Axonal and Dendritic Morphology of Excitatory Neurons in Layer 2/3 Mouse Barrel Cortex Imaged Through Whole-Brain Two-Photon Tomography and Registered to a Digital Brain Atlas

Abstract

Communication between cortical areas contributes importantly to sensory perception and cognition. On the millisecond time scale, information is signaled from one brain area to another by action potentials propagating across long-range axonal arborizations. Here, we develop and test methodology for imaging and annotating the brain-wide axonal arborizations of individual excitatory layer 2/3 neurons in mouse barrel cortex through single-cell electroporation and two-photon serial section tomography followed by registration to a digital brain atlas. Each neuron had an extensive local axon within the barrel cortex. In addition, individual neurons innervated subsets of secondary somatosensory cortex; primary somatosensory cortex for upper limb, trunk, and lower limb; primary and secondary motor cortex; visual and auditory cortical regions; dorsolateral striatum; and various fiber bundles. In the future, it will be important to assess if the diversity of axonal projections across individual layer 2/3 mouse barrel cortex neurons is accompanied by functional differences in their activity patterns.

Keywords: axonal morphology; barrel cortex; layer 2/3; mouse brain atlas; two-photon tomography.

Figure 1

Acquisition and analysis pipeline for single-cell reconstruction. (A) Shadow electroporation labels a single neuron by introducing GFP plasmids. After 3–5 days of expression time, the cell was viewed through a cranial window under a two-photon microscope to control for GFP expression and the absence of any sign of apoptosis. Animals were then perfused and the brain was cleared in a modified CUBIC solution. Finally, the sample was embedded in agarose and imaged under a two-photon tomographic microscope. (B) Example snapshots of an electroporation session. Left, fluorescent dye from the electroporation pipette fills the extracellular space revealing cell bodies as shadows in the image. The pipette approaches and contacts a randomly selected neuron. Right, following electroporation, the fluorescent dye rapidly fills the cell indicating successful pipette content delivery into the cell. (C) Quality check of the labeled neuron through the cranial window before perfusion. (D) Example image acquired from two-photon tomography. Left, a coronal section with a region of interest near the cell body. Right, the region of interest at higher resolution, showing the cell body, its dendrites, and its main descending axon. (E) Semi-automatic neuron reconstruction using Vaa3d. Left, an example region with axon in Vaa3d. Right, the same region with annotations (red) highlighting the axon. (F) Alignment with the Allen Mouse CCFv3. Left, a coronal section of the CCFv3 template. Middle, the template deformed to align with the sample space. Right, the CCFv3 atlas deformed in the same way as the template to match the sample space.

Figure 2

Reconstruction and quantification of example neuron AL110 with projections to the supplementary somatosensory cortex. (A) Serial overlays of GFP-labeled axon (red) and dendrites (green) in coronal views encompassing the anterior-posterior span of the axons of 1.5 mm. Each section represents a maximum projection of 300 μm. (B) Maximum projection of the reconstructed axon and dendrites in horizontal view, aligned to the Allen Mouse CCFv3 to indicate the boundaries between cortical regions. (C) Maximum projection of reconstructed axon and dendrites in coronal view overlaid with an anatomical section from the Allen Mouse CCFv3. (D) Maximum projection of reconstructed axon and dendrites in a tangential view (rotated 30 degrees) over the barrel field (blue). The cell is located in the D3 barrel column. (E) Quantification of axonal (top) and dendritic (bottom) length in respective brain regions identified by the Allen Mouse CCFv3. For (B) to (E): dendrites are shown in black, axon in gray matter is shown in red, and axon in white matter is shown in blue.

Figure 3

Reconstruction and quantification of example neuron AL126 with projections to the primary and secondary motor cortex. (A) Serial overlays of GFP-labeled axon (red) and dendrites (green) in coronal views encompassing the anterior-posterior span of the axons of 3.5 mm. Each section represents a maximum projection of 700 μm. (B) Maximum projection of the reconstructed axon and dendrites in horizontal view. (C) Maximum projection of the axon and dendrites in coronal view. (D) Maximum projection of the axon and dendrites in tangential view (rotated 30 degrees) over the barrel field. The neuron is located in the D1 barrel column. (E) Quantification of axonal (top) and dendritic (bottom) length in respective brain regions identified by the Allen Mouse CCFv3. For (B) to (E): dendrites are shown in black; axon in neocortical gray matter is shown in red; axon in white matter is shown in blue.

Figure 4

Reconstruction and quantification of example neuron AL157 with projections to the primary somatosensory cortex upper limb area. (A) Serial overlays of axon (red) and dendrites (green) in coronal views encompassing the anterior-posterior span of the axons of 2.125 mm. Each section represents a maximum projection of 425 μm. (B) Maximum projection of the reconstructed axon and dendrites in horizontal view. (C) Maximum projection of the axon and dendrites in coronal view. (D) Maximum projection of the axon and dendrites in tangential view (rotated 30 degrees) over the barrel field. The cell is located in the E2 barrel column. (E) Quantification of axonal (top) and dendritic (bottom) length in respective brain regions identified by the Allen Mouse CCFv3. For (B) to (E): dendrites are shown in black; axon in neocortical gray matter is shown in red; axon in white matter is shown in blue.

Figure 5

Reconstruction and quantification of example neuron GF243 with projections to an unassigned region of the primary somatosensory cortex and an anterior visual area. (A) Serial overlays of axon (red) and dendrites (green) in coronal views encompassing the anterior-posterior span of the axons of 1.25 mm. Each section represents a maximum projection of 250 μm. (B) Maximum projection of the reconstructed axon and dendrites in horizontal view. (C) Maximum projection of the axon and dendrites in coronal view. (D) Maximum projection of the axon and dendrites in tangential view (rotated 30 degrees) over the barrel field. The cell body is in the septa between the C1 and D1 barrel columns. (E) Quantification of axonal (top) and dendritic (bottom) length in respective brain regions identified by the Allen Mouse CCFv3. For (B) to (E): dendrites are shown in black; axon in neocortical gray matter is shown in red; axon in white matter is shown in blue.

Figure 6

Reconstruction and quantification of example neuron AL131 with projections to the caudoputamen and multiple visual areas. (A) Serial overlays of axon (red) and dendrites (green) in coronal views encompassing the anterior-posterior span of the axons of 4.125 mm. Each section represents a maximum projection of 825 μm. (B) Maximum projection of the reconstructed axon and dendrites in horizontal view. (C) Maximum projection of the axon and dendrites in coronal view. (D) Maximum projection of the axon and dendrites in tangential view (rotated 30 degrees) over the barrel field. The neuron is in the C2 barrel column. (E) Quantification of axonal (top) and dendritic (bottom) length in respective brain regions identified by the Allen Mouse CCFv3. For (B) to (E): dendrites are shown in black; axon in neocortical gray matter is shown in red; axon in the striatum is shown in green; axon in white matter is shown in blue.

Figure 7

Reconstruction and quantification of example neuron AL142 with projections to the lateral caudoputamen and towards the amygdala. (A) Serial overlays of axon (red) and dendrites (green) in coronal views encompassing the anterior-posterior span of the axons of 2.375 mm. Each section represents a maximum projection of 475 μm. (B) Maximum projection of the reconstructed axon and dendrites in horizontal view. (C) Maximum projection of the axon and dendrites in coronal view. (D) Maximum projection of the axon and dendrites in tangential view (rotated 30 degrees) over the barrel field. The cell is in the septa between the D2 and D3 barrel columns. (E) Quantification of axonal (top) and dendritic (bottom) length in respective brain regions identified by the Allen Mouse CCFv3. For (B) to (E): dendrites are shown in black; axon in neocortical gray matter is shown in red; axon in the striatum is shown in green; axon in white matter is shown in blue.

Figure 8

Summary of the ten reconstructed neurons. (A) Horizontal overlay of axons aligned to the Allen Mouse CCFv3. The axon of each neuron is shown in a different color. (B) Coronal overlay of axons. (C) Tangential view of axons aligned to the barrel map. (D) Quantification of axonal length in respective brain regions. The length of the axon in each layer of a specific region is summed up. The different brain regions receiving projections from the labeled neurons are sorted according to the mean length of the reconstructed axon across all neurons in each region of interest, from the longest axonal length on the left to the shortest axonal length on the right.