Ultrastructural localization of neurotensin‐like immunoreactivity within dense core vesicles in perikarya, but not terminals, colocalizing tyrosine hydroxylase in the rat ventral tegmental area

Abstract

Within the rat ventral tegmental area (VTA), the parabrachial pigmentosus and paranigral subdivisions are known to differ in their functional responses to injected neurotensin. These subdivisions also vary in their connections with other brain regions and in their number of neurotensin‐containing perikarya as seen by light microscopy. In both subdivisions, there may be intracellular as well as synaptic relations between dopamine and neurotensin. Dopaminergic neurons are known to be physiologically activated by neurotensin (NT) and may also contain this peptide. To characterize further the cellular relationships in each subdivision, we examined the ultrastructural immunocytochemical localization of a rat antiserum against NT and a rabbit antiserum against the catecholamine‐synthesizing enzyme tyrosine hydroxylase (TH) in single sections. The NT antiserum was raised against the entire peptide sequence. Immunoblots showed that the antiserum recognized the original antigen as well as the related peptides neuromedin N and lysine 8‐ arginine 9‐ neurotensin 10–13 (LANT‐6). In both the parabrachial pigmentosus and paranigral subdivisions, neurotensin‐like immunoreactivity (NTLI) was localized predominantly in the large (80–100 nm) dense core vesicles using the peroxidase anti‐peroxidase (PAP) method. In tissue labeled for NT by the PAP method and for TH by immunoautoradiography, serial section analysis revealed that all perikarya containing NTLI (n = 19) were also TH‐positive. Three times as many perikarya colocalized NTLI and TH in the parabrachial pigmentosus subdivision (n = 15) as in the paranigral subdivision (n = 4). Occasionally, a perikaryon containing TH and NTLI could be found in direct apposition to a TH‐labeled perikaryon without glial separation. In contrast to perikarya and dendrites, terminals showing NTLI (38 in parabrachial pigmentosus, 29 in paranigral) lacked detectable TH labeling. Of the terminals containing NTLI whose synaptic junctions could be identified, 48% were symmetric and 10% were asymmetric. The targets of these terminals included perikarya and dendrites lacking detectable immunoreactivity (69% in parabrachial pigmentosus, 55% in paranigral), immunolabeled for TH (26% in parabrachial pigmentosus, 38% in paranigral) or containing both NTLI and TH (5% in parabrachial pigmentosus, 7% in paranigral). Single terminals containing NTLI sometimes contacted more than one neuronal target, some of which were apposed to each other Without glial separation. TH‐labeled terminals synapsed onto double‐labeled perikarya in the paranigral subdivision, but were not observed to do so in the parabrachial pigmentosus subdivision. These results establish a direct synaptic basis for modulation of dopaminergic neurons in both paranigral and parabrachial pigmentosus subdivisions of the VTA by terminals containing NTLI and further establish a greater coexistence between TH and NTLI in the parabrachial pigmentosus subdivision by electron microscopy. These findings suggest that in the rat VTA: (1) the final processing of the neurotensin precursor occurs in the large dense core vesicles; (2) terminals containing NTLI, but not TH, arise from extrinsic, non‐catecholaminergic neurons; and (3) perikarya containingboth TH and NTLI project to targets outside the VTA.