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Double labeling for GFAP and TUNEL was performed by first staining sections by TUNEL (6), washing them thoroughly at room temperature in a solution of 0.01 M phosphate-buffered saline (PBS)-Triton X-100 (0.5%), and incubating them for 1 hour in the same solution with 1% bovine serum albumin. The sections were then incubated overnight at 4°C in the presence of monoclonal antibody against GFAP (1:400 dilution; G3893, Sigma). After PBS rinses, sections were incubated with goat antibody to mouse immunoglobulin G (IgG) (1:200; BODIPY, Molecular Probes) in PBS-Triton X-100 for 1 hour at room temperature. Microscopic fields were photographed with a Kodak digital camera under fluorescence illumination to detect localization of the BODIPY fluorescence probe and under ordinary illumination to detect the TUNEL diaminobenzidine reaction product. The two images from a given field were then superimposed with PhotoShop software to determine whether the GFAP and TUNEL labels were colocalized in the same cells.
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MK801 remains detectable in the rodent brain or cerebrospinal fluid for 3 to 4 hours after systemic administration. Elimination half-life of MK801 in rats was estimated to be 1.9 hours. The drug reaches maximal concentrations in the brain within 10 to 30 min after administration [A. Vezzani et al., J. Pharmacol. Exp. Ther. 249, 278 (1989)]. Phencyclidine (PCP), which freely penetrates blood-brain barriers, has a plasma elimination half-life of 3.9 hours and an even longer half-life in the rat brain [J. L. Valentine, L. W. Arnold, S. M. Owens, J. Pharmacol. Exp. Ther. 269, 1079 (1994)]. A single dose of PCP (10 mg/kg ip) was used because it produces behavioral symptoms indicative of NMDA receptor blockade lasting for 8 to 10 hours in 7-day-old rats. Ketamine freely penetrates the brain but is excreted rapidly and has a short half-life in the brain [P. F. White, M. P. Marietta, C. R. Pudwill W. L. Way, A. J. Trevor, J. Pharmacol. Exp. Ther. 196, 545 (1976) ]. It was administered in a series of seven injections spaced evenly over 9 hours, each injection delivering a dose of 20 mg/kg sc CPP penetrates blood-brain barriers poorly but has a long half-life in the brain [J. D. Kristensen, P. Hartvig, R. Karlsten, T. Gordh, M. Halldin, Br. J. Anaesth. 74, 193 (1995)]. To maintain a steady blockade of NMDA receptors for 8 hours, we administered CPP at a dose of 15 mg/kg ip at 0, 50, and 100 min and at 4 and 8 hours. Rat pups were killed at 24 hours after the first treatment.
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MK801 remains detectable in the rodent brain or cerebrospinal fluid for 3 to 4 hours after systemic administration. Elimination half-life of MK801 in rats was estimated to be 1.9 hours. The drug reaches maximal concentrations in the brain within 10 to 30 min after administration [A. Vezzani et al., J. Pharmacol. Exp. Ther. 249, 278 (1989)]. Phencyclidine (PCP), which freely penetrates blood-brain barriers, has a plasma elimination half-life of 3.9 hours and an even longer half-life in the rat brain [J. L. Valentine, L. W. Arnold, S. M. Owens, J. Pharmacol. Exp. Ther. 269, 1079 (1994)]. A single dose of PCP (10 mg/kg ip) was used because it produces behavioral symptoms indicative of NMDA receptor blockade lasting for 8 to 10 hours in 7-day-old rats. Ketamine freely penetrates the brain but is excreted rapidly and has a short half-life in the brain [P. F. White, M. P. Marietta, C. R. Pudwill W. L. Way, A. J. Trevor, J. Pharmacol. Exp. Ther. 196, 545 (1976) ]. It was administered in a series of seven injections spaced evenly over 9 hours, each injection delivering a dose of 20 mg/kg sc CPP penetrates blood-brain barriers poorly but has a long half-life in the brain [J. D. Kristensen, P. Hartvig, R. Karlsten, T. Gordh, M. Halldin, Br. J. Anaesth. 74, 193 (1995)]. To maintain a steady blockade of NMDA receptors for 8 hours, we administered CPP at a dose of 15 mg/kg ip at 0, 50, and 100 min and at 4 and 8 hours. Rat pups were killed at 24 hours after the first treatment.
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MK801 remains detectable in the rodent brain or cerebrospinal fluid for 3 to 4 hours after systemic administration. Elimination half-life of MK801 in rats was estimated to be 1.9 hours. The drug reaches maximal concentrations in the brain within 10 to 30 min after administration [A. Vezzani et al., J. Pharmacol. Exp. Ther. 249, 278 (1989)]. Phencyclidine (PCP), which freely penetrates blood-brain barriers, has a plasma elimination half-life of 3.9 hours and an even longer half-life in the rat brain [J. L. Valentine, L. W. Arnold, S. M. Owens, J. Pharmacol. Exp. Ther. 269, 1079 (1994)]. A single dose of PCP (10 mg/kg ip) was used because it produces behavioral symptoms indicative of NMDA receptor blockade lasting for 8 to 10 hours in 7-day-old rats. Ketamine freely penetrates the brain but is excreted rapidly and has a short half-life in the brain [P. F. White, M. P. Marietta, C. R. Pudwill W. L. Way, A. J. Trevor, J. Pharmacol. Exp. Ther. 196, 545 (1976) ]. It was administered in a series of seven injections spaced evenly over 9 hours, each injection delivering a dose of 20 mg/kg sc CPP penetrates blood-brain barriers poorly but has a long half-life in the brain [J. D. Kristensen, P. Hartvig, R. Karlsten, T. Gordh, M. Halldin, Br. J. Anaesth. 74, 193 (1995)]. To maintain a steady blockade of NMDA receptors for 8 hours, we administered CPP at a dose of 15 mg/kg ip at 0, 50, and 100 min and at 4 and 8 hours. Rat pups were killed at 24 hours after the first treatment.
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MK801 remains detectable in the rodent brain or cerebrospinal fluid for 3 to 4 hours after systemic administration. Elimination half-life of MK801 in rats was estimated to be 1.9 hours. The drug reaches maximal concentrations in the brain within 10 to 30 min after administration [A. Vezzani et al., J. Pharmacol. Exp. Ther. 249, 278 (1989)]. Phencyclidine (PCP), which freely penetrates blood-brain barriers, has a plasma elimination half-life of 3.9 hours and an even longer half-life in the rat brain [J. L. Valentine, L. W. Arnold, S. M. Owens, J. Pharmacol. Exp. Ther. 269, 1079 (1994)]. A single dose of PCP (10 mg/kg ip) was used because it produces behavioral symptoms indicative of NMDA receptor blockade lasting for 8 to 10 hours in 7-day-old rats. Ketamine freely penetrates the brain but is excreted rapidly and has a short half-life in the brain [P. F. White, M. P. Marietta, C. R. Pudwill W. L. Way, A. J. Trevor, J. Pharmacol. Exp. Ther. 196, 545 (1976) ]. It was administered in a series of seven injections spaced evenly over 9 hours, each injection delivering a dose of 20 mg/kg sc CPP penetrates blood-brain barriers poorly but has a long half-life in the brain [J. D. Kristensen, P. Hartvig, R. Karlsten, T. Gordh, M. Halldin, Br. J. Anaesth. 74, 193 (1995)]. To maintain a steady blockade of NMDA receptors for 8 hours, we administered CPP at a dose of 15 mg/kg ip at 0, 50, and 100 min and at 4 and 8 hours. Rat pups were killed at 24 hours after the first treatment.
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Supported in part by BMBF grant 01K095151 and grants from the National Institute of Aging (AG 11355), the National Institute of Drug Abuse (DA 05072), and the Research Scientist Award from the National Institute of Mental Health (MH 38894) to J.W.O.
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