Gian Carlo Demontis

Properties and functional roles of hyperpolarization‐gated currents in guinea‐pig retinal rods

1 The inward rectification induced by membrane hyperpolarization was studied in adult guinea‐pig rods by the perforated‐patch‐clamp technique. 2 CsCl blocked the rectification observed in both voltage‐ and current‐clamp recordings at voltages negative to −60 mV, while BaCl2 blocked the inward relaxation observed at voltages positive to −60 mV. The current activated at −90 mV had a low selectivity between sodium and potassium and reversed at −31.0 mV. 3 These observations suggest that two inward rectifiers are present in guinea‐pig rods: a hyperpolarization‐activated (Ih) and a hyperpolarization‐deactivated (Ikx) current. The functional roles of Ih and Ikx were evaluated by stimulating rods with currents sinusoidally modulated in time. 4 Rods behave like bandpass amplifiers, with a peak amplification of 1.5 at about 2 Hz. For hyperpolarizations that mainly gate Ikx, amplification and phase shifts are fully accounted for by a rod membrane analogue model that includes an inductance. For hyperpolarizations that also gate Ih, a harmonic distortion became apparent. 5 Bandpass filtering and amplification of rod signals, associated with Ih and Ikx gating by membrane hyperpolarization, are strategically located to extend, beyond the limits imposed by the slow phototransductive cascade, the temporal resolution of signals spreading to the rod synapse.

Adult human Müller glia cells are a highly efficient source of rod photoreceptors.

There is growing evidence that Müller glia cells (MGCs) might act as regenerative elements in injured retinas of fishes and amniotes. However, their differentiation potential in humans is yet unknown. We isolated Müller glia from adult human retinas and propagated them in vitro revealing for the first time their ability to differentiate into rod photoreceptors. These results were also confirmed with mice retinas. Here, we describe conditions by which human MGCs adopt a rod photoreceptor commitment with a surprising efficiency as high as 54%. Functional characterization of Müller glia‐derived photoreceptors by patch‐clamp recordings revealed that their electrical properties are comparable to those of adult rods. Interestingly, our procedure allowed efficient derivation of MGC cultures starting from both injured and degenerating and postmortem human retinas. Human transplanted Müller glia‐derived photoreceptors integrate and survive within immunodeficient mouse retinas. These data provide evidence that Müller glia retains an unpredicted plasticity and multipotent potential into adulthood, and it is therefore a promising source of novel therapeutic applications in retinal repair. STEM CELLS 2011;29:344–356

Apoptosis and adaptive responses to oxidative stress in human endothelial cells exposed to cyclosporin A correlate with BCL-2 expression levels.

Treatment of transplanted patients with cyclosporin A (CSA) may cause adverse effects such as nephrotoxicity and hypertension. As CSA is known to induce oxidative stress in several tissues, it may cause vascular problems by triggering oxidative stress in endothelial cells (EC). However, oxidative stress has been reported for acute exposure to CSA concentra¬tions exceeding its clinical range, whereas immunosuppression requires life‐long treatment with therapeutic concentrations. We therefore compared the effects of 21 h pharmacological (200 μM) vs. 8 days clinical (0.5–2.5 μM) doses of CSA on cultured human EC. Pharmacological doses of CSA cause a decrease in cell density via apoptosis and a down‐regulation of the antiapoptotic protein Bcl‐2. However, these effects are independent of CSA‐induced oxidative stress. In con¬trast, therapeutic concentrations of CSA cause Bcl‐2 up‐regulation and modification of EC morphology, both effects blocked by antioxidants. Therefore, a low level of oxidants may act in EC as second messengers that up‐regulate Bcl‐2, thus promoting survival of im¬paired EC. Our data suggest that the oxidative stress induced by clinical concentrations of CSA may be involved in the adverse effects of the drug on the vascular system of transplanted patients via an adaptive response involving Bcl‐2 up‐regulation rather than an apoptotic process.—Longoni, B., Boschi, E., Demontis, G. C., Ratto, G. M., Mosca, F. Apoptosis and adaptive responses to oxidative stress in human endothelial cells exposed to cyclosporin A correlate with BCL‐2 expres¬sion levels. FASEB J. 15, 731‐740 (2001)

Light sensitivity, adaptation and saturation in mammalian rods

Publisher Summary This chapter discusses the absolute sensitivity, background desensitization, and saturation in guinea-pig rods. The principal purpose of this study is to establish the extent and the conditions for rod adaptation in mammals and compare them with those of lower vertebrates. In agreement with a recent report, it was found that guinea pig rods possess adaptation properties qualitatively similar to those of amphibian rods. It was also found that light-adapted guinea-pig rods may efficiently signal light changes up to background levels that for a human subject would be equivalent to ambient illuminations of over l0 3 candles (cd)/m2. Considering that a dark-adapted rod may generate a detectable response to the absorption of a few photons, the range of light intensity over which a single rod may efficiently contribute to vision covers more than 4 log units.

Cellular mechanisms underlying the pharmacological induction of phosphenes

Visual sensations evoked by stimuli other than luminance changes are called phosphenes. Phosphenes may be an early symptom in a variety of diseases of the retina or of the visual pathways, but healthy individuals may perceive them as well. Phosphene‐like phenomena are perhaps the most common side effect reported in clinical pharmacology. Ivabradine, a novel anti‐anginal drug that reduces heart‐rate by inhibiting the hyperpolarization activated current expressed in cardiac sinoatrial node cells (If) induces phosphenes in some patients. One hypothesis is that ivabradine interacts with the visual system by inhibiting hyperpolarization‐activated current in retinal cells (Ih). An Ih current with properties similar to cardiac If has been reported in retinal neurones. Under normal circumstances most of the random fluctuations generated within the retinal circuits do not reach the level of conscious perception because they are filtered out. Presumably, filtering occurs mostly within the retina and one serious candidate for this action is the ability of Ih to act as a negative‐feedback mechanism. Ih activation in the membrane of visual cells causes dampening of responses to slow noisy inputs thus tuning the visual system to perceptually more relevant signals of higher frequency. Ih inhibition, by altering at the retinal synapses the filtering of signals generated by thermal breakdown of rhodopsin or other fluctuations, is expected to increase the probability of phosphene occurrence. It is the purpose of the present paper to outline and discuss the features of the visual system and the pharmacological conditions relevant to phosphene perception.

Mesenchymal Stem Cells Prevent Acute Rejection and Prolong Graft Function in Pancreatic Islet Transplantation

BACKGROUND Pancreatic islet transplantation is a promising cell-based therapy for type 1 diabetes (insulin-dependent diabetes mellitus), a disease triggered by the immune response against autoantigens of beta-cells. However, the recurrence of immune response after transplantation and the diabetogenic and growth-stunting side effects of immunosuppressants are major challenges to the application of islet transplantation. Mesenchymal stem cells (MSCs) have recently been reported to modulate the immune response in allogeneic transplantation. METHODS The ability of MSCs, either syngeneic or allogeneic to recipients, to prevent acute rejection and improve glycemic control was investigated in rats with diabetes given a marginal mass of pancreatic islets through the portal vein. RESULTS Reduced glucose levels and low-grade rejections were observed up to 15 days after transplantation upon triple-dose administration of MSCs, indicating that MSCs prolong graft function by preventing acute rejection. The efficacy of MSCs was associated with a reduction of pro-inflammatory cytokines and was independent of the administration route. Efficacy was similar for MSCs whether syngeneic or allogeneic to recipients and comparable to that of immunosuppressive therapy. CONCLUSIONS The results show that MSCs modulate the immune response through a down-regulation of pro-inflammatory cytokines, suggesting that MSCs may prevent acute rejection and improve graft function in portal vein pancreatic islet transplantation.

Functional characterisation and subcellular localisation of HCN1 channels in rabbit retinal rod photoreceptors

Gating of voltage‐dependent conductances in retinal photoreceptors is the first step of a process leading to the enhancement of the temporal performance of the visual system. The molecular components underlying voltage‐dependent gating in rods are presently poorly defined. In the present work we have investigated the isoform composition and the functional characteristics of hyperpolarisation‐activated cyclic nucleotide‐gated channels (HCN) in rabbit rods. Using immunocytochemistry we show the expression in the inner segment and cell body of the isoform 1 (HCN1). Electrophysiological investigations show that hyperpolarisation‐activated currents (Ih) can be measured only from the cell regions where HCN1 is expressed. Half‐activation voltage (–75.0 ± 0.3 mV) and kinetics (t1/2 of 101 ± 8 ms at –110 mV and 20 °C) of the Ih in rods are similar to those of the macroscopic current carried by homomeric rabbit HCN1 channels expressed in HEK 293 cells. The homomeric nature of HCN1 channels in rods is compatible with the observation that cAMP induces a small shift (2.3 ± 0.8 mV) in the half‐activation voltage of Ih. In addition, the observation that within the physiological range of membrane potentials, cAMP does not significantly affect the gain of the current‐to‐voltage conversion, may reflect the need to protect the first step in the processing of visual signals from changes in cAMP turnover.

High-Pass Filtering of Input Signals by the Ih Current in a Non-Spiking Neuron, the Retinal Rod Bipolar Cell

Hyperpolarization–activated cyclic nucleotide–sensitive (HCN) channels mediate the If current in heart and Ih throughout the nervous system. In spiking neurons Ih participates primarily in different forms of rhythmic activity. Little is known, however, about its role in neurons operating with graded potentials as in the retina, where all four channel isoforms are expressed. Intriguing evidence for an involvement of Ih in early visual processing are the side effects reported, in dim light or darkness, by cardiac patients treated with HCN inhibitors. Moreover, electroretinographic recordings indicate that these drugs affect temporal processing in the outer retina. Here we analyzed the functional role of HCN channels in rod bipolar cells (RBCs) of the mouse. Perforated–patch recordings in the dark–adapted slice found that RBCs exhibit Ih, and that this is sensitive to the specific blocker ZD7288. RBC input impedance, explored by sinusoidal frequency–modulated current stimuli (0.1–30 Hz), displays band–pass behavior in the range of Ih activation. Theoretical modeling and pharmacological blockade demonstrate that high–pass filtering of input signals by Ih, in combination with low–pass filtering by passive properties, fully accounts for this frequency–tuning. Correcting for the depolarization introduced by shunting through the pipette–membrane seal, leads to predict that in darkness Ih is tonically active in RBCs and quickens their responses to dim light stimuli. Immunohistochemistry targeting candidate subunit isoforms HCN1–2, in combination with markers of RBCs (PKC) and rod–RBC synaptic contacts (bassoon, mGluR6, Kv1.3), suggests that RBCs express HCN2 on the tip of their dendrites. The functional properties conferred by Ih onto RBCs may contribute to shape the retinas light response and explain the visual side effects of HCN inhibitors.

Effects of blocking the hyperpolarization-activated current (Ih) on the cat electroretinogram

The temporal properties of the electroretinogram (ERG) recorded from cat eyes were analyzed in the presence of either Cs+ or zatebradine which are known to inhibit the hyperpolarization activated current (Ih) in retinal rods. Both Cs+ and zatebradine reduce the ERG response to high-frequency sinusoidal stimuli of high mean luminance and contrast. Conversely, blockade of Ih has no effect on the frequency response characteristics of the isolated receptor component (PIII). These observations support the idea that Ih plays an important role in the transfer of signals from photoreceptors to second order neurons by suppressing the slow components originated in the phototransductive cascade. The result of this operation is an enhancement of the light response in a range of temporal frequencies relevant to vision.

Noggin Elicits Retinal Fate in Xenopus Animal Cap Embryonic Stem Cells

Driving specific differentiation pathways in multipotent stem cells is a main goal of cell therapy. Here we exploited the differentiating potential of Xenopus animal cap embryonic stem (ACES) cells to investigate the factors necessary to drive multipotent stem cells toward retinal fates. ACES cells are multipotent, and can be diverged from their default ectodermal fate to give rise to cell types from all three germ layers. We found that a single secreted molecule, Noggin, is sufficient to elicit retinal fates in ACES cells. Reverse‐transcription polymerase chain reaction, immunohistochemistry, and in situ hybridization experiments showed that high doses of Noggin are able to support the expression of terminal differentiation markers of the neural retina in ACES cells in vitro. Following in vivo transplantation, ACES cells expressing high Noggin doses form eyes, both in the presumptive eye field region and in ectopic posterior locations. The eyes originating from the transplants in the eye field region are functionally equivalent to normal eyes, as seen by electrophysiology and c‐fos expression in response to light. Our data show that in Xenopus embryos, proper doses of a single molecule, Noggin, can drive ACES cells toward retinal cell differentiation without additional cues. This makes Xenopus ACES cells a suitable model system to direct differentiation of stem cells toward retinal fates and encourages further studies on the role of Noggin in the retinal differentiation of mammalian stem cells. STEM CELLS 2009;27:2146–2152