Circuits that innervate excitatory-inhibitory cells in the inferior colliculus obtained with in vivo whole cell recordings.

Neurons excited by stimulation of one ear and suppressed by the other, called excitatory/inhibitory (EI) neurons, are sensitive to interaural intensity disparities, the cues animals use to localize high frequencies. EI neurons are first formed in lateral superior olive, which then sends excitatory projections to the dorsal nucleus of the lateral lemniscus and the inferior colliculus (IC), both of which contain large populations of EI cells. We evaluate herein the inputs that innervate EI cells in the IC of Mexican free-tailed bats (Tadarida brasilensis mexicana) with in vivo whole-cell recordings from which we derived excitatory and inhibitory conductances. We show that the basic EI property in the majority of IC cells is inherited from lateral superior olive, but that each type of EI cell is also innervated by the ipsilateral or contralateral dorsal nucleus of the lateral lemniscus, as well as additional excitatory and inhibitory inputs from monaural nuclei. We identify three EI types, each of which receives a set of projections that is different from the other types. To evaluate the role that the various projections played in generating binaural responses, we used modeling to compute a predicted response from the conductances. We then omitted one of the conductances from the computation to evaluate the degree to which that input contributed to the binaural response. We show that the formation of the EI property in the various types is complex, and that some projections exert such subtle influences that they could not have been detected with extracellular recordings or even from intracellular recordings of postsynaptic potentials.

[Using serial tachograms to measure the evoked impulse activity of isolated hippocampal neurons].

In these studies, we investigated the phenomenon of change in impulse activity of isolated hippocampal neurons during longtime recording. We described the use of serial tachograms during registering the electrical activity of neurons, analysis of which can improve the reliability of data. An analysis of the data identified three phases of changes in impulse activity of isolated neurons in the experimental registrations: a phase of increased activity, phase of stable activity and the phase of declining activity. It is established that in conditions of the perforated patch-clamp the phase of stable activity started at 10-15 minutes after formation of the tight junction and had an average duration of 30 minutes. It is shown that the use of the serial tahograms and phases of activity improves the quality of assessment in the measurement of the electrical activity of neurons.

Direct correction of non-space-clamped currents via Cole's theorem.

The currents measured during voltage clamp of a non-isopotential neuron reflect axial as well as membrane conductances. One wishes to remove the former in the hope that the latter will reveal quantitative information on the nature of the voltage gated channels at the clamp site. We here show that Cole's theorem can be used to directly remove the axial component from simulated voltage clamp recordings. It is direct in the sense that it requires neither simulation nor fitting. As it comes down to squaring the clamp current and then differentiating with respect to clamp voltage it may indeed be implemented in real time. When applied to synthetic potassium currents in straight fibers we find that our method accurately and robustly recovers both non-uniform conductances and non-uniform channel kinetics. We indicate the degree to which such accuracy is diminished for cells that taper and/or branch.

Postictal single-cell firing patterns in the hippocampus.

PURPOSE: Patients with epilepsy have varying degrees of postictal impairment including confusion and amnesia. This impairment adds substantially to the disease burden of epilepsy. However, the mechanism responsible for postictal cognitive impairment is unclear. The purpose of this study was to study single-cell firing patterns in hippocampal cells after spontaneous seizures in rats previously subjected to status epilepticus. METHODS: In this study, we monitored place cells and interneurons in the CA1 region of the hippocampus before and after spontaneous seizures in six epileptic rats with a history of status epilepticus. Place cells fire action potentials when the animal is in a specific location in space, the so-called place field. Place cell function correlates well with performance in tasks of visual-spatial memory and appears to be an excellent surrogate measure of spatial memory. RESULTS: Twelve spontaneous seizures were recorded. After the seizures, a marked decrease in firing rate of action potentials from place cells was noted, whereas interneuron firing was unchanged. In addition, when place cell firing fields persisted or returned, they had aberrant firing fields with reduced coherence and information content. In addition to postictal suppression of firing patterns, seizures led to the emergence of firing fields in previously silent cells, demonstrating a postictal remapping of the hippocampus. CONCLUSIONS: These findings demonstrate that postictal alterations in behavior are not due solely to reduced neuronal firing. Rather, the postictal period is characterized by robust and dynamic changes in cell-firing patterns resulting in remapping of the hippocampal map.

Decreased IH in hippocampal area CA1 pyramidal neurons after perinatal seizure-inducing hypoxia.

PURPOSE: The hyperpolarization-activated cation current (IH) has been proposed to play a role in some forms of epileptogenesis, as it critically regulates synaptic integration and intrinsic excitability of principal limbic neurons and can be pathologically altered after experimentally induced seizures. In hippocampal CA1 pyramidal neurons, IH is functionally decreased after kainate-induced status epilepticus in adult rats but is increased after hyperthermia-induced seizures in immature rat pups. This study aimed to determine whether and how IH may be altered in CA1 pyramidal neurons after seizure-inducing global hypoxia in the neonatal brain. METHODS: Seizures were induced in rat pups on postnatal day 10 by 14- to 16-min exposure to 5-7% O2. Whole-cell patch-clamp recordings were obtained from hippocampal CA1 pyramidal neurons in slices 30 min to 3 days after hypoxia treatment, and from control age-matched littermates. IH was isolated under voltage-clamp by subtracting current responses to hyperpolarizing voltage steps before and during application of the IH blocker ZD 7288 (100 microM). RESULTS: IH was significantly decreased in pyramidal neurons from the hypoxia-treated group compared with controls (p<0.001; 19 controls; 15 hypoxia). Analyses of tail currents and activation kinetics indicated no statistically significant differences between groups in the voltage dependence or time constants of activation. CONCLUSIONS: These data indicate that a single episode of neonatal hypoxia that induces seizures can persistently decrease IH in CA1 pyramidal neurons, raising this as a potential contributing mechanism to epileptogenesis in this setting. Our findings further indicate that the consequences of seizures for IH may depend more on seizure etiology than on maturational stage.

Biophysical properties of connexin-45 gap junction hemichannels studied in vertebrate cells.

Human HeLa cells transfected with mouse Cx45 and rat RIN cells transfected with chicken Cx45 were used to study the electrical and permeability properties of Cx45 gap junction hemichannels. With no extracellular Ca(2+), whole-cell recording revealed currents arising from hemichannels in both transfected cell lines. Multichannel currents showed a time-dependent activation or deactivation sensitive to voltage, V(m). These currents did not occur in non-transfected cells. The hemichannel currents were inhibited by raising extracellular Ca(2+) or by acidification with CO(2). The unitary conductance exhibited V(m) dependence (i.e., gamma(hc,main) increased/decreased with hyperpolarization/depolarization). Extrapolation to V(m) = 0 mV led to a gamma(hc,main) of 57 pS, roughly twice the conductance of an intact Cx45 gap junction channel. The open channel probability, P(o), was V(m)-dependent, declining at negative V(m) (P(o) < 0.11, V(m) < -50 mV), and increasing at positive V(m) (P(o) approximately 0.76, V(m) > 50 mV). Moreover, Cx45 nonjunctional hemichannels appeared to mediate lucifer yellow (LY) and propidium iodide (PI) dye uptake from the external solution when extracellular Ca(2+) level was reduced. Dye uptake was directly proportional to the number of functioning hemichannels. No significant dye uptake was detected in non-transfected cells. Cx45 transfected HeLa and RIN cells also allowed dye to leak out when preloaded with LY and then incubated in Ca(2+)-free external solution, whereas little or no dye leakage was observed when these cells were incubated with 2 mM external Ca(2+). Intact Cx45 gap junction channels allowed passage of either LY or PI dye, but their respective flux rates were different. Comparison of LY diffusion through Cx45 hemichannels and intact gap junction channels revealed that the former is more permeable, suggesting that gap junction channel pores exhibit more allosterical restriction to the dye molecules than the unopposed hemichannel. The data demonstrate the opening of Cx45 nonjunctional hemichannels in vertebrate cells when the external Ca(2+) concentration is reduced.

Properties of ATP-dependent K(+) channels in adrenocortical cells.

Bovine adrenocortical zona fasciculata (AZF) cells express a novel ATP-dependent K(+)-permeable channel (I(AC)). Whole cell and single-channel recordings were used to characterize I(AC) channels with respect to ionic selectivity, conductance, and modulation by nucleotides, inorganic phosphates, and angiotensin II (ANG II). In outside-out patch recordings, the activity of unitary I(AC) channels is enhanced by ATP in the patch pipette. These channels were K(+) selective with no measurable Na(+) or Ca(2+) conductance. In symmetrical K(+) solutions with physiological concentrations of divalent cations (M(2+)), I(AC) channels were outwardly rectifying with outward and inward chord conductances of 94.5 and 27.0 pS, respectively. In the absence of M(2+), conductance was nearly ohmic. Hydrolysis-resistant nucleotides including AMP-PNP and NaUTP were more potent than MgATP as activators of whole cell I(AC) currents. Inorganic polytriphosphate (PPP(i)) dramatically enhanced I(AC) activity. In current-clamp recordings, nucleotides and PPP(i) produced resting potentials in AZF cells that correlated with their effectiveness in activating I(AC). ANG II (10 nM) inhibited whole cell I(AC) currents when patch pipettes contained 5 mM MgATP but was ineffective in the presence of 5 mM NaUTP and 1 mM MgATP. Inhibition by ANG II was not reduced by selective kinase antagonists. These results demonstrate that I(AC) is a distinctive K(+)-selective channel whose activity is increased by nucleotide triphosphates and PPP(i). Furthermore, they suggest a model for I(AC) gating that is controlled through a cycle of ATP binding and hydrolysis.

Voltage-clamp analysis of membrane currents and excitation-contraction coupling in a crustacean muscle.

A single fibre preparation from the extensor muscle of a marine isopod crustacean is described which allows the analysis of membrane currents and simultaneously recorded contractions under two-electrode voltage-clamp conditions. We show that there are three main depolarisation-gated currents, two are outward and carried by K+, the third is an inward Ca2+ current, I(Ca). Normally, the K+ currents which can be isolated by using K+ channel blockers, mask I(Ca). I(Ca) activates at potentials more positive than -40 mV, is maximal around 0 mV, and shows strong inactivation at higher depolarisation. Inactivation depends on current rather than voltage. Ba2+, Sr2+ and Mg2+ can substitute for Ca2+. Ba2+ currents are about 80% larger than Ca2+ currents and inactivate little. The properties of I(Ca) characterise it as a high threshold L-type current. The outward current consists primarily of a fast, transient A current, I(K(A)) and a maintained, delayed rectifier current, I(K(V)). In some fibres, a small Ca2+-dependent K+ current is also present. I(K(A)) activates fast at depolarisation above -45 mV, shows pronounced inactivation and is almost completely inactivated at holding potentials more positive than -40 mV. I(K(A)) is half-maximally blocked by 70 microM 4-aminopyridine (4-AP), and 70 mM tetraethylammonium (TEA). I(K(V)) activates more slowly, at about -30 mV, and shows no inactivation. It is half-maximally blocked by 2 mM TEA but rather insensitive to 4-AP. Physiologically, the two K+ currents prevent all-or-nothing action potentials and determine the graded amplitude of active electrical responses and associated contractions. Tension development depends on and is correlated with depolarisation-induced Ca2+ influx mediated by I(Ca). The voltage dependence of peak tension corresponds directly to the voltage dependence of the integrated I(Ca). The threshold potential for contraction is at about -38 mV. Peak tension increases with increasing voltage steps, reaches maximum at around 0 mV, and declines with further depolarisation.

Directional rectification of gap junctional voltage gating between dieters cells in the inner ear of guinea pig.

Deiters cells (DCs) are the cochlear supporting cells in inner ear and contain multiple gap junction connexin genes, which when mutated can induce hearing loss. In the present study, the gap junctions between DCs were investigated by a double voltage clamp technique. Besides asymmetric responses to the polarities of transjunctional voltage (V(j)) and transmembrane potential (V(m)), the channels were also sensitive to which cell side was stimulated in a cell pair, i.e. voltage gating had directional dependence. The direction-dependent voltage gating could result in asymmetric current flow between the cells and influenced K(+) passage. Multiple connexins may constitute non-homotypic channels with directional dependence of voltage gating to mediate functional gap junction pathways in the cochlea. This may explain how a single connexin mutation can produce hearing loss.

Riluzole inhibits the persistent sodium current in mammalian CNS neurons.

The effects of 0.1-100 microM riluzole, a neuroprotective agent with anticonvulsant properties, were studied on neurons from rat brain cortex. Patch-clamp whole-cell recordings in voltage-clamp mode were performed on thin slices to examine the effects of the drug on a noninactivating (persistent) Na+ current (INa,p). INa,p was selected because it enhances neuronal excitability near firing threshold, which makes it a potential target for anticonvulsant drugs. When added to the external solution, riluzole dose-dependently inhibited INa,p up to a complete blocking of the current (EC50 2 microM), showing a significant effect at therapeutic drug concentrations. A comparative dose-effect study was carried out in the same cells for the other main known action of riluzole, the inhibitory effect on the fast transient sodium current. This effect was confirmed in our experiments, but we found that it was achieved at levels much higher than putative therapeutic concentrations. Only the effect on INa,p, and not that on fast sodium current, can account for the reduction in neuronal excitability observed in cortical neurons following riluzole treatment at therapeutic concentrations, and this might represent a novel mechanism accounting for the anticonvulsant and neuroprotective properties of riluzole.

LabPatch, an acquisition and analysis program for patch-clamp electrophysiology.

An acquisition and analysis program, "LabPatch," has been developed for use in patch-clamp research. LabPatch controls any patch-clamp amplifier, acquires and records data, runs voltage protocols, plots and analyzes data, and connects to spreadsheet and database programs. Controls within LabPatch are grouped by function on one screen, much like an oscilloscope front panel. The software is mouse driven, so that the user need only point and click. Finally, the ability to copy data to other programs running in Windows 95/98, and the ability to keep track of experiments using a database, make LabPatch extremely versatile. The system requirements include Windows 95/98, at least a 100-MHz processor and 16 MB RAM, a data acquisition card, digital-to-analog converter, and a patch-clamp amplifier. LabPatch is available free of charge at http://www.fhs.mcmaster.ca/huizinga/.

Adjustments for the display of quantized ion channel dwell times in histograms with logarithmic bins.

Dwell-time histograms are often plotted as part of patch-clamp investigations of ion channel currents. The advantages of plotting these histograms with a logarithmic time axis were demonstrated by, J. Physiol. (Lond.). 378:141-174), Pflügers Arch. 410:530-553), and, Biophys. J. 52:1047-1054). Sigworth and Sine argued that the interpretation of such histograms is simplified if the counts are presented in a manner similar to that of a probability density function. However, when ion channel records are recorded as a discrete time series, the dwell times are quantized. As a result, the mapping of dwell times to logarithmically spaced bins is highly irregular; bins may be empty, and significant irregularities may extend beyond the duration of 100 samples. Using simple approximations based on the nature of the binning process and the transformation rules for probability density functions, we develop adjustments for the display of the counts to compensate for this effect. Tests with simulated data suggest that this procedure provides a faithful representation of the data.

Morphological and physiological properties of the A17 amacrine cell of the rat retina.

In addition to the well-studied AII amacrine cell, there is another amacrine cell type participating in the rod pathway of the mammalian retina. In cat, this cell is called the A17 amacrine cell, and in rabbits, it is called the indoleamine-accumulating amacrine cell (S1 and S2); however, the presence of the corresponding cell type has not yet been described in detail for the rat retina. To this end, we injected amacrine cells with Neurobiotin in vertical retinal slices. After histological processing, we were able to reconstruct the morphology of a wide-field amacrine cell which showed characteristics of A17 and S1/S2 amacrine cells. The rat wide-field amacrine cells exhibited the same stratification pattern, their dendrites bore varicosities and ramified in sublamina 5 of the inner plexiform layer (IPL), and they were dye-coupled to other amacrine cells. To determine whether those amacrine cells shared electrophysiological characteristics as well, we performed whole-cell patch-clamp recordings and examined their voltage-activated currents and neurotransmitter-induced currents. We never observed voltage-gated Na+ currents and spike-like potentials upon depolarization by current injection in these cells. We identified GABA- and glycine-sensitive Cl- currents that could be blocked by bicuculline and strychnine, respectively. We also observed kainate- and AMPA-activated currents, which could be inhibited by the application of 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). Finally, a 400-ms full-field light stimulus was used to characterize the light responses of A17 amacrine cells. The light ON-induced inward current could be suppressed by the application of 2,3-Dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulphonamide (NBQX), while the majority of the light OFF-induced current was inhibited by bicuculline and reduced to a smaller extent by NBQX. CPP, an NMDA blocker, had no effect on the light response of rat A17 amacrine cells.

Sokolow-Lyon voltage criteria in sickle cell patients

The electrocardiogram remains the most commonly used method of cardiac assessment in developing countries. To determine the prevalence of electrocardiographic left ventricular hypertrophy (LVH) and the clinical significance of Sokolow-Lyon voltage criteria in sickle cell patients, echocardiographic and ECG findings were studied in 112 patients (71 with haemoglobin SS disease and 41 with haemoglobin SC disease). Electrocardiographic left ventricular hypertrophy (ECGLVH) defined as Sokolow-Lyon voltage greater than or equal to 35 mm was detected in 39 (55 percent) SS patients and 11 (27 percent) SC patients. This prevalence was higher in men than in women. There were statistically significant trends for increasing prevalence of ECGLVH with height (p<0.007 in SS, and p<0.01 in SC patients). But no significant trend was found with increasing posterior wall (PWT) or interventricular septal thickness (IVST). Sensitivity of Sokolow-Lyon criteria for detection of echocardiographic left ventricular hypertrophy was 63 percent and 33 percent in SS and SC patients, respectively, and specificity was 51 percent and 74 percent, respectively. Sokolow-Lyon voltage correlated with left ventricular mass in SS and SC patients (r = 0.44, p < 0.01 and r = 0.32, p < 0.05) and with left ventricular internal dimension (r = 0.2, p < 0.01 and r = 0.32, p < 0.05) but not significantly with PWT and IVST. We conclude that, in sickle cell patients, the electrocardiographic LVH mainly indicates the existence of an eccentric echocardiographic LVH with increase of left ventricular internal dimension.(AU)

The single-channel dose-response relation is consistently steep for rod cyclic nucleotide-gated channels: implications for the interpretation of macroscopic dose-response relations.

Cyclic nucleotide-gated channels contain four subunits, each with a C-terminal binding site for cGMP or cAMP. The dose-response relation for activation is usually fit with the Hill equation, I/I(max) = [cGMP]n/([cGMP]n + K(1/2)n, where I/I(max) is the fraction of maximal current, K(1/2) is the concentration of cGMP that gives a half-maximal current, and n is the Hill coefficient, taken as the minimum number of ligands required for significant activation. The dose-response relations in multichannel patches are often fit with Hill coefficients of

Errors in persistent inward currents generated by space-clamp errors: a modeling study.

1. The effects of imperfect space clamp on inactivating inward currents were examined with the use of a "ball-and-stick" neuronal model with uniform active and passive membrane properties. With poor space clamp, both transient and steady-state (persistent) components were distorted. The ratio of steady-state to peak current (i(s)/p), measured at the soma, was sometimes smaller but usually larger than would be the case with uniform space clamp. For a fast Na+ current, the anomalous persistent component was largest for large electrotonic lengths, low-conductance densities, and voltage-clamp potentials near the threshold of the current. Under some conditions, steady-state current could take one of two values, depending on the holding potential. 2. Membrane potential as a function of distance was examined, revealing a steady-state voltage gradient in which distal portions of the neuron were more positive than in the passive case, and often more positive than the command potential itself. These reversed voltage gradients, caused by the uncontrolled "window" Na+ current at remote electrotonic distances, produced steady-state axial current flow into the soma, thereby increasing the persistent current measured somatically. 3. The time at which the current peaked (tp) was sensitive to imperfections in the space clamp. This phenomenon made somatic membrane current and axial current at tp sensitive to the fidelity of space clamp as well. The ratio of steady-state axial current to that at t = tp was a good predictor of the degree of distortion of i(s)/p.(ABSTRACT TRUNCATED AT 250 WORDS)

[The effect of the transmembrane potential level on the dynamic extinction of the amplitude of the synaptic reactions in the snail under potential-fixation conditions]. / Vliianie urovnia transmembrannogo potentsiala na dinamiku ugasheniia amplitudy sinapticheskikh reaktsii ulitki v usloviiakh fiksatsii potentsiala.

The goal of this work was to describe a contribution of postsynaptic membrane in synaptic plasticity. To solve this problem the influence of the level of transmembrane potential on the dynamics of transmembrane synaptic current amplitude was studied during rhythmical stimulation of the nerve. It was shown that with increase of transmembrane potential value the slope of the function of transmembrane current decrease became steeper. It was also shown that with higher stimulation frequency the influence of transmembrane holding potential upon the dynamics of synaptic reaction increased. The data obtained can be considered as an evidence to the contribution of postsynaptic membrane in synaptic plasticity.