Prolonged Depolarizing Afterpotential Research Articles

Electrophysiological Methods for Measuring Photopigment Levels in Drosophila Photoreceptors.

The Drosophila G-protein-coupled photopigment rhodopsin (R) is composed of a protein (opsin) and a chromophore. The activation process of rhodopsin is initiated by photon absorption-inducing isomerization of the chromophore, promoting conformational changes of the opsin and resulting in a second dark-stable photopigment state (metarhodopsin, M). Investigation of this bi-stable photopigment using random mutagenesis requires simple and robust methods for screening mutant flies. Therefore, several methods for measuring reductions in functional photopigment levels have been designed. One such method exploits the charge displacements within the photopigment following photon absorption and the huge amounts of photopigment molecules expressed in the photoreceptors. This electrical signal, named the early receptor potential (or early receptor current), is measured by a variety of electrophysiological methods (e.g., electroretinogram and whole-cell recordings) and is linearly proportional to functional photopigment levels. The advantages of this method are the high signal-to-noise ratio, direct linear measurement of photopigment levels, and independence of phototransduction mechanisms downstream to rhodopsin or metarhodopsin activation. An additional electrophysiological method called prolonged depolarizing afterpotential (PDA) exploits the bi-stability of Drosophila photopigment and the absorption-spectral differences of fly R and M pigment states. The PDA is induced by intense blue light, converting saturating amounts of rhodopsin to metarhodopsin, resulting in the failure of light-response termination for an extended time in darkness, but it can be terminated by metarhodopsin to rhodopsin conversion using intense orange light. Since the PDA is a robust signal that requires massive photopigment conversion, even small defects in the biogenesis of the photopigment lead to readily detected abnormal PDA. Indeed, defective PDA mutants led to the identification of novel signaling proteins important for phototransduction.

Electroretinogram (ERG) Recordings from Drosophila.

Phototransduction is a process in which light is converted into electrical signals used by the central nervous system. Invertebrate phototransduction is a process mediated by the phosphoinositide signaling cascade, characterized by Phospholipase C (PLC) as the effector enzyme and the Transient Receptor Potential (TRP) channels as its target. The great advantage of using invertebrate photoreceptors is the simplicity of the preparation, the ease of light stimulation, the robust expression of key molecular components, and most importantly, the ability to apply the power of molecular genetics. This last feature is mainly attributed to Drosophila melanogaster as a preferred animal model. The Electroretinogram (ERG) is an extracellular voltage recording from the entire eye, which reflects the total electrical activity arising from the retina in response to a light stimulation. The Drosophila ERG light response is robust and easily obtained, thus making it a convenient method to identify defects in the light response as a result of mutations. The Prolonged Depolarizing Afterpotential (PDA) is a useful ERG phenomenon that can be recorded from white-eyed flies following intense blue light. It is induced by a massive photo-conversion of the photopigment rhodopsin to its dark stable state called metarhodopsin, due to failure of light response termination. Unlike the light coincident ERG recording, which declines quickly to the dark baseline after the cessation of the light stimulus, the PDA response continues long (hours) after light offset. However, this response can be suppressed to the dark baseline at any time by photo-conversion of metarhodopsin back to rhodopsin, by application of an intense orange light stimulus (see Figure 7; Minke, 2012). The PDA has been used as an important tool to screen for visual defective mutant (Minke, 2012).

Receptor demise from alteration of glycosylation site in Drosophila opsin: electrophysiology, microspectrophotometry, and electron microscopy.

In the delta Asn20 Drosophila stock, the N-linked glycosylation site of opsin in R1-6 receptors (Rh1) is absent. We used electroretinography (ERG), microspectrophotometry (MSP), and electron microscopy (EM) to quantify visual cell defects. Positive controls, w9, had wild type Rh1. MSP revealed minimal photopigment in delta Asn20 for 6 days posteclosion; w9 had near normal visual pigment. ERG sensitivity and prolonged depolarizing afterpotential (PDA) were compared for delta Asn20 and w9. Delta Asn20's R1-6 function is decreased 100-fold at eclosion and diminishes until only R7/8 functions at 11 days. What little rhodopsin is routed to the rhabdomere functions. Morphometry showed smaller R1-6 rhabdomeres in delta Asn20 for 8 days posteclosion. Rhabdomeres in w9 were normal. A negative control, ninaE(ol17), a deletion of the Rh1 gene, also has small rhabdomeres. Delta Asn20 and ninaE(ol17) lack the extreme rhabdomere elimination of ora (outer rhabdomeres absent), a nonsense mutant interrupting Rh1's coding sequence. Delta Asn20 and ora have surplus membrane while ninaE(ol17) does not. Freeze fracture reveals that delta Asn20's rhabdomeric P-face particle count is as low as for vitamin A deprivation, consistent with an opsin defect. High particle density, organized into rows, is present in adjacent plasmalemma where surplus membrane accumulates. In summary, delta Asn20 interferes with either synthesis, deployment, or maintenance of opsin.

Different ionic conductances are modulated during the late receptor potential and the prolonged depolarizing afterpotential in Hermissenda type A photoreceptors

Wavelength-dependent, bistable phenomena were found in the receptor potential of Hermissenda crassicornis type A photoreceptors. Short exposure to blue light induced a prolonged depolarizing afterpotential (PDA) following the cessation of the light stimulus. Stronger adaptation to blue light, as caused by prolonged exposure and/or high intensity stimulation, effected a reduction in the early depolarizing transient of the late receptor potential (LRP) as elicited by subsequent stimuli. Vast separation of LRP emergence and PDA emergence could be obtained in photoreceptors in which a strong cancellation of the LRP was accomplished but a PDA still emerged after cessation of the light stimulus. Short exposure to yellow light cancelled the PDA, and stronger adaptation restored the LRP (opposite effect to blue light). The initial depolarizing part of the LRP had earlier been demonstrated to be mediated by the light-dependent increase of an inward conductance. In contrast, in this study the PDA was found to be accompanied by the reduction of an outward conductance, most likely a K+ conductance. A bistable photopigment system is thought to control the bistable receptor potential phenomenology by regulating the different membrane conductances during the LRP and the PDA.

Fused rhabdomeres (fur) in Drosophila: an eye mutation that alters rhabdomere morphology and retinal function.

In the Drosophila compound eye, the photoreceptor cells are organized in highly precise units, the ommatidia. In each photoreceptor cell, the primary photopigment, opsin, is contained in the rhabdomere, an ordered array of densely packed microvilli. A genetic and phenotypic analysis of a new X-linked. P element-induced mutation, fur, (fused rhabdomeres) is presented. Light and electron microscope studies show that mutations at the fur locus result in the fusion of the adjacent rhabdomeres in the developing eye and the fusion takes place during the pupal stage of eye development. Electrophysiological experiments indicate that the fur mutant photoreceptors have reduced sensitivity to light and lack a PDA (prolonged depolarizing afterpotential), a response characteristic of normal photoreceptor cells. Recombination and deficiency mapping localize fur to the proximal region of the X chromosome. Reversion analysis indicates the fur mutant is the result of a P element insertion. These studies suggest that the fur locus encodes a gene that has specific roles in rhabdomere morphogenesis and retinal function.

Light and dark adaptation in fly photoreceptors: Duration and time integral of the impulse response

The properties of impulse responses of blowfly photoreceptors in light and dark adaptation were studied with brief (30 μsec) white light flashes applied with different repetition frequencies, thereby producing a range of interstimulus periods (ISPs) from 200 msec to 100 sec. The amplitude of the impulse response as intracellularly recorded membrane voltage recovered fully with ISP greater than 300 msec. The duration of the impulse response (as half-repolarization time) increased beyond that, saturating with ISPs of about 30 sec with durations of about 60 msec, which is to be compared with a duration of 14 msec with ISP of 200 msec. This finding is not associated with saturation of the membrane potential, as it is also found with subsaturating responses. It is also different from the prolonged depolarizing afterpotential (PDA). The findings could in principle be explained by calcium-activated potassium conductance in the photoreceptor membrane.

Spatial properties of the prolonged depolarizing afterpotential in barnacle photoreceptors. II. Antagonistic interactions.

In the preceding article, we investigated the spatial properties of the induction of the prolonged depolarizing afterpotential (PDA) by shifting visual pigment from the rhodopsin (R) to the metarhodopsin (M) state in the barnacle photoreceptor. In this work, we have studied the ranges within the cell of the antagonistic effects on the PDA of M-to-R transfer. When this transfer occurs during a PDA, it depresses the PDA; when it precedes PDA induction, it impedes that induction ("anti-PDA"). These ranges were previously shown (by a statistical technique) to be at least a few tens of nanometers within a half-second (D greater than 10(-13) cm2 s-1). We now demonstrate, with local illumination techniques in which a PDA was induced in one side of the cell and PDA depression or anti-PDA was induced in the other side, that both ranges are much smaller than the cell diameter (approximately 100 microns) within 30 s (D less than 10(-6)). We further show, using a less direct but shorter-range technique involving colored polarized light, that the interaction of the PDA with the anti-PDA is restricted to less than approximately 6 microns (D less than 6 X 10(-9)). This figure is quite low and suggests that the interaction may be confined to the pigment molecules, possibly in a complex of the type suggested in the preceding article.

Spatial properties of the prolonged depolarizing afterpotential in barnacle photoreceptors. I. The induction process.

In invertebrate photoreceptors, when the light stimulus results in substantial net transfer of the visual pigment from the rhodopsin (R) to the metarhodopsin (M) state, the ordinary late receptor potential (LRP) is followed by a prolonged depolarizing afterpotential (PDA). The dependence of the amplitude of the PDA on the amount of pigment conversion is strongly supralinear, and the PDA duration also depends on this amount. These observations indicate an interaction among the elements of the PDA induction process and also make possible a test of the range of this interaction. The test consists of a comparison of the PDA after localized pigment conversion, obtained by strong spot illumination, to that after weaker diffuse illumination converting a comparable total amount of pigment. The experiment was performed on the barnacle lateral eye. The effective spot size was measured by the early receptor potential (ERP), in seawater saturated with CO2, which considerably reduced the electrical coupling between the photoreceptors. The ERP was also used to determine whether there is diffusion of R molecules into the illuminated spot. The spot illumination induced a PDA with small amplitude and long duration, while no detectable PDA was induced by the diffuse light. This indicates that the range of the PDA interaction is much smaller than the entire cell. In addition, the ERP results showed that there was no detectable diffusion of R molecules into the illuminated spot area over 30 min. This measurement, with a calculated correction for the microvillar geometry of the photoreceptor, enabled us to put an upper limit on the diffusion coefficient of the pigment molecules in the inact, unfixed barnacle photoreceptor of D less than 6 X 10(-9) cm2 s-1.

Light-activated guanosinetriphosphatase in Musca eye membranes resembles the prolonged depolarizing afterpotential in photoreceptor cells.

Measurement of light-dependent GTPase (EC 3.1.5.1) activity in a paradigm guided by electrophysiological experiments was used to examine the involvement of a guanine nucleotide binding protein in fly phototransduction. Cell-free membrane preparations of Musca eyes responded to blue light by a 10- to 20-fold increase in GTP-hydrolyzing activity. This light-dependent GTPase had a low Km for GTP (0.5 microM) and was effectively inhibited by guanosine (5'----O3)-1-thiotriphosphate and guanosine 5'-[beta-gamma-imino]triphosphate but not by adenosine 5'-[beta-gamma-imino]triphosphate and ATP. The action spectrum of GTPase activity measured with intense light resembled closely the photoequilibrium spectrum of metarhodopsin. After illumination with blue (less than 480 nm) light, which converted rhodopsin to metarhodopsin, the GTPase remained highly active for at least 60 min in the dark. Similarly, rhodopsin-to-metarhodopsin conversion in intact cells induced a prolonged excitation in the dark, known as the prolonged depolarizing afterpotential (PDA). The persistent GTPase activity (like the PDA) was suppressed to the low basal activity of the unilluminated membranes after conversion of metarhodopsin to rhodopsin with red light (greater than 570 nm), whereas during illumination with red light, some GTPase activity was maintained. The magnitude of the persistent GTPase activity in the dark, like the PDA, depended in a supralinear manner on the amount of pigment conversion. Thus, the dependence of GTPase activity of Musca membrane preparations on photopigment conversion resembles the induction and suppression of the PDA measured in intact photoreceptors of Musca. These findings indicate that a guanine nucleotide binding protein is part of the chain of events leading to both the generation of the receptor potential and the PDA.

Non-local interactions between light induced processes inCalliphora photoreceptors

The prolonged depolarizing afterpotential (PDA) is a phenomenon which is tightly linked to visual pigment conversion. In order to determine whether processes underlying PDA induction and depression can spread in space, the PDA was recorded intracellularly in white-eyedCalliphora R1-6 photoreceptors and used to examine interactions between processes induced by activating statistically different photopigment molecules (Figs. 3–6). It was found that a PDA induced by converting some fraction of rhodopsin (R) molecules forward into the metarhodopsin (M) state can be completely depressed by equal or smaller amounts of pigment conversion, backward from metarhodopsin to rhodopsin even when largely different sets of pigment molecules were shifted in the respective directions, in agreement with previous experiments conducted on the barnacle. The characteristics of the afterpotentials obtained following the cessation of strong blue and green light stimuli which did not cause a net pigment conversion was examined (Figs. 7, 8). It was found that these afterpotentials, obtained when nonet R to M conversion took place, could not be depressed by an opposite net large M to R pigment conversion. Accordingly we propose to restrict the term PDA to an afterpotential which can be depressed by a net M to R pigment conversion. It is concluded: (a) that some processes underlying PDA induction and depression inCalliphora must interact at a distance which extends at least to the nearest neighboring pigment molecule, and (b) that inCalliphora photoreceptors net pigment conversion is required in order to induce and depress a PDA.

Drosophila locus with gene-dosage effects on rhodopsin

Mutations that decrease the amplitude of the prolonged depolarizing afterpotential (PDA) in Drosophila melanogaster have been shown to have reduced rhodopsin content in the rhabdomeres of photoreceptor cells. In the present study, a genetic analysis of a class of third chromosome PDA-defective mutants localized the ninaE locus to the salivary band region 92A-B. In flies with only one copy of this region instead of the normal two copies, and in ninaE heterozygotes, the rhodopsin content of the major class of photoreceptors is reduced. Three doses of this region increase the rhodopsin content of these photoreceptors. These characteristics of the ninaE locus are expected of the structural gene encoding the major species of opsin in the Drosophila compound eye.

The contribution of pigment transitions to sensitivity changes in the barnacle photoreceptor and the correlation with the prolonged depolarizing afterpotential.

A conditioning light can cause a decrease (adaptation) or an increase (facilitation) in the sensitivity of barnacle photoreceptors, as measured by the amplitude of the late receptor potential (LRP). We show that a net transfer of visual pigment from the rhodopsin (R) to the metarhodopsin (M) state induces a large facilitation whereas the reverse transfer results in a much smaller facilitation or even an adaptation. These effects were not due to the response to the conditioning light but to the pigment reactions. When the conditioning light did not alter the pigment population (i.e., M leads to M, R leads to R) it was followed by an intermediate degree of facilitation. These conclusions are correct for cells which have relatively low sensitivity. In sensitive cells, all pigment transitions produce adaptation. LRP facilitation and the prolonged depolarizing afterpotential (PDA) show several common characteristics with respect to pigment transitions: 1. Their magnitude increases with the amount of pigment transferred from R to M. 2. Both are depressed by the M leads to R transition. 3. Their production is impeded by the M leads to R transition. 4. The PDA itself is facilitated by the R leads to M transition and this facilitation decays with a time course comparable to that of LRP facilitation. These results suggest that there may be an underlying process common to LRP facilitation and PDA.

Mutation that selectively affects rhodopsin concentration in the peripheral photoreceptors of Drosophila melanogaster.

A Drosophila mutant (ninaAP228) that is low in rhodopsin concentration but identical to the wild-type fly in photoreceptor morphology has been isolated. R1-6 photoreceptors of the mutant differ from those of wild type in that (a) the prolonged depolarizing afterpotential (PDA) is absent, (b) concentrations of rhodopsin and opsin are substantially reduced, and (c) intramembrane particle density in the membranes of the rhabdomeres is low. Each of these traits is mimicked by depriving wild-type flies of vitamin A. The ninaAP228 mutation differs from vitamin A deprivation in that in the mutant (a) the rhabdomeric membrane particle density is reduced only in the R1-6 photoreceptors and not in R7 or R8, (b) the PDA can be elicited from the R7 photoreceptors, and (c) photoconversion of R1-6 rhodopsin to metarhodopsin by ultraviolet (UV) light is considerably more efficient than in vitamin A-deprived flies. The absorption properties of the mutant rhodopsin in the R1-6 photoreceptors appear to be identical to those of wild type as judged from rhodopsin difference spectra. The results suggest that the mutation affects the opsin, rather than the chromophore, component of rhodopsin molecules in the R1-6 photoreceptors. The interaction between the chromophore and R1-6 opsin, however, appears to be normal.

Transduction in photoreceptors with bistable pigments: Intermediate processes

Abstract. The prolonged depolarizing after potential (PDA) in the R1-6 receptors of the fly was used to isolate intermediate processes in phototransduction which are not manifested directly in the voltage response. It is first demonstrated that a pigment shift by light from metarhodopsin to rhodopsin in four species of the flies: Drosophila, Calliphora, Chrysomya and Musca induces an independent antagonistic process to the PDA, which is manifested in a strong inhibitory effect on PDA induction and is called the anti-PDA. By using mutants of Drosophila the existence of processes underlying the PDA were examined. The norpA(H52) and the trp mutant were used in which the voltage response of the photoreceptors could be reversibly abolished by elavated temperature and long intense light respectively. It is shown that the excitatory process underlying the PDA could be induced and depressed in conditions that block the voltage response of the photoreceptors, thus indicating the existance of intermediate processes which link the pigment activation by light to the PDA voltage response.

Properties of the on-transient of the intracellular response in the barnacle photoreceptor

We have studied the on-transient of the receptor potential of the barnacle photoreceptor. Its amplitude has previously been shown to depend on light intensity and state of light-dark adaptation. We have examined its dependence on 1) the presence of a prolonged depolarizing afterpotential (PDA), 2) a background light, 3) added alcohol, or 4) decreased K+ concentration in the bath. We find that the relative on-transient amplitude tends to increase initially with increasing depolarization arising from 1)-4) and then to decrease again at higher depolarization. This behavior is qualitatively explainable by the cell's current-voltage characteristics and by the adapting effect of the stimulus on the conductances arising from the PDA, the background light and the alcohol.

On the implications of bistability of visual pigment systems

The characteristics of different responses of invertebrate photoreceptors are reviewed. Invertebrate photopigment bistability has made possible the functional operational dissection of the pigment transition scheme. Outlasting the usual stimulus-coincident late receptor potential (LRP), additional antagonistic responses have been found: the prolonged depolarizing after-potential (PDA) arising from a net rhodopsin to metarhodopsin pigment shift, and a PDA-depression and an anti-PDA effect which arise from a reverse shift and cancel the PDA when induced during or closely before it. The characteristics of these aftereffects and of the LRP are reviewed, analyzed and compared. Both potentials require rhodopsin activation and they share the characteristics of a common ionic conductance-change mechanism. However, for the LRP response to weak stimuli, no antagonistic metarhodopsin-dependent effect has been found analogous to PDA-depression and the anti-PDA. However, this is just the response level where interactive effects would be weakest. For more intense stimuli, pigment-state effects on the shape of the LRP have been found, and net pigment shifts affect the strength of a facilitatory effect.

The distribution of bumps in the tail of the locust photoreceptor afterpotential.

An extended tail or prolonged depolarizing afterpotential (PDA) follows the receptor potential of a locust retinula cell when the stimulating light is in the intensity range that saturates the receptor potential. The amplitude and duration of this afterpotential depend on the intensity and duration of the stimulus. As the afterpotential decays, apparently exponentially, it becomes resolved into bumps, which we call light-induced dark bumps (LID bumps). The intervals between light-induced dark bumps are distributed in a way that is indistinguishable from a random (Poisson) distribution. As previously demonstrated, LID bumps are indistinguishable from bumps directly induced by low intensity light in light-adapted cells, which in turn grade into the slightly larger bumps produced, each by a single photon, in dark-adapted cells. The light-induced dark bumps continue for up to an hour in darkness, slowly becoming like dark-adapted bumps in amplitude and shape. To account for the random occurrence and discrete features of bumps after so long a latency, we propose that intense light generates a significant amount of an intermediate molecule or packet which decays slowly to start the same process that normally generates bumps with a short delay.

Lability of the prolonged depolarizing afterpotential in Balanus photoreceptors.

A large cell to cell variability of the prolonged depolarizing afterpotential (PDA) decay time constant (tau) has been measured in Balanus eberneus lateral ocelli. While 25% of the cells had PDA's of long duration, tau greater than 10 min. 45% of the cells tested showed either weak (tau less than 60 s) PDA or none at all. The variability was not reflected in the late receptor potential. All the cells showed normal light-coincident responses. The variability was not due to some alteration of the thermal stability of the pigment states, since after monochromatic adaptation the amplitude of the early receptor potential remained unchanged for at least 30 min. In addition, in some cells that initially showed PDAs of long duration, the decay time was either shortened or abolished after exposure to anoxia. Again, the late receptor potential and the stability of the pigments remained unaffected. These results indicate that the mechanisms which give rise to the PDA are not always tightly coupled to the direct chain of events that lead to the light-coincident response.

Hereditary retinal degeneration in Drosophila melanogaster. A mutant defect associated with the phototransduction process.

Two genes in Drosophila, rdgA and rdgB, which when defective cause retinal degeneration, were discovered by Hotta and Benzer (Hotta, Y., and S. Benzer. 1970. Proc. Natl, Acad. Sci. U. S, A. 67:1156-1163). These mutants have photoreceptor cells that are histologically normal upon eclosion but subsequently degenerate. The defects in the rdgA and rdgB mutants were localized by the study of genetic mosaics to the photoreceptor cells. In rdgB mutants retinal degeneration is light induced. It can be prevented by rearing the flies in the dark or by blocking the receptor potential with a no-receptor-potential mutation, norpA. Vitamin A deprivation and genetic elimination of the lysosomal enzyme acid phosphatase alsoprotect the photoreceptors of rdgB flies against light-induced damage. The photopigment kinetics of dark-reared rdgB flies appear normal in vitro by spectrophotometric measurements, and in vivo by measurements of the M potential. In normal Drosophila, a 1-s exposure to intense 470-nm light produces a prolonged depolarizing afterpotential (PDA) which can last for several hours. In dark-reared rdgB mutants the PDA lasts less than 2 min;; it appears to initiate the degeneration process, since the photoreceptors become permanently unresponsive after a single such exposure. Another mutant was isolated which prevents degeneration in rdgB flies but which has a normal receptor potential. This suppressor of degeneration is an allele of norpA. It is proposed that the normal norpA gene codes for a product which, when activated, leads to the receptor potential, and which is inactivated by the product of the normal rdgB gene.

Drosophila mutant with a transducer defect.

The trp is a conditional phototransduction mutant of Drosophila. Direct electrical measurements and shot noise analysis suggest that a prolonged intense light causes in the mutant a reduction in the quantum efficiency for quantum bump production that does not arise from bleaching of the visual pigment. This effect depends on the duration of the light and only weakly on its intensity. In the normal fly, an intense blue light that shifts the visual pigment from rhodopsin to metarhodopsin, induces an excitatory process manifested by a prolonged depolarizing after potential (PDA). In the mutant, the PDA has a small amplitude and bump noise is superimposed on the response. It can thus be shown that the excitatory process underlying the PDA is also present in those trp mutants where the PDA voltage response is small or absent. It is suggested that the absence of the PDA voltage response in the mutant is probably due to a defect in an intermediate process, which links the excitatory process to the membrane conductance change.