White noise analysis of Phycomyces light growth response system. I. Normal intensity range.

The Wiener-Lee-Schetzen method for the identification of a nonlinear system through white gaussian noise stimulation was applied to the transient light growth response of the sporangiophore of Phycomyces. In order to cover a moderate dynamic range of light intensity I, the imput variable was defined to be log I. The experiments were performed in the normal range of light intensity, centered about I0 = 10(-6) W/cm2. The kernels of the Wierner functionals were computed up to second order. Within the range of a few decades the system is reasonably linear with log I. The main nonlinear feature of the second-order kernel corresponds to the property of rectification. Power spectral analysis reveals that the slow dynamics of the system are of at least fifth order. The system can be represented approximately by a linear transfer function, including a first-order high-pass (adaptation) filter with a 4 min time constant and an underdamped fourth-order low-pass filter. Accordingly a linear electronic circuit was constructed to simulate the small scale response characteristics. In terms of the adaptation model of Delbrück and Reichardt (1956, in Cellular Mechanisms in Differentiation and Growth, Princeton University Press), kernels were deduced for the dynamic dependence of the growth velocity (output) on the "subjective intensity", a presumed internal variable. Finally the linear electronic simulator above was generalized to accommodate the large scale nonlinearity of the adaptation model and to serve as a tool for deeper test of the model.     

Light and dark adaptation in Phycomyces light-growth response

Sporangiophores of the fungus Phycomyces exhibit adaptation to light stimuli over a dynamic range of 10(10). This range applies to both phototropism and the closely related light-growth response; in the latter response, the elongation rate is modulated transiently by changes in the light intensity. We have performed light- and dark- adaptation experiments on growing sporangiophores using an automated tracking machine that allows a continuous measurement of growth velocity under controlled conditions. The results are examined in terms of the adaptation model of Delbruck and Reichardt (1956, Cellular Mechanisms in Differentiation and Growth, 3-44). The "level of adaptation," A, was inferred from responses to test pulses of light by means of a series of intensity-response curves. For dark adaptation to steps down in the normal intensity range (10(-6)-10(-2) W/m2), A decays exponentially with a time constant b = 6.1 +/- 0.3 min. This result is in agreement with the model. Higher-order kinetics are indicated, however, for dark adaptation in the high-intensity range (10(-2)-1 W/m2). Adaptation in this range is compared with predictions of a model relating changes in A to the inactivation and recovery of a receptor pigment. In response to steps up in intensity in the normal range, A was found to increase rapidly, overshoot the applied intensity level, and then relax to that level within 40 min. These results are incompatible with the Delbruck-Reichardt model or any simple generalizations of it. The asymmetry and overshoot are similar to adaptation phenomena observed in systems as diverse as bacterial chemotaxis and human vision. It appears likely that light and dark adaptation in Phycomyces are mediated by altogether different processes.     

System analysis of Phycomyces light-growth response. Photoreceptor and hypertropic mutants.

The light-growth responses of Phycomyces behavioral mutants, defective in genes madB, madC, and madH, were studied with the sum-of-sinusoids method of system identification. Modified phototropic action spectra of these mutants have indicated that they have altered photoreceptors (P. Galland and E.D. Lipson, 1985, Photochem. Photobiol. 41:331). In the two preceding papers, a kinetic model of the light-growth response system was developed and applied to wild-type frequency kernels at several wavelengths and temperatures. The present mutant studies were conducted at wavelength 477 nm. The log-mean intensity was 6 X 10(-2)W m-2 for the madB and madC night-blind mutants, and 10(-4)W m-2 for the madH hypertropic mutant. The prolonged light-growth responses of the madB and madC mutants are reflected in the reduced dynamic order of their frequency kernels. The linear response of the hypertropic mutant is essentially normal, but its nonlinear behavior shows modified dynamics. The behavior of these mutants can be accounted for by suitable modifications of the parametric model of the system. These modifications together support the hypothesis that an integrated complex mediates sensory transduction in the light responses and other responses of the sporangiophore.     

System analysis of Phycomyces light-growth response with sum-of-sinusoids test stimuli.

The light-growth response of Phycomyces has been studied with the sum-of-sinusoids method of nonlinear system identification (Victor, J.D., and R.M. Shapley, 1980, Biophys. J., 29:459). This transient response of the sporangiophore has been treated as a black-box system with one input (logarithm of the light intensity, I) and one output (elongation rate). The light intensity was modulated so that log I, as a function of time, was a sum of sinusoids. The log-mean intensity was 10(-4) W m-2 and the wavelength was 477 nm. The first- and second-order frequency kernels, which represent the linear and nonlinear behavior of the system, were obtained from the Fourier transform of the response at the appropriate component and combination frequencies. Although the first-order kernel accounts for most of the response, there remains a significant nonlinearity beyond the logarithmic transducer presumed to occur at the input of the sensory transduction chain. From the analysis of the frequency kernels, we have derived a dynamic nonlinear model of the light-growth response system. The model consists of a nonlinear subsystem followed by a linear subsystem. The model parameters were estimated from a combined nonlinear least-squares fit to the first- and second-order frequency kernels.     

The dynamic nonlinear behavior of fly photoreceptors evoked by a wide range of light intensities.

Fly photoreceptor cells were stimulated with steps of light over a wide intensity range. First- and second-order Volterra kernels were then computed from sequences of combined step responses. Diagonal values of the second-order Volterra kernels were much greater than the off-diagonal values, and the diagonal values were roughly proportional to the corresponding first-order kernels, suggesting that the response could be approximated by a static nonlinearity followed by a dynamic linear component (Hammerstein model). The amplitudes of the second-order kernels were much smaller in light-adapted than in dark-adapted photoreceptors. Hammerstein models constructed from the step input/output measurements gave reasonable approximations to the actual photoreceptor responses, with light-adapted responses being relatively better fitted. However, Hammerstein models could not account for several features of the photoreceptor behavior, including the dependence of the step response shape on step amplitude. A model containing an additional static nonlinearity after the dynamic linear component gave significantly better fits to the data. These results indicate that blowfly photoreceptors have a strong early gain control nonlinearity acting before the processes that create the characteristic time course of the response, in addition to the nonlinearities caused by membrane conductances.     

Increased intracellular sodium mimics some but not all aspects of photoreceptor adaptation in the ventral eye of Limulus

The effects of the intracellular iontophoretic injection of Na+ ions have been quantitatively compared with adaptation in ventral photoreceptors of Limulus. We find that: (a) both light adaptation and sodium injection are associated with a decrease in the variability of the threshold response amplitued; (b) both light adaptation and sodium injection are associated with a decrease in the absolute value of the temporal dispersion of the threshold response time delay; (c) the same template curve adequately fits the intensity response relationships measured under light adaptation and Na+ injection; (d) both light adaptation and Na+ injection produce a fourfold decrease in response time delay for a desensitization of 3 log units; (e) the time coures of light adaptation and dark adaptation is significantly faster than the onset of and recovery from desensitization produced by Na+ injection; (f) unlike local illumination, Na+ injection does not produce localized desensitization of the photoreceptor. These findings suggest that a rise in intracellular Na+ concentration makes at most only a minor contribution (probably less than 5%) to the total adaptation of these receptors in the intensity range we have examined (up to 3 log units above absolute threshold). However, changes in intracellular Na+ concentration may contribute to certain components of light and dark adaptation in these receptors.     

Light and dark adaptation in Phycomyces phototropism

Light and dark adaptation of the phototropism of Phycomyces sporangiophores were analyzed in the intensity range of 10(-7)-6 W X m- 2. The experiments were designed to test the validity of the Delbruck- Reichardt model of adaptation (Delbruck, M., and W. Reichardt, 1956, Cellular Mechanisms in Differentiation and Growth, 3-44), and the kinetics were measured by the phototropic delay method. We found that their model describes adequately only changes of the adaptation level after small, relatively short intensity changes. For dark adaptation, we found a biphasic decay with two time constants of b1 = 1-2 min and b2 = 6.5-10 min. The model fails for light adaptation, in which the level of adaptation can overshoot the actual intensity level before it relaxes to the new intensity. The light adaptation kinetics depend critically on the height of the applied pulse as well as the intensity range. Both these features are incompatible with the Delbruck-Reichardt model and indicate that light and dark adaptation are regulated by different mechanisms. The comparison of the dark adaptation kinetics with the time course of the dark growth response shows that Phycomyces has two adaptation mechanisms: an input adaptation, which operates for the range adjustment, and an output adaptation, which directly modulates the growth response. The analysis of four different types of behavioral mutants permitted a partial genetic dissection of the adaptation mechanism. The hypertropic strain L82 and mutants with defects in the madA gene have qualitatively the same adaptation behavior as the wild type; however, the adaptation constants are altered in these strains. Mutation of the madB gene leads to loss of the fast component of the dark adaptation kinetics and to overshooting of the light adaptation under conditions where the wild type does not overshoot. Another mutant with a defect in the madC gene shows abnormal behavior after steps up in light intensity. Since the madB and madC mutants have been associated with the receptor pigment, we infer that at least part of the adaptation process is mediated by the receptor pigment.     

Scanning CO2 laser bacterial inactivation systems

AIMS: The performance of three scanning CO(2) laser inactivation systems was assessed and included: a gantry system, a rapidly rotating mirror and a low-power hybrid system combining an oscillating mirror and rotary motion of the sample. METHODS AND RESULTS: Escherichia coli and Staphylococcus aureus were the target organisms on stainless steel, nutrient agar or moist collagen film and the laser power was varied from 2 to 1060 W (two laser sources). In general, a threshold energy density was identified, above which no inactivation was observed because the scanning velocity was too high (10 cm s(-1) for stainless steel, 660 W). Reducing the velocity increased the inactivation process until complete inactivation was observed at 1.3 cm s(-1) (E. coli, approximately 10(6) CFU per sample) and 0.82 cm s(-1) (S. aureus, approximately 10(8) CFU per sample); consequently, S. aureus organisms showed a greater resistance to laser irradiation. For the nutrient agar and collagen samples, the averages of the width of clearing were measured as a function of the translation velocity and the rates of inactivation (I(R), cm(2) s(-1)) were found; an optimum velocity was observed that produced the maximum rate of inactivation. At a laser power of 1060 W, the maximum value of I(R) was 140 cm(2) s(-1) ( approximately 10(7) CFU cm(-2)) for S. aureus on collagen and slightly less on nutrient agar (114 cm(2) s(-1), estimated from a best-fit polynomial, r(2) = 0.98). CONCLUSIONS: A comparison of the low- and high-power lasers produced values of 0.09 cm(2) s(-1) W(-1) (i.e. I(R) per Watt delivered) for S. aureus on nutrient agar with the low-power laser at 13 W and on collagen 0.13 cm(2) s(-1) W(-1) for 1060 W. The rate of inactivation was found to be a function of the laser power, translation velocity and properties of the substrate media. The three laser inactivation systems successfully demonstrated the potential speed, efficiency and application of such systems. SIGNIFICANCE AND IMPACT OF THE STUDY: Laser scanning systems offer the potential for rapid and efficient inactivation of surfaces, eliminating the need for chemical treatment.     

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.     

The sensitivity of chlamydomonas photoreceptor is optimized for the frequency of cell body rotation.

For phototactic migration, Chlamydomonas scans the surrounding light environment by rotating the cell body with an eyespot located on the equator. The intensity of the light signal received by the eyespot should therefore change cyclically at the frequency of the cell body rotation. In this study, the response of the photoreceptor to cyclically changing light stimuli was analyzed using immotile mutant cells. To simulate the light intensity change perceived by a rotating cell, light stimuli were applied that consisted of a light phase with the intensity changing similar to a half cycle of a sine wave and a dark phase of the same length. The fluence rate at the peak of the sine wave was of the order of 10(19) photons m(-2) s(-1), i.e. high intensity at which phototaxis is saturated. A photoreceptor current (PRC) was produced at the onset of each light phase. Interestingly, its amplitude varied depending on the frequency and was largest at 1-5 Hz, a frequency range similar to the frequency of cell body rotation. Experiments on the kinetics of the PRC indicate that the response was small at low frequency because of the inactivation of the PRC before full activation. In contrast, at high frequency the PRC was suppressed by adaptation to the repetitive stimuli. These characteristic kinetics of the PRC should be important for Chlamydomonas cells to extract information from the signals generated by the cell body rotation.     

Contrast gain, signal-to-noise ratio, and linearity in light-adapted blowfly photoreceptors

Response properties of short-type (R1-6) photoreceptors of the blowfly (Calliphora vicina) were investigated with intracellular recordings using repeated sequences of pseudorandomly modulated light contrast stimuli at adapting backgrounds covering 5 log intensity units. The resulting voltage responses were used to determine the effects of adaptational regulation on signal-to-noise ratios (SNR), signal induced noise, contrast gain, linearity and the dead time in phototransduction. In light adaptation the SNR of the photoreceptors improved more than 100-fold due to (a) increased photoreceptor voltage responses to a contrast stimulus and (b) reduction of voltage noise at high intensity backgrounds. In the frequency domain the SNR was attenuated in low frequencies with an increase in the middle and high frequency ranges. A pseudorandom contrast stimulus by itself did not produce any additional noise. The contrast gain of the photoreceptor frequency responses increased with mean illumination and the gain was best fitted with a model consisting of two second order and one double pole of first order. The coherence function (a normalized measure of linearity and SNR) of the frequency responses demonstrated that the photoreceptors responded linearly (from 1 to 150 Hz) to the contrast stimuli even under fairly dim conditions. The theoretically derived and the recorded phase functions were used to calculate phototransduction dead time, which decreased in light adaptation from approximately 5-2.5 ms. This analysis suggests that the ability of fly photoreceptors to maintain linear performance under dynamic stimulation conditions results from the high early gain followed by delayed compressive feed-back mechanisms.     

The effect of light adaptation on the dynamic properties of phototransduction in the fly,Phormia regina

The static and dynamic characteristics of phototransduction were studied in photoreceptors of the compound eye of the fly Phormia regina (Calliphoridae) using a green light emitting diode driven by a controlled current source. The LED provides sufficiently intense light to investigate the behaviour of the receptors over about half of the dark adapted range of the response versus log intensity curve. The effects of constant adapting light intensities upon the step response and upon the frequency response and coherence functions were examined. Using both methods the effect of light adaptation upon receptor sensitivity can be closely approximated by a similar linear dependence of log sensitivity upon log adapting intensity. However, there was no reliably detectable effect of light adaptation upon the time constant of the response over the range of adapting intensities used.Abbreviation LED Light Emitting Diode     

Early Receptor Potential Evidence for the Existence of Two Thermally Stable States in the Barnacle Visual Pigment

The early receptor potential (ERP) in the barnacle photoreceptor is shown by intracellular recording to exhibit a strong dependence on the color of the stimulus and of the preceding adaptation. The adaptation effects appear to be stable for at least 3 h in the dark. Most strikingly, the ERP is positive after red adaptation and mainly negative after blue adaptation. The simplest hypothesis which accounts for these observations is that two thermally stable pigment states with different absorption spectra contribute to the ERP. All ERP responses appear to be consistent with the sums of different ratios of the ERP's of the two pure states. The relative populations of the two states are shown to vary reciprocally, suggesting that the two are states of the same closed pigment cycle. Both states have approximately Dartnall nomogram-shaped absorption spectra, one peaked near 495 nm, and the other near 532 nm.     

Responses of crayfish photoreceptor cells following intense light adaptation

Summary After intense orange adapting exposures that convert 80% of the rhodopsin in the eye to metarhodopsin, rhabdoms become covered with accessory pigment and appear to lose some microvillar order. Only after a delay of hours or even days is the metarhodopsin replaced by rhodopsin (Cronin and Goldsmith 1984). After 24 h of dark adaptation, when there has been little recovery of visual pigment, the photoreceptor cells have normal resting potentials and input resistances, and the reversal potential of the light response is 10–15 mV (inside positive), unchanged from controls. The log V vs log I curve is shifted about 0.6 log units to the right on the energy axis, quantitatively consistent with the decrease in the probability of quantum catch expected from the lowered concentration of rhodopsin in the rhabdoms. Furthermore, at 24 h the photoreceptors exhibit a broader spectral sensitivity than controls, which is also expected from accumulations of metarhodopsin in the rhabdoms. In three other respects, however, the transduction process appears to be light adapted: (i) The voltage responses are more phasic than those of control photoreceptors. (ii) The relatively larger effect (compared to controls) of low extracellular Ca⁺⁺ (1 mmol/1 EGTA) in potentiating the photoresponses suggests that the photoreceptors may have elevated levels of free cytoplasmic Ca⁺⁺. (iii) The saturating depolarization is only about 30% as large as the maximal receptor potentials of contralateral, dark controls, and by that measure the log V-log I curve is shifted downward by 0.54 log units. The gain (change in conductance per absorbed photon) therefore appears to have been diminished.     

A quantitative comparison of the effects of intracellular calcium injection and light adaptation on the photoresponse of Limulus ventral photoreceptors

Calcium ions were iontophoretically injected into ventral photoreceptors of Limulus by passing current between two intracellular pipettes. Changes in sensitivity and photoresponse time course were measured for both light adaptation and Ca++ injection. We found for some photoreceptors that there was no significant difference in the photoresponse time course for desensitization produced by light adaptation or by Ca++ injection. In other photoreceptors, the time delay of photoresponse for Ca++ injection was slightly longer than for light adaptation. The variability of threshold response amplitude and time delay decreases when the photoreceptor is desensitized by either light adaptation or Ca++ injection. The peak amplitude versus log stimulus intensity relationships for controls, light adaptation, and Ca++ injection all could be described very closely by a single template curve shifted along the log intensity axis. A 40- to 50-fold change in sensitivity is associated with a 2-fold change in photoresponse time delay for both light adaptation and Ca++ injection.     

White noise analysis of Phycomyces light growth response system. III. Photomutants.

Wiener kernels have been measured for the light growth response of a number of mutants of Phycomyces which show abnormal phototropism (mad mutants). Representative mutants were chosen from the six complementation groups (madA to madF) associated with the light response pathway. One group, madA, associated with the input part of the pathway, exhibits an essentially normal response provided it is tested above its moderate threshold. The groups madB and madC appear more defective, in that their kernel amplitudes are very small even above their thresholds. Their similarity to each other suggests a close functional connection between the respective genes. The remaining three groups (madD, madE, and madF) have all been associated with the output of the pathway. Tbe kernels for all three indicate a gain reduction, which depends gradually on intensity. These three groups appear to have the same absolute threshold as wild-type. None of the mutants studied shows special behavior at high intensity that could be evidence of alterations in the photoreceptor complex.     

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.     

Wavelength dependence of dark adaptation in Phycomyces phototropism

The wavelength dependence of phototropic dark adaptation in Phycomyces was studied between 347 and 545 nm. Dark adaptation kinetics were measured for wavelengths of 383, 409, 477, and 507 nm in the intensity range from 6.2 X 10(-2) to 2 X 10(-7) W X m-2. At these wavelengths, dark adaptation follows a biexponential decay as found previously with broadband blue light (Russo, V. E. A., and P. Galland, 1980, Struct. Bonding., 41:71; Lipson, E. D., and S. M. Block, 1983, J. Gen. Physiol., 81:845). We have found that the time constants of the fast and slow components depend critically on the wavelength. At 507 nm, dark adaptation kinetics were found to be monophasic. The phototropic latency after a step down by a factor of 500 was measured for 19 different wavelengths. Maximal latencies were found at 383, 477, and 530 nm; minimal latencies were found at 409 and 507 nm. With irradiation programs that employ different wavelengths before and after the step down, the dark adaptation kinetics depend critically on the sequence in which the two wavelengths are given. We have found too that not only do the adaptation kinetics vary with wavelength, but so also do the phototropic bending rate and the phototropic latencies in experiments without intensity change. The results imply that more than one photoreceptor is mediating phototropism in Phycomyces and that sensory adaptation is regulated by these photoreceptors.     

Insect pupil mechanisms

Summary The hypothesis that the glow observable in dark adapted butterfly eyes is extinguished upon light adaptation by the action of migrating retinula cell pigment granules (Stavenga, 1975a) has been investigated. Experimental procedures applying optical methods to intact, living animals were similar to those used previously to investigate the migration of retinula cell pigment granules in Hymenoptera (Stavenga and Kuiper, 1977). The data obtained from nymphalid butterflies and Hymenoptera show close parallels, favouring the pigment migration hypothesis.The retinula cell pigment granules control the light flux in the butterfly rhabdom and hence are part of a pupil mechanism. The range of action of this pupil mechanism is about 3 log units of light intensity. The speed of pupil closure is slowed down with longer dark adaptation times. The way in which pupil processes can be distinguished from photochemical processes of the visual pigment is discussed.     

DARK ADAPTATION IN DINEUTES

The level of dark adaptation of the whirligig beetle can be measured in terms of the threshold intensity calling forth a response. The course of dark adaptation was determined at levels of light adaptation of 6.5, 91.6, and 6100 foot-candles. All data can be fitted by the same curve. This indicates that dark adaptation follows parts of the same course irrespective of the level of light adaptation. The intensity of the adapting light determines the level at which dark adaptation will begin. The relation between log aI ₀ (instantaneous threshold) and log of adapting light intensity is linear over the range studied.