5-HT_1A receptor antagonists acutely inhibit micturition in rats (Conley et al., 2001). Recently, studies have shown that after chronic administration, the 5-HT_1A receptor antagonists WAY-100635 (WAY) and robalzotan are unable to suppress micturition (Glew et al, 2003). Experiments were carried out to determine whether this was related to a change in the number of 5-HT_1A receptors in the brain.

Female Sprague Dawley rats (250-350g; n = 12) were implanted subcutaneously, under isoflurane anaesthesia with osmotic pumps (model 1002 ALZET; 14 day administration, 0.25 µl h^-1) connected to the jugular vein for constant i.v. administration of WAY to give a free plasma concentration of 10 nM, (which has previously been shown to attenuate micturition frequency) or saline. Brains were rapidly dissected from the rats which had undergone cystometry under terminal anaesthesia (Glew et al., 2003) on day 11 and 6 days after discontinuation of WAY administration (day 20) and frozen in chilled isopentane and stored at -80^oC. Multiple adjacent coronal sections (16 mm) at various levels of the brain were cut at -20^oC and thaw mounted onto poly-lysine coated slides. Autoradiographic incubations were performed with modifications according to Casanovas et al. (1999). 5-HT_1A receptors were labelled with 10µM [³H]WAY-100635 with non-specific binding (NSB) defined in the presence of 10µM 5-HT on duplicate slides. Sections were imaged using a Biospace 2000 imager and the counts per mm² (software package ß-vision) were converted into specific activity (fmol mg^-1 tissue) using brain paste standards. Specific binding was calculated by subtraction of NSB from total binding, with multiple readings (3) obtained for each region from each animal. ANOVA was used to compare treatment effects on each day, and also to compare responses between days.

Table 1: Binding of [³H]WAY-100635 (fmol mg^-1 tissue)in animals chronically pre-treated with vehicle or WAY (n = 2/3, multiple replicates). Values are mean ± s.e.mean. * P < 0.05.

These data indicate that after 11 days of chronic administration of WAY there is a significant increase in the number of 5-HT_1A receptors in the dorsal raphe, entorhinal cortex, hippocampus and amygdala. This upregulation is still present in the hippocampus and entorhinal cortex 6 days after administration was stopped. Thus the development of tolerance to 5-HT_1A receptor antagonist in the control of micturition is probably due an upregulation in the number of these receptors.

Casanovas, J.M. et al. (1999) J. Neurochem. 72, 262-272.
Conley, R.K. et al., (2001) Br. J. Pharmacol. 133, 61-72.
Glew, AG. et al., (2003) Br. J. Pharmacol. This meeting.

AGG is a MRC CASE student with Pfizer Ltd.