A NEW HIGHLY SENSITIVE PROCEDURE - A PREREQUISITE TO EVALUATE THE DENSITY OF GABA-A RECEPTORS 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 [H-3]muscimol in distinct areas of rat brain. This method had been optimized for the highest amount of [H-3]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 GABA(A) 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 GABA(A) receptor sites of the resulting membrane fractions are viable as muscimol binding had a higher affinity in cerebellum (CER) (K(D) = 6.9 +/- 1 nm) than frontal cortex (FC) (K(D) = 18.4 +/- 1.4 nM) which is in the range of highly purified receptors; CER was significantly more densily labelled than FC; the IC50 value of 2 x 10(-7) M for bicucullinmethiodide 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-a-pregnane-20-one (HPO) which was effective after a preincubation of only 30s as well as the threshold concentration of 10 nM HPO for enhancement of the binding of muscimol provided evidence for the high sensitivity of GABA(A) receptors to neuroactive steroids. The characterization of allosteric effects of HPO, 5-alpha-pregnane-3-alpha,21-diol-20-one (THDOC), alphaxalone and progesterone in FC revealed that the extent of neuromodulatory activity highly depended on the structure of steroids. With the exception of progesterone all the examined steroids enhanced receptor density as determined by Scatchard analysis but the magnitude of effects were significantly different. THDOC and HPO were most potent relative to control followed by alphaxalone enhancing the number of binding sites 2.6, 2.1 and 1.5-fold, respectively, at a concentration of 1 mum. In addition, the affinity was also affected selectively as being evident in the increase of K(D) by THDOC up to 30.6 +/- 2.3 nM and the decrease of K(D) by progesterone up to 15.1 +/- 2.3 nm. Thus the new procedure compared with alternative methods favourably as jugded by the density of GABA(A) receptors and their allosteric properties.