Comparing confocal and two-photon Ca2+ imaging of thin low-scattering preparations.

Ca2+ imaging provides insight into biological processes ranging from subcellular dynamics to neural network activity. Two-photon microscopy has assumed a dominant role in Ca2+ imaging. The longer wavelength infra-red illumination undergoes less scattering, and absorption is confined to the focal plane. Two-photon imaging can thus penetrate thick tissue ∼10-fold more deeply than single-photon visible imaging to make two-photon microscopy an exceptionally powerful method for probing function in intact brain. However, two-photon excitation produces photobleaching and photodamage that increase very steeply with light intensity, limiting how strongly one can illuminate. In thin samples, illumination intensity can assume a dominant role in determining signal quality, raising the possibility that single-photon microscopy may be preferable. We therefore tested laser scanning single-photon and two-photon microscopy side by side with Ca2+ imaging in neuronal compartments at the surface of a brain slice. We optimized illumination intensity for each light source to obtain the brightest signal without photobleaching. Intracellular Ca2+ rises elicited by one action potential had twice the signal/noise ratio with confocal as with two-photon imaging in axons, were 31% higher in dendrites, and about the same in cell bodies. The superior performance of confocal imaging in finer neuronal processes likely reflects the dominance of shot noise when fluorescence is dim. Thus, when out-of-focus absorption and scattering are not issues, single-photon confocal imaging can yield better quality signals than two-photon microscopy.

Direct synaptic excitation between hilar mossy cells revealed with a targeted voltage sensor.

The dentate gyrus not only gates the flow of information into the hippocampus, it also integrates and processes this information. Mossy cells (MCs) are a major type of excitatory neuron strategically located in the hilus of the dentate gyrus where they can contribute to this processing through networks of synapses with inhibitory neurons and dentate granule cells. Some prior work has suggested that MCs can form excitatory synapses with other MCs, but the role of these synapses in the network activity of the dentate gyrus has received little attention. Here, we investigated synaptic inputs to MCs in mouse hippocampal slices using a genetically encoded hybrid voltage sensor (hVOS) targeted to MCs by Cre-lox technology. This enabled optical recording of voltage changes from multiple MCs simultaneously. Stimulating granule cells and CA3 pyramidal cells activated well-established inputs to MCs and elicited synaptic responses as expected. However, the weak blockade of MC responses to granule cell layer stimulation by DCG-IV raised the possibility of another source of excitation. To evaluate synapses between MCs as this source, single MCs were stimulated focally. Stimulation of one MC above its action potential threshold evoked depolarizing responses in neighboring MCs that depended on glutamate receptors. Short latency responses of MCs to other MCs did not depend on release from granule cell axons. However, granule cells did contribute to the longer latency responses of MCs to stimulation of other MCs. Thus, MCs transmit their activity to other MCs both through direct synaptic coupling and through polysynaptic coupling with dentate granule cells. MC-MC synapses can redistribute information entering the dentate gyrus and thus shape and modulate the electrical activity underlying hippocampal functions such as navigation and memory, as well as excessive excitation during seizures.

An Inconvenient Truth: Calcium Sensors Are Calcium Buffers.

Recent advances in Ca2+ imaging have given neuroscientists a tool to follow the activity of large numbers of individual neurons simultaneously in vivo in the brains of animals as they are presented with sensory stimulation, respond to environmental challenges, and engage in behaviors. The Ca2+ sensors used to transduce changes in cellular Ca2+ into changes in fluorescence must bind Ca2+ to produce a signal. By binding Ca2+, these sensors can act as buffers, often reducing the magnitude of a Ca2+ change severalfold, and producing a proportional slowing of the rates of change. Ca2+ probes can thus distort the patterns of activity they are intended to study and modify ongoing Ca2+ signaling functions. Recognizing these factors will enhance the use of in vivo Ca2+ imaging in the investigation of neural circuit function.

Multiple cytosolic calcium buffers in posterior pituitary nerve terminals.

Cytosolic Ca(2+) buffers bind to a large fraction of Ca(2+) as it enters a cell, shaping Ca(2+) signals both spatially and temporally. In this way, cytosolic Ca(2+) buffers regulate excitation-secretion coupling and short-term plasticity of release. The posterior pituitary is composed of peptidergic nerve terminals, which release oxytocin and vasopressin in response to Ca(2+) entry. Secretion of these hormones exhibits a complex dependence on the frequency and pattern of electrical activity, and the role of cytosolic Ca(2+) buffers in controlling pituitary Ca(2+) signaling is poorly understood. Here, cytosolic Ca(2+) buffers were studied with two-photon imaging in patch-clamped nerve terminals of the rat posterior pituitary. Fluorescence of the Ca(2+) indicator fluo-8 revealed stepwise increases in free Ca(2+) after a series of brief depolarizing pulses in rapid succession. These Ca(2+) increments grew larger as free Ca(2+) rose to saturate the cytosolic buffers and reduce the availability of Ca(2+) binding sites. These titration data revealed two endogenous buffers. All nerve terminals contained a buffer with a Kd of 1.5-4.7 µM, and approximately half contained an additional higher-affinity buffer with a Kd of 340 nM. Western blots identified calretinin and calbindin D28K in the posterior pituitary, and their in vitro binding properties correspond well with our fluorometric analysis. The high-affinity buffer washed out, but at a rate much slower than expected from diffusion; washout of the low-affinity buffer could not be detected. This work has revealed the functional impact of cytosolic Ca(2+) buffers in situ in nerve terminals at a new level of detail. The saturation of these cytosolic buffers will amplify Ca(2+) signals and may contribute to use-dependent facilitation of release. A difference in the buffer compositions of oxytocin and vasopressin nerve terminals could contribute to the differences in release plasticity of these two hormones.

In situ Ca2+ titration in the fluorometric study of intracellular Ca2+ binding.

Imaging with Ca(2+)-sensitive fluorescent dye has provided a wealth of insight into the dynamics of cellular Ca(2+) signaling. The spatiotemporal evolution of intracellular free Ca(2+) observed in imaging experiments is shaped by binding and unbinding to cytoplasmic Ca(2+) buffers, as well as the fluorescent indicator used for imaging. These factors must be taken into account in the interpretation of Ca(2+) imaging data, and can be exploited to investigate endogenous Ca(2+) buffer properties. Here we extended the use of Ca(2+) fluorometry in the characterization of Ca(2+) binding molecules within cells, building on a method of titration of intracellular Ca(2+) binding sites in situ with measured amounts of Ca(2+) entering through voltage-gated Ca(2+) channels. We developed a systematic procedure for fitting fluorescence data acquired during a series of voltage steps to models with multiple Ca(2+) binding sites. The method was tested on simulated data, and then applied to 2-photon fluorescence imaging data from rat posterior pituitary nerve terminals patch clamp-loaded with the Ca(2+) indicator fluo-8. Focusing on data sets well described by a single endogenous Ca(2+) buffer and dye, this method yielded estimates of the endogenous buffer concentration and Kd, the dye Kd, and the fraction of Ca(2+) inaccessible cellular volume. The in situ Kd of fluo-8 thus obtained was indistinguishable from that measured in vitro. This method of calibrating Ca(2+)-sensitive fluorescent dyes in situ has significant advantages over previous methods. Our analysis of Ca(2+) titration fluorometric data makes more effective use of the experimental data, and provides a rigorous treatment of multivariate errors and multiple Ca(2+) binding species. This method offers a versatile approach to the study of endogenous Ca(2+) binding molecules in their physiological milieu.