SPONTANEOUS ELECTRICAL ACTIVITY IN DEVELOPING SPINAL MOTOR NEURONS OF ZEBRAFISH EMBRYOS

P.V. PLAZAS; N. C. SPITZER

Montreal

Congreso; Human Frontiers in Science Program 2011 Annual Meeting; 2011

Human Frontiers in Science Program

  Calcium (Ca2+) signals are key for many processes during the development of the nervous system. Spontaneous Ca2+ transients in embryonic spinal neurons of X. laevis have been shown to regulate growth cone motility and neurotransmitter specification. However, our understanding of the way in which electrical activity affects the structure and function of developing circuits is still quite limited.   We have taken advantage of the optical transparency and rapid development of zebrafish (Danio rerio) to study the patterns of intracellular Ca2+ signals in spinal motor neurons of intact embryos during the entire process of axonogenesis, pathfinding and establishment of early synaptic connections.   In each spinal hemisegment of zebrafish embryos three identified primary motor neurons (PMN), named CaP, MiP and RoP (for caudal, middle and rostral primary), are formed 9–10 hr post fertilization (hpf), and about 30 secondary motor neurons (SMN) are formed a few hr later. All PMN extend their axons within the spinal cord to a shared exit point, and then follow a common pathway to reach the horizontal myoseptum (HMS) that is marked by a set of specialized muscle pioneer cells. Here they pause before diverging to distinct targets. CaP axons extend ventrally to innervate the ventral myotome, MiP axons branch and extend along the dorsomedial myotome and RoP axons extend mediolaterally through the HMS.   We tracked PMN axon outgrowth and formation of connections in vivo in transgenic Hb9:eGFP zebrafish embryos between 17 hpf (beginning of axon extension) and 24 hpf (when early connections are established with muscle) and performed Ca2+ imaging with calcium green-1 dextran during pathfinding behavior to determine the timing of electrical activity. Midtrunk motor neurons from 17 to 24 hpf display two types of spontaneous intracellular Ca2+ transients: waves and spikes. Ca2+ waves are generated in both PMN and SMN, but only PMN exhibit Ca2+ spikes. In PMN, Ca2+ waves have an average duration of 52 ± 6 s and an average frequency that decreases from 4.2 ± 0.2 hr-1 at 17 hpf to 2.0 ± 0.6 hr-1 at 21-22 hpf. Ca2+ spikes have an average duration of 1.8 ± 0.7 s and 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 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 frequency activity (7.1 ± 0.4 min-1). Time lapse imaging for periods of 7 hr revealed that all four patterns are expressed in single neurons.    These results provide insight into when and where spontaneous electrical activity is expressed and its relation to the establishment of PMN precise stereotypical axonal branching patterns.