Modulation of the frequency of turning and the rate of migration of granule cells in vitro by altering the Ca²⁺ and cAMP signaling pathways. (A) Measurement of granule cell turning. In each photograph arrows and asterisks indicate the leading process and the soma of a granule cell, respectively. The numbers at the top of each photograph represent the elapsed time after in vitro. Bar: 10 μm.(B) Schematic representation showing how to identify and count the turning of granule cell. (C) Histograms showing the effects of the alterations of the Ca²⁺ and cyclic nucleotide signaling pathways on the frequency of turning (top histogram) and the rate of migration (bottom histogram) of granule cells. Each column represents the average frequency of turning and the average rate of migration obtained during a 10 hour-period of observation between 20–30 hours after in vitro. Each reagent was added to the culture medium at 20 hour after in vitro. Single (p <0.05) and double (p <0.01) asterisks indicate statistical significance. Bar: S.D.

Changes in the ratio of the 1^st and 2^nd modes of granule cell turning in vitro by altering the Ca²⁺ and cyclic nucleotide signaling pathways. (A) Schematic representation of four distinct modes of granule cell turning in vitro. The numbers in granule cell somata indicate the order in each mode of turning. (B) Histograms showing the effects of the alterations of the Ca²⁺ and cyclic nucleotide signaling pathways on the ratio of the 1^st and 2^nd modes of granule cell turning in vitro. Each column represents the ratio in the occurrences of the 1^st and 2^nd modes of granule cell turning during a 10 hour-period of observation between 20–30 hours after in vitro. Each reagent was added to the culture medium at 20 hour after in vitro. Single (p <0.05) and double (p <0.01) asterisks indicate statistical significance. Bar: S.D.

Migration and differentiation of cerebellar granule cells in vivo. (A) The three-dimensional representation of granule cell migration from the EGL to the IGL in the developing cerebellum. 1, Extension of two uneven horizontal processes near the top of the EGL; 2, Tangential migration in the middle and bottom of the EGL; 3, Bergmann glia-associated radial migration in the ML; 4, Stationary state in the PCL; 5, Glia-independent radial migration in the IGL; 6, Completion of migration in the middle or the bottom of the IGL. Abbreviations: P, Purkinje cell; B, Bergmann glia; G, Golgi cell, g, postmigratory granule cell; cf, climbing fiber; mft, mossy fiber terminal. (B) Micrographs showing the changes in the morphology of granule cells in the P10 mouse cerebellum revealed by Golgi-staining. a–j, granule cell precursors at the top of the EGL. k–z, tangentially migrating granule cells at the middle and bottom of the EGL. a1 and f1, Purkinje cells. b1, Bergmann glial cells. c1–e1 and g1–j1, radially migrating granule cells in the ML. k1 and r1–y1, postmigratory granule cells in the IGL. l1–q1, radially migrating granule cells in the IGL. Bar: 10 μm in a-y1.

Changes in the average number of turning granule cells at the EGL-ML border of the P10 mouse cerebella by the injection of saline, NMDA, caffeine, Rp-cAMP, or Br-cGMP. Each column represents the average number of turning granule cells at the 100 μm-length EGL-ML border obtained from at least 500 cells. Single (p <0.05) and double (p <0.01) asterisks indicate statistical significance. Bar: S.D.

Autonomous changes in the direction of granule cell migration in vitro. (A) Time-lapse series of images showing the turning of a granule cell in the microexplant cultures of P2 mouse cerebella over time. Elapsed time after in vitro is indicated on the bottom of each photograph. Black and white arrows indicate granule cell processes. Asterisks represent the granule cell soma. Bar: 15 μm. (B) Turning of the granule cell (shown in A) towards the left side. Seven pseudocolor images of the cell taken every 5 minutes during the period of 20:00–20:30 after in vitro are superimposed. (C) Turning of the granule cell (shown in A) towards the right side and then towards the bottom. Twenty-four pseudocolor images of the cell taken every 5 minutes during the period of 22:10–24:10 after in vitro are superimposed. (D) Reversing the direction of granule cell migration (shown in A). Twelve pseudocolor images of the cell taken every 5 minutes during the period of 24:40–25:35 after in vitro are superimposed. Bar: 12 μm in B–D. (E and F) Line drawings represent the trajectory of the migration of two granule cells in the microexplant cultures of P2–P3 mouse cerebella over time. The migration of these cells was observed in separate experiments. Black open circles represent the position of each granule cell at the beginning of observation (at 20:00 after in vitro). The interval between large filled circles (black and red) represents 30 minutes. The interval between small filled circles (black and red) represents 3 minutes. Red circles and red lines represent the frequent turning periods, while black circles and black lines represent the less frequent turning periods. The numbers in the figure represent the elapsed time after in vitro. Bar: 15 μm in E and F.

Four distinct modes of granule cell turning in vitro. (A)Sequential images of a granule cell represent the 1^st mode of turning. The tip of the leading process turned to the left side and then, the cell body followed the changes. (B, C) Sequential images of granule cells represent the 2^nd mode of turning. The tip of the leading process bifurcated, and both branches extended in the opposite direction. Subsequently, one of the branches collapsed and retracted, and the cell body followed the direction of extension of the remaining branch. (D) Sequential images of a granule cell represent the 1^st and 3^rd modes of turning. The tip of the leading process of the granule cell turned to the left side and then, the cell body followed the changes. Thereafter, the growth cone of the tip of the leading process collapsed. The trailing process then started to develop a new growth cone at the tip and converted into a new leading process. Sequentially, the soma started to migrate in the direction of the extension of the new leading process. (E and F)Sequential images of granule cells represent the 3^rd mode of turning. The leading process of the granule cells transformed to the trailing process and the trailing process became the leading process. Thereafter, the cells started to migrate in the reversed direction. (G) Sequential images of a granule cell represent the 4^th mode of turning. The granule cell completely withdrew the leading process, and extended a new leading process. Subsequently, the cell migrated toward the direction of the extension of the new leading process. Bar: 10 μm in A, D, E, F, 12 μm in B, and 8 μm in C, G. In A–G, asterisks and arrows indicate the granule cell somata and the leading processes, respectively. The numbers in each photograph represent the elapsed time after in vitro.

The summary of the effects of altering the Ca²⁺ and cyclic nucleotide signaling pathways on the 1^st and 2^nd modes of granule cell turning in vitro.

Micrographs showing three different modes of granule cell turning at the EGL-ML border of the P10 mouse cerebellum. (A) T-shape tuning of granule cells. a1–a25, Micrographs showing the extension of the vertical processes into the ML from the cell body of tangentially-oriented granule cells, which had two horizontally-extended axon-like processes. (B) L-shape turning of granule cells. b1–b34, Micrographs showing the turning of the tip of the horizontally-oriented leading process towards the ML. (C) Y-shape turning of granule cells. c1–c25, Micrographs showing the bifurcation of the tip of the horizontally-oriented leading process. Bar: 15 μm in a1-c25.

Changes in the ratio of the T-, L-, and Y-shape granule cell turning at the EGL-ML border of the P10 mouse cerebella by the application of saline, NMDA, caffeine, Rp-cAMP, or Br-cGMP. (A) Schematic representation of the T-, L-, and Y-shape granule cell turning in vivo. (B) Histograms showing the effects of the injection of saline, NMDA, caffeine, Rp-cAMP, or Br-cGMP on the ratio of the T-, L-, and Y-shape granule cell turning at the EGL-ML border. Single (p <0.05) and double (p <0.01) asterisks indicate statistical significance. Bar: S.D.