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Proc Natl Acad Sci U S A. Oct 13, 2009; 106(41): 17570–17575.
Published online Sep 30, 2009. doi:  10.1073/pnas.0809513106
PMCID: PMC2765163
Neuroscience

GABA uptake-dependent Ca2+ signaling in developing olfactory bulb astrocytes

Abstract

We studied GABAergic signaling in astrocytes of olfactory bulb slices using confocal Ca2+ imaging and two-photon Na+ imaging. GABA evoked Ca2+ transients in astrocytes that persisted in the presence of GABAA and GABAB receptor antagonists, but were suppressed by inhibition of GABA uptake by SNAP 5114. Withdrawal of external Ca2+ blocked GABA-induced Ca2+ transients, and depletion of Ca2+ stores with cyclopiazonic acid reduced Ca2+ transients by approximately 90%. This indicates that the Ca2+ transients depend on external Ca2+, but are mainly mediated by intracellular Ca2+ release, conforming with Ca2+-induced Ca2+ release. Inhibition of ryanodine receptors did not affect GABA-induced Ca2+ transients, whereas the InsP3 receptor blocker 2-APB inhibited the Ca2+ transients. GABA also induced Na+ increases in astrocytes, potentially reducing Na+/Ca2+ exchange. To test whether reduction of Na+/Ca2+ exchange induces Ca2+ signaling, we inhibited Na+/Ca2+ exchange with KB-R7943, which mimicked GABA-induced Ca2+ transients. Endogenous GABA release from neurons, activated by stimulation of afferent axons or NMDA application, also triggered Ca2+ transients in astrocytes. The significance of GABAergic Ca2+ signaling in astrocytes for control of blood flow is demonstrated by SNAP 5114-sensitive constriction of blood vessels accompanying GABA uptake. The results suggest that GABAergic signaling is composed of GABA uptake-mediated Na+ rises that reduce Na+/Ca2+ exchange, thereby leading to a Ca2+ increase sufficient to trigger Ca2+-induced Ca2+ release via InsP3 receptors. Hence, GABA transporters not only remove GABA from the extracellular space, but may also contribute to intracellular signaling and astrocyte function, such as control of blood flow.

Keywords: calcium-induced calcium release, GABA transporter, Neuron-glia interaction, sodium imaging, vasoconstriction

Glial cells are electrically inexcitable cells in the nervous system and have long been considered to be supporting cells with little direct impact on neuronal performance. It was only in the last decade when it was recognized that astrocytes, the major class of glial cells in the mammalian brain, participate in synaptic transmission and contribute to information processing. This led to the model of the “tripartite synapse” consisting of pre- and postsynaptic neuronal elements and glial processes (1). Astrocytes detect neuronal release of excitatory neurotransmitters such as glutamate and acetylcholine via G protein-coupled receptors and respond with cytosolic Ca2+ signaling (24). Astrocytes can then release “gliotransmitters” such as glutamate, D-serine and arachidonic acid in a Ca2+-dependent manner, thus modulating neuronal performance and local cerebrovascular blood flow (58). While most studies describe the integration of astrocytes in excitatory neuronal networks in great detail (reviewed in ref. 1), only little information exists about how astrocytes are affected by inhibitory neurotransmitters such as GABA and glycine. GABAA and GABAB receptor-mediated Ca2+ signaling has been reported in hippocampal astrocytes (911), where GABAB receptors mediate heterosynaptic depression (10). Only a minority of hippocampal astrocytes, however, respond to GABAB receptor activation with Ca2+ increases (11), raising the question how astrocytes are integrated in inhibitory neuronal networks.

Besides GABA receptors, astrocytes express GABA transporters (GATs), which significantly contribute to the clearance of GABA molecules from the synaptic cleft (12), but have not yet been considered to directly participate in neuronal signal processing. Glial GATs are also involved in pathological processes in the brain, for example, in patients with temporal lobe epilepsy, where astrocytic expression of GATs is increased (13), and inhibitors of glial GATs have anticonvulsant effects (14), indicating the involvement of glial GATs in the generation of epileptic seizures. It is not known, however, how glial GATs contribute to epileptic seizures.

In this study, we have investigated the role of GATs for neuron-glia signaling. We have chosen the olfactory bulb for this study, because astrocytic processes ensheath dendrodendritic, mostly GABAergic synapses (15, 16). Our results indicate a principle of GABAergic signaling between neurons and glial cells mediated by GATs. Activation of GABA uptake elicited an intracellular Na+ rise in olfactory bulb astrocytes. Our results suggest that this Na+ rise reduced Na+/Ca2+ exchange, thereby leading to a Ca2+ increase sufficient to trigger Ca2+-induced Ca2+ release via InsP3 receptors. Thus, in addition to clearance of GABA from the synaptic cleft, GABA uptake might serve as a mediator for neuron-glia signaling.

Results

GABAA Receptors Mediate Ca2+ Signaling in Neurons But Not in Astrocytes.

In brain slices of 2- to 7-day-old mice (P2–7), application of GABA (300 μM) evoked Ca2+ transients in 69% of olfactory bulb astrocytes (n = 899 cells/39 slices/29 animals) and 75% of periglomerular neurons (n = 261/10/6), with mean amplitudes of 128.7 ± 2.5% ΔF (n = 621/39/29) and 76.1 ± 3.2% ΔF (n = 195/10/6), respectively (Fig. 1A). In 3-week-old animals (P18–22), the fraction of responsive cells decreased to 34% of astrocytes (n = 47/8/4) and 29% of neurons (n = 306/8/3), with amplitudes of 76.8 ± 14.5% ΔF (n = 16/8/4) and 81.7 ± 5.4% ΔF (n = 88/8/3) (Fig. S1), indicating that GABAergic Ca2+ signaling in astrocytes and neurons is developmentally regulated.

Fig. 1.
GABA-mediated Ca2+ signaling in olfactory bulb astrocytes. (A) The GABAA receptor antagonist gabazine (60 μM) blocked Ca2+ transients induced by GABA (300 μM) in neurons (lower trace), but not in astrocytes (upper trace) at P2–7. ...

We further studied the mechanism underlying GABA-induced Ca2+ signaling using mice at an age of P2–7. In addition to GABA-evoked Ca2+ signaling, spontaneous Ca2+ transients or Ca2+ oscillations could also be measured in 68% of the astrocytes (n = 115/2/2; Fig. 1A, asterisks) and 25% of the periglomerular neurons (n = 56/2/2). If spontaneous Ca2+ signaling occurred shortly before the application of GABA, cells were not used for analysis.

Blocking GABAA receptors with gabazine suppressed GABA-induced Ca2+ transients in periglomerular neurons (n = 132/7/6; P < 0.005), but had no effect on the amplitude of Ca2+ transients in astrocytes (n = 28/5/4; Fig. 1 A and F). The GABAA receptor channel blocker picrotoxin (100 μM) reduced neuronal GABA-evoked Ca2+ transient amplitudes to 34.2 ± 4.1% (n = 49/3/2; P < 0.005), whereas astrocytic Ca2+ transients remained unaffected (n = 18/3/2; Fig. 1F). The GABAA receptor agonist muscimol (300 μM) evoked Ca2+ transients in all periglomerular neurons that responded to GABA (n = 77/4/3), but only in 18% of the GABA-sensitive astrocytes (n = 51/4/3; Fig. 1E), indicating that olfactory bulb astrocytes employ an additional pathway for GABA-mediated Ca2+ rises that neurons lack. This is supported by the delay of the Ca2+ response of 79.7 ± 3.4 s in astrocytes (n = 256/14/8) with respect to the Ca2+ response in periglomerular neurons (Fig. S2). In addition, the GABA sensitivity of astrocytes and neurons is strikingly different. The apparent EC50 value of the GABA-induced Ca2+ response, as calculated from measurements from at least three individual animals per data point, was 2.2 μM (n = 52–172 cells) in neurons and 100.9 μM (n = 15–124 cells) in astrocytes (Fig. 1B).

Application of baclofen (300 μM), a specific agonist of GABAB receptors, evoked Ca2+ transients in 39% of the astrocytes that responded to GABA (n = 80/7/6; Fig. 1 C and E). The GABAB antagonist CGP52432 (10 μM) entirely blocked the baclofen-induced Ca2+ transients (P < 0.005), but reduced the amplitude of GABA-induced Ca2+ transients in astrocytes by only 24 ± 4% (n = 29/3/3; P < 0.005; Fig. 1F). Hence, in the majority of astrocytes, GABA-induced Ca2+ signaling cannot be explained by GABAB receptor activation.

The results show that GABA receptors mediate Ca2+ signaling only in a minority of olfactory bulb astrocytes. They also argue against the possibility that Ca2+ transients in astrocytes depend on GABA-driven activity of neurons or any other neuronal activity, since inhibiting action potential firing with TTX (2 μM) only marginally reduced GABA-induced Ca2+ transient amplitudes in astrocytes by 9.5 ± 4.5% (n = 16/3/3; P = 0.011; Fig. 1D), and gabazine blocked Ca2+ responses in periglomerular neurons, but not in astrocytes. In neurons, GABA-induced Ca2+ transients were also suppressed in Ca2+-free solution (n = 86/4/3; P < 0.005) and in the presence of the voltage-gated Ca2+ channel blocker diltiazem (400 μM; n = 55/2/2; P < 0.005), suggesting Ca2+ influx via voltage-gated Ca2+ channels after GABA-evoked depolarization (Fig. S3).

Ca2+ Signaling Mediated by GABA Uptake.

Since GABA receptor antagonists only weakly reduced GABA-induced Ca2+ transients in olfactory bulb astrocytes, we tested the contribution of GATs to GABAergic signaling in astrocytes. Application of the GABA transporter substrate nipecotic acid at concentrations of 50 and 100 μM mimicked the effect of GABA and evoked Ca2+ transients in 54% and 68% of the GABA-sensitive astrocytes with amplitudes of 165.3 ± 5.8% ΔF and 167.1 ± 5.9% ΔF (n = 100/4/3), respectively (Fig. 2A). Next, we tested the effect of gabazine (60 μM) and CGP52432 (10 μM) on Ca2+ transients evoked by nipecotic acid. To prevent epileptiform activity of olfactory bulb neurons upon gabazine treatment, we added the glutamate receptor antagonists NBQX (30 μM) and D-AP5 (100 μM; Fig. 2A). In the presence of inhibitors of GABA receptors and ionotropic glutamate receptors, the Ca2+ transients evoked by 300 μM GABA and 100 μM nipecotic acid were reduced by 34.3 ± 3.8% (n = 116/7/6; P < 0.005) and 11.1 ± 7.2% (n = 23/2/2; P = 0.018), respectively (Fig. 2A). All of the following experiments were performed in the presence of the GABA receptor blockers and, unless otherwise noted, glutamate receptor blockers, to investigate GABA-induced Ca2+ signaling independent of GABA receptor activation.

Fig. 2.
GABA uptake mediates Ca2+ signaling in olfactory bulb astrocytes. (A) The GABA transporter substrate nipecotic acid (NPA) induced Ca2+ transients similar to GABA-induced Ca2+ transients. The Ca2+ transients were not blocked by GABA receptor antagonists ...

GATs are localized in the rat olfactory bulb, where neurons express GAT1 and astrocytes express GAT3 (17), which corresponds to the murine transporter mGAT4. The involvement of astrocytic GATs in GABA-induced Ca2+ signaling was tested by using the non-competitive mGAT4 inhibitor, SNAP 5114 (18). Incubation of the slice in 100 μM SNAP 5114 resulted in Ca2+ oscillations in most astrocytes, which made it difficult to distinguish between Ca2+ oscillations and putative GABA-induced Ca2+ transients in single cells (Fig. 2B, upper trace). Therefore, we analyzed the averaged signal derived from all astrocytes that responded to GABA under control conditions within a given slice preparation, thus reducing the randomly occurring Ca2+ oscillations, but conserving the Ca2+ transients evoked by GABA (Fig. 2B, lower trace). GABA-evoked Ca2+ responses were strongly reduced by 70.8 ± 1.5% (n = 60/5/5; P < 0.005) in the presence of SNAP 5114, indicating that GABA transporters are involved in mediating Ca2+ signaling in olfactory bulb astrocytes. The effect of SNAP 5114 on GABA-induced Ca2+ signaling was only partly reversible after washout for 30–40 min. The GAT1 inhibitor NNC 711 did not significantly reduce GABA-evoked Ca2+ transients (n = 24/3/3; Fig. S4). Hence, GABA-induced Ca2+ transients in olfactory bulb astrocytes are mediated by mGAT4, but not by GAT1.

Mechanism of GABA Uptake-Mediated Ca2+ Signaling.

GABA transporters use the inwardly directed Na+ gradient as the driving force (19), but do not cotransport Ca2+. This raises the question how GABA uptake is linked to cytosolic Ca2+ rises. We first investigated the source of Ca2+ to reveal the mechanism underlying GABA uptake-induced Ca2+ signaling in olfactory bulb astrocytes. Ca2+ transients evoked by GABA were entirely and reversibly suppressed in the absence of external Ca2+, indicating that Ca2+ influx is necessary for GABA-induced Ca2+ signaling (n = 33/4/3; P < 0.005; Fig. 3A). Upon re-addition of external Ca2+, a slow increase in intracellular Ca2+ was measured (Fig. 3A, arrowhead). On top of this slow Ca2+ increase, Ca2+ transients with a time course similar to GABA-induced Ca2+ transients were recorded. When intracellular Ca2+ stores were depleted by the endoplasmic Ca2+-ATPase inhibitor cyclopiazonic acid (CPA; 25 μM), GABA-induced Ca2+ transients were also largely reduced, on average by 87.2 ± 1.5% (n = 87/4/3; P < 0.005; Fig. 3B). Ca2+ store depletion by CPA resulted in an elevated baseline Ca2+ concentration, presumably due to store-operated Ca2+ entry (20). The results demonstrate that GABA-induced Ca2+ transients in olfactory bulb astrocytes require Ca2+ release from intracellular stores, but also depend on Ca2+ influx from the extracellular space.

Fig. 3.
GABA-induced Ca2+ signaling depends on external and internal sources and can be mimicked by NCX inhibition. All experiments were performed in the presence of GABA and glutamate receptor antagonists (gabazine, CGP52432, NBQX, D-AP5). (A) Withdrawal of ...

GABA-induced Ca2+ increases might result from reduced Na+/Ca2+ exchanger (NCX) efficacy due to elevated intracellular Na+ concentrations upon GABA uptake, which would lead to a reduced driving force for the NCX. To test whether GABA uptake indeed evoked significant Na+ rises, we measured changes in the Na+ concentration by two-photon imaging using the Na+ indicator SBFI (21). Application of 300 μM and 600 μM GABA evoked Na+ rises of 2.2 ± 0.1 mM (n = 74/3/2) and 3.2 ± 0.1 mM (n = 188/6/3), respectively, in the cell bodies (Fig. 3C), which corresponds to transporter-mediated Na+ transients in other glial cells (22). Larger Na+ transients are expected in the fine astrocyte processes due to the larger surface-to-volume ratio (22), but the detector noise rendered SBFI fluorescence recordings impossible in astrocytic processes. Concentrations of 100 μM and 600 μM nipecotic acid evoked Na+ rises with an amplitude of 1.8+/−0.1 mM (n = 47/3/2) and 2.3+/−0.1 mM (n = 84/3/2), respectively. SNAP 5114 (100 μM) entirely blocked GABA-induced Na+ transients (n = 240/6/4), indicating that they were mediated by GABA uptake via mGAT4 (Fig. 3D). To test whether activation of Na+-driven transporters were sufficient to evoke Ca2+ signaling in astrocytes, we applied L-asparagine, which is a substrate for the glial Na+-dependent neutral amino acid transporter SNAT3 (system N; 23). Asparagine has no significant effect on neurotransmitter receptors or Ca2+ release mechanisms, but increases intracellular Na+ when cotransported with Na+ by SNATs (23). Application of asparagine evoked Ca2+ transients comparable to GABA-induced Ca2+ transients in a dose-dependent manner (Fig. 3E). One hundred micromolar asparagine evoked Ca2+ transients in 25% (n = 16/2/2) of the astrocytes, whereas 300 μM and 1 mM asparagine evoked Ca2+ transients in 28% (n = 53/4/4) and 67% (n = 58/5/5) of GABA-sensitive astrocytes, respectively (Fig. 3D). The mean amplitudes of the asparagine-induced Ca2+ transients were 11.8 ± 5.2% ΔF (100 μM), 29.4 ± 5.8% ΔF (300 μM), and 71.8 ± 6.4% ΔF (1 mM). The mean amplitudes were significantly different from each other (P < 0.005, unpaired t test). The results are in line with the assumption that Na+ rises due to transporter activity in astrocytes are sufficient to induce Ca2+ signaling.

If Na+ rises induce Ca2+ transients in astrocytes by reducing NCX activity, pharmacological inhibition of NCX should mimic GABA-induced Ca2+ transients. Therefore, we checked whether reduced NCX activity can evoke Ca2+ transients by using the NCX inhibitor KB-R7943. Application of 50 μM KB-R7943 for 30 s and 60 s resulted in Ca2+ transients similar to GABA-induced Ca2+ transients in 58% and 82% of all astrocytes, with a mean amplitude of 105.3 ± 4.8% ΔF (n = 93/6/4) and 106.9 ± 6.5% ΔF (n = 45/4/4), respectively (Fig. 3F). Thus, inhibition of NCX activity can mimic GABA-induced Ca2+ transients, suggesting that reduced NCX activity mediates Ca2+ signaling evoked by GABA uptake into olfactory bulb astrocytes.

InsP3 Receptors Mediate Ca2+-Induced Ca2+ Release.

GABA-induced Ca2+ transients in olfactory bulb astrocytes appear to require a small Ca2+ increase dependent on external Ca2+, but are mainly attributable to Ca2+ release from intracellular stores, in line with Ca2+-induced Ca2+ release (CICR). Often, CICR is mediated by ryanodine receptors of the endoplasmic reticulum, but it has also been reported that inositol (1, 4, 5)-trisphosphate (InsP3) receptors can be stimulated by increases in cytosolic Ca2+ (24, 25). Ryanodine receptors can be blocked by ryanodine at concentrations >10 μM and by ruthenium red (26). In the present study, neither 100 μM ryanodine (n = 39/3/3) nor 10 μM ruthenium red (n = 21/3/2) reduced GABA-induced Ca2+ signaling (Fig. 4 A and B). These results suggest that ryanodine receptors were not involved in Ca2+ increases evoked by GABA.

Fig. 4.
InsP3 receptors, but not ryanodine receptors, mediate GABA-induced Ca2+ signaling in olfactory bulb astrocytes. (A) Ryanodine (100 μM) had no effect on GABA-induced Ca2+ transients. (B) Ruthenium red (10 μM) had no effect on Ca2+ transients ...

InsP3 receptors can be blocked by 2-APB at concentrations of several hundred micromolar (27, 28). However, 2-APB can also act on store-operated Ca2+ channels and transient receptor potential (TRP) channels (20, 29), thereby either blocking the channels (some TRPCs and TRPMs) or activating the channels (some TRPVs). Application of 600 μM 2-APB resulted in Ca2+ oscillations in olfactory bulb astrocytes, suggesting activation of TRP channels. Addition of 10 μM ruthenium red, which also acts as a TRPV inhibitor (30), suppressed the generation of Ca2+ oscillations, although an initial Ca2+ transient was elicited frequently upon incubation with 2-APB/ruthenium red (Fig. 4C). Under these conditions, GABA as well as ATP, which was used as control for InsP3-mediated Ca2+ release, failed to induce Ca2+ transients in astrocytes (n = 64/4/3; P < 0.005). Baseline Ca2+ slightly increased upon application of 2-APB, presumably due to Ca2+ leakage from internal stores (28). These results suggest that GABA uptake induces Ca2+ signaling that is mediated by 2-APB-sensitive InsP3 receptors.

Endogenous GABA Release Triggers Ca2+ Signaling in Astrocytes.

We asked whether endogenous GABA release after stimulation of GABAergic neurons by either NMDA/kainate application or electrical stimulation of receptor axons would activate astrocytic Ca2+ signaling. NMDA (100 μM) and kainate (100 μM) evoked Ca2+ transients in periglomerular neurons (n = 88/5/4) and astrocytes (n = 201/5/4; Fig. 5A). The V-ATPase inhibitor bafilomycin A1 suppressed NMDA-induced astrocytic Ca2+ transients (P < 0.005, unpaired t test), while ATP- and kainate-induced Ca2+ transients were not blocked (n = 47/3/2; Fig. 5B). Hence, kainate appears to induce Ca2+ transients in astrocytes directly, presumably via Ca2+-permeable AMPA/kainate receptors, as shown for other glial cells (31, 32). NMDA, in contrast, triggered vesicular neurotransmitter release from neurons and subsequent activation of astrocytes. To suppress Ca2+ signaling in astrocytes that was mediated by neurotransmitter receptors during NMDA application and receptor axon stimulation, we blocked GABA receptors, metabotropic glutamate receptors, AMPA/kainate receptors, P2Y1 receptors and A2A receptors (Fig. 5 C and D). Under these conditions, NMDA and receptor axon stimulation still evoked Ca2+ transients in astrocytes, which were significantly reduced by 56 ± 3% (n = 119/3/2; P < 0.005) and 51.8 ± 2.7% (n = 63/3/3; P < 0.005), respectively, by the mGAT4 inhibitor SNAP 5114, suggesting that GABA released by periglomerular neurons induced Ca2+ transients in astrocytes via mGAT4. In the presence of SNAP 5114, NMDA-evoked Ca2+ transients in neurons were not significantly reduced (n = 74/4/3), while stimulation-evoked Ca2+ transients in neurons were reduced by 13.4 ± 2.1% (n = 78/3/3; P = 0.005).

Fig. 5.
Endogenous GABA release triggers Ca2+ signaling in astrocytes. (A) Ca2+ transients evoked by ATP (30 μM), NMDA (100 μM) and kainate (100 μM) in neurons (upper trace) and astrocytes (lower trace). (B) Suppressing vesicular neurotransmitter ...

We also measured Ca2+ changes in neurons and astrocytes during synchronous (epileptiform) discharges of neurons induced by disinhibition using gabazine (20 μM) in the presence of glial receptor blockers (Fig. 5E). The Ca2+ transients had an average amplitude of 202 ± 13.8% ΔF (n = 66/4/4) in neurons and 141.9 ± 8.4% ΔF (n = 91/4/4) in astrocytes. SNAP 5114 reduced the amplitude of the Ca2+ transients by 16.9 ± 2.7% in neurons (n = 66/4/4); P < 0.005) and by 53.4 ± 2.0% in astrocytes (n = 91/4/4; P < 0.005).

Astrocytic GABA Uptake Triggers Vasoconstriction.

Ca2+ transients in astrocytes have been shown to evoke both vasoconstriction and vasodilation, depending on the metabolic state of the tissue (8, 33, 34). In the olfactory bulb, blood vessels that penetrate the nerve layer and enter the glomerular layer are densely covered by astrocytic processes as assessed by GFAP staining (Fig. 6 A and B). In contrast, only weak GFAP staining was detected around capillaries surrounding glomeruli. In a preparation of the intact olfactory bulb, we measured changes in the diameter of vessels in the nerve layer upon application of GABA in the presence of GABA receptor blockers and TTX. Because the intact pia mater and the nerve layer provide a major diffusion barrier (Fig. S5), we applied 3 mM GABA (1 min) to stimulate GABA uptake into astrocytes in deeper layers. After application of GABA, a constriction of blood vessels in the nerve layer was recorded (Fig. 6 C and D and Movie S1). On average, the diameter of blood vessels significantly decreased by 17.3 ± 2.3% (n = 8 preparations; P < 0.005; Fig. 6E). SNAP 5114 significantly reduced the GABA-induced vasoconstriction to 3.2 ± 0.7% (n = 8; P < 0.005).

Fig. 6.
Glial GABA uptake triggers vasoconstriction. (A) Immunostaining against GFAP (green), mouse IgG (blood vessels, red), and nuclear staining (Hoechst 33342, blue) in the nerve layer (NL) and glomerular layer (GL). (Scale bar, 40 μm.) (B) Area indicated ...

Discussion

In the present study, we have investigated GABA-induced Ca2+ signaling in olfactory bulb astrocytes and found a GAT-mediated Ca2+-signaling mechanism. GABA-induced Ca2+ transients were independent of GABA receptors, but were a consequence of Na+-driven GABA uptake. They could be mimicked by inhibition of Na+/Ca2+ exchange and depended on both extracellular Ca2+ and InsP3 receptor-mediated intracellular Ca2+ release. This suggests that GABA uptake-evoked cytosolic Na+ transients reduce Na+/Ca2+ exchange, leading to Ca2+ rises in astrocytes, which trigger Ca2+-induced Ca2+ release via InsP3 receptors (Fig. S6). This study demonstrates that GATs not only take up GABA, but also mediate intracellular signaling in astrocytes.

In immature neurons, which maintain a high intracellular Cl concentration, GABAA receptor activation can lead to depolarization and thereby Ca2+ influx via voltage-gated Ca2+ channels (35, 36). We studied olfactory bulbs of mice of postnatal days 2–7. At this age, mitral cells already maintain a low intracellular chloride concentration and are inhibited by GABA, whereas olfactory bulb granule cells are depolarized upon GABAA receptor activation and display Ca2+ influx via voltage-gated Ca2+ channels (37). Our results show that in the majority of periglomerular neurons, activation of GABAA receptors triggers Ca2+ influx via voltage-gated Ca2+ channels. In olfactory bulb astrocytes, GABAA receptors appear not to play a predominant role in Ca2+ signaling, which is in contrast to hippocampal astrocytes (11). The presence of voltage-gated Ca2+ channels in astrocytes is controversial, since Carmignoto et al. (38) failed to detect voltage-dependent Ca2+ signaling in hippocampal astrocytes. In olfactory bulb astrocytes, voltage-gated Ca2+ channels appear not to play a major role in Ca2+ signaling, since high K+ evoked much smaller Ca2+ transients in astrocytes as compared to neurons (Fig. S7). The GABA transporter substrate nipecotic acid was able to mimic the effect of GABA, and the mGAT4 inhibitor SNAP 5114 reduced GABA-induced Ca2+ transients, suggesting GABA uptake to be the mechanism underlying GABA-induced Ca2+ signaling.

Our results suggest that GABA uptake-mediated Na+ rises trigger Ca2+ signaling in astrocytes by reducing NCX efficacy. It has been shown that inhibition of NCX can result in increased Ca2+ concentrations in other cells, including glial cells (39). In olfactory bulb astrocytes, this increase in the cytosolic Ca2+ concentration depended on extracellular Ca2+. However, the major fraction of the GABA-induced Ca2+ transient was dependent on Ca2+ release from intracellular Ca2+ stores, and GABA evoked only small Ca2+ increases after Ca2+ stores had been depleted with CPA. Small Ca2+ increases as evoked by GABA in the presence of CPA can trigger Ca2+-induced Ca2+ release (CICR) from intracellular stores. This mechanism is different from the mechanism in NG2 cells, in which GABAA receptor activation leads to depolarization, Na+ influx through persistent Na+ channels and subsequent reversal of NCX, while CICR has not been reported in NG2 cells (40).

The mechanism presented in this study requires high concentrations of GABA, since the Ca2+ response saturated only at GABA concentrations in the millimolar range. Millimolar concentrations of GABA are expected in the vicinity of the synaptic cleft (36), where astrocytic processes, together with pre- and postsynaptic neuronal elements, contribute to the “tripartite synapse” (1). In the olfactory bulb, the neuropil of the glomeruli is strongly pervaded by processes of astrocytes (41), which ensheath dendrodendritic, putative GABAergic, synapses (15, 16). Thus, olfactory bulb astrocytes may detect GABA directly at the synaptic cleft and, as shown in the present study, respond to GABA released from periglomerular neurons and possibly granule cells with Ca2+ signaling. Strong stimulation of neurons triggered a robust global Ca2+ signal in the entire astrocyte. Under more physiological conditions such as odorant stimulation, local Ca2+ signals might be smaller and may be restricted to astrocyte processes ensheathing GABAergic synapses. A global Ca2+ signal could be evoked under pathophysiological conditions such as synchronized neuronal discharges occurring during epileptiform activity. Due to the GABA uptake-dependent mechanism, astrocytes would be able to respond not only to glutamatergic, but also to GABAergic neurotransmission with Ca2+ signaling, which might contribute to control of blood flow by vasodilation (34) and vasoconstriction (this study) and, hence, affect neuronal performance.

Materials and Methods

Solutions and Slice Preparation.

The standard artificial cerebrospinal fluid (aCSF) for acute brain slices contained (in mM): NaCl 125, KCl 2.5, CaCl2 2, MgCl2 1, D-glucose 25, NaHCO3 26, NaH2PO4 1.25, and L-lactate 0.5, 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 Ca2+-free saline, Ca2+ was replaced by 2 mM Mg2+, and 0.5 mM EGTA was added. In saline with altered sodium concentrations used for SBFl calibration (Fig. S8), K+-free saline (0K+) or high-K+ solution (50 mM K+, 50K+), KCl was exchanged by/for NaCl. Olfactory bulb slices were prepared from NMRI mice (P2–P22) as described before (42).

Confocal Ca2+ Imaging.

Acute brain slices were incubated in the dark at room temperature (21–24 °C) for 60 min in Ca2+-reduced aCSF containing 2 μM Fluo-4-AM, which labels glial cells and periglomerular neurons (41). Ca2+ changes in cells of an acute brain slice were measured with confocal laser scanning microscopes (Zeiss LSM 510 and Nikon eC1 plus). To measure Ca2+ dynamics, images were acquired in one focal plane at 0.3 Hz. Ca2+ signaling was evoked by either drug application or electrical stimulation of olfactory receptor axons using a bipolar tungsten electrode (MPI) connected to a stimulator (SD9K, Grass) and inserted into the olfactory nerve layer.

Data Analysis and Statistics.

For analysis of time series, Fluo-4-stained cell bodies were defined as regions of interest (ROIs), and the fluorescence intensity was measured in each ROI. Only cells of the glomerular layer and the external plexiform layer were chosen. Ca2+ changes are given as relative fluorescence changes (ΔF) with respect to the resting fluorescence that was normalized to 100%. Only cells that responded with an amplitude of at least 3% ΔF were considered as responsive. Measurements are given as mean values ± SEM, with n giving the number of cells/number of slices/number of animals tested. If not stated otherwise, significance of statistical difference was calculated using Student's paired t test, with P < 0.05.

Further information is given in the SI Text.

Supplementary Material

Supporting Information:

Acknowledgments.

We thank P. Meuth (Münster) for software programming, F. Barros (Valdivia, Chile) for helpful discussions, and K. Kaila (Helsinki, Finland) for critical comments on an earlier version of the manuscript. This work was funded by the Deutsche Forschungsgemeinschaft (LO 779/3 and SFB 530, TP B1) and Interdisziplinäres Zentrum für Klinische Forschung Münster (IZKF-FG6).

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

This article contains supporting information online at www.pnas.org/cgi/content/full/0809513106/DCSupplemental.

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