Mapping thalamic-anterior cingulate monosynaptic inputs in adult mice

Abstract

The anterior cingulate cortex (ACC) is located in the frontal part of the cingulate cortex, and plays important roles in pain perception and emotion. The thalamocortical pathway is the major sensory input to the ACC. Previous studies have show that several different thalamic nuclei receive projection fibers from spinothalamic tract, that in turn send efferents to the ACC by using neural tracers and optical imaging methods. Most of these studies were performed in monkeys, cats, and rats, few studies were reported systematically in adult mice. Adult mice, especially genetically modified mice, have provided molecular and synaptic mechanisms for cortical plasticity and modulation in the ACC. In the present study, we utilized rabies virus-based retrograde tracing system to map thalamic-anterior cingulate monosynaptic inputs in adult mice. We also combined with a new high-throughput VISoR imaging technique to generate a three-dimensional whole-brain reconstruction, especially the thalamus. We found that cortical neurons in the ACC received direct projections from different sub-nuclei in the thalamus, including the anterior, ventral, medial, lateral, midline, and intralaminar thalamic nuclei. These findings provide key anatomic evidences for the connection between the thalamus and ACC.

Keywords: Thalamus; anterior cingulate cortex; mapping; retrograde projection; volumetric imaging with synchronized on-the-fly-scan and readout.

Figure 1.

Schematic diagrams of afferents to the ACC neurons using rabies virus-based trans-monosynaptic tracing strategy. (a) The AAV helper virus with TVA receptor, RVG and EGFP; and the glycoprotein (G)-deleted rabies virus with EnVA and DsRed. (b) Experimental timeline of two viral injections. (c–d) Schematic diagrams of sagittal (c) and coronal (d) sections for showing the combination of two viruses micro-injected into the ACC and brain-wide labeling of monosynaptic inputs, especially the thalamus. The bottom figure in (c) indicates the enlarged schema of right thalamus. The green dots represent the neurons labeled by helper virus; the red dots represent the neurons sending presynaptic inputs to the ACC; the yellow dots represent the starter neurons, which are infected by both the helper virus and the rabies virus, and receive presynaptic inputs.

Figure 2.

VISoR imaging for rabies virus-injected into the unilateral ACC in adult mouse. (a) 3D-reconstructed whole brain in adult mouse. (b–d) The horizontal (b), sagittal (c) and coronal (d) views for one 3D-reconstructed whole brain (red: DsRed⁺; green: EGFP⁺). The blue rectangle in (c) represents the thalamus. Scale bar: 1 mm. (e–g) The horizontal (e), sagittal (f) and coronal (g) views for the thalamus. Scale bar: 200 μm. a: anterior; d: dorsal; l: left; r: right.

Figure 3.

Different sections of the thalamus on one 3D-reconstructed whole brain. (a–c) The horizontal, coronal, and sagittal sections are shown according to the intersection of two white lines. The thalamic nuclei at the intersection of two white lines are the AM (a), MD (b), and VM (c), respectively. Scale bar: 1 mm.

Figure 4.

The in-situ viral expression in the injection site of ACC. (a) Four different coronal slices (bregma: AP +1.21 mm, +0.85 mm, +0.25 mm, −0.23 mm) show that the helper virus and the rabies virus are both micro-injected into the right ACC (blue rectangle). The white dashed lines represent the anatomic location of the ACC. The red rectangle (DsRed⁺ neurons) represents presynaptic inputs in the left ACC that send direct projections to the starter neurons of the right ACC. Scale bar: 1 mm. (b) The enlarged views of the blue-boxed regions in (a). The white arrows indicate infected neurons. Scale bar: 50 μm. (c) The enlarged views of the red-boxed regions in (a). (d) Cell densities analysis of EGFP⁺ neurons and starter neurons at different coronal sections of the ACC (**p < 0.01 for AP +1.21 mm versus +0.85 mm; ^##p < 0.01 for AP +0.25 mm versus +0.85 mm; unpaired t-test; 3 slices for every mouse; n = 12 slices/4 mice). (e) Comparisons of the ratio of starter to EGFP⁺ neurons at different coronal sections of the ACC (*p < 0.05 for AP +1.21 mm versus +0.85 mm; ^##p < 0.01 for AP +0.25 mm versus +0.85 mm; unpaired t-test; n = 12 slices/4 mice). (f) Cell densities analysis of presynaptic inputs at different coronal sections of ipsi- and contra-ACC. n = 12 slices/4 mice. The approximate AP level from bregma is indicated. Error bars indicated SEM.

Figure 5.

Thalamic-anterior cingulate presynaptic inputs in one typical sample. (a–d) Four representative slices are chosen from different coronal slices (bregma: AP −0.83 mm, −1.07 mm, −1.55 mm, −2.03 mm) that contain different thalamic nuclei. These thalamic nuclei contain DsRed⁺ neurons and their cell densities are different. The enlarged views of the blue rectangle are shown in the right side. Left: scale bar: 1 mm; Right: scale bar: 200 μm. The white dashed lines are drawn according to the Mouse Brain in Stereotaxic Coordinates, 4nd edition, indicating different thalamic nuclei. The approximate AP level from bregma is indicated.

Figure 6.

Summary of thalamic-anterior cingulate presynaptic inputs from four adult mice. (a–d) Left: Four-slice overlay figures from four mice at different coronal sections (bregma: AP −0.83 mm, −1.07 mm, −1.55 mm, −2.03 mm). The number of DsRed⁺ neurons from different thalamic nuclei of every slice are counted and labeled in schematic graphs according to different coronal sections. Right: The histograms of the number of labeled neurons from four mice at different coronal sections (bregma: AP −0.83 mm, −1.07 mm, −1.55 mm, −2.03 mm). The approximate AP level from bregma is indicated. The red, blue, green, and brown dots (left) or columns (right) represent four sequence numbers from mouse 1 to mouse 4 separately.

Figure 7.

Statistical analysis of thalamic-anterior cingulate presynaptic inputs in adult mice. (a) Averaged number of input neurons in different thalamic nuclei. (b) Cell densities of input neurons in different thalamic nuclei. (c) Ratio of input neurons in different thalamic nuclei to starter neurons in the ACC. n = 4 mice. Error bars indicated SEM.