while psychostimulants have acute effects on brain dopamine systems , there is also evidence 
 from several lines of research for the involvement of the opioid system in psychostimulant 
 dependence .
for instance , positron emission tomography ( pet ) studies have revealed 
 functionally important changes in the brain opioid systems of cocaine users as compared to 
 controls ( gorelick et al . 

there is also pharmacological evidence from 
 animal studies showing that the opioid antagonist naltrexone attenuates amphetamine - induced 
 locomotor sensitization and reinstatement of drug - seeking in rats ( hggkvist et al . 

, naltrexone has been shown to significantly attenuate the 
 subjective effects of amphetamine in amphetamine - dependent subjects and healthy controls , as 
 well as reducing craving and risk of relapse to amphetamine use ( jayaram - lindstrm 
 et al . 

this is in line with studies linking the opioid system to the hedonic component of 
 reward ( berridge et al . 

a possible explanation of naltrexone 's effect could be that amphetamine causes an 
 activation of the opioid system and that this activation is inhibited by opioid antagonists , 
 which thereby attenuate the rewarding effects of amphetamine .
indeed , several studies have 
 shown evidence of such stimulant - induced opioid release in the ventral striatum of rodents 
 ( olive et al . 

2001 ; roth - deri et al . 
 2003 ; soderman & unterwald , 2009 ) .
recently , a dose of oral amphetamine was shown 
 to induce decreases in [ c]carfentanil binding in healthy control subjects . 

however , in this study there was no within - subject control for expectation effects and the 
 measurements were performed 3 h after dosing ( colasanti et al . 

administration of 
 amphetamine on the opioid system have not yet been investigated in humans . in this placebo controlled pet study of healthy , drug - naive subjects
amphetamine induces an acute opioid release in the brain , 
 specifically in the ventral striatum , measurable as a reduction in binding of the -opioid 
 receptor radioligand [ c]carfentanil .
the study was approved by the swedish medical products agency , the regional ethics review 
 board and the radiation safety committee and performed in accordance with ich guidelines for 
 good clinical practice .
ten healthy young men , aged 26.72.5 yr ( means.d . ) , were recruited by 
 advertising within the karolinska institutet .
screening procedures , including the alcohol 
 use disorders identification test , drug use disorders identification test and mini 
 international neuropsychiatric interview , were performed by a study physician in order to 
 exclude subjects with any somatic or psychiatric disease or a history of any substance use 
 disorders either themselves or in a first - degree relative . at screening and on the test 

days , subjects were tested for alcohol in exhaled air and with urine toxicology to exclude 
 use of any illicit drugs . a t1 weighted magnetic resonance ( mr ) scan ( 1.5 t )
was performed for every subject prior 
 to pet procedures to exclude intracranial pathology and obtain anatomical references for 
 definition of regions of interest ( rois ) . applying a double - blind ,
cross - over randomized design , three pet examinations were 
 performed for each subject .
the test days were approximately 1 wk apart , although three 
 examinations were delayed for technical reasons ( performed after an interval of 2040 d ) . 

the first examination served as a baseline measure , while the second and third 
 measurements were done after administration of an i.v .
the order of amphetamine or placebo was randomized and 
 double - blind to avoid confounding with expectation effects .
after the amphetamine and placebo sessions , subjects were asked to subjectively rate , on 
 a scale from 0 to 100 , how strongly they felt any effect of the drug and how strongly they 
 experienced any arousal or 
they were also asked to rate how strongly they 
 would like to have more of the drug .
we used the pet radioligand [ c]carfentanil , a selective -opioid receptor 
 agonist ( frost et al . 

[ c]carfentanil has been shown 
 to have excellent test  retest reliability , making it a suitable ligand for cross - over 
 experiments ( hirvonen et al . 

[ c]carfentanil is considered 
 sensitive to acute changes in opioid neurotransmission , based on previous findings of 
 reduced [ c]carfentanil binding potential ( bpnd ) in response to 
 behavioural or pharmacological challenges that have been interpreted as increased 
 endogenous opioid release ( hirvonen et al . 

the injected radioactivity was 306.815.1 
 mbq ( means.d . ) and injected mass 0.300 0.181 g ( mean s.d . ) .
there 
 were no significant differences in these variables between the baseline , placebo and 
 amphetamine conditions [ repeated measures analysis of variance ( anova ) for injected dose : 
 f2,18=0.41 , p=0.67 and for injected mass : 
 f2,18=0.14 , p=0.87 ] .
pet examinations were performed using the high resolution research tomograph ( siemens 
 molecular imaging ; germany ) . for each subject ,
an individual helmet was made and attached 
 to a holder on the coach to minimize head movement .
after a 6 min transmission scan using 
 a single cs source , the study drug ( amphetamine / placebo ) was injected into 
 an i.v .
two minutes later , the radioligand was 
 administered as a rapid bolus and flushed with saline .
list - mode data were acquired for 69 
 min , starting at the time of ligand injection .
pet images were reconstructed from a series 
 of 16 time frames ( 31 , 43 and 93 min ) , including modelling of the point spread 
 function , after correction for attenuation , randoms and scatter .
this reconstruction 
 procedure yields a spatial resolution of 1.5 mm ( varrone et al . 

pet images were corrected for head 
 movement using frame - by - frame realignment ( montgomery et al . 

rois were delineated on the mr images for 
 each subject individually , using the human brain atlas software ( fig .
ventral striatum was selected as the 
 primary roi , while a secondary , exploratory analysis included other brain regions involved 
 in drug abuse and reward , i.e. associative and sensorimotor striatum , prefrontal cortex 
 ( divided into orbitofrontal , dorsolateral and medial ) , anterior cingulate cortex , 
 hippocampus and amygdala .
the definitions of rois were based on previously published 
 guidelines ( abi - dargham et al . 

1.t1 weighted magnetic resonance image , coronal slice through the striatum with 
 superimposed regions of interest ( a ) .
high - resolution research 
 tomograph positron emission tomography image with [ c]carfentanil , 
 corresponding slice ( b ) .
t1 weighted magnetic resonance image , coronal slice through the striatum with 
 superimposed regions of interest ( a ) .
high - resolution research 
 tomograph positron emission tomography image with [ c]carfentanil , 
 corresponding slice ( b ) .
pet images were co - registered to the mr image using spm 5 and the parameters derived were 
 used to apply rois to the pet images in order to extract time 
quantitative analysis was performed using the 
 simplified reference tissue model with the occipital lobe as reference region , as has been 
 validated for [ c]carfentanil ( endres et al . 

the bpnd was the parameter of 
 interest , representing the ratio at equilibrium of specifically bound to that of 
 non - displaceable radioligand ( innis et al . 

bpnd data from the baseline , placebo and amphetamine conditions were analysed 
 with repeated measures anova for effect of treatment .
ratings of how much the 
 subjects  felt the drug effect ranged from 50 to 100 ( mean 8816.9 ) , feeling of 
( mean 6622.2 ) and how much they wanted more of the drug from 0 to 100 ( mean 
 6432.6 ) .
when analysing the pet data with repeated measures anova , we found no significant effects 
 of amphetamine compared to placebo on the [ c]carfentanil bpnd for 
 ventral striatum , which was the primary roi ( fig . 
 2 ) .
expressed in terms of difference between the placebo and amphetamine conditions , 
 mean bpnd was 0.037 in our sample , with s.d.=0.240 , giving us a 95% 
 confidence interval of 0.186 to 0.111 ( or 6.4 to 3.8% ) .
in other words , we are confident 
 that amphetamine does not reduce bpnd in the ventral striatum by > 6.4% , as 
 compared to placebo .
the same results were found when extending the analysis to 
 other regions of the striatum ( table 1 ) .
2.individual measures of [ c]carfentanil binding potential 
 ( bpnd ) for the ventral striatum . 

mean bpnd change from baseline for each condition in different regions 
 of the striatumregionbaselineplacebochange from baseline ( % ) amphetaminechange from baseline ( % ) 
 f 
 2,18 

 p 
 ventral striatum2.900.432.910.42 + 0.22.840.432.20.2920.750associative striatum1.840.341.940.21 + 5.11.860.29 + 0.60.2980.746sensorimotor striatum1.280.231.330.13 + 4.51.250.172.20.7930.468d.f .
, f and p values from repeated measures 
 analysis of variance of treatment effect , uncorrected for multiple comparisons . individual measures of [ c]carfentanil binding potential 
 ( bpnd ) for the ventral striatum .
mean [ c]carfentanil binding potential ( bpnd)s.d . 
 and mean bpnd change from baseline for each condition in different regions 
 of the striatum d.f .
, f and p values from repeated measures 
 analysis of variance of treatment effect , uncorrected for multiple comparisons .
an exploratory analysis of other brain regions , including the prefrontal cortex , amygdala 
 and hippocampus , did not reveal any effects of amphetamine on [ c]carfentanil 
 bpnd ( table 2 ) .
table 2.exploratory analysis of mean [ c]carfentanil binding potential in regions 
 of the prefrontal cortex and medial temporal loberegionbaselineplaceboamphetamine 
 f 
 2,18 

 p 
 dorsolateral prefrontal cortex1.1361.2521.2371.410.270medial prefrontal cortex1.1201.1761.1800.3230.728orbitofrontal cortex1.1851.2361.2120.4050.673anterior cingulate cortex1.1691.2701.2770.6260.626amygdala2.4752.3742.5670.1130.113hippocampus0.4530.5060.4551.3410.287d.f . , f and p values from repeated measures 
 analysis of variance of treatment effect , uncorrected for multiple comparisons .
exploratory analysis of mean [ c]carfentanil binding potential in regions 
 of the prefrontal cortex and medial temporal lobe d.f . , f and p values from repeated measures 
 analysis of variance of treatment effect , uncorrected for multiple comparisons .
measures of baseline [ c]carfentanil bpnd were found to be quite 
 similar to the values obtained in a previously published test 
the inter - individual variation in change 
 between different conditions as indicated by the standard deviation was higher than the 
 corresponding inter - individual variation in test 
we 
 therefore proceeded in analysing associations between individual bpnd changes and 
 subjective ratings of amphetamine effects .
however , we found no evidence of a correlation 
 between changes in [ c]carfentanil bpnd and the subjective effects of 
 amphetamine ( p>0.1 , data not shown ) .
in the present cross - over , randomized , placebo - controlled pet study , we found that an acute 
 i.v .
dose of amphetamine did not change [ c]carfentanil bpnd in the 
 striatum , nor in the prefrontal cortex , amygdala or hippocampus .
the dose of amphetamine administered in this study ( 0.3 mg / kg bodyweight ) is the one most 
 often used in human laboratory studies , causing strong , immediate subjective effects in 
 drug - naive individuals .
this dose has been shown to induce a significant dopamine release in 
 the ventral striatum , measurable in human pet studies as a decrease of the dopamine 
 d2-receptor radioligand [ c]raclopride bpnd by about 15% 
 ( drevets et al . 

importantly , a dose of 0.3 mg / kg bodyweight 
 was also used previously in studies showing that naltrexone blunts the subjective effects of 
 amphetamine ( jayaram - lindstrm et al . 

amphetamine - dependent patients often inject several 
 hundred mg and such a dose naturally gives rise to stronger pharmacological effects , 
 possibly including an activation of the opioid system .
however , due to the risk of adverse 
 reactions , such doses can not be administered to humans in an experimental setting .
animal 
 studies are therefore needed to investigate the opioid effects of amphetamine across a wider 
 dose range . in this study
, we started the pet measurement 2 min after the amphetamine / placebo injection 
 and continued for 69 min .
a possibility is that amphetamine does cause an endogenous opioid 
 release that either evaporates too fast or commences too late for us to capture it in this 
 time frame .
the 69 min time frame was chosen based on our previous studies , where 
 naltrexone 's effects were already evident after a couple of minutes and stayed significant 
 over several hours ( jayaram - lindstrm et al . 

amphetamine administration came somewhat 
 later , but was clearly evident during the first hour ( olive et al . 

evidence from a recent animal study suggests 
 that opioid activation following cocaine administration i.p .
peaks at 20 min ( soderman 
 & unterwald , 2009 ) . to test for signs of 
 such an early opioid activation , we did a post - hoc kinetic analysis using 
 only the first 39 min of the pet scan , a time frame that has previously been shown to have 
 good reliability with [ c]carfentanil ( hirvonen et al . 

this analysis did not reveal any trend of 
 early opioid release , suggesting that our original time frame was adequate .
several rodent studies have shown evidence of psychostimulant - induced opioid activation , a 
 finding that could not be translated to humans in this study .
dose of 2 mg amphetamine / kg bodyweight gives rise to an increase of 
 dialysate endorphin levels in the accumbens , peaking at 300% ( olive et al . 

2001 ) , and a dopamine increase peaking at 350% 
 ( butcher et al . 

doses in humans , the dose used in this study is probably less 
 potent , even though it did cause strong behavioural effects .
other species differences , 
 particularly in the anatomy of the dopamine system , might therefore be more important in 
 explaining these results ( berger et al . 

1991 ) . in human subjects , as opposed to other animals , it is important to acknowledge expectation 
 effects since they may involve opioid mechanisms ( scott et al . 

2008 ) . in this study , the subjects were aware that 
 they would not be administered any study drug during the first , baseline pet examination , 
 but in the second and third they would get either amphetamine or placebo intravenously . by 
 randomizing the order of placebo and amphetamine , we could control for expectation effects 
 and anova showed no effect of order on [ c]carfentanil bpnd .
neither 
 was there any significant difference between the baseline ( i.e. without study drug ) and 
 medication conditions , which means that the expectation of an amphetamine injection did not 
 give rise to any measurable opioid activation .
this study was performed with healthy , drug - naive subjects , but the effects of amphetamine 
 may be quite different in amphetamine - dependent patients .
for example , after three doses of 
 the same strength as in this study , a significant sensitization to amphetamine occurs with 
 an increased dopamine release in the striatum ( boileau et al . 

2006 ) . such mechanisms might also cause a downstream 
 recruitment of other neurotransmitter systems with repeated drug intake .
although we saw no 
 evidence of opioid activation in healthy subjects from this first dose of amphetamine , it is 
 possible that the opioid system becomes involved later in the development of stimulant 
 dependence , forming part of the neuro - adaptations in brain reward systems that create a 
 state of hedonic allostasis in the addicted brain ( koob & le moal , 2001 ) .
indeed , recent pet studies have shown 
 significant alterations of [ c]carfentanil bpnd in chronic cocaine 
 patients as compared to healthy controls , changes that predict treatment outcome and are 
 correlated with drug craving and risk of relapse to cocaine use ( ghitza et al . 

another recent study of alcohol - dependent 
 patients found that opiate - induced dopamine release in the ventral striatum was correlated 
 with the severity of alcohol dependence ( spreckelmeyer et al . 

2011 ) , providing further evidence of the important 
 interactions between these two neurotransmitter systems in substance use disorders .
we have previously shown that naltrexone pretreatment attenuates the subjective response to 
 an acute dose of amphetamine to a greater extent in amphetamine - dependent patients than in 
 healthy subjects ( jayaram - lindstrm et al . 

it is therefore possible that amphetamine does cause a 
 significant opioid release in the amphetamine - dependent , but not in the healthy , human 
 brain . genetic variability between samples , particularly in the prevalence of the asp40 
 single nucleotide polymorphism in the  opioid receptor gene that has been linked to 
 naltrexone response in alcohol dependence ,
might also have contributed to differences 
 between studies ; although we do not have genetic data to prove this ( dlugos et al . 

2012 ) published a study reporting a decrease in [ c]carfentanil 
 bpnd 3 h after an oral amphetamine dose of 0.5 mg / kg bodyweight .
there are 
 several differences between our studies that may help explain the differences in results .
administration of amphetamine in terms 
 of pharmacokinetics and also in the resulting subjective effects .
indeed , in colasanti 's 
 study , the high - dose group did not report any significant euphoria , while the subjects in 
 our study consistently reported strong effects of amphetamine as compared to placebo .
while a part of this interval is needed due to the slower 
 absorption of an oral amphetamine dose , it remains a significant difference between our 
 studies .
our choice of an immediate measurement was based on several lines of evidence , not 
 least previous studies showing a very fast reduction of ( 3)h - damgo binding after cocaine 
 administration in rodents ( soderman & unterwald , 2009 ) . also , the euphoric effects of an i.v . amphetamine dose are immediate and 
 start to wear off gradually after 12 h. of course , if opioid release plays any important 
 role in these acute rewarding effects , it has to be present within this time interval rather 
 than 3 or 4 h later .
there are also some technical differences between the studies that might have influenced 
 the results .
the increased resolution of the high - resolution research tomograph system 
 provides a higher signal recovery , which should provide additional sensitivity to changes in 
 binding ( schain et al . 

2012 ) . also , we had no bias in terms of differences 
 in injected mass or radioactivity between the different conditions . finally , a particular 
 strength of our study is the cross - over randomization , which allowed us to do within - subject 
 comparisons while still controlling for expectation effects .
the results of these two pet studies suggest another possible explanation for the fact that 
 naltrexone attenuates the subjective effects of amphetamine , namely , that weak unspecific 
 adverse effects of naltrexone ( e.g. nausea ) might somewhat blunt the immediate effects of 
 amphetamine , while its specific action as an opioid antagonist blocks the prolonged euphoric 
 effects possibly caused by opioid release 2 or 3 h later . to conclude , in this placebo - controlled pet study , an acute i.v .
dose of amphetamine did 
 not cause any opioid release in the healthy human brain reward system .
furthermore , possible 
 expectation effects were not mediated by opioid activation , since order of 
 placebo / amphetamine did not change [ c]carfentanil bpnd .
these 
 results are in contrast with earlier findings of stimulant - induced opioid activation in 
 rodents as well as recent data showing later effects of oral amphetamine using a slightly 
 different study design .
further studies are needed to more fully investigate this issue from 
 a translational perspective . with the recent development of small animal pet systems ,
the 
 effects of amphetamine on the opioid system could be studied across a wider dose range and 
 timescale than what is possible in humans .
studies of stimulant - dependent patients are also 
 needed to investigate the pathophysiological importance of the opioid system and its 
 potential as a therapeutic pharmacological target .
lars farde also holds a position as chief scientist , imed cns / pain , astrazeneca , 
 sweden .
 studies in rodents have shown that psychostimulant drugs such as cocaine and amphetamine 
 cause endorphin release in the brain reward system . 
 there is also evidence for the 
 involvement of the opioid system in human psychostimulant dependence . 
 the acute effects of 
 an i.v . 
 psychostimulant drug on the brain opioid system , however , have not yet been 
 investigated in humans . 
 we hypothesized that an i.v . 
 dose of amphetamine as compared to 
 placebo would cause an opioid release in the human brain reward system , measurable as a 
 reduction of the binding potential of the -opioid receptor radioligand 
 [ 11c]carfentanil . 
 ten healthy young men were examined using positron emission 
 tomography ( pet ) and [ 11c]carfentanil in three sessions : at baseline ; after 
 placebo ; after an i.v . 
 amphetamine dose of 0.3 mg / kg bodyweight . 
 the order of amphetamine 
 and placebo was double - blinded and randomized . 
 pet examinations were performed with a 
 siemens high resolution research tomograph . 
 data were analysed with the simplified 
 reference tissue model , applying manually drawn regions of interest for every subject . 
 using repeated measures analysis of variance , we found no significant differences in 
 [ 11c]carfentanil binding potential between amphetamine and placebo conditions 
 in any of the investigated brain regions . 
 in contrast to data from rodent studies and a 
 recent study of oral amphetamine administration in humans , an i.v . 
 dose of amphetamine 
 does not cause any acute opioid release in healthy human subjects . 
 the postulated role of 
 the opioid system in mediating the effects of amphetamine needs to be further investigated 
 in animal models of the disease as well as in patient populations . 
Introduction
Materials and method
Results
Discussion
Statement of Interest