Neurogenesis in postnatal rat spinal cord: A study in primary culture

Science

Volumn 276, Issue 5312, 1997, Pages 586-589

  Kehl, Lois J ^a   Fairbanks, Carolyn A ^a   Laughlin, Tinna M ^a   Wilcox, George L ^a  

^a UNIVERSITY OF MINNESOTA   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

BROXURIDINE;

ANIMAL CELL; ANIMAL TISSUE; ARTICLE; BRAIN ELECTROPHYSIOLOGY; FUNCTIONAL ASSESSMENT; IMMUNOCYTOCHEMISTRY; MITOSIS; NERVOUS SYSTEM DEVELOPMENT; NONHUMAN; POSTNATAL DEVELOPMENT; PRIORITY JOURNAL; RAT; SPINAL CORD INJURY;

ACTION POTENTIALS; ANIMALS; BROMODEOXYURIDINE; CELL DIFFERENTIATION; CELLS, CULTURED; CULTURE MEDIA; GLIAL FIBRILLARY ACIDIC PROTEIN; IMMUNOHISTOCHEMISTRY; MITOSIS; NEURONS; PHOSPHOPYRUVATE HYDRATASE; RATS; SPINAL CORD; TUBULIN;

EID: 0030971385     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.276.5312.586     Document Type: Article

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37. 2 (0.45 mm radius); 200× magnification] were quantified and averaged to the entire cover slip. Differences in numbers between TuJ1-and NSE-immunopositive cells may be attributed to differential protein expression or different antibody sensitivity.
38. A six-well culture plate of P16 rat spinal cord cells was established without fetal monolayers. At 4 DIV, two cover slips were selected; the attached cells were fixed, immunoreacted with TuJ1 or anti-NSE, and counted (25). Of the four remaining wells, two received supplements of FUDR (15 ng/ml) with each media change. At 12 DIV, FUDR-treated and control cover slips (no FUDR) were immunoreacted with TuJ1 and anti-NSE. Cells were counted and comparisons were made between neuronal cell numbers in FUDR-treated versus control cultures (no FUDR). Negative controls were performed as described (6).
39. NSE/BrdU double-immunocytochemistry was carried out as follows: P15 to P16 spinal cord cultures, without monolayers, were incubated with 1 μM BrdU (added with media changes every 2 to 3 days) for 7 days during the first, second, third, or fourth week in vitro. Cultures were fixed on the seventh day of each week (7, 14, 21, or 28 DIV) and double-immunostained for BrdU (1:1; Amersham) and NSE. TuJ1/ BrdU double-immunocytochemistry was as follows: BrdU (1 μM) was added to cultures with each media change (every 2 to 3 days) from 1 to 20 DIV. At 20 DIV, cultures were fixed and double-immunostained with TuJ1 (1:250) and sheep anti-BrdU (1:100; Fitzgerald). Acid denaturation (2N HCl), which is required for antigen detection with this antibody to BrdU, reduced TuJ1-immunoreactivity. Therefore, TuJ1/BrdU-immunoreactivity was visualized with confocal microscopy. Two independent observers scored TuJ1-labeled cells for BrdU content. TuJ1/BrdU cell counts performed with fluorescence microscopy on separate cover slips at 20 and 15 DIV yielded quantitatively similar results. Controls conducted with each experiment were as follows. TuJ1: mouse TuJ1 followed by anti-sheep secondary, sheep anti-BrdU followed by anti-mouse secondary, and TuJ1 primary omitted followed by co-application of anti-sheep and anti-mouse secondaries. NSE: rabbit anti-NSE followed by anti-mouse secondary, mouse anti-BrdU followed by anti-rabbit secondary, and anti-NSE primary omitted followed by co-application of anti-mouse and anti-rabbit secondaries. Control studies supported antibody specificity.
40. P15 to P16 cultures were established in six-well plates as described (6). Tissue culture inserts (0.45 μm per pore; Becton Dickinson) containing previously prepared fetal monolayers were placed in each well to condition media while ensuring fetal cells could not attach to postnatal cultures. To confirm fetal cells would not pass through insert membranes, 12 control wells were prepared without postnatal tissue. At 20 to 23 DIV, cover slips were tested for trypan blue exclusion. No viable cells were found to have passed through insert membranes. Postnatal cells were patch-clamped at 20 to 27 DIV. Those generating APs were injected with 0.2% dextran fluorescein (Molecular Probes; -500 pA, 10 Hz, 30 min) for later identification. Then cells were fixed, treated with 2N HCl (37°C for 30 min), 0.1 M borate buffer (25°C for 10 min), and immunoreacted with sheep anti-BrdU (1:100; Fitzgerald), followed by rhodamine-conjugated secondary antibody (1:40; Jackson Immuno-Research). Fluorescein-filled cells were identified with confocal microscopy and evaluated for BrdU-immunoreactivfty by two independent observers.
41. Studies approved by the University of Minnesota Animal Care and Use Committee.
42. We thank A. Frankfurter for the gift of TuJ1, P. Letourneau and I. Fischer for critical evaluation of the manuscript, L. Furcht for generously supplying laminin, D. Snow and S. Kilo for constructive suggestions, K. Hargreaves for encouragement, and M. Wessendorf, L. Stone, and G. Sedgewick for microscopic and photographic assistance. Supported by a National Institute of Dental Research Dentist-Scientist Award (L.J.K, grant DE00225), National Institute on Drug Abuse (NIDA) training grant (C.A.F. grant DA07234, and T.M.L., grant DA07097), and NIDA R01 and Research Scientist Development Awards (G.L.W., grants DA01933 and DA04274).