©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Long-lasting Changes of Calcium Oscillations in Astrocytes
A NEW FORM OF GLUTAMATE-MEDIATED PLASTICITY (*)

Lucia Pasti , Tullio Pozzan , Giorgio Carmignoto (§)

From the (1)Department of Biomedical Sciences and CNR Center for Biomembranes, University of Padova, Via Trieste 75, 35121 Padova, Italy

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Long-term changes of synaptic strength in the central nervous system are mediated by an increase of cytosolic calcium concentration ([Ca]) 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.


INTRODUCTION

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]()often followed by periodic oscillations and waves propagating from one cell to the other for hundreds of micrometers(14, 15) .

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] 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).

We provide evidence here for a long-lasting increase in the frequency of [Ca] 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.


MATERIALS AND METHODS

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]Measurements

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. 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 NaPO, 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).


RESULTS

Glutamate-mediated Increase in the Frequency of [Ca]Oscillations

Mixed cultures of astrocytes and neurons from the rat visual cortex were loaded with the Ca 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 ).

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, 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.

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 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.

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] 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 BtGMP, 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 BtGMP 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 BtGMP (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.

As to the source of NO, both neurons and astrocytes are known to express a constitutive Ca/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.

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.


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) .


DISCUSSION

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] 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.

The molecular mechanism underlying this long-term modification has not been clarified yet, although the results obtained with L-NAME and BtGMP 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.

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]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

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.



FOOTNOTES

*
This work was supported by grants from the Italian Research Council (CNR) ``Biotechnology'' and ``Oncology,'' ``Thelethon'' Italy, from the Italian Association for Cancer Research (AIRC), and from the ``AIDS Project'' of the Italian Health Ministry (to T. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed. Tel.: 39-49-828-6568; Fax: 39-49-828-6576.

The abbreviations used are: [Ca], 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; BtGMP, dibutyryl-cGMP; GFAP, glia fibrillary acidic protein; LY, lucifer yellow.


ACKNOWLEDGEMENTS

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.


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