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Much of our understanding of cell and tissue development, structure, and function stems from fluorescence microscopy. The acquisition of colorful and glowing images engages and excites users ranging from seasoned microscopists to STEM students. Fluorescence microscopes range in cost from several thousand to several hundred thousand US dollars. Therefore, the use of fluorescence microscopy is typically limited to well-funded institutions and biotechnology companies, research core facilities, and medical laboratories, but is financially impractical at many universities and colleges, primary and secondary schools (K-12), and in science outreach settings. In this study, we developed and characterized components that when used in combination with a smartphone or tablet, perform fluorescence microscopy at a cost of less than $50 US dollars per unit. We re-purposed recreational LED flashlights and theater stage lighting filters to enable viewing of green and red fluorophores including EGFP, DsRed, mRFP, and mCherry on a simple-to-build frame made of wood and plexiglass. These devices, which we refer to as glowscopes, were capable of 10 µm resolution, imaging fluorescence in live specimens, and were compatible with all smartphone and tablet models we tested. In comparison to scientific-grade fluorescence microscopes, glowscopes may have limitations to sensitivity needed to detect dim fluorescence and the inability to resolve subcellular structures. We demonstrate capability of viewing fluorescence within zebrafish embryos, including heart rate, rhythmicity, and regional anatomy of the central nervous system. Due to the low cost of individual glowscope units, we anticipate this device can help to equip K-12, undergraduate, and science outreach classrooms with fleets of fluorescence microscopes that can engage students with hands-on learning activities.

 

Oral Presentation Title: Evolution of the oligodendrocyte cell type and adaptive myelination phenotype (S10-03) 

Oligodendrocytes are multifunctional central nervous system (CNS) glia that are essential for neural function in gnathostomes. The evolutionary origins and specializations of the oligodendrocyte cell type are among the many remaining mysteries in glial biology and neuroscience. The role of oligodendrocytes as CNS myelinating glia is well established, but recent studies demonstrate that oligodendrocytes also participate in several myelin-independent aspects of CNS development, function, and maintenance. Furthermore, many recent studies have collectively advanced our understanding of myelin plasticity, and it is now clear that experience-dependent adaptations to myelination are an additional form of neural plasticity. These observations beg the questions of when and for which functions the ancestral oligodendrocyte cell type emerged, when primitive oligodendrocytes evolved new functionalities, and the genetic changes responsible for these evolutionary innovations. Here, I review recent findings and propose working models addressing the origins and evolution of the oligodendrocyte cell type and adaptive myelination. The core gene regulatory network (GRN) specifying the oligodendrocyte cell type is also reviewed as a means to probe the existence of oligodendrocytes in basal vertebrates and chordate invertebrates. 

Abstract Background In the developing central nervous system, pre-myelinating oligodendrocytes sample candidate nerve axons by extending and retracting process extensions. Some contacts stabilize, leading to the initiation of axon wrapping, nascent myelin sheath formation, concentric wrapping and sheath elongation, and sheath stabilization or pruning by oligodendrocytes. Although axonal signals influence the overall process of myelination, the precise oligodendrocyte behaviors that require signaling from axons are not completely understood. In this study, we investigated whether oligodendrocyte behaviors during the early events of myelination are mediated by an oligodendrocyte-intrinsic myelination program or are over-ridden by axonal factors. Methods To address this, we utilized in vivo time-lapse imaging in embryonic and larval zebrafish spinal cord during the initial hours and days of axon wrapping and myelination. Transgenic reporter lines marked individual axon subtypes or oligodendrocyte membranes. Results In the larval zebrafish spinal cord, individual axon subtypes supported distinct nascent sheath growth rates and stabilization frequencies. Oligodendrocytes ensheathed individual axon subtypes at different rates during a two-day period after initial axon wrapping. When descending reticulospinal axons were ablated, local spinal axons supported a constant ensheathment rate despite the increased ratio of oligodendrocytes to target axons. Conclusion We conclude that properties of individual axon subtypes instruct oligodendrocyte behaviors during initial stages of myelination by differentially controlling nascent sheath growth and stabilization. 

Oligodendrocytes (OLs), the myelinating cell type of the CNS, interact with a plethora of diverse neuronal subtypes but only wrap a select subset with myelin sheaths. Prior to initiating axon wrapping, OLs dynamically extend and retract membrane processes in order to contact and sample numerous axons. Whether neural activity-dependent mechanisms regulate exploratory axon sampling, target axon recognition, and stabilization of OL-axon interactions prior to initial axon wrapping is unknown. To test this, we directly observed interactions between pre-myelinating OL processes and individually labeled target axons in larval zebrafish using time-lapse confocal microscopy. In control larvae anesthetized with the neuromuscular blocker pancuronium bromide, we observed dynamic axon sampling characterized by frequent formation and turnover of OL-axon interactions. In contrast, treatment with the neural activity blocker tricaine methanesulfonate (MS-222) caused reduced frequency of new interaction formation, increased interaction duration, and reduced frequency of interaction retraction. Time-lapse imaging revealed differential effects on OL-axon interactions at axon varicosities and thin, intervening segments. Specifically, the destabilizing effects of neural activity on OL-axon interactions were heightened at axon varicosities. MS-222 increased contact durations at varicosities but not at neighboring intervening segments. Neural activity manipulations also influenced the dynamics of axon varicosity formation, lifetime, and turnover, raising the possibility that changes to axon morphology or local properties could direct OL-axon interactions and subsequent myelination. Taken together, we conclude that neural activity negatively regulates the duration of OL-axon interactions prior to initial axon wrapping and myelination. These findings support a mechanism whereby neural activity plays opposing roles on OL-axon interactions before and after initial myelin ensheathment. Prior to ensheathment, neural activity destabilizes interactions, which may serve to facilitate increased overall sampling of potential wrapping sites. After successful ensheathment, neural activity stabilizes OL-axon adhesion in order to promote continued growth and maturation of the myelin sheath. Current and future studies aim to understand the reciprocal effects between OL processes and axon morphology, and the effects of synaptic vesicle release during initial OL-axon interactions. 

In the developing central nervous system, pre-myelinating oligodendrocytes contact and sample candidate nerve axons by extending and retracting process extensions. Some contacts stabilize and mature, leading to the initiation of axon wrapping, myelin sheath formation, and sheath elongation by oligodendrocytes. Although axonal signals influence the overall process of myelination, which precise steps and oligodendrocyte cell behaviors require signaling from axons is incompletely understood. In this study, we investigated whether cell behaviors during the early events of myelination involve input from axons or are mediated by an oligodendrocyte-autonomous myelination program. To address this, we utilized in vivo time-lapse imaging in embryonic and larval zebrafish during the initial hours and days of axon wrapping and myelination. Transgenic reporter lines marked individual axon subtypes or oligodendrocyte membranes. In the larval zebrafish spinal cord, individual axon subtypes supported distinct nascent sheath growth rates and pruning frequencies. Oligodendrocytes ensheathed individual axon subtypes at different rates during a two-day period after initial axon wrapping. When the ratio of oligodendrocytes to target axons was increased by ablating spinal projection axons, local spinal neuron axons supported a constant ensheathment rate despite the increased ratio of oligodendrocytes to target axons. We conclude that properties of individual axon subtypes instruct oligodendrocyte behaviors during initial stages of myelination by differentially controlling nascent sheath growth and stabilization.