Decision letter: True S-cones are concentrated in the ventral mouse retina and wired for color detection in the upper visual field
2020-04-15

Article Figures and data Abstract eLife digest Introduction Results and discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Color, an important visual cue for survival, is encoded by comparing signals from photoreceptors with different spectral sensitivities. The mouse retina expresses a short wavelength-sensitive and a middle/long wavelength-sensitive opsin (S- and M-opsin), forming opposing, overlapping gradients along the dorsal-ventral axis. Here, we analyzed the distribution of all cone types across the entire retina for two commonly used mouse strains. We found, unexpectedly, that ‘true S-cones’ (S-opsin only) are highly concentrated (up to 30% of cones) in ventral retina. Moreover, S-cone bipolar cells (SCBCs) are also skewed towards ventral retina, with wiring patterns matching the distribution of true S-cones. In addition, true S-cones in the ventral retina form clusters, which may augment synaptic input to SCBCs. Such a unique true S-cone and SCBC connecting pattern forms a basis for mouse color vision, likely reflecting evolutionary adaptation to enhance color coding for the upper visual field suitable for mice’s habitat and behavior. eLife digest Many primates, including humans, can see color better than most other mammals. This difference is due to the variety of light-detecting proteins – called opsins – that are produced in the eye by cells known as cones. While humans have three, mice only have two different opsins, known as S and M, which detect blue/UV and green light, respectively. Mouse cones produce either S-opsins, M-opsins or both. Fewer than 10 percent of cone cells in mice produce just the S-opsin, and these cells are essential for color vision. Mice are commonly used in scientific research, and so their vision has been well studied. However, previous research has produced conflicting results. Some studies report that cone cells that contain only S-opsin are evenly spread out across the retina. Other evidence suggests that color vision in mice exists only for the upper field of their vision, in other words, that mice can only distinguish colors that appeared above them. Nadal-Nicolás et al. set out to understand how to reconcile these contrasting findings. Molecular tools were used to detect S- and M-opsin in the retina of mice and revealed large differences between the lower part, known as the ventral retina, and the upper part, known as the dorsal retina. The ventral retina detects light coming from above the animal, and about a third of cone cells in this region produced exclusively S-opsin, compared to only 1 percent of cones in the dorsal retina. These S-opsin cone cells in the ventral retina group into clusters, where they connect with a special type of nerve cells that transmit this signal. To better understand these findings, Nadal-Nicolás et al. also studied albino mice. Although albino mice have a different distribution of S-opsin protein in the retina, the cone cells producing only S-opsin are similarly clustered in the ventral retina. This suggests that the concentration of S-opsin cone cells in the ventral retina is an important feature in mouse sight. This new finding corrects the misconception that S-opsin-only cone cells are evenly spread throughout the retina and supports the previous evidence that mouse color vision is greatest in the upper part of their field of vision. Nadal-Nicolás et al. suggest this arrangement could help the mice to detect predators that may attack them from above during the daytime. Together, these new findings could help to improve the design of future studies involving vision in mice and potentially other similar species. Introduction Topographic representation of the visual world in the brain originates from the light-sensitive photoreceptors in the retina (Rhim et al., 2017). Although the neuronal architecture of the retina is similar among different vertebrates, the numbers and distributions of photoreceptors vary considerably (Hunt and Peichl, 2014). Such patterns have been evolutionarily selected, adapting to the animal’s unique behavior (diurnal or nocturnal) and lifestyle (prey or predator) for better use of the visual information in the natural environment (Dominy and Lucas, 2001; Gerl and Morris, 2008; Peichl, 2005). Color, an important visual cue for survival, is encoded by comparing signals carried by photoreceptors with different spectral preferences (Baden and Osorio, 2019). While amongst mammals, trichromatic color vision is privileged for some primates (Jacobs et al., 1996; Nathans et al., 1986; Yokoyama and Yokoyama, 1989), most terrestrial mammals are dichromatic (Marshak and Mills, 2014; Puller and Haverkamp, 2011; Jacobs, 1993). The mouse retina expresses two types of cone opsins, S- and M-opsin, with peak sensitivities at 360 nm and 508 nm, respectively (Jacobs et al., 1991; Nikonov et al., 2006). The expression patterns of these two opsins form opposing and overlapping gradients along the dorsal-ventral axis, resulting in a majority of cones expressing both opsins (herein either ‘mixed cones’ or M+S+) (Applebury et al., 2000; Ng et al., 2001; Wang et al., 2011). Thus, S-opsin enrichment in the ventral retina better detects short-wavelength light from the sky, and M-opsin in the dorsal retina perceives the ground (e.g., a grassy field) (Baden et al., 2013; Gouras and Ekesten, 2004; Osorio and Vorobyev, 2005; Szél et al., 1992), while co-expression of both opsins (herein either mixed cones or M+S+) (Röhlich et al., 1994) broadens the spectral range of individual cones and improves perception under varying conditions of ambient light (Chang et al., 2013). This unusual opsin expression pattern poses a challenge for color-coding, particularly so for mixed cones. However, it has been discovered that a small population of cones only expresses S-opsin (‘true S-cones’, or S+M-). These true S-cones are thought to be evenly distributed across the retina (Franke et al., 2019; Haverkamp et al., 2005; Szatko et al., 2019; Wang et al., 2011) and to be critical for encoding color, especially in the dorsal retina where they are quasi-evenly distributed in a sea of cones expressing only M-opsin (M+S-), a pattern akin to mammalian retinas in general (Haverkamp et al., 2005; Wang et al., 2011). Nonetheless, subsequent physiological studies revealed that color-opponent retinal ganglion cells (RGCs) are more abundant in the dorsal-ventral transition zone (Chang et al., 2013) and the ventral retina (Joesch and Meister, 2016). Recent large scale two-photon imaging results further demonstrated that color opponent cells were mostly located in the ventral retina (Szatko et al., 2019). Intriguingly, a behavior-based mouse study demonstrated that their ability to distinguish color is also restricted to the ventral retina (Denman et al., 2018). These results prompt us to study, at the single-cell level and across the whole retina, the spatial distributions of cone types with different opsin expression configurations and, more importantly, with regard to S-cone bipolar cell connections in order to better understand the anatomical base for the unique color-coding scheme of the mouse retina. Results and discussion True S-cones are highly concentrated in the ventral retina of pigmented mouse In mouse retina, the gradients of S- and M-opsin expression along the dorsal-ventral axis have been well documented (Figure 1A–B; Applebury et al., 2000; Calderone and Jacobs, 1995; Chang et al., 2013; Haverkamp et al., 2005; Jelcick et al., 2011; Lyubarsky et al., 1999; Ortín-Martínez et al., 2014; Szél et al., 1992; Wang et al., 2011), but the distribution of individual cone types with different combinations of opsin expression across the whole retina has not been characterized (but see Baden et al., 2013; Eldred et al., 2020), which we discuss below). We developed a highly reliable algorithm to automatically quantify the different opsins (S and M) and cone types (M+S-, true S, and mixed cones, Figure 2, Figure 2—figure supplement 1) based on high-resolution images of entire flat-mount retinas immunolabeled with S- and M-opsin antibodies (Figure 2—figure supplement 1). As demonstrated in examples of opsin labeling from dorsal, medial, and ventral retinal areas of the pigmented mouse (Figure 1B, left), while M opsin-expressing cones (M+: M+S+ + M+S-) were relatively evenly distributed across three regions, S opsin-expressing cones (S+: M+S+ + S+M-) showed considerable anisotropy, with a high density in the ventral retina and a precipitous drop in the dorsal retina, confirming previous observations (Haverkamp et al., 2005; Jelcick et al., 2011; Ortín-Martínez et al., 2014). Surprisingly, instead of finding an even distribution of true S-cones as previously presumed (Baden et al., 2013; Haverkamp et al., 2005; Wang et al., 2011), we found the ventral region had much more numerous true S-cones (~30% of the local cone population; Figure 1C left, Supplementary file 1A) than did the dorsal region (~1%). This result is evident from density maps of cone types from three examples of pigmented mice, showing highly concentrated true S-cones in the ventral retina (Figure 2A, left column, bottom row). In addition, M+S--cones were concentrated in the dorsal retina, whereas mixed cones dominated the medial and ventral retina (Figure 1C left and Figure 2A, left column, 4th and 5th rows). Figure 1 Download asset Open asset Cone outer segments across retinal areas. Immunodetection of M and S wavelength-sensitive opsins in retinal sections (A) and flat-mount retinas (B) in two mouse strains (pigmented and albino mice, left and right columns respectively). (C) Retinal scheme of S-opsin expression used for image sampling to quantify and classify cones in three different retinal regions. Pie graphs showing the percentage of cones manually classified as M+S- (green), S+M- (true S, magenta) and M+S+ (mixed, gray) based on the opsin expression in different retinal areas from four retinas per strain. Black mouse: pigmented mouse strain (C57BL6), white mouse: albino mouse strain (CD1). Figure 2 with 1 supplement see all Download asset Open asset Topography and total number of different opsins (M+, S+) and cone-type populations in the whole mouse retina. (A) Density maps depicting the distributions of different opsins expressing cones (M+ and S+) and different cone populations classified anatomically as: All, M+S+ (mixed), M+S-, S+M- (true S) cones in pigmented and albino mice (left and right side respectively). Each column shows different cone populations from the same retina and, at the bottom of each map is shown the number of quantified cones. Color scales are shown in the right panel of each row (from 0 [purple] to 17,300 [dark red] for all cone types except to 5000 cones/mm2 [dark red] for the true S-cones and M+S--cone in the albino strain). Retinal orientation depicted by D: dorsal, N: nasal, T: temporal, V: ventral. (B) Histogram showing the mean ± standard deviation of different cone subtypes for eight retinas per strain (Supplementary file 1B). The percentages of each cone subtype are indicated inside of each bar, where 100% indicates the total of the ‘all cones’ group. (C) Opsin expression profile across the different retinal quadrants (retinal scheme, DT: dorsotemporal, DN: dorsonasal, VT: ventrotemporal, VN: ventronasal). Line graphs show the spatial profile of relative opsins expression (mixed [gray], M+S- [green], true S-cones [magenta]), where the sum of these three cone populations at a given distance from the optic nerve (ON) head equals 100%. Black mouse: pigmented mouse strain, white mouse: albino mouse strain. Despite the vast difference in S-opsin expression pattern, the distribution of true S-cones is strikingly similar between the pigmented and albino mouse Such a highly skewed distribution of true S-cones conflicts with the general notion that true S-cones only account for ~5% of cones and are evenly distributed across the mouse retina (Baden et al., 2013; Franke et al., 2019; Haverkamp et al., 2005; Szatko et al., 2019; Wang et al., 2011); however, it is not unprecedented considering the diverse S-cone patterns seen in mammals (Ahnelt et al., 2000; Ahnelt and Kolb, 2000; Calderone et al., 2003; Hendrickson et al., 2000; Hendrickson and Hicks, 2002; Kryger et al., 1998; Müller and Peichl, 1989; Nadal-Nicolás et al., 2018; Ortín-Martínez et al., 2014; Ortín-Martínez et al., 2010; Peichl, 2005; Schiviz et al., 2008; Szél et al., 2000). Therefore, we also examined an albino mouse line to determine whether this observation persists across different mouse strains. Overall, albino retinas had slightly smaller cone populations (Figure 2B, Supplementary file 1B; Ortín-Martínez et al., 2014). Interestingly, while M-opsin expressing cones had similar distributions in both strains, S-opsin expression extended well into the dorsal retina of the albino mouse, exhibiting a greatly reduced gradient of S-opsin expression toward the dorsal retina compared to that seen in pigmented mice (Figure 1B–C, Figure 2A second row; Applebury et al., 2000; Ortín-Martínez et al., 2014). Consequently, most cones in the dorsal retina were mixed cones, and M+S- cones were very sparse (7%, compared to 97% in pigmented mouse, Figure 1C right, Supplementary file 1A, Figure 2A right). However, despite these differences, the percentage and distribution of true S-cones were remarkably conserved between strains. In both strains, true S-cones were extremely sparse in the dorsal retina (1%) but highly concentrated in the ventral retina (33% vs 29%, Figure 1C and Supplementary file 1A). Notably, the density maps of true S-cones are nearly identical in both strains (Figure 2A, bottom row). Evaluating the distribution of three main cone populations (mixed, M+S-, and true S-cone) in four retinal quadrants centered upon the optic nerve head reveals different profiles between pigmented and albino strain for mixed and M+S- cones (Figure 2C). For example, in the dorsotemporal (DT) quadrant, we observed an increase of M+S- cones from the center to the periphery (green line) in pigmented mice, compared to a majority of mixed cones (gray line) in albino mice. However, true S-cone profiles (magenta lines) were similar between the two strains in all quadrants, except for a slightly increased density along the edge of the ventronasal (VN) quadrant in pigmented mice. A recent study successfully modeled cone opsin expression and type determination according to graded thyroid hormone signaling in a pigmented mouse strain (C57BL/6) (Eldred et al., 2020). It would be interesting to see whether a different pattern of thyroid hormone and/or receptor distribution could recapitulate a similar true S-cone distribution with a very different form of S-opsin expression. S-cone bipolar cells exhibit a dorsal-ventral gradient with a higher density in the ventral retina One major concern regarding cone classification based on opsin immunolabeling is that some S+M- cones may instead be mixed cones with low M-opsin expression (Applebury et al., 2000; Baden et al., 2013; Nikonov et al., 2006; Röhlich et al., 1994). Even though a similar cone-type distributions have been observed in mouse retina, it has been assumed that only a fraction of the S+M- cones are ‘true’ S-cones (Baden et al., 2013; Eldred et al., 2020). Out of caution, S+M- cones were only referred to as ‘anatomical’ S-cones due to a lack of confirmation regarding their bipolar connections (Baden et al., 2013). Thus, both true S-cones and S-cone bipolar cells have been generally acknowledged to be evenly distributed across the retina (Haverkamp et al., 2005; Wang et al., 2011; Baden et al., 2013; Szatko et al., 2019; Franke et al., 2019; Eldred et al., 2020). In order to confirm the distribution of true S-cones, it is critical to uncover the distribution and dendritic contacts of S-cone bipolar cells (type 9, or SCBCs). Previously, SCBCs have only been identified among other bipolar, amacrine and ganglion cells in a Thy1-Clomeleon mouse line, rendering the quantification of their distribution across the entire retina impractical (Haverkamp et al., 2005). We generated a Copine9-Venus mouse line, in which SCBCs are specifically marked (Figure 2—figure supplement 1C), owing to the fact that Cpne9 is an SCBC-enriched gene (Shekhar et al., 2016). In retinal sections, these Venus+ bipolar cells have axon terminals narrowly ramified in sub-lamina 5 of IPL (Figure 3A), closely resembling type 9 BCs as identified in EM reconstructions (Behrens et al., 2016; Stabio et al., 2018a). In flat-mount view, these bipolar cells are often seen to extend long dendrites to reach true S-cones, bypassing other cone types (Figure 3B–C). The majority of dendritic endings formed enlarged terminals beneath true S-cones pedicles (Figure 3C–c’), but occasional slender ‘blind’ endings were present (arrow in Figure 3C–c”), which have been documented for S-cone bipolar cells in many species (Haverkamp et al., 2005; Herr et al., 2003; Kouyama and Marshak, 1992). Unexpectedly, we found that the distribution of SCBCs was also skewed toward VN retina, albeit with a shallower gradient (Figure 3D–E). To examine the connections between true S-cones and SCBCs, we immunolabeled S- and M-opsins in Copine9-Venus mouse retinas. Because M-opsin antibody signals did not label cone structures other than their outer segments, we first identified true S-cones at the outer segment level and then traced S-opsin labeling to their pedicles in the outer plexiform layer (OPL), where they connect with SCBCs (Figure 3C, for more details see material and methods). Although convergent as well divergent connections were found between true S-cones and SCBCs in both dorsal and ventral retina (see the source data), we noted different connectivity patterns. While in the dorsal retina, a single true S-cone connected to approximately 4 SCBCs (3.8 ± 0.2, see material and methods), in the ventral retina, a single SCBC contacted approximately 5 true S-cones (4.6 ± 0.4; Figure 3C, Supplementary file 2). These results agree well with the true S-cone to SCBC ratios calculated from cell densities in the DT and VN retina. Specifically, in the dorsal retina, the true S-cone to SCBC ratio was approximately 1:3.6, compared to 5.3:1 in the ventral retina (Supplementary file 3). Accordingly, both data sets support the presence of a prevalent divergence of true S-cone to SCBC connections in the dorsal retina, in comparison to a prominent convergence of contacts from true S-cones to SCBCs in VN retina. Critically, the specificity of wiring from true S-cones to SCBCs also confirms the identity of true S-cones as revealed by opsin labeling and further supports the finding that true S-cones are highly concentrated in VN mouse retina. Figure 3 Download asset Open asset S-cone Bipolar cells (SCBCs) in Cpne9-Venus mouse retina. (A) Retinal cross section showing the characteristic morphology of SCBCs (Behrens et al., 2016; Breuninger et al., 2011). (B) Detailed view of the selective connectivity between Venus+SCBCs and true S-cone terminals (yellow arrows). Note that SCBCs avoid contacts with cone terminals lacking S-opsin expression (M+S--cone pedicles, identified using cone arrestin), as well as a mixed cone pedicle, marked with an asterisk. In fact, on the contrary, the SCBCs prefer to develop multiple contacts to the same true S-cone pedicle. (C) Images from flat-mount retinas focused on the inner nuclear and outer plexiform layers (INL+OPL) or in the photoreceptor outer segment (OS) layer of the corresponding area. Magnifications showing divergent and convergent connectivity patterns from true S-cone pedicles in dorsal and ventral retinal domains, respectively. In the DT retina, six Venus+ SCBCs (cyan circles) contact a single true S-cone pedicle (magenta circle in DT); while one Venus+ SCBC contacts at four true S-cone pedicles in the VN retina (magenta in which to cones (yellow between true S-cones and SCBCs in DT and VN retina was as the number of true S-cone pedicles a single SCBC per retina (magenta or the number of SCBCs a single true S-cone pedicle per retina (cyan Detailed view of a SCBC two true S-cone Detailed view of a ‘blind’ SCBC Density maps depicting the distributions of SCBCs in Cpne9-Venus mice. Venus+ SCBCs along the axis from a flat-mount retina to the white in showing the increase of SCBCs towards the VN retina where true S-cone density row in Figure of Venus+ SCBC densities by the algorithm according to the number of other Venus+ SCBCs found an in two areas of and Venus+ SCBCs exhibit a sparse density forming they were in VN retina True S-cones in the ventral retina are not evenly distributed but form As demonstrated in the mouse retina, despite a large population of mixed cones, SCBCs connect with true S-cones, this mammalian color (Behrens et al., 2016; Breuninger et al., 2011; Haverkamp et al., 2005; et al., 2014). However, the increased density of SCBCs in the ventral retina not that of true S-cones Figure and Figure row). Thus, individual SCBCs in the ventral retina may be to develop more dendrites to the number of contacts with different S-cone terminals (Supplementary file 2, graphs in Figure Intriguingly, we discovered in both strains that true S-cones in the ventral retina appeared to than forming an even as revealed by (Figure Supplementary file 2). true S-cone may increase the availability of for individual SCBCs in a reduced Figure 4 with 1 supplement see all Download asset Open asset of true S-cones in the ventronasal (VN) retina. (A) Retinal from flat-mount retinas of true S-cones in the VN where true S-cone density of true S-cones that are not with mixed cones white outer segments in the (B) Retinal scheme of true S-cones used for two areas of along the axis. maps true S-cone in these regions. True S-cone are by the algorithm according to the number of other true S-cones found an of DT and VN populations of true S-cones to their (C) and true S-cone populations and their Images true S-cone (magenta and of their cells lines) from and cone the of other cone cone were used for all only their cone were The are examples of used in the in of for one albino VN retinal to of observed for cone Note that for true S-cones were lower than that of whereas between and distributed populations all cones were on the right show for all retinal using the mean and standard of their The range corresponding to standard from the mean that of is in of the for the in to for Note that the for all cones in this was to that by the ratio of true S-cones to all which in was to the for true S-cones for In the true S-cone was on the right shows true S-cone for all as for connectivity from a true S-cone to a single SCBC in the VN retina. Images of a true S-cone in a flat-mount retina, focused on the photoreceptor outer segment layer and the inner plexiform layers The upper left panel show the and of each true segment in the that the number the where outer segments contact the photoreceptor inner Each true S-cone pedicle to this is and color upper and are upon the SCBC dendritic profile upper To synaptic contacts between the SCBC and the cone pedicles the SCBC shown in lower left we single into dendritic for the contact with cone is shown in lower corresponding to the in lower left panel The lower right panel shows dendritic endings of this the marked cones It also contacts two cones of the field of view line the of the from other SCBCs are color for To quantify the spatial of true S-cone populations their lack we compared the observed true S-cone distributions 1 VN and DT retinal to generated populations (Figure To this we two one in which the between true S-cone was the set of for all cones, a relatively of true S-cones. the other cone were among observed cone (Figure of these generated distributions of for true S-cones (see that by the observed cone and of cone types for each 1 To the of true S-cone populations to their we first two of for true and and and Figure of these smaller in the between cones and more patterns. Interestingly, from true S-cone was and nearly from populations a toward lower than which may a toward Figure see To further the of true S-cone we the ratios of true S-cone for each cone as the S-cone ratio see for the of the for each retinal Intriguingly, were for true S-cones than for other cone which were to ratios due to so in ventral further a of true S-cones in areas (Figure Notably, a more form of of S-cones has been observed in the mouse et al., and with much lower densities in some (Ahnelt et al., 2000). Here, may the of true S-cone in the ventral retina, for example, by to high densities and 1996; et al., It is to that it may also the wiring of true S-cones with distributed SCBCs, which were not observed to in the ventral retina (Figure we observed examples of of true S-cones forming pedicles in the were in a and contacted by a SCBC (Figure true S-cones in the ventral retina may an anatomical base for mouse color vision Despite and a retina and et al., mice can detect color (Denman et al., 2018; et al., Although it whether the source of signals for color in or (Baden and Osorio, 2019; et al., 2000; and 2005; and Meister, 2016; et al., it is that true S-cones short-wavelength signals for color the notion that true S-cones are evenly distributed across the retina (Baden et al., 2013; Franke et al., 2019; Haverkamp et al., 2005; Szatko et al., 2019; Wang et al., 2011), whereas M+S- cones are