A bedside test to determine motion stereopsis using the Pulfrich phenomenon.

OBJECTIVE: Many diseases induce asymmetric delays in the visual pathway, resulting in a spontaneous Pulfrich phenomenon (PP). The PP is a visual stereoillusion that may cause difficulties in persons when traveling in cars, crossing the road, or playing ball games. The authors developed and tested a simple new bedside procedure to detect PP. DESIGN: A case series. PARTICIPANTS: Disease simulation in 2 normal subjects and 18 patients with optic neuritis (ON) was examined. Ninety normal subjects were studied to determine normal range of PP. INTERVENTION: The new test, called swinging pen test (SPT), is performed by oscillating a pen by hand. The SPT was compared to a gold standard, a mechanical pendulum (MP). MAIN OUTCOME MEASURES: The authors measured simulated PP in two normal subjects and PP in 18 patients with ON and 90 normal control subjects. The Pearson product-moment correlation (r) and the Spearman rank correlation (rs) between SPT and MP were calculated. RESULTS: The magnitudes of simulated PP determined with the SPT and the MP correlated well (r = 0.92, P < 0.005, and r = 0.96, P < 0.001). Correlation also was good in patients with ON (rs = 0.90, P < 0.05). The positive predictive value of the SPT was 100%, and the negative predictive value was 92%. The PP was absent in all control subjects testing with either pendulum. The normal range for PP varied from -1.40 to 1.52 msec. For the SPT, the intraobserver variability coefficient was 8.2%, and the interobserver variability coefficient was 10.5%. CONCLUSIONS: The authors believe that SPT will be of value to clinicians on bedside evaluation of motion stereopsis dysfunctions. The normal range of PP was approximately +/- -1.5 msec (approximately +/- -1.5 cm), corresponding to a 0.3-log unit neutral density filter).

Picrotoxin potentiates contraction while inhibiting Ca current but increasing birefringence signal in frog skeletal muscle fibers.

In single frog skeletal muscle fibers, picrotoxin (5 mM) potentiated the voltage-dependent component of contractions in response to 2-s depolarizing pulses while greatly inhibiting the simultaneously recorded tubular Ca current in a normal-Ca, Na- and CI-deficient solution, provided the contractions were generated at long time intervals (2 min). In normal Ringer's solution, picrotoxin reversibly increased the amplitude of the early large birefringence signal and the amplitude and duration of the simultaneously recorded twitch tension, suggesting that the drug may increase, directly or indirectly, the release of Ca from the SR.

Psychophysical determination of visual processing time by comparing depth seen in Pulfrich and Mach-Dvorak illusions.

A practical course for preclinical medical students was developed to illustrate aspects of binocular vision and mechanisms of primary visual transduction. It is based on a graphic analysis of two optical illusions, the Pulfrich and the Mach-Dvorak phenomena. A pendulum swinging in a plane perpendicular to the direction of observation appears to follow an elliptical path when viewed binocularly with a filter in front of one eye (Pulfrich illusion) or with alternating occlusion of the right and left eye above a critical frequency (Mach-Dvorak illusion). The Pulfrich phenomenon permits us to determine the relationship between perceived illusory depth and filter density. Analyzing the Mach-Dvorak phenomenon allows us to determine the dependence of illusory depth on interocular delay. Comparison of both determinations (depth against transmission and depth against time) permits us to establish, without complex calculations, the effect of luminescence on visual processing time. In addition, this course illustrates a general methodological concept mentioned by Popper: students make an unexpected observation, put forward a testable hypothesis, and try to falsify it.

Inhibition by 2,3-butanedione-monoxime of mitochondrial ADP-dependent respiration and muscle contraction.

2,3-Butanedione-monoxime (BDM) inhibits tension development in skeletal, cardiac and smooth muscle. Experiments on isolated single fibres of frog-muscle have shown a use dependent intermittent failure of twitching during continued stimulation. Our analysis of the effect of BDM on rabbit liver mitochondria and submitochondrial particles revealed a reduction of oxygen consumption in coupled mitochondria stimulated by adding of ADP, while in uncoupled mitochondria the oxygen consumption was not inhibited by BDM. Furthermore the substance did not inhibit ATP hydrolysis by submitochondrial particles. Based on these findings we suggest that BDM inhibits the activity of the mitochondrial ATP/ADP translocase.

Energetical considerations related to calcium release from the sarcoplasmic reticulum in skeletal muscle.

During the initial phase of activation (about 5 ms) of 1 g of muscle about 200 nmol of Ca++ are released from the SR which results in a current of about 7.7 A. Because at least 90% of Ca++ is released at a gradient below 1000/1 a free energy of about 3 mJ is available. Based on a SR-surface of approximately 2 m2/g of muscle a maximal inside negative membrane potential of -87 mV and a specific membrane capacitance of 1 microF/cm2 only about 9 nmol of Ca++ can be released without charge compensation before the calcium equilibrium potential is reached. This means that a countercurrent must compensate for practically all the calcium released in order to maintain a substantial driving force during release. The energy derived from the amount of Ca++ and its gradient or the caloric value of ATP required for Ca++-uptake (100 nmol) sets an upper limit for specific membrane resistance of the SR in the range of 200 omega cm2. This value is compatible with clearly lower numbers deduced from measurements on membranes obtained by fusing SR vesicles with lipid bilayers and the concept that the SR functions as a current source rather than as a voltage source. However, this value is much lower than estimates of specific membrane resistance based on indirect measurements.

Dynamic changes in plasma membrane properties of Semliki Forest virus infected cells related to cell fusion.

The mechanism of the processes leading to membrane fusion is as yet unknown. In this report we demonstrate that changes in membrane potential and potassium fluxes correlate with Semliki Forest virus induced cell-cell fusion at mildly acidic pH. The changes observed occur only at pH's below 6.2 corresponding to values required to trigger the fusion process. A possible role of these alterations of the plasma membrane related to membrane fusion phenomena is discussed.

Effects of repetitive activity, ruthenium red, and elevated extracellular calcium on frog skeletal muscle: implications for t-tubule conduction.

In this report we review evidence that indicates that experimental elevation of t-tubular calcium can lead to failure of action potential propagation within the t system and we present some new evidence suggesting that t-tubular calcium concentration may rise during repetitive activity. The evidence for t-tubular conduction failure consists of comparisons of the effects of high calcium and of ruthenium red on excitation and excitation-contraction coupling as well as morphological observations of wavy myofibrils in the axial core of fibers contracting tetanically in solutions containing elevated calcium concentrations. Evidence for elevation of t-tubular calcium concentration during repetitive activity comes from the following. During twitches, the early, large birefringence signal and force development are delayed in onset if the extracellular calcium and (or) potassium concentrations are above normal or if the fiber has been stimulated tetanically just prior to the test twitch. The delays that occur in twitches following tetanic contractions are attenuated when the extracellular and, therefore, the t-tubular calcium concentration is buffered with citrate.

Optical study of active ion transport in lipid vesicles containing reconstituted Na,K-ATPase.

A fluorescence method is described for the measurement of ATP-driven ion fluxes in lipid vesicles containing purified Na,K-ATPase. The membrane voltage of enzyme containing vesicles was measured by using a voltage-sensitive indocyanine dye. By addition of valinomycin the vesicle membrane is made selectively permeable to K+ so that the membrane voltage approaches the Nernst potential for K+. With constant external K+ concentration, the time course of internal K+ concentration can be continuously measured as change of the fluorescence signal after activation of the pump. The optical method has a higher time resolution than tracer-flux experiments and allows an accurate determination of initial flux rates. From the temperature dependence of active K+ transport its activation energy was determined to be 115 kJ/mol. ATP-stimulated electrogenic pumping can be measured as fast fluorescence change when the membrane conductance is low (i.e., at low or zero valinomycin concentration). In accordance with expectation, the amplitude of the fast signal change increases with decreasing passive ion permeability of the vesicle membrane. The resolution of the charge movement is so high that a few pump turnovers can be easily detected.

Birefringence signal and early mechanical changes at normal and increased tonicities in frog skeletal muscle.

Simultaneous measurements of the time course of early mechanical events and the early large birefringence signal were performed during activation of isolated frog skeletal muscle fibres bathed in iso- and hypertonic media. A piezo-electric transducer was used which had a resonance frequency of 3.3 kHz and a sensitivity of 740 mV/mN (0.5 mV noise peak to peak) and a compliance of 0.6 mu/mN. The delay from stimulus onset to the onset of the birefringence signal was estimated to be 0.78 +/- 0.03 ms in normal Ringer solution at room temperature (20-23 degrees C). The beginning of latency relaxation appeared subsequent to the birefringence signal after a delay of 0.36 +/- 0.03 ms. Increasing the tonicity retarded the time course of both birefringence signal and latency relaxation. In solutions of twofold greater tonicity an increase in tension (denoted pre-relaxation contraction) was observed to precede latency relaxation. Pre-relaxation contraction became more prominent with increasing tonicity and appeared to coincide with its onset with the onset of the birefringence signal. The pre-relaxation contraction was shown not to be a stimulus artifact and was not reduced in amplitude by D 600 or calcium-poor solutions. Lowering the temperature to 2.2 degrees C in normal Ringer solution delayed the onsets of the birefringence signal and latency relaxation, and increased the interval between the two signals but did not result in a tension increase before latency relaxation. The coincidence of the birefringence signal onset with the onset of pre-relaxation contraction suggests a common aetiology. The aetiology remains speculative but seems to require hypertonicity for expression of the mechanical signal.

Reconstitution of pure acetylcholine receptor in phospholipid vesicles and comparison with receptor-rich membranes by the use of a potentiometric dye.

Acetylcholine receptor, isolated in Triton X-100 on a cobra alpha-neurotoxin affinity column was incorporated into unilamellar phospholipid vesicles by a detergent depletion method using Amberlite XAD-2. Vesicles of an average diameter of 25 nm were formed, as verified by freeze-fracture electron microscopy and gel filtration. 85 to 95% of the alpha-bungarotoxin binding sites of the reconstituted acetylcholine receptor were oriented towards the outside of the vesicles. In the reconstituted receptor one molecule of residual Triton X-100 per 2.5 alpha-bungarotoxin binding sites on the receptor molecule could be assessed. The reconstituted protein was not accessible to papain digestion, whereas the pure acetylcholine receptor, solubilized by Triton X-100 was split into smaller polypeptides under the same condition. Reconstituted acetylcholine receptor and receptor-rich membranes did not exhibit the same behavior as measured by use of a potentiometric dye. This is interpreted as an irreversible alteration of at least 95% of the receptors purified in the presence of Triton X-100. Furthermore, it could be shown that the fluorescence intensity changes induced by carbamylcholine in receptor-rich membranes did not reflect ion fluxes, but conformational changes of the protein or a displacement of the dye from the protein.

Influence of sarcomere length, tonicity, and external sodium concentration on conduction velocity in frog muscle fibres.

1. Using an optical technique, conduction velocity in isolated frog muscle fibres has been measured at different sarcomere lengths and in solutions of altered tonicity and Na content. 2. Conduction velocity (in m/s) in normal Ringer solution is found to be independent of sarcomere length in the range of 2-5 microns. 3. Fibre cross-section appears to become circular with stretch to sarcomere lengths exceeding 4 microns. The data on fibre diameter and length are in agreement with the assumption that constant fibre volume is maintained during passive length changes. 4. In Na-deficient solutions, conduction velocity is reduced, in agreement with predictions based on action potential parameters. 5. In solutions of half or twice the normal tonicity, the conduction velocity is proportional to the square root of the measured fibre diameter. After correcting the bias involved in estimating fibre cross-section from only one measurement of fibre diameter, the data suggest an increase in specific internal resistance (Ri) by about 8% in twice hypertonic solution and a decrease by about 5% in half normal tonicity. 6. Releasing and stretching a fibre in hypertonic solution has no effect on conduction velocity as long as the initial sarcomere length is not exceeded. Stretching the fibre beyond the sarcomere length at which it was transferred to hypertonic solution reversibly increases conduction velocity.

Effect of 50% external sodium in solutions of normal and twice normal tonicity on internal sodium activity in frog skeletal muscle.

Neutral carrier based sodium-selective microelectrodes were used to monitor intracellular sodium activity in single frog skeletal muscle fibres during exposure to 50% external sodium solutions at normal and twice normal tonicity. Intracellular sodium activity in normal Ringer was 12.3 +/- 0.7 mM and was increased to 34.4 +/- 1.3 mM in hypertonic solution. Exposure to normotonic or hypertonic solutions containing only 50% sodium (NaCl) replaced by sucrose to maintain tonicity) did not affect the intracellular sodium activity during at least 20 min. Thus, in frog skeletal muscle, external sodium appears not to play a major role in regulating internal sodium, e.g. through ion exchange mechanisms as postulated for other excitable tissues.

Birefringence signals and tension development in single frog muscle fibres at short stimulus intervals.

The early large birefringence signal and mechanical activity were studied together in isolated single fibres of frog skeletal muscle with double stimulation at short stimulus intervals (2-60 msec) at room temperature and at 4--6 degrees C. In all fibres tested, extra tension and additional birefringence signal in response to the second stimulus appeared simultaneously and suddenly upon increasing the stimulus interval. The shape of the stimulus-interval versus tension-development curve makes it highly improbable that subthreshold calcium release occurs at shorter stimulus intervals; therefore, tension development reliably reflects Ca-release in these experiments. In contrast to the report by Suarez-Kurtz and Partker, birefringence signal and calcium release are shown not to be dissociated by double stimulation. This result supports the hypothesis that the early large birefringence signal is an intrinsic indicator of calcium release from the sr during EC-coupling in skeletal muscle.

A large birefringence signal preceding contraction in single twitch fibres of the frog.

1. When a single muscle fibre was externally stimulated to give a propagated action potential, a large decrease in light intensity was measured with the fibre positioned between crossed-polarizers oriented at +/- 45 degrees with respect to the fibre axis. This large optical signal begins just after stimulation and its man phase precedes the development of positive tension. 2. The peak (or plateau) amplitude of the signal, expressed as the peak (plateau) change in light intensity, delta I, divided by the resting light intensity, I, was typically (minus) 1 to 3x10-3 and the time-to-peak (plateau) was 4 to 6 ms (20 degrees C). 3. When mechanical activity was minimized by stretch or Ringer replacement with D2O or hypertonic solution, the peak tension response was reduced in far greater proportion than the peak optical response, suggesting that the early optical signal is not due to changes in tension or gross fibre movement. 4. The magnitude of the optical response was increased by nitrate and double-shock stimulatin, procedures which potentiate the twitch response. 5. The optical signal was shown to propagate along the fibre length with a conduction velocity appropriate to the surface action potential. 6. The above results suggest that the large, early birefringence signal reflects a step or steps in the sequence of events leading to contractile activation.

The optical properties of birefringence signals from single muscle fibres.

1. The optical retardation of single muscle fibres at rest and the optical properties of the large, early birefringence signal detectable during a twitch (Baylor & Oetliker, 1975, 1977a) were investigated. 2. The resting birefringence, B, which is the factor relating resting retardation, R, to the light path length through the fibre, L, was found to be 2.25 x 10-3 (i.e. R = 2.25 X 10-3 X L) and to be independent of wavelength ( lambda = 480-660 nm). 3. When the angle of incidence, psi, of the crossed polarizers with respect to the fibre axis was varied, the resting light intensity and large, early change in light intensity were related by the function sin2 psi-cos2psi. When the net phase shift, phi lambda, of a narrow longitudinal strip of fibre plus compensator was varied, the resting light intensity was described by the function (1-cos phi lambda), whereas the early change in light intensity followed sin phi lambda. These results make it likely that the optical mechanism underlying the early birefringence signal is a change in retardation. 4. When a narrow longitudinal strip of fibre was illuminated by monochromatic light in the range 480-690 nm, the magnitude of the signal varied approximately as expected if the retardation change is independent of wave-length. 5. The spatial characteristics of the signal were examined by moving a small slit of light across the fibre width as well as by measuring the signal collected from the entire fibre width as a function of wave-length. The results from both experiments support the idea that the large, early change in retardation is due to a volume-related rather than surface-related structure. 6. Under the assumption that the retardation change is distributed as fibre volume, its average magnitude was calculated. For fibres in normal Ringer the peak of the early retardation change compared with resting is about 1.7 x 10-3, and for fibres in D2O Ringer about 0.7 x 10-3.

Birefringence signals from surface and t-system membranes of frog single muscle fibres.

1. When the tonicity of Ringer is increased above 2-5 times normal and a single fibre stimulated externally, the large, early birefringence signal preceding twitch tension (Baylor & Oetliker, 1975, 1977 a,b) is sufficiently reduced and delayed so as to reveal a small but distinct signal ('1st component") preceding it. For an average-sized fibre, the deltaI/I of the 1st component was (minus) 1 to 2 x 10-5. 2. The time course of the 1st component superimposed with the surface action potential simultaneously recorded by an internal micro-electrode. The polarity of the 1st component reversed with compensation. 3. From these characteristics, the 1st component is thought to arise from a small change in optical retardation of the surface membrane due to the action potential. 4. When a fibre was impaled with two micro-electrodes, retardation changes accompanying small hyperpolarizing and depolarizing current steps were detected. In some cases the polarity of the observed signal was opposite in sign to that expected for a retardation change only from the surface membrane. 5. Because the anatomical orientation of the T-system appears to be primarily transverse rather than longitudinal, these signals of opposite polarity are probably, on balance, due to retardation changes from the membranes of the T-system. 6. The possible origin of the large birefringence signal preceding contraction is discussed.