Extended Data Fig. 8 |. Repeated activation of a random subset of neurons does not alter the performance of early-trained mice.

a, AAV injection paradigm to express DREADDs in a random subset of neurons and manipulate their activity with CNO. High-titer AAV is delivered to cFos^CreER mice, which express Cre in task-specific neurons following 4-hydroxytamoxifen administration. Low-titer AAV is delivered to Camk2a^CreER mice, which express Cre ubiquitously in excitatory neurons following 4-hydroxytamoxifen administration. cFos^CreER and Camk2a^CreER mice both express the same number of labeled neurons, but those in cFos^CreER mice are task-specific. b-d, Overlap between injected AAV (mCherry) and endogenous reporter (GFP) in early-trained mice at 13 weeks of age. Quantification of mCherry+ cells in the cortex (c) and hippocampus (d) of WT (n = 3) and Rett (n = 4) mice expressing cFos^CreER and WT (n = 4) and Rett (n = 4) expressing Camk2a^CreER. Scale bar, 200 μm. e-f, cFos reactivation in Camk2a^CreER mice expressing mCherry. Quantification of reactivation in the cortex and hippocampus of WT (n = 4) and Rett (n = 3) mice after Morris water maze training at 13 weeks of age (f). g-i, Morris water maze performance in early-trained WT and Rett mice expressing Camk2a^CreER and hM4Di-mCherry or mCherry. WT mCherry (n = 7), Rett mCherry (n = 8), WT hM4Di (n = 8), and Rett hM4Di (n = 6) mice were tested at 13 weeks of age and injected with CNO during testing (g). A probe trial on day 6 measured the time spent in target quadrant (h) and the number of platform area crossings (i). j-k, Morris water maze performance in early-trained WT and Rett mice expressing Camk2a^CreER and hM3Dq-mCherry or mCherry. A probe trial on WT mCherry (n = 7), Rett mCherry (n = 8), WT hM3Dq (n = 7), and Rett hM3Dq (n = 7) measured the time spent in the target quadrant (j) and the number of platform area crossings (k). The sample size (n) corresponds to the number of biologically independent mice. Data are represented as mean ± s.e.m. Statistical significance was determined using a two-way ANOVA with Tukey’s multiple comparisons test; ns (p>0.05).