[Show abstract][Hide abstract]ABSTRACT: Previous work has shown that oligodendrocytes (OLs) express both micro- and kappa-opioid receptors. In developing OLs, micro receptor activation increases OL proliferation, while the kappa-antagonist nor-binaltorphimine (NorBNI) affects OL differentiation. Because exogenous opioids were not present in our defined culture medium, we hypothesized that NorBNI blocked endogenous opioids produced by the OLs themselves. To test this, intact and partially processed proenkephalin and prodynorphin-derived peptides were assessed in OLs using immunocytochemistry or Western blot analysis, or both. Immature OLs possessed large amounts of intact and partially processed proenkephalin precursors, as well as posttranslational products of prodynorphin including dynorphin A (1-17). With maturation, however, intact or partially processed proenkephalin was expressed by only about 50% of OLs, while dynorphin A (1-17) was undetectable. To assess the function of OL-derived opioids, the effect of kappa-agonists/antagonists on OL differentiation and death was explored. kappa-Agonists alone had no effect. In contrast, NorBNI significantly increased OL death. Additive OL losses were evident when NorBNI was paired with toxic levels of glutamate, suggesting that kappa-receptor blockade alone is sufficient to induce OL death. Thus, the results indicate that OLs express proenkephalin and prodynorphin peptides in a developmentally regulated manner, and further suggest that opioids produced by OLs modulate OL maturation and survival through local (i.e., autocrine and/or paracrine) mechanisms.
[Show abstract][Hide abstract]ABSTRACT: Na,K-ATPase is an integral membrane enzyme responsible for maintenance of the transmembrane Na+/K+ gradient which generates membrane excitability. Previous studies showed that oligodendrocytes within the CNS robustly expressed the alpha2 isoform of the Na,K-ATPase while oligodendrocytes in isolated cultures did not. We tested whether the levels of this isoform might be modulated by interactions with neurons. Western blots showed alpha2 protein expression was very low in rat optic nerve immediately after birth, but that expression was greatly increased by days 5 and 14. In adult optic nerves, levels were barely detectable. Since the first myelinated axons are observed in rat optic nerve at day 5, and the next 2 weeks are considered the period of peak myelination, this timing suggested a relationship between oligodendrocyte-neuron contact, myelination onset and the upregulation of the alpha2 isoform. In further experiments we plated oligodendrocytes in isolation or in co-culture with neurons dissociated from cerebral cortex at the day of birth. After 6 days in vitro, 45% of oligodendrocytes co-cultured with neurons expressed abundant alpha2 protein which was detected by immunohistochemistry, a six-fold increase over cells expressing alpha2 protein in isolated cultures. Conditioned medium from neuronal cultures did not affect alpha2 levels in oligodendrocytes. These results suggest that neurons may play a role in upregulating glial expression of the alpha2 isoform during peak periods of myelination, and that the effect is likely to be dependent on contact.
No preview · Article · Jan 2001 · Developmental Brain Research
[Show abstract][Hide abstract]ABSTRACT: The present study describes transitions in myosin heavy chain expression in the extraocular muscles of rats between the ages of E17 and adult. The unique phenotype of the extraocular muscle is reflected in its fibre type composition, which is comprised by six distinct profiles, each defined by location (orbital versus global layer) and innervation pattern (single versus multiple terminals). During extraocular muscle myogenesis, developmental myosin heavy chains were expressed in both primary and secondary fibres from embryonic day E17 through the first postnatal week. At this time, the downregulation of developmental myosin heavy chain isoforms began in the global layer in a fibre type-specific manner, reaching completion only after the first postnatal month. By contrast, developmental isoforms were retained in the overwhelming majority of orbital layer fibres into adulthood and expressed differentially along the length of these fibres. Fast myosin heavy chain was detected pre- and postnatally in developing secondary fibres and in all of the singly innervated fibre types and one of the multiply innervated fibre types in the adult. As many as four fast isoforms were detected in maturing extraocular muscle, including the extraocular muscle-specific myosin heavy chain. Slow myosin heavy chain was expressed in primary fibres throughout development and in one of the multiply innervated fibre types in the adult. In contrast, the pure fast-twitch retractor bulbi initially expressed slow myosin heavy chain in fibres destined to switch to the fast myosin heavy chain developmental programme. Based upon spatial and temporal patterns of myosin heavy chain isoform transitions, we suggest that epigenetic influences, rather than purely myogenic stage-specific factors, are critical in determining the unique extraocular muscle phenotype.
No preview · Article · Jul 1996 · Journal of Muscle Research and Cell Motility
[Show abstract][Hide abstract]ABSTRACT: Purpose. Developmental mechanisms responsible for the unique extraocular muscle (EOM) phenotype are poorly understood. We developed a novel culture model for exploration of the regulatory roles of genetic and epigenetic factors in EOM development. Methods. Small, organotypic explant-like aggregates of adult rat skeletal muscle satellite cells grow well in culture and apparently mimic many aspects of normal development and regeneration. Satellite cells enzymatically released from newborn and adult rat EOM, masseter (Mas), and soleus (SOL) muscles were expanded in culture, formed into aggregates of 20-40,000 cells by centrifugation, and aggregates cultured on collagen-coated dishes for 1 to 3+ weeks. Fiber maturation was evaluated by morphological criteria and myosin immunocytochemistry. Results. In EOM and Mas aggregates, myotube formation began within 1 day and contractile myofibers were present within 1 week. Myotube formation in SOL aggregates is considerably slower. As cultures age, the primary myofibers slowly degenerate while a morphologically distinct set of secondary myofibers gradually appears. Myofibers in all cultures express embryonic and generic fast myosins. By contrast, the slow myosin isoform, normally seen in fibers derived from embryonic primary generation myoblasts, was rare in aggregate cultures of any of the muscles studied. Likewise, IIA myosin, an activity dependent isoform that appears late in the in vivo maturation of EOM, was rare in fibers formed in this culture system. Conclusions. Prior in vitro studies have shown that the trophic requirements of EOM are different from those of other skeletal muscle; an aggregate culture model was developed to pursue this variability. The aggregate culture system appears to reflect more accurately than dispersed cell culture techniques the normal progression of events occurring during skeletal muscle regeneration. Especially notable is the occurrence of two temporally distinct waves of myofiber formation. These data lend further support to the notion that EOM represents a distinctive muscle class in terms of phenotype and intrinsic/extrinsic regulatory mechanisms.