Organization and somatotopy of corticothalamic projections from L5B in mouse barrel cortex

Abstract

Neurons in cortical layer 5B (L5B) connect the cortex to numerous subcortical areas. Possibly the best-studied L5B cortico-subcortical connection is that between L5B neurons in the rodent barrel cortex (BC) and the posterior medial nucleus of the thalamus (POm). However, the spatial organization of L5B giant boutons in the POm and other subcortical targets is not known, and therefore it is unclear if this descending pathway retains somatotopy, i.e., body map organization, a hallmark of the ascending somatosensory pathway. We investigated the organization of the descending L5B pathway from the BC by dual-color anterograde labeling. We reconstructed and quantified the bouton clouds originating from adjacent L5B columns in the BC in three dimensions. L5B cells target six nuclei in the anterior midbrain and thalamus, including the posterior thalamus, the zona incerta, and the anterior pretectum. The L5B subcortical innervation is target specific in terms of bouton numbers, density, and projection volume. Common to all target nuclei investigated here is the maintenance of projection topology from different barrel columns in the BC, albeit with target-specific precision. We estimated low cortico-subcortical convergence and divergence, demonstrating that the L5B corticothalamic pathway is sparse and highly parallelized. Finally, the spatial organization of boutons and whisker map organization revealed the subdivision of the posterior group of the thalamus into four subnuclei (anterior, lateral, medial, and posterior). In conclusion, corticofugal L5B neurons establish a widespread cortico-subcortical network via sparse and somatotopically organized parallel pathways.

Keywords: corticothalamic projections; posterior medial thalamic nucleus; somatotopy; thalamus; whisker system.

Fig. 1.

Cortical bouton labeling and borders of subcortical target nuclei. Subcortical L5B boutons in the thalamus were labeled by virus-mediated expression of two different fluorescent proteins in barrel cortical neurons. (A) Schematic showing dual injection of viral particles encoding Synaptophysin–GFP (SP–GFP, green) and Synaptophysin–mOrange (SP–mOrange, magenta) into the BC and projections in the thalamus (modified from ref. 12). (B) Merged confocal fluorescence image of tangential sections of the BC at the level of layer 4, showing barrels (gray; Streptavidin staining) and deposits of SP–GFP (green) and SP–mOrange (magenta). (C) As in B, but at the L5B level with labeled L5B somata. Barrel outlines are from B. (D) Horizontal section (cytochrome c oxidase staining) containing L5B target nuclei in the thalamus: (POa, POm_medial, POm_lateral, PoT, and APT). The VPM, thalamic reticular nucleus (Rt), striatum (Str), and hippocampus (HC) are indicated for orientation. Zona incerta is shown in Fig. S1. (E) An image at a level similar to that shown in D but stained for neuronal somata by NeuroTrace. (Scale bars: 500 µm in B–E.)

Fig. 2.

Reconstructions of subcortical L5B bouton clouds. Shown are examples of confocal sections and bouton reconstructions. (A–C) Individual channels of horizontal sections through L5B target nuclei in the dorsal thalamus. (A) Somata (NeuroTrace staining; gray). (B) GFP-labeled L5B boutons (green). (C) mOrange-labeled L5B boutons (magenta). (D) Overlaid sections A–C. Dashed lines indicate borders of PO nuclei. (E) Reconstructed boutons from B and C (green and magenta, respectively). Bouton diameters are up-scaled twofold to increase visibility. Arrows indicate orientation (L, lateral; P, posterior). (F) Enlarged view of the boxed area in D but without the NeuroTrace signal. Arrowheads mark examples of putative giant L5B boutons (>1.5 µm). Small puncta (s, <1.5 µm) were excluded; “x” indicates examples of boutons that had their maximum brightness in an adjacent z-section. (G) 3D illustration of reconstructed boutons from experiments in A–F. Major grid lines are spaced by 500 µm. (Scale bars: 500 µm in A–D and F; 25 µm in E.)

Fig. S1.

Overview of deposits. Illustration of all approximate deposit locations and sizes (colored circles) overlaid over a consensus barrel field. Deposits with the same color were obtained from the same animal with one deposit leading to the expression of GFP and the other to the expression of mOrange.

Fig. S2.

Examples of horizontal slices of the ventral thalamus. (A) Ventral thalamus stained with cytochrome c oxidase. (B) Ventral thalamus stained with fluorescent Nissl stain (NeuroTrace). (Scale bars: 500 µm.) HC, hippocampus (for orientation); ZI, zona incerta.

Fig. 3.

Topology of cortico–subcortical projections. Two experiments with dual-color labeling (GFP, green, and mOrange, magenta) in the rowish (A–D) and arcish (E–H) direction resulted in different relative subcortical positions of bouton clouds. B–D and F–H are in horizontal orientation. (A) Rowish dual-color labeling in the BC approximately along the C row (green and magenta areas). (B–D) Reconstruction of bouton clouds (see the 3D illustration of subcortical bouton clouds in Fig. 2G) in the dorsal thalamus (PO; GFP-labeled boutons are located dorsally to mOrange-labeled boutons) (B), ZI (C), and APT (D). (E) As in A, but for an experiment with arcish dual-color labeling approximately along arcs 2–3 of the BC. (F–H) As B–D resulting from the dual-color labeling shown in E. (Scale bar: 500 µm.)

Fig. S3.

Comparison of small L6 and large L5B boutons in thalamus. Micrographs show boutons of different sizes in the VPM and POm_lateral. (A) Horizontal overview of a maximal intensity projection over eight slices showing the POm_lateral, VPM, and Rt. (B) A single z-section image of the POm_lateral from the yellow boxed region in A, showing many large boutons of L5B origin. The POm_lateral contains both large L5B and small L6 boutons. (C) A single z-section image of the VPM core area from the red boxed region in A showing a meshwork of small boutons of L6 origin. (D) A single z-section image of the posterior VPM from the blue boxed region in A showing some large boutons of L5B origin amid a meshwork of small L6 boutons. The VPM contains mostly small and dense L6 boutons (C) but also contains some large L5B boutons at the posterior end (D); in the Rt there are only small L6 boutons.

Fig. 4.

Quantification of projection parameters. Parameter estimates of L5B subcortical projections for each target nucleus. (A) Probability histogram of bouton diameters (only GFP-labeled) in the different nuclei as indicated by different colors. (B–F) The value for each experiment (both GFP and mOrange) is represented by black circles, and the median of all experiments is represented by gray lines. Black lines at the top of the panel indicate which nuclei are significantly different from each other (Wilcoxon signed rank test, P < 0.05). (B) The number of boutons in different nuclei, normalized by the number of labeled L5B neurons (median = 91 L5B neurons; Q₁: 50, Q₃: 141). (C) Bouton cloud volumes in different nuclei normalized by the number of labeled L5B neurons (median = 91 L5B neurons; Q₁: 50, Q₃: 141). (D) Bouton cloud densities (the ratio of bouton number to cloud volume). (E) Soma densities from NeuroTrace staining measured in a subsample of sections in two experiments. (F) The number of boutons per target cell (ratio of bouton densities to neuron densities).

Fig. S4.

Bouton and cloud quantifications. Bouton numbers (A) and cloud volumes (B) when the deposit size is normalized to an average column. The value for each experiment (both GFP and mOrange) is represented by black circles, and the median of all experiments is represented by gray lines. Black lines above the panels indicate which nuclei are significantly different from each other (Wilcoxon signed rank test, P < 0.05).

Fig. 5.

Overlap between GFP- and mOrange-labeled bouton clouds calculated with GLMs. (A) A low-overlap example from the POm_lateral showing a randomly selected subset of 1,000 boutons for clarity. The transparent gray surface illustrates the GLM-modeled surface that gives the best separation of bouton clouds (green and magenta dots). Overlap boutons are plotted in a dark shade of green and magenta, respectively. The percentage of boutons in the overlap volume is proportional to cloud overlap. (B) As in A but showing a high-overlap example from the PoT. (C) Estimated overlaps for all target nuclei. Each circle represents one experiment; gray bars show the median. Lines above the panel indicate which target nuclei are significantly different from each other (Wilcoxon signed rank test, P < 0.05).

Fig. S5.

A 1D illustration of model-based calculation of overlap. (A) Scatter plot of examples of boutons (green and magenta circles) in color over coordinate space. The black line illustrates the color gradient as predicted by the binomial model. (B) Boutons are illustrated as in A. Arrow lengths indicate the absolute difference between bouton color and the predicted color value. Black arrows indicate a difference <0.25; gray arrows indicate a difference >0.25. Boutons with absolute differences larger than 0.25 are defined as overlap boutons and are indicated by asterisks. In this example 8 of 26 boutons are overlap boutons, resulting in 30.8% overlap.

Fig. 6.

PO whisker projection maps. Schematic drawings of L5B-derived whisker maps in the PO in horizontal (Left) and coronal (Right) views showing the outside–in organization of whisker rows and the dorsoventral orientation of whisker arcs. Rows and arcs are shown in shades of gray.

Fig. S6.

Central gap in the PO. A horizontal view of subcortical boutons from dual deposits (Inset). Magenta-colored row E projections are located toward the middle of the PO but still leave a gap in the center. A, anterior; L, lateral.