Single K+ currents during differentiation of embryonic muscle cells in vitro.

After 3-7 days in culture, chicken myotubes possess five types of K+ channel: two high-conductance channels of 195 and 105 pS which are sensitive to tetraethylammonium (TEA), an ATP-sensitive channel of 64 pS and two low-conductance channels of 40 and 15 pS which are insensitive to TEA and ATP. The same population of channels is to be found in EGTA-treated muscle cells with blocked fusion and, with the exception of the ATP-sensitive channel, also in 1-day-old myoblasts. There are differences between myoblasts and myotubes in the percentage of incidence of individual channel types. High-conductance K+ channels are most frequently to be observed in myotubes, but they are rare in myoblasts and EGTA-treated cells where low-conductance K+ channels predominate.

Evidence that excitatory amino acids not only activate the receptor channel complex but also lead to use-dependent block.

The effects of fast application of excitatory amino acids N-methyl-D-aspartate (NMDA), L-aspartate (ASP), L-glutamate (GLU), quisqualate (QU) and kainate (KAIN) were studied in neurons from the embryonic spinal cord of the chick in monolayer cultures by employing the 'patch clamp' technique in the 'whole cell' mode. It was found that NMDA, ASP, GLU and QU, but not KAIN, induced responses that exhibited several components. The early component decayed with a time constant of 2 s to a lower level of membrane current and discontinuation of the application was followed by an after-current which returned to the base-line with a time constant of about 7 s. It is suggested that NMDA, ASP, GLU and QU, but not KAIN, not only activate the receptor channel complex but also induce use-dependent block.

Extracellular potassium concentration and focal electrical potentials elicited in the cerebral cortex of rat by interhemispheric stimulation.

All points in the time course of the evoked and self-sustained, DC-recorded potentials elicited by repetitive interhemispheric stimulation were found to remain negative by -1 mV and more with respect to the prestimulation DC level only when the coinciding, directly recorded, extracellular potassium concentration was 11 mM. A slow oscilloscope sweep displayed the -1 mV level as a virtual line limiting the initial positive waves of the interhemispheric response. This line, approximately parallel with the potassium concentration record of around 11 mM, probably expresses the potassium-dependent membrane depolarizations, which were not revealed at lower potassium concentrations. The evoked and self-sustained potentials coinciding with the latter were displayed in negative as well as positive values.

Slow potential changes in experimental neocortical propagated foci.

During and following interhemispheric stimulation (HS), characteristic negative slow potential changes (NSPCs) appeared which coincided with defined patterns of epileptic phasic field potentials (PFPs). Stimulus time-locked, high-amplitude, interhemispheric responses (IHRs), interictal and ictal patterns were accompanied by typical moderate NSPCs. In the wake of the latter, giant NSPCs occurred, concurrent with Van Harrevelds convulsions and/or either total or partial spreading, or local depressions. Partial depression of the PFPs and spreading of the moderate NSPCs were also present in the IHR stage. It is suggested that the moderate and giant NSPCs characterize two distinct types of experimental epileptogenesis which differ in the extent of alteration of the extracellular ion activities.

Interhemispheric field potentials, spreading depression and kindling.

The incidence of spreading depression, which was elicited by interhemispheric stimulation, increased with prolonged duration and strength of individual pulses and with longer stimulus trains. In the first trial spreading depression appeared in one-third of the experiments, in two-thirds it was activated by repeated stimulus trains. Both the activated and the first trial spreading depression were preceded by alternation of the interhemispheric response and often by self-sustained afterdischarges. All the phases of the interhemispheric response patterns contained inhibitory components either in the form of phasic focal polarizing waves of the evoked and self-sustained field potentials or giant slow spreading depolarizations. The stimulation parameters determine whether a permanent increase of excitability prevails, as in Goddard's kindling procedures, or whether inhibitory patterns, described in this paper, result from the stimulation.

Projection of tooth pulp afferents to the brainstem and to the cortex in the cat.

The projection of tooth pulp afferents in the spinal trigeminal nucleus (N.V.sp.) and the effect of dorsolateral medullotomy on cortical potentials evoked by electrical stimulation of Adelta tooth pulp nerve fibres were studied in cats. It was confirmed that antidromic responses were recorded in the tooth pulp nerve to stimulation of the ipsilateral but not the contralateral N.V.sp.[11]. Dorsolateral medullotomy at the level of the obex did not alter the cortical potentials induced by single pulses applied to the tooth pulp. It is concluded that Adelta tooth pulp afferents project exclusively to the ipsilateral trigeminal nucleus and that the impulse transmission to the cortex persists after transection of the pars caudalis of the N.V.sp.

Transepithelial potential in mesonephric nephrons of 7-day-old chick embryos in relation to the histochemically detected sodium pump.

In order to obtain basic information on the transport properties of differentiating embryonic nephrons, we examined the 7-day-old chick mesonephros by measuring the transtubular epithelial potential difference (TPD) and by histochemical detection of Na,K-ATPase activity. TPD as an indicator of the electrogenic transport was measured in individual segments of superficial nephrons in vivo. Their electric polarity was always lumen-negative. TPD was reduced by addition of 10 mM KCN applied to the mesonephric nephrons from the outside. In the proximal tubules, TPD was significantly lower (mean+/-SD: -1.0+/-0.5 mV) than in the distal and collecting tubules (-2.2+/-1.0 mV, p< or =0.05). Activity of the sodium pump was evaluated histochemically by detection of ouabain-sensitive potassium-dependent p-nitrophenyl phosphatase in cryostat sections of the mesonephros. The enzyme activity was demonstrated only in distal tubules and in the collecting ducts, but not in the proximal tubules. These findings have revealed significant differences between embryonic nephron segments: the distal tubule, in contrast to the proximal one, is supplied by the sodium pump and is able to generate higher TPD. Therefore, we consider that it is only the distal nephron, which possesses the ability of active transport.

Activation and modulation of ligand-gated ion channels.

Ligand-gated ionic channels are integral membrane proteins that enable rapid and selective ion fluxes across biological membranes. In excitable cells, their role is crucial for generation and propagation of electrical signals. This survey describes recent results from studies performed in the Department of Cellular Neurophysiology, Institute of Physiology ASCR, aimed at exploring the conformational dynamics of the acetylcholine, glutamate and vanilloid receptors during their activation, inactivation and desensitization. Distinct families of ion channels were selected to illustrate a rich complexity of the functional states and conformational transitions these proteins undergo. Particular attention is focused on structure-function studies and allosteric modulation of their activity. Comprehension of the fundamental principles of mechanisms involved in the operation of ligand-gated ion channels at the cellular and molecular level is an essential prerequisite for gaining an insight into the pathogenesis of many psychiatric and neurological disorders and for efficient development of novel specifically targeted drugs.

Sodium transport and electrical properties of the chick chorioallantoic membrane.

Transport and electrical properties of the chick chorioallantoic membrane (CAM) were studied in order to find the osmoregulatory organ which helps to compensate the renal filtration-reabsorption disbalance of chick embryos. It could be shown that CAM resembles Na+ transporting epithelia in that active Na+ absorption is responsible for the potential difference and short circuit current, which could be abolished by ouabain on the ectodermal and amiloride on the endodermal side. The transepithelial conductance rose with increasing sodium concentration in accordance with the Michaelis-Menten kinetics. The allantoic sac thus plays a role similar to the toad urinary bladder despite the low potential difference and resistance which indicate that CAM is a leaky epithelium. CAM is therefore not only a respiratory but also an osmoregulatory organ.

Single non-inactivating K+ channels in the myotubes of the chick embryo in tissue culture.

Single-channel K+ currents were studied in the myotubes from the chick embryo grown in tissue culture for 4--9 days by employing the patch clamp technique. The "cell attached" configuration was used and the pipette was filled with a solution containing 3 mmol.l-1 K+. The channels exhibited a high conductance of approximately 90 pS and the probability of finding them open increased by an e-fold factor for 13 mV depolarization for low levels of activity. The channels did not inactivate during long-lasting depolarization. These channels have been suggested to contribute to delayed rectification.

A new technique for studying potential changes in nephrons of chick embryo kidneys in vivo.

To make the chick embryo accessible to electrophysiological measurements in its mesonephric kidney during the period between embryonic days (e.d.) 5 and 10, a special "chick-embryo-incubation bath" was constructed. It consists of an aerated chamber covering the egg and maintaining the gas exchange across the shell, and of a warmed reservoir of the incubation medium, into which the embryo is pulled out of the egg through a window in the shell. The two compartments are separated with a rubber membrane tightly fitting to the edges of the shell-window. The incubation medium contains a modified Krebs-Henseleit-Ringer solution and anesthetic Tricaine (Sigma). Access to the mesonephric nephrons is achieved by surgical excision of the body wall on the right side performed at e.d. 5. On average only about 35 percent of the operated embryos survive till the third day after surgery but during the next two days a mortality rate recedes to zero. The tolerance of short-term survival of embryos placed in the incubation bath was tested for up to 4 1/2 h. It was very good in embryos of age 5 to 7 e.d. as assessed by a steady heart rate and the presence of arterio-venous differences. A modified differential amplifier containing circuits for frequency compensation of the two channels was used for high-fidelity registration of voltage changes in the embryonic nephron with a single double-barrel microelectrode.

Low impedance coaxial K+ selective microelectrodes.

A procedure for preparing coaxial K+ selective microelectrodes with a low longitudinal resistance (Re) of the liquid ion exchanger selective barrel is described. The low resistance was attained by inserting another microelectrode filled with 0.5 mol . 1-1 KCl into the ion-exchanger column (Corning 477317). The lower longitudinal resistance decreases the noise level and consequently increases the resolving power five times. This modification makes it possible to measure small and rapid K+ concentration changes.

Response to stretch of proprioceptors in adult rat muscles de-efferented at birth.

Hind-limb muscles of new-born rats were de-efferented by removing the lumbosacral spinal cord. Spinal ganglia remained intact, together with their peripheral axon. The presence of sensory terminals in limb spindles, induces the full ultrastructural differentiation of muscle spindles, as has been shown previously. In the present paper we have shown by integrating the sensory discharges in the whole nerve from chronically de-efferented muscles that even several months after birth, muscle proprioceptors (probably mostly spindles) still maintain their basic mechanoreceptor properties. Although the limbs were completely immobilized throughout the whole experimental period, spindles from these chronically de-efferented muscles still responded as slowly adapting receptors. The dynamic component was also present in the integrated neurogram response during stretching. It thus appears that basic functional properties of rat muscle proprioceptor persist even when these receptors differentiate and survive without motor innervation and any adequate functional stimuli, i.e. under conditions of permanent disuse.

Changes of extracellular potassium concentration induced by neuronal activity in the sinal cord of the cat.

1. Changes of extracellular K(+) concentration, [K](e), arising in the spinal cord of the cat in response to an afferent stimulation were studied by means of K(+)-specific micro-electrodes.2. In the most active areas of the spinal cord a single volley in a large afferent input like the common peroneal nerve or the posterior tibial nerve produced a transient increase in [K](e) of 0.05-0.1 mM, which reached its peak in 0.2-0.3 sec and it declined in about 3 sec.3. Much higher increases in [K](e) were found during repetitive stimulation of an afferent input. The highest increase (by 3 mM) was at 100 Hz, but even at 1 Hz a significant increase of 0.25 mM was observed. Equilibration of accumulated K(+) was slow with a time constant of about 6 sec, which is much longer than could be expected for the same process in free solution.4. A characteristic distribution of increased [K](e) was found in the spinal cord in response to 100 Hz afferent stimulation. The highest increase of 3 mM was found in and around the intermediate nucleus, but at depths between 0.9-1.8 mm the [K](e) increase exceeded 1 mM.5. In the ventral horns afferent stimulation (100 Hz) increased [K](e) by 0.25 mM, while the same stimulation of the ventral root resulted in a [K](e) increase of less than 0.05 mM.6. The consequences of K(e) (+) accumulation after neuronal discharge are discussed in respect to its possible role in the depolarization of primary afferent terminals.

The postnatal functional development of muscle stretch receptors in the rat.

The response to a 5-sec stretch of the triceps muscle was studied in dorsal root filaments L5 of 72 infant rats (1-19 days old) under urethane anesthesia. More than 50% of all units in 1-day-old rats responded by repetitive firing until the end of the 5-sec stretch (slowly adapting or SA receptors), while the rest ceased to fire earlier (relatively rapidly adapting or 1/2 SA receptors), or gave an "on" response only. The number of units exhibiting an SA response increased with age and attained 80% in 5-day-old rats. By the 10th day of life, almost 90% of endings behaved as SA receptors. During development, the maximal discharge frequencies at the peak of stretch increased markedly, and their values in 18-day-old rats were comparable to those in adult rats. The phasic component of the response to stretch, although less well defined in the younger animals, was already present even in 1-day-old rats. Adaptation of the static response during maintained stretch was relatively steep in all the age groups studied. The results indicate that, in the rat, large numbers of muscle stretch receptors are capable of responding to sustained stretch as SA receptors, even at an age when their morphological and ultrastructural maturation is not yet fully accomplished.

The measurement of K+e concentration changes in human muscles during volitional contractions.

Changes of extracellular potassium concentration [( K+]e) were measured in human muscles during volitional isometric contractions using liquid ion-exchanger electrodes. In principle, an intramuscular injection needle containing a microelectrode with a side-pore was inserted into the brachioradialis muscle. After insertion of the needle, the glass ion-selective microelectrode (ISM) could be moved out of the protective trocar shield into the muscle tissue. The average values of [K+]e in human muscles during maximal effort rose from 4.5 mmol/l K+ to 9.5 mmol/l K+. These values correspond closely to those previously found in muscles of experimental animals.

Work-induced potassium changes in skeletal muscle and effluent venous blood assessed by liquid ion-exchanger microelectrodes.

Using liquid ion-exchanger semimicroelectrodes with a side pore, we measured changes of extracellular potassium concentration (Ke+) in adult rabbit and cat gastrocnemius muscles and in venous effluent blood flowing from the cat gastrocnemius muscle during various bouts of activity induced by sciatic nerve stimulation. 1. Isometric tetanic contractions (at 50 Hz) of various durations caused transient accumulation of Ke+ which was non-linearly related to the duration of muscle activity. The peak values of Ke+ in response to muscle stimulation were analogous in rabbits and cats, attaining values, e.g. after a 20-sisometric tetanus, between 8-9 mEq/1K+ in both species. 2. Potassium concentration in venous effleunt blood (K+ven) was transiently increased after isometric tetani. Since blood flow was measured at the same time, it was possible to calculate the amount of K+ lost by the muscle after tetani of various durations. A 32 g gastrocnemius muscle of the cat, for example, loses 9.36 +/- 1.52 muEqK+ after a 20-s isometric tetanus, which corresponds roughly to 0.5% of the total muscle potassium content. The loss of K+ in this muscle was 29.3 pEq K+ /impulse/100 g fresh muscle tissue. 3. There was no evident difference between the amount of K+ released during isometric tetani, or tetanic contractions performed under isotonic conditions. Single twitches evoked by indirect stimulation at 1 HZ for several minutes also induced a small rise in K+ven. 4. If the loss of K+ from the muscle into the blood stream is transiently prevented by arterio-venous occlusion installed immediately before a 10-s isometric tetanus, most K+ is released subsequently when blood flow is renewed, if the occlusion lasts for 20-25 s. It is not until blood flow is occuded for 40-60 s that most K+ is apparently resorbed and only a minor portion is released and is to be found in the venous blood. 5. The transient accumulation of muscle extra-cellular potassium may locally affect nerve endings, skeletal and smooth muscle cells.