Subthreshold inward membrane currents in guinea-pig frontal cortex neurons

Current-clamp and single-electrode voltage-clamp recordings were used to study the inward currents activated in the subthreshold membrane potential range of cortical pyramidal neurons. The experiments were done on slices from guinea-pig frontal cortex and all recordings were obtained at a distance of 600–900 μm from the pial surface. In current-clamp recordings and from membrane potentials hyperpolarized to about −70 mV, the depolarization leading to spike firing was partially blocked by 1 μM tetrodotoxin, but not by calcium-free extracellular solution. The calcium-free solution only affected this depolarization when the membrane potential was held at a level more negative than −75 mV. Under voltage-clamp, an inward current was recorded between the resting membrane potential and the level of spike firing. This current was activated at about −60 mV and part of it was blocked by 1 μM tetrodotoxin; the remaining current was blocked by calcium-free extracellular solution. In five neurons both components were recorded and isolated in the same cell. The tetrodotoxin-sensitive component activated at close to −60 mV, was similar to the persistent sodium current (I_Na-p). The Ca²⁺-sensitive component activated at close to −60 or −65 mV, was less voltage-dependent than I_Na-p. This component was similar to the low threshold calcium current (I_T).These results suggest that the subthreshold depolarization which led to spike firing was dependent on I_Na-p and I_T, I_Na-p being the most important factor up to resting membrane potentials of −70 or −75 mV. A physiological role of this finding is revealed by the action of dopamine, which (at 10 μM) prevented the firing of action potentials from −60 mV, but not from −80 mV due to the inhibition of I_Na-p and the lack of effect on I_T.     

Calcium-dependent inward currents in voltage-clamped guinea-pig olfactory cortex neurones

Guinea-pig olfactory cortex neurones in vitro (23°C–25°C) were voltage clamped by means of a single microelectrode sample-and-hold technique. In most Cs⁺-loaded neurones (in the presence of tetrodotoxin), membrane depolarization beyond –60 mV elicited inward currents, which had rapid activation kinetics. The steady-state current-voltage relationship was N-shaped with a region of negative slope conductance between –50 mV and –20 mV. The rate of inactivation varied according to the holding potential and the command potential. The inward currents were maintained when external Ca²⁺ was replaced by Ba²⁺, and were blocked by Cd²⁺, suggesting that Ca²⁺ was the principal charge carrier. The results demonstrate the existence of calcium current in olfactory cortex neurones.     

Reoxygenation-induced arrhythmogenic transient inward currents in isolated cells of the guinea-pig heart

Transient inward currents (I _ti), activated by a rise in intracellular Ca concentration, are believed to trigger cardiac arrhythmias in reperfused hearts. In this report, I _ti in isolated cardiocytes from the guinea-pig were evoked by reoxygenation following a period of anoxia of between 4 min and 35 min. Reoxygenation was performed 1 min after the full development of an anoxia-induced time-independent K current. This current disappeared within 2–6 s and in the following 10 s I _ti developed to maximum amplitude. I _ti were evoked using a constant pulse pattern (holding potential V _h=–45 mV; test potential V _t=+10 mV; pulse duration 350 ms; frequency 1 Hz). In more than 95% of the cells, I _ti at the holding potential I _ti (–45 mV) declined with a time constant of =670±240 ms (mean±SD, n=17). In two cells, undamped oscillatory currents were observed. The amplitude of I _ti (-45 mV) was proportional to the amplitude and duration of the preceding depolarizing test pulse. Test pulses of long duration (500 ms and 1000 ms, mean ± SD) to potentials positive to +10 mV produced slowly decaying tail currents (=391±51 ms, mean ± SD), which superimposed with I _ti (–45 mV). The current/voltage relationship of I _ti peaked between –30 mV and –10 mV and approximated zero at the most positive potentials, i.e. no reversal of I _ti was found up to +80 mV. Using double-pulse protocols (prepulse potential +40 mV), I _ti were enhanced at potentials negative to –30 mV and were also present in the range of the normal resting potential of ventricular heart cells. The instantaneous current-voltage relationship was monotone between –50 mV and +40 mV. Because of the dependence of I _ti on the preceding depolarization, the instantaneous current-voltage relationship provides more reliable information on the voltage dependence of I _ti. The interval between two subsequent I _ti (–45 mV) values was 237±35 ms (mean ± SD, n=27) and depended on the amplitude of I _ti (–45 mV) to increase by 5.2±0.5% (mean ± SD) per 100 pA decrease in I _ti (–45 mV). A simple noise analysis showed that if one assumes that ionic channels are responsible for the generation of I _ti (–45 mV), their unitary conductance cannot exceed 0.36 pS. We conclude that reoxygenation-induced I _ti are triggered by a cyclic release of Ca from the sarcoplasmic reticulum and provide evidence that they are mediated by the electrogenic Na/Ca exchanger. The arrhythmogenic potency of reoxygenation-induced I _ti is demonstrated under current-clamp conditions.This work was supported by the Deutsche Forschungsgemeinschaft, Hi 137/10-1     

Anoxia on slow inward currents of immature hippocampal neurons

1. The effects of brief anoxia (2-4 min) on membrane currents--especially the tetrodotoxin (TTX)-insensitive, Cd2+-sensitive slow inward currents, presumed to be Ca2+ currents--were studied by single-electrode voltage clamp in CA1 and CA3 neurons in submerged hippocampal slices from adult and newborn Wistar rats (PN1-13). 2. In mature neurons, anoxia had no effect on Q-type inward relaxations, but slowly activating C-type outward currents were depressed. The most striking change was the suppression of Ca inward currents (especially the slowly inactivating L-type, by greater than 95%). This effect of anoxia was not sensitive to the N-methyl-D-aspartate (NMDA) receptor blocker, D-aminophosphonovalerate. Anoxia also reversibly abolished the NMDA-evoked inward current. 3. In neurons from newborn animals (PN1-6), Q-type inward relaxations and postanoxic outward currents were very small or undetectable. The slow inward (Ca) currents were smaller than in mature cells, but they showed a clearer separation between low-threshold, fast-inactivating and high-threshold, slowly inactivating currents. Both types of current were more resistant to anoxia (mean depression of L-type was by only 53.3 +/- 5.6%, mean +/- SE). 4. In such immature neurons, the NMDA-evoked inward currents were also more resistant to anoxia. 5. By PN7-13, increasing maturation was reflected in 1) larger voltage-dependent inward currents, 2) increasingly evident Q-type relaxations and postanoxic outward currents, and 3) near-complete blockade of inward currents by anoxia (at PN11-13, mean depression of L-type currents was by 98.5 +/- 1.5%).     

Spontaneous transient inward currents and rhythmicity in canine and guinea-pig tracheal smooth muscle cells

Spontaneous transient inward currents (STICs) were recorded in canine and guinea-pig tracheal myocytes held at negative membrane potentials. STICs were Cl^– selective since their reversal potential was dependent on the Cl^– gradient and they were blocked by the Cl^– channel blocker niflumic acid. STICs were insensitive to Cs⁺, charybdotoxin, and nifedipine. Ca²⁺-activated K⁺ currents often preceded STICs, suggesting that the STICs are Ca²⁺ dependent. In support of this suggestion, we found the Cl^– currents were: (1) abolished by depleting intracellular Ca²⁺ stores using caffeine, acetylcholine, histamine, or substance P; (2) enhanced by increasing external concentrations of Ca²⁺; (3) evoked by voltage-dependent Ca²⁺ influx. The channels responsible for this Cl^– current are of small unitary conductance (<20 pS). Decay of the STICs was described by a single exponential with a time constant of 94±9 ms at –70 mV; the time constant increased considerably at more positive potentials. Using Ca²⁺-dependent Cl^– currents and contractions as indices of internal levels of Ca²⁺, we found that isolated tracheal cells are capable of exhibiting rhythmic behaviour: bursts of currents and contractions with a periodicity of less than 0.1 Hz and which continued for more than 20 min. These rhythmic events were recorded at negative membrane potentials, suggesting that cyclical release of internally sequestered Ca²⁺ is responsible. We conclude that spontaneous release of Ca²⁺ from intracellular stores in tracheal muscle cells leads to transient currents in some cases accompanied by rhythmic contractions. Our studies provide evidence for a cellular mechanism that could underly myogenic oscillations of membrane potential in smooth muscle.     

Action potentials and sodium inward currents of developing neurons in Xenopus nerve-muscle cultures

Action potentials and voltage-gated Na+ inward currents from cultured embryonic neurons of Xenopus laevis were recorded using the patch-clamp technique in the whole cell configuration. Neurons together with muscle cells were dissociated from embryos shortly after completion of gastrulation. Under the voltage-clamp condition the voltage-gated Na+ inward current was isolated from other currents by pharmacological means and by ion substitution. A small Na+ current was observed in round cells without neurites (presumptive neurons). The mean amplitude of the peak Na+ current was 2.5 times larger in neurons with short processes than in presumptive neurons. As they developed further by extending longer processes, the maximum amplitude of the Na+ inward current recorded at the soma decreased. In varicosities, the Na+ inward current density was greater than that at the soma of neurons with extended neurites but kinetic properties and voltage-dependency were similar.     

Calcium-dependent potassium currents in neurons from cat sensorimotor cortex.

1. Ca(2+)-dependent K+ currents were studied in large pyramidal neurons (Betz cells) from layer V of cat sensorimotor cortex by use of an in vitro brain slice and single microelectrode voltage clamp. The Ca(2+)-dependent outward current was taken as the difference current obtained before and after blockade of Ca2+ influx. During step depolarizations in the presence of tetrodotoxin (TTX), this current exhibited a fast onset of variable amplitude and a prominent slowly developing component. 2. The Ca(2+)-dependent outward current first appeared when membrane potential was stepped positive to -40 mV. Downsteps from a holding potential of -40 mV revealed little or no time-, voltage-, or Ca(2+)-dependent current. When membrane potential was stepped positive to -40 mV, a prolonged Ca(2+)-dependent outward tail current followed repolarization. The decay of this tail current at -40 mV was best described by a single exponential function having a time constant of 275 +/- 75 (SD) ms. The tail current reversed at 96 +/- 5 mV in 3 mM extracellular K+ concentration ([K+]o) and at more positive potentials when [K+]o was raised, suggesting that it was carried predominantly by K+. 3. The Ca(2+)-dependent K+ current consisted of two pharmacologically separable components. The slowly developing current was insensitive to 1 mM tetraethylammonium (TEA), but a substantial portion was reduced by 100 nM apamin. Most of the remaining current was blocked by the addition of isoproterenol (20-50 microM) or muscarine (10-20 microM). 4. The time courses of the apamin- and transmitter-sensitive components were similar when activated by step depolarizations in voltage clamp, but they were quite different when activated by a train of action potentials. Applying the voltage clamp at the end of a train of 90 spikes (evoked at 100-200 Hz) resulted in an Ca(2+)-dependent K+ current with a prominent rapidly decaying portion (time constant approximately 50 ms at -64 mV) and a smaller slowly decaying portion (time constant approximately 500 ms at -64 mV). The rapidly decaying portion was blocked by apamin (50-200 nM), and the slowly decaying portion was blocked by isoproterenol (20-50 microM). 5. When recorded with microelectrodes containing 2 mM dimethyl-bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (dimethyl-BAPTA), which causes prolonged afterhyperpolarizations, the Ca(2+)-dependent K+ current evoked by step depolarizations had an extremely slow onset and decay. The current recorded after a train of evoked spikes had a similar slow decay.(ABSTRACT TRUNCATED AT 400 WORDS)     

Properties of an inward rectifying K channel in the membrane of guinea-pig atrial cardioballs

Single channel outward current fluctuations are recorded in excised (outside-out) membrane patches of isolated atrial cells in culture (cardioballs) from hearts of adult guinea-pigs. The ionic channel displays a high selectivity to K ions. Accordingly the reversal potential of the single channel current is close to the K equilibrium potential. The open channel conductance is unaffected by the membrane potential but depends on the K concentration of the outside solution (19.7pS at 2 mM K₀ to 30.7pS at 20 mM K₀). The open state probability (P₀) of the channel shows a marked voltage dependence. P₀ amounts to c.0.9 at –40mV and decreases to c.0.1 at +40mV.Under the assumption of no channel interaction a macroscopic steady state current voltage relationship is reconstructed from the single channel data. The relationship displays inward-going rectification. The rectification is due to the voltage dependence of P₀. TheI–V curve displays a negative slope at membrane potentials positive to –15 mV.In bathing solutions containing Ba ions (0.2 mM) P₀ is reduced by rapid closures which interrupt the open state events. The unit channel conductance is unaffected by Ba ions. The channel block exerted by Ba ions is augmented with increasing membrane hyperpolarization.The results suggest that the channel studied may represent a background K conductance.This work was supported by a grant to L.P. from the Ministerium für Wissenschaft und Forschung des Landes Nordrhein-Westfalen (AZ.IV B5-FA 8833)     

Glutamine-induced membrane currents in cultured chick spinal cord neurons

The effects of -glutamine (GLN) on cultured spinal cord neurons from the chick were studied in the whole cell mode of the patch clamp technique. GLN induced membrane currents rectified at positive membrane potentials (m.p.) and reversed polarity close to zero m.p. The dose-response curve was nearly linear at a semilogarithmic scale for concentrations of 10⁻⁵ M-10⁻² M. Summation of the responses evoked by GLN (10⁻³ M) and glycine (10⁻³ M) was observed when these two amino acids were applied together, while no significant increase of the responses was present when GLN was applied together with -glutamate (10⁻³ M) or kainate (10⁻³ M). It is suggested that GLN binds to the glutamate receptors and activates the same type of ionic channels as glutamate and kainate.     

Intracellular protons inhibit inward rectifier K+ channel of guinea-pig ventricular cell membrane

The effect of intracellular protons (H_i ⁺) on the inward rectifier K⁺ channel of the guinea-pig ventricular cell membrane was examined, using the patch-clamp technique. The inward single-channel current was recorded in inside-out and outside-out patch configurations, while the pH of the solution perfusing the intra and extracellular side, respectively, was varied. Low intracellular pH (pH_i), but not low extracellular pH, inhibited the channel. Low pH_i reduced the unit amplitude, which was about 20% smaller at pH_i 6.0 than that at pH_i 7.4 at every voltage tested. The slope conductance decreased from 41.7 pS at pH_i 7.4 to 35.1 pS at pH_i 6.0. Low pH_i also reduced the channel activity without apparent voltage dependence. The concentration/response curve indicated the half-maximum inhibition at pH_i 6.11 and a Hill coefficient of 2.52. Lowering the pH_i from 7.4 to 6.0 did not affect the distributions of the open times and the closed times below 50 ms, while the time constant of the histogram constructed from closings longer than 50 ms was approximately doubled. These results indicate that the inward rectifier K⁺ channel in ventricular myocytes is inhibited by H⁺ from the intracellular side. This might contribute to the depolarization of the resting membrane potential induced by intracellular acidosis during myocardial ischaemia.     

Temperature effects on neuronal membrane potentials and inward currents in rat hypothalamic tissue slices

Preoptic–anterior hypothalamic (PO/AH) neurones sense and regulate body temperature. Although controversial, it has been postulated that warm-induced depolarization determines neuronal thermosensitivity. Supporting this hypothesis, recent studies suggest that temperature-sensitive cationic channels (e.g. vanilloid receptor TRP channels) constitute the underlying mechanism of neuronal thermosensitivity. Moreover, earlier studies indicated that PO/AH neuronal warm sensitivity is due to depolarizing sodium currents that are sensitive to tetrodotoxin (TTX). To test these possibilities, intracellular recordings were made in rat hypothalamic tissue slices. Thermal effects on membrane potentials and currents were compared in PO/AH warm-sensitive, temperature-insensitive and silent neurones. All three types of neurones displayed slight depolarization during warming and hyperpolarization during cooling. There were no significant differences in membrane potential thermosensitivity for the different neuronal types. Voltage clamp recordings (at −92 mV) measured the thermal effects on persistent inward cationic currents. In all neurones, resting holding currents decreased during cooling and increased during warming, and there was no correlation between firing rate thermosensitivity and current thermosensitivity. To determine the thermosensitive contribution of persistent, TTX-sensitive currents, voltage clamp recordings were conducted in the presence of 0.5 μ m TTX. TTX decreased the current thermosensitivity in most neurones, but there were no resulting differences between the different neuronal types. The present study found no evidence of a resting ionic current that is unique to warm-sensitive neurones. This supports studies suggesting that neuronal thermosensitivity is controlled, not by resting currents, but rather by currents that determine rapid changes in membrane potential between successive action potentials.     

Properties of membrane currents in isolated smooth muscle cells from guinea-pig trachea

Tracheal smooth muscle cells were enzymatically isolated from guinea-pig trachea. These cells contracted in response to acetylcholine (0.01–10 M) in a concentration-dependent fashion. Under current-clamp conditions with 140 mM K⁺ in the pipette solution, the membrane potential oscillated spontaneously at around –30 mV. Under voltage-clamp conditions, there appeared spontaneous but steady oscillations of outward current (I _o). On depolarization from a holding potential at –40 mV, three components of outward current were elicited: transient outward current (I _T), steady-state outward current (I _s) and I _o. These three components of outward current reversed around the K⁺ equilibrium potential and were abolished by Cs⁺ in the pipette, indicating that K⁺ was the major charge carrier of these outward currents. All these three components were completely suppressed by extracellular tetraethylammonium (10 mM). Both I _T and I _o were depressed by quinidine (1 mM), 4-aminopyridine (10 mM) and nifedipine (100 nM), but I _s was not affected. I _T and I _o were suppressed by a Ca²⁺-free perfusate with less than 1 nM Ca²⁺ in the pipette, while with 10 nM Ca²⁺ in the pipette, only I _o was suppressed. In both conditions, I _s was not affected by the Ca²⁺-free perfusate. Therefore, it is suggested that I _o, I _T and I _s are separate types of K⁺ current. With Cs⁺ in the pipette, K⁺ currents were almost completely suppressed and a transient inward current was observed during depolarizing pulses. The inward current was not affected by tetrodotoxin and increased when the concentration of extracellular Ca²⁺ was raised, indicating that the current is a Ca²⁺ channel current. Even with a holding potential of –80 mV, the low-threshold inward current could not be observed. The high-threshold Ca²⁺ current was abolished by nifedipine (100 nM) and was enhanced by Bay K 8644 (100 nM). The order of permeation of divalent cations through the Ca²⁺ channel was Ba²⁺ >Sr²⁺ Ca²⁺. Cd²⁺ blocked the Ca²⁺ current more effectively than Ni²⁺. These results may indicate that the Ca²⁺ current of tracheal smooth muscle cells is mainly composed of the current through an L-type Ca²⁺ channel.     

Development of inward rectification and control of membrane excitability in mesencephalic v neurons

The present study was performed to assess the postnatal development and functional roles of inward rectifying currents in rat mesencephalic trigeminal (Mes V) neurons, which are involved in the genesis and control of oral-motor activities. Whole cell voltage-clamp recordings obtained from Mes V neurons in brain stem slices identified fast (I(KIR)) and slow (I(h)) inward rectifying currents, which were specifically blocked by BaCl(2) (300-500 microM) or 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyrimidinium chloride (ZD 7288, 10 microM), respectively. The whole cell current density for these channels increased between postnatal days 2 to 12 (P2-P12), and the time courses for I(h) activation and deactivation were each well described by two time constants. Application of ZD 7288 produced membrane hyperpolarization in the majority of cells and prolonged afterhyperpolarization repolarization. Additionally, in the presence of ZD 7288, spike frequency was decreased and adaptation was more pronounced. Interestingly, these neurons exhibited a voltage-dependent membrane resonance (<10 Hz) that was prominent around resting potential and more negative to rest and was blocked by ZD 7288. These results suggest that I(h) contributes to stabilizing resting membrane potential and controlling cell excitability. The presence of I(h) imparts the neuron with the unique property of low-frequency membrane resonance; the ability to discriminate between synaptic inputs based on frequency content.     

Subthreshold microstimulation in frontal eye fields updates spatial memories

The brain’s sensitivity to self-generated movements is critical for behavior, and relies on accurate internal representations of movements that have been made. In the present study, we stimulated neurons below saccade threshold in the frontal eye fields of monkeys performing an oculomotor delayed response task. Stimulation during, but not before, the memory period caused small but consistent displacements of memory-guided saccade endpoints. This displacement was in the opposite direction of the saccade that was evoked by stronger stimulation at the same site, suggesting that weak stimulation induced an internal saccade signal without evoking an actual movement. Consistent with this idea, the stimulation effect was nearly absent on a task where an animal was trained to ignore self-generated eye movements. These findings support a role for the frontal eye fields in accounting for self-generated movements, and indicate that corollary discharge signals can be manipulated independent of motor output. This work was supported by the EJLB Foundation, the Washington University Silvio Conte Center, and the National Eye Institute. R.W. was supported by grants from the National Institute of Health’s Medical Scientist Training Program (GM07200) and the National Eye Institute (EY13360).     

Nicotinic cholinoceptive neurons of the frontal cortex are reduced in Alzheimer's disease

The cellular distribution of nicotinic acetylcholine receptors was studied in the frontal cortex (area 10) of 1) Alzheimer patients and compared to 2) age-matched and 3) middle-aged controls using the monoclonal antibody WF 6 and an immunoperoxidase protocol. Statistical analysis revealed significant differences between the number of labeled neurons among all three groups tested (middle-aged controls greater than aged controls greater than Alzheimer cases). No differences were seen for cresyl violet-stained samples. These findings underline that the nicotinic receptor decrease found with radioligand binding may reflect a postsynaptic in addition to a presynaptic component.     

Glutamate receptor agonist-induced inward currents in spinal dorsal horn neurons dissociated from the adult rats.

Inward currents to glutamate receptor agonists, quisqualate (QA), kainate (KA) and N-methyl-D-aspartate (NMDA) were examined in spinal dorsal horn neurons by whole-cell voltage-clamp techniques after acute dissociation. Neurons were dissociated from the superficial dorsal horn (laminae I/II) of the adult rat (8-16 weeks old) spinal cords by enzymatic and mechanical treatment. The KA-induced current was sustained during KA application, while the QA- and NMDA-induced currents were attenuated. The NMDA response was augmented dose-dependently by addition of glycine (10(-7)-5 X 10(-6) M) and became obscure in the absence of glycine. The NMDA current was depressed by D-2-amino-5-phosphonovaleric acid (APV). Analyses of dose-response curves of these inward currents indicate that both the QA and KA currents were competitively blocked by 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), while the NMDA current was blocked non-competitively.     

Responses of cat vestibular neurons to stimulation of the frontal cortex

Summary To study the neural basis for the regulation of vestibulocollic reflexes during voluntary head movements, the effects of stimulation of the precruciate cortex near the presylvian sulcus (neck area of the motor cortex) and the frontal eye fields (FEF) on vestibular neurons were studied in cerebellectomized cats anesthetized with chloralose. Neurons were recorded in the medial and descending vestibular nuclei and antidromically identified from C₁. Stimulation of the FEF and precruciate cortex fired 29 and 13% of neurons that did not exhibit spontaneous activity. About 80% of spontaneously discharging neurons were influenced by stimulation of either of the two. Stimulation of the precruciate cortex or FEF suppressed or facilitated labyrinthine evoked monosynaptic activation of vestibulospinal neurons, suggesting that the frontal cortical neurons have the properties to regulate the vestibulocollic reflexes.