Amacrine Cells Forming Gap Junctions With Intrinsically Photosensitive Retinal Ganglion Cells: ipRGC Types, Neuromodulator Contents, and Connexin Isoform

Abstract

Purpose: Intrinsically photosensitive retinal ganglion cells (ipRGCs) signal not only centrally to non-image-forming visual centers of the brain but also intraretinally to amacrine interneurons through gap junction electrical coupling, potentially modulating image-forming retinal processing. We aimed to determine (1) which ipRGC types couple with amacrine cells, (2) the neuromodulator contents of ipRGC-coupled amacrine cells, and (3) whether connexin36 (Cx36) contributes to ipRGC-amacrine coupling.

Methods: Gap junction-permeable Neurobiotin tracer was injected into green fluorescent protein (GFP)-labeled ipRGCs in Opn4Cre/+; Z/EG mice to stain coupled amacrine cells, and immunohistochemistry was performed to reveal the neuromodulator contents of the Neurobiotin-stained amacrine cells. We also created Opn4Cre/+; Cx36flox/flox; Z/EG mice to knock out Cx36 in GFP-labeled ipRGCs and looked for changes in the number of ipRGC-coupled amacrine cells.

Results: Seventy-three percent of ipRGCs, including all six types (M1-M6), were tracer-coupled with amacrine somas 5.7 to 16.5 µm in diameter but not with ganglion cells. Ninety-two percent of the ipRGC-coupled somas were in the ganglion cell layer and the rest in the inner nuclear layer. Some ipRGC-coupled amacrine cells were found to accumulate serotonin or to contain nitric oxide synthase or neuropeptide Y. Knocking out Cx36 in M2 and M4 dramatically reduced the number of coupled somas.

Conclusions: Heterologous gap junction coupling with amacrine cells is widespread across mouse ipRGC types. ipRGC-coupled amacrine cells probably comprise multiple morphologic types and use multiple neuromodulators, suggesting that gap junctional ipRGC-to-amacrine signaling likely exerts diverse modulatory effects on retinal physiology. ipRGC-amacrine coupling is mediated partly, but not solely, by Cx36.

Figure 1.

All six types of ipRGCs were tracer-coupled to amacrine cells. (A) Neurobiotin staining patterns of six representative Opn4^Cre/+; Z/EG ipRGCs. Arrowheads highlight Neurobiotin-filled somas near each ipRGC. (B) None of the tracer-coupled somas were immunopositive for the ganglion cell marker RBPMS, indicating they were amacrine cells. Asterisks in the right panel mark the locations of the ipRGC-coupled somas shown in the left panel. Note that the asterisks do not colocalize with RBPMS⁺ somas. (C) Population-averaged numbers of amacrine cells tracer-coupled to each M1–M6 ipRGC, including ipRGCs lacking coupled somas. The number above each column is the number of ipRGCs analyzed for that ipRGC type. Error bars are SEM.

Figure 2.

ipRGCs were tracer-coupled with a wide range of soma sizes. (A) Frequency distribution of the soma diameters of all the cells tracer-coupled to all injected cells of every ipRGC type. (B) The average numbers of small, medium, and large somas that coupled with each M1–M6 ipRGC, including uncoupled ipRGCs.

Figure 3.

Some ipRGC-coupled amacrine cells had somas in the INL. (A) An M2 ipRGC that was tracer-coupled with three INL somas (arrowheads) and eight GCL somas (arrows). The rectangles mark the two regions that have been rotated 90° in the bottom panels to show their side views. (B) Frequency distribution of all GCL versus INL somas tracer-coupled to all injected M1–M6 ipRGCs.

Figure 4.

Neuromodulator contents of ipRGC-coupled amacrine cells. (A) An amacrine cell (arrowhead) coupled to an M3 ipRGC was bNOS immunopositive. The left panel shows this amacrine cell's Neurobiotin fill, and the right panel shows its bNOS immunoreactivity. (B) Two amacrine cells (arrowheads) coupled to another M3 ipRGC were NPY immunopositive. (C) One amacrine cell (arrowhead) coupled to an M2 ipRGC was serotonin immunopositive. Shortly after Neurobiotin injection, this retinal piece was incubated in 2 µM serotonin hydrochloride for 15 minutes to allow neurons to accumulate serotonin. (D) None of the five amacrine cells (asterisks) coupled to this M4 ipRGC were VIP immunopositive. The asterisks marking the coupled cells do not colocalize with any of the bright VIP-immunostained somas.

Figure 5.

ipRGC-amacrine coupling is mediated in part by Cx36. (A) Cx36 immunostaining confirms nonglobal Cx36 knockout in Opn4^Cre/+; Cx36^flox/flox; Z/EG retinas. A1: Cx36 immunostaining was imaged confocally in whole-mount retinas, and the z-stacks were rotated 90° to show these orthogonal views of Opn4^Cre/+; Cx36^+/+; Z/EG (left) and Opn4^Cre/+; Cx36^flox/flox; Z/EG (right) retinas. A2: Representative whole-mount images at focal planes within the outer (top) and inner (bottom) plexiform layers. (B) Neurobiotin staining patterns of six representative Opn4^Cre/+; Cx36^flox/flox; Z/EG ipRGCs. Arrowheads mark Neurobiotin-filled somas within the M3 ipRGC's dendritic field. (C) Population-averaged numbers of somas coupled to each Opn4^Cre/+; Cx36^flox/flox; Z/EG ipRGC of every type, including uncoupled ipRGCs (black columns). The number above each column is the number of ipRGCs analyzed for that ipRGC type. The Opn4^Cre/+; Z/EG control data (gray columns) have been replotted from Figure 1C. ***P < 0.001.