Frequency-dependent decay of calcium spikes in cultured spinal cord neurons

Calcium-dependent action potentials were recorded from cultured (dissociated) spinal cord neurons in the presence of extracellular tetrodotoxin and tetraethylammonium in the mouse. The plateau portion of these action potentials underwent pronounced frequency-dependent decay with repetitive depolarizing current injections. Interstimulus intervals as long as 5 min were often required to ensure reproducible spike durations. This decay was not due to recurrent inhibition or intracellular calcium accumulation. It may be due to accumulation of either voltage-dependent inactivation of the calcium spike or of voltage-dependent outward currents. Any manipulation that prolonged the duration of the spike resulted in an apparent enhancement of the decay; however, this may simply be a consequence of the hyperbolic relationship between spike duration and the slope of the spike decay. It does not necessarily imply an effect on the mechanisms underlying frequency-dependent decay. Caution should, therefore, be exercised in interpreting the effects of drugs upon these calcium-dependent spikes.     

Effects of nifedipine on nerve-evoked action potentials and consequent contractions in rat tail artery

The effects of nifedipine on the electrical and mechanical activity recorded from the rat tail artery were examined. Intracellular recordings were obtained from the smooth muscle and vessel diameter monitored during nerve stimulation. Nifedipine (0.1-10 microM) depressed contractions elicited by single and repetitive (2 Hz) stimulation by 32-100% but was ineffective in decreasing the amplitude of the associated action potentials. Concentrations greater than or equal to 10 microM caused a slowly developing membrane depolarization. No change in the amplitude of subthreshold excitatory junction potentials was observed at concentrations of nifedipine less than 50 microM when the membrane depolarization was less than 8 mV. At all concentrations examined nifedipine lowered the stimulus intensity required to initiate an action potential. The amplitude of the nerve-evoked action potential recorded in lowered external Ca2+ (1 mM) was slightly increased (5-10%) by nifedipine. However, nifedipine readily reversed the increased amplitudes of the nerve-evoked action potentials and contractions caused by the addition of 20 mM tetraethylammonium chloride in the tail artery. The action potential recorded in normal solution from the guinea pig vas deferens was selectively abolished by nifedipine.     

An inward calcium current underlying regenerative calcium potentials in rat striatal neurons in vitro enhanced by Bay K 8644

The single electrode voltage clamp technique was used to characterize the currents underlying the calcium potentials in rat caudate neurons in vitro. In current clamp experiments, long depolarizing current pulses evoked repetitive firing of fast somatic action potentials. These were abolished by tetrodotoxin (1 microM) and replaced by slow graded depolarizing potentials. These were preceded by a transient hyperpolarizing notch. Addition of 4-aminopyridine (100 microM) abolished the hyperpolarizing notch, enhanced the slow graded depolarizing response and induced the appearance of a slow all-or-nothing action potential. Both the slow graded response and the all-or-nothing action potential were abolished by cobalt (2 mM), suggesting the involvement of voltage-dependent calcium conductances. When the neurons were loaded intracellularly with caesium the action potential duration increased. Substitution of the extracellular calcium by barium (1-3 mM) or external addition of tetraethylammonium (5 mM) further prolonged spike duration and induced the appearance of long-lasting plateau potentials. These were insensitive to tetrodotoxin and were reversibly blocked by the calcium antagonists cobalt (2 mM), manganese (2 mM) or cadmium (500 microM). The calcium potentials were enhanced by the calcium 'agonist' BAY K 8644 (1-5 microM). In voltage clamp experiments when intracellular caesium was used to reduce outward currents and tetrodotoxin to block fast regenerative sodium currents, depolarizing voltage steps from a holding potential of -50, -40 mV activated an inward current. This current peaked in 50-80 ms and inactivated in two phases: an initial one at 150-200 ms followed by a second one after several hundred ms.(ABSTRACT TRUNCATED AT 250 WORDS)     

Membrane properties and synaptic responses of the guinea pig nucleus accumbens neurons in vitro

1. The membrane properties and synaptic responses of guinea pig nucleus accumbens neurons in vitro were studied with intracellular recording methods. 2. The population of neurons could be divided into groups of low (20-60 M omega, average 46.5 M omega) and high (60-180 M omega, average 96.5 M omega) input resistance. The resting membrane potential in both groups was approximately -70 mV. 3. Other membrane properties were quite similar in both groups. Inward rectification occurred at potentials more negative than -80 mV; this was blocked by Cs+ (2 mM). Membrane potential oscillations were observed at potentials between -65 and -55 mV; these were blocked by tetrodotoxin (TTX, 0.5 microM). Outward rectification occurred at potentials less negative than -45 mV; this was depressed by tetraethylammonium (TEA, 10 mM). 4. Action potentials elicited by small depolarizing current pulses (2-5 ms, 0.3-0.5 nA) were approximately 95 mV in amplitude and 1.0 ms in duration. The afterhyperpolarization following each action potential was less than 30 ms in duration, and no accommodation of action-potential discharge was seen at frequencies up to 40 Hz. The action potentials were reversibly blocked by TTX (0.3 microM). In addition, TTX-insensitive, Ca2+-dependent spikes were evoked by passing larger and more prolonged current pulses (greater than 40 ms, greater than 0.5 nA) across the membrane. 5. Focal electrical stimulation of the slice surface with low intensity (1 ms, less than 10 V) elicited excitatory postsynaptic potentials (EPSPs) in neurons of both high- and low-resistance groups. The reversal potential (+10.2 mV) for the EPSPs was close to the reversal potential (+7.7 mV) of the responses to glutamate applied in the superfusing solution. The N-methyl-D-aspartic acid (NMDA) receptor antagonists, D-alpha-aminoadipic acid (1 mM) and DL-2-amino-5-phosphonovaleric acid (DL-APV, 250 microM), reversibly depressed the EPSP; the glutamate uptake inhibitor, L-aspartic acid-beta-hydroxamate (50 microM), or removal of Mg2+ from the superfusate, augmented the EPSP. 6. When the intensity of the focal stimulus was increased (1 ms, greater than or equal to 10 V), a second larger depolarizing response (duration, 800 ms to 2 s) could be evoked in addition to the smoothly graded EPSP. This was seen only in cells of the high-resistance group (90-130 M omega).(ABSTRACT TRUNCATED AT 400 WORDS)     

Intrinsic membrane properties of neostriatal neurons can account for their low level of spontaneous activity

Electroresponsiveness of neostriatal neurons was studied by intracellular recording in a rat brain slice preparation maintained in standard solution or in solution containing K-channel blockers. In standard solution, the neurons fired repetitively at increasing frequencies with increasing amplitude of direct depolarization. The firing pattern was independent of the membrane potential from which firing was induced. In the presence of tetraethylammonium (20 mM), long-lasting (300-500 ms) plateau potentials could be elicited by the injection of short (5-10 ms) current pulses. Plateau potentials persisted in Na-free solution, in the presence of tetrodotoxin (1-3 microM) and if Ca in the perifusate was replaced by Ba. The plateau was blocked by Cd (500 microM). The plateaux were followed by depolarizing after-potentials. When the plateau potential failed due to fatigue, a small slow depolarization of short duration (10-30 ms) was elicited in Na-free or tetrodotoxin-containing solution, which increased in amplitude with membrane hyperpolarization. This slow depolarization was blocked by Cd, indicating that it was also mediated by Ca. By intrastriatal stimulation in the presence of 4-aminopyridine a long-lasting, voltage-dependent depolarization was triggered from the enhanced postsynaptic potential. In contrast, in the presence of tetraethylammonium, postsynaptic potentials were only slightly increased if they were compared at sizes subthreshold for the plateau potentials. It is concluded that neostriatal neurons, although being characterized as "silent" and "non-bursting", possess slow conductances for inward currents which they share with other mammalian central neurons. However, in contrast, to other central neurons, their Ca-spikes are suppressed by their K-conductances and, in contrast to oscillating neurons, low-threshold Ca-potentials are not prominent.     

Electrophysiological effects of tetracaine on the longitudinal muscle of rat uterus in various ionic environments

Electrical activity was recorded intracellularly from the uterine longitudinal muscle of estrogen-treated rat. Membrane potential was -59 mV. Action potential consisted of initial spike and the following plateau potential on which repetitive spike discharge rode. Tetracaine (0.1, 0.2 mM) reduced the maximum rates of rise and fall of initial spike and increased the duration of plateau potential. Sustained depolarization at -33 mV was produced by the application of 0.8 mM tetracaine. The membrane depolarization was accompanied with the increase in membrane resistance. Reduction of initial spike by tetracaine in the maximum rates of rise and fall was prevented by increasing the external Ca concentration and accelerated by decreasing it. In low K (1.2 mM) or isoprenaline (10(-8) M)-containing solution, tetracaine-induced prolongation of plateau potential was inhibited. Plateau potential was reduced in amplitude and was prolonged only slightly by tetracaine in low Na solution (sucrose substitution). Both the initial spike and plateau potentials were blocked completely by 0.2 mM tetracaine in the presence of Mn (0.5 mM) or verapamil (0.5 microM). It is speculated that tetracaine suppresses the K conductances responsible for resting membrane potential and the repolarizations of spike and plateau potentials. Ca influx during the generation of action potential is also supposed to be inhibited by tetracaine.     

Patch-clamp study of postnatal development of CA1 neurons in rat hippocampal slices: membrane excitability and K+ currents.

1. The postnatal development of membrane properties and outward K+ currents in CA1 neurons in rat hippocampal slices was studied with the use of whole-cell patch-clamp techniques. 2. Neurons at all postnatal ages (2-30 days; P2-30) were capable of generating tetrodotoxin (TTX)-sensitive action potentials in response to intracellular injection of depolarizing current pulses. There was a gradual increase in the amplitude and a decrease in the duration of these action potentials with age. Stable values for spike duration were reached by P15, whereas spike amplitude increased until P20-25. In P2-5 neurons, the duration of action potentials was greatly prolonged by depolarization from the resting membrane potential, indicating a weak spike repolarizing mechanism at depolarized potentials. In contrast, the duration of spikes evoked in P20-30 neurons was not affected by similar changes in the membrane potential. 3. Application of tetraethylammonium (TEA, 10 mM) had no effect on the duration of spikes in P3-5 neurons, whereas application of 4-aminopyridine (4-AP, 2 mM) produced large increases in spike duration. In contrast, the duration of spikes in P26 neurons was greatly increased after TEA application, whereas 4-AP had smaller effects on spike duration in these neurons. 4. The input resistance and membrane time constant decreased with age from P2 to P15. The values for both parameters were considerably greater than those reported with conventional intracellular recording electrodes in the immature hippocampus. The resting membrane potential became more hyperpolarized with age. When the recording pipettes contained KCl (140 mM), the resting potential of P3-4 neurons was 34 mV depolarized compared with resting potentials observed with potassium gluconate-filled pipettes. Only a 13-mV change in resting potential was observed during similar comparisons in P27-28 neurons. 5. Outward currents activated by depolarization were examined with the use of voltage-clamp techniques in P2-30 neurons. In P2-5 cells, a small, slowly inactivating outward current was evoked with depolarizing commands from holding potentials near -50 mV. By preceding the depolarizing commands with a hyperpolarizing prepulse, an additional early transient outward current was evoked. The sustained and transient outward currents were separated by their kinetic properties and their sensitivity to cobalt (Co2+), TEA, and 4-AP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Dichotomy of action-potential backpropagation in CA1 pyramidal neuron dendrites.

In hippocampal CA1 pyramidal neurons, action potentials are typically initiated in the axon and backpropagate into the dendrites, shaping the integration of synaptic activity and influencing the induction of synaptic plasticity. Despite previous reports describing action-potential propagation in the proximal apical dendrites, the extent to which action potentials invade the distal dendrites of CA1 pyramidal neurons remains controversial. Using paired somatic and dendritic whole cell recordings, we find that in the dendrites proximal to 280 microm from the soma, single backpropagating action potentials exhibit <50% attenuation from their amplitude in the soma. However, in dendritic recordings distal to 300 microm from the soma, action potentials in most cells backpropagated either strongly (26-42% attenuation; n = 9/20) or weakly (71-87% attenuation; n = 10/20) with only one cell exhibiting an intermediate value (45% attenuation). In experiments combining dual somatic and dendritic whole cell recordings with calcium imaging, the amount of calcium influx triggered by backpropagating action potentials was correlated with the extent of action-potential invasion of the distal dendrites. Quantitative morphometric analyses revealed that the dichotomy in action-potential backpropagation occurred in the presence of only subtle differences in either the diameter of the primary apical dendrite or branching pattern. In addition, action-potential backpropagation was not dependent on a number of electrophysiological parameters (input resistance, resting potential, voltage sensitivity of dendritic spike amplitude). There was, however, a striking correlation of the shape of the action potential at the soma with its amplitude in the dendrite; larger, faster-rising, and narrower somatic action potentials exhibited more attenuation in the distal dendrites (300-410 microm from the soma). Simple compartmental models of CA1 pyramidal neurons revealed that a dichotomy in action-potential backpropagation could be generated in response to subtle manipulations of the distribution of either sodium or potassium channels in the dendrites. Backpropagation efficacy could also be influenced by local alterations in dendritic side branches, but these effects were highly sensitive to model parameters. Based on these findings, we hypothesize that the observed dichotomy in dendritic action-potential amplitude is conferred primarily by differences in the distribution, density, or modulatory state of voltage-gated channels along the somatodendritic axis.     

Synaptic and intrinsic control of membrane excitability of neostriatal neurons. II. An in vitro analysis

1. The effects of intrinsic membrane properties on the spontaneous and synaptically evoked activity of neostriatal neurons were studied in an in vitro slice preparation with the use of intracellular recordings. The recorded neurons did not show spontaneous action potentials at rest; depolarizing current pulses triggered a tonic firing pattern. 2. Subthreshold spontaneous depolarizing potentials (SDPs) were observed in 52% of the recorded neurons. The amplitude of these potentials at rest ranged between 2 and 15 mV, and their duration between 4 and 100 ms. The frequency and the amplitude of the SDPs were functions of the membrane potential: membrane depolarization by constant positive current increased the frequency of the SDPs and reduced their amplitude; hyperpolarization of the membrane decreased their frequency and increased their amplitude. Often, at membrane potentials more negative than -90 mV, SDPs were completely suppressed. 3. SDPs were blocked by low calcium-cobalt containing solutions. In the presence of tetrodotoxin (TTX, 1-3 microM), SDPs were completely abolished in 50% of the tested neurons; in the remaining neurons, small (1-4 mV) TTX-resistant SDPs were observed. In most of the neurons, bicuculline (BIC, 10-100 microM) and low concentrations of tetanus toxin (5-10 micrograms/ml) did not clearly affect the SDPs. Higher concentrations of tetanus toxin (100 micrograms/ml) blocked the SDPs as well as the synaptic potentials evoked by intrastriatal stimulation. 4. At resting membrane potential, intrastriatal stimulation produced a fast depolarizing postsynaptic potential (EPSP) that was reduced by BIC (10-100 microM). The relationship between EPSP amplitude and membrane potential was studied either by utilizing K(+)-chloride electrodes or by the use of cesium-chloride electrodes. In both these cases, the reversal potential for the EPSPs was between 0 and -14 mV. In cesium-loaded neurons, the decrease of the EPSP, usually observed at negative membrane potentials (below -85 mV), was clearly reduced. Internal cesium prolonged the duration of the SDPs and the EPSPs evoked by intrastriatal stimulation. 5. The relationship between spontaneous and evoked synaptic activity and membrane potential was studied in the presence of different external potassium blockers. 4-Aminopyridine (4AP, 0.1-1 mM) increased the EPSP amplitude and the frequency of the SDPs, but did not decrease membrane rectification and the shunt of the EPSPs present at negative membrane potentials. On the contrary, rectification of the membrane and the shunt of the EPSPs below -85 mV were clearly reduced by tetraethylammonium (TEA, 10-20 mM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Backfiring of the isolated rat phrenic nerve does not collide with impulse propagation following repetitive nerve stimulation at 1–50 Hz

Acetylcholinesterase inhibition with neostigmine in the isolated rat phrenic nerve-hemidiaphragm preparation induced axonal backfiring and repetitive compound muscle action potentials following single nerve stimulation. The duration of backfiring and the repetitive compound muscle action potentials did not exceed 55 ms. With repetitive nerve stimulation at frequencies ranging from 1 to 50 Hz, backfiring was present only with the first stimulus and the amplitude of the second compound muscle action potential was maximally reduced, while the subsequent responses recovered gradually. However, the amplitudes of the concommitant antidromic nerve action potentials remained unchanged during the entire train of stimulation. Lack of nerve action potential amplitude changes and the short duration of backfiring of the first nerve action potential exclude a collision phenomenon of backfiring with the nerve action potential induced by the second stimulus. Moreover, the duration of the repetitive compound muscle action potentials did not exceed the duration of backfiring. Therefore, the prolongation of the muscle membrane refractory period by reexcitation following backfiring cannot explain the decrement of the second compound muscle action potential.     

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.     

Action-potential broadening and endogenously sustained bursting are substrates of command ability in a feeding neuron of Pleurobranchaea

1. The ventral white cells (VWC's) of the buccal ganglion of Pleurobranchaea, so named for their position and color, are a bilateral pair of neuron somata. Each sends a single axon out its contralateral stomatogastric nerve and has a dendritic field originating close to the soma. 2. The vwcs exhibit spontaneous episodes of prolonged depolarization (duration 1--4 min) accompanied by repetitive action-potential activity and separated by regular intervals of 3--30 min. Such prolonged burst episodes can be triggered by short pulses of depolarizing current. During the repetitive activity of the spontaneous bursts or that driven by imposed depolarization, the action potentials progressively broaden to 5--16 times their initial duration. 3. During spontaneous bursting or activity driven by imposed depolarization, the cyclic motor output of the feeding network is initiated or accelerated with a latency corresponding with the development of appreciable VWC spike broadening. When broadening of antidromic VWC spikes is suppressed by imposed hyperpolarization of the soma, the frequency of feeding cycles is significantly lower than when broadened spikes are allowed to develop. When trains of spikes are driven by depolarizing current, the motor output of the feeding network is not initiated until the VWC spikes have broadened to a repeatable "threshold" duration, regardless of the intensity of the depolarizing current. 4. The endogenous production of prolonged burst episodes, triggered by depolarizing current pulses, and progressive spike broadening can be demonstrated in the surgically isolated VWC soma. 5. The paired VWCs are strongly electrically coupled and display highly synchronous activity. They receive synaptic inputs from many previously identified interneurons of the feeding network and are thus reciprocally coupled within the network. 6. These results demonstrate that the capacity of this neuron to generate broadened action potentials during repetitive activity confers the ability to command coordinated motor-network output. The appropriate repetitive activity can be produced endogenously in the form of prolonged bursts of spikes.     

Modulation of Ca(2+) signaling by K(+) channels in a hypothalamic neuronal cell line (GT1-1)

The pulsatile release of gonadotropin releasing hormone (GnRH) is driven by the intrinsic activity of GnRH neurons, which is characterized by bursts of action potentials correlated with oscillatory increases in intracellular Ca(2+). The role of K(+) channels in this spontaneous activity was studied by examining the effects of commonly used K(+) channel blockers on K(+) currents, spontaneous action currents, and spontaneous Ca(2+) signaling. Whole-cell recordings of voltage-gated outward K(+) currents in GT1-1 neurons revealed at least two different components of the current. These included a rapidly activating transient component and a more slowly activating, sustained component. The transient component could be eliminated by a depolarizing prepulse or by bath application of 1.5 mM 4-aminopyridine (4-AP). The sustained component was partially blocked by 2 mM tetraethylammonium (TEA). GT1-1 cells also express inwardly rectifying K(+) currents (I(K(IR))) that were activated by hyperpolarization in the presence of elevated extracellular K(+). These currents were blocked by 100 microM Ba(2+) and unaffected by 2 mM TEA or 1.5 mM 4-AP. TEA and Ba(2+) had distinct effects on the pattern of action current bursts and the resulting Ca(2+) oscillations. TEA increased action current burst duration and increased the amplitude of Ca(2+) oscillations. Ba(2+) caused an increase in the frequency of action current bursts and Ca(2+) oscillations. These results indicate that specific subtypes of K(+) channels in GT1-1 cells can have distinct roles in the amplitude modulation or frequency modulation of Ca(2+) signaling. K(+) current modulation of electrical activity and Ca(2+) signaling may be important in the generation of the patterns of cellular activity responsible for the pulsatile release of GnRH.     

Electrogenic pump (Na+/K(+)-ATPase) activity in rat optic nerve

Rat optic nerves were studied in a sucrose gap chamber in order to study the origin of a late afterhyperpolarization that follows repetitive activity. The results provide evidence for electrogenic pump (Na+/K(+)-ATPase) activity in central nervous system myelinated axons and demonstrate an effect on axonal excitability. Repetitive stimulation (25-200 Hz; 200-5000 ms) led to a prolonged, temperature-dependent post-train afterhyperpolarization with duration up to about 40 s. The post-train afterhyperpolarization was blocked by the Na+/K(+)-ATPase blockers strophanthidin and ouabain, and the substitution of Li+ for Na+ in the test solution, which also blocks Na+/K(+)-ATPase. The peak amplitude of the post-train afterhyperpolarization was minimally changed by the potassium-channel blocker tetraethylammonium (10 mM), and the Ca2(+)-channel blocker CoCl2 (4 mM). Hyperpolarizing constant current did not reverse the afterhyperpolarization. The amplitude of the hyperpolarization was increased in the presence of the potassium-channel blocker 4-aminopyridine (1 mM). In the presence of 4-amino-pyridine, the post-train hyperpolarization was much reduced by strophanthidin, except for a residual early component lasting several hundred milliseconds which was blocked by the potassium-channel blocker tetraethylammonium. This finding indicates that after exposure to 4-aminopyridine, repetitive stimulation leads to activation of a tetraethylammonium-sensitive K(+)-channel that contributes during the first several hundred milliseconds to the post-train afterhyperpolarization. The amplitude of the compound action potential elicited by a single submaximal stimulus during the post-train hyperpolarization was smaller than that of the control response.The decrement in amplitude was not present under identical stimulation conditions when the post-train hyperpolarization was blocked by strophanthidin, indicating that the hyperpolarization associated with repetitive stimulation reduced excitability.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nimodipine increases excitability of rabbit CA1 pyramidal neurons in an age- and concentration-dependent manner.

1. Cellular properties were studied before and after bath application of the dihydropyridine L-type calcium channel antagonist nimodipine in aging and young rabbit hippocampal CA1 pyramidal cells in vitro. Various concentrations of nimodipine, ranging from 10 nM to 10 microM, were tested to investigate age- and concentration-dependent effects on cellular excitability. Drug studies were performed on a population of neurons at similar holding potentials to equate voltage-dependent effects. The properties studied under current-clamp conditions included steady-state current-voltage relations (I-V), the amplitude and integrated area of the postburst afterhyperpolarization (AHP), accommodation to a prolonged depolarizing current pulse (spike frequency adaptation), and single action-potential waveform characteristics following synaptic activation. 2. Numerous aging-related differences in cellular properties were noted. Aging hippocampal CA1 neurons exhibited significantly larger postburst AHPs (both the amplitude and the integrated area were enhanced). Aging CA1 neurons also exhibited more hyperpolarized resting membrane potentials with a concomitant decrease in input resistance. When cells were grouped to equate resting potentials, no differences in input resistance were noted, but the AHPs were still significantly larger in aging neurons. Aging CA1 neurons also fired fewer action potentials during a prolonged depolarizing current injection than young CA1 neurons. 3. Nimodipine decreased both the peak amplitude and the integrated area of the AHP in an age- and concentration-dependent manner. At concentrations as low as 100 nM, nimodipine significantly reduced the AHP in aging CA1 neurons. In young CA1 neurons, nimodipine decreased the AHP only at 10 microM. No effects on input resistance or action-potential characteristics were seen. 4. Nimodipine increased excitability in an age- and concentration-dependent manner by decreasing spike frequency accommodation (increasing the number of action potentials during prolonged depolarizing current injection). In aging CA1 neurons, this effect was significant at concentrations as low as 10 nM. In young CA1 neurons, nimodipine decreased accommodation only at higher concentrations (> or = 1.0 microM). 5. We conclude that aging CA1 neurons were less excitable than young neurons. In aging hippocampus, nimodipine restores excitability, as measured by size of the AHP and degree of accommodation, to levels closely resembling those of young adult CA1 neurons. These actions of nimodipine on aging CA1 hippocampal neurons may partly underlie the drug's notable ability to improve associative learning in aging rabbits and other mammals. Reversal of inhibitory postsynaptic potentials (IPSPs) by chloride ion and/or current injections into six motoneurons revealed the presence of inhibition during the period between phrenic bursts during fictive vomiting and also during the final phase of expulsion when phrenic discharge ceased by abdominal discharge continued. 3. Fictive coughing, evoked by repetitive electrical stimulation of superior laryngeal nerve afferents, was characterized by a large phrenic discharge followed immediately by a large abdominal nerve discharge. During fictive coughing, phrenic motoneurons retained their ramplike depolarizations throughout phrenic discharge; however, the amplitude of depolarization was greater than during inspiration. During the subsequent abdominal nerve discharge, the phrenic membrane potential usually underwent an initial rapid, transient hyperpolarization followed by a gradual repolarization associated with increased synaptic noise.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ionic basis of ON and OFF persistent activity in layer III lateral entorhinal cortical principal neurons

Principal neurons in layer III of the rat lateral entorhinal cortex (LEC) generate a self-sustained plateau potential and persistent spiking following the application of a brief suprathreshold excitatory stimulus delivered in the presence of the muscarinic receptor agonist carbachol. This persistent activity can be terminated by application of a second excitatory stimulus, and these cells can be repeatedly toggled between on and off states by consecutive excitatory stimuli. However, the ionic mechanisms that underlie the production of on and off states in layer III LEC neurons are unknown but seem to involve activity-dependent conductances, since they can be initiated by trains of action potentials evoked by either depolarizing current pulses applied to the cell or by repetitive spiking induced by activation of excitatory synaptic inputs. In this study, we obtained intracellular recordings from rat layer III LEC neurons in vitro, and a series of pharmacological and ionic substitution experiments were performed to identify mechanisms involved in the induction and termination of persistent spiking. Our data indicate that initiation of the on state depends on spike-evoked calcium influx and subsequent activation of calcium-activated nonselective cationic current. Moreover, we show that termination of persistent firing in response to an excitatory stimulus can be blocked by tetraethylammonium or iberiotoxin, suggesting that the activation of calcium-activated potassium current mediated by large conductance calcium-activated K(+) (i.e., BK) channels is required to induce the off state.     

Intrasomatic and intradendritic recordings of plateau potentials in slices of the dentate gyms maintained in vitro

Intrasomatic and intradendritic recordings were performed in slices of the dentate gyrus maintained in vitro. When barium ions (2.4 mM) were substituted for calcium ions in the perfusing medium, plateau potentials appeared with an amplitude of 20-40 mV which lasted from 40 ms to more than one min; during these plateau potentials, the input membrane resistance was decreased. In the soma, plateau potentials were also observed in a medium containing barium ions + tetrodotoxin (0.3 or 0.6 microM); whereas, in the dendrites, the barium-induced plateau potentials were abolished after addition of tetrodotoxin to the barium containing perfusion. The somatic plateau potentials had a duration which appeared to be dependent on the stimulus frequency. After being in contact with the barium-tetrodotoxin solution for a long period, the soma membrane potential was observed to jump between two relatively stable levels: a resting state and a depolarized state. In conclusion, calcium conductances appear to be present both at the soma and the dendrites of dentate granule cells; however, at the dendritic level, it appears that, when sodium channel permeability is blocked by tetrodotoxin, there is insufficient inward current to support the generation of action potentials.     

Contribution of potassium conductances to a time-dependent transition in electrical properties of a cockroach motoneuron soma.

Contribution of potassium conductances to a time-dependent transition in electrical properties of a cockroach motoneuron soma. The cell body of the cockroach (Periplaneta americana) fast coxal depressor motoneuron (Df) displays a time-dependent change in excitability. Immediately after dissection, depolarization evokes plateau potentials, but after several hours all-or-none action potentials are evoked. Because K channel blockers have been shown to produce a similar transition in electrical properties, we have used current-clamp, voltage-clamp and action-potential-clamp recording to elucidate the contribution of different classes of K channel to the transition in electrical activity of the neuron. Apamin had no detectable effect on the neuron, but charybdotoxin (ChTX) caused a rapid transition from plateau potentials to spikes in the somatic response of Df to depolarization. In neurons that already produced spikes when depolarized, ChTX increased spike amplitude but did not increase their duration nor decrease the amplitude of their afterhyperpolarization. 4-Aminopyridine (4-AP) (which selectively blocks transient K currents) did not cause a transition from plateau potentials to spikes but did enhance oscillations superimposed on plateau potentials. When applied to neurons that already generated spikes when depolarized, 4-AP could augment spike amplitude, decrease the latency to the first spike, and prolong the afterhyperpolarization. Evidence suggests that the time-dependent transition in electrical properties of this motoneuron soma may result, at least in part, from a fall in calcium-dependent potassium current (IK,Ca), consequent on a gradual reduction in [Ca2+ ]i. Voltage-clamp experiments demonstrated directly that outward K currents in this neuron do fall with a time course that could be significant in the transition of electrical properties. Voltage-clamp experiments also confirmed the ineffectiveness of apamin and showed that ChTX blocked most of IK,Ca. Application of Cd2+ (0.5 mM), however, caused a small additional suppression in outward current. Calcium-insensitive outward currents could be divided into transient (4-AP-sensitive) and sustained components. The action-potential-clamp technique revealed that the ChTX-sensitive current underwent sufficient activation during the depolarizing phase of plateau potentials to enable it to shunt inward conductances. Although the ChTX-sensitive conductance apparently makes little contribution to spike repolarization, the ChTX-resistant IK,Ca does make a significant contribution to this phase of the action potential. The 4-AP-sensitive current began to develop during the rising phase of both action potentials and plateau potentials but had little effect on the electrical activity of the neuron, probably because of its relatively small amplitude.     

Effects ofAnemonia sulcata toxin II on presynaptic currents and evoked transmitter release at neuromuscular junctions of the mouse

The effect ofAnemonia sulcata toxin II (ATX-II) on the amount of transmitter released by nerve impulses was investigated in motor end-plates of the mouse. ATX-II (80 nM) caused repetitive end-plate potentials in response to a single nerve stimulus and a 3- to 4-fold increase in the quantal content of the phasic end-plate potential. This increase is less than what would be expected if ATX-II induced plateau action potentials at the motor endings. To solve this discrepancy presynaptic currents were recorded by focal extracellular electrodes. It was found that the K current present at the endings is strong enough to prevent the development of presynaptic plateau action potentials, in contrast to what has been observed in other excitable membranes (unmyelinated axons, nodes of Ranvier and skeletal muscle fibres). By using tetraethylammonium and 3,4-diaminopyridine to block K channels and Co²⁺ to block Ca channels, ATX-II allowed the development of prolonged plateau responses at the endings upon motor nerve stimulation. These results suggest that the mouse motor endings are endowed with a relatively powerful K channel system, which effectively controls the amount of presynaptic depolarization.     

Die Wirkung von NiCl2 auf den isolierten Ranvierschen Schnürring

Summary 1. An attempt was made to explain the extreme prolongation of the nodal action potential by 0.1–1.0 mM NiCl₂ in terms of the ionic theory.2. The effects of NiCl₂ at room temperature are similar to those of temperature reduction: decreased maximum rate of rise of the action potential, lengthened action potential duration, elevated threshold, increased tendency for repetitive activity; in addition, the amplitude of the action potential is slightly increased.3. The long lasting plateaus of the responses obtained under the combined influence of NiCl₂ and temperature reduction are shortened by cathodal polarization, strong anodal polarization and decrease of [Na]_o. The plateau can be prematurely terminated by short anodal pulses of critical amplitude; short cathodal pulses reduce the duration of the plateau gradually with increasing pulse strength.4. Increased [K]_o prolongs plateau duration; the steep repolarization phase which normally terminates the plateau is replaced by a long lasting after-depolarization with stepwise potential decline. The amplitude of K-depolarization is not influenced by NiCl₂ or temperature reduction.5. 1.0 mM NiCl₂ changes the relation between maximum rate of rise and steady-state polarization; the potential change required for 50% sodium inactivation is +8 mV in normal Ringer's solution and +16 mV in the presence of 1.0 mM NiCl₂ (22° C).6. 1.0 mM NiCl₂ increases the time constant of delayed rectification as measured in Na-poor solutions at 4° C by a factor of about 2. The decrease of action potential under cathodal polarization is slightly delayed by NiCl₂.7. It is concluded that prolongation of the nodal action potential by NiCl₂ is due to delayed and reduced inactivation of sodium permeability and delayed increase of potassium permeability; part of the NiCl₂-effect could be explained by assuming competition between Ni⁺⁺ and Ca⁺⁺ for specific sites at the membrane.

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Mit 10 Textabbildungen

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Die Untersuchungen wurden mit Unterstützung der Deutschen Forschungsgemeinschaft ausgeführt.