QUANTITATIVE AUTORADIOGRAPHY OF HALOTHANE BINDING IN RAT-BRAIN

C-14-halothane direct photoaffinity labeling was used to characterize the distribution of halothane binding in rat brain to test the hypothe sis that anesthetics bind preferentially to a specific, heterogeneousl y distributed, receptor or channel. Slide-mounted sagittal rat brain s ections were placed in gas-tight quartz cuvettes with 100 mu M C-14-ha lothane in phosphate buffered saline with 0 to 7.5 mM unlabeled haloth ane, or unlabeled chloroform and isoflurane at 10 times the clinical E C50, and then exposed to UV light for 60 to 100 sec. Autoradiograms of nine brain regions (cortex, corpus callosum, hippocampal molecular an d pyramidal layers, dentate molecular and granule cell layers, and cer ebellar molecular, granular and white matter layers) were prepared and quantitated using Image 1.44. Total label incorporation was widesprea d, but exhibited subtle heterogeneity. There was significantly more to tal labeling in regions of high synaptic density than in regions conta ining primarily cell bodies or white matter. Most labeling (similar to 80%) was displaced by unlabeled halothane and can therefore be consid ered specific. Significantly more specific labeling was found in regio ns of high synaptic density. Isoflurane did not inhibit halothane phot olabeling significantly, but chloroform inhibited it by similar to 50% . In conclusion, halothane photolabeling distribution in the mammalian brain is widespread, saturable and selective, but does not mimic the distribution of any individual receptor or channel. Brain regions with high synaptic density displayed the greatest degree of specific bindi ng, consistent with transmission being an important functional target of volatile anesthetics. These results suggest a remarkably widespread individual target, or more likely, similar binding sites in multiple targets, and are consistent with the notion that anesthesia is the res ult of action at multiple sites.