Inhibitory inputs to an inhibitory interneuron: Spontaneous postsynaptic currents and GABAA receptors of A17 amacrine cells in the rat retina.

Amacrine cells constitute a large and heterogeneous group of inhibitory interneurons in the retina. The A17 amacrine plays an important role for visual signalling in the rod pathway microcircuit of the mammalian retina. It receives excitatory input from rod bipolar cells and provides feedback inhibition to the same cells. However, from ultrastructural investigations, there is evidence for input to A17s from other types of amacrine cells, presumably inhibitory, but there is a lack of information about the identity and functional properties of the synaptic receptors and how inhibition contributes to the integrative properties of A17s. Here, we studied the biophysical and pharmacological properties of GABAergic spontaneous inhibitory postsynaptic currents (spIPSCs) and GABAA receptors of A17 amacrines using whole-cell and outside-out patch recordings from rat retinal slices. The spIPSCs displayed fast onsets (10%-90% rise time ~740 µs) and double-exponential decays (τfast ~4.5 ms [43% of amplitude]; τslow ~22 ms). Ultra-fast application of brief pulses of GABA (3 mM) to patches evoked responses with deactivation kinetics best fitted by a triple-exponential function (τ1 ~5.3 ms [55% of amplitude]; τ2 ~48 ms [32% of amplitude]; τ3 ~187 ms). Non-stationary noise analysis of spIPSCs and patch responses yielded single-channel conductances of ~21 and ~25 pS, respectively. Pharmacological analysis suggested that the spIPSCs are mediated by receptors with an α1ßγ2 subunit composition and the somatic receptors have an α2ßγ2 and/or α3ßγ2 composition. These results demonstrate the presence of synaptic GABAA receptors on A17s, which may play an important role in signal integration in these cells.

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.

Elevated Glucose and Interleukin-1ß Differentially Affect Retinal Microglial Cell Proliferation.

Diabetic retinopathy is considered a neurovascular disorder, hyperglycemia being considered the main risk factor for this pathology. Diabetic retinopathy also presents features of a low-grade chronic inflammatory disease, including increased levels of cytokines in the retina, such as interleukin-1 beta (IL-1ß). However, how high glucose and IL-1ß affect the different retinal cell types remains to be clarified. In retinal neural cell cultures, we found that IL-1ß and IL-1RI are present in microglia, macroglia, and neurons. Exposure of retinal neural cell cultures to high glucose upregulated both mRNA and protein levels of IL-1ß. High glucose decreased microglial and macroglial cell proliferation, whereas IL-1ß increased their proliferation. Interestingly, under high glucose condition, although the number of microglial cells decreased, they showed a less ramified morphology, suggesting a more activated state, as supported by the upregulation of the levels of ED-1, a marker of microglia activation. In conclusion, IL-1ß might play a key role in diabetic retinopathy, affecting microglial and macroglial cells and ultimately contributing to neural changes observed in diabetic patients. Particularly, since IL-1ß has an important role in retinal microglia activation and proliferation under diabetes, limiting IL-1ß-triggered inflammatory processes may provide a new therapeutic strategy to prevent the progression of diabetic retinopathy.

Disruption of a neural microcircuit in the rod pathway of the mammalian retina by diabetes mellitus.

Diabetes leads to dysfunction of the neural retina before and independent of classical microvascular diabetic retinopathy, but previous studies have failed to demonstrate which neurons and circuits are affected at the earliest stages. Here, using patch-clamp recording and two-photon Ca(2+) imaging in rat retinal slices, we investigated diabetes-evoked changes in a microcircuit consisting of rod bipolar cells and their dyad postsynaptic targets, AII and A17 amacrine cells, which play an essential role in processing scotopic visual signals. AII amacrines forward their signals to ON- and OFF-cone bipolar cells and A17 amacrines provide GABAergic feedback inhibition to rod bipolar cells. Whereas Ca(2+)-permeable AMPA receptors mediate input from rod bipolar cells to both AII and A17 amacrines, diabetes changes the synaptic receptors on A17, but not AII amacrine cells. This was expressed as a change in pharmacological properties and single-channel conductance of the synaptic receptors, consistent with an upregulation of the AMPA receptor GluA2 subunit and reduced Ca(2+) permeability. In addition, two-photon imaging revealed reduced agonist-evoked influx of Ca(2+) in dendritic varicosities of A17 amacrine cells from diabetic compared with normal animals. Because Ca(2+)-permeable receptors in A17 amacrine cells mediate synaptic release of GABA, the reduced Ca(2+) permeability of these receptors in diabetic animals leads to reduced release of GABA, followed by disinhibition and increased release of glutamate from rod bipolar cells. This perturbation of neuron and microcircuit dynamics can explain the decreased dynamic range and sensitivity of scotopic vision that has been observed in diabetes.

Diabetic hyperglycemia reduces Ca2+ permeability of extrasynaptic AMPA receptors in AII amacrine cells.

There is increasing evidence that diabetic retinopathy is a primary neuropathological disorder that precedes the microvascular pathology associated with later stages of the disease. Recently, we found evidence for altered functional properties of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in A17, but not AII, amacrine cells in the mammalian retina, and the observed changes were consistent with an upregulation of the GluA2 subunit, a key determinant of functional properties of AMPA receptors, including Ca(2+) permeability and current-voltage (I-V) rectification properties. Here, we have investigated functional changes of extrasynaptic AMPA receptors in AII amacrine cells evoked by diabetes. With patch-clamp recording of nucleated patches from retinal slices, we measured Ca(2+) permeability and I-V rectification in rats with ∼3 wk of streptozotocin-induced diabetes and age-matched, noninjected controls. Under bi-ionic conditions (extracellular Ca(2+) concentration = 30 mM, intracellular Cs(+) concentration = 171 mM), the reversal potential (Erev) of AMPA-evoked currents indicated a significant reduction of Ca(2+) permeability in diabetic animals [Erev = -17.7 mV, relative permeability of Ca(2+) compared with Cs(+) (PCa/PCs) = 1.39] compared with normal animals (Erev = -7.7 mV, PCa/PCs = 2.35). Insulin treatment prevented the reduction of Ca(2+) permeability. I-V rectification was examined by calculating a rectification index (RI) as the ratio of the AMPA-evoked conductance at +40 and -60 mV. The degree of inward rectification in patches from diabetic animals (RI = 0.48) was significantly reduced compared with that in normal animals (RI = 0.30). These results suggest that diabetes evokes a change in the functional properties of extrasynaptic AMPA receptors of AII amacrine cells. These changes could be representative for extrasynaptic AMPA receptors elsewhere in AII amacrine cells and suggest that synaptic and extrasynaptic AMPA receptors are differentially regulated.

Diabetes causes transient changes in the composition and phosphorylation of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors and interaction with auxiliary proteins in the rat retina.

PURPOSE: The impairment of glutamatergic neurotransmission has been associated with diabetic complications in the central nervous system, such as diabetic retinopathy. Here, we investigated the effect of elevated glucose exposure and diabetes on α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor composition, subunit phosphorylation, and the association of the GluA2 subunit with accessory proteins in the retina. METHODS: The subunit composition of AMPA receptors and the association of the GluA2 subunit with modulatory proteins were evaluated with coimmunoprecipitation in retinal neural cell cultures and in the retina of experimentally induced-diabetic rats. The phosphorylation status of AMPA receptor subunits was evaluated with western blotting. RESULTS: In retinal neural cell cultures, elevated glucose did not significantly alter the composition of AMPA receptors, namely, the interactions between the GluA1, GluA2, and GluA4 subunits, but reduced GluA2 association with GRIP1. Moreover, elevated glucose did not cause changes on the level of GluA1 phosphorylated at serine residues 831 and 845. Diabetes induced early transitory changes in the interaction between AMPA receptor subunits GluA1, GluA2, and GluA4. At 8 weeks of diabetes, the content of GluA1 phosphorylated at serine 831 or serine 845 in the retina increased, compared to age-matched controls. CONCLUSIONS: Taken together, these results suggest that diabetes induces dynamic changes in AMPA receptor subunit composition, which could affect glutamatergic transmission in the rat retina.

High glucose and interleukin-1beta downregulate interleukin-1 type I receptor (IL-1RI) in retinal endothelial cells by enhancing its degradation by a lysosome-dependent mechanism.

Diabetic retinopathy has been considered a low-grade chronic inflammatory disease. The production of interleukin-1beta (IL-1beta) in the retina is increased, and this finding has been correlated with an increase in blood-retinal barrier permeability, suggesting that IL-1beta might have an important role in the pathogenesis of diabetic retinopathy. However, in this context, no attention has been given to interleukin-1 type I receptor (IL-1RI), which is the receptor responsible for IL-1beta triggered effects. Therefore, we investigated the effect of high glucose and IL-1beta on the IL-1RI regulation in retinal endothelial cells. A time-dependent downregulation of IL-1RI protein levels was detected in retinal endothelial cells exposed (1-24h) to high glucose, mannitol or IL-1beta. Long-term exposure (7days) to high glucose or mannitol also decreased IL-1RI protein content. IL-1RI downregulation was due to its activation by IL-1beta, since it was inhibited by the presence of anti-IL-1RI or anti-IL-1beta antibodies. Moreover, IL-1RI downregulation was prevented by lysosome inhibitors, chloroquine and ammonium chloride, but not by proteasome inhibitors, MG132 and lactacystin. We also found that IL-1RI translocates to the nucleus after high glucose or IL-1beta treatment. In conclusion, our results indicate that high glucose, probably due to osmotic stress, and IL-1beta downregulate IL-1RI in retinal endothelial cells. The downregulation of IL-1RI is triggered by its activation and is due, at least partially, to lysosomal degradation. High glucose and IL-1beta also enhance the translocation of IL-1RI to the nucleus.

High glucose and oxidative/nitrosative stress conditions induce apoptosis in retinal endothelial cells by a caspase-independent pathway.

Diabetic retinopathy (DR) is a leading cause of vision loss among working-age adults. Retinal endothelial cell apoptosis is an early event in DR, and oxidative stress is known to play an important role in this pathology. Recently, we found that high glucose induces apoptosis in retinal neural cells by a caspase-independent mechanism. Here, we investigated the mechanisms underlying retinal endothelial cell apoptosis induced by high glucose and oxidative/nitrosative stress conditions. Endothelial cells (TR-iBRB2 rat retinal endothelial cell line) were exposed to high glucose (long-term exposure, 7 days), or to NOC-18 (nitric oxide donor; 250microM) or H(2)O(2) (100microM) for 24h. Cell viability was assessed by the MTT assay and cell proliferation by [methyl-(3)H]-thymidine incorporation into DNA. Apoptotic cells were detected with Hoechst or Annexin V staining. Active caspases were detected by an apoptosis detection kit. Active caspase-3 and apoptosis-inducing factor (AIF) protein levels were assessed by Western blot or immunohistochemistry. High glucose, NOC-18 and H(2)O(2) increased apoptosis in retinal endothelial cells. High glucose and mannitol decreased cell proliferation, but mannitol did not induce apoptosis. Caspase activation did not increase in high glucose- or NOC-18-treated cells, but it increased in cells exposed to H(2)O(2). However, the protein levels of AIF decreased in mitochondrial fractions and increased in nuclear fractions, in all conditions. These results are the first demonstrating that retinal endothelial cell apoptosis induced by high glucose is independent of caspase activation, and is correlated with AIF translocation to the nucleus. NOC-18 and H(2)O(2) also activate a caspase-independent apoptotic pathway, although H(2)O(2) can also induce caspase-mediated apoptosis.

AMPA receptors at ribbon synapses in the mammalian retina: kinetic models and molecular identity.

In chemical synapses, neurotransmitter molecules released from presynaptic vesicles activate populations of postsynaptic receptors that vary in functional properties depending on their subunit composition. Differential expression and localization of specific receptor subunits are thought to play fundamental roles in signal processing, but our understanding of how that expression is adapted to the signal processing in individual synapses and microcircuits is limited. At ribbon synapses, glutamate release is independent of action potentials and characterized by a high and rapidly changing rate of release. Adequately translating such presynaptic signals into postsynaptic electrical signals poses a considerable challenge for the receptor channels in these synapses. Here, we investigated the functional properties of AMPA receptors of AII amacrine cells in rat retina that receive input at spatially segregated ribbon synapses from OFF-cone and rod bipolar cells. Using patch-clamp recording from outside-out patches, we measured the concentration dependence of response amplitude and steady-state desensitization, the single-channel conductance and the maximum open probability. The GluA4 subunit seems critical for the functional properties of AMPA receptors in AII amacrines and immunocytochemical labeling suggested that GluA4 is located at synapses made by both OFF-cone bipolar cells and rod bipolar cells. Finally, we used a series of experimental observables to develop kinetic models for AII amacrine AMPA receptors and subsequently used the models to explore the behavior of the receptors and responses generated by glutamate concentration profiles mimicking those occurring in synapses. These models will facilitate future in silico modeling of synaptic signaling and processing in AII amacrine cells.

Intensity-based volumetric registration of magnetic resonance images and whole-mount sections of the prostate.

OBJECTIVE: Magnetic Resonance Imaging (MRI) of the prostate provides useful in vivo diagnostic tissue information such as tumor location and aggressiveness, but ex vivo histopathology remains the ground truth. There are several challenges related to the registration of MRI to histopathology. We present a method for registration of standard clinical T2-weighted MRI (T2W-MRI) and transverse histopathology whole-mount (WM) sections of the prostate. METHODS: An isotropic volume stack was created from the WM sections using 2D rigid and deformable registration combined with linear interpolation. The prostate was segmented manually from the T2W-MRI volume and registered to the WM section volume using a combination of affine and deformable registration. The method was evaluated on a set of 12 patients who had undergone radical prostatectomy. Registration accuracy was assessed using volume overlap (Dice Coefficient, DC) and landmark distances. RESULTS: The DC was 0.94 for the whole prostate, 0.63 for the peripheral zone and 0.77 for the remaining gland. The landmark distances were on average 5.4 mm. CONCLUSION: The volume overlap for the whole prostate and remaining gland, as well as the landmark distances indicate good registration accuracy for the proposed method, and shows that it can be highly useful for registering clinical available MRI and WM sections of the prostate.

Long-term exposure to high glucose increases the content of several exocytotic proteins and of vesicular GABA transporter in cultured retinal neural cells.

Diabetic retinopathy is a leading cause of vision loss and blindness. Increasing evidence has shown that the neuronal components of the retina are affected even before the detection of vascular lesions. Hyperglycemia is considered the main pathogenic factor for the development of diabetic complications. Nevertheless, other factors like neuroinflammation, might also contribute for neural changes. To clarify whether hyperglycemia can be the main trigger of synaptic changes, we evaluated whether prolonged elevated glucose per se, mimicking chronic hyperglycemia, is able to change the content and distribution of several exocytotic proteins and vesicular glutamate and GABA transporters in retinal neurons. Moreover, we also tested the hypothesis that an inflammatory stimulus (interleukin-1ß) could exacerbate the effects induced by exposure to elevated glucose, contributing for changes in synaptic proteins in retinal neurons. Rat retinal neural cells were cultured for 9 days. Cells were exposed to elevated D-glucose (30 mM) or D-mannitol (osmotic control), for 7 days, or were exposed to interleukin-1ß (10 ng/ml) or LPS (1 µg/ml) for 24 h. The protein content and distribution of SNARE proteins (SNAP-25, syntaxin-1, VAMP-2), synapsin-1, synaptotagmin-1, rabphilin 3a, VGluT-1 and VGAT, were evaluated by western blotting and immunocytochemistry. The protein content and immunoreactivity of syntaxin-1, synapsin-1, rabphilin 3a and VGAT increased in retinal neural cells exposed to high glucose. No changes were detected when cells were exposed to interleukin-1ß, LPS or mannitol per se. Particularly, exposure to interleukin-1ß for 24 h did not exacerbate the effect of high glucose on the content and immunoreactivity of exocytotic proteins, suggesting the primordial role of hyperglycemia for neuronal changes. In summary, prolonged exposure to elevated glucose alters the total content of several proteins involved in exocytosis, suggesting that hyperglycemia per se is a fundamental factor for neuronal changes caused by diabetes.

Heme oxygenase-1 protects retinal endothelial cells against high glucose- and oxidative/nitrosative stress-induced toxicity.

Diabetic retinopathy is a leading cause of visual loss and blindness, characterized by microvascular dysfunction. Hyperglycemia is considered the major pathogenic factor for the development of diabetic retinopathy and is associated with increased oxidative/nitrosative stress in the retina. Since heme oxygenase-1 (HO-1) is an enzyme with antioxidant and protective properties, we investigated the potential protective role of HO-1 in retinal endothelial cells exposed to high glucose and oxidative/nitrosative stress conditions. Retinal endothelial cells were exposed to elevated glucose, nitric oxide (NO) and hydrogen peroxide (H(2)O(2)). Cell viability and apoptosis were assessed by MTT assay, Hoechst staining, TUNEL assay and Annexin V labeling. The production of reactive oxygen species (ROS) was detected by the oxidation of 2',7'-dichlorodihydrofluorescein diacetate. The content of HO-1 was assessed by immunobloting and immunofluorescence. HO activity was determined by bilirubin production. Long-term exposure (7 days) of retinal endothelial cells to elevated glucose decreased cell viability and had no effect on HO-1 content. However, a short-time exposure (24 h) to elevated glucose did not alter cell viability, but increased both the levels of intracellular ROS and HO-1 content. Moreover, the inhibition of HO with SnPPIX unmasked the toxic effect of high glucose and revealed the protection conferred by HO-1. Oxidative/nitrosative stress conditions increased cell death and HO-1 protein levels. These effects of elevated glucose and HO inhibition on cell death were confirmed in primary endothelial cells (HUVECs). When cells were exposed to oxidative/nitrosative stress conditions there was also an increase in retinal endothelial cell death and HO-1 content. The inhibition of HO enhanced ROS production and the toxic effect induced by exposure to H(2)O(2) and NOC-18 (NO donor). Overexpression of HO-1 prevented the toxic effect induced by H(2)O(2) and NOC-18. In conclusion, HO-1 exerts a protective effect in retinal endothelial cells exposed to hyperglycemic and oxidative/nitrosative stress conditions.

Müller cells do not influence leukocyte adhesion to retinal endothelial cells.

PURPOSE: Diabetic retinopathy is associated with inflammation. The authors investigated the influence of Müller cells on leukocyte adhesion to retinal endothelial cells. METHODS: ICAM-1 levels were assessed by Western blotting and immunocytochemistry. Leukocyte adhesion was quantified using a fluorescence assay. RESULTS: High glucose and oxidative/nitrosative stress conditions increased ICAM-1 levels in endothelial cells and leukocyte adhesion. Under the influence of Müller cells (co-cultures/conditioned medium), the effects were comparable to those found when endothelial cells were exposed, alone, to similar conditions. CONCLUSIONS: These results show that Müller cells do not influence leukocyte adhesion under the in vitro conditions used in this study.