Astrocytes going live: advances and challenges

Abstract

Astrocytes are one of the most numerous cell types in the CNS. They have emerged as sophisticated cells participating in a large and diverse variety of functions vital for normal brain development, adult physiology and pathology. Recent in vivo studies have provided exciting new insight into astrocyte physiology in the intact healthy brain. This review will summarize some of their most intriguing findings, discuss some of their implications, and look ahead at some of the challenges we face in studying astrocyte function in vivo.

Despite the wealth of information that has been gathered about astrocyte physiology using mainly cell cultures and tissue slices we know very little about astrocyte function in the intact normal brain. This can partly be attributed to conflicting results obtained from and the limitations associated with the various types of tissue preparation. For example, cultured astrocytes have been shown to differ significantly in their phenotypic and morphological characteristics from their in situ counterparts (Lovatt et al. 2007; Cahoy et al. 2008; Pivneva et al. 2008). Brain slices on the other hand lack many characteristics of intact circuits such as normal synaptic activity and metabolic demand, long-range modulatory inputs and vascular dynamics such as intraluminal flow. In vitro preparations offer a number of important advantages over in vivo approaches such as ease of access to astrocytes in all brain regions or efficient dissection of molecular pathways that might be difficult to determine and control in vivo given the overlapping receptor array of astrocytes and other cells. However, in vitro approaches are unable to determine, for example, what forms of the primarily chemically encoded excitation of astrocytes are germane to the normal brain, how astrocyte excitation may depend on sensory input or behavioural state, or how in turn it might influence normal brain physiology.

Fluorescence microscopy techniques, the most prominent being two-photon microscopy, enable studies in the intact brain. Brain volumes containing up to hundreds of cells can be studied at micrometre-scale spatial and up to video-rate temporal resolution (Gobel et al. 2007; Vucinic & Sejnowski, 2007; Duemani Reddy et al. 2008; Holekamp et al. 2008). In addition, fluorescence microscopy techniques now enable imaging in awake behaving subjects (Helmchen et al. 2001; Dombeck et al. 2007; Flusberg et al. 2008).

This review will summarize recent findings from in vivo fluorescence microscopy studies in the brain of vertebrate model systems and discuss how they have advanced our understanding of astrocyte function in the normal adult brain. Given the short format of this review, it will focus on the most well-studied form of astrocyte excitation involving changes in intracellular Ca²⁺ levels. It will discuss how astrocyte Ca²⁺ signalling might relate to neurophysiology and blood flow regulation, and dwell on some of the future technical challenges we face in studying astrocyte function in vivo.

What is an astrocyte?

The term astrocyte is an umbrella term for a heterogeneous population of cells that is highly tuned to its local environment. It includes, for example, protoplasmic astrocytes of the grey matter, fibrous astrocytes of the white matter, radial astrocytes of the retina and cerebellum, velate astrocytes of the cerebellum and olfactory bulb, as well as special types of astrocytes found exclusively in certain species, such as interlaminar astrocytes in the cortex of higher primates. In addition to their vastly different morphology and territorial organization across brain regions and species, astrocytes differ in their physiological properties such as membrane potential, potassium conductance, glutamate transporter and receptor expression, and the expression of proteins such as glial fibrillary acidic protein (GFAP). Nevertheless, astrocytes in the brain are generally characterized by one or more end feet contacting a basal lamina around blood vessels (Simard et al. 2003).

Astrocyte subtypes can be defined using overlapping parameters such as cell body location, process morphology, electrophysiological properties and gene expression profile. An alternative definition is functional (Luo et al. 2008): cells that perform the same function within the circuit belong to the same subtype. Hence, studying astrocyte excitation in different cell layers, brain regions and species in vivo may help to arrive at a refined definition of astrocyte subtypes.

Studying astrocyte excitation in vivo

Fluorescence microscopy approaches allow study of astrocytic subtypes located within several hundred micrometres below the cortical surface. These approaches can be considered non-invasive as long as intense illumination is avoided (Wang et al. 2006) and proper fluorescence labelling and surgical preparation is employed. Astrocytes in deeper brain regions can be reached using fluorescence microendoscopy (Jung et al. 2004; Levene et al. 2004). At present, however, it is unexplored to what extent insertion of microendoscopes into tissue might interfere with normal astrocyte physiology.

Study of astrocyte excitation is possible by means of endogenous or synthetic fluorescent ion indicators (Rose & Ransom, 1996; Hirase et al. 2004; Kasischke et al. 2004; Nimmerjahn et al. 2004). The currently most widely used ion indicators report changes in intracellular Ca²⁺ concentration. Ca²⁺ signalling is considered the primary form of astrocyte excitability. Using Ca²⁺ imaging, spontaneous and evoked Ca²⁺ excitation in astrocytes of the olfactory bulb (Petzold et al. 2008), and protoplasmic astrocytes in barrel cortex (Wang et al. 2006), visual cortex (Schummers et al. 2008), and other areas of somatosensory cortex has been studied primarily in rodents (Hirase et al. 2004; Nimmerjahn et al. 2004; Takano et al. 2006; Dombeck et al. 2007; Gobel et al. 2007; Winship et al. 2007; Bekar et al. 2008; Takata & Hirase, 2008). Nevertheless, it is important to realize that Ca²⁺ is just one out of many signalling molecules astrocytes use to mediate cell function.

Characteristics of astrocyte Ca²⁺ excitation

Astrocytes express a large variety of receptors allowing them to sense changes in extracellular fluid composition. Many different hormones and transmitters, including glutamate and ATP, stimulate Ca²⁺ elevation (Fiacco & McCarthy, 2006). Ca²⁺ imaging serves as a readout of such astrocyte excitation (Fig. 1).

Figure 1. Calcium imaging of astrocytic and neuronal network excitation in vivo.

A, two-photon fluorescence images of cells in layer 2 of rat neocortex, stained using the cell-permeant green fluorescent calcium indicator Oregon Green 488 BAPTA-1 acetoxymethyl ester (OGB-1-AM) (left) and the red fluorescent astrocyte marker Sulforhodamine 101 (SR101) (center). Overlay of green and red fluorescence images (right) allows separation of astrocytic (yellow) and neuronal (green) networks. Scale bar, 20 μm. B, maximum-intensity side-projection from a stack of fluorescence images showing astrocytes and neurons in layers 1 and 2 (L1/2) of rat neocortex. Note the relative abundance of astrocytes in layer 1. Scale bar, 50 μm. C and D, spontaneous calcium transients in two astrocytes (C) and one neuron (D) from a single recording measured as relative fluorescence change, ΔF(t)/F, in layer 2 of an anaesthetized rat over the time course of several minutes. Note the different amplitude and time course of astrocytic and neuronal transients. Scale bars in C apply to C and D. E, neuronal calcium transients during the highlighted period in D shown on an expanded time scale. Figure modified from Nimmerjahn et al. (2004), with permission from Macmillan Magazines Ltd, © 2004.

Various forms of Ca²⁺ excitation have been found in astrocytes including transient peak-like increases, multiple transient Ca²⁺ oscillations, and prolonged Ca²⁺ elevations showing a plateau (Hirase et al. 2004; Nimmerjahn et al. 2004; Takata & Hirase, 2008). Ca²⁺ increases typically occur in astrocytic microdomains (Grosche et al. 1999; Nett et al. 2002), i.e. portions of astrocytic processes, but can propagate to other parts of the cell as an intracellular Ca²⁺ wave under certain conditions. In addition, astrocyte cell culture data suggest that astrocytes possess the capacity to signal intercellularly via Ca²⁺ over long distances (Cornell-Bell et al. 1990). Such intercellular Ca²⁺ waves between astrocytes have been observed during development, in specialized tissues such as the retina, and in pathological conditions (Fiacco & McCarthy, 2006). Conclusive evidence for the existence of Ca²⁺ waves in the normal adult brain is currently lacking.

The precise signalling cascades underlying the various forms of Ca²⁺ elevations are not well understood. Generally, Ca²⁺ signals in astrocytes are determined by an intricate interplay between Ca²⁺ influx, amplification, buffering and extrusion pathways (Cotrina & Nedergaard, 2005). The primary mechanism of astrocytic Ca²⁺ increases, however, is inositol 1,4,5-trisphosphate (IP₃) receptor-mediated Ca²⁺ release from the endoplasmic reticulum (ER). Release of Ca²⁺ from the ER is mediated predominantly by activation of G protein-coupled receptors (GPCRs), although voltage-and ligand-gated ion channels are also expressed on astrocytes. A potential contribution of ryanodine receptor (RYR)-regulated Ca²⁺ stores has remained controversial (Fiacco & McCarthy, 2006; Agulhon et al. 2008). As a result of their dependence on intracellular stores, Ca²⁺ elevations in astrocytes are typically prolonged lasting several seconds.

Astrocyte Ca²⁺ excitation and its relationship to neuronal activity

Astrocytes interact with essentially all other cellular elements of the brain. In the cortex, an intricate relationship exists between astrocytes and neurons. Astrocytic processes ensheath a considerable portion of neuronal synapses, the vast majority of which (∼90%) are glutamatergic (Magistretti, 2006). The percentage of synapses ensheathed by astrocytic processes as well as the absolute number of synapses ensheathed by one astrocyte varies greatly with synapse type and brain region. For example, in the hippocampus ∼60% of synapses are ensheathed by astrocyte processes (Ventura & Harris, 1999) while in the cerebellum 67% of parallel fibre and 94% of climbing fibre synapses are apposed by astrocytic extensions (Xu-Friedman et al. 2001). In addition, species differences exist. In rodents, one astrocyte ensheathes thousands of synapses (Bushong et al. 2002) while in the human cortex it might be more than one million (Oberheim et al. 2006). Astrocyte ensheathment is subject to dynamic modulation (Hatton, 1997).

Astrocytes establish anatomical domains. Astrocytic domains are largely non-overlapping and their shape as well as astrocyte-to-neuron density can vary greatly with cell layer, brain region and species (Bushong et al. 2002; Oberheim et al. 2006; Halassa et al. 2007; Livet et al. 2007). Generally, synapses ensheathed within an astrocytic domain include synapses from multiple neurons but typically only restricted parts of each neuron. The interaction between astrocytes is primarily confined to their domain borders where they form gap-junctional connections. Given this close anatomical association of astrocytes with neurons and the repertoire of neurotransmitter receptors expressed on these cells astrocytes are in a position to sense changes in neuronal activity levels.

That indeed astrocytes can sense neural activity and respond to it with Ca²⁺ increases has been shown both in vitro and in vivo (Porter & McCarthy, 1996; Hirase et al. 2004). Two types of Ca²⁺ increases, spontaneous and evoked transients, can be distinguished. Spontaneous Ca²⁺ transients occur in both individual processes and cell bodies although Ca²⁺ excitation tends to be more frequent in astrocytic microdomains. In addition, studies in neocortical layers 1–3 of anaesthetized rodents indicate that spontaneous Ca²⁺ transients are temporally sparse (∼mHz) (Wang et al. 2006; Takata & Hirase, 2008). Frequency and number of spontaneous Ca²⁺ transients in astrocytes appear to be influenced by local cytoarchitecture (Takata & Hirase, 2008).

Sensory evoked Ca²⁺ increases in astrocytes tend to be more widespread (Wang et al. 2006). Evoked Ca²⁺ transients depend on strength and type of stimulus and typically show a delay of 0.5–6 s from stimulus onset (Wang et al. 2006; Dombeck et al. 2007; Winship et al. 2007; Bekar et al. 2008; Petzold et al. 2008; Schummers et al. 2008). Coordinated Ca²⁺ increases involving many neighbouring astrocytes in response to physiological stimuli appear rare in anaesthetized animals (Schummers et al. 2008). In the S1 hindlimb region of awake mice, coordinated Ca²⁺ increases in subsets of astrocytes and neurons can be observed following locomotor onset. However, astrocytic Ca²⁺ transients were described as qualitatively similar to that seen in anaesthetized mice (Dombeck et al. 2007), although a detailed analysis of the various forms of astrocyte Ca²⁺ excitation in awake mice was not presented.

What are the molecular mechanisms that underlie evoked astrocytic Ca²⁺ transients in vivo? Studies in cell culture and brain slices have shown that a number of different pathways potentially participate in neuron–astrocyte communication in the intact brain. So far, experiments performed in barrel cortex and olfactory bulb of anaesthetized rodents have confirmed the involvement of glutamatergic transmission in sensory evoked Ca²⁺ transients (Wang et al. 2006; Petzold et al. 2008). In addition, long-range neuromodulatory inputs from other brain regions may contribute to local Ca²⁺ excitation in astrocytes (Bekar et al. 2008). Astrocytes also show intrinsic Ca²⁺ excitability, i.e. Ca²⁺ increases in the absence of neuronal input (Parri et al. 2001; Nett et al. 2002; Takata & Hirase, 2008), indicating that astrocytes are not simple detectors of neural activity. The functional relevance of these intrinsic transients is currently unclear.

Does astrocyte Ca²⁺ excitation affect neuronal activity?In vitro studies have shown that astrocytes can modulate synaptic activity in many brain regions. In particular, Ca²⁺ increases in astrocytes can be coupled to regulated release of neuroactive molecules and neurotransmitters such as ATP, cytokines, vasoactive compounds, d-serine, and glutamate (Fiacco & McCarthy, 2006). The level and spatial pattern of such transmitter release may depend on spatial and temporal characteristics of astrocyte Ca²⁺ elevation as astrocytes seem capable of distinguishing and remembering the intensity and history of neuronal activity (Carmignoto, 2000). This suggests, that astrocytes might play an active role in modulating neuronal activity and behaviour (Perea & Araque, 2007; Navarrete & Araque, 2008).

What are the expected properties of Ca²⁺-dependent neuromodulation by astrocytes? Due to its dependence on intracellular stores, gliotransmitter release is likely to occur on a slow time scale and to be more sustained compared to Ca²⁺-dependent transmitter release by neuronal presynaptic terminals. In addition, given the lack of active zones and low density of vesicle-like structures found in astrocytic processes (Fiacco et al. 2009) pathways involving vesicular release may be expected to result in a more diffuse and low concentration release of gliotransmitter. Neuromodulatory effects, such as those seen during arousal, attention and memory, are slow and might involve gliotransmitter release (Pascual et al. 2005; Gibbs et al. 2008). However, the net effect of Ca²⁺-dependent gliotransmitter release on neurophysiology can be difficult to predict: released gliotransmitter can activate both synaptic and extrasynaptic receptors (Fellin et al. 2004). Transmitters such as ATP and its metabolic product adenosine can exert opposite effects on neuronal transmission (Pascual et al. 2005), and individual astrocytes influence only particular subsets of neuronal dendrites and synapses within their anatomical domain while neuronal integration takes into account input from many different cells across various dendritic arbors within a given time window.

A more basic question is whether gliotransmitter release does occur in the normal brain. In the hippocampus, it was found that genetic deletion of IP₃R2, the primary IP₃ receptor expressed by astrocytes in that region, results in complete loss of spontaneous and GPCR agonist-evoked astrocytic Ca²⁺ increases (Petravicz et al. 2008). In addition, IP₃R2 deletion does not affect baseline excitatory synaptic activity or ambient glutamate levels, or cause overt abnormalities in brain cytoarchitecture or behaviour (Fiacco et al. 2009). Selective and widespread astrocyte Ca²⁺ elevations through astrocyte-specific expression and agonist-induced activation of foreign Gq-coupled receptors also have no effect on baseline excitatory synaptic activity (Fiacco & McCarthy, 2006). Based on these findings, it was suggested that stimulation protocols and/or recording conditions that have been used in studies of Ca²⁺-dependent gliotransmitter release might be unphysiological (Agulhon et al. 2008; Fiacco et al. 2009).

Does that mean astrocytes do not play an active role in modulating synaptic transmission and behaviour? No. Apart from Ca²⁺-dependent pathways several Ca²⁺-independent pathways of gliotransmitter release exist. These include large pores such as gap junction hemichannels, purinergic P₂X₇ receptors, and volume-sensitive anion channels and transporters (Fiacco & McCarthy, 2006). Further, astrocytes might influence synaptic transmission on various time scales through changes in potassium channel or glutamate transporter activity (Anderson & Swanson, 2000) as well as dynamic changes in synapse ensheathment (Hatton, 1997).

Astrocyte Ca²⁺ excitation and its relationship to blood flow

Apart from ensheathing neuronal synapses, processes of cortical astrocytes form end feet onto the basal lamina around blood vessels (Fig. 2). In addition, some astrocytes form end feet on the pia mater constituting the glia limitans on which pial arteries reside. Astrocytes are therefore in a position to bridge two networks, neurons and blood vessels, that are largely disconnected from each other: cerebral blood vessels are surrounded by nerve fibres. Perivascular neuronal varicosities, however, are rare and abut primarily on astrocytic end feet, with a smaller proportion directly contacting smooth muscle cells (Hamel, 2006). In contrast, astrocyte end feet ensheath more than 99% of the cerebrovascular surface (Iadecola & Nedergaard, 2007). This intermediary role of astrocytes suggests that they might play a critical role in neuronal activity-dependent regulation of blood flow and energy metabolism, processes that provide the basis for functional brain imaging signals (Iadecola & Nedergaard, 2007; Pellerin et al. 2007).

Figure 2. Astrocytic ensheathment of cerebrovasculature.

A, two-photon fluorescence image of astrocytes (green) and blood vessels (red) in rat neocortex, stained using the astrocyte-specific marker SR101 and tail-vein injection of FITC-labelled dextran, respectively. Unstained neuronal cell bodies (arrowheads) appear as dark gaps. Scale bar, 20 μm. B, higher magnification fluorescence image showing how astrocytic end feet ensheath almost the entire cerebrovascular surface. Scale bar, 10 μm. Figure modified from Nimmerjahn et al. (2004), with permission from Macmillan Magazines Ltd, © 2004.

Control of blood flow to intraparenchymal arterioles is thought to occur at the level of pial arteries. Together with large cerebral arteries they are responsible for two-thirds of the vascular resistance (Iadecola & Nedergaard, 2007). Vascular adjustment underlying blood flow changes thus often involves changes in vessel diameter of both local arterioles and upstream arteries necessitating temporally coordinated cellular events. These events are likely to involve neurons, pericytes, astrocytes and vascular cells. Astrocytes and vascular cells are of particular relevance in this regard as they are able to propagate signals over larger areas through intercellular junctions (Xu et al. 2008).

Evidence that astrocytes contribute to blood flow regulation comes from both in vitro and in vivo studies. In vitro work has shown that evoking end feet Ca²⁺ excitation either directly using IP₃-or Ca²⁺-uncaging, or through neural stimulation leads to vasoconstriction or vasodilation (Zonta et al. 2003; Mulligan & MacVicar, 2004; Metea & Newman, 2006). These vasomotor responses are reduced in the presence of the Ca²⁺ chelator BAPTA and persist in the presence of tetrodotoxin, an inhibitor of Na⁺ channel based neural activity, suggesting that astrocytes alone can mediate vasomotor changes. Vasomotor responses as seen in vitro were found to depend on several factors, including metabolic state of the tissue and local nitric oxide (NO) levels (Metea & Newman, 2006; Gordon et al. 2008). However, as the precise interplay of signalling among neurons, astrocytes, vascular cells and other elements of the neurovascular unit leading to spatial heterogeneities in tissue oxygenation, NO level and other tissue properties is not well understood it has been difficult to predict vascular response patterns in the intact normal brain.

Studies in anaesthetized animals showed that evoking Ca²⁺ elevations in astrocyte end feet either directly or following neuronal stimulation leads primarily to vasodilations and only rarely to vasoconstrictions near the site of stimulation (Takano et al. 2006). However, vascular responses in more distant regions that more likely involve vasoconstrictions (Devor et al. 2007) were not investigated. Likewise, astrocyte Ca²⁺ excitation in brain regions undergoing distinct vasoconstriction in response to sensory stimulation has remained unexplored (Devor et al. 2008). Onset latencies reported for sensory evoked Ca²⁺ increases in astrocytes (∼0.5–3 s), as measured by fluorescence microscopy (Wang et al. 2006; Winship et al. 2007; Petzold et al. 2008; Schummers et al. 2008), and changes in cerebral blood flow (∼1–2 s), as assessed by laser Doppler flowmetry, laser speckle imaging and other techniques (Zonta et al. 2003; Martin et al. 2006; Devor et al. 2008), are largely consistent with a role of astrocytes in mediating blood flow changes. However, a detailed characterization of the precise timing relationship between neural activation, astrocytic Ca²⁺ levels and vasomotor response under physiological conditions is currently lacking.

What are the molecular mechanisms underlying evoked blood flow changes in vivo? Pharmacological manipulations leading to a preferential decrease in astrocytic Ca²⁺ elevations or glutamate transporter activity in astrocytes showed a reduction but not an elimination of evoked blood flow changes suggesting that in general several pathways contribute to blood flow regulation (Zonta et al. 2003; Gurden et al. 2006; Takano et al. 2006; Petzold et al. 2008; Schummers et al. 2008). A number of agents have been implicated in regulation of cerebral blood flow, including NO, H⁺, K⁺, neurotransmitters, adenosine and arachidonic acid (AA) metabolites (Koehler et al. 2006; Iadecola & Nedergaard, 2007). The cellular source of these agents is largely unknown as is the relative contribution of these agents in mediating blood flow changes in various brain regions. In the cerebellum, NO is thought to be a major mediator responsible for cerebral blood flow changes while in the neocortex NO seems to have a merely permissive role (Koehler et al. 2006; Iadecola & Nedergaard, 2007). While we currently know very little about the role of cerebellar astrocytes in mediating blood flow changes in vivo, neocortical and olfactory bulb astrocytes have been shown to contribute to blood flow changes via activation of cyclooxygenase 1, an enzyme acting downstream of AA (Takano et al. 2006; Petzold et al. 2008). AA is formed in astrocytes following Ca²⁺ dependent activation of phospholipase A₂ and metabolized to agents acting on vascular muscle cells. The type of AA metabolite produced differs between CNS regions and may depend on metabolic state of the tissue and other factors such as local level as demonstrated in vitro (Metea & Newman, 2006; Gordon et al. 2008). Vasomotor changes may also be induced by mobilization of Ca²⁺ activated K⁺ channels in astrocyte end feet leading to efflux of K⁺ hyperpolarizing and relaxing vascular muscle cells by acting on inward rectifier K⁺ channels (Filosa et al. 2006).

Future challenges

The above examples demonstrate that fluorescence microscopy studies in vivo have provided important insight into astrocyte physiology in the intact healthy brain. However, many mysteries regarding the role of astrocyte Ca²⁺ excitation remain. For example, how does Ca²⁺ excitation differ between astrocytic subtypes? What forms of Ca²⁺ excitation exist in a given subtype, and what is the functional relevance of the various forms of astrocytic Ca²⁺ excitation? Do they each function in a specific way to modulate brain activity? How are they influenced by anaesthesia or behaviour? Are there specific patterns of neuronal activity that trigger certain forms of astrocytic Ca²⁺ excitation? Does astrocytic Ca²⁺ excitation lead to release of gliotransmitter in the normal brain and if so, how does it affect neurophysiology and behaviour? What is the precise temporal relationship between neural activation, astrocytic Ca²⁺ levels and vasomotor response and how does it differ between brain regions? What is the effect if any of astrocyte Ca²⁺ excitation on other components of the neurovascular unit participating in local and upstream blood flow changes (Moore & Cao, 2008), and what is its effect on metabolism (Bernardinelli et al. 2004)? Further, given that astrocytes show intrinsic Ca²⁺ excitability (Parri et al. 2001; Nett et al. 2002) and may account for up to 30% of total energy consumption (Iadecola & Nedergaard, 2007), can the metabolic need of astrocytes itself drive vasomotor changes independently of neuronal activity? What are the respective implications for functional brain imaging signals? What is the relationship between astrocytic Ca²⁺ excitation and blood flow regulation in awake behaving compared to anaesthetized subjects given that anaesthetics can affect cerebral blood flow and its regulation either directly or through effects on parameters such as arterial pressure, respiration and body temperature (Iadecola & Nedergaard, 2007)?

Addressing these and other questions in the intact brain will require, at least in part, development and application of novel fluorescence microscopy approaches. For example, information about how astrocytes respond as a network to sensory stimulation may be obtained using 3D imaging (Gobel et al. 2007). Functional signals from such large-scale data sets might be efficiently extracted using automated cell sorting approaches (Mukamel et al. 2008). Functional interactions between neurons, astrocytes and blood flow in the brains of awake behaving animals may be recorded using miniaturized fluorescence microscopes or head-fixed preparations (Fig. 3) (Flusberg et al. 2008; Nimmerjahn et al. 2008). Spatial and temporal control over astrocyte Ca²⁺ excitation in vivo might be achieved using transgenic (Fiacco et al. 2007) or optogenetic approaches (Airan & Deisseroth, 2008). Long-term and deep brain imaging of Ca²⁺ excitation in subtypes of astrocytes may be possible through transgenic or viral expression of new genetically encoded Ca²⁺ sensors (Rochefort & Konnerth, 2008). Insight into the role of glial cells in neurophysiology and behaviour might be obtained using invertebrate model systems (Bacaj et al. 2008) in addition to studies in vertebrate systems.

Figure 3. Fluorescence microscopy approaches for imaging in awake behaving mice.

A, two-photon fluorescence microscopy in awake, head-restrained mice using an exercise ball. Fluorescence excitation is provided by a pulsed near-infrared laser whose beam is scanned across the sample; fluorescence detection is achieved using photomultiplier tubes (not shown). Mouse locomotion is tracked by measuring ball rotation and/or video. B, miniaturized one-photon fluorescence microscopy in freely behaving mice. Fluorescence excitation is provided by a mercury arc lamp; a high-speed electron-multiplying charge-coupled device (EM-CCD) camera detects emitted fluorescence. A fibre bundle delivers the arc lamp illumination to the miniature head-mounted microscope and returns a fluorescence image to the camera. A commutator allows the bundle to rotate as the mouse moves. Bundle rotations are tracked by an optical encoder for offline image stabilization. Mouse trajectory is tracked by video. Figure modified from Flusberg et al. (2008), with permission from Macmillan Magazines Ltd, © 2008. A and B, one main advantage of head-fixation is that it allows the use of a tabletop microscope with superior optical properties, while the use of a miniature head-mounted microscope enables a richer behavioural repertoire. Both fluorescence microscopy approaches rely on appropriate fluorescence labelling of the sample prior to imaging. Coloured arrows indicate illumination (red/blue) and light collection pathways (green).

In conclusion, advances in fluorescence microscopy and related techniques have yielded exciting new insight into astrocyte function in the normal brain and promise to continue broadening our understanding in this regard. Going live is more than just a question of verifying in vitro results. Rather, it provides a chance to obtain answers to some of the long-standing riddles in neuroscience!