Chromatic pupillometry isolation and evaluation of intrinsically photosensitive retinal ganglion cell-driven pupillary light response in patients with retinitis pigmentosa.

Purpose: The pupil light response (PLR) is driven by rods, cones, and intrinsically photosensitive retinal ganglion cells (ipRGCs). We aimed to isolate ipRGC-driven pupil responses using chromatic pupillometry and to determine the effect of advanced retinitis pigmentosa (RP) on ipRGC function. Methods: A total of 100 eyes from 67 patients with advanced RP and 18 healthy controls (HCs) were included. Patients were divided into groups according to severity of visual impairment: no light perception (NLP, 9 eyes), light perception (LP, 19 eyes), faint form perception (FFP, 34 eyes), or form perception (FP, 38 eyes). Pupil responses to rod-weighted (487 nm, -1 log cd/m2, 1 s), cone-weighted (630 nm, 2 log cd/m2, 1 s), and ipRGC-weighted (487 nm, 2 log cd/m2, 1 s) stimuli were recorded. ipRGC function was evaluated by the postillumination pupil response (PIPR) and three metrics of pupil kinetics: maximal contraction velocity (MCV), contraction duration, and maximum dilation velocity (MDV). Results: We found a slow, sustained PLR response to the ipRGC-weighted stimulus in most patients with NLP (8/9), but these patients had no detectable rod- or cone-driven PLR. The ipRGC-driven PLR had an MCV of 0.269 ± 0.150%/s and contraction duration of 2.562 ± 0.902 s, both of which were significantly lower than those of the rod and cone responses. The PIPRs of the RP groups did not decrease compared with those of the HCs group and were even enhanced in the LP group. At advanced stages, ipRGC responses gradually became the main component of the PLR. Conclusion: Chromatic pupillometry successfully isolated an ipRGC-driven PLR in patients with advanced RP. This PLR remained stable and gradually became the main driver of pupil contraction in more advanced cases of RP. Here, we present baseline data on ipRGC function; we expect these findings to contribute to evaluating and screening candidates for novel therapies.

Evaluation of the influences of low dose polybrominated diphenyl ethers exposure on human early retinal development.

Increasing evidence in animal models has suggested that polybrominated diphenyl ethers (PBDEs), a class of brominated flame retardants, can cause retinotoxicity. However, data on the influence of PBDE treatment on human retinal development are scarce due to the lack of appropriate models. In the present study, we report the utilization of human embryonic stem cell-derived retinal organoids (hESC-ROs) for toxicity assessment of the most common PBDE congener (BDE-47) during the early stages of retinal development. Exposure to BDE-47 decreased the thickness and area of the neural retina (NR) of hESC-ROs in a dose- and time-dependent manner. Abnormal retinal cell distributions, disordered NR structures, and neural rosette-like structures were found on hESC-ROs after low-level BDE-47 exposure. Moreover, BDE-47 exposure decreased cell proliferation, promoted cell apoptosis, and caused abnormal differentiation. Transcriptomic analysis demonstrated that differentially expressed genes, caused by BDE-47, were enriched in extracellular matrix organization. Metabolomic studies of hESC-ROs revealed significant changes in the metabolism of purine and glutathione after BDE-47 exposure for five weeks. This study clarifies the retinotoxicity of low-level BDE-47 treatment and highlights the powerfulness of the hESC-RO model, deepening our understanding of BDE-47-driven human early retina developmental toxicity.

Human Umbilical Cord Blood-Derived CD133+CD34+ Cells Protect Retinal Endothelial Cells and Ganglion Cells in X-Irradiated Rats through Angioprotective and Neurotrophic Factors.

Radiation retinopathy (RR) is a common complication following radiation therapy of globe, head, and neck malignancies, and is characterized by microangiopathy, neuroretinopathy, and the irreversible loss of visual function. To date, there is no effective treatment for RR. Stem cells have been clinically used to treat retinal degeneration. CD133+CD34+ cells from human umbilical cord blood (hUCB-CD133+CD34+ cells), a subpopulation of hematopoietic stem cells, were applied to determine their protective efficacy on irradiated rat retinas. After X-ray irradiation on the retinas, rats were intravitreally injected with hUCB-CD133+CD34+ cells. Transplantation of hUCB-CD133+CD34+ cells prevented retinal dysfunction 2 weeks post-operation and lasted at least 8 weeks. CD133+CD34+ cells were distributed along the retinal vessel and migrated to the ganglion cell layer. Moreover, grafted CD133+CD34+ cells reduced the apoptosis of endothelial and ganglion cells in irradiated rats and increased the number of survived CD31+ retinal endothelial cells and Brn3a+ ganglion cells at 2 and 4 weeks, respectively, post-operation. Co-culturing of CD133+CD34+ cells or supernatants with irradiated human retinal microvascular endothelial cells (hRECs) in vitro, confirmed that CD133+CD34+ cells ameliorated hREC apoptosis caused by irradiation. Mechanistically, we found that angioprotective mediators and neurotrophic factors were secreted by CD133+CD34+ cells, which might attenuate irradiation-induced injury of retinal endothelial cells and ganglion cells. hUCB-CD133+CD34+ cell transplantation, as a novel treatment, protects retinal endothelial and ganglion cells of X-irradiated rat retinas, possibly through angioprotective and neurotrophic factors.

Synaptic repair and vision restoration in advanced degenerating eyes by transplantation of retinal progenitor cells.

Stem cell transplantation shows enormous potential for treatment of incurable retinal degeneration (RD). To determine if and how grafts connect with the neural circuits of the advanced degenerative retina (ADR) and improve vision, we perform calcium imaging of GCaMP5-positive grafts in retinal slices. The organoid-derived C-Kit+/SSEA1- (C-Kit+) retinal progenitor cells (RPCs) become synaptically organized and build spontaneously active synaptic networks in three major layers of ADR. Light stimulation of the host photoreceptors elicits distinct neuronal responses throughout the graft RPCs. The graft RPCs and their differentiated offspring cells in inner nuclear layer synchronize their activities with the host cells and exhibit presynaptic calcium flux patterns that resemble intact retinal neurons. Once graft-to-host network is established, progressive vision loss is stabilized while control eyes continually lose vision. Therefore, transplantation of organoid-derived C-Kit+ RPCs can form functional synaptic networks within ADR and it holds promising avenue for advanced RD treatment.

Functional assessment of cryopreserved clinical grade hESC-RPE cells as a qualified cell source for stem cell therapy of retinal degenerative diseases.

The biosafety and efficiency of transplanting retinal pigment epithelial (RPE) cells derived from both human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have been evaluated in phase I and phase II clinical trials. For further large-scale application, cryopreserved RPE cells must be used; thus, it is highly important to investigate the influence of cryopreservation and thawing on the biological characteristics of hESC-RPE cells and their post-transplantation vision-restoring function. Here, via immunofluorescence, qPCR, transmission electron microscopy, transepithelial electrical resistance, and enzyme-linked immunosorbent assays (ELISAs), we showed that cryopreserved hESC-RPE cells retained the specific gene expression profile, morphology, ultrastructure, and maturity-related functions of induced RPE cells. Additionally, cryopreserved hESC-RPE cells exhibited a polarized monolayer, tight junction, and gap junction structure and an in vitro nanoparticle phagocytosis capability similar to those of induced hESC-RPE cells. However, the level of pigment epithelium-derived factor (PEDF) secretion was significantly decreased in cryopreserved hESC-RPE cells. Royal College of Surgeons rats with cryopreserved hESC-RPE cells engrafted into the subretinal space exhibited a significant decrease in the b-wave amplitude compared with rats engrafted with induced hESC-RPE cells at 4 weeks post transplantation. However, the difference disappeared at 8 weeks and 12 weeks post operation. No significant difference in the outer nuclear layer (ONL) thickness was observed between the two groups. Our data showed that even after cryopreservation and thawing, cryopreserved hESC-RPE cells are still qualified as a donor cell source for cell-based therapy of retinal degenerative diseases.

Improving cell survival and engraftment in vivo via layer-by-layer nanocoating of hESC-derived RPE cells.

BACKGROUND: Human embryonic stem cell-derived retinal pigment epithelial (hESC-RPE) cell transplants have served as a cell therapy for treating retinal degenerative diseases. However, how to optimize the survival and engraftment of hESC-RPE cells is a great challenge. METHODS: Here, we report hESC-RPE cells that are embedded with polyelectrolytes gelatin and alginate by layer-by-layer (LbL) self-assembly technique, based on the opposite charge of alternate layers. Cells were assessed for cell survival, immunogenicity, and function in vitro and in vivo. RESULTS: This strategy obviously decreased the immunogenicity of hESC-RPE cells without affecting its activity. LbL-RPE cell transplants into the subretinal space of Royal College of Surgeons (RCS) rats optimized cell engraftment and decreased immunogenicity compared to untreated RPE cell transplants (immunosuppression was not used during the 21-week study). Visual-functional assay with electroretinogram recordings (ERGs) also showed higher B wave amplitudes in RCS rats with LbL-RPE cell transplants. CONCLUSIONS: We demonstrate that transplanted LbL-RPE cells have better viability and grafting efficiency, optimized immunogenicity, and visual function. Therefore, LbL engineering is a promising method to increase the efficacy of hESC-RPE cell transplantation.

Exosomes derived from neural progenitor cells preserve photoreceptors during retinal degeneration by inactivating microglia.

Retinal degeneration (RD) is one of the most common causes of visual impairment and blindness and is characterized by progressive degeneration of photoreceptors. Transplantation of neural stem/progenitor cells (NPCs) is a promising treatment for RD, although the mechanisms underlying the efficacy remain unclear. Accumulated evidence supports the notion that paracrine effects of transplanted stem cells is likely the major approach to rescuing early degeneration, rather than cell replacement. NPC-derived exosomes (NPC-exos), a type of extracellular vesicles (EVs) released from NPCs, are thought to carry functional molecules to recipient cells and play therapeutic roles. In present study, we found that grafted human NPCs (hNPCs) secreted EVs and exosomes in the subretinal space (SRS) of RCS rats, an RD model. And direct administration of mouse neural progenitor cell-derived exosomes (mNPC-exos) delayed photoreceptor degeneration, preserved visual function, prevented thinning of the outer nuclear layer (ONL), and decreased apoptosis of photoreceptors in RCS rats. Mechanistically, mNPC-exos were specifically internalized by retinal microglia and suppressed their activation in vitro and in vivo. RNA sequencing and miRNA profiling revealed a set of 17 miRNAs contained in mNPC-exos that markedly inhibited inflammatory signal pathways by targeting TNF-α, IL-1ß, and COX-2 in activated microglia. The exosomes derived from hNPC (hNPC-exos) contained similar miRNAs to mNPC-exos that inhibited microglial activation. We demonstrated that NPC-exos markedly suppressed microglial activation to protect photoreceptors from apoptosis, suggesting that NPC-exos and their contents may be the mechanism of stem cell therapy for treating RD.

Validation and Safety of Visual Restoration by Ectopic Expression of Human Melanopsin in Retinal Ganglion Cells.

To study whether ectopic human melanopsin (hMel) in retinal ganglion cells (RGCs) could restore the visual function in end-stage retinal degeneration, AAV2/8-CMV-hMel/FYP was injected into the intravitreal space of Royal College of Surgeons (RCS) rats. It was observed that ectopic hMel/yellow fluorescent protein (YFP) was dominantly expressed in the RGCs of the RCS rat retinae. At 30-45 days after administration of AAV2/8-CMV-hMel/FYP in RCS rats, the flash visual evoked potentials and behavioral results demonstrated that visual function was significantly improved compared to that in the control group, while no improvement in flash electroretinography was observed at this time point. To translate this potential therapeutic approach to the clinic, the safety of viral vectors in the retinae of normal macaques was then studied, and the expression profile of exogenous hMel with/without internal limiting membrane peeling was compared before viral vector administration. The data revealed that there was no significant difference in the number of RGCs containing exogenous hMel/YFP between the two groups. Whole-cell patch-clamp recordings demonstrated that the hMel/YFP-positive RGCs of the macaque retinae reacted to the intense light stimulation, generating inward currents and action potentials. This result confirms that the ectopic hMel expressed in RGCs is functional. Moreover, the introduction of AAV2/8-CMV-hMel/FYP does not cause detectable pathological effects. Thus, this study suggests that AAV2/8-CMV-hMel/FYP administration without internal limiting membrane peeling is safe and feasible for efficient transduction and provides therapeutic benefits to restore the visual function of patients suffering photoreceptor loss.

Changes in intrinsic excitability of ganglion cells in degenerated retinas of RCS rats.

AIM: To evaluate the intrinsic excitability of retinal ganglion cells (RGCs) in degenerated retinas. METHODS: The intrinsic excitability of various morphologically defined RGC types using a combination of patch-clamp recording and the Lucifer yellow tracer in retinal whole-mount preparations harvested from Royal College of Surgeons (RCS) rats, a common retinitis pigmentosa (RP) model, in a relatively late stage of retinal degeneration (P90) were investigated. Several parameters of RGC morphologies and action potentials (APs) were measured and compared to those of non-dystrophic control rats, including dendritic stratification, dendritic field diameter, peak amplitude, half width, resting membrane potential, AP threshold, depolarization to threshold, and firing rates. RESULTS: Compared with non-dystrophic control RGCs, more depolarizations were required to reach the AP threshold in RCS RGCs with low spontaneous spike rates and in RCS OFF cells (especially A2o cells), and RCS RGCs maintained their dendritic morphologies, resting membrane potentials and capabilities to generate APs. CONCLUSION: RGCs are relatively well preserved morphologically and functionally, and some cells are more susceptible to decreased excitability during retinal degeneration. These findings provide valuable considerations for optimizing RP therapeutic strategies.

Proteomic profiling of early degenerative retina of RCS rats.

AIM: To identify the underlying cellular and molecular changes in retinitis pigmentosa (RP). METHODS: Label-free quantification-based proteomics analysis, with its advantages of being more economic and consisting of simpler procedures, has been used with increasing frequency in modern biological research. Dystrophic RCS rats, the first laboratory animal model for the study of RP, possess a similar pathological course as human beings with the diseases. Thus, we employed a comparative proteomics analysis approach for in-depth proteome profiling of retinas from dystrophic RCS rats and non-dystrophic congenic controls through Linear Trap Quadrupole - orbitrap MS/MS, to identify the significant differentially expressed proteins (DEPs). Bioinformatics analyses, including Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation and upstream regulatory analysis, were then performed on these retina proteins. Finally, a Western blotting experiment was carried out to verify the difference in the abundance of transcript factor E2F1. RESULTS: In this study, we identified a total of 2375 protein groups from the retinal protein samples of RCS rats and non-dystrophic congenic controls. Four hundred thirty-four significantly DEPs were selected by Student's t-test. Based on the results of the bioinformatics analysis, we identified mitochondrial dysfunction and transcription factor E2F1 as the key initiation factors in early retinal degenerative process. CONCLUSION: We showed that the mitochondrial dysfunction and the transcription factor E2F1 substantially contribute to the disease etiology of RP. The results provide a new potential therapeutic approach for this retinal degenerative disease.

Homeostatic Plasticity Mediated by Rod-Cone Gap Junction Coupling in Retinal Degenerative Dystrophic RCS Rats.

Rod-cone gap junctions open at night to allow rod signals to pass to cones and activate the cone-bipolar pathway. This enhances the ability to detect large, dim objects at night. This electrical synaptic switch is governed by the circadian clock and represents a novel form of homeostatic plasticity that regulates retinal excitability according to network activity. We used tracer labeling and ERG recording in the retinae of control and retinal degenerative dystrophic RCS rats. We found that in the control animals, rod-cone gap junction coupling was regulated by the circadian clock via the modulation of the phosphorylation of the melatonin synthetic enzyme arylalkylamine N-acetyltransferase (AANAT). However, in dystrophic RCS rats, AANAT was constitutively phosphorylated, causing rod-cone gap junctions to remain open. A further b/a-wave ratio analysis revealed that dystrophic RCS rats had stronger synaptic strength between photoreceptors and bipolar cells, possibly because rod-cone gap junctions remained open. This was despite the fact that a decrease was observed in the amplitude of both a- and b-waves as a result of the progressive loss of rods during early degenerative stages. These results suggest that electric synaptic strength is increased during the day to allow cone signals to pass to the remaining rods and to be propagated to rod bipolar cells, thereby partially compensating for the weak visual input caused by the loss of rods.

Functional ectopic neuritogenesis by retinal rod bipolar cells is regulated by miR-125b-5p during retinal remodeling in RCS rats.

Following retinal degeneration, retinal remodeling can cause neuronal microcircuits to undergo structural alterations, which particularly affect the dendrites of bipolar cells. However, the mechanisms and functional consequences of such changes remain unclear. Here, we used Royal College of Surgeon (RCS) rats as a model of retinal degeneration, to study structural changes in rod bipolar cells (RBCs) and the underlying mechanisms of these changes. We found that, with retinal degeneration, RBC dendrites extended into the outer nuclear layer (ONL) of the retina, and the ectopic dendrites formed synapses with the remaining photoreceptors. This ectopic neuritogenesis was associated with brain-derived neurotrophic factor (BDNF) - expression of which was negatively regulated by miR-125b-5p. Overexpression of miR-125b-5p in the retinae of RCS rats diminished RBC ectopic dendrites, and compromised the b-wave of the flash electroretinogram (ERG). In contrast, down-regulation of miR-125b-5p (or exogenous BDNF treatment) increased RBC ectopic dendrites, and improved b-wave. Furthermore, we showed that the regulation of ectopic neuritogenesis by BDNF occurred via the downstream modulation of the TrkB-CREB signaling pathway. Based on these findings, we conclude that ectopic dendrites are likely to be providing functional benefits and that, in RCS rats, miR-125b-5p regulates ectopic neuritogenesis by RBCs through modulation of the BDNF-TrkB-CREB pathway. This suggests that therapies that reduce miR-125b-5p expression could be beneficial in human retinal degenerative disease.

Connexin-based channels contribute to metabolic pathways in the oligodendroglial lineage.

Oligodendrocyte precursor cells (OPCs) undergo a series of energy-consuming developmental events; however, the uptake and trafficking pathways for their energy metabolites remain unknown. In the present study, we found that 2-NBDG, a fluorescent glucose analog, can be delivered between astrocytes and oligodendrocytes through connexin-based gap junction channels but cannot be transferred between astrocytes and OPCs. Instead, connexin hemichannel-mediated glucose uptake supports OPC proliferation, and ethidium bromide uptake or increase of 2-NBDG uptake rate is correlated with intracellular Ca(2+) elevation in OPCs, indicating a Ca(2+)-dependent activation of connexin hemichannels. Interestingly, deletion of connexin 43 (Cx43, also known as GJA1) in astrocytes inhibits OPC proliferation by decreasing matrix glucose levels without impacting on OPC hemichannel properties, a process that also occurs in corpus callosum from acute brain slices. Thus, dual functions of connexin-based channels contribute to glucose supply in oligodendroglial lineage, which might pave a new way for energy-metabolism-directed oligodendroglial-targeted therapies.

Light-evoked currents in retinal ganglion cells from dystrophic RCS rats.

PURPOSE: To study the electrophysiological properties of the light-evoked currents in ganglion cells in situations of retinal degeneration. METHODS: We investigated light-evoked currents in ganglion cells by performing whole-cell patch-clamp recordings from ganglion cells using a retina-stretched preparation from Royal College of Surgeons (RCS) rats, a model of retinal degeneration and congenic controls at different ages. Pharmacological inhibitors of the AMPA receptor (NBQX), GABA receptor (BMI), and sodium channels (TTX) were used to identify the components of the light-evoked currents in ON, OFF and ON-OFF retinal ganglion cells. RESULTS: We found that the light-evoked currents in ganglion cells from control rats were inhibited by NBQX, BMI and TTX, suggesting that AMPA receptors, GABA receptors and sodium channels contribute to these currents in ganglion cells. However, only AMPA receptor-mediated currents were recorded in RCS rats. Light-evoked inward currents were absent in the majority of ganglion cells from RCS rats, particularly at the later stages of retinal degeneration. At earlier stages of retinal degeneration, we found that both the timing and amplitude of light-evoked currents are significantly different in ganglion cells from RCS and control rats. CONCLUSIONS: Our study furthers the understanding of the electrophysiological characteristics of retinal ganglion cells during retinal degeneration, and provides insight into the optimal timing for the treatment of retinal degeneration.

Alterations of sodium and potassium channels of RGCs in RCS rat with the development of retinal degeneration.

All know that retinitis pigmentosa (RP) is a group of hereditary retinal degenerative diseases characterized by progressive dysfunction of photoreceptors and associated with progressive cells loss; nevertheless, little is known about how rods and cones loss affects the surviving inner retinal neurons and networks. Retinal ganglion cells (RGCs) process and convey visual information from retina to visual centers in the brain. The healthy various ion channels determine the normal reception and projection of visual signals from RGCs. Previous work on the Royal College of Surgeons (RCS) rat, as a kind of classical RP animal model, indicated that, at late stages of retinal degeneration in RCS rat, RGCs were also morphologically and functionally affected. Here, retrograde labeling for RGCs with Fluorogold was performed to investigate the distribution, density, and morphological changes of RGCs during retinal degeneration. Then, patch clamp recording, western blot, and immunofluorescence staining were performed to study the channels of sodium and potassium properties of RGCs, so as to explore the molecular and proteinic basis for understanding the alterations of RGCs membrane properties and firing functions. We found that the resting membrane potential, input resistance, and capacitance of RGCs changed significantly at the late stage of retinal degeneration. Action potential could not be evoked in a part of RGCs. Inward sodium current and outward potassium current recording showed that sodium current was impaired severely but only slightly in potassium current. Expressions of sodium channel protein were impaired dramatically at the late stage of retinal degeneration. The results suggested that the density of RGCs decreased, process ramification impaired, and sodium ion channel proteins destructed, which led to the impairment of electrophysiological functions of RGCs and eventually resulted in the loss of visual function.

Changes in glutamate homeostasis cause retinal degeneration in Royal College of Surgeons rats.

The aim of the present study was to investigate glutamate homeostasis in retinal degeneration-induced changes and the potential mechanisms of glutamate-mediated excitotoxicity in a rat model. The expression of vesicular glutamate transporter-1 (VGLUT-1) and protein kinase Cα (PKCα) in wild-type and Royal College of Surgeons (RCS) rat retinas, at postnatal Day 15 (P15), P30, P60 and P90, were detected using quantitative real-time polymerase chain reaction and immunohistochemistry. The levels of glutamine synthetase (GS) and L-glutamate/L-aspartate transporter (GLAST) were evaluated by western blotting. Compared with wild-type rats, outer nuclear layer thickness was significantly thinner and VGLUT-1 expression was upregulated in a time-dependent pattern in RCS rats. The ratio of VGLUT-1 to PKCα in RCS rats peaked at P60 (p<0.01) and subsequently decreased by P90 (p<0.01), while it remained constant in wild-type rats. The expression of GS increased gradually from P30 to P90 in RCS rats (p<0.01), while it remained constant in wild-type rats at various time-points. No significant difference in GLAST expression was found between RCS and wild-type rats at all stages of retinal degeneration. Our results confirm the occurrence of glutamate-mediated excitotoxicity to RCS rat retinas and provide an experimental foundation for safeguarding the remnant visual function in retinal degenerative disorders.

Optical control after transfection of channelrhodopsin-2 recombinant adenovirus in visual cortical cells.

Channelrhodopsin-2 ectopically expressed in the retina can recover the response to blue light in genetically blind mice and rats, but is unable to restore visual function due to optic nerve or optic tract lesions. Long Evans rats at postnatal day 1 were used for primary culture of visual cortical cells, and 24 hours later, cells were transfected with recombinant adenovirus carrying channelrhodopsin-2 and green fluorescent protein genes. After 2-4 days of transfection, green fluorescence was visible in the cultured cells. Cells were stimulated with blue light (470 nm), and light-induced action potentials were recorded in patch-clamp experiments. Our findings indicate that channelrhodopsin-2-recombinant adenovirus transfection of primary cultured visual cortical cells can control the production of action potentials via blue light stimulation.

Binocular form deprivation influences the visual cortex.

α-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors are considered to play a crucial role in synaptic plasticity in the developing visual cortex. In this study, we established a rat model of binocular form deprivation by suturing the rat binocular eyelids before eye-opening at postnatal day 14. During development, the decay time of excitatory postsynaptic currents mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors of normal rats became longer after eye-opening; however, the decay time did not change significantly in binocular form deprivation rats. The peak value in the normal group became gradually larger with age, but there was no significant change in the binocular form deprivation group. These findings indicate that binocular form deprivation influences the properties of excitatory postsynaptic currents mediated by α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors in the rat visual cortex around the end of the critical period, indicating that form stimulation is associated with the experience-dependent modification of neuronal synapses in the visual cortex.

The changes of potassium currents in RCS rat Müller cell during retinal degeneration.

Müller cells are the principal glial cells expressing membrane-bound potassium channel and predominantly mediating the homeostatic regulation of extracellular K+ produced by neuronal activity in retina. It's well known that Müller cells can be activated in many pathological conditions, but little is known about the change of potassium currents of Müller cells during the progression of retinitis pigmentosa. Herein, the Royal College of Surgeons rats (RCS rat) were employed to investigate some phenotypic and functional changes of Müller cells during retinal degeneration such as the expression of Kir4.1, membrane properties and K+ channel currents by using immunohistochemistry, RT-PCR, western blot and whole-cell patch clamping respectively. Compared with Müller cells in control retina, increased glutamine synthetase (GS) mRNA levels were seen at P30 and P60, and then decreased gradually in RCS rat retina. Morphologically, Müller cells showed significant hypertrophy and proliferation after p60. The increased expression of intermediate filament, glial fibrillary acidic protein (GFAP) and vimentin began at P30 and reached a peak at p60. Kir4.1 channels presented a peak expression at P30. Concomitantly, K(+) currents of Müller cells increased at P30 and decreased at P90 significantly. We concluded that retinal Müller cells of RCS rats underwent an activation initiated by the onset of retinal degeneration before p60 and then an obvious reactive gliosis, which led the basic membrane properties to suffer marked changes, and caused the Kir4.1 channels of Müller cells to occur a clear functional shift, even lose their normal electrophysiological properties. This process aggravates the impairment caused by the initial photoreceptor degeneration.

[The modification of electrophysiology affected by ectopic synapse in ON-retinal bipolar cells of RCS rats].

OBJECTIVE: To study the influence of the ectopic synapse for electrophysiological characteristics modification in ON retinal bipolar cells (ON-RBCs) of RCS rat. METHODS: Immunofluorescence of the retinal frozen section was taken in P60 d, P90 d of RCS rat (RCS) and control rat (CTR) with the anti-mGluR6 and anti-Synaptophysin, Lucifer Yellow staining solo ON-RBCs was taken in all the group. The whole cell recording was performed in the retinal slice of P60 d, P90 d in RCS and CTR. The modification of the passive membrane properties and the outward currents properties in RCS-ON-RBCs, CTR-ON-RBCs and CTR-OFF-RBCs were observed. RESULTS: RCS-ON-RBCs stretched out the ectopic neurite in different direction and the activity of synapse could be detected around the ectopic neurite. From Pn60d, passive membrane properties of RCS-ON-RBCs kept immature, The RMP in RCS-ON-RBCs and CTR-ON-RBCs were (-61.8 ± 3.07), (-50.44 ± 1.36) mV and (-63.1 ± 2.59), (-48.37 ± 3.69) mV when P60 d and P90 d, there ware significantly higher than CTR group (t = 2.191, 2.435, 5.817, 6.912;P < 0.05). The IR in RCS-ON-RBCs and CTR-ON-RBCs were (323.3 ± 42.6), (337.6 ± 71.3) MΩ and (321.2 ± 58.6), (340.3 ± 62.8) MΩ when P60 d and P90 d, there ware significantly higher than CTR group (t = 3.561, 1.987, 5.211, 4.034; P < 0.05). Outward currents were recorded when giving hyper- and depolarized voltage steps. In retinal degeneration, the amplitude of outward currents in RCS-ON-RBCs is significantly different with CTR-ON-RBCs (t = 5.561, 6.341; P < 0.05) or CTR-OFF-RBCs (t = 5.357, 6.997; P < 0.05). CONCLUSION: The ectopic neurite from RCS-ON-RBCs has the possibility for translating the signal. In retinitis pigmentosa, the modification of electrophysiology characteristics in RCS-ON-RBCs was significantly different with CTR-ON-RBCs and CTR-OFF-RBCs. Influence with the ectopic neurite is the possible cause.