3R-Project 44-95

Establishment of a micro technique for corticosteroid receptor binding studies

Mirza Hügin-Flores
Psychopharmacology Unit, Department of Psychiatry, University Hospital, Belle-Idée, 1211 Geneva, Switzerland
hugin-mirza@diogenes.hcuge.ch

Keywords: brain

Duration: 1 year Project Completion: 1999

Background and Aim
The measurement of corticosteroid receptors in brain structures of the rat often requires a large number of animals due to the small size of some of the areas of interest. The objective of this study was to establish a micro-technique for measurements of both corticosteroid receptors (mineralo- and glucocorticoid) with much smaller tissue samples, so that fewer animals would be required for such investigations.

Method and Results
Tissue (hippocampus or anterior pituitary) from rats was homogenized in 1.5 ml buffer TEMDG (25 mM Tris-Cl, 1 mM EDTA, 20 mM molybdate, 5 mM DTT, 10% glycerol, pH 7.4) using a motor-driven glass-teflon homogenizer. The homogenate was first spun in a microfuge at 10,000 x g at 4^oC for 15 min. The supernatant was then centrifuged at 105,000 x g at 4^oC for 60 min in an airfuge (Beckman) to obtain the cytosol fraction. The mineralocorticoid receptor (MR) was measured by incubating 6 nM [³H]-aldosterone in the presence of 0.5 microM RU-28362, a specific agonist that occupies glucocorticoid receptors. Non-specific binding was assessed by incubating 6 nM [³H]-aldosterone in the presence of 2.5 microM corticosterone. The level of glucocorticoid receptor (GR) was given by the difference between the binding of 10 nM [³H]-dexamethasone in the presence and in the absence of 0.5 microM RU-28362. The tubes containing a final volume of 100 microliters were incubated on ice for 24 h. Bound [³H]-steroid was separated from unbound [³H]-steroid by gel filtration on self-made Sephadex LH-20 mini-columns (1 ml). Binding levels were expressed as fmol/mg protein.

The results obtained show that the measurement in triplicate of both corticosteroid receptor types can be performed with 4 anterior pituitaries or 2 hippocampus, which is half the tissue required using the conventional standard method. The limit of detection of the micro-assay is 1.45 fmol/mg protein. Total bound radioactivity is 2% for MR and 6% for GR. Non-specific binding (in the presence of unlabeled ligand) is 10% of the total bound for MR and 5% for GR. The inter- and intraassay coefficients of variability are 6.3% and 4%, respectively (1).

Conclusions and Relevance for 3R
The refined technique developed for corticosteroid receptor binding in rat CNS tissue reduces the number of animals required per experiment by half.

References
1. Hügin-Flores M., Steimer T., Schulz P., Vallotton M.B. and Aubert M.L. (2000) Regulation of hippocampal corticosteroid receptors during chronic restraint stress ; effect of adrenalectomy and of CRH and AVP antagonists, submitted.