activation of mouse microglial cells affects p2 receptor signaling

Ž .Brain Research 853 2000 49–59www.elsevier.comrlocaterbres

Research report

Activation of mouse microglial cells affects P2 receptor signaling

Thomas Moller ) ,1, Oliver Kann 1, Alexej Verkhratsky, Helmut Kettenmann¨Max-Delbruck-Center for Molecular Medicine, Robert-Rossle-Straße 10, 13122 Berlin-Buch, Germany¨ ¨

Accepted 12 October 1999

Abstract

Microglial cells are the immunocompetent cells of the CNS, which are known to exist in several activation states. Here weŽw 2qx .investigated the impact of microglial activation on the P2 receptor-mediated intracellular calcium Ca signaling by means of fluo-3i

based Ca2q-imaging. Cultured mouse microglial cells were treated with either astrocyte-conditioned medium to induce a ramifiedŽ . w 2qxmorphology or LPS to shift the cells toward the fully activated stage. The extracellular application of ATP 100 mM induced a Ca i

elevation in 85% of both untreated and ramified microglial cells, whereas only 50% of the LPS-activated cells responded to the stimulus.w 2qxTo characterise the pharmacological profile of microglial P2 receptors we investigated the effects of various P2 agonists on Ca ini

cultured microglial cells. Untreated and ramified microglial cells demonstrated a very similar sensitivity to the different P2 agonists. Incontrast, in LPS-activated microglia, a sharp decrease of responses to P2 agonist stimulation was seen. This indicates that microglial

w 2qxactivation influences the capability of microglial cells to generate Ca signals upon P2 receptor activation. q 2000 Elsevier Sciencei

B.V. All rights reserved.

Keywords: Microglia; P2 receptor; Calcium signaling; Receptor modulation

1. Introduction

Microglial cells carry the important function of thew xintrinsic immunocompetent cells in the CNS 19,32 . In the

normal, healthy brain they are characterised by a small cellbody with elaborated processes and a low antigenic profile

w xand are termed resting microglia 51 . Under pathologicalconditions, such as brain tissue damage, stroke or infec-tion, these cells upregulate several surface molecules suchas MHCII and their morphological appearance is character-ized by fewer and shorter processes. At the peak of theiractivation microglial cells acquire features of phagocytic,

w xcytotoxic cells 32 . Various substances, including growthfactors, cytokines, chemoattractants and neurotransmittersaffect microglial properties and activation, which has been

Ždemonstrated in in vitro and in vivo studies for review:w x.Ref. 32 . Among these substances is ATP, which is

currently regarded as a widespread co-transmitter in thew xCNS 60 and might be released by injured or dying cells

w x58 .

) Corresponding author. Department of Neurology, University ofWashington, 1959 NE Pacific Street, Box 356465, Seattle, WA 98195,USA. Fax: q1-206-616-8272; e-mail: [email protected]

1 T. Moller and O. Kann contributed equally to this work.¨

ŽATP activates a broad spectrum of P2 receptors forw x.review: Refs. 43,46 . Recent cloning efforts have re-

vealed that the P2 receptors are encoded by two distinctw x Ž .gene families 8 . The mammalian P2X receptors P2X1 – 7

are ligand-gated ionic channels, differing in their ion selec-tivity, gating properties and sensitivity to purino nu-

w xcleotides 7,11,46 . Metabotropic P2 receptors are namedthe P2Y receptors, and based on molecular evidence five

Ž .mammalian members P2Y , P2Y , P2Y , P2Y and P2Y1 2 4 6 11Ž w x.have been identified so far for review: Ref. 46 . P2Y1

and P2Y preferentially respond to purino nucleotides,11

P2Y shows a preference for UTP and UDP, respectively,6

whereas P2Y and P2Y are sensitive to both, purino- and2 4w xpyrimidine nucleotides 46 . Matching the new molecular

data with ‘classical’ pharmacological studies is a difficulttask. The former distinction between P , P , P , P ,2D 2T 2U 2X

P and P receptors was solely based on pharmacologi-2Y 2Z

cal tools, and does not find a clear representation in theŽcloned receptors for discussion of this issue, see Ref.

w x.46 . Therefore, we decided to describe our results ob-tained by the application of five different P2 agonistsŽ .Ap A, ADPbS, bgMeATP, 2MeSADP and UTP as4

agonist specific rather then receptor specific.Activation of P2 receptors has been reported to alter

w 2qxintracellular free calcium concentration Ca in manyi

0006-8993r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved.Ž .PII: S0006-8993 99 02244-1

( )T. Moller et al.rBrain Research 853 2000 49–59¨50

w x 2q Žcell types 15,46 by either promoting Ca influx iono-. w 2qxtropic pathway or triggering Ca release from InsPi 3

Žsensitive intracellular calcium pools metabotropic path-.way . Cultured microglial cells express functionally active

w xP2 receptors 27 . ATP in submillimolar concentrationsactivates a non-selective current and leads to an increase inw 2qxCa , suggesting the involvement of P ionotropici 2X

w xreceptors 57 . However, calcium signals upon ATP appli-w xcation could not be recorded by another group 59 .

To address the question, whether microglia in situexpress P2 receptors, we used the only available model tostudy physiological properties of microglia in situ, the

w xcorpus callosum slice preparation 5,35 . To get a betterinsight into the mechanisms of ATP-triggered Ca2q signal-ing and into the pharmacological profile of the ATPresponse, we used cultured mouse microglia, the mostwidespread model to study the cellular properties of mi-croglia. Once isolated from brain and taken into culture,the cells show an intermediate form of activation in regardto their antigen profile and morphology. Addition of bacte-rial components such as lipopolysaccharides result in a

Ž .further stimulation of microglia like macrophages into aw xhighly phagocytic, cytotoxic variant 1 , which is also

accompanied by changes in the expression of ionic chan-w xnels 39,40 . On the other hand, recently developed culture

models provide us with highly ramified microglia, whichw xmorphologically resemble ‘resting microglia’ 16,24 . In

the present study, we have used the different culturemodels, namely ramified microglia, believed to be a modelfor resting-like cells, LPS-stimulated cells for fully acti-vated and unstimulated cells as an intermediate form. Weaddressed the question, whether microglial activation doesinfluence the expression of P2 receptors. By using severalP2 receptor agonists we found that microglial cells expressmultiple types of P2 receptors and that responses to stimu-lation of these receptors are downregulated upon LPS-mediated activation of these cells.

2. Material and methods

2.1. Culture of microglial cells

Microglia cells were prepared from cortex of newbornw xNMRI-mice essentially as described previously 18,20 . In

brief, mice were killed by decapitation, the brain wasremoved and cortical tissue was carefully freed from bloodvessels and meninges. Tissue was trypsinized for 2 min,carefully dissociated with a firepolished pipette and washedtwice. The cortical cells were cultured in Dulbecco’s modi-

Ž .fied Eagle’s Medium DMEM supplemented with 10%Ž .fetal calf serum FCS with change of medium every third

day. After 9–12 days, microglia were separated from theunderlying astrocytic monolayer by gentle agitation, usingtheir differential adhesive properties. The supernatant was

collected, centrifuged and the resulting pellet was resus-pended. Microglial cells were plated on poly-L-lysinecoated glass coverslips at a density of 2=104 cellsrcm2

and allowed to settle for 20 min. Non-adhesive cells wereŽ .removed by washing in phosphate-buffered saline PBS

Žand cells were kept in serum-free medium Macrophage-.SFM, Life Technologies, Germany supplemented with

20% astrocyte-conditioned medium for 1–2 days beforeŽ .being used for experiments. Purity of cultures )98%

was verified by staining sister cultures with Griffoniaw xsimplicifolia Isolectin-B 50 . Astrocyte-conditioned4

Ž .medium ACM was collected from astrocytic monolayersmaintained in DMEM without serum for 3 days. Super-natant was pooled, centrifuged, filtered and stored aty708C until use.

For preparation of ramified microglial cells we used aŽ .modified astrocyte-conditioned medium MACM accord-

w xing to the techniques originally described in Refs. 53,26 .Conditioned medium was prepared from astrocytic cultures

Ž . Žstimulated with bacterial lipopolysaccharide LPS 5 mgy1 .ml for 30 min. After incubation with LPS astrocytic

cultures were washed several times with PBS and culturedfor another 24 h in DMEM. MACM was collected andprimary microglial cultures plated on glass coverslips at adensity of 1=104 cells cmy2 were incubated with thismedium. The ramification of microglial cultures appeared3–6 days after plating. For activation of microglia, culturesprepared as described above were exposed to 100 ng mly1

LPS for 12–18 h.

[ 2 q]2.2. Ca measurements in cultured microglial cellsi

Cultured microglial cells were loaded with Ca2q indica-tor by incubation of glass coverslips with adhered cells innormal physiological bathing solution supplemented with 5

Ž .mM fluo-3 acetoxymethylester fluo-3rAM and 0.02%pluronic F-127 for 30 min at room temperature. For mea-suring the intracellular calcium concentration, a confocal

Ž . Žlaser-scanning microscope CLSM Sarastro 2000 Molec-.ular Dynamics, Sunnyvale, USA was used. The scanner

Žwas mounted on an upright microscope Axioscope, Zeiss,.Oberkochen, Germany equipped with a 20=r0.6 NA

and a 40=r0.75 NA water immersion objective. Fluo-3was excited at the 488 nm line of an argon laser and thefluorescence was measured at emission wavelength above510 nm selected with a longpass filter. Images were con-structed from 128=128 pixels and were acquired every3–5 s. Fluo-3 is a non-ratiometric Ca2q indicator and doesnot provide absolute Ca2q concentrations. Therefore, theCa2q concentration changes are depicted as fluorescenceintensity ratio, FrF . The resting fluorescence value F0 0

was determined at the beginning of each experiment by theaveraging of 10 images. Changes of FrF larger then 1.20

were considered a response. Acquisition of the fluores-cence data and image analysis were performed using Im-

( )T. Moller et al.rBrain Research 853 2000 49–59¨ 51

Ž .agespace Molecular Dynamics and standard PC evalua-tion software. Significance was determined using theANOVA test and Bonferroni’s multiple comparison posttest with a confidence interval of 95%.

[ 2 q]2.3. Ca recordings from microglial cells in braini

slices

The slices for recording from microglial cells in situwere prepared essentially as described by Brockhaus et al.w x5 . Briefly, forebrain hemispheres were taken from 6–9-

Ž .day-old mice and coronal slices 200 mm thick were cutŽ .using the vibratome Vibracut, Plano, Germany . For load-

2q Žing the cells with Ca sensitive dye, slices 1 h after. Žpreparation were incubated in carbogen 95% CO q5%2

.O gassed bicarbonate-buffered physiological solution2

supplemented with 5 mM fluo-3 acetoxymethylester and0.02% pluronic F-127 for 30 min at 378C. For microfluori-metric recordings slices were placed into a chambermounted on the stage of an upright microscope connectedwith a confocal laser scanner. The chamber was continu-ously perfused with bicarbonate-buffered solution; sub-stances were introduced by changing the perfusate. Changesin fluo-3 fluorescence were measured as described above.Cells were visualized with 40=r0.75 NA water immer-

sion objective. Microglial cells on the surface of corpuscallosum slice were identified by their morphological ap-

w xpearance as described before 5 .

2.4. Solutions and reagents

All solutions were freshly prepared from refrigeratedstock solutions. Standard bathing solution for experiments

Ž .with cultured cells was composed of in mM : NaCl —150; KCl — 5.4; CaCl — 2; MgCl — 1; HEPESrNaOH2 2

— 10; glucose — 10; pH 7.4. The bicarbonate-bufferedsolution for experiments with corpus callosum slices con-

Ž .tained in mM : NaCl — 135, KCl — 5.4, CaCl — 2.5,2

MgCl — 1, NaHCO , — 25, NaH PO — 1.6, glucose2 3 2 4

— 10, pH 7.4, when continuously gassed with 5%CO r95% O . To obtain calcium-free solution, CaCl was2 2 2

omitted, MgCl was increased to 2 mM and 5 mM EGTA2

was added. Fluo-3rAM and pluronic F-127 were obtainedŽ .from Molecular Probes Eugene, OR, USA .

X ŽP2 receptor agonists, Adenosine, Adenosine-5 -O- 2-. Ž .Thiodiphosphate ADPbS , b,g-Methylene-Adenosine-

X Ž . X5 -Triphosphate bgMeATP , 2-Methylthio-Adenosine-5 -Ž . XDiphosphate 2MeSADP , Uridine-5V -Triphosphate

Ž . 1 4 Ž .UTP , P ,P -Diadenosine-tetraphosphate Ap A , Adeno-4X Ž .sine-5 -Triphosphate ATP and thapsigargin were from

w 2qx Ž .Fig. 1. ATP-induced Ca signals in microglial cells in situ and in culture. A A microglial cell shown on the surface of a corpus callosum slice. As it isiŽ .typical for microglial cells after migrating to the slice surface, the cell has a round soma with fine processes. Scale bar denotes 5 mm. B The fluorescence

w 2qx Ž . Ž .ratio FrF corresponding to changes in intracellular Ca was recorded from the cell shown in A . ATP 200 mM was applied as indicated on the0 iŽ . Ž . Ž . w 2qxgraph. C Phase contrast image of microglial cell under standard culture conditions. Scale bar denotes 5 mm. D ATP 100 mM -induced Ca i

Ž .transient recorded from the cultured microglial cell shown in C .


Ž .RBI Natick, MA, USA . All other chemicals were ob-Ž .tained from Sigma Deisenhofen, Germany .

3. Results

[ 2 q]3.1. ATP-induced Ca transients in mouse microgliali

cells

To investigate the expression of functional P2 receptorsin microglial cells in situ we used acutely prepared corpuscallosum slices from early postnatal mice. Microglial cellsappearing on the slice surface in the region of the cingu-

Ž . 2qlum Fig. 1A were loaded with Ca -sensitive dye byincubating the slice with fluo-3rAM. Using a confocalmicroscope, we could selectively record fluorescencechanges in individual microglial cells, which had migratedonto the top of the slice. The application of 200 mM ATP

w 2qxfor 30 s caused a transient increase in Ca in eight outi

of 32 cells tested. Fig. 1B shows a representative examplew 2qxof an ATP-triggered Ca transient recorded from ani

individual microglial cell in an acute slice preparation.Ž .Similarly, bath application of ATP 100 mM, 30 s

w 2qxevoked Ca elevation in cultured mouse microglialiŽ . w 2qxcells Fig. 1D . ATP triggered Ca transients in 84.9%i

Ž .of the cells tested ns602 . Thus, the data obtaineddemonstrate that microglial cells both in situ and in vitroexpress ATP receptors linked to the generation of intra-cellular Ca2q signals.

3.2. Concentration-dependence of the ATP-induced[ 2 q]Ca eleÕationi

In order to determine the concentration-dependence ofw 2qxATP-induced Ca elevation, we measured changes ini

w 2qxCa in response to 30 s bath application of ATP ini

concentrations ranging from 0.5 to 300 mM. We per-formed these and the following experiments in cultures ofpurified microglial cells because this enabled us to analysea large number of cells in one experiment. External appli-

w 2qxcation of 0.5 mM ATP did not affect Ca significantly.iw 2qxThe threshold ATP concentration, which triggered Ca i

elevation was 1 mM, while the saturation of the amplitudesw 2qx Ž .of Ca transients was reached at 100 mM Fig. 2 .i

Further increases of ATP concentration to 300 mM did notw 2qxincrease the amplitude of the Ca transient. Repetitivei

application of 100 mM ATP did not lead to a significantw 2qx Ž .reduction in the elicited Ca signals data not shown .i

The EC determined from the averaged dose response50Žcurve pooled from 63 cells was 9.2 mM "0.7 mM

.S.E.M .

[ 2 q]3.3. Dependence of ATP-induced Ca responses oni

extracellular Ca2 q

As was mentioned above, ATP may trigger calciumsignals in microglial cells through activation of both

w 2q xFig. 2. Concentration dependence of the ATP-mediated Ca transientsiŽ . Žin cultured microglial cells. A Increasing concentrations 10, 30, 100

.and 300 mM of ATP were applied to a cultured microglial cell whileŽ .recording FrF . Interval between applications was 5 min. B Dose0

w 2q xresponse relationship of the amplitudes of Ca transients induced byiŽ .ATP application 1, 3, 10, 30, 100 and 300 mM . Values of 63 cells were

normalised to the amplitude of the response to 300 mM ATP. The barsdenote the S.E.M.

ionotropic and metabotropic P2 receptors. To clarify thew 2qxpossible routes of Ca elevation upon ATP stimulation,i

we compared ATP-induced calcium signals in normal,Ca2q-containing physiological solution, with those elicitedwhile bathing cells in Ca2q-free medium. When applying100 mM ATP to cultured microglial cells in Ca2q contain-

w 2qxing solution, we observed a transient Ca elevationi

which often had a clear biphasic shape, being comprised ofŽ .an initial peak followed by a plateau phase Fig. 3A . After

removal of extracellular Ca2q ATP was still able to triggerw 2qxan increase in Ca , but in a smaller population of cellsi

Ž .78.9%; ns228, Fig. 3A . After incubation for 5–10 min2q w 2qxin Ca -free medium, the Ca transients elicited withi

ATP became clearly monophasic, being limited to the peakw 2qxcomponent only. In 66.7% of responding cells, the Ca i

following ATP washout dropped below the prestimulatedŽw 2qx .level Ca undershoot . Thus, the sensitivity of ATP-i

w 2qx 2qinduced Ca transients to the extracellular Ca favoursi

the hypothesis that both Ca2q release from intracellularstores and a transmembrane Ca2q influx are operativeupon ATP stimulation.

3.4. Inhibition of the endoplasmatic reticulum Ca2 q pumpsaffects ATP-dependent calcium signaling

w 2qxThe fact that ATP-driven Ca transients in the ma-i

jority of ATP-sensitive microglial cells persisted in Ca2q-free medium implies the involvement of metabotropic P2receptors coupled with InsP -mediated intracellular Ca2q

3


w 2q xFig. 3. ATP-triggered Ca signals are modified by changes in extra-i2q Ž .cellular Ca and thapsigargin. A A response in fluorescence ratio

FrF from a cultured microglial cell was induced by application of ATP0Ž . 2q Ž .100 mM in Ca containing bath solution left . The initial fast peak isdistinct from the following plateau. The right panel shows the responsefrom the same cell to ATP in Ca2q-free medium. Note the lack of the

2q Ž .plateau component in Ca -free solution. B A 5-min incubation with500 nM thapsigargin abolishes the ATP-induced fluorescence signal in acultured microglial cell. Thapsigargin application alone triggers an in-

Ž .crease in FrF fluorescence. C In a second population thapsigargin0Ž .500 nM application did not increase the fluorescence signal, but ATPstill was able to elicit a response after 5 min of incubation withthapsigargin.

release. To substantiate this hypothesis we used thapsigar-gin, a specific blocker of the endoplasmatic reticulum

2q w x 2qCa pump 55 , to prevent Ca accumulation in theInsP -accessible intracellular Ca2q pool.3

The application of thapsigargin caused two differenttypes of responses in microglial cells. In one population of

Ž . Žcells 53.2%, ns109 the application of thapsigargin 500. w 2qxnM, 300 s induced a transient increase in Ca whichi

presumably reflects the discharge of intracellular stores by2q w 2qxmeans of Ca leakage. After the initial increase Ca i

recovered to a new, elevated steady-state level, and stayedat that level through the experiment. ATP applied 5 minafter the beginning of the thapsigargin application failed to

w 2qx Ž .trigger Ca elevation Fig. 3B . This implies that thosei

cells are equipped with metabotropic receptors only and donot possess functional, calcium-permeable ionotropic re-ceptors.

Ž . w 2qxIn the second population 46.8%, ns109 , no Ca i

elevation in response to thapsigargin application wasrecorded. In these cells, ATP was still able to generatew 2qxCa elevation after 5 min incubation with thapsigargin,i

w 2qxhowever, the amplitude of ATP-triggered Ca transientiŽ .was reduced by 15–30% Fig. 3C . These data indicate

that ionotropic receptors contribute to the Ca2q signal inthese cells.

3.5. P2 receptor subtypes in cultured microglial cells

To distinguish between the possible involvement of P1and P2 receptors, we tested the ability of the P1 agonist

w 2qxadenosine to generate Ca responses in cultured mi-i

croglia. External application of adenosine in the concentra-w 2qxtion range 100–1000 mM failed to affect Ca in alli

Ž .cells tested ns117, data not shown . Thus, it was possi-w 2qxble to conclude that ATP action on Ca in microgliali

cells is mediated exclusively through the activation of P2receptors.

The thapsigargin data indicate the involvement ofionotropic and metabotropic receptors in the ATP-inducedw 2qxCa signals in microglia. To investigate whether mi-i

croglial cells express several P2 receptor subtypes we usedŽfive different P2 receptor agonists bgMeATP, ADPbS,

.UTP, 2MeSADP and Ap A typically used in pharmaco-4w xlogical studies 15,60 . All substances were applied at 100

mM concentration for 30 s. As is shown in Fig. 4, mi-w 2qxcroglial cells responded by Ca elevation to all agonistsi

except Ap A. The application of 100 mM Ap A failed to4 4

w 2q xFig. 4. Representative examples of Ca transients induced by differ-i

ent P2 receptor agonists. The fluorescence ratio FrF was recorded in0

response to application of 100 mM of each Ap A, 2MeSADP, UTP,4w 2q xbgMeATP, ADPbS and ATP. While Ap A failed to affect Ca , all4 i

w 2q x Žother agonists elicited Ca elevation see text and Table 1 fori.percentage of cells responding to different agonists .


Ž .Fig. 5. Heterogeneity of P2 agonist sensitivity within microglial cells. A Changes in fluorescence ratio FrF of a cultured microglial cell in response to0Ž .100 mM of bgMeATP, ADPbS, 2MeSADP and UTP. B–D Responses of three other microglial cells from the same culture to the same paradigm as

Ž .described in A . Note the heterogeneity in responsiveness of the four different cells.

w 2qx Ž .affect Ca in all cells tested ns104 . A bgMeATP-iw 2qxinduced Ca elevation was seen in 28.5% of microgliali

Ž .cells ns591, S.E.M.s12.5%, six expt. . ADPbS in-w 2qx Žcreased Ca in 78.1% of the cells ns591, S.E.M.si

. w 2qx5.6%, six expt. . UTP triggered Ca elevation in 74.8%iŽ .of cells n s 591, S.E.M.s 7.5%, six expt. , and

w 2qx2MeSADP was able to initiate Ca transients in 60.7%iŽ .of cells ns591, S.E.M.s8.9%, six expt. . These data

Ž .give rise to the following two questions: 1 do microglialcells express sensitivities to several agonists, which mightreflect the expression of several subtypes of P2 receptors

Ž .and 2 are there certain patterns of P2 agonist sensitivity?

3.6. Heterogeneity of P2 agonist sensitiÕity

To address this question we applied the agonistŽbgMeATP, ADPbS, 2MeSADP and UTP 100 mM for 30

.s, D t;120 s in a sequence. In response to these agonistsw 2qxwe recorded Ca signals from all cells in the field ofi

w 2qxview. Some of the Ca response patterns are shown ini

Fig. 5 and all are summarised in Table 1. The data indicatethat all possible combinations of cellular response patternsto the P2 receptor subtypes are possible. Moreover, theappearance of these patterns varied between different mi-croglial cultures.

Table 1Heterogeneity of P2 agonist sensitivity in microglial cultures

w 2qx Ž w 2qx w 2qx .The table compares the occurrence of Ca transients I — Ca elevation, 0 — no Ca change in response to application of bgMeATP,i i iŽ .ADPbS, 2MeSADP and UTP measured in six different cultures nsnumber of recorded cells .

Ž .bgMeATP ADPbS 2MeSADP UTP ns156 ns120 ns70 ns84 ns70 ns91 n s591 Responder"S.E.M. %tot

0 I I I 92 17 32 49 42 – 232 39.5"10.6I I I I 5 48 – 20 12 20 105 17.7"6.00 I 0 I 8 7 28 4 1 – 48 9.5"6.20 I 0 0 7 17 2 – – – 26 3.6"2.2I 0 0 0 – – – – – 22 22 4.0"4.0I I 0 I 1 1 – – – 17 19 3.4"3.10 I I 0 8 5 1 – 1 – 15 2.0"0.90 0 0 I – – 5 3 5 – 13 3.0"1.4I I 0 0 – 1 – – – 9 10 1.8"1.6I 0 0 I – – – – – 5 5 0.9"0.9I I I 0 1 2 – – – 1 4 0.6"0.30 0 I I – – – – 2 1 3 0.7"0.5I 0 I I – – – – – 1 1 0.2"0.2I 0 I 0 – – – – – – – 0.0"0.00 0 I 0 – – – – – – – 0.0"0.00 0 0 0 34 22 2 8 7 15 88 13.2"2.8


3.7. P2 receptors in ramified microglial cells

ŽDespite their very different morphology Fig. 1 vs. Fig..6 ramified microglia did not differ from the standard

culture in their sensitivity to P2 receptor stimulation: 94.7%Ž . w 2qxns94 of the ramified cells responded with Ca i

elevations to a stimulus. However, there was one promi-nent but not significant difference between microglial cellsin standard culture conditions and ramified microglia: only

Ža small population of the ramified cells 1.3%, S.E.M.s. Ž .1.3%, four expt. responded to bgMeATP 100 mM . The

percentage of cells responsive to the other agonists was notsignificantly different compared to cells maintained in

Fig. 6. Examples of FrF transients induced by P2 receptor agonists in0Ž .ramified microglial cells. A Phase contrast image of cultured ramified

Ž . w 2q xmicroglial cells. Scale bar denotes 5 mm. B and C Ca transientsi

recorded from two different ramified microglial cells from the sameculture in response to bath application of 100 mM bgMeATP, ADPbS,2MeSADP and UTP as indicated on the graph.

Fig. 7. Downregulation of P2 receptor expression of LPS-treated mi-croglia. Examples of most frequently observed fluorescence ratio FrF0

responses to P2 receptor agonists observed in LPS-treated microglialŽ .cultures. The cell in A responds to ADPbS and UTP, whereas

Ž .bgMeATP and 2MeSADP were ineffective. B shows the trace of aLPS-treated microglial cell, which only responded to UTP.

Ž .standard culture conditions Fig. 8 . The majority of rami-Žfied microglial cells showed responses to ADPbS 96.9%,

. Žns57, S.E.M.s1.9%, four expt. , 2MeSADP 78.7%,. Žns57, S.E.M.s10.4%, four expt. and UTP 96.9%,

. Ž .ns57, S.E.M.s1.9%, four expt. Figs. 6 and 8 . NoneŽof the ramified microglia was sensitive to Ap A data not4

.shown .

3.8. LPS-induced actiÕation of microglial cells affects P2receptor expression

LPS is known to activate both macrophages and mi-w xcroglia 1 . LPS-induced activation of microglia is for

example accompanied with changes in the expression ofq w xK channels 39 . We therefore decided to investigate

whether LPS treatment affects the pattern of P2 agonistsensitivity in microglia. Microglial cultures were incubatedwith 100 ng mly1 LPS for 12 to 16 h. LPS did not affectthe microglial cell survival as was ascertained by vital dye

Ž .staining Liverdead kit L-3224 from Molecular Probes .After the LPS treatment we tested for the ability of the P2

w 2qxreceptor agonists to trigger Ca elevation. Overall, iniŽthe activated microglia only 51.1% ns290, S.E.M.s

. w 2qx3.6% three expt. responded with a transient Ca in-i

crease to P2 agonist application. The majority of LPS-


Fig. 8. Summary of the percentage of cells responding to the fourdifferent agonists in microglia in unstimulated standard culture, ramifiedmicroglial cells and LPS-treated microglial cells. Error bars indicateS.E.M. The table gives the level of significance for the differencebetween the three culture conditions in regard to a given agonist. Note thesignificant downregulation of ADPbS and 2MeSADP sensitivity and thesignificant increase in non-responding cells in LPS-stimulated microglia.

treated microglial cells, which retained their sensitivity toŽP2 agonists, responded to ADPbS 44.1%, ns290,

. ŽS.E.M.s3.2% three expt. and UTP 48.8%, ns290,. w 2qxS.E.M.s4.3%, three expt. , whereas Ca responsesi

Želicited by bgMeATP 1.8%, ns290, S.E.M.s1.8%,. Žthree expt. and 2MeSADP 1.8%, ns290, S.E.M.s

. Ž1.8%, three expt. were sharply downregulated Figs. 7 and.8 .

4. Discussion

4.1. P2 receptors are the most common transmitter recep-tors in glial cells

P2 receptors are widely distributed throughout the ner-vous system and are expressed in both neurons and glial

w xcells 43 . The P2 receptor family is the only commonreceptor for all three types of glial cells, namely astrocytes,oligodendrocytes and microglia. Both ionotropic andmetabotropic P2 receptors are described in glial cell linesw x w x w x14 , in Schwann cells 34,46 , in oligodendrocytes 31

w xand in astrocytes 30,49 . ATP-induced non-selective cur-rents, which reflect activation of ionotropic P2X receptors

w xwere detected in cultured microglial cells 17,41,57 asw xwell as in microglial cells in situ 21 . Thus, ATP is the

only known agent which has the potential to activate allmajor classes of glia.

4.2. Ca2 q signaling as a readout of P2 receptor actiÕation

In this study, we employed fluorometric Ca2q measure-ments to screen responses to P2 agonist stimulation. As aconsequence, we were only able to detect P2 receptorslinked to Ca2q signalling. The P2X receptors are non-

w x 2qselective cation channels 4,56 and are Ca -permeable2q w xproviding a pathway for Ca influx 47 . The G-protein

coupled metabotropic receptors of the P2Y family areclassical 7-transmembrane G-protein coupled proteinswhich are known to activate phospholipase C-driven InsP3

production and subsequent Ca2q release from internalw xstores 15,23,43,46 .

w 2qxThe first investigation of ATP-induced Ca signal-iw x 2qing in microglia 57 postulated the exclusive role of Ca

entry through P2X receptors. In the present study weconfirmed the existence of Ca2q-permeable P2X receptors

w 2qxin a population of microglial cells by the recorded Ca i

transients after blockade of calcium release from internalstores by thapsigargin. In addition, we report the presence

Ž .of metabotropic P2Y receptors by demonstrating that: 1w 2qx 2qATP-induced Ca responses could be evoked in Ca -i

free medium indicating release from intracellular sources;Ž . 2q2 inhibition of Ca accumulation in the intracellularstores by thapsigargin reduced or, in some cells, even

w 2qx Ž .inhibited ATP-driven Ca transients; 3 agonists showniŽto have a preference for metabotropic receptors UTP,

. w 2qxADPbS triggered a Ca elevation in the majority ofiw 2qxmicroglial cells. The Ca signals induced by activationi

of metabotropic P2 receptors usually consisted of an initialpeak followed by a plateau phase, which disappeared inCa2q-free bath solution. It is likely that this secondary

2q w xplateau phase represents a capacitative Ca influx 45which was shown to be active in microglia after

w xmetabotropic receptor activation 35,38 . A previous studyprobably missed the metabotropic P2 receptors due to along incubation time in Ca2q-free medium and the result-

w xing depletion of internal stores 57 . By using the prefer-able agonist of P1 receptors adenosine, we excluded thepossible involvement of these receptors in purino receptor

w 2qxmediated Ca signaling. In addition, by using the cor-i

pus callosum preparation, we were able to show thatmicroglial cells in situ possess functional P2 receptors.

4.3. Candidates for P2 receptor expression in culturedmicroglia

For our attempt to narrow down the possible P2 recep-tors expressed in microglia we will use the classificationand pharmacological profiles excellently reviewed in Refs.w x29,46 . We will use the terms ‘‘P2X rP2Y -like recep-n n

tors’’ for the endogenous receptors of microglial cells,which is supposed to indicate the problem of matching the

Žendogenous receptors with the clones. For extensive dis-w x .cussion of this issue, see Refs. 29,46 .


We know from earlier reports that microglia cells ex-w xpress ionotropic P2X receptors 41,57 . As most studies

were done before the cloning of the today-known recep-tors, no current classification can be derived from thoseexperiments. Nevertheless, recent reports have shown that

w xmicroglial cells express the P2X -like receptor 17 . The7

P2X receptor is unique in that it can exist in an additional7

conductance state, where it forms large transmembranepores, which can pass molecules with a molecular weight

w xof up to 1 kDa 52 . It was shown that an ATP concentra-tion in excess of 100 mM was necessary to activate the

w xP2X -like receptor in the channel and pore mode 9 . Since7

we used an ATP concentration of only 100 mM, theactivation of P2X receptors seems to be unlikely under7

our experimental conditions. In addition, since Ap A was4w xshown to activate P2X -like receptors in mast cells 547

and we never recorded a response to that agonist, itbecomes unlikely that in our preparation the P2X -like7

receptor is involved in the ATP-induced signal transduc-tion. The same is true for the P2X receptor as it was also2

w xshown to respond to Ap A 42 . In contrast to the exclu-4

sions by Ap A, the responses to bgMeATP might help to4

elucidate which P2X receptor might be involved.bgMeATP was reported to activate only P2X , P2X , and1 3

w xP2X rP2X heterodimer receptors 46 . As the P2X and2 3 1

P2X receptor are fast inactivating receptors and previous3

electrophysiological studies on microglia have shown sloww xinactivating currents after ATP application 41,57 , the

expression of P2X -like or P2X -like receptors becomes1 3

unlikely too. Nevertheless, we cannot exclude any otherP2X receptor or any combination thereof to be present inmicroglia. The presence of several P2X receptor subtypesseem even more likely as there is a discrepancy betweenthe percentage of cells which respond to bgMeATPŽ .28.5% and the percentage of cells which still produce aw 2qxCa signal after blocking the stores with thapsigarginiŽ .46.8% .

The non-responsiveness of microglia to Ap A might4

also be of some help in determining the metabotropic P2Yw 2qxreceptors involved in the ATP-induced Ca signaling.i

Ap A was shown to activate recombinant P2Y receptors4 1

from chick and human as well as the endogenous P2Y -like1

receptor of guinea-pig taenia caeci and rat colon muscu-w xlaris mucosae 25,42,48 . In addition, at the concentration

used in our study, it triggered responses in human P2Y2w x w x3,33 and rat, human and turkey P2Y 2,3,13 . Based on4

these studies, it seems unlikely that neither P2Y nor P2Y1 2w 2qxor P2Y participates in the P2 agonist-induced Ca4 i

signals in cultured microglia, but one has to keep in mindthat these studies were done on cloned receptors, whichmight not resemble the endogenous receptors expressed inmicroglia.

Nevertheless, if one would consider the argument madeabove, could the not excluded receptors P2Y , P2Y and6 11

Ž .the as-yet uncloned P2Y former P account for theADP 2T

responses seen? The preferred agonist for the cloned P2Y6

receptor is UDP. This receptor also responds with twoorders of magnitude less sensitivity but within the concen-trations used in our experiments, to UTP and 2MeSADPw x37 . A P2Y -like receptor might therefore be responsible6

w 2qxfor the UTP-induced Ca signals.i

The P2Y receptor is not activated by UTP and only11

shows a very weak activation by 2MeSADP, whereasw xADPbS was not tested 12 . The major agonist for the

P2Y is 2MeSADP, but it is also activated by ADPbSADPw x22 . Based on these considerations one could argue, thatthe 2MeSADP and the ADPbS elicited signals are due tothe P2Y receptor and may be the P2Y receptor. TheADP 11

involvement of an additional receptor other than the P2YADP

receptor would explain why some cells respond to ADPbSŽ .but not to 2MeSADP Table 1 . This receptor could be the

P2Y or an as-yet uncloned P2 receptor.11

As valid as the above-made conclusions might seem, itcannot be stressed enough that sole based on pharmaco-logical tools, it seems impossible at this time to determinewith certainty the endogenous receptor profile of culturedmicroglia. To address this question one might consider theuse of RT-PCR to test for the mRNA expressed in thecells. The high variability within cells would necessitatethe use of single cell RT-PCR, which at the number ofcells needed to get representative results, is outside thescope of this paper.

4.4. The culture models for the step-wise actiÕation ofmicroglia

We used the cultured ramified microglial cells as amodel for resting microglia but would like to point out thatthe model strongly relies on morphological criteria. Mostramified microglial cells express functional purinergic re-ceptors and are in that respect similar to microglial cells

Žcultured under standard conditions which, we believerepresent a form of an intermediate activated microglial

.cell . Both, ramified microglia and microglial cells cul-tured under standard conditions show responses to ADPbS,2MeSADP and UTP, while as a difference between thetwo populations, a smaller population of ramified cells

Ž .responded to bgMeATP Fig. 8 . As data from microgliain situ are not available, it remains to be shown whethercultured ramified microglia functionally represent the rest-ing microglial phenotype.

Activation of microglial cells with LPS, a classicalw xactivating agent for microglia 1 , triggered a significant

Ž .downregulation of P2 agonist sensitivity Fig. 8 . First, thetotal number of cells bearing functional P2 receptors linkedto Ca2q signaling decreased to about half. Second, inLPS-treated cells the responses to bgMeATP and

Ž .2MeSADP were almost completely absent Fig. 8 . Thisindicates a general as well as a subtype specific downregu-lation of P2 receptor function in the course of microglialactivation.


This is supported by our in situ observation. We studiedmicroglial cells in the corpus callosum, a model for ame-boid microglial cells. Functionally, these cells share fea-tures with activated microglial cells such as high motility

w xand phagocytic activity 6 . Only a minority of these cellshad functional purinergic receptors linked to Ca2q signal-ing. All these findings indicate that the expression of alarger repertoire of purinergic receptors is not a feature ofactivated microglia, but rather of microglial cells at lowactivation stage.

4.5. Possible functional role of the microglial P2 receptors

Resting microglial cells could employ their purinergicreceptors to sense neuronal damage. As ATP is massively

w xreleased from damaged cells after neuronal injury 58 , itmight serve as a signal to initiate microglia activation. Onemay speculate that microglia downregulate this responsechain in the course of their activation to prevent the cell

2q w 2qxfrom an overload of Ca . The ATP-induced Ca i

signalling is part of the intracellular signal transductionmechanism which couples plasmalemmal P2 receptors withthe microglial intracellular machinery. As non-activatedmicroglial cells do not express voltage-activated outwardpotassium currents, the activation of P2X receptors could

w xinitiate a long-lasting depolarisation 28 . Studies fromastrocytes indicate that the activation of metabotropic P2Yreceptors has long-term mitogenic consequences and af-

w xfects the DNA synthesis and proliferation 10,36 . In mi-croglial cells, ATP stimulation induces the expression of

w ximmediate early genes 44 , which may be regarded as akey step in triggering microglia activation.

Acknowledgements

This research was supported by Deutsche Forschungs-gemeinschaft. T.M. was supported by the Schmeil-Stif-tung, Heidelberg, Germany. The authors thank G. Muller¨for excellent technical assistance.

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