Previous Article | Next Article 
The Journal of Neuroscience, November 1, 2002, 22(21):9430-9444
Neuronal Activity Regulates Correlated Network Properties of
Spontaneous Calcium Transients in Astrocytes In Situ
Fernando
Aguado*,
Juan F.
Espinosa-Parrilla*,
María A.
Carmona, and
Eduardo
Soriano
Department of Cell Biology and Barcelona Science Park, University
of Barcelona, Barcelona E-08028, Spain
 |
ABSTRACT |
Spontaneous neuronal activity is essential to neural development.
Until recently, neurons were believed to be the only excitable cells to
display spontaneous activity. However, cultured astrocytes and, more
recently, astrocytes in situ are now known to exhibit spontaneous Ca2+ transients. Here we used
Ca2+ imaging of astrocytes from transgenic mice for
the simultaneous monitoring of [Ca2+]i
changes in large numbers of astrocytes. We found that spontaneous activity is a common property of most brain astrocytes that is lost in
response to a lesion. These spontaneous
[Ca2+]i oscillations require
extracellular and intracellular Ca2+. Moreover,
network analysis revealed that most astrocytes formed correlated
networks of dozens of these cells, which were synchronous with both
astrocytes and neurons. We found that decreasing spontaneous [Ca2+]i transients in neurons by TTX
does not alter the number of active astrocytes, although it impairs
their synchronous network activity. Conversely, bicuculline-induced
epileptic patterns of [Ca2+]i
transients in neurons cause an increase in the number of active astrocytes and in their network synchrony. Furthermore, activation of
non-NMDA and NMDA ionotropic glutamate receptors is required to
correlate astrocytic networks. These results show that spontaneous activity in astrocytes and neurons is patterned into correlated neuronal/astrocytic networks in which neuronal activity regulates the
network properties of astrocytes. This network activity may be
essential for neural development and synaptic plasticity.
Key words:
astrocyte; calcium; epilepsy; correlated networks; GFAP/GFP mice; glutamate receptors; injury; spontaneous activity; synchrony
| |
INTRODUCTION |
Spontaneous neuronal activity is an
important property of the developing brain. It is essential for
neuronal migration, axonal and dendritic growth, and the formation and
refinement of neural connections and synapses (Katz and Shatz, 1996
;
Komuro and Rakic, 1998; Feller, 1999; Spitzer et al., 2000; Stellwagen
and Shatz, 2002). Changes and oscillations in intracellular
Ca2+ concentrations
([Ca2+]i) are the
main mechanism by which spontaneous activity controls neural
development (Berridge, 1998). An additional feature of this activity is
the occurrence of synchronous patterns of coactive neurons, which
amplify activity functions in neural development by coordinating neural
activity and gene expression in vast numbers of cells (Yuste et al.,
1992; Buonanno and Fields, 1999; Feller, 1999; O'Donnovan, 1999; Wong,
1999; Ben-Ari, 2001).
Until recently, neurons were considered to be the only excitable cells
to participate in spontaneous and evoked neurotransmission and in the
synaptic control of brain excitability. However, cultured and in
situ astroglial cells respond to various stimuli such as neurotransmitters, hormones, and mechanical stress by increasing [Ca2+]i
(Verkhratsky et al., 1998). For instance, neuronal stimulation triggers
glutamate- and GABA-mediated
[Ca2+]i increases
in astrocytes in brain slices (Dani et al., 1992; Porter and McCarthy,
1996; Pasti et al., 1997; Kang et al., 1998); conversely, astrocyte
[Ca2+]i increases
modulate EPSCs and IPSCs in neurons through glial-released glutamate (Pasti et al., 1997, 2001; Araque et al., 1998a,b; Bezzi et
al., 1998; Parpura and Haydon, 2000). Thus, there may be reciprocal signaling between astrocytes and neurons, thereby dynamically regulating nerve excitability (Cooper, 1995; Vesce et al., 1999; Carmignoto, 2000; Araque et al., 2001; Bezzi and Volterra, 2001; Haydon, 2001).
Cortical astroglial cells in vitro display spontaneous
[Ca2+]i
oscillations (Fatatis and Rusell, 1992; Charles, 1994; Harris-White et
al., 1998). Recently, it has been shown that astrocytes from brain
slices also show spontaneous
[Ca2+]i
oscillations in situ (Parri et al., 2001; Nett et al.,
2002). Moreover, spontaneous astrocytic oscillations in the thalamus trigger neuronal excitation through the NMDA glutamate receptor (Parri
et al., 2001). By imaging large numbers of astrocytes in transgenic
mice targeted to identify these glial cells, here we show that
spontaneous
[Ca2+]i transients
are a common feature of resting astrocytes in all the brain regions
examined, but they are lost in reactive astrocytes. Most of these
spontaneously active astrocytes were synchronous, forming complex
correlated networks of up to dozens of astrocytes, which in turn were
synchronous with spontaneous neuronal networks. Most importantly, by
blocking action potentials with tetrodotoxin (TTX) or by regulating
neuronal activity with GABAA and ionotropic glutamate receptor antagonists, we show that spontaneous neuronal activity controls the properties and complexity of astrocytic network activity.
| |
MATERIALS AND METHODS |
Animals. Glial fibrillary acidic protein (GFAP)/green
fluorescent protein (GFP) transgenic mice (Zhuo et al.,
1997) were purchased from Jackson Laboratories (Bar Harbor, ME).
The GFAP/GFP colony was kept under controlled temperature (22 ± 2°C), humidity (40-60%), and light (12 hr cycles) and treated in
accordance with the European Community Council Directive
(86/609/EEC). Stab wound lesions were made as described previously
(Mathewson and Berry, 1985). In brief, postnatal day (P) 22 GFAP/GFP
mice were anesthetized with ketamine-xylazine injections (150 and 6 µg/g, respectively), and their heads were fixed to a
stereotactic frame. The cerebral cortex was then stabbed parasagittally
with a scalpel blade. The wounds were 1 mm deep and 3 mm long and ran 2 mm away from the midline over the right parietal cortex. Lesioned mice
were used 2 d later. Reactive astrocytes were recognized by the
increased GFP fluorescence signal and their typical hypertrophied
morphology (Latov et al., 1979; Mathewson and Berry, 1985; Salhia et
al., 2000; Nolte et al., 2001).
Materials. Fura-2 AM and pluronic acid were purchased from
Molecular Probes (Eugene, OR). (±)-2-amino-5-phosphonopentanoic acid
(APV), 6-cyano-7-nitroquinoxaline-2, 3-dione (CNQX),
(+)-
-methyl-4-carboxyphenylglycine (MCPG), (
)-bicuculline
methiodide (BMI), TTX, (±)-2-octanol, 1-heptanol, 18-glycyrrhetinic
acid (-GA),
trans-(1S,3R)-1-amino-1,3-cyclopentanedicarboxylic acid (t-ACPD), and EGTA were obtained from Sigma (St. Louis,
MO). Suramin, 4-chloro-m-cresol (4-CmC), and thapsigargin were from Calbiochem-Novabiochem Corporation (La Jolla, CA), and CGP55845 was
from Tocris Cookson (Buckhurst Hill, UK). Drugs were dissolved in
distilled water and DMSO.
Ca2+ imaging. P2-P24 and adult (3-11
month old) GFAP/GFP mice were anesthetized by hypothermia or
ketamine-xylazine injections (150 and 6 µg/g, respectively). Their
brains and spinal cords were removed and placed in cold artificial CSF
(ACSF) containing (in mM): NaCl 120, KCl 3, D-glucose 10, NaHCO3 26, NaH2PO4 2.25, CaCl2 2, MgSO4 1, pH 7.4, bubbled with 95% O2 and 5%
CO2. Coronal and horizontal tissue slices (300 µm thick) were cut with a vibratome and before imaging were kept for
at least 1 hr in a storage chamber containing ACSF bubbled continuously
with 95% O2 and 5% CO2 at room temperature (22-25°C).
[Ca2+]i in slices
was measured with the membrane-permeant acetoxymethyl ester of
fura-2 AM dissolved in DMSO with 0.001% pluronic acid. The tissue
slices were incubated in 3-5 µl of 5 mM fura-2 AM for 2 min and then in 3 ml of 10 µM dye in ACSF for 30 min, as
described previously (Schwartz et al., 1998; Aguiló et al.,
1999). Slices were always maintained in oxygenated ACSF.
Fura-2-loaded slices were transferred to a continuously superfused
recording chamber on the stage of a fluorescent upright microscope
(BX50WI; Olympus, Tokyo, Japan) equipped with 380 and 340 nm excitation
filters and differential interference contrast optics. Recordings of
[Ca2+]i changes
were imaged with 20× and 40× water-immersion objectives at room
temperature (22-25°C). Images were captured with a
silicon-intensifier tube camera (Hamamatsu C2400-08) and a frame
grabber (LG-3; Scion Corporation, Frederick, MD) connected to a
Macintosh computer (Apple Computers, Cupertino, CA). Fura-2
fluorescence images were collected at 4 sec intervals (15 frames were
averaged for each time point) at a single excitation wavelength using
the 380 nm bandpass filter over periods of up to 20 min controlled by
the NIH Image program. To prevent photobleaching, a shutter (UniBlitz) controlled by custom-written macros was used.
Network analysis. To analyze coactive networks of optical
recordings, we followed a methodology described previously in detail (Schwartz et al., 1998; Aguiló et al., 1999). Changes in the fluorescence in multiple cells were analyzed with a program written in
Interactive Data Language (Research Systems, Inc., Boulder, CO). The
fluorescence change over time was defined as 
F/F = (F0 F1)/(F0). The
onset of each calcium transient for every cell was determined using an
algorithm that defined the onset as the frame after which the
F/F change was larger than a given set threshold,
typically a change of three to five pixel value units per frame. This
threshold algorithm was very sensitive but produced false positives in
some cells in which noise baseline values were scored as transients, as
determined by visual inspection. The [Ca2+]i transients
detected by the program were inspected carefully, and spurious events
were canceled. The time of initiation of each calcium transient for
each cell was marked in a raster plot. These plots were used to
calculate the matrix of asymmetric correlation coefficients between all
cell pairs, i.e., the proportion of times that a cell becomes active
when another cell is also active. Contingency tables were then used,
and 
2 tests were run to detect which of
the correlation coefficients was significantly greater than expected.
Significant correlation coefficients were used to generate a
correlation map on which lines link neurons whose asymmetric
correlation coefficients are significant (p < 0.01) and on which the thickness of a line connecting any two cells
represents the size of the greater asymmetric correlation coefficient
between the cells.
To test whether the
[Ca2+]i transients
showed associations between the cells within a network, we measured the
number of simultaneous activations in a recording and used it as a
statistical test. To determine its p value, the distribution
of the statistics under the null hypothesis of independent transients
was computed by Monte Carlo simulation. The p value was then
calculated as the proportion of the 1000 replications in which the
statistical test exceeded the statistical test computed from the real
data. To simulate independent realizations of the transients, the
number of transients in each train was preserved, but the times were chosen at random. This approach is equivalent to assuming that the
distributions of the transients behave as Poisson processes with
varying underlying rates. We tested this assumption with a
control computation of p values using randomized starting
times for each train and wrapping around the ends and found no
different results. We also performed an additional test on each sample
to detect groups of cells that were activated simultaneously more than
once, and we used this number as the statistical test (Schwartz et al.,
1998; Aguiló et al., 1999). Monte Carlo simulations were again
used to estimate the significance of this statistical test.
The Student's t test was used to compare all
measurements. Data are expressed as mean ± SEM.
| |
RESULTS |
Resting, but not reactive astrocytes, display spontaneous
[Ca2+]i transients in the CNS
To examine whether astrocytes have spontaneous
[Ca2+]i
oscillations in situ, we used acute brain slices from
transgenic mice that express GFP under the control of the GFAP promoter
(Zhuo et al., 1997). GFAP/GFP-positive cells displayed the
typical size and morphology of astrocytes in all brain regions and ages
examined (Figs.
1A-C, 2 B,D,
4A,B), which was consistent with
earlier studies (Zhuo et al., 1997; Nolte
et al., 2001). When transgenic slices were loaded with the
Ca2+ indicator fura-2, neurons labeled
with this Ca2+ indicator did not express
GFP (Fig. 1A,B). Thus, fura-2
loading of GFAP/GFP transgenic slices allowed the unequivocal
identification of large numbers of astrocytes.
View larger version (81K):
[in this window]
[in a new window]
|
Figure 1.
Spontaneous
[Ca2+]i oscillations are a common
feature of astrocytes in situ. A,
B, Paired fluorescence photomicrographs showing the same
field under fura-2 (A) and GFP
(B) fluorescence, illustrating GFP/fura-2-labeled
astrocytes in layers II-III of the neocortex of P7 GFAP/GFP mice.
Double-labeled astrocytes are indicated by arrows;
pyramidal neurons lacking GFP fluorescence are indicated by
arrowheads. C, Fluorescence
photomicrograph illustrating fura-2-loaded Bergmann glial cells
(arrows) in a P25 cerebellar slice. Purkinje cells,
devoid of fura-2 loading, are labeled by asterisks.
D, Representative spontaneous changes of fura-2
fluorescent signal (F/F) over
time recorded in astrocyte somata located in distinct CNS regions of
P6-P25 GFAP/GFP mice. The spontaneous activity profile of the
neocortical astrocyte corresponds to the astrocyte shown in
A and B. Note bursting and oscillatory
Ca2+ changes in the examples shown in the thalamus
and hippocampus, respectively. Scale bars: A,
B, 20 µm; C, 12 µm.
Mol, Molecular layer; P, Purkinje cell
layer; GL, granule cell layer.
|
|
View larger version (59K):
[in this window]
[in a new window]
|
Figure 2.
Reactive astrocytes lack spontaneous
[Ca2+]i transients. A,
Schematic diagram illustrating the location of the stab wound lesion in
the parietal cortex (arrow) of P24 GFAP/GFP neocortex
and the areas in which spontaneous astrocytic activity was recorded 48 hr after the lesion (I, II, and
III). B, C,
Low-magnification photomicrographs showing GFAP/GFP-positive astrocytes
(arrows) in areas III (B, resting
astrocytes) and I (C, reactive astrocytes). Note that
the number of astrocytes and the intensity of GFP fluorescence are
markedly increased in astrocytes around the lesion.
D-G, Higher-magnification photomicrographs of typical
GFAP/GFP-positive resting astrocytes in area III (D, E)
and reactive GFAP/GFP-positive astrocytes in area I (F,
G). Intense GFP positive-reactive astrocytes have larger somata
and hypertrophied processes (F) than
resting GFP cells (D). In both resting
(E) and reactive (G)
astrocytes, the fura-2 indicator was loaded similarly.
H, [Ca2+]i profiles
over 800 sec of representative GFAP/GFP-positive astrocytes recorded
near the lesion site (reactive astrocyte; area I) and in
the contralateral hemisphere (resting astrocyte; area
III). Scale bars: A, 1 mm; B,
C, 40 µm; D-G, 5 µm.
|
|
We monitored spontaneous
[Ca2+]i changes
with a high-resolution imaging system described elsewhere (Schwartz et
al., 1998; Aguiló et al., 1999). Time-lapsed recordings revealed
spontaneous
[Ca2+]i changes in
numerous GFAP/GFP-positive cell bodies corresponding to astrocytes.
These spontaneous astrocytic
[Ca2+]i changes
occurred in various CNS regions, including the neocortex, hippocampus,
entorhinal cortex, striatum, cerebellum, thalamus, hypothalamus, and
spinal cord (Fig. 1A-D). Astrocytic
[Ca2+]i transients
were detected as early as P2 (the earliest stage examined) and were
common during the first 2 postnatal weeks. For instance, 71% of
GFAP/GFP-positive astrocytes recorded at P5-P9 (167 of 236 cells in 11 slices) showed spontaneous Ca2+ signaling
in the hippocampus (Figs. 1D,
4C,D). Because the number of fura-2-loaded cells
decreases with age (Yuste and Katz, 1991), fewer GFAP/GFP-positive
astrocytes were loaded with fura-2 after P15. However, almost half of
these fura-2-loaded astrocytes exhibited spontaneous
[Ca2+]i transients
at P24 (e.g., 44%; 24 of 55 cells in the neocortex) (Fig.
2F). Spontaneous
[Ca2+]i
oscillations were still detected in adult astrocytes, although less
frequently (e.g., 26%; 8 of 31 cells in four neocortical slices from
two animals) than in postnatal stages. Most of these spontaneous
[Ca2+]i events
were long (typically, 10-45 sec) (Figs. 1D,
2F, 3B-D, 4C) and variable in amplitude,
even in the same brain region. The frequency and patterns of activity
were variable and included random profiles, but also rhythmic
oscillations and bursting activity (Fig. 1D). We
conclude that spontaneous
[Ca2+]i transients
are a common feature of developing and adult astrocytes in the CNS.
View larger version (25K):
[in this window]
[in a new window]
|
Figure 3.
Pharmacological analysis of spontaneous
[Ca2+]i transients in hippocampal
astrocytes. A, Histograms illustrating the percentage of
hippocampal P2-P6 GFAP/GFP-positive astrocytes showing spontaneous
Ca2+ activity after administration of a range of
neurotransmitter receptor antagonists, TTX, and blockers of
Ca2+ mobilization. Significant reductions are
observed after EGTA, Co2+, and thapsigargin
treatments (*p < 0.05; **p < 0.01; ***p < 0.001). Each experimental condition
was performed in at least three slices. Data are expressed as
percentages of control values (mean ± SEM). B,
Representative plots of [Ca2+]i
oscillations recorded in the same GFAP/GFP-positive astrocyte from a P6
mouse perfused with normal ACSF (basal and washout),
Ca2+-free ACSF (2 mM EGTA, 0 mM [Ca2+]o), and
thapsigargin (2 µM). C, D,
[Ca2+]i oscillations recorded in P5
GFAP/GFP-positive astrocytes are abolished after perfusion of
Ca2+-free ACSF. Addition of t-ACPD
and 4-CmC (arrows) to nominally
Ca2+-free ACSF caused a transient and a progressive
increase in [Ca2+]i,
respectively. Abbreviations are defined in Materials and Methods.
|
|
View larger version (84K):
[in this window]
[in a new window]
|
Figure 4.
Spontaneous
[Ca2+]i oscillations in hippocampal
astrocytes belong to correlated neuronal/astrocytic networks.
A, B, Paired fluorescence photomicrographs illustrating
many GFAP/GFP-positive astrocytes (A) in the CA1
region of the hippocampus (P6) that are also labeled with fura-2 (e.g.,
cells in circles) (B).
Fura-2-loaded cells showing spontaneous fluorescence changes over time
are labeled by squares. Black squares
mark GFP-positive astrocytes, whereas GFP-negative neurons (located
mainly in the pyramidal layer) are labeled by white
squares. C, Representative plots of spontaneous
changes of fura-2 fluorescence
(F/F) over 800 sec in
astrocytes and neurons in the CA1 hippocampal region. The initiation of
each [Ca2+]i oscillation is labeled by
a thick mark at the bottom of the
plot. D, Histograms illustrating properties of
spontaneous [Ca2+]i oscillations in
astrocytes (white bars) and neurons (black
bars). Both the duration and amplitude of spontaneous
Ca2+ activations are higher in astrocytes than in
neurons, whereas the proportion of active cells and the rate of
oscillations are similar in both populations. E, Raster
plot representing the activation profile of each of the 80 active cells
shown in B over 800 sec. In the raster plot, each active
cell is represented by a line, and each thick
line marks the initiation of a
[Ca2+]i transient. Cells
1-27 correspond to astrocytes, and
28-80 correspond to neurons.
Dotted lines in the astrocyte raster plot indicate
simultaneous coactivation of at least two cells of the plot.
Synchronous coactivation among large numbers of neurons can also be
seen clearly. F, Correlation map illustrating each
active astrocyte of B (black squares), in
which cells with statistically significant correlation coefficients are
connected by lines. The thickness of the
lines is proportional to the degree of significance.
Most active astrocytes appear to belong to a correlated network.
G, Correlated map representing synchronous correlations
among all the astrocytes (black squares) shown in
F and seven representative neurons (white
squares, arrows in raster plot). Observe how
many astrocytes are synchronously connected with neuronal cells.
H, Distribution of pairwise correlations found in the
real data (arrow) and in 1000 simulations obtained by
the Monte Carlo test (bell-shaped curve) of the
astrocyte population shown in B. Note how the number of
correlated events in the real data set (arrow) exceeds
those expected by chance in simulated data. The corresponding
p value is shown in F. I,
Average of Monte Carlo p values showing the probability
that the number of times that any two cells had simultaneous onset of
activation was caused by chance. Although all cases in both
astrocytes (6 of 6; A) and neurons (6 of 6;
N) are significant (p < 0.05), astrocytic values are higher than neuronal ones.
J, Average of Monte Carlo p values
showing the probability that the number of times the same cells
were activated simultaneously at least twice was caused by chance. Each
set of neuronal populations gives a 0 p value (6 of 6;
N), whereas in astrocytic cells only four of six
cases are significant (p < 0.05)
(A). Both I and J
illustrate the higher synchronous correlation level of spontaneous
[Ca2+]i oscillations among neurons
than among astrocytes. K, Histograms summarizing the
proportion of spontaneous active cells with statistically significant
correlation coefficients: among astrocytes
(A A), among neurons
(NN), percentage of astrocytes
coactive with neurons (AN), and
percentage of neurons coactive with astrocytes
(NA). Statistical significance:
*p < 0.0001. Scale bar, 40 µm.
sr, Stratum radiatum; sp, stratum
pyramidale; so, stratum oriens.
|
|
Astrocytes have a number of functions, including homeostatic control of
the extracellular "milieu," the regulation of synaptic function,
and a protective role after CNS injury (Kettenmann and Ransom, 1995).
To study whether spontaneous
[Ca2+]i changes in
astrocytes depend on their functional state, we determined whether
these spontaneous oscillations persisted in reactive astrocytes. We
compared Ca2+ signaling in reactive and
resting astrocytes 48 hr after stab wound injuries (Mathewson and
Berry, 1985) in the neocortex of P22 GFAP/GFP transgenic mice (Fig.
2A). As described for endogenous GFAP and for
transgenic reporter expressions (Latov et al., 1979; Mathewson and
Berry, 1985; Salhia et al., 2000; Nolte et al., 2001), large numbers of
astrocytes located around the wound lesion (100-500 µm) exhibited
strong GFP expression in GFAP/GFP transgenic slices, in both the cell
bodies and processes (Fig. 2B,C).
Moreover, astrocytes in the hemisphere ipsilateral to the wound showed
the typical hypertrophy described for stab injury-induced reactive astrogliosis (Latov et al., 1979; Mathewson and Berry, 1985; Salhia et
al., 2000; Nolte et al., 2001). Thus, although resting astrocytes in the contralateral cortex were ~4 µm in size and showed extremely thin processes (Fig. 2D,E),
reactive astrocytes showed increased GFAP/GFP fluorescence and had
larger somata (~10 µm in size) and hypertrophied processes (Fig.
2F,G). Only 2 of 82 reactive
astrocytes (imaged at 100-200 µm from the lesion) had spontaneous
[Ca2+]i
oscillations, and these were of very low amplitudes (one of seven
slices from five animals). Those remaining reactive astrocytes showed
no spontaneous
[Ca2+]i changes
even after 15 min of recording (Fig. 2H).
Characteristically, other cell bodies located near the wound that were
GFAP/GFP negative and had the shape and size of neurons showed frequent
spontaneous [Ca2+]i transients
(data not shown). In contrast, the proportion of GFAP/GFP-positive
cells exhibiting these transients increased in proportion to their
distance from the lesion (Fig. 2A). For instance,
51% (19 of 33) and 44% (24 of 55) of astrocytes exhibited spontaneous
[Ca2+]i
oscillations at 2.5-3 mm ipsilateral to the lesion and in the contralateral hemisphere, respectively (six of six slices from four
animals) (Fig. 2H). These data indicate that
spontaneous [Ca2+]i
oscillations are a common functional property of resting astrocytes that is lost when astrocytes respond to a mechanical lesion.
Extracellular and intracellular Ca2+
mobilization is required for the generation of spontaneous oscillations
in hippocampal astrocytes
Next, we studied whether activation of neurotransmitter receptors
drives spontaneous
[Ca2+]i
oscillations in GFAP/GFP-positive astrocytes from P2-P6 hippocampal slices. Incubation with antagonists for ionotropic (APV/CNQX, 50 and 20 µM) and metabotropic (MCPG, 1 mM) glutamate
receptors did not alter the proportion of hippocampal astrocytes
displaying spontaneous
[Ca2+]i transients
(Fig. 3A). Moreover, no significant changes were observed
after addition of antagonists for ionotropic
GABAA (BMI, 30 µM) and
metabotropic GABAB (CGP55845, 6 µM) GABA receptors (Fig. 3A).
Similarly, incubation with the Na+ channel
blocker TTX (2 µM) did not significantly modify
spontaneous activity in astrocytes (Fig. 3A). In marked
contrast to these observations in astrocytes, we found that TTX and the
synaptic blockers altered spontaneous
[Ca2+]i transients
in neurons (see below). These data indicate that the generation of
spontaneous astrocytic activity depends neither on the activation of
the main neurotransmitter systems nor on action potentials.
Because ATP signaling is a relevant extracellular messenger that
activates astrocytes (Fields and Stevens, 2000), we evaluated the
participation of purinergic receptors in the generation of spontaneous
[Ca2+]i
oscillations by blocking P2 receptors with the antagonist suramin (100 µM) in the presence of TTX (2 µM) to block
suramin effects on neurons (Cunha and Ribeiro, 2000). Again, the
proportion of spontaneously active astrocytes was not altered
significantly under these conditions (Fig. 3A). Finally, we
studied the requirement of gap junctions in the generation of
spontaneous astrocytic activity (Giaume and McCarthy, 1996; Giaume and
Venance, 1998). After bath application of 100 µM -GA, a gap junction blocker (Davidson and Baumgarten, 1998; Venance et al., 1998), the percentage of
spontaneously active astrocytes was not altered (Fig. 3A).
Moreover, perfusion of the slices with the long-chain alcohols octanol
and heptanol (1 mM), which uncouple astrocyte gap
junctions (Venance et al., 1998), showed similar results (data not
shown). These data indicate that neither P2 receptor activation nor gap
junctions are essential for the generation of spontaneous astrocytic activity.
To investigate the cellular mechanisms that underlie spontaneous
[Ca2+]i
transients, we incubated hippocampal slices (P5-P9) with drugs influencing Ca2+ mobilization. Rapid
removal of extracellular Ca2+ by perfusion
with Ca2+-free ACSF containing 2 mM EGTA blocked spontaneous
[Ca2+]i
oscillations in 103 of 113 imaged astrocytes (four of six slices from
six animals) (Fig. 3A,B). This
blocking effect was partially reversed by washing EGTA containing
medium with normal ACSF (19 of 29 cells; three of three slices from
three animals) (Fig. 3B). Because the removal of
extracellular Ca2+ might deplete
intracellular Ca2+ stores, we evaluated
the levels of Ca2+ in intracellular stores
of astrocytes perfused with nominally Ca2+-free ACSF by activating
IP3 and ryanodine receptors. To activate IP3-mediated Ca2+
release from intracellular stores, we used the metabotropic glutamate receptor agonist t-ACPD (Porter and McCarthy, 1995).
Addition of t-ACPD (5 µM) to
Ca2+-free ACSF triggered a transient
increase of
[Ca2+]i in 52 of
54 identified astrocytes (two slices from two animals) (Fig.
3C). Moreover, perfusion of the ryanodine receptor agonist 4-CmC (6 mM) (Zorzato et al., 1993; Matyash et
al., 2002) in Ca2+-free ACSF elicited a
progressive increase of
[Ca2+]i in each of
the imaged astrocytes (42 astrocytes from two animals) (Fig.
3D). These results show that intracellular
Ca2+ stores are preserved in hippocampal
astrocytes perfused with nominally
Ca2+-free ACSF and therefore indicate that
extracellular Ca2+ is necessary to trigger
spontaneous activity in astrocytes.
We next examined whether voltage-gated
Ca2+ channels (VGCC) are involved in the
spontaneous increase of
[Ca2+]i in
astrocytes by addition of the nonselective VGCC blocker Co2+. After perfusion of GFAP/GFP
hippocampal slices with normal ACSF containing 1 mM
Co2+, only 1 of 32 astrocytes (three
slices from three animals) exhibited spontaneous
[Ca2+]i
oscillations (Fig. 3A). These data support the notion that spontaneous astrocyte Ca2+ activity
requires extracellular Ca2+ and operating
voltage-gated Ca2+ channels.
Finally, we analyzed the contribution of released intracellular
Ca2+ in spontaneous
[Ca2+]i transients
of astrocytes by application of thapsigargin, which depletes
intracellular Ca2+ stores by inhibiting
endoplasmic reticulum Ca2+-ATPase activity
(Thastrup et al., 1990). Addition of thapsigargin (2 µM)
to hippocampal slices decreased the number of astrocytes with
spontaneous
[Ca2+]i transients
(24 of 32 cells; four of four slices from four mice) by ~75% (Fig.
3A,B). During thapsigargin and EGTA
treatments, the astrocytes that maintained spontaneous
Ca2+ signaling had lower amplitudes and
frequencies of oscillation than in basal conditions (data not shown).
Moreover, although either nominally
Ca2+-free ACSF or 1 mM Co2+ abolished
[Ca2+]i
oscillations in virtually all imaged neurons (six slices), neuronal
[Ca2+]i transients
were mostly preserved (92.5%) after addition of 2 µM thapsigargin (25 of 27 neurons; two slices).
This indicates that in contrast to astrocytes, neuronal
oscillations depend almost exclusively on extracellular
Ca2+. Taken together, these observations
show that extracellular Ca2+ influx and
Ca2+ release from intracellular stores are
required to generate spontaneous [Ca2+]i transients
in hippocampal astrocytes in situ. Moreover, the similarities between these pharmacological findings and those in the
thalamus (Parri et al., 2001) suggest that spontaneous astrocytic
activity is generated by common mechanisms intrinsic to astrocytes in
distinct brain regions.
Astrocytes are integrated in complex spontaneous active networks of
neurons and glial cells
An important feature of spontaneous
[Ca2+]i
oscillations in developing neurons is the recruitment of active cells
into synchronous networks that amplify the functional consequences of
activity to control neuronal development (Yuste et al., 1992; Buonanno and Fields, 1999; Feller, 1999; O'Donovan, 1999, Wong, 1999, Ben-Ari, 2001). In our experiments, visual examination of the movies recorded in
the neocortex and hippocampus showed synchronous patterns of activity
in neuronal populations (Fig. 4), which have been described previously
(Ben-Ari et al., 1997; Garaschuk et al., 1998). Interestingly, spatiotemporal correlations of Ca2+
signaling between GFAP/GFP-positive astrocytes were also observed.
Because high numbers of astrocytes and neurons can be accurately
identified in GFAP/GFP-positive slices, we analyzed the occurrence of
patterned networks of correlated Ca2+
transients among astrocytic and neuronal cells in P5-P7
GFAP/GFP-positive hippocampal slices. First, we compared the astrocytes
and neurons present in the same fields for profiles of spontaneous
[Ca2+]i
transients. As above, astroglial cells were identified by the emission
of GFP fluorescence, whereas neurons were identified by the lack of GFP
expression and by their morphology and location in the hippocampal
layers (Figs. 4A,B). Spontaneous
[Ca2+]i changes
were monitored simultaneously in dozens of glial (Fig. 4B, 
) and neuronal (Fig. 4B,
) cells, and the onset of each calcium event in every active cell
was labeled in graphed plots by a mark (Fig. 4C). No
statistical differences were found in the percentages of active cells
or in the rates of
[Ca2+]i
oscillations between hippocampal astrocytes and neurons (Fig. 4D), indicating that astrocyte activity is a robust
phenomenon. In contrast, both the duration and the amplitude of these
oscillations were threefold higher in astrocytes than in neurons (Fig.
4D), as also illustrated in representative plots of
glial and neuronal activation profiles (Fig. 4C). This is
consistent with the analysis of the fast and slow calcium events in
neocortical slices (Badea et al., 2001; Thasiro et al., 2002).
Next we performed a synchrony network analysis among individual
astroglial cells to define spatiotemporal patterns of spontaneous Ca2+ oscillations in glial cells. We used
a recently developed statistical method that can identify and map the
simultaneous coactivations among vast numbers of individual cells
(Schwartz et al., 1998; Aguiló et al., 1999). First, we outlined
in raster plots the profile of the Ca2+
events of every active cell in each movie (Fig. 4E).
We then used contingency tables and 2
tests to identify the active cells with significant synchronous Ca2+ transients (Schwartz et al., 1998,
Aguiló et al., 1999). This analysis led to correlation maps
showing all the active cells recorded from a field in which each pair
of synchronous cells is connected by lines the thickness of which is
proportional to the degree of correlation (see Materials and
Methods) (Figs.
4F,G). Spatiotemporal analysis of
astrocytic spontaneous
[Ca2+]i changes
revealed complex networks of coactivated glial cells (Fig.
4F). Correlated Ca2+
oscillations were observed between neighboring astrocytes (Fig. 4F, e.g., cells 1 and 2 or
10 and 11) and also between distant cells (Fig.
4F, e.g., cells 1 and 24 or
7 and 24). In the hippocampus, single
networks of synchronous astrocytes included cells located in distinct
layers, such as the stratum oriens, the stratum radiatum, and the
pyramidal layer (Fig. 4B,F).
Quantitative analysis of correlated astroglial maps showed that ~81%
of the active astrocytes belonged to synchronous networks (Fig.
4K, AA).
We also quantified the overall degree of synchronous correlation in
each astroglial network. To calculate the level of coactivation present
in each recording, we compared the number of times that any two cells
had simultaneous onset activation times in each piece of real data
(number of coactivations) with the number of coactivation times present
in a distribution of 1000 random experiments created by Monte Carlo
simulation (Schwartz et al., 1998) (Fig. 4H). This
comparison gives a p value for the real data set that describes the probability that all of the coactivations present in each
sample were caused by chance (Fig. 4H). To
standardize the p value of each sample, Monte Carlo
simulations were created using the same number of cells, activations,
and time intervals as in the real data set, but the initiation of the
transients was randomly chosen (see Materials and Methods). Using this
analysis, we found an overall significant correlation
(p < 0.05; Monte Carlo simulation) in six of
six astroglial networks imaged from P5-P7 hippocampus (see
p value of the network illustrated in Fig.
4F) with an average p value of 0.002 (Fig.
4I). Taken together, these data show that astrocytic
spontaneous
[Ca2+]i transients
are organized into complex synchronous networks that recruit large
numbers of astrocytes, which are often located at great distance.
In the imaged movies that analyzed astrocytic
[Ca2+]i
oscillations, we also observed highly synchronous patterns of
spontaneous Ca2+ transients among
hippocampal neurons (Fig. 4E,G).
Postnatal hippocampal neurons displayed frequent synchronous events,
which corroborated previous studies (Garaschuk et al., 1998,
Ben-Ari, 2001). The highly synchronous patterns of hippocampal neuronal
network activity were easily observed in raster plots (Fig.
4E) and in correlation maps (Fig. 4G) and
recruited most active neurons. In fact, virtually all spontaneous
active neurons were synchronously interconnected (Fig.
4E,G, and illustrated as
NN in K). Thus, each
neuronal network displayed very significant overall synchrony, as shown by the p value 0 of the Monte Carlo test in each sample
analyzed (six cases of six) (Fig. 4I), which
indicated that the degree of synchrony was higher in neuronal than in
astrocytic networks (compare also the raster plots in Fig.
4E). To confirm this hypothesis, we then used a more
restrictive test to compare the number of times that synchronous cells
are simultaneously coactivated at least twice in each astroglial and
neuronal data set, with 1000 random experiments created by Monte Carlo
simulation (Schwartz et al., 1998, Aguiló et al., 1999).
p values were higher among astrocytic
(p < 0.05; four of six cases; average
0.078 ± 12) than neuronal networks (p < 0.05; six of six cases; average 0), indicating closer correlation in
the latter (Fig. 4J).
Next, we determined possible synchronous correlations of astrocytes and
neurons. Examination of mixed glial-neuronal correlation maps showed
that subsets of astrocytes and neurons were synchronously interconnected (Fig. 4G). For instance, the active glial
cells 1 and 2 were interconnected with neurons 70 and 80 (Fig.
4G). Quantitative analyses of correlation maps showed that
61% of astrocytes with spontaneous
[Ca2+]i transients
correlated with spontaneous active neurons (Fig. 4K,
AN). Conversely, most active neurons
(95.4%) were synchronous with active astrocytes (Fig.
4K, NA). We conclude that
spontaneous correlated network activity is not restricted to neuronal
populations, but rather is a common property of developing neurons and
astrocytes, which are interconnected into complex synchronous networks.
Network properties of spontaneous astroglial calcium transients are
regulated by neuronal activity
Neuronal activity may be affected by stimulated astroglial
[Ca2+]i
oscillations (Pasti et al., 1997, 2001; Araque et al., 1998a,b; Bezzi
et al., 1998: Parpura and Haydon, 2000; Parri et al., 2001). Conversely, there is evidence that astrocyte-evoked activity may increase after neuronal stimulation (Dani et al., 1992; Porter and
McCarthy, 1996; Pasti et al., 1997; Kang et al., 1998). We thus
investigated whether neuronal activity regulates spontaneous astrocytic
activity and its organization into spatiotemporal correlated networks.
First, we used TTX to abolish neuronal action potentials in GFAP/GFP
transgenic hippocampal slices (P5-P7). Incubation with the
Na+ channel blocker TTX (2 µM) impaired spontaneous
[Ca2+]i transients
in hippocampal neurons (Fig.
5A). Thus, TTX significantly decreased both the number of neurons showing
[Ca2+]i
oscillations and the activation rates of the remaining active neurons
(Fig. 5C,D). Furthermore, as shown in raster
plots and correlation maps, Na+ channel
blockade dramatically decorrelated the activity profiles of the
remaining active neurons, resulting in very simple neuronal networks
(Fig. 5A,B). These observations
were supported by a significant reduction in the number of coactive
neurons (Fig. 5E, NN) and by
the dramatic rise in the Monte Carlo p value, which reflect the overall network decorrelation (Fig. 5F).
View larger version (38K):
[in this window]
[in a new window]
|
Figure 5.
Spontaneous astrocytic correlated network
activity is impaired after TTX treatment. A, Raster plot
illustrating the activation profiles of astrocytes (cells
1-16) and neurons (cells
17-52) of a hippocampal CA1 field from a
P6 GFAP/GFP mouse before (basal) and after TTX
administration. Although major changes are not observed in the
astrocyte population, neuron activity is greatly impaired.
B, Correlation maps of all active astrocytes
(black squares) and a representative fraction of active
neurons (white squares, arrows in raster
plots) illustrated in A. After TTX treatment active
neurons and their correlations are almost absent, whereas correlated
astroglial activity persisted. C, Spontaneous active
astrocytes (white bar) and neurons (black
bar) in relation to basal conditions after TTX administration.
D, Activity rate in astrocytes (white
bar) and neurons (black bar) in relation to
control after TTX administration. E, Histograms
summarizing the proportion of spontaneous active cells with
statistically significant correlation coefficients after TTX: among
astrocytes (AA), among neurons
(NN), percentage of astrocytes
coactive with neurons (AN), and
percentage of neurons coactive with astrocytes
(NA). F, Average of
Monte Carlo p values showing the probability that the
number of times that any two cells had simultaneous onset of activation
was caused by chance. Both astrocytes (A) and
neurons (N) exhibit very significant values in
basal conditions, whereas TTX treatment decorrelates spontaneous
activity in both neural populations. Significant reductions
(*p < 0.05) are observed after TTX. Scale bar, 40 µm. sr, Stratum radiatum; sp, stratum
pyramidale; so, stratum oriens.
|
|
In contrast, TTX led to slight, although not significant, changes in
the number of active astrocytes and their
[Ca2+]i
oscillation rates (Fig.
5A,C,D). Moreover,
although the proportion of interconnected astrocytes was maintained
after Na+ channel blockade (Fig.
5E, AA), the patterns of
coactivation and overall network synchrony were strikingly impaired, as
illustrated by correlation maps (Fig. 5B) and higher Monte
Carlo p averages (Fig. 5F). In addition,
the percentage of astrocytes correlated with active neurons decreased
significantly after TTX administration (Fig. 5E,
AN). Thus, although
Na+ channels and neuronal activity are not
required for the generation of spontaneous astrocyte activity, their
correlated network properties with both neurons and astrocytes depend
dramatically on action potentials.
To substantiate these findings, we studied whether increased levels of
neuronal activity altered spontaneous astrocytic activity. For this, we
generated epileptiform discharges in P8-P10 hippocampal slices by
administering BMI (30 µM) to the bath. Elimination of GABAergic inhibitory neurotransmission by blocking ionotropic GABAA receptors by BMI induces hyperexcitability
and status epilepticus in cortical slices (Jones and Lambert, 1990;
Albowitz et al., 1997; Badea et al., 2001). After BMI application, we
observed an enhancement in the number of neurons showing spontaneous
[Ca2+]i transients
and in their rates of oscillations (Fig.
6A,C,D). Moreover, most of the active neurons were recruited into repetitive synchronous waves, which corroborated previous studies (Albowitz et
al., 1997; Badea et al., 2001), and led to highly coactive raster plots
and correlation maps (Fig. 6A,B).
Interestingly, the number of spontaneously active GFAP/GFP-positive
astrocytes and their activation rates also increased dramatically after
GABAA receptor blockade (Fig.
5A,C,D). More
importantly, the number of astrocytes coactive with other astrocytes
(AA) and with neurons (AN) after BMI incubation increased by 41 and 80%, respectively (Fig. 6E), which resulted in
very complex correlation maps (Fig. 6B) and in high
overall astrocyte synchrony (Fig. 6F). Neuronal epileptiform waves recruited many astrocytic
[Ca2+]i
oscillations, so that most active astrocytes were synchronous with
neuronal activation waves (see raster plot and correlation map in Fig.
6). Thus, BMI treatment not only increased spontaneous astrocytic
activity but also increased correlated activity between astrocytes and
between astrocytes and neurons.
View larger version (46K):
[in this window]
[in a new window]
|
Figure 6.
Spontaneous astrocytic correlated network activity
increases in BMI-induced epileptiform status. A, Raster
plot illustrating the activation profiles of astrocytes (cells
1-26) and neurons (cells
27-55) of a hippocampal CA1 field from a
P9 GFAP/GFP mouse before (basal) and after BMI
administration. BMI enhances spontaneous activity in both astrocytic
and neuronal populations. B, Correlation maps showing all active
astrocytes (black squares) and a representative fraction
of active neurons (white squares, arrows
in raster plots) shown in A. After BMI treatment,
correlated activity increased among astroglial and neuronal cells.
C, Percentage of spontaneous active astrocytes
(white bar) and neurons (black bar) in
relation to basal conditions after BMI administration.
D, Percentage of activity rate in astrocytes
(white bar) and neurons (black bar) in
relation to control after BMI administration. E,
Proportion of active astrocytes and neurons showing statistically
significant synchrony calculated from correlation maps after BMI
administration with respect to basal conditions. After BMI incubation,
astrocytes increase their synchronous correlation with both astrocytes
and neurons. F, Average of Monte Carlo p
values showing the probability that the number of times that any two
cells had simultaneous onset of activation was caused by chance. Both
astrocytes (A) and neurons
(N) exhibit very significant values in basal
conditions, whereas BMI treatment decreases p values in
astroglial populations. Statistical significance:
*p < 0.05. Scale bar, 40 µm. sr,
Stratum radiatum; sp, stratum pyramidale;
so, stratum oriens.
|
|
Overall, our results show that although the generation of spontaneous
astrocyte activity does not require neuronal activity, its network
properties are controlled, to a large extent, by neuronal excitation.
Activation of ionotropic glutamate receptors is required for the
generation of correlated astrocytic network activity
Gap junctions and extracellular messengers, such as glutamate,
ATP, and nitric oxide, have been proposed to control the propagation of
evoked Ca2+ activation in astrocyte
networks (Cornell-Bell et al., 1990; Dani et al., 1992; Finkbeiner,
1992; Venance et al., 1997; Guthrie et al., 1999; Willmott et
al., 2000; Newman, 2001; Schipke et al., 2002). To identify the
mechanisms that correlate spontaneous activity of astrocytic networks
in situ, we used several blocking agents on P5-P7 GFAP/GFP
hippocampal slices. Incubation with the gap junction blocker -GA
(100 µM) did not perturb the number of active
astrocytes (Figs. 3A,
7A-C,J).
Moreover, the p values reflecting the correlation degree
among the entire astrocyte population were totally preserved (Fig.
7A-C,J). Next, and because
neuronal activity was necessary to correlate spontaneous astrocyte
Ca2+ activation, we analyzed the
contribution of glutamate receptors. Addition of AMPA/kainate receptor
antagonists (20 µM CNQX) to the bath markedly
impaired astrocyte network correlation (Fig. 7J).
This effect was reverted by washing out the CNQX
(Fig. 7J). Moreover, perfusion with the NMDA receptor
antagonist APV (50 µM) also abolished
astrocytic network correlation (Fig.
7D,E,J). Again,
astrocyte coactivations were restored after washout (Fig. 7F). In contrast, the blockage of glutamate
metabotropic receptors by MCPG (1 mM) did not
decorrelate spontaneous astrocytic network activity (Fig.
7J). We conclude that activation of ionotropic glutamate receptors is not necessary for the generation of spontaneous astrocytic activity in situ, but that this activation is a
major regulator of their spatiotemporal coordination into correlated networks. Moreover, most of the blocking agents only slightly altered
the pattern of spontaneous
[Ca2+]i
oscillations in neurons (data not shown), whereas APV treatment reduced
the number of active neurons by 36.8%, in agreement with previous
reports (Ben-Ari et al., 1997, Garaschuk et al., 1998).
View larger version (53K):
[in this window]
[in a new window]
|
Figure 7.
Mechanisms that control network correlation of
spontaneous astrocytic activity in the hippocampus.
A-C, Correlation maps showing coordinated activity of a
P6 astrocyte network at basal conditions (A),
after incubation with the gap junction blocker -GA
(B), and after washout (wo) with
-GA (C). Note that the network correlation,
indicated by Monte Carlo p value (bottom
of the map), is not perturbed by blocking gap junctions.
D-F, Correlation maps showing the effect of NMDA
receptor blockade on a P5 astrocyte network. Correlated
Ca2+ events in nontreated astrocytes
(D) are reduced by APV incubation
(E) and recovered after washing the antagonist
(F). G-I, Network astrocyte
correlation in a BMI-treated hippocampal slice
(G) is markedly decreased by addition of CNQX
(H) and restored by washing the non-NMDA
antagonist (I). J, Histogram showing
the average of Monte Carlo p values (black
bars, left) and the percentage of active
astrocytes (white bars, right) after
addition of gap junction blockers and glutamate receptor antagonists in
basal GFAP/GFP hippocampal slices. K, Average of
astrocyte network correlations (black bars,
left) and percentage of active astrocytes (white
bars, right) after administration of ionotropic
glutamate receptor antagonists in BMI-treated hippocampal slices. Each
experimental condition was performed in at least three different
slices. Statistical significance: *p < 0.05. Scale
bar, 45 µm. sr, Stratum radiatum; sp,
stratum pyramidale; so, stratum oriens.
|
|
It is known that the lack of inhibitory
GABAA-mediated responses in BMI-treated
hippocampal slices markedly increases neuronal excitability and
glutamate release, which in turn produces epileptogenesis (Bradford,
1995). We analyzed the contribution of ionotropic glutamate receptors
in the generation of astrocytic network correlation in the BMI-induced
epilepsy model. Administration of BMI to P8-P11 GFAP/GFP hippocampal
slices increased correlated astrocytic activity (Figs. 6,
7G,K). Addition of CNQX (20 µM) to BMI-ACSF dramatically abolished the
correlation of astrocytic Ca2+ activity
(Fig. 7G,H,K).
This blockage was reverted by washing out the glutamate antagonist with
BMI-ACSF (Fig. 7I,K). The
decorrelation effect of CNQX on BMI-treated astrocytic networks was
accompanied by a significant 37.2% decrease in the number of
spontaneously active astrocytes (22 ± 2.7 vs 14.5 ± 1.5 active astrocytes; four slices; p < 0.05; paired
t test) (Fig.
7G,H,K). In
contrast, blockade of NMDA receptors in BMI-treated hippocampal slices
did not alter the correlation degree of astroglial networks (Fig.
7K). Moreover, the number of astrocytes exhibiting
[Ca2+]i changes in
BMI-ACSF (21 ± 3.8 active astrocytes; three slices) was not
perturbed by APV (18 ± 2.5 active astrocytes; three slices; p = 0.46; paired t test) (Fig.
7K). Similarly, incubation of BMI-induced epileptic
slices with CNQX decreased by 75.6 ± 8% the number of active
neurons (p < 0.05; paired t test),
whereas incubation with APV did not alter the number of active neurons
(p = 0.28; paired t test). These data
show that in conditions of neuronal hyperexcitability, such as
epilepsy, the correlation of spontaneous astrocytic network activity is
regulated mainly by AMPA/kainate receptors.
| |
DISCUSSION |
Spontaneous
[Ca2+]i
oscillations in astrocytes have been described in dissociated and
organotypic cultures (Fatatis and Rusell, 1992; Charles, 1994;
Harris-White et al., 1998). More recently, spontaneous
[Ca2+]i transients
have been reported in situ in microdomains of cerebellar Bergmann glia surrounding parallel fiber synapses and in the cell bodies of thalamic and hippocampal astrocytes (Grosche et al., 1999;
Parri et al., 2001; Nett et al., 2002). Here, we show that astrocytes
from a number of brain regions, including the cerebral cortex, display
spontaneous activity in situ in all the developmental stages
studied and in the adult. The finding that patterns of astrocyte
activation were variable, ranging from random events to bursting
activity, raises the possibility of correlations with development or
with region-specific patterns of neuronal activity.
Although resting astrocytes are related to the modulation of synaptic
transmission (Araque et al., 2001; Bezzi and Volterra, 2001), activated
astrocytes play essential roles in injury-related processes such as the
healing of scars, repair of the extracellular matrix, removal of
debris, and control of the blood-CNS interface (Raivich et al., 1999).
Thus, the finding that reactive astrocytes lack
[Ca2+]i transients
not only demonstrates that spontaneous astrocytic activity depends on
the functional state of these cells, but indicates a relationship with
the regulation of neural transmission. Because changes in
[Ca2+]i control
gene expression and cell differentiation (Spitzer et al., 2000), the
lack of spontaneous
[Ca2+]i transients
in astrocytes might contribute to the activation of astrocytes. Taken
together, the above data show that spontaneous [Ca2+]i
oscillations are a common property of most brain astrocytes that is
lost when they become activated after injury.
Although certain neurotransmitters, such as glutamate and ATP, trigger
increases in
[Ca2+]i and
oscillations (Cornell-Bell et al., 1990; Charles et al., 1991; Parpura
et al., 1994; Pasti et al., 1997; Porter and McCarthy, 1997; Reetz et
al., 1997; Verkhratsky et al., 1998; Guthrie et al., 1999), our results
show that the generation of spontaneous activity in hippocampal
astrocytes in situ is mostly independent of both ATP
signaling and activation of glutamatergic and GABAergic receptors, and
of neuronal activity, which is consistent with recent reports (Parri et
al., 2001; Nett et al., 2002; Thasiro et al., 2002). Moreover, we show
that spontaneous oscillations are critically dependent on extracellular
and intracellular Ca2+, which might cause
the typical large and long-lasting profiles of
[Ca2+]i
oscillations of astrocytes. Thus, it is conceivable that the removal of
extracellular Ca2+ may abolish a
Ca2+ influx-dependent baseline release of
neurotransmitters from nerve terminals that is necessary to
activate astroglial cells. However, treatment with the selective
V-ATPase inhibitor bafilomycin A1, which inhibits vesicular
neurotransmitter release (Araque et al., 2000; Zhou et al., 2000), does
not prevent astrocyte
[Ca2+]i
oscillations in situ (Nett et al., 2002), suggesting a
direct requirement of
[Ca2+]0 for the
generation of spontaneous astrocytic activity. Finally, the strikingly
similar pharmacological profiles observed for astrocytic [Ca2+]i
oscillations in the thalamus (Parri et al., 2001) and hippocampus (present results) support the notion that spontaneous astrocytic activity is triggered by mechanisms common to several brain regions.
The finding that the percentage of spontaneously active cells and their
oscillation rates are very similar in astrocytes and neurons indicates
that astrocytic activity is a robust phenomenon. Using an approach that allows the monitoring of
spontaneous
[Ca2+]i changes in
large numbers of astrocytes, we demonstrate large and complex networks
of correlated, synchronous astrocytes. We found that ~80% of
spontaneously active astrocytes in the hippocampus display synchronous
astrocyte-to-astrocyte
[Ca2+]i events,
forming complex networks that recruit dozens of astrocytes, often
located at great distances. Interestingly, the patterns of spontaneous
coactivation described here in the hippocampus in situ are
much more elaborate than those observed previously in dissociated,
organotypic cultures and neocortical slices (Dani et al., 1992;
Charles, 1998; Harris-White et al., 1998; Thasiro et al., 2002).
Similarly, in the thalamus in situ, Parri et al. (2001)
described spontaneous synchronous correlations among up to five
neighboring astrocytes. The highly synchronous activation characteristic of postnatal hippocampal neurons (Ben-Ari et al., 1997;
Garaschuk et al., 1998; Ben-Ari, 2001) might account for these
differences in network complexity. However, we favor the view that it
is the combined transgenic/imaging approach used in this study,
designed to screen activity in large numbers of astrocytes, that has
allowed the detection of these complex correlated networks. Finally,
because astrocytic networks occur at distinct postnatal stages in the
hippocampus (present data), the thalamus (Parri et al., 2001), and
other areas, such as the neocortex (F. Aguado, J. F. Espinosa-Parrilla, and E. Soriano, unpublished observations), network
activity may be a property of most astrocytes in vivo.
In our experiments, many spontaneously active astrocytes (61%) had
Ca2+ events correlated with hippocampal
neurons, which suggests a relationship between spontaneous neuronal and
glial activities. In fact, previous studies have described
structural and functional interactions between neurons and astrocytes,
such as gap junctions and extracellular messengers (Dani et al., 1992;
Porter and McCarthy, 1996; Pasti et al., 1997, 2001; Araque et al.,
1998a,b; Ventura and Harris, 1999; Alvarez-Maubecin et al.,
2000; Rouach et al., 2000; Rochon et al., 2001). Although the
percentage of active astrocytes and their rate of oscillation does not
change substantially after action potential blockade with TTX, the
overall synchronous activity of astrocytic networks in the postnatal
hippocampus is abolished, indicating that neuronal activity is required
to adjust the spatiotemporal pattern and synchrony of spontaneously
active astrocytes. Conversely, greater neuronal activity levels caused by epileptiform discharges with BMI not only raise the number of active
astrocytes (Tashiro et al., 2002), but dramatically increases
the network correlation between astrocytes and astrocytes and neurons.
In fact, in the bicuculline model of epilepsy, many astrocytes are
recruited into the highly synchronous epileptiform waves induced in
hippocampal neurons, demonstrating a fast spatiotemporal control of
glial spontaneous activity by neurons. Thus, astrocyte oscillations and
their degree of coactivation are regulated by neurons when the levels
of neuronal activity exceed a threshold, as in the epileptiform state.
Gap junctions control Ca2+ wave
propagation in cultured astrocytes (Giaume and McCarthy, 1996; Charles,
1998; Giaume and Venance, 1998). The present study, which shows that
correlation of spontaneous astrocytic Ca2+
activity is independent of gap junctions, together with the fact that
evoked Ca2+ waves in acute brain slices
are unaffected by gap junction blockers (Schipke et al., 2001),
suggests that different mechanisms correlate Ca2+ activity in cultured and in
situ astrocytes. In contrast, inhibition of NMDA and non-NMDA
glutamate receptors impairs spontaneous astrocytic activity, which is
consistent with other studies that show that activation of glutamate
receptors is involved in the propagation of astrocyte
Ca2+ signaling (Cornell-Bell et al., 1990;
Cornell-Bell and Finkbeiner, 1991; Dani et al., 1992; Finkbeiner, 1992;
Venance et al., 1997). Because, in addition to neurons, astrocytes
exhibit Ca2+-dependent glutamate release
(Bezzi et al., 1998, Araque et al., 2000; Innocenti et al., 2000),
astrocytic network activity could be controlled by glutamate derived
from both neurons and astrocytes. It is noteworthy that TTX and
ionotropic glutamate receptor antagonists decorrelate astrocytic
networks in a similar manner, without altering the number of active
astrocytes. These observations indicate that glutamate released from
depolarized nerve terminals contributes to the correlation of astrocyte
Ca2+ activity. This view is also in
agreement with data on the functional expression of NMDA and non-NMDA
receptors in astrocytes (Porter and McCarthy, 1995; Schipke et al.,
2001).
We also show that CNQX, but not APV, reverts the BMI-induced increased
levels of spontaneous activity of astrocytes and their spatiotemporal
coordination. Because activation of AMPA/kainite but not NMDA receptors
is the main mechanism involved in the generation and propagation of
epileptiform discharges in BMI-treated hippocampus (Jones and Lambert,
1990; Albowitz et al., 1997; Stoop and Pralong, 2000), the view that
neuronal-released glutamate correlates astrocytic network activity is
supported further. Taken together, the present data indicate that
neuronal glutamate modulates the network properties of spontaneous
astrocytic Ca2+ signaling by activating
ionotropic receptors.
In summary, we conclude that
[Ca2+]i
oscillations in astrocytes and their network properties are generated
by intrinsic mechanisms in vivo, which are modulated,
however, by neuronal activity and fast glutamate receptors. Thus,
together with data showing that astrocyte stimulation leads to neuronal
discharges (Pasti et al., 1997, 2001; Araque et al., 1998a,b; Bezzi et
al., 1998; Parpura and Haydon, 2000; Parri et al., 2001), our results
point to a complex functional scenario in which spontaneous astrocyte
and neuronal activities are regulated bi-directionally in a dynamic manner. Given the essential role of spontaneous and evoked neuronal activity in diverse events during neuronal development and in synaptic
plasticity (Katz and Shatz, 1996), a complex neuronal/astrocytic network may play a key role in these processes. In addition, our data
point to an unexplored function of astrocyte
[Ca2+]i signaling
in neuropathological processes such as epilepsy.
| |
FOOTNOTES |
Received Oct. 24, 2001; revised July 31, 2002; accepted Aug. 6, 2002.
*
F.A. and J.F.E.-P. contributed equally to this work.
This work was supported by grants from Comisión
Interministerial de Ciencia y Tecnología, Fondo de
Investigación Sanitaria, and Fondo Europeo de Desarróllo
Regional (FIS01-1684 and SAF01-3
TOP
ABSTRACT
INTRODUCTION
