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While the role of cholinergic neurotransmission from motoneurons is well established during neuromuscular development, whether it regulates central nervous system development in the spinal cord is unclear. Zebrafish presents a powerful model to investigate how the cholinergic system is set up and evolves during neural circuit formation. In this study, we carried out a detailed spatiotemporal analysis of the cholinergic system in embryonic and larval zebrafish. In 1-day-old embryos, we show that spinal motoneurons express presynaptic cholinergic genes including choline acetyltransferase ( chata ) , vesicular acetylcholine transporters ( vachta , vachtb ) , high-affinity choline transporter ( hacta ) and acetylcholinesterase ( ache ), while nicotinic acetylcholine receptor (nAChR) subunits are mainly expressed in interneurons. However, in 3-day-old embryos, we found an unexpected decrease in presynaptic cholinergic transcript expression in a rostral to caudal gradient in the spinal cord, which continued during development. On the contrary, nAChR subunits remained highly expressed throughout the spinal cord. We found that protein and enzymatic activities of presynaptic cholinergic genes were also reduced in the rostral spinal cord. Our work demonstrating that cholinergic genes are initially expressed in the embryonic spinal cord, which is dynamically downregulated during development suggests that cholinergic signaling may play a pivotal role during the formation of intra-spinal locomotor circuit.

Since the discovery of olfactory receptor (OR) genes, their expression in non-olfactory tissues have been reported in rodents and humans. For example, mouse OR23 (mOR23) is expressed in sperm and muscle cells and has been proposed to play a role in chemotaxis and muscle migration, respectively. In addition, mouse mesencephalic dopaminergic neurons express various ORs, which respond to corresponding ligands. As the OR genes comprise the largest multigene family of G protein-coupled receptors in vertebrates (over 400 genes in human and 1000 in rodents), it has been difficult to categorize the extent of their diverse expression in non-olfactory tissues making it challenging to ascertain their function. The zebrafish genome contains significantly fewer OR genes at around 140 genes, and their expression pattern can be easily analyzed by carrying out whole mount in situ hybridization (ISH) assay in larvae. In this study, we found that 31 out of 36 OR genes, including or104-2, or108-1, or111-1, or125-4, or128-1, or128-5, 133-4, or133-7, or137-3 are expressed in various tissues, including the trunk, pharynx, pancreas and brain in the larvae. In addition, some OR genes are expressed in distinct brain regions such as the hypothalamus and the habenula in a dynamic temporal pattern between larvae, juvenile and adult zebrafish. We further confirmed that OR genes are expressed in non-olfactory tissues by RT-PCR in larvae and adults. These results indicate tight regulation of OR gene expression in the brain in a spatial and temporal manner and that the expression of OR genes in non-olfactory tissues are conserved in vertebrates. This study provides a framework to start investigating the function of ORs in the zebrafish brain.

In Pisum sativum, the RAMOSUS genes RMS1, RMS2, and RMS5 regulate shoot branching via physiologically defined mobile signals. RMS1 is most likely a carotenoid cleavage enzyme and acts with RMS5 to control levels of an as yet unidentified mobile branching inhibitor required for auxin inhibition of branching. Our work provides molecular, genetic, and physiological evidence that RMS1 plays a central role in a shoot-to-root-to-shoot feedback system that regulates shoot branching in pea. Indole-3-acetic acid (IAA) positively regulates RMS1 transcript level, a potentially important mechanism for regulation of shoot branching by IAA. In addition, RMS1 transcript levels are dramatically elevated in rms3, rms4, and rms5 plants, which do not contain elevated IAA levels. This degree of upregulation of RMS1 expression cannot be achieved in wild-type plants by exogenous IAA application. Grafting studies indicate that an IAA-independent mobile feedback signal contributes to the elevated RMS1 transcript levels in rms4 plants. Therefore, the long-distance signaling network controlling branching in pea involves IAA, the RMS1 inhibitor, and an IAA-independent feedback signal. Consistent with physiological studies that predict an interaction between RMS2 and RMS1, rms2 mutations appear to disrupt this IAA-independent regulation of RMS1 expression.

The axon initial segment (AIS) is responsible for both the modulation of action potentials and the maintenance of neuronal polarity. Yet, the molecular mechanisms controlling its assembly are incompletely understood. Our study in single electroporated motor neurons in mouse embryos revealed that AnkyrinG (AnkG), the AIS master organizer, is undetectable in bipolar migrating motor neurons, but is already expressed at the beginning of axonogenesis at E9.5 and initially distributed homogeneously along the entire growing axon. Then, from E11.5, a stage when AnkG is already apposed to the membrane, as observed by electron microscopy, the protein progressively becomes restricted to the proximal axon. Analysis on the global motor neurons population indicated that Neurofascin follows an identical spatio-temporal distribution, whereas sodium channels and β4-spectrin only appear along AnkG + segments at E11.5. Early patch-clamp recordings of individual motor neurons indicated that at E12.5 these nascent AISs are already able to generate spikes. Using knock-out mice, we demonstrated that neither β4-spectrin nor Neurofascin control the distal-to-proximal restriction of AnkG.

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Modular organization of spinal neural circuits control dynamic regulation of locomotion. However, it is unknown when or how the distinct microcircuits emerge during development. We carried out high-resolution calcium imaging of neural activity driving the first motor behavior in one day old zebrafish embryo. During this period, at least two waves of neurogenesis occur to generate primary and secondary motoneurons. We found that embryos first display a single highly synchronized rhythmic neuronal circuit containing interneurons and motoneurons. Later, two distinct interneuron-motoneuron circuits emerge with one containing early-born motoneurons displaying low-frequency activity and the other containing later-born motoneurons with high-frequency activity. The results indicate a mode of birth order determined microcircuits where neurons that are born together are recruited together. Nicotine affected neuronal activity frequency, revealing a functional role for cholinergic signaling in the emergence of patterned spinal circuits. Indeed, we found aberrant arrhythmic synchronized activity in mutants for cholineacetyltransferase-a where acetylcholine is no longer synthesized. Overall, we reveal the sequential recruitment of birth order specified microcircuits during the emergence of the earliest motor behavior and highlight a conserved role for cholinergic signaling in regulating rhythmic neural activity in the embryonic spinal cord.

+/Ca++ action potentials. Unlike neurons, floor-plate action potentials relied primarily on the activation of voltage-gated T-type calcium channels (TTCCs). In situ hybridization showed that all 3 known subtypes of TTCCs are highly and predominantly expressed in the floor-plate. During SNA, we found that acetylcholine released by motoneurons recurrently trigger floor-plate action potentials by acting through nicotinic acetylcholine receptors. Finally, by expressing the genetically encoded calcium indicator GCaMP6f in the floor plate, we demonstrated that neuroepithelial action potentials are associated with calcium waves and propagate along the entire length of the spinal cord. By unraveling a novel physiological mechanism generating electrical signals which can propagate independently from neurons across a neural structure, our work significantly changes our understanding of the development, origin and extent of electrical signaling in the central nervous system. HIGHLIGHTS * Spinal neuroepithelial progenitors (NEP) are depolarized during spontaneous neural activity * NEPs form a single electrical syncytium connected by gap junctions * Floor-plate NEPs generate large Na+/Ca++ action potentials in response to acetylcholine * Neuroepithelial action potentials propagate across the entire spinal cord Competing Interest Statement The authors have declared no competing interest.