Ectopic vesicular neurotransmitter release alongsensory axons mediates neurovascular couplingvia glial calcium signalingAnne Thyssena,1, Daniela Hirnetb,c,1, Hartwig Wolburgd, Günther Schmalzinge, Joachim W. Deitmera,and Christian Lohra,b,c,2

aAbteilung für Allgemeine Zoologie, Technische Universität Kaiserslautern, 67653 Kaiserslautern, Germany; bAbteilung Tierphysiologie, Biozentrum Grindel,20146 Hamburg, Germany; cInterdisziplinäres Zentrum für Klinische Forschung, Institut für Physiologie I, Westfälische Wilhelms-Universität Münster, 48149Münster, Germany; dInstitut für Pathologie, Eberhard-Karls-Universität Tübingen, 72076 Tübingen, Germany; and eInstitut für Pharmakologie undToxikologie, Rheinisch-Westfälische Technische Hochschule Aachen, 52074 Aachen, Germany

Edited by Roger A. Nicoll, University of California, San Francisco, CA, and approved July 20, 2010 (received for review March 17, 2010)

Neurotransmitter release generally is considered to occur at activezones of synapses, and ectopic release of neurotransmitters hasbeen demonstrated in a few instances. However, themechanism ofectopic neurotransmitter release is poorly understood. We tookadvantage of the intimate morphological and functional proximityof olfactory receptor axons and specialized glial cells, olfactory en-sheathing cells (OECs), to study ectopic neurotransmitter release.Axonal stimulation evoked purinergic and glutamatergic Ca2+ re-sponses in OECs, indicating ATP and glutamate release. In axonsexpressing synapto-pHluorin, stimulationevokedan increase in syn-apto-pHluorinfluorescence, indicative of vesicle fusion. Transmitterrelease was dependent on Ca2+ and could be inhibited by bafilomy-cinA1andbotulinumtoxinA.Ca2+ transients inOECsevokedbyATP,axonal stimulation, and laser photolysis of NP-EGTA resulted in con-striction of adjacent blood vessels. Our results indicate that ATP andglutamate are released ectopically by vesicles along axons and me-diate neurovascular coupling via glial Ca2+ signaling.

neuron–glia interactions | olfactory bulb | olfactory ensheathing cells |purinergic signaling

In the conventional view, a neuron is divided into morpholog-ically and functionally distinct compartments, comprising

dendrites receiving synaptic input, axons conducting informationvia action potentials, and synaptic terminals transmitting in-formation by neurotransmitter release. Recent evidence, how-ever, indicates that neurotransmitters also can be released alongaxons (e.g., in optic nerve, olfactory nerve and corpus callosum)(1–4). Axonal neurotransmitter release may include thecotransmitter ATP, which is coreleased with glutamate fromaxons in optic and olfactory nerves (1, 2). However, the mech-anisms of this ectopic neurotransmitter release, and in particularthe mechanism of neuronal ATP release, are controversial. Earlystudies suggested vesicular release of ATP at varicosities of pe-ripheral nerves such as the vagus and enteric nerves (5). In fewinstances, vesicular ATP release could be demonstrated at cen-tral synapses (6, 7), and a vesicular ATP transporter has beendescribed recently (8). However, other studies question the hy-pothesis of vesicular ATP release. Release of ATP from pe-ripheral nerves, for example, is affected differently bypresynaptic modulation than is the release of vesicular nor-adrenaline (9), and suppressing vesicular release with botulinumtoxin and tetanus toxin does not inhibit ATP release from cho-linergic synaptosomes (10, 11). Alternative modes of ATP re-lease include pores formed by gap junctional hemichannels orP2X7 purinoceptors (12–14). Recent data demonstrate axonalATP release from cultured neurons through volume-regulatedanion channels (VRAC) upon electrical stimulation (15). Todate, it is unknown which mechanisms mediate ectopic ATPrelease in axon tracts such as optic and olfactory nerves.

The main targets of axonal neurotransmitter release are glialcell receptors, and functional neuron–glia interactions often aremediated by these neurotransmitters. In the corpus callosum andoptic nerve, for example, glutamate released by axons evokesAMPA receptor-mediated currents in NG2-positive glial pre-cursors (3, 4), whereas axonal glutamate and ATP release leads toCa2+ signals in glial cells in optic and olfactory nerves (1, 2).However, the function of these Ca2+ signals is still obscure. Insynaptic regions, transmitter-induced Ca2+ signaling in astrocyteslinks neuronal activity to changes in blood vessel diameter (neu-rovascular coupling) (16), but whether neurovascular couplingoccurs via glial cells other than astrocytes and in brain regionsdevoid of synapses, such as axon tracts, is not known. Therefore,we used olfactory ensheathing glial cells (OECs), which are closelyassociated with axons in the nerve layer of the olfactory bulb (17),as a monitor for glutamate and ATP to study (i) the mechanism ofectopic axonal transmitter release and (ii) the possibility of neu-rovascular coupling in the olfactory nerve via Ca2+ signaling inOECs. Our results show that ATP and glutamate release fromaxons is Ca2+-dependent and is sensitive to impairment of ves-icle fusion, suggesting ectopic transmitter release via vesicles. TheCa2+ increase in OECs evoked by these neurotransmitters trig-gered vasoconstriction in the nerve layer, demonstrating thatOECs are able to mediate neurovascular coupling.

ResultsRelease of ATP and Glutamate from Receptor Axons Stimulates Ca2+

Signaling in OECs. In the superficial layers of the mouse olfactorybulb, Fluo-4 acetoxymethyl ester (Fluo-4AM) preferentially labelsolfactory ensheathing cells (OECs), which can be distinguishedreadily from other cells by their position in the nerve layer, theirsize, and their shape (2). Electrical stimulation of receptor axons inthe nerve layer (individual pulse length: 1 ms; 20 Hz, 30 V) for 3 sevoked increases in the cytosolic Ca2+ concentration in OECs,reflected by an increase in fluorescence of 42.5± 3.1%ΔF (n=49)(Fig. 1A). A single stimulation pulse evoked a Ca2+ rise of 30.8 ±8.0%ΔF in 27% of the cells that responded to a 3-s train (n= 26),indicating that some OECs are able to detect the amount ofneurotransmitter that is released during a single compound actionpotential in the nerve layer (Fig. S1). Stimulation for 3 s elicited

Author contributions: A.T., D.H., J.W.D., and C.L. designed research; A.T., D.H., H.W., andC.L. performed research; G.S. contributed new reagents/analytic tools; A.T., D.H., H.W.,and C.L. analyzed data; and J.W.D. and C.L. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1A.T. and D.H. contributed equally to this work.2To whom correspondence should be addressed. E-mail: christian.lohr@uni-hamburg.de.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1003501107/-/DCSupplemental.

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inward currents in mitral cells of 806.7 ± 257.1 pA (n = 7); thestimulation-evoked responses in mitral cells and OECs were sup-pressed in the presence of tetrodotoxin (Fig. S1). After type 1metabotropic glutamate receptors (mGluR1s) were blocked with7-hydroxyiminocyclopropan[b]chromen-1a-carboxylic acid ethylester (CPCCOEt) (100 μM) or P2Y1 receptors were blocked withN(6)-methyl-2′-deoxyadenosine-3′,5′-bisphosphate (MRS2179)(60 μM) (Fig. S2), the stimulation-induced Ca2+ increase in OECswas reduced by 53.3 ± 4.6% (n = 49; P < 0.005) and 57.1 ± 4.1%(n = 33; P < 0.005), respectively (Fig. 1A). When both types ofreceptors were blocked simultaneously, the stimulation-inducedCa2+ transients in OECs were suppressed entirely (n = 64), in-dicating that glial responses are mediated by both glutamatergicand purinergic receptors. This result suggests that glutamate andATP are coreleased by receptor axons during stimulation.To verify the release of ATP from olfactory receptor axons, we

used P2X2 receptor-expressing HEK293 cells as “sniffer cells.”Puff application of ATP (100 μM) onto sniffer cells evoked aninward current of 382 ± 63 pA (n= 6), confirming the expressionofATP-sensitive receptors (Fig. 1B). To detectATP released fromaxons, sniffer cells were seeded onto exposed axon bundles ofwhole isolated olfactory bulbs, and whole-cell currents wererecorded. Electrical stimulation (30V) of an axon bundle attachedto the sniffer cell induced inward currents (Fig. 1C), whereasstimulation of adjacent axon bundles not directly attached to thesniffer cell did not evoke currents. This observation and the shortdelay between stimulus and current onset (Fig. 1C, Inset) suggestfast release of ATP from receptor axons and limited diffusion ofATP. The amplitudes of stimulation-induced currents in sniffercells increased with the number of stimulation pulses (Fig. 1C–E).

A single stimulation pulse evoked a current of 27 ± 9 pA (n= 7),whereas trains of 5 and 10 pulses (20 Hz) evoked currents of 128±14 pA (n= 12) and 322± 55 pA (n= 9), respectively. The currentamplitude evoked by 10 stimulation pulses was in the same rangeas the current amplitude following puff application of 100 μMATP, suggesting that the extracellular concentration of ATP re-ached at least several tens of micromoles upon electrical stim-ulation of receptor axons. In the presence of the P2X recep-tor antagonist pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate(PPADS), the stimulation-evoked currents were reduced by 95.1±2.4% (n = 5; P < 0.005), confirming that they were mediated byP2X2 receptors activated by ATP released upon electrical stimu-lation of receptor axons (Fig. 1E).

Neurotransmitter Release Is Ca2+ Dependent. To test whether therelease of glutamate and ATP is Ca2+ dependent, we suppressedvoltage-gated Ca2+ influx into receptor axons by the L-type Ca2+

channel blocker diltiazem (50 μM). The efficacy of diltiazem inblocking Ca2+ influx was verified by measurements of Ca2+

changes in Calcium Green-1 dextran-filled receptor axons in thenerve layer (Fig. 2A). In the presence of diltiazem, Ca2+ transientsinduced in receptor axons by electrical stimulation were reversiblyreduced by 94.8 ± 1.5% (n= 82; P < 0.005), as compared with thecontrol stimulation in the absence of diltiazem, indicating voltage-dependent Ca2+ influx into axons (Fig. 2B). OECs, in contrast,appear not to express voltage-gated Ca2+ channels, because de-polarization did not induce Ca2+ influx into OECs (Fig. S3A).Blocking axonal Ca2+ influx with diltiazem (Fig. 2C) or by with-drawal of external Ca2+ (Fig. S3B) reduced stimulation-inducedCa2+ transients in OECs by 89.3 ± 2.4% (n = 45; P < 0.005) and

Fig. 1. Glutamate and ATP release from olfactory receptor axons. (A)Electrical stimulation of receptor axons (20 Hz, 30 V, 3 s; indicated by anarrowhead) evokes Ca2+ transients in OECs that are reduced by CPCCOEt (100μM) and are completely blocked by additional application of MRS2719 (60μM). The response recovers after wash out of the drugs (Wash).(B) Mem-brane current evoked in a P2X2 receptor-expressing HEK293 cell (“sniffercell”) by puff application (arrowhead) of ATP (100 μM). (C) Single-pulsestimulation of receptor axons evokes an inward current in a sniffer cellseeded on top of an axon bundle in the nerve layer. Arrowheads indicatestimulation artefacts. (Inset) Expanded time scale showing onset of thecurrent response evoked by a single stimulation pulse. (D) Ionic current in-duced in a sniffer cell by a train of five stimulation pulses. (E) Inward currentsin sniffer cells are inhibited by PPADS.

Fig. 2. ATP and glutamate release is Ca2+-dependent but hemichannel-independent. (A) Electrical stimulation (20 Hz, 30 V, 3 s) induces Ca2+ influxinto receptor axons through diltiazem (Dilt)-sensitive Ca2+ channels. (B)Diltiazem almost entirely blocks stimulation-evoked Ca2+ transients in re-ceptor axons. ***P < 0.005. (C) Diltiazem suppresses Ca2+ signaling in OECsinduced by stimulation of receptor axons, whereas the amplitude of Ca2+

transients evoked by bath application of ATP is not affected. (D) Carbe-noxolone (CBX) (100 μM) has no effect on stimulation-induced Ca2+ tran-sients in OECs. (E) Ca2+ signaling in OECs is not affected by carbenoxolone(+Cbx). ns, not significant. In B and E, n = number of cells investigated.

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96.7 ± 1.0% (n = 23; P < 0.005), respectively, as compared withcontrol. The amplitude of ATP-evoked Ca2+ transients in OECswas unaffected by diltiazem. The results suggest that extrasynapticrelease of ATP and glutamate along olfactory receptor axonsdepends on Ca2+ influx via L-type voltage-gated Ca2+ channels, incontrast to synaptic transmitter release, which depends on N- andP/Q-type Ca2+ channels (18).Carbenoxolone blocks gap junction hemichannels and also

inhibits VRAC- and P2X7 receptor-mediated neurotransmitterrelease (19, 20). To test the involvement of gap junction hemi-channels, VRACs, and P2X7 receptors in ATP and/or glutamaterelease from olfactory receptor axons, we incubated the slices incarbenoxolone (100 μM). Carbenoxolone had no effect on theCa2+ transients in OECs evoked by the electrical stimulation ofreceptor axons, suggesting that gap junctional hemichannels,VRACs, and P2X7 receptors are not involved in axonal trans-mitter release (Fig. 2 D and E).

Localization of Synaptic Proteins and Vesicles in Receptor Axons. Thefusion of synaptic vesicles with the plasma membrane involvesnumerous proteins (21). If glutamate and ATP are releasedfrom olfactory receptor axons by vesicle exocytosis, these proteinsshould be located not only in the glomeruli, where olfactory re-ceptor axons terminate and synapse onto olfactory bulb neurons(Fig. 3A), but also in the nerve layer. Therefore, we stained ol-factory bulb slices with antibodies against the markers of thesynaptic vesicle fusion machinery, synaptophysin and bassoon,and the vesicular glutamate transporter VGluT2. Antibodiesagainst these markers predominantly labeled olfactory glomeruli(Fig. 3 B–D). In addition, significant antibody labeling was foundin the nerve layer, indicating that these proteins were located notonly in the synaptic terminals of receptor axons but also along theaxons, a prerequisite for vesicular release of glutamate and ATPalong olfactory receptor axons. The hypothesis of vesicular neu-rotransmitter release from axons also is supported by the presenceof vesicle-like structures in olfactory receptor axons in the directvicinity of OECs in electron micrographs (Fig. 3 E and F). How-ever, we did not find synaptic specializations such as active zonesand postsynaptic densities in electron micrographs, suggestingthat glutamate and ATP are released along olfactory receptoraxons in a paracrinemanner rather than at synaptiform structures.

ATP and Glutamate Are Released from Vesicles. The presence ofvesicles and vesicle-associated proteins in olfactory receptor axonscannot be considered a definite indicator of vesicular neuro-transmitter release in the nerve layer, because the vesicles couldbe transport vesicles carrying synaptic proteins to the axon ter-minals. To verify the presence of functional, releasable vesiclesthat can undergo vesicle fusion, we used a mouse strain in whicholfactory receptor axons express the vesicle fusion marker synapto-pHluorin (spH) (Fig. 4A) (22). Changes in spH fluorescence cor-relate linearly with the spike rate and hence with transmitter releasein receptor axons (23). Electrical stimulation of olfactory receptoraxon bundles (20 Hz, 30 V, 1 s) resulted in an increase in spHfluorescence of 15.0 ± 1.4% (n= 59) in the glomeruli, an increasethat is comparable to spH signals evoked by intense odor stimu-lation (23). In axon bundles, stimulation evoked spH signals of10.1 ± 1.2% (n = 22), indicative of vesicle fusion in axons (Fig.4B). The spH signals in glomeruli and axon bundles were sup-pressed in Ca2+-free, EGTA-buffered saline (P < 0.005; Fig. 4C)and by 1 μM tetrodotoxin (TTX) (P < 0.005) (Fig. S1D). Theseresults indicate Ca2+-dependent vesicle fusion not only in thesynaptic regions in the glomeruli but also along the axons in thenerve layer.Vesicular release of neurotransmitters can be suppressed by

preventing the loading of vesicles with transmitter molecules us-ing bafilomycin A1 or by cleaving the soluble N-ethylmaleimidesensitive factor attachment protein receptor (SNARE) complex

required for vesicle fusion using botulinum toxin A (24, 25). Wemonitored transmitter release evoked by receptor axon stimula-tion via Ca2+ changes in OECs before and after incubation ofolfactory bulb slices with either 10 μM bafilomycin A1 for 30 minor 150 μg/mL botulinum toxin A for 40 min (Fig. 4 D and E).Stimulation-induced Ca2+ transients were reduced after in-cubation with bafilomycin A1 and botulinum toxin by 87.2 ± 2.9%(n = 63; P < 0.005) and 92.6 ± 1.1% (n = 82; P < 0.005), re-spectively, whereas ATP- and dihydroxyphenylglycin (DHPG)-induced Ca2+ signaling was not affected or was affected onlyweakly (Fig. S4). These results demonstrate that release of bothATP and glutamate from olfactory receptor axons depends onfunctional vesicles and vesicle fusion.

Glial Ca2+ Signaling Mediates Vasoconstriction. Cerebral blood flowin gray matter has been shown to be controlled by Ca2+ signalingin astrocytes excited by synaptic release of neurotransmitters (16).We were interested in whether glial cells in the olfactory nerve,where neurotransmitters are released in the absence of synapticstructures, also can mediate neurovascular coupling. Fig. 5A and Bdemonstrates the tight morphological relationship between bloodvessels and OECs, which ensheath blood vessels with their pro-cesses. We bulk-loaded OECs in whole isolated olfactory bulbswith Fluo-4 AM using multicell bolus loading (26) and visualizedblood vessels by injecting sulforhodamine 101 (SR101) (Fig. S5).In OECs, puff application of ATP (1mM) evoked Ca2+ transients

Fig. 3. Synaptic vesicle markers in olfactory receptor axons. (A) Olfactoryreceptor axons (arrowhead) traced with 4-[4-(dihexadecylamino)styryl]-N-methylpyridinium iodide (DiA) (green) in the nerve layer (NL) terminate inglomeruli (asterisks) in the glomerular layer (GL). Nuclei are labeled withpropidium iodide (PI) (red). (B) Synaptophysin immunoreactivity (green) inthe glomeruli (asterisk), in the glomerular layer (GL), and in axons (arrow-heads) in the nerve layer (NL). (C) The vesicular glutamate transporterVGLUT2 (green) is detected in glomeruli (asterisks) and in axon bundles(arrowheads). (D) Bassoon immunoreactivity (green) is located in the glo-meruli (asterisks) and in the nerve layer (arrowhead). (Scale bar: A–D, 25 μm.)(E) Electron micrograph of the nerve layer. The cell body of an OEC ishighlighted in blue. (Scale bar: 0.5 μm.) (F) Region indicated by the square inE at higher magnification. Vesicle-like structures (arrowheads) are visible inolfactory receptor axons adjacent to an OEC. (Scale bar: 100 nm.)

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with a mean amplitude of 203.6 ± 10.7%ΔF (n= 64), which werefollowed by a constriction of adjacent blood vessels by 33.9 ±4.7% (n = 30) (Fig. 5 C and D and Movie S1). When olfactorybulbs were preincubated in MRS2179 (100 μM), ATP-inducedCa2+ transients in OECs were reduced by 79% (n = 32; P <0.005), and failed to evoke vasoconstriction (n = 13) (Fig. 5E).After washout of MRS2179, ATP evoked Ca2+ signaling andvasoconstriction recovered.To test whether a Ca2+ increase in OECs is sufficient to trigger

vasoconstriction, we released “caged Ca2+” in NP-EGTA–loadedOECs by photolysis. When the entire field of view was illuminatedwith 405-nm light, Ca2+ transients of 181.1 ± 22.5% ΔF (n = 23)were evoked in several OECs (Fig. 5F), leading to a global con-striction of blood vessels of 19.0 ± 2.6% (n = 16) (Fig. 5G andMovie S2). When the 405-nm illumination was focused on a 2 ×2 μm spot, Ca2+ was elevated only in single OECs (amplitude149.0 ± 30.6% ΔF; n = 7), followed by a local constriction ofadjacent blood vessels by 20.0 ± 2.8% (n= 10; Fig. 5 H and I andMovie S3). When the illumination spot was located outside theOEC, or when OECs not loaded with NP-EGTA were recorded,405-nm illumination failed to evoke Ca2+ transients in OECs, andno vascular response could be measured, ruling out nonspecificvascular effects by 405-nm illumination per se or out-of-focusphotolysis of NP-EGTA (e.g., in axons or epithelial cells) (Fig.S6). In addition, Ca2+ transients in OECs evoked by axon stim-ulation (20 Hz, 30 V, for 3 s) resulted in vasoconstriction of 38.2±

4.1% (n=10; P< 0.005) (Fig. 5 J andK andMovie S4), suggestingthat axonal activity can elicit neurovascular coupling in the ol-factory nerve layer, mediated by axon–glia communication andglial Ca2+ signaling.

DiscussionTwo recent studies showed that axons in white matter not onlyconduct action potentials but also release the neurotransmitterglutamate via vesicles at synaptiform structures and hence par-ticipate in information processing (3, 4). Both studies examinedaxons in the corpus callosum, where they demonstrate vesicularrelease of glutamate. It remained unclear, however, whether ve-sicular release is a general phenomenon in white matter through-out the brain or is restricted to the corpus callosum as a uniqueconductive structure transmitting a huge amount of informa-tion between the cortical hemispheres (27). In addition, it is notknown whether neurotransmitters other than glutamate can bereleased ectopically from vesicles along axons. In axons in thenerve layer of the olfactory bulb investigated in the present study,we demonstrated vesicular release not only of glutamate but alsoof ATP, which is an important cotransmitter (28, 29). In electronmicrographs we did not find synaptic specializations such aspostsynaptic densities, and immunoreactivity of proteins involvedin vesicle fusion was distributed evenly within the olfactory nervelayer, suggesting that olfactory receptor axons do not containactive zones. This even distribution is in contrast to the axons inthe corpus callosum, where punctate immunoreactivity againstthe vesicular glutamate transporter VGluT1 was demonstratedand where junctions between axons and NG2-positive cells pos-sess synaptic specializations (4).ATP is an important mediator in neuron–glia communication

(30, 31). ATP release from extrasynaptic sites involved in neuron–glia interactions has been reported. For example, in dorsalroot ganglion cells, Zhang et al. (32) described a quantal andCa2+-dependent release of ATP from somata of acutely dissoci-ated dorsal root ganglion cells; this ATP subsequently stimulatedadjacent satellite glial cells. Stevens and Fields (33) and Stevenset al (34) reported release of ATP from axons of cultured dorsalroot ganglion cells, causing Ca2+ signals in cocultured Schwanncells and oligodendrocyte precursors and thus inhibiting pro-liferation and maturation of Schwann cells but promoting myeli-nation by oligodendrocyte precursors. This result emphasizes thesignificance of axonal ATP release for the control of myelination(30). Glial cells themselves also can release ATP tomediate axon–glia communication. In rat optic nerve, mechanical and gluta-matergic stimulation of astrocytes leads to the release ofATP fromastrocytes, triggering Ca2+ waves covering adjacent astrocytes andoligodendrocytes (35). In the present study, glutamate-dependentATP release from OECs upon stimulation of receptor axonsappears not to be the primary source of ATP, because the ATP-mediated Ca2+ response in OECs remained when glutamate re-ceptors were inhibited (Fig. 1A). Only if both mGluRs and P2Y1receptors were blocked was the stimulation-evoked response en-tirely suppressed, indicating that the glutamatergic and the puri-nergic pathways proceed in parallel and not successively. Thus,simultaneous release of glutamate andATP from receptor axons isproposed here, as has been shown at olfactory receptor axon ter-minals in the neuropil of the olfactory bulb (36). The prominentrole of the purinergic system in the olfactory bulb, and in particularin the nerve layer, is emphasized by the high expression of ATP-degrading enzymes in the olfactory nerve layer (37).Neuronal activity has been shown to be linked to changes in

blood flow, a mechanism termed “neurovascular coupling” (16).The mechanism of neurovascular coupling in the olfactory bulb iscontroversial, because changes in blood flow of periglomerularblood capillaries has been shown to be triggered in an astrocyte-dependent as well as astrocyte-independent manner (23, 38). Inaddition, differences in astrocyte-mediated neurovascular coupling

Fig. 4. ATP and glutamate release from receptor axons depends on vesiclefusion. (A) Expression of the vesicle fusion marker spH in receptor axonbundles (arrowheads) and glomeruli (asterisks). The tip of the stimulationpipette is depicted in yellow (arrow). (Scale bar: 50 μm.) (B) Pseudocoloroverlay indicating regions of spH fluorescence increase upon electricalstimulation of axons. Warm colors represent large fluorescence increases;cool colors represent small fluorescence increases. (C) Electrical stimulation(Stim, arrowhead) (20 Hz, 30 V, 1 s) of receptor axons results in Ca2+-dependent increases in spH fluorescence in glomeruli and axons. (D) Ca2+

transients evoked by axon stimulation (20 Hz, 30 V, 3 s) were greatly reducedafter incubation of OECs with bafilomycin A1 (10 μM) for 30 min or (E) withbotulinum toxin A (150 μg/mL) for 40 min.

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have been found between in vivo and in vitro preparations. Acti-vation of Ca2+ transients in astrocytes of the somatosensory cortexand the olfactory bulb in vivo by stimulation of afferents was ac-companied by vasodilation and an increase in blood flow (23, 39,40). In brain-slice preparations, the situation appears more com-plicated, and both vasodilation and vasoconstriction could bemeasured (39, 41, 42), depending on the metabolic state of thetissue and the amplitude of astrocytic Ca2+ transients (43, 44). In allstudies mentioned above, glial cells are located in the neuropil andcouple neuronal activity to responses of the vasculature via de-tection of synaptically released neurotransmitters. It was notknown, however, whether glial Ca2+ signaling evoked by ectopicrelease of neurotransmitters is used also to regulate blood flow in

the brain. The vasoconstriction mediated by Ca2+ transients inOECs, which can be evoked by ectopic release of ATP and gluta-mate from receptor axons, demonstrates glial control of bloodvessels in an axon tract devoid of synapses. Hence, neurovascularcoupling probably is a major function of glial Ca2+ signaling notonly in gray matter but also in white matter regions in the brain.

MethodsPreparation of Brain Slices and Whole Olfactory Bulbs. Olfactory bulbs wereobtained from NMRI mice from postnatal days 0 to 17 (P0–P17) as describedpreviously (2). Animals were decapitated, and the olfactory bulbs werequickly removed from the brain. For whole-bulb preparations, olfactorybulbs were glued to round coverslips, placed in Ca2+-reduced artificial ce-rebrospinal fluid (ACSF), and allowed to recover at 30 °C for 1 h. Olfactory

Fig. 5. Ca2+ signaling in OECs mediates vasoconstriction. (A) Immunostaining against S100 calcium-binding protein (OECs, green) and mouse IgG [bloodvessels (red)]. Nuclei are counterstained with Hoechst (blue). The arrowhead indicates a blood vessel that is depicted at higher magnification in B. GL,glomerular layer; NL, nerve layer. (Scale bar: 50 μm.) (B) OEC somata (asterisks) are located in the direct vicinity of blood vessels, and OEC processes ensheathblood vessels (arrowheads). (Scale bar: 10 μm.) (C) Time series of Fluo-4–loaded OECs (green) and SR101-injected blood capillaries (red). ATP (1 mM) wasapplied to OECs via a puff pipette. Fluo-4 was measured in region of interest 1 (ROI 1), and SR101 was measured in ROI 2 (Movie S1). (Scale bar: 15 μm.) (D)Time course of a Ca2+ transient (upper trace) and the SR101 fluorescence (lower trace, to visualize vasoconstriction). (E) Analysis of Ca2+ response in OECs(Fluo-4) and vasoconstriction evoked by ATP in ACSF (ATP), in the presence of 100 μM MRS2179 (ATP+MRS) and after washout of MRS2179 (ATP wash).MRS2179 reduced ATP-mediated Ca2+ transients in OECs and completely inhibited vasoconstriction. ***P > 0.005. (F) Fluorescence of Fluo-4 (green) and SR101(red) before (Left) and after (Right) laser photolysis of NP-EGTA in OECs by illumination of the entire field of view with a 405-nm laser (Movie S2). (G) Timecourse of Ca2+ transients in OECs (upper trace) and vasoconstriction (lower traces) upon photolysis of NP-EGTA (arrow). (H) Spot illumination with 405 nmresults in a Ca2+ transient in a single OEC (Movie S3). (I) Time course of Ca2+ transients in OECs (upper trace) and vasoconstriction (lower traces) uponphotolysis of NP-EGTA (arrow). (J) Ca2+ response in OECs evoked by electrical stimulation (20 Hz, 30 V, 3 s) of axons (Movie S4). (K) Time course of Ca2+

transients and vasoconstriction upon axonal stimulation. (Scale bars in F, H, and J: 10 μm.) The rapid drop in SR101 fluorescence (arrow) is caused bya nonfluorescent erythrocyte that became stuck in the ROI in the blood vessel.

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bulbs were used within 3 h after preparation. To check the viability of OECsin whole olfactory bulbs, which might be impaired after storage for severalhours, we bath-applied ATP at different times after preparation and did notfind any sign of impairment in terms of generation and back-regulation ofATP-induced Ca2+ rises after storage up to 6 h. For brain slices, olfactorybulbs were glued to the stage of a Leica VT 1000s vibroslicer. Sagittal slices250 μm thick were cut in cooled, Ca2+-reduced ACSF and were stored at 30 °Cfor 1 h before the experiments.

Calcium Imaging. For bulk dye loading in brain slices, olfactory bulb slices wereincubated in ACSF containing 2 μM Fluo-4 AM for 1 h at room temperature.Dye-loaded slices were transferred into fresh ACSF and kept at room tem-perature for up to 6 h. In the nerve layer, Fluo-4 AM preferentially loads OECs(2). OECs inwhole olfactory bulbswere stained bymulticell bolus loading (26).ACSF containing 200 μM Fluo-4 AM was placed in a micropipette that wasinserted into the nerve layer under visual control. Fluo-4 AM was ejected bypressure of 0.4 bar for 10 s. After 30min OECs were brightly labeled by Fluo-4.In someexperiments, 1mMNP-EGTAAM (caged Ca2+)was added to the Fluo-4AM solution to coload OECs with Fluo-4 and NP-EGTA. Uncaging of NP-EGTAwas achieved by illumination with a 405-nm laser diode at 5 mW for 1–2 s.

Slices and whole olfactory bulbs were fixed in a perfusion chamber andcontinuously superfused with ACSF. Ca2+ imaging was performed using

confocal laser scanningmicroscopy (Zeiss LSM 510 and Nikon EC1). Changes inthe intracellular calcium concentration of OECs were induced either by bathor puff application of ATP and DHPG or by electrical stimulation of olfactoryreceptor axons using a stimulation pipette (tip resistance ∼0.5–2 MΩ).

Data Analysis. Changes in intracellular Ca2+ as well as changes in spH fluo-rescence are given as changes in fluorescence (ΔF) relative to the basal fluo-rescence at the beginning of an experiment, which was normalized to 100%.All values are given as means ± SEM. The number of evaluated cells is in-dicated by n. For each experiment and protocol, data from at least threeanimals were analyzed. Significance of statistical difference was calculatedusing Student’s t test for large data sets (n > 30) and the Mann–Whitney U testfor small data sets at 95% confidence level (P ≤ 0.05). Exact P values are givenbetween 0.05 and 0.005; P < 0.005 is stated for P values smaller than 0.005.

Further information is given in SI Methods.

ACKNOWLEDGMENTS. We thank P. Mombaerts and H. Spors (Frankfurt,Germany) for providing OMP-spH mice, P. Koch (Münster, Germany) fortechnical assistance, and H. Bigalke (Hannover, Germany) for providing bot-ulinum toxin A. Financial support by the Deutsche Forschungsgemeinschaft(Grants LO779/3 and SFB 530 TP B1) is gratefully acknowledged.

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