Figure 5.
Nuclear-targeting properties of the three β4 splice variants individually expressed in hippocampal neurons of lethargic mice. A, Quantitative RT-PCR revealed similar expression of CaV subunit isoforms in WT and lethargic adult hippocampus (mean ± SEM, n = 3). B, Double immunofluorescence labeling of WT and lethargic hippocampal cultures with anti-β4 and anti-vGLUT1 demonstrated the complete absence of β4 protein in the lethargic neurons. C, Cultured hippocampal neurons from lethargic mice reconstituted with βA-β4a, βA-β4b, or βA-β4e and WT controls immunolabeled with anti-β4. Immature neurons (DIV1, 2, and 3) showed strong nuclear targeting of β4b and, at a lower degree, of β4a; no nuclear targeting of β4e was observed at any developmental stage. D, The nucleus/cytoplasm ratio decreases to <1 within 5 d in culture. E, Frequency distribution analysis of nucleus/cytoplasm ratio of β4b in cultured hippocampal neurons from lethargic mice in different developmental stages showed nuclear targeting only in immature neurons. F, G, Blocking the spontaneous activity in mature neurons by 1 μm TTX for 12 h significantly increased nuclear targeting of β4a and β4b, but not β4e. *p < 0.05; **p < 0.001, unpaired t test. No significant nuclear targeting could be observed in cultures from wild-type hippocampal neurons at any stage of development and with or without TTX. Scale bar, 10 μm.