New evidence for purinergic signaling in the ... - The FASEB Journal

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New evidence for purinergic signaling in the olfactory bulb: A2A and P2Y1 receptors mediate intracellular calcium release in astrocytes Michael Doengi, Joachim W. Deitmer, and Christian Lohr1 Abteilung fu¨r Allgemeine Zoologie, University of Kaiserslautern, Kaiserslautern, Germany Purinergic receptors play a key role in neuron-glia and glia-neuron interactions. In the present study, we have recorded cytosolic Ca2ⴙ responses using confocal imaging in astrocytes of acute olfactory bulb slices from mice (postnatal days 3– 8). By application of agonists and antagonists, we identified two types of receptors, P2Y1 and A2A, that mediated Ca2ⴙ responses attributable to Ca2ⴙ release from intracellular stores in the astrocytes. Both receptor types were activated by application of ATP and ADP; however, when enzymatic ATP degradation was suppressed by the alkaline phosphatase inhibitor levamisole, ATP only activated MRS2179-sensitive P2Y1 but not ZM241385-sensitive A2A receptors. The dose-response curve for A2A receptors activated by adenosine revealed an EC50 of 0.3 ␮M, one order of magnitude smaller than the EC50 of 5 ␮M determined for P2Y1 receptors activated by ADP. Electrical stimulation of the olfactory nerve in the presence of glutamate receptor blockers to suppress excitation of postsynaptic neurons evoked Ca2ⴙ responses in most of the astrocytes, which were inhibited by blocking both P2Y1 and A2A receptors. Our results indicate that olfactory nerve terminals release not only glutamate, but also ATP, which activates P2Y1 receptors and, after degradation of ATP to adenosine, A2A receptors in astrocytes.—Doengi, M., Deitmer, J. W., Lohr, C. New evidence for purinergic signaling in the olfactory bulb: A2A and P2Y1 receptors mediate intracellular calcium release in astrocytes. FASEB J. 22, 2368 –2378 (2008) ABSTRACT

Key Words: neuron-glia interaction 䡠 adenosine triphosphate The purine atp is an important extracellular mediator in the physiological and pathophysiological processes of all vertebrate tissues, including the nervous system, and most if not all cells in the vertebrate brain carry purinergic receptors (1). Recent research has shown that purinergic signaling in the nervous system is by far more complex than other neurotransmitter systems. While enzymatic degradation of neurotransmitters such as acetylcholine and catecholamines in the synaptic cleft leads to inactivation of the transmitter molecule and the termination of neurotransmission, the degradation products of ATP, particularly ADP and adenosine, are potent agonists at different types of 2368

purinoceptors with different functions. Thus, the interplay of ATP release, its enzymatic degradation, and purinoceptor activation produces a large versatility of purinergic signaling modes, depending on the amount and type of ecto-nucleotidases and purinoceptors. Purinoceptors can be divided into P1 receptors (A1, A2A, A2B, and A3), which are adenosine-sensitive G-proteincoupled receptors; P2X receptors, which are ATP-gated cation channels; and P2Y receptors, which are Gprotein-coupled receptors activated by ATP, ADP, and other nucleotides. In glial cells, all three types of purinoceptors have been described previously. P2Y1 and P2Y2 receptors, for example, have been reported to link purinergic signaling to Ca2⫹ responses in many astrocytes, but other P2Y and P2X receptors have also been identified in astrocytes (see ref. 2 for review). ATP can evoke Ca2⫹ signaling not only in astrocytes but also in oligodendrocytes, olfactory ensheathing cells, and Schwann cells by P2Y and/or P2X receptor activation (3– 6). While P2 receptors frequently mediate cytosolic Ca2⫹ signaling, P1 receptors are most often linked to adenylate cyclase (1). P1-receptor-mediated calcium signaling in astrocytes has been investigated in only a few studies, the results of which are controversal: in astrocytes acutely isolated from the cerebellum and in astrocytes in hippocampal brain slices, adenosine elicited Ca2⫹ increases via A2B receptors (7, 8), whereas in cultured astrocytes from spinal cord and hippocampus, adenosine did not evoke Ca2⫹ increases (9, 10). P1 and P2 receptors are coexpressed in cultured rat cortical astrocytes, and activation of A2B receptors potentiated the Ca2⫹ rise evoked by P2Y receptor stimulation via adenylate cyclase stimulation, whereas A2B receptor activation per se did not elicit Ca2⫹ signals (11). Hence, it remains to be shown whether both P1 and P2 receptors can induce Ca2⫹ signaling in the same astroglial cell. We studied purinergic signaling in the olfactory bulb of mice, where astrocytes have been shown to contribute to the development of the glomeruli (12, 13) and to the signal processing in mature glomeruli (14). It is not 1

Correspondence: Abteilung fu¨r Allgemeine Zoologie, University of Kaiserslautern, POB 3049, D-67653 Kaiserslautern, Germany. E-mail: [email protected] doi: 10.1096/fj.07-101782 0892-6638/08/0022-2368 © FASEB

known, however, how neurons and astrocytes communicate in the olfactory bulb. Although purinoceptors have been demonstrated in the olfactory bulb by means of immunohistochemical and radioactive ligand binding techniques (15–17), the role of ATP as a neurotransmitter has not yet been described in that brain area. Therefore, we were interested in whether ATP acts as a neurotransmitter and mediates communication between neurons and astrocytes in the olfactory bulb. We have analyzed the effect of ATP on the Ca2⫹ concentration of olfactory bulb astrocytes. Our results show that not only ATP but also adenosine that is produced by ATP hydrolysis can evoke a Ca2⫹ rise in astrocytes. In addition, electrical stimulation of olfactory receptor axons resulted in Ca2⫹ transients in astrocytes, which were sensitive to purinoceptor blockage, suggesting that olfactory receptor axons released ATP that leads to purinoceptor activation in astrocytes.

MATERIALS AND METHODS Slice preparation We used juvenile mice at postnatal days (P) 3 to 8, because at that time astrocytes contribute to the development of the olfactory bulb (13), and purinergic signaling might be involved in this process. Olfactory bulb slices were prepared as described before (4). In brief, mice were decapitated, and the olfactory bulbs were quickly transferred into a chilled (4°C) Ca2⫹-reduced (0.5 mM Ca2⫹) artificial cerebrospinal fluid (aCSF, see below). Sagittal slices of the bulbs were cut using a vibratome (VTS1000; Leica, Bensheim, Germany). The brain slices (250 ␮m thick) were stored in Ca2⫹-reduced aCSF for 45 min at 30°C and 15 min at room temperature before dye loading. Ca2⫹-reduced aCSF was continuously gassed with carbogen (95% O2/5% CO2, buffered to pH 7.4 with CO2/ bicarbonate). Solutions The standard aCSF for acute brain slices contained (in mM): 125 NaCl, 2.5 KCl, 2 CaCl2, 1 MgCl2, 25 d-glucose, 26 NaHCO3, 1.25 NaH2PO4, and 0.5 l-lactate, gassed during the entire experiment by carbogen to adjust the pH to 7.4. In Ca2⫹reduced saline (0.5 mM), 1.5 mM CaCl2 was replaced by 1.5 mM MgCl2. In K⫹-free saline (0K⫹) or high-K⫹ solution (50 mM K⫹), KCl was exchanged for NaCl. Adenosine; the nucleotides ATP, ADP, UTP, UDP, and IDP; and the purinoceptor antagonist pyridoxalphosphate-6-azophenyl-2⬘,4⬘-disulfonic acid (PPADS) were obtained from Sigma-Aldrich (Taufkirchen, Germany). ATP-␥-S was from Jena Bioscience (Jena, Germany). The adenosine receptor blockers ZM241385, DPCPX, PSP 1115, and VUF 5574; the purinoceptor antagonists MRS2179; the purinergic agonist 2-methyl-thio-ATP (2MeSATP); and the metabotropic glutamate receptor blockers MPEP and JNJ16259685 were obtained from Tocris (Bristol, UK). The ionotropic glutamate receptor antagonists d-2-amino-5-phosphonopentanoic acid (D-AP5) and 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX) were obtained from Ascent Scientific (Weston-Super-Mare, UK). Cyclopiazonic acid and levamisole were obtained from Axxora (Gru¨nberg, Germany). All substances were stored as stock solutions at ⫺20°C according to the manufacturers description, except ATP-␥-S, which lost its resistance against hydrolysis when stored frozen. CALCIUM SIGNALING IN OLFACTORY BULB ASTROCYTES

ATP-␥-S solution was hence prepared immediately before use. ARC69931MX was a gift from the Medicines Company (Waltham, MA, USA) and was added to the aCSF directly before the experiment. Confocal Ca2ⴙ imaging and electrical stimulation Ca2⫹ imaging was performed as described previously (4, 18). For dye loading, Fluo-4-AM was dissolved in DMSO/20% pluronic to make 2 mM of stock solution. Acute brain slices were incubated in the dark at room temperature (21–24°C) for 60 min in Ca2⫹-reduced aCSF containing 1 ␮M Fluo-4-AM (Molecular Probes, Eugene, OR), which labels glial cells and interneurons but not olfactory receptor axons (4) or mitral cells (not shown), the principal output neurons of the olfactory bulb. After dye loading, slices were transferred onto a nylon mesh and kept dark in aCSF until the beginning of the experiment. All slices were treated equally to minimize possible artifacts resulting from different dye concentrations in the cells, which could affect the kinetics of evoked Ca2⫹ signals. Measurements of Ca2⫹ changes in cells of an acute brain slice were performed with a confocal laser scanning microscope (LSM 510; Zeiss, Oberkochen, Germany). The Ca2⫹sensitive dye Fluo-4 was excited using the 488 nm line of an argon laser. The emission signal was truncated by an optical bandpass filter at wavelengths of 505 and 550 nm. To measure Ca2⫹ dynamics, images were acquired in one focal plane with a frequency of 0.3–1 Hz. During the experiment, slices were continuously superfused with aCSF. Drugs were applied by switching from a drug-free to a drug-containing solution. Axons were stimulated by a glass pipette (tip resistance ⬃2.5 M⍀,), filled with aCSF, and connected to a stimulation unit (SD9K, Grass, West Warwick, RI) by a chlorided silver wire. A second silver wire at the outside of the glass electrode was grounded. The pipette tip was placed in the olfactory nerve layer. The following stimulation parameters were used: single pulse duration ⫽ 0.2 ms, frequency ⫽ 50 Hz, voltage ⫽ 30 – 60 V, and duration of stimulation train ⫽ 15 s. Antibody staining Antibody staining was performed as described in Rieger et al. (4). Briefly, olfactory bulbs were kept overnight at 4°C in 4% paraformaldehyde in phosphate buffered saline (PBS) containing in mM: 130 NaCl, 3 NaH2PO4, and 7 Na2HPO4, adjusted to pH 7.4. Slices were then sectioned with a vibratome and thoroughly rinsed 3⫻ with PBS and then incubated for 1 h in blocking solution (3% bovine serum albumin, 1% normal goat serum, and 0.1% Triton X-100 in PBS) to mask unspecific binding sites. Afterward, primary antibody (rbt-anti-GFAP, Dako Z0334, 1:1000; Dako, Glostrup, Denmark) and 5 ␮M propidium iodide were incubated overnight at 4°C. After being rinsed 3⫻ with PBS for 5 min, the secondary antibody (gt-anti-rabbit-IgG-Alexa488, Molecular Probes, A11008, 1:1000 in PBS) was incubated for 2 h, followed by repeated rinsing with PBS. Slices were mounted on slides with self-hardening embedding medium (30% glycerol, 12% polyvinyl alcohol, and 0.5% phenol in 0.1 M Tris). Data analysis and statistics Within a time series, Fluo-4-stained cell bodies were defined as regions of interest (ROIs), and the fluorescence intensity was measured in each ROI. Ca2⫹ changes were given as relative fluorescence changes (⌬F) with respect to the resting 2369

fluorescence, which was normalized to 100%. Measurements are given as mean ⫾ se, with n giving the number of cells. If not stated otherwise, cells from all developmental stages (P3–P8) were pooled. Significance of statistical differences was calculated using Student’s t test. Means were defined as statistically different at an error probability of P ⬍ 0.05.

RESULTS Cell identification Astrocytes and interneurons of the glomerular layer of the olfactory bulb were investigated. In fixed tissue, astrocytes can be visualized by an anti-GFAP antibody, showing the elaborate glial processes in the neuropil of the glomeruli (Fig. 1A). In the glomerular layer, cell bodies of both astrocytes and interneurons (juxtaglomerular cells) surround glomeruli. In Fluo-4-loaded acute brain slices, astrocytes were more intensely stained by Fluo-4 than interneurons. Physiologically, these two cell populations can be distinguished by their responses to low external K⫹ concentration (0K⫹). Astrocytes in the cerebellum, the hippocampus, and the brain stem, both in mice and rats, respond to 0K⫹ with increases in the intracellular Ca2⫹ concentration, due to Ca2⫹ influx through Kir4.1 channels (19 –21), while K⫹ withdrawal has no effect on neuronal Ca2⫹

(Fig. 1B, C). The identity of 0K⫹-sensitive cells as astrocytes has been confirmed by antibody staining in a previous study (21). We assume that cells in the olfactory bulb responding to 0K⫹ are astrocytes. In the rat embryonic olfactory bulb, vimentin-positive radial glial processes have been shown to contribute significantly to glial structures in the glomerular layer (12, 13). Antivimentin immunostaining disappears between P1 and P4 from the glomerular layer, and radial glial cell bodies are mainly located in deep layers in the olfactory bulb close to the subventricular zone (13). Since the animals used in the present study were mainly P4 and older and since we investigated only cell bodies in the superficial layers, it is unlikely that measurements were taken from radial glial cells. Another glial cell type, olfactory ensheathing cells, is located in the nerve layer and can readily be distinguished from astrocytes (4). ATP and ADP induce Ca2ⴙ release from intracellular stores in astrocytes Application of the purinoceptor ligands ATP or ADP evoked Ca2⫹ transients in all astrocytes investigated (Fig. 1B), while in interneurons, both ligands had no effect on cytosolic Ca2⫹ (Fig. 1C). ATP can activate both P2X and P2Y receptors, while ADP only acts on P2Y receptors (1). The efficacy of ADP to elicit Ca2⫹

Figure 1. Purinergic Ca2⫹ signaling in astrocytes of the olfactory bulb. A) Anti-GFAP immunostaining of astrocytes (green) in the glomerular layer of a mouse olfactory bulb. Astrocytes extend their processes into the neuropil of the glomeruli (asterisks). Nuclei of periglomerular astrocytes and interneurons were stained with propidium iodide (red). Scale bar ⫽ 20 ␮m. B, C) Ca2⫹ responses during application of ATP, ADP, and 50 mM K⫹ and during withdrawal of K⫹ (0K⫹) in astrocytes (B) and in juxtaglomerular interneurons (C). Withdrawal of K⫹ evokes Ca2⫹ signaling only in astrocytes but not in interneurons. D) Effect of Ca2⫹ store depletion by CPA on ATP-induced Ca2⫹ signaling in astrocytes. E) Mean amplitude of ATP-induced Ca2⫹ transients in astrocytes of different postnatal days (P3–P8). In this figure and the following figures, bars are mean ⫹ se, n indicates the number of cells investigated. Ca2⫹ transients at P7 were significantly smaller than at other postnatal days; **P ⬍ 0.01. 2370

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signaling in olfactory bulb astrocytes suggests the activation of P2Y receptors, which stimulate phospholipase C-mediated inositol trisphosphate (InsP3) production and subsequent Ca2⫹ release from intracellular stores (2). To check the contribution of intracellular Ca2⫹ stores, we depleted Ca2⫹ stores by application of cyclopiazonic acid (CPA), which blocks Ca2⫹-ATPases of the endoplasmic reticulum and hence prevents Ca2⫹ uptake into intracellular stores. In the presence of CPA, Ca2⫹ leakage from intracellular stores became apparent (Fig. 1D). After store depletion, the Ca2⫹ concentration in astrocytes remained at an elevated level. Similar results have been described in cerebellar astrocytes in culture and in situ, where the elevated Ca2⫹ concentration is due to store-operated Ca2⫹ entry (18, 22). When ATP (n⫽99) or ADP (n⫽75) was applied after Ca2⫹ store depletion by CPA, astrocytes did not respond with a Ca2⫹ transient (Fig. 1D). Withdrawal of external Ca2⫹, in contrast, had no major effect on the Ca2⫹ transients induced by ATP in astrocytes (n⫽61; not shown). These results show that application of ATP leads to Ca2⫹ transients that depend on intracellular Ca2⫹ stores and are due to intracellular Ca2⫹ release, while P2X receptor-mediated Ca2⫹ influx appears to play a minor role under our experimental conditions. Astrocytes in the olfactory bulb undergo morphological development during the first postnatal week (13), and we were interested in whether these developmental changes were accompanied by changes in the responsiveness to ATP. Application of ATP led to Ca2⫹ transients in all astrocytes in all developmental stages investigated. The mean amplitudes of the ATP-induced Ca2⫹ transients in astrocytes of animals from P3 to P8 were not significantly different except for P7, when the mean amplitude was slightly but statistically signifi-

cantly smaller than in other ages (Fig. 1E). However, this small decrease in amplitude may represent biological variability rather than developmental changes. Pharmacological receptor profile of purinergic Ca2ⴙ responses to nucleotides Ca2⫹ transients in olfactory bulb astrocytes are presumably mediated by P2Y receptors, since ADP does not activate P2X receptors and Ca2⫹ release is the predominant Ca2⫹ signaling pathway downstream of P2Y receptor activation (2). We used different agonists and antagonists of P2Y receptors to identify the subtypes of P2Y receptors that are involved in glial Ca2⫹ signaling in the olfactory bulb. UTP and UDP are potent agonists of P2Y2, P2Y4, and P2Y6 receptors (1, 23). UTP has also be shown to induce Ca2⫹ release by activation of P2Y11 receptors heterologously expressed in neuroblastoma cells by a mechanism independent of InsP3 production (24). Neither UTP nor UDP was able to induce Ca2⫹ signals in astrocytes, indicating that the involvement of these four receptor subtypes in mediating the Ca2⫹ responses is unlikely (Fig. 2A); 100 ␮M IDP, an agonist of P2Y13, but not of P2Y12 receptors (25), did not evoke Ca2⫹ transients in astrocytes (Fig. 2B). Ca2⫹ transients could also be elicited by 2MeSATP (30 ␮M; n⫽85), which acts on P2Y1, P2Y12, and P2Y13 as well as P2X receptors (Fig. 2C). 2MeSATP-induced Ca2⫹ signaling was abolished after store depletion with CPA, confirming Ca2⫹ stores to be the main source for the cytosolic Ca2⫹ responses on purinoceptor activation (n⫽38; not shown). Figure 2D summarizes the effects of the different purinoceptor agonists on the cytosolic Ca2⫹ concentra-

Figure 2. Effects of purinoceptor ligands. A, B) ADP (A), but not UTP, UDP, or IDP (B), evoke Ca2⫹ transients in astrocytes. C) ATP- and 2MeSATP-induced Ca2⫹ transients. D) Statistics of the effects of the P2Y receptor agonists. E, F) P2Y1 receptor antagonists PPADS (E) and MRS2179 (F) failed to block Ca2⫹ transients in most of the astrocytes. CALCIUM SIGNALING IN OLFACTORY BULB ASTROCYTES

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tion of olfactory bulb astrocytes. ATP, ADP, and 2MeSATP, but not UTP, UDP or IDP, elicited Ca2⫹ transients in all astrocytes investigated. The mean amplitudes of the Ca2⫹ transients were 125.9 ⫾ 2.0% ⌬F (n⫽979) for ATP, 120.6 ⫾ 1.4% ⌬F (n⫽1524) for ADP, and 128.1 ⫾ 9.2% ⌬F (n⫽85) for 2MeSATP. The results suggest that, with respect to the agonist profile, P2Y1 receptors and P2Y12 receptors are the likely receptors that mediate ATP/ADP-dependent Ca2⫹ signaling in olfactory bulb astrocytes. The P2Y12-specific antagonist ARC69931MX (20 –100 ␮M), however, did not significantly reduce the Ca2⫹ transients elicited by ADP in astrocytes (n⫽100; not shown). We also tested the P2Y1-specific antagonist MRS2179 and the nonspecific purinoceptor antagonist PPADS, which inhibits P2Y1, P2Y4, P2Y6, P2Y13, and P2X receptors. PPADS (10 ␮M) had no significant effect on the Ca2⫹ response to ADP application in 94% of the astrocytes (Fig. 2E) but entirely and reversibly blocked the Ca2⫹ transients in 6% of the cells (n⫽69). Similar results were obtained using MRS2179 (30 ␮M), which left the Ca2⫹ transients unchanged in 87% of the cells (Fig. 2F) but completely blocked the Ca2⫹ transients in 13% of the astrocytes (n⫽79). The results indicate that inhibiting P2Y1 receptors is not sufficient to suppress ADP-induced Ca2⫹ signaling in most of the astrocytes. Involvement of adenosine receptors In olfactory bulb astrocytes, the agonist profile of the purinoceptor-mediated Ca2⫹ signaling suggests the involvement of P2Y1 or P2Y12 receptors; however, P2Y1 and P2Y12 receptor antagonists failed to block the ADP-induced Ca2⫹ response in most of the astrocytes, suggesting the involvement of another receptor type.

Since ATP and ADP can be enzymatically degraded to AMP and adenosine (26), we tested the effect of adenosine on the Ca2⫹ concentration in olfactory bulb astrocytes. In 77% of the astrocytes (n⫽331), application of adenosine (10 ␮M) evoked Ca2⫹ increases with an average amplitude of 106.7 ⫾ 7.4% ⌬F. Adenosineinduced Ca2⫹ transients could be blocked by the A2A receptor antagonist ZM241385 (Fig. 3A) but were not affected by MRS2179 (Fig. 3B). Incubation of the cells with DPCPX (6 ␮M), PSB1115 (1 ␮M), and VUF 5574 (10 ␮M), antagonists of A1 receptors, A2B receptors, and A3 receptors, respectively, failed to block adenosine-evoked Ca2⫹ signaling or had only weak effects (Fig. 3C, D). We also tested the effect of the A2A antagonist ZM241385 on ATP- and ADP-evoked Ca2⫹ signaling. Ca2⫹ transients evoked by ATP or ADP were only slightly reduced by ZM241385 alone (Fig. 3A). However, they were entirely blocked by the combination of MRS2179 and ZM241385 (n⫽53), suggesting the expression of both P2Y1 receptors and A2A receptors in astrocytes (Fig. 3B–D). The results suggest that application of ATP and ADP results in the activation not only of P2Y1 receptors but also of A2A receptors. Two possible mechanisms can lead to the ATP-mediated activation of adenosine receptors: first, ATP could activate neurons that release adenosine, and second, ATP could be enzymatically degraded to adenosine. To test the latter hypothesis, we used the nonhydrolyzable ATP analog ATP-␥-S, and 2MeSATP. Ecto-nucleotidases are able to hydrolyze 2MeSATP to 2MeSADP and 2MeSAMP but not to 2MeS-adenosine (27). Hence, the 2MeSATP hydrolysis products do not activate A2A receptors. The Ca2⫹ transients elicited by 2MeSATP were blocked by MRS2179 alone in 81% of the astrocytes (n⫽47), while

Figure 3. Contribution of adenosine receptors. A) A2A adenosine receptor antagonist ZM241385 reversibly blocks Ca2⫹ transients evoked by adenosine (Ado) but has no effect on ADP-induced Ca2⫹ transients in astrocytes. B) When P2Y1 and A2A receptors are blocked by MRS2179 and ZM241385, ADP fails to induce Ca2⫹ transients in astrocytes. C) Statistics of the effects of MRS2179 and ZM241385 on the number of cells responding to ADP, ATP, and adenosine. D) MRS2179 and ZM241385, respectively, slightly reduced the amplitude of the Ca2⫹ transients evoked by ATP and ADP, while the combination of both compounds entirely blocked the response. Adenosine-induced Ca2⫹ transients were completely blocked by ZM241385. *P ⬍ 0.05; ***P ⬍ 0.005. 2372

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ATP-induced Ca2⫹ transients were only blocked by MRS2179 together with ZM241385 (Fig. 4A). Ca2⫹ transients evoked by ATP-␥-S, but not by ATP, were completely blocked by MRS2179 (n⫽39; Fig. 4B). Hence, ATP hydrolysis appears to be required for adenosine receptor activation during ATP application. It should be noted that ATP-␥-S lost its resistance against hydrolysis when the compound was stored as a stock solution at ⫺20°C for longer than 1 day, as ATP-␥-S-evoked Ca2⫹ transients were then only blocked by the combination of MRS2179 and ZM241385 (not shown). We used inhibitors of enzymes known to catalyze the hydrolysis of ATP in order to identify the enzymes involved in ATP degradation in the olfactory bulb. ARL67156 blocks the ecto-nucleotidases NTPDase1 and NTPDase 3, which can hydolyze ATP to ADP and AMP. In the presence of ARL67156, ATP-induced Ca2⫹ transients were only weakly reduced by MRS2179 (n⫽90), indicating that ARL67156 did not prevent the activation of A2A receptors (Fig. 4C). In contrast, the inhibitor of alkaline phosphatases, levamisole (10 mM), greatly reduced the ATP-evoked Ca2⫹ signaling in olfactory bulb astrocytes (Fig. 4D). Application of levamisole itself produced a slow, erratic Ca2⫹ increase. In the continuous presence of levamisole, only 26% of

the astrocytes responded to ATP with a Ca2⫹ rise, which had a mean amplitude of 6.9% ⌬F (n⫽93). Figure 4E, F summarizes the effects of MRS2179 on Ca2⫹ transients evoked by 2MeSATP, ATP-␥-S, and ATP in the presence of inhibitors of ATP-hydrolyzing enzymes. The results suggest that ATP application leads to activation of P2Y1 receptors and, by degradation of ATP to adenosine, of A2A receptors. We could not detect any heterogeneity of the astrocytes expressing only P2Y1 or A2A receptors or both types of receptors with respect to their location in the tissue or their morphology. Hence, it remains unclear whether astrocytes with different purinergic signaling represent different subclasses such as complex and passive astrocytes (28). Dose-response curves of ADP-induced Ca2ⴙ transients We applied different concentrations of adenosine and ADP to obtain the dose-response relationships of purinoceptor-dependent Ca2⫹ signaling in astrocytes (Fig. 5). Adenosine induced Ca2⫹ signaling in most of the cells at concentrations of 0.3 ␮M and higher with an EC50 of 0.23 ␮M and a Hill coefficient of 2.4 (n⫽66). ADP was less potent with an EC50 value of 1.1 ␮M but with a Hill coefficient similar to adenosine (1.9; n⫽44). Since we have shown that ADP application can lead to

Figure 4. Adenosine is produced by ATP hydrolysis. A) Ca2⫹ transients evoked by 2MeSATP in astrocytes are greatly reduced when P2Y1 receptors are blocked by MRS2179, whereas ATP-induced Ca2⫹ transients are only entirely blocked when P2Y1 receptors and A2A receptors are inhibited. B) Blockage of P2Y1 receptors with MRS2179 is sufficient to inhibit Ca2⫹ transients evoked by the nonhydrolyzable ATP analog ATP-␥-S. C) Blocking ecto-nucleotidases with ARL67156 in the presence of the P2Y1 receptor antagonist MRS2179 does not prevent ATP-induced Ca2⫹ signaling. D) Application of the alkaline phosphatase inhibitor levamisole induced small, irregularly shaped Ca2⫹ rises. In the presence of levamisole and MRS2179, ATP was not effective in evoking Ca2⫹ transients in astrocytes. E) Effects of MRS2179, ZM241385, ARL67156, and levamisole on the number of cells responding to 2MeSATP, ATP-␥-S, and ATP with an increase in Ca2⫹. F) Effects of MRS2179, ZM241385, ARL67156, and levamisole on the amplitude of the Ca2⫹ transients in olfactory bulb astrocytes induced by 2MeSATP, ATP-␥-S, and ATP. ***P ⬍ 0.005. CALCIUM SIGNALING IN OLFACTORY BULB ASTROCYTES

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Figure 5. Dose-response relationships of Ca2⫹ transients in astrocytes induced by adenosine and ADP. A–C) Ca2⫹ responses due to application of different concentrations of adenosine (A), ADP in the absence of ZM241385 (B), and in the presence of ZM241385 (C). D–F) Number of astrocytes responding to adenosine (D), ADP in the absence of ZM241385 (E), and in the presence of ZM241385 (F). G) Dose-response curves of adenosine and ADP receptor-mediated Ca2⫹ transients.

the activation of both P2Y1 and A2A receptors, we also studied the dose-response relationship of ADP-induced Ca2⫹ signaling in astrocytes in the presence of the A2A receptor antagonist ZM241385. This increased the EC50 value of the ADP-evoked Ca2⫹ response to 4.5 ␮M and decreased the Hill coefficient to 1.5 (n⫽126), suggesting that the dose-response relationship of ADP-induced Ca2⫹ signaling in the absence of ZM241385 is greatly influenced by the activation of A2A receptors and represents the responses mediated by both P2Y1 and A2A receptors. The resting extracellular ATP concentration in nervous tissue has been estimated to be ⬃1 ␮M (29). Since ATP can be stored in synaptic vesicles at a concentration up to 1 mM (30), synaptic release of ATP may result in increases of several micromolar, as measured with ATP-sensitive microelectrode biosensors (31). This could lead to strong activation of the purinoceptors with consideration of the EC50 values determined here. Ca2ⴙtransients in astrocytes evoked by receptor axon stimulation De Saint Jan and Westbrook (32) have recently shown that electrical stimulation of olfactory receptor axons can induce an inward current in glomerular astrocytes. To test whether receptor axon stimulation can also induce calcium signaling in olfactory bulb astrocytes, we electrically stimulated axons in the nerve layer and measured changes in cytosolic Ca2⫹ in astrocytes of nearby glomeruli (Fig. 6A). A single stimulation pulse evoked Ca2⫹ transients in 14% of the studied astrocytes (n⫽205), whereas stimulation for 5 s (50 Hz) and 15 s (50 Hz) elicited Ca2⫹ signaling in 63% (n⫽289) and 71% (n⫽205) of olfactory bulb astrocytes, respectively (Fig. 6B). The mean amplitude of stimulation-evoked 2374

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Ca2⫹ transients was 87.6 ⫾ 13.1% ⌬F (n⫽28) for single pulses, 103.5 ⫾ 4.4% ⌬F (n⫽181) for 5 s of stimulation, and 181.0 ⫾ 8.0% ⌬F (n⫽145) for 15 s of stimulation. The Ca2⫹ transients were abolished when action potential firing was prevented by the sodium channel blocker tetrodotoxin (TTX; n⫽41; Fig. 6C). The inward current elicited in astrocytes by single stimulation pulses is mainly mediated indirectly by glutamatergic excitation of postsysnaptic mitral/tufted cells and can be greatly reduced with the ionotropic glutamate receptor blockers NBQX and D-AP5 (32). In addition, activation of metabotropic glutamate receptors (mGluR) can induce depolarization of mitral cells and a Ca2⫹ increase in mitral cell tufts, which could also affect astrocytes indirectly (32, 33). To test whether glutamatergic excitation of olfactory bulb neurons contribute to the stimulation-evoked Ca2⫹ signaling in astrocytes, we blocked ionotropic glutamate receptors with NBQX and D-AP5 as well as mGluRs with JNJ16259685, a mGluR1 antagonist, and MPEP, a mGluR5 antagonist. Blockage of both ionotropic and metabotropic glutamate receptors completely suppresses the excitation and Ca2⫹ increase in mitral cells evoked by receptor axon stimulation and thereby indirect activation of astrocytes (32, 33). The efficacy of the glutamate receptor blockers was demonstrated by the significant reduction of the Ca2⫹ transients to 6.5 ⫾ 1.7% of the control (measured in the absence of the glutamate receptor blockers) in periglomerular interneurons, indicating that in the presence of the glutamate receptor blockers the excitation of postsynaptic neurons and thus indirect effects on astrocytes were almost completely suppressed (n⫽92; Fig. 6D, top trace). In the presence of the glutamate receptor blockers, the Ca2⫹ increase in astrocytes induced by a single

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Figure 6. Ca2⫹ signaling in astrocytes evoked by olfactory nerve stimulation. Olfactory receptor axons in the nerve layer were electrically stimulated. A) Astrocytes respond to a single stimulation pulse and to trains of pulses (50 Hz) of 5 and 15 s duration with Ca2⫹ transients. B) Relative number of astrocytes responding to a single stimulation pulse, a 5-s train of stimulation, and a 15-s train of stimulation with a Ca2⫹ transient. C) TTX blocks stimulation-induced Ca2⫹ transients in astrocytes. D) In the presence of 30 ␮M NBQX, 100 ␮M D-AP5, 5 ␮M JNJ16259685, and 2 ␮M MPEP, stimulation-evoked Ca2⫹ signaling in olfactory bulb interneurons was suppressed (top trace), while receptor axon stimulation induced Ca2⫹ transients in most of the astrocytes (bottom trace). Additional inhibition of P2Y1 receptors with MRS2179 and A2A receptors with ZM241385 entirely blocked stimulation-induced Ca2⫹ transients in all astrocytes. Statistical evaluation of the effect of the combination of NBQX/D-AP5/ JNJ16259685/MPEP (GluR-blocker) and NBQX/D-AP5/JNJ16259685/MPEP with MRS2179 and ZM241385 (GluR-blocker/ MRS/ZM) on the number of astrocytes responding to nerve stimulation (E) and the amplitude of the Ca2⫹ transients (F). G) In the presence of the ionotropic glutamate receptor blocker NBQX/D-AP5 and the purinoceptor blocker MRS/ZM, ⬃58% of the astrocytes still responded to stimulation with a Ca2⫹ increase, presumably due to metabotropic glutamate receptor activation.

stimulation pulse was entirely blocked, suggesting that glutamatergic transmission is requried for astrocytic Ca2⫹ signaling during weak stimulation (n⫽61; Fig. 6E). In contrast, 15 s of stimulation still evoked Ca2⫹ transients in 70% of the astrocytes that responded to stimulation in the absence of the glutamate receptor blockers (n⫽61; Fig. 6D, bottom trace). The amplitude of these Ca2⫹ transients was reduced to 54.8 ⫾ 5.8% of the control (n⫽61; Fig. 6F). When in addition P2Y1 receptors and A2A receptors were blocked by MRS2179 and ZM241385, the stimulation-induced Ca2⫹ transients were blocked in all astrocytes investigated (n⫽34). Our results show that receptor axon stimulation can induce purinergic Ca2⫹ signaling in olfactory bulb astrocytes, and that glutamatergic excitation of postsynaptic neurons is not required for this purinergic signaling. Astrocytes can also be excited by glutamate (32). To test whether activation of mGluRs without activation of ionotropic glutamate receptors and purinoceptors is sufficient to induce Ca2⫹ signaling in astrocytes, we stimulated receptor axons in the presence of NBQX, D-AP5, MRS2179, and ZM241385 (Fig. 6G). Under these conditions, stimulation of receptor axons (50 Hz, 15 s) elicited Ca2⫹ signaling in 58% of the astrocytes (n⫽37) with a mean amplitude of 54.2 ⫾ 8.2%, indiCALCIUM SIGNALING IN OLFACTORY BULB ASTROCYTES

cating that mGluR activation alone is able to induce Ca2⫹ transients in olfactory bulb astrocytes.

DISCUSSION In the present study, we have investigated the role of purinoceptors for calcium signaling in astrocytes of the developing mouse olfactory bulb. Our results demonstrate that astrocytes in the glomerular layer, but not interneurons in the glomerular layer, respond to ATP and ADP with Ca2⫹ release from intracellular stores. The pharmacological profile of the purinoceptors suggests the coexpression of P2Y1 receptors and A2A receptors in most of the astrocytes. The presence of functional purinoceptors in olfactory bulb glial cells and the activation of these receptors on receptor axon stimulation infer a role of ATP and adenosine as neurotransmitters in the mouse olfactory bulb. Our results show that olfactory receptor axon stimulation elicites purinergic Ca2⫹ signaling in astrocytes in the absence of glutamatergic excitation of postsynaptic neurons, suggesting that ATP is released from receptor axon terminals, as a cotransmitter together with glutamate, while adenosine is presumably generated by enzymatic degradation of ATP. 2375

Identification of P2 receptors Evidence for both P2X and P2Y receptors in the olfactory bulb has been reported by immunohistochemical and radioactive ligand binding studies (15– 17). P2Y1 receptor labeling with a radioactive receptor ligand, for example, has been demonstrated not only in the nerve layer, where P2Y1 receptor activation triggers Ca2⫹ signaling in olfactory ensheathing cells (4), but also in cells of the glomerular layer, containing astrocytes and juxtaglomerular interneurons (16). In the present study, we found P2Y1 receptor-mediated Ca2⫹ signaling in olfactory bulb astrocytes but not in juxtaglomerular interneurons. Hence, P2Y1-expressing astrocytes may mainly account for the P2Y1 receptor labeling in the glomerular layer. Anti-P2X2 receptor immunoreactivity was found in periglomerular interneurons (15, 17). P2X2 receptors are Ca2⫹-permeable cation channels; in our experiments, however, P2X receptor ligands such as ATP, ATP-␥-S, and 2MeSATP did not induce a Ca2⫹ influx into periglomerular interneurons. This discrepancy between P2X2 immunoreactivity in periglomerular interneurons and the lack of efficacy of ATP to produce Ca2⫹ signals in these cells may be explained by the difference in the age and species of the animals used for the studies: Kanjhan et al. (17) used adult rats, while in the present study juvenile mice were investigated. In the hippocampus, P2Y1 receptors are present in the astrocytes of juvenile as well as adult rats, while P2Y2 receptors are up-regulated on maturation, as studied by PCR and Ca2⫹ imaging (34). In the present study, however, P2Y2 receptor agonists did not induce a Ca2⫹ rise in astrocytes, and changes in the purinergic signaling during the developmental period investigated here were not found. P2Y1 receptor-mediated Ca2⫹ signaling has also been recorded in glial cells in other brain areas. Ca2⫹ waves in spinal cord astrocytes, for example, are triggered by P2Y1 receptor activation (35), and Ca2⫹ waves mediated by P2Y1 receptors in radial glial cells of the developing neocortex modulate glial cell proliferation (36). Since radial glial cells may be neural progenitors (37), Ca2⫹ waves in radial glial cells have been associated with neurogenesis. In specialized astrocytes of the cerebellum, the Bergmann glial cells, ATP evokes Ca2⫹ signaling presumably via P2Y1 receptor activation (38, 39). Thus, in most astrocytes studied so far, P2Y1 and P2Y2 receptors appear to be the predominant P2Y receptors mediating Ca2⫹ signaling. Olfactory bulb astrocytes express adenosine receptors In the olfactory bulb, three populations of astrocytes can be distinguished by the expression of P2Y receptors and A2A adenosine receptors: the P2Y1 receptor antagonist MRS2179 alone blocked ATP- and ADP-induced Ca2⫹ signaling in only ⬃15% of the cells but was not sufficient to block the Ca2⫹ transients in the remaining cells. However, when A2A receptors were antagonized 2376

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by ZM241385, the ATP- and ADP-mediated Ca2⫹ response was blocked by MRS2179 in all astrocytes, suggesting that during application of ATP and ADP both P2Y1 and A2A receptors are activated. Finally, ZM241385 alone was sufficient to block ATP-induced Ca2⫹ signaling in ⬃15% of the cells. Whether different types of astrocytes, as described in other brain areas (28), are represented by these differences in purinergic signaling is yet not known and needs further investigation. Activation of A2A receptors is most likely due to enzymatic degradation of ATP/ADP to adenosine (26), since A2A receptors were not activated by application of the nonhydrolyzable ATP analog ATP-␥-S or when ATP hydroloysis was blocked by the inhibitor of alkaline phosphatase, levamisole. Ca2⫹ signaling due to adenosine receptor activation has also been described in other types of glial cells. In oligodendrocyte progenitor cells, for example, activation of adenosine receptors has been shown to induce Ca2⫹ signaling, which regulates proliferation and differentiation of the cells (5, 40). Hippocampal astrocytes have been reported to express A2B receptors that are activated by adenosine resulting from ATP degradation, whereas in the same study, P2 receptor activation did not induce Ca2⫹ signaling (8). Often, adenosine mediates its effect on glial intracellular Ca2⫹ signaling by potentiating P2Y receptor-mediated Ca2⫹ release from intracellular stores (41, 42), presumably via adenylate cyclase, the main target of adenosine receptors (2, 43). In the present study, the main effect of adenosine appearently is not the modulation of P2Y1 receptor-mediated Ca2⫹ signaling but direct stimulation of Ca2⫹ release from intracellular stores, because A2A receptor activation elicited Ca2⫹ signaling without P2Y1 receptor activation. A2A receptors in the developing olfactory bulb have been demonstrated by means of radioligand labeling in the glomerular layer and external plexiform layer, both comprising astrocytes (44). In adult rats, the appearance of age-dependent deficits in odor discrimination and odor memory appears to be A2A receptor mediated, since it is prevented by administration of the A2A blocker ZM241385 (45). Whether A2A receptors of olfactory bulb astrocytes are involved in this process is unknown so far and needs further investigation. Besides P2Y1 and A2A receptors, no other purinoceptor subtypes could be identified in astrocytes by the pharmacological tools used here. Except for P2Y1, all P2Y receptors linked to intracellular Ca2⫹ release, P2Y2,4,6,11, are stimulated by UTP and/or UDP (1, 23), which were not effective in the present study. P2Y12 receptors are ADP-preferring receptors and have been described in oligodendrocytes (46) and in microglial cells, where they mediate chemotaxis (47). P2Y12 and P2Y13 receptors have been reported to act via the inhibition of adenylyl cyclase rather than via a signaling cascade that leads to Ca2⫹ release (1, 25). In addition, the P2Y12 receptor antagonist ARC69931MX did not reduce ADP-evoked Ca2⫹ responses. Ca2⫹ influx through P2X receptors does not appear to play a major

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role in the Ca2⫹ signaling investigated in the present study, since Ca2⫹ responses in astrocytes were due entirely to Ca2⫹ release from intracellular stores and were blocked by MRS2179 and ZM241385. Functional roles of purinergic receptors Recently, we have demonstrated release of ATP from receptor axons in the olfactory nerve, leading to P2Y1 receptor-mediated Ca2⫹ signaling in olfactory ensheathing cells (4). In the present study, we could show for the first time that olfactory receptor axon stimulation leads to release of ATP also in the glomeruli, which results in Ca2⫹ signaling in olfactory bulb astrocytes. The results were obtained from juvenile mice of an age of 3– 8 days, a time where astrocytes are involved in the maturation of the olfactory bulb (13). However, more experiments have to be done to examine the role of purinergic Ca2⫹ signaling in astrocytes for neuronal development. In various brain regions, Ca2⫹ signaling has been demonstrated to evoke release of glutamate and ATP from astrocytes, which affects nearby synapses (6, 48 –50) and support Ca2⫹ wave propagation (3, 51). In a recent study performed in the olfactory bulb, mechanical stimulation of astrocytes led to the release not only of glutamate but also of GABA, which induced ionic currents in adjacent mitral cells and granule cells (14). Whether this release of glutamate and GABA is Ca2⫹ dependent is yet not known; however, mechanical stimulation of astrocytes is able to induce cytosolic Ca2⫹ signaling (52, 53). As shown in the present study, purinoceptors are activated during receptor axon stimulation, and ATP is a transmitter activating astrocytes by inducing astrocytic Ca2⫹ signaling. Thereby, ATP may mediate release of “gliotransmitters” from astrocytes in the olfactory bulb, which affects neuronal performance (14), suggesting that purinergic signaling exerts its influence on the information processing in the neuronal network within a glomerulus via astrocytes.

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We thank Anne Rieger (Kaiserslautern, Germany) for providing the GFAP antibody staining. The German Research Council (DFG) is acknowledged for financial support (SFB 530/TP-B1 and LO779/2). We gratefully thank The Medicines Company (Waltham, MA, USA) for supplying ARC69931MX.

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Received for publication November 9, 2007. Accepted for publication January 31, 2008.

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