Extended Data Fig. 3: Validation of hippocampal feedback using different viral strategies.
From: Hippocampus shapes entorhinal cortical output through a direct feedback circuit
a. Top - Confocal image of a horizontal brain slice of a CamKII-Cre mouse injected in dorsal CA1 with AAV 2.1 EF1α-double floxed-ChR2-eYFP (41.5 nl/site with 3 week incubation), immunostained for GFP and DAPI (nuclear marker). Green hippocampal axons were observed in entorhinal cortex layers 5 and 2/3. Center – Expanded view of the hippocampal projection to ECL5 shows no infected neurons. Bottom – Expanded view the hippocampal projection to ECL2/3 shows a sample infected ECL2/3 neuron. b. Similar to (a), but using AAV 2.9 EF1α-double floxed-ChR2-eYFP. Hippocampal axons target ECL5 and ECL2/3. No infected neurons in ECL5; one infected neuron observed in ECL2/3. c. Similar to (a), but with AAV 2.5 EF1α-double floxed-ChR2-eYFP (82.8 nl/site, 3-week incubation). Axons in ECL5 and ECL2/3. No infected neurons detected in either layer. d. Similar to (a), but with a C57Bl/6 mouse injected with AAV 2.2 CAMKII-ChR2-eYFP. Axons innervate ECL5 and ECL2/3; no infected neurons in either layer. e. Quantification of the number of infected neurons observed in EC after 3 weeks of viral incubation period. 41.4 nl of virus was injected per site for viral serotypes AAV 2.1 (n = 8), 2.2 (n = 8), and 2.9 (n = 22), and 82.8 nl per site for AAV 2.5 (n = 11). Data represented as box and whisker plots (median, upper/lower quartiles, min to max whiskers). Each data point represents number of infected neurons in a mouse. Fitting a full model two-way ANOVA showed a significant effect of serotype used (p-value < 0.0001), EC layer (p-value < 0.0001), and significant interaction between the two factors (p-value < 0.0001). Post-hoc Šidák’s multiple comparison test shows a significant difference in the infection between ECL2/3 and ECL5 for AAV 2.9 (**** - p-value < 0.0001). Post-hoc Tukey’s multiple comparisons test showed significant difference in the number of infected cells between all serotypes except AAV 2.2 and AAV 2.5 (**** - p-value < 0.0001). f. Representative confocal image of a horizontal brain slice of a CamKII-Cre mouse injected with AAV 2.5 EF1α-double floxed-ChR2-eYFP into dorsal CA1, immunostained for GFP, MAP2 (marker for dendrites), and DAPI (nuclear marker). Green hippocampal axons were observed in ECL5 and ECL2/3. Enlarged views (f1–f3) show no GFP-MAP2 colocalization, confirming no EC neuronal infection (3 independent experiments). g. Schematic showing the injection strategy used to isolate MECL2/3 projecting dCA1 neurons. As proof of principle in one wild-type mouse, we injected AAV 2.retro Syn Cre along with AAV 2.1 Syn Flex GCaMP6s in MECL2/3. In the same animal, we injected a Cre-dependent virus expressing mCherry in dorsal CA1. h. (h1) Confocal image of a horizontal brain slice from a mouse stained for RFP showing injection site (dorsal CA1) of AAV 2.5 Syn DIO hM4D mCherry. (h2) Confocal image of horizontal section from the same mouse as in h1 stained for RFP showing the injection site (green neurons) for AAV 2.1 SynFlex GCaMP6s and AAV 2.retro Syn Cre in ECL2/3. While we targeted MECL2/3 projecting hippocampal neurons, we see projections to MECL5 as well. i. Confocal image of a horizontal brain slice from a mouse injected as described in (g). Green GCaMP+ MECL2/3 axons present in SLM of dCA1 at loci of our viral injection coordinates showing hM4D-mCherry positive CA1 PNs.
