Figure 1.
Specific fluorescent labeling of mossy cells. A, Low-magnification image of the left hilus showing cells labeled with DiO (green) labeled by a contralateral injection of retrograde tracer. Note that the axonal projections of the mossy cells to the inner molecular layer are clearly visible (arrowheads). Inset, Schematic of the hippocampus in situ shows the injection sites (green circles on the right) and the approximate level used for horizontal slices (green rectangle on the left). Scale bar, 60 μm. B, C, Histology from injected animals demonstrated that the DiO-labeled cells (B) were immunoreactive for GluR2/3 (C), which strongly stains only the mossy cells in the hilus, as indicated by the arrows. Scale bar, 30 μm. D, Top, Cell counts revealed that most of the DiO-labeled cells were GluR2/3+; therefore, the retrograde labeling was specific to mossy cells. Bottom, The two contralateral injections labeled the majority of the GluR2/3+ cells on the ipsilateral side, because most of these cells were DiO labeled. E, Camera lucida drawings of biocytin-filled processes from both a DiO-labeled mossy cell (top) and a mossy cell post hoc identified by GluR2/3 immunoreactivity (bottom) showed that both cells exhibit classic anatomical features of mossy cells, including large multipolar somata, dendrites mostly confined to the hilus, and axonal projections to the inner molecular layer (green). Scale bars, 30 μm. F, Representative whole-cell current-clamp traces from DiO-labeled (top; black) and GluR2/3+ (middle; blue) mossy cells showed similar responses to positive and negative current injections. Bottom, There were no significant differences between the current-voltage curves for the DiO-labeled (black circles) and GluR2/3+ (blue triangles) mossy cells. OML, Outer molecular layer; IML, inner molecular layer; GCL, granule cell layer; HIL, hilus.