Spatial and temporal aspects of neuronal calcium and sodium signals measured with low-affinity fluorescent indicators.

Low-affinity fluorescent indicators for Ca2+ or Na+ allow measuring the dynamics of intracellular concentration of these ions with little perturbation from physiological conditions because they are weak buffers. When using synthetic indicators, which are small molecules with fast kinetics, it is also possible to extract spatial and temporal information on the sources of ion transients, their localization, and their disposition. This review examines these important aspects from the biophysical point of view, and how they have been recently exploited in neurophysiological studies. We first analyze the environment where Ca2+ and Na+ indicators are inserted, highlighting the interpretation of the two different signals. Then, we address the information that can be obtained by analyzing the rising phase and the falling phase of the Ca2+ and Na+ transients evoked by different stimuli, focusing on the kinetics of ionic currents and on the spatial interpretation of these measurements, especially on events in axons and dendritic spines. Finally, we suggest how Ca2+ or Na+ imaging using low-affinity synthetic fluorescent indicators can be exploited in future fundamental or applied research.

Fast Synaptically Activated Calcium and Sodium Kinetics in Hippocampal Pyramidal Neuron Dendritic Spines.

An accurate assessment of the time course, components, and magnitude of postsynaptic currents is important for a quantitative understanding of synaptic integration and signaling in dendritic spines. These parameters have been studied in some detail in previous experiments, primarily using two-photon imaging of [Ca2+]i changes and two-photon uncaging of glutamate. However, even with these revolutionary techniques, there are some missing pieces in our current understanding, particularly related to the time courses of synaptically evoked [Ca2+]i and [Na+]i changes. In new experiments, we used low-affinity, linear Na+ and Ca2+ indicators, laser fluorescence stimulation, and a sensitive camera-based detection system, combined with electrical stimulation and two-photon glutamate uncaging, to extend measurements of these spine parameters. We found that (1) almost all synaptically activated Na+ currents in CA1 hippocampal pyramidal neuron spines in slices from mice of either sex are through AMPA receptors with little Na+ entry through voltage-gated sodium channels (VGSCs) or NMDA receptor channels; (2) a spectrum of sodium transient decay times was observed, suggesting a spectrum of spine neck resistances, even on the same dendrite; (3) synaptically activated [Ca2+]i changes are very fast and are almost entirely because of Ca2+ entry through NMDA receptors at the time when the Mg2+ block is relieved by the fast AMPA-mediated EPSP; (4) the [Ca2+]i changes evoked by uncaging glutamate are slower than the changes evoked by synaptic release, suggesting that the relative contribution of Ca2+ entering through NMDA receptors at rest following uncaging is higher than following electrical stimulation.

Mechanism of ArcLight derived GEVIs involves electrostatic interactions that can affect proton wires.

The genetically encoded voltage indicators ArcLight and its derivatives mediate voltage-dependent optical signals by intermolecular, electrostatic interactions between neighboring fluorescent proteins (FPs). A random mutagenesis event placed a negative charge on the exterior of the FP, resulting in a greater than 10-fold improvement of the voltage-dependent optical signal. Repositioning this negative charge on the exterior of the FP reversed the polarity of voltage-dependent optical signals, suggesting the presence of "hot spots" capable of interacting with the negative charge on a neighboring FP, thereby changing the fluorescent output. To explore the potential effect on the chromophore state, voltage-clamp fluorometry was performed with alternating excitation at 390 nm followed by excitation at 470 nm, resulting in several mutants exhibiting voltage-dependent, ratiometric optical signals of opposing polarities. However, the kinetics, voltage ranges, and optimal FP fusion sites were different depending on the wavelength of excitation. These results suggest that the FP has external, electrostatic pathways capable of quenching fluorescence that are wavelength specific. One mutation to the FP (E222H) showed a voltage-dependent increase in fluorescence when excited at 390 nm, indicating the ability to affect the proton wire from the protonated chromophore to the H222 position. ArcLight-derived sensors may therefore offer a novel way to map how conditions external to the ß-can structure can affect the fluorescence of the chromophore and transiently affect those pathways via conformational changes mediated by manipulating membrane potential.

Improvements in Simultaneous Sodium and Calcium Imaging.

High speed imaging of ion concentration changes in neurons is an important and growing tool for neuroscientists. We previously developed a system for simultaneously measuring sodium and calcium changes in small compartments in neurons (Miyazaki and Ross, 2015). We used this technique to analyze the dynamics of these ions in individual pyramidal neuron dendritic spines (Miyazaki and Ross, 2017). This system is based on high speed multiplexing of light emitting diodes (LEDs) and classic organic indicators. To improve this system we made additional changes, primarily incorporating lasers in addition to the LEDs, more sophisticated imaging protocols, and the use of newer sodium and calcium indicators. This new system generates signals with higher signal to noise ratio (S/N), less background fluorescence, and less photodynamic damage. In addition, by using longer wavelength indicators instead of indicators sensitive in the UV range, it allows for the incorporation of focal uncaging along with simultaneous imaging, which should extend the range of experiments.

Sodium Dynamics in Pyramidal Neuron Dendritic Spines: Synaptically Evoked Entry Predominantly through AMPA Receptors and Removal by Diffusion.

Dendritic spines are key elements underlying synaptic integration and cellular plasticity, but many features of these important structures are not known or are controversial. We examined these properties using newly developed simultaneous sodium and calcium imaging with single-spine resolution in pyramidal neurons in rat hippocampal slices from either sex. Indicators for both ions were loaded through the somatic patch pipette, which also recorded electrical responses. Fluorescence changes were detected with a high-speed, low-noise CCD camera. Following subthreshold electrical stimulation, postsynaptic sodium entry is almost entirely through AMPA receptors with little contribution from entry through NMDA receptors or voltage-gated sodium channels. Sodium removal from the spine head is through rapid diffusion out to the dendrite through the spine neck with a half-removal time of ∼16 ms, which suggests the neck has low resistance. Peak [Na+]i changes during single EPSPs are ∼5 mm Stronger electrical stimulation evoked small plateau potentials that had significant longer-lasting localized [Na+]i increases mediated through NMDA receptors.SIGNIFICANCE STATEMENT Dendritic spines, small structures that are difficult to investigate, are important elements in the fundamental processes of synaptic integration and plasticity. The main tool for examining these structures has been calcium imaging. However, the kinds of information that calcium imaging reveals is limited. We used newly developed, high-speed, simultaneous sodium and calcium imaging to examine ion dynamics in spines in hippocampal pyramidal neurons. We found that following single subthreshold synaptic activation most sodium entry was through AMPA receptors and not through NMDA receptors or through voltage-gated sodium channels and that the spine neck is not a significant resistance barrier. Most spine mechanisms are linear. However, regenerative NMDA conductances can be activated with stronger stimulation.

Hypocretin/Orexin Peptides Alter Spike Encoding by Serotonergic Dorsal Raphe Neurons through Two Distinct Mechanisms That Increase the Late Afterhyperpolarization.

UNLABELLED: Orexins (hypocretins) are neuropeptides that regulate multiple homeostatic processes, including reward and arousal, in part by exciting serotonergic dorsal raphe neurons, the major source of forebrain serotonin. Here, using mouse brain slices, we found that, instead of simply depolarizing these neurons, orexin-A altered the spike encoding process by increasing the postspike afterhyperpolarization (AHP) via two distinct mechanisms. This orexin-enhanced AHP (oeAHP) was mediated by both OX1 and OX2 receptors, required Ca(2+) influx, reversed near EK, and decayed with two components, the faster of which resulted from enhanced SK channel activation, whereas the slower component decayed like a slow AHP (sAHP), but was not blocked by UCL2077, an antagonist of sAHPs in some neurons. Intracellular phospholipase C inhibition (U73122) blocked the entire oeAHP, but neither component was sensitive to PKC inhibition or altered PKA signaling, unlike classical sAHPs. The enhanced SK current did not depend on IP3-mediated Ca(2+) release but resulted from A-current inhibition and the resultant spike broadening, which increased Ca(2+) influx and Ca(2+)-induced-Ca(2+) release, whereas the slower component was insensitive to these factors. Functionally, the oeAHP slowed and stabilized orexin-induced firing compared with firing produced by a virtual orexin conductance lacking the oeAHP. The oeAHP also reduced steady-state firing rate and firing fidelity in response to stimulation, without affecting the initial rate or fidelity. Collectively, these findings reveal a new orexin action in serotonergic raphe neurons and suggest that, when orexin is released during arousal and reward, it enhances the spike encoding of phasic over tonic inputs, such as those related to sensory, motor, and reward events. SIGNIFICANCE STATEMENT: Orexin peptides are known to excite neurons via slow postsynaptic depolarizations. Here we elucidate a significant new orexin action that increases and prolongs the postspike afterhyperpolarization (AHP) in 5-HT dorsal raphe neurons and other arousal-system neurons. Our mechanistic studies establish involvement of two distinct Ca(2+)-dependent AHP currents dependent on phospholipase C signaling but independent of IP3 or PKC. Our functional studies establish that this action preserves responsiveness to phasic inputs while attenuating responsiveness to tonic inputs. Thus, our findings bring new insight into the actions of an important neuropeptide and indicate that, in addition to producing excitation, orexins can tune postsynaptic excitability to better encode the phasic sensory, motor, and reward signals expected during aroused states.

Imaging with organic indicators and high-speed charge-coupled device cameras in neurons: some applications where these classic techniques have advantages.

Dynamic calcium and voltage imaging is a major tool in modern cellular neuroscience. Since the beginning of their use over 40 years ago, there have been major improvements in indicators, microscopes, imaging systems, and computers. While cutting edge research has trended toward the use of genetically encoded calcium or voltage indicators, two-photon microscopes, and in vivo preparations, it is worth noting that some questions still may be best approached using more classical methodologies and preparations. In this review, we highlight a few examples in neurons where the combination of charge-coupled device (CCD) imaging and classical organic indicators has revealed information that has so far been more informative than results using the more modern systems. These experiments take advantage of the high frame rates, sensitivity, and spatial integration of the best CCD cameras. These cameras can respond to the faster kinetics of organic voltage and calcium indicators, which closely reflect the fast dynamics of the underlying cellular events.

Simultaneous Sodium and Calcium Imaging from Dendrites and Axons.

Dynamic calcium imaging is a major technique of neuroscientists. It can reveal information about the location of various calcium channels and calcium permeable receptors, the time course, magnitude, and location of intracellular calcium concentration ([Ca(2+)]i) changes, and indirectly, the occurrence of action potentials. Dynamic sodium imaging, a less exploited technique, can reveal analogous information related to sodium signaling. In some cases, like the examination of AMPA and NMDA receptor signaling, measurements of both [Ca(2+)]i and [Na(+)]i changes in the same preparation may provide more information than separate measurements. To this end, we developed a technique to simultaneously measure both signals at high speed and sufficient sensitivity to detect localized physiologic events. This approach has advantages over sequential imaging because the preparation may not respond identically in different trials. We designed custom dichroic and emission filters to allow the separate detection of the fluorescence of sodium and calcium indicators loaded together into a single neuron in a brain slice from the hippocampus of Sprague-Dawley rats. We then used high-intensity light emitting diodes (LEDs) to alternately excite the two indicators at the appropriate wavelengths. These pulses were synchronized with the frames of a CCD camera running at 500 Hz. Software then separated the data streams to provide independent sodium and calcium signals. With this system we could detect [Ca(2+)]i and [Na(+)]i changes from single action potentials in axons and synaptically evoked signals in dendrites, both with submicron resolution and a good signal-to-noise ratio (S/N).

Ca2+ sparks and puffs are generated and interact in rat hippocampal CA1 pyramidal neuron dendrites.

1,4,5-Inositol trisphosphate receptors (IP3Rs) and ryanodine receptors (RyRs) mediate release of Ca(2+) from internal stores in many neurons. The details of the spatial and temporal characteristics of these signals and their interactions in dendrites remain to be clarified. We found that localized Ca(2+) release events, with no associated change in membrane potential, occurred spontaneously in the dendrites of rat hippocampal CA1 pyramidal neurons. Their rate, but not their amplitude or time course, could be modulated by changes in membrane potential. Together, these results suggest that the spontaneous events are similar to RyR-dependent Ca(2+) "sparks" found in cardiac myocytes. In addition, we found that we could generate another kind of localized Ca(2+) release event by either a synaptic tetanus in the presence of 3-((R)-2-carboxypiperazine-4-yl)-propyl-1-phosphonic acid and CNQX or by uncaging IP3. These events had slower rise times and decay times than sparks and were more heterogeneous. These properties are similar to Ca(2+) "puffs" found in oocytes. These two localized signals interact. Low-intensity tetanic synaptic stimulation or uncaging of IP3 increased the decay time of spontaneous Ca(2+) events without changing their rise time or amplitude. Pharmacological experiments suggest that this event widening is attributable to a delayed IP3R-mediated release of Ca(2+) triggered by the synergistic action of IP3 and Ca(2+) released by RyRs. The actions of IP3 appear to be confined to the main apical dendrite because uncaging IP3 in the oblique dendrites has no effect on the time course of localized events or backpropagating action potential-evoked Ca(2+) signals in this region.

Imaging calcium waves and sparks in central neurons.

Here we describe the use of wide-field charge-coupled device (CCD) camera-based imaging methods to detect the spatial and temporal aspects of calcium release from internal stores in dendrites of neurons in brain slice preparations. This approach is useful for revealing aspects of this signaling system, which is generally invisible to electrical recording. The changes in intracellular calcium ion concentrations, [Ca(2+)](i), sometimes occur as large-amplitude, propagating Ca(2+) waves or as much smaller, localized events (sparks). In this protocol, a cell is loaded with an indicator that responds to Ca(2+), waves or sparks are stimulated in the cell, and the spatial and temporal characteristics of calcium release from internal stores in the cell are detected using wide-field CCD camera-based imaging. Such camera systems have some advantages for detecting and analyzing these [Ca(2+)](i) changes because the waves are spatially extended and the sparks do not always occur at the same locations.

Developmental profile of localized spontaneous Ca(2+) release events in the dendrites of rat hippocampal pyramidal neurons.

Recent experiments demonstrate that localized spontaneous Ca(2+) release events can be detected in the dendrites of pyramidal cells in the hippocampus and other neurons (J. Neurosci. 29 (2009) 7833-7845). These events have some properties that resemble ryanodine receptor mediated "sparks" in myocytes, and some that resemble IP(3) receptor mediated "puffs" in oocytes. They can be detected in the dendrites of rats of all tested ages between P3 and P80 (with sparser sampling in older rats), suggesting that they serve a general signaling function and are not just important in development. However, in younger rats the amplitudes of the events are larger than the amplitudes in older animals and almost as large as the amplitudes of Ca(2+) signals from backpropagating action potentials (bAPs). The rise time of the event signal is fast at all ages and is comparable to the rise time of the bAP fluorescence signal at the same dendritic location. The decay time is slower in younger animals, primarily because of weaker Ca(2+) extrusion mechanisms at that age. Diffusion away from a brief localized source is the major determinant of decay at all ages. A simple computational model closely simulates these events with extrusion rate the only age dependent variable.

Understanding calcium waves and sparks in central neurons.

All cells use changes in intracellular calcium concentration ([Ca(2+)](i)) to regulate cell signalling events. In neurons, with their elaborate dendritic and axonal arborizations, there are clear examples of both localized and widespread Ca(2+) signals. [Ca(2+)](i) changes that are generated by Ca(2+) entry through voltage- and ligand-gated channels are the best characterized. In addition, the release of Ca(2+) from intracellular stores can result in increased [Ca(2+)](i); the signals that trigger this release have been less well-studied, in part because they are not usually associated with specific changes in membrane potential. However, recent experiments have revealed dramatic widespread Ca(2+) waves and localized spark-like events, particularly in dendrites. Here we review emerging data on the nature of these signals and their functions.

Synaptically activated Ca2+ waves and NMDA spikes locally suppress voltage-dependent Ca2+ signalling in rat pyramidal cell dendrites.

Postsynaptic [Ca(2+)](i) changes contribute to several kinds of plasticity in pyramidal neurons. We examined the effects of synaptically activated Ca(2+) waves and NMDA spikes on subsequent Ca(2+) signalling in CA1 pyramidal cell dendrites in hippocampal slices. Tetanic synaptic stimulation evoked a localized Ca(2+) wave in the primary apical dendrites. The [Ca(2+)](i) increase from a backpropagating action potential (bAP) or subthreshold depolarization was reduced if it was generated immediately after the wave. The suppression had a recovery time of 30-60 s. The suppression only occurred where the wave was generated and was not due to a change in bAP amplitude or shape. The suppression also could be generated by Ca(2+) waves evoked by uncaging IP(3), showing that other signalling pathways activated by the synaptic tetanus were not required. The suppression was proportional to the amplitude of the [Ca(2+)](i) change of the Ca(2+) wave and was not blocked by a spectrum of kinase or phosphatase inhibitors, consistent with suppression due to Ca(2+)-dependent inactivation of Ca(2+) channels. The waves also reduced the frequency and amplitude of spontaneous, localized Ca(2+) release events in the dendrites by a different mechanism, probably by depleting the stores at the site of wave generation. The same synaptic tetanus often evoked NMDA spike-mediated [Ca(2+)](i) increases in the oblique dendrites where Ca(2+) waves do not propagate. These NMDA spikes suppressed the [Ca(2+)](i) increase caused by bAPs in those regions. [Ca(2+)](i) increases by Ca(2+) entry through voltage-gated Ca(2+) channels also suppressed the [Ca(2+)](i) increases from subsequent bAPs in regions where the voltage-gated [Ca(2+)](i) increases were largest, showing that all ways of raising [Ca(2+)](i) could cause suppression.

Na+ imaging reveals little difference in action potential-evoked Na+ influx between axon and soma.

In cortical pyramidal neurons, the axon initial segment (AIS) is pivotal in synaptic integration. It has been asserted that this is because there is a high density of Na(+) channels in the AIS. However, we found that action potential-associated Na(+) flux, as measured by high-speed fluorescence Na(+) imaging, was about threefold larger in the rat AIS than in the soma. Spike-evoked Na(+) flux in the AIS and the first node of Ranvier was similar and was eightfold lower in basal dendrites. At near-threshold voltages, persistent Na(+) conductance was almost entirely axonal. On a time scale of seconds, passive diffusion, and not pumping, was responsible for maintaining transmembrane Na(+) gradients in thin axons during high-frequency action potential firing. In computer simulations, these data were consistent with the known features of action potential generation in these neurons.

Dendritic Kv3.3 potassium channels in cerebellar purkinje cells regulate generation and spatial dynamics of dendritic Ca2+ spikes.

Purkinje cell dendrites are excitable structures with intrinsic and synaptic conductances contributing to the generation and propagation of electrical activity. Voltage-gated potassium channel subunit Kv3.3 is expressed in the distal dendrites of Purkinje cells. However, the functional relevance of this dendritic distribution is not understood. Moreover, mutations in Kv3.3 cause movement disorders in mice and cerebellar atrophy and ataxia in humans, emphasizing the importance of understanding the role of these channels. In this study, we explore functional implications of this dendritic channel expression and compare Purkinje cell dendritic excitability in wild-type and Kv3.3 knockout mice. We demonstrate enhanced excitability of Purkinje cell dendrites in Kv3.3 knockout mice, despite normal resting membrane properties. Combined data from local application pharmacology, voltage clamp analysis of ionic currents, and assessment of dendritic Ca(2+) spike threshold in Purkinje cells suggest a role for Kv3.3 channels in opposing Ca(2+) spike initiation. To study the physiological relevance of altered dendritic excitability, we measured [Ca(2+)](i) changes throughout the dendritic tree in response to climbing fiber activation. Ca(2+) signals were specifically enhanced in distal dendrites of Kv3.3 knockout Purkinje cells, suggesting a role for dendritic Kv3.3 channels in regulating propagation of electrical activity and Ca(2+) influx in distal dendrites. These findings characterize unique roles of Kv3.3 channels in dendrites, with implications for synaptic integration, plasticity, and human disease.

IP(3) mobilization and diffusion determine the timing window of Ca(2+) release by synaptic stimulation and a spike in rat CA1 pyramidal cells.

Synaptically activated calcium release from internal stores in CA1 pyramidal neurons is generated via metabotropic glutamate receptors by mobilizing IP(3). Ca(2+) release spreads as a large amplitude wave in a restricted region of the apical dendrites of these cells. These Ca(2+) waves have been shown to induce certain forms of synaptic potentiation and have been hypothesized to affect other forms of plasticity. Pairing a single backpropagating action potential (bAP) with repetitive synaptic stimulation evokes Ca(2+) release when synaptic stimulation alone is subthreshold for generating release. We examined the timing window for this synergistic effect under conditions favoring Ca(2+) release. The window, measured from the end of the train, lasted 250-500 ms depending on the duration of stimulation tetanus. The window appears to correspond to the time when both IP(3) concentration and [Ca(2+)](i) are elevated at the site of the IP(3) receptor. Detailed analysis of the mechanisms determining the duration of the window, including experiments using different forms of caged IP(3) instead of synaptic stimulation, suggest that the most significant processes are the time for IP(3) to diffuse away from the site of generation and the time course of IP(3) production initiated by activation of mGluRs. IP(3) breakdown, desensitization of the IP(3) receptor, and the kinetics of IP(3) unbinding from the receptor may affect the duration of the window but are less significant. The timing window is short but does not appear to be short enough to suggest that this form of coincidence detection contributes to conventional spike timing-dependent synaptic plasticity in these cells.

Synaptic activation and membrane potential changes modulate the frequency of spontaneous elementary Ca2+ release events in the dendrites of pyramidal neurons.

In most neurons postsynaptic [Ca(2+)](i) changes result from synaptic activation opening voltage gated channels, ligand gated channels, or mobilizing Ca(2+) release from intracellular stores. In addition to these changes that result directly from stimulation we found that in pyramidal cells there are spontaneous, rapid, Ca(2+) release events, predominantly, but not exclusively localized at dendritic branch points. They are clearest on the main apical dendrite but also have been detected in the finer branches and in the soma. Typically they have a spatial extent at initiation of approximately 2 microm, a rise time of <15 ms, duration <100 ms, and amplitudes of 10-70% of that generated by a backpropagating action potential at the same location. These events are not caused by background electrical or synaptic activity. However, their rate can be increased by repetitive synaptic stimulation at moderate frequencies, mainly through metabotropic glutamate receptor mobilization of IP(3). In addition, their frequency can be modulated by changes in membrane potential in the subthreshold range, predominantly by affecting Ca(2+) entry through L-type channels. They resemble the elementary events ("sparks" and "puffs") mediated by IP(3) receptors and ryanodine receptors that have been described primarily in non-neuronal preparations. These spontaneous Ca(2+) release events may be the fundamental units underlying some postsynaptic signaling cascades in mature neurons.

Dendritic properties of turtle pyramidal neurons.

The six-layered mammalian neocortex evolved from the three-layered paleocortex, which is retained in present-day reptiles such as the turtle. Thus the turtle offers an opportunity to examine which cellular and circuit properties are fundamental to cortical function. We characterized the dendritic properties of pyramidal neurons in different cortical regions of mature turtles, Pseudemys scripta elegans, using whole cell recordings and calcium imaging from the axon, soma, and dendrites in a slice preparation. The firing properties, in response to intrasomatic depolarization, resembled those previously recorded with sharp electrodes in this preparation. Somatic spikes led to active backpropagating high-amplitude dendritic action potentials and intracellular calcium ion concentration ([Ca2+]i) changes at all dendritic locations, suggesting that both backpropagation and dendritic voltage-gated Ca2+ channels are primitive traits. We found no indication that Ca2+ spikes could be evoked in the dendrites, but fast Na+ spikes could be initiated there following intradendritic stimulation. Several lines of evidence indicate that fast, smaller-amplitude somatic spikes ("prepotentials") that are easily recorded in this preparation are generated in the axon. Most synaptically activated [Ca2+]i changes resulted from Ca2+ entry through voltage-gated channels. In some cells synaptic stimulation evoked a delayed Ca2+ wave due to release from internal stores following activation of metabotropic glutamate receptors. With some small differences these properties resemble those of pyramidal neurons in mammalian species. We conclude that spike backpropagation, dendritic Ca2+ channels, and synaptically activated Ca2+ release are primitive and conserved features of cortical pyramidal cells, and therefore likely fundamental to cortical function.

Priming of intracellular calcium stores in rat CA1 pyramidal neurons.

Repetitive synaptic stimulation evokes large amplitude Ca(2+) release waves from internal stores in many kinds of pyramidal neurons. The waves result from mGluR mobilization of IP(3) leading to Ca(2+)-induced Ca(2+) release. In most experiments in slices, regenerative Ca(2+) release can be evoked for only a few trials. We examined the conditions required for consistent release from the internal stores in hippocampal CA1 pyramidal neurons. We found that priming with action potentials evoked at 0.5-1 Hz for intervals as short as 15 s were sufficient to fill the stores, while sustained subthreshold depolarization or subthreshold synaptic stimulation lasting from 15 s to 2 min was less effective. A single episode of priming was effective for about 2-3 min. Ca(2+) waves could also be evoked by uncaging IP(3) with a UV flash in the dendrites. Priming was necessary to evoke these waves repetitively; 7-10 spikes in 15 s were again effective for this protocol, indicating that priming acts to refill the stores and not at a site upstream to the production of IP(3). These results suggest that normal spiking activity of pyramidal neurons in vivo should be sufficient to maintain their internal stores in a primed state ready to release Ca(2+) in response to an appropriate physiological stimulus. This may be a novel form of synaptic plasticity where a cell's capacity to release Ca(2+) is modulated by its average firing frequency.

Modulation of calcium wave propagation in the dendrites and to the soma of rat hippocampal pyramidal neurons.

Repetitive synaptic stimulation in the stratum radiatum (SR) evokes large amplitude Ca2+ waves in the thick apical dendrites of hippocampal CA1 pyramidal neurons. These waves are initiated by activation of metabotropic glutamate receptors (mGluRs), which mobilize inositol-1,4,5-trisphospate (IP3) and release Ca2+ from intracellular stores. We explored mechanisms that modulate the spatial properties of these waves. Higher stimulus current evoked waves of increasing spatial extent. Most waves did not propagate through the soma; the majority stopped close to the junction of the soma and apical dendrite. Pairing strong stimulation with one electrode and subthreshold stimulation with another (associative activation) extended the waves distally but failed to extend waves into the cell body. Pairing synaptic stimulation with backpropagating action potentials enhanced the likelihood of wave generation but did not extend the waves to the somatic region. Priming the stores with Ca2+ entry through voltage dependent channels modulated wave properties but did not extend them past the dendrites. These results are consistent with propagation failing due to the dilution of synaptically generated IP3 as it diffuses into the large volume of the soma (impedance mismatch). Synaptically activating waves in the presence of low concentrations of carbachol, which probably increased the tonic level of IP3 throughout the cell, enhanced the extent of propagation and generated waves that invaded the soma, as long as low-affinity indicators were used to detect the [Ca2+]i changes. Consistent with this explanation direct injection of IP3 into the soma promoted wave propagation into this region. Ca2+ waves that propagated through the cell body were interesting because they did not fill the volume of the soma, but passed through the centre, often with large amplitude. These waves may be particularly effective in activating gene expression and protein synthesis.