The clustered cell system is present before formation of the ACh patches in the intermediate gray layer of the cat superior colliculus.

Projection neurons from the superior colliculus (SC) to the cuneiform region (CFR) of the midbrain form distinct cell clusters in the intermediate gray layer which precisely overlap the cholinergic fiber patches in this region of the cat SC (Jeon and Mize, J. Comp. Neurol., 337 (1993) 127-150). The acetylcholine (ACh)-containing fiber patches do not express ACh until after birth. It is not known when the cell clusters first appear. In this study, we examined the development of the clusters by retrogradely labeling the cells with a tracer injected into the CFR. Kittens aged from embryonic day E34 to E57 and postnatal day P1 to P14 were perfused with aldehyde fixatives and crystals of the carbocyanine dye DiI were placed into the CFR. After 5- to 8-week incubations, the SC was sectioned and examined using epi-fluorescent microscopy. At E34, very few labeled cells and fibers were present in SC. Between E41 and E46, both labeled cells and fibers were found within the intermediate gray (IGL) and deep gray (DGL) layers of SC. In one E46 case, a few labeled cells were grouped together within the IGL, suggesting the onset of clusters seen at later ages. By E51, DiI labeling produced an obvious pattern of clustered cells. Up to 4 clusters were seen in a single section, each containing 4-12 labeled neurons. By E57, labeled cell clusters were conspicuous in the IGL in caudal sections and the number of cells per cluster was increased. This pattern was also present at birth (P1). Scattered labeled neurons were also found outside the clusters and throughout the deep layers as were fibers innervating this region of SC. In summary, the axonal projections of the IGL clustered cells appear to reach the cuneiform region as early as E46 and are well-developed by E57. The clustering of efferent neurons precedes the formation of cholinergic fiber patches in the cat SC which is first identifiable using ChAT antibodies around the second postnatal week of life.