Ultrastructural single‐ and double‐label immunohistochemical studies of substance P‐containing terminals and dopaminergic neurons in the substantia nigra in pigeons

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Abstract
The vast majority of striatonigral projection neurons in pigeons contain substance P (SP), and the vast majority of SP‐containing fibers terminating in the substantia nigra arise from neurons in the striatum. To help clarify the role of striatonigral projection neurons, we conducted electron microscopic single‐ and double‐label immunohistochemical studies of SP+ terminals and/or dopaminergic neurons (labeled with either anti‐dopamine, DA, or anti‐tyrosine hydroxylase, TH) in pigeons to determine: (1) the synaptic organization of SP+ terminals, (2) the synaptic organization of TH+ perikarya and/or dendrites, and (3) the synaptic relationship between SP+ terminals and TH+ neurons in the substantia nigra. Tissue single‐labeled for SP revealed numerous SP+ terminals contacting thin unlabeled dendrites in the substantia nigra, but few SP+ terminals were observed contacting perikarya or large‐diameter dendrites. SP+ terminals contained round, densely packed, clear vesicles, and often contained one or more dense‐core vesicles. Synaptic junctions between SP+ terminals and their targets were more often symmetric (86%) than asymmetric. In tissue single‐labeled for DA, we observed few terminals contacting DA+ perikarya, whereas terminals contacting DA+ dendrites were more abundant. Terminals contacting DA+ structures comprised at least four different morphologically distinct types based on the morphology of the clear synaptic vesicles and the type of synaptic junction. One type of terminal contained round clear vesicles and made symmetric synapses, and thus resembled the predominant type of SP+ terminal. The second type contained round clear vesicles and made asymmetric synapses, the third type contained medium‐size pleomorphic clear vesicles and made symmetric synapses, and the fourth type contained small pleomorphic clear vesicles and made symmetric synapses. The presence of contacts between SP+ terminals and dopaminergic dendrites in the substantia nigra was directly demonstrated in tissue double‐labeled for SP (by the peroxidase‐antiperoxidase procedure, or PAP, with diaminobenzidine) and TH (by either the silver‐intensified immunogold procedure or the PAP procedure with benzidine dihydrochloride). SP+ terminals commonly contacted thin TH+ dendrites in the substantia nigra, but few SP + terminals contacted large‐diameter TH+ dendrites or perikarya. Synapses between SP+ terminals and TH+ neurons were always symmetric. TH+ dendrites also were contacted by terminals not labeled for SP, which were more abundant than were SP+ terminals. Non‐TH+ neurons were also contacted by both SP+ terminals and non‐SP+ terminals. These results indicate that (1) SP+ striatonigral projection neurons have a monosynaptic influence on the activity of nigral dopaminergic neurons, (2) SP+ striatonigral neurons and non‐SP+ neurons have convergent input onto individual dopaminergic nigral neurons and (3) SP+ striatonigral neurons have a direct influence on the activity of nondopaminergic nigral neurons.