Light-induced voltage noise in the photoreceptor of Drosophila melanogaster

The Drosophila photoreceptor potential is thought to be composed of discrete unit potentials called bumps. The steady-state receptor potential and the accompanying voltage fluctuations were recorded intracellularly under steady illumination. The occurrence rate, effective amplitude, and duration of the bumps were deduced by assuming a shot noise model. Over a wide range of light intensity, the duration of bumps remained essentially constant (25-30 ms). Below the saturation intensity for the receptor potential, the bump rate was roughly proportional to the intensity, and the adjustment of bumps to smaller size at higher intensity was mainly responsible for the nonlinear behavior of the receptor potential. The reduction in size of bumps at increasing light intensity was found to be due mainly to the diminishing magnitude of the bump current, and not to some other secondary effects. The bump rate saturated at about 3 x 105-106 events/s.     

Light reduces the excitation efficiency in the nss mutant of the sheep blowfly Lucilia

The nss (no steady state) phototransduction mutant of the sheep blowfly Lucilia was studied electrophysiologically using intracellular recordings. The effects of the nss mutation on the receptor potential are manifested in the following features of the light response. (a) The responses to a flash or to dim lights are close to normal, but the receptor potential decays close to the baseline level during prolonged illumination after a critical level of light intensity is reached. (b) The decline of the response is accompanied by a large reduction in responsiveness to light that recovers within 20 s in the dark. (c) The full reduction in responsiveness to light is reached when approximately 13% of the photopigment molecules are converted from rhodopsin (R) to metarhodopsin (M). (d) A maximal net pigment conversion from R to M by blue light induces persistent inactivation in the dark, without an apparent voltage response. This inactivation could be abolished at any time by M-to-R conversion with orange light. The above features of the mutant indicate that the effect of the nss mutation on the light response of Lucilia is very similar to the effects of the transient receptor potential (trp) mutation on the photoreceptor potential of Drosophila. Noise analysis and voltage measurements indicate that the decay of the receptor potential is due to a severe reduction in the rate of occurrence of the elementary voltage responses (bumps). The bumps are only slightly modified in shape and amplitude during the decline of the response to light of medium intensity. There is also a large increase in response latency during intense background illumination. These results are consistent with the hypothesis that separate, independent mechanisms determine bump triggering and bump shape and amplitude. The nss mutation affects the triggering mechanism of the bump.     

Photoconvertible pigment states and excitation in Calliphora; the induction and properties of the prolonged depolarising afterpotential

1. The proposed models of two independent groups, which relate the different states of the visual pigment to the excitation of the membrane in invertebrate photoreceptors (with particular reference to the prolonged depolarising afterpotential, the PDA) are compared and evaluated. 2. The validity of the late receptor potential (the "normal" receptor response) as an index of photoreceptor sensitivity, i.e., an index of the number of rhodopsin to metarhodopsin transitions, is verified by concurrent spectrophotometry. 3. Electrophysiological observations alone allow the calculation of 1.3 x 10(8) photopigment molecules in the rhabdom of an R1-6 photoreceptor of a vitamin A-bred Calliphora. 4. The PDA is shown to be quantifiable in terms of the number of rhodopsin to metarhodopsin conversions by the absorption of single light quanta. 5. The comparison of discrete membrane fluctuations (quantum bumps) during the PDA and during exposure to sustained light stimuli that mimic the PDA suggest that, the PDA, similar to the late receptor potential, may be due to the summation of quantum bumps.     

Frequency characteristics in the visual system of drosophila. Genetic dissection of electroretinogram components

Various drosophila mutants were used to dissect the electroretinogram (ERG) frequency response into components of different origins. The ommochrome granules in the receptor cell body are known to migrate in response to light, limiting the amount of light entering the rhabdomere. Comparison between the ERG frequency responses of the wild type and the mutant lacking the ommochrome granules indicates that the pigment migration reduces the amplitude gain at frequencies below 0.5 Hz. The ERG of drosophila compound eyes consists of contributions from receptor cells and the second-order cells in the lamina. Mutants with defective laminae showed a high-frequency cutoff with a corner frequency of about 20 Hz, while in wild type the response peaked in that frequency region. These results suggest that the lamina contributes mainly to the high-frequency components of the ERG transfer function. The shot noise model (Dodge et al., 1968) has been tested in drosophila by comparing the frequency response of the superimposed on the intracellular receptor potential. The results are consistent with the hypothesis that the receptor potential consists of a summation of small discrete potentials (bumps). In a mutant in which the bumps exhibit latency dispersion in response to a dim flash, the receptor showed a poor high-frequency response, the corner frequency being lowered to about 1-2 Hz. The slope of the cutoff was approximately 20 dB/dec indicating that the latency dispersion in this mutant is the major limiting factor in temporal resolution. Light-evoked high frequency oscillations have been observed in the ERG of another mutant. The oscillation was found sharply turned to light flickering at about 55 Hz.     

The roles of receptor noise and cone oil droplets in the photopic spectral sensitivity of the budgerigar,Melopsittacus undulatus

Individual budgerigars (Melopsittacus undulatus) were taught to detect narrow bands of wavelengths under ambient illumination of known spectral composition. Because the cone pigments of this species of bird have been identified and data on carotenoid absorbance present in the cone oil droplets are available, predictions of the Vorobyev-Osorio equations can be calculated with reasonable confidence. Based on more than 27,600 individual choices made by several birds at 10 wavelengths, the photopic sensitivity (i.e., color thresholds) of these birds is found to be consistent with the hypothesis that threshold discrimination of colored targets is limited by receptor noise and that high sensitivity to near-ultraviolet wavelengths is in harmony with the relatively small number of ultraviolet cones present in the retina. The pronounced fine structure of the sensitivity spectrum is caused by the absorption of cone oil droplets. Under natural sunlight, containing more energy in the near-ultraviolet than is present in artificial indoor lighting, the birds' peak of sensitivity in the ultraviolet should be much less prominent than it is in laboratory experiments.     

Cell responses to single pheromone molecules may reflect the activation kinetics of olfactory receptor molecules

Olfactory receptor cells of the silkmoth Bombyx mori respond to single pheromone molecules with "elementary" electrical events that appear as discrete "bumps" a few milliseconds in duration, or bursts of bumps. As revealed by simulation, one bump may result from a series of random openings of one or several ion channels, producing an average inward membrane current of 1.5 pA. The distributions of durations of bumps and of gaps between bumps in a burst can be fitted by single exponentials with time constants of 10.2 ms and 40.5 ms, respectively. The distribution of burst durations is a sum of two exponentials; the number of bumps per burst obeyed a geometric distribution (mean 3.2 bumps per burst). Accordingly the elementary events could reflect transitions among three states of the pheromone receptor molecule: the vacant receptor (state 1), the pheromone-receptor complex (state 2), and the activated complex (state 3). The calculated rate constants of the transitions between states are k(21)=7.7 s(-1), k(23)=16.8 s(-1), and k(32)=98 s(-1).     

Ultraviolet and green receptors in principal eyes of jumping spiders

Spectral sensitivities of cells in principal eyes of the jumping spider Phidippus reqius were measured using techniques of intracellular recording. Three types of cells were found. UV cells had peak sensitivities at 370 nm and were over 4 log units less sensitive at wavelengths longer than 460 nm. Green-sensitive cells had spectral sensitivities which were well fit by nomogram curves peaking at 532 nm. UV-green cells had dual peaks of sensitivity at about 370 and 525 nm, but the ratios of UV-to-green sensitivities varied over a 40: 1 range from cell to cell. Moreover, responses of UV-green cells to flashes of UV light were slower than to flashes of green light. Segregation of receptor types into the known layers of receptors in these eyes could not be shown. It is concluded that jumping spiders have the potential for dichromatic color vision.     

Estimation of leaf nitrogen content from spectral characteristics of rice canopy

Ground-based remotely sensed reflectance spectra of hyperspectral resolution were monitored during the growing period of rice under various nitrogen application rates. It was found that reflectance spectrum of rice canopy changed in both wavelength and reflectance as the plants developed. Fifteen characteristic wavebands were identified from the apparent peaks and valleys of spectral reflectance curves, in accordance with the results of the first-order differentiation, measured over the growing season of rice. The bandwidths and center wavelengths of these characteristic wavebands were different among nitrogen treatments. The simplified features by connecting these 15 characteristic wavelengths may be considered as spectral signatures of rice canopy, but spectral signatures varied with developmental age and nitrogen application rates. Among these characteristic wavebands, the changes of the wavelength in band 11 showed a positive linear relationship with application rates of nitrogen fertilizer, while it was a negative linear relationship in band 5. Mean reflectance of wavelengths in bands 1, 2, 3, 5, 11, and 15 was significantly correlated with application rates. Reflectance of these six wavelengths changed nonlinearly after transplanting and could be used in combination to distinguish rice plants subjected to different nitrogen application rates. From the correlation analyses, there are a variety of correlation coefficients for spectral reflectance to leaf nitrogen content in the range of 350-2400 nm. Reflectance of most wavelengths exhibited an inverse correlation with leaf nitrogen content, with the largest negative value (r = -0.581) located at about 1376 nm. Changes in reflectance at 1376 nm to leaf nitrogen content during the growing period were closely related and were best fitted to a nonlinear function. This relationship may be used to estimate and to monitor nitrogen content of rice leaves during rice growth. Reflectance of red light minimum and near-infrared peak and leaf nitrogen content were correlated nonlinearly.     

Graded and discrete receptor potentials in the compound eye of the Australian Bulldog-ant (Myrmecia gulosa)

1. Graded and discrete receptor potentials are recorded from the visual cells of the Australian Bulldog-ant. The intensity dependence of the graded responses is described by a new formula [Eq. (3)]. While the frequency of the discrete potentials in relation to the number of light quanta fits best a Poisson distribution, the graded potentials are best described by a logarithmic Gaussian distribution. 2. It is shown that the non-linear summation of single bumps and the reduction of the bump amplitude lead to a logarithmic intensity dependence. 3. The frequency spectrum of single bumps is measured with a Fast-Fourier-Analysis. It is observed that the harmonic frequencies have a negative slope around 12 dB/octave. 4. A difference is found in the higher harmonics of bumps generated at lower light intensities from those generated at higher light intensities. It is shown that this difference becomes more obvious if the bumps are further divided into short and longer latency groups. 5. From these results, it is concluded, that there is a mutual influence between neighbouring visual cells. Using this influence as a basis, a model of the low electric coupling between the cells is discussed.     

Visual spectral sensitivity of hatchling loggerhead (Caretta caretta L.) and leatherback (Dermochelys coriacea L.) sea turtles,as determined by single-flash electroretinography

Electroretinographic recordings were made from hatchling loggerhead and leatherback sea turtle eyecup preparations to generate dark-adapted spectral sensitivity curves. Both species were maximally sensitive to wavelengths between 500 and 540 nm, with a secondary peak near 380 nm. The spectral sensitivity curve for leatherbacks was attenuated at the long wavelength end of the spectrum relative to that of the loggerheads. This difference may reflect adaptations to lighting available at the relatively shallow (loggerhead) versus deeper (leatherback) sites where each species forages. The broad spectrum of wavelengths detected by both species (near UV to yellow–orange) indicates that vision is likely mediated by more than one photopigment, potentially rendering these turtles capable of color vision.     

Spectral composition and visual foraging in the three‐spined stickleback (Gasterosteidae: Gasterosteus aculeatus L.): elucidating the role of ultraviolet wavelengths

Visual signalling can be affected by both the intensity and spectral distribution of environmental light. In shallow aquatic habitats, the spectral range available for visually mediated behaviour, such as foraging, can reach from ultraviolet (UV) to long wavelengths in the human visible range. However, the relative importance of different wavebands in foraging behaviour is generally unknown. Here, we test how the spectral composition of ambient light influences the behaviour of three‐spined sticklebacks (Gasterosteus aculeatus) when foraging for live cladoceran Daphnia magna. Although paying particular attention to the UV waveband, we measured the foraging preferences of sticklebacks for prey presented under four different spectral conditions. These conditions selectively removed UV (UV–), short‐wave (SW–), mid‐wave (MW–) or long‐wave (LW–) light from the entire spectrum. The absence of UV and long wavelengths strongly reduced prey attractiveness for G. aculeatus compared with conditions without short‐wave and mid‐wave light. To control for potential light habitat preferences in the main experiment, we conducted a further choice experiment without prey stimuli. Fish in these trials did not discriminate significantly between the different spectral conditions. When comparing both experiments, it was observed that, although filter preferences for MW– and LW– conditions were virtually consistent, they differed at shorter wavelengths, with a reduced preference for UV– conditions and, at the same time, an increased preference for SW– conditions in the presence of prey. Thus, prey choice seems to be strongly affected by visual information at the short‐wave end of the spectrum. The foraging preferences were also mirrored by the chromatic contrast values between prey and the experimental background, as calculated for each lighting condition using a series of physiological models on stickleback perception. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2012, 105 , 359–368.     

Limulus ventral eye. Physiological properties of photoreceptor cells in an organ culture medium

Ventral photoreceptor cells bathed in an organ culture medium typically have resting potentials of -85 mV and membrane resistances of 35 Momega and, when dark-adapted, exhibit large potential fluctuations (LPFs) of 60 mV and small potential fluctuations (SPFs) of less than 30 mV. LPFs appear to be regenerative events triggered by SPFs, the well-known quantum bumps. In the dark, SPFs and LPFs occur spontaneously. At intensities near threshold, the rate of occurrence is directly proportional to light intensity, indicating that SPFs and LPFs are elicited by single photon events. At higher intensities, SPFs and LPFs sum to produce a receptor potential that is graded over approximately a 9-log-unit range of light intensity. Amplitude histograms of the discrete potential waves are bimodal, reflecting the SPF and LPF populations. Histograms of current waves are unimodal. SPFs and LPFs are insensitive to 1 microgram tetrodotoxin. I-V characteristics show initial inward currents of approximately 15 nA for voltage clamps to - 40 mV and steady-state outward currents for all clamp potentials. Photoreceptor cells bathed in organ culture medium retain these properties for periods of at least 75 days.     

Electroretinogram Characteristics and the Spectral Mechanisms of the Median Ocellus and the Lateral Eye in Limulus polyphemus

Electrical responses (ERG) to light flashes of various wavelengths and energies were obtained from the dorsal median ocellus and lateral compound eye of Limulus under dark and chromatic light adaptation. Spectral mechanisms were studied by analyzing (a) response waveforms, e.g. response area, rise, and fall times as functions of amplitude, (b) slopes of amplitude-energy functions, and (c) spectral sensitivity functions obtained by the criterion amplitude method. The data for a single spectral mechanism in the lateral eye are (a) response waveforms independent of wavelength, (b) same slope for response-energy functions at all wavelengths, (c) a spectral sensitivity function with a single maximum near 520 mµ, and (d) spectral sensitivity invariance in chromatic adaptation experiments. The data for two spectral mechanisms in the median ocellus are (a) two waveform characteristics depending on wavelength, (b) slopes of response-energy functions steeper for short than for long wavelengths, (c) two spectral sensitivity peaks (360 and 530–535 mµ) when dark-adapted, and (d) selective depression of either spectral sensitivity peak by appropriate chromatic adaptation. The ocellus is 200–320 times more sensitive to UV than to visible light. Both UV and green spectral sensitivity curves agree with Dartnall's nomogram. The hypothesis is favored that the ocellus contains two visual pigments each in a different type of receptor, rather than (a) various absorption bands of a single visual pigment, (b) single visual pigment and a chromatic mask, or (c) fluorescence. With long duration light stimuli a steady-state level followed the transient peak in the ERG from both types of eyes.     

Circadian rhythms in Limulus photoreceptors. II. Quantum bumps

The light response of the lateral eye of the horseshoe crab, Limulus polyphemus, increases at night, while the frequency of spontaneous discrete fluctuations of its photoreceptor membrane potential (quantum bumps) decreases. These changes are controlled by a circadian clock in the brain, which transmits activity to the eye via efferent optic nerve fibers (Barlow, R. B., S. J. Bolanski, and M. L Brachman. 1977. Science. 197:86-89). Here we report the results of experiments in which we recorded from single Limulus photoreceptors in vivo for several days and studied in detail changes in their physiological and membrane properties. We found that: (a) The shape of (voltage) quantum bumps changes with the time of day. At night, spontaneous bumps and bumps evoked by dim light are prolonged. The return of the membrane potential to its resting level is delayed, but the rise time of the bump is unaffected. On average, the area under a bump is 2.4 times greater at night than during the day. (b) The rate of spontaneous bumps decreases at night by roughly a factor of 3, but their amplitude distribution remains unchanged. (c) The resting potential and resistance of the photoreceptor membrane do not change with the time of day. (d) the relationship between injected current and impulse rate of the second order neuron, the eccentric cell, also remains unchanged with the time of day. Thus the efferent input from the brain to the retina modulates some of the membrane properties of photoreceptor cells. Our findings suggest that the efferent input acts on ionic channels in the membrane to increase the sensitivity of the photoreceptor to light.     

Aminoquinolines as fluorescent labels for hydrophilic interaction liquid chromatography of oligosaccharides

Abstract In this study, we investigated the potential of four different aminoquinoline (AQ) compounds as fluorescent labels for glycan analysis using hydrophilic interaction liquid chromatography (HILIC) and fluorescence detection (FLD). We confirmed the optimal excitation and emission wavelengths of 3-AQ and 6-AQ conjugated to glycan standards using three-dimensional fluorescent spectral scanning. The optimal excitation and emission wavelengths for 6-AQ were confirmed at λex=355 nm and λem=440 nm. We concluded that the optimal wavelengths for 3-AQ were λex=355 nm and λem=420 nm, which differed considerably from the wavelengths applied in previous reports. HILIC-FLD chromatograms using experimentally determined wavelengths were similar to 2-aminobenzamide controls, but the peak capacity and resolution differed significantly when published 3-AQ λex/em values were applied. Furthermore, we found that 5-AQ and 8-AQ labeled maltohexaose did not display any fluorescent pro\xadperties when used as a carbohydrate tag for HPLC analysis. Finally, we applied experimentally determined wavelengths to 3-AQ labeled N-glycans released from human IgG to illustrate changes in retention time as well as to demonstrate that AQ labeling is applicable to complex sample analysis via exoglycosidase sequencing.     

Fluorescence emission spectral shift measurements of membrane potential in single cells.

Previous measurements of transmembrane potential using the electrochromic probe di-8-ANEPPS have used the excitation spectral shift response by alternating excitation between two wavelengths centered at voltage-sensitive portions of the excitation spectrum and recording at a single wavelength near the peak of the emission spectrum. Recently, the emission spectral shift associated with the change in transmembrane potential has been used for continuous membrane potential monitoring. To characterize this form of the electrochromic response from di-8-ANEPPS, we have obtained fluorescence signals from single cells in response to step changes in transmembrane potentials set with a patch electrode, using single wavelength excitation near the peak of the dye absorption spectrum. Fluorescence changes at two wavelengths near voltage-sensitive portions of the emission spectrum and shifts in the complete emission spectrum were determined for emission from plasma membrane and internal membrane. We found that the fluorescence ratio from either dual-wavelength recordings, or from opposite sides of the emission spectrum, varied linearly with the amplitude of the transmembrane potential step between -80 and +60 mV. Voltage dependence of difference spectra exhibit a crossover point near the peak of the emission spectra with approximately equal gain and loss of fluorescence intensity on each side of the spectrum and equal response amplitude for depolarization and hyperpolarization. These results are consistent with an electrochromic mechanism of action and demonstrate how the emission spectral shift response can be used to measure the transmembrane potential in single cells.     

Electroretinogram and the spectral sensitivity of the compound eyes in the firefly Photuris versicolor (Coleoptera-Lampyridae): A correspondence between green sensitivity and species bioluminescence emission

ERGs were recorded from the dorsal sector of dark- and chromatic-adapted compound eyes in the dark-active firefly Photuris versicolor ♀ and ♂ at different wavelengths across the spectrum ranging from 320 nm to 700 nm over 4.5 log units of change in the stimulus intensity. ERG elicited by white light stimulus was an on-negative monophasic wave typical of scotopic eyes. ERGs elicited by chromatic stimuli differed in their waveform characteristics in the short (near-u.v. and violet) and long (green-yellow) wavelengths. The slope of the intensity-response curves at different stimulus wavelengths were similar for phasic response and differed for the plateau component of the ERG. The spectral sensitivity curves obtained under dark- and chromatic-adapted conditions revealed peaks in the near-u.v. (λ_max, 380 nm) and in the green (λ_max 550 nm), suggesting the presence of at least two receptor types in the dorsal sector of the compound eyes of P. versicolor. The green (550 nm) peak corresponds with the species bioluminescence emission peak (552 nm).     

Two components of the receptor current are developed from distinct elementary signals in Limulus ventral nerve photoreceptor

Transient elementary currents, bumps, stimulated by short dim light flashes were measured in ventral nerve photoreceptors of Limulus. It is demonstrated that light activates two types of bumps, which form two distinct components of the receptor current at higher light intensities. The two bump types, which are both assumed to be activated by single absorbed photons, differ in current amplitude and kinetic parameters. The current amplitude of one bump type is smaller than 0.3 nA and that of the other type is in the usual current range of up to several nanoamperes. The average latency of small bumps measured from the short stimulus flash is shorter than that of the large bumps. The small bumps have slower activation kinetics than the large bumps. It is demonstrated that with increasing flash intensity the small bumps overlap first and form a macroscopic current, on top of which the large bumps are superimposed. Results indicate that a single absorbed photon selectively activates only one kind of the enzyme cascades evoking one bump type. We conclude that the active meta conformation of a rhodopsin molecule selectively binds a specific type of G-protein, which is involved in the stimulation of one of the transduction cascades. The two bump types, which are the elements of two macroscopic current components support the previous assumption that light activates different transduction mechanisms in Limulus photoreceptors.     

Recognition of cysteine-containing peptides through prompt fragmentation of the 4-dimethylaminophenylazophenyl-4'-maleimide derivative during analysis by MALDI-MS

Under specified UV-MALDI conditions, the 4-dimethylaminophenylazophenyl-4'-maleimide (DABMI) derivative of cysteine-containing peptides undergoes prompt fragmentation to produce a characteristic mass spectral pattern. As reported previously by others, derivatization with chromophoric DABMI allows cysteine-containing peptides and proteins to be tracked during HPLC by absorbance at upper UV and visible wavelengths. Here, we describe methodology by which these same peptide derivatives can be recognized by their distinctive MALDI mass spectral fragmentation pattern, then mass mapped, allowing for easy, rapid identification of cysteine-containing peptides.     

Multiple Mechanisms of Photoreceptor Spectral Tuning in Heliconius Butterflies

The evolution of color vision is often studied through the lens of receptor gain relative to an ancestor with fewer spectral classes of photoreceptor. For instance, in Heliconius butterflies, a genus-specific UVRh opsin duplication led to the evolution of UV color discrimination in Heliconius erato females, a rare trait among butterflies. However, color vision evolution is not well understood in the context of loss. In Heliconius melpomene and Heliconius ismenius lineages, the UV2 receptor subtype has been lost, which limits female color vision in shorter wavelengths. Here, we compare the visual systems of butterflies that have either retained or lost the UV2 photoreceptor using intracellular recordings, ATAC-seq, and antibody staining. We identify several ways these butterflies modulate their color vision. In H. melpomene, chromatin reorganization has downregulated an otherwise intact UVRh2 gene, whereas in H. ismenius, pseudogenization has led to the truncation of UVRh2. In species that lack the UV2 receptor, the peak sensitivity of the remaining UV1 photoreceptor cell is shifted to longer wavelengths. Across Heliconius, we identify the widespread use of filtering pigments and co-expression of two opsins in the same photoreceptor cells. Multiple mechanisms of spectral tuning, including the molecular evolution of blue opsins, have led to the divergence of receptor sensitivities between species. The diversity of photoreceptor and ommatidial subtypes between species suggests that Heliconius visual systems are under varying selection pressures for color discrimination. Modulating the wavelengths of peak sensitivities of both the blue- and remaining UV-sensitive photoreceptor cells suggests that Heliconius species may have compensated for UV receptor loss.