Rapid communication QUANTITATIVE AUTORADIOGRAPHY OF NEUROTRANSMITrER RECEPTORS USING TRITIUM-SENSITIVE FILM * JOHN B. PENNEY *, KIRK FREY and ANNE

In the past 3 years techniques have been developed for autoradiographic localization of diffusable compounds including neurotransmitter receptor ligands (Young and Kuhar, 1979; Herkenham and Pert, 1980). The high spatial resolution of these techniques has provided elegant demonstrations of the regional heterogeneity of receptor densities within the CNS, however, this is achieved at the expense of the direct quantitation capabilities of homogenate binding methods. We have, therefore, developed an autoradiographic technique using tritium sensitive film (LKB Ultrofilm 3H) to achieve rapid high resolution quantification of benzodiazepine, GABA and glycine receptor numbers and affinities in multiple areas of the central nervous system. For the receptor assays, frozen sections of brain and spinal cord were mounted on gelatin coated slides at room temperature and stored at -20°C. Prior to assay, the slides were given three consecutive 5 min washes in 50 mM Tris-citrate buffer (pH 7.2) at 0°C. They were then incubated for 30 min at 0°C in solutions of either [3H]flunitrazepain (1-15 nM, 88 Ci/mmol) or [3H]muscimol (5-80 nM, 19 Ci/mmol) in 50 mM Tris citrate buffer pH 7.2, or [3H]strychnine (5-80 nM, 15 Ci/mmol) in 50 mM Na-K phosphate buffer (pH 7.0). Adjacent sections were incubated with excess diazepam (2>(10 -6 M), GABA (10 -3 M) or