Extended Data Fig. 5: Effects of unilateral auditory deprivation on the visual system activation, and validation of FosTRAP labeling system in AUDd, related to main Figs. 3 and 4.

a-b, Random unilateral ear sealing does not disrupt cFos expression across hemispheres in VISp. a, cFos expression across hemispheres in VISp was quantified in the same group of animals that underwent random unilateral ear sealing while performing SFI and then sacrificed. b, cFos immunohistochemistry for exemplar showing VISp cortical region for an animal sacrificed on day 21 of SFI training with one ear sealed (left) and one ear open (middle). Scale bars denote 50 μm. Comparison of percent activation according to cFos counts across hemispheres for VISp cortical region (right) ipsilateral versus contralateral to the sealed ear of animals sacrificed upon completion of SFI at day 21 (n = 5) of training. c-g, FosTRAP expression recapitulates cFos protein expression pattern in AUDd. c, FosTRAP animals were trained for 21 days on SFI and split into two groups. One group (top)(n = 4) was induced with 4-OHT after being injected with a Cre-dependent AAV expressing GFP in AUDd and eventually sacrificed immediately after performing SFI again with ears open. The other group (bottom)(n = 4) performed the no lever version of the task also with ears open and immediately sacrificed following session completion. d, Percent distribution across the cortical layers in AUDd of cFos protein and cFosCreERT2 (as visualized via Cre-dependent GFP expression) in FosTRAP animals that were induced (green) while performing SFI with their ears open and later in another SFI session immediately sacrificed upon completion (black), again with ears open. These distributions were compared to the cFos protein percent distribution of FosTRAP animals that were sacrificed upon session completion in the no lever context with their ears open (red)(effect of interaction F_(8,45) = 2.335, P = 0.0343; no lever layer V cFos protein vs. SFI layer V cFosCreERT2/cFos protein, P = 0.0172 and P = 0.0496, respectively) with ears open (n = 4). e-f, AUDd cortical layer expression pattern of endogenous cFos protein for FosTRAP animals sacrificed immediately after performing SFI (e) and being in the no lever context (f) with their ears open. g, FosTRAP cFosCreERT2 expression as visualized via Cre-dependent GFP expression in AUDd of an animal induced with its ears open while performing SFI. Scale bars denote 100 μm. h, Fiber placement in AUDd of FosTRAP animals injected with an AAV expressing Cre-dependent ChR2-EYFP and induced with its ears open while performing SFI. Scale bar denotes 500 μm. For VISp cFos expression analysis, data were analyzed using a paired t-test. Bars denote mean percentage across hemispheres. For FosTRAP validation and AUDd laminar analysis, data were analyzed using two-way ANOVA followed by Sidak post hoc comparisons. Values are mean percentages across layers. Error bars denote s.e.m. For all immunohistochemistry images, ‘D’ and ‘L’ denote dorsal and lateral, respectively. *P < 0.05; NS, not significant.