Poster
# 110
Poster |
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6th Internet World Congress for Biomedical Sciences |
Trevor Sharp(1), Elena Castro(2)
(1)Department of Clinical Pharmacology. University of Oxford - Oxford. United Kingdom
(2) Clinical Pharmacology. University of Oxford - Oxford. United Kingdom
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[Pharmacology] |
[Psychiatry] |
Animal brains were obtained from adult male rats (250-275 g, Sprague-Dawley, Harlan-Olac, Bicester, U.K.) and stored at -20°C. Human brain tissue was obtained at autopsy from four subjects (two men and two women, mean age=67±9 years, postmortem delay=24.8±3.6 h) with no history of neurological or psychiatric disorders. Sequential 14 µm cryostat sections from hippocampus and dorsal raphé nucleus (DRN) of each rat and human brain were thaw-mounted on to gelatinised slides and stored at -20°C until use.
5-HT1A receptor binding was determined autoradiographically using [3H]WAY-100635. Brain sections were preincubated for 30 min in 50 mM Tris-HCl buffer (pH 7.5). For the inhibition studies, sections were then incubated for 2 h in either 50 mM Tris-HCl buffer containing 10 µM pargyline with 3 nM [3H]WAY-100635 in the presence or absence of (±)-pindolol at 7 different concentrations (0.1 nM-1 µM). Each drug concentration was tested in triplicate (rat) or duplicate (human). Inhibition curves for the DRN were always matched with inhibition curves for the corresponding hippocampus.
Non-specific binding was determined using 10 µM unlabeled 5-HT. In saturation studies, conditions used were identical to those described above, except that the sections were incubated with increasing concentrations of [3H]WAY-100635 (0.25 - 12 nM).
Following incubation, sections were washed for 2 X 2 min in ice-cold buffer, briefly dipped in de-ionised water at 4°C, and then air-dried. Autoradiograms were generated by apposing labelled tissues to microscale tritium standards and tritium-sensitive film (Hyperfilm, Amersham plc, UK) for 6 weeks. Autoradiograms were quantified using a computerized image analysis system (MCID system, Image Research, St. Catharine´s, Canada).
Ki values were calculated using the IC50 values determined in the inhibition studies and KD values calculated in saturation studies. For rat tissue, we used the KD of [3H]WAY-100635 estimated in our saturation experiments (1.5-1.7 nM). For human tissue, we used the KD previously determined for human hippocampus by Burnet et al. (3). The data were paired in that each Ki value for the DRN was matched with the Ki value of the corresponding hippocampus.
Data are presented as mean ± s.e.m. values. Binding data were analysed using the programme InPlot (GraphPad Software). Ki values were compared across species and brain areas using one-way ANOVA followed post hoc by Dunnett´s t-test.
[3H]WAY-100635 (specific activity 80 Ci/mmol) was supplied by Wyeth Research UK Ltd. The following drugs were used: (±)-pindolol HCl (Sigma), 5-HT creatinine sulphate (Sigma) and pargyline (Sigma).
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[Pharmacology] |
[Psychiatry] |
