.3 or 1 nmol in 10 µl saline/rat) on ICI and MVP were also investigated.

Intracerebroventricularly administered EP dose-dependently prolonged ICI (Fig. 1A) without changing MVP (data not shown) compared with the vehicle-1-treated control group.  On the other hand, intravenously administered EP showed no significant effect on ICI or MVP (data not shown).  ICI prolongation induced by intracerebroventricularly administered EP was significantly attenuated by intracerebroventricularly pretreated MEC (data not shown), MLA (Fig. 1B), SR (Fig. 2A) and SCH (Fig. 2B), but not by DHβE (data not shown).  Intracerebroventricularly adminisrered PHA dose-dependently prolonged ICI (Fig. 1C) without changing MVP (data not shown) compared with the vehicle-3-treated control group, similar to EP.

Since EP can penetrate the blood-brain barrier, we compared effects of intracerebroventricularly and intravenously administered EP to investigate whether the intracerebroventricularly administered EP-induced changes were central or peripheral effects due to diffusion out into the systemic circulation.  In this study, ICI prolongation was induced by intracerebroventricular, but not intravenous, administration of EP.  The effects of EP after intracerebroventricular administration on ICI occurred within 15 min, therefore, the time of onset seems to be insufficient for direct action on the peripheral nervous system after diffusion out from the brain into systemic circulation.  In addition, the intracerebroventricularly administered EP-induced ICI prolongation was attenuated by intracerebroventricularly pretreated MEC and MLA, but not by DHβE.  These results indicate that EP centrally inhibits the micturition reflex through brain α7 nAChRs.  In fact, effects of intracerebroventricularly administered PHA on ICI and MVP were similar to EP, supporting the involvement of brain α7 nAChRs in inhibitory regulation of the micturition reflex.  And, our present data indicate that intracerebroventricularly administered EP centrally inhibits the micturition reflex through brain GABAA and GABAB receptors as shown by the EP-induced ICI prolongation was attenuated by intracerebroventricularly pretreated SR and SCH.  Therefore, stimulation of brain α7 nAChRs might inhibit the micturition reflex through these receptors-mediated enhancement of GABA release and stimulation of brain GABAA and GABAB receptors.

Brain GABAA and GABAB receptors are involved in brain α7 nAChR-mediated inhibition of the rat micturition reflex.  Therefore, brain alpha7 nAChRs could be novel therapeutic targets for patients with LUTDs attributed to neurogenic bladder overactivity.

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