Abstract
In neurons, local translation of mRNAs contributes to axon outgrowth and activity-dependent synaptic plasticity. The identification and visualization of individual localized mRNAs is critical for understanding these processes. Here, we describe a sensitive fluorescence in situ hybridization (FISH) method that provides high-resolution information on mRNA localization using in vitro cultured rat hippocampal neurons. The method employs digoxigenin (DIG)-labeled RNA probes and fluorescent tyramide signal amplification for detection of mRNAs. It enables not only the visualization but also quantification of mRNA granules, and changes in their localization in response to various stimuli. The method is also compatible with immunocytochemistry, which allows for the co-visualization of RNAs and proteins in fixed cells.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Kandel ER (2001) The molecular biology of memory storage: a dialogue between genes and synapses. Science 294:1030–1038
Torre ER, Steward O (1992) 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. J Neurosci 12:762–772
Steward O, Wallace CS, Lyford GL, Worley PF (1998) Synaptic activation causes the mRNA for the IEG Arc to localize selectively near activated postsynaptic sites on dendrites. Neuron 21:741–751
Holt CE, Schuman EM (2013) The central dogma decentralized: new perspectives on RNA function and local translation in neurons. Neuron 80:648–657
Darnell RB (2013) RNA protein interaction in neurons. Annu Rev Neurosci 36:243–270
Mikl M, Vendra G, Kiebler MA (2011) Independent localization of MAP2, CaMKIIα and β-actin RNAs in low copy numbers. EMBO Rep 12:1077–1084
Schenborn ET, Mierendorf RC Jr (1985) A novel transcription property of SP6 and T7 polymerases: dependence on template structure. Nucleic Acids Res 13:6223–6236
Goslin K, Asmussen H, Banker G (1998) Rat hippocampal neurons in low-density culture. In: Banker G, Goslin K (eds) Culturing nerve cells, 2nd edn. MIT, Cambridge, MA
Goetze B, Tuebing F, Yunli X, Dorostkar M, Thomas S, Pehl U, Boehm S, Macchi P, Kiebler MA (2006) The brain-specific double-stranded RNA-binding protein Staufen2 is required for dendritic spine morphogenesis. J Cell Biol 172:221–231
Heraud-Farlow JE, Sharangdhar T, Li X, Pfeifer P, Tauber S, Orozco D, Hörmann A, Thomas S, Bakosova A, Farlow AR, Edbauer D, Lipshitz HD, Morris QD, Bilban M, Doyle M, Kiebler MA (2013) Staufen2 regulates neuronal target RNAs. Cell Rep 5(6):1511–1518
Femino AM, Fay FS, Fogarty K, Singer RH (1998) Visualization of single RNA transcripts in situ. Science 280:585–590
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Heraud-Farlow, J.E., Sharangdhar, T., Kiebler, M.A. (2015). Fluorescent In Situ Hybridization in Primary Hippocampal Neurons to Detect Localized mRNAs. In: Hauptmann, G. (eds) In Situ Hybridization Methods. Neuromethods, vol 99. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2303-8_16
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2303-8_16
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2302-1
Online ISBN: 978-1-4939-2303-8
eBook Packages: Springer Protocols