Altered Retinal Dopamine Levels in a Melatonin-proficient Mouse Model of Form-deprivation Myopia.

Reduced levels of retinal dopamine, a key regulator of eye development, are associated with experimental myopia in various species, but are not seen in the myopic eyes of C57BL/6 mice, which are deficient in melatonin, a neurohormone having extensive interactions with dopamine. Here, we examined the relationship between form-deprivation myopia (FDM) and retinal dopamine levels in melatonin-proficient CBA/CaJ mice. We found that these mice exhibited a myopic refractive shift in form-deprived eyes, which was accompanied by altered retinal dopamine levels. When melatonin receptors were pharmacologically blocked, FDM could still be induced, but its magnitude was reduced, and retinal dopamine levels were no longer altered in FDM animals, indicating that melatonin-related changes in retinal dopamine levels contribute to FDM. Thus, FDM is mediated by both dopamine level-independent and melatonin-related dopamine level-dependent mechanisms in CBA/CaJ mice. The previously reported unaltered retinal dopamine levels in myopic C57BL/6 mice may be attributed to melatonin deficiency.

Cell-Subtype-Specific Remodeling of Intrinsically Photosensitive Retinal Ganglion Cells in Streptozotocin-Induced Diabetic Mice.

Recent evidence suggests that melanopsin-expressing intrinsically photosensitive retinal ganglion cells (ipRGCs), a neuronal class regulating nonimage 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.

nGnG Amacrine Cells and Brn3b-negative M1 ipRGCs are Specifically Labeled in the ChAT-ChR2-EYFP Mouse.

Purpose: Experimental access to specific cell subtypes is essential for deciphering the complexity of retinal networks. Here, we characterized the selective labeling, caused by ectopic transgene expression, of two atypical retinal neurons in the ChAT-Channelrhodopsin-2 (ChR2)-EYFP mouse. Methods: Retinal sections and flat-mounts were prepared for double-staining immunohistochemistry with antibodies against EYFP and various neuronal markers. Sagittal/coronal brain slices were made to visualize EYFP signals in central nuclei. Whole-cell recordings were conducted to test the functionality of ChR2. Results: Two populations of EYFP-positive retinal cells were observed. The inner nuclear layer (INL)-located one (type I cell) distributed regularly throughout the entire retina, whereas the ganglion cell layer (GCL)-residing one (type II cell) was restricted ventrally. None of them was cholinergic, as evidenced by the complete absence of ChAT immunoreactivity. Type I cells were immunolabeled by the amacrine marker syntaxin. However, the vast majority of them were neither positive to GABA/GAD65, nor to GlyT1/glycine, suggesting that they were non-GABAergic non-glycinergic amacrine cells (nGnG ACs), which was confirmed by double-labeling with the nGnG AC marker PPP1R17. Type II cells were immunopositive to melanopsin, but not to Brn3a or Brn3b. They possessed dendrites stratifying in the outermost inner plexiform layer (IPL) and axons projecting to the suprachiasmatic nucleus (SCN) rather than the olivary pretectal nucleus (OPN), suggesting that they belonged to a Brn3b-negative subset of M1-type intrinsically photosensitive retinal ganglion cells (ipRGCs). Glutamatergic transmission-independent photocurrents were elicited in EYFP-positive cells, indicating the functional expression of ChR2. Conclusions: The ChAT-ChR2-EYFP retina exhibits ectopic, but functional, transgene expression in nGnG ACs and SCN-innervating M1 ipRGCs, thus providing an ideal tool to achieve efficient labeling and optogenetic manipulation of these cells.

Transgene is specifically and functionally expressed in retinal inhibitory interneurons in the VGAT-ChR2-EYFP mouse.

Ectopic transgene expression in the retina has been reported in various transgenic mice, indicating the importance of characterizing retinal phenotypes. We examined transgene expression in the VGAT-ChR2-EYFP mouse retina by fluorescent immunohistochemistry and electrophysiology, with special emphasis on enhanced yellow fluorescent protein (EYFP) localization in retinal neuronal subtypes identified by specific markers. Strong EYFP signals were detected in both the inner and outer plexiform layers. In addition, the ChR2-EYFP fusion protein was also expressed in somata of the great majority of inhibitory interneurons, including horizontal cells and GABAergic and glycinergic amacrine cells. However, a small population of amacrine cells residing in the ganglion cell layer were not labeled by EYFP, and a part of them were cholinergic ones. In contrast, no EYFP signal was detected in the somata of retinal excitatory neurons: photoreceptors, bipolar and ganglion cells, as well as Müller glial cells. When glutamatergic transmission was blocked, bright blue light stimulation elicited inward photocurrents from amacrine cells, as well as post-synaptic inhibitory currents from ganglion cells, suggesting a functional ChR2 expression. The VGAT-ChR2-EYFP mouse therefore could be a useful animal model for dissecting retinal microcircuits when targeted labeling and/or optogenetic manipulation of retinal inhibitory neurons are required.