Changes in membrane currents of hippocampal neurons evoked by brief anoxia

1. Effects of anoxia (2-4 min of 95% N2-5% CO2) on membrane currents of CA1 neurons were studied by single-electrode voltage clamp in hippocampal slices (from Sprague-Dawley rats) kept in an interface-type chamber at 33.5 degree. 2. When recording with KCl electrodes at a holding potential (VH) near-70 mV, anoxia evoked a slow outward current [0.18 +/- 0.06 (SE) nA], accompanied by a conductance increase ( + 46 +/- 20%, mean +/- SE). The difference current evoked by N2 had a reversal potential near-100 mV. It was much smaller in presence of 2-4 mM extracellular Cs, and any remaining outward current was abolished by 10 mM tetraethylammonium (TEA). Only inward currents were observed when recording with CsCl electrodes. 3. Inward relaxations evoked by large hyperpolarizing pulses from VH less than or equal to - 70 mV (Q-type) were not significantly depressed by anoxia (-1.5 +/- 6.0%). 4. Some voltage-dependent outward currents (evoked by 200-ms depolarizing pulses) were depressed during anoxia: 1) a fast-inactivating (A-like) current, obtained at VH less than or equal to -70 mV and suppressed by 200 microM 4-AP, was reduced by 25.6 +/- 7.3% (n = 5); 2) a slower, noninactivating (C-like) current, suppressed by TEA, was reduced by 52 +/- 7.2% (n = 16). Neither of these currents (1 or 2) was observed when recording with 2- to 3-M CsCl electrodes; and 3) small (M-like) inward relaxations, observed at VH approximately -40 mV 5. Net inward currents could be evoked after blockage of GK with 10 mM TEA when recording with KCl electrodes or by recording with CsCl electrodes. At VH less than or equal to -70 mV, large, transient, and incompletely controlled currents were evoked by depolarizing pulses; at VH less than or equal to -50 mV, smaller and more persistent currents were evoked by depolarizing pulses (L-like), and transient currents (T-like?) were seen immediately after hyperpolarizing pulses. 6.L-type currents (at VH less than or equal to -50 mV) were nearly abolished after 1-2 min anoxia (by approximately 90%). This was equally true of the currents evoked by constant pulses or peak currents in I-V plots. After reoxygenation, recovery was biphasic, with a quick early phase (to 50-80% in 2 min) and then a much slower one (to 60-90% by 10-15 min).(ABSTRACT TRUNCATED AT 400 WORDS)     

Anoxia produces smaller changes in synaptic transmission, membrane potential, and input resistance in immature rat hippocampus

1. The reversible blocking effect of brief anoxia (2-4 min) on synaptic transmission was studied in submerged hippocampal slices (kept mostly at 34 degrees), obtained from adult (greater than 120 g) and very young (6-50 g) Wistar rats. Excitatory postsynaptic potentials (EPSPs) were recorded with extra- and intracellular electrodes, sometimes simultaneously: in CA1, they were evoked by stratum radiation stimulation, in CA3 by hilar stimulation. 2. In slices from adults, EPSPs in CA1 were depressed by 90% after 2 min of anoxia, and postanoxic recovery was relatively slow (one-half recovery times 4.0 +/- 0.23 min, mean +/- SE). EPSPs in CA3 were consistently more resistant, especially those generated by mossy fibers; after 2 min of anoxia, these were reduced by only 14.7 +/- 5.4%. 3. In newborn animals (PN1-4), both intra- and extracellular EPSPs (but no population spikes) could be recorded in CA1. Although smaller and more fatigable than in the adult, they were much more resistant to anoxia, after 2 min being reduced by only 44.1 +/- 8.8%; and they were not abolished even after 6-7 min. On the other hand, postanoxic recovery was very rapid, being one-half complete in 2.4 +/- 0.48 min. Only large and very prolonged (giant) depolarizing PSPs [probably inhibitory postsynaptic potentials (IPSPs)] could be recorded in CA3 neurons; they were rapidly blocked by anoxia. 4. In older pups (PN6-21), the CA1 EPSPs became progressively more sensitive to anoxia. At the end of the second week, they were as rapidly blocked as in slices from adults; but postanoxic recovery remained quicker throughout this period. In CA3, EPSPs could now be evoked that were as resistant to anoxia as in adult slices. 5. In both CA1 and CA3 neurons from adult rats, anoxia (for 2-3 min) reduced the input resistance (RN) by 45.7 +/- 6.25%. In CA1 neurons, there was most often some hyperpolarization (-7.2 +/- 1.8 mV), which was less consistent in CA3 cells. The return of O2 typically led to a second (postanoxic) phase of hyperpolarization (-7.9 +/- 1.93 mV). 6. At PN1-4, the resting potential (Vm) of most cells had to be maintained by current injection; the input resistance (RN) of CA1 neurons was 70% higher than in mature cells, and there was little time-dependent inward rectification. Anoxia produced no regular changes in Vm, and reductions in RN were very small (by only 9.6 +/- 5.0%). A postanoxic hyperpolarization was seen in only 2 neurons out of 11.(ABSTRACT TRUNCATED AT 400 WORDS)     

Pharmacological and biophysical characterization of voltage-gated calcium currents in the endopiriform nucleus of the guinea pig

The endopiriform nucleus (EPN) is a well-defined structure that is located deeply in the piriform region at the border with the striatum and is characterized by dense intrinsic connections and prominent projections to piriform and limbic cortices. The EPN has been proposed to promote synchronization of large populations of neurons in the olfactory cortices via the activation of transient depolarizations possibly mediated by Ca(2+) spikes. It is known that principal cells in the EPN express both a low- and high-voltage-activated (HVA) Ca(2+) currents. We further characterized HVA conductances possibly related to Ca(2+)-spike generation in the EPN with a whole cell, patch-clamp study on neurons acutely dissociated from the EPN of the guinea pig. To study HVA currents in isolation, experiments were performed from a holding potential of -60 mV, using Ba(2+) as the permeant ion. Total Ba(2+) currents (I(Ba)) evoked by depolarizing square pulses peaked at 0/+10 mV and were completely abolished by 200 microM Cd(2+). The pharmacology of HVA I(Ba)s was analyzed by applying saturating concentrations of specific Ca(2+)-channel blockers. The L-type blocker nifedipine (10 microM; n = 11), the N-type-channel blocker omega-conotoxin GVIA (0.5 microM; n = 24), and the P/Q-type blocker omega-conotoxin MVIIC (1 microM; n = 16) abolished fractions of total I(Ba)s equal on average to 24.7 +/- 5.4%, 27.1 +/- 3.4%, and 22.2 +/- 2.4%, respectively (mean +/- SE). The simultaneous application of the three blockers reduced I(Ba) by 68.5 +/- 6.6% (n = 10). Nifedipine-sensitive currents and most N- and P/Q-type currents were slowly decaying, the average fractional persistence after 300 ms of steady depolarization being 0.77 +/- 0.02, 0.60 +/- 0.06, and 0.68 +/- 0.04, respectively. The residual, blocker-resistant (R-type) currents were consistently faster inactivating, with an average fractional persistence after 300 ms of 0.30 +/- 0.08. Fast-decaying R-type currents also displayed a more negative threshold of activation (by about 10 mV) than non-R-type HVA currents. These results demonstrate that EPN neurons express multiple pharmacological components of the HVA Ca(2+) currents and point to the existence of an R-type current with specific functional properties including fast inactivation kinetics and intermediate threshold of activation.     

Transient outward currents in cochlear ganglion neurons of the chick embryo.

Cochlear ganglion neurons were isolated from chick embryos and membrane currents recorded using the patch-clamp technique. Depolarizing voltage steps elicited transient outward currents whose inactivation was best fitted by a double-exponential function with time constants < 30 ms and > 100 ms. The fast inactivating transient outward current (Ito,f) had a threshold for activation of -61 +/- 5.5 mV; steady-state inactivation was voltage-dependent between -90 and -60 mV, with half-inactivation near -75 mV. The slowly inactivating outward current (Ito,s) showed an activation threshold of 34 +/- 4 mV. Half-inactivation was at -67 +/- 3 mV. Ito,f was blocked by 4-aminopyridine which did not affect Ito,s. The effect was concentration- and voltage-dependent. Tetraethylammonium had no effect on either fast or slow transient currents but reduced the amplitude of the non-inactivating outward current in a dose-dependent manner. Ito,f was strongly inhibited by removing Ca2+ from the extracellular bathing solution. Cobalt ions inhibited Ito,f in a dose-dependent manner between 2 and 20 mM. The inhibitory effect of Co2+ was voltage-dependent, displaying a bell-shaped inhibition curve as a function of membrane voltage, maximal inhibition occurring between -20 and 0 mV. Ca2+ removal did not affect Ito,s and partially reduced the amplitude of the steady-state current. These results provide kinetic and pharmacological evidence for the presence of two distinct transient outward currents in cochlear neurons. These currents may play a role in the first synaptic relay of sound transmission.     

Characteristics of potassium and calcium currents of hepatic stellate cells (ito) in rat

Hepatic stellate cells (HSCs) are known to play a role in the pathogenesis of the increased intrahepatic vascular resistance found in chronic liver diseases. The aim of this study was to evaluate the K+ and Ca2+ currents in cultured HSCs from rat liver, through the patch-clamp technique. Most cells were positive for desmin immunostain after isolation and in alpha-smooth muscle actin immunostain after 10 - 14 days of culturing. Outward and inward rectifying K+ currents were confirmed. Two different types of K+ currents were distinguished: one with the inward rectifying current and the other without. The outward K+ currents consisted of at least four components: tetraethylammonium (TEA)-sensitive current, 4-aminopyridine (4-AP)-sensitive current, pimozide-sensitive current and three blocker-resistant current. The peaks of the outward K+ currents evoked by a depolarizing pulse were decreased to 32.0 +/- 3.0, 62.8 +/- 3.7 and 32.8 +/- 3.5% by 5 mM TEA, 2 mM 4-AP and 15 micro M pimozide, respectively. Moreover, the combined application of three blockers caused 86.6 +/- 4.8% suppression. The inward currents evoked hyperpolarizing pulses were inwardly rectifying and almost blocked by Ba2+. Elevation of external K+ increased the inward current amplitude and positively shifted its reversal potential. Voltage- dependent Ca2+ currents which were completely abolished by Cd2+ and nimodipine were detected in 14 day cultured HSCs. In this study, the cultured HSCs were found to express outward K+ currents composed of multiple pharmacological components, Ba2+-sensitive inward rectifying K+ current and L-type Ca2+ current.     

Pharmacology of a slowly inactivating outward current in hippocampal CA3 pyramidal neurons

The pharmacology of a slowly inactivating outward current was examined using whole cell patch-clamp recordings in CA3 pyramidal cells of guinea pig hippocampal slices. The current had a low activation threshold (about -60 mV) and inactivated slowly (time constant of 3.4 +/- 0.5 s at -50 mV) and completely at membrane voltages depolarized to -50 mV. The slowly inactivating outward current was mainly mediated by K+ with a reversal potential close to the equilibrium potential for K+. The slowly inactivating outward current had distinct pharmacological properties: its time course was not affected by extracellular Cs+ (1 mM) or 4-AP (1-5 mM)-broad spectrum inhibitors of K+ currents and of inactivating K+ currents, respectively. The presence of extracellular Mn2+ (0.5-1 mM), which suppresses several Ca2+ -dependent K+ currents, also did not affect the slowly inactivating outward current. The current was partially suppressed by TEA (50 mM) and was blocked by intracellular Cs+ (134 mM). In addition, intracellular QX-314 (5 mM), a local anesthetic derivative, inhibited this current. The slowly inactivating outward current with its low activation threshold should be operational at the resting potential. Our results suggest that the transient outward current activated at subthreshold membrane potentials in hippocampal pyramidal cells consists of at least three components. In addition to the well-described A- and D-currents, the slowest decaying component reflects the time course of a distinct current, suppressible by QX-314.     

Calcium inward currents in internally perfused giant axons

1. Voltage clamp experiments were carried out on squid axons perfused with an isotonic solution of 25 mM-CsF + sucrose and placed in a Na-free solution of 100 mM-CaCl(2) + sucrose.2. Depolarizing voltage steps produced inward currents of 4-6 muA/cm(2) peak amplitude which decayed slightly during a 60 msec pulse; the inward current disappeared when the internal potential reached +50 to +60 mV and became outward for larger depolarizations.3. Tetrodotoxin completely blocked the inward current and part of the outward current. No inward currents were seen with 100 mM-MgCl(2) + sucrose as the external solution. Substituting acetate for external Cl(-) did not abolish the tetrodotoxin-sensitive outward currents.4. It is concluded that the inward current is carried by Ca and the tetrodotoxin-sensitive outward current by Cs ions, both moving through the Na channel.5. The reversal potential of the tetrodotoxin-sensitive current was in the average +54 mV. Raising the external Ca concentration or adding NaCl to the external solution increased the reversal potential; lowering the external Ca concentration or replacing the internal CsF by a Na salt decreased the reversal potential.6. From the reversal potentials of the tetrodotoxin-sensitive current measured with varying external and internal solutions the relative permeabilities of the Na channel were calculated as P(Ca)/P(Cs) = 1/0.6, P(Ca)/P(Na) = 1/10 to 1/7 and P(Cs)/P(Na) = 1/22 to 1/9 by means of the constant field equation. The permeability ratios suggest that under these experimental conditions the Na channel is still primarily permeable to Na ions, although its selectivity is relatively small.7. The time course of the tetrodotoxin-sensitive Ca inward current was different from the time course of the Na inward current. The Na current consisted of an initial peak followed by a more slowly decaying component, the Ca current showed only the slow component.8. The slowly inactivating tetrodotoxin-sensitive Ca inward currents give rise to the long lasting action potentials which have first been observed by Tasaki and coworkers under similar conditions.     

K+ currents generated by NMDA receptor activation in rat hippocampal pyramidal neurons

Long lasting outward currents mediated by Ca2+-activated K+ channels can be induced by Ca2+ influx through N-methyl-D-aspartate (NMDA)-receptor channels in voltage-clamped hippocampal pyramidal neurons. Using specific inhibitors, we have attempted to identify the channels that underlie these outward currents. At a holding potential of -50 mV, applications of 1 mM NMDA to the soma of cultured hippocampal pyramidal neurons induced the expected inward currents. In 44% of cells tested, these were followed by outward currents (average amplitude 60 +/- 7 pA) that peaked 2.5 s after the initiation of the inward NMDA currents and decayed with a time constant of 1.4 s. In 43% of those cells exhibiting an outward current, SK channel inhibitors, UCL 1848 (100 nM) and apamin (100 nM) abolished the outward current. In the remainder of the cells, the outward currents were either insensitive or only partly inhibited (44 +/- 4%) by 100 nM UCL 1848. In these cells, the outward currents were reduced by the slow afterhyperpolarization (sAHP) inhibitors, muscarine (3 microM; 43 +/- 9%), UCL 1880 (3 microM; 34 +/- 10%), and UCL 2027 (3 microM; 57 +/- 6%). Neither the BK channel inhibitor, charybdotoxin (100 nM), nor the Na+/K+ ATPase inhibitor, ouabain (100 microM), reduced these outward currents. Irrespective of the pharmacology, the time course of the outward current did not differ. Interestingly, no correlation was observed between the presence of a slow apamin-insensitive afterhyperpolarization and an outward current insensitive to SK channel blockers following NMDA-receptor activation. It is concluded that an NMDA-mediated rise in [Ca2+]i can result in the activation of apamin-sensitive SK channels and of the channels that underlie the sAHP. The activation of these channels may, however, depend on their location relative to NMDA receptors as well as on the spatial Ca2+ buffering within individual neurons.     

Ca2+-activated outward currents in neostriatal neurons

Whole-cell voltage-clamp recordings of outward currents were obtained from acutely dissociated neurons of the rat neostriatum in conditions in which inward Ca2+ current was not blocked and intracellular Ca2+ concentration was lightly buffered. Na+ currents were blocked with tetrodotoxin. In this situation, about 53 +/- 4% (mean +/- S.E.M.; n = 18) of the outward current evoked by a depolarization to 0 mV was sensitive to 400 microM Cd2+. A similar percentage was sensitive to high concentrations of intracellular chelators or to extracellular Ca2+ reduction (<500 microM); 35+/-4% (n=25) of the outward current was sensitive to 3.0 mM 4-aminopyridine. Most of the remaining current was blocked by 10 mM tetraethylammonium. The results suggest that about half of the outward current is activated by Ca2+ entry in the present conditions. The peptidic toxins charybdotoxin, iberotoxin and apamin confirmed these results, since 34 +/- 5% (n = 14), 29 5% (n= 14) and 28 +/- 6% (n=9) of the outward current was blocked by these peptides, respectively. The effects of charybdotoxin and iberotoxin added to that of apamin, but their effects largely occluded each other. There was additional Cd2+ block after the effect of any combination of toxins. Therefore, it is concluded that Ca2+-activated outward currents in neostriatal neurons comprise several components, including small and large conductance types. In addition, the present experiments demonstrate that Ca2+-activated K+ currents are a very important component of the outward current activated by depolarization in neostriatal neurons.     

Development of Na(+)- and K(+)-currents in the cochlear ganglion of the chick embryo.

The development of Na(+)- and K(+)-currents in the primary afferent neurons of the cochlear ganglion was studied using the patch-clamp technique. Cells were dissociated between days 6 and 17 of development and membrane currents recorded within the following 24 h. Outward currents were the first to appear between days 6 and 7 of embryonic development and their magnitude increased throughout development from 200 pA on day 7 to 900 pA on days 14-16. Threshold for activation decreased by 20 mV between days 8 and 14. Outward currents were absent when Cs+ replaced K+ in the pipette and were partially blocked by external tetraethylammonium. Outward currents contained at least three components: (i) a non-inactivating outward current, similar to the delayed-rectifier, predominating in mature neurons; (ii) a slowly inactivating current (tau about 200 ms), most evident in early and intermediate stages (days 7-10); and (iii) a rapidly inactivating outward current (tau about 20 ms) similar to the A-current (IA) described in other neurons, which was distinctly expressed in mature neurons. Sodium currents were identified as fast transient inward currents, sensitive to tetrodotoxin and extracellular Na(+)-removal. They appeared later than K(+)-currents and increased in size from about 100 pA between days 9-11 to 600 pA by days 13-16. The development of membrane currents in cochlear ganglion neurons corresponded to defined stages of the innervation pattern of the chick cochlea [Whitehead and Morest (1985) Neuroscience 14, 255-276]. These currents could be functionally related to the establishment of synaptic connections between transducing cells and primary afferent neurons.     

TTX-sensitive action potentials and excitability of adult rat sensory neurons cultured in serum- and exogenous nerve growth factor-free medium

The excitability of adult rat dorsal root ganglion (DRG) neurons cultured in the absence of serum and exogenously added nerve growth factor (NGF) was studied. Current-clamp recordings revealed the presence of tetrodotoxin (TTX)-sensitive action potentials. Voltage-clamp recordings demonstrated the presence of both inward and outward currents. The inward Na+ current had a maximal amplitude near -10 mV and was completely blocked by TTX. A sustained Ca2+ inward current and a slowly activating outward K+ current were also observed. TTX-sensitive and TTX-resistant action potentials have been observed in previous studies in DRG neurons cultured in the presence of serum. By contrast, in the study reported here, only TTX-sensitive action potentials and Na+ currents were found in the neurons cultured in the absence of serum and nerve growth factor.     

Inhibition of T-type calcium currents by dihydropyridines in mouse embryonic dorsal root ganglion neurons.

The effects of dihydropyridines (DHPs) normally considered to be specific for L-type calcium channels were studied on the T-type Ca channel current of acutely isolated dorsal root ganglion (DRG) neurons taken from 13-day-old (E13) mouse embryos. Potent but reversible inhibitory effects of the DHP nicardipine were found in the micromolar range. For example, 5 microM nicardipine suppressed 93 +/- 5% of T-type currents. In comparison, other classical DHPs such as nifedipine, PN 200-110 and nitrendipine had only weak effects (less than 20% inhibition) at the same concentration. The inhibition by nicardipine was found slightly to be voltage dependent and the drug induced a leftward shift in the steady-state inactivation. The DHP agonist (-)-Bay K 8644, which dramatically increased the L-type current, weakly decreased T-type Ca currents (17 +/- 8% at 5 microM). In conclusion, neuronal T-type Ca channels may be potential targets for some dihydropyridines. This property is not only a feature of the central nervous system (J. Physiol., 412 (1989) 181-195) and can be extended to peripheral neurons.     

Block of potassium outward currents in the crayfish stretch receptor neurons by 4-aminopyridine, tetraethylammonium chloride and some other chemical substances.

The effects of 4-aminopyridine (4-AP) and tetraethylammonium (TEA) on the outward potassium currents in the rapidly and slowly adapting stretch receptor neurons (SRNs) of the crayfish (Pacifastacus leniusculus) were studied using a two micro-electrode voltage-clamp technique. The leakage current was not affected by either 4-AP or TEA. External 4-AP blocked the peak outward current in a dose-dependent manner (1:1 stoichiometry) with an apparent dissociation constant (Kd) of 2.3 +/- 0.2 mM (mean +/- SEM) in the slowly and 1.4 +/- 0.2 mM in the rapidly adapting SRN, the block being voltage dependent. External application of TEA resulted in a block of the steady state current enhancing the transient characteristics of the current response. The block appeared to deviate from a 1:1 stoichiometry and the apparent Kd for TEA was 9.6 +/- 3.4 mM with a cooperativity factor n = 0.43 +/- 0.03 in the slowly adapting SRN and 34.5 +/- 9.2 mM and 0.37 +/- 0.03 respectively in the rapidly adapting SRN. Low Ca2+, apamin and charybdotoxin, which are known to block Ca(2+)-dependent K-currents, had no effects on the outward current as was also the case with catechol. It is concluded that the different effects of TEA and 4-AP on the outward current in the two types of SRNs can be explained by the presence of at least two, probably heteromultimeric, channel populations having similar sensitivity to 4-AP but different sensitivity to TEA. One channel has a high affinity (Kd = 0.8-1.6 mM) for TEA and the other a low affinity (Kd = 173-213 mM) for TEA. The low-affinity channel seems to dominate in the slowly adapting SRN while both channels are equally common in the rapidly adapting SRN. Further, the present results do not support the existence of a macroscopic Ca(2+)-dependent K+ current in the SRNs.     

Calcium-dependent chloride current induced by axotomy in rat sympathetic neurons.

1. Seven to ten days after sectioning their axons, rat sympathetic neurons were studied using intracellular recording techniques in an in vitro preparation of the superior cervical ganglion. 2. In 75% of axotomized cells, an after-depolarization (ADP) was observed following spike firing or depolarization with intracellular current pulses. Discontinuous single-electrode voltage-clamp techniques were employed to study the ADP. When the membrane potential was clamped at the resting level just after an action potential, a slow inward current was recorded in cells that showed an ADP. 3. In the presence of TTX and TEA, inward peaks and outward currents were recorded during depolarizing voltage jumps, followed by slowly decaying inward tail currents accompanied by large increases in membrane conductance. The inward peak and tail currents activated between -10 and -20 mV and reached maximum amplitudes around 0 mV. With depolarizing jumps to between +40 and +50 mV, net outward currents were recorded during the depolarizing jumps but inward tail currents were still activated. 4. In the presence of the Ca2+ channel blocker cadmium, or when Ca2+ was substituted by Mg2+, the ADP disappeared. In voltage-clamped cells, cadmium blocked the inward tail currents. The reversal potential for the inward tail current was approximately -15 mV. Substitution of the extracellular NaCl by sucrose or sodium isethionate increased the amplitude of the inward tail current, and displaced its equilibrium potential to more positive values. Changes in extracellular [K+] did not appreciably affect the inward tail current amplitude or equilibrium potential. Niflumic acid, a blocker of chloride channels activated by Ca2+, almost completely blocked the tail current. 5. No ADPs were observed in non-axotomized neurons, and when depolarizing pulses were applied while in voltage clamp no inward tail currents were evoked in these normal cells. 6. It is concluded that axotomy of sympathetic ganglion cells produces the appearance of a Ca(2+)-dependent chloride current responsible for the ADP observed following spike firing.     

Characterisation of the ionic currents in freshly isolated rat ureter smooth muscle cells: evidence for species-dependent currents

To better understand excitability, and hence contraction, the ionic currents underlying the action potential were identified and characterised in enzymatically isolated smooth muscle cells of the rat ureter. Using the whole-cell patch-clamp, under voltage-clamp conditions with K(+) in the pipette, three types of responses occurred to depolarisation: (1) sustained outward current and spontaneous transient outward currents (STOCs); (2) inward current; and (3) fast outward current. Investigation using different voltage protocols and pharmacological blockers and agonists revealed the presence of three outward and two inward currents. The outward currents were: (1) a sustained BK current, sensitive to low concentrations of tetraethylammonium (TEA) and featuring bursts of STOCs superimposed on it; (2) a fast, transient, A-type K current sensitive to 4-aminopyridine; and (3) a TEA and Ca(2+)-insensitive, late K(+) rectifier current. The inward currents were: (1) a fast L-type Ca(2+) channel current sensitive to nifedipine, Cd(2+) and potentiated by Ba(2+); and (2) a Ca(2+)-sensitive Cl(-) channel, which was inhibited by niflumic acid and Ba(2+), and produced a large tail current upon repolarisation at the end of the voltage step. The I- V relationships and peak amplitudes of all the currents are described. The finding of a K(+) rectifier and Ca(2+)-activated Cl(-) channel distinguish the rat ureteric cells from those of the guinea-pig. Thus, as well as the previously established difference in sarcoplasmic reticulum Ca(2+)-release mechanisms, there is also a species difference in ion channel expression in this tissue. We relate these currents to their possible contribution to the characteristically extremely long lasting action potential in the rat ureter.     

Ionic currents in crustacean neurosecretory cells.

1. The patterns of electrical activity and membrane characteristics of a population of neurosecretory-cell somata in the X-organ of the crayfish were investigated with microelectrodes and whole-cell, voltage-clamp techniques. Some neurons (56%) were silent but could be excited by intracellular current injection: other cells showed spontaneous tonic activity (35%), and some had spontaneous bursting activity (9%). The spiking activity was abolished by tetrodotoxin (TTX) exposure and by severing the axon near the cell body. After axotomy, only a small, slow, regenerative depolarization remained that could be blocked by Cd2+. 2. Under voltage clamp the steady-state I-V curve in low [Ca2+]i (9 X 10(-9) M) showed a slope conductance of 16.7 +/- 3.9 (SD) nS (n = 10) at -50 mV and zero current potential of -50.1 +/- 7.7 mV. In current-clamp mode these neurons were either silent or fired tonically. With high [Ca2+]i (1.7 X 10(-6) M) both the slope conductance and inward and outward currents were reduced. In some neurons high [Ca2+]i reveals a negative slope resistance in the range of -46 to -41 mV. It could be supressed by removing [Na+]o, but it was TTX insensitive. These are the neurons that under current clamp showed bursting activity. 3. The main inward current in cell somata was a Ca2+ current of 2 +/- 0.6 nA (n = 18), activated at -40 mV and peaking at 20 mV. It showed relaxation with prolonged pulses. No Na(+)-dependent, TTX-sensitive inward currents were recorded with short (100-ms) pulses in axotomized neurons. 4. Two outward currents could be distinguished.(ABSTRACT TRUNCATED AT 250 WORDS)     

Multiple conductances are modulated by 5-HT receptor subtypes in rat subthalamic nucleus neurons

Firing patterns of subthalamic nucleus (STN) neurons influence normal and abnormal movements. The STN expresses multiple 5-HT receptor subtypes that may regulate neuronal excitability. We used whole-cell patch-clamp recordings to characterize 5-HT receptor-mediated effects on membrane currents in STN neurons in rat brain slices. In 80 STN neurons under voltage-clamp (-70 mV), 5-HT (30 microM) evoked inward currents in 64%, outward currents in 17%, and biphasic currents in 19%. 5-HT-induced outward current was caused by an increased K(+) conductance (1.4+/-0.2 nS) and was blocked by the 5-HT(1A) antagonist WAY 100135. The 5-HT-evoked inward current, which was blocked by antagonists at 5-HT(2C) and/or 5-HT(4) receptors, had two types of current-voltage (I-V) relations. Currents associated with the type 1 I-V relation showed negative slope conductance at potentials <-110 mV and were occluded by Ba(2+). In contrast, the type 2 I-V relation appeared linear and had positive slope conductance (0.64+/-0.11 nS). Type 2 inward currents were Ba(2+)-insensitive, and the reversal potential of -19 mV suggests a mixed cation conductance. In STN neurons in which 5-HT evoked inward currents, 5-HT potentiated burst firing induced by N-methyl-d-aspartate (NMDA). But in neurons in which 5-HT evoked outward current, 5-HT slowed NMDA-dependent burst firing. We conclude that 5-HT receptor subtypes can differentially regulate firing pattern by modulating multiple conductances in STN neurons.     

Mechanisms of spreading depression and hypoxic spreading depression-like depolarization.

Spreading depression (SD) and the related hypoxic SD-like depolarization (HSD) are characterized by rapid and nearly complete depolarization of a sizable population of brain cells with massive redistribution of ions between intracellular and extracellular compartments, that evolves as a regenerative, "all-or-none" type process, and propagates slowly as a wave in brain tissue. This article reviews the characteristics of SD and HSD and the main hypotheses that have been proposed to explain them. Both SD and HSD are composites of concurrent processes. Antagonists of N-methyl-D-aspartate (NMDA) channels or voltage-gated Na(+) or certain types of Ca(2+) channels can postpone or mitigate SD or HSD, but it takes a combination of drugs blocking all known major inward currents to effectively prevent HSD. Recent computer simulation confirmed that SD can be produced by positive feedback achieved by increase of extracellular K(+) concentration that activates persistent inward currents which then activate K(+) channels and release more K(+). Any slowly inactivating voltage and/or K(+)-dependent inward current could generate SD-like depolarization, but ordinarily, it is brought about by the cooperative action of the persistent Na(+) current I(Na,P) plus NMDA receptor-controlled current. SD is ignited when the sum of persistent inward currents exceeds persistent outward currents so that total membrane current turns inward. The degree of depolarization is not determined by the number of channels available, but by the feedback that governs the SD process. Short bouts of SD and HSD are well tolerated, but prolonged depolarization results in lasting loss of neuron function. Irreversible damage can, however, be avoided if Ca(2+) influx into neurons is prevented.     

Biophysical and pharmacological characterization of voltage-sensitive calcium currents in neonatal rat inferior colliculus neurons

Calcium conductances have been found in neonatal inferior colliculus neurons, however the biophysical and pharmacological profiles of the underlying calcium currents have not yet been characterized. In this study, we examined which types of voltage-activated calcium currents comprise the whole-cell inward current of neonatal inferior colliculus neurons (10-22microm in diameter). On the basis of their voltage-dependence and pharmacological sensitivities, three major components of barium currents were identified. A low threshold voltage-activated current that activated around -70mV, a mid threshold voltage-activated current that activated near -50mV, and a high threshold voltage-activated current that activated around -40mV. Low and mid threshold voltage-activated currents were present in 33% and 41% of the recordings, respectively, whereas high threshold voltage-activated currents were recorded in all inferior colliculus neurons tested. Nickel chloride (50microM) and U-92032 (1microM), which both block low threshold voltage-activated currents, reduced the amplitude of low threshold voltage-activated peak currents at a test potential of -60mV by 72% and 10%, respectively. In addition, 50microM nickel chloride and 1microM U-92032 reduced the amplitude of mid threshold voltage-activated peak currents measured at -20mV by 55% and 21%, respectively. Further pharmacological analysis indicated the presence of multiple types of high threshold voltage-activated currents in neonatal inferior colliculus neurons. The dihydropyridine nimodipine (1microM), a selective L-type current antagonist, reduced the amplitude of high threshold voltage-activated peak currents by 25%. In addition, FPL 64176 (1microM), a non-dihydropyridine L-type current agonist caused a dramatic 534% increase in the amplitude of the slow sustained component of the tail current measured at -40mV. These data indicate that inferior colliculus neurons express L-type channels. omega-Conotoxin GVIA (1microM), a selective blocker of N-type current, inhibited high threshold voltage-activated peak currents by 28% indicating the presence of N-type channels. omega-Agatoxin IVA (300nM), a potent P/Q-type antagonist, reduced high threshold voltage-activated peak currents by 27%, suggesting that inferior colliculus neurons express P/Q-type channels. Concomitant application of nimodipine (1microM), omega-conotoxin GVIA (1microM) and omega-agatoxin IVA (300nM) onto inferior colliculus neurons decreased the control high threshold voltage-activated peak currents only by 62%.Thus, inferior colliculus neurons may express at least one more type of calcium current in addition to low and mid threshold voltage-activated currents and L-type, N-type and P/Q-type high threshold currents.     

HERG-Like potassium current regulates the resting membrane potential in glomus cells of the rabbit carotid body

Direct evidence for a specific K(+) channel underlying the resting membrane potential in glomus cells of the carotid body has been absent. The product of the human ether-a-go-go-related gene (HERG) produces inward rectifier currents that are known to contribute to the resting membrane potential in other neuronal cells. The goal of the present study was to determine whether carotid body glomus cells express HERG-like K(+) current, and if so, to determine whether a HERG-like current regulates the resting membrane potential. Freshly dissociated rabbit glomus cells under whole cell voltage clamp exhibited slowly decaying outward currents that activated 20-30 mV positive to the resting membrane potential. Raising extracellular K(+) revealed a slowly deactivating inward tail current indicative of HERG-like K(+) current. HERG-like currents were not found in cells resembling type II cells. The HERG-like current was blocked by dofetilide (DOF) in a concentration-dependent manner (IC(50) = 13 +/- 4 nM, mean +/- SE) and high concentrations of Ba(2+) (1 and 10 mM). The biophysical and pharmacological characteristics of this inward tail current suggest that it is conducted by a HERG-like channel. The steady-state activation properties of the HERG-like current (V(h) = -44 +/- 2 mV) suggest that it is active at the resting membrane potential in glomus cells. In whole cell, current-clamped glomus cells (average resting membrane potential, - 48 +/- 4 mV), DOF, but not tetraethylammonium, caused a significant (13 mV) depolarizing shift in the resting membrane potential. Using fluorescence imaging, DOF increased [Ca(2+)](i) in isolated glomus cells. In an in-vitro carotid body preparation, DOF increased basal sensory discharge in the carotid sinus nerve in a concentration-dependent manner. These results demonstrate that glomus cells express a HERG-like current that is active at, and responsible for controlling the resting membrane potential.