Fig 1.
The morphology of pyramidal and granule cells at DIV 13 in dissociated culture.
(A) Typical morphology of pyramidal cells, granule cells and interneurons. The cell type was characterized by immunostaining with Math2, Prox1 (magenta) and GAD67, respectively. Pyramidal and granule cells were visualized by GFP transfection. Interneurons were visualized by immunostaining with GAD67. Scale bar: 40 μm. (B) Histogram distribution of somal size in pyramidal and granule cells of DIV 10–13. n = 40 for each cell type. (C) Axon/dendrite specification as demarcated by Ankyrin G expression. Cells were immunostained with Ankyrin G (magenta) and Math2 or Prox1 (blue) at DIV 7. Asterisks indicate axon. Scale bar: 20 μm. (D) Expression of synaptic markers at DIV 16. Boxed regions in the left panel are shown in the right panels. PSD95 (red) was localized in protrusions in both principal (upper) and minor (lower) dendrites. Some PSD95 signals in dendritic protrusions (asterisks) were apposed to Synapsin I signals (green). Dendrites were visualized by GFP expression. Scale bar: 20 μm (left), 2 μm (right). (E-G) Quantification of total dendritic length (E), the number of primary dendrites (F) and branch terminals (G). n = 20. Student's t-test: *P<0.05, **P<0.03, ***P<0.001. (H) Ratio of length of each dendrite arbor to the longest dendrite. All dendrite arbors in a neuron were ranked from the longest to the shortest. n = 20.
Fig 2.
Population of pyramidal and granule cells in dissociated culture.
(A) Proportion of neuronal species in cultures from different aged pups. Whole neurons, pyramidal cells and granule cells were identified as NeuN-, Math2- and Prox1-positive cells, respectively. n = 722 (E17), 508 (P0), 634 (P4). (B) Maturation of granule cells. Each point shows the ratio of each marker-positive cells to Prox1-positive cells. n (DIV 3, DIV 7, DIV 11, DIV 16) = (371, 202, 258, 95) for calretinin, (395, 186, 208, 123) for calbindin. (C) Maturation marker expression of granule cells. Cells were immunostained with calretinin or calbindin (green) and Prox1 (magenta). Scale bar: 30 μm.
Fig 3.
Morphology change in pyramidal and granule cells.
(A-B) Typical morphology of pyramidal and granule cells at DIV 7 (A) and DIV 16 (B) of cultures prepared from P0 mice. Cells were visualized by GFP transfection. Cell types were identified by Math2 and Prox1 immunostaining (magenta). The principal dendrite and axon were indicated by white arrowhead and asterisk, respectively. Scale bar: 40 μm. (C-E) Quantification of total dendritic length (C), the number of branch terminals (D) and primary dendrites (E) during dendrite development. n = 20. Student's t-test: *P<0.05, **P<0.03, ***P<0.001.
Fig 4.
Dynamics of dendrite development.
(A) Typical morphology change in pyramidal and granule cells cultured from P0 mice. Blue lines indicate newly extended dendrites, red lines indicate retracted dendrites within a 12 hr period of observation. (B) The number of dendrites during dendritic development. Each bar indicates the number of stable, appeared or disappeared dendrites within a 12 hr period of observation in 30 min-interval movies. n = 10 (DIV 7, 10), 16 (DIV 13).
Fig 5.
The principal dendrite determination.
(A) Typical morphology of the pyramidal and granule cell at DIV 4. Cells were isolated from P0 hippocampi and visualized by GFP transfection. Cell types were identified by Math2 and Prox1 immunostaining (magenta). The principal dendrite and axon were indicated by white arrowhead and asterisk, respectively. Scale bar: 20 μm. (B-E) Time-lapse imaging of dendritic development in GFP-transfected pyramidal and granule cells. Cell types were identified by Math2 and Prox1 immunostaining (magenta) after imaging. Asterisks indicate axon; arrows and arrowheads indicate the principal dendrites at the time points of observation. Scale bar: 30 μm. (F) The frequency of replacement of the principal dendrite observed from DIV 4. The replacement of the principal dendrite was counted every 10 hr in 30 min-interval movies. n = 22.
Fig 6.
Localization of the Golgi apparatus.
(A) Immunofluorescence of the Golgi apparatus with anti-GM130 in pyramidal and granule cells transfected with GFP at DIV 7. Cell morphology and the Golgi were visualized by GFP expression (green) and anti-GM130 immunostaining (magenta). Cell types were identified by Math2 and Prox1 immunostaining (cyan). Scale bar: 10 μm. (B-C) Golgi distribution at DIV 7 and DIV 16 in pyramidal (B) and granule cells (C). Defining the centroid of the nucleus as the origin of a polar coordinate system, the soma was separated into three regions as shown. n = 10.
Fig 7.
Translocation of the Golgi apparatus.
(A) Golgi localization in a neuron transfected with TdTomato (magenta) and AcGFP-Golgi (green). Scale bar: 10 μm. (B) Golgi dynamics during the principal dendrite replacement. Neurons were transfected with TdTomato (magenta) and AcGFP-Golgi (green). White arrowheads indicate the tip of AcGFP-Golgi. Scale bar: 20 μm. (C) The frequency of simultaneous Golgi relocation to the principal dendrite. n = 17. (D) Golgi localization was visualized with anti-GM130 (green) in a neuron overexpressing GRASP65-GFP. Cell was visualized by TdTomato expression (magenta). The Golgi was fragmented and dispersed in the cell soma. (E) Polarity index (ratio of the 2nd to the 1st dendritic length) of control neurons and neurons overexpressing GRASP65-GFP at DIV 10. n = 20 (control), 10 (GRASP65-GFP); Student’s t-test: *P< 0.001. (F) Time-lapse imaging of dendrites and Golgi (inset) in a neuron overexpressing GRASP65-GFP. TdTomato (magenta) and GRASP65-GFP (green) were transfected at DIV 4 and images were taken from DIV 5.