Characterization of spontaneous electrical activity in developing spinal motor neurons of zebrafish embryos

PAOLA V. PLAZAS; NICHOLAAS C SPITZER

San Diego

Congreso; Society for Neuroscience 40th Annual Meeting; 2010

Society for Neuroscience

Calcium signals are key for many processes during embryonic development. In the nervous system, fluctuations in intracellular calcium levels play important roles in proliferation, migration and neuronal differentiation. Moreover, spontaneous calcium transients in embryonic spinal neurons of X. laevis have been shown to regulate processes including 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.    In this work, we have taken advantage of the optical transparency and rapid development of zebrafish (Danio rerio) to study the patterns of intracellular calcium signals in spinal primary motor neurons (PMN) of intact embryos during the entire process of axonogenesis, pathfinding and establishment of early connections.    We simultaneously tracked axon outgrowth and formation of connections in vivo in transgenic Hb9:eGFP zebrafish embryos between 16 and 24 hpf and performed calcium imaging with calcium green-1 dextran during pathfinding behavior to determine the timing of electrical activity. Embryos were paralyzed by exposure to 10 µM a-bungarotoxin. Midtrunk PMN from 16 hpf (beginning of axon extension) to 24 hpf (when early connections are established with muscle) displayed spontaneous intracellular calcium transients that are restricted to single neurons. The calcium transients had an average peak amplitude of 35 ± 3% above baseline and a duration of 42 ± 2 s. The average frequency increased from 1.5 ± 0.4 per hour at 16 hpf to 6.2 ± 0.3 per hour at 18 hpf, when axons are reaching the horizontal myoseptum. 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. We are now focusing on the activity patterns between 18 and 24 hpf. Supported by NIH NS15918 to NCS.