Multiple Posterior Insula Projections to the Brainstem Descending Pain Modulatory System

Abstract

The insular cortex is an important hub for sensory and emotional integration. It is one of the areas consistently found activated during pain. While the insular's connections to the limbic system might play a role in the aversive and emotional component of pain, its connections to the descending pain system might be involved in pain intensity coding. Here, we used anterograde tracing with viral expression of mCherry fluorescent protein, to examine the connectivity of insular axons to different brainstem nuclei involved in the descending modulation of pain in detail. We found extensive connections to the main areas of descending pain control, namely, the periaqueductal gray (PAG) and the raphe magnus (RMg). In addition, we also identified an extensive insular connection to the parabrachial nucleus (PBN). Although not as extensive, we found a consistent axonal input from the insula to different noradrenergic nuclei, the locus coeruleus (LC), the subcoereuleus (SubCD) and the A5 nucleus. These connections emphasize a prominent relation of the insula with the descending pain modulatory system, which reveals an important role of the insula in pain processing through descending pathways.

Keywords: A5; LC; anterograde tracing; mCherry; noradrenergic nuclei; nucleus raphe magnus; parabrachial nucleus; periaqueductal gray; posterior insular cortex; spinal cord dorsal horn.

Figure 1

Virus-mediated mCherry expression in the right posterior insular cortex. (A) Coronal schematic diagrams showing the extend of mCherry expression at different anterior-posterior levels. The colored shading is the superimposed average of expression from all mice (n = 4) used in this study. The bulk of the expression was between levels of −0.3 mm and −1.0 mm from bregma. (B) A representative immunostaining of a coronal section for mCherry (red), showing expression within the insular cortex. Green autofluorescence is used for anatomical feature reference (scale bar: 1 mm). (C) Higher magnification image of layer 5 posterior insula pyramidal neurons expressing mCherry (red). Nuclear DAPI staining is shown in blue. Immunofluorescent cell somata, dendrites and some axons are visible (scale bar: 30 μm).

Figure 2

Posterior insular projections to the PAG. (A) Axonal projections labeled with immunofluorescence for mCherry (magenta) in the vlPAG ipsilateral to the injected insula. Nuclei are stained with DAPI (grey). (B) A higher magnification of the section in (A). Insula emanating axons with en-passant varicosities are visible. (C) Posterior insula emanating axons (magenta) in the contralateral vlPAG. (D) A higher magnification of the section in (C). (E) Immunofluorescence for mCherry (red), and TH (green) shows posterior insular projections ventral of the Aqueduct (Aq) within the dopamine neuron containing dorsal raphe nucleus. DAPI staining is shown in blue. (F) A higher magnification of the section in (E). (G) Insula axonal projections in the ipsilateral dlPAG. (H) A higher magnification of the section in (G). Scale bars: 50 μm (A,C,E,G), 10 μm (B,D,F,H). Arrows denote varicosities.

Figure 3

Retrograde labelling of Insula–PAG projections. (A) CTB-labeled cell bodies in the posterior insula (red) lie mainly in layers V and VI in both the agranular (aIC) and granular/dysgranular (g/dIC) subareas of the posterior insular cortex (blue: nuclear DAPI staining; cl: claustrum; scale bar 100 μm). Upper inset: The CTB fluorescence (red) at the injection site is shown (green: 488 nm autofluorescence for anatomical reference; scale bar: 300 μm), Lower inset: of CTB labelled somata scale bar: 20 μm. (B) CTB (red) injection site from a different animal (dashed line delineates the PAG, scale bar: 500 μm). (C) CTB-labeled cell bodies in the posterior insula (red) from the example in (B) (scale bar: 250 μm).

Figure 4

Posterior insular projections to the RMg. (A) Axonal projections expressing mCherry (magenta) in the ipsilateral RMg. Blue dashed lines delineate the RMg. (B) Higher magnification of the image in (A), showing axons and varicosities. (C) Axonal projections expressing mCherry (magenta) in the ipsilateral RMg. Blue dashed line denotes the midline, dividing the ipsilateral and the contralateral site. Axons crossing from the ipsilateral to the contralateral site are visible. (D) Higher magnification of the image in (C). Nuclei are stained with DAPI (grey). Scale bars: 50 μm (A,C), 10 μm (B,D). Arrows denote varicosities.

Figure 5

Posterior insular projections to noradrenergic brainstem nuclei. (A) Axonal projections from the insula labeled with immunofluorescence for mCherry (red) to the ipsilateral LC. Noradrenergic cells are labelled with TH (green) and nuclei with DAPI (blue; scale bar: 50 μm). The inset shows higher magnification of insula-emanating axons with varicosity-like structures (scale bar: 5 μm). (B) Orthogonal view of a high resolution (100×) single imaging plane showing a noradrenergic neuron (green) labelled with anti-TH antibodies and the respective x–z, y–z planes reconstructed from a 3D stack. Varicosities (denoted by the crossing of the yellow lines) from insular projections (red) are shown in close contact around the TH-positive cell (DAPI stain is in blue; scale bar: 5 μm). (C) Axonal projections from the insula labeled with immunofluorescence for mCherry (red) to the contralateral LC. Noradrenergic cells are labelled with TH (green) and nuclei with DAPI (blue; scale bar: 50 μm). The inset shows higher magnification of insula-emanating axons with varicosity-like structures (scale bar: 5 μm). (D) Axonal projections from the insula labeled with immunofluorescence for mCherry (red) to the ipsilateral SubCD. Noradrenergic cells are labelled with TH (green) and nuclei with DAPI (blue; scale bar: 50 μm). The inset shows higher magnification of insula-emanating axons with varicosity-like structure (scale bar: 5 μm). (E) Axonal projections from the insula labeled with immunofluorescence for mCherry (red) to the ipsilateral A5 nucleus. Noradrenergic cells are labelled with TH (green) and nuclei with DAPI (blue; scale bar: 50 μm). The inset shows higher magnification of insula-emanating axons with varicosity-like structures (scale bar: 5 μm). (F) Axonal projections from the insula labeled with immunofluorescence for mCherry (red) to the ipsilateral A7 nucleus. Noradrenergic cells are labelled with TH (green) (scale bar: 50 μm). The inset shows higher magnification of insula-emanating axons with varicosity-like structures (scale bar: 5 μm). Arrows denote varicosities.

Figure 6

Posterior insular projections to the PBN. (A) Insular axonal projections to the ipsilateral lPBN immunolabeled for mCherry (magenta). (B) Higher magnification of (A). Insula en-passant axonal varicosities are visible. (C) Insular axonal projections to the contralateral lPBN immunolabeled for mCherry (magenta). (D) Higher magnification of (A). Insula en-passant axonal varicosities are visible. scp: Superior Cerebellar Peduncle, grey: DAPI staining, scale bar: 50 μm (A,C), 10 μm (B,D). Arrows denote varicosities.

Figure 7

Lack of axonal projection from the insula to the spinal dorsal horn projections. (A) Lumbar section of the spinal cord stained with anti-mCherry (red) and with DAPI (blue); green autofluorescence was used for anatomical feature recognition (scale bar: 250 μm). (B) Thoracic level of the spinal cord stained with anti-mCherry (red) and with DAPI (blue); green autofluorescence was used for anatomical feature recognition (scale bar: 250 μm). (C) Higher resolution of the contralateral superficial lumbar dorsal horn. In grey, the nuclear staining is shown (DAPI), while in magenta, the anti-mCherry staining for insular axons is imaged. No evidence of axonal shafts or varicosities was identified. Dashed lines denote the borders between different dorsal horn laminas (I, II, III; scale bar: 50 μm).

Figure 8

Comparison of axonal length densities for insular projections to different regions of the brainstem. Blue rhombi: means ± SEM. Grey circles: individual values for the different mice. PAG: ventrolateral periaqueductal grey, RMg: nucleus raphe magnus, LC: locus coeruleus, SubCD: dorsal subcoeruleus nucleus, A5: noradrenergic nucleus A5, PBN: lateral parabrachial nucleus. Axonal densities between the brainstem nuclei differed statistically (rmANOVA, F(5, 15) = 7.71, p = 0.019).