Human behaviour is influenced by social norms but norms can entail two types of information. Descriptive norms refer to what others do in this context, while injunctive norms refer to what ought to be done to ensure social approval. In many real-world situations these norms are often presented concurrently meaning that their independent effects on behaviour are difficult to establish. Here we used an online Dictator Game to test how descriptive and injunctive norms would influence dictator donations when presented independently of one another. In addition, we varied the cost of complying with the norm: By stating that $0.20 or $0.50 cent donations from a $1 stake were normal or suggested, respectively. Specifying a higher target amount was associated with increased mean donation size. In contrast to previous studies, descriptive norms did not seem to influence giving behaviour in this context, whereas injunctive norms were associated with increased likelihood to give at least the target amount to the partner. This raises the question of whether injunctive norms might be more effective than descriptive norms at promoting prosocial behaviour in other settings. It is well documented that human behaviour deviates consistently from predictions based on economically-rational, agents. One domain in which the departure from expected behaviour is particularly pronounced is in social interactions with other individuals. A slew of laboratory and field studies have shown that people are often more helpful than would be predicted based on short-term, income-maximising strategies see . ExplainOne factor underlying how individuals are expected to behave in a given context is how others behave in that setting. The tendency to copy the behaviour of others is widespread in the animal kingdom and might reduce the costs of gathering and processing information required to behave optimally in a given environment is done in a given situation, injunctive norms give information about what ought to be done In other situations, the appropriate social behaviour may be made apparent by the use of injunctive norms. Whereas descriptive norms give individuals information about what Although several studies have tested the relative impact of descriptive and injunctive norms when presented together, fewer studies have asked which type of norm has the largest effect on behaviour. In order to assess the independent effects of descriptive and injunctive norms on behaviour they have to be presented in isolation. Some studies attempting to assess the independent influence of descriptive versus injunctive norms have been conducted in the health sphere and have shown that descriptive norms are often more effective than injunctive norms at eliciting the desired behaviour e.g. . For exaIn the context of social behaviour, a recent study attempted to disentangle the influence of descriptive and injunctive norms on behaviour in the Dictator Game While the study above provides a useful starting point for investigating the influence of descriptive versus injunctive norms on prosocial behaviour, there are still many gaps in our understanding. First, the study above did not compare behaviour compared to a baseline where no norm information was given to subjects. Thus, we do not know the extent to which norm information of each type influenced behaviour relative to a neutral control. Second, the study above only measured whether dictators split the endowment fairly with the receiver or not, rather than norm compliance. Crucially, individuals who were given the ‘selfish’ norm information could still have complied with the norm by giving the ‘selfish’ amount but this was not measured. Thus, it is not known from this study whether norm compliance was higher when complying with the norm was relatively cheap versus when it was relatively costly. Finally, it is possible that specifying a high level of prosocial behaviour might have the perverse effect of making individuals less likely to comply. Such an effect might arise because individuals who are unwilling to bear the costs associated with the suggested level of giving might not experience a warm glow of giving Here we used the Dictator Game, to test the impact of descriptive versus injunctive norms on voluntary donations. A previous metastudy of Dictator Games has shown that dictators typically transfer around 28% of the stake to receivers www.mturk.com). We recruited workers from the USA to play in an online Dictator Games . Of the 2,400 workers recruited to play the game, 1,200 were randomly assigned to the active role of ‘dictator’ and the remaining 1,200 to the passive role of ‘receiver’ . Since only Player 1 is active in this game, all analyses were restricted to data from Player 1 only. Prior to taking part in the Dictator Game, subjects were first asked to provide some background demographic information on their age, gender, education and income levels . The treatments varied with respect to the information that Player 1 received prior to playing the game (see below). In all treatments, Player 1 was informed that Player 2 would see the information and was required to answer two comprehension question correctly to indicate that they understood the rules of the game. Players who answered either of these questions incorrectly were not permitted to take part in the experiment.Treatment 1: Most Player 1s give $0.20 or more to Player 2 (n = 198).Treatment 2: Most Player 1s give $0.50 or more to Player 2 (n = 200).Treatment 3: It is suggested that Player 1 give $0.20 or more to Player 2 (n = 197).Treatment 4: It is suggested that Player 1 give $0.50 or more to Player 2 (n = 196).Treatment 5 (control 1): Most people on MTurk are at least 20 years old (n = 192).Treatment 6 (control 2): Most people on MTurk are less than 50 years old (n = 199).Treatment 1 and 2 were designed to test whether a descriptive norm about behaviour in the game would affect Player 1′s behaviour, and whether the cost involved in adhering to the norm ($0.20 versus $0.50) affected whether Player 1 would conform to the norm. Treatments 3 and 4 were designed to test whether injunctive norms or descriptive norms, respectively, were more effective at influencing Player 1′s behaviour. Treatments 5 and 6 were included as control treatments. We included the number ‘20’ in treatment 5 and the number ‘50’ in treatment 6 to control for the possibility that these numbers might have acted as an anchor We first asked whether descriptive or injunctive norms, respectively, were associated with increased compliance relative to a neutral control. We coded the propensity to comply with the norm information as ‘1’ if Player 1 gave at least the target amount  to Player 2 and ‘0’ if Player 1 gave less than the target amount to Player 2. We analysed data using two generalized linear models (GLM) with binomial error structure. Separate models for the $0.20 target amount and the $0.50 target amount were produced. For each analysis, a global model was specified which included the following terms: age, gender and treatment . For players in the control conditions, we specified the response as ‘1’ if the player gave at least the target amount specified by the matched norm amount , and ‘0’ otherwise. Thus, we can compare how many players give at least $0.20 or $0.50 (respectively) in the norm treatments compared to players in the matched control conditions.Next, we asked whether mean donations varied according to the norm information  or the target amount specified . Control data were not included in this analysis as no target amount was specified in the control treatments. The amount given by Player 1 (‘donation’) was set as the response term in a linear model (LM) with Gaussian error structure. The global model included the terms age, gender, target amount, treatment and the 2-way interaction between treatment and amount.Finally, we tested the idea that specifying a large target donation ($0.50 versus $0.20) might have the perverse effect of making Player 1 more likely to give nothing at all to Player 2. If Player 1 gave nothing to Player 2 we coded this as ‘1’ and if Player 1 gave a non-zero donation to Player 2 then this was coded as ‘0’. Data were analysed using a GLM with binomial error structure, with the following explanatory terms: age, gender, treatment (‘descriptive’/‘injunctive’), target amount (‘$0.20’/‘$0.50’), and the 2-way interaction between treatment and amount.All data were analysed in the statistical package, R 2 = 19.2, df = 1, p<0.001). This was apparently not an artefact of a larger number acting as an anchor in the $0.50 target condition since we found that players were slightly more likely to give $0.50 or more in the ‘age 20’ control condition  than in the ‘age 50’ control condition , which is the opposite of what we would have expected if the number 50 was anchoring players towards making larger donations . As expected, compliance was reduced when a higher target amount was specified. Players were less likely to give a target amount of at least $0.50  than a target amount of at least $0.20 .The most common donation across all conditions (excluding controls) was $0.50 . PoolingInjunctive norms were associated with increased compliance relative to controls, both when the target amount was $0.20  might have the perverse effect of making players more likely to give nothing at all. Of the 1,182 subjects available for analysis, 341 (28.8%) gave none of the endowment to Player 2. The results from the GLM indicated that males were more likely than females to give none of the endowment to Player 2; and there was a small negative effect of age on propensity to keep the entire endowment  & 7. TheWe have shown that donations in an online Dictator Game are influenced by injunctive but not descriptive norms. In contrast to a previous study investigating the effect of norms on Dictator Game giving People are exposed to both injunctive and descriptive norms of behaviour in several real-world settings. Often, however, these different types of norm may be presented together and may either be congruent or might contradict one another It may be the case that we found lower compliance for descriptive versus injunctive norms in this setting because people simply did not believe the descriptive norm information. Subject belief is important in this study - unlike most real world settings where subjects can directly observe how others behave in that situation. By contrast, since our injunctive norm entailed an instruction rather than information, compliance should not be affected by subject beliefs. We did not include a question in this study to ask subjects whether they believed the descriptive norm information, though previous data indicate that subjects do typically trust that the game instructions are accurate and that they are not deceived . Moreover, we did not use deception in this or any previous studies, meaning that our Requester ID (the name under which experiments are posted) is not known for deceiving participants. Finally, the amounts we specified in the norm information are consistent with patterns observed in previous Dictator Games in this setting e.g. : most pePrevious work has argued that descriptive and injunctive norms are concerned with fundamentally different goals, namely choosing the appropriate behaviour and gaining social approval, respectively Some previous work has shown not only that descriptive norms are more effective motivators of the desired behaviour but that injunctive norms may have a counter-productive effect and make individuals more inclined to do the opposite of the behaviour that is socially demanded To conclude, we have shown that injunctive norms are effective at motivating increasing donations in an anonymous online Dictator Game, whereas descriptive norms did not appear to affect dictator donations. As expected, players were less likely to comply with the norm when doing so involved greater costs ($0.50 compared to $0.20). Nevertheless, higher target amounts did result in increased mean donations. Our data suggest injunctive norms might be used to great effect to motivate prosocial behaviour in real-world settings, for example by museums when soliciting donations from visitors. Future work should explore whether the source of the injunctive norm and the likely beneficiary of the behaviour have an effect on prosocial behaviour.S1 FileSupplementary online material including game instructions, subject information, additional analyses and R code.(DOC)Click here for additional data file.S2 FileSupplementary online material containing data used for analysis.(XLSX)Click here for additional data file.


We investigated the role of NRPs in the rat carotid balloon injury model, in which endothelial denudation and arterial stretch induce neointimal hyperplasia involving VSMC migration and proliferation.Neuropilins 1 and 2 (NRP1 and NRP2) play crucial roles in endothelial cell migration contributing to angiogenesis and vascular development. Both NRPs are also expressed by cultured vascular smooth muscle cells (VSMCs) and are implicated in VSMC migration stimulated by PDGF-BB, but it is unknown whether NRPs are relevant for VSMC function in vivo. NRP knockdown also reduced the phosphorylation of PDGFα and PDGFβ receptors in rat VSMC, which mediate VSMC migration and proliferation.NRP1 and NRP2 mRNAs and proteins increased significantly following arterial injury, and immunofluorescent staining revealed neointimal NRP expression. Down-regulation of NRP1 and NRP2 using shRNA significantly reduced neointimal hyperplasia following injury. Furthermore, inhibition of NRP1 by adenovirally overexpressing a loss-of-function NRP1 mutant lacking the cytoplasmic domain (ΔC) reduced neointimal hyperplasia, whereas wild-type (WT) NRP1 had no effect. NRP-targeted shRNAs impaired, while overexpression of NRP1 WT and NRP1 ΔC enhanced, arterial re-endothelialization 14 days after injury. Knockdown of either NRP1 or NRP2 inhibited PDGF-BB-induced rat VSMC migration, whereas knockdown of NRP2, but not NRP1, reduced proliferation of cultured rat VSMC and neointimal VSMC in vivo by modulating VSMC migration (via NRP1 and NRP2) and proliferation (via NRP2), independently of the role of NRPs in re-endothelialization.NRP1 and NRP2 play important roles in the regulation of neointimal hyperplasia  The principal reason for failure of PTCA is restenosis, particularly in diabetics. A major underlying cause of restenosis, as well as of stenosis following coronary bypass and other vascular grafting, is the abnormal accumulation of neointimal vascular smooth muscle cells (VSMCs) resulting from media-to-intima migration and proliferation. These processes, together with deposition of extracellular matrix, lead to neointimal hyperplasia, causing luminal narrowing, thereby limiting the beneficial effects of intervention. The development of drug-eluting stents has considerably improved the outcome and is now the preferred method of revascularization, but restenosis remains an important cause for the failure of revascularization therapy.Percutaneous transluminal coronary angioplasty (PTCA) continues to be used for coronary revascularization for emergency heart attack and for angina refractory to standard medical therapy, with ∼2 million procedures worldwide every year  in the endothelium and mediates VEGF signalling important for directed cell migration, a function at least partly dependent on the NRP1 cytoplasmic domain.9 Furthermore, NRP1 is also able to mediate the cellular functions of non-canonical NRP1 ligands involving signalling and complexation with other growth factor receptors, including PDGF/PDGF receptors(R), fibroblast growth factor-2 (FGF-2),10 and transforming growth factor β (TGF-β) and its receptors.16 NRPs are highly expressed in VSMCs, mesenchymal stem cells (MSC), and hepatic stellate cells and mediate migration of these cells in response to PDGF by associating with PDGFRα and β, thereby regulating their phosphorylation state and downstream chemotactic signalling.17 It is so far unclear whether NRP1 plays a role in VSMC function in vivo. PDGF signalling is known to be a key regulator of VSMC migration following endovascular injury;18 PDGF-BB and its receptors, PDGFRα and PDGFRβ, are differentially up-regulated in a time-dependent manner after angioplasty.19 Other growth factors, such as FGF-2, are also strongly implicated in migration and proliferation of VSMC leading to neointima formation induced by endovascular injury.20 We examined whether NRPs play a role in neointimal remodelling induced by balloon angioplasty in the rat carotid artery, a well-characterized model of arterial remodelling following endothelial denudation. We found that endogenous NRP1 and NRP2 were up-regulated following arterial injury, and that disruption of NRP function either by overexpressing a NRP1 mutant lacking the cytoplasmic domain (NRP1 ΔC) or by targeted knockdown of NRP1 and NRP2, significantly reduced neointimal hyperplasia following arterial injury. Interestingly, NRP2 knockdown also selectively reduced neointimal VSMC proliferation and decreased the migration and proliferation of cultured VSMC, whereas NRP1 knockdown inhibited only VSMC migration. NRP1 and NRP2 inhibition also impaired re-endothelialization and reduced endothelial cell migration. These findings indicate novel roles for NRPs in mediating pathological VSMC accumulation, neointimal thickening, and endothelial regeneration in response to endovascular injury.Neuropilin 1 (NRP1) is a receptor for Class 3 semaphorins which regulate axonal guidance and neuronal patterning during embryogenesis,2.Supplementary material online.An expanded Methods section is available in the 2.1Supplementary material online).21 Adenoviruses (Ad) were delivered to injured carotid arteries in pluronic® gel F-127 (Sigma-Aldrich) containing 1010 adenoviral particles applied to the adventitial surface of the artery. Specimen retrieval was done following perfusion under terminal anaesthesia using pentobarbital via intraperitoneal injection. Two hundred millilitres of 0.9% saline solution was perfused at a rate of 20 mL/min via the abdominal aorta. The specimens were then carefully dissected through neck incision. Neointimal hyperplasia was assessed by determining the ratios of the intimal to medial areas (I/M) in haematoxylin- and eosin-stained paraffin-embedded sections using ImageJ.23 Re-endothelialization was assessed by immunohistochemical staining of endothelial nitric oxide synthase (eNOS)26 and also using Evan's blue staining of the whole artery.All animal experiments were performed in compliance with the Animals (Scientific Procedures) Act (1986) and in accordance with Home Office and University College London guidelines. Animals were anaesthetized using a combination of Midazolam (625 μg/100 g) and Fentanyl (40 μg/100 g) supported by Halothan 0.5% at 2 L/min oxygen flow. Carotid injury in anaesthetized male Sprague-Dawley rats (Charles River UK) was performed using an embolectomy catheter as previously described , western blotting, immunofluorescent and immunohistochemical staining, generation of adenoviruses encoding NRP1 constructs and shRNAs targeted to NRP1 and NRP2, culture of rat aortic SMC (RAoSMC) and rat aortic endothelial cells (RAoEC), and assays of cell migration was performed as previously described29 .Measurement of gene expression by absolute quantitative RT-PCR (qPCR)  labelling. BrdU solution (Sigma-Aldrich) was injected in rats intraperitoneally 24 h prior to tissue harvest. BrdU-labelled cells were detected by immunostaining paraffin-embedded arterial sections using anti-BrdU antibody . Proliferation of cultured RAoEC was determined in 96-well plates (seeding density of 7000 cells per well) by assessing cell confluence in living cells using an IncuCyte™ Zoom (Essen Bioscience) for up to 3 days.2.4P < 0.05.Results are presented as means ± SEM. Statistical analysis was performed using GraphPad Prism Version 5 by using either one-way or two-way ANOVA with Bonferroni post tests, comparing a treatment with control conditions. Repeated-measures two-way ANOVA was made for cell proliferation assays and cell migration scratch assays performed using the IncuCyte™ Zoom (Essen Bioscience). The number of experiments or animals used in each study is specified for each figure. Statistical significance was validated at *3.3.1Figure ). Assessment of VEGFR2 (Flk1) mRNA showed a nearly two-fold increase in the gene copy number 14 and 28 days after injury, but the mRNA copy number was very low relative to that of NRPs, and VEGFR2 up-regulation was not statistically significant . VEGFR2 up-regulation probably reflects endothelial cell proliferation and repopulation of the lumen after injury. NRP1 and NRP2 protein expression increased significantly after injury with a maximum up-regulation at 7 and 14 days . IF staining of arterial sections revealed expression of NRP1 and NRP2 after arterial injury in the medial and neointimal layers of the carotid 14 days after angioplasty . Immunofluorescent staining of NRP1 and NRP2 showed extensive neointimal staining suggestive that NRP1 and NRP2 were at least partly localized to VSMC . Immunostaining of VSMC with antibody to smooth muscle-specific α-actin (SMA) also showed extensive neointimal staining, though for technical reasons we were unable to quantify the extent of co-staining with NRPs. Some luminal cells were positive for NRP1 and NRP2 immunostaining, and these luminal cells showed co-staining for endothelial-specific eNOS . It was further noted that sparse cells within the neointima were positive for both NRP1 and the monocyte/macrophage marker, CD68, as well as for NRP2 and CD68, indicating that some neointimal NRP positive cells were macrophages . For NRP1, 5.9 and 2.7% immunofluorescent staining co-localized with eNOS and CD68, respectively, and corresponding values for NRP2 were 12.9 and 2.2%.Assessment of NRP1 gene regulation during the development of neointimal hyperplasia by qPCR at 7, 14, and 28 days after injury of the left carotid artery (LCA) showed that NRP1 mRNA copy number was significantly increased at all time points in injured arteries compared with the uninjured right carotid arteries (RCA). NRP2 mRNA expression was also significantly elevated 7 days after angioplasty thereafter declining to control levels by 28 days after injury .13 Overexpression of Ad.shRNA constructs targeted to NRPs in carotid arteries significantly and selectively decreased NRP1 and NRP2 arterial mRNA and protein expression in vivo and protein expression in RAoSMC . Morphometric analysis of the transduced vessels revealed that overexpression of either shNRP1 or shNRP2 significantly decreased neointimal hyperplasia, evaluated by determining I/M, compared with the shScrambled control at all time points . NRP2 knockdown reduced neointima formation to a somewhat greater extent than NRP1 shRNA, though not significantly so (P = 0.1261). It was also examined whether combined knockdown of NRP1 and NRP2 could inhibit neointima formation more profoundly. The results of this experiment confirmed the inhibitory effects of targeted knockdown of either NRP alone, with NRP2 knockdown again exerting a marginally greater effect, but showed that the combined knockdown caused no greater inhibition than NRP2 shRNA alone .The up-regulation of NRP1 and NRP2 in the carotid artery following injury suggested a possible role of NRPs in increased VSMC migration contributing to neointimal thickening and arterial remodelling. To address this possibility, we used two approaches: targeted knockdown of NRP expression using small hairpin (sh) RNAs specific for NRP1 and NRP2 and overexpression of adenoviral constructs encoding either a WT NRP1 or a mutant NRP1 lacking the cytoplasmic domain (NRP1 ΔC) previously shown to inhibit PDGF-BB-induced migration in human coronary artery VSMC and VEGF-induced endothelial cell migration  and LacZ (Ad.LacZ) by PCR and immunostaining 7 days after injury and infection with vectors . Adenovirally delivered GFP and LacZ were strongly expressed in injured rat carotid arteries, with substantial expression found in the media, adventitia, and neointima, as indicated by PCR (GFP only)  and immunohistochemical staining . At 7 and 14 days post-injury, protein expression of adenovirally delivered GFP or LacZ was detected, as indicated by western blot of whole carotid artery lysates, but expression decreased markedly at 28 days after injury .Delivery of Ad.NRP1 WT and Ad.NRP1 ΔC was performed in pluronic gel applied to the adventitial surface of carotid arteries at the time of injury as described previously.Figure  and D; see Table S3Supplementary material online, ). At 28 days after injury, I/M ratios were modestly reduced compared with earlier times consistent with the observation that intimal hyperplasia reaches a peak after ∼2–3 weeks following injury and thereafter remains relatively constant.31 At this time point, there was no statistically significant difference between arteries transduced with LacZ, NRP1 WT, and NRP1 ΔC adenoviruses. The lack of a significant inhibitory effect of Ad.NRP1 ΔC at 28 days in part may reflect reduced adenoviral expression of this molecule at later times after delivery as indicated by LacZ and GFP expression .Morphometric analysis of the vessels revealed that overexpression of the NRP1 ΔC mutant significantly decreased the formation of neointimal hyperplasia compared with the Ad.LacZ and Ad.NRP1 WT at 7 and 14 days post-injury , whereas staining was almost completely absent immediately after injury  and progressively increased thereafter to ∼75% luminal coverage 28 days following injury . Quantification of endothelial eNOS staining showed that endothelial coverage was significantly reduced by Ad.shRNA targeted to NRP1 at 14 days and by Ad.shRNA to NRP2 at 7 and 14 days, but it was not significantly affected by Ad.shRNA at 28 days . In addition, en face staining of arteries using Evans blue dye at 14 days after injury also revealed decreased re-endothelialization of vessels following knockdown of NRP1 and NRP2, though this effect was significant only for Ad.shNRP1 vs. Ad.shScrambled . The reduced re-endothelialization values obtained from Evan's blue staining compared with immunohistochemical staining of eNOS may be due to leakage of dye in the mitotic-regenerating endothelium.32 Overexpression of Ad.NRP1 WT or Ad.NRP1 ΔC had no significant effect on re-endothelialization at 7 days, but at 14 days after injury, overexpression of both constructs enhanced re-endothelialization compared with control Ad.LacZ-transduced arteries . Treatment with Ad.NRP1 WT also caused a significant enhancement in re-endothelialization at 28 days compared with Ad.LacZ (90 vs. 74%), while Ad.NRP1 ΔC had no significant effect at this time. Given these results, we evaluated effects of manipulating NRP expression on migration and proliferation in primary cultures of RAoEC in response to VEGF. Overexpression of Ad.NRP1 WT significantly enhanced VEGF-induced RAoEC migration (from 32 h onwards) and Ad.NRP1 ΔC significantly reduced RAoEC migration (from 48 h onwards), while overexpression of NRP1 WT and NRP1 ΔC did not alter RAoEC proliferation . In contrast, Ad.shRNAs targeted to either NRP1 or NRP2 significantly inhibited RAoEC migration   and proliferation  .Given that NRP1 has been shown to play an important role in endothelial cell migration, we investigated whether reduced neointimal hyperplasia consequent upon NRP knockdown or overexpression of NRP1 ΔC could be due to an effect on re-endothelialization. Endothelial luminal coverage was determined by immunostaining of eNOS at six different levels across the whole length of the injured artery. Continuous specific staining of the endothelium was observed in uninjured carotid arteries . Relaxation of injured carotids was also significantly less than that of uninjured arteries. We observed a trend towards reduced relaxation in vessels treated with either Ad.shNRP1 or Ad.shNRP2 compared with Ad.shScrambled, but the differences were not statistically significant .The effect of NRP knockdown on endothelial function of carotid arteries 14 days after angioplasty was assessed in organ bath studies of vascular reactivity. These experiments demonstrated a significantly reduced contractile response in the injured arteries compared with the uninjured arteries, but indicated no effect of Ad.shNRP1 or Ad.shNRP2, compared with Ad.shScrambled , Ad.shNRP1 had no significant effect on neointimal BrdU labelling, whereas Ad.shNRP2 caused a significant reduction in neointimal cell proliferation . Furthermore, neither Ad.NRP1 WT nor Ad.NRP1 ΔC had any significant effect on VSMC proliferation within neointimal lesions . In vivo proliferation was also assessed at 14 days post-injury, but very little proliferation was observed at this time (data not shown).Two major mechanisms could explain the inhibitory effects of NRP inhibition on neointima formation: inhibition of neointimal cell migration and/or reduced cell proliferation. Analysis of BrdU labelling in injured carotid arteries 7 days after angioplasty, a time when neointimal cell proliferation peaks in the rat carotid injury model,3.5Figure 6; see Figure S3CSupplementary material online, ). NRP1 knockdown caused a significant decrease in cell migration in response to PDGF-BB, as determined in Transwell assays of chemotaxis and in scratch assays of wound closure (chemokinesis), while NRP2 shRNA also significantly inhibited the migratory response to PDGF-BB, although to a lesser extent . We next examined the effects of adenoviral overexpression of NRP1 WT and the NRP1 ΔC mutant on PDGF-BB-induced RAoSMC migration . Infection of RAoSMC with Ad.NRP1 WT did not significantly alter the migration of RAoSMC towards a gradient of PDGF-BB, compared with control cells infected with Ad.LacZ. In contrast, Ad.NRP1 ΔC infection caused a significant inhibition of cell migration induced by the PDGF-BB gradient , indicating a role for the NRP1 cytoplasmic domain in downstream signalling mediating the chemotactic response of these cells, consistent with our previous findings in human VSMC.11 VSMC migration, as assessed by scratch assay, was not affected by the overexpression of the Ad.NRP1 WT or ΔC constructs (data not shown).Our results indicated that inhibition of NRP1 function in arterial VSMC by either targeted knockdown or overexpression of NRP1 ΔC had little effect on VSMC proliferation and, therefore, was likely to act primarily by inhibiting directed cell migration. This possibility was evaluated directly by examining the effect of shRNA-mediated knockdown of NRP1 and NRP2 on the migratory response of primary cultures of rat arterial SMC (RAoSMC) to PDGF-BB. Infection of RAoSMC with shRNAs targeted to rat NRP1 and NRP2 significantly reduced NRP protein expression in RAoSMC .3.6Figure 6, PDGF treatment of RAoSMC induced a marked increase in tyrosine phosphorylation of PDGFRβ and PDGFRα. NRP1- and NRP2-targeted knockdown using shRNA infection in these cells significantly decreased PDGFRα and PDGFRβ tyrosine phosphorylation without affecting total PDGFR levels . Adenoviral overexpression of NRP1 WT or NRP1 ΔC had no significant effect on phosphorylation of either PDGFRβ or PDGFRα in RAoSMC (results not shown).The mechanisms mediating the role of NRPs in VSMC migration and proliferation were addressed by investigating the effects of NRP knockdown and adenoviral overexpression on PDGF receptor activation in RAoSMC. As shown in 4.33 and in VSMC as well as endothelial cells in human coronary arteries and aortae used for coronary artery bypass grafting.34 However, hitherto, the role of NRPs in VSMC functions in vivo has not been investigated. A major conclusion of the present study is that NRP1 and NRP2 are both expressed in the VSMC of the carotid artery wall and are significantly up-regulated at mRNA and protein levels in the carotid artery after endothelial balloon injury. Both NRPs were most strongly expressed at 7 and 14 days post-injury, coincident with the most active phase of neointimal VSMC accumulation and with the maximum extent of neointimal thickening in this model. Further consistent with the notion that NRPs are involved in neointimal VSMC accumulation, immunofluorescent staining revealed strong neointimal NRP1 and NRP2 expression in the neointima further suggestive of expression in neointimal VSMC. It was noted that whereas total arterial NRP1 and NRP2 protein expression declined at 28 days, NRP1 mRNA expression remained elevated at this time, perhaps reflecting a later phase of NRP1 expression in the endothelial repopulation of injured arteries.NRPs are expressed in arterial VSMC and have been shown to mediate PDGF signalling and migration in VSMC. NRP1 is also reported to be most strongly expressed in late mouse embryos in the VSMC of large vessels11 and rat arterial VSMC, we propose that the inhibitory effect of interfering with NRP function on neointima formation results primarily from inhibition of VSMC migration, probably due to inhibition of PDGF-BB signalling, though whether PDGF signalling is the only or major target for the action of NRP1 inhibition is not clear. For example, NRP1 also regulates signalling by TGF-β and its receptors, which are important mediators of VSMC proliferation.36 The inhibitory effect of Ad.NRP1 ΔC on neointimal hyperplasia is most likely due to a dominant negative effect of this construct on NRP1 functions in VSMC dependent on its cytoplasmic domain, as we previously demonstrated in human VSMC.11 A role for the NRP1 cytoplasmic domain in arteriogenesis has been demonstrated in mice with a global knock-in of NRP1 lacking its cytoplasmic region. These mice exhibit normal developmental angiogenesis, but impaired neonatal arteriogenesis in the heart, kidney, and hindlimb.37 The NRP1 cytoplasmic domain is thought to function at least in part through interaction of its C-terminal PDZ domain binding motif to the PDZ domain protein, synectin, and this and other possible protein interactions with the NRP1 cytoplasmic domain could play a scaffolding role in cell migration.37 The role of the NRP2 cytoplasmic domain is less clear and warrants further investigation. A key role for NRP1 specifically in neointimal VSMC migration is also inferred from the finding that neither NRP1 shRNA nor NRP1 ΔC caused inhibition of neointimal cell proliferation measured by in vivo BrdU labelling. NRP1 knockdown also had no effect on RAoSMC proliferation. Furthermore, previously published work indicates an important role for NRP1 in directed cell migration induced by chemotactic cues such as PDGF-BB and VEGF, but that NRP1 is unimportant for cell proliferation.38 While we cannot preclude NRP1 cytoplasmic domain playing a role in other cellular processes contributing to neointimal hyperplasia, our present findings strongly indicate that inhibition of NRP1 function in the rat carotid artery selectively interferes with VSMC migration underlying neointimal VSMC accumulation.The second important conclusion of this paper is that NRP1 and NRP2 both contribute to neointimal VSMC hyperplasia and neointima formation in the injured rat carotid injury model. This conclusion is based on two major lines of evidence: (i) targeted shRNAs to either NRP1 or NRP2, effective in reducing NRP expression in cultured rat VSMC and in the carotid artery, significantly inhibited neointimal thickening at 7, 14, and 28 days after endothelial balloon injury; (ii) adenoviral overexpression of an NRP1 ΔC mutant lacking the cytoplasmic domain, previously shown to inhibit VEGF- and PDGF-BB-induced migration of, respectively, endothelial cells and VSMC, also strongly inhibited neointima formation at 7 and 14 days after injury. Given that NRP inhibition using siRNA- or shRNA-mediated NRP1 and NRP2 knockdown, or by overexpression of NRP1 ΔC, inhibits PDGF-BB-induced migration of cultured primary human11 and rat VSMC, our results are consistent with NRP2 playing a role in directed VSMC migration contributing to neointimal expansion. This may be mediated either independently of NRP1 by, for example, a direct interaction between NRP2 and PDGFRα or β, and/or could arise from an interaction between NRP1 and NRP2. Our previous findings indicate that NRP1 and NRP2 readily associate with each other, consistent with the conclusion that these molecules heterodimerize or form mixed oligomers.9 The finding that the combination of shRNAs to NRP1 and NRP2 had no additive inhibitory effect on neointima formation suggests that the two NRPs lie on the same pathway. Since Ad.shNRP2 also significantly reduced in vivo neointimal BrdU labelling and caused a small decrease in RAoSMC proliferation, NRP2 may also contribute to the proliferation as well as migration of VSMC, though further work is required to support this conclusion.In contrast to NRP1, the cellular functions of NRP2 are less well understood. Our finding that shRNA-mediated NRP2 knockdown inhibited neointima formation indicates a novel role for NRP2 in pathological vascular remodelling. Since NRP2 knockdown also inhibited PDGF-BB-induced chemotaxis of human15 Herein we demonstrate that NRP1 and NRP2 knockdown significantly reduced PDGFRα and PDGFRβ tyrosine phosphorylation in rat VSMC, consistent with our previous findings that NRP1 knockdown inhibited PDGFR activation in human coronary artery VSMC.11 These results suggest that impaired PDGFR activation consequent upon NRP knockdown contributes to the inhibitory effects of NRP knockdown on VSMC migration in vivo after carotid injury and in cultured VSMC. In contrast to the effects of NRP1 knockdown, overexpression of NRP1 ΔC had no significant effect on PDGFR activity, consonant with our previous finding that this mutant had no effect on activation of PDGFR or VEGFR in U87 glioma cells,6 and indicating that the NRP1 cytosolic domain is not required for PDGFR activity in rat VSMC. The differential roles of NRP1 and NRP2 in VSMC proliferation, and the underlying mechanisms involved, warrant further study.The mechanism through which NRPs regulate PDGF-induced VSMC chemotaxis has not been fully defined yet, but previous findings show that NRP1 knockdown inhibits PDGF-induced PDGFRα activation.9 NRP1 is hypothesized to be essential for endothelial cell migration from contiguous uninjured endothelium. This study is the first to examine the role of NRPs in endothelial regeneration of a large artery and demonstrates that targeted depletion of NRP1 or NRP2 using Ad.shRNAs markedly reduced re-endothelialization at 7 and 14 days after balloon injury. The retardation of re-endothelialization caused by NRP1 and NRP2 knockdowns is likely mediated by inhibition of endothelial cell migration and proliferation, a conclusion supported by the finding that shRNAs targeted to NRP1 and NRP2 also reduced RAoEC migration and proliferation. Conversely, overexpression of NRP1 WT significantly enhanced endothelial re-coverage 14 and 28 days after endothelial denudation and also augmented RAoEC migration. These data are consistent with a large body of evidence supporting a role for NRPs in mediating endothelial cell migration in vivo and in cells, and support the conclusion that NRP1 and NRP2 play a significant role in re-endothelialization.In the rat balloon injury model, endothelial denudation triggers vascular remodelling, resulting in rapid neointima formation due to the proliferation and migration of VSMC, which is followed by a slower regeneration of the endothelium. In this latter process, based on genetic and biochemical evidence pointing to a key role of endothelial NRP1 in angiogenesis and directed endothelial cell migration,Reduced re-endothelialization is predicted to enhance neointima formation, whereas knockdown of NRPs strikingly decreased the I/M ratio. This finding strongly argues that the inhibitory effect of NRP1 and NRP2 depletion on neointima formation is independent of the role of NRPs in re-endothelialization and is instead mediated via a direct inhibitory effect on VSMC migration. Furthermore, the inhibitory effects of Ad.NRP1 ΔC on neointimal hyperplasia at 7 days after injury are unlikely to be due to its effects on re-endothelialization, since it had no significant effect on re-endothelialization at this time.11 However, though Ad.NRP1 ΔC significantly reduced RAoEC migration, it had no significant effect on RAoEC proliferation. These data suggest that endothelial cell proliferation and enhancement of re-endothelialization by overexpression of NRP1 are largely dependent on the NRP1 extracellular domain and do not require the intracellular domain. In addition, re-endothelialization in vivo relies more on endothelial proliferation, and consequently, inhibition of RAoEC migration by Ad.NRP1 ΔC may not impact so significantly upon re-endothelialization. The significant reduction in neointimal thickening caused by Ad.NRP1 ΔC is likely due to its direct inhibitory effects on VSMC migration. Studies of endothelial contractility in injured carotid arteries showed reduced responses following injury consistent with previous findings,39 but showed no effect of NRP1 or NRP2 knockdown on the contractile response mediated via α1-adrenegric receptor stimulation. We observed a tendency towards attenuated endothelium-dependent relaxation in the carotid arteries treated with Ad.shNRP1/2 compared with Ad.shScrambled, which, though not significant, is consistent with our finding of reduced re-endothelialization in response to NRP knockdowns.In contrast, increased re-endothelialization is expected to reduce neointimal hyperplasia, but Ad.NRP1 WT did not have such an effect. We propose that this is because the enhancement of re-endothelialization caused by Ad.NRP1 WT may not be sufficient to significantly impact upon neointimal thickening. The trend towards enhancement of re-endothelialization by Ad.NRP1 ΔC was unexpected, since previous studies showed that this construct inhibited migration of HUVECs.These findings argue that a major role of NRP1 and NRP2 in early arterial remodelling following endothelial damage is as a mediator of VSMC migration contributing to neointimal expansion. NRPs are required for optimum endothelial regeneration, as indicated by the inhibitory effects of NRP knockdown on re-endothelialization, but this effect does not appear to be strong enough to counteract the inhibitory effect of targeted shRNAs on neointimal growth. This novel role for NRP1 and NRP2 in mediating pathological arterial neointimal hyperplasia in the rat injured carotid artery model suggests that these receptors may be suitable targets for novel therapies designed to prevent or reduce excessive VSMC accumulation in vascular proliferative disorders. In particular, the ability of NRP1 ΔC to both reduce neointimal hyperplasia, without impairing and even modestly enhancing re-endothelialization, suggests that selective targeting of the NRP1 C-terminal domain could reduce neointimal hyperplasia while sparing endothelial function.Cardiovascular Research online.Supplementary material is available at British Heart Foundation (BHF) programme grant RG/06/003 (I.E. and M.M.) and BHF Project grant PG/12/65/29840 (to I.Z. and C.P-M.), a BHF studentship (to V.L.), and by funding from the European Union's Seventh Program for research, technological development and demonstration under grant agreement no. 278313, ‘BIOMAGSCAR’ (V.M. and L.F.).This work was supported by


Background and Objective: In order to characterize the β-adrenoceptor (AR) subtypes involved in agonist-stimulated relaxation of murine urinary bladder we studied the effects of (-)-isoprenaline and CL 316,243 on tonic contraction and spontaneous contractions in detrusor strips of wild-type (WT) and β2-AR knockout (β2-AR KO) mice.Materials and Methods: Urinary bladders were isolated from male WT and β2-AR KO mice. β-AR subtype expression was determined with quantitative real-time PCR. Intact muscle strips pre-contracted with KCl (40 mM) were exposed to cumulatively increasing concentrations of (-)-isoprenaline or β3-AR agonist CL 316,243 in the presence and absence of the subtype-selective β-AR blockers CGP 20712A (β1-ARs), ICI 118,551 (β2-ARs), and L748,337 (β3-ARs).Results: Quantitative real-time PCR confirmed lack of β2-AR expression in bladder tissue from β2-AR KO mice. In isolated detrusor strips, pre-contraction with KCl increased basal tone and enhanced spontaneous activity significantly more in β2-AR KO than in WT. (-)-Isoprenaline relaxed tonic tension and attenuated spontaneous activity with similar potency, but the concentrations required were two orders of magnitude higher in β2-AR KO than WT. The concentration-response curves (CRCs) for relaxation were not affected by CGP 20712A (300 nM), but were shifted to the right by ICI 118,551 (50 nM) and L748,337 (10 μM). The -logEC50 values for (-)-isoprenaline in WT and β2-AR KO tissue were 7.98 and 6.00, respectively, suggesting a large receptor reserve of β2-AR. (-)-CL 316,243 relaxed detrusor and attenuated spontaneous contractions from WT and β2-AR KO mice with a potency corresponding to the drug’s affinity for β3-AR. L743,337 shifted the CRCs to the right.Conclusion: Our findings in β2-AR KO mice suggest that there is a large receptor reserve for β2-AR in WT mice so that this β-AR subtype will mediate relaxation of tone and attenuation of spontaneous activity under physiological conditions. Nevertheless, upon removal of this reserve, β3-AR can also mediate murine detrusor relaxation. Our results confirm the importance of β2-ARs for murine detrusor relaxation and attenuation of spontaneous contractions, but also attest contribution of β3-ARs.Therefore, we have examined which β-AR subtype mediates inhibition of murine detrusor tone and spontaneous contractions. To address this, we have used the general β-AR agonist (-)-isoprenaline and the β2-AR KO mice. The mice were bred in the Department of Pharmacology and Toxicology, University of Würzburg, Germany. All experiments were performed in accordance with the local authorities  and comply with the European Commission Directive 86/609/EEC regarding the protection and welfare of animals used for experimental as well as scientific purposes.The control experiments of the present study were performed in FVB/N-WT mice, which match the genetic background of β2-AR KO mice (24–40 weeks) were killed by cervical dislocation under CO2 anesthesia, and urinary bladders and lungs were removed. The bladders were cut open and detrusor tissue and mucosa were dissected with sharp scissors and further processed separately. RNA was isolated from the tissue samples using the RNeasy®-Kit (Qiagen) and total RNA was reverse transcribed using Superscript II reverse transcriptase (Invitrogen). For cDNA amplification of β1-ARs, β2-ARs, β3-ARs and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a reaction mixture was used containing SsoFast EvaGreen Supermix (BioRad). Quantitative real-time PCR was performed using the C1000 Thermal Cycler CFX96 (BioRad) and the data was analyzed as previously described  and 28 ± 4 g for β2-AR KO mice (n = 49). Strips of mouse urinary bladder were dissected as described previously  were mounted in an organ bath filled with 5 ml of modified Tyrode solution of the following composition (in mM): NaCl 126.9, KCl 5.4, MgCl2 1.05, CaCl2 1.8, NaH2HPO4 0.45, NaHCO3 22, EDTA 0.04, ascorbic acid 0.2, glucose 5.6. Phentolamine (3 μM) and prazosin (1 μM) were added to block α-ARs. The solution in the bath was maintained at 37°C, and was oxygenated by vigorously bubbling with carbogen . The pH was 7.4. All drugs were obtained from the same sources and dissolved either in distilled water of dimethyl sulfoxide as in our previous study  and preloaded with 5 mN. After 30 min in the organ bath, tension was readjusted to 5 mN. Force of contraction was recorded with Chart 4.0TM . Tonic tension was analyzed as the increase of force produced by 40 mM KCl, measured from the lower limit of the “noise” produced by spontaneous activity under baseline conditions and in the presence of KCl. The amplitudes and the time integral of spontaneous contractions were analyzed during the 2-min period before the next concentration increase, using Chart software. Agonist-induced attenuation of spontaneous activity was expressed as integral in percent of control.The preparations were allowed to equilibrate for at least 60 min. During this period, they were stimulated two consecutive times with KCl . After another 45 min of washout, the strips were pre-contracted by depolarization with 40 mM KCl. Relaxation was induced with cumulatively increasing concentrations of (-)-isoprenaline, CL 316,243 or forskolin. Relaxation was measured as the difference between minimum force prior to addition of agonist (steady state force) and force in the presence of the agonist, and was expressed in percent of the response to 10 μM forskolin added at the end of each experiment (= 100%). All β-AR subtype-selective blockers were added 30 min before the start of KCl pre-contraction and remained in the bath solution throughout the remainder of the experiment. The concentrations of antagonists were CGP 20712A 300 nM, ICI 118,551 50 nM , and L74®. The negative logarithm to the base of 10 of the molar concentration producing 50% of the maximum response (-logEC50 [M]) as well as the maximum response (Emax) were calculated and expressed as mean ± SD. Please note that the non-linear regression curves depicted in the figures were fitted to the mean values of the data.Concentration-response curves were constructed by non-linear regression for each individual experiment by using Prism 5.050) in the presence of the antagonist divided by the EC50 of in the absence of antagonist  was plotted versus log(molar concentration of antagonist), where CR stands for concentration ratio, i.e., the agonist concentration producing 50% of the maximum effect . Analysis of variance (ANOVA) was used for multiple group comparison, followed by an additional Bonferroni comparison test where appropriate. P < 0.05 was regarded as significant.The results are represented as mean ± standard deviation (mean ± SD). A two-tailed 2-ARs and to check for any compensatory changes in expression of β1- and β3-ARs. Figure 1 shows that β2-ARs were only detectable in detrusor tissue from WT but not from β2-AR KO mice, and the same results were obtained in the respective lung tissues, which served as controls. Furthermore, β1- and β3-ARs were expressed in bladder and lung tissue from all animals . Between WT and β2-AR KO mice there were no differences in expression levels of β1- and β3-ARs .Expression of the three β-AR subtypes was determined by RT-PCR and quantitative real-time PCR in intact detrusor tissue from WT and genetically modified animals in order to verify knock-out of βFigure 2A). Mean values of peak force (Fpeak) increases were greater in strips from β2-AR KO  than WT mice ; steady-state tone (Fss) was 1.57 ± 0.76 mN/mg w.w. β2-AR KO and 0.76 ± 0.28 mN/mg w.w. in WT (P < 0.05).Depolarization of mouse detrusor strips with 40 mM KCl induced a rapid increase in tone and spontaneous activity, which stabilized within 45 min ∗s, and 6.26 ± 3.74 (mN/mg w.w.)∗s, P < 0.05]. After addition of KCl the integral for spontaneous detrusor activity increased significantly to 41.60 ± 39.78 (mN/mg w.w.)∗s in β2-AR KO and to 10.30 ± 8.77 (mN/mg w.w.)∗s in WT .In the same detrusor strips the baseline amplitudes of spontaneous contractions and their time integrals were greater in β1-AR blocker CGP 20712A (300 nM) had little effect on KCl-induced Fpeak and Fss in either mouse strain , except for Fss in WT mice . CGP 20712A did not affect spontaneous contraction integral neither at baseline nor after KCl . Exposure to the β2-AR blocker ICI 118,551 (50 nM) increased Fpeak and baseline spontaneous activity in strips from β2-AR KO mice. Responses to the β3-AR blocker L748,337 (100 nM) exhibited great variability, but appeared to increase Fpeak and Fss rather than spontaneous activity.The β2-AR KO mice . However, strips from β2-AR KO mice were significantly less sensitive by almost 2 log units. The -logEC50 values for (-)-isoprenaline-induced relaxation of tone were 7.98 for WT and 6.00 for β2-AR KO mice (see Table 1). The respective values for the (-)-isoprenaline-induced attenuation of integral for spontaneous contractions were 8.39 ± 1.06 and 6.34 ± 0.63, and were not significantly different from the effect on relaxation of tone.Increasing concentrations of (-)-isoprenaline caused almost complete relaxation of tone, and attenuation of spontaneous activity in intact strips from WT and β2-AR blocker ICI 118,551 (50 nM) shifted the CRCs to higher (-)-isoprenaline concentrations by about 1.4 log units . In β2-AR KO mice, the three β-AR blockers produced little effect on the CRCs of (-)-isoprenaline , and the small shift to the right with 100 nM L748,337 did not reach significance. All -logEC50 and Emax values are summarized in Table 1. Spontaneous activity was attenuated by (-)-isoprenaline to a somewhat larger extent in β2-AR KO than WT strips . The effects of (-)-isoprenaline on spontaneous activity were not influenced by blocking β1-AR with CGP 20712A . There was a trend for a shift in the CRC by the β2-AR blocker ICI 118,551 in WT strips  and by the β3-AR blocker L748,337 in β2-AR KO strips . Again, due to the large variability, the effects on spontaneous contractions were less clear than on tonic contraction.In detrusor strips from WT mice, CGP 20712A (300 nM) and L748,337 (100 nM) had little effect on the CRCs of (-)-isoprenaline, whereas the β2-ARs are the major β-AR subtype mediating relaxation in WT detrusor strips. However, given the low affinity of L748,337 for murine β3-ARs -isoprenaline-mediated relaxation in WT detrusor . With 10 μM L478,337, the CRC was clearly shifted to the right . The results with 1 and 3 μM L748,557 were less consistent . Nevertheless, these findings suggest that β3-ARs may be involved to a larger extent than previously anticipated by us  clearly deviated from unity suggesting a more complex mechanism than simple competition of (-)-isoprenaline and L748,337 for a single binding site. Fitting a linear regression of slope 1 to the data points, yielded a pA2 value of 6.08 for L748,337 in strips from WT mice.These findings confirm our previous observations , 2015a te β3-ARs , the coned by us , but as 3-AR-selective agonist CL 316,243 that was employed by 2-AR KO with similar potency and efficacy . In comparison with (-)-isoprenaline, CL 316,243 tended to be less potent in strips from WT than β2-AR KO mice . Again, more vigorous spontaneous activity was observed in β2-AR KO than WT strips . The complete CRCs revealed that detrusor strips relaxed less completely  and that attenuation of spontaneous activity failed to reach significance both in WT and β2-AR KO strips . Concentrations of 100–300 nM L748,337 had no significant effect on the CRCs for CL 316,243 under any experimental condition, but concentrations between 1 and 10 μM L748,337 induced complex changes in relaxation . In strips from WT mice, L748,337 did not shift the CRCs of CL 316,243, but with 3 and 10 μM L748,337 relaxation became more complete. In β2-AR KO mice, L748,337 caused a shift of the CRCs to higher concentrations of CL 316,243, in addition to more complete relaxation. Again, the Schild plot  deviated from unity indicating a complex mechanism of interaction also between CL 316,243 and L748,337. Using a slope factor of 1, the pA2 value was 6.70 for L748,337 in strips from β2-AR KO mice.In order to resolve these different interpretations, we next investigated the effects of the β2-AR KO mice . Forskolin completely relaxed tonic tension  and attenuated spontaneous contractions  and there were no differences in sensitivity between strips from WT and β2-AR KO mice (Table 1).In order to estimate receptor-independent relaxation we have also studied the responses to adenylyl cyclase activation with forskolin  in WT an2-AR KO mice and β-AR subtype-selective ligands as pharmacological tools  via Case MLCK, . Severalcle tone . β-AR-mechannels . However2-AR subtype, whilst compensatory expression of β3-(or β1-)ARs is absent in detrusor tissue from β2-AR KO mice.Constitutive systemic knockout of a particular receptor subtype may cause compensatory changes in expression of other receptors or proteins . Therefo2-AR KO mice were bred against a genetic background different from the previously used C57Bl6 mice, new control experiments had to be performed with FVB/N-WT mice. The results were similar as in C57Bl6 mice -isoprenaline to the right, confirming that β2-ARs are involved in this relaxing effect. The affinity estimate calculated for ICI 118,557 based on these shifts  was in good agreement with its known affinity at β2-AR -isoprenaline could be mediated by β1- or β3-AR. Since β1-ARs are predominant only in guinea-pig urinary bladder (2-AR KO mice must have been mediated via β3-ARs. The (-)-isoprenaline concentrations required for half-maximum relaxation were about ∼90-fold higher in detrusor from β2-AR KO than WT mice. Given the affinity of murine β3-ARs for (-)-isoprenaline, e.g., -logEC50 5.57 -isoprenaline for relaxation of WT mouse detrusor, i.e., 7.98 was about 1.5 orders of magnitude lower than its known affinity at mammalian β2-AR . This finding suggests that there is a large receptor reserve for detrusor relaxation via β2-AR. When β2-AR are blocked with ICI 118,551 or are absent as in β2-AR KO mice, (-)-isoprenaline is able to relax mouse detrusor via β3-AR stimulation, but spare receptors do not appear to play a role in this case.The -logEC2-ARs, because only ICI 118,551 (50 nM) shifted the CRCs for (-)-isoprenaline to the right, whereas the β1-AR blocker CGP 20712A (300 nM) and the β3-AR blocker L748,337 (100 nM) were without effect -isoprenaline  in C57Bl6 murine detrusor and between L748,337 and (-)-isoprenaline  in human detrusor -isoprenaline- and CL 316,243-induced relaxation of detrusor from WT and βdetrusor . However2+ oscillations but appears to be generated by different mechanism as evidenced by different pharmacological responses -isoprenaline occurred in the same concentration ranges in WT and β2-AR KO strips as relaxation of tonic tension, and the CRC was only shifted to the right by ICI118,551 suggesting a dominant role for β2-AR in this process. Nevertheless, the small attenuation of spontaneous activity by CL 316,243 indicates a modulating effect of β3-AR as well. Our findings do not confirm that suppression of phasic contractions by (-)-isoprenaline is most sensitive to β1-AR blockers (2- and β3-AR appear to be involved.Although, spontaneous activity of detrusor muscle is not fully understood, increasing evidence suggests that smooth muscle cells posses intrinsic mechanisms for spontaneous contractions and that these are synchronized and modulated by interstitial cells distributed throughout the bladder wall . Spontanesponses . Here, wblockers , because2-AR KO mice were similar between the two groups. Furthermore, also after forskolin, spontaneous activity was suppressed less completely than tonic tension.Relaxation of tonic and phasic detrusor contractions after receptor-independent activation of adenylyl cyclase with forskolin in WT and β2-AR KO mice suggest that there is a large receptor reserve for β2-AR so that this β-AR subtype will be activated preferentially by physiological ligands. Nevertheless β3-AR can also mediate relaxation and attenuate spontaneous contractions in the absence of β2-AR, when β2-AR are blocked or when selective β3-AR agonists are used.We have reported an example how false extrapolation of drug affinities for a given receptor subtype from different species can lead to an incomplete picture. Our novel findings in βSP: experimental procedure, result analysis, evaluation of results, writing the manuscript; KL: experimental procedure, result analysis, evaluation of results, writing the manuscript; MM: result analysis, evaluation of results, revision of the manuscript; MW: result analysis, evaluation of results, revision of the manuscript; UR: result analysis, evaluation of results, writing and revision of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


FHM2) is caused by loss‐of‐function mutations in α2 Na+,K+ATPase (α2NKA), an isoform almost exclusively expressed in astrocytes in adult brain. Cortical spreading depression (CSD), the phenomenon that underlies migraine aura and activates migraine headache mechanisms, is facilitated in heterozygous FHM2‐knockin mice with reduced expression of α2NKA. The mechanisms underlying an increased susceptibility to CSD in FHM2 are unknown. Here, we show reduced rates of glutamate and K+ clearance by cortical astrocytes during neuronal activity and reduced density of GLT‐1a glutamate transporters in cortical perisynaptic astrocytic processes in heterozygous FHM2‐knockin mice, demonstrating key physiological roles of α2NKA and supporting tight coupling with GLT‐1a. Using ceftriaxone treatment of FHM2 mutants and partial inhibition of glutamate transporters in wild‐type mice, we obtain evidence that defective glutamate clearance can account for most of the facilitation of CSD initiation in FHM2‐knockin mice, pointing to excessive glutamatergic transmission as a key mechanism underlying the vulnerability to CSD ignition in migraine.Migraine is a common disabling brain disorder. A subtype of migraine with aura (familial hemiplegic migraine type 2:  In a third of patients, the headache is preceded by transient focal symptoms that are most frequently visual (migraine with aura: MA). Increasing evidence supports a key role of cortical spreading depression (CSD) in migraine pathogenesis in that CSD underlies migraine aura   clearance is suggested by its colocalization with the Glu transporters (GluTs) GLT‐1 and GLAST in astrocytic processes surrounding glutamatergic synapses in adult somatic sensory cortex  is caused by mutations in ATP1A2, the gene encoding the α2 NKA  mice carrying the pathogenic W887R FHM2 mutation and is halved in the brain of heterozygous W887R/+ mutants  mice, we provide evidence that the defective Glu clearance can account for most of the facilitation of CSD initiation in the FHM2 mouse model.Here, to test these hypotheses and to gain insights into the physiological role of the α2 NKA in heterozygous W887R/+ FHM2‐KI mice causes a reduced rate of Glu clearance by cortical astrocytes during neuronal activity, we took advantage of the fact that i) Glu uptake into astrocytes by GluTs is electrogenic, and therefore can be monitored electrophysiologically, and ii) the time course of the Glu transporter current elicited in astrocytes upon extracellular neuronal stimulation in hippocampal slices  reflects, to some extent, the time course of Glu clearance by astrocytes and provides a relative indication of how rapidly synaptically released Glu is taken up from extracellular space  by the stimulation of neuronal afferents with an extracellular electrode located in layer 1, 200 μm from the patch‐clamped astrocyte, in acute slices of the somatic sensory cortex from P22‐23 WT and FHM2‐KI mice, in the presence of antagonists of Glu and GABA receptors Fig . The inwτ = 6.80 ± 0.19 ms . The decay kinetics of the STC elicited in cortical astrocytes of heterozygous W887R/+ FHM2‐KI mice were slower compared to those in WT astrocytes, as shown by the significantly larger time constant of decay of both the STC isolated pharmacologically (τ = 7.97 ± 0.31 ms) and the STC isolated nonpharmacologically in a larger number of cells (τ = 7.82 ± 0.16 ms)  Fig  and the Diamond, , other f, the decay kinetics of the STC elicited by the 10th pulse in the train should be slower than those of the STC elicited by a single pulse stimulation , was 7.56 ± 0.16 ms (n = 23), while that elicited by a single pulse in the same cells was τ1 = 6.45 ± 0.15 ms  and the average ratio τ10/τ1 (50 Hz) was 1.18 ± 0.02 (n = 23). The slowing of the STC after the train was larger in FHM2‐KI compared to WT mice (in KI: τ10/τ1 (50 Hz) = 1.26 ± 0.03, n = 21; unpaired t‐test, P = 0.021). The relative slowing and the difference between WT and KI mice were more pronounced after a 100‐Hz train (τ10/τ1 (100 Hz) = 1.25 ± 0.03, n = 18, in WT versus τ10/τ1 (100 Hz) = 1.40 ± 0.05, n = 14, in KI, P = 0.009). As a consequence, the slowing down of Glu clearance in FHM2‐KI compared to WT mice was quantitatively larger after repetitive stimulation than after single pulse stimulation and increased with increasing stimulation frequency  = 9.82 ± 0.24; τ10(100 Hz) = 11.08 ± 0.41 ms) were 30 and 37% larger than those in WT mice, respectively (τ10(50 Hz) = 7.56 ± 0.16 ms; τ10(100 Hz) = 8.09 ± 0.23 ms) . GLT‐1 is the quantitatively dominant GluT in the brain and mediates the majority of Glu clearance in the adult murine neocortex  and the αet al, et al, et al, et al, +) ir puncta (green) that overlapped with VGLUT1+ ir puncta (red) was reduced by 18% in FHM2‐KI mice (0.47 ± 0.03 μm2) compared to WT (0.57 ± 0.03 μm2) . As a change in size of ir puncta has been considered strongly suggestive of a change in protein expression , as revealed by triple‐labeling immunofluorescence of GLT‐1a, VGLUT1, and α2 NKA in WT cortical sections  in cerebral cortex sections to study the density of GLT‐1a gold particles associated with the membrane of PAPs. In line with previous studies  as a consequence of the very high expression of K+ channels that are open at negative resting voltages, the astrocyte passive conductance is essentially K+ selective and the astrocyte membrane potential behaves as a good K+ electrode, rendering astrocytes useful [K+]e biosensors   provides a measure of the rate of K+ clearance, which has been shown to be equivalent to that obtained with [K+]e‐sensitive microelectrodes , represent a major source of K+ efflux during neuronal activity  in WT astrocytes decayed with a time constant of 2.36 ± 0.10 s, more than two orders of magnitude more slowly than the STC   and FHM2‐KI astrocytes  confirms this conclusion. In both genotypes, the time constants of decay of IK are similar in the absence and presence of Glu receptors blockers, and the slowing of K+ clearance in FHM2‐KI cortical slices is also similar in the two conditions.Therefore, to investigate the effect of the FHM2 mutation on Ket al, T = 30°C), CSD induced in acute cortical slices by brief pulses of high K+  is at least in part due to the younger age of the animals and the lower temperature. In fact, the facilitation of CSD was larger in cortical slices from older (P34 ‐ 35) mice: the threshold for CSD induction (T = 30°C) was 39% lower and the velocity of CSD propagation 26% higher in FHM2‐KI relative to WT mice  rescued by systemic treatment with ceftriaxone (Cef), a drug that increases the membrane expression of GLT‐1 in neocortex  and/or to some other effect of Cef not related to GLT‐1 expression. Mechanisms involving changes in expression of the α2 NKA or the glial Glu‐cystine antiporter xCT .As a second approach to study whether there is a causative relationship between reduced rate of Glu clearance by astrocytes and CSD facilitation, we investigated whether pharmacological reduction of the rate of Glu clearance in WT mice to a value similar to that in FHM2‐KI mice reduces the threshold for CSD induction and increases the velocity of CSD propagation to values similar to those in the FHM2 mutants. To identify which concentration of drug produced a slowing of the rate of Glu clearance in WT mice close to that produced by the FHM2 mutation and obtain information on the time necessary to reach steady‐state inhibition, we measured the STC in cortical slices from P22‐23 WT mice before and after the application of different subsaturating concentrations of DL‐TBOA. We identified a concentration of DL‐TBOA (2.5 μM) that increased by 32 ± 2% the time constant of decay of the STC elicited by single pulse stimulation, thus producing a slowing of the rate of Glu clearance close to  that produced by the FHM2 mutation Fig A. In the2 NKA pump in heterozygous W887R/+ FHM2‐KI mice on the rate of Glu clearance by astrocytes during neuronal activity, as measured from the decay kinetics of the synaptically activated Glu transporter current recorded in cortical astrocytes. We have shown that Glu clearance by astrocytes during neuronal activity is slower in FHM2‐KI compared to WT mice and that the density of GLT‐1a in the membrane of astrocytic processes surrounding cortical excitatory synapses is about 50% reduced in the FHM2 mutants, a reduction that mirrors the reduced expression of the α2 NKA protein. Interestingly, the relative impairment of Glu clearance in FHM2‐KI mice is activity dependent. In fact, when synaptic Glu release was induced by a train of pulses, the slowing down of the rate of Glu clearance in the FHM2 mutants was larger than that observed with a single pulse and was larger with a 100‐Hz compared to 50‐Hz train. This is consistent with and likely reflects the decreased Glu binding capacity of GluTs in the KI mice due to the reduced density of GLT‐1a in PAPs. As a consequence, a relatively lower number of GluTs remain available to bind Glu at the end of the train in KI compared to WT astrocytes, resulting in a relatively larger slowing of the rate of Glu clearance.We have studied the functional consequences of the reduced membrane expression of the α2 NKA pump in the clearance of synaptically released Glu during neuronal activity. Our findings suggest that this key role is based on a necessary tight coupling between the α2 pump and GLT‐1 in cortical PAPs. These findings include (i) the strict colocalization of α2 NKA and GLT‐1a in astrocytic processes in the vicinity of glutamatergic synapses; (ii) the 48% reduction in the membrane density of GLT‐1a in KI PAPs, quantitatively similar to the reduction in the expression of α2 NKA, and, in contrast, the unaltered membrane density of GLT‐1a in AxTs where α2 is not expressed; (iii) the insignificant increase in membrane density of GLT‐1a in PAPs in Cef‐treated FHM2‐KI mice, in contrast with the increased density of GLT‐1a in AxTs.These data provide the first direct experimental evidence, to our knowledge, for a key role of the αe biosensors, we have also studied the functional consequences of the reduced expression of α2 NKA in heterozygous W887R/+ KI mice on the rate of K+ clearance, as measured from the decay kinetics of the [K+]e‐dependent slow inward current elicited in astrocytes by neuronal activity and the ensuing K+ efflux. The finding that the rate of K+ clearance after a train of pulses at 50 Hz is slower in FHM2‐KI mice provides the first direct experimental support for an important role of the α2 NKA pump in K+ clearance following neuronal activity.By using astrocytes as [K]+ clearance during neuronal activity explain the facilitation of initiation and propagation of experimental CSD in the FHM2 mouse model and the episodic vulnerability to CSD ignition in FHM2? Despite important progress, the mechanisms underlying the initiation of experimental CSD remain incompletely understood and controversial  fraction of the facilitation of CSD propagation, suggesting that other mechanisms contribute. The observation that also Cef treatment in FHM2‐KI mice had a differential effect on CSD threshold and velocity appears consistent with the interpretation that increased reuptake of Glu in AxTs might account for the small rescue of facilitation of CSD induction produced by Cef.et al, 2 NKA pumps exert a dampening role owing mainly to their key role in Glu clearance by astrocytes. There is evidence that cooperative activation of postsynaptic NMDARs by independent excitatory synapses due to Glu spillover occurs with, for example, high‐frequency stimulation or by increasing the probability of Glu release or with pharmacological inhibition of GluTs, that is, in conditions in which the binding capacity of GluTs is overwhelmed by high extracellular Glu  could result from slowing  of Na+‐dependent transporters such as the electrogenic Na+/HCO3− cotransporters or the Na+/Ca2+ exchangers , thus creating the conditions for initiation of the positive feedback cycle that ignites CSD. Similar mechanisms may underlie the susceptibility to CSD ignition in common forms of migraine for which there is indirect evidence consistent with enhanced cortical excitatory transmission  or reduced Glu clearance (FHM2). In contrast with the enhanced excitatory synaptic transmission at pyramidal cell synapses, inhibitory transmission at fast‐spiking and other multipolar interneuron synapses was unaltered in FHM1‐KI mice  mice harboring the W887R FHM2 mutation . No statistical methods were used to choose sample sizes that were estimated based on previous experience and are in line with those in the literature. No animals were excluded from the analysis.After assessing for normal distribution (using the Shapiro–Wilk or the Kolmogorov–Smirnov test), comparison between two groups was made using two‐tailed unpaired or paired t‐test for normal distributed data or the Mann–Whitney et al , very negative resting membrane potential , inability to generate action potentials and linear current–voltage relationships, typical of so‐called passive astrocytes  of either sex as described in Tottene et al  and in A2, 1 mM CaCl2, 25 mM NaHCO3, 1.25 mM NaH2PO4, 25 mM glucose, saturated with 95% O2 and 5% CO2. For the recording of Glu transporter‐mediated currents, the extracellular solution also contained antagonists of AMPA receptors (10 μM NBQX), NMDA receptors ‐MK801), and GABAA receptors (20 μM (+)‐bicuculline or 5 μM gabazine) to block neuronal postsynaptic current flow  115 K‐gluconate, 6 KCl, 4 MgATP, 0.3 NaGTP, 10 Na‐phosphocreatine, 10 HEPES, 5 glucose . The extracellular solution contained 125 mM NaCl, 2.5 mM KCl, 1 mM MgCln = 66; KI: 16.0 ± 0.8 MΩ, n = 47; P = 0.15). Pipette resistance was 5–6 MΩ. Currents were sampled at 10 kHz and filtered at 2 kHz.Currents were evoked in astrocytes  by passing constant current pulses  every 15 s (or 20 s with train of pulses) through a concentric bipolar tungsten electrode  placed in layer 1 at least 200 μm away from the recorded astrocyte ‐3‐[[3‐[[4‐(trifluoromethyl)benzoyl]amino]phenyl]methoxy]‐L‐aspartic acid  (0.5–15 μM). The STC was obtained by subtracting the residual current remaining in the presence of TFB‐TBOA (TBOA) from the total control current ) with τrise = 2.35 ms ) with τrise = 2.35 ms and with A equal to the maximal current measured in individual astrocytes at about 60 ms after stimulation when the transient transporter current had decayed to zero .Synaptically activated Glu transporter currents (STCs) elicited by single pulse stimulation were isolated pharmacologically using saturating concentrations of the GluTs inhibitor (3 UK) 0.5–5 μM. TheSweeps are averages of at least five responses. Stimulation artifacts have been truncated.et al  and perfused transcardially with a flush of saline solution followed by 4% freshly depolymerized paraformaldehyde in 0.1 M phosphate buffer . Brains were removed, postfixed in the same fixative , and cut in coronal sections on a vibratome in 50‐μm sections that were collected in PB until processing  and decapitated. Cerebral cortex was quickly separated, tissue was homogenized , an isoform almost exclusively expressed in astrocytes in the adult brain.Migraine is a common disabling brain disease. Cortical spreading depression (CSD) is thought to play a key role in migraine pathogenesis in that it underlies migraine aura and may trigger migraine headache. The mechanisms of the primary brain dysfunction underlying the susceptibility to CSD ignition in the human brain and the onset of a migraine attack remain largely unknown and are a major open issue in the pathophysiology of migraine. To gain insights into this question, we studied the mechanisms underlying facilitation of CSD in a knockin (KI) mouse model of familial hemiplegic migraine type 2 (FHM2), a rare monogenic form of migraine with aura caused by loss‐of‐function mutations in the gene encoding the α+ clearance by cortical astrocytes during neuronal activity are reduced in heterozygous KI mice that carry the W887R FHM2 mutation and have 50% reduced α2 NKA protein in the brain, thus providing direct evidence for a key role of α2 NKA in clearance of synaptically released glutamate and in extracellular K+ buffering during neuronal activity and (ii) the density of GLT‐1a glutamate transporters in the membrane of astrocytic processes surrounding cortical excitatory synapses is about 50% reduced in the FHM2 mouse model, pointing to a specific tight coupling between α2 NKA and GLT‐1a in the astrocytic processes. Measurements of CSD threshold and velocity in cortical slices from FHM2‐KI mice treated with ceftriaxone and from wild‐type mice after pharmacological inhibition of a fraction of glutamate transporters provide evidence that the defective glutamate clearance by astrocytes underlies most of the facilitation of CSD initiation (and a large fraction of the facilitation of CSD propagation) in the FHM2 mouse model.Combining patch‐clamp recordings from astrocytes in acute cortical slices with immunohistochemistry and immunogold electron microscopy, we show that (i) the rates of glutamate and KBy uncovering the key mechanism underlying the facilitation of CSD in the FHM2 mouse model, this study moves forward our understanding of the molecular and cellular mechanisms that may underlie the brain susceptibility to CSD ignition in migraine and point out a direction in which to search for novel migraine therapies. In particular, it points to excessive cortical glutamatergic synaptic transmission as a key mechanism underlying vulnerability to CSD ignition in migraine and to the enhancement of astrocytic glutamate transporter function as a possible new therapeutic strategy.AppendixClick here for additional data file.Expanded View Figures PDFClick here for additional data file.Source Data for Expanded View and AppendixClick here for additional data file.Review Process FileClick here for additional data file.Source Data for Figure 1Click here for additional data file.Source Data for Figure 2Click here for additional data file.Source Data for Figure 3Click here for additional data file.Source Data for Figure 4Click here for additional data file.Source Data for Figure 5Click here for additional data file.Source Data for Figure 6Click here for additional data file.Source Data for Figure 7Click here for additional data file.Source Data for Figure 8Click here for additional data file.Source Data for Figure 9Click here for additional data file.


Gender inequality and sexist behaviour is prevalent in almost all workplaces and rampant in online environments. Although there is much research dedicated to understanding sexist behaviour, we have almost no insight into what triggers this behaviour and the individuals that initiate it. Although social constructionist theory argues that sexism is a response towards women entering a male dominated arena, this perspective doesn’t explain why only a subset of males behave in this way. We argue that a clearer understanding of sexist behaviour can be gained through an evolutionary perspective that considers evolved differences in intra-sexual competition. We hypothesised that female-initiated disruption of a male hierarchy incites hostile behaviour from poor performing males who stand to lose the most status. To test this hypothesis, we used an online first-person shooter video game that removes signals of dominance but provides information on gender, individual performance, and skill. We show that lower-skilled players were more hostile towards a female-voiced teammate, especially when performing poorly. In contrast, lower-skilled players behaved submissively towards a male-voiced player in the identical scenario. This difference in gender-directed behaviour became more extreme with poorer focal-player performance. We suggest that low-status males increase female-directed hostility to minimize the loss of status as a consequence of hierarchical reconfiguration resulting from the entrance of a woman into the competitive arena. Higher-skilled players, in contrast, were more positive towards a female relative to a male teammate. As higher-skilled players have less to fear from hierarchical reorganization, we argue that these males behave more positively in an attempt to support and garner a female player’s attention. Our results provide the clearest picture of inter-sexual competition to date, highlighting the importance of considering an evolutionary perspective when exploring the factors that affect male hostility towards women. Gender equality is currently one of the most hotly discussed topics across all scientific disciplines, political and religious ideologies, and workplaces –3. AlthoFrom an evolutionary perspective, intrasexual competition is common and is one of the strongest forces shaping sex differences . This isDespite these historical sex-differences in competition, men and women currently interact and compete for the same professional positions and accolades ,3. The mVideo games perfectly blend biologically and socially-constructed competitive environments thus providing a unique opportunity to probe how intersexual competition shapes individual behaviours and attitudes in humans. For example, a recent survey by the Entertainment Software Association states men and women are equally likely to play competitive video games . BecauseHalo 3 .Neither written nor verbal consent to participate in this study was collected by the authors specifically because all individuals that participated in the study were anonymous (as players use pseudonyms) and had already agreed to the terms of Xbox Live (which state that conversations can be recorded). Because of these aspects, and that data for this study was obtained from a prior study, the Ohio University Institutional Review Board (IRB) determined that this study was exempt from IRB review under Category 4, research involving collection or study of existing data.Halo 3, teams cooperate to kill members of the opposing team outside of a dedicated storyline such as that seen in a single player campaign within the game. As a result, the multiplayer games can be argued to be direct competitions outside of any sexualized storyline or content as they are only associated with the goal of eliminating opponents. Killing opponents results in a positive outcome for the team, while dying results in a negative outcome. In addition, improved performance within a game allows players to attain higher ranks and improve their skill rating, a long-term measurement of player status (i.e. dominance) that is publicly exhibited. We examine how individuals behave toward a male or female-voiced teammate as a function of positive (number of kills) and negative (number of deaths) player performance, as well as player status (maximum skill achieved).In multiplayer first-person shooters like Halo 3 is that players are covered head-to-toe with armor and identified by armor color, rather than facial features or body type. Additionally, player controlled avatars are not the hypersexualized males normally seen in many other video games, hopefully decreasing the overt sexism seen in most online competitive games and minimizing the effect of the game environment.A secondary benefit of using Halo 3. The audio/video output of an Xbox 360 console was routed to a television and to a digital video converter connected to a computer. The converter allowed each multiplayer match to be recorded as a video file to the computer, capturing exactly what the researcher experienced while in the game. In addition, the researchers connected an audio playback device to the Xbox controller, which allowed them to broadcast pre-recorded audio clips to other players, as if they were speaking to each other through the real-time voice channel.Data collection involved analyzing video recordings generated from Kuznekoff and Rose’s (2013) original study. In that study, the authors played in and recorded actual multiplayer matches of the video game Halo 3 as one normally would; however, this condition did not use the real-time voice channel to communicate with other players, and as a result, was not further analyzed here. For the male and female manipulations, the researchers also played in the matches as expected, but also developed a list of roughly a dozen phrases that were recorded separately by a male and female voice (Three Xbox LIVE (XBL) accounts were created and assigned to a respective experimental manipulation: control, male, and female. The control condition simply played in matches of le voice . These pHalo 3 XBL matchmaking system, up to 8 individuals (4 on each team) are matched up against other players, by the system, according to their skill level. While players may form a party of up to four people beforehand, players have no control over the other players assigned to that game by the matchmaking system. The player skill level is an objective indicator  of how good or bad that particular player is in the specific playlist selected. This skill level ranges from 1–50, with a higher score indicating a greater skill level. While being matched, players can leave the game up until the game starts and any vacancies may be filled with other players before starting. During the matches, we broadcasted the pre-recorded audio clips to other players at appropriate times (In the te times . For exaN = 126) were then transcribed by a professional transcription company (www.pacifictranslation.com.au) by individuals blind to the true purpose of the study and the experimental manipulations used. The authors randomly spot-checked 10% of the files for accuracy. One independent coder and one of the authors coded the comments each player made. Coders were provided with a codebook that explained the coding procedures. Both coders looked for comments and questions that appeared to be directed toward the experimental player and classified them as positive, negative, or neutral. Comments and questions in the positive categories were those that coders perceived as supportive or friendly, while those in the negative categories were those that the coders perceived to be aggressive or condescending  as a starting point, the current study focused on individual gamers instead of each overall game. First, we only used the recordings from the male and female manipulations in which other players spoke. Following this selection phase, the selected video files  statistics, 10% of the total transcript files (N = 263), one file for each individual player/participant, were randomly selected and coded by both coders. Cohen’s kappa was calculated for each category and kappa values between 0.61 and 0.80 represent substantial agreement . The kapHalo 3 matchmaking system, which analyzes the performance of the player in each game and adjusts that player’s skill level over time. This skill level provides a standard metric by which player skill (i.e. dominance) can be compared against each other.In addition to coding comments made by focal players, we also examined focal-player performance for the games they played in. These data included the overall game outcome  from the perspective of the focal player, the focal player’s maximum skill level achieved, and the focal player’s number of kills and deaths. Lastly, these performance statistics were also recorded for the experimentally controlled player, which allowed us to calculate a relative performance metric for each focal player . The skill level is calculated automatically by the We examined whether the number of positive, negative and neutral statements by a player were correlated with the outcome of the game, the experimental manipulation, and individual performance metrics . We also added interactions between manipulations and the three performance metrics as we hypothesized that performance would differentially affect behaviour of focal individuals in the different manipulations.Rather than individual performance alone, we also examined whether positive, negative and neutral statements were correlated with performance of the focal player relative to the experimental player (i.e. the experimenter controlled player). For this examination, we created a model with the experimental manipulation and each of the relative performance metrics. We also added the interactions between the manipulation and each of the relative performance metrics.https://github.com/latrodektus/VG_Sexism).We used separate GLMs with a Poisson distribution and a log link to test the six models regarding positive, negative, and neutral statements outlined above. To examine whether individual performance relative to the experimental player was correlated with the use of sexist statements, we used a GLM with a binomial distribution and a logit link. All statistics were completed in R 3.1.1, mean values are followed with the standard error, and we provide standardized beta coefficients for significant effects. We used the effects package  to plot We played a total of 163 games of Halo 3 in the two manipulations. We stopped at 163 as this is a substantial time effort. Data could not be analyzed during the experiment as the transcripts needed to be transcribed. Of the 163 games, 82 were in the female manipulation and 81were in the male manipulation and players only spoke within 102 of the games. A total of 189 players spoke in these 102 games; all of them were male. This is not to say that women did not play, just that they did not speak. This does, however, reinforce the fact that women are entering a very male dominated environment. Of these individuals, 147 individuals were teammates of the experimental player and 42 individuals were opponents. For our analyses, we focused on teammates as these individuals interact with the experimental player for the duration of the game. Opponents, in contrast, only interacted with the experimental player for a short period (less than 3 minutes) in the lobby after the game. Of the 147 teammates, 82 individuals were in the female manipulation and 65 were in the male manipulation. The range of players that spoke within an individual game varied from 1–3. To test whether the ‘Game’ affected the statement type and number, we added game as a random variable in our initial analyses. As ‘Game’ did not explain any of the variation in the type and number of statements made, it was removed from further analyses.2 = 14.61, p<0.0001, 2 = 7.01, p = 0.008) on the number of positive statements  such that focal players that had a relatively greater number of deaths than the experimental player made more positive statements. Focal players with a higher skill achieved relative to the experimental player also made more positive comments ; there was a near-significant interaction with manipulation such that the experimental player in the female-voiced treatment received more positive comments when focal players were of a higher skill level, but fewer positive comments when the experimental player was higher in skill  such that focal players that had relatively fewer kills than the experimental player made more positive statements. There was no effect of the manipulation or any of the other manipulation by performance interactions . There was also a significant interaction of the manipulation with both measures of focal player performance. Overall, there were a greater number of negative comments made by focal players in both manipulations as they died more often (i.e. their team performance decreased). However, focal players made a greater number of negative statements in the female-voiced manipulation when dying less (i.e. greater performance), and a greater number of negative statements in the male-voiced manipulation when dying extremely often (i.e. very poor performance) . As a result, we removed them from our analysis to ensure they did not skew our results towards significance. Focal players stated more negative statements in the female-voiced (2.66±0.36) compared to the male-voiced manipulation . There was a positive correlation between the difference in deaths and the number of negative statements  with focal players that died relatively more often than the experimental player (i.e. relatively poorer team performance) making more negative comments. There was also a negative correlation between the relative difference in kills and the number of negative statements made  such that focal players that had relatively more kills than the experimental player made fewer negative comments. There was no effect of any other factors on the number of negative statements . In the model examining how relative performance and skill affected neutral statements, there was only a negative correlation between the number of neutral statements made by focal players and the difference in kills between the focal and experimental player  such that players that had a greater number of kills relative to the experimental player made fewer neutral comments. No other factors affected the number of neutral statements made by focal players.The number of neutral comments stated by focal players was only affected by the number of deaths. Focal players that died more stated more neutral statements , the number of deaths  or the number of kills  relative to the experimental player.Since there were a greater number of negative statements within the female manipulation, we examined whether these statements could be considered hostile sexism . Of the The goal of this study was to examine the moderating effect of performance and skill on the frequency of positive and negative statements towards a female- or male-voiced teammate in an online first-person shooter video game—a metric providing insight into sexism. We found that skill determined the frequency of positive and negative statements spoken towards both male- and female-voiced teammates. In addition, poorer performance (fewer kills and more deaths) resulted in more negative statements specifically in the female-voiced manipulation. We thus argue that our results best support an evolutionary explanation of female-directed aggression. Low-status males that have the most to lose due to a hierarchical reconfiguration are responding to the threat female competitors pose. High-status males with the least to fear were more positive, suggesting they were switching to a supportive, and potentially, mate attraction role.Our results are particularly important because the ecological design observed players in their natural state rather than in a laboratory or other foreign environment. The large online community of players using pseudonyms means that policing individual behaviour is almost impossible. Individuals thus generally behave expressively and honestly since there is no fear of retribution. Additionally, our use of an online environment avoided certain biases that can occur with human research, specifically the social desirability bias, where participants engage in or report more pro-social behaviour and under report or refrain from anti-social behaviour. Our study thus provides a unique perspective into female-directed hostility.While playing with male teammates, men generally follow rules associated with navigating hierarchies. Skill did not moderate focal player positivity towards a male-voiced teammate, but higher skilled individuals were less negative. In addition, when performing poorly, players increased the number of positive and neutral statements , and werMales behaved in the opposite manner when playing with a female-voiced teammate. Overall, the female-voiced manipulation experienced a greater frequency of negative comments, but female-directed negativity decreased as focal-player performance improved . In addiOur results support an evolutionary argument for why low-status, low-performing males are hostile towards female competitors. Dominance is tightly linked to fitness in men as studies from hunter-gatherer societies demonstrate that dominance rank increases fitness through offspring number  and resoApart from restructuring the hierarchy, a high status female poses a secondary threat to relatively lower status males: as women are attracted to dominance , a high-There are two alternative social explanations for our results. First, players could be responding to the novelty of having a female voiced teammate in a male-dominated environment. Indeed, a female speaking while playing first-person shooter games is rare as none of the 189 players recorded were female. However, novelty is not the driver of the differences in behaviour as performance and skill mediated the number of positive and negative statements. Second, individuals may simply be responding more aggressively towards individuals with a higher pitched voice , as dominance is predictable by voice pitch . AlthougOur study demonstrates that video games offer a unique opportunity to examine variation in sexist behaviours and the situations that result in changes in sexist attitudes. By demonstrating that female-directed hostility primarily originates from low-status, poorer-performing males, our results suggest that a way to counter it may be through teaching young males that losing to the opposite sex is not socially debilitating. This can be most easily done using video games as the gaming population is nearly equally split between men (52%) and women (48%) . HoweverS1 Dataset(CSV)Click here for additional data file.S1 Text(DOCX)Click here for additional data file.S2 Text(DOCX)Click here for additional data file.