Half-maximal Amplitude Research Articles

Mitochondrial Ca2+ Uptake Increases Ca2+ Release from Inositol 1,4,5-Trisphosphate Receptor Clusters in Smooth Muscle Cells

Smooth muscle activities are regulated by inositol 1,4,5-trisphosphate (InsP(3))-mediated increases in cytosolic Ca2+ concentration ([Ca2+](c)). Local Ca2+ release from an InsP(3) receptor (InsP(3)R) cluster present on the sarcoplasmic reticulum is termed a Ca2+ puff. Ca2+ released via InsP(3)R may diffuse to adjacent clusters to trigger further release and generate a cell-wide (global) Ca2+ rise. In smooth muscle, mitochondrial Ca2+ uptake maintains global InsP(3)-mediated Ca2+ release by preventing a negative feedback effect of high [Ca2+] on InsP(3)R. Mitochondria may regulate InsP(3)-mediated Ca2+ signals by operating between or within InsP(3)R clusters. In the former mitochondria could regulate only global Ca2+ signals, whereas in the latter both local and global signals would be affected. Here whether mitochondria maintain InsP(3)-mediated Ca2+ release by operating within (local) or between (global) InsP(3)R clusters has been addressed. Ca2+ puffs evoked by localized photolysis of InsP(3) in single voltage-clamped colonic smooth muscle cells had amplitudes of 0.5-4.0 F/F(0), durations of approximately 112 ms at half-maximum amplitude, and were abolished by the InsP(3)R inhibitor 2-aminoethoxydiphenyl borate. The protonophore carbonyl cyanide 3-chloropheylhydrazone and complex I inhibitor rotenone each depolarized DeltaPsi(M) to prevent mitochondrial Ca2+ uptake and attenuated Ca2+ puffs by approximately 66 or approximately 60%, respectively. The mitochondrial uniporter inhibitor, RU360, attenuated Ca2+ puffs by approximately 62%. The "fast" Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acted like mitochondria to prolong InsP(3)-mediated Ca2+ release suggesting that mitochondrial influence is via their Ca2+ uptake facility. These results indicate Ca2+ uptake occurs quickly enough to influence InsP(3)R communication at the intra-cluster level and that mitochondria regulate both local and global InsP(3)-mediated Ca2+ signals.

Mitochondria Act Within InsP3R Clusters To Maintain Ca2+ Release In Smooth Muscle

Many smooth muscle activities including contraction, transcription, growth and apoptosis are regulated by transient inositol 1,4,5-trisphosphate (InsP3)-mediated increases in cytosolic Ca2+ concentration ([Ca2+]c). InsP3 binds to receptors (InsP3R) present on the sarcoplasmic reticulum to evoke Ca2+ release. InsP3R exist in clusters and Ca2+ released from one receptor may activate nearby InsP3R within this cluster in a CICR-like process to evoke a ‘puff’. Ca2+ released may also diffuse to adjacent clusters to trigger further Ca2+ release and generate a Ca2+ rise throughout the cell. Mitochondrial Ca2+ uptake limits a negative feedback process operative on InsP3R to maintain Ca2+ release. Inhibition of mitochondrial Ca2+ uptake decreases InsP3-mediated Ca2+ waves by ≥50%. We addressed whether mitochondria act to maintain release by operating within or between InsP3R clusters. Ca2+ puffs were evoked by localized photolysis of InsP3 in single voltage-clamped colonic smooth muscle cells in which [Ca2+]c and ΔΨM were measured simultaneously. EGTA, a slow Ca2+ buffer, was used to functionally uncouple puff sites to prevent the formation of Ca2+ waves. EGTA was used at a concentration ([300 μM]) which does not affect the magnitude or kinetics of Ca2+ puffs. InsP3-evoked Ca2+ puffs had amplitudes of 0.5-5.0 F/F0 and durations of ∼200 ms at half-maximum amplitude. Puffs were abolished by the InsP3R inhibitor 2-APB. The protonophore CCCP and the mitochondrial inhibitor rotenone, each used with oligomycin, depolarized the mitochondrial membrane potential (ΔΨM) and prevented mitochondrial Ca2+ uptake. Depolarizing ΔΨM with CCCP attenuated Ca2+ puffs by ∼65% while rotenone inhibited them by ∼60%. These results indicate mitochondrial Ca2+ uptake occurs quickly enough to influence InsP3R communication at the intra-cluster level. Supported by the Wellcome Trust and British Heart Foundation.

Pentobarbital Enhances γ-Aminobutyric Acid-Mediated Excitation Without Altering Synaptic Plasticity in Rat Hippocampus

Synaptic plasticity is thought to provide a molecular mechanism for learning and memory. N-methyl-d-aspartate receptor-mediated plasticity requires that N-methyl-d-aspartate receptor activation coincides with postsynaptic depolarizing potentials (DPSP(A)'s). Pentobarbital, in high concentrations, enhances DPSP(A)'s, but high concentrations suppress synaptic plasticity, probably by impairing glutamatergic transmission. Here we tested the hypothesis that low concentrations of pentobarbital can enhance DPSP(A)'s and modify the induction of synaptic plasticity. Studies were performed in vitro on rat hippocampal slices. With glutamate transmission blocked, intracellular recording from CA1 neurons was used to investigate the influence of 5 microM pentobarbital on DPSP(A)'s and neuron excitability evoked by high frequency (100 Hz) stimulation. With glutamate transmission intact, extracellular recording was used to examine the effect of 5 microM pentobarbital on the induction of long-term depression and long-term potentiation of synaptic transmission by conditioning stimuli applied to the Schaffer collateral pathway. High frequency stimulation generated typical DPSP(A)'s that were mediated by gamma-aminobutyric acid(A) receptors and dependent upon HCO3-. Pentobarbital (5 microM) increased the amplitude, but not the width, at half-maximal amplitude of DPSPA's (P < 0.01). Pentobarbital increased the probability of action potential generation during the DPSP(A)'s. Pentobarbital did not alter the induction of long-term depression or long-term potentiation. Despite increasing the amplitude of DPSP(A)'s, 5 microM pentobarbital did not alter the induction of synaptic plasticity by a range of conventional conditioning stimuli. These results do not support the hypothesis that excitatory effects of pentobarbital may alter synaptic plasticity.

SparkMaster: automated calcium spark analysis with ImageJ

Ca sparks are elementary Ca-release events from intracellular Ca stores that are observed in virtually all types of muscle. Typically, Ca sparks are measured in the line-scan mode with confocal laser-scanning microscopes, yielding two-dimensional images (distance vs. time). The manual analysis of these images is time consuming and prone to errors as well as investigator bias. Therefore, we developed SparkMaster, an automated analysis program that allows rapid and reliable spark analysis. The underlying analysis algorithm is adapted from the threshold-based standard method of spark analysis developed by Cheng et al. (Biophys J 76: 606-617, 1999) and is implemented here in the freely available image-processing software ImageJ. SparkMaster offers a graphical user interface through which all analysis parameters and output options are selected. The analysis includes general image parameters (number of detected sparks, spark frequency) and individual spark parameters (amplitude, full width at half-maximum amplitude, full duration at half-maximum amplitude, full width, full duration, time to peak, maximum steepness of spark upstroke, time constant of spark decay). We validated the algorithm using images with synthetic sparks embedded into backgrounds with different signal-to-noise ratios to determine an analysis criteria at which a high sensitivity is combined with a low frequency of false-positive detections. Finally, we applied SparkMaster to analyze experimental data of sparks measured in intact and permeabilized ventricular cardiomyocytes, permeabilized mammalian skeletal muscle, and intact smooth muscle cells. We found that SparkMaster provides a reliable, easy to use, and fast way of analyzing Ca sparks in a wide variety of experimental conditions.

High-Density Presynaptic Transporters Are Required for Glutamate Removal from the First Visual Synapse

Reliable synaptic transmission depends not only on the release machinery and the postsynaptic response mechanism but also on removal or degradation of transmitter from the synaptic cleft. Accumulating evidence indicates that postsynaptic and glial excitatory amino acid transporters (EAATs) contribute to glutamate removal. However, the role of presynaptic EAATs is unclear. Here, we show in the mouse retina that glutamate is removed from the synaptic cleft at the rod to rod bipolar cell (RBC) synapse by presynaptic EAATs rather than by postsynaptic or glial EAATs. The RBC currents evoked by electrical stimulation of rods decayed slowly after pharmacological blockade of EAATs. Recordings of the evoked RBC currents from EAAT subtype-deficient mice and the EAAT-coupled anion current reveal that functional EAATs are localized to rod terminals. Model simulations suggest that rod EAATs are densely packed near the release site and that rods are equipped with an almost self-sufficient glutamate recollecting system.

Effects of B and N implantation on optical absorption and photoluminescence and comparison to the effects of implanting Si, Ge, O, and Ar in silica

Silica samples were implanted at multiple energies with B and N ions, which resulted in a constant concentration implanted layer ∼600 nm thick starting ∼100 nm below the sample surface. The energies to produce the constant concentration layer ranged from 35 to 320 keV and the concentrations were 0.03–2.1 at.% for B and 0.024–2.5% for N. The optical absorption of the suite of samples was measured from 2.8 to 6.5 eV. No bands were detected at energies <2.8 eV. Bands which have been identified in the literature as intrinsic to SiO 2 were fitted to the data with only one free parameter, the amplitudes of the maxima of the bands. This fit was within ±2% from 2.8 to 5.4 eV. To fit the data from 5.4 to 6.6 eV we assumed that there was a single band with an energy >6.5 eV. The nonlinear fitting program calculated a band at 6.76 ± 0.06 eV with a width at half maximum amplitude of 0.81 ± 0.08 eV, for all concentrations of B and a band at 6.65 ± 0.06 eV with a full width at half maximum of 0.67 ± 0.08 eV for the N implanted samples to achieve a fit to ±2% from 5.4 to 6.5 eV. In the range of energies from 2.0 to 6.5 eV no bands attributable to either B or N were required to fit the observed optical absorption. Excitation of the samples with 5 eV photons produced a photoluminescence [PL] band at 4.43 ± 0.02 eV with a full width at half maximum amplitude [FWHM] of 0.53 ± 0.02 eV. When measured at 4.43 eV the photoluminescence excitation [PLE] band had a maximum at 5.00 ± 0.02 eV with a FWHM of 0.40 ± 0.02 eV. Bands with approximately the same energies and FWHMs were observed in the N samples as observed in the B samples. The correlation of the amplitudes of these PL bands with the amplitudes of the absorption bands at 5.01 and 5.17 eV is less than one. Based on a comparison of the absorption amplitudes deduced from the linear and nonlinear fitting procedure, and PL and PLE amplitudes in the B, Si, N, O, and Ar samples, an absorption band at 5.00 ± 0.02 eV producing the PL at 4.43 ± 0.02 eV is due to a Si related defect state. In the Ge case PL bands were observed at 4.38 ± 0.02 and 3.23 ± 0.02 eV with PLE bands at 4.98 ± 0.2 and 5.18 ± 0.02 eV, respectively. An absorption band at 5.17 eV is the source of the 3.23 eV PL band and is, therefore Ge related. The fitted absorption spectra in the Ge case required another absorption band at 5.54 ± 0.07 eV as well as a band at 6.87 ± 0.04 eV to fit the spectra in the Ge case for all concentrations, to within the ±2%. PL bands at other energies were not detected in the PL spectra of any of the samples when excited with energies in the 4.5–6.4 eV range.

Characteristics of Prolonged Ca 2+ Release Events Associated With the Nuclei in Adult Cardiac Myocytes

Confocal microscopy was used to study the properties of nuclear Ca2+ regulation in adult ventricular myocytes. Prolonged nuclear Ca2+ release (PNCR) events were identified in both intact and permeabilized rat myocytes. PNCR occurred spontaneously and was restricted to localized regions at the ends of the elongated nuclei. Typically, PNCR took the form of a rapid rise in [Ca2+] followed by a maintained plateau. The mean duration of PNCR (1.78+/-0.19 seconds) was markedly greater than the half decay time for cytosolic Ca2+ sparks (31.2+/-0.56 ms) obtained under the same conditions. The PNCR width at half maximum amplitude (5.0+/-0.2 microm) was also significantly greater than that of cytosolic Ca2+ sparks (2.6+/-0.05 microm) obtained under the same conditions. Experiments involving the use of syto-11 to accurately locate the nuclei demonstrated that PNCR originates from the nuclear envelope or a closely associated structure. The spatial spread of PNCR was asymmetrical, with greater diffusion of Ca2+ toward the center of the nucleus than the cytosol. Both PNCR and Ca2+ sparks were abolished by interventions that deplete SR Ca2+ stores or inhibit RYR activation. Experiments on intact, electrically stimulated cells revealed that diffusion of Ca2+ from the ends of the nucleus toward the center is a prominent feature of the nucleoplasmic Ca2+ transient. The possibility that recruitment of Ca2+ release sites involved in PNCR might influence the temporal and spatial characteristics of the nucleoplasmic [Ca2+] transient is considered.

Localised calcium release events in cells from the muscle of guinea-pig gastric fundus.

After enzymatic dispersion of the muscle of the guinea-pig gastric fundus, single elongated cells were observed which differed from archetypal smooth muscle cells due to their knurled, tuberose or otherwise irregular surface morphology. These, but not archetypal smooth muscle cells, consistently displayed spontaneous localized (i.e. non-propagating) intracellular calcium ([Ca(2+)](i)) release events. Such calcium events were novel in their magnitude and kinetic profiles. They included short transient events, plateau events and events which coalesced spatially or temporally (compound events). Quantitative analysis of the events with an automatic detection programme showed that their spatio-temporal characteristics (full width and full duration at half-maximum amplitude) were approximately exponentially distributed. Their amplitude distribution suggested the presence of two release modes. Carbachol application caused an initial cell-wide calcium transient followed by an increase in localized calcium release events. Pharmacological analysis suggested that localized calcium release was largely dependent on external calcium entry acting on both inositol trisphosphate receptors (IP(3)Rs) and ryanodine receptors (RyRs) to release stored calcium. Nominally calcium-free external solution immediately and reversibly abolished all localized calcium release without blocking the initial transient calcium release response to carbachol. This was inhibited by 2-APB (100 microm), ryanodine (10 or 50 microm) or U-73122 (1 microm). 2-APB (100 microm), xestospongin C (XeC, 10 microm) or U-73122 (1 microm) blocked both spontaneous localized calcium release and localized release stimulated by 10 microm carbachol. Ryanodine (50 microm) also inhibited spontaneous release, but enhanced localized release in response to carbachol. This study represents the first characterization of localized calcium release events in cells from the gastric fundus.

Transmitter characteristics of cutaneous, renal and skeletal muscle small arteries in the rat.

We studied transmitter characteristics of proximal and distal arteries supplying skin (saphenous artery and its medial tarsal branch), kidneys (terminal branches of renal artery and interlobar arteries) and skeletal muscle (proximal and distal sections of external sural artery). Artery segments were mounted in an isometric myograph and intramural nerves were activated by electrical field stimulation. Adrenergic and purinergic components of the neurogenic response were blocked using phenoxybenzamine and alpha,beta-methylene adenosine triphosphate (mATP), respectively. Arteries from skin or kidney developed rapid and prominent neurogenic contractile responses, with half-maximal amplitude reached within 5-15 s; responses in proximal vessels were greater than in distal vessels. Arteries from skeletal muscle responded to sympathetic stimulation with a moderate contraction developing over 1 min or more, the response of distal segments was greater than that of proximal segments. In skeletal muscle vessels the sympathetically evoked contraction was completely blocked by phenoxybenzamine, whereas in skin and renal vessels it was the combined effect of noradrenaline and adenosine triphosphate (ATP). Given alone, mATP did not change the magnitude of the response to nerve stimulation, but increased its latency and also potentiated the response to exogenous noradrenaline. In all vascular beds, distal vessels were more sensitive to noradrenaline and mATP. It thus appears that the noradrenaline/ATP ratio of the sympathetic vasoconstrictor response differs between vascular beds in a way that is consistent with known differences in the selective regulation of regional vascular resistance by the sympathetic nervous system.

Effect of amiodarone on the dynamics of mechanical restitution of rat papillary muscles.

We studied the effect of amiodarone (class III antiarrhythmic drug) on the dynamics of mechanical restitution of rat papillary muscle. Amiodarone produced a weak negative inotropic effect and stimulated potentiation of contractility of the muscle preparation after short-term (4-60 sec) cessation of its electrical stimulation. On the other hand, the time of attaining half-maximum amplitude of contractions after amiodarone treatment did not differ from the control. Analysis of curves presenting the drop of potentiation of muscle preparation contractility after resumption of regular electrical stimulation after 60-sec arrest until attaining a stable level showed that the amplitude returned to the initial level by the 9thcontraction-relaxation cycle both in the control and after amiodarone treatment. Coefficient of the drop of contraction amplitude potentiation was virtually the same in the two groups. Presumably, amiodarone does not modulate calcium-binding capacity of the sarcoplasmic reticulum, but improves Ca retention in the sarcoplasmic reticulum stores.

Crosstalk between ryanodine receptors and IP(3) receptors as a factor shaping spontaneous Ca(2+)-release events in rabbit portal vein myocytes.

In smooth muscle cells freshly isolated from rabbit portal vein, there was only one site discharging the majority of spontaneous Ca(2+)-release events; the activity of this single site was studied using laser scanning confocal imaging after loading the cells with the fluorescent Ca(2+) indicator fluo-4 acetoxymethyl ester. Localised spontaneous Ca(2+)-release events visualised by line-scan imaging revealed two predominant spatiotemporal patterns: (i) small-amplitude, fast events similar to Ca(2+) sparks in cardiomyocytes and (ii) larger and slower events. The sum of two Gaussian profiles was well fitted to the amplitude histogram (peak frequencies at 1.8 and 3.2 F/F(0)) and spatial spread (full width at half-maximal amplitude) histogram (peak frequencies at 2 and 3.8 microm) for the 230 localised Ca(2+)-release events analysed. The existence of two populations of Ca(2+)-release events was also supported by the histograms of the rise times and half-decay times, which revealed modes at 38 and 65 ms, respectively. Shifting the scan line along the z-axis during imaging from a single discharge site suggested that the appearance of two populations of Ca(2+)-release events is not due to out-of-focus imaging. Both small and large events persisted upon 3-5 min exposure to 1-5 microM nicardipine, but were abolished after 10-15 min exposure to 50-100 microM ryanodine, 0.1 microM thapsigargin or 10 microM cyclopiazonic acid. Only small-amplitude, fast events persisted in the presence of inhibitors of inositol 1,4,5-trisphosphate (IP(3))-induced Ca(2+) release, 10 microM xestospongin C or 30 microM 2-aminoethoxy-diphenylborate (2-APB), or in the presence of 2.5 microM U-73122 (a phospholipase C (PLC) inhibitor). Coupling between neighbouring Ca(2+)-release domains giving rise to spontaneous [Ca(2+)](i) waves was abolished in the presence of 2-APB. Examination of the saltatory propagation of the waves suggested that the critical factor that determines propagation between domains is a time-dependent change in the sensitivity of ryanodine receptors and/or IP(3) receptors to Ca(2+), which can give rise to 'loose coupling' between release sites. These results suggest that activation of IP(3) receptors (due to the tonic activity of PLC and ongoing production of IP(3)) recruits neighbouring domains of ryanodine receptors, leading to larger Ca(2+) releases and saltatory propagation of [Ca(2+)](i) waves in portal vein myocytes.

Background adaptation in a rat model of retinopathy of prematurity.

Low dark-adapted, scotopic retinal and visual sensitivity in retinopathy of prematurity (ROP) could be due to disease of the inner retina, or the recently described rod photoreceptor abnormalities. Receptoral disease decreases catch of quanta from both test flashes and steady background lights; increment threshold functions are shifted up and right. In diseases with normal receptors but low retinal sensitivity due to abnormal post receptoral processing, the increment threshold functions are shifted up with no horizontal translation. Herein we test the hypothesis that the rod photoreceptors are the site of ROP disease which causes low dark adapted b-wave sensitivity. The effect of steady background light on the ERG b-wave in a rat model of ROP is studied. ERG stimulus/response functions were obtained using full-field stimuli in the dark-adapted state, and in the presence of a steady background light. In each adaptation condition, log sigma, the test flash intensity that produced a half-maximum b-wave amplitude, was calculated. In pilot experiments, the background light selected had raised log sigma about a log unit in controls. In dark-adapted ROP rats log sigma was significantly higher, 0.35 log unit, than in controls. In the presence of the background light, log sigma in ROP and control rats did not differ significantly indicating a relative shift, up and right, of the increment sensitivity function for the less sensitive ROP rats. The effect of the background light is consistent with receptoral disease causing low dark adapted b-wave sensitivity in ROP rats.

FALCON test results from the Bathurst Mining camp

BHP Billiton commenced exploration surveying with the world's first fully operational, airborne gravity gradiometer in October 1999. This gradiometer (called Einstein), together with a later one called Newton, was developed in conjunction with Lockheed Martin by BHP Billiton's FALCON project. FALCON data are acquired by Sander Geophysics Ltd., flying a Cessna Grand Caravan to survey specifications typical of aeromagnetic surveys. The first FALCON survey was flown over a portion of the Bathurst mining camp in New Brunswick, Canada in order to compare system performance with existing extensive and detailed ground-gravity data. The ground-gravity data, supplied courtesy of Noranda Minerals Exploration Ltd., were upward continued to the flying height and vertically differentiated to provide vertical gravity gradient data suitable for comparison with the airborne data. The two data sets compare very well and the results demonstrate that the FALCON airborne gravity gradiometer is capable of detecting sources with a vertical gravity gradient signal of greater than 10 Eo and a full-width at half-maximum amplitude of 500 m.

Regulation of calcium sparks and spontaneous transient outward currents by RyR3 in arterial vascular smooth muscle cells.

Intracellular Ca(2+) levels control both contraction and relaxation in vascular smooth muscle cells (VSMCs). Ca(2+)-dependent relaxation is mediated by discretely localized Ca(2+) release events through ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR). These local increases in Ca(2+) concentration, termed sparks, stimulate nearby Ca(2+)-activated K(+) (BK) channels causing BK currents (spontaneous transient outward currents or STOCs). STOCs are hyperpolarizing currents that oppose vasoconstriction. Several RyR isoforms are coexpressed in VSMCs; however, their role in Ca(2+) spark generation is unknown. To provide molecular information on RyR cluster function and assembly, we examined Ca(2+) sparks and STOCs in RyR3-deficient freshly isolated myocytes of resistance-sized cerebral arteries from knockout mice and compared them to Ca(2+) sparks in cells from wild-type mice. We used RT-PCR to identify RyR1, RyR2, and RyR3 mRNA in cerebral arteries. Ca(2+) sparks in RyR3-deficient cells were similar in peak amplitude (measured as F/F(0)), width at half-maximal amplitude, and duration compared with wild-type cell Ca(2+) sparks. However, the frequency of STOCs (between -60 mV and -20 mV) was significantly higher in RyR3-deficient cells than in wild-type cells. Ca(2+) sparks and STOCs in both RyR3-deficient and wild-type cells were inhibited by ryanodine (10 micromol/L), external Ca(2+) removal, and depletion of SR Ca(2+) stores by caffeine (1 mmol/L). Isolated, pressurized cerebral arteries of RyR3-deficient mice developed reduced myogenic tone. Our results suggest that RyR3 is part of the SR Ca(2+) spark release unit and plays a specific molecular role in the regulation of STOCs frequency in mouse cerebral artery VSMCs after decreased arterial tone.

Falcon Test Results from the Bathurst Mining Camp

BHP Billiton commenced exploration surveying with the world’s first fully operational, airborne gravity gradiometer in October 1999. This gradiometer (called Einstein), together with a later one called Newton, was developed in conjunction with Lockheed Martin by BHP Billiton’s FALCON project. FALCON data are acquired by Sander Geophysics Ltd., flying a Cessna Grand Caravan to survey specifications typical of aeromagnetic surveys.The first FALCON survey was flown over a portion of the Bathurst mining camp in New Brunswick, Canada in order to compare system performance with existing extensive and detailed ground-gravity data.The ground-gravity data, supplied courtesy of Noranda Minerals Exploration Ltd., were upward continued to the flying height and vertically differentiated to provide vertical gravity gradient data suitable for comparison with the airborne data.The two data sets compare very well and the results demonstrate that the FALCON airborne gravity gradiometer is capable of detecting sources with a vertical gravity gradient signal of greater than 10 Eö and a full-width at half-maximum amplitude of 500 m.

Development of germanium gallium sulphide glass fibres for the [formula omitted] praseodymium-doped fibre amplifier

We report on our current progress in the development of germanium gallium sulphide glass fibres for the praseodymium-doped fibre amplifier (PDFA). Praseodymium-doped glasses with compositions (GeS x ) 98(GaS 3) 2 ( x: 2.25, 2.5, 3) are melted from germanium and gallium metals and sulphur in sealed silica ampoules at 1000°C and then quenched in air. The critical step in the fibre manufacturing process is the fabrication of the fibre preform out of glass rods. The `rod in tube' method was selected for preform fabrication. A new hot deformation process for manufacturing cladding tubes using the visco-elastic properties of the glass is described. A measure for the thermal stability of the glasses is the difference between the glass transition temperature, T g, and the crystallisation onset temperature, T x. The thermal stability was evaluated using dilatometry and combined differential thermal analysis/thermogravimetry (DTA/TG) analysis for determining T g and T x. For the selected germanium gallium sulphide glasses the difference between the crystallisation temperature and glass transition temperature is more than 200°C. Both the fibre drawing temperature and the hot deformation temperature (to produce cladding glass tubes) are less than the crystallisation onset temperature. The germanium gallium sulphide host glasses are transparent in the 0.5–10 μm wavelength region. The wavelength of the maximum of the stimulated emission band with full width at half maximum (FWHM) amplitude of 80 nm is 1.34 μm , the emission lifetime is 390 μs .

Calcium sparks in human coronary artery smooth muscle cells resolved by confocal imaging.

The observation of local 'elementary' Ca2+ release events (Ca2+ sparks) through ryanodine receptor (RyR) channels in the sarcoplasmic reticulum (SR) has changed our understanding of excitation-contraction (EC) coupling in cardiac and smooth muscle. In arterial smooth muscle, Ca2+ sparks have been suggested to oppose myogenic vasoconstriction and to influence vasorelaxation by activating co-localized Ca2+ activated K+ (K(Ca)) channels (STOCs). However, all prior studies on Ca2+ sparks have been performed in non-human tissues. In order to understand the possible significance of Ca2+ sparks to human cardiovascular function, we used high spatial resolution confocal imaging to record Ca2+ sparks in freshly-isolated, individual myocytes of human coronary arteries loaded with the Ca2+ indicator fluo-3. Local SR Ca2+ release events recorded in human myocytes were similar to 'Ca2- sparks' recorded previously from non-human smooth muscle cells. In human myocytes, the peak [Ca2+]i amplitudes of Ca2+ sparks (measured as F/F0) and width at half-maximal amplitude were 2.3 and 2.27 microm, respectively. The duration of Ca2+ sparks was 62 ms. Ca2+ sparks were completely inhibited by ryanodine (10 micromol/l). Ryanodine-sensitive STOCs could be identified with typical properties of K(Ca) channels activated by Ca2+ sparks. Our data implies that modern concepts suggesting an essential role of Ca2+ spark generation in EC coupling recently derived from non-human muscle are applicable to human cardiovascular tissue. Although the basic properties of Ca2+ sparks are similar, our results demonstrate that Ca2+ sparks in coronary arteries in humans, have features distinct from non-arterial smooth muscle cells of other species.

Effects of ionotropic glutamate receptor blockade and 5-HT1A receptor activation on spreading depression in rat neocortical slices.

The effect of the AMPA antagonist NBQX (10 microM), NMDA antagonist ketamine (100 microM) and 5-HT1A agonist 8-OH-DPAT (1, 10 and 100 microM) on the properties of a KCl-induced spreading depression (SD) was studied in parietal cortical slices of adult rats. Whereas NBQX did not significantly affect the SD, ketamine significantly (p < 0.01) reduced the amplitude of the first SD peak (12.8 +/- 4.6 mV) and blocked the second SD peak when compared with the controls (19.8 +/- 5.2 mV and 25 +/- 5 mV, respectively). Ketamine also decreased the SD duration at half maximal amplitude from 34.9 +/- 12.4 s to 22.2 +/- 12 s (p < 0.05). 8-OH-DPAT attenuated the duration of the SD from 42 +/- 15.6 s to 21.2 +/- 10.6 s (p < 0.05, 100 microM). These data indicate that not only NMDA receptor blockade, but also activation of the 5-HT1A receptor attenuates the SD and may be beneficial in the reduction of ischemic injury following focal cerebral ischemia.

Identification of different putative neuronal subtypes in cultures of the superior region of the hippocampus using electrophysiological parameters

Cultured neurons offer many advantages over a slice preparation for whole-cell patch-clamp studies, such as better control over the environment and space clamp control. However, heterogeneous cultures of neurons present problems in distinguishing the cell type from which recordings are made. The present study uses correlations with data obtained in the hippocampal slice preparation to determine the feasibility of “identifying” different neuronal subtypes in cultures obtained from the superior region of postnatal two- to 13-day-old rat hippocampus. Whole-cell patch-clamp recording in the current-clamp mode after 24–96 h in culture was used to determine if the action potential duration would be a useful criterion in distinguishing cell types. Single action potentials were elicited by a 0.1–0.2 ms, 2–4 nA depolarizing pulse. The average membrane potential and input resistance were −46.8±1.2 mV ( n=58) and 576±56 MΩ ( n=57), respectively. A frequency distribution of the action potential duration measured at half-maximal amplitude showed four distinct groups of neurons (group 1, 1.36±0.03 ms, n=17; group 2, 2.19±0.05 ms, n=20; group 3, 3.17±0.10 ms, n=16; group 4, 4.36±0.13, n=5). Based on correlations with previous studies using intracellular recording in identified cells in slices, the data suggest that group 1 represents basket cells, group 2 represents vertical cells, group 3 represents a combination of stellate cells and pyramidal cells, and group 4 represents another unidentified class of cells. Further analysis of the fast afterhyperpolarization allows distinction between pyramidal cells and stellate cells in group 3. In contrast to the interneurons in a slice preparation, these cells offer good voltage control and environmental control. Future studies will record from these cells in current-clamp mode to quickly characterize the action potential before switching to voltage-clamp recording to characterize the currents present in the different types of interneurons.

The voltage-dependent conductances of rat neocortical layer I neurons.

Whole cell patch-clamp techniques were used to study voltage-dependent sodium (Na+), calcium (Ca2+), and potassium (K+) conductances in acutely isolated neurons from cortical layer I of adult rats. Layer I cells were identified by means of gamma-aminobutyric acid (GABA) immunocytochemistry. Positive stainings for the Ca2+-binding protein calretinin in a subset of cells, indicated the presence of Cajal-Retzius (C-R) cells. All investigated cells displayed a rather homogeneous profile of voltage-dependent membrane currents. A fast Na+ current activated at about -45 mV, was half-maximal steady-state inactivated at -66.6 mV, and recovery from inactivation followed a two-exponential process (tau1 = 8.4 ms and tau2 = 858.8 ms). Na+ currents declined rapidly with two voltage-dependent time constants, reaching baseline current after some tens of milliseconds. In a subset of cells (< 50%) a constant current level of < 65 pA remained at the end of a 90 ms step. A transient outward current (Ifast) activated approximately -40 mV, declined rapidly with a voltage-insensitive time constant (tau approximately 350 ms) and was relatively insensitive to tetraethylammonium (TEA, 20 mM). Ifast was separated into two components based on their sensitivity to 4-aminopyridine (4-AP): one was blocked by low concentrations (40 microM) and a second by high concentrations (6 mM). After elimination of Ifast by a conditioning prepulse (50 ms to -50 mV), a slow K+ current (I(KV)) could be studied in isolation. I(KV) was only moderately affected by 4-AP (6 mM), while TEA (20 mM) blocked most (> 80%) of the current. I(KV) activated at about -40 mV, declined monoexponentially in a voltage-dependent manner (tau approximately 850 ms at -30 mV), and revealed an incomplete steady-state inactivation. In addition to Ifast and I(KV), indications of a Ca2+-dependent outward current component were found. When Na+ currents, Ifast, and I(KV) were blocked by tetrodotoxin (TTX, 1 microM), 4-AP (6 mM) and TEA (20 mM) an inward current carried by Ca2+ was found. Ca2+ currents activated at depolarized potentials at about -30 mV, were completely blocked by 50 microM cadmium (Cd2+), were sensitive to verapamil (approximately 40% block by 10 microM), and were not affected by nickel (50 microM). During current clamp recordings, isolated layer I neurons displayed fast spiking behaviour with short action potentials (approximately 2 ms, measured at half maximal amplitude) of relative small amplitude (approximately 83 mV, measured from the action potential threshold).