Biomedical subjects
A Vyshedskiy
Publications and source records attributed to A Vyshedskiy.
Transpulmonary speed of sound input into the supraclavicular space.
The transpulmonary speed of sound input at the mouth has been shown to vary with lung volume. To avoid the disadvantages that exist in certain clinical situations in inputting sound at the mouth, we input sound in the supraclavicular space of 21 healthy volunteers to determine whether similar information on the relationship of sound speed to lung volume could be obtained. We measured the transit time at multiple microphones placed over the chest wall using a 16-channel lung sound analyzer (Stethographics). There was a tight distribution of transit times in this population of subjects. At functional residual capacity, it was 9 +/- 1 (SD) ms at the apical sites and 13 +/- 1 ms at the lung bases. The sound speed at total lung capacity was 24 +/- 2 m/s and was 22 +/- 2 m/s at residual volume (P < 0.001). In all subjects, the speed of sound was faster at higher lung volume. This improved method of studying the mechanism of sound transmission in the lung may help in the development of noninvasive tools for diagnosis and monitoring of lung diseases.
Sound transmission in the lung as a function of lung volume.
We were interested in how the transmission of sound through the lung was affected by varying air content in intact humans as a method of monitoring tissue properties noninvasively. To study this, we developed a method of measuring transthoracic sound transit time accurately. We introduced a "coded" sound at the mouth and measured the transit time at multiple microphones placed over the chest wall by using a 16-channel lung sound analyzer (Stethographics). We used a microphone placed over the neck near the trachea as our reference and utilized cross-correlation analysis to calculate the transit times. The use of the coded sound, composed of a mix of frequencies from 130 to 150 Hz, greatly reduced the ambiguity of the cross-correlation function. The measured transit time varied from 1 ms at the central locations to 5 ms at the lung bases. Our results also indicated that transit time at all locations decreased with increasing lung volume. We found that these results can be described in terms of a model in which sound transmission through the lung is treated as a combination of free-space propagation through the trachea and a propagation through a two-phase system in the parenchyma.
Analysis of presynaptic Ca2+ influx and transmitter release kinetics during facilitation at the inhibitor of the crayfish neuromuscular junction.
The inhibitory synapse of the crayfish neuromuscular junction was used to examine mechanisms underlying the F2 component of synaptic facilitation. Because previous studies have shown accelerated transmitter release during facilitation, we examined whether an activity-dependent plasticity in I(Ca) could underlie this acceleration. We established that fluorescent transients generated by Magnesium Green can resolve small differences in presynaptic Ca(2+) influx that correlate with changes in IPSC waveform. However, there was no change in Ca(2+) transients associated with the accelerated release. Analyzing the initial rise of IPSC and the duration of the presynaptic spike yielded a depolarization-release coupling plot that captures the impact of spike waveform on the initial rate of release. We conclude that accelerated release during F2 facilitation cannot be attributed to plasticity of I(Ca) or modulation of spike waveform. Kinetic analysis showed a reduction in synaptic delay during facilitation only when broad action potentials were used. In unfacilitated release, synaptic delay increased as spike duration lengthened. We propose that small single Ca(2+) channel currents during the plateau phase of broad action potentials raise local Ca(2+) concentration only enough to fill a high-affinity site. Occupation of this site in itself, or events downstream, would convert a vesicle from control to facilitated state. If the conversion were a slow process, it could explain the changes in synaptic delay reported here. This hypothesis can also account for a number of observations related to Ca(2+) cooperativity and synaptic facilitation.
Presynaptic Ca(2+) influx at the inhibitor of the crayfish neuromuscular junction: a photometric study at a high time resolution.
Presynaptic calcium influx at the inhibitor of the crayfish neuromuscular junction was investigated by measuring fluorescence transients generated by calcium-sensitive dyes. This approach allowed us to correlate presynaptic calcium influx with transmitter release at a high time resolution. Systematic testing of the calcium indicators showed that only low-affinity dyes, with affinities in the range of micromolar, should be used to avoid saturation of dye binding and interference with transmitter release. Presynaptic calcium influx was regulated by slowly increasing the duration of the action potential through progressive block of potassium channels. The amplitude of the calcium transient, measured from a cluster of varicosities, was linearly related to the duration of the action potential with a slope of 1.2. Gradual changes in potassium channel block allowed us to estimate the calcium cooperativity of transmitter release over a 10-fold range in presynaptic calcium influx. Calcium cooperativity measured here exhibited one component with an average value of 3.1. Inspection of simultaneously recorded presynaptic calcium transients and inhibitory postsynaptic currents (IPSCs) showed that prolonged action potentials were associated with a slow rising phase of presynaptic calcium transients, which were matched by a slow rate of rise of IPSCs. The close correlation suggests that fluorescence transients provide information on the rate of calcium influx. Because there is an anatomic mismatch between the presynaptic calcium transient, measured from a cluster of varicosities, and IPSC, measured with two-electrode voltage clamp, macropatch recording was used to monitor inhibitory postsynaptic responses from the same cluster of varicosities from which the calcium transient was measured. Inhibitory postsynaptic responses recorded with the macropatch method exhibited a faster rising phase than that recorded with two-electrode voltage clamp. This difference could be attributed to slight asynchrony of transmitter release due to action potential conduction along fine branches. In conclusion, this report shows that fluorescence transients generated by calcium-sensitive dyes can provide insights to the properties of presynaptic calcium influx, and its correlation with transmitter release, at a high time resolution.
Neuromodulators enhance transmitter release by two separate mechanisms at the inhibitor of crayfish opener muscle.
A presynaptic voltage control method has been used to investigate the modulatory effects of serotonin (5-HT) and okadaic acid (OA) on the inhibitory junction of the crayfish opener muscle. Instead of using action potentials, we used 20 msec pulses depolarized to 0 mV to activate transmitter release. This approach allowed us to monitor two separate physiological parameters related to the release process. The first parameter, transmitter release kinetics, is characterized as the delay when inhibitory postsynaptic conductance reaches its half-maximum (IPSG50). The second parameter, the total area of IPSG (IPSGarea), estimates total transmitter output. We have reported previously that the F2 component of synaptic facilitation is associated with a decrease in IPSG50 but without a change in IPSGarea. These results raised the possibility that IPSG50 and IPSGarea could be mediated by separate mechanisms that were modulated independently. To explore this possibility, we investigated the effects of 5-HT (100-200 nM) and OA (2.5 microM) on the two parameters. 5-HT and OA enhanced IPSG neither by changing the sensitivity of postsynaptic receptors, as tested by iontophoretically ejected GABA, nor by elevating resting and action potential-activated presynaptic free calcium, as monitored by fura-2 imaging. 5-HT and OA decreased IPSG50 by 3.0 +/- 1.4 and 3.6 +/- 1.1 msec, respectively, and increased IPSGarea by 50 +/- 21 and 37 +/- 6%, respectively. The ability of F2 facilitation to accelerate release kinetics was reduced in the presence of the modulators, suggesting that the mechanism underlying the accelerated release kinetics was shared by the two modes of synaptic enhancement. This report demonstrates that the acceleration in release kinetics and the increase in total release are two separate mechanisms for enhancing transmitter output and that these two mechanisms can be activated without changes in presynaptic calcium dynamics.
Study of the inhibitor of the crayfish neuromuscular junction by presynaptic voltage control.
The inhibitor of the crayfish opener muscle was investigated by a presynaptic voltage control method. Two microelectrodes were inserted into the inhibitor and the amplitude and duration of presynaptic depolarization were controlled by a voltage-clamp amplifier. The inhibitory postsynaptic potential (IPSP) was measured from a muscle fiber located near the presynaptic voltage electrode. Nonlinear summation of IPSP amplitudes was corrected after chloride equilibrium potential was measured. With the use of 5-ms presynaptic pulses, the depolarization-release coupling (D-R) curve constructed from IPSP peak amplitudes (IPSPcor) had a threshold of about -35 mV and reached its maximal level at -5 to -10 mV. Depolarization beyond the maximum led to a suppression of neurotransmitter release. When transmitter release during a presynaptic pulse was completely suppressed, IPSPs activated by tail current could be identified with an average synaptic delay of 2.5 ms. Transmitter secretion triggered by a calcium current activated during the 5-ms pulses (IPSPon) was also measured on the rising phase of an IPSP, at 2.5 ms after the end of the 5-ms pulses. D-R coupling plots measured from IPSPon exhibited a more pronounced suppression than that obtained from IPSPcor. The effect of presynaptic pulse duration on the level of transmitter release was analyzed. Transmitter release increased with increasing duration and was nearly saturated by 20-ms pulses depolarized to 0 mV. The following conditions were identified as necessary to obtain a consistent D-R curve with a clear suppression: 1) small animals, 3.8 cm head to tail, 2) 15 degrees C, 3) 40 mM tetraethylammonium and 1 mM 4-aminopyridine, 4) an extracellular calcium concentration of < or = 10 mM. In addition, a consistent correlation was found among the branching pattern of the inhibitor, the placement of the presynaptic electrode, and the characteristics of the D-R curves. An ideal presynaptic electrode configuration involved placing the voltage electrode in a secondary branch, approximately 100 microns from the main branch point, and placing the current electrode at the branch point. Postsynaptically, optimal recordings were obtained from muscle fibers innervated by a single branch of the inhibitor that originated from a point near the presynaptic voltage electrode. A cable-release model was constructed to evaluate the relationship between the shape of the D-R coupling curves and the space constants of the presynaptic terminals. A comparison between the model and the D-R coupling curves suggested that the space constant of an inhibitor branch on a muscle fiber is > or = 8 times longer than its actual length. Therefore the upper limit estimate of the space constant of a typical preparation is approximately 3 mm. Results reported here outline morphological and physiological conditions needed to achieve optimal control of the presynaptic branch of the crayfish inhibitor. The cable-release model quantitatively defines the extent of presynaptic voltage control.
Activation and detection of facilitation as studied by presynaptic voltage control at the inhibitor of the crayfish opener muscle.
Facilitation at the crayfish neuromuscular inhibitor synapse was investigated with the use of a presynaptic voltage control method in which 5-ms presynaptic pulses were used to activate and monitor facilitation. A single 5-ms pulse was able to activate facilitation with a decay time constant similar to that of the F2 component of facilitation activated by action potentials. The quality of the control of presynaptic potential during F2 facilitation was evaluated by measuring the amplitude of presynaptic pulses and by analyzing the shape of the depolarization-release coupling plot during facilitation. Both approaches suggested that neither the amplitude of presynaptic depolarizations nor the space clamp of the presynaptic axon was changed during F2 facilitation. The activation of facilitation was examined by changing the amplitude of conditioning pulses systematically and using a test pulse of a constant amplitude to monitor facilitation. We found that a significant amount of facilitation could be activated by conditioning pulses that were subthreshold to the activation of transmitter release. Facilitation plateaued before the inhibitory postsynaptic potentials (IPSPs) activated by conditioning pulses reached their maximum. A double logarithm plot of facilitation magnitude against the conditioning IPSP amplitude yielded a slope of 0.34, which implies that the calcium ion cooperativity of activating facilitation is about one third of the secretion process. These findings enabled us to activate near maximal facilitation, by a burst of subthreshold conditioning pulses, without any conditioning transmitter release, and, therefore, to avoid complications associated with previous transmitter release. The detection of facilitation was examined by changing test pulse amplitude systematically to evaluate the ability of the test pulse to detect a constant level of facilitation. The magnitude of normalized facilitation decreased with increasing test pulse amplitude. The magnitude of absolute facilitation (the amplitude of the facilitated minus the control IPSP) increased with increasing test pulse amplitude. A double logarithm plot between facilitated and control IPSPs gave rise to a slope of 0.77, which suggests that the calcium cooperativity of transmitter release was decreased during facilitation.
Change of transmitter release kinetics during facilitation revealed by prolonged test pulses at the inhibitor of the crayfish opener muscle.
A presynaptic voltage-control method was used to study synaptic facilitation at the inhibitory neuromuscular synapse of the crayfish opener muscle. The expression of the F2 component of facilitation, monitored 150 ms after a conditioning stimulus, was examined by systematically changing the duration of the presynaptic test pulse. (Test pulses in all experiments were depolarized to 0 mV.) Control and facilitated inhibitory postsynaptic potentials (IPSPs) exhibited identical time courses when test pulse duration was brief (approximately 2 ms). When the duration of the test pulse was increased beyond 2 ms, the transmitter release time course shifted to an earlier point in time during facilitation. Meanwhile, the increase in total transmitter release, measured from inhibitory postsynaptic conductance (IPSG) area (total release), became less pronounced with increasing duration of the test pulse. With a 20-ms test pulse, facilitation did not cause any detectable change in total release but the half-maximal point of the facilitated IPSG shifted by 3 ms (release shift). This change in release kinetics was not associated with a decrease in minimal synaptic delay. Furthermore, the relationship between total release and presynaptic pulse duration suggested that the transmitter release activated by a 20-ms pulse can be defined as a distinct component of continuous transmitter release (early component). The facilitation process accelerated the release kinetics of the early component but did not modify its total transmitter content. To test the hypothesis that the release shift is indeed mediated by the same mechanism that increases IPSP amplitude during facilitation, we investigated the correlation between the release shift and IPSP amplitude change. The two parameters were significantly correlated when the magnitude of facilitation was changed 1) during the decay of facilitation and 2) by varying the strength of the conditioning stimulus. The experimental approach reported here provides two new physiological parameters, release shift and total release, for the analysis of synaptic facilitation.