Abstract
Most neurons elaborate a characteristic dendritic arbor which is physiologically important for receiving and processing of synaptic inputs. Pathologically, disturbances in the regulation of dendritic tree complexity are often associated with mental retardation and other neurological deficits. Rho GTPases are major players in the regulation of dendritic tree complexity. They are involved in many signal transduction cascades, activated at the neuronal plasma membrane, and relayed to intracellular proteins that directly rearrange the cytoskeleton. The use of siRNA technology combined with morphometric and imaging techniques allows the roles of individual Rho GTPases, such as Rac1, in dendritic branching to be examined. In this chapter we describe the establishment, transfection, and processing of a primary hippocampal cell culture. Methods to assess the complexity of dendritic arbors like the Sholl analysis, and techniques to investigate Rac1 activity in hippocampal cells, and specifically in neuronal dendrites, such as fluorescence resonance energy transfer (FRET) imaging are presented.
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Jaffe AB, Hall A (2005) Rho GTPases: biochemistry and biology. Annu Rev Cell Dev Biol 21:247–269
Heasman SJ, Ridley AJ (2008) Mammalian rho GTPases: new insights into their functions from in vivo studies. Nat Rev Mol Cell Biol 9:690–701
Luo L (2002) Actin cytoskeleton regulation in neuronal morphogenesis and structural plasticity. Annu Rev Cell Dev Biol 18:601–613
Jan YN, Jan LJ (2010) Branching out: mechanisms of dendritic arborisation. Nat Rev Neurosci 11:316–328
Schulz J, Franke K, Frick M, Schumacher S (2016) Different roles of the small GTPases Rac1, Cdc42, and RhoG in CALEB/NGC-induced dendritic tree complexity. J Neurochem 139:26–39
Franke K, Otto W, Johannes S, Baumgart J, Nitsch R, Schumacher S (2012) miR-124-regulated RhoG reduces neuronal process complexity via ELMO/Dock180/Rac1 and Cdc42 signalling. EMBO J 31:2908–2921
Sholl DA (1953) Dendritic organization in the neurons of the visual and motor cortices of the cat. J Anat 87:387–406
Sander EE, van Delft S, ten Klooster JP, Reid T, van der Kammen RA, Michiels F, Collard JG (1998) Matrix-dependent TIAM1/Rac signaling in epithelial cells promotes either cell-cell adhesion or cell migration and is regulated by phosphatidylinositol 3-kinase. J Cell Biol 143:1385–1398
Spiering D, Bravo-Cordero JJ, Moshfegh Y, Miskolci M, Hodgson L (2013) Quantitative ratiometric imaging of FRET-biosensors in living cells. Methods Cell Biol 114:593–609
Fritz RD, Menshykau D, Martin K, Reimann A, Pontelli V, Pertz O (2015) SrGAP2-dependent integration of membrane geometry and Slit-Robo-repulsive cues regulates fibroplast contact inhibition of locomotion. Dev Cell 35:78–92
Chen JG, Rasin MR, Kwan KY, Sestan N (2005) Zfp312 is required for subcortical axonal projections and dendritic morphology of deep-layer pyramidal neurons of the cerebral cortex. Proc Natl Acad Sci U S A 102:17792–17797
Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, Preibisch S, Rueden C, Saalfeld S, Schmid B, Tinevez J-Y, White JD, Hartenstein V, Eliceiri K, Tomancak P, Cardona A (2012) Fiji: an open-source platform for biological-image analysis. Nat Methods 9:676–682
Ferreira T, Blackman A, Oyrer J, Jayabal A, Chung A, Watt A, Sjöström J, van Meyel D (2014) Neuronal morphometry directly from bitmap images. Nat Methods 11:982–984
Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to ImageJ: 25 years of image analysis. Nat Methods 9:671–675
Green MR, Sambrook J (2012) Molecular cloning: a laboratory manual, 4th edn. CSHL Press, New York
Kardash E, Bandemer J, Raz E (2011) Imaging protein activity in live embryos using fluorescence resonance energy transfer biosensors. Nat Protoc 6:1835–1846
Aoki K, Matsuda M (2009) Visualization of small GTPase activity with fluorescence resonance energy transfer-based biosensors. Nature Protoc 4:1623–1631
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Schulz, J., Schumacher, S. (2018). Methods to Study the Roles of Rho GTPases in Dendritic Tree Complexity. In: Rivero, F. (eds) Rho GTPases. Methods in Molecular Biology, vol 1821. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8612-5_21
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DOI: https://doi.org/10.1007/978-1-4939-8612-5_21
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