Synaptic efficacy enhanced by glial cells in vitro

Science

Volumn 277, Issue 5332, 1997, Pages 1684-1687

  Pfrieger, Frank W ^a,b   Barres, Barbara A ^a  

^a STANFORD UNIVERSITY SCHOOL OF MEDICINE   (United States)

^b MAX DELBRÜCK CENTER FOR MOLECULAR MEDICINE   (Germany)

Author keywords

[No Author keywords available]

Indexed keywords

ANIMAL CELL; ARTICLE; BRAIN MATURATION; CELL SURVIVAL; CONTROLLED STUDY; GLIA CELL; NERVE CELL MEMBRANE POTENTIAL; NONHUMAN; PRIORITY JOURNAL; RAT; RETINA GANGLION CELL; SYNAPTOGENESIS; ULTRASTRUCTURE;

ACTION POTENTIALS; ANIMALS; ASTROCYTES; CELL SURVIVAL; CELLS, CULTURED; COCULTURE TECHNIQUES; MICROGLIA; NEUROGLIA; OLIGODENDROGLIA; RATS; RATS, SPRAGUE-DAWLEY; RETINAL GANGLION CELLS; SYNAPSES; SYNAPTIC TRANSMISSION;

ANIMALIA;

EID: 0030773837     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.277.5332.1684     Document Type: Article

References (33)

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14. 2. All results in this study were obtained from at least three independent culture preparations unless otherwise indicated. Care and handling of Sprague-Dawley rats were in accordance with institutional guidelines.
15. Membrane currents were recorded at room temperature (18° to 22°C) and at a holding potential of -70 mV with the whole-cell configuration of the patch-clamp technique as described (7). Postsynaptic currents were analyzed with a computer program (Lab-view software, National Instruments Corp.). We visually checked whether detected events resembled postsynaptic currents.
16. Collicular glia were prepared as described [K. D. McCarthy and J. De Vellis, J. Cell Biol. 85, 890 (1980)]. Briefly, trypsin-digested and triturated superior colliculi (0.5 mm thick) from P2 rats were cultured in poly-D-lysine-coated tissue culture flasks (Falcon), in a medium that does not support survival of neurons, containing Dulbecco's minimum essential medium, fetal bovine serum (10%), penicillin (100 U/ml), streptomycin (100 mg/ml), glutamine (2 mM), and sodium pyruvate (1 mM; all supplied by Gibco). After 5 days, the culture flasks were shaken, and the non-adherent cells were washed off. The adherent cells were detached enzymatically and cultured with RGCs at a ratio of 5:1 in serum-free medium (8).
17. To purify collicular neurons, we trypsinized and triturated superficial layers of superior colliculi from P7 rats. Non-neuronal cells were removed from the cell suspension with the Griffonia simplicifolia lectin I (GSL I) isolectin-B4 (microglia, endothelial cells; Vector Labs) and the O4 antibody (oligodendrocyte lineage). Collicular neurons were selected with a L1-specific antibody (ASCS4; Developmental Studies Hybridoma Bank) and cultured for 10 to 15 days at a ratio of 1:3 with RGCs in the absence of glial cells. For cocultures of collicular neurons, glia, and RGCs, the collicular cell suspension was plated together with purified RGCs. To record from collicular neurons, we labeled RGCs with Dil (7 μM in Earle's balanced salt solution for 10 min at 36°C; Molecular Probes), before they were added to collicular cells.
18. F. W. Pfrieger and B. A. Barres, data not shown.
19. Glia-conditioned medium was obtained from purified collicular glia (10) that were cultured at high density in the same serum-free medium as RGCs (8). The medium was added every second day to RGC cultures at a dilution of 1:1.5 together with fresh medium.
20. -1; n = 6). Both components were active, as they fasciculated neurites of RGCs.
21. Astrocytes (7) and oligodendrocytes [B. A. Barres et al., Cell 70, 31 (1992)] were purified by immunopanning from postnatal rat optic nerves as described, because these glial cell types cannot be purified from other brain areas. Microglial cells were purified from the superior colliculus of P8 rats by immunopanning with a goat anti-rat immunoglobulin G antibody (Jackson ImmunoResearch Laboratories) and a rat anti-mouse Thy 1.2 antibody (Boehringer). RGCs were cultured with each glial cell type at a ratio of three to five glial cells per neuron in the defined serum-free medium (8). In order to avoid contact between neurons and glial cells, we cultured RGCs on cover slips above glial feeding layers.
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23. Miniature EPSCs were isolated by adding TTX (10 μM, Sigma), bicuculline methylchloride (50 μM, RBI), and D-2-amino-5-phosphonopentanoic acid (50 μM, RBI) to the extracellular recording solution.
24. The average whole-cell resting membrane potential in RGCs cultured for 10 to 15 days without (-48 ± 1 mV, n = 22) or with glial cells (-49 ± 1 mV, n = 19) was not different. This, however, does not rule out that glial cells increased quantal release by depolarizing nerve terminals.
25. For electron microscope analysis, cultures were fixed with glutaraldehyde (2.5% in phosphate-buffered saline for 30 min), treated with osmium tetroxide (1% for 30 min), dehydrated, and embedded in Epon 812 (Polysciences). For each culture, ultrathin sections were cut from at least two blocks with a glass knife, stained with lead citrate and uranyl acetate, and examined with a Phillips EM 410. Ultrastructural criteria to identify synapses were a pre- and a postsynaptic density, a synaptic cleft, and more than two synaptic vesicles in the presynaptic active zone. We counted synapses in photomicrographs (×18,500 magnification) from randomly chosen areas in one to seven sections per block. The number of neurons in glia-free cultures was estimated by counting neuronal cell bodies in the sections. Because the density of neurons was similar in glia-free and in cocultures, we could directly compare the number of synapses in equal numbers of micrographs from each culture condition.
26. In three glia-free cultures, we counted 168, 104, and 43 synapses in 109, 60, and 61 micrographs with approximately 20, 29, and 8 neurons. In the corresponding cocultures with glia, we found 373, 179, and 87 synapses in 104, 57, and 44 micrographs, respectively. Thus, glial cells increased the number of synapses per micrograph by 2.3, 1.8, and 2.8, respectively.
27. EPSCs were evoked by extracellular stimulation as described [F. W. Pfrieger, K. Gottmann, H. D. Lux, Neuron 12, 97 (1994)]. Briefly, a glass pipette (tip diameter: 1 μm), filled with extracellular solution was placed near a neurite, where brief (100 μs) current pulses (0.2 mA) evoked EPSCs in the postsynaptic neuron clamped at a holding potential of -70 mV. At each stimulation site, two trains of 20 stimuli were applied at 1, 5, 10, and 25 Hz, respectively. Action potentials in the postsynaptic neuron were blocked by including the lidocaine derivate QX-314 (10mM, RBI) in the intracellular recording solution. For the analysis of EPSCs, membrane currents were integrated over a time window of 8 ms beginning at the EPSC onset. For each stimulus, the presence of an EPSC or a stimulation failure was determined by the experimenter. In order to normalize failure rates at 5 and 10 Hz, for each RGC we subtracted the failure percentage at 1 Hz and divided by the rate at 25 Hz. To test the reliability of extracellular stimulation, we performed current-clamp recordings with 6,7-dinitroquinoxaline-2,3-dione (10 μM) added extracellularly to exclude action-potential induction by synaptic currents.
28. -glia are 2.8 at 1 Hz, 4.0 at 5 Hz, and 4.8 at 10 and 25 Hz. The ratios at higher frequencies exceeded the observed twofold increase in the synapse number, indicating that glial cells enhanced the efficacy of individual release sites.
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33. Supported by the Human Frontier Science Program Organization and the Deutsche Forschungsgemeinschaft (F.W.P.) and by The Searle Scholar Program/ The Chicago Community Trust (B.A.B.). We thank T. L. Schwarz and R. W. Tsien for valuable comments on the manuscript, S. Shen for ultrathin sections, F. Thomas for kind help with the electron microscope, and R. Scheller for generously providing an anti-synaptotagmin antibody. We also thank Regeneron for the kind gift of recombinant BDNF and CNTF.