Characterization of a hyperpolarization-activated inward current in Cajal-Retzius cells in rat neonatal neocortex

Cajal-Retzius cells are among the first neurons appearing during corticogenesis and play an important role in the establishment of cortical lamination. To characterize the hyperpolarization-activated inward current (I(h)) and to investigate whether I(h) contributes to the relatively positive resting membrane potential (RMP) of these cells, we analyzed the properties of I(h) in visually identified Cajal-Retzius cells in cortical slices from neonatal rats using the whole cell patch-clamp technique. Membrane hyperpolarization to -90 mV activated a prominent inward current that was inhibited by 1 mM Cs(+) and was insensitive to 1 mM Ba(2+). The activation time constant for I(h) was strongly voltage dependent. In Na(+)-free solution, I(h) was reduced, indicating a contribution of Na(+). An analysis of the tail currents revealed a reversal potential of -45.2 mV, corresponding to a permeability coefficient (pNa(+)/pK(+)) of 0. 13. While an increase in the extracellular K(+) concentration ([K(+)](e)) enhances I(h), it was reduced by a [K(+)](e) decrease. This [K(+)](e) dependence could not be explained by an effect on the electromotive force on K(+) but suggested an additional extracellular binding site for K(+) with an apparent dissociation constant of 7.2 mM. Complete Cl(-) substitution by Br(-), I(-), or NO(3)(-) had no significant effect on I(h), whereas a complete Cl(-) substitution by the organic compounds methylsulfate, isethionate, or gluconate reduced I(h) by approximately 40%. The I(h) reduction observed in gluconate could be abolished by the addition of Cl(-). The analysis of the [Cl(-)](e) dependence of I(h) revealed a dissociation constant of 9.8 mM and a Hill-coefficient of 2.5, while the assumption of a gluconate-dependent I(h) reduction required an unreasonably high Hill-coefficient >20. An internal perfusion with the lidocaine derivative lidocaine N-ethyl bromide blocks I(h) within 1 min after establishment of the whole cell configuration. An inhibition of I(h) by 1 mM Cs(+) was without an effect on RMP, action potential amplitude, threshold, width, or afterhyperpolarization. We conclude from these results that Cajal-Retzius cells express a prominent I(h) with characteristic properties that does not contribute to the RMP.     

Release of monoamines and nitric oxide is involved in the modulation of hyperpolarization-activated inward current during acute thalamic hypoxia

Using slices of the dorsal lateral geniculate nucleus, it has been shown that, in the presence of excitatory and inhibitory amino acid antagonists, brief periods of hypoxia (3-4 min of 95% N(2)/5% CO(2)) induce in thalamocortical neurons an increase in instantaneous input conductance (G(N)) accompanied by an inward shift in baseline holding current (I(BH)). These effects have been suggested to be mediated, at least in part, by a positive shift in the voltage-dependence of the hyperpolarization-activated, mixed Na(+)/K(+) current (I(h)) and a change in its activation kinetics which transforms it into an almost instantaneously activated current. In this study, using the whole-cell patch-clamp technique, the contribution of an increased Ca(2+)-dependent transmitter release to the hypoxic response of thalamocortical neurons was further investigated using (i) blockers of calcineurin, a Ca(2+)/calmodulin-activated phosphatase that selectively regulates Ca(2+)-dependent release, and (ii) antagonists of neurotransmitters that are known to modulate I(h). Thalamocortical neurons (n = 23) recorded with electrodes filled with calcineurin autoinhibitory fragment (30-250 microM), a membrane impermeable blocker of calcinuerin, showed no difference either in resting, or in the hypoxia-induced changes in, G(N), I(BH) and I(h), when compared to thalamocortical cells patched with electrodes that did not contain calcineurin autoinhibitory fragment. In contrast, in 18 of these neurons recorded with calcineurin autoinhibitory fragment-filled electrodes, bath application either of cyclosporin-A (20 microM) or tacrolimus (50-100 microM), two membrane permeable blockers of calcineurin, abolished the effects of hypoxia on G(N), I(BH), and I(h). Separate application of noradrenaline, serotonin, histamine and nitric oxide antagonists produced only a small depression of the hypoxic response, while concomitant bath application of these antagonists decreased the hypoxia-induced changes in G(N) and I(BH) by 55 and 42%, respectively (n = 12). Concomitant bath application of 8-bromo-adenosine-3'5'-cyclicmonophosphate and 8-bromo-guanosine-3'5'-cyclicmonophosphate (both 1mM), which are known to mediate the action of these transmitters on I(h), increased G(N) (40%), decreased I(h) time-constant of activation (30%) and significantly occluded (50%) the hypoxia-induced effect on G(N) and I(BH). Thalamocortical neurons (n = 6) patched with electrodes filled with 8-bromo-adenosine-3'5'-cyclicmonophosphate and 8-bromo-guanosine-3'5'-cyclicmonophosphate (both 1 mM) showed a larger G(N) than the one recorded with the standard internal solution, and a significant depression of the hypoxia-induced changes in G(N) and I(BH).These results indicate that during acute thalamic hypoxia an increased release of noradrenaline, serotonin, histamine and nitric oxide is responsible for transforming I(h) into an instantaneously activating current via cyclic AMP- and cyclic GMP-mediated mechanisms.     

Activation characteristics of the calcium-dependent outward potassium current in Helix

The activation of calcium-dependent outward potassium current [I_K(Ca)] by shortlasting Ca²⁺ inward currents was studied. These Ca²⁺ currents were produced either by small depolarizing pulses preceding the larger depolarizations or by interposed repolarizations (I.R.s) starting from depolarized membrane potentials. I_K(Ca) then develops with a potential-invariant time course (half time 6–12 ms) and the normally bell-shaped isochronal I_K(Ca)/V curve, measured at between 30 and 300 ms, is straightened. However, these Ca²⁺-injecting pulses, of any amplitude and duration, do not increase the steady-state conductance to values beyond those measured with single step depolarizations to lower potentials. varied in length, activation of I_K(Ca) increased linearly with the during depolarization to near the supposed calcium equilibrium potential with no further Ca²⁺ influx. When I.R.s are varied in length, activation of I_(Ca) increases linearly with the amount of Ca²⁺ current. Fading of activation during I.R. follows a time course nearly ten times slower than activation and is not expressed in tail currents. The time course of I_K(Ca) is described by a function defined only by voltage parameters of activation combined with a minimum activation time constant which is similar to that found in tail currents. Peak location and general form of the I_K(Ca)/V relationship for times up to several hundred milliseconds are well predicted without the necessity to explicitly account for actual calcium entry.     

Bistable behavior originating in the axon of a crustacean motor neuron

Both vertebrate and invertebrate motor neurons can display bistable behavior in which self-sustained tonic firing results from a brief excitatory stimulus. Induction of the bistability is usually dependent on activation of intrinsic conductances located in the somatodendritic area and is commonly sensitive to action of neuromodulators. We have observed bistable behavior in a neuromuscular preparation from the foregut of the crab Cancer borealis that consists of the gastric mill 4 (gm4) muscle and the nerve that innervates it, the dorsal gastric nerve (dgn). Nerve-evoked contractions of enhanced amplitude and long duration (>30 s) were induced by extracellular stimulation when the stimulus voltage was above a certain threshold. Intracellular and extracellular recordings showed that the large contractions were accompanied by persistent firing of the dorsal gastric (DG) motor neuron that innervates gm4. The persistent firing could be induced only by stimulating a specific region of the axon and could not be triggered by depolarizing the soma, even at current amplitudes that induced high-frequency firing of the neuron. The bistable behavior was abolished in low-Ca²⁺ saline or when nicardipine or flufenamic acid, blockers of L-type Ca²⁺ and Ca²⁺-activated nonselective cation currents, respectively, was applied to the axonal stimulation region of the dgn. Negative immunostaining for synapsin and synaptotagmin argued against the presence of synaptic/modulatory neuropil in the dgn. Collectively, our results suggest that bistable behavior in a motor neuron can originate in the axon and may not require the action of a locally released neuromodulator.     

Characterization of a transient outward current in a rapidly adapting insect mechanosensory neuron

This paper describes the first voltage-clamp recordings from an arthropod cuticular sensory neuron. In the femoral tactile spine neuron of the cockroachPeriplaneta americana, a rapidly activating and inactivating outward current,I _A, appeared when the neuron was hyperpolarized for a short period before a depolarizing test pulse.I _A could be separated from the other outward currents using 5 mM 4-aminopyridine (4-AP), which specifically blocked it. Tetraethylammonium (TEA), (50 mM) did not removeI _A, but decreased the steady-state outward current by about 50%. The threshold forI _A activation was about -75 mV. The minimum activation and inactivation time constants were approximately 0.2 ms and 15 ms, respectively. The voltage dependencies of activation and inactivation were well fit-ted by Boltzmann distributions, giving values of membrane potential at halfmaximal activation (V ₅₀) equal to −56.5 mV and an equivalent gating charge ofn=3.9 for activation andV ₅₀=-86.7 mV andn=3.4 for inactivation. In current-clamp recordings, 4-AP reversibly reduced the cell's normal adaptation by lowering the threshold for action potentials, but did not affect the amplitude or duration of single action potentials. These results indicate thatI _A plays a role in short-term adaptation by opposing membrane depolarization and reducing the spike frequency during maintained stimulation.     

Serotonergic stretch receptors induce plateau properties in a crustacean motor neuron by a dual-conductance mechanism.

1. The mechanisms for induction of bistable plateau potential properties by a set of serotonergic/cholinergic peripheral stretch receptor cells [gastropyloric receptor (GPR) cells] were examined in the crab stomatogastric ganglion (STG) with the use of intracellular recording techniques. 2. GPR cell stimulation evoked nicotinic excitatory postsynaptic potentials (EPSPs) and induced plateau potential capability in the dorsal gastric (DG) motor neuron. The plateau potential could be triggered during a GPR train either by the summating nicotinic EPSPs or by brief intracellular current injection. After pharmacological blockade of nicotinic and muscarinic receptors, a slow depolarization in response to GPR stimulation was revealed. Prolonged plateau potentials could still be evoked after this treatment. Local application of serotonin (5-HT; 10 microM to 1 mM) mimicked the noncholinergic plateau inducing effects of GPR stimulation in the DG motor neuron. 3. The synergistic action of acetylcholine (ACh) and 5-HT was examined by stimulating the GPR cells at different frequencies (1-20 Hz). The plateau induction was present down to 2 Hz. The time to onset for triggering a plateau during a GPR train was determined by the co-released ACh. 4. The 5-HT-evoked slow depolarization persisted in tetrodotoxin (TTX; 0.1-1 microM), and the DG motor neuron could still produce a plateau potential on brief depolarization in the absence of the spike-generating mechanism. 5. In normal TTX-containing saline, the 5-HT-evoked depolarization was accompanied by a weak and variable decrease in apparent input conductance. After substituting one-half of the extracellular sodium with either Trisma-HCl or choline, the decrease in apparent input conductance became more pronounced. This decrease was converted to an increase in apparent input conductance when extracellular Ca2+ was replaced with Mg2+. 6. Under voltage-clamp conditions, local application of 5-HT caused a slow inward current of prolonged duration in DG. The current versus voltage relationship had an inverted U-shape with no apparent reversal potential in the entire voltage range investigated (-90 to -5 mV). The 5-HT-induced changes in input conductance showed a complex voltage dependence, with a conductance decrease from moderately depolarized voltages. 7. Extracellular Cs+ (2-4 mM) caused the DG to hyperpolarize 2-4 mV from rest, whereas lowering extracellular Ca2+ caused it to depolarize 7-15 mV. The combined action of low extracellular Ca2+ and 2-4 mM Cs+ caused an almost complete block of the slow 5-HT-evoked depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)     

Intracellular alkalinization potentiates slow inward current and prolonged bursting in a molluscan neuron

1. The bilaterally paired ventral white cells (VWCs) of the buccal ganglion of Pleurobranchaea drive the cyclic motor output of ingestive feeding behavior during prolonged and endogenously sustained burst episodes (7). The capacity to support burst episodes is specifically induced by appetitive (food) stimulation of chemosensory pathways (5). Cyclic 3',5'-adenosine monophosphate (cAMP) and its agonists also induce prolonged burst episodes (8) through potentiation of a slow inward current (6). 2. Intracellular alkalinization of the VWC by externally applied ammonium ion and methylamine (5-20 mM) induces bursting and enhances slow inward current measured under voltage-clamp conditions. The enhancement of slow inward current is seen in the induction or augmentation of a negative slope resistance region in the current-voltage relation and in the enhancement of slowly decaying inward current tails recorded near the K+ equilibrium potential following depolarizing voltage commands. 3. Intracellular injection of alkalinizing agents, bicarbonate ion and a strong buffer solution at pH 8.1, also enhance the inward current. In ammonium saline, enhancement of inward current is dependent on NH3 content, not NH4+; NH3 is the intracellular alkalinizing agent of ammonium saline. Therefore, the change in slow inward current is an effect specific to intracellular pH. 4. The time courses of inward current enhancement and intracellular pH change in NH4+ saline are similar. The results of this study suggest that normal fluctuations in intracellular pH may be significant determinants of the excitability and consequent activity of these and perhaps other neurons. The potential interaction of intracellular pH and cyclic AMP metabolism is discussed.     

Human Kir2.1 channel carries a transient outward potassium current with inward rectification

We have previously reported a depolarization-activated 4-aminopyridine-resistant transient outward K⁺ current with inward rectification (I _to.ir) in canine and guinea pig cardiac myocytes. However, molecular identity of this current is not clear. The present study was designed to investigate whether Kir2.1 channel carries this current in stably transfected human embryonic kidney (HEK) 293 cells using whole-cell patch-clamp technique. It was found that HEK 293 cells stably expressing human Kir2.1 gene had a transient outward current elicited by voltage steps positive to the membrane potential (around −70 mV). The current exhibited a current–voltage relationship with intermediate inward rectification and showed time-dependent inactivation and rapid recovery from inactivation. The half potential (V _0.5) of availability of the current was −49.4 ± 2.1 mV at 5 mM K⁺ in bath solution. Action potential waveform clamp revealed two components of outward currents; one was immediately elicited and then rapidly inactivated during depolarization, and another was slowly activated during repolarization of action potential. These properties were similar to those of I _to.ir observed previously in native cardiac myocytes. Interestingly, inactivation of the I _to.ir was strongly slowed by increasing intracellular free Mg²⁺ (Mg²⁺ _i , from 0.03 to 1.0, 4.0, and 8.0 mM). The component elicited by action potential depolarization increased with the elevation of Mg²⁺ _i . Inclusion of spermine (100 μM) in the pipette solution remarkably inhibited both the I _to.ir and steady-state current. These results demonstrate that the Mg²⁺ _i -dependent current carried by Kir2.1 likely is the molecular identity of I _to.ir observed previously in cardiac myocytes.     

Block and activation of the hyperpolarization-activated inward current by Ba and Cs in frog sinus venosus

Voltage clamp experiments were performed on isolated frog sinus venosus trabeculae using the double mannitol gap voltage clamp technique.On hyperpolarization from the holding potential (–30, –50 mV) to various potential levels slow activation of inward current was recorded. Several basic features of this current system resemble those of the currenti _f in mammalian pace-maker tissues. The current activates from a threshold ranging between –50, –70 mV and increases in the inward direction with the negative pulse amplitude. Conductance measurements during current development show a conductance increase. The current is strongly reduced during perfusion with Na-free medium.However, there were several important differences in its properties from those of thei _f current in other preparations. Ba in concentrations of 0.3–5 mM reduces the amplitude of the inward current in a concentration-dependent manner. Cs in low concentration range (1–10 mM) fails to have any effect on the time dependent current. Cs concentrations higher than 10 mM increase the current amplitude in a dose-dependent manner. The current increase induced by Cs still remains in Na-free solution and is not affected by Cl replacement. These results suggest that Cs may carry inward current. The identity of the ionic mechanism responsible for the observed current is discussed.     

5-HT modulation of multiple inward rectifiers in motoneurons in intact preparations of the neonatal rat spinal cord

This study introduces novel aspects of inward rectification in neonatal rat spinal motoneurons (MNs) and its modulation by serotonin (5-HT). Whole cell tight-seal recordings were made from MNs in an isolated lumbar spinal cord preparation from rats 1-2 days of age. In voltage clamp, hyperpolarizing step commands were generated from holding potentials of -50 to -40 mV. Discordant with previous reports involving slice preparations, fast inward rectification was commonly expressed and in 44% of the MNs co-existed with a slow inward rectification related to activation of I(h). The fast inward rectification is likely caused by an I(Kir). Thus it appeared around E(K) and was sensitive to low concentrations (100-300 microM) of Ba2+ but not to ZD 7288, which blocked I(h). Both I(Kir) and I(h) were inhibited by Cs2+ (0.3-1.5 mM). Extracellular addition of 5-HT (10 microM) reduced the instantaneous conductance, most strongly at membrane potentials above E(K). Low [Ba2+] prevented the 5-HT-induced instantaneous conductance reduction below, but not that above, E(K). This suggests that 5-HT inhibits I(Kir), but also other instantaneous conductances. The biophysical parameters of I(h) were evaluated before and under 5-HT. The maximal I(h) conductance, G(max), was 12 nS, much higher than observed in slice preparations. G(max) was unaffected by 5-HT. In contrast, 5-HT caused a 7-mV depolarizing shift in the activation curve of I(h). Double-exponential fits were generally needed to describe I(h) activation. The fast and slow time constants obtained by these fits differed by an order of magnitude. Both time constants were accelerated by 5-HT, the slow time constant to the largest extent. We conclude that spinal neonatal MNs possess multiple forms of inward rectification. I(h) may be carried by two spatially segregated channel populations, which differ in kinetics and sensitivity to 5-HT. 5-HT increases MN excitability in several ways, including inhibition of a barium-insensitive leak conductance, inhibition of I(Kir), and enhancement of I(h). The quantitative characterization of these effects should be useful for further studies seeking to understand how neuromodulation prepares vertebrate MNs for concerted behaviors such as locomotor activity.     

Inhibition of the slow inward current and the time-dependent outward current of mammalian ventricular muscle by gentamicin

The aminoglycoside antibiotic, gentamicin (GM), depressed the plateau phase and shortened the duration of the action potential in guinea pig papillary muscle. Its effect on the membrane currents was studied by a single sucrose gap voltage clamp method. The slow inward current (i_s) was remarkably diminished by GM with little change in its time course, in the voltage-dependency of the steady-state inactivation and activation or in its reversal potential. The maximal amplitude of i_s, obtained by subtracting the Co²⁺-resistant current, was reduced to 57% by 0.1 mmol/l GM and almost reduced to zero by 1 mmol/l GM. The efficacy of GM in inhibiting i_s was reduced by increasing the external Ca²⁺ concentration from 1.8 to 5.4 or 10.8 mmol/l, but not by the application of adrenaline. The time-dependent outward current (i_K) was also decreased by GM but only at higher concentrations. It is proposed that the depressant action of GM on i_s was due to a blockade of slow channels, whereby GM may have dislocated Ca from the binding sites at slow channels on the external surface of the membrane.     

Effects of calcium and calcium-chelating agents on the inward and outward current in the membrane of mollusc neurones.

1. Effects of internal and external Ca and Ca-chelating agents, EGTA and EDTA on transmembrane ionic currents were studied in isolated, internally dialysed neurones from the molluscs, Helix pomatia and Limnea stagnalis.2. The possible pharmacological effect of internally applied EGTA was investigated on the background of constant free Ca concentration (5.3 x 10(-9)M). EGTA had no effect on Ca and Na inward currents but considerably depressed the delayed K outward current. No effective removal of this action could be achieved by the elevation of intracellular free Ca.3. In the absence of divalent cations in the external medium, EGTA (as well as EDTA) applied either intra- or extracellularly caused the appearance of a very large Na inward current with kinetics similar to those of Ca inward current and with the reversal potential around 10 mV. Effective concentrations of chelating agents were 0.1 mM (extracellular) and 1.0 mM (intracellular).4. Increase in intracellular Ca in the absence of EGTA (by dialysis of the cell with Ca-saturated solutions) did not produce any significant effect on the delayed K outward current. The small change observed in this current could be evaluated as a depression of maximum slope conductance and a shift to more negative membrane potential.5. Ca inward current has been found extremely sensitive to internal Ca. 5.8 x 10(-8)M of internal free Ca produced its complete depression. This effect was reversible. Na inward current could be inhibited with 3.5 x 10(-7)M intracellular Ca.     

β-Adrenergic modulation of transient inward current in guinea-pig cardiac myocytes

Transient inward current (I_ti) indicating Ca²⁺-release from the sarcoplasmic reticulum and L-type Ca²⁺-current(I_Ca) were studied in atrial and ventricular myocytes from hearts of adult guinea-pigs by means of whole-cell voltage-clamp. The increase of I_Ca caused by -adrenergic stimulation using isoprenaline (ISO) or related experimental manoeuvres such as superfusion with forskoline (FORSK) was used as a qualitative monitor of an increase of intracellular cAMP. Changes of I_ti were used to manifest changes of sarcoplasmic Ca²⁺-release. In myocytes dialysed with citrate-based (60 mM) pipette filling solution containing 100 M EGTA spontaneous transient inward currents were recorded at a constant holding potential of –50 mV in the majority of myocytes. Superfusion with a solution containing ISO (5·10^–8M) increased the amplitude of spontaneous I_ti and reduced its time-to-peak. The effects of ISO on I_ti developed in parallel to stimulation of I_Ca. In myocytes which did not show spontaneous cyclic Ca²⁺-release in the above condition, this could be evoked de novo by ISO. Spontaneous I_ti was suppressed in the majority of cells by increasing the concentration of EGTA in the dialysing solution to 200 M. Brief (50 ms) activation of I_Ca by voltage steps from –50 to +10 mV usually failed to trigger Ca²⁺-release from the SR. The increase of I_Ca-amplitude upon administration of ISO went ahead with the induction of Ca²⁺-release by brief activation of I_Ca. The effects of ISO could be mimicked by FORSK or intracellular dialysis with 35-cyclic adenosine monophosphate. The effects on I_Ca and SR Ca²⁺-release were dependent on the concentration of the stimulating substance. In a given cell changing superfusion from a low to a high concentration of ISO or FORSK resulted in an increase of the number of Ca²⁺-release events per number of Ca²⁺-currents elicited and a shortening of time-to-peak of I_ti's. The stimulating effects of ISO or FORSK on Ca²⁺-release were only partially due to an increase of the triggering I_Ca. Ca²⁺-currents too small to trigger Ca²⁺-release before -adrenergic stimulation could evoke Ca²⁺-release after augmentation of intracellular cAMP. Whereas the effects of ISO and FORSK on I_Ca were reversible, the stimulatory effects on Ca²⁺-release persisted after washing out the substances. The results give support to the hypothesis that -adrenoceptor-mediated positive inotropic and arrhythmogenic effects are, at least partly, due to a cyclic AMP-dependent regulatory mechanism modulating sarcoplasmic Ca²⁺-release.This work was supported by the Deutsche Forschungsgemeinschaft (FG Konzell)     

Inhibition of transient outward current by intracellular ion substitution unmasks slow inward calcium current in cardiac Purkinje fibers

The ionophore nystatin was used to replace intracellular K⁺ with Cs⁺ in calf Purkinje fibers. Such replacement inhibited the transient outward current, and unmasked a large slow inward current (I_si). These results support the involvement of K⁺ as a charge carrier of the transient outward current, and suggest a method for better analysis of I_si.     

牵张刺激对乳大鼠心房肌细胞瞬时外向钾电流和内向整流钾电流的影响

 目的：探究牵张刺激对乳鼠心房肌细胞瞬时外向钾电流（Ito）、内向整流钾电流（IK1）和动作电位时程（APD）的影响。方法：取1日龄SD乳大鼠心脏,分离、消化获得心房肌细胞。于细胞牵引装置培养24 h分组：对照组不予牵张刺激;牵张组予增加12%硅胶膜面积牵张刺激24 h。膜片钳全细胞记录方法记录细胞膜Ito、IK1和APD的变化。结果：在+60 mV刺激电压水平,牵张组Ito密度与对照组相比明显降低［(16±04）pA/pF vs (121±29) pA/pF,P<001］。在-120 mV刺激电压下,牵张组IK1密度较对照组增大［（-108± 08） pA/pF vs (-88±09)pA/pF,P<001］。牵张组动作电位复极50%（APD50）和复极90％（APD90）均较对照组明显缩短［(105±14）ms vs (155±24) ms,(300± 28) ms vs (563±36) ms,P<001］。结论：牵张刺激可降低乳大鼠心房肌细胞Ito密度,增大IK1密度,缩短APD,这可能是压力负荷增大致心房电重构的基础之一。