Use of zebrafish to study and affect activity-dependent myelination in vivo
Funded by the Ministry of Innovation, Science and Research of the State of North Rhine-Westphalia, the following work is planned in cooperation with the University of Bonn at the Anatomy Institute. Disorders of white matter »myelin« are the cause of many common neurological diseases such as multiple sclerosis (MS), Charcot-Marie-Tooth (CMT) or Pelizaeus-Merzbacher disease. More recently, a variety of psychiatric disorders such as schizophrenia, bipolar disorder, autism, or chronic depression have also been linked to CNS white matter defects (Fields, 2008), with abnormalities in the expression of many genes involved in the myelination process. In addition, up to 25% of preterm low birth weight infants (<1,500 g) have been shown to develop periventricular white matter injury (PWMI) due to altered oligodendrocyte development. This white matter damage is often associated with motor and cognitive deficits that are later observed in these infants. Failure of oligodendrocyte differentiation and remyelination is also a major cause of permanent paralysis after spinal cord injury.
Despite this tremendous involvement of oligodendrocytes in CNS myelination in health and disease, there is still a great deal of uncertainty about the various factors that drive oligodendrocytes to provide proper initial myelination, remyelination, or even fine-tuning of myelination that enables CNS plasticity for learning and memory. Much of the work to date has focused on autoimmune responses against the myelin sheath and cell signaling factors involved in oligodendrocyte proliferation, growth, and differentiation. To date, most of this work has been performed in vitro, and little is known about how neuronal activity and activity in the surrounding glial tissue, e.g., changes in cell membrane potential, additionally influence the myelination process.
To gain a better understanding of the influence of different cell activities on the myelination process, we aim to simultaneously observe cell activity and myelination in zebrafish in vivo. Zebrafish are commonly used as a vertebrate model organism for neural development and myelination (Buckley et al., 2008). The basic properties of myelin are common in vertebrates, and zebrafish have been shown to be a powerful system for studying the myelination process in vivo using fluorescent proteins (Yoshida and Macklin, 2005). Zebrafish are also well suited for genetic manipulations, overexpression studies, Morpholino knockdown experiments, and targeted generation of knock-out and knock-in fish lines using the recently described zinc finger technique. A large number of mutant fish lines, including many with myelination phenotypes, are freely available as part of various screening projects. In addition, the transparency of zebrafish larvae and the presence of various albino lines make zebrafish an ideal model for all types of noninvasive in vivo imaging.
Several neurotransmitter receptors have been found in oligodendrocytes; e.g., unlike conventional neurons, administration of glutamate and even GABA leads to their depolarization. Like neurons and astrocytes, oligodendrocytes also increase their internal Ca2+ levels through neurotransmitter receptors and voltage-gated channels (Kirischuk et al., 1995), which in turn can be activated by an increase in extracellular K+ concentration. Monitoring internal Ca2+ concentrations with the fluorescent GCaMP reporter or variants thereof (Dreosti et al., 2009) will be one way to detect activity levels in oligodendrocytes and other surrounding glial or neuronal cells by in vivo 2-photon imaging techniques. Intracellular Ca2+ concentration plays an important role in processes such as induction of synaptic plasticity, pathophysiology of various diseases, other intracellular signaling, and transmitter release. Transmitter release, in turn, as a further form of cellular activity, can be monitored very closely using sypHy or variants thereof (Granseth et al., 2006; Odermatt et al., 2012). The ability to generate mosaic zebrafish expressing different fluorescent reporters for myelination and/or cell activity in different cells, through cell transplants from different transgenic fish lines (Carmany and Moens, 2006), will provide very powerful tools to study their interaction in vivo. In the long term, we hope to be able to manipulate cell activity even remotely by expressing light-gated channels such as channelrhodopsin and halorhodopsin (Zhang et al., 2007) in these cells.
Oligodendrocytes remove K+ from the extracellular space through inward rectifier K+ channels and connexin channels (Menichella et al., 2006; Odermatt et al., 2003). When the oligodendrocyte is depolarized, the K+ influx into the oligodendrocyte decreases because the conductance through these channels is reduced, so the extracellular K+ concentration increases significantly because the extracellular space is small. Therefore, neuronal action potentials can be evoked more easily at the junctions, and conduction velocity increases due to depolarization of oligodendrocytes. In this case, oligodendrocytes affect not only the axons that they themselves surround, but also those that are currently crossing their action field (Yamazaki et al., 2010).
In summary, myelination and oligodendrocyte function in health and disease depend on a very complex interplay of many different factors, one of which is certainly the activity level of the cells involved themselves. A better understanding of these activity-dependent regulations in vivo should certainly help to provide new insights into the process of myelination.