Electrophysiology of the mammillary complex in vitro. II. Medial mammillary neurons.

1. The electrophysiological properties of guinea pig medial mammillary body (MMB) neurons were studied using an in vitro slice preparation. 2. The neurons (n = 80) had an average resting potential of -57 +/- 5.5 (SD) mV, an input resistance of 176 +/- 83 M omega, and a spike amplitude of 58 +/- 15.7 mV. Most of the neurons were silent at rest (n = 52), but some fired spontaneous single spikes (n = 16) or spike bursts (n = 14). 3. The main electrophysiological characteristic of MMB neurons was the ability to generate Ca(2+)-dependent regenerative events, which resulted in very robust burst responses. However, this regenerative event was not the same for all neurons, ranging from typical low-threshold Ca2+ spikes (LTSs) to intermediate-threshold plateau potentials (ITPs). 4. The ITPs were distinct from the LTSs in that they lasted > or = 100 ms and were not inactivated at membrane potentials at or positive to -55 mV. 5. Some cells with a prominent ITP and no LTS (n = 36) displayed repetitive, usually rhythmic, bursting (n = 14). This ITP could be powerful enough to maintain rhythmic membrane potential oscillations after pharmacological block of Na+ conductances. 6. A group of 32 MMB neurons displayed complex bursting that was generated by activation of both LTSs and ITPs. This was established on the basis of their distinct time- and voltage-dependent characteristics. In a group of neurons (n = 14), the burst responses were exclusively generated by an LTS; however, a Ca(2+)-dependent plateau potential contributed to the generation of rebound-triggered oscillatory firing. 7. In addition to the Ca(2+)-dependent LTS and/or ITP, MMB neurons always displayed high-threshold Ca2+ spikes after reduction of K+ conductances with tetraethylammonium. 8. MMB neurons display one of the richer varieties of voltage-dependent Ca2+ conductances so far encountered in mammalian CNS. We propose that the very prominent endogenous bursting and oscillatory properties of MB neurons allow this nuclear complex to function as an oscillatory relay for the transmission of low-frequency rhythmic activities throughout the limbic circuit.     

Bursting activity in leech Retzius neurons induced by low external chloride

We have investigated the bursting activity of Retzius neurons in the central nervous system of the leech Hirudo medicinalis as induced in Cl(-)-free saline by measuring membrane potential, membrane current and the intracellular calcium concentration ([Ca2+]i), using fura-2 or Oregon-Green488-Bapta-1. The Retzius neurons changed their low tonic firing to rhythmical bursting activity when the extracellular Cl- concentration ([Cl-]o) was lowered to 1 mM or less. In Cl(-)-free saline (Cl- exchanged by gluconate), bursting was accompanied by a rise in intracellular Ca2+ in both cell body and axon, which oscillated in synchrony with the bursts. The Ca2+ transients depended on the amplitude and duration of the depolarization underlying the burst, and were presumably due to Ca2+ influx through voltage-dependent Ca2+ channels. In Ca(2+)-free, EGTA-buffered saline or in the presence of Ca2+ channel blockers verapamil (1 mM) or diltiazem (500 microM) the depolarizations underlying the bursts in Cl(-)-free saline were enhanced in amplitude and duration. Bursting was not affected by depleting the intracellular Ca2+ stores with cyclopiazonic acid. The depolarization in Cl(-)- and Ca(2+)-free saline did not evoke intracellular Ca2+ changes. The burst-underlying membrane depolarization induced by Cl- removal was found to be due to a Na(+)-dependent persistent inward current and could be inhibited by saxitoxin (25-50 microM). The results suggest that a persistent Na+ current is generated in Cl(-)-free saline and induces the depolarization underlying rhythmic activity, and that presumably Ca(2+)-induced K+ currents modulate the bursting behaviour.     

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.     

Electrophysiology of the mammillary complex in vitro. I. Tuberomammillary and lateral mammillary neurons.

1. The electrophysiological properties of the tuberomammillary and lateral mammillary neurons in the guinea pig mammillary body were studied using an in vitro brain slice preparation. 2. Tuberomammillary (n = 79) neurons were recorded mainly ventral to the lateral mammillary body as well as ventromedially to the fornix within the rostral part of the medial mammillary nucleus. Intracellular staining with horseradish peroxidase (n = 9) and Lucifer yellow (n = 3) revealed that these cells have several thick, long, spiny dendrites emerging from large (20-35 microns) fusiform somata. 3. Most tuberomammillary neurons (66%) fired spontaneously at a relatively low frequency (0.5-10 Hz) at the resting membrane potential. The action potentials were broad (2.3 ms) with a prominent Ca(2+)-dependent shoulder on the falling phase. Deep (17.8 mV), long-lasting spike afterhyperpolarizations were largely Ca(2+)-independent. 4. All tuberomammillary neurons recorded displayed pronounced delayed firing when the cells were activated from a potential negative to the resting level. The cells also displayed a delayed return to the baseline at the break of hyperpolarizing pulses applied from a membrane potential level close to firing threshold. Analysis of the voltage- and time dependence of this delayed rectification suggested the presence of a transient outward current similar to the A current (IA). These were not completely blocked by high concentrations of 4-aminopyridine, whereas the delayed onset of firing was always abolished when voltage-dependent Ca2+ conductances were blocked by superfusion with Cd2+. 5. Tuberomammillary neurons also displayed inward rectification in the hyperpolarizing and, primarily, depolarizing range. Block of voltage-gated Na(+)-dependent conductances with tetrodotoxin (TTX) selectively abolished inward rectification in the depolarizing range, indicating the presence of a persistent low-threshold sodium-dependent conductance (gNap). In fact, persistent TTX-sensitive, plateau potentials were always elicited following Ca2+ block with Cd2+ when K+ currents were reduced by superfusion with tetraethylammonium. 6. The gNap in tuberomammillary neurons may subserve the pacemaker current underlying the spontaneous firing of these cells. The large-amplitude spike afterhyperpolarization of these neurons sets the availability of the transient outward rectifier, which, in conjunction with the pacemaker current, establishes the rate at which membrane potential approaches spike threshold. 7. Repetitive firing elicited by direct depolarization enhanced the spike shoulder of tuberomammillary neurons. Spike trains were followed by a Ca(2+)-dependent, apamine-sensitive, slow afterhyperpolarization. 8. Lateral mammillary neurons were morphologically and electrophysiologically different from tuberomammillary neurons. All lateral mammillary neurons neurons recorded (n = 44) were silent at rest (-60 mV).(ABSTRACT TRUNCATED AT 400 WORDS)     

5-HT and dopamine modulates CaV1.3 calcium channels involved in postinhibitory rebound in the spinal network for locomotion in lamprey

Postinhibitory rebound (PIR) can play a significant role for producing stable rhythmic motor patterns, like locomotion, by contributing to burst initiation following the phase of inhibition, and PIR may also be a target for modulatory systems acting on the network. The current aim was to explore the PIR in one type of interneuron in the lamprey locomotor network and its dependence on low voltage-activated (LVA) calcium channels, as well as its modulation by 5-HT and dopamine. PIR responses in commissural interneurons, mediating reciprocal inhibition and left-right alternation in the network, were significantly larger than in motoneurons. The L-type calcium channel antagonist nimodipine reduced PIR amplitude by ～ 50%, whereas the L-channel agonist BAY K 8644 enhanced PIR amplitude, suggesting that LVA calcium channels of the L-subtype (Ca(V)1.3) participate in the PIR response. The remainder of the response was blocked by nickel, indicating that T-type (Ca(V)3) LVA calcium channels also contribute. No evidence was obtained for the involvement of a hyperpolarization-activated current. Furthermore, 5-HT, acting via 5-HT(1A) receptors, reduced PIR, as did dopamine, acting via D(2) receptors. Coapplication of nimodipine caused no further PIR reduction, indicating that these modulators target Ca(V)1.3 channels specifically. These results suggest that PIR may play a prominent role in the generation of alternating network activity and that the Ca(V)1.3 and Ca(V)3 subtypes of LVA calcium channels together underlie the PIR response. 5-HT and dopamine both target PIR via Ca(V)1.3 channels, which may contribute significantly to their modulatory influence on locomotor network activity.     

Persistent calcium current in rat suprachiasmatic nucleus neurons

The suprachiasmatic nuclei contain the primary circadian clock, and suprachiasmatic nuclei neurons exhibit a diurnal modulation of spontaneous firing rate. The present study examined the voltage-gated persistent Ca(2+) current, in acutely isolated rat suprachiasmatic nuclei neurons using a ramp-type voltage-clamp protocol. Slow triangular voltage-clamp commands from a holding potential of -85 mV to +5 mV elicited inward current (100-400 pA) that was completely blocked by Cd(2+). This current showed little or no hysteresis, and was identified as persistent Ca(2+) current. The threshold for persistent Ca(2+) current ranged between -60 and -45 mV, and it was maximal at about -8 mV. Nifedipine at 10-20 microM blocked 80-100%. To assess the role of persistent Ca(2+) current in the generation of spontaneous action potentials in both acutely isolated and intact suprachiasmatic nuclei neurons, the effect of Cd(2+) and nifedipine on firing rate was studied using on-cell recording. Application of Cd(2+) exerted a weak excitatory effect and nifedipine had no significant effect on the spontaneous firing rate of isolated suprachiasmatic nuclei neurons. In all intact suprachiasmatic nuclei neurons in slice preparations (n=15), Cd(2+) slowly inhibited spontaneous firing; in high-frequency firing cells (four of 15), a transient increase of firing rate preceded inhibition. No significant effect of nifedipine on firing rate of intact suprachiasmatic nuclei neurons was found. Therefore, persistent Ca(2+) current itself (as carrier of charge) does not appear to contribute significantly to spontaneous firing of suprachiasmatic nuclei neurons. A slowly developing inhibitory effect of Cd(2+) on spontaneous firing of intact suprachiasmatic nuclei neurons in slice preparations may be due to penetration of Cd(2+) through Ca(2+) channels, and its subsequent effect on intracellular mechanisms, while the transient increase of firing rate in high-frequency firing neurons is probably due to inhibition of Ca(2+)-activated K(+) current.     

A Ba(2+)-sensitive K(+) current contributes to the resting membrane potential of neurons in rat suprachiasmatic nucleus

A Ba(2+)-sensitive K(+) current was studied in neurons of the suprachiasmatic nucleus (SCN) using the whole cell patch-clamp technique in acutely prepared brain slices. This Ba(2+)-sensitive K(+) current was found in approximately 90% of the SCN neurons and was uniformly distributed across the SCN. Current-clamp studies revealed that Ba(2+) (500 microM) reversibly depolarized the membrane potential by 6.7 +/- 1.3 mV (n = 22) and concomitantly Ba(2+) induced an increase in the spontaneous firing rate of 0.8 +/- 0.2 Hz (n = 12). The Ba(2+)-evoked depolarizations did not depend on firing activity or spike dependent synaptic transmission. No significant day/night difference in the hyperpolarizing contribution to the resting membrane potential of the present Ba(2+)-sensitive current was observed. Voltage-clamp experiments showed that Ba(2+) (500 microM) reduced a fast-activating, voltage-dependent K(+) current. This current was activated at levels below firing threshold and exhibited outward rectification. The Ba(2+)-sensitive K(+) current was strongly reduced by tetraethylammonium (TEA; 20 and 60 mM) but was insensitive to 4-aminopyridine (4-AP; 5 mM) and quinine (100 microM). A component of Ba(2+)-sensitive K(+) current remaining in the presence of TEA exhibited no clear voltage dependence and is less likely to contribute to the resting membrane potential. The voltage dependence, kinetics and pharmacological properties of the Ba(2+)- and TEA-sensitive K(+) current make it unlikely that this current is a delayed rectifier, Ca(2+)-activated K(+) current, ATP-sensitive K(+) current, M-current or K(+) inward rectifier. Our data are consistent with the Ba(2+)- and TEA-sensitive K(+) current in SCN neurons being an outward rectifying K(+) current of a novel identity or belonging to a known family of K(+) channels with related properties. Regardless of its precise molecular identity, the current appears to exert a significant hyperpolarizing effect on the resting potential of SCN neurons.     

Origin of the slow afterhyperpolarization and slow rhythmic bursting in striatal cholinergic interneurons

Striatal cholinergic interneurons recorded in slices exhibit three different firing patterns: rhythmic single spiking, irregular bursting, and rhythmic bursting. The rhythmic single-spiking pattern is governed mainly by a prominent brief afterhyperpolarization (mAHP) that follows single spikes. The mAHP arises from an apamin-sensitive calcium-dependent potassium current. A slower AHP (sAHP), also present in these neurons, becomes prominent during rhythmic bursting or driven firing. Although not apamin sensitive, the sAHP is caused by a calcium-dependent potassium conductance. It is not present after blockade of calcium current with cadmium or after calcium is removed from the media or when intracellular calcium is buffered with bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. It reverses at the potassium equilibrium potential. It can be generated by subthreshold depolarizations and persists after blockade of sodium currents by tetrodotoxin. It is slow, being maximal approximately 1 s after depolarization onset, and takes several seconds to decay. It requires >300-ms depolarizations to become maximally activated. Its voltage sensitivity is sigmoidal, with a half activation voltage of -40 mV. We conclude the sAHP is a high-affinity apamin-insensitive calcium-dependent potassium conductance, triggered by calcium currents partly activated at subthreshold levels. In combination with those calcium currents, it accounts for the slow oscillations seen in a subset of cholinergic interneurons exhibiting rhythmic bursting. In all cholinergic interneurons, it contributes to the slowdown or pause in firing that follows driven activity or prolonged subthreshold depolarizations and is therefore a candidate mechanism for the pause response observed in cholinergic neurons in vivo.     

Identification of two calcium currents in acutely dissociated neurons from the rat lateral geniculate nucleus

1. Intracellular recording in the in vitro slice preparation and whole-cell, patch-clamp recording of acutely dissociated neurons from the rat lateral geniculate nucleus (LGN) were combined to study the Ca currents underlying their electrical responses. In slices from young animals (postnatal days 13-16), we found that dorsal LGN neurons have responses similar to those of adult preparations, including the presence of a low-threshold Ca spike (LTS). After enzymatic isolation of LGN neurons from the same animals, the firing properties appeared well preserved, as indicated by whole-cell, current-clamp recordings from dissociated multipolar cells (presumably geniculocortical relay neurons). 2. Two types of Ca currents were identified in voltage-clamped, isolated LGN neurons on the basis of their voltage dependency, pharmacology, and selectivity properties. These two currents resemble the low-voltage-activated (LVA) and high-voltage-activated (HVA) Ca channels found in rat sensory neurons (9). 3. The LVA current component required negative potentials (less than -80 mV) to deinactivate completely, started to activate around -60 mV and reached a plateau level around -25 mV. It peaked within 30-6 ms and decayed with a single time constant of approximately 24 ms at -20 mV. Its inactivation curve ranged from -100 to -40 mV, with a half-inactivation near -60 mV. The HVA current component could be isolated by holding the membrane potential positive to -60 mV, activated at potentials positive to -30 mV and peaked around +5 mV. The time-to-peak ranged from 30 to 6 ms in the voltage range from -30 to +35 mV and decayed very slowly with sustained depolarizing pulses (time constant ranged between 1,600 and 40 ms over the same voltage range). 4. The inactivation of LVA Ca current during depolarizing voltage steps was consistent with a voltage-dependent process. The recovery from inactivation after short (100 ms), inactivating prepulses displayed two exponential phases. The slower phase was predominant under conditions that induce large current flow through the membrane, suggesting a Ca-mediated mechanism. 5. The LVA current was preferentially blocked by 50 microM Ni2+, leaving the HVA currents almost unaltered. Fifty micromolars Cd2+, in contrast, seemed more effective in blocking the HVA component of the Ca current.(ABSTRACT TRUNCATED AT 400 WORDS)     

Subthreshold inward membrane currents in guinea-pig frontal cortex neurons

Current-clamp and single-electrode voltage-clamp recordings were used to study the inward currents activated in the subthreshold membrane potential range of cortical pyramidal neurons. The experiments were done on slices from guinea-pig frontal cortex and all recordings were obtained at a distance of 600-900 microm from the pial surface. In current-clamp recordings and from membrane potentials hyperpolarized to about -70 mV, the depolarization leading to spike firing was partially blocked by 1 microM tetrodotoxin, but not by calcium-free extracellular solution. The calcium-free solution only affected this depolarization when the membrane potential was held at a level more negative than -75 mV. Under voltage-clamp, an inward current was recorded between the resting membrane potential and the level of spike firing. This current was activated at about -60 mV and part of it was blocked by 1 microM tetrodotoxin; the remaining current was blocked by calcium-free extracellular solution. In five neurons both components were recorded and isolated in the same cell. The tetrodotoxin-sensitive component activated at close to -60 mV, was similar to the persistent sodium current (I(Na-p)). The Ca2+-sensitive component activated at close to -60 or -65 mV, was less voltage-dependent than I(Na-p). This component was similar to the low threshold calcium current (I(T)). These results suggest that the subthreshold depolarization which led to spike firing was dependent on I(Na-p) and I(T), I(Na-p) being the most important factor up to resting membrane potentials of -70 or -75 mV. A physiological role of this finding is revealed by the action of dopamine, which (at 10 microM) prevented the firing of action potentials from -60 mV, but not from -80 mV due to the inhibition of I(Na-p) and the lack of effect on I(T).     

Slowly inactivating sodium current (I(NaP)) underlies single-spike activity in rat subthalamic neurons

One-half of the subthalamic nucleus (STN) neurons switch from single-spike activity to burst-firing mode according to membrane potential. In an earlier study, the ionic mechanisms of the bursting mode were studied but the ionic currents underlying single-spike activity were not determined. The single-spike mode of activity of STN neurons recorded from acute slices in the current clamp mode is TTX-sensitive but is not abolished by antagonists of ionotropic glutamatergic and GABAergic receptors, blockers of calcium currents (2 mM cobalt or 40 microM nickel), or intracellular Ca(2+) ions chelators. Tonic activity is characterized by a pacemaker depolarization that spontaneously brings the membrane from the peak of the afterspike hyperpolarization (AHP) to firing threshold (from -57.1 +/- 0.5 mV to -42.2 +/- 0.3 mV). Voltage-clamp recordings suggest that the Ni(2+)-sensitive, T-type Ca(2+) current does not play a significant role in single-spike activity because it is totally inactivated at potentials more depolarized than -60 mV. In contrast, the TTX-sensitive, I(NaP) that activated at -54.4 +/- 0.6 mV fulfills the conditions for underlying pacemaker depolarization because it is activated below spike threshold and is not fully inactivated in the pacemaker range. In some cases, the depolarization required to reach the threshold for I(NaP) activation is mediated by hyperpolarization-activated cation current (I(h)). This was directly confirmed by the cesium-induced shift from single-spike to burst-firing mode which was observed in some STN neurons. Therefore, a fraction of I(h) which is tonically activated at rest, exerts a depolarizing influence and enables membrane potential to reach the threshold for I(NaP) activation, thus favoring the single-spike mode. The combined action of I(NaP) and I(h) is responsible for the dual mode of discharge of STN neurons.     

Calcium-dependent fast depolarizing afterpotentials in vasopressin neurons in the rat supraoptic nucleus

Oxytocin (OT) and vasopressin (VP) synthesizing magnocellular cells (MNCs) in the supraoptic nucleus (SON) display distinct firing patterns during the physiological demands for these hormones. Depolarizing afterpotentials (DAPs) in these neurons are involved in controlling phasic bursting in VP neurons. Our whole cell recordings demonstrated a Cs(+)-resistant fast DAP (fDAP; decay tau = approximately 200 ms), which has not been previously reported, in addition to the well-known Cs(+)-sensitive slower DAP (sDAP; decay tau = approximately 2 s). Immunoidentification of recorded neurons revealed that all VP neurons, but only 20% of OT neurons, expressed the fDAP. The activation of the fDAP required influx of Ca(2+) through voltage-gated Ca(2+) channels as it was strongly suppressed in Ca(2+)-free extracellular solution or by bath application of Cd(2+). Additionally, the current underlying the fDAP (I(fDAP)) is a Ca(2+)-activated current rather than a Ca(2+) current per se as it was abolished by strongly buffering intracellular Ca(2+) with BAPTA. The I-V relationship of the I(fDAP) was linear at potentials less than -60 mV but showed pronounced outward rectification near -50 mV. I(fDAP) is sensitive to changes in extracellular Na(+) and K(+) but not Cl(-). A blocker of Ca(2+)-activated nonselective cation (CAN) currents, flufenamic acid, blocked the fDAP, suggesting the involvement of a CAN current in the generation of fDAP in VP neurons. We speculate that the two DAPs have different roles in generating after burst discharges and could play important roles in determining the distinct firing properties of VP neurons in the SON neurons.     

Contribution of persistent Na+ current and M-type K+ current to somatic bursting in CA1 pyramidal cells: combined experimental and modeling study

The intrinsic firing modes of adult CA1 pyramidal cells vary along a continuum of "burstiness" from regular firing to rhythmic bursting, depending on the ionic composition of the extracellular milieu. Burstiness is low in neurons exposed to a normal extracellular Ca(2+) concentration ([Ca(2+)](o)), but is markedly enhanced by lowering [Ca(2+)](o), although not by blocking Ca(2+) and Ca(2+)-activated K(+) currents. We show, using intracellular recordings, that burstiness in low [Ca(2+)](o) persists even after truncating the apical dendrites, suggesting that bursts are generated by an interplay of membrane currents at or near the soma. To study the mechanisms of bursting, we have constructed a conductance-based, one-compartment model of CA1 pyramidal neurons. In this neuron model, reduced [Ca(2+)](o) is simulated by negatively shifting the activation curve of the persistent Na(+) current (I(NaP)) as indicated by recent experimental results. The neuron model accounts, with different parameter sets, for the diversity of firing patterns observed experimentally in both zero and normal [Ca(2+)](o). Increasing I(NaP) in the neuron model induces bursting and increases the number of spikes within a burst but is neither necessary nor sufficient for bursting. We show, using fast-slow analysis and bifurcation theory, that the M-type K(+) current (I(M)) allows bursting by shifting neuronal behavior between a silent and a tonically active state provided the kinetics of the spike generating currents are sufficiently, although not extremely, fast. We suggest that bursting in CA1 pyramidal cells can be explained by a single compartment "square bursting" mechanism with one slow variable, the activation of I(M).     

Afterhyperpolarization current in myenteric neurons of the guinea pig duodenum

Whole cell patch and cell-attached recordings were obtained from neurons in intact ganglia of the myenteric plexus of the guinea pig duodenum. Two classes of neuron were identified electrophysiologically: phasically firing AH neurons that had a pronounced slow afterhyperpolarization (AHP) and tonically firing S neurons that lacked a slow AHP. We investigated the properties of the slow AHP and the underlying current (I(AHP)) to address the roles of Ca(2+) entry and Ca(2+) release in the AHP and the characteristics of the K(+) channels that are activated. AH neurons had a resting potential of -54 mV and the AHP, which followed a volley of three suprathreshold depolarizing current pulses delivered at 50 Hz through the pipette, averaged 11 mV at its peak, which occurred 0.5-1 s following the stimulus. The duration of these AHPs averaged 7 s. Under voltage-clamp conditions, I(AHP)'s were recorded at holding potentials of -50 to -65 mV, following brief depolarization of AH neurons (20-100 ms) to positive potentials (+35 to +50 mV). The null potential of the I(AHP) at its peak was -89 mV. The AHP and I(AHP) were largely blocked by omega-conotoxin GVIA (0.6-1 microM). Both events were markedly decreased by caffeine (2-5 mM) and by ryanodine (10-20 microM) added to the bathing solution. Pharmacological suppression of the I(AHP) with TEA (20 mM) or charybdotoxin (50-100 nM) unmasked an early transient inward current at -55 mV following step depolarization that reversed at -34 mV and was inhibited by niflumic acid (50-100 microM). Mean-variance analysis performed on the decay of the I(AHP) revealed that the AHP K(+) channels have a mean chord conductance of ~10 pS, and there are ~4,000 per AH neuron. Spectral analysis showed that the AHP channels have a mean open dwell time of 2.8 ms. Cell-attached patch recordings from AH neurons confirmed that the channels that open following action currents have a small unitary conductance (10-17 pS) and open with a high probability (相似文献   

Development and serotonergic modulation of NMDA bursting in rat trigeminal motoneurons

The development of N-methyl-D-aspartate (NMDA)-induced burst discharge in rat trigeminal motoneurons (TMNs) between postnatal days P1 and P10 was examined using whole cell patch-clamp recording methods in brain slices. Bath application of NMDA (50 microM) induced a Mg(2+)-dependent rhythmical bursting activity starting around P8. Prior to the onset of bursting, the membrane potential depolarized and the input resistance increased. Hyperpolarization of the membrane potential with extrinsic current demonstrated a narrow window of membrane potential where maintained rhythmical burst discharge was evident. In P1-P4 neurons, NMDA application produced membrane depolarization and a minimal change in input resistance, but no burst activity at any membrane potential. Voltage-clamp analysis indicated that the bursting activity was related to the presence or absence of a voltage-dependent Mg(2+) block and induction of a negative slope conductance (NSC) region in the I(NMDA)-V relationship. Regardless of age, reduction of extracellular Mg(2+) from 1 mM to 30 microM enhanced I(NMDA) at voltages negative to -60 mV. However, in 1 mM Mg(2+), P1-P4 neurons were devoid of a prominent NSC region compared with P8-P10 neurons, suggesting that the efficacy of depolarization in unblocking the NMDA receptors increased with age. NMDA bursting was not dependent on calcium influx through voltage-gated calcium channels (VGCC) but did require a minimal concentration of Ca(2+) in the bath. Intracellular bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid application suppressed burst discharge completely, suggesting that intracellular Ca(2+) directly, or via second-messenger systems, regulates NMDA receptor activity and bursting. Interestingly, NMDA bursting could be induced in P1-P4 neurons by simultaneous bath application of serotonin (5-HT, 10 microM), which by itself did not produce bursting, suggesting an "enabling" role for 5-HT. Voltage-clamp analysis demonstrated that the NMDA/5-HT bursting resulted from induction of an NSC in the I-V relationship of total membrane current. 5-HT by itself produced no such effect. The mechanisms for this effect were due to an enhancement of the NSC region of the I(NMDA)-V relationship and reduction of a presumed leak current by 5-HT. These data indicate that NMDA bursting in trigeminal motoneurons is developmentally regulated and subject to neuromessenger modulation. Control of the Mg(2+) sensitivity of the NMDA receptor and voltage-dependent block by neuromessengers could be an effective means to control the efficacy of glutamatergic synaptic drive to motoneurons during rhythmical oral-motor activity at early postnatal ages.     

Calcium dynamics underlying pacemaker-like and burst firing oscillations in midbrain dopaminergic neurons: a computational study

A mathematical model of midbrain dopamine neurons has been developed to understand the mechanisms underlying two types of calcium-dependent firing patterns that these cells exhibit in vitro. The first is the regular, pacemaker-like firing exhibited in a slice preparation, and the second is a burst firing pattern sometimes exhibited in the presence of apamin. Because both types of oscillations are blocked by nifedipine, we have focused on the slow calcium dynamics underlying these firing modes. The underlying oscillations in membrane potential are best observed when action potentials are blocked by the application of TTX. This converts the regular single-spike firing mode to a slow oscillatory potential (SOP) and apamin-induced bursting to a slow square-wave oscillation. We hypothesize that the SOP results from the interplay between the L-type calcium current (I(Ca,L)) and the apamin-sensitive calcium-activated potassium current (I(K,Ca,SK)). We further hypothesize that the square-wave oscillation results from the alternating voltage activation and calcium inactivation of I(Ca,L). Our model consists of two components: a Hodgkin-Huxley-type membrane model and a fluid compartment model. A material balance on Ca(2+) is provided in the cytosolic fluid compartment, whereas calcium concentration is considered constant in the extracellular compartment. Model parameters were determined using both voltage-clamp and calcium-imaging data from the literature. In addition to modeling the SOP and square-wave oscillations in dopaminergic neurons, the model provides reasonable mimicry of the experimentally observed response of SOPs to TEA application and elongation of the plateau duration of the square-wave oscillations in response to calcium chelation.     

Effect of lamotrigine on the Ca(2+)-sensing cation current in cultured hippocampal neurons.

Concentrations of extracellular calcium ([Ca(2+)](e)) in the CNS decrease substantially during seizure activity. We have demonstrated previously that decreases in [Ca(2+)](e) activate a novel calcium-sensing nonselective cation (csNSC) channel in hippocampal neurons. Activation of csNSC channels is responsible for a sustained membrane depolarization and increased neuronal excitability. Our study has suggested that the csNSC channel is likely involved in generating and maintaining seizure activities. In the present study, the effects of anti-epileptic agent lamotrigine (LTG) on csNSC channels were studied in cultured mouse hippocampal neurons using patch-clamp techniques. At a holding potential of -60 mV, a slow inward current through csNSC channels was activated by a step reduction of [Ca(2+)](e) from 1.5 to 0.2 mM. LTG decreased the amplitude of csNSC currents dose dependently with an IC(50) of 171 +/- 25.8 (SE) microM. The effect of LTG was independent of membrane potential. In the presence of 300 microM LTG, the amplitude of csNSC current was decreased by 31 +/- 3% at -60 mV and 29 +/- 2.9% at +40 mV (P > 0.05). LTG depressed csNSC current without affecting the potency of Ca(2+) block of the current (IC(50) for Ca(2+) block of csNSC currents in the absence of LTG: 145 +/- 18 microM; in the presence of 300 microM LTG: 136 +/- 10 microM. n = 5, P > 0.05). In current-clamp recordings, activation of csNSC channel by reducing the [Ca(2+)](e) caused a sustained membrane depolarization and an increase in the frequency of spontaneous firing of action potentials. LTG (300 microM) significantly inhibited csNSC channel-mediated membrane depolarization and the excitation of neurons. Fura-2 ratiometric Ca(2+) imaging experiment showed that LTG also inhibited the increase in intracellular Ca(2+) concentration induced by csNSC channel activation. The effect of LTG on csNSC channels may partially contribute to its broad spectrum of anti-epileptic actions.     

Ca2+-activated K+-current density is correlated with soma size in rat vestibular-afferent neurons in culture

Vestibular-afferent neurons (VANs) transmit information about linear and angular accelerations during head movements from vestibular end organs to vestibular nuclei. In situ, these neurons show heterogeneous discharge patterns that may be produced by differences in their intrinsic properties. However, little is known about the ionic currents underlying their different firing patterns. Using the whole cell patch-clamp technique, we analyzed the expression of Ca(2+) and Ca(2+)-activated K(+) currents (I(KCa)) in primary cultured neurons isolated from young rats (p7-p10). We found two overlapping subpopulations of VANs classified according to low-threshold Ca(2+)-current [low-voltage-activated (LVA)] expression; LVA (-) neurons, formed by small cells, and LVA (+) neurons composed of medium to large cells. The I(KCa) in both cell-groups was carried through channels of high (BK), intermediate (IK), and low conductance (SK), besides a resistant channel to classical blockers (IR). BK was expressed preferentially in LVA (+) cells, whereas IR expression was preferentially in LVA (-) cells. No correlation between SK and IK expression with the soma size was found. Current-clamp experiments showed that BK participates in the adaptation of discharge and in the duration of the action potential, whereas SK and IK did not show a significant contribution to electrical discharge of cultured VANs. However, because of the low number of VANs in culture with repetitive firing it is difficult to interpret our results in terms of discharge patterns. Our results demonstrate that vestibular-afferent neurons possess different Ca(2+)-activated K(+) (K(Ca)) channels and that their expression, heterogeneous among the cells, would contribute to explain some of the differences in the electrical-firing properties of these neurons.     

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

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

Role of membrane potential in calcium signaling during rhythmic bursting in tritonia swim interneurons

Rhythmic bursting in neurons is accompanied by dynamic changes in intracellular Ca(2+) concentration. These Ca(2+) signals may be caused by membrane potential changes during bursting and/or by synaptic inputs. We determined that membrane potential is responsible for most, if not all, of the cytoplasmic Ca(2+) signal recorded during rhythmic bursting in two neurons of the escape swim central pattern generator (CPG) of the mollusk, Tritonia diomedea: ventral swim interneuron B (VSI) and cerebral neuron 2 (C2). Ca(2+) signals were imaged with a confocal laser scanning microscope while the membrane potential was recorded at the soma. During the swim motor pattern (SMP), Ca(2+) signals in both neurons transiently increased during each burst of action potentials with a more rapid decay in secondary than in primary neurites. VSI and C2 were then voltage-clamped at the soma, and each neuron's own membrane potential waveform recorded during the SMP was played back as the voltage command. In all regions of VSI, this completely reproduced the amplitude and time course of Ca(2+) signals observed during the SMP, but in C2, the amplitude was lower in the playback experiments than during the SMP, possibly due to space clamp problems. Therefore in VSI, the cytoplasmic Ca(2+) signal during the SMP can be accounted for by its membrane potential excursions, whereas in C2 the membrane potential excursions can account for most of the SMP Ca(2+) signal.