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. 2013 Sep 9;23(17):1696–1701. doi: 10.1016/j.cub.2013.07.010

Nonuniform Distribution and Spectral Tuning of Photosensitive Retinal Ganglion Cells of the Mouse Retina

Steven Hughes 1, Thomas S Watson 1, Russell G Foster 1,∗, Stuart N Peirson 1,∗∗, Mark W Hankins 1
PMCID: PMC3770899  PMID: 23954426

Summary

Melanopsin-expressing photosensitive retinal ganglion cells (pRGCs) represent a third class of retinal photoreceptor [1–3]. These cells are intrinsically photosensitive, but also receive inputs from rod and cone photoreceptors [4–7], acting as the primary sensory conduit mediating non-image-forming responses to light [8–11]. Multiple subtypes of pRGC have been described in the mouse retina with characteristic morphologies and functional properties, and which perform distinct physiological roles [12–15]. Here, we examine the levels of melanopsin expression and distribution of pRGC subtypes across the mouse retina, identifying a previously unreported anatomical and functional specialization of the melanopsin system. Our results show a dorsal-ventral gradient in the expression of melanopsin and the distribution of pRGCs, which, combined with dorsal-ventral gradients in ultraviolet-sensitive and medium-wavelength-sensitive cone opsin expression, produce dramatic variations in the ratio of cone opsins and pRGCs across the retina. Using c-fos expression as a marker of light activation in vivo [16–18], we show that the responses of pRGCs are spectrally tuned by gradients in cone opsin expression depending on their location in the retina. These data illustrate the importance of classical photoreceptors in providing spectral tuning of pRGC light responses and have important implications for the complexity of non-image-forming responses to light.

Highlights

  • •

    Melanopsin-expressing pRGCs show a nonuniform distribution across the mouse retina

  • •

    Different pRGC subtypes show distinct patterns of distribution

  • •

    Gradients in cone opsin expression lead to a spectral tuning of pRGC light responses

Results and Discussion

The Melanopsin System Is Spatially Specialized in the Mouse Retina

Immunostaining with a highly sensitive melanopsin antibody revealed a nonuniform pattern of staining across the wild-type mouse retina (Figure 1 and Figure S1 available online). The total number of melanopsin-immunoreactive cells identified was typically 1,600–1,800 cells per retina, consistent with previous estimates of total photosensitive retinal ganglion cell (pRGC) numbers detected with melanopsin antibodies [19]. These include brightly stained M1-type cells with processes located in the OFF layer of the inner plexiform layer (IPL) and also weakly stained M2-type cells with process located in ON layer of the IPL. However, the density of melanopsin-positive cells and the extent of dendritic processes are both significantly higher in the dorsal retina compared to the ventral retina (Figure 1). In the dorsal retina, both M1 and M2 cells are clearly visible, with extensive and highly overlapping dendritic networks observed in both the OFF and ON layers of the IPL. By contrast, the ventral retina is more sparsely populated and dominated by intensely stained M1-type cells (Figure S1). There was little noticeable variation in the pattern of staining observed between the temporal and nasal regions. The orientation of retinae was confirmed by double labeling for ultraviolet-sensitive (UVS) cone opsin (see below).

Figure 1.

Figure 1

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Nonuniform Patterning of Melanopsin Expression across the Mouse Retina

(A) Whole retina image showing the nonuniform pattern of melanopsin staining and distribution of melanopsin-positive pRGCs across the wild-type C57/BL6 retina. Staining in the dorsal retina shows higher cell densities and increased coverage of the retina surface with dendritic processes when compared to the ventral retina. A total of 1,641 melanopsin-positive cells were identified.

(B and C) Images collected from the dorsal and ventral retina as indicated in the main image.

(D) Analysis of pRGC cell density confirms an ∼1.6-fold increase in the density of cells in the dorsal retina compared to the ventral retina (t test, p = 0.004).

(E) Threshold analysis of image pixel intensity confirms an ∼1.8-fold increase in the area of the retina covered by melanopsin cell bodies and dendrite processes in the dorsal and ventral retina (t test, p = 1.5 × 10−9). For (D) and (E), analysis is based on n = 5 nonoverlapping images (0.49 mm2 each) collected from the dorsal and ventral retina.

Data are shown as mean ± SEM. See also Figure S1.

A similar pattern of melanopsin staining was observed for all retina examined (>50), including retina from multiple strains of wild-type mice (C57/Bl6 and C3H/He), degenerate rd/rd cl retina lacking outer retina photoreceptors, Opn4−/− mice lacking melanopsin, and also retinae collected throughout postnatal development (Figure S1). Collectively, these observations suggest that this patterning of pRGC distribution is a fundamental property of the mouse retina.

Distribution of pRGC Subtypes

The distribution of specific pRGC subtypes was determined by cell density and nearest-neighbor analysis (Figures 2 and S2). Both approaches show a significant increase in density of M1 cells and M2 cells in the dorsal retina compared to the ventral retina. There are, however, subtle differences in the distribution of these cell types. The highest density of M1 cells is observed in the upper dorsal retina, whereas the highest density of M2 cells is observed in the middorsal retina. No significant differences in density of either M1 or M2 cells were observed between the temporal and nasal regions (for detailed nearest-neighbor analysis, see Figure S2). The dorsal-ventral distribution of M1-type pRGCs was further confirmed by analysis of retina from Opn4−/− (tau-LacZ+/+) mice (Figure S2) that selectively report M1-type pRGCs [20]. Due to the rarity of bistratified M3 cells [19], no attempt was made to characterize the distribution of these cells.

Figure 2.

Figure 2

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Differential Distribution of pRGC Subtypes across the Mouse Retina

(A, C, and D) Graphs showing the location and distribution of M1- and M2-type pRGCs across the mouse retina. A total of 1,747 melanopsin cells were detected, 920 M1 cells and 827 M2 cells. Both M1- and M2-type pRGCs show a similar, although not identical, dorsal-ventral pattern of distribution, with the density of both cell types higher in the dorsal retina compared to the ventral retina.

(B) Manual counting of cell density confirms a dorsal-ventral gradient in distribution of M1-type pRGCs (dorsal 75.4 ± 9.1 and ventral 43.3 ± 7.9 cells/mm2, t test, p = 0.011) and M2-type pRGCs (dorsal 81.7 ± 8.8 and ventral 36.9 ± 4.1 cells/mm2 (t test, p = 0.0009). Analysis of cell density based on counts from n = 6 nonoverlapping images (0.49 mm2) from the dorsal and ventral retina. Data are shown as mean ± SEM (for detailed nearest-neighbor analysis of M1 and M2 distribution see Figure S2). Due to rarity of M3 cells [19], no attempt was made to characterize the distribution of these cells, and they are likely included as M1 cells.

(E) Graph showing the uniform distribution of EYFP-positive cells across the retina of Opn4.Cre+/−EYFP+/+ mice. A total of 4,705 EYFP cells were detected in the retina shown (range 4,415–4,705 cells per retina, n = 2), with expression of EYFP largely restricted to cells located in the GCL and INL, consistent with the detection of M1–M5-type pRGCs (Figure S2).

(F) Analysis of cell density shows that EYFP cells (M1–M5-type pRGCs) show a uniform density in the dorsal and ventral retina (275.3 ± 19.6 and 306.6 ± 14.9 cells/mm2 respectively, n = 3 retina, t test, p = 0.22), melanopsin-positive cells (M1- and M2-type pRGCs) show a significant dorsal-ventral gradient with higher densities of cells detected in the dorsal retina (t test, p = 4.1 × 10−9). EYFP-positive melanopsin-negative cells (M4 and M5 cells) show an opposing dorsal-ventral gradient with significantly higher density of cells observed in the ventral retina (t test, p = 0.002). Analysis shown in (F) is based on manual counting from n = 5 regions (0.25 mm2) from the dorsal and ventral retina of n = 3 mice. Data are shown as mean ± SEM. (For a detailed nearest-neighbor analysis of EYFP cell distribution, see Figure S2.)

(G and H) Double labeling with EYFP (green) and melanopsin (red) antibodies shows a significant difference in levels of colocalization observed in the dorsal and ventral retina.

See also Figure S2.

In addition to M1–M3-type cells, there also exist at least two other subtypes of pRGC, M4- and M5-type cells, in which the levels of melanopsin expression are too low to be reliably detected with melanopsin antibodies [19], but can be detected using Opn4.Cre-based reporter mice [21]. Here, we have used the same Opn4.Cre mouse with an EYFP reporter to determine the distribution of M1–M5-type pRGCs. For a full description of this model and a discussion of potential limitations, see Figure S2. In contrast to the distribution of M1 and M2 cells, EYFP-expressing cells were found to be uniformly distributed across the retina (n = 3 retina, t test, p = 0.22) (Figures 2 and S2). Double labeling with melanopsin and EYFP antibodies again confirmed the dorsal-ventral distribution of melanopsin-immunoreactive M1 and M2 cells (n = 3 retina, t test, p = 4.1 × 10−9). Interestingly, levels of melanopsin and EYFP coexpression varied in dorsal-ventral manner, with significantly lower densities of melanopsin-negative EYFP-positive M4–M5 cells detected in the dorsal retina (n = 3 retina, t test, p = 0.002) (Figure 2). Collectively, our data indicate that melanopsin-expressing cells as a whole are uniformly distributed across the mouse retina, but the distribution of specific subtypes of pRGCs varies. Both M1- and M2-type cells show higher densities in the dorsal retina, whereas melanopsin-negative EYFP-positive M4–M5 cells are more numerous in the ventral retina.

Cone Opsin Gradients and Spectral Tuning of pRGC Light Responses

In addition to the dorsal-ventral gradients in melanopsin expression and distribution of pRGC subtypes that we have described, there also exist significant dorsal-ventral gradients in the expression of cone opsins within M cones (95% of all cones) of the mouse retina [22–25]. Levels of medium-wavelength-sensitive (MWS) opsin are highest in the dorsal retina, whereas levels of ultraviolet-sensitive (UVS) opsin are highest in the ventral retina. The rarer S cones (5% of cones) express only UVS opsin and show no such gradients in expression (Figure S3). When combined, these gradients result in dramatic variations in the ratio of cone opsins and pRGC subtypes present across the retina (Figure 3). Recent studies have confirmed that the changeable ratio of UVS and MWS opsin leads to a spectral tuning of light responses recorded from bipolar cells and ganglion cells of the mouse retina [26, 27]. Using the expression of c-fos as a marker of cellular light activation in vivo [16–18], we confirm the spectral tuning of retinal ganglion cells by cone opsin gradients and demonstrate that the responses of pRGCs are spectrally tuned depending on their location in the retina (Figures 4 and S4).

Figure 3.

Figure 3

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Changing Ratios of Cone Opsins and Melanopsin-Expressing pRGCs across the Mouse Retina

(A) Double labeling with MWS (red) and UVS cone opsin (green) antibodies highlights the opposing dorsal-ventral gradients of MWS and UVS cone opsin expression present within M cones (∼95% of all cones) of the mouse retina. M cones in the dorsal retina express high levels of MWS opsin and lack UVS opsin, M cones in the medial retina express high levels of both MWS and UVS opsin, whereas M cones in the dorsal retina express low levels of MWS and high levels of UVS opsin. S cones (∼5% of all cones) are evenly distributed across the retina and express only UVS cone opsin (shown in circles). For a full characterization of cone opsin expression in the mouse retina, see Figure S3.

(B) Double labeling for melanopsin (red) and UVS cone opsin (green) highlights the changing ratio of these photopigments in the dorsal and ventral retina and is most evident in the mid dorsal retina at the transition from high to low UVS expression. Overall, the dorsal retina contains higher densities of M1- and M2-type pRGCs, high MWS opsin expression, and low UVS opsin expression, whereas the ventral retina contains lower densities of M1- and M2-type pRGCs and lower MWS opsin expression but significantly increased expression of UVS opsin.

See also Figure S3.

Figure 4.

Figure 4

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The Responses of M1- and M2-Type pRGCs Are Spectrally Tuned by Gradients in Cone Opsin Expression Present within M Cones of the Mouse Retina

(A and B) Images showing the levels of c-fos expression (red) detected within pRGCs (green) located in the dorsal and ventral regions of wild-type C57/Bl6 mice, degenerate rd/rd cl mice lacking rod and cone photoreceptors, and Opn4−/− mice lacking melanopsin expression, following white light (A) and UV light pulses (B).

(C and D) Graphs showing the percentage of M1- and M2-type pRGCs showing detectable levels of c-fos expression following white light pulses (C) and UV light pulses (D). The responses of M1- and M2-type pRGCs are spectrally tuned by gradients in MWS and UVS cone opsin expression present within M cones of the mouse retina. Spectral tuning of M1-type pRGCs is evident from Opn4−/− mice receiving white light pulses (t test, p = 0.004) and wild-type mice receiving UV light pulses (t test, p = 0.03). The responses of M2-type pRGCs are spectrally tuned in the wild-type retina following white light (t test, p = 0.0032) and UV light pulses (t test, p = 0.038). In all cases, the direction of enhanced responses is consistent with significant excitatory input from M cones and the opposing gradients of MWS and UVS cone opsin expression. No significant gradients in c-fos expression were observed in the rd/rd cl retina for either M1-type pRGCs or M2-type pRGCs following either white light or UV light pulses. Interestingly, for UV light pulses, the percentage of responsive M1 cells and the intensity of c-fos staining were lower in the wild-type retina compared to rd/rd cl retina (t test, dorsal p = 0.03, ventral p = 0.21). Analysis is based on manual counting from n = 8–10 nonoverlapping regions (0.25 mm2) in the dorsal and ventral regions of each retina; values shown are the mean of n = 3–4 retina per group. Identification of M2 cells is not permitted using Opn4−/− mice [20] (Figure S2). Orientation of retina from Opn4−/− and wild-type mice was performed by colabeling with UVS cone opsin (shown for Opn4−/− only). Orientation of rd/rd cl retina was performed based on the patterning of melanopsin expression. Data are shown as mean ± SEM.

See also Figure S4 for analysis of light-induced c-fos expression in EYFP cells from the retina of Opn4.Cre+/−EYFP+/+ mice.

Spectral Tuning of M1-Type pRGCs

In wild-type mice, white light pulses resulted in a near saturating activation of M1-type cells in both the dorsal and ventral retina (dorsal, 96.7% ± 1.4% and ventral, 92.8% ± 3.7%, n = 3 retina, t test, p = 0.31) (Figure 4). Robust responses to white light were also observed for M1 cells in the rd/rd cl retina lacking rod and cone photoreceptors (dorsal, 81.5% ± 1.0% and ventral, 86.8% ± 1.7%, n = 3 retina, t test, p = 0.54), indicating that melanopsin-based photoresponses are sufficient to drive near maximal responses in M1 cells under these conditions. Analysis of light-induced c-fos expression in the retina of Opn4−/− mice [17], where pRGCs lack endogenous photoresponses, confirmed a significant gradient in the excitatory inputs to M1 pRGCs with a higher number of cells responding in the dorsal retina (dorsal, 78.2% ± 3.0% and ventral, 48.5% ± 4.1%, n = 3 retina, t test, p = 0.004). Overall, the pattern of c-fos expression detected in M1 pRGCs from Opn4−/− mice receiving white light pulses is consistent with a significant excitatory input from M cones and the gradient of MWS cone opsin expression. We could also expect a significant contribution to M1 responses to white light from rod-based input [11, 28–31], yet it is likely this is reduced under these intensities of light [32].

In contrast to white light, a significant gradient of responses was observed for M1-type pRGCs in wild-type retina following UV light stimuli, with increased numbers of responsive cells detected in the ventral retina compared to the dorsal retina (dorsal, 56.0% ± 5.4% and ventral, 71.9% ± 6.8%, n = 3 retina, t test, p = 0.03) (Figure 4). The direction of this gradient is again consistent with an excitatory input from M cones and the gradient of UVS opsin. However, despite the more striking gradient in expression of UVS opsin compared to MWS opsin, the gradient of UV light responses observed for M1 cells in Opn4−/− mice were less obvious than those observed in responses to white light (dorsal, 47.4% ± 4.3% and ventral, 54.6% ± 4.6%, n = 4 retina, t test, p = 0.29). Interestingly, the percentage of M1-type cells responding to UV light was significantly higher in the rd/rd cl retina compared to wild-type retina (WT versus rd/rd cl t test, dorsal p = 0.03, ventral p = 0.21). Thus, it would seem that M1-type pRGCs show significantly greater responses to UV light in the absence of outer retina photoreceptors. White light activates M cones, rods, and melanopsin, whereas UV light is most efficiently absorbed by UVS opsin (expressed in M cones and S cones) (Figure S4). It is therefore possible that S cones provide additional inhibitory signals to M1 cells under UV illumination. This conclusion is supported by recent data showing that both M cones and S cones provide excitatory inputs to pRGCs, and that S cones also mediate a marked OFF inhibition of M1 pRGC-driven responses [30].

Spectral Tuning of M2-Type pRGCs

In the wild-type retina, M2-type pRGCs show significant gradients in c-fos expression following both white light and UV light exposure. Following white light stimuli, greater numbers of responsive M2 cells are detected in the dorsal retina, (dorsal, 84.4% ± 1.7% and ventral, 56.9% ± 6.5%, n = 3 retina, t test, p = 0.0032), whereas following UV light stimuli higher numbers of responsive M2 cells are detected in the ventral retina (dorsal, 50.8% ± 8.4% and ventral, 75.0% ± 9.3%, n = 3 retina, t test, p = 0.038) (Figure 4). The pattern of these responses is consistent with a significant excitatory input from M cones and the opposing gradients of MWS and UVS cone opsin expression. No such gradients were observed for M2 cells in the rd/rd cl retina lacking rod and cone photoreceptors (Figure 4). For both white light and UV light stimuli, the number of responsive M2 cells was higher in the wild-type retina compared to the rd/rd cl retina, indicating an additive effect of melanopsin and outer retina-driven signals. Gradients of light-induced c-fos expression were also observed for EYFP cells in retina from Opn4.Cre.EYFP mice following UV light (n = 3 retina, t test, p = 0.0016) and to a lesser extent white light pulses (n = 3 retina, t test, p = 0.14) (Figure S4). Collectively our data indicate that gradients in cone opsin expression influence the responses of M4–M5-type cells, consistent with recent studies of M4-type cells [13].

Differential Spectral Tuning of pRGC Subtypes and Non-Image-Forming Responses to Light

Overall, we show that the gradients of cone opsin expression within M cones of the mouse retina lead to the spectral tuning of pRGC light responses depending on their location in the retina. However, given the high levels of endogenous photosensitivity observed for M1-type cells, it seems probable that under physiological conditions the gradients in cone opsin expression may do more to spectrally tune the responses of M2-type cells, and M4–M5 cells, than M1-type cells. These conclusions are consistent with the current understanding of the different pRGC subtypes. The photoresponses of M1 cells are driven primarily by melanopsin phototransduction, whereas M2 cells, and also M4–M5 cells, exhibit lower levels of endogenous photosensitivity and rely upon greater excitatory inputs from the outer retina [6, 7, 11, 15, 21, 33, 34]. Specific subclasses of pRGCs innervate specific areas of the brain [12, 21, 35–37] and participate in different non-image-forming response to light [12, 13]. Based on our data, it seems highly likely that different classes of pRGC, and therefore different non-image-forming responses, are spectrally tuned to different wavelengths of light. Given their higher density in the dorsal retina it is likely that the majority of M2 cells show a green light bias, driven by the high levels of MWS cone opsin expression (λmax 508 nm). By contrast, EYFP-positive melanopsin-negative M4–M5 cells are more numerous in the ventral retina and likely receive predominantly UVS-driven inputs (λmax 360 nm). Responses from M1-type pRGCs are likely to be closer to the peak sensitivity of melanopsin (λmax 479 nm) [38].

Conclusions

In summary, our results show a previously unreported anatomical and functional specialization of the murine melanopsin system. This study provides the first evidence for a differential distribution of pRGC subtypes and illustrates the importance of classical photoreceptor input in providing spectral tuning of pRGC light responses. These data have clear implications for the complexity of sensory irradiance detection and the study of different non-image-forming responses to light.

Acknowledgments

This work was supported by a Wellcome Trust Programme Grant awarded to R.G.F., S.N.P., and M.W.H. and by BBSRC grants awarded to M.W.H. and R.G.F. and to S.N.P. and R.G.F. Opn4 tau-LacZ mice were a generous gift from K.-W. Yau, Johns Hopkins University. Opn4.Cre mice were a generous gift from S. Hattar, Johns Hopkins University. MWS cone opsin antibody (RJ492) was a kind gift from J. Nathans, Johns Hopkins University.

Published: August 15, 2013

Footnotes

This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License, which permits non-commercial use, distribution, and reproduction in any medium, provided the original author and source are credited.

Supplemental Information includes Supplemental Results and Discussion, Supplemental Experimental Procedures, and four figures and can be found with this article online at http://dx.doi.org/10.1016/j.cub.2013.07.010.

Contributor Information

Russell G. Foster, Email: russell.foster@eye.ox.ac.uk.

Stuart N. Peirson, Email: stuart.peirson@eye.ox.ac.uk.

Supplemental Information

Document S1. Supplemental Results and Discussion, Supplemental Experimental Procedures, and Figures S1–S4
mmc1.pdf (726.9KB, pdf)

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Associated Data

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Supplementary Materials

Document S1. Supplemental Results and Discussion, Supplemental Experimental Procedures, and Figures S1–S4
mmc1.pdf (726.9KB, pdf)

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