cereb. astrocyte calcium signal and gliotransmission in human brain tissue...

7/27/2019 Cereb. Astrocyte Calcium Signal and Gliotransmission in Human Brain Tissue Cortex-2012-Navarrete-Cercor-bhs122

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Astrocyte Calcium Signal and Gliotransmission in Human Brain Tissue

Marta Navarrete1,, Gertrudis Perea1,3,, Laura Maglio1, Jess Pastor2, Rafael Garca de Sola2 and Alfonso Araque1

1Instituto Cajal, Consejo Superior de Investigaciones Cientcas. Madrid 28002, Spain and 2Clinical Neurophysiology Service,Hospital Universitario La Princesa, Madrid, Spain3Present address: Picower Institute for Learning and Memory, Massachusetts Institute of Technology, Cambridge, MA, USA

Address correspondence to Alfonso Araque, Instituto Cajal (CSIC), Doctor Arce 37, Madrid 28002, Spain. Email: [email protected]. N. and G. P. contributed equally to this work.

Brain function is recognized to rely on neuronal activity and signal-

ing processes between neurons, whereas astrocytes are generally

considered to play supportive roles for proper neuronal function.

However, accumulating evidence indicates that astrocytes sense

and control neuronal and synaptic activity, indicating that neuron

and astrocytes reciprocally communicate. While this evidence has

been obtained in experimental animal models, whether this bidirec-

tional signaling between astrocytes and neurons occurs in human

brain remains unknown. We have investigated the existence of

astrocyte

neuron communication in human brain tissue, using elec-trophysiological and Ca2+ imaging techniques in slices of the

cortex and hippocampus obtained from biopsies from epileptic

patients. Cortical and hippocampal human astrocytes displayed

spontaneous Ca2+ elevations that were independent of neuronal

activity. Local application of transmitter receptor agonists or nerve

electrical stimulation transiently elevated Ca2+ in astrocytes, indi-

cating that human astrocytes detect synaptic activity and respond

to synaptically released neurotransmitters, suggesting the exist-

ence of neuron-to-astrocyte communication in human brain tissue.

Electrophysiological recordings in neurons revealed the presence of

slow inward currents (SICs) mediated by NMDA receptor acti-

vation. The frequency of SICs increased after local application of

ATP that elevated astrocyte Ca2+. Therefore, human astrocytes are

able to release the gliotransmitter glutamate, which affect neuronalexcitability through activation of NMDA receptors in neurons.

These results reveal the existence of reciprocal signaling between

neurons and astrocytes in human brain tissue, indicating that astro-

cytes are relevant in human neurophysiology and are involved in

human brain function.

Keywords:gliotransmission, glutamate, human astrocytes, intracellular

Ca2+, neuron-glia communication

Astrocytes, classically considered supportive cells forneurons without being directly involved in brain informationprocessing, are emerging as important actors in brain physi-

ology. They exhibit cellular excitability based on intracellularCa2+variations that occur spontaneously or evoked by differentstimuli, such as mechanical stimulation, exogenous ligands, orneurotransmitters released from synaptic terminals (Porter andMcCarthy 1996; Perea and Araque 2005a,b; Navarrete and

Araque 2008; Shigetomi et al. 2008). In addition, astrocytesmay release neuroactive substances, called gliotransmitters,such as glutamate, ATP, D-serine, adenosine, etc. that can inu-ence neuronal excitability and synaptic transmission and plas-ticity (Serrano et al. 2006;Martn et al. 2007;Perea and Araque2007;Navarrete and Araque 2008,2010;Shigetomi et al. 2008;Henneberger et al. 2010; Santello et al. 2011; Navarrete et al.2012). These ndings have led to the establishment of the tri-partite synapse concept, in which astrocytes and neurons

reciprocally communicate, suggesting that brain physiologyresults from the coordinated signaling between neurons andastrocytes (Volterra and Meldolesi 2005; Haydon and Car-mignoto 2006;Perea et al. 2009). While these ideas are primar-ily founded on observations made in rodent brainpreparations, the existence of astrocyteneuron bidirectionalcommunication in human brain remains largely unknown.

We have therefore investigated the existence and propertiesof this astrocyteneuron communication in human brain tissue,

using electrophysiological and Ca

2+

imaging techniques inslices of the cortex and the hippocampus obtained from biop-sies from pharmacologically intractable epileptic patients un-dergoing surgical treatment. We found that cortical andhippocampal astrocytes exhibit Ca2+-based intrinsic excitability,and that they respond with transient Ca2+ elevationsto neurotransmitters released during synaptic activity. TheseCa2+ elevations affect neuronal excitability because they stimu-late the release of glutamate from astrocytes that activatingNMDARs evoke slow inward currents (SICs) in neurons. There-fore, human astrocytes detect synaptic activity and are capableof release gliotransmitters that act on neuronal receptors.

These ndings indicate the existence of bidirectional com-munication between astrocytes and neurons in human tissue,

and suggest that astrocytes may be more relevant cellularelements than previously thought in human brain function.

Materials and Methods

Human Brain Slice Preparation

Human brain tissue was obtained from biopsies from patients (11males and 15 females; age: 2859 and 2151 years old, respectively)diagnosed of drug-resistant temporal lobe epilepsy for more than 7years (cf. Arellano et al. 2004; Pastor et al. 2010) that underwentsurgery to control their seizures. Patients consent was obtained ac-cording to the Declaration of Helsinki, and protocols were approvedby the institutional ethical committee (Hospital de la Princesa,

Madrid, Spain). Slices (350400m thickness) were prepared in coldACSF containing (in mM): KCl 3, MgCl2 10, NaHCO3 25, CaCl2 1,glucose 10, and sucrose 250; and then incubated during >1 h at roomtemperature (2124C) in ACSF containing (in mM): NaCl 124, KCl2.69, KH2PO4 1.25, MgSO4 2, NaHCO3 26, CaCl2 2, and glucose 10,continuously gassed with 95% O2/5% CO2 (pH 7.3). The timebetween the tissue removal from the patient and the slice preparationwas typically 45 min and tissue was kept in cold ACSF-high sucroseduring transportation. They were then transferred to an immersion re-cording chamber and continuously perfused with gassed ACSF at 2mL/min. Cells were visualized under an Olympus BX50WI micro-scope or an Olympus FV300 laser-scanning confocal microscope(Olympus Optical). Astrocyte calcium signal and electrophysiologicalrecordings were performed in CA1 and CA3 regions from hippo-campus and layers 2/35 from the cortex. Because similar results

were obtained, data collected from each area were pulled together.

The Author 2012. Published by Oxford University Press. All rights reserved.For Permissions, please e-mail: [email protected]

Cerebral Cortexdoi:10.1093/cercor/bhs122

Cerebral Cortex Advance Access published May 10, 2012


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Electrophysiology

Electrophysiological recordings were made using the whole-cellpatch-clamp technique. For neuronal recordings, patch electrodes hadresistances of 310 M when lled with the internal solution (inmM): KGluconate 135, KCl 10, HEPES 10, MgCl2 1, ATP-Na2 2 (pH7.3); and for astrocyte recordings, electrodes had resistances of 49Mand lled with an intracellular solution (in mM): MgCl21, NaCl 8,ATP-Na2 2, GTP-Na2 0.4, HEPES 10, titrated with KOH to pH 7.27.25, and adjusted to 275285 mOsm. Recordings were obtained with

PC-ONE ampliers (Dagan Instruments). Fast and slow whole-cell ca-pacitances were neutralized and series resistance was compensated(70%), and the membrane potential was held at 70 mV. Signalswere fed to a Pentium-based PC through a DigiData 1440A interfaceboard. Signals were ltered at 1 kHz and acquired at 10 KHz samplingrate. The pCLAMP 10.2 (Axon instruments) software was used forstimulus generation, data display, acquisition, and storage. Thetacapillaries (25 m tip) lled with ACSF were used for bipolar synap-tic stimulation. The electrodes were connected to a stimulator S-900through an isolation unit and placed 75150 m away from therecording cells.

SICs were distinguished from miniature synaptic currents(mEPSCs) by their relatively slower time courses (mEPSCs: on =2.90.3 ms, off fast= 3.6 0.5 ms, off slow = 24.9 3.1 ms, n = 18; SICs:on = 8.9 1.4 ms, off= 84.6 13.8 ms, n = 35). Neuronal responseswere considered SICs when they showed a off 2 times slower thanthe off, slow of mEPSCs (cf. Perea and Araque 2005a;Navarrete andAraque 2008;Shigetomi et al. 2008).

Ca2+ imaging

Human tissue was prepared as described previously for rodent tissue(Araque et al. 2002; Nimmerjahn et al. 2004; Perea and Araque2005a). Briey, Ca2+ levels in human astrocytes were monitored byuorescence microscopy using the Ca2+ indicator uo-4. Slices wereincubated with uo-4-AM (25 L of 2 mM dye were dropped over thetissue, attaining a nal concentration of 210 M and 0.01% of pluro-nic) and Sulforhodamine 101 (100 M) for 3060 min at room temp-erature. In these conditions, most of the Fluo-4-loaded cells wereastrocytes as indicated by their SR101 staining (Nimmerjahn et al.

2004; Dombeck et al. 2007; Ka

tz et al. 2008; Takata and Hirase2008), and conrmed in some cases by their electrophysiologicalproperties (see Fig. 2B). Astrocytes were imaged with an OlympusFV300 laser-scanning confocal microscope or a CCD camera (RetigaEX) attached to the Olympus BX50WI microscope. Cells were illumi-nated during 200500 ms with a xenon lamp at 490 nm using a mono-chromator Polychrome V (TILL Photonics), and images were acquiredevery 12 s. Polychrome V and CCD camera were controlled and syn-chronized by the IP Lab software (BD Biosciences) that was also usedfor quantitative epiuorescence measurements. Ca2+ variations re-corded at the soma of the cells were estimated as changes of the uor-escence signal over baseline (F/F0), and regions of interest wereconsidered to respond to the stimulation when F/F0 increased 3times the standard deviation of the baseline for at least 2 consecutiveimages and with a delay15 s after the stimulation. Local applicationof WIN (300 M), ATP (20 mM), and glutamate (0.8 mM) was deliv-

ered by pressure pulses or by iontophoresis through a micropipette(25 s). Mean Ca2+ wave velocity was estimated from the ratio of thedistance and the time delay of the calcium elevations between thenearest and the farthest responding cells relative to the stimulatingpipette.

Experiments were performed at room temperature (2124C). Dataare expressed as mean SEM. Results were compared using a 2-tailedStudentst-test (= 0.05). Statistical differences were established withP< 0.05 (*),P< 0.01 (**), andP< 0.001 (***).

Drugs and chemicals

D-(-)-2-Amino-5-phosphonopentanoic acid (D-AP5) and thapsigarginwere purchased from Tocris. Fluo-4-AM was from Molecular Probes.All other drugs were purchased from Sigma-Aldrich Co.

Results

We monitored intracellular Ca2+ levels in astrocytes in hippo-campal and cortical slices (Fig. 1). Human astrocytes in bothhippocampal and cortical slices displayed spontaneous Ca2+

elevations, but with differential Ca2+ activity manifested asdifferent number of active cells and oscillation frequencies(Fig. 1A and B). These spontaneous Ca2+ elevations wereabolished by perfusion with 1 M thapsigargin for 3045 min(3 slices), which depletes the internal stores by inhibiting theCa2+ ATPase (Araque et al. 1998), but were insensitive to 1M TTX, which prevents action potential generation (Fig. 1B),indicating that they were mediated by Ca2+ release frominternal stores and were independent of action potential-mediated neuronal activity. Therefore, human astrocytesdisplay Ca2+ excitability that is due to intrinsic propertiesindependent of neuronal network activity.

Murine astrocytes express a wide variety of neurotransmit-ter receptors coupled with intracellular Ca2+ signaling (Verkh-

ratsky et al. 1998; Perea and Araque 2005b; Volterra and

Meldolesi 2005; Haydon and Carmignoto 2006; Perea et al.

2009). We then asked whether the Ca2+ signal in human astro-

cytes could be triggered by activation of neurotransmitterreceptors that are known to mediate neuronastrocyte com-

munication in rodents. We locally applied either ATP (20

mM), glutamate (0.8 mM), or the CB1 receptor agonist WIN

(300 M), from a micropipette (tip diameter: 2 m) by ionto-

phoresis or pressure pulses (25 s duration). Local application

of these agonists evoked transient Ca2+ elevations in astro-

cytes in both hippocampal and cortical slices (Fig.1Cand D).

While the Ca2+ signal evoked by WIN was restricted to a small

area under the delivery micropipette, those evoked by gluta-

mate or ATP propagated concentrically as waves to larger dis-

tances at similar rates (30 m/s) in hippocampal and cortical

slices (Fig. 1D and E) (cf. Oberheim et al. 2009; Kuga et al.

2011). Both the Ca2+

signal extension and the propagationspeed were unaffected by TTX (Fig. 1E) but abolished by

thapsigargin (5.7 0.8% from control; 3 slices). Although an

indirect effect mediated by neuronal activation cannot be

totally ruled out due to the lack of receptor specicity, the

insensitivity of the astrocytic responses evoked by these trans-

mitters to TTX suggests a direct activation of astrocytic recep-

tors. Taken together, these results indicate that human

astrocytes responded to transmitter receptor activation with

Ca2+ elevations mediated by Ca2+ mobilization from internal

stores, and suggest that they could respond to synaptically

released neurotransmitters.To test this hypothesis, we recorded astrocyte Ca2+ levels in

hippocampal and cortical slices while electrically stimulatingaxons with electrodes placed 75150 m away from therecorded cells. Electrical simulation effectively elicited neuro-transmitter release, as conrmed by the excitatory postsyn-aptic potentials (EPSPs) and currents (EPSCs) recorded inneurons (Fig.2A,C, F). Trains of electrically stimuli (30 Hz, 5s) reliably evoked Ca2+ elevations in 76% of recorded astro-cytes (n = 29 astrocytes; 11 slices) (Fig. 2D and E). In somecases, the Ca2+ signal was also monitored in patch-clamp re-corded cells identied as astrocytes by their electrophysiologi-cal properties, that is, high membrane conductance and theabsence of action potentials (Fig. 2Band D), conrming thatresponding cells were astrocytes (cf. Bordey and Sontheimer1998; Hinterkeuser et al. 2000; Schrder et al. 2000). These

2 AstrocyteNeuron Signaling in Human Brain Tissue Navarrete et al.


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Figure 1. Ca2+ signal in human astrocytes in situ. (A) Pseudocolor images ofuo-4-lled hippocampal and cortical slices, and representative Ca2+ levels showing spontaneousCa2+ elevations from hippocampal and cortical astrocytes. Scale bar: 60 m (left), 40 m (right). (B) Relative number of active astrocytes and oscillation frequency in controland TTX (n = 46 and 39 astrocytes for the hippocampus and the cortex, respectively; n 6 slices for each bar). (C) Fluorescence images of a uo-4-loaded hippocampal slicedepicting the experimental arrangement (left), and Ca2+ levels 10 s before and after ATP application. Scale bar 60 m. (D) Astrocyte Ca2+ responses (at the regions shown inC) to 2 s application of WIN (300 M), glutamate (0.8 mM), or ATP (20 mM). (E) Astrocyte Ca 2+ wave extension and speed in control and TTX (n 4 slices for each bar). Errorbars indicate SEM. *P < 0.05.

Figure 2. Human astrocytes respond with Ca2+ elevations to synaptic activity. (A) Depolarization of recorded neurons (left) elicits action potential ring (right). Scale bar 15 m.(B) Typical passive electrophysiological responses of astrocytes to voltage pulses, and I-V relationships of neurons and astrocytes (n 5 cells for each bar). (C) Representativetraces of evoked EPSPs (top) and EPSCs (bottom) in recorded neurons by electrical stimulation. (D) Representative uorescence images of Ca2+ levels of a patch-clampedastrocyte lled with uo-4 before and after a train (30 Hz, 5 s) of axonal stimulation. Scale bar 10 m. (E) Astrocyte Ca 2+ responses in 7 astrocytes elicited by 2 consecutivetrains of electrical stimulation (30 Hz, 5 s; S1, S2; arrows). (F) Representative traces of evoked EPSCs in recorded neurons before and after a train of electrical stimulation (30 Hz,5 s). (G) Astrocyte Ca2+ responses of 9 astrocytes to trains of electrical stimulation (arrows) before (Control) and after perfusion with TTX. (H) Relative changes from controlrecordings of astrocyte Ca2+ signal evoked by electrical stimulation in the presence TTX (1 M; n = 14 astrocytes from 4 slices). Error bars indicate SEM. ***P < 0.001.

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astrocyte responses were abolished by TTX (11.4 4.0% fromcontrol values, n = 4 slices; Fig. 2G and H), indicating thatthey were unlikely due to direct astrocyte stimulation butrather depended on synaptic activity. Furthermore, althoughtrains of high frequency nerve stimulation were used toclearly reveal the astrocyte responsiveness to synaptic activity(cf.Araque et al. 2002,Perea and Araque 2005a,b;Haas et al.2006), the sensitivity of the astrocytic responses to TTX

(Fig. 2G and H), the fact that synaptic currents could bereliably evoked after train stimulation (Fig. 2F) and that suc-cessive trains of electrical stimuli could reliably induce astro-cytic responses (Fig.2E) indicate that they are mediated by aphysiological process, that is, action potential generation,rather than resulting from artifact damage. Therefore, humanastrocytes respond to synaptic activity with Ca2+ elevations,indicating the existence of neuron-to-astrocyte communicationin human brain tissue.

We next investigated the consequences of astrocyte Ca2+

signal on human neurons. In hippocampal slices, local appli-cation of ATP evoked astrocyte Ca2+ elevations that propa-gated as a wave throughout the Stratum radiatum reaching

the Stratum pyramidale, and then evoking Ca2+

elevations inpyramidal neurons after a conspicuous delay from the initialastrocyte Ca2+elevations (Fig. 3A), suggesting that astrocyteCa2+ stimulates the release of gliotransmitters that acting ontransmitter receptors affect the intracellular Ca2+ levels inhuman neurons.

Murine astrocytes can release the gliotransmitter glutamate,which activating NMDARs evokes SICs in neurons (Parri et al.2001;Fellin et al. 2004;Perea and Araque 2005a;Haydon andCarmignoto 2006; Navarrete and Araque 2008; Shigetomiet al. 2008;Perea et al. 2009;Bardoni et al. 2010;Sasaki et al.2011), we asked whether this signaling also occurred inhuman brain slices. We recorded hippocampal and cortical

whole-cell currents from neurons in the absence of extracellu-

lar Mg2+ to maximize NMDAR activation. In these conditions,the presence of spontaneous SICs, which were distinguishedfrom mEPSCs by their relatively slower time courses (seeMaterials and Methods section) (Fig. 3BD; cf. Perea and

Araque 2005a;Shigetomi et al. 2008;Sasaki et al. 2011), wasobserved (mean amplitude: 23.3 4.3pA; n = 46 from 6neurons; Fig. 3BD; cf. Perea and Araque 2005a; Shigetomiet al. 2008; Sasaki et al. 2011). Local application of ATP,

which elevated Ca2+ levels in astrocytes (Fig. 3A), also in-creased the frequency of SICs in both hippocampal and corti-cal neurons (Fig. 3E and F). While SIC frequency wasinsensitive to TTX (n = 3 neurons), SICs were abolished by 50M AP5, indicating that they were independent of action

potential-evoked neurotransmitter release and that they weremediated by NMDARs (Fig. 3E and F). Therefore, in agree-ment with compelling evidence obtained in rodents (Parriet al. 2001;Fellin et al. 2004;Perea and Araque 2005a;Navar-rete and Araque 2008; Shigetomi et al. 2008; Bardoni et al.2010;Sasaki et al. 2011), Ca2+ elevations in human astrocytesstimulate the release of glutamate that activates NMDARs inneurons, indicating the existence of gliotransmission andastrocyte-to-neuron communication in human brain tissue.

Discussion

Present results indicate that human astrocytes in situ displayspontaneous Ca2+ elevations, respond with Ca2+ elevations to

applied neurotransmitter receptor agonists, and, more impor-tantly, to synaptically released neurotransmitters. Human as-trocytes are also able to release the gliotransmitter glutamate,

which activates postsynaptic NMDARs and evoke SICs inneurons. Taken together, these results demonstrate thathuman astrocytes not only sense synaptic activity, but alsoregulate neuronal excitability through glutamate release,showing the existence of bidirectional communication

between neurons and astrocytes in human brain tissue andsuggesting that astrocytes are playing active roles in human

brain function. Furthermore, the fact that astrocytes andneurons establish functional units in human brain tissue indi-cate that Tripartite Synapses, as functional entities originallypostulated in rodents, are also present in more phylogeneti-cally evolved brains, perhaps as an intrinsic propertycommon to all nervous systems.

Cell physiology studies on human brain tissue are necess-ary limited and present several constraints that restrict thefeasible experimental procedures. Furthermore, the accessiblesamples are far from ideal control samples. For example,present work is based on tissue obtained from patients that

usually suffered long-lasting epileptic conditions and under-went pharmacological treatments. Nevertheless, present studyshows that the basic principles of the neuronastrocyte com-munication previously described in animal models, that is,astrocyte responsiveness to synaptic activity and astrocyteability to release gliotransmitters that act on neurons, exist inhuman brain. Further studies are required to fully characterizethe properties of the astrocyteneuron signaling in the humanepileptic tissue and their role in the pathophysiologicalprocess of the temporal lobe epilepsy.

One potential concern about the physiological relevanceof data obtained in human brain tissue derives from the factthat the biological sample may correspond to unhealthy orabnormal tissue, which could render results more related to

pathological rather than physiological phenomena. Further-more, normal physiological conditions may be altered by theprevious pharmacological treatment of patients as well as bythe procedures involved, from the clinical surgery until thetissue samples reach the experimental bench. However,

besides the well-documented morphological alterations ofthe epileptic tissue (e.g. Honavar and Meldrum 1997;Mathern et al. 2000; Arellano et al. 2004; Alonso-Nanclareset al. 2011), the cellular properties of the recorded cells inthe present study indicate that the cellular viability is largelypreserved in these cells. Indeed, recordings of calcium-basedcellular activity as well as electrophysiological parameters ofneurons and astrocytes (membrane resting potential, I-V

curves, action potential amplitudes, synaptic currents, etc.;Fig. 2A and B) suggest that the observed phenomena do notresult from a damaged tissue. Furthermore, present resultsshow similar properties of the spontaneous astrocyte calciumsignal in human and rodent tissues (Aguado et al. 2002;Nettet al. 2002; Takata and Hirase 2008; Kuchibhotla et al. 2009;Sasaki et al. 2011), suggesting that they reect normal phys-iological characteristics. Nevertheless, we cannot discard thatthe normal properties were actually altered due to the patho-logical conditions.

The presence of NMDAR-mediated SICs induced by gluta-mate released from astrocytes have been shown in differentrodent brain areas, specically, CA1 hippocampal area, ven-trobasal thalamus, nucleus accumbens, and neocortex (Parri



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et al. 2001;Angulo et al. 2004;Fellin et al. 2004;Fellin et al.2006;Perea and Araque 2005a;Ding et al. 2007;Nestor et al.2007;Navarrete and Araque 2008;Shigetomi et al. 2008;Pirt-timaki et al. 2011; Sasaki et al. 2011). Whether these eventsare general or specic of certain brain regions remainsunknown. Perhaps, the existence of SICs requires specicstructural and functional interactions, such as spatial locationof glutamate release sources, spatial distribution of glutamatetransporters, or reduced glutamate accessibility to postsyn-aptic NMDA receptors. The physiological meaning of theseSICs in human tissue is unknown, as in rodents, althoughthese currents have been shown to depolarize postsynapticneurons and to serve as a synchronizing mechanism for

neuronal activity (Angulo et al. 2004; Fellin et al. 2004). Incontrast to the available data obtained in rodent brain slices,the presence of SICs and their role in vivo still need exper-imental conrmation. Nevertheless, the presence of SICs inhuman brain tissue indicates the existence of structural andfunctional relationships between astrocytes and neurons inhuman brain, and demonstrates the ability of human astro-cytes to release gliotransmitters that can activate neuronalreceptors.

Studies performed in animal models reveal a high richnessin the signaling processes and physiological consequences ofthe astrocyteneuron communication (Volterra and Meldolesi2005; Haydon and Carmignoto 2006; Perea et al. 2009). It is

Figure 3. Human astrocytes release glutamate that activates NMDA receptors in postsynaptic neurons. (A) Left: uorescence images of a uo-4-loaded hippocampal sliceshowing Ca2+ levels before (basal) and after (5 and 10 s) ATP application. Right: ATP application (arrow) evoked Ca2+ elevations in astrocytes (red traces corresponding toastrocytes marked with red circles in A) and delayed Ca2+ elevations in neurons (blue traces corresponding to neurons marked with blue circles in A). Scale bar 30 m. (B)Whole-cell currents from a human neuron showing spontaneous SICs (red asterisks; expanded at bottom) and mEPSCs (blue asterisks; expanded at bottom) in control and in thepresence of 50 M AP5. Note that SICs were absent in AP5. (C) Time course parameters of mEPSCs recorded in human neurons (n 18 for each bar from 5 neurons). mEPSCsdecay time courses were tted to 2 exponential functions with 2 time constants ( fast and slow ). (D) Time course parameters of spontaneous and ATP-evoked SICs (n 35 foreach bar from 6 neurons). (E) Whole currents from human neurons. ATP application (arrows) evoked a SIC (expanded at bottom) in control but not in AP5. (F) Mean frequency ofneuronal SICs in control, after ATP, and in AP5 (n 5 neurons for each bar). Results from hippocampal and cortical neurons were similar and pooled together. Error bars indicateSEM. *P < 0.05, ***P < 0.001.

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noteworthy that due to technical limitations, the analysis ofthe astrocyte calcium signal in the present study was restrictedto global calcium events occurring at the astrocytic cell body.However, the actual calcium signaling events in astrocytes areunderestimated in this analysis because recent studies have re-

vealed the existence of important calcium signals limited toastrocytic processes that may have important implications insynaptic physiology (Di Castro et al. 2011; Panatier et al.

2011). The fact that the basic cellular machinery is present inhuman brain tissue, that is, astrocyte Ca2+ signal triggered byneurotransmitter receptors activated by neurotransmitters re-leased during synaptic activity, astrocytic gliotransmitterrelease, and neuronal receptor activation by gliotransmitters,suggests that the more complex properties described inrodent brains, such as astrocyte processing of synaptic infor-mation and complex modulation of synaptic activity and plas-ticity by multiple astrocytic signals (Perea and Araque 2005a,2007;Serrano et al. 2006;Henneberger et al. 2010;Navarreteand Araque 2010;Santello et al. 2011;Navarrete et al. 2012),may also take place in human brain. Perhaps even more intri-cate relationships between astrocytes and neurons may exist

considering the higher structural complexity of human astro-cytes (Ramn y Cajal 1913; Oberheim et al. 2006,2009;Matyash and Kettenmann 2010).

Our current knowledge of the morphological and physio-logical properties of human astrocytes is largely incomplete(see Matyash and Kettenmann 2010). However, Ramn yCajal (1913) reported that human cerebral cortex differedfrom other animals in the higher number of astrocytes andtheir richer arborizations, and more recent studies have ele-gantly provided further insights in the morphological proper-ties of human astrocytes, revealing their higher complexityand heterogeneity as well as unique characteristics (Oberheimet al. 2006,2009). Little is also known about the physiologicalproperties of human astrocytes. Some studies have explored

their electrophysiological properties (e.g.Bordey and Sonthei-mer 1998; Hinterkeuser et al. 2000; Schrder et al. 2000;Seifert et al. 2004, 2006; Black et al. 2010; Matyash and Ket-tenmann 2010), and their Ca2+-based excitability has been re-cently reported (Oberheim et al. 2009). However, nothing isknown about the possible functional interactions withneurons, that is, their ability to respond to synaptic activityand to release gliotransmitters that act on neuronal receptors.

To our knowledge, this study represents the rst demon-stration of the existence of these functional properties in as-trocytes interactions, suggesting the presence of bidirectionalcommunication between astrocytes and neurons in the human

brain.

While recent reports have questioned the relevance of as-trocyte Ca2+ signaling on neurophysiology (seeAgulhon et al.2008), accumulating evidence continue to conrm the rel-evance of this signaling in the detection and control of neur-onal and synaptic activity (Haydon and Carmignoto 2006;Serrano et al. 2006;Perea and Araque 2007;Perea et al. 2009;Henneberger et al. 2010;Navarrete and Araque 2010;Panatieret al. 2011;Santello et al. 2011;Navarrete et al. 2012). Presentresults add further evidence that supports such relevance,indicating that Tripartite Synapses also exist in human brain.

In conclusion, present data show that human astrocytesdetect synaptic activity and release gliotransmitter that affectneuronal function, indicating the existence of functional bidir-ectional communication between neurons and astrocytes in

human brain tissue. These results indicate that astrocytes arerelevant in human neurophysiology and that astrocyteneuron signaling is involved in human brain function.

Conict of Interest: None declared.

Funding

This work was supported by grants from Ministerio deCiencia e Innovacin, Spain (BFU2010-15832, CSD2010-00045), European Union (HEALTH-F2-2007-202167), andCajal Blue Brain to AA, and ISCIII (PS09/02116), Spain, to J.P.and R.G.S. G.P. is supported by a Marie Curie InternationalOutgoing Fellowship (FP7-253635).

Notes

We thank W. Buo, E.D. Martin, and A. Perez-Alvarez for helpfulcomments, and Ivn Rodrguez for technical assistance with tissuesamples.

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