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. 2015 Dec 2;35(48):15996–16011. doi: 10.1523/JNEUROSCI.2068-15.2015

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Copyright © 2015 Stephen et al.

This article is freely available online through the J Neurosci Author Open Choice option.

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Figure 6. — Mutant Miro1 (Miro1^ΔEF) expression, in situ, disrupts the ability of mitochondria to respond to neuronal activity and alters activity-driven astrocyte mitochondrial positioning near synapses. A, Schematic representation of the biolistic triple-transfection system used to label astrocytes in slices. DNA of either Miro1^WT-ires-mtdsRed2 or Miro1^ΔEF-ires-mtdsRed2 was mixed with GFAP-driven GFP DNA and this mix was used to coat gold bullets. The standard biolistic transfection protocol for slices using a gene gun was subsequently followed. In this way sparse astrocytes express GFAP-driven GFP and either Miro1^WT or Miro1^ΔEF-ires-mtdsRed2. B, Example confocal images of hippocampal slices biolistically transfected with GFAP-driven GFP and either Miro1^WT or Miro1^ΔEF-ires-mtdsRed2 imaged 2–3 d post-shooting (slices were shot at 7 DIV). Scale bars, 15 μm. C, Mitochondria moving before (control) and after 4-AP treatment (100 μm, 5 min) with Miro1^WT and Miro1^ΔEF overexpression (OE) [Miro1^WT (control and 4-AP): n = 3 cells, 3 slices; Miro1^ΔEF (control and 4-AP): n = 3 cells, 3 slices]. D, Velocity of moving mitochondria before and after 4-AP treatment with Miro1^WT or Miro1^ΔEF OE (Miro1^WT control: n = 11 mitochondria, 3 slices; Miro1^WT + 4-AP: n = 10 mitochondria, 3 slices; Miro1^ΔEF control and Miro1^ΔEF + 4-AP: n = 10 mitochondria, 3 slices). E, Mitochondrial length (morphology) pre-4-AP treatment and post-4-AP treatment with Miro1^WT or Miro1^ΔEF OE (Miro1^WT control and Miro1^WT + 4-AP: n = 3 cells, 3 slices; Miro1^ΔEF control and Miro1^ΔEF + 4-AP: n = 3 cells, 3 slices). F, Representative images of mitochondrial distribution in relation to VGLUT1-labeled synapses with Miro1^WT and Miro1^ΔEF OE before and following 4-AP treatment (5 min, 100 μm). Scale bars, 5 μm. G, Distance between mitochondria and their nearest VGLUT1-labeled synapse under basal conditions and with 4-AP treatment (Miro1^WT control: n = 7 ROIs, 3 slices; Miro1^WT + 4-AP: n = 6 ROIs, 3 slices; Miro1^ΔEF control: n = 7 ROIs, 3 slices; Miro1^ΔEF + 4-AP: n = 7 ROIs, 3 slices). H, Number of mitochondria and VGLUT1 puncta in analyzed ROIs (per μm) with Miro1^WT or Miro1^ΔEF OE (Miro1^WT control: n = 7 ROIs, 3 slices; Miro1^WT + 4-AP: n = 6 ROIs, 3 slices; Miro1^ΔEF control: n = 7 ROIs, 3 slices; Miro1^ΔEF + 4-AP: n = 7 ROIs, 3 slices). *p < 0.05, **p < 0.01, ***p < 0.001.