Fine gating properties of channels responsible for persistent sodium current generation in entorhinal cortex neurons

The gating properties of channels responsible for the generation of persistent Na(+) current (I(NaP)) in entorhinal cortex layer II principal neurons were investigated by performing cell-attached, patch-clamp experiments in acutely isolated cells. Voltage-gated Na(+)-channel activity was routinely elicited by applying 500-ms depolarizing test pulses positive to -60 mV from a holding potential of -100 mV. The channel activity underlying I(NaP) consisted of prolonged and frequently delayed bursts during which repetitive openings were separated by short closings. The mean duration of openings within bursts was strongly voltage dependent, and increased by e times per every approximately 12 mV of depolarization. On the other hand, intraburst closed times showed no major voltage dependence. The mean duration of burst events was also relatively voltage insensitive. The analysis of burst-duration frequency distribution returned two major, relatively voltage-independent time constants of approximately 28 and approximately 190 ms. The probability of burst openings to occur also appeared largely voltage independent. Because of the above "persistent" Na(+)-channel properties, the voltage dependence of the conductance underlying whole-cell I(NaP) turned out to be largely the consequence of the pronounced voltage dependence of intraburst open times. On the other hand, some kinetic properties of the macroscopic I(NaP), and in particular the fast and intermediate I(NaP)-decay components observed during step depolarizations, were found to largely reflect mean burst duration of the underlying channel openings. A further I(NaP) decay process, namely slow inactivation, was paralleled instead by a progressive increase of interburst closed times during the application of long-lasting (i.e., 20 s) depolarizing pulses. In addition, long-lasting depolarizations also promoted a channel gating modality characterized by shorter burst durations than normally seen using 500-ms test pulses, with a predominant burst-duration time constant of approximately 5-6 ms. The above data, therefore, provide a detailed picture of the single-channel bases of I(NaP) voltage-dependent and kinetic properties in entorhinal cortex layer II neurons.     

Lidocaine suppresses subthreshold oscillations by inhibiting persistent Na(+) current in injured dorsal root ganglion neurons

The aim of this study was to determine the effect and mechanism of low concentration of lidocaine on subthreshold membrane potential oscillations (SMPO) and burst discharges in chronically compressed dorsal root ganglion (DRG) neurons. DRG neurons were isolated by enzymatic dissociation method. SMPO, burst discharges and single spike were elicited by whole cell patch-clamp technique in current clamp mode. Persistent Na(+) current (I(NaP)) and transient Na(+) current (I(NaT)) were elicited in voltage clamp mode. The results showed that SMPO was suppressed and burst discharges were eliminated by tetrodotoxin (TTX, 0.2 micromol/l) in current clamp mode, I(NaP) was blocked by 0.2 micromol/l TTX in voltage clamp mode. SMPO, burst discharges and I(NaP) were also suppressed by low concentration of lidocaine (10 micromol/l) respectively. However, single spike and I(NaT) could only be blocked by high concentration of lidocaine (5 mmol/l). From these results, it is suggested that I(NaP) mediates the generation of SMPO in injured DRG neurons. Low concentration of lidocaine (10 micromol/l) suppresses SMPO by selectively inhibiting I(NaP), but not I(NaT), in chronically compressed DRG neurons.     

Mechanism of graded persistent cellular activity of entorhinal cortex layer v neurons

Working memory is an emergent property of neuronal networks, but its cellular basis remains elusive. Recent data show that principal neurons of the entorhinal cortex display persistent firing at graded firing rates that can be shifted up or down in response to brief excitatory or inhibitory stimuli. Here, we present a model of a potential mechanism for graded firing. Our multicompartmental model provides stable plateau firing generated by a nonspecific calcium-sensitive cationic (CAN) current. Sustained firing is insensitive to small variations in Ca2+ concentration in a neutral zone. However, both high and low Ca2+ levels alter firing rates. Specifically, increases in persistent firing rate are triggered only during high levels of calcium, while decreases in rate occur in the presence of low levels of calcium. The model is consistent with detailed experimental observations and provides a mechanism for maintenance of memory-related activity in individual neurons.     

Biophysical properties and slow voltage-dependent inactivation of a sustained sodium current in entorhinal cortex layer-II principal neurons: a whole-cell and single-channel study.

The functional and biophysical properties of a sustained, or "persistent," Na(+) current (I(NaP)) responsible for the generation of subthreshold oscillatory activity in entorhinal cortex layer-II principal neurons (the "stellate cells") were investigated with whole-cell, patch-clamp experiments. Both acutely dissociated cells and slices derived from adult rat entorhinal cortex were used. I(NaP), activated by either slow voltage ramps or long-lasting depolarizing pulses, was prominent in both isolated and, especially, in situ neurons. The analysis of the gating properties of the transient Na(+) current (I(NaT)) in the same neurons revealed that the resulting time-independent "window" current (I(NaTW)) had both amplitude and voltage dependence not compatible with those of the observed I(NaP), thus implying the existence of an alternative mechanism of persistent Na(+)-current generation. The tetrodotoxin-sensitive Na(+) currents evoked by slow voltage ramps decreased in amplitude with decreasing ramp slopes, thus suggesting that a time-dependent inactivation was taking place during ramp depolarizations. When ramps were preceded by increasingly positive, long-lasting voltage prepulses, I(NaP) was progressively, and eventually completely, inactivated. The V(1/2) of I(NaP) steady state inactivation was approximately -49 mV. The time dependence of the development of the inactivation was also studied by varying the duration of the inactivating prepulse: time constants ranging from approximately 6.8 to approximately 2.6 s, depending on the voltage level, were revealed. Moreover, the activation and inactivation properties of I(NaP) were such as to generate, within a relatively broad membrane-voltage range, a really persistent window current (I(NaPW)). Significantly, I(NaPW) was maximal at about the same voltage level at which subthreshold oscillations are expressed by the stellate cells. Indeed, at -50 mV, the I(NaPW) was shown to contribute to >80% of the persistent Na(+) current that sustains the subthreshold oscillations, whereas only the remaining part can be attributed to a classical Hodgkin-Huxley I(NaTW). Finally, the single-channel bases of I(NaP) slow inactivation and I(NaPW) generation were investigated in cell-attached experiments. Both phenomena were found to be underlain by repetitive, relatively prolonged late channel openings that appeared to undergo inactivation in a nearly irreversible manner at high depolarization levels (-10 mV), but not at more negative potentials (-40 mV).     

Microcircuits of functionally identified neurons in the rat medial entorhinal cortex

Extracellular recordings have elucidated spatial neural representations without identifying underlying microcircuits. We labeled neurons juxtacellularly in medial entorhinal cortex of freely moving rats with?a friction-based, pipette-stabilization system. In a linear maze novel to the animals, spatial firing of superficial layer neurons was reminiscent of grid cell activity. Layer 2 stellate cells showed stronger theta modulation than layer 3 neurons, and both fired during the ascending phase of field potential theta. Deep-layer neurons showed little or no activity. Layer 2 stellate cells resided in hundreds of small patches. At the dorsomedial entorhinal border, we identified larger (putative parasubicular) patches, which contained polarized head-direction selective neurons firing during the descending theta phase. Three axon systems interconnected patches: centrifugal axons from superficial cells to single large patches, centripetal axons from large-patch cells to single small patches, and circumcurrent axons interconnecting large patches. Our microcircuit analysis during behavior reveals modularity of entorhinal processing. VIDEO ABSTRACT:     

Theoretical considerations underlying Na(+) uptake mechanisms in freshwater fishes

Ion and acid-base regulating mechanisms have been studied at the fish gill for almost a century. Original models proposed for Na(+) and Cl(-) uptake, and their linkage with H(+) and HCO(3)(-) secretion have changed substantially with the development of more sophisticated physiological techniques. At the freshwater fish gill, two dominant mechanisms for Na(+) uptake from dilute environments have persisted in the literature. The use of an apical Na(+)/H(+) exchanger driven by a basolateral Na(+)/K(+)-ATPase versus an apical Na(+) channel electrogenically coupled to an apical H(+)-ATPase have been the source of debate for a number of years. Advances in molecular biology have greatly enhanced our understanding of the basic ion transport mechanisms at the fish gill. However, it is imperative to ensure that thermodynamic principles are followed in the development of new models for gill ion transport. This review will focus on the recent molecular advances for Na(+) uptake in freshwater fish. Emphasis will be placed on thermodynamic constraints that prevent electroneutral apical NHE function in most freshwater environments. By combining recent advances in molecular and functional physiology of fish gills with thermodynamic considerations of ion transport, our knowledge in the field should continue to grow in a logical manner.     

A voltage-dependent persistent sodium current in mammalian hippocampal neurons

Currents generated by depolarizing voltage pulses were recorded in neurons from the pyramidal cell layer of the CA1 region of rat or guinea pig hippocampus with single electrode voltage-clamp or tight-seal whole-cell voltage-clamp techniques. In neurons in situ in slices, and in dissociated neurons, subtraction of currents generated by identical depolarizing voltage pulses before and after exposure to tetrodotoxin revealed a small, persistent current after the transient current. These currents could also be recorded directly in dissociated neurons in which other ionic currents were effectively suppressed. It was concluded that the persistent current was carried by sodium ions because it was blocked by TTX, decreased in amplitude when extracellular sodium concentration was reduced, and was not blocked by cadmium. The amplitude of the persistent sodium current varied with clamp potential, being detectable at potentials as negative as -70 mV and reaching a maximum at approximately -40 mV. The maximum amplitude at -40 mV in 21 cells in slices was -0.34 +/- 0.05 nA (mean +/- 1 SEM) and -0.21 +/- 0.05 nA in 10 dissociated neurons. Persistent sodium conductance increased sigmoidally with a potential between -70 and -30 mV and could be fitted with the Boltzmann equation, g = gmax/(1 + exp[(V' - V)/k)]). The average gmax was 7.8 +/- 1.1 nS in the 21 neurons in slices and 4.4 +/- 1.6 nS in the 10 dissociated cells that had lost their processes indicating that the channels responsible are probably most densely aggregated on or close to the soma. The half-maximum conductance occurred close to -50 mV, both in neurons in slices and in dissociated neurons, and the slope factor (k) was 5-9 mV. The persistent sodium current was much more resistant to inactivation by depolarization than the transient current and could be recorded at greater than 50% of its normal amplitude when the transient current was completely inactivated. Because the persistent sodium current activates at potentials close to the resting membrane potential and is very resistant to inactivation, it probably plays an important role in the repetitive firing of action potentials caused by prolonged depolarizations such as those that occur during barrages of synaptic inputs into these cells.     

Kinetic Comparisons of Heart and Kidney Na(+),K(+)-ATPases

Most kinetic measurements of the partial reactions of Na⁺,K⁺-ATPase have been conducted on enzyme from mammalian kidney. Here we present a kinetic model that is based on the available equilibrium and kinetic parameters of purified kidney enzyme, and allows predictions of its steady-state turnover and pump current in intact cells as a function of ion and ATP concentrations and the membrane voltage. Using this model, we calculated the expected dependence of the pump current on voltage and extracellular Na⁺ concentration. The simulations indicate a lower voltage dependence at negative potentials of the kidney enzyme in comparison with heart muscle Na⁺,K⁺-ATPase, in agreement with experimental results. The voltage dependence is enhanced at high extracellular Na⁺ concentrations. This effect can be explained by a voltage-dependent depopulation of extracellular K⁺ ion binding sites on the E2P state and an increase in the proportion of enzyme in the E1P(Na⁺)₃ state in the steady state. This causes a decrease in the effective rate constant for occlusion of K⁺ by the E2P state and hence a drop in turnover. Around a membrane potential of zero, negligible voltage dependence is observed because the voltage-independent E2(K⁺)₂ → E1 + 2K⁺ transition is the major rate-determining step.     

DHA improves cognition and prevents dysfunction of entorhinal cortex neurons in 3xTg-AD mice

Defects in neuronal activity of the entorhinal cortex (EC) are suspected to underlie the symptoms of Alzheimer's disease (AD). Whereas neuroprotective effects of docosahexaenoic acid (DHA) have been described, the effects of DHA on the physiology of EC neurons remain unexplored in animal models of AD. Here, we show that DHA consumption improved object recognition (↑12%), preventing deficits observed in old 3xTg-AD mice (↓12%). Moreover, 3xTg-AD mice displayed seizure-like akinetic episodes, not detected in NonTg littermates and partly prevented by DHA (↓50%). Patch-clamp recording revealed that 3xTg-AD EC neurons displayed (i) loss of cell capacitance (CC), suggesting reduced membrane surface area; (ii) increase of firing rate versus injected current (F-I) curve associated with modified action potentials, and (iii) overactivation of glutamatergic synapses, without changes in synaptophysin levels. DHA consumption increased CC (↑12%) and decreased F-I slopes (↓21%), thereby preventing the opposite alterations observed in 3xTg-AD mice. Our results indicate that cognitive performance and basic physiology of EC neurons depend on DHA intake in a mouse model of AD.     

Kinetic diversity of Na+ channel bursts in frog skeletal muscle

Individual Na+ channels of dissociated frog skeletal muscle cells at 10 degrees C fail to inactivate in 0.02% of depolarizing pulses, thus producing bursts of openings lasting hundreds of milliseconds. We present here a kinetic analysis of 87 such bursts that were recorded in multi-channel patches at four pulse potentials. We used standard dwell-time histograms as well as fluctuation analysis to analyze the gating kinetics of the bursting channels. Since each burst contained only 75-150 openings, detailed characterization of the kinetics from single bursts was not possible. Nevertheless, at this low kinetic resolution, the open and closed times could be well fitted by single exponentials (or Lorentzians for the power spectra). The best estimates of both the open and closed time constants produced by either technique were much more broadly dispersed then expected from experimental or analytical variability, with values varying by as much as an order of magnitude. Furthermore, the values of the open and closed time constants were not significantly correlated with one another from burst to burst. The bursts thus expressed diverse kinetic behaviors, all of which appear to be manifestations of a single type of Na+ channel. Although the opening and closing rates were dispersed, their average values were close to those of alpha m and 2 beta m derived from fits to the early transient Na+ currents over the same voltage range. We propose a model in which the channel has both primary states (e.g., open, closed, and inactivated), as well as "modes" that are associated with independent alterations in the rate constants for transition between each of these primary states.     

Kinetic analysis of acetylcholine-induced chloride current in isolated snail neurons

1. Kinetics of activation and desensitization phases of the acetylcholine (ACh)-induced chloride current (ICI) were studied using isolated single neurons of Japanese land snail and the "concentration clamp" technique. 2. The dose-response curve for the peak ICI gave a dissociation constant of 7.1 x 10(-6) M and a Hill coefficient of 1.8. 3. The current-voltage relationship was linear in the voltage range examined (-60 to +10 mV) and the reversal potential (EACh) was -7.2 +/- 1.5 mV (N = 10). The value was close to the calculated equilibrium potential for chloride ions (ECI). 4. Both activation and desensitization phases of the ACh-induced ICI consisted of a single exponential at concentrations less than 3 x 10(-6) M and a double exponential at higher concentrations. The time constants of both phases decreased with increasing ACh concentrations but showed no potential dependency. 5. The recovery from desensitization of the ICI induced by 5 x 10(-6) M ACh proceeded double exponentially, with time constants of 11 and 114 sec at a holding potential of -30 mV. 6. Noise analysis was performed on a steady-state current induced by 3 x 10(-7) to 2 x 10(-6) M ACh. The mean open time was about 60 msec at 10(-6) M ACh and the single-channel conductance was 14 PS. 7. These results suggest that the ACh receptor-Cl channel complex in snail neurons has two binding sites with the dissociation constant of 7.1 x 10(-6) M and is rapidly activated and desensitized to a steady level in the presence of the agonist.     

Cellular mechanisms underlying stimulus-dependent gain modulation in primary visual cortex neurons in vivo

Gain modulation is a widespread neuronal phenomenon that modifies response amplitude without changing selectivity. Computational and in vitro studies have proposed cellular mechanisms of gain modulation based on the postsynaptic effects of background synaptic activation, but these mechanisms have not been studied in vivo. Here, we used intracellular recordings from cat primary visual cortex to measure neuronal gain while changing background synaptic activity with visual stimulation. We found that increases in the membrane fluctuations associated with increases in synaptic input do not obligatorily result in gain modulation in vivo. However, visual stimuli that evoked sustained changes in resting membrane potential, input resistance, and membrane fluctuations robustly modulated neuronal gain. The magnitude of gain modulation depended critically on the spatiotemporal properties of the visual stimulus. Gain modulation in vivo may thus be determined on a moment-to-moment basis by sensory context and the consequent dynamics of synaptic activation.     

Parvalbumin-immunoreactive neurons in the entorhinal cortex of the rat: localization,morphology, connectivity and ultrastructure

Brain Cell Biology - We studied the distribution, morphology, ultrastructure and connectivity of parvalbumin-immunoreactive neurons in the entorhinal cortex of the rat. Immunoreactive cell bodies...     

Two septal-entorhinal GABAergic projections differentially control coding properties of spatially tuned neurons in the medial entorhinal cortex

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Zn2(+)-induced subconductance events in cardiac Na+ channels prolonged by batrachotoxin. Current-voltage behavior and single-channel kinetics

The mechanism of voltage-dependent substate production by external Zn2+ in batrachotoxin-modified Na+ channels from canine heart was investigated by analysis of the current-voltage behavior and single-channel kinetics of substate events. At the single-channel level the addition of external Zn2+ results in an increasing frequency of substate events with a mean duration of approximately 15-25 ms for the substate dwell time observed in the range of -70 to +70 mV. Under conditions of symmetrical 0.2 M NaCl, the open state of cardiac Na+ channels displays ohmic current-voltage behavior in the range of -90 to +100 mV, with a slope conductance of 21 pS. In contrast, the Zn2(+)-induced substate exhibits significant outward rectification with a slope conductance of 3.1 pS in the range of -100 to -50 mV and 5.1 pS in the range of +50 to +100 mV. Analysis of dwell-time histograms of substate events as a function of Zn2+ concentration and voltage led to the consideration of two types of models that may explain this behavior. Using a simple one-site blocking model, the apparent association rate for Zn2+ binding is more strongly voltage dependent (decreasing e-fold per +60 mV) than the Zn2+ dissociation rate (increasing e-fold per +420 mV). However, this simple blocking model cannot account for the dependence of the apparent dissociation rate on Zn2+ concentration. To explain this result, a four-state kinetic scheme involving a Zn2(+)-induced conformational change from a high conductance conformation to a substate conformation is proposed. This model, similar to one introduced by Pietrobon et al. (1989. J. Gen. Physiol. 94:1-24) for H(+)-induced substate behavior in L-type Ca2+ channels, is able to simulate the kinetic and equilibrium behavior of the primary Zn2(+)-induced substate process in heart Na+ channels. This model implies that binding of Zn2+ greatly enhances conversion of the open, ohmic channel to a low conductance conformation with an asymmetric energy profile for Na+ permeation.