it consists of at least two g - protein - coupled cannabinoid receptors cb1 and cb2 ( cb1r and cb2r ) , their intrinsic ligands ( endocannabinoids ) such as n - arachidonoyl ethanolamine ( anandamide , aea )   and 2-arachidonoyl glycerol ( 2-ag ) , and their associated proteins involved in synthesis , transport , and degradation . the cb1r , which mediates the psychoactive effects of marijuana , is widely expressed and is considered one of the most abundant g - protein - coupled receptors in the brain . in the central nervous system , endocannabinoids are released from postsynaptic sites and , by activation of the presynaptically located cb1r , inhibit the release of several neurotransmitters such as gaba , glutamate , dopamine , and acetylcholine . in vitro immunohistochemical   and autoradiography   studies in rats have shown that the cb1r is highly expressed in the basal ganglia ( lateral caudate - putamen , globus pallidus , entopeduncular nucleus , and substantia nigra pars reticulata ) , cerebellum ( molecular layer ) , and hippocampus ( ca1 , ca3 , and dentate gyrus molecular layer ) . moderate levels are found throughout the cortical regions , whereas low levels are observed in the brainstem ( midbrain , pons ) and spinal cord . the cb1r has been shown to be involved in various physiological functions like nociception , control of movement , memory , neuroendocrine regulation , brain development , and maturation [ 16 , 17 ] . biochemical and functional alterations of cb1r have been shown to be implicated in the pathophysiology of distinct neurological and psychiatric disorders   including schizophrenia [ 1921 ] . it is known that cannabis and its derivatives can trigger psychotic - like symptoms in normal individuals , and numerous epidemiological studies have demonstrated that consuming cannabis during adolescence ( particularly early adolescence ) constitutes a risk factor for schizophrenia onset later in life [ 2325 ] . the ages associated with adolescence are commonly considered in humans to be approximately 12 to 2025 years of age and postnatal day ( pnd ) 2855 in rodents . the adolescent brain undergoes both progressive and regressive changes providing the biological basis for the unique adolescent behaviors and their associated changes during maturation to adulthood . at the cellular level , these changes correspond to the marked overproduction of axon and synapses in early puberty and rapid pruning in late adolescence . to date , most developmental studies of the cannabinoid system [ 2831 ] have focused on the embryonic and early postnatal stages . in vitro autoradiographic studies significant increases in cb1r density appeared regionally in the developing brain until pnd 21   or pnd 30 , and the maximum adult level was reached at pnd 60 . in contrast , rodriguez de fonseca et al .   reported slight decreases in binding between pnd 30 and 40 and adulthood ( pnd 70 ) . recently , the development of new efficient radiotracers has enabled the study of cb1r in vivo using positron emission tomography ( pet ) . burns et al .   demonstrated that the selective , high - affinity inverse agonist for the cb1r , named [ f]mk-9470 had the potential to be a valuable tool for the in vivo study of cb1r biology and pharmacology . several in vivo preclinical [ 3541 ] and clinical studies [ 4245 ] have used this compound successfully .   we have recently reported higher levels of dopamine d1 and d2 receptors , both serotonin 5ht1a receptor binding and mrna expression , and gabaa receptor binding   in adolescent rats ( pnd 39 ) compared to adults ( pnd 70 ) , that is , in accordance with the regressive elimination of synapses and receptors that occurs during the transition from adolescence to adulthood . in the present study , we have undertaken two objectives : first , to demonstrate the feasibility of imaging cb1r in vivo in adolescence and adulthood using small animal pet with [ f]mk-9470 ; second , to compare the level of expression / regional distribution of cb1r in adolescent and adult rats obtained in vivo with pet and in vitro with autoradiography using [ h]cp55,940 . the aim was to test the hypothesis whether cb1r pruning occurs during the transition from adolescence to adulthood as it has been indicated for other neuroreceptor systems . cb1r imaging was performed in all animals using the radioligand [ f]mk-9470 ( n-[2-(3-cyanophenyl)-3-(4-(2-[f]fluoroethoxy)phenyl)-1-methylpropyl]-2-(5-methyl-2-pyridyloxy)-2-methylpropanamide ) , a high specificity , high - affinity inverse agonist at the cb1r . the precursor for radiotracer synthesis and the authentic [ f]mk-9470 standard were obtained from merck research labs ( west point , pa , usa ) . radiolabelling was performed using a two - step semiautomated procedure following the method outlined by burns et al .   with some modifications . in the first step , 2-bromo-1[f]fluoroethane ( [ f]brfe ) was synthesised using a nuclear interface fdg synthesizer ( ge medical system ) . f - fluoroalkylation of the mk-9470 precursor was then manually carried out using cs2co3 as a base . an aliquot of [ f]brfe was added , and [ f]mk-9470 was obtained in up to 8% overall yield ( not corrected for decay ) after high - performance liquid chromatography ( hplc ) and sep - pak purification . [ f]mk-9470 product was confirmed by coinjection with the [ f]mk-9470 standard . the final product obtained had a radiochemical purity > 95% and specific activity averaging 6000  ci / mmole ( 222  gbq/mole ) . ltd ( perth , australia ) and were housed in polyethylene boxes with wire lids ( 489  343  240  mm ) in groups of two - three per cage . all handling of animals and procedures was carried out in accordance with the guidelines established by the animal care and ethics committee at the australian nuclear science and technology organisation ( ansto ) . the animals were kept at a constant temperature of 22  2c on a 1212  h light - dark cycle with lights on at 9 am and were handled during the seven days preceding the experiment . the adult cohort consisted of 6 rats with body weights ranging between 381  22  g at 10 weeks of age ( pnd 7072 ) , and the adolescent cohort consisted of 6 rats with body weights ranging between 148  22  g at 7 weeks of age ( pnd 3537 ) . animals were fasted for at least 6 hours before the start of the experiment . pet imaging with [ anaesthesia was induced by exposing rats to 4% isoflurane in oxygen and then maintained by reducing the ratio to 1.52.5% for the duration of the studies . isoflurane anesthesia has been shown not to have any significant effects on absolute [ f]mk-9470 binding as compared to control conditions . heart rate ( 333.2  25.9  beats / min ) , respiratory rate ( 41.6  9.2  cycles / min ) , and saturation in oxygen ( > 95% ) were measured with a pulse oximeter ( starr , life sciences corp , usa ) . we also monitored the respiratory rate under the ct part of the scanner with a pressure sensor connected to a computer ( biovet , m2 m imaging crop , usa ) . after anaesthesia and placing of the animal in the scanner with the help of laser guidance , a catheter was placed in a lateral tail vein of the rat and connected to an infusion pump ( harvard apparatus , usa ) . a 60  min pet scan was started at the same time of the start of the one - minute injection of [ f]mk-9470 at a constant tracer mass ( 65.2  1.5  pmoles ) . a 15  min ct scan activity volumes were reconstructed with an iterative reconstruction ( osem / map )   including attenuation and scatter correction , achieving a reconstructed spatial resolution of 1.5  mm . a previously developed magnetic - resonance - imaging- ( mri-)based rat brain atlas was coregistered to the pet volume , using the ct information of the skull ( anatomist / brainvisa , v3.1.4 , http://brainvisa.info/ ) . in detail , all pet acquisitions ( 12 animals ) were coregistered with their respective ct ( see figure 1 ) . all cts in the adolescent cohort were manually / visually coregistered to one adolescent ct ( adolescence reference ct ) . finally , the  reference  cts were manually / visually coregistered to the mri - based rat brain atlas encompassing eleven volumes of interest ( voi ) ( figure 2(a ) ) . transformation matrixes were then created from the mri - based rat brain atlas to each pet image in each group . previous studies in rats with [ f]mk-9470 have used the last 20  min of a 60  min acquisition period ( 40 to 60  min ) for quantification purposes [ 3537 ] . in this study , we used percentage of injected dose ( activity concentration ( mbq / ml ) divided by injected dose ( mbq ) ) of the last 20 minutes of acquisition ( % id4060 ) as absolute cb1r binding measure . twenty - four hours after in vivo imaging , the animals ( 6 adolescents and 5 adults ) were euthanized , their brain was dissected , frozen in liquid nitrogen , and stored at 80c . coronal brain sections ( 16  m ) were cut with a cryostat and thaw - mounted onto microscope slides . autoradiography was carried out based on the method previously described in dalton et al . . all sections were processed simultaneously to minimize experimental variance . on the day of the experiment , sections were taken out of the 80c freezer and allowed to come to room temperature for approximately 60  min or until dry . sections were preincubated for 30  min at room temperature in 50  mm tris - hcl ( ph 7.4 ) containing 5% bovine serum albumin ( bsa ) in order to equilibrate the tissue to the assay conditions and remove any endogenous ligand . radioligand binding was measured using single - point saturation analysis which provides a good estimate of receptor density . the kd of rat brain cb1r has been evaluated at 5.2  nm . in order to ensure saturation of cb1r , sections were then incubated for 2  h at room temperature in the same buffer as preincubation with the addition of 10  nm [ h]cp55,940 ( specific activity 139.6  ci / mmole , perkin elmer , usa ) . nonspecific binding was determined by incubating adjacent sections in the presence of 10  m cp55,940 . the concentration of [ h]cp55,940 was measured in 10  l aliquots taken from the incubation mixture . after the incubation , sections were washed for 1  h at 4c in 50  mm tris - hcl ( ph 7.4 ) containing 1% bsa , and a second wash was then carried out for 3  h in the same buffer at 4c . the third wash was in 50  mm tris - hcl ( ph 7.4 ) for 5  min at 4c . dried sections were apposed to kodak biomax mr films , together with autoradiographic tritium standards ( [ h ] microscales from amersham ) , in x - ray film cassettes . films were developed after 35 days using kodak gbx developer and fixed with kodak gbx fixer . films were analysed using a computer - assisted image analysis system , multianalyst , connected to a gs-690 imaging densitometer ( bio - rad , usa ) . eleven brain regions of interest ( roi ) were manually drawn with the help of a stereotaxic atlas of the rat brain   and corresponded to the 11 voi analysed in vivo ( figure 4 ) . quantification of receptor binding in each brain region was performed by measuring the average optical density in adjacent brain sections . optical density measurements for specific binding were then converted into fmoles of [ h]cp55,940 per mg of tissue equivalent ( fmol / mg te ) according to the calibration curve obtained from the [ h]-labelled standards . statistical tests were performed using pasw statistics ( version 18.0.0 ) and graphpad prism ( version 5.04 ) . data were analysed for significant outliers ( 2  sd ) , and none were detected . the mass and injected dose of [ f]mk-9470 between the adolescent and adult cohorts were compared using unpaired student 's t - tests . pearson correlations were used to examine the relationship between % id4060 and body weight and between [ f]mk-9470 cb1r binding in vivo and [ h]cp55,940 cb1r binding in vitro . analysis of covariance ( ancova ) controlling for body weight was used to determine if there was an effect of age and/or region on cb1r absolute binding measured in vivo . in vitro data were analysed using two - way anova ( age  region ) followed by least significant difference ( lsd ) tests . adolescent rats showed the regional distribution that corresponds to the previously published regional distribution of cb1r [ 11 , 53 ] , but adult rats unexpectedly demonstrated a more uniform regional distribution of the pet radioligand ( figures 1 and 2 ) . cerebellum , striatum , cortical regions , and ( moderately ) hippocampus showed higher in vivo cb1r absolute binding compared to other brain regions . regions known to have fewer cb1r like the thalamus and especially the brainstem ( midbrain , pons ) presented relatively high cb1r absolute binding in vivo ( figure 2 ) . time - activity curves ( expressed in % id4060 ) showed that [ f]mk-9470 entered the brain with a slow kinetic and reached a peak at approximately 20  min after - injection . there were no statistically significant differences in the mass of [ f]mk-9470 injected between the adolescent and adult cohort ( mean  sem : 64.8  1.5  pm and 66.2  2.9  pm , resp . no statistical differences were found in the injected doses ( i d ) ( t(10 ) = 0.56 , p = 0.59 ) between the adolescents ( 8.22  2.07  mbq ) and the adults ( 7.02  0.59  mbq ) . animal weights were found to be significantly different ( t(10 ) = 18.16 , p < 0.0001 ) between adolescent ( 148  9  g ) and adult animals ( 382  9  g ) , and pearson 's correlation showed that weight was strongly and negatively correlated to % id4060  ( r = 0.921 , p < 0.0001 ) . two - way ancova ( age  region ) controlling for weight showed a significant main effect of age ( f(1,109 ) = 4.95 , p = 0.028 ) with adults having higher cb1r absolute binding compared to adolescents ( + 44.4% over 11 voi ) ( figure 2(b ) ) . a significant effect of region was also found ( f(10,109 ) = 2.41 , p = 0.012 ) . no interaction was observed between age and region ( f(10,109 ) = 0.84 , p = 0.59 ) . table 1 presents cb1r absolute binding levels in adolescents and adults before ( unadjusted values ) and after controlling for animal body weight ( adjusted values ) . two - way anova ( age  region ) showed a statistically significant main effect of age ( f(1,99 ) = 17.323 , p < 0.0001 ) with the adults having higher cb1r binding than the adolescents ( figure 4 ) . a significant main effect of region ( f(10,99 ) = 140.1 , p < 0.0001 ) was also found . no interaction between age and region ( f(10,99 ) = 1.62 , p = 0.113 ) was observed . the significant main effect of age was further analysed by lsd post hoc tests revealing that cb1r - specific binding was significantly higher in the adults compared to adolescents in the frontal cortex ( + 23.4% ; p = 0.024 ) , the cortex ( + 27.1% ; p = 0.020 ) , the hippocampus ( + 15.4% ; 0.018 ) , and the cerebellum ( + 15.2% , p = 0.002 ) ( table 2 and figure 4 ) . correlations were not statistically significant between absolute cb1r binding evaluated with [ f]mk-9470 in vivo and specific cb1r binding calculated with [ h]cp55,940 in vitro , ( r = 0.1816 , p = 0.41 ) . in the present study , we used two complementary techniques to examine potential developmental differences in cb1r binding in the brain of adolescent and adult rats . after controlling for body weight , cb1r absolute binding measured in vivo with pet and [ f]mk-9470 was significantly higher in the adult animals compared to adolescents over 11 brain regions . noteworthy , the percentage of increase observed in the adult compared to the adolescent cohort with the 2 complementary techniques was not of the same magnitude ( 44% in vivo versus 11% in vitro over the 11 regions of interest ) , and no significant correlation was found between the data obtained with the two techniques . the comparison between in vitro and in vivo results ( no correlation ) and the apparent discrepancies may relate to a number of factors . the percentage of injected dose ( % i d ) that we used here gives an absolute index of binding in vivo . this means that it reflects specific and nonspecific binding in the brain , radioligand present in the brain blood circulation , and possible radioactive metabolites crossing the blood - brain barrier . we did not calculate standardised uptake values ( absolute index normalising for weight ) because it would have biased our results as our two groups had significantly different weight means . also , the absence of a brain region devoid of cb1r prevented us from implementing a simplified reference tissue model . we chose to use an atlas - based analysis of our data , with predefined voi , over a statistical parametric mapping approach because we wanted to compare the same regions with the in vivo and in vitro methodologies . to our knowledge , the metabolism of [ f]mk-9470 in the male ( adult and adolescent ) rat brain has not been assessed ; therefore , the presence of active metabolites that cross the blood - brain barrier can not be ruled out . indeed , a metabolite is likely to cross the blood - brain barrier of adult female wistar rats ( casteels et al . , oral communication ) . radiometabolites produced in adults ( but not in adolescents ) could potentially cross the blood brain barrier , affect the % i d we calculated , and in turn contribute to the uniform regional distribution of the pet radioligand we observed in adults compared to adolescents ( figures 1 and 2 ) . differences in radioligand present in the brain blood circulation ( e.g. , difference in blood flow ) between the adolescent and adult cohorts that would have affected our measures can not be ruled out either . to be in line with previous studies in rats with [ f]mk-9470 [ 3537 ] , we have used the last 20  min of a 60  min acquisition period ( 40 to 60  min ) for quantification purposes . a recent study however indicated that the distribution volume ( vt ) of [ f]mk-9470 as quantitative outcome evaluated by full kinetic modelling was reasonably correlated with standardised uptake values between 60 and 80  min ( casteels et al . longer acquisitions periods ( at least 80  min ) in future studies using this radioligand would ensure that equilibrium is reached . the second factor that could explain discrepancies between in vitro and in vivo results is the drug phenotype . indeed , [ f]mk-9470 is an inverse agonist at cb1r , whereas [ h]cp55,940 is an agonist at both cb1r and cb2r [ 54 , 55 ] . the concentration of cb2r in the rat brain is supposed to be small in comparison to cb1r [ 3 , 56 , 57 ] . inverse agonists will preferentially bind to receptors uncoupled from their g - protein , whereas agonists will preferentially bind to receptors that are coupled to their g - protein . this means that in vivo we would have preferentially bound cb1r uncoupled to their g - protein , whereas in vitro the g - protein - coupled ones would have been targeted . in vitro assays typically reflect all receptors that are available to bind to radioligand , whereas in vivo , only a subset of these receptors are available to bind to radioligand since some may be compartmentalised , some in a low affinity state and some occupied by endogenous ligand . finally , another factor affecting the comparison between in vitro and in vivo measures is the difference in concentration of the radioligand used . theory of pet experiment is based upon the injection of a radioligand at tracer concentration that is not supposed to trigger any biological effect . in order to meet this requirement , the radioligand should not bind to more than 510% of the total receptors concentration ( bmax ) . based on previously reported bmax in rat brain ( 0.51.1  pmol / mg prot ) , we calculated that the mass of ligand needed to be approximately 0.10.7  nmoles . on the other hand , quantitative in vitro autoradiography studies need saturation of the available binding sites ( at least 3 times greater than the kd ) . thus , by saturating a different proportion of receptors in vivo and in vitro , differential outcomes must be cautiously interpreted . our main results , showing an increase in cb1r in adults ( pnd 7072 ) compared to adolescents ( pnd 3537 ) in vivo and in vitro , are in accordance with in vitro studies that have looked at cb1r expression over time in development . belue and collaborators   have found significant regional increases in the numbers of cb1r ( bmax ) in the developing rat brain ( pnd 0 , 7 , 14 , 21 , and 60 ) using in vitro autoradiography with [ h]cp55,940 . although cb1r density was not measured during adolescence , this study suggested that cb1r binding continuously increased until the maximum adult level was reached at pnd 60 . they observed that cortical regions ( mainly posterior cortex ) and hippocampus showed a statistically significant increase in binding between pnd 21 and pnd 60 . according to the authors , the increase in cb1r could be an indication of either an increased differentiation of neurones into cells harbouring cb1r or an induction of the expression of cb1r in cells already differentiated . another in vitro study using the same radioligand ( [ h]cp55,940 ) showed that cb1rs are transiently expressed in white matter areas during embryonic and early postnatal periods , progressively  shift  to their adult localization at pnd 30 , and increase between pnd 30 and adulthood in the hippocampus , nucleus accumbens , and cerebral cortex . in addition , ellgren et al .   reported an increase in cb1r protein expression in the nucleus accumbens shell and no changes in prefrontal cortex between mid-(pnd 38 ) and late-(pnd 49 ) adolescence . in humans , an in vitro study found an increase in cb1r density between children / infant age ( n = 5 , 3 months to 8 years old ) and adults ( n = 5 , 22 to 73 years old ) in frontal cortex , hippocampus ca1 and dg , caudate putamen , globus pallidus , and cerebellum . interestingly , a recent pet study using [ f]mk-9470 found an increase in cb1r binding in the basal ganglia , lateral temporal cortex , and limbic system of aged female but not male humans . another pet study using [ c]omar in healthy males showed an age - associated decline in cb1r volume of distribution that was significant in globus pallidus only . to allow comparison with other studies from our group [ 46 , 51 ] , we chose to evaluate the ontogeny of cb1r in adolescent and adult male rodents . recent experiments have shown that female wistar rats presented a high ~3539% intersubject variability in cb1r binding evaluated as [ f]mk-9470 standard uptake values between 60 and 80 minutes ( casteels et al . , oral communication , 2011 ) . intersubject variability in our study with males only was of 17% in the adult group and 18% in the adolescent group . future in vivo animal studies looking at the ontogeny of cb1r in female rats as well as during aging would help in clarifying the relationships between gender , aging , and the endocannabinoid system ( figure 3 ) . in the mammalian brain , synapses and receptors within most regions the first one occurs just before birth , after completion of the brain innervation , and witnesses the apoptosis ( programmed cell death ) of 50% of neurones in order to increase efficiency of synaptic transmission [ 64 , 65 ] . the second one occurs during the periadolescence period with a tremendous overproduction of synapses and receptors followed by their progressive elimination or pruning . this pattern of expression  overproduction followed by elimination  is shared among mammalian brains and part of a fundamental developmental strategy called  functional validation  . teicher et al .   reported an overproduction of d1 and d2 from pnd 25 to 40 followed by a pruning to reach adulthood . we have recently reported higher levels of dopamine d1 and d2 receptors , both serotonin 5ht1a receptor binding and mrna expression , and gabaa receptor binding   in adolescent rats ( pnd 39 ) compared to adults ( pnd 70 ) that is in line with the regressive elimination of synapses and receptors that occurs during the transition from adolescence to adulthood . in contrast , the results of the present study indicate that cb1rs are not undergoing a dramatic elimination between adolescence ( pnd 3537 ) and adulthood ( pnd 7072 ) and continue to increase , at least until pnd 7072 . our study does not rule out the possibility that the cb1rs are undergoing pruning at a later developmental  aging  stage . since a homeostatic and modulatory role is attributed to endocannabinoids , the cb1r upregulation could be related to a compensation of functional losses in other monaminergic or gabaergic systems . in addition , changes in cb1r may reflect changes in endocannabinoid markers such as aea and 2-ag . limited information is available regarding endogenous cannabinoid ligands levels during the transition from adolescence to adulthood ; however , a recent study has shown an increase of aea but not 2-ag levels from early to late adolescence in the prefrontal cortex of the rats . studies looking at the developmental profile of endocannabinoid ligands in different brain regions and their correlations with cb1r levels would help in elucidating the developmental and morphogenic roles of this system during the transition from adolescence to adulthood . the present study demonstrated the feasibility of imaging cb1r in vivo with pet and [ f]mk-9470 in adolescent and adult rats . our results suggest that cb1rs are increased during the transition from adolescence ( pnd 3537 ) to adulthood ( pnd 7072 ) , a pattern that is opposite of most other neuroreceptor systems that have already started undergoing pruning during this time window . availability of new radioligands such as [ f]mk-9470 in combination with pet would offer a unique opportunity to gain insights into the role of the endocannabinoid system during critical stages of development using longitudinal and within - subjects experimental designs and understand the consequences of its alterations after pharmacological challenges as well as in neurodevelopmental animal models of psychosis .
despite the important role of cannabinoid cb1 receptors ( cb1r ) in brain development , little is known about their status during adolescence , a critical period for both the development of psychosis and for initiation to substance abuse . in the present study , we assessed the ontogeny of cb1r in adolescent and adult rats in vivo using positron emission tomography with [ 18f]mk-9470 . 
 analysis of covariance ( ancova ) to control for body weight that would potentially influence [ 18f]mk-9470 values between the two groups revealed a main effect of age ( f(1,109)=5.0 , p = 0.02 ) on [ 18f]mk-9470 absolute binding ( calculated as percentage of injected dose ) with adult estimated marginal means being higher compared to adolescents amongst 11 brain regions . 
 this finding was confirmed using in vitro autoradiography with [ 3h]cp55,940 ( f(10,99)=140.1 , p < 0.0001 ) . 
 this ontogenetic pattern , suggesting increase of cb1r during the transition from adolescence to adulthood , is the opposite of most other neuroreceptor systems undergoing pruning during this period .