Electrophysiological properties of early postnatal rat septal neurons in short-term culture.

Electrophysiological properties of septal neurons dissociated from PN1-PN7 rats were examined between 1 and 5 days in vitro (DIV) with whole-cell patch-clamp recording. The neurons had RMPs in the range -40 to -80 mV, resistances 0.5-1.5 G omega, and 50-90 mV action potentials. By 2-3 DIV, most neurons were spontaneously active, with some cells exhibiting rhythmic firing patterns. Depolarizations from -80 mV holding potential elicited TTX-sensitive inward Na+ currents, and transient and sustained outward K+ currents. Pharmacological dissection of the outward currents in PN6/7 neurons suggest the presence of multiple types of K+ currents (IA, IC, IK). L-type Ca2+ currents were observed in all PN6/7 neurons examined but were not always detectable in PN1/2 neurons. All PN1/2 and PN6/7 neurons were sensitive to glutamate and GABA but did not respond to ACh or NE applications. Responses to GABA were excitatory i.e., characteristic of immature neurons. Comparison of the results obtained in this study with the properties of adult neurons characterized in vivo or in brain slice preparations in vitro, suggest that septal neurons from PN1-PN7 rats are still in the process of differentiation of their mature electrical and chemosensitive membrane properties.     

Different Types of Potassium Outward Current in Relay Neurons Acutely Isolated from the Rat Lateral Geniculate Nucleus

Different classes of potassium (K+) outward current activated by depolarization were characterized in relay neurons acutely isolated from the rat lateral geniculate nucleus (LGN), using the whole-cell version of the patch-clamp technique. A fast-transient current (IA), activated at around - 70 mV, declined rapidly with a voltage-dependent time constant (tau=6 ms at + 45 mV), was 50% steady-state inactivated at - 70 mV, and rapidly recovered from inactivation with a monoexponential time course (tau=21 ms). IA was blocked by 4-aminopyridine (4-AP, 2 - 8 mM) and was relatively insensitive to tetraethylammonium (TEA, 2 - 10 mM). After elimination of IA by a conditioning prepulse (30 ms to - 50 mV), a slow-transient K+ current could be studied in isolation, and was separated into three components, IKm, IKs and a calcium (Ca2+)-dependent current, IK[Ca]. The slow-transient current was not consistently affected by 4-AP (up to 8 mM), while TEA (2 - 10 mM) predominantly blocked IKs and IK[Ca]. The component IKm persisted in a solution containing TEA and 4-AP, activated at around - 55 mV, declined monoexponentially during maintained depolarization (tau=98 ms at + 45 mV), was 50% inactivated at - 39 mV, and recovered with tau=128 ms from inactivation. IKs activated at a similar threshold, but declined much slower with tau=2662 ms at + 45 mV. Steady-state inactivation of IKs was half-maximal at - 49 mV, and recovery from inactivation occurred relatively fast with tau=116 ms. From these data and additional current-clamp recordings it is concluded that the K+ currents, due to their wide range of kinetics and dependence on membrane voltage or internal Ca2+ concentration, are capable of cooperatively controlling the firing threshold and of shaping the different states of electrophysiological behaviour in LGN relay cells.     

Voltage-gated ionic currents in an identified modulatory cell type controlling molluscan feeding

An important modulatory cell type, found in all molluscan feeding networks, was investigated using two-electrode voltage- and current-clamp methods. In the cerebral giant cells of Lymnaea, a transient inward Na+ current was identified with activation at -58 +/- 2 mV. It was sensitive to tetrodotoxin only in high concentrations (approximately 50% block at 100 microm), a characteristic of Na+ channels in many molluscan neurons. A much smaller low-threshold persistent Na+ current (activation at < -90 mV) was also identified. Two purely voltage-sensitive outward K+ currents were also found: (i) a transient A-current type which was activated at -59 +/- 4 mV and blocked by 4-aminopyridine; (ii) a sustained tetraethylammonium-sensitive delayed rectifier current which was activated at -47 +/- 2 mV. There was also evidence that a third, Ca2+-activated, K+ channel made a contribution to the total outward current. No inwardly rectifying currents were found. Two Ca2+ currents were characterized: (i) a transient low-voltage (-65 +/- 2 mV) activated T-type current, which was blocked in NiCl2 (2 mm) and was completely inactivated at approximately -50 mV; (ii) A sustained high voltage (-40 +/- 1 mV) activated current, which was blocked in CdCl2 (100 microm) but not in omega-conotoxin GVIA (10 microm), omega-agatoxin IVA (500 nm) or nifedipine (10 microm). This current was enhanced in Ba2+ saline. Current-clamp experiments revealed how these different current types could define the membrane potential and firing properties of the cerebral giant cells, which are important in shaping the wide-acting modulatory influence of this neuron on the rest of the feeding network.     

Ionic mechanism of the outward current induced by intracellular injection of inositol trisphosphate into Aplysia neurons

Inositol 1,4,5-trisphosphate (InsP3) has been proposed to be the intracellular second messenger in the mobilization of Ca2+ from intracellular stores in a variety of cell types. The ionic mechanism of the effect of intracellularly injected InsP3 on the membrane of identified neurons (R9-R12) of Aplysia kurodai was investigated with conventional voltage-clamp, pressure-injection, and ion-substitution techniques. Brief pressure injection of InsP3 into a neuron voltage-clamped at -40 mV reproducibly induced an outward current (10-60 sec in duration, 20-60 nA in amplitude) associated with a conductance increase. The current was increased by depolarization and decreased by hyperpolarization up to -80 mV, where it disappeared. Extracellular application of tetraethylammonium (TEA; 5 mM) blocked the InsP3-induced outward current, and the current was not affected by the presence of bath-applied 4-aminopyridine (4-AP; 5 mM). The InsP3-induced outward current recorded at a holding potential of -40 mV increased in amplitude in low-K+ solutions and decreased in amplitude in high-K+ solutions. Alteration of [Cl-]0, as well as perfusion with Ca2+ free plus 2 mM EGTA solution, did not affect the outward current. The InsP3-induced outward current was found to disappear when the neuron was injected with the Ca2+ chelator EGTA. The outward current evoked by repeated InsP3 injection at low doses exhibited summation and facilitation and, at high doses, was shown to desensitize. The calmodulin inhibitor N-(6-amino-hexyl)-5-chloro-1-naphthalene sulfonamide (W-7; 20-50 microM), inhibited both the InsP3-induced and the Ca2+-activated outward currents. An intracellular pressure injection of Ca2+ ions into the same identified neuron was shown to produce an outward current associated with a K+ conductance increase similar to the InsP3-induced current, and the current was blocked by bath-applied TEA (5mM). These results suggest that brief pressure injection of InsP3 into certain identified neurons of Aplysia induces a 4-AP-resistant, TEA-sensitive K+ current activated by increased intracellular free Ca2+ concentration, and this increase might be the result of the mobilization of Ca2+ from intracellular stores by InsP3.     

Brain-derived neurotrophic factor induces long-lasting Ca2+-activated K+ currents in rat visual cortex neurons

Brain-derived neurotrophic factor (BDNF) increases postsynaptic intracellular Ca2+ and modulates synaptic transmission in various types of neurons. Ca2+-activated K+ currents, opened mainly by intracellular Ca2+ elevation, contribute to hyperpolarization following action potentials and modulate synaptic transmission. We asked whether BDNF induces Ca2+-activated K+ currents by postsynaptic elevation of intracellular Ca2+ in acutely dissociated visual cortex neurons of rats. Currents were analysed using the nystatin-perforated patch clamp technique and imaging of intracellular Ca2+ mobilization with fura-2. At a holding potential of -50 mV, BDNF application (20 ng/mL) for 1-2 min induced an outward current (IBDNF-OUT; 80.0 +/- 29.0 pA) lasting for more than 90 min without attenuation in every neuron tested. K252a (200 nm), an inhibitor of Trk receptor tyrosine kinase, and U73122 (3 microm), a specific phospholipase C (PLC)-gamma inhibitor, suppressed IBDNF-OUT completely. IBDNF-OUT was both charybdotoxin- (600 nm) and apamin- (300 nm) sensitive, suggesting that this current was carried by Ca2+-activated K+ channels. BAPTA-AM (150 microm) gradually suppressed IBDNF-OUT. Fura-2 imaging revealed that a brief application of BDNF elicited a long-lasting elevation of intracellular Ca2+. These results show that BDNF induces long-lasting Ca2+-activated K+ currents by sustained intracellular Ca2+ elevation in rat visual cortex neurons. While BDNF, likely acting through the Trk B receptor, was necessary for the induction of long-lasting Ca2+-activated K+ currents via intracellular Ca2+ elevation, BDNF was not necessary for the maintenance of this current.     

Fluoxetine inhibits A-type potassium currents in primary cultured rat hippocampal neurons

The effects of fluoxetine (Prozac) on the transient A-currents (IA) in primary cultured hippocampal neurons were examined using the whole-cell patch clamp technique. Fluoxetine did not significantly decrease the peak amplitude of whole-cell K+ currents, but it accelerated the decay rate of inactivation, and thus decreased the current amplitude at the end of the pulse. For further analysis, IA and delayed rectifier K+ currents (IDR) were isolated from total K+ currents. Fluoxetine decreased IA (the integral of the outward current) in a concentration-dependent manner with an IC50 of 5.54 microM. Norfluoxetine, the major active metabolite of fluoxetine, was a more potent inhibitor of IA than was fluoxetine, with an IC50 of 0.90 microM. Fluoxetine (3 microM) inhibited IA in a voltage-dependent manner over the whole range of membrane potentials tested. Analysis of the time dependence of inhibition gave estimates of 34.72 microM(-1) s(-1) and 116.39 s(-1) for the rate constants of association and dissociation, respectively. The resulting apparent Kd was 3.35 microM, similar to the IC50 value obtained from the concentration-response curve. In current clamp configuration, fluoxetine (3 microM) induced depolarization of resting membrane potential and reduced the rate of action potential. Our results indicate that fluoxetine produces a concentration- and voltage-dependent inhibition of IA, and that this effect could affect the excitability of hippocampal neurons.     

Voltage-dependent calcium channels regulate melatonin output from cultured chick pineal cells

Chick pineal cells maintained in primary culture display a circadian rhythm of melatonin production and release, and the nocturnal increase in melatonin output is enhanced by elevating extracellular K+. The divalent cations, Co2+, Cd2+, and Mn2+, each reduce nocturnal melatonin output. Nitrendipine and nifedipine also prevent the nocturnal rise in melatonin output, while Bay K 8644 increases it, suggesting a role for voltage-dependent Ca2+ channels in regulating melatonin output. The whole-cell patch-clamp technique was used to record from individual chick pineal cells. Under conditions designed to isolate currents through voltage-dependent Ca2+ channels, biphasic inward currents are elicited by large depolarizing commands (e.g., to 0 mV) from a holding potential of -90 mV; from a holding potential of -40 mV, only a sustained inward current is elicited by steps to 0 mV. Both components of the inward current are blocked by Co2+ or Cd2+. The sustained current is increased in amplitude by Bay K 8644 and blocked by nifedipine, while the transient current is unaffected. Since there is no evidence for vesicular release of melatonin, the "L-type" calcium channels mediating the sustained calcium current appear to be involved in the pathways regulating melatonin synthesis in chick pineal cells.     

Calcium action on the membrane currents possessing the properties of mechano-electric transducer currents in inner hair cells of the Guinea-pig cochlea

In free-standing hair bundle, depolarization to +80 mV evoked a stable outward current and repolarization to -80 mV evoked a transient inward current attributable to the opening of mechano-electric transducer channels. The study investigated the Ca2+ dependence of this transducer-like membrane current in isolated inner hair cells of guinea-pig cochlea. The amplitude of outward currents increased and the rate of inward current decay, corresponding to adaptation kinetics, decreasing as the extracellular Ca2+ concentrations lessened, whereas the amplitude of outward current decreased and an adaptation accelerated as the extracellular Ca2+ elevated. Treatment with the cAMP agonist, 8-bromo-cAMP, induced an effect similar to that caused by elevating the extracellular Ca2+.     

Dopamine enhances a voltage-dependent transient K+ current in the MMQ cell, a clonal pituitary line expressing functional D2 dopamine receptors

The influence of dopamine on voltage-dependent K+ current (IK) was studied in cultured MMQ cells using the whole-cell patch-clamp technique. IK in nearly all MMQ cells revealed a transient outward current component and inactivated during maintained depolarization lasting 60 ms. The transient component was inhibited by prepulse potentials more positive than -40 mV or by addition of 4 mM 4-aminopyridine to the bathing solution and was insensitive to the external Ca2+ concentration. Thus, this transient K+ current resembled the A-current (IA) found in other cells. Dopamine at 1 microM increased by 50% (P less than 0.001) the peak of IK evoked by a test potential to +80 mV and the response was prevented by pretreatment with 100 nM haloperidol, a D2 receptor antagonist. These data suggest that MMQ clonal pituitary cells possess a voltage-gated K+ A-current and that this current can be modulated by dopamine via D2 receptors.     

Analysis of FMRF-amide effects on Aplysia bursting neurons

The peptide L-phenylalanyl-L-methionyl-L-arginyl-L-phenylalaninamide (FMRF-amide) was pressure-applied onto the somata of bursting neurons L4 and L6 in the Aplysia abdominal ganglion. FMRF-amide causes a biphasic response, first depolarizing and then hyperpolarizing the neuron. In voltage-clamp experiments, FMRF-amide induces an inward current that begins 100-200 msec after applying the peptide and peaks in 2-10 sec. This is followed by an outward current that begins with a latency of 2-5 sec and peaks in 15-65 sec. The entire response lasts 1-5 min. Experiments were done to separate the two currents induced by FMRF-amide on the basis of ion selectivity and kinetics and to determine their I(V) relationships. The currents were studied using a method to quickly measure I(V) curves. The inward current is caused by a conductance increase and has a reversal potential of approximately +18 mV. This current depends on the concentration of extracellular Na ions but not Ca, Cl, or K ions and is insensitive to tetrodotoxin, hexamethonium, and curare. The outward current is caused by a conductance increase and has a reversal potential of approximately -61 mV, which is similar to the reversal potential of the fast, transient K current (IA) in the same cells. This current is sensitive to changes in the external K ion concentration but not to changes in Cl, Ca, or Na concentration. The outward current is partially blocked by 1 mM 4-aminopyridine but not TEA or curare. Neither current is significantly voltage dependent within the range from -70 to -40 mV.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemokine modulation of high-conductance Ca(2+)-sensitive K(+) currents in microglia from human hippocampi

During acute pathological processes, microglia transform into an activated state characterized by a defined morphology and current profile, and are recruited to injury sites by chemokines. No information is available on the ion channels and the mode of action of chemokines in microglia in brain slices from humans with a chronic pathology. Thus, patch-clamp recordings of microglia were performed in hippocampal slices from seven patients who underwent surgery for pharmaco-resistant epilepsy. Cells were identified as microglia by positive labelling with fluorescein-conjugated tomato lectin before recording. All the recorded cells had an ameboid morphology characteristic of activated microglia. However, they had a high input resistance (3.6 G omega), a zero-current resting potential of -16 mV, and lacked Na+ currents, inwardly rectifying and delayed rectifying K+ currents such as non-activated microglia. Importantly, recorded cells expressed Ca2+-sensitive outward currents that activated at 0 mV with non-buffered intracellular Ca2+ and were sensitive to 1 mm tetraethylammonium (TEA). The estimated single-channel conductances were 187 pS in cell-attached and 149 pS in outside-out patches, similar to those of high-conductance Ca2+-dependent K+ channels. The chemokine MIP1-alpha increased whole-cell outward current amplitudes measured at +60 mV by a factor of 3.3. Thus, microglia in hippocampi from epileptic patients express high-conductance Ca2+-dependent K+ channels that are modulated by the chemokine MIP1-alpha. This modulation may contribute to the migratory effect of MIP1-alpha on microglia.     

Astrocytes in the hippocampus of patients with temporal lobe epilepsy display changes in potassium conductances

Functional properties of astrocytes were investigated with the patch-clamp technique in acute hippocampal brain slices obtained from surgical specimens of patients suffering from pharmaco-resistant temporal lobe epilepsy (TLE). In patients with significant neuronal cell loss, i.e. Ammon's horn sclerosis, the glial current patterns resembled properties characteristic of immature astrocytes in the murine or rat hippocampus. Depolarizing voltage steps activated delayed rectifier and transient K+ currents as well as tetrodotoxin-sensitive Na+ currents in all astrocytes analysed in the sclerotic human tissue. Hyperpolarizing voltages elicited inward rectifier currents that inactivated at membrane potentials negative to -130 mV. Comparative recordings were performed in astrocytes from patients with lesion-associated TLE that lacked significant histopathological hippocampal alterations. These cells displayed stronger inward rectification. To obtain a quantitative measure, current densities were calculated and the ratio of inward to outward K+ conductances was determined. Both values were significantly smaller in astrocytes from the sclerotic group compared with lesion-associated TLE. During normal development of rodent brain, astroglial inward rectification gradually increases. It thus appears reasonable to suggest that astrocytes in human sclerotic tissue return to an immature current pattern. Reduced astroglial inward rectification in conjunction with seizure-induced shrinkage of the extracellular space may lead to impaired spatial K+ buffering. This will result in stronger and prolonged depolarization of glial cells and neurons in response to activity-dependent K+ release, and may thus contribute to seizure generation in this particular condition of human TLE.     

A slowly inactivating potassium current truncates spike activity in ganglion cells of the tiger salamander retina

Voltage-gated ganglion cell membrane currents were studied under whole-cell patch clamp in isolation and in retinal slices. The cells were identified by (1) backfilling their axons with rhodamine and later identifying them by their fluorescence in the slice or the mix of isolated cells or (2) by filling them with Lucifer yellow during recording in retinal slices. Both methods yielded cells with similar currents. In some cases, isolated cells lacked processes yet showed currents similar to other cells, suggesting that voltage-gated currents in all cells were located primarily at the soma. Both a conventional inactivating sodium current and a sustained calcium current were found. We describe 3 inactivating outward currents, ordered in their rate of inactivation. The fastest current resembled IA reported by Connor and Stevens (1971a, b). A slower current labeled IB inactivated with a time constant of 339 msec at 0 mV. The current with slowest inactivation is labeled IC here, inactivating with a time constant of 4.03 sec at 0 mV. An additional outward current was sustained and calcium dependent labeled IK(Ca). IB was the largest of these currents. It was slower than IA, was not blocked by 4AP, and inactivated over a much more positive potential range. IB appears to play an important role in spike generation, different from that of IA: Its inactivation leads to a slow depolarizing shift of the membrane during a current step, truncating spike activity after about 300-700 msec as the membrane potential enters the region of sodium inactivation. We analyze how the inactivating outward current acts to ensure a graded spiking response and to truncate the spiking output in the presence of large excitatory inputs.     

Evidence for voltage-activated outward currents in the neuropilar membrane of locust nonspiking local interneurons

Outward currents activated by depolarization were studied in the neuropilar membrane of locust nonspiking local interneurons, using the single-electrode voltage-clamp technique in situ. Preliminary observation of these currents in 272 neurons revealed two families. The first and most commonly observed (85% of recordings) showed a large transient current followed by a slowly decaying/late current. The second (15% of recordings) showed an additional outward current with a slow rate of activation, a peak within 100-150 msec, and a slow rate of inactivation. Only neurons of the first type were studied further. The transient current was activated by depolarization around -60 mV, with a time to peak of approximately 11 msec at -50 mV and less than 3 msec at -20 mV. This current decayed exponentially, with a time constant of 8.1 +/- 1.6 msec (n = 8 interneurons) at -30 mV. This time constant of inactivation did not appear to depend strongly on membrane voltage, in the range in which it was studied. A second and longer time constant of inactivation of 50-400 msec could not be assigned to either of the transient and late components of the outward current. The ratio of transient-to-late current varied between 1.6 and 5.4, with a mean of about 2.5. The reversal potential for the transient current could, on average, be shifted by 14 mV by a threefold increase in the bath K+ concentration, indicating that K+ is a charge carrier for the current. The transient current became inactivated with maintained depolarization and appeared half-inactivated at about -60 mV (slope factor k1/2 = 8 mV). This current was thus not fully inactivated at "resting" potential (average, -58 mV). Recovery from inactivation followed a single exponential time course, with a time constant of approximately 100 msec at -80 mV. The time course of recovery from inactivation of the transient current was well correlated with that of the recovery of transient outward rectification, as measured in current-clamp recording. Tetraethylammonium, at a bath concentration of 10 mM reduced the transient current by 70% and the delayed current by 60%. 4-Aminopyridine, at a bath concentration of 5 mM, had a significant effect in only two of five interneurons, reducing the transient current by approximately 85% and the late current by approximately 15%. Quinidine at a bath concentration of 100 microM was ineffective. Although these blockers did not allow a clear pharmacological separation of the currents, they were effective in reducing the outward rectification observed in current clamp during step depolarization.(ABSTRACT TRUNCATED AT 400 WORDS)     

Three types of voltage-dependent calcium current in cultured rat hippocampal neurons

Voltage-dependent calcium (Ca2+) currents in cultured rat hippocampal neurons were studied with the whole-cell recording mode of the patch-clamp technique. On the basis of the voltage-dependence of activation, kinetics of inactivation and pharmacology, 3 types of Ca2+ currents were distinguished. The low-threshold Ca2+ current (Il) was activated at -60 mV, and completely inactivated during a 100-ms depolarization to -40 mV (time constant: tau = 16 +/- 1 ms). The high-threshold currents (Ih), which were activated at -20 mV, could be separated into two types. The high-threshold, fast inactivating current (Ih,f) decayed quickly during a maintained depolarization (tau = 33 +/- 3 ms at 0 mV), whereas the high-threshold, slowly inactivating current (Ih,s) decayed with a much slower time constant (tau = 505 +/- 42 ms at 0 mV). The inactivations of Ih,f and Ih,s exhibited different time- and voltage-dependencies. Nickel ions (Ni2+, 25 microM) markedly suppressed Il, but little affected Ih. Cadmium ions (Cd2+, 10 microM) almost completely suppressed Ih, but left a small amount of Il. Lanthanum ions (La3+, 10 microM) almost completely suppressed both Il and Ih. Ih,s was sensitive to block by the dihydropyridine antagonist nicardipine (10 microM).     

Interaction of phencyclidine with voltage-dependent potassium channels in cultured rat hippocampal neurons: comparison with block of the NMDA receptor-ionophore complex

Whole-cell voltage-clamp recording techniques were used to investigate the blockade of voltage-dependent K+ channels by phencyclidine (PCP) in cultured rat hippocampal neurons. All recordings were carried out in the presence of tetrodotoxin (1-2 microM) to eliminate Na+ currents. Step depolarization from a holding potential of -40 mV activated a slowly rising, minimally inactivating K+ current (IK). PCP (0.5-1000 microM) caused a reduction in the maximum conductance of IK [IC50(+30 mV), 22 microM] without altering its voltage dependency. The PCP block of IK diminished at depolarized potentials. Analysis according to the scheme of Woodhull (1973) suggested that block occurs via binding to an acceptor site (presumably within the channel pore) that senses 40-50% of the transmembrane electrostatic field. PCP had no effect on the kinetic properties of IK and the block failed to show use dependency, suggesting that PCP may bind to the IK channel via a hydrophobic mechanism not requiring open channels. For comparison, we also investigated the effect of PCP on the transient K+ current, IA, activated by step depolarization following a 200 msec prepulse to -90 mV (20 mM tetraethylammonium was present in the bathing solution to reduce IK). In contrast to the potent blocking action of PCP on IK, the drug only affected IA at high concentrations [IC50(+30 mV), 224 microM]. At concentrations causing substantial block (300-500 microM), PCP produced an acceleration in the IA inactivation rate, and, for brief (5-6 msec) depolarizing steps, the suppression of IA was use dependent. These observations suggest that PCP block of IA requires open channels. PCP reduced inward current responses induced by the excitatory amino acid agonist N-methyl-D-aspartate (NMDA) at substantially lower concentrations than those required for its effects on K+ channels [IC50(-60 mV), 0.45 microM]. The PCP-like dioxadrol stereoisomer dexoxadrol (10 microM) blocked NMDA-evoked inward current responses, while its behaviorally inactive enantiomer levoxadrol did not. Dexoxadrol and levoxadrol also blocked IK in a stereoselective fashion (IC50's, 73 and 260 microM, respectively), whereas the sigma ligands (+)- and (-)-SKF 10,047 and (+)-3-[3-hydroxyphenyl]-N-(1-propyl)piperidine [(+)-3-PPP] had little effect on the current (IC50's, greater than 300-500 microM). We conclude that PCP causes a selective, voltage-dependent block of IK in hippocampal neurons via a PCP- and not a sigma-type acceptor site.(ABSTRACT TRUNCATED AT 400 WORDS)     

Phencyclidine blocks two potassium currents in spinal neurons in cell culture

We studied the effects of phencyclidine (PCP) on the transient and delayed outward K+ currents recorded from spinal cord neurons grown (10-20 days) in cell culture. Sodium channels were blocked with tetrodotoxin (1 microM) and solutions containing low calcium concentrations in the presence of Mg2+ or Co2+ (5 mM) were used to reduce Ca2+ currents. PCP decreased the amplitude and prolonged the decay phase of the action potentials recorded at a holding potential of -70 mV. PCP (0.1-0.5 mM) was more effective than tetraethylammonium (TEA) or 4-aminopyridine (4-AP) in reducing both transient and delayed currents. The amplitude of the transient current during control experiments was always larger than that of the delayed current. It appeared that 4-AP (5 mM) was more potent in blocking the transient current, while TEA (10 mM) modified the delayed current more effectively. Both currents were also reduced by about 10% when the cell soma was perfused with Co2+. This suggested that a small fraction of the total outward current is a Ca2+-activated K+ current. The PCP-induced blockade of K+ currents in central neurons coupled with the profound synaptic effects of the drug may provide the basis for explaining the psychopathology of this hallucinogenic agent.     

Expression of membrane currents in rat neocortical neurons in serum-free culture. II. Outward currents

The timing of expression and properties of outward membrane currents in cultured neocortical pyramidal-shaped neurons have been investigated using the gigaseal whole-cell voltage clamp and single-channel recording techniques. Dissociated primary cultures of synchronized (same cell cycle), growth arrested (G1 phase) and birth-dated cells from fetal rat (E18) were maintained in a serum-free medium. The earliest appearing membrane current in the soma is a voltage-dependent outward current carried by K+. The current consists of two components, one rapidly rising component, resembling those associated with the transient outward current (IA) and the other similar to the delayed rectifier current (IK). The ratio between the peak IA and IK was about 0.3 at all membrane voltages. The magnitude of both IA and IK increased with time in culture but the ratio remained unchanged. Direct measurements of unitary currents showed the presence of two voltage-activated outward conductances, 32 pS and 120 pS. The small conductance channel was sparse. The large conductance channel is K+-selective and was sensitive to both voltage and internal Ca2+.     

Voltage clamp analysis of intact stomatogastric neurons

Two-electrode voltage clamp of intact, identified pyloric neurons of the spiny lobster stomatogastric ganglion reveals two major outward currents. A rapidly inactivating, tetraethylammonium- (TEA) insensitive, 4-aminopyridine- (4AP) sensitive, outward current resembles IA of molluscan neurons; it activates rapidly on depolarizations above rest (e.g. -45 mV), delaying both the axonal-sodium and the neuropil-calcium spikes which escape voltage-clamp control. We infer that A-current is distributed both in a space clamped region (on or near the soma) and in a non-space clamped region with access to the generators for sodium and calcium spikes. A calcium-dependent outward current, IO(Ca), activates rapidly at clamp steps above -25 mV and inactivates at depolarizing holding voltages. Increasing depolarization results in an increase in both IO(Ca) and firing rate but a reduction in the amplitude of the sodium spike current. Blockage of IO(Ca) with Cd2+ causes little change in spike firing pattern. These observations are consistent with IO(Ca) being activated primarily in the soma and nearby regions which are under good control with a soma voltage clamp (and distant from the Na(+)-spike trigger zone). While the lack of space clamp limits resolution of charging transients and tail currents, the identification of the major current subgroups can still be readily accomplished, and inferences about the location and function of currents can be made which would not be possible if the cells were space clamped or truncated.     

Calcium-activated potassium current clamps the dark potential of vertebrate rods

Vertebrate photoreceptors respond to light with a graded hyperpolarization from a membrane potential in the dark of approximately -35 mV. The present work investigates the physiological role of the Ca2+-activated K+ current in the photovoltage generation in mechanically isolated rods from salamander retina. Membrane current or voltage in isolated rods was recorded from light- and dark-adapted rods under voltage- or current-clamp conditions, respectively. In light-adapted rods of the salamander, selective blockade of Ca2+-activated K+ channels by means of charybdotoxin depolarized the plasma membrane of current-clamped rods by approximately 30 mV, from a resting potential of approximately -35 mV. A similar depolarization was observed if external Ca2+ (1 mM) was substituted with Ba2+ or Sr2+. Under control conditions, the injection of currents of increasing amplitude (up to -100 pA, to mimic the current entering the rod outer segment) could not depolarize the membrane potential beyond a saturating value of approximately -20 mV. However, in the presence of charybdotoxin, rods depolarized up to +20 mV. In experiments with dark-adapted current-clamped rods, charybdotoxin perfusion lead to transient depolarizations up to 0 mV and steady-state depolarizations of approximately 5 mV above the dark resting potential. Finally, the recovery phase of the voltage response to a flash of light in the presence of charybdotoxin showed a transient overshoot of the membrane potential. It was concluded that Ca2+-activated K+ current is necessary for clamping the rod photovoltage to values close to the dark potential, thus allowing faithful single photon detection and correct synaptic transmission.