MEMBRANE
CAPACITANCE MEASUREMENTS ACETYLCHOLINE ACTION
BORING
B. B. DUNNINGand XENIAMACI~NE Department of Pharmacology,
University of Minnesota. Minneapolis, ~inncsofa
55455
Summary--Capacitance of the membrane of H&x aspersu neurones was measured by frequency analysis. The specific membrane capacitance in resting conditions was 10 f 4.7 pF/cm’. No capacitance changes were observed during acetyl~boline-induced potential changes.
Cell diameter was estimated during an experiment using a calibrated micrometer disc inserted into the eyepiece of the dissecting microscope. This arrangement allowed resolution to &20 pm. Cell diameters ranged from lQQ_-200w. No attempt was made to estimate any infolding that might be present in the surface membrane of these neuranes (see MIROLLIand TALBOTL1972) in arriving at the value for cell surface area. Membrane potentials were recorded using KClfilled microelectrodes with a high input impedance differ~ut~~l amplifier (PH~LBRIGK RESEARCHES, 1966, p. 82). Input capacitance neutralization was added to this basic circuit to extend the useful frequency range of the ampli~~r when using microelectrodes. Current was injected via an intracellular microelectrode from a constant current generator (PHILBRICKRESEARCEKS, 1966. p. 66) and was recorded by a current-to-voltage transducer (GAGE and EISENBERG,1969). Capacitance was measured using sine wave analysis on the assumption that the membrane is equivalent to a circuit containing a parallel resistance and capacitance (RC circuit). The sine wave voltage applied METHODS to the constant current generator was derived from Experiments were conducted on single neurones of a multigenerator (Exact Model 124). Frequencies the isolated subesophageal ganglionic mass of H&.x between 50 and 1000 Hz were used. The current inaspcrsa. The preparation was continuously perfused tensity was kept below 10 nA (peak to peak) so that with physiological saline at room temperature and the resulting voltage fluctuations would be subthresadjusted to pH 7.65. The saline had the following hold (less than 5 mV peak to peak). To increase the composition in mmol/l: NaCl, 70; KCl, 4; C&i,, accuracy of our measurements, the signal-to-noise 7; MgCl,, 5; Tris-HCI, 5. Acetylcholine was applied ratio was improved by either of two methods dependto the soma membrane in two ways. Most usually ing on the aim of the experiment. The voltage changes iontophoretic application from a micropipette was of interest were either passed through a band-pass filter or averaged using a Waveform Eductor (Princeused with ejection pulses of 200-500 msec duration. ton Applied Research). In two experiments drops of 1 M iodoacetylcholine Capacitance measurements made using microelecwere added to the bath perfusing the ganglion. This resulted in ACh con~eutrations of about 10 mM in trodes are subject to errors due to capacitative crossthe bath. Observations were limited to neurones talk between voltage and current electrodes. This whichrespondedtoAChwitha transient hyperpolarizaerror was minimized by shielding the current election. trode with a grounded concentric shieId. There are 73 Few studies in recent years have been directed toward m~suring the capacitance of nerve membranes (TAYLOR, 1965; MATSIJMOTU, INOUEand KISH~MOTO, 1970; TAKASHI~ and SCHWANN,1974). Of these, only two have been able to demonstrate changes in membrane Gapacitance as a result of modifications in the environment of the preparation. Using the squid giant axon it has been shown that membrane capacitance increases as the temperature is increased (TAYLOR, 1965) and that membrane capacitance decreases when external potassium is raised to 100 mM (MA~suMO~ Ct af., 1970). It would be expected that major structural changes occurring in the cell membrane would lead to alterations in membrane capacitance. It has been suggested that acetylcholine-induced permeability changes are the result of macromolecular perturbations occurring within the activated cell membrane (BLOOM,1970). The experiments performed here were designed to determine whether such structural alterations result in membrane capacitance changes during acetylcholine {ACh) action.
74
B. B. DUNNINGand XENIA MACHNE
additional sources of error in the estimation of membrane capacitance. There was some scatter in the unaveraged data points (at a given frequency) as a result of minor fluctuations in the magnitude of the injected current. Signal averaging over many cycles reduced the scatter to less than 2;;. In the derivation of the alternating current-to-voltage relation for a parallel RC circuit, it is found that if the frequency of the impressed current is much greater than 1/27cRC then an approximation to the actual current-to-voltage relation can be derived which does not involve the resistance R (HAGIWAKA and SAITO, 1959). The error in this approximation due to ignoring resistance changes of the order observed in this preparation during ACh action are less than 1% in the frequency range between 400 and 1000 Hz. A difficulty inherent in our experimental set-up arises from the fact that the topography of somatic ACh receptors is unknown and that presumably the receptors occupy only a small portion of the total membrane area. If any capacitance changes were observed, several assumptions on receptor topography would be required to reach an estimate of the actual change within the activated membrane. RESULTS On the assumption that the cell membrane is equivalent to a parallel RC circuit, the peak to peak amplitude of the potential change across the membrane should be inversely proportional to the frequency of the applied alternating current, provided that the current is maintained constant. The results obtained on the resting membrane of a single neurone are shown in Figure 1 (filled circles). The amplitude of the sine wave voltage developed across the cell membrane was
plotted as a function of the reciprocal frequency of the alternating current. The current intensity was 5 nA for all frequencies. The graph illustrates a straight line relationship for frequencies greater than 150 Hz. The straight line relationship demonstrates that the membrane capacitance is not frequency dependent over the frequency range tested and that our assumption of a parallel RC mode1 of the cell membrane is justified. The total cell capacitance can be calculated from the slope of the straight line using the following equation: C = [(Slope)2z]/1, where I is electric current. In the cell of Figure 1, C was equal to 6 x lO_‘F. By measuring the diameter of the cell and assuming a spherical shape, the specific membrane capacitance, C,. is calculated to be 13 pF/cm’. For 12 cells, C, equalled 10 k 4.7 pF/ cm2. Consistent C, values were obtained within the first hour after impaling a cell. Thereafter. C,,, tended to increase in spite of the fact that no change in resting membrane conductance occurred. It is our impression that this resulted from a slight osmotic imbalance leading to swollen cells having stretched cell membranes. Membrane capacitance was determined during the rising phase or the peak of the ACh-induced potential change using a signal averager to improve the signalto-noise ratio of the potential changes measured. No change from the resting value of C, occurred at these times (See Fig. 1). In addition, the sine wave voltage developed across the cell membrane was observed during the entire ACh response using band-pass filters (465 and 950 Hz) to selectively suppress unwanted signals. At no time during the action of ACh, whether applied iontophoretically or by perfusion. was a change in this voltage observed. Thus. there was no indication of capacitance changes within the limits of our technique. DISCUSSION
Fig. I. Relation between peak amplitude of potential change and reciprocal frequency of applied alternating current for resting membrane (0) and for ACh-activated membrane (0). Measurements made during ACh action were taken during the peak of the response. Current intensity was 5 nA at all frequencies. The solid line is fitted by eye to the data points and the broken line illustrates the slope of the linear portion of the relation. The data points taken during ACh action deviate at low frequency from the points obtained on the resting membrane due to the reduction in cell input resistance during the action of ACh.
The capacitance values found for the resting membrane of Helix asprm neurones are much smaller than those reported for Helix ponmtia (MAISKY, 1963). The discrepancy may be due to the fact that the capacitance values for the latter preparation were obtained by square pulse analysis. The values of C, measured in our preparation are in the same range as those reported for neurones of the puffer fish (HAGIWARA and SAITO, 1959). The fact that we were unable to detect any ACh-induced capacitance changes suggests that no major structural alterations in the lipid component of the membrane are associated with the action of ACh. REFERENCES BLOOM. B. M. (1970). Receptor theories. In: Medicinal Chrmistry. Part I. 3rd Edition (BL’RGLR. A.. Ed.) pp. 108-l 17. Wiley-Interscience, New York.
ACh and membrane capacitance GAGE, P. W. and EISENBERG. R. S. (1969). Capacitance of the surface and transverse tubular membrane of frog sartorius muscle fibers. J. CJUI.Physiol. 53: 265-278. HACIWARA,S. and SAITO,N. (1959). Membrane potential change and membrane current in sup~arneduilar~ nerve cell of puffer. J. ~~,Z~~~~~?~Sj~~. 22: 104-271. MAISKY.V. A. (1963). Electrical characteristics of the surface membrane of giant nerve cells of Nrlir pomrrrir~. Fi-_i#l. z/z. (Musk.) 49: 1468-1474. MA~SUMOIY). N., IN&Y. I. and KISFZIMOTO. U. (1970). The electrical impedance of the squid axon membrane measured between internal and external electrodes. Jup. J. Pizpsio/. 20: 516 536.
75
MIROLLI.M. and TALBOTT,S. R. (1972). The geometrical factors determining the electrical properties of a molluscan neurone. J. Physiol., Lmd. 227: 19-34. PHILBRICKR~SEAKCHES ENGINEERING STAFF(1966). Ap$~~~j~~~~ ~~~}~~~~~ jirr C~777~7if~~77~~ ~~77~~~~~~~s. 115 pp. Philbrick Researches Inc.. Dedham. Mass. TAXASHIPIIA. S. and SCI-IWANN.H. P. (1974). Passive electricaf properties of squid axon membrane. f. ntrrtzl~~i/ii~Bioi. 17: 51 -68. T41 LOR.R. E. ( 1965).Impedance of the squid axon membrane. J. ceil trtlizj~.Plt@clf. 66: :!l-26.
CAPACITANCE MEASUREMENTS ACETYLCHOLINE ACTION
BORING
B. B. DUNNINGand XENIAMACI~NE Department of Pharmacology,
University of Minnesota. Minneapolis, ~inncsofa
55455
Summary--Capacitance of the membrane of H&x aspersu neurones was measured by frequency analysis. The specific membrane capacitance in resting conditions was 10 f 4.7 pF/cm’. No capacitance changes were observed during acetyl~boline-induced potential changes.
Cell diameter was estimated during an experiment using a calibrated micrometer disc inserted into the eyepiece of the dissecting microscope. This arrangement allowed resolution to &20 pm. Cell diameters ranged from lQQ_-200w. No attempt was made to estimate any infolding that might be present in the surface membrane of these neuranes (see MIROLLIand TALBOTL1972) in arriving at the value for cell surface area. Membrane potentials were recorded using KClfilled microelectrodes with a high input impedance differ~ut~~l amplifier (PH~LBRIGK RESEARCHES, 1966, p. 82). Input capacitance neutralization was added to this basic circuit to extend the useful frequency range of the ampli~~r when using microelectrodes. Current was injected via an intracellular microelectrode from a constant current generator (PHILBRICKRESEARCEKS, 1966. p. 66) and was recorded by a current-to-voltage transducer (GAGE and EISENBERG,1969). Capacitance was measured using sine wave analysis on the assumption that the membrane is equivalent to a circuit containing a parallel resistance and capacitance (RC circuit). The sine wave voltage applied METHODS to the constant current generator was derived from Experiments were conducted on single neurones of a multigenerator (Exact Model 124). Frequencies the isolated subesophageal ganglionic mass of H&.x between 50 and 1000 Hz were used. The current inaspcrsa. The preparation was continuously perfused tensity was kept below 10 nA (peak to peak) so that with physiological saline at room temperature and the resulting voltage fluctuations would be subthresadjusted to pH 7.65. The saline had the following hold (less than 5 mV peak to peak). To increase the composition in mmol/l: NaCl, 70; KCl, 4; C&i,, accuracy of our measurements, the signal-to-noise 7; MgCl,, 5; Tris-HCI, 5. Acetylcholine was applied ratio was improved by either of two methods dependto the soma membrane in two ways. Most usually ing on the aim of the experiment. The voltage changes iontophoretic application from a micropipette was of interest were either passed through a band-pass filter or averaged using a Waveform Eductor (Princeused with ejection pulses of 200-500 msec duration. ton Applied Research). In two experiments drops of 1 M iodoacetylcholine Capacitance measurements made using microelecwere added to the bath perfusing the ganglion. This resulted in ACh con~eutrations of about 10 mM in trodes are subject to errors due to capacitative crossthe bath. Observations were limited to neurones talk between voltage and current electrodes. This whichrespondedtoAChwitha transient hyperpolarizaerror was minimized by shielding the current election. trode with a grounded concentric shieId. There are 73 Few studies in recent years have been directed toward m~suring the capacitance of nerve membranes (TAYLOR, 1965; MATSIJMOTU, INOUEand KISH~MOTO, 1970; TAKASHI~ and SCHWANN,1974). Of these, only two have been able to demonstrate changes in membrane Gapacitance as a result of modifications in the environment of the preparation. Using the squid giant axon it has been shown that membrane capacitance increases as the temperature is increased (TAYLOR, 1965) and that membrane capacitance decreases when external potassium is raised to 100 mM (MA~suMO~ Ct af., 1970). It would be expected that major structural changes occurring in the cell membrane would lead to alterations in membrane capacitance. It has been suggested that acetylcholine-induced permeability changes are the result of macromolecular perturbations occurring within the activated cell membrane (BLOOM,1970). The experiments performed here were designed to determine whether such structural alterations result in membrane capacitance changes during acetylcholine {ACh) action.
74
B. B. DUNNINGand XENIA MACHNE
additional sources of error in the estimation of membrane capacitance. There was some scatter in the unaveraged data points (at a given frequency) as a result of minor fluctuations in the magnitude of the injected current. Signal averaging over many cycles reduced the scatter to less than 2;;. In the derivation of the alternating current-to-voltage relation for a parallel RC circuit, it is found that if the frequency of the impressed current is much greater than 1/27cRC then an approximation to the actual current-to-voltage relation can be derived which does not involve the resistance R (HAGIWAKA and SAITO, 1959). The error in this approximation due to ignoring resistance changes of the order observed in this preparation during ACh action are less than 1% in the frequency range between 400 and 1000 Hz. A difficulty inherent in our experimental set-up arises from the fact that the topography of somatic ACh receptors is unknown and that presumably the receptors occupy only a small portion of the total membrane area. If any capacitance changes were observed, several assumptions on receptor topography would be required to reach an estimate of the actual change within the activated membrane. RESULTS On the assumption that the cell membrane is equivalent to a parallel RC circuit, the peak to peak amplitude of the potential change across the membrane should be inversely proportional to the frequency of the applied alternating current, provided that the current is maintained constant. The results obtained on the resting membrane of a single neurone are shown in Figure 1 (filled circles). The amplitude of the sine wave voltage developed across the cell membrane was
plotted as a function of the reciprocal frequency of the alternating current. The current intensity was 5 nA for all frequencies. The graph illustrates a straight line relationship for frequencies greater than 150 Hz. The straight line relationship demonstrates that the membrane capacitance is not frequency dependent over the frequency range tested and that our assumption of a parallel RC mode1 of the cell membrane is justified. The total cell capacitance can be calculated from the slope of the straight line using the following equation: C = [(Slope)2z]/1, where I is electric current. In the cell of Figure 1, C was equal to 6 x lO_‘F. By measuring the diameter of the cell and assuming a spherical shape, the specific membrane capacitance, C,. is calculated to be 13 pF/cm’. For 12 cells, C, equalled 10 k 4.7 pF/ cm2. Consistent C, values were obtained within the first hour after impaling a cell. Thereafter. C,,, tended to increase in spite of the fact that no change in resting membrane conductance occurred. It is our impression that this resulted from a slight osmotic imbalance leading to swollen cells having stretched cell membranes. Membrane capacitance was determined during the rising phase or the peak of the ACh-induced potential change using a signal averager to improve the signalto-noise ratio of the potential changes measured. No change from the resting value of C, occurred at these times (See Fig. 1). In addition, the sine wave voltage developed across the cell membrane was observed during the entire ACh response using band-pass filters (465 and 950 Hz) to selectively suppress unwanted signals. At no time during the action of ACh, whether applied iontophoretically or by perfusion. was a change in this voltage observed. Thus. there was no indication of capacitance changes within the limits of our technique. DISCUSSION
Fig. I. Relation between peak amplitude of potential change and reciprocal frequency of applied alternating current for resting membrane (0) and for ACh-activated membrane (0). Measurements made during ACh action were taken during the peak of the response. Current intensity was 5 nA at all frequencies. The solid line is fitted by eye to the data points and the broken line illustrates the slope of the linear portion of the relation. The data points taken during ACh action deviate at low frequency from the points obtained on the resting membrane due to the reduction in cell input resistance during the action of ACh.
The capacitance values found for the resting membrane of Helix asprm neurones are much smaller than those reported for Helix ponmtia (MAISKY, 1963). The discrepancy may be due to the fact that the capacitance values for the latter preparation were obtained by square pulse analysis. The values of C, measured in our preparation are in the same range as those reported for neurones of the puffer fish (HAGIWARA and SAITO, 1959). The fact that we were unable to detect any ACh-induced capacitance changes suggests that no major structural alterations in the lipid component of the membrane are associated with the action of ACh. REFERENCES BLOOM. B. M. (1970). Receptor theories. In: Medicinal Chrmistry. Part I. 3rd Edition (BL’RGLR. A.. Ed.) pp. 108-l 17. Wiley-Interscience, New York.
ACh and membrane capacitance GAGE, P. W. and EISENBERG. R. S. (1969). Capacitance of the surface and transverse tubular membrane of frog sartorius muscle fibers. J. CJUI.Physiol. 53: 265-278. HACIWARA,S. and SAITO,N. (1959). Membrane potential change and membrane current in sup~arneduilar~ nerve cell of puffer. J. ~~,Z~~~~~?~Sj~~. 22: 104-271. MAISKY.V. A. (1963). Electrical characteristics of the surface membrane of giant nerve cells of Nrlir pomrrrir~. Fi-_i#l. z/z. (Musk.) 49: 1468-1474. MA~SUMOIY). N., IN&Y. I. and KISFZIMOTO. U. (1970). The electrical impedance of the squid axon membrane measured between internal and external electrodes. Jup. J. Pizpsio/. 20: 516 536.
75
MIROLLI.M. and TALBOTT,S. R. (1972). The geometrical factors determining the electrical properties of a molluscan neurone. J. Physiol., Lmd. 227: 19-34. PHILBRICKR~SEAKCHES ENGINEERING STAFF(1966). Ap$~~~j~~~~ ~~~}~~~~~ jirr C~777~7if~~77~~ ~~77~~~~~~~s. 115 pp. Philbrick Researches Inc.. Dedham. Mass. TAXASHIPIIA. S. and SCI-IWANN.H. P. (1974). Passive electricaf properties of squid axon membrane. f. ntrrtzl~~i/ii~Bioi. 17: 51 -68. T41 LOR.R. E. ( 1965).Impedance of the squid axon membrane. J. ceil trtlizj~.Plt@clf. 66: :!l-26.
