Selective PDE inhibitors rolipram and sildenafil improve object retrieval performance in adult cynomolgus macaques Rationale Selective phosphodiesterase -LRB- PDE -RRB- inhibitors improve the formation of hippocampus-dependent memories in several rodent models of cognition . However , studies evaluating the effects of PDE inhibition on prefrontal cortex-dependent cognition and in monkeys are rare . Introduction Second messenger cyclic nucleotides , i.e. , cyclic adenosine monophosphate -LRB- cAMP -RRB- and cyclic guanosine monophosphate -LRB- cGMP -RRB- , have been strongly implicated in processes of synaptic long-term potentiation -LRB- LTP ; Frey et al. 1993 ; Zhuo et al. 1994 -RRB- and have cognition-enhancing properties -LRB- Bernabeu et al. 1996 ; Blokland et al. 2006 ; Prickaerts et al. 2002a ; Prickaerts et al. 2005 -RRB- . cAMP and cGMP are hydrolyzed by phosphodiesterase -LRB- PDE -RRB- enzymes , and inhibitors of PDEs -LRB- PDE-Is -RRB- produce cognition-enhancing effects in animal models of cognition . In this field , research has focused on PDE4 and PDE5 inhibitors -LRB- Blokland et al. 2006 ; Rose et al. 2005 ; Rutten et al. 2006 -RRB- and more recently PDE2 -LRB- Boess et al. 2004 ; Rutten et al. 2007b -RRB- and PDE10 -LRB- Rodefer et al. 2005 -RRB- inhibitors . For example , inhibition of PDE2 , PDE4 , and PDE5 can improve memory in the object recognition task in rodents -LRB- Boess et al. 2004 ; Prickaerts et al. 2004 ; Rutten et al. 2007b -RRB- . Moreover , the cAMP-selective PDE4-I rolipram is an effective performance enhancer in the passive avoidance task -LRB- Egawa et al. 1997 ; Imanishi et al. 1997 -RRB- , fear-conditioning test -LRB- Barad et al. 1998 -RRB- , and radial arm maze -LRB- Zhang and O'Donnell 2000 -RRB- . The cGMP-selective PDE5-I sildenafil has been shown to enhance performance in the object recognition task , in the T-maze , and spatial plus maze -LRB- Devan et al. 2006 ; Devan et al. 2004 ; Patil et al. 2006 ; Prickaerts et al. 2004 -RRB- . In addition , sildenafil improved object recognition memory in mice -LRB- Rutten et al. 2005 -RRB- , and another PDE5-I , zaprinast , improved performance in a passive avoidance task in neonatal chicks -LRB- Campbell and Edwards 2006 -RRB- . Taken together , these results confirm the beneficial effects of PDE inhibition on cognition involving the hippocampus . The present study assesses the effects of PDE inhibition on object retrieval -LRB- OR -RRB- performance , a task of prefrontal cognition , in monkeys . OR -LRB- also known as the detour reaching task -RRB- involves attention , response inhibition , and planning -LRB- i.e. , executive function ; Diamond et al. 1989 -RRB- . Because the prefrontal cortex is not well represented in rodents -LRB- especially the dorsolateral prefrontal cortex -RRB- and considerable debate exists on whether the rodent prefrontal cortex subserves the same behavioral functions as the primate/human prefrontal cortex -LRB- Brown and Bowman 2002 ; Uylings et al. 2003 -RRB- , tests of prefrontal functioning -LRB- i.e. , executive function -RRB- are preferentially carried out in monkeys . Successful completion of the OR task requires various hypothetical steps . An animal must appreciate the contradiction between visual and tactile input , ignore the incorrect input , remember the previous incorrect approach , and formulate a novel approach to obtain the reward , which acts against the innate tendency of perseveration toward sustained sensory stimulation -LRB- Lipina and Colombo 2001 -RRB- . Direct support for the involvement of the frontal cortex in OR comes from studies in which lesions of the frontal cortex impaired , while lesions of the hippocampus did not impair , performance in the task -LRB- Diamond et al. 1989 ; Wilkinson et al. 1997 -RRB- . Furthermore , deficits in OR tasks have been documented in monkeys after treatment with methylphenyl tetrahydropyridine -LRB- Schneider and Roeltgen 1993 ; Taylor et al. 1990a ; Taylor et al. 1990b -RRB- , phencyclidine -LRB- Jentsch et al. 2000 ; Jentsch et al. 1999a ; Jentsch et al. 1999b -RRB- , and after excitotoxic lesioning with quinolinic acid of the striatum -LRB- Roitberg et al. 2002 -RRB- . The effect of PDE-Is in the OR task have , to our knowledge , never been evaluated before in unimpaired monkeys . As mentioned above , improvement in hippocampus-dependent memory tasks in rodents is well established through PDE inhibition . However , no evidence exists on the effects of rolipram or sildenafil on prefrontal cortex-dependent tests of executive function . As the PDE4 enzyme is omnipresent throughout the brain and PDE5 is expressed in the hippocampus , cortex , and cerebellum -LRB- van Staveren et al. 2004 -RRB- , we hypothesized that OR performance would be improved by the PDE4 and PDE5 inhibitors rolipram and sildenafil , respectively . Materials and methods Subjects Fourteen adult -LRB- age 5 -- 12 years ; weights 6 -- 9 kg -RRB- male cynomolgus macaques -LRB- Macaca fascicularis -RRB- were housed in same-sex pairs in a colony room maintained at 21 ± 2 °C , 40 ± 10 % humidity , and on a normal 12-h light/dark cycle -LRB- lights on at 7:00 a.m. -RRB- . Food -LRB- Purina High Protein no. 5045 -RRB- and water were available ad libitum . All experimental procedures were approved by the Institutional Animal Care and Use Committee of Roche Palo Alto and were in accordance with National Institutes of Health guidelines . Object retrieval task The OR task has been previously described by Diamond et al. -LRB- 1989 -RRB- . Briefly , this task requires a monkey to reach into a clear acrylic box -LRB- dimensions = 5 × 5 × 5 cm -RRB- with one open side , to retrieve a food treat -LRB- cubes of apple or pear , 1 -- 2 cm2 -RRB- . The box was positioned in front of the monkey and outside of the home cage , with the open side facing left , right , or toward the monkey . Food treats were placed on the outer edge , inner edge , or deep within the box . A test session consisted of 17 trials with nine `` easy '' food retrievals -LRB- i.e. , placement of the food reward on the inner or outer edges of the box or when the opening is toward the monkey -RRB- and eight `` difficult '' food retrievals -LRB- i.e. , placement of the food reward deep within the box and the open side facing left or right , see Fig. 1 -RRB- . The order of presentation never varied -LRB- Table 1 -RRB- , there were no contingencies for incorrect reaches -LRB- i.e. , monkeys typically acquired the treat after the incorrect reach -RRB- or dropped treats , and trials were terminated if there were no reaches within 3 min . The box was cleaned diligently between trials to minimize cues that could influence the task and subsequent task performance . After an initial period -LRB- 1 week -RRB- to acclimatize the monkeys to the apparatus and procedure , tests were conducted twice a week . Fig. 1A schematic overview of possible trials in the object retrieval -LRB- detour-reaching -RRB- task . The transparent box containing a food reward is depicted form the monkey 's point of view . The top five orientations depict easy trials , and the bottom two orientations depict difficult trialsTable 1Primate object retrieval : trial orderTrial numberDescriptionLevel1LOS -- line of sightEasy2LOS -- line of sightEasy3RO -- right outsideEasy4RD -- right deepDifficult5RO -- right outsideEasy6RI -- right insideEasy7RD -- right deepDifficult8LO -- left outsideEasy9LD -- left deepDifficult10LO -- left outsideEasy11li -- left insideEasy12ld -- left deepDifficult13ld -- left deepDifficult14rd -- right deepDifficult15ld -- left deepDifficult16rd -- right deepDifficult17los -- line of sightEasy Drugs Rolipram -LRB- 0.003 , 0.01 , 0.03 , and 0.1 mg/kg ; Sigma Chemicals , St Louis , MO -RRB- and sildenafil -LRB- 0.3 , 1 , and 3 mg/kg ; Sequoia Research Products Limited , Pangbourne , UK -RRB- were prepared fresh daily in a suspension of 10 % cremaphore/90 % saline and administered 30 or 60 min before testing , respectively . Drugs were administered via intramuscular injection -LRB- i.m. -RRB- in a volume of 0.1 ml/kg . Compounds were typically administered on Tuesday and Friday each week . Weekly test sessions were comprised of a vehicle session and a drug test session . Drug administration and behavioral measurement were completed blind , and each drug -- dose combination was tested once . Drug doses were administered in a pseudorandom order . Data analysis The mean percent correct first reaches for easy and difficult food retrievals were analyzed with a one-factor -LRB- Dose -RRB- repeated-measures analysis of variance . When significant effects were demonstrated , a post-hoc Dunnett analysis comparing dose conditions to vehicle performance was performed . For all tests , the significance level was 0.05 -LRB- two-tailed -RRB- . Results During vehicle sessions , monkeys readily reached for the food treats , successfully acquiring the treat on the first reach 100 ± 0 % during easy trials and 52 ± 3 % during difficult trials . Over the course of the experiment -LRB- 2 months -RRB- , there were no changes in vehicle performance . The PDE4 inhibitor rolipram -LRB- 0.003 -- 0.1 mg/kg , i.m. -RRB- dose-dependently increased correct first reaches during difficult trials -LRB- F -LSB- 3 , 55 -RSB- = 13.5 , p < 0.05 -RRB- , reaching significance at 0.01 mg/kg -LRB- Fig. 2a -RRB- . At the highest dose tested without side effects -LRB- 0.03 mg/kg -RRB- , the percentage correct first reaches were increased approximately 20 to 71 ± 3 % . Note that at 0.1 mg/kg -LRB- data not shown -RRB- , emesis was observed in all but two monkeys ; these results were therefore excluded from analysis . Fig. 2The effects of PDE-I on object retrieval -LRB- OR -RRB- performance -LRB- mean values and SEM -RRB- a The effects of the PDE4 inhibitor rolipram and the PDE5 inhibitor sildenafil -LRB- b -RRB- on the percentage correct first reaches on difficult trials in the OR task . Asterisks indicate significant differences from baseline -LRB- P < 0.05 -RRB- The PDE5 inhibitor sildenafil -LRB- 0.3 -- 3 mg/kg , i.m. -RRB- dose-dependently increased correct first reaches during difficult trials -LRB- F -LSB- 3 , 50 -RSB- = 8.6 , p < 0.05 -RRB- , reaching significance at 1 mg/kg -LRB- Fig. 2b -RRB- . At the highest dose tested -LRB- 3 mg/kg -RRB- , correct reaches were increased approximately 20 to 73 ± 3 % . No side effects were observed . Neither rolipram nor sildenafil altered performance during the easy trials , with the exception of the high dose of rolipram -LRB- 0.1 mg/kg -RRB- , in which monkeys failed to perform the task -LRB- data not shown -RRB- because of emetic side effects . Discussion The present study demonstrates performance-enhancing effects of two selective PDE-Is on OR performance in monkeys . OR is subserved by the prefrontal cortex and/or fronto-striatal pathways , within which modulation of dopamine and acetylcholine transmission are involved in attention , response inhibition -LRB- i.e. , executive function -RRB- , and working memory in rodents and primates -LRB- Jentsch et al. 2000 ; Lipina and Colombo 2001 ; Palfi et al. 1996 ; Ramos et al. 2003 ; Wilkinson et al. 1997 -RRB- . To our knowledge , this is the first study to investigate the effects of PDE5 inhibition on executive function in monkeys . Of note , two previous studies have investigated the effects of sildenafil on cognition-related variables in humans . In these studies , sildenafil was shown to enhance simple reaction times and to some extent enhancement of focused attention -LRB- Grass et al. 2001 ; Schultheiss et al. 2001 -RRB- . However , these findings have not been further described in the literature . The effects of the PDE4 inhibitor rolipram on executive function have yet to be assessed in humans . Thus , the current results complement and extend to the cognition-enhancing effects of PDE inhibition . Previous studies have repeatedly shown that PDE-Is can have cognition-enhancing effects , mainly in hippocampus-dependent memory tasks , in rodents . For example , the PDE4 inhibitor rolipram improved long-term memory in the object recognition task , in passive avoidance learning , and fear conditioning -LRB- Barad et al. 1998 ; Rutten et al. 2006 ; Zhang et al. 2005 -RRB- . In addition , rolipram had performance-enhancing effects in a prefrontal cortex-dependent working memory task , i.e. , delayed alternation , in young rats and young monkeys -LRB- Ramos et al. 2003 -RRB- . Results from the present study corroborate the cognition-enhancing effects of low-dose -LRB- 0.01 mg/kg -RRB- rolipram treatment in a prefrontal cortex-dependent task , although the present OR task does not involve working memory but requires attention and response inhibition -LRB- i.e. , executive function ; Diamond et al. 1989 -RRB- . Furthermore , in aged mice , rolipram ameliorated the age-related deficits in the passive avoidance task , a test of hippocampus-dependent memory . In contrast , rolipram impaired prefrontal cortex-dependent working memory performance in aged rodents and aged monkeys . With advancing age , opposite profiles between the function of protein kinase A -LRB- PKA -RRB- in the hippocampus and prefrontal cortex were suggested to explain these results ; that is , the prefrontal cortex showed indices of increased PKA activity , while the hippocampus exhibited evidence of decreased PKA activity -LRB- Ramos et al. 2003 -RRB- . Although in the present study , cognition-enhancing effects on executive function were observed after rolipram treatment in young monkeys , the possible cognition-impairing effects of rolipram on prefrontal cortex-dependent tests in aged monkeys should be further investigated . Compared to PDE4 inhibition , the cognition-enhancing effects of PDE5 inhibition have not been studied as extensively . However , a growing number of studies have shown cognition-enhancing effects of PDE5-Is in multiple tests and in multiple species . PDE5-Is improved cognitive performance in object recognition and inhibitory and passive avoidance tasks -LRB- Baratti and Boccia 1999 ; Prickaerts et al. 2005 ; Prickaerts et al. 2004 ; Prickaerts et al. 2002b ; Rutten et al. 2005 ; Shafiei et al. 2006 ; Singh and Parle 2003 -RRB- . In addition , the inhibition of PDE5 reversed memory deficits induced by scopolamine , diabetic neuropathy , or nitric oxide -LRB- NO -RRB- synthase inhibitors in rats -LRB- Devan et al. 2006 ; Devan et al. 2004 ; Patil et al. 2004 ; Prickaerts et al. 1997 -RRB- . To our knowledge , no literature exists on the possible cognition-enhancing effects of PDE5 inhibitors in nonhuman primates . Furthermore , the effects of PDE5 inhibition on prefrontal cortex-depending cognition , i.e. , working memory and executive function , are unknown . The underlying mechanisms of PDE-Is and cognition enhancement are still elusive , but several possible pathways have been described . Possible mechanisms of action for rolipram and sildenafil are based on the proposed underlying signaling pathways of LTP . Both cAMP and cGMP have been strongly implicated in hippocampal LTP -LRB- Frey et al. 1993 -RRB- . Activation of LTP-related signaling pathways of cAMP/PKA/cAMP response element-binding protein -LRB- CREB -RRB- and cGMP/protein kinase G -LRB- PKG -RRB- / CREB have been implicated as the underlying mechanisms for the cognition-enhancing effects of PDE4 and PDE5 inhibitors -LRB- Bernabeu et al. 1996 ; Blokland et al. 2006 ; Lu and Hawkins 2002 ; Prickaerts et al. 2002a ; Rutten et al. 2007b -RRB- . A very recent study suggested another pathway through which cGMP can influence cognitive processes . This study showed that the hippocampal NO/cGMP pathway directly stimulates the postsynaptic cAMP/PKA/CREB pathway -LRB- Matsumoto et al. 2006 -RRB- . Alternatively , cGMP has also been found to maintain LTP via a presynaptic cGMP/PKG pathway -LRB- Zhuo et al. 1994 -RRB- . However , the present study involves prefrontal cortex-dependent behavior , and it remains to be proven that the above mentioned hippocampal LTP mechanisms of synaptical strengthening are also applicable in the prefrontal cortex . Of note , the effects of rolipram on prefrontal cortex performance were explained via activation of the postsynaptic PKA pathway -LRB- Ramos et al. 2003 -RRB- . Thus , both cGMP and cAMP can activate cellular cascades resulting in CREB phosphorylation , which could induce long-lasting changes in neuronal signaling and may thereby explain the performance enhancement observed in the present study . Apart from LTP-related mechanisms of action , the effects of rolipram and sildenafil on prefrontal functioning in the OR may be explained by their stimulatory effect on neurotransmission . Because rolipram is independent of a specific receptor system , it is suggested that rolipram may ameliorate physiological abnormalities that occur secondary to alterations in dopaminergic , glutamatergic , serotonergic , and/or adrenergic transmission -LRB- Maxwell et al. 2004 ; Rutten et al. 2007a -RRB- . Elevated cAMP levels are thought to excite noradrenergic and dopaminergic neurotransmitter systems -LRB- Schoffelmeer et al. 1985 -RRB- , thus enhancing their availability , hence also enhancing synaptic transmission . Similar to rolipram , sildenafil is a drug that is not linked to specific neurotransmitter systems . Modulatory activity of sildenafil was suggested on central dopaminergic pathways -LRB- Ferrari et al. 2002 -RRB- . Dopaminergic dysfunction in prefrontal cortex may subserve at least a component of the impaired OR/detour performance in deficit models -LRB- Jentsch et al. 1999a -RRB- . Moreover , serotonin depletions of the prefrontal cortex in the common marmoset result in deficits in the acquisition of the OR task -LRB- Walker et al. 2006 -RRB- . Hence , activation of cGMP or cAMP may improve dopaminergic turnover and prefrontal functioning in the OR task . Thus far , it remains unclear which of these abovementioned mechanisms are involved in the prefrontal cortex and how they explain cognition enhancement by inhibition of PDEs in the OR task . To summarize , previous research demonstrated the cognition - and largely memory-enhancing effects of PDE4 and PDE5 inhibition in rodents . The results from the current study extend these cognition-enhancing effects of PDE-Is in rodents to nonhuman primates . More specifically , rolipram and sildenafil improve OR performance , thus demonstrating that PDE4 and PDE5 inhibition enhance performance in a cognitive domain involving executive function -LRB- and attention -RRB- mediated by the prefrontal cortex .

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

attention
executive function
phosphodiesterase
pde4
rolipram
pde5
sildenafil
cognition
object retrieval
monkey


Vectors for expression of proteins with single or combinatorial fluorescent protein and tandem affinity purification tags in Dictyostelium We constructed a series of expression vectors for purification of native proteins and protein complexes in Dictyostelium . Protein purification is achieved by either a C-terminal or N-terminal fusion of the protein of choice to the tandem affinity purification -LRB- TAP -RRB- tag . The TAP tag consists of a protein A tag and a calmodulin binding peptide -LRB- CBP -RRB- and has been successfully used for purification of native protein complexes from yeast and animal cells . Protein expression is driven by the constitutive actin 15 promoter and the vectors optionally carry additional green - or yellow fluorescent protein -LRB- GFP or YFP -RRB- tags for fusion at either a C - or N-terminal location . Tandem affinity purification of native Dictyostelium protein complexes was tested by using pArc-34 , one of the members of the well characterized Dictyostelium Arp2/3 complex , as bait . After denaturation and SDS -- PAGE separation of the pArc-34 associated proteins all members of the Arp2/3 complex could be identified . Dictyostelium discoideum is a genetically tractable model system for studying remodeling of the actin cytoskeleton during cell locomotion , cell division , phagocytosis and vesicle trafficking and for elucidation of signaling pathways that control chemotaxis and development . Several metazoan-type cytoskeletal components were identified for the first time in Dictyostelium -LSB- 1 -- 3 -RSB- , and the process of pleckstrin homology domain mediated recruitment that plays such crucial roles in leukocyte chemotaxis and phagocytosis was also first demonstrated in the Dictyostelids -LSB- 4 -RSB- . The Dictyostelium genome is now completely sequenced and each gene is therefore available for functional analysis by gene disruption , overexpression and mutagenesis -LSB- 5 -RSB- . Genetic approaches to create tagged mutations , such as REMI -LRB- restriction enzyme mediated integration -RRB- , have identified numerous genes with crucial roles in developmental or cellular processes -LSB- 6 -RSB- . However in only a few cases has it been possible to use genetic approaches , such as screens for suppressor mutations , to determine epistatic relationships between genes -LSB- 7 -RSB- . Or , in other words , to determine in what order their cognate proteins act on each other in a pathway . In other genetically tractable systems , such as the fruit fly and the nematode , epistatic relationships are usually determined by crossing in loss - and gain-of-function mutants of genes in the pathway . The poor accessibility of the Dictyostelid sexual stage , the macrocyst , has prohibited this approach . Other genetic screens , such as the yeast two-hybrid system or the split-ubiquitin method are being successfully used to identify interacting proteins in a pathway -LSB- 8,9 -RSB- . However , these methods depend on successful heterologous expression of the proteins in yeast , which is not possible for many proteins . The TAP tag was developed about seven years ago to purify native protein complexes from yeast -LSB- 10,11 -RSB- and was used for systematic identification of protein complexes in yeast -LSB- 12 -- 15 -RSB- and metazoans -LSB- 16,17 -RSB- . For C-terminal tagging , the TAP tag contains in tandem , a calmodulin binding peptide tag , a TEV protease cleavage site and a protein A tag . For N-terminal tagging the order of these units is reversed . We have constructed a series of vectors for C - and N-terminal tagging of proteins expressed in Dictyostelium . Protein expression is driven by the strong constitutive actin15 promoter . Additional cloning sites were introduced to allow replacement of this promoter by an inducible promoter . A third green - or yellow fluorescent protein tag was introduced for C - or N-terminal expression in some of the vectors for rapid assessment of the levels of protein expression and the cellular location of the expressed proteins . Materials and methods Construction of plasmid vectors Gene fragments were amplified by polymerase chain reaction -LRB- PCR -RRB- 2 with a 4:1 mixture of the Taq and Pfu DNA polymerases -LRB- Promega , Madison , WI -RRB- . All oligonucleotide primers used in this study are listed in Table 1 . The integrating Dictyostelium vector EXP4 -LRB- + -RRB- -LSB- 18 -RSB- was used as starting material . An XbaI and a HindIII site upstream of the actin15 promoter were first successively destroyed by filling in the overhanging sites after digestion with the Klenow fragment of polymerase I , followed by religation , resulting in vector EXP4 -LRB- - XH -RRB- . This also generated a unique Nhe1 site replacing the HindIII site . A long multiple cloning site -LRB- MCS -RRB- was generated by deleting the existing polylinker by BglII/XhoI digestion of the vector and by ligating a duplex of primers MCS1 and MCS2 that carries compatible sticky ends into the BglII and XhoI sites . The resulting vector , EXP5 -LRB- + -RRB- was used as the backbone for all subsequent constructs containing TAP and/or EGFP/EYFP fragments -LRB- Table 2 -RRB- . The N-terminal TAP fragment -LRB- NTAP -RRB- was amplified from the plasmid pBS1761 -LSB- 11 -RSB- using primers NTAP1 and NTAP2 and ligated into the BamHI and HindIII sites of EXP5 -LRB- + -RRB- , yielding pDV-NTAP . The C-terminal TAP fragment -LRB- CTAP -RRB- was amplified from plasmid pBS1479 -LSB- 10 -RSB- using primers CTAP1 and CTAP2 and ligated into the ClaI and XhoI sites of EXP5 -LRB- + -RRB- , yielding vector pDV-CTAP . Enhanced yellow - and green fluorescent protein genes -LRB- YFP -RRB- and -LRB- GFP -RRB- were amplified from the vectors pEYFP-N1 and pEGFP-C3 -LRB- Clontech , Mountain View , CA -RRB- , respectively . For N-terminal location of YFP , primers tapYFP1 and tapYFP2 were used and insertion occurred in the HindIII and SpeI sites of EXP5 -LRB- + -RRB- , yielding pDV-NYFP . For C-terminal location of YFP or GFP , primers tapYFP3 and tapYFP4 were used and insertion occurred into the EcoRI and ClaI sites of EXP5 -LRB- + -RRB- , yielding pDV-CYFP or pDV-CGFP . All constructs were validated by sequencing across the entire tag and MCS and are listed in Table 2 . Generation and transformation of construct p34-ArcCTAP The Dictyostelium p34-Arc protein is encoded by the ArpE gene -LSB- 5,19 -RSB- . The 1251 bp ArpE coding region was amplified by PCR from genomic DNA isolated from wild-type AX2 cells , using primers p34Arc1 and p34Arc2 -LRB- Table 1 -RRB- . The BamHI/XbaI digested PCR product was ligated into the BamHI and XbaI sites of vector pDV-CTAP , yielding vector p34-ArcCTAP . The p34-ArcCTAP , pDV-CGFP-CTAP and pDV-CTAP constructs were transformed by electroporation into D. discoideum strain AX2 and transformants were selected by growth in the presence of 20 μg/ml G418 . Purification of TAP-tagged protein complexes TAP purification was performed as described previously -LSB- 10,11 -RSB- with some modifications . Briefly , 2 × 109 exponentially growing AX2 cells expressing TAP constructs were harvested , washed twice in 10 mM Na/K-phosphate buffer , pH 6.5 and resuspended in 10 ml IPP -LRB- 150 mM NaCl in 10 mM Tris , pH 8.0 -RRB- , containing one tablet of Complete Protease inhibitor cocktail -LRB- Roche , Lewes , UK -RRB- per 50 ml . Cells were lysed by addition of Triton X-100 to 1 % -LRB- v/v -RRB- and incubated for 30 min on ice . Lysates were cleared by centrifugation for 20 min at 20000g and 4 °C . The supernatant was rotated for 1 h at 4 °C with 0.1 % -LRB- v/v -RRB- Nonidet P-40 -LRB- NP-40 -RRB- and 200 μl bed volume of IgG-agarose beads -LRB- Sigma , St Louis , MO -RRB- in Polyprep columns -LRB- Bio-rad , Hercules , CA -RRB- . The beads were washed three times with IPPN -LRB- 0.1 % NP-40 in IPP -RRB- , and once with 10 ml TEV cleavage buffer -LRB- 1 mM DTT and 0.5 mM EDTA in IPPN -RRB- . Bound complexes were liberated by rotating the column for 2 h at 16 °C with 100 units of TEV protease -LRB- Invitrogen , Carlsbad , CA -RRB- in 1 ml TEV cleavage buffer and recovered by elution . Subsequently 3 μl of 1 M CaCl2 , 3 ml CaM binding buffer -LRB- 1 mM imidazole , 1 mM Mg-acetate , 10 mM β-mercaptoethanol and 2 mM CaCl2 in IPPN -RRB- and 200 μl of bed volume Calmodulin Affinity Resin -LRB- Stratagene , La Jolla , CA -RRB- were added per milliliter of eluate . The mixture was rotated for 1 h at 4 °C in a second Polyprep column . Beads were washed 3 times with 10 ml CaM binding buffer and bound material was eluted in 5 fractions of 200 μl each with CaM elution buffer -LRB- 1 mM imidazole , 1 mM Mg-acetate , 10 mM β-mercaptoethanol and 2 mM EGTA in IPPN -RRB- . Proteins were separated by SDS -- PAGE on 4 -- 12 % Bis -- Tris gels -LRB- Invitrogen , Carlsbad , CA -RRB- and visualized with a Colloidal Blue Staining kit -LRB- Invitrogen , Carlsbad , CA -RRB- . Protein mass fingerprinting Protein mass fingerprint data was obtained by MALDI-TOF-TOF -LRB- MS/MS -RRB- analysis performed at the University of Dundee ` Fingerprints ' Proteomics Facility using an Applied Biosystems 4700 Proteomic Analyser . Protein bands were excised , in-gel reductively alkylated and digested in 20 mM NH4HCO3 containing 0.1 % N-octylglucoside and 12.5 μg/ml trypsin -LSB- 20 -RSB- . One-tenth of each digest was applied to a 192 well MALDI sample plate -LRB- Applied Biosystems -LRB- AB -RRB- -RRB- , allowed to air dry and mixed with 0.5 μl of a solution of 5 mg/ml α-cyano-4-hydroxy-trans-cinnamic acid matrix -LRB- Sigma , StLouis , USA -RRB- , 50 % -LRB- v/v -RRB- acetonitrile , 0.1 % -LRB- v/v -RRB- trifluoroacetic acid in 10 mM NH4H2PO4 , and then allowed to air dry prior to analysis . The mass spectrometer was internally calibrated using the AB 4700 Proteomic Analyser Calibration Mix . Using the 4000 series Explorer Software -LRB- AB -RRB- , MS spectral data were acquired from the samples and an MS/MS list was automatically generated for further analysis based on the top 5 most intense ions present -LRB- trypsin and major keratin ions were excluded -RRB- . The MS and MS/MS spectral data obtained were exported from the 4700 using the Global Proteome Server -LRB- GPS -RRB- Explorer Software -LRB- AB -RRB- . The data were then submitted to a local MASCOT search engine -LRB- Matrixscience , London , UK -RRB- for protein database searching against the Uniprot database -LRB- http://www.ebi.ac.uk/swissprot/ -RRB- for identification . Results and discussion Cloning strategies The integrating Dictyostelium vector EXP4 -LRB- + -RRB- was chosen as the backbone for all constructs . This vector is derived from pATSP -LSB- 21 -RSB- , itself a derivative of the standard cloning vector pAT153 . EXP4 -LRB- + -RRB- carries the Act6NeoR cassette for G418 selection in Dictyostelium , and a cassette for protein expression consisting of the constitutive actin 15 -LRB- A15 -RRB- promoter , the 2H3 terminator and a small polylinker with 6 restriction sites -LSB- 18 -RSB- . Because some of the cloning steps used to make this vector could not be retraced , we sequenced about 900 bp each around the points of fusion of the 2H3 terminator with the actin6 promoter , and the NeoR terminal region with the original pAT153 vector backbone . This revealed that the 2H3 terminator was preceded by 257 bp of the 2H3 coding region . However , since this coding sequence is downstream of the stopcodon in EXP4 -LRB- + -RRB- and all derived vectors , it is not incorporated in the expressed tagged proteins . The complete sequence of EXP -LRB- 4 + -RRB- was reconstructed and a vector map is presented in Fig. 1 . To obtain a larger multiple cloning region , we first successively destroyed the XbaI and HindIII sites upstream of the A15 promoter , creating a novel NheI site . In addition to the existing SalI site , this site can be used to exchange promoters or to insert genes with their own promoter . The existing polylinker was removed by BglII and XhoI digestion and replaced by an adaptor duplex of oligonucleotides MCS1 and MCS2 -LRB- Table 1 -RRB- , yielding vector EXP5 -LRB- + -RRB- -LRB- Fig. 1 -RRB- . The new multiple cloning site -LRB- MCS -RRB- contains an ATG start codon , preceded by 5 adenine residues to generate the Dictyostelium Kozak sequence and followed by 7 unique restriction sites : The MCS was subsequently used to clone in the TAP tags and enhanced YFP or GFP tags that were amplified by PCR . This is possible in any configuration i.e. TAP alone , YFP -LRB- GFP -RRB- alone or combinations of TAP and YFP -LRB- GFP -RRB- at the same site or at opposing sites . When cloned at the same site , the YFP -LRB- GFP -RRB- is in between the gene of interest and the TAP sequence as indicated above . Here , NTAP and CTAP stand for N - and C-terminal TAP , respectively , while NYFP and CYFP -LRB- GFP -RRB- stand for N - and C-terminal GFPs and YFPs . Vectors with single NTAP , CTAP , NYFP or CYFP -LRB- GFP -RRB- tags were created first and validated by DNA sequencing across the entire tag and MCS . The CTAP fragment contained a stopcodon before the XhoI restriction site . Vectors with TAP + GFP -LRB- YFP -RRB- tags were then generated by removing a tag from one of these vectors and cloning it in the appropriate position in the other vectors . Some restriction sites can not be used anymore : BglII will digest both the NTAP and the CTAP tags , while EcoRI will digest the CTAP tag . NheI will digest the CTAP tag , which then leaves only SalI to introduce a different promoter . All vectors prepared in the course of this work are listed in Table 2 and their sequences are deposited in GenBank . Purification of the Dictyostelium Arp2/3 complex with TAP-tagged p34-Arc To validate that the TAP tag can be used to purify native protein complexes in Dictyostelium we used a TAP tagged D. discoideum p34-Arc protein as bait for purification of members of the well-characterized Arp2/3 complex . The Arp2/3 complex is a complex of 7 proteins , including the p34-Arc protein , involved in the nucleation of actin polymerization -LSB- 19 -RSB- . We prepared a C-terminal fusion of the TAP tag with D. discoideum p34-Arc , as was previously done for yeast and human p34-Arc -LRB- named Arc35 and ARP2C , respectively -RRB- -LSB- 14 -RSB- . The p34-Arc gene was amplified from D. discoideum genomic DNA using the primers p34Arc1 and p34Arc2 -LRB- Table 1 -RRB- , which harbour BamH1 and XbaI sites , respectively , and cloned into the BamHI and XbaI digested pCTAP vector . This yielded the vector p34-Arc-CTAP , which was transformed into D. discoideum AX2 cells . AX2 cells transformed with the pDV-CTAP vector and with a pDV-CGFP-CTAP vector served as controls . For each transformed cell line , lysates were prepared by detergent treatment from 2 × 109 cells and adsorbed to IgG agarose . After extensive washing , the 4.8 kD CBP moiety of the TAP tag with fused `` bait '' and complexed `` prey '' were released from IgG agarose by TEV protease treatment . The proteins or protein complexes were further purified over calmodulin resin , eluted with EGTA , denatured and size-fractionated on SDS -- PAGE gels . Fig. 2 shows that a few faint bands were visible in preparations from cells that contained the empty pDV-CTAP vector . Extracts from pDV-CGFP-CTAP transformed cells showed a strong band at 32 kD , the expected size of the CGFP -- CBP fusion -LRB- 26.8 + 4.8 kD -RRB- after successful cleavage . Extracts of cells transformed with p34-Arc-CTAP displayed a pattern of 8 bands , similar to that observed after purification of the Dictyostelium Arp2/3 complex by traditional chromatography -LSB- 19 -RSB- . Bands 1 -- 8 were cut out from the gel , proteins were digested in gel with trypsin and sequences of the extracted peptides were determined by MALDI-TOF/TOF mass spectrometry . This yielded 12 -- 18 different peptide sequences for each of the 4 larger bands and 7 -- 11 different peptide sequences for each of the four smaller bands . Query of the Uniprot protein database with the peptide sequences yielded all members of the D. discoideum Arp2/3 complex for bands 1 -- 4 and 6 -- 8 -LRB- Fig. 2 -RRB- . Band 5 , which is also present in the pDV-CTAP and pDV-CGFP-CTAP extracts was identified as discoidin B , a highly abundant Dictyostelium lectin , which is most likely a non-specific contaminant . Conclusions We have constructed eleven integrating plasmid vectors for protein expression in Dictyostelium that carry the TAP tag for tandem affinity purification of native proteins or protein complexes . In addition to the TAP tag for N-terminal or C-terminal fusion , a subset of vectors carry an additional enhanced GFP or enhanced YFP tag at either the same or opposite location as the TAP tag . The vectors are constructed in such a manner that the existing constitutive actin15 promoter can easily be swapped for an inducible promoter . Alternatively the pDV-CTAP vectors also allow cloning of a gene under its own promoter . In addition , the neomycin selection cassette can be exchanged for a different selection marker e.g. blasticidin resistance , which requires only a single copy of the bsr gene . Preferentially , the `` bait '' gene of choice should be expressed under its own promoter from a single copy vector in a `` bait '' null mutant . This will avoid artefacts due to ectopic expression and competition of the endogenous `` bait '' with the tagged `` bait '' for binding to cellular proteins . Moreover , rescue of the null mutant phenotype by the tagged `` bait '' will ensure that the tags are not interfering with protein function . However , it may often be desirable to overexpress the `` bait '' to obtain a sufficient yield of protein complexes , although this also increases the probability of pulling down unrelated proteins . We show that for the pDV-CGFP-CTAP vector , the highly fluorescent GFP protein is efficiently released and purified by the two affinity steps and the TEV protease treatment . We also show that by using a p34-Arc-CTAP fusion protein as bait , the 6 associated members of the Arp2/3 complex could be isolated with negligible contamination . We therefore expect that these vectors will prove to be useful tools for identification of native protein complexes in Dictyostelium .

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

tandem affinity purification
yellow fluorescent protein
dictyostelium discoideum
native protein complexes


Acute neuropsychological effects of MDMA and ethanol -LRB- co - -RRB- administration in healthy volunteers Rationale In Western societies , a considerable percentage of young people expose themselves to 3,4-methylenedioxymethamphetamine -LRB- MDMA or `` ecstasy '' -RRB- . Commonly , ecstasy is used in combination with other substances , in particular alcohol -LRB- ethanol -RRB- . MDMA induces both arousing as well as hallucinogenic effects , whereas ethanol is a general central nervous system depressant . Introduction In Western societies , a considerable proportion of young people expose themselves to 3,4-methylenedioxymethamphetamine -LRB- MDMA or ` ecstasy ' ; Gross 2002 ; Parrott 2001 ; Tancer and Johanson 2007 -RRB- . Ecstasy has gained widespread use in the ` club ' scene , typically all-night parties with loud music and intense lights -LRB- Winstock et al. 2001 -RRB- . The average dose of ecstasy used recreationally is reported to be around 80 -- 90 mg of MDMA with considerable individual variation -LRB- Tanner-Smith 2006 -RRB- . Ecstasy users are generally multidrug users who have experience with various recreational drugs and use these in combination with ecstasy -LRB- Gouzoulis-Mayfrank and Daumann 2006b -RRB- . Probably due to its availability , alcohol remains one of the most co-used substances -LRB- Barrett et al. 2005 -RRB- . As the use of alcohol is known to induce impairment of cognitive function and decrease the awareness of this impairment , this can lead to dangerous behaviour like driving under influence -LRB- Lamers and Ramaekers 2001 ; Riley et al. 2001 -RRB- . MDMA acts primarily by releasing serotonin -LRB- 5-hydroxytryptamine -LRB- HT -RRB- -RRB- from pre-synaptic 5-HT terminals . It reverses the direction of the reuptake transporter and increases 5-HT levels at the post-synaptic receptors -LRB- Liechti and Vollenweider 2000 ; Mlinar and Corradetti 2003 ; Pifl et al. 1995 -RRB- . MDMA is also a potent releaser of dopamine and -LRB- nor -RRB- adrenaline -LRB- Colado et al. 2004 ; Liechti and Vollenweider 2001 -RRB- . MDMA is rapidly absorbed following oral administration . Within 30 min , MDMA is detectable in the blood . Plasma levels peak at 1 -- 2 h after drug administration , and maximum behavioural and subjective effects occur around 1 -- 2 h and have declined by 4 h in spite of persisting plasma levels -LRB- de la Torre et al. 2004 ; Green et al. 2003 -RRB- . Increasing the dose does not result in a proportional rise in plasma concentrations , which is indicative of non-linear pharmacokinetics -LRB- de la Torre et al. 2000 -RRB- . The behavioural effects of MDMA resemble but are not restricted to effects of psychostimulants -LRB- e.g. amphetamines or ` speed ' -RRB- as well as hallucinogenics -LRB- e.g. lysergic acid or ` lysergic acid diethylamide ' -RRB- , although MDMA 's most characteristic effects are described as an increase in empathy and friendliness -LRB- Vollenweider et al. 2002 -RRB- . This led to MDMA being categorized as an ` entactogen , ' as coined by Nichols and Oberlender -LRB- 1990 -RRB- . Most research into the cognitive effects of MDMA in humans has focused on the long-term effects , where only memory was consistently found to be impaired -LRB- Verbaten 2003 ; Verkes et al. 2001 -RRB- . Our review of the acute effects of MDMA in humans showed that cognitive effects were assessed only in a limited number of studies , using diverse tests and generally addressing only certain aspects of neuropsychological function . As such , no consensus on MDMA 's cognitive effects could be reached -LRB- Dumont and Verkes 2006 -RRB- . Since then , reports on the effects of MDMA generally confirmed previous findings -LRB- Kuypers et al. 2006 ; Kuypers et al. 2007 ; Ramaekers et al. 2006 ; Tancer and Johanson 2007 -RRB- . Interestingly , two studies reported effects of MDMA on memory , which had not been assessed previously . These reports showed acute impairment of immediate and delayed recall of words as well as spatial memory by MDMA -LRB- Kuypers and Ramaekers 2005 , 2007 -RRB- . Drinks containing ethanol , commonly referred to as alcohol , are widely available and regularly used in Western society . Ethanol is chiefly a central nervous system -LRB- CNS -RRB- depressant . It inhibits both excitatory and inhibitory post-synaptic potentials by potentiating the action of gamma-aminobutyric acid at its receptor -LRB- Suzdak et al. 1988 -RRB- . Reports of the cognitive effects of combined use of MDMA and ethanol in humans have been sparse in the literature . Studies that were performed assessed psychomotor function , attentional performance and subjective effects -LRB- Hernandez-Lopez et al. 2002 ; Kuypers et al. 2006 ; Ramaekers et al. 2006 -RRB- . In general , MDMA and ethanol had no or opposite effects on effect measures , and as such co-administration did not exacerbate single-drug effects . In the current study , we employed a series of tests sensitive to changes in all common neuropsychological domains induced by several pharmacological compounds , including amphetamines -LRB- Wezenberg et al. 2004 -RRB- . It is generally acknowledged that the combined use of alcohol with other CNS-depressant drugs may enhance the effects of ethanol or of the other drugs . MDMA , however , has stimulant effects while ethanol is a sedative agent , suggesting that the effects of co-administration are diminished rather than augmented compared to the effects following single administration . This hypothesis was investigated during acute co-administration of MDMA and ethanol in healthy volunteers . Materials and methods Study design This study utilised a four-way , double-blind , randomised , crossover , placebo-controlled design . Sixteen volunteers were randomly assigned to one of four treatment sequences . Each volunteer received a capsule containing either 100-mg MDMA or placebo and an ethanol -- placebo infusion -LRB- target blood alcohol concentration -LRB- BAC -RRB- of 0.6 ‰ -RRB- with a washout of 7 days between each treatment . Study outline Subjects arrived in the morning and were admitted to the study after a negative urine drug screen -LRB- opiates , cocaine , benzodiazepines , amphetamines , methamphetamines and delta-9-tetrahydrocannabinol -RRB- , as well as a negative alcohol breath test and recording of signs and symptoms of possible health problems . A light breakfast was offered . Drug administration was scheduled at 1030 hours and the alcohol infusion was started at 1100 hours for a duration of 3 h . At 1130 hours , subjects performed the psychological test battery as described below . Specific test times are reported in Table 1 . Subjects received lunch at 1400 hours and were sent home at 1700 hours after a medical check . Adverse events where recorded throughout the study day . Vital signs were monitored using a Datascope ® Accutorr Plus ™ cardiovascular monitor and Braun ® type 6021 ThermoScan during the study day . The data presented in this report are a subset of a larger data set , which will be reported elsewhere . Table 1TimelineNeuropsychological testsDescriptionTime -LRB- h : m -RRB- Drug administration0 : 0018-word list immediate recallImmediate recall of 18-word list1 : 00SDSTTranslate symbols to digits with key present in 90 s1 : 05SDRTTranslate symbols to digits from memory1 : 08Pursuit taskKeep dot within moving circle1 : 10Tangles taskTangled line leads to which target ? 1:13 Switch taskFollow , possibly conflicting , instructions -LRB- choice between left or right -RRB- 1:1718-word list delayed recallDelayed recall of 18-word list1 : 2218-word list delayed recognitionRecognise words of 18-word list memorised earlier among 18 distracters1 : 23Point taskKeep pen steady in air , measures tremor1 : 25Visual analogue scales16 100-mm scales for subjective experiences1 : 30Times are relative to drug administration . Subjects Sixteen healthy volunteers -LRB- nine male , seven female -RRB- , regular users of ecstasy and alcohol , aged 18 -- 29 years and within 80 -- 130 % of their ideal bodyweight , were recruited through advertisement on the internet and at local drug testing services . They were all in good physical and mental health as determined by assessment of medical history , a medical , electrocardiogramme and clinical , haematological and chemical blood examination . Previous drug use was assessed using a structured interview . Fifteen volunteers were right handed and one was left handed . The study was approved by the local Medical Ethics Committee . All subjects gave their written informed consent before participating in the study and were compensated for their participation . Subject demographics and drug history are reported in Table 2 . Table 2Volunteer demographics -- drug history MeanSDMinMaxAge -LRB- years -RRB- 22.12.918.029.0 Education -LRB- years -RRB- 16.51.61218 Height -LRB- cm -RRB- 174.712.3147.0189.1 Weight -LRB- kg -RRB- 67.512.445.788.4 Opiates0 .10.301 LSD2 .56.6025 Amphetamines37 .381.10250 Ecstasy94 .6138.414431 Cannabis1 ,174.31,665.5205,840 Cocaine33 .7105.70400 Alcohol2 ,367.91,981.6505,200 Solvents3 .613.3050 Barbiturates0000Benzodiazepines18 .657.30216 Psilocybin6 .910.4030 Drug quantities mentioned are lifetime drug exposures , not further specified . One subject had a mild adverse reaction -LRB- local vascular reaction -RRB- to the alcohol infusion and one subject did not refrain from drug use ; both -LRB- one male , one female -RRB- were excluded from further participation and results obtained were not included in the data analysis . Drugs and dosages MDMA -LRB- or matched placebo -RRB- was given as a capsule in a single dose of 100 mg via oral administration -LRB- dose range ; 1.1 -- 2.2 mg/kg -RRB- . MDMA was obtained from Lipomed AG , Arlesheim , Switzerland and encapsuled according to Good Manufacturing Practise by the Department of Clinical Pharmacy , UMC St Radboud , Nijmegen , the Netherlands . MDMA 100 mg orally is a relevant dose in the range of normal single recreational dosages . Previous experiments in humans used doses up to 150 mg without serious adverse events . Ethanol -LRB- or matched placebo -RRB- was administered continuously by intravenous infusion of 10 % ethanol in glucose solution resulting in an ethanol blood concentration of 0.6 ‰ with a duration of 3 h as described below . Alcohol clamping To standardise alcohol delivery and maintain a constant alcohol blood concentration over time , an intravenous ethanol clamp was used . Ethanol was administered by infusion of a 10 % ethanol in glucose solution for a duration of 3 h . The infusion rate was calculated using frequent breath alcohol concentrations measurements , according to a previously designed algorithm -LRB- Amatsaleh et al. 2006a -RRB- . Breath alcohol concentration was assessed using a HONAC AlcoSensor IV ® Intoximetre . An intravenous administration route was chosen , ensuring standardisation of the rate and bioequivalence of ethanol administration . This is an important pre-requisite for predictable pharmacokinetics of ethanol . The process was semi-automated using a computer spreadsheet programme , which uses measured breath alcohol concentrations to calculate the infusion rate needed to maintain the ethanol level at 0.6 mg/mL . This is a relevant dose equivalent to peak levels of approximately two to three units of alcoholic beverages . In many European countries driving is prohibited at BAC above 0.5 ‰ . This limit has been confirmed by a report that shows that at an average BAC of 0.6 ‰ psychomotor performance is significantly impaired -LRB- Amatsaleh et al. 2006b -RRB- . A BAC of 0.6 ‰ is equivalent to approximately two to three alcoholic beverages commonly used in social settings in Western society , which is considered to be a safe and relatively moderate dose , despite its significant CNS effects . MDMA blood analysis For the assessment of serum levels of MDMA , blood samples were collected 90 min after drug administration from each subject on each study day . Venous blood samples -LRB- 10 ml -RRB- were collected into heparinised tubes , centrifuged immediately at 4 °C for 15 min . Plasma was split into aliquots of 2 mL -LRB- to prevent over-freezing -- thawing -RRB- , frozen rapidly using liquid nitrogen and stored at − 80 °C . Samples were analysed for MDMA and MDA concentration by the Toxicology unit of the Leyenburg hospital , The Hague , the Netherlands . Neuropsychological tests , apparatus and procedure The performance on all neuropsychological tests was recorded by means of a digitising tablet -LRB- WACOM UD-1218-RE -RRB- , a laptop computer , a pressure-sensitive pen -LRB- which could also be used as a cursor -RRB- and test forms . The x and y coordinates of the pen tip on and up to 5 mm above the digitiser were sampled with a frequency of 200 Hz and a spatial accuracy of 0.2 mm . The time schedule of the tests is summarised in Table 1 . To familiarise the subjects with the tests and procedures , they were invited to the hospital to perform a practise session within 1 week before the actual study days . All tests had five equivalent versions for four test days and one practise day , test versions were counterbalanced over test days . Executive function Switch task This test is a reaction time task measuring simple as well as complex reaction time , assessing executive performance -LRB- Baker and Letz 1986 -RRB- . After a random period of 0.75 to 1.75 s , two rectangular fields appeared on both sides of a circle in the centre of the screen . Only one of the two fields provided the subjects with information , either a colour , an arrow or both . The other non-informative field always had a neutral grey colour . Five conditions were subsequently presented to subjects . If only green fields appeared , subjects had to move as fast as possible into the green field . If green and red fields appeared , subjects had to move into the green field and away from the red field as soon as they appeared . If green fields with a left or right arrow were presented , subjects were to move into the direction of the arrow . Green and red fields with a left or right arrow indicated that subjects were to follow the direction of the arrows in the green field , but the opposite direction of the arrows in the red field . Finally , the first condition was repeated . All conditions contained 20 trials except condition four in which there were 40 trials -LRB- total = 120 trials -RRB- . The outcome measures were the mean reaction times per condition . The last condition is a repetition of the first to check for possible changes in attention . Memory Eighteen-word list A verbal memory test based on the classic Auditory Verbal Learning Test -LRB- Vakil and Blachstein 1993 -RRB- was used . A variant was made consisting of a list of 18 words . The classic test uses 15 words . A longer wordlist was chosen , however , to prevent ceiling effects . The list was presented verbally three times . Under normal circumstances , subjects are supposed to remember an increasing number of words after each trial . Directly after each presentation , and after an interval of 20 min , subjects were asked to recall as many words as possible . After the delayed recall trial , a list of 36 words was presented from which they were asked to recognise the 18 words previously presented . The incorrect words were distracters and resembled the correct words in a semantic or phonologic manner . Responses were either correct positive -LRB- when a word that was recognised was indeed part of the list presented during immediate recall -RRB- or false positive -LRB- when a word was recognised but was not part of the list presented during immediate recall , e.g. the word was a distracter -RRB- . The outcome measure was the number of correctly recalled or recognised words for the average of the three immediate recall trials , the delayed recall trial and the delayed recognition trial . Symbol digit recall test The symbol digit recall test -LRB- SDRT -RRB- followed directly after the Symbol Digit Substitution test -LRB- SDST -RRB- , which is discussed in the last paragraph of this section . After subjects had finished the SDST , they were shown the symbols of the SDST without the translation key , one at a time , and asked to produce the corresponding numbers . This test is based on an extended procedure of the SDST to measure incidental learning -LRB- Kaplan et al. 1991 -RRB- . The outcome measure was the number of correctly translated symbols . Psychomotor function Pursuit task To measure implicit procedural learning , a computerised version of the rotor pursuit task was used . This test is based on the classic rotary pursuit task -LRB- Ammons 1951 -RRB- . It is a continuous motor task . Subjects had to follow the movement of a large target stimulus on the computer screen with a cursor by moving the pen over the XY tablet . The speed of the target gradually increased when the cursor was contained within the target but decreased considerably when it was not . The target followed a spatially predictable circular path over the screen . The outcome measure for this test was the total number of rotations within 2 min . Point task The point task , a measure for tremor , required subjects to try to keep the cursor inside a very small circle for 1 min , while avoiding contact between the pen and the test form . The outcome measure for this test was the deviation from the target . Visuospatial and visuomotor function Tangle task The tangle task required the subject to visually track a particular line winding through two to four other lines . On subsequent trials , the tangles increased in complexity ; they got longer and made more 90 ° turns . The paper form had a start area and five target areas , numbered 1 to 5 , which reflect the maximum target areas on the screen , starting with only three target areas . This test is modelled after the visualisation test from the ` kit for factor referenced cognitive tests . ' It was selected by the US NAVY to study environmental and other time-course effects and has good task stability and reliability -LRB- Bittner et al. 1986 -RRB- . The outcome measures are the reaction time per trial and the number of correct trials in 2 min . Attention Symbol Digit Substitution test This test is a version of the subtest from the Wechsler Adult Intelligence Scale -LRB- Wechsler 1981 -RRB- . Subjects had to substitute the nine symbols for the digits 1 -- 9 on the basis of a given translation key . The outcome measure was the total number of digits completed in 90 s . According to Hege et al. -LRB- 1997 -RRB- this task measures many cognitive components , e.g. visuospatial scanning , intermediate memory , perceptual motor speed and speed of cognitive processing . Therefore , subsequent analyses were performed in order to attempt and disentangle these cognitive processes . Based on pen pressure , movement trajectories were defined as either pen-up periods or pen-down periods . This allowed for subsequent analysis of matching times and movement -LRB- writing -RRB- times in the Symbol Digit Substitution test . For the motor component , the mean writing times were computed . For the more cognitive component , the mean matching times were computed . These analyses have been previously performed -LRB- Sabbe et al. 1999 ; Wezenberg et al. 2005 -RRB- . Subjective Subjective effects were recorded using the Bond and Lader -LRB- Visual Analogue -RRB- Mood Rating Scale -LRB- BLMRS -RRB- . This inventory was completed at the end of each neuropsychological test battery on each study day . The BLMRS scale consisted of 16 lines , each 10 cm in length , with opposite terms at each end of the line -LRB- alert -- drowsy , calm -- excited , strong -- feeble , muzzy -- clear-headed , well coordinated -- clumsy , lethargic -- energetic , contented -- discontented , troubled -- tranquil , mentally slow -- quick witted , tense -- relaxed , attentive -- dreamy , incompetent -- proficient , happy -- sad , antagonistic -- amicable , interested -- bored , withdrawn -- gregarious -RRB- . Subjects were asked to indicate which item was more appropriate by marking the line . The outcome measure was the distance to the marker on each scale . These scale scores were then aggregated to scores for ` calmness , ' ` alertness ' and ` contentedness ' as described by Bond et al. -LRB- 1974 -RRB- . Statistical analyses Statistical evaluation -LRB- using SPSS 11.5 for Windows -RRB- was performed with general linear model repeated-measures analysis of variance . Main and interaction effects were tested using a two-factor -LRB- ` ethanol ' and ` MDMA ' -RRB- , two-level -LRB- absent versus present -RRB- multivariate model . The analysis of the data was based on Maxwell and Delaney -LRB- 2004 -RRB- and Kirk -LRB- 1995 -RRB- . First the presence of interaction -LRB- non-additivity -RRB- was tested with alfa = 0.05 . When the interaction was not statistically significant we proceeded by testing the main effects , each at alfa = 0.05 . In the case of a significant interaction , we proceeded by testing simple main effects of each drug , i.e. MDMA vs. placebo and ethanol vs. placebo . Results Subject demographics are summarised in Table 2 . Out of 16 subjects , 14 completed the study procedure . One subject had a mild adverse reaction -LRB- local vascular reaction which subsided with infusion stop -RRB- to the alcohol infusion and one subject did not refrain from drug use ; both were discontinued from study participance and data already obtained were not included in statistical analysis . Only significant results are mentioned in this section , unless stated otherwise . MDMA blood concentration 90 min after administration did not differ for MDMA single vs. MDMA and ethanol co-administration and was on average 196 μg/L -LRB- SD = 83 μg/L -RRB- . Blood alcohol concentration was maintained at an average of 0.54 ‰ -LRB- SD = 0.07 ‰ -RRB- . Executive function Executive function -LRB- switch task -RRB- did not show any significant main or interaction effects . Memory function Memory function was assessed by the 18-word list -LRB- outcome measures were ` immediate recall , ' ` delayed recall ' and ` recognition , ' see Fig. 1 -RRB- as well as the SDRT . Immediate recall was impaired only by ethanol -LRB- F -LRB- 1 , 12 -RRB- = 8.71 , p = 0.011 -RRB- . Fig. 1Memory effects -LRB- 18-word list -RRB- , Immediate : immediate recall , average score of three trials of correctly recalled verbally presented words , Delayed : correctly recalled verbally presented words 20 min after presentation , Recognition : correctly recognised verbally presented words containing 18 distracters , 20 min after presentation -LRB- mean and SEM -RRB- . Immediate recall was impaired only by ethanol -LRB- F -LRB- 1 , 12 -RRB- = 8.71 , p = 0.011 -RRB- . Delayed recall was impaired by MDMA -LRB- F -LRB- 1 , 12 -RRB- = 10.447 , p = 0.007 -RRB- as well as by ethanol -LRB- F -LRB- 1 , 12 -RRB- = 16.031 , p = 0.002 -RRB- ; recognition was not affected by any drug condition Delayed recall as assessed by the 18-word list was impaired by MDMA -LRB- F -LRB- 1 , 12 -RRB- = 10.447 , p = 0.007 -RRB- as well as by ethanol -LRB- F -LRB- 1 , 12 -RRB- = 16.031 , p = 0.002 -RRB- . The SDRT , also a test for delayed recall , showed a similar pattern of impairment by MDMA -LRB- F -LRB- 1 , 12 -RRB- = 5.300 , p = 0.038 -RRB- as well as by ethanol -LRB- F -LRB- 1 , 12 -RRB- = 7.654 , p = 0.016 -RRB- . Psychomotor function Psychomotor function was assessed with tests for tremor -LRB- point task -RRB- , accuracy -LRB- pursuit task -RRB- and speed -LRB- SDST motor time , see Fig. 2 -RRB- ; other SDST results are reported in the section `` Attention . '' Ethanol impaired psychomotor speed as reflected in the increase in SDST motor time -LRB- F -LRB- 1 , 12 -RRB- = 9.295 , p = 0.009 -RRB- . Fig. 2Psychomotor effects : SDST writing time -LRB- mean , SEM -RRB- . Ethanol increased writing times -LRB- F -LRB- 1 , 12 -RRB- = 9.295 , p = 0.009 -RRB- Visuospatial and visuomotor function Visuospatial and visuomotor function were measured with the tangle task , subdivided into ` total number correctly solved ' and ` reaction time , ' and did not show any significant effects , although a trend of impairment by MDMA -LRB- F -LRB- 1 , 12 -RRB- = 3.966 , p = 0.068 -RRB- was observed . Attention Attention was assessed with the SDST task ; the outcome measures were ` motor time ' -LRB- see `` Psychomotor function '' -RRB- , ` matching time ' -LRB- Fig. 3 -RRB- and ` total number correctly substituted . ' The time required to match symbols to the corresponding numbers showed a significant MDMA and ethanol interaction -LRB- F -LRB- 1 , 12 -RRB- = 6.214 , p = 0.027 -RRB- . Tests for simple main effects revealed that both single-drug conditions reduced attention compared to placebo -LRB- ethanol F -LRB- 1 , 13 -RRB- = 6.248 , p = 0.027 ; MDMA F -LRB- 1 , 13 -RRB- = 6.822 , p = 0.022 ; see Fig. 3 -RRB- . Fig. 3Attention effects : SDST matching time , i.e. time needed for translation -LRB- mean and SEM -RRB- . A significant MDMA by ethanol interaction was found -LRB- F -LRB- 1 , 12 -RRB- = 6.214 , p = 0.027 -RRB- Subjective effects Subjective effects are depicted in Fig. 4 . Feelings of ` contentedness ' where increased significantly by MDMA only -LRB- F -LRB- 1 , 12 -RRB- = 4.710 , p = 0.049 -RRB- . Fig. 4Subjective effects -LRB- aggregated Bond and Lader scores , mean and SEM -RRB- . Feelings of ` Contentedness ' where increased significantly by MDMA only -LRB- F -LRB- 1 , 12 -RRB- = 4.710 , p = 0.049 -RRB- A significant interaction effect -LRB- F -LRB- 1 , 12 -RRB- = 7.358 , p = 0.018 -RRB- was found for feelings of ` Alertness . ' Feelings of ` Calmness ' were reduced only by MDMA -LRB- F -LRB- 1 , 12 -RRB- = 20.259 , p = 0.001 -RRB- A significant interaction effect -LRB- F -LRB- 1 , 12 -RRB- = 7.358 , p = 0.018 -RRB- was found for feelings of ` alertness . ' Tests for simple main effects revealed that ethanol but not MDMA significantly decreased feelings of alertness compared to placebo -LRB- F -LRB- 1 , 13 -RRB- = 50.613 , p < 0.001 -RRB- . Feelings of ` calmness ' were reduced only by MDMA -LRB- F -LRB- 1 , 12 -RRB- = 20.259 , p = 0.001 -RRB- . Discussion This study demonstrates that the effects of 100-mg MDMA , commonly known as ecstasy , on cognitive function are no greater than the effects of a relatively low dose of ethanol . This is remarkable as these results suggest that the effects of 100-mg MDMA are comparable to the peak effects of two to three alcoholic beverages . Co-administration of these compounds did not result in any significant cognitive impairments beyond those observed after administration of only ethanol . The use of moderate amounts of alcohol is common in Western societies and , although impairing cognitive function , socially accepted , while ecstasy use remains very controversial . Of course , our findings only relate to the acute neuropsychological implications of ecstasy use and not to the physiological and long-term effects , which rightfully remain topics of discussion -LRB- Gouzoulis-Mayfrank and Daumann 2006a ; Nutt 2006 ; Parrott 2007 -RRB- . Drug effects observed in this placebo-controlled crossover study were moderate . Co-administration was well tolerated as indicated by the subjective scores , which were comparable to those found after single administration of MDMA . An interaction of MDMA and ethanol was found for subjective alertness scores . Ethanol , as expected , reduced subjective alertness , while MDMA co-administration reversed the reduction of subjective alertness by ethanol . In the present study , MDMA by itself did not significantly affect subjective alertness , although this effect has been consistently reported in other studies and is a well-known effect of amphetamines . However , MDMA did significantly reduce subjective calmness , i.e. subjects felt more excited after MDMA use . Probably , the Bond and Lader Mood Rating Scale is not well suited for the assessment of subjective effects of psychoactive drug effects and future studies should employ more appropriate subjective drug effect measures such as the Profile Of Mood States -LRB- de Wit et al. 2002 -RRB- . When considering the results for each neuropsychological domain , executive function was not affected by any drug condition . A previous study showed impairment of executive function by ethanol but not MDMA , although ethanol impaired performance in only one out of three tests of executive function -LRB- Lamers et al. 2003 -RRB- . The BAC in this study was 0.3 ‰ at the time of testing compared to 0.56 ‰ in our current study , suggesting a lack of sensitivity of the test employed in the current study . The above-mentioned previous study also reported visuospatial and visuomotor impairment by MDMA but not by ethanol . Although not significant , our current results show a similar pattern where MDMA showed a trend of impairment of visuospatial and visuomotor function , whereas ethanol did not . Psychomotor function was impaired only after ethanol administration -LRB- SDST motor time , see Fig. 2 -RRB- . The majority of studies addressed in our review of acute effects of MDMA in humans -LRB- Dumont and Verkes 2006 -RRB- did not report any change in psychomotor function after MDMA either . However , increased psychomotor function after MDMA has also been found -LRB- Lamers et al. 2003 ; Ramaekers et al. 2006 -RRB- . These studies administered 75 mg instead of 100 mg . Possibly , the effects of MDMA are biphasic , with a low dose of MDMA exhibiting more amphetamine-like effects , e.g. arousal , increasing performance , whereas higher doses may elicit more hallucinogenic effects and impair performance -LRB- Liechti et al. 2001 ; Solowij et al. 1992 -RRB- . As mentioned above , MDMA co-administration reversed the ethanol-induced feelings of sedation , although MDMA was unable to reverse the psychomotor impairment induced by ethanol . This dissociation between subjective and objective sedation confirms previous findings by Hernandez-Lopez et al. -LRB- 2002 -RRB- . Several studies assessed MDMA 's effect on attention using the Digit Symbol Substitution Task -LRB- DSST -RRB- , although no significant effects were found -LRB- Cami et al. 2000 ; Farre et al. 2004 ; Kuypers and Ramaekers 2005 -RRB- . One study reported decreased DSST performance after ethanol as well as after ethanol and MDMA co-administration but no effect of MDMA -LRB- Hernandez-Lopez et al. 2002 -RRB- . Our findings confirm these findings to a large extent . We found no main effects of MDMA or ethanol on attention , although an interaction of ethanol and MDMA for ` matching time ' -LRB- time required to match the number to the corresponding symbol -RRB- was found . Co-administration of MDMA and ethanol increased ` matching time ' comparable to the increase observed after both MDMA and ethanol single administration , compliant with our hypothesis of competitive mechanisms of action of both drugs -LRB- see Fig. 3 -RRB- . Studies investigating the long-term effects of MDMA consistently found memory to be affected -LRB- Verbaten 2003 -RRB- . In the present report , almost all memory measures showed quantitatively comparable impairment for each drug condition -LRB- see Fig. 1 -RRB- , although the effect of MDMA on immediate recall did not reach statistical significance . Only delayed recognition was not impaired in any drug condition . These findings suggest a deficit in the retrieval of verbal information encoded in memory , rather than impairment in the storage of information . Our findings are similar to the results of a previous study on MDMA-induced effects on memory -LRB- Kuypers and Ramaekers 2005 -RRB- . In this previous study , no memory impairment was observed after methylphenidate administration , a pronounced dopamine and norepinephrine releaser , suggesting the involvement of serotonin in memory impairment . Several other studies also have shown serotonin-mediated modulation of memory function through interaction with the cholinergic neurotransmitter system , although the details of this complicated interaction remain elusive -LRB- Cassel and Jeltsch 1995 ; Garcia-Alloza et al. 2006 ; Meneses 2007 -RRB- . Generally , subjects stated that they were well aware of their impaired memory after MDMA . BAC was on average 0.56 ‰ . At this level , driving is prohibited by law in many European countries because of its interference with normal functioning . Although the effects were moderate , ethanol impaired cognitive performance in various tests . Similar moderate effects were observed with MDMA 100 mg , considered to be slightly above the average recreational dose -LRB- Tanner-Smith 2006 -RRB- . This might be considered surprising for a drug with reported robust subjective stimulating and hallucinogenic properties . However , because the effects caused by a single dose of 100-mg MDMA were comparable to the effects of a BAC of 0.56 ‰ , this dose should by inference be considered unacceptable in motorised traffic . Arguably , the moderate drug effects as found in this study could be explained by ` missing ' the time of the maximal drug effects . Although the average MDMA blood concentration reported here -LRB- 196 μg/L -RRB- is comparable to Cmax of 100-mg MDMA -LRB- 199.8 μg/L -RRB- as reported by de la Torre et al. -LRB- 2000 -RRB- , MDMA concentration was assessed at the end of the testing procedure . However , Hernandez-Lopez et al. -LRB- 2002 -RRB- found significant effects at 60 min as well as 90 min after drug administration , arguing against the suggestion of ` missing ' peak drug effects . The circumstances in which these substances are normally used can not be fully recreated in the laboratory and this may have suppressed the effects of both substances . It is not unlikely that these substances show enhanced effects when tested under typical circumstances and surroundings . Recently , Parrott et al. -LRB- 2006 -RRB- concluded that the increase in physical activity and body temperature typically experienced when using MDMA enhance MDMA effects . Ball et al. -LRB- 2006 -RRB- demonstrated that a familiar surrounding increased MDMA-induced locomotor response as well as single-neuron activity in rats , compared to unfamiliar surroundings . Therefore , the psychosocial context in which MDMA is used , along with the different expectations and behaviour , probably influences its effects -LRB- Sumnall et al. 2006 -RRB- . It is unlikely , however , that this affects the quality of the interactions of MDMA and ethanol . In conclusion , co-administration of MDMA and ethanol did not impair cognitive function significantly more than MDMA or ethanol administration alone . The most prominent effect of -LRB- co - -RRB- administration of MDMA and ethanol was an impairment of memory . Ethanol also impaired psychomotor function . Although the impairment of performance by each drug condition was relatively moderate , this significant impairment of cognitive function should be considered intolerable in motorised traffic and other cognitively demanding situations as confirmed by previous research and as defined by law . However , the effects of these drugs in the concentrations used in the present study on established neuropsychological tests appear to be smaller than one would assume based on their reputation .

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

mdma
ecstasy
alcohol
ethanol
interaction
acute
effects
healthy volunteers