Ca2+ imaging in the mammalian brain in vivo

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Abstract

Changes in intracellular free calcium ion concentration ([Ca2+]i) have been visualized over more than two decades using fluorescent dyes and optical microscopy. So far, however, most imaging studies have been performed on isolated cells or brain tissue. Here, we review approaches to measure cellular [Ca2+]i changes in vivo, i.e. within the intact brain of a living animal. In particular we describe the application of two-photon microscopy to the mammalian central nervous system, which has recently enabled studies of Ca2+ dynamics in individual dendrites in anaesthetized rats. New developments in microscopy and labeling techniques are creating further opportunities to study Ca2+ dynamics in vivo and are likely to make measurements of spatio-temporal [Ca2+]i distributions feasible even in awake, behaving mammals.

Introduction

Ca2+ acts as intracellular second messenger controlling a variety of cellular phenomena including secretion, contraction, excitability, and neuronal plasticity. Astonishingly, Ca2+ is able to control several different processes in an individual cell simultaneously. This is possible due to compartmentation of Ca2+ signals Helmchen, 1999, Wang and Augustine, 1999. An important step in understanding the role of Ca2+ in a particular phenomenon is therefore to determine the size, spatial spread, and time course of [Ca2+]i changes in the relevant region of the cell. In excitable cells, Ca2+ fluxes are closely linked to the cells' electrical activity. Action potentials and/or activation of synaptic receptor channels can cause Ca2+ influx, which is electrogenic and may itself contribute to regenerative membrane potential changes (such as Ca2+ action potentials). Thus measurements of [Ca2+]i changes can subserve a second function: the monitoring of electrical activity of individual neurones or of a whole region of the brain, the latter following bulk-labelling with indicator.

Measurements of spatio-temporal [Ca2+]i distributions became possible with the development of fluorescent Ca2+ indicators (Tsien, 1989). Using these indicators and light microscopy techniques, a wealth of information about Ca2+ signaling mechanisms has been collected over the last two decades in many different cell types. In the mammalian central nervous system (CNS) Ca2+ influx into dendrites and its role in synaptic integration and plasticity have been studied extensively (for reviews see Regehr and Tank, 1994, Denk et al., 1996, Eilers and Konnerth, 1997, Mainen et al., 1999, Sabatini et al., 2001, Yuste et al., 2000). Most experiments, however, were carried out in vitro, on either isolated cells or extracted tissue such as brain slices. One of the reasons is that light scattering typically restricts the use of conventional (wide-field) and confocal microscopy to the study of cells near the surface of brain tissue. Deeper within the tissue resolution is compromised and signal-to-noise deteriorates.

From in vitro models, one can draw only limited conclusions about neuronal function in the intact brain. One cannot observe [Ca2+]i changes as they occur during natural brain activity or during interactions with the outside world, either in response to sensory inputs or preceding an appropriate behavioural response. Furthermore, the properties and/or expression of proteins involved in Ca2+ handling (Ca2+-permeable channels, Ca2+ buffers or pumps) may be modulated in vivo according to the behavioural state of the animal. Hence their properties could be different in vitro. It is therefore necessary to study Ca2+ signaling in vivo, both to establish the presence and characteristics of signaling mechanisms found in vitro and to establish their relevance for animal behaviour.

Until recently in vivo Ca2+ imaging of individual cells was feasible only in a few favorable preparations, mostly insects and lower vertebrates (for reviews, see Borst and Egelhaaf, 1994, Fetcho and O'Malley, 1997, Galizia and Menzel, 2000). These pioneering studies characterized Ca2+ signals that result from sensory input or precede activity of the animal. The eventual goal is to measure activity under conditions that mimic the animal's experiences as closely as possible (e.g. Kern et al., 2001). These studies constitute a benchmark for what we should like to achieve in the mammalian CNS.

With the development of two-photon laser scanning microscopy (Denk et al., 1990), we now have a tool that permits visualization of cellular compartments up to 600 μm deep within biological tissue (Denk and Svoboda, 1997). Due to its depth penetration, two-photon microscopy has contributed substantially to the current trend to study cells within their native context (Lichtman and Fraser, 2001). It has been applied in a number of in vivo studies, including work on skin (Masters et al., 1997), cortical blood flow (Kleinfeld et al., 1998), embryos (Squirrell et al., 1999), dendritic spine morphology (Lendvai et al., 2000), Alzheimer plaques (Christie et al., 2001), and tumors (Brown et al., 2001). Two-photon microscopy for the first time enabled measurements of dendritic Ca2+ dynamics in neocortical pyramidal neurones of anaesthetized rats (Svoboda et al., 1997), opening a new field for the investigation of the intact mammalian brain on the cellular and sub-cellular scale.

In this review, we focus on the use of two-photon microscopy for imaging neuronal [Ca2+]i changes in anaesthetized rodents. Following a brief summary of previous in vivo approaches in non-mammalian preparations, we discuss some technical aspects, in particular the combination of two-photon microscopy with in vivo electrophysiology. We then give examples of [Ca2+]i measurements in rat somatosensory cortex and olfactory bulb and finally present an outlook on new developments that we anticipate will further extend the opportunities to study Ca2+ signaling in living animals.

Section snippets

Imaging in non-mammalian species

While in vivo Ca2+ imaging in the mammalian brain was established only recently, measurements in intact insects and lower vertebrates have a longer history. Table 1 summarizes experiments in which Ca2+ signals have been measured during sensory input or motor output, including several semi-intact in vitro preparations in which the sensory organ and its associated region of the brain were both intact.

Ca2+ indicators have been employed for two distinct purposes in vivo: to measure bulk activity in

In vivo two-photon microscopy

Two-photon laser scanning microscopy (2PLSM) is based on two-photon excitation of fluorophores using near-infrared pulsed laser light (Denk et al., 1990). The key advantages of 2PLSM, relative to wide-field and confocal microscopies, are (1) reduced light scattering due to the longer excitation wavelength and (2) confinement of excitation to the focal volume due to the nonlinear dependence of two-photon excitation rate on illumination intensity (Denk et al., 1995). As a consequence, 2PLSM

Ca2+ imaging of spines

One exciting new possibility is the resolution of synaptic activation patterns using sensory stimuli. Natural stimuli could cause clustered activation of synapses, perhaps on individual dendritic branches, they could cause activation of synapses distributed throughout the dendritic tree or perhaps the reality is somewhere in between. This is very much an open question at present. These input patterns can greatly influence information processing in dendrites (Poirazi and Mel, 2001). In the case

Summary

The remarkable depth penetration of two photon microscopy now enables neuroscientists to conduct types of cellular [Ca2+]i measurements in the intact mammalian brain that previously were possible only in insects and lower vertebrates. To date most experiments have examined the responses of single cells in anaesthetized rodents to artificial or simple sensory inputs. New labelling techniques should enable small groups of neurones to be imaged in real time at cellular resolution, without recourse

Acknowledgments

We thank W. Denk, B. Sakmann, and D.W. Tank for their generous support and R. Friedrich for critically reading the manuscript. Part of this work was supported by a postdoctoral fellowship to F.H. from the Human Frontier Science Program (LT0067-1998-B) and an Individual Marie-Curie fellowship to J.W. by the E.C. (QLGA-CT-2001-50999).

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