The photovoltage of rods and cones in the dark-adapted mouse retina.

Research on photoreceptors has led to important insights into how light signals are detected and processed in the outer retina. Most information about photoreceptor function, however, comes from lower vertebrates. The large majority of mammalian studies are based on suction pipette recordings of outer segment currents, a technique that doesn't allow examination of phenomena occurring downstream of phototransduction. Only a small number of whole-cell recordings have been made, mainly in the macaque. Due to the growing importance of the mouse in vision research, we have optimized a retinal slice preparation that allows the reliable collection of perforated-patch recordings from light responding rods and cones. Unexpectedly, the frequency of cone recordings was much higher than their numeric proportion of ∼3%. This allowed us to obtain direct functional evidence suggestive of rod­cone coupling in the mouse. Moreover, rods had considerably larger single photon responses than previously published for mammals (3.44 mV, SD 1.37, n = 19 at 24°C; 2.46 mV, SD 1.08, n = 10 at 36°C), and a relatively high signal/noise ratio (6.4, SD 1.8 at 24°C; 6.8, SD 2.8 at 36°C). Both findings imply a more favourable transmission at the rod­rod bipolar cell synapse. Accordingly, relatively few photoisomerizations were sufficient to elicit a half-maximal response (6.7, SD 2.7, n = 5 at 24°C; 10.6, SD 1.7, n = 3 at 36°C), leading to a narrow linear response range. Our study demonstrates new features of mammalian photoreceptors and opens the way for further investigations into photoreceptor function using retinas from mutant mouse models.

Prolonged illumination up-regulates arrestin and two guanylate cyclase activating proteins: a novel mechanism for light adaptation.

Light adaptation in vertebrate photoreceptors is mediated by multiple mechanisms, one of which could involve nuclear feedback and changes in gene expression. Therefore, we have investigated light adaptation-associated changes in gene expression using microarrays and real-time PCR in isolated photoreceptors, in cultured isolated retinas and in acutely isolated retinas. In all three preparations after 2 h of an exposure to a bright light, we observed an up-regulation of almost 100% of three genes, Sag, Guca1a and Guca1b, coding for proteins known to play a major role in phototransduction: arrestin, GCAP1 and GCAP2. No detectable up-regulation occurred for light exposures of less than 1 h. Functional in vivo electroretinographic tests show that a partial recovery of the dark current occurred 1-2 h after prolonged illumination with a steady light that initially caused a substantial suppression of the photoresponse. These observations demonstrate that prolonged illumination results in the up-regulation of genes coding for proteins involved in the phototransduction signalling cascade, possibly underlying a novel component of light adaptation occurring 1-2 h after the onset of a steady bright light.

Involvement of Autophagic Pathway in the Progression of Retinal Degeneration in a Mouse Model of Diabetes.

The notion that diabetic retinopathy (DR) is essentially a micro-vascular disease has been recently challenged by studies reporting that vascular changes are preceded by signs of damage and loss of retinal neurons. As to the mode by which neuronal death occurs, the evidence that apoptosis is the main cause of neuronal loss is far from compelling. The objective of this study was to investigate these controversies in a mouse model of streptozotocin (STZ) induced diabetes. Starting from 8 weeks after diabetes induction there was loss of rod but not of cone photoreceptors, together with reduced thickness of the outer and inner synaptic layers. Correspondingly, rhodopsin expression was downregulated and the scotopic electroretinogram (ERG) is suppressed. In contrast, cone opsin expression and photopic ERG response were not affected. Suppression of the scotopic ERG preceded morphological changes as well as any detectable sign of vascular alteration. Only sparse apoptotic figures were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay and glia was not activated. The physiological autophagy flow was altered instead, as seen by increased LC3 immunostaining at the level of outer plexiform layer (OPL) and upregulation of the autophagic proteins Beclin-1 and Atg5. Collectively, our results show that the streptozotocin induced DR in mouse initiates with a functional loss of the rod visual pathway. The pathogenic pathways leading to cell death develop with the initial dysregulation of autophagy well before the appearance of signs of vascular damage and without strong involvement of apoptosis.

Time course of neurotrophic factor upregulation and retinal protection against light-induced damage after optic nerve section.

PURPOSE: To assess neurotrophic factor upregulation in the retina after damage to the optic nerve and relate that regulation to changes in photoreceptor stability and function. METHODS: Retinas of adult pigmented (Long-Evans) rats were examined at successive times (1-60 days) after unilateral optic nerve section. The distribution and expression of ciliary neurotrophic factor (CNTF) and basic fibroblast growth factor (FGF-2) and their receptor elements FGFR1 and CNTFRalpha were studied with immunohistochemistry and Western blot analysis. FGF-2 and CNTF mRNA levels were also assessed, with semiquantitative reverse transcription-PCR. Levels and localization of the intracellular signaling molecule ERK and its activated, phosphorylated form pERK, were examined by immunohistochemistry. To assess the correlation between neurotrophic factor levels and their protective effect against light damage, albino (Sprague-Dawley) rats were exposed to bright continuous light (1000 lux) for 24 or 48 hours at successive times after nerve section. The TUNEL technique was used to visualize neuronal cell death in the retina. RESULTS: CNTF upregulation was detected 1 week after optic nerve section, peaked at 2 weeks, and fell to control levels at 4 weeks. CNTF appeared first in the inner retina in the ganglion cells, then in the Muller cells in which it became prominent at the outer limiting membrane (OLM) and in the outer segment (OS) region of photoreceptors. FGF-2 upregulation became prominent, particularly in photoreceptors, 21 to 28 days after surgery, continued to 2 months, and slowly declined thereafter. Double labeling with antibodies to ligand and the receptor showed colocalization of CNTF to its receptor at the OS region, whereas FGF-2-to-FGFR1 binding was found in the outer nuclear (ONL) and outer plexiform (OPL) layers. Optic nerve section provided a significant protective effect against light-induced damage in the first 2 weeks. There was no protection when animals were exposed to damaging light 1 month after nerve section. CONCLUSIONS: The upregulation of CNTF 7 to 14 days after nerve section correlates with a reduction in the a-wave described previously. Colocalization of CNTF and CNTFRalpha on the outer segments suggests that CNTF acts at the photoreceptor membrane. The slower upregulation of FGF-2 correlates with a reduction of the b-wave. FGF-2/FGFR1 colocalization in the OPL suggests that this factor acts at the synaptic terminals of photoreceptors, modulating the release of neurotransmitters. The time course of pERK upregulation suggests that the successive upregulation of CNTF and FGF-2 activates the ERK pathway. Based on the time course of protection against bright continuous light, it seems that CNTF plays a major role in this effect, and FGF-2 has a less important role in the protection against light-induced damage.

A simple and inexpensive light source for research in visual neuroscience.

Investigating the properties of light responsive neurons and their networks requires appropriate control of stimulus parameters, such as intensity, spectral composition, spatial and temporal profile. In the present paper, we describe how to build a simple, versatile and low-cost light source for use in visual neuroscience. The light source is a InGaN-based ultrabright light-emitting diode (LED), which may generate conventional light flashes as well as a variety of time varying stimuli to be used in quantitative studies of the visual system. In particular, with this instrument one may generate light stimuli sinusoidally modulated in time at frequencies ranging from 0.05 to 50 Hz, with less than 1% harmonic distortion at a contrast exceeding 85%. The relationship between applied voltage and energy emitted by the source is linear over an intensity range that exceeds 4.5 log-units, up to the full suppression of the light-sensitive currents in mammalian rods. The light source has minimal space requirement and does not generate appreciable radiating heat and hum, allowing its use for single cell work "in vitro" as well as for "in vivo" recording of the electroretinogram (ERG).

New N-n-propyl-substituted 3-aryl- and 3-cyclohexylpiperidines as partial agonists at the D4 dopamine receptor.

We have previously reported that compounds dimethyl-substituted on the phenyl ring of N-n-propyl-3-phenylpiperidines (PPEs) have a high (nM) affinity and selectivity toward the D(4) dopamine receptor (D(4) DAR) with m,p-dimethyl PPE (1) having the highest affinity and selectivity. In the present paper we have investigated the role of the methyl substitution by the synthesis of monomethylated (2a-c) and nonmethylated (2d) PPEs followed by the characterization of their biological properties using receptor binding assays. Our findings reveal that the methyl substitution of the phenyl ring is not necessary for a high and selective binding affinity to the D(4) DAR. Moreover, we have also synthesized cyclohexylpiperidines (CHPEs, 3a-d), which all showed higher binding affinities for the D(4) DAR than their aromatic counterparts. These results indicate that a pi-pi type interaction of the phenyl ring of PPEs with the D(4) DAR might not be essential, whereas a simple hydrophobic attraction between the cyclohexyl substituent of CHPEs and a hypothesized lipophilic pocket of the receptor might be crucial. Furthermore, functional assays indicate that 3d, as well as 1, are partial agonist at the D(4) DAR and therefore might represent new pharmacological tools to investigate the role of D(4) DAR activation in the control of cognitive functions and emotional states in health and disease.

Processing of retinal signals in normal and HCN deficient mice.

This study investigates the role of two different HCN channel isoforms in the light response of the outer retina. Taking advantage of HCN-deficient mice models and of in vitro (patch-clamp) and in vivo (ERG) recordings of retinal activity we show that HCN1 and HCN2 channels are expressed at distinct retinal sites and serve different functions. Specifically, HCN1 operate mainly at the level of the photoreceptor inner segment from where, together with other voltage sensitive channels, they control the time course of the response to bright light. Conversely, HCN2 channels are mainly expressed on the dendrites of bipolar cells and affect the response to dim lights. Single cell recordings in HCN1⁻/⁻ mice or during a pharmacological blockade of I(h) show that, contrary to previous reports, I(kx) alone is able to generate the fast initial transient in the rod bright flash response. Here we demonstrate that the relative contribution of I(h) and I(kx) to the rods' temporal tuning depends on the membrane potential. This is the first instance in which the light response of normal and HCN1- or HCN2-deficient mice is analyzed in single cells in retinal slice preparations and in integrated full field ERG responses from intact animals. This comparison reveals a high degree of correlation between single cell current clamp data and ERG measurements. A novel picture emerges showing that the temporal profile of the visual response to dim and bright luminance changes is separately determined by the coordinated gating of distinct voltage dependent conductances in photoreceptors and bipolar cells.

Effect of HCN channel inhibition on retinal morphology and function in normal and dystrophic rodents.

PURPOSE: To elucidate short- and long-term effects of ivabradine, an inhibitor of the hyperpolarization-activated current (I(f)) recently approved for treatment of stable angina, on retinal function and integrity. As careful ivabradine administration is recommended for patients with retinitis pigmentosa, an additional objective was to test the consequences of repeated ivabradine delivery on retinal integrity in the rd10 mouse, an animal model of the human degenerative disease. METHODS: The electroretinogram (ERG) was recorded in intact anesthetized animals in response to flashes or time-varied sinusoidal light stimuli of different frequency. Retinal integrity and hyperpolarization-activated cyclic nucleotide-gated (HCN) channel distribution were assessed by immunocytochemistry, confocal microscopy, and Western blot analysis. RESULTS: Neither a- nor b-waves of the flash-ERG were significantly affected by ivabradine administration. Conversely, reversible changes in the response to sinusoidal stimuli were observed during both acute and continued treatment. HCN inhibition enhanced the gain of frequency-response curves (FRCs) at the lowest stimulus frequencies and reduced it in the 1- to 7-Hz range. These effects were dose dependent and reverted to normal 1 week after discontinuation of ivabradine. Retinal morphology and distribution of HCN were preserved and no signs of retinal damage were observed in healthy animals. HCN inhibition in dystrophic mice had no effect on either extent or progression of retinal degeneration. CONCLUSIONS: The results are consistent with the hypothesis that the visual symptoms reported by patients during prolonged treatment with ivabradine are due only to a reversible pharmacologic effect.

Selective Hcn1 channels inhibition by ivabradine in mouse rod photoreceptors.

PURPOSE: To evaluate in mammalian rod photoreceptors the selectivity for hyperpolarization-activated cyclic nucleotide-gated (Hcn1, coded by Hcn1) over potassium-selective (Kir 2.4, coded by Kcnj14) channels of ivabradine, a selective inhibitor of the cardiac "funny" current (I(f)). METHODS: Rods were isolated from the mouse retina and voltage clamped by the perforated-patch technique. The hyperpolarization-activated current (I(h)) was blocked by ivabradine during repetitive stimulation with activating/deactivating voltage steps from -80 to -30 mV, from a holding of -35 mV. RESULTS: Full inhibition was observed at a high concentration of ivabradine (30 microM), with intermediate effects at 3 and 0.3 microM. Steady state activation and activation kinetics of the ivabradine- and CsCl-blocked currents were similar, consistent with the block by ivabradine of ion permeation through Hcn1 channels. Hcn1 blockade was also consistent with the lack of current reactivation during long steps at -110 mV. At doses that fully block I(h), ivabradine does not affect the inward rectifier current through potassium-selective Kir 2.4 channels or the outward currents evoked by stepping up from -80 to 50 mV. CONCLUSIONS: In mammalian rods, ivabradine is a selective inhibitor of Hcn1 channels. Phosphenes perception in response to abrupt changes in luminance, which has been transiently reported in a dose-dependent way by few patients treated with ivabradine, was consistent with Hcn1 inhibition in rods.

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 I(f) current in heart and I(h) throughout the nervous system. In spiking neurons I(h) 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 I(h) 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 I(h), 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 I(h) activation. Theoretical modeling and pharmacological blockade demonstrate that high-pass filtering of input signals by I(h), 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 I(h) 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 I(h) onto RBCs may contribute to shape the retina's light response and explain the visual side effects of HCN inhibitors.

Vision: how to catch fast signals with slow detectors.

The visual system is equipped with highly sensitive but slow detectors, yet it can resolve light changes up to 60 Hz. Processes taking place in retinal circuits go beyond the intrinsic limits of the transduction machinery by an unconventional exploitation of voltage-dependent conductances, cleverly lined up to generate a cascade of band-pass amplification stages.

Correlation between ERG changes and FGF2 mRNA Up-regulation in patients with choroidal melanoma.

To study electroretinographic (ERG) response to light flashes in patients with choroidal melanoma and to define possible factors involved in the modification of both a- and b-wave.ISCEV standard flash-ERG was recorded from both affected and control eyes on 24 patients before surgical operation (local excision or enucleation). The choroidal melanomatous mass ranged from 33 to 2880mm(3). Tissues from both melanomatous retina-choroid complex and areas far from the melanoma (unaffected) were taken from 13 enucleated eyes to measure the level of FGF2 mRNA, utilizing the technique of semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Tissues from 10 normal eyes were used as control. The majority of patients showed a marked a- and b-wave attenuation in the affected eye with respect to the fellow eye. In 10 retinal specimens, the expression of FGF2 mRNA showed an increase in retinal regions far from the melanoma compared to control eyes. Many patients present an increase in the expression of FGF2 mRNA in the unaffected part of the retina and a clear attenuation in both a- and b-ERG components. The size of melanoma does not predict the amount of reduction in the ERG response, at least for sizes less than 1000mm(3). We suggest that the melanoma triggers a process leading to an up-regulation of FGF2 in the human eye and this up-regulation might be responsible for the ERG attenuation.

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 (I(h)) can be measured only from the cell regions where HCN1 is expressed. Half-activation voltage (-75.0 +/- 0.3 mV) and kinetics (t(1/2) of 101 +/- 8 ms at -110 mV and 20 degrees C) of the I(h) 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 I(h). 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.