Figure 4

Inhibiting contralateral hippocampus via activation of hM4D blocks AD development induced by optogenetic train stimulation in CaMKIIa-ChR2 mice. (a) Illustration of unilateral blue light stimulation with bilateral field recording in horizontal section, targeting ventral CA3/DG at DV-2.1. hM4D(Gi)-mCherry was expressed by AAV vector contralateral to stimulation in the hM4D group. (b–d) Representative example of hM4D expression in composite images of horizontal sections from three levels of the hippocampus: dorsal, middle and ventral, respectively. hM4D is visible by mCherry autofluorescence (red; magenta when co-localized with nuclear counterstain (Hoechst, blue)) and ChR2 by YFP (green). (e) Representative section of hemisphere ipsilateral to optogenetic stimulation (opposite side of c), lacking hM4D expression. Post-experiment burn-in (electrode location) is visible in the hilus. (f,g) Traces of bilateral field recordings in control and hM4D mouse, respectively (I: ipsi, C: contra). Timeline arrow indicates number of stimulation trains from IP injection (dashed line) of vehicle or CNO. Bilateral progression of ADs is visibly abrogated after CNO injection in the hM4D-expressing mouse (g, bottom). (h,i) Counting contralateral intra-train s-LFPs and aligning bins of three stimulations according to the first ipsilateral progressive AD (see Fig. 2f–g), a 37% increase in s-LFPs (h, repeated measures ANOVA with Dunnett’s post-hoc test) is seen, coinciding in time with contralateral progressive AD onset (i, Friedman test with Dunn’s post-hoc test). (j) Quantification of AD durations, which were remarkably similar between ipsilateral (left graph) and contralateral sides (right graph). AD durations did not increase further in hM4D-expressing animals injected with CNO (red line) (repeated measures ANOVA with Dunnett’s post-hoc test, binned data, 5 stimulations per bin (see Results)). (k) AD durations of hM4D and control groups in three 5 stimulation bins (Post1-3) covering 75 minutes post-injection were directly compared. Durations were reduced after injection for the hM4D group both ipsi- and contralaterally (left and right graph, respectively) (One-way ANOVA with Sidak’s post-hoc test, see Table 2 for exact P values). Scale bars: (b–e) 100 µm; (f,g) 5 mV, 5 s. Error bars: ± SD. *P < 0.05, **P < 0.01, ns: non-significant.