Ethanol-Induced Apoptotic Neurodegeneration and Fetal Alcohol Syndrome

  • Mani J
  • Allen J
  • Clark J
 et al. 
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P. Green-gard, P. B. Allen, A. C. Nairn, Neuron 23, 435 (1999). 5. Ovariectomized female rats were prescreened for sexual receptivity (6) by subcutaneous (sc) adminis-tration of EB (2 ␮g) followed by P (100 ␮g) 48 hours later. Stereotaxic surgery was performed on sexually receptive rats (6) and mice (3), which were then used in experiments to compare the effects of P, D 1 agonist, and serotonin. Behavioral testing was per-formed during the dark phase of the reversed light/dark cycle as described (3, 6). The experimen-tal observer was blind to the treatment conditions and mouse genotypes. 9. EB-induced hypothalamic cytosol PRs in mice carry-ing the wild-type gene encoding DARPP-32 (ϩ/ϩ) and the null mutation (Ϫ/Ϫ) were assayed by one-point binding analysis as described previously (3). The following PR concentrations (in fmol/mg of protein) were obtained: vehicle (ϩ/ϩ) ϭ 2.5 Ϯ 1.2; vehicle (Ϫ/Ϫ) ϭ 2.6 Ϯ 0.87; EB (ϩ/ϩ) ϭ 8.75 Ϯ 1.5; EB (Ϫ/Ϫ) ϭ 8.4 Ϯ 1.8. Each value is the mean ϮSEM of six independent determinations. 10. A. C. Nairn and S. Shenolikar, Curr. Opin. Neurobiol. 2, 296 (1992). 11. P. B. Allen et al., in preparation. 12. Mice homozygous for the I-1 and DARPP-32 targeted mutations were generated by cross-breeding the two mutant lines. Resulting double heterozygotes were then crossed to yield mouse lines homozygous for each mutation and also the corresponding wild-type controls. Additional wild-type and double mutant mice were then generated by inbreeding wild types and double mutants. 13. Rats were decapitated and the brains removed. The hypothalamus was dissected from coronal sections submerged in oxygenated ice-cold artificial cerebro-spinal fluid without Ca 2ϩ or Mg 2ϩ , and the tissues were processed for cAMP and PKA assays. Sample processing and cAMP assays were performed accord-ing to the procedures of Moore et al. (20). PKA assays using Kemptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) as a substrate were carried out with 5 ␮g of protein as described (21). The amount of cAMP and PKA activity in each sample was normalized to the total amount of protein in the homogenate. Preincubation of pro-tein extracts with the PKA-specific peptide inhibitor Walsh peptide demonstrated a concentration-depen-dent inhibition of substrate phosphorylation (a sub-strate:inhibitor ratio of 1:10 ϭ 85% inhibition; 1:100,000 ϭ 15%), confirming that the phosphoryl-ation of the substrate peptide was specific to PKA. 17. Mice were killed by exposure of the head to micro-wave irradiation for 900 ms, for which a Muromachi Microwave Applicator (Stoelting, Wood Dale, IL) set at 1.5 power was used. The brains were isolated from the crania, and 1-mm coronal sections bracketing the hypothalamic area were prepared with the aid of a mouse brain matrix (Activational Systems, Ann Ar-bor, MI), following the stereotaxic coordinates of Franklin and Paxinos (22). The microdissection of the hypothalamus was performed as described (23), and the tissues were stored at Ϫ80°C until processed. Frozen samples were processed and immunoblotted for phospho–DARPP-32 and total DARPP-32 (24). Phospho–DARPP-32 and total DARPP-32 bands were quantified by densitometry with the use of a Phos-phoimager:SF (Molecular Dynamics). The phospho– DARPP-32 values were normalized for the amount of total DARPP-32 present in the samples, and data were represented as percent of vehicle controls. The linear range of signals for densitometry was obtained by exposing the chemiluminescent membranes to x-ray film for varying periods of time. The linearity of measurements was confirmed by calibrating the val-ues obtained to a standard range of phospho–DARPP-32 concentrations in striatal tissues under conditions of basal and D 1 activation. The exposure conditions

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  • J S K M C Mani

  • J H Allen

  • J D Clark

  • B W Blaustein

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