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. 2003 Jul 2;23(13):5887–5896. doi: 10.1523/JNEUROSCI.23-13-05887.2003

Figure3.

Figure3.

Expression of ali1 mRNA is tightly associated with depolarization-dependent survival and/or NMDA-dependent survival of cerebellar granule neurons.A,Northern blotting of ali1 mRNA expression in granule neurons. Total RNA samples (10μg) extracted from granule neurons cultured in the presence of 5 mm KCl, 30μm AP-5; 15 mm KCl, 30μm AP-5; 25 mm KCl, 30μm AP-5; or 150 μm NMDA, 15 mm KCl were subjected to Northern blotting. The cDNA obtained by differential display analysis (which corresponds to the 3′-untranslated region of rat ali1 cDNA) was used as a probe. The bottom panel depicts expression levels of G6PDH mRNA. B, Quantification of data shown in A. Granule neurons were cultured in the presence of 5 mm KCl,30μm AP-5(▵); 15 mm KCl,30μm AP-5 (○); 25 mm KCl, 30μm AP-5 (▴); or 15 mm KCl, 150μm NMDA (•). C, Rapid downregulation of ali1 mRNA expression by blocking the NMDA receptor with AP-5. Granule neurons were cultured for 4.5 d in medium containing 15 mm KCland150μm NMDA, and the NMDA receptor was blocked by the addition of AP-5 to a final concentration of 300μm. Total RNA (10μg) extracted at the indicated times was subjected to Northern blotting analyses. The bottom panel displays G6PDH mRNA expression levels. D, The data shown in C were quantified. E, Effects of various inhibitors on ali1 mRNA expression. Cerebellar granule neurons were cultured for 4 d in medium containing 25 mm KCl, as described in Materials and Methods. The medium was replaced with one containing 5 mm KCl, and the culture was continued for another 24 hr, after which cells were stimulated with 5 mm KCl, 30μm AP-5 (5); 15 mm KCl, 30μm AP-5 (15); 25 mm KCl, 30μm AP-5 (25); or 15 mm KCl, 150μm NMDA (N). Inhibitors were added to the culture medium 30 min before stimulation with KCl and/or NMDA. After stimulation (24 hr), total RNA was extracted and subjected to competitive PCR analysis. The inhibitors used included 35 μm cycloheximide (CHX), 4.0 μm actinomycin D (AcD), and 0.2 μm nifedipine (NFD). Ethanol was used as vehicle for nifedipine. F, The data shown in E were quantified.