In JoVE (1)

Other Publications (23)

Articles by Angeles B. Ribera in JoVE

Other articles by Angeles B. Ribera on PubMed

In Vivo Analysis of Kvbeta2 Function in Xenopus Embryonic Myocytes

The Journal of Physiology. Jun, 2002  |  Pubmed ID: 12068032

Kv1 potassium channels consist of pore-forming alpha subunits as well as auxiliary beta subunits. In heterologous systems, Kv1alpha subunits suffice for induction of voltage-dependent potassium current (I(Kv)). Although Kv1 channels can be expressed without auxiliary subunits in heterologous systems, coexpression with Kvbeta subunits has dramatic effects on surface expression and kinetic properties. Much less is known about the functional roles of Kvbeta subunits in vivo, despite their presence in the majority of native Kv1 channel complexes. We used an antisense approach to probe the contribution of Kvbeta2 subunits to native Kv1 channel function in embryonic myocytes. We compared the effects of antisense Kvbeta2 treatment on the whole cell I(Kv) to those produced by overexpression of a dominant-negative Kv1alpha subunit. The reductions in the maximal potassium conductance produced by antisense Kvbeta2 treatment and elimination of Kv1alpha subunit function were not significantly different from each other. In addition, simultaneous elimination of Kv1alpha and Kvbeta2 subunit function resulted in no further reduction of the maximal conductance. The Kv channel complexes targeted by Kvbeta2 and/or Kv1alpha subunit elimination contributed to action potential repolarization because elimination of either or both subunits led to increases in the duration of the action potential. As for potassium conductance, the effects of elimination of both alpha and beta subunits on the duration of the action potential were not additive. Taken together, the results suggest that Kv1 potassium channel complexes in vivo have a strong requirement for both alpha and beta subunits.

Selective Regulation of XSlo Splice Variants During Xenopus Embryogenesis

Journal of Neurophysiology. Nov, 2003  |  Pubmed ID: 12867527

Calcium-activated potassium channels regulate excitability of the adult nervous system. In contrast, little is known about the contribution of calcium-activated potassium channels to excitability of the embryonic nervous system when electrical membrane properties and intracellular calcium levels show dramatic changes. Embryonic Xenopus spinal neurons exhibit a well-characterized developmental program of excitability that involves several different currents including calcium-activated ones. Here, we show that a molecular determinant of calcium-activated potassium channels, xSlo, is expressed during Xenopus embryogenesis even prior to differentiation of excitable tissues. Five different xSlo variants are expressed in embryonic tissues as a consequence of alternative exon usage at a single splice site. One of these variants, xSlo59, is neural-specific, and its expression is limited to late stages of neuronal differentiation. However, expression of the four other variants occurs in both muscle and neurons at all stages of development examined. Electrophysiological analysis of recombinant xSlo channels reveals that the xSlo59 exon serves as a gain-of-function module and allows physiologically relevant levels of membrane potential and intracellular calcium to activate effectively the resultant channel. These results suggest that xSlo59 channels play a unique role in sculpting the excitable membrane properties of Xenopus spinal neurons.

Immunocytochemistry As a Tool for Zebrafish Developmental Neurobiology

Methods in Cell Science : an Official Journal of the Society for In Vitro Biology. 2003  |  Pubmed ID: 14739591

Two methods are presented here that allow clear visualization of antibody localization in zebrafish whole mount preparations, both for immunocytochemistry (ICC) alone and in combination with in situ hybridization (ISH). The first protocol describes ICC performed using a modified permeabilization technique and the chromogen AEC (3-Amino-9-ethylcarbazole). The second protocol describes the co-localization of transcriptional and translational products using a combined ISH/ICC protocol. A fluorescing chromogen (Fast Red, FR) is used to detect mRNA transcripts by ISH, and is combined with ICC that uses a secondary antibody conjugated to a different fluorescent molecule (Alexa 488). These procedures allow the identification of gene expression patterns in cell types identifiable with known antibodies.

Carboxyl Tail Region of the Kv2.2 Subunit Mediates Novel Developmental Regulation of Channel Density

Journal of Neurophysiology. Dec, 2004  |  Pubmed ID: 15306626

Molecular mechanisms underlying the acquisition of stable electrical phenotypes in developing neurons remain poorly defined. As Xenopus embryonic spinal neurons mature, they initially exhibit dramatic changes in excitability due to a threefold increase in voltage-gated potassium current (IKv) density. Later when mature neurons begin synapse formation, IKv density remains stable. Elevation of Kv1.1 and Kv2.1 RNA levels indicates that excess transcript levels of these Kv genes can increase current density in both young and mature neurons. In contrast, Kv2.2 overexpression increases IKv density in young but not mature neurons despite the presence of protein translated from injected RNA at this stage. Because protein domains can determine biophysical as well as subcellular localization properties of channel subunits, we tested whether a region of the Kv2.2 subunit regulated functional expression in mature neurons. We focused on the large cytoplasmic carboxy tail, a region that differs most between Kv2.2 and the structurally related Kv2.1 subunit. Chimeric Kv2 subunits were generated in which different regions of the large cytoplasmic carboxyl tail were exchanged between Kv2.1 and Kv2.2 subunits. All chimeric Kv2 subunits induced voltage-gated potassium currents when expressed heterologously in oocytes. In vivo chimeric subunits increased IKv density in young neurons on overexpression in the developing embryo. In contrast, in mature neurons, only those chimeras lacking a domain in the proximal carboxy terminus, proxC, increased IKv density when overexpressed. Thus the proxC domain mediates developmental and subunit-specific regulation of IKv and identifies a novel function for protein domains.

Developmental, Molecular, and Genetic Dissection of INa in Vivo in Embryonic Zebrafish Sensory Neurons

Journal of Neurophysiology. Jun, 2005  |  Pubmed ID: 15673553

The presence of multiple Nav1 isotypes within a neuron and the lack of specific blockers hamper identification of the in vivo roles of sodium current (INa) components, especially during embryonic stages. To identify the functional properties of INa components in vivo in developing neurons, we took a molecular genetic approach. Embryonic zebrafish Rohon-Beard (RB) mechanosensory neurons express two different sodium channel isotypes: Nav1.1 and Nav1.6. To examine the properties of Nav1.1- and Nav1.6-encoded currents in RB cells at different developmental stages, we eliminated the contribution of Nav1.6 and Nav1.1 channels, respectively, using an antisense morpholino (MO) approach. MOs were injected into one-cell stage embryos, and RB sodium currents were recorded using patch-clamp techniques in both conventional whole cell mode as well from nucleated patches. Only a subset of RB cells appeared to be affected by the Nav1.1MO. Overall, the effect of the Nav1.1MO was a small 25% average reduction in current amplitude. Further, Nav1.1MO effects were most pronounced in RB cells of younger embryos. In contrast, the effects of the Nav1.6 MO were observed in all cells and increased as development proceeded. These results indicated that developmental upregulation of RB INa entailed an increase in the number of functional Nav1.6 channels. In addition, analysis of voltage-dependent steady-state activation and inactivation parameters revealed that specific functional properties of channels were also developmentally regulated. Finally, analysis of macho mutants indicated that developmental upregulation of INa was absent in RB cells. These results indicate that MOs are a useful tool for the molecular dissection and analysis of ion channel function in vivo.

Embryonic and Larval Expression of Zebrafish Voltage-gated Sodium Channel Alpha-subunit Genes

Developmental Dynamics : an Official Publication of the American Association of Anatomists. Jul, 2006  |  Pubmed ID: 16615064

Whereas it is known that voltage-gated calcium channels play important roles during development, potential embryonic roles of voltage-gated sodium channels have received much less attention. Voltage-gated sodium channels consist of pore-forming alpha-subunits (Na(v)1) and auxiliary beta-subunits. Here, we report the embryonic and larval expression patterns for all eight members of the gene family (scna) coding for zebrafish Na(v)1 proteins. We find that each scna gene displays a distinct expression pattern that is temporally and spatially dynamic during embryonic and larval stages. Overall, our findings indicate that scna gene expression occurs sufficiently early during embryogenesis to play developmental roles for both muscle and nervous tissues.

Gene Duplications and Evolution of Vertebrate Voltage-gated Sodium Channels

Journal of Molecular Evolution. Aug, 2006  |  Pubmed ID: 16830092

Voltage-gated sodium channels underlie action potential generation in excitable tissue. To establish the evolutionary mechanisms that shaped the vertebrate sodium channel alpha-subunit (SCNA) gene family and their encoded Nav1 proteins, we identified all SCNA genes in several teleost species. Molecular cloning revealed that teleosts have eight SCNA genes, compared to ten in another vertebrate lineage, mammals. Prior phylogenetic analyses have indicated that the genomes of both teleosts and tetrapods contain four monophyletic groups of SCNA genes, and that tandem duplications expanded the number of genes in two of the four mammalian groups. However, the number of genes in each group varies between teleosts and tetrapods, suggesting different evolutionary histories in the two vertebrate lineages. Our findings from phylogenetic analysis and chromosomal mapping of Danio rerio genes indicate that tandem duplications are an unlikely mechanism for generation of the extant teleost SCNA genes. Instead, analyses of other closely mapped genes in D. rerio as well as of SCNA genes from several teleost species all support the hypothesis that a whole-genome duplication was involved in expansion of the SCNA gene family in teleosts. Interestingly, despite their different evolutionary histories, mRNA analyses demonstrated a conservation of expression patterns for SCNA orthologues in teleosts and tetrapods, suggesting functional conservation.

Knockdown of Nav1.6a Na+ Channels Affects Zebrafish Motoneuron Development

Development (Cambridge, England). Oct, 2006  |  Pubmed ID: 16943272

In addition to rapid signaling, electrical activity provides important cues to developing neurons. Electrical activity relies on the function of several different types of voltage-gated ion channels. Whereas voltage-gated Ca2+ channel activity regulates several aspects of neuronal differentiation, much less is known about developmental roles of voltage-gated Na+ channels, essential mediators of electrical signaling. Here, we focus on the zebrafish Na+ channel isotype, Nav1.6a, which is encoded by the scn8a gene. A restricted set of spinal neurons, including dorsal sensory Rohon-Beard cells, two motoneuron subtypes with different axonal trajectories, express scn8a during embryonic development. CaP, an early born primary motoneuron subtype with ventrally projecting axons expresses scn8a, as does a class of secondary motoneurons with axons that project dorsally. To test for developmental roles of scn8a, we knocked down Nav1.6a protein using antisense morpholinos. Na+ channel protein and current amplitudes were reduced in neurons that express scn8a. Furthermore, Nav1.6a knockdown altered axonal morphologies of some but not all motoneurons. Dorsally projecting secondary motoneurons express scn8a and displayed delayed axonal outgrowth. By contrast, CaP axons developed normally, despite expression of the gene. Surprisingly, ventrally projecting secondary motoneurons, a population in which scn8a was not detected, displayed aberrant axonal morphologies. Mosaic analysis indicated that effects on ventrally projecting secondary motoneurons were non cell-autonomous. Thus, voltage-gated Na+ channels play cell-autonomous and non cell-autonomous roles during neuronal development.

Dorsal-ventral Gradient for Neuronal Plasticity in the Embryonic Spinal Cord

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Apr, 2008  |  Pubmed ID: 18385340

Within the developing Xenopus spinal cord, voltage-gated potassium (Kv) channel genes display different expression patterns, many of which occur in opposing dorsal-ventral gradients. Regional differences in Kv gene expression would predict different patterns of potassium current (I(Kv)) regulation. However, during the first 24 h of postmitotic differentiation, all primary spinal neurons undergo a temporally coordinated upregulation of I(Kv) density that shortens the duration of the action potential. Here, we tested whether spinal neurons demonstrate regional differences in I(Kv) regulation subsequent to action potential maturation. We show that two types of neurons, I and II, can be identified in culture on the basis of biophysical and pharmacological properties of I(Kv) and different firing patterns. Chronic increases in extracellular potassium, a signature of high neuronal activity, do not alter excitability properties of either neuron type. However, elevating extracellular potassium acutely after the period of action potential maturation leads to different changes in membrane properties of the two types of neurons. I(Kv) of type I neurons gains sensitivity to the blocker XE991, whereas type II neurons increase I(Kv) density and fire fewer action potentials. Moreover, by recording from neurons in vivo, we found that primary spinal neurons can be identified as either type I or type II. Type I neurons predominate in dorsal regions, whereas type II neurons localize to ventral regions. The findings reveal a dorsal-ventral gradient for I(Kv) regulation and a novel form of neuronal plasticity in spinal cord neurons.

Localization of Kv2.2 Protein in Xenopus Laevis Embryos and Tadpoles

The Journal of Comparative Neurology. Oct, 2008  |  Pubmed ID: 18680201

Voltage-gated potassium (Kv) channels sculpt neuronal excitability and play important developmental roles. Kv channels consist of pore-forming alpha- and auxiliary subunits. For many Kv alpha-subunits, existing mRNA probes and antibodies have allowed analysis of expression patterns, typically during adult stages. Here, we focus on the Kv2.2 alpha-subunit, for which the mRNA shows broad expression in the embryo and adult. A lack of suitable antibodies, however, has hindered detailed analysis of Kv2.2 protein localization, especially during development. We developed an antibody that specifically recognizes Kv2.2 protein in Xenopus laevis, a vertebrate well suited for study of early developmental stages. The Kv2.2 antibody recognized heterologously expressed Kv2.2 but not the closely related Kv2.1 protein. Immunodetection of the protein showed its presence at St 32 in ventrolateral regions of the hindbrain and spinal cord. At later stages, several sensory tissues (retina, otic, and olfactory epithelia) also expressed Kv2.2 protein. As development progressed in the central nervous system, Kv2.2 protein distribution expanded in close association with the cytoskeletal marker alpha-tubulin, consistent with growth of neuronal tracts. We analyzed the subcellular distribution of Kv2.2 protein within single cultured neurons. In addition to a surface membrane presence, Kv2.2 protein also resided intracellularly closely associated with alpha-tubulin, as in vivo. Furthermore, in contrast to Kv2.1, Kv2.2 protein localized to long, axonal-like processes, consistent with its in vivo location in tracts. Despite their primary sequence similarity, the contrasting localizations of Kv2.1 and Kv2.2 support different roles for the two during development and neuronal signaling.

Kv1 Potassium Channel Complexes in Vivo Require Kvbeta2 Subunits in Dorsal Spinal Neurons

Journal of Neurophysiology. Oct, 2008  |  Pubmed ID: 18684900

Whereas Kvbeta2 subunits modulate potassium current properties carried by Kv1 channel complexes in heterologous systems, little is known about the contributions of Kvbeta2 subunits to native potassium channel function. Using antisense approaches and in situ recordings from Xenopus embryo spinal cord neurons, we tested the in vivo roles of Kvbeta2 subunits in modulation of voltage-dependent potassium current (IKv). We focused on 1) two different populations of dorsal spinal neurons that express both Kvbeta2 and Kv1 alpha-subunit genes and 2) the 24- and 48-h developmental period, during which IKv undergoes developmental regulation. At both 24 and 48 h, antisense methods produced efficient knock-down of both Kvbeta2 protein and IKv. At both times, dominant negative suppression of Kv1 channels also eliminated IKv, indicating that Kv1 channels require Kvbeta2 subunits to function in dorsal spinal neurons. Even though Kv1 channels determined the IKv values of both dorsal neuron types, comparisons of their IKv properties revealed important differences at both developmental stages. The latter results support the notion that different Kv1 alpha-subunits and/or posttranslational modifications underlie the IKv values of the two dorsal neuron types. Overall, the results demonstrate that Kvbeta2 subunits function in vivo as obligatory subunits of Kv1 channels in at least two neuron types and two different developmental stages.

Sensory Neuron Sodium Current Requires Nongenomic Actions of Thyroid Hormone During Development

Journal of Neurophysiology. Nov, 2008  |  Pubmed ID: 18799597

Development of the embryonic nervous system requires thyroid hormone. However, the underlying mechanisms and targets of thyroid hormone action are not well defined. To identify embryonic roles for thyroid hormone we tested for effects on a key neuronal trait, voltage-gated sodium current (I(Na)), in the zebrafish model system. We recorded from Rohon-Beard sensory neurons (RBs) using whole cell voltage-clamp methods. Here, we provide in vivo evidence for thyroid hormone regulation of I(Na). Chronic thyroid hormone application increased RB peak I(Na) density 1.4-fold. However, I(Na) density showed a similar increase within 5 min of an acute hormone application, a time course not expected for a genomic mechanism. Tetraiodothyroacetic acid (tetrac), a thyroid hormone blocker, blocked both chronic and acute effects. Further, the thyroid hormone precursor thyroxine (T4) affected I(Na), yet the traditionally active form triiodothyronine did not. Consequently, we tested for a nonconventional T4 receptor. LM609, a selective antagonist of integrin alphaVbeta3, occluded the rapid effect of T4, implicating a specific integrin dimer as a T4 receptor. Chronic application of either tetrac or LM609 significantly reduced sodium conductance, demonstrating an in vivo requirement for T4-integrin regulation of I(Na). Further, removing endogenous T4 levels via yolkectomy reduced sodium conductance, an effect that was partially rescued by T4 supplementation following surgery. Because RBs mediate the embryonic touch response, we tested for behavioral effects. Tetrac and LM609 significantly reduced the percentage of touch trials eliciting a normal touch response. T4's rapid effect on RB I(Na) highlights the importance of embryonic T4 availability and nongenomic T4 signaling.

Scn1bb, a Zebrafish Ortholog of SCN1B Expressed in Excitable and Nonexcitable Cells, Affects Motor Neuron Axon Morphology and Touch Sensitivity

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Nov, 2008  |  Pubmed ID: 19020043

Voltage-gated Na(+) channels initiate and propagate action potentials in excitable cells. Mammalian Na(+) channels are composed of one pore-forming alpha-subunit and two beta-subunits. SCN1B encodes the Na(+) channel beta1-subunit that modulates channel gating and voltage dependence, regulates channel cell surface expression, and functions as a cell adhesion molecule (CAM). We recently identified scn1ba, a zebrafish ortholog of SCN1B. Here we report that zebrafish express a second beta1-like paralog, scn1bb. In contrast to the restricted expression of scn1ba mRNA in excitable cells, we detected scn1bb transcripts and protein in several ectodermal derivatives including neurons, glia, the lateral line, peripheral sensory structures, and tissues derived from other germ layers such as the pronephros. As expected for beta1-subunits, elimination of Scn1bb protein in vivo by morpholino knock-down reduced Na(+) current amplitudes in Rohon-Beard neurons of zebrafish embryos, consistent with effects observed in heterologous systems. Further, after Scn1bb knock-down, zebrafish embryos displayed defects in Rohon-Beard mediated touch sensitivity, demonstrating the significance of Scn1bb modulation of Na(+) current to organismal behavior. In addition to effects associated with Na(+) current modulation, Scn1bb knockdown produced phenotypes consistent with CAM functions. In particular, morpholino knock-down led to abnormal development of ventrally projecting spinal neuron axons, defasciculation of the olfactory nerve, and increased hair cell number in the inner ear. We propose that, in addition to modulation of electrical excitability, Scn1bb plays critical developmental roles by functioning as a CAM in the zebrafish embryonic nervous system.

Molecular Components Underlying Nongenomic Thyroid Hormone Signaling in Embryonic Zebrafish Neurons

Neural Development. Jun, 2009  |  Pubmed ID: 19505305

Neurodevelopment requires thyroid hormone, yet the mechanisms and targets of thyroid hormone action during embryonic stages remain ill-defined. We previously showed that the thyroid hormone thyroxine (T4) rapidly increases voltage-gated sodium current in zebrafish Rohon-Beard cells (RBs), a primary sensory neuron subtype present during embryonic development. Here, we determined essential components of the rapid T4 signaling pathway by identifying the involved intracellular messengers, the targeted sodium channel isotype, and the spatial and temporal expression pattern of the nongenomic alphaVbeta3 integrin T4 receptor.

Zebrafish Motor Neuron Subtypes Differ Electrically Prior to Axonal Outgrowth

Journal of Neurophysiology. Oct, 2009  |  Pubmed ID: 19692510

Different muscle targets and transcription factor expression patterns reveal the presence of motor neuron subtypes. However, it is not known whether these subtypes also differ with respect to electrical membrane properties. To address this question, we studied primary motor neurons (PMNs) in the spinal cord of zebrafish embryos. PMN genesis occurs during gastrulation and gives rise to a heterogeneous set of motor neurons that differ with respect to transcription factor expression, muscle targets, and soma location within each spinal cord segment. The unique subtype-specific soma locations and axonal trajectories of two PMNs-MiP (middle) and CaP (caudal)-allowed their identification in situ as early as 17 h postfertilization (hpf), prior to axon genesis. Between 17 and 48 hpf, CaPs and MiPs displayed subtype-specific electrical membrane properties. Voltage-dependent inward and outward currents differed significantly between MiPs and CaPs. Moreover, by 48 hpf, CaPs and MiPs displayed subtype-specific firing behaviors. Our results demonstrate that motor neurons that differ with respect to muscle targets and transcription factor expression acquire subtype-specific electrical membrane properties. Moreover, the differences are evident prior to axon genesis and persist to the latest stage studied, 2 days postfertilization.

Developmental Regulation of Subtype-specific Motor Neuron Excitability

Annals of the New York Academy of Sciences. Jun, 2010  |  Pubmed ID: 20536935

At early embryonic stages, zebrafish spinal neuron subtypes can be distinguished and accessed for physiological studies. This provides the opportunity to determine electrophysiological properties of different spinal motor neuron subtypes. Such differences have the potential to then regulate, in a subtype-specific manner, activity-dependent developmental events such as axonal outgrowth and pathfinding. The zebrafish spinal cord contains a population of early born neurons. Our recent work has revealed that primary motor neuron (PMN) subtypes in the zebrafish spinal cord differ with respect to electrical properties during early important periods when PMNs extend axons to their specific targets. Here, we review recent findings regarding the development of electrical properties in PMN subtypes. Moreover, we consider the possibility that electrical activity in PMNs may play a cell nonautonomous role and thus influence the development of later developing motor neurons. Further, we discuss findings that support a role for a specific sodium channel isoform, Nav1.6, expressed by specific subtypes of spinal neurons in activity-dependent processes that impact axonal outgrowth and pathfinding.

In Vivo Evidence for Transdifferentiation of Peripheral Neurons

Development (Cambridge, England). Sep, 2010  |  Pubmed ID: 20685733

It is commonly thought that differentiated neurons do not give rise to new cells, severely limiting the potential for regeneration and repair of the mature nervous system. However, we have identified cells in zebrafish larvae that first differentiate into dorsal root ganglia sensory neurons but later acquire a sympathetic neuron phenotype. These transdifferentiating neurons are present in wild-type zebrafish. However, they are increased in number in larvae that have a mutant voltage-gated sodium channel gene, scn8aa. Sodium channel knock-down promotes migration of differentiated sensory neurons away from the ganglia. Once in a new environment, sensory neurons transdifferentiate regardless of sodium channel expression. These findings reveal an unsuspected plasticity in differentiated neurons that points to new strategies for treatment of nervous system disease.

Brain-derived Neurotrophic Factor Mediates Non-cell-autonomous Regulation of Sensory Neuron Position and Identity

The Journal of Neuroscience : the Official Journal of the Society for Neuroscience. Oct, 2010  |  Pubmed ID: 20980609

During development, neurons migrate considerable distances to reside in locations that enable their individual functional roles. Whereas migration mechanisms have been extensively studied, much less is known about how neurons remain in their ideal locations. We sought to identify factors that maintain the position of postmigratory dorsal root ganglion neurons, neural crest derivatives for which migration and final position play an important developmental role. We found that an early developing population of sensory neurons maintains the position of later born dorsal root ganglia neurons in an activity-dependent manner. Further, inhibiting or increasing the function of brain-derived neurotrophic factor induces or prevents, respectively, migration of dorsal root ganglia neurons out of the ganglion to locations where they acquire a new identity. Overall, the results demonstrate that neurotrophins mediate non-cell-autonomous maintenance of position and thereby the identity of differentiated neurons.

Genetic Analysis of the Touch Response in Zebrafish (Danio Rerio)

International Journal of Comparative Psychology. Mar, 2010  |  Pubmed ID: 26456999

Both mammals and zebrafish possess mechanosensory neurons that detect tactile sensation via free nerve endings. However, the basis for mechanotransduction and the unique cellular properties of these sensory neurons are poorly understood. We review the advantages of zebrafish for studies of the biological mechanisms involved in touch sensitivity. Importantly, Granato and colleagues (1996) demonstrated that a simple touch assay efficiently recovers mutations that affect sensory neurons.

Disruption of Eaat2b, a Glutamate Transporter, Results in Abnormal Motor Behaviors in Developing Zebrafish

Developmental Biology. Feb, 2012  |  Pubmed ID: 22094018

Analysis of zebrafish mutants that have defects in motor behavior can allow entrée into the hindbrain and spinal cord networks that control locomotion. Here, we report that zebrafish techno trousers (tnt) locomotor mutants harbor a mutation in slc1a2b, which encodes Eaat2b, a plasma membrane glutamate transporter. We used tnt mutants to explore the effects of impaired glutamate transporter activity on locomotor network function. Wild-type larvae perform robust swimming behavior in response to touch stimuli at two and four days after fertilization. In contrast, tnt mutant larvae demonstrate aberrant, exaggerated body bends beginning two days after fertilization and they are almost paralyzed four days after fertilization. We show that slc1a2b is expressed in glial cells in a dynamic fashion across development, which may explain the abnormal sequence of motor behaviors demonstrated by tnt mutants. We also show that tnt larvae demonstrate enhanced excitation of neurons, consistent with the predicted effects of excessive glutamate. These findings illustrate the dynamic regulation and importance of glutamate transporters during development. Since glutamate toxicity caused by EAAT2 dysfunction is thought to promote several different neurological disorders in humans, including epilepsy and neurodegenerative diseases, tnt mutants hold promise as a new tool to better understand these pathologies.

α3Na+/K+-ATPase Deficiency Causes Brain Ventricle Dilation and Abrupt Embryonic Motility in Zebrafish

The Journal of Biological Chemistry. Mar, 2013  |  Pubmed ID: 23400780

Na(+)/K(+)-ATPases are transmembrane ion pumps that maintain ion gradients across the basolateral plasma membrane in all animal cells to facilitate essential biological functions. Mutations in the Na(+)/K(+)-ATPase α3 subunit gene (ATP1A3) cause rapid-onset dystonia-parkinsonism, a rare movement disorder characterized by sudden onset of dystonic spasms and slow movements. In the brain, ATP1A3 is principally expressed in neurons. In zebrafish, the transcripts of the two ATP1A3 orthologs, Atp1a3a and Atp1a3b, show distinct expression in the brain. Surprisingly, targeted knockdown of either Atp1a3a or Atp1a3b leads to brain ventricle dilation, a likely consequence of ion imbalances across the plasma membrane that cause accumulation of cerebrospinal fluid in the ventricle. The brain ventricle dilation is accompanied by a depolarization of spinal Rohon-Beard neurons in Atp1a3a knockdown embryos, suggesting impaired neuronal excitability. This is further supported by Atp1a3a or Atp1a3b knockdown results where altered responses to tactile stimuli as well as abnormal motility were observed. Finally, proteomic analysis identified several protein candidates highlighting proteome changes associated with the knockdown of Atp1a3a or Atp1a3b. Our data thus strongly support the role of α3Na(+)/K(+)-ATPase in zebrafish motility and brain development, associating for the first time the α3Na(+)/K(+)-ATPase deficiency with brain ventricle dilation.

Connexin 35b Expression in the Spinal Cord of Danio Rerio Embryos and Larvae

The Journal of Comparative Neurology. Mar, 2014  |  Pubmed ID: 23939687

Electrical synapses are expressed prominently in the developing and mature nervous systems. Unlike chemical synapses, little is known about the developmental role of electrical synapses, reflecting the limitations imposed by the lack of selective pharmacological blockers. At a molecular level, the building blocks of electrical synapses are connexin proteins. In this study, we report the expression pattern for neuronally expressed connexin 35b (cx35b), the zebrafish orthologue of mammalian connexin (Cx) 36. We find that cx35b is expressed at the time of neural induction, indicating a possible early role in neural progenitor cells. Additionally, cx35b localizes to the ventral spinal cord during embryonic and early larval stages. We detect cx35b mRNA in secondary motor neurons (SMNs) and interneurons. We identified the premotor circumferential descending (CiD) interneuron as one interneuron subtype expressing cx35b. In addition, cx35b is present in other ventral interneurons of unknown subtype(s). This early expression of cx35b in SMNs and CiDs suggests a possible role in motor network function during embryonic and larval stages.

Spinal Neurons Require Islet1 for Subtype-specific Differentiation of Electrical Excitability

Neural Development. Aug, 2014  |  Pubmed ID: 25149090

In the spinal cord, stereotypic patterns of transcription factor expression uniquely identify neuronal subtypes. These transcription factors function combinatorially to regulate gene expression. Consequently, a single transcription factor may regulate divergent development programs by participation in different combinatorial codes. One such factor, the LIM-homeodomain transcription factor Islet1, is expressed in the vertebrate spinal cord. In mouse, chick and zebrafish, motor and sensory neurons require Islet1 for specification of biochemical and morphological signatures. Little is known, however, about the role that Islet1 might play for development of electrical membrane properties in vertebrates. Here we test for a role of Islet1 in differentiation of excitable membrane properties of zebrafish spinal neurons.

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