Affiliation:
1. Stanford University School of Medicine, Department of Neurobiology, Fairchild Science Building, Stanford, CA 94305–5125, USA.
Abstract
Although astrocytes constitute nearly half of the cells in our brain, their function is a long-standing neurobiological mystery. Here we show by quantal analyses, FM1-43 imaging, immunostaining, and electron microscopy that few synapses form in the absence of glial cells and that the few synapses that do form are functionally immature. Astrocytes increase the number of mature, functional synapses on central nervous system (CNS) neurons by sevenfold and are required for synaptic maintenance in vitro. We also show that most synapses are generated concurrently with the development of glia in vivo. These data demonstrate a previously unknown function for glia in inducing and stabilizing CNS synapses, show that CNS synapse number can be profoundly regulated by nonneuronal signals, and raise the possibility that glia may actively participate in synaptic plasticity.
Publisher
American Association for the Advancement of Science (AAAS)
Reference35 articles.
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5. Purification and culture of RGCs. Step-by-step protocols for all procedures are available on request to barres@stanford.edu. 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.
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