Extended Data Fig. 4: Specificity of Double-virus Rabies Tracing.
From: Hippocampus shapes entorhinal cortical output through a direct feedback circuit
a-b. Sections from two different transgenic animals showing the extent of 2A label, a proxy for starter cells. Dotted line is lamina dessicans, the border between L3 and deep layers of EC. Note the two distinct needle tracks, the restriction of 2A label to ECL3, and the fact that there is a “hole” in the 2A expression pattern (and cell death, as judged by NeuN staining) corresponding to the actual AAV injection sites. c. Zoom of the 4th section from the right in panel b, showing (c1) 2A label, (c2) rabies GFP expression, (c3) NeuN expression, and (c4) merged of the 3, with GFP in green, NeuN in purple, and 2A label in blue. Note that CA1 is a major input, nearly on par with the known major inputs, the deep layers of EC and Pre/Postsubiculum, as shown in Table 2. d. Zoom of c1-c4, respectively, showing the only instance where the two injections were visible in the same section, with the rabies track visible on the right (open yellow arrowhead) and the area of cell death resulting from the large bolus of the AAV injection on the left (solid yellow arrowhead). Note that rabies-GFP label tends to erase the 2 A label (circles), making the distinction between L3 starter cells and L3 presynaptic cells difficult to judge precisely. So our data cannot speak definitively on the interconnectivity of neurons within L3. The variability of the number of starter cells in our dataset comes from this fact, as well as the fact that the overlap between the two injections varies between animals, but they all point to the same basic results. All scale bars are 100 µm. The experiment was repeated in 4 animals with similar results.
