Effects of lysophosphatidylcholine on resting potassium conductance of isolated guinea pig ventricular cells

We studied the effects of lysophosphatidylcholine (LPC), a toxic metabolite of ischemia, on the inward rectifier potassium channel current in isolated guinea pig ventricular cells. LPC (10-50 microM) added to the external solution decreased the resting membrane potential and occasionally induced repetitive action potential discharges, with or without loss of repolarization. In voltage clamp studies, LPC (20 microM) decreased the conductance at the levels of resting potentials (approximately equal to -80 mV) from 26 +/- 8 nS to 16 +/- 3 nS (mean and SD, n = 4) within 10 min. Prolonged application of LPC (greater than 12 min) produced transient inward currents after depolarizing clamp pulses, thereby suggesting that the LPC elevated intracellular Ca2+ concentrations. The effect of LPC on the single inward rectifier K channel current was examined using the patch clamp technique in a cell-attached mode. LPC decreased the single channel conductance, depending on the concentration (5-100 microM). The slope conductance in the presence of 150 mM K+ in the pipette decreased from 45 +/- 7 pS (control) to 32 +/- 17, 20 +/- 19, and 14 +/- 10 pS for 5, 20 and 100 microM LPC, respectively. LPC induced little change with regard to probability of the channel opening. These results suggest that LPC depolarizes membrane by decreasing single channel conductance of the inward rectifier K channel. This reduction partially contributes to the alleged LPC-induced abnormal automaticities and conduction disturbances in the heart.     

Studies of solitary semicircular canal hair cells in the adult pigeon. II. Voltage-dependent ionic conductances

1. The ionic conductances present in putative type II hair cells enzymatically dissociated from the anterior, posterior, and lateral semicircular canal cristae of the white king pigeon (Columba livia) vestibule were studied under whole cell voltage clamp. 2. Two classes of voltage-dependent potassium conductances were distinguishable on the basis of the time course of activation and inactivation and pharmacologic sensitivity. The rapid potassium conductance, IA, as inhibited by 6 mM 4-aminopyridine (4-AP), whereas the slow potassium conductance, IK, was inhibited by 50 mM tetraethylammonium (TEA). These conductances were not affected by extracellular calcium removal. IA was quite similar to the rapidly-inactivating A-current of molluscan soma, whereas IK was more like the delayed rectifier of molluscan soma. 3. The steady-state inactivation of IA occurred over a potential range from -100 to -40 mV. The threshold for activation of IA occurred between -60 and -50 mV. The slope conductance of the I-V curve over a range of -50 to -20 mV was 13.7 nS when the conditioning pulse was -100 mV, and we estimate it to be approximately 1-2 nS from the resting membrane potential of -56 mV. 4. The steady-state inactivation of IK was approximately 60% at -40 mV and was completely removed at -80 mV. The threshold for activation of IK was between -50 and -40 mV. The slope conductance of the I-V curve over a range of -50 to -20 mV was 10.5 nS when the conditioning pulse was -80 mV, and we estimate it to be approximately 6-7 nS from the resting potential of -56 mV. 5. At -56 mV (the average resting membrane potential of putative type II semicircular canal hair cells), approximately 10-14% of IA channels and approximately 57-70% of IK channels were not inactivated: thus IA and IK can contribute to the outward current during small depolarizations from rest. 6. A small calcium-dependent outward current, IK(Ca), could be elicited during step depolarizations from a holding potential of -40 mV. This calcium-dependent current was active over the range of -20 to +40 mV. 7. Inward currents could not be detected when the cells were exposed to normal physiological solutions. However, when the outward currents were blocked with internal cesium and the external solution contained 20 mM barium, sustained inward currents with rapid activation kinetics could be detected. The threshold for activation of the inward current occurred at -40 mV, and the I-V relationship peaked at -10 mV.(ABSTRACT TRUNCATED AT 400 WORDS)     

Voltage-dependent calcium currents in dissociated granule cells from rat cerebellum.

Voltage-dependent calcium currents were investigated by the patch-clamp technique in whole-cell recording configuration in cultures from 8-day-old rat cerebella, which contained greater than or equal to 90% granule cells. In solutions designed to minimize the sodium and potassium conductances and in 20 mM barium, an inward current activated around -25 mV, reached a peak amplitude at +20 mV and reversed around +80 mV. In 20 mM calcium, this current was approximately 50% of that in barium. From one to three days in vitro only 16% of the cells tested (n = 20) had a current exceeding 50 pA in maximum amplitude, while after four days in vitro the current reached 100 pA in all neurons tested (n greater than 70). Verapamil (50-100 microM) reversibly depressed this current. The dihydropyridine agonist Bay K 8644 (1 microM) enhanced the maximum conductance by 25 +/- 8% (n = 4), caused a negative shift in the activation of 21 +/- 5 mV and a prolongation of the deactivation time course as the voltage was stepped back from +20 to -80 mV. The GABAB agonist baclofen (50 microM) reversibly depressed the current by 27 +/- 8% in 80% of the cells. The effect was similar to that of GABA (10 microM), when the GABAA response (chloride current) was partially blocked by bicucculine. This current can be classified as a dihydropyridine-sensitive high-voltage-activated calcium current. The modulation by GABAB agonists is likely to be significant for presynaptic inhibition.     

Characterization of spontaneous and evoked inhibitory postsynaptic potentials in rat supraoptic neurosecretory neurons in vitro

Intracellular recordings from 52 supraoptic nucleus neurosecretory neurons in perfused explants of rat hypothalamus revealed abundant spontaneous inhibitory postsynaptic potentials (sIPSPs) and a compound evoked inhibitory postsynaptic potential (eIPSP) following electrical stimulation in the diagonal band of Broca (DBB). These IPSPs were characterized in terms of the magnitude and ionic specificity of the underlying current and in terms of the transmitter responsible for their activation. sIPSPs rose rapidly to peak within 3-5 ms and decayed exponentially with a mean time constant of 20.2 +/- 1.9 ms (mean +/- SE), a value 1.6-fold greater than the mean cell time constant of 13.8 +/- 1.0 ms. The eIPSPs rose rapidly to peak within 3-10 ms and decayed exponentially over 60-100 ms with a mean time constant of 37.0 +/- 2.8 ms, which is 2.6-fold greater than the mean cell time constant. These features imply a brief persistence of the conductance underlying the IPSPs. In recordings with KAcetate-filled micropipettes, sIPSPs were hyperpolarizing at membrane potentials in the range of -50 to -70 mV and reversed polarity when the membrane was hyperpolarized beyond -80 mV. The mean reversal potential (EsIPSP) was -72.4 +/- 1.1 mV. eIPSPs were hyperpolarizing at resting membrane potential and could be reversed by membrane hyperpolarization beyond a mean reversal potential (EIPSP) of -67.4 +/- 1.4 mV. In recordings with KCl-filled micropipettes, sIPSPs were depolarizing at all membrane potentials more negative than -50 mV. Under these conditions, EsIPSP was estimated at -44 mV. sIPSPs were absent when chloride ions were removed from the perfusion medium. eIPSPs were depolarizing at all membrane potentials and often evoked action potentials; mean EeIPSP was 43.2 mV. Reversal potentials of spontaneous and evoked IPSPs were similar. At a given membrane potential, sIPSP amplitudes varied widely between 1 and 20 mV. The conductance increase underlying individual sIPSPs was estimated to vary between 0.17 and 3.0 nS (avg 0.6 nS) against a mean resting input conductance of 3.78 +/- 0.41 nS. Estimates of the conductance underlying eIPSPs varied widely between cells, from 0.8 to 22.0 nS (mean 72 nS). Accordingly, the ratio of evoked to spontaneous IPSP conductance varied from 1.6 to 43.7 (mean 13.1). The reversal potential of evoked IPSPs shifted with the extracellular concentration of Cl- ions ([Cl-]0) with a mean slope of 41 mV/log [Cl-]0.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hyperpolarization-activated, mixed-cation current (I(h)) in octopus cells of the mammalian cochlear nucleus

Octopus cells in the posteroventral cochlear nucleus of mammals detect the coincidence of synchronous firing in populations of auditory nerve fibers and convey the timing of that coincidence with great temporal precision. Earlier recordings in current clamp have shown that two conductances contribute to the low input resistance and therefore to the ability of octopus cells to encode timing precisely, a low-threshold K(+) conductance and a hyperpolarization-activated mixed-cation conductance, g(h). The present experiments describe the properties of g(h) in octopus cells as they are revealed under voltage clamp with whole-cell, patch recordings. The hyperpolarization-activated current, I(h), was blocked by extracellular Cs(+) (5 mM) and 4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino) pyridinium chloride (50-100 nM) but not by extracellular Ba(2+) (2 mM). The reversal potential for I(h) in octopus cells under normal physiological conditions was -38 mV. Increasing the extracellular potassium concentration from 3 to 12 mM shifted the reversal potential to -26 mV; lowering extracellular sodium concentration from 138 to 10 mM shifted the reversal potential to -77 mV. These pharmacological and ion substitution experiments show that I(h) in octopus cells is a mixed-cation current that resembles I(h) in other neurons and in heart muscle cells. Under control conditions when cells were perfused intracellularly with ATP and GTP, I(h) had an activation threshold between about -35 to -40 mV and became fully activated at -110 mV. The maximum conductance associated with hyperpolarizing voltage steps to -112 mV ranged from 87 to 212 nS [150 +/- 30 (SD) nS, n = 36]. The voltage dependence of g(h) obtained from peak tail currents is fit by a Boltzmann function with a half-activation potential of -65 +/- 3 mV and a slope factor of 7. 7 +/- 0.7. This relationship reveals that g(h) was activated 41% at the mean resting potential of octopus cells, -62 mV, and that at rest I(h) contributes a steady inward current of between 0.9 and 2.1 nA. The voltage dependence of g(h) was unaffected by the extracellular application of dibutyryl cAMP but was shifted in hyperpolarizing direction, independent of the presence or absence of dibutyryl cAMP, by the removal of intracellular ATP and GTP.     

Hyperpolarization-activated inward current in histaminergic tuberomammillary neurons of the rat hypothalamus.

1. Intracellular recordings were obtained from histaminergic tuberomammillary (TM) neurons of rat hypothalamus in an in vitro slice preparation. The properties of a time- and voltage-dependent inward current activated on hyperpolarization, Ih, were studied by use of the single-electrode voltage-clamp technique. 2. The activation curve of Ih was well fit by a sigmoidal function, with half-maximal activation occurring at -98 +/- 6 mV. 3. The estimated reversal potential of Ih (Eh) in TM neurons was -35 +/- 9 (SD) mV. 4. The time constant of activation was well fit by a single exponential function and exhibited marked voltage dependence: at -90 mV, Ih activated with a time constant of 823 +/- 35 ms, whereas at -130 mV, Ih activated with a time constant of 280 +/- 65 ms. The time constant of deactivation of Ih at -60 mV was 302 +/- 35 ms. 5. Raising the extracellular potassium concentration ([K+]o) to 10 mM shifted Eh to a more depolarized value, while lowering the extracellular sodium concentration [( Na+]o) shifted Eh in the negative direction. Altering the extracellular chloride concentration ([Cl-]o) had little effect on Eh. 6. Increasing [K+]o to 10 mM increased the amplitude of both Ih and its underlying conductance gh, while reducing [Na+]o caused a small reduction in the amplitude of Ih with no measurable effect on gh. 7. The time constant of activation of Ih became shorter in raised [K+]o and longer in lowered [Na+]o. 8. Extracellularly applied cesium blocked Ih in a voltage-dependent manner. Extracellular barium reduced Ih but was less effective than cesium. 9. We conclude that Ih, carried by sodium and potassium ions, accounts for inward rectification of TM neurons. By increasing the whole-cell conductance during periods of prolonged hyperpolarization, Ih may act as an ionic shunt, decreasing the efficacy of synaptic inputs. This effect would be most apparent during rapid-eye-movement sleep, when TM neurons fall silent.     

Angiotensin II induces calcium-dependent rhythmic activity in a subpopulation of rat hypothalamic median preoptic nucleus neurons

Whole cell patch-clamp recordings revealed a subpopulation (16%, n = 18/112) of rat median preoptic nucleus (MnPO) neurons responded to bath-applied angiotensin II (Ang II; 100 nM to 5 microM; 30-90 s) with a prolonged TTX-resistant membrane depolarization and rhythmic bursting activity. At rest, cells characteristically displayed relatively low input resistance and negative resting potentials. Ang-II-induced responses featured increased input resistance, a reversal potential of -95 +/- 2 mV, an increase in action potential duration from 2.9 +/- 0.5 to 4.3 +/- 0.8 ms, and the appearance of a rebound excitation at the offset of membrane responses to hyperpolarizing current injection. The latter was sensitive to Ni2+ (0.5-1 mM; n = 5), insensitive to extracellular Cs+ (1 mM, n = 7), and intracellular QX-314 (4 mM, n = 5), consistent with activation of a T-type Ca2+ conductance. Coincident with the Ang-II-induced depolarization was the appearance of rhythmic depolarizing shifts at a frequency of 0.14 +/- 0.09 Hz with superimposed bursts of 4-22 action potentials interspersed with silent periods persisting for >1 h after washout. These TTX-resistant depolarizing shifts increased in amplitude and decreased in frequency with membrane hyperpolarization with activity ceasing beyond approximately -80 mV, and were abolished in low-Ca(2+)/high-Mg2+ bathing medium (n = 6), Co2+ (1 mM; n = 6), or Ni2+ (0.5-1 mM; n = 8). Thus in a subpopulation of MnPO neurons, Ang II induces "pacemaker-like" activity by reducing a K(+)-dependent leak conductance that contributes to resting membrane potential and promoting of Ca(2+)-dependent regenerative auto-excitation mediated, in part, by a T-type Ca2+ conductance.     

Orexin-induced modulation of state-dependent intrinsic properties in thalamic paraventricular nucleus neurons attenuates action potential patterning and frequency

The thalamic paraventricular nucleus (PVT) receives a dense innervation from orexin-synthesizing lateral hypothalamic neurons. Since PVT neurons display state-dependent tonic or low threshold spike-driven burst firing patterns, we examined how the response to exogenously applied orexins might modulate these features. Data were obtained with whole-cell patch clamp recording techniques in rat brain slices prepared during the subjective lights-on period. PVT neurons displayed a mean resting membrane potential of -61+/-6 mV and input conductance of 1.3+/-0.1 nS (n=60). The majority (90/107) of cells tested responded to orexin A and/or orexin B peptides (100-1000 nM), each inducing similar slowly rising and prolonged membrane depolarizations. We next evaluated associated changes in firing patterns and action potential frequency. Of 17 spontaneously silent neurons, 5 were induced into tonic firing and 4 into burst firing modes. Of nine spontaneously bursting neurons, three displayed an increase in burst frequency and in the number of action potentials within a burst. By contrast, another six cells were induced into tonic firing mode, with a marked decrease in instantaneous firing frequency and a shift in their excitatory postsynaptic potential-evoked responses from burst firing patterns to single action potentials. Under voltage clamp, orexins induced inward current (-21.8+/-2.4 pA at -60 mV) in 20/22 cells. In 13 cells, current-voltage (I-V) plots revealed a decrease in net conductance and reversal at -110+/-9 mV, while 3 cells displayed an increase in net conductance that reversed at -26+/-8 mV. These observations imply suppression of potassium and/or induction of nonselective cationic conductances in orexin-induced depolarization in PVT neurons, permitting these peptides to modulate intrinsic state-dependent properties. In vivo, such changes in firing patterns and frequency of action potential discharges could influence neurotransmission through PVT and activity-dependent synaptic plasticity at target sites of these neurons.     

Effects of methylphenidate on the membrane potential and current in neurons of the rat locus coeruleus

Effects of methylphenidate (MPH), a therapeutic agent used in children presenting the attention deficit hyperactivity disorder (ADHD), on the membrane potential and current in neurons of the rat locus coeruleus (LC) were examined using intracellular and whole cell patch-clamp recording techniques. Application of MPH (30 microM) to artificial cerebrospinal fluid (ACSF) produced a hyperpolarizing response with amplitude of 12 +/- 1 mV (n = 29). Spontaneous firing of LC neurons was blocked during the MPH-induced hyperpolarization. Superfusion of LC neurons with ACSF containing 0 mM Ca(2+) and 11 mM Mg(2+) (Ca(2+)-free ACSF) produced a depolarizing response associated with an increase in spontaneous firing of the action potential. The MPH-induced hyperpolarization was blocked in Ca(2+)-free ACSF. Yohimbine (1 microM) and prazosin (10 microM), antagonists for alpha(2) and alpha(2B/2C) receptors, respectively, blocked the MPH-induced hyperpolarization in LC neurons. Tetrodotoxin (TTX, 1 microM) produced a partial depression of the MPH-induced hyperpolarization in LC neurons. Under the whole cell patch-clamp condition, MPH (30-300 microM) produced an outward current (I(MPH)) with amplitude of 110 +/- 6 pA (n = 17) in LC neurons. The I(MPH) was blocked by Co(2+) (1 mM). During prolonged application of MPH (300 microM for 45 min), the hyperpolarization gradually decreased in the amplitude and eventually disappeared, possibly because of depression of norepinephrine (NE) release from noradrenergic nerve terminals. At a low concentration (1 microM), MPH produced no outward current but consistently enhanced the outward current induced by NE. These results suggest that the MPH-induced response is mediated by NE via alpha(2B/2C)-adrenoceptors in LC neurons. I(MPH) was associated with an increase in the membrane conductance of LC neurons. The I(MPH) reversed its polarity at -102 +/- 6 mV (n = 8) in the ACSF. The reversal potential of I(MPH) was changed by 54 mV per decade change in the external K(+) concentration. Current-voltage relationship showed that the I(MPH) exhibited inward rectification. Ba(2+) (100 microM) suppressed the amplitude and the inward rectification of the I(MPH.) These results suggest that the I(MPH) is produced by activation of inward rectifier K(+) channels in LC neurons.     

Potassium currents in octopus cells of the mammalian cochlear nucleus.

Octopus cells in the posteroventral cochlear nucleus (PVCN) of mammals are biophysically specialized to detect coincident firing in the population of auditory nerve fibers that provide their synaptic input and to convey its occurrence with temporal precision. The precision in the timing of action potentials depends on the low input resistance (approximately 6 MOmega) of octopus cells at the resting potential that makes voltage changes rapid (tau approximately 200 micros). It is the activation of voltage-dependent conductances that endows octopus cells with low input resistances and prevents repetitive firing in response to depolarization. These conductances have been examined under whole cell voltage clamp. The present study reveals the properties of two conductances that mediate currents whose reversal at or near the equilibrium potential for K(+) over a wide range of extracellular K(+) concentrations identifies them as K(+) currents. One rapidly inactivating conductance, g(KL), had a threshold of activation at -70 mV, rose steeply as a function of depolarization with half-maximal activation at -45 +/- 6 mV (mean +/- SD), and was fully activated at 0 mV. The low-threshold K(+) current (I(KL)) was largely blocked by alpha-dendrotoxin (alpha-DTX) and partially blocked by DTX-K and tityustoxin, indicating that this current was mediated through potassium channels of the Kv1 (also known as shaker or KCNA) family. The maximum low-threshold K(+) conductance (g(KL)) was large, 514 +/- 135 nS. Blocking I(KL) with alpha-DTX revealed a second K(+) current with a higher threshold (I(KH)) that was largely blocked by 20 mM tetraethylammonium (TEA). The more slowly inactivating conductance, g(KH), had a threshold for activation at -40 mV, reached half-maximal activation at -16 +/- 5 mV, and was fully activated at +30 mV. The maximum high-threshold conductance, g(KH), was on average 116 +/- 27 nS. The present experiments show that it is not the biophysical and pharmacological properties but the magnitude of the K(+) conductances that make octopus cells unusual. At the resting potential, -62 mV, g(KL) contributes approximately 42 nS to the resting conductance and mediates a resting K(+) current of 1 nA. The resting outward K(+) current is balanced by an inward current through the hyperpolarization-activated conductance, g(h), that has been described previously.     

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.     

4-Aminopyridine produces epileptiform activity in hippocampus and enhances synaptic excitation and inhibition

Using extra- and intracellular recording techniques, we investigated the epileptiform activity induced by low concentrations (5 and 10 microM) of bath-applied 4-aminopyridine (4-AP) in the CA3 subfield of rat hippocampal slices. We also studied the effects of 4-AP on the excitatory and inhibitory synaptic conductance changes in CA3 neurons produced by mossy fiber stimulation. Low concentrations of 4-AP induced spontaneously occurring epileptiform discharges at extracellular potassium concentrations between 1 and 10 mM. In contrast, picrotoxin and bicuculline produced spontaneous epileptiform discharges at extracellular potassium concentrations between 5 and 10 mM. The paroxysmal depolarizing shift (PDS) induced by 4-AP was also investigated. At potentials between -40 and -10 mV, the waveform of the PDS consisted of a depolarizing component enveloped by a hyperpolarizing component. The amplitude of the depolarizing component of the PDS was a monotonic function of the membrane potential, and the mean measured reversal potential was -25.7 mV. Under voltage-clamp conditions, the measured conductance associated with the depolarizing component of the PDS averaged 110 nS, with a reversal potential of -14.1 mV. Application of 5 microM 4-AP produced an increase in the inhibitory synaptic conductance change calculated from currents measured 15 ms following mossy fiber stimulation. The mean value increased from 35.2 to 58.1 nS (P less than 0.05) without a significant change in reversal potential. A concentration of 10 microM 4-AP also produced an increase in this inhibitory synaptic conductance change (from 53.3 to 66.3 nS, P less than 0.05) but caused a significant depolarization of the reversal potential (from -66.5 to -61.6 mV, P less than 0.05). This change in reversal potential may reflect a prolongation of the excitatory synaptic currents produced by 4-AP that contributes to the current measured 15 ms from the stimulus. Following application of either 5 or 10 microM 4-AP, there were no significant changes in the resting potential or input resistance of the neurons studied. Application of 5 microM 4-AP also significantly increased the amplitude of the measured excitatory synaptic conductance change produced by mossy fiber stimulation (from 27.9 to 44.1 nS, P less than 0.05) without producing a change in the reversal potential. In 5 of 21 neurons studied, a long-lasting outward synaptic current was present at holding potentials near rest following mossy fiber stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Epileptiform activity in the hippocampus produced by tetraethylammonium

1. The epileptiform discharges in the CA3 region of the rat hippocampal slice produced by bath application of the potassium channel blocker tetraethylammonium (TEA) were investigated. The effects of a convulsant (5 mM) and subconvulsant (0.5 mM) concentration of TEA on the mossy fiber-evoked synaptic currents were studied by the use of voltage-clamp techniques to determine whether TEA, like 4-aminopyridine (4-AP), another potassium channel blocker and convulsant, increased both inhibitory and excitatory components of the synaptic response. 2. At extracellular potassium concentrations of 2.5 mM, TEA (5 mM) was found to produce spontaneously occurring epileptiform discharges that could be recorded extracellularly. The intracellular correlate of the epileptiform discharge, the paroxysmal depolarizing shift (PDS), could be reversed in polarity by depolarizing the membrane and was associated with a large increase in membrane conductance. These results suggest that a synaptically mediated potential underlies the generation of the epileptiform discharge. 3. The reversal potential for the PDS was dependent on the time, relative to the extracellularly recorded field discharge, at which the measurement was made. In current clamp the mean reversal potential of the PDS measured at the midpoint of the extracellular discharge was -3.3 +/- 2.9 (SE) mV (n = 9). The reversal potential of the PDS was considerably more negative when measured either before or after the midpoint of the extracellular discharge, suggesting the presence of an inhibitory synaptic component. In voltage clamp similar results were obtained and a large conductance change was found to be associated with the PDS. These results suggest that the synaptic conductance associated with the PDS has both inhibitory and excitatory components. 4. TEA increased significantly the mossy fiber-evoked, early-inhibitory conductance. A convulsant concentration (5 mM) increased the conductance measured 15 ms after the stimulus from 39.7 +/- 8.7 to 87.2 +/- 8.0 nS (n = 6). The reversal potential associated with the conductance depolarized from -68.3 +/- 3.4 to -58.3 +/- 4.0 mV after 5 mM TEA. A subconvulsant concentration of TEA (0.5 mM) also increased the conductance of the mossy fiber-evoked response at 15 ms after the stimulus from 49.5 +/- 3.1 to 63.1 +/- 6.1 nS (n = 4) without an associated shift in reversal potential. 5. The late-inhibitory component of the mossy fiber-evoked response, when present, was increased by 5 mM TEA and unchanged by 0.5 mM TEA. 6. The excitatory mossy fiber-evoked synaptic current was studied in the presence of picrotoxin and was found to be increased and prolonged by 5 mM TEA.(ABSTRACT TRUNCATED AT 400 WORDS)     

Single channel analysis of the inward rectifier K current in the rabbit ventricular cells

The inward rectifier K channel in rabbit ventricular cells was studied by the patch-clamp method. Single channel currents were recorded in giga-sealed cell-attached patches with 150 mM K+ in the pipette. The slope conductance in the membrane potential range from -140 to -40 mV was 46.6 +/- 6.7 pS (mean +/- S.D., n = 16), and was reduced by decreasing [K+] in the pipette (20 or 50 mM). The channel was blocked by an application of Cs+ or Ba2+ (0.04-1 mM) in the pipette. Outwardly directed current, recorded with 50 mM K+ in the pipette, revealed the inward rectification of the single channel current. The probability of the channel being open was 0.33 +/- 0.05 (n = 10) at the resting potential (RP=-81.7 +/- 1.7 mV, n = 16) with 150 mM K+ in the pipette, and it decreased with hyperpolarization. The mean open time of the channel was 178 +/- 25 msec (n = 6) at RP. The closed time of the channel seemed to have two exponential components, with time constants of 11.0 +/- 2.0 msec and 1.92 +/- 0.52 sec (n = 6) at RP. The slower time constant was increased with hyperpolarization. The averaged patch current recorded upon hyperpolarizing pulses demonstrated a time-dependent current decay as expected from the single channel kinetics. The results indicated that the inward rectifier K+ current has time- and voltage-dependent kinetics.     

Rat hippocampal neurons in culture: potassium conductances

Two-electrode voltage-clamp methodology was used to analyze voltage-dependent ionic conductances in 81 rat hippocampal neurons grown in culture for 4-6 wk. Pyramidal and multipolar cells with 15- to 20-micron-diameter cell bodies were impaled with two independent KCl electrodes. The cells had resting potentials of -30 to -60 mV and an average input resistance of about 30 M omega. A depolarizing command applied to a cell maintained in normal medium invariably evoked a fast (2-10 ms) inward current that saturated the current-passing capacity of the system. This was blocked in a reversible manner by application of tetrodotoxin (TTX) (0.1-1.0 microM) near the recorded cell. In the presence of TTX, a depolarizing command evoked a rapidly rising (3-5 ms), rapidly decaying (25 ms) transient outward current reminiscent of "IA" reported in molluscan neurons. This was followed by a more slowly activating (approximately 100 ms) outward current response of greater amplitude that decayed with a time constant of about 2-3 s. These properties resemble those associated with the K+ conductance, IK, underlying delayed rectification described in many excitable membranes. IK was blocked by extracellular application of tetraethylammonium (TEA) but was insensitive to 4-aminopyridine (4-AP) at concentrations that effectively eliminated IA. IA, in turn, was only marginally depressed by TEA. Unlike IK, IA was completely inactivated when the membrane was held at potentials positive to -50 mV. Inactivation was completely removed by conditioning hyperpolarization at -90 mV. A brief hyperpolarizing pulse (10 ms) was sufficient to remove 95% of the inactivation. IA activated on commands to potentials more positive than -50 mV. The inversion potential of the ionic conductance underlying IA and IK was in the range of the K+ equilibrium potential, EK, as measured by the inversion of tail currents; and this potential was shifted in a depolarizing direction by elevated [K+]0. Thus, both current species reflect activation of membrane conductance to K+ ions. Hyperpolarizing commands from resting potentials revealed a time- and voltage-dependent slowly developing inward current in the majority of cells studied. This membrane current was observed in cells exhibiting "anomalous rectification" and was therefore labeled IAR. It was activated at potentials negative to -70 mV with a time constant of 100-200 ms and was not inactivated. A return to resting potential revealed a tail current that disappeared at about EK. IAR was blocked by extracellular CS+ and was enhanced by elevating [K+]0. It thus appears to be carried by inward movement of K+ ions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Conductance changes underlying a late synaptic hyperpolarization in hippocampal CA3 neurons

1. Single-electrode current- and voltage-clamp techniques were employed to study properties of the conductance underlying an orthodromically evoked late synaptic hyperpolarization or late inhibitory postsynaptic potential (IPSP) in CA3 pyramidal neurons in the rat hippocampal slice preparation. 2. Late IPSPs could occur without preceding excitatory postsynaptic potentials at the resting membrane potential and were graded according to the strength of the orthodromic stimulus. The membrane hyperpolarization associated with the late IPSP peaked within 140-200 ms after orthodromic stimulation of mossy fiber afferents. The late IPSP returned to base line with a half-decay time of approximately 200 ms. 3. As determined from constant-amplitude hyperpolarizing-current pulses, the membrane conductance increase during the late IPSP, and the time course of its decay, were similar whether measurements were made near the resting membrane potential or when the cell was hyperpolarized by approximately 35 mV. 4. When 1 mM cesium was added to the extracellular medium to reduce inward rectification, late IPSPs could be examined over a range of membrane potentials from -60 to -140 mV. For any given neuron, the late IPSP amplitude-membrane potential relationship was linear over the same range of membrane potentials for which the slope input resistance was constant. The late IPSP reversed symmetrically near -95 mV. 5. Intracellular injection of ethyleneglycol-bis-(beta-aminoethylether)-N,N'-tetraacetic acid or extracellular application of forskolin, procedures known to reduce or block certain calcium-dependent potassium conductances in CA3 neurons, had no significant effect on the late IPSP. 6. Single-electrode voltage-clamp techniques were used to analyze the time course and voltage sensitivity of the current underlying the late IPSP. This current [the late inhibitory postsynaptic current (IPSC)] began as early as 25 ms after orthodromic stimulation and reached a peak 120-150 ms following stimulation. 7. The late IPSC decayed with a single exponential time course (tau = 185 ms). 8. A clear reversal of the late IPSC at approximately -99 mV was observed in a physiological concentration of extracellular potassium (3.5 mM).(ABSTRACT TRUNCATED AT 400 WORDS)     

Glycine binding site of the synaptic NMDA receptor in subpostremal NTS neurons

The nucleus of the tractus solitarius (NTS) plays an important role in the control of several autonomic reflex functions and has glutamate and GABA as main neurotransmitters. In this work, we used patch-clamp recordings in transverse slice preparations from rats to study whether the glycine binding site of the N-methyl-D-aspartate (NMDA) receptor is saturated or not in neurons of the subpostremal NTS. Except at hyperpolarized voltages and close to the reversal potential, glycine potentiated the NMDA responses in a concentration-dependent manner. The total charge transferred by glutamatergic currents was enhanced by glycine (500 microM; from 28 +/- 13 to 42 +/- 18 pC at +50 mV, n = 7, P < 0.05). Glycine increased the conductance of the postsynaptic membrane, without altering its reversal potential, both in the presence (from 2.4 +/- 0.06 to 3.4 +/- 0.09 nS; n = 7) and absence (from 3.1 +/- 0.06 to 4.4 +/- 0.10 nS; n = 8) of Mg2+ in the bathing solution. d-serine, in the presence of strychnine, also increased the amplitude of the NMDA component (by 68 +/- 19%, P < 0.05, n = 5). The membrane potential was hyperpolarized (16 +/- 6 mV, n = 8) by glycine, suggesting the presence of inhibitory glycinergic receptors. Our results indicate that the glycine site of the NMDA receptor in neurons of the subpostremal NTS is not saturated and that glycine may act as a modulator of the NMDA transmission in this nucleus.     

Voltage dependence of excitatory postsynaptic potentials of rat neocortical neurons

1. The properties of excitatory postsynaptic potentials (EPSPs) of rat neocortical neurons were investigated with a fast single-electrode current-voltage clamp in vitro. Typically, apparently pure EPSPs were obtained by selection of electric stimuli of low intensity. 2. The amplitude and time integral of the EPSP increased when the neuron was depolarized. At threshold for generation of action potentials, the amplitude of EPSPs was increased by approximately 30% [from 5.0 +/- 2.1 to 6.3 +/- 1.0 (SD) mV, n = 12]. The integral of EPSPs was maximally about fourfold (3.7 +/- 1.5, n = 16) larger than at resting membrane potential (Em). The mechanisms involved in this augmentation of EPSPs were further investigated. 3. The amplitude and the time integral of excitatory postsynaptic currents (EPSCs) decreased linearly with shifts in command potential from -100 to -60 mV. The decrease of the EPSC integral with depolarization indicates that the enhancement of the EPSP may be brought about by recruitment of a voltage-dependent inward current. 4. Evoking EPSPs at various delays after the onset of small depolarizing current pulses (0.3-0.6 nA, 600 ms) revealed that augmentation decays with time. The integral of EPSPs evoked approximately 80 ms after the onset of the current pulse was 3.7 (+/- 1.5, n = 16) times larger than at Em. The integral of EPSPs evoked at 480 ms. however, were only twofold (+/- 0.7, n = 16) larger. Hence EPSPs evoked after a delay of 80 ms were 1.7-fold (+/- 0.4, n = 24) larger than EPSPs evoked after 480 ms. EPSCs were independent of the delay of stimulation at all potentials. 5. Intracellular application of the lidocaine derivative N-(2,6-dimethyl-phenylcarbamoylmethyl) triethylammonium bromide (QX 314) at 100 mM from pipettes rapidly abolished fast action potentials and inward rectification. During comparable depolarizations the increase in EPSP integrals was much smaller in QX 314-treated neurons than in controls. On average, the integral of EPSPs evoked at 70-90 ms was 1.7 times (+/- 1.0) larger than at Em, and the integral of EPSPs evoked with larger delays was close to the value obtained at resting Em (0.9 +/- 0.3, n = 8). The ratio of EPSP integrals early versus late (1.8 +/- 0.5) is comparable to controls, suggesting that QX 314-sensitive currents are unlikely to be involved in the time-dependent enhancement. 6. Mimicking EPSPs by brief depolarizations atop long depolarizations revealed a time- and voltage-dependent enhancement comparable to that of EPSPs.(ABSTRACT TRUNCATED AT 400 WORDS)     

Suprachiasmatic nucleus communicates with anterior thalamic paraventricular nucleus neurons via rapid glutamatergic and gabaergic neurotransmission: state-dependent response patterns observed in vitro

The hypothalamic suprachiasmatic nucleus uniquely projects to the midline thalamic paraventricular nucleus. To characterize this projection, patch clamp techniques applied in acute rat brain slice preparations examined responses of anterior thalamic paraventricular nucleus neurons to focal suprachiasmatic nucleus stimulation. Whole cell recordings from slices obtained during daytime (n=40) revealed neurons with a mean membrane potential of -66+/-1.2 mV, input conductance of 1.5+/-0.1 nS and state-dependent tonic or burst firing patterns. Electrical stimulation (one or four pulses) in suprachiasmatic nucleus elicited monosynaptic excitatory postsynaptic potentials (mean latency of 12.6+/-0.6 ms; n=12), featuring both AMPA and N-methyl-D-aspartate-glutamate receptor-mediated components, and monosynaptic bicuculline-sensitive inhibitory postsynaptic potentials (mean latency of 16.6+/-0.6 ms; n=7) reversing polarity at -72+/-2.6 mV, close to the chloride equilibrium potential. Glutamate microstimulation of suprachiasmatic nucleus also elicited transient increases in spontaneous excitatory or inhibitory postsynaptic currents in anterior thalamic paraventricular neurons. Recordings from rats under reverse light/dark conditions (n=22) yielded essentially similar responses to electrical stimulation. At depolarized membrane potentials, suprachiasmatic nucleus-evoked excitatory postsynaptic potentials triggered single action potentials, while evoked inhibitory postsynaptic potentials elicited a silent period in ongoing tonic firing. By contrast, after manual adjustment of membrane potentials to hyperpolarized levels, neuronal response to the same "excitatory" stimulus was a low threshold spike and superimposed burst firing, while responses to "inhibitory" stimuli paradoxically elicited excitatory rebound low threshold spikes and burst firing. These data support the existence of glutamatergic and GABAergic efferents from the suprachiasmatic nucleus to its target neurons. Additionally, in thalamic paraventricular nucleus neurons, responses to activation of their suprachiasmatic afferents may vary in accordance with their membrane potential-dependent intrinsic properties, a characteristic typical of thalamocortical neurons.