scholarly journals Simultaneous monitoring of changes in magnesium and calcium concentrations in frog cut twitch fibers containing antipyrylazo III.

1989 ◽  
Vol 93 (4) ◽  
pp. 585-608 ◽  
Author(s):  
M Irving ◽  
J Maylie ◽  
N L Sizto ◽  
W K Chandler
Keyword(s):  
Action Potential ◽  
Voltage Clamp ◽  
Rate Constant ◽  
Time Course ◽  
Average Rate ◽  
Wavelength Dependence ◽  
Phenol Red ◽  
Single Action ◽  
Ph Indicators ◽  
Time Courses

Antipyrylazo III was introduced into frog cut twitch fibers (17-19 degrees C) by diffusion. After action potential stimulation, the change in indicator absorbance could be resolved into two components that had different time courses and wavelength dependences. The first component was early and transient and due to an increase in myoplasmic free [Ca] (Maylie, J., M. Irving, N.L. Sizto, and W.K. Chandler, 1987, Journal of General Physiology, 89:83-143). The second component, usually measured at 590 nm (near the isosbestic wavelength for Ca), developed later than the Ca transient and returned towards baseline about 100 times more slowly. Although the wavelength dependence of this component is consistent with an increase in either free [Mg] or pH, its time course is clearly different from that of the signals obtained with the pH indicators phenol red and 4',5'-dimethyl-5-(and -6-) carboxyfluorescein, suggesting that it is mainly due to an increase in free [Mg]. After a single action potential in freshly prepared cut fibers that contained 0.3 mM antipyrylazo III, the mean peak amplitude of delta A (590) would correspond to an increase in free [Mg] of 47 microM if all the signal were due to a change in [Mg] and all the intracellular indicator reacted with Mg as in cuvette calibrations. With either repetitive action potential stimulation or voltage-clamp depolarization, the delta A (590) signal continued to develop throughout the period when free [Ca] was elevated and then recovered to within 40-90% of the prestimulus baseline with an average rate constant between 0.5 and 1.0 s-1. With prolonged voltage-clamp depolarization, both the amplitude and rate of development of the delta A(590) signal increased with the amplitude of the depolarization and appeared to saturate at levels corresponding to an increase in free [Mg] of 0.8-1.4 mM and a maximum rate constant of 3-4 s-1, respectively. These results are consistent with the idea that the delta A(590) signal is primarily due to changes in myoplasmic free [Mg] produced by a change in the Mg occupancy of the Ca,Mg sites on parvalbumin that results from the Ca transient.

scholarly journals Perturbation of sarcoplasmic reticulum calcium release and phenol red absorbance transients by large concentrations of fura-2 injected into frog skeletal muscle fibers.

1990 ◽  
Vol 96 (3) ◽  
pp. 493-516 ◽  
Author(s):  
P C Pape ◽  
M Konishi ◽  
S Hollingworth ◽  
S M Baylor
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Action Potential ◽  
Time Course ◽  
Calcium Release ◽  
Free Calcium ◽  
Phenol Red ◽  
Charge Balance ◽  
Ph Change ◽  
Single Action ◽  
Fura 2

Intact single twitch fibers from frog muscle were studied on an optical bench apparatus after micro-injection with two indicator dyes: phenol red, to monitor a previously described signal (denoted delta pHapp; Hollingworth and Baylor. 1990. J. Gen. Physiol. 96:473-491) possibly reflective of a myoplasmic pH change following action potential stimulation; and fura-2, to monitor the associated change in the myoplasmic free calcium concentration (delta[Ca2+]). Additionally, it was expected that large myoplasmic concentrations of fura-2 (0.5-1.5 mM) might alter delta pHapp, since it was previously found (Baylor and Hollingworth. 1988. J. Physiol. 403:151-192) that the Ca2(+)-buffering effects of large fura-2 concentrations: (a) increase the estimated total concentration of Ca2+ (denoted by delta[CaT]) released from the sarcoplasmic reticulum (SR), but (b) reduce and abbreviate delta[Ca2+]. The experiments show that delta pHapp was increased at the larger fura-2 concentrations; moreover, the increase in delta pHapp was approximately in proportion to the increase in delta[CaT]. At all fura-2 concentrations, the time course of delta pHapp, through time to peak, was closely similar to, although probably slightly slower than, that of delta[CaT]. These properties of delta pHapp are consistent with an hypothesis proposed by Meissner and Young (1980. J. Biol. Chem. 255:6814-6819) and Somlyo et al. (1981. J. Cell Biol. 90:577-594) that a proton flux from the myoplasm into the SR supplies a portion of the electrical charge balance required as Ca2+ is released from the SR into the myoplasm. A comparison of the amplitude of delta pHapp with that of delta[CaT] indicates that, in response to a single action potential, 10-15% of the charge balance required for Ca2+ release may be carried by protons.

Calcium Dynamics and Compartmentalization in Leech Neurons

2005 ◽  
Vol 94 (6) ◽  
pp. 4430-4440 ◽  
Author(s):  
Sofija Andjelic ◽  
Vincent Torre
Keyword(s):  
Information Processing ◽  
Action Potential ◽  
Action Potentials ◽  
Time Course ◽  
Ccd Camera ◽  
Calcium Dynamics ◽  
Single Action ◽  
Single Action Potential ◽  
Calcium Indicator ◽  
Mechanosensory Neurons

Calcium dynamics in leech neurons were studied using a fast CCD camera. Fluorescence changes (Δ F/ F) of the membrane impermeable calcium indicator Oregon Green were measured. The dye was pressure injected into the soma of neurons under investigation. Δ F/ F caused by a single action potential (AP) in mechanosensory neurons had approximately the same amplitude and time course in the soma and in distal processes. By contrast, in other neurons such as the Anterior Pagoda neuron, the Annulus Erector motoneuron, the L motoneuron, and other motoneurons, APs evoked by passing depolarizing current in the soma produced much larger fluorescence changes in distal processes than in the soma. When APs were evoked by stimulating one distal axon through the root, Δ F/ F was large in all distal processes but very small in the soma. Our results show a clear compartmentalization of calcium dynamics in most leech neurons in which the soma does not give propagating action potentials. In such cells, the soma, while not excitable, can affect information processing by modulating the sites of origin and conduction of AP propagation in distal excitable processes.

Time course of postnatal changes in rat heart action potential and in transient outward current is different

1994 ◽  
Vol 267 (3) ◽  
pp. H1157-H1166 ◽  
Author(s):  
G. M. Wahler ◽  
S. J. Dollinger ◽  
J. M. Smith ◽  
K. L. Flemal
Keyword(s):  
Action Potential ◽  
Rat Heart ◽  
Action Potentials ◽  
Time Course ◽  
Outward Current ◽  
Transient Outward Current ◽  
Papillary Muscles ◽  
Isolated Cells ◽  
Time Courses ◽  
Action Potential Shortening

The rat ventricular action potential shortens after birth. The contribution of increases in the transient outward current (Ito) to postnatal action potential shortening was assessed by measuring Ito in isolated cells and by determining the effect of 2 mM 4-aminopyridine (4-AP) on the action potentials of papillary muscles. 4-AP had no effect on 1-day action potential duration at 25% repolarization (APD25), and 1-day cells had little Ito. In 8- to 10-day muscles, 4-AP caused a small, but significant, increase in APD25. Ito increased slightly between day 1 and days 8-10, but this increase was not significant. Most of the increase in Ito (79%) and in the response to 4-AP (64%) occurred between days 8-10 and adult; however, approximately 75% of the APD25 shortening took place by days 8-10. Thus, while Ito may contribute to repolarization in late neonatal and adult cells, the different time courses of action potential shortening and increases in Ito suggest that changes in Ito are unlikely to be responsible for most of the postnatal action potential shortening.

scholarly journals Properties of tetraethylammonium ion-resistant K+ channels in the photoreceptor membrane of the giant barnacle.

1981 ◽  
Vol 77 (6) ◽  
pp. 629-646 ◽  
Author(s):  
D R Edgington ◽  
A E Stuart
Keyword(s):  
Action Potential ◽  
Action Potentials ◽  
Cardiac Glycosides ◽  
Time Course ◽  
Photoreceptor Membrane ◽  
K Channels ◽  
Na Pump ◽  
Tetraethylammonium Ion ◽  
Electrogenic Na Pump ◽  
Time Courses

After the offset of illumination, barnacle photoreceptors undergo a large hyperpolarization that lasts seconds or minutes. We studied the mechanisms that generate this afterpotential by recording afterpotentials intracellularly from the medial photoreceptors of the giant barnacle Balanus nubilus. The afterpotential has two components with different time-courses: (a) an earlier component due to an increase in conductance to K+ that is not blocked by extracellular tetraethylammonium ion (TEA+) or 3-aminopyridine (3-AP) and (b) a later component that is sensitive to cardiac glycosides and that requires extracellular K+, suggesting that it is due to an electrogenic Na+ pump. The K+ conductance component increases in amplitude with increasing CA++ concentration and is inhibited by extracellular Co++; the Co++ inhibition can be overcome by increasing the Ca++ concentration. Thus, the K+ conductance component is Ca++ dependent. An afterpotential similar to that evoked by a brief flash of light is generated by depolarization with current in the dark and by eliciting Ca++ action potentials in the presence of TEA+ in the soma, axon, or terminal regions of the photoreceptor. The action potential undershoot is generated by an increase in conductance to K+ that is resistant to TEA+ and 3-AP and inhibited by Co++. The similarity in time-course and pharmacology of the hyperpolarization afterpotentials elicited by (a) a brief flash of light, (b) depolarization with current, and (c) an action potential indicates that Ca++-dependent K+ channels throughout the photoreceptor membrane are responsible for all three hyperpolarizing events.

scholarly journals Changes in phenol red absorbance in response to electrical stimulation of frog skeletal muscle fibers.

1990 ◽  
Vol 96 (3) ◽  
pp. 473-491 ◽  
Author(s):  
S Hollingworth ◽  
S M Baylor
Keyword(s):  
Time Course ◽  
Polarized Light ◽  
Weighted Average ◽  
Wavelength Dependence ◽  
Fiber Axis ◽  
Phenol Red ◽  
Ph Indicator ◽  
Similar Time ◽  
Average Value ◽  
Absorbance Changes

Intact single twitch fibers from frog muscle were stretched to long sarcomere length, micro-injected with the pH indicator dye phenol red, and activated by action potential stimulation. Indicator-related absorbance changes (denoted by delta A0 and delta A90) were measured with 0 degree and 90 degrees polarized light (oriented, respectively, parallel and perpendicular to the fiber axis). Two components of delta A were detected that had generally similar time courses. The "isotropic" component, calculated as the weighted average (delta A0 + 2 delta A90)/3, had the wavelength dependence expected for a change in myoplasmic pH. If calibrated in pH units, this signal's peak amplitude, which occurred 15-20 ms after stimulation, corresponded to a myoplasmic alkalization of average value 0.0025 +/- 0.0002 (+/- SEM; n = 9). The time course of this change, as judged from a comparison with that of the fibers' intrinsic birefringence signal, was delayed slightly with respect to that of the myoplasmic free [Ca2+] transient. On average, the times to half-peak and peak of the phenol red isotropic signal lagged those of the birefringence signal by 2.4 +/- 0.2 ms (+/- SEM; n = 8) and 8.4 +/- 0.5 ms (+/- SEM; n = 4), respectively. The other component of the phenol red signal was "dichroic," i.e., detected as a difference (delta A0-delta A90 greater than 0) between the two polarized absorbance changes. The wavelength dependence of this signal was similar to that of the phenol red resting dichroic signal (Baylor and Hollingworth. 1990. J. Gen. Physiol. 96:449-471). Because of the presence of the active dichroic signal, and because approximately 80% of the phenol red molecules appear to be bound in the resting state to either soluble or structural sites, the possibility exists that myoplasmic events other than a change in pH underlie the phenol red isotropic signal.

Propagation of action potentials in the dendrites of neurons from rat spinal cord slice cultures

1996 ◽  
Vol 75 (1) ◽  
pp. 154-170 ◽  
Author(s):  
M. E. Larkum ◽  
M. G. Rioult ◽  
H. R. Luscher
Keyword(s):  
Spinal Cord ◽  
Action Potential ◽  
Calcium Imaging ◽  
Action Potentials ◽  
Synaptic Activity ◽  
Rat Spinal Cord ◽  
Voltage Sensitive Dye ◽  
Single Action ◽  
Before And After ◽  
Time Courses

1. We examined the propagation of action potentials in the dendrites of ventrally located presumed motoneurons of organotypic rat spinal cord cultures. Simultaneous patch electrode recordings were made from the dendrites and somata of individual cells. In other experiments we visualized the membrane voltage over all the proximal dendrites simultaneously using a voltage-sensitive dye and an array of photodiodes. Calcium imaging was used to measure the dendritic rise in Ca2+ accompanying the propagating action potentials. 2. Spontaneous and evoked action potentials were recorded using high-resistance patch electrodes with separations of 30-423 microm between the somatic and dendritic electrodes. 3. Action potentials recorded in the dendrites varied considerably in amplitude but were larger than would be expected if the dendrites were to behave as passive cables (sometimes little or no decrement was seen for distances of > 100 microm). Because the amplitude of the action potentials in different dendrites was not a simple function of distance from the soma, we suggest that the conductance responsible for the boosting of the action potential amplitude varied in density from dendrite to dendrite and possibly along each dendrite. 4. The dendritic action potentials were usually smaller and broader and arrived later at the dendritic electrode than at the somatic electrode irrespective of whether stimulation occurred at the dendrite or soma or as a result of spontaneous synaptic activity. This is clear evidence that the action potential is initiated at or near the soma and spreads out into the dendrites. The conduction velocity of the propagating action potential was estimated to be 0.5 m/s. 5. The voltage time courses of previously recorded action potentials were generated at the soma using voltage clamp before and after applying 1 microM tetrodotoxin (TTX) over the soma and dendrites. TTX reduced the amplitude of the action potential at the dendritic electrode to a value in the range expected for dendrites that behave as passive cables. This indicates that the conductance responsible for the actively propagating action potentials is a Na+ conductance. 6. The amplitude of the dendritic action potential could also be initially reduced more than the somatic action potential using 1-10 mM QX-314 (an intracellular sodium channel blocker) in the dendritic electrode as the drug diffused from the dendritic electrode toward the soma. Furthermore, in some cases the action potential elicited by current injection into the dendrite had two components. The first component was blocked by QX-314 in the first few seconds of the diffusion of the blocker. 7. In some cells, an afterdepolarizing potential (ADP) was more prominent in the dendrite than in the soma. This ADP could be reversibly blocked by 1 mM Ni2+ or by perfusion of a nominally Ca2+-free solution over the soma and dendrites. This suggests that the back-propagating action potential caused an influx of Ca2+ predominantly in the dendrites. 8. With the use of a voltage-sensitive dye (di-8-ANEPPS) and an array of photodiodes, the action potential was tracked along all the proximal dendrites simultaneously. The results confirmed that the action potential propagated actively, in contrast to similarly measured hyperpolarizing pulses that spread passively. There were also indications that the action potential was not uniformly propagated in all the dendrites, suggesting the possibility that the distribution of Na+ channels over the dendritic membrane is not uniform. 9. Calcium imaging with the Ca2+ fluorescent indicator Fluo-3 showed a larger percentage change in fluorescence in the dendrites than in the soma. Both bursts and single action potentials elicited sharp rises in fluorescence in the proximal dendrites, suggesting that the back-propagating action potential causes a concomitant rise in intracellular calcium concentration...

scholarly journals Calcium signals recorded from cut frog twitch fibers containing antipyrylazo III.

1987 ◽  
Vol 89 (1) ◽  
pp. 83-143 ◽  
Author(s):  
J Maylie ◽  
M Irving ◽  
N L Sizto ◽  
W K Chandler
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Action Potential ◽  
Time Course ◽  
Wavelength Dependence ◽  
Difference Spectrum ◽  
Half Width ◽  
Arsenazo Iii ◽  
Transverse Tubular System ◽  
Low Amplitude ◽  
Regulatory Sites

The Ca indicator antipyrylazo III was introduced into cut frog twitch fibers by diffusion (Maylie, J., M. Irving, N. L. Sizto, and W. K. Chandler. 1987. Journal of General Physiology. 89:41-81). Like arsenazo III, antipyrylazo III was largely bound to or sequestered by intracellular constituents; on average, a fraction 0.68 was so immobilized. After action potential stimulation, there was an early change in absorbance, with a wavelength dependence that nearly matched a cuvette Ca-difference spectrum. As with arsenazo III, this signal became prolonged as experiments progressed. In a freshly prepared cut fiber containing 0.3 mM indicator, the absorbance change had an average half-width of 10 ms at 18 degrees C. The peak amplitude of this Ca signal depended on the indicator concentration in a roughly parabolic manner, which is consistent with a 1:2 stoichiometry for Ca:indicator complexation and, for indicator concentrations less than or equal to 0.4 mM, constant peak free [Ca]. If all the antipyrylazo III inside a fiber can react normally with Ca, peak free [Ca] is 3 microM at 18 degrees C. If only freely diffusible indicator can react, the estimate is 42 microM. The true amplitude probably lies somewhere in between. The time course of Ca binding to intracellular buffers and of Ca release from the sarcoplasmic reticulum is estimated from the 3- and 42-microM myoplasmic [Ca] transients. After action potential stimulation, the release waveform is rapid and brief; its latency after the surface action potential is 2-3 ms and its half-width is 2-4 ms. This requires rapid coupling between the action potential in the transverse tubular system and Ca release from the sarcoplasmic reticulum. The peak fractional occupancy calculated for Ca-regulatory sites on troponin is 0.46 for the 3-microM transient and 0.93 for the 42-microM transient. During a 100-ms tetanus at 100 Hz, the corresponding fractional occupancies are 0.56 and 0.94. The low value of occupancy associated with the low-amplitude [Ca] calibration seems inconsistent with a brief tetanus being able to produce near-maximal activation (Blinks, J. R., R. Rudel, and S. R. Taylor. 1978. Journal of Physiology. 277:291-323; Lopez J. R., L. A. Wanck, and S. R. Taylor. 1981. Science. 214:47-82).

Role of calcium influx and buffering in the kinetics of Ca(2+)-activated K+ current in rat vagal motoneurons

1992 ◽  
Vol 68 (6) ◽  
pp. 2237-2247 ◽  
Author(s):  
P. Sah
Keyword(s):  
Action Potential ◽  
Time Constant ◽  
Potassium Current ◽  
Time Course ◽  
Calcium Influx ◽  
Outward Current ◽  
Resting Potential ◽  
Motor Nucleus ◽  
Simple Diffusion ◽  
Single Action

1. Intracellular recordings were obtained from neurons of the dorsal motor nucleus of the vagus (DMV) in transverse slices of the rat medulla maintained in vitro. These neurons had a resting potential of -59.8 +/- 8.7 (SD) mV. Single action potentials elicited by brief depolarizing current pulses were followed by a prolonged afterhyperpolarization (AHP). Under voltage clamp, the current underlying the AHP was found to be a calcium-activated potassium current. 2. The outward current (GkCa,1) was voltage insensitive and was not blocked by tetraethylammonium (TEA) (10 mM). Unlike the slower time course calcium-activated potassium current recorded in some other neurons, GkCa,1 was blocked by apamin (25-100 nM), indicating that SK type calcium-activated potassium channels underlie this current. 3. GkCa,1 was maximal within 10 ms of the action potential and its decay was well described by a single exponential. After a single action potential the time constant of decay of GkCa,1 was 155 +/- 66 (+/- SD) ms. 4. Calcium influx was increased by adding TEA to the extracellular solution or by firing more than one action potential. As the calcium load was increased, both the peak amplitude and the time constant of decay of GkCa,1 increased. In cells impaled with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA)-filled electrodes, the time constant of decay of GkCa,1 after a single action current was 71 +/- 19 ms. 5. A simple diffusion-based model that incorporates two intrinsic calcium buffers is developed that accounts for many of the properties of GkCa,1. It is concluded that the decay of GkCa,1 reflects the time course of removal of calcium that has entered the cell during the action potential.

scholarly journals Effects of Partial Sarcoplasmic Reticulum Calcium Depletion on Calcium Release in Frog Cut Muscle Fibers Equilibrated with 20 mM EGTA

1998 ◽  
Vol 112 (3) ◽  
pp. 263-295 ◽  
Author(s):  
Paul C. Pape ◽  
De-Shien Jong ◽  
W. Knox Chandler
Keyword(s):  
Sarcoplasmic Reticulum ◽  
Action Potential ◽  
Calcium Release ◽  
Charge Movement ◽  
Ca Channel ◽  
Phenol Red ◽  
Fractional Rate ◽  
Normal Physiological Condition ◽  
Additional Release ◽  
Time Courses

Resting sarcoplasmic reticulum (SR) Ca content ([CaSR]R) was varied in cut fibers equilibrated with an internal solution that contained 20 mM EGTA and 0–1.76 mM Ca. SR Ca release and [CaSR]R were measured with the EGTA–phenol red method (Pape et al. 1995. J. Gen. Physiol. 106:259–336). After an action potential, the fractional amount of Ca released from the SR increased from 0.17 to 0.50 when [CaSR]R was reduced from 1,200 to 140 μM. This increase was associated with a prolongation of release (final time constant, from 1–2 to 10–15 ms) and of the action potential (by 1–2 ms). Similar changes in release were observed with brief stimulations to −20 mV in voltage-clamped fibers, in which charge movement (Qcm) could be measured. The peak values of Qcm and the fractional rate of SR Ca release, as well as their ON time courses, were little affected by reducing [CaSR]R from 1,200 to 140 μM. After repolarization, however, the OFF time courses of Qcm and the rate of SR Ca release were slowed by factors of 1.5–1.7 and 6.5, respectively. These and other results suggest that, after action potential stimulation of fibers in normal physiological condition, the increase in myoplasmic free [Ca] that accompanies SR Ca release exerts three negative feedback effects that tend to reduce additional release: (a) the action potential is shortened by current through Ca-activated potassium channels in the surface and/or tubular membranes; (b) the OFF kinetics of Qcm is accelerated; and (c) Ca inactivation of Ca release is increased. Some of these effects of Ca on an SR Ca channel or its voltage sensor appear to be regulated by the value of [Ca] within 22 nm of the mouth of the channel.

Calcium transients in intact rat skeletal muscle fibers in agarose gel

1995 ◽  
Vol 269 (1) ◽  
pp. C28-C34 ◽  
Author(s):  
S. L. Carroll ◽  
M. G. Klein ◽  
M. F. Schneider
Keyword(s):  
Action Potentials ◽  
Time Course ◽  
Calcium Transients ◽  
Agarose Gel ◽  
Similar Time ◽  
Single Action ◽  
Fura 2 ◽  
Calcium Indicator ◽  
Flexor Digitorum Brevis ◽  
Time Courses

Intact single fibers enzymatically dissociated from rat flexor digitorum brevis muscle were suspended in 0.5% low-melting-temperature agarose gel to minimize fiber movement during action potentials or trains of action potentials. Resting Ca2+ concentration ([Ca2+]) and changes in [Ca2+] were monitored using the fluorescent calcium indicator fura 2. The time course and waveform of [Ca2+] transients during an action potential or trains of action potentials in fibers in agarose were calculated using kinetic parameters previously determined to correct for the calcium-fura 2 kinetic delay. Half times of the calculated calcium transients for single action potentials were 30-fold briefer than the original fura 2 signals. To confirm the time course and waveform of the calculated calcium transients, changes in [Ca2+] were monitored using the more rapidly equilibrating calcium indicator mag-fura 2. [Ca2+] transients for fibers containing fura 2 had very similar time courses and waveforms as mag-fura 2 signals from other fibers, indicating that the corrections for the calcium-fura 2 kinetic delay were accurate. The advantages of the agarose gel suspension are discussed.

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