Neuroscience Letters, 91 (1988) 315-320 315 Elsevier Scientific Publishers Ireland Ltd. NSL 05520 Differential distribution of 5-hydroxytryptamine and substance P in synaptosomal vesicles of rat ventral spinal cord Gabriel Fried, Johan Franck and Ernst Brodin Departments of Physiology and Pharmacology, Karolinska Institute, Stockholm (Sweden) (Received 22 February 1988; Revised version received 2 May 1988; Accepted 2 May 1988) Key words." Serotonin; Substance P; Synaptosomal vesicle; Ventral spinal cord; Rat The subcellular distribution of 5-hydroxytryptamine (5-HT) and substance P (SP) was investigated in rat ventral spinal cord using differential and density gradient centrifugation. 5-HT was determined by high performance liquid chromatography (HPLC) and SP by radioimmunoassay (RIA). Both substances were found to be stored within synaptosomes at high densities. After partial lysis of the synaptosomes, 5-HT was also recovered in low density fractions, while substance P was found also in a fraction of intermediate density. The results indicate that within synaptosomes from ventral spinal cord, most of the 5-HT is stored in small synaptic vesicles, while SP is only stored in large synaptic vesicles. A minor part of the 5-HT may in addition also be stored in large vesicles, as previously suggested by ultrastructural studies. Serotonin (5-HT) and substance P (SP) coexist in neurons with their cell bodies in the medullary raphe nuclei projecting to the ventral spinal cord [8, 15, 17]. Subcel- lular studies have shown codistribution of 5-HT and SP in synaptosomal fractions of rat ventral spinal cord [7], and ultrastructural studies using electron microscopic immunocytochemistry have shown presence of both 5-HT and SP in large dense- cored vesicles in rat spinal cord [22]. The extent of 5-HT and SP colocalization in large synaptic vesicles is not clear, neither is the functional significance of their coex- istence. However, the neurons that project to the ventral spinal cord have nerve end- ings close to ~-motoneurons, and there are reports of pre- and postsynaptic interac- tions between SP and 5-HT [3, 21, 25, 26]. Furthermore, 5-HT potentiated SP release from rat ventral spinal cord [16] and 5-HT uptake inhibitor treatment increased tis- sue levels and potassium-induced release of SP [4, 6, 16]. An increased knowledge of how coexisting transmitters are stored and released should be of great importance for the understanding of their pre- and postsynaptic interactions and the mechanisms of action of certain drugs. In order to study the subcellular storage sites of 5-HT and Correspondence." G. Fried, Department of Physiology I, Karolinska Institute, S-104 01 Stockholm, Swe- den. 0304-3940/88/$ 03.50 O 1988 Elsevier Scientific Publishers Ireland Ltd.