Guo-Zhong Xu

Gene expression and protein distribution of orexins and orexin receptors in rat retina.

Orexins, composed of orexin A and orexin B, are identified as endogenous ligands of two orphan G-protein-coupled receptors: orexin 1 and orexin 2 receptors (OX1R and OX2R). Orexins are implicated in regulating wake/sleep states, feeding behaviors, etc. Using reverse transcription-polymerase chain reactive (RT-PCR) analysis and immunofluorescence double labeling, we investigated the distributions of orexin A, orexin B, OX1R and OX2R in rat retina. RT-PCR analysis revealed the presence of mRNAs of prepro-orexin, OX1R and OX2R in rat retina. Immunostaining for orexin A and orexin B was observed in many cells in the inner nuclear layer and the ganglion cell layer. In the outer retina, horizontal cells, labeled by calbindin, and bipolar cells, labeled by homeobox protein Chx10, were orexin A- and orexin B-positive. In the inner retina, two orexins were both found in GABAergic amacrine cells (ACs), including dopaminergic and cholinergic ones, stained by tyrosine hydroxylase and choline acetyltransferase respectively. Glycinergic ACs, including AII ACs, also expressed orexins. Weak to moderate labeling for orexin A and orexin B was diffusely distributed in the inner plexiform layer. Additionally, orexins were expressed in almost all ganglion cells (GCs) retrogradely labeled by cholera toxin B subunit. Specifically, double-labeling experiments demonstrated that melanopsin-positive GCs (intrinsically photosensitive retinal GCs, ipRGCs) were labeled by two orexins. Morever, OX1R immunoreactivity was observed in most of GCs and all dopaminergic ACs, as well as in both outer and inner plexiform layers. In contrast, no obvious OX2R immunostaining was detectable in the rat retina. These results suggest that orexins may modulate the function of neurons, especially in the inner retina. We further hypothesize that the orexin signaling via ipRGCs may be involved in setting the suprachiasmatic nucleus (SCN) circadian clock.

The Role of Retinal Dopamine in C57BL/6 Mouse Refractive Development as Revealed by Intravitreal Administration of 6-Hydroxydopamine.

Purpose Although retinal dopamine (DA) has been long implicated in myopia development, current studies demonstrate that retinal DA levels are unaltered in C57BL/6 mice with form-deprivation myopia. This work was undertaken to explore whether and how refractive development is perturbed in this mouse strain when retinal DA levels are reduced by 6-hydroxydopamine (6-OHDA) administration. Methods On two successive days, 6-OHDA was injected into the vitreous of P18 mice. Retinal DA levels were measured by HPLC and TH levels analyzed by quantitative Western blotting. To choose appropriate 6-OHDA doses that significantly reduce retinal DA levels, but cause minimal disturbance of overall retinal physiology, ERG analysis was performed. Refractive errors were measured using a photorefractor, and ocular biometry performed with optical coherence tomography and photokeratometry. Results Administration of 6-OHDA of 6.25 μg and 12.5 μg significantly reduced retinal levels of DA and TH, but without affecting ERG a- and b-wave amplitudes. With normal visual experience, 6-OHDA induced myopic refractive shifts in a dose-dependent fashion. Form deprivation induced further myopic shifts in 6-OHDA-injected eyes, but did not cause further decline in retinal DA. Furthermore, 6-OHDA administration resulted in a shorter axial length and a steeper cornea, whereas form deprivation led to a longer axial length, without changing the corneal radius of curvature. Conclusions Reducing retinal DA levels led to myopic refractive shifts in C57BL/6 mice, which mainly resulted from a steeper cornea. In addition to the DA-independent mechanism for form-deprivation myopia, there is a DA-dependent mechanism in parallel that underlies myopic refractive shifts under normal laboratory conditions in this mouse strain.

Natriuretic peptide receptors are expressed in rat retinal ganglion cells.

Natriuretic peptides (NPs) exert their actions through three membrane-bound receptors, which are known as NP receptors (NPRs: NPR-A, NPR-B and NPR-C). In this work we examined the expression of three NPRs in rat retinal ganglion cells (GCs), retrogradely labeled and intracellularly dye-injected, by double immunofluorescence labeling. In vertical sections, almost all GCs, retrogradely labeled by cholera toxin B, were stained by antibodies against the three NPRs. The labeling for three NPRs was observed mainly on the membranes of the somata of GCs, whereas the staining for NPR-A was also seen in the cytoplasm. Moreover, with tangential sections, almost all cells located in the ganglion cell layer were NPR-A, B, C immunoreactive. By combining with intracellular injection of Neurobiotin into GCs in whole mount retinas that enables to identify ON-, OFF- and ON-OFF-types of GCs according to arborization of their dendrites in the inner plexiform layer, we further demonstrated that NPRs were expressed in these major types of GCs.

Orexin-B modulates synaptic transmission of rod bipolar cells in rat retina

ABSTRACT Orexin‐A, ‐B play a crucial role in arousal and feeding by activating two G‐protein‐coupled receptors: orexin receptor 1 (OX1R) and orexin receptor 2 (OX2R). Orexins, along with orexin receptors, are expressed in retinal neurons, and they have been shown to differentially modulate excitatory AMPA receptors of amacrine and ganglion cells in the inner retina. In this work we report that orexin‐B modulates the activity of rod bipolar cells (RBCs) located in the outer retina of rat. Intravitreal injection of orexin‐B increased the amplitude of the scotopic electroretinographic b‐wave, a reflection of RBC activity, recorded in vivo. Patch clamp recordings in rat retinal slices showed that orexin‐B did not change glutamatergic excitatory component of the RBC response driven by photoreceptors. Effects of orexin‐B on GABA receptor‐mediated synaptic transmission of RBCs were then examined. In retinal slice preparations orexin‐B suppressed GABA receptor‐mediated inhibitory postsynaptic currents of RBCs in the inner plexiform layer. Furthermore, using whole‐cell recordings in isolated RBCs it was shown that orexin‐B suppressed GABAC receptor‐, but not GABAA receptor‐, mediated currents of the RBCs, an effect that was blocked by OX1R and OX2R antagonists. The orexin‐B‐induced inhibition of GABAC currents was likely mediated by a Gi/o/PC‐PLC/Ca2+‐independent PKC signaling pathway, as such inhibition was absent when each step of the above‐pathway was blocked with GDP‐&bgr;‐S/pertussis toxin (for Gi/o), D609 (for PLC), bisindolylmaleimide IV (for PKC)/rottlerin (for PKC&dgr;), respectively. The orexin‐B‐induced potentiation of RBC activity may improve visual acuity and contrast sensitivity of the animal during the dark period (wake phase). HighlightsOrexin‐B potentiates the rod‐driven responses of rat retinal bipolar cells (BCs).Such modulation is induced by reducing GABAergic feedback from amacrine cells to BCs.Orexin‐B suppresses GABAergic inhibitory postsynaptic currents (IPSCs) of RBCs.Orexin‐B suppresses GABA currents of RBCs via OX1R and OX2R activation.Gi/o/PC‐PLC/Ca2+‐independent PKC signaling pathway mediates the orexin effect on BCs.

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.