Quantitative Autoradiography of Halothane Binding in Rat Brain

C-halothane direct photoaffinity labeling was used to characterize the distribution of halothane binding in rat brain to test the hypothesis that anesthetics bind preferentially to a specific, heterogeneously distributed, receptor or channel. Slidemounted sagittal rat brain sections were placed in gas-tight quartz cuvettes with 100 mM C-halothane in phosphate buffered saline with 0 to 7.5 mM unlabeled halothane, or unlabeled chloroform and isoflurane at 10 times the clinical EC50, and then exposed to UV light for 60 to 100 sec. Autoradiograms of nine brain regions (cortex, corpus callosum, hippocampal molecular and pyramidal layers, dentate molecular and granule cell layers, and cerebellar molecular, granular and white matter layers) were prepared and quantitated using Image 1.44. Total label incorporation was widespread, but exhibited subtle heterogeneity. There was significantly more total labeling in regions of high synaptic density than in regions containing primarily cell bodies or white matter. Most labeling (;80%) was displaced by unlabeled halothane and can therefore be considered specific. Significantly more specific labeling was found in regions of high synaptic density. Isoflurane did not inhibit halothane photolabeling significantly, but chloroform inhibited it by ;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 binding, 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 result of action at multiple sites. Recent studies on targets of anesthetic action have focused on specific receptors or channels (Forman and Miller, 1989; Bazil and Minneman, 1989; Martin et al., 1990; Franks and Lieb, 1994; Harris et al., 1995) which have generally been found to be heterogeneously distributed in the brain (Mohler et al., 1980; Palacios et al., 1981; Halpain et al., 1984; Clarke et al., 1985; Waksman et al., 1986; Savasta et al., 1986; Frey and Howland, 1992). If anesthetics act preferentially through individual targets, a rational next question is whether anesthetic binding is distributed heterogeneously in the brain in accordance with the distribution of these targets. Determination of specific binding of inhalational anesthetics at the necessary level of resolution has been difficult due to the low affinity and rapid kinetics of these small volatile compounds. However, C-halothane direct photoaffinity labeling, developed in this laboratory (Eckenhoff and Shuman, 1993) to study inhalational anesthetic binding, has permitted characterization of binding at the amino acid level of resolution (Eckenhoff, 1996a), and thus should also be a reasonable way to characterize binding at the macromolecular level. By combining photolabeling with quantitative autoradiography, we examined the distribution of halothane labeling in rat brain to test the hypothesis that anesthetics bind preferentially to a specific, heterogeneously distributed, receptor or channel.