Figure 2.

Evidence that the dendrites of M4 cells stratify just distal (sclerad) to those of M2 cells within the ON sublayer of the IPL. A, B, Top, Fluorescence images from wholemount retinae showing an M4 cell (A) and an M2 cell (B) filled with Lucifer yellow (green). Bottom, Digitally rotated views of the sample zones indicated above (rectangular outlines) to show the stratification of M4 (A) and M2 (B) distal dendrites in relation to the ChAT-immunoreactive processes (red). Both types ramify slightly below the ON ChAT band, but the M2 cell appears to have a slightly greater downward displacement. C, Direct comparison of the dendritic stratification of an M2 cell (green) and an overlapping M4 cell (red) filled with contrasting fluorescent dyes by sharp electrode intracellular injections. The rotated image below is as in A and B, except that the anti-ChAT immunofluorescence is shown in blue, M2 dendrites in green, and M4 dendrites in red. Scale bars: A–C, 50 μm. D, E, Quantification of confocal data collected as in A and B for a population of M4 cells (D; n = 5 cells, 2 spots sampled per terminal dendrite, 38–54 dendritic locations per cell) and M2 cells (E; n = 4 cells, 2 spots sampled per terminal dendrite, 8–36 dendritic locations per cell; see Materials and Methods). The intensity of the ChAT signal (red curve) is plotted as a function of its displacement from the peak of the dendritic signal of the ipRGC (normalized to z = 0; black curve). The offset in depth between ipRGC terminal dendrites and the ON ChAT band (which appears as the major peak in the red curve) is ∼1 μm for M4 cells and twice that for M2 cells. Error bars indicate SEM. LY, Lucifer yellow. F, Bar graph indicating the relative stratification of M4 and M2 dendrites revealed by analysis of points of overlap in the distal dendritic arbors of paired M2 plus M4 cells, filled with contrasting dyes as in C. M4 dendrites nearly always lay sclerad to the overlapping M2 dendrite (“M4>M2”). Less commonly, they appeared to stratify at the same depth (“M4 = M2”), and only rarely lay vitread to the M2 process (“M4<M2”). Data are from 69 points of overlap among 5 paired M4–M2 injections. G, schematic representation of the dendritic stratification of M4 cells (black) within the IPL, with terminal dendrites lying just below the ON ChAT band (red) and just above the dendrites of M2 cells (gray). inl, Inner nuclear layer; ipl, inner plexiform layer; gcl, ganglion cell layer.