Fast Calcium Transients Research Articles

Fast calcium transients in dendritic spines driven by extreme statistics.

Fast calcium transients (<10 ms) remain difficult to analyse in cellular microdomains, yet they can modulate key cellular events such as trafficking, local ATP production by endoplasmic reticulum-mitochondria complex (ER-mitochondria complex), or spontaneous activity in astrocytes. In dendritic spines receiving synaptic inputs, we show here that in the presence of a spine apparatus (SA), which is an extension of the smooth ER, a calcium-induced calcium release (CICR) is triggered at the base of the spine by the fastest calcium ions arriving at a Ryanodyne receptor (RyR). The mechanism relies on the asymmetric distributions of RyRs and sarco/ER calcium-ATPase (SERCA) pumps that we predict using a computational model and further confirm experimentally in culture and slice hippocampal neurons. The present mechanism for which the statistics of the fastest particles arriving at a small target, followed by an amplification, is likely to be generic in molecular transduction across cellular microcompartments, such as thin neuronal processes, astrocytes, endfeets, or protrusions.

Fast calcium transients in dendritic spines driven by extreme statistics.

Fast calcium transients in dendritic spines driven by extreme statistics.

Fast calcium transients translate the distribution and conduction of neural activity in different regions of a single sensory neuron.

In the present study, cytosolic calcium concentration changes were recorded in response to various forms of excitations, using the fluorescent calcium indicator dye OG-BAPTA1 together with the current or voltage clamp methods in stretch receptor neurons of crayfish. A single action potential evoked a rise in the resting calcium level in the axon and axonal hillock, whereas an impulse train or a large saturating current injection would be required to evoke an equivalent response in the dendrite region. Under voltage clamp conditions, amplitude differences between axon and dendrite responses vanished completely. The fast activation time and the modulation of the response by extracellular calcium concentration changes indicated that the evoked calcium transients might be mediated by calcium entry into the cytosol through a voltage-gated calcium channel. The decay of the responses was slow and sensitive to extracellular sodium and calcium concentrations as well as exposure to 1-10mM NiCl2 and 10-500µM lanthanum. Thus, a sodium calcium exchanger and a calcium ATPase might be responsible for calcium extrusion from the cytosol. Present results indicate that the calcium indicator OG-BAPTA1 might be an efficient but indirect way of monitoring regional membrane potential differences in a single neuron.

N- and L-Type Voltage-Gated Calcium Channels Mediate Fast Calcium Transients in Axonal Shafts of Mouse Peripheral Nerve.

In the peripheral nervous system (PNS) a vast number of axons are accommodated within fiber bundles that constitute peripheral nerves. A major function of peripheral axons is to propagate action potentials along their length, and hence they are equipped with Na+ and K+ channels, which ensure successful generation, conduction and termination of each action potential. However little is known about Ca2+ ion channels expressed along peripheral axons and their possible functional significance. The goal of the present study was to test whether voltage-gated Ca2+ channels (VGCCs) are present along peripheral nerve axons in situ and mediate rapid activity-dependent Ca2+ elevations under physiological circumstances. To address this question we used mouse sciatic nerve slices, Ca2+ indicator Oregon Green BAPTA-1, and 2-photon Ca2+ imaging in fast line scan mode (500 Hz). We report that transient increases in intra-axonal Ca2+ concentration take place along peripheral nerve axons in situ when axons are stimulated electrically with single pulses. Furthermore, we show for the first time that Ca2+ transients in peripheral nerves are fast, i.e., occur in a millisecond time-domain. Combining Ca2+ imaging and pharmacology with specific blockers of different VGCCs subtypes we demonstrate that Ca2+ transients in peripheral nerves are mediated mainly by N-type and L-type VGCCs. Discovery of fast Ca2+ entry into the axonal shafts through VGCCs in peripheral nerves suggests that Ca2+ may be involved in regulation of action potential propagation and/or properties in this system, or mediate neurotransmitter release along peripheral axons as it occurs in the optic nerve and white matter of the central nervous system (CNS).

Analysis of spontaneous and nerve-evoked calcium transients in intact extraocular muscles in vitro

Extraocular muscles (EOMs) have unique calcium handling properties, yet little is known about the dynamics of calcium events underlying ultrafast and tonic contractions in myofibers of intact EOMs. Superior oblique EOMs of juvenile chickens were dissected with their nerve attached, maintained in oxygenated Krebs buffer, and loaded with fluo-4. Spontaneous and nerve stimulation-evoked calcium transients were recorded and, following calcium imaging, some EOMs were double-labeled with rhodamine-conjugated alpha-bungarotoxin (rhBTX) to identify EOM myofiber types. EOMs showed two main types of spontaneous calcium transients, one slow type (calcium waves with 1/2max duration of 2–12s, velocity of 25–50μm/s) and two fast “flash-like” types (Type 1, 30–90ms; Type 2, 90–150ms 1/2max duration). Single pulse nerve stimulation evoked fast calcium transients identical to the fast (Type 1) calcium transients. Calcium waves were accompanied by a local myofiber contraction that followed the calcium transient wavefront. The magnitude of calcium-wave induced myofiber contraction far exceeded those of movement induced by nerve stimulation and associated fast calcium transients. Tetrodotoxin eliminated nerve-evoked transients, but not spontaneous transients. Alpha-bungarotoxin eliminated both spontaneous and nerve-evoked fast calcium transients, but not calcium waves, and caffeine increased wave activity. Calcium waves were observed in myofibers lacking spontaneous or evoked fast transients, suggestive of multiply-innervated myofibers, and this was confirmed by double-labeling with rhBTX. We propose that the abundant spontaneous calcium transients and calcium waves with localized contractions that do not depend on innervation may contribute to intrinsic generation of tonic functions of EOMs.

Spatiotemporal properties of nicotine induced activation in the rat somatosensory cortex

Event Abstract Back to Event Spatiotemporal properties of nicotine induced activation in the rat somatosensory cortex Gabor Molnar1*, Balazs Rozsa2 and Gabor Tamas1 1 Research Group for Cortical Microcircuits of the Hungarian Academy of Sciences, University of Szeged, Hungary 2 Institute of Experimental Medicine, Hungarian Academy of Sciences, Hungary Nicotine activates individual neurons as well as glial cells of young animals, but network effects of nicotine are not clear. We applied high speed single and two-photon network imaging combined with whole cell recordings to characterize the spatial and temporal pattern of nicotine induced activity in the neocortical microcircuit. Neurons and astrocytes were identified using differential staining patterns of bulk injected Oregon Green BAPTA-1 (OGB-1) and sulforhodamine 101 in slices taken from the rat somatosensory cortex (P8-14). The staining properties corresponded to the kinetics of calcium transients in neurons versus glial cells with faster and slower rise and decay kinetics, respectively. Nicotine (10 µM) increased the frequency of calcium transients corresponding to action potentials in neurons and induced longer lasting intracellular calcium responses in neurons and astrocytes. We detected closely placed active groups of cells consisting of neurons intermingled by astrocytes in response to nicotine. Clusters of cells responding to nicotine showed a relatively stereotyped activation pattern in which neurons with fast calcium transients were followed by glial cells and neurons with slower calcium signals. In conclusion, nicotine drives activation sequences in functionally grouped neurons and astrocytes in he juvenile neocortex. Conference: 12th Meeting of the Hungarian Neuroscience Society, Budapest, Hungary, 22 Jan - 24 Jan, 2009. Presentation Type: Poster Presentation Topic: Research on the cerebral cortex and related structures Citation: Molnar G, Rozsa B and Tamas G (2009). Spatiotemporal properties of nicotine induced activation in the rat somatosensory cortex. Front. Syst. Neurosci. Conference Abstract: 12th Meeting of the Hungarian Neuroscience Society. doi: 10.3389/conf.neuro.01.2009.04.214 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 09 Mar 2009; Published Online: 09 Mar 2009. * Correspondence: Gabor Molnar, Research Group for Cortical Microcircuits of the Hungarian Academy of Sciences, University of Szeged, Szeged, Hungary, molnarg@bio.u-szeged.hu Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Gabor Molnar Balazs Rozsa Gabor Tamas Google Gabor Molnar Balazs Rozsa Gabor Tamas Google Scholar Gabor Molnar Balazs Rozsa Gabor Tamas PubMed Gabor Molnar Balazs Rozsa Gabor Tamas Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Endogenous Brain-Derived Neurotrophic Factor Triggers Fast Calcium Transients at Synapses in Developing Dendrites

Brain-derived neurotrophic factor (BDNF) is involved in many aspects of the formation of functional neuronal networks. BDNF signaling regulates neuronal development not only globally, at the level of entire neurons or networks, but also at a subcellular level and with high temporal specificity; however, the spatiotemporal characteristics of intrinsic BDNF signaling are essentially unknown. Here, we used calcium imaging to directly observe intrinsic BDNF signaling in developing hippocampal neurons. We found that blocking intrinsic BDNF signaling with function-blocking BDNF antibodies (alphaBDNF) or K252-a reduced the frequency of spontaneously occurring fast and localized calcium rises in dendrites. Conversely, focal application of BDNF evoked fast and local dendritic calcium transients, which required activation of TrkB (tropomyosin-related kinase B) receptors as well as activation of voltage-gated sodium and calcium channels. Virus-mediated expression of PSD-95:CFP (postsynaptic density-95 tagged with cyan fluorescent protein) revealed that spontaneous local calcium transients occurred frequently at postsynaptic sites along the dendrite. The frequency of synaptically localized calcium transients was specifically reduced by blocking intrinsic BDNF signaling, whereas nonsynaptic calcium rises were not affected. Furthermore, focal BDNF delivery evoked localized and fast calcium elevations specifically at postsynaptic sites. Together, our results demonstrate that BDNF-dependent calcium signaling in developing hippocampal neurons is fast and occurs at synapses. These temporal and spatial characteristics of intrinsic BDNF signaling as well as its relative abundance renders BDNF an ideal signaling molecule in the establishment of specific synaptic connectivity and functional neuronal networks.

Calbindin in Cerebellar Purkinje Cells Is a Critical Determinant of the Precision of Motor Coordination

Long-term depression (LTD) of Purkinje cell-parallel fiber synaptic transmission is a critical determinant of normal cerebellar function. Impairment of LTD through, for example, disruption of the metabotropic glutamate receptor-IP3-calcium signaling cascade in mutant mice results in severe deficits of both synaptic transmission and cerebellar motor control. Here, we demonstrate that selective genetic deletion of the calcium-binding protein calbindin D-28k (calbindin) from cerebellar Purkinje cells results in distinctly different cellular and behavioral alterations. These mutants display marked permanent deficits of motor coordination and sensory processing. This occurs in the absence of alterations in a form of LTD implicated in the control of behavior. Analysis of synaptically evoked calcium transients in spines and dendrites of Purkinje cells demonstrated an alteration of time course and amplitude of fast calcium transients after parallel or climbing fiber stimulation. By contrast, the delayed metabotropic glutamate receptor-mediated calcium transients were normal. Our results reveal a unique role of Purkinje cell calbindin in a specific form of motor control and suggest that rapid calcium buffering may directly control behaviorally relevant neuronal signal integration.

Calcium imaging of epileptiform events with single-cell resolution.

Epileptic discharges propagate through apparently normal circuits, although it is still unclear how this recruitment takes place. To understand the role of different classes of neurons in neocortical epilepsy, we have developed a novel imaging assay that detects which neurons participate in epileptiform discharges. Using calcium imaging of neuronal populations during bicuculline-induced spontaneous epileptiform events in slices from juvenile mouse somatosensory cortex, we find that fast calcium transients correlate with epileptiform field potentials and intracellular depolarizing shifts and can be used as an optical signature that a given neuron has participated in an epileptiform event. Our results demonstrate a novel method to characterize epileptiform events with single-cell resolution. In addition, our data are consistent with an important role for layer 5 in generating neocortical seizures and indicate that subgroups of neurons are particularly prone to epileptiform recruitment.

GABA- and glutamate-mediated network activity in the hippocampus of neonatal and juvenile rats revealed by fast calcium imaging

In the rat hippocampus, during the first postnatal week, network activity is characterized by GABA-driven giant depolarizing potentials (GDPs) associated with calcium signals that are readily blocked when the GABAAantagonist bicuculline is applied to the bath. Towards the end of the first postnatal week, in concomitance with the shift of GABA responses from the depolarizing to the hyperpolarizing direction, functional glutamatergic connections start appearing. At this developmental stage, application of bicuculline blocks GABAA-mediated inhibition and induces the appearance of interictal epileptiform discharges. In the present experiments, we have used a high spatio-temporal resolution imaging system to compare, on a time scale of tens of ms, the onset and propagation of fast calcium transients generated within a GABAergic or glutamatergic network. We found that, during the first postnatal week, calcium signals associated to evoked GDPs arise from the activation of a local circuitry of neurons spanning the stratum radiatum and the pyramidal layer. Similar activation patterns were elicited by focal application of GABA in the presence of kynurenic acid, a broad spectrum ionotropic glutamatergic antagonist, and were blocked by bicuculline. During the second postnatal week, in the presence of bicuculline, calcium signals associated with interictal discharges evoked by stimulation of glutamatergic fibres propagated along the well-defined three-synaptic pathway from the dentate gyrus to the CA1 hippocampal area.

N-Type Calcium Channels and Their Regulation by GABABReceptors in Axons of Neonatal Rat Optic Nerve

Axons of neonatal rat optic nerves exhibit fast calcium transients in response to brief action potential stimulation. In response to one to four closely spaced action potentials, evoked calcium transients showed a fast-rising phase followed by a decay with a time constant of approximately 2-3 sec. By selective staining of axons or glial cells with calcium dyes, it was shown that the evoked calcium transient originated from axons. The calcium transient was caused by influx because it was eliminated when bath calcium was removed. Pharmacological profile studies with calcium channel subtype-specific peptides suggested that 58% of the evoked calcium influx was accounted for by N-type calcium channels, whereas L- and P/Q-type calcium channels had little, if any, contribution. The identity of the residual calcium influx remains unclear. GABA application caused a dramatic reduction of the amplitude of the action potential and the associated calcium influx. When GABAA receptors were blocked by bicuculline, the inhibitory effect of GABA on the action potential was eliminated, whereas that on the calcium influx was not, indicating involvement of GABAB receptors. Indeed, the calcium influx was inhibited by the GABAB receptor agonist baclofen. This baclofen effect was occluded by a previous block of N-type calcium channels and was unaffected by the broad-spectrum K+ channel blocker 4-AP. We conclude that neonatal rat optic nerve axons express N-type calcium channels, which are subjected to regulation by G-protein-coupled GABAB receptors. We suggest that receptor-mediated inhibition of axonal calcium channels plays a protective role in neonatal anoxic and/or ischemic injury.

Imaging of calcium transients during excitation-contraction coupling in skeletal muscle fibers.

Action potential stimulation of skeletal muscle fibers activates the release of Ca2+ from the sarcoplasmic reticulum (SR), thus leading to a fast and transient change in myoplasmic calcium concentration ([Ca2+]i). Until very recently these calcium transients have been only recorded by means of space averaging photodetectors, such as photomultipliers and photodiodes, in combination with the use of several intracellular calcium indicators1–8. The high frequency response exhibited by this kind of photodevices is ideal to track the fast calcium transients, characteristic of the muscle cells; however, their physical construction make them inherently unable to provide information about the topological distribution of the release process. Given the structural complexity of the muscle cells, it is possible that the limited knowledge gained so far has omitted important features of this physiological process. In an attempt to surmount these limitations we have adapted video microscopy techniques to perform rapid Ca2+-imaging in this preparation9. This has been a difficult task because the muscle fiber is a thick specimen (100–150 µm in diameter) able to undergo very fast changes in shape and volume as a consequence of its electrical activation.

Fast calcium transients in rat peritoneal mast cells are not sufficient to trigger exocytosis.

The calcium concentration, [Ca]i, in single rat peritoneal mast cells was measured by means of the new Ca indicator dye fura-2. Upon stimulation with antigen or compound 48/80, [Ca]i rose for seconds to values greater than 5 microM. These Ca transients did not depend on the presence of extracellular Ca, and they sometimes occurred spontaneously, especially in the presence of exogenous phosphatidylserine. Calcium transients did not necessarily lead to degranulation. Degranulation usually occurred during periods of somewhat elevated [Ca] (0.5-1 microM) following transients but was sometimes observed at [Ca]i less than or equal to 250 nM. We found no evidence that an antigen-induced Ca influx is required for degranulation.