Crayfish Photoreceptor Research Articles

Electrical response hysteresis of crayfish photoreceptors to light intensity. Dependence on circadian time.

We studied the light-triggered current of crayfish photoreceptors. We found that when a train of light flashes of either increasing or decreasing intensity is applied, the current waveform presents the non-linear behavior known as hysteresis. Additionally, we observed that the extent of this response depends on the circadian time at which the pulses are applied. We hypothesize that positive feedback loops of biochemical networks underlying light energy transduction are responsible of the observed behavior. It has been demonstrated that a dynamical system hysteresis provides a mechanism that enhances its robustness against random perturbations. Taking into account this characteristic we hypothesize that the electrical-response hysteresis of crayfish photoreceptors: 1) makes the visual system more stable to environmental noise, and hence 2) adds stability to circadian clock oscillations.

Desensitization and Recovery of Crayfish Photoreceptors Upon Delivery of a Light Stimulus.

A method to study desensitization and recovery of crayfish photoreceptors is presented. We performed intracellular electrical recordings of photoreceptor cells in isolated eyestalks using the discontinuous single electrode-switched voltage-clamp configuration. First, with a razor blade we made an opening in the dorsal cornea to get access to the retina. Thereafter, we inserted a glass electrode through the opening, and penetrated a cell as reported by the recording of a negative potential. Membrane potential was clamped at the photoreceptor's resting potential and a light-pulse was applied to activate currents. Finally, the two light-flash protocol was employed to measure current desensitization and recovery. The first light-flash triggers, after a lag period, the transduction ionic current, which after reaching a peak amplitude decays towards a desensitized state; the second flash, applied at varying time intervals, assesses the state of the light-activated conductance. To characterize the light-elicited current, three parameters were measured: 1) latency (the time elapsed between light flash delivery and the moment in which current achieves 10% of its maximum value); 2) peak current; and 3) desensitization time constant (exponential time constant of the current decay phase). All parameters are affected by the first pulse. To quantify recovery from desensitization, the ratio p2/p1 was employed versus time between pulses. p1 is the peak current evoked by the first light-pulse, and p2 is the peak current evoked by the second pulse. These data were fitted to a sum of exponential functions. Finally, these measurements were carried out as function of circadian time.

Desensitization and Recovery of Crayfish Photoreceptors Upon Delivery of a Light Stimulus

Desensitization and Recovery of Crayfish Photoreceptors Upon Delivery of a Light Stimulus

Desensitization and recovery of crayfish photoreceptors. Dependency on circadian time, and pigment-dispersing hormone

In this work, we studied the characteristics of recovery from desensitization of the light-elicited current of crayfish. Applying a two-flash protocol, we found that the first flash triggers a current that activates with a noticeable latency, reaches a peak value, and thereafter decays along a single exponential time course. In comparison with the first-elicited current, the current elicited by the second flash not only presents an expected smaller peak current, depending on the time between flashes, but it also displays a different latency and decay time constant. Recovery of the first flash values of these current parameters depends on the circadian time at which the experiments are conducted, and on the presence of pigment-dispersing hormone. Our data also suggest the existence of distinctive desensitized states, whose induction depends on circadian time and the presence of pigment-dispersing hormone.

Visual adaptation modulates a potassium conductance in retinular cells of the crayfish.

Crayfish photoreceptors exhibit a voltage-dependent potassium conductance, G(K), that is generally similar to the delayed rectifier channel described in neurons and other arthropod retinular cells. G(K) activation (i.e. the apparent threshold, Vth) occurs near the resting potential and G(K) is substantially reduced by 25 mM extracellular tetraethylammonium (TEA) and by intracellular Cs+ injections. Light exposure, sufficient to reduce visual sensitivity 100-fold, increases Vth (shifts it in the depolarizing direction) by about 20 mV. The light-dependent change in Vth does not depend upon the corresponding increase (depolarization) of the steady-state membrane potential nor does it depend upon inward calcium currents. Vth is slightly influenced by fluctuations in Ko associated with the light-elicited currents. During light exposure Ko (measured with K+-sensitive electrodes) increases by 2.1 mM (equivalent to an 8 mV increase in E(K)). This increase in E(K) makes only a modest contribution to the light-dependent change in Vth as determined by perfusion with high potassium salines. Intracellular calcium injections increase Vth by 10 to 20 mV and reduce visual sensitivity by 5- to 10-fold. The results imply that during exposure to high levels of illumination, K+ currents at the steady-state membrane potential are diminished by a calcium-dependent change in G(K) gating and, to a smaller degree, by a reduced K+ concentration gradient. It is notable that Ca2+ appears to inhibit both G(K) and the light-elicited conductance from both inside and outside the plasma membrane. As a consequence of the light-dependent change in Vth, G(K) makes only modest contributions to the changes in sensitivity and speed normally associated with light adaptation. These functions are regulated by the transduction pathway and are revealed at the resting potential in the time course and magnitude of the light-elicited currents.

Tachykinin-Related Peptide and GABA-Mediated Presynaptic Inhibition of Crayfish Photoreceptors

Off-axis illumination elicits lateral inhibition at the primary visual synapse in crustacea and insects. The evidence suggests that the inhibitory action is presynaptic (i.e., on the photoreceptor terminal) and that the amacrine neurons of the lamina ganglionaris (the first synaptic layer) may be part of the inhibitory pathway. The neurotransmitters and the synaptic mechanisms are unknown. We show by immunocytochemistry that GABA and a tachykinin-related peptide (TRP) are localized in the amacrine neurons of the crayfish lamina ganglionaris. Indirect evidence suggests that GABA and TRP may be colocalized in these neurons. The extensive processes of the amacrine neurons occupy lamina layers containing the terminals of photoreceptors. Application of exogenous GABA and TRP to photoreceptor terminals produces a short-latency, dose-dependent hyperpolarization with a decay time constant on the order of a few seconds. TRP also exhibits actions that evolve over several minutes. These include a reduction of the receptor potential (and the light-elicited current) by approximately 40% and potentiation of the action of GABA by approximately 100%. The mechanisms of TRP action in crayfish are not known, but a plausible pathway is a TRP-dependent elevation of intracellular Ca(2+) that reduces photoreceptor sensitivity in arthropods. Although the mechanisms are not established, the results indicate that in crayfish photoreceptors TRP displays actions on two time scales and can exert profound modulatory control over cell function.

Light-regulated localization of the beta-subunit of Gq-type G-protein in the crayfish photoreceptors.

In crayfish photoreceptor cells, Gq-type G-protein plays a central role in the phototransduction pathway, and the translocation of Gq alpha has been proposed as one of the molecular mechanisms to control photoreceptor sensitivity. We here investigated beta subunit of Gq and its localization profiles under various light conditions in the crayfish photoreceptor cells to understand the functional characteristic of visual Gq in the phototransduction pathway. An immunoprecipitation experiment was performed using an anti-Gq alpha antibody and a thiol-cleavable crosslinker. A 39 kDa protein was co-immunoprecipitated with Gq alpha, but not by irradiation, in the presence of GTP gamma S. The partial amino acid sequence of the 39 kDa protein was similar to G beta e in Drosophila photoreceptors, indicating that the crayfish G beta which combines with Gq alpha is a G beta e homologue. Immunohistochemical and immunoblot analyses revealed that the amount of the G beta decreased in the rhabdomeric membranes and increased in the cytoplasm in the light, compared with that in the dark. The profile of the translocation was similar to that reported for Gq alpha. Since both alpha and beta gamma subunits are necessary for G-proteins to be activated by rhodopsin in the rhabdom, the light-modulated translocation of a G beta e homologue possibly controls the amount of Gq which can be activated by light-stimulated rhodopsin.

Circadian clock function in isolated eyestalk tissue of crayfish.

Electrical mass response of crayfish photoreceptors (electroretinogram) was recorded continuously for up to seven days in isolated preparations that consisted of the retina and lamina ganglionaris. Electroretinogram amplitude varied in a circadian manner with a nocturnal acrophase and a period of 22-23 h in preparations kept in darkness. Acclimatization of animals to reversed light/dark cycles resulted in a phase reversal of the rhythm in vitro. The per (period) gene of Drosophila has been implicated in the genesis of rhythms in insects and in vertebrates. Immunocytochemical staining with an antibody against the PER gene product revealed immunoreactivity in the retinal photoreceptors, as well as in cell bodies in the lamina ganglionaris. Labelled axons run distally towards the photoreceptors and proximally to other areas of the lamina.

Light-modulated subcellular localization of the alpha-subunit of GTP-binding protein Gq in crayfish photoreceptors.

Gq-type GTP-binding protein (Gq) plays an important role in invertebrate visual phototransduction. The subcellular localization of the alpha subunit of visual Gq in crayfish photoreceptor was investigated immunocytochemically and biochemically to demonstrate the details of the rhodopsin-Gq interaction. The localization of Gq(alpha) changed depending on the light condition. In the dark, Gq(alpha) was localized in the whole rhabdoms as the membrane-bound form. In the light, half of the Gq(alpha) was localized in the cytoplasm as the soluble form. The translocation of Gq(alpha) was reversible. The light-modulated translocation possibly controls the amount of Gq that can be activated by rhodopsin. In vitro hydroxylamine treatment of rhabdomeric membranes suggested that the translocation was regulated by the fatty-acid modification of Gq(alpha).

The Subcellular Distribution of Calcium in the Visual Cells of Crayfish, Observed with the Combined Oxalate-Pyroantimonate Technique, Depends on Extracellular Calcium

1. With the combined oxalate-pyroantimonate method we determined the subcellular distribution of calcium in the photoreceptor cells of the crayfish Orconectes limosus. 2. The calcium antimonate deposits were identified with the electron microscope. Energydispersion X -ray analysis verified that the electron dense deposits contained calcium and antimony. 3. Calcium antimonate deposits were found in the submicrovillar cisternae, mitochondria, multivesicular bodies, and pigment granules, as well as in the cytoplasm . 4. The calcium deposits in the cytoplasm of the retinula cell and in the pigment granules were reduced after incubation of the retinae in a solution with a lowered calcium concentration and even more after we permeabilized the plasma membrane with the saponin β-escin. 5. We suppose that the calcium -storing pigment granules participate in the regulation of the cytosolic calcium concentration of the crayfish photoreceptor cells.

Interaction of GTP-binding protein Gq with photoactivated rhodopsin in the photoreceptor membranes of crayfish

Interaction of G-protein with photoactivated rhodopsin (Rh ∗) in crayfish photoreceptor membranes was investigated by immunoprecipitation using an antibody against rhodopsin. Two kinds of protein were co-precipitated with rhodopsin. One is an α subunit of class-q G-protein (42 kDa, CGqα) which showed light-induced, dose-dependent binding to rhodopsin, and the other is an actin-like protein (44 kDa) with light-independent binding. Most of the CGqα was available for binding to Rh ∗ but was dissociated from Rh ∗ in the presence of GTPγS. These findings demonstrate that, in the crayfish photoreceptor, a Gq class of G-protein is activated by Rh ∗.

Effects of chloroquine on the photosensory membrane turnover and the ultrastructure of lysosome-related bodies of the crayfish photoreceptor.

The effect of chloroquine in combination with bright light on the ultrastructure of crayfish (Orconectes limosus R.) photoreceptors was investigated in vivo. Chloroquine had several effects upon the crayfish retina. Multivesicular bodies (MVB) that are involved in lysosomal degradation of photosensory membrane were altered in ultrastructure. MVB frequently contained smaller MVB in a state of more advanced membrane degradation. Additionally screening pigment-like granules appeared in MVB. MVB accumulated in and filled the retinular cell cytoplasm just proximal or distal to the basement membrane which indicated inhibition of photosensory membrane degradation. Under chloroquine treatment rhabdom degradation appeared to be inhibited, as rhabdom diameter was less reduced under these conditions. Also chloroquine caused accumulation of screening pigment granules in glial cells within the lamina ganglionaris.

Evidence for a pathway of distal screening pigment granules across the basement membrane of the crayfish photoreceptor.

The distal pigment cells of Orconectes limosus contain two layers of large electron lucent vacuoles that are separated by layers of small right-angled platelets adjacent to the crystalline cones. The crystalline cones of the dioptric apparatus of this species have evaginations into the distal pigment cell cytoplasm. In photoreceptors of Orconectes limosus and Procambarus clarkii a dark pigment accumulation site was detected just distal to the basement membrane at the edges of each retina. These pigment accumulations occurred independent of the state of light adaptation. Ultrastructurally the pigment granules at this accumulation site resemble distal screening pigment granules according to their size (up to 1.2 microns in diameter) and fibrous structure. Distal screening pigment granules were also found in tube-like cell processes or extracellularly within and proximal to the retinal basement membrane, indicating pigment transport to and across the basement membrane. Proximal to the basement membrane screening pigment granules were also observed disintegrated to a gravel-like electron dense material in widely branched cells. Evidence was found that an electron dense material, probably resulting from disintegrating screening pigment granules, was incorporated in the integument of the eyestalk. Four hours after injection of gold particles into the eye stalk distal to the retina they were detected inside and proximal to the retinal basement membrane.

Conditional inhibition of screening-pigment aggregation by lidocaine in crayfish photoreceptors and frog retinal pigment epithelium.

Lidocaine, at concentrations equal to or lower than those that inhibit fast axoplasmic transport, was found to interfere with the dark-adapting migration of the screening pigments along crayfish photoreceptors and within the cells of the frog retinal pigment epithelium (RPE). The effects of the anesthetic on pigment movements were studied in isolated eyes incubated under light or dark conditions in media of different ionic compositions. Treatment of crayfish eyes with 25 mmol l-1 lidocaine in normal Van Harreveld's saline arrested pigment migration to the dark-adapted position or caused migration towards the light-adapted position in the dark. Similar results were obtained with frog eyecups exposed to 5 mmol l-1 lidocaine in Ringer's solution. In each case, the inhibition of dark adaptation was reversible and dependent on the levels of Na+ and Ca2+ in the incubation medium. A dark-adapted position of both pigments was compatible with lidocaine treatment provided that low-Na+, or high-Ca2+ or Co(2+)-containing solutions were used. These results indicate that light-adapted and dark-adapted pigment positions in both types of retinal cells can occur in the absence of local nervous input. Further, the data suggest a direct effect of lidocaine upon the photoreceptors or RPE cells. The inhibition of pigment aggregation is interpreted to be a consequence of an anesthetic-induced increase in the permeability of the plasma membrane, which in turn affects the intracellular ionic balance that controls pigment position.

Isolation of the rhabdomeral microvillar cytoskeleton of the crayfish ( Orconectes limosus) photoreceptor by a crosslinking reagent

1. 1. Rhabdomeral microvilli of photoreceptor cells of invertebrates contain a labile central cytoskeleton. For stabilization of the rhabdomeral cytoskeleton of the crayfish Orconectes limosus the crosslinking reagent suberic acid bis ( N- hydroxysuccinimide ester ) was used. 2. 2. It was found that this crosslinking reagent can be successfully used to stabilize and isolate the microvillar cytoskeleton of crayfish photoreceptors. 3. 3. After detergent treatment cytosolic proteins and the cell membranes were removed. 4. 4. By the combined use of crosslinker and detergent the accessibility of antibodies or other markers to the microvillar cytoskeleton is possible. 5. 5. This method may be useful, because at present little is known about the proteins associated with the central filament of invertebrate photoreceptors.

Na +/K +-ATPase regulation by neurotransmitters

A long period of experimental work has led to the conclusion that Na +/K +-ATPase is the enzymatic version of the Na +/K + pump. This enzymatic system is in charge of various important cell functions. Among them cationic equilibrium and recovering of resting membrane potential in neurons is relevant. A tetrameric ensemble of peptides conform the system known as α and β subunits. The α subunit is subdivided in α1, α2 and α3, according to different location and properties. Regulatory factors intrinsic to the Na +/K +-ATPase system are: ATP, Na + and Mg 2+ concentrations inside the cell, and K + outside. The enzyme activity is also regulated by extrinsic factors like some hormones (insulin and thyroxine). Induction of gene expression or post-translational modifications of the preexisting pool of the enzyme are the basic mechanisms of regulation proposed. Other extrinsic factors that seem to regulate the enzyme activity are some neurotransmitters. Among them the most extensively studied are catecholamines, mainly norepinephrine (NE) and lately serotonin (5-HT). The mechanism suggested for NE activation of the enzyme seems to involve specific receptors or a non-specific chelating action related to the catechol group that would relieve the inhibition by divalent cations. Another possibility is that NE removes an endogenous inhibitory factor present in the cytoplasm. The Na +/K +-ATPase is activated also by 5-HT. In vivo pharmacological and nutriological manipulations of brain 5-HT are accompanied by parallel responses of Na +/K +-ATPase activity. Serotonin agonists do activate the enzyme and antagonists neutralize the activation. In vitro there is a different dose dependent activation, according to the brain region. The mechanism involved seems to implicate a specific receptor system. Serotonin Na +/K +-ATPase interaction in the rat brain is probably of functional relevance because it disappears in amygdaloid kindling. Also it seems to influence the ionic regulation of the pigment transport mechanism in crayfish photoreceptors. In relation to other neurotransmitters, a weak response to histamine was observed with acetylcholine, GABA and glutamic acid, the results were negative.

Peroxidase and tyrosinase are present in secondary lysosomes that degrade photosensory membranes of the crayfish photoreceptor: possible role in pigment granule formation.

Three enzymes (acid phosphatase, peroxidase, and tyrosinase) were localized by electron microscopy within the retina of crayfish Orconectes limosus. Peroxidase activity was observed only in lamellar bodies, which are secondary lysosomes and degrade photosensory membrane. After H2O2 was omitted from the reaction medium, peroxidase activity in lamellar bodies was partly inhibited but was not missing completely. After addition of sodium pyruvate, which inhibits endogenous generation of H2O2, staining of lamellar bodies was absent. Tyrosinase activity was found in lamellar bodies and in small vesicles within the rhabdoms similar to those found positive for acid phosphatase. Granules (500-700 nm in diameter) with an electron opaque matrix and mature screening pigment granules showed tyrosinase activity. Moreover, lamellar structures within membrane-bound organelles that additionally contained screening pigment-like granules were electron dense because of tyrosinase activity. After addition of phenylthiourea (PTU) to the incubation medium, lamellar bodies did not generally contain electron dense deposits, although weak staining of single membranes still was sometimes observed. After addition of sodium pyruvate in combination with PTU, no staining was detected. The possible role of tyrosinase in ommochrome synthesis within secondary lysosomes that degrade photosensory membrane is discussed.

Light‐adapting migration of the screening‐pigment in crayfish photoreceptors is a two‐stage movement comprising an all‐or‐nothing initial phase

The light-adapting response of the screening-pigment in crayfish retinal photoreceptors, previously described as a monophasic movement, was found to consist of two stages with different properties: (1) a rapid initial expansion that once started proceeds for at least half of the full distance, and (2) a slower and more variable continuation of the movement. The two components were resolved in isolated eyes stimulated under conditions expected to restrict Na+ influx into the photoreceptors. Only the second stage of the response to light was inhibited when Na+ was substituted with choline, or if the normal saline contained amiloride, a diuretic that hinders Na+ entry across many cell membranes. Amiloride in a medium without Na+ delayed, but did not curb, the first stage, whereas the rest of the movement was markedly restrained. Partial replacement of Na+ with Li+ blocked the second stage without affecting the rapid initial shift triggered by light. Exposure of dark-adapted eyes to high Na+ levels or to ouabain in the presence of Na+ in the dark also elicited a two-staged pigment dispersion to the light-adapted position. Low Na+ concentrations or amiloride affected the latency, but not the rate or extent, of the first stage of migration in ouabain-treated eyes. Consistent though less significant results were obtained with cyanide and the Na+ ionophore monensin. These observations suggest a differential control of pigment position over two defined domains along the photoreceptors, probably to integrate a double mechanism of light-adaptation: an all-or-nothing partial shift of the pigment screen as a safety factor against overexposure, followed by a regulated adjustment according to stimulation intensity.

Motion detection and adaptation in crayfish photoreceptors. A spatiotemporal analysis of linear movement sensitivity.

Impulse and sine wave responses of crayfish photoreceptors were examined to establish the limits and the parameters of linear behavior. These receptors exhibit simple low pass behavior which is well described by the transfer function of a linear resistor-capacitor cascade of three to five stages, each with the same time constant (tau). Additionally, variations in mean light intensity modify tau twofold and the contrast sensitivity by fourfold. The angular sensitivity profile is Gaussian and the acceptance angle (phi) increases 3.2-fold with dark adaptation. The responses to moving stripes of positive and negative contrast were measured over a 100-fold velocity range. The amplitude, phase, and waveform of these responses were predicted from the convolution of the receptor's impulse response and angular sensitivity profile. A theoretical calculation based on the convolution of a linear impulse response and a Gaussian sensitivity profile indicates that the sensitivity to variations in stimulus velocity is determined by the ratio phi/tau. These two parameters are sufficient to predict the velocity of the half-maximal response over a wide range of ambient illumination levels. Because phi and tau vary in parallel during light adaptation, it is inferred that many arthropods can maintain approximately constant velocity sensitivity during large shifts in mean illumination and receptor time constant. The results are discussed relative to other arthropod and vertebrate receptors and the strategies that have evolved for movement detection in varying ambient illumination.

Ethanol-induced extension of screening-pigment migration range in crayfish photoreceptors

1. Ethanol 5–10% extends the normal range of screening-pigment migration along the photoreceptors of crayfish compound eyes, the effect being accounted for by a supramaximal pigment aggregation in the dark. 2. Excised eyes incubated in the presence of ethanol showed a faster and enhanced pigment aggregation to the dark-Actapted position, whereas the kinetics of the reverse movement in response to illumination was little affected. Methanol and propanol also facilitated pigment aggregation, although not with the same effectiveness as ethanol at equivalent concentrations. 3. The ethanol-induced facilitation of pigment aggregation persisted for some time after thorough washing of the eyes, and counteracted the inhibition of dark-Actaptation normally caused by an increaScin temperature. 4. Two possible explanations for this peculiar effect of ethanol are discussed: (a) alterations in photoreceptor membrane permeability that, in turn, may affect the ionic mechanisms controlling pigment position; and (b) promotion of local release of neurotransmitters involved in the modulation of light/dark adaptation.