Time Course Of Currents Research Articles

Accumulation of cytoplasmic calcium, but not apamin‐sensitive afterhyperpolarization current, during high frequency firing in rat subthalamic nucleus cells

The autonomous firing pattern of neurons in the rat subthalamic nucleus (STN) is shaped by action potential afterhyperpolarization currents. One of these is an apamin-sensitive calcium-dependent potassium current (SK). The duration of SK current is usually considered to be limited by the clearance of calcium from the vicinity of the channel. When the cell is driven to fire faster, calcium is expected to accumulate, and this is expected to result in accumulation of calcium-dependent AHP current. We measured the time course of calcium transients in the soma and proximal dendrites of STN neurons during spontaneous firing and their accumulation during driven firing. We compared these to the time course and accumulation of AHP currents using whole-cell and perforated patch recordings. During spontaneous firing, a rise in free cytoplasmic calcium was seen after each action potential, and decayed with a time constant of about 200 ms in the soma, and 80 ms in the dendrites. At rates higher than 10 Hz, calcium transients accumulated as predicted. In addition, there was a slow calcium transient not predicted by summation of action potentials that became more pronounced at high firing frequency. Spike AHP currents were measured in voltage clamp as tail currents after 2 ms voltage pulses that triggered action currents. Apamin-sensitive AHP (SK) current was measured by subtraction of tail currents obtained before and after treatment with apamin. SK current peaked between 10 and 15 ms after an action potential, had a decay time constant of about 30 ms, and showed no accumulation. At frequencies between 5 and 200 spikes s(-1), the maximal SK current remained the same as that evoked by a single action potential. AHP current did not have time to decay between action potentials, so at frequencies above 50 spikes s(-1) the apamin-sensitive current was effectively constant. These results are inconsistent with the view that the decay of SK current is governed by calcium dynamics. They suggest that the calcium is present at the SK channel for a very short time after each action potential, and the current decays at a rate set by the deactivation kinetics of the SK channel. At high rates, repetitive firing was governed by a fast apamin-insensitive AHP current that did not accumulate, but rather showed depression with increases in activation frequency. A slowly accumulating AHP current, also insensitive to apamin, was extremely small at low rates but became significant with higher firing rates.

Active Dendritic Conductances Dynamically Regulate GABA Release from Thalamic Interneurons

Inhibitory interneurons in the dorsal lateral geniculate nucleus (dLGN) process visual information by precisely controlling spike timing and by refining the receptive fields of thalamocortical (TC) neurons. Previous studies indicate that dLGN interneurons inhibit TC neurons by releasing GABA from both axons and dendrites. However, the mechanisms controlling GABA release are poorly understood. Here, using simultaneous whole-cell recordings from interneurons and TC neurons and two-photon calcium imaging, we find that synchronous activation of multiple retinal ganglion cells (RGCs) triggers sodium spikes that propagate throughout interneuron axons and dendrites, and calcium spikes that invade dendrites but not axons. These distinct modes of interneuron firing can trigger both a rapid and a sustained component of inhibition onto TC neurons. Our studies suggest that active conductances make LGN interneurons flexible circuit-elements that can shift their spatial and temporal properties of GABA release in response to coincident activation of functionally related subsets of RGCs.

Different Roles for AMPA and NMDA Receptors in Transmission at the Immature Retinogeniculate Synapse

The relay of information at the retinogeniculate synapse, the connection between retina and visual thalamus, begins days before eye opening and is thought to play an important role in the maturation of neural circuits in the thalamus and visual cortex. Remarkably, during this period of development, the retinogeniculate synapse is immature, with single retinal ganglion cell inputs evoking an average peak excitatory postsynaptic current (EPSC) of only about 40 pA compared with 800 pA in mature synapses. Yet, at the mature synapse, EPSCs >400 pA are needed to drive relay neuron firing. This raises the question of how small-amplitude EPSCs can drive transmission at the immature retinogeniculate synapse. Here we find that several features of the immature synapse, compared with the mature synapse, contribute to synaptic transmission. First, although the peak amplitude of EPSC is small, the decay time course of both alpha-amino-3-hydroxy-5-methyl-4isoxazolepropionic acid receptor (AMPAR) and N-methyl-d-aspartate receptor (NMDAR) currents is significantly slower. The prolonged time course of NMDAR currents is a result of the presence of both NR2B and NR2C/D subunits. In addition, the extended presence of neurotransmitter released prolongs the synaptic current time course. Second, reduced sensitivity to magnesium block results in significantly greater synaptic charge transfer through NMDAR. Third, AMPAR currents contribute to the spike latency, but not to temporal precision, at the immature synapse. Furthermore, intrinsic excitability is greater. These properties enable immature synapses with predominantly NMDARs and little or no AMPARs to contribute to the relay of information from retina to visual cortex.

Temperature dependence of NR1/NR2B NMDA receptor channels

N-methyl- d-aspartate (NMDA) receptors are highly expressed in the CNS, mediate the slow component of excitatory transmission and play key roles in synaptic plasticity and excitotoxicity. These ligand-gated ion channels are heteromultimers composed of NR1 and NR2 subunits activated by glycine and glutamate. In this study, patch-clamp recordings were used to study the temperature sensitivity of recombinant NR1/NR2B receptors expressed in human embryonic kidney (HEK) 293 cells. Rate constants were assessed by fitting a six-state kinetic scheme to time courses of transient macroscopic currents induced by glutamate at 21.9–46.5 °C. Arrhenius transformation of the rate constants characterizing NMDA receptor channel activity indicates that the most sensitive were the rate constants of desensitization (temperature coefficient Q 10=10.3), resensitization (Q 10=4.6) and unbinding (Q 10=3.6). Other rate constants and the amplitude of single-channel currents were less temperature sensitive. Deactivation of responses mediated by NR1/NR2B receptors after a brief application of glutamate was best fit by a double exponential function (τ fast: Q 10=3.7; τ slow: Q 10=2.7). From these data, we conclude that desensitization/resensitization of the NMDA receptor and glutamate unbinding are especially temperature sensitive and imply that at physiological temperatures the channel kinetics play an important role in determining amplitude and time course of NMDA receptor-mediated postsynaptic currents and these receptors mediated synaptic plasticity.

Microscopic kinetic determinants of macroscopic currents: insights from coupling and uncoupling of GABAA receptor desensitization and deactivation

The time course of inhibitory postsynaptic currents (IPSCs) reflects GABA(A) receptor deactivation, the process of current relaxation following transient activation. Fast desensitization has been demonstrated to prolong deactivation, and these processes have been described as being 'coupled'. However, the relationship between desensitization and deactivation remains poorly understood. We investigated the 'uncoupling' of GABA(A) receptor macroscopic desensitization and deactivation using experimental conditions that affected these two processes differently. Changing agonist affinity preferentially altered deactivation, changing agonist concentration preferentially altered macroscopic desensitization, and a pore domain mutation prolonged deactivation despite blocking fast desensitization. To gain insight into the mechanistic basis for coupling and uncoupling, simulations were used to systematically evaluate the interplay between agonist affinity, gating efficacy, and desensitized state stability in shaping macroscopic desensitization and deactivation. We found that the influence of individual kinetic transitions on macroscopic currents depended not only on model connectivity, but also on the relationship among transitions within a given model. In addition, changing single rate constants differentially affected macroscopic desensitization and deactivation, thus providing parsimonious kinetic explanations for experimentally observed uncoupling. Finally, these findings permitted development of an algorithmic framework for kinetic interpretation of experimental manipulations that alter macroscopic current properties.

Melatonin inhibits nicotinic currents in cultured rat cerebellar granule neurons

There is limited data regarding the effects of melatonin on the activity of neuronal acetylcholine receptors (nAChRs) themselves. This study analyzes the effects of low concentrations of melatonin on nicotine-evoked currents from cerebellar granule neurons (CGNs) in culture. Using electrophysiological and Ca(2+)-imaging techniques, it was found a subset of rat CGNs to which nicotine application elicited both intracellular Ca(2+) transients and inward whole-cell currents. These responses were mediated by heteromeric nAChRs, as assessed by their sensitivity to nicotine and time constant of current decay. Preincubating the cells with low melatonin concentrations (down to 1 pm) significantly reduced the current amplitude in a dose-dependent manner, without affecting the receptor's apparent affinity and voltage-dependency, nor the current's rise and decay time course. The inhibitory effect of melatonin was significantly reduced by luzindole, a competitive antagonist of both MT(1) and MT(2) melatonin receptors. In conclusion, melatonin inhibits nicotinic currents through non-alpha7 heteromeric nAChRs expressed by CGNs in culture, an effect that appears to be at least partially mediated by melatonin membrane receptors. Direct modulation of nicotinic receptors is accomplished at doses that are likely to be physiologically relevant, thus providing a mechanism through which melatonin circadian rhythmic levels could modulate cholinergic activity.

Lysosomal Dysfunction Produces Distinct Alterations in Synaptic α-Amino-3-Hydroxy-5-Methylisoxazolepropionic Acid and N-Methyl-D-Aspartate Receptor Currents in Hippocampus

The early processes that lead to synaptic dysfunction during aging are not clearly understood. Dysregulation of alpha-amino-3-hydroxy-5-methylisoxazolepropionic acid (AMPA) and N-methyl-D-aspartate (NMDA) receptors may cause age-related cognitive decline. Using hippocampal slice cultures exhibiting lysosomal dysfunction, an early marker of brain aging that is linked to protein accumulation, we identified alterations to AMPA and NMDA receptor-mediated synaptic currents. The miniature and spontaneous excitatory postsynaptic currents that were examined after 3, 6, and 9 days of lysosomal disruption showed progressive changes in amplitude, frequency, and rise and decay kinetics. To investigate whether modifications in specific channel properties of single synaptic receptors contributed to changes in the amplitude and time course of synaptic currents, we examined the single channel properties of synaptic AMPA and NMDA receptors. The channel open probability and the mean open times showed decreases in both receptor populations, whereas the closed times were increased without any change in the channel conductance. The Western blot analysis revealed a progressive decline in synaptic markers including glutamate receptor subunits. These results indicate that lysosomal dysfunction leads to progressive functional perturbation of AMPA and NMDA receptors in this slice model of protein accumulation, suggesting that age-related cognitive decline could result from altered glutamate receptor function before reductions in synaptic density.

Neurons Skip a Beat during Fast Ripples

Fast ripples are EEG transients emanating from epileptic foci, but fast-ripple frequencies far exceed maximal neuronal firing rates. In this issue of Neuron, Menendez de la Prida and coworkers propose that out-of-phase firing of a subpopulation of neurons during physiological ripple activity effectively doubles the ripple frequency to produce fast ripples.

TARP Subtypes Differentially and Dose-Dependently Control Synaptic AMPA Receptor Gating

A family of transmembrane AMPA receptor regulatory proteins (TARPs) profoundly affects the trafficking and gating of AMPA receptors (AMPARs). Although TARP subtypes are differentially expressed throughout the CNS, it is unclear whether this imparts functional diversity to AMPARs in distinct neuronal populations. Here, we examine the effects of each TARP subtype on the kinetics of AMPAR gating in heterologous cells and in neurons. We report a striking heterogeneity in the effects of TARP subtypes on AMPAR deactivation and desensitization, which we demonstrate controls the time course of synaptic transmission. In addition, we find that some TARP subtypes dramatically slow AMPAR activation kinetics. Synaptic AMPAR kinetics also depend on TARP expression level, suggesting a variable TARP/AMPAR stoichiometry. Analysis of quantal synaptic transmission in a TARP gamma-4 knockout (KO) mouse corroborates our expression data and demonstrates that TARP subtype-specific gating of AMPARs contributes to the kinetics of native AMPARs at central synapses.

Electrostatic Interaction of Internal Mg2+ with Membrane PIP2 Seen with KCNQ K+ Channels

Activity of KCNQ (Kv7) channels requires binding of phosphatidylinositol 4,5-bisphosphate (PIP2) from the plasma membrane. We give evidence that Mg2+ and polyamines weaken the KCNQ channel–phospholipid interaction. Lowering internal Mg2+ augmented inward and outward KCNQ currents symmetrically, and raising Mg2+ reduced currents symmetrically. Polyvalent organic cations added to the pipette solution had similar effects. Their potency sequence followed the number of positive charges: putrescine (+2) < spermidine (+3) < spermine (+4) < neomycin (+6) < polylysine (≫+6). The inhibitory effects of Mg2+ were reversible with sequential whole-cell patching. Internal tetraethylammonium ion (TEA) gave classical voltage-dependent block of the pore with changes of the time course of K+ currents. The effect of polyvalent cations was simpler, symmetric, and without changes of current time course. Overexpression of phosphatidylinositol 4-phosphate 5-kinase Iγ to accelerate synthesis of PIP2 attenuated the sensitivity to polyvalent cations. We suggest that Mg2+ and other polycations reduce the currents by electrostatic binding to the negative charges of PIP2, competitively reducing the amount of free PIP2 available for interaction with channels. The dose–response curves could be modeled by a competition model that reduces the pool of free PIP2. This mechanism is likely to modulate many other PIP2-dependent ion channels and cellular processes.

Allosteric modulation of nicotinic acetylcholine receptors

Allosteric modulation refers to the concept that proteins could exist in multiple conformational states and that binding of allosteric ligands alters the energy barriers or “isomerization coefficients” between various states. In the context of ligand gated ion channels such as nicotinic acetylcholine receptors (nAChRs), it implies that endogenous ligand acetylcholine binds at the orthosteric site, and that molecules that bind elsewhere on the nAChR subunit(s) acts via allosteric interactions. For example, studies with the homomeric α7 nAChRs indicate that such ligand interactions can be well described by an allosteric model, and that positive allosteric effectors can affect energy transitions by (i) predominantly affecting the peak current response (Type I profile) or, (ii) both peak current responses and time course of agonist-evoked response (Type II profile). The recent discovery of chemically heterogeneous group of molecules capable of differentially modifying nAChR properties without interacting at the ligand binding site illustrates the adequacy of the allosteric model to predict functional consequences. In this review, we outline general principles of the allosteric concept and summarize the profiles of novel compounds that are emerging as allosteric modulators at the α7 and α4β2 nAChR subtypes.

OK, Everyone Keep Quiet

Glutamate transporters in glial cells and postsynaptic neurons are believed to accumulate synaptically released glutamate; however, the role of presynaptic glutamate transporters has been unclear (see Wadiche and von Gersdorff). Veruki et al . combined electrophysiological and pharmacological analysis to investigate the role of presynaptic glutamate transporters that mediate a prominent anion current uncoupled from glutamate uptake in retinal rod bipolar (RB) cells. After showing that glutamate transporter-mediated inward currents recorded from RB cells in rat retinal slices were largest and fastest when elicited by puffs of glutamate delivered near axon terminals in the inner plexiform layer, the authors confirmed the presynaptic location of the transporters by recording from isolated patches. Dual recordings from RB cells and synaptically connected AII amacrine cells, as well as a comparison of the time course of transporter currents recorded in RB cells in response to released glutamate to currents recorded from patches, indicated that transporters on RB cells were located perisynaptically (and farther from the site of release than were the postsynaptic receptors). Moreover, transporter currents in RB cells were activated not only by auto-released glutamate but also by glutamate released from neighboring RB cells. Transporter activation led to a decrease in transmitter release and thus a reduction in the postsynaptic response. Thus, presynaptic RB glutamate transporters appear to mediate both auto- and lateral inhibition of transmitter release. M. L. Veruki, S. H. Mørkve, E. Hartveit, Activation of a presynaptic glutamate transporter regulates synaptic transmission through electrical signaling. Nat. Neurosci. 9 , 1388-1396 (2006). [PubMed] J. I. Wadiche, H. von Gersdorff, Long-distance signaling via presynaptic glutamate transporters. Nat. Neurosci. 9 , 1352-1353 (2006). [PubMed]

Tandem-Pore K+ Channels Mediate Inhibition of Orexin Neurons by Glucose

Glucose-inhibited neurons orchestrate behavior and metabolism according to body energy levels, but how glucose inhibits these cells is unknown. We studied glucose inhibition of orexin/hypocretin neurons, which promote wakefulness (their loss causes narcolepsy) and also regulate metabolism and reward. Here we demonstrate that their inhibition by glucose is mediated by ion channels not previously implicated in central or peripheral glucose sensing: tandem-pore K(+) (K(2P)) channels. Importantly, we show that this electrical mechanism is sufficiently sensitive to encode variations in glucose levels reflecting those occurring physiologically between normal meals. Moreover, we provide evidence that glucose acts at an extracellular site on orexin neurons, and this information is transmitted to the channels by an intracellular intermediary that is not ATP, Ca(2+), or glucose itself. These results reveal an unexpected energy-sensing pathway in neurons that regulate states of consciousness and energy balance.

Effect of extracellular pH on recombinant α1β2γ2 and α1β2 GABA A receptors

Recently, we have reported that extracellular protons allosterically modulated neuronal GABA A receptors [Mozrzymas, J.W., Zarnowska, E.D., Pytel, M., Mercik, K., 2003a. Modulation of GABA(A) receptors by hydrogen ions reveals synaptic GABA transient and a crucial role of desensitiztion process. Journal of Neuroscience 23, 7981–7992]. However, GABA ARs in neurons are heterogeneous and the effect of hydrogen ions depends on the receptor subtype. In particular, γ2 subunit sets the receptor sensibility to several modulators including protons. However, the mechanisms whereby protons modulate γ2-containing and γ2-free GABA ARs have not been fully elucidated. To this end, current responses to ultrafast GABA applications were recorded for α1β2γ2 and α1β2 receptors at different pH values. For both receptor types, increase in pH induced a decrease in amplitudes of currents elicited by saturating [GABA] but this effect was stronger for α1β2 receptors. In the case of α1β2γ2 receptors, protons strongly affected the current time course due to a down regulation of binding and desensitization rates. This effect was qualitatively similar to that described in neurons. Protons strongly influenced the amplitude of α1β2 receptor-mediated currents but the effect on their kinetics was weak suggesting a predominant direct non-competitive inhibition with a minor allosteric modulation. In conclusion, we provide evidence that extracellular protons strongly affect GABA A receptors and that, depending on the presence of the γ2 subunit, the modulatory mechanisms show profound quantitative and qualitative differences.

State-Dependent Verapamil Block of the Cloned Human Cav3.1 T-Type Ca2+ Channel

Verapamil is a potent phenylalkylamine antihypertensive believed to exert its therapeutic effect primarily by blocking high-voltage-activated L-type calcium channels. It was the first clinically used calcium channel blocker and remains in clinical use, although it has been eclipsed by other calcium channel blockers because of its short half-life and interactions with other channels. In addition to blocking L-type channels, it has been reported to block T-type (low-voltage activated) calcium channels. This type of cross-reactivity is likely to be beneficial in the effective control of blood pressure. Although the interactions of T channels with a number of drugs have been described, the mechanisms by which these agents modulate channel activity are largely unknown. Most calcium channel blockers exhibit state-dependence (i.e., preferential binding to certain channel conformations), but little is known about state-dependent verapamil block of T channels. We stably expressed human Ca(v)3.1 T-type channels in human embryonic kidney 293 cells and studied the state-dependence of the drug with macroscopic and gating currents. Verapamil blocked currents at micromolar concentrations at polarized potentials similar to those reported for L-type channels, although unlike for L-type currents, it did not affect current time course. The drug exhibited use-dependence and significantly slowed the apparent recovery from inactivation. Current inhibition was dependent on potential. This dependence was restricted to negative potentials, although all data were consistent with verapamil binding in the pore. Gating currents were unaffected by verapamil. We propose that verapamil achieves its inhibitory effect via occlusion of the channel pore associated with an open/inactivated conformation of the channel.

Developmental Regulation of Whole Cell Capacitance and Membrane Current in Identified Interneurons inC. elegans

Postembryonic developmental changes in electrophysiological properties of the AIY interneuron class were investigated using whole cell voltage clamp. AIY interneurons displayed an increase in cell capacitance during larval development, whereas steady-state current amplitude did not increase. The time course of the outward membrane current, carried at least in part by K+ ions, matured, from a slowly activating, sustained current to a rapidly activating, decaying current. We also investigated how the development of capacitance and outward current was altered by loss-of-function mutations in genes expressed in AIY. One such gene, the LIM homeobox gene ttx-3, is known to be involved in the specification of the AIY neuronal subtype. In ttx-3 mutants, capacitance and outward current matured precociously. In mutants of the gene wrk-1, an immunoglobulin superfamily (IgSF) member whose expression is regulated by ttx-3, capacitance matured normally, whereas outward current matured precociously. We conclude that AIY interneurons contain distinct pathways for regulating capacitance and membrane current.

Monte Carlo Simulation of Release of Vesicular Content in Neuroendocrine Cells

The release of transmitter from the vesicle, its diffusion through the fusion pore, and the cleft and its interaction with the carbon electrode were simulated using the Monte Carlo method. According to the simulation the transmitter release is largely determined by geometric factors--the ratio of the fusion pore cross-sectional and vesicular areas, if the diffusion constant is as in the aqueous solution--but the speed of transmitter dissociation from the gel matrix plays an important role during the rise phase of release. Transmitter is not depleted near the entrance to the fusion pore and there is no cleft-to-vesicle feedback, but the depletion becomes evident if the diffusion constant is reduced, especially if the pore is wide. In general, the time course of amperometric currents closely resembles the time course of the simulated transmitter concentration in the cleft and the time course of release. Surprisingly, even a tenfold change of the electrode efficiency has only a marginal effect on the amplitude or the time course of amperometric currents. Greater electrode efficiency however lowers the cleft concentration, but only if the cleft is narrow. As the cleft widens the current amplitudes diminish and rise times lengthen, but the decay times are less affected. Moreover, the amplitude dependence of the rise and decay times becomes steeper as the cleft widens and/or as the release kinetics slows. Finally, lower diffusion constant of transmitter in the narrow cleft does not further prolong the amperometric currents, whose slow time course reflects slow release kinetics.

Inhibitory Synaptic Transmission Governs Inspiratory Motoneuron Synchronization

Neurons within the intact respiratory network produce bursts of action potentials that cause inspiration or expiration. Within inspiratory bursts, activity is synchronized on a shorter timescale to generate clusters of action potentials that occur in a set frequency range and are called synchronous oscillations. We investigated how GABA and glycine modulate synchronous oscillations and respiratory rhythm during postnatal development. We recorded inspiratory activity from hypoglossal nerves using the in vitro rhythmically active mouse medullary slice preparation from P0-P11 mice. Average oscillation frequency increased with postnatal development, from 17 +/- 12 Hz in P0-P6 mice (n = 15) to 38 +/- 7 Hz in P7-P11 mice (n = 37) (P < 0.0001). Bath application of GABAA and GlyR antagonists significantly reduced oscillation power in neonates (P0-P6) and juveniles (P7-P10) and increased peak integrated activity in both age groups. To test whether elevating slice excitability is sufficient to reduce oscillation power, Substance P was bath applied alone. Substance P, although increasing peak integrated activity, had no significant effect on oscillation power. Prolonging the time course of GABAergic synaptic currents with zolpidem decreased the median oscillation frequency in P9-P10 mouse slices. These data demonstrate that oscillation frequency increases with postnatal development and that both GABAergic and glycinergic transmission contribute to synchronization of activity. Further, the time course of synaptic GABAergic currents is a determinant of oscillation frequency.

Intrinsic kinetics determine the time course of neuronal synaptic transporter currents

Efficient clearance of synaptically released glutamate from the extracellular space is an absolute requirement for maintaining information processing in the central nervous system. In the cerebellum, clearance of glutamate relies on uptake by Bergmann glial cells and Purkinje cells (PCs). Uptake by PCs can be monitored by recording the synaptic transport current (STC) mediated by the PC-specific transporter excitatory amino acid transporter 4 (EAAT4). The slow time course of the PC STC has been used to argue that glutamate clearance is protracted. We find, however, that the time course of the STC is not affected by altering the amount of glutamate released at individual synapses or by partial transporter blockade, manipulations that would be expected to change the duration of the extracellular glutamate transient. Ion substitution experiments and kinetic modeling of the PC transporter current suggest that physiological levels of intracellular Na(+) and glutamate slow the cycling rate of transporters and thereby lengthen the time course of STCs. The model predicts that PC transporters bind glutamate quickly but that the actual cycling rate of EAAT4 in physiological conditions is slow; therefore, the STC reflects the intrinsic kinetics of the glutamate transporter, not the rate of glutamate clearance.

Sodium Currents Activate without a Hodgkin and Huxley-Type Delay in Central Mammalian Neurons

Hodgkin and Huxley established that sodium currents in the squid giant axons activate after a delay, which is explained by the model of a channel with three identical independent gates that all have to open before the channel can pass current (the HH model). It is assumed that this model can adequately describe the sodium current activation time course in all mammalian central neurons, although there is no experimental evidence to support such a conjecture. We performed high temporal resolution studies of sodium currents gating in three types of central neurons. The results show that, within the tested voltage range from -55 to -35 mV, in all of these neurons, the activation time course of the current could be fit, after a brief delay, with a monoexponential function. The duration of delay from the start of the voltage command to the start of the extrapolated monoexponential fit was much smaller than predicted by the HH model. For example, in prefrontal cortex pyramidal neurons, at -46 mV and 12 degrees C, the observed average delay was 140 micros versus the 740 micros predicted by the two-gate HH model and the 1180 micros predicted by the three-gate HH model. These results can be explained by a model with two closed states and one open state. In this model, the transition between two closed states is approximately five times faster than the transition between the second closed state and the open state. This model captures all major properties of the sodium current activation. In addition, the proposed model reproduces the observed action potential shape more accurately than the traditional HH model.