Role of spontaneous electrical activity in spinal motor neuron axonal outgrowth and pathfinding

P.V. PLAZAS; N. C. SPITZER

Washington DC

Congreso; Society for Neuroscience 41st Annual Meeting; 2011

Society for Neuroscience

   Neuronal circuits become wired during development via mechanisms that direct axons to make synaptic connections with their appropriate targets. The role of genetic programs in this process is increasingly well understood. However, evidence for a role of electrical activity in the assembly of developing circuits is quite limited.    In zebrafish embryos, each spinal hemisegment contains 3 primary motor neurons (PMN), named CaP, MiP and RoP (for caudal, middle and rostral primary) and ~30 secondary motor neurons (SMN). During the first 24 hr of development, PMN exit the spinal cord and then follow a common pathway to reach the horizontal myoseptum (HMS), where they pause before diverging to distinct targets. CaP innervates the ventral myotome, MiP innervates the dorsomedial myotome and RoP axons extend mediolaterally through the HMS.    We tracked axon outgrowth in vivo in transgenic Hb9:eGFP zebrafish embryos between 17 hr when PMN begin axon extension and 24 hr post fertilization (hpf) when early connections are established with muscle, and performed Ca2+ imaging during pathfinding behavior to determine the timing of spontaneous electrical activity. PMN display two types of spontaneous intracellular Ca2+ transients: waves and spikes. Ca2+ waves are generated in both PMN and SMN, with similar durations (55±7 s) and frequencies (3.2±0.9 h-1). In contrast, only PMN exhibit Ca2+ spikes (2.5±0.7 s), which are generated in specific patterns at different developmental stages. At 17-18 hpf spikes are single events at a frequency of 6.5±0.5 min-1. Between 19 and 20 hpf (when axons are reaching the HMS) PMN exhibit bursts of activity at higher spike frequencies (18.3±0.5 min-1) and beyond this developmental stage spikes occur both as high frequency single events (15.3±0.3 min-1) and as bursts of lower spike frequency activity (7.1±0.4 min-1). Time-lapse imaging reveals that all four patterns are expressed sequentially in single neurons.     Suppression of Ca2+ spiking activity by stochastic expression of human inward rectifier K+ channels (hKir2.1) in single PMN led to substantial errors in MiP and RoP axon growth. Errors comprise aberrant branching in 30% of MiPs and intraspinal pathfinding mistakes made by 26% of RoPs. Misguided RoP growth cones either orient away from the endogenous spinal cord exit point or extend towards the endogenous exit point but bypass it. These results provide insight into when and where spontaneous electrical activity is expressed and its relation to the establishment of PMN precise stereotypical axonal trajectories.    Supported by NIH NS15918 and MH074702 to NCS.