Difference between revisions of "Neurotransporter Atlas: GLT1"

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== Contributing laboratories ==
 
== Contributing laboratories ==

Revision as of 10:45, 17 February 2015

Welcome to NT-atlas, a part of the Rodent Brain WorkBench

NT-atlas is an online public neuroscience data repository for extensive documentation of the distribution of glutamate transporters (Excitatory Amino Acid Transporters: EAATs). It provides access to collections of high resolution image data showing the distribution of glutamate receptors in mouse and rat brains.

NT-atlas is part of the Rodent Brain WorkBench, a new research and development project funded by The Research Council of Norway and the Centre for Molecular Biology and Neuroscience.

This website is under development. The first data presented concern distributions of the EAAT2 (GLT1) subtype and are based on data published in:

Holmseth, Silvia; Scott, Heather A; Real, Katia; Lehre, Knut Petter Dæhlin; Leergaard, Trygve Brauns; Bjaalie, Jan G. & Danbolt, Niels Christian (2009). The concentrations and distributions of three C-terminal variants of the GLT1 (EAAT2; slc1a2) glutamate transporter protein in rat brain tissue suggest differential regulation. Neuroscience.  ISSN 0306-4522.  162(4), s 1055- 1071 . doi:10.1016/j.neuroscience.2009.03.048

Use the links on this page to access the repository. Selected images can be viewed side-by-side in a viewer tool. 


Goals and background

The primary goal of the NT-atlas project is :

  • To better understand the system of neurotransporters by mapping the distributions of the glutamate transporters across the entire mouse and rat brain


NT-atlas is a part of the Rodent Brain WorkBench project. The goals of this project are:

  • to construct a comprehensive database, or several interoperable database systems, that will include structure and structure-function data from whole brain and selected brain regions
  • to develop tools for visualization and analysis linked to the databases and allowing detailed investigations to be performed on the data
  • to generate a digital atlas system for multiple categories of image data from rat and mouse brain

 

Background:

Glutamate is the major excitatory transmitter in the central nervous system, and is inactivated by cellular uptake catalyzed by the glutamate transporters (or Excitatory Amino Acid Transporters, EAATs): EAAT1 (GLAST), (EAAT2 (GLT1), EAAT3 (EAAC), EAAT4 and EAAT5. The EAATs belong to Solute Carrier Family (slc) number 1 (for review and references, see, Danbolt, 2001).

EAAT2 (slc1a2) is the quantitatively and functionally most important of the five EAAT subtypes in the mature brain. It accounts for more than 90 % of the total glutamate uptake activity in the forebrain and is essential for normal brain function (Danbolt et al., 1992; Haugeto et al., 1996; Tanaka et al., 1997). EAAT2 protein is expressed in astrocytes in the normal and mature nervous system (Levy et al., 1993; Chaudhry et al., 1995; Lehre et al., 1995).

Although it has been clear that EAAT2 protein is expressed in neurons in the normal and mature brain, it has been equally clear that EAAT2 mRNA is present in both neurons and astroglia (Torp et al., 1994, 1997; Berger and Hediger, 1998). It now turns out that EAAT2 protein is expressed in synaptic terminals, but at 10 times lower densities than in astroglia, and that the glutamate uptake into terminals is fully dependent on the EAAT2 gene (Furness et al. 2008).

EAAT2 is variably spliced in many different ways and is found with alternative N-termini or C-termini, as well as with other modifications (for references see: Meyer et al., 1999; Danbolt, 2001). Three C-terminal variants of EAAT2 exist:

  • GLT1a (Pines et al., 1992)
  • GLT1b (Utsunomiya-Tate et al., 1997)
  • GLT1c (Rauen et al., 2004)  

 

The major neuronal mRNA isoform is GLT1a (Berger et al., 2005) and GLT1a is the only form found in terminals (Furness et al. 2008; Holmseth et al., 2009). The question of whether GLT1b is present in neurons or not has been controversial (for references and discussion see: Holmseth et al., 2009).


The repository contains now two series of sections:

  • NM01: Mouse cryo-sections processed according to Procedure #1 (see Experimental procedures) with detergent (to maximize tissue penetation). This reveal regional differences in expression levels, but comes at the expense of cellular details.
  • R4372: Rat vibratome sections processed according to Procedure #2 (see Experimental procedures) as free-floating sections that have neither been frozen nor exposed to detergents. This procedure is best for visualizing cellular details.

 For more information, see: http://www.neurotransporter.org/

Experimental procedures

For methodological details, see Holmseth and coworkers (2009). Animals were perfusion fixed with 4 % formaldehyde and 0.2 % glutaraldehyde in 0.1 M sodium phosphate buffer.

Procedure 1: Sections were cut from frozen tissue and processed with Triton X-100. Free floating sections (40 μm thick) were treated with 1M ethanolamine–HCl (pH 7.4), blocked with 10% newborn calf serum TBS (300 mM NaCl and 100 mM Tris–HCl pH 7.4) with 0.25% Triton X-100, and incubated overnight with primary antibodies diluted in blocking solution, followed by biotinylated secondary antibodies also diluted in blocking solution and developed with the biotin–streptavidin–peroxidase system and diaminobenzidine as described (Danbolt et al., 1998). The GLT1a antibody (Ab#355) was used at 0.06 μg/ml and the GLT1b antibody (Ab#357) at 0.08 μg/ml. 

Procedure 2: Sections were cut on a Vibratome and the tissue was not frozen. Free floating sections (40 μm thick) were treated with 1M ethanolamine–HCl (pH 7.4), blocked with 10% newborn calf serum TBS (300 mM NaCl and 100 mM Tris–HCl pH 7.4), and incubated overnight with primary antibodies diluted in blocking solution, followed by biotinylated secondary antibodies also diluted in blocking solution and developed with the biotin–streptavidin–peroxidase system and diaminobenzidine as described (Danbolt et al., 1998). The GLT1a antibody (Ab#355) was used at 0.3 μg/ml and the GLT1b antibody (Ab#357) at 3 μg/ml.

 

General histochemistry

Neighboring sections were counterstained with a standard thionine stain or with a combined stain for myelin (according to Woelche, 1942) and cytoarchitecture (standard Cresyl Violet counterstain)  

 

Atlas coordinate values

For all sections, Bregma-related stereotaxic coordinates (“Bregma” in image repository) were found by anchoring selected sections to atlas diagrams using multiple anatomical landmarks. For the remaining sections, coordinates were interpolated from serial number and section thickness. Nearest matching atlas diagrams (“Atlas bregma” and corresponding “Atlas interaural” in image repository) were determined from closest matching bregma values in Franklin and Paxinos (2007, mouse brain atlas) and Paxinos and Watson (2008, rat brain atlas), respectively.  


Access image repository

The present  viewing tool allows the user to navigate within and across pairs of coronal or sagittal section images obtained from normal mice and rats. The EAAT atlas project is currently exploring different approaches for viewing of microscopic images via the web. The viewing tool is used for rapid inspection of large series of sections with images of higher resolution. 

Re-use of data from this repository is allowed provided that reference is given to the following publication:

Holmseth, Silvia; Scott, Heather A; Real, Katia; Lehre, Knut Petter Dæhlin; Leergaard, Trygve Brauns; Bjaalie, Jan G. & Danbolt, Niels Christian (2009). The concentrations and distributions of three C-terminal variants of the GLT1 (EAAT2; slc1a2) glutamate transporter protein in rat brain tissue suggest differential regulation. Neuroscience.  ISSN 0306-4522.  162(4), s 1055- 1071 . doi:10.1016/j.neuroscience.2009.03.048


Name Species CuttingDirection BlckDate Comments
NM01  MOUSE  CORONAL  2006-07-07  GLT1a, GLT1b, thionin, myelin 
R4372  RAT  SAGITTAL  2009-01-07  thionin,GLT1a,GLT1b, Right hemisphere 


Below, selected images are available for inspection.

To view the high resolution images, choose from the table above.


A: R4372s036_GLT1A

B: R4372s037_GLT1B

B: R4372s046_GLT1A

B: R4372s047_GLT1B

Template:Gallery

Contributing laboratories

The Neurotransporter Group - Knut Petter Lehre, Niels Chr. Danbolt
Centre for Molecular Biology and Neuroscience &
Institute of Basic Medical Sciences - Anatomy
University of Oslo
P.O. Box 1105 Blindern
N - 0317 Oslo
Norway
http://www.neurotransporter.org
http://www.cmbn.no


Neural Systems and Graphics Computing Laboratory - Trygve B. Leergaard, Jan G. Bjaalie
Centre for Molecular Biology and Neuroscience &
Institute of Basic Medical Sciences - Anatomy
University of Oslo
P.O. Box 1105 Blindern
N - 0317 Oslo
Norway
http://www.nesys.uio.no
http://www.rbwb.org


Credits

Histology

Anna Torbjørg Bore
Saurabh Jain


Immunohistochemistry

Silvia Holmseth
Henriette Danbolt
Knut Petter Lehre
Niels Christian Danbolt


NT-atlas web-application development team:

Jan Olav Kjøde
Ivar Andre Moene 
Dmitri Darine 
Saurabh Jain
Trygve B. Leergaard
Jan G. Bjaalie


References

Berger UV, Hediger MA (1998)
Comparative analysis of glutamate transporter expression in rat brain using differential double in situ hybridization.
Anat Embryol (Berl) 198:13-30.

Berger UV, Desilva TM, Chen WZ, Rosenberg PA (2005) 
Cellular and subcellular mRNA localization of glutamate transporter isoforms GLT1a and GLT1b in rat brain by in situ hybridization.
J Comp Neurol 492:78- 89.

Chaudhry FA, Lehre KP, Campagne MV, Ottersen OP, Danbolt NC, Storm-Mathisen J (1995)
Glutamate transporters in glial plasma membranes: highly differentiated localizations revealed by quantitative ultrastructural immunocytochemistry.
Neuron 15:711-720

Danbolt NC (2001)
Glutamate uptake.
Prog Neurobiol 65: 1-105.

Danbolt NC, Storm-Mathisen J, Kanner BI (1992)
An [Na+ + K+]coupled L-glutamate transporter purified from rat brain is located in glial cell processes.
Neuroscience 51:295-310.

Danbolt NC, Lehre KP, Dehnes Y, Chaudhry FA, Levy LM (1998)
Localization of transporters using transporter-specific antibodies.
Methods Enzymol 296:388-407.

Franklin K, Paxinos G (2007)
The mouse brain in stereotaxic coordinates (San Diego, Elsevier Academic Press)

Furness D, Dehnes Y, Akhtar A, Rossi D, Hamann M, Grutle N, Gundersen V, Holmseth S, Lehre K, Ullensvang K, Wojewodzic M, Zhou Y, Attwell D, Danbolt N (2008) 
A quantitative assessment of glutamate uptake into hippocampal synaptic terminals and astrocytes: New insights into a neuronal role for excitatory amino acid transporter 2 (EAAT2).
Neuroscience 157:80-94.

Holmseth S, Scott HA, Real K, Lehre KP, Leergaard TB, Bjaalie JG and Danbolt NC (2009) 
The concentrations and distributions of three C-terminal variants of the GLT1 (EAAT2; slc1a2) glutamate transporter protein in rat brain tissue suggests differential regulation.
Neuroscience, 2009; doi:10.1016/j.neuroscience.2009.03.048

Haugeto Ø, Ullensvang K, Levy LM, Chaudhry FA, Honoré T, Nielsen M, Lehre KP, Danbolt NC (1996)
Brain glutamate transporter proteins form homomultimers.
J Biol Chem 271:27715-27722.

Lehre KP, Levy LM, Ottersen OP, Storm-Mathisen J, Danbolt NC (1995)
Differential expression of two glial glutamate transporters in the rat brain: quantitative and immunocytochemical observations.
J Neurosci 15:1835-1853.

Levy LM, Lehre KP, Rolstad B, Danbolt NC (1993)
A monoclonal antibody raised against an [Na+ - K+]coupled L- glutamate transporter purified from rat brain confirms glial cell localization.
FEBS Lett 317:79-84.

Meyer T, Fromm A, Münch C, Schwalenstöcker B, Fray AE, Ince PG, Stamm S, Gron G, Ludolph AC, Shaw PJ (1999)
The RNA of the glutamate transporter EAAT2 is variably spliced in amyotrophic lateral sclerosis and normal individuals.
J Neurol Sci 170:45-50.

Paxinos G, Watson C (2008)
The rat brain in stereotaxic coordinates (San Diego, Elsevier Academic Press)

Pines G, Danbolt NC, Bjørås M, Zhang Y, Bendahan A, Eide L, Koepsell H, Storm-Mathisen J, Seeberg E, Kanner BI (1992)
Cloning and expression of a rat brain L-glutamate transporter.
Nature 360:464-467.

Rauen T, Wiessner M, Sullivan R, Lee A, Pow DV (2004)
A new GLT1 splice variant: cloning and immunolocalization of GLT1c in the mammalian retina and brain. 
Neurochem Int 45:1095-1106.

Tanaka K, Watase K, Manabe T, Yamada K, Watanabe M, Takahashi K, Iwama H, Nishikawa T, Ichihara N, Hori S, Takimoto M, Wada K (1997)
Epilepsy and exacerbation of brain injury in mice lacking the glutamate transporter GLT- 1.
Science 276:1699-1702.

Torp R, Danbolt NC, Babaie E, Bjørås M, Seeberg E, Storm-Mathisen J, Ottersen OP (1994)
Differential expression of two glial glutamate transporters in the rat brain: an in situ hybridization study.
Eur J Neurosci 6:936-942.

Torp R, Hoover F, Danbolt NC, Storm-Mathisen J, Ottersen OP (1997)
Differential distribution of the glutamate transporters GLT1 and rEAAC1 in rat cerebral cortex and thalamus: an in situ hybridization analysis.
Anat Embryol (Berl) 195:317- 326.

Utsunomiya-Tate N, Endou H, Kanai Y (1997)
Tissue specific variants of glutamate transporter GLT-1.
FEBS Lett 416:312-316.

Woelche M (1942)
Eine neue Methode der Markscheidenfarbung.
Psychol. Neurol. 51:199-202.