Activity-dependent competition regulates motor neuron axon pathfinding via PlexinA3

Abstract

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. Spikes arose in the distal axon of PMN and were propagated to the cell body. Suppression of Ca 2+ spiking activity in single PMN led to stereotyped errors, but silencing all electrical activity had no effect on axon guidance, indicating that an activitybased competition rule regulates this process. This competition was not mediated by synaptic transmission. Combination of PlexinA3 knockdown with suppression of Ca 2+ activity in single PMN produced a synergistic increase in the incidence of pathfinding errors. However, expression of PlexinA3 transcripts was not regulated by activity. Our results provide an in vivo demonstration of the intersection of spontaneous electrical activity with the PlexinA3 guidance molecule receptor in regulation of axon pathfinding.calcium transients | spontaneous activity | stochastic expression D evelopment of the nervous system involves the outgrowth of a complex network of axons to specific synaptic targets. Axon pathfinding to the target area is directed by interactions between the growth cone and guidance cues present in the environment (1, 2). Electrical activity has been considered to have a role by contributing to the fine tuning of connections (3, 4). However, evidence for a role of activity during early pathfinding decisions has been emerging (5-7). The mechanisms underlying such regulation remain unclear, but the response of neurons to chemotropic molecules in vitro is modulated by electrical activity (7,8).Here, we use the zebrafish embryo as an in vivo model to address the role of electrical activity in axon pathfinding. Each spinal hemisegment contains 3 primary motor neurons (PMN), named caudal primary (CaP), middle primary (MiP), and rostral primary (RoP), and ∼30 secondary motor neurons (SMN). During the first day of development, all three PMN axons pioneer into the periphery through a shared exit point, follow a common pathway to reach the horizontal myoseptum (HMS) and then diverge onto cell subtype-specific trajectories (9) (Fig. 1A).We characterize early calcium (Ca 2+ ) signals in PMN of intact zebrafish embryos during axon outgrowth and investigate their role in pathfinding behavior. We show that spontaneous electrical activity is expressed in developing PMN during the entire process of axon pathfinding. Specific patterns of Ca 2+ spiking activity are present at different developmental stages and expressed sequentially in CaP, MiP, and then RoP, beginning with the onset of axonogenesis. To investigate the role of Ca 2+ activity, mosaic expression of an exogenous potassium channel (hKir2.1) was used to suppress electrical activity in single PMN. The results indicate that an activity-based competition rule regulates e...

The assembly of developing motor neurons depends on an interplay between spontaneous activity, type II cadherins and gap junctions

“…Our observations show similarities with the correlated SA that has been reported in the developing retina (Feller et al, 1996;Graeber et al, 2013), and in chick and zebrafish spinal motor neurons (Law et al, 2014;Plazas et al, 2013;Wang et al, 2009), where in one case activity was thought to be regulated by recurrent synaptic excitation.…”

Promoting axon regeneration in the adult CNS by modulation of the melanopsin/GPCR signaling

“…Although we lack direct evidence that the regenerated axons belong to the neurons that are more active, silencing these neurons suppresses mTOR activation and axonal regeneration. During development, neuronal activity is essential for axon growth and wiring (27). In vitro, postnatal RGCs respond to growth factors by elongating axons after depolarization (28).…”

Optogenetic-mediated increases in in vivo spontaneous activity disrupt pool-specific but not dorsal-ventral motoneuron pathfinding

“…Periodic activity of the appropriate frequency is required for Drosophila motoneurons to retract from inappropriate muscles in response to a semaphorinmediated signal (39). A recent in vivo study also showed that zebrafish primary motoneurons required spontaneous Ca 2+ transients to pathfind accurately (40). However, both the characteristics of the spontaneous activity and the downstream cellular/molecular mechanisms differ from those in chick cord, suggesting possible species and cell-type diversity and the need to study these events in multiple organisms.…”