bluuebunny/crossref_metadata_2025 huggingface dataset:
Endogenous calcium buffering in motoneurones of the nucleus hypoglossus from mouse
Authors: Lips Mario B. , Keller Bernhard U.
Published: September 2004

Simultaneous patch clamp and rapid microfluorometric calcium measurements were performed on sixty‐five motoneurones in slices of the nucleus hypoglossus in the brainstem of 2‐ to 6‐day‐old mice.

Hypoglossal motoneurones were particularly vulnerable to mechanical or metabolic stress during isolation of in vitro slice preparations. Therefore, experimental conditions were optimized for functional integrity, as judged by spontaneous rhythmic activity of hypoglossal nerves (XII).

Calcium concentrations in the cell soma were monitored with a temporal resolution in the millisecond time domain during depolarizing voltage steps. Ratiometric fluorescence measurements were made using a rapid monochromator (switching τ < 10 ms), a photomultiplier tube and the calcium sensitive dyes fura‐2 and mag‐fura‐5.

Dynamics of somatic calcium transients were investigated as a function of the concentration of calcium indicator dye in the cell. Decays of calcium transients were approximated to a single exponential component and decay time constants showed a linear dependence on dye concentration. The extrapolated decay time in the absence of indicator dye was 0·7 ± 0·2 s, suggesting rapid somatic calcium dynamics under physiological conditions.

By a process of back‐extrapolation, the ‘added buffer’ method, a calcium binding ratio of 41 ± 12 (9 cells) was obtained indicating that 98 % of the calcium ions entering a hypoglossal motoneurone were bound by endogenous buffers.

Endogenous calcium binding ratios in hypoglossal motoneurones were small compared with those of other neurones with comparable size or geometry. Accordingly, our measurements suggest that the selective vulnerability of hypoglossal motoneurones to calcium‐related excitotoxicity might partially result from low concentrations of calcium buffers in these cells.
Differential regulation of potassium currents by FGF‐1 and FGF‐2 in embryonic Xenopus laevis myocytes
Authors: Chauhan‐Patel R. , Spruce A. E.
Published: September 2004

Fibroblast growth factors (FGFs) are involved in the regulation of many aspects of muscle development. This study investigated their role in regulating voltage‐dependent K+ currents in differentiating Xenopus laevis myocytes. Both FGF‐1 and FGF‐2 are expressed by developing muscle cells, so their actions were compared. Experiments were performed on cultured myocytes isolated from stage 15 embryos.

Long‐term exposure of the embryonic myocytes to FGF‐1 downregulated inward rectifier K+ current (IK(IR)) density as well as both sustained and inactivating voltage‐dependent outward K+ currents (IK,S and IK,I, respectively) and their densities. In contrast, FGF‐2 upregulated these currents, although, because of an increase in capacitance caused by FGF‐2, current density did not change with this factor.

The regulation of IK(IR) by FGF‐1 was prevented by the cytoplasmic tyrosine kinase inhibitor herbimycin A, but that of IK,S and IK,I was unaffected, indicating that FGF‐1 achieves its regulatory effects on electrical development via separate signalling pathways. The receptor tyrosine kinase inhibitor genistein in isolation suppressed K+ currents, but this may have occurred through a channel‐blocking mechanism.

In many cells, IK,S was found to be composed of two components with differing voltage dependencies of activation. The FGFs brought about an alteration in the amount of total IK,S by equal effects on each component. Conversely, herbimycin A increased the proportion of low voltage‐activated current without affecting total current amplitude. Therefore, we suggest that a single species of channel whose voltage dependence is shifted by tyrosine phosphorylation generates IK,S.

In summary, FGF‐1 and FGF‐2 exert opposite effects on voltage‐dependent K+ currents in embryonic myocytes and, furthermore, FGF‐1 achieves its effects on different K+ currents via separate second messenger pathways.
α‐Adrenergic effects on Na+‐K+ pump current in guinea‐pig ventricular myocytes
Authors: Wang Y. , Gao J. , Mathias R. T. , Cohen I. S. , Sun X. , Baldo G. J.
Published: September 2004

The whole‐cell patch clamp was employed to study Na+‐K+ pump current (Ip) in acutely isolated myocytes. α‐Adrenergic receptors were activated with noradrenaline (NA) after blocking β‐adrenergic receptors with propranolol. Ip was measured as the current blocked by strophanthidin (Str).

Activation of α‐receptors by NA increased Ip in a concentration‐dependent manner. The K0.5 depended on intracellular calcium ([Ca2+]i), however maximal stimulation did not. At 15 nm[Ca2+]i the K0.5 was 219 nm NA whereas at 1.4 μm [Ca2+]i it was 3 nm.

The voltage dependence of Ip was not shifted by NA at either high or low [Ca2+]i. At each voltage, maximal stimulation of Ip was 14‐15 %.

Staurosporine (St), an inhibitor of protein kinase C (PKC), eliminated the α‐receptor‐mediated stimulation of Ip at either high or low[Ca2+]i.

The stimulation of Ip was independent of changes in intracellular sodium or external potassium concentrations, and did not reflect a change in affinity for Str.

Phenylephrine, methoxamine and metaraminol, three selective α1‐adrenergic agonists, stimulate Ip in a similar manner to NA. Stimulation of Ip by NA was eliminated by prazosin, an α1‐antagonist, but was unaffected by yohimbine, an α2‐antagonist.

We conclude noradrenaline activates ventricular α1‐receptors, which are specifically coupled via PKC to increase Na+‐K+ pump current. The sensitivity of the coupling is [Ca2+]i dependent, however the maximal increase in pump current is [Ca2+]i and voltage independent.
Cholinergic activation and tonic excitation induce persistent gamma oscillations in mouse somatosensory cortex in vitro
Authors: Buhl Eberhard H. , Tamás Gabor , Fisahn André
Published: September 2004

Concomitant application of the cholinergic agonist carbachol and nanomolar doses of kainate can elicit persistent gamma frequency oscillations in all layers of the mouse somatosensory cortex in vitro. Receptor pharmacology with bath‐applied antagonists indicated that oscillatory network activity depended crucially on the participation of cholinergic muscarinic, (S)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA)/kainate and GABAA receptors.

The timing of action potentials and the occurrence of excitatory as well as inhibitory postsynaptic events was highly correlated with the phasic change of extracellularly recorded population activity. Firing probability was lowest during the peak negativity of IPSPs and gradually increased during their ensuing decay. In conjunction with the effect of a barbiturate to decrease the frequency of gamma oscillations, this suggests a crucial role of IPSPs in phasing the suprathreshold activity of principal neurons.

At nearby (< 1 mm) sites contained within any given cortical layer, oscillatory extra‐ and intracellular activity was highly synchronous with no apparent phase lag. However, interlaminar mapping experiments demonstrated a phase reversal of both extra‐ and intracellularly recorded activity near the lower border of thalamo‐recipient layer 4, thus corroborating findings that have been obtained in vivo.

In conclusion, a modest increase of tonic excitatory drive in conjunction with the activation of cholinergic muscarinic receptors can elicit persistent gamma frequency network oscillations in the rodent somatosensory cortex. These findings (re)emphasize the role of the cholinergic ascending system in the cortical processing of sensory information.
Divalent ion block of inward rectifier current in human capillary endothelial cells and effects on resting membrane potential
Authors: Jow Flora , Numann Randy
Published: September 2004

Cultured human capillary endothelial cells (HCEC) contain a large inward rectifier current, IK(IR), that can be abolished by removing external K+ or by adding 50 μm Ba2+.

We show that IK(IR) is responsible for maintaining the hyperpolarized potential (−60.6 ± 0.5 mV, n= 83) of HCEC. Blocking IK(IR) with 50 μm Ba2+ shifts the zero current level and depolarizes HCEC by 36.5 ± 1.3 mV (n= 4).

Increasing external Ca2+ concentration ([Ca2+]o) from 0.5 to 7 mm reduces the magnitude of IK(IR) by 36.5 ± 2.3 % (n= 5) and depolarizes the cells by 10.33 ± 2.4 mV (n= 3), whereas decreasing [Ca2+]o from 1.8 to 0.5 mm increases the amplitude of IK(IR) by 6.9 ± 1.9 % (n= 4). The relationship between [Ca2+]o and the percentage block of IK(IR) gives a Kd value of 5.4 ± 0.6 mm at −120 mV.

I
K(IR) is also blocked by other divalent ions, with Ba2+ >> Sr2+ > Mg2+ > Mn2+= Ca2+, and the block of peak current at −120 mV being 85.3 ± 3.2 % (n= 5) for 50 μm Ba2+, 62.9 ± 2.2 % (n= 5) for 5 mm Sr2+, 40.7 ± 2.5 % (n= 9) for 5 mm Mg2+, 33.4 ± 2.1 % (n= 5) for 5 mm Mn2+ and 32.9 ± 2.1 % (n= 5) for 5 mm Ca2+.

The voltage dependence of Sr2+ block of peak IK(IR) occurred with a Kd value of 1.0 ± 0.09 mm for −140 mV, 1.9 ± 0.16 mm for −130 mV, 3.1 ± 0.28 mm for −120 mV, 4.6 ± 0.34 mm for −110 mV and 6.4 ± 0.5 mm for −100 mV (n= 5), with a calculated electrical distance (δ) of 0.44 from the outside.
Presynaptic inhibition by 5‐HT1B receptors of glutamatergic synaptic inputs onto serotonergic caudal raphe neurones in rat
Authors: Li Yu‐Wen , Bayliss Douglas A.
Published: September 2004

Autonomous, pacemaker‐like activity of serotonergic raphe neurones and its autoregulation by somatodendritic 5‐HT1A receptors are well described, but little is known of synaptic inputs onto raphe neurones or their modulation. Therefore, we recorded unitary excitatory postsynaptic currents (EPSCs) in caudal raphe neurones (raphe obscurus and pallidus) following local electrical stimulation in a neonatal rat brainstem slice preparation; most neurones (79 %; n= 72/91) recovered following post hoc immunohistochemistry were tryptophan hydroxylase‐immunoreactive, indicating that they were serotonergic.

Evoked EPSCs occurred at relatively constant latency with variable amplitude and apparent ‘failures’ at fixed suprathreshold stimulus intensity. At −60 mV, EPSCs were wholly due to CNQX‐sensitive, non‐NMDA glutamate receptors; at depolarized potentials, a small AP‐5‐sensitive NMDA component was often observed.

EPSCs were potently and reversibly inhibited by 5‐HT with an EC50 of 0.1 μM. This effect was mimicked by 5‐HT1B agonists (CP‐93,129 and anpirtoline), but not by a 5‐HT1A agonist (8‐OH‐DPAT), indicating that 5‐HT1B receptors mediate the inhibition of EPSCs.

Multiple lines of evidence indicate that inhibition of EPSCs by 5‐HT was mediated presynaptically. First, currents evoked by exogenous glutamate application were unaffected by 5‐HT and/or 5‐HT1B agonists. In addition, the frequency of spontaneous glutamatergic miniature EPSCs was diminished by CP‐93,129 and paired‐pulse facilitation of EPSCs was enhanced by 5‐HT. Finally, the 5‐HT1B receptor agonists that blocked synaptic transmission had no effect on resting membrane properties of raphe neurones.

These data indicate that serotonergic caudal raphe neurones receive glutamatergic inputs that are inhibited by presynaptic 5‐HT1B receptors; inhibition of excitatory synapses onto raphe cells may represent a novel mechanism for autoregulation of serotonergic neuronal activity by 5‐HT.
Activation of bovine tracheal chloride channels by amino group‐specific reagents
Authors: Duszyk Marek , Shu Yali , Ho Anthony K. , Man S. F. Paul
Published: September 2004

The predominant Cl− channel in bovine tracheal epithelial cells has a conductance of ∼71 pS and accounts for more than 80 % of the total chloride conductance. We examined the effects of protein‐modifying reagents on channel function and found that amino groups are critically involved in gating.

Patch clamp studies showed that lysine‐specific reagents, such as dimethyl adipimidate (DMA), significantly increased the channel open probability, but not its conductance. This suggests that modified residues are involved in the gating mechanism, but are distant from the channel permeation pathway.

Kinetic analysis of channel activity showed that histograms of open and closed durations could be well fitted by double exponential distributions, suggesting that the channel has at least two open and two closed states. DMA did not change the number of open or closed states, but increased channel mean open time.

Since membrane impermeant reagents were effective only from the extracellular side, we conclude that lysine residues in the extracellular domain of the channel are critically involved in gating. These residues may present an important target for site‐directed mutagenesis and pharmacological activation of Cl− channels in epithelial cells.
Changes in [Ca2+]i and membrane currents during impaired mitochondrial metabolism in dissociated rat hippocampal neurons
Authors: Nowicky Alex V. , Duchen Michael R.
Published: September 2004

This study was designed to establish the basis for altered membrane excitability during the inhibition of mitochondrial metabolism in central mammalian neurons. Perforated whole‐cell patch clamp and fluorimetric techniques were combined to examine changes in membrane currents, intracellular calcium ([Ca2+]i) and mitochondrial potential (ΔΨm) in neurons dissociated from the CA1 subfield of the hippocampus of young rats.

On application of the mitochondrial inhibitor NaCN, or the uncoupler carbonyl cyanide p‐trifluoromethoxyphenylhydrazone (FCCP), the membrane potential hyperpolarized and membrane conductance increased. Under voltage clamp, an outward current was seen. The reversal potential of the current at −83 mV and its dependence on extracellular [K+] confirmed that this was a K+ conductance.

Simultaneous recordings of [Ca2+]i and current showed a striking correlation between a rise in [Ca2+]i and the developed outward current. Flash photolysis of the caged Ca2+ chelator, diazo‐2, reversed both the rise in [Ca2+]i and the outward current. The current was reduced by 80% by charybdotoxin, was attenuated by 10 mm TEA+ but was unaffected by apamin or by the KATP channel blocker tolbutamide (400 μm−1 mm). These data suggest strongly that the current is carried by Ca2+‐dependent K+ channels.

Simultaneous recordings of membrane current, ΔΨm and [Ca2+]i revealed the sequence of events in response to impaired mitochondrial function (CN, FCCP or anoxia): ΔΨm depolarized, followed rapidly by an increase in [Ca2+]i followed in turn by the outward current. [Ca2+]i and membrane current recovered only after mitochondrial repolarization.

The rise in [Ca2+]i appeared to result from an increased Ca2+ influx through voltage‐gated Ca2+ channels. It was dependent on extracellular Ca2+ and was much reduced by methoxyverapamil (D600). The rate of Mn2+ quench of fura‐2 fluorescence was increased by the inhibitors, and the inhibitors induced a small inward current when K+ channels were blocked that preceded the rise in [Ca2+]i. However, the increase in [Ca2+]i showed no obvious dependence on membrane potential in cells clamped at a range of holding potentials from −90 to −45 mV.

Thus, removal of oxygen, uncoupling mitochondrial oxidative phosphorylation or inhibition of respiration, all lead to mitochondrial depolarization, an increased Ca2+ influx through (voltage‐gated) channels, even at hyperpolarized membrane potentials, raising [Ca2+]i which in turn drives an increased K+ conductance that modulates membrane excitability.
Control of pulsatile 5‐HT/insulin secretion from single mouse pancreatic islets by intracellular calcium dynamics
Authors: Barbosa Rui M. , Silva Amélia M. , Tomé Angelo R. , Stamford Jonathan A. , Santos Rosa M. , Rosário Luís M.
Published: September 2004

Glucose‐induced insulin release from single islets of Langerhans is pulsatile. We have investigated the correlation between changes in cytosolic free calcium concentration ([Ca2+]i) and oscillatory insulin secretion from single mouse islets, in particular examining the basis for differences in secretory responses to intermediate and high glucose concentrations. Insulin release was monitored in real time through the amperometric detection of the surrogate insulin marker 5‐hydroxytryptamine (5‐HT) via carbon fibre microelectrodes. The [Ca2+]i was simultaneously recorded by whole‐islet fura‐2 microfluorometry.

In 82 % of the experiments, exposure to 11 mM glucose evoked regular high‐frequency (average, 3.4 min−1) synchronous oscillations in amperometric current and [Ca2+]i. In the remaining experiments (18 %), 11 mM glucose induced an oscillatory pattern consisting of high‐frequency [Ca2+]i oscillations that were superimposed on low‐frequency (average, 0.32 min−1) [Ca2+]i waves. Intermittent high‐frequency [Ca2+]i oscillations gave rise to a similar pattern of pulsatile 5‐HT release.

Raising the glucose concentration from 11 to 20 mM increased the duration of the steady‐state [Ca2+]i oscillations without increasing their amplitude. In contrast, both the duration and amplitude of the associated 5‐HT transients were increased by glucose stimulation. The amount of 5‐HT released per secretion cycle was linearly related to the duration of the underlying [Ca2+]i oscillations in both 11 and 20 mM glucose. The slopes of the straight lines were identical, indicating that there is no significant difference between the ability of calcium oscillations to elicit 5‐HT/insulin release in 11 and 20 mM glucose.

In situ 5‐HT microamperometry has the potential to resolve the high‐frequency oscillatory component of the second phase of glucose‐induced insulin secretion. This component appears to reflect primarily the duration of the underlying [Ca2+]i oscillations, suggesting that glucose metabolism and/or access to glucose metabolites is not rate limiting to fast pulsatile insulin release.
Evidence for an electrogenic Na+‐HCO3− symport in rat cardiac myocytes
Authors: Aiello Ernesto A. , Petroff Martín G. Vila , Mattiazzi Alicia R. , Cingolani Horacio E.
Published: September 2004

The perforated whole‐cell configuration of patch clamp and the pH fluorescent indicator SNARF were used to determine the electrogenicity of the Na+‐HCO3− cotransport in isolated rat ventricular myocytes.

Switching from Hepes buffer to HCO3− buffer at constant extracellular pH (pHo) hyperpolarized the resting membrane potential (RMP) by 2.9 ± 0.4 mV (n= 9, P < 0.05). In the presence of HCO3−, the anion blocker SITS depolarized RMP by 2.6 ± 0.5 mV (n= 5, P < 0.05). No HCO3−‐induced hyperpolarization was observed in the absence of extracellular Na+. The duration of the action potential measured at 50 % of repolarization time (APD50) was 29.2 ± 6.1 % shorter in the presence of HCO3− than in its absence (n= 6, P < 0.05).

Quasi‐steady‐state currents were evoked by voltage‐clamped ramps ranging from −130 to +30 mV, during 8 s. The development of a novel component of Na+‐dependent and Cl−‐independent steady‐state outward current was observed in the presence of HCO3−. The reversal potential (Erev) of the Na+‐HCO3− cotransport current (INa,Bic) was measured at four different levels of extracellular Na+. A HCO3−:Na+ ratio compatible with a stoichiometry of 2:1 was detected. INa,Bic was also studied in isolation in standard whole‐cell experiments. Under these conditions, INa,Bic reversed at −96.4 ± 1.9 mV (n= 5), being consistent with the influx of 2 HCO3− ions per Na+ ion through the Na+‐HCO3− cotransporter.

In the presence of external HCO3−, after 10 min of depolarizing the membrane potential (Em) with 45 mm extracellular K+, a significant intracellular alkalinization was detected (0.09 ± 0.03 pH units; n= 5, P < 0.05). No changes in pHi were observed when the myocytes were pre‐treated with the anion blocker DIDS (0.001 ± 0.024 pH units; n= 5, n.s.), or when exposed to Na+‐free solutions (0.003 ± 0.037 pH units; n= 6, n.s.).

The above results allow us to conclude that the cardiac Na+‐HCO3− cotransport is electrogenic and has an influence on RMP and APD of rat ventricular cells.
Calcium current during a single action potential in a large presynaptic terminal of the rat brainstem
Authors: Borst J. G. G. , Sakmann B.
Published: September 2004

The calcium current of a ‘giant’ synaptic terminal (the calyx of Held) was studied using two‐electrode voltage clamp in slices of the rat brainstem.

In terminals with a long axon (length > 100 μm), the passive current transient decayed bi‐exponentially following voltage steps. In terminals with a short axon (length < 30 μm), the slow component was reduced or absent. These terminals also had small slow calcium tail currents following long depolarizing voltage steps, suggesting that these are largely due to axonal calcium channels.

Terminals were voltage clamped with action potential waveform commands. At both 24 and 36°C the calcium current began shortly after the peak of the action potential and ended before the terminal was fully repolarized.

The calcium current during the repolarization phase was 69 ± 1 % (n= 3) of maximal, judged from the increase in this current when a plateau phase was added to the action potential waveform.

A Hodgkin‐Huxley m2 model, based on the measured activation and deactivation of the calcium current, reproduced both the time course and the amplitude increase of the calcium currents during the different action potential waveforms well.

The fast gating of the calcium channels in the terminal ensures that they are effectively opened during the repolarization phase of an action potential. This implies that the distance between open calcium channels is minimized, which is in agreement with the view that multiple calcium channels are needed to release a vesicle in this synapse.
Intracellular Ca2+ regulation by the leech giant glial cell
Authors: Nett Wolfgang , Deitmer Joachim W.
Published: September 2004

We have measured the intracellular Ca2+ concentration, [Ca2+]i, and the intracellular Na+ concentration, [Na+]i, with the fluorescent dyes fura‐2 (for Ca2+) and SBFI (for Na+) in situ in giant glial cells of the central nervous system of the leech Hirudo medicinalis.

The basal [Ca2+]i was 79 ± 35 nm (n= 27) in cells voltage clamped at −70 to −80 mV, and 75 ± 29 nm (mean ±s.d., n= 82) in unclamped cells at a mean membrane potential of −67 ± 6 mV.

Removal of external Na+ evoked a small reversible [Ca2+]i increase of 29 ± 21 nm (n= 27) in cells voltage clamped at −70 to −80 mV, and of 35 ± 18 nm (n= 37) in unclamped cells. This [Ca2+]i increase, and the time constant of the subsequent [Ca2+]i recovery after Na+ re‐addition, did not change significantly with the holding potential between −110 and −60 mV.

The basal [Na+]i was 5.6 ± 1.3 mm (n= 18). Increasing [Na+]i by inhibiting the Na+‐K+ pump with 100 μm ouabain had no effect on the [Ca2+]i rise upon removal of external Na+.

The time course of recovery from a [Ca2+]i load mediated by voltage‐dependent Ca2+ influx during depolarization in high K+ was unaffected by the removal of external Na+.

Cyclopiazonic acid (10 μm), an inhibitor of the endoplasmic reticulum Ca2+‐ATPase, caused a transient increase in [Ca2+]i of 28 ± 11 nm (n= 5), and significantly slowed the recovery from imposed [Ca2+]i loads.

Iontophoretic injection of orthovanadate, an inhibitor of P‐type ATPases including the plasma membrane Ca2+‐ATPase, caused a persistent increase in the basal [Ca2+]i of 163 ± 101 nm (n= 5) in standard saline, and of 427 ± 338 nm in Na+‐free saline (n= 5). Vanadate injection significantly slowed the recovery from [Ca2+]i loads. Removal of external Na+ during vanadate injection induced an additional, reversible [Ca2+]i increase of 254 ± 64 nm (n= 3).

The results suggest that the low basal [Ca2+]i in these glial cells is predominantly maintained by a Ca2+‐ATPase in the plasma membrane. This ATPase is also the main Ca2+ extruder after an intracellular Ca2+ load, while intracellular stores appear to contribute little to this recovery. A Na+‐Ca2+ exchanger seems to play a minor role in the maintenance of basal [Ca2+]i in these cells, but becomes prominent when the plasma membrane Ca2+‐ATPase is blocked.
Vagally evoked synaptic currents in the immature rat nucleus tractus solitarii in an intact in vitro preparation
Authors: Smith Bret N. , Dou Ping , Barber William D. , Dudek F. Edward
Published: September 2004

Whole‐cell voltage‐clamp recordings in an in vitro brainstem‐cranial nerve explant preparation were used to assess the local circuitry activated by vagal input to nucleus tractus solitarii (NTS) neurones in immature rats.

All neurones that responded to vagal stimulation displayed EPSCs of relatively constant latency. Approximately 50 % of these also demonstrated variable‐latency IPSCs, and ∼31 % also displayed variable‐latency EPSCs to vagal stimulation. All neurones also had spontaneous EPSCs and IPSCs.

Evoked and spontaneous EPSCs reversed near 0 mV and were blocked by the glutamate AMPA/kainate receptor antagonists 6,7‐nitroquinoxaline‐2,3‐dione (DNQX) or 6‐cyano‐7‐nitroquinoxaline‐2,3‐dione (CNQX) at rest. Evoked EPSCs had rapid rise times (< 1 s) and decayed monoexponentially (τ= 2.04 ± 0.03 ms) at potentials near rest.

At holding potentials positive to ∼−50 mV, a slow EPSC could be evoked in the presence of DNQX or CNQX. This current peaked at holding potentials near −25 mV and was blocked by the NMDA receptor antagonist dl‐2‐amino‐5‐phosphonovaleric acid (AP5). It was therefore probably due to activation of NMDA receptors by vagal afferent fibres.

Fast IPSCs reversed near −70 mV and were blocked by the GABAA receptor antagonist bicuculline. In addition, bicuculline enhanced excitatory responses to vagal stimulation and increased spontaneous EPSC frequency. Antagonists to AMPA/kainate receptors reversibly blocked stimulus‐associated IPSCs and also decreased the frequency of spontaneous IPSCs.

These findings suggest that glutamate mediates synaptic transmission from the vagus nerve to neurones in the immature NTS by acting at non‐NMDA and NMDA receptors. NTS neurones may also receive glutamatergic and GABAergic synaptic input from local neurones that can be activated by vagal input and/or regulated by amino acid inputs from other brainstem neurones.1. Whole‐cell voltage‐clamp recordings in an in vitro brainstem‐cranial nerve explant preparation were used to assess the local circuitry activated by vagal input to nucleus tractus solitarii (NTS) neurones in immature rats.
Regulation of Na+ channels by luminal Na+ in rat cortical collecting tubule
Authors: Palmer Lawrence G. , Sackin Henry , Frindt Gustavo
Published: September 2004

The idea that luminal Na+ can regulate epithelial Na+ channels was tested in the cortical collecting tubule of the rat using whole‐cell and single‐channel recordings. Here we report results consistent with the idea of Na+ self‐inhibition.

Macroscopic amiloride‐sensitive currents (INa) were measured by conventional whole‐cell clamp. INa was a saturable function of external Na+ concentration ([Na+]o) with an apparent Km of 9 mm. Single channel currents (iNa) were measured in cell‐attached patches. iNa increased with pipette Na+ concentration with an apparent Km of 48 mm. Since INa= (iNa)NPo, the different Km values imply that the channel density (N) and/or open probability (Po) increase as [Na+]o decreases. Reduction of [Na+]o after increasing intracellular Na+ concentration also increased the outward amiloride‐sensitive conductance, consistent with activation of the Na+ channels.

The underlying mechanism was studied by changing pipette Na+ concentration while recording from cell‐attached patches. No increase in NPo was observed, suggesting that the effect is not a direct interaction between [Na+]o and the channel.

[Na+]o was varied outside the patch‐clamp pipette while recording from cell‐attached patches. When amiloride was in the bath to prevent Na+ entry, no change in NPo was observed.

Activation of the channels by hyperpolarization was observed with 140 mm Na+o but not with 14 mm Na+o.

The results are consistent with the concept of self‐inhibition of Na+ channels by luminal Na+. Activation of the channels by lowering [Na+]o is not additive with that achieved by hyperpolarization.
Detection and functional characterization of ryanodine receptors from sea urchin eggs
Authors: Lokuta Andrew J. , Darszon Alberto , Beltrán Carmen , Valdivia Hector H.
Published: September 2004

Immunoblot analysis, [3H]ryanodine binding, and planar lipid bilayer techniques were used to identify and characterize the functional properties of ryanodine receptors (RyRs) from Lytechinus pictus and Strongylocentrotus purpuratus sea urchin eggs.

An antibody against mammalian skeletal RyRs identified an ≈400 kDa band in the cortical microsomes of sea urchin eggs while a cardiac‐specific RyR antibody failed to recognize this protein. [3H]Ryanodine binding to cortical microsomes revealed the presence of a high‐affinity (Kd= 13 nM), saturable (maximal density of receptor sites, Bmax= 1.56 pmol (mg protein)−1) binding site that exhibited a biphasic response to Ca2+.

Upon reconstitution of cortical microsomes into lipid bilayers, only sparse and unstable openings of a high‐conductance cation channel were detected. Addition of crude sea urchin egg homogenate to the cytosolic (cis side) of the channel increased the frequency of openings and stabilized channel activity. The homogenate‐activated channels were Ca2+ sensitive, selective for Ca2+ over Cs+, and driven by ryanodine into a long‐lived subconductance state that represented ≈40 % of the full conductance level. Homogenate dialysed in membranes with a molecular weight cut‐off <= 2000 lacked the capacity to increase the frequency of RyR openings and to stabilize channel activity.

Direct application of cyclic adenosine diphosphoribose (cADPR) or photolysis of NPE‐cADPR (‘caged’ cADPR) by ultraviolet laser pulses produced transient activation of sea urchin egg RyRs. Calmodulin (CaM) failed to activate reconstituted RyRs; however, channel activity was inhibited by the CaM blocker trifluoroperazine, suggesting that CaM was necessary but not sufficient to sustain RyR activity.

These findings suggest that a functional Ca2+ release unit in sea urchin eggs is a complex of several molecules, one of which corresponds to a protein functionally similar to mammalian RyRs.
Multimodal output mapping of human central motor representation on different spatial scales
Authors: Classen Joseph , Knorr Uwe , Werhahn Konrad J. , Schlaug Gottfried , Kunesch Erwin , Cohen Leonardo G. , Seitz Rüdiger J. , Benecke Reiner
Published: September 2004

Non‐invasive mapping by focal transcranial magnetic stimulation (TMS) is frequently used to investigate cortical motor function in the intact and injured human brain. We examined how TMS‐derived maps relate to the underlying cortical anatomy and to cortical maps generated by functional imaging studies.

The centres of gravity (COGs) of TMS maps of the first dorsal intersosseus muscle (FDI) were integrated into 3‐D magnetic resonance imaging (MRI) data sets in eleven subjects. In seven of these subjects the TMS‐derived COGs were compared with the COG of regional cerebral blood flow increases using positron emission tomography (PET) in an index finger flexion protocol.

Mean TMS‐derived COG projections were located on the posterior lip of the precentral gyrus and TMS‐derived COG projections were in close proximity to the mean PET‐derived COG, suggesting that the two methods reflect activity of similar cortical elements.

Criteria for a reliable assessment of the COG and the number of positions with a minimum amplitude of two‐thirds of the maximum motor‐evoked potential (T3Ps) were determined as a function of the number of stimuli and extension of the stimulation field. COGs and T3Ps were compared with an estimate of the size of the human motor cortex targeting α‐motoneurons of forearm muscles. This comparison suggests that TMS can retrieve spatial information on cortical organization below the macroanatomic scale of cortical regions.

Finally, we studied the cortical representation of hand muscles in relation to facial and foot muscle representations and investigated hemispherical asymmetries. We did not find any evidence for a different ipsi‐ or contralateral representation of the mentalis muscle. Also, no difference was found between FDI representations on the dominant versus the non‐dominant hemisphere.
Role of domain I of neuronal Ca2+ channel α1 subunits in G protein modulation
Authors: Stephens Gary J. , Cantí Carles , Page Karen M. , Dolphin Annette C.
Published: September 2004

We studied the G protein inhibition of heteromultimeric neuronal Ca2+ channels by constructing a series of chimeric channels between the strongly modulated α1B subunit and the α1E(rbEII) subunit, which showed no modulation.

In parallel studies, α1 subunit constructs were co‐expressed together with the accessory Ca2+ channel α2‐δ and β2a subunits in mammalian (COS‐7) cells and Xenopus oocytes. G protein inhibition of expressed Ca2+ channel currents was induced by co‐transfection of Gβ1 and Gγ2 subunits in COS‐7 cells or activation of co‐expressed dopamine (D2) receptors by quinpirole (100 nm) in oocytes.

The data indicate that transfer of the α1B region containing the N‐terminal, domain I and the I‐II loop (i.e. the α1B1‐483 sequence), conferred G protein modulation on α1E(rbEII), both in terms of a slowing of activation kinetics and a reduction in current amplitude.

In contrast, the data are not consistent with the I‐II loop and/or the C‐terminal forming a unique site for G protein modulation.
Caesium blocks depolarizing after‐potentials and phasic firing in rat supraoptic neurones
Authors: Ghamari‐Langroudi Masoud , Bourque Charles W.
Published: September 2004

The effects of Cs+ on the action potential, post‐train after‐hyperpolarization (AHP), Ca2+‐dependent post‐spike depolarizing after‐potential (DAP) and phasic firing were examined during intracellular recordings from magnocellular neurosecretory cells (MNCs) in superfused rat hypothalamic explants.

Extracellular Cs+ reversibly inhibited (IC50, ≈1 mM) DAPs, and associated after‐discharges, that followed brief spike trains in each of sixteen cells tested. Although bath application of Cs+ also provoked a small reversible depolarization, inhibition of the DAP was retained when membrane voltage was kept constant by current injection.

Application of Cs+ had no significant effects on spike duration (n= 8), frequency‐dependent spike broadening (n= 8), spike hyperpolarizing after‐potentials (n= 14), or the amplitude of the isolated AHP (n= 7). Caesium‐evoked inhibition of the DAP, therefore, does not result from diminished spike‐evoked Ca2+ influx, and may reflect direct blockade of the conductance underlying the DAP.

Inhibition of the DAP was associated with an enhancement of the amplitude and duration of the AHP, indicating that the currents underlying the AHP and the DAP overlap in time following a train of action potentials, and that the relative magnitude of these currents is an important factor in determining the shape and time course of post‐train after‐potentials.

Bath application of Cs+ reversibly abolished phasic firing in each of seven cells tested. This effect was reversible and persisted at all subthreshold voltages tested. These results indicate that the current underlying the DAP is necessary for the genesis of plateau potentials and phasic firing in MNCs.
Effects of rapid shortening on rate of force regeneration and myoplasmic [Ca2+] in intact frog skeletal muscle fibres
Authors: Vandenboom R. , Claflin D. R. , Julian F. J.
Published: September 2004

The effect of rapid shortening on rate of force regeneration (dF/dtR) was examined in single, intact frog (Rana temporaria) skeletal muscle fibres (3·0 °C). Step releases leading to unloaded shortening were applied after 500 ms of stimulation, during the plateau of an isometric tetanus. Initial mean sarcomere length ranged from 2·05 to 2·35 μm; force regeneration after shortening was at 2·00 μm.

Values for dF/dtR following a 25 nm half‐sarcomere−1 release were 3·17 ± 0·17 (mean ± s.e.m., n= 8) times greater than the initial rate of rise of force before release (dF/dtI). As release size was increased from 25 to 175 nm half‐sarcomere−1, the relationship between release size and dF/dtR decreased sharply before attaining a plateau value that was 1·34 ± 0·09 times greater than dF/dtI. Despite wide variations in dF/dtR, the velocity of unloaded shortening remained constant (2·92 ± 0·08 μm half‐sarcomere−1 s−1; n= 8) for the different release amplitudes used in this study.

To investigate its role in the attenuation of dF/dtR with increased shortening, the effects of rapid ramp (constant velocity) shortening on intracellular free Ca2+ concentration ([Ca2+]i) were monitored using the Ca2+‐sensitive fluorescent dye furaptra. Compared with an isometric contraction, rapid fibre shortening was associated with a transient increase in [Ca2+]i while force regeneration after shortening was associated with a transient reduction in [Ca2+]i. The greatest reductions in [Ca2+]i were associated with the largest amplitude ramps.

Cross‐bridge‐mediated modifications of the Ca2+ affinity of troponin C (TnC) may explain the fluctuations in [Ca2+]i observed during and after ramps. Associated fluctuations in TnC Ca2+ occupancy could play a role in the reduction of dF/dtR with increasing release size.
The transient outward current in mice lacking the potassium channel gene Kv1.4
Authors: London Barry , Wang Dao W. , Hill Joseph A. , Bennett Paul B.
Published: September 2004

The transient outward current (Ito) plays a prominent role in the repolarization phase of the cardiac action potential. Several K+ channel genes, including Kv1.4, are expressed in the heart, produce rapidly inactivating currents when heterologously expressed, and may be the molecular basis of Ito.

We engineered mice homozygous for a targeted disruption of the K+ channel gene Kv1.4 and compared Ito in wild‐type (Kv1.4+/+), heterozygous (Kv1.4+/‐) and homozygous ‘knockout’ (Kv1.4−/−) mice. Kv1.4 RNA was truncated in Kv1.4−/− mice and protein expression was absent.

Adult myocytes isolated from Kv1.4+/+, Kv1.4+/− and Kv1.4−/− mice had large rapidly inactivating outward currents. The peak current densities at 60 mV (normalized by cellular capacitance, in pA pF−1; means ± s.e.m.) were 53.8 ± 5.3, 45.3 ± 2.2 and 44.4 ± 2.8 in cells from Kv1.4+/+, Kv1.4+/− and Kv1.4−/− mice, respectively (P < 0.02 for Kv1.4+/+vs. Kv1.4−/−). The steady‐state values (800 ms after the voltage clamp step) were 30.9 ± 2.9, 26.9 ± 3.8 and 23.5 ± 2.2, respectively (P < 0.02 for Kv1.4+/+vs. Kv1.4−/−). The inactivating portion of the current was unchanged in the targeted mice.

The voltage dependence and time course of inactivation were not changed by targeted disruption of Kv1.4. The mean best‐fitting V½ (membrane potential at 50 % inactivation) values for myocytes from Kv1.4 +/+, Kv1.4+/− and Kv1.4−/− mice were ‐53.5 ± 3.7, ‐51.1 ± 2.6 and ‐54.2 ± 2.4 mV, respectively. The slope factors (k) were ‐10.1 ± 1.4, ‐8.8 ± 1.4 and ‐9.5 ± 1.2 mV, respectively. The fast time constants for development of inactivation at ‐30 mV were 27.8 ± 2.2, 26.2 ± 5.1 and 19.6 ± 2.1 ms in Kv1.4+/+, Kv1.4+/− and Kv1.4−/− myocytes, respectively. At +30 mV, they were 35.5 ± 2.6, 30.0 ± 2.1 and 28.7 ± 1.6 ms, respectively. The time constants for the rapid phase of recovery from inactivation at ‐80 mV were 32.5 ± 8.2, 23.3 ± 1.8 and 39.0 ± 3.7 ms, respectively.

Nearly the entire inactivating component as well as more than 60 % of the steady‐state outward current was eliminated by 1 mm 4‐aminopyridine in Kv1.4+/+, Kv1.4+/− and Kv1.4−/− myocytes.

Western blot analysis of heart membrane extracts showed no significant upregulation of the Kv4 subfamily of channels in the targeted mice.

Thus, Kv1.4 is not the molecular basis of Ito in adult murine ventricular myocytes.
Role of myosin heavy chain composition in kinetics of force development and relaxation in rat myocardium
Authors: Fitzsimons Daniel P. , Patel Jitandrakumar R. , Moss Richard L.
Published: September 2004

The effects of ventricular myosin heavy chain (MHC) composition on the kinetics of activation and relaxation were examined in both chemically skinned and intact myocardial preparations from adult rats. Thyroid deficiency was induced to alter ventricular MHC isoform expression from ∼80 %α‐MHC/20 %β‐MHC in euthyroid rats to 100 %β‐MHC, without altering the expression of thin‐filament‐associated regulatory proteins.

In single skinned myocytes, increased expression of β‐MHC did not significantly affect either maximal Ca2+‐activated tension (P0) or the Ca2+ sensitivity of tension (pCa50). However, unloaded shortening velocity (V0) decreased by 80 % due to increased β‐MHC expression.

The kinetics of activation and relaxation were examined in skinned multicellular preparations using the caged Ca2+ compound DM‐nitrophen and caged Ca2+ chelator diazo‐2, respectively. Myocardium expressing 100 %β‐MHC exhibited apparent rates of submaximal and maximal tension development (kCa) that were 60 % lower than in control myocardium, and a 2‐fold increase in the half‐time for relaxation from steady‐state submaximal force.

The time courses of cell shortening and intracellular Ca2+ transients were assessed in living, electrically paced myocytes, both with and without β‐adrenergic stimulation (70 nm isoproterenol (isoprenaline)). Thyroid deficiency had no affect on either the extent of myocyte shortening or the resting or peak fura‐2 fluorescence ratios. However, induction of β‐MHC expression by thyroid deficiency was associated with increased half‐times for myocyte shortening and relengthening and increased half‐time for the decay of the fura‐2 fluorescence ratio. Qualitatively similar results were obtained in both the absence and the presence of β‐adrenergic stimulation although the β‐agonist accelerated the kinetics of the twitch and the Ca2+ transient.

Collectively, these data provide evidence that increased β‐MHC expression contributes significantly to the observed depression of contractile function in thyroid deficient myocardium by slowing the rates of both force development and force relaxation.
Calcium cycling and contractile activation in intact mouse cardiac muscle
Authors: Gao Wei Dong , Perez Nestor Gustavo , Marban Eduardo
Published: September 2004

Excitation‐contraction coupling in mouse cardiac muscle remains poorly characterized, despite the fact that the mouse is the mammalian species of choice for genetic manipulation. In this study, we characterized the relationship between internal calcium concentration ([Ca2+]i) and contraction in intact mouse ventricular muscle loaded with fura‐2 salt at 20–22°C.

Both Ca2+transient amplitude and twitch force increased monotonically as external Ca2+concentration ([Ca2+]o) was increased up to 8.0 mm, with no changes in diastolic levels or in the times to peak of either Ca2+transients or force. The decay of Ca2+transients was accelerated as [Ca2+]oincreased, while relaxation was prolonged. Both Ca2+transient amplitude and twitch force increased as stimulation rate increased from 0.2 to 4 Hz, but the increase in force was much greater than the underlying increase in [Ca2+]i.

The steady‐state force‐[Ca2+]irelationship revealed an [Ca2+]irequired for 50% of maximal activation (Ca50) of 0.95 ± 0.08 μm, a Hill coefficient of 9.9 ± 2.6, and a maximal Ca2+‐activated force (Fmax) of 60 ± 5 mN mm−2.

Unlike rat ventricular myocardium, mouse cardiac muscle resists supraphysiological [Ca2+]o. The strong positive force‐frequency relationship in mouse cardiac muscle, with increases of force disproportionate to the increases in Ca2+transients, suggests frequency‐dependent ‘sensitization’ of the myofilaments. During steady‐state activation, mouse muscle exhibits decreased Ca2+responsiveness relative to other species, but high co‐operativity.

These physiological features of mouse cardiac muscle merit consideration when interpreting the phenotypic consequences of genetic manipulations
Mathematical model of vertebrate gap junctions derived from electrical measurements on homotypic and heterotypic channels
Authors: Vogel Rolf , Weingart Robert
Published: September 2004

A mathematical model has been developed which describes the conductive and kinetic properties of homotypic and heterotypic gap junction channels of vertebrates.

The model consists of two submodels connected in series. Each submodel simulates a hemichannel and consists of two conductances corresponding to a high (H) and low (L) conductance state and a switch, which simulates the voltage‐dependent channel gating.

It has been assumed that the conductances of the high state and low state vary exponentially with the voltage across the hemichannel.

The parameters of the exponentials can be derived from data of heterotypic or homotypic channels. As a result, the behaviour of heterotypic channels can be predicted from homotypic channel data and vice versa.

The two switches of a channel are governed by the voltage drop across the respective hemichannel. The switches of a channel work independently, thus giving rise to four conformational states, i.e. HH, LH, HL and LL.

The computations show that the dogma of a constant conductance for homotypic channels results from the limited physiological range of transjunctional voltages (Vj) and the kinetic properties of the channel, so a new fitting procedure is presented.

Simulation of the kinetic properties at the multichannel level revealed current time courses which are consistent with a contingent gating.

The calculations have also shown that the channel state LL is rare and of short duration, and hence easy to miss experimentally.

The design of the model has been kept flexible. It can be easily expanded to include additional features, such as channel substates or a closed state.
Demonstration of facilitatory I wave interaction in the human motor cortex by paired transcranial magnetic stimulation
Authors: Ziemann Ulf , Tergau Frithjof , Wassermann Eric M. , Wischer Stephan , Hildebrandt Jörg , Paulus Walter
Published: September 2004

Transcranial magnetic stimulation (TMS) of the human motor cortex results in multiple discharges (D and I waves) in the corticospinal tract. We tested whether these volleys can be explored non‐invasively with paired TMS. The intensity of the first stimulus (S1) was set to produce a motor‐evoked potential (MEP) of 1 mV in the resting contralateral abductor digiti minimi (ADM) muscle; the second stimulus (S2) was set to 90 % of the resting motor threshold. At interstimulus intervals of 1·1‐1·5, 2·3‐2·9 and 4·1‐4·4 ms the MEP elicited by S1 plus S2 was larger than that produced by S1 alone.

Varying the S1 intensity between 70 and 130 % resting motor threshold with S2 held constant at 90 % resting motor threshold showed that the threshold for the first MEP peak was <= 70 % resting motor threshold. The second and third MEP peaks appeared only at higher S1 intensities. The latency of all peaks decreased with increasing S1 intensity.

Varying the S2 intensity with S1 held constant to produce a MEP of 1 mV on its own showed that the amplitude of all MEP peaks increased with S2 intensity, but that their timing remained unchanged.

Paired TMS in the active ADM (S1 clearly suprathreshold, S2 just above threshold; interstimulus interval, 1 ms) produced strong MEP facilitation. The onset of this facilitation occurred later by about 1·5 ms than the onset of the MEP evoked by S2 alone. No MEP facilitation was seen if the magnetic S2 was replaced by anodal or cathodal transcranial electrical stimulation.

It is concluded that the MEP facilitation after paired TMS, at least for the first MEP peak, is due to facilitatory interaction between I waves, and takes place in the motor cortex at or upstream from the corticospinal neurone.