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1.  Interaural timing difference circuits in the auditory brainstem of the emu (Dromaius novaehollandiae) 
In the auditory system, precise encoding of temporal information is critical for sound localization, a task with direct behavioral relevance. Interaural timing differences are computed using axonal delay lines and cellular coincidence detectors in nucleus laminaris (NL). We present morphological and physiological data on the timing circuits in the emu, Dromaius novaehollandiae, and compare these results with those from the barn owl (Tyto alba) and the domestic chick (Gallus gallus). Emu NL was composed of a compact monolayer of bitufted neurons whose two thick primary dendrites were oriented dorsoventrally. They showed a gradient in dendritic length along the presumed tonotopic axis. The NL and nucleus magnocellularis (NM) neurons were strongly immunoreactive for parvalbumin, a calcium-binding protein. Antibodies against synaptic vesicle protein 2 and glutamic acid decarboxlyase revealed that excitatory synapses terminated heavily on the dendritic tufts, while inhibitory terminals were distributed more uniformly. Physiological recordings from brainstem slices demonstrated contralateral delay lines from NM to NL. During whole-cell patch-clamp recordings, NM and NL neurons fired single spikes and were doubly-rectifying. NL and NM neurons had input resistances of 30.0 ± 19.9 MΩ and 49.0 ± 25.6 MΩ, respectively, and membrane time constants of 12.8 ± 3.8 ms and 3.9 ± 0.2 ms. These results provide further support for the Jeffress model for sound localization in birds. The emu timing circuits showed the ancestral (plesiomorphic) pattern in their anatomy and physiology, while differences in dendritic structure compared to chick and owl may indicate specialization for encoding ITDs at low best frequencies.
doi:10.1002/cne.20862
PMCID: PMC2948976  PMID: 16435285
avian; nucleus laminaris; nucleus magnocellularis; dendrite; coincidence detection; sound localization
2.  Developmental Changes Underlying the Formation of the Specialized Time Coding Circuits in Barn Owls (Tyto alba) 
The Journal of Neuroscience  2002;22(17):7671-7679.
Barn owls are capable of great accuracy in detecting the interaural time differences (ITDs) that underlie azimuthal sound localization. They compute ITDs in a circuit in nucleus laminaris (NL) that is reorganized with respect to birds like the chicken. The events that lead to the reorganization of the barn owl NL take place during embryonic development, shortly after the cochlear and laminaris nuclei have differentiated morphologically. At first the developing owl’s auditory brainstem exhibits morphology reminiscent of that of the developing chicken. Later, the two systems diverge, and the owl’s brainstem auditory nuclei undergo a secondary morphogenetic phase during which NL dendrites retract, the laminar organization is lost, and synapses are redistributed. These events lead to the restructuring of the ITD coding circuit and the consequent reorganization of the hindbrain map of ITDs and azimuthal space.
PMCID: PMC3260528  PMID: 12196590
avian development; morphogenesis; auditory; laminaris; evolution; interaural time difference
3.  Maps of interaural time difference in the chicken’s brainstem nucleus laminaris 
Biological cybernetics  2008;98(6):541-559.
Animals, including humans, use interaural time differences (ITDs) that arise from different sound path lengths to the two ears as a cue of horizontal sound source location. The nature of the neural code for ITD is still controversial. Current models differentiate between two population codes: either a map-like rate-place code of ITD along an array of neurons, consistent with a large body of data in the barn owl, or a population rate code, consistent with data from small mammals. Recently, it was proposed that these different codes reflect optimal coding strategies that depend on head size and sound frequency. The chicken makes an excellent test case of this proposal because its physical pre-requisites are similar to small mammals, yet it shares a more recent common ancestry with the owl. We show here that, like in the barn owl, the brainstem nucleus laminaris in mature chickens displayed the major features of a place code of ITD. ITD was topographically represented in the maximal responses of neurons along each isofrequency band, covering approximately the contralateral acoustic hemisphere. Furthermore, the represented ITD range appeared to change with frequency, consistent with a pressure gradient receiver mechanism in the avian middle ear. At very low frequencies, below400 Hz, maximal neural responses were symmetrically distributed around zero ITD and it remained unclear whether there was a topographic representation. These findings do not agree with the above predictions for optimal coding and thus revive the discussion as to what determines the neural coding strategies for ITDs.
doi:10.1007/s00422-008-0220-6
PMCID: PMC3170859  PMID: 18491165
Auditory; Hearing; Sound localization; Sensory
4.  Npc1 Acting in Neurons and Glia Is Essential for the Formation and Maintenance of CNS Myelin 
PLoS Genetics  2013;9(4):e1003462.
Cholesterol availability is rate-limiting for myelination, and prior studies have established the importance of cholesterol synthesis by oligodendrocytes for normal CNS myelination. However, the contribution of cholesterol uptake through the endocytic pathway has not been fully explored. To address this question, we used mice with a conditional null allele of the Npc1 gene, which encodes a transmembrane protein critical for mobilizing cholesterol from the endolysosomal system. Loss of function mutations in the human NPC1 gene cause Niemann-Pick type C disease, a childhood-onset neurodegenerative disorder in which intracellular lipid accumulation, abnormally swollen axons, and neuron loss underlie the occurrence of early death. Both NPC patients and Npc1 null mice exhibit myelin defects indicative of dysmyelination, although the mechanisms underlying this defect are incompletely understood. Here we use temporal and cell-type-specific gene deletion in order to define effects on CNS myelination. Our results unexpectedly show that deletion of Npc1 in neurons alone leads to an arrest of oligodendrocyte maturation and to subsequent failure of myelin formation. This defect is associated with decreased activation of Fyn kinase, an integrator of axon-glial signals that normally promotes myelination. Furthermore, we show that deletion of Npc1 in oligodendrocytes results in delayed myelination at early postnatal days. Aged, oligodendocyte-specific null mutants also exhibit late stage loss of myelin proteins, followed by secondary Purkinje neuron degeneration. These data demonstrate that lipid uptake and intracellular transport by neurons and oligodendrocytes through an Npc1-dependent pathway is required for both the formation and maintenance of CNS myelin.
Author Summary
The myelin sheath in the central nervous system is a specialized extension of the oligodendrocyte plasma membrane that serves as an electrical insulator to ensure proper nerve conduction. To accomplish this, myelin is enriched in lipids, particularly unesterified cholesterol, which is an essential and limiting component for myelin formation. Here we determine the contribution of exogenously derived cholesterol to myelination by using a conditional null mutant of the mouse Npc1 gene. Npc1 encodes a transmembrane protein critical for mobilizing exogenously derived cholesterol from late endosomes and lysosomes, and is mutated in patients with Niemann-Pick type C disease, a degenerative disorder caused by impaired intracellular lipid trafficking. We show that mice lacking Npc1 in either neurons or oligodendrocytes exhibit a defect in myelin formation in selected brain regions, with an arrest in oligodendrocyte maturation. In addition, mice with Npc1 deficiency in oligodendrocytes, when aged, show progressive motor dysfunction with myelin breakdown and secondary Purkinje neuron loss. Taken together, our findings demonstrate the role of Npc1 in mediating reciprocal signaling between neurons and glia, and highlight the importance of exogenous cholesterol for CNS myelin formation and maintenance.
doi:10.1371/journal.pgen.1003462
PMCID: PMC3623760  PMID: 23593041
5.  Myelin Gene Regulatory Factor Is Required for Maintenance of Myelin and Mature Oligodendrocyte Identity in the Adult CNS 
The Journal of Neuroscience  2012;32(36):12528-12542.
Although the transcription factors required for the generation of oligodendrocytes and CNS myelination during development have been relatively well established, it is not known whether continued expression of the same factors is required for the maintenance of myelin in the adult. Here, we use an inducible conditional knock-out strategy to investigate whether continued oligodendrocyte expression of the recently identified transcription factor myelin gene regulatory factor (MRF) is required to maintain the integrity of myelin in the adult CNS. Genetic ablation of MRF in mature oligodendrocytes within the adult CNS resulted in a delayed but severe CNS demyelination, with clinical symptoms beginning at 5 weeks and peaking at 8 weeks after ablation of MRF. This demyelination was accompanied by microglial/macrophage infiltration and axonal damage. Transcripts for myelin genes, such as proteolipid protein, MAG, MBP, and myelin oligodendrocyte glycoprotein, were rapidly downregulated after ablation of MRF, indicating an ongoing requirement for MRF in the expression of these genes. Subsequently, a proportion of the recombined oligodendrocytes undergo apoptosis over a period of weeks. Surviving oligodendrocytes gradually lose the expression of mature markers such as CC1 antigen and their association with myelin, without reexpressing oligodendrocyte progenitor markers or reentering the cell cycle. These results demonstrate that ongoing expression of MRF within the adult CNS is critical to maintain mature oligodendrocyte identity and the integrity of CNS myelin.
doi:10.1523/JNEUROSCI.1069-12.2012
PMCID: PMC3752083  PMID: 22956843
6.  Modeling coincidence detection in nucleus laminaris 
Biological Cybernetics  2003;89(5):388-396.
A biologically detailed model of the binaural avian nucleus laminaris is constructed, as a two-dimensional array of multicompartment, conductance-based neurons, along tonotopic and interaural time delay (ITD) axes. The model is based primarily on data from chick nucleus laminaris. Typical chick-like parameters perform ITD discrimination up to 2 kHz, and enhancements for barn owl perform ITD discrimination up to 6 kHz. The dendritic length gradient of NL is explained concisely. The response to binaural out-of-phase input is suppressed well below the response to monaural input (without any spontaneous activity on the opposite side), implicating active potassium channels as crucial to good ITD discrimination.
doi:10.1007/s00422-003-0444-4
PMCID: PMC3269635  PMID: 14669019
7.  Biophysical basis of the sound analog membrane potential that underlies coincidence detection in the barn owl 
Interaural time difference (ITD), or the difference in timing of a sound wave arriving at the two ears, is a fundamental cue for sound localization. A wide variety of animals have specialized neural circuits dedicated to the computation of ITDs. In the avian auditory brainstem, ITDs are encoded as the spike rates in the coincidence detector neurons of the nucleus laminaris (NL). NL neurons compare the binaural phase-locked inputs from the axons of ipsi- and contralateral nucleus magnocellularis (NM) neurons. Intracellular recordings from the barn owl's NL in vivo showed that tonal stimuli induce oscillations in the membrane potential. Since this oscillatory potential resembled the stimulus sound waveform, it was named the sound analog potential (Funabiki et al., 2011). Previous modeling studies suggested that a convergence of phase-locked spikes from NM leads to an oscillatory membrane potential in NL, but how presynaptic, synaptic, and postsynaptic factors affect the formation of the sound analog potential remains to be investigated. In the accompanying paper, we derive analytical relations between these parameters and the signal and noise components of the oscillation. In this paper, we focus on the effects of the number of presynaptic NM fibers, the mean firing rate of these fibers, their average degree of phase-locking, and the synaptic time scale. Theoretical analyses and numerical simulations show that, provided the total synaptic input is kept constant, changes in the number and spike rate of NM fibers alter the ITD-independent noise whereas the degree of phase-locking is linearly converted to the ITD-dependent signal component of the sound analog potential. The synaptic time constant affects the signal more prominently than the noise, making faster synaptic input more suitable for effective ITD computation.
doi:10.3389/fncom.2013.00102
PMCID: PMC3821004  PMID: 24265615
phase-locking; sound localization; auditory brainstem; periodic signals; oscillation; owl
8.  ERK1/ERK2 MAPK signaling is required to increase myelin thickness independent of oligodendrocyte differentiation and initiation of myelination 
Wrapping of the myelin sheath around axons by oligodendrocytes is critical for the rapid conduction of electrical signals, required for the normal functioning of the central nervous system (CNS). Myelination is a multistep process where oligodendrocytes progress through a well-coordinated differentiation program regulated by multiple extracellular growth and differentiation signals. The intracellular-transduction of the extracellular signals that regulate myelination is poorly understood. Here we demonstrate a critical role for two important signaling molecules, extracelluar-signal-regulated-kinases-1 and -2 (ERK1/ERK2), downstream mediators of mitogen-activated protein kinases (MAPK), in the control of CNS myelin thickness. We generated and analyzed two lines of mice lacking both ERK1/ERK2 function specifically in oligodendrocyte-lineage cells. In the absence of ERK1/ERK2 signaling oligodendrocyte progenitor cells (OPC) proliferated and differentiated on schedule. Mutant oligodendrocytes also ensheathed axons normally and made a few wraps of compact myelin. However, the subsequent increase in myelination that correlated myelin thickness in proportion to the axon caliber failed to occur. Furthermore, although the numbers of differentiated oligodendrocytes in the adult mutants were unchanged, they showed an inability to upregulate the transcription of major myelin genes that normally occurs during active myelination. Similarly, in vitro ERK1/ERK2 deficient NG2+ oligodendrocytes differentiated normally but failed to form typical myelin-like membrane sheets. None of these effects were observed in single ERK1 or ERK2 mutants. These studies suggest that the predominant role of ERK1/ERK2 signaling in vivo is in promoting rapid myelin growth to increase its thickness, subsequent to oligodendrocyte differentiation and the initiation of myelination.
doi:10.1523/JNEUROSCI.0137-12.2012
PMCID: PMC3521511  PMID: 22745486
oligodendrocyte; myelin
9.  Sustained Activation of ERK1/2 MAPK in Oligodendrocytes and Schwann Cells Enhances Myelin Growth and Stimulates Oligodendrocyte Progenitor Expansion 
Myelin is a biologically active membrane receiving and processing signals from axons. Whereas, much is known about its structure and molecular composition, the intracellular signal transduction pathways, active during specific phases of myelinogenesis for regulating myelin formation remain poorly understood. Recent genetic loss-of-function studies have suggested a key role of extracelluar-signal-regulated-kinases-1 and -2 (ERK1/2), downstream mediators of mitogen-activated-protein-kinases (MAPKs), in promoting CNS and PNS myelination. In contrast, other studies, largely in vitro, have suggested that activation of ERK1/2 pathway can be detrimental for glial cell function and myelination. Given these conflicting reports, we investigated the effects of cell-autonomous activation of ERK1/2 in glial cells during developmental myelination in the intact CNS and PNS. Two lines of transgenic mice with sustained activation of ERK1/2 in oligodendrocyte progenitors (OPCs), oligodendrocytes, and Schwann cells were generated. Consistent with our loss-of-function studies, gain of ERK1/2 function in oligodendrocyte-lineage cells significantly increased myelin thickness, independent of oligodendrocyte differentiation or initiation of myelination. Additionally, increased activation of ERK1/2 in OPCs during early development resulted in transient hyper-proliferation and overproduction of OPCs, but generation of normal numbers of myelinating oligodendrocytes. Thus, these in vivo studies suggest a beneficial biphasic requirement of ERK1/2 during developmental myelination in the CNS, deployed first during early stages of the oligodendrocyte lineage for promoting OPC expansion and then redeployed later in myelinating oligodendrocytes for promoting myelin growth. Furthermore, Schwann cells with activated ERK1/2 hypermyelinate PNS axons, suggesting that ERK1/2 signaling is a conserved mechanism that promotes both CNS and PNS developmental myelination.
doi:10.1523/JNEUROSCI.4403-12.2013
PMCID: PMC3711773  PMID: 23283332
oligodendrocyte; myelin; Schwann cells
10.  MYRF Is a Membrane-Associated Transcription Factor That Autoproteolytically Cleaves to Directly Activate Myelin Genes 
PLoS Biology  2013;11(8):e1001625.
Oligodendrocyte development and myelination rely on an unusual membrane-associated transcription factor that shares functional domains with bacteriophage proteins.
The myelination of axons is a crucial step during vertebrate central nervous system (CNS) development, allowing for rapid and energy efficient saltatory conduction of nerve impulses. Accordingly, the differentiation of oligodendrocytes, the myelinating cells of the CNS, and their expression of myelin genes are under tight transcriptional control. We previously identified a putative transcription factor, Myelin Regulatory Factor (Myrf), as being vital for CNS myelination. Myrf is required for the generation of CNS myelination during development and also for its maintenance in the adult. It has been controversial, however, whether Myrf directly regulates transcription, with reports of a transmembrane domain and lack of nuclear localization. Here we show that Myrf is a membrane-associated transcription factor that undergoes an activating proteolytic cleavage to separate its transmembrane domain-containing C-terminal region from a nuclear-targeted N-terminal region. Unexpectedly, this cleavage event occurs via a protein domain related to the autoproteolytic intramolecular chaperone domain of the bacteriophage tail spike proteins, the first time this domain has been found to play a role in eukaryotic proteins. Using ChIP-Seq we show that the N-terminal cleavage product directly binds the enhancer regions of oligodendrocyte-specific and myelin genes. This binding occurs via a defined DNA-binding consensus sequence and strongly promotes the expression of target genes. These findings identify Myrf as a novel example of a membrane-associated transcription factor and provide a direct molecular mechanism for its regulation of oligodendrocyte differentiation and CNS myelination.
Author Summary
Oligodendrocytes are a highly specialized cell type that surround axons of the vertebrate central nervous system with myelin, electrically insulating them and allowing rapid and energy-efficient propagation of nerve signals. We previously identified a protein, MYRF, that is required for the final stages of oligodendrocyte differentiation and myelination. Although we proposed that MYRF might act as a transcription factor, it remains uncertain whether this is true, given that MYRF and related proteins contain a transmembrane domain that might preclude localization to the nucleus. Here, we show that the MYRF protein undergoes an activating cleavage event to release the functional transcription factor from the transmembrane domain that otherwise anchors it to the endoplasmic reticulum. Unexpectedly, this cleavage event is mediated by a portion of MYRF that is related to a self-cleaving domain found in bacteriophage proteins. This distinguishes it from other membrane-associated transcription factors that are cleaved via regulated proteolysis within the membrane bilayer. We find that the N-terminal product of MYRF cleavage directly binds to a wide range of genes involved in myelination, stimulating their expression. Many of these MYRF binding sites identify previously uncharacterized enhancers for these myelin genes.
doi:10.1371/journal.pbio.1001625
PMCID: PMC3742440  PMID: 23966833
11.  Bilateral matching of frequency tuning in neural cross-correlators of the owl 
Biological cybernetics  2009;100(6):521-531.
Sound localization requires comparison between the inputs to the left and right ears. One important aspect of this comparison is the differences in arrival time to each side, also called interaural time difference (ITD).A prevalent model of ITD detection, consisting of delay lines and coincidence-detector neurons, was proposed by Jeffress (J Comp Physiol Psychol 41:35–39, 1948). As an extension of the Jeffress model, the process of detecting and encoding ITD has been compared to an effective cross-correlation between the input signals to the two ears. Because the cochlea performs a spectrotemporal decomposition of the input signal, this cross-correlation takes place over narrow frequency bands. Since the cochlear tonotopy is arranged in series, sounds of different frequencies will trigger neural activity with different temporal delays. Thus, the matching of the frequency tuning of the left and right inputs to the cross-correlator units becomes a ‘timing’ issue. These properties of auditory transduction gave theoretical support to an alternative model of ITD-detection based on a bilateral mismatch in frequency tuning, called the ‘stereausis’ model. Here we first review the current literature on the owl’s nucleus laminaris, the equivalent to the medial superior olive of mammals, which is the site where ITD is detected. Subsequently, we use reverse correlation analysis and stimulation with uncorrelated sounds to extract the effective monaural inputs to the cross-correlator neurons. We show that when the left and right inputs to the cross-correlators are defined in this manner, the computation performed by coincidence-detector neurons satisfies conditions of cross-correlation theory. We also show that the spectra of left and right inputs are matched, which is consistent with predictions made by the classic model put forth by Jeffress.
doi:10.1007/s00422-009-0312-y
PMCID: PMC2719282  PMID: 19396457
Barn owl; Interaural time difference; Cross-correlation; Coincidence detection; Cochlear delays; Sound localization; Nucleus laminaris; Stereausis
12.  Oligodendrocytes as Regulators of Neuronal Networks during Early Postnatal Development 
PLoS ONE  2011;6(5):e19849.
Oligodendrocytes are the glial cells responsible for myelin formation. Myelination occurs during the first postnatal weeks and, in rodents, is completed during the third week after birth. Myelin ensures the fast conduction of the nerve impulse; in the adult, myelin proteins have an inhibitory role on axon growth and regeneration after injury. During brain development, oligodendrocytes precursors originating in multiple locations along the antero-posterior axis actively proliferate and migrate to colonize the whole brain. Whether the initial interactions between oligodendrocytes and neurons might play a functional role before the onset of myelination is still not completely elucidated. In this article, we addressed this question by transgenically targeted ablation of proliferating oligodendrocytes during cerebellum development. Interestingly, we show that depletion of oligodendrocytes at postnatal day 1 (P1) profoundly affects the establishment of cerebellar circuitries. We observed an impressive deregulation in the expression of molecules involved in axon growth, guidance and synaptic plasticity. These effects were accompanied by an outstanding increase of neurofilament staining observed 4 hours after the beginning of the ablation protocol, likely dependent from sprouting of cerebellar fibers. Oligodendrocyte ablation modifies localization and function of ionotropic glutamate receptors in Purkinje neurons. These results show a novel oligodendrocyte function expressed during early postnatal brain development, where these cells participate in the formation of cerebellar circuitries, and influence its development.
doi:10.1371/journal.pone.0019849
PMCID: PMC3093406  PMID: 21589880
13.  Noise Reduction of Coincidence Detector Output by the Inferior Colliculus of the Barn Owl 
A recurring theme in theoretical work is that integration over populations of similarly tuned neurons can reduce neural noise. However, there are relatively few demonstrations of an explicit noise reduction mechanism in a neural network. Here we demonstrate that the brainstem of the barn owl includes a stage of processing apparently devoted to increasing the signal-to-noise ratio in the encoding of the interaural time difference (ITD), one of two primary binaural cues used to compute the position of a sound source in space. In the barn owl, the ITD is processed in a dedicated neural pathway that terminates at the core of the inferior colliculus (ICcc). The actual locus of the computation of the ITD is before ICcc in the nucleus laminaris (NL), and ICcc receives no inputs carrying information that did not originate in NL. Unlike in NL, the rate-ITD functions of ICcc neurons require as little as a single stimulus presentation per ITD to show coherent ITD tuning. ICcc neurons also displayed a greater dynamic range with a maximal difference in ITD response rates approximately double that seen in NL. These results indicate that ICcc neurons perform a computation functionally analogous to averaging across a population of similarly tuned NL neurons.
doi:10.1523/JNEUROSCI.0220-06.2006
PMCID: PMC2492673  PMID: 16738236
interaural time difference; sound localization; inferior colliculus; nucleus laminaris; barn owl; pooling
14.  Mechanisms for Adjusting Interaural Time Differences to Achieve Binaural Coincidence Detection 
Understanding binaural perception requires detailed analyses of the neural circuitry responsible for the computation of interaural time differences (ITDs). In the avian brainstem, this circuit consists of internal axonal delay lines innervating an array of coincidence detector neurons that encode external ITDs. Nucleus magnocellularis (NM) neurons project to the dorsal dendritic field of the ipsilateral nucleus laminaris (NL) and to the ventral field of the contralateral NL. Contralateral-projecting axons form a delay line system along a band of NL neurons. Binaural acoustic signals in the form of phase-locked action potentials from NM cells arrive at NL and establish a topographic map of sound source location along the azimuth. These pathways are assumed to represent a circuit similar to the Jeffress model of sound localization, establishing a place code along an isofrequency contour of NL. Three-dimensional measurements of axon lengths reveal major discrepancies with the current model; the temporal offset based on conduction length alone makes encoding of physiological ITDs impossible. However, axon diameter and distances between Nodes of Ranvier also influence signal propagation times along an axon. Our measurements of these parameters reveal that diameter and internode distance can compensate for the temporal offset inferred from axon lengths alone. Together with other recent studies these unexpected results should inspire new thinking on the cellular biology, evolution and plasticity of the circuitry underlying low frequency sound localization in both birds and mammals.
doi:10.1523/JNEUROSCI.3464-09.2010
PMCID: PMC2822993  PMID: 20053889
Sound; Localization; Auditory; Brainstem; Axon; Conduction; Velocity
15.  Localization of KCNC1 (Kv3.1) Potassium Channel Subunits in the Avian Auditory Nucleus Magnocellularis and Nucleus Laminaris during Development 
Journal of Neurobiology  2003;55(2):165-178.
The KCNC1 (previously Kv3.1) potassium channel, a delayed rectifier with a high threshold of activation, is highly expressed in the time coding nuclei of the adult chicken and barn owl auditory brainstem. The proposed role of KCNC1 currents in auditory neurons is to reduce the width of the action potential and enable neurons to transmit high frequency temporal information with little jitter. Because developmental changes in potassium currents are critical for the maturation of the shape of the action potential, we used immunohistochemical methods to examine the developmental expression of KCNC1 subunits in the avian auditory brainstem. The KCNC1 gene gives rise to two splice variants, a longer KCNC1b and a shorter KCNC1a that differ at the carboxy termini. Two antibodies were used: an antibody to the N-terminus that does not distinguish between KCNC1a and b isoforms, denoted as panKCNC1, and another antibody that specifically recognizes the C terminus of KCNC1b. A comparison of the staining patterns observed with the pan-KCNC1 and the KCNC1b specific antibodies suggests that KCNC1a and KCNC1b splice variants are differentially regulated during development. Although pan-KCNC1 immunoreactivity is observed from the earliest time examined in the chicken (E10), a subcellular redistribution of the immunoproduct was apparent over the course of development. KCNC1b specific staining has a late onset with immunostaining first appearing in the regions that map high frequencies in nucleus magnocellularis (NM) and nucleus laminaris (NL). The expression of KCNC1b protein begins around E14 in the chicken and after E21 in the barn owl, relatively late during ontogeny and at the time that synaptic connections mature morphologically and functionally.
doi:10.1002/neu.10198
PMCID: PMC3268178  PMID: 12672015
chicken; barn owl; ontogeny; time coding; outward current; high threshold
16.  Early Intervention for Spinal Cord Injury with Human Induced Pluripotent Stem Cells Oligodendrocyte Progenitors 
PLoS ONE  2015;10(1):e0116933.
Induced pluripotent stem (iPS) cells are at the forefront of research in regenerative medicine and are envisaged as a source for personalized tissue repair and cell replacement therapy. Here, we demonstrate for the first time that oligodendrocyte progenitors (OPs) can be derived from iPS cells generated using either an episomal, non-integrating plasmid approach or standard integrating retroviruses that survive and differentiate into mature oligodendrocytes after early transplantation into the injured spinal cord. The efficiency of OP differentiation in all 3 lines tested ranged from 40% to 60% of total cells, comparable to those derived from human embryonic stem cells. iPS cell lines derived using episomal vectors or retroviruses generated a similar number of early neural progenitors and glial progenitors while the episomal plasmid-derived iPS line generated more OPs expressing late markers O1 and RIP. Moreover, we discovered that iPS-derived OPs (iPS-OPs) engrafted 24 hours following a moderate contusive spinal cord injury (SCI) in rats survived for approximately two months and that more than 70% of the transplanted cells differentiated into mature oligodendrocytes that expressed myelin associated proteins. Transplanted OPs resulted in a significant increase in the number of myelinated axons in animals that received a transplantation 24 h after injury. In addition, nearly a 5-fold reduction in cavity size and reduced glial scarring was seen in iPS-treated groups compared to the control group, which was injected with heat-killed iPS-OPs. Although further investigation is needed to understand the mechanisms involved, these results provide evidence that patient-specific, iPS-derived OPs can survive for three months and improve behavioral assessment (BBB) after acute transplantation into SCI. This is significant as determining the time in which stem cells are injected after SCI may influence their survival and differentiation capacity.
doi:10.1371/journal.pone.0116933
PMCID: PMC4311989  PMID: 25635918
17.  Fibroblast Growth Factor Receptor Signaling in Oligodendrocytes Regulates Myelin Sheath Thickness 
The Journal of Neuroscience  2012;32(19):6631-6641.
Formation of the central nervous system (CNS) white matter is developmentally tightly regulated, but the molecules and mechanisms of myelination control in the postnatal CNS are poorly understood. Here, we show that myelin growth is controlled by Fibroblast Growth Factor (FGF) signaling, originally identified as a proliferative signal for oligodendrocyte precursor cells (OPC) in vitro. We created two lines of mice lacking both FGF-receptor 1 (Fgfr1) and Fgfr2 in oligodendrocyte lineage cells but found that in these mice OPC proliferation and differentiation were unaffected. Also axonal ensheathment and the initiation of myelination was on time. However, the rapid growth of CNS myelin, normally occurring in the second postnatal week, was strongly inhibited. Throughout adulthood, the myelin sheath remained disproportionately thin relative to the axon caliber. In adult mice, mutant oligodendrocytes were normal in number, whereas the transcription of major myelin genes was reduced. This FGF-receptor mediated stimulation of mature oligodendrocytes could also be modeled in vitro, demonstrating that enhanced expansion of oligodendroglial processes requires signaling by extracellular-signal regulated kinases-1 and -2 (Erk1/2), downstream mediaters of Mitogen-Activated Protein Kinase (MAPK). Also in vivo, Erk1/2-MAPK activity was reduced in the hypomyelinated CNS of Fgfr1/Fgfr2 mutant mice. These studies reveal a previously unrecognized function of FGF-receptor signaling in oligodendrocytes that contributes to the regulation of myelin sheath thickness, and which uncouples the initiation of ensheathment from the later phase of continued myelin growth.
doi:10.1523/JNEUROSCI.6005-11.2012
PMCID: PMC3367512  PMID: 22573685
oligodendrocyte; myelin
18.  Transplantation of human neural stem cells transduced with Olig2 transcription factor improves locomotor recovery and enhances myelination in the white matter of rat spinal cord following contusive injury 
BMC Neuroscience  2009;10:117.
Background
Contusive spinal cord injury is complicated by a delayed loss of oligodendrocytes, resulting in chronic progressive demyelination. Therefore, transplantation strategies to provide oligodendrocyte lineage cells and to enhance the extent of myelination appear to be justified for spinal cord repair. The present study investigated whether transplantation of human neural stem cells (NSCs) genetically modified to express Olig2 transcription factor, an essential regulator of oligodendrocyte development, can improve locomotor recovery and enhance myelination in a rat contusive spinal cord injury model.
Results
HB1.F3 (F3) immortalized human NSC line was transduced with a retroviral vector encoding Olig2, an essential regulator of oligodendrocyte development. Overexpression of Olig2 in human NSCs (F3.Olig2) induced activation of NKX2.2 and directed differentiation of NSCs into oligodendrocyte lineage cells in vitro. Introduction of Olig2 conferred higher proliferative activity, and a much larger number of F3.Olig2 NSCs were detected by 7 weeks after transplantation into contused spinal cord than that of parental F3 NSCs. F3.Olig2 NSCs exhibited frequent migration towards the white matter, whereas F3 NSCs were mostly confined to the gray matter or around the lesion cavities. Most of F3.Olig2 NSCs occupying the spared white matter differentiated into mature oligodendrocytes. Transplantation of F3.Olig2 NSCs increased the volume of spared white matter and reduced the cavity volume. Moreover, F3.Olig2 grafts significantly increased the thickness of myelin sheath around the axons in the spared white matter. Finally, animals with F3.Olig2 grafts showed an improvement in the quality of hindlimbs locomotion.
Conclusion
Transplantation of NSCs genetically modified to differentiate into an oligodendrocytic lineage may be an effective strategy to improve functional outcomes following spinal cord trauma. The present study suggests that molecular factors governing cell fate decisions can be manipulated to enhance reparative potential of the cell-based therapy.
doi:10.1186/1471-2202-10-117
PMCID: PMC2758886  PMID: 19772605
19.  The Axonal Membrane Protein Caspr, a Homologue of Neurexin IV, Is a Component of the Septate-like Paranodal Junctions That Assemble during Myelination  
The Journal of Cell Biology  1997;139(6):1495-1506.
We have investigated the potential role of contactin and contactin-associated protein (Caspr) in the axonal–glial interactions of myelination. In the nervous system, contactin is expressed by neurons, oligodendrocytes, and their progenitors, but not by Schwann cells. Expression of Caspr, a homologue of Neurexin IV, is restricted to neurons. Both contactin and Caspr are uniformly expressed at high levels on the surface of unensheathed neurites and are downregulated during myelination in vitro and in vivo. Contactin is downregulated along the entire myelinated nerve fiber. In contrast, Caspr expression initially remains elevated along segments of neurites associated with nascent myelin sheaths. With further maturation, Caspr is downregulated in the internode and becomes strikingly concentrated in the paranodal regions of the axon, suggesting that it redistributes from the internode to these sites. Caspr expression is similarly restricted to the paranodes of mature myelinated axons in the peripheral and central nervous systems; it is more diffusely and persistently expressed in gray matter and on unmyelinated axons. Immunoelectron microscopy demonstrated that Caspr is localized to the septate-like junctions that form between axons and the paranodal loops of myelinating cells. Caspr is poorly extracted by nonionic detergents, suggesting that it is associated with the axon cytoskeleton at these junctions. These results indicate that contactin and Caspr function independently during myelination and that their expression is regulated by glial ensheathment. They strongly implicate Caspr as a major transmembrane component of the paranodal junctions, whose molecular composition has previously been unknown, and suggest its role in the reciprocal signaling between axons and glia.
PMCID: PMC2132621  PMID: 9396755
20.  The Role of Myelin in Theiler's Virus Persistence in the Central Nervous System 
PLoS Pathogens  2007;3(2):e23.
Theiler's virus, a picornavirus, persists for life in the central nervous system of mouse and causes a demyelinating disease that is a model for multiple sclerosis. The virus infects neurons first but persists in white matter glial cells, mainly oligodendrocytes and macrophages. The mechanism, by which the virus traffics from neurons to glial cells, and the respective roles of oligodendrocytes and macrophages in persistence are poorly understood. We took advantage of our previous finding that the shiverer mouse, a mutant with a deletion in the myelin basic protein gene (Mbp), is resistant to persistent infection to examine the role of myelin in persistence. Using immune chimeras, we show that resistance is not mediated by immune responses or by an efficient recruitment of inflammatory cells into the central nervous system. With both in vivo and in vitro experiments, we show that the mutation does not impair the permissiveness of neurons, oligodendrocytes, and macrophages to the virus. We demonstrate that viral antigens are present in cytoplasmic channels of myelin during persistent infection of wild-type mice. Using the optic nerve as a model, we show that the virus traffics from the axons of retinal ganglion cells to the cytoplasmic channels of myelin, and that this traffic is impaired by the shiverer mutation. These results uncover an unsuspected axon to myelin traffic of Theiler's virus and the essential role played by the infection of myelin/oligodendrocyte in persistence.
Author Summary
Theiler's virus persists in the central nervous system of mice and causes a chronic disease that resembles multiple sclerosis, a common demyelinating disease of humans. The virus infects neurons for one to two weeks, but later on it persists in the white matter, in oligodendrocytes and also in macrophages. Oligodendrocytes are the myelin-making cells of the central nervous system. Strikingly, in mice with a genetic defect of myelin, the virus infects neurons normally but is unable to persist. Understanding the reason for the lack of persistence in this mutant mouse should pinpoint an essential step in the complex process resulting in persistence. In this article, we show that resistance to persistent infection is not mediated by the immune system and is not due to inefficient viral replication in oligodendrocytes or macrophages. Instead, we show that virus transported in axons traffics into the myelin, and that this traffic is interrupted by the myelin mutation. This unsuspected axon to myelin traffic of Theiler's virus is necessary for viral persistence. Our results warrant looking for a similar phenomenon in other persistent infections of the nervous system, including in humans.
doi:10.1371/journal.ppat.0030023
PMCID: PMC1797621  PMID: 17305428
21.  Mutation of 3-Hydroxy-3-Methylglutaryl CoA Synthase I Reveals Requirements for Isoprenoid and Cholesterol Synthesis in Oligodendrocyte Migration Arrest, Axon Wrapping, and Myelin Gene Expression 
The Journal of Neuroscience  2014;34(9):3402-3412.
Myelin membrane, which ensheaths axons, has an unusually high amount of cholesterol. Cholesterol influences membrane fluidity and assembles lipid-rich microdomains within membranes, and some studies have shown that cholesterol is important for myelination. How cholesterol influences the development and differentiation of oligodendrocytes, glial cells that make myelin, is not known nor is clear whether isoprenoids, which also are products of the cholesterol biosynthetic pathway, contribute to myelination. Through a forward genetic screen in zebrafish we discovered that mutation of hmgcs1, which encodes an enzyme necessary for isoprenoid and cholesterol synthesis, causes oligodendrocyte progenitor cells (OPCs) to migrate past their target axons and to fail to express myelin genes. Drawing on a combination of pharmacological inhibitor and rescue experiments, we provide evidence that isoprenoids and protein prenylation, but not cholesterol, are required in OPCs to halt their migration at target axons. On the other hand, cholesterol, but not isoprenoids, is necessary both for axon ensheathment and myelin gene expression. Our data reveal that different products of the cholesterol biosynthetic pathway have distinct roles in oligodendrocyte development and that they together help to coordinate directed migration, axon wrapping, and gene expression.
doi:10.1523/JNEUROSCI.4587-13.2014
PMCID: PMC3935092  PMID: 24573296
cholesterol; isoprenoid; myelin; prenylation; zebrafish
22.  Myelination of the corpus callosum in male and female rats following complex environment housing during adulthood 
Brain research  2009;1288:9-17.
Myelination is an important process in brain development, and delays or abnormalities in this process have been associated with a number of conditions including autism, developmental delay, attention deficit disorder, and schizophrenia. Myelination can be sensitive to developmental experience; however, although the adult brain remains highly plastic, it is unknown whether myelination continues to be sensitive to experience during adulthood. Male and female rats were socially housed until four months of age, at which time they were moved into either a complex or “enriched” environment (EC) or an isolated condition (IC). Although the area of the splenium (posterior 20% of the callosum, which contains axons from visual cortical neurons) increased by about 10% following two months of EC housing, the area occupied by myelinated axons was not influenced by adult housing condition. Instead, it was the area occupied by glial cell processes and unmyelinated axons which significantly increased following EC housing. Neither the size nor the myelin content of the genu (anterior 15% of the callosum) was sensitive to manipulations of adult housing condition, but males had more area occupied by myelinated axons in both callosal regions. Finally, the inability of two months of complex environment housing during adulthood to impact the number of myelinated axons in the splenium was confirmed in a subset of animals using quantitative electron microscopy. We conclude that the sensitivity of myelination to experience is reduced in adulthood relative to development in both sexes.
doi:10.1016/j.brainres.2009.06.087
PMCID: PMC2737079  PMID: 19596280
enrichment; EC; electron microscopy; sex differences; splenium; genu
23.  Erythropoietin Amplifies Stroke-Induced Oligodendrogenesis in the Rat 
PLoS ONE  2010;5(6):e11016.
Background
Erythropoietin (EPO), a hematopoietic cytokine, enhances neurogenesis and angiogenesis during stroke recovery. In the present study, we examined the effect of EPO on oligodendrogenesis in a rat model of embolic focal cerebral ischemia.
Methodology and Principal Findings
Recombinant human EPO (rhEPO) at a dose of 5,000 U/kg (n = 18) or saline (n = 18) was intraperitoneally administered daily for 7 days starting 24 h after stroke onset. Treatment with rhEPO augmented actively proliferating oligodendrocyte progenitor cells (OPCs) measured by NG2 immunoreactive cells within the peri-infarct white matter and the subventricular zone (SVZ), but did not protect against loss of myelinating oligodendrocytes measured by cyclic nucleotide phosphodiesterase (CNPase) positive cells 7 days after stroke. However, 28 and 42 days after stroke, treatment with rhEPO significantly increased myelinating oligodendrocytes and myelinated axons within the peri-infarct white matter. Using lentivirus to label subventricular zone (SVZ) neural progenitor cells, we found that in addition to the OPCs generated in the peri-infarct white matter, SVZ neural progenitor cells contributed to rhEPO-increased OPCs in the peri-infarct area. Using bromodeoxyuridine (BrdU) for birth-dating cells, we demonstrated that myelinating oligodendrocytes observed 28 days after stroke were derived from OPCs. Furthermore, rhEPO significantly improved neurological outcome 6 weeks after stroke. In vitro, rhEPO increased differentiation of adult SVZ neural progenitor cells into oligodendrocytes and enhanced immature oligodendrocyte cell proliferation.
Conclusions
Our in vivo and in vitro data indicate that EPO amplifies stroke-induced oligodendrogenesis that could facilitate axonal re-myelination and lead to functional recovery after stroke.
doi:10.1371/journal.pone.0011016
PMCID: PMC2884017  PMID: 20552017
24.  Astrocytic TIMP-1 Promotes Oligodendrocyte Differentiation and Enhances CNS Myelination 
Tissue inhibitor of metalloproteinase-1 (TIMP-1) is an extracellular protein and endogenous regulator of matrix metalloproteinases (MMPs) secreted by astrocytes in response to CNS myelin injury. We have previously reported that adult TIMP-1KO mice exhibit poor myelin repair following demyelinating injury. This observation led us to hypothesize a role for TIMP-1 in oligodendrogenesis and CNS myelination. Herein, we demonstrate that compact myelin formation is significantly delayed in TIMP-1KO mice which coincided with dramatically reduced numbers of white matter astrocytes in the developing CNS. Analysis of differentiation in CNS progenitor cells (neurosphere) cultures from TIMP-1KO mice revealed a specific deficit of NG2+ oligodendrocyte progenitor cells. Application of rmTIMP-1 to TIMP-1KO neurosphere cultures evoked a dose-dependent increase in NG2+ cell numbers, while treatment with GM6001, a potent broad spectrum MMP inhibitor did not. Similarly, administration of recombinant murine TIMP-1 (rmTIMP-1) to A2B5+ immunopanned oligodendrocyte progenitors significantly increased the number of differentiated O1+ oligodendrocytes, while antisera to TIMP-1 reduced oligodendrocyte numbers. We also determined that A2B5+ oligodendrocyte progenitors grown in conditioned media derived from TIMP-1KO primary glial cultures resulted in reduced differentiation of mature O1+ oligodendrocytes. Finally, we report that addition of rmTIMP-1 to primary glial cultures resulted in a dose-dependent proliferative response of astrocytes. Together, these findings describe a previously uncharacterized role for TIMP-1 in the regulation of oligodendrocytes and astrocytes during development and provide a novel function for TIMP-1 on myelination in the developing CNS.
doi:10.1523/JNEUROSCI.5474-10.2011
PMCID: PMC3090636  PMID: 21508247
metalloproteinase; astrocyte; oligodendrocyte progenitor cell
25.  Cyclin dependent kinase 5 is required for the normal development of oligodendrocytes and myelin formation 
Developmental biology  2013;378(2):94-106.
The development of oligodendrocytes, the myelinating cells of the vertebrate CNS, is regulated by a cohort of growth factors and transcription factors. Less is known about the signaling pathways that integrate extracellular signals with intracellular transcriptional regulators to control oligodendrocyte development. Cyclin dependent kinase 5 (Cdk5) and its co-activators play critical roles in the regulation of neuronal differentiation, cortical lamination, neuronal cell migration and axon outgrowth. Here we demonstrate a previously unrecognized function of Cdk5 in regulating oligodendrocyte maturation and myelination. During late embryonic development Cdk5 null animals displayed a reduction in the number of MBP+ cells in the spinal cord, but no difference in the number of OPCs. To determine whether the reduction of oligodendrocytes reflected a cell-intrinsic loss of Cdk5, it was selectively deleted from Olig1+ oligodendrocyte lineage cells. In Olig1Cre/+; Cdk5fl/fl conditional mutants, reduced levels of expression of MBP and PLP mRNA were observed throughout the CNS and ultrastructural analyses demonstrated a significant reduction in the proportion of myelinated axons in the optic nerve and spinal cord. Pharmacological inhibition or RNAi knockdown of Cdk5 in vitro resulted in the reduction in oligodendrocyte maturation, but had no effect on OPC cell proliferation. Conversely, over-expression of Cdk5 promoted oligodendrocyte maturation and enhanced process outgrowth. Consistent with this data, Cdk5−/− oligodendrocytes developed significantly fewer primary processes and branches than control cells. Together, these findings suggest that Cdk5 function as a signaling integrator to regulate oligodendrocyte maturation and myelination.
doi:10.1016/j.ydbio.2013.03.023
PMCID: PMC3686511  PMID: 23583582
conditional Cdk5 knockout; OPC; oligodendrocytes; differentiation; myelination

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