Individuals with Attention-Deficit/Hyperactive Disorder (ADHD) have unexplained difficulties on tasks requiring speeded processing of colored stimuli. Color vision mechanisms, particularly short-wavelength (blue-yellow) pathways, are highly sensitive to various diseases, toxins and drugs that alter dopaminergic neurotransmission. Thus, slow color processing might reflect subtle impairments in the perceptual encoding stage of stimulus color, which arise from hypodopaminergic functioning.1) Color perception of blue-yellow (but not red-green) stimuli is impaired in ADHD as a result of deficient retinal dopamine; 2) Impairments in the blue-yellow color mechanism in ADHD contribute to poor performance on speeded color naming tasks that include a substantial proportion of blue-yellow stimuli; and 3) Methylphenidate increases central dopamine and is also believed to increase retinal dopamine, thereby normalizing blue-yellow color perception, which in turn improves performance on the speeded color naming tasks.Requires three approaches, including:1) direct assessment of color perception in individuals with ADHD to determine whether blue-yellow color perception is selectively impaired; 2) determination of relationship between performance on neuropsychological tasks requiring speeded color processing and color perception; and 3) randomized, controlled pharmacological intervention with stimulant medication to examine the effects of enhancing central dopamine on color perception and task performanceIf substantiated, the findings of color perception problems would necessitate a re-consideration of current neuropsychological models of attention-deficit/hyperactivity disorder, guide psycho-education, academic instruction, and require consideration of stimulus color in many of the widely used neuropsychological tests. Attention-Deficit/Hyperactivity Disorder (ADHD) is a common and impairing childhood-onset disorder characterized by inattention, hyperactivity, and impulsiveness that affects 3–7% of school-aged children and tends to persist into adulthood . ConvergTo date, there is no adequate explanation to account for either the observed color processing problems in ADHD or their selective amelioration by psychostimulant medication. We propose that the color processing problems may reflect subtle detrimental effects of dopaminergic deficiency in the central nervous system on retinal dopamine, which in turn impedes the efficiency of the short-wavelength (blue-cone) color vision mechanism as well as of other visual functions. Prior to elaborating on this hypothesis, we first review the evidence of problems in color processing associated with ADHD and the existing explanatory accounts. We then outline possible empirical approaches to test this hypothesis, and discuss the theoretical and clinical significance of the hypothesis should it be substantiated.perceptual deficit in selection of visual stimuli on the basis of color (red versus blue) as well as in later semantic stages of visual selective attention [Several lines of evidence indicate impaired performance on tasks requiring rapid and/or continuous processing of colored stimuli in ADHD. For example, in a seminal study on rapid automatized naming speed, Denckla  describettention .Currently, two classes of explanation  are discernible in the literature for the observed problems in rapid processing of colored stimuli in individuals with ADHD. These theoretical accounts are not mutually exclusive, but rather they overlap and reflect perspectives taken from different research approaches and different levels of measurement.Psychological accounts attribute slow color naming to developmental immaturity. For example, the development of color naming appears to be much more difficult for young children than naming shapes or animals -27. AlsoAn existing neurobiological account of color processing problems in ADHD associates poor color naming or poor color processing performance to alterations in the underlying neural substrate. For example, slower RAN color naming speed  was found to be related to smaller anterior superior white matter volumes (both right and left hemispheres) in ADHD compared to healthy controls . DecreasCurrent explanations referring to poor sustained attention, problems in set shifting or semantic processing, or differences in white matter volumes associated with ADHD can not easily account for the dissociation between rapid naming of colored stimuli and rapid naming of letters, digits, or words. Nor can they adequately account for the indications that color processing problems appear to be greater for yellow and blue stimuli. Moreover, from a developmental perspective, the neurodevelopmental immaturity hypothesis would predict greater problems in rapid color naming in children with ADHD compared to adolescents, with possible attenuation of the deficits in adults with ADHD. To date this hypothesis has not been tested directly, but meta-analyses of cognitive deficits in children, adolescents, and young adults with ADHD indicate color naming deficits of moderate to large effect size (d = .58–.62) across the life span with no evidence of age-related changes ,40.Color perception is controlled, at least in part, by retinal dopaminergic neurons . We propTo present the detailed hypothesis, we first provide the necessary background on color perception and the role of retinal dopamine, then argue how hypo-dopaminergic functioning in ADHD will give rise to impairments in the tritan color mechanism, which in turn will influence speeded color naming on tasks involving a substantial proportion of blue and yellow stimuli.Color perception is based on the three cone photoreceptor types maximally sensitive to long, middle, and short wavelengths in the perceived light spectrum that constitute two functionally and anatomically distinct systems at the retina and lateral geniculate nucleus; a 'red-green' system with a foveal specialization in which long and middle wavelength cone signals are antagonistic, and a 'blue-yellow' pathway in which short wavelength cones are opposed by a combined signals from long and middle wavelength cones without such a foveal overrepresentation . Normal Most color perception defects  are congenital and arise from altered sensitivity defects of the L and M cones. By contrast to these red-green color vision deficits, which respectively affect 2% and 6% of the male population, congenital defects in S-cone sensitivity are rare (about 0.01%) but affect both sexes equally . AcquireColor perception problems, particularly involving the blue-yellow (tritan) mechanism, have also been linked with alterations in retinal dopamine: dopamine is a major neurotransmitter in the mammalian retina ,53. DopaAlterations in the level of retinal dopamine are reflected particularly in deficits in the short-wavelength chromatic pathway that is responsible for blue-yellow color discrimination, a system that appears to be especially vulnerable to the effects of disorders and drugs ,50,56-58The fundamental mechanisms causing a specific retinal impairment of color discrimination along the blue/yellow axis in dopaminergic disorders and acquired dyschromatopsias remain unclear. Short wavelength sensitive cones may be more fragile than long and medium wavelength sensitive cones or their relative scarcity and anatomical distribution may be responsible for the greater vulnerability of the blue-yellow perception by alterations of the dopaminergic system ,47,53,69Hypo-dopaminergic functioning has been postulated in ADHD ,70 and aWe propose that the observed impairments in individuals with ADHD on tasks requiring speeded color processing of blue-yellow stimuli might be attributable in part to hypo-functioning of both central and retinal dopamine. Critical to this hypothesis is the premise that alterations in central and retinal dopamine occur in parallel . To our knowledge there is no direct evidence that this is the case. Rather, we draw inferences from the following evidence: 1) cerebrospinal concentrations of a metabolite of CNS dopamine, homovanillic acid, correlates positively with electroretinogram blue-cone amplitude ; 2) in PBLUE in yellow ink) decrease the strength of Stroop interference compared to non-opponent color pairs  [According to this retinal dopaminergic hypothesis, deficits in blue-yellow color perception result in poor performance on many of the standard neuropsychological tasks  that include a substantial proportion of blue-yellow stimuli. Thus poor task performance may reflect subtle impairments in color vision as well as, or instead of, impairments in higher-order cognitive function. Consistent with this prediction are the recent findings that individual differences in color perception influence performance on the classic Stroop color-word task and that incongruent opponent color pairs  . A neurared ink) . Also, ired ink) . The prered ink) .Stimulant-induced increases in the availability of dopamine would be expected to be reflected in retinal dopaminergic tone and thus have therapeutic effects on the short-wavelength mechanism. Thus, methylphenidate would be expected to improve visual functioning, including the speed of color processing and naming. Indeed, there is preliminary evidence of beneficial effects of methylphenidate on color naming in children with ADHD ,23.Alterations in short-wavelengths mechanism may be gender-related. Notably, estrogen has been found to have a modulatory influence on dopamine activity  and a feThe limited data available on developmental changes in retinal and central dopaminergic mechanisms, color perception, and color naming, suggest that the retinal dopamine hypothesis will likely hold for children, adolescents, and adults with ADHD. For example, animal research indicates that dopaminergic neurons are among the first neurochemical systems to appear in the developing retina and that the neural retina and dopaminergic system interact closely in a two-way manner throughout developmental period . It is oOn the other hand, the increasing efficiency of the mature (adult) brain permits the development and use of compensatory strategies. Thus, adults with ADHD, although impaired in color perception relative to healthy peers, may be able to call upon compensatory attentional strategies to enhance performance on tasks requiring rapid perception of blue and yellow stimuli, thereby exhibiting better performance than children or adolescents with ADHD.Three strategies are proposed to provide a rigorous test of the hypothesis: 1) direct assessment of color vision pathways in individuals with ADHD versus a comparison group of healthy peers; 2) evaluation of the relationship between color vision (particularly for blue-yellow stimuli) and performance on tasks requiring speeded color processing; and 3) investigation of stimulant effects on both color vision and performance on those tasks in individuals with ADHD. Color vision can be assessed directly using clinical measures that are sensitive to problems with stimuli along the blue-yellow axis -85 as weConvergent findings from empirical investigations and neural network simulation support the hypothesis that color perception contributes to performance on neuropsychological tasks requiring speeded color naming ,71. PrelIf substantiated in larger well-controlled studies, evidence that blue-yellow color perception problems contribute to poor performance on neuropsychological tasks of executive function requiring speeded color processing, would necessitate careful consideration to stimulus color when interpreting performance on many of the standard neuropsychological tests. Also, evidence of specific impairments in blue-yellow color perception  would necessitate a reconsideration of current neuropsychological models of ADHD, which posit the core deficits to be in higher-order executive functioning and not at the level of sensory and perceptual processing. From a clinical perspective, this retinal-dopaminergic hypothesis might indicate the need to include a visual examination in the assessment of ADHD, and raises the possibility that electroretinogram blue cone amplitudes may also be a possible neurobiologic marker related to central dopamine function in ADHD, as well as in cocaine-dependent patients .ADHD: Attention-Deficit/Hyperactivity DisorderRAN: Rapid Automatized Naming TestVEP: Visual evoked potentialThe author(s) declare that they have no competing interests.RT developed the hypothesis and prepared the initial draft of the manuscript; TB refined it and conducted a preliminary test of it; DG conducted an in-depth literature search. All authors helped revising the manuscript and have read and approved the final manuscript.


Since vaccinations are an effective prevention tool for maintaining the health of society, the monitoring of immunization coverage allows us to identify areas where disease outbreaks are likely to occur, and possibly assist us in predicting future outbreaks. The aim of this study is the investigation of the coverage achieved for compulsory  and recommended  vaccinations between 1998 and 2002 in the municipality of Bologna and the identification of the subjects not complying with compulsory and recommended vaccinations.The statistics regarding vaccinal coverage were elaborated from the data supplied by the Bologna vaccinal registration system (1998–2000) and the IPV4 program (2001–2002). To calculate the coverage for compulsory vaccinations and cases of non-compliance reference was made to the protocol drawn up by the Emilia Romagna Regional Administration. The reasons for non-compliance were divided into various categoriesrd year and for anti-dyphteria, tetanus, pertussis at 6 years. The frequency of subjects with total non-compliance (i.e. those who have not begun any compulsory vaccinations by the age of one year) is generally higher in Bologna than in the region, with a slight increase in 2002 (2.52% and 1.06% in the city and the region respectively). The frequency of the anti-measles vaccination is higher than that of mumps and rubella, which means that the single vaccine, as opposed to the combined MMR (measles-mumps-rubella) was still being used in the period in question.In Bologna the levels of immunization for the four compulsory vaccinations are satisfactory: over 95% children completed the vaccinal cycle, receiving the booster for anti-polio foreseen in their 3The most common reason for non compliance is objection of parents and is probably due to reduction of certain diseases or anxiety about the possible risks.In Bologna the frequency of children aged 12 and 24 months who have achieved compulsory vaccination varied, in 2002, between 95% and 98%. As regards recommended vaccinations the percentage of coverage against Haemophilus influenzae is 93.3%, while the levels for measles, mumps and pertussis range from 84% to approx. 92%. Although these percentages are higher if compared to those obtained by other Italian regions, every effort should be made to strengthen the aspects that lead to a successful vaccinal strategy. Vaccinations are an effective prevention tool for maintaining the health of the individual and society as a whole. In Italy, however, certain diseases that are preventable with vaccines whose efficiency and safety have been fully tested and proven, still present a public health issue.Monitoring immunization coverage allows us to identify areas where disease outbreaks are likely to occur, and possibly assist us in predicting future outbreaks. Also, monitoring can help keep immunization coverage high. One of the main aims of public health services throughout the world is therefore to obtain reliable data about the level of immunization with a view to supporting the implementation and monitoring of vaccination programs .Vaccinal coverage is normally referred to in terms of the percentage of subjects vaccinated out of the total target population. In pediatric age the target population is made up of the cohorts of children who were under one year old as of 31 December in a given locality, while the vaccinal state is defined as the number of doses required to bring about immunity. To determine vaccinal coverage, the WHO , on a yearly basis, considers the percentage of children who have received three doses of vaccines against diphtheria, tetanus, polio, hepatitis B, pertussis and Haemophilus influenzae (Hib), and one dose of anti-measles, mumps and rubella (MMR) by the age of 24 months.In Italy the vaccination coverage of newborns  is estimated every year by the regional authorities. The data are then sent to the Ministry of Health. In order to have a more detailed picture of the national situation , a cluster survey, called ICONA, was made in 1998 by means of interviews carried out at the subjects' home. The results showed a considerable difference between the coverage achieved for compulsory  and for recommended  vaccines .After five years the national survey was repeated ICONA 2003) [ [3], witIn the wake of the approval of the National Health Plan (NHP) to eliminate measles and congenital Rubella (13 November 2003) and considering the current attempt to overcome the existing division between compulsory and recommended vaccinations, we decided to undertake a study with the following objectives:1. To investigate the coverage achieved for compulsory and recommended vaccinations between 1998 (the year when the NHP set the objective of 95% coverage)  and 20022. To identify the categories of subjects not complying with compulsory and recommended vaccinationsIn Bologna the vaccination of subjects under 18 years of age is carried out almost exclusively by the Community Pediatric Service . In the current make-up of Bologna Health Services, this operative Unit is situated within the Department of Primary Care, and is divided into two districts: Bologna East and Bologna West.Until 2000 the registration of vaccinations was carried out by the Public Health Department by means of a computerized system run by the Municipality of Bologna. The office had access to registration records (they could check the issuing of vaccination certificates), but not to the more detailed data processing system .Details of births or new residents under the age of 6 years were passed on to the subject's local pediatric consultation centre by the municipal Registry Office, with the issuing of a vaccination booklet. At the end of the year the Office printed a list of non-compliances and sent it to the respective Pediatric Centres for a further check. Finally, full details were sent yearly to the Regional offices by the Community Pediatric Service. During the year health workers could check the extent of vaccinal coverage only by means of the paper documents (files) sent at the same time as the vaccination booklets. The only way that the employees who registered the vaccinations on the files could find out about subjects who had moved or died was by going directly to the municipal registry office."for the purposes of the management of vaccinal registration, the municipality periodically transmits details of population variations  to the Local Health Offices" .Since the beginning of 2001 the registration of vaccinations has been run exclusively by the Local Health Services of the city of Bologna, using a special computer program , Alongside the installation of the vaccinal registration program, a project was launched to set up an on-line link between the various centres administrating the vaccines at pediatric age. This allows the immediate registration of vaccinal data at the moment of vaccination and the possibility to consult in real time the vaccinal situation of each subject, including details of any migration and access to the so-called "notes" .The great advantage of the revised system is that it is now possible to monitor vaccinal coverage at any time, obtain information pertaining to moves or deaths, and contact residents who have not been vaccinated.The statistics regarding vaccinal coverage were elaborated from the data supplied by the vaccinal registration system of the Municipality of Bologna (1998–2000) and the IPV4 program (2001–2002).To calculate the coverage for compulsory vaccinations and cases of non-compliance reference was made to the protocol drawn up by the Emilia Romagna Regional Administration:residents who reached one year during the year of reference and received at least 2 doses of each compulsory vaccine by the age of 12 months;residents who reached two years during the year of reference and received at least 3 doses of each compulsory vaccine by the age of 24 months.subjects who did not receive any compulsory vaccination by the age of one year or received only one dose of the compulsory vaccinations.The reasons for non-compliance are categorized matching those used in the collection of regional data for coverage:- living abroad: children resident in Bologna but living abroad for a long period- refusals or objectors: children whose parents were explicitly against at least one compulsory vaccination- immigrants: children from other regions of Italy or from other countries- delays: children who received at least one vaccination after the pre-established dates- other: children with health contra-indications, momentarily exempt for health reasons, not traceable or without fixed abode.For all recommended vaccinations  the extent of coverage was calculated at 24 months and also at 13 years for anti-measles and anti-rubella.The levels of immunization for the four compulsory vaccinations (taken as a whole) achieved during the five-year period in question are shown in table Data for the assessment of each separate vaccination are available only from 2001 .In the city of Bologna, as in the region taken as a whole, over 95% children completed the vaccinal cycle, receiving the booster for anti-polio foreseen in their 3The frequency of subjects with total non-compliance (i.e. those who have not begun any compulsory vaccinations by the age of one year) is generally higher in Bologna  than in the region, with a slight increase in 2002 (2.52% and 1.06% in the city and the region respectively) table .The vaccination registration system provides us with information about the reasons for non-compliance for the compulsory vaccinations: some can be attributed to the conditions of the children , others to the parents . At 12 months the most common reason for non-compliance is "parents who object to and refuse the vaccinations" (1.01–1.56% of children resident in Bologna) table . At 24 mData regarding the coverage at 24 months for the recommended vaccinations confirm the decline in the vaccination of children in Bologna as compared to the rest of the region table , a trendThe underlying trend for such coverage tends to vary over the five-year period. The level of protection against pertussis remains more or less constant, even though it falls a little below the regional average. However, a steady rise can be seen for the other recommended vaccinations, especially evident in the case of anti-Hib . The notable increase in anti-Hib vaccination can probably be attributed to the availability of a combined vaccine.At 12 months a similar consistent increase in the number of subjects receiving the anti-Hib vaccination can again be seen in Bologna (from 26.7% to 91.6%) and throughout the whole of the region (from 40.9% to 96.0%). In Bologna the target percentage of 95% coverage for the recommended vaccinations has yet to be reached.Table The principal infant vaccinations have now been included in the Essential Levels of Assistance that the Region must guarantee free of charge to all citizens. Despite this, however, the levels of coverage for compulsory and recommended vaccinations have not yet reached homogeneous levels. In the municipality of Bologna the frequency of children aged 12 and 24 months who have been vaccinated against DTP, tetanus, polio, hepatitis B and pertussis varied, in 2002, between 95% and 98%. As far as the recommended vaccinations are concerned, the percentage of coverage against Hib is 93.3%, while the levels for measles, mumps and pertussis range from 84% to approx. 92%. Although these percentages are generally lower than the regional levels, they can nevertheless be considered fairly good if compared to those obtained by certain other Italian regions ,7 the raThe data for Bologna in 2002 show a general rise in the level of coverage for recommended vaccinations in comparison to previous years, especially for anti-Hib. However, even though an increase can be seen in the levels of anti-measles and rubella and the situation in Bologna is on average better than that reported by the rest of the country, the target coverage of 95% envisaged by the National Plan for the Elimination of Measles and Rubella has not yet been achieved. The objectives set by the Plan, to be reached by 2007, are:- to reduce and maintain the incidence of Congenital Rubella Syndrome (CRS) at levels below 1 case in every 100.000 live-borns.- to achieve and maintain the elimination of measles at a national level, interrupting its indigenous transmission.It should be stressed that the inadequate vaccinal coverage achieved for anti-measles does not interrupt the circulation of the virus and may even bring about a greater probability of complications and outbreaks; it prolongs the interval between epidemics and delays the outbreaks until later in adult life, as presumably occurred in the Campania region . It has As far as the data on non-compliance (compulsory vaccinations) is concerned, it is interesting to note that the frequency is higher in Bologna than the regional average and, in particular, Bologna East has a higher percentage than Bologna West both at 12 and 24 months. The percentage of total non-compliance is also greater in the East district than the West. Furthermore, the number of children in this category is steadily increasing: passing from 0.88% in 1998  to 2.52% in 2002 .The most common reason for non compliance is objection of parents. Objection to compulsory vaccinations, especially in Bologna , is on the rise, even though vaccinal coverage remains above 95%. There is an increasing tendency not to comply with vaccinal obligations, probably because the disappearance  or limited incidence  of certain diseases has led some people to believe that the relative vaccines are obsolete and there is less concern about the risk of contracting infectious diseases. However, recent reports  indicateA further reason for the refusal of vaccination is the anxiety about the possible risks associated with receiving the vaccines. In other words there is the pressure, especially in industrialised countries, from organised groups opposing the practice of vaccination and spreading alarmist and often inaccurate information ,14. For In Bologna the frequency of children aged 12 and 24 months who have achieved compulsory vaccination varied, in 2002, between 95% and 98%; As regards recommended vaccinations the percentage of coverage against Hib is 93.3%, while the levels for measles, mumps and pertussis range from 84% to approx. 92%. Although these percentages are higher if compared to those obtained by other Italian regions, it is necessary to take into account that in Italy, during the 1998–2002 period the mean number of cases of measles, rubella and mumps was 5456, 3685 and 24663 respectively ,20.The author(s) declare that they have no competing interests.SS participated in the design and coordination of the study, in the interpretation of results, in revising and editing the manuscript. RR participated in the conception of the study, in the interpretation of the results and in writing the first draft. IR collected the data and performed statistical analyses. FZ participated in the interpretation of the results, the writing of the manuscript and assisted with the statistical analyses and surveysThe pre-publication history for this paper can be accessed here:


Drosophila PGRPs have lost this enzymatic activity and serve as microbe sensors through peptidoglycan recognition. Other PGRP family members, such as Drosophila PGRP-SC1 or mammalian PGRP-L, have conserved the amidase function and are able to cleave peptidoglycan in vitro. However, the contribution of these amidase PGRPs to host defense in vivo has remained elusive so far. Using an RNA-interference approach, we addressed the function of two PGRPs with amidase activity in the Drosophila immune response. We observed that PGRP-SC1/2–depleted flies present a specific over-activation of the IMD (immune deficiency) signaling pathway after bacterial challenge. Our data suggest that these proteins act in the larval gut to prevent activation of this pathway following bacterial ingestion. We further show that a strict control of IMD-pathway activation is essential to prevent bacteria-induced developmental defects and larval death.Peptidoglycan-recognition proteins (PGRPs) are evolutionarily conserved molecules that are structurally related to bacterial amidases. Several  Drosophila immune pathways. The contribution of PGRPs which have maintained enzymatic activity to host defense has remained elusive so far. Here, the authors investigate in vivo data on the role of Drosophila PGRPs with enzymatic activity. Their results suggest that these proteins are required in the larval gut to negatively regulate the immune response, thus preventing bacterially induced developmental defects and death.It has long been known that the mammalian immune response needs to be kept under tight control. Responses that are delayed or of insufficient vigor can lead to a failure to control infection. However, excessive or inappropriate inflammation can be harmful or event fatal. Using the fruit fly as a model, evidence is presented that such an immuno-modulation is also essential in invertebrates and is mediated by peptidoglycan-recognition proteins (PGRPs). PGRPs are evolutionarily conserved molecules derived from enzymes that cleave bacterial peptidoglycan. It has been shown previously that some PGRPs have lost this enzymatic activity and function as sensors of bacteria upstream of the  Drosophila involves rapid synthesis of small-sized cationic peptides by the fat body [The antimicrobial host defense of fat body ,2. Thesefat body ,4. The sfat body ,6. An effat body , PGRP-SDfat body , and grafat body ,10. On tfat body –13 and Pfat body ,15.Drosophila to discriminate between gram-positive and gram-negative bacteria relies on the specific recognition of different forms of peptidoglycan (PGN) [N-acetylglucosamine and N-acetylmuramic acid connected via stem peptides [l-lysine in the third position of the stem peptide (Lys-PGN) and are recognized by PGRP-SA and PGRP-SD [The ability of an (PGN) ,17. Bactpeptides   [N-acetylmuramic acid and the l-alanine in the stem peptide of PGN. In some of these PGRP molecules, the amidase function is conserved, as documented for Drosophila PGRP-SC1 [PGRP-L mutant mice show no immune phenotype. On the basis of in vitro experiments, it has been proposed that amidase PGRPs could act as scavenging molecules. Indeed, degradation of PGN by Drosophila PGRP-SC1b markedly reduces its immuno-stimulatory potency in cell-culture assays [Drosophila PGRPs with amidase activity. We show that two PGRPs with described amidase activity, namely PGRP-SC1 and PGRP-SC2, control the intensity of the Drosophila immune response. We also present evidence that in the absence of such a control, infection-induced IMD-pathway over-activation can cause developmental defects and larval death.PGRPs form a large group of proteins present in insects and mammals –25, whic(NAMLAA) . These bPGRP-SC1  and PGRPPGRP-SC1 , and forPGRP-SC1 ,30. In oPGRP-SC1 . On the e assays . We repoDrosophila with reduced PGRP-SC1 and PGRP-SC2 levels. The Drosophila genome contains a cluster of two tandemly arranged PGRP-SC1 loci (named a and b) and a single PGRP-SC2 locus [PGRP-SC1 mRNAs differ by only three nucleotides and translate into a unique protein that is 70% identical to the PGRP-SC2 polypeptide  exhibited a 90% reduction of both PGRP-SC1 and PGRP-SC2 mRNA levels  with gram-negative bacteria and measured diptericin transcript levels as a conventional readout for IMD-pathway activation. Six hours after infection with Enterobacter cloacae or Escherichia coli (a time-point that corresponds to the peak of diptericin mRNA kinetics in wild-type flies), no differences were noted between the levels of diptericin mRNA in UAS iPGRP-SC and DaGal4;UAS iPGRP-SC flies , it remained high in DaGal4;UAS iPGRP-SC flies. Similar results were obtained with another m-DAP-PGN–containing bacteria (Bacillus subtilis), although the differences between UAS iPGRP-SC and DaGal4;UAS iPGRP-SC could be detected as soon as 6 h after infection  and an independent UAS iPGRP-SC insertion generated identical results (data not shown). When gram-positive bacteria were used as inducers, two of them  did not activate the IMD pathway above clean injury levels. A third one, Staphylococcus aureus, triggered slightly higher diptericin-transcription levels in PGRP-SC–depleted flies than in controls, although the induction was mild, as expected from gram-positive bacteria  are not able to compensate for the absence of PGRP-SCs in vivo. In the absence of loss-of-function mutants for each of the three PGRP-SC genes, we can obviously not rule out the possibility that only one or two of them are responsible for the observed phenotype. To obtain additional proof that the effects obtained in PGRP-SC–depleted flies were specific, we performed similar experiments in flies in which the non-amidase bacterial receptor PGRP-SA had been depleted by RNA interference. Whereas DaGal4;UAS iPGRP-SA flies showed an expected reduced ability to respond to infection by the gram-positive M. luteus, their response to E. coli remained wild-type . This suggests that the over-response observed in PGRP-SC–depleted flies did not result from an uncontrolled bacterial growth in the hemolymph, and that the role of PGRP-SC proteins in vivo is not to scavenge bacteria from the circulating hemolymph.Since PGRP-SC1 has been proposed to act as a scavenger molecule , we test E. coli C and 2D.drosomycin by gram-positive or gram-negative bacteria  were similar in UAS iPGRP-SC and DaGal4;UAS iPGRP-SC flies. This difference between the role of PGRP-SC1/2 on Toll- and IMD-pathway activation could reflect functional redundancy between amidases for Lys-type PGN which might not exist for m-DAP-type PGN-cleaving enzyme. Alternatively, this could pertain to the difference in the mode of activation of the transmembrane receptors upstream of each pathway.It is notable that non-enzymatic PGRPs, such as PGRP-SA, PGRP-LC, or PGRP-LE are able to discriminate between Lys-type and m-DAP-type PGN –17 Figu. Recent PGRP-SC1 and PGRP-SC2 genes are transcribed in almost identical patterns and mostly in the gut cells [PGRP-SC1 and PGRP-SC2 mRNA and represents the main site of PGRP-SC amidase synthesis at this developmental stage  [CadGal4 is not a very strong driver[PGRP SC1/2 mRNA levels in the gut were not as pronounced in CadGal4;UAS iPGRP-SC than in DaGal4;UAS iPGRP-SC larvae  . Consistng driver, we noteC larvae A. Howeve on Toll-pathway activation [picky flies are mutant only for PGRP-SC1a and PGRP-SC1b, whereas PGRP-SC2 is also affected in our PGRP-SC–depleted flies.Taken together, our data provide novel insights into the physiological roles of PGRPs in tivation , a phenoSalmonella typhimurium–infected flies produce a tumor necrosis factor (TNF)–like cytokine which has been shown to be damaging for the host [2O2) must be tightly balanced to avoid larval lethality [Our results indicate that the gut is the main tissue in which the regulation by PGRP-SC proteins is taking place. However, the fact that IMD-pathway over-activation was also detected when bacteria were introduced directly into the circulating hemolymph suggests that these secreted proteins could also be present in the blood or in the circulating hemocytes. We further show that in the absence of a control of the immune response, infection can lead to developmental defects or death by over-activation of the immune pathway. Interestingly, recent reports indicate that other immune-induced pathways can have a harmful effect on fly survival. the host . In addiethality . In thisethality .diptericin expression) and as a JNK  kinase [Drosophila IMD pathway and the developmental defects which we observed; in particular, a role of the apoptosis pathways in this process should be considered. Finally, it will be of interest to investigate whether amidases or other PGN-modifying enzymes are involved in modulating bacteria-induced immune response in mammals. In this respect, it is intriguing that one human PGRP family member (PGRP-Iβ) is expressed in the esophagus [The results presented here are consistent with previous data showing that over-expression of some components of the IMD pathway are larval lethal. However, the molecular mechanisms by which over-activation of the IMD pathway leads to lethality remain unknown. A number of observations may provide clues about this issue: (i) several components of the IMD pathway are homologous to mammalian proteins involved in signaling through the TNF receptor, a pathway known to trigger apoptosis ; and (ii) kinase . It is s) kinase . Furthersophagus , which eE. coli, M. luteus, E. carotovora carotovora 15, B. subtilis, E. cloacae, E. faecalis, S. aureus, and E. coli AmpR.The following microorganisms were used: key1IKKγ is a loss-of-function mutation allele. ΔPGRP-LCE is a complete deletion of the PGRP-LC locus. Flies carrying either of these mutations are unable to activate the IMD pathwayseml. PGRP-SA is a point-mutation null allele which prevents Toll-pathway activation by some bacteria. All these alleles have been previously described [ and Caudal Gal4  are transgenic strains in which all  or only the gut  cells express the yeast Gal4 transcription factor. In HsGal4 flies, the production of the Gal4 protein is inducible by a heat pulse. DaGal4;UAS iPGRP-SC are strains in which all the cells produced PGRP-SC dsRNA targeting the endogenous PGRP-SC transcript to degradation by RNA interference. Drosophila strains produced include: PGRP-SA, PGRP-SC1a, PGRP-SC1b, PGRP-SC2, PGRP-SD, PGRP-LB, PGRP-LC, PGRP-LE, IKKγ, and RpL32.escribed . Daughteg for 10 min at room temperature. The supernatant was discarded and the pellet was resuspended in fresh Luria-Bertani (LB) media. Cell suspensions were serially diluted in PBS, and the concentration of cells was determined by optical-density measurement. Flies were anaesthetized with CO2 and were infected by pricking the dorsal thorax with a thin tungsten needle that had previously been dipped into cultures of the appropriate bacterial strains.Cells from overnight bacterial cultures were recovered by centrifugation at 3,000 E. coli or with E. cloacae. Twenty-four hours later, flies were homogenized in 400 μl of LB media, and various dilutions were spread on LB plates containing ampicillin for E. coli (50 μg/ml) or on LB plates for E. cloacae. The number of colony-forming units per fly was determined through overnight growth on plates. For survival experiments, 25 flies of each tested genotype were pricked and transferred each day into new vials with fresh medium. Survival assays were repeated four times.In bacterial-growth experiments, five 4-d-old adults were infected with UAS iPGRP-SC plasmid was constructed by inserting a 558-bp PCR fragment corresponding to the PGRP-SC1a coding sequence flanked by BamHI and NheI sites between the NheI–BamHI sites (sense) and the XbaI–BglII sites (antisense), respectively, into the RNAi vector [UAS iPGRP-SA by inserting a 612-bp fragment corresponding to the PGRP-SA coding sequence; primers were as follows: forward 5′- GGGGGATCCATGCAGCCGGTTCGATTCGGA-3′, reverse 5′- GGGG CTAGCTCCGA TGGAAGTTTATCCACA-3′. For plasmid UAS PGRP-SC1b, the wild-type PGRP-SC1b cDNA was obtained by PCR using the EST GH07464  as a template. This fragment was then sub-cloned into the pUAST vector. Primers were as follows: forward 5′-GGGGGAATTCATGGTTTCCAAAGTGGCTCT-3′, reverse 5′-GGGGTCTAGACACTCTAACCAGACCAGTGG-3′. After sequencing, the constructs were injected into 1118w embryos.The i vector ; primersdiptericin transcription by real-time Q-PCR 6 and 24 h after infection. For observation of developmental defects, larvae were transferred to standard cornmeal-agar medium and incubated at 25 °C until hatching.Wandering third-instar larvae were placed in a tube containing a mixture of a concentrated overnight bacterial culture with 5 % sucrose, and were incubated for 30 min at 25 °C. For controls, larvae were incubated in LB broth supplemented by 5% sucrose. Larvae were then transferred to grape-juice plates and incubated at 25 °C. Larvae were monitored for PGRP-SC1, forward 5′-aagcgatcgtcaactattacagc-3′, reverse 5′-gagagccactttggaaacCA-3′; PGRP-SC1b (for over-expression checking), forward 5′-AGCTTCCTGGGCAACTACAA-3′, reverse 5′-GAGATCATGTTCGGCTCCAG-3′; PGRP-SC2, forward 5′-TGACCATCATCTCCAAGTCG-3′, reverse 5′-CAGCGAGGTCTTGCTCGT-3′; PGRP-SA, forward 5′-GCTTCGTTGGGACTCCACTA-3′; reverse 5′-CGTGTGATGGATG ACCACAT-3′. RpL32, diptericin, drosomycin, and PGRP-SD primers are described in [Real-time Q-PCR was performed as described . Primersribed in ,11.Imaginal discs were dissected in PBS, incubated in a 5 μg/ml AO solution for 1 min, rinsed three times for 5 min in fresh PBS, and mounted in glycerol.Figure S1(578 KB PDF)Click here for additional data file.Figure S2(1.4 MB PDF)Click here for additional data file.http://flybase.bio.indiana.edu) accession numbers for the Drosophila strains produced include IKKγ (CG16910), PGRP-LB (CG14704), PGRP-LC (CG4432), PGRP-LE (CG8995), PGRP-SA (CG11709), PGRP-SC1a (CG14746), PGRP-SC1b (CG8577), PGRP-SC2 (CG14745), PGRP-SD (CG7496), and RpL32 (CG7939).The FlyBase (http://www.expasy.org/enzyme) accession number for NAMLAA is EC3.5.1.28.The Swiss-Prot Enzyme Nomenclature database (


Attention Deficit Hyperactivity Disorder (ADHD) is a prevalent and highly heritable childhood disorder. The dopamine D4 receptor (DRD4) gene has shown a genetic association with ADHD in Caucasian populations with meta-analysis indicating a small but significant effect across datasets. It remains uncertain whether this association can be generalised to non-Caucasian ethnic groups. Here we investigate two markers within the DRD4 gene in a Taiwanese population, the exon 3 variable number tandem repeat (VNTR) and a 5' 120 base-pair duplication.Within-family transmission disequilibrium tests of association of the 5' 120 base-pair duplication, and exon 3 VNTR in a Taiwanese population.No evidence of association of ADHD with either polymorphism in this population was observed.The DRD4 gene markers investigated were not found to be associated with ADHD in this Taiwanese sample. Further work in Taiwanese and other Asian populations will therefore be required to establish whether the reports of association of DRD4 genetic variants in Caucasian samples can be generalised to Asian populations. Attention Deficit Hyperactivity Disorder (ADHD) is one of the most prevalent and heritable childhood behavioural disorders. Progress in identifying some of the genes involved in ADHD susceptibility has been relatively fruitful over the past decade by screening genetic variants that lie within or close to genes that regulate neurotransmitter systems, particularly dopamine pathway genes . One of Other genetic variants in the DRD4 5'-regulatory region have also been reported to be associated with ADHD. Of particular interest is a 120-bp duplication that has been associated with ADHD in two studies ,26 althoBoth the 120 bp duplication ,31 and tlong repeat alleles  in a Han Chinese population. This association, however, was not supported using within family tests of association and the association was not specific to any single allele. More recent data from Leung and colleagues does however support this hypothesis by observing a significant increase in prevalence of the 2-repeat allele in a Han Chinese ADHD sample in comparison to a control sample (p = 0.015) )Fourth, it has been noted that for within family tests of association, dropped genotypes or genotyping errors for risk alleles with population frequency of less than 0.5 may give rise to false negative findings . AlthougOne final explanation may be due to hypothesized reciprocity between ADHD associations with the dopamine transporter and DRD4. As hypothesised by Swanson and colleagues  the riskThese results taken together with other reports of Taiwanese and Asian samples find no consistency in the association between genetic variants of DRD4 and ADHD. Further work in Taiwanese and other Asian populations will therefore be required to establish whether the reports of association of DRD4 genetic variants in Caucasian samples can be generalised to Asian populations.DRD4 – Dopamine Receptor D4, ADHD – Attention Deficit Hyperactivity Disorder, TDT – Transmission Disequilibrium Test, HHRR – Haplotype-based Haplotype Relative Risk, VNTR – Variable Number Tandem Repeat.The author(s) declare that they have no competing interests.Keeley Brookes and Philip Asherson selected the markers for analysis. Keeley Brookes carried out the genotyping of the population and performed statistical analysis under the supervision of Philip Asherson. DNA was provided by Chih-Ken Chen, Yu-Shu Huang and Yu-Yu Wu. DNA was organised by Xiaohui Xu. Philip Asherson directed the study.The pre-publication history for this paper can be accessed here:


The behaviour of children with Attention-Deficit / Hyperactivity Disorder is often described as highly variable, in addition to being hyperactive, impulsive and inattentive. One reason might be that they do not acquire complete and functional sequences of behaviour. The dynamic developmental theory of ADHD proposes that reinforcement and extinction processes are inefficient because of hypofunctioning dopamine systems, resulting in a narrower time window for associating antecedent stimuli and behaviour with its consequences. One effect of this may be that the learning of behavioural sequences is delayed, and that only short behavioural sequences are acquired in ADHD. The present study investigated acquisition of response sequences in the behaviour of children with ADHD.Fifteen boys with ADHD and thirteen boys without, all aged between 6–9 yr, completed a computerized task presented as a game with two squares on the screen. One square was associated with reinforcement. The task required responses by the computer mouse under reinforcement contingencies of variable interval schedules. Reinforcers were cartoon pictures and small trinkets. Measures related to response location  and to response timing  were analyzed by autocorrelations of consecutive responses across five lags. Acquired response sequences were defined as predictable responding shown by high explained variance.Children with ADHD acquired shorter response sequences than comparison children on the measures related to response location. None of the groups showed any predictability in response timing. Response sequencing on the measure related to the discriminative stimulus was highly related to parent scores on a rating scale for ADHD symptoms.The findings suggest that children with ADHD have problems with learning long sequences of behaviour, particularly related to response location. Problems with learning long behavioural sequences may ultimately lead to deficient development of verbally governed behaviour and self control. The study represents a new approach to analyzing the moment-to-moment dynamics of behaviour, and provides support for the theory that reinforcement processes are altered in ADHD. Attention-Deficit/Hyperactivity Disorder (ADHD)  is a behThere is some support for the notion that ADHD behaviour may be characterized by hampered acquisition of complete and functional sequences of behaviour. First, children with ADHD did not perform sequences of arm movements as one functional unit, but were slower, showed greater variability in movement timing, and demonstrated longer inter-segment intervals than children without ADHD, who appeared to program the entire arm movements and executed the sequence as one functional unit that was temporally coordinated . The chiThe DDT suggests that dysfunctioning reinforcement and extinction processes can explain why symptomatic ADHD behaviour is acquired through dynamic interaction between the child and the environment throughout development ,3. ReinfOn a behavioural level, reinforcers select responses by increasing the probability of repeating responses that produce reinforcers. Reinforcement as a process operates within a limited time window from the occurrence of the behaviour to the perception of the consequences of this behaviour. Altered reinforcement processes in ADHD may be described as a narrower time window than normal for associating behaviour with its consequences. A narrow time window may theoretically be depicted as a shorter and steeper delay-of-reinforcement gradient  Figure . The delThere may be many behavioural consequences of a shorter delay gradient in ADHD  schedules, where responses result in a reinforcer after varying time intervals. With VI schedules a possible confusion of reinforcement effects with timing problems is avoided, as reinforcers are presented at unpredictable times . All mouThe present study analyzed response data from 28 boys in the age range of 6:2–9:0 (yr:mo), 15 of whom had an ADHD diagnosis and 13 were healthy controls. These boys represented the young age group from the previous study. The older children (aged 9.5–12 yr) from that study were not included, as the comparison group showed to be inadequate  [Participants in the ADHD group were referred from different clinical sources  and were included if they met the following criteria: 1) DSM-IV diagnosis of ADHD, of either three subcategories; 2) Full Scale IQ of at least 80; 3) no evidence of neurological disorder, psychosis, or pervasive developmental disorder; and 4) not taking any medication within the last 48 hours prior to testing. A diagnosis was confirmed after thorough clinical evaluation. Eight of the children had ADHD combined type, six had ADHD hyperactive / impulsive type, and one child had ADHD inattentive type. In addition, inclusion in the ADHD group warranted a score at or above the 95e (DBRS)  home or Comparison children were recruited from schools in urban and suburban areas of Oslo, the capital of Norway. Inclusion criteria were the same as for the ADHD group, except that no DSM-IV diagnosis should be present. In addition, they had to score below the sub-clinical range on the DBRS.Intellectual ability was assessed by screening all children with four subtests  of the WISC-R  demogra.The study was approved of by the Regional Medical Committee of Research Ethics. The parents of all the participants received written information about the study and gave written consent for their child to take part. All children were tested in quiet rooms, using the same tasks, apparatus, and test procedures . If you want to point, you can click with one of these buttons (experimenter points to the mouse buttons). You may talk while you are playing, but I will not answer any questions about the game. I will sit back here and write a little while you play. Do you understand your task? You may start now."The task was designed as a computer game, and was presented to the child with the following instruction (translated from Norwegian): The task was run on a Toshiba Pentium 300 CDT laptop connected to a colour monitor  schedules of reinforcement, where responses may produce reinforcers after the passage of varying time intervals , were usThere were two sessions, each of five segments. Each segment consisted of four short and four long intervals, and was terminated by a response and the delivery of a reinforcer. In the first session, the child would see a total of 40 reinforcers (cartoons). In the second session, the child received a small tangible reinforcer  in addition to the cartoon picture. This was done in order to maintain reinforcer value. The entire task, including instruction, break between sessions, and a final, short interview with the child, took less than 30 min to complete.Data were recorded by the laptop. Response side (left or right), response coordinates , and interresponse times (IRTs) were the recorded dependent measures. The individual IRT distributions were highly skewed with a long tail towards long IRTs. IRTs were therefore normalized by log transformations prior to analysis (logIRT = log10 (IRT/1000 + 0.001)).side response pattern, i.e., whether consecutive responses were on the left or right side of the screen. Highly predictable responding would probably be related to the side where the correct target was positioned, and would thus be a complementary measure of stimulus control. Likewise, low predictability implies that responses are equally distributed on the two sides and is thus also a measure of low stimulus control. Next, due to the fact that reinforcers affect more responses than the one that produces it .Data from the VI 20s condition was used to study response sequences, as the short schedule only allowed for one or a few responses before a reinforcer was delivered. Predictability of responses over long sequences could theoretically be found according to different aspects of the behaviour, and in order to explore the different possibilities we computed three measures related to the spatial dimension and one measure related to timing. The first measure was a general t Figure , predictThe ordering of responses spatially and temporally was assessed by autocorrelations. Autocorrelations  of each measure were correlations of consecutive values over five lags . The autocorrelations were computed for each individual over sessions and segments. We predicted that the behaviour of children with ADHD would be characterized by lower autocorrelations and that their autocorrelation curves across lags would be steeper compared to the behaviour of healthy comparison children.Data were analyzed by means of SPSS 11.0 for Windows (SPSS) and Statistica 6.1  program Demographic and diagnostic measures of the ADHD and the comparison group were tested with two-tailed t-tests for equality of means and are displayed in Table In general, acquisition of predictable response sequences was found in the spatial measures and not in the temporal measure. The ADHD group had significantly lower autocorrelations than the comparison group on two of the three spatial measures. Both groups had very low autocorrelations related to response timing. There was no Session effect on the three spatial measures, but the main effect of Lag was significant for all four measures. Results of the planned analyses with ANOVA and MANOVA are shown in Table 2 > 0.22, median R2 = 0.26), while it was in the upper range for the comparison group  . Highly predictable responding over lags would imply that behaviour was ordered in sequences related to side. Less variance was accounted for in the ADHD group than in the comparison group. For the ADHD group, explained variance in the first lag across segments was low , indicating that there was rather low predictability from one response to the next. For the comparison group, explained variance in the first lag was higher .This measure assessed to what degree the children tended to respond in any predictable pattern in terms of the distance between responses, anchored to the centre of the square that responses were placed within (whether it was the correct response target or not). Highly predictable responding would imply that behaviour was ordered in sequences of similar distances between responses. Again, the explained variance for the ADHD group was lower than for the comparison group Figure . ExplainThere were significant main effects of Group and Lag, but not of Session and Segment Table . A signi2 > 0.27; median R2 = 0.40), and slightly higher for the comparison group  . The significant main effect of Session showed that explained variance was lower in the second session than in the first, particularly for the comparison group. The main effect of Lag was significant, while the main effect of Segment was not. There was a significant interaction effect between Group and Session, showing that while the ADHD group had lower explained variance than the comparison group in the first session, the comparison group had lower explained variance than the ADHD group in the second session. The significant interaction between Group, Session, and Lag in the ANOVA was not confirmed by the MANOVA.The development of patterns in response timing was investigated by means of consecutive interresponse times (IRTs). Explained variance of the first lag was generally very low and not significantly different between the groups  argues that a shortened delay gradient probably relates more to the hyperactive / impulsive and the combined subtypes of ADHD than to the inattentive subtype . The preside response pattern and square response pattern, but not according to target response pattern or timing response pattern. This was supported by the high correlations between variance accounted for in consecutive response lags of these two spatial measures and the scores on both hyperactive/impulsive and inattentive clinical dimensions  of the screen, irrespective of on which side the correct target was displayed. The significant group difference implied a highly predictable pattern in the comparison group, indicating that these children varied their responding between sides almost only to the extent that the correct target square switched sides on the screen. This was supported by their mean percent correct responding at 87% during stable state [The le state , demonstle state . The comHowever, there was a drop in explained variance in the comparison group at the end of each session  in the first lag of the two groups indicates high predictability from response n to n+1 when in the correct square, while the ADHD group varied more on consecutive responses as indicated by the significant interaction between group and lag.The  the DDT , predictside response pattern. Therefore, it might be argued that the reduced predictability found in both square response pattern and target response pattern is a consequence of larger arm movements in the ADHD group because of more varied responding from side to side, rather than an effect of inefficient reinforcement of response placement within a square. The present analysis did not allow identifying predictability of response placements when consecutive responding was within the same square, which involves smaller movements. However, disentangling the variability related to larger arm movements and the variability related to decreased stimulus control  may not be feasible. Further, it may be speculated that, since the striatum, which is involved in the planning and execution of motor actions, and the nucleus accumbens, which is involved in learning and reinforcement, both receive important dopaminergic afferents, these functional processes may both be impaired in an individual with ADHD [Increased variability is a consequence of reduced stimulus control. This is seen in the ADHD group in terms of the ith ADHD .Acquisition of functional behavioural sequences may be related to processes involved in habit learning. Habit learning is characterized by a transition from response-consequence associations that are flexible and sensitive to reinforcement devaluation, to stimulus-response associations that are less flexible and sensitive (e.g. ). Thus, timing response pattern. Speculatively, this lack of a predictable timing pattern could be related to the visuo-spatial nature of the task, both in terms of response alternatives  and in terms of the reinforcer. There is some evidence that striatal neurons involved in sequential habit learning may encode visuo-spatial information rather than temporal [Explained variance of first lag (and following lags) was too low to conclude that there was any predictability in IRTs. Thus, there was no temporal .The present findings suggest that the learning of coherent and predictable behavioural sequences will be difficult in children with ADHD, probably because the time window available for reinforcers to work is narrower in ADHD compared to normal. Skilled performance is characterized by responses emitted with brief interresponse intervals, smooth transitions between responses, and efficient coordination of consecutive movements so that a whole sequence is conducted in a predictable manner (e.g. ). ReinfoOther interpretations are possible, however. It has been suggested that the length of delay gradients may be dependent on working memory (WM) capacity because the ability to relate sensory information to responses and reinforcing stimuli seems to correlate with ongoing neuronal activity in prefrontal cortex . BehavioThere might be alternative motivational explanations (than a shorter delay gradient) for the behaviour observed in the ADHD group. Rather than a reduced effect of positive reinforcement , the behaviour may be a result of reduced negative control . However, non-compliance would have involved refusal to complete the task, which none of the children did, and most of them even reported that the task was fun. In addition, the instruction was non-directive, excluding the possibility of non-compliance to any instructed response pattern. A group of children aged 9–12 yr also participated in the original study, but the results of the comparison group indicated that their responding was mainly compliant and not controlled by reinforcers, as shown by lack of schedule control . The behThe present study provides new insights into the recently started discussion of intra-individual ADHD-related variability . CastelSome limitations of the present study could be improved in future studies. First, the findings need to be replicated in a larger sample of children. New samples, including girls and children of other ages, need to be analyzed in order to verify if this behavioural style is general to ADHD, whether it may be identified at an earlier age, and whether it continues to constitute an important factor in the behaviour of older children. Children with other psychopathologies should be added in order to establish the specificity of this learning style. Second, the present task may be criticized for its lack of ecological validity. On the other hand, the present task may have been optimal for studying ordering and sequencing of responses, as no earlier experience or learning history would interfere. In the future, a wide range of serial or sequential tasks should be investigated because the dynamic developmental theory of ADHD predicts that the presently-observed learning style should be found in the behaviour of people with ADHD across tasks and activities. Finally, various serial tasks could be conducted during brain imaging in order to investigate the brain areas involved.The present findings provide support for the dynamic developmental theory of ADHD predicting that a short and steep delay-of-reinforcement gradient will result in fewer responses in a predictable sequence than when the delay gradient is normal . The hypThe present study represents a new approach to analyzing the micro-dynamics of consecutive responses in a moment-to-moment manner. A majority of the responses were emitted with IRT < 1s. The study of behavioural processes and their environmental correlates may thus approach the time-scale of brain processes and we may get closer to directly measure brain-behavioural interactions. In this perspective, concepts like working memory, inhibition, and even timing problems may be too wide concepts to identify the underlying learning style, because a learning style characterized by inefficient chunking or development of entire behavioural sequences may be manifest in different domains in different individuals. One might speculate that sequencing of motor action and sequencing of speech and thought may be of the same functional origin . Thus, The author(s) declare that they have no competing interests.HA participated in the development of the study design, development of the reinforcement task used, carried out the data collection, prepared the data, performed the statistical analyses, and wrote the manuscript. TS participated in the development of the study design, development of the reinforcement task, wrote the programs for statistical analyses, participated in data analyses, read the manuscript and approved the final draft.


Limited data from positron emission tomography (PET) studies of subjects with attention-deficit/hyperactivity disorder (ADHD) indicate alterations in brain dopamine neurotransmission. However, these studies have used conventional univariate approaches that are less sensitive to detect complex interactions that may exist between different brain dopamine pathways and individual symptoms of ADHD. We aimed to investigate these potential interactions in adolescents with ADHD.11C]-DOPA to map dopamine presynaptic function in various cortical, striatal and midbrain regions in a group of 8 male adolescents with ADHD and 6 age matched controls. To evaluate the interactions between the studied brain regions, multivariate statistical methods were used.We used a 3D PET scan to measure utilization of native L-[11C]-DOPA utilization in adolescent with ADHD as compared to control subjects, especially in subcortical regions. This pattern of dopaminergic activity was correlated specifically with symptoms of inattention.Abnormal dopaminergic function was found in multiple brain regions of patients with ADHD. A main finding was lower L-[Dopamine signalling in the brain plays an important modulatory role in a variety of motor and cognitive functions. We have identified region-specific functional abnormalities in dopaminergic function, which may help better account for the symptoms of ADHD. Attention deficit hyperactivity disorder (ADHD) is a common neurodevelopmental disorder characterized by difficulties associated with attention, motor hyperactivity and impulsivity. The inappropriate behaviour is related to deficits of executive functions including working memory, response inhibition and motor timing -7. Neura18F]fluorodopa in two PET studies [18F]fluorodopa in the prefrontal cortex in comparison with control subjects. In a second study, they instead reported an increased level of [18F]fluorodopa in the right midbrain in adolescents with ADHD. Three independent groups, using SPECT, have reported increased density of the dopamine transporter (DAT) in the striatum in adults and children with ADHD DOPA, , which is transported into the presynaptic neurons. There it is converted by the enzyme DOPA decarboxylase to L-[11C]dopamine and stored in storage vesicles. Therefore, PET data obtained by L-[11C]DOPA reflect DOPA decarboxylase activity and dopamine storage processes. The properties of the radiotracer L-[11C]DOPA differ in several important aspects from its more commonly used fluorine labelled analogue [11C]DOPA is state-dependently modulated by drugs that affect dopaminergic systems and is thus also a measure of the functional state of the dopaminergic neurons [In the present study we hypothesized that the behavioural problems of subjects with ADHD are associated with altered dopamine synthesis. We used the radio L-[analogue . The tur neurons -29.11C]DOPA for multivariate analysis have many potential applications, such as in the understanding of drug effects DOPA Ki values, required further analysis to determine whether there were indeed differences between the adolescents with and without ADHD. Thus, the data sets were explored using the statistical technique partial least squares regression (PLS), which is closely related to principal components analysis (PCA) DOPA Ki values. In most brain areas in the subjects with ADHD the rate of dopamine synthesis was lower than in the healthy controls, with the values being particularly low in the subcortical regions. The low synthesis in the subcortical areas correlates with the severity of the attention deficit symptoms, assessed according to the DSM-IV. These findings are in good agreement with extensive experimental and clinical research supporting the notion that dopamine system plays a modulatory role in attention processes, while impulse control is more linked to other neurotransmitter systems, e.g., serotonin DOPA Ki values suggest that children with ADHD differ from controls in their metabolism of DOPA over a range of cortical and subcortical regions. It is most likely that the low values reflect aberrations in the presynaptic synthesis and release of dopamine. These results support earlier theories that a hypofunction of the dopamine system underlies the behavioural symptoms in ADHD [18F]fluorodopa have demonstrated that there is a decreased uptake in the frontal cortex and an increased uptake in the midbrain [11C]DOPA. The differences might be attributable to the different specificity of the radioligands. As a radiotracer, L-[11C]DOPA seems to detect changes in the functional tone in the dopaminergic systems more readily than its fluorine labelled analogue PE2I, that binds selectively to the dopamine transporter [Other SPECT and PET studies of dopamine transporter binding in ADHD have reported increased binding capacity in the striatum of adults and children with ADHD -23,47. Hnsporter ,49. We cnsporter . From ounsporter . In somensporter . Recentlnsporter . The datregion-specific pattern emerges. The most profound decreases are seen in subcortical areas  and different radioligands, as well as differences in the age and medication history of the populations studied. A fundamental problem exists in addition to this, which is that the ADHD construct is based on behavioural symptoms and therefore includes heterogeneous populations of children with different cognitive and behavioural dysfunctions. This functional variation is probably associated with certain molecular variations in specific brain areas that may be large in relation to a more subtle general difference between ADHD subjects and controls. In the work reported here, it was only possible to reveal a general pathological pattern of the complex dopamine system that is shared by subjects with core ADHD symptoms by conducting multivariate analysis on data obtained by PET.Attempts to create a single theory explaining ADHD have failed so far. This is not remarkable in view of the present study, revealing the complexity of the interaction between the dopamine synthesis in the various subcortical and cortical areas and between this interactive pattern and the symptoms of attention deficit. We have only studied one component in the complex chain of dopamine signalling. It is obvious that the neurobiological mechanisms underlying ADHD are numerous and interactive. The challenge for future research is to characterize various subtypes of ADHD and, in particular, to determine the underlying cognitive dysfunctions and their interaction with various neural and neurotransmitter systems.R-Caudd, R-Caudint, R-Caudv are the dorsal, intermediate and ventral sections of the right caudate nucleus; R-Putd, R-Putint, R-Putd are the dorsal, intermediate and ventral sections of right putamen; R-Mpfd, R-Mpfi, R-Mpfv are the dorsal, intermediate and ventral sections of the right mesial prefrontal cortex; R-Pfd, R-Pfi, R-Pfv are the dorsal, intermediate and ventral sections of the right dorsolateral prefrontal cortex; R-Acc is the right nucleus accumbens; R-Mbrain is the right midbrain; the prefix L indicates the same areas on the left side.HF is shareholder and scientific adviser for COGMED Cognitive Medical Systems AB, which develops cognitive training programmes aimed to improve working memory function.HF participated in the conception, design and writing of the study. EF participated in the planning of the study recruited patients and performed the clinical assessments. SW and NW performed post processing of PET data and the statistical analyses. JW participated in the design of the study, acquisition and analyses of PET data and helped to draft the manuscript. All authors read and approved the final manuscript.


In their article, “Exposures to Environmental Toxicants and Attention Deficit Hyperactivity Disorder [ADHD] in U.S. Children,” p < 0.2) for inclusion of factors and variables associated with ADHD on univariate analysis was generous compared with the commonly used 0.1 or 0.05, and very close to the p-value of the lead–ADHD association of 0.19. The lead–ADHD relationship also exhibited a significant monotonic dose response, so it would have been helpful to know how the authors developed their exposure metric. Why, for example, were quintiles selected rather than another interval scheme, and why were they not of uniform size? Was the reported dose response the only model considered, or did the authors investigate other models, as some have done in studying the relationship of lead exposure and cognition (In the logistic model used by ognition ?


The purpose of this study was to examine the association of exposures to tobacco smoke and environmental lead with attention deficit hyperactivity disorder (ADHD).Data were obtained from the National Health and Nutrition Examination Survey 1999–2002. Prenatal and postnatal tobacco exposure was based on parent report; lead exposure was measured using blood lead concentration. ADHD was defined as having current stimulant medication use and parent report of ADHD diagnosed by a doctor or health professional.p = 0.22). If causally linked, these data suggest that prenatal tobacco exposure accounts for 270,000 excess cases of ADHD, and lead exposure accounts for 290,000 excess cases of ADHD in U.S. children.Of 4,704 children 4–15 years of age, 4.2% were reported to have ADHD and stimulant medication use, equivalent to 1.8 million children in the United States. In multivariable analysis, prenatal tobacco exposure  and higher blood lead concentration  were significantly associated with ADHD. Postnatal tobacco smoke exposure was not associated with ADHD (OR = 0.6; 95% CI, 0.3–1.3; We conclude that exposure to prenatal tobacco and environmental lead are risk factors for ADHD in U.S. children. Attention deficit hyperactivity disorder (ADHD) is one of the most common childhood disorders, with an estimated prevalence ranging from 3 to 8% . ChildreNumerous studies have found a significant association between prenatal environmental tobacco smoke (ETS) exposure and ADHD or ADHD-related behaviors, even after controlling for postnatal ETS exposures and familial psychopathology . In caseEnvironmental lead exposure, measured in blood or dentin, has been associated with higher rates of inattention and impulsivity , 2001. AStudies investigating the effect of environmental exposures on neurobehavioral outcomes have been complicated by evidence from both animal and human studies that toxicants may have differing effects in male and female subjects . The preThe purpose of this study was to explore the relationship between exposure to ETS and environmental lead with ADHD using a large nationally representative sample of children. We also explored whether sex modifies the relationships between these neurotoxicants and ADHD. Finally we provide estimates of population attributable fraction of prenatal ETS exposure and lead exposure for ADHD in U.S. children.The data for this analysis came from the National Health and Nutrition Examination Survey (NHANES), conducted from 1999 to 2002. The NHANES is a cross-sectional household survey of the noninstitutionalized civilian population. NHANES used a complex, multistage probability sampling design, with oversampling of children (< 5 years of age), Mexican Americans, and non-Hispanic blacks . DetailsThe primary dependent variables that we used to assess ADHD status were parent report of a diagnosis of ADHD and stimulant medication use. Parent-reported ADHD was based on the parent or guardian’s response to the question “Has a doctor or health professional ever told you that your child had attention deficit disorder?” To improve the specificity of the diagnosis of parent-reported ADHD, we used parent-reported ADHD and stimulant medication use as the main outcome. Stimulant medication use was based on National Drug Codes 03700, 17900, 39500, and 82000 . These cWe used parent report to measure children’s exposure to tobacco products. Measurement of prenatal ETS exposure consisted of the question “Did the child’s biological mother smoke at any time while she was pregnant with him/her?” No information on the quantity or brand of cigarettes smoked during pregnancy was collected. We assessed postnatal ETS exposure using parent-reported exposure to household ETS by asking “Does anyone who lives here smoke cigarettes, cigars, or pipes anywhere inside this home?” In a secondary analysis, we explored using the child’s serum cotinine concentration, a metabolite of nicotine, as a biomarker of ETS exposure . All chiWe determined blood lead concentration by graphite furnace atomic absorption spectrophotometry . The limWe examined multiple covariates and potential confounders for the association of prenatal ETS exposure and lead exposure with ADHD. Demographic variables included the child’s age, sex, race, and socioeconomic status [as measured by poverty-to-income ratio (PIR)]. PIR is the ratio of family income to the poverty threshold for the year of the interview. Children with PIR values < 1 are considered to be living below the poverty level. Health insurance coverage was also included as a covariate. In addition, a review of the literature suggested that preschool attendance, low birth weight, and ferritin levels (an indicator of iron status) should be considered potential confounders because of their prior documented associations with child behavioral problems and environmental toxicants . Child’sp < 0.2) in bivariable analyses were included in the logistic regression analyses. Postnatal ETS exposure was forced into all multivariable models. Because NICU admission and birth weight may be acting as intervening variables on the pathway from prenatal ETS exposure to ADHD, we included these two variables in secondary analyses to examine whether their inclusion altered our findings . Regression diagnostics were conducted to identify influential observations and collinearity. Influential observations were excluded from analyses to examine whether their inclusion altered the results (n = 9). The exclusion of these outliers did not significantly influence the estimates of prenatal ETS exposure or environmental lead exposure. After excluding outliers and children without routine access to health care, 4,704 children were available for bivariate analyses; of those children, 3,879 had complete data available for multivariate analyses.Among children 4–15 years of age, 5,171 were available for analysis. We found that children who did not have routine access to health care were unlikely to be treated with stimulant medications; therefore, we excluded these children from the main analysis , exposed females, unexposed males, and exposed males . We alsoAnalyses were performed using the SUDAAN statistical package to account for the multistage, complex sampling design . Sample This study was approved by the National Center for Health Statistics Institutional Review Board, Cincinnati Children’s Hospital Medical Center Institutional Review Board, and the University of Milwaukee College of Nursing Institutional Review Board. Consent was obtained from all participants.p = 0.023), preschool attendance (p = 0.003), male sex (p < 0.001), increasing age in years (p < 0.001), and health insurance coverage (p < 0.001) (p = 0.001).Of the 4,704 eligible children 4–15 years of age, 344 (8.2%) had only parent-reported ADHD and 154 (4.3%) reported stimulant medication use, equivalent to 3.8 million and 2.0 million U.S. children and adolescents, respectively. Of the 4,704 children, 135 (4.2% weighted percent) had parent report of both ADHD and stimulant medication use, equivalent to 1.8 million children in the United States. . In biva< 0.001) . Non-Hisp = 0.224). The risk for ADHD was significantly associated with preschool attendance .In multivariable analysis, prenatal ETS exposure and blood lead concentration were significant predictors of ADHD . The adjp = 0.141). Compared with unexposed females, females who were pre-natally exposed to ETS were at a 4.6-fold higher risk for ADHD compared with unexposed females , whereas exposed males were at an almost significant 2-fold higher risk for ADHD than unexposed males  (p = 0.242) or blood lead levels by maternal smoking (p = 0.837).Next, we tested for interactions between sex and prenatal exposure to ETS, sex and blood lead concentration, and prenatal exposure to ETS and blood lead concentration. We did not find a significant interaction between prenatal ETS exposure and sex using a formal interaction term (= 0.073) . There wWe conducted secondary analyses to examine the effects of lead exposure at blood lead levels < 5 μg/dL and to test the stability of our results. When the sample was restricted to children with concurrent blood lead concentrations ≤ 5 μg/dL, there was still a significant association between higher blood lead levels and ADHD. Compared with children in the lowest quintile (nondetectable to 0.7 μg/dL), children with blood lead levels in the highest quintile (2.0–5 μg/dL) had a 4.5-fold  higher risk for ADHD.p = 0.055) and 4.5 , respectively. Consistent with postnatal ETS exposure measured by parent report, postnatal ETS exposure using serum cotinine was not associated with ADHD . Using the same multivariable model, we found that when the ADHD outcome was defined simply by parent report or by stimulant medication use, rather than the combination of the two, the AOR of maternal smoking and blood lead level did not differ appreciably. Finally, there was no substantive change in the relationship of either prenatal ETS exposure or blood lead concentration when we included children without routine access to health care in the model.When birth weight and NICU were added to the primary model, the adjusted OR for prenatal ETS exposure and the fifth quintile of blood lead levels did not change, remaining at 2.2 , corresponding to 270,000 cases of ADHD in children 4–15 years of age . AlthougOverall, 4.2% of children surveyed had parent-reported ADHD and were taking stimulant medication, equivalent to approximately 1.8 million U.S. children in the 4- to 15-year-old population. This rate is consistent with previous estimates of ADHD prevalence, which have ranged from 3 to 8% . Our anaIn this sample, the overall adjusted risk for ADHD was 2.5-fold higher for children exposed prenatally to ETS, which is consistent with previous case–control studies , 1998. PAlthough the difference in risks by sex was not statistically significant, these results suggest that females may be more susceptible to tobacco-associated ADHD. Females exposed prenatally to ETS had a 4.6-fold increased risk of ADHD compared with unexposed females, whereas males exposed prenatally to ETS were at a 2.1-fold increased risk for ADHD compared with unexposed males. When females are exposed prenatally to ETS, their risk of ADHD becomes equivalent to that of unexposed males.Postnatal ETS exposure was not associated with ADHD in this analysis. We used parent report of household ETS and serum cotinine, but neither was associated with parent-reported ADHD. In previous research, investigators found a significant association of current tobacco consumption of the mother and externalizing behavior problems in children using continuous measures of behavior . It is pWe found a significant dose–response relationship of higher blood lead levels and ADHD. Compared with the lowest quintile of blood lead levels, children with blood lead levels > 2.0 μg/dL were at a 4.1-fold increased risk of ADHD. When we limited the analysis to children with blood lead levels ≤ 5 μg/dL, the association between increased blood lead levels and ADHD remained. These results are consistent with previous reports that have found significant associations between blood or dentin lead levels and behavior problems , 2001. OAn interesting finding in this study was the increased likelihood of ADHD for children who attended preschool or child care. Presumably, children who attend preschool or child care are more likely to have problem behaviors noted by staff, thus increasing their chance for referral and diagnosis of ADHD by a health professional. Results from the NICHD child care study also suggest that children who are more aggressive and disobedient are more likely to be placed in child care at younger ages and for longer periods of time . RegardlThis study has several limitations. First, the cross-sectional nature of our data makes it difficult to infer a causal relationship from our observed associations. Second, although some might argue that concurrent blood lead tests are not an adequate biomarker of a child’s lifetime exposure, recent studies indicate that concurrent blood lead level is a stronger predictor of lead-associated IQ decrements than blood lead measured during early childhood . Still, An additional limitation of our cross-sectional data is susceptibility to recall bias. Mothers of children with behavior problems may be more likely to recall gestational intake of potentially harmful substances, such as tobacco, owing to a drive to identify a cause of their child’s disorder. On the other hand, mothers may fail to report tobacco use during pregnancy due to guilt or social stigma . The reliance of our outcome variable on parent report is also a potential limitation. Nevertheless, by combining parent report of ADHD with stimulant medication use for our main outcome, we maximized the likelihood that all children counted as ADHD cases had been evaluated and diagnosed by a medical professional. On the other hand, these criteria required us to limit our analyses to children with routine access to health care services. Excluding children without routine access to health care enhances the specificity of the diagnosis of ADHD, but it also likely underestimates the total number of ADHD cases that are attributable to prenatal ETS exposure and environmental lead exposure.Diagnostic and Statistical Manual of Mental Disorders)-based ADHD diagnostic instrument, which would offer increased specificity to detect behavior problems and specific ADHD subtypes, as well as the possibility of behavioral symptom counts. More continuous measures of ADHD symptoms or of specific underlying neurobehavioral domains may be the most powerful way to detect lead and tobacco effects, because ADHD may represent the tail of a continuum of behavioral effects produced by environmental chemical exposures and other etiologic factors (Using a dichotomous measure of ADHD is less preferable than the administration of a DSM ( factors . However factors .This study confirms the previously observed association of prenatal ETS exposure and ADHD. We also found a significant dose–response relationship between childhood lead exposure and ADHD. This analysis indicates that 270,000 ADHD cases in children 4–15 years of age are attributable to pre-natal ETS exposure, and 290,000 cases of ADHD among U.S. children 4–15 years of age are attributable to environmental lead exposure. The findings of this study underscore the profound behavioral health impact of these prevalent exposures, and highlight the need to strengthen public health efforts to reduce prenatal ETS exposure and childhood lead exposure.