Recent evidence suggests that melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs), a neuronal class regulating non-image forming (NIF) vision and generally thought to be injury resistant, are dysfunctional in certain neurodegenerative diseases. Although disrupted NIF visual functions have been reported in patients and animals with diabetes, it remains controversial whether ipRGCs exhibit remodeling during diabetes and if so, whether such remodeling is variable among ipRGC subtypes. Here, we demonstrate that survival, soma-dendritic profiles, and melanopsin-based functional activity of M1 ipRGCs were unaltered in streptozotocin-induced 3-month diabetic mice. Such resistance remained at 6 months after streptozotocin administration. In contrast, M2/M3 ipRGCs underwent significant remodeling in diabetic mice, manifested by enlarged somata and increased dendritic branching complexity. Consistent with the unaltered melanopsin levels, the sensitivity of melanopsin-based activity was unchanged in surviving M2 cells, but their response gain displayed a compensatory enhancement. Meanwhile, the pupillary light reflex, a NIF visual function controlled by M2 cells, was found to be impaired in diabetic animals. The resistance of M1 cells might be attributed to the adjacency of their dendrites to capillaries, which makes them less disturbed by the impaired retinal blood supply at the early stage of diabetes.

Melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs) are a class of ganglion cell (GC) photoreceptors capable of responding to light independent of rod/cone input (1,2) and mediating a wide range of non-image forming (NIF) visual functions, including circadian photoentrainment and pupillary light reflex (PLR) (3). It has long been known that ipRGCs are resistant to various experimental injuries and retinal diseases, such as optic nerve crush and retinal degeneration (47). However, there is recent evidence that ipRGCs may be less resilient to certain other injuries/diseases. For example, ipRGC loss has been observed in patients with Alzheimer disease, which may explain the circadian rhythm dysfunction in Alzheimer disease (8).

Diabetic retinopathy, a primary complication of diabetes mellitus (DM), causes visual dysfunction or blindness by influencing various retinal neurons, including GCs (9,10). Reduced GC density (11), remodeled GC dendritic branching patterns (11,12), and altered intrinsic/light-evoked GC activities (12,13) have been reported in various animal models of DM. Although NIF vision abnormalities were revealed in patients and animals with DM (14,15), the susceptibility of ipRGCs to diabetic retinopathy has been a subject of debate. In streptozotocin (STZ)-induced DM rats, no differences have been detected in melanopsin-positive cell number, melanopsin expression level, or retinal projection to NIF visual centers, suggesting the capacity for resistance (16). By contrast, in Ins2Akita DM mice, dendritic swelling and axonal varicosity were observed in melanopsin-containing cells, implying ipRGC damage (11). Consistently, ipRGCs in C57 mice appear sensitive to STZ treatment, as demonstrated by increased dendritic branching of melanopsin-immunoreactive cells, elevated melanopsin transcript levels, and decreased circadian sensitivity (15,17). It should be noted that all these previous studies have regarded ipRGCs as a single, homogeneous population, which may obscure cell-subtype-specific damage, increasing the complexity of data explanation and sometimes leading to conflicting conclusions.

ipRGCs have been divided into at least six distinct subtypes (M1–M6) according to melanopsin expression level, soma-dendritic morphology, and physiological activity (18,19). Studies have revealed that different ipRGC subtypes may be differentially susceptible to certain types of injury (4,20). Here, on the basis of an STZ-induced model, we explored whether and how mouse ipRGCs are altered by DM in a cell-subtype-specific manner.

An expanded version of this section can be found in the Supplementary Material. Male C57BL/6 and Opn4-tdTomato mice were maintained under a 12-h light/12-h dark photoperiod. DM was induced at 7–8 weeks of age with an intraperitoneal injection of STZ (85 mg/kg body weight) for 3 consecutive days. Mice with fasting blood glucose levels >11.1 mmol/L were considered to be diabetic (Supplementary Fig. 1). Avidin-biotin-peroxidase reaction-enhanced immunostaining and soma-dendritic profile reconstruction were conducted, following the method developed by Berson et al. (21), with two rabbit melanopsin antibodies: PA1-780, which primarily probes M1 cells but labels few, if any, non-M1 ipRGCs in mice (7,22), and UF006, which clearly labels M1–M3 ipRGCs while faintly staining a few other ipRGCs in mice (21). Whole-retina cell counting was performed according to the procedure described previously (23) using a 10× objective lens (numerical aperture [N.A.] 0.25). The density of Brn3a-positive conventional GCs and melanopsin (UF006)-positive M1–M3 ipRGCs were assessed under a 20× objective lens (N.A. 0.8) by counting cells in eight 518 × 518 μm (for Brn3a) or 694 × 520 μm (for melanopsin) squares. Blood vessels were labeled by fluorescein-conjugated isolectin B4 (IB4). The whole-retina area was delineated using the Polygon tool in ImageJ software. Immunofluorescence intensity measurement was conducted using an Olympus FV1000 confocal microscope under a 20× objective lens (N.A. 0.75). One-micrometer-thick optical sections along the z-axis were captured in a single retinal region and were processed with maximum intensity projection to create a composite. The same acquisition settings were used for all image sets, with each region imaged only once. Offline gray-level measurement of the composite image was performed using Fiji software after background subtraction. Western blot analysis was performed using freshly extracted retinal lysates (50 μg/lane). Immunoblots were visualized by enhanced chemiluminescence and captured using the ChemiDoc XRS System with Image Lab software. Multielectrode array (MEA) recording of melanopsin-based photoresponses using USB-MEA60-Inv-BC-System and MC_Rack software has been described in detail previously (24). The retina was stimulated with a series of 10-s 480-nm full-field light flashes at 3.42 × 1011–3.42 × 1014 photons/cm2/s. Cluster analysis of the spikes was performed using a detection threshold of three to four times the SD of the voltage with Offline Sorter software. Whole-cell patch clamp recording was carried out using a MultiClamp 700B amplifier. Signals were low-pass filtered at 2.4 kHz and sampled at 10 kHz in I = 0 mode. Full-field, 5-s light stimuli at 1.5 × 109–1.5 × 1015 photons/cm2/s were generated using a 100-W halogen lamp, band-pass filtered at 480 nm with a narrowband filter, and regulated using a logic-controlled electromechanical shutter. PLR was recorded using a NeurOptics A-2000 pupillometer. Statistical analyses were performed using OriginPro 2015 software. Data are presented as mean ± SD when normally distributed and as median ± interquartile range when nonnormally distributed. Unless otherwise specified, P values represented the results of unpaired Student t test. P < 0.05 was considered statistically significant.

Data and Resource Availability

The data generated in this study are available from the corresponding author upon request. No applicable resources were generated during this study.

DM Did Not Alter the Number or Morphology of M1 ipRGCs

To determine the total number of M1 cells and reconstruct their soma-dendritic profiles in 3-month DM mice, avidin-biotin-peroxidase reaction-enhanced immunohistochemistry technique was used on whole-mount retinal preparations with a polyclonal melanopsin antibody (PA1-780) known to preferentially label M1 cells while labeling few, if any, non-M1 ipRGCs in mice (7,22). In both control (Fig. 1A and C) and DM (Fig. 1B and D) mice, immunoperoxidase labeling clearly revealed a sparse array of somata in the GC layer (GCL) and a few somata in the inner nuclear layer. A complete cell count gave 850.143 ± 66.336 and 792.200 ± 115.639 cells per retina for control and DM mice, respectively; these values were not statistically different (P = 0.253) (Fig. 1G), suggesting an absence of M1 cell death in DM animals. By contrast, the density of cells positive for Brn3a, a pan-GC marker that does not label ipRGCs (25), was significantly lower in DM retinas (2,595.743 ± 180.318 cells/mm2) than in control retinas (3,030.185 ± 225.895 cells/mm2, P = 0.024) (Fig. 1E, F, and H), suggesting increased apoptosis in conventional GCs. The reduced density is unlikely caused by retinal area enlargement as a result of DM because mean retinal areas were similar between the control and DM groups (P = 0.645) (Fig. 1I).

Figure 1

DM did not cause loss of M1 ipRGCs but reduced density of conventional GCs. A and B: Representative photomicrographs captured from a small area within whole-mount retinas stained by enhanced immunohistochemistry with a melanopsin antibody (PA1-780) preferentially labeling M1 ipRGCs in control and DM mice. Focal planes lie in the GCL. C and D: Whole-retina mapping of immunostained cells shown in A and B; each dot represents a melanopsin-immunoreactive soma. E and F: Representative images showing Brn3a-positive conventional GCs in whole-mount retinas collected from control (E) and DM (F) mice. G: No significant difference was detected in whole-retina melanopsin-positive (Mel+) cell number between control and DM mice. H: DM led to a significantly lower density of Brn3a-positive GCs. I: Retinal areas did not differ significantly in DM mice. Sample sizes are given in parentheses. Scale bar = 100 μm in B and F and 1 mm in D. Ctrl, control; D, dorsal; N, nasal; T, temporal; V, ventral.

Figure 1

DM did not cause loss of M1 ipRGCs but reduced density of conventional GCs. A and B: Representative photomicrographs captured from a small area within whole-mount retinas stained by enhanced immunohistochemistry with a melanopsin antibody (PA1-780) preferentially labeling M1 ipRGCs in control and DM mice. Focal planes lie in the GCL. C and D: Whole-retina mapping of immunostained cells shown in A and B; each dot represents a melanopsin-immunoreactive soma. E and F: Representative images showing Brn3a-positive conventional GCs in whole-mount retinas collected from control (E) and DM (F) mice. G: No significant difference was detected in whole-retina melanopsin-positive (Mel+) cell number between control and DM mice. H: DM led to a significantly lower density of Brn3a-positive GCs. I: Retinal areas did not differ significantly in DM mice. Sample sizes are given in parentheses. Scale bar = 100 μm in B and F and 1 mm in D. Ctrl, control; D, dorsal; N, nasal; T, temporal; V, ventral.

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Figure 2A and B are representative microphotographs showing immunoperoxidase labeling at the outermost level of the inner plexiform layer (IPL), where the dendritic arbors of M1 cells reside (21). Using dozens of such images and on the basis of a strategy developed by Berson et al. (21), we reconstructed the soma-dendritic morphology (Fig. 2C and D) of individual M1 cells randomly chosen from all four retinal quadrants for morphometric analysis. Sholl analysis revealed no significant changes in the number of dendritic intersections at any distance from the soma (all P > 0.05) (Fig. 2G) between the two groups, implying similar degrees of dendritic complexity. No significant differences were detected in any of the morphological parameters analyzed between control and DM mice (all P > 0.05) (Fig. 2H–L). Thus, 3-month DM likely did not modify the soma-dendritic profiles of M1 ipRGCs.

Figure 2

DM did not cause morphological changes in M1 ipRGCs. A and B: Representative areas of whole-mount retinas stained with PA1-780 melanopsin antibody using immunoperoxidase method in control and DM mice. Focal planes lie in the outermost level of the IPL to reveal the dendritic network of M1 ipRGCs. C and D: Reconstructed mosaic comprising all M1 cells in the areas shown in A and B. E and F: Dendritic field areas of each fully reconstructed M1 cell in C and D indicated as minimal polygons. G: Sholl analysis of M1 cells from control and DM mice revealed no significant changes in dendritic complexity. Inset is a bar graph comparing the total number of intersections between groups. H–L: No significant difference was detected in any major soma-dendritic parameters between control and DM mice. Sample sizes are given in parentheses. Scale bars = 200 μm. Ctrl, control.

Figure 2

DM did not cause morphological changes in M1 ipRGCs. A and B: Representative areas of whole-mount retinas stained with PA1-780 melanopsin antibody using immunoperoxidase method in control and DM mice. Focal planes lie in the outermost level of the IPL to reveal the dendritic network of M1 ipRGCs. C and D: Reconstructed mosaic comprising all M1 cells in the areas shown in A and B. E and F: Dendritic field areas of each fully reconstructed M1 cell in C and D indicated as minimal polygons. G: Sholl analysis of M1 cells from control and DM mice revealed no significant changes in dendritic complexity. Inset is a bar graph comparing the total number of intersections between groups. H–L: No significant difference was detected in any major soma-dendritic parameters between control and DM mice. Sample sizes are given in parentheses. Scale bars = 200 μm. Ctrl, control.

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DM Caused Cell Loss and Morphological Changes in M2 ipRGCs

To test whether non-M1 ipRGC subtypes were also morphologically resilient to DM, retina whole mounts were stained with another melanopsin antibody, UF006. This antibody is reported to be capable of clearly labeling not only M1 cells but also M2 and M3 cells, which have lower levels of melanopsin expression (21). In addition to the “outer” melanopsin-positive plexus in the OFF sublamina of the IPL arising mainly from M1 cells, an “inner” network of melanopsin-immunoreactive dendrites in the ON sublamina, presumably derived from non-M1 ipRGCs, were clearly discernible (Supplementary Fig. 2 and Fig. 4A–D). Therefore, tracing UF006-immunopositive signals allowed us to identify and further characterize the morphology of M2 cells, which stratify in the ON sublayer, and M3 cells, which are bistratified neurons.

Figure 4

DM severely changed soma-dendritic profiles of M2 ipRGCs. A and B: Representative areas of whole-mount retinas stained with UF006 melanopsin antibody using immunoperoxidase method in control and DM mice. Focal planes lie in the inner part of the IPL, where the dendritic networks of M2 ipRGCs reside. C and D: Reconstruction of the mosaic comprising all M2 cells in the areas shown in panels A and B. E and F: Dendritic field areas of each fully reconstructed M2 cell in C and D, indicated as minimal polygons. G: Sholl analysis revealed a significant increase in the number of dendritic intersections in multiple dendritic regions, indicating enhanced dendritic branching. Inset is a bar graph comparing the total number of intersections between the two groups. H–L: Bar graphs comparing major soma-dendritic parameters between control and DM mice. Soma swelling (H), dendritic field enlargement (I), increased total branch points (K), and increased dendritic length (L) were detected in DM mice. Sample sizes are given in parentheses. Scale bars = 100 μm. *P < 0.05, **P < 0.01. Ctrl, control.

Figure 4

DM severely changed soma-dendritic profiles of M2 ipRGCs. A and B: Representative areas of whole-mount retinas stained with UF006 melanopsin antibody using immunoperoxidase method in control and DM mice. Focal planes lie in the inner part of the IPL, where the dendritic networks of M2 ipRGCs reside. C and D: Reconstruction of the mosaic comprising all M2 cells in the areas shown in panels A and B. E and F: Dendritic field areas of each fully reconstructed M2 cell in C and D, indicated as minimal polygons. G: Sholl analysis revealed a significant increase in the number of dendritic intersections in multiple dendritic regions, indicating enhanced dendritic branching. Inset is a bar graph comparing the total number of intersections between the two groups. H–L: Bar graphs comparing major soma-dendritic parameters between control and DM mice. Soma swelling (H), dendritic field enlargement (I), increased total branch points (K), and increased dendritic length (L) were detected in DM mice. Sample sizes are given in parentheses. Scale bars = 100 μm. *P < 0.05, **P < 0.01. Ctrl, control.

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As expected, UF006 revealed much more somata than PA1-780 did (Fig. 3A–D). A complete count yielded 1,152.400 ± 114.837 in DM retinas, whereas significantly more (P = 0.003) (Fig. 3E) were counted in control retinas (1,421.875 ± 204.672). Given that the M1 cell number was not reduced (Fig. 1), we speculated that DM resulted in loss of non-M1 ipRGCs.

Figure 3

DM caused loss of M2 ipRGCs. A and B: Representative photomicrographs captured from a small area within whole-mount retinas in the control and DM groups, which were stained by enhanced immunohistochemistry with a melanopsin antibody (UF006) labeling both M1 and non-M1 ipRGCs. C and D: Distribution maps of melanopsin-positive (Mel+) cells shown in A and B; each dot represents a melanopsin-immunoreactive soma. E: Counting of whole-retina cells positive for UF006 melanopsin antibody revealed a significant decline among DM mice. F: A comparison of density of M1, M2, and M3 cells, distinguished by distinct stratification patterns in the IPL, between control and DM mice. DM significantly reduced M2 cell density but did not cause M1 or M3 cell loss. Sample sizes are given in parentheses. Scale bars = 100 μm in B and 1 mm in D. Ctrl, control; D, dorsal; N, nasal; T, temporal; V, ventral.

Figure 3

DM caused loss of M2 ipRGCs. A and B: Representative photomicrographs captured from a small area within whole-mount retinas in the control and DM groups, which were stained by enhanced immunohistochemistry with a melanopsin antibody (UF006) labeling both M1 and non-M1 ipRGCs. C and D: Distribution maps of melanopsin-positive (Mel+) cells shown in A and B; each dot represents a melanopsin-immunoreactive soma. E: Counting of whole-retina cells positive for UF006 melanopsin antibody revealed a significant decline among DM mice. F: A comparison of density of M1, M2, and M3 cells, distinguished by distinct stratification patterns in the IPL, between control and DM mice. DM significantly reduced M2 cell density but did not cause M1 or M3 cell loss. Sample sizes are given in parentheses. Scale bars = 100 μm in B and 1 mm in D. Ctrl, control; D, dorsal; N, nasal; T, temporal; V, ventral.

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This speculation was further explored by separately calculating the densities of M1, M2, and M3 cells (distinguished by tracing their unique dendritic stratification in the IPL), which was achieved by cell counting in eight 691 × 518-μm regions in each retina. Although densities of M1 (38.365 ± 5.221 vs. 39.761 ± 6.320 cells/mm2, P = 0.660) and M3 (1.188 ± 0.628 vs. 0.891 ± 0.393 cells/mm2, P = 0.310) cells were similar between control and DM mice, M2 cell density was markedly lower in the DM group (50.125 ± 6.775 vs. 39.289 ± 8.545 cells/mm2, P = 0.022), suggesting that DM-caused ipRGC loss is restricted to M2 cells (Fig. 3F).

Sholl analysis detected significantly higher numbers of dendritic intersections at 90, 110, 130, 150, 170, 190, 210, 230, 310, 390, and 410 μm from the soma (P < 0.05 or < 0.01) (Fig. 4G) of DM mice; the total number of intersections for each individual M2 cell was also significantly increased (90.229 ± 35.020 vs. 124.065 ± 57.085, P = 0.009) (Fig. 4G, inset). Most morphometric parameters, including soma diameter, dendritic field diameter, total branch points, and total dendritic length, were significantly increased by DM (P = 0.014, 0.034, 0.015, and 0.002, respectively), whereas only the number of primary dendrites showed no significant difference (P = 0.801) between control and DM (Fig. 4H–L), suggesting a severe morphological remodeling.

DM Resulted in Laminar-Specific Morphological Remodeling of M3 ipRGCs

Since M3 cells are much fewer than M1/M2 cells (21), morphological analysis of them was conducted using a smaller data set (Fig. 5A and B). Although not altering the number of primary dendrites (P = 0.500) (Fig. 5I), DM caused significant soma enlargement (P = 0.005) (Fig. 5E), and more interestingly, DM appeared to alter dendritic structure in a laminar-specific manner in M3 cells. Sholl analysis revealed a significant increase in intersection number for ON sublayer dendrites at 130, 150, 170, 190, 210, and 230 μm from the soma in DM mice (P < 0.05 or < 0.01) (Fig. 5C). Consistently, the total intersection number of the ON sublayer was significantly larger in DM animals (37.111 ± 14.174 vs. 52.405 ± 14.858, P = 0.002) (Fig. 5C, inset). By contrast, for OFF sublayer dendrites, no change in intersection number was observed, except at 190 μm from the soma (P < 0.05) (Fig. 5D), and total intersection numbers were similar between the two groups (38.972 ± 9.921 vs. 43.310 ± 8.606, P = 0.152) (Fig. 5D, inset). Dendritic field diameter (Fig. 5F) and total branch points (Fig. 5G) were increased for the ON (P = 0.043 and 0.002) but not for the OFF (P = 0.400 and 0.089) sublayer. For both ON and OFF sublayers, total dendritic length exhibited an increasing tendency (Fig. 5H); however, these effects were not statistically significant (P = 0.081 and 0.057).

Figure 5

DM differentially affected ON and OFF sublayer dendritic arbors of M3 ipRGCs. A and B: Representative examples of soma-dendritic profiles of M3 cells in control and DM mice reconstructed from whole-mount retinas stained with UF006 melanopsin antibody using immunoperoxidase method. C and D: Sholl analysis of ON and OFF sublayer dendritic arbors of M3 cells. DM significantly increased the number of dendritic intersections in multiple regions of ON arbors but only did so at 190 μm from the soma of OFF arbors. Insets are bar graphs comparing the total number of intersections between groups. E–I: Bar graphs comparing major soma-dendritic parameters between control and DM mice. Soma swelling (E), dendritic field enlargement (F), and increased total branch points (G) of ON sublayer arbors were observed in DM mice. Sample sizes are given in parentheses. Scale bars = 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001. Ctrl, control.

Figure 5

DM differentially affected ON and OFF sublayer dendritic arbors of M3 ipRGCs. A and B: Representative examples of soma-dendritic profiles of M3 cells in control and DM mice reconstructed from whole-mount retinas stained with UF006 melanopsin antibody using immunoperoxidase method. C and D: Sholl analysis of ON and OFF sublayer dendritic arbors of M3 cells. DM significantly increased the number of dendritic intersections in multiple regions of ON arbors but only did so at 190 μm from the soma of OFF arbors. Insets are bar graphs comparing the total number of intersections between groups. E–I: Bar graphs comparing major soma-dendritic parameters between control and DM mice. Soma swelling (E), dendritic field enlargement (F), and increased total branch points (G) of ON sublayer arbors were observed in DM mice. Sample sizes are given in parentheses. Scale bars = 100 μm. *P < 0.05, **P < 0.01, ***P < 0.001. Ctrl, control.

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DM Hardly Altered Overall Melanopsin-Based Spiking Activity

To test whether DM led to functional abnormalities in ipRGCs, we performed high-throughput evaluation of the melanopsin-driven light response using MEA technique. The melanopsin-based photoresponse, isolated by a pharmacological cocktail that blocked glutamatergic transmission (26), increased as a function of light intensity (Fig. 6A1 and B4). Irradiance–response (I–R) curves of major parameters describing melanopsin-based activity, such as total spike number during stimulation and peak firing rate, did not differ significantly between control and DM retinas (P > 0.05 for all four parameters, two-way ANOVA) (Fig. 6C–F). Moreover, no significant difference in response threshold (the intensity generating 5% maximal response) was detected between the two groups (P = 0.699, two-sample Mann-Whitney U test) (Fig. 6G and H). Thus, DM had minor, if any, impact on overall melanopsin-based retinal outputs to higher centers.

Figure 6

DM did not alter overall melanopsin-based retinal outputs. A1–B4: Representative raster plots of spiking activity in response to a series of full-field, 480-nm light pulses with increasing intensity (blue bars), which were obtained from one control (A1–4) and one DM (B1–4) retina through MEA recording. Each trace represents the spike train from a clearly distinguishable single unit identified by offline spike sorting. Photoresponses were recorded in the presence of the glutamatergic cocktail and typical of melanopsin-based activity, being sluggish and persistent. C–F: Group data comparing I–R functions of four major parameters (total spike number during stimulation, peak firing rate, peak latency, and half decay time) of melanopsin-based light response between control and DM retinas. No significant changes in any of these parameters were detected in DM mice. G: Michaelis-Menten equation-fitted I–R curves on the basis of peak firing rate data. H: Box plot showing no significant difference in photoresponse threshold between control and DM retinas. Sample sizes are given in parentheses. Ctrl, control.

Figure 6

DM did not alter overall melanopsin-based retinal outputs. A1–B4: Representative raster plots of spiking activity in response to a series of full-field, 480-nm light pulses with increasing intensity (blue bars), which were obtained from one control (A1–4) and one DM (B1–4) retina through MEA recording. Each trace represents the spike train from a clearly distinguishable single unit identified by offline spike sorting. Photoresponses were recorded in the presence of the glutamatergic cocktail and typical of melanopsin-based activity, being sluggish and persistent. C–F: Group data comparing I–R functions of four major parameters (total spike number during stimulation, peak firing rate, peak latency, and half decay time) of melanopsin-based light response between control and DM retinas. No significant changes in any of these parameters were detected in DM mice. G: Michaelis-Menten equation-fitted I–R curves on the basis of peak firing rate data. H: Box plot showing no significant difference in photoresponse threshold between control and DM retinas. Sample sizes are given in parentheses. Ctrl, control.

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DM Caused Functional Alternations in M2 but Not M1 ipRGCs

The MEA experiment assessed population network activity produced by all ipRGC subtypes and possibly a number of displaced spiking amacrine cells electrically coupled to ipRGCs (27). Therefore, significant DM-induced changes of a particular ipRGC subtype may have been concealed by unaltered responses of other cell types. To explore whether DM modifies melanopsin-based responses of M1/M2 cells to a different extent, we performed whole-cell current clamp recording in individual ipRGCs on whole-mount retinas harvested from STZ-treated Opn4-tdTomato mice (28). In these mice, somata of M1–M3 cells are brightly labeled with the fluorescent protein tdTomato, allowing targeted recording. The neurobiotin tracer was included in the patch pipette for offline confirmation of the identity of M1 and M2 cells on the basis of their distinct soma-dendritic profiles and stratification levels in the IPL (Fig. 7B and E).

Figure 7

DM boosted melanopsin-based photoresponses of surviving M2 ipRGCs and impaired pupillary light reflex. A and D: Representative melanopsin-based light responses of M1 and M2 cells to an intensity series of full-field, 480-nm light pulses recorded in current-clamp mode in control and DM Opn4-tdTomato retinas in the presence of the glutamatergic cocktail. B and E: Confocal photomicrographs of whole-mount retinas showing M1 and M2 ipRGCs with typical dendritic branching characteristics revealed by neurobiotin included in the patch pipette. C and F: Comparison of I–R curves, derived from peak depolarization (Depol.) amplitudes, revealed that the melanopsin-based photoresponse was unchanged in M1 cells but significantly boosted in M2 cells by DM. G: Representative images of pupillary constriction from control and DM mice captured during 463-nm light pulses of various intensities. H: I–R curves of PLRs plotted on the basis of normalized pupil area as a function of light intensity in the control and DM groups. I: Mean light intensity required for half-maximal constriction (EC50) (extracted from the sigmoidal curve fits for each mouse) was significantly increased in the DM group. Scale bar = 100 μm. Sample sizes are given in parentheses. Ctrl, control.

Figure 7

DM boosted melanopsin-based photoresponses of surviving M2 ipRGCs and impaired pupillary light reflex. A and D: Representative melanopsin-based light responses of M1 and M2 cells to an intensity series of full-field, 480-nm light pulses recorded in current-clamp mode in control and DM Opn4-tdTomato retinas in the presence of the glutamatergic cocktail. B and E: Confocal photomicrographs of whole-mount retinas showing M1 and M2 ipRGCs with typical dendritic branching characteristics revealed by neurobiotin included in the patch pipette. C and F: Comparison of I–R curves, derived from peak depolarization (Depol.) amplitudes, revealed that the melanopsin-based photoresponse was unchanged in M1 cells but significantly boosted in M2 cells by DM. G: Representative images of pupillary constriction from control and DM mice captured during 463-nm light pulses of various intensities. H: I–R curves of PLRs plotted on the basis of normalized pupil area as a function of light intensity in the control and DM groups. I: Mean light intensity required for half-maximal constriction (EC50) (extracted from the sigmoidal curve fits for each mouse) was significantly increased in the DM group. Scale bar = 100 μm. Sample sizes are given in parentheses. Ctrl, control.

Close modal

In the presence of the glutamatergic cocktail, a 480-nm full-field light pulse elicited membrane depolarization in an intensity-dependent manner in M1 ipRGCs in both the control and the DM group (Fig. 7A). The I–R curve of the DM group highly mimicked that of the control group (P = 0.600, two-way ANOVA) (Fig. 7C), suggesting unchanged melanopsin phototransduction. By contrast, the melanopsin-based response of M2 ipRGCs was enhanced in the DM group as evidenced by the modest, but significant upward scaling of the I–R curve (P = 0.006, two-way ANOVA) (Fig. 7D and F), suggesting an increased response gain.

M2 cells are known to provide the majority ipRGC input to the olivary pretectal nucleus, the nucleus controlling the PLR (29). Therefore, we examined the PLR using 463-nm light pulses to test whether NIF vision was disturbed when M2 cells were injured by DM. Both control and DM mice showed a light-dependent pupillary constriction, but the I–R curve for the DM group exhibited a significant rightward shift (P = 0.008, two-way ANOVA) (Fig. 7G and H). Consistently, the light intensity to elicit 50% constriction was significantly increased in DM mice (1.630 × 1012 ± 6.190 × 1011 photons/cm2/s vs. 4.998 × 1011 ± 3.531 × 1011 photons/cm2/s, P = 0.003) (Fig. 7I). Thus, hyperglycemia-induced M2 loss attenuated the PLR; the boosted light response and remodeled morphology observed are likely compensatory responses to such cell loss.

DM Did Not Change Melanopsin Expression Levels

For both M1 and M2 cells, the thresholds of melanopsin-based response were not significantly changed by DM (P = 0.888 and 0.678, Mann-Whitney U test) (Supplementary Fig. 3), implying unaltered melanopsin expression levels because the light sensitivity of a photoreceptor is directly proportional to its photopigment concentration (30). Indeed, quantitative Western blot analysis of retinal protein extracts using the PA1-780 melanopsin antibody detected no significant difference between control and DM animals (P = 0.105 for 53-kDa band, P = 0.228 for 85-kDa band) (Supplementary Fig. 4A1 and 2). Moreover, quantifying the UF006 melanopsin antibody immunofluorescence signals in the inner (containing mainly M2 dendrites) and outer (containing mainly M1 dendrites) melanopsin-immunopositive plexuses in the IPL revealed comparable values for both plexuses (P = 0.243 and 0.058 for ON [inner] and OFF [outer] sublamina, respectively) (Supplementary Fig. 4B1–C3).

Prolonged Resistance of M1 Cells

To test whether duration of DM, a chronic condition, would affect the resistance of M1 cells, melanopsin immunostaining using the PA1-780 antibody was conducted on C57BL/6 retinas harvested at 180–195 days after STZ injection. No significant difference in cell number (P = 0.082) or in any major morphological parameters (all P > 0.05) was detected between DM and control mice (Supplementary Figs. 5 and 6). Furthermore, in Opn4-tdTomato retinas, melanopsin-based responses of M1 cells were recorded at 155–180 days after DM induction. No appreciable DM-induced changes in I–R curve (P = 0.479) or response threshold (P = 0.810) were detected (Supplementary Fig. 7). Thus, the resistance of M1 cells seems to be a long-duration capability, lasting for at least 6 months.

Spatial Relationships Between ipRGC Dendrites and Retinal Capillaries Are Cell-Subtype-Dependent

Consistent with previous reports (31,32), the overall vascular densities, quantified by IB4-stained areas, were not significantly changed in 3-month DM retinas (P > 0.05 for all three laminar capillary plexuses) (Fig. 8A1 and 2), suggesting no gross vascular remodeling. We also assessed the local density of superficial capillary plexuses, which nourish the GCL (33), surrounding individual cell bodies. No significant difference was found among the four groups (control-M1, control-M2, DM-M1, and DM-M2) analyzed (P = 0.128, Kruskal-Wallis test) (Fig. 8B1–3), ruling out the possibility that the soma of a specific subtype is more heavily surrounded by blood vessels. However, measurement of distances between melanopsin immunofluorescence peaks and IB4 peak demonstrated that the intermediate capillary plexus, which nourishes the IPL (33), was more adjacent to the OFF melanopsin-positive plexus than to the ON one in both control and DM mice (both P < 0.0001, Holm-Šídák multiple comparison test), but DM did not change either ON melanopsin–IB4 or OFF melanopsin–IB4 peak distance (both P = 0.959, Holm-Šídák multiple comparison test) (Fig. 8C1–5). Thus, M1 dendrites, residing in the outer OFF melanopsin plexus, are closer to intermediate capillaries than M2 dendrites. Therefore, M1 cells might suffer less from perturbed supply caused by reduced retinal blood flow at early DM (34), resulting in the resistance capability.

Figure 8

Cell-subtype-specific spatial relationship between ipRGC dendritic arbors and retinal capillaries. A1: Representative photomicrographs of IB4 staining in control and DM retinas, revealing three (superficial, intermediate, and deep) capillary plexuses. A2: Bar graphs showing that the overall vascular density, determined by measuring IB4-positive area, was not changed by DM in any of the three capillary plexuses. B1 and B2: Representative collapsed confocal stacks of whole-mount retinas double-stained with UF006 melanopsin antibody and IB4 from control and DM mice, showing the spatial relationship between somata of M1/M2 cells (identified by differentially stratified dendrites) and the superficial capillary plexus. B3: The density of capillaries surrounding individual ipRGCs, calculated by measuring IB4-positive area size in an 83-μm diameter region centered at each soma (dashed circles in B1 and B2), is similar among all four experimental groups. C1 and C3: Digitally rotated side views of representative confocal z-stacks showing ON (inner) and OFF (outer) melanopsin-positive plexuses in relation to IB4-labeled intermediate capillary plexus. Numbers denote relative depth of the IPL (GCL = 0, inner nuclear layer = 100). C2 and C4: Fluorescence intensity profiles, averaged from dozens of images as C1 and C3, of melanopsin and IB4 signals versus normalized (Norm.) depth of the IPL. C5: Bar graph showing that the average distance between fluorescence peaks of OFF melanopsin plexus and IB4 plexus is much shorter than ON melanopsin–IB4 peak distance in both control and DM retinas; for both of the two distances, the DM group is not different from control. Scale bars = 40 μm in A1 and 30 μm in B2 and C3. Sample sizes are given in parentheses. Ctrl, control; Mel, melanopsin.

Figure 8

Cell-subtype-specific spatial relationship between ipRGC dendritic arbors and retinal capillaries. A1: Representative photomicrographs of IB4 staining in control and DM retinas, revealing three (superficial, intermediate, and deep) capillary plexuses. A2: Bar graphs showing that the overall vascular density, determined by measuring IB4-positive area, was not changed by DM in any of the three capillary plexuses. B1 and B2: Representative collapsed confocal stacks of whole-mount retinas double-stained with UF006 melanopsin antibody and IB4 from control and DM mice, showing the spatial relationship between somata of M1/M2 cells (identified by differentially stratified dendrites) and the superficial capillary plexus. B3: The density of capillaries surrounding individual ipRGCs, calculated by measuring IB4-positive area size in an 83-μm diameter region centered at each soma (dashed circles in B1 and B2), is similar among all four experimental groups. C1 and C3: Digitally rotated side views of representative confocal z-stacks showing ON (inner) and OFF (outer) melanopsin-positive plexuses in relation to IB4-labeled intermediate capillary plexus. Numbers denote relative depth of the IPL (GCL = 0, inner nuclear layer = 100). C2 and C4: Fluorescence intensity profiles, averaged from dozens of images as C1 and C3, of melanopsin and IB4 signals versus normalized (Norm.) depth of the IPL. C5: Bar graph showing that the average distance between fluorescence peaks of OFF melanopsin plexus and IB4 plexus is much shorter than ON melanopsin–IB4 peak distance in both control and DM retinas; for both of the two distances, the DM group is not different from control. Scale bars = 40 μm in A1 and 30 μm in B2 and C3. Sample sizes are given in parentheses. Ctrl, control; Mel, melanopsin.

Close modal

Cell-Subtype-Specific Susceptibility

Susceptibility to a specific neuronal injury can differ substantially among closely related GC subtypes. A well-known example is α-GCs. When subjected to experimental glaucoma, transient OFF-type α-GCs exhibit higher death rates and much more dramatic morphological-physiological remodeling than the other α-GC subtypes (35,36). In the R6/2 Huntington disease mouse model, M1 ipRGCs undergo more severe apoptosis than non-M1 ipRGCs (20). In axotomized mouse retinas, >70% of M1 cells survive, whereas very few M2 cells are spared (4). Deciphering the mechanisms underlying such differential vulnerabilities may contribute to developing novel diagnostic and neuroprotective strategies against various optic neuropathies.

Two previous studies, one conducted in mice (17) and the other in rats (16), reported no significant loss of melanopsin-stained ipRGCs at 3 months postonset of DM. These studies made no attempt to discriminate ipRGC subtypes, but the total numbers of melanopsin-positive cells revealed in them (∼600–700 in mice [17] and 1,000–1,200 in rats [16]) were much smaller than those of the entire ipRGC population (M1–M6) (19,37). Therefore, ipRGCs described in these works might correspond to M1 cells, which have the highest melanopsin levels among all ipRGC subtypes; our finding that severe apoptosis did not occur in M1 ipRGCs is consistent with the findings of both studies.

The vulnerability of GCs to certain retinal diseases may depend on their dendritic stratification levels in the IPL. There is now mounting evidence that GCs ramifying in the vitreal half of the IPL are more susceptible at the early stage of hyperglycemia. In STZ-induced DM mice, significantly increased spontaneous activity was observed in ON-type, but not in OFF-type, GCs (13); morphological parameters and electrophysiological properties were also preferentially affected in ON-type GCs (12). These findings are indeed consistent with our observation that M2/M3 cells, which have dendritic fields in the ON sublamina, were remodeled, whereas M1 cells, which are functionally ON-type but stratify mainly in the OFF sublamina, were not.

Morphological and physiological alternations have been observed in mouse ON α-GCs (ON RGA2), which are virtually M4 cells (38), in STZ-induced DM mice (12). In the current study, just as previously reported (21), an array of large, weakly melanopsin-immunopositive somata characteristic of M4 cells could be revealed under higher magnification (Supplementary Fig. 8A). The density of these M4-like cells was significantly lower in the DM than in the control group (P = 0.002) (Supplementary Fig. 8B), suggesting DM-induced cell loss. Thus, it is likely that M4, another ON cell, is significantly affected by DM. It is of particular interest that for the bistratified M3 ipRGCs, ON dendritic arbors were more vulnerable to DM than OFF arbors. Similar laminar specificities were previously seen in ON-OFF direction-selective GCs (RGD2 GCs) in STZ-treated mice (12), whereas in experimental glaucoma, which preferentially affects OFF-type GCs, the opposite specificity was observed in ON-OFF GCs (36).

Functional Implications

At first glance, the boosted melanopsin-based light response in M2 ipRGCs in 3-month DM mice seems at odds with the impaired PLR observed because increased ipRGC activity enhances rather than suppresses pupillary constriction (39). However, the modestly boosted response, together with the increased dendritic size/branching, might serve as a compensatory response to the loss of M2s, thereby increasing the strength of survived M2 driving forces to NIF visual centers. In mice with ipRGCs partially ablated, the PLR was attenuated at lower but not higher light intensity (16,17), a phenotype similar to that seen in the DM mice. Our PLR results are also in line with two previous DM studies, which showed that the rat PLR and mouse circadian entrainment in response to lower irradiance were selectively impaired (16,17).

In the current study, melanopsin protein expression levels were found to be unaltered in 3-month DM mice. This finding is consistent with that of a rat study in which 15 weeks of DM induction did not affect melanopsin protein levels (16) and is further supported by electrophysiological results that the melanopsin-based photoresponse threshold, determined primarily by melanopsin expression (40), was unchanged by DM. This suggests that the enhanced melanopsin-based activity of M2 ipRGCs in DM mice was caused by abnormalities in downstream effectors of melanopsin phototransduction and/or intrinsic membrane properties of M2 cells rather than by an upregulation of photopigment levels. However, in an earlier work, increased melanopsin mRNA levels were detected in mice at 4 weeks after STZ injection (15), which seemingly conflicts with those of the current study. Increased melanopsin transcript expression may not have been transferred to final protein products. Alternatively, melanopsin protein levels in STZ-treated mice may have increased only temporarily.

ipRGCs are driven by not only melanopsin but also rod/cone photoreceptors (24,41), which play an important role in ipRGC photoresponses and NIF vision under lower irradiance. Various second-order neurons are known to be injured in DM animals (42,43). Since many of these interneurons form synapses with ipRGCs (41), their loss or lesion is likely to impair rod/cone-driven responses of ipRGCs, leading to NIF visual function abnormalities under lower irradiance. Further studies are needed to better understand the effect of DM on this rod/cone-driven component.

Mechanisms Underlying the Resistance Ability of M1 Cells

Although diabetic retinopathy is manifested by microvascular damages, gross retinal vascular structure in rodents are largely intact at the early DM stage (31,32), and neuronal injures might not be associated with severe disruption of the blood-retina barrier, which is seen at the advanced stage. However, recent evidence has shown early dysfunction of retinal hemodynamics in DM rodents (34). Since the retina is a highly metabolically demanding organ, even small changes in blood supply may lead to lesion of neurons. Although the somata of M1 and M2 cells are surrounded by capillaries of similar density, dendritic arbors of M1s are spatially much closer to the intermediate capillary plexus than M2s (Fig. 8). This advantage may make M1s less affected by impaired supply, providing a mechanism underlying its resistance ability, and is consistent with the result that the ON dendrites were more severely remodeled than the OFF dendrites in M3s (Fig. 5).

A number of other factors may contribute to the resistance ability of M1 ipRGCs. Intercellular electrical communications contribute substantially to neuronal injury and even cause secondary cell death (44). Among all known ipRGC subtypes, M1s are the most weakly coupled (4,45). Furthermore, Cx30.2, a connexin closely associated with retinal cell survival under hyperglycemic conditions (46), seems to be expressed by M2 cells but not M1 cells (44,47). In sum, their relatively weak coupling capacity and lack of injury-promoting connexin channels may render M1 ipRGCs less vulnerable to diabetic retinopathy. Besides, cell-subtype-specific remodeling may also result from the distinct central targets of different ipRGC subtypes. M1 and M2 cells project to a series of higher centers, and not all of them are overlapped (18,48). M1 cells may be supported by extra neurotrophins from their specific targets (devoid of M2 axons), an advantage over M2 cells that enables their resistance against DM.

This article contains supplementary material online at https://doi.org/10.2337/figshare.13724092.

W.-Y.C. and X.H. contributed equally to this work.

Acknowledgments. The authors thank Dr. Tian Xue (University of Science and Technology of China) for kindly providing the Opn4-tdTomato mice and Wen-Hao Chen (Fudan University) and Wei Zhou (Fudan University) for insightful discussion of the manuscript.

Funding. This work was supported by the National Natural Science Foundation of China (81790640, 82070993, 31571072, 32070989, 31872766, 31571075, 81430007, and 81470661), the Ministry of Science and Technology of China (2011CB504602 and 2015AA020512), Shanghai Municipal Science and Technology Major Project (No. 2018SHZDZX01), ZJLab, and Sanming Project of Medicine in Shenzhen (SZSM202011015).

Duality of Interest. No potential conflicts of interest relevant to this article were reported.

Author Contributions. W.-Y.C., X.H., L.-J.C., C.-X.Y., W.-L.S., and J.Y. performed the experiments. W.-Y.C., X.H., C.-X.Y., F.Y., Y.-M.Z., and S.-J.W. analyzed and interpreted the data. W.-Y.C., X.H., and S.-J.W. wrote the manuscript. Y.-M.Z., X.-L.Y., and S.-J.W. designed the experiment. All authors read and approved the final manuscript. W.-Y.C. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

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