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J Histochem Cytochem. 2016 May;64(5):323-33. doi: 10.1369/0022155416641604. Epub 2016 Mar 29.

Monitoring mRNA Translation in Neuronal Processes Using Fluorescent Non-Canonical Amino Acid Tagging.

Author information

1
Department of Cognitive Neuroscience, Radboud University Medical Centre, 6500 HB Nijmegen, The Netherlands (AK)
2
Donders Institute for Brain, Cognition, and Behaviour, Centre for Neuroscience, 6525 AJ Nijmegen, The Netherlands (AK, KAW)
3
Language and Genetics Department, Max Planck Institute for Psycholinguistics, 6525 XD Nijmegen, The Netherlands (KAW)
4
Laboratory of Molecular Biology, National Institute of Mental Health, National Institutes of Health, Bethesda, Maryland (AG, BBK, AA)
5
Department of Molecular Animal Physiology, Radboud University, Nijmegen, The Netherlands (GJM)

Abstract

A steady accumulation of experimental data argues that protein synthesis in neurons is not merely restricted to the somatic compartment, but also occurs in several discrete cellular micro-domains. Local protein synthesis is critical for the establishment of synaptic plasticity in mature dendrites and in directing the growth cones of immature axons, and has been associated with cognitive impairment in mice and humans. Although in recent years a number of important mechanisms governing this process have been described, it remains technically challenging to precisely monitor local protein synthesis in individual neuronal cell parts independent from the soma. This report presents the utility of employing microfluidic chambers for the isolation and treatment of single neuronal cellular compartments. Furthermore, it is demonstrated that a protein synthesis assay, based on fluorescent non-canonical amino acid tagging (FUNCAT), can be combined with this cell culture system to label nascent proteins within a discrete structural and functional domain of the neuron. Together, these techniques could be employed for the detection of protein synthesis within developing and mature neurites, offering an effective approach to elucidate novel mechanisms controlling synaptic maintenance and plasticity.

KEYWORDS:

FUNCAT; axon; click assay; local protein synthesis; microfluidic chambers; neurons; synapse

PMID:
27026294
PMCID:
PMC4851274
DOI:
10.1369/0022155416641604
[Indexed for MEDLINE]
Free PMC Article

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