Biomedical subjects

H D Lux

Publications and source records attributed to H D Lux.

At least 37 records · Page 2Linked to original sources

PubMed1992

Pharmacological characterization of calcium currents and synaptic transmission between thalamic neurons in vitro.

We recorded from pairs of cultured, synaptically connected thalamic neurons. Evoked excitatory postsynaptic currents (EPSCs) reversed at +17 mV and were blocked reversibly by 1 mM kynurenic acid, a glutamate receptor antagonist. NMDA and non-NMDA receptors mediated excitatory post-synaptic responses, as shown by selective block of EPSC components with 50 microM (+/-)-2-amino-5-phosphonopentanoic acid and 10 microM 6,7-dinitroquinoxaline-2,3-dione, respectively. Inhibitory postsynaptic responses were evoked less frequently and were blocked by the GABAA receptor antagonist (-)-bicuculline methochloride. The pharmacological profiles of whole-cell calcium currents and evoked EPSCs were compared. With 50 microM cadmium chloride (Cd), whole-cell low voltage-activated (LVA) calcium currents were reduced in amplitude and high voltage-activated (HVA) calcium currents and excitatory synaptic transmission were completely blocked. This suggests that the residual calcium influx through LVA channels into the presynaptic terminal does not suffice to trigger transmitter release. A saturating concentration of omega-conotoxin GVIA (omega-CgTx) (2.5 microM) blocked one-third of whole-cell HVA calcium currents and evoked EPSCs. The dihydropyridine nifedipine (50 microM) reversibly reduced whole-cell HVA calcium currents in a voltage-dependent manner but not excitatory synaptic transmission. Cd and omega-CgTx did not alter amplitude distributions of miniature EPSCs, demonstrating that the inhibition of synaptic transmission was due to block of presynaptic calcium channels. We conclude that excitatory glutamatergic transmission in thalamic neurons in vitro was mediated mainly by HVA calcium currents, which were insensitive to omega-CgTx and nifedipine.

Animals↗

PubMed1991

Calmodulin antagonists and protein phosphatase inhibitor okadaic acid fasten the 'run-up' of high-voltage activated calcium current in rat hippocampal neurones.

Voltage-activated Ca2+ channel currents were recorded from cultured rat hippocampal neurones using the whole-cell clamp technique with Ba2+ as a charge carrier. After breaking into the cell the amplitude of low-voltage activated Ca2+ channel current increased to a new steady value within 1 min whereas several minutes were required for a full development of the high-voltage activated current (IHVA). Pretreatment of cells with calmodulin antagonists (trifluoperazine or W-13) or protein phosphatase inhibitor, okadaic acid, fastened the development of IHVA. Trifluoperazine (6-40 microM) also increased IHVA when applied after breaking into the cell in standard external solution. Incubation of cells in the presence of permeable precursor of Ca2+ chelator, BAPTA, was without effect. The effects of all inhibitors studied allow to suggest that IHVA in intact cells is largely masked due to activity of calmodulin-activated protein phosphatase.

Animals↗

PubMed1991

Cytoplasmic alkalinization increases high-threshold calcium current in chick dorsal root ganglion neurones.

Changes of calcium currents with intracellular pH (pHi) were investigated in chick dorsal root ganglion (DRG) neurones. High-threshold calcium currents decreased after extracellular application of a permeable weak acid, sodium acetate (CH3COONa), and increased when applying a permeable weak base, ammonium chloride (NH4Cl), whereas both compounds were ineffective against the low-threshold calcium current. These weak electrolytes, employed to change pHi, did not alter the kinetic and steady-state parameters of activation and inactivation of the calcium current. Extracellular application of concanavalin A (Con A) and wheat germ agglutinin to elevate pHi increased the high-threshold calcium current. Their effect developed within 2-5 min and was independent of lectin concentration varied from 0.1 to 1 mg/ml. The lectin effects were greatly diminished if Na/H exchange was blocked by amiloride or suppressed by low external sodium. Succinilated Con A and Con A in the presence of D-mannose were less effective. Calcium currents were recorded simultaneously with the pHi, monitored with a proton-sensitive microelectrode. It was found that 50% inhibition of the calcium current occurred at pHi of 6.5. Histidine-specific reagents--diethylpyrocarbonate, Rose Bengal and Methylene Blue--prevented the modulation of the calcium conductance by CH3COONa and NH4Cl. Extracellular baclofen and theophylline or intracellular phorbol esters, staurosporine, calmodulin antagonists R24571 and W-13, and neomycine failed to prevent the modulation of the calcium current by weak electrolytes. These observations are consistent with an interaction between intracellular protons and calcium channels.

Animals↗

PubMed1991

Do calcium channel classifications account for neuronal calcium channel diversity?

Calcium (Ca2+) ions are involved in the development and control of a variety of neuronal properties and functions such as channel expression, synaptic transmission and neurosecretion. The main pathway by which Ca2+ enters the intracellular space is through voltage-activated Ca2+ channels that can be classified according to their different biophysical and pharmacological properties. Identification and characterization of these channel types are prerequisites for understanding the mechanisms that underlie Ca2(+)-controlled processes. In this article we summarize the efforts made to identify neuronal Ca2+ channel types, and we attempt to evaluate how useful existing classifications are in assigning specific properties and functions to distinct channel types in neurons.

Animals↗

PubMed1991

Distribution of Ca2+ and Na+ conductances during neuronal differentiation of chick DRG precursor cells.

The distribution of Ca2+ and Na+ conductances on neuronal precursor cells was investigated during differentiation. Ionic conductances on the soma or on the growth cone were isolated by superfusing all other parts of the cells with sucrose. Conductances on the neuritic shaft were detected as additional conductances after removing sucrose from the neuritis shaft. Neuronal precursor cells were isolated from chick dorsal root ganglia by selectively killing differentiated neurons. Cultured precursor cells differentiated into morphological and functional mature neurons. Functionally undifferentiated precursor cells (during the first 10 hr in culture) expressed only low-voltage-activated (LVA) Ca2+ currents. High-voltage-activated (HVA) Ca2+ and Na+ currents appeared delayed after more than 10 hr in culture. Voltage-dependent conductances, if expressed by a cell, were present on all parts of the surface membrane at all stages of differentiation. LVA Ca2+ conductances were well represented on the growth cone as well as on the soma in functionally undifferentiated precursor cells. During differentiation of precursor cells, LVA Ca2+ and HVA Ca2+ as well as Na+ conductances were expressed on the somatic membrane, on the neuritic shaft, and on the growth cone. These results demonstrate the expression of Ca2+ channels on growth cones during differentiation.

Animals↗

PubMed1990

Low- and high-voltage-activated Ca2+ conductances in electrically excitable growth cones of chick dorsal root ganglion neurons.

Growth cone Ca2+ currents of chick dorsal root ganglion (DRG) neurons were recorded by a patch pipette located on the cell soma. Somatic and neuritic conductances were selectively blocked either with TTX and Cd2+ or by superfusing with isotonic sucrose using a laminar flow perfusion system. DRG growth cones were electrically excitable and growth cone Ca2+ currents were similar to Ca2+ currents described in DRG somata. In particular low-voltage-activated (LVA) Ca2+ conductances were well represented contrary to previous suggestions in other cell types.

Animals↗

PubMed1990

Calcium current inactivation during nerve-growth-factor-induced differentiation of PC12 cells.

The inactivation of calcium currents during nerve growth factor (NGF)-induced differentiation of rat pheochromocytoma (PC12) cells was investigated. Whole cell calcium and barium currents were recorded in PC12 cells using the patch-clamp method. A shift of the steady-state inactivation curve towards more negative potentials, as well as an increase in the strength of time-dependent inactivation, was observed in differentiating PC12 cells (+NGF) compared to undifferentiated cells (-NGF). The fraction of current inactivated after 200 ms normalized to the peak current amplitude significantly increased from 0.27 +/- 0.01 (n = 39) to 0.39 +/- 0.01 (n = 109) following NGF treatment. The increase in the strength of inactivation preceded the increase in the peak calcium current amplitude observed in PC12 cells during NGF treatment. In differentiating cells, regional differences in the strength of inactivation paralleled differences in current density. Barium currents recorded from growth cones, where the current density was high, showed 30% more inactivation than soma currents. The strength of inactivation of calcium currents in individual cells was not correlated to the effectiveness of nifedipine, nor was the effect of nifedipine on calcium currents altered during differentiation. These results are discussed, suggesting a redistribution of calcium channels during the differentiation of PC12 cells.

Adrenal Gland Neoplasms↗

PubMed1990

Neurons sensitive to pH in slices of the rat ventral medulla oblongata.

The effects of extracellular pH changes on neurons in slices of the rat ventral medulla oblongata were investigated by extracellular recording. Changes in discharge rate were correlated with pH changes in the tissue next to the recorded cell, as measured by H(+)-selective microelectrodes. pH was altered by varying the bicarbonate concentration ([HCO3-]) in the superfusion solution. In 136 out of 316 neurons, the number of spontaneous or electrically evoked discharges per unit time increased with decreasing pH and decreased with increasing pH. Changes of only 0.01-0.04 pH unit were effective in these pH-sensitive neurons. The response was transient; the discharge rate returned to the control value within a few minutes. The pH sensitivity persisted in the presence of 0.5 microM atropine, 20 microM bicuculline and after replacing Ca2+ by Mg2+ in the superfusion solution to reduce synaptic transmission. The response to the same pH decrease was stronger when increasing PCO2 than when reducing [HCO3-]0. The pH-induced response significantly increased during hypoxia. The results show that in the ventral medulla oblongata neurons exist that transiently respond to small decreases and increases of pH. The pH sensitivity is an intrinsic property of these neurons; it is not due to a synaptic mechanism but is modulated by PCO2 and PO2.

Animals↗

PubMed1990

Temperature sensitivity of Ca currents in chick sensory neurones.

We have investigated the effects of temperature on the Ca currents of chick sensory neurones. Raising the temperature from 17 to 37 degrees C, caused low-threshold (LVA, T) and high-threshold (HVA, L and N) Ca currents to show a marked amplitude increase and a drastic acceleration of their activation-inactivation gatings. Compared to HVA channels, the LVA type showed a weaker temperature sensitivity. Its average Q10 values were closer to those of other voltage-operated ion channels: 1.7 (permeability), 1.9 (activation) and 2.2 (inactivation). Alternatively, the activation kinetics and peak permeability of HVA Ca channels showed maximal Q10 values of about 5 and 2.8, respectively. HVA channel deactivation was less sensitive to temperature (Q10 1.8). Inactivation of these channels was slow and monoexponential between 17 and 22 degrees C, but faster and double exponential above 30 degrees C, uncovering a fast temperature-sensitive decaying phase. The size and rate of decay of this component decreased with increasing membrane depolarizations and persisted at holding potentials positive to -80 mV, suggesting the involvement of temperature-sensitive Ca-mediated processes in the mechanism of HVA channel inactivation. Our data are consistent with the view that heating from 17 to 37 degrees C causes both an increased probability of Ca channels to open and a drastic acceleration of their activation-inactivation kinetics.

Animals↗

PubMed1990

Na+ currents through low-voltage-activated Ca2+ channels of chick sensory neurones: block by external Ca2+ and Mg2+.

1. Whole-cell currents through low-voltage-activated (LVA) Ca2+ channels carried by monovalent cations were studied in chick dorsal root ganglion (DRG) cells. 2. With 120 mM [Na+] on both sides of the membrane and [Ca2+]o less than or equal to 100 microM, the currents reversed at 0 mV. Their half-times of activation and inactivation were strictly voltage-dependent and decreased to near-constant values of 0.6-0.85 and 40 ms, respectively, at positive membrane potentials. The longer activation times were observed with [Ca2+]o greater than or equal to 50 microM. 3. The selectivity of the Ca2+ channel for monovalent ions with reference to internal Na+ was evaluated from the reversal potential. The Li+ and Na+ permeabilities were similar. The permeability ratios of K+ and Rb+ were 0.45, and 0.33 for Cs+. 4. Micromolar increases in [Ca2+]o produced small voltage shifts of half-times of activation (less than or equal to +3 mV at 10 microM and +10 mV at 500 microM), but strongly depressed the Na+ current. The Ca2(+)-induced block of Na+ current satisfied a 1:1 stoichiometry with an apparent KD of 1.8 microM at -20 mV. The block was, however, relieved with more positive and negative potentials, with KDs of 55 and 8.5 microM at +90 and -110 mV, respectively. 5. Relaxation time constants of block and unblock of Na+ currents through the LVA Ca2+ channel were measured on step changes to and from membrane potentials at which pronounced Ca2(+)-induced block occurred. 6. At -20 mV, the time constants of block decreased with micromolar increase in [Ca2+]o in line with a blocking rate coefficient of 1.9 x 10(8) M-1 s-1, but settled to values of 0.18 ms at [Ca2+]o beyond 50 microM. The Na+ currents were unblocked with time constant (tau u) of around 0.25 ms at strongly positive and negative membrane potentials at 22 degrees C. 7. Tau u failed to show any obvious dependence on [Ca2+]o up to the millimolar range. This finding contradicts suggestions that removal of the block occurs in a [Ca2+]o-dependent manner as a result of an increased probability of Ca2+ ion mobilization by repulsive forces with increased Ca2+ occupation of the channel. 8. The time course of unblock of Na+ currents was strongly temperature-dependent showing a Q10 of 2.5 for tau u. 9. The voltage dependence of the Na+ current block by Ca2+ ions is best accounted for by a single, centrally located Ca2+ binding site.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1989

Activity dependent alkaline and acid transients in guinea pig hippocampal slices.

Changes of extracellular proton concentration ([H+]o) and K+ activity ([K+]o) were simultaneously measured by ion-sensitive microelectrodes in the CA3 region of guinea pig hippocampal slices. Repetitive electrical stimulation and application of glutamate or GABA were associated with prominent alkaline transients of up to 0.2 pH units lasting 2-10 s followed by smaller acid transients lasting up to 4 min. About 10-fold smaller alkaline transients were induced by spontaneous field discharges in the presence of bicuculline. The time to the maximal amplitude of the alkaline transients and the time to maximal increases of [K+]o were in the same range, concurring with the assumption that alkaline transients are due to a proton influx through cationic channels. However, spontaneous field discharges in low-calcium solution in which synaptic transmission is reduced were associated with acid transients of up to 0.02 pH units lasting 2-20 s. An alkaline transient was superimposed on the acid transient only when increases of [K+]o exceeded 1.5 mM. The effects of changing [H+]o on electrically evoked field potentials and spontaneous field discharges were studied in the range from pH 7.00 to 7.80. Electrically evoked field potentials were markedly depressed from pH 7.15 to 7.00 and enhanced from pH 7.60 to 7.80. The frequency of spontaneous field discharges in the presence of bicuculline significantly decreased by reducing pH from 7.40 to 7.30 and continuously increased from pH 7.40 to 7.80. In the same way, the frequency and the amplitude of spontaneous field discharges in low-calcium solution decreased from pH 7.40 to 7.15 and increased from pH 7.40 to 7.80.(ABSTRACT TRUNCATED AT 250 WORDS)

Acid-Base Equilibrium↗

PubMed1989

Voltage-dependent GABA-induced modulation of calcium currents in chick sensory neurons.

Externally applied gamma-aminobutyric acid (GABA) quickly and reversibly reduces by 60% voltage activated Ca2+ currents in chick dorsal root ganglion cells. This action is antagonized by depolarization, with characteristic time and voltage requirements. Intracellular perfusion with guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) or guanosine 5'-O-(2-thiodiphosphate) (GDP beta S) mimicks and blocks the GABA effect, respectively. A 3-state model describing the reactions involved is proposed.

Animals↗

PubMed1989

Expression of depolarizing voltage- and transmitter-activated currents in neuronal precursor cells from the rat brain is preceded by a proton-activated sodium current.

The early expression of amiloride-sensitive proton-activated sodium currents (INa(H] was demonstrated using the giga-seal whole-cell voltage clamp technique in cells from the primordial tectum of E12 rat embryos. Less than 10% of these cells stained for tetanus toxin receptors after 2 h in vitro. However, after 10 h in vitro all cells with neuronal geometry were tetanus toxin-positive and capable of generating voltage-activated Na currents (INa(V] and high-voltage activated Ca2+-currents (ICa(HV]. INa(H) was expressed roughly in parallel with INa(V) and ICa(HV), but exceeded the former currents in amplitude by 50-100 times, reaching 600 pA and more. In 25% of the cells tested within the first 5 h in vitro INa(H) was, in fact, the only cationic inward current resolved. Responses to quisqualate and kainate appeared only after 3 days in vitro, and responses to N-methyl-D-aspartate/glycine were seen only after 4 days in vitro. These results suggest that the channels carrying INa(H) are present at the earliest stages of neuronal development.

Animals↗

PubMed1989

Proton-induced Na+ current develops prior to voltage-dependent Na+ and Ca2+ currents in neuronal precursor cells from chick dorsal root ganglion.

Proton-induced Na+ currents (INa(H] were measured in precursor cells from chick dorsal root ganglia with whole-cell patch-clamp recording. Precursor cells were isolated using the method of Rohrer et al. (EMBO. J., 4 (1985) 1709-1714) and fast pH changes were applied with a technique developed by Davies et al. (J. Physiol. (Lond.), 400 (1988) 159-187). Proton-induced transient Na+ currents showing the same properties as in more mature neurons could be elicited already early in differentiation, before high-voltage activated Ca2+ currents and voltage-dependent Na+ currents develop.

Animals↗

PubMed1989

gamma-Aminobutyric acid-induced ion movements in the guinea pig hippocampal slice.

gamma-Aminobutyric acid (GABA)-induced regional changes of extracellular Cl, K and Na concentration ([Cl]o, [K]o, [Na]o), as well as of the extracellular space were measured with ion-sensitive microelectrodes in guinea pig hippocampal slices. Microdrop application of GABA to the pyramidal cell layer of CA3 or CA1 induced a decrease of [Cl]o, while application to the dendritic layer of CA3 or CA1 induced an increase of [Cl]o in addition. All changes of [Cl]o persisted in the presence of TTX and were blocked by bath-applied bicuculline. The GABA-induced decrease of [Cl]o was reduced by bicuculline application to the pyramidal cell layer. The increase of [Cl]o was blocked by bicuculline application to the dendritic layer. Additionally, GABA induced an increase of [K]o and decreases/increases of [Na]o. Changes of [Cl]o, [K]o and [Na]o together were approximately electroneutral. [Cl]o increases were exaggerated and [Cl]o decreases partly masked by shrinkage of the extracellular space after GABA application. Changing [K] in the superfusate transiently changed GABA-induced [Cl]o movements in a way predicted from a change in driving force due to the effect of [K] on membrane potential. Then a partial recovery followed towards the original [Cl]o change. We conclude that inward and outward Cl transports maintain [Cl]i below equilibrium in CA3 and CA1 pyramidal somata and above equilibrium in CA3 and CA1 dendrites. The significance of this Cl-distribution for hippocampal inhibition is discussed.

Animals↗

PubMed1989

A diacylglycerol analogue reduces neuronal calcium currents independently of protein kinase C activation.

Diacylglycerol analogues (for example 1,2-oleoylacetylglycerol, OAG) and phorbol esters are activators of protein kinase C, and have been widely used to study the function of this enzyme in both intact cells and cell-free preparations. Electrophysiological studies have shown that these activators can either depress or increase Ca2+ currents, or decrease K+ currents when applied outside the cell. It has been assumed that these effects are mediated by protein kinase C activation. Here we report that micromolar levels of OAG and phorbol esters depress Ca2+ currents in chick sensory neurons independently of their effect as activators of protein kinase C. The depression of the Ca2+ current is rapid and is unaffected by intracellular application of the protein kinase C inhibitors staurosporin, sphingosine and H-7. Furthermore, the activators were ineffective when applied intracellularly, indicating that their site of action is on the outside of the membrane.

Action Potentials↗

PubMed1989

Proton-induced chloride current and voltage-activated Na+ and Ca2+ currents in embryonic neurons from the medicinal leech (Hirudo medicinalis).

Voltage-activated ionic currents and currents induced by step changes in proton concentration (pH 7.9-6.7) were studied in early embryonic neurons from Hirudo medicinalis. Ganglia were dissociated at embryonic day (E) 8-15, and the largest neurons were investigated using whole-cell patch clamp recording. All cells studied displayed voltage-activated Na+ and Ca2+ currents. Step changes in pH induced sustained currents which reversed at the equilibrium potential for Cl- and were blocked by substituting Cl- with acetate or sulfate. These currents thus differ in their ion selectivity from proton-induced currents in vertebrate neurons which are carried by Na+.

Animals↗

PubMed1989

Relations between slow extracellular potential changes, glial potassium buffering, and electrolyte and cellular volume changes during neuronal hyperactivity in cat brain.

The aim of this investigation is to estimate the contribution of spatial glial K+ buffer currents to extracellular K+ homeostasis during enhanced neuronal activity. Neuronal hyperactivity was induced by electrical stimulation of the cortical surface or the ventrobasal thalamic nuclei of cats (5-50 Hz, 0.1-0.2 ms, two to three times threshold stimulation intensity, 5-20 s). The accompanying slow field potential changes were recorded simultaneously across the grey matter with vertical assemblies of eight micropipettes glued 300 microns apart. Using the Poisson equation, the amplitudes of the underlying current sources and sinks were calculated. The current source densities depended on the depth of recording, frequency, strength, and duration of the stimulation. Current sinks, corresponding to a removal of 0.1-0.5 mmoles of monovalent cations per liter of brain tissue and second from the extracellular space, were observed in middle cortical layers, whereas sources appeared at superficial and deeper sites. These sinks and sources might represent K+ moved across glial membranes by spatial buffer currents. The consequences of glial buffer currents of this magnitude were investigated with model calculations. It turned out that measurements of electrolyte and volume changes of the extracellular space (Dietzel et al. Exp. Brain Res. 40:432-439, 1980; Exp. Brain Res. 46:73-84, 1982) could only partially be explained by spatial buffer currents of this magnitude. Comparison of the calculated values with intracellular measurements in neurons and glial cells (Coles et al. Ann. N.Y. Acad. Sci. 481:303-317, 1986; Ballanyi et al. J. Physiol. 382:159-174, 1987) suggests that spatial buffering combines with an approximately equimolar KCl transport and, depending on the preparation, also K+/Na+-exchange across glial membranes.

Action Potentials↗