Afterdepolarization mechanism in the in vitro, cesium-loaded, sympathetic preganglionic neuron of the cat

Intracellular recordings were performed in Cs-loaded sympathetic preganglionic neurons (SPNs) of the intermediolateral nucleus, identified by antidromic stimulation, in the slice of the T2 or T3 segment of the cat spinal cord. Loading the neurons with Cs resulted in broadening of the action potential, depression of the fast component of the afterhyperpolarization (AHP), and appearance of an afterdepolarization (ADP). A typical ADP in a Cs-loaded neuron had time to peak of 45-110 ms, half-decay time of 70-250 ms, and amplitude of 2-10 mV at membrane potentials between -60 and -70 mV and at a Ca and K concentration of 2.5 and 3.6 mM, respectively, in the superfusion medium. The ADP was associated with a decrease in neuron input resistance and increased in magnitude with hyperpolarization of the cell membrane. The relation between peak ADP amplitude and membrane potential was linear within the range of membrane potentials from -60 to -100 mV. The ADP was reversibly suppressed by the Ca-channel blocker cobalt (2 mM) or by low Ca Krebs solution (0.25 mM). Superfusion with BaCl2 (1.0 mM) or tetraethylammonium (TEA) (10-20 mM) caused an increase in amplitude of the ADP and an increase in action potential duration. Hyperpolarizing pulses, delivered during the course of the spike shoulder, resulted in a decrease of spike duration and ADP amplitude. The ADP was not affected by tetrodotoxin, at a dose blocking the Na-spike, and was enhanced, in association with an increase in action potential duration, when NaCl in the Krebs solution was replaced with choline chloride. Increasing intracellular Cl concentration or decreasing extracellular Cl concentration had no effect on the ADP. Changes in external K concentration from 3.6 to 10 or 0.36 mM increased and decreased, respectively, the amplitude of the ADP. In the absence of Cs, and ADP, with similar time course to that recorded in Cs-loaded SPNs, was recorded when CaCl2 was replaced by BaCl or NaCl was replaced by TEAC1. It is concluded that the SPN afterpotential includes a Ca-dependent inward current, in addition to the already described fast and slow outward K currents of the AHP.     

Effects of sodium depletion on contractions evoked in intact and skinned muscles of the guinea-pig mesenteric artery

In the guinea-pig mesenteric artery, reduction in [Na]o by 30 mM (substituted by choline or sucrose; 137 mM [Na]o in Krebs solution) generated contraction with no change in membrane potential. In NaCl-free solution (15 mM [Na]o), the amplitude of phasic contraction reached 0.8 times the contraction evoked by 118 mM [K]o with only a slight depolarization. In NaCl-free solution, the amplitude of phasic contraction evoked by noradrenaline (NA) 5 X 10(-5) M or caffeine 5 mM increased to roughly twice the amplitude of the contraction evoked in the control solution. In Ca-free solution, the K-, NaCl-free- or Na-free-induced contractions rapidly ceased, but NA-induced contraction ceased within 5 min and the caffeine-induced contraction persisted for more than 15 min. In a skinned fiber, increase of [Na]o from 10 to 60 mM suppressed the pCa-tension relationship in the ranges of 10(-7) and 10(-5) M free Ca but not with a dose of 30 mM [Na]o. NA (10(-5) M) had no effect on skinned fibers. Increase in Na concentration (60 mM) had no effect on Ca accumulation in the store site or on Ca release by caffeine. Possible Na-related mechanisms on the development of mechanical response are discussed in relation to Ca on the surface and in the internal membrane structure. The NaCl-free-induced contraction in smooth muscles of the guinea-pig mesenteric artery is postulated to be due to influx of Ca through the Na channel, rather than the Ca channel.     

Afterhyperpolarization mechanisms in cat sympathetic preganglionic neuron in vitro

A long-lasting afterhyperpolarization (AHP) follows the antidromic or current-induced action potential of sympathetic preganglionic neurons (SPNs) studied in slices of cat spinal cord maintained in vitro. Duration and amplitude of the AHP that follows a single spike were 2.8 +/- 0.3 s and 16.0 +/- 0.7 mV (mean +/- SE), respectively. In most cases two components could be distinguished, an initial faster and usually larger component [fast (F) AHP] followed by a slowly decaying component [slow (S) AHP]. An increase in membrane conductance was associated with the AHP. The amplitude of both components increased with membrane depolarization and decreased with hyperpolarization. Both fast and slow component were nullified at a voltage of -90 mV in 3.6 mM K+. Peak AHP amplitude decreased as K+ was increased from 1.5 to 7.0 mM. The null point for both fast (F) AHP and slow (S) AHP shifted in the depolarizing or hyperpolarizing direction when K+ was increased or decreased, respectively. These data suggest that an increase in K+-conductance is the mechanism underlying the AHP. The two components of the AHP could be separated by their differential sensitivity to superfusion with the Ca2+-channel blocker cobalt (2 mM) or with low Ca2+ (0.25 mM). These procedures resulted in an AHP of much shorter duration (330 ms, range 150-600), presumably the FAHP. These observations indicate that a Ca2+-activated K+-conductance is likely to be involved in the generation of the SAHP. The FAHP was depressed during superfusion with tetraethylammonium (TEA) (20 mM) and intracellular cesium injection. The SAHP was enhanced by TEA and enhanced or depressed by cesium. In 3.6 mM K+ the FAHP reversed in polarity at membrane voltages more negative than -90 mV. This component had an approximately linear relation of amplitude to membrane potential. The SAHP did not reverse in most cells. In the few cases in which it reversed, the change in amplitude for a given change in membrane voltage was much smaller on the negative than on the positive side of the null potential. Thus the SAHP shows voltage-dependent, nonlinear characteristics. This difference in behavior of the two components was also observed when the null point was displaced in high or low K+. In the presence of tetrodotoxin (TTX) the AHP persisted in temporal association with a high-threshold, TTX-resistant, cobalt-sensitive spike. During the time course of the AHP the efficacy of synaptic input decreased, suggesting that the AHP has an important role in regulating the firing rate of the SPN.     

Long-lasting reduction of excitability by a sodium-dependent potassium current in cat neocortical neurons

1. The function and ionic mechanism of a slow outward current were studied in large layer V neurons of cat sensorimotor cortex using an in vitro slice preparation and single microelectrode voltage clamp. 2. With Ca2+ influx blocked, a slow relaxation ("tail") of outward current followed either (1) repetitive firing evoked for 1 s or (2) a small 1-s depolarizing voltage clamp step that activated the persistent Na+ current of neocortical neurons, INaP. When a depolarization that activated INaP was maintained, an outward current gradually developed and increased in amplitude over a period of tens of seconds to several minutes. An outward tail current of similar duration followed repolarization. The slow outward current was abolished by TTX, indicating it depended on Na+ influx. 3. With Ca2+ influx blocked, the onset of the slow Na+-dependent outward current caused spike frequency adaptation during current-evoked repetitive firing. Following the firing, the decay of the Na+-dependent current caused a slow afterhyperpolarization (sAHP) and a long-lasting reduction of excitability. It also was responsible for habituation of the response to repeated identical current pulses. 4. The Na+-dependent tail current had properties expected of a K+ current. Membrane chord conductance increased during the tail, and tail amplitude was reduced or reversed by membrane potential hyperpolarization and raised extracellular K+ concentration [( K+]0). 5. The current tail was reduced reversibly by the K+ channel blockers TEA (5-10 mM), muscarine (5-20 microM), and norepinephrine (100 microM). These agents also resulted in a larger, more sustained inward current during the preceding step depolarization. Comparison of current time course before and after the application of blocking agents suggested that, in spite of its capability for slow buildup and decay, the onset of the Na+-dependent outward current occurs within 100 ms of an adequate step depolarization. 6. With Ca2+ influx blocked, extracellular application of dantrolene sodium (30 microM) had no clear effect on the current tail or the corresponding sAHP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cellular properties of lateral spinal nucleus neurons in the rat L6-S1 spinal cord.

Conventional intracellular recordings were made from 26 lateral spinal nucleus (LSN) neurons in slices of L6-S1 spinal cord from 10- to 15-day-old rats. At rest, LSN neurons did not fire spontaneous action potentials. With injection of a positive current pulse, action potentials had an amplitude of 72 +/- 7 (SD) mV and duration at half-peak height of 0.75 +/- 0.22 ms. Action potentials were followed by an afterpotential. Most LSN neurons (13/17) exhibited only an afterhyperpolarization (AHP); four neurons exhibited both a fast and a slow AHP separated by an afterdepolarization (ADP). For LSN neurons that exhibited only an AHP, a slow ADP could be identified during bath application of apamin (100 nM). Four of 11 LSN neurons showed a postinhibitory rebound (PIR). Two types of PIR were noted, one with high threshold and low amplitude and the other with low threshold and high amplitude. The PIR with high amplitude was partially blocked in 0 mM Ca2+/high Mg2+ (10 mM) recording solution. Repetitive firing properties were examined in 17 LSN neurons. On the basis of the ratio of the slopes between initial instantaneous firing and steady-state firing frequencies, neurons with low spike frequency adaptation (SFA, 8/17) and high SFA (4/17) were identified. In addition, 2/17 LSN neurons exhibited biphasic repetitive firing patterns, which were composed of a fast SFA, delayed excitation, and low SFA; another two neurons showed only delayed excitation. Plateau potentials also were found in two LSN neurons. Dorsal root stimulation revealed that most LSN neurons (12/13) had polysynaptic postsynaptic potentials (PSP); only one neuron exhibited a monosynaptic PSP. Electrical stimulation of the dorsal root evoked prolonged discharges in low SFA neurons and a short discharge in high SFA neurons. Intrinsic properties were modulated by bath application of substance P (SP). Membrane potentials were depolarized in all eight LSN neurons tested, and membrane resistance was either increased (n = 3) or decreased (n = 2). Both instantaneous firing and steady-state firing were facilitated by SP. In addition, oscillation of membrane potentials were induced in three LSN neurons. These results demonstrate that LSN neurons exhibit a variety of intrinsic properties, which may significantly contribute to sensory processing, including nociceptive processing.     

Charybdotoxin and apamin sensitivity of the calcium-dependent repolarization and the afterhyperpolarization in neostriatal neurons.

1. Intracellular recordings from neostriatal neurons in an in vitro slice preparation of the rat brain were used to analyze the pharmacological sensitivity of the action potential (AP) repolarization and the afterhyperpolarization (AHP) that follows a single action potential. The interspike voltage trajectory and the AHP could be divided into two main parts: a fast component lasting a few milliseconds and better observed during a train of spikes, and a slow component lasting approximately 250 ms and that comprises the main portion of the AHP. In some cells, a slow (up to 1 s) component of low amplitude was also detected. 2. Single APs were elicited at two imposed membrane potentials (around -60 and around -80 mV). The AP amplitude was larger, the repolarization rate was faster, and the duration was shorter when spikes were evoked at -80 mV. When measured from the -60 mV holding potential, the afterpotential was an AHP with peak amplitude of -5 mV. The afterpotential became a delayed depolarization (DD) at -80 mV. 3. Firing frequency adaptation was voltage sensitive. The firing of APs induced by long intracellular current pulses from a holding potential of -80 mV exhibited only a slow-frequency adaptation (time constant of seconds). However, at -60 mV, an initial and faster frequency adaptation was evident (time constant of tens of milliseconds). 4. The Ca2+ channel blocker Cd2+ retarded AP repolarization rate. This effect correlated with a significant block of the fast and slow components of the AHP. In contrast, Ni2+ had no significant effects on the same parameters.(ABSTRACT TRUNCATED AT 250 WORDS)     

Action potential afterdepolarization mediated by a Ca2+-activated cation conductance in myenteric AH neurons

We investigated the nature of afterdepolarizing potentials in AH neurons from the guinea-pig duodenum using whole-cell patch-clamp recordings in intact myenteric ganglia. Afterdepolarizing potentials were minimally activated following action-potential firing under normal conditions, but after application of charybdotoxin (40 nM) or tetraethyl ammonium (TEA; 10-20 mM) to the bathing solution, prominent afterdepolarizing potentials followed action potentials. The whole-cell current underlying afterdepolarizing potentials (I(ADP)) in the presence of TEA (10-20 mM) reversed at -38 mV and was not voltage-dependent. Reduction of NaCl in the bathing (Krebs) solution to 58 mM shifted the reversal potential of the I(ADP) to -58 mV, suggesting that the current underlying the afterdepolarizing potential was carried by a mixture of cations. The relative contributions of Na(+) and K(+) to this current were estimated to be about 1:5. Substitution of external Na(+) with N-methyl D-glucamine blocked the current while replacement of internal Cl(-) with gluconate did not block the I(ADP). The I(ADP) was also inhibited when CsCl-filled patch pipettes were used. The I(ADP) was blocked or substantially decreased in amplitude in the presence of N-type Ca(2+) channel antagonists, omega-conotoxin GVIA and omega-conotoxin MVIIC, respectively, and was eliminated by external Cd(2+), indicating that it was dependent on Ca(2+) entry. The I(ADP) was also inhibited by ryanodine (10-20 microM), indicating that Ca(2+)-induced Ca(2+) release was involved in its activation. Niflumic acid consistently inhibited the I(ADP) with an IC(50) of 63 microM. Using antibodies against the pore-forming subunits of L-, N- and P/Q-type voltage-gated Ca(2+) channels, we have demonstrated that myenteric AH neurons express N- and P/Q, but not L-type voltage-gated Ca(2+) channels. We conclude that the ADP in myenteric AH neurons, in the presence of an L-type Ca(2+)-channel blocker, is generated by the opening of Ca(2+)-activated non-selective cation channels following action potential-mediated Ca(2+) entry mainly through N-type Ca(2+) channels. Ca(2+) release from ryanodine-sensitive stores triggered by Ca(2+) entry contributes significantly to the activation of this current.     

Effects of thymol on the electrical and mechanical properties of the guinea-pig taenia coli

Effects of thymol (0.02-2 mM) on the electrical and mechanical activities of the smooth muscle cells of the guinea-pig taenia coli were investigated with either micro-electrode or double sucrose gap methods.1. Thymol, in a concentration of more than 0.03 mM, reduced the amplitude and maximum rate of rise of the spikes without any change of the membrane potential. When the concentration was increased to 0.3 mM, thymol completely blocked the spontaneous and evoked spike activities. In a concentration of more than 0.1 mM, thymol reduced the membrane resistance in proportion to the concentration without any change of the membrane potential.2. Ionic mechanisms involved in the effects of thymol on the membrane resistance were investigated in various ionic environments. The results showed that in concentrations below 0.5 mM thymol might selectively increase the Cl-conductance of the membrane. Participations of Na and K ion in the effects of thymol on the membrane resistance could be eliminated. However, at more than 1 mM, thymol increased the membrane conductance non-selectively. Excess Ca in the external solution partly suppressed the action of thymol on the taenia coli.3. Potentiation of the twitch tension was not observed on treatment with any concentration of thymol.4. After pre-treatment with thymol (0.5 mM), isotonic K Krebs solution depolarized the membrane and reduced the membrane resistance as observed in the absence of thymol. However, thymol completely suppressed the K-induced contracture.5. Application of excess Ca and acetylcholine during the maintained contracture evoked by isotonic K Krebs solution induced further development of the contracture. However, on pre-treatment with thymol, neither excess Ca nor acetylcholine could evoke a mechanical response in isotonic K Krebs solution.6. The results obtained from the present experiments are discussed in relation to the roles of Ca on the smooth muscle cells.     

Na+-activated K+ current contributes to postexcitatory hyperpolarization in neocortical intrinsically bursting neurons

The ionic mechanisms underlying the termination of action-potential (AP) bursts and postburst afterhyperpolarization (AHP) in intrinsically bursting (IB) neocortical neurons were investigated by performing intracellular recordings in thin slices of rat sensorimotor cortex. The blockade of Ca(2+)-activated K(+) currents enhanced postburst depolarizing afterpotentials, but had inconsistent and minor effects on the amplitude and duration of AHPs. On the contrary, experimental conditions resulting in reduction of voltage-dependent Na(+) entry into the cells caused a significant decrease of AHP amplitude. Slice perfusion with a modified artificial cerebrospinal fluid in which LiCl (40 mM) partially replaced NaCl had negligible effects on the properties of individual APs, whereas it consistently increased burst length and led to an approximately 30% reduction in the amplitude of AHPs following individual bursts or short trains of stimulus-induced APs. Experiments performed by partially replacing Na(+) ions with choline revealed a comparable reduction in AHP amplitude associated with an inhibition of bursting activity. Moreover, in voltage-clamp experiments carried out in both in situ and acutely isolated neurons, partial substitution of extracellular NaCl with LiCl significantly and reversibly reduced the amplitude of K(+) currents evoked by depolarizing stimuli above-threshold for Na(+)-current activation. The above effect of Na(+)-to-Li(+) substitution was not seen when voltage-gated Na(+) currents were blocked with TTX, indicating the presence of a specific K(+)-current component activated by voltage-dependent Na(+) (but not Li(+)) influx. The above findings suggest that a Na(+)-activated K(+) current recruited by the Na(+) entry secondary to burst discharge significantly contributes to AHP generation and the maintenance of rhythmic burst recurrence during sustained depolarizations in neocortical IB neurons.     

A calcium-activated hyperpolarization follows repetitive firing in hippocampal neurons

1. A long-lasting afterhyperpolarization (AHP) follows current-induced repetitive firing in hippocampal CA1 neurons studied in vitro. A 10-25% increase in membrane slope conductance occurs during the AHP, suggesting that it may be mediated by an increased conductance to either K+ or Cl-. 2. Intracellular Cl- iontophoresis does not alter the AHP but does attenuate the IPSP. In contrast Ba2+, a cation that can decrease K+ conductance, eliminates the AHP but not the IPSP. These findings suggest the AHP is produced by a long-lasting increased conductance to K+, and is distinct from the IPSP. 3. Mn2+, a Ca2+-channel blocker, eliminates the AHP. In comparison, the AHP persists in the presence of the Na+-channel blocker, tetrodotoxin (TTX), and appears to be temporally associated with TTX-resistant "Ca2+ spikes." It is concluded that AHP is probably activated by Ca2+ influx. 4. These observations indicate that the AHP may be produced by a Ca2+ activated K+ current. A balance between cellular depolarization produced by Ca2+ entry and repolarization generated by a Ca2+-activated K+ current appears to operate to control excitability in some mammalian cortical neurons as it does in molluscan neurons. Disruption of this balance by Ba2+ produces spontaneous membrane-potential oscillations and recurrent burst firing in hippocampal neurons. Increases in the magnitude and duration of Ca2+ depolarization and/or decreases in the Ca2+-activated, K+-mediated repolarization may be mechanisms that lead to spontaneous, epileptiform bursting in mammalian cortical neurons.     

The effect of sodium and calcium on the action potential of the smooth muscle of the guinea-pig taenia coli

1. Spontaneous spike activity and action potentials evoked by external field stimulation were recorded, intracellularly and with the double sucrose gap method, from the smooth muscle of guinea-pig taenia coli.2. Replacement of external NaCl with sucrose (leaving 10 mM-Na in the buffer) caused hyperpolarization and stopped spontaneous activity within 10 min. Spikes could, however, be evoked for 2-3 hr. The amplitude, the overshoot and rate of rise of the spike were increased.3. In 10 mM-[Na](o) the intracellular Na concentration was reduced from 35 to 24 mM, shifting the Na-equilibrium potential from +34 to -22 mV.4. Excess Ca (12.5 mM) caused hyperpolarization and increased membrane conductance. The amplitude and the rate of rise of the spike were increased, the threshold was raised and the latency of the spike evoked by threshold stimulation became shorter.5. The effect of reducing the external Ca concentration depended on the Na concentration present, being greater with higher external [Na](o). When the membrane was depolarized and spikes deteriorated in low Ca (0.2-0.5 mM) reduction of Na to 10 mM caused repolarization and recovery of the action potential.6. Mn (0.5-1.0 mM) blocked spontaneous spike discharge after 20 min. Higher concentrations (more than 2.0 mM) were required to block the evoked action potential.7. The results indicate that the smooth muscle spike in taenia is due to Ca-entry and that Na influences spike activity indirectly by competing with Ca in controlling the membrane potential.     

A Ca2+-independent slow afterhyperpolarization in substantia nigra compacta neurons

The discharge properties of dopaminergic neurons in substantia nigra are influenced by slow adaptive responses, which have not been fully identified. The present study describes, in a slice preparation from the rat, a complex afterhyperpolarization (AHP), elicited by action potential trains. The AHP could be subdivided into a fast component (AHP(f)), which was generated near action potential threshold, relaxed within approximately 1 s, and had highest amplitude when evoked by short-lasting (0.1 s) depolarizations, and a slow component (AHP(s)), which lasted several seconds, was evoked from subthreshold potentials, and required prolonged depolarizing stimuli (>0.1 s). A large proportion of the AHP(f) was sensitive to (i) 0.1 microM apamin, (ii) the Ca(2+) antagonists, Cd(2+) (0.2 mM) and Ni(2+) (0.3 mM), (iii) low (0.2 mM) extracellular Ca(2+) concentration, and (iv), Ca(2+) chelation with intracellular EGTA. The AHP(s) was resistant to the above treatments, and it was insensitive to 25 microM dantrolene or prolonged exposure to 1 microM thapsigargin. The reversal potential of the AHP(s) (-97 mV) was close to the K(+) equilibrium potential. It was significantly inhibited by 5 mM 4-aminopyridine, 5 microM haloperidol, 10 microM terfenadine, or high extracellular Mg(2+) (10 mM), but not by 30 mM tetraethylammonium chloride, 50 microM carbachol, 0.5 microM glipizide, 2 microM (-)sulpiride, 100 microM N-allyl-normetazocine, or 100 microM pentazocine. Haloperidol reduced the post-stimulus inhibitory period seen during spontaneous discharge, but had no detectable effect on spike frequency adaptation. It is concluded that the SK-type Ca(2+)-activated K(+) channels underlies a major component of the AHP(f), whereas the AHP(s) is Ca(2+)-independent and relies, in part, on a voltage-dependent K(+) current with properties resembling the ether-a-go-go-related gene K(+) channel. The latter component exerts a slow, spike-independent, inhibitory influence on repetitive discharge and contributes to the prolonged decrease in excitability following sustained depolarizing stimuli.     

Postnatal changes in membrane properties of mice trigeminal ganglion neurons

Intracellular recordings from neurons in the mouse trigeminal ganglion (TG) in vitro were used to characterize changes in membrane properties that take place from early postnatal stages (P0-P7) to adulthood (>P21). All neonatal TG neurons had uniformly slow conduction velocities, whereas adult neurons could be separated according to their conduction velocity into Adelta and C neurons. Based on the presence or absence of a marked inflection or hump in the repolarization phase of the action potential (AP), neonatal neurons were divided into S- (slow) and F-type (fast) neurons. Their passive and subthreshold properties (resting membrane potential, input resistance, membrane capacitance, and inward rectification) were nearly identical, but they showed marked differences in AP amplitude, AP overshoot, AP duration, rate of AP depolarization, rate of AP repolarization, and afterhyperpolarization (AHP) duration. Adult TG neurons also segregated into S- and F-type groups. Differences in their mean AP amplitude, AP overshoot, AP duration, rate of AP depolarization, rate of AP repolarization, and AHP duration were also prominent. In addition, axons of 90% of F-type neurons and 60% of S-type neurons became faster conducting in their central and peripheral branch, suggestive of axonal myelination. The proportion of S- and F-type neurons did not vary during postnatal development, suggesting that these phenotypes were established early in development. Membrane properties of both types of TG neurons evolved differently during postnatal development. The nature of many of these changes was linked to the process of myelination. Thus myelination was accompanied by a decrease in AP duration, input resistance (R(in)), and increase in membrane capacitance (C). These properties remained constant in unmyelinated neurons (both F- and S-type). In adult TG, all F-type neurons with inward rectification were also fast-conducting Adelta, suggesting that those F-type neurons showing inward rectification at birth will evolve to F-type Adelta neurons with age. The percentage of F-type neurons showing inward rectification also increased with age. Both F- and S-type neurons displayed changes in the sensitivity of the AP to reductions in extracellular Ca(2+) or substitution with Co(2+) during the process of maturation.     

Large-conductance calcium-activated potassium channels in neonatal rat intracardiac ganglion neurons

The properties of single Ca2+-activated K+ (BK) channels in neonatal rat intracardiac neurons were investigated using the patch-clamp recording technique. In symmetrical 140 mM K+, the single-channel slope conductance was linear in the voltage range -60/+60 mV, and was 207+/-19 pS. Na+ ions were not measurably permeant through the open channel. Channel activity increased with the cytoplasmic free Ca2+ concentration ([Ca2+]i) with a Hill plot giving a half-saturating [Ca2+] (K0.5) of 1.35 microM and slope of approximately equals 3. The BK channel was inhibited reversibly by external tetraethylammonium (TEA) ions, charybdotoxin, and quinine and was resistant to block by 4-aminopyridine and apamin. Ionomycin (1-10 microM) increased BK channel activity in the cell-attached recording configuration. The resting activity was consistent with a [Ca2+]i <100 nM and the increased channel activity evoked by ionomycin was consistent with a rise in [Ca2+]i to > or =0.3 microM. TEA (0.2-1 mM) increased the action potential duration approximately equals 1.5-fold and reduced the amplitude and duration of the afterhyperpolarization (AHP) by 26%. Charybdotoxin (100 nM) did not significantly alter the action potential duration or AHP amplitude but reduced the AHP duration by approximately equals 40%. Taken together, these data indicate that BK channel activation contributes to the action potential and AHP duration in rat intracardiac neurons.     

Slow wave activity in rabbit jejunum

A study of slow wave activity present in rabbit jejunum was carried out using intracellular recording techniques and the double sucrose-gap apparatus. Two different indications of changes in membrane potential previously postulated to be pacemaker activity were seen. Slow wave activity was dependent on membrane potential, decreasing in amplitude and increasing in frequency with depolarization, and increasing in amplitude with hyperpolarization. There was no significant change in frequency with hyperpolarization. Slow waves could be entrained using short depolarizing pulses.The ionic dependence of slow waves was studied. In 15 mM Na, slow wave activity was maintained and small oscillations were seen with 5 mM Na. Removing K abolished slow wave activity, although Cs could act as a substitute for K. Removing Ca abolished slow wave activity; Sr ions were able to replace Ca in the generation of slow waves. Agents which blocked or reduced the transmembrane flux of Ca ions (Mn, lanthanum and high concentrations of verapamil) produced a decrease in slow wave amplitude and frequency. Small oscillations in membrane potential could be produced in Na-free-40 mM Ca solution following application of short duration depolarizing current pulses.The results obtained could not be readily explained in terms of an oscillating electrogenic Na-K pump. Rather they support the hypothesis of an oscillating ionic conductance change associated with alterations in the distribution of Ca ions across the membrane (Tomita and Watanabe, 1973).     

Calcium-dependent potentials in mammalian red nucleus neurons in vitro

Intracellular recordings were made from red nucleus (RN) neurons in guinea-pig slice preparations. The slow afterhyperpolarization (AHP) following an action potential was reversibly abolished by Co2+ or Mn2+. Its amplitude was dependent on the extracellular K+ concentration. When tetraethylammonium was added to the perfusing solution, a tetrodotoxin-resistant regenerative depolarization was evoked which was blocked by Co2+ or Mn2+. There results suggest that the slow AHP is produced by an increase in Ca2+-dependent K+ conductance and that RN neurons have a voltage-dependent Ca2+ conductance.     

Nature of anthopleurin-B-induced release of norepinephrine from adrenergic nerves

Anthopleurin-B (AP-B), a new polypeptide from sea anemone (Anthopleura xanthogrammica), markedly increased the amount of norepinephrine (NA) released from the guinea pig isolated vas deferens. The AP-B-induced release of NA was inhibited or abolished by pretreatment with reserpine and by guanethidine or procaine but remained almost unaffected by mecamylamine. D 600, nifedipine, diltiazem, Mn2+, and Mg2+ markedly inhibited the NA releasing action of AP-B. The AP-B-induced release of NA increased in a linear fashion with increasing Na+ concentrations (85-150 mM). Also the NA release by AP-B increased with an increase in the concentration of external Ca2+ from 0 to 0.8 mM but decreased with an increase in the Ca2+ concentration from 0.8 to 2.0 mM. These results suggest that AP-B increases the permeability across the nerve cell membrane to both Na+ and Ca2+ and that this plays an important role in the NA release from the adrenergic nerve.     

Activity-dependent increase of the AHP amplitude in T sensory neurons of the leech

We identified a new form of activity-dependent modulation of the afterhyperpolarization (AHP) in tactile (T) sensory neurons of the leech Hirudo medicinalis. Repetitive intracellular stimulation with 30 trains of depolarizing impulses at 15-s inter-stimulus interval (ISI) led to an increase of the AHP amplitude (~60% of the control). The enhancement of AHP lasted for >/=15 min. The AHP increase was also elicited when a T neuron was activated by repetitive stimulation of its receptive field. The ISI was a critical parameter for the induction and maintenance of AHP enhancement. ISI duration had to fit within a time window with the upper limit of 20 s to make the training effective to induce an enhancement of the AHP amplitude. After recovery from potentiation, AHP amplitude could be enhanced once again by delivering another training session. The increase of AHP amplitude persisted in high Mg(2+) saline, suggesting an intrinsic cellular mechanism for its induction. Previous investigations reported that AHP of leech T neurons was mainly due to the activity of the Na(+)/K(+) ATPase and to a Ca(2+)-dependent K(+) current (I(K/Ca)). In addition, it has been demonstrated that serotonin (5HT) reduces AHP amplitude through the inhibition of the Na(+)/K(+) ATPase. By blocking the I(K/Ca) with pharmacological agents, such as cadmium and apamin, we still observed an increase of the AHP amplitude after repetitive stimulation, whereas 5HT application completely inhibited the AHP increment. These data indicate that the Na(+)/K(+) ATPase is involved in the induction and maintenance of the AHP increase after repetitive stimulation. Moreover, the AHP increase was affected by the level of serotonin in the CNS. Finally, the increase of the AHP amplitude produced a lasting depression of the synaptic connection between two T neurons, suggesting that this activity-dependent phenomenon might be involved in short-term plasticity associated with learning processes.     

Sodium action potentials induced by calcium chelation in rat uterine smooth muscle

Muscular strips of rat myometrium were exposed to Ca-free solutions in the presence of low concentrations of EGTA (0.1–1 mM). A study of electrical activity was carried out using intracellular recording techniques and the double sucrose-gap apparatus. The results were as followed:

1. After 5–15 min the resting membrane potential was depolarized to about ?35 mV und a rhythmic activity appeared. The potentials had no overshoot and their duration was noticeably prolonged showing a sustained plateau close to zero potential. Rhythmic activity was observed in a definite membrane potential range. The effects were essentially reversed upon Ca-replacement.
2. The maximal amplitude of the potentials was strongly dependent on the external Na-concentration. At low Na-concentrations (13 mM), the membrane repolarized to its normal resting value (about ?50 mV).
3. The potentials were unaffected by tetrodotoxin (2μM). They were inhibited by Mn (0.1 mM) and D600 (0.1–1 μM) and by increasing Mg concentration (1.24 mM) or Ca concentration (10^?3 mM) after 1 h in the presence of EGTA. Higher concentrations of Mn (1–2.5 mM) also repolarized the membrane to its normal resting potential. Tetraethylammonium ions (20 mM) increased both amplitude and duration of the responses but did not blocked the repolarization.
4. Inward Na currents characterized by a slow inactivation were recorded on voltage-clamped preparations.
5. Inhibition of Na-pumping with K-free solution or ouabain (0.1–1 mM) blocked the rhythmic activity and maintained the membrane potential at a steady-state depolarized value. Action potentials reappeared by application of inward current pulses which repolarized the membrane to about ?45 mV.

These results suggest that Ca-chelation induces membrane permeability changes resulting essentially in: 1) an increase in resting Na-conductance, 2) a prolonged Na-inward current entering through channels normally used by Ca ions, and 3) a reduction in K-conductance. However, active Na pumping does indeed participate to the generation of rhythmic activity.     

The pathway for the slow inhibitory postsynaptic potential in bullfrog sympathetic ganglia

Intracellular and sucrose gap recording techniques were used to examine synaptically evoked potentials and the response of neurons in bullfrog paravertebral sympathetic ganglia to muscarinic agonists. These neurons were defined as either B or C cells on the basis of the conduction velocity of antidromically evoked action potentials. Following stimulation of preganglionic C-fibers in the rostral portion of the VIIIth spinal nerve, a fast nicotinic excitatory postsynaptic potential (EPSP) and a slow atropine-sensitive inhibitory postsynaptic potential (IPSP) could be recorded intracellularly in C cells of the IXth and Xth paravertebral ganglia treated with 70 microM d-tubocurarine chloride (dTC). Under these conditions, local iontophoretic application of acetylcholine (ACh) could produce a slow hyperpolarization of C cell membrane potential. ACh hyperpolarizations or slow IPSPs were not detected in ganglionic B cells. Stimulation of the preganglionic B-fibers in the sympathetic chain produced a fast nicotinic EPSP and a slow muscarinic EPSP in ganglionic B cells. A small population of C cells also received cholinergic B-fiber innervation from the sympathetic chain and exhibited a slow IPSP upon tetanic stimulation of this pathway. When curarized ganglia were examined by means of sucrose gap recording, superfusion of the muscarinic agonist, methacholine (MCh), produced an initial hyperpolarization (MChH) followed by a depolarization (MChD). Both responses were blocked by atropine and therefore presumably reflect the activation of muscarinic receptors involved in the generation of the slow IPSP and the slow EPSP, respectively. Although synaptic transmission was blocked by Ringer solution containing 4 mM Co2+, neither this solution nor 10 microM tetrodotoxin reduced the amplitude of the MChH. The MChH was slightly reduced by Ringer solution containing 0.1 mM Ca2+, however, the response could be restored by the addition of 6 mM Mg2+. These results indicate that the MChH in curarized bullfrog sympathetic ganglia results from a direct muscarinic action on ganglionic cells. This suggests that the slow IPSP is mediated by ACh released from cholinergic preganglionic fibers that make synaptic contact with ganglionic C cells.