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. 2022 Feb 14;9(1):ENEURO.0381-21.2021. doi: 10.1523/ENEURO.0381-21.2021

Figure 1.

Figure 1.

Selective expression of hM4Di-DREADD in CaMKIIα-positive forebrain excitatory neurons in CaMKIIα-tTA::TRE-hM4Di bigenic mice. A, Shown are representative confocal images indicating expression of the HA-tagged hM4Di-DREADD in the hippocampus as identified by HA/CaMKIIα double immunofluorescence. B, C, HA-tagged hM4Di-DREADD expression was not observed in either PV-positive inhibitory interneurons (B) or GFAP-positive astrocytes (C). D, HA-tagged hM4Di-DREADD in the cortex was also observed in the CaMKIIα-positive neurons as identified with HA/CaMKIIα double immunofluorescence. E, F, Immunofluorescence experiments indicate the absence of expression of HA-tagged hM4Di-DREADD in subcortical brain regions, namely the periaqueductal gray (E) and pallidum (F). G, Shown is a schematic of the experimental paradigm for harvesting cortex and hippocampus at P7 for Western blotting analysis. H, I, HA expression was clearly noted in the cortex (H) as well as the hippocampus (I) in Western blots from CaMKIIα-tTA::TRE-hM4Di bigenic pups (P7). J, Shown are representative Western blots for c-Fos along with their respective actin loading controls at P7 half an hour after vehicle (Veh) or CNO treatment for cortex (top panel) and hippocampus (bottom panel). K, L, Quantitative densitometry indicated a significant reduction in c-Fos protein levels in the cortex (K) as well as the hippocampus (L) of PNCNO-treated bigenic pups at P7 compared with their vehicle-treated controls. M, Shown is a schematic of the experimental paradigm for harvesting cortex and hippocampus at P35 in the juvenile window for Western blotting analysis. N, O, HA expression was noted in the cortex (N) as well as the hippocampus (O) of CaMKIIα-tTA::TRE-hM4Di bigenic juvenile mice (P35). P, Shown are representative Western blots for c-Fos along with their respective actin loading controls at P35 half an hour after vehicle (Veh) or CNO treatment for cortex (top panel) and hippocampus (bottom panel). Q, R, Quantitative densitometry indicated a significant reduction in c-Fos protein levels in the cortex (Q) but not in the hippocampus (R) of JCNO-treated bigenic mice at P35 compared with their vehicle-treated controls. All immunofluorescence experiments and Western blotting experiments were performed on n = 3–5/group. Results are expressed as the mean ± SEM. *p < 0.05, compared with vehicle-treated controls using the two-tailed, unpaired Student’s t test.