Development of layer-specific axonal arborizations in mouse primary somatosensory cortex

Abstract

In the developing neocortex, pyramidal neurons use molecular cues to form axonal arbors selectively in the correct layers. Despite the utility of mice for molecular and genetic studies, little work has been done on the development of layer-specific axonal arborizations of pyramidal neurons in mice. We intracellularly labeled and reconstructed the axons of layer 2/3 and layer 5 pyramidal neurons in slices of primary somatosensory cortex from C57Bl6 mice on postnatal days 7-21. For all neurons studied, the development of the axonal arborizations in mice follows a pattern similar to that seen in other species; laminar specificity of the earliest axonal branches is similar to that of mature animals. At P7, pyramidal neurons are very simple, having only a main descending axon and few primary branches. Between P7 and P10, there is a large increase in the total number of axonal branches, and axons continue to increase in complexity and total length from P10 to P21. Unlike observations in ferrets, cats, and monkeys, two types of layer 2/3 pyramidal neurons are present in both mature and developing mice; cells in superficial layer 2/3 lack axonal arbors in layer 4, and cells close to the layer 4 border have substantial axonal arbors within layer 4. We also describe axonal and dendritic arborization patterns of three pyramidal cell types in layer 5. The axons of tall-tufted layer 5 pyramidal neurons arborize almost exclusively within deep layers while tall-simple, and short layer 5 pyramidal neurons also project axons to superficial layers.

Figure 1

Photomicrographs illustrating examples of typical intracellular labeling results at P10 (A, B) and P14 (C, D). For each age the labeled neurons are visualized with strepavidin-Cy3, which binds the biocytin (A, C) and laminar borders are determined by a DAPI counterstain (B, D). The reconstruction of the neuron at P10 (A, B) is in figure 5 (first column for P10). The scale bar is 250 microns and applies to all panels.

Figure 2

Bar graphs indicate the number of primary (white bars) and total (black bars) axonal branch points per cell (A, B) and the total axonal length per cell (C, D) for layer 2/3 pyramidal neurons (A, C) and layer 5 pyramidal neurons (B, D). Primary axonal branches are defined as branches from the main descending axon. All values are mean ± SEM. Asterisks denote statistical significance between ages, with the grey asterisks for primary axonal branches and black asterisks for total axonal branches and axonal length (* = p<0.05 ** = p<0.001).

Figure 3

Representative examples of reconstructions of both type I (two columns on the left) and type II (two columns on the right) layer 2/3 pyramidal neurons. Neurons are illustrated with axons depicted in gray and dendrites in black. Ages are indicated to the left. Lines define the laminar boundaries for each cortical layer. The upper line indicates the top of the slice, with layer 1 included with layer 2/3. The type I neurons show the classic pattern with the descending axon crossing layer 4 with a few or no branches. In contrast the type II neurons show a large number of axonal branches within layer 4. The scale bar is 250 microns and applies to all drawings.

Figure 4

Representative examples of reconstructions of tall-tufted layer 5 pyramidal neurons. The tall-tufted layer 5 pyramidal neurons have little, if any, axonal projections to the superficial layers. Conventions are the same as figure 3. The scale bar is 250 microns and applies to all drawings.

Figure 5

Representative examples of reconstructions of tall-simple layer 5 pyramidal neurons. The tall-simple layer 5 pyramidal neurons have a significant axonal projection to the superficial layers in contrast to the pattern observed in the tall-tufted layer 5 pyramidal neurons. Conventions are the same as figure 3. The scale bar is 250 microns and applies to all drawings.

Figure 6

Representative examples of reconstructions of short layer 5 pyramidal neurons. The short layer 5 pyramidal neurons have a significant axonal projection to the superficial layers in contrast to the pattern observed in the tall-tufted layer 5 pyramidal neurons. Conventions are the same as figure 3. The scale bar is 250 microns and applies to all drawings.

Figure 7

Bar graphs indicate, for all layer 2/3 pyramidal neurons, the number of axonal branch points (A) and the axonal length (B) per cell present in each cortical layer (mean ± SEM).

Figure 8

Each layer 2/3 pyramidal neuron reconstructed in this study is plotted as a function of its distance from the top of layer 4 to the bottom of the soma against the percentage of axonal branches located in layer 4 (A) or the percentage of basal dendritic length located in layer 4 (B). Based on these plots we separated the neurons into two groups according to the distance from the top of layer 4, with neurons found at a distance more than 60 microns being termed type I neurons and neurons found at a distance less than 60 microns being termed type II neurons (see text). The cut-off point for the cell types are denoted by the black line in both graphs A and B.

Figure 9

Bar graphs indicate, for type I layer 2/3 pyramidal neurons (A, B) and type II layer 2/3 pyramidal neurons (C, D), the number of axonal branch points (A, C) and the axonal length (B, D) per cell present in each cortical layer (mean ± SEM). Panels E and F indicate the percentage of axonal branches per cell in each cortical layer (mean ± SEM) for type I and type II layer 2/3 pyramidal neurons respectively. Asterisks denote statistical significance between type I and type II neurons at indicated ages (* = p<0.05, ** = p<0.001). Diamond denotes statistical significance between ages for type II neurons (p<0.05).

Figure 10

Each layer 5 pyramidal neuron reconstructed in this study is plotted as a function of its total apical dendritic length against the axonal length in layer 2/3. From this plot the separation of tall-simple and tall-tufted layer 5 pyramidal neurons can be seen with the tall-tufted neurons having greater apical dendritic length and less axonal length in layer 2/3. The vertical line denotes the cut-off between the tall-tufted and tall-simple layer 5 neurons. Note that the y-axis is split for one short layer 5 neuron with a very long axon in layer 2/3. There is a significant difference in total apical dendritic length between tall-tufted layer 5 neurons and both tall-simple (at all ages except P7; p < 0.05, t-test) and short (p < 0.001, t-test) layer 5 pyramidal neurons.

Figure 11

Bar graphs indicate the number of axonal branch points (A, C, E) and the axonal length (B, D, F) per cell present in each cortical layer (mean ± SEM) for tall-tufted layer 5 pyramidal neurons (A, B), tall-simple layer 5 pyramidal neurons (C, D), and short layer 5 pyramidal neurons (E, F). Statistical significance (p<0.05) between groups at the indicated ages is denoted by either an asterisk for tall-tufted vs. short, a triangle for tall-tufted vs. tall-simple, or a diamond for tall-simple vs. short layer 5 pyramidal neurons.