A Promising Combination: PACAP and PARP Inhibitor Have Therapeutic Potential in Models of Diabetic and Hypertensive Retinopathies.

Diabetes and hypertension are complex pathologies with increasing prevalence nowadays. Their interconnected pathways are frequently manifested in retinopathies. Severe retinal consequences and their tight connections as well as their possible treatments are particularly important to retinal research. In the present, work we induced diabetes with streptozotocin in spontaneously hypertensive rats and treated them either with PACAP or olaparib and alternatively with both agents. Morphological and immunohistochemical analyses were carried out to describe cell-specific changes during pathologies and after different treatments. Diabetes and hypertension caused massive structural and cellular changes especially when they were elicited together. Hypertension was crucial in the formation of ONL and OPL damage while diabetes caused significant differences in retinal thickness, OPL thickness and in the cell number of the GCL. In diabetes, double neuroprotective treatment ameliorated changes of calbindin-positive cells, rod bipolar cells and dopaminergic amacrine cells. Double treatment was curative in hypertensive diabetic rat retinas, especially in the case of rod bipolar and parvalbumin-positive cells compared to untreated or single-treated retinas. Our results highlighted the promising therapeutic benefits of olaparib and PACAP in these severe metabolic retinal disorders.

Orexin-A differentially modulates inhibitory and excitatory synaptic transmission in rat inner retina.

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.

Differential Contribution of Gap Junctions to the Membrane Properties of ON- and OFF-Bipolar Cells of the Rat Retina.

Gap junctions are ubiquitous within the retina, but in general, it remains to be determined whether gap junction coupling between specific cell types is sufficiently strong to mediate functionally relevant coupling via electrical synapses. From ultrastructural, tracer coupling and immunolabeling studies, there is clear evidence for gap junctions between cone bipolar cells, but it is not known if these gap junctions function as electrical synapses. Here, using whole-cell voltage-clamp recording in rat (male and female) retinal slices, we investigated whether the gap junctions of bipolar cells make a measurable contribution to the membrane properties of these cells. We measured the input resistance (RN) of bipolar cells before and after applying meclofenamic acid (MFA) to block gap junctions. In the presence of MFA, RN of ON-cone bipolar cells displayed a clear increase, paralleled by block of the electrical coupling between these cells and AII amacrine cells in recordings of coupled cell pairs. For OFF-cone and rod bipolar cells, RN did not increase in the presence of MFA. The results for rod bipolar cells are consistent with the lack of gap junctions in these cells. However, for OFF-cone bipolar cells, our results suggest that the morphologically identified gap junctions between these cells do not support a junctional conductance that is sufficient to mediate effective electrical coupling. Instead, these junctions might play a role in chemical and/or metabolic coupling between subcellular compartments.

Electroretinogram of the Cone-Dominant Thirteen-Lined Ground Squirrel during Euthermia and Hibernation in Comparison with the Rod-Dominant Brown Norway Rat.

Purpose: The majority of small animal species used in research are nocturnal, with retinae that are anatomically and functionally dissimilar from humans, complicating their use as disease models. Herein we characterize the retinal structure and electrophysiological function of the diurnal, cone-dominant 13-lined ground squirrel (13-LGS) retina during euthermia and in hibernation. Methods: Full-field electroretinography (ERG) was performed in 13-LGS and Brown Norway (BN) rat models to establish baseline values for retinal function in each species, including following intravitreal injection of pharmacologic agents to selectively block the contributions of ON- and OFF-bipolar cells. The effect of hibernation-associated retinal remodeling on electrophysiological function was assessed in 13-LGS during torpor and emergence, with correlative histology performed using transmission electron microscopy. Results: Under light-adapted conditions, the a-, b-, and d-wave amplitude of the 13-LGS was significantly greater than that of the BN rat. Retinal function was absent in the 13-LGS during hibernation and correlated to widespread disruption of photoreceptor and RPE structure. Remarkably, both retinal function and structure recovered rapidly on emergence from hibernation, with ERG responses reaching normal amplitude within 6 hours. Conclusions: ERG responses for both BN rats and 13-LGS reflect the relative proportions of cone photoreceptors present within the retinae, indicating that the cone-dominant 13-LGS may be a potentially useful model for studying human central retinal function and disease. That retinal remodeling and restoration of electrophysiological function occurs rapidly on emergence from hibernation implies the 13-LGS may also be a useful tool for studying aspects of retinal physiology and recovery from injury.

Cannabinoid Signaling Selectively Modulates GABAergic Inhibitory Input to OFF Bipolar Cells in Rat Retina.

Purpose: In the mammalian retina, cannabinoid type 1 receptors (CB1Rs) are well-positioned to alter inhibitory synaptic function from amacrine cells and, thus, might influence visual signal processing in the inner retina. However, it is not known if CB1R modulates amacrine cells feedback inhibition at retinal bipolar cell (BC) terminals. Methods: Using whole-cell voltage-clamp recordings, we examined the pharmacological effect of CB1R activation and inhibition on spontaneous inhibitory postsynaptic currents (sIPSCs) and glutamate-evoked IPSCs (gIPSCs) from identified OFF BCs in light-adapted rat retinal slices. Results: Activation of CB1R with WIN55212-2 selectively increased the frequency of GABAergic, but not glycinergic sIPSC in types 2, 3a, and 3b OFF BCs, and had no effect on inhibitory activity in type 4 OFF BCs. The increase in GABAergic activity was eliminated in axotomized BCs and can be suppressed by blocking CB1R with AM251 or GABAA and GABAρ receptors with SR-95531 and TPMPA, respectively. In all OFF BC types tested, a brief application of glutamate to the outer plexiform layer elicited gIPSCs comprising GABAergic and glycinergic components that were unaffected by CB1R activation. However, blocking CB1R selectively increased GABAergic gIPSCs, supporting a role for endocannabinoid signaling in the regulation of glutamate-evoked GABAergic inhibitory feedback to OFF BCs. Conclusions: CB1R activation shape types 2, 3a, and 3b OFF BC responses by selectively regulate GABAergic feedback inhibition at their axon terminals, thus cannabinoid signaling might play an important role in the fine-tuning of visual signal processing in the mammalian inner retina.

Inhibitory components of retinal bipolar cell receptive fields are differentially modulated by dopamine D1 receptors.

During adaptation to an increase in environmental luminance, retinal signaling adjustments are mediated by the neuromodulator dopamine. Retinal dopamine is released with light and can affect center-surround receptive fields, the coupling state between neurons, and inhibitory pathways through inhibitory receptors and neurotransmitter release. While the inhibitory receptive field surround of bipolar cells becomes narrower and weaker during light adaptation, it is unknown how dopamine affects bipolar cell surrounds. If dopamine and light have similar effects, it would suggest that dopamine could be a mechanism for light-adapted changes. We tested the hypothesis that dopamine D1 receptor activation is sufficient to elicit the magnitude of light-adapted reductions in inhibitory bipolar cell surrounds. Surrounds were measured from OFF bipolar cells in dark-adapted mouse retinas while stimulating D1 receptors, which are located on bipolar, horizontal, and inhibitory amacrine cells. The D1 agonist SKF-38393 narrowed and weakened OFF bipolar cell inhibitory receptive fields but not to the same extent as with light adaptation. However, the receptive field surround reductions differed between the glycinergic and GABAergic components of the receptive field. GABAergic inhibitory strength was reduced only at the edges of the surround, while glycinergic inhibitory strength was reduced across the whole receptive field. These results expand the role of retinal dopamine to include modulation of bipolar cell receptive field surrounds. Additionally, our results suggest that D1 receptor pathways may be a mechanism for the light-adapted weakening of glycinergic surround inputs and the furthest wide-field GABAergic inputs to bipolar cells. However, remaining differences between light-adapted and D1 receptor-activated inhibition demonstrate that non-D1 receptor mechanisms are necessary to elicit the full effect of light adaptation on inhibitory surrounds.

Effect of Proteasome Inhibitors on the AAV-Mediated Transduction Efficiency in Retinal Bipolar Cells.

BACKGROUND: Adeno-associated Virus (AAV) vectors are the most promising vehicles for therapeutic gene delivery to the retina. To develop a practical gene delivery tool, achieving high AAV transduction efficiency in specific cell types is often required. AAV-mediated targeted expression in retinal bipolar cells is needed in certain applications such as optogenetic therapy, however, the transduction efficiency driven by endogenous cell-specific promoters is usually low. Methods that can improve AAV transduction efficiency in bipolar cells need to be developed. OBJECTIVE: The study aimed to examine the effect of proteasome inhibitors on AAV-mediated transduction efficiency in retinal bipolar cells. METHODS: Quantitative analysis of fluorescent reporter protein expression was performed to assess the effect of two proteasome inhibitors, doxorubicin and MG132, on AAV-mediated transduction efficiency in retinal bipolar cells in mice. RESULTS: Our results showed that doxorubicin can increase the AAV transduction efficiency in retinal bipolar cells in a dose-dependent manner. We also observed doxorubicin-mediated cytotoxicity in retinal neurons, but the cytotoxicity could be mitigated by the coapplication of dexrazoxane. Three months after the coapplication of doxorubicin (300 µM) and dexrazoxane, the AAV transduction efficiency in retinal bipolar cells increased by 33.8% and no cytotoxicity was observed in all the layers of the retina. CONCLUSION: Doxorubicin could enhance the AAV transduction efficiency in retinal bipolar cells in vivo. The potential long-term cytotoxicity caused by doxorubicin to retinal neurons could be partially mitigated by dexrazoxane. The coapplication of doxorubicin and dexrazoxane may serve as a potential adjuvant regimen for improving AAV transduction efficiency in retinal bipolar cells.

Rod bipolar cells dysfunction occurs before ganglion cells loss in excitotoxin-damaged mouse retina.

Progressive degeneration of retinal ganglion cells (RGCs) will cause a blinding disease. Most of the study is focusing on the RGCs itself. In this study, we demonstrate a decline of the presynaptic rod bipolar cells (RBCs) response precedes RGCs loss and a decrease of protein kinase Cα (PKCα) protein expression in RBCs dendrites, using whole-cell voltage-clamp, electroretinography (ERG) measurements, immunostaining and co-immunoprecipitation. We present evidence showing that N-methyl D-aspartate receptor subtype 2B (NR2B)/protein interacting with C kinase 1 (PICK1)-dependent degradation of PKCα protein in RBCs contributes to RBCs functional loss. Mechanistically, NR2B forms a complex with PKCα and PICK1 to promote the degradation of PKCα in a phosphorylation- and proteasome-dependent manner. Similar deficits in PKCα expression and response sensitivity were observed in acute ocular hypertension and optic never crush models. In conclusion, we find that three separate experimental models of neurodegeneration, often used to specifically target RGCs, disrupt RBCs function prior to the loss of RGCs. Our findings provide useful information for developing new diagnostic tools and treatments for retinal ganglion cells degeneration disease.

Extrasynaptic NMDA Receptors on Rod Pathway Amacrine Cells: Molecular Composition, Activation, and Signaling.

In the rod pathway of the mammalian retina, axon terminals of glutamatergic rod bipolar cells are presynaptic to AII and A17 amacrine cells in the inner plexiform layer. Recent evidence suggests that both amacrines express NMDA receptors, raising questions concerning molecular composition, localization, activation, and function of these receptors. Using dual patch-clamp recording from synaptically connected rod bipolar and AII or A17 amacrine cells in retinal slices from female rats, we found no evidence that NMDA receptors contribute to postsynaptic currents evoked in either amacrine. Instead, NMDA receptors on both amacrine cells were activated by ambient glutamate, and blocking glutamate uptake increased their level of activation. NMDA receptor activation also increased the frequency of GABAergic postsynaptic currents in rod bipolar cells, suggesting that NMDA receptors can drive release of GABA from A17 amacrines. A striking dichotomy was revealed by pharmacological and immunolabeling experiments, which found GluN2B-containing NMDA receptors on AII amacrines and GluN2A-containing NMDA receptors on A17 amacrines. Immunolabeling also revealed a clustered organization of NMDA receptors on both amacrines and a close spatial association between GluN2B subunits and connexin 36 on AII amacrines, suggesting that NMDA receptor modulation of gap junction coupling between these cells involves the GluN2B subunit. Using multiphoton Ca2+ imaging, we verified that activation of NMDA receptors evoked an increase of intracellular Ca2+ in dendrites of both amacrines. Our results suggest that AII and A17 amacrines express clustered, extrasynaptic NMDA receptors, with different and complementary subunits that are likely to contribute differentially to signal processing and plasticity.SIGNIFICANCE STATEMENT Glutamate is the most important excitatory neurotransmitter in the CNS, but not all glutamate receptors transmit fast excitatory signals at synapses. NMDA-type glutamate receptors act as voltage- and ligand-gated ion channels, with functional properties determined by their specific subunit composition. These receptors can be found at both synaptic and extrasynaptic sites on neurons, but the role of extrasynaptic NMDA receptors is unclear. Here, we demonstrate that retinal AII and A17 amacrine cells, postsynaptic partners at rod bipolar dyad synapses, express extrasynaptic (but not synaptic) NMDA receptors, with different and complementary GluN2 subunits. The localization of GluN2A-containing receptors to A17s and GluN2B-containing receptors to AIIs suggests a mechanism for differential modulation of excitability and signaling in this retinal microcircuit.

Contribution of Bipolar Cells of Cone ON and OFF Pathways to Electroretinograms Elicited by Ultraviolet and Middle Wavelength Stimuli.

PURPOSE: To determine the contribution of the ON and OFF cone bipolar cell pathways to the electroretinograms (ERGs) elicited by ultraviolet (UV) and middle wavelength light in mice. MATERIALS AND METHODS: The experiments were performed on 8- to 10-week-old C57BL/6J mice. The ERGs elicited by single-flash and flickering UV light stimuli were compared to those elicited by green light stimuli under photopic conditions. Pharmacological agents were used to selectively block the ON and OFF pathways contributing to the ERGs. Saline was used as a control. The flicker ERGs elicited by UV light were compared to the ERGs elicited by green light after the injection of the pharmacological agents to determine the contribution of the cone ON and OFF pathways to the ERGs. RESULTS: The photopic single-flash and flicker ERGs were more sensitive to the UV light stimuli than to those elicited by green light stimuli. The flicker ERG responses elicited by both UV and green light stimuli at stimulus frequencies lower than 15-Hz decreased after L-2-amino-4-phosphobutyric acid was injected. The ERGs elicited by UV light at 30-Hz and by green light at frequencies lower than 15-Hz decreased significantly after the intravitreal injection of cis-2, 3-piperidine-dicarboxylic acid. An analysis of the ON and OFF components of the flicker ERGs showed that there might be pharmacological differences between the UV light-sensitive responses and the green light-sensitive responses. CONCLUSIONS: These results suggest that the UV light-sensitive cones connect to both the ON and OFF bipolar cells differently than that of the green light-sensitive cones.

Inhibition of non-NMDA ionotropic glutamate receptors delays the retinal degeneration in rd10 mouse.

Retinitis pigmentosa (RP) is a hereditary blinding disease characterized by neurodegeneration of photoreceptors. Retinal ganglion cells (RGCs) in animal models of RP exhibit an abnormally high spontaneous activity that interferes with signal processing. Blocking AMPA/Kainate receptors by bath application of CNQX decreases the spontaneous firing, suggesting that inhibiting these receptors in vivo may help maintain the function of inner retinal neurons in rd10 mice experiencing photoreceptor degeneration. To test this, rd10 mice were i.p. injected with CNQX or GYKI 52466 (an AMPA receptor antagonist) for 1-2 weeks, and examined for their retinal morphology (by immunocytochemistry), function (by MEA recordings) and visual behaviors (using a black/white box). Our data show that iGluRs were up-regulated in the inner plexiform layer (IPL) of rd10 retinas. Application of CNQX at low doses both in vitro and in vivo, attenuated the abnormal spontaneous spiking in RGCs, and increased the light-evoked response of ON RGCs, whereas GYKI 52466 had little effect. CNQX application also improved the behavioral performance. Interestingly, in vivo administration of CNQX delayed photoreceptor degeneration, evidenced by the increased cell number and restored structure. CNQX also improved the structure of bipolar cells. Together, we demonstrated that during photoreceptor degeneration, blockade of the non-NMDA iGluRs decelerates the progression of RGCs dysfunction, possibly by dual mechanisms including slowing photoreceptor degeneration and modulating signal processing within the IPL. Accordingly, this strategy may effectively extend the time window for treating RP.

Trehalose reduces retinal degeneration, neuroinflammation and storage burden caused by a lysosomal hydrolase deficiency.

The accumulation of undegraded molecular material leads to progressive neurodegeneration in a number of lysosomal storage disorders (LSDs) that are caused by functional deficiencies of lysosomal hydrolases. To determine whether inducing macroautophagy/autophagy via small-molecule therapy would be effective for neuropathic LSDs due to enzyme deficiency, we treated a mouse model of mucopolysaccharidosis IIIB (MPS IIIB), a storage disorder caused by deficiency of the enzyme NAGLU (alpha-N-acetylglucosaminidase [Sanfilippo disease IIIB]), with the autophagy-inducing compound trehalose. Treated naglu-/ - mice lived longer, displayed less hyperactivity and anxiety, retained their vision (and retinal photoreceptors), and showed reduced inflammation in the brain and retina. Treated mice also showed improved clearance of autophagic vacuoles in neuronal and glial cells, accompanied by activation of the TFEB transcriptional network that controls lysosomal biogenesis and autophagic flux. Therefore, small-molecule-induced autophagy enhancement can improve the neurological symptoms associated with a lysosomal enzyme deficiency and could provide a viable therapeutic approach to neuropathic LSDs. ABBREVIATIONS: ANOVA: analysis of variance; Atg7: autophagy related 7; AV: autophagic vacuoles; CD68: cd68 antigen; ERG: electroretinogram; ERT: enzyme replacement therapy; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFAP: glial fibrillary acidic protein; GNAT2: guanine nucleotide binding protein, alpha transducing 2; HSCT: hematopoietic stem cell transplantation; INL: inner nuclear layer; LC3: microtubule-associated protein 1 light chain 3 alpha; MPS: mucopolysaccharidoses; NAGLU: alpha-N-acetylglucosaminidase (Sanfilippo disease IIIB); ONL: outer nuclear layer; PBS: phosphate-buffered saline; PRKCA/PKCα: protein kinase C, alpha; S1BF: somatosensory cortex; SQSTM1: sequestosome 1; TEM: transmission electron microscopy; TFEB: transcription factor EB; VMP/VPL: ventral posterior nuclei of the thalamus.

Functional and Morphological Analysis of OFF Bipolar Cells.

Retinal first-order neurons, photoreceptors, receive visual inputs and convert them to neural signals. The second-order neurons, bipolar cells then sort out the visual signals and encode them through multiple neural streams. Approximately 15 morphological types of bipolar cells have been identified, which are thought to encode different aspects of visual signals such as motion and color (Ichinose et al. J Neurosci 34(26):8761-8771, 2014; Euler et al. Nat Rev Neurosci 15(8):507-519, 2014). To investigate functional aspects of OFF bipolar cells, single cell recordings are preferred; however, bipolar cells in the mouse retina are small and hard to distinguish from other types of cells. Here, we describe our methodology and tips for immunohistochemistry and patch clamp recordings for analyzing light-evoked responses in each type of OFF bipolar cell.

Quantifying the effect of light activated outer and inner retinal inhibitory pathways on glutamate release from mixed bipolar cells.

Inhibition mediated by horizontal and amacrine cells in the outer and inner retina, respectively, are fundamental components of visual processing. Here, our purpose was to determine how these different inhibitory processes affect glutamate release from ON bipolar cells when the retina is stimulated with full-field light of various intensities. Light-evoked membrane potential changes (ΔVm ) were recorded directly from axon terminals of intact bipolar cells receiving mixed rod and cone inputs (Mbs) in slices of dark-adapted goldfish retina. Inner and outer retinal inhibition to Mbs was blocked with bath applied picrotoxin (PTX) and NBQX, respectively. Then, control and pharmacologically modified light responses were injected into axotomized Mb terminals as command potentials to induce voltage-gated Ca2+ influx (QCa ) and consequent glutamate release. Stimulus-evoked glutamate release was quantified by the increase in membrane capacitance (ΔCm ). Increasing depolarization of Mb terminals upon removal of inner and outer retinal inhibition enhanced the ΔVm /QCa ratio equally at a given light intensity and inhibition did not alter the overall relation between QCa and ΔCm . However, relative to control, light responses recorded in the presence of PTX and PTX + NBQX increased ΔCm unevenly across different stimulus intensities: at dim stimulus intensities predominantly the inner retinal GABAergic inhibition controlled release from Mbs, whereas the inner and outer retinal inhibition affected release equally in response to bright stimuli. Furthermore, our results suggest that non-linear relationship between QCa and glutamate release can influence the efficacy of inner and outer retinal inhibitory pathways to mediate Mb output at different light intensities.

The association between laminin and microglial morphology in vitro.

Microglia are immune cells in the central nervous system (CNS) that contribute to primary innate immune responses. The morphology of microglia is closely associated with their functional activities. The majority of microglial studies have focused on the ramified or amoeboid morphology; however, bipolar/rod-shaped microglia have recently received much attention. Bipolar/rod-shaped microglia form trains with end-to-end alignment in injured brains and retinae, which is proposed as an important mechanism in CNS repair. We previously established a cell culture model system to enrich bipolar/rod-shaped microglia simply by growing primary microglia on scratched poly-D-lysine (PDL)/laminin-coated surfaces. Here, we investigated the role of laminin in morphological changes of microglia. Bipolar/rod-shaped microglia trains were transiently formed on scratched surfaces without PDL/laminin coating, but the microglia alignment disappeared after 3 days in culture. Amoeboid microglia digested the surrounding laminin, and the gene and protein expression of laminin-cleaving genes Adam9 and Ctss was up-regulated. Interestingly, lipopolysaccharide (LPS)-induced transformation from bipolar/rod-shaped into amoeboid microglia increased the expression of Adam9 and Ctss, and the expression of these genes in LPS-treated amoeboid-enriched cultures remained unchanged. These results indicate a strong association between laminin and morphological transformation of microglia, shedding new light on the role of bipolar/rod-shaped microglia in CNS repair.

Complexin 3 Increases the Fidelity of Signaling in a Retinal Circuit by Regulating Exocytosis at Ribbon Synapses.

Complexin (Cplx) proteins modulate the core SNARE complex to regulate exocytosis. To understand the contributions of Cplx to signaling in a well-characterized neural circuit, we investigated how Cplx3, a retina-specific paralog, shapes transmission at rod bipolar (RB)→AII amacrine cell synapses in the mouse retina. Knockout of Cplx3 strongly attenuated fast, phasic Ca(2+)-dependent transmission, dependent on local [Ca(2+)] nanodomains, but enhanced slower Ca(2+)-dependent transmission, dependent on global intraterminal [Ca(2+)] ([Ca(2+)]I). Surprisingly, coordinated multivesicular release persisted at Cplx3(-/-) synapses, although its onset was slowed. Light-dependent signaling at Cplx3(-/-) RB→AII synapses was sluggish, owing largely to increased asynchronous release at light offset. Consequently, propagation of RB output to retinal ganglion cells was suppressed dramatically. Our study links Cplx3 expression with synapse and circuit function in a specific retinal pathway and reveals a role for asynchronous release in circuit gain control.

Pituitary Adenylate Cyclase Activating Polypeptide, A Potential Therapeutic Agent for Diabetic Retinopathy in Rats: Focus on the Vertical Information Processing Pathway.

Pituitary adenylate cyclase activating polypeptide (PACAP) is a neurotrophic and neuroprotective peptide that has been shown to exert protective effects in different neuronal injuries, such as retinal degenerations. Diabetic retinopathy (DR), the most common complication of diabetes, affects the microvasculature and neuronal architecture of the retina. We have proven earlier that PACAP is also protective in a rat model of DR. In this study, streptozotocin-induced DR was treated with intravitreal PACAP administration in order to further analyze the synaptic structure and proteins of PACAP-treated diabetic retinas, primarily in the vertical information processing pathway. Streptozotocin-treated Wistar rats received intravitreal PACAP injection three times into the right eye 2 weeks after the induction of diabetes. Morphological and molecular biological (qRT-PCR; Western blot) methods were used to analyze retinal synapses (ribbons, conventional) and related structures. Electron microscopic analysis revealed that retinal pigment epithelium, the ribbon synapses and other synaptic profiles suffered alterations in diabetes. However, in PACAP-treated diabetic retinas more bipolar ribbon synapses were found intact in the inner plexiform layer than in DR animals. The ribbon synapse was marked with C-terminal binding protein 2/Bassoon and formed horseshoe-shape ribbons, which were more retained in PACAP-treated diabetic retinas than in DR rats. These results are supported by molecular biological data. The selective degeneration of related structures such as bipolar and ganglion cells could be ameliorated by PACAP treatment. In summary, intravitreal administration of PACAP may have therapeutic potential in streptozotocin-induced DR through maintaining synapse integrity in the vertical pathway.

Increased proliferation of late-born retinal progenitor cells by gestational lead exposure delays rod and bipolar cell differentiation.

PURPOSE: Studies of neuronal development in the retina often examine the stages of proliferation, differentiation, and synaptic development, albeit independently. Our goal was to determine if a known neurotoxicant insult to a population of retinal progenitor cells (RPCs) would affect their eventual differentiation and synaptic development. To that end, we used our previously published human equivalent murine model of low-level gestational lead exposure (GLE). Children and animals with GLE exhibit increased scotopic electroretinogram a- and b-waves. Adult mice with GLE exhibit an increased number of late-born RPCs, a prolonged period of RPC proliferation, and an increased number of late-born rod photoreceptors and rod and cone bipolar cells (BCs), with no change in the number of late-born Müller glial cells or early-born neurons. The specific aims of this study were to determine whether increased and prolonged RPC proliferation alters the spatiotemporal differentiation and synaptic development of rods and BCs in early postnatal GLE retinas compared to control retinas. METHODS: C57BL/6N mouse pups were exposed to lead acetate via drinking water throughout gestation and until postnatal day 10, which is equivalent to the human gestation period for retinal neurogenesis. RT-qPCR, immunohistochemical analysis, and western blots of well-characterized, cell-specific genes and proteins were performed at embryonic and early postnatal ages to assess rod and cone photoreceptor differentiation, rod and BC differentiation and synaptic development, and Müller glial cell differentiation. RESULTS: Real-time quantitative PCR (RT-qPCR) with the rod-specific transcription factors Nrl, Nr2e3, and Crx and the rod-specific functional gene Rho, along with central retinal confocal studies with anti-recoverin and anti-rhodopsin antibodies, revealed a two-day delay in the differentiation of rod photoreceptors in GLE retinas. Rhodopsin immunoblots supported this conclusion. No changes in glutamine synthetase gene or protein expression, a marker for late-born Müller glial cells, were observed in the developing retinas. In the retinas from the GLE mice, anti-PKCα, -Chx10 (Vsx2) and -secretagogin antibodies revealed a two- to three-day delay in the differentiation of rod and cone BCs, whereas the expression of the proneural and BC genes Otx2 and Chx10, respectively, increased. In addition, confocal studies of proteins associated with functional synapses (e.g., vesicular glutamate transporter 1 [VGluT1], plasma membrane calcium ATPase [PMCA], transient receptor potential channel M1 [TRPM1], and synaptic vesicle glycoprotein 2B [SV2B]) revealed a two-day delay in the formation of the outer and inner plexiform layers of the GLE retinas. Moreover, several markers revealed that the initiation of the differentiation and intensity of the labeling of early-born cells in the retinal ganglion cell and inner plexiform layers were not different in the control retinas. CONCLUSIONS: Our combined gene, confocal, and immunoblot findings revealed that the onset of rod and BC differentiation and their subsequent synaptic development is delayed by two to three days in GLE retinas. These results suggest that perturbations during the early proliferative stages of late-born RPCs fated to be rods and BCs ultimately alter the coordinated time-dependent progression of rod and BC differentiation and synaptic development. These GLE effects were selective for late-born neurons. Although the molecular mechanisms are unknown, alterations in soluble neurotrophic factors and/or their receptors are likely to play a role. Since neurodevelopmental delays and altered synaptic connectivity are associated with neuropsychiatric and behavioral disorders as well as cognitive deficits, future work is needed to determine if similar effects occur in the brains of GLE mice and whether children with GLE experience similar delays in retinal and brain neuronal differentiation and synaptic development.

Morphological and physiological analysis of type-5 and other bipolar cells in the Mouse Retina.

Retinal bipolar cells are second-order neurons in the visual system, which initiate multiple image feature-based neural streams. Among more than ten types of bipolar cells, type-5 cells are thought to play a role in motion detection pathways. Multiple subsets of type-5 cells have been reported; however, detailed characteristics of each subset have not yet been elucidated. Here, we found that they exhibit distinct morphological features as well as unique voltage-gated channel expression. We have conducted electrophysiological and immunohistochemical analysis of retinal bipolar cells. We defined type-5 cells by their axon terminal ramification in the inner plexiform layer between the border of ON/OFF sublaminae and the ON choline acetyltransferase (ChAT) band. We found three subsets of type-5 cells: XBCs had the widest axon terminals that stratified at a close approximation of the ON ChAT band as well as exhibiting large voltage-gated Na(+) channel activity, type-5-1 cells had compact terminals and no Na(+) channel activity, and type-5-2 cells contained umbrella-shaped terminals as well as large voltage-gated Na(+) channel activity. Hyperpolarization-activated cyclic nucleotide-gated (HCN) currents were also evoked in all type-5 bipolar cells. We found that XBCs and type-5-2 cells exhibited larger HCN currents than type-5-1 cells. Furthermore, the former two types showed stronger HCN1 expression than the latter. Our previous observations (Ichinose et al., 2014) match the current study: low temporal tuning cells that we named 5S corresponded to 5-1 in this study, while high temporal tuning 5f cells from the previous study corresponded to 5-2 cells. Taken together, we found three subsets of type-5 bipolar cells based on their morphologies and physiological features.

Mitochondrial Protection by Exogenous Otx2 in Mouse Retinal Neurons.

OTX2 (orthodenticle homeobox 2) haplodeficiency causes diverse defects in mammalian visual systems ranging from retinal dysfunction to anophthalmia. We find that the retinal dystrophy of Otx2(+/GFP) heterozygous knockin mice is mainly due to the loss of bipolar cells and consequent deficits in retinal activity. Among bipolar cell types, OFF-cone bipolar subsets, which lack autonomous Otx2 gene expression but receive Otx2 proteins from photoreceptors, degenerate most rapidly in Otx2(+/GFP) mouse retinas, suggesting a neuroprotective effect of the imported Otx2 protein. In support of this hypothesis, retinal dystrophy in Otx2(+/GFP) mice is prevented by intraocular injection of Otx2 protein, which localizes to the mitochondria of bipolar cells and facilitates ATP synthesis as a part of mitochondrial ATP synthase complex. Taken together, our findings demonstrate a mitochondrial function for Otx2 and suggest a potential therapeutic application of OTX2 protein delivery in human retinal dystrophy.