Long-lived zebrafish Rohon-Beard cells.

During normal development of the nervous system, extensive neuronal proliferation as well as death occurs. The extent of development death varies considerably between neuronal populations from little to almost 100%. Early born somatosensory neurons, known as Rohon-Beard cells, have served as an example of neurons that disappear during early developmental stages, presumably as their function is taken over by later developing dorsal root ganglion neurons. However, recent studies have raised questions about the extent to which zebrafish Rohon-Beard cells die during embryogenesis. While Rohon-Beard cells have distinguishing morphological features during embryonic stages development, they subsequently undergo substantial changes in their shape, size and position that hinder their unambiguous identification at later stages. To overcome this obstacle, we identify Rohon-Beard cells at one day, and using a combination of mosaic and stable transgenic labeling and repeated observation, follow them for 13-16 days post fertilization. We find that about 40% survive to late larval stages. Our studies also reveal that Rohon-Beard cells display an unusual repertoire of cell death properties. At one day, about 25% Rohon-Beard cells expose phosphatidyl serine at the surface membrane, but less than one Rohon-Beard cell/embryo expresses activated-caspase-3. Further, the temporal delay between detection of cell death markers and loss of the soma ranges from

Zebrafish as a Model System for the Study of Severe CaV2.1 (α1A) Channelopathies.

The P/Q-type CaV2.1 channel regulates neurotransmitter release at neuromuscular junctions (NMJ) and many central synapses. CACNA1A encodes the pore-containing α1A subunit of CaV2.1 channels. In humans, de novo CACNA1A mutations result in a wide spectrum of neurological, neuromuscular, and movement disorders, such as familial hemiplegic migraine type 1 (FHM1), episodic ataxia type 2 (EA2), as well as a more recently discovered class of more severe disorders, which are characterized by ataxia, hypotonia, cerebellar atrophy, and cognitive/developmental delay. Heterologous expression of CaV2.1 channels has allowed for an understanding of the consequences of CACNA1A missense mutations on channel function. In contrast, a mechanistic understanding of how specific CACNA1A mutations lead in vivo to the resultant phenotypes is lacking. In this review, we present the zebrafish as a model to both study in vivo mechanisms of CACNA1A mutations that result in synaptic and behavioral defects and to screen for effective drug therapies to combat these and other CaV2.1 channelopathies.

Investigation of Islet2a function in zebrafish embryos: Mutants and morphants differ in morphologic phenotypes and gene expression.

Zebrafish primary motor neurons differ from each other with respect to morphology, muscle targets and electrophysiological properties. For example, CaP has 2-3-fold larger densities of both inward and outward currents than do other motor neurons. We tested whether the transcription factor Islet2a, uniquely expressed in CaP, but not other primary motor neurons, plays a role in specifying its stereotypic electrophysiological properties. We used both TALEN-based gene editing and antisense morpholino approaches to disrupt Islet2a function. Our electrophysiology results do not support a specific role for Islet2a in determining CaP's unique electrical properties. However, we also found that the morphological phenotypes of CaP and a later-born motor neuron differed between islet2a mutants and morphants. Using microarrays, we tested whether the gene expression profiles of whole embryo morphants, mutants and controls also differed. Morphants had 174 and 201 genes that were differentially expressed compared to mutants and controls, respectively. Further, islet2a was identified as a differentially expressed gene. To examine how mutation of islet2a affected islet gene expression specifically in CaPs, we performed RNA in situ hybridization. We detected no obvious differences in expression of islet1, islet2a, or islet2b in CaPs of mutant versus sibling control embryos. However, immunolabeling studies revealed that an Islet protein persisted in CaPs of mutants, albeit at a reduced level compared to controls. While we cannot exclude requirement for some Islet protein, we conclude that differentiation of the CaP's stereotypic large inward and outward currents does not have a specific requirement for Islet2a.

Zebrafish In Situ Spinal Cord Preparation for Electrophysiological Recordings from Spinal Sensory and Motor Neurons.

Zebrafish, first introduced as a developmental model, have gained popularity in many other fields. The ease of rearing large numbers of rapidly developing organisms, combined with the embryonic optical clarity, served as initial compelling attributes of this model. Over the past two decades, the success of this model has been further propelled by its amenability to large-scale mutagenesis screens and by the ease of transgenesis. More recently, gene-editing approaches have extended the power of the model. For neurodevelopmental studies, the zebrafish embryo and larva provide a model to which multiple methods can be applied. Here, we focus on methods that allow the study of an essential property of neurons, electrical excitability. Our preparation for the electrophysiological study of zebrafish spinal neurons involves the use of veterinarian suture glue to secure the preparation to a recording chamber. Alternative methods for recording from zebrafish embryos and larvae involve the attachment of the preparation to the chamber using a fine tungsten pin1,2,3,4,5. A tungsten pin is most often used to mount the preparation in a lateral orientation, although it has been used to mount larvae dorsal-side up4. The suture glue has been used to mount embryos and larvae in both orientations. Using the glue, a minimal dissection can be performed, allowing access to spinal neurons without the use of an enzymatic treatment, thereby avoiding any resultant damage. However, for larvae, it is necessary to apply a brief enzyme treatment to remove the muscle tissue surrounding the spinal cord. The methods described here have been used to study the intrinsic electrical properties of motor neurons, interneurons, and sensory neurons at several developmental stages6,7,8,9.

Spinal neurons require Islet1 for subtype-specific differentiation of electrical excitability.

BACKGROUND: 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. RESULTS: We focus our studies on the role of Islet1 in two populations of early born zebrafish spinal neurons: ventral caudal primary motor neurons (CaPs) and dorsal sensory Rohon-Beard cells (RBs). We take advantage of transgenic lines that express green fluorescent protein (GFP) to identify CaPs, RBs and several classes of interneurons for electrophysiological study. Upon knock-down of Islet1, cells occupying CaP-like and RB-like positions continue to express GFP. With respect to voltage-dependent currents, CaP-like and RB-like neurons have novel repertoires that distinguish them from control CaPs and RBs, and, in some respects, resemble those of neighboring interneurons. The action potentials fired by CaP-like and RB-like neurons also have significantly different properties compared to those elicited from control CaPs and RBs. CONCLUSIONS: Overall, our findings suggest that, for both ventral motor and dorsal sensory neurons, Islet1 directs differentiation programs that ultimately specify electrical membrane as well as morphological properties that act together to sculpt neuron identity.

Connexin 35b expression in the spinal cord of Danio rerio embryos and larvae.

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.

α3Na+/K+-ATPase deficiency causes brain ventricle dilation and abrupt embryonic motility in zebrafish.

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.

Disruption of Eaat2b, a glutamate transporter, results in abnormal motor behaviors in developing zebrafish.

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.

Brain-derived neurotrophic factor mediates non-cell-autonomous regulation of sensory neuron position and identity.

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.

In vivo evidence for transdifferentiation of peripheral neurons.

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.

Developmental regulation of subtype-specific motor neuron excitability.

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.

Genetic Analysis of the Touch Response in Zebrafish (Danio rerio).

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.

Zebrafish motor neuron subtypes differ electrically prior to axonal outgrowth.

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.

Molecular components underlying nongenomic thyroid hormone signaling in embryonic zebrafish neurons.

BACKGROUND: 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. RESULTS: We first tested which signaling pathways mediate T4's rapid modulation of sodium current (I(Na)) by perturbing specific pathways associated with nongenomic thyroid hormone signaling. We found that pharmacological blockade of protein phosphatase 1 and the mitogen-activated protein kinase p38 isoform decreased and increased tonic sodium current amplitudes, respectively, and blockade of either occluded rapid responses to acute T4 application. We next tested for the ion channel target of rapid T4 signaling via morpholino knock-down of specific sodium channel isotypes. We found that selective knock-down of the sodium channel alpha-subunit Na(v)1.6a, but not Na(v)1.1la, occluded T4's acute effects. We also determined the spatial and temporal distribution of a nongenomic T4 receptor, integrin alphaVbeta3. At 24 hours post fertilization (hpf), immunofluorescent assays showed no specific integrin alphaVbeta3 immunoreactivity in wild-type zebrafish embryos. However, by 48 hpf, embryos expressed integrin alphaVbeta3 in RBs and primary motoneurons. Consistent with this temporal expression, T4 modulated RB I(Na) at 48 but not 24 hpf. We next tested whether T4 rapidly modulated I(Na) of caudal primary motoneurons, which express the receptor (alphaVbeta3) and target (Na(v)1.6a) of rapid T4 signaling. In response to T4, caudal primary motoneurons rapidly increased sodium current peak amplitude 1.3-fold. CONCLUSION: T4's nongenomic regulation of sodium current occurs in different neuronal subtypes, requires the activity of specific phosphorylation pathways, and requires both integrin alphaVbeta3 and Na(v)1.6a. Our in vivo analyses identify molecules required for T4's rapid regulation of voltage-gated sodium current.

scn1bb, a zebrafish ortholog of SCN1B expressed in excitable and nonexcitable cells, affects motor neuron axon morphology and touch sensitivity.

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.

Sensory neuron sodium current requires nongenomic actions of thyroid hormone during development.

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.

Localization of Kv2.2 protein in Xenopus laevis embryos and tadpoles.

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.

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.

Dorsal-ventral gradient for neuronal plasticity in the embryonic spinal cord.

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.