Membrane Potentials of the Lobster Giant Axon Obtained by Use of the Sucrose-Gap Technique

A method similar to the sucrose-gap technique introduced be Stäpfli is described for measuring membrane potential and current in singly lobster giant axons (diameter about 100 micra). The isotonic sucrose solution used to perfuse the gaps raises the external leakage resistance so that the recorded potential is only about 5 per cent less than the actual membrane potential. However, the resting potential of an axon in the sucrose-gap arrangement is increased 20 to 60 mv over that recorded by a conventional micropipette electrode when the entire axon is bathed in sea water. A complete explanation for this effect has not been discovered. The relation between resting potential and external potassium and sodium ion concentrations shows that potassium carries most of the current in a depolarized axon in the sucrose-gap arrangement, but that near the resting potential other ions make significant contributions. Lowering the external chloride concentration decreases the resting potential. Varying the concentration of the sucrose solution has little effect. A study of the impedance changes associated with the action potential shows that the membrane resistance decreases to a minimum at the peak of the spike and returns to near its initial value before repolarization is complete (a normal lobster giant axon action potential does not have an undershoot). Action potentials recorded simultaneously by the sucrose-gap technique and by micropipette electrodes are practically superposable.     

Double Sucrose-Gap Method Applied to Single Muscle Fiber of Xenopus laevis

Passive electrical properties (internal conductance, membrane conductance, low frequency capacity, and high frequency capacity obtained from the foot of the action potential) of normal and glycerol-treated muscle of Xenopus were determined with the intracellular microelectrode technique. The results show that the electrical properties of Xenopus muscle are essentially the same as those of frog muscle. Characteristics of the action potential of Xenopus muscle were also similar to those of frog muscle. Twitch tension of glycerol-treated muscle fibers of Xenopus recovered partially when left in normal Ringer for a long time (more than 6 h). Along with the twitch recovery, the membrane capacity increased. Single isolated muscle fibers of Xenopus were subjected to the double sucrose-gap technique. Action potentials under the sucrose gap were not very different from those obtained with the intracellular electrode, except for the sucrose-gap hyperpolarization and a slight tendency toward prolongation of the shape of action potential. Twitch contraction of the artificial node was recorded as a change of force from one end of the fiber under the sucrose gap. From the time-course of the recorded force and the sinusoidal stress-strain relationship at varying frequencies of the resting muscle fiber, the time-course of isotonic shortening of the node was recovered by using Fourier analysis. It was revealed that the recorded twitch force can approximately be regarded as isotonic shortening of the node.     

Vestibulo-ocular reflexes in the adult sea lamprey

Vestibulo-ocular reflexes were elicited in isolated preparations of small adult sea lampreys (Petromyzon marinus). Mechanical stimulation of the labyrinths or electrical stimulation of the vestibular nerves produced stereotyped, conjugated eye movements (Table 1) and appropriate electrical activities in individual extraocular muscles (Fig. 1). No inhibition of discharges in motor nerves was observed during stimulation of opposing reflexes. Likewise, nystagmus was never seen during continued stimulation. The primitive eye reflexes of the lamprey probably correspond to the simple excitatory pathways from single ampullae to individual eye muscles of higher vertebrates.     

Transmission pathways in the rat superior cervical ganglion

On isolated preparations of the superior cervical ganglion (SCG, n = 8) taken from 21-day-old rats, we studied the intraganglion pathways and mechanisms underlying generation of synaptic responses of SCG neurons to antidromic stimulation. One of the three nerves connected with the SCG was stimulated, and compound action potentials were recorded simultaneously from the other two nerves; then, the order of stimulated and recorded nerves was changed. Orthodromic stimulation of the cervical sympathetic nerve (CSN) evoked responses in the internal carotid nerve (ICN), which could be completely blocked by hexamethonium, and responses in the external carotid nerve (ECN), which contained a component that was not blocked by this of the ECN caused responses in the CSN, which were not blocked by hexamethonium. Effects of superfusion of the SCG with a Ca²⁺-free solution allowed us to conclude that the hexamethonium-insensitive component of the responses in the CSN and ECN and ECN-CSN conduction can be explained by the presence of direct fibers going from the CSN to the ECN with no synaptic relay. Possible mechanisms underlying antidromic stimulation-induced synaptic responses in SCG neurons are discussed. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 396–399, July–October, 2007.     

Electrophysiological Actions of Oxytocin on the Rabbit Myometrium

The electrical activities of myometrial cells of the pregnant rabbit uterus have been studied by means of sucrose-gap and intracellular micro-electrode recording techniques. The resting potential of the myometrial cell was about -50 mv, and it is unaffected by the duration of pregnancy or placental attachment. Action potentials of the myometrium, although dependent on external Na⁺, were not always of the regenerative type; preparations from nonparturient uteri often produce mainly small spikes. The mean spike amplitude was 35 mv, rising at a mean maximum rate of 3 v/sec. Oxytocin, in concentrations less than 500 µU/ml, increased the mean spike amplitude to 48 mv and the mean maximum rate of rise to 7 v/sec, without affecting the resting potential. The relation between membrane potential and dV/dt of the spike was steepened by oxytocin, suggesting that oxytocin increased the number of normally sparse sodium gates in the myometrial membrane. By this action, oxytocin is believed to increase the probability of successful regenerative spikes and thereby initiate electrical activity in quiescent preparations, increase the frequency of burst discharges, the number of spikes in each burst, and the amplitude of spikes in individual cells.     

Ultrasound-induced changes in synaptic processes with different transmitters in smooth muscles

The effect of therapeutic-intensity ultrasound on neuromuscular transmission and spontaneous electrical and contractile activity in smooth muscles of the gastrointestinal tract of guinea pig was studied by a modified sucrose-gap technique. The action of ultrasound was found to facilitate the acetylcholinergic neuromuscular transmission (mainly by increasing the amplitude of excitatory postsynaptic potentials). The higher efficiency of the nonadrenergic neuromuscular transmission was manifested as an increase (nearly twofold) in the total duration, but not in the amplitude, of inhibitory postsynaptic potentials. Modulations of the first and second components of the potentials caused respectively by the action of ATP and of nitric oxide as possible transmitters, were different. Concurrently with enhancing the synaptic transmission efficiency, ultrasound exerted an opposite, inhibitory, effect on generation of spontaneous action potentials and contraction of smooth muscles. All the ultrasound effects were fully reversible. The findings permit assuming a special mechanism of modification of the synaptic transmission in smooth muscles under the action of ultrasound.Neirofiziologiya/Neurophysiology, Vol. 25, No. 4, pp. 297–302, July–August, 1993.     

Neural inhibition of egg-laying in the locust, Locusta migratoria

The role of the oviducal nerves during egg-laying in Locusta migratoria has been examined. Section of the oviducal nerves did not inhibit egg-laying in any observable way. Electrical stimulation of the oviducal nerves resulted in a contraction of the common and lower lateral oviducts which propelled ovulated eggs up towards the ovaries. Recordings from oviducal nerves using chronically implanted electrodes showed that electrical activity was low during actual egg-laying, but high at times when egg-laying was not occurring (i.e. during digging behaviour, or following interruption of egg-laying). During these periods of high activity recurrent bursts of action potentials occurred. Similar patterns of electrical activity were recorded in semi-intact preparations using suction electrodes applied to exposed oviducal nerves of locusts which had been interrupted during the process of egg-laying. High frequency bursts of activity were recorded simultaneously from both left and right oviducal nerves.It is concluded that one function of the oviducal nerves is to inhibit egg-laying at inappropriate times, by inducing contractions of the oviducts which propel eggs back towards the ovaries. These nerves therefore provide a physiological basis for part of the adaptive ovipositional activities of locusts.     

Funnel web spider venom produces spontaneous action potentials in nerve

Venom from the lethal Australian spider, $\text{Atrax robustus}$, causes fasciculation of muscles $\text{in vivo}$ and in isolated diaphragms in mice. Spontaneous end-plate potentials were recorded in muscle fibres exposed to the venom and associated spontaneous electrical activity could also be recorded from the phrenic nerve. It was proposed that the venom produces muscle fasciculation by causing abnormal, spontaneous, repetitive firing of motor nerves. The mechanism of this action was investigated in aplysia neurones. The venom produced abnormal, spontaneous, repetitive inward currents in voltage clamped neurones and changed the current-voltage characteristics of the surface membrane. It is suggested that the basic mode of action of Funnel-web venom is to change the electrical field in nerve membrane.     

Effects of haloperidol and clotrimazole on synaptic transmission in and contractile activity of smooth muscles of the guinea-pig intestine

In muscle strips of the guinea-pig large intestine, haloperidol and clotrimazole increased spontaneous electrical and contractile activities and decreased ATP-evoked hyperpolarization of smooth-muscle cells and the amplitude of inhibitory synaptic potentials. The pattern of effects of haloperidol on hyperpolarization induced by intramural stimulation of muscle strips was preserved under conditions of pre-incubation of the preparations in Krebs solutions containing pyridoxal-5′-phosphate, Nω-nitro-L-arginine, or apamin, as well as both apamin and tetraethylammonium. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 412–415, July–October, 2007.     

Studies on the mechanisms of the effects of protein kinase C activation on electrical and contractile activity of smooth muscle

Experiments have been carried out with guinea-pig smooth muscles taenia coli by the use of the double sucrose-gap technique. Phorbol esters (PE), activating protein kinase C (PcC) suppressed the spontaneous and induced (by turning on of depolarizing pulses, or turning off a long lasting hyperpolarizing step) electrical and contractile activity of a muscle. The inactive analog of PE did not affect the muscle activity. Na/H-ionophore monensin imitated the effects of active PE. Blockade of K+ channels by 10 mM TEA greatly decreased or in some cases even removed the inhibitory effects of PE. A treatment of the muscle by Na+/H+ exchange inhibitor ethyl isopropyl amiloride (EIPA) increased the amplitude of action potentials during membrane depolarization and markedly weakened the PE-induced suppression of muscle electrical activity. The data obtained suggested that inhibitory effects of PE on smooth muscle electrical and contractile activity resulted from an increase in potassium permeability of the membrane. Na+/H+ exchange seems to be involved in PE-induced K+ channels activation.     

Toxic concentrations of selenite shortens repolarization phase of action potential in rat papillary muscle

Selenium (Se) has long been recognized as both an essential mammalian nutrient and a hazardous element. Sodium selenite is commonly used as a dietary supplement for the treatment of Se deficiency. On the other hand, chronic Se toxicity has been demonstrated to affect the major organs, including the heart, in experimental animals. This study examines the effects of high concentrations of extracellular selenite (in the range of 0.001–1 mM) application into the recording bath on the electrical properties of rat papillary muscles. Conventional glass semifloating microelectrodes were used to record intracellular action potentials (APs) in isolated rat papillary muscles. The amplitude of APs and the resting membrane potential of papillary muscles were not changed following a 20-min selenite (1 mM) application compared to the first minute of its application. Freshly isolated ventricular myocytes by an enzymatic method were used to determine the selenite-induced alterations in Na⁺ currents. Na⁺ currents, measured at 22°C, by the whole-cell patch-clamp technique, decreased by 38±8% in the presence of 1 mM selenite for 5 min. These selenite-induced effects were not reversed, but are restored by dithiothreitol (1 mM). These results demonstrated that toxic concentrations of selenite induced a significant shortening in AP duration as a result of an increase in the rate of repolarization. Our findings also suggest that a decrease in Na⁺ currents, in addition to Ca²⁺ currents, may play a role in the shortening of AP duration in rat papillary muscles.     

Effects of haloperidol, clotrimazole, and pyridoxal-5′-phosphate on synaptic transmission in smooth muscles of the human colon

In strips of smooth muscles of the human colon, haloperidol (Hal) and clotrimazole (Clo), in contrast to pyridoxal-5′-phosphate (PP), suppressed spontaneous electrical and contractile activities of these strips and also post-inhibitory excitation developing after inhibitory synaptic potentials (ISPs). Haloperidol, Clo, PP, and PP applied against the background of the action of Nω-nitro-L-arginine noticeably changed the parameters of ISPs. The pattern of effect of Hal on synaptic inhibition in smooth muscles was preserved against the background of the action of PP, and that of PP was preserved against the background of the action of Hal. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 408–411, July–October, 2007.     

Effects of Contralateral Afferent Stimulation on H Reflex in Humans

We studied changes of the H reflex recorded from the m. soleus, which were evoked by conditioning transcutaneous stimulation of the n. tibialis and n. peroneous comm. of the contralateral leg. In both cases, rather similar two-phase changes in the amplitude of the tested H reflex were observed. After a latent period (50 to 60 msec), the reflex was facilitated for about 300 msec, with the maximum at an about 100-msec-long interval. Then, facilitation was replaced by inhibition; the time course of the latter at test intervals longer than 500 msec could be satisfactorily approximated by a logarithmic curve. The mean durations of inhibition calculated with the use of a least-square technique were 4.0 and 2.7 sec in the cases of stimulation of n. tibialis and n. peroneous comm., respectively. Facilitation of the reflex was initiated with the intensity of conditioning stimulation corresponding to the threshold for excitation of cutaneous receptors. Facilitation could also be evoked by electrical stimulation of the skin in the contralateral popliteal dimple outside the projections of the above-mentioned nerves. Inhibition of the H reflex was evoked only with greater intensities of transcutaneous stimulation of the contralateral nerves corresponding to activation of low-threshold afferents of the above-mentioned nerves. The examined inhibition of the H reflex is probably of a presynaptic nature because it was not eliminated by tonic activation of the motoneurons of the tested muscle evoked by voluntary sole flexion. Long-lasting contralateral presynaptic inhibition can play a noticeable role in redistribution of the tone of skeletal muscles in the course of the motor activity. Neirofiziologiya/Neurophysiology, Vol. 37, No. 4, pp. 372–378, July–August, 2005.     

Suppressive effect of an activator of ATP-dependent potassium channels, flocalin, on electrical and contractile activities of smooth muscles of the guinea-pig ureter

In experiments on isolated segments or strips obtained from the guinea-pig ureter, we showed, using a sucrose-gap technique, that application of an activator of ATP-dependent potassium channels (K_ATP), (flocalin (PF-5), suppresses generation of action potentials (APs) by ureter smooth muscle cells (SMCs). Pre-incubation of the ureter preparations under study in Krebs solution containing 1 to 10 μM PF-5 results initially in a decrease in the frequency of oscillations preceding an AP plateau, shortening of this plateau, and, later on, complete inhibition of AP generation. In the presence of PF-5, spikes induced by hyperpotassium depolarization were also inhibited, while a tonic component of such depolarization underwent a mild decrease. The data obtained indicate that PF-5 modulates the entry of Ca²⁺ ions through L-type voltage-dependent channels in the SMC membrane. Shortening of the plateau and suppression of the spikes initiated by application of an activator of voltage-dependent L-type potassium channels, Bay K 8644, can be considered a confirmation of the modulatory influence of PF-5 on voltage-dependent L-type potassium channels. It seems possible that Bay K 8644 can be used under experimental conditions for initiation and long-lasting modulation of APs generated by the ureter SMC instead of corresponding neurotransmitters. We hypothesize that voltage-dependent entry of Ca²⁺ ions into SMCs depends significantly on the PF-5-induced activation of K_ATP channels of the ureter SMCs. Neirofiziologiya/Neurophysiology, Vol. 37, Nos. 5/6, pp. 403–409, September–December, 2005.     

4-Aminopyridine-induced changes in the electrical and contractile activities of the gastric smooth muscle

Effects of 4-aminopyridine (4-AP) on the electrical and contractile activities of the fundus and antrum of the cat stomach were studied using the sucrose-gap technique. In the fundus, low concentrations of 4-AP (up to 1 mmol/l) induced membrane depolarization and appearance of spike potentials and phasic contractions. After preliminary administration of atropine, 4-AP produced an opposite effect: hyperpolarization and relaxation. On the background of tetrodotoxin (TTX) plus antagonists of cholinergic and adrenergic receptors, high concentrations of 4-AP (greater than 5 mmol/l) caused membrane depolarization and appearance of spike potentials and phasic contractions. In the antrum, 4-AP in low concentrations (up to 1 mmol/l) decreased both the amplitude and the duration of the second component of the plateau-action potential, as well as those of the phasic contractions. This effect decreased in the presence of adrenergic receptor antagonists and was abolished by TTX. On this background, high concentrations of 4-AP (greater than 5 mmol/l) led to the appearance of spike potentials superimposed on the second component of the plateau-action potentials, and to a further increase in the phasic contraction amplitudes. The present data suggest that 4-AP exerts its effects via an increase in neurotransmitter release (low concentrations) and/or directly on the smooth muscle cell membrane (high concentrations).     

The relationship between body size and the field potentials generated by swimming crayfish

Aquatic animals generate electrical field potentials which may be monitored by predators or conspecifics. Many crustaceans use rapid, forceful contractions of the flexor and extensor muscles to curl and extend their abdomens during swimming in escape and locomotion. When crayfish swim they generate electrical field potentials that can be recorded by electrodes nearby in the water. In general, it is reasonable to assume that larger bodied crayfish will generate signals of greater amplitude because they have larger muscles. It is not known, however, how activity in particular muscles and nerves combines to produce the compound electrical waveform recorded during swimming. We therefore investigated the relationship between abdominal muscle, body size and the amplitude of nearby tailflip potentials in the freshwater crayfish (Cherax destructor). We found that amplitude was correlated positively with abdominal muscle mass. The mean amplitude recorded from the five smallest and five largest individuals differed by 440 microV, a difference sufficiently large to be of significance to predators and co-inhabitants in the wild.     

Nerves in the endodermal canals of hydromedusae and their role in swimming inhibition

N eoturris breviconis (Anthomedusae) has a nerve plexus in the walls of its endodermal canals. The plexus is distinct from the ectodermal nerve plexuses supplying the radial and circular muscles in the ectoderm and no connections have been observed between them. Stimulation of the endodermal plexus evokes electrical events recorded extracellularly as “E” potentials. These propagate through all areas where the plexus has been shown by immunohistology to exist and nowhere else. When Neoturris is ingesting food, trains of “E” potentials propagate down the radial canals to the margin and cause inhibition of swimming. This response is distinct from the inhibition of swimming associated with contractions of the radial muscles but both may play a part in feeding and involve chemoreceptors. Preliminary observations suggest that the “E” system occurs in other medusae including Aglantha digitale (Trachymedusae) where the conduction pathway was previously thought to be an excitable epithelium.     

An intracellular study of pudendal afferent inputs onto tail motoneurons in the spinalized cat

Postsynaptic potentials, elicited by stimulation of the sensory pudendal (SPud) and superficial perineal nerves (SPeri) on both sides, were recorded from motoneurons innervating tail muscles in the non-anaesthetized and spinalized cat. The stimulation of SPud and SPeri on both sides predominantly produced excitatory postsynaptic potentials (EPSPs) in all kinds of tail motoneurons (70-95%). The inhibitory postsynaptic potentials (IPSPs) were often observed in motoneurons innervating ventral tail muscles (30-33%). The means of averaged central latencies of EPSPs and IPSPs ranged from 4.3 to 7.3 ms, and from 4.6 to 8.4 ms, respectively. The findings suggests that polysynaptic neuronal pathways from pudendal nerve to tail motoneurons produce tonic activities of all tail muscles to raise the tail in micturation, defecation and sexual movements which are induced by stimulation of pudendal nerves.     

Calcium dependency and the effect of calcium antagonists on molluscan proboscis smooth muscles from the whelk,Buccinum undatum

In all four proboscis muscles of the whelk Buccinum undatum, the potassium-induced depolarization response was acutely dependent upon extracellular calcium, being eliminated in calcium-free conditions. The responses to acetylcholine were found to be partly dependent upon intracellular calcium. Responses to the peptides phenylalanine-methionine-arginine-phenylalanine-NH₂ and phenylalanine-leucine-arginine-phenylalanine-NH₂ were much more resistant to calcium-free conditions and appeared to engage the excitation-contraction coupling mechanism by mobilizing stored intracellular calcium. Sucrose-gap studies of radular retractor muscles showed that the organic calcium “antagonist” nifedipine enhanced potassium-induced depolarization responses, initiating spike-like action potentials and associated fast twitch activity. The inorganic calcium antagonist gadolinium exerted concentration-dependent inhibitory actions on these muscles. Basal tonus and fast twitch activity in response to potassium-induced depolarization were eliminated as were the spike-like action potentials of the membrane electrical response. The inorganic calcium “antagonist” cadmium greatly enhanced potassium-induced contractures in all four muscles, and on its own it induced tonic force and fast twitches in all the muscles. It seems likely that cadmium may have displaced stored intracellular calcium to induce myofilament activation. While these molluscan smooth muscles appear to possess calcium channels with fast and slow characteristics, their behaviour and pharmacological manipulation is very different from their more well known mammalian transient and long-lasting channel counterparts.     

Nerve-muscle networks in the accessory gland tubules of a male insect: Structural and physiological properties

The accessory gland tubules of the cockroach Leucophaea maderae, (F.) are innervated by the phallic nerves which arise from the terminal ganglion. Tubule preparations stained with methylene blue showed that a fine network of nerve fibres covers the entire surface of the tubule with putative nerve cells at various points. Muscle fibres of the tubules were arranged in an irregular lattice and appeared as flattened elipses approx 8–10 μm across and 1.5 μm in thickness. These muscles were monomyofibrillar with a poorly developed T-tubule system and a diffuse Z band. Innervation of the muscles showed abundant evidence of neurosecretomotor-type terminals. Clear evidence was also obtained for a multiterminal-type innervation. Accessory gland tubules had a basic wave-like pattern of motion in vitro which showed a wide variation in the time-course of each swing (1–50 s). A twisting and curling motion of the tubules was also recorded. Spontaneous junctional potentials were observed in accessory gland preparations isolated from the terminal ganglion. The duration for junction potential ranged between 30–140 ms. Action potentials recorded from muscles had a slower rise time and a longer duration (230–530 ms).