PMID- 22649026
OWN - NLM
STAT- MEDLINE
DCOM- 20130307
LR  - 20211021
IS  - 1552-4930 (Electronic)
IS  - 1552-4922 (Print)
IS  - 1552-4922 (Linking)
VI  - 81
IP  - 8
DP  - 2012 Aug
TI  - Quantitative analysis of BDNF/TrkB protein and mRNA in cortical and striatal 
      neurons using alpha-tubulin as a normalization factor.
PG  - 704-17
LID - 10.1002/cyto.a.22073 [doi]
AB  - The neurotrophin brain-derived neurotrophic factor (BDNF) and its receptor 
      tyrosine kinase TrkB serve important regulatory roles for multiple aspects of the 
      biology of neurons including cell death, survival, growth, differentiation, and 
      plasticity. Regulation of the local availability of BDNF/TrkB at distinct 
      subcellular domains such as soma, dendrites, axons, growth cones, nerve 
      terminals, and spines appears to contribute to their specific functions. In view 
      of the variance in size and shape of neurons and their compartments, previous 
      quantitative studies of the BDNF/TrkB protein and mRNA lacked a robust 
      normalization procedure. To overcome this problem, we have established methods 
      that use immunofluorescence detection of alpha-tubulin as a normalization factor for 
      the quantitative analysis of protein and mRNA in primary rat cortical and 
      striatal neurons in culture. The efficacy of this approach is demonstrated by 
      studying the dynamic distribution of proteins and mRNA at different growth stages 
      or conditions. Treatment of cultured neurons with KCl resulted in increased 
      levels of TrkB protein, reduced levels of BDNF mRNA (composite of multiple 
      transcripts) and a slight reduction in BDNF protein levels in the dendrites from 
      the cortex. The KCl treatment also lowered the percentage of BDNF and TrkB 
      proteins in the soma indicative of protein transport. Finally, analysis of the 
      rat cortical and striatal neurons demonstrated comparable or even higher levels 
      of BDNF/TrkB protein and BDNF mRNA in the neurons from the striatum. Thus, in 
      contrast to previous observations made in vivo, striatal neurons are capable of 
      synthesizing BDNF mRNA when cultured in growth media in vitro. The analytical 
      approach presented here provides a detailed understanding of BDNF/TrkB levels in 
      response to a variety of neuronal activities. Our methods could be used broadly, 
      including applications in cell and tissue cytometry, to yield accurate 
      quantitative data of gene expression in cellular and subcellular contexts. (c) 2012 
      International Society for Advancement of Cytometry.
CI  - Copyright (c) 2012 International Society for Advancement of Cytometry.
FAU - Ma, Bin
AU  - Ma B
AD  - Department of Microbiology, New York University School of Medicine, New York, New 
      York 10016, USA.
FAU - Savas, Jeffrey N
AU  - Savas JN
FAU - Chao, Moses V
AU  - Chao MV
FAU - Tanese, Naoko
AU  - Tanese N
LA  - eng
GR  - R01 NS061917/NS/NINDS NIH HHS/United States
GR  - S10 RR017970/RR/NCRR NIH HHS/United States
GR  - NS061917/NS/NINDS NIH HHS/United States
PT  - Journal Article
PT  - Research Support, N.I.H., Extramural
DEP - 20120530
PL  - United States
TA  - Cytometry A
JT  - Cytometry. Part A : the journal of the International Society for Analytical 
      Cytology
JID - 101235694
RN  - 0 (Brain-Derived Neurotrophic Factor)
RN  - 0 (RNA, Messenger)
RN  - 0 (Tubulin)
RN  - 147336-22-9 (Green Fluorescent Proteins)
RN  - EC 2.7.10.1 (Receptor, trkB)
SB  - IM
MH  - Animals
MH  - Blotting, Western
MH  - Brain-Derived Neurotrophic Factor/*genetics/metabolism/pharmacology
MH  - Cells, Cultured
MH  - Cerebral Cortex/*cytology
MH  - Densitometry
MH  - Fluorescence
MH  - Gene Expression Regulation
MH  - Green Fluorescent Proteins/metabolism
MH  - In Situ Hybridization, Fluorescence
MH  - Microscopy, Confocal
MH  - Neostriatum/*cytology
MH  - Neurons/*metabolism
MH  - RNA, Messenger/genetics/metabolism
MH  - Rats
MH  - Receptor, trkB/*genetics/metabolism
MH  - Tubulin/*metabolism
PMC - PMC3549458
MID - NIHMS432377
EDAT- 2012/06/01 06:00
MHDA- 2013/03/08 06:00
PMCR- 2013/01/20
CRDT- 2012/06/01 06:00
PHST- 2011/12/11 00:00 [received]
PHST- 2012/03/20 00:00 [revised]
PHST- 2012/04/26 00:00 [accepted]
PHST- 2012/06/01 06:00 [entrez]
PHST- 2012/06/01 06:00 [pubmed]
PHST- 2013/03/08 06:00 [medline]
PHST- 2013/01/20 00:00 [pmc-release]
AID - 10.1002/cyto.a.22073 [doi]
PST - ppublish
SO  - Cytometry A. 2012 Aug;81(8):704-17. doi: 10.1002/cyto.a.22073. Epub 2012 May 30.