Cal-590™, sodium salt

Glutamate imaging reveals several random release sites on CA3 giant algae fiber beads.

One of the most studied central synapses that has provided fundamental insights into the cellular mechanisms of nerve conduction is the “giant” excitatory connection between hippocampal mossy fibers (MF) and CA3 pyramidal cells. The large presynaptic box features multiple release sites and is densely packed with thousands of synaptic vesicles, to support very easy “explosive” transmission. , is still not well understood. This knowledge is essential for a better understanding of the mechanisms that underpin presynaptic plasticity and the rules for postsynaptic signal integrity. Here, we used the optical glutamate sensor SF-iGluSnFR and the intracellular Ca indicator Cal-590 to monitor spike-induced glutamate release and presynaptic calcium influx into MF boutons. Multiplexed images reveal that different sites on individual MF giant buds are highly likely to release glutamate, also showing short-term use-dependent facilitation. The current approach provides new insights into the underlying mechanisms of neurotransmitter delivery at excitatory synapses.

Quantitative glutamate release by multiplex imaging correlates with presynaptic CA2+ homeostasis at multiple synapses in situ.

Information processing by brain circuits depends on CA2+-dependent stochastic regulation of the excitatory neurotransmitter glutamate. While optical glutamate sensors have enabled the detection of synaptic discharges, understanding the presynaptic mechanism requires simultaneous reading of glutamate release and presynaptic Ca2+ nanostructures in situ. Here, we find that the fluorescence lifetime of the red kinetic Ca2+ tracer Cal-590 is sensitive to nanoscale Ca2+ and uses it in conjunction with green glutamate sensors to correlate quantitative neurotransmission with presynaptic Ca kinetics. Specific axonal circuitry reveal that the efficiency of glutamate release, but not its short-term plasticity, varies with time-dependent fluctuations in resting or resting presynaptic Ca2+. Within individual presynaptic granules, we found no nanocoupling of induced presynaptic Ca influx with the dominant glutamate release site, indicating weak coupling between the two. This approach allows for a better understanding of the editing mechanism at central synapses.

Calcium two-photon enhanced deep imaging in vivo.

Two-photon laser calcium imaging has emerged as a useful method for probing neuronal structure and function at the cellular and subcellular level in vivo. Applications range from imaging of subcellular parts such as dendrites, spines, and axonal plants to functional analysis of groups of neurons or macroglia. However, the depth of penetration is often limited to a few hundred micrometers, which corresponds, for example, to the upper cortical layers of the mouse brain. Light scattering and aberrations resulting from tissue refractive index inhomogeneity are the reasons for these limitations. The deep penetration of two-photon imaging can be enhanced by various methods, such as the implementation of adaptive optics, the use of three-photon excitation, and/or the labeling of cells with genetically encoded red fluorescent sensors. However, most of the methods used to date require the implementation of new devices and/or time-consuming staining protocols. Here we present a simple method that can be easily implemented in combination with standard two-photon microscopes. The method includes an improved depth-limited labeling protocol that uses the red-shifted fluorescent calcium label Cal-590 and leverages the use of ultrashort laser pulses. This in vivo approach allows functional imaging of neuronal populations at single-cell resolution in all six layers of the mouse cortex. We have shown that stable recordings in deep cortical layers are not limited to anesthetized animals, but are highly feasible in awake and intact mice. We anticipate that the enhanced depth penetration will be beneficial for two-photon functional imaging and

In vivo two-photon deep imaging of neuronal circuitry using the fluorescent tracer Ca2+ Cal-590.

In vivo, two-photon Ca2+ imaging has become an effective approach for the functional analysis of neuronal populations, individual neurons, and subcellular neuronal compartments in the healthy brain. When imaging single-labeled neurons, the depth of penetration can often reach 1 mm below the cortical surface. However, for densely labeled neuronal populations, imaging at single cell resolution is largely limited to the upper cortical layers of the mouse brain. Here, we review recent two-photon deep Ca2+ imaging developments and the use of redshifted fluorescent Ca2+ tracers as a promising strategy for augmenting depth imaging, taking advantage of reduced photon scattering at long wavelengths of excitation and emission. We describe results demonstrating that the newly introduced Ca2+-sensitive dye can be used to record neuronal activity in vivo in isolated cortical neurons and in neurons within populations at depths up to 900 μm below the Bial surface. Therefore, the new approach allows a comprehensive functional mapping of all six cortical layers of the mouse brain. Specific features of Cal-590-based two-photon imaging include good signal-to-noise ratio, fast kinetics, and linear dependence of Ca2+ transients on the number of action potentials. Another field of application is obtaining dichromatic images by combining Cal-590 with other shorter wavelength Ca2+ indicators, such as OGB-1. Overall, Cal-590-based two-photon microscopy appears to be a promising tool for recording neuronal activity at previously inaccessible depths for functional imaging of neural circuitry.

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Two-photon deep brain imaging with variable fluorometric index red Ca2+.

In vivo, imaging of neuronal assemblies with Ca2+ in deep cortical layers remains a major challenge, as the depth of microscopic photon recording is limited by photon scattering and absorption in brain tissue. One possible strategy to increase the depth of the image is to use redshift fluorescent dyes, in which the scattering of photons at long wavelengths is reduced. Here, we tested the red mutant fluorescent Ca2+ reporter Cal-590 for deep tissue experiments in the mouse cortex in vivo. In experiments involving massive loading of neurons with Cal-590 acetoxymethyl ester (AM) transcription, cell-connected, two-photon composite recordings revealed that, despite the relatively low affinity of Cal-590 for Ca2+ ( Kd = 561 nm), it was possible to discern a transient Ca2+ single-action potential in most neurons with a good signal-to-noise ratio. Voltage-dependent Ca2+ transients were recorded in neurons of all six cortical layers at depths down to -900 μm below the cortical surface. We show that Cal-590 is also suitable for multicolor functional imaging experiments in conjunction with other Ca indices. Ca2+ transients in single neuron dendrites labeled with 1,2-bis(o-aminophenoxy)ethane-N,N,N ‘,N’-tetraacetic acid-1 (OGB-1) from Oregon green and the surrounding Cal population were record -590 cells simultaneously labeled in two spectrally separate channels. We conclude that the red mutant Ca indicator Cal-590 is well suited for in vivo two-photon Ca2+ imaging experiments in all layers of the mouse cortex. In combination with spectrally different Ca indices such as OGB-1, Cal-590 can be easily used in simultaneous multi-color functional imaging experiments.

Interaction between light and heat in the bleaching of rhodopsin.

Rhodopsin, the pigment in retinal rods, can be bleached by light or high temperatures. Previous work showed that when white light is used, the bleaching rate is not dependent on temperature and therefore must be independent of the internal energy of the molecule.

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