Localization of Inositol 1, 4, 5–Trisphosphate Receptors in Mouse Retina Ganglion Cells

Abstract

Abstract: : Purpose: Inositol–1, 4, 5–triphosphate receptors (IP₃Rs) are intracellular Ca²⁺ channels known to be involved in several intracellular signaling pathways. IP₃R mediated changes in cytosolic Ca²⁺ concentrations control neuronal functions ranging from synaptic transmission to differentiation and apoptosis. Similarly, cytosolic Ca2+ transients determine retinal ganglion cell physiology and pathophysiology. Determining the distribution of biophysically distinct IP₃Rs in retina ganglion cells provides necessary information on the molecular substrates of IP₃R mediated Ca²⁺ signaling in these interneurons. Methods:Primary cultures were prepared following a modified protocol (Luo et al., 2001) using acute isolation of retinal ganglion cells from adult mouse followed by enzymatic and mechanical dissociation. Cell cultures were maintained for 14 days. Cryo–sections of adult mouse retina were prepared as described previously (Koulen and Brandstätter, 2002). Immunocytochemical labeling of IP₃Rs in cultured retinal ganglion cells and of retinal ganglion cells in vertical sections of the retina was carried out using isoform specific antibodies and was detected with fluorescence microscopy. Retinal ganglion cells were identified by the use of morphological criteria and retinal ganglion cell specific immunocytochemical markers, neurofilament 68 kDa and Thy1.1. Results: Retinal ganglion cell morphology and immunoreactivity to neurofilament 68 kDa and Thy1.1 were identified in both the retinal ganglion cell primary cultures and tissue cryosections. Retinal ganglion cells showed differential distribution of IP₃R isoforms 1, 2 and 3. The IP₃R types 1 and 3 were detected intracellularly throughout the cell, whereas type 2 was localized mainly in the soma. Conclusions:Expression of all three IP₃Rs by retinal ganglion cells indicates that all IP₃R types potentially play a role in Ca²⁺ homeostasis and Ca²⁺ signaling in these cells. Differential localization of IP₃ receptor subtypes may be an important molecular mechanism by which retinal ganglion cells organize their cytosolic Ca²⁺ signals. These data will help to delineate the potential involvement of IP₃R channels in signal processing and neuroprotection of retinal ganglion cells. References: Luo X, Heidinger V, Picaud S, Lambrou G, Dreyfus H, Sahel J, Hicks D. Invest Ophthalmol Vis Sci. 2001;42:1096–1106. Koulen P, Brandstatter JH. Invest Ophthalmol Vis Sci. 2002;43:1933–1940. Supported by NIH grant EY14227 (PK).

Keywords: retina: proximal (bipolar, amacrine, and ganglion cells) • retina • retina: neurochemistry