Graphene oxide nanosheets modulate spinal glutamatergic transmission and modify locomotor behaviour in an in vivo zebrafish model.

Graphene oxide (GO), an oxidised form of graphene, is widely used for biomedical applications, due to its dispersibility in water and simple surface chemistry tunability. In particular, small (less than 500 nm in lateral dimension) and thin (1-3 carbon monolayers) graphene oxide nanosheets (s-GO) have been shown to selectively inhibit glutamatergic transmission in neuronal cultures in vitro and in brain explants obtained from animals injected with the nanomaterial. This raises the exciting prospect that s-GO can be developed as a platform for novel nervous system therapeutics. It has not yet been investigated whether the interference of the nanomaterial with neurotransmission may have a downstream outcome in modulation of behaviour depending specifically on the activation of those synapses. To address this problem we use early stage zebrafish as an in vivo model to study the impact of s-GO on nervous system function. Microinjection of s-GO into the embryonic zebrafish spinal cord selectively reduces the excitatory synaptic transmission of the spinal network, monitored in vivo through patch clamp recordings, without affecting spinal cell survival. This effect is accompanied by a perturbation in the swimming activity of larvae, which is the locomotor behaviour generated by the neuronal network of the spinal cord. Such results indicate that the impact of s-GO on glutamate based neuronal transmission is preserved in vivo and can induce changes in animal behaviour. These findings pave the way for use of s-GO as a modulator of nervous system function.

Motor Control: The Curious Case of Cerebrospinal-Fluid-Contacting Neurons.

Since their discovery almost a century ago, the functions of the cerebrospinal-fluid-contacting neurons have remained elusive: a new study paves the way towards understanding how these unusual spinal cord neurons regulate motor activity.

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

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.

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.

Pacemaker and plateau potentials shape output of a developing locomotor network.

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 (I(NaP)). 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.