From the
Long-term changes of synaptic strength in the central nervous
system are mediated by an increase of cytosolic calcium concentration
([Ca
Astrocytes, the mayor class of glial cells in the mammalian
brain, serve a series of important functions in the central nervous
system, participating to the regulation of the ionic composition of the
extracellular space, the formation of the blood-brain barrier, and
guiding the migration of neurons in the developing
embryo(1, 2) . Astrocytes also possess an efficient
uptake system for several neurotransmitters(3, 4) ,
thereby contributing to their removal from the synaptic cleft,
voltage-dependent ion channels, and a variety of neurotransmitter
receptors, including ionotropic and metabotropic glutamate receptor
subtypes(5, 6, 7, 8) . The function of
these receptors is still unclear, but their activation, both in
vitro(9, 10, 11) and in brain
slices(12, 13) , causes a rapid elevation in the
intracellular calcium concentration
[Ca
The sensitivity of astrocytes
to glutamate is of particular interest since glutamate represents the
main excitatory neurotransmitter in the central nervous system, and it
is responsible for phenomena as diverse as neurodegeneration (16) and long-term potentiation and depression of synaptic
connections(17, 18) . These latter phenomena are thought
to represent a form of memory function at the cellular
level(19, 20) . In this scenario, the sensitivity of
astrocytes to glutamate is generally believed to be of secondary
importance, at least for the processing of information in the neural
network. However, recent findings suggest a revision of the role played
by these glial cells in the brain and lead to the hypothesis that
[Ca
We provide evidence here for a
long-lasting increase in the frequency of
[Ca
The long-term change in the oscillatory
response upon repetitive glutamate stimulation was observed also in the
presence of 50 µM CNQX, a selective antagonist of the
AMPA/kainate glutamate receptor(30) , indicating that the
activation of AMPA/kainate receptors is not necessary for the induction
of this phenomenon. When glutamate was added in the absence of
extracellular Ca
We next investigated whether the induction of
this phenomenon is specific to the mGluR or whether it could be induced
also by stimulation of other receptors coupled to the production of
IP3. Indeed, oscillations in nonexcitable cells largely depend on the
production of IP3 and on regenerative cycles of Ca
Another consequence of the mGluR stimulation in astrocytes is the
activation of a constitutive NO synthase and production of
NO(39) . This membrane-permeant compound is an intercellular
messenger thought to be involved in a variety of cellular
functions(40) , including long term potentiation and long term
depression in neurons(41, 42) . We, therefore, tested
whether NO is involved in the induction of the increase in oscillation
frequency in astrocytes. The NO synthase inhibitor L-NAME (20
µM) (43) abolished the increase in oscillation frequency
otherwise observed following repetitive stimulation with glutamate (Fig. 3A and ), without significantly
affecting the amplitude of the
[Ca
As to the source of NO, both neurons and astrocytes
are known to express a constitutive
Ca
The potentiation of
the response using the 1 Hz protocol was observed mainly in astrocytes
having an oscillation frequency lower than 8 during the first series of
glutamate pulses (Fig. 6). In other words, cells oscillating at
low frequency during the first glutamate pulse showed a net
potentiation during the second pulse, while those oscillating at high
frequency were not potentiated and, in some cases, were inhibited.
Long-term changes in neurotransmitter-mediated responses have
been previously thought to be an exclusive property of neuronal cells.
The finding here reported that glutamate can modulate the frequency of
[Ca
The molecular
mechanism underlying this long-term modification has not been clarified
yet, although the results obtained with L-NAME and
Bt
Despite the fact that much interest has been devoted in
the last few years to the understanding of the physiological
significance and biochemical mechanism of these
[Ca
The stimulus regime for each
experimental condition is described in detail in the figure legends.
The time interval between agonist applications, unless specified, was
10 min. Values in the third pulse column indicate the change in
frequency of the third with respect to the first pulse; the level of
significance is, however, calculated comparing the values of
oscillation frequency obtained in the third stimulation with those
obtained in the second. Statistical analysis was performed by paired t-test.
We thank M. Santato and G. Ronconi for technical
assistance, M. Murgia for helpful discussions, M. J. Berridge, V.
Gallo, J. Meldolesi, C. Montecucco, and R. Rizzuto for critically
reading of the manuscript and for suggestions.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
]
) following
activation of excitatory neurotransmitter receptors. These phenomena,
which represent a possible cellular basis for learning and memory
processes in eukaryotes, are believed to be restricted to neurons. Here
we provide evidence for a long-term change of the response elicited by
the excitatory neurotransmitter glutamate in a non-neuronal cell
population of the central nervous system, i.e. visual cortical
astrocytes in culture. Stimulation with glutamate induces in astrocytes
a regular pattern of [Ca
]
oscillations. A second stimulation, after an interval
ranging from 2 to 60 min, induces an oscillatory response characterized
by an increased frequency. Induction of this change in the astrocyte
response is abolished by a specific inhibitor of the nitric oxide
synthase and recovers upon exogenous nitric oxide generation or
addition of a permeant cGMP analogue. Local brief pulses of glutamate
to individual astrocytes, at a rate of 1 Hz, also elicit
[Ca
]
oscillations
whose frequency increases following a second series of pulses. The
long-lasting modification in the
[Ca
]
oscillatory
response induced by glutamate in astrocytes demonstrates that in the
central nervous system cellular memory is not a unique feature of
neurons.
]
(
)often followed by periodic oscillations and waves
propagating from one cell to the other for hundreds of
micrometers(14, 15) .
]
oscillations and
waves in astrocytes represent a Ca
-based form of
excitability which serves as a long-range signaling system in the
central nervous system(10, 21, 22) . In
particular: i) oscillations of
[Ca
]
in astrocytes can
be elicited by impulse activity in adjacent
neurons(23, 12) ; ii) stimulated astrocytes can send
messages back to neurons, in as much as
[Ca
]
elevations in
astrocytes have been observed to trigger significant
[Ca
]
increases in
neighboring neurons, either by diffusion of Ca
through gap junctions (24) or as a consequence of local
release of glutamate (25).
]
oscillations in
visual cortical astrocytes upon repetitive stimulation with glutamate,
but not with other agonists linked to IP3 generation and
[Ca
]
oscillations.
These findings add new information about the signals governing the
interactions between neurons and astrocytes and, more importantly,
demonstrate the existence of a novel long-lasting modification of the
response to glutamate in a non-neuronal cell population of the central
nervous system.
Cell Cultures
Primary cultures of cells from the
visual cortex were prepared from neonatal Wistar rats (Charles River
Italia, Como). Pups were sacrificed by cervical dislocation, and blocks
of tissue, 2 3 mm, from the occipital cortex were removed and
cut into small pieces. Cells were dissociated in trypsin (0.8 mg/ml),
then plated on poly-L-lysine-coated (10 µg/ml; Sigma)
glass coverslips of 24-mm diameter at a density of 10
cells
per dish (35 mm). The growth medium consisted of Eagle's basal
medium (BME; ICN, Milan, Italy) containing 2 mM glutamine, 20
mM NaHCO
, 25 mM KCl, 10% fetal calf
serum, and streptomycin (0.1 mg/ml; Life Technologies, Inc.).
Experiments were performed on 8-15-day-old cultures. At day 11,
78% of the cells were identified as astrocytes and 21% as neurons by an
indirect immunofluorescent technique utilizing anti-GFAP (Boehringer)
and anti-200-kDa neurofilament antibodies(26) , respectively.
[Ca
Fura-2 loading was performed as
described(27) . Briefly, cells were loaded with 5
µM fura-2/acetoxymethyl ester in their culture medium for
40 min at 37 °C. Coverslips were then mounted in a thermostated
chamber (37 °C) (Medical System Corp., Greenvale, NY) on the stage
of an inverted epifluorescence microscope (Zeiss Axiovert 100TV)
equipped with the imaging apparatus (Analytical Imaging Concepts,
Atlanta, GA) for fura-2 measurements. The ratio images (1 ratio image
every 2 s, unless otherwise stated) were computed off-line after
subtraction of the background. Background was calculated at the end of
each experiment after adding 2 mM ionomycin followed by 1
mM Mn]
Measurements
. R
and R
and
were calculated in situ as
described(27) . Drugs were either added in the perfusate or
directly into the chamber dissolved in a volume of Krebs-Ringer
modified buffer (KRB) equal to that of the chamber (0.5 ml). No
differences between the two procedures were observed. The composition
of KRB is (in mM): 125 NaCl, 5 KCl, 1
Na
PO
, 1 MgSO
, 1 CaCl
,
5.5 glucose, 0.2 sulfinpyrazone, 20 HEPES (pH 7.4, 37 °C). The
frequency of oscillations (number of
[Ca
]
peaks per min)
was calculated from the initial 60-120 s of the cell response. To
investigate the role of extracellular Ca
, 2 min
before the addition of glutamate, the perfusion medium was changed with
a Ca
-free KRB supplemented with 1 mM EGTA.
Stimulation with glutamate was carried out in this medium. After the
removal of the stimulus, cells were incubated for 10 min in KRB with 1
mM CaCl
, followed by the procedure described
above.
Local Applications of Glutamate
Glutamate was
applied by means of a 0.1-µm tip diameter glass pipette positioned
50-100 µm from the cell of interest and connected to a
microinjection pressure system (Microinjector 5242, Eppendorf, Hamburg,
Germany). The recording chamber was continuously perfused with KRB (5
ml/min) through a large diameter pipette positioned several millimeters
from the glutamate pipette. LY fluorescence was measured at a single
excitation wavelength (440 nm; emitted light long-pass filtered at 540
nm) providing a fast temporal resolution (33 ms per frame).
Chemicals
CNQX and 1S,3R-ACPD
were from Tocris (Bristol, Great Britain), SIN-1 from BIOMOL (Plymouth
Meeting, PA); and fura-2 from Molecular Probes. All other chemicals
were from Sigma (Milan, Italy).
Glutamate-mediated Increase in the Frequency of
[Ca
Mixed cultures of astrocytes and neurons
from the rat visual cortex were loaded with the Ca]
Oscillations
indicator fura-2 and analyzed by the fluorescence video imaging
technique(28, 29) . The addition of 20 µM glutamate caused an abrupt rise of
[Ca
]
in both neurons
and astrocytes. The pattern of the
[Ca
]
response in the
two cell populations was, however, different. In neurons, the
[Ca
]
rise consisted of
a rapid peak followed by a return to a lower steady state level (data
not shown); only occasionally (5 out of 52), the first peak was
followed by irregular oscillations above an elevated plateau. In
astrocytes, the addition of 20 µM glutamate induced
sustained, periodic [Ca
]
oscillations eventually followed by
[Ca
]
waves propagating
from one cell to the other. Besides 20 µM, three
additional concentrations were tested. At 1 mM, astrocytes (n = 33) responded with an initial spike followed by a
long-lasting elevation of [Ca
]
with no oscillations; at 100 µM, astrocytes (n = 58) showed a pattern of mixed response, from a
step rise of [Ca
]
to
periodic oscillations; at 5 µM (n = 15),
they responded with a single transient
[Ca
]
elevation. Fig. 1A shows that astrocytes exposed to a second
application of 20 µM glutamate, 10 min after the first,
had a clearly increased frequency of
[Ca
]
oscillations,
while their amplitude and shape did not change significantly.
Subsequent exposures to glutamate resulted in further increases in the
frequency of oscillations. The bar graph of Fig. 1A reports the mean frequency of
[Ca
]
oscillations
during three consecutive stimulations. The washout of glutamate, which
blocked oscillations, and the subsequent reapplication of the stimulus
were necessary for inducing the increase in oscillation frequency.
Indeed, prolongation (over 10 min) of the first glutamate pulse
resulted in a decrease in both the amplitude and frequency of
oscillations.
Figure 1:
Changes
in [Ca] oscillation frequency induced by
glutamate stimulation. A, [Ca
]
oscillations in a single astrocyte loaded with fura-2 following
repetitive applications of 20 µM glutamate. The continuous line at the bottom of the traces indicates the
application of glutamate. &cjs0606; indicates a time interval of 10 min
during which cells were first washed with, and then maintained in, KRB
without glutamate. Roman numerals indicate subsequent
glutamate applications. The bar graph on the right presents the mean frequency ± S.E. of
[Ca
] oscillations following three
consecutive stimulations. The statistical significance, calculated by
paired t-test, is reported in Table I. The efficacy of a
fourth glutamate pulse was tested in a few cells (n =
7). B and C, increase in
[Ca
] oscillation frequency induced by the
second stimulation of glutamate performed 2 min and 60, respectively,
after the first. Conditions and labels are as in A. Details of
fura-2 loading, drug application, and fluorescence data acquisition are
given under ``Materials and
Methods.''
Two temporal aspects of the phenomenon were then
investigated: i) the length of the first glutamate stimulation and ii)
how long cells maintain the modification of their response after the
first stimulation. Results indicated that the increase in oscillation
frequency represents a rapidly developing and relatively long-lasting
event. In fact, the frequency of oscillations at the second glutamate
stimulation increased even when the length of the first stimulation was
reduced to 2 min, i.e. the minimum time necessary for a
reliable calculation of the frequency of oscillations and the interval
between two consecutive glutamate applications was as short as 2 min (Fig. 1B) or as long as 60 min (Fig. 1C;
see also ).
, oscillations were still observed,
although, in most astrocytes, two glutamate exposures were necessary
for inducing the increase in oscillation frequency (). A
possible explanation of this finding is that oscillations, in most
astrocytes, slowed down quite rapidly and subsided in 3-5 min,
thus not providing a proper stimulation for triggering the increase in
oscillation frequency. A similar response pattern was observed
following the selective stimulation of the mGluR (31) by 10
µM 1S,3R-ACPD (32) (). The lower efficacy of
1S,3R-ACPD with respect to glutamate may be due to a
differential activation by these two compounds of the various mGluRs
expressed by astrocytes. Taken together, these latter results indicate
that the activation of mGluRs is sufficient for the generation of the
long-term change of the [Ca
]
response in astrocytes and that Ca
entry
is not an absolute requirement for its induction, although, as already
reported(10) , it is crucial for the maintenance of the
oscillatory response.
release from internal stores(33, 34) . Other
agonists that engage the phosphoinositide signaling pathway might,
therefore, induce an increase in oscillation frequency following
repetitive applications. Four stimuli were tested: the
neurotransmitters NAdr and acetylcholine, the peptide bradykinin, and
ATP(35) . Acetylcholine, bradykinin, and ATP induced an increase
in [Ca
]
through
Ca
mobilization, but
[Ca
]
oscillations were
rare, and, when present, they were not as regular as those induced by
glutamate, at least in our experimental conditions (data not shown).
The level of the [Ca
]
peaks and the general shape of the
[Ca
]
transients
following repetitive stimulation with the same agonist were, however,
remarkably similar, recalling the ``fingerprint'' phenomenon
already described in insulinoma cells(36) . Comparable responses
following repetitive stimulation with 100 nM NAdr were
observed in the majority of astrocytes (Fig. 2A), while
in some of them (9 out of 57), NAdr induced a pattern of regular
[Ca
]
oscillations. In
none of these cells, however, the frequency of oscillations increased
following the second stimulation (Fig. 2B and ). Taken together, these results suggest that in astrocytes
the long-term change in oscillation frequency upon repetitive
stimulation is specifically linked to activation of the mGluR. Further
experiments will be necessary to clarify whether it represents a
peculiar property of the astrocyte response to glutamate or whether a
similar phenomenon can be observed with different stimuli also in other
cells, thereby representing a general modulatory mechanism of the
[Ca
]
oscillatory
response.
Figure 2:
[Ca]
transients in response to NAdr stimulation. Nonoscillatory (A)
and oscillatory (B) response in single astrocytes following
two consecutive applications of 100 nM NAdr. The time interval
between stimulations was 10 min. Conditions and labels are as in Fig.
1.
Role of Nitric Oxide in the Long-term Change of the
Astrocyte Response
In order to obtain some clues as to the
mechanism(s) underlying the increase in oscillation frequency, we used
a number of pharmacological tools known to affect some of the signaling
pathways modulated by stimulation of the mGluR(37) . We first
examined the possible influence of phosphorylation processes.
Staurosporine, which in the nanomolar range is considered a specific
inhibitor of protein kinase C(38) , was without effect up to 50
nM (). Above this concentration, probably through
effects other than the inhibition of protein kinase C, this drug
induced a decrease in oscillation amplitude and frequency even during
the first glutamate stimulation. Although not conclusive, these results
suggest that activation of protein kinase C is not required for the
increase in oscillation frequency induced by glutamate stimulation. The
oscillatory response was, however, abolished by activators of either
protein kinase C or cAMP-dependent protein kinase, PMA (100
nM), and dibutyryl cAMP (100 µM), respectively
(data not shown). This inhibitory effect, although suggesting a
negative modulation of the oscillatory response per se by
activation of protein kinase C or cAMP-dependent protein kinase,
complicates a proper analysis of the possible role of phosphorylation
processes in the induction of the increase in oscillation frequency.
]
spikes. The
effect was stereospecific, the inactive optical isomer D-NAME
(20 µM) (43) being without effect (Fig. 3B and ). The involvement of NO in the
modulation of oscillation frequency was further supported by
experiments with the NO donor SIN-1(44) . When added to L-NAME-treated cells during the glutamate applications, this
compound (100 µM) partially reversed the inhibitory effect
of L-NAME (Fig. 3C and ), without
affecting frequency and amplitude of oscillations. A possible role of
an elevation of the cGMP levels was then investigated, since a well
known action of NO is the stimulation of the soluble cGMP-synthesizing
enzyme, guanylate cyclase(45) . The addition of 20 µM Bt
GMP, a permeant analogue of cGMP, could, in part,
restore, in L-NAME treated cells, the increase in oscillation
frequency observed following repetitive glutamate applications (Fig. 3D). The value of this increase is similar to that
observed with the NO donor, although the treatment with
Bt
GMP also caused a clear reduction in the amplitude of the
[Ca
]
peaks (Fig. 3D).
Figure 3:
The
induction mechanism of the increase in oscillation frequency involves
the production of NO. A and B, oscillatory response
to 20 µM glutamate in single astrocytes following
preincubation for 10 min with either L-NAME (A) or D-NAME (B), 40 min before the first glutamate
application. Note that the NO synthase inhibitor L-NAME
induced a decrease in oscillation frequency that is statistically
significant following the third glutamate challenge (Table I). C and D, reversal by SIN-1 and BtGMP (dbcGMP) of the L-NAME inhibitory effect on
glutamate-mediated increase in oscillation frequency. The continuous lines at the bottom of the traces indicate
the application of 20 µM glutamate (thick line)
and either SIN-1 or Bt
GMP (thin line). Conditions
and labels are as in Fig. 1.
At the level of the nervous system, cGMP
exerts multiple effects(46, 47) . Concerning
[Ca]
oscillations, the
recent finding that cGMP affects the levels of cADP-ribose is of
particular interest(48) . In fact, cADP-ribose is considered a
modulator of the ryanodine receptor, an intracellular
Ca
-release channel expressed in a variety of cell
types(49, 50, 51) . In order to test the
possibility that the effects of NO and cGMP on
[Ca
]
oscillations
could be, at least in part, mediated through cADP-ribose, cells were
treated with 10 µM ryanodine. This ryanodine concentration
is known to block the channel in a low conductance open
conformation(52) . If ryanodine receptors are involved in the
mechanism of oscillations in astrocytes, ryanodine is expected to
either block oscillations or change their pattern. However, the
addition of 10 µM ryanodine, either before the first
glutamate stimulation or during oscillations, was ineffective. Further
experiments are, therefore, necessary to clarify the action of cGMP on
oscillations.
/calmodulin-dependent NO
synthase(53, 39) . In neurons, the production of NO is
triggered by the activation of the N-methyl-D-aspartate receptor(54) . The
increase in oscillation frequency in astrocytes was, however, observed
also in the presence of 10 µM MK-801, an open channel
blocker of the N-methyl-D-aspartate glutamate
receptor(55) , suggesting that the production of NO by neurons
is not crucial for the induction of the astrocyte plasticity. The
possibility that neuronal-derived NO cooperates in the regulation of
oscillation frequency in astrocytes cannot, however, be excluded.
Local Brief Application of Glutamate
Prolonged
exposures to constant levels of glutamate, such as those described
above, do not occur in normal physiological conditions. During synaptic
activity, astrocyte processes, which are in intimate association with
nerve terminals in the synaptic cleft(56) , are probably exposed
to concentrations of glutamate higher than 20 µM, but for
very short time periods(57) . We, therefore, investigated
whether brief local applications of glutamate to individual astrocytes
could induce a change in [Ca]
and, possibly, oscillations. Glutamate (100 µM)
was applied through a capillary pipette connected to a microinjection
pressure system, under continuous perfusion of the chamber. As shown in Fig. 4, an astrocyte, immunocytochemically identified by
anti-GFAP antibody staining at the end of the recording experiment (Fig. 4D), responded to a single 100 µM glutamate pulse with a transient elevation of
[Ca
]
(Fig. 4, A, B, and C), whose kinetics is shown in Fig. 4H; closely located astrocytes hardly responded to
the brief glutamate pulse. In order to visualize the spatial diffusion
and the duration of the pulse, in all the experiments, the fluorescent
dye LY was included in the pipette solution, and its fluorescence was
detected with a time resolution of 33 ms per frame. The duration of the
glutamate pulse which elicited the response shown in Fig. 4, A, B, and C was estimated to be less than
100 ms, since LY fluorescence increased to a maximum within 66 ms and
then decreased to the background level within the following 33 ms (Fig. 4, E, F, and G). A series of
brief applications of glutamate (100 µM) at low frequency
(0.1 Hz) induced only single transient
[Ca
]
elevations after
each pulse (Fig. 5A). After a time interval of 10 min,
the [Ca
]
response
following a second series of pulses at 0.1 Hz did not change
significantly. However, if the frequency of the pulses was increased to
1 Hz, astrocytes failed to respond to each pulse (Fig. 5B, inset) and started to oscillate with
a periodicity that was characteristic of the cell under study (Fig. 5B and ). The majority of astrocytes
(18 out of 32) showed at least a 10% increase in their frequency of
oscillations following a second train of glutamate pulses at 1 Hz (). A third series of pulses was performed in some cells
and a typical example is shown in Fig. 5B. In this and
other 5 out of 9 cells tested, the frequency of oscillations clearly
increased also during the third series of pulses ().
Figure 4:
[Ca] changes
induced in an individual astrocyte by a local brief application of
glutamate. A, B, and C, pseudo-color images
of fura-2-loaded astrocytes before (A), at the peak of the
[Ca
] change (B), and 9 s after (C) the glutamate pulse. The gain of the camera was set to a
level that allowed us to detect part of the fine astrocyte processes.
Thus, the fluorescence at 340 nm at the center of the responding cell
body reached saturation at the peak of the response, and the ratio was
slightly underestimated. The position of the stimulating pipette is
shown as dashed lines in A.
[Ca
] levels (nM) are shown by the vertical bar in C. D, GFAP immunostaining of the same
region. E, F, and G, images of LY
fluorescence taken at the maximal time resolution of our measuring
system. Images taken at (E) 33 (F) and 66 (G) ms after the onset of the pulse. The position of the
pipette (arrow) and that of the cell (dashed lines)
which undergoes the large [Ca
] increase are
indicated in E. Bars in A and E, 10 µm. H, kinetics of [Ca
] changes of the
responding cell shown in A, B, and C. The
[Ca
] values are the means calculated over
the cell body and the largest processes. A, B, and C in the plot refer to the pseudo-color images in A, B, and C.
Figure 5:
[Ca] changes
induced by local brief applications of glutamate. A,
[Ca
] oscillations in an individual
astrocyte following glutamate stimulation at a slow rate (0.1 Hz); the dots indicate the timing of glutamate applications. The 10-min
interval is indicated by &cjs0606;. B,
[Ca
] oscillations in an individual
astrocyte following the application of the glutamate pulse at a
frequency of 1 Hz, as indicated by the continuous line on the bottom of the trace. The inset shows the kinetics of
the first two [Ca
] spikes with an expanded
time scale; the vertical bars on the bottom indicate
the timing of each pulse; 1 ratio image per second was acquired; bar = 5 s. In both cells (A and B),
the duration of the glutamate pulse, as measured by LY fluorescence,
was 66 ms.
The
reliability of this experimental approach is based on a series of
controls related to the glutamate pulse. As already mentioned, in all
the experiments, the glutamate pipette also contained the fluorescent
dye LY, thereby allowing us to determine the duration of the pulse. The
following results were obtained. i) The duration of the pulse, as
measured at the beginning and at the end of each train of glutamate
pulses, was unchanged, suggesting that a larger release of glutamate
(and of LY) from the pipette in the second series of pulses with
respect to the first, cannot account for the increase in oscillation
frequency. The duration of the pulse in the various experiments varied
from 66 to 166 ms. ii) The basal level of LY fluorescence in the
chamber was measured before and at the end of each experiment and found
not to be increased, indicating that repetitive trains of pulses did
not lead to an increased basal concentration of glutamate in the
chamber. iii) A possible increase of glutamate, after each pulse, in
the microenvironment surrounding the cell of interest was also
investigated. The following test was performed: in a first pulse the
gain of the camera was set to detect, as maximal fluorescence level,
the concentration of LY corresponding to that contained in the pipette
(25 µg/ml). A second pulse was then performed with a concentration
of LY in the pipette 200-fold higher than in the first pulse (5 mg/ml),
maintaining the same gain of the camera. In these latter conditions, a
value detectable just below the saturation level would, therefore,
correspond to a concentration of LY less than 25 µg/ml, i.e. 1:200 dilution. The LY fluorescence after the pulse reached this
value in less than 100 ms (3 frames). Assuming similar constants of
dilution for LY and glutamate, the kinetics of the pulse indicates that
the concentration of glutamate decreased to 0.5 µM, i.e. 200-fold dilution, within 100 ms.
Figure 6:
Changes in
[Ca] oscillation frequency following local
glutamate stimulation. The [Ca
] oscillation
frequency in each cell, as measured in the first series of pulses, is
plotted as a function of the relative change in oscillation frequency
in the second with respect to the first series of pulses. The
statistical significance, as calculated by paired t-test, is
reported in Table I. The line is the regression fit to the data (r = 0.71).
The brief repetitive glutamate applications used in these latter
experiments are probably a reasonable approximation of the situation of
an astrocyte adjacent to a neuron firing brief repetitive trains of
action potentials. Indeed, [Ca]
oscillations and waves in astrocytes have been reported to
occur following neuronal stimulation both in culture (23) and in
brain slices(12) .
]
oscillations in
astrocytes challenges this view and represents, to our knowledge, the
first example of a long-term change induced by a neurotransmitter in a
non-neuronal cell population. This form of cellular memory function in
astrocytes, that is triggered by repetitive activation of mGluRs, is a
rapidly inducible and relatively long-lasting event.
GMP suggest that NO, while it is without effects on
oscillations themselves, could be involved in the mechanism controlling
the frequency of oscillations.
[Ca
]
oscillations in
astrocytes, as in many other cell types, depend on cycles of
[Ca
]
release,
presumably mediated by IP3, and
[Ca
]
uptake from
intracellular stores(34) . It is, therefore, reasonable to
speculate that an increase in oscillation frequency should result from
a modification of processes related to either the mGluR, i.e. number of receptors, coupling to the G-protein, and/or changes in
the [Ca
]
release
mechanism.
]
oscillations,
many questions remain, however, unanswered or are still at the level of
working hypotheses. Periodic [Ca
]
increases following stimulation with hormones and growth
factors have been observed in a variety of nonexcitable cells and have
been linked to the control of many cellular events such as secretion,
cell growth, contraction, and fertilization (33, 34). One attractive
possibility is that Ca
may exert its action as second
messenger through a frequency rather than an amplitude-dependent
mechanism(33, 58, 59) . As mentioned above, the
frequency of oscillations in many cells, including
astrocytes(10) , is dependent on the concentration of the
agonist(33) , and a positive correlation between frequency of
[Ca
]
oscillations and
secretion process was demonstrated in pituitary cells(60) . In
other words, the information carried by the intensity of the stimulus
is preserved and converted into a defined frequency of oscillations. In
astrocytes, [Ca
]
oscillations and waves propagating from cell to cell by
means of gap junctions create a functional network and have been
proposed to represent a Ca
-based form of
excitability(10, 22) . Our results, demonstrating a
long-lasting increase in the frequency of oscillations without changes
in their amplitude, suggest that also in astrocytes the information may
be encoded through a change in the frequency of oscillations. Changes
in oscillation frequency might affect a number of functional features
of astrocytes, for example the synthesis and/or release of neuroactive
substances, such as NO itself (40) and
neurotrophins(61, 62, 63) , which participate in
experience-dependent neuronal plasticity in the visual
cortex(64, 65, 66) . Interestingly, one of the
intercellular messengers that is proposed to be involved in the
induction of long-term modifications in neurons, i.e. NO(41, 42) , appears to also be involved in the
induction of the long-term modifications in the astrocyte response.
Further studies in intact neural tissue, where
[Ca
]
oscillations in
astrocytes have been observed recently(13) , are necessary to
evaluate the physiological relevance of this astrocyte property.
Nevertheless, the present results raise the intriguing possibility that
intense synaptic activity may induce long-lasting changes in both
neurons and astrocytes, i.e. an increased synaptic strength in
neurons and an increased frequency of
[Ca
]
oscillations in
astrocytes.
Table: Frequency of oscillations and its relative
change in astrocytes following three (I, II, III) consecutive
applications of various agonists
], intracellular calcium concentration;
CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; AMPA,
-amino-3-hydroxy-5-methylisoaxole-4-propionic acid; mGluR,
glutamate metabotropic receptor; 1S,3R-ACPD,
1-aminocyclopentane-1,3-dicarboxylic acid; IP3, inositol
(1,4,5)-trisphosphate; NAdr, noradrenaline; PMA, phorbol 12-myristate
13-acetate; L-NAME, N
-nitro-L-arginine methyl ester; D-NAME, N
-nitro-D-arginine
methyl-ester; SIN-1, 3-morpholinosydnonimine hydrochloride;
Bt
GMP, dibutyryl-cGMP; GFAP, glia fibrillary acidic
protein; LY, lucifer yellow.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.