Ca(2+) signaling in dendritic spines.

Dendritic spines are cellular microcompartments that are isolated from their parent dendrites and neighboring spines. Recently, imaging studies of spine Ca(2+) dynamics have revealed that Ca(2+) can enter spines through voltage-sensitive and ligand-activated channels, as well as through Ca(2+) release from intracellular stores. Relationships between spine Ca(2+) signals and induction of various forms of synaptic plasticity are beginning to be elucidated. Measurements of spine Ca(2+) concentration are also being used to probe the properties of single synapses and even individual calcium channels in their native environment.

Analysis of calcium channels in single spines using optical fluctuation analysis.

Most synapses form on small, specialized postsynaptic structures known as dendritic spines. The influx of Ca2+ ions into such spines--through synaptic receptors and voltage-sensitive Ca2+ channels (VSCCs)--triggers diverse processes that underlie synaptic plasticity. Using two-photon laser scanning microscopy, we imaged action-potential-induced transient changes in Ca2+ concentration in spines and dendrites of CA1 pyramidal neurons in rat hippocampal slices. Through analysis of the large trial-to-trial fluctuations in these transients, we have determined the number and properties of VSCCs in single spines. Here we report that each spine contains 1-20 VSCCs, and that this number increases with spine volume. We are able to detect the opening of a single VSCC on a spine. In spines located on the proximal dendritic tree, VSCCs normally open with high probability (approximately 0.5) following dendritic action potentials. Activation of GABA(B) receptors reduced this probability in apical spines to approximately 0.3 but had no effect on VSCCs in dendrites or basal spines. Our studies show that the spatial distribution of VSCC subtypes and their modulatory potential is regulated with submicrometre precision.

Estimating intracellular calcium concentrations and buffering without wavelength ratioing.

We describe a method for determining intracellular free calcium concentration ([Ca(2+)]) from single-wavelength fluorescence signals. In contrast to previous single-wavelength calibration methods, the proposed method does not require independent estimates of resting [Ca(2+)] but relies on the measurement of fluorescence close to indicator saturation during an experiment. Consequently, it is well suited to [Ca(2+)] indicators for which saturation can be achieved under physiological conditions. In addition, the method requires that the indicators have large dynamic ranges. Popular indicators such as Calcium Green-1 or Fluo-3 fulfill these conditions. As a test of the method, we measured [Ca(2+)] in CA1 pyramidal neurons in rat hippocampal slices using Oregon Green BAPTA-1 and 2-photon laser scanning microscopy (BAPTA: 1,2-bis(2-aminophenoxy)ethane-N,N,N', N'-tetraacetic acid). Resting [Ca(2+)] was 32-59 nM in the proximal apical dendrite. Monitoring action potential-evoked [Ca(2+)] transients as a function of indicator loading yielded estimates of endogenous buffering capacity (44-80) and peak [Ca(2+)] changes at zero added buffer (178-312 nM). In young animals (postnatal days 14-17) our results were comparable to previous estimates obtained by ratiometric methods (, Biophys. J. 70:1069-1081), and no significant differences were seen in older animals (P24-28). We expect our method to be widely applicable to measurements of [Ca(2+)] and [Ca(2+)]-dependent processes in small neuronal compartments, particularly in the many situations that do not permit wavelength ratio imaging.

Timing of synaptic transmission.

Many behaviors require rapid and precisely timed synaptic transmission. These include the determination of a sound's direction by detecting small interaural time differences and visual processing, which relies on synchronous activation of large populations of neurons. In addition, throughout the brain, concerted firing is required by Hebbian learning mechanisms, and local circuits are recruited rapidly by fast synaptic transmission. To achieve speed and precision, synapses must optimize the many steps between the firing of a presynaptic cell and the response of its postsynaptic targets. Until recently, the behavior of mammalian synapses at physiological temperatures was primarily extrapolated from studies at room temperature or from the properties of invertebrate synapses. Recent studies have revealed some of the specializations that make synapses fast and precise in the mammalian central nervous system at physiological temperatures.

Optical measurement of presynaptic calcium currents.

Measurements of presynaptic calcium currents are vital to understanding the control of transmitter release. However, most presynaptic boutons in the vertebrate central nervous system are too small to allow electrical recordings of presynaptic calcium currents (I(Ca)pre). We therefore tested the possibility of measuring I(Ca)pre optically in boutons loaded with calcium-sensitive fluorophores. From a theoretical treatment of a system containing an endogenous buffer and an indicator, we determined the conditions necessary for the derivative of the stimulus-evoked change in indicator fluorescence to report I(Ca)pre accurately. Matching the calcium dissociation rates of the endogenous buffer and indicator allows the most precise optical measurements of I(Ca)pre. We tested our ability to measure I(Ca)pre in granule cells in rat cerebellar slices. The derivatives of stimulus-evoked fluorescence transients from slices loaded with the low-affinity calcium indicators magnesium green and mag-fura-5 had the same time courses and were unaffected by changes in calcium influx or indicator concentration. Thus both of these indicators were well suited to measuring I(Ca)pre. In contrast, the high-affinity indicator fura-2 distorted I(Ca)pre. The optically determined I(Ca)pre was well approximated by a Gaussian with a half-width of 650 micros at 24 degrees C and 340 micros at 34 degrees C.

Control of neurotransmitter release by presynaptic waveform at the granule cell to Purkinje cell synapse.

The effect of changes in the shape of the presynaptic action potential on neurotransmission was examined at synapses between granule and Purkinje cells in slices from the rat cerebellum. Low concentrations of tetraethylammonium were used to broaden the presynaptic action potential. The presynaptic waveform was monitored with voltage-sensitive dyes, the time course and amplitude of presynaptic calcium entry were determined with fluorescent calcium indicators, and EPSCs were measured with a whole-cell voltage clamp. Spike broadening increased calcium influx primarily by prolonging calcium entry without greatly affecting peak presynaptic calcium currents, indicating that the majority of calcium channels reach maximal probability of opening in response to a single action potential and that spike broadening increases the open time of these channels. EPSCs were exquisitely sensitive to elevations of calcium influx produced by spike broadening; there was a high power relationship between calcium influx and release such that a 23% increase in spike width led to a 25% increase in total calcium influx, which in turn doubled synaptic strength. The finding that even small changes in spike width influence neurotransmitter release suggests that altering the presynaptic waveform may be an important means of modifying the strength of this synapse. Waveform changes do not, however, contribute significantly to presynaptic modulation via activation of adenosine A1 or GABAB receptors. Furthermore, greatly reducing presynaptic calcium influx did not alter the presynaptic waveform, indicating that calcium channels and calcium-activated channels do not participate in shaping the presynaptic waveform.

Timing of neurotransmission at fast synapses in the mammalian brain.

Understanding the factors controlling synaptic delays has broad implications. On a systems level, the speed of synaptic transmission limits the communication rate between neurons and strongly influences local circuit dynamics. On a molecular level, the delay from presynaptic calcium entry to postsynaptic responses constrains the molecular mechanism of vesicle fusion. Previously it has not been possible to elucidate the determinants of synaptic delays in the mammalian central nervous system, where presynaptic terminals are small and difficult to study. We have developed a new approach to study timing at rat cerebellar synapses: we used optical techniques to measure voltage and calcium current simultaneously from presynaptic boutons while monitoring postsynaptic currents electrically. Here we report that the classic view that vesicle release is driven by calcium entry during action-potential repolarization holds for these synapses at room temperature, but not at physiological temperatures, where postsynaptic responses commence just 150 micros after the start of the presynaptic action potential. This brisk communication is a consequence of rapid calcium-channel kinetics, which allow significant calcium entry during the upstroke of the presynaptic action potential, and extremely fast calcium-driven vesicle fusion, which lags behind calcium influx by 60 micros.

Detecting changes in calcium influx which contribute to synaptic modulation in mammalian brain slice.

The control of neurotransmitter release by modulation of presynaptic calcium influx was investigated at the granule cell to Purkinje cell synapse in rat cerebellar slices. Excitatory post-synaptic currents were measured using whole cell voltage clamp, and changes in presynaptic Ca influx were determined with the Ca-sensitive dye mag-fura-5. Single stimuli of the parallel fibers evoked rapid changes in mag-fura-5 fluorescence which increased from 10 to 90% in 1.4 msec, and then decayed within hundreds of milliseconds to prestimulus levels. These fluorescence changes were unaffected by disruption of internal stores with ryanodine or thapsigargin, and were reduced by 79% by the calcium channel toxin omega-conotoxin-MVIIC. We conclude that these signals result from calcium entry into presynaptic terminals through voltage gated calcium channels opened by action potentials. These fluorescence signals allow us to quantitate changes in calcium influx. We used this approach to study the enhancement of stimulus-evoked synaptic currents by 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor and antagonist of adenosine receptors. Both enhancement of calcium influx into presynaptic terminals, and reduction in the firing threshold of the parallel fibers, were found to contribute to IBMX-mediated synaptic enhancement. Changes in presynaptic calcium influx were also quantified with a novel method, which is unaffected by changes in fiber threshold. These studies illustrate some of the difficulties encountered when determining the factors responsible for synaptic enhancement and demonstrate how measurements of presynaptic calcium influx can contribute to our understanding of synaptic modulation. The approach described here promises to be widely useful in elucidating the role of calcium influx in the modulation of synapses in brain slice.

Calcium control of transmitter release at a cerebellar synapse.

The manner in which presynaptic Ca2+ influx controls the release of neurotransmitter was investigated at the granule cell to Purkinje cell synapse in rat cerebellar slices. Excitatory postsynaptic currents were measured using whole-cell voltage clamp, and changes in presynaptic Ca2+ influx were determined with the Ca(2+)-sensitive dye furaptra. We manipulated presynaptic Ca2+ entry by altering external Ca2+ levels and by blocking Ca2+ channels with Cd2+ or with the toxins omega-conotoxin GVIA and omega-Aga-IVA. For all of the manipulations, other than the application of omega-Aga-IVA, the relationship between Ca2+ influx and release was well approximated by a power law, n approximately 2.5. When omega-Aga-IVA was applied, release appeared to be more steeply dependent on Ca2+ (n approximately 4), suggesting that omega-Aga-IVA-sensitive channels are more effective at triggering release. Based on interactive effects of toxins on synaptic currents, we conclude that multiple types of Ca2+ channels synergistically control individual release sites.