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The enormous advances made over the last 50 years in materials science, microelectronics, and nanoelectronics, together with the acknowledgment that substrate-integrated planar multielectrode arrays (MEA) are limited to recording of extracellular field potentials (FPs) rather than the entire electrophysiological signaling repertoire of the brain, have prompted a number of laboratories to merge the advantages of planar MEA technologies (non-damaging and durable) with those of the classical sharp and patch electrodes for intracellular recordings. Unlike extracellular planar electrode-based MEAs, the new generation of three-dimensional (3D) vertical nanoelectrodes are designed to functionally penetrate the plasma membrane of cultured cells and operate in a similar manner to classical intracellular microelectrodes. Although only approximately 10 years has elapsed since the development of the first vertical 3D nanostructure-based MEAs, this technology has progressed to enable recordings of attenuated intracellular action potentials (APs) and synaptic potentials from individual neurons, cardiomyocytes, and striated myotubes. Furthermore, recently the scaling advantages of nanochip/microchip fabrication technologies enabled simultaneously intracellular recordings of APs from hundreds of cultured cardiomyocytes, thus heralding a new milestone in MEA technology.In this chapter we present the earliest and today's cutting-edge achievements of this "young vertical nano-sensors MEA technology" at the single-cell and network levels, explain the biophysical principles and the various configurations used to form functional nanoelectrode/cell hybrids, and describe the quality and characteristic features of the recorded intracellular APs and subthreshold synaptic potentials by the vertical nanoelectrode-based MEA. Basic cell-biological mechanisms that curtail the length of time intracellular access by the nanoelectrodes are discussed, and approaches to overcome this problem are offered.Recent development of biotechnologies that use induced human pluripotent stem cells taken from healthy subjects and patients, and in vitro drug screening for the development of personalized medicine as well as basic brain research will benefit tremendously from the use of MEAs that record the entire brain electrophysiological signaling repertoire from individual cells within an operational network rather than only extracellular FPs.

Multielectrode arrays (MEA) are used extensively for basic and applied electrophysiological research of neuronal- and cardiomyocyte-networks. Whereas immense progress has been made in realizing sophisticated MEA platforms of thousands of addressable, high-density, small diameter, low impedance sensors, the quality of the interfaces formed between excitable cells and classical planar sensor has not improved. As a consequence in vitro and in vivo MEA are “blind” to the rich and important “landscape” of sub-threshold synaptic potentials generated by individual neurons. Disregarding this essential fraction of network signaling repertoire has become the standard and almost the “scientific ideology” of MEA users. To overcome the inherent limitations of substrate integrated planar MEA platforms that only record extracellular field potentials, a number of laboratories have developed in vitro MEA for intracellular recordings. Most of these novel devices use vertical nano-rods or nano-wires that penetrate the plasma membrane of cultured cells and record the electrophysiological signaling in a manner similar to sharp intracellular microelectrodes. In parallel, our laboratory began to develop a bioinspired approach in-which cell biological energy resources are harnessed to self-force a cell into intimate contact with extracellular gold mushroom-shaped microelectrodes to record attenuated synaptic- and action-potentials with characteristic features of intracellular recordings. Here we describe some of the experiments that helped evolve the approach and elaborate on the biophysical principles that make it possible to record intracellular potentials by an array of extracellular electrode. We illustrate the qualities and limitations of the method and discuss prospects for further improvement of this technology.

On May 10th 2010 Platon Grigorevitch Kostyuk sadly left us at the age of 85. He was a talented scientist, a brilliant experimenter, an outstanding organizer of science and an excellent teacher. Platon Kostyuk was born in 1924 in Kiev, Ukraine. He obtained a double education: a graduate of the Kiev University Department of Biology in 1946 and the Kiev Medical Institute in 1949, he became a pioneer in neuroscience, the first in the Soviet Union to use microelectrodes for intracellular recording of electrical signals in neurons. Despite the difficulties for international travel for those living behind the Iron Curtain, he was able to present his work at the International Congress of Physiology in Buenos Aires in 1959 and here met Prof. John Eccles who invited him to work at the University of Canberra in Australia in 1960–1961. This was the start of an outstanding international career, complementing his creative achievements in the Soviet Union. In 1966 P.G. Kostyuk became director of the Bogomoletz Institute of Physiology in Kiev, which he headed for nearly 45 years. Under his direction this Institute became a leading centre for neuroscience, renowned not only in the Soviet Union but also internationally. New directions of research were developed in cell physiology, molecular biophysics and neurophysiology. Several important discoveries were made including the development of a method for intracellular perfusion, evidence for a calcium-dependent conductance in nerve cells and the discovery of new types of ion channels. Elected to the Ukraine Academy of Science in 1969 and Grand Academician of the Soviet Academy of Science in 1974, Kostyuk has also been honoured by many international societies. He is the author of more than 650 articles, 17 monographs and 7 discoveries and was the creator and editor of two scientific journals: “Neurophysiology” and “Neuroscience”. The outstanding career and multifaceted activities of Academician Platon Kostyuk form a pyramid of achievement, at the top of which ride his research in cognition and his enthusiasm for the discovery of the secrets of Nature.

Non-technical summary What is known about gastric electrophysiology and used in motility clinics throughout the world is mostly deduced from animal studies and extracellular recordings from human patients. Extracellular recording from gastrointestinal muscles, however, is prone to extensive motion artifact, and it is not clear that animal models can be translated directly to human physiology. Therefore, we have performed a detailed analysis of electrical activity from carefully mapped specimens of gastric muscle removed from humans during surgery for gastric cancers. Our data show several important differences in electrical activity recorded with intracellular microelectrodes and accepted gastric electrophysiological dogma. We observed ongoing electrical slow wave activity in the gastric fundus; we also found no evidence for a slow wave frequency gradient. Muscles from all regions through the thickness of the muscularis demonstrated intrinsic pacemaker activity, and this corresponded with the widespread distribution of pacemaker cells.

Amoeba proteus may drive a current through themselves, and this current may determine the direction of cytoplasmic streaming. Cytoplasm streams from the more contracted tail or uroid region to the extending pseudopods. The positions of the uroid and pseudopods change as the amoeba changes direction. The membrane potential of the amoeba may change with location in the cell. This voltage gradient may regulate the magnitude and direction of cytoplasmic streaming. Bingley and Thompson (1962), recorded from amoebae using intracellular microelectrodes and reported that the membrane potential of the uroid region was more negative than that of the pseudopods. In contrast, Nuccitelli et al. (1977), using a vibrating probe, observed that current entered the uroid region and exited through the pseudopods. They concluded that the uroid region was more depolarized than the pseudopods. We revisited this issue by recording membrane potential simultaneously from two regions of the amoeba and by recording ionic currents using the two-electrode voltage clamp. Our data support the findings of Nuccitelli et al. Cytoplasm may stream from more depolarized to more hyperpolarized regions. Moreover, overshooting depolarizations of the cell may produce global cytoplasmic contractions and rounding of the amoeba.

Although KATP channels have been proposed as playing a role in most types of myocardial damage associated with ischemia/reperfusion, the potential benefits of KATP channel modulators against the biochemical and electrical disturbances observed during ischemia remain unclear. We have thus studied the effects of glibenclamide and cromakalim, KATP channel blocker and opener respectively, on membrane lipid injury and arrhythmias, in a model of ischemic-reperfused guinea-pig myocardium. Ventricular strips were prelabeled with [3H] arachidonic acid, then subjected to normal conditions (Time-related Control) or to simulated ischemic-reperfused conditions in absence of drug (Control) or in presence of glibenclamide 10 microM or cromakalim 10 microM. The release of radioactive compounds was counted by liquid scintillation spectrometry, while action potentials (AP) were recorded with intracellular microelectrodes. Reperfusion induced a significant increase of arachidonic acid release (P0.05 versus Time-related Control). Glibenclamide inhibited the reperfusion-induced arachidonic acid release while cromakalim only delayed it (respectively 483 +/- 87 dpm/g, P0.05 and 790 +/- 143 dpm/g. NS versus 838 +/- 80 dpm/g for Control, after 30 min of reperfusion). Unlike glibenclamide, cromakalim was proarrhythmic during reperfusion (in 100% of preparations versus 33% in Control or in presence of glibenclamide, P0.05). This in vitro study shows that glibenclamide prevented the reperfusion-induced membrane arachidonic acid release, without proarrhythmic effect, whereas cromakalim, associated with proarrhythmicity, was unable to protect myocardium from cell lipid damage.

At present, the prime methodology for studying neuronal circuit-connectivity, physiology and pathology under in vitro or in vivo conditions is by using substrate-integrated microelectrode arrays. Although this methodology permits simultaneous, cell-non-invasive, long-term recordings of extracellular field potentials generated by action potentials, it is 'blind' to subthreshold synaptic potentials generated by single cells. On the other hand, intracellular recordings of the full electrophysiological repertoire (subthreshold synaptic potentials, membrane oscillations and action potentials) are, at present, obtained only by sharp or patch microelectrodes. These, however, are limited to single cells at a time and for short durations. Recently a number of laboratories began to merge the advantages of extracellular microelectrode arrays and intracellular microelectrodes. This Review describes the novel approaches, identifying their strengths and limitations from the point of view of the end users--with the intention to help steer the bioengineering efforts towards the needs of brain-circuit research.

Cardiological research greatly rely on the use of cultured primary cardiomyocytes (CMs). The prime methodology to assess CM network electrophysiology is based on the use of extracellular recordings by substrate-integrated planar Micro-Electrode Arrays (MEAs). Whereas this methodology permits simultaneous, long-term monitoring of the CM electrical activity, it limits the information to extracellular field potentials (FPs). The alternative method of intracellular action potentials (APs) recordings by sharp- or patch-microelectrodes is limited to a single cell at a time. Here, we began to merge the advantages of planar MEA and intracellular microelectrodes. To that end we cultured rat CM on micrometer size protruding gold mushroom-shaped microelectrode (gMμEs) arrays. Cultured CMs engulf the gMμE permitting FPs recordings from individual cells. Local electroporation of a CM converts the extracellular recording configuration to attenuated intracellular APs with shape and duration similar to those recorded intracellularly. The procedure enables to simultaneously record APs from an unlimited number of CMs. The electroporated membrane spontaneously recovers. This allows for repeated recordings from the same CM a number of times (>8) for over 10 days. The further development of CM-gMμE configuration opens up new venues for basic and applied biomedical research.

The cellular mechanisms of cocaine-induced arrhythmias are not clear. Production of early afterdepolarizations (EADs) in single myocytes have been proposed as a possible mechanism of cocaine arrhythmogenesis. The objective of this investigation was to determine whether cocaine exerts arrhythmogenic or antiarrhythmic actions related to the production or inhibition of afterdepolarizations in multicellular preparations. Rat and guinea pig papillary muscles superfused in vitro with Tyrode's solution at 37°C were stimulated at 1 Hz. Standard intracellular microelectrodes were used to record membrane potentials. Cocaine administered at 2.9–58 μM never induced EADs; also, overdrive in the presence of cocaine never induced delayed afterdepolarizations (DADs). On the other hand, cocaine administered at 58 μM suppressed DADs and triggered activity induced by overdrive in the presence of ouabain octahydrate and isoproterenol. Thus, cocaine acting on multicellular ventricular preparations in vitro did not induce EADs or DADs. On the contrary, cocaine inhibited DADs and triggered activity induced by other drugs.

We used intracellular microelectrodes to study the effects of histamine on both normal and abnormal automaticity in sheep Purkinje fibers. Histamine, dimaprit and 4-methylhistamine caused a similar reduction of the action potential duration in driven Purkinje fibers. Histamine (10-6 M) induced spontaneous activity in p previously quiescent preparations more often than did equimolar concentrations of dimaprit and 4-methylhistamine. The effects of histamine on automaticity were enhanced in the presence of barium In fact histamine, at concentrations which were unable to induce automaticity in normal preparations, induced it in the presence of barium. In Purkinje fibers manifesting barium-induced automatic activity, histamine (10-7--10-6M) significantly increased the average number of spontaneous action potentials and shortened the time of their appearance. In the same range of concentrations, histamine dose-dependently increased the iological manifestation of calcium overload. Histamine (10-6--10-4M) increased the OAP amplitude of strophanthidin -treated Purkinje fibers, eventually inducing triggered extrabeats. All these previously described effects were selectively blocked by cimetidine (10-5 M). It is concluded that histamine may induce cardiac arrhythmias under conditions of calcium overload and that this effect may be due to induction or enhancement of oscillatory afterpotentials.

We studied the actions and interactions of ethanol and imipramine on the sinus node. Strips of the right rat atrium including the sinus node were superfused with Tyrode's solution at 37°C while beating spontaneously. The preparations were exposed to imipramine or ethanol alone as well as to the two drugs in combination while recording membrane potentials with standard intracellular microelectrodes. The results obtained show that ethanol 0.8 and 2.4 g/l exerted a positive chronotropic action. On the other hand, imipramine 0.25 mg/l did not modify the sinus node rate. However, it reduced significantly the positive chronotropic action of ethanol. The sinus node rate decreased under the action of a higher concentration of imipramine (1 mg/l). When ethanol was tested in combination with this concentration of imipramine, the effect of the latter prevailed. In conclusion, a concentration of imipramine that did not affect the sinus node rate antagonized the positive chronotropic action of ethanol. In addition, the negative chronotropic action of a higher concentration of imipramine prevailed over the positive action of ethanol. The results obtained provide additional support to the notion that the use of ethanol and cardioactive drugs in combination may result in significant changes in the actions of either of the two, or both. This is of clinical relevance, since at least some of the individuals under treatment with cardioactive drugs will be alcoholics and/or social drinkers.

1. Intracellular microelectrodes were used to identify three major electrophysiological categories of neurone in both the internal and external submucous plexuses of the porcine small intestine. 2. Two classes of neurone with a long-lasting after-hyperpolarization following their action potential were differentiated by the presence or absence of fast excitatory synaptic inputs (EPSPs) and were termed AH neurones. S neurones received fast EPSPs but did not display after-hyperpolarizations. 3. The mean resting membrane potentials of the three groups of neurones showed a similar trend in both plexuses, with significantly higher values for the two populations of AH neurone than for S neurones. No significant variation of input resistance with cell type was detected. Neuronal input resistance was significantly greater in the internal submucous plexus than in the external submucous plexus. 4. Over 80% of AH neurones in the internal submucous plexus displayed fast EPSPs but a similar percentage of AH neurones in the external submucous plexus did not show fast EPSPs. S neurones constituted 60% of cells studied in the internal submucous plexus but less than 30% of the cell population in the external submucous plexus. 5. This study of porcine submucous neurones has revealed both similarities and differences to previous work in the guinea-pig small intestine. The most contrasting features are the relative abundance and subclassification of AH neurones in the pig in addition to the apparent paucity of slow synaptic potentials. The differences in the neuronal profiles of the internal and external submucous plexuses may reflect a differentiation of function between the two enteric nerve networks.

The physicochemical characteristics of the terbium-adriamycin complex (terbomycin) were studied. Perturbations in the visible absorption spectrum of adriamycin by terbium (Tb3+) was indicative of formation of the terbomycin complex. The absorption maximum of free adriamycin at 479 nm shifted towards the absorption maximum of terbomycin at 539 nm. The binding of Tb3+ to adriamycin was negligible at acidic pH. At alkaline pH, the affinity of Tb3+ for adriamycin increased. The stoichiometry of binding was estimated to be 0.5; one Tb3+ ion per two adriamycin molecules. Thermodynamic analysis revealed that the spontaneous formation of terbomycin was due to an increase in the entropy of the system. The effects of adriamycin, Tb3+ and terbomycin on sinus node automaticity were studied using sinus node from rats, superfused with modified mammalian Tris-Tyrode's solution (37 degrees C). The sinus node rate was monitored with intracellular microelectrodes. 25 microM Tb3+ increased the sinus node rate. Adriamycin (50 microM) depressed sinus node automaticity. Terbomycin also reduced the sinus node rate. There was no difference between the effects of adriamycin and terbomycin. The chronotropic effect of terbomycin persisted in the presence of atropine.

The effects of lindane, a gamma-isomer of hexachlorocyclohexane, were studied on transmembrane potentials and currents of frog atrial heart muscle using intracellular microelectrodes and the whole cell voltage-clamp technique. Lindane (0.34 microM to 6.8 microM) dose-dependently shortened the action potential duration (APD). Under voltage-clamp conditions, lindane (1.7 microM) increased the amplitude of the outward current (Iout) which developed in Ringer solution containing TTX (0.6 microM), Cd2+ (1 mM) and TEA (10 mM). The lindane-increased Iout was not sensitive to Sr2+ (5 mM). It was blocked by subsequent addition of quinidine (0.5 mM) or E-4031 (1 microM). E-4031 lengthened the APD; it prevented or blocked the lindane-induced APD shortening. In conclusion, our data revealed that lindane increased the quinidine and E-4031-sensitive rapid delayed outward K+ current which contributed to the AP repolarization in frog atrial muscle.

1. Bovine ventricular myocytes isolated according to isenberg and Klockner (1982a) were superfused with Tyrode solution ([Ca]0-3.6 mM, 35°C) and investigated with intracellular microelectrodes. The properties of the action potential (AP) were compared to those published for multicellular ventricular tissue. 2. At 1 Hz stimulation, the AP had 4 clearly discernible phases with a trapezoidal configuration and a duration of 276 ±38 ms. At [Ca]0=1.8 mM the 4 phases were less clear. Following a 1 min rest period, 1 Hz stimulation shortened the AP by 28% ([Ca]0=3.6 mM) or by 12% ([Ca]0=1.8 mM). 3. The AP started with a sodium spike which rose with\(\dot V_{\max }\) of 250±60 V/s to an overshoot of +40±5 mV. The sodium spike was blocked by 60 μM TTX.\(\dot V_{\max }\) was modified with 2s conditioning pre-pulses; the resultingh∞ curve showed half maximal inactivation atV0.5=−65 mV and a slope factork=5.5 mV. Reduction of [Ca]0 to 1.8 mM shiftedh∞ by −5 mV but did not modifyk. However, large shifts inh∞ curves were measured in myocytes depolarized by 20 mM or 0 mM [K]0. 4. Slow AP's were studied when the sodium spike was inactivated by high [K]0 or blocked by TTX. The slow AP's rose with\(\dot V_{\max }\) of 11±5 V/s to a maximum plateau potential of 36±4 mV;\(\dot V_{\max }\) increased up to 24 V/s when the latency was shortened by increased stimulus strength.\(\dot V_{\max }\), plateau and duration were increased by 0.1 μM adrenaline, reduced by 1.8 mM [Ca]0 and blocked by Ni, Co or D600. 5. Changes in [K]0 modified the resting potential (Vm) as discribed in multicellular ventricular tissue.Vmax was −84 ±4 mV at [K]0=5.4 mM and −54±3 mV at [K]0=20 mM; the latter value suggests [K]i=152±8 mM. The increase in [K]0 abolished the sodium spike and shortened the AP, but did not change the maximum plateau potential. In voltage clamp experiments the i-v relation of the repolarizing late outward currents was N-shaped. The sensitivity of these currents to [Cs]0 and [K]0 indicated that the inward rectifying potassium conductance declines with [K]0. 6. The experimental interventions used in the present study changed the AP of the isolated myocyte in a way similar to that reported for multicellular ventricular tissue, i.e. no electrophysiological peculiarity was observed. We conclude that the enzymatic isolation procedure did not significantly alter the properties of the Na-, Ca- or K-channels.

1. Spike propagation across the nonhomogeneous section of the giant axon in ganglion T3 of the cockroach was analyzed by intracellular microelectrodes recording at the posterior and anterior ends of T3. Ascending and descending potentials were evoked by stimulation of A5-A6 and T2-T3 connectives. 2. At high frequencies, descending and ascending impulses exhibit the following: a) consecutive reduction in the spike amplitude, b) a decrease in the afterhyperpolarization; c) gradual appearance of a prepotential together with an increase in delay of spike initiation; d) failure of full spike invasion into the recording area, showing only a decremental potential. 3. The duration of a train required to block spike propagation when the whole connective is stimulated is much shorter (about 6 times) than that required when a single giant axon is stimulated. 4. The conduction block is associated with a marked decrease in effective membrane resistance, greater than that expected from depolarization and delayed rectification. 5. Synaptic potentials could be recorded in the giant axons in the caudal base of ganglion T3 after stimulation of either the ipsilateral or contralateral connectives at both ends of the ganglion. These synaptic potentials could be blocked by d-tubocurarine (d-TC) or low Ca2+-high Mg2+. 6. Activation of these synapses produces a marked increase in membrane conductance, blocking propagation of spike trains through the ganglion. 7. After these synapses are blocked by d-TC or low Ca2+-high Mg2+, high-frequency stimulation still produces a conduction block. 8. It seems that conduction of spike during repetitive stimulation is affected both by accumulation of extracellular potassium, which depolarizes the membrane and causes sodium inactivation, and by activation of synaptic inputs to shunt the membrane in this region. 8. Each of these two mechanisms by itself can produce conduction block along the giant axons in ganglion T3.

The electrophysiological properties of reabsorptive sweat duct (RSD) cells from normal and cystic fibrosis (CF) subjects were studied using intracellular microelectrodes. The apical membrane potential (Va) of CF duct cells was reversed in "polarity" (+28.0 +/- 2.4 mV, n = 46) compared with normal duct cells (-24.9 +/- 0.4 mV, n = 145), and the basolateral membrane potential (Vb) of CF cells was hyperpolarized significantly (-50.1 +/- 1.2 mV, n = 46) in comparison to normal cells (-34.6 +/- 0.4 mV, n = 145). The substitution of the impermeant anion gluconate for Cl- in the lumen of the normal duct depolarized Va from -24.9 +/- 1.1 to 8.9 +/- 3.1 mV (n = 18) and hyperpolarized Vb from -34.3 +/- 1.1 to -55.6 +/- 3.7 mV (n = 18), which mimicked the cell electrical potential profile of CF ducts even in the presence of Cl-. Cl- substitution in the bath depolarized Vb of normal ducts by 22.5 +/- 2.6 mV (n = 24), while hyperpolarizing Va by -3.4 +/- 1.6 mV (n = 24). The response of the electrical profiles of CF cells to Cl- substitution in either the lumen or the bath was significantly reduced compared with normal cells. The effect of the Na+ conductance blocker amiloride (10(-4) M) on Vb was not significantly different in CF (delta Vb = -26.4 +/- 3.8 mV, n = 9) vs. normal (delta Vb = -27.6 +/- 2.5 mV, n = 30) cells.(ABSTRACT TRUNCATED AT 250 WORDS)

In the following, an attempt will be made to discuss the role of the K+ conductive pathway in the nephron. The aim of Table 1 is to give a systematic summary of what is known about K+-conductive pathways along the nephron. This table provides references to pertinent experimental work. In addition, this table clearly documents that a K+ conductive pathway is present in most, if not all cells of the different nephron segments; its functional role, however, may be quite different. Rather than giving a systematic overview, the present text seeks to provide new insights into the functional role of the K+-conductive pathways in several segments of the nephron. After an introduction into the two most important methods to examine the conductive properties of the nephron, namely the use of intracellular microelectrodes and the patch clamp technique, we shall focus on three issues: 1) the need for K+ recycling across the basolateral membrane in proximal tubule segments; 2) the specific role of the apical K+-conductance in the diluting segment as a means to recycle K+; and 3) the role of the apical K+-conductance in the collecting tubule for net K+ secretion.

Electrical properties of the egg membrane of Drosophila melanogaster were examined using intracellular microelectrodes. Unfertilized eggs and fertilized eggs for the period up to the syncytial blastoderm stage were used. Among Na, K, and Cl, K was most permeant to the membrane. The K permeability, however, did not completely determine the membrane potential. The resting potential in standard solution was −63.5 ± 8.0 mV (mean ± SD) in unfertilized eggs collected within 2–3 days after virgin flies started to lay eggs. The resting potential in fertilized eggs was −27.0 ± 8.4 mV within 20 min after egg deposition, while it was −55.1 ± 6.5 mV at the syncytial blastoderm stage. These changes at different developmental stages were associated with changes in the K-dependence of the membrane. The larger amplitude of the resting potential was suggested to be due to increased K permeability but not to decreased nonspecific leakage. The current-voltage relation was linear throughout the stages examined. Action potentials, such as those in eggs of other animals, were not observed. High Ca media significantly increased the amplitude of the resting potential associated with increase in the membrane resistance. A remarkable nonlineality in the I–V relation appeared in high Ca media, which caused continuously increasing hyperpolarization during sustained inward current. Eggs of temperature-sensitive mutants, shi ts 1 and para ts 1 showed properties similar to those in wild-type eggs with transient temperature changes.

The electrical membrane properties of the marine (brackish-water) ciliate Paramecium calkinsi were investigated under constant-current and voltage-clamp conditions, using two intracellular microelectrodes. The action potential and membrane currents were extremely tolerant to changes in the salinity of the bathing medium. Current-voltage relationships exhibited a moderate inward-going rectification of the membrane upon hyperpolarization, and a prominent outward-going rectification upon depolarization of the membrane. Ion substitution experiments showed that the electrically excitable response is a graded Ca2+-action potential, similar to that found in freshwater ciliates.

1. The relative spectral sensitivities of identified large second order neurons (L neurons) of the dragonfly ocellus have been examined with dye filled intracellular microelectrodes placed in the brain. 2. Representative L neurons of the four geometric classes present in the median ocellar nerve show essentially similar waveforms in response to white, green and UV light. 3. Details of the retinal projections of these four neuron classes have been established. 4. L neurons exhibit a marked reverse Purkinje shift at low light intensities. Green sensitivity (485 nm) decreased relative to UV sensitivity (360 nm) as the intensity of illumination increased. At a mean intensity of 1.78 × 1012 photons cm−2 s−1 the L neurons are equally sensitive to UV and green light. 5. The reverse Purkinje shift is discussed in relation to data previously obtained for dragonfly receptor cell spectral sensitivity and in relation to its possible functional significance.

The construction and use of tissue holders that allow simultaneous long-term recordings of mechanical and intracellular electrical activity from a variety of smooth-muscle preparations is described. The tissue holders permit adjustment of tissue stretch following its fixation for recording purposes. By this means intracellular microelectrodes can be kept in position despite marked changes in the mechanical activity of the tissue. Since simultaneous recordings of electrical and mechanical activity of up to 2 hrs duration can be made, studies of the relationships between the membrane potential changes and mechanical changes evoked by drugs and neurotransmitters are greatly facilitated.

The influence of light in a spectrum range of 350–500 nm 20 W m-2 (20,000 erg · cm-2 · s-1) has been studied in the mycelial cells of Neurospora crassa. Light-induced input resistance and membrane potential changes can be measured by means of intracellular microelectrodes. The value of the input resistance reached maximum after a 2–5 min illumination. The maximum hyperpolarization of the cell membrane reaching 30–40 mV was observed after 20–25 min illumination, when the input resistance values did not differ significantly in the illuminated and non-illuminated cells.

Isolated sartorius nerve-muscle preparations from Rana temporaria were irrigated in a chamber. Resting muscle membrane potentials and evoked end-plate potentials were recorded via intracellular microelectrodes. Multiple end-plate potentials appeared after the preparation had been exposed to a suspension, in Ringer's solution, of the toxic synthetic pyrethroid: 5-benzyl-3-furylmethyl (+)-cis-chrysanthemate (NRDC 119). It is thought that this effect was due to repetitive activity arising in the motor nerve fibers. The pyrethroid had no effect on the muscle resting potential nor on the amplitude or time course of the initial evoked end-plate potential.

Light-initiated two component receptor potentials and the transmembrane currents which generate them were recorded from ventral nerve photoreceptors ofLimulus polyphemus using intracellular microelectrodes. The magnitude and temporal separation of the two components could be manipulated by using pairs of light pulses of variable duration, repetition rate and intensity; the optimal values of these parameters for component separation varied among preparations, but two components could be obtained fromall ventral nerve photoreceptors tested.

The electrophysiological properties of subendocardial Purkinje fibers surviving in myocardial infarcts were studied with intracellular microelectrodes in isolated superfused preparations and correlated with subsequent light and electron microscopic studies. Transmembrane action potentials could always be recorded from one or two cell layers of subendocardial Purkinje fibers in infarcted regions 3 days to 7 weeks after coronary artery occlusion; ventricular muscle action potentials were rarely found. Microscopic studies also demonstrated several layers of intact subendocardial Purkinje fibers; the subjacent ventricular muscle cells were irreversibly injured and replaced by scar. At all time intervals, surviving Purkinje fibers had significantly reduced maximum diastolic potentials, action potential amplitudes, and depolarization velocities as well as prolonged action potential durations. These parameters normalized between 24 hours and 7 weeks after coronary artery occlusion. Surviving Purkinje fibers with electrophysiological abnormalities at 24 hours and 3 days contained vast lipid deposits. Lipid was less prevalent at 10 days when action potential characteristics had improved. By 7 weeks, action potentials were nearly normal and lipid was absent. Subendocardial Purkinje fibers surviving in infarcts are subject to conditions which cause electrophysiological and ultrastructural abnormalities. Persistent abnormalities in the electrophysiological properties of these surviving Purkinje fibers may cause persistent altered electrophysiological properties of the infarcted heart.

1. 1. The electrical properties of the neurosecretory cells lying on the link nerve in Carausius morosus have been examined using intracellular microelectrodes. 2. 2. Intrasomatic recordings, confirmed by injection of Procion Yellow, show that the cell bodies are electrically excitable giving overshooting action potentials of up to 70 mV in height. 3. 3. The action potentials have a positive overshoot of 10–20 mV and an undershoot of 8 mV. Action potential duration is long, being 7–20 msec at 50% spike height. 4. 4. The passive electrical properties of the membrane have also been examined.

Though the ultrafine short tips provided by our micropipette puller are helpful for many preparations, longer tips are still needed for some cases where target cells for intracellular work are deeply embedded in tissue. Because the original 'loop' type of heating filament proved unsuitable for widening to provide the longer tips, we studied simpler forms of heating filaments. By comparison with the common loop design, a rectangular trough filament proved to have many advantages without any significant disadvantage. In particular, this type of filament may be used at varying widths to provide long tips as well as short ones. Thus our micropipette puller can now provide tip lengths that extend upward by continuous gradations from about 6 to 27 mm, while maintaining tip diameter in the ultrafine range of 0.1 micrometer or less. These long tips have proved more reliable and free from bending than long tips traditionally provided by the Livingston puller. By using narrow filaments in conjunction with other parameters that influence tip size, tips have also formed with diameters up to 1.5 micrometers and lengths of only 2-3 mm.

The propagation process of the atrial excitation was investigated during ectopic atrial rhythms evoked by electrical pacing through stimulation at several sites in 35 isolated rabbit atria using intracellular microelectrodes. Excitation sequences was correlated with the atrial deflections in a distant bipolar electrogram representing a cranio-caudal atrial activation. The conduction velocity was faster along the crista terminalis but slower across the coronary sinus ostium than in the ordinary atrial muscle. These differences in conduction velocity resulted in the upward artial deflection on the distant bipolar electrogram in the rhythm which was produced by stimulation at the right side of the lower interatrial septum. On a function as a preferential conduction pathway in the case of left to right direction of excitation, as in left atrial rhythm. On stimulation of the postero-inferior region of the left atrium, the region around the coronary sinus ostium was the first area in the right atrium to be activated. Inversely, the impulse originating from the coronary sinus ostium wasconducted relatively fast to the postero-inferior region of the left atrium. These findings suggested that there might be a preferential conduction pathway along the coronary sinus vein or its neighbouring tissues.

Right atria were excised from the hearts of 20 young dogs (15 +/- 5 weeks old) and maintained in vitro in order to perform microelectrode impalements. The atria contracted spontaneously for at least 6 hours at a stable rate of 127/min when they were perfused with Krebs-Ringer bicarbonate solution through the sinus node artery. Cells within a small area near the sinus node artery and between the superior vena cava and right atrium consistently yielded transmembrane potentials typical of pacemaker cells. This area was verified histologically to be the midportion of the sinus node. Electrical characteristics of these sinus node pacemaker cells were: maximum diastolic potential, -56 +/- 7 mV (mean +/- 1 SD); action potential amplitude, 56 +/- 8 mV; overshoot, 0 +/- 2 mV. Smooth transitions from phase 4 to phase 0 were always present. From these experiments we conclude that the excised perfused canine right atrium provides a useful new experimental cardiac preparation because it maintains stable spontaneous activity for many hours in vitro and the sinus node within it is readily accessible for study by intracellular microelectrodes.

1. E.p.s.p.s and terminal potentials (t.p.s) produced by the action of single medial gastrocnemius Ia afferent fibres were examined with intracellular microelectrodes in cat triceps surae motoneurones. 2. Simple terminal potentials (t.p.s) appeared as positive-negative diphasic waves with a single positive peak. Shape indices of excitatory post-synaptic potentials (e.p.s.p.s) recorded in conjunction with simple t.p.s indicate that these e.p.s.p.s were generated at an electrotonically confined compartment on the motoneurone somadendritic membrane. 3. Compound t.p.s. were similar ones, except that they had two or more positive peaks. Shape indices of e.p.s.p.s recorded in conjunction with compound t.p.s indicate that these e.p.s.p.s were generated at two or more electrotonically separate compartments. 4. E.p.s.p. latency and post-synaptic electrotonic delay were determined for a group of single fibre e.p.s.p.s with simple t.p.s. The shortest recorded latency was 0.26 msec. By subtracting estimated electrotonic delay, the shortest synaptic delay obtained for a medial gastrocnemius Ia single fibre e.p.s.p. was 0.17 msec. 5. The finding of a significant synaptic delay supports the hypothesis that the Ia-motoneurone synapse is a chemically mediated synapse.

Individual murine spinal cord neurons were grown in dissociated tissue cultures. Using either one or two conventional intracellular microelectrodes neurons were voltage-clamped and current-voltage (I–V) curves were constructed using command pulses.l-Aspartic acid was then applied and I–V curves repeated in the presence of this excitatory amino acid.l-Aspartic acid induced a region of negative slope conductance (RNSC) in the steady-state I–V relationships of these neurons. Such a RNSC accounts for the apparent reduction of conductance observed in response to excitatory amino acids5,7,8 and its instability likely contributes to the electrogenesis of bursting in central neurons.

(1) Depolarizing after-potentials (DAPs) of hippocampal pyramidal cells were studied with intracellular microelectrodes in rabbits anesthetized with Nembutal and, in addition, curarized. (2) DAPs were classified into two types. One was linearly related in magnitude to the spike and the membrane potential of the cell body while the other was not. Therefore, the former was tentatively called the true DAP and the latter the pseudo-DAP. (3) In accordance with reduction in the membrane potential, the magnitude of the true DAP diminished and at a membrane potential of −60 to −50 mV, the true DAP disappeared. On further reduction in the membrane potential, a hyperpolarization-going dip appeared in place of the true DAP. (4) The pseudo-DAP usually appeared when the membrane potential of the cell body was reduced beyond −60 to −50 mV. But the pseudo-DAP was not quantitatively related to the membrane potential. It was shown that the pseudo-DAP was a nonsynaptic process, and its possible relation to the dendritic spike was discussed. (5) It was demonstrated that it was the pseudo-DAP that led to the genesis of the so-called inactivation response,i.e. a large and prolonged depolarization of the cell membrane with a strong tendency to spike inactivation.

The action of veratridine on the resting membrane potential of intact crayfish axons in the presence of different extracellular alkaline-earth cations has been studied using intracellular microelectrodes. At extracellular pH values of 7, 6 and 5, the apparent binding sequence was consistently Ca2+ ⩾Sr2+ >Mg2+ ≈Ba2+. This particular selectivity sequence and the insensitivity of binding order to changes in extracellular pH have been noted for other biological preparations and provide structural information about the veratridine - interaction sites to which the alkaline earths bind.

Experiments were performed on the hearts of open-chest anesthetized dogs and on isolated rabbit hearts. Intracellular microelectrodes and bipolar surface electrodes were used for recording from and for stimulating the atria, AV node, bundle of His, bundle branches and ventricles. Atrial or ventricular premature beats which were usually blocked or delayed within the AV node were facilitated when ectopic activity caused preexcitation and early recovery of the site of block or delay. Ectopic atrial, ventricular, nodal, and bundle of His responses allowed previously blocked or delayed antegrade and retrograde beats to be conducted only when the ectopic activity arrived at an appropriate time at the site of block within the AV node. Therefore, apparent supernormal AV conduction may occur in the human heart by this mechanism, and some reported cases of supernormal conduction may be due to the preexcitation and early recovery of a refractory barrier by ectopic activity.

The hearts of 7–12 week old human embryos were investigated by means of intracellular microelectrodes. Average heart rate was 91 beats per minute. The cardiac pacemaker was localized in the sinus venosus. The mean amplitude of action potentials amounted in the pacemaker fibres to 58 mV, in the atrium to 90 mV and in the ventricles to 110 mV. The isolated left auricle exhibits spontaneous activity. The atria are electrically isolated from the ventricles and excitation is conducted by a preferential pathway, most probably by the atrio-ventricular node and bundle.

Electrocardiograms, ventricular monophasic action potentials registered with suction and intracellular microelectrodes, and pressure curves were recorded simultaneously in isolated rabbit hearts perfused with oxygenated Krebs-Henscleit solution containing varying calcium/magnesium ratios. The duration of the action potential and Q-T interval was not affected by low and high magnesium concentrations unless calcium was omitted or significantly reduced. When Mg was completely absent, solutions free of calcium or with one-sixteenth of the "normal" Ca concentration caused a progressive prolongation of the action potential and Q-T interval until they occupied the entire cardiac cycle. When Mg concentration was low (0.075 to 0.6 mM/L.), the prolongation occurred at a slower rate and was less pronounced. With higher Mg concentrations (1.2 to 19.2 mM/L.), the initial lengthening of the action potential and Q-T interval was followed by a gradual shortening. The higher the Mg concentration employed, the shorter was the period of the initial lengthening, and the more pronounced was the subsequent shortening. These observations can be best explained by assuming that calcium-like action of Mg on the plateau of the action potential becomes apparent only when Ca is absent or significantly decreased. The depression of contractility by calcium deficiency proceeded at the same rate at all studied Mg concentrations. The findings are discussed in relation to the electrocardiographic patterns encountered in clinical cal cium and magnesium deficiency.

The mode of action of tetrodotoxin on the frog muscle fiber membrane has been analyzed with the aid of intracellular microelectrodes. Tetrodotoxin of 10–7 concentration made the applied cathodal current ineffective in producing action potential, whereas the resting potential and resting membrane resistance underwent little or no change. With 10–8 tetrodotoxin the muscle fibers responded with the small action potentials at high critical depolarizations. These results can be explained on the basis of the membrane being stabilized by inactivation of the sodium-carrying mechanism. Although delayed rectification was not observed in normal muscle fibers, it became apparent in the fibers rendered inexcitable by tetrodotoxin. This finding, together with other evidence in the existing literature, supports an applicability of the sodium theory to the frog muscle fibers.

DEVELOPLTENT of electrophysiological technique has provided a means for direct determination of the site of neutropic drug action at cellular level. Intracellular microelectrodes are effective tools for investigation of the mechanism of action of substances affecting synaptic and junctional transmission. The experimental results presented here are concerned with the mechanism of conduction in the neuro-muscular junction.

Effects of Carcholin, Mecholyl, neostigmine and strychnine on spontaneously beating isolated rabbit auricles were studied by recording the transmembrane potential changes with intracellular microelectrodes and the contractions by means of a sensitive mechano-transducer. Electrical recordings were taken in both pacemaker and nonpacemaker areas. Carcholin (10–5 m) and Mecholyl (10–4 m) consistently shortened the duration of the action potential and diminished the developed tension. At higher concentrations Carcholin (10–3 m) and Mecholyl (10–2 m) arrested all auricular activity. Atropine (10–3 m) and epinephrine (10–3 m) restored activity. Following atropine or epinephrine, the inhibitory capacity of the anticholinesterases was abolished. Neostigmine and strychnine had variable effects.

1. The action of three thiamine antagonists (Neopyrithiamine, Oxythiamine, Amprolium) have been studied with intracellular microelectrodes on isolated heart tissue of rat, calf and sheep.

Intracellular microelectrodes have been used to study the site of origin and direction of spread of activity in the rabbit atrium. In this study a number of fiber groups have been found which possess specialized electrophysiological characteristics and a consistent anatomical localization. A pacemaker potentiality has been found only in tissues derived from embryologically distinct structures such as the sinus venosus, the venosus valves and the lower segment of the auricular canal. Activity normally spreads slowly from the S-A node and excites the crista terminalis along a broad front. Spread is then rapid through the crista, the pectinate muscles and the fibers of the atrial roof. The septum is normally excited from the crista terminalis. Excitation reaches the A-V node almost simultaneously from the crista terminalis and the right segment of the S-A ring bundle. This latter shows many characteristics of specialized conducting tissue. Around the A-V ring conduction velocity slows markedly in fibers which have many of the electrophysiological characteristics of A-V nodal fibers.