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. 2022 Aug 30;11:e79848. doi: 10.7554/eLife.79848

Figure 4. High-throughput retrograde labeling with CVS-N2c enables identification of non-canonical projections to DGCs.

(A) Schematic illustration and representative confocal images, describing the injection scheme designed to target DGCs for retrograde labeling in an interneuron reporter line. (B) Representative confocal images (left) of the regions highlighted in (A) showing retrogradely-labeled neurons along specific hippocampal layers and their overlay with the interneuron-specific marker. (C) Summary bar plot showing the distribution of DG-projecting hippocampal neurons outside of the DG (magenta) and of them, the fraction of double-labeled neurons (grey). Calculation of cell numbers in the dendritic cell layers combined cells along the entire proximo-distal hippocampal axis, from CA3 to CA1. N=189 cells from 3 animals. (D) Representative parasagittal sections of the hippocampus following retrograde labeling from the DG with CVS-N2c-tdTomato, along with immunolabeling of parvalbumin (Pvalb, top) and Somatostatin (Sst, bottom). Expanded view of the S.O. and S.LM. are shown to the right of each image. (E) Summary plot describing the proportion of Pvalb- or Sst-positive neurons of the total CVS-N2c labeled neurons in the S.O. or S.LM. of the CA1. N=125 cells from three animals.

Figure 4—source data 1. Cell count for experimental designs and ratio calculations.
Tables showing the cell count for tdTomato only and tdTomato +EGFP + cells per layer/section/animal along with the calculated normalized averages used to plot Figure 4C. Below, are two tables showing the average cell count per layer/slice in three individual animals, following immunolabeling for PV and Sst, used to plot Figure 4E.

Figure 4.

Figure 4—figure supplement 1. Cross validation of novel excitatory and inhibitory intrahippocampal projections to DGCs.

Figure 4—figure supplement 1.

(A and B) Both injection of AAV-mDLX-EGFP, for specific expression in inhibitory neurons, into the CA1 (A), as well as injection of AAV-CaMKII-EGFP, for specific expression in excitatory neurons, into the subiculum (B) reveal axonal arborizations in the molecular layer of the DG. (C) Retrograde labeling from a sparse population of adult-born DGCs (as shown in Figure 2B) also reveals projections from the subiculum (top) and S.O. (bottom). (D) Retrograde labeling from the ventral DG (vDG) using the Prox1cre line (left panel) shows projection neurons along the superficial most layer of the ventral subiculum (middle and right panels).