Regional localization of specific [ 125I]leptin binding sites in rat forebrain

Specific [ 125I]leptin receptor binding sites have been identified in choroid plexus (CP), but have eluded regional localization within the brain parenchyma. To optimize specific [ 125I]leptin binding in brain loci, we ran experiments varying the pH of incubation buffers. We found that specific [ 125I]leptin binding in CP was strikingly pH dependent with the most acidic buffer, pH 5.5, resulting in a greater than 100% increase over the amount of specific binding measured at pH 7.5. While low pH permitted detection of specific binding in parenchymal loci, clear pH dependency was only observed in the CP. In the caudate putamen (CauP), a locus with low specific binding, values for specific binding did not differ significantly across the range of pH conditions tested. Using incubation buffers at pH 6.0 in subsequent binding experiments, we localized specific [ 125I]leptin binding in several brain loci including thalamus and hypothalamus. In CP and thalamus, where the range of OD permitted analysis of binding parameters, [ 125I]leptin binding was saturable with increasing concentrations of unlabelled leptin. In all loci, specific [ 125I]leptin binding was insensitive to competition by high concentrations of other unlabelled compounds. Our results varying pH conditions of the incubation buffer suggest leptin receptors may be divided into subclassifications based on pH sensitivity of the specific binding. Furthermore, our results suggest that although densities are low, high affinity leptin receptors are present in neural loci implicated in food intake and energy balance, and are more widespread in the forebrain than previously determined.