Morphological study of the tegmental pedunculopontine nucleus, substantia nigra and subthalamic nucleus, and their interconnections in rat organotypic culture

The morphological organization of the tegmental pedunculopontine nucleus, m idbrain extrapyramidal area, substantia nigra and subthalamic nucleus and t heir interrelationships were studied in rat organotypic culture using immun ohistochemistry and NADPH-diaphorase histochemistry. Three coronal sections , one containing the tegmental pedunculopontine nucleus/midbrain extrapyram idal area, another with the substantia nigra and the third with the subthal amic nucleus, were obtained from postnatal 1-2-day-old rats. These sections were co-cultured for 3-4 weeks using the roller-tube technique. In the teg mental pedunculopontine nucleus/midbrain extrapyramidal area, the distribut ion pattern of cholinergic neurons was similar to that found in the in vivo study. We could, therefore, identify the subdivisions of the tegmental ped unculopontine nucleus (i.e., pars compacta and pars dissipata) and the midb rain extrapyramidal area. As in the in vivo situation, glutamate immunoreac tive neurons were also located in these areas. Approximately 10% of NADPH-d iaphorase positive neurons in the tegmental pedunculopontine nucleus, were glutamate immunoreactive. In the substantia nigra, as in the in vivo, tyros ine hydroxylase immunoreactive (putative dopaminergic) neurons were identif ied predominantly in the substantia nigra pars compacta, and parvalbumin im munoreactive neurons (putative GABAergic) mainly in the substantia nigra pa rs reticulata. The subthalamic nucleus was ladened with glutamate immunorea ctive neurons. NADPH-diaphorase stained axons originating from the tegmenta l pedunculopontine nucleus were traced into the substantia nigra and subtha lamic nucleus. They were often in close apposition to tyrosine hydroxylase immunoreactive neurons in the substantia nigra. Parvalbumin immunoreactive fibers from the substantia nigra projected heavily to the midbrain extrapyr amidal area, but only sparsely to the tegmental pedunculopontine nucleus an d the subthalamic nucleus. These findings indicate that the tegmental pedun culopontine nucleus/midbrain extrapyramidal area, substantia nigra and subt halamic nucleus in the organotypic culture have retained a basic morphologi cal organization and connectivity similar to those seen in the in vivo situ ation. Therefore, this preparation could be a useful model to conduct furth er studies to investigate functional circuits among the structures represen ted.