The role of reflecting pigment cells in the turnover of crayfish photoreceptors

Summary The photoreceptors of the crayfish Procambarus clarkii undergo an extensive cycle of turnover in the late afternoon. Quantitative light and electron microscopy reveal a sharp increase in the fractional volume (i.e., density) of reflecting-pigment-cell granules and vacuoles shortly following late-afternoon photoreceptor turnover. The reflecting pigment cells (RPCs), which permanently reside within the crayfish retina, are shown to serve much the same function as the vertebrate pigment epithelium. The RPCs phagocytose partially digested photoreceptive microvilli and the ingested debris is degraded further into the granules and vacuoles which characterize these cells. Phagosome degradation appears to be mediated by Golgi complexes. Acid phosphatase appears to be involved in the initial rhabdom breakdown but not in the final reduction of RPC granules.     

Fatty acid composition and ultrastructure of photoreceptive membranes in the crayfish Procambarus clarkii under conditions of thermal and photic stress

The ultrastructural state of the crayfish visual membrane is correlated with its fatty acid composition during times of photic and thermal stress and the period over which the dynamic events occur is investigated. Crayfish kept at 4 °C under constant darkness contain in their rhabdoms significantly increased amounts of unsaturated fatty acids such as 16:1, 18:1, 20:5, and 22:6 compared with individuals kept at 25 °C. The ratio of unsaturated/saturated fatty acids (UFA/SFA-ratio) amounts to 2.17 in the cold-water- and 1.46 in the warm water-acclimated animals. The visual membranes of crayfish suddenly transferred from 4 °C to 25 °C exhibited ultrastructural modifications such as membrane collapse and disappearance of microvillar dense␣core-filaments most clearly 3 h post-transfer. Parallel to the structural changes a significant increase in fatty acid 18:0 was observed, while the amounts of 16:1 and 20:1 decreased. When 4 °C, dark-adapted crayfish were exposed to light alone and not a temperature increase, only fatty acid 22:6 showed a significant reduction to 10% of its pre-experimental level within 2 h of exposure. Thereafter, it slowly increased again. In cold water-acclimated crayfish that had been exposed to light of 5000 lx for 3␣weeks no significant change of the UFA/SFA ratio was observed, although fatty acid species 18:0, 20:4, and 20:5 had increased at the expense of fatty acids 14:0, 16:0, 16:1, 18:1, 20:1, and 22:6. The total amount of fatty acids, however, had become significantly smaller (from 0.058 ng g⁻¹ body weight in the dark-adapted to 0.048 ng g⁻¹ in the light-adapted crayfish). Morphologically the rhabdom volume had decreased by approx. 20%, but ultrastructurally rhabdom microvilli remained almost unchanged. The amount of peroxidized lipids in the retina following irradiation with bright white light in the cold-adapted crayfish fell during the first 2 h of exposure from 0.4 nmol g⁻¹ to 0.32 nmol g⁻¹, but after 12 h of exposure had reached a level of 0.48 nmol g⁻¹. Greatest structural abnormalities to the visual membranes occurred when dark-adapted, cold-acclimated crayfish were suddenly subjected to bright light and an increase in water temperature. Under such conditions the microvillar arrangement was disrupted and membrane collapse and disappearance of core-filaments were apparent. Our results provide evidence that the fatty acid composition of the membranes determines to a considerable extent the structural integrity of the photoreceptor, but that it is too simplistic a model to think that peroxidation of membrane lipids alone is responsible for the disintegration of the photoreceptive membranes in the crayfish eye following exposure to bright light. Accepted: 4 July 1996     

Evidence for putative photoreceptor axon terminals in the medulla externa of the crayfish

Summary Within the medulla externa of the crayfish compound eye a class of axonal endings with similar characteristics to the photoreceptor terminals of the lamina ganglionaris were studied with light and electron microscopic techniques. These terminals are restricted to the superficial layers of the medulla externa and each is marked by a rod-shaped inclusion selectively impregnated with reduced silver methods.Electron microscopy of the medullary terminals confirms the presence of a rod inclusion composed of fine regularly arranged filaments. These inclusions are often closely associated with mitochondria and glycogen deposits within the endings. Synaptic contacts made by these terminals are characterized by a presynaptic ribbon density which is in contact with two or three postsynaptic elements. Often one postsynaptic element participates in more than one synaptic complex. Numerous invaginated processes, microtubules, synaptic vesicles, and ER cisternae are also present in the medullary terminals.The eighth retinula cell in the retina of the crayfish studied here resembles that previously observed by Nässel (1976). The similarity of the medullary terminals to the photoreceptor endings in the lamina suggest that they may belong to one of the eight photoreceptor cells forming an ommatidium.This work was supported in part by grants from the National Science Foundation (BNS77-15803) and National Institute of Health (NS08964)The authors wish to acknowledge the technical assistance of Ms. Georgia Hammond-Soltis     

Intracellular Ca2+ concentration and latency of light-induced Ca2+ changes in photoreceptors of the honeybee drone

We have measured Ca_i at rest and upon light stimulation in the photoreceptors of the honeybee drone microfluorometrically with the fluorescent Ca²⁺ indicator dyes fura-2, fluo-3 and Ca-green 5N.In darkness, Ca_i was 90 nM after 5 min of dark adaptation. A saturating light step caused Ca_i to rise in the bulk cytoplasm to 750 nM within 1 s. Our measurements with the low affinity dye Ca-green 5N showed that bright 1-s light flashes cause a rapid increase in Ca_i which was graded with stimulus intensity. Ca-green 5N fluorescence reached a peak in about 200 ms, and then decayed to a slightly lower sustained plateau. The fluorescence signal peaked, when the receptor potential was repolarizing from its peak to the plateau. This observation is in agreement with the proposal that the peak-to-plateau transition of the receptor potential is caused by the rise in Ca_i From our Fluo-3 measurements it appears that the latency of the Ca²⁺ increase is by 3–4 ms longer than the latency of the receptor potential elicited by bright 100-ms light flashes. This result provides no support for the proposal that Ca²⁺ mediates the opening of those membrane channels responsible for the upstroke of the receptor potential.Abbreviations ER endoplasmic reticulum - IP₃ Inositol 1,4,5-trisphosphate - SMC submicrovillar cisternae     

Factors influencing the degradation of photoreceptor membrane in the crayfish,Procambarus clarkii

Summary Light-induced degradation of photoreceptor membrane in the crayfish was studied by quantitative light and electron microscopy. The production of lysosomal organelles within the photoreceptor cells was enhanced by presenting the light stimulus intermittently (i.e., flicker) or by doubling its intensity. The enhancement was seen primarily as an increase in the number and size of multivesicular bodies. As these stimulus conditions are likely to facilitate intracellular Ca⁺⁺ fluxes, the results are compatibl with recent speculations that Ca⁺⁺ ions may regulate membrane degradation. To test the possibility that Ca⁺⁺ acts as a signal coupling receptor stimulation with membrane loss, retinas were incubated in the dark with the ionophore A23187 in the presence or absence of external Ca⁺⁺. The results demonstrate that A23187 produces a Ca⁺⁺-dependent increase in lysosomal organelles, predominantly multivesicular bodies. These data are consistent with a role for intracellular Ca⁺⁺ in the degradative process; however, the exact locus of the effect is unclear.Supported by a grant (BNS 8004587) from the National Science Foundation to G.S.H. The authors gratefully acknowledge the helpful discussions and expert technical assistance of Thomas R. Tokarski     

Multivesicular body formation and function in the light-adapted crayfish retina: a new interpretation

Summary Quantitative TEM refutes the hypothesis of multivesicular body formation at dawn via the degradation of photoreceptor. No significant relationship exists between multivesicular bodies and rhabdom diameter or between multivesicular bodies and coated vesicles. The density of multivesicular bodies is related to the vesicle-producing Golgi bodies. A new theory concerning the formation and possible function of multivesicular bodies is discussed.     

Immunocytochemical localization of taurine in the fish retina under light and dark adaptations

Summary. Previously we have observed the lack of immunoreactivity of taurine in the rod outer segments from light-adapted fish, such as the ayu Plecoglossus altivelis and lefteye flounder Paralichthys olivaceus. This finding prompted us to investigate if there is a difference in the immunocytochemical localization of taurine in the rod outer segments between the dark- and light-adapted states. In the retinas of the glass eel Anguilla japonica and the young goldfish Carassius auratus, extremely intense immunostaining was found in the cone outer segments, rod inner segments, photoreceptor supranuclear region and outer plexiform layer. The rod outer segments were not immunostained in the light-adapted state, while they were intensely immunostained in the dark-adapted state. Consequently, it was suggested that the lack of immunoreactivity in the rod outer segment may depend on light stimulation. In addition, the conspicuous immunocytochemical localization of taurine was discussed with the possible functional roles for taurine in the fish retina. Received January 25, 2000/Accepted January 31, 2000     

Rhabdom breakdown in the eye of Cirolana borealis (Crustacea) caused by exposure to daylight

Summary The effect of daylight on the compound eye was investigated in the deep-water crustacean isopod Cirolana borealis Lilljeborg. The animals were captured and fixed at night (dark-exposed, i.e. not exposed to light) and day (daylight-exposed), respectively. Changes in light and darkness have an effect on the retinula cells; the ultrastructure of dark-exposed eyes is characterized by well-preserved organelles and cytoplasm. The photoreceptor membranes covering the microvilli are regularly aligned, and the outline of the villi is smooth. Electron-dense pigment granules are evenly distributed in the cytoplasm of the retinula cell outside the rhabdom. Daylight-exposed eyes differ from the dark-exposed eyes in the following aspects: (i) the microvilli are disrupted, (ii) retinula-cell pigment is found in the rhabdom, and (iii) the cytoplasm of retinula cells is vesiculated. These results are interpreted as retinal damage caused by excess exposure to light.     

Patch clamp study and kinetic modeling on the most abundant chloride channel on distal fibers of crayfish opener muscle

Whereas the inhibitory innervation of the deep extensor abdominal muscle in crayfish is mediated by a weakly acting common inhibitor, the opener muscle exhibits a stronger inhibition. In the present study the most abundant γ-aminobutyric acid-activated chloride channel on distal fibers of crayfish opener muscle was characterized by measuring the current responses after applying pulses of γ-aminobutyric acid to outside-out patches. The results were compared to those obtained earlier with the chloride channel on the deep extensor abdominal muscle of the same species. The double logarithmic plot of the dose-response relationship had a slope of n _H = 2.2 in contrast to n _H = 5.3 for the channel on the deep extensor abdominal muscle. The rise time of the current response declined to 1 ms at a γ-aminobutyric acid concentration of 50 mmol · l⁻¹. With lower concentrations the rise time increased to a maximal value of 280 ms. No peak of the rise time at low γ-aminobutyric acid concentrations, as observed for the channel on the deep extensor abdominal muscle, was obvious. The open and closed times were similar to those of the channel of the deep extensor abdominal muscle. Different reaction schemes were discussed to describe the kinetics of the chloride channel of the opener muscle. Accepted: 12 August 1996     

Physiological characterization of antennular flicking reflexes in the crayfish

The cellular substrates of antennular flicking behavior in the crayfish Procambarus clarkii were investigated. Flicking involves fast downward movements of the external filament of each biramous antennule (1st antenna), and is mediated by phasic contractions of a short muscle, the external filament depressor. Phasic contractions of the external filament depressor depend upon stereotyped impulse bursts in a single motorneuron (P1). These bursts have a characteristic impulse frequency profile that is consistent upon successive occurrences. The temporal characteristics of the impulse burst suggest that the central depolarizations generating each burst may be similar to driver potentials described for motor neurons in crustacean cardiac ganglia. Responses of the external filament to odorants have a long latency and are characterized by repetitive bursts and tonic activity in some external filament depressor fibers. Tonic activity in a slowly contracting muscle, the antennular depressor muscle, is also evoked by chemical stimulation. Flicking is consistently evoked only by mechanical or hydrodynamic stimulation of the cephalothorax, antennae and antennules. The sensitivity and short latency of the hydrodynamic antennule-generated flick reflex is consistent with the sensitivity of rapidly conducting, hydrodynamically activated mechanoreceptor neurons in both antennular filaments. I propose that antennular flicking, which has been shown to enhance the dynamic response characteristics of olfactory receptor neurons on the external antennular filament, has evolved as a response to the turbulence associated with fluid movement, within which chaotic odorant concentration fronts may be imbedded. Accepted: 23 October 1996     

Electron microscopy of severed motor fibers in the crayfish

Summary Cut and crushed crayfish claw nerves were examined with the electron microscope at intervals up to 6 months after lesion. In sections 1 centimeter distal to the lesion there were no signs of degeneration among the giant motor axons even after many months. Swelling of glial wrappings was observed within 48 hours of nerve severance and was particularly notable in the innermost glial layer, the adaxonal layer. Golgi elements, rough endoplasmic reticulum, and mitochondria accumulated in the glia. These changes were perhaps indicative of a greater supportive role required by the severed axons. Regeneration from the proximal stumps of the giant axons began within one week and had proceeded across the lesion gap by 4 weeks. Axon sprouts appeared to travel toward the terminals within the glial sheaths of the distal giant axon segments. Before regeneration was complete, as determined by a simple behaviour test, the regenerating axons occupied increasing proportions of the sheath space. After regeneration was complete occasional degenerations were seen among the sprouts. These degenerations may have occurred in regenerating axons which had grown to the incorrect muscles. The original distal giant axons probably degenerated, as well, after regeneration was complete. There was no evidence of rehealing of proximal and distal segments of the axons.This work was supported by NIH postdoctoral fellowship number 1F2 NB 32, 723 N RB awarded to RHN and grants in aid from the Multiple Sclerosis Society, The American Cancer Society and The National Institutes of Health.     

Enhancement of sensitivity in photoreceptors of the honey bee drone by light and by Ca2+

Summary Deeply dark adapted (1 h) photoreceptor cells of the honey bee drone show a light-induced enhancement of sensitivity (facilitation) as an aftereffect of illumination or in the presence of dim backgrounds.The Ca²⁺-dependency of this effect was studied: Reduction of extracellular Ca²⁺ to 0.1 mM decreases the sensitivity of a dark adapted cell, and the light-induced increase in sensitivity due to repetitive, dim, 20 ms test flashes is slower than in normal saline. After a sensitizing conditioning light, the sensitivity drops faster in low-calcium saline. The light-induced enhancement of sensitivity is mimicked by pressure injections of low amounts of Ca²⁺ (Ca²⁺/EGTA-buffers; 0.15 M free Ca²⁺) into a dark adapted cell. Injection of EGTA alone decreases the sensitivity. Injection of a solution containing ca1 mM free Ca²⁺ sequentially decreases and later increases the sensitivity transiently.These results suggest a model in which a progressive increase in intracellular Ca²⁺ concentration by light first increases (facilitates), and, at higher concentrations, decreases (light adapts) the sensitivity of the cells. One possible site of action for this positive and negative feedback control of cell sensitivity by Ca²⁺ is the endoplasmic reticulum.     

Cardiac development in crayfish: ontogeny of cardiac physiology and aerobic metabolism in the red swamp crayfish Procambarus Clarkii

The cardiovascular system performs key physiological functions even as it develops and grows. The ontogeny of cardiac physiology was studied throughout embryonic and larval development in the red swamp crayfish Procambarus clarkii using videomicroscopic dimensional analysis. The heart begins to contract by day 13 of development (at 25°C, 20 kPa O₂). Prior to eclosion, heart rate (ƒH) decreases significantly. Previous data suggests that the decrease in cardiac parameters prior to hatching may be due to an oxygen limitation of the embryo. Throughout development, metabolizing mass and embryonic oxygen consumption primarily increased while egg surface area remains constant. The limited area for gas exchange of the egg membrane, in combination with the increasing oxygen demand of the embryo could result in an inadequate diffusive supply of oxygen to developing tissues. To determine if the decrease in cardiac function was the result of an internal hypoxia experienced during late embryonic development, early and late stage embryos were exposed to hyperoxic water (PO₂ =40 kPa O₂). The ƒH in late stage embryos increased significantly over control values when exposed to hyperoxic water suggesting that the suppression in cardiac function observed in late stage embryos is likely due to a limited oxygen supply.     

Parallel processing of proprioceptive information in the terminal abdominal ganglion of the crayfish

The processing of proprioceptive information from the exopodite-endopodite chordotonal organ in the tailfan of the crayfish Procambarus clarkii (Girard) is described. The chordotonal organ monitors relative movements of the exopodite about the endopodite. Displacement of the chordotonal strand elicits a burst of sensory spikes in root 3 of the terminal ganglion which are followed at a short and constant latency by excitatory postsynaptic potentials in interneurones. The afferents make excitatory monosynaptic connections with spiking and nonspiking local interneurones and intersegmental interneurones. No direct connections with motor neurones were found.Individual afferents make divergent patterns of connection onto different classes of interneurone. In turn, interneurones receive convergent inputs from some, but not all, chordotonal afferents. Ascending and spiking local interneurones receive inputs from afferents with velocity thresholds from 2–400°/s, while nonspiking interneurones receive inputs only from afferents with high velocity thresholds (200–400°/s).The reflex effects of chordotonal organ stimulation upon a number of uropod motor neurones are weak. Repetitive stimulation of the chordotonal organ at 850°/s produces a small reduction in the firing frequency of the reductor motor neurone. Injecting depolarizing current into ascending or non-spiking local interneurones that receive direct chordotonal input produces a similar inhibition.     

Assembly of rhabdomeric membrane from smooth endoplasmic reticulum can be activated by light in chromophore-deprived photoreceptors of Manduca sexta

Summary Deficiency of the photopigment chromophore, resulting from carotenoid/retinoid (vitamin A) deprivation, that severely impairs the visual function of Manduca sexta also leads to the hypertrophy of smooth endoplasmic reticulum in the photoreceptors. The excess endomembrane accumulates in the stacked cisternae of myeloid bodies. Although 11-cis retinal promotes substantial recovery of function in the retinas of deprived moths maintained in darkness, the myeloid bodies remain. When such recovering photoreceptors were exposed to light of moderate intensities, the amount of endomembrane diminished to normal levels over a period of several hours, while rhabdomeres grew larger. Since there was no endocytolysis, the myeloid bodies must have provided the membrane for rhabdomere enlargement. Bright light similarly mobilized the myeloid bodies in deprived receptors. Thus the persistence of myeloid bodies in moderately illuminated chromophoredeficient receptors is a consequence of their insensitivity. However, the initial hypertrophy of endomembrane does not appear to result from the lack of adequate stimulation: normal, chromophore-replete photoreceptors maintained in darkness from before the period of retinal development had large rhabdomeres and no myeloid bodies. The development of myeloid bodies during the differentiation of vitamin A-deprived photoreceptors appears to entail an influence of the chromophore at another level of receptor cell function.     

The distribution of polarization sensitivity in the crayfish retinula

In many arthropod eyes the ommatidia contain two classes of retinular cells with orthogonally oriented microvilli. These receptors provide the basis for two-channel polarization vision. In several contexts such as navigation or the detection of polarization contrast, two channels may be insufficient. While solutions to this problem are known (e.g. in insects and stomatopod crustaceans) none have been found in the majority of decapods. To examine this issue further, the polarization sensitivity and the E-vector angle eliciting a maximum response (theta (max)) were measured at over 300 loci on the crayfish retinula. The polarization response ratio (which is proportional to polarization sensitivity) was similar at all locations on the retinula. Around the central pole of the eye, theta (max) was distributed about the vertical and horizontal axes. Along the dorsal rim, the distribution of theta (max) exhibits modes at 0 degrees , 45 degrees and 90 degrees and a small mode at 135 degrees relative to the dorso-ventral axis of the eyestalk (0 degrees ). Smaller numbers of cells (20 to 25%) with theta (max )near the diagonal were also found in anterior and posterior retinula areas. Thus crayfish visual interneurons, which integrate signals from multiple ommatidia may have access to a multi-channel polarization analyzer.     

Diurnal changes of lysosome-related bodies in the crayfish photoreceptor cells

Summary The effect of illumination on the degradation of microvillar membrane in the invertebrate photoreceptor cell has been correlated with the appearance in the cytoplasm of certain distinct lysosome-related bodies. Three types of organelles were distinguished in the retinula cell cytoplasm of the crayfish, multivesicular bodies (MVB), both large (4.20-1.50 m) and small (1.49-0.30 m), combination bodies (CB), and lamellar bodies (LB). Under diurnal lighting conditions significant temporal differences were found in the appearance of these three classes of organelles in the retinula cell. Small MVB are present at a consistent level throughout most of the diurnal cycle but show peak numbers at 30 min after light onset and again after 6 h of dark adaptation. Large MVB increase significantly 1 h after light onset and remain elevated through 4 h in the light. After 4 h the large MVB decline gradually for the remaining light period. Combination bodies and LB do not begin to increase until 1 h after light onset and are at peak levels between 4 and 6 h into the light period. The minimum rhabdome diameter coincides with the peak levels of large MVB, CB, and LB. These data support the hypothesis that light causes microvillar membrane breakdown, resulting in the initial production of MVB which in turn undergo degradation to form CB and finally LB. This primary degradative response appears to be completed within the first 8 h of the light period.Supported by a grant from the National Science Foundation (BNS77-15803) and PHS Grant S05-RR-7031     

Transduction in photoreceptors: Determination of the pigment transition or state coupled to excitation

Several recent reports have shown that light absorption by metarhodopsin does not contribute to the excitation of invertebrate photoreceptors at low intensities. Where the pigment transition scheme is known, this result may be used to exclude some or most of the transitions and states of the pigment as sources of the coupling to excitation. The methodology of this approach is described and illustrated.Based on material presented at the European Neurosciences Meeting, Florence, September 1978     

Cross-connections coordinate postural motoneuron activity in the crayfish abdomen

Intracellular recordings and dye injections were used to examine mutual coupling among slow abdominal postural motoneurons in the 4th abdominal ganglion in crayfish (Procambarus clarkii). Intracellular current injection into one motoneuron altered the spike firing rate of some of its synergists. Depending on the polarity of the injected current, the premotor effect on the synergists was excitatory or inhibitory. The magnitude of the effect was intensity dependent. No dye coupling was found among the motoneurons following injection of Lucifer yellow. The morphological basis of the coupling was examined by differential filling of motoneuron pairs, one with horseradish peroxidase and the other with Lucifer yellow. The stained motoneurons were simultaneously visualized under light microscopy to determine the proximity of their differently colored dendrites. It was thus possible to locate the site of the presumed monosynaptic contacts between them. Combined physiological and morphological evidence suggests that these neurons are mutually coupled, forming part of an integrative system for abdominal posture control in crayfish.