Jonathan R. McDearmid

Development of the locomotor network in zebrafish

The zebrafish is a leading model for studies of vertebrate development and genetics. Its embryonic motor behaviors are easy to assess (e.g. for mutagenic screens), the embryos develop rapidly (hatching as larvae at 2 days) and are transparent, permitting calcium imaging and patch clamp recording in vivo. We review primarily the recent advances in understanding the cellular basis for the development of motor activities in the developing zebrafish. The motor activities are generated largely in the spinal cord and hindbrain. In the embryo these segmented structures possess a relatively small number of repeating sets of identifiable neurons. Many types of neurons as well as the two types of muscle cells have been classified based on their morphologies. Some of the molecular signals for cellular differentiation have been identified recently and mutations affecting cell development have been isolated. Embryonic motor behaviors appear in sequence and consist of an early period of transient spontaneous coiling contractions, followed by the emergence of twitching responses to touch, and later by the ability to swim. Coiling contractions are generated by an electrically coupled network of a subset of spinal neurons whereas a chemical (glutamatergic and glycinergic) synaptic drive underlies touch responses and swimming. Swimming becomes sustained in larvae once the neuromodulatory serotonergic system develops. These results indicate many similarities between developing zebrafish and other vertebrates in the properties of the synaptic drive underlying locomotion. Therefore, the zebrafish is a useful preparation for gaining new insights into the development of the neural control of vertebrate locomotion. As the types of neurons, transmitters, receptors and channels used in the locomotor network are being defined, this opens the possibility of combining cellular neurophysiology with forward and reverse molecular genetics to understand the principles of locomotor network assembly and function.

Steps during the development of the zebrafish locomotor network.

This review summarizes recent data from our lab concerning the development of motor activities in the developing zebrafish. The zebrafish is a leading model for studies of vertebrate development because one can obtain a large number of transparent, externally and rapidly developing embryos with motor behaviors that are easy to assess (e.g. for mutagenic screens). The emergence of embryonic motility was studied behaviorally and at the cellular level. The embryonic behaviors appear sequentially and include an early, transient period of spontaneous, alternating tail coilings, followed by responses to touch, and swimming. Patch clamp recording in vivo revealed that an electrically coupled network of a subset of spinal neurons generates spontaneous tail coiling, whereas a chemical (glutamatergic and glycinergic) synaptic drive underlies touch responses and swimming and requires input from the hindbrain. Swimming becomes sustained in larvae once serotonergic neuromodulatory effects are integrated. We end with a brief overview of the genetic tools available for the study of the molecular determinants implicated in locomotor network development in the zebrafish. Combining genetic, behavioral and cellular experimental approaches will advance our understanding of the general principles of locomotor network assembly and function.

Early interneuron dysfunction in ALS: Insights from a mutant sod1 zebrafish model

To determine, when, how, and which neurons initiate the onset of pathophysiology in amyotrophic lateral sclerosis (ALS) using a transgenic mutant sod1 zebrafish model and identify neuroprotective drugs.

Investigating the contribution of VAPB/ALS8 loss of function in amyotrophic lateral sclerosis

The mutations P56S and T46I in the gene encoding vesicle-associated membrane protein-associated protein B/C (VAPB) cause ALS8, a familial form of amyotrophic lateral sclerosis (ALS). Overexpression of mutant forms of VAPB leads to cytosolic aggregates, suggesting a gain of function of the mutant protein. However, recent work suggested that the loss of VAPB function could be the major mechanism leading to ALS8. Here, we used multiple genetic and experimental approaches to study whether VAPB loss of function might be sufficient to trigger motor neuron degeneration. In order to identify additional ALS-associated VAPB mutations, we screened the entire VAPB gene in a cohort of ALS patients and detected two mutations (A145V and S160Δ). To directly address the contribution of VAPB loss of function in ALS, we generated zebrafish and mouse models with either a decreased or a complete loss of Vapb expression. Vapb knockdown in zebrafish led to swimming deficits. Mice knocked-out for Vapb showed mild motor deficits after 18 months of age yet had innervated neuromuscular junctions (NMJs). Importantly, overexpression of VAPB mutations were unable to rescue the motor deficit caused by Vapb knockdown in zebrafish and failed to cause a toxic gain-of-function defect on their own. Thus, Vapb loss of function weakens the motor system of vertebrate animal models but is on its own unable to lead to a complete ALS phenotype. Our findings are consistent with the notion that VAPB mutations constitute a risk factor for motor neuron disease through a loss of VAPB function.

Als2 mRNA splicing variants detected in KO mice rescue severe motor dysfunction phenotype in Als2 knock-down zebrafish

Recessive ALS2 mutations are linked to three related but slightly different neurodegenerative disorders: amyotrophic lateral sclerosis, hereditary spastic paraplegia and primary lateral sclerosis. To investigate the function of the ALS2 encoded protein, we generated Als2 knock-out (KO) mice and zAls2 knock-down zebrafish. The Als2(-/-) mice lacking exon 2 and part of exon 3 developed mild signs of neurodegeneration compatible with axonal transport deficiency. In contrast, zAls2 knock-down zebrafish had severe developmental abnormalities, swimming deficits and motor neuron perturbation. We identified, by RT-PCR, northern and western blotting novel Als2 transcripts in mouse central nervous system. These Als2 transcripts were present in Als2 null mice as well as in wild-type littermates and some rescued the zebrafish phenotype. Thus, we speculate that the newly identified Als2 mRNA species prevent the Als2 KO mice from developing severe neurodegenerative disease and might also regulate the severity of the motor neurons phenotype observed in ALS2 patients.

Firing dynamics and modulatory actions of supraspinal dopaminergic neurons during zebrafish locomotor behavior.

Summary Background Dopamine (DA) has long been known to have modulatory effects on vertebrate motor circuits. However, the types of information encoded by supraspinal DAergic neurons and their relationship to motor behavior remain unknown. Results By conducting electrophysiological recordings from awake, paralyzed zebrafish larvae that can produce behaviorally relevant activity patterns, we show that supraspinal DAergic neurons generate two forms of output: tonic spiking and phasic bursting. Using paired supraspinal DA neuron and motoneuron recordings, we further show that these firing modes are associated with specific behavioral states. Tonic spiking is prevalent during periods of inactivity while bursting strongly correlates with locomotor output. Targeted laser ablation of supraspinal DA neurons reduces motor episode frequency without affecting basic parameters of motor output, strongly suggesting that these cells regulate spinal network excitability. Conclusions Our findings reveal how vertebrate motor circuit flexibility is temporally controlled by supraspinal DAergic pathways and provide important insights into the functional significance of this evolutionarily conserved cell population.

Pacemaker and Plateau Potentials Shape Output of a Developing Locomotor Network

Summary Background During development, spinal networks undergo an intense period of maturation in which immature forms of motor behavior are observed. Such behaviors are transient, giving way to more mature activity as development proceeds. The processes governing age-specific transitions in motor behavior are not fully understood. Results Using in vivo patch clamp electrophysiology, we have characterized ionic conductances and firing patterns of developing zebrafish spinal neurons. We find that a kernel of spinal interneurons, the ipsilateral caudal (IC) cells, generate inherent bursting activity that depends upon a persistent sodium current (INaP). We further show that developmental transitions in motor behavior are accompanied by changes in IC cell bursting: during early life, these cells generate low frequency membrane oscillations that likely drive “coiling,” an immature form of motor output. As fish mature to swimming stages, IC cells switch to a sustained mode of bursting that permits generation of high-frequency oscillations during locomotion. Finally, we find that perturbation of IC cell bursting disrupts motor output at both coiling and swimming stages. Conclusions Our results suggest that neurons with unique bursting characteristics are a fundamental component of developing motor networks. During development, these may shape network output and promote stage-specific reconfigurations in motor behavior.

Nitric Oxide Synthase Regulates Morphogenesis of Zebrafish Spinal Cord Motoneurons

Nitric oxide (NO) is a signaling molecule that is synthesized in a range of tissues by the NO synthases (NOSs). In the immature nervous system, the neuronal isoform of NOS (NOS1) is often expressed during periods of axon outgrowth and elaboration. However, there is little direct molecular evidence to suggest that NOS1 influences these processes. Here we address the functional role of NOS1 during in vivo zebrafish locomotor circuit development. We show that NOS1 is expressed in a population of interneurons that lie close to nascent motoneurons of the spinal cord. To determine how this protein regulates spinal network assembly, we perturbed NOS1 expression in vivo with antisense morpholino oligonucleotides. This treatment dramatically increased the number of axon collaterals formed by motoneuron axons, an effect mimicked by pharmacological inhibition of the NO/cGMP signaling pathway. In contrast, exogenous elevation of NO/cGMP levels suppressed motor axon branching. These effects were not accompanied by a change in motoneuron number, suggesting that NOS1 does not regulate motoneuron differentiation. Finally we show that perturbation of NO signaling affects the ontogeny of locomotor performance. Our findings provide evidence that NOS1 is a key regulator of motor axon ontogeny in the developing vertebrate spinal cord.

Effects of nitric oxide on neuromuscular properties of developing zebrafish embryos

Nitric oxide is a bioactive signalling molecule that is known to affect a wide range of neurodevelopmental processes. However, its functional relevance to neuromuscular development is not fully understood. Here we have examined developmental roles of nitric oxide during formation and maturation of neuromuscular contacts in zebrafish. Using histochemical approaches we show that elevating nitric oxide levels reduces the number of neuromuscular synapses within the axial swimming muscles whilst inhibition of nitric oxide biosynthesis has the opposite effect. We further show that nitric oxide signalling does not change synapse density, suggesting that the observed effects are a consequence of previously reported changes in motor axon branch formation. Moreover, we have used in vivo patch clamp electrophysiology to examine the effects of nitric oxide on physiological maturation of zebrafish neuromuscular junctions. We show that developmental exposure to nitric oxide affects the kinetics of spontaneous miniature end plate currents and impacts the neuromuscular drive for locomotion. Taken together, our findings implicate nitrergic signalling in the regulation of zebrafish neuromuscular development and locomotor maturation.

A zebrafish model exemplifies the long preclinical period of motor neuron disease

To the Editor, The article ‘Amyotrophic lateral sclerosis: a long preclinical period?’’ is an interesting and thought-provoking review.1 We commend the authors for raising the profile of early embryonic and preclinical stages that are currently poorly explored in amyotrophic lateral sclerosis (ALS) research. In relation to these concepts, we would like to highlight recent, important work on a zebrafish model of ALS that contributes to the concept of preclinical change. Specifically, we have developed a sod1 zebrafish model of ALS/motor neuron disease (MND) and demonstrated that zebrafish, like mice and humans, show hallmark features of ALS, suggesting that the zebrafish provides an excellent model system to study motor neuron disease.2 Additionally, the transparency and ex vivo development of embryos enables one to trace early embryonic changes in disease pathogenesis. As a follow-up to the original study, our further studies have established that the temporal changes in neuronal populations and circuitry were evident from the earliest embryonic …