Abstract: The Drosophila phototransduction cascade transforms light into depolarizations that are further shaped by activation of voltage-dependent K+ (Kv) channels. In whole-cell recordings of isolated photoreceptors, we show that light selectively modulated the delayed rectifier (Shab) current. Shab currents were increased by light with similar kinetics to the light-induced current itself (latency approximately 20 ms), recovering to control values with a t(1/2) of approximately 60 s in darkness. Genetic disruption of PLCbeta4, responsible for light-induced PIP(2) hydrolysis, abolished this light-dependent modulation. In mutants of CDP-diaclyglycerol synthase (cds(1)), required for PIP(2) resynthesis, the modulation became irreversible, but exogenously applied PIP(2) restored reversibility. The modulation was accurately and reversibly mimicked by application of PIP(2) to heterologously expressed Shab channels in excised inside-out patches. The results indicate a functionally implemented mechanism of Kv channel modulation by PIP(2) in photoreceptors, which enables light-dependent regulation of signal processing by direct coupling to the phototransduction cascade.

Abstract: Response variability is a fundamental issue in neural coding because it limits all information processing. The reliability of neuronal coding is quantified by various approaches in different studies. In most cases it is largely unclear to what extent the conclusions depend on the applied reliability measure, making a comparison across studies almost impossible. We demonstrate that different reliability measures can lead to very different conclusions even if applied to the same set of data: in particular, we applied information theoretical measures (Shannon information capacity and Kullback-Leibler divergence) as well as a discrimination measure derived from signal-detection theory to the responses of blowfly photoreceptors which represent a well established model system for sensory information processing. We stimulated the photoreceptors with white noise modulated light intensity fluctuations of different contrasts. Surprisingly, the signal-detection approach leads to a safe discrimination of the photoreceptor response even when the response signal-to-noise ratio (SNR) is well below unity whereas Shannon information capacity and also Kullback-Leibler divergence indicate a very low performance. Applying different measures, can, therefore, lead to very different interpretations concerning the system's coding performance. As a consequence of the lower sensitivity compared to the signal-detection approach, the information theoretical measures overestimate internal noise sources and underestimate the importance of photon shot noise. We stress that none of the used measures and, most likely no other measure alone, allows for an unbiased estimation of a neuron's coding properties. Therefore the applied measure needs to be selected with respect to the scientific question and the analyzed neuron's functional context.

Abstract: Determining the contribution of a single type of ion channel to information processing within a neuron requires not only knowledge of the properties of the channel but also understanding of its function within a complex system. We studied the contribution of slow delayed rectifier K+ channels to neural coding in Drosophila photoreceptors by combining genetic and electrophysiological approaches with biophysical modeling. We show that the Shab gene encodes the slow delayed rectifier K+ channel and identify a novel voltage-gated K+ conductance. Analysis of the in vivo recorded voltage responses together with their computer-simulated counterparts demonstrates that Shab channels in Drosophila photoreceptors attenuate the light-induced depolarization and prevent response saturation in bright light. We also show that reduction of the Shab conductance in mutant photoreceptors is accompanied by a proportional drop in their input resistance. This reduction in input resistance partially restores the signaling range, sensitivity, and dynamic coding of light intensities of Shab photoreceptors to those of the wild-type counterparts. However, loss of the Shab channels may affect both the energy efficiency of coding and the processing of natural stimuli. Our results highlight the role of different types of voltage-gated K+ channels in the performance of the photoreceptors and provide insight into functional robustness against the perturbation of specific ion channel composition.

Abstract: The compound eyes of insects contain photoreceptors in small eyelets, ommatidia. The photoreceptors generally vary very little from ommatidium to ommatidium. However, in the large compound eyes of the cockroach (Periplaneta americana), previous studies have shown large differences in the optical structure between the ommatidia. The anatomy suggests pooling of 6-20 photoreceptor signals into one second-order cell in the first synapse. Here, we show and characterize an unexpectedly large and seemingly random functional variability in the cockroach photoreceptors in terms of sensitivity, adaptation speed, angular sensitivity, and signal-to-noise ratio. We also investigate the implications of action potentials, triggered by the light-induced membrane depolarization in the photoreceptor axons. The combination of the functional features reported here is unique among the compound eyes. Recordings from the proximal parts of the thin and long photoreceptor axons or small and distant second-order neurons are not practical with the present methods. To alleviate this lack of data, we used computer simulations mimicking the functional variability, spike coding, and pooling of 12 photoreceptor signals, on the basis of our recordings from the photoreceptor somata and distal axons. The predicted responses of a simulated second-order cell follow surprisingly reliably the simulated light stimuli when compared with a simulation of functionally identical photoreceptors. We hypothesize that cockroach photoreceptors use action potential coding and a kind of population coding scheme for making sense of the inherently unreliable light signals at low luminance and for optimization of vision in its mainly dim living conditions.

Abstract: Calcineurin, a Ca(2+)-calmodulin-dependent protein phosphatase (PP2B) is one of the links between Ca(2+) signals and regulation of gene transcription in cardiac muscle. We studied the Ca(2+) signal specificity of calcineurin activation experimentally and with modelling. In the rat atrial preparation, an increase in pacing frequency increased nuclear activity of the calcineurin-sensitive transcription factor, nuclear factor of activated T-cells (NFAT), 2-fold in a cyclosporin A (CsA)-sensitive manner. In line with this, modelling results predicted that the frequency of cardiac Ca(2+) transients encodes the stimulus for calcineurin activation. We further observed experimentally that calcineurin inhibition by CsA modulated Ca(2+) release in a Ca(2+)-dependent manner. CsA had no effect on [Ca(2+)](i) at a pacing frequency of 1 Hz but it significantly suppressed the amplitude of Ca(2+) transients, systolic [Ca(2+)](i) and time averaged [Ca(2+)](i) at 6 Hz. Calcineurin had a differential role in the expression of immediate-early genes B-type natriuretic peptide (BNP) and c-fos. CsA inhibited the pacing-induced BNP gene expression, whereas pacing alone had no effect on the expression of c-fos. However, in the presence of CsA, c-fos mRNA levels were significantly augmented by increased pacing frequency. These results show that frequency-dependent calcineurin activation has a specific role in [Ca(2+)](i) regulation and gene expression, constantly recruited by varying cardiac Ca(2+) signals.

Abstract: In order to clarify the role of inactivating and noninactivating K+ conductances in nonspiking neurons, we developed an isopotential model of the Drosophila photoreceptor membrane based on Hodgkin-Huxley-type equations. The model includes voltage dependent potassium conductances, the shaker (gKA) and the delayed rectifier (gKs). The model parameters were derived from published results by Hardie and coworkers and nearly identical model was used also in our previous work (J. E. Niven, M. Vähäsöyrinki, M. Kauranen, R. C. Hardie, M. Juusola, and M. Weckström. The Contribution of shaker K+ channels to the information capacity of Drosophila photoreceptors. Nature. 421:630-634, 2003). The model explains how the two types of channels function together to define the voltage dependent properties of the photoreceptor membrane. Additionally the model enables us to run simulations of conditions which are difficult to achieve in patch clamp, like prolonged membrane depolarizations by light adaptation. Effects of the activation of the delayed rectifier type conductance were found to be in accordance with published experimental work but the inactivation of the shaker channels, in addition to its importance in the determination of the resting potential, produced voltage amplification over equivalent passive membrane under dark adapted conditions. This phenomenon was not present in light adapted conditions. The modulation of the voltage dependence of the conductances as reported by serotonin (5-HT) caused the shaker to act essentially like the delayed rectifier conductance.

Abstract: An array of rapidly inactivating voltage-gated K+ channels is distributed throughout the nervous systems of vertebrates and invertebrates. Although these channels are thought to regulate the excitability of neurons by attenuating voltage signals, their specific functions are often poorly understood. We studied the role of the prototypical inactivating K+ conductance, Shaker, in Drosophila photoreceptors by recording intracellularly from wild-type and Shaker mutant photoreceptors. Here we show that loss of the Shaker K+ conductance produces a marked reduction in the signal-to-noise ratio of photoreceptors, generating a 50% decrease in the information capacity of these cells in fully light-adapted conditions. By combining experiments with modelling, we show that the inactivation of Shaker K+ channels amplifies voltage signals and enables photoreceptors to use their voltage range more effectively. Loss of the Shaker conductance attenuated the voltage signal and induced a compensatory decrease in impedance. Our results demonstrate the importance of the Shaker K+ conductance for neural coding precision and as a mechanism for selectively amplifying graded signals in neurons, and highlight the effect of compensatory mechanisms on neuronal information processing.

Abstract: Activation of the contractile machinery in skeletal muscle is initiated by the action-potential-induced release of Ca2+ from the sarcoplasmic reticulum (SR). Several proteins involved in SR Ca2+ release are affected by calmodulin kinase II (CaMKII)-induced phosphorylation in vitro, but the effect in the intact cell remains uncertain and is the focus of the present study. CaMKII inhibitory peptide or inactive control peptide was injected into single isolated fast-twitch fibres of mouse flexor digitorum brevis muscles, and the effect on free myoplasmic [Ca2+] ([Ca2+]i) and force during different patterns of stimulation was measured. Injection of the inactive control peptide had no effect on any of the parameters measured. Conversely, injection of CaMKII inhibitory peptide decreased tetanic [Ca2+]i by ~25 %, but had no significant effect on the rate of SR Ca2+ uptake or the force-[Ca2+]i relationship. Repeated tetanic stimulation resulted in increased tetanic [Ca2+]i, and this increase was smaller after CaMKII inhibition. In conclusion, CaMKII-induced phosphorylation facilitates SR Ca2+ release in the basal state and during repeated contractions, providing a positive feedback between [Ca2+]i and SR Ca2+ release.

Abstract: Effects of chelerythrine and bisindolylmaleimide I on action potential duration and on voltage-activated K(+) and Ca(2+) currents in rat ventricular myocytes were studied using perforated patch-clamp technique. The action potentials were markedly prolonged after application of 20 microM chelerythrine or 100 nM bisindolylmaleimide I. Chelerythrine and bisindolylmaleimide I reduced the amplitude of sustained current (I(K,sus)) significantly. Transient K(+) current (I(to)) was inhibited only by chelerythrine. Ca(2+) current was reduced only with highest chelerythrine concentration (50 microM). Application of chelerythrine and bisindolylmaleimide I inhibited outward K(+) currents significantly also in ruptured patch-clamp configuration. Bisindolylmaleimide V, an inactive analogue of bisindolylmaleimide I, decreased I(K,sus) substantially. However, I(to) and I(K,sus) were not affected by calphostin C. Direct protein kinase C activators resulted in decrease of outward K(+) currents. Chelerythrine blocked I(to) in a use-dependent manner and the block did not recover during a 4-min washout. I(K,sus) was not blocked by this mechanism by either inhibitor. We conclude that chelerythrine and bisindolylmaleimide I inhibit outward K(+) currents independently of protein kinase C inhibition.

Abstract: Selective estrogen receptor modulators (SERMs), tamoxifen (Tam) and toremifene (Tor), are widely used in the treatment of breast cancer. In addition, they have been demonstrated to prevent estrogen deficiency-induced bone loss in postmenopausal women. These effects are thought to be caused by the interaction of the SERMs with the estrogen receptor, although SERMs have also been shown to conduct non-receptor-mediated effects such as rapid changes in membrane functions. We compared the effects of Tam, Tor, and 17beta-estradiol (E2) on the viability of rat osteoclasts and osteoblasts. Both Tam and Tor were found to cause osteoclast apoptosis in in vitro cultures, which was reversed by E2. In addition, at higher concentration (10 microM), both SERMs had an estrogen receptor-independent effect, which involved interaction with the plasma membrane as demonstrated with UMR-108 osteosarcoma cells by Tam and Tor, but not E2. A leak of protons leading to changes in intracellular pH was shown both in medullary bone derived membrane vesicles and in intact cells. These effects were followed by a rapid loss of cell viability and subsequent cell lysis. Our results show that both Tam and Tor have an ionophoric effect on the plasma membranes of bone cells and that these SERMs differed in this ability: Tor induced rapid membrane depolarization only in the presence of high concentration of potassium. These non-receptor-mediated effects may be involved in therapeutic responses and explain some clinical side effects associated with the treatment of patients with these SERMs.

Abstract: Ca(2+) influx through the L-type Ca(2+) channels is the primary pathway for triggering the Ca(2+) release from the sarcoplasmic reticulum (SR). However, several observations have shown that Ca(2+) influx via the reverse mode of the Na(+)-Ca(2+) exchanger current (I(Na-Ca)) could also trigger the Ca(2+) release. The aim of the present study was to quantitate the role of this alternative pathway of Ca(2+) influx using a mathematical model. In our model 20% of the fast sodium channels and the Na(+)-Ca(2+) exchanger molecules are located in the restricted subspace between the sarcolemma and the SR where triggering of the calcium-induced calcium release (CICR) takes place. After determining the strengths of the alternative triggers with simulated voltage-clamps in varied membrane voltages and resting [Na](i) values, we studied the CICR in simulated action potentials, where fast sodium channel current contributes [Na](i) of the subspace. In low initial [Na](i) the Ca(2+) influx via the L-type Ca(2+) channels is the major trigger for Ca(2+) release from the SR, and the Ca(2+) influx via the reverse mode of the Na(+)-Ca(2+) exchanger cannot trigger the CICR. However, depending on the initial [Na](i), the contribution of the Ca(2+) entry via the exchanger may account for 25% (at [Na](i) = 10 mM) to nearly 100% ([Na](i) = 30 mM) of the trigger Ca(2+). The shift of the main trigger from L-type calcium channels to the exchanger reduced the delay between the action potential upstroke and the intracellular calcium transient. This may contribute to the function of the myocyte in physiological situations where [Na](i) is elevated. These main results remain the same when using different estimates for the most crucial parameters in the modeling or different models for the exchanger.

Abstract: We simulated mechanisms that increase Ca2+ transients with two models: the Luo-Rudy II model for guinea pig (GP) ventricle (GP model) representing long action potential (AP) myocytes and the rat atrial (RA) model exemplifying myocytes with short APs. The interventions were activation of stretch-gated cationic channels, increase of intracellular Na+ concentration ([Na+]i), simulated bet-adrenoceptor stimulation, and Ca2+ accumulation into the sarcoplasmic reticulum (SR). In the RA model, interventions caused an increase of AP duration. In the GP model, AP duration decreased except in the simulated beta-stimulation where it lengthened APs as in the RA model. We conclude that the changes in the APs are significantly contributed by the increase of the Ca2+ transient itself. The AP duration is controlled differently in cardiac myocytes with short and long AP durations. With short APs, an increase of the Ca2+ transient promotes an inward current via Na+/Ca2+-exchanger lengthening the AP. This effect is similar regardless of the mechanism causing the increase of the Ca2+ transient. With long APs the Ca2+ transient increase decreases the AP duration via inactivation of the L-type Ca2+ current. However, L-type current increase (as with beta-stimulation) increases the AP duration despite the simultaneous Ca2+ transient augmentation. The results explain the dispersion of AP changes in myocytes with short and long APs during interventions increasing the Ca2+ transients.

Abstract: In heart muscle a mechanical stimulus is sensed and transformed into adaptive changes in cardiac function by a process called mechanotransduction. Adaptation of heart muscle to mechanical load consists of neurohumoral activation and growth, both of which decrease the initial load. Under prolonged overload this process becomes maladaptive, leading to the development of left ventricular hypertrophy and ultimately to heart failure. Widespread synergism and crosstalk among a variety of molecules and signals involved in hypertrophic signaling pathways make the prevention or treatment of left ventricular hypertrophy and heart failure a challenging task. Therapeutic strategies should include either a complete and continuous reduction of load or normalization of left ventricular mass by interventions aimed at specific targets involved in mechanotransduction.

Abstract: Sexual communication between male and female fireflies involves the visual detection of species-specific bioluminescent signals. Firefly species vary spectrally in both their emitted light and in the sensitivity of the eye, depending on the time when each is active. Tuning of spectral sensitivity in three firefly species that occupy different photic niches was investigated using light and electron microscopy, microspectrophotometry, and intracellular recording to characterize the location and spectral absorption of the screening pigments that filter incoming light, the visual pigments that receive this filtered light, and the visual spectral sensitivity. Twilight-active species had similar pink screening pigments, but the visual pigment of Photinus pyralis peaked near 545 nm, while that of P. scintillans had a lambdamax near 557 nm. The night-active Photuris versicolo, had a yellow screening pigment that was uniquely localized, while its visual pigment was similar to that of P. pyralis. These results show that both screening and visual pigments vary among species. Modeling of spectral tuning indicates that the combination of screening and visual pigments found in the retina of each species provides the best possible match of sensitivity to bioluminescent emission. This combination also produced model sensitivity spectra that closely resemble sensitivities measured either with electroretinographic or intracellular techniques. Vision in both species of Photinus appears to be evolutionarily tuned for maximum discrimination of conspecific signals from spectrally broader backgrounds. Ph. versicolor, on the other hand, appears to have a visual system that offers a compromise between maximum sensitivity to, and maximum discrimination of, their signals.

Abstract: The stretch-induced changes in contraction force, cAMP and cGMP in isolated rat left atrium were studied. Increasing the diastolic intra-atrial pressure from 1 cmH2O to 8 cmH2O caused an immediate (<500 ms) increase in contraction force, the magnitude of which was 2.24+/-0.29 (n=6) times the force elicited by 1 cmH2O. This was followed by a slower, gradual increase of the force, which was maximal 8 min after the stretch (4.33+/-0.31, n=6). These phenomena were not accompanied by changes in the cAMP (n=24) or cGMP (n=24) concentrations within the tissue at any duration of stretch tested (2, 8, 20 and 36 min, n=6 at each time point). Furthermore, it was estimated that if the beta-adrenergic receptor agonist isoprenaline (100 nM) was used to produce an increase of the contraction force of the same magnitude as that induced by stretch, the cAMP concentration was greater (4.20+/-0.29 pmol/mg, n=5, P<0.001) when compared to that produced after 20 min of stretch (2.69+/-0.12 pmol/mg, n=6). Even without significantly changing the cGMP concentration, isoprenaline significantly increased the [cAMP]/[cGMP] ratio (3.4+/-0.36, n=5, P<0.01) compared to stretch (1.95+/-0.14, n=6). This result shows that in the rat atrium stretch does not regulate the production or breakdown of cyclic nucleotides (cAMP or cGMP). Thus it seems very unlikely that the effects of stretch on rat atrium function are caused by cAMP or cGMP.

Abstract: In the first visual synapse of the insect compound eye, both the presynaptic and postsynaptic signals are graded, nonspiking changes in membrane voltage. The synapse exhibits tonic transmitter release (even in dark) and strong adaptation to long-lasting light backgrounds, leading to changes also in the dynamics of signal transmission. We have studied these adaptational properties of the first visual synapse of the blowfly Calliphora vicina. Investigations were done in situ by intracellular recordings from the presynaptic photoreceptors, photoreceptor axon terminals, and the postsynaptic first order visual interneurons (LMCs). The dark recovery, the shifts in intensity dependence, and the underlying processes were studied by stimulating the visual system with various adapting stimuli while observing the recovery (i.e., dark adaptation). The findings show a transient potentiation in the postsynaptic responses after intense light adaptation, and the underlying mechanisms seem to be the changes in the equilibrium potential of the transmitter-gated conductance (chloride) of the postsynaptic neurons. The potentiation by itself serves as a mechanism that after light adaptation rapidly recovers the sensitivity loss of the visual system. However, this kind of mechanism, being an intrinsic property of graded potential transmission, may be quite widespread among graded synapses, and the phenomenon demonstrates that functional plasticity is also a property of graded synaptic transmission.

Abstract: In this study our aims were to investigate the presence and source of catecholamines in pericardial fluid of normotensive, reserpine-treated and spontaneously hypertensive rats. We found that noradrenaline is the only detectable catecholamine present in rat pericardial fluid. The effect of reserpine 6, 12, and 214 h after pre-treatment with 5 mg kg(-1) (8.2 micromol kg(-1)) i.p. shows that the concentration of noradrenaline in pericardial fluid reflects the amount of noradrenaline released within the heart rather than the amount of noradrenaline in plasma. Using spontaneously hypertensive rats (SHR) as a model for primary hypertension we could show that the level of pericardial noradrenaline is approximately threefold in the pericardial fluid of the SHRs when compared to respective values of age-matched normotensive Wistar-Kyoto rats (WKY), suggesting that there was an increased noradrenaline overflow in the hearts of the SHRs. In conclusion, determination of the noradrenaline concentration in the pericardial fluid might provide a new method for estimating the release of noradrenaline in the heart.

Abstract: By inducing a small reduction of the intracellular pH (0.18 units) with 20 mmol L-1 propionate we demonstrated that acidification changed the responses of isolated rat atria to stretch. Stretch (increase of the intra-atrial pressure) in normal pH increased the Ca2+ transients' amplitude (Indo-1 fluorescence) from 0.26 +/- 0.09 in 1 mmHg to 0.36 +/- 0.13 in 4 mmHg (P < 0.05, n=6), without affecting the diastolic [Ca2+]i level (n.s. n=6). The changes in Ca2+ balance during stretch were accompanied by a biphasic increase in the contraction force. Five minutes of continuous stretch increased the action potential duration (APD90%, P < 0.01, n=13) and decreased the APD15% (P < 0.001, n=13). During acidosis, the stretch-induced increase of the Ca2+ transient amplitude (0.4 +/- 0. 13 vs. 0.3 +/- 0.08, P < 0.05, n=6) was accompanied by the increase of the diastolic [Ca2+]i (1.16 +/- 0.07, P < 0.05, n=6) compared with non-acidotic control (1.06 +/- 0.06, n=6). Acidic intracellular pH also inhibited the stretch-induced changes in the action potentials (n=10) and slowed down the development of the contractile force during stretch. The results showed that acidosis modulates the mechanotransduction. It does this by interfering with the intracellular Ca2+ balance, inhibiting the Ca2+ extrusion mechanisms and reducing the Ca2+-buffering power of the cells. The physiological and pathological processes associated with stretch are therefore modulated by intracellular pH owing to its concerted effects on intracellular Ca2+ handling caused by a competitive inhibition of various Ca2+-binding molecules.

Abstract: We have investigated the role of sarcoplasmic reticulum (SR) in the modulation on rat action potentials by stretch. The action potentials were recorded intracellularly from rat atrial myocytes in an isolated atrial preparation with small, physiological stretch produced by pressure (1-3 mmHg) inside the atria. The SR function was inhibited by pharmacological interventions, either with ryanodine (100 nmol L-1), thapsigargin (10 nmol L-1) or caffeine (1 mmol L-1). The duration of action potentials was increased by stretch from 1 to 3 mmHg. The repolarization indices APD30% (P < 0.05), APD60% (P < 0.01), and APD90% (P < 0.01) were all increased significantly (n=10). Ryanodine, thapsigargin, and caffeine inhibited this prolongation, or even reversed the effect with repolarization indices APD30% (P < 0.05) and APD60% (P < 0.05) which decreased in stretch with thapsigargin treatment. As a conclusion, we suggest that the SR and the intracellular calcium balance play an important role in the modulation of the shape of the rat atrial action potential during stretch.

Abstract: We sought to investigate whether atrial myocyte contraction and secretion of the atrial natriuretic peptide (ANP) are affected in the same manner by intervention in intracellular Ca(2+) handling by acidosis. The effects of propionate (20 mM)-induced intracellular acidosis on the stretch-induced changes in ANP secretion, contraction force, and intracellular Ca(2+) concentration ([Ca(2+)](i)) were studied in the isolated rat atrium. The stretch of the atrium was produced by increasing the intra-atrial pressure of the paced and superfused preparation. Contraction force was estimated from pressure pulses generated by the contraction of the atrium. Intracellular Ca(2+) was measured from indo 1-AM-loaded atria, and ANP was measured by radioimmunoassay from the perfusate samples collected during interventions. Intracellular pH of the atrial myocytes was measured by a fluorescent indicator (BCECF)-based imaging system. Intracellular acidification caused by 20 mM propionic acid (0.18 pH units) potentiated the stretch-induced (intra-atrial pressure from 1 to 4 mmHg) ANP secretion, causing a twofold secretion compared with nonacidotic controls. Simultaneously, the responsiveness of the atrial contraction to stretch was reduced (P < 0.05, n = 7). Stretch augmented the systolic indo 1-AM transients in acidic (P < 0.05, n = 6) and nonacidic atria (P < 0.05, n = 6). However, during acidosis this was accompanied by an increase of the diastolic indo 1-AM ratio (P < 0.05, n = 6). Cooccurrence of stretch and acidosis caused an increase in systolic and diastolic [Ca(2+)](i) and potentiated the stretch-induced ANP secretion, whereas the contraction force and its stretch sensitivity were decreased. This mechanism may be involved in ischemia-induced ANP secretion, suggesting a role for ANP secretion as an indicator of contractile dysfunction.

Abstract: To study the effects of stretch on the function of rat left atrium, we recorded contraction force, calcium transients, and intracellular action potentials (APs) during stretch manipulations. The stretch of the atrium was controlled by intra-atrial pressure. The Frank-Starling behavior of the atrium was manifested as a biphasic increase of the contraction force after increasing the stretch level. The development of the contraction force after step increase of the stretch (intra-atrial pressure from 1 to 3 mm Hg) was accompanied by the increase in the amplitude of the calcium transients (P<0.05, n=4) and decrease in the time constant of the Ca2+ transient decay. The APs of the individual myocytes were also affected by stretch; the duration of the AP was decreased at positive voltages (AP duration at 15% repolarization level, P<0.001; n=13) and increased at negative voltages (AP duration at 90% repolarization level, P<0. 01; n=13). To study the mechanisms causing these changes we developed a mathematical model describing [Ca2+]i and electrical behavior of single rat atrial myocytes. Stretch was simulated in the model by increasing the troponin (TnC) sensitivity and/or applying a stretch-activated (SA) calcium influx. We mimicked the Ca2+ influx by introducing a nonselective cationic conductance, the SA channels, into the membrane. Neither of the 2 plausible mechanosensors (TnC or SA channels) alone could produce similar changes in the Ca2+ transients or APs as seen in the experiments. The model simulated the effects of stretch seen in experiments best when both the TnC affinity and the SA conductance activation were applied simultaneously. The SA channel activation led to gradual augmentation of Ca2+ transients, which modulated the APs through increased Na+/Ca2+-exchanger inward current. The role of TnC affinity change was to modulate the Ca2+ transients, stabilize the diastolic [Ca2+]i, and presumably to produce the immediate increase of the contraction force after stretch seen in experiments. Furthermore, we found that the same mechanism that caused the normal physiological responses to stretch could also generate arrhythmogenic afterpotentials at high stretch levels in the model.

Abstract: BACKGROUND: Adrenomedullin (ADM), a new vasorelaxing and natriuretic peptide, may function as an endogenous regulator of cardiac function, because ADM and its binding sites have been found in the heart. We characterize herein the cardiac effects of ADM as well as the underlying signaling pathways in vitro. METHODS AND RESULTS: In isolated perfused, paced rat heart preparation, infusion of ADM at concentrations of 0.1 to 1 nmol/L for 30 minutes induced a dose-dependent, gradual increase in developed tension, whereas proadrenomedullin N-20 (PAMP; 10 to 100 nmol/L), a peptide derived from the same gene as ADM, had no effect. The ADM-induced positive inotropic effect was not altered by a calcitonin gene-related peptide (CGRP) receptor antagonist, CGRP8-37, or H-89, a cAMP-dependent protein kinase inhibitor. ADM also failed to stimulate ventricular cAMP content of the perfused hearts. Ryanodine (3 nmol/L), a sarcoplasmic reticulum Ca2+ release channel opener, suppressed the overall ADM-induced positive inotropic effect. Pretreatment with thapsigargin (30 nmol/L), which inhibits sarcoplasmic reticulum Ca2+ ATPase and depletes intracellular Ca2+ stores, attenuated the early increase in developed tension produced by ADM. In addition, inhibition of protein kinase C by staurosporine (10 nmol/L) and blockade of L-type Ca2+ channels by diltiazem (1 micromol/L) significantly decreased the sustained phase of ADM-induced increase in developed tension. Superfusion of atrial myocytes with ADM (1 nmol/L) in isolated left atrial preparations resulted in a marked prolongation of action potential duration between 10 and -50 mV transmembrane voltage, consistent with an increase in L-type Ca2+ channel current during the plateau. CONCLUSIONS: Our results show that ADM enhances cardiac contractility via cAMP-independent mechanisms including Ca2+ release from intracellular ryanodine- and thapsigargin-sensitive Ca2+ stores, activation of protein kinase C, and Ca2+ influx through L-type Ca2+ channels.

Abstract: 1. Atrial arrhythmias, like atrial fibrillation and extrasystoles, are common in clinical situations when atrial pressure is increased. Although cardiac mechanoelectrical feedback has been under intensive study for many years, the mechanisms of stretch-induced arrhythmias are not known in detail. This is partly due to methodological difficulties in recording intracellular voltage during stretch stimulation. In this study we investigated the effects of gadolinium (Gd3+), a blocker of stretch-activated (SA) channels, on stretch-induced changes in rat atrial action potentials and contraction force. 2. By intracellular voltage recordings from rat isolated atria we studied the effects of Gd3+ (80 microM) on stretch-induced changes in action potentials. The stretch was induced by increasing pressure inside the atrium (1 mmHg to 7 mmHg). An elastic electrode holder that moved along the atrial tissue was used in the recordings. Thus the mechanical artifacts were eliminated and the cell-electrode contact was made more stable. To examine the influence of Gd3+ on atrial contraction we stretched the atria at different diastolic pressure levels (1 to 7 mmHg) with Gd3+ application of (80 microM) or diltiazem (5.0 microM). Contraction force was monitored by recording the pressure changes generated by the atrial contractions. 3. Our results show that: (1) atrial stretch induces delayed afterdepolarizations (DADs), increase in action potential amplitude and increase in relative conduction speed; (ii) Gd3+ blocks stretch-induced DADs and action potential changes; (iii) Gd3+ inhibits pressure-stimulated increase in the atrial contraction force, while similar inhibition is not observed with diltiazem, a blocker of L-type calcium channels. 4. This study suggests that Gd3+ inhibits stretch-induced changes in cell electrophysiology and contraction in the rat atrial cells and that the effects of gadolinium are due to rather specific block of stretch-activated ion channels with only a small effect on voltage-activated calcium channels.

Abstract: To investigate the role of Ca2+ in stretch-induced synthesis and release of atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP) isolated superfused rat atria were stretched by raising intra-atrial pressure. The immunoreactive (ir-) ANP and BNP concentrations were analysed by radioimmunoassay and the corresponding mRNA levels were quantified by Northern blot and dot blot analyses. Stretch-induced ir-ANP release and a rise in BNP mRNA levels increased at high (3.0 mM) compared to low (0.5 mM) extracellular Ca2+ concentration ([Ca2+]o). Moreover, the adaptation of stretch-induced ir-ANP release was dependent on [Ca2+]o. Atrial BNP mRNA levels were increased by stretch also in non-paced, electrically silent atria, where voltage-activated Ca2+ channels are not activated. The stretch-induced rise in BNP mRNA was blocked by gadolinium (80 microM), but not by the L-type channel blocker diltiazem (3.0 microM). This study indicates that both the stretch-secretion coupling of ir-ANP release and the pressure-stimulated synthesis of BNP mRNA are Ca2+-dependent processes. Gadolinium inhibits the stretch-stimulated rise in BNP mRNA levels in contracting and non-contracting atria, which is similar to its ability to block stretch-activated ir-ANP release, suggesting the involvement of Ca2+-permeable stretch-activated channels.

Abstract: Many neurons use graded membrane-potential changes, instead of action potentials, to transmit information. Traditional synaptic models feature discontinuous transmitter release by presynaptic action potentials, but this is not true for synapses between graded-potential neurons. In addition to graded and continuous transmitter release, they have multiple active zones, ribbon formations and L-type Ca2+ channels. These differences are probably linked to the high rate of vesicle fusion required for continuous transmitter release. Early stages of sensory systems provide some of the best characterized graded-potential neurons, and recent work on these systems suggests that modification of synaptic transmission by adaptation is a powerful feature of graded synapses.

Abstract: When BDM is added to the perfusion solutions used during the preparation of single, enzymatically dispersed mammalian cardiomyocytes, higher yields of calcium-tolerant cells are obtained. As the principal component of a storage solution, BDM also improves the survival of myocytes maintained in cold storage. These data support the hypothesis that BDM can act as a cardioprotective agent under certain conditions.

Abstract: To characterize the transfer of graded potentials and the properties of the associated noise in the photoreceptor-interneuron synapse of the blowfly (Calliphora vicina) compound eye, we recorded voltage responses of photoreceptors (R1-6) and large monopolar cells (LMC) evoked by: (a) steps of light presented in the dark; (b) contrast steps; and (c) pseudorandomly modulated contrast stimuli at backgrounds covering 6 log intensity units. Additionally, we made recordings from photoreceptor axon terminals. Increased light adaptation gradually changed the synaptic signal transfer from low-pass to band-pass filtering. This was accompanied by decreased synaptic delay and increased contrast gain, but the overall synaptic gain and the intrinsic noise (i.e., transmission noise) were reduced. Based on these results, we describe a descriptive synaptic model, in which the kinetics of the tonic transmitter (histamine) release from the photoreceptor axon terminals change with mean photoreceptor depolarization. During signal transmission, tonic transmitter release is augmented by voltage-dependent contrast-enhancing mechanisms in the photoreceptor axons that produce fast transients from the rising phases of the photoreceptor responses and add these enhanced voltages to the original photoreceptor responses. The model can predict the experimental findings and it agrees with the recently proposed theory of maximizing sensory information.

Abstract: That particular membrane conductances are selected for expression to enable the efficient coding of biologically relevant signals is illustrated by recent work on insect photoreceptors. These studies exploit the richness of insect vision and the accessibility of insect photoreceptors to cellular analysis in both intact animal and isolated cell preparations. The distribution of voltage-gated conductances among photoreceptors of different species correlates with visual ecology. Delayed-rectifier K+ channels are found in the rapidly responding photoreceptors of fast-flying flies. The conductance's activation range and dynamics match light-induced signals, and enable a rapid response by reducing the membrane time constant. Slow-moving flies have slowly responding photoreceptors that lack the delayed rectifier, but express an inactivating K+ conductance that is metabolically less demanding. Complementing these findings, locust photoreceptor membranes are modulated diurnally. The delayed rectifier is exhibited during the day and the inactivating K+ current is exhibited at night. Insect photoreceptors also demonstrate the amplification of signals by voltage-gated Na+ channels. In drone-bee photoreceptors, voltage-gated Na+ channels combine with K+ channels to enhance the small transient signals produced by the image of a queen bee passing over the retina. This subthreshold amplifier operates most effectively over the range of light intensities at which drones pursue queens.

Abstract: Light-adapted fly photoreceptor cells were stimulated with brief positive and negative contrast flashes (contrast = delta I/I, I = intensity). Membrane potential responses to a wide range of flash intensities were well-fitted by a static nonlinearity followed by a compartmental model represented by a gamma function. However, the agreement improved if one parameter of the gamma function, n, varied quadratically with input light intensity. Response amplitude and time to peak were estimated from the fitted parameters. Response amplitude varied approximately linearly with contrast but showed nonlinear compression with the largest negative flashes. Reducing the background light level by 3 decades or hyperpolarizing the cell electrically produced stronger nonlinear compression with both contrast polarities. This is probably due to fast voltage-activated K+ channels. Responses to double flashes with varying time separations were well-fitted by summed gamma functions, allowing separation of the individual flash responses. There was no detectable time-dependent interaction between paired positive flashes at all separations. However, the response to two negative flashes was greater than the linear prediction at short separations, and this facilitatory nonlinearity decayed with a time constant of about 1 msec. The facilitation is probably related to resonant behavior in light-adapted photoreceptors and may be due to an IP3-induced intracellular Ca2+ release.

Abstract: We determined concentrations and molecular sizes of natriuretic peptides in rat pericardial fluid and plasma by use of specific radioimmunoassays (RIA) and gel filtration HPLC. Our study shows that pericardial fluid forms a local extracellular storage of immunoreactive (ir) atrial natriuretic peptide (irANP) and brain natriuretic peptide (irBNP) near the heart where these peptides can be found in high concentrations in vivo. The concentrations of irANP, irBNP and NH2-terminal fragment of proANP (irNT-proANP) in pericardial fluid were 9.8 +/- 3.7, 0.49 +/- 0.47 and 28.9 +/- 11.8 nmol/l, respectively. IrBNP had the lowest (20 +/- 11) and irANP the highest (90 +/- 32) concentration ratio between pericardial fluid and plasma. The elution positions of irANP, irBNP and irNT-proANP in pericardial fluid and plasma were similar as examined by gel filtration HPLC. Furthermore, we show that the reduction of noradrenaline content of the heart muscle by reserpine reduces concentration of irANP in pericardial fluid by 39.6% and in plasma by 30.3% when compared to respective control group values. The concentration of irBNP is reduced by 44.1% in pericardial fluid but in plasma its reduction was not statistically significant. Vasoactive peptides released into the interstitial space and from there into pericardial fluid may have a more active role in the regulation of cardiac function than previously considered.

Abstract: 1. We studied the graded and spiking properties of the "non-spiking" first-order visual interneurons of the fly compound eye in situ with the use of intracellular recordings. Iontophoretical QX-314 injections, Lucifer yellow marking, and (discontinuous) current-clamp method together with transfer function analysis were used to characterize the neural signal processing mechanisms in these neurons. 2. A light-OFF spike was seen in one identified anatomic subtype (L3, n = 6) of the three first-order visual interneurons (L1, L2, and L3, or LMCs) when recorded from synaptic region (i.e., in the 1st visual ganglion, lamina ganglionaris) in dark-adapted conditions. Hyperpolarization of the membrane potential by current caused the identified L1 (n = 4), as well as L3 (n = 6), to produce an OFF spike, a number of action potentials, and some subthreshold depolarizations after the light-ON response. In L2 the OFF spike or action potentials could not be elicited. 3. To produce action potentials in L1 and L3, it was found to be necessary to hyperpolarize the cells approximately 35-45 mV (n = 43) below the resting potential (RP) in the synaptic zone. Recordings from the axons of these cells revealed that near the second neuropil (chiasma) the threshold of these spikes was near to (approximately 10 mV below, n = 16) or even at the RP when an ON spike was also produced (n = 4). 4. The recorded spikes were up to 54 mV in amplitude, appeared with a maximum frequency of up to 120 impulses/s, and had a duration of approximately 8 ms. In L1 and L3 the spikes were elicited either after a light pulse (L3) or after a negative current step that was superimposed on a hyperpolarizing steady-state current (L3 and L1). A positive current step (similarly superimposed on a hyperpolarizing steady-state current) also triggered the spikes during the step. 5. Iontophoretic injection of a potent intracellularly effective blocker of voltage-gated sodium channels, QX-314, irreversibly eradicated the spikes and subthreshold depolarizations (n = 5). In addition, further injections elongated the light-ON responses and decreased or even abolished the light-OFF response. 6. Negative prepulses followed by positive current steps were applied from the RP, to test the activation-inactivation properties of the channels responsible for the OFF spike.(ABSTRACT TRUNCATED AT 400 WORDS)

Abstract: 1. Randomly modulated light stimuli were used to characterize the nonlinear dynamic properties of the synapse between photoreceptors and large monopolar neurons (LMC) in the fly retina. Membrane potential fluctuations produced by constant variance contrast stimuli were recorded at eight different levels of background light intensity. 2. Representation of the photoreceptor-LMC input-output data in the form of traditional characteristic curves indicated that synaptic gain was reduced by light adaptation. However, this representation did not include the time-dependent properties of the synaptic function, which are known to be nonlinear. Therefore nonlinear systems analysis was used to characterize the synapse. 3. The responses of photoreceptors and LMCs to random light fluctuations were characterized by second-order Volterra series, with kernel estimation by the parallel cascade method. Photoreceptor responses were approximately linear, but LMC responses were clearly nonlinear. 4. Synaptic input-output relationships were measured by passing the light stimuli to LMCs through the measured photoreceptor characteristics to obtain an estimate of the synaptic input. The resulting nonlinear synaptic functions were well characterized by second-order Volterra series. They could not be modeled by a linear-nonlinear-linear cascade but were better approximated by a nonlinear-linear-nonlinear cascade. 5. These results support two possible structural models of the synapse, the first having two parallel paths for signal flow between the photoreceptor and LMC, and the second having two distinct nonlinear operations, occurring before and after chemical transmission. 6. The two models were cach used to calculate the synaptic gain to a brief change in photoreceptor membrane potential. Both models predicted that synaptic gain is reduced by light adaptation.

Abstract: Voltage-activated and spontaneous chloride-channel activity was studied in melanoma cell-line A2058 by patch-clamp technique. Whole-cell and inside-out patch recordings carried out with leak subtraction show voltage-activated chloride-conductance. In addition, a large leak-type conductance typical of epithelial cells was found in whole-cell experiments. This current was carried mostly by chloride-ions but also a leak-type potassium conductance was found showing KCl fluxes to be possible. Cell-attached and inside-out patch recordings showed at least two types of spontaneous chloride-channel activity. Bursting, flickering-type channels were found only in cell-attached recordings. That led to the conclusion that some intracellular factors are needed for that kind of activity. A second spontaneous, 30 pS chloride-channel with slow kinetics was found both in cell-attached and inside-out patch configuration. A voltage-activated chloride-channel found had a conductance of approximately 25 pS. In our experiments these channels did not need external calcium for activation. Voltage ramp recordings in cell-attached configuration gives the intracellular chloride concentration of 163 mM on the basis of chloride reversal potential (extracellular 146 mM in our experiments). Intracellular chloride concentration thus seems to be maintained slightly higher than the extracellular one.

Abstract: 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.

Abstract: The effects of salicylaldoxime, 2-(OH)C6H4CH = NOH, on the action potential duration, transient outward K+ current and slow inward Ca2+ current were studied in isolated rat ventricular myocytes. The application of salicylaldoxime (0.1-2.0 mM) reversibly increased the action potential duration and reduced in a dose-dependent manner both the transient outward K+ and the slow inward Ca2+ currents. The effect of salicylaldoxime on these two ionic currents was similar to that of 2,3-butanedione monoxime, but was about ten times more potent. Compounds which block both K+ and Ca2+ currents may represent a new type of Class III antiarrhythmic agent which counteracts arrhythmias initiated by re-entry with reduced proarrhythmic risk via triggered activity.

Abstract: 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.

Abstract: To elucidate the mechanism involved in the release of atrial natriuretic peptide (ANP), we studied the importance of ryanodine-sensitive Ca2+ release in stretch-secretion coupling. The experiments were made with a left atrial preparation, where the stretch of myocytes was induced by changing the intra-atrial pressure. When external pacing was not applied, the atrial preparation was not spontaneously contracting, and it was therefore possible to investigate the secretory mechanism in the quiescent atrium. The superfusate was collected in 2-min fractions and assayed for ANP immunoreactivity. Filtration analysis revealed that the major fraction in the superfusate in all experimental situations had a similar molecular weight as the ANP 1-28. Ryanodine (1.0 microM and 0.1 microM) inhibited stretch-stimulated ANP secretion dose dependently both in paced and non-paced atrium, but did not have any effect on basal secretion. The present results support the notion that intracellular Ca2+ transients from the intracellular stores are essential for stretch-stimulated ANP secretion, independently from excitation and contraction. Basal ANP secretion is not inhibited by blocking ryanodine-sensitive Ca2+ channels, either in contracting or in non-contracting atria. In addition our results confirm that the principal stimulus for ANP secretion in response to atrial distension is the stretch of myocytes. Length shortening of myocytes is not essential for ANP release.

Abstract: 1. We have used intracellular recordings and ionophoretic injections in vivo to investigate the ion exchange mechanisms responsible for the maintenance of the ion gradients in the large monopolar cells (LMCs) of the first optic ganglion of the blowfly, Calliphora vicinia. 2. Ionophoretic chloride injections caused a rapid approximately 20-mV depolarization of the resting potential (Erp) and abolished or even reversed the light-ON response (OR), which is caused by histamine-gated chloride conductance, as the chloride equilibrium potential (ECl) was increased beyond the Erp, i.e., 50 mV upward. Ionophoretic sodium injections were found to mimic the action of the ionophoretic chloride injections and thus also to cause chloride accumulation inside the cell. 3. Ionophoretic injections of bicarbonate only had the effect of hyperpolarizing the Erp by 5-15 mV for 1-25 s, but chloride gradient, i.e., ECl remained unchanged. Intracellular proton load caused depolarization of the Erp by 15 +/- 5 mV (mean +/- SE) for 20-25 s and a slight 15 +/- 5-mV decrease of the peak OR. Ionophoretic injections of potassium, acetate, and furosemide failed to cause any physiological effect. 4. The time constant for the recovery of the peak OR after sodium load increased linearly as a function of injected charge whereby the time constant for the recovery after chloride accumulation increased slowly up to 50 nC of injected charge, after which it increased rapidly, possibly indicating substrate inhibition. The time constant for the recovery of peak OR after sodium load was from 5 to 65 nC greater than that of chloride.(ABSTRACT TRUNCATED AT 250 WORDS)

Abstract: 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.

Abstract: 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.

Abstract: The rpa (receptor potential absent) mutation of the blowfly, Calliphora erythrocephala, reduces the light-evoked responses of photoreceptor cells and renders the fly blind. This phenotype is similar to the phenotype caused by norpA mutations in Drosophila which have been shown to occur within a gene encoding phospholipase C. In Western blots, norpA antiserum stains a protein in homogenates of wild-type Calliphora eye and head that is similar in molecular weight to the norpA protein. Very little staining of this protein is observed in similar homogenates of rpa mutant. Moreover, norpA antiserum strongly stains retina in immunohistochemical assays of wild-type adult head, but not in rpa mutant. Furthermore, eyes of rpa mutant have a reduced amount of phospholipase C activity compared to eye of wild-type Calliphora. These data suggest that the rpa mutation occurs in a phospholipase C gene of the blowfly that is homologous to the norpA gene of Drosophila.

Abstract: 1. Isolated superfused rat atrial preparations were used to study the mechanism of stretch-induced atrial natriuretic peptide (ANP) secretion. The stretch of the atrial myocytes was induced by raising the intra-atrial pressure. The secretion rates were analysed by measuring ANP concentrations from the superfusate fractions by radioimmunoassay. 2. The effect of gadolinium, a blocker of stretch-activated ion channels, on stretch-induced and basal ANP secretion was investigated by superfusing the atrial preparation with 5, 20 or 80 microM GdCl3. Gadolinium decreased stretch-induced ANP secretion in a dose-dependent manner, but did not affect basal secretion. 3. Because high concentrations of gadolinium may block voltage-gated calcium channels, we tested whether the selective blockers of L-type (diltiazem) and T-type (NiCl2) calcium channels affect the stretch-stimulated ANP release. Neither diltiazem at 3 microM nor NiCl2 at 50 microM affected stretch-induced ANP release in paced atrial preparation. 4. Gadolinium, but not diltiazem, also inhibited stretch-stimulated ANP secretion in non-paced, quiescent atria. 5. The findings that ANP release is inhibited by Gd3+, but not by diltiazem or NiCl2, and that the stretch-induced secretion in quiescent atria is also inhibited by Gd3+, suggest that stretch-activated ion channels are involved in the regulation of stretch-induced ANP release.

Abstract: 1. The voltage responses to light of dark-adapted cockroach photoreceptors were recorded from the somata in the retina and the axons below the two basement membranes. 2. One or more spike-like fast depolarizations superimposed on the graded receptor potential were recorded in photoreceptor axons identified by Lucifer yellow injections. These spikes are voltage dependent in as much as they could be elicited with depolarizing current pulses as well as with light stimuli. In photoreceptor somata only graded receptor potentials were recorded. 3. The physiological function of these axonal spikes may be to serve as an amplification mechanism that counteracts the unfavorable combination of photoreceptor geometry and electrical properties.

Abstract: The membrane properties of short type blowfly photoreceptors (R1-6) were investigated in dark and light adaptation with single electrode current and voltage clamp techniques. The impedance of the cells was defined in frequency domain by using discontinuous current clamp and white-noise-modulated current injection. We found that the slow activation and relaxation of the voltage-dependent K+ conductance transform the photoreceptor membrane effectively into a band-pass filter. This behaviour could be observed under current clamp as voltage-dependent outward and inward rectification of the membrane. The voltage-dependent band-pass filtering is likely to be present in all neurons with graded potentials and voltage-dependent membrane conductances.

Abstract: 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.

Abstract: 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.

Abstract: 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.

Abstract: 1. The membrane properties of the photoreceptors of the blowfly (Calliphora vicina) were investigated in situ by making intracellular recordings in the intact retina, using discontinuous single-electrode current and voltage clamp techniques. Single channels were investigated using inside-out patches from dissociated photoreceptors. 2. Photoreceptors have a resting potential in darkness of -60.4 +/- 6.6 mV (mean +/- S.D.; n = 43), a resting input resistance of 32 +/- 3 M omega (n = 11) and membrane time constant of 4.1 +/- 1 ms (n = 9). These values give a total cell capacitance of 0.13 nF and an effective membrane area of 1.3 x 10(-4) cm2. 3. Single-electrode voltage clamp reveals a voltage-sensitive outward current with an activation threshold at approximately -75 mV. This conductance has two kinetic components, the slower component activating at more depolarized levels. On the basis of its kinetics, a reversal potential of -85 +/- 6 mV (n = 6), sensitivity to intracellularly injected tetraethylammonium chloride (TEA), and its slow and partial inactivation (approximately 25%) this mechanism is classified as a delayed rectifier potassium conductance. 4. Voltage-sensitive potassium channels showing similar properties were found in excised inside-out patches from dissociated photoreceptors. Single-channel conductances are ca 20 pS for both fast and slow kinetic components, indicating a channel density in the intact cell of ca 2 microns -2. The reversal potential follows the Nernst slope for potassium ions. 5. The voltage dependence of the conductance was determined in patches containing channels of predominantly one or the other kinetic component. The midpoint of the activation curve is -65 mV for the fast and -50 mV for the slow component. Activation time constants (measured from a holding potential of -100 mV) are voltage dependent, and in the range 1-10 ms for the fast and 5-40 ms for the slow component. Both kinetic components are blocked by TEA (greater than 2.5 mM). The slow component is more sensitive to quinidine (greater than 200 microM), and the fast component to 4-aminopyridine (4-AP; greater than 200 microM). 6. In the intact preparation the outward current shows no dependence on light stimulation in the studied ranges of voltage (up to -25 mV) and intensity (up to 5.5 x 10(4) effective photons). Ensemble averages of channel openings in perfused inside-out patches show no dependence on calcium concentration in the range 10 nM-1.8 mM.(ABSTRACT TRUNCATED AT 400 WORDS)

Abstract: The properties of impulse responses of blowfly photoreceptors in light and dark adaptation were studied with brief (30 mu sec) 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.

Abstract: 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.

Abstract: A special shape-factor intensity (SFI) quantification method for analysing of macro- and microrecordings of brain activity during surgery for epilepsy is illustrated. Comparative analyses show that epileptic activity may be characterized as different from normal brain activity by both macro-and microtechniques. The possible advantages of brain slice technique in comparison with direct peroperative microrecording are discussed; absence of artifacts from respiratory and pulsatory movements of the brain is stressed. The slice technique cannot be advocated for routine use in epilepsy surgery as it requires advanced neurophysiological knowledge.

Abstract: Latency as a time difference between the stimulating light-pulse and the recorded potential response was studied with intracellular recording techniques on blowfly (Calliphora) visual receptors. Latency was shown to be a meaningful parameter in analysing the information content of the recorded receptor potential responses by comparing latency vs. amplitude functions using different intensities of stimulating light, stimulating angles of the incident light, ambient temperatures, and flies of different developmental stages. The maximum amplitude of the response is not sufficient to describe stimulus-response functions. With the help of the linear correlation analysis the most useful latency criterion among three widely used latency parameters (viz. the amplitude threshold (1 mV), the visual estimation (detectable voltage) and the maximum slope intersection with the base-line) was investigated. These were studied by correlating them with other commonly used response parameters. An arbitrarily chosen latency parameter that shows the lowest correlation coefficient is considered to contain more additional information from the photoreceptor voltage response than a latency parameter with high correlation. According to our analysis the most useful latency determination is proposed to be the time from the stimulus onset to the intersection of the maximum tangent at the base line, a criterion which also allows one to use concepts derived from systems analysis. The concept of latency is discussed and a definition proposed in the light of this and other recent findings.

Abstract: 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.