Figure 2. Brain-wide retrograde tracing identifies monosynaptic inputs of prelimbic cortex (PL) engram cells.
(A–B) Experimental design and timeline. Fos-CreERT2 animals were first injected in the PL with helper adeno-associated viruses (AAVs) expressing GFP, TVA receptor, and oG (rabies optimized glycoprotein) in a Cre-dependent manner. Tamoxifen (or vehicle for control) was injected right after contextual fear conditioning (CFC) to trigger recombination in cFos+ cells. Three weeks later, a modified rabies virus (RV∆G-mCherry with EnvA coating) was injected in PL where it infected TVA-expressing cells, replicated in oG-expressing cells, and was retrogradely transsynaptically transported. A week later, brains were collected to quantify monosynaptic inputs of PL engram cells labelled with mCherry. (C, D) Representative images of the PL injection site (scale 400 µm) and magnified view of starter cells (scale 20 µm) with tamoxifen (C) or vehicle (D) injection. (E) Percentage of starter cells over DAPI in PL. (F) Representative images of traced cells throughout the brain (scale 500 µm). (G) Magnified views of traced cells (scale 100 µm) in claustrum (CLA) (inset 1), insular cortex (INS) (1), basolateral amygdala (BLA) (2), retrosplenial cortex (RSP) (3), ventral CA1 (vCA1) (4), and entorhinal cortex (EC) (5). (H) Brain-wide quantification of traced cells, normalized by the number of starter cells for each animal, in the medial prefrontal cortex (mPFC) subregions (left) and the rest of the brain (right). Tamoxifen: n=3 animals; vehicle: n=2 animals.
Figure 2—figure supplement 1. Raw quantifications of the rabies tracing experiment.
