Technical Reference #1645

1645.

Up-Regulation of Astrocyte Metabotropic Glutamate Receptor 5 by Amyloid0Beta Peptide Christopher Casley; Viktor Lakics; Hyoung-gon Lee; Lisa Broad; Theresa Day; Tricia Cluett; Mark Smith; Michael O;Neill; Ann Kingston, Eli Lilly & Company, Brain Research, 1260(1645), (2009)
Link To Paper

Abstract:
The effects of amyloid-beta peptide (Aβ) on astrocyte responses to activation of mGlu5receptors have been investigated using calcium imaging. Pre-incubation with Aβ1–40peptide for up to 72 h produced a time- and concentration-dependent 2–4 fold enhanc

Keywords:
Amyloid beta protein; mGlu5; AD; astrocytes; calcium mobilization

Materials & Methods:
4.1. Materials (S)-35-Dihydroxyphenylglycine (DHPG) 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP) (RS)-α-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid hydrobromide (AMPA) 6-chloro-34-dihydro-3-(5-norbornen-2-yl)-2H- 124-benzothiazidiazine-7-sulfonamide-11-dioxide (cyclothiazide) were obtained fromTocris Cookson Ltd. (Bristol UK). Fluo- 3-AM and fura-2-AM were obtained from Molecular Probes (Leiden The Netherlands). Basic FGF (bFGF) was obtained from R&D Biologicals (UK). All other chemicals were obtained from Sigma-Aldrich(DorsetUK).Cell culturemedia and supplements were purchased fromLife Technologies Ltd. (UK). Poly-D-lysinecoated (35 mm) glass-bottomed microwell dishes were from MatTek Corporation (Ashland MA) and 96-well poly-D-lysinecoated FLIPR plates were from Becton Dickenson UK. 4.2. Tissue culture Astrocyte-rich cultures were prepared from neonatal C57BL/6J mouse cortices (McCarthy and de Vellis 1980). Briefly cortices were dissected and meninges removed before tissue was triturated and grown in 75 cm2 tissue culture flasks in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% foetal calf serum (FCS) and antibiotic–antimycotic solution (Sigma). Cells were maintained in culture for 21 days at which point they were typically 90% confluent. Cells were then plated at a density of 3×105 cells/cm2 into: 35 mm poly- D-lysine-coated glass-bottomed microwell dishes (calcium imaging studies) or poly-D-lysine-coated 96-well optical plates (FLIPR assay) or 6-well plates (for RT-PCR studies). Th cultures were then treated with L-leucine-methyl-ester (LLME 20 mM for 2 h) an agent known to selectively kill microglia (Giulian and Baker 1986). Following LLME treatment cultures were washed and used for experiments 24 h later. Purity of cultures obtained using this method was evaluated by immunohistochemistry for glial fibrillary acidic protein (GFAP) or CD11b a selective marker for microglia and macrophages. Cultures were typically found to be 95% astrocytes with 5% microglia. 4.3. Preparation of Aβ peptides Aβ1–40 and Aβ40–1 were obtained from Bachem (Merseyside UK) and dissolved in 0.01% acetic acid at 200 μM initial concentration. Peptides were incubated at room temperature for 72 h. The aggregation state of peptides was verified by thioflavin-T assay (LeVine 1993). Peptide solutions were then diluted appropriately in DMEM for cell treatments. 4.4. Fluorescence imaging detection of calcium responses Cells were loaded in growth media supplemented with 2 μM fura-2-AM at 37 °C for 30 min and were then washed and incubated for a further 30 min at room temperature in HEPESbuffered saline solution in the absence of fura-2. The composition of HEPES-buffered saline solution was: 135 mM NaCl 5 mM KCl 2.5 mM CaCl2 1.2 mM MgCl2 10 mM glucose 10 mM HEPES (pH 7.3). After this time dishes containing cells were mounted on the stage of an inverted epifluorescence microscope (Axiovert 100TV Zeiss Oberkochen Germany) and viewed using a ×10 (air) or ×40 (oil immersion) fluorescence objective. Cells loaded with fura-2 were excited by light of wavelength 340 and 380 nm which was provided by a polychrome II housing a xenon lamp and monochromator from T.I.L.L. photonics (Planegg Germany). Emitted light was passed through a dichroic mirror (400 nm) and high-pass barrier filter (480 nm) and subsequently captured by a SensiCam cooled CCD camera (PCO CCD Imaging Kelheim Germany). Digitized images were stored and processed using Axon Imaging Workbench software (Version 2.2 and 4 Axon Instruments Inc. Union City CA USA). Traces obtained using CCD imaging were corrected for background and data were normalized to baseline and expressed as ratios of excitation at 340 nm and 380 nm. Calcium responses were also assessed using a FLIPR (Fluorescent Imaging Plate Reader) system (Molecular Devices Winnersh UK). In these experiments cells were loaded with the calcium-sensitive dye fluo-3-AM at 10 μM in Tyrode's buffer [137 mM NaCl 2.5 mM CaCl2 2.7 mM KCl 1 mM MgCl2 0.2 mM NaHPO4 12 mM NaHCO3 and 5 mM glucose] for 1 h at room temperature. The media was then removed and replaced with Tyrode's buffer. In a subset of experiments the media was replaced with calcium-free Tyrode's buffer which contained 100 μM EGTA. 96-well plates were then transferred to a FLIPR system for analysis. The dye was excited by light of 488 nm wavelength from an Argon laser and the emitted fluorescence passed through a 510–570 nm bandpass interference filter before detection with a cooled CCD camera (Princeton Instruments). Fluorescence was recorded every second for the first minute following agonist addition with additional readings every 6 s for a further 2 min. Data files were saved to a Dell Optiplex GX110 computer and stored for offline analysis using FLIPR system software and Origin 6.1 (Origin-Lab). Agonists were added to all 96 wells simultaneously by means of an inbuilt automated pipetting system. Results are expressed as relative fluorescence units (RFU) (maximum fluorescence value after agonist addition minus the minimum fluorescence value obtained prior to addition). All data are presented as the mean±SEM of quadruplicate cultures. Non-linear regression curve fitting and statistical analyses were performed with GraphPad software San Diego CA USA. 4.5. Reverse transcriptase PCR Total RNA was extracted from astrocytes in 6-well plates using the RNAquaeous 4PCR™ total RNA isolation kit (Ambion Austin TX). First strand cDNA synthesis was carried out using 1 μg of DNA-free total RNA 37°C for 60 min. PCR of mGlu5 cDNA was carried out using primers and conditions as described in Minoshima and Nakanishi (1999). Primer sequences were as follows: 5′ mGlu5 primer 5′-GTC TCC TGA TGT CAA GTG GTT-3′ 3′ mGlu5 primer 5′-GGA CCA CAC TTC GTC ATC ATC-3′. The optimum number of cycles was determined experimentally and was defined as the number of cycles producing detectable PCR-amplified product under non-saturating conditions. For mGlu5 32 PCR cycles were performed (1 min 94 °C 1 min 60 °C and 2 min 72 °C). PCR products were sequenced and confirmed to be mGlu5 and quantified by densitometry of the ethidium-bromide-stained gels after electrophoresis using a Kodak EDAS-120 digital gel documentation system and 1D Image Analysis Software (Eastman Kodak Company Rochester NY). To determine the magnitude of change in gene expression densities of PCR products were compared to standard curves generated from serially diluted samples known to be positive for the transcript of interest. Expression of the reference genes β-actin and S18 were also evaluated in each test sample as described above. Quantum RNA™ β-actin and S18 internal standard kits (Ambion Austin TX) were used to normalize PCR product levels according to the manufacturers' instructions. mGluR5 expression in untreated astrocyte cultures was assigned a value of 1 and gene expression in the rest of the test samples was calculated relative to this arbitrary value.

Microscopic Technique
Fluroescence Microscopy, Epifluorescence

Cell Type(s)
glial