(a) Schematic of calcium imaging protocol. (b) Specimen images (5 hours apart) of tg(mbp: mCherry-CAAX) (hotter colours indicate more fluorescence, left scale) in developing oligodendrocytes. Red, blue and white arrows indicate sheaths that became longer, shorter or did not change length, respectively, over 5 hours. Experiments were repeated six times with similar results. (c-e) Sheath behaviour over time in 3 cells to illustrate diversity of behaviour. Pie charts show percentage of sheaths growing (red; 75% in c, 42.9% d, 37.5% e), growing then shortening (purple; 25% c, 14.3% d), shortening (blue; 14.3% d, 62.5% e), and not changing length (black; 28.6% d). (f) Change of sheath length over 5 hours as a function of mean [Ca²⁺]_i transient rate (see Online Methods) in 68 sheaths. (g) Increase of sheath length per hour and [Ca²⁺]_i transient rate in 14 sheaths which were of constant length for at least 1 hour before lengthening started (at t=0). Data are mean±SEM. (h) Protocols for experiments in i (top) and j (bottom). Large lines: structural stack acquisition; small red lines: stimulation. (i-j) Sheath growth over 5 hours in (i) 78 sheaths in 5 sham-stimulated fish and 71 sheaths in 5 stimulated fish, and (j) 99 sheaths in 6 sham-injected fish and 93 sheaths in 6 TTX-injected fish (box and whisker plots show median, 25th and 75th centiles, and most extreme data values). (k) Sheath growth over 3 hours for oligodendrocytes briefly whole-cell clamped with internal solution containing 1 mM EGTA (Ctrl, 24 sheaths from 6 fish) or 30 mM BAPTA (21 sheaths from 5 fish). (l) Percentage of sheaths with different growth behaviour (G, growth; 0, no change, G->S, growing then shortening, S, shortening) exhibiting >1 Ca²⁺ transients in the 63 mins imaged over 5 hours (black, left axis) or showing [Ca²⁺]_i bursts (red, right axis). Insets show specimen transients and bursts (scale bars 1 min and ∆F/F=1). Different growth states are associated with significantly different numbers of transients (Chi-squared p=10^-5, n=68 sheaths from 6 animals) and bursts (p=9x10^-4). Because [Ca²⁺]_i was only sampled for 10.5 mins per hour, and bursts are rare, it is possible that all (rather than 33% of) shortening sheaths experience [Ca²⁺]_i bursts. Two-tailed statistical tests used: linear regression t-statistic in f; Mann-Whitney in i, j; unpaired t-test in k; Chi-squared test in l.