A New Highly Sensitive Procedure — a Prerequisite to Evaluate the Density of GABAAReceptors and to Study Their Allosteric Properties in Distinct Areas of Rat Brain
التفاصيل البيبلوغرافية
العنوان:
A New Highly Sensitive Procedure — a Prerequisite to Evaluate the Density of GABAAReceptors and to Study Their Allosteric Properties in Distinct Areas of Rat Brain
A new procedure for tissue preparation is described for the study of modulatory effects of neuroactive steroids and barbiturates on the in vitro binding of [3H]muscimol in distinct areas of rat brain. This method had been optimized for the highest amount of [3H]muscimol binding and a maximal effect of pentobarbital on radioligand binding. The following steps like tissue freezing, initial homogenization in alkaline triethanolamine buffer (pH 9.0), fractionated centrifugations and washings of the resulting membrane fractions in disodium piperazine-N-N'-bis[2-hydroxypropanesulfonic acid] buffer (pH 7.4) provided the best results. In spite of a shorter time of preparation compared to preparative techniques traditionally used our procedure yields GABAA receptor sites with a higher sensitivity to the binding of muscimol without affecting their allosteric properties thereby allowing the use of minute brain tissue samples as little as 50mg wet weight. The GABAA receptor sites of the resulting membrane fractions are viable as muscimol binding had a higher affinity in cerebellum (CER) (KD = 6.9 +/- 1nM) than frontal cortex (FC) (KD = 18.4 +/- 1.4nM) which is in the range of highly purified receptors; CER was significantly more densely labelled than FC; the IC50 value of 2 x 10(-7) M for bicucullin-methiodide and the magnitude of the effect of pentobarbital are similar to published results. The rapid modulatory onset of the action of 3-alpha-hydroxy-5-alpha-pregnane-20-one (HPO) which was effective after a preincubation of only 30s as well as the threshold concentration of 10nM HPO for enhancement of the binding of muscimol provided evidence for the high sensitivity of GABAA receptors to neuroactive steroids.(ABSTRACT TRUNCATED AT 250 WORDS)
