Drug-dependent ion channel gating by application of concentration jumps using U-tube technique.

The rapid application system described has been used to study a variety of ionic channels in several different types of single cells. The system is inexpensive, easy to install, and can be used repeatedly. The consumption of UTS, i.e., drugs or agonists, is low. The time interval between switching the valve and the expected effect is often shorter than 150 ms for cells about 8-15 microns in diameter and is about 20-25 ms for patches positioned in the hole of the U-tube. Time and duration of substance application can be controlled by a computer connected to a digital-analog (D/A) output.

Electrophysiological demonstration of N-methyl-D-aspartate receptors at the afferent synapse of catfish electroreceptor organs.

An excitatory amino acid, most probably L-glutamate, acts as a neurotransmitter at the receptor cell--afferent fibre synapses in the ampullary electroreceptor organs of the freshwater catfish Ictalurus nebulosus. In the present study, we have used an electrophysiological approach to investigate the presence of N-methyl-D-aspartate receptors at this level. N-Methyl-D-aspartate, dissolved in an Mg(2+)-containing (normal) solution, had no effect on afferent activity, not even at 5 mM. However, addition of 5 mM N-methyl-D-aspartate to an Mg(2+)-free solution evoked an enduring increase in firing rate. The application of N-methyl-D-aspartate combined with electrical sine wave stimulation produced a firing increase in the primary afferents, even in the presence of Mg2+ (1.5 mM). Glycine (0.01-0.001 mM) significantly potentiated the N-methyl-D-aspartate responses. Addition of antagonists of the actions of N-methyl-D-aspartate, 7-chlorokynurenic acid, DL-2-amino-5-phosphonovaleric acid and ketamine in concentrations of 0.5-2.0 mM led to a decrease in resting and stimulus-evoked activity. 7-Chlorokynurenic acid also blocked the responses to application of N-methyl-D-aspartate. The glycine agonist D-serine (0.01 mM) prevented the 7-chlorokynurenic inhibitory effect. These results suggest the involvement of N-methyl-D-aspartate receptors in mediating the actions of L-glutamate at the afferent synapses of the electroreceptor organs of the catfish.

Synaptic noise in spike trains of normal and denervated electroreceptor organs.

The sequence of interspike intervals of the spontaneous activity in denervated electroreceptor organs of the catfish is analysed with several statistical techniques: visual inspection of dot displays, interval histograms, serial correlograms, tests for stationary and trends, and tests for renewal properties, based on the spectrum of intervals. The interspike interval train of primary afferents can usually be treated as a renewal process. Following denervation, a number of non-renewal properties emerge. The interval histogram of the renewal spike trains can be fitted well with a gamma probability density function; non-renewal cases need a more complex approach. We propose that the stochastic fluctuations in interval duration arise from randomly occurring quantal depolarizations (Stein's model of stochastic neuronal excitations. Two important properties with regard to synaptic transduction are: afferent firing results from capture of a limited number of depolarizing quanta; and the quantal input rate and the threshold for firing appear to be correlated.

The emergence of electroreceptor organs in regenerating fish skin and concurrent changes in their transduction properties.

The process of regeneration of skin patch denervated empullary electroreceptor organs of the African catfish Clarias gariepinus has been investigated at an ambient temperature of 28 degrees C with both electrophysiological and histological methods. At day 1 after denervation none of the receptor organs on the skin patch showed afferent activity. At this stage none of the ampullary organs previously recorded showed a normal appearance. Degenerative changes consisted of a decreased number of receptor cells and an often invisible lumen. At day 7 regeneration seems to start with a high density of primordial ampullary organs, more than a seven-fold increase compared to controls. In these units, the level of spontaneous activity is very low: compared to controls, more than a two-fold increase in mean interspike interval. At this stage, the sensitivity to electrical stimuli is already at the level of untreated control organs. At day 15 there is a lower, i.e. approximately normal, density of ampullary organs with a normal morphology. In these units both spontaneous firing and sensitivity returned to normal. It can be concluded that the functional dichotomy between spontaneous firing and sensitivity that was found in degenerating ampullary electroreceptor organs is also found during the process of their regeneration, although the underlying cellular changes may be totally different. The speed of recovery suggests that only regeneration of the distal part of the sectioned nerve fibers takes place.

Quantitative aspects of transduction in an electroreceptor organ studied by means of experimental manipulation of the interspike interval.

The interspike interval histogram of spontaneously firing electroreceptor organs of freshwater catfish (Ictalurus nebulosus LeS.) fits well with the probability density function of a gamma distribution. The shape parameter r of this probability density function can be used as a measure for the firing threshold of the spike generator, and the scale parameter lambda provides a measure for the input rate of synaptic quanta. Here, we have studied the physiological meaning of the two parameters of the fitted gamma probability density function by manipulating the mean firing rate. This was done in two ways: "frequency clamp" stimulation and a change in temperature. Frequency clamp stimulation, compensating for adaptation of the response, keeps the afferent firing rate at a fixed value for a limited period. We show that within such a period not only the mean spike rate remains constant, but these spike trains can be regarded as renewal series: the spike generator is functionally uncoupled from the frequency-dependent parts of the transduction path. Concerning the gamma parameters, two types of responses are found: at strong excitation lambda increases, r does not change; with small stimuli r is inversely proportional to the stimulus strength, lambda remains unchanged. This is difficult to explain when stimulus transduction acts via the synaptic input rate only. Stimulation seems to influence the firing threshold of the spike generator directly. At strong inhibition a regular firing pattern suddenly takes over; the major part of the variability suddenly disappears from the spike train. A change in temperature causes a shift in lambda (Q10 approximately 2.3), but not in r. This points to a temperature effect on the input rate only.

Caffeine reduces the efficacy of electroreceptor cell synapses: an electrophysiological single-unit in vivo study.

Ampullary electroreceptor organs of catfish, Ictalurus melas, were exposed apically to caffeine solutions at concentrations of 0, 5, 7.5, 10, and 15 mM. Recording sinusoidally-modulated activity of single-unit afferents reveals a dose-dependent decrease in mean afferent activity and sensitivity. A rebound effect of average activity occurs after caffeine is washed out. After 25 min exposure to 15 mM caffeine the peak of the gain curve shifts from 8 Hz to 4 Hz. The corresponding phase characteristic shows an increased phase lag with a maximum shift of 35 degrees at 20 Hz. The latency between stimulus and response increases from 12 to 19 ms; the recovery time after onset of the pulse decreases with 60 ms. The most probable explanation for the recorded effects is that caffeine reduces the availability of intracellular Ca2+ by blocking of the inositol triphosphate receptors in the endoplasmic reticulum. This in turn would affect many intracellular properties and processes. The unavailability of Ca2+ could reduce the synaptic efficacy and increase latency by suppressing fusion of synaptic vesicles with the presynaptic membrane and by depressing vesicle transport. The change in frequency response corresponds in part to reduction of the apical membrane surface area of the receptor cells, and in part to the increased latency. Accumulation of glutamate-containing vesicles could account for the higher mean activity and modulation amplitude in the lower frequency range after caffeine is washed out. Caffeine might act postsynaptically by inducing hyperpolarization of the terminals of the primary afferents.

Converging electroreceptor cells improve sensitivity and tuning.

We studied the effect of convergent clustering of ampullary electroreceptor organs on stimulus transduction and transmission in the catfish Icalurus melas by electrophysiologically recording primary afferent activity of single ampullae (singlets) and pairs (doublets) innervated by the same afferent. Doublets were twice as sensitive as singlets, and showed sharper tuning around the best frequency. The slope of the phase curve in the doublets was slightly steeper than in the singlets. The spontaneous activity and scatter in interspike interval were not correlated with clustering. The implications of these findings for signal averaging in sensory neurons and their relevance for behaviour are discussed.

Identification of AMPA receptors in catfish electroreceptor organs.

Single afferent unit recording in microampullae of the catfish revealed that bath-applied AMPA increases both resting discharge frequency and electrically evoked responses. The potency of AMPA is of the order of 10 microM. DNQX strongly inhibits the excitatory effects of AMPA. The results suggest the presence of AMPA receptors at the synaptic membrane of ampullary electroreceptor organs in the catfish.

In vitro electroreceptor organs for pharmacological studies.

Electroreception is a well-established sensory faculty in aquatic vertebrates. The general physiology of the receptor organs is comprehensively documented. The transduction mechanism of the receptor cells and the synaptic transmission mechanism are less well understood. Research has been hampered by the inaccessibility of the synaptic site. This paper describes how to prepare an in vitro preparation of ampullary electroreceptor organs which allows exposure of both the mucosal and the serosal sides of the receptor cells to superfusion of test solutions. The preparation is quite robust and has been shown to function reliably for more than 8 h. Furthermore, the use of in vitro electroreceptors organs as a model for pharmacological studies is evaluated.

The electrosensitivity of the lateral line of the European eel, Anguilla anguilla L.

The lateral line neuromast of the eel (Anguilla anguilla) are slightly sensitive to electric current (threshold current density about 1 x 10(-6) A/cm2). A comparison is made with threshold values found by several authors by means of conditioning experiments.

Functioning of catfish electroreceptors: relation between skin potential and receptor activity.

Properties of catfish electroceptors were investigated by simultaneous recording of the skin potential and the activity of an afferent nerve. 1. The normal threshold stimulus intensity induces a potential amplitude of about 10 to 30 muV across the skin (Table I). 2. The average spike frequency in the nerve increases approximately with the logarithm of the stimulus intensity (Fig. 1). 3. The direct current restoring the receptor activity in calcium deficient media makes the skin potential more negative. 4. Presumably, not the skin potential itself but a difference between the skin potential and the e.m.f. generated by the receptor epithelium influences receptor functioning. 5. Amplitude and phase characteristics can be described by a filter circuit (Fig. 4 and 5).

Nonselective cationic currents elicited by extracellular ATP in human B-lymphocytes.

Adenosine 5'-triphosphate-(ATP)-induced whole-cell currents were studied in human B-lymphocytes, transformed by the Epstein-Barr virus, by means of the tight-seal voltage-clamp technique. During bath application of ATP, the membrane conductance was increased. The change of membrane conductance occurred within milliseconds. The dose response relationship for the ATP(4-)-elicited membrane current (Ip) was fitted by the Hill function with a Hill coefficient of 1 and a KD value of 0.2 mmol/l. Adenosine, as well as the Mg(2+)-bound form of ATP, did not effect the membrane conductance. Ip did not desensitize within 1 min and could be evoked repeatedly up to 100 times in 1 cell in the presence of the G-protein blocker Guanosine 5'-o-(2-thiodiphosphate) (GDP [beta S]). Therefore, it seems that ion channels in form of P2Z-purinoceptors are involved in the observed effects. The permeability (P) sequence for cations carrying Ip was PCa:PK:PCs:PNa:PTRIS = 35:2:1.2:1:0.1. The reversal potential of IP was not changed by substitution of intracellular Cl- for aspartate, indicating that anions are not involved in the purinoceptor-dependent conductance. A single-channel conductance of P2Z-receptor-dependent ion channels of about 3 pS was determined by noise analysis of Ip.

Spontaneous nerve activity and sensitivity in catfish ampullary electroreceptor organs after tetanus toxin application.

The functioning of electroreceptor organs of Ictalurus sp. was investigated by inhibiting synaptic transmission by the administration of tetanus toxin in vitro. A piece of Ictalurus skin of about 20 mm diameter was mounted in an Ussing-type chamber. After establishing the normal functioning of the organ, tetanus toxin (TeTx) was applied basolaterally for 150 min in 66.7 pM and 400 pM concentrations, while the single unit nerve activity was recorded extracellularly. Spontaneous spike activity and the sensitivity of the electroreceptor organs were measured. The results show that TeTx reduces sensitivity to less then 20% of its original value, whereas the spontaneous activity is unaffected by the treatment. This indicates that the afferent nerve is capable of generating impulses independent of receptor cell neurotransmitter release. In the discussion we suggest two alternative mechanisms for the emergence of the spontaneous spike activity.

External tetraethylammonium as a molecular caliper for sensing the shape of the outer vestibule of potassium channels.

External tetraethylammonium (TEA+) blocked currents through Kv1.1 channels in a voltage-independent manner between 0 and 100 mV. Lowering extracellular pH (pHo) increased the Kd for TEA+ block. A histidine at position 355 in the Kv1.1 channel protein (homologous to Shaker 425) was responsible for this pH-dependent reduction of TEA+ sensitivity, since the TEA+ effect became independent of pHo after chemical modification of the Kv1.1 channel at H355 and in the H355G and H355K mutant Kv1.1 channels. The Kd values for TEA+ block of the two mutant channels (0.34 +/- 0.06 mM, n = 7 and 0.84 +/- 0. 09 mM, n = 13, respectively) were as expected for a vestibule containing either no or a total of four positive charges at position 355. In addition, the pH-dependent TEA+ effect in the wt Kv1.1 channel was sensitive to the ionic strength of the solution. All our observations are consistent with the idea that lowering pHo increased protonation of H355. This increase in positive charge at H355 will repel TEA+ electrostatically, resulting in a reduction of the effective [TEA+]o at the receptor site. From this reduction we can estimate the distance between TEA+ and each of the four histidines at position 355 to be approximately 10 A, assuming fourfold symmetry of the channel and assuming that TEA+ binds in the central axis of the pore. This determination of the dimensions of the outer vestibule of Kv1.1 channels confirms and extends earlier reports on K+ channels using crystal structure data as well as peptide toxin/channel interactions and points out a striking similarity between vestibules of Kv1.1 and KcsA channels.

Expression in mammalian cells and electrophysiological characterization of two mutant Kv1.1 channels causing episodic ataxia type 1 (EA-1).

Episodic ataxia type 1 (EA-1) is a rare neurological disorder and was the first ionic channel disease to be associated with defects in a potassium channel. Until now 10 different point mutations in the KCNA1-gene have been reported to cause this disorder. We have investigated the functional consequences of two mutations leading to amino acid substitutions in the first and sixth transmembrane segments of a Kv1.1 channel subunit, by means of the patch-clamp technique; we injected cRNA coding for, respectively, F184C and V408A mutant Kv1.1 channels into mammalian cells and compared the resulting currents with those in the wild-type. The expression levels of F184C and V408A mutant channels relative to that of the wild-type was 38 and 68%, respectively. Since the single-channel conductance of the F184C mutant was similar to that of the wild-type (12 pS) without an apparent change in the maximum open probability, we conclude that the lower expression level in the F184C mutant channels is due to a reduced number of functional channels on the cell surface. F184C activated slower, and at more depolarized potentials, and deactivated faster compared with the wild-type. V408A channels deactivated and inactivated faster compared with the wild-type. Studies with different extracellular cations and tetraethylammonium gave no indication that the pore structure was changed in the mutant channels. Acetazolamide, that is helpful in some patients suffering from EA-1, was without effect on Kv1.1 wild-type or mutant channels. This study confirms and extends earlier studies on the functional consequences of Kv1.1 mutations associated with EA-1, in an attempt to understand the pathophysiology of the disease.

Ultrastructure of free nerve endings in respiratory and squamous epithelium on the rat nasal septum.

The distribution of nerve fibres in the mucosa of the nasal septum of the rat was investigated by means of transmission electron microscopy on transverse and tangential ultrathin sections. Near the basement membrane of respiratory and squamous epithelium, a rather dense network of unmyelinated nerve fibres occurs. Some fibres in the respiratory epithelium ascend between the epithelial cells to reach up to the tight junctions. These fibres appeared in transverse sections to end as hooks or boutons, sometimes with branches. These shapes resemble the free nerve endings that are considered to act as nociceptors. The small intraepithelial fibres, with diameters of about 0.5-1 microns, contain both dense granules and clear vesicles comparable to synaptic vesicles. Substance P was found in dense granules in basal fibres; vasoactive intestinal peptide was absent throughout the epithelium. Acetylcholinesterase activity was observed closely associated with the basal fibres; the apical fibres showed little if any activity. Membrane specializations pointing to an efferent function as well as structures usually associated with mechanoreceptive functions were lacking in both respiratory and squamous epithelium.

Simultaneous measurements of calcium mobilization and afferent nerve activity in electroreceptor organs of anesthetized Kryptopterus bicirrhis.

The transduction pathway of ampullary electroreceptor organs involves ionic currents. It has been shown that calcium, as well as sodium and potassium play important parts in this process. In this study we examine the stimulus-evoked changes in the Fura-2 ratio in electroreceptor cells. Furthermore, we recorded stimulus-evoked Fura-2 ratio changes while Na+ and K+ channels were blocked by amiloride and TEA. Simultaneously, extracellular recordings of the afferent spike activity were made. The results show the presence of stimulus evoked fluctuations in the Fura-2 ratio. These fluctuations can be abolished by the application of Cd2+, TEA, and amiloride. The stimulus-evoked activity of the afferent nerve was decreased due to application of these drugs. We conclude that the transduction current is carried by Na+, K+, and probably Ca2+. This fits the existing model on transduction in electroreceptors.

Ampullary electroreceptors in catfish (Teleostei): temperature dependence of stimulus transduction.

The response properties of ampullary electroreceptors have been studied in the catfish Ictalurus nebulosus at skin temperatures between 5 and 35 degrees C. A unimodal relationship between spontaneous activity and temperature was obtained. Mean (+/- SEM) peak discharge rate was 57.3 +/- 1.8 impulses s-1 at 25 degrees C; the receptors were active at 5 degrees C (15.0 impulses s-1) and at 35 degrees C (31.5 impulses s-1). There were no dynamic responses to temperature changes in either the warming or cooling direction. The shape of the frequency characteristic depended on temperature: the peak of the gain curve shifted to low frequencies at low temperatures. There was a concomitant change of the phase characteristic: the intersection at zero degree phase angle shifted to higher frequencies with an increase of temperature, thus increasing the lead at lower frequencies and decreasing the lag at higher frequencies. Latency after combined excitatory and inhibitory impulse stimulation was temperature dependent, ranging from 16.4 ms (5 degrees C) to 5.6 ms (35 degrees C). Application of the specific calcium channel blocker menthol (0.2 mM) suppressed spontaneous activity, the effect becoming more prominent at higher temperatures. Sensitivity to sinusoidal electrical stimulation was also impaired, but to a lesser degree and mainly at lower temperatures. We conclude that the filter properties of the receptor organ can be modelled by a band-pass filter in series with a latency, both of which are temperature dependent. These filter properties might be partially based on the activation kinetics of the transduction channels.

Biomonitoring: cadmium deteriorates electro-orientation performance in catfish.

1. Exposure of catfish, Ictalurus nebulosus, to sublethal concentrations of cadmium deteriorates electro-orientation performance. 2. Cadmium, at a concentration of 40 micrograms/l, doubles the behavioural threshold for electric stimuli within 48 hr of exposure; both prolonged exposure and higher concentrations result in higher thresholds. The effect is reversible. 3. Electro-orientation performance can be used to monitor the quality of surface water.