Neuronal activity promotes oligodendrogenesis and adaptive myelination in the mammalian brain

Abstract

Myelination of the central nervous system requires the generation of functionally mature oligodendrocytes from oligodendrocyte precursor cells (OPCs). Electrically active neurons may influence OPC function and selectively instruct myelination of an active neural circuit. In this work, we use optogenetic stimulation of the premotor cortex in awake, behaving mice to demonstrate that neuronal activity elicits a mitogenic response of neural progenitor cells and OPCs, promotes oligodendrogenesis, and increases myelination within the deep layers of the premotor cortex and subcortical white matter. We further show that this neuronal activity-regulated oligodendrogenesis and myelination is associated with improved motor function of the corresponding limb. Oligodendrogenesis and myelination appear necessary for the observed functional improvement, as epigenetic blockade of oligodendrocyte differentiation and myelin changes prevents the activity-regulated behavioral improvement.

Figures

Fig. 1. Neuronal activity that elicits complex motor behavior stimulates OPC proliferation in the premotor cortex

(A) Schematic of optogenetic stimulation with simplified M2 circuit projections; expanded view shows the quantified area. (B) Proliferating oligodendroglial lineage cells are found near active neuronal cell bodies in the deep layers of the optogenetically stimulated M2 motor cortex. Confocal micrograph (split view on the left; composite image on the right) of the M2 deep cortical layers V and VIa 3 hours after optogenetic stimulation in a Thy1::ChR2 mouse. The immediate early gene c-Fos (blue) indicates recent neuronal activity. MAP2 (white) indicates neuronal cell bodies and apical dendrites. Proliferating cells marked by EdU (red) are seen throughout the deep cortical layers and frequently colocalize with the oligodendroglial lineage marker Olig2 (green; colocalized EdU+/Olig2+ cells are indicated by the white arrowheads.) Scale bars, 100 mm. (C) Total EdU+ cells in M2 of P35 mice (n = 3 per group) 3 hours after optogenetic stimulation. The M2 regions ipsilateral (Ipsi) and contralateral (Contra) to the site of the optical fiber are quantified. (D) Representative confocal micrograph of EdU+ cells (red) and Olig2+ (green), together with the total number of EdU+/Olig2+ cells in M2 3 hours after optogenetic manipulation. Scale bar, 10 mm. (E) Representative confocal micrograph of an EdU-marked (red) PDGFRa+ (green) OPC, together with the total number of EdU-marked OPCs in M2 3 hours after optogenetic manipulation. Scale bar, 10 mm. In all graphs, red bars indicate Thy1::ChR2 mice; blue bars denote identically manipulated WT littermate controls. Mice in whom seizures were induced rather than ambulatory behavior are shown on the right-hand side of the graphs (n = 3 per group). *P < 0.05, **P < 0.01, ***P < 0.001. Error bars indicate SEM.

Fig. 2. Oligodendroglial lineage cells in the corpus callosum respond to premotor circuit activity

(A) Schematic of optogenetic stimulation with simplified M2 circuit projections; expanded view shows the area quantified in the corpus callosum (shaded gray). (B) Confocal micrograph from a stimulated Thy1::ChR2 corpus callosum overlaid on a differential interference contrast background to illustrate regional tissue architecture. Activated microglial marker CD68, green; EdU, red. No EdU+ activated microglia were detected in the corpus callosum of stimulated or unstimulated mice. Scale bar, 20 mm. (C to E) Total EdU+ (C), EdU+/Olig2+ (D), and EdU/Iba1+/CD68+ cells (E) in the indicated region of corpus callosum of P35 stimulated (Stim; n = 3 per group) or unstimulated (Unstim; n = 3 per group) mice 3 hours after optogenetic stimulation. (F) Total number of EdU+/CC1+ mature oligodendrocytes 4 weeks after premotor cortex optogenetic stimulation (or no stimulation) at P35 in Thy1::ChR2 (n = 4 per group) or WT (n = 3 per group) mice. In all graphs, red bars indicate Thy1::ChR2 mice; blue bars denote identically manipulated WT littermate controls. As the corpus callosum is a single structure, the contralateral structure does not serve as an internal control for stimulation. Thus, an unstimulated group, in which surgery was performed and the optical-neural interface was placed but no light exposure occurred, was included in corpus callosum analyses. Mice in whom seizures were induced rather than ambulatory behavior are shown on the right-hand side of the graph (n = 3 per group). *P < 0.05, ***P < 0.001. Error bars indicate SEM.

Fig. 3. Microglial inflammatory response to optical fiber manipulation is equivalent in ::ChR2 and WT mice

(A) The majority of reactive microglia are localized to the superficial cortex (layer I), near the site of the optical fiber placement. Representative confocal micrographs illustrating CD68+ activated microglia in the superficial M2 cortex in comparison to largely CD68− microglia in the deep cortex (layer V/VI). EdU, red; pan-microglial marker Iba1, green; activated microglial marker CD68, white. Scale bars, 50 mm. (B) Percentage of EdU-marked activated microglia (Iba1+/CD68+) in superficial versus deep layers of M2 cortex ipsilateral to the optical fiber, 3 hours after blue light stimulation in Thy1::ChR2 and WT littermate control mice. (C) Representative confocal micrograph illustrating a CD68+ activated microglia marked with EdU. EdU, red; Iba1, green; CD68, blue. Scale bar, 10 mm. (D) Total numbers of EdU-marked activated microglia throughout the M2 cortex in Thy1::ChR2 and WT littermate control mice 3 and 24 hours after optogenetic manipulation ipsilateral and contralateral to the optical fiber placement. (E) Density of total CD68 microglia throughout the M2 cortex ipsilateral and contralateral to the optical fiber placement at 3 and 24 hours after a single 30-min session of optogenetic manipulation in P35 Thy1::ChR2 and WT mice. (F) Density of total CD68+ microglia throughout the M2 ipsilateral and contralateral to the optical fiber placement after a 7-day stimulation paradigm (10 min per day for 7 days) was measured on day 7 (after stimulation from P29 to P35) or 4 weeks after the completion of stimulation (from P35 to P42) in Thy1::ChR2 and WT mice. In all graphs, red bars indicate Thy1::ChR2 mice; blue bars denote identically manipulated WT littermate controls. Mice in whom seizures were induced rather than ambulatory behavior are shown to the right of the 3-hour graphs (n = 3 per group). n.s. (not significant) indicates P > 0.05. *P < 0.05, **P < 0.01. Error bars indicate SEM.

Fig. 4. Neuronal activity initiates differentiation

(A) Total number of EdU+ cells in Thy1::ChR2 and WT mice at 24 hours (n = 4 per group) and 4 weeks (n = 7 per group) poststimulation. (B to D) Representative confocal micrographs illustrating colocalization of EdU (red) with markers (green) of cell mitosis (Ki67), apoptosis (cleaved caspase-3), or histone modification markers histone H3 lysine 9 trimethylation (H3K9me3) and histone H3 acetylation (AcH3). In all graphs, red bars indicate Thy1::ChR2 mice; blue bars denote identically manipulated WT littermate controls. *P < 0.05, **P < 0.01,***P < 0.001. n.s. denotes P > 0.05. Error bars indicate SEM. Scale bars, 10 mm. Value of (—) over x axis = 0 ± 0.

Fig. 5. Neuronal activity promotes oligodendrogenesis

Thy1::ChR2 and WT optogenetically stimulated mice sacrificed 4 weeks after a 7-day stimulation paradigm (P35 to P42) with (n = 4 Thy1::ChR2 and 3 WT) or without (n = 7 per group) exposure to TSA (10 mg/kg). (A to F) Cell identity markers of EdU-marked surviving cells quantified in the M2 cortex reveal that the majority of the remaining EdU+ cells are in the oligodendroglial lineage [Olig2+, (A)], with diminution of the oligodendrocyte precursor cell (PDGFRα+) population (B) and concomitant appearance of EdU+ immature oligodendrocytes [(D); nuclear Olig1+; red and dark blue bars] and mature oligodendrocytes marked by perinuclear Olig1+ [(C and D), pink and light blue bars] and CC1 (E and F). (C and E) Representative confocal micrographs illustrating colocalization of EdU (red) with perinuclear Olig1 [green (C)] or CC1 [green (E)]. Scale bars, 10 μm. (D and F) Administration of the histone deacetylase inhibitor TSA during the week of stimulation results in a blockade of mature oligodendrogenesis. TSA-treated groups are shown on the right halves of these graphs. Increased numbers of immature (Olig1nuclear+) oligodendrocytes in the TSA group are consistent with an epigenetic blockade of precursor differentiation. In all graphs, red or pink bars indicate Thy1::ChR2 mice; blue bars denote identically manipulated WT littermate controls. *P < 0.05, **P < 0.01, ***P < 0.001. n.s. represents P > 0.05. Error bars indicate SEM. Value of (—) over x axis = 0 ± 0.

Fig. 6. Neuronal activity increases myelin thickness

(A) Transmission electron microscopy was performed in the projections from the premotor cortex entering the corpus callosum at the level indicated (red box). A representative TEM image of a myelinated axon at this level ipsilateral to optogenetic stimulation is shown at right (8000×, scale bar, 200 nm). (B to D) Scatter plots of g-ratio as a function of axon caliber in the cortical and subcortical projections from the ipsilateral (B) and contralateral (C) M2 in Thy1::ChR2 (n =4 mice) and WT (n = 3 mice). Black squares, Thy1::ChR2; red circles, WT. (D) Scatter plot of g-ratios in TSA-treated Thy1::ChR2 (black squares; n = 3 mice) and WT (red circles; n = 3 mice) ipsilateral to stimulation. n.s. represents P > 0.05. Error bars indicate SEM.

Fig. 7. Activity-regulated oligodendrogenesis and myelin remodeling correlate with improved behavioral function

CatWalk gait analysis was used to evaluate motor performance during normal gait 4 weeks after the stimulation paradigm described above. (A) Schematic of the CatWalk apparatus. Experimental mice walk through a long, narrow corridor on a touch-sensitive glass platform, under which a digital camera records paw print contours, allowing for software-guided reconstruction and analysis of gait. LF, left forepaw; LH, left hindpaw; RF, right forepaw; RH, right hindpaw. (B) Interlimb forelimb swing speed (in centimeters per second) difference of left forelimb minus right forelimb (n = 5 Thy1::ChR2 and 9 WT; n = 6 TSA-treated Thy1::ChR2 and 5 TSA-treated WT). (C and D) No alterations to paw pressure (C) or stride length (D) were observed in optogenetically stimulated Thy1::ChR2 mice or their WT littermate controls. The same is true in animals treated with the histone deacetylase inhibitor TSA (C and D). Untreated: n = 5 Thy1::ChR2 mice and 9 WT mice; TSA-treated: n = 4 Thy1::ChR2 mice and 5 WT mice. n.s. denotes P > 0.05. *P < 0.05. Error bars indicate SEM. AU, arbitrary units.