Calcium waves

Waves through living systems are best characterized by their speeds at 20 degrees C. These speeds vary from those of calcium action potentials to those of ultraslow ones which move at 1-10 and/or 10-20 nm s(-1). All such waves are known or inferred to be calcium waves. The two classes of calcium waves which include ones with important morphogenetic effects are slow waves that move at 0.2-2 microm s(-1) and ultraslow ones. Both may be propagated by cycles in which the entry of calcium through the plasma membrane induces subsurface contraction. This contraction opens nearby stretch-sensitive calcium channels. Calcium entry through these channels propagates the calcium wave. Many slow waves are seen as waves of indentation. Some are considered to act via cellular peristalsis; for example, those which seem to drive the germ plasm to the vegetal pole of the Xenopus egg. Other good examples of morphogenetic slow waves are ones through fertilizing maize eggs, through developing barnacle eggs and through axolotl embryos during neural induction. Good examples of ultraslow morphogenetic waves are ones during inversion in developing Volvox embryos and across developing Drosophila eye discs. Morphogenetic waves may be best pursued by imaging their calcium with aequorins.     

Stretch-activated calcium channels relay fast calcium waves propagated by calcium-induced calcium influx

For nearly 30 years, fast calcium waves have been attributed to a regenerative process propagated by CICR (calcium-induced calcium release) from the endoplasmic reticulum. Here, I propose a model containing a new subclass of fast calcium waves which is propagated by CICI (calcium-induced calcium influx) through the plasma membrane. They are called fast CICI waves. These move at the order of 100 to 1000 microm/s (at 20 degrees C), rather than the order of 3 to 30 microm/s found for CICR. Moreover, in this proposed subclass, the calcium influx which drives calcium waves is relayed by stretch-activated calcium channels. This model is based upon reports from approx. 60 various systems. In seven of these reports, calcium waves were imaged, and, in five of these, evidence was presented that these waves were regenerated by CICI. Much of this model involves waves that move along functioning flagella and cilia. In these systems, waves of local calcium influx are thought to cause waves of local contraction by inducing the sliding of dynein or of kinesin past tubulin microtubules. Other cells which are reported to exhibit waves, which move at speeds in the fast CICI range, include ones from a dozen protozoa, three polychaete worms, three molluscs, a bryozoan, two sea urchins, one arthropod, four insects, Amphioxus, frogs, two fish and a vascular plant (Equisetum), together with numerous healthy, as well as cancerous, mammalian cells, including ones from human. In two of these systems, very gentle local mechanical stimulation is reported to initiate waves. In these non-flagellar systems, the calcium influxes are thought to speed the sliding of actinomyosin filaments past each other. Finally, I propose that this mechanochemical model could be tested by seeing if gentle mechanical stimulation induces waves in more of these systems and, more importantly, by imaging the predicted calcium waves in more of them.     

On the conservation of fast calcium wave speeds

Calcium waves were first seen about 25 years ago as the giant, 10 micro m/s wave or tsunami which crosses the cytoplasm of an activating medaka fish egg [J Cell Biol 76 (1978) 448]. By 1991, reports of such waves with approximately 10 micro m/s velocities through diverse, activating eggs and with approximately 30 micro m/s velocities through diverse, fully active systems had been compiled to form a class of what are now called fast calcium waves [Proc Natl Acad Sci USA 88 (1991) 9883; Bioessays 21 (1999) 657].This compilation is now updated to include organisms from algae and sponges up to blowflies, squid and men and organizational levels from mammalian brains and hearts as well as chick embryos down to muscle, nerve, epithelial, blood and cancer cells and even cell-free extracts. Plots of these data confirm the narrow, 2-3-fold ranges of fast wave speeds through activating eggs and 3-4-fold ones through fully active systems at a given temperature. This also indicate Q(10)'s of 2.7-fold per 10 degrees C for both activating eggs and for fully activated cells.Speeds through some ultraflat preparations which are a few-fold above the conserved range are attributed to stretch propagated calcium entry (SPCE) rather than calcium-induced calcium release (CICR).     

Classes and mechanisms of calcium waves

The best known calcium waves move at about 5–30 μm/s (at 20°C) and will be called fast waves to distinguish them from slow (contractile) ones which move at 0.1-1 μm/s as well as electrically propagated, ultrafast ones. Fast waves move deep within cells and seem to underlie most calcium signals. Their velocity and hence mechanism has been remarkably conserved among all or almost all eukaryotic cells. In fully active (but not overstimulated) cells of all sorts, their mean speeds lie between about 15–30 μm/s at 20°C. Their amplitudes usually lie between 3–30 μM and their frequencies from one per 10–300 s. They are propagated by a reaction diffusion mechanism governed by the Luther equation in which Ca²⁺ ions are the only diffusing propagators, and calcium induced calcium release, or CICR, the only reaction; although this reaction traverses various channels which are generally modulated by IP₃ or cADPR. However, they may be generally initiated by a second, lumenal mode of CICR which occurs within the ER. Moreover, they are propagated between cells by a variety of mechanisms. Slow intracellular waves, on the other hand, may be mechanically propagated via stretch sensitive calcium channels.     

Excitation-contraction coupling and extracellular calcium transients in rabbit atrium: reconstruction of basic cellular mechanisms

Interactions of electrogenic sodium-calcium exchange, calcium channel and sarcoplasmic reticulum in the mammalian heart have been explored by simulation of extracellular calcium transients measured with tetramethylmurexide in rabbit atrium. The approach has been to use the simplest possible formulations of these mechanisms, which together with a minimum number of additional mechanisms allow reconstruction of action potentials, intracellular calcium transients and extracellular calcium transients. A 3:1 sodium-calcium exchange stoichiometry is assumed. Calcium-channel inactivation is assumed to take place by a voltage-dependent mechanism, which is accelerated by a rise in intracellular calcium; intracellular calcium release becomes a major physiological regulator of calcium influx via calcium channels. A calcium release mechanism is assumed, which is both calcium- and voltage-sensitive, and which undergoes prolonged inactivation. 200 microM cytosolic calcium buffer is assumed. For most simulations only instantaneous potassium conductances are simulated so as to study the other mechanisms independently of time- and calcium-dependent outward current. Thus, the model reconstructs extracellular calcium transients and typical action-potential configuration changes during steady-state and non-steady-state stimulation from the mechanisms directly involved in trans-sarcolemmal calcium movements. The model predicts relatively small trans-sarcolemmal calcium movements during regular stimulation (ca. 2 mumol kg-1 fresh mass per excitation); calcium current is fully activated within 2 ms of excitation, inactivation is substantially complete within 30 ms, and sodium-calcium exchange significantly resists repolarization from approximately -30 mV. Net calcium movements many times larger are possible during non-steady-state stimulation. Long action potentials at premature excitations or after inhibition of calcium release can be supported almost exclusively by calcium current (net calcium influx 5-30 mumol kg-1 fresh mass); action potentials during potentiated post-stimulatory contractions can be supported almost exclusively by sodium-calcium exchange (net calcium efflux 4-20 mumol kg-1 fresh mass). Large calcium movements between the extracellular space and the sarcoplasmic reticulum can take place through the cytosol with virtually no contractile activation. The simulations provide integrated explanations of electrical activity, contractile function and trans-sarcolemmal calcium movements, which were outside the explanatory range of previous models.     

Studying the isolated central nervous system; a report on 35 years: more inquisitive than acquisitive

1. The CNS from invertebrate animals such as slugs, snails, leeches, and cockroaches, can be isolated and kept alive for many hours. 2. The electrical and pharmacological properties of invertebrate CNS neurons have many similarities and it is probable that the basic rules governing the CNS evolved more than 600 million years ago. 3. The nerve cells can show sodium action potentials, calcium action potentials, EPSP, IPSP, biphasic potentials, electrogenic sodium pump potentials, and a variety of potassium, sodium, calcium and chloride currents. 4. Invertebrate CNS ganglia contain identifiable individual nerve cells whose properties and responses to neurotransmitters and drugs are constant and repeatable from preparation to preparation. 5. It was possible to set up an isolated CNS-nerve trunk-muscle preparation and study the transport of radioactive material from the CNS to the muscle and from muscle to CNS. This has provided information about axoplasmic transport in both invertebrate and vertebrate preparations. 6. The methods developed from studies of invertebrate isolated CNS preparations have been applied to vertebrate isolated CNS preparations. 7. In addition to thin slices of the mammalian brain, it is possible to keep 5 cm lengths of the whole mammalian spinal cord and brain stem alive for many hours. 8. The isolated mammalian spinal cord has functional ipsilateral and contralateral reflexes, ascending and descending pathways, extensive sensory integrative local area networks, and inhibitory interneuron circuits. Much of the in vivo circuitry is functional in vitro. 9. The isolated mammalian spinal cord and brain stem can be developed to include functional higher brain circuits that will provide increased understanding of the control and integrative action of the mammalian central nervous system.     

Fast calcium waves

Calcium waves are propagated in five main speed ranges which cover a billion-fold range of speeds. We define the fast speed range as 3-30μm/s after correction to a standard temperature of 20°C. Only waves which are not fertilization waves are considered here. 181 such cases are listed here. These are through organisms in all major taxa from cyanobacteria through mammals including human beings except for those through other bacteria, higher plants and fungi. Nearly two-thirds of these speeds lie between 12 and 24μm/s. We argue that their common mechanism in eukaryotes is a reaction-diffusion one involving calcium-induced calcium release, in which calcium waves are propagated along the endoplasmic reticulum. We propose that the gliding movements of some cyanobacteria are driven by fast calcium waves which are propagated along their plasma membranes. Fast calcium waves may drive materials to one end of developing embryos by cellular peristalsis, help coordinate complex cell movements during development and underlie brain injury waves. Moreover, we continue to argue that such waves greatly increase the likelihood that chronic injuries will initiate tumors and cancers before genetic damage occurs. Finally we propose numerous further studies.     

Non-propagated potentials in the electromyogram of mammalian ocular muscles]

Electromyograms of mammalian extraocular muscles were recorded by means of a coaxial electrode. Besides normal extracellular spike potentials (1-2 msec duration), monophasic waves (with a decline lasting up to 7 msec) were recorded. As to the interpretation of these potential changes in terms of a potential drop that is produced by local currents flowing from the resting region of a fibre towards the active region consideration is given to two cases. First, a propagated active region (spike potentials, at least diphasic) and second, a stationary active region (with resulting monophasic waves). In the EMGs spontaneous monophasic potentials recruit at a lower threshold than spike potentials; frequency changes were observed when head position was altered. The latter are interpreted as local depolarizations occurring at neuromuscular junctions of multiple innervated muscle fibres among those fibre types that compose extraocular muscles.     

Initiation and propagation of a neuronal intracellular calcium wave

The ability to image calcium movement within individual neurons inspires questions of functionality including whether calcium entry into the nucleus is related to genetic regulation for phenomena such as long term potentiation. Calcium waves have been initiated in hippocampal pyramidal cells with glutmatergic signals both in the presence and absence of back propagating action potentials (BPAPs). The dendritic sites of initiation of these calcium waves within about 100 μm of the soma are thought to be localized near oblique junctions. Stimulation of synapses on oblique dendrites leads to production of inositol 1,4,5-trisphosphate (IP₃) which diffuses to the apical dendrite igniting awaiting IP₃ receptors (IP₃Rs) and initiating and propagating catalytic calcium release from the endoplasmic reticulum. We construct a reduced mathematical system which accounts for calcium wave initiation and propagation due to elevated IP₃. Inhomogeneity in IP₃ distribution is responsible for calcium wave initiation versus subthreshold or spatially uniform suprathreshold activation. However, the likelihood that a calcium wave is initiated does not necessarily increase with more calcium entering from BPAPs. For low transient synaptic stimuli, timing between IP₃ generation and BPAPs is critical for calcium wave initiation. We also show that inhomogeneity in IP₃R density can account for calcium wave directionality. Simulating somatic muscarinic receptor production of IP₃, we can account for the critical difference between calcium wave entry into the soma and failure to do so.     

Evidence for glutamate-mediated activation of hippocampal neurons by glial calcium waves

Communication from astrocytes to neurons has recently been reported by two laboratories, but different mechanisms were thought to underlie glial calcium wave activation of associated neurons. Neuronal calcium elevation by glia observed in the present report is similar to that reported previously, where an increase in neuronal calcium was demonstrated in response to glial stimulation. In the present study hippocampal neurons plated on a confluent glial monolayer displayed a transient increase in intracellular calcium following a short delay after the passage of a wave of increased calcium in underlying glia. Activated cells displayed action potentials in response to glial waves and showed antineurofilament immunoreactivity. Finally, the N-methyl-D -aspartate glutamate receptor antagonist DL -2-amino-5-phosphonovaleric acid and the non-NMDA glutamate receptor antagonist 6,7-dinitroquinoxaline-2,3-dione significantly reduced the responsiveness of neurons to glial calcium waves. Our results indicate that hippocampal neurons growing on hippocampal or cortical astrocytes respond to glial calcium waves with elevations in calcium and increased electrical activity. Furthermore, we show that in most cases this communication appears to be mediated by ionotropic glutamate receptor channels. © 1995 John Wiley & Sons, Inc.     

Calcium waves and oscillations in eggs

Eggs from several protostomes (molluscs, annelids, nemerteans, etc.) and two deuterostomes (mammals and ascidians) display repetitive calcium signals. Oscillations in the level of intracellular calcium concentration are occasionally triggered by maturing hormones (as in some molluscs) and mostly observed after fertilization which occurs at different stages of the meiotic cell cycle (oocytes are arrested in prophase, metaphase I or metaphase II). In most eggs examined so far, calcium oscillations last until the end of meiosis just before male and female pronuclei form. This ability depends on the sensitivity of InsP3 channels and on the permeability of the plasma membrane to extracellular calcium. In eggs that undergo cytoplasmic reorganization at fertilization (annelids, nemerteans, ascidians, etc.) the repetitive calcium signals are waves that originate from localized cortical sites that become calcium waves pacemakers. In ascidians we have identified the site of initiation of repetitive calcium waves as an accumulation of endoplasmic reticulum sandwiched between the plasma membrane and an accumulation of mitochondria. We compare and discuss the generation of calcium signals in the different eggs, their relationship with the cell cycle and the possible roles they play during development.     

Spontaneous calcium transients regulate neuronal plasticity in developing neurons

Calcium ions play critical roles in neuronal differentiation. We have recorded transient, repeated elevations of calcium in embryonic Xenopus spinal neurons over periods of 1 h in vitro and in vivo, confocally imaging fluo 3-loaded cells at 5 s intervals. Calcium spikes and calcium waves are found both in neurons in culture and in the intact spinal cord. Spikes rise rapidly to approximately 400% of baseline fluorescence and have a double exponential decay, whereas waves rise slowly to approximately 200% of baseline fluorescence and decay slowly as well. Imaging of fura 2-loaded neurons indicates that intracellular calcium increases from 50 to 500 nM during spikes. Both spikes and waves are abolished by removal of extracellular calcium. Developmentally, the incidence and frequency of spikes decrease, whereas the incidence and frequency of waves are constant. Spikes are generated by spontaneous calcium-dependent action potentials and also utilize intracellular calcium stores. Waves are produced by a mechanism that does not involve classic voltage-dependent calcium channels. Spikes are required for expression of the transmitter GABA and for potassium channel modulation. Waves in growth cones are likely to regulate neurite extension. The results demonstrate the roles of a novel signaling system in regulating neuronal plasticity, that operates on a time scale 10⁴ times slower than that of action potentials. © 1995 John Wiley & Sons, Inc.     

Slow wave and spike action potentials recorded in cell cultures from the muscularis externa of the guinea pig small intestine.

Intracellular recordings were obtained to investigate whether slow wave and spike type action potentials are present in cell cultures of the muscularis externa from the guinea pig small intestine. The muscularis externa of the small intestine was dissociated by using specific purified enzymes and gentle mechanical dissociation. Cells were plated on cover slips and maintained in culture for up to 4 weeks. Dissociated cells obtained in this way reorganized themselves in a few days to form small cell clumps showing spontaneous movements. Intracellular recordings of these clumps displayed both spike and slow wave type action potentials. Spikes were observed on top of some slow waves and were abolished by the addition of nifedipine or the removal of extracellular calcium. Slow waves, however, were nifedipine insensitive and temperature sensitive, and were abolished by octanol (a gap junction blocker) and forskolin (an adenyl cyclase activator). Slow waves were never observed in small clumps (<50 microm), suggesting that a critical mass of cells might be required for their generation. These observations demonstrated for the first time the presence of nifedipine-insensitive slow waves in cell cultures of the muscularis externa from the guinea pig small intestine. Cell cultures allow rigorous control of the immediate environment for the cells and this should facilitate future studies on the molecular and cellular mechanisms responsible for the slow waves in the gastrointestinal tract.     

Retinal waves: mechanisms and function in visual system development

A characteristic feature of developing neural networks is spontaneous periodic activity. In the developing retina, retinal ganglion cells fire bursts of action potentials that drive large increases in intracellular calcium concentration with a periodicity of minutes. These periodic bursts of action potentials propagate across the developing inner retina as waves, driving neighboring retinal ganglion cells to fire in a correlated fashion. Here we will review recent progress in elucidating the mechanisms in mammals underlying retinal wave propagation and those regulating the periodicity with which these retinal waves occur. In addition, we will review recent experiments indicating that retinal waves are critical for refining retinal projections to their primary targets in the central visual system and may be involved in driving developmental processes within the retina itself.     

Dynamics of retinal waves are controlled by cyclic AMP

Waves of spontaneous activity sweep across the developing mammalian retina and influence the pattern of central connections made by ganglion cell axons. These waves are driven by synaptic input from amacrine cells. We show that cholinergic synaptic transmission during waves is not blocked by TTX, indicating that release from starburst amacrine cells is independent of sodium action potentials. The spatiotemporal properties of the waves are regulated by endogenous release of adenosine, which sets intracellular cAMP levels through activation of A2 receptors present on developing amacrine and ganglion cells. Increasing cAMP levels increase the size, speed, and frequency of the waves. Conversely, inhibiting adenylate cyclase or PKA prevents wave activity. Together, these results imply a novel mechanism in which levels of cAMP within an immature retinal circuit regulate the precise spatial and temporal patterns of spontaneous neural activity.     

Characterization and subcellular targeting of GCaMP-type genetically-encoded calcium indicators

Genetically-encoded calcium indicators (GECIs) hold the promise of monitoring [Ca(2+)] in selected populations of neurons and in specific cellular compartments. Relating GECI fluorescence to neuronal activity requires quantitative characterization. We have characterized a promising new genetically-encoded calcium indicator-GCaMP2-in mammalian pyramidal neurons. Fluorescence changes in response to single action potentials (17+/-10% DeltaF/F [mean+/-SD]) could be detected in some, but not all, neurons. Trains of high-frequency action potentials yielded robust responses (302+/-50% for trains of 40 action potentials at 83 Hz). Responses were similar in acute brain slices from in utero electroporated mice, indicating that long-term expression did not interfere with GCaMP2 function. Membrane-targeted versions of GCaMP2 did not yield larger signals than their non-targeted counterparts. We further targeted GCaMP2 to dendritic spines to monitor Ca(2+) accumulations evoked by activation of synaptic NMDA receptors. We observed robust DeltaF/F responses (range: 37%-264%) to single spine uncaging stimuli that were correlated with NMDA receptor currents measured through a somatic patch pipette. One major drawback of GCaMP2 was its low baseline fluorescence. Our results show that GCaMP2 is improved from the previous versions of GCaMP and may be suited to detect bursts of high-frequency action potentials and synaptic currents in vivo.     

Development of evolutionary biomedicine as a novel direction of biological science

This review analyzes the data obtained for the last decade on invertebrate animals (insects, round worms, molluscs) as models of various human diseases. As illustration, there are considered advances in studying of the invertebrate system of insulin-like peptides and signaling mechanisms of their action—evolutionary conservative homologues of the mammalian insulin/IGF-1 system. Results are presented of studies on the nematode Caenorhabditis elegans, Drosophila Drosophila melanogaster, and mollusks, which “model” individual aspects of some human diseases—diabetes, neurodegenerative dysfunction, carcinogenesis, as well as aging. A conclusion is made about the development of a new, synthetic direction combining evolutionary science and molecular biology and medicine, which we call evolutionary biomedicine. The roots of the appearance of this synthetic direction are to be searched for in L.A. Orbeli’s evolutionary ideas, methods, and scientific predictions.     

Spatial trigger waves: positive feedback gets you a long way

Trigger waves are a recurring biological phenomenon involved in transmitting information quickly and reliably over large distances. Well-characterized examples include action potentials propagating along the axon of a neuron, calcium waves in various tissues, and mitotic waves in Xenopus eggs. Here we use the FitzHugh-Nagumo model, a simple model inspired by the action potential that is widely used in physics and theoretical biology, to examine different types of trigger waves—spatial switches, pulses, and oscillations—and to show how they arise.     

Capacitative calcium entry channels

In the phospholipase C signaling system, Ca(2+) is mobilized from intracellular stores by an action of inositol 1,4,5-trisphosphate. The depletion of intracellular calcium stores activates a calcium entry mechanism at the plasma membrane called capacitative calcium entry. The signal for activating the entry is unknown but likely involves either the generation or release, or both, from the endoplasmic reticulum of some diffusible signal. Recent research has focused on mammalian homologues of the Drosophila TRP protein as potential candidates for capacitative calcium entry channels. This review summarizes current knowledge about the nature of capacitative calcium entry signals, as well as the potential role of mammalian TRP proteins as capacitative calcium entry channel molecules.