Control of myelination by specific patterns of neural impulses

Abstract

A cell culture preparation equipped with stimulating electrodes was used to investigate whether action potential activity can influence myelination of mouse dorsal root ganglia axons by Schwann cells. Myelination was reduced to one-third of normal by low-frequency impulse activity (0.1 Hz), but higher-frequency stimulation (1 Hz) had no effect. The number of Schwann cells and the ultrastructure of compact myelin were not affected. The frequency of stimulation that inhibited myelination decreased expression of the cell adhesion molecule L1, and stimulation under conditions that prevented the reduction in L1 blocked the effects on myelination. This link between myelination and functional activity in the axon at specific frequencies that change axonal expression of L1 could have important consequences for the structural and functional relationship of myelinating axons.

Fig. 1.

Experimental preparation for studying the effects of action potentials on myelination of DRG neurons in vitro. A, Mouse DRG neurons were cultured in the side compartments of multi-compartment cell culture chambers placed in 35-mm-diameter dishes. After 3–4 weeks in culture, axons extend into the central compartment by growing under the high-resistance barriers between compartments. Stimulating electrodes on opposite sides of the barrier were used to control the pattern of action potential firing for up to 5 d in an incubator. Purified Schwann cells (SC) were added to DRG neuron (N) cultures, and myelination was initiated by the addition of ascorbic acid. B, Compact myelin (arrows) was detected by Sudan black staining or immunocytochemical localization of myelin basic protein (green) (C) 12–14 d after initiating myelination. C, Confocal microscopy of preparations double-staining for MBP (green) and L1 (red) shows the disappearance of L1 from the axon and Schwann cell after the fiber becomes myelinated (also see Martini and Schachner, 1986). Scale bars, 50 μm.

Fig. 2.

The ultrastructure of myelinated axons was normal in cultures stimulated at 0.1 Hz (A–C) and 1 Hz (D–F), and cultures under both stimulus conditions showed axons undergoing several stages of myelination. Note several loose wraps of Schwann cell cytoplasm ensheathing axons (A, D, arrows). Higher magnification of a myelinated axon is shown from cultures stimulated at 0.1 Hz (C, arrow) and 1 Hz (F, arrow). Multiple layers (up to 19) of compact myelin, with ∼10 nm between major dense lines (C, arrows), were evident in myelinated axons stimulated at 0.1 Hz (C) and 1 Hz (F). Scale bars: A, B, E, 500 nm; C, F, 50 nm.

Fig. 3.

Myelination is inhibited by 0.1 Hz electrical stimulation. Myelin profiles were identified by immunocytochemical staining for myelin basic protein (MBP) in cultures stimulated at 0 (A), 0.1 (C), and 1 (E) Hz for 5 d. Hoechst nuclear stain was used to count the number of Schwann cells in each preparation (B, D, F).G, The number of MBP-positive myelin profiles was significantly lower in cultures stimulated at 0.1 Hz. **p < 0.001 versus 0 Hz; p < 0.02 ANOVA on 0, 0.1, and 1 Hz, respectively; n = 44 experiments. H, The total number of Schwann cells was not significantly different in stimulated or unstimulated cultures, indicating an inhibitory effect on the myelination process. Scale bars, 50 μm.

Fig. 4.

A, L1 mRNA levels were compared in DRG neurons and Schwann cells (SC) using RT-PCR. Stimulation at a frequency of 0.1 Hz for 5 d significantly lowered L1 expression in DRG neurons (136 bp PCR product, lane 1vs lane 2), but stimulation at 1 Hz had no effect on L1 levels (lane 3 vs lane 1). Schwann cells express a short-splice isoform of L1 mRNA (Takeda et al., 1996) (124 bp PCR product), which was not detected in these DRG cultures. Stimulation of cultures containing only Schwann cells failed to alter their L1 expression level (lane 4 vs lane 5). B, L1 mRNA levels were normalized to neuron-specific enolase (NSE) mRNA levels in DRG cultures and cyclophilin (CYC) in Schwann cell cultures.

Fig. 5.

Stimulation at 0.1 Hz had no effect on myelination when the stimulus-induced change in L1 levels was blocked by adding NGF. Top panel, The number of MBP-positive myelin profiles was not significantly different in cultures stimulated at 0.1 Hz compared with unstimulated controls in the presence of 50 ng/ml NGF.Bottom panel, The downregulation of L1 mRNA (136 bp) levels produced by 0.1 Hz stimulation was prevented by the addition of 50 ng/ml NGF during stimulation, which is known to upregulate L1 expression in PC12 cells (McGuire and Greene, 1978) and DRG neurons (Itoh et al., 1995b). NSE mRNA (535 bp) was used as a control for variation in cell density and PCR efficiency.