mGluR5 stimulation increases myelin sheath length without affecting cell number.

a, Protocol to label individual oligodendrocytes, manipulate mGluR5 activity and assess myelination. b, Relative frequency of sheath lengths (DMSO n = 427, mean 16.79 µm; MTEP n = 201, mean 18.41 µm; CHPG n = 470, mean 30.44 µm). c, Individual oligodendrocytes at 4 dpf, labeled with mbp:eGFP–CAAX, after treatment with DMSO, mGluR5 antagonist MTEP and allosteric agonist CHPG. Scale bar, 15 µm. d, Mean sheath length per oligodendrocyte (one OL/fish) post treatment (one-way analysis of variance (ANOVA), P < 0.0001; Holm–Šídák’s multiple comparisons test: DMSO versus CHPG P = 0.0062, DMSO versus MTEP P = 0.0047; CHPG versus MTEP P < 0.0001). 1% DMSO (N = 23, 26.90 µm ± 6.031), CHPG (N = 28, 31.66 µm ± 4.27) and MTEP (N = 12, 20.15 µm ± 8.77). e, Number of myelin sheaths produced by single oligodendrocytes (one-way ANOVA; P = 0.2090, Kruskal–Wallis DMSO versus CHPG P = 0.2477; DMSO versus MTEP P > 0.9999; CHPG versus MTEP P > 0.9999). DMSO, N = 23, 18.04 ± 4.995; MTEP, N = 12, 16.75 ± 3.89; CHPG N = 28, 15.32 ± 6.08). Scale bar, 50 µm. f, Representative images of Tg(olig1:nls–mApple) after pharmacological manipulation of mGluR5. Scale bar, 50 µm. g, Number OPCs in the spinal cord (one-way ANOVA; P = 0.7774; Tukey’s multiple comparisons test, DMSO versus MTEP P = 0.9908; DMSO versus CHPG P = 0.8579; CHPG versus MTEP P = 0.7781), DMSO (N = 15); CHPG (N = 17); MTEP (N = 16). DMSO 371.1 ± 62.39; MTEP 368.5 ± 54.37; CHPG 381.8 ± 53.13. h, Representative images of Tg(mbp:nls–eGFP) after pharmacological manipulation of mGluR5. i, Number of myelinating oligodendrocytes in the spinal cord (one-way ANOVA; P = 0.5155; Tukey’s multiple comparisons test: DMSO versus MTEP P = 0.9141; DMSO versus CHPG P = 0.7359; CHPG versus MTEP P = 0.5037), DMSO (N = 22); CHPG (N = 25); MTEP (N = 18). DMSO, 252 ± 33.24; MTEP, 257.3 ± 43.61; CHPG, 243 ± 45.46. Scale bar, 50 µm. Data indicate mean ± s.d.

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Mutations in genes encoding mGluR5 reduce myelin sheath length without affecting sheath number.

a, HCR detection of grm5a and grm5b messenger RNA in OPCs at 5 dpf. Scale bar, 5 μm. b: HCR detection of grm5a and grm5b mRNA in OLs at 5 dpf. Scale bar, 5 μm. c,d, Schematic showing organization of human and zebrafish genes encoding mGluR5 and their CRISPR/Cas9 based editing that led to identification of mutants with premature STOP codons in exons 2 and 1 of grm5a (c) and grm5b (d), respectively. e, Images of wild-type (N = 35) and grm5a^−/−grm5b^−/− (N = 22) oligodendrocytes expressing mbp:eGFP–CAAX. f, Images of wild-type (N = 18) and grm5a^−/−grm5b^−/− (N = 12) oligodendrocytes expressing mbp:eGFP–CAAX at 4 dpf. g, Images of wild-type (N = 10) and grm5a^−/−grm5b^−/− (N = 14) oligodendrocytes expressing mbp:eGFP–CAAX at 7 dpf. Scale bars 15 μm (e–g). h, Mean sheath length of wild-type (N = 35, 21.11 ± 5.149) and grm5a^−/−grm5b^−/− (N = 22, 16.34 ± 5.38) oligodendrocytes, in 3 dpf old animals (two-sided Mann–Whitney U-test, P = 0.0035). i, Mean sheath length of wild-type (N = 18, 28.8 ± 6.5) and grm5a^−/−grm5b^−/− (N = 12, 19.47 ± 3.67) oligodendrocytes, in 4 dpf old animals (two-sided unpaired t-test: P = 0.0001). j, Mean sheath length of wild-type (N = 10, 31.79 ± 4.723) and grm5a^−/−grm5b^−/− (N = 14, 22.68 ± 4.40) oligodendrocytes, in 7 dpf old animals (two-sided unpaired t-test: P < 0.0001). k, Comparison of mean sheath length in wild-type animals between 3, 4 and 7 dpf animals (one-way ANOVA, Kruskal–Wallis test P < 0.0001; Dunn’s multiple comparisons test: wild-type 3 dpf versus 4 dpf P = 0.0002; wild-type 4 dpf to 7 dpf P = 0.7173, wild-type 3 dpf versus 7 dpf P < 0.0001). (3 dpf, 21.11 ± 5.149; 4 dpf, 28.8 ± 6.52; 7 dpf, 31.79 ± 4.72). l, Number of myelin sheath per oligodendrocyte in 3 dpf wild-type (16.2 ± 4.8) and grm5a^−/−grm5b^−/− (15.36 ± 3.94) (two-sided unpaired t-test; P = 0.4964). m, Number of sheaths per oligodendrocyte in 4 dpf wild-type (16.06 ± 4.03) and grm5a^−/−grm5b^−/− (15.92 ± 34.46) (two-sided unpaired t-test; P = 0.9301). n, Number of myelin sheath per oligodendrocyte in 7 dpf wild-type (21.2 ± 5.35) and grm5a^−/−grm5b^−/− (19.5 ± 8.91) (two-sided unpaired t-test; P = 0.5971). o, Comparison of the mean sheath length in grm5a^−/−grm5b^−/− animals between 3, 4 and 7 dpf animals (one-way ANOVA, Kruskal–Wallis test P < 0.0001; Dunn’s multiple comparisons test: grm5a^−/−grm5b^−/− 3 dpf versus 4 dpf P = 0.1675; grm5a^−/−grm5b^−/− 4 dpf to 7 dpf P = 0.2070, grm5a^−/−grm5b^−/− 3 dpf versus 7 dpf P < 0.0009) (3 dpf, 16.34 ± 5.38; 4 dpf, 19.47 ± 3.67; 7 dpf, 22.68 ± 4.40). Data show mean ± s.d.

Source data

Oligodendrocyte-restricted expression of grm5a promotes myelin sheath elongation.

a, Construct used to express mGluR5 tethered to eGFP, alongside a membrane anchored reporter mScarlet, in myelinating oligodendrocytes. b, Control construct used to express fluorescent reporter in myelinating oligodendrocytes. c,c′, Images of oligodendrocytes expressing control mbp:memScarlet in wild-type (WT) (c) (N = 15) and grm5a^−/− (c′) (N = 20) animals at 4 dpf. d,d′, Oligodendrocytes expressing mbp:memScarlet-P2A–grm5a–eGFP in WT (d) (N = 12) and grm5a^−/− (d′) (N = 12) animals at 4 dpf. Scale bar, 10 μm. e, Relative frequency distribution of individual myelin sheath lengths. WT mbp:memScarlet n = 265 (mean 26.49 µm); WT mbp:memScarlet-P2A–grm5a–eGFP n = 124 (mean 31.55 µm); grm5a^−/− mbp:memScarlet n = 365 (mean 19.54 µm); grm5a^−/− mbp:memScarlet-P2A–grm5a–eGFP n = 138 (mean 31.58 µm). f, Mean sheath length of WT oligodendrocytes expressing mbp:memScarlet-P2A–grm5a–eGFP (N = 12, 32.19 ± 8.41) or mbp:memScarlet (N = 15, 26.92 ± 4.28) (two-sided unpaired t-test, P = 0.0445). g, Mean myelin sheath length of grm5a^−/− oligodendrocytes expressing mbp:memScarlet-P2A–grm5a–eGFP (N = 12, 33.25 ± 6.57) or mbp:memScarlet (N = 20, 20.05 ± 3.45) (two-sided unpaired t-test, P = <0.0001). h, Sheath number per oligodendrocytes expressing mbp:memScarlet-P2A–grm5a–eGFP (12.67 ± 3.46) or mbp:memScarlet (17.13 ± 5.17) (two-sided unpaired t-test, P = 0.0164). i, Sheath number per oligodendrocytes in grm5a^−/− mutants expressing mbp:memScarlet-P2A–grm5a–eGFP (N = 12, 11.25 ± 4.33) or mbp:memScarlet (N = 20, 17.1 ± 5.21) (two-sided unpaired t-test, P = 0.0027). Scale bar, 10 μm. Data show mean ± s.d.

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Endogenous mGluR5 is required for CHPG-induced high-amplitude myelin Ca²⁺ transients to occur.

a, Schematic of treatment of larvae and live imaging of Ca²⁺ activity in myelin sheaths. b,b′, Top shows representative maximum time series projections of Tg(mbp:memGCaMP7s) fluorescence with Ca²⁺ transients detected by AQuA2 false-colored automatically; bottom show single time-point images from the time-lapse series and highlight transients identified within yellow boxes. WT (N = 14) (b) and grm5a^−/−grm5b^−/− animals (b′) (N = 16) treated with vehicle and CHPG (WT N = 15; grm5a^−/−grm5b^−/−N = 17). Scale bar, 10 µm. c, Median amplitude (dF/F) of Ca²⁺ transients per fish (one-way ANOVA; P = 0.0079; Tukey’s multiple comparison test; WT DMSO versus CHPG P = 0.111, WT DMSO versus grm5a^−/−grm5b^−/− DMSO P = 0.9633; WT DMSO versus grm5a^−/−grm5b^−/− CHPG P = 0.9210, WT CHPG versus grm5a^−/−grm5b^−/− DMSO P = 0.0310, WT CHPG versus grm5a^−/−grm5b^−/− CHPG P = 0.0407; grm5a^−/−grm5b^−/− CHPG versus grm5a^−/−grm5b^−/− DMSO P = 0.9987) (WT DMSO N = 14, 0.94 ± 0.24, WT CHPG N = 15, 1.21 ± 0.31, grm5a^−/−grm5b^−/− DMSO N = 16, 0.98 ± 0.16, grm5a^−/−grm5b^−/− CHPG N = 17, 0.99 ± 0.19). d, Frequency of myelin Ca²⁺ transients in CHPG and DMSO-treated grm5a^−/−grm5b^−/− mutant and WT animals (one-way ANOVA P = 0.1658; Holm–Šídák’s multiple comparisons tests: WT DMSO versus WT CHPG P = 0.3022; WT DMSO versus grm5a^−/−grm5b^−/− DMSO P = 0.2591; WT CHPG versus grm5a^−/−grm5b^−/− DMSO P = 0.9840; WT CHPG versus grm5a^−/−grm5b^−/− CHPG P = 0.6868; WT DMSO versus grm5a^−/−grm5b^−/− CHPG P = 0.6868). WT DMSO 138 ± 149.2, WT CHPG 74.39 ± 48.68, grm5a^−/−grm5b^-/, DMSO 75.04 ± 82.95, grm5a^−/−grm5b^−/− CHPG 108.9 ± 95.43. Scale bar, 10 µm. e, Median duration of Ca²⁺ transients per fish (one-way ANOVA P = 0.2687; Tukey’s multiple comparisons test: WT DMSO versus WT CHPG P = 0.4745; WT DMSO versus grm5a^−/−grm5b^−/− DMSO P = 0.9864; WT DMSO versus grm5a^−/−grm5b^−/− CHPG P = 0.8633; WT CHPG versus grm5a^−/−grm5b^−/− DMSO P = 0.2638, WT CHPG versus grm5a^−/−grm5b^−/− CHPG P = 0.08864; grm5a^−/−grm5b^−/− DMSO versus grm5a^−/−grm5b^−/− CHPG P = 0.6516). WT DMSO 12.76 ± 6.964, WT CHPG 9.495 ± 5.044, grm5a^−/−grm5b^−/−, DMSO 13.52 ± 5.936, grm5a^−/−grm5b^−/−, CHPG 11.06 ± 6.233. f, Ca²⁺ transient dF/F traces of the events shown in corresponding conditions in b and b'. Data show mean ± s.d.

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Opto-stimulation of neuronal activity induces mGluR5-dependent high-amplitude myelin Ca²⁺ transients.

a, Opto-stimulation of ChRimsonR-expressing Chx10 interneurons is carried out during continuous time-lapse imaging of myelin Ca²⁺ transients in Tg(mbp:memGCaMP7s) animals. b, Optogenetic-stimulation paradigm, consisting of the pre-stimulation period ‘pre’ (gray), stimulation period ‘stim’ (black; single opto-stimulation/light pulse (red) per min, ending with 4× stimulations within 10 s) and post-stimulation period (green). c, Maximum projection of AQuA2-detected Ca²⁺ transients in a WT Tg(chx10:Gal4 UAS:ChRimsonR–tdTomato;mbp:memGCaMP7s) animal at 4 dpf. c′, Individual frames of time series showing the increase in fluorescence associated with myelin Ca²⁺ transient indicated by boxed area in c. d, Maximum projection of AQuA2-detected Ca²⁺ transients in a grm5a^−/−grm5b^−/−, Tg(chx10:Gal4 UAS:ChRimsonR–tdTomato; mbp:memGCaMP7s) animal at 4 dpf. Scale bar, 10 µm. d′, Individual frames of time series showing the increase in fluorescence associated with myelin Ca²⁺ transient indicated by boxed area in d. e, Frequency of myelin Ca²⁺ transients in WT animals across pre, stim and post-stim periods (two-sided Friedman test P = 0.0103; multiple comparisons test: pre versus stim P = 0.1017; pre versus post P = 0.0140; stim. versus post P > 0.9999) (WT N = 9, WT pre 2.55 ± 1.83, WT stim 7.51 ± 7.95; WT post 14.97 ± 14.75). f, Frequency of myelin Ca²⁺ transients per grm5a^−/−grm5b^−/− animals over time across pre, stim and post-stim periods (two-sided Friedman test P = 0.008; multiple comparisons test: pre versus stim P > 0.9999; pre versus post: P = 0.0179; stim versus post P = 0.0373) (grm5a^−/−grm5b^−/−N = 8, grm5a^−/−grm5b^−/− pre 1.28 ± 0.53; grm5a^−/−grm5b^−/− stim 1.93 ± 1.39; grm5a^−/−grm5b^−/− post 6.34 ± 5.06). g, Individual WT Ca²⁺ amplitudes separated into three periods, pre (n = 65), stim (n = 175) and post-stimulation (n = 173) (One-way ANOVA, P = <0.0001, Kruskal–Wallis test multiple comparison: pre versus stim P > 0.9999; pre versus post P < 0.0001, stim versus post P < 0.0001) WT pre 0.76 ± 0.25, WT stim 0.88 ± 0.61 WT post 1.22 ± 0.78. h, Individual grm5a^−/−grm5b^−/− Ca²⁺ amplitudes separated into the three periods, pre (n = 50), stim (n = 45) and post-stimulation (n = 45) (one-way ANOVA, Kruskal–Wallis test P = 0.6768, Dunn’s multiple comparison, pre versus stim P > 0.9999; pre versus post P > 0.9999, stim versus post P > 0.9999) grm5a^−/−grm5b^−/− pre 0.63 ± 0.19, grm5a^−/−grm5b^−/− stim 0.69 ± 0.34 grm5a^−/−grm5b^−/− post 0.66 ± 0.20. Scale bar, 10 µm. Data are shown as mean ± s.d.

Source data

mGluR5 mediates myelin sheath growth in response to opto-stimulation of chx10 interneurons.

a, Experimental setup for long-term (16 h) opto-stimulation of individual ChRimsonR-positive and -negative Tg(mbp:memGCaMP7s) fish. Fish were screened for transgenesis, responsiveness to opto-stimulation and placed in individual wells of a 96-well plate and optically stimulated for 16 h before myelin morphology was assessed. b, Representative images, following 16 h of opto-stimulation, of a WT (left) and grm5a^−/−grm5b^−/− mutant (right) expressing Tg(chx10:ChRimsonR–tdTomato;mbp:memGCaMP7s) (ChR⁺) (WT N = 21, grm5a^−/−grm5b^−/−N = 18) (top). Representative images of Tg(mbp:memGCaMP7s), ChRimsonR–tdTomato negative (ChR⁻) WT (left) and grm5a^−/−grm5b^−/− mutants (right) following 16 h of opto-stimulation (WT N = 13, grm5a^−/−grm5b^−/−N = 9) (bottom). Scale bar, 10 µm. c, Avg. myelin sheath length per WT and grm5a^−/−grm5b^−/− ChR⁺ and ChR⁻ animals (one-way ANOVA; P < 0.0001; Tukey’s multiple comparisons; WT ChR⁺ versus WT ChR⁻P = 0.0032; WT ChR⁺ versus grm5a^−/−grm5b^−/− ChR⁺P < 0.0001; WT ChR⁺ versus grm5a^−/−grm5b^−/− ChR⁻P < 0.0001; WT ChR⁻ versus grm5a^−/−grm5b^−/− ChR⁺P = 0.1636; WT ChR⁻ versus grm5a^−/−grm5b^−/− ChR⁻P = 0.0488; grm5a^−/−grm5b^−/− ChR⁺ versus grm5a^−/−grm5b^−/− ChR⁻P = 0.7810) WT ChR⁻N = 13, 18.02 ± 4.73 WT ChR⁺N = 21, 22.24 ± 3.16 grm5a^−/−grm5b^−/− ChR⁻N = 18, 14.24 ± 2.44 grm5a^−/−grm5b^−/− ChR⁺N = 18, 15.51 ± 2.40. d: Relative frequency distribution of individual sheath lengths, measured post-stimulation (WT ChR⁺n = 462, 21.67 µm; WT ChR⁻n = 315, 16.23 µm; grm5a^−/−grm5b^−/− ChR⁺n = 271, 14.04 µm; grm5a^−/−grm5b^−/− ChR⁻n = 271, 15.11 µm). Data are shown as mean ± s.d.

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