Effects of Nitric Oxide on Neuromuscular Properties of Developing Zebrafish Embryos

Abstract

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.

NO-sGC Pathway Modulates Ca2+ Release and Muscle Contraction in Zebrafish Skeletal Muscle

“…Zebrafish express two types of NOS: NOS1 and NOS2, which are highly conserved in evolution, showing similar amino acid sequences, cofactor binding domains, specific domains for each isoform and intron position and phases (Andreakis et al, 2011 ). Although, both larvae and adult zebrafish express NOS1 in the brain and peripheral nervous system (Holmqvist et al, 2000 , 2004 ; Shin et al, 2000 ; Poon et al, 2003 ), where NO/cGMP can modulate formation and maturation of neuromuscular junctions (Jay et al, 2014 ), this isoform has not yet been characterized in skeletal muscle of the zebrafish. NOS2 is widely expressed in all zebrafish tissues in two different isoforms: NOS2a, which is expressed after LPS injection in adult zebrafish and NOS2b, which presents a N-terminal myristoylation sequence and is constitutively expressed in all tested tissues, suggesting that NOS2a behaves as a classical iNOS and NOS2b is similar to the mammalian eNOS (Lepiller et al, 2009 ).…”

“…Based on previous works, we hypothesized that in skeletal muscle of zebrafish larvae NO exerts its effects on Ca 2+ homeostasis during muscle contraction mainly through negative modulation of Ca 2+ release and Ca 2+ uptake via the NO-sGC-PKG pathway. To address this, we used 5–7 days-post fecundation-larvae of zebrafish, a well-established animal model to study formation, development, and motor innervation as well as to comprehend molecular and cellular aspect of several myopathies (Schredelseker et al, 2010 ; Muto et al, 2011 ; Jay et al, 2014 ). In our study, we observed that nNOS is expressed in skeletal muscle and the Ca 2+ transients recorded were limited to the sarcolemmal.…”

Characterisation and comparison of temporal release profiles of nitric oxide generating donors

“…Unlike conventional neurotransmitters, NO is not constrained by cellular membranes and diffuses in three dimensions from its source of production ( Garthwaite and Boulton, 1995 ). Particularly in the nervous system, the concentration gradient associated with this diffusion is important in signalling mechanisms such as regulation of plasticity ( Hardingham and Fox, 2006; Hardingham et al, 2013 ) and development ( Bradley et al, 2010; Jay et al, 2014 ). Among the many unknowns are the exact levels of NO generated by different sources ( i .…”

Nitrate (NO3−) and nitrite (NO2−) are endocrine disruptors to downregulate expression of tyrosine hydroxylase and motor behavior through conversion to nitric oxide in early development of zebrafish

“…Dopamine receptor antagonist decreases the generation of motor neuron and reduces the response to tail-touch response test and swimming activity, suggesting those behaviors are under control of DA neuron [35]. On the other hand, it has been reported in zebrafish that NO signaling is involved in neuromuscular development and locomotor maturation [36], and perturbation of NO signaling affects the ontogeny of locomotor performance [37]. Although further studies are required to clarify whether NO produced by exposure to nitrate and nitrite acts directly on motor neurons, our data reveal that nitrate and nitrite act as an environmental toxicant on DA neurons.…”