Large and long-lasting cytosolic calcium surges in astrocytes have been described in cultured cells and acute slice preparations. The mechanisms that give rise to these calcium events have been extensively studied in vitro. However, their existence and functions in the intact brain are unknown. We have topically applied Fluo-4 AM on the cerebral cortex of anesthetized rats, and imaged cytosolic calcium fluctuation in astrocyte populations of superficial cortical layers in vivo, using two-photon laser scanning microscopy. Spontaneous [Ca 2+ ] i  events in individual astrocytes were similar to those observed in vitro. Coordination of [Ca 2+ ] i  events among astrocytes was indicated by the broad cross-correlograms. Increased neuronal discharge was associated with increased astrocytic [Ca 2+ ] i  activity in individual cells and a robust coordination of [Ca 2+ ] i  signals in neighboring astrocytes. These findings indicate potential neuron–glia communication in the intact brain.

Introduction Astrocytes are nonneuronal cells of the brain with some known and hypothesized functions (Kettenmann and Ransom 1995;  Fields and Stevens-Graham 2002 ). Traditionally, astrocytes have been considered to mediate supportive and protective functions in the central nervous system because of their strategic placement relative to the vasculature, and because they lack fast sodium action potentials. It is only recently that this family of glial cells has been implicated in controlling the dynamics of the neuronal networks in the central nervous system ( Nedergaard 1994 ;  Parpura et al. 1994 ;  Kang et al. 1998 ;  Parri et al. 2001 ). Although the membrane potential of unidentified glial cells shows correlated changes with neuronal activity in vivo ( Amzica and Steriade 2000 ;  Amzica and Massimini 2002 ), most of our knowledge on neuron–glia and glia–glia communication comes from studies in vitro. In cultured and acutely prepared astrocytes, free calcium concentration ([Ca 2+ ] i ) in the cytosol undergoes large changes spontaneously or in response to various physiological and pharmacological manipulations, such as mechanical stimulation, membrane potential depolarization, and activation of metabotropic glutamate receptors ( Cornell-Bell et al. 1990a ;  Pasti et al. 1997 ). These slow events are mediated by release of Ca 2+  from intracellular stores ( Charles et al. 1993 ;  Venance et al. 1997 ). The [Ca 2+ ] i  surges can be evoked by strong neuronal activity ( Dani et al. 1992 ;  Porter and McCarthy 1996 ), suggesting a potential homeostatic role of astrocytes in the regulation of extracellularly accumulating neurotransmitters ( Verkhratsky et al. 1998 ). Conversely, spontaneous [Ca 2+ ] i  changes in astrocytes have been shown to influence neuronal excitability ( Parpura et al. 1994 ;  Kang et al. 1998 ;  Pasti et al. 2001 ). The mechanism of activity propagation among astrocytes is controversial. In tissue cultures, [Ca 2+ ] i  events can propagate among a network of astrocytes via gap junction or by elevation of adenosine triphosphate level ( Cornell-Bell et al. 1990b ;  Charles et al. 1991 ;  Nedergaard 1994 ;  Reetz et al. 1997 ;  Newman 2001 ). In the in vitro slice preparation, coordination of [Ca 2+ ] i  activity appears independent of gap junctions but may require transmitter activation of N-methyl-D-aspartic acid (NMDA) and/or metabotropic glutamate receptors ( Parri et al. 2001 ;  Aguado et al. 2002 ;  Nett et al. 2002;  Tashiro et al. 2002 ). Moreover, the extent and magnitude of these network effects vary as a function of the preparation used, and can involve correlated [Ca 2+ ] i  changes in no, or only a few, neighboring astrocytes, or the whole population ( Porter and McCarthy 1996 ;  Verkhratsky et al. 1998 ). Whether and how the observations in the various in vitro situations apply to the intact brain have yet to be determined. We have used two-photon laser scanning microscopy (2-PLSM) to monitor cytosolic Ca 2+  concentration in astrocytes labeled with Fluo-4 acetoxymethyl (AM) ester in juvenile rats in vivo. We find that [Ca 2+ ] i  dynamics in astrocytes is rather quiescent during baseline anesthesia. However, increased population bursting, brought about by attenuating γ-aminobutyric acid (GABA A ) receptor-mediated neurotransmission, leads to increased magnitude [Ca 2+ ] i  surges, and the [Ca 2+ ] i  changes become more strongly coordinated in neighboring astrocytes.
Introduction
Astrocytes are nonneuronal cells of the brain with some known and hypothesized functions (Kettenmann and Ransom 1995;  Fields and Stevens-Graham 2002 ). Traditionally, astrocytes have been considered to mediate supportive and protective functions in the central nervous system because of their strategic placement relative to the vasculature, and because they lack fast sodium action potentials. It is only recently that this family of glial cells has been implicated in controlling the dynamics of the neuronal networks in the central nervous system ( Nedergaard 1994 ;  Parpura et al. 1994 ;  Kang et al. 1998 ;  Parri et al. 2001 ). Although the membrane potential of unidentified glial cells shows correlated changes with neuronal activity in vivo ( Amzica and Steriade 2000 ;  Amzica and Massimini 2002 ), most of our knowledge on neuron–glia and glia–glia communication comes from studies in vitro.In cultured and acutely prepared astrocytes, free calcium concentration ([Ca 2+ ] i ) in the cytosol undergoes large changes spontaneously or in response to various physiological and pharmacological manipulations, such as mechanical stimulation, membrane potential depolarization, and activation of metabotropic glutamate receptors ( Cornell-Bell et al. 1990a ;  Pasti et al. 1997 ). These slow events are mediated by release of Ca 2+  from intracellular stores ( Charles et al. 1993 ;  Venance et al. 1997 ). The [Ca 2+ ] i  surges can be evoked by strong neuronal activity ( Dani et al. 1992 ;  Porter and McCarthy 1996 ), suggesting a potential homeostatic role of astrocytes in the regulation of extracellularly accumulating neurotransmitters ( Verkhratsky et al. 1998 ). Conversely, spontaneous [Ca 2+ ] i  changes in astrocytes have been shown to influence neuronal excitability ( Parpura et al. 1994 ;  Kang et al. 1998 ;  Pasti et al. 2001 ). The mechanism of activity propagation among astrocytes is controversial. In tissue cultures, [Ca 2+ ] i  events can propagate among a network of astrocytes via gap junction or by elevation of adenosine triphosphate level ( Cornell-Bell et al. 1990b ;  Charles et al. 1991 ;  Nedergaard 1994 ;  Reetz et al. 1997 ;  Newman 2001 ). In the in vitro slice preparation, coordination of [Ca 2+ ] i  activity appears independent of gap junctions but may require transmitter activation of N-methyl-D-aspartic acid (NMDA) and/or metabotropic glutamate receptors ( Parri et al. 2001 ;  Aguado et al. 2002 ;  Nett et al. 2002;  Tashiro et al. 2002 ). Moreover, the extent and magnitude of these network effects vary as a function of the preparation used, and can involve correlated [Ca 2+ ] i  changes in no, or only a few, neighboring astrocytes, or the whole population ( Porter and McCarthy 1996 ;  Verkhratsky et al. 1998 ). Whether and how the observations in the various in vitro situations apply to the intact brain have yet to be determined.We have used two-photon laser scanning microscopy (2-PLSM) to monitor cytosolic Ca 2+  concentration in astrocytes labeled with Fluo-4 acetoxymethyl (AM) ester in juvenile rats in vivo. We find that [Ca 2+ ] i  dynamics in astrocytes is rather quiescent during baseline anesthesia. However, increased population bursting, brought about by attenuating γ-aminobutyric acid (GABA A ) receptor-mediated neurotransmission, leads to increased magnitude [Ca 2+ ] i  surges, and the [Ca 2+ ] i  changes become more strongly coordinated in neighboring astrocytes.Results Loading of Calcium-Sensitive Dye To examine the depth of penetration of the Fluo-4 AM, coronal brain slices (300 μm thick) were acutely prepared after the residual dye was washed off from the craniotomy. A large number of cells below the craniotomy showed fluorescence labeling ( Figure 1 ). On the basis of morphological appearance (see also  Videos S1-S4 ), most brightly labeled cells were astrocytes, in accordance with recent observations using a pressure application of the indicator ( Stosiek et al. 2003 ). The large overlap between Fluo-4 AM-loaded cells and astrocytes identified by S100B immunoreactivity provided confidence that most of the loaded cells were astrocytes ( Video S5 ). In addition to astrocytes, capillary endothelial cells and pericytes, outlining microvessels, were also observed, albeit less regularly. Some processes of astrocytes contacted local vessels. To quantify the dye penetration, mean bulk fluorescence intensity was plotted for different depths from the pial surface. Most intensive labeling occurred between 50–150 μm below the surface (i.e., layers I/II), but labeled cells could be visualized at greater than 300 μm as well ( Figure 1 C). The decreased fluorescence on the surface is likely due to the diluting effect of the washout procedure in the superficial tissue. Like the histological appearance, in vivo imaging revealed numerous astrocytes ( Figure 1 E). Although the labeling was dense, the somata and several associated processes, including vessel-contacting end feet, of single astrocytes could be clearly revealed ( Figure 2 ). Figure 1 In Vivo Loading and Imaging of Astrocytes Using Fluo-4 AM (A) Acute slice prepared 1 h after dye loading. Scale bar, 200 μm. (B) Higher magnification reveals cells with typical astrocyte morphology. Scale bar, 20 μm. (C) Average bulk fluorescence as a function of the depth from the pial surface. (D) Schematic drawing of the experimental arrangement. Abbreviations: EKG, electrocardiogram. PMT, photomultiplier. LFP, glass micropipe for local field potential and multiple unit recording. The same pipette was used to deliver bicuculline. (E) Image taken 50–150 μm below pial surface in vivo. Flattened xyz stack. (F) Fluo-4 AM loaded cells (left) were stained for S100B immunoreactivity (right), and the images were merged (center). See  Video S3  for large-scale staining. Scale bar, 20 μm. Figure 2 Time-Lapse Imaging of Astrocytes In Vivo Four astrocytes, from which fluorometric Ca 2+  imaging (0.5 Hz) was made, are outlined. A blood vessel, outlined by the astrocyte end feet, runs diagonally across the viewed area. White arrows show the end foot connected to the imaged astrocyte. Spontaneous Calcium Events in Astrocytes In our initial experiments, we made a large number of line scans (sampling rate ∼200 Hz) of dye-loaded cells to examine whether some of them were neurons. We never observed short-lasting [Ca 2+ ] i  transients (less than 200 ms;  Svoboda et al. 1997 ;  Garaschuk et al. 2000 ), suggesting that the brightly loaded cells were likely to be non-neuronal ( Parri et al. 2001 ;  Stosiek et al. 2003 ). In subsequent experiments ( n  = 8 rats), cells with astrocytic appearance ( n  = 185) were selected for long-term (10–20 min) monitoring. For quantitative studies, three states of [Ca 2+ ] i  activity were distinguished: (a) quiescent state with very slow (less than 0.025 Hz) oscillations of baseline fluorescence level, (b) [Ca 2+ ] i  spikes (greater than or equal to 20% increase in  ΔF/F 0  between 5–50 s), and (c) [Ca 2+ ] i  plateau potentials (greater than or equal to 20% increase in  ΔF/F 0  for greater than 50 s). [Ca 2+ ] i  spikes and [Ca 2+ ] i  plateau potentials were automatically detected. In the control (baseline) condition, 11% of astrocytes had at least one spike event, and 52% had at least one plateau event in 10 min. The mean frequency of [Ca 2+ ] i  spikes among the cells that had at least one [Ca 2+ ] i  spike was 0.121 ± 0.098 per minute (mean width at greater than or equal to 20%  ΔF/F 0 : 25.1 ± 10.31 s) and the mean frequency of [Ca 2+ ] i  plateau was 0.118 ± 0.058 per minute (mean duration: 160.4 ± 114.9 s). To investigate whether the baseline values of [Ca 2+ ] i  dynamics were affected by increasing neuronal activity, we induced regularly occurring population bursts by local application of bicuculline ( Schwartz and Bonhoeffer 2001 ;  n  = 7 rats). Large amplitude (0.69 ± 0.26 mV) synchronous field events (approximately 100 ms) occurred at relatively regular frequency (0.15 ± 0.06 Hz), associated with multiple unit discharges. No significant difference was observed in average heartbeat frequency between the control sessions and bicuculline sessions (4.51 ± 0.54 Hz and 4.36 ± 0.74 Hz, respectively; paired t-test,  p  = 0.13). We used two methods to evaluate the effect of neuronal activity on [Ca 2+ ] i  in astrocytes ( n  = 214 cells). First, the incidence of [Ca 2+ ] i  spikes and plateau events was counted in the absence and presence of bicuculline-induced population bursts. Under bicuculline condition significantly more astrocytes had [Ca 2+ ] i  spikes (11% versus 24%;  p  < 0.001; Fisher's exact test), whereas the probability (52% versus 54%) of plateau potentials did not differ significantly. The mean duration of plateau potentials, however, was significantly longer (160.4 ± 114.9 s versus 211.12 ± 152.175 s; t-test,  p  < 0.001) after bicuculline treatment. Among the cells that exhibited at least one spike or plateau event, there was not a significant difference in frequency of the event occurrences (spike: 0.121 ± 0.098/min versus 0.098 ± 0.068/min; t-test,  p  = 0.24; plateau 0.118 ± 0.058/min versus 0.112 ± 0.049/min; t-test,  p  = 0.46). Thus, the major difference between control and bicuculline conditions was the higher proportion of active astrocytes under bicuculline. The second method examined [Ca 2+ ] i  changes in the frequency domain. The  ΔF/F 0  trace was considered as a continuous process, and the power spectrum estimate was calculated with a multi-taper method for each astrocyte and averaged across cells. There was a general increase of power at all frequencies in bicuculline-treated animal. The most consistent significant increase ( p  < 0.05) of power appeared in the frequency range of 0.10–0.24Hz, reflecting the increased incidence of [Ca 2+ ] i  spikes. Short-term cross-correlation of neuronal field bursts and [Ca 2+ ] i  signals (± 10 s) did not show a significant time-locked relationship ( Figure 3 ). Figure 3 Frequency Domain Analysis of Population Dynamics of Fluorescence in Astrocytes in Control State and during Bicuculline-Induced Neuronal Hyperactivity Insets show local field potentials in a control animal and regular spiking in a bicuculline treated mouse (scale bar: 2.0 s, 500 μV). Asterisks show significant differences ( p  < 0.05) between groups at various frequencies. Spatio-Temporal Dynamics of [Ca 2+ ] i  Events In individual experiments, propagation of synchronous activity could be observed visually ( Figure 4 A;  Video S6 ) but the spatio-temporal relationship of [Ca 2+ ] i  dynamics among astrocytes varied across experiments. To quantify the magnitude and spatial extent of this population effect, pair-wise cross-correlograms of  ΔF/F 0  intensity were calculated separately for nearby cell pairs (local: less than or equal to 50 μm) and distant cell pairs (greater than 50 μm). In control conditions, the temporal correlation of [Ca 2+ ] i  signals in neighboring pairs was somewhat larger than in distant pairs, but this difference was not significant ( n  = 374 neighbor pairs and  n  = 1,138 distant pairs). Nevertheless, [Ca 2+ ] i  signals in astrocytes were not completely random, since the cross-correlograms had wide central peaks at the 10–100 s scale ( Figure 4 B). In contrast to the baseline condition, the temporal correlation of [Ca 2+ ] i  changes in local and distant pairs were significantly different after large population bursts were brought about by bicuculline ( Figure 4 C). Correlation of distant pairs under bicuculline ( n  = 433 pairs) was similar to those in the control condition. However, synchrony between local pairs ( n  = 1,282) increased several-fold relative to both distant pairs under the same condition ( t -test,  p  < 0.0001) and to local pairs in the baseline condition ( t -test,  p  < 0.0001). Figure 4 Spatio-Temporal Dynamics of Astrocyte Ca 2+  Activity (A) Definition of nearby (less than 50 μm) and distant (greater than 50 μm) cell pairs. (B) Fluorescence changes in two nearby astrocytes. (C) Cross-correlogram of fluorescent intensity. (D) Mean cross-correlation of  ΔF/F 0  in all nearby (thick line) and distant (thin line) cell pairs in control condition (left) and in the presence of bicuculline (right). Note large increase of  ΔF/F 0  correlation in nearby cell pairs in the bicuculline condition (error bar: standard error of the mean). (E) Relationship between distance of the two cells and the magnitude of correlation at zero timelag. Note lack of a reliable relationship in the control condition (left). Note also the significant negative correlation between the distance and correlated  ΔF/F 0  changes in cell pairs in the bicuculline-treated cortex (right). Using a different approach, the magnitude of the zero-timelag correlation coefficient for each cell pair was plotted against distance between the cell pairs. Under control condition, no notable relationship was observed between these variables ( Figure 5 ;  n  = 1,512 cell pairs,  r  = 0.019,  p  = 0.46). In contrast, a significant negative correlation was found between the synchrony of [Ca 2+ ] i  signals in the bicuculline condition ( n  = 1,715;  r  = −0.281;  p  < 0.0001).
Results
Loading of Calcium-Sensitive Dye To examine the depth of penetration of the Fluo-4 AM, coronal brain slices (300 μm thick) were acutely prepared after the residual dye was washed off from the craniotomy. A large number of cells below the craniotomy showed fluorescence labeling ( Figure 1 ). On the basis of morphological appearance (see also  Videos S1-S4 ), most brightly labeled cells were astrocytes, in accordance with recent observations using a pressure application of the indicator ( Stosiek et al. 2003 ). The large overlap between Fluo-4 AM-loaded cells and astrocytes identified by S100B immunoreactivity provided confidence that most of the loaded cells were astrocytes ( Video S5 ). In addition to astrocytes, capillary endothelial cells and pericytes, outlining microvessels, were also observed, albeit less regularly. Some processes of astrocytes contacted local vessels. To quantify the dye penetration, mean bulk fluorescence intensity was plotted for different depths from the pial surface. Most intensive labeling occurred between 50–150 μm below the surface (i.e., layers I/II), but labeled cells could be visualized at greater than 300 μm as well ( Figure 1 C). The decreased fluorescence on the surface is likely due to the diluting effect of the washout procedure in the superficial tissue. Like the histological appearance, in vivo imaging revealed numerous astrocytes ( Figure 1 E). Although the labeling was dense, the somata and several associated processes, including vessel-contacting end feet, of single astrocytes could be clearly revealed ( Figure 2 ). Figure 1 In Vivo Loading and Imaging of Astrocytes Using Fluo-4 AM (A) Acute slice prepared 1 h after dye loading. Scale bar, 200 μm. (B) Higher magnification reveals cells with typical astrocyte morphology. Scale bar, 20 μm. (C) Average bulk fluorescence as a function of the depth from the pial surface. (D) Schematic drawing of the experimental arrangement. Abbreviations: EKG, electrocardiogram. PMT, photomultiplier. LFP, glass micropipe for local field potential and multiple unit recording. The same pipette was used to deliver bicuculline. (E) Image taken 50–150 μm below pial surface in vivo. Flattened xyz stack. (F) Fluo-4 AM loaded cells (left) were stained for S100B immunoreactivity (right), and the images were merged (center). See  Video S3  for large-scale staining. Scale bar, 20 μm. Figure 2 Time-Lapse Imaging of Astrocytes In Vivo Four astrocytes, from which fluorometric Ca 2+  imaging (0.5 Hz) was made, are outlined. A blood vessel, outlined by the astrocyte end feet, runs diagonally across the viewed area. White arrows show the end foot connected to the imaged astrocyte.
Loading of Calcium-Sensitive Dye
To examine the depth of penetration of the Fluo-4 AM, coronal brain slices (300 μm thick) were acutely prepared after the residual dye was washed off from the craniotomy. A large number of cells below the craniotomy showed fluorescence labeling ( Figure 1 ). On the basis of morphological appearance (see also  Videos S1-S4 ), most brightly labeled cells were astrocytes, in accordance with recent observations using a pressure application of the indicator ( Stosiek et al. 2003 ). The large overlap between Fluo-4 AM-loaded cells and astrocytes identified by S100B immunoreactivity provided confidence that most of the loaded cells were astrocytes ( Video S5 ). In addition to astrocytes, capillary endothelial cells and pericytes, outlining microvessels, were also observed, albeit less regularly. Some processes of astrocytes contacted local vessels. To quantify the dye penetration, mean bulk fluorescence intensity was plotted for different depths from the pial surface. Most intensive labeling occurred between 50–150 μm below the surface (i.e., layers I/II), but labeled cells could be visualized at greater than 300 μm as well ( Figure 1 C). The decreased fluorescence on the surface is likely due to the diluting effect of the washout procedure in the superficial tissue. Like the histological appearance, in vivo imaging revealed numerous astrocytes ( Figure 1 E). Although the labeling was dense, the somata and several associated processes, including vessel-contacting end feet, of single astrocytes could be clearly revealed ( Figure 2 ).Spontaneous Calcium Events in Astrocytes In our initial experiments, we made a large number of line scans (sampling rate ∼200 Hz) of dye-loaded cells to examine whether some of them were neurons. We never observed short-lasting [Ca 2+ ] i  transients (less than 200 ms;  Svoboda et al. 1997 ;  Garaschuk et al. 2000 ), suggesting that the brightly loaded cells were likely to be non-neuronal ( Parri et al. 2001 ;  Stosiek et al. 2003 ). In subsequent experiments ( n  = 8 rats), cells with astrocytic appearance ( n  = 185) were selected for long-term (10–20 min) monitoring. For quantitative studies, three states of [Ca 2+ ] i  activity were distinguished: (a) quiescent state with very slow (less than 0.025 Hz) oscillations of baseline fluorescence level, (b) [Ca 2+ ] i  spikes (greater than or equal to 20% increase in  ΔF/F 0  between 5–50 s), and (c) [Ca 2+ ] i  plateau potentials (greater than or equal to 20% increase in  ΔF/F 0  for greater than 50 s). [Ca 2+ ] i  spikes and [Ca 2+ ] i  plateau potentials were automatically detected. In the control (baseline) condition, 11% of astrocytes had at least one spike event, and 52% had at least one plateau event in 10 min. The mean frequency of [Ca 2+ ] i  spikes among the cells that had at least one [Ca 2+ ] i  spike was 0.121 ± 0.098 per minute (mean width at greater than or equal to 20%  ΔF/F 0 : 25.1 ± 10.31 s) and the mean frequency of [Ca 2+ ] i  plateau was 0.118 ± 0.058 per minute (mean duration: 160.4 ± 114.9 s). To investigate whether the baseline values of [Ca 2+ ] i  dynamics were affected by increasing neuronal activity, we induced regularly occurring population bursts by local application of bicuculline ( Schwartz and Bonhoeffer 2001 ;  n  = 7 rats). Large amplitude (0.69 ± 0.26 mV) synchronous field events (approximately 100 ms) occurred at relatively regular frequency (0.15 ± 0.06 Hz), associated with multiple unit discharges. No significant difference was observed in average heartbeat frequency between the control sessions and bicuculline sessions (4.51 ± 0.54 Hz and 4.36 ± 0.74 Hz, respectively; paired t-test,  p  = 0.13). We used two methods to evaluate the effect of neuronal activity on [Ca 2+ ] i  in astrocytes ( n  = 214 cells). First, the incidence of [Ca 2+ ] i  spikes and plateau events was counted in the absence and presence of bicuculline-induced population bursts. Under bicuculline condition significantly more astrocytes had [Ca 2+ ] i  spikes (11% versus 24%;  p  < 0.001; Fisher's exact test), whereas the probability (52% versus 54%) of plateau potentials did not differ significantly. The mean duration of plateau potentials, however, was significantly longer (160.4 ± 114.9 s versus 211.12 ± 152.175 s; t-test,  p  < 0.001) after bicuculline treatment. Among the cells that exhibited at least one spike or plateau event, there was not a significant difference in frequency of the event occurrences (spike: 0.121 ± 0.098/min versus 0.098 ± 0.068/min; t-test,  p  = 0.24; plateau 0.118 ± 0.058/min versus 0.112 ± 0.049/min; t-test,  p  = 0.46). Thus, the major difference between control and bicuculline conditions was the higher proportion of active astrocytes under bicuculline. The second method examined [Ca 2+ ] i  changes in the frequency domain. The  ΔF/F 0  trace was considered as a continuous process, and the power spectrum estimate was calculated with a multi-taper method for each astrocyte and averaged across cells. There was a general increase of power at all frequencies in bicuculline-treated animal. The most consistent significant increase ( p  < 0.05) of power appeared in the frequency range of 0.10–0.24Hz, reflecting the increased incidence of [Ca 2+ ] i  spikes. Short-term cross-correlation of neuronal field bursts and [Ca 2+ ] i  signals (± 10 s) did not show a significant time-locked relationship ( Figure 3 ). Figure 3 Frequency Domain Analysis of Population Dynamics of Fluorescence in Astrocytes in Control State and during Bicuculline-Induced Neuronal Hyperactivity Insets show local field potentials in a control animal and regular spiking in a bicuculline treated mouse (scale bar: 2.0 s, 500 μV). Asterisks show significant differences ( p  < 0.05) between groups at various frequencies.
Spontaneous Calcium Events in Astrocytes
In our initial experiments, we made a large number of line scans (sampling rate ∼200 Hz) of dye-loaded cells to examine whether some of them were neurons. We never observed short-lasting [Ca 2+ ] i  transients (less than 200 ms;  Svoboda et al. 1997 ;  Garaschuk et al. 2000 ), suggesting that the brightly loaded cells were likely to be non-neuronal ( Parri et al. 2001 ;  Stosiek et al. 2003 ). In subsequent experiments ( n  = 8 rats), cells with astrocytic appearance ( n  = 185) were selected for long-term (10–20 min) monitoring. For quantitative studies, three states of [Ca 2+ ] i  activity were distinguished: (a) quiescent state with very slow (less than 0.025 Hz) oscillations of baseline fluorescence level, (b) [Ca 2+ ] i  spikes (greater than or equal to 20% increase in  ΔF/F 0  between 5–50 s), and (c) [Ca 2+ ] i  plateau potentials (greater than or equal to 20% increase in  ΔF/F 0  for greater than 50 s). [Ca 2+ ] i  spikes and [Ca 2+ ] i  plateau potentials were automatically detected. In the control (baseline) condition, 11% of astrocytes had at least one spike event, and 52% had at least one plateau event in 10 min. The mean frequency of [Ca 2+ ] i  spikes among the cells that had at least one [Ca 2+ ] i  spike was 0.121 ± 0.098 per minute (mean width at greater than or equal to 20%  ΔF/F 0 : 25.1 ± 10.31 s) and the mean frequency of [Ca 2+ ] i  plateau was 0.118 ± 0.058 per minute (mean duration: 160.4 ± 114.9 s).nnΔF/F 0ΔF/F 0ΔF/F 0To investigate whether the baseline values of [Ca 2+ ] i  dynamics were affected by increasing neuronal activity, we induced regularly occurring population bursts by local application of bicuculline ( Schwartz and Bonhoeffer 2001 ;  n  = 7 rats). Large amplitude (0.69 ± 0.26 mV) synchronous field events (approximately 100 ms) occurred at relatively regular frequency (0.15 ± 0.06 Hz), associated with multiple unit discharges. No significant difference was observed in average heartbeat frequency between the control sessions and bicuculline sessions (4.51 ± 0.54 Hz and 4.36 ± 0.74 Hz, respectively; paired t-test,  p  = 0.13).npWe used two methods to evaluate the effect of neuronal activity on [Ca 2+ ] i  in astrocytes ( n  = 214 cells). First, the incidence of [Ca 2+ ] i  spikes and plateau events was counted in the absence and presence of bicuculline-induced population bursts. Under bicuculline condition significantly more astrocytes had [Ca 2+ ] i  spikes (11% versus 24%;  p  < 0.001; Fisher's exact test), whereas the probability (52% versus 54%) of plateau potentials did not differ significantly. The mean duration of plateau potentials, however, was significantly longer (160.4 ± 114.9 s versus 211.12 ± 152.175 s; t-test,  p  < 0.001) after bicuculline treatment. Among the cells that exhibited at least one spike or plateau event, there was not a significant difference in frequency of the event occurrences (spike: 0.121 ± 0.098/min versus 0.098 ± 0.068/min; t-test,  p  = 0.24; plateau 0.118 ± 0.058/min versus 0.112 ± 0.049/min; t-test,  p  = 0.46). Thus, the major difference between control and bicuculline conditions was the higher proportion of active astrocytes under bicuculline.nppppThe second method examined [Ca 2+ ] i  changes in the frequency domain. The  ΔF/F 0  trace was considered as a continuous process, and the power spectrum estimate was calculated with a multi-taper method for each astrocyte and averaged across cells. There was a general increase of power at all frequencies in bicuculline-treated animal. The most consistent significant increase ( p  < 0.05) of power appeared in the frequency range of 0.10–0.24Hz, reflecting the increased incidence of [Ca 2+ ] i  spikes. Short-term cross-correlation of neuronal field bursts and [Ca 2+ ] i  signals (± 10 s) did not show a significant time-locked relationship ( Figure 3 ).ΔF/F 0pSpatio-Temporal Dynamics of [Ca 2+ ] i  Events In individual experiments, propagation of synchronous activity could be observed visually ( Figure 4 A;  Video S6 ) but the spatio-temporal relationship of [Ca 2+ ] i  dynamics among astrocytes varied across experiments. To quantify the magnitude and spatial extent of this population effect, pair-wise cross-correlograms of  ΔF/F 0  intensity were calculated separately for nearby cell pairs (local: less than or equal to 50 μm) and distant cell pairs (greater than 50 μm). In control conditions, the temporal correlation of [Ca 2+ ] i  signals in neighboring pairs was somewhat larger than in distant pairs, but this difference was not significant ( n  = 374 neighbor pairs and  n  = 1,138 distant pairs). Nevertheless, [Ca 2+ ] i  signals in astrocytes were not completely random, since the cross-correlograms had wide central peaks at the 10–100 s scale ( Figure 4 B). In contrast to the baseline condition, the temporal correlation of [Ca 2+ ] i  changes in local and distant pairs were significantly different after large population bursts were brought about by bicuculline ( Figure 4 C). Correlation of distant pairs under bicuculline ( n  = 433 pairs) was similar to those in the control condition. However, synchrony between local pairs ( n  = 1,282) increased several-fold relative to both distant pairs under the same condition ( t -test,  p  < 0.0001) and to local pairs in the baseline condition ( t -test,  p  < 0.0001). Figure 4 Spatio-Temporal Dynamics of Astrocyte Ca 2+  Activity (A) Definition of nearby (less than 50 μm) and distant (greater than 50 μm) cell pairs. (B) Fluorescence changes in two nearby astrocytes. (C) Cross-correlogram of fluorescent intensity. (D) Mean cross-correlation of  ΔF/F 0  in all nearby (thick line) and distant (thin line) cell pairs in control condition (left) and in the presence of bicuculline (right). Note large increase of  ΔF/F 0  correlation in nearby cell pairs in the bicuculline condition (error bar: standard error of the mean). (E) Relationship between distance of the two cells and the magnitude of correlation at zero timelag. Note lack of a reliable relationship in the control condition (left). Note also the significant negative correlation between the distance and correlated  ΔF/F 0  changes in cell pairs in the bicuculline-treated cortex (right). Using a different approach, the magnitude of the zero-timelag correlation coefficient for each cell pair was plotted against distance between the cell pairs. Under control condition, no notable relationship was observed between these variables ( Figure 5 ;  n  = 1,512 cell pairs,  r  = 0.019,  p  = 0.46). In contrast, a significant negative correlation was found between the synchrony of [Ca 2+ ] i  signals in the bicuculline condition ( n  = 1,715;  r  = −0.281;  p  < 0.0001).
Spatio-Temporal Dynamics of [Ca 2+ ] i  Events
In individual experiments, propagation of synchronous activity could be observed visually ( Figure 4 A;  Video S6 ) but the spatio-temporal relationship of [Ca 2+ ] i  dynamics among astrocytes varied across experiments. To quantify the magnitude and spatial extent of this population effect, pair-wise cross-correlograms of  ΔF/F 0  intensity were calculated separately for nearby cell pairs (local: less than or equal to 50 μm) and distant cell pairs (greater than 50 μm). In control conditions, the temporal correlation of [Ca 2+ ] i  signals in neighboring pairs was somewhat larger than in distant pairs, but this difference was not significant ( n  = 374 neighbor pairs and  n  = 1,138 distant pairs). Nevertheless, [Ca 2+ ] i  signals in astrocytes were not completely random, since the cross-correlograms had wide central peaks at the 10–100 s scale ( Figure 4 B). In contrast to the baseline condition, the temporal correlation of [Ca 2+ ] i  changes in local and distant pairs were significantly different after large population bursts were brought about by bicuculline ( Figure 4 C). Correlation of distant pairs under bicuculline ( n  = 433 pairs) was similar to those in the control condition. However, synchrony between local pairs ( n  = 1,282) increased several-fold relative to both distant pairs under the same condition ( t -test,  p  < 0.0001) and to local pairs in the baseline condition ( t -test,  p  < 0.0001).ΔF/F 0nnnntptpUsing a different approach, the magnitude of the zero-timelag correlation coefficient for each cell pair was plotted against distance between the cell pairs. Under control condition, no notable relationship was observed between these variables ( Figure 5 ;  n  = 1,512 cell pairs,  r  = 0.019,  p  = 0.46). In contrast, a significant negative correlation was found between the synchrony of [Ca 2+ ] i  signals in the bicuculline condition ( n  = 1,715;  r  = −0.281;  p  < 0.0001).nrpnrpDiscussion Astrocytes in superficial cortical layers were successfully loaded using Fluo-4 AM by surface application up to 350 μm from the pial surface in juvenile rats. In agreement with previous literature ( Parri et al. 2001 ;  Dallwig and Deitmer 2002 ;  Simard et al. 2003 ), the majority of the Fluo-4-loaded cells exhibited astrocytic morphology with multipolar branching and bushy microprocesses impinging on local vasculature. 2-PLSM imaging revealed spontaneous [Ca 2+ ] i  events in individual astrocytes in vivo. Some coordination of these events was indicated by the broad cross-correlograms in the baseline condition. Increased neuronal discharge was associated with increased astrocytic activity and a robust coordination of [Ca 2+ ] i  signals in neighboring astrocytes, providing evidence for neuron–glia communication in the intact brain. The magnitude, frequency and pattern of [Ca 2+ ] i  events observed here are qualitatively similar to those described in tissue cultures ( Dani et al. 1992 ;  Charles 1998 ) and acute hippocampal, neocortical, and thalamic slice preparations ( Parri et al. 2001 ;  Aguado et al. 2002 ;  Nett et al. 2002;  Tashiro et al. 2002 ). It has been reported that the percentage of active astrocytes in brain slices showed a 2- to 3-fold decrease from early postnatal days to juvenile age ( Parri et al. 2001 ;  Aguado et al. 2002 ). In our experiments, a large portion of the imaged astrocytes were active, showing either [Ca 2+ ] i  or plateau potentials. It is unlikely that the elevated activity in vivo is due to anesthesia because urethane is known to suppress transmitter release from presynaptic vesicles and attenuate both α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and NMDA receptors ( Hara and Harris 2002 ). Since blockade of these receptors decreases astrocytic [Ca 2+ ] i  activity in vitro ( Parri et al. 2001 ;  Aguado et al. 2002 ), it is expected that in the drug-free animal the percentage of active cells will be even higher. A different explanation for the lower percentage of active astrocytes in the slice, relative to the in vivo situation and tissue culture preparation, is that the trauma of brain slicing attenuates spontaneous [Ca 2+ ] i  activity. Reactive astrocytes in a stab wound area show very limited [Ca 2+ ] i  activity ( Aguado et al. 2002 ). In addition, the temperature at which the cells are kept may be playing an important role. In the absence of provoking conditions, spontaneous [Ca 2+ ] i  activity in individual astrocytes does not spread among astrocytes as an intercellular Ca 2+  wave ( Nett et al. 2002). In baseline condition, the magnitude of correlated activity in nearby and distant astrocytes was quite similar. Nevertheless, the presence of zero-timelag correlation suggests that activity in the astrocytic syncytium in vivo is not random, but is under some coordinated control. Widespread but limited coordination of glial cells can be brought about by common synchronizing inputs in the intact brain, such as vascular and vegetative nervous system control or large-scale slow changes of neuronal excitability. The latter possibility is supported by the observation that ionotropic glutamate receptor antagonists and tetrodotoxin effectively decorrelated the astrocytic network without altering the number of active astrocytes ( Aguado et al. 2002 ). Furthermore, the intact corticothalamic system displays substantial excitability fluctuation at the time scale of the astrocytic [Ca 2+ ] i  events ( Jando et al. 1995 ). Although neuronal activity is not needed to generate [Ca 2+ ] i  surges in astrocytes ( Aguado et al. 2002 ;  Nett et al. 2002), neurotransmitters can enhance the frequency of such events. The impact of neuronal activity on the glial network is illustrated by the increased activity and enhanced local correlation of [Ca 2+ ] i  signal in astrocytes after regular population bursting of neurons was brought about by the GABA A -receptor blocker bicuculline. These changes shared similarities to those observed in hippocampal and neocortical slices ( Aguado et al. 2002 ;  Tashiro et al. 2002 ). In contrast to the slice situation, we did not find a time-locked triggering of astrocytic events to the neuronal bursts (see also  Nett et al. 2002). This discrepancy may be explained by the magnitude of the evoked neuronal bursts. Bicuculline in vitro evoked rare (greater than 30 s intervals), but very large bursts or afterdischarges ( Tashiro et al. 2002 ;  Aguado et al. 2002 ). In vivo, synchronous events of moderate size occurred frequently (approximately 0.3 Hz). The enhanced bursts, associated with large field potentials, can be regarded as interictal epileptic spikes ( Schwartz and Bonhoeffer 2001 ), but seizures were never observed. Although the exact mechanisms of neuron–astrocyte signaling remain to be disclosed, our findings indicate that neuronal and glial networks are coupled in the intact brain. Many of the imaged astrocytes had processes (end feet) in close contact with small brain vessels ( Peters et al. 1970 ). It has been shown that surges of [Ca 2+ ] i  in astrocytes trigger the release of vasoactive compounds ( Bezzi et al. 1998 ). Furthermore, stimulation of single astrocytes in cortical slices led to delayed (greater than 30 s) and protracted dilation of the contacted arteriole ( Zonta et al. 2003 ). These findings support the view that a cardinal function of astrocytes in the intact brain is to regulate local circulation according to the metabolic needs of neurons. Overall, the approach introduced in this paper will be a potent tool to investigate these issues in vivo.
Discussion
Astrocytes in superficial cortical layers were successfully loaded using Fluo-4 AM by surface application up to 350 μm from the pial surface in juvenile rats. In agreement with previous literature ( Parri et al. 2001 ;  Dallwig and Deitmer 2002 ;  Simard et al. 2003 ), the majority of the Fluo-4-loaded cells exhibited astrocytic morphology with multipolar branching and bushy microprocesses impinging on local vasculature. 2-PLSM imaging revealed spontaneous [Ca 2+ ] i  events in individual astrocytes in vivo. Some coordination of these events was indicated by the broad cross-correlograms in the baseline condition. Increased neuronal discharge was associated with increased astrocytic activity and a robust coordination of [Ca 2+ ] i  signals in neighboring astrocytes, providing evidence for neuron–glia communication in the intact brain.The magnitude, frequency and pattern of [Ca 2+ ] i  events observed here are qualitatively similar to those described in tissue cultures ( Dani et al. 1992 ;  Charles 1998 ) and acute hippocampal, neocortical, and thalamic slice preparations ( Parri et al. 2001 ;  Aguado et al. 2002 ;  Nett et al. 2002;  Tashiro et al. 2002 ). It has been reported that the percentage of active astrocytes in brain slices showed a 2- to 3-fold decrease from early postnatal days to juvenile age ( Parri et al. 2001 ;  Aguado et al. 2002 ). In our experiments, a large portion of the imaged astrocytes were active, showing either [Ca 2+ ] i  or plateau potentials. It is unlikely that the elevated activity in vivo is due to anesthesia because urethane is known to suppress transmitter release from presynaptic vesicles and attenuate both α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) and NMDA receptors ( Hara and Harris 2002 ). Since blockade of these receptors decreases astrocytic [Ca 2+ ] i  activity in vitro ( Parri et al. 2001 ;  Aguado et al. 2002 ), it is expected that in the drug-free animal the percentage of active cells will be even higher. A different explanation for the lower percentage of active astrocytes in the slice, relative to the in vivo situation and tissue culture preparation, is that the trauma of brain slicing attenuates spontaneous [Ca 2+ ] i  activity. Reactive astrocytes in a stab wound area show very limited [Ca 2+ ] i  activity ( Aguado et al. 2002 ). In addition, the temperature at which the cells are kept may be playing an important role.In the absence of provoking conditions, spontaneous [Ca 2+ ] i  activity in individual astrocytes does not spread among astrocytes as an intercellular Ca 2+  wave ( Nett et al. 2002). In baseline condition, the magnitude of correlated activity in nearby and distant astrocytes was quite similar. Nevertheless, the presence of zero-timelag correlation suggests that activity in the astrocytic syncytium in vivo is not random, but is under some coordinated control. Widespread but limited coordination of glial cells can be brought about by common synchronizing inputs in the intact brain, such as vascular and vegetative nervous system control or large-scale slow changes of neuronal excitability. The latter possibility is supported by the observation that ionotropic glutamate receptor antagonists and tetrodotoxin effectively decorrelated the astrocytic network without altering the number of active astrocytes ( Aguado et al. 2002 ). Furthermore, the intact corticothalamic system displays substantial excitability fluctuation at the time scale of the astrocytic [Ca 2+ ] i  events ( Jando et al. 1995 ).Although neuronal activity is not needed to generate [Ca 2+ ] i  surges in astrocytes ( Aguado et al. 2002 ;  Nett et al. 2002), neurotransmitters can enhance the frequency of such events. The impact of neuronal activity on the glial network is illustrated by the increased activity and enhanced local correlation of [Ca 2+ ] i  signal in astrocytes after regular population bursting of neurons was brought about by the GABA A -receptor blocker bicuculline. These changes shared similarities to those observed in hippocampal and neocortical slices ( Aguado et al. 2002 ;  Tashiro et al. 2002 ). In contrast to the slice situation, we did not find a time-locked triggering of astrocytic events to the neuronal bursts (see also  Nett et al. 2002). This discrepancy may be explained by the magnitude of the evoked neuronal bursts. Bicuculline in vitro evoked rare (greater than 30 s intervals), but very large bursts or afterdischarges ( Tashiro et al. 2002 ;  Aguado et al. 2002 ). In vivo, synchronous events of moderate size occurred frequently (approximately 0.3 Hz). The enhanced bursts, associated with large field potentials, can be regarded as interictal epileptic spikes ( Schwartz and Bonhoeffer 2001 ), but seizures were never observed. Although the exact mechanisms of neuron–astrocyte signaling remain to be disclosed, our findings indicate that neuronal and glial networks are coupled in the intact brain.Many of the imaged astrocytes had processes (end feet) in close contact with small brain vessels ( Peters et al. 1970 ). It has been shown that surges of [Ca 2+ ] i  in astrocytes trigger the release of vasoactive compounds ( Bezzi et al. 1998 ). Furthermore, stimulation of single astrocytes in cortical slices led to delayed (greater than 30 s) and protracted dilation of the contacted arteriole ( Zonta et al. 2003 ). These findings support the view that a cardinal function of astrocytes in the intact brain is to regulate local circulation according to the metabolic needs of neurons. Overall, the approach introduced in this paper will be a potent tool to investigate these issues in vivo.Materials and Methods Subjects and surgery Male and female rats, 12–16 d postnatal (P12– P16), of the Sprague–Dawley strain were used in these experiments. Animals were deeply anesthetized with 1.7 g/kg urethane. An outline of the craniotomy above the primary somatosensory (barrel) cortex was marked with a dental drill. A metal frame, similar to what has been described in  Kleinfeld and Denk (2000 ), was attached to the skull with cyanoacrylic. A craniotomy (3–4 mm diameter), centered at 1.5 mm posterior to bregma and 2.5 mm from midline, was performed and the dura mater was surgically removed. Care was taken to avoid any damage to pial vessels or the cortex. Dye loading Fluo-4 AM (F-14201, 50 μg; Molecular Probes, Eugene, Oregon, United States) was mixed with 2 μl of Pluronic (P-3000, Molecular Probes) and 5 μl of dymethyl sulfoxide (D-8779; Sigma, St. Louis, Missouri, United States) for 15 min. The solution was then diluted in 18 μl of artificial cerebrospinal fluid (ACSF) (125 mM NaCl, 3 mM KCl, 10 mM glucose, 26 mM NaHCO 3 , 1.1 mM NaH 2 PO 4 , 2 mM CaCl 2 , 1 mM MgSO 4 ; pH adjusted to 7.4) and mixed for a further 15 min. A small volume (up to 12 μl) of the dye-containing solution was applied to the cortical surface by a micropipette. The solution was retained in place by a small piece gelfoam. The unbound dye was removed 45–60 min after the surface application of Fluo-4 AM by irrigating the exposed surface with ACSF for at least 10 min. The craniotomy was then covered with 1% agar dissolved in phosphate-buffered saline (pH 7.4), and a glass coverslip was placed on a metal frame. This arrangement allowed access for a glass recording electrode from the side. Juvenile rats (P13–P15) were used because we found in preliminary experiments that in adult animals, mostly vascular cells were loaded with the current protocol. Electrophysiological recording During the recording session, a heating blanket was placed under the rat to maintain body temperature at approximately 37°C. The electrocardiogram (EKG) was monitored continuously. The R wave of EKG was used to monitor brain pulsation-derived movement of artifacts during imaging. Population bursts of cortical neurons (“interictal” spikes;  Schwartz and Bonhoeffer 2001 ) were induced by inserting a large-tip (20–50 μm tip diameter) glass pipette, containing 2 mM bicuculline in 0.9% (w/v) NaCl, into the deep layers of the somatosensory cortex. This electrode also served to record local field potential and multiple unit activity. Large population bursts were reliably induced 10–30 min after the insertion of the pipette. Imaging A custom-made 2-PLSM was constructed as described earlier ( Majewska et al. 2000 ). In brief, a Ti:S laser (Mira 800F; Coherent, Santa Clara, California, United States) was pumped by a solid state CW laser (Verdi 8; Coherent) to produce a mode-locked beam (840 nm; approximately 100 fs pulse width at 76 MHz repetition rate). The beam was directed to a modified confocal scanhead (Fluoview 300; Olympus, Tokyo, Japan). The fluorescent signal was first filtered with an emission filter (HQ525, passband 525 ± 25 nm; Chroma, Rockingham, Vermont, United States) and detected by an external photo-multiplier tube (R-3896, Hamamatsu Photonics, Hamamatsu City, Japan) with a built-in preamplifier board (F-5 PSU-B; Olympus). Data analysis Fluorescence signal was quantified by measuring the mean pixel value of a manually selected somatic area for each frame of the image stack using ImageJ software. The values were exported to MatLab and the fluorescence change  ΔF/F 0  was computed, where  F 0  is the mean of the lowest 20% of the somatic fluorescence signals. Sessions that had visible drifts when image sequences were replayed as animation (the majority of the cells showed correlated activity [ |r|  > 0.6], or greater than 10% fluorescence change due to the heartbeat when the cell was imaged in line scan [approximately 200 Hz]) were excluded from the analysis. For display purposes, the signal was convolved with a Hanning window of order three to smooth the signal trace. Power spectra of fluorescent signals were computed using the multi-taper method  (NW = 4) . For the calcium event detection,  ΔF/F 0  signal was convolved with a Hanning window of order 15. “Spike” events were defined as transient increase of  ΔF/F 0  signal exceeding 20%, lasting 5–50 s. “Plateau” events were defined as sustained increase of  ΔF/F 0  (greater than 20%) signal longer than 50 s. Peak amplitudes of both spike and plateau events required an increase of at least 50%  ΔF/F 0  from the onset of events. Calcium events were automatically detected with the above detection. Cross-correlation between cell pairs was computed by normalizing the  ΔF/F 0  signals to unity (zero mean, unity standard deviation) so that the computed values represent the correlation coefficient between the two signals at a given timelag. All numbers are indicated as mean ± standard deviation, unless otherwise noted. Immunocytochemisty Since Fluo-4 AM loading was best visible in the somatic region of the putative astrocytes, we chose S100B antibody (A5110; DakoCytomation, Glostrup, Denmark) because this antibody stains the somatic region of astrocytes as well as its processes ( Ren et al. 1992 ). Following Fluo-4 AM loading, acute brain slices (300 μm thickness) were cut coronally around the dye-loaded area using standard procedures. Fluo-4 in cells of the acute brain slices were fixed by incubating the acute brain slices in freshly made saline containing 40 mg/ml 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDAC, E7750; Sigma) for 30 min. Next, the slices were incubated in formalin-based fixative (4% formaldehyde, 0.1 M phosphate buffer, [pH ∼7.1]) for 30 min. Once the fixation procedures were completed, the sections were mounted on a glass slide and imaged with 2-PLSM (z-stack; wavelength, 840 nm). After imaging of calcium-loaded cells, and three subsequent washes in phosphate-buffered saline (PBS) (1.06 mM KH 2 PO 4 , 155.17 mM NaCl, 2.96 mM NaHPO 4 , pH approximately 7.4), the slices were treated with S100B antibody (made in rabbit, 1:50 dilution) in Triton X-PBS (0.5% Triton X in PBS) overnight. The sections were subsequently washed three times in PBS, followed by incubation with the secondary fluorescent antibody (1:1000 dilution, 711-166-152, CY3 Anti-Rabbit IgG [H + L]; Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, United States) in Triton X-PBS solution for 2 h. Simultaneous viewing of the two image stacks allowed a systematic comparison of the extent of overlap between Fluo-4 loading and S100B immunoreactivity ( Video S5 ).
Materials and Methods
Subjects and surgery Male and female rats, 12–16 d postnatal (P12– P16), of the Sprague–Dawley strain were used in these experiments. Animals were deeply anesthetized with 1.7 g/kg urethane. An outline of the craniotomy above the primary somatosensory (barrel) cortex was marked with a dental drill. A metal frame, similar to what has been described in  Kleinfeld and Denk (2000 ), was attached to the skull with cyanoacrylic. A craniotomy (3–4 mm diameter), centered at 1.5 mm posterior to bregma and 2.5 mm from midline, was performed and the dura mater was surgically removed. Care was taken to avoid any damage to pial vessels or the cortex. Dye loading Fluo-4 AM (F-14201, 50 μg; Molecular Probes, Eugene, Oregon, United States) was mixed with 2 μl of Pluronic (P-3000, Molecular Probes) and 5 μl of dymethyl sulfoxide (D-8779; Sigma, St. Louis, Missouri, United States) for 15 min. The solution was then diluted in 18 μl of artificial cerebrospinal fluid (ACSF) (125 mM NaCl, 3 mM KCl, 10 mM glucose, 26 mM NaHCO 3 , 1.1 mM NaH 2 PO 4 , 2 mM CaCl 2 , 1 mM MgSO 4 ; pH adjusted to 7.4) and mixed for a further 15 min. A small volume (up to 12 μl) of the dye-containing solution was applied to the cortical surface by a micropipette. The solution was retained in place by a small piece gelfoam. The unbound dye was removed 45–60 min after the surface application of Fluo-4 AM by irrigating the exposed surface with ACSF for at least 10 min. The craniotomy was then covered with 1% agar dissolved in phosphate-buffered saline (pH 7.4), and a glass coverslip was placed on a metal frame. This arrangement allowed access for a glass recording electrode from the side. Juvenile rats (P13–P15) were used because we found in preliminary experiments that in adult animals, mostly vascular cells were loaded with the current protocol. Electrophysiological recording During the recording session, a heating blanket was placed under the rat to maintain body temperature at approximately 37°C. The electrocardiogram (EKG) was monitored continuously. The R wave of EKG was used to monitor brain pulsation-derived movement of artifacts during imaging. Population bursts of cortical neurons (“interictal” spikes;  Schwartz and Bonhoeffer 2001 ) were induced by inserting a large-tip (20–50 μm tip diameter) glass pipette, containing 2 mM bicuculline in 0.9% (w/v) NaCl, into the deep layers of the somatosensory cortex. This electrode also served to record local field potential and multiple unit activity. Large population bursts were reliably induced 10–30 min after the insertion of the pipette. Imaging A custom-made 2-PLSM was constructed as described earlier ( Majewska et al. 2000 ). In brief, a Ti:S laser (Mira 800F; Coherent, Santa Clara, California, United States) was pumped by a solid state CW laser (Verdi 8; Coherent) to produce a mode-locked beam (840 nm; approximately 100 fs pulse width at 76 MHz repetition rate). The beam was directed to a modified confocal scanhead (Fluoview 300; Olympus, Tokyo, Japan). The fluorescent signal was first filtered with an emission filter (HQ525, passband 525 ± 25 nm; Chroma, Rockingham, Vermont, United States) and detected by an external photo-multiplier tube (R-3896, Hamamatsu Photonics, Hamamatsu City, Japan) with a built-in preamplifier board (F-5 PSU-B; Olympus). Data analysis Fluorescence signal was quantified by measuring the mean pixel value of a manually selected somatic area for each frame of the image stack using ImageJ software. The values were exported to MatLab and the fluorescence change  ΔF/F 0  was computed, where  F 0  is the mean of the lowest 20% of the somatic fluorescence signals. Sessions that had visible drifts when image sequences were replayed as animation (the majority of the cells showed correlated activity [ |r|  > 0.6], or greater than 10% fluorescence change due to the heartbeat when the cell was imaged in line scan [approximately 200 Hz]) were excluded from the analysis. For display purposes, the signal was convolved with a Hanning window of order three to smooth the signal trace. Power spectra of fluorescent signals were computed using the multi-taper method  (NW = 4) . For the calcium event detection,  ΔF/F 0  signal was convolved with a Hanning window of order 15. “Spike” events were defined as transient increase of  ΔF/F 0  signal exceeding 20%, lasting 5–50 s. “Plateau” events were defined as sustained increase of  ΔF/F 0  (greater than 20%) signal longer than 50 s. Peak amplitudes of both spike and plateau events required an increase of at least 50%  ΔF/F 0  from the onset of events. Calcium events were automatically detected with the above detection. Cross-correlation between cell pairs was computed by normalizing the  ΔF/F 0  signals to unity (zero mean, unity standard deviation) so that the computed values represent the correlation coefficient between the two signals at a given timelag. All numbers are indicated as mean ± standard deviation, unless otherwise noted. Immunocytochemisty Since Fluo-4 AM loading was best visible in the somatic region of the putative astrocytes, we chose S100B antibody (A5110; DakoCytomation, Glostrup, Denmark) because this antibody stains the somatic region of astrocytes as well as its processes ( Ren et al. 1992 ). Following Fluo-4 AM loading, acute brain slices (300 μm thickness) were cut coronally around the dye-loaded area using standard procedures. Fluo-4 in cells of the acute brain slices were fixed by incubating the acute brain slices in freshly made saline containing 40 mg/ml 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDAC, E7750; Sigma) for 30 min. Next, the slices were incubated in formalin-based fixative (4% formaldehyde, 0.1 M phosphate buffer, [pH ∼7.1]) for 30 min. Once the fixation procedures were completed, the sections were mounted on a glass slide and imaged with 2-PLSM (z-stack; wavelength, 840 nm). After imaging of calcium-loaded cells, and three subsequent washes in phosphate-buffered saline (PBS) (1.06 mM KH 2 PO 4 , 155.17 mM NaCl, 2.96 mM NaHPO 4 , pH approximately 7.4), the slices were treated with S100B antibody (made in rabbit, 1:50 dilution) in Triton X-PBS (0.5% Triton X in PBS) overnight. The sections were subsequently washed three times in PBS, followed by incubation with the secondary fluorescent antibody (1:1000 dilution, 711-166-152, CY3 Anti-Rabbit IgG [H + L]; Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, United States) in Triton X-PBS solution for 2 h. Simultaneous viewing of the two image stacks allowed a systematic comparison of the extent of overlap between Fluo-4 loading and S100B immunoreactivity ( Video S5 ).

Subjects and surgery Male and female rats, 12–16 d postnatal (P12– P16), of the Sprague–Dawley strain were used in these experiments. Animals were deeply anesthetized with 1.7 g/kg urethane. An outline of the craniotomy above the primary somatosensory (barrel) cortex was marked with a dental drill. A metal frame, similar to what has been described in  Kleinfeld and Denk (2000 ), was attached to the skull with cyanoacrylic. A craniotomy (3–4 mm diameter), centered at 1.5 mm posterior to bregma and 2.5 mm from midline, was performed and the dura mater was surgically removed. Care was taken to avoid any damage to pial vessels or the cortex.
Subjects and surgery
Male and female rats, 12–16 d postnatal (P12– P16), of the Sprague–Dawley strain were used in these experiments. Animals were deeply anesthetized with 1.7 g/kg urethane. An outline of the craniotomy above the primary somatosensory (barrel) cortex was marked with a dental drill. A metal frame, similar to what has been described in  Kleinfeld and Denk (2000 ), was attached to the skull with cyanoacrylic. A craniotomy (3–4 mm diameter), centered at 1.5 mm posterior to bregma and 2.5 mm from midline, was performed and the dura mater was surgically removed. Care was taken to avoid any damage to pial vessels or the cortex.Dye loading Fluo-4 AM (F-14201, 50 μg; Molecular Probes, Eugene, Oregon, United States) was mixed with 2 μl of Pluronic (P-3000, Molecular Probes) and 5 μl of dymethyl sulfoxide (D-8779; Sigma, St. Louis, Missouri, United States) for 15 min. The solution was then diluted in 18 μl of artificial cerebrospinal fluid (ACSF) (125 mM NaCl, 3 mM KCl, 10 mM glucose, 26 mM NaHCO 3 , 1.1 mM NaH 2 PO 4 , 2 mM CaCl 2 , 1 mM MgSO 4 ; pH adjusted to 7.4) and mixed for a further 15 min. A small volume (up to 12 μl) of the dye-containing solution was applied to the cortical surface by a micropipette. The solution was retained in place by a small piece gelfoam. The unbound dye was removed 45–60 min after the surface application of Fluo-4 AM by irrigating the exposed surface with ACSF for at least 10 min. The craniotomy was then covered with 1% agar dissolved in phosphate-buffered saline (pH 7.4), and a glass coverslip was placed on a metal frame. This arrangement allowed access for a glass recording electrode from the side. Juvenile rats (P13–P15) were used because we found in preliminary experiments that in adult animals, mostly vascular cells were loaded with the current protocol.
Dye loading
Fluo-4 AM (F-14201, 50 μg; Molecular Probes, Eugene, Oregon, United States) was mixed with 2 μl of Pluronic (P-3000, Molecular Probes) and 5 μl of dymethyl sulfoxide (D-8779; Sigma, St. Louis, Missouri, United States) for 15 min. The solution was then diluted in 18 μl of artificial cerebrospinal fluid (ACSF) (125 mM NaCl, 3 mM KCl, 10 mM glucose, 26 mM NaHCO 3 , 1.1 mM NaH 2 PO 4 , 2 mM CaCl 2 , 1 mM MgSO 4 ; pH adjusted to 7.4) and mixed for a further 15 min. A small volume (up to 12 μl) of the dye-containing solution was applied to the cortical surface by a micropipette. The solution was retained in place by a small piece gelfoam. The unbound dye was removed 45–60 min after the surface application of Fluo-4 AM by irrigating the exposed surface with ACSF for at least 10 min. The craniotomy was then covered with 1% agar dissolved in phosphate-buffered saline (pH 7.4), and a glass coverslip was placed on a metal frame. This arrangement allowed access for a glass recording electrode from the side. Juvenile rats (P13–P15) were used because we found in preliminary experiments that in adult animals, mostly vascular cells were loaded with the current protocol.Electrophysiological recording During the recording session, a heating blanket was placed under the rat to maintain body temperature at approximately 37°C. The electrocardiogram (EKG) was monitored continuously. The R wave of EKG was used to monitor brain pulsation-derived movement of artifacts during imaging. Population bursts of cortical neurons (“interictal” spikes;  Schwartz and Bonhoeffer 2001 ) were induced by inserting a large-tip (20–50 μm tip diameter) glass pipette, containing 2 mM bicuculline in 0.9% (w/v) NaCl, into the deep layers of the somatosensory cortex. This electrode also served to record local field potential and multiple unit activity. Large population bursts were reliably induced 10–30 min after the insertion of the pipette.
Electrophysiological recording
During the recording session, a heating blanket was placed under the rat to maintain body temperature at approximately 37°C. The electrocardiogram (EKG) was monitored continuously. The R wave of EKG was used to monitor brain pulsation-derived movement of artifacts during imaging. Population bursts of cortical neurons (“interictal” spikes;  Schwartz and Bonhoeffer 2001 ) were induced by inserting a large-tip (20–50 μm tip diameter) glass pipette, containing 2 mM bicuculline in 0.9% (w/v) NaCl, into the deep layers of the somatosensory cortex. This electrode also served to record local field potential and multiple unit activity. Large population bursts were reliably induced 10–30 min after the insertion of the pipette.Imaging A custom-made 2-PLSM was constructed as described earlier ( Majewska et al. 2000 ). In brief, a Ti:S laser (Mira 800F; Coherent, Santa Clara, California, United States) was pumped by a solid state CW laser (Verdi 8; Coherent) to produce a mode-locked beam (840 nm; approximately 100 fs pulse width at 76 MHz repetition rate). The beam was directed to a modified confocal scanhead (Fluoview 300; Olympus, Tokyo, Japan). The fluorescent signal was first filtered with an emission filter (HQ525, passband 525 ± 25 nm; Chroma, Rockingham, Vermont, United States) and detected by an external photo-multiplier tube (R-3896, Hamamatsu Photonics, Hamamatsu City, Japan) with a built-in preamplifier board (F-5 PSU-B; Olympus).
Imaging
A custom-made 2-PLSM was constructed as described earlier ( Majewska et al. 2000 ). In brief, a Ti:S laser (Mira 800F; Coherent, Santa Clara, California, United States) was pumped by a solid state CW laser (Verdi 8; Coherent) to produce a mode-locked beam (840 nm; approximately 100 fs pulse width at 76 MHz repetition rate). The beam was directed to a modified confocal scanhead (Fluoview 300; Olympus, Tokyo, Japan). The fluorescent signal was first filtered with an emission filter (HQ525, passband 525 ± 25 nm; Chroma, Rockingham, Vermont, United States) and detected by an external photo-multiplier tube (R-3896, Hamamatsu Photonics, Hamamatsu City, Japan) with a built-in preamplifier board (F-5 PSU-B; Olympus).Data analysis Fluorescence signal was quantified by measuring the mean pixel value of a manually selected somatic area for each frame of the image stack using ImageJ software. The values were exported to MatLab and the fluorescence change  ΔF/F 0  was computed, where  F 0  is the mean of the lowest 20% of the somatic fluorescence signals. Sessions that had visible drifts when image sequences were replayed as animation (the majority of the cells showed correlated activity [ |r|  > 0.6], or greater than 10% fluorescence change due to the heartbeat when the cell was imaged in line scan [approximately 200 Hz]) were excluded from the analysis. For display purposes, the signal was convolved with a Hanning window of order three to smooth the signal trace. Power spectra of fluorescent signals were computed using the multi-taper method  (NW = 4) . For the calcium event detection,  ΔF/F 0  signal was convolved with a Hanning window of order 15. “Spike” events were defined as transient increase of  ΔF/F 0  signal exceeding 20%, lasting 5–50 s. “Plateau” events were defined as sustained increase of  ΔF/F 0  (greater than 20%) signal longer than 50 s. Peak amplitudes of both spike and plateau events required an increase of at least 50%  ΔF/F 0  from the onset of events. Calcium events were automatically detected with the above detection. Cross-correlation between cell pairs was computed by normalizing the  ΔF/F 0  signals to unity (zero mean, unity standard deviation) so that the computed values represent the correlation coefficient between the two signals at a given timelag. All numbers are indicated as mean ± standard deviation, unless otherwise noted.
Data analysis
Fluorescence signal was quantified by measuring the mean pixel value of a manually selected somatic area for each frame of the image stack using ImageJ software. The values were exported to MatLab and the fluorescence change  ΔF/F 0  was computed, where  F 0  is the mean of the lowest 20% of the somatic fluorescence signals. Sessions that had visible drifts when image sequences were replayed as animation (the majority of the cells showed correlated activity [ |r|  > 0.6], or greater than 10% fluorescence change due to the heartbeat when the cell was imaged in line scan [approximately 200 Hz]) were excluded from the analysis. For display purposes, the signal was convolved with a Hanning window of order three to smooth the signal trace. Power spectra of fluorescent signals were computed using the multi-taper method  (NW = 4) . For the calcium event detection,  ΔF/F 0  signal was convolved with a Hanning window of order 15. “Spike” events were defined as transient increase of  ΔF/F 0  signal exceeding 20%, lasting 5–50 s. “Plateau” events were defined as sustained increase of  ΔF/F 0  (greater than 20%) signal longer than 50 s. Peak amplitudes of both spike and plateau events required an increase of at least 50%  ΔF/F 0  from the onset of events. Calcium events were automatically detected with the above detection. Cross-correlation between cell pairs was computed by normalizing the  ΔF/F 0  signals to unity (zero mean, unity standard deviation) so that the computed values represent the correlation coefficient between the two signals at a given timelag. All numbers are indicated as mean ± standard deviation, unless otherwise noted.ΔF/F 0F 0|r|(NW = 4)ΔF/F 0ΔF/F 0ΔF/F 0ΔF/F 0ΔF/F 0Immunocytochemisty Since Fluo-4 AM loading was best visible in the somatic region of the putative astrocytes, we chose S100B antibody (A5110; DakoCytomation, Glostrup, Denmark) because this antibody stains the somatic region of astrocytes as well as its processes ( Ren et al. 1992 ). Following Fluo-4 AM loading, acute brain slices (300 μm thickness) were cut coronally around the dye-loaded area using standard procedures. Fluo-4 in cells of the acute brain slices were fixed by incubating the acute brain slices in freshly made saline containing 40 mg/ml 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDAC, E7750; Sigma) for 30 min. Next, the slices were incubated in formalin-based fixative (4% formaldehyde, 0.1 M phosphate buffer, [pH ∼7.1]) for 30 min. Once the fixation procedures were completed, the sections were mounted on a glass slide and imaged with 2-PLSM (z-stack; wavelength, 840 nm). After imaging of calcium-loaded cells, and three subsequent washes in phosphate-buffered saline (PBS) (1.06 mM KH 2 PO 4 , 155.17 mM NaCl, 2.96 mM NaHPO 4 , pH approximately 7.4), the slices were treated with S100B antibody (made in rabbit, 1:50 dilution) in Triton X-PBS (0.5% Triton X in PBS) overnight. The sections were subsequently washed three times in PBS, followed by incubation with the secondary fluorescent antibody (1:1000 dilution, 711-166-152, CY3 Anti-Rabbit IgG [H + L]; Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, United States) in Triton X-PBS solution for 2 h. Simultaneous viewing of the two image stacks allowed a systematic comparison of the extent of overlap between Fluo-4 loading and S100B immunoreactivity ( Video S5 ).
Immunocytochemisty
Since Fluo-4 AM loading was best visible in the somatic region of the putative astrocytes, we chose S100B antibody (A5110; DakoCytomation, Glostrup, Denmark) because this antibody stains the somatic region of astrocytes as well as its processes ( Ren et al. 1992 ). Following Fluo-4 AM loading, acute brain slices (300 μm thickness) were cut coronally around the dye-loaded area using standard procedures. Fluo-4 in cells of the acute brain slices were fixed by incubating the acute brain slices in freshly made saline containing 40 mg/ml 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDAC, E7750; Sigma) for 30 min. Next, the slices were incubated in formalin-based fixative (4% formaldehyde, 0.1 M phosphate buffer, [pH ∼7.1]) for 30 min. Once the fixation procedures were completed, the sections were mounted on a glass slide and imaged with 2-PLSM (z-stack; wavelength, 840 nm). After imaging of calcium-loaded cells, and three subsequent washes in phosphate-buffered saline (PBS) (1.06 mM KH 2 PO 4 , 155.17 mM NaCl, 2.96 mM NaHPO 4 , pH approximately 7.4), the slices were treated with S100B antibody (made in rabbit, 1:50 dilution) in Triton X-PBS (0.5% Triton X in PBS) overnight. The sections were subsequently washed three times in PBS, followed by incubation with the secondary fluorescent antibody (1:1000 dilution, 711-166-152, CY3 Anti-Rabbit IgG [H + L]; Jackson ImmunoResearch Laboratories, West Grove, Pennsylvania, United States) in Triton X-PBS solution for 2 h. Simultaneous viewing of the two image stacks allowed a systematic comparison of the extent of overlap between Fluo-4 loading and S100B immunoreactivity ( Video S5 ).Supporting Information Video S1 Visualization of Loaded Astrocytes (Low Magnification) The primary somatosensory cortex (P15) was stained with Fluo-4 AM in vivo and subsequently imaged in vitro. Acute slices (approximately 300 μm thickness) were cut in cold ACSF after the cells were loaded in vivo. (Z step = 1 μm; scale bar = 50 μm). (49 MB AVI). Click here for additional data file. Video S2 Visualization of Loaded Astrocytes (High Magnification, Layer I) Same slice as shown in  Videos S1 , but with higher magnification. Z step = 1 μm; scale bar = 20 μm. (48 MB AVI). Click here for additional data file. Video S3 Visualization of Loaded Astrocytes (High Magnification, Layers II/III) Detailed imaging of in vivo-loaded acute slice preparation of the primary somatosensory cortex (P15; approximately 270 μm below the pial surface). Z step = 1 μm; scale bar = 20 μm. (39 MB AVI). Click here for additional data file. Video S4 High-Contrast Image Upper Layers (I to II/III) of the Fluo-4 AM-Loaded Somatosensory Cortex (P15) Empty circles in layers II/III, presumed unloaded neurons (note their absence in layer I). The loaded cells have typical glial morphological appearance. Z step = 1 μm; scale bar = 50 μm. (50 MB AVI). Click here for additional data file. Video S5 Double-Labeling of Fluo-4 AM-Loaded Astrocytes with S100B Antibody Acute slices (300 μm thickness) were prepared from the in vivo Fluo-4 AM-loaded somatosensory cortex. The slices were subsequently incubated in EDAC containing saline followed by formalin fixation. The loaded astrocytes were identified by examination at various depths and numbered (left). Next, the slices were processed for immunocytochemistry with astrocyte marker S100B. Depth scans (1 μm between the frames) were taken again to determine immunoreactivity of cells with S100B (right movie). An overlapping set of the cells was identified to be S100B-immunoreactive, indicating that nearly all Fluo-4 AM-loaded cells were astrocytes. (5 MB AVI). Click here for additional data file. Video S6 Imaging of Fluo-4 AM Fluorescence Activity in Astrocytes In Vivo Movie taken from a P14 rat. Image was taken with 2 Hz sampling rate for 10 min and compressed to 36 s for display purposes. Note spatial- and light-emission-stability of the recorded cells. Note also that at frames approximately 9 s and 15 s, two of the astrocytes in the middle display transient increased fluorescence. Scale bar 50 micro μ. (55 MB AVI). Click here for additional data file.
Supporting Information
Visualization of Loaded Astrocytes (Low Magnification)
The primary somatosensory cortex (P15) was stained with Fluo-4 AM in vivo and subsequently imaged in vitro. Acute slices (approximately 300 μm thickness) were cut in cold ACSF after the cells were loaded in vivo. (Z step = 1 μm; scale bar = 50 μm).(49 MB AVI).Click here for additional data file.Visualization of Loaded Astrocytes (High Magnification, Layer I)
Same slice as shown in  Videos S1 , but with higher magnification. Z step = 1 μm; scale bar = 20 μm.(48 MB AVI).Click here for additional data file.Visualization of Loaded Astrocytes (High Magnification, Layers II/III)
Detailed imaging of in vivo-loaded acute slice preparation of the primary somatosensory cortex (P15; approximately 270 μm below the pial surface). Z step = 1 μm; scale bar = 20 μm.(39 MB AVI).Click here for additional data file.High-Contrast Image Upper Layers (I to II/III) of the Fluo-4 AM-Loaded Somatosensory Cortex (P15)
Empty circles in layers II/III, presumed unloaded neurons (note their absence in layer I). The loaded cells have typical glial morphological appearance. Z step = 1 μm; scale bar = 50 μm.(50 MB AVI).Click here for additional data file.Double-Labeling of Fluo-4 AM-Loaded Astrocytes with S100B Antibody
Acute slices (300 μm thickness) were prepared from the in vivo Fluo-4 AM-loaded somatosensory cortex. The slices were subsequently incubated in EDAC containing saline followed by formalin fixation. The loaded astrocytes were identified by examination at various depths and numbered (left). Next, the slices were processed for immunocytochemistry with astrocyte marker S100B. Depth scans (1 μm between the frames) were taken again to determine immunoreactivity of cells with S100B (right movie). An overlapping set of the cells was identified to be S100B-immunoreactive, indicating that nearly all Fluo-4 AM-loaded cells were astrocytes.(5 MB AVI).Click here for additional data file.Imaging of Fluo-4 AM Fluorescence Activity in Astrocytes In Vivo
Movie taken from a P14 rat. Image was taken with 2 Hz sampling rate for 10 min and compressed to 36 s for display purposes. Note spatial- and light-emission-stability of the recorded cells. Note also that at frames approximately 9 s and 15 s, two of the astrocytes in the middle display transient increased fluorescence. Scale bar 50 micro μ.(55 MB AVI).Click here for additional data file.


Background Adenoid cystic carcinoma is a rare malignant parotid tumor. Metastasis can occur even a decade or more after initial treatment of the primary.
Background
Adenoid cystic carcinoma is a rare malignant parotid tumor. Metastasis can occur even a decade or more after initial treatment of the primary.Case presentation We report a 60 year old female patient who presented with adenoid cystic carcinoma of the parotid gland. She underwent a total conservative parotidectomy followed by adjuvant radiotherapy. While on follow up, patient developed multiple liver metastases which manifested three years later. Patient lived for another two years before she died of her disease.
Case presentation
We report a 60 year old female patient who presented with adenoid cystic carcinoma of the parotid gland. She underwent a total conservative parotidectomy followed by adjuvant radiotherapy. While on follow up, patient developed multiple liver metastases which manifested three years later. Patient lived for another two years before she died of her disease.Conclusions Although distant metastases of adenoid cystic carcinoma develop frequently, isolated metastasis to liver is unusual. Even after manifestation of distant metastasis, patients can be expected to live for a number of years. Palliative chemotherapy can be considered in symptomatic cases while the usefulness of metastatectomy is controversial.
Conclusions
Although distant metastases of adenoid cystic carcinoma develop frequently, isolated metastasis to liver is unusual. Even after manifestation of distant metastasis, patients can be expected to live for a number of years. Palliative chemotherapy can be considered in symptomatic cases while the usefulness of metastatectomy is controversial.

Background Adenoid cystic carcinoma (ACC) is a rare malignant neoplasm of the salivary gland. Salivary gland neoplasms constitute 3% of cancers of all sites, of which, 10–15% are malignant [ 1 , 2 ]. Though ACC is the most common malignant tumor of the submandibular, sublingual and minor salivary glands, it accounts for only 15% of parotid cancers [ 3 ]. They are generally slow growing and spread relentlessly to adjacent structures. Hematogenous spread is more common than lymphatic spread, the common sites of metastasis being the lung, bone and viscera [ 4 , 5 ]. We present a case of multiple liver metastases occurring 3 years after surgery for ACC of the parotid gland. The primary therapy, metastasis and outcome of ACC are discussed.
Background
Adenoid cystic carcinoma (ACC) is a rare malignant neoplasm of the salivary gland. Salivary gland neoplasms constitute 3% of cancers of all sites, of which, 10–15% are malignant [ 1 , 2 ]. Though ACC is the most common malignant tumor of the submandibular, sublingual and minor salivary glands, it accounts for only 15% of parotid cancers [ 3 ]. They are generally slow growing and spread relentlessly to adjacent structures. Hematogenous spread is more common than lymphatic spread, the common sites of metastasis being the lung, bone and viscera [ 4 , 5 ]. We present a case of multiple liver metastases occurring 3 years after surgery for ACC of the parotid gland. The primary therapy, metastasis and outcome of ACC are discussed.Case presentation A 60 year old woman presented with a small swelling beneath the right ear lobe of 4 months duration. The swelling measured 2 × 1 cm placed in the superficial part of the parotid and was not fixed. There was no facial nerve palsy or palpable cervical nodes. A fine needle aspiration cytology (FNAC) was carried out which showed the lesion to be ACC [ 6 ]. The clinical staging was T1, N0, M0. The patient underwent a total conservative parotidectomy after metastatic work up. Histopathology revealed ACC with cribriform pattern and perineural invasion (Figure  1 ). 60 Gy adjuvant external beam radiotherapy was administered post-operatively to the parotid area and the neck. Patient was placed on regular follow-up. Three years after primary surgery, patient presented with heaviness and pain in the right hypochondrium of 15 days duration. Patient was anicteric and abdominal examination revealed firm nodular and non tender enlargement of the liver. There was no ascites. Surgical site and neck were clinically normal. Chest roentgenogram was normal. Ultrasonography (US) of the abdomen revealed multiple metastatic lesions scattered in both lobes (Figure  2 ). Liver function tests were normal and an US guided FNAC revealed metastatic ACC (Figure  3 ). Since the lesions were multiple and scattered over both lobes of liver, surgical option was not considered and the patient was offered palliative chemotherapy which she declined. She developed pedal edema and abdominal distention 20 months after detection of liver metastasis. On clinical examination, patient was anicteric but liver had increased in size and abdomen showed evidence of a little free fluid. Chest CT scan was normal. Bone scan did not suggest any metastatic focus. Patient died a month later still without evidence of local recurrence or pulmonary metastasis.
Case presentation
A 60 year old woman presented with a small swelling beneath the right ear lobe of 4 months duration. The swelling measured 2 × 1 cm placed in the superficial part of the parotid and was not fixed. There was no facial nerve palsy or palpable cervical nodes. A fine needle aspiration cytology (FNAC) was carried out which showed the lesion to be ACC [ 6 ]. The clinical staging was T1, N0, M0. The patient underwent a total conservative parotidectomy after metastatic work up. Histopathology revealed ACC with cribriform pattern and perineural invasion (Figure  1 ). 60 Gy adjuvant external beam radiotherapy was administered post-operatively to the parotid area and the neck. Patient was placed on regular follow-up. Three years after primary surgery, patient presented with heaviness and pain in the right hypochondrium of 15 days duration. Patient was anicteric and abdominal examination revealed firm nodular and non tender enlargement of the liver. There was no ascites. Surgical site and neck were clinically normal. Chest roentgenogram was normal. Ultrasonography (US) of the abdomen revealed multiple metastatic lesions scattered in both lobes (Figure  2 ). Liver function tests were normal and an US guided FNAC revealed metastatic ACC (Figure  3 ). Since the lesions were multiple and scattered over both lobes of liver, surgical option was not considered and the patient was offered palliative chemotherapy which she declined. She developed pedal edema and abdominal distention 20 months after detection of liver metastasis. On clinical examination, patient was anicteric but liver had increased in size and abdomen showed evidence of a little free fluid. Chest CT scan was normal. Bone scan did not suggest any metastatic focus. Patient died a month later still without evidence of local recurrence or pulmonary metastasis.Conclusions Although ACC is the second most common malignant salivary gland neoplasm and constitutes approximately one third of all salivary gland malignancies it constitutes only 2% of parotid neoplasms [ 3 ]. As ACC is neurotropic, frozen section analysis of nerve margins is suggested specially when nerve is grossly involved by the tumor [ 7 ]. A total conservative or a radical parotidectomy is advocated for ACC though the main intent is to obtain a tumor free area of at least 1 cm [ 8 ]. ACC, with its often unusually slow biologic growth, tends to have a protracted course and ultimately a poor outcome, with a 10-year survival reported to be less than 50% for all grades [ 9 , 10 ]. These carcinomas typically show frequent recurrences and late distant metastases [ 11 ]. In a retrospective review of 92 cases, a tumor size greater than 4 cm was associated with an unfavorable clinical course [ 12 ]. Cribriform and solid patterns seen histologically were thought to predict more biological aggressiveness while tubular pattern represented more differentiated pattern of ACC. Over long periods of patient follow-up such grade based prognostication is less valid. Currently, stage and tumor location are the only factors considered prognostically significant [ 13 ]. Radiotherapy has been used as a primary modality for patients with surgical contraindications and in those with unresectable neoplasms. Though no improvement in survival is reported, the use of adjuvant radiation improves locoregional control and disease free survival. This patient received adjuvant radiation and did not have any locoregional recurrence. Regional metastasis is less common occurring in about 17% while systemic failure occurs in 33 to 50% of the patients [ 4 , 8 ]. Though involvement with distant metastases are unpredictable, organs involved in the order of decreasing frequency are lung, bone, brain and the liver [ 3 ]. Other rare metastatic sites of parotid and non parotid ACC include stomach, toe, choroids, brain and skin [ 14 - 18 ]. The initial site of metastasis is usually the organ containing the first capillary bed (first filter) and hence lungs would be the common site of metastasis [ 19 ]. Clinical observations from various malignancies have indicated that metastasis from certain types of tumor tend to occur in specific target organs leading to the famous 'soil and seed' hypothesis where metastatic cells 'home' to the organ [ 20 ]. Though liver metastasis has been reported, most of the liver metastases reported are of non parotid ACC [ 3 , 5 ]. The occurrence is usually metachronous or synchronous with metastasis to other organs like the lung as it is the first filter. In the series of Spiro, of the 74 patients developing metastasis from salivary gland ACC, 23 did so without loco-regional recurrence while 5 had isolated bone metastasis [ 5 ]. Sung and colleagues found metastases in 46 out of 94 head and neck ACC [ 21 ]. In that study, only one patient developed liver metastasis and that patient had metastasis to both lung and bone. In this case, the patient manifested with multiple metastatic foci in the liver as the first and only metastatic organ which is very unusual. Surgical options of metastatectomy were not explored as patient had multiple metastases involving both lobes of liver. Although the patient lived with disease for a further two years, she did not show any evidence of lung metastasis or loco-regional recurrence. In this patient, the liver metastasis could have occurred prior to treatment as an organ of preference; evidenced by the fact that there were no other organs showing metastasis nor was there a loco-regional recurrence later. Studies in ACC have shown long tumor doubling times of pulmonary metastasis and late recurrences up to 10 years after primary treatment [ 22 ]. The estimated doubling time of lung metastasis in ACC ranges from 200 to 600 days [ 22 ]. There is even a suggestion that metastasis at the cellular level could occur many years prior to clinical presentation of primary tumor. FNAC was done to prove the metastatic foci in this patient and can be a useful tool for diagnosis [ 23 ]. Although this patient declined chemotherapy, chemotherapeutic responses have been reported in ACC [ 24 ]. ACC carries a mortality of 75–80% over a 30 year period and most patients who die of their disease do so between 5 and 10 years after initial treatment [ 10 ]. Our patient died 5 years from diagnosis with metastatic disease that developed 3 years after initial treatment. ACC is a rare malignant tumor of parotid gland. Metastasis can manifest very late and hence a long term follow-up and a high index of suspicion is necessary to diagnose them early. An annual ultrasound study of abdomen would be desirable on follow-up. Unlike metastasis from other malignancies, these grow indolently and long term survival can be expected even with multiple metastases as also evidenced in the present case. Chemotherapy could be considered in selected patients as a therapeutic option in metastatic disease.
Conclusions
Although ACC is the second most common malignant salivary gland neoplasm and constitutes approximately one third of all salivary gland malignancies it constitutes only 2% of parotid neoplasms [ 3 ]. As ACC is neurotropic, frozen section analysis of nerve margins is suggested specially when nerve is grossly involved by the tumor [ 7 ]. A total conservative or a radical parotidectomy is advocated for ACC though the main intent is to obtain a tumor free area of at least 1 cm [ 8 ]. ACC, with its often unusually slow biologic growth, tends to have a protracted course and ultimately a poor outcome, with a 10-year survival reported to be less than 50% for all grades [ 9 , 10 ]. These carcinomas typically show frequent recurrences and late distant metastases [ 11 ]. In a retrospective review of 92 cases, a tumor size greater than 4 cm was associated with an unfavorable clinical course [ 12 ]. Cribriform and solid patterns seen histologically were thought to predict more biological aggressiveness while tubular pattern represented more differentiated pattern of ACC. Over long periods of patient follow-up such grade based prognostication is less valid. Currently, stage and tumor location are the only factors considered prognostically significant [ 13 ].Radiotherapy has been used as a primary modality for patients with surgical contraindications and in those with unresectable neoplasms. Though no improvement in survival is reported, the use of adjuvant radiation improves locoregional control and disease free survival. This patient received adjuvant radiation and did not have any locoregional recurrence.Regional metastasis is less common occurring in about 17% while systemic failure occurs in 33 to 50% of the patients [ 4 , 8 ]. Though involvement with distant metastases are unpredictable, organs involved in the order of decreasing frequency are lung, bone, brain and the liver [ 3 ]. Other rare metastatic sites of parotid and non parotid ACC include stomach, toe, choroids, brain and skin [ 14 - 18 ]. The initial site of metastasis is usually the organ containing the first capillary bed (first filter) and hence lungs would be the common site of metastasis [ 19 ]. Clinical observations from various malignancies have indicated that metastasis from certain types of tumor tend to occur in specific target organs leading to the famous 'soil and seed' hypothesis where metastatic cells 'home' to the organ [ 20 ]. Though liver metastasis has been reported, most of the liver metastases reported are of non parotid ACC [ 3 , 5 ]. The occurrence is usually metachronous or synchronous with metastasis to other organs like the lung as it is the first filter. In the series of Spiro, of the 74 patients developing metastasis from salivary gland ACC, 23 did so without loco-regional recurrence while 5 had isolated bone metastasis [ 5 ]. Sung and colleagues found metastases in 46 out of 94 head and neck ACC [ 21 ]. In that study, only one patient developed liver metastasis and that patient had metastasis to both lung and bone. In this case, the patient manifested with multiple metastatic foci in the liver as the first and only metastatic organ which is very unusual. Surgical options of metastatectomy were not explored as patient had multiple metastases involving both lobes of liver. Although the patient lived with disease for a further two years, she did not show any evidence of lung metastasis or loco-regional recurrence. In this patient, the liver metastasis could have occurred prior to treatment as an organ of preference; evidenced by the fact that there were no other organs showing metastasis nor was there a loco-regional recurrence later. Studies in ACC have shown long tumor doubling times of pulmonary metastasis and late recurrences up to 10 years after primary treatment [ 22 ]. The estimated doubling time of lung metastasis in ACC ranges from 200 to 600 days [ 22 ]. There is even a suggestion that metastasis at the cellular level could occur many years prior to clinical presentation of primary tumor. FNAC was done to prove the metastatic foci in this patient and can be a useful tool for diagnosis [ 23 ]. Although this patient declined chemotherapy, chemotherapeutic responses have been reported in ACC [ 24 ]. ACC carries a mortality of 75–80% over a 30 year period and most patients who die of their disease do so between 5 and 10 years after initial treatment [ 10 ]. Our patient died 5 years from diagnosis with metastatic disease that developed 3 years after initial treatment.ACC is a rare malignant tumor of parotid gland. Metastasis can manifest very late and hence a long term follow-up and a high index of suspicion is necessary to diagnose them early. An annual ultrasound study of abdomen would be desirable on follow-up. Unlike metastasis from other malignancies, these grow indolently and long term survival can be expected even with multiple metastases as also evidenced in the present case. Chemotherapy could be considered in selected patients as a therapeutic option in metastatic disease.List of abbreviations used ACC:  Adenoid cystic carcinoma FNAC:  Fine needle aspiration cytology Gy:  Gray
List of abbreviations used
ACC:  Adenoid cystic carcinomaACC:FNAC:  Fine needle aspiration cytologyFNAC:Gy:  GrayGy:Competing interests None declared
Competing interests
None declaredAuthors' contributions KH  was the principal clinician who planned the evaluation and procedure, in addition to conceptualizing and drafting the article. SRMG  was the pathologist. Both the authors read and approved the final manuscript.
Authors' contributions
KH  was the principal clinician who planned the evaluation and procedure, in addition to conceptualizing and drafting the article.KHSRMG  was the pathologist.SRMGBoth the authors read and approved the final manuscript.Pre-publication history The pre-publication history for this paper can be accessed here:
Pre-publication history
The pre-publication history for this paper can be accessed here: