The release and uptake of excitatory amino acids. regions of EAAT1, EAAT2, and EAAT3. EAAT2 mRNA Yunaconitine was abundant in human fetal white matter during the period of peak incidence of PVL and virtually PRPH2 disappeared by 2 postnatal months. Its developmental profile differed significantly from that of both EAAT1 and EAAT3 mRNA. Immunoblotting exhibited that EAAT2 protein was highly expressed in early development relative to adult values. Double-label immunocytochemistry detected EAAT2 in OLs but not astrocytes or axons in the human fetal white matter. We conclude that transient expression of EAAT2 occurs during the windows of peak vulnerability for PVL, suggesting that this developmentally up-regulated transporter may be a major source of extracellular glutamate in ischemic injury to the cerebral white matter of the preterm infant. J. Comp. Neurol. 501:879C890, 2007. values decided from Wilcoxon assessments: EAAT1 (0.003), EAAT2 (0.014), and EAAT3 (0.003). EAAT1 and EAAT3 mRNA levels were higher after than before birth; in contrast, EAAT2 mRNA Yunaconitine levels were higher before than after birth (Fig. 4). Open in a separate windows Fig. 2. Nonisotopic in situ hybridization of EAAT2/GLT1, EAAT1/GLAST, and EAAT3/EAAC1 mRNA in human fetal cerebral white matter. C shows a section of white matter (WM) and cortex (CTX) of a 32-week-old subject, demonstrating a prominent amount of EAAT2 mRNA in the white matter. In contrast, a 7-month-old subject (D) expresses almost no EAAT2 mRNA in the white matter. Cortex Yunaconitine serves as a positive control, showing EAAT2 labeling in both subjects. EAAT1 (A,B) and EAAT3 (E,F) expression was present and increased with development, in contrast to EAAT2. Level bars &50 m. Open in a separate windows Fig. 4. Comparison of Yunaconitine preterm (40 weeks) and postterm ( 40 weeks) expression of EAAT1, EAAT2, and EAAT3 mRNA. EAAT1 and EAAT3 levels were significantly higher after term, whereas EAAT2 levels were significantly lower after birth. Statistical analysis of preterm and postterm expression using the Wilcoxon test gives values of 0.003 (EAAT1), 0.014 (EAAT2), and 0.003 (EAAT3). *% 0.05, **% 0.01. Evaluation of EAAT2 protein expression in the human fetus and infant Because the in situ results (Figs. 2, ?,3)3) demonstrated that EAAT2 is usually developmentally up-regulated during the peak of vulnerability for premature infants to PVL, we analyzed the expression of EAAT2 at the protein level by using an antibody raised against the N terminus of GLT1/EAAT2, known to be shared by the two known variant forms of GLT1/EAAT2. To test the specificity of the antinGLT1 antibody in human brain tissue, immunoblot analysis was performed on lysates prepared from normal human motor cortex (Fig. 5). A dense band appeared at 67 kDa (Fig. 5, lane 2). A light band was also observed above the 120-kDa marker (Fig. 5, lane 2), probably representing dimers of the transporter. These data are consistent with results obtained with this antibody in rodent brain lysates (Chen et al., 2002, 2004). To demonstrate the specificity of the Yunaconitine immune response to the antigenic N-terminus GLT1 peptide, the immunoreactivity of purified anti-nGLT1 antibody was compared with that of the preimmune serum (Fig. 5, lane 1). In addition, the ability of the peptide against which the antibody was generated to block immunoreactivity was tested (Fig. 5, lane 3). Preimmune serum was not immunoreactive (Fig. 5, lane 1). Antigenic peptide nearly completely blocked the immunoreactivity of the anti-nGLT1 antibody against human brain lysate (Fig. 5, lane 3). Thus, the anti-nGLT1 antibody specifically detected protein made up of the epitope against which it was generated in human lysates. Previous studies have shown that this immunreactivity produced by this antibody is not present in brain lysates or in tissue sections from GLT1 knockout mice (Chen et al., 2004). Open in a separate windows Fig. 5. Western blot analysis of the specificity of the anti-nGLT1 antibody in human brain tissue. To characterize the anti-nGLT1 antibody, it was tested on lysates prepared from normal human motor cortex and appears as a band at 67 kDa (lane 2). A lighter band is also observed above the 120-kDa marker (lane 2). To demonstrate the specificity of the immune response to the nGLT1 peptide, the immunoreactivity of the anti-nGLT1 antibody was compared with that of the preimmune serum (lane 1). In addition, the ability of the peptide against which the antibody was generated to block immunoreactivity was tested (lane 3). To assess whether the transient expression of EAAT2 mRNA occurs at the protein level, an immunoblot using anti-nGLT1 antibody in parietooccipital cerebral white matter lysates was performed (Fig. 6). Lane 1 (Fig. 6) contains adult human standard cerebral white matter.