The Journal of Neuroscience, February 1, 2002, 22(3):931-945
Mechanisms of the Release of Anterogradely Transported
Neurotrophin-3 from Axon Terminals
XiaoXia
Wang1,
Rafal
Butowt1,
Michael R.
Vasko2, and
Christopher S.
von
Bartheld1
1 Department of Physiology and Cell Biology, University
of Nevada School of Medicine, Reno, Nevada 89557, and
2 Department of Pharmacology and Toxicology, University of
Indiana School of Medicine, Indianapolis, Indiana 46202-5120
Neurotrophins have profound effects on synaptic function and
structure. They can be derived from presynaptic, as well as
postsynaptic, sites. To date, it has not been possible to measure the
release of neurotrophins from axon terminals in intact tissue. We
implemented a novel, extremely sensitive assay for the release and
transfer of anterogradely transported neurotrophin-3 (NT-3) from a
presynaptic to a postsynaptic location that uses synaptosomal
fractionation after introduction of radiolabeled NT-3 into the
retinotectal projection of chick embryos. Release of the anterogradely
transported NT-3 in intact tissue was assessed by measuring the amount
remaining in synaptosomal preparations after treatment of whole tecta
with pharmacological agents. Use of this assay reveals that release of
NT-3 from axon terminals is increased by depolarization, calcium influx
via N-type calcium channels, and cAMP analogs, and release is most
profoundly increased by excitation with kainic acid or mobilization of
calcium from intracellular stores. NT-3 release depends on
extracellular sodium, CaM kinase II activity, and requires intact
microtubules and microfilaments. Dantrolene inhibits the high
potassium-induced release of NT-3, indicating that release of calcium
from intracellular stores is required. Tetanus toxin also inhibits NT-3
release, suggesting that intact synaptobrevin or synaptobrevin-like
molecules are required for exocytosis. Ultrastructural autoradiography and immunolabel indicate that NT-3 is packaged in
presumptive large dense-core vesicles. These data show that release of
NT-3 from axon terminals depends on multiple regulatory proteins and
ions, including the mobilization of local calcium. The data provide
insight in the mechanisms of anterograde neurotrophins as synaptic modulators.
Key words:
neurotrophic factors; secretion; presynaptic terminals; calcium; axonal transport; neurotrophin; synapse; synaptic
transmission
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