Membrane permeabilities determining resting,action and mechanoreceptor potentials inStentor coeruleus

Summary In response to mechanical stimuli the protozoan,Stentor coeruleus, contracts in an all-or-none fashion and simultaneously reverses the direction of its ciliary beat. These behaviors have previously been shown to be correlated with the presence of a mechanoreceptor potential and all-or-none action potential (Wood 1970, 1973a). In the studies reported below the ionic bases of the resting, receptor and action potentials ofStentor were determined by use of intracellular microelectrodes penetrating animals chilled to 8.5–10 °C. The resting potential is most dependent on the extracellular concentration of KCl but some dependence on CaCl₂ concentration was also observed. If allowance is made for the large increases in membrane conductance observed in solutions containing 2–8 mM KCl it is found that the resting potential data are well described by a modified form of the Goldman equation whereP _Ca/P _K = 0.068 andP _Cl/P _K = 0.072. The intracellular ionic activities (K _i ⁺ = 13.1 mM, Cl _i ^– = 9.9 mM, Ca _i ⁺ = 0 mM) which provide the best fit of this equation to the resting potential data are in close agreement with the intracellular concentration values measured by flame microspectrophotometry (K_i=12.4 mM, Cl_i = 9.4 mM) except in the case of Ca_i where most of the intracellular concentration is presumed to be bound. 65 to 75 mV action potentials are produced by suprathreshold depolarizations but contractions were not generally seen in these chilled animals, only ciliary reversals. The action potential peak varies with CaCl₂ concentration with a slope of 12.6 mV/10 fold change but varies only slightly with KCl or Cl^– concentration. These peak potentials are well described by assuming that theP _Ca/P _k = 7.9 andP _Cl/P _K=1.0 at the time of the action potential peak. Depolarizing receptor potentials and brief inward receptor currents were observed for all forms of punctate and gross bodily mechanical stimulation employed. No evidence was found for any form of hyperpolarizing mechanoreceptor potentials as observed in some other ciliates. The reversal potential of the mechanoreceptor current varied with CaCl₂ concentration in a manner similar to that of the action potential peak. As in the case of the action potential both theP _Ca/P _k andp _cl/p _k ratios appear to increase as a result of mechanical stimulation to 9.3–15 and 1.2–1.95 respectively. Mechanoreceptor currents are voltage dependent being increased when the membrane is depolarized above resting potential and decreased when the membrane is hyperpolarized. In general the electrophysiological characteristics ofStentor appear similar to those ofParamecium andStylonychia, but its resting membrane appears more selectively permeable to K⁺, it produces only depolarizing receptor potentials when mechanically stimulated and the initial action potential elicited by depolarizing current pulses can be all-or-none even in culture medium.     

Non-propagated potentials in the electromyogram of mammalian ocular muscles]

Electromyograms of mammalian extraocular muscles were recorded by means of a coaxial electrode. Besides normal extracellular spike potentials (1-2 msec duration), monophasic waves (with a decline lasting up to 7 msec) were recorded. As to the interpretation of these potential changes in terms of a potential drop that is produced by local currents flowing from the resting region of a fibre towards the active region consideration is given to two cases. First, a propagated active region (spike potentials, at least diphasic) and second, a stationary active region (with resulting monophasic waves). In the EMGs spontaneous monophasic potentials recruit at a lower threshold than spike potentials; frequency changes were observed when head position was altered. The latter are interpreted as local depolarizations occurring at neuromuscular junctions of multiple innervated muscle fibres among those fibre types that compose extraocular muscles.     

Relations of single semicircular canals to the pontine reticular formation.

The effects of afferent vestibular impulses on single pontine reticular formation units and on a small filament of the IIIrd cranial nerve were recorded with tungsten microelectrodes in 40 curarized guinea pigs. Single-shock and repetitive electrical stimulations were applied by means of stimulating electrodes inserted bilaterally into the perilymphatic space of single ampullae of the anterior and lateral semicircular canals. The reticular unitary response consisted mainly in excitation of the resting discharge rate: most units showed vestibular convergence being affected by separate stimulation of the single four ampullae. the reticular evoked field and unitary potentials accounted for latency values ranging from 0.3 to 2.5 msec. As for the early latencies they can be interpreted as responses mediated by direct vestibulo-reticular fibres. A delimited vestibular projection field in the parameidan pontine reticular formation was not identified.     

FUNCTIONAL ANALYSIS OF SWIM-BLADDER MUSCLES ENGAGED IN SOUND PRODUCTION OF THE TOADFISH

A functional analysis of the striated swim-bladder muscles engaged in the sound production of the toadfish has been performed by simultaneous recording of muscle action potentials, mechanical effects, and sound. Experiments with electrical nerve stimulation were made on excised bladder, while decerebrate preparations were used for studies of reflex activation of bladders in situ. The muscle twitch in response to a single maximal nerve volley was found to be very fast. The average contraction time was 5 msec. with a range from 3 to 8 msec., the relaxation being somewhat slower. The analysis of muscle action potentials with surface electrodes showed that the activity of the muscle fibers running transversely to the long axis of the muscle was well synchronized both during artificial and reflex activation. With inserted metal microelectrodes monophasic potentials of 0.4 msec. rise time and 1.2 to 1.5 msec. total duration were recorded. The interval between peak of action potential and onset of contraction was only 0.5 msec. Microphonic recordings of the characteristic sound effect accompanying each contraction showed a high amplitude diphasic deflection during the early part of the contraction. During relaxation a similar but smaller deflection of opposite phase could sometimes be distinguished above the noise level. The output from the microphone was interpreted as a higher order derivative function of the muscle displacement. This interpretation was supported by complementary experiments on muscle sound in mammalian muscle. The dependence of the sound effects on the rate of muscle contraction was demonstrated by changing the temperature of the preparation and, in addition, by a special series of experiments with repeated stimulation at short intervals. Results obtained by varying the pressure within the bladder provided further evidence for the view that the sound initiated in the muscle is reinforced by bladder resonance. Analysis of spontaneous grunts confirmed the finding of a predominant sound frequency of about 100 per second, which was also found in reflexly evoked grunts. During these, muscle action potentials of the same rate as the dominant sound frequency were recorded, the activity being synchronous in the muscles on both sides. Some factors possibly contributing to rapid contraction are discussed.     

Electrophysiological studies of the smooth muscle cell membrane of the rabbit common carotid artery

The electrical responses of the smooth muscle cells of the rabbit common carotid artery to extracellular stimulation were studied in isotonic and hypertonic solution (1.7 times normal tonicity) with microelectrodes. No spontaneous electrical or mechanical activity was recorded when the tissue was in either isotonic or hypertonic solution. The voltage-current relation of smooth muscle cells in the common carotid artery showed marked rectification in both isotonic and hypertonic solutions. In isotonic and hypertonic solutions mean values for membrane potentials were -44.5 and -51.5 mv, for space constants 1.13 and 1.21 mm, and for time constants 212.2 and 238.2 msec, respectively. Addition of 34.3 mM TEA to the solutions caused spontaneous action potentials in the common carotid artery. The action potentials recorded simultaneously from two microelectrodes showed good synchronization. It was concluded that there is electrical transmission between cells of this artery.     

Insemination of rabbit eggs is associated with slow depolarization and repetitive diphasic membrane potentials

The plasma membrane of the rabbit egg allows only one sperm to enter the egg during fertilization, but the mechanism of this block to polyspermy is unknown. Electrophysiology and in vitro fertilization techniques were employed in this study to investigate the possibility that a voltage block to polyspermy exists in rabbit eggs. Ovulated zona-intact eggs had a mean membrane potential of -71 +/- 2.1 mV (interior negative). A stereotypic response occurred 12-135 min following in vitro insemination in 19 of 40 eggs. Association of this stereotypic response with the appearance of pronuclei suggested that the electrical response was related to some interaction of gametes. This response consisted of a slow transient 8 +/- 1.5 mV depolarization upon which were superimposed up to 36 repetitive diphasic insemination potentials. Each potential consisted of a brief 2.0 +/- 0.44 mV hyperpolarization followed by a slow 2.5 +/- 0.45 mV depolarization. The small amplitude of the stereotypic response when compared with the large variation of resting potentials suggested that the response was insufficient to block polyspermy by a mechanism dependent upon the magnitude of the rabbit egg membrane potential.     

Diphasic synaptic potentials and prolonged poststimulus hyperpolarization in Helix pomatia neurons

Synaptic activity of neurons giving diphasic excitatory-inhibitory potentials in response to orthodromic stimulation was recorded intracellularly. In response to stimulation of nerves by a single short pulse these neurons developed only the excitatory component of the diphasic potential, but with a longer stimulus a prolonged inhibitory phase, partly suppressing the initial excitatory component, was added. The excitatory phase appeared only when the resting potential reached a certain level. In their response to repetitive stimulation, neurons with a diphasic potential are divided into habituating and nonhabituating. The diphasic potential can also arise in response to application of acetylcholine to the soma of these neurons. It is postulated that this potential reflects the response of different receptors of the postsynaptic membrane to the same mediator. Prolonged poststimulus hyperpolarization can be obtained after repetitive orthodromic or direct stimulation of some neurons. However, as analysis of the results showed, poststimulus hyperpolarization is endogenous in origin and differs in its mechanisms from the diphasic potential.M. V. Lomonosov Moscow State University. Translated from Neirofiziologiya, Vol. 5, No. 2, pp. 193–200, March–April, 1973.     

Reversal of Photoreceptor Polarity Recorded during the Graded Receptor Potential Response to Light in the Eye of Limulus

Intracellular electrodes were inserted into single photoreceptor units of the excised lateral eye of Limulus, and preparations were selected from which graded receptor potentials of relatively large amplitude could be recorded in response to light stimuli. The experimental data indicated that the graded receptor potential does not arise solely from a collapse of the resting membrane potential of the sensory cells of the eye, since a reversal of polarity of the photoreceptor unit could be demonstrated when the eye was stimulated by light. In the recovery period following stimulation, characteristic changes in the so-called resting potential were recorded. It is suggested that these changes in the so-called resting membrane potential are electrical signs of recovery processes occurring in the photoreceptor, because the potential changes were recorded when the eye was in darkness and because the magnitudes of the potential changes were a predictable function of the intensity and duration parameters of the preceding light stimulus.     

An Example of Information Retention in Rabbit Ventricular Muscle Fibres

The preparation was stimulated externally and transmembrane action potentials were recorded with intracellular microelectrodes. The relationship between the area of the first action potential after a pause in stimulation and the duration of the pause was examined. It was found that the area retained its dependence on the pattern of stimulation prior to the pause. These experiments confirm one of the predictions of a mathematical model (Gibbs et al., 1963) which describes the relationship between the area of action potentials and the pattern of stimulation.     

Light- and dark-induced action potentials in Physcomitrella patens

Glass microelectrodes were inserted into Physcomitrella patens gametophyte leaves and action potentials (APs) were recorded in response to sudden illumination as well as after darkening, i.e., when the dark-induced membrane depolarization crossed a threshold. Application of 5 mM La³⁺ (a calcium channel inhibitor), 10 mM TEA⁺ (a potassium channel inhibitor) and increased free Ca²⁺ resulted in a loss of excitability. Lack of Ca²⁺ in the external medium did not prevent APs from occurring. It was concluded that during light- dark-induced excitation of Physcomitrella patens, APs might rely upon calcium influxes from the intracellular compartments. APs were not blocked by the proton pump inhibitors (DES, DCCD), although the resting potential (RP) diminished significantly.Key words: action potential, calcium, moss, Physcomitrella patens, plant     

Receptor potentials and action potentials in Drosera tentacles

Summary Voltage fluctuations identified as receptor potentials can be detected with electrodes applied to the mucilage surrounding the head of a tentacle of Drosera intermedia if the head is stimulated by contact with a live insect, by the touch of a clean, inert object, or by application of salt solutions. Associated with a low receptor potential are action potentials, which occur at a frequency dependent on the magnitude of the receptor potential. These action potentials can be detected with electrodes applied to any region of the stalk of the tentacle. Inflection of the lower stalk follows the occurrence of action potentials. Inflection is minute for isolated action potentials but large and rapid when several occur within a brief interval.The apparent amplitude of action potentials recorded from the stalk is independent of receptor potential amplitude, but that of action potentials recorded from the mucilage commonly decreases as the receptor potential deviates from the baseline and increases as it returns. It is suggested that variation of apparent amplitude of the action potentials may result from postulated variation in the resistance of receptor membranes.     

Electrophysiological analysis of the effect of cortisone on the rat neuromuscular apparatus]

The influence of cortisone (1.5 mg per 100 g of body weight, daily, for 10 days) on the neuro-muscular system was studied in rats in situ. The action potentials of the nerve and muscle were recorded with the extracellular electrodes. The rest potentials (RP) of the muscle fibers and the miniature end-plate potentials (MEPP) were recorded with the intracellular glass microelectrodes. A decrease of the RP and the MEPP amplitude, and an increase of the MEPP frequency and prolongation of the neuromuscular transmission time were revealed in rats given daily doses of cortisone, 1.5 mg/100 g of body weight, during 10 days; reliability of the neuro-muscular transmission (acceleration of the fall of the muscle action potential amplitude during tetanus) proved to decrease under the action of cortisone.     

Origin of Axon Membrane Hyperpolarization under Sucrose-Gap

One of the disadvantages of the sucrose-gap method for measuring membrane potentials with extracellular electrodes is a membrane hyperpolarization of the order of 30 to 60 mv, as compared with the resting potential obtained with intracellular microelectrodes in the absence of a sucrose-gap. In the present study the contribution of the sucrose-sea water junction potential to this hyperpolarization effect has been evaluated by comparing the effects on the resting potential of several anion and cation substitutions in the sea water bathing the lobster giant axon under sucrose-gap. Measurements with microelectrodes demonstrate a significant liquid junction potential between sucrose and standard artificial sea water. The value of this liquid junction potential as well as the measured resting membrane potential varies as a function of the anions and cations substituted in the sea water. Both the liquid junction potential and the sucrose-gap-induced hyperpolarization can be eliminated by using a low mobility anion to replace most of the chloride in sea water while the normal cation content remains unchanged. These data provide evidence that loop currents at the sucrose-sea water-axon junctions are at least partly responsible for membrane hyperpolarization under a sucrose gap.     

Electrical Properties of Hypothalamic Neuroendocrine Cells

Goldfish hypothalamic neuroendocrine cells have been investigated with intracellular recordings. The cells showed resting potentials of 50 mv and action potentials up to 117 mv followed by a long lasting and prominent diphasic hyperpolarizing afterpotential. The action potential occurred in two steps indicating sequential invasion. "Total" neuron (input) resistance was measured to be 3.3 x 10⁷ Ω and total neuron time constant was 42 msec. Orthodromic volleys, produced by olfactory tract stimulation, generated graded excitatory postsynaptic potentials. These neuroendocrine cells seem, therefore, to have electrical membrane properties that are similar to those of other central neurons. Antidromic volleys (pituitary stimulation) produced inhibitory post-synaptic potentials whose latency was only slightly longer than that of the antidromic spike indicating the presence of recurrent collaterals. This finding suggests that the concept of the neuroendocrine cell as a neuron whose axon forms contacts only on blood vessels and not on other neurons or effector cells is too restrictive. Perfusion of the gills with dilute (0.3 per cent) sea water produced an inhibition of spontaneous activity. This inhibition is discussed in relation to recent work which demonstrates that goldfish hypothalamic hormones facilitate Na⁺ influx across the gill membrane.     

Electrophysiological analysis of cortico-tectal connections in the turtle

Two types of evoked potentials are recorded in the tectum mesencephali in response to electrical stimulation of the forebrain surface of the turtleEmys orbicularis. The results of a layer-by-layer analysis show that evoked potentials of type I in response to stimulation of the hippocampal and piriform cortex are generated outside the tectum. Evoked potentials of type II, consisting of two surface-negative components, are recorded in the tectum in response to stimulation of the rostro-central surface of the forebrain. The first component appeared after a latent period of 20 msec and lasted 40–60 msec; the second component appeared after 80–100 msec and lasted 100–300 msec. Layer-by-layer and pharmacological analysis showed that the first component of the type II evoked potential is generated in the tegmental structures of the mesencephalon, whereas the second (long-latency) is generated in the tectum. The tectal origin of the second component is confirmed by its interaction with the tectal response to photic stimulation or to electrical stimulation of the optic nerve, evidence that these evoked potentials are generated by common structures. The efferent pathway from the dorsal cortex to the primary visual center is unilateral and has features of polysynaptic projections (long latent period, low lability).     

A comparative study of the physiological properties of the inner ear in Doppler shift compensating bats (Rhinolophus rouxi andPteronotus parnellii)

Summary Cochlear microphonic (CM) and evoked neural (N-1) potentials were studied in two species of Doppler shift compensating bats with the aid of electrodes chronically implanted in the scala tympani. Potentials were recorded from animals fully recovered from the effects of anesthesia and surgery. InPteronotus p. parnellii andRhinolophus rouxi the CM amplitude showed a narrow band, high amplitude peak at a frequency about 200 Hz above the resting frequency of each species. InPteronotus the peak was 25–35 dB higher in amplitude than the general CM level below or above the frequency of the amplitude peak. InRhinolophus the amplitude peak was only a few dB above the general CM level but it was prominent because of a sharp null in a narrow band of frequencies just below the peak. The amplitude peak and the null were markedly affected by body temperature and anesthesia. InPteronotus high amplitude CM potentials were produced by resonance, and stimulated cochlear emissions were prominent inPteronotus but they were not observed inRhinolophus. InPteronotus the resonance was indicated by a CM afterpotential that occurred after brief tone pulses. The resonance was not affected by the addition of a terminal FM to the stimulus and when the ear was stimulated with broadband noise it resulted in a continual state of resonance. Rapid, 180 degree phase shifts in the CM were observed when the stimulus frequency swept through the frequency of the CM amplitude peak inPteronotus and the frequency of the CM null inRhinolophus. These data indicate marked differences in the physiological properties of the cochlea and in the mechanisms responsible for sharp tuning in these two species of bats.     

Electrophysiological studies of the isolated posterior moiety of the electroplax of the electric eel

Intracellular microelectrodes were used to measure resting and action potentials of electric eel electroplax cells from which the middle portion of the anterior moiety had been removed. External electrode studies of this preparation have been described by Chagas and Esquibel (C. R. Acad. Sc., Paris, 260: 3172 ('65)). In the present studies it was found that the resting and action potentials were almost as large as normal in the intact portion of the preparation, whereas these potentials were greatly reduced in the areas from which the anterior moiety had been removed. The reduction in amplitude was greater the farther the microelectrode was from the intact portion, indicating the possibility of decremental spread from the intact portions. The action potentials measured with external electrodes appeared to be an average of potentials from the various areas of the preparation. In other experiments the entire anterior moiety of the electroplax was removed, leaving only the innervated plasma membrane plus some adhering cytoplasm and extracellular material. Various high K⁺ solutions were used to bathe the inner surface. No action potentials could be elicited from these preparations, but resting potentials as high as 51 mV were observed using external electrodes. The resting potential could be reduced reversibly by carbamylcholine applied to the outer surface. Carbamylcholine applied to the inner surface had no effect.     

Electrophysiological properties ofDictyostelium derived from membrane potential measurements with microelectrodes

Summary Electrical membrane properties of the cellular slime moldDictyostelium discoideum were investigated with the use of intracellular microelectrodes. The rapid potential transients (1 msec) upon microelectrode penetration of normal cells had a negative-going peak-shaped time course. This indicates that penetration of a cell with a microelectrode causes a rapid depolarization, which can just be recorded by the microelectrode itself. Therefore, the initial (negative) peak potential transient valueE _p (–19 mV) should be used as an indicator of the resting membrane potentialE _m ofD. discoideum before impalement, rather than the subsequent semistationary depolarized valueE _n (–5 mV). Using enlarged cells such as giant mutant cells (E _p=–39 mV) and electrofused normal cells (E _p=–30 mV) improved the reliability ofE _p as an indicator ofE _m. From the data we concluded thatE _m ofD. discoideum cells bathed in (mm) 40 NaCl, 5 KCl and 1 CaCl₂ is at least –50 mV. This potential was shown to be dependent on extracellular potassium. The average input resistanceR _i of the impaled cells was 56 M for normalD. discoideum. However, our analysis indicates that the membrane resistance of these cells before impalement is >1 G. Specific membrane capacitance was 1–3 pF/cm². Long-term recording of the membrane potential showed the existence of a transient hyperpolarization following the rapid impalement transient. This hyperpolarization was associated with an increase inR _i of the impaled cell. It was followed by a depolarization, which was associated with a decrease inR _i. The depolarization time was dependent on the filling of the microelectrode. The present characterization of the electrical membrane properties ofDictyostelium cells is a first step in a membrane electrophysiological analysis of signal transduction in cellular slime molds.     

Responses of neuroblastoma cells to iontophoretically applied acetylcholine

Dissociated mouse neuroblastoma cells were studied in vitro by using intracellular microelectrodes for electrical stimulation and recording. Some, but not all cells, which exhibited well developed action potentials to electrical stimulation also showed changes in membrane potential to iontophoretically applied acetylcholine (ACh). The types of responses to ACh varied. Short latency depolarizing responses to pulses of ACh (similar to those obtained with skeletal muscle) as well as sustained depolarization to steady ACh application (D response) occurred. A longer latency prolonged hyperpolarizing response (H response) and bi- and triphasic combinations of H and D responses were also seen. Pairs of cells showing morphologic contact were tested for the occurrence of effective synaptic coupling by placing intracellular microelectrodes in each cell. In none of 95 cases tested did spike activity produced by direct electrical stimulation of one cell elicit a synaptic potential of 200 μv or more in the other.     

Development of electrical coupling and action potential synchrony between paired aggregates of embryonic heart cells

Summary Pairs of spheroidal aggregates of embryonic chick heart cells, held in suction pipettes were brought into contact and allowed to synchronize their spontaneous action potentials. Contractions were suppressed with cytochalasin B. Both intracellular and extracellular electrodes were used to analyze the development of synchrony. Electric coupling occurred in three phases. During phase I electrical interactions were absent despite close physical contact. Phase II was characterized by partial synchrony. Action potentials in the faster aggregate (F) induced small depolarizations in the other member of the pair (S). These depolarizations sometimes triggered action potentials inS depending on when during the diastolic depolarization inS they occurred. In these cases both the latency between the action potentials (L) and the fluctuations in latency (V _L) were large. At the end of phase II the aggregates often passed through a brief period when fluctuation in interbeat interval in both increased noticeably. In phase III, beginning about 8 min after initial contact, action potentials were completely entrained at a certainL. During the subsequent 20–40 minL fell along an approximately exponential time course from about 130 to <1 msec, whileV _L declined in parallel. When well-coupled aggregates were pulled apart and immediately pressed back together, they re-established synchronization according to the usual three-phase time course. Synchronized aggregates could be partially decoupled by separating them just far enough to reduce the area of mutual contact. Pairs joined only by cellular strands maintained entrained action potentials with long latencies for many minutes. These results indicate that electronic junctions form between the paired heart cell aggregates causing the gradual development of action potential synchrony.