Control of Synapse Number by Glia
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Purification and culture of RGCs. Step-by-step protocols for all procedures are available on request to [email protected]. RGCs were purified by sequential immunopanning to greater than 99.5% purity from P6 Sprague-Dawley rats (Simonsen Labs Gilroy CA) as described (4). About 15 000 RGCs were cultured per well in 24-well plates (Falcon) on glass (Assistant) or Aclar 22C (Allied Signal) cover slips coated with poly- d -lysine (10 μg/ml) followed by merosin (2 μg/ml) or laminin (2 μg/ml). RGCs were cultured in 600 μl of serum-free medium modified from Bottenstein and Sato (34) containing Neurobasal (Gibco) bovine serum albumin selenium putrescine triiodo-thyronine transferrin progesterone pyruvate (1 mM) glutamine (2 mM) ciliary neurotrophic factor (CNTF) (10 ng/ml) brain-derived neurotrophic factor (BDNF) (50 ng/ml) insulin (5 μg/ml) and forskolin (10 μM). Recombinant human BDNF and CNTF were provided by Regeneron Pharmaceuticals. Tetrodotoxin (TTX) and picrotoxin were from RBI. Antibodies were obtained as follows: Anti-synaptophysin (Sigma) anti-GluR2/3 (Upstate Biotech) and anti–PSD-95 (Chemicon). An antibody to synaptotagmin was generated by immunization of a rabbit with a peptide corresponding to the NH 2 -terminal lumenal portion of synaptotagmin (22). All other reagents were obtained from Sigma.
K. Christopherson C. Harrington S. Venkatapathy B. A. Barres data not shown.
Preparation of astrocytes. Collicular glia were prepared as described (23). Briefly p1-2 SCs were digested with trypsin and plated in tissue culture flasks (Falcon) in a medium that does not allow neurons to survive [Dulbucco's minimum essential medium fetal bovine serum (10%) penicillin (100 U/ml) streptomycin (100 mg/ml) glutamine (2 mM) and Na-pyruvate (1 mM)]. After 4 days nonadherent cells were shaken off flasks and remaining cells were removed enzymatically and cultured in contact with RGCs unless otherwise stated.
Electrophysiology. Membrane currents were recorded by whole-cell patch clamping at room temperature (18° to 22°C) at a holding potential of −70 mV unless otherwise specified. Patch pipettes (3 to 10 megohm) were pulled from borosilicate capillary glass (WPI). For recordings of synaptic currents the bath solution contained (in mM) 120 NaCl 3 CaCl 2 2 MgCl 2 5 KCl and 10 Hepes (pH 7.3). The internal pipet solution contained (in mM) 100 K-gluconate 10 KCl 10 EGTA (Ca 2+ -buffered to 10 −6 ) and 10 Hepes (pH 7.3). For recordings of autaptic currents the internal pipet solution contained (in mM) 122.5 K-gluconate 8 NaCl 10 Hepes 0.2 EGTA 2 Mg-ATP 0.3 Na-GTP 20 K 2 -creatine phosphate and phosphocreatine kinase (50 U/ml). For recordings of calcium currents the bath solution contained 5 mM BaCl 2 160 mM tetraethylammonium-Cl 10 mM Hepes and 1 μM TTX. The internal solution contained (in mM) 108 CsMeSO 3 4.5 MgCl 2 9 EGTA 24 Hepes 4 Na-ATP and 0.3 Mg-GTP. All data were analyzed with Sigma Stat (SPSS) Origin (Microcal Software) or Mini Analysis Program (Synaptosoft). All tests were Student t tests unless otherwise stated.
E. M. Ullian S. K. Sapperstein K. Christopherson B. A. Barres data not shown.
Imaging. Cover slips were mounted in a rapid-switching perfusion chamber. Cultures were perfused at room temperature in a saline solution consisting of (in mM) 120 NaCl 2.5 KCl 2 CaCl 2 2 MgCl 2 25 Hepes (pH 7.4) 10 μM CNQX (6-cyano-7-nitroquinoxaline-2 3-dione) and 50 μM APV ( d l -2-amino-5-phosphonovaleric acid). Synaptic terminals were loaded in the presence of FM1-43 (15 μM) and 90 mM KCl. After loading a field containing labeled puncta was chosen. A 4 pixel by 4 pixel area around the center of mass of each of these puncta was imaged with a cooled charge-coupled device camera through a 60 by 1.3 numerical aperture objective mounted on a Nikon diaphot inverted microscope and Metamorph imaging software (Universal Imaging). The FM1-43 was unloaded with 90-s stimulation with 90 mM KCl in FM1-43–free perfusion solution.
Western blot analysis. RGC cell lysates were prepared by extraction with 2% SDS. Proteins were resolved by SDS–polyacrylamide gel electrophoresis and transferred onto polyvinylidene difluoride (Millipore). Membranes were incubated in blocking buffer [phosphate-buffered saline (PBS) containing 0.1% Tween-20 and 5% nonfat milk] for 30 min at room temperature followed by incubation for 1 hour in blocking buffer containing either affinity-purified rabbit antibodies to synaptotagmin lumenal domain (1.0 μg/ml) mouse anti-synaptophysin (1:1000; Sigma) or mouse anti-p115 (1:2000) (provided by M. G. Waters Princeton University). Immunoreactive proteins were detected with horseradish peroxidase (HRP)–conjugated anti-rabbit or anti-mouse immunoglobulin G (1:40 000; Jackson Immunoresearch) and visualized with a chemiluminescent substrate for HRP (SuperSignal West Pico Pierce Chemicals).
Immunocytochemistry. RGC cultures were fixed in 4% paraformaldehyde in PBS or phosphate buffer (pH 7.4) for 5 min at room temperature and washed in PBS containing 0.3% Triton X-100. Primary antibodies were applied at various concentrations overnight in staining buffer [0.5% bovine serum albumin 0.5 % Triton X-100 30 mM NaPO 4 (pH 7.4) 750 mM NaCl 5% normal goat serum and 0.4% NaN 3 ]. Secondary antibodies goat anti-mouse and anti-rabbit Alexa 488 and Alexa 594 conjugates (Molecular Probes) were used at a dilution of 1:200 for 1 hour at room temperature. All washes were done with PBS containing 0.1% Triton X-100.
Target SC neurons were purified as described (6). Briefly SC neurons were dissociated in papain and microglia macrophages and oligodendrocytes were removed by panning with the Griphonia Symplicifolia lectin 1 (Vector Labs) and the O4 antibody. Neurons were then selected with an ASCS4 monoclonal antibody (DSHB).
Supplementary data are available on Science Online at www.sciencemag.org/cgi/content/full/291/5504/657/DC1.
Electron microscopy. Cells were prepared for EM as described (6). Briefly cells were washed in 0.1 M phosphate buffer (pH 7.2) then fixed for 30 min in 2% glutaraldehyde buffered with 0.1 M sodium phosphate (pH 7.2) at 4°C. After rinsing with buffer specimens were stained en bloc with 2% aqueous uranyl acetate for 15 min dehydrated in ethanol and embedded in poly/bed812 for 24 hours. Fifty-nanometer sections were poststained with uranyl acetate and lead citrate and viewed with a Philips Electronic Instruments CM-12 transmission electron microscope.
Reversal experiments. RGCs were cultured with glia for 5 days after which the glia were removed and the media exchanged for fresh media. Control RGCs were recorded after 5 days of coculture with glia and exchange of media for astrocyte-conditioned media.
Bottenstein J. E., Sato G. H., J. Neurosci. 76, 514 (1979).
We thank E. Kavalali for advice on FM1-43 experiments J. Waters and P. De Koninck for critically reading a draft of the manuscript and M. Hernandez for technical assistance. Supported by a Mcknight Investigator Award (B.A.B.) a Kirsch Investigator Award from the Steven and Michele Kirsch Foundation (B.A.B.) and NIH grant NS10784 (E.M.U.).