Distinct mechanisms of signal processing by lamina I spino-parabrachial neurons.

Lamina I spino-parabrachial neurons (SPNs) receive peripheral nociceptive input, process it and transmit to the supraspinal centres. Although responses of SPNs to cutaneous receptive field stimulations have been intensively studied, the mechanisms of signal processing in these neurons are poorly understood. Therefore, we used an ex-vivo spinal cord preparation to examine synaptic and cellular mechanisms determining specific input-output characteristics of the neurons. The vast majority of the SPNs received a few direct nociceptive C-fiber inputs and generated one spike in response to saturating afferent stimulation, thus functioning as simple transducers of painful stimulus. However, 69% of afferent stimulation-induced action potentials in the entire SPN population originated from a small fraction (19%) of high-output neurons. These neurons received a larger number of direct Aδ- and C-fiber inputs, generated intrinsic bursts and efficiently integrated a local network activity via NMDA-receptor-dependent mechanisms. The high-output SPNs amplified and integrated the nociceptive input gradually encoding its intensity into the number of generated spikes. Thus, different mechanisms of signal processing allow lamina I SPNs to play distinct roles in nociception.

Perturbed Ca2+-dependent signaling of DYT2 hippocalcin mutant as mechanism of autosomal recessive dystonia.

A recent report of autosomal-recessive primary isolated dystonia (DYT2 dystonia) identified mutations in HPCA, a gene encoding a neuronal calcium sensor protein, hippocalcin (HPCA), as the cause of this disease. However, how mutant HPCA leads to neuronal dysfunction remains unknown. Using a multidisciplinary approach, we demonstrated the failure of dystonic N75K HPCA mutant to decode short bursts of action potentials and theta rhythms in hippocampal neurons by its Ca2+-dependent translocation to the plasma membrane. This translocation suppresses neuronal activity via slow afterhyperpolarization (sAHP) and we found that the N75K mutant could not control sAHP during physiologically relevant neuronal activation. Simulations based on the obtained experimental results directly demonstrated an increased excitability in neurons expressing N75K mutant instead of wild type (WT) HPCA. In conclusion, our study identifies sAHP as a downstream cellular target perturbed by N75K mutation in DYT2 dystonia, demonstrates its impact on neuronal excitability, and suggests a potential therapeutic strategy to efficiently treat DYT2.

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.

The effect of nimodipine on calcium homeostasis and pain sensitivity in diabetic rats.

1. The pathogenesis of diabetic neuropathy is a complex phenomenon, the mechanisms of which are not fully understood. Our previous studies have shown that the intracellular calcium signaling is impaired in primary and secondary nociceptive neurons in rats with streptozotocin (STZ)-induced diabetes. Here, we investigated the effect of prolonged treatment with the L-type calcium channel blocker nimodipine on diabetes-induced changes in neuronal calcium signaling and pain sensitivity. 2. Diabetes was induced in young rats (21 p.d.) by a streptozotocin injection. After 3 weeks of diabetes development, the rats were treated with nimodipine for another 3 weeks. The effect of nimodipine treatment on calcium homeostasis in nociceptive dorsal root ganglion neurons (DRG) and substantia gelatinosa (SG) neurons of the spinal cord slices was examined with fluorescent imaging technique. 3. Nimodipine treatment was not able to normalize elevated resting intracellular calcium ([Ca(2+)]( i )) levels in small DRG neurons. However, it was able to restore impaired Ca(2+) release from the ER, induced by either activation of ryanodine receptors or by receptor-independent mechanism in both DRG and SG neurons. 4. The beneficiary effects of nimodipine treatment on [Ca(2+)]( i ) signaling were paralleled with the reversal of diabetes-induced thermal hypoalgesia and normalization of the acute phase of the response to formalin injection. Nimodipine treatment was also able to shorten the duration of the tonic phase of formalin response to the control values. 5. To separate vasodilating effect of nimodipine Biessels et al., (Brain Res. 1035:86-93) from its effect on neuronal Ca(2+) channels, a group of STZ-diabetic rats was treated with vasodilator - enalapril. Enalapril treatment also have some beneficial effect on normalizing Ca(2+) release from the ER, however, it was far less explicit than the normalizing effect of nimodipine. Effect of enalapril treatment on nociceptive behavioral responses was also much less pronounced. It partially reversed diabetes-induced thermal hypoalgesia, but did not change the characteristics of the response to formalin injection. 6. The results of this study suggest that chronic nimodipine treatment may be effective in restoring diabetes-impaired neuronal calcium homeostasis as well as reduction of diabetes-induced thermal hypoalgesia and noxious stimuli responses. The nimodipine effect is mediated through a direct neuronal action combined with some vascular mechanism.

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.

Isoproterenol evokes extracellular Ca2+ spikes due to secretory events in salivary gland cells.

Secretory cells should in principle export substantial amounts of calcium via exocytosis since Ca2+ is sequestered in secretory granules. Based on a new technique for measurements of the extracellular calcium concentration in the vicinity of the cell membrane and on the droplet techniques we have monitored the rate of calcium extrusion from salivary gland acinar cells. Isoproterenol (ISP), a beta-adrenergic agonist and powerful secretogogue, evoked no change in the cytosolic free Ca2+ concentration ([Ca+]i but induced vigorous extracellular Ca+ concentration ([Ca2+]o) spiking. The absence of [Ca2+]i elevation and the pulsatile nature of the changes in [Ca2+]o indicate that these spikes are most likely due to calcium release from secretory granules. The cholinergic agonist acetylcholine (ACh), which induces moderate secretion, evoked a marked rise in [Ca2+]i and a smooth rise in [Ca2+]o, most likely induced by plasma membrane calcium pumps, on which shortlasting [Ca2+]o spikes were superimposed. The rate of ISP-induced calcium efflux was very substantial. The calculated calcium loss during the first 100 s of supramaximal stimulation corresponded to a reduction of the total cellular calcium concentration of approximately 0.4 mm. We conclude that in salivary glands, calcium release via exocytosis is one of the main mechanisms extruding calcium from cells to the extracellular milieu.

Isoproterenol evokes extracellular Ca2+ spikes due to secretory events in salivary gland cells.

Secretory cells should in principle export substantial amounts of calcium via exocytosis since Ca2+ is sequestered in secretory granules. Based on a new technique for measurements of the extracellular calcium concentration in the vicinity of the cell membrane and on the droplet technique, we have monitored the rate of calcium extrusion from salivary gland acinar cells. Isoproterenol (ISP), a beta-adrenergic agonist and powerful secretogogue, evoked no change in the cytosolic free Ca2+ concentration ([Ca2+]i) but induced vigorous extracellular Ca2+ concentration ([Ca2+]i) spiking. The absence of [Ca2+]i elevation and the pulsatile nature of the changes in [Ca2+]i indicate that these spikes are most likely due to calcium release from secretory granules. The cholinergic agonist acetylcholine (ACh), which induces moderate secretion, evoked a marked rise in [Ca2+]i and a smooth rise in [Ca2+]i, most likely induced by plasma membrane calcium pumps, on which shortlasting [Ca2+]i spikes were superimposed. The rate of ISP-induced calcium efflux was very substantial. The calculated calcium loss during the first 100 s of supramaximal stimulation corresponded to a reduction of the total cellular calcium concentration of approximately 0.4 mM. We conclude that in salivary glands, calcium release via exocytosis is one of the main mechanisms extruding calcium from cells to the extracellular milieu.

Distribution of Ca2+ extrusion sites on the mouse pancreatic acinar cell surface.

The localizations of Ca2+ extrusion sites in mouse pancreatic acinar cells during elevation of the intracellular free calcium concentration ([Ca2+]i) have been studied. During an agonist stimulated calcium elevation as well as when intracellular calcium is released from a 'caged compound', Ca2+ is primarily extruded from the apical secretory pole of the cells in spite of different spatial patterns of [Ca2+]i different sources of Ca2+, and the presence or absence of agonist. This is most likely due to a relatively high density of calcium pumps in the secretory granule region, although it could be explained by calcium pumps in this part of the cell having different characteristics from those in the basal membrane. The intensity of Ca2+ extrusion in the apical secretory pole is such that substantial (several millimoles per litre) changes of the free calcium concentration in the lumen of the acinus can occur during agonist stimulation.

[Advantages of secured nailing]. / Prednosti jisteného hrebování.

At the traumatological department of the Surgical Clinic in Prague 5-Motol up to 1986 practically all operations of the skeleton were made by the open method, i.e. under direct visual control. After the Clinic had bought an X-ray apparatus with an amplifier and pulsed skiascopy the number of closed operations increased. The time taken by the operation is shorter and the operations are less risky, in particular for patients of the more advanced age groups. At present the department prefers secured nailing of the tibia, femur and humerus. The introduction of this surgical technique caused a radical change in the surgical approach and therefore the department does not use any longer splint osteosynthesis for diaphyseal fractures of the long bones.

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.

Calcium clamp in isolated neurones of the snail Helix pomatia.

1. Intracellular free calcium concentration ([Ca2+]i) in isolated non-identified Helix pomatia neurones has been clamped at different physiologically significant levels by a feedback system between the fluorescent signal of fura-2 probe loaded into the cell and ionophoretic injection of Ca2+ ions through a CaCl2-loaded microelectrode. The membrane potential of the neurone has also been clamped using a conventional two-microelectrode method. 2. Special measurements have shown that the transport indices of injecting microelectrodes filled with 50 mM CaCl2 are quite variable (0.11 +/- 0.06, mean +/- S.D.). However, for each electrode the transport indices remained stable during several injection trials into a solution drop having the size of a neurone. The spread of calcium ions from the tip of the microelectrode across the cytosol of the neurone terminated within 2-4 s. The spatial difference in [Ca2+]i at this time did not exceed 10%. 3. Clamping of [Ca2+]i at a new increased level was accompanied by a transient of the Ca(2+)-injecting current. To increase [Ca2+]i by 0.1 microM, the amount of calcium ions injected during this stage had to be 36 +/- 20 microM Ca2+ per cell volume. Obviously, this transient represents the filling of a fast cytosolic buffer which has to be saturated to reach a new increased level of [Ca2+]i. It was followed by a steady component of Ca(2+)-injecting current, which was quite low (corresponding to injection of 0.39 +/- 0.20 microM s-1 for a 0.1 microM change of [Ca2+]i). This may represent the functioning of Ca(2+)-eliminating systems and corresponds to a similar amount of Ca2+ extruded from the cytoplasm. 4. Changes in the injection current also developed when Ca2+ influx through the membrane was triggered by the activation of voltage-gated calcium channels. The amount of Ca2+ entering the cell during the first seconds of depolarization to--15 mV was equal to 0.59 +/- 0.31 microM s-1 per cell volume. 5. No activation of Ca(2+)-dependent potassium current was observed during the changes in [Ca2+]i to levels exceeding the basal one by several times. Obviously, to activate this current, a much stronger increase in [Ca2+]i is needed in the immediate vicinity of the corresponding channels.

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.