CONVERGENCE OF SYNAPTIC INPUTS FROM THE STRIATUM AND THE GLOBUS-PALLIDUS ONTO IDENTIFIED NIGROCOLLICULAR CELLS IN THE RAT - A DOUBLE ANTEROGRADE LABELING STUDY

Two major sources of afferent synaptic inputs to projection neurons in the rat substantia nigra reticulata are the striatum and the globus pallidus. In order to understand better the functional relationships between these two afferents in the control of the activity of nigrofugal neurons, experiments have been performed to test the possibility that single nigrofugal cells receive convergent synaptic inputs from the striatum and the globus pallidus. To address this question we have used two different approaches. First, we have developed a double anterograde labelling technique suitable for both light and electron microscopy and combined this procedure with the retrograde transport of lectin-conjugated horseradish peroxidase in order to retrogradely label the nigrocollicular cells. Second, we have combined the anterograde transport of Phaseolus vulgaris-leucoagglutinin from the globus pallidus and immunocytochemistry for DARPP-32 as a marker for the striatal terminals, with the retrograde transport of lectin-conjugated horseradish peroxidase from the superior colliculus. In the double anterograde labelling experiment, biocytin was injected in the striatum, Phaseolus vulgaris-leucoagglutinin in the globus pallidus and lectin-conjugated horseradish peroxidase in the superior colliculus. Following these injections, rich plexuses of biocytin- and Phaseolus vulgaris-leucoagglutinin-labelled terminals were found in the ventral two-thirds of the substantia nigra. The biocytin-positive terminals (striatonigral) were generally small and formed rich plexuses without any apparent neuronal association whereas the Phaseolus vulgaris-leucoagglutinin-labelled terminals (pallidonigral) were much larger and formed baskets around the perikarya of retrogradely and non retrogradely labelled cells in the substantia nigra reticulata. In areas of the substantia nigra reticulata where the fields of biocytin- and Phaseolus vulgaris-leucoagglutinin-labelled terminals overlapped, the perikarya and the proximal dendrites of retrogradely and non retrogradely labelled cells were found to be apposed by numerous Phaseolus vulgaris-leucoagglutinin-immunoreactive pallidonigral terminals and a few biocytin-labelled striatonigral terminals. In the sections prepared for electron microscopy, the biocytin was localized using 3,3'-diaminobenzidine tetrahydrochloride whereas Phaseolus vulgaris-leucoagglutinin was localized using benzidine dihydrochloride. It was thus possible to distinguish the biocytin- from the Phaseolus vulgaris-leucoagglutinin-labelled terminals in the electron microscope by the texture of the reaction product associated with them. Examination of 231 biocytin-labelled (striatonigral) terminals and 105 Phaseolus vulgaris-leucoagglutinin-immunoreactive (pallidonigral) terminals revealed that the striatonigral terminals were generally small, contained few mitochondria and formed symmetric synapses predominantly with the distal dendrites (77%) and far less frequently with the perikarya (3%) of substantia nigra reticulata cells. On the other hand, most of the pallidonigral terminals were large, contained many mitochondria and formed symmetric synapses predominantly with the perikarya (54%) and proximal dendrites (32%) of nigral cells. Five of the 12 retrogradely labelled nigrocollicular cells examined in the electron microscope were found to receive synaptic inputs from both striatal and pallidal terminals. In the same material, a total of 15 unlabelled dendrites and six unlabelled perikarya were also found to receive convergent inputs from the striatum and the globus pallidus. The results of the second series of experiments, combining immunocytochemistry for DARPP-32 with the anterograde transport of Phaseolus vulgaris-leucoagglutinin and the retrograde transport of lectin-conjugated horseradish peroxidase, revealed that most of the DARPP-32-immunoreactive terminals in the substantia nigra displayed morphological features similar to the striatonigral terminals and formed symmetric synapses predominantly with dendrites (78%) and less frequently with perikarya (22%) of nigral cells. In many cases the nigral structures postsynaptic to the DARPP-32-immunoreactive terminals were retrogradely labelled with lectin-conjugated horseradish peroxidase thus identifying them as nigrocollicular neurons. In some cases, DARPP-32-immunoreactive terminals (presumed striatonigral) and Phaseolus vulgaris-leucoagglutinin-labelled (pallidonigral) terminals were found to converge on the same nigrocollicular cells. A small population of DARPP-32-immunoreactive terminals forming asymmetrical synaptic contacts was also detected in the substantia nigra reticulata.