Analytical Subcellular Fractionation of Rat Cortex: Resolution of Serotonergic Nerve Endings and Receptors

Abstract: An analytical procedure for the subcellular fractionation of rat brain cortex is presented; it consists of a two-step procedure involving a differential centrifuga-tion using the five-fraction scheme and an isopycnic cen-trifugation in continuous sucrose gradients. All fractions obtained were analyzed for their content of various constituents, such as receptor binding, uptake, and several marker enzymes. Special attention was paid to the subcellular distribution of the serotonin S₂ receptors; they were mainly recovered in the microsomal P fraction, but a significant amount was also associated with the mito-chondrial (M and L) fractions. After equilibration in density gradients, serotonin S₂ receptors revealed two peaks, which were similarly affected after treatment with ami-triptyline and/or yohimbine. There is no evidence to suggest that serotonin S₂ receptors are associated with nerve endings containing the neurotransmitter serotonin. Although three main profiles, a microsomal, a mitochondrial, and a mixed one, clearly appear from the differential centrifugation, subgroups of these main profiles were also found. For instance, the microsomal distribution patterns of serotonin S₂ receptors and 5′-nucleoti-dase are very similar, but differ from that of UDP-galactosyltransferase. Similarly, the mitochondrial profiles of cytochrome oxidase and 5-HT (serotonin) uptake are different. An analytical approach for brain fractionation, when performed with appropriate measurements (cytochrome oxidase, amine uptake, 5′-nucleotidase, and receptor binding), is rapid and clearly differentiates pre-and postsynaptic constituents.