Pericytes in capillaries are contractile in vivo, butarterioles mediate functional hyperemia in themouse brainFrancisco Fernández-Kletta,b,c, Nikolas Offenhauserb, Ulrich Dirnaglb,d, Josef Prillera,b,c,d,1,2, and Ute Lindauerb,1,3

aDepartment of Neuropsychiatry and Laboratory of Molecular Psychiatry, Charité-Universitätsmedizin Berlin, D-10117 Berlin, Germany; bDepartment ofExperimental Neurology and Center for Stroke Research Berlin, Charité-Universitätsmedizin Berlin, D-10117 Berlin, Germany; cBerlin–Brandenburg Center forRegenerative Therapies, Charité-Universitätsmedizin Berlin, D-13353 Berlin, Germany; and dNeuroCure Research Center, D-10117 Berlin, Germany

Edited by Marcus E. Raichle, Washington University, St. Louis, MO, and approved November 11, 2010 (received for review August 13, 2010)

Modern functional imaging techniques of the brain measure localhemodynamic responses evoked by neuronal activity. Capillarypericytes recently were suggested to mediate neurovascular cou-pling in brain slices, but their role in vivo remains unexplored. Weused two-photonmicroscopy to study in real time pericytes and thedynamic changes of capillary diameter and blood flow in the cortexof anesthetized mice, as well as in brain slices. The thromboxaneA2 analog, 9,11-dideoxy-9α,11α-methanoepoxy Prostaglandin F2α(U46619), induced constrictions in the vicinity of pericytes in a frac-tion of capillaries, whereas others dilated. The changes in vesseldiameter resulted in changes in capillary red blood cell (RBC) flow.In contrast, during brief epochs of seizure activity elicited by localadministration of the GABAA receptor antagonist, bicuculline, capil-lary RBCflow increasedwithout pericyte-induced capillary diameterchanges. Precapillary arterioles were the smallest vessels to dilate,together with penetrating and pial arterioles. Our results provide invivo evidence that pericytes can modulate capillary blood flow inthe brain, which may be important under pathological conditions.However, our data suggest that precapillary and penetrating arte-rioles, rather than pericytes in capillaries, are responsible for theblood flow increase induced by neural activity.

cerebral blood flow | neurovascular coupling | cortical spreadingdepolarization | electrophysiology

Gray matter of the brain has a very high metabolic activity,maintaining the energy requirements of information trans-

fer and processing (1). In the brain, adaptation of regional bloodflow to local increases in neuronal activity guarantees a spatiallyand temporally matched supply of substrates. In addition, this“neurovascular coupling” underlies noninvasive brain mappingtechniques like functional magnetic resonance imaging (fMRI)and positron emission tomography (PET) (2). The spatial andtemporal acuity of these techniques depends on the degree bywhich the hemodynamic response overlaps with the activation ofneural tissue and, ultimately, on the disposition of blood flowregulating elements.The smooth muscle cells (SMCs) of arteries and arterioles are

the canonical effectors of blood flow regulation. However, it hasbeen proposed that control of blood flow may be exerted bypericytes located in the walls of capillaries, at the place where themetabolic demand occurs and could be most rapidly sensed (3).The contractility of cultured pericytes is well established and, ofall organs, capillaries in the central nervous system (CNS) areendowed with the highest number of pericytes (4). Pericytes ofthe isolated retina have been shown to react to vasoactive sub-stances (5). Recently, pericytes in cerebellar slice preparationswere found to react with dilatations and constrictions to the ap-plication of different neurotransmitters, suggesting a general ca-pacity of capillaries to increase blood supply locally (6). However,the behavior of brain pericytes in vivo has not been investigatedand is difficult to predict from experiments performed in isolatedtissue, in which the concerted response of the vascular network

is disrupted, no transluminal pressure gradients exist, and tissuehomeostasis is independent of blood supply. We used intravitaltwo-photon laser scanning microscopy (TPLSM) of the braincortex of green fluorescent protein (GFP) transgenic mice (7), inwhich the expression of GFP by endothelial cells and pericytesenabled the visualization of the capillary vessel wall.We studied inreal time the reaction of capillary pericytes to application of thevasoconstrictor 9,11-dideoxy-9α,11α-methanoepoxy Prostaglan-din F2α (U46619) and during functional hyperemia associatedwith bicuculline-induced neuronal activity or cortical spreadingdepolarization (CSD). We find that pericytes are contractile andcapable of modulating cerebral blood flow (CBF) at the capillarylevel, but they do not play a major role in the process of neuro-vascular coupling.

ResultsPericytes in Acute Brain Slice Preparations Constrict in Response toU46619. Vascular cells in the brains of GFP transgenic mice werediscernedbymeans of theirGFPexpression (Fig. 1). Pericyteswereidentified morphologically on the basis of their typical fusiform,protruding cell body and their expression of the marker pro-teins aminopeptidase-N (APN), platelet-derived growth factor-β(PDGFR-β), and α-smoothmuscle actin (α-SMA) (Fig. 1A,D, andE). GFP-expressing pericytes were negative for the endothelial cellmarker platelet-endothelial cell adhesionmolecule-1 (PECAM-1)(Fig. 1B) and the astrocyte marker glial fibrillary acidic protein(GFAP) (Fig. 1F). Pericytes were enclosed within the basementmembrane of capillaries, but separated from endothelial cells bya membrane sheet (Fig. 1C). These features are hallmarks of brainpericytes (8). The characteristic morphology of a GFP-positivepericyte with circumferential processes arising from the cell body isshown in Fig. S1. To elucidate whether brain cortical pericytespossess contractile properties, we imaged capillaries in brain slicesexposed to U46619, a TBXA2 receptor agonist and potent vaso-constrictor (Fig. 2A and Movies S1 and S2). The reaction toU46619 was concentration dependent, resulting in constrictionsof up to 75 ± 12% of basal capillary diameter (P < 0.001, n = 11,Fig. 2B). The mean diameter changes were negatively correlatedwith the logarithmic concentration of U46619 (r= 0.99, P < 0.05).The U46619-induced constriction was partially reversed by addi-

Author contributions: F.F.-K., N.O., U.D., J.P., and U.L. designed research; F.F.-K. and N.O.performed research; F.F.-K., U.D., and J.P. contributed new reagents/analytic tools; F.F.-K.,N.O., J.P., and U.L. analyzed data; and F.F.-K., N.O., and J.P. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.1J.P. and U.L. contributed equally to this work.2To whom correspondence should be addressed. E-mail: josef.priller@charite.de.3Present address: Department of Neurosurgery, Klinikum rechts der Isar, Technical Uni-versity Munich, 81675 Munich, Germany.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1011321108/-/DCSupplemental.

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tion of 1 μMSQ 29,548, a competitive TBXA2 receptor antagonist(Fig. 2D and Movies S1 and S2). Preincubation with SQ 29,548completely prevented the effects of U46619 (Fig. 2C). The con-strictions were localized in discrete sections of the capillaries. Theyeither were sphincter-like (<10 μm, 53% of the cases) or encom-passed longer vessel segments. Fifty-seven percent of the con-strictions were localized to capillary bifurcations. Occasionally,a bulging of pericyte cell bodies occurred after superfusion withU46619 (Fig. 2A, Insets, and Movies S1 and S2). Importantly,capillary constrictions at pericyte bodies exceeded constrictionsmeasured halfway between two pericyte bodies (median 84% vs.95% of basal diameter, P < 0.005, n= 18 slices, Fig. 2E). In threecases, discrete capillary segments dilated adjacent to the constric-tion sites (Fig. 2E and Movie S2), suggesting increased capillaryintraluminal pressure due to overall constriction. Our results in-dicate that pericytes in brain capillaries constrict in response toactivation of TBXA2 receptors.

Pericyte Constrictions Induced by U46619 Occur in Vivo and AreParalleled by Red Blood Cell (RBC) Perfusion Changes. We nextsought to determine whether similar changes could also be ob-served in vivo. To this end, we superfused 10 μMU46619 througha closed cranial window located over the parietal sensory cortexof anesthetized GFP transgenic mice. To study the dynamics ofcapillary reactivity, we imaged capillaries containing pericytes at2-min intervals with TPLSMover a total period of∼1 h before andduring the superfusion with 10 μM U46619 (five animals). In 7 of14 capillaries thus investigated, we observed constrictions withmorphological features similar to the constrictions found in theslice preparations (Movies S3 and S4).To determine the efficacy of capillary diameter changes on

capillary flow, we measured capillary diameters, RBC velocities,and flux before and after the superfusion with either vehicle (44capillaries from five mice) or 10 μM U46619 (37 capillaries fromfive mice). Fig. 3A shows the constriction of a capillary at the siteof a pericyte body. Changes in all parameters differed signifi-

cantly between vehicle- and U46619-treated capillaries (medianof the diameter changes, 104% vs. 96%, P < 0.001; RBC flux,101% vs. 17%, P < 0.0001; RBC velocity, 97% vs. 27%, P <0.001; Fig. 3B). Of note, all U46619-treated animals showedcapillary dilatations and constrictions, which were accompaniedby increases and decreases in flow parameters. We found a sig-nificant negative linear correlation between the pooled absolutedifferences in capillary diameters and the changes in RBC flow(r = 0.49, P < 0.001) and velocity (r = 0.38, P < 0.001), re-spectively (Fig. 3C). Flux and velocity differences were stronglycorrelated (r = 0.73, P < 0.001; Fig. 3C). Next, we compared theU46619-induced capillary diameter changes at pericyte bodiesand halfway between them. The constrictions were found to besignificantly more pronounced at pericyte bodies (median 83% vs.96% of basal diameter, P < 0.0005, n = 19, Fig. 3D), indicatingthat they reflected changes in pericyte contractile tone. In dilatingcapillaries, diameter changes did not differ significantly betweensites populated by pericyte bodies and halfway between them,although they tended to be larger at pericyte bodies (median119% vs. 108% of basal, n = 6).U46619 did not have consistent effects on the diameters of

arteries and arterioles at the pial surface. Five of 10 imaged

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Fig. 1. Pericytes can be visualized in the brains of GFP transgenic mice. (A)The small (∼10 μm) spindle-shaped cell bodies protruding from the capillarywall and their processes were positive for the pericyte marker APN (arrow-head). (B) Negative immunoreactivity of the protruding cell body associatedto the vessel wall for PECAM-1, an endothelial cell marker (arrowhead).Insets in A–C show reconstructions of the z-sections at the planes marked bythe dashed lines. (C) Immunoreactivity for laminin reveals the basal mem-brane, which completely encloses the capillary vascular GFP-positive struc-tures. Spindle-shaped cells are separated by basal membrane from theunderlying endothelium (Inset). (D) Protruding, spindle-shaped vascular cellbodies and their processes are positive for the pericyte marker PDGFR-β. (E)Some of these cells express also the contractile protein α-smooth muscleactin (α-SM actin). (F) Pericyte bodies are contacted by GFAP-positive astro-cyte processes, but separated from them by the vascular basal membrane(Inset). (Scale bars: 10 μm; Insets, 5 μm.)

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Fig. 2. U46619 induces pericyte contraction in brain slice preparations. (A)TPLSM images of capillaries in slice, before or after superfusion with 100 nMU46619. Pericyte bodies (p) can be identified. Discrete constrictions alongthe vessel can be observed (arrowheads), and bulging of a pericyte cell bodyis evident (Inset). (Scale bars: 10 μm; Inset, 5 μm.) (B) Effect of differentconcentrations of U46619 on capillary diameters at the segments of maximaleffect (*P < 0.05, **P < 0.001, n = 11; b, basal). (C) Effects of superfusion withvehicle (n = 7), 100 nM U46619 (n = 15), or 100 nM U46619 plus pre-incubation with 1 μM of SQ 29,548 (n = 5) (*P < 0.0001; **P < 0.00001). (D)Partial reversal of the U46619-induced constriction (100 nM) by 1 μM SQ29,548 (n = 6). (E) The constrictions elicited by 100 nM U46619 at pericytebodies are greater than constrictions at vessels halfway between pericytebodies (connected circles represent mean constrictions for one experiment ateither location; n = 19; *P < 0.005).

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arterioles (1 per animal) showed constrictions (75 ± 10% ofbaseline), 4 showed dilatations (130 ± 19% of baseline), and 1artery did not visibly react. Nevertheless, we always measureda decrease in CBF by laser Doppler flowmetry (LDF) in responseto U46619 (75 ± 8% of baseline, n = 9). This discrepancy, to-gether with the heterogeneous capillary diameter response afterU46619 administration, the correlation of capillary diameter andperfusion changes, and the enhanced constriction at pericytebodies, suggests that pericytes in capillaries are effective regu-lators of blood flow.

Capillary Dilatation Is Not Required for Functional Hyperemia. Next,we sought to elucidate whether similar localized capillary di-ameter changes, attributable to the activity of pericytes, werepresent during neuronal activity-induced hyperemia. We inserteda micropipette into the parietal cortex of mice, which was filledwith 10 mM of the GABAA receptor antagonist, bicuculline (n =8, Fig. 4G). Leakage of bicuculline from themicropipette tip led tosharp recurring bursts of neuronal spike activity (Fig. 4A and ref.9). We categorized cortical vessels located within ∼100 μm fromthe micropipette tip into pial, penetrating, precapillary arteriolesor capillaries on the basis of topology, vessel wall morphology,diameter, and RBC velocity (Fig. 4 C and D). Precapillary arte-rioles and pericyte-containing capillaries were imaged at 150–300

μmdepth in cortical layer II. The distributions of average diameterand RBC velocity differed significantly between all vessel types(Fig. 4 C and D), indicating that we had identified distinct vesseltypes. Diameter increases associated with neuronal activity oc-curred consistently in pial, penetrating, or precapillary arterioles,but not at pericytes in capillaries. Penetrating arterioles showedthe most pronounced reactions. RBC velocity increases weredetected in all vessel types. Fig. 4A and Movies S5, S6, S7, andS8 provide typical tracings of the diameter and RBC velocityresponses. When averaged, diameter responses of pial, penetrat-ing, and parenchymal arterioles peaked at 102.4 ± 3.3%, 103.4 ±3.5%, and 102.3 ± 5% of preburst basal values, respectively.Capillary diameter responses reached only 100.1 ± 2.5% of pre-spike basal values, measured at pericytes at the time of maximalRBC velocity increase (Fig. 4B). No correlation was found be-tween diameter and RBC velocity or between diameter and fluxchanges in capillaries (Fig. 4E and F). None of the different vesseltypes showed significant differences in the time to rise or time topeak of the diameter or RBC velocity changes (Fig. S2). Theseresults suggest that relaxation of pericytes in capillaries neither isrequired for nor substantially contributes to the development ofthe functional hyperemic response. Pial, penetrating, and pre-capillary arterioles were the effectors of flow changes duringneurovascular coupling. Low-frequency oscillations (∼0.1–0.2Hz)of vessel diameter were observed in arterioles, but not in capil-laries with pericytes (Fig. S3). Although the frequency wouldmatch vasomotor oscillations (10), which have been linked tofluctuations in the electrical andmetabolic activity of the “resting”brain (11), the oscillations we observed in arterioles were clearlyassociated with periodic changes in the frequency of bicuculline-induced spiking bursts (Fig. S3). Incidentally, we observed onecapillary in which a pericyte contraction led to a cessation of RBCflow irrespective of spike burst activity (Movie S9).

Capillaries Dilate Passively During CSD. To test whether a moreprolonged vasodilatory stimulus is able to provoke responsesat the level of capillaries, we induced CSD, a spreading wave ofneuronal and glial depolarization associated with hyperemia.We elicited 15 CSDs in eight animals by electrical stimulationof the frontal cortex, while recording epidural electrocorticogram(ECoG), measuring LDF blood flow, and performing TPLSMimaging over the parietal cortex (Fig. 5A). Single tissue volumes ofthe parietal cortex containing precapillary arterioles and down-stream capillaries were imaged. After electrical stimulation, thepassage of CSD was detected by the typical transient decreasein spontaneous ECoG activity, negative direct current (DC)-potential shift, and increase in CBF (Fig. 5B). A dilatory responsewas observed in precapillary arterioles, which coincided with thedilatation of capillaries (Fig. 5C). Importantly, capillary dilata-tions occurred irrespective of the presence of pericyte bodies(median 124% of basal at pericyte bodies vs. 126% betweenpericyte bodies, n = 12, P = 0.33; Fig. 5D). These results suggestthat capillaries dilate passively due to increased perfusion pres-sure during CSD-induced hyperemia. However, even under theseconditions, localized constrictions of some capillaries weredetected (Movie S10). The repeated finding of capillary con-strictions throughout different experimental setups suggests thatpericytes mediate local capillary vasoactivity in the CNS, albeitunrelated to neurovascular coupling.

DiscussionPericytes are contractile cells that surround capillaries in the brainat high density. The traditional view that CBF is regulated solelyby precapillary arterioles has recently been challenged by studiesin retina and cerebellar slices (6), as well as in ischemic braintissue (12). However, there is still no direct evidence that pericytescontrol CBF. In part, this may be due to the difficulties of visu-alizing CNS pericytes in vivo. Making use of GFP transgenic mice,

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Fig. 3. U46619 induces pericyte contraction in vivo, paralleled by RBC ve-locity and flux changes. (A) Capillary of the sensory cortex of anesthetizedmouse, imaged before and after the application of 10 μM U46619. Plasmawas labeled by TRITC-dextran (red). Dark stripes correspond to the passageof RBCs. Arrowheads point at constrictions in the vicinity of the pericytebody (p) (Scale bar: 10 μm.) (B) Box plots of the changes in diameter, RBCflux, and velocity (Top to Bottom, expressed as percentage of basal) after thesuperfusion of 10 μM U46619 (n = 37 capillaries, five animals) or in controlssuperfused with vehicle (n = 45 capillaries, five animals). Outliers are notrepresented in the box plot (one animal exhibited increases in velocity incapillaries of between 300 and 600% after treatment with U46619). (C)Scatter plots of the maximal absolute differences from basal after super-fusion with U46619 (red dots) or in the control experiments (blue dots)expressed in micrometers, RBC·s−1 or, mm·s−1 for diameter, flux, and velocitydifferences (Top to Bottom). A strong positive linear correlation is foundbetween the differences in RBC velocity and flux (two groups pooled).Positive correlations are also found for the pair diameter vs. RBC flux anddiameter vs. RBC velocity. (D) The constrictions elicited by superfusion withU46619 at pericyte bodies were greater than constrictions halfway betweenpericyte bodies (connected circles represent mean constrictions for eachcapillary network, measured at either location; n = 18; *P < 0.0005).

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we demonstrate in real time the contractile function of pericytesin brain capillaries and its effect on capillary flow. In experimentalstroke, capillary constrictions obstruct RBC flow for hours despitesuccessful reopening of the occluded vessel (12), suggesting thatpericytes may be involved in the “no-reflow” phenomenon after

cerebral ischemia. We asked whether TBXA2, a classical media-tor of vasoconstriction, could affect pericyte tone and causecapillary flow disturbances. Topical administration of the TBXA2

receptor agonist U46619 caused constrictions of capillaries invivo. Although the impact of U46619 on CBF measured by LDF

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Fig. 4. Capillary dilatation does not partake in the hyperemic response induced by bicuculline. (A) Original LFP, diameter, and RBC velocity traces of dif-ferent vascular segments. The LFP traces (blue) show typical bicuculline-induced activity bursts. (Upper) In the pial, penetrating, or precapillary arterioles, butnot at pericytes in the capillaries, neuronal activity bursts are associated with increases in diameter. High burst frequency caused summation of the diameteror flow responses. (Lower) RBC velocity changes of the same vessels, demonstrating brief increases associated with increased neuronal activity. (B) Averagetraces ± SD (gray areas) of the pooled diameter (Upper) or RBC velocity responses (Lower) of the different vessel types, binned in 200-ms segments. The bluevertical lines at t = 0 represent the time point of the maximal neuronal depolarization during the spike bursts. Whereas an average dilatation is present inarteriolar segments, capillaries do not respond at all. Velocity increases are detectable in each vessel type studied. Because activity bursts recurred fre-quently, the fall before the burst reflects the decline of the preceding response. No RBC velocity data are available for penetrating arterioles. (C and D)Distribution of the basal diameters (C) and RBC velocities (D) of the different vessel types investigated (*P < 0.0001, see E for sample n). (E and F) Scatterplots of the observed absolute diameter changes in capillaries and the observed changes in the RBC velocity or flux, respectively; no significant correlationsare found. (G) Scheme of the preparation: A micropipette filled with 10 mM bicuculline was inserted in the parietal cortex through a partially closed cranialwindow and served for LFP recordings. (H) Topology of cortical microvasculature. Pial arterioles run at the cortical surface and dive into the cortex to becomepenetrating arterioles. Precapillary arterioles arise from penetrating arterioles radially into the parenchyma, where they give rise to the capillary networkprovided with pericytes.

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Fig. 5. Capillaries dilate passively during CSD. (A) Scheme of the preparation. A bipolar stimulation electrode placed over the frontal cortex is used to elicitCSDs. TPLSM imaging, ECoG/DC, and LDF recording are performed through a closed parietal window. (B) Electrophysiological and LDF tracings of the passageof a CSD showing a transient decrease of ECoG activity, DC potential shift, and transient elevation of CBF (LDF trace). (C, Left) Cortical volumes were imagedcontaining parenchymal arterioles (art) and their downstream capillaries. p, pericytes. (C, Right) Diameter tracings at different vessel segments, populated ornot by pericyte bodies, showing simultaneous dilatation of all vessel segments. (D) Paired maximal diameter changes measured either at pericyte bodies orhalfway between pericyte bodies do not differ significantly.

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was moderate (∼25% decrease), tissue hypoperfusion may lead toedema of astrocyte end feet and compromise capillary patencyindependent of pericyte function (13). However, constrictionswere more pronounced at pericyte bodies, indicating that theywere caused by active pericyte contractility. Furthermore, wereplicated U46619-induced pericyte contractions ex vivo in acutebrain slices, where, in the absence of blood flow, tissue homeostasisis guaranteed by superfusion with oxygenated artificial (a)CSF. Invivo, RBC flow in capillaries is facilitated by the presence of a thinfluid layer between the RBC and the endothelium. Exhaustion ofthis layer by subtle decreases in capillary diameter might havea profound impact on resistance to RBC flow (14). In our study,RBC flow changes in single capillaries correlated with diameterchanges. Thus, it seems likely that minor changes in capillarydiameters, effected by pericyte contractility, could play a crucialrole under pathological conditions of the brain, e.g., ischemia,where TBXA2 is produced endogenously (15).One of the challenging questions remains whether pericytes also

participate in neurovascular coupling, i.e., the process that linksneural activity to local increases in blood flow. Dilatation ofcapillaries has recently been describedduring prolonged functionalstimulation of the rat whisker barrel cortex in vivo (16). However,our CSD data implicate that capillaries may dilate passively in re-sponse to increased perfusion pressure from upstream arteriolardilatation. Thus, it would appear that direct visualization of peri-cyte function in vivo is mandatory to address their role in neuro-vascular coupling. Because vasodilatation propagates to feedingvessels during functional hyperemia (17, 18), we speculated thatshort hyperemic stimuli could prevent the recruitment of largervessels and unveil the relaxation of pericytes in capillaries. Un-fortunately, we were unable to obtain reproducible and stable he-modynamic responses to repeated, 15-s whisker stimulations inanesthetized mice, which would have allowed for sequential ac-quisition of diameter and perfusion changes in different segmentsof the vascular tree. To overcome this obstacle, we used focal ap-plication of bicuculline, an inhibitor of GABAA receptors thatinduces synchronized neuronal depolarizations of brief duration(∼100 ms) and high metabolic demand (9). In our experiments,the RBC velocity changes in response to bicuculline were smallerthan those previously reported during somatosensory or olfactorystimulation (19, 20). Nevertheless, we did observe a functionalhyperemic response with dilatation of precapillary, penetrating,and pial arterioles. In contrast, pericytes in capillaries showedno changes.Although the domains of pial or penetrating arterioles do not

match the boundaries of neuronal functional columns in the so-matosensory cortex, neuronal columns exhibit localized hyper-emic responses, at least during the initial seconds after stimulation(21). Thus, daughter vessels must allocate the blood flow increaseto the activated neural parenchyma. Our data support the notionthat precapillary arterioles carry out this function (22).We find noevidence for neurovascular coupling at the level of capillaries, aswas hypothesized on the basis of ex vivo findings (6). Hence, thedistribution of precapillary arterioles limits the spatial resolutionof the functional hyperemic response, which is the basis of brainfunctional imaging techniques like fMRI or PET.It could be argued that GABAergic inhibition by bicuculline

may have interfered with pericyte function in our experiments.GABAergic signaling has been shown to influence the tone ofcortical vessels ex vivo and mediates the cortical hyperemia in-duced by stimulation of the basal forebrain (23, 24). In fact,GABAergic terminals arising from local interneurons innervatethe cortical parenchymal microvasculature, including arteriolesand capillaries (25). However, precapillary arterioles, which wereonly slightly wider than capillaries (but functionally distinct, asdemonstrated by their arteriolar RBC velocity, see ref. 26), wereable to dilate in the presence of bicuculline. Further, whereascerebellar pericytes are indeed contacted by GABAergic termi-

nals (27), inhibition of GABAergic signaling by bicuculline in thecerebellum does not affect basal blood flow or neuronal activity-induced functional blood flow responses (28, 29).In summary, we performed real-time imaging of pericytes in the

cerebral cortex of anesthetizedmice to evaluate their contributionto blood flow control in vivo. Our findings suggest that pericytescan modulate CBF under pathological conditions, which mayhave important implications for CNS disorders. In contrast, wefind no evidence for neurovascular coupling by capillary pericytesin the mouse brain.

MethodsDetailed methods are provided in SI Methods.

Animals. We used heterozygous adult β-actin-GFP transgenic mice of eithersex (7) bred in our facilities. All animal procedures were performed in ac-cordance with the standards for animal care of our institution and permis-sion was obtained according to the national regulations.

TPLSM Vessel Imaging and Analysis. Two-photon images (either stacks orsingle focal planes) from GFP-positive vessels were acquired using a Leica TCSSP2 microscope. Outer diameters were measured manually using ImageJ(Wayne Rasband, US National Institutes of Health, Bethesda) or automaticallyusing custom written MATLAB software (MathWorks). RBC velocity and fluxmeasurements were performed as described previously (30, 31). In theU46619 experiments, the capillary diameter was measured at the points ofmaximal constriction/dilatation. Separately, diameter changes at pericytebodies or at capillaries halfway between two neighboring pericytes wereassessed. In the bicuculline experiments, one single focal plane of the vesselunder study was imaged, and diameter was measured in segments popu-lated by SMCs or pericytes. In the CSD experiments, diameter measurementswere undertaken at SMCs in arterioles and, in capillaries, at segments eitherpopulated by pericyte bodies or halfway between them.

Acute Brain Slice Experiments. Coronal sections (300 μm thick) of the sensorycortex were cut and placed in artificial cerebrospinal fluid bubbled withcarbogen. We imaged capillaries running parallel to the slice surface at 70–100 μmdepth. U46619 or SQ 29,548 (Cayman Chemical) was dissolved in aCSF.

In Vivo Experiments. Throughout imaging, thiopental-anesthetized mice(initial dosage 75 mg/kg body weight i.v.) were mechanically ventilated andphysiological parameters were monitored. All mice received 100 μL of 5%TRITC-dextran (molecular mass 70,000 kDa, wt/vol) in saline to label bloodplasma. In the U46619 in vivo experiments, the drug (10 μM) or vehicle wassuperfused through a closed cranial window, and tissue volumes containingthe vessel were scanned. CBF was monitored with a LDF probe over theexposed cortex. In the bicuculline experiments, the drug (10 mM) leakedfrom the tip of a micropipette inserted in the cortex, which served as a localfield potential (LFP)-recording electrode. Vessels were imaged at a singlefocal plane at a frequency of ∼0.5–0.8 Hz. Isolated activity bursts (separatedfrom the previous and the next by at least 2 s) were detected off-line and thediameter or velocity responses pooled and averaged. For the CSD experi-ments, an additional open cranial window was prepared over the frontalcortex. A bipolar electrode was placed over the exposed dura at the frontalwindow, and a train of five current pulses (1 mA, 100 ms, 1 Hz) was deliveredto provoke CSD. An epidural electrode in the imaging window recorded theDC potential and ECoG. CBF was recorded simultaneously by LDF. Tissuevolumes containing precapillary arterioles and their downstream capillarieswere imaged at 1-min lapses for assessment of vessel diameter changes.

Statistical Analysis. Unless otherwise stated, data are presented as means ±SD. Where tested distributions were not distinct from normal, Student’s ttest, one-way ANOVA, or one-way ANOVA for repeated measurements withBonferroni correction was used. Otherwise, the Mann–Whitney–Wilcoxon orthe Wilcoxon signed ranks test was chosen.

ACKNOWLEDGMENTS. We thank G. Royl for his help with the automatedcapillary flow measurement routine and Jörg Rösner for his excellent sup-port at the TPLSM facility. We also thank C. Schaffer and N. Nishimura forkindly making their software available. This work was supported by theDeutsche Forschungsgemeinschaft, the Bundesministerium für Bildung undForschung, and the Hermann and Lilly Schilling Foundation.

22294 | www.pnas.org/cgi/doi/10.1073/pnas.1011321108 Fernández-Klett et al.

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