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Review
, 7 (1), e13861

From Bernstein's Rheotome to Neher-Sakmann's Patch Electrode. The Action Potential

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Review

From Bernstein's Rheotome to Neher-Sakmann's Patch Electrode. The Action Potential

Edward Carmeliet. Physiol Rep.

Abstract

The aim of this review was to provide an overview of the most important stages in the development of cellular electrophysiology. The period covered starts with Bernstein's formulation of the membrane hypothesis and the measurement of the nerve and muscle action potential. Technical innovations make discoveries possible. This was the case with the use of the squid giant axon, allowing the insertion of "large" intracellular electrodes and derivation of transmembrane potentials. Application of the newly developed voltage clamp method for measuring ionic currents, resulted in the formulation of the ionic theory. At the same time transmembrane measurements were made possible in smaller cells by the introduction of the microelectrode. An improvement of this electrode was the next major (r)evolution. The patch electrode made it possible to descend to the molecular level and record single ionic channel activity. The patch technique has been proven to be exceptionally versatile. In its whole-cell configuration it was the solution to measure voltage clamp currents in small cells. See also: https://doi.org/10.14814/phy2.13860 & https://doi.org/10.14814/phy2.13862.

Keywords: Conduction; ionic theory; pacemaker; patch.

Figures

Figure 1
Figure 1
(A) Photograph of Julius Bernstein at the time of his rectorship at the University of Halle (1890). With permission reproduced from (Seyfarth 2006). (B) Drawing of the first action potential in nerve. The negative variation was measured using the rheotome build by Bernstein. With permission reproduced from (Nilius 2003).
Figure 2
Figure 2
(A) Schematic of the Bernstein's rheotome (Seyfarth 2006). (B) Photograph of the real instrument (width 20 cm) build in Heidelberg circa 1870 according to (Nilius 2003). (C) Using very short sampling intervals the rheotome due to its inertia acts as integrator (Hoff and Geddes 1957). Reproduced with permission.
Figure 3
Figure 3
(A) Ernest Overton (1865–1933) known for his contributions on membrane permeability to lipid‐soluble anesthetics and for the study of ionic effects on membrane excitability. (B) title page of the study on the indispensability of sodium‐(or lithium) ions for the muscle contraction (Overton, 1902). Reproduced with permission.
Figure 4
Figure 4
(A) J.Z.Young (1907–1997), professor of Anatomy University College London. Was the enthusiastic promotor of the use of giant axons of the squid and cuttlefish by neurophysiologists. (B) photo of Loligo forbesi. Reproductions from (Schwiening 2012) with permission. (C) L.W. Williams (1875–1912), professor of Anatomy at Harvard Medical School, started studies on the anatomy of the squid but died prematurely in an elevator accident at Harvard in 1912. (D) frontispiece of Williams’ thesis on the anatomy of the common squid. Photograph of Williams reproduced from Kingsley (1913), with permission.
Figure 5
Figure 5
(A) K.S. Cole, physicist by training but performed many experiments with a physiological impact. He shifted definitively in the biological direction when he was promoted Professor of Physiology at the Columbia College of Physicians and Surgeons. Reproduced from (Goldman 1985) with permission. (B) His first experiment using the squid giant axon was to measure the impedance change during the action potential. Reproduced from (Cole and Curtis 1949) with permission. The impedance measurement was not only a scientific success but had also an artistic dimension. The picture was taken by the Biophysics section of the Biophysical Society as their logo. In Sweden it appeared in modern apartments in a slightly modified form with the time axis and the impedance axis exchanged. The result was highly appreciated.
Figure 6
Figure 6
(A) August 1939, edge of the second world war. Measurement of resting and action potential in the squid giant axon with an intracellular electrode. Demonstration of a large overshoot (Hodgkin and Huxley 1939). (B) Postwar 1947 after the damage reconstruction of the Plymouth laboratory. Confirmation of the sodium hypothesis: the action potential amplitude of the squid giant axon changes as expected for a sodium electrode (Hodgkin and Katz 1949). Reproduced with permission.
Figure 7
Figure 7
Left: Authors of the voltage clamp saga, winners of the Nobel Prize at work. The cover of the 1963 Nobel Prize Programme, made available by Deborah Hodgkin to Schwiening. With permission. Right: Photograph of Katz B. by Weidmann Silvio. Courtesy Weidmann.
Figure 8
Figure 8
Ionic theory. (A) Compilation of the essential steps from measurement of currents at different voltages, inward current shown as upward deflection; (B) dissection in Na+ and K+ current, (C) current–voltage relation of the two currents and (D) time evolution of conductance at different voltages, resulting in the formulation of equations and calculation of the action potential (Hodgkin 1958). With permission.
Figure 9
Figure 9
Left: The historical hand‐operated machine, also called “Brains of steel” by the advertisers, which replaced the Cambridge University Computer out of order for 6 months. Photograph taken by Schwiening 2012. With permission. Right: Original voltage clamp currents, recorded by Cole in 1947, published in 1949 (Cole, 1949). Reproduced from (Cole 1979), with permission. Compare with currents of the Cambridge group in Figure 8.
Figure 10
Figure 10
Two Ph.D. students at the Chicago University Judith Graham (1946) and Gilbert Ning Ling (1949) were at the start of the microelectrode saga. Their director at the Department of Physiology was Ralph W. Gerard. The photograph of Graham was granted by the Stanford Medical History Center; photograph of Ling by Wikipedia.
Figure 11
Figure 11
(A) The microelectrode message spread over the world. In Europe Weidmann and Coraboeuf learned the technique during their stay in Cambridge, via A. Hodgkin. Their first recordings were probably too hasty and did not show an overshoot (Coraboeuf and Weidmann 1949a,b). (B) The wrong message was corrected 2 weeks later, showing a strong and beautiful overshoot (Coraboeuf and Weidmann 1949a,b). Reproduced with permission. (C) Photograph of Coraboeuf reproduced with permission from (Escande 1999); (D) photograph of Weidmann, courtesy of Ruth Weidmann.
Figure 12
Figure 12
(A) Impedance changes during the course of the Purkinje action potential. Injection of short hyperpolarizing pulses. Voltage calibration in steps of 10 mV. Duration of the flyback of sweep cycle approximately 12 msec (Weidmann 1951a). (B) Membrane reversal and resting potential as a function of percentage normal sodium (Draper and Weidmann 1951).(C) Relationship between “clamp” potential and maximal rate of rise of action potential. Open circles: Tyrode solution, crosses: 25% normal sodium, full circles after changing back to normal.(Weidmann 1955a). With permission.
Figure 13
Figure 13
(A) Dependence of action potential amplitude on external sodium concentration in frog ventricle. From right to left: 100, 75, 50, and 25% of normal. The slope of the relationship between overshoot and change in external sodium was only 17.3 mV for a tenfold change (Niedergerke and Orkand 1966a). (B) Effect of change in external calcium concentration: from left to right, 0.3, 1, 3, and 5 mmol/L Ca2 + ‐Ringer (Niedergerke and Orkand 1966b). (C) Action potential recorded in calf Purkinje fibers, presence of adrenaline (5.5 × 10−6 mol/L). The sodium current was progressively blocked with increasing doses of TTX. (a): control, (b): 3 × 10−8, (c): 3 × 10−7, (d) and (e): 3 × 10−6 mol/L. (Carmeliet and Vereecke 1969). (D): Action potentials in chloride Tyrode are compared with action potentials in acetylglycinate and nitrate solutions (Carmeliet 1961a). With permission.
Figure 14
Figure 14
(A) Current–voltage relations in 4 and 141 mM external K+ concentration in sheep cardiac Pu fibers. The lines cutting the voltage axis are resistance measurements during the change in potential between the two extremes. Application of long rectangular currents (Hall et al. 1963). (B) Relative membrane slope resistance of sheep cardiac Purkinje fibers as a function of the membrane potential in two external K+ concentrations. Short pulses were superimposed on long polarizing pulses to depolarized and hyperpolarized levels (Carmeliet 1961a). With permission.
Figure 15
Figure 15
(A) Pulsatile K+ concentration changes in the extracellular space measured by K+ sensitive electrodes at different rates of stimulation in frog ventricle (Kline and Morad 1976). (B) Effect of external K+ concentration on the rate of 42K+ efflux in sheep Purkinje fibers (Carmeliet, 1961a). With permission.
Figure 16
Figure 16
(A) The two‐microelectrode voltage clamp. Current is injected through the electrode in the middle of the preparation; membrane potential is measured between an intra‐ and extracellular electrode (Deck et al. 1964). (B) Sucrose gap method (Rougier et al. 1968). Current is injected in the left compartment, passes through the fibers of the preparation in the sucrose gap, through the membrane of the cells in the right compartment and is measured. Membrane potential is recorded between an intracellular and extracellular electrode. Figure 16A and B are reproduced from Fozzard and Beeler (1975), with permission. (C) Tests of voltage stability in time. Dog ventricular trabecula in sucrose gap, single intracellular microelectrode. Potential difference between two internal microelectrodes (middle trace) during the flow of a small inward current (upper trace), excited by a 10 mV depolarizing current from holding potential of −40 mV to inactivate the INa. (D) potential difference between an intracellular and an extracellular electrode (middle trace) during a large rapid inward current (INa upper trace) showing escape; holding potential −80 mV (Beeler and Reuter 1970b). With permission.
Figure 17
Figure 17
Comparison of voltage clamp results in squid giant axon (long and large intracellular current and voltage electrodes) and in dog ventricle (two microelectrodes). (A) Current–voltage relation of the Na+ and K+ currents in the squid giant axon (Hodgkin et al. 1952). (B) Current–voltage relation of the peak Na+ current in normal Tyode (x) and in solution with 31% of normal Na+ concentration (o). Positive (outward) currents were measured at the end of the 500 msec clamp step. Dog ventricle. Sucrose gap and single intracellular microelectrode (Beeler and Reuter 1970b). With permission.
Figure 18
Figure 18
Analysis of the positive dynamic current, Iqr. Effect of 4‐AP and caffeine on transient outward currents elicited in a sheep cardiac Purkinje fiber by depolarizing pulses from a holding potential of −55 mV to −15 mV. (A): Control (a) and 1 mmol/L 4‐AP (b). (B): 10 mmol/L caffeine in the absence (a) and presence (b) 1 mmol/L 4‐AP (b). (Coraboeuf and Carmeliet 1982). Reproduced with permission.
Figure 19
Figure 19
(A) Photograph of a single canine Purkinje cell in the suction‐perfusion pipette. (B): Current response to a voltage clamp depolarization from −150 mV to −40 mV imposed through the suction pipette (Makielski et al. 1987). With permission.
Figure 20
Figure 20
(A) Current–voltage relationships of sodium current obtained using single suction pipette (left) and double suction pipette (right). Holding potential was −80 mV. Same cell in both cases (Brown et al. 1981b). (B) Current traces illustrate separation between capacitive current transients and activation of the Na+ inward current. Voltage and current records using two suction pipettes for voltage clamp of a rat ventricular cell. Notice minimal overlapping of capacitive and current signal (Brown et al. 1981b). With permission.
Figure 21
Figure 21
Photograph of Erwin Neher‐1944 and Bert Sakmann‐1942. Permission to use the photographs was granted by the Max Planck Institute of Biophysical Chemistry. The Nobel Prize in Physiology or Medicine 1991 was awarded jointly to Erwin Neher and Bert Sakmann “for their discoveries concerning the function of single ion channels in cells.” (A) Scanning electron micrograph of a tip‐on view of the pipette opening. The darker ring represents the rim of the pipette. Tip opening diameter is 1.1 μm. The width of the rim is 0.2 μm. (B) side‐on view of the same pipette (Sakmann and Neher 1985). (C) Single channel currents from denervated frog (Rana pipiens) cutaneous pectoris muscle. Two microelectrodes to measure membrane potential; below: patch electrode. The patch pipette contained suberyldicholine 0.2 μmole; membrane potential −120 mV (Neher and Sakmann 1976b). With permission.
Figure 22
Figure 22
(A) Gigaseal formation between pipette and frog muscle sarcolemma. Schematic diagram showing a pipette pressed against the membrane. The seal resistance is between 50 and 100 MΩ. Applying suction resulted in the formation of a gigaseal; part of the membrane is drawn into the pipette. (B) Current record before, during, and after suction. Suction caused the seal to increase from 150 MΩ to 60 gigaΩ. Notice the fall in noise. Short channel openings are caused by the presence of suberyldicholine. (C) A multitude of possible configurations to measure channels and whole‐cell currents (Hamill et al. 1981). With permission.
Figure 23
Figure 23
(A) Study of channels in intracellular organelles in vivo. The patch electrode is introduced via a large microelectrode (sharp), which is withdrawn after the patch electrode has entered the cell (Jonas et al. 1997). (B) Study of bacterial ionic channels using the spheroplast. A sheroplast is a structure which is molded from bacterial plasma membranes via culturing in the presence of antibiotics that inhibit cell wall synthesis. (C) On the way to automation. Planar PDMS (polydimethyl siloxane) patch electrode recording of channel currents. Schematic of planar patch recording system. The 200 μm thick, oxidized PDMS partition is sandwiched between bath (upper) and electrode (lower) chambers. A devitellinized oocyte is dropped onto the aperture (8 μm). Gigaseals are possible. Planar system allows for creating arrays of such setup in parallel. High throughput (Klemic et al. 2002). With permission.
Figure 24
Figure 24
(A) Current–voltage relations for TTX‐sensitive Na+ current from single ventricular myocytes of neonatal rats. Whole‐cell records of currents in 40 mmol/L Na+ outside (see inset) (Kunze et al. 1985). (B) Bovine single ventricular myocytes. Two microelectrode voltage clamp on single cells. The clamp step depolarized the cell from −50 mV to 0 mV. Measurement of L‐type calcium current and effect of adrenaline 0.2 μmole (Isenberg and Klöckner 1982). Permission granted.

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References

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