INGEBI   02650
INSTITUTO DE INVESTIGACIONES EN INGENIERIA GENETICA Y BIOLOGIA MOLECULAR "DR. HECTOR N TORRES"
Unidad Ejecutora - UE
congresos y reuniones científicas
Título:
SPONTANEOUS ELECTRICAL ACTIVITY IN DEVELOPING SPINAL MOTOR NEURONS OF ZEBRAFISH EMBRYOS
Autor/es:
P.V. PLAZAS; N. C. SPITZER
Lugar:
Montreal
Reunión:
Congreso; Human Frontiers in Science Program 2011 Annual Meeting; 2011
Institución organizadora:
Human Frontiers in Science Program
Resumen:
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 910 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.