Negative Slope Resistance Research Articles

Tl+-induced μs Gating of Current Indicates Instability of the MaxiK Selectivity Filter as Caused by Ion/Pore Interaction

Patch clamp experiments on single MaxiK channels expressed in HEK293 cells were performed at high temporal resolution (50-kHz filter) in asymmetrical solutions containing 0, 25, 50, or 150 mM Tl+ on the luminal or cytosolic side with [K+] + [Tl+] = 150 mM and 150 mM K+ on the other side. Outward current in the presence of cytosolic Tl+ did not show fast gating behavior that was significantly different from that in the absence of Tl+. With luminal Tl+ and at membrane potentials more negative than −40 mV, the single-channel current showed a negative slope resistance concomitantly with a flickery block, resulting in an artificially reduced apparent single-channel current Iapp. The analysis of the amplitude histograms by β distributions enabled the estimation of the true single-channel current and the determination of the rate constants of a simple two-state O-C Markov model for the gating in the bursts. The voltage dependence of the gating ratio R = Itrue/Iapp = (kCO + kOC)/kCO could be described by exponential functions with different characteristic voltages above or below 50 mM Tl+. The true single-channel current Itrue decreased with Tl+ concentrations up to 50 mM and stayed constant thereafter. Different models were considered. The most likely ones related the exponential increase of the gating ratio to ion depletion at the luminal side of the selectivity filter, whereas the influence of [Tl+] on the characteristic voltage of these exponential functions and of the value of Itrue were determined by [Tl+] at the inner side of the selectivity filter or in the cavity.

Saturation and Microsecond Gating of Current Indicate Depletion-induced Instability of the MaxiK Selectivity Filter

Patch clamp experiments on single MaxiK channels expressed in HEK293 cells were performed with a high temporal resolution (50-kHz filter) in symmetrical solutions with 50, 150, or 400 mM KCl and 2.5 mM CaCl2 and 2.5 mM MgCl2. At membrane potentials >+100 mV, the single-channel current showed a negative slope resistance, concomitantly with a flickery block, which was not influenced by Ca2+ or Mg2+. The analysis of the amplitude histograms by beta distributions revealed that current in this voltage range was reduced by two effects: rate limitation at the cytosolic side of the pore and gating with rate constants 10–20-fold higher than the cutoff frequency of the filter (i.e., dwell times in the microsecond range). The data were analyzed in terms of a model that postulates a coupling between both effects; if the voltage over the selectivity filter withdraws ions from the cavity at a higher rate than that of refilling from the cytosol, the selectivity filter becomes instable because of ion depletion, and current is interrupted by the resulting flickering. The fit of the IV curves revealed a characteristic voltage of 35 mV. In contrast, the voltage dependence of the gating factor R, i.e., the ratio between true and apparent single-channel current, could be fitted by exponentials with a characteristic voltage of 60 mV, suggesting that only part of the transmembrane potential is felt by the flux through the selectivity filter.

Effects of rolipram on induction of action potential bursts in central snail neurons

Effects of rolipram, a selective inhibitor of phosphodiesterases (PDE) IV, on induction of action potential bursts were studied pharmacologically on the RP4 central neuron of the giant African snail ( Achatina fulica Ferussac). Oscillations of membrane potential bursts were elicited by rolipram and forskolin. The bursts of potential elicited by rolipram were not inhibited after administration with (a) calcium-free solution, (b) high-magnesium solution (30 mM) or (c) U73122. However, the bursts of potential elicited by rolipram were inhibited by pretreatment with KT-5720 (10 μM). Voltage-clamp studies revealed that rolipram decreased the total inward current and steady-state outward currents of the RP4 neuron. The negative slope resistance (NSR) was not detectable in control or rolipram treated RP4 neurons. TEA elicited action potential bursts and an NSR at membrane potential between −50 mV and −30 mV. It is suggested that the bursts of potential elicited by rolipram were not due to (1) synaptic effects of neurotransmitters; (2) NSR of steady-state I– V curve; (3) phospholipase activity of the neuron. The rolipram-elicited bursts of potential were dependent on the phosphodiesterases inhibitory activity and the cAMP signaling pathway in the neuron.

Effects of 2,3-Butanedione Monoxime on Induction of Action Potential Bursts in Central Snail Neurons: Direct and Indirect Modulations of Ionic Currents

The effects of 2,3-butanedione monoxime (BDM) on induction of action potential bursts were studied pharmacologically on the RP4 central neuron of giant African snail (Achatina fulica Ferussac). The effect of okadaic acid on the neuron was also tested. The RP4 neuron showed a spontaneous firing of action potential. Okadaic acid (1 µmol/l) did not alter the frequency of spontaneous action potential while BDM (3 mmol/l) reversibly elicited bursts of potential (BoP) of the RP4 neuron. The BoP elicited by BDM (3 mmol/l) were reversed 20 min after incubation with diazoxide (500 µmol/l) while the BoP were not altered in preparations treated with okadaic acid and BDM. The BDM-elicited BoP were not inhibited after administration with (a) hexamethonium (100 µmol/l), (b) atropine (1 mmol/l), (c) d-tubocurarine (100 µmol/l), (d) prazosin (100 µmol/l), (e) propranolol (100 µmol/l), (f) calcium-free solution, (g) high K⁺ (12 mmol/l) or (h) with high Mg²⁺ (30 mmol/l) solutions. The BDM-elicited BoP were inhibited by pretreatment with KT-5720 (10 µmol/l) or H89 (10 µmol/l), the protein kinase A inhibitors. However, the BoP were not affected after application of chelerythrine (10 µmol/l) or Ro 31-8220 (10 µmol/l), the protein kinase C inhibitors. Voltage-clamped studies revealed that BDM elicited a negative slope resistance (NSR) at membrane potentials between –50 and –10 mV. The NSR was not detectable at the same membrane potential in control RP4 neuron. It is suggested that the BoP elicited by BDM were not due to (1) the synaptic effects of neurotransmitters; (2) the activation of cholinergic, adrenergic receptors, or (3) phosphatase activity of the neuron. The BDM-elicited BoP were dependent on the protein kinase A related cAMP in the neuron and the delayed outward K⁺ current may contribute to the BDM-elicited BoP.

Persistent sodium and calcium currents in rat hypoglossal motoneurons.

Voltage-dependent persistent inward currents are thought to make an important contribution to the input-output properties of alpha-motoneurons, influencing both the transfer of synaptic current to the soma and the effects of that current on repetitive discharge. Recent studies have paid particular attention to the contribution of L-type calcium channels, which are thought to be widely distributed on both the somatic and the dendritic membrane. However, the relative contribution of different channel subtypes as well as their somatodendritic distribution may vary among motoneurons of different species, developmental stages, and motoneuron pools. In this study, we have characterized persistent inward currents in juvenile (10- to 24-day-old) rat hypoglossal (HG) motoneurons. Whole-cell, voltage-clamp recordings were made from the somata of visualized rat HG motoneurons in 300-microm brain stem slices. Slow (10 s), triangular voltage-clamp commands from a holding potential of -70 to 0 mV and back elicited whole-cell currents that were dominated by outward, potassium currents, but often showed a region of negative slope resistance on the rising phase of the command. In the presence of potassium channel blockers (internal cesium and external 4-aminopyridine and tetraethylammonium), net inward currents were present on both the rising and falling phases of the voltage-clamp command. A portion of the inward current present on the ascending phase of the command was mediated by TTX-sensitive sodium channels, whereas calcium channels mediated the remainder of the current. We found roughly the same relative contributions of P-, N-, and L-type channels to the calcium currents recorded at the soma that had previously been found in neonatal rat HG motoneurons. In most cells, the somatic voltage thresholds for calcium current onset and offset were similar and the peak current was largest on the ascending phase of the clamp command. However, about one-third of the cells exhibited a substantial clockwise current hysteresis, i.e., inward currents were present at lower voltages on the descending phase of the clamp command. In the same cells, 1-s depolarizing voltage-clamp commands were followed by prolonged tail currents, consistent with a prominent contribution from dendritic channels. In contrast to previous reports on turtle and mouse motoneurons, blocking L-type calcium channels did not eliminate these presumed dendritic currents.

AMRP peptides modulate a novel K(+) current in pleural sensory neurons of Aplysia.

Modulation of Aplysia mechanosensory neurons is thought to underlie plasticity of defensive behaviors that are mediated by these neurons. In the past, identification of modulators that act on the sensory neurons and characterization of their actions has been instrumental in providing insight into the functional role of the sensory neurons in the defensive behaviors. Motivated by this precedent and a recent report of the presence of Aplysia Mytilus inhibitory peptide-related (AMRP) neuropeptides in the neuropile and neurons of the pleural ganglia, we sought to determine whether and how pleural sensory neurons respond to the AMRPs. In cultured pleural sensory neurons under voltage clamp, AMRPs elicited a relatively rapidly developing, then partially desensitizing, outward current. The current exhibited outward rectification; in normal 10 mM K(+), it was outward at membrane potentials more positive than -80 mV but disappeared without reversing at more negative potentials. When external K(+) was elevated to 100 mM, the AMRP-elicited current reversed around -25 mV; the shift in reversal potential was as expected for a current carried primarily by K(+). In the high-K(+) solution, the reversed current began to decrease at potentials more negative than -60 mV, creating a region of negative slope resistance in the I-V relationship. The AMRP-elicited K(+) current was blocked by extremely low concentrations of 4-aminopyridine (4-AP; IC(50) = 1.7 x 10(-7) M) but was not very sensitive to TEA. In cell-attached patches, AMRPs applied outside the patch-thus presumably through a diffusible messenger-increased the activity of a K(+) channel that very likely underlies the macroscopic current. The single-channel current exhibited outward rectification, and the open probability of the channel decreased with hyperpolarization; together, these two factors accounted for the outward rectification of the macroscopic current. Submicromolar 4-AP included in the patch pipette blocked the channel by reducing its open probability without altering the single-channel current. Based on the characteristics of the AMRP-modulated K(+) current, we conclude that it is a novel current that has not been previously described in Aplysia mechanosensory neurons. In addition to this current, two other AMRP-elicited currents, a slow, 4-AP-resistant outward current and a Na(+)-dependent inward current, were occasionally observed in the cultured sensory neurons. Responses consistent with all three currents were observed in sensory neurons in situ in intact pleural ganglia.

Amphetamine elicited potential changes in vertebrate and invertebrate central neurons

The effects of amphetamine on potential changes in both vertebrate and invertebrate central neurons and factors affecting the potential changes were tested. The animals studied included mice, newborn rat and African snail. Seizure was elicited after lethal doses of d-amphetamine (75 mg/kg, i.p.) administration in mice. Repetitive firing of the action potentials were elicited after d-amphetamine (1-30 microM) administration in thin thalamic brain slices of newborn rat. Bursting firing of action potentials in the giant African central RP4 neuron were also elicited after d-amphetamine or l-amphetamine (0.27 mM) administration. The amphetamine elicited bursting firing of action potentials was not blocked even after high concentrations of d-tubocurarine, atropine, haloperidol, hexamethonium administration. Therefore, the amphetamine elicited potential changes may not be directly related to the activation of the receptors of the neuron. The bursting firing of action potentials elicited by amphetamine occurred 20-30 min after amphetamine administration extracellularly, even after high concentrations of d-amphetamine administration (0.27, 1 mM). However, the bursting firing of potentials occurred immediately if amphetamine was administrated intracellularly at lower concentration. Extracellular application of ruthenium red, the calcium antagonist, abolished the amphetamine elicited bursting firing of action potentials. If intracellular injection of EGTA, a calcium ion chelator, or injection with high concentrations of magnesium, the bursting firing of potentials were immediately abolished. These results suggested that the active site of amphetamine may be inside of the neuron and the calcium ion in the neuron played an important role on the bursting of potentials. In two-electrode voltage clamped RP4 neuron, amphetamine, at 0.27 mM, decreased the total inward and steady outward currents of the RP4 neuron. d-Amphetamine also decreased the calcium, Ia and the steady-state outward currents of the RP4 neuron. Besides, amphetamine elicited a negative slope resistance (NSR) if membrane potential was in the range of -50 to -10 mV. The NSR was decreased in cobalt substituted calcium free and sodium free solution. The effects of secondary messengers on the amphetamine elicited potential changes were tested. The bursting firing of action potentials elicited by amphetamine in central snail neurons decreased following extracellular application of H8 (N-(2-methyl-amino) ethyl-3-isoquinoline sulphonamide dihydrochloride), a specific protein kinase A inhibitor and anisomycin, a protein synthesis inhibitor. However, the bursting firing of action potentials were not affected after extracellular application of H7 (1,(5-isoquinolinesulphonyl)-2-methylpiperasine dihydrochloride), a specific protein kinase C (PKC) inhibitor, or intracellular application of GDPbetaS, a G protein inhibitor. The oscillation of membrane potential of the bursting activity was blocked after intracellular injection of 3'-deoxyadenosine, an adenylyl-cyclase inhibitor. These results suggested that the bursting firing of action potentials elicited by d-amphetamine in snail neuron may be associated with the cyclic AMP second messenger system; on the other hand, it may not be associated with the G protein and protein kinase C activity. It is concluded that amphetamine elicited potential changes in both vertebrate and invertebrate central neurons. The changes are closely related to the ionic currents and second messengers of the neurons.

Action potential bursts in central snail neurons elicited by d-amphetamine: roles of ionic currents

The roles of the ionic currents in the firing of potential bursts elicited by d-amphetamine in central snail neurons were studied in the identified RP4 neuron of the African snail, Achatina fulica Ferussac, using the two-electrode voltage-clamp method. Oscillations of membrane potential bursts were elicited by d-amphetamine. The action potential bursts elicited by d-amphetamine decreased following intracellular injection of either EDTA or magnesium, or extracellular application of lanthanum. Voltage-clamped studies revealed that d-amphetamine decreased the fast Na +, Ca 2+ and transient outward K + currents of the RP4 neuron. It also decreased the steady-state K + current and elicited a negative slope resistance in the steady-state I–V curve between −50 and −10 mV. The amplitude of negative slope resistance was decreased if either Na +-free saline or Co 2+-substituted Ca 2+-free saline was perfused. d-Amphetamine did not increase the amplitude of the slowly inactivating Ca 2+ current or the persistent Na + currents of RP4 neuron. Tetraethylammonium, a blocker of the delayed outward K + current, elicited action potential bursts and negative slope resistance in the RP4 neuron, while 4-aminopyridine, an inhibitor of transient outward K + current (I A), did not. These results demonstrate that the delayed outward K + current and the negative slope resistance in steady-state I–V curve elicited by d-amphetamine may be responsible for the action potential bursts in central snail neurons elicited by d-amphetamine.

Evidence for a Novel Bursting Mechanism in Rodent Trigeminal Neurons

We investigated bursting behavior in rodent trigeminal neurons. The essential mechanisms operating in the biological systems were determined based on testable predictions of mathematical models. Bursting activity in trigeminal motoneurons is consistent with a traditional mechanism employing a region of negative slope resistance in the steady-state current-voltage relationship (Smith, T. G. 1975. Nature. 253:450–452). However, the bursting dynamics of trigeminal interneurons is inconsistent with the traditional mechanisms, and is far more effectively explained by a new model of bursting that exploits the unique stability properties associated with spike threshold (Baer, S. M., T. Erneux, and J. Rinzel. 1989. SIAM J. Appl. Math. 49:55–71).

Ionic Basis for Serotonin-Induced Bistable Membrane Properties in Guinea Pig Trigeminal Motoneurons

Intracellular recordings and pharmacological manipulations were employed to investigate the ionic basis for serotonin-induced bistable membrane behaviors in guinea pig trigeminal motoneurons (TMNs). In voltage clamp, 10 microM serotonin (5-HT) induced a region of negative slope resistance (NSR) in the steady-state current-voltage (I-V) relationship at potentials less negative than -58 mV, creating the necessary conditions for membrane bistability. The contributions of sustained Na+ and Ca2+ currents to the generation of the NSR were investigated using specific ion channel antagonists and agonists. The NSR was eliminated by the L-type Ca2+ channel antagonist nifedipine (5-10 microM), indicating the contribution of L channels. In nifedipine, inward rectification was present in the I-V relationship in a similar voltage range (greater than -58 mV). This region was subsequently linearized by tetrodotoxin (TTX), indicating the presence of a persistent Na+ current. When the 5-HT-induced NSR was eliminated by perfusion in low Ca2+ solution (0.4 mM), it was restored by the Na+ channel agonist veratridine (10 microM). Commensurate with bistability, in current clamp during bath application of 5-HT, plateau potentials were elicited by transient depolarizing or hyperpolarizing stimuli. Plateau potentials evoked by depolarization were observed under control and TTX conditions, but were blocked by nifedipine, suggesting the participation of an L-type Ca2+ current. Plateau potentials initiated after release from hyperpolarization (anode break) were blocked by 300 microM Ni2+, suggesting the responses relied on deinactivation of a T-type Ca2+ current. Conditional bursting was also observed in 5-HT. Nifedipine or low Ca2+ solutions blocked bursting, and the L-channel agonist Bay K 8644 (10 microM) extended the duration of individual bursts, demonstrating the role of L-type Ca2+ currents. Interestingly, when bursting was blocked by nifedipine or low Ca2+, it could be restored by veratridine application via enhancement of the persistent Na+ current. We conclude that bistable membrane behaviors in TMNs are mediated by L-type Ca2+ and persistent Na+ currents. 5-HT is associated with enhancement of TMN activity during oral-motor activity; the induction of bistable membrane properties by 5-HT represents a cellular mechanism for this enhancement.

Vasopressin/oxytocin-related conopressin induces two separate pacemaker currents in an identified central neuron of Lymnaea stagnalis.

The molluscan vasopressin/oxytocin analogue Lys-conopressin excites neurons in the anterior lobe of the right cerebral ganglion of the snail Lymnaea stagnalis. Persistent inward currents that underlie the excitatory response were studied with the use of voltage-ramp protocols in the identified neuron RCB1 and other anterior lobe neurons. Under whole cell voltage-clamp conditions, two types of conopressin-activated current could be distinguished on the basis of their voltage dependence: 1) a pacemaker-like current that was activated at potentials above -40 mV (high-voltage-activated current, I(HVA)) and 2) an inward current that was activated at all potentials between -90 and +10 mV (low-voltage-activated current, I(LVA)). Ion substitution experiments indicate that sodium is the main charge carrier for I(HVA) and I(LVA). Both currents are differentially affected by cadmium. I(HVA) and I(LVA) differ in dose dependence, with median effective concentration values of 7.7 x 10(-8) M and 2.2 x 10(-7) M, respectively. Vasopressin and oxytocin act as weak agonists for the conopressin responses. The kinetics of desensitization and washout of I(HVA) and I(LVA) are different. The HVA response shows little desensitization, whereas the LVA response desensitizes within minutes (time constant 80 +/- 28 s, mean +/- SD). The time constant of washout on removal of conopressin is 159 +/- 63 s for I(HVA) and 36 +/- 13 s for I(LVA). These results suggest that two distinct conopressin receptors are involved in the activation of both currents. The conopressin-activated currents induce or enhance a region of negative slope resistance in the steady-state current-voltage relation. They differ from a third persistent inward current that is carried by calcium and completely blocked by cadmium. The presumed functional roles of these currents, possibly including autoregulation, are discussed.

Veratridine-enhanced persistent sodium current induces bursting in CA1 pyramidal neurons.

The mechanism of veratridine-induced bursting activity was studied in rat hippocampal CA1 pyramidal neurons. Veratridine (0.1-0.3 microM) induces bursting in previously normal pyramidal neurons. The current-voltage curves of untreated neurons show a slight deviation from the linear Ohmic relation; this deviation is known as the "depolarizing rectification". Veratridine markedly accentuates the depolarizing rectification so that a zero slope or negative slope appears in the current-voltage curve of these neurons. Both the veratridine-induced bursting activity and negative slope resistance are blocked by small concentrations of tetrodotoxin or by raising the calcium concentration of the superfusion medium. Under single-electrode voltage clamping, a subthreshold persistent (slowly inactivating) sodium current, which can be recorded in untreated neurons, is found to be enhanced in the veratridine-treated neurons. This current is thought to be responsible for the slow depolarizing phase of bursting activity and the development of negative slope resistance in the current-voltage relationship. The present results demonstrate that veratridine enhances the slowly inactivating sodium current, leading to the development of negative slope resistance and induction of bursting in rat hippocampal CA1 pyramidal neurons.

Intracellularly applied anti-P70 antibody blocks the induction of abnormal membrane properties by pentylenetetrazole in identified Euhadra neurons

Using the voltage-clamp technique combined with pressure injection, we have studied the action of pentylenetetrazole (PTZ) on identified Euhadra neurons by examining how the PTZ-induced changes in membrane properties are affected by an antibody against P70, a protein found in the experimentally-induced epileptogenic cortex of rats. Intracellular injection of anti-P70 antibody blocked the induction by PTZ; bursting activity with both of development of negative slope resistance region in the steady state I–V curve and a reduction in the delayed outward potassium current. These results suggest a novel mechanism of action for PTZ, involving intracellular protein(s) which react with anti-P70 antibody.

Properties of inward and outward potassium currents in cultured mouse motoneurons

Inward rectifier potassium currents and calcium-dependent potassium currents have been studied in cultured embryonic mouse motoneurons. Sustained unitary inward rectifier potassium currents were recorded from cell-attached patches and the channel conductance was dependent on external K+ concentration with a value of 25 pS when external K+ was 140 mM. The channel open probability exhibited a sigmoidal dependence on potential with the largest values (near 0.7) at depolarizing patch potentials. Inactivating inward rectifier potassium currents were also recorded in some cell-attached patches following voltage steps to hyperpolarizing potentials with the rate of inactivation faster with larger hyperpolarizing steps. Whole-cell inward rectifier potassium currents increased from an initial level to a steady-state level with hyperpolarizing steps to -120 mV from a holding potential of -60 mV; with larger hyperpolarizing commands the peak currents decayed to the steady-state. The steady-state current-voltage relation exhibited a region of negative slope resistance. External Cs+ (0.5-1 mM) reduced the amplitudes of macroscopic currents and diminished the open times of unitary currents consistent with block of open rectifying channels with an estimated KD for channel block of 1 mM. A large conductance calcium-dependent potassium channel was isolated in inside-out patches with a conductance of 240 pS with symmetrical 140 mM K+ across the patches and a conductance of 110 pS when the external K+ was reduced to 5 mM. With symmetrical K+ the channel open probability exhibited a sigmoid dependence on potential with the largest values, in excess of 0.8, associated with patch depolarization. The dependence of open probability on potential was dependent on the concentrations of internal Ca2+ and external K+. Properties of inward rectifier and calcium-dependent K+ channels, such as the voltage dependence of open probability, are involved in the establishment of cellular excitability in motoneurons. Future studies will be useful to investigate whether channel properties of motoneurons are altered after cell treatment with neurotoxic agents including oxygen radicals or excitotoxic amino acids.

Characterization of the membrane ion currents of a model molluscan muscle, the accessory radula closer muscle of Aplysia california. I. Hyperpolarization-activated currents.

1. The simple neuromuscular circuit consisting of the accessory radula closer (ARC) muscle of the mollus Aplysia californica together with its innervating motor and modulatory neurons has been extensively studied as a model preparation in which it might be possible to reach an integrated understanding of the neural and cellular mechanisms of behavioral plasticity, in this case of a component of Aplysia feeding behavior. Previous work has suggested that much of the plasticity of this behavior is implemented by appropriate release of modulatory neurotransmitters and peptide cotransmitters that modulate several parameters of the contractions of the ARC muscle. However, little is as yet known about the underlying cellular mechanisms. 2. We have begun to study single, functionally intact fibers dissociated from the ARC muscle to assess to what extent the modulation of its contraction might be mediated by one candidate mechanism, modulation of its membrane ion currents. First, however, it was necessary to gain a thorough understanding of the unmodulated currents and their likely roles in normal contraction. Using voltage-clamp techniques, we have therefore identified and characterized the major currents present in the ARC muscle fibers. We describe these currents in this and the following two papers. These results constitute the first detailed description of ion currents in a molluscan muscle and lay the foundation for further study, to be presented in subsequent papers, of the roles of two currents that we have indeed found to be modulated in ways likely to contribute to the modulation of contraction. 3. In this paper we first describe the general electrophysiological characteristics of the dissociated fibers and present evidence that the fibers can be adequately space clamped. 4. The physiological operating voltage range of the nonspiking ARC muscle most likely extends from about -80 to about -25 mV. The steady-state current-voltage (I-V) relation of total membrane current rectifies inwardly in the negative and outwardly in the positive portion of this voltage range, with a plateau region of high or even negative slope resistance separating the two regions of rectification. 5. The current responsible for the inward rectification at negative voltages is a classical inwardly rectifying K current. It is activated by hyperpolarization with quasi-instantaneous kinetics; its whole I-V relation shifts along the voltage axis in a Nernstian manner with altered extracellular K+ concentration; it is blocked by low extracellular Ba2+ and Cs+, and the block is promoted by hyperpolarization.(ABSTRACT TRUNCATED AT 400 WORDS)

Ionic contributions to the oscillatory firing activity of rat Purkinje cells in vitro

Oscillatory firing activity in cerebellar Purkinje cells (PCs) can be maintained by intrinsic ionic conductances in the apparent absence of excitatory and inhibitory synaptic input as demonstrated by application of TTX or antagonists of amino acid-mediated transmission or both. Bursting activity in these cells is associated with a region of ZSR (zero slope resistance, the beginning part of a negative slope resistance region) of the whole cell quasi-steady-state I–V relationship. Blockade of Na + current by TTX unmasked the ZSR region in all PCs tested. Based on current and voltage clamp experiments, hyperpolarization-activated cation current ( I h) participates in the rhythmic firing activity by influencing the amplitude and duration of the interburst interval and the resultant pattern of the burst generation. Blockade of I h with cesium (Cs +) retards the membrane rebound from the after-hyperpolarization and results in longer and more negative hyperpolarization between bursts. However, Cs + did not affect the presence and characteristic of the ZSR region of the whole cell quasi-steady-state I–V curve.

Augmentation of bursting pacemaker activity by serotonin in an identified Achatina fulica neurone: an increase in sodium- and calcium-activated negative slope resistance via cyclic-AMP-dependent protein phosphorylation.

The mechanism of serotonin (5-HT) action on bursting activity was examined in a bursting pacemaker neurone of the snail Achatina fulica. 5-HT augmented both the depolarizing and post-burst-hyperpolarizing phases of the bursting cycle in a dose-dependent manner. This biogenic amine also enhanced the negative slope resistance (NSR), which was normally detectable at membrane potentials between -40 and -20 mV, and produced another NSR at voltages between -20 and 0 mV. The former NSR disappeared in Na(+)-free saline and the latter was abolished by replacement with Co(2+)-substituted Ca(2+)-free saline. Both isobutylmethylxanthine, extracellular applied, and intracellularly applied cyclic AMP simulated a 5-HT effect on the current-voltage relationships. In contrast, the 5-HT effect was suppressed in a dose-dependent manner by prior treatment with a cyclic-AMP-dependent protein kinase inhibitor, isoquinoline sulphonamide. Similar suppression was observed after intracellular injection of a cyclic-AMP-dependent protein kinase inhibitor isolated from bovine muscle. These results suggest that 5-HT may augment the bursting pacemaker activity by its stimulatory effect on both the slow Na+ channels and the Ca2+ channels through cyclic-AMP-dependent protein phosphorylation.

Plateau potentials drive axonal impulse bursts in insect motoneurons

Intracellular recordings were made from the soma of the `fast' coxal depressor motoneuron of the hind leg of the cockroach Periplaneta americana under current- and voltage-clamp. In response to depolarizing current injection, this neuron was able to generate plateau potentials that were able to far outlive the duration of the applied depolarization. These events constitute an inherent membrane property of the neuron because they could be evoked in somata that had been surgically isolated from other parts of the neuron (the soma is devoid of synaptic contacts); these experiments also showed that the soma of this neuron can participate in the generation of plateau potentials. The amplitude of these events at the spike-initiating zone was sufficient to evoke action potentials that correlated 1:1 with axonal impulses recorded extracellularly from the axon of the neuron. The ability to generate plateau potentials is associated with a region of negative slope resistance in the current--voltage relation of the neuron. Ion substitution experiments showed that the plateau potentials recorded from this neuron are calcium dependent. Our observations show for the first time that an insect motoneuron can produce plateau potentials. Accordingly, bistable membrane properties may play a role in shaping motor output in insects.

A specific 70K protein found in epileptic rat cortex: Induction of bursting activity and negative resistance by its intracellular application in Euhadra neurons

The effect of 70-kD protein (P70, a specific protein found in cobalt-induced epileptogenic focus of rat cerebral cortex) on membrane properties was examined in identified neurons of the snail, Euhadra peliomphala, using the pressure injection method combined with the voltage-clamp technique. In neurons that normally exhibited spontaneous regular firing, intracellular injection of P70 elicited bursting activity and a negative slope resistance (NSR) region in their current-voltage (I-V) curve in a manner corresponding to the duration of its injection. These responses were suppressed by prior injection of an antibody to P70 into the neurons, and were markedly inhibited by a reduction of extracellular Na+ ions and the anticonvulsant agent phenytoin, but not by Co(2+)-substituted Ca(2+)-free saline. In addition, intracellularly applied P70 potentiated both bursting activity and the NSR induced by a Na channel activator, veratridine. However, prior application of a saturating dose of this activator occluded the effect of P70. These results suggest that P70 elicits a Na(+)-dependent negative resistance, which may contribute to the generation of bursting activity.

Cyclic AMP-dependent protein phosphorylation is involved in the development of negative slope resistance and a reduction of the potassium current induced by pentylenetetrazole in identified snail neurons

The role of cAMP-dependent protein phosphorylation in the pentylenetetrazole (PTZ)-induced bursting activity was examined in snail neurons, using the voltage clamp method in combination with the pressure injection technique. The cAMP-dependent protein kinase inhibitors, protein kinase inhibitor isolated from rabbit muscle and isoquinolinesulfonamide, inhibited the PTZ-induced negative slope resistance (NSR) in the steady state I– V curve. These inhibitors also suppressed the action of PTZ on the delayed outward potassium current ( I KD). This suppression was transiently abolished by intracellular injection of the catalytic subunit of cAMP-dependent protein kinase. These findings suggest that cAMP-dependent protein phosphorylation may be involved in both the development of the NSR and a reduction of the I KD by PTZ, leading to depolarizing phase of a bursting cycle.