Figure 4. DREADD-activated cells have increased myelination. a, b, Maximum projection confocal images of representative biocytin-filled hM3Dq-mCherry+ (a; red) and GFP+ (b; yellow) cells. Scale bars, 50 μm. c, Left, Post hoc confirmation demonstrating that putative hM3Dq-mCherry+ biocytin-labeled cells (red) were hM3Dq-mCherry+ (purple). Right, Post hoc confirmation that putative GFP+ biocytin-labeled cells (yellow) were GFP+ (gray). Scale bars, 10 μm. d, Reconstructed cells were located at similar cortical depths (hM3Dq-mCherry, red diamonds: 394.5 ± 42.3 μm; GFP, green diamonds: 369.8 ± 36.5 μm; U = 61.000, p = 0.832). e, Top, Low-magnification confocal image of a biocytin-labeled PV+ interneuron (red), with myelinated internodes revealed by MBP immunofluorescence (green). Arrowheads indicate myelinated internodes belonging to the biocytin-labeled cell. Scale bar, 10 μm. Bottom, High-magnification confocal maximum projection image of a biocytin-labeled (red), myelinated (MBP; green) axonal segment. Scale bar, 5 μm. f, g, Proximal axonal reconstruction (left) with indicated segmental myelination of hM3Dq-mCherry+ (f) and GFP+ (g) cells shown in (a) and (b), respectively, with their corresponding schematic dendrogram representationa (right). Soma and axon are shown in gray, myelin in green. Horizontal axis is in micrometers. h, All examined hM3Dq-mCherry+ (red, n = 12) and GFP+ cells (green, n = 14) show myelination. i, hM3Dq-mCherry+ cells show a higher total amount of myelin per cell compared with GFP+ cells. hM3Dq-mCherry: 230.3 ± 42.4 μm myelin/cell; GFP: 117.9 ± 25.1 μm myelin/cell; t(24) = 2.359, p = 0.027. j, hM3Dq-mCherry+ cells show a greater number of internodes per cell compared with GFP+ cells (hM3Dq-mCherry: 10.4 ± 1.3 internodes/cell; GFP: 5.6 ± 1.0 internodes/cell; t(24) = 3.008, p = 0.006). k, Internode length is similar between hM3Dq-mCherry+ and GFP+ cells (hM3Dq-mCherry: 23.2 ± 1.2 μm, n = 99 internodes; GFP: 19.9 ± 1.3 μm, n = 75 internodes; t(172) = 1.888, p = 0.061). l, hM3Dq-mCherry+ cells have an increased number of internodes beginning from fourth branch order axonal segments distally (F(12,240) = 2.890, p < 0.001, repeated-measures ANOVA). m, hM3Dq-mCherry+ and GFP+ cells have a similar myelination onset (hM3Dq-mCherry: 1.7 ± 0.4; GFP: 1.9 ± 0.5; U = 89.000, p = 0.574). n, In contrast, the most distal branch order showing myelination is located further along the axon in hM3Dq-mCherry+ cells (hM3Dq-mCherry: 7.6 ± 0.4; GFP: 5.3 ± 0.3; t(21) = 4.387, p < 0.001). ***p < 0.001 **p < 0.01, *p < 0.05, n.s., Nonsignificant. Unpaired Student's two-tailed t test was used in i, j, k, and n; Mann–Whitney two-tailed U test was in d and m because of non-normality. Scale bars indicate mean ± SEM.