Construction of a two-photon microscope for video-rate Ca(2+) imaging.

We describe the construction of a video-rate two-photon laser scanning microscope, compare its performance to a similar confocal microscope, and illustrate its use for imaging local Ca(2+) transients from cortical neurons in brain slices. Key features include the use of a Ti-sapphire femtosecond laser allowing continuous tuning over a wide (700-1000 nm) wavelength range, a resonant scanning mirror to permit frame acquisition at 30 Hz, and efficient wide-field fluorescence detection. Two-photon imaging provides compelling advantages over confocal microscopy in terms of improved imaging depth and reduced phototoxicity and photobleaching, but the high cost of commercial instruments has limited their widespread adoption. By constructing one's own system the expense is greatly reduced without sacrifice of performance, and the microscope can be more readily tailored to specific applications.

Subcellular mechanisms of presenilin-mediated enhancement of calcium signaling.

Mutations in presenilin-1 (PS1), the leading cause of early-onset, autosomal-dominant familial Alzheimer's disease (FAD), enhance calcium signaling mediated by inositol 1,4,5-trisphosphate (IP3). To elucidate the subcellular mechanisms underlying this enhancement, we used high resolution line-scanning confocal microscopy to image elementary calcium release events ("puffs") in Xenopus oocytes expressing wild-type or mutant PS1. Here we report that mutant PS1-rendered puffs more sensitive to IP3 and increased both the magnitude and the rate of calcium release during each event. These effects were not attributable to quantitative changes in the levels of IP3 receptors or their distribution on the ER, but were instead associated with an abnormal elevation of ER calcium stores. Together, our results suggest that the effects of mutant PS1 on calcium signaling are manifested predominantly at the level of the regulation of calcium stores rather than via perturbations in the numbers or activity of IP3-activated calcium release channels.

Phasic characteristic of elementary Ca(2+) release sites underlies quantal responses to IP(3).

Ca(2+) liberation by inositol 1,4,5-trisphosphate (IP(3)) is 'quantal', in that low [IP(3)] causes only partial Ca(2+) release, but further increasing [IP(3)] evokes more release. This characteristic allows cells to generate graded Ca(2+) signals, but is unexpected, given the regenerative nature of Ca(2+)-induced Ca(2+) release through IP(3) receptors. Two models have been proposed to resolve this paradox: (i) all-or-none Ca(2+) release from heterogeneous stores that empty at varying [IP(3)]; and (ii) phasic liberation from homogeneously sensitive stores. To discriminate between these hypotheses, we imaged subcellular Ca(2+) puffs evoked by IP(3) in Xenopus oocytes where release sites were functionally uncoupled using EGTA. Puffs were little changed by 300 microM intracellular EGTA, but sites operated autonomously and did not propagate waves. Photoreleased IP(3) generated flurries of puffs-different to the prolonged Ca(2+) elevation following waves in control cells-and individual sites responded repeatedly to successive increments of [IP(3)]. These data support the second hypothesis while refuting the first, and suggest that local Ca(2+) signals exhibit rapid adaptation, different to the slower inhibition following global Ca(2+) waves.

Ca(2+)-dependent activation of Cl(-) currents in Xenopus oocytes is modulated by voltage.

Ca(2+)-activated Cl(-) currents (I(Cl,Ca)) were examined using fluorescence confocal microscopy to monitor intracellular Ca(2+) liberation evoked by flash photolysis of caged inositol 1,4, 5-trisphosphate (InsP(3)) in voltage-clamped Xenopus oocytes. Currents at +40 mV exhibited a steep dependence on InsP(3) concentration ([InsP(3)]), whereas currents at -140 mV exhibited a higher threshold and more graded relationship with [InsP(3)]. Ca(2+) levels required to half-maximally activate I(Cl,Ca) were about 50% larger at -140 mV than at +40 mV, and currents evoked by small Ca(2+) elevations were reduced >25-fold. The half-decay time of Ca(2+) signals shortened at increasingly positive potentials, whereas the decay of I(Cl,Ca) lengthened. The steady-state current-voltage (I-V) relationship for I(Cl,Ca) exhibited outward rectification with weak photolysis flashes but became more linear with stronger stimuli. Instantaneous I-V relationships were linear with both strong and weak stimuli. Current relaxations following voltage steps during activation of I(Cl,Ca) decayed with half-times that shortened from about 100 ms at +10 mV to 20 ms at -160 mV. We conclude that InsP(3)-mediated Ca(2+) liberation activates a single population of Cl(-) channels, which exhibit voltage-dependent Ca(2+) activation and voltage-independent instantaneous conductance.

Initiation of IP(3)-mediated Ca(2+) waves in Xenopus oocytes.

Inositol (1,4,5)-trisphosphate (IP(3)) evokes Ca(2+) liberation in Xenopus oocytes as elementary events (Ca(2+) puffs) that become coupled to propagate Ca(2+) waves with increasing [IP(3)]. To investigate this transition between local and global Ca(2+) signaling, we developed an optical method for evoking rapid subcellular Ca(2+) elevations, while independently photoreleasing IP(3) and simultaneously recording confocal Ca(2+) images. Focal Ca(2+) elevations triggered waves within 100 ms of photoreleasing IP(3), compared with latencies of seconds following photorelease of IP(3) alone. Wave velocity varied with [IP(3)] but was independent of time after photorelease of IP(3), indicating that delayed wave initiation did not involve slow binding of IP(3) to its receptors. The amount of Ca(2+) required to trigger a wave was approximately 10-fold greater than the average size of puffs, and puffs showed no progressive increase in magnitude before waves initiated. Instead, Ca(2+) puffs contributed to a slow rise in basal free [Ca(2+)], which further increased puff frequency and sensitized IP(3) receptors so that individual events then triggered waves. Because the wave threshold is much greater than the size of the elementary puff, cells can employ both local and global signaling mechanisms, and the summation of stochastic behavior of elementary events allows generation of reproducible periodic waves.

Radial localization of inositol 1,4,5-trisphosphate-sensitive Ca2+ release sites in Xenopus oocytes resolved by axial confocal linescan imaging.

The radial localization and properties of elementary calcium release events ("puffs") were studied in Xenopus oocytes using a confocal microscope equipped with a piezoelectric focussing unit to allow rapid (>100 Hz) imaging of calcium signals along a radial line into the cell with a spatial resolution of <0.7 micrometer. Weak photorelease of caged inositol 1,4,5-trisphosphate (InsP3) evoked puffs arising predominantly within a 6-micrometer thick band located within a few micrometers of the cell surface. Approximately 25% of puffs had a restricted radial spread, consistent with calcium release from a single site. Most puffs, however, exhibited a greater radial spread (3.25 micrometer), likely involving recruitment of radially neighboring release sites. Calcium waves evoked by just suprathreshold stimuli exhibited radial calcium distributions consistent with inward diffusion of calcium liberated at puff sites, whereas stronger flashes evoked strong, short-latency signals at depths inward from puff sites, indicating deep InsP3-sensitive stores activated at higher concentrations of InsP3. Immunolocalization of InsP3 receptors showed punctate staining throughout a region corresponding to the localization of puffs and subplasmalemmal endoplasmic reticulum. The radial organization of puff sites a few micrometers inward from the plasma membrane may have important consequences for activation of calcium-dependent ion channels and "capacitative" calcium influx. However, on the macroscopic (hundreds of micrometers) scale of global calcium waves, release can be considered to occur primarily within a thin, essentially two-dimensional subplasmalemmal shell.

Construction of a confocal microscope for real-time x-y and x-z imaging.

We describe the construction of a simple 'real-time' laser-scanning confocal microscope, and illustrate its use for rapid imaging of elementary intracellular calcium signaling events. A resonant scanning galvanometer (8 kHz) allows x-y frame acquisition rates of 15 or 30 Hz, and the use of mirrors to scan the laser beam permits use of true, pin-hole confocal detection to provide diffraction-limited spatial resolution. Furthermore, use of a piezoelectric device to rapidly focus the objective lens allows axial (x-z) images to be obtained from thick specimens at similar frame rates. A computer with image acquisition and graphics cards converts the output from the microscope to a standard video signal, which can then be recorded on videotape and analyzed by regular image processing systems. The system is largely made from commercially available components and requires little custom construction of mechanical parts or electronic circuitry. It costs only a small fraction of that of comparable commercial instruments, yet offers greater versatility and similar or better performance.

Hemispheric asymmetry of macroscopic and elementary calcium signals mediated by InsP3 in Xenopus oocytes.

1. The mechanisms underlying hemispheric asymmetry of the inositol 1, 4,5-trisphosphate (InsP3)-calcium signalling pathway in Xenopus oocytes were examined by fluorescence imaging of calcium signals and recording calcium-activated Cl- currents (ICl,Ca) evoked by intracellular calcium injections and photorelease of InsP3. 2. The maximal ICl,Ca evoked by strong photorelease of InsP3 was 8 times greater in the animal than the vegetal hemisphere, but the average threshold amounts of InsP3 required to evoke detectable currents were similar in each hemisphere. 3. Currents evoked by injections of calcium were about 2.5 times greater near the animal pole than near the vegetal pole, whereas fluorescence signals evoked by injections were similar in each hemisphere. 4. Calcium waves were evoked by photolysis flashes of similar strengths in both hemispheres of albino oocytes, but peak calcium levels evoked by supramaximal stimuli were 70 % greater in the animal hemisphere. 5. Elementary calcium release events (puffs) in the animal hemisphere had amplitudes about double that in the vegetal hemisphere, and more often involved coupled release from adjacent sites. Calcium release sites were more closely packed in the animal hemisphere, with a mean spacing of about 1.5 micro m compared with 2.25 micro m in the vegetal hemisphere. 6. The larger amplitude of currents mediated by InsP3 in the animal hemisphere, therefore, involves an increased flux of calcium at individual release units, a more dense packing of release units and a higher density of Cl- channels.

A continuum of InsP3-mediated elementary Ca2+ signalling events in Xenopus oocytes.

1. The elementary release events underlying inositol 1,4, 5-trisphosphate (InsP3)-mediated calcium signalling were investigated in Xenopus oocytes by means of high-resolution confocal linescan imaging together with flash photolysis of caged InsP3. 2. Weak photolysis flashes evoked localized, transient calcium signals that arose at specific sites following random latencies of up to several seconds. The duration, spatial spread and amplitude of these elementary events varied widely. Event durations (at half-maximal amplitude) were distributed exponentially between about 100 and 600 ms. Fluorescence magnitudes (F/F0 of Oregon Green 488 BAPTA-1) showed a skewed distribution with a peak at about 1.5 and a tail extending as high as 3.5. 3. Individual release sites exhibited both small events (blips) and large events (puffs). The spatiotemporal distribution of calcium signals during puffs was consistent with calcium diffusion from a point source (< a few hundred nanometres), rather than with propagation of a microscopic calcium wave. 4. Estimates of the calcium flux associated with individual events were made by integrating fluorescence profiles along the scan line in three dimensions to derive the 'signal mass' at each time point. The smallest resolved events corresponded to liberation of < 2 x 10-20 mol Ca2+, and large events to about 2 x 10-18 mol Ca2+. The rise of signal mass was more prolonged than that of the fluorescence intensity, suggesting that calcium liberation persists even while the fluorescence begins to decline. Rates of rise of signal mass corresponded to Ca2+ currents of 0.4-2.5 pA. 5. Measurements of signal mass from different events showed a continuous, exponential distribution, arising through variability in magnitude and duration of calcium flux. 6. We conclude that localized calcium transients in the oocyte represent a continuum of events involving widely varying amounts of calcium liberation, rather than falling into separate populations of 'fundamental' and 'elementary' events (blips and puffs) involving, respectively, single and multiple InsP3 receptor channels. This variability probably arises through stochastic variation in both the number of channels recruited and the duration of channel opening.

Activation and co-ordination of InsP3-mediated elementary Ca2+ events during global Ca2+ signals in Xenopus oocytes.

1. The activation of elementary calcium release events ('puffs') and their co-ordination to generate calcium waves was studied in Xenopus oocytes by confocal linescan imaging together with photorelease of inositol 1,4,5-trisphosphate (InsP3) from a caged precursor. 2. Weak photolysis flashes evoked no responses or isolated calcium puffs, whereas flashes of increasing strength evoked more frequent puffs, often occurring in flurries as abortive waves, and then a near-simultaneous calcium liberation originating at multiple sites. The numbers of sites activated increased initially as about the fourth power of photoreleased [InsP3]. 3. Following repeated, identical photolysis flashes, puffs arose after stochastically varying latencies of a few hundred milliseconds to several seconds. The cumulative number of events initially increased as about the third power of time. No rise in free [Ca2+] was detected preceding the puffs, suggesting that this co-operativity arises through binding of multiple InsP3 molecules, rather than through calcium feedback. 4. The mean latency to onset of calcium liberation shortened as about the square of the flash strength, and the dispersion in latencies between events reduced correspondingly. 5. Weak stimuli often evoked coupled puffs involving adjacent sites, and stronger flashes evoked saltatory calcium waves, propagating with non-constant velocity. During waves, [Ca2+] rose slowly between puff sites, but more abruptly at active sites following an initial diffusive rise in calcium. 6. Initial rates of rise of local [Ca2+] at release sites were similar during puffs and release induced by much (> 10-fold) greater [InsP3]. In contrast, macroscopic calcium measurements averaged over the scan line showed a graded dependence of rate of calcium liberation upon [InsP3], due to recruitment of additional sites and decreasing dispersion in activation latencies. 7. We conclude that the initiation of calcium liberation depends co-operatively upon [InsP3] whereas the subsequent regenerative increase in calcium flux depends upon local calcium feedback and is largely independent of [InsP3]. Wave propagation is consistent with the diffusive spread of calcium evoking regenerative liberation at heterogeneous discrete sites, the sensitivity of which is primed by InsP3.

A high-resolution, confocal laser-scanning microscope and flash photolysis system for physiological studies.

We describe the construction of a high-resolution confocal laser-scanning microscope, and illustrate its use for studying elementary Ca2+ signalling events in cells. An avalanche photodiode module and simple optical path provide a high efficiency system for detection of fluorescence signals, allowing use of a small confocal aperture giving near diffraction-limited spatial resolution (< 300 nm lateral and < 400 nm axial). When operated in line-scan mode, the maximum temporal resolution is 1 ms, and the associated computer software allows complete flexibility to record line-scans continuously for long (minutes) periods or to obtain any desired pixel resolution in x-y scans. An independent UV irradiation system permits simultaneous photolysis of caged compounds over either a uniform, wide field (arc lamp source) or at a tightly focussed spot (frequency-tripled Nd:YAG laser). The microscope thus provides a versatile tool for optical studies of dynamic cellular processes, as well as excellent resolution for morphological studies. The confocal scanner can be added to virtually any inverted microscope for a component cost that is only a small fraction of that of comparable commercial instruments, yet offers better performance and greater versatility.

Inositol 1,4,5-trisphosphate receptors in Xenopus laevis oocytes: localization and modulation by Ca2+.

Inositol 1,4,5-trisphosphate receptors (InsP3R) in Xenopus laevis oocytes were localized and their regulation by Ca2+ was investigated. Antibodies raised against the C-terminal region of the mouse cerebellar InsP3R (cAb) cross-reacted with a 255 kD protein in Western blots of Xenopus microsomal membranes. Immunolocalization of this protein in cryosections of oocytes revealed diffuse staining of the cytoplasm, intense staining of the sub-plasma membrane region of the animal hemisphere, and punctate staining in association with the germinal vesicle. In the presence of 40 microM free Ca2+, isolated oocyte membranes exhibited a high affinity binding site for Ins 1,4,5-P3 (KD = 5nM) and a binding capacity of 450 fmol/mg protein. The specific binding capacity of oocyte membranes for [3H]-Ins 1,4,5-P3 increased as the level of free Ca2+ present in binding assays was raised from < 0.1 nM to 4.0 microM, with an apparent EC50 of 60 nM. Increasing the concentration of free Ba2+ failed to facilitate [3H]-Ins1,4,5-P3 binding. Other inositol phosphates competed for Ins1,4,5-P3 binding sites with approximate IC50 values of: Ins1,3,4,5-P4 = 79 nM, Ins2,4,5-P3 = 455 nM and L-Ins1,4,5-P3 = 20 microM. In addition, 150 micrograms/ml (approximately 12 microM) heparin displaced 50% of bound [3H]-Ins1,4,5-P3, whereas caffeine (10 mM) had little effect. Functional reconstitution of solubilized InsP3Rs into lipid bilayers revealed that Ca2+ was a necessary co-agonist for activation of the InsP3R. When InsP3 (5 microM) and Ca2+ (5 microM) were applied together, conductance steps were observed. InsP3 or Ca2+ alone had little effect. These results suggest that the subcellular organization of InsP3Rs and the facilitation of InsP3 binding and channel opening by Ca2+ contribute to the Ins1,4,5-P3-mediated Ca2+ spikes, waves, and oscillations observed in Xenopus oocytes.