Amacrine cells coupled to ganglion cells via gap junctions are highly vulnerable in glaucomatous mouse retinas

Abstract

We determined whether the structural and functional integrity of amacrine cells (ACs), the largest cohort of neurons in the mammalian retina, are affected in glaucoma. Intraocular injection of microbeads was made in mouse eyes to elevate intraocular pressure as a model of experimental glaucoma. Specific immunocytochemical markers were used to identify AC and displaced (d)ACs subpopulations in both the inner nuclear and ganglion cell layers, respectively, and to distinguish them from retinal ganglion cells (RGCs). Calretinin- and γ-aminobutyric acid (GABA)-immunoreactive (IR) cells were highly vulnerable to glaucomatous damage, whereas choline acetyltransferase (ChAT)-positive and glycinergic AC subtypes were unaffected. The AC loss began 4 weeks after initial microbead injection, corresponding to the time course of RGC loss. Recordings of electroretinogram (ERG) oscillatory potentials and scotopic threshold responses, which reflect AC and RGC activity, were significantly attenuated in glaucomatous eyes following a time course that matched that of the AC and RGC loss. Moreover, we found that it was the ACs coupled to RGCs via gap junctions that were lost in glaucoma, whereas uncoupled ACs were largely unaffected. Our results suggest that AC loss in glaucoma occurs secondary to RGC death through the gap junction-mediated bystander effect. J. Comp. Neurol., 2016. © 2016 Wiley Periodicals, Inc.

Targeting neuronal gap junctions in mouse retina offers neuroprotection in glaucoma

“…We therefore extended our study to determine whether blockade of GJs could offer significant protection to these vulnerable AC subpopulations in glaucomatous eyes. Consistent with previous reports (22,33), antibodies against GABA and CR labeled neuronal cell bodies in the INL and GCL of control retinas with dendritic processes forming characteristic bands in the IPL (Figure 4, A and F). Eight weeks after initial microbead injection we observed a significant reduction in the number of both GABA-and CR-positive cells in the INL and GCL of CxWT mice, which was accompanied by marked changes in the density of labeled dendrites in the IPL (Figure 4 Expression of Cx36 is upregulated in experimental glaucoma.…”

“…To distinguish between RGCs and dACs in terms of their loss in glaucoma, we immunostained vertical retinal sections taken from midperipheral retina with BRN3A to identify RGCs and then counterstained with the nuclear dye DAPI to assess the total number of cells in the GCL ( Figure 3G). The number of dACs was computed as the difference between DAPI-positive nuclei and BRN3A-positive cells in the GCL as we described in a recent study (22). We observed a significant decrease in both BRN3A-positive RGCs and dACs in C57BL/6 mice measured at 8 weeks after the initial microbead injection ( Figure 3I).…”

“…Our recent finding that dACs are lost in microbead-injected control mice (22) is consistent with studies showing that certain subpopulations of ACs are injured in animal models of glaucoma (20,21). Moreover, ACs that are coupled to RGCs via GJs, including GABAergic and calretinin-positive (CR-positive) cells, are particularly vulnerable, suggesting that their loss in glaucoma reflects secondary degeneration via the bystander effect (22). We therefore extended our study to determine whether blockade of GJs could offer significant protection to these vulnerable AC subpopulations in glaucomatous eyes.…”

“…Most RGCs also couple heterologously to ACs (23), suggesting that primary RGC loss in glaucomatous eyes can lead to AC loss through GJ-mediated secondary cell death (12). Indeed, a recent study showed that the most vulnerable ACs in experimental glaucoma are those that are coupled to RGCs (22). Together, these data suggest that GJ-mediated secondary cell death may ultimately be responsible for the majority of neuronal loss and the resultant impaired visual function associated with glaucoma.…”

“…Damage to the optic nerve due to elevated IOP and the loss of retinal ganglion cells (RGCs) are key sequelae of glaucoma, but amacrine cells (ACs), interneurons critical to retinal signaling, also appear to show significant injury (18)(19)(20)(21)(22). In mice, nearly all the RGC subtypes are homologously coupled via GJs (23), thereby providing the structural substrate for progressive, bystander-mediated cell death.…”