ABSTRACT
ABSTRACT: Diabetic retinopathy is a prominent cause of blindness in adults, with early retinal ganglion cell (RGC) loss contributing to visual dysfunction or blindness. In the brain, defects in y-aminobutyric acid (GABA) synaptic transmission are associated with pathophysiological and neurodegenerative disorders, whereas glucagon-like peptide-1 (GLP-1) has demonstrated neuroprotective effects. However, it is not yet clear whether diabetes causes alterations in inhibitory input to RGCs and whether and how GLP-1 protects against neurodegeneration in the diabetic retina through regulating inhibitory synaptic transmission to RGCs. In the present study, we used the patch-clamp technique to record GABA subtype A receptor-mediated miniature inhibitory postsynaptic currents (mIPSCs) in RGCs from streptozotocin-induced diabetes model rats. We found that early diabetes (4 weeks of hyperglycemia) decreased the frequency of GABAergic mIPSCs in RGCs without altering their amplitude, suggesting a reduction in the spontaneous release of GABA to RGCs. Topical administration of GLP-1 eyedrops over a period of 2 weeks effectively countered the hyperglycemia-induced downregulation of GABAergic mIPSC frequency, subsequently enhancing the survival of RGCs. Concurrently, the protective effects of GLP-1 on RGCs in diabetic rats were eliminated by topical administration of exendin-9-39, a specific GLP-1 receptor antagonist, or SR95531, a specific antagonist of the GABA subtype A receptor. Furthermore, extracellular perfusion of GLP-1 was found to elevate the frequencies of GABAergic mIPSCs in both ON- and OFF-type RGCs. This elevation was shown to be mediated by activation of the phosphatidylinositol-phospholipase C/inositol 1,4,5-trisphosphate receptor/Ca2+/protein kinase C signaling pathway downstream of GLP-1 receptor activation. Moreover, multielectrode array recordings revealed that GLP-1 functionally augmented the photoresponses of ON-type RGCs. Optomotor response tests demonstrated that diabetic rats exhibited reductions in visual acuity and contrast sensitivity that were significantly ameliorated by topical administration of GLP-1. These results suggest that GLP-1 facilitates the release of GABA onto RGCs through the activation of GLP-1 receptor, leading to the de-excitation of RGC circuits and the inhibition of excitotoxic processes associated with diabetic retinopathy. Collectively, our findings indicate that the GABA system has potential as a therapeutic target for mitigating early-stage diabetic retinopathy. Furthermore, the topical administration of GLP-1 eyedrops represents a non-invasive and effective treatment approach for managing early-stage diabetic retinopathy.
ABSTRACT
Progressive damage of retinal ganglion cells (RGCs) is observed in early diabetic retinopathy. Intracellular Ca2+ overload mediated by Ca2+ influx through voltage-gated Ca2+ channels (VGCCs) is involved in neurodegeneration, whereas glucagon-like peptide-1 (GLP-1) provides neuroprotection. However, whether GLP-1 plays a neuroprotective role in diabetic retinas by modulating VGCCs remains unknown. We found that eye drops of exendin-4, a long-acting GLP-1 receptor (GLP-1R) agonist, prevented the increase of L-type Ca2+ current (ILCa) densities of RGCs induced by 4-week hyperglycemia and promoted RGC survival by suppressing L-type VGCC (L-VGCC) activity in streptozotocin-induced diabetic rats. Moreover, exendin-4-induced suppression of ILCa in RGCs may be mediated by a GLP-1R/Gs/cAMP-PKA/ryanodine/Ca2+/calmodulin/calcineurin/PP1 signaling pathway. Furthermore, exendin-4 functionally improved the light-evoked spiking ability of diabetic RGCs. These results suggest that GLP-1R activation enhances cAMP to PP1 signaling and that PP1 inactivates L-VGCCs by dephosphorylating them, thereby reducing Ca2+ influx, which could protect RGCs against excitotoxic Ca2+ overload.
ABSTRACT
Light modulates mood through various retina-brain pathways. We showed that mice treated with short-term acute bright light exposure displayed anxiety-related phenotypes in a prolonged manner even after the termination of the exposure. Such a postexposure anxiogenic effect depended upon melanopsin-based intrinsically photosensitive retinal ganglion cell (ipRGC) activities rather than rod/cone photoreceptor inputs. Chemogenetic manipulation of specific central nuclei demonstrated that the ipRGC-central amygdala (CeA) visual circuit played a key role in this effect. The corticosterone system was likely to be involved in this effect, as evidenced by enhanced expression of the glucocorticoid receptor (GR) protein in the CeA and the bed nucleus of the stria terminalis and by the absence of this effect in animals treated with the GR antagonist. Together, our findings reveal a non-image forming visual circuit specifically designed for "the delayed" extinction of anxiety against potential threats, thus conferring a survival advantage.
Subject(s)
Central Amygdaloid Nucleus , Retinal Ganglion Cells , Mice , Animals , Retinal Ganglion Cells/metabolism , Retina , Retinal Cone Photoreceptor Cells , Photoreceptor Cells, Vertebrate/metabolism , LightABSTRACT
The increasing global prevalence of myopia calls for elaboration of the pathogenesis of this disease. Here, we show that selective ablation and activation of intrinsically photosensitive retinal ganglion cells (ipRGCs) in developing mice induced myopic and hyperopic refractive shifts by modulating the corneal radius of curvature (CRC) and axial length (AL) in an opposite way. Melanopsin- and rod/cone-driven signals of ipRGCs were found to influence refractive development by affecting the AL and CRC, respectively. The role of ipRGCs in myopia progression is evidenced by attenuated form-deprivation myopia magnitudes in ipRGC-ablated and melanopsin-deficient animals and by enhanced melanopsin expression/photoresponses in form-deprived eyes. Cell subtype-specific ablation showed that M1 subtype cells, and probably M2/M3 subtype cells, are involved in ocular development. Thus, ipRGCs contribute substantially to mouse eye growth and myopia development, which may inspire novel strategies for myopia intervention.
Subject(s)
Myopia , Retinal Ganglion Cells , Animals , Mice , Myopia/etiology , Photoreceptor Cells, Vertebrate , Retinal Ganglion Cells/physiology , Vision, OcularABSTRACT
Glucagon-like peptide-1 (GLP-1) is expressed in retinal neurons, but its role in the retina is largely unknown. Here, we demonstrated that GLP-1 or the GLP-1 receptor (GLP-1R; a G protein-coupled receptor) agonist exendin-4 suppressed γ-aminobutyric acid receptor (GABAR)-mediated currents through GLP-1Rs in isolated rat retinal ganglion cells (GCs). Pre-incubation with the stimulatory G protein (Gs) inhibitor NF 449 abolished the exendin-4 effect. The exendin-4-induced suppression was mimicked by perfusion with 8-Br-cAMP (a cAMP analog), but was eliminated by the protein kinase A (PKA) inhibitor Rp-cAMP/KT-5720. The exendin-4 effect was accompanied by an increase in [Ca2+]i of GCs through the IP3-sensitive pathway and was blocked in Ca2+-free solution. Furthermore, when the activity of calmodulin (CaM) and CaM-dependent protein kinase II (CaMKII) was inhibited, the exendin-4 effect was eliminated. Consistent with this, exendin-4 suppressed GABAR-mediated light-evoked inhibitory postsynaptic currents in GCs in rat retinal slices. These results suggest that exendin-4-induced suppression may be mediated by a distinct Gs/cAMP-PKA/IP3/Ca2+/CaM/CaMKII signaling pathway, following the activation of GLP-1Rs.
Subject(s)
Glucagon-Like Peptide 1 , Retinal Ganglion Cells , Animals , Calcium-Calmodulin-Dependent Protein Kinase Type 2/metabolism , Exenatide/metabolism , Exenatide/pharmacology , Glucagon-Like Peptide 1/pharmacology , Rats , Receptors, GABA/metabolism , Retinal Ganglion Cells/physiology , Signal TransductionABSTRACT
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.
Subject(s)
Melatonin , Myopia , Animals , Disease Models, Animal , Dopamine , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Retina , Sensory DeprivationABSTRACT
In this work, modulation by orexin-A of the release of glutamate and GABA from bipolar and amacrine cells respectively was studied by examining the effects of the neuropeptide on miniature excitatory postsynaptic currents (mEPSCs) and miniature inhibitory postsynaptic currents (mIPSCs) of rat retinal ganglion cells (GCs). Using RNAscope in situ hybridization in combination with immunohistochemistry, we showed positive signals for orexin receptor-1 (OX1R) mRNA in the bipolar cell terminals and those for orexin receptor-2 (OX2R) mRNA in the amacrine cell terminals. With whole-cell patch-clamp recordings in rat retinal slices, we demonstrated that application of orexin-A reduced the interevent interval of mEPSCs of GCs through OX1R. However, it increased the interevent interval of mIPSCs, mediated by GABAA receptors, through OX2R. Furthermore, orexin-A-induced reduction of mEPSC interevent interval was abolished by the application of PI-PLC inhibitors or PKC inhibitors. In contrast, orexin-A-induced increase of GABAergic mIPSC interevent interval was mimicked by 8-Br-cAMP or an adenylyl cyclase activator, but was eliminated by PKA antagonists. Finally, application of nimodipine, an L-type Ca2+ channel blocker, increased both mEPSC and mIPSC interevent interval, and co-application of orexin-A no longer changed the mEPSCs and mIPSCs. We conclude that orexin-A increases presynaptic glutamate release onto GCs by activating L-type Ca2+ channels in bipolar cells, a process that is mediated by an OX1R/PI-PLC/PKC signaling pathway. However, orexin-A decreases presynaptic GABA release onto GCs by inhibiting L-type Ca2+ channels in amacrine cells, a process that is mediated by an OX2R/cAMP-PKA signaling pathway.
Subject(s)
Amacrine Cells/metabolism , Excitatory Postsynaptic Potentials/genetics , Inhibitory Postsynaptic Potentials/genetics , Orexin Receptors/genetics , Orexins/metabolism , Retinal Bipolar Cells/metabolism , Retinal Ganglion Cells/metabolism , Synaptic Transmission/genetics , Amacrine Cells/drug effects , Animals , Excitatory Postsynaptic Potentials/drug effects , Glutamic Acid/drug effects , Glutamic Acid/metabolism , Inhibitory Postsynaptic Potentials/drug effects , Orexin Receptors/metabolism , Orexins/pharmacology , Patch-Clamp Techniques , Phosphoinositide Phospholipase C/antagonists & inhibitors , Phosphoinositide Phospholipase C/metabolism , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/metabolism , Rats , Receptors, GABA-A/genetics , Receptors, GABA-A/metabolism , Retinal Bipolar Cells/drug effects , Retinal Ganglion Cells/drug effects , Synaptic Transmission/drug effects , gamma-Aminobutyric Acid/metabolismABSTRACT
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.
Subject(s)
Diabetes Mellitus, Experimental/chemically induced , Diabetes Mellitus, Experimental/metabolism , Retina/metabolism , Retinal Ganglion Cells/drug effects , Retinal Ganglion Cells/metabolism , Rod Opsins/metabolism , Streptozocin/toxicity , Animals , Mice , Retina/drug effectsABSTRACT
We show for the first time that the neuropeptide orexin modulates pupillary light response, a non-image-forming visual function, in mice of either sex. Intravitreal injection of the orexin receptor (OXR) antagonist TCS1102 and orexin-A reduced and enhanced pupillary constriction in response to light, respectively. Orexin-A activated OX1Rs on M2-type intrinsically photosensitive retinal ganglion cells (M2 cells), and caused membrane depolarization of these cells by modulating inward rectifier potassium channels and nonselective cation channels, thus resulting in an increase in intrinsic excitability. The increased intrinsic excitability could account for the orexin-A-evoked increase in spontaneous discharges and light-induced spiking rates of M2 cells, leading to an intensification of pupillary constriction. Orexin-A did not alter the light response of M1 cells, which could be because of no or weak expression of OX1Rs on them, as revealed by RNAscope in situ hybridization. In sum, orexin-A is likely to decrease the pupil size of mice by influencing M2 cells, thereby improving visual performance in awake mice via enhancing the focal depth of the eye's refractive system.SIGNIFICANCE STATEMENT This study reveals the role of the neuropeptide orexin in mouse pupillary light response, a non-image-forming visual function. Intravitreal orexin-A administration intensifies light-induced pupillary constriction via increasing the excitability of M2 intrinsically photosensitive retinal ganglion cells by activating the orexin receptor subtype OX1R. Modulation of inward rectifier potassium channels and nonselective cation channels were both involved in the ionic mechanisms underlying such intensification. Orexin could improve visual performance in awake mice by reducing the pupil size and thereby enhancing the focal depth of the eye's refractive system.
Subject(s)
Orexins/administration & dosage , Photic Stimulation/methods , Pupil/drug effects , Reflex, Pupillary/drug effects , Retinal Ganglion Cells/cytology , Retinal Ganglion Cells/drug effects , Animals , Benzimidazoles/administration & dosage , Female , Intravitreal Injections , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Orexin Receptors/agonists , Orexin Receptors/metabolism , Orexins/antagonists & inhibitors , Pupil/physiology , Pyrrolidines/administration & dosage , Reflex, Pupillary/physiology , Retinal Ganglion Cells/metabolismABSTRACT
Correlated spontaneous activity in the developing retina (termed "retinal waves") plays an instructive role in refining neural circuits of the visual system. Depolarizing (ON) and hyperpolarizing (OFF) starburst amacrine cells (SACs) initiate and propagate cholinergic retinal waves. Where cholinergic retinal waves stop, SACs are thought to be driven by glutamatergic retinal waves initiated by ON-bipolar cells. However, the properties and function of cholinergic and glutamatergic waves in ON- and OFF-SACs still remain poorly understood. In the present work, we performed whole-cell patch-clamp recordings and Ca2+ imaging from genetically labeled ON- and OFF-SACs in mouse flat-mount retinas. We found that both SAC subtypes exhibited spontaneous rhythmic depolarization during cholinergic and glutamatergic waves. Interestingly, ON-SACs had wave-induced action potentials (APs) in an age-dependent manner, but OFF-SACs did not. Simultaneous Ca2+ imaging and patch-clamp recordings demonstrated that, during a cholinergic wave, APs of an ON-SAC appeared to promote the dendritic release of acetylcholine onto neighboring ON- and OFF-SACs, which enhances their Ca2+ transients. These results advance the understanding of the cellular mechanisms underlying correlated spontaneous activity in the developing retina.
Subject(s)
Acetylcholine/metabolism , Action Potentials/physiology , Amacrine Cells/metabolism , Amacrine Cells/physiology , Glutamates/metabolism , Retina/physiology , Animals , Animals, Newborn , Calcium/metabolism , Cholinergic Agents/metabolism , Female , Male , Mice , Retina/metabolismABSTRACT
Aerobic exercise has been shown to play a crucial role in preventing neurological diseases and improving cognitive function. In the present study, we investigated the effect of treadmill training on retinal ganglion cells (RGCs) following optic nerve transection in adult rats. We exercised the rats on a treadmill for 5 d/week (30 min/d at a rate of 9 m/min) or placed control rats on static treadmills. After 3 weeks of exercise, the left optic nerve of each rat was transected. After the surgery, the rat was exercised for another week. The percentages of surviving RGCs in the axotomized eyes of inactive rats were 67% and 39% at 5 and 7 days postaxotomy, respectively. However, exercised rats had significant more RGCs at 5 (74% survival) and 7 days (48% survival) after axotomy. Moreover, retinal brain-derived neurotrophic factor (BDNF) protein levels were significantly upregulated in response to exercise compared with those in the axotomized eyes of inactive rats. Blocking BNDF signaling during exercise by intraperitoneal injections of ANA-12, a BDNF tropomyosin receptor kinase (TrkB) receptor antagonist, reduced the number of RGCs in exercised rats to the level of RGCs in the inactive rats, effectively abolishing the protection of RGCs afforded by exercise. The results suggest that treadmill training effectively rescues RGCs from neurodegeneration following optic nerve transection by upregulating the expression of BDNF.
Subject(s)
Optic Nerve Injuries/pathology , Optic Nerve/pathology , Physical Conditioning, Animal/methods , Animals , Brain-Derived Neurotrophic Factor/metabolism , Cell Death , Disease Models, Animal , Male , Optic Nerve Injuries/metabolism , Optic Nerve Injuries/therapy , Rats , Up-RegulationABSTRACT
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.
Subject(s)
Amacrine Cells/metabolism , Homeodomain Proteins/metabolism , Retinal Ganglion Cells/metabolism , Transcription Factor Brn-3B/metabolism , Transgenes/physiology , Animals , Channelrhodopsins/metabolism , Choline O-Acetyltransferase/metabolism , Female , Gene Expression , Male , Mice, Inbred C57BL , Mice, Transgenic , Transgenes/geneticsABSTRACT
Higher visual centers could modulate visually-guided ocular growth, in addition to local mechanisms intrinsic to the eye. There is evidence that such central modulations could be species (even subspecies)-dependent. While the mouse has recently become an important experimental animal in myopia studies, it remains unclear whether and how visual centers modulate refractive development in mice, an issue that was examined in the present study. We found that optic nerve crush (ONC), performed at P18, could modify normal refractive development in the C57BL/6 mouse raised in normal visual environment. Unexpectedly, sham surgery caused a steeper cornea, leading to a modest myopic refractive shift, but did not induce significant changes in ocular axis length. ONC caused corneal flattening and re-calibrated the refractive set-point in a bidirectional manner, causing significant myopic (<-3 D, 54.5%) or hyperopic (>+3 D, 18.2%) shifts in refractive error in most (totally 72.7%) animals, both due to changes in ocular axial length. ONC did not change the density of dopaminergic amacrine cells, but increased retinal levels of dopamine and DOPAC. We conclude that higher visual centers are likely to play a role in fine-tuning of ocular growth, thus modifying refractive development in the C57BL/6 mouse. The changes in refractive error induced by ONC are accounted for by alternations in multiple ocular dimensions, including corneal curvature and axial length.
Subject(s)
Myopia/physiopathology , Optic Nerve/growth & development , Retina/growth & development , Visual Pathways/growth & development , 3,4-Dihydroxyphenylacetic Acid/metabolism , Amacrine Cells/metabolism , Animals , Cornea/growth & development , Cornea/pathology , Dopamine/metabolism , Mice, Inbred C57BL , Myopia/metabolism , Myopia/pathology , Nerve Crush , Retina/metabolism , Retina/pathology , Tyrosine 3-Monooxygenase/metabolism , Visual Pathways/metabolismABSTRACT
In this work, we investigated changes in the morphology of intrinsically photosensitive retinal ganglion cells (ipRGCs), M1 subtype, and pupillary light reflex following local and selective ablation of photoreceptors in mice. Laser photocoagulation was used to selectively destroy four patches of photoreceptors per eye at around 4 papillary diameters from the optic disc and at the 3, 6, 9, and 12 o'clock positions between the retinal vessels in the adult mouse retina, leaving cells in the inner retina intact. Morphological parameters of individual M1 cells specifically labeled by the antibody against melanopsin (PA1-780), including dendritic field size, total dendritic length, and dendritic branch number, were examined 1, 2, 4, and 8 weeks after photocoagulation with Neurolucida software. A considerable reduction in these parameters in M1 cells in the "lesioned areas" was found at all the four time points after photocoagulation, as compared with those in the "unlesioned areas". Although M1 cells in the lesioned areas showed significant changes as early as 1 week after laser treatment and the changes gradually increased, reaching a peak value at 2 weeks, morphological restoration was clearly seen in these cells over time. However, no difference in the morphological parameters of M1 cells was observed between the unlesioned areas of laser-treated mice and the corresponding areas of age-matched normal mice without laser lesions. Fluorescence intensity of the somata of melanopsin-positive M1 cells located inside the lesioned areas was significantly decreased at all the four time points after photocoagulation, whereas no changes in pupillary light reflex were detected at different light irradiations, indicating that photocoagulation-induced local photoreceptor loss and alterations of ipRGCs may be insufficient to cause abnormalities in non-image-forming (NIF) visual functions. The results suggest that intact photoreceptors could be crucial for maintaining the expression levels of melanopsin and normal morphology of M1 cells.
Subject(s)
Laser Coagulation , Reflex, Pupillary/physiology , Retina/surgery , Retinal Ganglion Cells/pathology , Animals , Light , Male , Mice , Mice, Inbred C57BL , Photoreceptor Cells, Vertebrate/metabolism , Retinal Ganglion Cells/metabolism , Retinal Ganglion Cells/radiation effects , Rod Opsins/metabolismABSTRACT
Purpose: We investigate morphologic and physiologic alterations of ganglion cells (GCs) in a streptozocin (STZ)-induced diabetic mouse model. Methods: Experiments were conducted in flat-mount retinas of mice 3 months after the induction of diabetes. Changes in morphology of four subtypes of GCs (ON-type RGA2 [ON-RGA2], OFF-type RGA2 [OFF-RGA2], ON-type RGC1 [ON-RGC1], and ON-OFF type RGD2 [ON-OFF RGD2]) were characterized in Thy1-YFP transgenic mice. Using whole-cell patch-clamp recording, passive membrane properties and action potential (AP) firing properties were further investigated in transient ON- and OFF-RGA2 cells. Results: Morphologic parameters were significantly altered in the dendrites branching in the ON sublamina of the inner plexiform layer (IPL) for ON-RGA2 cells and ON-OFF RGD2 cells. Much less significant changes, if any, were seen in those arborizing in the OFF sublamina of the IPL for OFF-RGA2 and ON-OFF RGD2 cells. No detectable changes in morphology were seen in RGC1 cells. Electrophysiologically, increased resting membrane potentials and decreased membrane capacitance were found in transient ON-RGA2 cells, but not in transient OFF-RGA2 cells. Similar alterations in AP firing properties, such as an increase in AP width and reduction in maximum spiking rate, were shared by these two subtypes. Furthermore, in response to depolarizing current injections, both cells generated more APs suggesting an enhanced excitability of these cells in diabetic conditions. Conclusions: These differential changes in morphology and electrophysiology in subtypes of GCs may be responsible for reduced contrast sensitivity known to occur during the early stage of diabetic retinopathy.
Subject(s)
Diabetes Mellitus, Experimental/pathology , Diabetic Retinopathy/pathology , Retinal Ganglion Cells/pathology , Animals , Bacterial Proteins/metabolism , Blood Glucose/metabolism , Diabetes Mellitus, Experimental/blood , Diabetic Retinopathy/blood , Disease Models, Animal , Female , Luminescent Proteins/metabolism , Male , Membrane Potentials , Mice , Mice, Inbred C57BL , Mice, Transgenic , Patch-Clamp Techniques , Photic Stimulation , StreptozocinABSTRACT
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-ß-S/pertussis toxin (for Gi/o), D609 (for PLC), bisindolylmaleimide IV (for PKC)/rottlerin (for PKCδ), 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).
Subject(s)
Inhibitory Postsynaptic Potentials/drug effects , Orexins/pharmacology , Retina/cytology , Retinal Rod Photoreceptor Cells/drug effects , Synaptic Transmission/drug effects , Animals , Animals, Newborn , Calcium/metabolism , Excitatory Amino Acid Agents/pharmacology , GABA Agents/pharmacology , Glycine/pharmacology , Light , Male , Membrane Potentials/drug effects , Orexin Receptors/metabolism , Patch-Clamp Techniques , Propionates/pharmacology , Rats , Rats, Sprague-Dawley , gamma-Aminobutyric Acid/pharmacologyABSTRACT
Purpose: The neuropeptides orexin-A and orexin-B are widely expressed in the vertebrate retina; however, their role in visual function is unclear. This study investigates whether and how orexins modulate signal transmission to dopaminergic amacrine cells (DACs) from both outer retinal photoreceptors (rods and cones) and inner retinal photoreceptors (melanopsin-expressing intrinsically photosensitive retinal ganglion cells [ipRGCs]). Methods: A whole-cell voltage-clamp technique was used to record light-induced responses from genetically labeled DACs in flat-mount mouse retinas. Rod and cone signaling to DACs was confirmed pharmacologically (in wild-type retinas), whereas retrograde melanopsin signaling to DACs was isolated either pharmacologically (in wild-type retinas) or by genetic deletion of rod and cone function (in transgenic mice). Results: Orexin-A attenuated rod/cone-mediated light responses in the majority of DACs and inhibited all DACs that exhibited melanopsin-based light responses, suggesting that exogenous orexin suppresses signal transmission from rods, cones, and ipRGCs to DACs. In addition, orexin receptor 1 antagonist SB334867 and orexin receptor 2 antagonist TCS OX229 enhanced melanopsin-based DAC responses, indicating that endogenous orexins inhibit signal transmission from ipRGCs to DACs. We further found that orexin-A inhibits melanopsin-based DAC responses via orexin receptors on DACs, whereas orexin-A may modulate signal transmission from rods and cones to DACs through activation of orexin receptors on DACs and their upstream neurons. Conclusions: Our results suggest that orexins could influence visual function via the dopaminergic system in the mammalian retina.
Subject(s)
Amacrine Cells/metabolism , Dopamine/metabolism , Orexins/pharmacology , Photoreceptor Cells, Vertebrate/metabolism , Retinal Ganglion Cells/metabolism , Rod Opsins/metabolism , Signal Transduction/drug effects , Animals , Benzoxazoles/pharmacology , Female , Isoquinolines/pharmacology , Male , Mice , Mice, Inbred C57BL , Mice, Transgenic , Naphthyridines , Orexin Receptor Antagonists/pharmacology , Patch-Clamp Techniques , Pyridines/pharmacology , Urea/analogs & derivatives , Urea/pharmacologyABSTRACT
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.
Subject(s)
Interneurons/metabolism , Optogenetics/methods , Recombinant Fusion Proteins/biosynthesis , Retina/metabolism , Animals , Bacterial Proteins/metabolism , Luminescent Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Transgenic , Models, Animal , Rhodopsin/metabolism , Vesicular Inhibitory Amino Acid Transport Proteins/metabolismABSTRACT
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.
Subject(s)
Dopamine/metabolism , Myopia/metabolism , Oxidopamine/administration & dosage , Refraction, Ocular , Retina/metabolism , Tomography, Optical Coherence/methods , Adrenergic Agents/administration & dosage , Animals , Blotting, Western , Chromatography, High Pressure Liquid , Disease Models, Animal , Dose-Response Relationship, Drug , Electroretinography , Follow-Up Studies , Intravitreal Injections , Male , Mice , Mice, Inbred C57BL , Myopia/diagnosis , Retina/pathology , Retina/physiopathologyABSTRACT
Sigma receptor (σR), a unique receptor family, is classified into three subtypes: σR1, σR2 and σR3. It was previously shown that σR1 activation induced by 1µM SKF10047 (SKF) suppressed N-methyl-d-aspartate (NMDA) receptor-mediated responses of rat retinal ganglion cells (GCs) and the suppression was mediated by a distinct Ca(2+)-dependent phospholipase C (PLC)-protein kinase C (PKC) pathway. In the present work, using whole-cell patch-clamp techniques in rat retinal slice preparations, we further demonstrate that SKF of higher dosage (50µM) significantly suppressed AMPA receptor (AMPAR)-mediated light-evoked excitatory postsynaptic currents (L-EPSCs) of retinal ON-type GCs (ON GCs), and the effect was reversed by the σR1 antagonist BD1047, suggesting the involvement of σR1. The SKF (50µM) effect was unlikely due to a change in glutamate release from bipolar cells, as suggested by the unaltered paired-pulse ratio (PPR) of AMPAR-mediated EPSCs of ON GCs. SKF (50µM) did not change L-EPSCs of ON GCs when the G protein inhibitor GDP-ß-S or the protein kinase G (PKG) inhibitor KT5823 was intracellularly infused. Calcium imaging further revealed that SKF (50µM) did not change intracellular calcium concentration in GCs and persisted to suppress L-EPSCs when intracellular calcium was chelated by BAPTA. The SKF (50µM) effect was intact when protein kinase A (PKA) and phosphatidylinostiol (PI)-PLC signaling pathways were both blocked. We conclude that the SKF (50µM) effect is Ca(2+)-independent, PKG-dependent, but not involving PKA, PI-PLC pathways.