A digital feedback controller application for studying photoreceptor adaptation by 'voltage clamp by light'.

We present a new digital feedback application for the study of the sensitivity characteristics of photoreceptors. The amplitude of the recorded membrane voltage of a cell is steered by changing the incoming light intensity with a motor-driven circular, linear neutral-density wedge (CFW). The voltage response is sampled and fed to a software position controller of the CFW. The controller determines the position of the wedge according to the desired (command) value of the response. The light intensity changes during steady-state represent the sensitivity change, the time-course of adaptation.

Tonic transmitter release in a graded potential synapse.

1. We studied graded synaptic transmission in the fly photoreceptor-interneuron synapse by using intracellular in situ recordings from pre- and postsynaptic cells. 2. A large presynaptic hyperpolarization after light adaptation, caused by the activation of the electrogenic Na+/K+ pump, drastically reduced the conspicuous postsynaptic dark noise. At the same time, the postsynaptic neurons depolarized, with an increase of input resistance of 5-10 M omega. 3. The spectral characteristics of the postsynaptic membrane noise in dark and during noise reduction, together with the other results, suggested that the transmitter release decreased dramatically approximately 12 mV below the resting potential of the presynaptic photoreceptors. 4. During the postsynaptic noise reduction, the saturated and subsaturated first-order visual interneuron responses were increased up to 9 mV with a time constant of recovery of approximately 10 s. This increase was shown to be caused by the negative shift of the reversal potential of the transmitter-gated (mainly Cl-) conductance, caused apparently by the reduced transmitter input. 5. The results strongly suggest that the photoreceptor transmitter release in fly is tonic, even in dark, and further support the modulation of the synaptic voltage transfer by postsynaptic Cl- extrusion.

A method for determining photoreceptor signal-to-noise ratio in the time and frequency domains with a pseudorandom stimulus.

We have developed a method that utilizes repeated sequences of pseudorandomly modulated stimuli for calculation of the SNR either in the time or frequency domains. The method has the advantage that the distribution of SNR over relevant frequencies is readily observed. In addition, a SNR value, calculated as the ratio of the corresponding variances, is an estimate of the true SNR because it has been weighted by the cell's frequency response. The procedure offers significant advantages when studying signal transmission in nonspiking cells like photoreceptors.

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 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.

A digital feedback controller application: a light response control system to reveal photoreceptor adaptation.

We present a new digital feedback application for the study of sensitivity characteristics of biological photoreceptors. The voltage response of a cell is controlled by feeding back to the stimulating LED--via the controller--the recorded membrane voltage. The light intensity changes represent the change in the sensitivity of the photoreceptors, revealing the time course of adaptation.

Measurement of cell impedance in frequency domain using discontinuous current clamp and white-noise-modulated current injection.

A method is described for the determination of cellular input impedance of non-spiking neurones. The input impedance is important when cellular geometry and the effects of voltage-dependent channels are considered. Cells are impaled with a single glass microelectrod and current is injected using a time-sharing technique. The cell's impedance is measured by randomly modulating the injected current and calculating the impedance as a transfer function between current and recorded membrane voltage. Corresponding coherence functions can also be calculated for estimating the signal-to-noise ratio, and also linearity (i.e. possible activation of voltage-dependent conductances) of the membrane.

Non-linearities in response properties of insect visual cells: an analysis in time and frequency domain.

Intracellularly recorded voltage responses of the visual cells of the blowfly (Calliphora erythrocephala) were analysed in the time and frequency domains. The photoreceptors were stimulated with pulse (impulse), sine, sine-sweep and pseudorandomly (white noise) modulated green light. The blowfly photoreceptor responses, as analysed from the linear transfer functions, seem to arise from a system similar to that of cascaded low-pass filters, with a corner frequency at about 63 Hz (SD +/- 12 Hz). The system is likely to have at least five poles, including one linear second order term, and a pure delay element. Arising from the non-linearities a second harmonic can be seen in the power spectra of responses elicited by sine modulated light. This non-linearity is at least partly explained by the self-shunting property of the membrane voltage response. Light adaptation increases the non-linearities in frequencies lower than 20 Hz, as seen in the decrease of the coherence function with the signal-to-noise ratio remaining constant. Light adaptation also accelerates the transduction process and it appears in the linear transfer function in a form typical to negative feedback. With low stimulus frequencies it causes a 'phase lead'-type non-linearity. In addition, the sine-sweep responses show quite different frequency characteristics in respect of depolarization and repolarization. Lateral inhibition between photoreceptor responses recorded from retinular cell axons in the lamina appears as a drop in gain and as an increasing phase-lag in frequencies from 30 Hz upwards in linear transfer functions. The source of this capacitive-like coupling can be considered to be in the high resistance barriers compartmentalizing the second optic ganglion into discrete anatomical units.

Time constant of isovolumic pressure fall in the intact canine left ventricle.

The two standard methods of computation of the time constant of the isovolumic single exponential decay of the left ventricular pressure were compared in anaesthetised, artificially ventilated, closed chest dogs in 87 experiments. In 23 additional experiments, carried out under basal steady state conditions, the time constant attained a mean (SD) value of 32(5) ms when computed according to the method with zero pressure asymptote assumption (Tz) and 39(7) ms when estimated according to a fit with variable pressure asymptote (Tv). Phenylephrine infusion significantly prolonged Tz from 31(6) to 46(12) and Tv from 39(7) to 92(52) ms. The increase in Tv was significantly greater than that in Tz. Propranolol increased Tv by 50% but Tz remained unaltered. Isoproterenol significantly decreased Tz from 33(2) to 18(3) and Tv from 38(3) to 29(3) ms. The decrease in Tz was significantly greater than that in Tv. Calcium chloride and atrial pacing decreased time constants, but volume loading by dextran infusion did not affect them. With regard to the whole material, the linear regression analysis yielded 0.776 as the coefficient of correlation between Tz and Tv. The two methods of time constant calculation appeared to diverge significantly always when changes in afterload or sympathetic activity were involved. These results indicate the dependence of relaxation on afterload but the independence on preload or ejection timing. In the light of these results the zero pressure asymptote method is recommended for the time constant computation as the practical choice when relaxation of the intact left ventricle is assessed.

The automatic computation of pressure-derived maximal shortening velocity (Vmax) of the unloaded contractile element in the intact canine heart left ventricle.

Estimation of the maximum velocity (Vmax) of the contractile element of the intact left ventricular wall muscle demands extrapolation of the force-velocity curve to zero load. The present paper describes our on-line computation system for measuring and analysing pressure derived Vmax using both linear (Vmax-lin) and exponential (Vmax-exp) extrapolation methods. The developed pressure during isovolumetric phase of systole was used as an equivalent of the force. Testing on anaesthetized artificially ventilated dogs showed the exponential function to fit pressure-velocity data better than the straight line did. The Vmax-exp attained 15-35% greater values than Vmax-lin, but both responded almost equally when considered on the basis of linear regression analysis (r = 0.991, n = 725). Changes of contractility caused by i.v. infusion of isoproterenol, calcium chloride or propranolol were practically similar when assessed by either method of Vmax computation, or by dP/dtmax. Volume loading by dextran infusion increased not only dP/dtmax, by 33 +/- 13%, but also Vmax, up to 24 +/-. When arterial pressure was raised by phenylephrine infusion, or heart rate by atrial pacing, dP/dtmax increased significantly while Vmax remained unaltered. Hence, the linear and exponential dP/dtmax increased significantly while Vmax remained unaltered. Hence, the linear and exponential extrapolation procedures provided comparable values for Vmax, but the linear one due to its simplicity is more suited for on-line computation. The Vmax thus obtained is, however, not independent of the changes in preload.

Electromechanical delay in the intact dog heart.

A computer method was developed for the determination of electromechanical delay defined as the time between the onset of Q-wave and the onset of the left ventricular systolic pressure rise. It was validated for heart catheterization studies on 56 intact anaesthetized beagle dogs in 86 sessions. The mean basal value of the electromechanical delay was 22 +/- 4 msec. Heart rate, contractility, preload and afterload were changed by atrial pacing and by infusions of calcium chloride, isoproterenol, propranolol, dextran and phenylephrine. Increase of heart rate by pacing from the spontaneous rate of 90 per min to 240 per min prolonged the electromechanical delay from 21 +/- 5 to 33 +/- 14 msec (P less than 0.001). Otherwise the duration of electromechanical delay changed independently of the heart rate. If it changed, the direction of the change followed that of the pre-ejection period. Its proportion of the pre-ejection period varied from 26 to 52%. The electromechanical delay shortened when a positive inotropic effect was noticed or the presystolic fibre length increased.

Stewart-Hamilton and gamma variate methods in computer analysis of dye curves.

The conventional Stewart-Hamilton (SH) principle was compared to the gamma variate function fitting (G) method in on-line computer determination of cardiac output from dye dilution curves on anaesthetized animals (dogs, cats and reindeer), and on an artificial circulation model with known flows. In animal experiments, indicator was injected into the pulmonary artery (n = 674), the coefficient of linear correlation (Rlin) between G and SH was 0.982, and the regression line of G was 1.101 x SH-0.072 l/min. When the right atrium was the site of indicator injection (n = 88), Rlin was 0.982, and the regression line nearly agreed with the line of unity. In the artificial circulation model with the single circulation arrangement (n = 81), Rlin between SH and known flow (Q) was 0.959, and between G and Q it was 0.951; both methods over-estimated the flows, with the over-estimation being greater in the case of G. In the artificial model with recirculation effect (n = 31), Rlin between SH and Q was 0.953, and between G and Q 0.942, G again over-estimating the real flow essentially more than SH. Thus, in most cases the Stewart-Hamilton principle seems to be the more reliable way of analysis of dye dilution curves also in modern computer practice.

Spectral sensitivity in insect photoreceptors at a range of temperatures.

The function of the visual receptors of blowflies (Calliphora erythrocephala) was studied using intracellular recording techniques where by the eye was kept at various temperatures below room temperature. Cooling was found to reduce the response amplitude, decrease the time course, and increase the latency. The Q10 as calculated from different recordings and from various temperature steps was found to be dependent both on the temperature range and the stimulation intensity, although more on the latter. From different kinds of spectral sensitivities of the receptor cells those with a double peak (UV and green) were studied with regard to the effect of the temperature change. We found a difference in the relative sensitivity change of the two peaks if the temperature was lowered. The sensitivity in UV remained enhanced in relation to green after cooling. This finding can be considered as supporting the sensitizing pigment theory, according to which the double peaked spectral sensitivity is explained by assuming a photostable pigment transferring the energy to the photopigment. This support is valid if the UV sensitivity at room temperature is assumed to be higher, and is limited by the reduced energy transfer to the photopigment caused by the increased mobility of the sensitizing pigment.