Reliability of Spike Timing in Neocortical Neurons

screen of the microscope. Between 150 to 300 nerve fibers were analyzed per cross section. 13. C. F. Eldridge, M. Bartlett, R. P. Bunge, P. M. Wood, J. Cell Biol. 105, 1023 (1987); C. F. Eldridge, M. B. Bunge, R. P. Bunge, J. Neurosci. 9, 625 (1989). 14. Addition of progesterone (dissolved in ethanol; final concentration of ethanol, 0.1 %) daily to a final concentration of 20 nM to culture medium for 2 weeks did not increase the area occupied by the neurite network extending around DRG explants (control, 20.6 + 1.4; progesterone, 19.7 + 1.2 mm2), the density of neurites (control, 422 + 24; progesterone, 397 + 35 mm/mm2), or the number of Schwann cells (control, 2500 + 193; progesterone, 2625 + 104 cells per mm2), measured after staining the explants with toluidine blue. Measurements were made with an imaging system (Biocom, RAG version 2). Results are means + SEM of five culture dishes, each containing 10 DRG explants. 15. U. Suter et al., Nature 356, 241 (1992). 16. L. M. Bolin and E. M. Shooter, J. Cell Biol. 123, 237 (1993). 17. B. Droz, L. Di Giamberardino, H. L. Koenig, Brain Res. 219, 57 (1981); A. D. Toews, R. Armstrong, R. M. Gould, P. Morell, J. Neurosci. 8, 593 (1988). 18. H. L. Koenig et al., unpublished observations. 19. I. Jung-Testas, M. Schumacher, P. Robel, E.-E. Baulieu, J. Steroid Biochem. Mol. Biol. 48, 145 (1994). 20. J. M. Verdi and A. T. Campagnoni, J. Biol. Chem. 265, 20314 (1990). 21. C. Hildebrand, C. M. Bowe, I. Nilsson Remahl, Prog. Neurobiol. 43, 85 (1994); 0. A. Bizzozero and L. K. Good, J. Biol. Chem. 266,17092 (1991). 22. The 313-HSD may also be expressed by some neurons within the nervous system (R. Guennoun, R. J. Fiddes, M. Gouezu, M. Lombes, E.-E. Baulieu, Mol. Brain Res., in press). 23. R. Morfin et al., Proc. Natl. Acad. Sci. U.S.A. 89, 6790 (1992). 24. Steroids (pregnenolone, progesterone, and corticosterone) in sciatic nerve and plasma were measured by RIA. Fourteen nerves were pooled for each sample. The RIA procedures have been described and validated (4) [C. Corpechot et al., Endocrinology 133, 1003 (1993)]. The progesterone antiserum cross-reacts with 5o-dihydroprogesterone (32%); therefore, our assay includes both progestins, which are potent activators of gene expression via the intracellular progesterone receptor [R. Rupprecht et al., Neuron 11, 523 (1993)]. 25. Rat Schwann cells were prepared from DRG cultures as described (13), with minor modifications. During the first week, DRG explants were cultured in Dulbecco's modified Eagle's medium-Ham's F12 (50:50, v/v) containing 100% fetal bovine serum and nerve growth factor (30 ng/ml), and were treated for 3 days with the antimitotic agent cytosine arabinoside (10 puM) to eliminate all cells other than sensory neurons and Schwann cells. Subsequently, the explants were grown for an additional 3 weeks in defined serum-free medium (8). The outgrowth of axons induces Schwann cell growth [J. L. Saizer and R. P. Bunge, J. Cell Biol. 84, 739 (1980)], resulting in the generation of large numbers of surrounding Schwann cells. The ganglia were then cut out of the cultures with a microscalpel. The remaining pure Schwann cells were stained as described [S. M. Hsu, L. Raine, H. Fanger, J. Histochem. Cytochem. 29, 577 (1981 )] with a polyclonal rabbit antiserum to a synthetic peptide common to the four known isoforms of rat 313-HSD (22). 26. Schwann cells from DRG explants were harvested by exposure to trypsin and seeded in new culture dishes. After 48 hours, the cells were incubated in triplicate with 100 nM [7-3H(N)]pregnenolone (NEN, 925 GBq/mmol) at 37°C for 24 hours. Steroids were extracted from the incubation medium with ethyl acetate:isooctane (1 1, v/v) and separated by thin-layer chromatography (TLC) on silica gel plates developed once in chloroform:ethyl acetate (4: 1, v/v). Quantitation of radioactive areas on TLC plates was performed with an automatic TLC linear analyzer. Ste roids were further characterized by high-pressure liquid chromatography on a reversed-phase C18 octadecylsilane column and by recrystallization (4) [Y. Akwa et al., J. Cell Biol. 121, 135 (1993)]. 27. Neuron-Schwann cell cultures of rat DRG were prepared as described (25). After 4 weeks in defined medium, cells were cultured for an additional 2 weeks in myelination-promoting medium [defined medium containing 10% fetal bovine serum and L-ascorbic acid (50 pLg/ml)], in which Schwann cells differentiate rapidly and myelinate the DRG axons. Ascorbic acid is necessary for the Schwann cells to assemble a basal lamina, a prerequisite for myelination (13). 28. Myelinated fibers were stained with Sudan black after fixing the cultures with paraformaldehyde (4%) and osmium (0.1%) [N. Kleitman, P. M. Wood, R. P. Bunge, in Culturing Nerve Cells, G. Banker and K. Goslin, Eds. (Bradford Books, London, 1991), pp. 337-377]. Myelin formation was quantified as described (13) with an imaging system (Biocom, RAG version 2). Electron microscopic studies confirmed that the myelin formed in vitro was morphologically normal. 29. We thank Roussel-Uclaf for antisera to corticosterone (Ab 524) and progesterone (Ab 1093); J. Fiet for the antiserum to pregnenolone; B. Delespierre for technical assistance; C. Corpechot for help with the RIAs; V. Meremans for help with the analysis of axon diameters; and J.-C. Lambert and L. Outin for editorial assistance. Supported in part by the Association Francaise contre les Myopathies.