Diverse Long-Range Axonal Projections of Excitatory Layer 2/3 Neurons in Mouse Barrel Cortex

Abstract

Excitatory projection neurons of the neocortex are thought to play important roles in perceptual and cognitive functions of the brain by directly connecting diverse cortical and subcortical areas. However, many aspects of the anatomical organization of these inter-areal connections are unknown. Here, we studied long-range axonal projections of excitatory layer 2/3 neurons with cell bodies located in mouse primary somatosensory barrel cortex (wS1). As a population, these neurons densely projected to secondary whisker somatosensory cortex (wS2) and primary/secondary whisker motor cortex (wM1/2), with additional axon in the dysgranular zone surrounding the barrel field, perirhinal temporal association cortex and striatum. In three-dimensional reconstructions of 6 individual wS2-projecting neurons and 9 individual wM1/2-projecting neurons, we found that both classes of neurons had extensive local axon in layers 2/3 and 5 of wS1. Neurons projecting to wS2 did not send axon to wM1/2, whereas a small subset of wM1/2-projecting neurons had relatively weak projections to wS2. A small fraction of projection neurons solely targeted wS2 or wM1/2. However, axon collaterals from wS2-projecting and wM1/2-projecting neurons were typically also found in subsets of various additional areas, including the dysgranular zone, perirhinal temporal association cortex and striatum. Our data suggest extensive diversity in the axonal targets selected by individual nearby cortical long-range projection neurons with somata located in layer 2/3 of wS1.

Keywords: axonal structure; barrel cortex; layer 2/3 pyramidal neuron; neocortex; projection neurons.

Figure 1

Selective labeling of wS1 L2/3 neurons and their long-range axonal projections. (A) L2/3 neurons selectively expressed tdTomato in Rasgrf2-dCre mice crossed with LSL-tdTomato mice. (B) An example injection of AAV-FLEX-tdTomato targeted to the C2 whisker representation of Rasgrf2-dCre mice to express tdTomato in L2/3 neurons of wS1. (C–I) In the same mouse as panel B, increasing the camera exposure time allowed axonal fluorescence to be observed in wS2 (C), wM1 (D), AM (E), wM2 (F), CM and striatum (G), a posterior region (PP) presumably overlapping with visual cortex (H), and PM, PL1/2 and TeA (I).

Figure 2

Hotspots of axonal projections of wS1 L2/3 neurons in dorsal sensorimotor cortex. (A) Schematic drawing showing the approximate locations of the major hotspots of long-range axon in the dorsal cortex from wS1 L2/3. (B) An oblique view of (A) showing schematic locations of long-range projections. (C) The estimated center of each projection in sensorimotor cortex relative to the center of the injection site in wS1. (D) The estimated center of each projection in sensorimotor cortex relative to the Bregma (Paxinos and Franklin, 2001).

Figure 3

Morphology of an individual S2p neuron. (A) The dendrites, soma and local axonal arborisations in wS1 of an example neuron (AP049) viewed at low magnification (left) with 3D reconstruction of neurites in that section superimposed (right; red: axon; white: dendrite; green: soma). (B) At higher magnification in wS1, spines become obvious on dendrites, and the axon can be seen to be labeled with high-contrast at a specific focal plane (left). The 3D tracing of the whole section was superimposed (right). (C) Same as (A), but in wS2. (D) Same as (B), but for wS2 (E) Same as (A), but in dorsolateral striatum. (F) Same as (B), but in dorsolateral striatum. (G) Local axon, dendrite and soma of this neuron. (H) Coronal projection of this neuron's structure. (I) Horizontal projection of this neuron's structure. The wS1 barrel field is schematically indicated.

Figure 4

Axonal and dendritic structure of neurons retrogradely-labeled from wS2. (A–G) Dendritic (black) and axonal (red in A–F, blue in G) arborisations of different individual neurons viewed locally in wS1 (left), in coronal projection (center) and in horizontal projection (right). The wS1 barrel field is schematically indicated (right).

Figure 5

Morphology of an individual M1p neuron. (A) The dendrites, soma and local axonal arborisations in wS1 of an example neuron (TY308) (left) overlaid with 3D reconstruction of neurites (right; blue: axon; white: dendrite; green: soma). (B) Example axonal arborisations in wM1 from this neuron (left) overlaid with 3D reconstruction (right). (C) Local axon, dendrites and cell body of this neuron (left). Coronal (middle) and horizontal (right) projection of this neuron's structure together with the schematic wS1 barrel field (right).

Figure 6

Axonal and dendritic structure of neurons retrogradely-labeled from wM1. (A–H) Dendritic (black) and axonal (blue) arborisations of different individual neurons viewed locally in wS1 (left), in coronal projection (center) and in horizontal projection with the schematic wS1 barrel field (right).

Figure 7

Comparison of the structures of S2p and M1p neurons. (A) Overlay of dendrites (black) and local axon (red for S2p, blue for M1p) from all S2p (left) and M1p (right) singly-labeled neurons in individual brains. The superimposed neurons were vertically aligned to the pia and horizontally aligned with respect to the main descending axon. (B) Overlay of dendrites (black) and axonal structures (red for S2p, blue for M1p) for S2p (left) and M1p (right) neurons shown in coronal projection. (C) Same as (B), but in horizontal projection with the schematic wS1 barrel field. (D) Quantification of dendritic length (left), number of dendrites emanating from the cell body (center) and number of dendritic branch points (right). See also Supplementary Data File 1. (E) Quantification of total axonal length for S2p and M1p neurons. See also Supplementary Data File 1. (F) Thresholded analysis of cell-by-cell axon length in specific targets. Each column represents the axon length of one neuron. Some S2p neurons (red) strongly projected to wS2 and striatum. Some M1p neurons strongly projected to wM1, striatum and CM. Dark colors indicate > 10 mm of branching axon in the target region. Light shading indicates regions with < 10 mm and > 1 mm of axon. See also Supplementary Data File 1. (G) Quantification of branching axon length in wS1, wS2, wM1/2, PL1/2, AM, CM, PM, PP, TeA, and striatum. See also Supplementary Data File 1.