Fig. 6: Camkk2 and AMPK are required for the compartment-specific mitochondrial morphology in CA1 PNs in vivo.

a–i High magnification representative images of mitochondrial morphology within isolated secondary or tertiary hippocampal CA1 (a) basal, (d) apical oblique, and (g) apical tuft dendrites in which a mitochondrial matrix-targeted fluorescent protein (mt-YFP or mt-DsRed) and cell fill (tdTomato or mGreenLantern—not shown) were co-expressed by IUE with or without Cre recombinase (+/-Cre). AMPKα1^F/Fα2^F/F double conditional mice were in utero electroporated with the same mitochondrial markers/cell fills and either no Cre (AMPK^FF/FF - Cre) or Cre (AMPK^FF/FF + Cre). Camkk2^-/- constitutive knock-out mice were electroporated with the same mitochondrial markers/cell fills as above (Camkk2^-/-). Quantification of mitochondrial length and occupancy in the (b, c) basal, (e, f) apical oblique, and (h, i) apical tuft dendritic compartments reveals a significant increase in mitochondrial length (b) and occupancy (c) in basal dendrites when knocking out AMPK or Camkk2 when compared to their controls, with Camkk2 KO having a much bigger effect (WT: n = 32 dendritic segments, 313 individual mitochondria, mean length = 1.065 μm ± 0.029 (SEM), mean occupancy = 33.01% ± 1.84%; WT + Cre: n = 46 dendritic segments, 627 individual mitochondria, mean length = 1.437 μm ± 0.040 (SEM), mean occupancy = 40.95% ± 1.94%; AMPK^FF/FF - Cre: n = 42 dendritic segments, 463 individual mitochondria, mean length = 1.418 μm ± 0.039 (SEM), mean occupancy = 39.24% ± 1.89%; AMPK^FF/FF + Cre: n = 36 dendritic segments, 381 individual mitochondria, mean length = 2.832 μm ± 0.116 (SEM), mean occupancy = 50.72% ± 2.36%, length increase = 97.08%, occupancy increase = 23.86%; Camkk2^-/-: n = 46 dendritic segments, 449 individual mitochondria, mean length = 4.975 μm ± 0.233 (SEM), mean occupancy = 73.36% ± 2.03%, length increase = 367%, occupancy increase = 122%). A significant increase in mitochondrial length (e) and occupancy (f) is also seen in apical oblique dendrites when knocking out Camkk2, but only an increase in length is seen when knocking out AMPK. (WT: n = 28 dendritic segments, 184 individual mitochondria, mean length = 1.569 μm ± 0.104 (SEM), mean occupancy = 42.38% ± 2.98%; WT + Cre: n = 44 dendritic segments, 525 individual mitochondria, mean length = 1.791 μm ± 0.052 (SEM), mean occupancy = 48.50% ± 2.12%; AMPK^FF/FF - Cre: n = 45 dendritic segments, 440 individual mitochondria, mean length = 1.885 μm ± 0.075 (SEM), mean occupancy = 45.82% ± 2.29%; AMPK^FF/FF + Cre: n = 52 dendritic segments, 499 individual mitochondria, mean length = 2.645 μm ± 0.098 (SEM), mean occupancy = 47.79% ± 1.62%, length increase = 47.68%; Camkk2^-/-: n = 29 dendritic segments, 288 individual mitochondria, mean length = 5.366 μm ± 0.346 (SEM), mean occupancy = 78.64% ± 2.07%, length increase = 242%, occupancy increase = 85.56%). Note that in Camkk2-null CA1 PNs we observe a significant increase in mitochondrial length (h) and occupancy (i) in the apical tuft compared to WT controls (WT: n = 33 dendritic segments, 226 individual mitochondria, mean length = 6.562 μm ± 0.437 (SEM), mean occupancy = 84.19% ± 1.44%; WT + Cre: n = 29 dendritic segments, 189 individual mitochondria, mean length = 5.206 μm ± 0.052 (SEM), mean occupancy = 79.40% ± 1.38%; AMPK^FF/FF - Cre: n = 38 dendritic segments, 229 individual mitochondria, mean length = 6.498 μm ± 0.368 (SEM), mean occupancy = 87.00% ± 1.67%; AMPK^FF/FF + Cre: n = 47 dendritic segments, 370 individual mitochondria, mean length = 5.723 μm ± 0.300 (SEM), mean occupancy = 80.65% ± 1.71%; Camkk2^-/-: n = 35 dendritic segments, 199 individual mitochondria, mean length = 11.14 μm ± 0.758 (SEM), mean occupancy = 95.03% ± 1.73%, length increase = 69.77%, occupancy increase = 12.88%). p values are indicated in the figure following a one way ANOVA with Sidak’s multiple comparisons test. Data are shown as individual points on box plots with 25^th, 50^th and 75^th percentiles indicated with whiskers indicating min and max values. Scale bar, 5 μm.