In vivo biosynthesis of [35S]- and [3H]substance P in the striatum of the rat and their axonal transport to the substantia nigra

Abstract

The biosynthesis and axonal transport of the neuropeptide substance P (SP) in the striatonigral tract of the rat was examined using an in vivo radiolabeling of the rostral corpus striatum and a series of high performance liquid chromatography (HPLC) steps for the purification of radiolabeled SP. The corpus striatum of unrestrained rats was continuously infused via indwelling cannulae for 16 hr with [35S]methionine or a mix of [3H]leucine and [3H]proline. Radiolabeled SP was acid extracted from discrete regions of this striatonigral SP projection--corpus striatum (SP-immunopositive cell bodies), ansa lenticularis (striatonigral SP axons), and substantia nigra (striatonigral SP terminals)--and was purified to a constant specific activity by sequential HPLC. The radiochemical purity of SP was verified by chemical derivative formation (SP-Met11-sulfoxide) and further HPLC. The in vivo labeling procedure resulted in a high level of incorporation of the amino acids into tissue protein and peptide pools. [35S]SP and [3H]SP were positively identified in all three regions of this peptidergic projection. The amount of [35S]SP harvested in each region accounted for 0.0015%, 0.003%, and 0.071% of the total tissue 35S present in the striatum, striatonigral fibers, and substantia nigra, respectively. The amount of [3H]SP harvested in each region accounted for 0.0025%, 0.011%, and 0.27% of the total tissue 3H present in the three regions, respectively. The amount of radiolabeled SP in the striatonigral regions for both isotopic infusion studies was highly correlated with the immunoassayable SP content in those regions, suggesting rapid equilibration of de novo biosynthesized SP with striatonigral tissue pools of SP. Tritium autoradiography of the striatonigral projection after 3H-amino acid infusion provided further support for the specificity of the radiolabeling procedures. Heavily labeled fibers were seen leaving the striatal infusion site caudally, forming a distinct fiber bundle. This bundle projected caudoventrally and formed a dense terminal plexus primarily within the reticulata portion of the substantia nigra. These results demonstrate that SP biosynthesis in the corpus striatum and its transport to the substantia nigra can be studied in discrete striatonigral regions obtained from individual unrestrained rats. This preparation should allow for studies on the dynamics of SP biosynthesis, axonal transport, and turnover in the striatonigral projection.