Single-axon level morphological analysis of corticofugal projection neurons in mouse barrel field

Abstract

Corticofugal projection neurons are key components in connecting the neocortex and the subcortical regions. In the barrel field, these neurons have various projection targets and play crucial roles in the rodent whisker sensorimotor system. However, the projection features of corticofugal projection neurons at the single-axon level are far from comprehensive elucidation. Based on a brain-wide positioning system with high-resolution imaging for Thy1-GFP M-line mice brains, we reconstructed and analyzed more than one hundred corticofugal projection neurons in both layer V and VI of barrel cortex. The dual-color imaging made it possible to locate the neurons' somata, trace their corresponding dendrites and axons and then distinguish the neurons as L5 type I/II or L6 type. The corticofugal projection pattern showed significant diversity across individual neurons. Usually, the L5 type I neurons have greater multi-region projection potential. The thalamus and the midbrain are the most frequent projection targets among the investigated multidirectional projection neurons, and the hypothalamus is particularly unique in that it only appears in multidirectional projection situations. Statistically, the average branch length of apical dendrites in multi-region projection groups is larger than that of single-region projection groups. This study demonstrated a single-axon-level analysis for barrel corticofugal projection neurons, which could provide a micro-anatomical basis for interpreting whisker sensorimotor circuit function.

Figure 1

Location of the barrel field and the local morphology of the pyramidal neurons. (A) The merged image of the GFP-channel (green, thickness of projection: 300 μm) and the PI-channel (red, thickness of projection: 2 μm). The superimposed outline is used for brain area localization. SSp-bfd indicates barrel-related primary somatosensory cortex. TH represents the thalamus. Scale Bar = 1 mm. (B) Enlarged view of the yellow bounding box in (A) with rotation, which illustrates the cortical cytoarchitecture and the fine morphology of pyramidal neurons. Scale Bar = 100 μm.

Figure 2

Three typical morphology-based types of pyramidal neurons (A) are reconstructed and encoded with different colors. (B) Branch number of the apical tuft versus apical dendrite height in L5 type I and II. (C) Distribution boxes of relative soma locations of L5 type I and II, where the red dotted line represents the mean value. L5 type I: n = 67; L5 type II: n = 9. (D) The general spatial projection of 65 pyramidal neurons from the same hemisphere of one brain dataset. L5 type I: green, n = 39; L5 type II: purple, n = 5; L6: blue, n = 21.

Figure 3

Pie charts on the fractional occurrence frequency of a certain projection target region to all regions observed by the axon tip. Results of L5 type I neurons (A) and L6 neurons (B). The frequency values ranked in descending order in a clockwise direction. Abbreviations: cst – corticospinal tract; MB – Midbrain; TH –Thalamus; P – Pons; HY – Hypothalamus; STR – Striatum; PAL – Pallidum; MY – Medulla; ml – medial lemniscus; alv – alveus; cc – corpus callosum.

Figure 4

Quantification of the projection mode of L5 type I neurons. (A) The collaboration matrix quantifies the “posterior probability” of another projection region given the neuron projection to the current region. Taking the value in the second row and the third column as an example: 0.25 = Num _ST/Num _S, Num _ST represents the number of multidirectional projection neurons to include the mode “ST”. Num _S represents the number of neurons that project to the striatum. Similarly, the value in the third row and the second column can be calculated by: 0.10 = Num _ST/Num _T. (B) The coexistence matrix measures the occurrence frequency of two directional projection modes. Every element of the matrix represents the ratio of some neurons projecting to the two regions to all the neurons having at least two projection regions (n = 28).

Figure 5

Comparative complexity of dendrites and axons between the single-region projection group (SPG) and the multi-region projection group (MPG). (A) Reconstruction samples of single-region projection neurons (n = 3, lavender) and multi-region projection neurons (n = 2, purple). Comparisons of apical dendrite length (B), basal dendrite length (C), axon length (D), total number of apical dendrite branches (E), total number of basal dendrite branches (F), and total number of axon branches (G) between the SPG and the MPG. SPG: n = 39; MPG: n = 28. *Represents p-value < 0.05, *** Represents p-value < 0.001.

Figure 6

Linear correlations of the axonal and dendritic complexity parameters of L5 type I neurons, where R represents the Pearson correlation coefficient and p represents the p-value (two-tailed).