Spike Repolarization Research Articles

Contribution of potassium conductances to a time-dependent transition in electrical properties of a cockroach motoneuron soma.

Contribution of potassium conductances to a time-dependent transition in electrical properties of a cockroach motoneuron soma. The cell body of the cockroach (Periplaneta americana) fast coxal depressor motoneuron (Df) displays a time-dependent change in excitability. Immediately after dissection, depolarization evokes plateau potentials, but after several hours all-or-none action potentials are evoked. Because K channel blockers have been shown to produce a similar transition in electrical properties, we have used current-clamp, voltage-clamp and action-potential-clamp recording to elucidate the contribution of different classes of K channel to the transition in electrical activity of the neuron. Apamin had no detectable effect on the neuron, but charybdotoxin (ChTX) caused a rapid transition from plateau potentials to spikes in the somatic response of Df to depolarization. In neurons that already produced spikes when depolarized, ChTX increased spike amplitude but did not increase their duration nor decrease the amplitude of their afterhyperpolarization. 4-Aminopyridine (4-AP) (which selectively blocks transient K currents) did not cause a transition from plateau potentials to spikes but did enhance oscillations superimposed on plateau potentials. When applied to neurons that already generated spikes when depolarized, 4-AP could augment spike amplitude, decrease the latency to the first spike, and prolong the afterhyperpolarization. Evidence suggests that the time-dependent transition in electrical properties of this motoneuron soma may result, at least in part, from a fall in calcium-dependent potassium current (IK,Ca), consequent on a gradual reduction in [Ca2+ ]i. Voltage-clamp experiments demonstrated directly that outward K currents in this neuron do fall with a time course that could be significant in the transition of electrical properties. Voltage-clamp experiments also confirmed the ineffectiveness of apamin and showed that ChTX blocked most of IK,Ca. Application of Cd2+ (0.5 mM), however, caused a small additional suppression in outward current. Calcium-insensitive outward currents could be divided into transient (4-AP-sensitive) and sustained components. The action-potential-clamp technique revealed that the ChTX-sensitive current underwent sufficient activation during the depolarizing phase of plateau potentials to enable it to shunt inward conductances. Although the ChTX-sensitive conductance apparently makes little contribution to spike repolarization, the ChTX-resistant IK,Ca does make a significant contribution to this phase of the action potential. The 4-AP-sensitive current began to develop during the rising phase of both action potentials and plateau potentials but had little effect on the electrical activity of the neuron, probably because of its relatively small amplitude.

Intracellular pH buffering shapes activity-dependent Ca2+ dynamics in dendrites of CA1 interneurons.

Voltage-gated calcium (Ca) channels are highly sensitive to cytosolic H+, and Ca2+ influx through these channels triggers an activity-dependent fall in intracellular pH (pHi). In principle, this acidosis could act as a negative feedback signal that restricts excessive Ca2+ influx. To examine this possibility, whole cell current-clamp recordings were taken from rat hippocampal interneurons, and dendritic Ca2+ transients were monitored fluorometrically during spike trains evoked by brief depolarizing pulses. In cells dialyzed with elevated internal pH buffering (high beta), trains of >15 action potentials (Aps) provoked a significantly larger Ca2+ transient. Voltage-clamp analysis of whole cell Ca currents revealed that differences in cytosolic pH buffering per se did not alter baseline Ca channel function, although deliberate internal acidification by 0.3 pH units blunted Ca currents by approximately 20%. APs always broadened during a spike train, yet this broadening was significantly greater in high beta cells during rapid but not slow firing rates. This effect of internal beta on spike repolarization could be blocked by cadmium. High beta also 1) enhanced the slow afterhyperpolarization (sAHP) seen after a spike train and 2) accelerated the decay of an early component of the sAHP that closely matched a sAHP conductance that could be blocked by apamin. Both of these effects on the sAHP could be detected at high but not low firing rates. These data suggest that activity-dependent pHi shifts can blunt voltage-gated Ca2+ influx and retard submembrane Ca2+ clearance, suggesting a novel feedback mechanism by which Ca2+ signals are shaped and coupled to the level of cell activity.

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

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

Synaptic Current at the Rat Ganglionic Synapse and Its Interactions With the Neuronal Voltage-Dependent Currents

The membrane current activated by fast nicotinic excitation of intact and mature rat sympathetic neurons was studied at 37 degrees C, by using the two-microelectrode voltage-clamp technique. The excitatory postsynaptic current (EPSC) was modeled as the difference between two exponentials. A fast time constant (tau2; mean value 0.57 ms), which proves to be virtually voltage-independent, governs the current rise phase and a longer time constant (tau1; range 5.2-6.8 ms in 2 mM Ca2+) describes the current decay and shows a small negative voltage dependence. A mean peak synaptic conductance of 0.58 muS per neuron is measured after activation of the whole presynaptic input in 5 mM Ca2+ external solution (0.40 muS in 2 mM Ca2+). The miniature EPSCs also rise and decay with exponential time constants very similar to those of the compound EPSC recorded at the same voltage. A mean peak conductance of 4.04 nS is estimated for the unitary event. Deconvolution procedures were employed to decompose evoked macrocurrents. It is shown that under appropriate conditions the duration of the driving function describing quantal secretion can be reduced to <1 ms. The shape of the EPSC is accurately mimicked by a complete mathematical model of the sympathetic neuron incorporating the kinetic properties of five different voltage-dependent current types, which were characterized in a previous work. We show that IA channels are opened by depolarizing voltage steps or by synaptic potentials in the subthreshold voltage range, provided that the starting holding voltage is sufficiently negative to remove IA steady-state inactivation (less than -50 mV) and the voltage trajectories are sufficiently large to enter the IA activation range (greater than -65 mV). Under current-clamp conditions, this gives rise to an additional fast component in the early phase of membrane repolarization-in response to voltage pulses-and to a consistent distortion of the excitatory postsynaptic potential (EPSP) time course around its peak-in response to the synaptic signal. When the stimulation initiates an action potential, IA is shown to significantly increase the synaptic threshold conductance (up to a factor of 2 when IA is fully deinactivated), compared with that required when IA is omitted. The voltage dependence of this effect is consistent with the IA steady-state inactivation curve. It is concluded that IA, in addition to speeding up the spike repolarization process, also shunts the excitatory drive and delays or prevents the firing of the neuron action potential.

Intrinsic membrane characteristics distinguish two subsets of nociceptive modulatory neurons in rat RVM.

Pain modulating neurons of the rostral ventromedial medulla (RVM) include three physiologically distinct classes of neurons in intact, anesthetized animals: and cells that change their activity before the onset of withdrawal reflexes and cells, which have activity unrelated to withdrawal reflexes. A previous in vitro intracellular study demonstrated that the RVM contains two types of neurons that are distinguished by their action-potential characteristics. The present in vivo intracellular study examined whether these intracellularly recorded action-potential characteristics are correlated with the physiological response properties of RVM neurons recorded. RVM neurons exhibited two distinct types of action potentials in vivo. Fast-spike (FS) neurons (n = 30) had short-duration action potentials (0.27 +/- 0.02 (SE) ms at half amplitude) and biphasic afterhyperpolarizations with a characteristic rapid overshooting spike repolarization. Slow-spike (SS) neurons (n = 25) had longer duration action potentials (0.44 +/- 0.02 ms at half-amplitude) due to a slower-spike repolarization rate and monophasic afterhyperpolarization. and cell classes included both FS and SS neurons. FS and neurons had an early onset response to noxious heat stimulation. SS and cells showed a delayed onset response to noxious heat. cells (n = 13) were all SS cells. Among the SS neurons, only cells had action potentials longer than 0. 45 ms (n = 9). FS and SS neurons were intermingled throughout the RVM. The majority of intracellularly labeled cells (n = 15) had fusiform somata with two to five fine caliber primary dendrites and a predominantly mediolateral orientation of the long axis of their dendritic tree. All labeled FS cells (n = 5) had large, multipolar somata with four to nine large caliber primary dendrites. The present study defines in vivo membrane and morphological characteristics of RVM neurons that correlate with physiological differences and may be used for identification of nociceptive modulatory RVM neurons in slice preparations.

Synaptic activation of Ca2+ action potentials in immature rat cerebellar granule cells in situ.

Although numerous Ca2+ channels have been identified in cerebellar granule cells, their role in regulating excitability remained unclear. We therefore investigated the excitable response in granule cells using whole cell patch-clamp recordings in acute rat cerebellar slices throughout the time of development (P4-P21, n = 183), with the aim of identifying the role of Ca2+ channels and their activation mechanism. After depolarizing current injection, 46% of granule cells showed Ca2+ action potentials, whereas repetitive Na+ spikes were observed in an increasing proportion of granule cells from P4 to P21. Because Ca2+ action potentials were no longer observed after P21, they characterized an immature granule cell functional stage. Ca2+ action potentials consisted of an intermediate-threshold spike (ITS) activating at -60/-50 mV and sensitive to voltage inactivation and of a high-threshold spike (HTS), activating at above -30 mV and resistant to voltage inactivation. Both ITS and HTS comprised transient and protracted Ca2+ channel-dependent depolarizations. The Ca2+ action potentials could be activated synaptically by excitatory postsynaptic potentials, which were significantly slower and had a proportionately greater N-methyl-D-aspartate (NMDA) receptor-mediated component than those recorded in cells with fast repetitive Na+ spikes. The NMDA receptor current, by providing a sustained and regenerative current injection, was critical for activating the ITS, which was not self-regenerative. Moreover, NMDA receptors determined temporal summation of impulses during repetitive mossy fiber transmission, raising membrane potential into the range required for generating protracted Ca2+ channel-dependent depolarizations. The nature of Ca2+ action potentials was considered further using selective ion channel blockers. N-, L-, and P-type Ca2+ channels generated protracted depolarizations, whereas the ITS and HTS transient phase was generated by putative R-type channels (R(ITS) and R(HTS), respectively). R(HTS) channels had a higher activation threshold and were more resistant to voltage inactivation than R(ITS) channels. At a mature stage, most of the Ca2+-dependent effects depended on the N-type current, which promoted spike repolarization and regulated the Na+-dependent discharge frequency. These observations relate Ca2+ channel types with specific neuronal excitable properties and developmental states in situ. Synaptic NMDA receptor-dependent activation of Ca2+ action potentials provides a sophisticated mechanism for Ca2+ signaling, which might be involved in granule cell development and plasticity.

Mechanisms for signal transformation in lemniscal auditory thalamus.

1. During alertness, lemniscal thalamocortical neurons in the ventral medial geniculate body (MGBv) encode sound signals by firing action potentials in a tonic mode. When they are in a burst firing mode, characteristic of thalamic neurons during some sleep states, the same stimuli may have an alerting function, leading to conscious perception of sound. We investigated the intrinsic membrane properties of MGBv neurons in search of mechanisms that enable them to convert from burst to tonic firing modes, allowing accurate signal coding of sensory stimuli. 2. We studied thalamocortical relay neurons and identified neurons morphologically with injected N-(2-aminoethyl) biotinamide hydrochloride in in vitro slice preparations of young rats. With the use of the whole cell recording method, we examined the contributions of distinct conductances to voltage responses evoked by current pulses. The neurons (n = 74) displayed a narrow range of resting potentials (-68 +/- 4 mV, mean +/- SD) and an average input resistance of 226 +/- 100 M omega. The membrane time constant was 40 +/- 17.6 ms and the action potential threshold was -51.6 +/- 3 mV. 3. Injections of hyperpolarizing current pulses from rest revealed an inward rectification produced by two voltage-dependent components. A fast component, sensitive to blockade with Ba2+ (100-200 microM), was attributed to an inward rectifier, IIR. Such applications also increased input resistance and depolarized neurons, consistent with a blockade of various K+ conductances. Application of Ba2+ often unmasked another voltage-dependent rectification with a slower time course. The second component was sensitive to blockade with Cs+ (1.5 mM), reminiscent of a hyperpolarization-activated current, IH. 4. Depolarizing pulses from rest produced ramp-shaped voltage responses that led to delayed tonic firing. Blockade of Na+ conductances by tetrodotoxin (TTX, 300-600 nM), or extracellular replacement of Ca2+ with Mg2+ (with TTX present), reduced the slope of the ramp and the overall depolarizing response. Application of 4-aminopyridine (4-AP, 100 microM), a blocker of A-type K+ conductances, increased input resistance and the overall depolarizing response. The voltage ramp therefore represents a complex rectification due to voltage-dependent contributions of persistent Na-, Ca2+, and K+ conductances. 5. Depolarizing pulses from potentials of less than -75 mV evoked phasic burst responses, consisting of one to seven action potentials riding on a low-threshold spike (LTS). The LTS was absent in low extracellular Ca2+ conditions and was blocked by application of Ni2+ (0.6 mM), but not by Cd2+ (50 microM). Similar depolarization from less than -80 mV evoked several action potentials, often followed by a TTX-resistant high-threshold spike (HTS) of longer duration. Firing of HTSs always occurred during 4-AP (100 microM) application, inferring that, normally, A-type K+ conductances may control ability to fire an HTS. As in the LTS, a Ca2+ current is a major participant in the HTS because extracellular replacement of Ca2+ with Mg2+ or application of Cd2+ (50 microM) blocked its genesis. After TTX blockade of Na+ conductances, "tonic firing" of HTSs occurred during depolarization above -45 mV. 6. During tonic firing evoked by current pulses, the second and subsequent spikes were longer in duration than the initial action potentials. Low extracellular concentrations of Ca2+ or Cd2+ (50 microM) application reduced the durations of the nonprimary spikes, inferring a contribution of high-threshold voltage-dependent Ca2+ conductances to their repolarizing phase. Also, K+ conductances may contribute to spike repolarization, because 4-AP (100 microM) or tetraethylammonium (2 mM) application led to prolonged action potentials and the generation of plateau potentials. A fast afterhyperpolarization, likely mediated by a Ca(2+)-dependent K+ conductance, limited the tonic firing. Such conductances, therefore, may regulate the re

Characterization of voltage-sensitive Na+ and K+ currents recorded from acutely dissociated pelvic ganglion neurons of the adult rat.

1. Electrophysiological properties of acutely dissociated neurons from the major pelvic ganglion (MPG) of the adult male rat were studied with whole cell patch-clamp recording techniques. The MPG neurons innervating the urinary bladder were labeled by retrograde axonal tracing methods with the use of a fluorescent dye, Fast Blue (FB) injected into the bladder wall and identified with a fluorescent microscope. 2. Passive and active membrane properties such as resting membrane potential, input resistance, duration of action potentials, thresholds for spike activation, or duration of afterhyperpolarization in unidentified MPG neurons were comparable with those of FB-labeled neurons innervating the urinary bladder. The action potential in both unidentified and bladder efferent MPG neurons was reversibly abolished by tetrodotoxin (TTX, 1 microM). The afterhyperpolarization of the TTX-sensitive action potential in both groups was reduced by application of Cd2+ (0.1 mM) and further suppressed by tetraethylammonium (TEA, 10 mM). Extracellularly applied TEA increased the duration of the action potential, and 4-aminopyridine (4-AP, 1 or 2 mM) also reduced the spike afterhyperpolarization and increased the spike duration. The duration of the action potential was decreased and the rate of spike repolarization was increased by approximately 2.5-fold with negative shift of membrane potential from -40 to -80 mV. 3. The isolated Na+ current was reversibly blocked by 1 microM TTX and had a mean peak amplitude of 127.3 pA/pF when activated from a holding potential of -70 mV in the external solution containing 100 mM Na+. The Na+ conductance reached half-maximal activation at a membrane potential of -21.5 mV with a slope factor of 4.9 mV. The steady-state inactivation of Na+ conductance occurred at membrane potentials more depolarized than -90 mV, and the half-maximal inactivation was obtained at -57.5 mV with a slope factor of 8.8 mV. 4. The fast-transient A-type K+ current (IA) was activated at membrane potentials more depolarized than -60 mV from a holding membrane potential of -100 mV, reached a peak amplitude within 10 ms after the onset of depolarizing voltage steps, and decayed within 20-30 ms at membrane potential depolarizations to +20 to +30 mV. The IA current activated by a voltage step to +20 mV from a holding potential of -100 mV averaged 102.1 pA/pF. The half-maximal activation of the IA conductance was obtained at a membrane potential of -21.2 mV with a slope factor of 9.9 mV. In steady-state inactivation of IA current, the half-maximal inactivation occurred at -76.5 mV and the slope factor was 8.0 mV. 5. The delayed K+ current was reduced by 25-35% by bath application of Cd2+ or the elimination of extracellular Ca2+ ions. The bath application of 4-AP (2 mM) suppressed the IA current by 75% and the delayed K+ current by 60%. Extracellularly applied TEA (10 mM) suppressed the delayed K+ current by 90%, but suppressed the IA current by only 16%. 6. These results indicate that bladder neurons and unidentified neurons in the MPG have similar properties including a TTX-sensitive Na+ current and three distinct types of voltage-sensitive K+ currents-IA current, Ca(2+)-activating K+ current, and delayed rectifier K+ current-that contribute to the repolarization phase of the action potential. These electrical properties of the MPG neurons resemble those of sympathetic neurons in the superior cervical and inferior mesenteric ganglia.

Potassium currents and membrane excitability of neurons in the rat's dorsal nucleus of the lateral lemniscus.

1. The contribution of voltage-activated outward potassium currents to membrane excitability of neurons in the rat's dorsal nucleus of the lateral lemniscus (DNLL) was studied in a brain slice preparation using whole cell patch-clamp and intracellular recordings. Voltage-clamp methods and pharmacological manipulations were used to examine the currents regulating membrane dynamics in DNLL. 2. A delayed sustained outward current was evoked by applying depolarizing voltage steps across the cell membrane from a holding potential of -50 mV. An additional transient outward current was evoked when the depolarizing steps were preceded by a hyperpolarizing prepulse of -110 or -120 mV. 3. The transient outward current peaked within 6.8 ms of the onset of a depolarizing pulse. It decayed with a time constant of 12.3 ms for a 60-mV depolarizing voltage shift. Half-inactivation of this current occurred at -81.3 mV. The time constant for removal of the inactivation was 17.4 ms. The transient current had a high sensitivity to 4-aminopyridine (4-AP). 4. The sustained current was activated more slowly and was more sensitive to tetraethylammonium (TEA) than the transient current. The sustained current had both Ca2+-dependent and Ca2+-independent components. The Ca2+-dependent portion emerged at potentials of about -35 mV and was activated fully at +10 mV. The Ca2+-independent component was activated at potentials more positive than -40 mV and increased in magnitude with further depolarization. Inactivation of the Ca2+-independent component was voltage dependent. Also, TEA suppressed the Ca2+-independent compound. 5. The transient current in DNLL neurons closely resembled the A current (IA) described for hippocampal and other neurons in both kinetics and pharmacology. The Ca2+-independent component of the sustained current resembled the K current (IK) described for other neurons in both its properties of activation and inactivation and its pharmacology. 6. The outward current of some DNLL neurons was found to contain a dendrotoxin-sensitive component. This component reached its peak at 6.8 ms and had voltage-sensitive time constants of decay of 25.5 and 8.5 ms with voltage steps of 40 and 60 mV, respectively. 7. Application of 4-AP and TEA markedly prolonged the spike width, abolished the fast component of the after hyperpolarization and depolarized the cell membrane. Also, the number of action potentials produced by positive current injection increased under the influence of 4-AP and TEA. Membrane excitability and spike repolarization were dependent on both 4-AP-sensitive transient and TEA-sensitive sustained currents. 8. Neurons in DNLL typically exhibit a steady discharge of action potentials in response to sustained membrane depolarization. The rate and temporal pattern of production of action potentials in these cells are determined by the combination of transient and sustained potassium channels.

The Role of K+Currents in Frequency-Dependent Spike Broadening inAplysiaR20 Neurons: A Dynamic-Clamp Analysis

The R20 neurons of Aplysia exhibit frequency-dependent spike broadening. Previously, we had used two-electrode voltage clamp to examine the mechanisms of this spike broadening (Ma and Koester, 1995). We identified three K+ currents that mediate action-potential repolarization: a transient A-type K+ current (I(Adepol)), a delayed rectifier current (IK-V), and a Ca(2+)-sensitive K+ current(IK-CA). A major constraint in that study was the lack of completely selective blockers for I(Adepol) and I(K-V), resulting in an inability to assess directly the effects of their activation and inactivation on spike broadening. In the present study, the dynamic-clamp technique, which employs computer simulation to inject biologically realistic currents into a cell under current-clamp conditions (Sharp et al., 1993a,b), was used either to block I(Adepol) or I(K-V) or to modify their inactivation properties. The data in this paper, together with earlier results, lead to the following hypothesis for the mechanism of spike broadening in the R20 cells. As the spike train progresses, the primary responsibility for spike repolarization gradually shifts from I(Adepol) to I(K-V) to I(K-Ca). This sequence can be explained on the basis of the relative rates of activation and inactivation of each current with respect to the constantly changing spike durations, the cumulative inactivation of I(Adepol) and I(K-V), and the progressive potentiation of I(K-Ca). Positive feedback interactions between spike broadening and inactivation contribute to the cumulative inactivation of both I(Adepol) and I(K-V). The data also illustrate that when two or more currents have similar driving forces and partially overlapping activation characteristics, selectively blocking one current under current-clamp conditions can lead to a significant underestimate of its normal physiological importance.

Slow inactivation of Na+ current and slow cumulative spike adaptation in mouse and guinea-pig neocortical neurones in slices.

1. Spike adaptation of neocortical pyramidal neurones was studied with sharp electrode recordings in slices of guinea-pig parietal cortex and whole-cell patch recordings of mouse somatosensory cortex. Repetitive intracellular stimulation with 1 s depolarizing pulses delivered at intervals of < 5 s caused slow, cumulative adaptation of spike firing, which was not associated with a change in resting conductance, and which persisted when Co2+ replaced Ca2+ in the bathing medium. 2. Development of slow cumulative adaptation was associated with a gradual decrease in maximal rates of rise of action potentials, a slowing in the post-spike depolarization towards threshold, and a positive shift in the threshold voltage for the next spike in the train; maximal spike repolarization rates and after-hyperpolarizations were unchanged. 3. The data suggested that slow adaptation reflects use-dependent removal of Na+ channels from the available pool by an inactivation process which is much slower than fast, Hodgkin-Huxley-type inactivation. 4. We therefore studied the properties of Na+ channels in layer II-III mouse neocortical cells using the cell-attached configuration of the patch-in-slice technique. These had a slope conductance of 18 +/- 1 pS and an extrapolated reversal potential of 127 +/- 6 mV above resting potential (Vr) (mean +/- S.E.M.; n = 5). Vr was estimated at -72 +/- 3 mV (n = 8), based on the voltage dependence of the steady-state inactivation (h infinity) curve. 5. Slow inactivation (SI) of Na+ channels had a mono-exponential onset with tau on between 0.86 and 2.33 s (n = 3). Steady-state SI was half-maximal at -43.8 mV and had a slope of 14.4 mV (e-fold)-1. Recovery from a 2 s conditioning pulse was bi-exponential and voltage dependent; the slow time constant ranged between 0.45 and 2.5 s at voltages between-128 and -68 mV. 6. The experimentally determined parameters of SI were adequate to simulate slow cumulative adaptation of spike firing in a single-compartment computer model. 7. Persistent Na+ current, which was recorded in whole-cell configuration during slow voltage ramps (35 mV s-1), also underwent pronounced SI, which was apparent when the ramp was preceded by a prolonged depolarizing pulse.

Spike after-depolarization and burst generation in adult rat hippocampal CA1 pyramidal cells.

1. Intracellular recordings in adult rat hippocampal slices were used to investigate the properties and origins of intrinsically generated bursts in the somata of CA1 pyramidal cells (PCs). The CA1 PCs were classified as either non-bursters or bursters according to the firing patterns evoked by intrasomatically applied long ( > or = 100 ms) depolarizing current pulses. Non-bursters generated stimulus-graded trains of independent action potentials, whereas bursters generated clusters of three or more closely spaced spikes riding on a distinct depolarizing envelope. 2. In all PCs fast spike repolarization was incomplete and ended at a potential approximately 10 mV more positive than resting potential. Solitary spikes were followed by a distinct after-depolarizing potential (ADP) lasting 20-40 ms. The ADP in most non-bursters declined monotonically to baseline ('passive' ADP), whereas in most bursters it remained steady or even re-depolarized before declining to baseline ('active' ADP). 3. Active, but not passive, ADPs were associated with an apparent increase in input conductance. They were maximal in amplitude when the spike was evoked from resting potential and were reduced by mild depolarization or hyperpolarization (+/- 2 mV). 4. Evoked and spontaneous burst firing was sensitive to small changes in membrane potential. In most cases maximal bursts were generated at resting potential and were curtailed by small depolarizations or hyperpolarizations (+/- 5 mV). 5. Bursts comprising clusters of spikelets ('d-spikes') were observed in 12% of the bursters. Some of the d-spikes attained threshold for triggering full somatic spikes. Gradually hyperpolarizing these neurones blocked somatic spikes before blocking d-spikes, suggesting that the latter are generated at more remote sites. 6. The data suggest that active ADPs and intrinsic bursts in the somata of adult CA1 PCs are generated by a slow, voltage-gated inward current. Bursts arise in neurones in which this current is sufficiently large to generate suprathreshold ADPs, and thereby initiate a regenerative process of spike recruitment and slow depolarization.

Potassium conductances underlying repolarization and after‐hyperpolarization in rat CA1 hippocampal interneurones.

1. The roles of multiple potassium conductances underlying action potential repolarization and after-hyperpolarization (AHP) in visually identified st. oriens-alveus (st. O-A) inhibitory interneurones of neonatal rat CA1 hippocampal slices were determined using whole-cell patch clamp techniques. 2. 4-Aminopyridine dose-dependently prolonged the action potential repolarization. The effects of 4-AP persisted in Ca(2+)-free conditions. Action potentials evoked from hyperpolarized potentials possessed an increased rate of repolarization. These data suggest an involvement of the rapidly activating transient current, IA, in spike repolarization. 3. Action potential duration was increased in the presence of Ca(2+)-free, Cd(2+)-containing solution, iberiotoxin or 1 mM TEA. The fast component of the AHP was attenuated by these agents suggesting that the Ca(2+)-activated K+ conductance, IC, underlies both the spike repolarization and fast AHP. 4. In Ca(2+)-free conditions, TEA (> 1 mM) dose-dependently prolonged the action potential duration by blocking a late conductance in action potential repolarization, suggesting a role for the sustained current, IK. 5. The slow AHP was attenuated by Ca(2+)-free medium, apamin or the Ca2+ chelator EGTA, suggesting a role for the Ca(2+)-activated K+ conductance, IAHP. 6. We conclude that action potential repolarization and AHP of st. O-A interneurones result from the activation of pharmacologically distinct, temporally overlapping potassium conductances. These findings are discussed with reference to the voltage clamp data presented in the preceding manuscript.

Consequences and mechanisms of spike broadening of R20 cells in Aplysia californica

We studied frequency-dependent spike broadening in the two electrically coupled R20 neurons in the abdominal ganglion of Aplysia. The peptidergic R20 cells excite the R25/L25 interneurons (which trigger respiratory pumping) and inhibit the RB cells. When fired at 1-10 Hz, the duration of the falling phase of the action potential in R20 neurons increases 2-10 fold during a spike train. Spike broadening recorded from the somata of the R20 cells affected synaptic transmission to nearby follower cells. Chemically mediated synaptic output was reduced by approximately 50% when recorded trains of nonbroadened action potentials were used as command signals for a voltage-clamped R20 cell. Electrotonic EPSPs between the R20 cells, which normally facilitated by two- to fourfold during a high frequency spike train, showed no facilitation when spike broadening was prevented under voltage-clamp control. To examine the mechanism of frequency-dependent spike broadening, we applied two-electrode voltage-clamp and pharmacological techniques to the somata of R20 cells. Several voltage-gated ionic currents were isolated, including INa, a multicomponent ICa, and three K+ currents--a high threshold, fast transient A-type K+ current (IAdepol), a delayed rectifier K+ current (IK-V), and a Ca(2+)-sensitive K+ current (IK-Ca), made up of two components. The influences of different currents on spike broadening were determined by using the recorded train of gradually broadening action potentials as the command for the voltage clamp. We found the following. (1) IAdepol is the major outward current that contributes to repolarization of nonbroadened spikes. It undergoes pronounced cumulative inactivation that is a critical determinant of spike broadening. (2) Activity-dependent changes in IK-V, IK-Ca, and ICa have complex effects on the kinetics and extent of broadening. (3) The time integral of ICa during individual action potentials increases approximately threefold during spike broadening.

Diverse neuronal populations mediate local circuit excitation in area CA3 of developing hippocampus.

1. Studies were undertaken to better understand why the developing hippocampus has a marked capacity to generate prolonged synchronized discharges when exposed to gamma-aminobutyric acid-A (GABAA) receptor antagonists. 2. Excitatory synaptic interactions were studied in small microdissected segments of hippocampal area CA3. Slices were obtained from 10- to 16-day-old rats. Application of the GABAA receptor antagonist penicillin produced prolonged synchronized discharges in minislices that were very similar, if not identical, to those recorded in intact slices. The sizes of minislices were systematically varied. Greater than 90% of those that measured 600 microns along the cell body layer produced prolonged synchronized discharges, whereas most minislices measuring 300 microns produced only brief interictal spikes. 3. Action potentials in the majority (75%, 158 of 254) of cells impaled with microelectrodes were able to entrain the entire CA3 population. They were also able to increase (on average 26%) the frequency of spontaneous population discharges. The population discharges were followed by a refractory period that lasted 5-60 s, during which single cells were unable to initiate a population discharge. 4. The majority (87%) of neurons with intrinsic burst properties were found to entrain the CA3 population. The electrophysiological characteristics of these cells were reminiscent of recordings obtained from more mature rats. Action potentials were quite prolonged and demonstrated a secondary shoulder or hump on the down-slope of the spike. 5. When bursting cells were filled with Lucifer yellow and imaged during recording sessions by videomicroscopy and later using confocal microscopy, they showed the anatomic features of CA3 hippocampal pyramidal cells. Confocal microscopy permitted detailed characterization of individual neurons and showed substantial variation in cellular microanatomy. 6. Another class of cells that were found to entrain the CA3 population but did not demonstrate intrinsic bursts were termed regular-firing cells. These cells possessed many of the anatomic and physiological features of bursting cells with the exception of burst firing. They were rarely encountered in intracellular recordings. 7. The third physiological class of cells was termed fast-spiking cells. These had action potentials that were shorter in duration than the other two cell types. They were distinct in the rapid rate of spike repolarization. They demonstrated modest degrees of spike frequency adaptation and fired repeatedly and at relatively high frequencies. Compared with reports on fast-spiking cells in mature hippocampus and neocortex, action potentials appear to be slower and repetitive discharging appeared to be of a lower frequency.(ABSTRACT TRUNCATED AT 400 WORDS)

Regenerative properties of pyramidal cell dendrites in area CA1 of the rat hippocampus.

1. Intracellular recordings were obtained from 184 distal apical dendrites and twenty-six somata of CA1 pyramidal neurones in the rat hippocampal slice preparation. In the presence of 3.25 mM K+ 200 ms suprathreshold current pulses evoked three different types of firing patterns in the apical dendrites, all of which were distinct from regular somatic firing. Fast tetrodotoxin (TTX)-sensitive spiking was evoked in 38.8% of the dendrites. Compound spiking, consisting of an initial fast spike followed by one or more secondary slow spikes of variable amplitude and duration, was seen in 44.1% of dendrites. 'Classical' burst firing, resembling intrinsic somatic bursts, was evoked in 17.1% of the dendrites. 2. In fast spiking dendrites, the spikes evoked by long depolarizing pulses were rarely overshooting, showed prominent accommodation and declined progressively to about one-third of the initial amplitude. The amplitude of single dendritic fast spikes (50.6 +/- 1.5 mV; mean +/- S.E.M.) was smaller than that of somatic spikes (82.2 +/- 1.9 mV) and their rate of rise (81.3 +/- 4.3 V s-1) was markedly slower than that of somatic spikes (291.5 +/- 17.8 V s-1). However, the thresholds were not significantly different (dendrites, -49.8 +/- 0.8 mV; somata, -50.8 +/- 1.3 mV). These results indicate that fast spikes in the distal parts of apical dendrites are generated by a local regenerative Na+ current. 3. 4-Aminopyridine (4-AP, 0.1-0.5 mM) caused a dose-dependent slowing of the repolarization of the fast spikes, while tetraethylammonium (TEA, 2 mM) and Co2+ (2 mM) induced a slowing of the late phase of the repolarization. These results indicate that the transient outward K+ current, IA, and the Ca(2+)-activated K+ current, IC, are involved in the repolarization of dendritic Na(+)-dependent spikes. 4. Compound spiking was completely blocked by TTX (0.5-1 microM). The secondary slow spikes within the complex were blocked by Co2+ (2 mM), nifedipine (10 microM) and high concentrations (> 50 microM) of verapamil, while Ni2+ (100-300 microM) had no effect. Thus, compound spiking consists of an initial Na(+)-dependent spike followed by one or more slow Ca(2+)-dependent spikes mediated by L-type Ca2+ channels located in the apical dendrites. 5. In fast spiking dendrites, 4-AP (0.5-2.5 mM) changed the firing pattern from regular fast spiking to compound spiking. In the presence of 4-AP (0.1-0.5 mM), the single fast spike evoked by a short (20 ms), threshold current pulse, was followed by secondary slow spikes of variable amplitude and duration.(ABSTRACT TRUNCATED AT 400 WORDS)

Serotonergic modulation of locust motor neurons.

1. The effects of the putative endogenous neuromodulator serotonin (5-HT) on the fast extensor and flexor tibiae motor neurons in the locust (Schistocerca gregaria) metathoracic ganglion, were analyzed. 2. 5-HT consistently increased the duration of the fast extensor spike and usually reduced the afterhyperpolarization, although this effect was less consistent. The spike broadening in the fast extensor was associated with an increase in the amplitude of the excitatory postsynaptic potential (EPSP) evoked monosynaptically in the flexor motor neurons by fast extensor stimulation. 5-HT also increased the membrane resistance of the fast extensor and flexor tibiae motor neurons. 3. The effects of 5-HT were mimicked by bath application of the 5-HT uptake inhibitor imipramine, and blocked by the 5-HT receptor antagonist ketanserin. The effects were also mimicked by dibutryl cyclic AMP, a membrane permeant analogue of cyclic AMP, and by the phosphodiesterase inhibitor 3-isobutyl-1-methyl-xanthine, but not by dibutryl cyclic GMP. The 5-HT-dependent modulation was blocked by the protein kinase A inhibitor H8. In addition, injection of cyclic AMP into the fast extensor or a flexor motor neuron could mimic the effects of 5-HT on these neurons. 4. 5-HT probably broadened the FETi action potential by modulating potassium conductances responsible for spike repolarization. 5. These results show that 5-HT modulates both the fast extensor and flexor tibiae motor neurons, resulting in potentiation of synaptic transmission between these neurons. In addition, the increase in flexor membrane resistance will potentiate other inputs onto these cells, which will affect the output of the motor neurons during locomotion.

Ionic conductances contributing to spike repolarization and after-potentials in rat medial vestibular nucleus neurones.

1. Intracellular recordings were made from 123 tonically active medial vestibular nucleus (MVN) neurones in a horizontal slice preparation of the dorsal brainstem of the rat. On the basis of their averaged action potential shapes, the cells were classified as either type A, having a single deep after-hyperpolarization (AHP; 40/123 cells, 33%), or type B, having an early fast AHP and a delayed slow AHP (83/123 cells, 67%). The two cell types were distributed throughout the rostrocaudal extent of the MVN. 2. In type A cells TEA reduced the single deep AHP and decreased the rate of spike repolarization. Depolarizing current pulses from a hyperpolarized membrane potential elicited spikes with short plateau potentials in TEA. These persisted in Ca(2+)-free medium but were abolished along with the spontaneous activity in TTX. Ca(2+)-free medium did not affect the initial rate of repolarization but reduced the deep AHP. Apamin and carbachol had little effect. 4-Aminopyridine (4-AP) slowed spike repolarization and the AHP amplitude by a small amount. Thus, in type A cells spike repolarization and AHP appear to be mediated largely by a TEA-sensitive potassium current (presumably IK) and an apamin-insensitive Ca(2+)-activated potassium current (presumably IC). 3. The early fast AHP in type B cells was readily abolished in TEA. In seven of ten type B cells tested, the spontaneous spikes developed plateau potentials of 100-120 ms duration in 10 mM TEA, which then became 7-9 s long in Ca(2+)-free medium. In the remaining three cells, the spontaneous plateaux were 1.75-2 s long in TEA, and were reduced to 30-100 ms in Ca(2+)-free medium. TTX abolished the spontaneous spikes and plateaux. The delayed AHP was abolished by apamin, which induced irregular firing. 4-AP slowed spike repolarization and abolished the fast AHP, but did not induce plateaux. Thus, in type B cells spike repolarization involves a TEA-sensitive current (presumably IK) as well as IC and the 4-AP-sensitive potassium current IA, while the apamin-sensitive potassium current IAHP is responsible for the delayed AHP. 4. The tonic activity in type B cells appears to be regulated mainly by interactions between a persistent Na+ current, which in most cells is large enough to generate plateaux when repolarization is impeded in TEA, and the hyperpolarization mediated by IAHP. About 30% of type B cells have an additional inward Ca2+ current.(ABSTRACT TRUNCATED AT 400 WORDS)

CAMP-independent effects of 8-(4-parachlorophenylthio)-cyclic AMP on spike duration and membrane currents in pleural sensory neurons of Aplysia

1. The serotonergic modulation of pleural sensory neurons in Aplysia is mediated via two second messenger systems: the adenosine cyclic monophosphate/protein kinase A (cAMP/PKA) and diacylglycerol/protein kinase C systems. Often membrane permeable derivatives of cAMP, such as 8-(4-parachlorophenylthio)-cAMP (pcpt-cAMP), have been used to investigate the role of cAMP/PKA in modulating sensory neurons. In light of recent findings that pcpt-cAMP may have cAMP-independent actions, we have reexamined the effects of pcpt-cAMP on the action potential and membrane currents of the sensory neurons. 2. Although pcpt-cAMP (500 microM to 1 mM) and serotonin (5-HT; 10 microM) induced comparable measures of spike broadening (an average increase above baseline of 29 and 40%, respectively), the broadening produced by the two was qualitatively different. Serotonin-induced broadening developed slowly over 9-12 min, was most prominent during later phases of the spike repolarization, and reduced the spike afterhyperpolarization. In contrast, pcpt-cAMP-induced broadening developed rapidly, was rather uniform throughout the repolarization phase of the spike, delayed the peak of the action potential, and increased the afterhyperpolarization. 3. Preexposure of sensory neurons to 5-HT did not occlude further spike broaden by subsequent application of pcpt-cAMP. Indeed the effects of the two were additive. In addition, the effects of pcpt-cAMP were not mimicked by another analogue of cAMP, 8-bromo-cAMP. Interestingly, most of the effects of pcpt-cAMP on the action potential were mimicked by 8-(4-parachlorophenyl-thio)-guanosine cyclic monophosphate (pcpt-cGMP), but not by 8-bromo-cGMP. 4. During voltage-clamp pulses to 20 mV, pcpt-cAMP reduced the membrane current throughout the voltage-clamp pulse, which was qualitatively different from the modulation of the membrane current by 5-HT. In addition, the pcpt-cAMP-induced reduction in the membrane current at the beginning of the pulse was much greater than that induced by 5-HT. Moreover, preexposure of sensory neurons to 5-HT did not occlude further reduction in the membrane current by subsequent application of pcpt-cAMP. 5. These results suggest that pcpt-cAMP has some mechanisms of action that are not shared by 5-HT or cAMP but are shared by pcpt-cGMP. In addition, these findings provide further evidence that results obtained with this compound should be interpreted with caution.

Electrophysiological properties of guinea pig trigeminal motoneurons recorded in vitro.

1. Intracellular recording and stimulation were made from guinea pig trigeminal motoneurons (TMNs) in brain stem slices. Electrophysiological properties were examined and the underlying currents responsible for motoneuron excitability were investigated by the use of current clamp and single electrode voltage clamp (SEVC) techniques. 2. The voltage responses to subthreshold hyperpolarizing or depolarizing current pulses showed voltage- and time-dependent inward rectification. SEVC analysis demonstrated that the hyperpolarizing inward rectification resulted from the development of a slowly occurring voltage-dependent inward current activated at hyperpolarized membrane potentials. This current persisted in solutions containing low Ca2+/Mn2+, tetraethylammonium (TEA), and Ba2+, whereas it was reduced by 1-3 mM cesium. The depolarizing inward rectification was mediated by a persistent sodium current (INa-P) that was completely abolished by bath application of tetrodotoxin (TTX). 3. Action potential characteristics were studied by intracellular stimulation with brief current pulses (< 3 ms) in combination with ionic substitutions or application of specific ionic conductance blocking agents. Bath application of TTX abolished the action potential, whereas 1-10 mM TEA or 0.5-2 mM 4-aminopyridine (4-AP) increased, significantly, the spike duration, suggesting participation of the delayed rectifier and A-current type conductances in spike repolarization. SEVC analysis revealed a TEA-sensitive sustained outward current and a fast, voltage-dependent, transient current with properties consistent with their roles in spike repolarization. 4. TMN afterhyperpolarizing potentials (AHPs) that followed a single spike consisted of fast and slow components usually separated by a depolarizing hump [afterdepolarization (ADP)]. The fast component was abolished by TEA or 4-AP but not by Mn2+, Co2+, or the bee venom apamin. In contrast, the slow AHP was readily reduced by Mn2+, Co2+, or apamin, suggesting participation of an apamin-sensitive, calcium-dependent K+ conductance in the production of the slow AHP. SEVC analysis and ionic substitutions demonstrated a slowly activating and deactivating calcium-dependent K+ current with properties that could account for the slow AHP observed in these neurons. 5. Repetitive discharge was examined with long depolarizing current pulses (1 s) and analysis of frequency-current plots. When evoked from resting potential (about -55 mV), spike onset from rheobase occurred rapidly and was maintained throughout the current pulse. At higher current intensities, early and late adaptations in spike discharge were observed. Frequency-current plots exhibited a bilinear relationship for the first interspike interval (ISI) in approximately 50% of the neurons tested and in most neurons tested during steady-state discharge (SS).(ABSTRACT TRUNCATED AT 400 WORDS)