Decoding glutamate receptor activation by the Ca2+ sensor protein hippocalcin in rat hippocampal neurons.

Hippocalcin is a Ca(2+)-binding protein that belongs to a family of neuronal Ca(2+)sensors and is a key mediator of many cellular functions including synaptic plasticity and learning. However, the molecular mechanisms involved in hippocalcin signalling remain illusive. Here we studied whether glutamate receptor activation induced by locally applied or synaptically released glutamate can be decoded by hippocalcin translocation. Local AMPA receptor activation resulted in fast hippocalcin-YFP translocation to specific sites within a dendritic tree mainly due to AMPA receptor-dependent depolarization and following Ca(2+)influx via voltage-operated calcium channels. Short local NMDA receptor activation induced fast hippocalcin-YFP translocation in a dendritic shaft at the application site due to direct Ca(2+)influx via NMDA receptor channels. Intrinsic network bursting produced hippocalcin-YFP translocation to a set of dendritic spines when they were subjected to several successive synaptic vesicle releases during a given burst whereas no translocation to spines was observed in response to a single synaptic vesicle release and to back-propagating action potentials. The translocation to spines required Ca(2+)influx via synaptic NMDA receptors in which Mg(2+) block is relieved by postsynaptic depolarization. This synaptic translocation was restricted to spine heads and even closely (within 1-2 microm) located spines on the same dendritic branch signalled independently. Thus, we conclude that hippocalcin may differentially decode various spatiotemporal patterns of glutamate receptor activation into site- and time-specific translocation to its targets. Hippocalcin also possesses an ability to produce local signalling at the single synaptic level providing a molecular mechanism for homosynaptic plasticity.

Estimating transmitter release rates and quantal amplitudes in central synapses from postsynaptic current fluctuations.

Several approaches recently introduced to analyze release rates in central synapses advanced our understanding of synaptic neurotransmission, however, leaving many questions still unresolved. In this work we present evidence that a new method recently developed by Sakaba and Neher to study neurotransmission in calyx of Held, a giant glutamatergic synapse, could be also applied for estimating release rate functions and averaged quantal sizes in small central synapses. By means of different simulation approaches applied to reproduce GABAergic neurotransmission in the hippocampus we have shown that possible problems with a spatial voltage clamp which can occur in synaptic connections distributed over a large area of dendritic tree are not crucial for applicability of the method when synapses are compactly distributed or located proximally and when release rates are below 1 ms(-1). In another set of simulations we have also shown that at above mentioned release rates desensitization and/or saturation of postsynaptic GABAA receptors does not prevent accurate estimates of release rate and averaged quantal size. Thus, we conclude that the new approach based on analysis of fluctuations of postsynaptic currents under conditions of stationary release or moderately nonstationary conditions might be applicable to studies of small central synapses.

Glutamate-receptor-induced modulation of GABAergic synaptic transmission in the hippocampus.

There is a large body of evidence about the short- and long-term changes in GABAergic transmission in the hippocampus produced by the action of different endogenous neuromodulators and in particular neurotransmitters. Both intrinsic hippocampal cells and afferent fibres coming into the hippocampus from various parts of the CNS release substances that are capable of changing inhibitory transmission. This review surveys current understanding of the action of glutamate on the inhibitory transmission mediated in the hippocampus via GABA(A) receptors. Here we pay special attention to the molecular and cellular mechanisms leading to spatio-temporal changes of the glutamate concentration in the extracellular space and to the localization and identity of glutamate receptors involved in this direct modulation of inhibition.

Postsynaptic mechanism may contribute to inhibitory acetylcholine effect on GABAergic synaptic transmission in hippocampal cell cultures.

The effect of acetylcholine (ACh) on evoked GABAergic inhibitory postsynaptic currents (IPSCs) was studied in cell cultures of dissociated hippocampal neurons with established synaptic connections. Spontaneous IPSCs and IPSCs evoked by extracellular stimulation of a single presynaptic neuron were recorded. ACh inhibited the evoked IPSCs in most of the connections, although facilitation was also observed. Regardless of inhibitory or facilitatory effects on the evoked IPSCs, an enhanced spontaneous synaptic input to the postsynaptic neurons was usually observed. ACh-induced changes in the evoked IPSCs were usually accompanied by changes in paired pulse depression (PPD), which are thought to reflect presynaptic mechanisms of modulation. However, the time course of PPD changes did not always match that of the IPSC changes, suggesting a contribution of other, possibly postsynaptic, mechanism(s). To analyze this possibility, effects of ACh on responses to direct application of exogenous GABA were studied. In a proportion of the neurons (40%) ACh reversibly decreased GABA responses, indicating that postsynaptic mechanisms may also contribute to the inhibitory ACh effect on GABAergic transmission. We conclude that several different modulatory mechanisms of ACh action participate in the regulation of GABAergic transmission at the level of synaptic connection of a single GABAergic neuron.

Rat hippocampal neurons maintain their own GABAergic synaptic transmission in culture.

The whole-cell patch-clamp technique was used to record monosynaptic inhibitory postsynaptic currents (IPSCs) from pairs of hippocampal neurons cultured for 2-3 weeks. The application of fresh physiological solution for 2-3 min reversibly reduced the amplitude of evoked GABAergic IPSCs to 72.5% of control value. The amplitude and frequency of spontaneous IPSCs decreased too. The depression of evoked IPSCs was significantly smaller or absent if conditioned solution was applied (physiological solution which had been previously in contact with neurons for 30 min). Currents evoked by exogenously applied GABA were unaffected by fresh solution. These results suggest that hippocampal neurons release some endogenous substance(s), by which they up regulate presynaptically their own inhibitory synaptic transmission.

A new technique for assessing the microscopic distribution of cellular calcium exit sites.

This paper contains a description of a new method designed to monitor the distribution of Ca2+ efflux from cells or small cellular aggregates. The idea behind this method is to use a fluorescent Ca2+ indicator bound to dextrans of high molecular weight to slow down Ca2+ diffusion. Due to the decrease in diffusion rate, Ca2+ ions should be held close to the site of their release from the cells for a relatively long time, enough for the confocal microscope to detect such a local increase in Ca2+ concentration. This paper gives a detailed description of the method, illustrated with results of measurements of agonist-dependent and agonist-independent Ca2+ extrusion from pancreatic acinar cells. An appendix provides the mathematical background that should allow selection of the concentration of buffer which is necessary to achieve a particular Ca2+ diffusion coefficient.

Localization of Ca2+ extrusion sites in pancreatic acinar cells.

We have investigated the localization of Ca2+ extrusion sites in mouse pancreatic acinar cells. Employing a new technique, in which high resolution localization of cellular Ca2+ exit is achieved by confocal microscopy and a Ca2+-sensitive fluorescent probe coupled to heavy dextran to slow down diffusion of extracellular Ca2+, it is shown directly that the secretory pole (secretory granule area) is the major site for Ca2+ extrusion following agonist stimulation. This Ca2+ extrusion appears not to be a consequence of exocytosis, as assessment of secretion under our experimental conditions (low external Ca2+ concentration, room temperature) using the technique of monitoring quinacrine fluorescence shows little loss of secretory granules in spite of sustained Ca2+ exit. We conclude that Ca2+ is primarily extruded by Ca2+ pumps from the secretory pole and propose that this process is useful for maintaining a high Ca2+ concentration in the acinar lumen, which is necessary for promotion of endocytosis.

Inositol trisphosphate and cyclic ADP-ribose-mediated release of Ca2+ from single isolated pancreatic zymogen granules.

In pancreatic acinar cells low (physiological) agonist concentrations evoke cytosolic Ca2+ spikes specifically in the apical secretory pole that contains a high density of secretory (zymogen) granules (ZGs). Inositol 1,4,5-trisphosphate (IP3) is believed to release Ca2+ from the endoplasmic reticulum, but we have now tested whether the Ca(2+)-releasing messengers IP3 and cyclic ADP-ribose (cADPr) can liberate Ca2+ from AGs. In experiments on single isolated ZGs, we show using confocal microscopy that IP3 and cADPr evoke a marked decrease in the free intragranular Ca2+ concentration. Using a novel high resolution method, we have measured changes in the Ca2+ concentration in the vicinity of an isolated AG and show that IP3 and cADPr cause rapid Ca2+ release from the granule, explaining the agonist-evoked cytosolic Ca2+ rise in the secretory pole.

The effect of acetylcholine and serotonin on calcium transients and calcium currents in identified Helix pomatia L. neurons.

The results presented demonstrate that in D neurons of the snail Helix pomatia L., acetylcholine (ACh) (10 divided by 100 microM) and serotonin (5-HT) (0.1 divided by 1000 microM) applications reduce both the basal intracellular concentration level ([Ca2+]in) and the amplitudes of calcium transients induced by membrane depolarization. It is likely that the mechanism of [Ca2+]in changes in the suppression of calcium inward currents (ICa). Influences of Ach and 5-HT on ICa were studied. Both effects were dose-dependent (ACh--0.01 divided by 100 microM and 5-HT--0.1 divided by 1000 microM). The half-maximal effects (IC50) were evoked by ACh concentration of 0.15 microM and 5-HT--15 microM. Furthermore we have also shown that in some cells 5-HT could evoke a transient increase in ICa (IC50 = 2 microM). The effects of Ach and 5-HT were nonadditive--the subsequent application of ACh after 5-HT, and vice versa, produced no inhibitory effects. This may indicate that both substances act through a common intermediate (possibly, G-protein).

Calcium clamp in single nerve cells.

Free calcium concentration in isolated single neurons was clamped using a new technical approach based on a feed-back connection between the Fura-2 fluorescence signal measuring the intracellular Ca2+ concentration ([Ca2+]i) and iontophoretic current injecting Ca2+ into the cell. Beginning of [Ca2+]i clamping at a level above the basal one triggered fast (few seconds) current transients equal to injection of 36 +/- 20 microM Ca2+ (for a 0.1 microM change of [Ca2+]i), representing the filling of a fast cytosolic buffer. Continuation of clamping required very small clamping currents (corresponding to injection of 0.39 +/- 0.20 microM.s-1 Ca2+). This value increased proportionally to the magnitude of the change of [Ca2+]i above basal level, indicating the activation of calcium-dependent mechanisms for Ca2+ removal from the cytosol. The described approach allowed measurement, under physiological conditions, of the capacitative and kinetic properties of different Ca-regulating systems functioning in a single nerve cell as well as other types of cells.

Effect of Ag+ on membrane permeability of perfused Helix pomatia neurons.

1. Isolated, non-identified neurons were voltage clamped using the internal perfusion technique. 2. Ions of Ag+ (1-100 microM) introduced into the bathing solution activated a steady-state inward current (IAg) in the soma. The effect of Ag+ was reversible when the concentration of Ag+ was less than 75 microM or the time of application was shorter than 10 min. 3. IAg was observed both in the presence and absence of Na+ ions in the extracellular saline. It could also be activated when Cs+ ions were substituted for Na+ ions. 4. The current-voltage characteristics were linear in the voltage range -100 to 0 mV. The reversal potential in control saline was an average of 1.19 +/- 5.1 mV. 5. The application of Ag+ ions induces an elevation of intracellular free Ca2+ concentration by 10-20 times in both Ca(2+)-containing and Ca(2+)-free extracellular salines, as revealed by Fura-2 measurements. 6. Agents that increase the intracellular free Ca2+ concentration ([Ca2+]i), like thymol, caffeine and dinitrophenol, increased the amplitude of IAg. The effect was additive. Ruthenium Red, which blocks the release of Ca2+ from intracellular stores, decreased the Ag+ effect. 7. It is concluded that extracellularly applied Ag+ ions increase the cytoplasmic free Ca2+ concentration, which in turn activates non-specific cationic channels. 8. Ag+ ions in 1-10 microM concentration were able to decrease the voltage-activated Ca2+ current amplitude. This decrease, however, was due to the increase of [Ca2+]i which caused Ca(2+)-dependent inactivation.

Extrusion of calcium from a single isolated neuron of the snail Helix pomatia.

Simultaneous optical measurements of extra- and intracellular Ca2+ concentrations were carried out on isolated snail neurons injected iontophoretically with Ca2+. The fluorescent indicator Fura-2 was used to measure intracellular concentration of free Ca, and the absorbant indicator Antipyrylazo III to measure changes in extracellular calcium concentration in the micro-chamber containing the cell. The velocity of Ca2+ extrusion from a single cell has been shown to be in accordance with the level of free Ca in the neuronal cytoplasm. After an increase in intracellular free Ca by iontophoretic injection from a microeletrode to 0.2-0.5 microM, the velocity of Ca2+ extrusion from the neuron was approximately 0.3-4.6 microM/sec per cell volume. During caffeine-induced calcium-dependent calcium release of Ca2+ from intracellular stores a stimulation of calcium extrusion took place, reaching the velocity of 5.0 microM/sec per cell volume.

Blocking effect of La3+ ions on transmembrane ionic current evoked by intracellular cyclic AMP injection in identified Helix pomatia neurons.

Using the fluorescent probe fura-2 for measurements of the cytoplasmic concentration of free Ca2+ ions [( Ca]in) in combination with conventional current- or voltage-clamp methods, we studied the effects of La3+ ions on the cellular responses evoked by intracellular cAMP (adenosine 3',5'-cyclic monophosphate) injections into isolated Helix pomatia neurons. La3+ ions in submillimolar concentrations decreased cAMP-evoked [Ca]in transients. Transmembrane ionic currents evoked by cAMP injections were completely blocked by La3+ ions (1.0 mM) in all neurons under investigation. La(3+)-induced effects essentially differ from each other in RPa1 (right parietal 1) and LPa3 (left parietal 3) neurons. La3+ ions in milli- and submillimolar concentrations strongly affect different subcellular systems, especially, those which regulate an intracellular calcium concentration.

[Tocopherol modulation of acetylcholine-induced current in mollusk neurons]. / Moduliatsiia tokoferolom atsetilkholinindutsiruemogo toka v neironakh molliuska.

It is shown that vitamin E (tocopherol) and arachidonic acid are antagonists at the cellular level in regulation of Ca-calmodulin-dependent acetylcholine-induced current in molluscan neurons.

Free calcium transients and oscillations in nerve cells.

Changes in the intracellular level of free calcium induced by different influences in neurones of the snail Helix pomatia have been measured by changes in Fura-2 fluorescence. Thymol in submillimolar concentrations induced the release of stored intracellular calcium. This effect was similar to xanthine-induced release. IP3 and Gpp[NH]p injections also released intracellular calcium. The response to cAMP injections was more complicated and included, probably, both the release of stored calcium and its influx through membrane channels. Oscillations of intracellular free calcium are described. It has been suggested that oscillations can occur only in cases where the mechanism of Ca-dependent calcium release is activated.

Inositol-1,4,5-trisphosphate and non-hydrolysable GTP analogue induced calcium release from intracellular stores of the Helix pomatia neurons.

1. Cytoplasmic Ca2+ concentration ICaIin and membrane potential of single Helix pomatia neurons was studied by Fura-2 fluorescence measurement and conventional current clamp methods. 2. Intracellular injection of inositol-1,4,5-trisphosphate (IP3) and nonhydrolysable GTP analogue (Gpp/NH/p) led to a rise of ICaIin; in contrast, GTP injection did not cause significant ICaIin changes. 3. We suggest that both IP3 and Gpp/NH/p directly activated Ca release from intracellular stores.

[Use of a microfluorometric method for measuring free calcium in the cytoplasm of isolated cultured rat cardiomyocytes]. / Primenenie metoda mikrofliuorometrii dlia izmereniia svobodnogo ka'ltsiia v tsitoplazme odinochnykh ku'ltiviruemykh kardiomiotsitov krysy.

Dual wavelength microfluorometry was used to measure the cytoplasmic free calcium concentration [( Ca2+]in) in single cultured cells from ventricular myocytes of neonatal rats loaded with the indicator fura-2. At 2.5 nmol/l extracellular Ca2+ in the resting cells [Ca2+]in was between 80 and 110 nmol/l. Sometimes, spontaneous low-frequency (approximately 0.1 Hz) [Ca2+]in oscillations were observed. High-potassium depolarization led to a Ca2+-antagonists-sensitive rise of [Ca2+]in. Both caffeine++ (5-10 mmol/l) and thymol (lmmol/l) initialized transient increase of [Ca2+]in. Mechanisms of [Ca2+]in homeostasis in heart muscle cells were discussed.

[Calcium release from the intracellular stores of the soma of the mollusk neuron under the action of inositol triphosphate and its nonhydrolyzable analog GTP]. / Osvobozhdenie kal'tsiia iz vnutrikletochnykh depo somy neirona molliuska pod deistviem inozitoltrifosfata i negidrolizuemogo analoga GTF.

The effects of intracellularly injected inositol trisphosphate (IP3) and nonhydrolyzed GTP analogue (Gpp[NH]p) on intracellular concentration of calcium ions [Ca2+]in was determined by fluorescence signals obtained from Helix pomatia neurons. At the same time these neurons were studied under the current clamp conditions. The IP3 and Gpp[NH]p injection caused a long-lasting [Ca2+]in increase in all neurons. Removal of the Ca2+ ions from external solution did not change the [Ca2+]in values. It is suggested that there is IP3- and GTP-dependent release of Ca2+ from intraneuronal stores in the Helix pomatia neurons.

[Relation between the surface potential of mouse neuroblastoma clone C1300 cells and the phase of the cell cycle]. / Zavisimost' poverkhnostnogo potentsiala kletok neiroblastomy myshi klona C1300 ot fazy kletochnogo tsikla.

The surface charge of neuroblastoma cells in different phases of the cell cycle was studied by the microelectrophoresis method. The surface charge increased by 50% on the average after addition of colchicine to the culture medium, by 20-30% after addition of dibutyryl-cAMP or removal of the serum from the medium and decreased by 30% after addition of DMSO. These changes correlated well with variations of the protein content per cell.