Background It has been estimated that approximately 12% of women consume alcohol at some time during their pregnancy, and as many as 5% of children born in the United States are impacted by prenatal alcohol exposure (PAE). The range of physical, behavioral, emotional, and social dysfunctions that are associated with PAE are collectively termed fetal alcohol spectrum disorder (FASD). Methods Using a saccharin-sweetened ethanol solution, we developed a limited access model of PAE. C57BL/6J mice were provided access to a solution of either 10% (w/v) ethanol and 0.066% (w/v) saccharin or 0.066% (w/v) saccharin (control) for 4 h/d. After establishing consistent drinking, mice were mated and continued drinking during gestation. Following parturition, solutions were decreased to 0% in a stepwise fashion over a period of 6 days. Characterization of the model included measurements of maternal consumption patterns, blood ethanol levels, litter size, pup weight, maternal care, and the effects of PAE on fear-conditioned and spatial learning, and locomotor activity. Results Mothers had mean daily ethanol intake of 7.17 ± 0.17 g ethanol/kg body weight per day, with average blood ethanol concentrations of 68.5 ± 9.2 mg/dl after 2 hours of drinking and 88.3 ± 11.5 mg/dl after 4 hours of drinking. Food and water consumption, maternal weight gain, litter size, pup weight, pup retrieval times, and time on nest did not differ between the alcohol-exposed and control animals. Compared with control offspring, mice that were exposed to ethanol prenatally displayed no difference in spontaneous locomotor activity but demonstrated learning deficits in 3 hippocampal-dependent tasks: delay fear conditioning, trace fear conditioning, and the delay nonmatch to place radial-arm maze task. Conclusions These results indicate that this model appropriately mimics the human condition of PAE and will be a useful tool in studying the learning deficits seen in FASD.
The mouse model should be a useful tool in testing hypotheses about the neural mechanisms underlying the learning deficits present in fetal alcohol spectrum disorders. Moreover, a mouse prenatal ethanol model should increase the opportunity to use the power of genetically defined and genetically altered mouse populations.
In RBL-2H3 tumor mast cells, cross-linking the high affinity IgE receptor (Fc⑀RI) with antigen activates cytosolic tyrosine kinases and stimulates Ins(1,4,5)P 3 production. Using immune complex phospholipase assays, we show that Fc⑀RI cross-linking activates both PLC␥1 and PLC␥2. Activation is accompanied by the increased phosphorylation of both PLC␥ isoforms on serine and tyrosine in antigen-treated cells. We also show that the two PLC␥ isoforms have distinct subcellular localizations. PLC␥1 is primarily cytosolic in resting RBL-2H3 cells, with low levels of plasma membrane association. After antigen stimulation, PLC␥1 translocates to the plasma membrane where it associates preferentially with membrane ruffles. In contrast, PLC␥2 is concentrated in a perinuclear region near the Golgi and adjacent to the plasma membrane in resting cells and does not redistribute appreciably after Fc⑀RI cross-linking. The activation of PLC␥1, but not of PLC␥2, is blocked by wortmannin, a PI 3-kinase inhibitor previously shown to block antigen-stimulated ruffling and to inhibit Ins(1,4,5)P 3 synthesis. In addition, wortmannin strongly inhibits the antigen-stimulated phosphorylation of both serine and tyrosine residues on PLC␥1 with little inhibition of PLC␥2 phosphorylation. Wortmannin also blocks the antigen-stimulated translocation of PLC␥1 to the plasma membrane. Our results implicate PI 3-kinase in the phosphorylation, translocation, and activation of PLC␥1. Although less abundant than PLC␥2, activated PLC␥1 may be responsible for the bulk of antigen-stimulated Ins(1,4,5)P 3 production in RBL-2H3 cells. INTRODUCTIONIn mast cells and basophils, cross-linking the high affinity cell surface receptors for IgE (Fc⑀RI) 1 activates the tyrosine kinases Lyn and Syk (Eiseman and Bolen, 1992;Hutchcroft et al., 1992) and initiates a signaling cascade that leads to the secretion of histamine and other granule constituents, to changes in adhesive properties, cell shape, and surface topography and to the de novo synthesis of lipid mediators and cytokines (reviewed in Beaven & Metzger, 1993;Oliver et al., 1997). Tyrosine kinase activation by the Fc⑀RI and related members of the multisubunit immunoreceptor family, which includes the T cell antigen receptor, the B cell antigen receptor, and several Fc␥ receptors, depends on immunoreceptor tyrosine-based activation motifs (ITAMs) located in the cytoplasmic domains of individual receptor subunits (Cambier, 1995). The heterotrimeric (␣␥ 2 ) Fc⑀RI of RBL-2H3 mast cells contains ITAM motifs in both the ␥ and  subunit cytoplasmic domains (Metzger, 1992 to ITAM phosphorylation and Syk activation by its association with the ␥ subunit phospho-ITAM. Syk activation, resulting in the phosphorylation of multiple protein substrates, in turn initiates the signaling cascade Oliver et al., 1994;Wilson et al., 1995;Rivera and Brugge, 1995). Pharmacological studies have established that Sykdependent tyrosine phosphorylation is required for the antigen-stimulated synthesis of inositol (1,4,5) trisphosphate (I...
We have synthesized phosphatidylinositol 3-phosphate from phosphatidylinositol 4-phosphate by using dilsopropylcarbodilmide to promote migration of the 4-phosphate via a cyclic phosphodiester intermediate. The product was isolated by a thin-layer chromatographic method that depends on the ability of phosphatidylinositol 4-phosphate, but not phosphatidylinositol 3-phosphate, to form complexes with boric acid. The rmal yield of the procedure was 8% phosphatidylinositol 3-phosphate, which was 40% pure. ethylamine, and 2,6-di-tert-butyl-4-methylphenol (BHT) were from Aldrich. Technical grade ethoxyquin used for TLC was from Sigma, whereas purified ethoxyquin used in the PtdIns3P synthesis was from Crescent Chemical (Hauppauge, NY). Silica gel 60 plates (0.25 mm thick, without fluorescent indicator) were from Merck. Solvents were from Fisher. Fatty acid and globulin-free bovine albumin and trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) were from Sigma. Silicic acid (Bio-Sil A, 100-200 mesh) was from Bio-Rad. PtdIns3P 3-phosphatase was purified from rat brains as described (7). PtdIns[32P]3P was prepared by phosphorylation of soybean phosphatidylinositol with anti-platelet-derived growth factor receptor immunoprecipitates with [y-32P]ATP as described (7). PtdIns-[32P]4P was a gift of Linda Pike (Department of Biochemistry and Molecular Biophysics, Washington University, St. Louis). All other materials used in this work were obtained as described (7).TLC of Phosphatidylinositol Phosphates. Phosphatidylinositol phosphates were separated in the presence of boric acid as follows. Silica gel 60 plates (5 x 20 cm) were immersed face up for 10 sec with gentle swirling in CDTA solution. This solution was prepared by stirring a mixture of 4.55 g of disodium CDTA-H2O, 165 ml of H20, 330 m.l of ethanol, and 3.0 ml of 10 M NaOH until the CDTA was dissolved. The plates were allowed to air-dry by standing up for 1 hr and then baked at 100'C for 10 min. Lipids were spotted 1.7 cm from the bottom of the plate. The immersion depth in the developing solution was 0.7 cm. The TLC developing solution was prepared by stirring together methanol (75 ml), CHC13 (60 ml), pyridine (45 ml), and boric acid (12 g) until the boric acid was dissolved. Water (7.5 ml), 88% (vol/vol) formic acid (3.0 ml), BHT (0.375 g), and technical grade ethoxyquin (75 dl) were then added. The plates were developed in a saturated tank and were run to the top (=3 hr). PtdIns4P migrated with an Rf of 0.46, whereas the Rf of PtdIns3P was 0.51 (Fig. 1).Two secondary fronts were observed with Rf values of 0.66 (boric acid) and 0.22 (formate). The relative migrations of several other lipids in this system are given in Table 1.Synthesis of PtdIns3P. PtdIns4P (5 mg, 5.0 pumol) was dissolved in 2.5 ml of CHCl3/methanol, 3:2 (vol/vol). Addition of 5 ,.l of 1.0 M HCl was required to achieve solution.Five microcuries (1 Ci = 37 GBq) of f3H]PtdIns4P and 5 "I of ethoxyquin (purified grade) were then added. This mixture was passed over a 0.5-ml column of ...
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