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The role of Ca2+ in the generation of spontaneous astrocytic Ca2+ oscillations
Neuroscience 120 (2003) 979 –992
THE ROLE OF Ca2ⴙ IN THE GENERATION OF SPONTANEOUS ASTROCYTIC Ca2ⴙ OSCILLATIONS H. R. PARRI* AND V. CRUNELLI
Astrocytes in the CNS are postulated to respond to neuronal activity and form a parallel network of signalling in the brain (Cornell-Bell et al., 1990). There is now increasing evidence that glia interact with neurons, responding to neurotransmitter release (Porter and McCarthy, 1996) and affecting postsynaptic activity (Pasti et al., 1997; Araque et al., 1998; Kang et al., 1998; Castonguay and Robitaille, 2001). Astrocytes can however be spontaneously active, displaying [Ca2⫹]i oscillations which are independent of neuronal activity. Cultured cortical (Fatatis and Russell, 1992) and suprachiasmatic nucleus (van den Pol et al., 1992) astrocytes displayed spontaneous, tetrodotoxin (TTX)-insensitive [Ca2⫹]i oscillations. In in situ slice preparations, spontaneous [Ca2⫹]i oscillations were originally reported in the ventrobasal (VB) thalamus (Parri et al., 2001), and recently in other brain regions including the hippocampus and cortex (Nett et al., 2002; Tashiro et al., 2002; Aguado et al., 2002). Whilst neuron-astrocyte interactions are beginning to be elucidated, the function of, and the impact of spontaneous astrocytic [Ca2⫹]i oscillations are unknown. A pathological role has been suggested in the cortex since spontaneous oscillations were only observed in cortical slices in models of epilepsy (Tashiro et al., 2002). In support of this hypothesis is the observation that astrocytes cultured from a patient with Rasmussen’s encephalitis displayed spontaneous oscillations (Manning and Sontheimer, 1997). In the thalamus there seems to be a functional role for spontaneous astrocytic oscillations. Oscillations in the VB thalamus are correlated between astrocytes suggesting communication by a released factor, and recordings from thalamocortical neurons in this thalamic nucleus show that large NMDA-mediated inward currents are correlated to [Ca2⫹]i increases in neighbouring astrocytes (Parri et al., 2001; Parri and Crunelli, 2002a), indicating that astrocytes can affect or generate neuronal activity without prior neuronal input. In the hippocampus, synchronised astrocytic and neuronal oscillations are seen, and the interaction between the neuronal and astrocytic elements seems dependent on glutamate (Aguado et al., 2002). The spontaneous oscillations observed in different brain regions share many features including the duration of the individual [Ca2⫹]i elevations, the periodicity of the oscillations, and a dependence on intracellular [Ca2⫹]i release (Parri et al., 2001; Nett et al., 2002; Tashiro et al., 2002; Aguado et al., 2002). However, the steps leading to [Ca2⫹]i release are poorly understood. Though seemingly not requiring the eliciting of neurotransmitter release, it is not known if the activation of a membrane bound receptor
School of Biosciences, Cardiff University, Museum Avenue, PO Box 911, Cardiff, CF10 3US, Wales, UK
Abstract—Astrocytes in the rat thalamus display spontaneous [Ca2ⴙ]i oscillations that are due to intracellular release, but are not dependent on neuronal activity. In this study we have investigated the mechanisms involved in these spontaneous [Ca2ⴙ]i oscillations using slices loaded with Fluo-4 AM (5 M) and confocal microscopy. Bafilomycin A1 incubation had no effect on the number of spontaneous [Ca2ⴙ]i oscillations indicating that they were not dependent on vesicular neurotransmitter release. Oscillations were also unaffected by ryanodine. Phospholipase C (PLC) inhibition decreased the number of astrocytes responding to metabotropic glutamate receptor (mGluR) activation but did not reduce the number of spontaneously active astrocytes, indicating that [Ca2ⴙ]i increases are not due to membrane-coupled PLC activation. Spontaneous [Ca2ⴙ]i increases were abolished by an IP3 receptor antagonist, whilst the protein kinase C (PKC) inhibitor chelerythrine chloride prolonged their duration, indicating a role for PKC and inositol 1,4,5,-triphosphate receptor activation. BayK8644 increased the number of astrocytes exhibiting [Ca2ⴙ]i oscillations, and prolonged the responses to mGluR activation, indicating a possible effect on storeoperated Ca2ⴙ entry. Increasing [Ca2ⴙ]o increased the number of spontaneously active astrocytes and the number of transients exhibited by each astrocyte. Inhibition of the endoplasmic reticulum Ca2ⴙ ATPase by cyclopiazonic acid also induced [Ca2ⴙ]i transients in astrocytes indicating a role for cytoplasmic Ca2ⴙ in the induction of spontaneous oscillations. Incubation with 20 M Fluo-4 reduced the number of astrocytes exhibiting spontaneous increases. This study indicates that Ca2ⴙ has a role in triggering Ca2ⴙ release from an inositol 1,4,5,-triphosphate sensitive store in astrocytes during the generation of spontaneous [Ca2ⴙ]i oscillations. © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: ventrobasal thalamocortical.
thalamus,
glia,
development,
*Corresponding author. Tel: ⫹44-29-2087-5150; fax: ⫹44-29-20874986. E-mail address: [email protected] (H. R. Parri). Abbreviations: ACSF, artificial cerebrospinal fluid; 2-APB, 2-aminoethoxydiphenylborate; CCE, capacitive Ca2⫹ entry; CICR, calcium induced Ca2⫹ release; CPA, cyclopiazonic acid; DHP, dihydropyridine; DHPG, (S)-3,5-dihydroxyphenylglycine; IP3, inositol 1,4,5,-triphosphate; IP3-R, inositol 1,4,5,-triphosphate receptor; mGluR, metabotropic glutamate receptor; PKC, protein kinase C; PLC, phospholipase C; RyR, ryanodine receptor; SERCA, sarco(endo)plasmic reticulum Ca2⫹ ATPase; SOCC, store-operated calcium current; TC, thalamocortical; trans-ACPD, (⫾)-1-aminocyclopentane-trans1,3-dicarboxylic acid; TTX, tetrodotoxin; VB thalamus, ventrobasal thalamus.
0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00379-8
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Fig. 1. Spontaneous [Ca2⫹]i oscillations are intrinsic. A. Pseudocolour images of control and bafilomycin A1-treated slices. Circles indicate positions of spontaneously active astrocytes during a 600 s recording period. B. Spontaneous [Ca2⫹]i oscillations from two astrocytes in the control slice (upper traces) and from the slice following treatment with bafilomycin A1 (lower traces), showing that oscillations occur in both conditions. C. Bar graph of the number of spontaneously active astrocytes in the two conditions. D. Currents recorded from a TC neuron during lemniscal afferent synaptic stimulation in control and following incubation with bafilomycin A1, showing that whilst oscillations are unaffected, synaptic transmission was abolished. Arrows indicate timings of synaptic stimulations.
is required for their generation. There is also debate about the signal and mechanism of [Ca2⫹]i release. Calcium induced Ca2⫹ release (CICR) via ryanodine receptors (RyRs) has been suggested as the mechanism in cortical astrocytes (Tashiro et al., 2002), whilst inositol 1,4,5,triphosphate receptors (IP3-Rs) are implicated in the hippocampus (Nett et al., 2002). In this study, we investigated the mechanism leading to the generation of spontaneous astrocytic [Ca2⫹]i oscillations in the rat VB thalamus. The oscillations have many of the properties associated with agonist-receptor-mediated astrocytic Ca2⫹ increases but are intrinsic to astrocytes and are not dependent on vesicular transmitter release or on membrane receptor-mediated phospholipase C (PLC) activation. PLC is however implicated in the generation as is CICR via IP3-Rs. Some of these results were published in abstract form (Parri and Crunelli, 2002b).
EXPERIMENTAL PROCEDURES All procedures involving experimental animals were carried out in accordance with the U.K. Animals (Scientific Procedure) Act 1986 and local ethics committee guidelines. Slices of rat VB thalamus were prepared as described previously (Parri et al., 2001) from 5 to 17 day old rats. After a recovery
period of 1 h, slices were loaded with fluo-4 AM (Molecular Probes, Eugene, OR, USA) by incubating for 40 – 60 min at 28 °C with 5 M of the indicator dye and 0.01% pluronic acid. Under these conditions, astrocytes were preferentially loaded with the fluorescent indicator, whereas loading of neurons was rarely observed (Parri et al., 2001). All experiments were performed in standard artificial cerebrospinal fluid (ACSF) of composition (in mM): NaCl 120, NaHCO3 16, KCl 2, KH2PO4 1.25, MgSO4 1, CaCl2 2 (Turner et al. 1994). TTX (1 M) was also present in all experiments except the synaptic stimulation experiments described in Fig. 1. Experiments were conducted at room temperature (20 –24 °C). Chemicals were obtained from Sigma (St. Louis, MO, USA) unless otherwise stated. (S)-3,5-dihydroxyphenylglycine (DHPG) and (⫾)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) were obtained from Tocris (Bristol, UK). 2-Aminoethoxydiphenylborate (2-APB) and chelerythrine chloride were obtained from Calbiochem (Nottingham, UK). Pharmacological compounds were bath applied unless otherwise stated. Following a control recording period in normal ACSF, pharmacological agents were included in the perfusing ACSF and washed over the preparation. The number of active astrocytes seen during this experimental period was then compared with the number seen during the control period. For focal application of compounds, patch pipettes were filled with the compound dissolved in ACSF, the pipette tips were placed about 20 m above the astrocyte and the drug applied by use of a pressure ejection system (Neurophore; Medical Systems Corporation, NY, USA).
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Confocal fluorescence imaging The recording chamber and manipulators were mounted on a moveable top plate platform (MP MTP-01; Scientifica, Harpenden, UK). Fluorescence was measured using a Noran Odyssey confocal (Thermo Noran, USA) fitted to a Nikon E600FN (Nikon, UK, Kingston, UK) upright microscope. Averages of eight whole field images (206 m⫻158 m) were routinely acquired every 0.8 –5 s with a ⫻40 objective lens (NA⫽0.8). Acquisition and image analysis were performed using Noran Intervision software. Fluorescence values over time for specific regions of interest were exported into Sigmaplot (SPSS Inc., Chicago, IL, USA) for further analysis and ⌬F% plot production. ⌬F% is the increase of fluorescence, either spontaneous or evoked and is calculated by dividing the fluorescence change by the basal⫻100%. Displayed monochrome images showing slice and cellular morphology were produced by averaging in Intervision; contrast and brightness were also adjusted to enhance morphological information.
Measurement of spontaneous activity For experiments where the effect of a compound on spontaneous activity had to be compared in different slices (e.g. bafilomycin A1 experiments), control images were typically acquired for 600 s, and treated slices then imaged for 600 s, and the properties of the spontaneous oscillations in the two groups compared. Six hundred seconds was chosen as the maximum recording period to avoid dye bleaching affecting the observation of [Ca2⫹]i increases. Where activity could be compared in the same slice, acquisition times for both conditions were typically 300 s. To ease comparison between different experiments data were normalised, so that the number of astrocytes displaying Ca2⫹ transients during an experiment is quoted as a value per 100 s, e.g. in a defined slice area where 10 astrocytes displayed spontaneous transients in a 600 s period the number of spontaneously active astrocytes is quoted as 1.67 ast/100 s. To control for variability in the display of spontaneous oscillations, experimental and control slices were taken from the same animal and experiments performed on the same day. The frequency of Ca2⫹ oscillations in astrocytes exhibiting multiple Ca2⫹ increases is expressed as the number of increases per 100 s.
Evoked astrocytic [Ca2ⴙ]i increases [Ca2⫹]i increases evoked by drug application, e.g. caffeine and DHPG, are expressed as the absolute number of astrocytes displaying responses immediately following drug application in the defined slice area.
Electrophysiology Patch clamp recordings were made using sylgarded pipettes (2– 4 M⍀) containing an internal solution of composition (in mM): KMeSO4 120, HEPES 10, EGTA 10, Na2ATP 4, GTP 0.5. Currents were recorded using an Axopatch 200B amplifier, and acquired and analysed using Pclamp8 software (Axon Instruments, Foster City, CA, USA). Synaptic stimulation was achieved with a constant current isolated stimulator (Digitimer, Welwyn Garden City, UK) and a bipolar electrode. Data are displayed as mean⫾S.E.M. Significance was calculated using two tail unpaired and paired Student’s t-test as indicated in the text. ANOVA and Bonferroni tests were used for comparisons of more than two groups.
RESULTS Spontaneous astrocytic [Ca2ⴙ]i oscillations are intrinsic Spontaneous astrocytic [Ca2⫹]i oscillations in the VB thalamus display a variety of patterns of [Ca2⫹]i increase,
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including single transient [Ca2⫹]i increases or multiple [Ca2⫹]i increases within a recording period. Astrocytes displaying multiple increases have a periodicity of around 70 s (Parri and Crunelli, 2001). The [Ca2⫹]i oscillations are not dependent on neuronal activity since they are not blocked by TTX (Parri et al., 2001). This, however, does not exclude the possibility that they are sustained by spontaneous neurotransmitter release which could activate astrocytic metabotropic receptors to sustain [Ca2⫹]i oscillations. Experiments were thus performed using bafilomycin A1, which depletes neurotransmitter vesicles and so blocks neuronal and glial transmitter release by inhibition of vacuolar H⫹-ATPases (Araque et al., 2000; Pasti et al., 2001). Astrocytes displayed spontaneous [Ca2⫹]i oscillations in control conditions (Fig. 1B, upper traces) and following incubation with 4 M bafilomycin A1 (Fig. 1B, lower traces). Following bafilomycin A1 treatment the number of astrocytes displaying spontaneous [Ca2⫹]i oscillations was 1.29⫾0.3/100s (n⫽4 slices; see Experimental Procedures) compared with 1.21⫾0.1/100s (n⫽4 slices) in control conditions (Fig. 1C). The amplitude of spontaneous fluorescence increases was 114⫾11% (n⫽31) in control and 108⫾14% in bafilomycin A1 (n⫽29), no effect of bafilomycin A1 on the number of [Ca2⫹]i transients displayed by each astrocyte was seen. Bafilomycin A1 was shown to have caused vesicular depletion by comparing the synaptic responses of thalamocortical (TC) neurons in control and bafilomycin A1 conditions (Fig. 1D). Synaptic stimulation in control untreated slices evoked inward currents of amplitude 240⫾70pA (n⫽4 neurons). Following incubation with bafilomycin A1, synaptically induced currents were completely abolished (n⫽5 neurons). These results therefore show that spontaneous astrocytic oscillations are not dependent on transmitter release from neurons, or potential vesicular transmitter release from astrocytes (Araque et al., 2000; Pasti et al., 2001), and demonstrate that the generation of spontaneous [Ca2⫹]i oscillations is an intrinsic ability of astrocytes. Involvement of PLC and IP3-Rs activation As spontaneous astrocytic oscillations arise from the release of Ca2⫹ from intracellular stores (Parri et al., 2001), we investigated whether release was due to activation of RyRs or IP3-Rs. Caffeine (20 mM) application elicited [Ca2⫹]i increases in spontaneously active VB thalamus astrocytes (Fig. 2A), indicating the presence of RyRs. Experiments in bafilomycin A1-treated slices (n⫽3) confirmed that the caffeine effect was directly on the astrocytes and not on presynaptic neuronal elements. Incubation of slices for 15 min with 20 M ryanodine did not affect the number of astrocytes displaying spontaneous [Ca2⫹]i oscillations (Fig. 2B; control: 1.6⫾0.5/100 s, n⫽3 slices; ryanodine: 1.5⫾0.5/100 s, n⫽3 slices). The frequency of oscillations exhibited by individual astrocytes was also not affected by ryanodine (0.6⫾0.09/100 s, n⫽16, three slices in control and 0.63⫾0.06/100 s, n⫽16, three slices in ryanodine). The amplitude of the spontaneous fluorescence increases was unaffected by ryanodine treatment being
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Fig. 2. RyR blockade and spontaneous [Ca2⫹]i oscillations. A. traces from two spontaneously active astrocytes showing that caffeine causes [Ca2⫹]i release. B1. Traces of ⌬F% for two astrocytes in the presence of ryanodine, showing that astrocytes still display spontaneous [Ca2⫹]i oscillations but caffeine is ineffective in eliciting [Ca2⫹]i release. B2 Bar graphs showing astrocytic spontaneous activity in control conditions and in ryanodine (top) and the number of astrocytes responding to caffeine application in both conditions (bottom).
208.9⫾22% (n⫽18, three slices) in control and 246⫾27% in the presence of ryanodine. In control conditions, application of caffeine evoked [Ca2⫹]i increases in 12⫾3.1 astrocytes (n⫽3 slices), whilst after ryanodine incubation caffeine evoked [Ca2⫹]i increases in only 1.15⫾0.48 (P⬍0.05) astrocytes. Thus, whilst the RyRs
which cause the [Ca2⫹]i increase in response to caffeine are blocked, spontaneous oscillations continue unaffected, indicating that the observed oscillations are not due to activation of RyRs. Application of the IP3-R activator thimerosal (50 M) resulted in a 270% increase in the number of astrocytes
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exhibiting [Ca2⫹]i oscillations, from 0.77⫾0.3/100 s (n⫽3 slices) to 3.87⫾1.1/100 s (same slices, P⬍0.005; Fig. 3A), showing that IP3-Rs are present on thalamic astrocytes and that their activation causes [Ca2⫹]i increases. To further investigate the possible role of IP3-Rs, the cell permeable IP3-R antagonist 2-aminoethoxydiphenylborate (2-APB) was used (Fig. 3B–D). As a control to test that the IP3-Rs were blocked, we used (S)-3,5-dihydroxyphenylglycine (DHPG), a specific agonist of the metabotropic glutamate receptor (mGluR) mGluR1 which is coupled via PLC to the generation of inositol 1,4,5,-triphosphate (IP3) and [Ca2⫹]i release (Bernstein et al., 1998). In control conditions DHPG application evoked [Ca2⫹]i increases in 10⫾2.7 (n⫽4 slices) astrocytes (Fig. 3B, C). A 10 min perfusion of 100 M 2-APB resulted in a decrease in the ability of DHPG to evoke astrocytic [Ca2⫹]i increases, the number of astrocytes responding to DHPG being 3.25⫾2.9 (P⬍0.05, n⫽4 slices; Fig. 3C), indicating that astrocytic IP3-Rs were inhibited. Spontaneous astrocytic [Ca2⫹]i increases were also greatly reduced from 1.0⫾0.1/ 100 s in control to 0.1⫾0.25/ 100 s in 2-APB (P⬍0.005, n⫽5 slices). The fluorescence increases during spontaneous oscillations was 133⫾18% (n⫽20, 3 slices) in control and 1⫾0.9% (P⬍0.001; Fig. 3D) following 2-APB treatment, and DHPG induced fluorescence increases were reduced from 181⫾15.8% to 34.5⫾8.9% (P⬍0.001, paired t-test). To further investigate the possible role of the PLC–IP3-R pathway, we used the PLC inhibitor U73122 (Fig. 4A). The number of astrocytes displaying spontaneous [Ca2⫹]i increases in control conditions was 1.42⫾0.28/100 s (n⫽11 slices) whilst following treatment with 20 M U73122 for 15 min, the number was 1.36⫾0.24/100 s (n⫽11). The frequency of astrocytic oscillations was unaffected by U73122 being 0.8⫾0.19/100 s in control and 1.05⫾0.10/100 s following U73122. The amplitude of increases was also unaffected (140⫾26% in control and 164⫾25% in U73122). In control conditions, trans-ACPD application evoked [Ca2⫹]i increases in 13.36⫾1.39 (n⫽8) astrocytes (fluorescence increase 313.9⫾37%) and 3.91⫾1.2 (P⬍0.001, paired t-test; fluorescence increase 6.9⫾3%) following U73122 treatment, showing that U73122 had significantly inhibited receptor-mediated IP3-R activation. These data also show that receptor-mediated activation of PLC is not required to sustain spontaneous astrocytic [Ca2⫹]i oscillations. To further test for the involvement of the products of PLC activation, we investigated the effect of the protein kinase C (PKC) inhibitor chelerythrine chloride. Application of 10 M chelerythrine chloride resulted in transient spontaneous [Ca2⫹]i increases being transformed to sustained [Ca2⫹]i elevations (4⫾1.2 astrocytes, n⫽3 slices; Fig. 4B). Amplitude of spontaneous activity was 86⫾56% and amplitude of sustained increases in chelerythrine chloride 193⫾58% (n⫽3 astrocytes, P⬍0.05, paired ttest). This suggests an important role for PKC in the termination of spontaneous astrocytic [Ca2⫹]i increases. DHP-sensitive channels Although spontaneous astrocytic [Ca2⫹]i oscillations in the VB thalamus are due to [Ca2⫹]i release from intracellular stores they are also inhibited by dihydropyridine (DHP) antagonists (Parri et al., 2001). We therefore
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looked at the effect of up-regulating this apparent DHPsensitive Ca2⫹ entry pathway with the DHP agonist BayK8644 (Fig. 5). Inclusion of 10 M BayK8644 in the perfusate increased the number of astrocytes displaying spontaneous [Ca2⫹]i increases from 1.33⫾0.5/100 s to 2.67⫾0.5/100 s (P⬍0.05, n⫽3 slices). The amplitude of spontaneous increases in control was 81⫾13% (n⫽9 astrocytes) and in BayK8644, 140⫾7% (n⫽18 astrocytes, P⬍0.05). The frequency of transients exhibited by individual astrocytes (Fig. 5A) also significantly increased. The number of oscillations per astrocyte in control conditions was 0.4⫾0.13/100 s (n⫽18) in control and 1.07⫾0.18/100 s (P⬍0.05, paired t-test) in BayK8644. For astrocytes spontaneously active in both conditions the frequency was not different. The effect of BayK8644 is therefore to increase the number of astrocytes exhibiting oscillations. The existence of voltage-gated Ca2⫹ channels in astrocytes is controversial, with studies showing their occurrence (MacVicar, 1984; Duffy and MacVicar, 1994) whilst others show that astrocytes in situ do not possess voltage-gated Ca2⫹ channels, (Carmignoto et al., 1998). Studies in arteriolar smooth muscle cells (Curtis and Scholfield, 2001) have suggested that the current induced by Ca2⫹ store depletion, the store-operated calcium current (SOCC), is a DHP-sensitive non-selective cation current, though capacitive Ca2⫹ entry (CCE) was unaffected by nifedipine in rat cerebellar astrocytes (Lo et al., 2002). We investigated whether such an effect of DHPs could explain our observations. DHPG was applied focally to cause [Ca2⫹]i release and activate the store depletion Ca2⫹ entry pathway. In the presence of BayK8644, the DHPG-elicited [Ca2⫹]i increase was significantly greater than in control conditions at each time point from 6 to 17 s from its start (P⬍0.05, n⫽10 astrocytes in five slices; Fig. 5B). The findings with BayK8644 suggest a role for Ca2⫹ entry via DHP-sensitive Ca2⫹ channels in the manifestation of spontaneous astrocytic [Ca2⫹]i oscillations. To further investigate the role of Ca2⫹, therefore, [Ca2⫹]o was varied. The role of Ca2ⴙ Changing [Ca2⫹]o showed that the number of astrocytes displaying oscillations, and the amount of activity that they displayed, was proportional to [Ca2⫹]o (Fig. 6A). The number of astrocytes exhibiting spontaneous [Ca2⫹]i transients increased from 0.93⫾0.26/100 s in 1 mM [Ca2⫹]o to 1.53⫾0.18/100 s in 2.5 mM [Ca2⫹]o and 3.11⫾0.22/100 s in 5 mM [Ca2⫹]o (P⬍0.0005 ANOVA, n⫽5 slices). The Bonferroni post hoc test showed a significance of P⬍0.016 between 1 mM and 5 mM [Ca2⫹]o and between 2.5 mM and 5 mM [Ca2⫹]o. The number of transients exhibited by spontaneously active astrocyte was 1.64⫾0.32 in 1 mM, 1.5⫾0.12 in 2.5 mM and 2.46⫾0.34 in 5 mM [Ca2⫹]o (P⬍0.05, ANOVA). The Bonferroni post hoc test showed a significance of P⬍0.016 between 2.5 mM and 5 mM [Ca2⫹]o. The frequency of oscillations displayed by astrocytes that were active in all three conditions was not significantly different (n⫽4). Fluorescence increases in the
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Fig. 3. IP3-Rs in spontaneous [Ca2⫹]i oscillations. A. Traces from two astrocytes showing the effect of thimerosal application. Bar graph illustrates number of astrocytes displaying [Ca2⫹]i increases in control period and following thimerosal application. B. Traces from two astrocytes showing spontaneous [Ca2⫹]i oscillations and [Ca2⫹]i increases evoked by DHPG before and after incubation with 2-APB. C. Bar graphs illustrate the number of spontaneously active astrocytes, and the number of astrocytes that respond to DHPG, in control and 2-APB. D. Bar graph of the mean fluorescence increases from spontaneous activity and DHPG activation in control and 2-APB-treated conditions.
three [Ca2⫹]o concentrations (1, 2.5 and 5 mM) were not significantly different (243⫾50%, 316⫾49 and 256⫾24, respectively). These experiments show that increasing
[Ca2⫹]o enhances the exhibition of spontaneous astrocytic activity, showing that Ca2⫹ has a role in spontaneous activity generation.
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Fig. 4. Role of PLC–IP3-R pathway in spontaneous [Ca2⫹]i oscillations. A. Traces from two astrocytes showing [Ca2⫹]i in control conditions and following U73122 incubation for 15 min. Trans-ACPD responses are inhibited following U73122 treatment whilst spontaneous [Ca2⫹]i increases are still seen. Upper bar graph illustrates number of astrocytes exhibiting spontaneous [Ca2⫹]i increases in the two conditions; the lower bar graph shows the number of astrocytes responding to mGluR activation in the same slices. B. Effect of 10 M chelerythrine chloride on kinetics of spontaneous [Ca2⫹]i increases. Traces show ⌬F% from two astrocytes in a slice during chelerythrine chloride application. Transient [Ca2⫹]i increases become sustained [Ca2⫹]i increases.
Glial cells can exhibit spatially restricted [Ca2⫹]i signals which do not propagate throughout the cell (Grosche et al., 1999; Nett et al. 2002). Fluorescent measurements from astrocytes where individual astrocytic processes could be visualised showed that not all [Ca2⫹]i increases that occurred in the processes were propagated to the soma (Fig. 6B) and that the degree of correlation between events in the processes decreased with distance from the soma (Fig. 6C, open circles; n⫽11 astrocytes). This result indicates that spontaneous increases can be restricted to regions or compartments of thalamic astrocytes. When recordings were made with 5 mM [Ca2⫹]o (Fig. 6C, filled circles; n⫽9 astro-
cytes), the degree of correlation increased from 73⫾5.2– 92⫾2.7 (P⬍0.005), showing that in addition to having a role in the generation of spontaneous [Ca2⫹]i transients, the amount of Ca2⫹ also affects the ability of intracellular Ca2⫹ waves to propagate intracellularly. If spontaneous astrocytic [Ca2⫹]i transients are influenced by the level of cytoplasmic Ca2⫹, then sarco(endo) plasmic reticulum Ca2⫹-ATPase (SERCA) inhibition may also induce sufficient cytoplasmic Ca2⫹ increases to cause [Ca2⫹]i release from the ER. Application of the SERCA blocker cyclopiazonic acid (CPA) results in an emptying of intracellular stores and activation of CCE (Simpson
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Fig. 5. Effect of BayK8644 on spontaneous [Ca2⫹]i oscillations. A. Trace from an astrocyte showing effect of BayK8644 application. Bar graphs show the number of astrocytes displaying spontaneous [Ca2⫹]i increases before and during BayK8644 application (left), and the frequency of oscillations displayed in spontaneously active astrocytes (expressed as number of increases/100 s) in the two conditions. B1. Pair of traces on left show the effect of focal DHPG application on [Ca2⫹]i in the same astrocyte in control conditions and in the presence of BayK8644. B2 Plot displays data from such experiments for 10 astrocytes with fluorescence normalised to the peak for each astrocyte. Filled circles show fluorescence in control conditions, open circles in the presence of 10 M BayK8644. The asterisk denotes a significance of P⬍0.05, and the arrows denote the range of time for which the difference in fluorescence in the two conditions is significant.
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Fig. 6. [Ca2⫹]o and spontaneous oscillations. A. Traces of ⌬F% from two astrocytes showing the effect of modifying [Ca2⫹]o. Hatched bar indicates time of perfusion with ACSF containing different [Ca2⫹]o. Bar graph summarises the data from n⫽3–5 experiments. B. Traces of spontaneous [Ca2⫹]i from the soma (s) and two regions of the astrocyte processes (a and b) as indicated in the displayed image of the astrocyte (right). ⌬F% plots from region “a” show a [Ca2⫹]i increase which is independent of that occurring in process “b” or the soma. C. Plot of the degree of correlation of [Ca2⫹]i increases between areas on the processes and in the astrocyte soma against the distance from the soma. Open circles show data from experiments performed with 2 mM [Ca2⫹]o and filled circles show data from experiments with 5 mM [Ca2⫹]o.
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Fig. 7. CPA and [Ca2⫹]i release. A. Effect of CPA on [Ca2⫹]i in astrocytes. Upper traces show that CPA induced transients in a proportion of astrocytes (see text) whilst in other astrocytes (lower traces) a slow accumulation of [Ca2⫹]i occurred. B. The same protocols applied in the absence of [Ca2⫹]o. Traces show that transient increases are still seen, whilst slow increases in [Ca2⫹]i are not.
and Russell, 1997; Lo et al., 2002) (Fig. 7A). In our preparation acute CPA addition resulted in a slow increase in astrocytic [Ca2⫹]i levels in 56⫾23% of astrocytes (n⫽3 slices), and also induced [Ca2⫹]i transients in 68⫾22% more astrocytes (n⫽3 slices) than in the previous control period, with the time to [Ca2⫹]i transient generation following CPA addition being 104⫾8 s (n⫽19 astrocytes). The mean slow fluorescence increase 400 s after CPA addition was 99⫾18% (n⫽16 astrocytes), consistent with a chronic activation of CCE by long-term store depletion. This explanation is supported by the findings that in 0 mM [Ca]o CPA still resulted in [Ca2⫹]i increases in VB astrocytes, but only 10% of the astrocytes displayed slow [Ca2⫹]i increases (Fig. 7B). The time to transient generation in 0 mM [Ca2⫹]o was 166⫾11 s (n⫽15 astrocytes). Previous findings (Parri et al., 2001) show that astrocytes display spontaneous [Ca2⫹]i increases 3– 4 min following perfusion with 0 mM [Ca2⫹]o suggesting that it can take quite a long time for ER luminal Ca2⫹ to leak out, and that stores are still able to release Ca2⫹ in a spontaneous manner for this period.
Ca2ⴙ buffering and generation of [Ca2ⴙ]i oscillations The data therefore show that spontaneous [Ca2⫹]i oscillations can be modulated by cytoplasmic Ca2⫹. To determine whether the level of [Ca2⫹]i was important for the generation of spontaneous activity, we manipulated the amount of [Ca2⫹]i buffering in the astrocytes by incubating in different concentrations of Fluo-4AM (Fig. 8). The number of astrocytes displaying spontaneous [Ca2⫹]i oscillations was 2.29⫾0.71/100 s (n⫽3 slices) in 1 M, 1.67⫾0.23/100 s (n⫽3 slices) with 5 M and 0.53⫾0.16/ 100 s (n⫽3 slices) with 20 M Fluo-4 (Fig. 8B) (P⬍0.05, ANOVA; P⬍0.016 between 20 M and 5 M, Fluo-4 and between 20 M and 1 M Fluo-4, Bonferroni post hoc test). The frequency of oscillations in individual astrocytes was 0.22⫾0.03/100 s for 20 M, 0.58⫾0.11/100 s for 5 M and 0.50⫾0.05/100 s for 1 M (P⬍0.05, ANOVA; P⬍0.001 between 20 M and 5 M Fluo-4 and between 20 M and 1 M Fluo-4, Bonferroni post hoc test). The magnitude of spontaneous fluorescence increases was not different between the groups: 20 M, 145.6⫾23.6%; 5 M, 139⫾16%; 1 M, 109⫾13.8%. The number of astrocytes displaying evoked [Ca2⫹]i increases in response to trans-ACPD was
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Fig. 8. Effect of [Ca2⫹]i buffering on spontaneous oscillations. A. Plots of ⌬F% showing spontaneous [Ca2⫹]i increases in slices incubated with 1 M Fluo-4AM and the effect of the mGluR agonist trans-ACPD, and from astrocytes in slices incubated with 20 M Fluo-4AM, where the number of spontaneous increases are reduced but ACPD is still able to release [Ca2⫹]i. B. Summary of data with varying Fluo-4AM concentration. C. Spontaneous increase acquired at 7 Hz, showing that the transient [Ca2⫹]i increase is preceded by a slow rise in cytosolic Ca2⫹. F units are absolute increases in fluorescence.
not different between the different indicator concentrations (ANOVA), showing that the astrocytes were still able to release [Ca2⫹]i but that the initiation of the spontaneous events was affected by the buffering of [Ca2⫹]i. Imaging of spontaneous transients at a rate of 7 Hz confirmed the apparent role of Ca2⫹ in triggering release, in that a slow rise in [Ca2⫹]i is seen before the transient [Ca2⫹]i increase: an increase in fluorescence of 0.83⫾ 0.4%/s (n⫽3) occurred before the transient, which is consistent with the triggering of [Ca2⫹]i release when cytoplasmic Ca2⫹ reaches a threshold level (Bootman et al., 1997; Thomas et al., 2000).
DISCUSSION The major findings of this study on the mechanism of generation of spontaneous astrocytic [Ca2⫹]i oscillations which in the VB thalamus are associated with NMDA in-
ward currents in TC neurons are: 1) spontaneous astrocytic [Ca2⫹]i oscillations are intrinsic, being independent of vesicular transmitter release and membrane receptor activation, 2) PKC activation and IP3-R involvement are however implicated, and 3) cytoplasmic Ca2⫹ seems to be the trigger for astrocytic [Ca2⫹]i release. Intrinsic nature of astrocytic [Ca2ⴙ]i oscillations We previously showed that the [Ca2⫹]i oscillations are independent of neuronal activity since they are resistant to TTX. Here we have extended these findings by showing that they are not sustained by spontaneous neurotransmitter release, since blocking of neurotransmitter release by depletion of presynaptic vesicles does not affect the occurrence of spontaneous astrocytic [Ca2⫹]i increases. Taken together these observations indicate that astrocytic oscillations are not dependent on agonist
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stimulation. The oscillations therefore are intrinsic to the astrocytes.
tivation of PKC by the PLC-catalysed production of diacylglycerol.
Intracellular stores and activation pathways
Ca2ⴙ and the generation and intracellular synchronisation of [Ca2ⴙ]i oscillations
The presence of RyRs on VB astrocytes is indicated by the ability of caffeine to elicit [Ca2⫹]i increases. It has been shown in the neocortex that astrocytes display increased oscillation frequency upon the application of caffeine (Tashiro et al., 2002), which lead the authors to conclude that RyRs were responsible for generating the oscillations. Since ryanodine had no effect on spontaneous oscillation in the VB thalamus, RyRs are not involved in the generation of spontaneous [Ca2⫹]i oscillations. Our experiments on the PLC–IP3-R pathway indicate that this pathway is indeed involved in the generation of the spontaneous [Ca2⫹]i transients and oscillations observed in the VB thalamus. Thimerosal, which sensitises IP3-Rs (Peuchen et al., 1996), increased the number of astrocytes displaying [Ca2⫹]i increases. The IP3-R antagonist 2-APB abolished both trans-ACPD responses and spontaneous [Ca2⫹]i oscillations, indicating an IP3-R locus for oscillation generation. Recent studies, however, have shown that both 2-APB and the antagonist xestospongin C can have other effects on SOCC (Bishara et al., 2002) and SERCA pumps (Castonguay and Robitaille, 2002) which could affect the degree of intracellular store filling (Solovyova et al., 2002) and, at least partly, explain our observations. As expected, inhibition of PLC by U73122 inhibited mGluR-activated [Ca2⫹]i increases, but did not block spontaneous [Ca2⫹]i activity. This result agrees with those seen with bafilomycin A1, since the effect of bafilomycin A1 would also be expected to greatly reduce the amount of neuroactive compounds activating astrocyte receptors. On the other hand, it does not exclude the possibility that PLC activation is necessary for spontaneous oscillation manifestation since i) there could conceivably be residual PLC activation to continue IP3 production and/or ii) PLC is being activated by Ca2⫹ ions. PLC-␦1, which is activated in the range 0.1–1 M Ca2⫹ (Allen et al., 1997), is preferentially expressed in astrocytes at around 14 days postnatally (Yamada et al., 1991). In addition, PLC activation by Ca2⫹ was suggested to be important in the propagation of intracellular Ca2⫹ waves in cultured striatal astrocytes (Venance et al., 1997), and modelling studies indicate that PLC-␦1 could have a pivotal role in the regenerative stage of wave propagation (Hofer et al., 2002). A role for PLC activation is indicated by experiments with the PKC inhibitor chelerythrine chloride. Following receptor activation of PLC, IP3 and diacylglycerol are produced. IP3 causes the release of [Ca2⫹]i and diacylglycerol activates PKC, which then phosphorylates the mGluR and PLC to cause a negative feedback. This negative feedback by PKC is important in determining the oscillatory period following agonist stimulation in astrocytes (Codazzi et al., 2001). The switching of spontaneous [Ca2⫹]i increases into sustained increases in the presence of chelerythrine chloride indicates that PKC negative feedback on PLC has a role in controlling the duration of the spontaneous [Ca2⫹]i oscillations, and also suggests a previous ac-
It is clear from our experiments that Ca2⫹ has an important role in the generation of spontaneous [Ca2⫹]i oscillations. The BayK8644 experiments support the findings of our previous studies that showed that nifedipine reduced the number of spontaneously active astrocytes. As oscillations are due to intracellular Ca2⫹ release, this suggested that Ca2⫹ entry across the plasma membrane, and more specifically via L-type Ca2⫹ channels was triggering intracellular Ca2⫹ release via a CICR mechanism. The findings in this present study that BayK8644 increases the number of astrocytes exhibiting spontaneous [Ca2⫹]i oscillations confirm the previously reported DHP-sensitivity but also strongly indicate that this effect may be on the store refilling component, as has been seen in other cell types (Curtis and Scholfield, 2001). This, in turn, suggests that the observed effect of DHPs is to affect the filling state of the ER, and so an increased luminal Ca2⫹ level could be acting to sensitise the IP3-Rs for release (Sienaert et al., 1998). IP3-Rs are Ca2⫹ dependent (Bezprozvanny et al., 1991), requiring cytoplasmic Ca2⫹ binding for IP3 activation and being able to be activated by Ca2⫹ in low levels of IP3. Our experiments with different extracellular and cytoplasmic Ca2⫹ concentrations support a mechanism whereby Ca2⫹ is acting on IP3-Rs in generating spontaneous astrocytic [Ca2⫹]i oscillations. Using low agonist concentrations to activate PLC and IP3 production, it has been established that the generation of “global” (i.e. cellwide) Ca2⫹ signals depends on the spatiotemporal recruitment of “elementary” Ca2⫹ release events (Bootman et al., 1997; Thomas et al., 2000). The recruitment of elementary release sites provides the “pacemaker” Ca2⫹ rise necessary to trigger a regenerative response (Bootman et al., 1997). Our data show that areas of single astrocytes as close as 5 m could display independent Ca2⫹ increases, that do not always lead to global events. This is consistent with studies in cultured astrocytes where it has been shown that release sites were situated 5–7 m apart (Laskey et al., 1998). The increase in the number of astrocytes generating spontaneous [Ca2⫹]i oscillations with increasing [Ca2⫹]i indicates a role for cytoplasmic Ca2⫹ in triggering [Ca2⫹]i release: this is echoed by the apparent correlating effect on the astrocytic “compartments” of increasing [Ca2⫹]o. The data are consistent with a scenario where the action of BayK8644 is on Ca2⫹ entry following store depletion and therefore fills stores to a point where they are more likely to release [Ca2⫹]i. Increasing [Ca2⫹]o could have the effect of filling stores and also increasing cytoplasmic Ca2⫹ which increases the excitability of the ER by bringing the IP3-Rs closer to CICR threshold (Bootman et al., 1997; Simpson and Russell, 1997). In Hela cells activated by histamine, increasing [Ca2⫹]o has also been shown to increase the frequency of oscillations (Bootman et al., 1996).
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A triggering effect of cytoplasmic Ca2⫹ on IP3-Rs is supported by our data with SERCA inhibition. CPA caused transients in VB astrocytes in our study, an effect similar to that observed in cultured astrocytes (Simpson and Russell, 1997), where thapsigargin resulted in propagation of Ca2⫹ waves without the need for IP3 activation. The suggested mechanism was that Ca2⫹ leak through IP3-Rs at specific initiation sites high in IP3-Rs and SERCA triggered CICR from IP3-Rs at these points (Simpson and Russell, 1997). Our results therefore indicate that such a mechanism underlies spontaneous astrocytic [Ca2⫹]i oscillations and confirm the generation of [Ca2⫹]i transients by cytoplasmic Ca2⫹. The role of cytoplasmic Ca2⫹ levels in spontaneous [Ca2⫹]i oscillations generation is supported by the results obtained in slices incubated with increasing concentrations of Fluo-4AM. The reduction in spontaneous activity, and the lack of effect on trans-ACPD-elicited responses, indicate that the increased Ca2⫹ buffering effect of the indicator decreases the free cytoplasmic Ca2⫹ and hence lowers the CICR triggering effect on IP3-Rs. This effect is perhaps analogous to that reported for the effects of indicators on inter-astrocytic wave propagation in culture (Wang et al. 1997). The results of this study on the mechanism of spontaneous [Ca2⫹]i oscillations in VB astrocytes show that their properties are similar to agonist-evoked oscillations in astrocytes which have a dependence on IP3 and [Ca2⫹]i. However, in the VB thalamus, oscillations are spontaneous, requiring no agonist application. These results therefore point to a scenario where there is a level of constitutive PLC activation, possibly by Ca2⫹, which produces sufficient IP3 to put the IP3-Rs in a primed state for activation by Ca2⫹, and that spontaneous oscillations occur by Ca2⫹ triggering CICR from the astrocytic IP3-Rs. Acknowledgements—We would like to thank T. M. Gould for technical/image processing help and advice. This work was supported by the Wellcome Trust (Grant 37089-98).
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(Accepted 30 April 2003)
THE ROLE OF Ca2ⴙ IN THE GENERATION OF SPONTANEOUS ASTROCYTIC Ca2ⴙ OSCILLATIONS H. R. PARRI* AND V. CRUNELLI
Astrocytes in the CNS are postulated to respond to neuronal activity and form a parallel network of signalling in the brain (Cornell-Bell et al., 1990). There is now increasing evidence that glia interact with neurons, responding to neurotransmitter release (Porter and McCarthy, 1996) and affecting postsynaptic activity (Pasti et al., 1997; Araque et al., 1998; Kang et al., 1998; Castonguay and Robitaille, 2001). Astrocytes can however be spontaneously active, displaying [Ca2⫹]i oscillations which are independent of neuronal activity. Cultured cortical (Fatatis and Russell, 1992) and suprachiasmatic nucleus (van den Pol et al., 1992) astrocytes displayed spontaneous, tetrodotoxin (TTX)-insensitive [Ca2⫹]i oscillations. In in situ slice preparations, spontaneous [Ca2⫹]i oscillations were originally reported in the ventrobasal (VB) thalamus (Parri et al., 2001), and recently in other brain regions including the hippocampus and cortex (Nett et al., 2002; Tashiro et al., 2002; Aguado et al., 2002). Whilst neuron-astrocyte interactions are beginning to be elucidated, the function of, and the impact of spontaneous astrocytic [Ca2⫹]i oscillations are unknown. A pathological role has been suggested in the cortex since spontaneous oscillations were only observed in cortical slices in models of epilepsy (Tashiro et al., 2002). In support of this hypothesis is the observation that astrocytes cultured from a patient with Rasmussen’s encephalitis displayed spontaneous oscillations (Manning and Sontheimer, 1997). In the thalamus there seems to be a functional role for spontaneous astrocytic oscillations. Oscillations in the VB thalamus are correlated between astrocytes suggesting communication by a released factor, and recordings from thalamocortical neurons in this thalamic nucleus show that large NMDA-mediated inward currents are correlated to [Ca2⫹]i increases in neighbouring astrocytes (Parri et al., 2001; Parri and Crunelli, 2002a), indicating that astrocytes can affect or generate neuronal activity without prior neuronal input. In the hippocampus, synchronised astrocytic and neuronal oscillations are seen, and the interaction between the neuronal and astrocytic elements seems dependent on glutamate (Aguado et al., 2002). The spontaneous oscillations observed in different brain regions share many features including the duration of the individual [Ca2⫹]i elevations, the periodicity of the oscillations, and a dependence on intracellular [Ca2⫹]i release (Parri et al., 2001; Nett et al., 2002; Tashiro et al., 2002; Aguado et al., 2002). However, the steps leading to [Ca2⫹]i release are poorly understood. Though seemingly not requiring the eliciting of neurotransmitter release, it is not known if the activation of a membrane bound receptor
School of Biosciences, Cardiff University, Museum Avenue, PO Box 911, Cardiff, CF10 3US, Wales, UK
Abstract—Astrocytes in the rat thalamus display spontaneous [Ca2ⴙ]i oscillations that are due to intracellular release, but are not dependent on neuronal activity. In this study we have investigated the mechanisms involved in these spontaneous [Ca2ⴙ]i oscillations using slices loaded with Fluo-4 AM (5 M) and confocal microscopy. Bafilomycin A1 incubation had no effect on the number of spontaneous [Ca2ⴙ]i oscillations indicating that they were not dependent on vesicular neurotransmitter release. Oscillations were also unaffected by ryanodine. Phospholipase C (PLC) inhibition decreased the number of astrocytes responding to metabotropic glutamate receptor (mGluR) activation but did not reduce the number of spontaneously active astrocytes, indicating that [Ca2ⴙ]i increases are not due to membrane-coupled PLC activation. Spontaneous [Ca2ⴙ]i increases were abolished by an IP3 receptor antagonist, whilst the protein kinase C (PKC) inhibitor chelerythrine chloride prolonged their duration, indicating a role for PKC and inositol 1,4,5,-triphosphate receptor activation. BayK8644 increased the number of astrocytes exhibiting [Ca2ⴙ]i oscillations, and prolonged the responses to mGluR activation, indicating a possible effect on storeoperated Ca2ⴙ entry. Increasing [Ca2ⴙ]o increased the number of spontaneously active astrocytes and the number of transients exhibited by each astrocyte. Inhibition of the endoplasmic reticulum Ca2ⴙ ATPase by cyclopiazonic acid also induced [Ca2ⴙ]i transients in astrocytes indicating a role for cytoplasmic Ca2ⴙ in the induction of spontaneous oscillations. Incubation with 20 M Fluo-4 reduced the number of astrocytes exhibiting spontaneous increases. This study indicates that Ca2ⴙ has a role in triggering Ca2ⴙ release from an inositol 1,4,5,-triphosphate sensitive store in astrocytes during the generation of spontaneous [Ca2ⴙ]i oscillations. © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: ventrobasal thalamocortical.
thalamus,
glia,
development,
*Corresponding author. Tel: ⫹44-29-2087-5150; fax: ⫹44-29-20874986. E-mail address: [email protected] (H. R. Parri). Abbreviations: ACSF, artificial cerebrospinal fluid; 2-APB, 2-aminoethoxydiphenylborate; CCE, capacitive Ca2⫹ entry; CICR, calcium induced Ca2⫹ release; CPA, cyclopiazonic acid; DHP, dihydropyridine; DHPG, (S)-3,5-dihydroxyphenylglycine; IP3, inositol 1,4,5,-triphosphate; IP3-R, inositol 1,4,5,-triphosphate receptor; mGluR, metabotropic glutamate receptor; PKC, protein kinase C; PLC, phospholipase C; RyR, ryanodine receptor; SERCA, sarco(endo)plasmic reticulum Ca2⫹ ATPase; SOCC, store-operated calcium current; TC, thalamocortical; trans-ACPD, (⫾)-1-aminocyclopentane-trans1,3-dicarboxylic acid; TTX, tetrodotoxin; VB thalamus, ventrobasal thalamus.
0306-4522/03$30.00⫹0.00 © 2003 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/S0306-4522(03)00379-8
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Fig. 1. Spontaneous [Ca2⫹]i oscillations are intrinsic. A. Pseudocolour images of control and bafilomycin A1-treated slices. Circles indicate positions of spontaneously active astrocytes during a 600 s recording period. B. Spontaneous [Ca2⫹]i oscillations from two astrocytes in the control slice (upper traces) and from the slice following treatment with bafilomycin A1 (lower traces), showing that oscillations occur in both conditions. C. Bar graph of the number of spontaneously active astrocytes in the two conditions. D. Currents recorded from a TC neuron during lemniscal afferent synaptic stimulation in control and following incubation with bafilomycin A1, showing that whilst oscillations are unaffected, synaptic transmission was abolished. Arrows indicate timings of synaptic stimulations.
is required for their generation. There is also debate about the signal and mechanism of [Ca2⫹]i release. Calcium induced Ca2⫹ release (CICR) via ryanodine receptors (RyRs) has been suggested as the mechanism in cortical astrocytes (Tashiro et al., 2002), whilst inositol 1,4,5,triphosphate receptors (IP3-Rs) are implicated in the hippocampus (Nett et al., 2002). In this study, we investigated the mechanism leading to the generation of spontaneous astrocytic [Ca2⫹]i oscillations in the rat VB thalamus. The oscillations have many of the properties associated with agonist-receptor-mediated astrocytic Ca2⫹ increases but are intrinsic to astrocytes and are not dependent on vesicular transmitter release or on membrane receptor-mediated phospholipase C (PLC) activation. PLC is however implicated in the generation as is CICR via IP3-Rs. Some of these results were published in abstract form (Parri and Crunelli, 2002b).
EXPERIMENTAL PROCEDURES All procedures involving experimental animals were carried out in accordance with the U.K. Animals (Scientific Procedure) Act 1986 and local ethics committee guidelines. Slices of rat VB thalamus were prepared as described previously (Parri et al., 2001) from 5 to 17 day old rats. After a recovery
period of 1 h, slices were loaded with fluo-4 AM (Molecular Probes, Eugene, OR, USA) by incubating for 40 – 60 min at 28 °C with 5 M of the indicator dye and 0.01% pluronic acid. Under these conditions, astrocytes were preferentially loaded with the fluorescent indicator, whereas loading of neurons was rarely observed (Parri et al., 2001). All experiments were performed in standard artificial cerebrospinal fluid (ACSF) of composition (in mM): NaCl 120, NaHCO3 16, KCl 2, KH2PO4 1.25, MgSO4 1, CaCl2 2 (Turner et al. 1994). TTX (1 M) was also present in all experiments except the synaptic stimulation experiments described in Fig. 1. Experiments were conducted at room temperature (20 –24 °C). Chemicals were obtained from Sigma (St. Louis, MO, USA) unless otherwise stated. (S)-3,5-dihydroxyphenylglycine (DHPG) and (⫾)-1-aminocyclopentane-trans-1,3-dicarboxylic acid (trans-ACPD) were obtained from Tocris (Bristol, UK). 2-Aminoethoxydiphenylborate (2-APB) and chelerythrine chloride were obtained from Calbiochem (Nottingham, UK). Pharmacological compounds were bath applied unless otherwise stated. Following a control recording period in normal ACSF, pharmacological agents were included in the perfusing ACSF and washed over the preparation. The number of active astrocytes seen during this experimental period was then compared with the number seen during the control period. For focal application of compounds, patch pipettes were filled with the compound dissolved in ACSF, the pipette tips were placed about 20 m above the astrocyte and the drug applied by use of a pressure ejection system (Neurophore; Medical Systems Corporation, NY, USA).
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Confocal fluorescence imaging The recording chamber and manipulators were mounted on a moveable top plate platform (MP MTP-01; Scientifica, Harpenden, UK). Fluorescence was measured using a Noran Odyssey confocal (Thermo Noran, USA) fitted to a Nikon E600FN (Nikon, UK, Kingston, UK) upright microscope. Averages of eight whole field images (206 m⫻158 m) were routinely acquired every 0.8 –5 s with a ⫻40 objective lens (NA⫽0.8). Acquisition and image analysis were performed using Noran Intervision software. Fluorescence values over time for specific regions of interest were exported into Sigmaplot (SPSS Inc., Chicago, IL, USA) for further analysis and ⌬F% plot production. ⌬F% is the increase of fluorescence, either spontaneous or evoked and is calculated by dividing the fluorescence change by the basal⫻100%. Displayed monochrome images showing slice and cellular morphology were produced by averaging in Intervision; contrast and brightness were also adjusted to enhance morphological information.
Measurement of spontaneous activity For experiments where the effect of a compound on spontaneous activity had to be compared in different slices (e.g. bafilomycin A1 experiments), control images were typically acquired for 600 s, and treated slices then imaged for 600 s, and the properties of the spontaneous oscillations in the two groups compared. Six hundred seconds was chosen as the maximum recording period to avoid dye bleaching affecting the observation of [Ca2⫹]i increases. Where activity could be compared in the same slice, acquisition times for both conditions were typically 300 s. To ease comparison between different experiments data were normalised, so that the number of astrocytes displaying Ca2⫹ transients during an experiment is quoted as a value per 100 s, e.g. in a defined slice area where 10 astrocytes displayed spontaneous transients in a 600 s period the number of spontaneously active astrocytes is quoted as 1.67 ast/100 s. To control for variability in the display of spontaneous oscillations, experimental and control slices were taken from the same animal and experiments performed on the same day. The frequency of Ca2⫹ oscillations in astrocytes exhibiting multiple Ca2⫹ increases is expressed as the number of increases per 100 s.
Evoked astrocytic [Ca2ⴙ]i increases [Ca2⫹]i increases evoked by drug application, e.g. caffeine and DHPG, are expressed as the absolute number of astrocytes displaying responses immediately following drug application in the defined slice area.
Electrophysiology Patch clamp recordings were made using sylgarded pipettes (2– 4 M⍀) containing an internal solution of composition (in mM): KMeSO4 120, HEPES 10, EGTA 10, Na2ATP 4, GTP 0.5. Currents were recorded using an Axopatch 200B amplifier, and acquired and analysed using Pclamp8 software (Axon Instruments, Foster City, CA, USA). Synaptic stimulation was achieved with a constant current isolated stimulator (Digitimer, Welwyn Garden City, UK) and a bipolar electrode. Data are displayed as mean⫾S.E.M. Significance was calculated using two tail unpaired and paired Student’s t-test as indicated in the text. ANOVA and Bonferroni tests were used for comparisons of more than two groups.
RESULTS Spontaneous astrocytic [Ca2ⴙ]i oscillations are intrinsic Spontaneous astrocytic [Ca2⫹]i oscillations in the VB thalamus display a variety of patterns of [Ca2⫹]i increase,
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including single transient [Ca2⫹]i increases or multiple [Ca2⫹]i increases within a recording period. Astrocytes displaying multiple increases have a periodicity of around 70 s (Parri and Crunelli, 2001). The [Ca2⫹]i oscillations are not dependent on neuronal activity since they are not blocked by TTX (Parri et al., 2001). This, however, does not exclude the possibility that they are sustained by spontaneous neurotransmitter release which could activate astrocytic metabotropic receptors to sustain [Ca2⫹]i oscillations. Experiments were thus performed using bafilomycin A1, which depletes neurotransmitter vesicles and so blocks neuronal and glial transmitter release by inhibition of vacuolar H⫹-ATPases (Araque et al., 2000; Pasti et al., 2001). Astrocytes displayed spontaneous [Ca2⫹]i oscillations in control conditions (Fig. 1B, upper traces) and following incubation with 4 M bafilomycin A1 (Fig. 1B, lower traces). Following bafilomycin A1 treatment the number of astrocytes displaying spontaneous [Ca2⫹]i oscillations was 1.29⫾0.3/100s (n⫽4 slices; see Experimental Procedures) compared with 1.21⫾0.1/100s (n⫽4 slices) in control conditions (Fig. 1C). The amplitude of spontaneous fluorescence increases was 114⫾11% (n⫽31) in control and 108⫾14% in bafilomycin A1 (n⫽29), no effect of bafilomycin A1 on the number of [Ca2⫹]i transients displayed by each astrocyte was seen. Bafilomycin A1 was shown to have caused vesicular depletion by comparing the synaptic responses of thalamocortical (TC) neurons in control and bafilomycin A1 conditions (Fig. 1D). Synaptic stimulation in control untreated slices evoked inward currents of amplitude 240⫾70pA (n⫽4 neurons). Following incubation with bafilomycin A1, synaptically induced currents were completely abolished (n⫽5 neurons). These results therefore show that spontaneous astrocytic oscillations are not dependent on transmitter release from neurons, or potential vesicular transmitter release from astrocytes (Araque et al., 2000; Pasti et al., 2001), and demonstrate that the generation of spontaneous [Ca2⫹]i oscillations is an intrinsic ability of astrocytes. Involvement of PLC and IP3-Rs activation As spontaneous astrocytic oscillations arise from the release of Ca2⫹ from intracellular stores (Parri et al., 2001), we investigated whether release was due to activation of RyRs or IP3-Rs. Caffeine (20 mM) application elicited [Ca2⫹]i increases in spontaneously active VB thalamus astrocytes (Fig. 2A), indicating the presence of RyRs. Experiments in bafilomycin A1-treated slices (n⫽3) confirmed that the caffeine effect was directly on the astrocytes and not on presynaptic neuronal elements. Incubation of slices for 15 min with 20 M ryanodine did not affect the number of astrocytes displaying spontaneous [Ca2⫹]i oscillations (Fig. 2B; control: 1.6⫾0.5/100 s, n⫽3 slices; ryanodine: 1.5⫾0.5/100 s, n⫽3 slices). The frequency of oscillations exhibited by individual astrocytes was also not affected by ryanodine (0.6⫾0.09/100 s, n⫽16, three slices in control and 0.63⫾0.06/100 s, n⫽16, three slices in ryanodine). The amplitude of the spontaneous fluorescence increases was unaffected by ryanodine treatment being
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Fig. 2. RyR blockade and spontaneous [Ca2⫹]i oscillations. A. traces from two spontaneously active astrocytes showing that caffeine causes [Ca2⫹]i release. B1. Traces of ⌬F% for two astrocytes in the presence of ryanodine, showing that astrocytes still display spontaneous [Ca2⫹]i oscillations but caffeine is ineffective in eliciting [Ca2⫹]i release. B2 Bar graphs showing astrocytic spontaneous activity in control conditions and in ryanodine (top) and the number of astrocytes responding to caffeine application in both conditions (bottom).
208.9⫾22% (n⫽18, three slices) in control and 246⫾27% in the presence of ryanodine. In control conditions, application of caffeine evoked [Ca2⫹]i increases in 12⫾3.1 astrocytes (n⫽3 slices), whilst after ryanodine incubation caffeine evoked [Ca2⫹]i increases in only 1.15⫾0.48 (P⬍0.05) astrocytes. Thus, whilst the RyRs
which cause the [Ca2⫹]i increase in response to caffeine are blocked, spontaneous oscillations continue unaffected, indicating that the observed oscillations are not due to activation of RyRs. Application of the IP3-R activator thimerosal (50 M) resulted in a 270% increase in the number of astrocytes
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exhibiting [Ca2⫹]i oscillations, from 0.77⫾0.3/100 s (n⫽3 slices) to 3.87⫾1.1/100 s (same slices, P⬍0.005; Fig. 3A), showing that IP3-Rs are present on thalamic astrocytes and that their activation causes [Ca2⫹]i increases. To further investigate the possible role of IP3-Rs, the cell permeable IP3-R antagonist 2-aminoethoxydiphenylborate (2-APB) was used (Fig. 3B–D). As a control to test that the IP3-Rs were blocked, we used (S)-3,5-dihydroxyphenylglycine (DHPG), a specific agonist of the metabotropic glutamate receptor (mGluR) mGluR1 which is coupled via PLC to the generation of inositol 1,4,5,-triphosphate (IP3) and [Ca2⫹]i release (Bernstein et al., 1998). In control conditions DHPG application evoked [Ca2⫹]i increases in 10⫾2.7 (n⫽4 slices) astrocytes (Fig. 3B, C). A 10 min perfusion of 100 M 2-APB resulted in a decrease in the ability of DHPG to evoke astrocytic [Ca2⫹]i increases, the number of astrocytes responding to DHPG being 3.25⫾2.9 (P⬍0.05, n⫽4 slices; Fig. 3C), indicating that astrocytic IP3-Rs were inhibited. Spontaneous astrocytic [Ca2⫹]i increases were also greatly reduced from 1.0⫾0.1/ 100 s in control to 0.1⫾0.25/ 100 s in 2-APB (P⬍0.005, n⫽5 slices). The fluorescence increases during spontaneous oscillations was 133⫾18% (n⫽20, 3 slices) in control and 1⫾0.9% (P⬍0.001; Fig. 3D) following 2-APB treatment, and DHPG induced fluorescence increases were reduced from 181⫾15.8% to 34.5⫾8.9% (P⬍0.001, paired t-test). To further investigate the possible role of the PLC–IP3-R pathway, we used the PLC inhibitor U73122 (Fig. 4A). The number of astrocytes displaying spontaneous [Ca2⫹]i increases in control conditions was 1.42⫾0.28/100 s (n⫽11 slices) whilst following treatment with 20 M U73122 for 15 min, the number was 1.36⫾0.24/100 s (n⫽11). The frequency of astrocytic oscillations was unaffected by U73122 being 0.8⫾0.19/100 s in control and 1.05⫾0.10/100 s following U73122. The amplitude of increases was also unaffected (140⫾26% in control and 164⫾25% in U73122). In control conditions, trans-ACPD application evoked [Ca2⫹]i increases in 13.36⫾1.39 (n⫽8) astrocytes (fluorescence increase 313.9⫾37%) and 3.91⫾1.2 (P⬍0.001, paired t-test; fluorescence increase 6.9⫾3%) following U73122 treatment, showing that U73122 had significantly inhibited receptor-mediated IP3-R activation. These data also show that receptor-mediated activation of PLC is not required to sustain spontaneous astrocytic [Ca2⫹]i oscillations. To further test for the involvement of the products of PLC activation, we investigated the effect of the protein kinase C (PKC) inhibitor chelerythrine chloride. Application of 10 M chelerythrine chloride resulted in transient spontaneous [Ca2⫹]i increases being transformed to sustained [Ca2⫹]i elevations (4⫾1.2 astrocytes, n⫽3 slices; Fig. 4B). Amplitude of spontaneous activity was 86⫾56% and amplitude of sustained increases in chelerythrine chloride 193⫾58% (n⫽3 astrocytes, P⬍0.05, paired ttest). This suggests an important role for PKC in the termination of spontaneous astrocytic [Ca2⫹]i increases. DHP-sensitive channels Although spontaneous astrocytic [Ca2⫹]i oscillations in the VB thalamus are due to [Ca2⫹]i release from intracellular stores they are also inhibited by dihydropyridine (DHP) antagonists (Parri et al., 2001). We therefore
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looked at the effect of up-regulating this apparent DHPsensitive Ca2⫹ entry pathway with the DHP agonist BayK8644 (Fig. 5). Inclusion of 10 M BayK8644 in the perfusate increased the number of astrocytes displaying spontaneous [Ca2⫹]i increases from 1.33⫾0.5/100 s to 2.67⫾0.5/100 s (P⬍0.05, n⫽3 slices). The amplitude of spontaneous increases in control was 81⫾13% (n⫽9 astrocytes) and in BayK8644, 140⫾7% (n⫽18 astrocytes, P⬍0.05). The frequency of transients exhibited by individual astrocytes (Fig. 5A) also significantly increased. The number of oscillations per astrocyte in control conditions was 0.4⫾0.13/100 s (n⫽18) in control and 1.07⫾0.18/100 s (P⬍0.05, paired t-test) in BayK8644. For astrocytes spontaneously active in both conditions the frequency was not different. The effect of BayK8644 is therefore to increase the number of astrocytes exhibiting oscillations. The existence of voltage-gated Ca2⫹ channels in astrocytes is controversial, with studies showing their occurrence (MacVicar, 1984; Duffy and MacVicar, 1994) whilst others show that astrocytes in situ do not possess voltage-gated Ca2⫹ channels, (Carmignoto et al., 1998). Studies in arteriolar smooth muscle cells (Curtis and Scholfield, 2001) have suggested that the current induced by Ca2⫹ store depletion, the store-operated calcium current (SOCC), is a DHP-sensitive non-selective cation current, though capacitive Ca2⫹ entry (CCE) was unaffected by nifedipine in rat cerebellar astrocytes (Lo et al., 2002). We investigated whether such an effect of DHPs could explain our observations. DHPG was applied focally to cause [Ca2⫹]i release and activate the store depletion Ca2⫹ entry pathway. In the presence of BayK8644, the DHPG-elicited [Ca2⫹]i increase was significantly greater than in control conditions at each time point from 6 to 17 s from its start (P⬍0.05, n⫽10 astrocytes in five slices; Fig. 5B). The findings with BayK8644 suggest a role for Ca2⫹ entry via DHP-sensitive Ca2⫹ channels in the manifestation of spontaneous astrocytic [Ca2⫹]i oscillations. To further investigate the role of Ca2⫹, therefore, [Ca2⫹]o was varied. The role of Ca2ⴙ Changing [Ca2⫹]o showed that the number of astrocytes displaying oscillations, and the amount of activity that they displayed, was proportional to [Ca2⫹]o (Fig. 6A). The number of astrocytes exhibiting spontaneous [Ca2⫹]i transients increased from 0.93⫾0.26/100 s in 1 mM [Ca2⫹]o to 1.53⫾0.18/100 s in 2.5 mM [Ca2⫹]o and 3.11⫾0.22/100 s in 5 mM [Ca2⫹]o (P⬍0.0005 ANOVA, n⫽5 slices). The Bonferroni post hoc test showed a significance of P⬍0.016 between 1 mM and 5 mM [Ca2⫹]o and between 2.5 mM and 5 mM [Ca2⫹]o. The number of transients exhibited by spontaneously active astrocyte was 1.64⫾0.32 in 1 mM, 1.5⫾0.12 in 2.5 mM and 2.46⫾0.34 in 5 mM [Ca2⫹]o (P⬍0.05, ANOVA). The Bonferroni post hoc test showed a significance of P⬍0.016 between 2.5 mM and 5 mM [Ca2⫹]o. The frequency of oscillations displayed by astrocytes that were active in all three conditions was not significantly different (n⫽4). Fluorescence increases in the
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Fig. 3. IP3-Rs in spontaneous [Ca2⫹]i oscillations. A. Traces from two astrocytes showing the effect of thimerosal application. Bar graph illustrates number of astrocytes displaying [Ca2⫹]i increases in control period and following thimerosal application. B. Traces from two astrocytes showing spontaneous [Ca2⫹]i oscillations and [Ca2⫹]i increases evoked by DHPG before and after incubation with 2-APB. C. Bar graphs illustrate the number of spontaneously active astrocytes, and the number of astrocytes that respond to DHPG, in control and 2-APB. D. Bar graph of the mean fluorescence increases from spontaneous activity and DHPG activation in control and 2-APB-treated conditions.
three [Ca2⫹]o concentrations (1, 2.5 and 5 mM) were not significantly different (243⫾50%, 316⫾49 and 256⫾24, respectively). These experiments show that increasing
[Ca2⫹]o enhances the exhibition of spontaneous astrocytic activity, showing that Ca2⫹ has a role in spontaneous activity generation.
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Fig. 4. Role of PLC–IP3-R pathway in spontaneous [Ca2⫹]i oscillations. A. Traces from two astrocytes showing [Ca2⫹]i in control conditions and following U73122 incubation for 15 min. Trans-ACPD responses are inhibited following U73122 treatment whilst spontaneous [Ca2⫹]i increases are still seen. Upper bar graph illustrates number of astrocytes exhibiting spontaneous [Ca2⫹]i increases in the two conditions; the lower bar graph shows the number of astrocytes responding to mGluR activation in the same slices. B. Effect of 10 M chelerythrine chloride on kinetics of spontaneous [Ca2⫹]i increases. Traces show ⌬F% from two astrocytes in a slice during chelerythrine chloride application. Transient [Ca2⫹]i increases become sustained [Ca2⫹]i increases.
Glial cells can exhibit spatially restricted [Ca2⫹]i signals which do not propagate throughout the cell (Grosche et al., 1999; Nett et al. 2002). Fluorescent measurements from astrocytes where individual astrocytic processes could be visualised showed that not all [Ca2⫹]i increases that occurred in the processes were propagated to the soma (Fig. 6B) and that the degree of correlation between events in the processes decreased with distance from the soma (Fig. 6C, open circles; n⫽11 astrocytes). This result indicates that spontaneous increases can be restricted to regions or compartments of thalamic astrocytes. When recordings were made with 5 mM [Ca2⫹]o (Fig. 6C, filled circles; n⫽9 astro-
cytes), the degree of correlation increased from 73⫾5.2– 92⫾2.7 (P⬍0.005), showing that in addition to having a role in the generation of spontaneous [Ca2⫹]i transients, the amount of Ca2⫹ also affects the ability of intracellular Ca2⫹ waves to propagate intracellularly. If spontaneous astrocytic [Ca2⫹]i transients are influenced by the level of cytoplasmic Ca2⫹, then sarco(endo) plasmic reticulum Ca2⫹-ATPase (SERCA) inhibition may also induce sufficient cytoplasmic Ca2⫹ increases to cause [Ca2⫹]i release from the ER. Application of the SERCA blocker cyclopiazonic acid (CPA) results in an emptying of intracellular stores and activation of CCE (Simpson
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Fig. 5. Effect of BayK8644 on spontaneous [Ca2⫹]i oscillations. A. Trace from an astrocyte showing effect of BayK8644 application. Bar graphs show the number of astrocytes displaying spontaneous [Ca2⫹]i increases before and during BayK8644 application (left), and the frequency of oscillations displayed in spontaneously active astrocytes (expressed as number of increases/100 s) in the two conditions. B1. Pair of traces on left show the effect of focal DHPG application on [Ca2⫹]i in the same astrocyte in control conditions and in the presence of BayK8644. B2 Plot displays data from such experiments for 10 astrocytes with fluorescence normalised to the peak for each astrocyte. Filled circles show fluorescence in control conditions, open circles in the presence of 10 M BayK8644. The asterisk denotes a significance of P⬍0.05, and the arrows denote the range of time for which the difference in fluorescence in the two conditions is significant.
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Fig. 6. [Ca2⫹]o and spontaneous oscillations. A. Traces of ⌬F% from two astrocytes showing the effect of modifying [Ca2⫹]o. Hatched bar indicates time of perfusion with ACSF containing different [Ca2⫹]o. Bar graph summarises the data from n⫽3–5 experiments. B. Traces of spontaneous [Ca2⫹]i from the soma (s) and two regions of the astrocyte processes (a and b) as indicated in the displayed image of the astrocyte (right). ⌬F% plots from region “a” show a [Ca2⫹]i increase which is independent of that occurring in process “b” or the soma. C. Plot of the degree of correlation of [Ca2⫹]i increases between areas on the processes and in the astrocyte soma against the distance from the soma. Open circles show data from experiments performed with 2 mM [Ca2⫹]o and filled circles show data from experiments with 5 mM [Ca2⫹]o.
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Fig. 7. CPA and [Ca2⫹]i release. A. Effect of CPA on [Ca2⫹]i in astrocytes. Upper traces show that CPA induced transients in a proportion of astrocytes (see text) whilst in other astrocytes (lower traces) a slow accumulation of [Ca2⫹]i occurred. B. The same protocols applied in the absence of [Ca2⫹]o. Traces show that transient increases are still seen, whilst slow increases in [Ca2⫹]i are not.
and Russell, 1997; Lo et al., 2002) (Fig. 7A). In our preparation acute CPA addition resulted in a slow increase in astrocytic [Ca2⫹]i levels in 56⫾23% of astrocytes (n⫽3 slices), and also induced [Ca2⫹]i transients in 68⫾22% more astrocytes (n⫽3 slices) than in the previous control period, with the time to [Ca2⫹]i transient generation following CPA addition being 104⫾8 s (n⫽19 astrocytes). The mean slow fluorescence increase 400 s after CPA addition was 99⫾18% (n⫽16 astrocytes), consistent with a chronic activation of CCE by long-term store depletion. This explanation is supported by the findings that in 0 mM [Ca]o CPA still resulted in [Ca2⫹]i increases in VB astrocytes, but only 10% of the astrocytes displayed slow [Ca2⫹]i increases (Fig. 7B). The time to transient generation in 0 mM [Ca2⫹]o was 166⫾11 s (n⫽15 astrocytes). Previous findings (Parri et al., 2001) show that astrocytes display spontaneous [Ca2⫹]i increases 3– 4 min following perfusion with 0 mM [Ca2⫹]o suggesting that it can take quite a long time for ER luminal Ca2⫹ to leak out, and that stores are still able to release Ca2⫹ in a spontaneous manner for this period.
Ca2ⴙ buffering and generation of [Ca2ⴙ]i oscillations The data therefore show that spontaneous [Ca2⫹]i oscillations can be modulated by cytoplasmic Ca2⫹. To determine whether the level of [Ca2⫹]i was important for the generation of spontaneous activity, we manipulated the amount of [Ca2⫹]i buffering in the astrocytes by incubating in different concentrations of Fluo-4AM (Fig. 8). The number of astrocytes displaying spontaneous [Ca2⫹]i oscillations was 2.29⫾0.71/100 s (n⫽3 slices) in 1 M, 1.67⫾0.23/100 s (n⫽3 slices) with 5 M and 0.53⫾0.16/ 100 s (n⫽3 slices) with 20 M Fluo-4 (Fig. 8B) (P⬍0.05, ANOVA; P⬍0.016 between 20 M and 5 M, Fluo-4 and between 20 M and 1 M Fluo-4, Bonferroni post hoc test). The frequency of oscillations in individual astrocytes was 0.22⫾0.03/100 s for 20 M, 0.58⫾0.11/100 s for 5 M and 0.50⫾0.05/100 s for 1 M (P⬍0.05, ANOVA; P⬍0.001 between 20 M and 5 M Fluo-4 and between 20 M and 1 M Fluo-4, Bonferroni post hoc test). The magnitude of spontaneous fluorescence increases was not different between the groups: 20 M, 145.6⫾23.6%; 5 M, 139⫾16%; 1 M, 109⫾13.8%. The number of astrocytes displaying evoked [Ca2⫹]i increases in response to trans-ACPD was
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Fig. 8. Effect of [Ca2⫹]i buffering on spontaneous oscillations. A. Plots of ⌬F% showing spontaneous [Ca2⫹]i increases in slices incubated with 1 M Fluo-4AM and the effect of the mGluR agonist trans-ACPD, and from astrocytes in slices incubated with 20 M Fluo-4AM, where the number of spontaneous increases are reduced but ACPD is still able to release [Ca2⫹]i. B. Summary of data with varying Fluo-4AM concentration. C. Spontaneous increase acquired at 7 Hz, showing that the transient [Ca2⫹]i increase is preceded by a slow rise in cytosolic Ca2⫹. F units are absolute increases in fluorescence.
not different between the different indicator concentrations (ANOVA), showing that the astrocytes were still able to release [Ca2⫹]i but that the initiation of the spontaneous events was affected by the buffering of [Ca2⫹]i. Imaging of spontaneous transients at a rate of 7 Hz confirmed the apparent role of Ca2⫹ in triggering release, in that a slow rise in [Ca2⫹]i is seen before the transient [Ca2⫹]i increase: an increase in fluorescence of 0.83⫾ 0.4%/s (n⫽3) occurred before the transient, which is consistent with the triggering of [Ca2⫹]i release when cytoplasmic Ca2⫹ reaches a threshold level (Bootman et al., 1997; Thomas et al., 2000).
DISCUSSION The major findings of this study on the mechanism of generation of spontaneous astrocytic [Ca2⫹]i oscillations which in the VB thalamus are associated with NMDA in-
ward currents in TC neurons are: 1) spontaneous astrocytic [Ca2⫹]i oscillations are intrinsic, being independent of vesicular transmitter release and membrane receptor activation, 2) PKC activation and IP3-R involvement are however implicated, and 3) cytoplasmic Ca2⫹ seems to be the trigger for astrocytic [Ca2⫹]i release. Intrinsic nature of astrocytic [Ca2ⴙ]i oscillations We previously showed that the [Ca2⫹]i oscillations are independent of neuronal activity since they are resistant to TTX. Here we have extended these findings by showing that they are not sustained by spontaneous neurotransmitter release, since blocking of neurotransmitter release by depletion of presynaptic vesicles does not affect the occurrence of spontaneous astrocytic [Ca2⫹]i increases. Taken together these observations indicate that astrocytic oscillations are not dependent on agonist
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stimulation. The oscillations therefore are intrinsic to the astrocytes.
tivation of PKC by the PLC-catalysed production of diacylglycerol.
Intracellular stores and activation pathways
Ca2ⴙ and the generation and intracellular synchronisation of [Ca2ⴙ]i oscillations
The presence of RyRs on VB astrocytes is indicated by the ability of caffeine to elicit [Ca2⫹]i increases. It has been shown in the neocortex that astrocytes display increased oscillation frequency upon the application of caffeine (Tashiro et al., 2002), which lead the authors to conclude that RyRs were responsible for generating the oscillations. Since ryanodine had no effect on spontaneous oscillation in the VB thalamus, RyRs are not involved in the generation of spontaneous [Ca2⫹]i oscillations. Our experiments on the PLC–IP3-R pathway indicate that this pathway is indeed involved in the generation of the spontaneous [Ca2⫹]i transients and oscillations observed in the VB thalamus. Thimerosal, which sensitises IP3-Rs (Peuchen et al., 1996), increased the number of astrocytes displaying [Ca2⫹]i increases. The IP3-R antagonist 2-APB abolished both trans-ACPD responses and spontaneous [Ca2⫹]i oscillations, indicating an IP3-R locus for oscillation generation. Recent studies, however, have shown that both 2-APB and the antagonist xestospongin C can have other effects on SOCC (Bishara et al., 2002) and SERCA pumps (Castonguay and Robitaille, 2002) which could affect the degree of intracellular store filling (Solovyova et al., 2002) and, at least partly, explain our observations. As expected, inhibition of PLC by U73122 inhibited mGluR-activated [Ca2⫹]i increases, but did not block spontaneous [Ca2⫹]i activity. This result agrees with those seen with bafilomycin A1, since the effect of bafilomycin A1 would also be expected to greatly reduce the amount of neuroactive compounds activating astrocyte receptors. On the other hand, it does not exclude the possibility that PLC activation is necessary for spontaneous oscillation manifestation since i) there could conceivably be residual PLC activation to continue IP3 production and/or ii) PLC is being activated by Ca2⫹ ions. PLC-␦1, which is activated in the range 0.1–1 M Ca2⫹ (Allen et al., 1997), is preferentially expressed in astrocytes at around 14 days postnatally (Yamada et al., 1991). In addition, PLC activation by Ca2⫹ was suggested to be important in the propagation of intracellular Ca2⫹ waves in cultured striatal astrocytes (Venance et al., 1997), and modelling studies indicate that PLC-␦1 could have a pivotal role in the regenerative stage of wave propagation (Hofer et al., 2002). A role for PLC activation is indicated by experiments with the PKC inhibitor chelerythrine chloride. Following receptor activation of PLC, IP3 and diacylglycerol are produced. IP3 causes the release of [Ca2⫹]i and diacylglycerol activates PKC, which then phosphorylates the mGluR and PLC to cause a negative feedback. This negative feedback by PKC is important in determining the oscillatory period following agonist stimulation in astrocytes (Codazzi et al., 2001). The switching of spontaneous [Ca2⫹]i increases into sustained increases in the presence of chelerythrine chloride indicates that PKC negative feedback on PLC has a role in controlling the duration of the spontaneous [Ca2⫹]i oscillations, and also suggests a previous ac-
It is clear from our experiments that Ca2⫹ has an important role in the generation of spontaneous [Ca2⫹]i oscillations. The BayK8644 experiments support the findings of our previous studies that showed that nifedipine reduced the number of spontaneously active astrocytes. As oscillations are due to intracellular Ca2⫹ release, this suggested that Ca2⫹ entry across the plasma membrane, and more specifically via L-type Ca2⫹ channels was triggering intracellular Ca2⫹ release via a CICR mechanism. The findings in this present study that BayK8644 increases the number of astrocytes exhibiting spontaneous [Ca2⫹]i oscillations confirm the previously reported DHP-sensitivity but also strongly indicate that this effect may be on the store refilling component, as has been seen in other cell types (Curtis and Scholfield, 2001). This, in turn, suggests that the observed effect of DHPs is to affect the filling state of the ER, and so an increased luminal Ca2⫹ level could be acting to sensitise the IP3-Rs for release (Sienaert et al., 1998). IP3-Rs are Ca2⫹ dependent (Bezprozvanny et al., 1991), requiring cytoplasmic Ca2⫹ binding for IP3 activation and being able to be activated by Ca2⫹ in low levels of IP3. Our experiments with different extracellular and cytoplasmic Ca2⫹ concentrations support a mechanism whereby Ca2⫹ is acting on IP3-Rs in generating spontaneous astrocytic [Ca2⫹]i oscillations. Using low agonist concentrations to activate PLC and IP3 production, it has been established that the generation of “global” (i.e. cellwide) Ca2⫹ signals depends on the spatiotemporal recruitment of “elementary” Ca2⫹ release events (Bootman et al., 1997; Thomas et al., 2000). The recruitment of elementary release sites provides the “pacemaker” Ca2⫹ rise necessary to trigger a regenerative response (Bootman et al., 1997). Our data show that areas of single astrocytes as close as 5 m could display independent Ca2⫹ increases, that do not always lead to global events. This is consistent with studies in cultured astrocytes where it has been shown that release sites were situated 5–7 m apart (Laskey et al., 1998). The increase in the number of astrocytes generating spontaneous [Ca2⫹]i oscillations with increasing [Ca2⫹]i indicates a role for cytoplasmic Ca2⫹ in triggering [Ca2⫹]i release: this is echoed by the apparent correlating effect on the astrocytic “compartments” of increasing [Ca2⫹]o. The data are consistent with a scenario where the action of BayK8644 is on Ca2⫹ entry following store depletion and therefore fills stores to a point where they are more likely to release [Ca2⫹]i. Increasing [Ca2⫹]o could have the effect of filling stores and also increasing cytoplasmic Ca2⫹ which increases the excitability of the ER by bringing the IP3-Rs closer to CICR threshold (Bootman et al., 1997; Simpson and Russell, 1997). In Hela cells activated by histamine, increasing [Ca2⫹]o has also been shown to increase the frequency of oscillations (Bootman et al., 1996).
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A triggering effect of cytoplasmic Ca2⫹ on IP3-Rs is supported by our data with SERCA inhibition. CPA caused transients in VB astrocytes in our study, an effect similar to that observed in cultured astrocytes (Simpson and Russell, 1997), where thapsigargin resulted in propagation of Ca2⫹ waves without the need for IP3 activation. The suggested mechanism was that Ca2⫹ leak through IP3-Rs at specific initiation sites high in IP3-Rs and SERCA triggered CICR from IP3-Rs at these points (Simpson and Russell, 1997). Our results therefore indicate that such a mechanism underlies spontaneous astrocytic [Ca2⫹]i oscillations and confirm the generation of [Ca2⫹]i transients by cytoplasmic Ca2⫹. The role of cytoplasmic Ca2⫹ levels in spontaneous [Ca2⫹]i oscillations generation is supported by the results obtained in slices incubated with increasing concentrations of Fluo-4AM. The reduction in spontaneous activity, and the lack of effect on trans-ACPD-elicited responses, indicate that the increased Ca2⫹ buffering effect of the indicator decreases the free cytoplasmic Ca2⫹ and hence lowers the CICR triggering effect on IP3-Rs. This effect is perhaps analogous to that reported for the effects of indicators on inter-astrocytic wave propagation in culture (Wang et al. 1997). The results of this study on the mechanism of spontaneous [Ca2⫹]i oscillations in VB astrocytes show that their properties are similar to agonist-evoked oscillations in astrocytes which have a dependence on IP3 and [Ca2⫹]i. However, in the VB thalamus, oscillations are spontaneous, requiring no agonist application. These results therefore point to a scenario where there is a level of constitutive PLC activation, possibly by Ca2⫹, which produces sufficient IP3 to put the IP3-Rs in a primed state for activation by Ca2⫹, and that spontaneous oscillations occur by Ca2⫹ triggering CICR from the astrocytic IP3-Rs. Acknowledgements—We would like to thank T. M. Gould for technical/image processing help and advice. This work was supported by the Wellcome Trust (Grant 37089-98).
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(Accepted 30 April 2003)