Costorage of serotonin binding protein with serotonin in the rat CNS

Previous studies have identified two neurectoderm-specific serotonin binding proteins (SBP), one with an apparent Mr of 45 kDa, and one of 56 kDa. The current experiments were undertaken to test the hypothesis that these proteins are specific components of serotonergic neurons. Since actin has been found to bind serotonin, the relationship of the 2 forms of SBP to actin was also investigated. Antisera against purified 45 and 56 kDa SBP were raised in rabbits and shown by analyses of immunoblots and differential absorption to be monospecific and not cross-reactive. Neither antiserum reacted with purified actin and none of 3 different anti-actin sera reacted with purified 45 or 56 kDa SBP. The antisera to 45 and 56 kDa were used for immunocytochemical localization of the proteins, which was compared to that of serotonin. SBP immunoreactivity was found in rat brain and spinal cord; however, no significant differences were observed in the pattern of distribution of 45 and 56 kDa SBP-immunoreactive structures. Immunostaining of neuronal perikarya by either SBP antiserum required pretreatment of animals with colchicine. The distribution of neurons and terminals labeled by each antiserum to SBP was similar to that of neurons and terminals labeled by anti-5-HT sera. SBP-immunoreactive neuronal perikarya were present in the nuclei raphe dorsalis, raphe centralis superior, raphe medianus, raphe magnus, raphe obscurus, raphe pallidus, dorsal to the medial lemniscus in the region of the B9 cell group, near the interpeduncular nucleus, in the area postrema, the pars compacta of the substantia nigra, the dorsomedial nucleus of the hypothalamus, and the arcuate nucleus. SBP-immunoreactive fibers and terminals were present in many additional areas of the brain, as well as the spinal cord, where they paralleled those that were immunostained with antibodies to 5-HT. When double-immunostaining was used, serotonin and 45 and 56 kDa SBP immunoreactivities were found to be colocalized in both the brain and spinal cord. Cells and fibers found to be stained by one immunoreagent were also stained by the others; therefore, serotonergic neurons of the CNS probably contain both 45 and 56 kDa SBP. Moreover, it also seems likely that nonserotonergic neurons contain neither form of SBP. These data strongly suggest that SBP is an intrinsic and specific component of serotonergic neurons that can serve as a serotonergic marker.