immunohistochemical-calcium-imaging-methods-wholemount-rat

Immunohistochemical and Calcium Imaging Methods in Wholemount Rat Retina

In this paper we describe the tools, reagents, and the practical steps that are needed for: 1) successful preparation of wholemount retinas for ...

University of California, Los Angeles

Aberrant calcium regulation has been implicated as a causative factor in the degeneration of retinal ganglion cells (RGCs) in numerous injury models of optic neuropathy. Since calcium has dual roles in maintaining homeostasis and triggering apoptotic pathways in healthy and injured cells, respectively, investigation of voltage-gated Ca channel (VGCC) regulation as a potential strategy to reduce the loss of RGCs is warranted. The accessibility and structure of the retina provide advantages for the investigation of the mechanisms of calcium signalling in both the somata of ganglion cells as well as their unmyelinated axons. The goal of the present study was to determine the distribution of VGCC subtypes in the cell bodies and axons of ganglion cells in the normal retina and to define their contribution to calcium signals in these cellular compartments. We report L-type Ca channel α1C and α1D subunit immunoreactivity in rat RGC somata and axons. The N-type Ca channel α1B subunit was in RGC somata and axons, while the P/Q-type Ca channel α1A subunit was only in the RGC somata. We patch clamped isolated ganglion cells and biophysically identified T-type Ca channels. Calcium imaging studies of RGCs in wholemounted retinas showed that selective Ca channel antagonists reduced depolarization-evoked calcium signals mediated by L-, N-, P/Q- and T-type Ca channels in the cell bodies but only by L-type Ca channels in the axons. This differential contribution of VGCC subtypes to calcium signals in RGC somata and their axons may provide insight into the development of target-specific strategies to spare the loss of RGCs and their axons following injury.

Differential calcium signaling mediated by voltage-gated calcium channels in rat retinal ganglion cells and their unmyelinated axons.

We report that the most common retinal ganglion cell type that remains after optic nerve transection is the M1 melanopsin ganglion cell. M1 ganglion cells are members of the intrinsically photosensitive retinal ganglion cell population that mediates non-image-forming vision, comprising ∼2.5% of all ganglion cells in the rat retina. In the present study, M1 ganglion cells comprised 1.7±1%, 28±14%, 55±13% and 82±8% of the surviving ganglion cells 7, 14, 21 and 60 days after optic nerve transection, respectively. Average M1 ganglion cell somal diameter and overall morphological appearance remained unchanged in non-injured and injured retinas, suggesting a lack of injury-induced degeneration. Average M1 dendritic field size increased at 7 and 60 days following optic nerve transection, while average dendritic field size remained similar in non-injured retinas and in retinas at 14 and 21 days after optic nerve transection. These findings demonstrate that M1 ganglion cells are more resistant to injury than other ganglion cell types following optic nerve injury, and provide an opportunity to develop pharmacological or genetic therapeutic approaches to mitigate ganglion cell death and save vision following optic nerve injury.

Melanopsin ganglion cells are the most resistant retinal ganglion cell type to axonal injury in the rat retina.

The α2δ auxiliary subunits of voltage-gated Ca2+ channels (VGCCs) are important modulators of VGCC function. Gabapentin interacts with α2δ1 and α2δ2 subunits and is reported to reduce Ca2+ channel current amplitude (ICa). This study aimed to determine the effects of gabapentin on VGCCs in retinal ganglion cells (RGCs). Whole cell patch clamp was used to record ICa in isolated RGCs, and calcium imaging was used to measure Ca2+ transients from RGCs in situ. Immunohistochemistry was used to detect the presence of α2δ1-containing VGCCs in isolated RGCs in the absence and presence of gabapentin pretreatment. Acute administration of gabapentin reduced ICa and Ca2+ transients compared to control conditions. In isolated RGCs, pretreatment with gabapentin (4-18 h) reduced ICa, and cell surface α2δ1 staining was reduced compared to nonpretreated cells. Acute administration of gabapentin to isolated RGCs that had been pretreated further reduced ICa. These results show that gabapentin has both short-term and long-term mechanisms to reduce ICa in isolated RGCs. Some Ca2+ channel blockers have been shown to protect RGCs in retinal trauma suggesting that modulation of VGCCs by gabapentin may prevent the deleterious effects of elevated Ca2+ levels in RGCs in trauma and disease.

Article	Year
Differential calcium signaling mediated by voltage-gated calcium channels in rat retinal ganglion cells and their unmyelinated axons. PloS one\| PubMed ID: 24416240	2014
Melanopsin ganglion cells are the most resistant retinal ganglion cell type to axonal injury in the rat retina. PloS one\| PubMed ID: 24671191	2014
Modulation of voltage-gated Ca2+ channels in rat retinal ganglion cells by gabapentin. Visual neuroscience\| PubMed ID: 24801623	2014

Allison Sargoy

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