A method for isolating cortical interneurons sharing the same birthdays for gene expression studies

Experimental Neurology
Hui Xuan NgSeong-Seng Tan

Abstract

The two neuronal populations in the cortex, pyramidal neurons and interneurons, can be separated based on neurotransmitter identity, however, within this segregation a large degree of diversity exists. Investigations into the molecular diversity of neurons are impeded by the inability to isolate cell populations born at different times for gene expression analysis. Developing interneurons may be distinguished by the expression of Glutamic Acid Decarboxylase-67 (GAD67). Neuronal birthdating using nucleoside analogs is an effective means of identifying coetaneous interneurons. Using these two features, neurotransmitter identity and birthdating, we have developed a method to isolate migrating interneurons using fluorescent-activated cell sorting (FACS) for RNA extraction and gene expression analysis. We utilized 5-ethynyl-2'-deoxyuridine (EdU) to birthdate interneuron cohorts and the GAD67 knock-in GFP transgenic mice to identify interneurons. In combination, we achieved simultaneous detection of GFP and EdU signals during FACS sorting of coetaneous interneurons with minimum loss of RNA integrity. RNA quality was deemed to be satisfactory by quantitative polymerase chain reaction (qPCR) for the interneuron-specific transcript Gad67.

References

Jul 27, 1992·FEBS Letters·B Halliwell, J M Gutteridge
Jun 1, 1990·Journal of Clinical Pathology·D P JacksonP Quirke
Aug 25, 1991·Analytical Biochemistry·A SchneiderbauerD Ernst
Apr 1, 1984·The Biochemical Journal·B Halliwell, J M Gutteridge
Jul 1, 1997·Developmental Biology·L ZhuoA Messing
Feb 16, 2002·Methods : a Companion to Methods in Enzymology·K J Livak, T D Schmittgen
Jun 7, 2002·Cerebral Cortex·Stewart A AndersonSusan K McConnell
Mar 11, 2003·Annual Review of Neuroscience·Oscar Marín, John L R Rubenstein
Jun 29, 2004·Trends in Neurosciences·Arnold R Kriegstein, Stephen C Noctor
Jun 21, 2005·Developmental Dynamics : an Official Publication of the American Association of Anatomists·Anthony ActonMatthew W Grow
Feb 3, 2006·The Journal of Neuroscience : the Official Journal of the Society for Neuroscience·Vicki HammondSeong-Seng Tan
Jan 25, 2007·The Journal of Comparative Neurology·Vladimir V Rymar, Abbas F Sadikot
Jan 4, 2008·Developmental Neurobiology·Eric D MarshJeffrey A Golden
Feb 15, 2008·Proceedings of the National Academy of Sciences of the United States of America·Adrian Salic, Timothy J Mitchison
Nov 11, 2008·Journal of Neuroscience Methods·Fatemah ChehrehasaAlan Mackay-Sim
Dec 17, 2009·The Journal of Neuroscience : the Official Journal of the Society for Neuroscience·Emmanuelle BitounKay E Davies
Oct 14, 2010·The Journal of Histochemistry and Cytochemistry : Official Journal of the Histochemistry Society·Yoko MatsudaZenya Naito
Dec 15, 2010·The Journal of Neuroscience : the Official Journal of the Society for Neuroscience·Julia MahlerWolfgang Driever
Dec 25, 2010·American Journal of Physiology. Gastrointestinal and Liver Physiology·Richard J von FurstenbergSusan J Henning
Jun 21, 2011·Analytical Biochemistry·Dezhong QuBiliang Zhang
Sep 17, 2011·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Brenton L CavanaghAdrian C B Meedeniya
Nov 23, 2011·The European Journal of Neuroscience·Mary AntypaWilliam D Andrews
Apr 28, 2012·The Journal of Neuroscience : the Official Journal of the Society for Neuroscience·Naoko InamuraFujio Murakami
Aug 30, 2012·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Yanli SunJinming Li

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