Dynamics of glutamatergic signaling in the mushroom ... - HAL - ESPCI

Sinakevitch et al. Neural Development 2010, 5:10
http://www.neuraldevelopment.com/content/5/1/10
Page 18 of 20
Tris-SMB supplemented with 0.5% (v/v) Triton X-100
(Tris-SMB-Tx), then pre0incubated with 5% (v/v) normal
swine serum (Dako, Glostrup, Denmark) in Tris-SMB-Tx
for 1 hour. Brain sections were then simultaneously incubated
with rabbit Glu antiserum (1:1,000) and chicken
anti-GFP (1:1,000) in Tris-SMB-Tx overnight at room
temperature. After a wash in Tris-Tx, sections were incubated
overnight at room temperature with conjugated
secondary antibodies diluted 1:250. After a final wash in
Tris buffer, sections were embedded in 80% glycerol.
For marker proteins, Drosophila brains were dissected
and fixed overnight in 4% paraformaldehyde in PBS
made from tablets (Sigma P4417). The fixed brains were
washed in PBS, embedded in 8% agarose and cut in
serial 60-μm sections. Sections were washed four times
in PBS containing 0.5% (v/v) Triton X-100 (PBS-Tx),
then blocking solution was applied consisting of 10% (v/v)
swine serum in PBS-Tx. After one night incubation with
primaryantibodiesinPBS-Tx,thesectionswerethoroughly
washed in PBS and then incubated with the
secondary antibodies at 1:500. After a final wash in PBS,
sections were embedded in 80% glycerol.
Microscopy
Images were collected on a Zeiss LSM 510 confocal
microscope (Carl Zeiss, Oberkochen, Germany). Series
of four to ten 0.5-μm optical sections (1,024 × 1,024 at
12-bit color depth) were scanned using 40 × 1.0 or 63 ×
1.4 oil iris Plan-Apochromat objectives. The images
were stored as TIFF files and edited in Adobe Photoshop
CS2. The Glu and dNR1 antibodies gave only
superficial staining in sections so the images were composed
of two to five optical sections for these antigens.
For DVGluT, GS and ubiquitin, images were made from
up to ten optical sections from a stack. The contrast
and intensity were adjusted accordingly.
Abbreviations
a/bc: a/b core; BSA: bovine serum albumin; FXS: fragile X syndrome; GFP:
green fluorescent protein; Glu: L-glutamate; GS: glutamine synthetase; iGluR:
ionotropic receptor; KC: Kenyon cell; MB: mushroom body; mGluR:
metabotropic receptor; NMDAR: NMDA receptor; PBS: phosphate-buffered
saline; SMB: sodium metabisulfite; VGluT: vesicular Glu transporter.
Acknowledgements
We thank Drs G Romano, L Restifo and J-M Dura for fly stocks, Drs A
DiAntonio, AS Chiang and D Kalderon for antibodies, and Drs SM Brown and
M-T Besson for valuable discussions. We are grateful to to Drs. M. Adams, B.
H. Smith, C. Durch and to J Patterson (Ms) for providing technical and
material support to IS for the experiments of Figure 4. This work benefited
from a two-year Programme d’Urgence FRM/Postdocs UFP20060306548
from the Fondation pour la Recherche Médicale to IS and a research grant
MNM2-11627 from the Association Française contre les Myopathies to SB.
Author details
1 Laboratoire de Neurobiologie, CNRS UMR 7637, ESPCI ParisTech, 10 rue
Vauquelin, 75231 Paris cedex 5, France.
2 Institut de Biologie du
Développement de Marseille-Luminy, CNRS UMR 6216, Université de la
Méditerranée, Campus de Luminy case 907, 13288 Marseille cedex 9, France.
3 Institut de Génomique Fonctionnelle, CNRS UMR 5203, INSERM U661,
Université de Montpellier I et II, 141 rue de la Cardonille, 34094 Montpellier
cedex 5, France.
4 Center for Insect Science and Department of Neuroscience,
University of Arizona, Tucson, Arizona 85721, USA.
5 Current address: Arizona
State University, School of Life Sciences, Tempe, AZ 85287-4501, USA.
Authors’ contributions
IS designed and performed the immunohistochemistry experiments,
analyzed the data and wrote the manuscript. YG performed the targeted
transposition. NJS supervised initial experiments and wrote the manuscript.
SB supervised the experiments and wrote the manuscript. All authors
approved the final manuscript.
Competing interests
The authors declare that they have no competing interests.
Received: 12 September 2009 Accepted: 6 April 2010
Published: 6 April 2010
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