GABA uptake-dependent Ca2؉ signaling in developing olfactory bulb

Michael Doengia,b,c, Daniela Hirnetb,c, Philippe Coulonc, Hans-Christian Papec, Joachim W. Deitmera, and Christian Lohra,b,c,1

aAbteilung fu¨r Allgemeine Zoologie, Universita¨t Kaiserslautern, Postfach 3049, D-67653 Kaiserslautern, Germany; and bInterdisziplina¨res Zentrum fu¨r Klinische Forschung and cInstitut fu¨r Physiologie I, Westfa¨lische Wilhelms-Universita¨t Mu¨ nster, D-48149 Mu¨nster, Germany

Edited by Tullio Pozzan, University of Padua, Padua, Italy, and approved August 21, 2009 (received for review November 25, 2008) We studied GABAergic signaling in astrocytes of olfactory bulb slices (11), raising the question how astrocytes are integrated in .using confocal Ca2؉ imaging and two-photon Na؉ imaging. GABA inhibitory neuronal networks evoked Ca2؉ transients in astrocytes that persisted in the presence of Besides GABA receptors, astrocytes express GABA transporters GABAA and GABAB antagonists, but were suppressed by (GATs), which significantly contribute to the clearance of GABA inhibition of GABA uptake by SNAP 5114. Withdrawal of external molecules from the synaptic cleft (12), but have not yet been .Ca2؉ blocked GABA-induced Ca2؉ transients, and depletion of Ca2؉ considered to directly participate in neuronal signal processing ,stores with cyclopiazonic acid reduced Ca2؉ transients by approxi- Glial GATs are also involved in pathological processes in the -mately 90%. This indicates that the Ca2؉ transients depend on for example, in patients with temporal lobe , where astro external Ca2؉, but are mainly mediated by intracellular Ca2؉ release, cytic expression of GATs is increased (13), and inhibitors of glial conforming with Ca2؉-induced Ca2؉ release. Inhibition of ryanodine GATs have effects (14), indicating the involvement ,receptors did not affect GABA-induced Ca2؉ transients, whereas the of glial GATs in the generation of epileptic . It is not known 2؉ InsP3 receptor blocker 2-APB inhibited the Ca transients. GABA also however, how glial GATs contribute to epileptic seizures. -induced Na؉ increases in astrocytes, potentially reducing Na؉/Ca2؉ In this study, we have investigated the role of GATs for ,exchange. To test whether reduction of Na؉/Ca2؉ exchange induces signaling. We have chosen the olfactory bulb for this study Ca2؉ signaling, we inhibited Na؉/Ca2؉ exchange with KB-R7943, because astrocytic processes ensheath dendrodendritic, mostly NEUROSCIENCE which mimicked GABA-induced Ca2؉ transients. Endogenous GABA GABAergic (15, 16). Our results indicate a principle of GABAergic signaling between and glial cells mediated by release from neurons, activated by stimulation of afferent axons or ϩ 2؉ GATs. Activation of GABA uptake elicited an intracellular Na NMDA application, also triggered Ca transients in astrocytes. The ϩ significance of GABAergic Ca2؉ signaling in astrocytes for control of rise in olfactory bulb astrocytes. Our results suggest that this Na rise reduced Naϩ/Ca2ϩ exchange, thereby leading to a Ca2ϩ in- blood flow is demonstrated by SNAP 5114-sensitive constriction of crease sufficient to trigger Ca2ϩ-induced Ca2ϩ release via InsP blood vessels accompanying GABA uptake. The results suggest that 3 receptors. Thus, in addition to clearance of GABA from the GABAergic signaling is composed of GABA uptake-mediated Na؉ rises synaptic cleft, GABA uptake might serve as a mediator for neuron- that reduce Na؉/Ca2؉ exchange, thereby leading to a Ca2؉ increase .2؉ 2؉ glia signaling sufficient to trigger Ca -induced Ca release via InsP3 receptors. Hence, GABA transporters not only remove GABA from the extracel- Results lular space, but may also contribute to intracellular signaling and 2؉ GABAA Receptors Mediate Ca Signaling in Neurons But Not in function, such as control of blood flow. Astrocytes. In brain slices of 2- to 7-day-old mice (P2–7), application of GABA (300 ␮M) evoked Ca2ϩ transients in 69% of olfactory ͉ ͉ calcium-induced calcium release GABA transporter bulb astrocytes (n ϭ 899 cells/39 slices/29 animals) and 75% of Neuron-glia interaction ͉ sodium imaging ͉ vasoconstriction periglomerular neurons (n ϭ 261/10/6), with mean amplitudes of 128.7 Ϯ 2.5% ⌬F(n ϭ 621/39/29) and 76.1 Ϯ 3.2% ⌬F(n ϭ lial cells are electrically inexcitable cells in the nervous 195/10/6), respectively (Fig. 1A). In 3-week-old animals (P18–22), Gsystem and have long been considered to be supporting cells the fraction of responsive cells decreased to 34% of astrocytes (n ϭ with little direct impact on neuronal performance. It was only in 47/8/4) and 29% of neurons (n ϭ 306/8/3), with amplitudes of 76.8 Ϯ the last decade when it was recognized that astrocytes, the major 14.5% ⌬F(n ϭ 16/8/4) and 81.7 Ϯ 5.4% ⌬F(n ϭ 88/8/3) (Fig. S1), ϩ class of glial cells in the mammalian brain, participate in synaptic indicating that GABAergic Ca2 signaling in astrocytes and neu- transmission and contribute to information processing. This led rons is developmentally regulated. to the model of the ‘‘tripartite ’’ consisting of pre- and We further studied the mechanism underlying GABA-induced ϩ postsynaptic neuronal elements and glial processes (1). Astro- Ca2 signaling using mice at an age of P2–7. In addition to ϩ ϩ cytes detect neuronal release of excitatory GABA-evoked Ca2 signaling, spontaneous Ca2 transients or 2ϩ such as glutamate and acetylcholine via G protein-coupled Ca oscillations could also be measured in 68% of the astrocytes ϩ ϭ receptors and respond with cytosolic Ca2 signaling (2–4). (n 115/2/2; Fig. 1A, asterisks) and 25% of the periglomerular ϭ 2ϩ Astrocytes can then release ‘‘gliotransmitters’’ such as gluta- neurons (n 56/2/2). If spontaneous Ca signaling occurred mate, D-serine and arachidonic acid in a Ca2ϩ-dependent man- shortly before the application of GABA, cells were not used for ner, thus modulating neuronal performance and local cerebro- analysis. vascular blood flow (5–8). While most studies describe the integration of astrocytes in excitatory neuronal networks in great Author contributions: M.D., D.H., J.W.D., and C.L. designed research; M.D., D.H., and P.C. detail (reviewed in ref. 1), only little information exists about performed research; M.D., D.H., and C.L. analyzed data; and H.-C.P., J.W.D., and C.L. wrote how astrocytes are affected by inhibitory neurotransmitters such the paper. as GABA and . GABAA and GABAB receptor-mediated The authors declare no conflict of interest. 2ϩ Ca signaling has been reported in hippocampal astrocytes This article is a PNAS Direct Submission. (9–11), where GABAB receptors mediate heterosynaptic de- 1To whom correspondence should be addressed. E-mail: [email protected]. pression (10). Only a minority of hippocampal astrocytes, how- This article contains supporting information online at www.pnas.org/cgi/content/full/ 2ϩ ever, respond to GABAB receptor activation with Ca increases 0809513106/DCSupplemental.

www.pnas.org͞cgi͞doi͞10.1073͞pnas.0809513106 PNAS Early Edition ͉ 1of6 Downloaded by guest on September 29, 2021 Fig. 2. GABA uptake mediates Ca2ϩ signaling in olfactory bulb astrocytes. (A) The GABA transporter substrate (NPA) induced Ca2ϩ transients similar to GABA-induced Ca2ϩ transients. The Ca2ϩ transients were not blocked by GABA receptor antagonists (60 ␮M , 10 ␮M CGP52432). NBQX (30 ␮M) Fig. 1. GABA-mediated Ca2ϩ signaling in olfactory bulb astrocytes. (A) The and D-AP5 (100 ␮M) were applied to suppress epileptiform activity induced by GABA gabazine (60 ␮M) blocked Ca2ϩ transients induced by A gabazine. (B) Upper trace, the GAT inhibitor SNAP 5114 evoked Ca2ϩ oscillations GABA (300 ␮M) in neurons (lower trace), but not in astrocytes (upper trace) at in astrocytes, rendering analysis of putative GABA-induced Ca2ϩ transients in P2–7. Asterisks indicate spontaneous Ca2ϩ signaling. (B) Dose-response curve of single astrocytes difficult. Lower trace, averaged Ca2ϩ signal from 13 individual GABA-induced Ca2ϩ transients in neurons (open squares) and astrocytes (closed astrocytes of one experiment to reduce unsynchronized Ca2ϩ oscillations and squares). (C) The GABA receptor antagonist CGP52432 (10 ␮M) blocked Ca2ϩ B unmask GABA-evoked Ca2ϩ transients. Inhibiting GABA uptake with SNAP 5114 transients elicited by (Bac, 300 ␮M), but not Ca2ϩ transients elicited by (100 ␮M) suppressed GABA-induced Ca2ϩ signaling. GABA (300 ␮M). (D) TTX (2 ␮M) only weakly reduced GABA-induced Ca2ϩ tran- sients in astrocytes. (E) Agonist profile of Ca2ϩ signaling in astrocytes. (F) Antag- 2ϩ ␮ ␮ onist profile of GABA-induced Ca signaling. CGP52432 (10 M) and TTX (2 M) 2ϩ ϩ GABA-induced Ca signaling cannot be explained by GABA significantly reduced GABA-induced Ca2 transients in astrocytes. *,PϽ0.05; ***, B P Ͻ 0.005. Bars, means ϩ error of the mean; n, the number of cells investigated. receptor activation. The results show that GABA receptors mediate Ca2ϩ signaling only in a minority of olfactory bulb astrocytes. They also argue 2ϩ Blocking GABAA receptors with gabazine suppressed GABA- against the possibility that Ca transients in astrocytes depend on induced Ca2ϩ transients in periglomerular neurons (n ϭ 132/7/6; GABA-driven activity of neurons or any other neuronal activity, P Ͻ 0.005), but had no effect on the amplitude of Ca2ϩ transients since inhibiting firing with TTX (2 ␮M) only 2ϩ in astrocytes (n ϭ 28/5/4; Fig. 1 A and F). The GABAA receptor marginally reduced GABA-induced Ca transient amplitudes in channel blocker (100 ␮M) reduced neuronal GABA- astrocytes by 9.5 Ϯ 4.5% (n ϭ 16/3/3; P ϭ 0.011; Fig. 1D), and 2ϩ evoked Ca2ϩ transient amplitudes to 34.2 Ϯ 4.1% (n ϭ 49/3/2; P Ͻ gabazine blocked Ca responses in periglomerular neurons, but 2ϩ 0.005), whereas astrocytic Ca2ϩ transients remained unaffected not in astrocytes. In neurons, GABA-induced Ca transients were 2ϩ ϭ Ͻ (n ϭ 18/3/2; Fig. 1F). The GABA receptor agonist (300 also suppressed in Ca -free solution (n 86/4/3; P 0.005) and A 2ϩ ␮ 2ϩ in the presence of the voltage-gated Ca channel blocker diltiazem M) evoked Ca transients in all periglomerular neurons that ϩ ϭ (400 ␮M; n ϭ 55/2/2; P Ͻ 0.005), suggesting Ca2 influx via responded to GABA (n 77/4/3), but only in 18% of the GABA- 2ϩ sensitive astrocytes (n ϭ 51/4/3; Fig. 1E), indicating that olfactory voltage-gated Ca channels after GABA-evoked depolarization bulb astrocytes employ an additional pathway for GABA-mediated (Fig. S3). 2ϩ Ca rises that neurons lack. This is supported by the delay of the 2؉ 2ϩ Ca Signaling Mediated by GABA Uptake. Since GABA receptor Ca response of 79.7 Ϯ 3.4 s in astrocytes (n ϭ 256/14/8) with ϩ antagonists only weakly reduced GABA-induced Ca2 transients in 2ϩ response in periglomerular neurons (Fig. S2). respect to the Ca olfactory bulb astrocytes, we tested the contribution of GATs to In addition, the GABA sensitivity of astrocytes and neurons is GABAergic signaling in astrocytes. Application of the GABA strikingly different. The apparent EC50 value of the GABA-induced 2ϩ transporter substrate nipecotic acid at concentrations of 50 and 100 Ca response, as calculated from measurements from at least ␮M mimicked the effect of GABA and evoked Ca2ϩ transients in ␮ ϭ three individual animals per data point, was 2.2 M(n 52–172 54% and 68% of the GABA-sensitive astrocytes with amplitudes of ␮ ϭ cells) in neurons and 100.9 M(n 15–124 cells) in astrocytes 165.3 Ϯ 5.8% ⌬F and 167.1 Ϯ 5.9% ⌬F(n ϭ 100/4/3), respectively (Fig. 1B). (Fig. 2A). Next, we tested the effect of gabazine (60 ␮M) and ␮ ϩ Application of baclofen (300 M), a specific agonist of GABAB CGP52432 (10 ␮M) on Ca2 transients evoked by nipecotic acid. ϩ receptors, evoked Ca2 transients in 39% of the astrocytes that To prevent epileptiform activity of olfactory bulb neurons upon ϭ responded to GABA (n 80/7/6; Fig. 1 C and E). The GABAB gabazine treatment, we added the antagonists antagonist CGP52432 (10 ␮M) entirely blocked the baclofen- NBQX (30 ␮M) and D-AP5 (100 ␮M; Fig. 2A). In the presence of induced Ca2ϩ transients (P Ͻ 0.005), but reduced the amplitude of inhibitors of GABA receptors and ionotropic glutamate receptors, GABA-induced Ca2ϩ transients in astrocytes by only 24 Ϯ 4% (n ϭ the Ca2ϩ transients evoked by 300 ␮M GABA and 100 ␮M 29/3/3; P Ͻ 0.005; Fig. 1F). Hence, in the majority of astrocytes, nipecotic acid were reduced by 34.3 Ϯ 3.8% (n ϭ 116/7/6; P Ͻ

2of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0809513106 Doengi et al. Downloaded by guest on September 29, 2021 0.005) and 11.1 Ϯ 7.2% (n ϭ 23/2/2; P ϭ 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 Ca2ϩ signaling independent of GABA receptor activation. GATs are localized in the rat olfactory bulb, where neurons express GAT1 and astrocytes express GAT3 (17), which corre- sponds to the murine transporter mGAT4. The involvement of astrocytic GATs in GABA-induced Ca2ϩ signaling was tested by using the non-competitive mGAT4 inhibitor, SNAP 5114 (18). Incubation of the slice in 100 ␮M SNAP 5114 resulted in Ca2ϩ oscillations in most astrocytes, which made it difficult to distinguish between Ca2ϩ oscillations and putative GABA-induced Ca2ϩ tran- sients 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 Ca2ϩ oscillations, but con- serving the Ca2ϩ transients evoked by GABA (Fig. 2B, lower trace). GABA-evoked Ca2ϩ responses were strongly reduced by 70.8 Ϯ 1.5% (n ϭ 60/5/5; P Ͻ 0.005) in the presence of SNAP 5114, indicating that GABA transporters are involved in mediating Ca2ϩ Fig. 3. GABA-induced Ca2ϩ signaling depends on external and internal sources signaling in olfactory bulb astrocytes. The effect of SNAP 5114 on and can be mimicked by NCX inhibition. All experiments were performed in the GABA-induced Ca2ϩ signaling was only partly reversible after presence of GABA and glutamate receptor antagonists (gabazine, CGP52432, NBQX, D-AP5). (A) Withdrawal of external Ca2ϩ reversibly abolished GABA- washout for 30–40 min. The GAT1 inhibitor NNC 711 did not 2ϩ 2ϩ 2ϩ 2ϩ ϭ induced Ca transients. Re-addition of Ca evoked a slow Ca increase (ar- significantly reduce GABA-evoked Ca transients (n 24/3/3; rowhead), on top of which Ca2ϩ transients or Ca2ϩ oscillations were recorded. A 2ϩ Fig. S4). Hence, GABA-induced Ca transients in olfactory bulb spontaneous Ca2ϩ transient is indicated by an asterisk. (B) Cyclopiazonic acid astrocytes are mediated by mGAT4, but not by GAT1. (CPA, 25 ␮M) reduced GABA-induced Ca2ϩ transients, unmasking a Ca2ϩ signaling ϩ component independent of internal Ca2 release (arrowhead). (C) Changes in NEUROSCIENCE Mechanism of GABA Uptake-Mediated Ca2؉ Signaling. GABA trans- intracellular Naϩ measured by SBFI. GABA (600 ␮M) and nipecotic acid (NPA, 600 porters use the inwardly directed Naϩ gradient as the driving force ␮M) evoked Naϩ transients in astrocytes. (D) SNAP 5114 (100 ␮M) abolished 2ϩ GABA-induced Naϩ transients. (E) GABA- and asparagine-induced Ca2ϩ tran- (19), but do not cotransport Ca . This raises the question how ϩ ϩ 2ϩ sients. (F) Inhibition of Na /Ca2 exchange by KB-R7943 mimics GABA-induced GABA uptake is linked to cytosolic Ca rises. We first investigated 2ϩ the source of Ca2ϩ to reveal the mechanism underlying GABA Ca transients. uptake-induced Ca2ϩ signaling in olfactory bulb astrocytes. Ca2ϩ transients evoked by GABA were entirely and reversibly suppressed ϩ 2ϩ 2ϩ (Fig. 3D). To test whether activation of Na -driven transporters in the absence of external Ca , indicating that Ca influx is were sufficient to evoke Ca2ϩ signaling in astrocytes, we applied 2ϩ ϭ Ͻ necessary for GABA-induced Ca signaling (n 33/4/3; P L-asparagine, which is a substrate for the glial Naϩ-dependent 2ϩ 0.005; Fig. 3A). Upon re-addition of external Ca , a slow increase neutral SNAT3 (system N; 23). Asparagine 2ϩ in intracellular Ca was measured (Fig. 3A, arrowhead). On top of has no significant effect on receptors or Ca2ϩ 2ϩ 2ϩ this slow Ca increase, Ca transients with a time course similar release mechanisms, but increases intracellular Naϩ when cotrans- 2ϩ to GABA-induced Ca transients were recorded. When intracel- ported with Naϩ by SNATs (23). Application of asparagine evoked 2ϩ 2ϩ lular Ca stores were depleted by the endoplasmic Ca -ATPase Ca2ϩ transients comparable to GABA-induced Ca2ϩ transients in ␮ 2ϩ inhibitor cyclopiazonic acid (CPA; 25 M), GABA-induced Ca a dose-dependent manner (Fig. 3E). One hundred micromolar transients were also largely reduced, on average by 87.2 Ϯ 1.5% 2ϩ ϭ ϩ asparagine evoked Ca transients in 25% (n 16/2/2) of the (n ϭ 87/4/3; P Ͻ 0.005; Fig. 3B). Ca2 store depletion by CPA ␮ 2ϩ ϩ astrocytes, whereas 300 M and 1 mM asparagine evoked Ca resulted in an elevated baseline Ca2 concentration, presumably transients in 28% (n ϭ 53/4/4) and 67% (n ϭ 58/5/5) of GABA- due to store-operated Ca2ϩ entry (20). The results demonstrate that ϩ sensitive astrocytes, respectively (Fig. 3D). The mean amplitudes of GABA-induced Ca2 transients in olfactory bulb astrocytes require the asparagine-induced Ca2ϩ transients were 11.8 Ϯ 5.2% ⌬F (100 ϩ ϩ Ca2 release from intracellular stores, but also depend on Ca2 ␮M), 29.4 Ϯ 5.8% ⌬F (300 ␮M), and 71.8 Ϯ 6.4% ⌬F (1 mM). The influx from the . mean amplitudes were significantly different from each other (P Ͻ ϩ ϩ GABA-induced Ca2 increases might result from reduced Na / 0.005, unpaired t test). The results are in line with the assumption ϩ ϩ Ca2 exchanger (NCX) efficacy due to elevated intracellular Na that Naϩ rises due to transporter activity in astrocytes are sufficient concentrations upon GABA uptake, which would lead to a reduced to induce Ca2ϩ signaling. driving force for the NCX. To test whether GABA uptake indeed If Naϩ rises induce Ca2ϩ transients in astrocytes by reducing ϩ ϩ evoked significant Na rises, we measured changes in the Na NCX activity, pharmacological inhibition of NCX should mimic ϩ concentration by two-photon imaging using the Na indicator SBFI GABA-induced Ca2ϩ transients. Therefore, we checked whether ϩ (21). Application of 300 ␮M and 600 ␮M GABA evoked Na rises reduced NCX activity can evoke Ca2ϩ transients by using the NCX of 2.2 Ϯ 0.1mM(n ϭ 74/3/2) and 3.2 Ϯ 0.1mM(n ϭ 188/6/3), inhibitor KB-R7943. Application of 50 ␮M KB-R7943 for 30 s and respectively, in the cell bodies (Fig. 3C), which corresponds to 60sresultedinCa2ϩ transients similar to GABA-induced Ca2ϩ transporter-mediated Naϩ transients in other glial cells (22). Larger transients in 58% and 82% of all astrocytes, with a mean amplitude Naϩ transients are expected in the fine astrocyte processes due to of 105.3 Ϯ 4.8% ⌬F(n ϭ 93/6/4) and 106.9 Ϯ 6.5% ⌬F(n ϭ 45/4/4), the larger surface-to-volume ratio (22), but the detector noise respectively (Fig. 3F). Thus, inhibition of NCX activity can mimic rendered SBFI fluorescence recordings impossible in astrocytic GABA-induced Ca2ϩ transients, suggesting that reduced NCX processes. Concentrations of 100 ␮M and 600 ␮M nipecotic acid activity mediates Ca2ϩ signaling evoked by GABA uptake into evoked Naϩ rises with an amplitude of 1.8ϩ/Ϫ0.1mM(n ϭ 47/3/2) olfactory bulb astrocytes. and 2.3ϩ/Ϫ0.1mM(n ϭ 84/3/2), respectively. SNAP 5114 (100 ϩ 2؉ 2؉ ␮M) entirely blocked GABA-induced Na transients (n ϭ 240/6/4), InsP3 Receptors Mediate Ca -Induced Ca Release. GABA-induced indicating that they were mediated by GABA uptake via mGAT4 Ca2ϩ transients in olfactory bulb astrocytes appear to require a

Doengi et al. PNAS Early Edition ͉ 3of6 Downloaded by guest on September 29, 2021 Fig. 4. InsP3 receptors, but not ryanodine receptors, mediate GABA-induced Ca2ϩ signaling in olfactory bulb astrocytes. (A) Ryanodine (100 ␮M) had no effect on GABA-induced Ca2ϩ transients. (B) Ruthenium red (10 ␮M) had no effect on 2ϩ Ca transients evoked by GABA and ATP. (C) Inhibition of InsP3 receptors with 2-APB (600 ␮M) blocked GABA- and ATP-induced Ca2ϩ transients almost com- pletely. Ruthenium red (10 ␮M) was added to suppress 2-APB-mediated Ca2ϩ oscillations.

small Ca2ϩ increase dependent on external Ca2ϩ, but are mainly attributable to Ca2ϩ release from intracellular stores, in line with Ca2ϩ-induced Ca2ϩ 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 Fig. 5. Endogenous GABA release triggers Ca2ϩ signaling in astrocytes. (A)Ca2ϩ 2ϩ can be stimulated by increases in cytosolic Ca (24, 25). Ryanodine transients evoked by ATP (30 ␮M), NMDA (100 ␮M) and kainate (100 ␮M) in receptors can be blocked by ryanodine at concentrations Ͼ10 ␮M neurons (upper trace) and astrocytes (lower trace). (B) Suppressing vesicular and by ruthenium red (26). In the present study, neither 100 ␮M neurotransmitter release by 10 ␮M bafilomycin A1 abolished NMDA-mediated ryanodine (n ϭ 39/3/3) nor 10 ␮M ruthenium red (n ϭ 21/3/2) Ca2ϩ signaling in astrocytes (lower trace), but not in neurons (upper trace). (C) reduced GABA-induced Ca2ϩ signaling (Fig. 4 A and B). These Inhibiting astrocytic neurotransmitter receptors by a mixture of antagonist [30 ␮ ␮ ␮ ␮ results suggest that ryanodine receptors were not involved in Ca2ϩ M MRS2179 (P2Y1), 1 M SCH58261 (A2A), 10 M CGP52432 (GABAB), 60 M gabazine (GABAA), 10 ␮M MPEP (mGluR5), and 30 ␮M NBQX (AMPAR)] did not increases evoked by GABA. affect NMDA-induced Ca2ϩ transients in astrocytes. Addition of SNAP 5114 (100 InsP3 receptors can be blocked by 2-APB at concentrations of ␮M) reduced NMDA-induced Ca2ϩ transients in astrocytes (average of 13 cells), several hundred micromolar (27, 28). However, 2-APB can also act but not in neurons (average of 41 cells). (D)Ca2ϩ transients evoked by electrical ϩ on store-operated Ca2 channels and transient receptor potential stimulation of receptor axons (50 Hz, 10 s) in the presence of the antagonist (TRP) channels (20, 29), thereby either blocking the channels mixture were reduced by SNAP 5114 in astrocytes (lower trace, average of 42 (some TRPCs and TRPMs) or activating the channels (some cells), but not in neurons (upper trace, average of 35 cells). (E) Synchronous TRPVs). Application of 600 ␮M 2-APB resulted in Ca2ϩ oscilla- neuronal discharges (*, upper trace) induced by 20 ␮M gabazine. Using a receptor antagonist mixture as above, but with 50 ␮M IEM 1460 instead of NBQX to block tions in olfactory bulb astrocytes, suggesting activation of TRP 2ϩ ␮ only GluR2-containing, Ca -permeable AMPA receptors while allowing for fast channels. Addition of 10 M ruthenium red, which also acts as a glutamatergic synaptic transmission maintained neuronal discharges, which 2ϩ TRPV inhibitor (30), suppressed the generation of Ca oscilla- were accompanied by SNAP 5114-sensitive Ca2ϩ transients in astrocytes (lower 2ϩ tions, although an initial Ca transient was elicited frequently upon trace). incubation with 2-APB/ruthenium red (Fig. 4C). Under these conditions, GABA as well as ATP, which was used as control for 2ϩ 2ϩ ␮ ␮ 2ϩ InsP3-mediated Ca release, failed to induce Ca transients in signaling. NMDA (100 M) and kainate (100 M) evoked Ca astrocytes (n ϭ 64/4/3; P Ͻ 0.005). Baseline Ca2ϩ slightly increased transients in periglomerular neurons (n ϭ 88/5/4) and astrocytes upon application of 2-APB, presumably due to Ca2ϩ leakage from (n ϭ 201/5/4; Fig. 5A). The V-ATPase inhibitor A1 internal stores (28). These results suggest that GABA uptake suppressed NMDA-induced astrocytic Ca2ϩ transients (P Ͻ 0.005, 2ϩ 2ϩ induces Ca signaling that is mediated by 2-APB-sensitive InsP3 unpaired t test), while ATP- and kainate-induced Ca transients receptors. were not blocked (n ϭ 47/3/2; Fig. 5B). Hence, kainate appears to induce Ca2ϩ transients in astrocytes directly, presumably via Ca2ϩ- Endogenous GABA Release Triggers Ca2؉ Signaling in Astrocytes. We permeable AMPA/kainate receptors, as shown for other glial cells asked whether endogenous GABA release after stimulation of (31, 32). NMDA, in contrast, triggered vesicular neurotransmitter GABAergic neurons by either NMDA/kainate application or elec- release from neurons and subsequent activation of astrocytes. To trical stimulation of receptor axons would activate astrocytic Ca2ϩ suppress Ca2ϩ signaling in astrocytes that was mediated by neuro-

4of6 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0809513106 Doengi et al. Downloaded by guest on September 29, 2021 AB C vessels in the nerve layer was recorded (Fig. 6 C and D and Movie S1). On average, the diameter of blood vessels significantly de- creased by 17.3 Ϯ 2.3% (n ϭ 8 preparations; P Ͻ 0.005; Fig. 6E). SNAP 5114 significantly reduced the GABA-induced vasoconstric- tion to 3.2 Ϯ 0.7% (n ϭ 8; P Ͻ 0.005). a b Discussion ϩ D E In the present study, we have investigated GABA-induced Ca2 signaling in olfactory bulb astrocytes and found a GAT-mediated Ca2ϩ-signaling mechanism. GABA-induced Ca2ϩ transients were independent of GABA receptors, but were a consequence of Naϩ-driven GABA uptake. They could be mimicked by inhibition of Naϩ/Ca2ϩ exchange and depended on both extra- 2ϩ 2ϩ cellular Ca and InsP3 receptor-mediated intracellular Ca release. This suggests that GABA uptake-evoked cytosolic Naϩ transients reduce Naϩ/Ca2ϩ exchange, leading to Ca2ϩ rises in 2ϩ 2ϩ astrocytes, which trigger Ca -induced Ca release via InsP3 Fig. 6. Glial GABA uptake triggers vasoconstriction. (A) Immunostaining receptors (Fig. S6). This study demonstrates that GATs not only against GFAP (green), mouse IgG (blood vessels, red), and nuclear staining take up GABA, but also mediate intracellular signaling in (Hoechst 33342, blue) in the nerve layer (NL) and glomerular layer (GL). (Scale bar, astrocytes. 40 ␮m.) (B) Area indicated in A at higher magnification. Blood vessels penetrating In immature neurons, which maintain a high intracellular ClϪ the nerve layer are densely covered by astrocytic processes (arrows), while blood concentration, GABAA receptor activation can lead to depolariza- capillaries surrounding glomeruli are only weakly contacted by GFAP-positive 2ϩ 2ϩ astrocyte endfeet (arrowheads). (Scale bar, 15 ␮m.) (C) Blood vessel in the nerve tion and thereby Ca influx via voltage-gated Ca channels (35, layer in an intact olfactory bulb before (a) and after (b) application of 3 mM 36). We studied olfactory bulbs of mice of postnatal days 2–7. At GABA. Arrowheads indicate the center of vasoconstriction. (Scale bar, 10 ␮m.) (D) this age, mitral cells already maintain a low intracellular Changes in blood vessel diameter after GABA application in the absence (black) concentration and are inhibited by GABA, whereas olfactory bulb and presence (green) of SNAP 5114. Arrowheads a and b indicate time points granule cells are depolarized upon GABAA receptor activation and 2ϩ 2ϩ when the images in C were taken. (E) SNAP 5114 significantly blocked the display Ca influx via voltage-gated Ca channels (37). Our NEUROSCIENCE GABA-induced reduction in blood vessel diameter (***, P Ͻ 0.005). results show that in the majority of periglomerular neurons, acti- 2ϩ vation of GABAA receptors triggers Ca influx via voltage-gated 2ϩ Ca channels. In olfactory bulb astrocytes, GABAA receptors transmitter receptors during NMDA application and receptor axon appear not to play a predominant role in Ca2ϩ signaling, which is stimulation, we blocked GABA receptors, metabotropic glutamate in contrast to hippocampal astrocytes (11). The presence of voltage- receptors, AMPA/kainate receptors, P2Y1 receptors and A2A re- gated Ca2ϩ channels in astrocytes is controversial, since Carmi- ceptors (Fig. 5 C and D). Under these conditions, NMDA and 2ϩ 2ϩ gnoto et al. (38) failed to detect voltage-dependent Ca signaling receptor axon stimulation still evoked Ca transients in astrocytes, in hippocampal astrocytes. In olfactory bulb astrocytes, voltage- Ϯ ϭ Ͻ which were significantly reduced by 56 3% (n 119/3/2; P gated Ca2ϩ channels appear not to play a major role in Ca2ϩ Ϯ ϭ Ͻ 0.005) and 51.8 2.7% (n 63/3/3; P 0.005), respectively, by the signaling, since high Kϩ evoked much smaller Ca2ϩ transients in mGAT4 inhibitor SNAP 5114, suggesting that GABA released by astrocytes as compared to neurons (Fig. S7). The GABA trans- 2ϩ periglomerular neurons induced Ca transients in astrocytes via porter substrate nipecotic acid was able to mimic the effect of 2ϩ mGAT4. In the presence of SNAP 5114, NMDA-evoked Ca GABA, and the mGAT4 inhibitor SNAP 5114 reduced GABA- ϭ ϩ transients in neurons were not significantly reduced (n 74/4/3), induced Ca2 transients, suggesting GABA uptake to be the 2ϩ while stimulation-evoked Ca transients in neurons were reduced mechanism underlying GABA-induced Ca2ϩ signaling. Ϯ ϭ ϭ ϩ by 13.4 2.1% (n 78/3/3; P 0.005). Our results suggest that GABA uptake-mediated Na rises 2ϩ We also measured Ca changes in neurons and astrocytes trigger Ca2ϩ signaling in astrocytes by reducing NCX efficacy. It has during synchronous (epileptiform) discharges of neurons induced been shown that inhibition of NCX can result in increased Ca2ϩ by disinhibition using gabazine (20 ␮M) in the presence of glial ϩ concentrations in other cells, including glial cells (39). In blockers (Fig. 5E). The Ca2 transients had an average bulb astrocytes, this increase in the cytosolic Ca2ϩ concentration amplitude of 202 Ϯ 13.8% ⌬F(n ϭ 66/4/4) in neurons and 141.9 Ϯ depended on extracellular Ca2ϩ. However, the major fraction of the 8.4% ⌬F(n ϭ 91/4/4) in astrocytes. SNAP 5114 reduced the GABA-induced Ca2ϩ transient was dependent on Ca2ϩ release ϩ amplitude of the Ca2 transients by 16.9 Ϯ 2.7% in neurons (n ϭ from intracellular Ca2ϩ stores, and GABA evoked only small Ca2ϩ 66/4/4); P Ͻ 0.005) and by 53.4 Ϯ 2.0% in astrocytes (n ϭ 91/4/4; increases after Ca2ϩ stores had been depleted with CPA. Small P Ͻ 0.005). Ca2ϩ increases as evoked by GABA in the presence of CPA can trigger Ca2ϩ-induced Ca2ϩ release (CICR) from intracellular ϩ Astrocytic GABA Uptake Triggers Vasoconstriction. Ca2 transients in stores. This mechanism is different from the mechanism in NG2 astrocytes have been shown to evoke both vasoconstriction and cells, in which GABAA receptor activation leads to depolarization, vasodilation, depending on the metabolic state of the tissue (8, 33, Naϩ influx through persistent Naϩ channels and subsequent re- 34). In the olfactory bulb, blood vessels that penetrate the nerve versal of NCX, while CICR has not been reported in NG2 cells (40). layer and enter the glomerular layer are densely covered by The mechanism presented in this study requires high concentra- astrocytic processes as assessed by GFAP staining (Fig. 6 A and B). tions of GABA, since the Ca2ϩ response saturated only at GABA In contrast, only weak GFAP staining was detected around capil- concentrations in the millimolar range. Millimolar concentrations laries surrounding glomeruli. In a preparation of the intact olfactory of GABA are expected in the vicinity of the synaptic cleft (36), bulb, we measured changes in the diameter of vessels in the nerve where astrocytic processes, together with pre- and postsynaptic layer upon application of GABA in the presence of GABA receptor neuronal elements, contribute to the ‘‘tripartite synapse’’ (1). In the blockers and TTX. Because the intact pia mater and the nerve layer olfactory bulb, the neuropil of the glomeruli is strongly pervaded by provide a major diffusion barrier (Fig. S5), we applied 3 mM processes of astrocytes (41), which ensheath dendrodendritic, pu- GABA (1 min) to stimulate GABA uptake into astrocytes in tative GABAergic, synapses (15, 16). Thus, olfactory bulb astro- deeper layers. After application of GABA, a constriction of blood cytes may detect GABA directly at the synaptic cleft and, as shown

Doengi et al. PNAS Early Edition ͉ 5of6 Downloaded by guest on September 29, 2021 in the present study, respond to GABA released from periglomeru- Confocal Ca2؉ Imaging. Acute brain slices were incubated in the dark at room lar neurons and possibly granule cells with Ca2ϩ signaling. Strong temperature (21–24 °C) for 60 min in Ca2ϩ-reduced aCSF containing 2 ␮M Fluo- ϩ stimulation of neurons triggered a robust global Ca2ϩ signal in the 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 micro- entire astrocyte. Under more physiological conditions such as 2ϩ 2ϩ scopes (Zeiss LSM 510 and Nikon eC1 plus). To measure Ca dynamics, images odorant stimulation, local Ca signals might be smaller and may be were acquired in one focal plane at 0.3 Hz. Ca2ϩ signaling was evoked by either restricted to astrocyte processes ensheathing GABAergic synapses. application or electrical stimulation of olfactory receptor axons using a 2ϩ A global Ca signal could be evoked under pathophysiological bipolar tungsten electrode (MPI) connected to a stimulator (SD9K, Grass) and conditions such as synchronized neuronal discharges occurring inserted into the olfactory nerve layer. during epileptiform activity. Due to the GABA uptake-dependent mechanism, astrocytes would be able to respond not only to Data Analysis and Statistics. For analysis of time series, Fluo-4-stained cell bodies glutamatergic, but also to GABAergic neurotransmission with were defined as regions of interest (ROIs), and the fluorescence intensity was Ca2ϩ signaling, which might contribute to control of blood flow by measured in each ROI. Only cells of the glomerular layer and the external ϩ vasodilation (34) and vasoconstriction (this study) and, hence, affect plexiform layer were chosen. Ca2 changes are given as relative fluorescence ⌬ neuronal performance. changes ( F) with respect to the resting fluorescence that was normalized to 100%. Only cells that responded with an amplitude of at least 3% ⌬F were Ϯ Materials and Methods 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 Solutions and Slice Preparation. The standard artificial cerebrospinal fluid (aCSF) stated otherwise, significance of statistical difference was calculated using Stu- for acute brain slices contained (in mM): NaCl 125, KCl 2.5, CaCl2 2, MgCl2 1, dent’s paired t test, with P Ͻ 0.05. D-glucose 25, NaHCO3 26, NaH2PO4 1.25, and L-lactate 0.5, gassed during the Further information is given in the SI Text. entire experiment by carbogen to adjust the pH to 7.4. In Ca2ϩ-reduced saline (0.5 mM), 1.5 mM CaCl was replaced by 1.5 mM MgCl .InCa2ϩ-free saline, Ca2ϩ was 2 2 ACKNOWLEDGMENTS. We thank P. Meuth (Mu¨nster) for software program- replaced by 2 mM Mg2ϩ, and 0.5 mM EGTA was added. In saline with altered ϩ ϩ ming, F. Barros (Valdivia, Chile) for helpful discussions, and K. Kaila (Helsinki, sodium concentrations used for SBFl calibration (Fig. S8), K -free saline (0K )or Finland) for critical comments on an earlier version of the manuscript. This work ϩ ϩ ϩ high-K solution (50 mM K , 50K ), KCl was exchanged by/for NaCl. Olfactory was funded by the Deutsche Forschungsgemeinschaft (LO 779/3 and SFB 530, TP bulb slices were prepared from NMRI mice (P2–P22) as described before (42). B1) and Interdisziplina¨res Zentrum fu¨r Klinische Forschung Mu¨nster (IZKF-FG6).

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