Involvement of P2X and P2Y receptors in microglial activation in vivo Microglial cells are the primary immune effector cells in the brain . Extracellular ATP , e.g. , released after brain injury , may initiate microglial activation via stimulation of purinergic receptors . In the rat nucleus accumbens -LRB- NAc -RRB- , the involvement of P2X and P2Y receptors in the generation of microglial reaction in vivo was investigated . A stab wound in the NAc increased immunoreactivity -LRB- IR -RRB- for P2X1 ,2,4,7 and P2Y1 ,2,4,6,12 receptors on microglial cells when visualized with confocal laser scanning microscopy . A prominent immunolabeling of P2X7 receptors with antibodies directed against the ecto - or endodomain was found on Griffonia simplicifolia isolectin-B4-positive cells . Additionally , the P2X7 receptor was colocalized with active caspase 3 but not with the anti-apoptotic marker pAkt . Four days after local application of the agonists α , βmeATP , ADPβS , 2MeSATP , and BzATP , an increase in OX 42 - and G. simplicifolia isolectin-IR was observed around the stab wound , quantified both densitometrically and by counting the number of ramified and activated microglial cells , whereas UTPγS appeared to be ineffective . The P2 receptor antagonists PPADS and BBG decreased the injury-induced increase of these IRs when given alone and in addition inhibited the agonist effects . Further , the intra-accumbally applied P2X7 receptor agonist BzATP induced an increase in the number of caspase-3-positive cells . These results indicate that ATP , acting via different P2X and P2Y receptors , is a signaling molecule in microglial cell activation after injury in vivo . The up-regulation of P2X7-IR after injury suggests that this receptor is involved in apoptotic rather than proliferative effects . Introduction Microglial cells are the principal intrinsic immune competent cells of the brain . Resting microglial cells with a ramified morphology become activated under almost all pathological conditions , e.g. , mechanical or chemical injury or inflammation . They change into ameboid-shaped motile phagocytotic and cytotoxic cells , which invade the site of injury and initiate repair or defense processes by secreting proteases , neurotrophic factors , and inflammatory mediators -LSB- 1 , 2 -RSB- . This suggests that microglial cells are able to sense signal molecules entering the extracellular space by disturbances of brain homeostasis and promoting their transformation into the active state -LSB- 3 , 4 -RSB- . Further , microglia express major histocompatibility complex antigen class II and may perform phagocytotic activity to clear cellular debris -LSB- 4 -- 7 -RSB- . One of the most prominent compounds released by injury is the purine nucleotide ATP . Extracellularly applied ATP induces the activation of a cation conductance and a subsequent increase in intracellular Ca2 + -LSB- 8 -RSB- . ATP was shown to stimulate microglial cells to respond with rapid movements of their processes -LSB- 9 -RSB- . Further , ATP also induced the outgrowth of processes and membrane ruffling in cultured systems -LSB- 10 , 11 -RSB- . Dependent on the concentration of ATP applied to in vitro systems , microglial cells secrete various biologically active substances , such as the cytokines tumor necrosis factor-α -LRB- TNF-α -RRB- -LSB- 12 -RSB- , interleukin -LRB- IL -RRB- -1 β -LSB- 13 , 14 -RSB- , or IL-6 -LSB- 15 -RSB- . Boucsein and colleagues -LSB- 16 -RSB- have shown that in the presence of purinergic ligands , the lipopolysaccharide-induced release of TNF-α , IL-6 , IL-12 , and macrophage inflammatory protein-1α was attenuated . In patch-clamp investigations with ATP-induced Ca2 + wave recordings of astrocytes , a current response in an adjacent microglial cell was observed , suggesting that microglia can sense the activity of astrocytes -LSB- 17 -RSB- . All these data suggest that a variety of ATP-sensitive ionotropic -LRB- P2X -RRB- and metabotropic -LRB- P2Y -RRB- receptors is expressed at resting and/or activated microglial cells -LSB- 18 -- 22 -RSB- . The functional expression of ATP receptors on cultured microglia is well known -LSB- 18 , 23 , 24 -RSB- . The basic problem in studying this type of glial cells is that any manipulation ex vivo or in vitro will promote their transformation into the active form . We used an in vivo model of a stab wound injury in the rat nucleus accumbens -LRB- NAc -RRB- to observe the P2 receptor-mediated response of microglial cells to ATP released by injury . This technique enables local administration of receptor-specific purinergic ligands by microinfusion . Furthermore , our investigations focused especially on the P2X7 receptor subtype on microglial cells in vivo and the functional consequences of injury-induced stimulation . The possible involvement of P2X7 receptors in apoptotic and proliferative processes in microglia and astrocytes , respectively , was also investigated . Material and methods Materials Ketamine hydrochloride -LRB- Ketanest ; Ratiopharm , Ulm , Germany -RRB- , xylazine hydrochloride -LRB- Rompun ; Bayer , Leverkusen , Germany -RRB- , and thiopental natrium -LRB- Trapanal , Altana Pharma , Konstanz , Germany -RRB- were used for anesthesia . For intra-accumbal microinjection , 2-methylthioATP -LRB- 2MeSATP -RRB- , α,β-methyleneATP -LRB- α , βmeATP , both RBI ; Natick , MA , USA -RRB- ; adenosine 5 ′ - -LRB- β-thio -RRB- diphosphate -LRB- ADPβS -RRB- , 2 ′ ,3 ′ - O - -LRB- 4-benzoyl-benzoyl -RRB- ATP -LRB- BzATP -RRB- , brilliant blue G -LRB- BBG -RRB- , and 5-bromo-2 ′ - deoxyuridine -LRB- BrdU -RRB- -LRB- all from Sigma , Deisenhofen , Germany -RRB- ; uridine 5 ′ - O - -LRB- γ-thio -RRB- triphosphate -LRB- UTPγS , Inspire Pharmaceuticals Inc. , Durham , NC , USA -RRB- ; pyridoxal-5 ′ - phosphate-6-azophenyl-2 ′ ,4 ′ - disulphonic acid -LRB- PPADS ; Biotrend , Köln , Germany -RRB- ; and artificial cerebrospinal fluid -LSB- ACSF -LRB- in mM -RRB- : 126 NaCl ; 2.5 KCl ; 1.2 NaH2PO4 ; 1.3 MgCl2 , and 2.4 CaCl2 ; pH 7.4 -RSB- were used . Antibodies directed against glial fibrillary acidic protein -LRB- GFAP ; mouse anti-GFAP , Sigma , Deisenhofen , Germany ; rabbit anti-cow GFAP , DakoCytomation , Hamburg , Germany -RRB- ; BrdU -LRB- mouse anti-BrdU , Clone Bu20a ; DakoCytomation -RRB- , active caspase 3 -LRB- rabbit , Promega , Madison , WI , USA -RRB- ; OX 42 -LRB- CD 11b , mouse anti-rat , complement receptor type 3 ; Serotec , Oxford , UK -RRB- ; -LRB- a -RRB- lectin from Bandeiraea simplicifolia -LRB- Griffonia simplicifolia -RRB- isolectin B4 -LRB- BSI-B4 , peroxidase labeled -RRB- and -LRB- b -RRB- lectin from B. simplicifolia -LRB- G. simplicifolia -RRB- isolectin B4 -LRB- GSA-B4 , biotin conjugated ; Sigma , Deisenhofen , Germany , each -RRB- ; phosphorylated serine/threonine kinases -LRB- pAkt1/2/3 ; rabbit , Ser 473 , raised against the short amino acid sequence containing phosphorylated Ser 473 of Akt 1 , Akt 2 , and Akt 3 of mouse origin , recommended for the detection of Ser 473 phosphorylated Akt 1 and corresponding phosphorylated Akt 2 and Akt 3 -LRB- Santa Cruz Biotechnology , Inc. , Santa Cruz , CA , USA -RRB- ; and the following P2 receptor antisera were used : rabbit anti-P2X1 , anti-P2X2 , and anti-P2X4 ,5,6 -LRB- Alomone Labs , Jerusalem , Israel -RRB- as well as rabbit anti-P2X7 receptor-subtype -LRB- intracellular C-terminus binding , Alomone Labs -RRB- , anti-P2X3 -LRB- guinea pig ; Neuromics Inc. , Northfield , MN , USA -RRB- . Furthermore , a goat anti-rat ecto-P2X7 receptor antibody -LRB- a kind gift of Dr. M. Kim ; -LSB- 25 -RSB- -RRB- was used for immunofluorescence double-labeling studies . Additionally , anti-P2Y1 , anti-P2Y2 , anti-P2Y4 , anti-P2Y12 -LRB- rabbit ; Alomone Labs , Jerusalem , Israel -RRB- , anti-P2Y6 -LRB- rabbit ; Santa Cruz Biotechnology , Santa Cruz , CA , USA -RRB- , and anti-P2Y1 -LRB- rabbit , GlaxoSmithKline , Brentford , Middlesex , UK -RRB- receptor antibodies were tested . The secondary carbocyanine -LRB- Cy -RRB- 2 - and Cy3-conjugated IgGs as well as Cy2 - and Cy3-conjugated streptavidin -LRB- Jackson ImmunoResearch , West Grove , PA , USA -RRB- were used . For histochemical detection , 3,3 ′ - diaminobenzidine hydrochloride -LRB- DAB ; Sigma Chemical Co. , St. Louis , USA -RRB- was applied . Animals Male Wistar rats -LRB- 280 -- 320 g -RRB- were housed under a 12-h light , 12 h-dark cycle and allowed access to lab food and water ad libitum . All procedures using animals were approved by the committee of Animal Care and Use of the relevant local governmental body in accordance with the law of experimental animal protection . Surgery/microinjection Anesthetized rats were fixed in a stereotaxic frame . At the coordinates AP = 1.7 mm -LRB- rostral to bregma -RRB- , L = 1.5 mm -LRB- lateral to the sagittal suture -RRB- , and V = 6.5 mm -LRB- below the surface of the hemisphere -RRB- , the injection cannulae connected to a syringe pump via an FEP tubing was inserted into the NAc -LSB- 26 -RSB- . By microinfusion , rats received ACSF , which was used as control as well as vehicle for the following P2 receptor agonists : 2MeSATP -LRB- nonselective -RRB- , α , βmeATP -LRB- P2X1 ,3 -RRB- , ADPβS -LRB- P2Y1 ,11,12,13 -RRB- , UTPγS -LRB- P2Y4 ,6 -RRB- , given as 0.1 nmol each , and BzATP -LRB- preferential P2X7 -RRB- 0.3 nmol . As antagonists , PPADS -LRB- 30 pmol ; nonselective -RRB- and BBG -LRB- 1 pmol , P2X7 -RRB- were applied . When the effects of the P2 receptor agonists were pharmacologically proven , the microinfusion of the respective antagonists preceded infusion of the agonist and antagonist mixture . As a proliferation marker , BrdU -LRB- 0.1 nmol -RRB- was applied together with the antagonist , the agonist , or ACSF alone . Test substances were injected in a volume of 1 μl at a rate of 12 μl/h . Immunocytochemical studies and double-immunofluorescence studies Immunocytochemical and double-immunofluorescence studies were done as previously described -LSB- 26 , 27 -RSB- . After a postinjection time of 2 h and 4 days , the rats were transcardially perfused under thiopental sodium anesthesia with paraformaldehyde -LRB- 2 % -RRB- in sodium acetate buffer -LRB- pH 6.5 ; solution A -RRB- followed by paraformaldehyde -LRB- 2 % -RRB- / glutaraldehyde -LRB- 0.1 % -RRB- in sodium borate buffer -LRB- pH 8.5 ; solution B -RRB- . The brains were immediately removed and stored overnight in solution B without glutaraldehyde . Serial coronal sections -LRB- 50-μm thick -RRB- from the NAc were obtained using a vibratome -LRB- Leica , Typ VT 1000S , Nussloch , Germany -RRB- and collected as free-floating slices -LRB- 0.1 M Tris ; pH 7.6 -RRB- . Immunocytochemical studies BSI immunoreactivity Free-floating sections were rinsed with 0.05 M Tris-buffered saline -LRB- TBS , pH 7.6 -RRB- and were treated with 1 % hydrogen peroxide for 30 min to inactivate endogenous peroxide activity . Immunolabeling was performed with lectin from BSI-B4 -LRB- 1:200 -RRB- in TBS containing 2 % bovine serum albumin overnight at 4 °C , followed by washing in 0.05 M Tris buffer -LRB- pH 8.0 -RRB- . Peroxidase activity was visualized with DAB -LRB- 0.07 % -RRB- containing nickel ammonium sulphate -LRB- 1 % -RRB- and hydrogen peroxide , which renders a black reaction product . Active caspase 3 immunoreactivity Free-floating sections were rinsed with 0.1 M TBS -LRB- pH 7.4 -RRB- and treated with 1 % hydrogen peroxide for 25 min to inactivate endogenous peroxide activity . Immunoreactivity -LRB- IR -RRB- was studied with rabbit anti-active caspase 3 -LRB- 1:500 -RRB- in TBS containing 10 % normal horse serum -LRB- NHS -RRB- and 0.1 % Triton X-100 overnight at 4 °C , followed by biotinylated horse anti-rabbit IgG -LRB- 1:100 , Vector Labs . Burlingame , CA , USA -RRB- and preformed streptavidin/biotin-peroxidase complex -LRB- 1:125 , StreptABComplex ; DakoCytomation -RRB- for 2 h. DAB -LRB- 0.05 % -RRB- served as chromogen . GFAP/BrdU immunoreactivity The procedure was applied to free-floating slices as previously described -LSB- 26 -RSB- . Briefly , the GFAP staining procedure was carried out with rabbit anti-cow GFAP antiserum -LRB- 1:600 -RRB- and biotinylated protein A -LRB- 1:400 ; Calbiochem , La Jolla , CA , USA -RRB- . To detect the astroglial marker , the streptavidin/biotin technique based on a StreptABComplex -LRB- 1:125 -RRB- and DAB -LRB- 0.05 % -RRB- as chromogen were used . Mitotic astrocytes were identified by immunostaining of the incorporated BrdU . After DNA denaturation -LRB- 2 N HCl -RRB- and neutralization -LRB- borate buffer ; 0.15 M ; pH 8.5 -RRB- , the slices were incubated with a mouse monoclonal antibody against BrdU -LRB- 1:75 -RRB- , followed by incubation with horse biotinylated anti-mouse immunoglobulins -LRB- 1:100 ; Vector Labs. , Burlingame , CA , USA -RRB- and with ABC Elite Kit -LRB- 1:50 ; Vectastain ; Vector -RRB- . Peroxidase activity was visualized with DAB -LRB- 0.07 % -RRB- containing nickel ammonium sulphate -LRB- 1 % -RRB- plus cobalt chloride -LRB- 1 % -RRB- -LRB- DAB-Ni/Co -RRB- and hydrogen peroxide , which renders a black reaction product . For GFAP/BrdU double-staining experiments to characterize mitogenic changes , slices were first processed for anti-GFAP labeling followed by BrdU immunolabeling . Double-immunofluorescence studies After washing with TBS -LRB- 0.05 M ; pH 7.6 -RRB- and blocking with normal goat serum -LRB- 1 % NGS -RRB- in TBS , slices were incubated in an antibody mixture of mouse anti-OX 42 -LRB- CD11b ; 1:100 -RRB- and rabbit anti-P2X receptor antibodies -LRB- P2X1 1:400 ; P2X2 1:400 ; P2X4 1:400 ; P2X5 1:400 ; P2X6 1:400 ; P2X7 1:1,000 -RRB- or P2Y receptor antibodies -LSB- P2Y1 1:400 ; P2Y2 1:1,000 ; P2Y4 1:1,000 ; P2Y12 1:400 -LRB- Alomone Labs -RRB- ; P2Y1 : 1:1,500 -LRB- GlaxoSmithKline -RRB- ; P2Y6 1:200 -LRB- Santa Cruz Biotechnology , Inc. -RRB- -RSB- with 0.1 % Triton X-100 in 1 % NGS in TBS for 48 h at 4 °C . The secondary antibodies employed for simultaneous localization of the two primary antisera were Cy2-conjugated goat anti-mouse IgG -LRB- 1:500 -RRB- and Cy3-conjugated goat anti-rabbit IgG -LRB- 1:800 -RRB- , respectively . Cy2-conjugated goat anti-guinea pig IgG -LRB- 1:400 -RRB- and Cy3-conjugated goat anti-mouse IgG -LRB- 1:1,000 -RRB- were used for visualization of guinea pig anti-P2X3 -LRB- 1:1,000 -RRB- . Sections were washed three times for 5 min each in 1 % NGS -LSB- or 5 % fetal calf serum -LRB- FCS -RRB- for guinea pig anti-P2X3 -RSB- in TBS and then incubated for 2 h in a solution containing a mixture of the secondary antibodies with 1 % NGS -LRB- 5 % FCS -RRB- in TBS . GSA-B4 and P2X or P2Y double labeling was performed in two steps : first the incubation of the slices with GSA-B4 -LRB- 1:300 -RRB- , followed by treatment with Cy2-conjugated streptavidin -LRB- 1:350 -RRB- ; and second , by incubation with rabbit P2X/Y antibodies followed by Cy3-conjugated goat anti-rabbit IgG -LRB- 1:800 -RRB- as described above . The ecto-P2X7 receptor antibody -LRB- goat anti-rat ecto-P2X7 receptor , 0.12 μg/ml -LSB- 25 -RSB- -RRB- was combined with rabbit anti-caspase 3 -LRB- 1:500 -RRB- , rabbit anti-pAkt -LRB- 1:600 -RRB- , or rabbit anti-P2X7 -LRB- C-terminal , 1:1,000 -RRB- and subsequent detection with Cy2-conjugated donkey anti-goat IgG -LRB- 1:1,000 -RRB- and Cy3-donkey anti-rabbit IgG -LRB- 1:1,000 -RRB- . Incubation of slices with goat anti-rat ecto-P2X7 -LRB- 1:600 -RRB- , rabbit anti-active caspase 3 -LRB- 1:500 -RRB- , or rabbit anti-pAkt -LRB- 1:600 -RRB- and GSA-B4 -LRB- 1:300 -RRB- was followed by treatment with Cy3-conjugated donkey anti-goat -LRB- 1:1,000 -RRB- or Cy3-conjugated goat anti-rabbit IgG -LRB- 1:400 -RRB- and Cy2-conjugated streptavidin -LRB- 1:350 -RRB- . Control experiments were carried out without the primary P2 receptor antibody or by preadsorption of the antibody with the peptides used for their generation . When slices were incubated with PBS instead of the primary antibody or with primary antibody serum , which had been preabsorbed with peptide antigen for 1 h before use , no immunofluorescence with either of the control procedures was observed . After intensive washing and mounting on glass slides , all stained sections were dehydrated in a series of graded ethanol , processed through n-butylacetate , coverslipped with entellan -LRB- Merck , Darmstadt , Germany -RRB- , and analyzed by light microscopy or confocal laser scanning microscopy . Confocal microscopy Double immunofluorescence was investigated by a scanning confocal microscope -LRB- LSM 510 , Zeiss , Oberkochen , Germany -RRB- equipped with an argon laser emitting at 488 nm -LRB- yellow-green Cy2-immunofluorescence -RRB- and a helium/neon laser emitting at 543 nm -LRB- red Cy3-immunofluorescence -RRB- . Quantification and statistical analysis Changes in BSI-B4-IR were determined by densitometry -LRB- in regions 1 and 2 , Fig. 1a -RRB- using a digital image analyzer -LRB- Diana II -RRB- in combination with an advanced image data analyzer -LRB- AIDA 2.0 -RRB- . Results are expressed as percentage of the BSI-B4-IR of the ACSF-treated sides in the same regions . Fig. 1a A rat brain slice including needle tracts and regions of interest within the nucleus accumbens -LRB- NAc -RRB- in which immunoreactivity -LRB- IR -RRB- and cells were evaluated -LRB- 1 core 1 ; 2 core 2 ; according to -LSB- 29 -RSB- -RRB- . b BSI-B4-marked microglial cells after stab-wound injury -LRB- overview -RRB- . c Quantification of the number of activated microglial cells after injury and microinjection of P2-receptor-agonists 2MeSATP , ADPβS , α , βmeATP -LRB- 0.1 nmol each -RRB- , and BzATP -LRB- 0.3 nmol -RRB- after a postinjection time of 4 days . Values are expressed as percentage of ACSF-treated controls and represent mean ± SEM of six animals per group . d -- f BSI-B4-IR of microglial cells in the NAc of rats , illustrating changes in the glial morphology : d A great number of resting -LRB- process-bearing -RRB- microglial cells -LRB- arrows -RRB- under control conditions ; e , f changes in the number of resting microglial cells -LRB- arrow -RRB- and activated microglial cells -LRB- arrowhead -RRB- after different treatment conditions -LRB- scale bar = 200 μm -RRB- . g Quantification of the effects of agonists 2MeSATP , ADPβS , α , βmeATP -LRB- 0.1 nmol each -RRB- , and BzATP -LRB- 0.3 nmol -RRB- alone and in combination with PPADS -LRB- 0.1 nmol/0 .03 nmol each -RRB- or BBG -LRB- 1 pmol -RRB- on BSI-B4-IR of microglial cells in brain slices of the NAc of the rat after a postinjection time of 4 days . Data are expressed as percentage of the ACSF-treated side and represent the mean ± SEM of six animals per group -LRB- * p < 0.05 , vs. ACSF group ; + p < 0.05 , agonist vs. antagonist/agonist group -RRB- Additionally , the number of BSI-B4-labeled ramified and activated microglial cells as well as GFAP and GFAP/BrdU double-labeled cells were counted under a light microscope -LRB- Axioskop ; Zeiss -RRB- with a 20x objective within a square -LRB- 0.5 × 0.5 mm -RRB- in region 1 -LRB- Fig. 1a -RRB- . Each value represents five replications for each condition . The results were expressed as a percentage of ACSF-treated sides of the same regions . The individual groups were compared with one-way analysis of variance -LRB- ANOVA -RRB- using Bonferroni t test . A probability level of 0.05 or less was considered to be statistically significant . Results Immunocytochemistry After mechanical injury -LRB- Fig. 1a , b -RRB- , a characteristic BSI-B4 immunolabeling around the stab wound was observed in comparison with untreated controls , decreasing in relation to the distance from the lesion . In untreated rats , a great number of resting -LRB- process-bearing -RRB- microglial cells was present -LRB- Fig. 1d , arrows -RRB- , whereas after stab-wound injury , changes in the number of resting microglial cells with retracted processes -LRB- Fig. 1e , arrow -RRB- and activated microglial cells -LRB- Fig. 1f , arrowhead -RRB- were observed . Quantification of BSI-B4-immunolabeling in the NAc -LRB- region 1 ; Fig. 1c -RRB- of rats 4 days after stab-wound injury indicated a variable influence of P2 receptor agonists on the number of activated cells . Whereas α , βmeATP had no effect , 2MeSATP was more potent in tendency than was ADPβS in increasing the number of activated microglial cells . BzATP , which was injected into the NAc in a higher dose than were the previous ligands -LSB- 28 -RSB- , also induced a marked enhancement of activated microglial cells -LRB- Fig. 1c -RRB- . UTPγS was without effect , both on the number of activated microglia and on resting microglia -LRB- not shown -RRB- . Quantification of BSI-B4-IR in the NAc -LRB- region 1 plus 2 -RRB- by densitometry showed a significant increase for all agonists used with the exception of UTPγS -LRB- not shown -RRB- when compared with ACSF-treated animals . The effects of α , βmeATP , ADPβS , and 2MeSATP could be inhibited by pretreatment with PPADS and those of BzATP by pretreatment with BBG -LRB- Fig. 1g -RRB- . PPADS and BBG given alone inhibited and failed to alter BSI-B4-IR , respectively . Double-immunofluorescence studies at P2X and P2Y receptor subtypes Multiple immunofluorescence labeling in combination with laser scanning microscopy was used to characterize the P2X/Y receptor subtype expression after injury in comparison with untreated controls . Under control conditions , no P2X or P2Y -LRB- with the exception of P2Y1 -RRB- receptor-IR was coexpressed on single GSA-B4-positive cells . The mechanical injury associated with the introduction of the injection cannula resulted in the expression of P2X1 ,2,4,7 and P2Y1 ,2,4,6,12 receptors in the lesioned area , observed earliest 2 h until 4 days after injury . An up-regulation of immunolabeling of P2X1 ,2,4,7 receptor subtypes was unequivocal ; examples are shown in Fig. 2 -LRB- a -- i -RRB- . In the affected area , P2Y1 ,2 and P2Y12 fluorescence labeling was observed on GSA-B4-positive cells with low intensity . P2Y4 labeling appeared preferentially on process-bearing microglial cells -LRB- not shown -RRB- . P2X2-IR -LRB- Fig. 2c , d -RRB- , P2X4 -LRB- Fig. 2e , f -RRB- , and P2Y6-IR -LRB- Fig. 2l , m -RRB- were found on both resting and especially on activated microglial cells , the latter being identified by the lack of processes . No immunofluorescence was observed for P2X3 ,5,6 receptor subtypes on either type of microglial cells before and after stab wounding . Fig. 2Confocal images of examples of double immunofluorescence labeling to characterize the colocalization of P2X1 ,2,4,7 and P2Y1 ,6,12 receptor subtypes on a -- f OX42 - or g -- o GSA-B4-labeled microglial cells in the NAc of rats 4 days after stab-wound injury -LRB- thin arrow process-bearing microglial cell ; thick arrow activated microglial cell -RRB- -LRB- scale bars : a , b = 20 μm ; c , d = 100 μm ; e -- i = 20 μm ; j , k = 100 μm ; l , m = 20 μm ; n , o = 50 μm -RRB- Injury-induced prominent expression of the P2X7 receptor was observed on microglial cells by double labeling with GSA-B4 -LRB- Fig. 2g -- i -RRB- and also on astrocytes , as described earlier -LSB- 29 -RSB- . The two different P2X7 receptor antibodies , one directed against the extracellular domain of the rat P2X7 receptor -LSB- 25 -RSB- and the other against its C terminus -LRB- C-terminal P2X7 -RRB- -LRB- Alomone Labs -RRB- labeled the same cells -LRB- Fig. 3a , b ; thin arrow -RRB- but also different cells -LRB- Fig. 3c , d ; thick arrow -RRB- . Additionally , the ecto-P2X7 antibody labeled GSA-positive cells -LRB- data not shown -RRB- . Fig. 3Apoptosis or proliferation after P2X7 receptor stimulation after stab-wound injury in the NAc of rats 4 days after injury . a -- d Colocalization of P2X7-IR detected by antibodies directed against the C-terminus -LRB- C-term -RRB- -LRB- b , d -RRB- and the ectodomain -LRB- a -- d -RRB- of the rat P2X7 . e -- j Confocal images of double-labeling studies : Coexpression of active caspase 3 on single GSA-B4-positive cells -LRB- e , f -RRB- and active caspase 3 and the ecto-P2X7 receptor subtype -LRB- g , h -RRB- . No double labeling of pAkT on GSA-B4-labeled microglial cells 4 days after injury -LRB- i , j -RRB- -LRB- scale bars : b -- g = 20 μm -RRB- . k Quantification of the number of active caspase 3-positive cells in region 1 -LRB- see Fig. 1a -RRB- . Values are expressed as percentage of controls and represent the mean ± SEM of six animals per group . -LRB- * p < 0.05 , vs. ACSF group ; + p < 0.05 , agonist vs. antagonist/agonist group ; # p < 0.05 , antagonist vs. antagonist/agonist group -RRB- Possible role of the P2X7 receptor after injury in vivo To find out whether the P2X7 receptor is related to apoptotic and/or proliferative processes after injury , we used the early apoptotic marker active caspase 3 and the proliferation marker BrdU . After BzATP microinjection , a significant increase in the number of active caspase-3-positive cells was found in comparison with ACSF-treated animals -LRB- Fig. 3k -RRB- . This effect was reduced by pretreatment with the P2X7 receptor antagonist BBG . Further , BBG given alone decreased the number of active caspase 3-immunopositive cells in comparison with ACSF-treated rats -LRB- Fig. 3k -RRB- . Double immunofluorescence-labeling showed colocalization of active caspase 3 at single GSA-B4-positive cells in the NAc of rats -LRB- Fig. 3e , f -RRB- . Furthermore , colocalization of the ecto-P2X7 receptor subtype on caspase-3-positive cells was also documented -LRB- Fig. 3g , h -RRB- . Additionally , a possible coexpression of the anti-apoptotic and possibly proliferation-promoting pAkt on GSA-B4-positive cells was studied 4 days after injury . However , no colocalization of pAkt with GSA-B4-positive cells -LRB- Fig. 3i , j -RRB- was found by immunofluorescence labeling . Eventually , no colocalization of P2X7 and pAkt was observed to this time point -LRB- data not shown -RRB- . A possible influence of P2X7 receptor stimulation on the increase in cell number of activated and ramified microglial cells after BzATP stimulation was also studied . Changes in the number of activated microglia are shown in Fig. 1c . By quantification of ramified -LRB- resting -RRB- microglial cells in region 1 -LRB- Fig. 1a -RRB- , an increase to 123.8 ± 5.3 % in comparison with ACSF-treated rats was found , similar to that after treatment with 2MeSATP -LRB- 124.0 ± 7.8 % -RRB- . For comparison , no characteristic changes were found after microinfusion of ADPβS -LRB- 108.3 ± 11,6 % -RRB- and α , βmeATP -LRB- 101.7 ± 6.9 % -RRB- . Besides the effects of BzATP on the number of active and resting microglial cells , no influence of BzATP treatment on the number of GFAP -LRB- specific marker for fibrous astrocytes -RRB- - positive cells -LRB- BzATP 1.7 ± 7.3 % ; BBG 0.4 ± 3.0 % ; BzATP/BBG 0.96 ± 4.8 % -RRB- was found in comparison with ACSF-treated rats . There was also no effect on the number of GFAP/BrdU double-labeled cells -LRB- proliferating cells -RRB- -LRB- BzATP 14.8 ± 4.5 % ; BBG 8.8 ± 11.2 % ; BzATP/BBG 15.9 ± 5.9 % -RRB- in comparison with ACSF-treated rats . Discussion The results presented here demonstrate the involvement of various P2 receptors in microglial response to the placement of cannulas and microinjection , both resulting in tissue damage and thereby in high extracellular ATP concentrations -LSB- 30 -RSB- . Microglial cells stimulated by ATP may participate both in neurodegeneration -LRB- apoptosis and/or necrosis -RRB- and neuroprotection . Cultured microglial cells from mouse or rat brain were shown to respond to ATP with the activation of a cationic conductance , accompanied by an increase in cytosolic Ca2 + -LSB- 8 -RSB- , followed by changes of microglial morphology -LSB- 31 , 32 -RSB- . The exposition of cultured microglial cells to ADP or ATP further induced membrane ruffling and markedly enhanced chemokinesis , a prerequisite to perform tissue surveillance in the brain -LSB- 33 -RSB- . In vivo , microglial cells are homogenously distributed within the NAc characterized by rod-shaped somata with thin and highly ramified processes extended in all directions . Injury by stab wound caused microglial cell activation around the lesion track , characterized by swollen somata and thicker processes . The additional administration of P2X and P2Y receptor agonists enhanced the occurrence of activated microglial cells detected by their BSI-IR . The observed changes suggest that the stimulation of P2X1 ,3 receptors has only minor relevance in the process of microglia activation , whereas in agreement with the subsequent pharmacological investigations , involvement of P2X7 and P2Y1 ,2,4,6,12 receptors in this process is highly probable . Whereas P2X1 -- 7 receptor-IR was absent in the nonaffected NAc , the expression of P2X1 ,2,3,4,7 receptors but not of P2X5 ,6 receptors was detected after the stab-wound lesion . P2X2 ,4,6 receptors are widely expressed in central and peripheral neurones as well as other excitable cells -LSB- 22 , 34 , 35 -RSB- , and the P2X7 receptor is expressed in macrophage-type cells such as microglia but also mediates purinergic responses in astrocytes and neurons -LSB- 36 -RSB- . Changes in microglial expression of P2X receptors -LRB- P2X1 -- 7 -RRB- during postnatal development of the rat have been described -LSB- 37 -RSB- . Obviously , microglial cells are involved in long-term trophic events such as cell proliferation , differentiation , movement , and death during development -LSB- 37 -RSB- . Upregulation of P2X1-IR at microglial cells , as found in this study , was described at activated astrocytes after stab-wound injury in the adult rat NAc -LSB- 29 -RSB- . Further , the P2X2 receptor was found to be situated at activated microglia in our study and previously at astroglial cells -LSB- 29 -RSB- . After ischemia in vivo , P2X2 and P2X4-receptor-IR in the hippocampus became up-regulated in different cellular phenotypes -LSB- 38 -RSB- . P2X2 was expressed on neuronal cell bodies and fibers in hippocampal pyramidal cells , whereas intensive P2X4-IR was localized at microglial cells . Confocal microscopic analysis with OX 42 -LSB- 39 -RSB- at organotypic cultures showed that P2X4 and P2X7 are expressed on microglia , whereas P2X1 and P2Y1 ,2,12 , although present in the slices , do not colocalize at microglia , and the P2X6 receptor is absent . P2X4 receptors seem to be functionally relevant for microglial chemotaxis , which was suppressed by the knockdown of the P2X4 receptor in cultured microglia by ribonucleic acid -LRB- RNA -RRB- interference through the lentivirus vector system -LSB- 33 -RSB- . After spinal-nerve injury , P2X4 receptor expression strikingly increased at the ipsilateral site in hyperactive microglia but not in neurons or astrocytes -LSB- 40 -RSB- . Intraspinal administration of P2X4 receptor antisense oligodeoxynucleotide decreased induction of P2X4 receptors and suppressed tactile allodynia after nerve injury . The major P2X receptor expressed at microglial cells is the P2X7 receptor , characterized in several reports in vitro -LSB- 13 , 16 , 41 -RSB- . This receptor subtype appeared to be strongly coexpressed with the microglial marker GSA-B4 after the mechanical lesion used in our study , suggesting an outstanding role of this receptor in injury-induced microglial responses . It is thought to be closely related to the immune functions of microglia , such as production of inflammatory cytokines , superoxide , and nitric oxide -LSB- 42 -RSB- . The P2X7 receptor is reported to be little susceptible to desensitization in the presence of ATP , which makes it quite different from other purinergic receptors , including the P2X4 receptor , which can be desensitized within a few minutes -LSB- 23 , 43 -RSB- . Up-regulation of microglial P2X7 receptors has been observed in several pathological models , such as in vivo ischemia -LSB- 41 , 44 , 45 -RSB- , as well as multiple sclerosis and amyotrophic lateral sclerosis of the human spinal cord -LSB- 46 -RSB- . The P2X7 receptors were also specifically up-regulated around β-amyloid plaques in a mouse model of Alzheimer 's disease -LRB- Tg2576 -RRB- -LSB- 47 -RSB- and in human proliferative vitreoretinopathy on Müller glial cells -LSB- 48 -RSB- . Within the P2Y receptor family , in our study , the P2Y1 receptor was markedly expressed on microglial cells of untreated rats and was up-regulated in concert with P2Y2 ,4,6,12 receptors after injury . In the rabbit retina , microglial cells express functional P2Y1 receptors -LSB- 49 -RSB- . Activation of these receptors stimulates phenotypic alterations characteristically for microglial activation . Our data also demonstrate a strong colocalization of the P2Y6 receptor with GSA-IR . In addition to the microglial function to build a barrier between injured and healthy tissue , these cells are able to clear dead cells or dangerous debris from brain tissue . This phagocytotic process was shown to be triggered by activation of P2Y6 receptors by the endogenous agonist UDP -LSB- 50 -RSB- . Several authors report on ATP-induced migration of microglial cells to the affected sites of the brain -LSB- 9 , 10 , 51 -- 53 -RSB- . Expression of the P2Y12 receptor on microglial cells after injury seems to be strongly related to the motility of this cell type . In primary cultured microglial cells , exposition to ADP or ATP induced membrane ruffling and markedly enhanced chemokinesis , which could be blocked by the P2Y12 receptor antagonist cangrelor -LRB- AR-C69931MX -RRB- , suggesting that Gi/o-coupled P2Y receptors are involved -LSB- 10 -RSB- . The high dynamic of intact microglial processes as well as their chemotactic response to injury or local injection of ATP was shown in mouse cortex using time-lapse , two-photon laser imaging -LSB- 9 -RSB- . Investigations in P2Y12 receptor-deficient mice gave further support that P2Y12 receptors are required for the nucleotide-evoked chemotaxis -LSB- 33 , 54 -RSB- . Eventually , expression of P2Y12 receptors was observed in native and axotomized facial nuclei , and the number of P2Y12-expressing cells increased following facial nerve axotomy , considered to be a crucial component in the regeneration cascades of motor neurons -LSB- 55 -RSB- . The published data confirm that not all known P2 receptors can be detected by their immunoreactivity . However , the mRNA of the P2X1 -- 7 -LSB- 29 -RSB- and P2Y1 ,2,4,6,12 subtypes -LSB- 27 -RSB- was detectable in the NAc of untreated rats . The P2 receptors present on microglia may be differently involved in the processes of their activation , as shown by the inhibition of BSI-B4-IR using PPADS and BBG . It can be suggested by pharmacological studies that P2Y1 and P2X7 receptors may play a predominant role in the response of microglia to the consequences of a stab wound in the NAc . Retinal detachment and application of ADPßS onto control retinas induced phenotype alterations of microglial cells -LRB- decrease of soma size , retraction of cell processes -RRB- and had no influence on microglial cell density -LSB- 49 -RSB- . In the stab-wound model , stimulation of P2 receptors with agonists preferring P2Y1 and P2X7 receptor subtypes promoted the morphological changes of resting to activated microglial cells . P2X7 receptors are ATP-gated ion channels ; their sustained activation leads to cytolysis in an apoptotic fashion in the microglia -LSB- 56 -RSB- . P2X7 receptor-mediated apoptosis involves activation of the proteolytic pathway of caspase activation , which leads to nuclear DNA damage -LSB- 57 -RSB- . In our study , immunocytochemistry showed expression of P2X7 and active caspase 3 at GSA-positive cells after injury without agonist application . The active caspase 3 has been defined as the main executioner of programmed cell death and as an early apoptotic marker -LSB- 58 -RSB- . The microinfusion of BzATP increased the number of caspase-3-positive cells , suggesting an involvement of P2X7 receptors in apoptotic changes after traumatic events . The BzATP-induced increase in ramified and activated microglia -LRB- and proliferating astrocytes -RRB- was only marginal . The phosphoinositide-3-kinase / serine-threonine-kinase Akt -LRB- PI3-K/Akt -RRB- pathway is associated with cell proliferation , promoting cell survival and inhibiting apoptosis -LSB- 59 -RSB- . Because in the present study performed at 4 days after injury no coexpression of pAkt with GSA and P2X7 was found , the evidence for a preferential apoptotic pathway triggered by P2X7 receptors is unlikely . Up-regulation of P2 receptors after mechanical injury might be both the cause and the result of the induction of apoptotic/necrotic cell death activating microglial cells with harmful or beneficial effects . The present results from in vivo investigations implicate that ATP via both P2X and P2Y receptor-stimulation acts as a signaling molecule in microglial cells in vivo . An up-regulation of P2X7-IR after injury suggests involvement of this receptor subtype in apoptotic rather than in proliferative events .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O


Acute and constitutive increases in central serotonin levels reduce social play behaviour in peri-adolescent rats Rationale Serotonin is an important modulator of social behaviour . Individual differences in serotonergic signalling are considered to be a marker of personality that is stable throughout lifetime . While a large body of evidence indicates that central serotonin levels are inversely related to aggression and sexual behaviour in adult rats , the relationship between serotonin and social behaviour during peri-adolescence has hardly been explored . There is a large body of literature that describes the association between serotonin -LRB- 5-hydroxytryptamine , 5-HT -RRB- signalling , social status , aggression and sexual behaviour in humans and animals . For instance , cerebrospinal fluid -LRB- CSF -RRB- 5-hydroxyindoleacetic acid -LRB- 5-HIAA ; the major metabolite of 5-HT -RRB- levels are inversely related to aggression in humans , primates and rodents -LRB- Higley and Linnoila 1997 ; Fairbanks et al. 2001 -RRB- . Selective serotonin reuptake inhibitors -LRB- SSRIs -RRB- , which are well-known anti-depressants , reduce impulsive aggression as well as sexual behaviour in humans -LRB- New et al. 2004 ; Olivier et al. 2006 -RRB- . Furthermore , depending on the social structure of the community , serotonergic drugs are able to interchange the dominant and subordinate status of community members -LRB- Edwards and Kravitz 1997 ; Larson and Summers 2001 -RRB- . It has been proposed that individual differences in 5-HT neurotransmission are an important neural underpinning of personality -LRB- Serretti et al. 2006 -RRB- , because central 5-HT levels are relatively stable throughout lifetime -LRB- Higley and Linnoila 1997 -RRB- . Studies showing that polymorphisms in 5-HT-related genes are linked to impulsive aggression in humans -LRB- Ferrari et al. 2005 ; Haberstick et al. 2006 ; Popova 2006 -RRB- support the idea that the serotonergic modulation of social behaviour is heritable -LRB- Higley and Linnoila 1997 -RRB- . An important form of social behaviour is social play behaviour , which is the earliest form of non-mother-directed social behaviour in young mammals . Social play behaviour consists of behaviours found in adult sexual , affiliative and aggressive encounters -LRB- Bolles and Woods 1964 ; Baenninger 1967 ; Poole and Fish 1975 ; Meaney and Stewart 1981 -RRB- . However , during social play , these behaviours are displayed in an exaggerated and/or out-of-context fashion -LRB- Poole and Fish 1975 -RRB- . In rats , a bout of social play behaviour starts with one rat soliciting another animal , by attempting to nose or rub the nape of its neck . If the animal that is solicited upon rotates to its dorsal surface , ` pinning ' is the result , i.e. one animal lying with its dorsal surface on the floor with the other animal standing over it . From this position , the supine animal can initiate another bout of play , by trying to gain access to the other animal 's neck . Thus , during social play , pinning , which is considered to be the most obvious posture in social play behaviour in rats , is not an endpoint but rather functions as a releaser of a prolonged play bout -LRB- Poole and Fish 1975 ; Pellis and Pellis 1987 ; Pellis 1988 -RRB- . The animal that is pounced upon can also respond by evading or by turning around to face the other animal . In the latter situation , a brief period of boxing/wrestling may follow , in which the animals try to push each other away . If the solicited animal evades , the other animal may start to chase it , thus making another attempt to launch a play bout -LRB- see Vanderschuren et al. 1995a , for a detailed analysis of the temporal structure of social play behaviour in rats -RRB- . It is thought that social play sub-serves the facilitation of social and cognitive development because play deprivation , i.e. social isolation during 2 weeks in peri-adolescence when social play is most abundant -LRB- i.e. from postnatal day 21 until postnatal day 35 -RRB- , leads to behavioural disturbances , most prominently in the social domain -LRB- e.g. Hol et al. 1999 ; Van den Berg et al. 1999 -RRB- . These behavioural changes are more selective than those of isolation rearing , which entails continuous isolation from weaning throughout further lifetime . This not only affects social behaviour -LRB- Ferdman et al. 2007 -RRB- but also induces behavioural disturbances related to exploratory behaviour -LRB- Heidbreder et al. 2000 -RRB- , cognition -LRB- Heidbreder et al. 2000 ; Dalley et al. 2002 ; Bianchi et al. 2006 -RRB- and reward sensitivity -LRB- Howes et al. 2000 -RRB- , suggesting that social play plays a rather selective role in the development of social skills . There is evidence indicating that 5-HT modulates social play in non-human primates . For instance , low CSF 5-HIAA levels are associated with increased solitary play -LRB- Maestripieri et al. 2006 ; Higley and Linnoila 1997 -RRB- , although these findings are difficult to interpret in terms of central 5-HT levels . The neurobiology of social play has been widely investigated in rats -LRB- Vanderschuren et al. 1997 ; Siviy 1998 -RRB- , but surprisingly , little attention has been paid to the role of the serotonergic system in rat social play itself . While treatment with para-chloro-phenylalanine or a low tryptophan diet , resulting in a decrease in central 5-HT levels , did not affect social play , fenfluramine and the SSRI fluoxetine , agents that increase extra-neuronal 5-HT levels , inhibited play -LRB- Panksepp et al. 1987 ; Knutson et al. 1996 -RRB- . Further , the 5-HT receptor agonist quipazine reduced social play -LRB- Normansell and Panksepp 1985 -RRB- . These data suggest that there is an inverse relationship between 5-HT signalling and social play . Furthermore , the findings that 5-HT modulates social behaviour in peri-adolescent animals indicates that 5-HT affects social behaviour throughout development , consistent with the proposed trait-like relationship between 5-HT and social behaviour . The serotonin transporter -LRB- SERT -RRB- critically regulates extracellular 5-HT levels by its re-uptake into pre-synaptic terminals -LRB- Lesch et al. 1996 -RRB- , and SERT knockout models would therefore be very useful to test the hypothesis that central 5-HT levels and social play behaviour are inversely related in a trait-like manner . SERT knockout mice are available -LRB- e.g. Bengel et al. 1998 -RRB- , which display reduced aggression -LRB- Holmes et al. 2002a -RRB- and social interaction -LRB- Kalueff et al. 2007 -RRB- . However , because social play behaviour in mice is much less developed than in rats -LRB- Poole and Fish 1975 ; Pellis and Pasztor 1999 -RRB- , mice are not the preferred rodent species to study age-specific patterns of social behaviour during development . As social play behaviour in rats has been well described -LRB- Bolles and Woods 1964 ; Baenninger 1967 ; Poole and Fish 1975 ; Meaney and Stewart 1981 ; Pellis and Pellis 1987 ; Pellis 1988 ; Vanderschuren et al. 1997 -RRB- , we measured social play in the recently generated SERT knockout rat -LRB- Smits et al. 2006 -RRB- , which displays a ninefold increase in extra-neuronal 5-HT levels -LRB- Homberg et al. 2007 -RRB- . In line with the findings that increases in 5-HT levels reduce aggressive and sexual behaviour , we have observed that both aggressive and sexual behaviour -LRB- Homberg et al. , unpublished observations -RRB- are reduced in the SERT knockout rat . The constitutive absence of the SERT in mice causes changes in the functioning of 5-HT receptors -LRB- Fabre et al. 2000 ; Bouali et al. 2003 -RRB- , so that changes in behaviour may be the result of adaptations that have occurred during development . Therefore , we also investigated social play behaviour in wild-type rats after acute treatment with compounds that increase central 5-HT levels , i.e. the SERT-blocker fluoxetine and 3,4-methylenedioxymethamphetamine -LRB- MDMA or `` ecstasy '' -RRB- , which causes 5-HT release by reversing 5-HT transport . Materials and methods Subjects All experiments were conducted with the approval of the animal ethics committees of the VU Medical Center , Amsterdam , The Netherlands , and the University Medical Center Utrecht , The Netherlands . Both commercially supplied and SERT knockout rats were used in this study . Male Wistar rats -LRB- Harlan , Horst , the Netherlands -RRB- arrived at the age of 3 weeks in the animal facility . SERT knockout rats , which have been generated by ethylnitrosourea-induced mutagenesis in a Wistar background -LRB- for detailed description , see Smits et al. 2006 -RRB- , were bred by crosses between outcrossed -LRB- six generations -RRB- SERT + / − rats . At the age of 2 weeks , ear cuts were taken under anaesthesia and used for genotyping . At the age of 3 weeks , male SERT − / − and SERT + / + rats -LRB- littermates served as controls -RRB- were transported from the breeding room to the experimental room . The animals were socially housed -LRB- four rats per type IV macrolon cage -RRB- under a normal day/light cycle -LRB- lights off at 7 p.m. -RRB- at controlled room temperature -LRB- 21 ± 2 °C -RRB- and relative humidity of 60 ± 15 % . Food and water were available ad libitum . Three days after arrival , the rats were weighed and handled daily until testing . Behavioural procedure Testing was performed as described previously -LRB- Vanderschuren et al. 1995b -RRB- . Rats , aged 28 -- 35 days , were tested in an acrylic plastic cage -LRB- 40 × 40 × 60 cm -LSB- l × w × h -RSB- -RRB- with approximately 2 cm of wood shavings covering the floor . The test cage was illuminated by a 25-W red light bulb mounted 60 cm above the test cage . Background noise , produced by a radio , was used to minimize the influence of extraneous sounds . Two days preceding the test , the animals were habituated to the test cage during 10 min . The animals of a test pair did not differ more than 10 g in body weight and had no previous common social experience . On the test day , test pairs were isolated for 3.5 h before the test to induce a half-maximal increase in the amount of social play behaviour -LRB- Niesink and Van Ree 1989 -RRB- . Thirty minutes before the test , pairs of animals in group 1 were treated with 0 or 1 mg/kg fluoxetine -LRB- subcutaneous -LSB- s.c. -RSB- -RRB- , animals in group 2 were pre-treated with 0 or 10 mg/kg fluoxetine and group 3 received 0 , 0.5 , 2 or 5 mg/kg MDMA -LRB- s.c. -RRB- . Pairs of similarly treated rats were tested for 15 min in the test cage and the test order was randomized across treatments . Behaviour of the animals was recorded on video tape , and analysis from the video tape recordings was performed afterwards . Coding of the drug solutions ensured that both during experimentation and analysis , the experimenter was unaware of the treatment of the animals . Using Observer 4.0 -LRB- Noldus Information Technology B.V. , Wageningen , The Netherlands -RRB- , frequencies and durations of the following behaviours was scored -- pinning : one of the animals lying with its dorsal surface on the floor of the test cage with the other animal standing over it ; pouncing : play soliciting by nosing the partner 's nape ; boxing/wrestling : facing each other in vertical position and struggling using the forepaws ; following/chasing : moving in the direction of or pursuing the test partner , who moves away ; social grooming and social exploration : sniffing or licking any body part of the test partner . Behaviour was assessed per pair of animals . Animals were used only once . Drugs Fluoxetine was purchased from Sigma-Aldrich -LRB- Schnelldorf , Germany -RRB- , and MDMA was purchased from O.P.G. -LRB- Utrecht , The Netherlands -RRB- . Both drugs were freshly dissolved in saline on the day of the experiment and injected s.c. in the flank in a volume of 2 ml/kg . Statistical analyses Data were analysed using one-way analysis of variance -LRB- ANOVA -RRB- , followed by Student -- Newman -- Keuls post-hoc tests where appropriate . The level of probability for statistically significant effects was set at p < 0.05 -LRB- n.s. = non-significant -RRB- . Results SERT − / − pairs of rats , as compared to SERT + / + pairs , showed a strong reduction in pinning frequency -LRB- Fig. 1a ; F -LRB- 2 , 13 -RRB- = 5.78 , p < 0.05 -RRB- , pouncing frequency -LRB- F -LRB- 2 , 13 -RRB- = 7.08 , p < 0.05 -RRB- and boxing/wresting frequency -LRB- F -LRB- 2 , 11 -RRB- = 9.91 , p < 0.01 -RRB- . The duration of following/chasing was significantly increased in SERT − / − rats -LRB- Fig. 1b ; F -LRB- 2 , 13 -RRB- = 6.5 , p < 0.05 -RRB- , while social grooming and social exploration were not affected by deletion of the SERT gene -LRB- social exploration : F -LRB- 2 , 12 -RRB- = 0.22 , n.s. ; social grooming : F -LRB- 2 , 12 -RRB- = 0.76 , n.s. -RRB- . Fig. 1Social play behaviour in peri-adolescent male SERT − / − and SERT + / + rats . The data represent mean ± SEM of the number of pins , pounces and boxing/wresting episodes -LRB- a -RRB- and mean ± SEM of the duration of social exploration and following/chasing -LRB- b -RRB- during a 15 min test . Asterisk , p < 0.05 SERT − / − vs SERT + / + As illustrated in Fig. 2a , 1 mg/kg fluoxetine did not affect pinning -LRB- F -LRB- 2 , 13 -RRB- = 1.33 , n.s. -RRB- , pouncing -LRB- F -LRB- 2 , 13 -RRB- = 2.56 , n.s. -RRB- and boxing/wrestling -LRB- F -LRB- 2 , 13 -RRB- = 1.75 , n.s. -RRB- . One-way ANOVA did not also reveal effects of 1 mg/kg fluoxetine on social exploration -LRB- F -LRB- 2 , 13 -RRB- = 0.55 , n.s. -RRB- , following/chasing -LRB- F -LRB- 2 , 13 -RRB- = 0.69 , n.s. -RRB- or social grooming -LRB- F -LRB- 2 , 13 -RRB- = 0.00 , n.s. ; Fig. 2b -RRB- . A higher dose of fluoxetine , 10 mg/kg , on the other hand , significantly reduced pinning -LRB- F -LRB- 2 , 14 -RRB- = 15.86 , p < 0.005 -RRB- , pouncing -LRB- F -LRB- 2 , 14 -RRB- = 36.18 , p < 0.0001 -RRB- , boxing/wrestling -LRB- F -LRB- 2 , 14 -RRB- = 23.82 , p < 0.0005 ; Fig. 2c -RRB- and following/chasing -LRB- F -LRB- 2 , 14 -RRB- = 20.17 , p < 0.001 -RRB- but not social exploration -LRB- F -LRB- 2 , 14 -RRB- = 3.03 , n.s. -RRB- and social grooming -LRB- F -LRB- 2 , 14 -RRB- = 0.03 , n.s. ; Fig. 2d -RRB- . Fig. 2Effect of fluoxetine on social play behaviour in peri-adolescent male wild-type rats . Thirty minutes before the 15-min test , the animals were treated with 1 -LRB- a , b -RRB- or 10 mg/kg -LRB- c , d -RRB- fluoxetine -LRB- s.c. -RRB- . The data represent mean ± SEM of the number of pins , pounces and boxing/wresting episodes -LRB- a , c -RRB- and mean ± SEM of the duration of social exploration , following/chasing and social grooming -LRB- b , d -RRB- . Asterisk , p < 0.05 vs saline MDMA was tested in three different doses : 0.5 , 2.0 and 5.0 mg/kg . One-way ANOVA indicated that MDMA treatment dose-dependently suppressed pinning -LRB- Fig. 3a ; F -LRB- 2 , 30 -RRB- = 28.8 , p < 0.0001 , post-hoc testing : all MDMA groups vs saline p < 0.05 -RRB- , pouncing -LRB- F -LRB- 2 , 29 -RRB- = 31.77 , p < 0.0001 , post-hoc testing : all MDMA groups vs saline p < 0.05 -RRB- and boxing -LRB- F -LRB- 2 , 29 -RRB- = 22.07 , p < 0.0001 , post-hoc testing : all MDMA groups vs saline p < 0.05 -RRB- . In addition , MDMA decreased following/chasing -LRB- Fig. 3b ; F -LRB- 2 , 29 -RRB- = 8.81 , p < 0.0005 , post-hoc testing : all MDMA groups vs saline p < 0.05 -RRB- . Social exploration -LRB- F -LRB- 2 , 30 -RRB- = 6.41 , p < 0.005 , post-hoc testing : 2 and 5 mg/kg MDMA vs saline p < 0.05 -RRB- and social grooming -LRB- F -LRB- 2 , 28 -RRB- = 24.89 , p < 0.0001 , post-hoc testing : 2 and 5 mg/kg MDMA groups vs saline p < 0.05 ; Fig. 3b -RRB- were also attenuated by MDMA treatment . Fig. 3Effect of MDMA on social play behaviour in peri-adolescent male wild-type rats . Thirty minutes before the 15-min test , the animals were treated with 0 , 0.5 , 2 or 5 mg/kg MDMA -LRB- s.c. -RRB- . The data represent mean ± SEM of the number of pins , pounces and boxing/wresting episodes -LRB- a -RRB- and mean ± SEM of the duration of social exploration , following/chasing and social grooming -LRB- b -RRB- . Asterisk , p < 0.05 vs saline ; double cross , p < 0.05 vs 0.5 mg/kg MDMA Discussion In this study , we show that increasing extra-neuronal 5-HT levels , either constitutively by deletion of the SERT gene or transiently by pharmacological manipulation -LRB- i.e. treatment with fluoxetine or MDMA -RRB- , decreases social play behaviour in peri-adolescent rats . These effects were specific for playful social behaviours , i.e. pinning , pouncing and boxing/wrestling . Mixed effects were observed for following/chasing , social grooming and social exploration . The similarities between the genetic and pharmacological manipulations of the serotonergic system suggest that reduced social play behaviour in SERT − / − rats is the result of increased extracellular 5-HT levels -LRB- Homberg et al. 2007 -RRB- . Fluoxetine and MDMA are well known to increase central 5-HT levels , but they affect dopaminergic neurotransmission as well , raising the possibility that reduced social play is due to an effect on the dopaminergic system . Fluoxetine , at 10 mg/kg as used in the present study , increases dopamine release in the frontal cortex -LRB- Tanda et al. 1994 -RRB- . MDMA is a potent releaser of 5-HT , but it releases dopamine as well , albeit at higher concentrations -LRB- for reviews , see White et al. 1996 ; Green et al. 2003 -RRB- . Although psychostimulant drugs that increase central dopaminergic neurotransmission have been found to suppress social play behaviour -LRB- e.g. Beatty et al. 1982 ; for review , see Vanderschuren et al. 1997 -RRB- , these effects are most likely mediated through non-dopaminergic mechanisms because the selective dopamine reuptake inhibitor GBR-12909 and the dopamine receptor agonist apomorphine did not suppress play -LRB- Vanderschuren et al. 2005 -RRB- . Moreover , the pre-synaptic functioning of dopaminergic , as well as noradrenergic neurons , was not altered in SERT − / − rats -LRB- Homberg et al. 2007 -RRB- . Therefore , the present findings are most likely the result of altered function of the serotonergic system , rather than the dopaminergic system . Indirect 5-HT agonists such as MDMA and parachloroamphetamine enhance locomotor activity -LRB- Geyer 1996 -RRB- , but the doses required to enhance locomotor activity in peri-adolescent rats are higher than those that suppress play in the present study -LRB- see Åberg et al. 2007 -RRB- . Fluoxetine does not alter locomotor activity -LRB- Homberg et al. 2004 -RRB- , and locomotor activity does not differ between SERT + / + and SERT − / − rats under both a novel and habituated conditions -LRB- Homberg et al. , unpublished observations -RRB- . In the present study , following/chasing , the social parameter most closely related to locomotor activity , was increased in SERT − / − rats and decreased by fluoxetine and MDMA . Thus , there is no parallel in the effects of fluoxetine , MDMA and SERT knockout on locomotor activity and following/chasing . This indicates that the reduction in social play found in SERT − / − rats and after treatment with fluoxetine and MDMA is not likely to be secondary to effects on locomotor activity . An alternative explanation of the present findings is that the decrease in social play behaviour is due to increased anxiety . Although the animals were habituated to the test cage and tested under red light conditions , reluctance to contact an unfamiliar conspecific may have interfered with our findings . SERT − / − mice display anxiety-related symptoms and reduced social interaction -LRB- Holmes et al. 2002b ; Kalueff et al. 2007 -RRB- , MDMA and fluoxetine have anxiogenic effects in a variety of tests -LRB- Lin et al. 1999 ; Morley and McGregor 2000 ; Drapier et al. 2007 -RRB- and the SSRI citalopram reduces social interaction -LRB- Dekeyne et al. 2000 -RRB- . MDMA has also been reported to decrease social investigation and aggression and to increase non-social exploration , which was interpreted as attempts to escape from the test arena , i.e. an anxiogenic-like effect of MDMA -LRB- Maldonado and Navarro 2001 -RRB- . On the other hand , pro-social effects of MDMA have also been reported -LRB- Morley and McGregor 2000 -RRB- . SERT − / − rats spent more time on following and chasing than SERT + / + rats . This suggests that while social play was reduced , SERT − / − rats were still interested in their playing partner , which argues against social anxiety as an explanation of reduced play behaviour in SERT − / − rats . Moreover , social exploratory behaviour , which is the parameter used in the social interaction test of anxiety -LRB- File and Seth 2003 -RRB- , was not affected in SERT − / − and in fluoxetine-treated rats , whereas MDMA only mildly reduced social exploration at doses that completely eliminated social play . Thus , the observed effects were relatively selective for social play , and increases in extra-neuronal 5-HT levels do not induce a generalized aversion to contact an unfamiliar rat . Long-term SSRI treatment is associated with increased extra-neuronal 5-HT levels and several compensatory adaptations -LRB- Dawson et al. 2002 -RRB- that are strikingly similar to those seen in SERT − / − rats , like desensitization of pre - and post-synaptic 5-HT1A receptors -LRB- Homberg et al. , unpublished observations -RRB- . Chronic SSRI treatment decreases aggressive -LRB- Fuller 1997 -RRB- and sexual behaviour -LRB- Olivier et al. 2006 -RRB- in rats , phenotypes we also observed in SERT − / − rats -LRB- Homberg et al. , unpublished observations -RRB- . Because acute fluoxetine and MDMA treatment cause an immediate increase in extra-neuronal 5-HT levels without compensatory adaptations , it is likely that reduced social play in SERT − / − rats is due to the increased extra-neuronal 5-HT levels , rather than compensatory adaptations . Ansorge et al. -LRB- 2004 -RRB- showed that early chronic SSRI treatment in wild-type mice induces anxiety - and depression-like phenotypes that resembles those of SERT − / − mice . It may follow that SERT − / − rats behave as being chronically treated with SSRIs , but given that acute and chronic SSRI treatment induce opposite effects -LRB- Silva and Brandao 2000 -RRB- and that the effects of anti-depressants on the immature developing brain may differ as opposed to the effects on the mature brain of adults -LRB- Ansorge et al. 2004 ; Taravosh-Lahn et al. 2006 -RRB- , further research is required to support this suggestion . Anti-depressants are frequently prescribed to children with major depressive disorder and anxiety -LRB- Cheung et al. 2006 -RRB- , while there is only sparse knowledge of the effects of SSRIs on the developing brain -LRB- Spear and Brake 1983 ; Ansorge et al. 2004 -RRB- . Because social behaviour is reduced in peri-adolescent and adult SERT − / − rats , it is likely that changes in 5-HT homeostasis found at adulthood in SERT − / − rats -LRB- Homberg et al. 2007 -RRB- reflect the situation in the peri-adolescent SERT − / − brain . We thus propose that insight into the function of the serotonergic system in the SERT − / − -LRB- peri-adolescent -RRB- brain may be helpful to gain insight into the effects of SSRIs on the developing brain . 5-HT is considered to be an important marker of personality traits such as harm avoidance -LRB- Gerra et al. 2000 -RRB- , social dominance -LRB- Edwards and Kravitz 1997 ; Larson and Summers 2001 -RRB- , social attachment -LRB- Beech and Mitchell 2005 -RRB- and social impulsivity -LRB- Fairbanks et al. 2001 -RRB- , which are relatively stable within individuals . These traits may be the result of changes in SERT function because a polymorphism in the SERT gene of humans and non-human primates , which decreases SERT expression and function -LRB- Lesch et al. 1996 -RRB- , has quite consistently been associated with childhood -LRB- Beitchman et al. 2006 ; Haberstick et al. 2006 -RRB- and adult aggression -LRB- Sakai et al. 2006 -RRB- and childhood -LRB- Nobile et al. 2004 -RRB- and adult depression -LRB- Caspi et al. 2003 -RRB- . Furthermore , this polymorphism has been associated with reduced free play behaviour in monkeys -LRB- Bethea et al. 2004 -RRB- . Anti-social behaviour in children has been suggested to be related to developmental abnormalities in the serotonergic system , and altered social behaviour during childhood likely extends into adulthood -LRB- Van Goozen and Fairchild 2006 -RRB- . Thus , the observations that SERT − / − rats display reduced social play behaviour , reduced aggression and reduced sexual behaviour is likely to be the result of constitutive disruptions in 5-HT function . On the other hand , because social play sub-serves the development of an adequate social repertoire -LRB- Vanderschuren et al. 1997 ; Hol et al. 1999 ; Van den Berg et al. 1999 -RRB- , the reduction in social play in SERT − / − rats could also itself cause changes in adult social behaviour , independent of 5-HT function . Whichever possibility holds true , we think that because of their lifelong altered 5-HT homeostasis , the SERT knockout rat model may contribute to our understanding of the role of 5-HT during the development of social behaviour .

O O O O O O B O O B I O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O 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O O O O O O O O

serotonin
social play
fluoxetine
knockout rat
mdma