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 (I(f)) 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 I(f) 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.

The impact of organic inhibitors of the hyperpolarization activated current (Ih) on the electroretinogram (ERG) of rodents.

We have compared the effect of two distinct Ih inhibitors on the temporal properties of the ERG response that, as previously shown, correlates well with the HCN activation in rods. The present results confirm the notion that cilobradine is more effective than zatebradine in inducing bradycardia. Importantly, the doses of cilobradine that reduce the heart rate to values comparable to, or lower than, those obtained with higher doses of zatebradine have little effect on the frequency response of the ERG. While more potent than zatebradine in its bradycardic action, cilobradine appears comparatively less effective on the visual response. A possible explanation is that the affinity of cilobradine for the HCN channels in the heart is higher than that for the HCN channels of retinal neurons.

Electroretinogram changes associated with retinal upregulation of trophic factors: observations following optic nerve section.

The purpose of the present work was to assess whether upregulation of trophic factors and protection from damage induced in the retina by optic nerve section are associated with changes in the flash electroretinogram (ERG). We have examined the ERG in adult pigmented rat at different survival times over a period of 3 months following section of the optic nerve. The a-wave was analyzed using the Lamb-Pugh model and the parameters of best fit were estimated in control animals and at successive survival times. The amplitudes of the a- and b-waves were reduced over the first 7 days after nerve section. The a-wave recovered its relative amplitude by 21 days, but the b-wave remained depressed 5 weeks following nerve section. Analysis of the a-wave indicated a 20-30% reduction in the dark current of sectioned eyes at 7 days survival. A significant reduction of the amplification constant was observed in both nerve-sectioned and nerve-intact eyes, relative to normal and sham-operated controls. This reduction persisted to the longest survival time examined. The reduction of the a-wave at 7 days after nerve section coincides with a period of upregulation of ciliary nerve trophic factor. The amplification factor is influenced over a longer time course, which corresponds with a period of up-regulation of basic fibroblast growth factor. These changes in growth factor expression and ERG parameters are in turn associated with protection of photoreceptors against light damage. Present results suggest that the sensitivity of the retina to light may be regulated by mechanisms which protect photoreceptors against stress.

Synthesis and dopaminergic properties of the two enantiomers of 3-(3,4-dimethylphenyl)-1-propylpiperidine, a potent and selective dopamine D4 receptor ligand.

The synthesis of the two enantiomers of 3-(3,4-dimethylphenyl)-1-propylpiperidine 1, a potent and selective D4 dopaminergic ligand, was performed. The 3-(3,4-dimethylphenyl)- 1-propylpiperidine with the R configuration showed an affinity for the D4 receptors 6-fold higher than the corresponding enantiomer with the S configuration. Furthermore, the (R)-1 enantiomer proved to be highly selective for D4 receptors with respect to D2-D3 receptors, with a Ki ratio higher than 25,000, while the (S)-1 enantiomer was about 100-fold less selective than the (R)-1 one.

Mechanisms of photoreceptor death and survival in mammalian retina.

The mammalian retina, like the rest of the central nervous system, is highly stable and can maintain its structure and function for the full life of the individual, in humans for many decades. Photoreceptor dystrophies are instances of retinal instability. Many are precipitated by genetic mutations and scores of photoreceptor-lethal mutations have now been identified at the codon level. This review explores the factors which make the photoreceptor more vulnerable to small mutations of its proteins than any other cell of the body, and more vulnerable to environmental factors than any other retinal neurone. These factors include the highly specialised structure and function of the photoreceptors, their high appetite for energy, their self-protective mechanisms and the architecture of their energy supply from the choroidal circulation. Particularly important are the properties of the choroidal circulation, especially its fast flow of near-arterial blood and its inability to autoregulate. Mechanisms which make the retina stable and unstable are then reviewed in three different models of retinal degeneration, retinal detachment, photoreceptor dystrophy and light damage. A two stage model of the genesis of photoreceptor dystrophies is proposed, comprising an initial "depletion" stage caused by genetic or environmental insult and a second "late" stage during which oxygen toxicity damages and eventually destroys any photoreceptors which survive the initial depletion. It is a feature of the model that the second "late" stage of retinal dystrophies is driven by oxygen toxicity. The implications of these ideas for therapy of retinal dystrophies are discussed.

Temporal fidelity in the visual system.

The temporal properties of the visual system have been analyzed by recording the ERG and its isolated components in response to light stimuli whose luminance was varied sinusoidally. The performance of the visual system to periodic light stimuli was tested in human subjects psychophysically. The comparison of the results in control conditions and after administration of drugs that specifically block the hyperpolarization activated current (Ih) suggest that the inner rectifying properties of the inner segment membrane of rods is involved in a process of temporal differentiation of the visual signals whereby high frequency components of the response especially relevant for the visual performance are enhanced. It is proposed that the temporal fidelity of the visual system is the results of an elaboration starting at early level of the signal generated by the phototransductive cascade.

The impact of basic fibroblast growth factor on photoreceptor function and morphology.

PURPOSE: To assess the impact of basic fibroblast growth factor (bFGF) on photoreceptor function and morphology. METHODS: Impact was assessed in two models. In one, the endogenous expression of bFGF in photoreceptors was raised by sectioning one optic nerve of rats 3 to 4 weeks before study. In the other, bFGF was injected into the vitreous chamber in rats and cats. Retinal function was assessed from the electroretinogram (ERG), and retinal morphology was studied using DNA dyes, immunolabeling, and in situ hybridization. RESULTS: In both models of bFGF upregulation, the ERG b-wave was suppressed over a wide stimulus range and in light- and dark-adapted conditions. The a-wave was not suppressed by either procedure and at the brightest intensities was enhanced by both procedures. In nerve-sectioned eyes, outer retina appeared normal histologically, but levels of bFGF protein in the inner and outer nuclear layers were raised, whereas bFGF mRNA levels remained unchanged. In both models, levels of synaptophysin in the outer plexiform layer and of cytochrome oxidase in inner segments were raised in association with increases in bFGF protein levels. CONCLUSIONS: bFGF increased the ability of photoreceptors to respond to light but attenuated the transmission of this response to inner retinal cells, presumably by blocking the photoreceptor-bipolar synapse. If the expression of bFGF protein is upregulated in human photoreceptor dystrophies, it may contribute a reversible component to the loss of vision. The relationship between these actions of bFGF and its ability to protect photoreceptors from stress remains to be established.

Analysis of pharmacologically isolated components of the ERG.

An harmonic analysis was applied to the electroretinogram (ERG) measured in intact cat eyes in control conditions and after pharmacological isolation of the components attributed to photoreceptors (PIII) and bipolar neurons (PII). The frequency response curves obtained in various conditions showed that the bandwidth of the PII component extends over a range of stimulus frequencies higher than the bandwidth of PIII. The enhancement of the PII response to stimuli of high temporal frequency suggests the presence of a frequency dependent gain control located either pre- and/or post-synaptically in the transmission line between the phototransductive cascade and bipolar neurons. A possible role of these processes is to enhance relevant visual information whilst selectively attenuating low frequency signals originating in the transductive cascade.

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.

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.

N-n-Propyl-substituted 3-(dimethylphenyl)piperidines display novel discriminative properties between dopamine receptor subtypes: synthesis and receptor binding studies.

3-Phenylpiperidines (PPEs) have been thoroughly investigated in view of their interesting dopaminergic activity, and the N-n-propyl substitution has been suggested as the most effective among several PPEs differently substituted on the phenyl ring. In previous studies, we found that the dimethyl substitution on the phenyl ring of N-unsubstituted PPEs provided compounds active toward alpha2-adrenergic receptors (alpha2-ARs), which proved to possess interesting selectivity properties. The high degree of homology between the binding domains of alpha2-ARs and D4-dopaminergic receptors (D4-DARs) prompted us to verify whether this kind of substitution on the aromatic ring might prove to be active against retinal DARs of the D4 subtype. On the basis of these premises, we synthesized the dimethylphenyl-substituted PPEs 4a-f, in which an n-propyl chain is present on the aminic nitrogen. Radioligand binding assays on bovine retina and striatum membranes for D1-like and D2-like DARs indicated that PPEs 4a, 4b, and 4f possess a high affinity and selectivity for the D4-DAR subtype of bovine retina.

The energetic cost of photoreception in retinal rods of mammals.

The effects of temperature on rod sensitivity and adaptation are analysed in the general context of the energy requirements of photoreception. The dependence of adaptation on the [Na]i turn-over appears to be critical in mammalian rods where the metabolic load is particularly heavy because of both temperature conditions and large Na+ influx. Estimates of the energy dissipated by rods in darkness and during bright illumination show that the metabolic load is reasonably well distributed. From this analysis it also results that most of the energy, which a rod dissipates in both darkness and light, is needed to keep [Na]i and [Ca]i low.

Effect of blocking the Na+/K+ ATPase on Ca2+ extrusion and light adaptation in mammalian retinal rods.

Membrane current and light response were recorded from rods of monkey and guinea pig by means of suction electrodes. The correlation between adaptation and the Na+/K+ pump was investigated by measuring light-dependent changes in sensitivity with and without inhibition of Na+/K+ ATPase by strophanthidin. Strophanthidin was found to reduce the dark current, to slow the time course of the photoresponse, and to increase light sensitivity. At concentrations between 20 and 500 nM, the pump inhibitor suppressed in a reversible way the current re-activation occurring during prolonged illumination and modified the light-dependent decrease in sensitivity, which in control conditions approximates to a Weber-Fechner function. The effects of the pump inhibitor on the adaptive properties of rods are associated with an increased time constant of the membrane current attributed to the operation of the Na+:Ca2+,K+ exchanger. The effects of rapid application of the pump inhibitor on the current re-activation are consistent with the idea that significant changes in the internal sodium occur in rods of mammals during background illumination and that they play an important role in the process of light adaptation.

Calcium homeostasis in the outer segments of retinal rods from the tiger salamander.

1. The processes regulating intracellular calcium in the outer segments of salamander rods have been investigated. The main preparation used was the isolated rod loaded with the Ca(2+)-sensitive photoprotein aequorin, from which outer segment membrane current and free [Ca2+]i could be recorded simultaneously. Two other preparations were also used: outer segment membrane current was recorded from intact, isolated rods using a suction pipette, and from detached outer segments using a whole-cell pipette. 2. Measurements of free intracellular [Ca2+] in Ringer solution were obtained from two aequorin-loaded rods. Mean [Ca2+]i in darkness was 0.41 microM, and after a bright flash [Ca2+]i fell to below detectable levels ( < 0.3 microM). No release of intracellular Ca2+ by a bright flash of light could be detected ( < 0.2 microM). 3. Application of the phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) caused an increase in the size of the light-sensitive current and a rise in [Ca2+]i, but application of IBMX either when the light-sensitive channels had been closed by a bright light or in the absence of external Ca2+ caused no detectable rise in [Ca2+]i. It is concluded that IBMX increases [Ca2+]i by opening light-sensitive channels, and does not release Ca2+ from stores within the outer segment. 4. Removal of external Na+ caused a rise in [Ca2+]i to around 2 microM and completely suppressed the light-sensitive current. 5. The Na(+)-Ca2+, K+ exchange current in aequorin-loaded rods was activated in first-order manner by internal free calcium, with a mean Michaelis constant, KCa, of 1.6 microM. 6. The KCa of the Na(+)-Ca2+, K+ exchange was increased by elevating internal [Na+]. 7. The Michaelis relation between [Ca2+]i and the activity of the Na(+)-Ca2+, K+ exchange was used to calculate the change in [Ca2+]i occurring during the response to a bright light. In aequorin-loaded rods in Ringer solution the mean change in free [Ca2+]i after a bright flash was 0.34 microM. In these rods 10% of the dark current was carried by Ca2+. 8. Most of the calcium entering the outer segment was taken up rapidly and reversibly by buffer systems. The time constant of equilibration between free and rapidly bound Ca2+ was less than 20 ms. No slow component of calcium uptake was detected. 9. Two components of calcium buffering could be distinguished in the outer segments of aequorin-loaded rods.(ABSTRACT TRUNCATED AT 400 WORDS)

Extrusion of calcium from rod outer segments is driven by both sodium and potassium gradients.

Calcium is transported across the surface membrane of both nerve and muscle by a Na+-dependent mechanism, usually termed the Na:Ca exchange. It is well established from experiments on rod outer segments that one net positive charge enters the cell for every Ca2+ ion extruded by the exchange, which is generally interpreted to imply an exchange stoichiometry of 3 Na+:1 Ca2+. We have measured the currents associated with the operation of the exchange in both forward and reversed modes in isolated rod outer segments and we find that the reversed mode, in which Ca2+ enters the cell in exchange for Na+, depends strongly on the presence of external K+. The ability of changes in external K+ concentration ([K+]o) to perturb the equilibrium level of [Ca2+]i indicates that K+ is co-transported with calcium. From an examination of the relative changes of [Ca2+]o, [Na+]o, [K+]o and membrane potential required to maintain the exchange at equilibrium, we conclude that the exchange stoichiometry is 4 Na+:1 Ca2+, 1 K+ and we propose that the exchange should be renamed the Na:Ca, K exchange. Harnessing the outward K+ gradient should allow the exchange to maintain a Ca2+ efflux down to levels of internal [Ca2+] that are considerably lower than would be possible with a 3 Na+:1 Ca2+ exchange.

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.

Ion transport by the Na-Ca exchange in isolated rod outer segments.

The inward membrane current generated by the coupled exchange of external sodium for internal calcium has been investigated in isolated rod outer segments. The exchange rate is sensitive to voltage, with a reduction by a factor of e occurring for a 70-mV depolarization in normal Ringer's solution. The voltage sensitivity is not a constant property of the exchange, as it is reduced by an increase in external Na+ or by the removal of external Ca2+, Mg2+, or K+. Changes in membrane potential do not appear to affect the affinity of the exchange mechanism for internal Ca2+, but hyperpolarization increases the affinity for external Na+. When the external Na+ concentration is raised sufficiently to saturate the exchange mechanism, the voltage sensitivity is no longer apparent. We propose that the voltage dependence of the exchange is due to the external Na+-binding site being sensitive to membrane potential, perhaps because it is located within the membrane electric field.