Shi-Jun Weng

Unaltered retinal dopamine levels in a C57BL/6 mouse model of form-deprivation myopia.

PURPOSE Retinal dopamine has been long implicated in the signaling pathway regulating eye growth, as evidenced by its reduced levels in myopic eyes in various species. We examined whether and how retinal dopamine levels were changed in a C57BL/6 mouse model of experimental myopia. METHODS Form-deprivation myopia (FDM) was induced in C57BL/6 mice by wearing monocular occluder for 4 weeks. Refractive errors were measured using an infrared photorefractor. Retinal dopamine/DOPAC and vitreal DOPAC levels were assessed by high-performance liquid chromatography (HPLC). Extracellular dopamine concentrations were examined by Western blot analysis of dopamine transporter (DAT) expression levels. The intactness of retinal dopaminergic system was evaluated by counting tyrosine hydroxylase (TH) immunoreactive cells, measuring the areas occupied by processes of these cells, and quantifying retinal TH expression at both protein and transcription levels. RESULTS Form-deprivation myopia was successfully induced in C57BL/6 mice with the refractive status of deprived eyes being significantly different from fellow eyes. Unlike most of the previous results obtained in other myopic animal models, however, no significant changes in retinal dopamine, DOPAC, DAT, and vitreal DOPAC levels were detected in deprived eyes, either in the daytime or at night. Furthermore, neither the number of dopaminergic amacrine cells, the area size occupied by the processes of these cells, nor retinal TH expression, were altered in deprived eyes. CONCLUSIONS The retinal dopamine system remains intact in C57BL/6 mice with FDM, and retinal dopamine levels are not associated with the development of FDM in this mouse strain.

Orexin-A differentially modulates AMPA-preferring responses of ganglion cells and amacrine cells in rat retina

By activating their receptors (OX1R and OX2R) orexin-A/B regulate wake/sleeping states, feeding behaviors, but the function of these peptides in the retina remains unknown. Using patch-clamp recordings and calcium imaging in rat isolated retinal cells, we demonstrated that orexin-A suppressed α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)-preferring receptor-mediated currents (AMPA-preferring currents) in ganglion cells (GCs) through OX1R, but potentiated those in amacrine cells (ACs) through OX2R. Consistently, in rat retinal slices orexin-A suppressed light-evoked AMPA-preferring receptor-mediated excitatory postsynaptic currents in GCs, but potentiated those in ACs. Intracellular dialysis of GDP-β-S or preincubation with the Gi/o inhibitor pertussis toxin (PTX) abolished both the effects. Either cAMP/the protein kinase A (PKA) inhibitor Rp-cAMP or cGMP/the PKG blocker KT5823 failed to alter the orexin-A effects. Whilst both of them involved activation of protein kinase C (PKC), the effects on GCs and ACs were respectively eliminated by the phosphatidylinositol (PI)-phospholipase C (PLC) inhibitor and phosphatidylcholine (PC)-PLC inhibitor. Moreover, in GCs orexin-A increased [Ca(2+)]i and the orexin-A effect was blocked by intracellular Ca(2+)-free solution and by inositol 1,4,5-trisphosphate (IP3) receptor antagonists. In contrast, orexin-A did not change [Ca(2+)]i in ACs and the orexin-A effect remained in intracellular or extracellular Ca(2+)-free solution. We conclude that a distinct Gi/o/PI-PLC/IP3/Ca(2+)-dependent PKC signaling pathway, following the activation of OX1R, is likely responsible for the orexin-A effect on GCs, whereas a Gi/o/PC-PLC/Ca(2+)-independent PKC signaling pathway, following the activation of OX2R, mediates the orexin-A effect on ACs. These two actions of orexin-A, while working in concert, provide a characteristic way for modulating information processing in the inner retina.

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.

Hyperactivity of ON-Type Retinal Ganglion Cells in Streptozotocin-Induced Diabetic Mice

Impairment of visual function has been detected in the early stage of diabetes but the underlying neural mechanisms involved are largely unknown. Morphological and functional alterations of retinal ganglion cells, the final output neurons of the vertebrate retina, are thought to be the major cause of visual defects in diabetes but direct evidence to support this notion is limited. In this study we investigated functional changes of retinal ganglion cells in a type 1-like diabetic mouse model. Our results demonstrated that the spontaneous spiking activity of ON-type retinal ganglion cells was increased in streptozotocin-diabetic mice after 3 to 4 months of diabetes. At this stage of diabetes, no apoptotic signals or cell loss were detected in the ganglion cell layer of the retina, suggesting that the functional alterations in ganglion cells occur prior to massive ganglion cell apoptosis. Furthermore, we found that the increased activity of ON-type ganglion cells was mainly a result of reduced inhibitory signaling to the cells in diabetes. This novel mechanism provides insight into how visual function is impaired in diabetic retinopathy.

Co-expression of two subtypes of melatonin receptor on rat M1-type intrinsically photosensitive retinal ganglion cells.

Intrinsically photosensitive retinal ganglion cells (ipRGCs) are involved in circadian and other non-image forming visual responses. An open question is whether the activity of these neurons may also be under the regulation mediated by the neurohormone melatonin. In the present work, by double-staining immunohistochemical technique, we studied the expression of MT1 and MT2, two known subtypes of mammalian melatonin receptors, in rat ipRGCs. A single subset of retinal ganglion cells labeled by the specific antibody against melanopsin exhibited the morphology typical of M1-type ipRGCs. Immunoreactivity for both MT1 and MT2 receptors was clearly seen in the cytoplasm of all labeled ipRGCs, indicating that these two receptors were co-expressed in each of these neurons. Furthermore, labeling for both the receptors were found in neonatal M1 cells as early as the day of birth. It is therefore highly plausible that retinal melatonin may directly modulate the activity of ipRGCs, thus regulating non-image forming visual functions.

Signaling mechanism for modulation by ATP of glycine receptors on rat retinal ganglion cells.

ATP modulates voltage- and ligand-gated channels in the CNS via the activation of ionotropic P2X and metabotropic P2Y receptors. While P2Y receptors are expressed in retinal neurons, the function of these receptors in the retina is largely unknown. Using whole-cell patch-clamp techniques in rat retinal slice preparations, we demonstrated that ATP suppressed glycine receptor-mediated currents of OFF type ganglion cells (OFF-GCs) dose-dependently, and the effect was in part mediated by P2Y1 and P2Y11, but not by P2X. The ATP effect was abolished by intracellular dialysis of a Gq/11 protein inhibitor and phosphatidylinositol (PI)-phospholipase C (PLC) inhibitor, but not phosphatidylcholine (PC)-PLC inhibitor. The ATP effect was accompanied by an increase in [Ca2+]i through the IP3-sensitive pathway and was blocked by intracellular Ca2+-free solution. Furthermore, the ATP effect was eliminated in the presence of PKC inhibitors. Neither PKA nor PKG system was involved. These results suggest that the ATP-induced suppression may be mediated by a distinct Gq/11/PI-PLC/IP3/Ca2+/PKC signaling pathway, following the activation of P2Y1,11 and other P2Y subtypes. Consistently, ATP suppressed glycine receptor-mediated light-evoked inhibitory postsynaptic currents of OFF-GCs. These results suggest that ATP may modify the ON-to-OFF crossover inhibition, thus changing action potential patterns of OFF-GCs.

Signalling mechanism for somatostatin receptor 5-mediated suppression of AMPA responses in rat retinal ganglion cells

Somatostatin (SRIF) is involved in a variety of physiological functions via the activation of five subtypes of specific receptors (sst1-5). Here, we investigated the effects of SRIF on AMPA receptor (AMPAR)-mediated currents (AMPA currents) in isolated rat retinal ganglion cells (GCs) using patch-clamp techniques. Immunofluorescence double labelling demonstrated the expression of sst5 in rat GCs. Consistent to this, whole cell AMPA currents of GCs were dose-dependently suppressed by SRIF, and the effect was reversed by the sst5 antagonist BIM-23056. Intracellular dialysis of GDP-β-S or pre-incubation with the Gi/o inhibitor pertussis toxin (PTX) abolished the SRIF effect. The SRIF effect was mimicked by the administration of either 8-Br-cAMP or forskolin, but was eliminated by the protein kinase A (PKA) antagonists H-89/KT5720/Rp-cAMP. Moreover, SRIF increased intracellular Ca(2+) levels and did not suppress the AMPA currents when GCs were infused with an intracellular Ca(2+)-free solution or in the presence of ryanodine receptor modulators caffeine/ryanodine. Furthermore, the SRIF effect was eliminated when the activity of calmodulin (CaM), calcineurin and protein phosphatase 1 (PP1) was blocked with W-7, FK-506 and okadaic acid, respectively. SRIF persisted to suppress the AMPA currents when cGMP-protein kinase G (PKG) and phosphatidylinositol (PI)-/phosphatidylcholine (PC)-phospholipase C (PLC) signalling pathways were blocked. In rat flat-mount retinas, SRIF suppressed AMPAR-mediated light-evoked excitatory postsynaptic currents (L-EPSCs) in GCs. We conclude that a distinct Gi/o/cAMP-PKA/ryanodine/Ca(2+)/CaM/calcineurin/PP1 signalling pathway comes into play due to the activation of sst5 to mediate the SRIF effect on GCs.

Functional Assessment of Melanopsin-Driven Light Responses in the Mouse: Multielectrode Array Recordings

Intrinsically photosensitive retinal ganglion cells (ipRGCs) are a special subset of retinal output neurons capable of detecting and responding to light via a unique photopigment called melanopsin. Melanopsin activation is essential to a wide array of physiological functions, especially to those related to non-image-forming vision. Since ipRGCs only constitute a very small proportion of retinal ganglion cells, targeted recording of melanopsin-driven responses used to be a big challenge to vision researchers. Multielectrode array (MEA) recording provides a noninvasive, high throughput method to monitor melanopsin-driven responses. When synaptic inputs from rod/cone photoreceptors are silenced with glutamatergic blockers, extracellular electric signals derived from melanopsin activation can be recorded from multiple ipRGCs simultaneously by tens of microelectrodes aligned in an array. In this chapter we describe how our labs have approached MEA recording of melanopsin-driven light responses in adult mouse retinas. Instruments, tools and chemical reagents routinely used for setting up a successful MEA recording are listed, and a standard experimental procedure is provided. The implementation of this technique offers a useful paradigm that can be used to conduct functional assessments of ipRGCs and NIF vision.

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.