CD39 and control of cellular immune responses CD39 is the cell surface-located prototypic member of the ecto-nucleoside triphosphate diphosphohydrolase -LRB- E-NTPDase -RRB- family . Biological actions of CD39 are a consequence -LRB- at least in part -RRB- of the regulated phosphohydrolytic activity on extracellular nucleotides . This ecto-enzymatic cascade in tandem with CD73 -LRB- ecto-5 -- nucleotidase -RRB- also generates adenosine and has major effects on both P2 and adenosine receptor signalling . Despite the early recognition of CD39 as a B lymphocyte activation marker , little is known of the role of CD39 in humoral or cellular immune responses . There is preliminary evidence to suggest that CD39 may impact upon antibody affinity maturation . Pericellular nucleotide/nucleoside fluxes caused by dendritic cell expressed CD39 are also involved in the recruitment , activation and polarization of naïve T cells . We have recently explored the patterns of CD39 expression and the functional role of this ecto-nucleotidase within quiescent and activated T cell subsets . Our data indicate that CD39 , together with CD73 , efficiently distinguishes T regulatory cells -LRB- Treg -RRB- from other resting or activated T cells in mice -LRB- and humans -RRB- . Furthermore , CD39 serves as an integral component of the suppressive machinery of Treg , acting , at least in part , through the modulation of pericellular levels of adenosine . We have also shown that the coordinated regulation of CD39/CD73 expression and of the adenosine receptor A2A activates an immunoinhibitory loop that differentially regulates Th1 and Th2 responses . The in vivo relevance of this network is manifest in the phenotype of Cd39-null mice that spontaneously develop features of autoimmune diseases associated with Th1 immune deviation . These data indicate the potential of CD39 and modulated purinergic signalling in the co-ordination of immunoregulatory functions of dendritic and Treg cells . Our findings also suggest novel therapeutic strategies for immune-mediated diseases . Introduction Our research interests have been in the purinergic modulation of vascular inflammatory and cellular immune responses in transplantation settings -LSB- 1 , 2 -RSB- . It is generally accepted that extracellular nucleotides -LSB- e.g. , ATP , uridine triphosphate -LRB- UTP -RRB- , adenosine diphosphate -LRB- ADP -RRB- -RSB- , and the derivative nucleosides -LRB- e.g. , adenosine from ATP -RRB- , are released in a regulated manner by most all cells to provide the primary components for purinergic responses -LSB- 3 -RSB- . High levels of ATP may be released by CD4 + and CD8 + T cells upon stimulation with Con A or anti-CD3 mAb and serve to activate cells -LSB- 4 -RSB- . Importantly , ATP -LSB- or uridine diphosphate -LRB- UDP -RRB- -RSB- stimulation of monocytes , lymphocytes and endothelium induces largely proinflammatory responses , such as the release of interleukin -LRB- IL -RRB- -1 -LRB- or IL-8 -RRB- -LSB- 4 -- 6 -RSB- . On dendritic cells -LRB- DC -RRB- , exposure to extracellular ATP induces migration and differentiation to drive cellular immune responses -LSB- 7 -RSB- . Adenosine is also recognized as a bioactive agent in vascular inflammatory states , with effects mediated on both vascular cells and leukocytes -LSB- 8 -RSB- . In addition , adenosine has known anti-thrombotic effects , modulates the expression of anti-apoptotic genes and is immunosuppressive -LSB- 9 -RSB- . Adenosine is constitutively present in the extracellular space at low concentrations , but under metabolically stressful and hypoxic conditions , the levels rise dramatically -LSB- 10 -RSB- . Primary release of the mediator could occur ab initio , or this might follow conversion of released nucleotides to adenosine -LRB- see later -RRB- . The nucleotide/nucleoside mediators alluded to above bind specific purinergic receptors that comprise the second requirement for this complicated signalling network . Almost all cells carry cell-surface type 2 purinergic -LRB- P2 -RRB- receptors for nucleotides and adenosine or type 1 purinergic -LRB- P1 -RRB- receptors -LSB- 11 -RSB- . There are seven ionotropic -LRB- P2X -RRB- , at least eight metabotropic -LRB- P2Y -RRB- and four adenosine receptor subtypes that have been identified and characterized to date -LSB- 12 -RSB- . Multiple P2X and P2Y receptor subtypes are expressed by monocytes and dendritic cells , whereas lymphocytes express only P2Y receptors -LSB- 11 -RSB- . These various receptors operate in both auto - and paracrine loops and are considered to play a complex , important role in the regulation of vascular and immune cell-mediated responses . Depending on the P2 or adenosine receptor subtype , the cell types and signalling pathway involved , these receptors might preferentially trigger and mediate short-term -LRB- acute -RRB- processes that affect metabolism , adhesion , activation or migration . However , purinergic signalling also has profound impacts upon other more protracted reactions , including cell proliferation , differentiation and apoptosis , such as seen in several chronic inflammatory states -LSB- 12 -RSB- . These mechanisms could be also implicated in immune memory -LSB- 9 , 13 -RSB- . The third , and final , component of purinergic signalling systems comprises ecto-nucleotidases -LSB- 2 , 14 -RSB- . These ecto-enzymes hydrolyze extracellular nucleotides to generate nucleosides that in turn activate adenosine receptors , often with opposing effects to those seen with P2-mediated effects . Within the past decade , ecto-nucleotidases belonging to several enzyme families have been discovered , cloned and functionally characterized by pharmacological means . In this review , we will focus on CD39 , the prototype of the ecto-nucleoside triphosphate diphosphohydrolase -LRB- E-NTPDase -RRB- family -LRB- EC 3.6.1.5 -RRB- -LSB- 15 , 16 -RSB- . These proteins comprise a group of ecto-enzymes that hydrolyze extracellular nucleoside tri - and diphosphates . The ecto-nucleotidase chain or cascade , as initiated by NTPDases , is terminated by ecto-5 -- nucleotidase -LRB- CD73 ; EC 3.1.3.5 -RRB- -LSB- 2 , 17 -RSB- . Together , ecto-5 -- nucleotidase and adenosine deaminase -LRB- ADA ; EC 3.5.4.4 -RRB- , another ecto-enzyme that is involved in purine salvage pathways by converting adenosine to inosine , closely regulate local and pericellular extracellular concentrations of adenosine -LSB- 2 -RSB- . Most notably , however , in many tissues and cells , NTPDases also comprise dominant parts of a complex cell surface-located nucleotide hydrolyzing and interconverting machinery . Such ensembles also include the ecto-nucleotide pyrophosphatase phosphodiesterases -LRB- E-NPPs -RRB- , NAD-glycohydrolases , CD38/NADase activity , alkaline phosphatases , diadenosine polyphosphate hydrolases , adenylate kinase , nucleoside diphosphate kinase , and potentially ecto-F1-Fo ATP synthases -LSB- 2 -RSB- . Many aspects of these ecto-nucleotidase families and detailed expositions of structure/function relationships of the E-NTPDases and their role in the vasculature and nervous systems have been reviewed recently . The interested reader is referred to the recent special issue of Purinergic Signalling entitled ` Ecto-nucleotidases -- -LRB- PUSI 2 , 2 : 2006 -RRB- . This review will address only the immunobiology of CD39 , focusing on T lymphocytes and alluding briefly to co-expression of CD73 by immunosuppressive T cell subsets . CD39 in the immune system Recently , almost all of the published investigative focus on CD39 has been on the thromboregulatory properties of CD39 with respect to platelet and endothelial activation -LSB- 1 -RSB- . However , CD39 was first described as a B lymphocyte activation marker -LSB- 18 -RSB- . This ecto-nucleotidase is also expressed on natural killer -LRB- NK -RRB- cells , monocytes , DC and subsets of activated T cells -LSB- 19 -RSB- . The relevance of the expression of CD39 by these cells is not yet clear . Development of mutant mice either null for Cd39 or where human CD39 has been over-expressed have provided useful models to study the role of this ecto-nucleotidase in immune responses -LSB- 20 , 21 -RSB- . In the Cd39-null mouse -LSB- 20 -RSB- , B cell numbers -LSB- anti-CD45RA 14.8 ; anti-CD22.2 -LRB- Lyb-8.20 ; PharMingen , San Diego CA , USA -RRB- -RSB- in unstimulated blood and spleen have been noted to be normal -LRB- not shown -RRB- . Somewhat surprisingly , elicited IgG xenoantibody responses to solubilized xenoantigens administered to Cd39-null mice were found to be markedly suppressed -LSB- 14 -RSB- . With Dr. M. Cascalho -LRB- Mayo Clinic , Rochester , MN , USA -RRB- , we have investigated whether Cd39-null mice are capable of appropriate antibody responses . We have noted that Cd39-null mice exhibit impaired B cell memory responses to T-dependent antigens . Unexpectedly , however , these mutant mice show a significant increase in the frequency of somatic mutations post-immunization -LRB- not shown -RRB- . These observations suggest that CD39 function may contribute to the affinity maturation of antibody responses and to facilitate post-germinal center terminal B cell differentiation -LRB- M. Cascalho et al. , manuscript in preparation -RRB- . Although CD39 has been shown to be the dominant ecto-nucleotidase expressed by NK and NK-T cells and to impact upon cytokine production -LRB- -LSB- 19 -RSB- ; G. Beldi , unpublished -RRB- , the relevance of this has not yet been fully elucidated . CD39 is also the major NTPDase expressed by monocyte-macrophages . Upregulation of tissue factor expression by these cells in vitro and alterations in splenic macrophage populations in vivo have been observed in Cd39-null mice . P2Y receptors that are impacted upon by CD39 are critically linked to monocyte and endothelial cell responsiveness -LSB- 22 , 23 -RSB- . P2Y receptors on monocytes could be also subject to desensitization , comparable to what we have observed in platelet P2Y1 receptors in Cd39-null mice -LSB- 20 -RSB- . Therefore , we were interested to observe major defects in monocycle entry and migration into the substance of Matrigel plugs injected into the subcutaneous tissue of Cd39-null mice . In parallel , we also evaluated parameters of monocyte transendothelial migration influenced by ATP in vitro and noted failure of Cd39-null cells to migrate in response to exogenous nucleotides . This defect could be overcome by co-stimulation with serotonin , suggesting a degree of P2Y receptor desensitization in Cd39-null monocytes -LSB- 14 , 24 -RSB- . Ecto-enzymes , specifically ecto-nucleotidases , are known to play an important role in leukocyte trafficking but this complex area will not be dealt with here -LRB- for an excellent review on this topic , see -LSB- 25 -RSB- -RRB- . However , CD39 has been recognized as a critical control point in the regulation of leukocyte accumulation within hypoxic tissues and a sixfold increase in CD39/CD73 tandem functioning in the setting of hypoxia has been demonstrated -LSB- 26 , 27 -RSB- . Langerhans cells -LRB- LC -RRB- are members of the DC family of antigen-presenting cells residing in the skin . NTPDase1 enzymatic function on antigen presenting DC is involved in the recruitment , activation and polarization of naïve T cells . Mizumoto et al. established that LC from the Cd39-null mice do not hydrolyze ATP and ADP , unlike wild-type cells -LSB- 28 -RSB- . Cd39-null DC are also markedly unresponsive to ATP and are susceptible to cell death , but only after prolonged exposure to nucleotides -LSB- 28 -RSB- . Mutant mice null for Cd39 have amplified inflammatory responses to irritant chemicals . In these Cd39-null mice , there are major defects in dendritic cell formation , antigen presentation and T cell responses to haptens . These result in markedly attenuated responses to contact allergens in type IV hypersensitivity cutaneous responses that are also seen in inflammatory bowel disease models following haptenic stimulation -LRB- not shown -RRB- . These data suggest that Cd39 expression is required for optimal stimulation of hapten-reactive T cells in mice -LSB- 28 -RSB- . Somewhat paradoxically , CD39 also appears to function as an additional recognition structure on haptenated target immunocytes for HLA-A1-restricted , hapten-specific cytotoxic T cells -LSB- 29 -RSB- . Curiously , following deletion of Cd39 , LC are fully functional with respect to homing and phenotypic maturation . However , the major defect is that these cells appear less able to stimulate T cells . These abnormalities are potentially also relevant to allograft rejection processes -LSB- 14 -RSB- . Sequelae of these putative immune abnormalities include the relative failure of Cd39-null mice to reject allografts under limited co-stimulation blockade -LSB- 30 -RSB- . These data indicate a previously unrecognized role of CD39 and the effects on nucleotide-mediated signalling in immunological responses -LSB- 28 -RSB- . Localization of CD39 within lipid rafts implies that this ecto-enzyme may be involved in cell-cell contacts and signalling -LSB- 31 -RSB- . Such membrane micro-domains serve as the ` message center -- in which numerous signalling molecules are concentrated and scaffolding developed -LSB- 32 -RSB- . The Cd39-null DC might exhibit defective functions because of putative defects in initiating and maintaining cell-cell contacts . This mechanism speculatively involves facilitating integrin associations by purinergic signals within the ` immunological synapse -- and would have parallels to the way chemokines have been implicated in this process -LSB- 33 -- 36 -RSB- . CD39 on DC and/or T cells may be likewise translocated to the immunological synapse during antigen presentation to facilitate intercellular signalling . More typically , CD39 is considered to play a cellular immunoregulatory role by hydrolyzing ATP -LRB- and perhaps ADP -RRB- released by T cells during antigen presentation and thereby generating adenosine , a known immunosuppressive molecule . Recent work has also indicated that regulatory CD4 + ve CD25 + ve T cells -LRB- Treg cells -RRB- play important roles in the suppression of immunological reactivity and maintenance of tolerance -LSB- 37 , 38 -RSB- . Patterns of expression of CD39 by Treg and the possibility that the balance of extracellular nucleotides/nucleosides influence -LRB- s -RRB- the function of these interesting cells have been a major focus of investigation in our laboratories of late . CD39 expression by immunosuppressive regulatory T cells Treg are central to the acquisition of immunological tolerance . Here the immune system does not mount cellular responses against specific antigens while reactivity towards other antigens is maintained . The precise mechanisms underlying the acquisition of tolerance are not fully understood . Transplanted graft outcomes in the absence of heavy immunosuppression depend on consistent balances between cytopathic effector cells and Treg -LSB- 39 -RSB- . Depletion of the cytopathic T cell clone has been proposed as one mechanism for the induction of tolerance . However , it is now clear that the long-term maintenance of tolerance is also dependent on self-perpetuating immunoregulatory mechanisms that limit or constrain alloresponses . The suppressive Treg populations are considered key to the development and maintenance of peripheral tolerance -LSB- 33 , 40 -RSB- . Significant deficiencies are apparent in identifying Treg . Although traditionally defined by CD4 + CD25 + expression , the latter marker along with other membrane proteins such as GITR and CTLA4 becomes non-specific and somewhat redundant following activation . Such markers are widely upregulated on other cells thereby losing specificity for the Treg population . The forkhead winged transcription factor FoxP3 is specific for Treg ; however , its intracellular location limits its usefulness in the study of this population . Moreover , it is now apparent that CD25 does not encompass all Treg as defined by FoxP3 + expression -LSB- 41 -RSB- . Similarly , the mechanisms of Treg action are poorly defined . Putative mechanisms of suppression by Treg include cell-to-cell contact predominant in vitro and the release of soluble mediators that may predominate in vivo . As an example , IL-10 and transforming growth factor β have been identified as soluble factors that may mediate Treg suppression . Our recent studies and data from other groups have indicated that adenosine is an important mediator generated by Treg cells , and appears responsible for , at least in part , their functions -LSB- 42 -RSB- . Patterns of immune expression of CD39 by Treg were determined using standard techniques ; using the Cd39-null mouse cells as negative controls . CD3 + , CD8 + , NK1 .1 + , B220 + , CD11b + and CD11c + cells were positively selected from spleens and lymph nodes of 8 - to 10-week-old C57BL6 mice through the use of MACS Sort magnetic beads in MACS LS Separation columns -LRB- Miltenyi Biotec , Bergisch Gladbach , Germany -RRB- . T cells enriched for CD4 were obtained from lymph node and spleen preparations using CD4 T cell columns -LRB- R&D Systems , Minneapolis , MN , USA -RRB- . CD4 + / CD25 + and CD4 + / CD39 + were positively selected using the relevant antibody and MACS Sort magnetic beads in MACS MS Separation columns -LRB- Miltenyi Biotech , Bergisch Gladbach , Germany -RRB- . Where indicated , the same subsets were also sorted after staining the purified CD4 + cells with CD25 or CD39 , using the MoFlo cell sorter -LRB- BD Biosciences , San Jose , CA , USA -RRB- . In selected experiments , CD4 + / CD25 + cells were purified using the murine CD4 + CD25 + T regulatory cell isolation kit from Miltenyi Biotech -LRB- Bergisch Gladbach , Germany -RRB- . The purity of the different cell populations was verified by flow cytometry on FACSort -LRB- BD Biosciences , San Jose , CA , USA -RRB- . Anti-mouse and anti-human CD4 , CD8 , CD19 , B220 , NK1 .1 and CD25 were from eBiosciences -LRB- San Diego , CA , USA -RRB- , as were anti-mouse CD5 , CD62L , CD45RB , interferon -LRB- IFN -RRB- - γ and IL-4 . Purified anti-mouse CD3 and CD28 were from PharMingen -LRB- BD Biosciences , San Diego CA , USA -RRB- . Rabbit anti-mouse CD39 polyclonal antibody was used to stain cells purified from wild-type lymph nodes and from spleen , as described -LSB- 43 -RSB- . Using cells harvested from naïve C57BL6 mice , CD39 was found to be expressed by the majority of monocytes and by subsets of lymphocytes , also inferred by gating on forward scatter -LRB- FSC -RRB- and side scatter -LRB- SSC ; not shown -RRB- . Among node-derived lymphocytes , the majority of CD39 + cells are found in CD19 + -LRB- or B220 + -RRB- B cells . The remaining CD39 + cells were shown to reside almost totally within the CD4 + subset -LRB- Fig. 1a -RRB- . There , they consistently range from 8 to 12 % of all CD4 + cells in lymph node -LRB- and spleen -RRB- . Further characterization of resting C57BL6 lymphocytes revealed that CD39 is selectively expressed on CD4 + / CD25 + T cells -LRB- Fig. 1a -RRB- . CD39 is consistently and abundantly expressed in CD4 + / CD25high T cells , while the CD4 + / CD25dim populations show a dichotomic expression pattern of CD39 , with ~ 50 % of the cells positive . Less than 1 % of CD39 + cells were found in the CD4 + / CD25 − compartment . The majority of peripheral CD4 + / CD39 + cells are also CD45RBlow and mostly CD62Llow , with similar expression patterns observed in BALB/c mice -LRB- not shown -RRB- . Fig. 1 a Expression of CD39 on mouse lymphoid cells . Lymph node cell suspensions were prepared from 8-week-old C57/BL6 mice and were gated based on FSC and SSC parameters -LRB- not shown -RRB- . CD4 + cells were gated based on CD25 expression . CD39 expression on the different subsets is shown in the histograms -LRB- open profiles -RRB- plotted against an irrelevant control -LRB- gray profiles -RRB- . Differential expression of CD39 on CD4 + cells can be shown to be closely associated with CD25 . b RT-PCR analysis of foxp3 mRNA expression . This was done in sorted cells with the following markers : Foxp3 + / CD39 + , Foxp3 + / CD39 - , Foxp3 - / CD39 + and Foxp3 - / CD39 - . CD4 + cells obtained from the Foxp3-GFP ` knockin -- animals that had been generated by Mohamed Oukka and Vijay K. Kuchroo with colleagues -LRB- Ref -RRB- . B lymphocytes were used as CD39 + control and included for comparison -LRB- striped bars -RRB- . c RT-PCR analysis of CD73 mRNA expression . This was done in sorted cells with the following markers : Foxp3 + / CD39 + , Foxp3 + / CD39 - , Foxp3 - / CD39 + and Foxp3 - / CD39 - . B lymphocytes were used as CD39 + control and included for comparison -LRB- striped bars -RRB- . CD73 is also a useful immunophenotypic marker for Treg cells -LRB- not shown -RRB- ; and when combined with CD39 provides near-concordance with foxp3 expression . The Foxp3 - / CD39 + subset does not express CD73 and these cells resemble the memory phenotype -LRB- not shown -RRB- These results were confirmed by reverse transcription polymerase chain reaction -LRB- RT-PCR -RRB- analysis of selected cell populations . Both CD11b + and CD11c + cells expressed high levels of CD39 mRNA , indicating that macrophages and dendritic cells constitutively express CD39 . Within the T cell compartment , the highest expression levels of CD39 transcripts were found in CD4 + cells . Gene expression profiling of CD4 + T cells , sorted on the basis of CD39 cell surface expression , indicated that the CD4 + / CD39 + , but not CD4 + / CD39 − , T cells robustly express markers of Treg cells -LSB- 40 -RSB- , viz. . Foxp3 , CD25 , GITR and CTLA-4 . Further , CD4 + / CD39 + cells are anergic in the absence of IL-2 -LRB- not shown -RRB- and suppress T effector -LRB- CD4 + / CD25 − -RRB- proliferation with an efficacy similar to that observed with classic CD4 + / CD25 + Treg -LRB- S. Deaglio et al. , submitted manuscript -RRB- . We have also confirmed the utility of CD39 in defining human Treg populations as comparable patterns of CD39 antigen expression in human cells are observed . The majority of human CD19 + B cells express CD39 ; however , within the T cell compartment CD39 + cells are present in the CD4 + subset -LRB- not shown -RRB- . As in the mouse , CD39 is highly expressed in the CD4 + / CD25high T cells , with only ~ 50 % of cells CD39 + within the CD4 + / CD25dim population . Negligible CD39 expression is found in the CD4 + / CD25 − population -LRB- not shown -RRB- . Gene expression profiling of human CD4 + / CD39 + T cells also confirms that these cells express Foxp3 , GITR and CD25 in a pattern analogous to that of traditional CD4 + / CD25 + Treg -LRB- K. Dwyer et al. , submitted -RRB- . Our analysis demonstrates that CD39 expression in both murine and human T cells is restricted to a subpopulation of CD4 + / CD25 + cells that expresses markers associated with T regulatory function . We have further examined the exact relationship between CD39 expression and the regulatory phenotype by using T cells from mutant mice with the green fluorescent protein -LRB- GFP -RRB- reporter gene introduced into the endogenous Foxp3 locus -LSB- designated as ` Foxp3 + -LRB- GFP + -RRB- knockin -- cells ; kindly provided for these experiments by M. Oukka and V. K. Kuchroo , Boston , MA , USA -RSB- -LSB- 44 -RSB- . Four populations could be defined by differential CD39 and Foxp3 expression : Foxp3 + / CD39 + , Foxp3 + / CD39 − -LRB- minor population grouping -RRB- , Foxp3 − / CD39 + and Foxp3 − / CD39 − . These populations were sorted and gene expression profiles determined . The GFP + / CD39 + fraction was shown to mirror the genetic profile of Treg , as defined by the presence of Foxp3 transcripts in this positive control -LRB- Fig. 1b -RRB- . Interestingly , the next major subpopulation Foxp3 -- / CD39 + cells contain T lymphocytes that are not classic Treg and yet appear to be associated with the memory compartment -LRB- W. Gao , manuscript in preparation ; Fig. 1b -RRB- . Foxp3 + -LRB- GFP + -RRB- Treg however were also found to co-express the ecto-nucleotidase CD73 , a unique situation amongst T lymphocytes -LRB- Fig. 1c -RRB- . Consistent with other phenotypic data -LRB- not shown -RRB- , RT-PCR analysis confirms that Foxp3 + / CD39 + cells have high levels of gene expression of both CD39 and CD73 -LRB- Fig. 1b -RRB- . CD73 , which converts AMP to adenosine downstream of CD39 , has been independently identified on CD25 + -LRB- FoxP3 + -RRB- Treg and CD25 − uncommitted primed precursor Th cells -LSB- 42 -RSB- . This recent work further supports our observations that the expression of ecto-nucleotidases and consequent adenosine generation play a role in the mediation of some of the suppressive capabilities of Treg cells . Hence , CD73 is co-expressed with CD39 as a cell surface marker of murine Tregs -LRB- Fig. 1c -RRB- . Adenosine as a Treg effector molecule Tregs from mutant mice deficient in CD39 have impaired regulatory function manifesting as a 50 % decrease in the ability of Cd39-null Tregs to modulate effector T cell function in vitro and in vivo . These results indicate that CD39 expressed by Treg is the major and rate limiting ecto-nucleotidase responsible for the generation of adenosine and suggest that a putative CD39/CD73-adenosinergic axis -LRB- i.e. , generating adenosine -RRB- may contribute to the immunoregulatory function of Treg -LRB- S. Deaglio et al. , manuscript submitted ; also Fig. 2 -RRB- . Fig. 2Schematic representation of Treg markers . The cellular phenotype of these suppressive T cells can be defined by FoxP3 + / CD39 + / CD73 + expression . Phosphohydrolysis of extracellular nucleotides by CD39 and CD73 generates adenosine , which exerts a component of the immunosuppressive effect Adenosine plays a central and direct role in the regulation of inflammatory responses and limiting inflammatory tissue destruction -LSB- 9 , 10 , 45 -RSB- . Potentially , close cell-cell contacts with pericellular generation of adenosine and regulated expression of adenosine receptors may be important modulatory factors directly suppressing T cell responses . Early in the immune response , adenosine favors recruitment of DC , which initiates specific immune responses -LSB- 9 , 10 , 28 -RSB- . The immunosuppressive effects of adenosine on T cells are thought to be mediated primarily through the A2A receptor . Adenosine inhibits the production of proinflammatory cytokines and superoxide anions . Adenosine activation of A2A receptor also induces heterologous desensitization of chemokine receptors , which are critical in leukocyte trafficking , through the activation of protein kinase A -LSB- 46 -RSB- . It is also feasible that the suppression mediated via Treg-generated adenosine could be exerted indirectly through downregulation of co-stimulatory molecules on DC or by competition for other non-characterized signal molecules -LSB- 47 -RSB- . Irrespective of the mechanism of action , we have shown that FoxP3 + Tregs are the only T cells that contain the full enzymatic machinery necessary to generate adenosine . This effect , in conjunction with the expression of the adenosine A2A receptor on effector -LRB- CD4 + / CD25 − -RRB- T cells , generates immunosuppressive loops limiting effector cell proliferation both in vitro and in vivo -LRB- S. Deaglio et al. , submitted -RRB- . Immune deviation and autoimmune diathesis in Cd39-null mice The pathophysiological relevance of the CD39-adenosinergic loop was further tested by examining effects of exogenous , pharmacological adenosine A2A receptor agonists on T cells , committed to either Th1 or Th2 lineages -LSB- 48 -RSB- . Adenosine might contribute to the resolution of inflammation by facilitating Th2 pathways by the inhibition of Th1 cell functions -LSB- 49 -RSB- ; this property has been addressed further in the Cd39-null mice . Subsets of naïve CD4 + T cells were polarized to a Th1 or Th2 phenotype through the addition of IL-12 -LRB- 10 ng/ml -RRB- and anti-IL-4 -LRB- 10 μg/ml -RRB- or IL-4 -LRB- 10 ng/ml -RRB- and anti-IFN-γ -LRB- 10 μg/ml -RRB- , respectively , following stimulation with plate-bound anti-CD3 -LRB- 5 μg/ml -RRB- and soluble anti-CD28 -LRB- 2.5 μg/ml -RRB- . The polarization of cells was confirmed by intracellular cytokine staining and gene profiling via RT-PCR analysis . Heightened expression of the A2A receptor , as measured by RT-PCR , could be detected in CD4 + / CD25 − cells polarized in vitro to Th1 , but not Th2 , phenotypes at day 3 -LRB- not shown -RRB- . In keeping with this observation , the addition of the adenosine A2A receptor agonist , ATL146e -LRB- kind gift from J. Linden , Adenosine Therapeutics -RRB- , at day 3 of culture , when all cells are already committed to either a Th1 or Th2 phenotype , selectively inhibits Th1 proliferation . This suggests once more that the A2A receptor is the critical immunomodulatory adenosine receptor -LSB- 9 -RSB- . No inhibition of the Th2 proliferation response by ATL146e -LSB- 50 -RSB- could be noted -LRB- Fig. 3a -RRB- . Fig. 3CD39 generation of adenosine preferentially regulates Th1 immune responses . a 3H-thymidine incorporation of CD4 + T cells purified from WT mice as polarized towards a Th1 or a Th2 phenotype for 5 days . The selective adenosine A2A agonist ATL146e was added after 3 days of culture . Data are expressed as % of inhibition and are the mean of duplicates ; error bars represent the SEM of three independent experiments . b CD4 + T cells were purified from Cd39-null -LRB- filled histograms -RRB- or WT -LRB- open histograms -RRB- mice and polarized towards a Th1 phenotype . The mRNA was extracted at day 3 and assayed for IFN-g -LRB- left panel -RRB- by RT-PCR . Data are representative of more than four independent experiments . c CD4 + T cells were purified from Cd39-null -LRB- filled histograms -RRB- or WT -LRB- open histograms -RRB- mice and polarized towards a Th1 phenotype . The indicated adenosine receptor agonists and antagonists were added at the beginning of the experiment -LRB- left panel -RRB- . d This panel shows the effects of apyrase on IFN-g production by Cd39-null CD4 + T cells polarized towards a Th1 phenotype . For these experiments , mRNA was extracted at day 3 and assayed for IFN-g -LRB- RT-PCR -RRB- . Representative data are shown from three independent experiments for each . e Representative images of Cd39-null mouse on C57BL/6/129 SVJ -LRB- upper panel -RRB- or BALB/c backgrounds -LRB- lower panel -RRB- manifesting alopecia . Histology of skin samples obtained from Cd39-null mice affected by alopecia . Uninvolved skin areas from the same animals as well as samples from WT mice were used for comparison . Biopsies were fixed and stained for CD4 cells . Original magnifications : × 10 for the upper middle panel , × 40 for the upper right and × 20 for the lower panels . f Skin samples were obtained from Cd39-null mice affected by alopecia , the tissue homogenized and mRNA extracted . Cytokine profiling was performed by RT-PCR , using a preamplification technique . Uninvolved skin from the same animal or a matched WT mouse used as controls . Representative data are from three animals Consistent with these results , in vitro polarization of CD4 + T cells towards a Th1 phenotype results in the increased production of IFN-γ by Cd39-null T cells , with a Th1-deviated phenotype -LRB- Fig. 3b -RRB- . Addition of ATL146e -LSB- 50 -RSB- , or soluble NTPDases -LRB- apyrase -RRB- , strongly inhibits the Th1 response in both the wild-type and Cd39-null polarized cells confirming that A2A receptors are present and functional in both groups . These data suggest lack of substrate in the Cd39-null cells to be responsible for the observed Th1 bias -LRB- Fig. 3c -RRB- . The A2A antagonist 8 - -LRB- 3-chloro-styryl -RRB- caffeine was shown to augment IFN-γ production in wild-type cells polarized to a Th1 phenotype -LRB- Fig. 3c -RRB- , further supporting this hypothesis . In addition , reconstitution of Cd39-null cells with apyrase restores the catalytic potential of the cells , producing adenosine and inhibiting IFN-γ production in a dose-dependent manner , effects similar to that of ATL146e -LRB- Fig. 3d -RRB- . Additional evidence that the perturbation in adenosine generation causes Th1 deviation , is that Cd39-null mice spontaneously develop autoimmune alopecia . Fifteen per cent -LRB- 9/59 -RRB- of designated Cd39-null animals develop skin lesions characterized by extensive and well-demarcated hair loss -LRB- Fig. 3e -RRB- . No lesions are observed in age-matched WT controls -LRB- 0/28 -RRB- . Similar lesions were observed in Cd39-null mice on the BALB/c background suggesting that the phenotype is strain independent -LRB- Fig. 3e -RRB- . The alopecia typically appears at ~ 20 -- 0 weeks of age in the facial region and thereafter extends out to include the trunk . The skin lesions are characterized by the presence of a population of CD4 + and CD8 + lymphocytes within the damaged hair follicles -LRB- Fig. 3e -RRB- . Moreover , sixfold increases in IFN-γ transcript levels are noted in areas of skin with active disease , when compared to areas of uninvolved skin from the same animal or from wild type controls -LRB- Fig. 3f -RRB- . In addition , there is increased expression of IL-2 , Foxp3 and CTLA-4 , consistent with the cellular infiltration . IL-4 and IL-10 were not detected -LRB- Fig. 3f -RRB- . Autoimmune manifestations impacting other organ systems and on renal function are under evaluation -LRB- D. Friedman , not shown -RRB- . These data validate the importance of adenosine in directing T cell subset differentiation and support a role for CD39 in orchestrating Treg cell suppressive responses under both in vitro and in vivo conditions . Summation This review summarizes components of extracellular nucleotide-mediated signalling pathway in T cells that are impacted upon largely by CD39 , the prototypic member of the E-NTPDase family of ecto-nucleotidases . Modulated , distinct NTPDase expression appears to regulate nucleotide - and nucleoside-mediated signalling in the immune system . As the vasculature uses similar mediators to regulate blood fluidity and hemostasis , expression of CD39 on either endothelial or immune cells might allow for full integration of vascular inflammatory and immune cell reactions at sites of injury . There is a wide field for future investigations of the role of nucleotides , nucleosides and ecto-nucleotidases in immune-mediated diseases . Increasing interest in this field may open up new avenues for investigation and the development of new treatment modalities for a large variety of illnesses , including atherosclerosis and the vascular or immune inflammation seen in transplant-related diseases .

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

apyrase
b cells
cd39
dendritic cells
ntpdase
platelet
t cells
vasculature

ecto-atpase e-ntpd immunology kidney liver


Effects of the noradrenergic agonist clonidine on temporal and spatial attention Rationale Recent theories posit an important role for the noradrenergic system in attentional selection in the temporal domain . In contrast , the spatially diffuse topographical projections of the noradrenergic system are inconsistent with a direct role in spatial selection . Introduction The locus coeruleus -- norepinephrine -LRB- LC -- NE -RRB- system is one of several brainstem neuromodulatory nuclei with widely distributed , ascending projections to the neocortex -LRB- Berridge and Waterhouse 2003 -RRB- . The conventional view of NE is that it has broad , nonspecific functions such as regulating arousal -LRB- Jouvet 1969 -RRB- . However , recent studies suggest that NE may have more specific cognitive functions , which are nevertheless closely related to the concept of arousal -LRB- Robbins 1997 ; Aston-Jones et al. 2000 ; Yu and Dayan 2005 ; Chamberlain et al. 2006 -RRB- . In particular , it has been proposed that NE has a key role in facilitating the responses to decision-making processes and in regulating the balance between exploitation and exploration -LRB- Aston-Jones and Cohen 2005 -RRB- . Recent views on the role of the LC -- NE system in decision making have been based on neuronal recordings of the primate LC during the performance of simple decision-making tasks . These recordings have indicated that under alert conditions -LRB- i.e. , conditions typical of human subjects in our experiments -RRB- , LC phasic responses are selectively observed after task-relevant and otherwise motivationally significant -LRB- e.g. , highly salient or infrequent -RRB- stimuli -LRB- Aston-Jones et al. 2000 -RRB- . Furthermore , stimulus-elicited phasic LC bursts reliably precede -LRB- by ∼ 200 ms -RRB- and are closely coupled in time with the behavioral response -LRB- Bouret and Sara 2004 ; Clayton et al. 2004 -RRB- . The noradrenergic innervation associated with such LC responses results in a system-wide , transient increase in the responsivity of efferent target neurons , which is thought to facilitate processing in response to the eliciting stimulus -LRB- Berridge and Waterhouse 2003 -RRB- . These observations have led to the suggestion that the LC phasic response can be thought of as a temporal attentional filter -LRB- 1 -RRB- that selects for the occurrence -LRB- i.e. , timing -RRB- of task-relevant -LRB- Aston-Jones and Cohen 2005 -RRB- or unexpected -LRB- Dayan and Yu 2006 -RRB- events , much like cortical attentional systems filter the content -LRB- e.g. , spatial characteristics or color -RRB- of stimuli and -LRB- 2 -RRB- that facilitates the processing of and responding to these stimuli to help optimize task performance -LRB- Aston-Jones and Cohen 2005 ; Nieuwenhuis et al. 2005a ; see also Coull 1994 -RRB- . In the current study , we evaluated the following two hypotheses : -LRB- 1 -RRB- According to the temporal filtering hypothesis outlined above , the LC -- NE system should be involved in temporal attention modulations operating at the phasic time scale -LRB- e.g. , in the order of tens of seconds -RRB- ; -LRB- 2 -RRB- In contrast , because of the spatially nonspecific pattern of LC projections to the forebrain , the LC -- NE system can not be directly involved in spatial attention shifts . That is , although the LC -- NE system projects to multiple brain areas , including many known to be involved in spatial attention , the diffuse character of these projections is inconsistent with the direct biasing of attention in any specific direction -LRB- i.e. , the LC does not `` know '' the relevant spatial location -RRB- . To test these two hypotheses , we contrasted , in human subjects , the effects of a noradrenergic drug on measures of temporal and spatial attention . Most previous studies examining the functional significance of the LC -- NE system have employed drugs acting on α2 receptors . In this study , we used the α2 adrenoceptor agonist clonidine , which at low doses decreases LC firing and attenuates the release of NE from axon terminals -LRB- Svensson et al. 1975 -RRB- . Previous studies have examined the effects of clonidine on temporal and spatial attention in a visuospatial cueing task . In this task , subjects are required to respond as quickly as possible to a series of stimuli presented to the left or right of fixation . Each stimulus is preceded by a cue indicating at which location the imperative stimulus is most likely to appear , and subjects are instructed to make use of this information by switching their attention to the location indicated by the cue . The comparison of reaction times on validly versus invalidly cued trials -LRB- the `` validity effect '' -RRB- provides an index of the efficiency of cue-induced spatial attention shifts . Fernandez-Duque and Posner -LRB- 1997 -RRB- extended this paradigm by including and contrasting reaction times on neutral-cue trials and no-cue trials -LRB- the `` alerting effect '' -RRB- , which provides an index of the transient increase in responsivity brought about by the mere presentation of the cue . The alerting effect thus serves as a measure of temporal -LRB- cue-evoked -RRB- changes in attention to the imperative stimulus . Clonidine has been found to reduce the size of the alerting effect in humans -LRB- Coull et al. 2001 -RRB- and monkeys -LRB- Witte and Marrocco 1997 -RRB- , suggesting that the temporal attentional effects of cues are mediated in part by the noradrenergic system . In contrast , studies have reported mixed results regarding the influence of clonidine on the validity effect : Two studies have reported a reduced validity effect -LRB- Clark et al. 1989 ; Coull et al. 2001 -RRB- , and one study has reported no effect of clonidine -LRB- Witte and Marrocco 1997 -RRB- . However , it is hard to interpret clonidine-related reductions in the validity effect , because they may reflect either a direct involvement of the noradrenergic system in spatial attention processes or an interaction between the alerting and attention-directing -LRB- i.e. , spatial -RRB- effects of cues . Specifically , by increasing the responsivity of cortical representations after cue presentation , LC-induced phasic NE release can enhance the effects of target selection by location -LRB- e.g. , the top-down modulation by prefrontal cortex -RRB- , resulting in an increased validity effect . The effects of this amplification mechanism would be diminished by clonidine . In contrast to previous studies , we used separate tasks for measuring the effects of clonidine on temporal and spatial attention : an attentional blink task , which requires the selection of targets in time and has no spatial component , and a visual search task , which requires the selection of targets in space but without the help of alerting cues . In the attentional blink task , each trial starts with a rapid serial visual presentation -LRB- RSVP -RRB- stream containing two target stimuli -LRB- T1 and T2 -RRB- and multiple distractors , each presented for about 100 ms. At the end of the RSVP stream , subjects are required to report the identity of T1 and T2 . The critical finding in this task is that subjects are heavily impaired at the identification of T2 for a few hundred milliseconds after correct identification of T1 . This deficit , known as the `` attentional blink , '' is usually most severe around 200 -- 400 ms -LRB- or two to four items -RRB- after T1 , after which , performance gradually recovers -LRB- Raymond et al. 1992 ; Chun and Potter 1995 -RRB- . In contrast , if T2 follows T1 without intervening distractors -LRB- at `` lag 1 '' -RRB- , performance on T2 is often -LRB- partially -RRB- spared -LRB- Raymond et al. 1992 ; Hommel and Akyürek 2005 -RRB- ; this phenomenon has been labeled `` lag-1 sparing . '' These and other findings suggest that the attentional blink task reveals important clues about the time course of fine-scale fluctuations in attention -LRB- Olivers 2007 -RRB- . Recently , a theory has been proposed that explains the attentional blink in terms of the temporal dynamics of the LC -- NE system -LRB- Nieuwenhuis et al. 2005b -RRB- . The theory was inspired by the observation that the timing of the attentional blink coincides with the refractory period in LC activity that follows an LC phasic response to target stimuli . Specifically , although NE potentiates processing in cortical areas , local NE release within the LC is autoinhibitory because of the noradrenergic action at presynaptic and dendritic α2 autoreceptors -LRB- Aghajanian et al. 1977 -RRB- . This autoinhibition results in a period after the LC phasic response -LRB- ∼ 200 -- 450 ms after the eliciting stimulus -RRB- , during which subsequent LC phasic discharge is rarely observed . According to the theory , the attentional blink may be mediated by this momentary unavailability of the LC phasic response -LRB- and attendant noradrenergic potentiation of information processing -RRB- after the LC response to T1 . In other words , if T2 is presented during the LC refractory period associated with T1 , it will not receive the benefit of NE-mediated facilitation and is therefore more likely to remain unidentified . A computational model implementing this theory was shown to accurately simulate the time course of the attentional blink , including lag-1 sparing -LRB- Nieuwenhuis et al. 2005b -RRB- . In addition , the theory offers an account of the close relationship of the attentional blink to the P3 component of the event-related potential , an electrophysiological correlate of the phasic NE release -LRB- Nieuwenhuis et al. 2005a -RRB- . To assess the effect of clonidine on spatial attention , we used a visual search task . On each trial , subjects searched for a target among multiple distractors in a visual search array and indicated as quickly as possible whether the target was present or absent . The number of distractors -LRB- i.e. , set size -RRB- was systematically varied across trials . In one condition -LRB- single-feature search -RRB- , the target was defined by a salient basic feature , such that search was efficient and the number of distractors had little influence on reaction times . In another condition -LRB- conjunction search -RRB- , the target was defined by a conjunction of two features , such that search was inefficient , and reaction times increased linearly with the number of distractors -- as if each item needed to be examined in turn . In this task condition , the efficiency of spatial attention processes is expressed in the function relating search reaction times to set size : The more visual attention is impaired , the steeper the slope of this function -LRB- as expressed in an increased effect of set size ; Tales et al. 2002 -RRB- . Our predictions with regard to the effects of clonidine were the following : First , we expected that clonidine would impair performance on the attentional blink task , in particular through its stimulation of inhibitory α2 autoreceptors in the LC . In accordance with the temporal filtering hypothesis , we expected that the clonidine-induced reduction in noradrenergic activity would lead to an impaired identification of T1 but especially of T2 , because it should presumably exacerbate the lack of NE associated with T2 processing under normal conditions -LRB- Nieuwenhuis et al. 2005b -RRB- . Second , aside from the well-documented main effect of clonidine on response speed -LRB- i.e. , general slowing ; e.g. , Tiplady et al. 2005 -RRB- , we expected no drug effects on visual search efficiency , as indexed by the effects of distractor set size . This latter prediction was based on the notion that the effects of NE are topographically diffused and hence should not affect the efficiency of selection in the spatial domain . Materials and methods Subjects Thirty-two healthy young adults , aged 18 -- 25 years , took part in a single experimental session in return for 60 . Only subjects with a systolic blood pressure above 100 mmHg and a diastolic blood pressure above 70 mmHg were included in the study . All subjects underwent a medical screening and were considered to be in satisfactory health . The use of medication that could interfere with clonidine was stopped the day before . All subjects with color blindness were excluded from the study . Subjects received an oral dose of 150 μg of clonidine or placebo in a double-blind , between-subjects design . Clonidine has well-established antihypertensive properties ; therefore , blood pressure and heart rate were monitored for subject safety . Measurements were taken every 15 min starting from t = − 15 until t = 120 and every 30 min from t = 120 until t = 240 . The clonidine group -LRB- N = 16 , eight women , M age 21.3 -RRB- and the control group -LRB- N = 16 , eight women , M age 21.3 -RRB- had similar mean ages . We also verified that the groups were similar in terms of intellectual functioning as assessed with the three-subtest version of the Groninger Intelligence Test -LRB- GIT -RRB- , a reliable indicator of the full-scale IQ -LRB- Luteijn 1966 -RRB- . Estimated IQ scores were 117.1 for the clonidine group and 116.8 for the control group . The visual-search task data from one subject from the clonidine group were discarded because of an accidental failure to comply with the task instructions . The study was approved by the medical ethics committee of the VU University Medical Center and was conducted according to the Declaration of Helsinki . Informed consent was obtained from all subjects before their inclusion in the study . Procedure Each subject was tested at approximately the same time of the day -LRB- afternoon -RRB- . Subjects were instructed to abstain from caffeine , nicotine , and other psycho-active substances from 15 h before the start of the session and to abstain from alcohol from 15 h before the start of the session until the day after the session . After the medical screening , subjects received a lactose-filled capsule with either clonidine or placebo . Immediately after that , the subtests of the GIT were administered . The two attentional tasks , described in detail below , were performed between t = 60 and t = 120 min posttreatment . Half of the subjects in each group started with the attentional blink task . The other half started with the visual search task . After completion of the tasks , the subjects were debriefed and paid . At t = 240 , subjects were reevaluated and returned home by taxi if blood pressure was -LRB- near -RRB- normal . Attentional blink task Stimulus generation and response recording in both tasks were controlled by E-Prime software -LRB- Psychology Software Tools , Pittsburgh , PA -RRB- . Stimuli were presented in black against a light grey background . Each trial started with a fixation cross measuring 0.5 × 0.5 ° , presented for 1,000 ms in the center of the display . Subsequently , the fixation cross was replaced by an RSVP stream of 15 -- 22 uppercase letters , each measuring approximately 0.9 × 0.9 ° . Each letter was randomly drawn -LRB- without replacement -RRB- from the alphabet and presented for 50 ms , followed by a 30-ms blank interval . This relatively rapid pace of stimulus presentation -LRB- i.e. , 80 ms between two consecutive stimulus onsets -RRB- was chosen to avoid ceiling levels of performance and consequently to increase the sensitivity of the task to individual and group differences . `` I , '' `` O , '' `` Q , '' and `` S '' were left out as they resemble digits too much . On each trial , two of the letters were replaced with digits , randomly drawn without replacement from the set 2 to 9 . T2 was presented three to six temporal positions from the end of the stream . The temporal distance between T1 and T2 was systematically varied from one to five items , corresponding to lags of 80 , 160 , 240 , 320 , and 400 ms. The subject 's task was to identify both T1 and T2 by typing the digits in order on a standard keyboard after the end of the RSVP stream . Subjects were instructed to guess whenever they failed to identify a digit . The two keyboard entries were followed by the presentation of a feedback stimulus for 150 ms -LRB- e.g. , ` + , − ' to indicate that T1 was correct and T2 was incorrect -RRB- . After a 1,000-ms blank screen , the next trial started . Each subject started with 15 practice trials , three with each condition , randomly intermixed . This was followed by four blocks of 50 trials each with each block containing ten repetitions of each lag . All task instructions were automated and presented on screen . Visual search task On each trial , subjects searched for a target among multiple distractors in a visual search array . On half of the trials , the target , a vertical red bar , was present in the array . On the other half of the trials , the target was absent . In one condition -LRB- single-feature search -RRB- , the distractors in the search display were vertical green bars of equal size as the target . Because in this case the target is defined by a unique feature -LRB- color -RRB- , it `` pops out '' from the display , resulting in a fast and efficient search process . In another condition -LRB- conjunction search -RRB- , the distractors were vertical green bars and horizontal red bars . In this case , the target does not consist of a single identifying feature but is defined by a specific conjunction of features -LRB- color and orientation -RRB- , resulting in a more time-consuming and error-prone search process . Each trial started with a white fixation cross measuring 0.9 × 0.9 ° against a dark background , presented for 500 ms in the center of the display . Subsequently , the fixation cross was replaced by the search display , which consisted of four , eight , or 16 items that were randomly plotted in the cells of an imaginary 6 × 6 matrix -LRB- 8.7 ° horizontally × 9.6 ° vertically -RRB- with some random jitter within the cells . The subject 's task was to report whether or not the target -LRB- 0.7 × 1.3 ° -RRB- was present by giving a response with their left or right index finger using the ` z ' and ` m ' keys on the computer keyboard . The keyboard entry was immediately followed a 1,000-ms blank screen after which the next trial started . Subjects performed four blocks of 96 trials each , with each block containing 16 repetitions of the factorial combination of set size -LRB- 4 , 8 , or 16 -RRB- and trial type -LRB- target present or absent -RRB- presented in random order . Search condition -LRB- single-feature search or conjunction search -RRB- was varied across blocks in an ABBA-order . Half of the subjects started with single-feature search , and the other half started with conjunction search . Subjects received written instructions and 12 practice trials before entering the experimental phase . The task instructions encouraged subjects to respond as quickly as possible while minimizing the number of errors . Performance feedback was provided at the end of each block . All task instructions were automated and presented on screen . Results Physiological measures Figure 1 presents for both groups the absolute values of heart rate , systolic , and diastolic blood pressure as observed throughout the experimental session . All three measures showed a significant effect of time -LRB- all P < 0.001 -RRB- . Systolic -LSB- F -LRB- 1 , 30 -RRB- = 11.8 , P = 0.002 -RSB- and diastolic blood pressure -LSB- F -LRB- 1 , 30 -RRB- = 6.3 , P = 0.018 -RSB- both showed significant main effects of group . The main effects of time and group were qualified by significant time X group interactions for systolic -LSB- F -LRB- 13 , 390 -RRB- = 9.8 , P < 0.001 -RSB- and diastolic blood pressure -LSB- F -LRB- 13 , 390 -RRB- = 6.1 , P < 0.001 -RSB- . Fig. 1Systolic and diasystolic blood pressure and heart rate following administration of placebo or 150 μg of clonidine -LRB- time = 0 -RRB- . The grey shaded area marks the time period during which the two cognitive tasks were administered Attentional blink task Figure 2 shows the average T1 accuracy -LRB- left panel -RRB- and T2 accuracy -LRB- right panel ; contingent on correct T1 identification -RRB- as a function of lag and group . A similar pattern of results was found if T2 accuracy was averaged across correct and incorrect T1 trials . Trials on which T1 and T2 were accurately identified but in the wrong order were treated as correct . As an analysis of variance -LRB- ANOVA -RRB- showed no significant effects of the order in which the two tasks were performed , this factor was omitted from the reported analyses . Fig. 2Average identification accuracy for the first -LRB- T1 ; left panel -RRB- and second -LRB- T2 ; right panel -RRB- of the two targets in the attentional blink task as a function of group -LRB- clonidine vs placebo -RRB- and the lag between T1 and T2 . As is usual , T2 accuracy is reported contingent on accurate identification of T1 T1 and T2 accuracy were entered in separate two-way mixed ANOVAs with lag and time-on-task -LRB- blocks 1,2 vs 3,4 -RRB- as within-subject factors and group as a between-subject factor . The variable time-on-task was included to assess the possibility that a group effect emerged over time . T1 accuracy showed an increasing trend with lag -LSB- F -LRB- 4 , 120 -RRB- = 12.3 , P < 0.001 -RSB- . The clonidine group -LRB- 79.5 % correct -RRB- performed slightly worse on T1 than the placebo group -LRB- 84.6 % -RRB- , but the main effect of group did not reach significance -LSB- F -LRB- 1 , 30 -RRB- = 2.0 , P = 0.17 -RSB- . The lag X group interaction was not significant -LSB- F -LRB- 4 , 120 -RRB- = 1.0 , P = 0.39 -RSB- . The two T2 accuracy curves show a pattern that is characteristic of attentional blink research : lag-1 sparing , followed by a drop in performance for lags 2 , 3 , and 4 -LRB- i.e. , the attentional blink -RRB- , and followed by the initial recovery of performance at lag 5 , at least in the clonidine group . This pattern was expressed in a significant effect of lag -LSB- F -LRB- 4 , 120 -RRB- = 43.2 , P < 0.001 -RSB- . The most important for the present purposes is the finding that T2 accuracy did not reliably differ between the two groups -LSB- F -LRB- 1 , 30 -RRB- < 1 -RSB- . Furthermore , although at lag 4 there was a sizeable numerical effect of group in the expected direction , the lag X group interaction was not significant -LSB- F -LRB- 4 , 120 -RRB- = 1.2 , P = 0.30 -RSB- . Time-on-task did not reliably affect T1 or T2 performance or interact with other variables . In a separate analysis , we calculated the average T2 performance across lags 3 -- 5 as a summary measure of attentional-blink magnitude and examined group differences in this measure . The two groups did not differ in attentional-blink magnitude , and performance at lags 3 -- 5 did not improve with time-on-task -LSB- both F -LRB- 1 , 30 -RRB- < 1 -RSB- . Interestingly , there was a reliable interaction between group and time-on-task -LSB- F -LRB- 1 , 30 -RRB- = 4.9 , P = 0.034 -RSB- : Whereas performance of the placebo group deteriorated over time -LRB- 66.6 vs 62.2 % correct ; P = 0.65 -RRB- , performance of the clonidine group improved slightly -LRB- 60.3 vs 62.6 % ; P = 0.28 -RRB- . In a final analysis , we examined the percentage of order reversals at lag 1 , the phenomenon that the two targets , when immediately succeeding each other , are often identified correctly but reported in the wrong order -LRB- Hommel and Akyürek 2005 -RRB- . The percentage of such order reversals was roughly the same in the placebo group -LRB- 34.4 % -RRB- and in the clonidine group -LSB- 35.4 % ; t -LRB- 30 -RRB- < 1 -RSB- . Visual search task : single-feature search The total number of items in the search display -LRB- set size -RRB- was varied from trial to trial , allowing us to derive the function relating reaction time -LRB- RT -RRB- to set size . The slope of this function measures the cost for adding additional items to the display and is often interpreted as `` search efficiency , '' with steeper slopes indicating slower , less efficient search . As expected and illustrated in Fig. 3 , slopes in the single-feature search condition were close to zero -LSB- average 1.6 ms/item ; t -LRB- 30 -RRB- = 2.6 , P = 0.014 -RSB- . Fig. 3Average correct reaction times for the clonidine and placebo groups in the visual search task . Top panels show data from the single-feature search condition , and bottom panels show data from the conjunction search condition . Data for target-present trials are plotted in the left-hand panels , data for target-absent trials are plotted in the right-hand panels Correct RTs were entered in a three-way mixed ANOVA with target presence -LRB- target present vs target absent -RRB- and set size -LRB- 4 , 8 , or 16 -RRB- as within-subject factors and group as between-subject factor . Despite the relatively shallow RT slopes , the main effect of set size was significant -LSB- F -LRB- 2 , 58 -RRB- = 6.3 , P = 0.006 -RSB- . Furthermore , the clonidine group was overall slower than the placebo group -LSB- F -LRB- 1 , 29 -RRB- = 5.6 , P = 0.025 -RSB- . Group did not interact with target presence -LSB- F -LRB- 1 , 29 -RRB- = 1.2 , P = 0.29 -RSB- or set size -LSB- F -LRB- 2 , 58 -RRB- = 2.5 , P = 0.10 -RSB- . The three-way interaction was also not significant -LSB- F -LRB- 2 , 58 -RRB- < 1 -RSB- . Error rates were generally low -LRB- average 2.3 % -RRB- . An ANOVA yielded only a significant main effect of target presence -LSB- F -LRB- 1 , 29 -RRB- = 8.9 , P = 0.006 -RSB- , with errors occurring more often on target-present trials -LRB- 2.9 % -RRB- than on target-absent trials -LRB- 1.8 % -RRB- . Visual search task : conjunction search As expected , slopes in the conjunction-search condition were sizeable -LSB- average 25.0 ms/item ; t -LRB- 30 -RRB- = 13.8 , P < 0.001 -RSB- , indicating that visual search was time consuming and inefficient . A three-way mixed ANOVA yielded significant main effects of target presence -LSB- F -LRB- 1 , 29 -RRB- = 31.5 , P < 0.001 -RSB- and set size -LSB- F -LRB- 2 , 58 -RRB- = 152.9 , P < 0.001 -RSB- and a significant interaction of these two variables -LSB- F -LRB- 2 , 58 -RRB- = 26.7 , P < 0.001 -RSB- , indicating that set-size effects were larger for target-absent trials . Importantly , the main effect of group was not significant -LSB- F -LRB- 1 , 29 -RRB- = 2.1 , P = 0.16 -RSB- , and group did not reliably interact with any of the task variables -LRB- all F < 1 -RRB- . Error rates were again low -LRB- average 3.3 % -RRB- . An ANOVA yielded the same pattern of effects as the RT analysis , indicating that there was no speed -- accuracy trade-off , and that there were no significant group differences . Discussion The main findings of the current study can be summarized as follows . First , in contrast to our predictions , clonidine did not have a deleterious effect on attentional blink performance . Although the numerical group differences in T1 and T2 accuracy were in the expected direction , these differences were small compared to the sizeable performance differences between the individuals within each group -LRB- for a discussion of these individual differences , see Martens et al. 2006 -RRB- . The results from the visual search task were as expected : Although clonidine slowed the overall response speed , it did not affect the efficiency of the visual search for a target in a two-dimensional array of stimuli . Below , we will discuss the implications of these principal findings . The absence of a significant drug effect on attentional blink performance appears to be at odds with the temporal filtering hypothesis , which suggests that phasic activity of the LC -- NE system is important for the selection of important or infrequent stimuli in the temporal domain -LRB- Aston-Jones and Cohen 2005 ; Dayan and Yu 2006 -RRB- . The attentional blink results also seem inconsistent with the predictions of a recent theory that explains the attentional blink in terms of the temporal dynamics of the LC -- NE system -LRB- Nieuwenhuis et al. 2005b -RRB- . One possible explanation for these discrepancies is that the predicted group differences do in fact exist , but that our task and experimental design were not sufficiently sensitive to reveal them . Some critical factors in this regard may be the choice of a between-subject design and the sample size of 16 subjects per group . However , previous studies using between-subject designs , similar group sizes , the same subject population , and the same task have had no difficulty detecting group-levels effect on attentional blink performance of various manipulations aimed at distracting subjects from the RSVP stream -LRB- Olivers and Nieuwenhuis 2005 , 2006 -RRB- . Indeed , our choice of design was based on these previous studies and on the assumption that the effect of clonidine would be of at least the same size as the effect of these behavioral manipulations . In this context , it is worth noting that the interindividual variance in attentional blink performance was comparable to that in the Olivers and Nieuwenhuis studies , discounting increased variance as a reason for our failure to detect group differences . A more principled reason for the use of a between-subject design was that subjects can learn to adopt a ` mental state ' that greatly benefits attentional blink performance -LRB- Olivers and Nieuwenhuis 2006 -RRB- , and we wanted to avoid the transfer of such learning between drug conditions to exclude a potential contaminating source of variance . In addition , Coull et al. -LRB- 1995a , b -RRB- have found that in within-subject designs , the order of treatments -LRB- i.e. , clonidine before or after placebo -RRB- can show complicated interactions with task performance , another source of variance that we aimed to exclude . Another design feature that may have led to a failure to detect an existing effect is the timing of test administration relative to the treatment -LRB- t = 60 -- 120 min -RRB- . In particular , the systolic and diastolic blood-pressure time series reveal a maximal drug effect after the test period , suggesting that cognitive effects may have peaked later too . However , there are at least three arguments against this possibility . First , Tiplady et al. -LRB- 2005 -RRB- have investigated the time course of effects of 150 μg of orally administered clonidine on cognitive test variables and found that such effects were already present at t = 45 min but were decreased at t = 135 min . Second , our task-block comparisons show that , if anything , the group difference in attentional blink performance became smaller over time , a trend opposite from that expected if the effects of clonidine needed more time to develop . A third and related argument is that we found no significant interaction between group and task order , corroborating the notion that group effects did not become larger over time . Alternatively , we can not rule out the possibility that group effects peaked earlier than our test period . However , our choice of test period seems justified by the finding of significant cognitive effects of clonidine in various previous studies using similar test periods -LRB- Halliday et al. 1989 , 1994 ; Coull et al. 2001 -RRB- . Thus , our methods and results do not seem to contain any clear indication that the observed null effects reflect a lack of sensitivity . However , it is of course possible that a higher drug dose would yield more robust group differences . This is a possibility that can be addressed in future research -LRB- note that such research should take into account that the primary mode of action of clonidine changes with increasing dose ; -LRB- Arnsten and Cai 1993 -RRB- . At low doses , presynaptic receptors and LC receptors are stimulated , leading to a reduced NE activity . In contrast , at higher doses , postsynaptic receptors are activated , resulting in a boost of NE activity . This has important implications for the expected effects of clonidine on cognitive function -RRB- . Another possible explanation for the absence of significant drug effects on attentional blink performance is that the attentional blink may not be mediated by the LC -- NE system . This possibility would be particularly bothersome for the theory of Nieuwenhuis et al. -LRB- 2005b -RRB- , which directly relates the attentional blink to a refractory period in activity of the LC -- NE system . Although there is substantial indirect evidence in support of this theory -LRB- e.g. , the relationship between the attentional blink and the P3 -RRB- , the basic tenets of the theory are based on cell recordings in animals and are hard to validate directly in humans . Indeed , the current study is perhaps the most direct test to date of a relationship between the attentional blink and the LC -- NE system . In this context , it is important to note that another research group has reported preliminary evidence consistent with such a relationship -LRB- De Martino et al. 2005 -RRB- . This study , using a between-subject design , found that attentional blink performance was significantly impaired after intake of the ß-adrenergic receptor antagonist propranolol -LRB- 40-mg oral dose compared to placebo -RRB- . Although this finding is not predicted by the theory of Nieuwenhuis et al. -LRB- 2005a , b -RRB- , which relates the attentional blink to the dynamics of inhibitory α2 autoreceptors in the LC , it will be informative to determine to what extent the current findings generalize to other task designs and other noradrenergic drugs . A further avenue for future research will be to test and evaluate the effects of noradrenergic drugs on temporal attention using alternative experimental paradigms , such as the temporal analogue of the spatial cueing paradigm -LRB- Posner et al. 1980 ; Coull and Nobre 1998 ; see Coull et al. 2001 -RRB- . Eventually , accumulating evidence obtained in such human psychopharmacological studies can be used to accommodate theories about the role of NE in temporal attention . The results from the visual search task were consistent with our predictions : Although clonidine led to a general slowing of response speed , we found no evidence for drug effects on visual search efficiency as indexed by the effects of the distractor set size . This pattern of results was expected based on the notion that the effects of NE are topographically diffuse -LRB- i.e. , not selective with regard to any specific spatial representations -RRB- and hence should not affect the efficiency of selection in the spatial domain . Previous studies using visuospatial cueing tasks have found that clonidine reduces the cue validity effect -LRB- Clark et al. 1989 ; Coull et al. 2001 -RRB- , which has led to the suggestion that the LC -- NE system is directly involved in visuospatial orienting . For example , it has been proposed that the LC -- NE system plays an important role in the disengagement of visuospatial attention from invalidly cued spaces -LRB- Clark et al. 1989 ; Posner and Petersen 1990 -RRB- . This proposal is consistent with the little available evidence , which suggests that the reduced validity effect after clonidine administration is mainly due to attenuated performance costs on invalidly cued trials . However , we propose an alternative interpretation for these results to reconcile them with our theoretical framework . According to our interpretation , the presentation of the cue causes a spatially nonspecific , transient LC -- NE response that boosts the processing of any stimuli presented for a brief period thereafter -LRB- i.e. , the alerting effect -RRB- . Although not supported by the current attentional blink findings , this assumption is based on various sources of evidence in the animal literature -LRB- Aston-Jones and Cohen 2005 ; Witte and Marrocco 1997 -RRB- . The cue-induced LC -- NE response speeds up cortical spatial selection processes involved in localizing the cue and/or subsequent imperative stimulus . This improves performance on validly cued trials and impairs performance on invalidly cued trials thus increasing the validity effect . Administration of clonidine counteracts this amplifying effect of phasic LC -- NE activity thereby reducing the validity effect -LRB- note that this hypothesis predicts both improved performance on invalid trials and impaired performance on valid trials -RRB- . This type of interaction between the alerting effect and spatial selection processes is absent in the visual search task , which has no clear alerting component . Therefore , the current results are consistent with the notion that the LC -- NE system is not directly involved in visuospatial orienting but can only indirectly modulate the effects of spatial attention mechanisms implemented elsewhere in the brain . One drawback of the current study is that we did not include a task that has already been shown to be sensitive to clonidine in previous studies . Therefore , a possible interpretation of the reported null findings is that the administered dose of clonidine failed to affect general cognitive function in our subjects , and that the main effect of drug on visual search RT reflects drug-induced motor slowing rather than a central cognitive deficit . Although we deem this possibility unlikely , given that previous studies using similar doses of clonidine have found significant effects on cognitive function -LRB- Coull et al. 2001 ; Tiplady et al. 2005 -RRB- , a more extensive study with a larger task battery is needed to adequately address this issue . There is rapidly growing interest in the specific role of the LC -- NE system in human cognition -LRB- Cohen and Aston-Jones 2005 -RRB- . Psychopharmacological data from humans will be of critical importance in testing and further developing the increasingly sophisticated hypotheses that are based on neurophysiological observations in animals -LRB- e.g. , Yu and Dayan 2005 -RRB- . The current research provides a valuable contribution toward a better understanding of the role of the LC -- NE system in human attention , both in the spatial and the temporal domain .

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norepinephrine
clonidine
locus coeruleus
attention
attentional blink
visual search