Luigi Cervetto

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.

Electrical responses of rods in the retina of Bufo marinus

1. Intracellular responses to flashes and steps of light have been recorded from the outer segment and the cell body of rods in the retina of the Bufo marinus. The identification of the origin of recorded responses has been confirmed by intracellular marking.

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.

Ionic movements through light-sensitive channels of toad rods.

Electrical photoresponses of rods in the isolated toad retina were recorded during ionic manipulations of the Na+‐free extracellular medium. In the presence of a concentration of external Ca2+ above 10(‐5) M, voltage photoresponses were observed only in the presence of external Na+ or Li+. When external Ca2+ was reduced below 10(‐6) M, voltage photoresponses of normal polarity could be detected even in the absence of Na+ or Li+, but in the presence of external Mg2+. In the presence of normal extracellular Ca2+ hyperpolarizing photoresponses were observed even in the absence of Na+ or Li+, provided small amounts of phosphodiesterase inhibitors (IBMX, RO 20‐1724, papaverine, caffeine, theophylline) were added to the perfusate. Responses obtained in low‐Na+ IBMX solutions required the presence of millimolar amounts of a variety of divalent cations, among which Mn2+ and Ba2+ were the most effective. When the concentration of both external Ca2+ and Mg2+ was reduced to micromolar amounts, depolarizing photoresponses were observed. In these conditions measurements with radioactive tracers showed a light‐modulated efflux of 42K+ or 86Rb+. The light‐modulated 42K+ or 86Rb+ efflux was halved by 2 X 6 mM‐external K+ and was completely blocked when K+ was raised above 10 mM. These results show that ionic movements through light‐sensitive channels are controlled by Ca2+ and Mg2+ and possibly also be the intracellular level of cyclic nucleotides. Moreover, the movement of ions through the light‐sensitive channel, does not obey the independence principle.

The effect of phosphodiesterase inhibitors on the electrical activity of toad rods.

The membrane potential of toad rods was recorded during addition of small amounts of phosphodiesterase inhibitors to the extracellular medium. Separate application of 3‐isobutyl‐1‐methylxanthine (IBMX), caffeine, theophylline, papaverine and RO 20‐1724 slowed down the time course of rod photo‐response to dim flashes of light. These changes were associated with a two to six‐fold increase in the amplitude of photoresponse. The effects on kinetics may be described simply by an expansion of the photoresponse time scale. When the drug concentration was raised above a certain level, the rods showed supralinear behaviour whereby doubling of the intensity of a dim flash could increase the response more than two‐fold. Under similar conditions rods also showed light sensitization whereby responses to dim flashes were enhanced in the presence of dim backgrounds. Taking the drug concentration that induced a two‐fold increase in the time‐to‐peak, IBMX was found the most effective compound, followed by papaverine, RO 20‐1724, theophylline and caffeine with relative effectivities 1, 1/2, 1/7, 1/40 and 1/100. Sensitivity, kinetics and supralinear behaviour may be restored to normal by steady background illumination while still in the presence of IBMX. However the intensity of the steady light, needed to restore the sensitivity to control levels, is not sufficient to accelerate the kinetics back to control values. In the presence of 50 microM‐IBMX a dim steady background of light enhanced the response to dim flashes. When the intensity of the light background was increased rods were desensitized and the supralinear behaviour disappeared. The antagonism between the effects of IBMX and the effects of background illumination on the kinetics of photoresponse suggests that phosphodiesterase activity controls the time course of light response in vertebrate rods.

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.

The modulation of the ionic selectivity of the light-sensitive current in isolated rods of the tiger salamander.

1. By using the method of Hodgkin, McNaughton & Nunn (1985) for rapidly changing the extracellular medium, we analysed the effect of the organic compound IBMX (3‐isobutyl‐1‐methylxanthine) on the movement of divalent cations through the light‐sensitive channels of isolated retinal rods of the tiger salamander. 2. When the rod is treated with 0.5 mM‐IBMX it is possible to observe photocurrents larger than 50 pA carried by Ba2+, Sr2+, Ca2+, Mg2+ and Mn2+. Under these conditions Ca2+, Mg2+ and Mn2+ carry photocurrents of similar amplitude, while Ba2+ and Sr2+ usually carry larger photocurrents. 3. The movement of Mn2+ through the light‐sensitive channel, which is hardly detected under normal conditions, can also be observed after treating the rod for a few seconds with a solution containing 35 mM[Na+]o and 10(‐7) M[Ca2+]o. Under these conditions the photocurrent carried by Mn2+ is fully saturated in the presence of 1 mM‐extracellular Mn2+. 4. When the rod is pre‐treated with an extracellular solution containing 0.5 mM‐IBMX the maximal photocurrent which can be carried by 10 mM [Ca2+]o increases from about 10 pA to approximately 200 pA. In these conditions the half‐activation of the Ca2+ current is between 1 and 10 mM, that is 20‐50 times higher than in normal conditions (Menini, Rispoli & Torre, 1988). 5. When the rod is pre‐treated with an extracellular solution containing 0.5 mM‐IBMX the half‐activation of the photocurrent which can be carried by Mg2+, Ba2+ and Sr2+ is equivalent to or greater than 10 mM. In the absence of pre‐treatment with IBMX the half‐activation of the photocurrent carried by Mg2+, Ba2+ and Sr2+ is less than 5 mM. 6. We conclude that the light‐sensitive channel can exist in at least two distinct open states. The selectivity of the channel in the first open state is as described in a previous paper (Menini et al. 1988). Mn2+, which is hardly permeable through the light‐sensitive channel in the first open state, can move through the light‐sensitive channel in the second open state. Ca2+, Mg2+, Ba2+ and Sr2+ permeate more freely through the light‐sensitive channel in the second open state, probably because the electrostatic interactions between these ions and the channel are less strong.