Glial and neuronal expression of the Inward Rectifying Potassium Channel Kir7.1 in the adult mouse brain

Abstract

Inward Rectifying Potassium channels (Kir) are a large family of ion channels that play key roles in ion homeostasis and neuronal excitability. The most recently described Kir subtype is Kir7.1, which is known as a K⁺ transporting subtype. Earlier studies localised Kir7.1 to subpopulations of neurones in the brain. However, the pattern of Kir7.1 expression across the brain has not previously been examined. Here, we have determined neuronal and glial expression of Kir7.1 in the adult mouse brain, using immunohistochemistry and transgenic mouse lines expressing reporters specific for astrocytes [glial fibrillary acidic protein-enhanced green fluorescent protein (GFAP-EGFP], myelinating oligodendrocytes (PLP-DsRed), oligodendrocyte progenitor cells (OPC, Pdgfra-creER^T2 /Rosa26-YFP double-transgenic mice) and all oligodendrocyte lineage cells (SOX10-EGFP). The results demonstrate significant neuronal Kir7.1 immunostaining in the cortex, hippocampus, cerebellum and pons, as well as the striatum and hypothalamus. In addition, astrocytes are shown to be immunopositive for Kir7.1 throughout grey and white matter, with dense immunostaining on cell somata, primary processes and perivascular end-feet. Immunostaining for Kir7.1 was observed in oligodendrocytes, myelin and OPCs throughout the brain, although immunostaining was heterogeneous. Neuronal and glial expression of Kir7.1 is confirmed using neurone-glial cortical cultures and optic nerve glial cultures. Notably, Kir7.1 have been shown to regulate the excitability of thalamic neurones and our results indicate this may be a widespread function of Kir7.1 in neurones throughout the brain. Moreover, based on the function of Kir7.1 in multiple transporting epithelia, Kir7.1 are likely to play an equivalent role in the primary glial function of K⁺ homeostasis. Our results indicate Kir7.1 are far more pervasive in the brain than previously recognised and have potential importance in regulating neuronal and glial function.

Keywords: Kir7.1; astrocytes; immunohistochemistry; neurones; oligodendrocytes.

Figure 1

Validation of Kir7.1 immunostaining. (A‐C) To serve as positive controls, immunostaining for Kir7.1 is demonstrated in Purkinje neurones (A; adult PLP‐DsRed mouse cerebellum, in which white matter tracts appear magenta) and choroid plexus epithelium (B; counterstained with Hoechst Blue to visualise the cell nuclei). Immunostaining was absent following pre‐incubation in blocking peptide (A, Inset), and in adult mouse skeletal muscle, which does not express Kir7.1 (C; counterstained for collagen, which appears magenta). (D) Western blot analysis of protein lysates from mouse cerebellum (CBL) and cortex (CTX) confirmed robust Kir7.1 protein expression with a predicted band at 54 kDa. Positive bands were absent in the presence of the competitive peptide. In some samples, very dim bands were observed at approximately 15 kDa, which corresponds to protein proteolysed during sampling. Scale bars: (A,C) 50 μm; (B) 10 μm; (A, Inset) 100 μm.

Figure 2

Kir7.1 immunostaining in the adult mouse cerebellum. (A) Immunostaining for Kir7.1 (green) in cerebellum from PLP‐DsRed mouse for myelin staining (red), where colocalisation appears yellow in the overlay (Ai); individual channels are illustrated for Kri7.1 (Aii) and PLP (Aiii). Higher magnification (Aiv) and isosurface imaging (Av) illustrate the apposition of Kir7.1 and PLP. (B) High magnification of the molecular layer (MCL) showing Kir7.1 (green) and GFAP (magenta), illustrating immunostaining of Bergmann glia cell bodies, where colocalisation appears white in the overlay (Bi); individual channels are illustrated for Kir7.1 (Bii) and GFAP (Biii). (Ci) Kir7.1‐immunopositive Purkinje cell somata, together with axons (Ci, arrows) and dendritic trees (Cii). Scale bars: 50 μm in all panels.

Figure 3

Kir7.1 immunostaining in the adult mouse forebrain. (A) Overview of the pattern of Kir7.1 expression in the adult mouse forebrain. Kir7.1 immunostaining was absent in negative controls pre‐incubated with peptide (A, Inset). (B,C) Double immunofluorescence labelling for Kir7.1 (green) and Tuj1 (red) in the cortex (B) and hippocampus (C). (B) Layer 2/3 cortical neurones expressing Kir7.1 on their cell bodies (asterisks) and axons (arrows); co‐expression appears yellow in the overlay (Bi) and Kir7.1 immunopositivity can be seen in Tuj1‐negative cells, which are likely to be astrocytes (arrowheads); individual channels are illustrated for Kir7.1 (Bii) and Tuj1 (Biii), together with isosurface images showing the close apposition of Kir7.1 and Tuj1 voxels (Biv). (C) Hippocampal pyramidal cells express Kir7.1 on cell somata and axons (Ci); individual channels are illustrated for Kir7.1 (Cii) and Tuj1 (Ciii), together with higher magnification of the overlay image (Cv) and isosurface image (Civ), illustrating the close apposition of Kir7.1 and Tuj1 voxels. Scale bars: (A) 300 μm; (A, Inset) 100 μm; (Bi‐iii and Ci‐iii)  25 μm; (Biv) 1 square unit = 35.42 μm; (Civ‐v) 1 square unit = 16.64 μm.

Figure 4

Astrocytic expression of Kir7.1. (A) Kir7.1 immunostaining (red) in cortical astrocytes (green) from adult GFAP‐EGFP mice, indicated by arrows in the overlay (Ai) and individual channels for Kir7.1 (Aii) and GFAP‐EGFP (Aiii). (B) High‐magnification 3D rendered isosurface images of a cortical Layer 1 astrocyte (Bi) and a perivascular astrocyte(Bii), showing the close apposition of Kir7.1 and EGFP voxels. (C) Double immunofluorescence labelling for Kir7.1 (red) and GFAP (green), illustrating Kir7.1‐immunopositive astrocytes in the hippocampus, where co‐localisation appears yellow in the overlay (Ci). Kir7.1 immunostaining is strongest in molecular (mo) and polymorph (po) layers, while the granule cell layer (sg) is less populated by astrocytes; individual channels are illustrated for Kir7.1 (Cii) and GFAP (Ciii), together with high‐magnification overlay (Cv) and isosurface image (Civ). Scale bars: (Ai‐iii) 30 μm; (Bi) 1 square unit = 5.47 μm; (Bii) 1 square unit = 25 μm; (Ci‐iii) 50 μm; (Civ‐v) 1 square unit = 6.06 μm.

Figure 5

Kir7.1 expression in the corpus callosum (CC). (A) Kir7.1 immunostaining (red) in astrocytes (green) from adult GFAP‐EGFP mice, indicated by arrows in the overlay (Ai) and individual channels for Kir7.1 (Aii) and EGFP (Aiii); Kir7.1 immunostaining can also be seen in GFAP‐eGFP‐negative cells, which most likely are oligodendroglial cells (arrowheads) and striatal neurones (asterisks). High magnification of the overlay (Aiv) and isosurface image (Av) illustrates the close apposition of Kir7.1 and EGFP voxels (Av). (B) Sagittal section from an adult Sox10‐eGFP reporter mouse, in which some oligodendrocytes (green) are seen to be immunopositive for Kir7.1 (red), indicated by arrowheads (Bi), together with Sox10‐eGFP‐negative cells, which most likely are astrocytes (Bi, arrows), and the strongest immunopositivity in striatal neurones (Bi, asterisks); individual channels are illustrated for Kir7.1 (Bii) and SOX10 (Biii). High magnification of the overlay (Biv) and isosurface image (Bv) shows the close apposition of Kir7.1 and EGFP voxels. (C) Colocalisation of Kir7.1 immunostaining (red) in PdgfRa‐YFP positive OPCs (green, some indicated by arrows) in the corpus callosum of 15‐day‐old mice; colocalisation appears yellow in the overlay (Ci) and individual channels are illustrated for Kir7.1 (Cii) and PdgfRa‐YFP (Ciii). High magnification of the overlay (Civ) and isosurface image (Cv), showing the close apposition of Kir7.1 and PdgfRa‐YFP voxels. Scale bars: (Ai‐iii, Bi‐iii, Ci‐iii) 25 μm; (Aiv‐v) 1 square unit = 16.43 μm; (Biv‐v) 1 square unit = 37.36 μm; (Civ‐v) 1 square unit = 11.17 μm.

Figure 6

Kir7.1 expression in the mouse hindbrain. (A) Low‐magnification confocal image of sagittal section from adult PLP‐DsRed (red) reporter mouse immunolabelled for Kir7.1 (green). Kir7.1 is widely expressed in the pons, with intense expression in the choroid plexus of the third ventricle (CP), whereas the corticospinal tract appears immunonegative for Kir7.1 (arrows). (B) Higher magnification images from the area indicated by the white square in (A), immunostained for MBP (red) and Kir7.1 (green), showing neuronal expression of Kir7.1 (asterisks) and some co‐localisation of Kir7.1 and MBP(appears yellow in Bi); individual channels are illustrated for MBP (Bii) and Kir7.1 (Biii), together with the colocalisation channel (Biv) and isosurfacing image (Bv), which does not reveal very close apposition of Kir7.1 and MBP voxels (Bv), suggesting that Kir7.1 may be expressed by axons and not by myelin. (Bi‐inset) Double immunolabelling for Kir7.1 (green) and GFAP (magenta) did not detect expression of Kir7.1 in astrocytes of the pons. Scale bars: (A) 300 μm; (Bi‐v)  50 μm; (Bi, Inset) 25 μm.

Figure 7

Neurons and glia express Kir7.1 in vitro. (A, B) Cells were isolated from P1‐2 mouse cortex and analysed after 14 days in vitro (DIV) by double immunofluorescence labelling for Kir7.1 (green) and the astrocyte marker GFAP (red, A) or neuronal marker Tuj1 (red, B, asterisks indicate Kir7.1+Tuj1‐ cells that are most likely astrocytes (C‐F) Optic nerve glial explant cultures from P7‐12 mice were analysed at 10DIV by double immunofluorescence labelling for Kir7.1 (green), with the plasmalemmal markers Na⁺/K⁺‐ATPase (C) and PSD95 (D), or the glial Kir channels Kir4.1 (E) and Kir5.1 (F), In all cases, overlays are illustrated (Ai, Bi, Ci, Di, Ei, Fi, co‐expression appears yellow), together with individual channels (Aii‐iii, Bii‐iii, Ci‐iii, Di‐iii, Ei‐iii, Fi‐iii). Scale Bars: A‐B = 50μm; C‐F = 20µm. [Colour figure can be viewed at wileyonlinelibrary.com]