Measurement of the rate of myelination using a fluorescent analogue of ceramide

Abstract

Fluorescence digital imaging microscopy was used to investigate the process of myelin formation by Schwann cells in neuronal cocultures. The uptake of the fluorescent ceramide analogue N-[5-(5,7-dimethyl BODIPY)-1-pentanoyl]D-erythro-sphingosine (C5-DMB-ceramide) and its return to the plasma membrane as the corresponding fluorescent sphingomyelin and galactocerebroside analogues were measured. Through observation of this process it was possible to determine the rate of lipid synthesis in myelin internodes. The highest rate of synthesis of fluorescent sphingomyelin and galactocerebroside analogues was observed between days 3 and 7 after induction of myelination. This rate was approximately 5-fold greater than the steady-state rate of synthesis in fully myelinated internodes and 10-fold higher than the rate observed prior to myelination. The internode diameter increased during the first 3 days of myelination, but this was followed by a reduction in diameter and then an increase until the myelin sheath formation was completed. Internodes were found to be heterogeneous in terms of lipid distribution, with fluorescence intensities ranging 5-fold in myelinating cultures. Additionally, the rate of lipid transport along the internode was slow since there was a quicker increase in fluorescence intensity near the cell body of the Schwann cell than near the nodes of Ranvier. The results show that fluorescence digital imaging microscopy can be used to study the process of myelin formation and to determine the rate of formation, lipid transport, and heterogeneity of the myelin membrane.

Glial Cell Line-Derived Neurotrophic Factor Alters Axon Schwann Cell Units and Promotes Myelination in Unmyelinated Nerve Fibers

“…To eliminate the possible contribution of endogenous NGF secreted by Schwann cells, in parallel experiments, neutralizing anti-NGF antibody (50 ng/ml; Boehringer Mannheim, Indianapolis, IN) was added to the coculture. Myelination was assessed by adding a fluorescent ceramide analog, N-(4,4-difluoro-5,7-dimethyl-4-boro-3a,4a-diaza-5-indacene-3-pentanoyl)sphingosine (BODIPY FL C5)-ceramide (Bilderback et al, 1997) (Molecular Probes, Eugene, OR). These experiments were done at least in triplicate with DRG neurons from three different sets of embryos.…”

Glucocorticoids and progestins signal the initiation and enhance the rate of myelin formation

“…C5-DMB-hexadecanoic acid (0.75 M) was added directly to the cocultures, and the rate of fluorescent lipid incorporation at various stages in the myelination process was continuously measured. By monitoring the synthesis of myelinspecific lipids (galactocerebroside, sulfatide, and sphingomyelin) by TLC, active myelin formation was found to occur between 4 and 7 days after induction with ascorbate (12). By measuring the fluorescent lipid incorporation throughout the myelination process by fluorescence digital imaging microscopy, the peak rate of myelin formation was also found to occur between 4 and 7 days after induction.…”

“…A method has been developed that allows the continuous monitoring of a single internode throughout the myelination process. Using the fluorescent ceramide analogue, N- [5-(5,7-dimethyl BODIPY)-1-pentanoyl]D-erythrosphingosine (C5-DMB-ceramide), cocultures of dorsal root ganglia neurons and Schwann cells were observed to metabolize the fluorescent ceramide and incorporate it into myelin lipids (12). The rate of incorporation increased substantially when lipid synthesis was induced during active myelin formation and, consequently, this is a measure of the actual rate of myelin formation.…”

“…Cells were labeled using essentially the conditions of Bilderback et al (12). Brief ly, 250 nmol of the C5-DMBhexadecanoic acid was dissolved in 200 l of ethanol and injected into 10 ml of 1.7 mg/ml delipidated BSA in Hepesbuffered DMEM.…”

“…The labeled cocultures were excited at wavelengths between 420 and 480 nm, whereas the emission was monitored at wavelengths greater than 515 nm. The fluorescence intensity of an internode was corrected for background fluorescence and the mean intensity of the internode was calculated (12). The fluorescence intensity and the internode diameters were measured using the IPLab Spectrum program (Signal Analytics Corporation, Vienna, VA).…”