Hyperpolarization-activated inward current in neurons of the rat's dorsal nucleus of the lateral lemniscus in vitro

Hyperpolarization-activated inward current in neurons of the rat's dorsal nucleus of the lateral lemniscus in vitro. J. Neurophysiol. 78: 2235-2245, 1997. The hyperpolarization-activated current (Ih) underlying inward rectification in neurons of the rat's dorsal nucleus of the lateral lemniscus (DNLL) was investigated using whole cell patch-clamp techniques. Patch recordings were made from DNLL neurons of young rats (21-30 days old) in 400 micro;m tissue slices. Under current clamp, injection of negative current produced a graded hyperpolarization of the cell membrane, often with a gradual sag in the membrane potential toward the resting value. The rate and magnitude of the sag depended on the amount of hyperpolarizing current. Larger current resulted in a larger and faster decay of the voltage. Under voltage clamp, hyperpolarizing voltage steps elicited a slowly activating inward current that was presumably responsible for the sag observed in the voltage response to a steady hyperpolarizing current recorded under current clamp. Activation of the inward current (Ih) was voltage and time dependent. The current just was seen at a membrane potential of -70 mV and was activated fully at -140 mV. The voltage value of half-maximal activation of Ih was -78.0 +/- 6.0 (SE) mV. The rate of Ih activation was best approximated by a single exponential function with a time constant that was voltage dependent, ranging from 276 +/- 27 ms at -100 mV to 186 +/- 11 ms at -140 mV. Reversal potential (Eh) of Ih current was more positive than the resting potential. Raising the extracellular potassium concentration shifted Eh to a more depolarized value, whereas lowering the extracellular sodium concentration shifted Eh in a more negative direction. Ih was sensitive to extracellular cesium but relatively insensitive to extracellular barium. The current amplitude near maximal-activation (about -140 mV) was reduced to 40% of control by 1 mM cesium but was reduced to only 71% of control by 2 mM barium. When the membrane potential was near the resting potential (about -60 mV), cesium had no effect on the membrane potential, current-evoked firing rate and input resistance but reduced the spontaneous firing. When the membrane potential was more negative than -70 mV, cesium hyperpolarized the cell, decreased current-evoked firing and increased the input resistance. Ih in DNLL neurons does not contribute to the normal resting potential but may enhance the extent of excitation, thereby making the DNLL a consistently powerful inhibitory source to upper levels of the auditory system.     

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.     

Bursting and tonic discharges in two classes of reticular thalamic neurons

1. Two types of cat reticular (RE) thalamic cells were disclosed by means of intracellular recordings under urethan anesthesia. The RE neurons were identified by their typical depolarizing spindle oscillations in response to synchronous stimulation of the internal capsule. 2. In type I neurons (n = 41), depolarizing current pulses induced tonic firing at the resting or slightly depolarized membrane potential (Vm) and triggered high-frequency spike bursts at a Vm more negative than -75 mV. As well, these cells discharged rebound bursts at the break of a hyperpolarizing current pulse. Internal capsule stimulation elicited spindle sequences made off by depolarizing waves giving rise to spike bursts. 3. Type II cells (n = 9) did not discharge spike bursts to large depolarizing current pulses even when the Vm reached -100 mV, nor did they fire rebound bursts after long-lasting hyperpolarizing current pulses or spike bursts riding on the rhythmic depolarizing components of spindle sequences. 4. Compared with type I cells, type II cells showed less frequency accommodation during tonic firing. The latter neuronal class discharged at high frequencies (40 Hz) with slight DC depolarization, approximately 8-10 Hz at the resting Vm, and no underlying synaptic or subthreshold oscillatory events could be detected when the firing was blocked by DC hyperpolarization. 5. The presence of two cell classes in the RE nucleus challenges the common view that this nucleus consists of a single neuronal class. We suggest that a different set of conductances is present in type II RE neurons, thus preventing the low-threshold Ca2+ current from dominating the behavior of these cells.     

Dynamic clamp study of Ih modulation of burst firing and delta oscillations in thalamocortical neurons in vitro

The dynamic clamp technique was used in thalamocortical neurons of the rat and cat dorsal lateral geniculate nucleus in vitro to investigate the effects of the hyperpolarization-activated cation current, Ih, and of its neuromodulation on burst firing and delta oscillations. Specific block of endogenous Ih using 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)pyridinium chloride (ZD7288) (300 microM) abolished the depolarizing "sag" response to negative current steps, markedly increased the latency and shortened the duration of the low-threshold Ca2+ potentials, and decreased the number of action potentials in the burst evoked by the low-threshold Ca2+ potential. Subsequent introduction of artificial Ih using the dynamic clamp re-instated the "sag" and all the original properties of the low-threshold Ca2+ potential. In the absence of ZD7288, introduction of artificial outward Ih with the intention of abolishing endogenous Ih removed the depolarizing "sag" and produced similar effects on the low-threshold Ca2+ potentials as those observed during the pharmacological block of Ih. Application of ZD7288 to thalamocortical neurons displaying delta oscillations led to a reduction in the voltage range of their existence or to a complete cessation of this behaviour. A subsequent introduction of artificial Ih re-enabled the generation of delta oscillations. In the presence of ZD7288, physiologically relevant positive shifts in the voltage-dependence of artificial Ih increased the amplitude and duration of the low-threshold Ca2+ potential and increased the likelihood of delta oscillations while negative shifts had opposite effects. These results highlight the important difference between the dependence of burst firing and oscillations on membrane potential and their dependence on the properties of Ih, and demonstrate that the modulation by Ih of low-threshold Ca2+ potentials and burst firing in thalamocortical neurons, as well as the ability of these neurons to generate delta oscillations, is more elaborate than previously described.     

Inhibition of a hyperpolarization-activated current by clonidine in rat dorsal root ganglion neurons

Whole cell voltage- and current-clamp recordings were carried out to investigate the effects of clonidine, an alpha 2-adrenoceptor agonist, in L4 and L5 dorsal root ganglion (DRG) neurons of the rat. In voltage-clamp mode, application of 20 microM clonidine reversibly reduced the inward current evoked by hyperpolarizing voltage steps. The "clonidine-sensitive current" was obtained by subtracting the current during clonidine application from the control current, and its properties were as follows. 1) It was a slowly activating inward current evoked by hyperpolarization. 2) The reversal potential in the standard extracellular solution ([K+]o = 5 mM, [Na+]o = 151 mM) was -38.3 mV, and reduction of [Na+]o shifted it to a more negative potential, whereas an increase of [K+]o shifted it to a more positive potential, indicating that the current was carried by Na+ and K+ (PNa/PK = 0.22). 3) The relationship between the chord conductance underlying the clonidine-sensitive current and voltage could be fitted by a Boltzmann equation. These results indicate that the clonidine-sensitive current corresponds to a hyperpolarization-activated current (Ih), i.e., clonidine inhibits Ih in rat DRG neurons. DRG neurons were classified as small (15.9-32.9 microns diam), medium-sized (33-42.9 microns), and large (43-63.6 microns), and 7 of 19, 24 of 25, and 22 of 22 of these types exhibited Ih with mean +/- SE clonidine-induced inhibition values of 36.1 +/- 3.5% (n = 7), 43.1 +/- 3.7% (n = 24), and 35.1 +/- 2.7% (n = 22), respectively. Clonidine application to L4 and L5 DRG neurons excised from rats the sciatic nerves of which had been transected 14-35 days previously (transected DRG neurons) also reduced Ih. In current-clamp mode, 9 of 13 intact and 4 of 6 transected medium-sized DRG neurons that exhibited Ih responded to clonidine with hyperpolarization (> 2 mV). Some medium-sized DRG neurons exhibited repetitive action potentials in response to a depolarizing current pulse, and clonidine reduced the firing discharge frequencies in 8 of 11 intact and 3 of 4 transected neurons tested. Injection of a hyperpolarizing current pulse produced time-dependent rectification in DRG neurons that exhibited Ih, and clonidine blocked this rectification in all intact and transected neurons tested. These results suggest that inhibition of Ih due to alpha 2-adrenoceptor activation contributes to modulation of DRG neuronal activity in rats. On the basis of our findings, we discuss the possible mechanisms whereby sympathetically released norepinephrine modulates the abnormal activity of DRG neuronal cell bodies after nerve injury.     

Electrophysiological properties and their modulation by norepinephrine in the ambiguus neurons of the guinea pig

Electrophysiological properties of guinea pig ambiguus (AMB) neurons were studied in a brainstem slice preparation. During subthreshold depolarization AMB neurons displayed an early slow depolarization and a late outward rectification both of which were blocked by replacing Ca2+ with Co2+ in the extracellular solution. AMB neurons showed hyperpolarizing inward rectification which was blocked by extracellular Cs+ and is likely caused by the activation of Ih: In 58% (n = 49) of AMB neurons spike firing was restricted to the early phase of a long-lasting depolarizing current injection (phasic firing). The remaining AMB neurons showed repetitive firing throughout the depolarization (tonic firing). A Ca(2+)-mediated K+ current (IK(Ca)) caused an afterhyperpolarization that followed both single and repetitive spike firing. IK(Ca) also controlled the firing pattern in both types of firing, especially in the phasic firing. Norepinephrine (NE) blocked both the hyperpolarizing inward rectification and the Ca(2+)-dependent AHP. These effects of NE were antagonized by propranolol. It is proposed that the blockade of IK(Ca) and Ih contribute to the improvement of the 'signal-to-noise ratio' by NE in AMB neurons.     

Calcium current and inactivation in identified neurons in Hermissenda crassicornis

1. N-type (omega-conotoxin sensitive) calcium currents (ICa) were recorded in identified neurons in Hermissenda crassicornis using low-resistance patch electrodes (0.7 +/- 0.3 M omega; n = 101) under conditions that eliminated inward Na+ currents (choline ions substitution) and suppressed outward K+ currents (Cs+, tetraethylammonium, and 4-AP). Step depolarization from a holding potential of -60 mV to potentials above -30 mV elicited ICa, which peaked approximately 20 mV and declined with increasing depolarizations. 2. Evidence for a low-threshold current was present. Step depolarization from a more hyperpolarizing potentials (e.g., -90 mV) revealed a small shoulder (< 100 pA) at -60 to -40 mV that was sensitive to Co2+ and Ni2+. However, under the conditions examined here (holding potential of -60 mV), the high-voltage-activated current predominated. 3. Barium (Ba2+) and strontium (Sr2+) permeate the Ca2+ channel with similar activation kinetics (ease of permeation; Ba2+ > Ca2+ > Sr2+). Steady-state activation of permeability versus membrane potentials for Ca2+, Ba2+, and Sr2+ as charge carriers could be fitted with the Boltzmann equation, with half-activation voltage and slope factor of 2.9 and 7.7 mV for ICa, -13.1 mV and 7.8 for Ba2+ current (IBa) and -2.3 mV and 7.8 for Sr2+ current (ISr). The time course of activation was monotonic with time constant (tau) for ICa ranging from 2 to 8 ms. 4. The inactivation profile was complex. At negative step potentials (e.g., -20 mV), inactivation of the current was slow. Depolarization steps to relatively positive voltages (e.g., 10 mV) showed more rapid inactivation than those at more positive potentials (e.g., 40 mV). When extracellular Ca2+ was raised from 5 to 10 mM, a biphasic decay (tau fast of 25 +/- 4 ms; and tau slow of 473 +/- 64 ms; mean +/- SD, n = 9) was seen. Such an observation suggested a current-mediated inactivation. 5. With a pulse duration of approximately 350 ms, ISr showed inactivation whereas Ba2+ virtually removed the decay. However, IBa turned off with more prolonged depolarization. 6. A twin-pulse protocol was used to assess the voltage dependence of inactivation: an incomplete U-shaped inactivation curve was observed for ICa, IBa, and ISr. Channels available for inactivation were increased in the presence of Ca2+ ions. 7. Inactivation was further studied with the Ca2+ chelators, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid and bis(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA). With 10 mM of BAPTA, in the pipette, inactivation was reduced but not removed.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Prevalence of extracranial carotid and vertebral artery disease in Chinese patients with coronary artery disease

Electrotonic responses recorded extra- or intracellularly from peripheral nerve preparations show a "sag" to hyperpolarizing current pulses. The biophysical nature of this "inward rectification" is still under discussion since the phenomenon has not been noted at voltage-clamped single nerve fibres, and since Cs+, which reduces inward rectification, is not a specific ion channel blocker. In this study, we found that low micromolar concentrations of ZD 7288, a specific blocker of the hyperpolarization-activated cationic current (Ih) in the soma of central mammalian neurons, result in a complete block of inward rectification in the electrotonic responses of isolated rat spinal dorsal roots. In addition, ZD 7288 enhanced the activity-dependent slowing of conduction seen in compound C fibre action potentials of isolated rat vagus nerves and augmented the post-tetanic hyperpolarization following trains of action potentials in unmyelinated and myelinated axons. The data suggest that ZD 7288 is a potent blocker and a useful research tool for the study of hyperpolarization-activated inward rectification (Ih) of peripheral nerve preparations.     

Synaptically activated low-threshold muscarinic inward current sustains tonic firing in rabbit prevertebral sympathetic neurons

Whole-cell patch-clamp experiments were performed on non-dissociated rabbit coeliac sympathetic neurons in the presence of nicotinic blockers. Coeliac neurons were classified as either silent or spontaneously active (pacemaker) cells. Under voltage-clamp conditions, pacemaker cells exhibited a steady-state N-shaped current-voltage relationship due to the presence of a persistent voltage-dependent inward current in the potential range of -100 to approximately -20 mV. This inward current sustained the regular firing activity of pacemaker cells and was absent from quiescent neurons. It disappeared in the presence of tetrodotoxin and in low Ca(2+)-high Mg2+ external solutions and was enhanced by eserine. Splanchnic nerve stimulation induced slow regenerative depolarizations and firing discharges in silent neurons by activating a low-threshold voltage-sensitive inward current. The synaptic current had a U-shaped voltage-dependence from -96 to approximately -20 mV and exhibited the dynamic properties of the muscarinic voltage-dependent inward current INa,M. It gave the current-voltage relationship an N shape similar to that observed in spontaneously active cells. The muscarinic antagonists atropine and pirenzepine abolished the inward current present in pacemaker cells and that induced by nerve stimulation in silent neurons. These data provide evidence that both spontaneous firing activity and nerve-evoked depolarizing responses in coeliac neurons are sustained by the activation of the muscarinic Na,M current. The tonic activation of INa,M in spontaneously firing cells results from a sustained Ca(2+)-dependent tetrodotoxin-sensitive release of acetylcholine. This study provides evidence that the role of the muscarinic receptors is not purely a neuromodulatory one, but that these receptors are directly involved in ganglionic neurotransmission.     

Therapeutic options provided by new antiepileptic drugs]

Intracellular recordings were obtained from antidromically identified motoneurons in an embryonic chick spinal cord slice preparation at two developmental stages (embryonic days 12 and 18, E12 and E18) which bracket a critical period in spinal cord growth. The resting membrane potential of chick motoneurons did not change significantly between E12 and E18, but there was a significant decrease in neuronal input resistance. A small inward rectification was present in cells of both ages, although a lower proportion of E12 motoneurons exhibited inward rectification compared to E18 motoneurons. Injection of depolarizing current pulses revealed that most E12 motoneurons exhibited spike adaptation, while the majority of E18 motoneurons showed high frequency tonic firing. Bath application of serotonin (5-HT) and its agonists 5-carboxamido-tryptamine (5-CT, a 5-HT1 agonist) and alpha-methyl 5-HT (a 5-HT2 agonist) produced hyperpolarizing responses accompanied by decreased input resistance in all E12 motoneurons studied. The same three agonists produced depolarizing responses and increased input resistance in all E18 motoneurons studied. The effects of serotonergic agonists on motoneuronal excitability were tested using depolarizing current pulses. In most cases, serotonergic agonists caused a decrease in firing frequency during the hyperpolarizing response in E12 neurons. At E18, bath application of 5-HT, 5-CT or alpha-methyl 5-HT produced an increase in firing frequency in all motoneurons during the depolarizing response. Our results indicate that both 5-HT1 and 5-HT2 receptor subtypes contribute to modulation of chick motoneuron excitability and appear to reverse the polarity of their effects on membrane potential after a critical period in development of the spinal cord.     

Endoscopic features of the columnar-lined esophagus

Electrophysiological characterization of neurons within the rat subiculum was carried out with intracellular recordings in an in vitro slice preparation. Subicular neurons responded to threshold pulses of depolarizing current delivered at a resting membrane potential (RMP) of 45.7+/-5.8 mV (mean+/-SD, n=85) with an initial burst of three to five fast action potentials that rode on a depolarizing envelope and was terminated by an afterhyperpolarization (burst AHP) (duration 113+/-35 ms; peak amplitude 2.7+/-0.6 mV, n=10). Tonic firing replaced the bursting mode at membrane potential less negative than -55 mV. Suprathreshold depolarizing pulses evoked at RMP both an initial burst and successive tonic firing. Intracellular staining with biocytin showed morphological features typical of pyramidal cells (n=8). The relationship between frequency of repetitive firing and injected current (f-I) revealed that the burst firing frequency (250-300 Hz) was only slightly influenced by the amount of injected current. By contrast, the f-I curve of the tonic firing phase depended upon current intensity: it displayed an initial segment that increased at first linearly and then turned into a plateau for both the early and the late inter-spike intervals. The frequency of the tonic firing declined only slightly with time, thus suggesting a lack of adaptation. During tonic firing, each single action potential was followed by a fast AHP and a depolarizing afterpotential. Termination of repetitive firing was followed by an AHP (spike-train AHP; duration 223+/-101 ms, peak amplitude 5.6+/-2.4 mV, n=17). Fast spike-train and burst AHPs were reduced by bath application of the Ca2+-channel blockers Co2+ (2 mM) and Cd2+ (1 mM) (n=8), thus suggesting the participation of Ca2+-dependent K+ conductances in these AHPs. Subicular bursting neurons generated persistent, subthreshold voltage oscillations at 5.3+/-1 Hz (n=20) during steady depolarization positive to -60 mV; at values positive to -55 mV, the oscillatory activity could trigger clusters of single action potentials with a periodicity of 0.9-2 Hz. Oscillations were not prevented by application of excitatory amino acid receptor and GABA(A) receptor antagonists (n=5), Ca2+-channel blockers (n=5), or Cs+ (3 mM; n=4), but were abolished by the Na+-channel blocker tetrodotoxin (1 microM; n=6). Our findings demonstrate that pyramidal-like subicular neurons generate both bursting and non-adapting tonic firing, depending upon their membrane potential. These neurons also display oscillatory activity in the range of theta frequency that depends on the activation of a voltage-gated Na+ conductance. These electrophysiological properties may play a role in the process of signals arising from the hippocampal formation before being funnelled towards other limbic structures.     

Electrophysiological characterization of different types of neurons recorded in vivo in the motor cortex of the cat. II. Membrane parameters, action potentials, current-induced voltage responses and electrotonic structures

1. Electrical properties of four functional classes [inactivating bursting (ib), noninactivating bursting (nib), fast spiking (fsp), and regular spiking (rsp)] of neurons in the motor cortex of conscious cats were studied with the use of intracellular voltage recording and single-electrode voltage-clamp (SEVC) techniques. Evaluations were made of action potentials and afterpotentials, current-voltage (I-V) relationships, and passive cable properties. Values of membrane potential (Vm), input resistance (RN), membrane time constant (T0), and firing threshold (T50) were also measured. The data were used to extend the electrophysiological classifications of neurons described in the companion paper. 2. Average values of Vm (from -63 to -66 mV), action-potential amplitudes (from 72 to 77 mV), and firing threshold (-54 mV) were not statistically different in different types of neurons. However, the magnitude of intracellularly injected depolarizing current required to induce spike discharge at 50% probability varied significantly (from 0.6 to 1.1 nA) among cell types. The mean RN and T0 measured at Vm varied between 8.3 and 19.8 M omega, and 7.2 and 15.1 ms, respectively, in the cell classes. 3. Action potentials were overshooting. Their mean duration at half amplitude varied from 0.25 to 0.73 ms among different cell types. Three types of action-potential configurations were distinguished. Type I action potentials found in nib and rsp neurons were relatively fast and had a depolarizing afterpotential (DAP) as well as fast and slow after hyperpolarizations (fAHPs, sAHPs). Type II action potentials found in ib and rsp cells had relatively slow rise and decay phases, DAPs, and sAHPs. Their fAHPs were small or absent. Type III action potentials were found exclusively in fsp cells, had very short durations, prominent fAHPs, but no sAHPs. 4. Steady-state I-V relationships were determined by measuring voltage responses to 0.2- to 1.0-nA hyperpolarizing, rectangular current pulses at different membrane potentials. Both RN and T0 exhibited nonlinear behavior over wide ranges of membrane potential; however, between -65 and -75 mV, the I-V relationships varied little, and they appeared constant in most cells. The steady-state values of RN increased with decreasing, and decreased with increasing the membrane potential in all but fsp cells. The I-V relationships were virtually linear in fsp neurons. 5. Transient I-V relationships were studied by measuring voltage responses to depolarizing and hyperpolarizing, rectangular current pulses of increasing amplitude from a preset membrane potential of -70 mV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Inactivation of macroscopic late Na+ current and characteristics of unitary late Na+ currents in sensory neurons

Na+ currents in adult rat large dorsal root ganglion neurons were recorded during long duration voltage-clamp steps by patch clamping whole cells and outside-out membrane patches. Na+ current present >60 ms after the onset of a depolarizing pulse (late Na+ current) underwent partial inactivation; it behaved as the sum of three kinetically distinct components, each of which was blocked by nanomolar concentrations of tetrodotoxin. Inactivation of one component (late-1) of the whole cell current reached equilibrium during the first 60 ms; repolarizing to -40 or -50 mV from potentials of -30 mV or more positive gave rise to a characteristic increase in current (tau >/= 5 ms), attributed to removal of inactivation. A second component (late-2) underwent slower inactivation (tau > 80 ms) at potentials more positive than -80 mV, and steady-state inactivation appeared complete at -30 mV. In small membrane patches, bursts of brief openings (gamma = 13-18 pS) were usually recorded. The distribution of burst durations indicated that two populations of channel were present with inactivation rates corresponding to late-1 and late-2 macroscopic currents. The persistent Na+ current in the whole cell that extended to potentials more positive than -30 mV appeared to correspond to sporadic, brief openings that were recorded in patches (mean open time approximately 0.1 ms) over a wide potential range. None of the three types of gating described corresponded to activation/inactivation gating overlap of fast transient currents.     

A family of hyperpolarization-activated mammalian cation channels

Pacemaker activity of spontaneously active neurons and heart cells is controlled by a depolarizing, mixed Na+/K+ current, named Ih (or I(f) in the sinoatrial node of the heart). This current is activated on hyperpolarization of the plasma membrane. In addition to depolarizing pacemaker cells, Ih is involved in determining the resting membrane potential of neurons and provides a mechanism to limit hyperpolarizing currents in these cells. Hormones and neurotransmitters that induce a rise in cyclic AMP levels increase Ih by a mechanism that is independent of protein phosphorylation, and which involves direct binding of the cyclic nucleotide to the channel that mediates Ih. Here we report the molecular cloning and functional expression of the gene encoding a hyperpolarization-activated cation channel (HAC1) that is present in brain and heart. This channel exhibits the general properties of Ih channels. We have also identified full-length sequences of two related channels, HAC2 and HAC3, that are specifically expressed in the brain, indicating the existence of a family of hyperpolarization-activated cation channels.     

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.     

Electroresponsive properties and membrane potential trajectories of three types of inspiratory neurons in the newborn mouse brain stem in vitro

1. The electrophysiological properties of inspiratory neurons were studied in a rhythmically active thick-slice preparation of the newborn mouse brain stem maintained in vitro. Whole cell patch recordings were performed from 60 inspiratory neurons within the rostral ventrolateral part of the slice with the aim of extending the classification of inspiratory neurons to include analysis of active membrane properties. 2. The slice generated a regular rhythmic motor output recorded as burst of action potentials on a XII nerve root with a peak to peak time of 11.5 +/- 3.4 s and a duration of 483 +/- 54 ms (means +/- SD, n = 50). Based on the electroresponsive properties and membrane potential trajectories throughout the respiratory cycle, three types of inspiratory neurons could be distinguished. 3. Type-1 neurons were spiking in the interval between the inspiratory potentials (n = 9) or silent with a resting membrane potential of -48.6 +/- 10.1 mV and an input resistance of 306 +/- 130 M omega (n = 15). The spike activity between the inspiratory potentials was burst-like with spikes riding on top of an underlying depolarization (n = 11) or regular with no evidence of bursting (n = 12). Hyperpolarization of the neurons below threshold for spike initiation did not reveal any underlying phasic synaptic activity, that could explain the bursting behavior. 4. Type-1 neurons showed delayed excitation after hyperpolarizing square current pulses or when the neurons were depolarized from a hyperpolarized level. This membrane behavior resembles the response seen in other CNS neurons expressing an IA. The response to 1-s long depolarizing pulses with a large current strength showed signs of activation of an active depolarizing membrane response leading to a transient reduction in the spike amplitude. The relationship between the membrane potential and the amplitude of square current pulses (Vm-I) showed a small upward rectification below -70 mV, and spike adaptation throughout a 1-s pulse had a largely linear time course. 5. Type-1 neurons depolarized and started to fire spikes 398 +/- 102 ms (n = 20) before the upstroke of the integrated XII nerve discharge. The inspiratory potential was followed by fast hyperpolarization, a short fast-repolarizing phase (1,040 +/- 102 ms, n = 5) and a longer slow-repolarizing phase (lasting until the next inspiratory discharge). 6. Type-2 neurons were spiking in the interval between the inspiratory potentials with no evidence of bursting behavior and had an input resistance of 296 +/- 212 M omega (n = 26). The response to hyperpolarizing pulses revealed an initial sag and postinhibitory rebound depolarization. This membrane behavior resembles the response seen in other CNS neurons expressing an Ih. The Vm-I relationship was linear at depolarized potentials and showed a marked upward rectification below -60 mV. Spike trains elicited by 1-s long pulses showed a pronounced early and late adaptation. 7. Type-2 neurons depolarized and started to fire spikes 171 +/- 87 ms (n = 23) before the upstroke of the integrated XII nerve discharge. The inspiratory potential had a variable amplitude from cell to cell and was followed by a short hyperpolarization in the cells displaying a large amplitude inspiratory potential.(ABSTRACT TRUNCATED AT 250 WORDS)     

Neuroactive steroids induce GABA(A) receptor-mediated depolarizing postsynaptic potentials in hippocampal CA1 pyramidal cells of the rat

Intracellular recordings were performed in area CA1 pyramidal cells of rat hippocampal slices to determine the effects of certain steroids on inhibitory postsynaptic potentials/currents (IPSP/Cs) mediated by GABA(A) receptors. Following application of the steroids 5alpha-pregnan-3alpha,21-diol-20-one (5alpha-THDOC), alphaxalone and 5beta-pregnan-3alpha-ol-20-one (pregnanolone) hyperpolarizing PSPs developed into biphasic responses consisting of an early hyperpolarizing and a late depolarizing PSP sequence. Steroid-induced depolarizing PSPs could be elicited in the presence of antagonists to non-NMDA, NMDA, and GABA(B) receptors, indicating that these receptor types do not contribute significantly to the initiation of these responses. Depolarizing PSPs were completely blocked by both GABA(A) receptor antagonists bicuculline and t-butylbicyclophosphorothionat (TBPS) providing evidence for their mediation by GABA(A) receptors. The reversal potential of steroid-induced late inward PSCs, measured in single-electrode voltage clamp, was -29.9+/-5.3 mV, whereas the early outward current, which corresponded to the early hyperpolarizing component of PSPs, reversed at -68.2+/-1.5 mV. Depolarizing PSPs and late inward PSCs were sensitive to reduction of extracellular [HCO3-] and block of carbonic anhydrase by application of acetazolamide. The results suggest that certain neuroactive steroids can induce GABA(A) receptor-mediated depolarizing PSPs, which are dependent on HCO3-.     

K+ channel blocking actions of flecainide compared with those of propafenone and quinidine in adult rat ventricular myocytes

The K+ channel blocking action of the class Ic antiarrhythmic agent flecainide was compared with that of propafenone and quinidine in isolated adult rat ventricular myocytes by using the whole-cell patch-clamp technique. In rat ventricular myocytes, depolarization activates both an inactivating (ITO) and a maintained (IK) outward K+ current. Flecainide, propafenone and quinidine all were potent inhibitors of ITO with IC50s of 3.7, 3.3 and 3.9 microM, respectively. Flecainide and quinidine were less potent inhibitors of IK than was propafenone with IC50s of 15 and 14 microM compared with an IC50 of 5 microM for propafenone. By contrast with their effects on outward currents, these agents produced little or no inhibition of the inward rectifier K+ current, even when present at 300 microM. All three agents produced a concentration-dependent increase in the rate of inactivation of ITO but they only produced minor hyperpolarizing shifts (approximately 3 mV) in the voltage dependence of steady-state inactivation. Although propafenone had little effect on the rate of ITO recovery from inactivation (tau CONTROL = 64 +/- 5 ms; tau PROPAFENONE = 84 +/- 9 ms), ITO recovery in the presence of flecainide and quinidine was biexponential; it exhibited an additional slow component (tau FAST = 67 +/- 5 ms and tau SLOW = 2580 +/- 1500 ms for flecainide; tau FAST = 55 +/- 5 ms and tau SLOW = 871 +/- 99 ms for quinidine). Consistent with these observations, flecainide and quinidine, but not propafenone, produced use-dependent block of ITO at a stimulation frequency of 1 Hz.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hyperpolarization induces a rise in intracellular sodium concentration in dopamine cells of the substantia nigra pars compacta

We investigated the effect of changes in membrane-voltage on intracellular sodium concentration ([Na+]i) of dopamine-sensitive neurons of the substantia nigra pars compacta in a slice preparation of rat mesencephalon. Whole-cell patch-clamp techniques were combined with microfluorometric measurements of [Na+]i using the Na+-sensitive probe, sodium-binding benzofuran isophthalate (SBFI). Hyperpolarization of spontaneously active dopamine neurons (recorded in current-clamp mode) caused the cessation of action potential firing accompanied by an elevation in [Na+]i. In dopamine neurons voltage-clamped at a holding potential of -60 mV elevations of [Na+]i were induced by long-lasting (45-60 s) voltage jumps to more negative membrane potentials (-90 to -120 mV) but not by corresponding voltage jumps to -30 mV. These hyperpolarization-induced elevations of [Na+]i were depressed during inhibition of I(h), a hyperpolarization-activated inward current, by Cs+. Hyperpolarization-induced elevations in [Na+]i might occur also in other cell types which express a powerful I(h) and might signal lack of postsynaptic activity.