Neuronal co–expression of Kir4.1 and aquaporin–4: switch from horizontal cells to Müller cells during retinal development.

Abstract

Abstract: : Purpose: The inwardly rectifying potassium channel Kir4.1 and the water channel aquaporin–4 (AQP4) perform extracellular K⁺ siphoning and associated water redistribution in the retina. Recently, we reported the expression of AQP4 in retinal horizontal cells from E18.5 until P15, at which time AQP4 is detectable in Müller cells as well as in horizontal cells. We now describe the ontogeny of Kir4.1 and AQP4 immunoexpression in the mouse retina, both of which co–localized in horizontal cells from P0 to P12. Methods: Triple–immunofluorescence staining was performed on retinal whole mounts and cryosections, followed by confocal microscopy. Labeling for Kir4.1, AQP4 and selective neuronal and glial markers (calbindin; cellular retinal–binding protein, glial fibrillary associated protein; neurofilament; recoverin) were revealed with secondary antibodies coupled to Alexa Fluor. Results: In P0 retinas, Kir4.1 and AQP4 selectively co–localized in horizontal cell somata and processes. P9 retinas showed co–expression of Kir4.1 and AQP4 throughout whole layers of retina: in Müller endfeet, major processes in both inner and outer plexiform layers, outer limiting membrane, as well as in the inner nuclear layer. Strong immunoreactivity of Kir4.1 deprived of AQP4 was noticed in some calbindin–positive amacrine and ganglion cells. No staining of perivascular astrocytes was visualized. At P12, horizontal cells maintain their expression of Kir4.1 and AQP4. At P15, AQP4 labeling in horizontal cells persisted, while Kir4.1 labeling was undetectable. Amacrine cells also did not show Kir4.1 labeling at this age. Ganglion cells strongly labeled for Kir4.1, but not for AQP4. Müller cells showed a staining pattern similar to P9. Kir4.1 and AQP4 prominently co–localized on perivascular astrocytes in the vitreal surface, but not in the OPL blood vessels, where only AQP4 was present. Because of the strong labeling of Muller cell processes and of presumably ganglion cells in the vitreal surface it is not clear whether astrocytes contain Kir4.1 or not. Conclusions: This report of co–expression of Kir4.1 and AQP4 in horizontal cells during retinal development suggests the involvement of these neurons in water and extracellular K⁺ homeostasis.

Keywords: retinal development • ion channels • horizontal cells