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Suppression of Ca 2+ spiking activity in single PMN leads to errors in axon pathfinding. (A) Ca 2+ spikes are blocked by stochastic expression of hKir2.1 or exposure to 0.02% tricaine for 15 min. n = 20 for each condition. (B) Percentage of PMN exhibiting Ca 2+ spiking activity at 19 hpf. n = 20 for each group; values are means ± SEM. *P < 0.0005 compared with control. (C) RoP and MiP neurons expressing hKir2.1 bypass the exit point or project rostrally and exhibit extra branching. Neither tricaine nor Kir2.1 + tricaine leads to errors in axon pathfinding. Control, embryos injected with Hb9:Gal4 and UAS:DsRed plasmids; Kir2.1, embryos injected with Hb9:Gal4 and UAS: DsRed::UAS:hKir2.1 plasmids; Tricaine, embryos raised in the presence of 0.02% tricaine; Kir2.1 + Tricaine, embryos expressing hKir2.1 and raised in the presence of tricaine. (D) Tetanus toxin expression does not cause pathfinding errors. Control, embryos injected with Hb9:Gal4 and UAS:eGFP plasmids; TeNT-LC, embryos injected with Hb9:Gal4 and UAS:TeNT-LC:eGFP plasmids. (C and D) Dorsal is to the top and rostral is to the left. Dot-dash lines mark lateral edges of the myotomes; dotted lines mark the ventral edge of the spinal cord. n ≥ 30 cells from ≥30 24-hpf embryos for each group. Fig. 7. Synergistic interaction of hKir2.1 and PlexinA3. Percentage of (A) RoPs extending their axons rostrally or bypassing the endogenous exit point or (B) MiP axons with aberrant branching, including data from Combination of a subthreshold concentration of PlexinA3 MO and hKir2.1 expression generates an increase in the incidence of pathfinding errors. Control, embryos injected with Hb9:Gal4 and UAS:DsRed plasmids; Kir, embryos injected with Hb9:Gal4 and UAS:DsRed::UAS:hKir2.1 plasmids; MO 0.5 mM, embryos injected with 0.5 mM PlexinA3 MO and Hb9:Gal4 and UAS: DsRed plasmids; MO 0.3 mM, embryos injected with 0.3 mM PlexinA3 MO and Hb9:Gal4 and UAS:DsRed plasmids; MO 0.3 mM + Kir, embryos injected with 0.3 mM PlexinA3 MO and Hb9:Gal4 and UAS:DsRed::UAS:hKir2.1 plasmids; 5 mm MO + Kir, embryos injected with 0.3 mM MO with five mismatched bases based on PlexinA3 MO and Hb9:Gal4 and UAS:DsRed::UAS: hKir2.1 plasmids. n = 30-40 neurons from ≥30 24-hpf embryos for each condition. *P < 0.05 using a Fisher exact test comparing PlexinA3 MO + Kir-injected with Kir-injected alone. (C-E) Single PMN expressing hKir2.1 and DsRed and exhibiting a pathfinding error expresses PlexinA3 mRNA. (C) A DsRed immunopositive RoP extending its axon rostrally. (D) In situ hybridization shows strong expression of PlexinA3 mRNA. (E) Merge of C and D. Dorsal is to the top and rostral is to the left. Dot-dash lines mark lateral edges of the myotomes; dotted lines mark the ventral edge of the spinal cord. n = 4 MiPs, and n = 4 RoPs.
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The role of electrical activity in axon guidance has been extensively studied in vitro. To better understand its role in the intact nervous system, we imaged intracellular Ca(2+) in zebrafish primary motor neurons (PMN) during axon pathfinding in vivo. We found that PMN generate specific patterns of Ca(2+) spikes at different developmental stages....
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... per- turbations. Coinjections of PlexinA3 MO (0.3 mM) with Hb9: Gal4 and UAS:hKir2.1::UAS:DsRed constructs induced a signif- icant increase in the incidence of pathfinding errors in PMN compared with embryos coinjected with 0.3 mM PlexinA3 MO and Hb9:Gal4 and UAS:DsRed constructs or coinjected with Hb9:Gal4 and UAS:hKir2.1::UAS:DsRed constructs (Fig. 7 A and B). As a control, hKir2.1 was coinjected with a five-base mismatch PlexinA3 MO (0.3 mM) (14), which elicited aberrant growth of motor axons comparable with the abnormal axon out- growth generated by the expression of hKir2.1 alone. The syner- gistic effects observed in these experiments suggest an intersection of PlexinA3 signaling with ...
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Embryonic development requires tight coordination between tissues as they frequently grow at different rates. Such differential growth rates can cause shifts between neighboring tissues, and are a particular challenge for individual cells that span multiple tissues, in part because mechanical tension on such cells is predicted to be...
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... In control conditions, we detected the presence of sporadic spontaneous transients in the telencephalon of the three transgenic backgrounds we used ( Fig. 1D-O, blue symbols). The analyzed parameters were in the range of what has been reported in the Xenopus brain (Dulcis and Spitzer, 2008;Demarque and Spitzer, 2010) and longer than what has been reported for the embryonic zebrafish spinal cord (Warp et al., 2012;Plazas et al., 2013). ...
... Ca 21 transients were already reported in the spinal cord of zebrafish embryos around 24 hpf (Warp et al., 2012;Plazas et al., 2013). Here, recordings were performed in the subpallium and olfactory bulb of embryonic zebrafish at around 48 hpf, a time at which the transition toward synaptic network activity has already occurred in the spinal cord (Warp et al., 2012). ...
... The duration of Ca 21 transients we measured in the subpallium (7.8 6 2.5 s) were similar to what has been reported in Xenopus (7.5 6 1.0 s; Gu et al., 1994) and longer than what has been described in the zebrafish spinal cord (3.3 6 1.6 s; Plazas et al., 2013). The duration of these individual transients was increased by both the veratridine and the TCNF treatments reported here, while these treatments led to diametrically opposite effects on DA specification and high-speed locomotion. ...
During the embryonic period, neuronal communication starts before the establishment of the synapses with alternative forms of neuronal excitability, called here embryonic neural excitability (ENE). ENE has been shown to modulate the unfolding of development transcriptional programs, but the global consequences for developing organisms are not all understood. Here, we monitored calcium (Ca ²⁺ ) transients in the telencephalon of zebrafish embryos as a proxy for ENE to assess the efficacy of transient pharmacological treatments to either increase or decrease ENE. Increasing or decreasing ENE at the end of the embryonic period promoted an increase or a decrease in the numbers of dopamine (DA) neurons, respectively. This plasticity of dopaminergic specification occurs in the subpallium (SP) of zebrafish larvae at 6 d postfertilization (dpf), within a relatively stable population of vMAT2-positive cells. Nondopaminergic vMAT2-positive cells hence constitute an unanticipated biological marker for a reserve pool of DA neurons that can be recruited by ENE. Modulating ENE also affected larval locomotion several days after the end of the treatments. In particular, the increase of ENE from 2 to 3 dpf promoted hyperlocomotion of larvae at 6 dpf, reminiscent of zebrafish endophenotypes reported for attention deficit hyperactivity disorders (ADHDs). These results provide a convenient framework for identifying environmental factors that could disturb ENE as well as to study the molecular mechanisms linking ENE to neurotransmitter specification.
... Indeed, it involves crosstalk among several cues or receptors that are specific of the axonal path [13][14][15] . Guidance molecules act in addition to other factors such as the molecular machinery specific to each neuronal population 16 , the substrate stiffness 17 or the neuronal activity 18,19 . Furthermore, over the past years several molecules have been described to be involved in axon guidance such as morphogens (Shh, Wnt) 20,21 , growth factors (TGFβ, BMP) 22 and adhesion molecules (L1CAM, NrCAM) 23 . ...
In the injured adult central nervous system (CNS), activation of pro-growth molecular pathways in neurons leads to long-distance regeneration. However, most regenerative fibers display guidance defects, which prevent reinnervation and functional recovery. Therefore, the molecular characterization of the proper target regions of regenerative axons is essential to uncover the modalities of adult reinnervation. In this study, we use mass spectrometry (MS)-based quantitative proteomics to address the proteomes of major nuclei of the adult visual system. These analyses reveal that guidance-associated molecules are expressed in adult visual targets. Moreover, we show that bilateral optic nerve injury modulates the expression of specific proteins. In contrast, the expression of guidance molecules remains steady. Finally, we show that regenerative axons are able to respond to guidance cues ex vivo, suggesting that these molecules possibly interfere with brain target reinnervation in adult. Using a long-distance regeneration model, we further demonstrate that the silencing of specific guidance signaling leads to rerouting of regenerative axons in vivo. Altogether, our results suggest ways to modulate axon guidance of regenerative neurons to achieve circuit repair in adult.
... The zebrafish was also instrumental in investigating how living axons are guided in the peripheral nervous system (PNS). For instance, studies showed that motor axons exiting the central nervous system (CNS) are guided by a synergetic mechanism involving calcium waves and PlexinA3 (Plazas et al., 2013). Once in the periphery, motor axons are guided towards prepatterned AChR clusters on muscles (Panzer et al., 2006). ...
... They will certainly open new avenues in many different animal models and guidance systems for further characterization and understanding of molecular mechanisms of axon guidance. Moreover, combinations of these approaches with reporter tools have already greatly helped to visualize the cytoskeletal or second messenger dynamics, for instance by using reporters or manipulations of the guidance system at the growth cone level via optogenetic approaches (Plazas et al., 2013;Medioni et al., 2015;Endo et al., 2016;Nichols and Smith, 2019;Harris et al., 2020;. Today's possibilities to investigate in detail and in real time the mechanisms underlying growth cone navigation are basically unlimited, given the number of tools that are now available. ...
The molecular mechanisms of neural circuit formation have been of interest to Santiago Ramón y Cajal and thousands of neuroscientists sharing his passion for neural circuits ever since. Cajal was a brilliant observer and taught us about the connections and the morphology of neurons in the adult and developing nervous system. Clearly, we will not learn about molecular mechanisms by just looking at brain sections or cells in culture. Technically, we had to come a long way to today’s possibilities that allow us to perturb target gene expression and watch the consequences of our manipulations on navigating axons in situ. In this review, we summarize landmark steps towards modern live-imaging approaches used to study the molecular basis of axon guidance.
... Given the fact that scn8ab is a main modulator of neural activity, we speculate that scn8ab-mediated electrical signals in fin neurons are crucial for axon regeneration upon fin amputation. Multiple studies in developing embryos have provided evidence that neuronal activity can affect neurite development and further establishment of neural networks (53,54). For instance, knockdown of scn8aa in developing zebrafish embryos leads to aberrant axonal trajectories and delayed growth of secondary motor neurons (53). ...
... For instance, knockdown of scn8aa in developing zebrafish embryos leads to aberrant axonal trajectories and delayed growth of secondary motor neurons (53). Decreased neural activity in developing zebrafish larvae increases the incidence of pathfinding errors in primary motor neurons (54). Our data suggest that the voltage-gated channel activity of Scn8ab is required for nerve reconstruction and for nerves to reach a sufficient threshold number in zebrafish fins, which enable normal blastema formation. ...
Teleost fishes and urodele amphibians can regenerate amputated appendages, whereas this ability is restricted to digit tips in adult mammals. One key component of appendage regeneration is reinnervation of the wound area. However, how innervation is regulated in injured appendages of adult vertebrates has seen limited research attention. From a forward genetics screen for temperature-sensitive defects in zebrafish fin regeneration, we identified a mutation that disrupted regeneration while also inducing paralysis at the restrictive temperature. Genetic mapping and complementation tests identify a mutation in the major neuronal voltage-gated sodium channel (VGSC) gene scn8ab. Conditional disruption of scn8ab impairs early regenerative events, including blastema formation, but does not affect morphogenesis of established regenerates. Whereas scn8ab mutations reduced neural activity as expected, they also disrupted axon regrowth and patterning in fin regenerates, resulting in hypoinnervation. Our findings indicate that the activity of VGSCs plays a proregenerative role by promoting innervation of appendage stumps.
... With its anatomically distinct and well localized axonal pathways, contained in small narrow volumes, confocal and two photon microscopy studies are facilitated (Akin and Zipursky, 2016;Bak and Fraser, 2003;Gaynes et al., 2015;Hutson and Chien, 2002;Mason and Wang, 1997;Nagel et al., 2015;Pittman et al., 2008;Sretavan and Reichardt, 1993). In the spinal cord, individual sensory Rohon-Beard axonal growth has been followed live in vivo by widefield and confocal microscopy in the zebrafish (Andersen and Halloran, 2012;Lee et al., 2017;Liu and Halloran, 2005), and individual motoneurons by confocal microscopy in the zebrafish and Xenopus (Bremer and Granato, 2016;Plazas et al., 2013) and widefield in Drosophila (Murray and Whitington, 1999). Ex vivo cultures of chicken spinal cord (Dumoulin et al., 2021), a refinement of the openbook preparation (Bovolenta and Dodd, 1990;Yaginuma et al., 1991), have provided live data on axonal growth and guidance, but live in vivo studies of axonal growth and guidance in the chicken model system remain unfeasible with current technology and both genetic and molecular perturbation possibilities are also limited. ...
Axonal growth and guidance at the ventral floor plate is here followed $\textit{in vivo}$ in real time at high resolution by light-sheet microscopy along several hundred micrometers of the zebrafish spinal cord. The recordings show the strikingly stereotyped spatio-temporal control that governs midline crossing. Commissural axons are observed crossing the ventral floor plate midline perpendicularly at about 20 microns/h, in a manner dependent on the Robo3 receptor and with a growth rate minimum around the midline, confirming previous observations. At guidance points, commissural axons are seen to decrease their growth rate and growth cones increase in size. Commissural filopodia appear to interact with the nascent neural network, and thereby trigger immediate plastic and reversible sinusoidal-shaped bending movements of neighboring commissural shafts. Ipsilateral axons extend concurrently, but straight and without bends, at three to six times higher growth rates than commissurals, indicating they project their path on a substrate-bound surface rather than relying on diffusible guidance cues. Growing axons appeared to be under stretch, an observation that is of relevance for tension-based models of cortical morphogenesis. The \textit{in vivo} observations provide for a discussion of the current distinction between substrate-bound and diffusible guidance cues. The study applies the transparent zebrafish model that provides an experimental model system to explore further the cellular, molecular and physical mechanisms involved during axonal growth, guidance and midline crossing through a combination of $\textit{in vitro}$ and $\textit{in vivo}$ approaches.
... Indeed, it is becoming clearer that guidance molecules activity is finely modulated by a number of co-factors: identity of the responding neuronal population (Nédelec et al., 2012), crosstalk of other cues or receptors (Charoy et al., 2012;Delloye-Bourgeois et al., 2015;Kuwajima et al., 2012), substrate stiffness (Koser et al., 2016), neuronal activity (Nicol et al., 2007;Plazas et al., 2013). In addition, over the past few years others molecules have been described to be involved in axon guidance such as morphogens (Shh, Wnt), growth factors (TGFb, BMP) (Yam and Charron, 2013) or adhesion molecules (L1CAM, NrCAM) (Pollerberg et al., 2013). ...
Long-distance regeneration of the central nervous system (CNS) has been achieved from the eye to the brain through activation of neuronal molecular pathways or pharmacological approaches. Unexpectedly, most of the regenerative fibers display guidance defects, which prevents reinnervation and further functional recovery. Therefore, characterizing the mature neuronal environment is essential to understand the adult axonal guidance in order to complete the circuit reconstruction. To this end, we used mass spectrometry to characterize the proteomes of major nuclei of the adult visual system: suprachiasmatic nucleus (SCN), ventral and dorsal lateral geniculate nucleus (vLGN, dLGN) and superior colliculus (SC)), as well as the optic chiasm. These analyses revealed the presence of guidance molecules and guidance-associated factors in the adult visual targets. Moreover, by performing bilateral optic nerve crush, we showed that the expression of some proteins was significantly modulated by the injury in the visual targets, even in the ones most distal to the lesion site. On another hand, we found that the expression of guidance molecules was not modified upon injury. This implies that these molecules may possibly interfere with the reinnervation of the brain targets. Together, our results provides an extensive characterization of the molecular environment in intact and injured conditions. These findings open new ways to correct regenerating axon guidance notably by manipulating the expression of the corresponding guidance receptors in the nervous system.
... During early zebrafish development, beginning at around 16 hpf, PMNs (including CaPs) extend their axons out from their soma located within the spinal cord to make contact with the developing trunk musculature (Plazas et al., 2013). In the case of CaPs, their axons extend downwards to initially make contact with a specific class of slow skeletal muscle cells known as the muscle pioneer cells (Fig. 1A) (Myers et al., 1986). ...
The role of TPC2-mediated Ca2+ release was recently characterized in zebrafish during the establishment of the early spinal circuitry, one of the key events in the coordination of neuromuscular activity. Here, we extend our study to investigate the in vivo role of TPC2 in the regulation of caudal primary motor neuron (CaP) axon extension. We used a combination of: TPC2 knock-down with a translational-blocking morpholino antisense oligonucleotide (MO); TPC2 knock-out via the generation of a tpcn2dhkz1a mutant line of zebrafish using CRISPR/Cas9 gene-editing; and pharmacological inhibition of TPC2 via incubation with bafilomycin A1 (an H+-ATPase inhibitor) or trans-ned-19 (an NAADP receptor antagonist), and showed that these treatments attenuated CaP Ca2+ signaling and inhibited axon extension. We also characterized the expression of an arc1-like transcript in CaPs grown in primary culture. MO-mediated knock-down of ARC1-like in vivo led to an attenuation of the Ca2+ transients in the CaP growth cones, and an inhibition of axon extension. Together our new data suggest a link between ARC1-like, TPC2 and Ca2+ signaling during axon extension in zebrafish.
... Primary motoneurons act as leader cells in early circuit assembly for locomotion (Wan et al., 2019). CaP motoneurons axons initiated axonogenesis at 17hpf, while activity-dependent competition between primary motoneurons regulated axon pathfinding (Plazas et al., 2013). In this study, the axons of CaP motoneurons was selected to study, we found that the axons were truncated in 10 ng/L and 50 ng/L 17btrenbolone. ...
17β-trenbolone (17β-TBOH) is one of the dominant metabolites of trenbolone acetate, which is widely applied in beef cattle operations around the globe. The effects of environmental concentrations of 17β-trenbolone on the early development of zebrafish embryos have received very little attention. Melatonin could regulate sleep-wake cycle and plays a protective role in various adverse conditions. Here, environmentally realistic concentrations of 17β-trenbolone (1 ng/L, 10 ng/L, 50 ng/L) has been exposure to zebrafish embryos at 2 h postfertilization (hpf). The results showed that 10 ng/L and 50 ng/L 17β-trenbolone disturbed the distribution of caudal primary motoneurons and downregulated expression of motoneuron development related genes along with locomotion decreasing. While melatonin could recover the detrimental effects caused by 17β-trenbolone. Interestingly, 17β-trenbolone exposure increased waking activity and decreased rest even in a low dose (1 ng/L). Moreover, it upregulated hypocretin/orexin (Hcrt) signaling which promotes wakefulness. Melatonin restored the insomnia-like alternation induced by 17β-trenbolone exposure. Collectively, we conclude that 17β-trenbolone disturbed motoneuron development and altered sleep/wake behavior, while melatonin could alleviate the deleterious influence on motoneuron development and recover the circadian rhythm.
... With its anatomically distinct and well localized axonal pathways, contained in small narrow volumes, confocal and two photon microscopy studies are facilitated (Akin and Zipursky, 2016;Bak and Fraser, 2003;Gaynes et al., 2015;Hutson and Chien, 2002;Mason and Wang, 1997;Nagel et al., 2015;Pittman et al., 2008;Simpson et al., 2013;Sretavan and Reichardt, 1993). In the spinal cord, individual sensory Rohon-Beard axonal growth has been followed live in vivo by widefield and confocal microscopy in the zebrafish (Andersen and Halloran, 2012;Lee et al., 2017;Liu and Halloran, 2005), and individual motoneurons by confocal microscopy in the zebrafish and Xenopus (Bremer and Granato, 2016;Plazas et al., 2013) and widefield in Drosophila (Murray and Whitington, 1999). Though there are compelling reasons to study axon guidance with the Xenopus model (reviewed by (Erdogan et al., 2016)), a live imaging study of Xenopus spinal cord floor plate commissurals was based mostly on fixed samples (Moon and Gomez, 2005) because the closely apposed notochord obscured live imaging, just as mouse commissural axon studies also rely on fixed samples (e.g. ...
How axons are wiring the vertebrate spinal cord has in particular been studied at the ventral floor plate using fixed samples or looking at single growing axons with various microscopy techniques. Thereby may remain hidden important live organismal scale information concerning dynamics and concurrent timing of the many axons simultaneously crossing the floor plate. Here then, applying light-sheet microscopy, axonal growth and guidance at the floor plate are followed in vivo in real time at high resolution along several hundred micrometers of the zebrafish spinal cord by using an interneuron expressing GFP as a model axon. The commissural axons are observed crossing the ventral floor plate midline perpendicularly at about 20 microns/h and in a manner dependent on the Robo3 receptor. Commissural growth rate reaches a minimum at the midline, confirming previous observations. Ipsilateral axons extend concurrently, at three to six times higher growth rates. At guidance points, commissural axons are seen to decrease their growth rate and growth cones increase in size. Commissural filopodia appear to interact with the nascent neural network, and thereby trigger immediate plastic and reversible sinusoidal-shaped bending movements of neighboring commissural shafts. A simple protocol isolating single neuronal cells from the spinal cord is developed to facilitate further molecular characterization. The recordings show the strikingly stereotyped spatio-temporal control that governs midline crossing. The live observations give renewed perspective on the mechanisms of axonal guidance in the spinal cord that provide for a discussion of the current distinction between diffusible long-range versus substrate-bound short-range guidance cues.
... In developing spinal networks of chick embryos, depolarization and discharge of MNs were found to occur earliest at the onset of each cycle of rhythmic activity (O'Donovan et al., 1994) developmental defect in the embryonic zebrafish spinal cord, although synchronized activity still exists across distant segments in the spinal cord (Warp et al., 2012). Activity-dependent competition between primary MN subtypes was also suggested to regulate MN axon pathfinding in the embryonic zebrafish spinal cord (Plazas et al., 2013). In a developing circuit, early maturation of MNs, combined with their ability to retrogradely control network excitability and activity through electrical coupling with pre-motor INs, may provide a robust mechanism for generating reliable behavioral output while regulating network activity at the same time. ...
Animal survival requires a functioning nervous system to develop during embryogenesis. Newborn neurons must assemble into circuits producing activity patterns capable of instructing behaviors. Elucidating how this process is coordinated requires new methods that follow maturation and activity of all cells across a developing circuit. We present an imaging method for comprehensively tracking neuron lineages, movements, molecular identities, and activity in the entire developing zebrafish spinal cord, from neurogenesis until the emergence of patterned activity instructing the earliest spontaneous motor behavior. We found that motoneurons are active first and form local patterned ensembles with neighboring neurons. These ensembles merge, synchronize globally after reaching a threshold size, and finally recruit commissural interneurons to orchestrate the left-right alternating patterns important for locomotion in vertebrates. Individual neurons undergo functional maturation stereotypically based on their birth time and anatomical origin. Our study provides a general strategy for reconstructing how functioning circuits emerge during embryogenesis. VIDEO ABSTRACT.
