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Journal of Neuroscience, Vol 12, 762-772, Copyright © 1992 by Society for Neuroscience
Demonstration of local protein synthesis within dendrites using a new cell culture system that permits the isolation of living axons and dendrites from their cell bodies
ER Torre and O Steward
Department of Neuroscience, University of Virginia Health Science Center, Charlottesville 22908.
The presence of polyribosomes within dendrites suggests a capability for
local dendritic protein synthesis. However, local synthesis is difficult to
evaluate because of rapid somatodendritic protein transport. The present
study describes a two-surfaced culture system that allowed the separation
of living axons and dendrites from their cell bodies of origin. Because
this system eliminates the transport of proteins produced in the cell body,
it was possible to study the extent of dendritic protein synthesis
directly. Hippocampal neurons were plated on a Nucleopore polycarbonate
membrane that was mounted on a thick matrix of proteins (Matrigel) fixed on
a coverslip. As the neurons grew, axons and dendrites grew through the
membrane into the Matrigel. To evaluate local protein synthesis within
dendrites, the membrane with the cell bodies was removed, leaving a dense
array of transected dendrites and axons on the coverslip with few
contaminant cell bodies. Absence of cell bodies was confirmed by staining
with the nuclear stain Hoechst 33258. Coverslips with isolated neurites
were pulse labeled with 3H-leucine for 30 min, and fixed for
autoradiography to identify sites of protein synthesis. Autoradiographic
analyses revealed that isolated dendrites (immunochemically identified
using antibodies against MAP2) became heavily labeled, whereas axons
exhibited little if any labeling. The labeling was essentially eliminated
when the neurites were pulse labeled with 3H-leucine in the presence of
puromycin, whereas labeling was affected only minimally by chloramphenicol.
The puromycin-sensitive incorporation of 3H-leucine in dendrites
demonstrates that the polyribosomes previously described are active in
protein synthesis. This system will allow a characterization of synthetic
activity within isolated neurites and provide a new approach to identifying
proteins that are produced within dendrites.
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