The voltage-dependent conductances of rat neocortical layer I neurons

Whole cell patch-clamp techniques were used to study voltage-dependent sodium (Na+), calcium (Ca2+), and potassium (K+) conductances in acutely isolated neurons from cortical layer I of adult rats. Layer I cells were identified by means of gamma-aminobutyric acid (GABA) immunocytochemistry. Positive stainings for the Ca2+-binding protein calretinin in a subset of cells, indicated the presence of Cajal-Retzius (C-R) cells. All investigated cells displayed a rather homogeneous profile of voltage-dependent membrane currents. A fast Na+ current activated at about -45 mV, was half-maximal steady-state inactivated at -66.6 mV, and recovery from inactivation followed a two-exponential process (tau1 = 8.4 ms and tau2 = 858.8 ms). Na+ currents declined rapidly with two voltage-dependent time constants, reaching baseline current after some tens of milliseconds. In a subset of cells (< 50%) a constant current level of < 65 pA remained at the end of a 90 ms step. A transient outward current (Ifast) activated approximately -40 mV, declined rapidly with a voltage-insensitive time constant (tau approximately 350 ms) and was relatively insensitive to tetraethylammonium (TEA, 20 mM). Ifast was separated into two components based on their sensitivity to 4-aminopyridine (4-AP): one was blocked by low concentrations (40 microM) and a second by high concentrations (6 mM). After elimination of Ifast by a conditioning prepulse (50 ms to -50 mV), a slow K+ current (I(KV)) could be studied in isolation. I(KV) was only moderately affected by 4-AP (6 mM), while TEA (20 mM) blocked most (> 80%) of the current. I(KV) activated at about -40 mV, declined monoexponentially in a voltage-dependent manner (tau approximately 850 ms at -30 mV), and revealed an incomplete steady-state inactivation. In addition to Ifast and I(KV), indications of a Ca2+-dependent outward current component were found. When Na+ currents, Ifast, and I(KV) were blocked by tetrodotoxin (TTX, 1 microM), 4-AP (6 mM) and TEA (20 mM) an inward current carried by Ca2+ was found. Ca2+ currents activated at depolarized potentials at about -30 mV, were completely blocked by 50 microM cadmium (Cd2+), were sensitive to verapamil (approximately 40% block by 10 microM), and were not affected by nickel (50 microM). During current clamp recordings, isolated layer I neurons displayed fast spiking behaviour with short action potentials (approximately 2 ms, measured at half maximal amplitude) of relative small amplitude (approximately 83 mV, measured from the action potential threshold).     

Characterization of outward currents in neurons of the avian nucleus magnocellularis

Characterization of outward currents in neurons of the avian nucleus magnocellularis. J. Neurophysiol. 80: 2824-2835, 1998. Neurons of the nucleus magnocellularis (NM) preserve the timing of auditory signals through the convergence of a variety of voltage- and ligand-gated ion channels. To understand better how these channels interact, we have characterized the kinetics, voltage sensitivity, and pharmacology of outward currents of NM neurons in brain slices. The reversal potential (Erev) of outward currents varied with potassium concentration as expected for currents carried by potassium. However, Erev was consistently more positive than the Nernst potential for potassium (EK). Deviation of Erev from the calculated EK most likely arose from potassium accumulation in extracellular spaces by potassium conductances active at rest and during depolarizing steps. Three outward potassium currents were studied that varied in voltage and pharmacological sensitivity. A tetraethylammonium (TEA)-sensitive, high-threshold current was activated within 1-5 ms of the onset of depolarization, with a half-maximal activation voltage (V1/2) of -19 mV. It was blocked partially by 4-aminopyridine (4-AP) and was the dominant ionic conductance of NM neurons. A dendrotoxin-I (DTX) and 4-AP-sensitive, low-threshold current had a V1/2 of -58 mV, rapid activation kinetics, and only partial inactivation, with decay time constants between 20 and 100 ms. A rapidly inactivating current was observed that was resistant to TEA and DTX and was blocked by intracellular Cs+. The transient current was inactivated almost completely at the resting potential. The onset of inactivation was fastest at potentials negative to those that caused activation. When intracellular K+ was replaced by Cs+, large inward and outward currents were obtained that corresponded respectively to the above-mentioned DTX- and TEA-sensitive currents. Outward, TEA-sensitive current was carried by Cs+, with a PCs/PK of approximately 0.1. In current-clamped neurons, DTX induced repetitive firing and increased membrane time constant near rest but had little effect on action potential duration. These studies indicate that a low-threshold, DTX-sensitive current plays a key role in making NM neurons highly responsive to the onset and offset of synaptic stimuli.     

Morphological and membrane properties of rat magnocellular basal forebrain neurons maintained in culture

Morphological and electrophysiological characteristics of magnocellular neurons from basal forebrain nuclei of postnatal rats (11-14 days old) were examined in dissociated cell culture. Neurons were maintained in culture for periods of 5-27 days, and 95% of magnocellular (>23 micron diam) neurons stained positive with acetylcholinesterase histochemistry. With the use of phase contrast microscopy, four morphological subtypes of magnocellular neurons could be distinguished according to the shape of their soma and pattern of dendritic branching. Corresponding passive and active membrane properties were investigated with the use of whole cell configuration of the patch-clamp technique. Neurons of all cell types displayed a prominent (6-39 mV; 6.7-50 ms duration) spike afterdepolarization (ADP), which in some cells reached firing threshold. The ADP was voltage dependent, increasing in amplitude and decreasing in duration with membrane hyperpolarization with an apparent reversal potential of -59 +/- 2.3 (SE) mV. Elevating [Ca2+]o (2.5-5.0 mM) or prolonging spike repolarization with 10 mM tetraethylammonium (TEA) or 1 mM 4-aminopyridine (4-AP), potentiated the ADP while it was inhibited by reducing [Ca2+]o (2.5-1 mM) or superfusion with Cd2+ (100 microM). The ADP was selectively inhibited by amiloride (0.1-0.3 mM or Ni2+ 10 microM) but unaffected by nifedipine (3 microM), omega-conotoxin GVIA (100 nM) or omega-agatoxin IVA (200 nM), indicating that Ca2+ entry was through T-type Ca2+ channels. After inhibition of the ADP with amiloride (300 microM), depolarization to less than -65 mV revealed a spike afterhyperpolarization (AHP) with both fast and slow components that could be inhibited by 4-AP (1 mM) and Cd2+ (100 microM), respectively. In all cell types, current-voltage relationships exhibited inward rectification at hyperpolarized potentials >/=EK (approximately -90 mV). Application of Cs+ (0.1-1 mM) or Ba2+ (1-10 microM) selectively inhibited inward rectification but had no effect on resting potential or cell excitability. At higher concentrations, Ba2+ (>10 microM) also inhibited an outward current tonically active at resting potential (VH -70 mV), which under current-clamp conditions resulted in small membrane depolarization (3-10 mV) and an increase in cell excitability. Depolarizing voltage commands from prepulse potential of -90 mV (VH -70 mV) in the presence of tetrodotoxin (0.5 microM) and Cd2+ (100 microM) to potentials between -40 and +40 mV cause voltage activation of both transient A-type and sustained delayed rectifier-type outward currents, which could be selectively inhibited by 4-AP (0.3-3 mM) and TEA (1-3 mM), respectively. These results show that, although acetylcholinesterase-positive magnocellular basal forebrain neurons exhibit considerable morphological heterogeneity, they have very similar and characteristic electrophysiological properties.     

Characteristics of a fast transient outward current in guinea pig trigeminal motoneurons

A fast transient voltage dependent outward current (TOC) in trigeminal motoneurons (TMNs) was studied in guinea pig brainstem slices by use of sharp electrodes in combination with single electrode voltage clamp techniques. In solutions containing TTX, low Ca2+/Mn2+ and 20 mM TEA this current activated around -55 to -60 mV from holding potentials negative to resting potential, obtained its peak amplitude within 5 ms and decayed as a single exponential with a time constant of 6-8 ms. Half maximal values for inactivation and activation were -72 and -37 mV, respectively. Bath application of 5 mM 4-AP suppressed this current by approximately 90% and eliminated the early depolarizing transient membrane rectification observed in response to a constant depolarizing current pulse, prolonged the action potential duration, and reduced the threshold voltage and delay to onset of the action potential. It is suggested that this current resembles the typical A-current observed in many CNS neurons and, as a result of its voltage and time dependent properties, could contribute to control of motoneuronal discharge and timing of burst onset during rhythmical jaw movements. Therefore, any cellular models of masticatory activity should include the properties of this current.     

Effects of intracellular 4-aminopyridine and tetraethylammonium load on action potentials in the rabbit atrial myocardium

Investigated are rabbit atrial trabeculae (2-4 mm length, 150-800 micrometers diameter). When using a standard microelectrode technique, the action potentials after an intracellular 4-AP and TEA load are prolonged. Only the TEA load significantly diminishes the resting potential of the beating preparations. The typical configuration of the action potential following a period of rest (rapid initial repolarization, secondary depolarization and a late retarded repolarization) is not changed qualitatively. During a repetitive stimulation after a pacing pause the action potentials' restitution is accelerated at 4-AP. After 4-AP and TEA load the secondary depolarization of the first post rest action potential is more accentuated. By reconstructing the post rest action potentials a 4-AP sensitive current is determined. At constant drive this current shows a threshold potential at -40 and a reversal potential at -10 mV. The current is inward directed between -40 and -10 mV and is outward directed at more positive potentials than -10 mV. After a pacing pause the current-voltage relation is shifted to more negative potentials. The 4-AP sensitive current is modelled by consideration of a slow activated (during the action potential) and deactivated (during the pacing pause) conductivity. Processes of accumulation are discussed.     

Whole-cell analysis of ionic currents underlying the firing pattern of neurons in the gustatory zone of the nucleus tractus solitarii

1. Previous work from this laboratory has shown that rostral nucleus tractus solitarii (rNTS) neurons can be separated into four different classes on the basis of responses to a current injection paradigm consisting of membrane hyperpolarization immediately followed by a depolarizing pulse. These classes have been termed Group I, II, III, and IV neurons. The regular repetitive firing discharge pattern of Group I cells is changed into an irregular spike train by membrane hyperpolarization. Hyperpolarization of Group II neurons delays the firing discharge induced by depolarization. Hyperpolarization had the least effect on the discharge pattern of Group III neurons. The discharge pattern of Group IV neurons consisted of a short burst of spikes. We used whole-cell recordings and pharmacological channel blockers in an in vitro brain stem slice preparation to determine the ionic basis for the repetitive firing properties of rNTS neurons. 2. Application of 4-aminopyridine (4-AP, 1 mM) decreased input resistance and increased action potential duration in all groups of neurons. However, the discharge pattern of Group I, III, and IV neurons was either unaltered or slightly modified by 4-AP. In contrast the delay in firing of Group II cells induced by hyperpolarization was strongly reduced and in some cases completely suppressed by application of 4-AP. This suggests that a 4-AP-sensitive conductance primarily underlies the firing pattern of Group II cells. 3. Voltage-clamp recordings revealed that the delay in Group II neurons is due to a transient outward potassium current that is partially inactivated around the resting membrane potential. Hyperpolarization removed this inactivation, causing a delay in the firing of the cell. The potassium current was blocked by 4-AP. A similar current was occasionally seen in neurons of the other groups. On the basis of its voltage and pharmacological dependence this current was presumed to be an A-current (IKA). 4. Blockade of calcium currents by a low-calcium (0.5 mM) saline containing 2 mM Co2+ depressed the excitability of rNTS cells. For Group II neurons the delay in firing activity was increased. In the other groups the repetitive firing pattern was suppressed. In addition the amplitude of the afterhyperpolarization occurring after a short train of action potentials was substantially reduced. This indicates that calcium currents (ICa) and calcium-activated potassium currents (IKCa) contribute to the repetitive firing properties of rNTS neurons. 5. In about half of Group I, III, and IV neurons an additional property was found.(ABSTRACT TRUNCATED AT 400 WORDS)     

The pathophysiology of anti-neutrophil cytoplasmic antibodies (ANCA) and their clinical relevance

Intracellular recordings were made from complex spike firing neurons of the mouse dorsal cochlear nucleus (DCN) in vitro. The whole cochlear nucleus was dissected out and maintained submerged in rapidly flowing artificial cerebrospinal fluid (CSF). Recordings were made with current clamp techniques in the presence or absence of ion channel blocking drugs tetrodotoxin (TTX, 1 microM), tetraethylammonium (TEA, 20 mM), 4-aminopyridine (4-AP, 5 mM) or verapamil (50, 100, 150, 250 microM). The cells showed both spontaneous firing and responses to injections of depolarising current consisting of a mixture of a tall single action potential and complexes of 2 to 3 smaller wider action potentials superimposed on a plateau depolarisation. The membrane properties were: resting membrane potential -68.8 +/- 8.5 mV, cell resistance 54.1 +/- 26.5 M omega, time constant 9.6 +/- 5.4 ms and capacitance 0.25 +/- 0.5 nF; the first three variables had bimodel distributions. The current/voltage (I/V) relationship at membrane below resting was non-linear. Previously published histological evidence from the mouse DCN has shown that both cartwheel cells and Purkinje-like neurons are present. Both DCN cartwheel cells and cerebellar Purkinje cells are known to fire both tall single action potentials and complexes of smaller wider action potentials. It is therefore possible that the recordings shown here were made from these neuron types. TTX (1 microM) abolished both the tall single and the complexes of smaller action potentials, suggesting that the tall single action potentials are sodium dependent and possibly that a TTX sensitive sodium channel is responsible for the plateau as is suggested for Purkinje cells in the cerebellum. Verapamil (100 microM) abolished only the complex action potentials and the plateau leaving the tall narrow action potentials intact, which is consistent with the smaller complexes being calcium dependent. Higher concentrations abolished all spiking activity. TEA and 4-AP used separately both caused marked depolarisation to around -20 mV, suggesting that there is a large potassium current active at and near resting.     

Electrical membrane properties of trapezoid body neurons in the rat auditory brain stem are preserved in organotypic slice cultures

The medial nucleus of the trapezoid body (MNTB) is a conspicuous structure in the mammalian auditory brain stem. It is a major component of the superior olivary complex and is involved in sound localization. Recently, organotypic slice culture preparations of the superior olivary complex were introduced to investigate the development of inhibitory and excitatory projections (Sanes and Hafidi, 1996; Lohmann et al., 1998). In the present article, we further assessed the organotypicity of our culture system (Lohmann et al., 1998) and examined electrical membrane properties of MNTB neurons expressed under culture conditions. To do so, MNTB neurons from early postnatal rats (P3-5) were studied after 3-6 days in vitro (DIV) by whole-cell patch-clamp recordings. Their mean resting potential was -59 mV, the input resistance averaged 171 Momega, and the average time constant was 3 ms. Four types of voltage-activated conductances were observed in voltage-clamp recordings. All cells expressed a tetrodotoxin (TTX)-sensitive sodium current. Two types of potassium currents could be characterized: a tetraethylammonium (TEA) -sensitive and a 4-aminopyridine (4-AP)-sensitive conductance, both of which are composed of a transient and a sustained component. Finally, an inwardly rectifying current, activated by hyperpolarizing voltage steps, was found. In current-clamp recordings, depolarizing current pulses typically elicited a single action potential. In the presence of 4-AP, however, these current pulses induced a train of action potentials. The duration of action potentials was increased by 4-AP and the afterhyperpolarization was reduced. Hyperpolarizing current injections induced a "sag" in the membrane potential, indicating the influence of an inwardly rectifying current. Our results demonstrate that MNTB neurons in slice cultures have electrical membrane properties comparable to those of their counterparts in acute slices.     

Isolation and characterization of a persistent potassium current in neostriatal neurons

1. Depolarization-activated, calcium-independent potassium (K+) currents were studied with the use of whole cell voltage-clamp recording from neostriatal neurons acutely isolated from adult (> or = 4 wk old) rats. The whole cell K+ current was composed of transient and persistent components. The aims of the experiments were to isolate the persistent component and then to characterize its voltage dependence and kinetics. 2. Application of 10 mM 4-aminopyridine (4-AP) completely blocked the transient currents while reducing the persistent current by approximately 40% [50% inhibitory concentration (IC50), of blockable current = 125 microM]. The persistent K+ current also was reduced by tetraethylammonium (TEA). Two components to the TEA block were present, having IC50s of 125 microM (23% of the blockable current) and 5.9 mM (77% of the blockable current). Collectively, these results suggested that the persistent components of the total K+ current was pharmacologically heterogeneous. The properties of the 4-AP-resistant, persistent K+ current (IKrp) were subsequently studied. 3. The kinetics of activation and deactivation of IKrp were voltage dependent. Examination of the entire activation/deactivation time constant profile showed that it was bell shaped, with time constants being moderately rapid (tau approximately 50 ms) at membrane potentials corresponding to the resting potential of neostriatal cells (approximately -80 mV), becoming considerably longer (tau approximately 100 ms) at potentials near the cells' spike thresholds (approximately -45 mV), and decreasing to a minimum (tau approximately 5 ms) at potentials associated with the peak of the cells' action potentials (approximately +20 mV). The inactivation kinetics of IKrp also were voltage dependent. The time constants of inactivation varied between 1 and 8 s at potentials between -10 and +35 mV. 4. Unlike persistent K+ currents in many other cell types, IKrp activated at relatively hyperpolarized membrane potentials (approximately -70 mV). The Boltzmann function describing activation had a half-activation voltage of -13 mV and a slope factor of 12 mV. In addition, the Boltzmann function describing the voltage dependence of inactivation of IKrp had a relatively depolarized half-inactivation voltage of -55 and a large slope factor of 19 mV, indicating that this current was available over a broad range of membrane potentials (between -100 and -10 mV). 5. Neostriatal neurons recorded in vivo exhibit subthreshold shifts in membrane potential of variable duration (tens of ms to s) from a hyperpolarized resting state to a depolarized state that is limited in amplitude just below spike threshold. The voltage dependence of activation and inactivation of IKrp indicates that it will be available on depolarization from the hyperpolarized state. However, the slow activation rate of this current suggests that it will contribute little either to limiting the amplitude of the initial depolarization associated with entry into the depolarized state or to depolarizing episodes of short duration (e.g., < 50 ms). However, IKrp should limit the amplitude of membrane depolarizations associated with prolonged excursions into the depolarized state.     

Firing properties of spherical bushy cells in the anteroventral cochlear nucleus of the gerbil

In gerbils, spherical bushy cells (SBCs) encode low frequency sound signals into a temporal firing pattern. To investigate the support for the timing in this temporal code, we characterized the membrane electrical properties of visually identified SBCs in brainstem slices. A brief depolarizing subthreshold transient potential (TP) triggered, with relatively invariant latency, a single spike at the onset of a response to depolarizing current pulses. The activation of a subthreshold Na+-conductance, sensitive to blockade with tetrodotoxin, and a high threshold Ca2+-conductance, sensitive to blockade with Co2+ or Cd2+, accelerated the rising phase and amplified the TP. A K+-conductance, sensitive to blockade by 4-aminopyridine (4-AP, 50 microM), shaped the decay of the TP. Following a single spike, voltage-gated activation of transient and sustained K+-conductances suppressed any tendency to repetitively discharge. A reduction in either K+-conductance due to application of 4-AP or tetraethylammonium (TEA, 10 mM), converted the single spike mode to repetitive firing during the depolarizing pulses. A persistent, tetrodotoxin-sensitive Na+-conductance amplified steady-state depolarizing responses. A hyperpolarization-activated conductance, greatly decreased by extracellular Cs+ (3 mM) but resistant to Ba2+ (up to 1 mM), filtered the responses to hyperpolarizing current inputs. A depolarized membrane potential promoted repetitive firing in SBCs. This state, expected in pathophysiological conditions, would corrupt the temporal code.     

Three kinetically distinct Ca2+-independent depolarization-activated K+ currents in callosal-projecting rat visual cortical neurons

Three kinetically distinct Ca2+-independent depolarization-activated K+ currents in callosal-projecting rat visual cortical neurons. J. Neurophysiol. 78: 2309-2320, 1997. Whole cell, Ca2+-independent, depolarization-activated K+ currents were characterized in identified callosal-projecting (CP) neurons isolated from postnatal day 7-16 rat primary visual cortex. CP neurons were identified in vitro after in vivo retrograde labeling with fluorescently tagged latex microbeads. During brief (160-ms) depolarizing voltage steps to potentials between -50 and +60 mV, outward K+ currents in these cells activate rapidly and inactivate to varying degrees. Three distinct K+ currents were separated based on differential sensitivity to 4-aminopyridine (4-AP); these are referred to here as IA, ID, and IK, because their properties are similar (but not identical) K+ currents termed IA, ID, and IK in other cells. The current sensitive to high (>/=100 mu M) concentrations of 4-AP (IA) activates and inactivates rapidly; the current blocked completely by low (相似文献   

Developmental stages of clinical research programs, Part I

Whole-cell, voltage-clamp recordings were made from rat cerebellar granule cells in culture under experimental conditions designed to study voltage-gated Na+ currents that were elicited by depolarizing commands from a holding potential of -60 mV up to +20 mV. These tetrodotoxin-sensitive inward currents were reduced in a dose-related manner by bath application of the structurally novel, anticonvulsant drug topiramate (10-1000 microM; n = 16). Dose-response analysis of this effect revealed an IC50 of 48.9 microM. Topiramate also made the steady-state inactivation curve of this current shift toward more negative values (midpoint of the inactivation curve -46.9 mV under control conditions and -56.5 mV during topiramate application; n = 5). We propose that these effects may contribute to control the sustained depolarizations with repetitive firing of action potentials that occur within neuronal networks during seizure activity. Therefore they may represent a mechanism of action for this novel anticonvulsant drug.     

Modulation of multiple potassium currents by metabotropic glutamate receptors in neurons of the hypothalamic supraoptic nucleus

We studied the effects of activation of the metabotropic glutamate receptors on intrinsic currents of magnocellular n urons of the supraoptic nucleus (SON) with whole cell patch-clamp and conventional intracellular recordings in coronal slices (400 micron) of the rat hypothalamus. Trans-(+/-)-1-amino-1,3-cyclopentane dicarboxylic acid (trans-ACPD, 10-100 microM), a broad-spectrum metabotropic glutamate receptor agonist, evoked an inward current (18.7 +/- 3.45 pA) or a slow depolarization (7.35 +/- 4.73 mV) and a 10-30% decrease in whole cell conductance in approximately 50% of the magnocellular neurons recorded at resting membrane potential. The decrease in conductance and the inward current were caused largely by the attenuation of a resting potassium conductance because they were reduced by the replacement of intracellular potassium with an equimolar concentration of cesium or by the addition of potassium channel blockers to the extracellular medium. In some cells, trans-ACPD still elicited a small inward current after blockade of potassium currents, which was abolished by the calcium channel blocker, CdCl2. Trans-ACPD also reduced voltage-gated and Ca2+-activated K+ currents in these cells. Trans-ACPD reduced the transient outward current (IA) by 20-70% and/or the IA-mediated delay to spike generation in approximately 60% of magnocellular neurons tested. The cells that showed a reduction of IA generally also showed a 20-60% reduction in a voltage-gated, sustained outward current. Finally, trans-ACPD attenuated the Ca2+-dependent outward current responsible for the afterhyperpolarization (IAHP) in approximately 60% of cells tested. This often revealed an underlying inward current thought to be responsible for the depolarizing afterpotential seen in some magnocellular neurons. (RS)-3,5-dihydroxyphenylglycine, a group I receptor-selective agonist, mimicked the effects of trans-ACPD on the resting and voltage-gated K+ currents. (RS)-alpha-methyl-4-carboxyphenylglycine, a group I/II metabotropic glutamate receptor antagonist, blocked these effects. A group II receptor agonist, 2S,1'S,2'S-2carboxycyclopropylglycine and a group III receptor agonist, (+)-2-amino-4-phosphonobutyric acid, had no effect on the resting or voltage-gated K+ currents, indicating that the reduction of K+ currents was mediated by group I receptors. About 80% of the SON cells that were labeled immunohistochemically for vasopressin responded to metabotropic glutamate receptor activation, whereas only 33% of labeled oxytocin cells responded, suggesting that metabotropic receptors are expressed preferentially in vasopressinergic neurons. These data indicate that activation of the group I metabotropic glutamate receptors leads to an increase in the postsynaptic excitability of magnocellular neurons by blocking resting K+ currents as well as by reducing voltage-gated and Ca2+-activated K+ currents.     

Comprehensive study on the membrane currents of porcine granulosa cells in culture

The membrane currents of primary cultured porcine granulosa cells have been studied using the whole-cell configuration of the patch-clamp technique. And effects of K+ channel blockers upon progesterone production of the cells have been also studied. The author has identified and characterized two types of K+ currents, transient outward current (Ito) and a delayed rectifier K+ current (Ik), and Ca2+ current (Ica). Ito and Ik were voltage -and calcium-dependent. Both of the currents were blocked by 4-aminopyridine (4-AP), a K+ channel blocker, but only Ik was sensitive to tetraethylammonium (TEA), another K+ channel blocker. Ica was inactivated within 50 ms of the test pulse. Nifedipine and verapamil, L-type Ca2+ channel blockers, did not suppress Ica even at a concentration of 10 microM. Tetramethrin (1 microM), a T-type Ca2+ channel blocker, decreased Ica. These findings suggested that the current was T-type Ca2+ current. LH and dibutyryl cAMP, potent stimulants of steroid production, attenuated Ito by 13.9 +/- 1.8% (n = 7) and 21.0 +/- 1.5% (n = 4), respectively. However, they did not affect Ik and Ica. These results indicated that LH did not modulate Ca2+ current directly, but it suppressed Ito through cAMP. 4-AP (0.2-5 mM) suppressed basal and LH-induced progesterone production of porcine granulosa cells dose-dependently, but TEA (2-10 mM) did not influence progesterone production. These data suggest that Ito may play a role in steroid secretion or other functions in granulosa cells.     

Control of the delayed outward potassium currents in bursting pace-maker neurones of the snail, Helix pomatia

The net outward current in bursting pace-maker neurones of the snail (Helix pomatia) during sustained and repeated voltage clamp pulses was studied. The properties of currents remaining in cobalt-Ringer or after TEA injection were compared with those in untreated cells. 2. With sustained voltage clamp depolarizations the net outward current first increases to a maximum at 150 msec and then declines to 60% or less of its peak intensity. This depression, which is greater during repetition of short pulses (e.g. 100 msec pulses at 0-5 sec intervals), represents a true decrease in the outward flow of K (designated IK) and is not due to a decreased driving force resulting from extracellular K accumulation. The steady-state current-voltage (I-V) relationship for IK is N-shaped (Heyer & Lux, 1976). 3. A component of IK persists when Ca and Mg in the medium are replaced by Co (ICo-res). With voltage clamp depolarizations ICo-res increases rapidly to a maximum and then partially inactivates with voltage dependent time constants of hundredths or tenths of seconds. Repolarization removes the inactivation. Thus, repeated stimulation with short pulses does not increase the depression of ICo-res-ICo-res (e.g. measured during voltage steps from holding potentials of -50 to near 0 mV) is smaller in test pulses preceded by depolarization and larger in pulses preceded by hyperpolarization. The steady state I-V relationship is not N-shaped. ICo-res is blocked by intracellular injection of tetraethylammonium (TEA). 4. Repeated voltage clamp depolarization to near 0 mV with 100 msec pulses for neurones with large Ca currents in normal Ringer produces a long-term depression which is maximal with 300-400 msec repolarizations (to -50 mV) between pulses. This corresponds with stimulus parameters for the maximum Ca current (Heyer & Lux, 1976). Intracellular injection of Ca2+ (also Ba2+ and Co2+) likewise reduces the total net outward current and especially the delayed outward current under voltage clamp. 5. The component of IK which is removed by Co is identified as Ca dependent and designated IK(Ca). With single voltage clamp pulses IK(Ca) follows the approximate time course and voltage dependence of the slow inward Ca current (Iin slow; Heyer & Lux, 1976). Several lines of evidence suggest that Ca ions moving through the membrane activate IK(Ca). 6. Part of IK cannot be blocked by intracellular TEA injection. In different neurones the magnitude of the IK component resistant to TEA (ITEA-res) is approximately proportional to the relative magnitudes of Iin slow.ITEA-res does not inactivate with sustained depolarization and shows pronounced long-term depression with repetitive stimulation at intermediate intervals and an increased outward current at the onset of the second and subsequent pulses following short repolarizations. The steady-state I-V relationship is N-shaped. ITEA-res is abolished by extracellular Co. 7. A net inward current with low depolarizations can be measured after TEA injection...     

Selective modulation of GABAA receptors by aluminum

Aluminum has been implicated in several neurodegenerative conditions including Alzheimer's disease. Because the mammalian olfactory system has an unusual capacity for the uptake and transneuronal spread of inhaled substances such as aluminum, whole cell recording techniques were used to examine the actions of aluminum on basic membrane properties and amino acid receptors on rat olfactory bulb mitral/tufted (M/T) neurons in culture. Aluminum had little direct effects on M/T neurons. Aluminum (100 microM) did not evoke a membrane current or alter action-potential shape or duration. Aluminum also had no marked effects on the family of voltage-gated membrane currents evoked by a series of 10-mV, 50-ms depolarizing steps. However, aluminum dramatically potentiated the current evoked by 30 microM gamma-aminobutyric acid (GABA) at concentrations <100 microM. Conversely, higher concentrations of aluminum blocked the GABA-evoked current. The effects of aluminum on GABA-evoked currents were not voltage dependent. Aluminum (100 microM) equally potentiated both inward currents at -30 mV and outward currents at + 30 mV. At 300 microM, aluminum blocked both inward and outward currents to a similar extent. In some neurons, aluminum only blocked the current and potentiation was not observed. The biphasic action of aluminum on GABA-evoked currents suggests separate binding sites: a high-affinity potentiating site and a low-affinity inhibiting site. Despite its effects on GABA-evoked currents, aluminum did not alter membrane currents evoked by glutamate, N-methyl-D-aspartate, kainate, or glycine. Aluminum also did not reduce spontaneous excitatory synaptic activity, suggesting little, if any, effect on glutamate release. Although a causal role for aluminum in Alzheimer's disease and other neuropathological conditions remains controversial, it is clear that elevated aluminum concentrations in the brain are associated with a variety of cognitive impairments. The present results indicate that aluminum can alter the function of GABAA receptors and may suggest that aluminum can contribute to cognitive impairment through disruption of inhibitory circuits.     

Metabotropic glutamate receptor inhibition of visceral afferent potassium currents

The effects of metabotropic glutamate receptor activation (mGluR) on voltage-gated potassium currents have been characterized in visceral sensory afferent neurons. L-Glutamate is known to be a primary neurotransmitter in visceral afferents which terminate at the level of the nucleus of the solitary tract (NTS). Synaptic communication between these afferents and the NTS has been shown to involve both postsynaptic ionotropic and presynaptic metabotropic glutamate receptor activation. The purpose of the present study was to determine the effects of mGluR activation on voltage-gated potassium currents in visceral sensory neurons. Application of mGluR agonist t-ACPD inhibited both the peak and the steady state voltage-gated potassium current in 39 out of 56 visceral afferent neurons tested (70%) by 22.0 +/- 3 and 22.8 +/- 2%, respectively. Voltage and pharmacological protocols were utilized to isolate the potassium current affected by mGluR activation. Increasing the holding potential from -100 mV to -30 mV only partially attenuated the inhibitory effects of t-ACPD (decreased effect by 11%), suggesting that t-ACPD modulates both a voltage insensitive and a voltage-sensitive potassium current. In addition, 4-aminopyridine (5 microM) was applied to eliminate the 4-AP sensitive transient current. Also, this protocol only partially attenuated the inhibitory effects of t-ACPD (decreased effect by 6.3%), suggesting that mGluR activation inhibits both a 4-AP-sensitive and 4-AP-insensitive potassium current in visceral afferent neurons. Results from this study suggest that mGluRs may regulate visceral sensory afferent neuronal activity through inhibition of voltage-gated potassium channels.     

Occupational health services and OSHA compliance

Two different types of action potentials were observed among the pyramidal cells and interneurons in cat motor cortex: the narrow action potentials and the wide action potentials. These two types of action potentials had similar rising phases (528.8 +/- 77.0 vs 553.1 +/- 71.8 mV/ms for the maximal rising rate), but differed in spike duration (0.44 +/- 0.09 vs 1.40 +/- 0.39 ms) and amplitude (57.31 +/- 8.22 vs 72.52 +/- 8.31 mV), implying that the ionic currents contributing to repolarization of these action potentials are different. Here we address this issue by pharmacological manipulation and using voltage-clamp technique in slices of cat motor cortex. Raising extracellular K+ concentration (from 3 mM to 10 mM), applying a low dose of 4-aminopyridine (2-200 microM) or administering a low concentration of tetraethylammonium (0.2-1.0 mM) each not only broadened the narrow action potentials, but also increased their amplitudes. In contrast, high K+ medium or low dose of tetraethylammonium only broadened the wide action potentials, leaving their amplitudes unaffected, and 4-aminopyridine had only a slight broadening effect on the wide spikes. These results implied that K+ currents were involved in the repolarization of both types of action potentials, and that the K+ currents in the narrow action potentials seemed to activate much earlier than those in the wide spikes. This early activated K+ current may counteract the rapid sodium current, yielding the extremely brief duration and small amplitude of the narrow spikes. The sensitivity of the narrow spikes to 4-aminopyridine may not be mainly attributed to blockade of the classical A current (IA), because depolarizing the membrane potential to inactivate IA did not reproduce the effects of 4-aminopyridine. Blockade of Ca2+ influx slowed the last two-thirds repolarization of the wide action potentials. On the contrary, the narrow action potentials were not affected by Ca(2+)-current blockers, but if they were first broadened by 4-aminopyridine or tetraethylammonium, subsequent application of Ca(2+)-free medium caused further broadening, suggesting that the narrow action potentials were too brief to activate the Ca(2+)-activated potassium currents for their repolarization. Therefore, the effects of low concentrations of tetraethylammonium on the narrow spikes appeared to be mainly due to blockade of an outward current that was different from the tetraethylammonium-sensitive Ca(2+)-activated potassium current (IC). In the neurons with the narrow spikes, voltage-clamp experiments revealed two voltage-gated outward currents that were sensitive to tetraethylammonium and 4-aminopyridine, respectively. Both currents were activated rapidly following the onset of depolarizing steps. Interestingly, the tetraethylammonium-sensitive current was a transient outward current that inactivated rapidly (tau < or = 5 ms), while the 4-aminopyridine-sensitive current was relatively persistent during maintained depolarization. The 4-aminopyridine-sensitive current did not show obvious inactivation even at membrane potential of -40 mV, which completely inactivated the transient tetraethylammonium-sensitive, current. The results indicate that different potassium currents are involved in the repolarization of the narrow and wide action potentials in cat motor cortex. A novel tetraethylammonium-sensitive transient outward current and a 4-aminopyridine-sensitive outward current are responsible for the short duration and small amplitude of the narrow action potentials in the interneurons and some of the layer V pyramidal cells. These two currents are voltage-gated and Ca(2+)-independent. For the wide action potentials that characterize most pyramidal neurons, a Ca(2+)-independent tetraethylammonium-sensitive outward current and a Ca(2+)-activated potassium current are the main contributors to their repolarization.     

Mutagenicity and mutation spectra of 2-acetylaminofluorene at frameshift and base-substitution alleles in four DNA repair backgrounds of Salmonella

Neurons in the superior vagal (jugular) ganglion relay afferent information from thoracic visceral organs and may be important in inflammatory processes due to the peripheral release of bioactive neuropeptides such as substance P. We characterized the excitable properties and underlying voltage-gated Na+ (INa) and K+ (IKv) currents in acutely dissociated guinea pig jugular ganglion neurons with microelectrode and whole-cell patch-clamp recording techniques. Current clamp recordings revealed a resting potential of approx. -55 mV and input resistance of approx. 100 M ohms. Brief depolarizing steps evoked an overshooting action potential (approx. 2 ms duration), fast (< 20 ms duration) afterhyperpolarization (AHPF) sequence in all neurons, followed by a slow (> 1 s) Cd(2+)-sensitive afterhyperpolarization (AHPS) in 45% of the neurons. The AHPS was implicated in limiting repetitive action potential firing during maintained depolarizing steps. The action potential in 15/17 neurons, and a major component of the whole cell INa in 13/13 neurons were insensitive to TTX (1-10 microM), indicating that jugular neurons express predominantly a TTX-resistant type of INa. Cd2+ (200 microM) did not affect action potential repolarization, while tetraethylammonium (TEA; 10 mM) in the presence of Cd2+ markedly prolonged action potential repolarization, and blocked the AHPF in 11/11 neurons. This suggested that the action potential repolarization and the AHPF are mediated by IKv, with little contribution by Ca(2+)-dependent IK (IK(Ca)). Whole cell IKv activated rapidly (tau < 1.5 ms), and inactivated variably over a time period of seconds. IKv activation and inactivation voltage dependencies and TEA sensitivity were compatible with its availability during the action potential and AHPF. Only 1/26 neurons exhibited current with the rapid inactivation kinetics and voltage-dependencies characteristic of classic IA-type current. These results highlight differences in the properties of jugular neurons (e.g., deficiency of rapid IA, and lack of a TTX-sensitive subpopulation), relative to those known for other visceral and somatic afferents, and thus provide a basis for further functional studies.     

Voltage-clamp analysis and computer simulation of a novel cesium-resistant A-current in guinea pig laterodorsal tegmental neurons

Increased firing of cholinergic neurons of the laterodorsal tegmental nucleus (LDT) plays a critical role in generating the behavioral states of arousal and rapid eye movement sleep. The majority of these neurons exhibit a prominent transient potassium current (IA) that shapes firing but the properties of which have not been examined in detail. Although IA has been reported to be blocked by intracellular cesium, the IA in LDT neurons appeared resistant to intracellular cesium. The present study compared the properties of this cesium-resistant current to those typically ascribed to IA. Whole cell recordings were obtained from LDT neurons (n = 67) in brain slices with potassium- or cesium-containing pipette solutions. A transient current was observed in cells dialyzed with each solution (KGluc-85%; CsGluc-79%). However, in cesium-dialyzed neurons, the transient current was inward at test potentials negative to about -35 mV. Extracellular 4-aminopyridine (4-AP; 2-5 mM) blocked both inward and outward current, suggesting the inward current was reversed IA rather than an unmasked transient calcium current as previously suggested. This conclusion was supported by increasing [K]o from 5 to 15 mM, which shifted the reversal potential positively for both inward and outward current (+17.89 +/- 0.41 mV; mean +/- SE). Moreover, recovery from inactivation was rapid (tau = 15.5 +/- 4 ms; n = 4), as reported for IA, and both inward and outward transient current persisted in calcium-free solution [0 calcium/4 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N', N'-tetraacetic acid; n = 4] and during cadmium-blockade of calcium currents (n = 3). Finally, the transient current was blocked by intracellular 4-AP indicating that adequate dialysis occurred during the recordings. Thus the Cs-resistant current is a subthreshold IA. We also estimated the voltage-dependence of activation (V1/2 = -45.8 +/- 2 mV, k = 5.21 +/- 0.62 mV, n = 6) and inactivation (V1/2 = -59. 0 +/- 2.38 mV, k = -5.4 +/- 0.49 mV, n = 3) of this current. Computer simulations using a morphologically accurate model cell indicated that except for the extreme case of only distal A-channels and a high intracellular resistivity, our parameter estimates were good approximations. In conclusion, guinea pig LDT neurons express subthreshold A-channels that are resistant to intracellular cesium ions. This suggests that these channels differ fundamentally in their ion permeation mechanism from those previously studied. It remains to be determined if Cs+ resistance is common among brain A-channels or if this property is conferred by known A-channel subunits.