Fig. 6: Suppression of adolescent ACAVIS neuron activity results in their excessive loss of local excitatory inputs.
a Experimental approach to test the impact of ACAVIS neuron activity during adolescent or late-adolescent period on maintaining local excitatory input drive onto ACAVIS neurons into adulthood through optogenetic interrogation of local excitatory input onto ACAVIS neurons. (Left) RetroAAV2-cre was injected into the VIS, while AAV-DIO- hM4D(Gi)-(iDREADD)-mCherry or AAV-DIO-mCherry was injected into ACA at p12 to selectively express iDREADD-mCherry or mCherry in ACAVIS neurons by p25. (middle) Mice were injected with 10 mg/kg CNO in normal saline twice a day from p25 to p34 in adolescent iDREADD and adolescent mCherry groups, or from p45 to p54 in late-adolescence iDREADD group. Additional injections of AAV-Flpo and AAV-CreOff/FlpOn-ChR2-YFP in ACA at p60.4 ± 2.2 in average (p52–p80) enable ChR2-GFP expression in all but ACAVIS neurons in ACA. (right) Slice patch clamp recordings were performed from ACAVIS neuron at p85.7 ± 2.3 in average (p72–107) while optogenetically activating ChR2-expressing local excitatory inputs. b (Left) Representative image showing ChR2-YFP expressing local inputs (green) and mCherry expressing ACAVIS neurons (magenta) in ACA. (right) High magnification of boxed area showing mCherry + ACAVIS neuron does not express ChR2-YFP and is surrounded by ChR2 + terminals. Scale bars: 200 μm on the left and 20 μm on the right. Experimental images were obtained from 24 mice with similar results obtained. c The quantification of the spread and intensity of the YFP signal showed no significant differences among groups (one-way ANOVA, area: F2,30 = 1.334, P = 0.8962, mean intensity: F2,28 = 1.334, P = 0.1612, n = 13 slices from 9 biologically independent mice for adolescent iDREADD. n = 15 slices from 10 biologically independent mice for adolescent mCherry, and n = 7 slices from 5 biologically independent mice for late-adolescent iDREADD). d Examples of light-evoked EPSC in ACAVIS neurons in the presence of strontium to produced delayed asynchronous release of neurotransmitters for each groups. Arrows indicate detected qEPSCs. e, f Averaged qEPSC (e) frequency and (f) amptitude for adolescent iDREADD, adolescent mCherry and late-adolescent iDREADD groups (frequency: one-way ANOVA F2,43 = 8.010, P = 0.0011, post hoc Tukey test **PAdol.iDREADD-Adol.mCherry = 0.009, **PAdol.iDREADD-Lateadol.iDREADD = 0.002; mean amplitude: one-way ANOVA, F2,43 = 1.352, P = 0.2697, n = 15 cells from four biologically independent mice for adolescent iDREADD. n = 15 cells from 4 biologically independent mice for adolescent mCherry, and n = 16 cells from 4 biologically independent mice for late-adolescent iDREADD). Data in c, e–f are presented as mean ± s.e.m. See related Supplementary Figs. 8 and 9. Source data are provided as a Source data file.
