Cortical GABAergic interneurons transiently assume a sea urchin-like nonpolarized shape before axon initiation

Abstract

Mature neurons polarize by extending an axon and dendrites. In vitro studies of dissociated neurons have demonstrated that axons are initiated from a nonpolarized stage. Dissociated hippocampal neurons form four to five minor neurites shortly after plating but then one of them starts to elongate rapidly to become the future axon, whereas the rest constitutes the dendrites at later stages. However, neuroepithelial cells as well as migrating neurons in vivo are already polarized, raising the possibility that mature neurons inherit the polarities of immature neurons of neuroepithelial or migrating neurons. Here we show that the axon of interneurons in mouse cortical explant emerges from a morphologically nonpolarized shape. The morphological maturation of cortical interneurons labeled by electroporation at an embryonic stage was analyzed by time-lapse imaging during the perinatal stage. In contrast to earlier stages, most interneurons at this stage show sea urchin-like nonpolarized shapes with alternately extending and retracting short processes. Abruptly, one of these processes extends to give rise to an outstandingly long axon-like process. Given that the interneurons exhibit typical polarized shapes during embryonic development, the present results suggest that axon-dendrite polarity develops from a nonpolarized intermediate stage.

Figure 1.

Sea urchin-like cells alternately extend and retract short processes. A, B, Time sequence of confocal images of a sea urchin-like cell. Imaging began at P0.5. Elapsed time after start of observation (hours) is indicated on the top right corner of each photograph. C, Length of processes of cell shown in B plotted against elapsed time. Different colors represent different processes. Extensions and retractions can be seen. Scale bar, 30 μm.

Figure 2.

Extension of an axon-like process from a sea urchin-like cell. A, Time sequence of confocal images of a cell that initiated an axon-like process during time-lapse imaging. Time-lapse imaging began at P0.5. Panels in top and bottom rows show the same cell, but in the bottom row, an axon-like process is marked in pink. Elapsed time after the onset of imaging (hours) is indicated on the top right corner of the bottom panels. Dashed lines indicate the pial surface. B–E, Growing tips of axon-like processes. High magnification of the areas demarcated by rectangles in A. B corresponds to the blue rectangle in A and shows a sea urchin like-cell that has just initiated an axon-like process headed by a growth cone. C–E, Growing tips of the axon-like process demarcated by green rectangles in A. F, Time course of the extension of several processes from a sea urchin-like cell. Many short processes repeatedly extend and retract, but one continues to extend almost monotonically (thick black line). Note that two other processes extend up to 150–200 μm but fail to extend farther. This cell corresponds to the one shown in supplemental Movie 4 (available at www.jneurosci.org as supplemental material). G, H, Low-magnification images of the lateral cortex showing the morphologies of interneurons extending axon-like processes (magenta). G, Image taken 32 h after the onset of imaging; H, image taken 40 h after the onset of imaging. Comparison of these two images demonstrates extension of long processes. Scale bars: A, 30 μm; B–E, 10 μm; G, H, 100 μm. D, Dorsal; L, lateral.

Figure 3.

GABAergic interneurons observed in perinatal mouse neocortex in vivo. Neurons in the CP and marginal zone are shown. A–D, Confocal images of samples fixed in coronal sections at E18.5 (A), P0.5 (B), P1.5 (C), and P2.5 (D). Most labeled cells in the marginal zone show migrating neurons with a bipolar shape (A, bottom left), but those in the CP primarily showed a multipolar morphology at E18.5 (A, bottom right). At P0.5, although labeled cells extend longer processes, many show multipolar morphology. At later stages, cellular morphologies become more complex (D). In A–D, the largest panel in each shows a low-magnification view of the representative samples. The bottom two panels in A and right panels in B–D show high-power pictures. E, Length of the longest process becomes larger as development proceeds. F, Proportion of labeled cells bearing a long (>200 μm) process markedly increases during postnatal development. In E and F, numbers of neurons analyzed were 833 at E18.5, 745 at P0.5, 915 at P1.5, and 702 at P2.5. Scale bars: top panel in A, left panels in B–D, 50 μm; bottom panels in A, right panels in B–D, 10 μm.

Figure 4.

Schematic showing transitions of cortical interneuron morphology during perinatal development. Most cortical interneurons labeled by electroporation tangentially migrated in the MZ at E18.5. At this stage, they showed morphologies typical for migrating neurons extending a leading and a trailing process (gray). As development proceeds, they become situated in the CP, in which many of them alternately extend and retract of short processes (blue). These cells had a low motility of somata. Some of these cells then began to extend a long axon-like process (magenta) primarily toward the ventricle.