Cyclic AMP modulates electrical signaling in a weakly electric fish

Many species of electric fish show diurnal or socially elicited variation in electric organ discharge amplitude. In Sternopygus macrurus, activation of protein kinase A by 8-bromo-cAMP increases electrocyte sodium current magnitude. To determine whether the behavioral plasticity in electric organ discharge amplitude is controlled by electrocyte biophysical properties, we examined whether the effects of phosphorylation on ion currents in the electric organ translate directly into electric organ discharge changes. We injected the electric organ of restrained fish with 8-bromo-cAMP and monitored the electric organ discharge. The effect of protein kinase A activation on electrocyte action potentials was examined in isolated electric organ using two-electrode current clamp. Electric organ discharge and action potential amplitude and pulse duration increased in response to 8-bromo-cAMP. Pulse and action potential duration both increased by about 25%. However, the increase in electric organ discharge amplitude (approximately 400%) was several-fold greater than the action potential amplitude increase (approximately 40%). Resting membrane resistance decreased in electrocytes exposed to 8-bromo-cAMP. We propose that in the Thevenin equivalent circuit of the electric organ a moderate increase in action potential amplitude combined with a decrease in internal resistance produces a greater voltage drop across the external resistance (the water around the fish), accounting for the large increase in the externally recorded electric organ discharge.     

Waveform generation in Rhamphichthys rostratus (L.) (Teleostei,Gymnotiformes)

Rhamphichthys rostratus (L.) emits brief pulses (2 ms) repeated very regularly at 50 Hz. The electric organ shows a heterogeneous distribution of the electrocyte tubes and the occurrence of three electrocyte types (caudally innervated, rostrally innervated and marginallycaudally innervated). In the sub-opercular region the electric organ consists of a pair of tubes containing only caudally innervated electrocytes. At the abdominal region the EO consists of three pairs of tubes. Each pair contains one of the described electrocyte types. The number of electrocyte tubes increases toward the tail to reach nine or ten pairs in the most caudal segments. In the intermediate region most tubes contain doubly innervated electrocytes except the ventral pair that contains caudally innervated electrocytes. The caudal 25% contains exclusively caudally innervated electrocytes. The electric organ discharge consists of five wave components (V1 to V5). Electrophysiological data are consistent with the hypothesis that V1 results from the activity of the rostral faces of rostrally innervated electrocytes. V2 results from the activities of rostral faces of marginally-caudally innervated electrocytes while V3 results from the activities of caudal faces of most electrocytes. Curarization experiments demonstrated that V4 and V5 result from action potential invasion and are not directly elicited by neural activity.Abbreviations AEN1 anterior electromotor nerve 1 - AEN2 anterior electromotor nerve 2 - BMB boraxic methylene blue - CIE caudally innervated electrocytes - EMF electromotive force - EO electric organ - EOD electric organ discharge - I current amplitude - MCIE marginally-caudally innervated electrocytes - MT medial tubes - PEN posterior electromotor nerve - R _n internal impedance - RIE rostrally innervated electrocytes - Rl load resistor - SAT short abdominal tubes - V voltage amplitude     

Sodium-dependent plateau potentials in electrocytes of the electric fish <Emphasis Type="Italic">Gymnotus carapo</Emphasis>

The weakly electric fish Gymnotus carapo emits a triphasic electric organ discharge generated by muscle-derived electrocytes, which is modified by environmental and physiological factors. Two electrode current clamp recordings in an in vitro preparation showed that Gymnotus electrocytes fired repetitively and responded with plateau potentials when depolarized. This electrophysiological behavior has never been observed in electrocytes from related species. Two types of plateaus with different thresholds and amplitudes were evoked by depolarization when Na⁺-dependent currents were isolated in a K⁺- and Ca²⁺-free solution containing TEA and 4-AP. Two electrode voltage clamp recordings revealed a classical fast activating–inactivating Na⁺ current and two persistent Na⁺-dependent currents with voltage-dependencies consistent with the action potential (AP) and the two plateaus observed under current clamp, respectively. The three currents, the APs and the plateaus were reduced by TTX, and were absent in Na⁺-free solution. The different Na⁺-dependent currents in Gymnotus electrocytes may be targets for the modifications of the electric organ discharge mediated by environmental and physiological factors.     

Androgens alter electric organ discharge pulse duration despite stability in electric organ discharge frequency.

Weakly electric fish in the genus Sternopygus emit a sinusoidal, individually distinct, and sexually dimorphic electric organ discharge (EOD) that is used in electrolocation and communication. Systemically applied androgens decrease EOD frequency, which is set by a medullary pacemaker nucleus, and increase pulse duration, which is determined by the cells of the electric organ (the electrocytes), in a coordinated fashion. One possibility is that androgens broaden the EOD pulse duration by acting on the pacemaker neurons, thereby effecting a change in pacemaker firing frequency, and that the change in EOD pulse duration is due to an activity-dependent process. To determine whether androgens can alter pulse duration despite a stable pacemaker nucleus firing frequency, we implanted small doses of dihydrotestosterone in the electric organ. We found that androgen implants increased EOD pulse duration, but did not influence EOD frequency. In addition, using immunocytochemistry, we found that electrocytes label positively with an androgen receptor antibody. While it is not known on which cells androgens act directly, together these experiments suggest that they likely act on the electrocytes to increase EOD pulse duration. Since pulse duration is determined by electrocyte action potential duration and ionic current kinetics, androgens may therefore play a causative role in influencing individual variation and sexual dimorphism in electrocyte electrical excitability, an important component of electrocommunicatory behavior.     

A temporal analysis of testosterone-induced changes in electric organs and electric organ discharges of mormyrid fishes

The electric organ discharge (EOD) of several species of mormyrid fishes within the genus Brienomyrus is sexually dimorphic during the breeding season: the duration of the male's EOD is much longer than the duration of the female's (for a review see Hopkins, 1986). The mormyrid used here, Brienomyrus sp., exhibits similar alterations in the duration of the triphasic EOD after treatment with testosterone, as do other members of this genus (for reviews see Bass, 1986a,b). In this experiment, animals were intraperitoneally implanted with pellets of either 11-ketotestosterone or 17 a-methyltestosterone, and the time course of the changes in the duration of each of the three phases of the EOD were quantified. Additionally, the time course of changes in the morphology of the electric organ, after testosterone treatment, was also quantified using electron microscopic techniques. The results suggest that the change in the duration of the first phase of the EOD is due exclusively to the change in the thickness of the electrocyte body: this is consistent with a model proposed by Bennett and Grundfest (1961) for the electrogenesis of a triphasic EOD. Changes in the duration of the second and third phases of the EOD are highly correlated with the changes in the surface area of the posterior and anterior faces of the electrocyte, respectively. The results support the hypothesis that gonadal steroid hormone-induced changes in the EOD are due to structural changes in the electrocyte's membranes, and that all of the observed changes in the discharge of this system can be explained by the action of steroid hormones on the peripheral target cells (electrocytes).     

Electric organ morphology of Sternopygus macrurus, a wave-type, weakly electric fish with a sexually dimorphic EOD.

In several species of electric fish with a sex difference in their pulse-type electric organ discharge (EOD), the action potential-generating cells of the electric organ (electrocytes) of males are larger and more invaginated compared to females. Androgen treatment of females and juveniles produces a longer-duration EOD pulse that mimics the mature male EOD, with a concurrent increase in electrocyte size and/or membrane infolding. In Sternopygus macrurus, which generates a wave-type EOD, androgen also increases EOD pulse duration. To investigate possible morphological correlates of hormone-dependent changes in EOD in Sternopygus, we examined electric organs from both fish collected in the field, and untreated and androgen-treated specimens in the laboratory. The electrocytes are cigar shaped, with prominent papillae on the posterior, innervated end. Electrocytes of field-caught specimens were significantly larger in all parameters than were electrocytes of specimens maintained in the laboratory. EOD pulse duration and frequency were highly correlated, and were significantly different between the sexes in sexually mature fish. Nevertheless, no sex difference in electrocyte morphology was observed, nor did any parameters of electrocyte morphology correlate with EOD pulse duration or frequency. Further, whereas androgen treatment significantly lowered EOD frequency and broadened EOD pulse duration, there was no difference in electrocyte morphology between hormone-treated and control groups. Thus, in contrast to results from studies on both mormyrid and gymnotiform pulse fish, electrocyte morphology is not correlated with EOD waveform characteristics in the gymnotiform wave-type fish Sternopygus. The data, therefore, suggest that sex differences in EOD are dependent on changes in active electrical properties of electrocyte membranes.     

兔肺静脉肌袖心肌细胞动作电位的特性和一些离子流机制

研究兔肺静脉肌袖心肌细胞（cardiomyocytes from rabbit pulmonary vein sleeves, PVC）动作电位的特性和一些离子流机制——内向整流钾电流（IKl）、瞬时外向钾电流（ITo）和非选择性阳离子流（I NSCC）,并与左心房心肌细胞（left atrial cardiomyocytes,LAC）进行比较。采用全细胞膜片钳技术,记录动作电位和上述各离子流。发现PVC动作电位时程（action potential duration,APD）较LAC的明显延长,并可以诱发出第二平台反应。PVC上存在I NSCC. PVC的IKl、I To和I NSCC电流密度均较LAC的明显减小。PVC和LAC存在复极离子流的差异,这种差异构成了两者动作电位差异的基础,进而可能成为肺静脉肌袖致心律失常特性的重要离子流机制。     

Electric organ morphology of Sternopygus macrurus,a wave-type,weakly electric fish with a sexually dimorphic EOD

In several species of electric fish with a sex difference in their pulse-type electric organ discharge (EOD), the action potential-generating cells of the electric organ (electrocytes) of males are larger and more invaginated compared to females. Androgen treatment of females and juveniles produces a longer-duration EOD pulse that mimics the mature male EOD, with a concurrent increase in electrocyte size and/or membrane infolding. In Sternopygus macrurus, which generates a wave-type EOD, androgen also increases EOD pulse duration. To investigate possible morphological correlates of hormone-dependent changes in EOD in Sternopygus, we examined electric organs from both fish collected in the field, and untreated and androgen-treated specimens in the laboratory. The electrocytes are cigar shaped, with prominent papillae on the posterior, innervated end. Electrocytes of field-caught specimens were significantly larger in all parameters than were electrocytes of specimens maintained in the laboratory. EOD pulse duration and frequency were highly correlated, and were significantly different between the sexes in sexually mature fish. Nevertheless, no sex difference in electrocyte morphology was observed, nor did any parameters of electrocyte morphology correlate with EOD pulse duration or frequency. Further, whereas androgen treatment significantly lowered EOD frequency and broadened EOD pulse duration, there was no difference in electrocyte morphology between hormone-treated and control groups. Thus, in contrast to results from studies on both mormyrid and gymnotiform pulse fish, electrocyte morphology is not correlated with EOD waveform characteristics in the gymnotiform wave-type fish Sternopygus. The data, therefore, suggest that sex differences in EOD are dependent on changes in active electrical properties of electrocyte membranes. © 1992 John Wiley & Sons, Inc.     

Effect of sotalol on transmembrane ionic currents responsible for repolarization in cardiac ventricular myocytes from rabbit and guinea pig

The effects of sotalol, a β-adrenoceptor blocker and class III antiarrhythmic agent, on transmembrane ionic currents were examined in single rabbit and guinea pig ventricular myocytes using whole-cell voltage-clamp techniques. In neither of these species did 60 μM sotalol appreciably effect the inward rectifier, the transient outward or the inward calcium currents. In addition, sotalol did not elicit a slowly inactivating component of the sodium current as did 1 μg/ml veratrine. In guinea pig ventricular myocytes, sotalol also significantly depressed the outward delayed rectifier current. An outward delayed rectifier current was not observed in rabbit ventricular myocytes examined at room temperature; and, under these conditions sotalol did not lengthen action potential duration. Sotalol induced lengthening of cardiac action potential duration can, therefore, be explained by depression the outward delayed rectifier current.     

Plasmalemmal,voltage-dependent ionic currents from excitable pulvinar motor cells ofMimosa pudica

Summary Plasmalemmal ionic currents from excitable motor cells of the primary pulvinus ofMimosa pudica were investigated by patch-clamp techniques. In almost all of the enzymatically isolated protoplasts, a delayed rectifier potassium current was activated by depolarization, while no currents were detected upon hyperpolarization. This sustained outward current was reversibly blocked by Ba and TEA and serves to repolarize the membrane potential. Outward single channel currents that very likely underly the macroscopic outward potassium current had an elementary conductance of 20 pS. In addition, in a few protoplasts held at hyperpolarized potentials, depolarization-activated transient inward currents were observed, and under current clamp, action potential-like responses were triggered by depolarizing current injections or by mechanical perturbations. The activation characteristics of both inward currents and spikes showed striking similarities compared to those of action potentialsin situ.     

Ionic currents that contribute to a sexually dimorphic communication signal in weakly electric fish

Weakly electric fish produce a communication signal, the electric organ discharge, that is species specific, and in many species, sexually dimorphic. Because the neural circuit that controls the electric organ discharge is relatively simple, it is an excellent model in which to study both the biophysical mechanisms underlying a rhythmic behavior and the neuroendocrine control of a sexually dimorphic behavior. By studying the effects of ion channel blockers on neurons in the medullary pacemaker nucleus, I pharmacologically characterized three ionic currents that influence the pacemaker rhythm, and thus electric organ discharge frequency, in the gymnotiform fish, Apteronotus leptorhynchus. These currents included a tetrodotoxin-sensitive sodium current; a potassium current that was sensitive to 4-aminopyridine; and a calcium current that was sensitive to nickel and cadmium, but resistant to specific blockers of L-, N-, P-, and Q-type calcium currents. The pharmacological profiles of the ionic currents in the pacemaker nucleus are similar to those of ionic currents involved in pacemaking in other neuronal oscillators. Because these ionic currents dramatically influence pacemaker firing frequency, which is directly related to electric organ discharge frequency, these ionic currents are likely targets of steroid hormone action in producing sexual dimorphisms in electric organ discharge frequency. Additional studies are needed to determine how these ionic currents interact to generate the electric organ discharge rhythm and to investigate the possibility that sexual dimorphism in the electric organ discharge results from the actions of gonadal steroids on these ionic currents. Accepted: 3 June 1999     

血管紧张素Ⅱ对缺血心肌细胞钾离子通道的作用

实验用胶原酶酶解法急性分离豚鼠心室肌细胞,利用全细胞膜片钳的方法记录心室肌细胞的延迟整流钾电流(Ik）、内向整流钾电流（Ik1）和ATP敏感钾电流（IKATP）。采用低氧、无糖、高乳酸和酸中毒综合方式模拟缺血灌流,造成细胞的模拟缺血,并在缺血的基础上继续用含100nmol/L AngⅡ灌流细胞,观察Ang Ⅱ对模拟缺血心室肌细胞钾离子通道的影响。实验结果显示：（1）模拟缺血时,Ik明显减小;Ang Ⅱ能进一步抑制Ik。（2）模拟缺血条件下,Ik1受到抑制,并且以内向电流的抑制为主;Ang Ⅱ可加强对Ik1内向电流的抑制,而对部分外向电流则有增加的作用。（3）模拟缺血使IKATP外向电流略有增加;Ang Ⅱ则明显加强IKATP外向电流,此效应能被优降糖所阻断。     

Slow components of potassium tail currents in rat skeletal muscle

The kinetics of potassium tail currents have been studied in the omohyoid muscle of the rat using the three-microelectrode voltage-clamp technique. The currents were elicited by a two-pulse protocol in which a conditioning pulse to open channels was followed by a test step to varying levels. The tail currents reversed at a single well-defined potential (VK). At hyperpolarized test potentials (-100 mV and below), tail currents were inward and exhibited two clearly distinguishable phases of decay, a fast tail with a time constant of 2-3 ms and a slow tail with a time constant of approximately 150 ms. At depolarized potentials (-60 mV and above), tail currents were outward and did not show two such easily separable phases of decay, although a slow kinetic component was present. The slow kinetic phase of outward tail currents appeared to be functionally distinct from the slow inward tail since the channels responsible for the latter did not allow significant outward current. Substitution of Rb for extracellular K abolished current through the anomalous (inward-going) rectifier and at the same time eliminated the slow inward tail, which suggests that the slow inward tail current flows through anomalous rectifier channels. The amplitude of the slow inward tail was increased and VK was shifted in the depolarizing direction by longer conditioning pulses. The shift in VK implies that during outward currents potassium accumulates in a restricted extracellular space, and it is suggested that this excess K causes the slow inward tail by increasing the inward current through the anomalous rectifier. By this hypothesis, the tail current slowly decays as K diffuses from the restricted space. Consistent with such a hypothesis, the decay of the slow inward tail was not strongly affected by changing temperature. It is concluded that a single delayed K channel is present in the omohyoid. Substitution of Rb for K has little effect on the magnitude or time course of outward current tails, but reduces the magnitude and slows the decay of the fast component of inward tails. Both effects are consistent with a mechanism proposed for squid giant axon (Swenson and Armstrong, 1981): that (a) the delayed potassium channel cannot close while Rb is inside it, and (b) that Rb remains in the channel longer than K.     

Augmentation and suppression of action potentials by estradiol in the myometrium of pregnant rat.

The purpose of this study was to investigate the actions of estradiol on spontaneous and evoked action potentials in the isolated longitudinal smooth muscle cells of the pregnant rat. Single cells were obtained by enzymatic digestion from pregnant rat longitudinal myometrium. Action potentials and currents were recorded by whole-cell current-clamp and voltage-clamp methods, respectively. The acute effects of 17beta-estradiol on action potentials and inward and outward currents were investigated. The following results were obtained. The average resting membrane potential of single myometrial cells was -54 mV (n = 40). In many cells, an electrical stimulation evoked a membrane depolarization, and action potentials were superimposed on the depolarization. In some cells, spontaneous action potentials were observed. Estradiol (30 microM) slightly depolarized the membrane (ca. 5 mV) and attenuated the generation of action potentials by reducing the frequency and amplitude of the spikes. Afterhyperpolarization was also attenuated by estradiol (30 microM). On the other hand, in 5 of 35 cells, estradiol increased the first spike amplitude and action potential duration, while frequency of the spike generation and afterhyperpolarization were inhibited. In voltage-clamped muscle cells, estradiol inhibited both inward and outward currents. Acute inhibition or augmentation of spike generation by estradiol is due to the balance of inhibition of inward and outward currents. Inhibition of both currents also prevented afterhyperpolarization, causing potential-dependent block of Ca spikes.     

Current clamp and voltage clamp study of the inhibitory action of DNP on membrane electrical properties of frog auricular heart muscle.

Current clamp studies showed that after 10 minutes under DNP 10(-4) M the membrane potential does not change significantly while an important shortening of the action potential duration and a diminished amplitude are observed. Voltage clamp studies have been performed on the slow inward and delayed outward currents. DNP 10(-4) M induced a marked decrease of the slow inward current related to the reduction in both conductance and driving force, and a decrease in the amplitude of the delayed current. The decrease of the slow inward current seems to be mainly responsible for the suppression of the plateau of the action potential during metabolic inhibition.     

Melanocortins regulate the electric waveforms of gymnotiform electric fish

The hypothalamic-pituitary-adrenal/interrenal axis couples serotonergic activity in the brain to the peripheral regulators of energy balance and response to stress. The regulation of peripheral systems occurs largely through the release of peptide hormones, especially the melanocortins (adrenocorticotropic hormone [ACTH] and alpha melanocyte stimulating hormone [α-MSH]), and beta-endorphin. Once in circulation, these peptides regulate a wide range of processes; α-MSH in particular regulates behaviors and physiologies with sexual and social functions. We investigated the role of the HPI and melanocortin peptides in regulation of electric social signals in the gymnotiform electric fish, Brachyhypopomus pinnicaudatus. We found that corticotropin releasing factor, thyrotropin-releasing hormone, and α-MSH, three peptide hormones of the HPI/HPA, increased electric signal waveform amplitude and duration when injected into free-swimming fish. A fourth peptide, a synthetic cyclic-α-MSH analog attenuated the normal circadian and socially-induced EOD enhancements in vivo. When applied to the electrogenic cells (electrocytes) in vitro, only α-MSH increased the amplitude and duration of the electrocyte discharge similar to the waveform enhancements seen in vivo. The cyclic-α-MSH analog had no effect on its own, but blocked or attenuated α-MSH-induced enhancements in the single-cell discharge parameters, demonstrating that this compound functions as a silent antagonist at the electrocyte. Overall, these results strongly suggest that the HPI regulates the EOD communication signal, and demonstrate that circulating melanocortin peptides enhance the electrocyte discharge waveform.     

Effects of quinidine and lidocaine on action potential and membrane currents of frog ventricles

The effects of quinidine and lidocaine on frog ventricle were studied by using a single sucrose gap voltage clamp technique. In Ca2+-Ringer, quinidine (80 microM) caused slight prolongation of action potential duration (APD50) and significant inhibition of twitch tension. Lidocaine (40 microM) shortened APD50 without significant effect on twitch tension. In tetrodotoxin (TTX)-treated preparations, quinidine caused significant prolongation of APD50 from 529 +/- 19 msec to 597 +/- 11 msec, (n = 9) and inhibition of twitch tension, but lidocaine did not affect APD50 and twitch tension. Under voltage clamp condition, quinidine reduced peak inward current in the absence of TTX, but enhanced peak inward current in the presence of TTX. The steady state outward current was increased by quinidine. Lidocaine didn't affect peak inward current in the absence or in the presence of TTX. Membrane current through the inward rectifier (IK1) was slightly increased by lidocaine, but significantly inhibited by quinidine. The enhancement of peak inward current by quinidine was retarded or reversed in preparation bathed with Sr2+-Ringer. When Ni2+ was added to a preparation bathed in Ca2+-Ringer, an inhibition of calcium inward current and action potential plateau was observed. The spike amplitude of the action potential was, however, unaffected by Ni2+. In this Ni2+-treated preparation, lidocaine (20 microM) caused significant shortening of APD50 without significant effect on action potential amplitude. The shortening of APD50 was associated with a slight increase of steady state outward current. The increase of steady state outward current by lidocaine was absent in the TTX-treated preparation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ionic channels in a line of embryonal carcinoma cells induced to undergo neuronal differentiation.

The gigaseal patch clamp technique was used to investigate the electrophysiological properties of a line of embryonal carcinoma cells (PCC4) that were induced to undergo neuronal differentiation. A large increase in number of voltage-dependent potassium and sodium channels was observed during differentiation. The pharmacology and kinetics of the macroscopic sodium and potassium currents in the differentiated cells closely resembled those of the rapid inward sodium current and the delayed rectifier, respectively. The kinetic behavior of single-channel potassium currents was consistent with the properties of the macroscopic delayed rectifier current.     

巨孔匙(虫戚)(Megathura)未受精卵细胞膜离子流的特征

用双微电极电压钳技术在巨孔匙(虫戚)(Megathura)未受精卵细胞膜上记录到多种离子流。主要有一种内向的两价离子流和几种钾离子流:包括钡离子激活的钾离子流,迅速激活又迅速失活的钾离子流(类似于I_A)和异常整流钾离子流。不同细胞的离子流大小不同。在一些卵可能会缺少其中某一种离子流。此外,还观察到浴槽溶液中氯和钠离子浓度改变对膜电位及膜电导的影响。