Article Figures & Data
Figures
- Figure 1.
Ontogenic changes in granule cells in organotypic cultures. A, Schematic drawing of morphological and locational changes in granule cells at the postnatal period in vivo [cited and modified from Ramón y Cajal (2000)]. Granule cells are classified into four stages according to their morphology and location: stage I, a germinal and horizontal stage; stage II, a descending stage; stage III, a multipolar stage; stage IV, a mature stage. B, A well preserved cerebellar architecture at DIV 10 is shown by Nissl staining of a representative organotypic culture. C, An EGFP-expressing granule cell and Purkinje cells in a representative organotypic culture at DIV 10 are shown by EGFP fluorescence (green) and calbindin D immunostaining (red), respectively. D, Morphological changes of representative granule cells at four stages in organotypic slice cultures. Confocal images were obtained from EGFP-expressing granule cells, indicating stage I at DIV 2, stage II at DIV 3, stage III at DIV 4, and stage IV at DIV 10. In D–I, two adjacent EGLs were separated as marked by a solid line. PCL, Purkinje cell layer. E, Time-dependent changes in a cell population of four developmental stages of granule cells in organotypic slice cultures. Three cerebellar slices were placed on a culture membrane and subjected to organotypic culture in a six-well plate. EGFP-positive granule cells in six slices were classified at each time point, and the numbers of different stages of granule cells in these slices were counted and averaged. Experiments were performed three times, and data of a representative experiment are presented. The symbols and bars represent the mean ± SEM, respectively. F–H, Synaptic maturation of granule cells at stage IV. Expression of postsynaptic PSD-95 and presynaptic synaptophysin and GAD was visualized by immunostaining with the respective antibodies (red) and observed over or adjacent to the claw-like structure of a granule cell dendrite (green).
- Figure 2.
Changes in electrophysiological properties of developing granule cells in organotypic slice cultures. A, B, Resting membrane potentials (Vrest) and membrane resistances (Rm) of granule cells at four stages are indicated. The resting membrane potential and membrane resistance progressively decreased from stage I to stage IV. C, D, Action potentials evoked by injecting indicated currents for 600 ms were measured from granule cells at the four stages at the holding potential of −70 mV. Representative voltage traces of stages II and IV (C) and frequencies of action potentials at the four stages (D) are indicated. E–G, Average peak amplitudes of inward currents (E) and those of outward currents at the peak (transient; F) and at the plateau phase (sustained; G) were determined for granule cells at the four stages by changing the membrane potential from the holding potential of −70 mV to the test potential of −30 mV (E) or 10 mV (F, G). Statistical analysis was done by one-way ANOVA, followed by Tukey's multiple-comparison tests. The symbols and bars represent the mean ± SEM, respectively. The numbers in parentheses are numbers of cells examined. In all analyses, *p < 0.05, **p < 0.01, ***p < 0.001 compared with stage I; •p < 0.05, ••p < 0.01, •••p < 0.001 compared with stage II; ■■■p < 0.001 compared with stage III.
- Figure 3.
Ontogenic expression of marker proteins in organotypically cultured granule cells. A, Confocal images of expression of TAG-1, Homer 2, and GABAAα6 in EGFP-expressing cells at four stages as analyzed by EGFP fluorescence (green) and immunostaining with the respective antibodies (red). The insets indicate immunostained Homer 2 and GABAAα6 at dendrites of EGFP-expressing granule cells. B, Quantification of immunostained marker proteins in granule cells. The distribution of immunoreactivity is expressed as either percentages of immunoreactive area (TAG-1) or numbers of immunofluorescent puncta (Homer 2 and GABAAα6). The black and white columns indicate numbers of fluorescent puncta at the dendrites and somas of the EGFP-expressing granule cells, respectively. The columns and bars represent the mean ± SEM, respectively. Numbers analyzed for dendritic and somatic distributions were 4–14 and 3–9, respectively. The distribution of immunoreactivity in somas was statistically analyzed for all four stages, and that in dendrites was done for stages II, III, and IV. Statistical analysis was performed by one-way ANOVA, followed by Tukey's multiple-comparison tests. In all analyses, *p < 0.05, **p < 0.01 compared with stage I; •p < 0.05, ••p < 0.01, •••p < 0.001 compared with stage II; ■p < 0.05, ■■p < 0.01 compared with stage III.
- Figure 4.
Blockade of the maturation of granule cells by membrane depolarization with high KCl via the CaN signaling. A–D, Organotypic slices were cultured under different conditions from DIV 3 to DIV 10, and images of EGFP-expressing granule cells were obtained by confocal microscopy at DIV 10: Non-Depo, 5 mm KCl (A); Depo, 25 mm KCl (B); FK506/Depo, 25 mm KCl with 10 μm FK506 (C); Rap/Depo, 25 mm KCl with 10 μm rapamycin (D). E, F, Time courses of granule cell development and maturation under the culture conditions with 25 mm KCl in the presence (F) and absence (E) of 10 μm FK506. Percentages of different stages of granule cells were determined as described in Figure 1E. G, Percentages of stage IV granule cells at DIV 10 when cultures were maintained in media containing 5, 9, 12, 15, 25, or 35 mm KCl. H, Percentages of stage IV granule cells at DIV 10 when cultures were maintained in media containing 25 mm KCl with various concentrations of FK506. I, Percentages of stage III and stage IV granule cells at DIV 10 when the cells were cultured at 5, 25, or 25 mm KCl in the presence of either 10 μm FK506 or 10 μm rapamycin. In E–I, experiments of organotypic cultures were performed three times, and data shown are from a representative experiment. The symbols/columns and bars represent the mean ± SEM, respectively.
- Figure 5.
Expression of marker proteins in granule cells cultured under the depolarized condition in the presence and absence of FK506. A, C, E, G, Confocal images of expression of GABAAα6 (A), Homer 2 (C), PSD-95 (E), and synaptophysin (G) as analyzed by EGFP fluorescence (green) and immunostaining with the respective antibodies (red) at DIV 10. Immunostained GABAAα6 at dendrites is indicated in the insets. B, D, Quantification of immunostained GABAAα6 (B) and Homer 2 (D) in granule cells. The black and white bars indicate the number of immunostained puncta of GABAAα6 and Homer 2 at the dendrites (n = 7–10) and somas (n = 3–4), respectively, of EGFP-expressing granule cells at DIV 10. F, Quantification of immunostained PSD-95 in granule cells at DIV 10 under the indicated conditions (n = 6–10). Statistical analysis was done by using unpaired Student's t test for B and D, and by one-way ANOVA, followed by Tukey's multiple-comparison tests for F. The columns and bars represent the mean ± SEM, respectively. **p < 0.01; ***p < 0.001.
- Figure 6.
Effects of high KCl and FK506 on electrophysiological properties of organotypically cultured granule cells. A, B, Organotypic slices were cultured at 25 mm KCl in the presence or absence of 10 μm FK506. Resting membrane potentials (Vrest) and action potentials evoked by injecting the indicated currents for 600 ms were measured from granule cells at DIV 10–14 as described in Figure 2, except that recording was performed at 25 mm KCl. Representative voltage traces are indicated in B. C–E, Organotypic slices were cultured at 5 mm KCl or 25 mm KCl with or without 10 μm FK506. Resting membrane potential (C), membrane resistance (Rm) (D), and action potentials (E) of granule cells at DIV 10–14 were measured as described in Figure 2 by recording these cells at 5 mm KCl within 1 min after formation of the whole-cell mode. The numbers in parentheses (A, C, D) are numbers of cells examined.
- Figure 7.
Recovery of GABAergic transmission by the addition of FK506 to depolarized granule cells. Granule cells were cultured at 5 mm KCl (A) or 25 mm KCl without (B) or with (C) 10 μm FK506. Spontaneous IPSCs were recorded at the holding potential of −20 mV from granule cells at DIV 10 in the presence and absence of 20 μm bicuculline methochloride. Representative current traces and an extended timescale of IPSCs are indicated in the top and bottom traces, respectively.
