Journal of Psychiatric Research 76 (2016) 121e127
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Chemosensory processing in children with attention-deficit/ hyperactivity disorder Anna Lorenzen a, *, 1, 2, Deborah Scholz-Hehn a, 1, Christian D. Wiesner a, Stephan Wolff b, Til O. Bergmann c, d, e, Thilo van Eimeren f, g, Luisa Lentfer a, Lioba Baving a, Alexander Prehn-Kristensen a a
Department of Child and Adolescent Psychiatry and Psychotherapy, University Hospital Schleswig-Holstein, Niemannsweg 147, 24105, Kiel, Germany Department of Neuroradiology, University Hospital Schleswig-Holstein, Arnold- Heller-Str. 3, 24105, Kiel, Germany Department of Neurology & Stroke, and Hertie Institute for Clinical Brain Research, University of Tübingen, Ottfried-Müller-Str. 25, 72076, Tübingen, Germany d Institute for Medical Psychology and Behavioral Neurobiology, University of Tübingen, Ottfried-Müller-Str. 25, 72076, Tübingen, Germany e Institute of Psychology, Christian-Albrechts University of Kiel, Olshausenstr. 62, 24118, Kiel, Germany f Department of Nuclear Medicine, University Hospital, Kerpenerstr. 62, 50937, Cologne, Germany g Department of Neurology, University Hospital, Kerpenerstr. 62, 50937, Cologne, Germany b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 August 2015 Received in revised form 12 February 2016 Accepted 12 February 2016
Background: In attention-deficit/hyperactivity disorder (ADHD) not only deficits in dopamine-related cognitive functioning have been found but also a lower dopamine-sensitive olfactory threshold. The aim of the present study was to proof that only olfactory but not trigeminal sensitivity is increased in ADHD. Structural magnetic resonance imaging (MRI) was used to show increased olfactory bulb (OB) volume- a structure which is strongly shaped by olfactory performance through the mechanism of neuroplasticity (e.g. synaptogenesis). To elucidate whether cortical mechanisms are involved in altered olfaction in ADHD, functional MRI (fMRI) was introduced. Methods: A total of 18 boys with ADHD and 17 healthy controls (aged 7e12) were included in the study. Olfactory as well as trigeminal detection thresholds were examined. OB sizes were measured by means of structural MRI and an analysis of effective functional (fMRI) coupling of primary olfactory cortex was conducted. The frontal piriform cortex (fPIR) was chosen as seed region because of its importance in processing both trigeminal and olfactory stimuli as well as having profound influence on inner OB-signaling. Results: Increased olfactory sensitivity as well as an increase in OB volume was found in ADHD. There were no group differences in sensitivity towards a trigeminal stimulus. Compared to healthy controls, the fPIR in ADHD was more positively coupled with structures belonging to the salience network during olfactory and, to a lesser extent, during trigeminal stimulation. Conclusions: Olfactory functioning is superior in subjects with ADHD. The observed increase in OB volume may relate to higher olfactory sensitivity in terms of neuroplasticity. During the processing of chemosensory stimuli, the primary olfactory cortex in ADHD is differently coupled to higher cortical structures which might indicate an altered top-down influence on OB structure and function. © 2016 Elsevier Ltd. All rights reserved.
Keywords: ADHD Olfactory processing Trigeminal processing Sensory threshold Olfactory bulb fMRI
* Corresponding author. E-mail addresses: [email protected] (A. Lorenzen), [email protected] (D. Scholz-Hehn), [email protected] (C.D. Wiesner), [email protected] (S. Wolff), [email protected] (T.O. Bergmann), [email protected] (T. van Eimeren), [email protected] (L. Lentfer), [email protected] (L. Baving), a.prehn@ zip-kiel.de (A. Prehn-Kristensen). 1 Anna Lorenzen and Deborah Scholz-Hehn contributed equally to this work. 2 Present address: Experimental Pediatric Neuroimaging, University Children's Hospital, Dept. III, (Pediatric Neurology), Hoppe-Seyler-Str. 1, 72076 Tübingen, Germany. http://dx.doi.org/10.1016/j.jpsychires.2016.02.007 0022-3956/© 2016 Elsevier Ltd. All rights reserved.
1. Introduction Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder with a world-wide prevalence of 5e7% (Willcutt, 2012). Alterations in dopaminergic pathways are thought to be involved in the underlying pathology, which not only leads to symptoms of inattention and/or hyperactivity (American Psychiatric Association, 2000) but possibly also to alterations in
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olfactory processing. It was shown that children with ADHD show higher sensitivity towards olfactory stimuli when compared to both healthy controls (HC) and children with ADHD being treated with methylphenidate (MPH) (Romanos et al., 2008). MPH is regarded as the first-line treatment in ADHD and blocks dopamine reuptake in the mesolimbic system and the prefrontal cortex (PFC) (Heal et al., 2009; Wilens, 2008). The orbitofrontal cortex (OFC), amygdala, hippocampus, and pirifom cortex (PIR) are core structures of the human olfactory system (Benarroch, 2010; Zald and Pardo, 2000). Contrary to all other sensory systems (e.g. trigeminal or visual) olfactory processing does not rely on thalamic gating: The olfactory bulb (OB) is the critical relay in stimulus processing (Kay and Sherman, 2007) and the olfactory threshold is linked to OB size (Buschhüter et al., 2008). The trigeminal system is also involved in the perception of chemosensory stimuli with its free nerve endings extending into the nasal mucosa. It mediates sensations like burning, stinging or cooling via the brainstem and thalamus before reaching the somatosensory/insular cortex and converging with the olfactory system (Hummel and Livermore, 2002). There is strong evidence for lower OB volume being accompanied by reduced olfactory sensitivity in patients suffering from a loss of nigrostriatal dopaminergic neurons (e.g. Parkinson) (Brodoehl et al., 2012; Wang et al., 2011). It remains unclear, however, why ADHD is accompanied with higher olfactory sensitivity. It was proposed that striatal dopaminergic dysregulation may cause a decrease in inner-bulb-mediated inhibition of neuronal signals (Romanos et al., 2008; Schecklmann et al., 2011a,b). They assume an indirect mechanism of decreased neurogenesis of inhibitory dopaminergic interneurons. In addition, there is also direct cortical influence on inner-bulb circuits. Animal studies have shown that so-called centrifugal structures (PIR and anterior olfactory nucleus), which are dopamine sensitive in part (Fallon and Moore, 1978; Ikemoto, 2007), exert tonic inhibitory influence on the OB by dampening neuronal signals (Boyd et al., 2012). Alterations in the cortical dopamine system might lead to a cascade of decreased centrifugal downscaling on the OB transmission, resulting in higher excitation and increased olfactory sensitivity. By means of neuroplastic processes this may cause increased OB volume. To test the hypothesis that children suffering from ADHD show a higher olfactory sensitivity, detection thresholds and OB volumes were measured. By introducing a trigeminal stimulus it was intended to show that higher sensitivity in ADHD is restricted to the olfactory system and not due to unspecific hypersensitivity (Ghanizadeh, 2011). To test the hypothesis of alterations in cortical olfactory circuits exerting direct centrifugal influence on OB, explorative functional magnetic resonance imaging (fMRI) was conducted to evaluate the effective connectivity of PIR during intensity-controlled chemosensory stimulus processing. 2. Methods and materials 2.1. Participants 18 patients with ADHD and 17 healthy controls (HC) aging from 7.9 to 12.8 years were examined. All participants were diagnosed by the Kiddie-SADS-PL, a semi-structured diagnostic interview (Kaufman et al., 1997) according to the DSM-IV classification system. Eleven patients fulfilled ADHD-criteria for the predominantly hyperactive subtype and seven patients fulfilled criteria for the inattentive subtype. Eight patients had a co-morbid oppositional defiant disorder (three of the inattentive and five of the hyperactive subtype). Participants were also screened for psychopathological symptoms by parental ratings based on the Child Behavior
Checklist (CBCL) (Achenbach, 1991). Exclusion criterion for HC was a T-value of 60 in any of the syndrome- and summed scales. Intellectual ability was assessed with the German version of the Culture Fair Intelligence Test (CFT-20) (Weiß, 2006) and the CFT-1 (Weiß and Osterland, 2013). Exclusion criterion was an IQ under 85. Most participants were in prepubescent phase, two patients and two HC were in early puberty as measured with the German version of the Pubertal Development Scale (Watzlawik, 2009). There was no statistical significant group difference in age but a marginally significant difference in IQ. For details see Table 1. Exclusion criteria for all participants were acute or chronic disease of the respiratory tract or usual MRI exclusion criteria. Fifteen patients were MPH-naïve. Three patients had received MPH at least one year ago and for a period not longer than six months. All families gave informed consent. The study was approved by the Ethics Committee of the Medical Faculty of the University of Kiel.
2.2. Chemosensory threshold Individual olfactory as well as trigeminal detection thresholds were assessed using the “two-alternative-staircase-detection”method (Doty et al., 1995). Two serial dilutions were prepared: one with the olfactory stimulus phenylethyl alcohol (PEA, rose-like) and one with the trigeminal stimulus L-menthol (peppermint-like). The chemical preparation and testing procedure were conducted according to Pause et al. (2001). The Substances were diluted 1:2 in propylene glycol (v/v). This stock solution was further diluted in 16 half-decimal logarithmic steps [lowest concentration 1:63000000 (v/v)]. Finally, each of the sixteen brown glass bottles contained 6.5 ml chemical solution. Both tests on olfactory as well as trigeminal thresholds were conducted in the same session separated by a half an hour. The order of the presentation was counterbalanced across the groups. Participants were instructed to decide which of the two bottles smelled more intensive (one containing the chemosensory stimulus and one containing only the solvent). A higher threshold score (in terms of dilution step) corresponded to higher sensitivity. To avoid habituation, there was an intertrial interval (ITI) of 20 s. The procedure took place in an air-conditioned room at a constant 19 C. Participants were not allowed to eat a half an hour before chemosensory testing and were instructed to avoid spicy foods like garlic prior to testing that day.
2.3. Volumetry of OB The MRI measurements were performed with a 3 T Philips Intera Achieva (Best, The Netherlands) using a 32-channel head coil. A T2-weighted turbo-spin-echo-sequence (TR ¼ 3000 m, TE ¼ 80 m, 220 mm (FH) 175 mm (RL), slice thickness 2 mm, 30 coronal slices, in plane resolution 0.43 0.43 mm2, 5:42 scan time) was applied. A feature of the DRIVE (driven equilibrium radiofrequency reset pulse)-sequence is the relatively short acquisition time with high resolution. Data was analyzed independently by two experimenters who were blind to the groups (final results as mean value of both). Inter-rater reliability for the right OB was r ¼ .926 and for the left r ¼ .939. Manual contouring of the left and right OB in each coronal slice was performed using the ITK-Snap software (www.itksnap.org). A sudden change in diameter marked the beginning of the olfactory tract and therefore the proximal end of the OB (Abolmaali et al., 2002). Individual OB volumes were also calculated as a percentage of the whole brain volume which was defined as the sum of the grey and white matter (SPM Toolbox Easy Volume, http://www.sbirc.ed.ac.uk/LCL/LCL_M1.html).
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Table 1 Sample description and statistical evaluation.
Age (years) Intelligence (IQ) Internal (CBCL-T-value) External (CBCL-T-value) Sum (CBCL-T-value) PEA threshold Menthol threshold OB volume (in mm3)
ADHD [Mean ± SD]
HC [Mean ± SD]
Statistic for group comparison
10.0 ± 1.7 99.5 ± 12.8 63.4 ± 8.0 65.9 ± 8.8 68.9 ± 7.8 10.3 ± 1.4 7.2 ± 1.0 110.8 ± 22.3
10.5 ± 0.93 107.7 ± 12.4 50.6 ± 5.4 43.7 ± 7.8 45.5 ± 5.4 8.0 ± 2.2 7.0 ± 2.3 96.7 ± 10.0
.315 .066 .001 .001 .001 .002 .738 .033a
a ANOVA main effect Group. ADHD ¼ Attention-deficit/hyperactivity disorder, HC ¼ healthy controls, OB ¼ olfactory bulb, PEA ¼ phenylethyl alcohol, SD ¼ standard deviation.
2.4. Functional MRI 2.4.1. Design and procedure Chemosensory stimuli were delivered by a custom-made MRIcompatible olfactometer in accordance to Prehn-Kristensen et al. (2009). The application allowed a constant total airflow of 3 L/ min divided into a carrier current (1 L/min) as well as a second current (2 L/min) which passed either an empty bottle during “clean air” condition or one of two bottles with 20 ml of the chemosensory solvent [PEA 20 ml undiluted or L-menthol 20 ml 70% (v/v)]. All components were made of odor-free materials (Teflon, glass) except of the one-way valves (polystyrol) as well as the silicon oxygen mask delivering the stimuli to participant's nose. The event-related fMRI-paradigm was an adapted version of the one used by Prehn-Kristensen et al. (2009). The paradigm was programmed in E-Prime (Psychology Software Tools, Inc., Sharpsburg, PA). Visual instructions prompted the participants to inhale while the stimuli were delivered. Instructions appeared on a backprojected screen mounted 70 cm behind the scanner bore and could be seen via a mirror attached to the head coil. Stimuli were presented in a pseudo-randomized order (avoiding presentation of two similar stimuli in succession) for a duration of 1.5 s and an average ISI of 20.5 s (range: 18.8e26.2 s). In every session, the three conditions (PEA, menthol, clean air) were repeated 7 times, resulting in 21 trials per session. For more details see Fig. S1 (available online). 2.4.2. Subjective ratings of chemosensory stimuli To avoid analyzing simple group effects of different thresholds in the fMRI part, subjective stimulus intensity was individually adjusted to an equal level for all participants. Therefore, participants had to evaluate the stimuli prior to the MRI measurement on a unipolar visual analogue scale ranging from 0 (not at all) to 5 (maximum) regarding intensity, pleasantness, unpleasantness and familiarity (Laudien et al., 2008; Adolph and Pause, 2012). By manually regulating the flow rate of the odorous air, it was assured that all participants perceived PEA and menthol with medium intensity (between 1.5 and 3.5). 2.4.3. fMRI data acquisition and analysis For functional imaging, T2*-weighted echo-planar images (EPIs) were obtained [repetition time (TR) ¼ 2500 ms; echo time (TE) ¼ 35.5 ms; flip angle ¼ 90 ; FOV ¼ 216 216 mm2; matrix 64 64; 38 axial slices; slice thickness ¼ 3 mm; gap ¼ 0.3 mm; 184 volumes; 7:50 scan time]. A high-resolution, T1-weighted scan was used for structural MRI of the whole brain (TR ¼ 8.2 ms; TE ¼ 3.8 ms; flip-angle ¼ 8 ; FOV 240 240 mm2; 160 sagittal slices, voxel size ¼ 1 1 1mm3; 5:35 scan time). Data was preprocessed and statistically analyzed using the Statistical Parametric Mapping software SPM8 (Welcome Department of Imaging
Neuroscience, London). After spatial realignment to mean EPI, all functional volumes were co-registered to participant's T1weighted image. Spatial normalization was performed according to the DARTEL-approach (Ashburner, 2007). Because data was based on a pediatric study group, an age/gender-specific tissue probability map was created with the Template-O-Matic toolbox implemented in SPM (Wilke et al., 2008). After normalization voxel size was 2 2 2 mm. The functional data was smoothed with a Gaussian kernel of 6 mm FWHM. For statistical evaluation, on single-subject level nine regressors for every session were entered into the design matrix modelling the three conditions (PEA, menthol, clean air) as well as six movement regressors. The three regressors of interest were convolutions of a box-car with the canonical synthetic hemodynamic response function (HRF), timelocked to event onset. Contrasts of interest were created on the first-level (subject-level) and then entered into second-level (group-level) t-testing. The resulting T-maps were based on whole brain analysis at a p-level of < .001 and a minimum cluster size of 10 voxels (uncorrected for multiple comparisons). 2.4.4. Psychophysiological interaction analysis Psychophysiological Interaction (PPI-) analysis was performed in order to investigate task-specific functional connectivity of the olfactory cortex during chemosensory stimulation. The choice of the seed-region was based on literature, showing the PIR not only as largest structure and main component of primary olfactory cortex in humans (Benarroch, 2010; Zelano et al., 2005) but also as a region exerting direct feed-back influence on OB (Boyd et al., 2012). In contrast to the temporal PIR, the frontal PIR (fPIR) is a region where of olfactory and trigeminal stimuli are co-processed (Chevy and Klingler, 2014) and is a target of top-down modulation resulting in higher-order processing (Zelano et al., 2005). Grouplevel one-sample t-tests of contrasts “PEA vs. clean air” and “menthol vs. clean air” both revealed significant activation within bilateral PIR (Fig. 1A, B). We decided to use left fPIR as a seed-region because, in the contrast of the olfactory stimulus vs. clean air as well as trigeminal vs. clean air, the peak activation inside the bilateral fPIR across both groups was on the left side. Consequently, we consider the left fPIR to be more representative than the right fPIR in terms of olfactory and trigeminal processing. ROI mask was created manually using MRIcron (www.mccauslandcenter.sc.edu/ mricro/mricron/using) and its anatomical boundaries were derived from Atlas of the Human Brain Second Edition (Mai et al., 2004). The first eigenvariate of the BOLD signal was extracted from the 260 voxels within the left fPIR ROI mask. On the 1st-level, two separate PPI models were defined per subject, using either PEA vs. implicit baseline or menthol vs. implicit baseline as psychological terms with the chemosensory stimulus set to one and every other regressor in the design set to zero (Beucke et al., 2012; Cremers et al., 2014). Additionally, for the direct comparison
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data was available) and 15 HC (data of one HC could not be analyzed due to extreme movement). Computations were conducted using IBM Statistical Package for the Social Sciences (SPSS) 22.0 for Windows. 3. Results 3.1. Chemosensory detection threshold The ANOVA revealed a main effect for GROUP, indicating that ADHD patients displayed higher sensitivity scores (lower threshold) [F (1,32) ¼ 6.60, p ¼ .015], as well as a main effect of STIMULUS with lower threshold in detection of PEA than of menthol [F (1,32) ¼ 23.98, p < .001]. More importantly, there was an interaction effect of GROUP STIMULUS [F (1,32) ¼ 5.82, p ¼ .022]. As predicted, the PEA detection threshold was lower in ADHD than in HC [ADHD: M ¼ 10.3, SD ¼ 1.4, HC: M ¼ 8.0, SD ¼ 2.2; t (32) ¼ 3.44; p ¼ .002]. There was no difference between groups in the menthol threshold [ADHD: M ¼ 7.2, SD ¼ 1.0; HC: M ¼ 7.0, SD ¼ 2.3; t (32) ¼ .34; p ¼ .738] (Table 1, Fig. 2). When including IQ as covariate in an ANCOVA the interaction still was significant (p ¼ .007). 3.2. Volumetry of the OB
Fig. 1. Brain activation across all subjects. Whole brain analysis projected on DARTELtemplate in neurological convention (p < .001, minimum cluster size ¼ 10 voxels, uncorrected). A: PEA vs. air B: Menthol vs. air. OFC ¼ orbitofrontal cortex, PIR ¼ piriform cortex.
between the stimuli, the psychological term was built as PEA vs. menthol. PPI-interaction term [physiological BOLD signal (eigenvariate) x HRF convolved task main-effect] was entered as regressor of interest into single-subject analysis. In addition, the psychological (task main effect) and the physiological term (BOLD signal eigenvariate), as well as 6 movement regressors, were added as regressors of no interest into the design matrix. On the 2nd-level, resulting parameter estimates of the PPI term were entered into a random effects analysis to contrast PPI effects of ADHD and HC groups. Resulting T-maps were based on explorative, whole-brain analysis at a p-level of .001 and a minimum cluster size of 10 voxels (uncorrected for multiple comparisons).
As revealed by the ANOVA main effect for GROUP [F (1,29) ¼ 5.0, p ¼ .033], children with ADHD displayed a higher absolute OB volume (M ¼ 110.8 3, SD ¼ 22.3) than HC (M ¼ 96.7 3, SD ¼ 10.0). Neither interaction [F (1,29) ¼ 1.3, p ¼ .270] nor main effect of SIDE [F (1,29) ¼ .08, p ¼ .782] were significant. The same was true for the relative OB volumes: there was a significant main effect of GROUP revealing a higher OB volume in ADHD than HC [F (1,29) ¼ 5.3, p ¼ .029], but no main effect for SIDE [F (1,29) ¼ .05, p ¼ .820] and no significant interaction [F (1,29) ¼ 1.2, p ¼ .284] (Table 1, Fig. 2). When including IQ as a covariate the main effect of GROUP was still significant with regard to the absolute (p ¼ .040) as well as the relative values (p ¼ .045). 3.3. fMRI 3.3.1. Subjective ratings of chemosensory stimuli ANOVAs on subjective ratings only revealed main effects for STIMULUS indicating that menthol was rated as being more intense than PEA [PEA: M ¼ 2.68, SD ¼ 0.64; menthol: M ¼ 3.03, SD ¼ 0.63; F (1,29) ¼ 4.2, p ¼ .050], marginally more pleasant [PEA: M ¼ 2.59,
2.5. Statistical analysis Threshold and subjective rating data was both analyzed using a 2 2 analysis of variance (ANOVA) with GROUP as the betweensubject factor and STIMULUS (PEA vs. menthol) as within-subject factor. Statistical analysis of chemosensory detection performance was carried out on data of 17 patients and 17 HC (one patient did not participate due to onset of MPH-therapy). Rating data obtained in the fMRI part were based on 15 patients and 16 HC (three patients and one HC refused to participate in the fMRI-measurement). OB volume (absolute and relative to whole brain volume) was analyzed using a 2 2 ANOVA with GROUP as the between-subject factor and SIDE (left vs. right) as within-subject factor. Analysis of OB volumetry was carried out on 16 patients (due to lack of compliance during fMRI paradigm, in one of the patients only OB-
Fig. 2. Chemosensory detection performance and OB volume in ADHD and HC. A: *ANOVA interaction effect p < .05; ** t-test p < .01. B: * ANOVA main effect p < .05. HC ¼ healthy controls, M ¼ mean, OB ¼ olfactory bulb, PEA ¼ phenylethyl alcohol, SD ¼ standard deviation.
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SD ¼ 1.23; menthol: M ¼ 3.13, SD ¼ 1.27; F (1,29) ¼ 3.6, p ¼ .065], and more familiar [PEA: M ¼ 1.58, SD ¼ 1.38; menthol: M ¼ 4.0, SD ¼ 1.17; F (1,29) ¼ 41.2 p < .001]. All other main effects and interactions were not significant (p > .178) implicating no group differences in subjective ratings of chemosensory stimuli (see Table 2 for descriptive results).
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between the frontal piriform cortex (fPIR) and the anterior insula, ACC, SN and SMG. Group contrast of the PPI with respect to the trigeminal condition only revealed a significant cluster in the ACC. Olfactory threshold performance is in line with former results revealing a significantly higher sensitivity towards the olfactory stimulus PEA in children with ADHD (Romanos et al., 2008). Moreover, no differences in the chemosensory threshold were found regarding perception of the trigeminal stimulus menthol in our study. These data pinpoint the specific olfactory-related alteration in chemosensory sensitivity in ADHD. As reported by Romanos et al. (2008), the olfactory threshold performance can be normalized by MPH. In fact, the olfactory system depends on dopaminergic processing (Westermann et al., 2008), making it vulnerable in dopaminergic diseases. In Parkinson's disease an early-onset decrease in olfactory performance was found in 90% of patients (Doty, 2012) with underlying dopaminergic pathology (Wang et al., 2011) and concomitant decreased OB volumes (Brodoehl et al., 2012). Other researchers could not find an increase in olfactory sensitivity in ADHD vs. HC. But this could possibly be explained by an insufficient MPH-washout time (Schecklmann et al., 2011a,b; Ghanizadeh et al., 2012) or normalization of olfactory sensitivity in adults (like the ontogenetic decay of impulsivity) (Schecklmann et al., 2011b). In addition to a higher olfactory sensitivity, OB volumes were found to be higher in ADHD than in in controls. Olfactory sensitivity is often predicted by OB volume (Buschhüter et al., 2008; Hummel et al., 2011). Therefore, the higher OB volume and the higher olfactory sensitivity in ADHD, both, suggest a common underlying mechanism. Perception of PEA induced clear activations in the primary and secondary olfactory cortices (PIR, OFC, amygdala and hippocampus) across both groups. ADHD patients and controls did not differ with respect to BOLD activations during the inhalation of olfactory stimuli. This might be expectable because stimulus intensity was individually adjusted to control for group differences in chemosensory thresholds. In children with ADHD the primary olfactory cortex (frontal PIR) showed altered functional connectivity with structures belonging to the salience network (including the insula, ACC, SMG, and SN) (Downar et al., 2002; Seeley et al., 2007) which is highly sensitive to dopaminergic disruptions (Christopher et al., 2015; Palaniyappan and Liddle, 2012). More precisely, patients showed positive coupling of the fPIR with mentioned regions, whereas this coupling was negative in HC. The salience network is involved in bottom-up integration of incoming sensory stimuli, which prevents the organism from being flooded with irrelevant sensory information. Tagging an event as being salient enables access to attention and deeper processing (Menon and Uddin, 2010). Functional and structural alterations in the salience network in ADHD as well as alterations in bottom-up sensory processing are supported by recently published findings (Yu, 2013; Li et al., 2015; Tegelbeckers et al., 2015). There are not only topdown projections to the primary olfactory cortex (Garcia-Cabezas and Barbas, 2014; Wilson and Sullivan, 2011) but also extensive centrifugal back projections from the PIR to the OB (Gray and
3.3.2. Activation maps Contrasting PEA and clean air across both groups showed activity in the bilateral PIR (here, as part of the bilateral activated amygdala cluster), bilateral insula, as well as bilateral amygdala and left orbitofrontal cortex (OFC) (Fig. 1A). For details see Table S1 (available online). The contrast menthol vs. clean air showed activity in two large bilateral clusters including the amygdala, insula, as well as the PIR (Fig. 1B). Contrasting clean air and PEA as well as clean air and menthol showed significant clusters in the motor cortex and superior frontal gyrus indicating that inhalation of clean air triggered additional cognitive processes. Because of that, clean air was excluded as a control condition from further analysis. Twosample t-testing of group differences in PEA and menthol revealed no significant activations (p < .001, k > 10 voxels, uncorrected). 3.3.3. PPI-analysis PPI of the PEA vs. implicit baseline condition in ADHD vs. HC revealed clusters in the ACC, anterior insula, supramarginal gyrus (SMG) and substantia nigra (SN). Additionally, there were significant clusters in the angular gyrus, temporal pole, precuneus, lingual gyrus, posterior cingulate gyrus and posterior insula (Fig. 3A). See Table S2 for details (available online). Further analysis indicated positive coupling between the fPIR and the mentioned regions in ADHD compared to negative coupling in HC. Contrasting HC vs. ADHD revealed no significantly activated clusters. PPI of the menthol vs. implicit baseline condition in ADHD vs. HC revealed a cluster in the right ACC only (Fig. 3B). Here, in the peak voxel, children with ADHD showed positive coupling with fPIR compared to negative coupling in HC. Again, contrasting HC vs. ADHD revealed no significantly activated clusters. The additional direct PPI-comparison between PEA and menthol contrasting ADHD vs. HC revealed clusters in bilateral posterior as well as left anterior insula, ACC and putamen. In all these regions, the PPI-contrast estimates were shown to be positive in the patients and negative in HC. 4. Discussion As expected, increased sensitivity was found in children with ADHD only in the olfactory but not in trigeminal threshold performance when compared to healthy controls (HC). Furthermore, an increased volume of the OB was observed in ADHD. There were no group differences in neuronal activation towards olfactory and trigeminal stimuli (PEA and menthol). However, analysis of psychophysiological interactions (PPI) during olfactory stimulus processing revealed group differences in the functional connectivity
Table 2 Descriptive statistics of subjective ratings of chemosensory stimuli, rated on a visual analogue scale ranging from 0 (not at all) to 5 (maximum). PEA [Mean ± SD] ADHD Intensity Pleasantness Unpleasantness Familiarity
2.69 2.65 0.98 1.6
± ± ± ±
0.63 1.23 1.07 1.53
HC 2.81 2.52 1.76 1.54
± ± ± ±
0.46 1.26 1.46 1.28
Menthol [Mean ± SD] Total 2.75 2.58 1.38 1.57
± ± ± ±
0.54 1.22 1.33 1.38
ADHD 3.03 3.12 1.31 3.93
± ± ± ±
.63 1.26 1.61 1.17
Note: ADHD ¼ Attention-deficit/hyperactivity disorder, HC ¼ healthy controls, PEA ¼ phenylethyl alcohol, SD ¼ standard deviation.
HC 3.0 3.14 1.45 4.1
± ± ± ±
0.68 1.33 1.44 1.22
Total 3.02 3.13 1.38 4.0
± ± ± ±
0.65 1.27 1.5 1.18
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Fig. 3. Functional connectivity (PPI) in ADHD vs. HC. Whole brain analysis projected on DARTEL-template in neurological covention (p < .001, minimum cluster size ¼ 10 voxels, uncorrected). A: PEA B: Menthol. ACC ¼ anterior cingulate, SMG ¼ supramarginal gyrus, SN ¼ substantia nigra.
Skinner, 1988). Thus, cortical processes in terms of olfactory learning and attention (Huart et al., 2013) but also altered salience attribution could possibly influence the chemosensory circuits of the OB. The piriform cortex exerts a net inhibitory effect on bulbar excitation (Boyd et al., 2012). In the extreme case of disrupted centrifugal fibers, OB excitation could be enhanced by dampening of these inhibitory influences (Gray and Skinner, 1988). The OBvolume is shaped through mechanisms of synaptic plasticity as well as neurogenesis as a function of olfactory sensitivity (Huart et al., 2013). A higher olfactory performance due to increased bulbar excitability could thus lead to increased OB volume in ADHD. Whether or not dysregulations in dopamine-sensitive networks cause decreased neurogenesis of dopaminergic inhibitory interneurons leading to increased excitability of OB (Romanos et al., 2008; Schecklmann et al., 2011a,b) remains speculative. There is also the possibility of a peripheral mechanism since Hegg and Lucero (2004) have observed dopaminergic suppression through D2-receptors in olfactory receptor neurons. However, our fMRI results indicate changes in central mechanisms rather than pure peripheral effects. But we cannot rule out that peripheral mechanisms might play an additional role. Consequently, PETstudies (e.g. tracing dopaminergic signaling in the OB and olfactory/salient network) as well as the application of MPH in further fMRI-studies are required to elucidate the exact dopaminergic mechanisms. Processing of trigeminal menthol also involves connectivity changes between the fPIR and the salience network, albeit to a lesser extent than seen in olfactory processing. This indicates a global sensory alteration and not limited to the olfactory domain. Superior sensitivity in the olfactory domain in ADHD may be unique due to direct cortical feedback loops to the OB (bypassing thalamic relay). A possible confounding influence of IQ (marginal significant group difference) was controlled by adopting intelligence as a covariate in behavioral and volumetric analysis. Lower IQ scores in ADHD (but distinctly above cut-off of 85) may reflect the observed inability to sustain attention during the test procedure. Considerable overlap of olfactory and trigeminal stimulation in the olfactory cortex was observed. This is not surprising, considering that CO2 is the only chemo-stimulus which does not stimulate the olfactory system (Hummel and Livermore, 2002). In the present study, menthol was used as a trigeminal stimulus because this substance is more child-oriented than pungent CO2. Lack of additional somatosensory activation in the contrast menthol vs. clean air may be due to stimulus intensity not strong enough to elicit an effect of pungency. In addition, we already had to choose a liberal significance level because olfactory compared to e.g. visual stimuli are known to produce low intensity activations, high variability and low contrast-to-noise ratio (Morrot et al., 2013). Also, PPI-analyses tend to lack power and bear the risk for beta errors (O'Reilly et al., 2012). Since PEA and menthol were presented intermixed in an
event-related design, there might be a concern of possible interactions between the olfactory and trigeminal stimulus processing. Although the ITI in the fMRI measurement was considerably long (20.5 s on average), the ITI itself was variable (jitter: 18.8e26.2 s), and the order of chemosensory stimuli was pseudorandomized, we still cannot exclude such possible interactions. For example, an events-in-blocks design (Albrecht et al., 2009) with longer ITIs might help not only to increase the signal power but also to reduce possible interactions between different kinds of chemosensory stimuli. Taken together, children with ADHD displayed a selective superior olfactory sensitivity, accompanied by an increased OB volume. Functional connectivity between the primary olfactory cortex and regions of the dopamine-sensitive salience network were altered in ADHD, suggesting a hyper-sensitive olfactory processing that might trigger processes of OB neuroplasticity. Conflicts of interest None Contributions Anna Lorenzen: Data collection, analysis of the data, article preparation. Deborah Scholz-Hehn: Data collection, analysis of the data, article preparation. Christian D. Wiesner: Contributed in data analysis and interpretation. Author has approved final article Stephan Wolff: Contributed in data acquisition. Author has approved final article Til O. Bergmann: Contributed in data analysis and interpretation. Author has approved final article Thilo van Eimeren: Contributed in data analysis and interpretation. Author has approved final article Luisa Lentfer: Diagnostics. Author has approved final article Lioba Baving: Contributed in data analysis and interpretation. Author has approved final article Alexander Prehn-Kristensen: Supervision of data acquisition, analysis and article preparation. Acknowledgements We would like to thank Oliver Granert (Department of Neurology, University Hospital Schleswig-Holstein, Kiel, Germany) for his valuable advice concerning the fMRI design as well as Susanne Kell and Petra Schneckenburger (Department of Child and
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Journal of Psychiatric Research journal homepage: www.elsevier.com/locate/psychires
Chemosensory processing in children with attention-deficit/ hyperactivity disorder Anna Lorenzen a, *, 1, 2, Deborah Scholz-Hehn a, 1, Christian D. Wiesner a, Stephan Wolff b, Til O. Bergmann c, d, e, Thilo van Eimeren f, g, Luisa Lentfer a, Lioba Baving a, Alexander Prehn-Kristensen a a
Department of Child and Adolescent Psychiatry and Psychotherapy, University Hospital Schleswig-Holstein, Niemannsweg 147, 24105, Kiel, Germany Department of Neuroradiology, University Hospital Schleswig-Holstein, Arnold- Heller-Str. 3, 24105, Kiel, Germany Department of Neurology & Stroke, and Hertie Institute for Clinical Brain Research, University of Tübingen, Ottfried-Müller-Str. 25, 72076, Tübingen, Germany d Institute for Medical Psychology and Behavioral Neurobiology, University of Tübingen, Ottfried-Müller-Str. 25, 72076, Tübingen, Germany e Institute of Psychology, Christian-Albrechts University of Kiel, Olshausenstr. 62, 24118, Kiel, Germany f Department of Nuclear Medicine, University Hospital, Kerpenerstr. 62, 50937, Cologne, Germany g Department of Neurology, University Hospital, Kerpenerstr. 62, 50937, Cologne, Germany b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 20 August 2015 Received in revised form 12 February 2016 Accepted 12 February 2016
Background: In attention-deficit/hyperactivity disorder (ADHD) not only deficits in dopamine-related cognitive functioning have been found but also a lower dopamine-sensitive olfactory threshold. The aim of the present study was to proof that only olfactory but not trigeminal sensitivity is increased in ADHD. Structural magnetic resonance imaging (MRI) was used to show increased olfactory bulb (OB) volume- a structure which is strongly shaped by olfactory performance through the mechanism of neuroplasticity (e.g. synaptogenesis). To elucidate whether cortical mechanisms are involved in altered olfaction in ADHD, functional MRI (fMRI) was introduced. Methods: A total of 18 boys with ADHD and 17 healthy controls (aged 7e12) were included in the study. Olfactory as well as trigeminal detection thresholds were examined. OB sizes were measured by means of structural MRI and an analysis of effective functional (fMRI) coupling of primary olfactory cortex was conducted. The frontal piriform cortex (fPIR) was chosen as seed region because of its importance in processing both trigeminal and olfactory stimuli as well as having profound influence on inner OB-signaling. Results: Increased olfactory sensitivity as well as an increase in OB volume was found in ADHD. There were no group differences in sensitivity towards a trigeminal stimulus. Compared to healthy controls, the fPIR in ADHD was more positively coupled with structures belonging to the salience network during olfactory and, to a lesser extent, during trigeminal stimulation. Conclusions: Olfactory functioning is superior in subjects with ADHD. The observed increase in OB volume may relate to higher olfactory sensitivity in terms of neuroplasticity. During the processing of chemosensory stimuli, the primary olfactory cortex in ADHD is differently coupled to higher cortical structures which might indicate an altered top-down influence on OB structure and function. © 2016 Elsevier Ltd. All rights reserved.
Keywords: ADHD Olfactory processing Trigeminal processing Sensory threshold Olfactory bulb fMRI
* Corresponding author. E-mail addresses: [email protected] (A. Lorenzen), [email protected] (D. Scholz-Hehn), [email protected] (C.D. Wiesner), [email protected] (S. Wolff), [email protected] (T.O. Bergmann), [email protected] (T. van Eimeren), [email protected] (L. Lentfer), [email protected] (L. Baving), a.prehn@ zip-kiel.de (A. Prehn-Kristensen). 1 Anna Lorenzen and Deborah Scholz-Hehn contributed equally to this work. 2 Present address: Experimental Pediatric Neuroimaging, University Children's Hospital, Dept. III, (Pediatric Neurology), Hoppe-Seyler-Str. 1, 72076 Tübingen, Germany. http://dx.doi.org/10.1016/j.jpsychires.2016.02.007 0022-3956/© 2016 Elsevier Ltd. All rights reserved.
1. Introduction Attention-deficit/hyperactivity disorder (ADHD) is a neurodevelopmental disorder with a world-wide prevalence of 5e7% (Willcutt, 2012). Alterations in dopaminergic pathways are thought to be involved in the underlying pathology, which not only leads to symptoms of inattention and/or hyperactivity (American Psychiatric Association, 2000) but possibly also to alterations in
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olfactory processing. It was shown that children with ADHD show higher sensitivity towards olfactory stimuli when compared to both healthy controls (HC) and children with ADHD being treated with methylphenidate (MPH) (Romanos et al., 2008). MPH is regarded as the first-line treatment in ADHD and blocks dopamine reuptake in the mesolimbic system and the prefrontal cortex (PFC) (Heal et al., 2009; Wilens, 2008). The orbitofrontal cortex (OFC), amygdala, hippocampus, and pirifom cortex (PIR) are core structures of the human olfactory system (Benarroch, 2010; Zald and Pardo, 2000). Contrary to all other sensory systems (e.g. trigeminal or visual) olfactory processing does not rely on thalamic gating: The olfactory bulb (OB) is the critical relay in stimulus processing (Kay and Sherman, 2007) and the olfactory threshold is linked to OB size (Buschhüter et al., 2008). The trigeminal system is also involved in the perception of chemosensory stimuli with its free nerve endings extending into the nasal mucosa. It mediates sensations like burning, stinging or cooling via the brainstem and thalamus before reaching the somatosensory/insular cortex and converging with the olfactory system (Hummel and Livermore, 2002). There is strong evidence for lower OB volume being accompanied by reduced olfactory sensitivity in patients suffering from a loss of nigrostriatal dopaminergic neurons (e.g. Parkinson) (Brodoehl et al., 2012; Wang et al., 2011). It remains unclear, however, why ADHD is accompanied with higher olfactory sensitivity. It was proposed that striatal dopaminergic dysregulation may cause a decrease in inner-bulb-mediated inhibition of neuronal signals (Romanos et al., 2008; Schecklmann et al., 2011a,b). They assume an indirect mechanism of decreased neurogenesis of inhibitory dopaminergic interneurons. In addition, there is also direct cortical influence on inner-bulb circuits. Animal studies have shown that so-called centrifugal structures (PIR and anterior olfactory nucleus), which are dopamine sensitive in part (Fallon and Moore, 1978; Ikemoto, 2007), exert tonic inhibitory influence on the OB by dampening neuronal signals (Boyd et al., 2012). Alterations in the cortical dopamine system might lead to a cascade of decreased centrifugal downscaling on the OB transmission, resulting in higher excitation and increased olfactory sensitivity. By means of neuroplastic processes this may cause increased OB volume. To test the hypothesis that children suffering from ADHD show a higher olfactory sensitivity, detection thresholds and OB volumes were measured. By introducing a trigeminal stimulus it was intended to show that higher sensitivity in ADHD is restricted to the olfactory system and not due to unspecific hypersensitivity (Ghanizadeh, 2011). To test the hypothesis of alterations in cortical olfactory circuits exerting direct centrifugal influence on OB, explorative functional magnetic resonance imaging (fMRI) was conducted to evaluate the effective connectivity of PIR during intensity-controlled chemosensory stimulus processing. 2. Methods and materials 2.1. Participants 18 patients with ADHD and 17 healthy controls (HC) aging from 7.9 to 12.8 years were examined. All participants were diagnosed by the Kiddie-SADS-PL, a semi-structured diagnostic interview (Kaufman et al., 1997) according to the DSM-IV classification system. Eleven patients fulfilled ADHD-criteria for the predominantly hyperactive subtype and seven patients fulfilled criteria for the inattentive subtype. Eight patients had a co-morbid oppositional defiant disorder (three of the inattentive and five of the hyperactive subtype). Participants were also screened for psychopathological symptoms by parental ratings based on the Child Behavior
Checklist (CBCL) (Achenbach, 1991). Exclusion criterion for HC was a T-value of 60 in any of the syndrome- and summed scales. Intellectual ability was assessed with the German version of the Culture Fair Intelligence Test (CFT-20) (Weiß, 2006) and the CFT-1 (Weiß and Osterland, 2013). Exclusion criterion was an IQ under 85. Most participants were in prepubescent phase, two patients and two HC were in early puberty as measured with the German version of the Pubertal Development Scale (Watzlawik, 2009). There was no statistical significant group difference in age but a marginally significant difference in IQ. For details see Table 1. Exclusion criteria for all participants were acute or chronic disease of the respiratory tract or usual MRI exclusion criteria. Fifteen patients were MPH-naïve. Three patients had received MPH at least one year ago and for a period not longer than six months. All families gave informed consent. The study was approved by the Ethics Committee of the Medical Faculty of the University of Kiel.
2.2. Chemosensory threshold Individual olfactory as well as trigeminal detection thresholds were assessed using the “two-alternative-staircase-detection”method (Doty et al., 1995). Two serial dilutions were prepared: one with the olfactory stimulus phenylethyl alcohol (PEA, rose-like) and one with the trigeminal stimulus L-menthol (peppermint-like). The chemical preparation and testing procedure were conducted according to Pause et al. (2001). The Substances were diluted 1:2 in propylene glycol (v/v). This stock solution was further diluted in 16 half-decimal logarithmic steps [lowest concentration 1:63000000 (v/v)]. Finally, each of the sixteen brown glass bottles contained 6.5 ml chemical solution. Both tests on olfactory as well as trigeminal thresholds were conducted in the same session separated by a half an hour. The order of the presentation was counterbalanced across the groups. Participants were instructed to decide which of the two bottles smelled more intensive (one containing the chemosensory stimulus and one containing only the solvent). A higher threshold score (in terms of dilution step) corresponded to higher sensitivity. To avoid habituation, there was an intertrial interval (ITI) of 20 s. The procedure took place in an air-conditioned room at a constant 19 C. Participants were not allowed to eat a half an hour before chemosensory testing and were instructed to avoid spicy foods like garlic prior to testing that day.
2.3. Volumetry of OB The MRI measurements were performed with a 3 T Philips Intera Achieva (Best, The Netherlands) using a 32-channel head coil. A T2-weighted turbo-spin-echo-sequence (TR ¼ 3000 m, TE ¼ 80 m, 220 mm (FH) 175 mm (RL), slice thickness 2 mm, 30 coronal slices, in plane resolution 0.43 0.43 mm2, 5:42 scan time) was applied. A feature of the DRIVE (driven equilibrium radiofrequency reset pulse)-sequence is the relatively short acquisition time with high resolution. Data was analyzed independently by two experimenters who were blind to the groups (final results as mean value of both). Inter-rater reliability for the right OB was r ¼ .926 and for the left r ¼ .939. Manual contouring of the left and right OB in each coronal slice was performed using the ITK-Snap software (www.itksnap.org). A sudden change in diameter marked the beginning of the olfactory tract and therefore the proximal end of the OB (Abolmaali et al., 2002). Individual OB volumes were also calculated as a percentage of the whole brain volume which was defined as the sum of the grey and white matter (SPM Toolbox Easy Volume, http://www.sbirc.ed.ac.uk/LCL/LCL_M1.html).
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Table 1 Sample description and statistical evaluation.
Age (years) Intelligence (IQ) Internal (CBCL-T-value) External (CBCL-T-value) Sum (CBCL-T-value) PEA threshold Menthol threshold OB volume (in mm3)
ADHD [Mean ± SD]
HC [Mean ± SD]
Statistic for group comparison
10.0 ± 1.7 99.5 ± 12.8 63.4 ± 8.0 65.9 ± 8.8 68.9 ± 7.8 10.3 ± 1.4 7.2 ± 1.0 110.8 ± 22.3
10.5 ± 0.93 107.7 ± 12.4 50.6 ± 5.4 43.7 ± 7.8 45.5 ± 5.4 8.0 ± 2.2 7.0 ± 2.3 96.7 ± 10.0
.315 .066 .001 .001 .001 .002 .738 .033a
a ANOVA main effect Group. ADHD ¼ Attention-deficit/hyperactivity disorder, HC ¼ healthy controls, OB ¼ olfactory bulb, PEA ¼ phenylethyl alcohol, SD ¼ standard deviation.
2.4. Functional MRI 2.4.1. Design and procedure Chemosensory stimuli were delivered by a custom-made MRIcompatible olfactometer in accordance to Prehn-Kristensen et al. (2009). The application allowed a constant total airflow of 3 L/ min divided into a carrier current (1 L/min) as well as a second current (2 L/min) which passed either an empty bottle during “clean air” condition or one of two bottles with 20 ml of the chemosensory solvent [PEA 20 ml undiluted or L-menthol 20 ml 70% (v/v)]. All components were made of odor-free materials (Teflon, glass) except of the one-way valves (polystyrol) as well as the silicon oxygen mask delivering the stimuli to participant's nose. The event-related fMRI-paradigm was an adapted version of the one used by Prehn-Kristensen et al. (2009). The paradigm was programmed in E-Prime (Psychology Software Tools, Inc., Sharpsburg, PA). Visual instructions prompted the participants to inhale while the stimuli were delivered. Instructions appeared on a backprojected screen mounted 70 cm behind the scanner bore and could be seen via a mirror attached to the head coil. Stimuli were presented in a pseudo-randomized order (avoiding presentation of two similar stimuli in succession) for a duration of 1.5 s and an average ISI of 20.5 s (range: 18.8e26.2 s). In every session, the three conditions (PEA, menthol, clean air) were repeated 7 times, resulting in 21 trials per session. For more details see Fig. S1 (available online). 2.4.2. Subjective ratings of chemosensory stimuli To avoid analyzing simple group effects of different thresholds in the fMRI part, subjective stimulus intensity was individually adjusted to an equal level for all participants. Therefore, participants had to evaluate the stimuli prior to the MRI measurement on a unipolar visual analogue scale ranging from 0 (not at all) to 5 (maximum) regarding intensity, pleasantness, unpleasantness and familiarity (Laudien et al., 2008; Adolph and Pause, 2012). By manually regulating the flow rate of the odorous air, it was assured that all participants perceived PEA and menthol with medium intensity (between 1.5 and 3.5). 2.4.3. fMRI data acquisition and analysis For functional imaging, T2*-weighted echo-planar images (EPIs) were obtained [repetition time (TR) ¼ 2500 ms; echo time (TE) ¼ 35.5 ms; flip angle ¼ 90 ; FOV ¼ 216 216 mm2; matrix 64 64; 38 axial slices; slice thickness ¼ 3 mm; gap ¼ 0.3 mm; 184 volumes; 7:50 scan time]. A high-resolution, T1-weighted scan was used for structural MRI of the whole brain (TR ¼ 8.2 ms; TE ¼ 3.8 ms; flip-angle ¼ 8 ; FOV 240 240 mm2; 160 sagittal slices, voxel size ¼ 1 1 1mm3; 5:35 scan time). Data was preprocessed and statistically analyzed using the Statistical Parametric Mapping software SPM8 (Welcome Department of Imaging
Neuroscience, London). After spatial realignment to mean EPI, all functional volumes were co-registered to participant's T1weighted image. Spatial normalization was performed according to the DARTEL-approach (Ashburner, 2007). Because data was based on a pediatric study group, an age/gender-specific tissue probability map was created with the Template-O-Matic toolbox implemented in SPM (Wilke et al., 2008). After normalization voxel size was 2 2 2 mm. The functional data was smoothed with a Gaussian kernel of 6 mm FWHM. For statistical evaluation, on single-subject level nine regressors for every session were entered into the design matrix modelling the three conditions (PEA, menthol, clean air) as well as six movement regressors. The three regressors of interest were convolutions of a box-car with the canonical synthetic hemodynamic response function (HRF), timelocked to event onset. Contrasts of interest were created on the first-level (subject-level) and then entered into second-level (group-level) t-testing. The resulting T-maps were based on whole brain analysis at a p-level of < .001 and a minimum cluster size of 10 voxels (uncorrected for multiple comparisons). 2.4.4. Psychophysiological interaction analysis Psychophysiological Interaction (PPI-) analysis was performed in order to investigate task-specific functional connectivity of the olfactory cortex during chemosensory stimulation. The choice of the seed-region was based on literature, showing the PIR not only as largest structure and main component of primary olfactory cortex in humans (Benarroch, 2010; Zelano et al., 2005) but also as a region exerting direct feed-back influence on OB (Boyd et al., 2012). In contrast to the temporal PIR, the frontal PIR (fPIR) is a region where of olfactory and trigeminal stimuli are co-processed (Chevy and Klingler, 2014) and is a target of top-down modulation resulting in higher-order processing (Zelano et al., 2005). Grouplevel one-sample t-tests of contrasts “PEA vs. clean air” and “menthol vs. clean air” both revealed significant activation within bilateral PIR (Fig. 1A, B). We decided to use left fPIR as a seed-region because, in the contrast of the olfactory stimulus vs. clean air as well as trigeminal vs. clean air, the peak activation inside the bilateral fPIR across both groups was on the left side. Consequently, we consider the left fPIR to be more representative than the right fPIR in terms of olfactory and trigeminal processing. ROI mask was created manually using MRIcron (www.mccauslandcenter.sc.edu/ mricro/mricron/using) and its anatomical boundaries were derived from Atlas of the Human Brain Second Edition (Mai et al., 2004). The first eigenvariate of the BOLD signal was extracted from the 260 voxels within the left fPIR ROI mask. On the 1st-level, two separate PPI models were defined per subject, using either PEA vs. implicit baseline or menthol vs. implicit baseline as psychological terms with the chemosensory stimulus set to one and every other regressor in the design set to zero (Beucke et al., 2012; Cremers et al., 2014). Additionally, for the direct comparison
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data was available) and 15 HC (data of one HC could not be analyzed due to extreme movement). Computations were conducted using IBM Statistical Package for the Social Sciences (SPSS) 22.0 for Windows. 3. Results 3.1. Chemosensory detection threshold The ANOVA revealed a main effect for GROUP, indicating that ADHD patients displayed higher sensitivity scores (lower threshold) [F (1,32) ¼ 6.60, p ¼ .015], as well as a main effect of STIMULUS with lower threshold in detection of PEA than of menthol [F (1,32) ¼ 23.98, p < .001]. More importantly, there was an interaction effect of GROUP STIMULUS [F (1,32) ¼ 5.82, p ¼ .022]. As predicted, the PEA detection threshold was lower in ADHD than in HC [ADHD: M ¼ 10.3, SD ¼ 1.4, HC: M ¼ 8.0, SD ¼ 2.2; t (32) ¼ 3.44; p ¼ .002]. There was no difference between groups in the menthol threshold [ADHD: M ¼ 7.2, SD ¼ 1.0; HC: M ¼ 7.0, SD ¼ 2.3; t (32) ¼ .34; p ¼ .738] (Table 1, Fig. 2). When including IQ as covariate in an ANCOVA the interaction still was significant (p ¼ .007). 3.2. Volumetry of the OB
Fig. 1. Brain activation across all subjects. Whole brain analysis projected on DARTELtemplate in neurological convention (p < .001, minimum cluster size ¼ 10 voxels, uncorrected). A: PEA vs. air B: Menthol vs. air. OFC ¼ orbitofrontal cortex, PIR ¼ piriform cortex.
between the stimuli, the psychological term was built as PEA vs. menthol. PPI-interaction term [physiological BOLD signal (eigenvariate) x HRF convolved task main-effect] was entered as regressor of interest into single-subject analysis. In addition, the psychological (task main effect) and the physiological term (BOLD signal eigenvariate), as well as 6 movement regressors, were added as regressors of no interest into the design matrix. On the 2nd-level, resulting parameter estimates of the PPI term were entered into a random effects analysis to contrast PPI effects of ADHD and HC groups. Resulting T-maps were based on explorative, whole-brain analysis at a p-level of .001 and a minimum cluster size of 10 voxels (uncorrected for multiple comparisons).
As revealed by the ANOVA main effect for GROUP [F (1,29) ¼ 5.0, p ¼ .033], children with ADHD displayed a higher absolute OB volume (M ¼ 110.8 3, SD ¼ 22.3) than HC (M ¼ 96.7 3, SD ¼ 10.0). Neither interaction [F (1,29) ¼ 1.3, p ¼ .270] nor main effect of SIDE [F (1,29) ¼ .08, p ¼ .782] were significant. The same was true for the relative OB volumes: there was a significant main effect of GROUP revealing a higher OB volume in ADHD than HC [F (1,29) ¼ 5.3, p ¼ .029], but no main effect for SIDE [F (1,29) ¼ .05, p ¼ .820] and no significant interaction [F (1,29) ¼ 1.2, p ¼ .284] (Table 1, Fig. 2). When including IQ as a covariate the main effect of GROUP was still significant with regard to the absolute (p ¼ .040) as well as the relative values (p ¼ .045). 3.3. fMRI 3.3.1. Subjective ratings of chemosensory stimuli ANOVAs on subjective ratings only revealed main effects for STIMULUS indicating that menthol was rated as being more intense than PEA [PEA: M ¼ 2.68, SD ¼ 0.64; menthol: M ¼ 3.03, SD ¼ 0.63; F (1,29) ¼ 4.2, p ¼ .050], marginally more pleasant [PEA: M ¼ 2.59,
2.5. Statistical analysis Threshold and subjective rating data was both analyzed using a 2 2 analysis of variance (ANOVA) with GROUP as the betweensubject factor and STIMULUS (PEA vs. menthol) as within-subject factor. Statistical analysis of chemosensory detection performance was carried out on data of 17 patients and 17 HC (one patient did not participate due to onset of MPH-therapy). Rating data obtained in the fMRI part were based on 15 patients and 16 HC (three patients and one HC refused to participate in the fMRI-measurement). OB volume (absolute and relative to whole brain volume) was analyzed using a 2 2 ANOVA with GROUP as the between-subject factor and SIDE (left vs. right) as within-subject factor. Analysis of OB volumetry was carried out on 16 patients (due to lack of compliance during fMRI paradigm, in one of the patients only OB-
Fig. 2. Chemosensory detection performance and OB volume in ADHD and HC. A: *ANOVA interaction effect p < .05; ** t-test p < .01. B: * ANOVA main effect p < .05. HC ¼ healthy controls, M ¼ mean, OB ¼ olfactory bulb, PEA ¼ phenylethyl alcohol, SD ¼ standard deviation.
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SD ¼ 1.23; menthol: M ¼ 3.13, SD ¼ 1.27; F (1,29) ¼ 3.6, p ¼ .065], and more familiar [PEA: M ¼ 1.58, SD ¼ 1.38; menthol: M ¼ 4.0, SD ¼ 1.17; F (1,29) ¼ 41.2 p < .001]. All other main effects and interactions were not significant (p > .178) implicating no group differences in subjective ratings of chemosensory stimuli (see Table 2 for descriptive results).
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between the frontal piriform cortex (fPIR) and the anterior insula, ACC, SN and SMG. Group contrast of the PPI with respect to the trigeminal condition only revealed a significant cluster in the ACC. Olfactory threshold performance is in line with former results revealing a significantly higher sensitivity towards the olfactory stimulus PEA in children with ADHD (Romanos et al., 2008). Moreover, no differences in the chemosensory threshold were found regarding perception of the trigeminal stimulus menthol in our study. These data pinpoint the specific olfactory-related alteration in chemosensory sensitivity in ADHD. As reported by Romanos et al. (2008), the olfactory threshold performance can be normalized by MPH. In fact, the olfactory system depends on dopaminergic processing (Westermann et al., 2008), making it vulnerable in dopaminergic diseases. In Parkinson's disease an early-onset decrease in olfactory performance was found in 90% of patients (Doty, 2012) with underlying dopaminergic pathology (Wang et al., 2011) and concomitant decreased OB volumes (Brodoehl et al., 2012). Other researchers could not find an increase in olfactory sensitivity in ADHD vs. HC. But this could possibly be explained by an insufficient MPH-washout time (Schecklmann et al., 2011a,b; Ghanizadeh et al., 2012) or normalization of olfactory sensitivity in adults (like the ontogenetic decay of impulsivity) (Schecklmann et al., 2011b). In addition to a higher olfactory sensitivity, OB volumes were found to be higher in ADHD than in in controls. Olfactory sensitivity is often predicted by OB volume (Buschhüter et al., 2008; Hummel et al., 2011). Therefore, the higher OB volume and the higher olfactory sensitivity in ADHD, both, suggest a common underlying mechanism. Perception of PEA induced clear activations in the primary and secondary olfactory cortices (PIR, OFC, amygdala and hippocampus) across both groups. ADHD patients and controls did not differ with respect to BOLD activations during the inhalation of olfactory stimuli. This might be expectable because stimulus intensity was individually adjusted to control for group differences in chemosensory thresholds. In children with ADHD the primary olfactory cortex (frontal PIR) showed altered functional connectivity with structures belonging to the salience network (including the insula, ACC, SMG, and SN) (Downar et al., 2002; Seeley et al., 2007) which is highly sensitive to dopaminergic disruptions (Christopher et al., 2015; Palaniyappan and Liddle, 2012). More precisely, patients showed positive coupling of the fPIR with mentioned regions, whereas this coupling was negative in HC. The salience network is involved in bottom-up integration of incoming sensory stimuli, which prevents the organism from being flooded with irrelevant sensory information. Tagging an event as being salient enables access to attention and deeper processing (Menon and Uddin, 2010). Functional and structural alterations in the salience network in ADHD as well as alterations in bottom-up sensory processing are supported by recently published findings (Yu, 2013; Li et al., 2015; Tegelbeckers et al., 2015). There are not only topdown projections to the primary olfactory cortex (Garcia-Cabezas and Barbas, 2014; Wilson and Sullivan, 2011) but also extensive centrifugal back projections from the PIR to the OB (Gray and
3.3.2. Activation maps Contrasting PEA and clean air across both groups showed activity in the bilateral PIR (here, as part of the bilateral activated amygdala cluster), bilateral insula, as well as bilateral amygdala and left orbitofrontal cortex (OFC) (Fig. 1A). For details see Table S1 (available online). The contrast menthol vs. clean air showed activity in two large bilateral clusters including the amygdala, insula, as well as the PIR (Fig. 1B). Contrasting clean air and PEA as well as clean air and menthol showed significant clusters in the motor cortex and superior frontal gyrus indicating that inhalation of clean air triggered additional cognitive processes. Because of that, clean air was excluded as a control condition from further analysis. Twosample t-testing of group differences in PEA and menthol revealed no significant activations (p < .001, k > 10 voxels, uncorrected). 3.3.3. PPI-analysis PPI of the PEA vs. implicit baseline condition in ADHD vs. HC revealed clusters in the ACC, anterior insula, supramarginal gyrus (SMG) and substantia nigra (SN). Additionally, there were significant clusters in the angular gyrus, temporal pole, precuneus, lingual gyrus, posterior cingulate gyrus and posterior insula (Fig. 3A). See Table S2 for details (available online). Further analysis indicated positive coupling between the fPIR and the mentioned regions in ADHD compared to negative coupling in HC. Contrasting HC vs. ADHD revealed no significantly activated clusters. PPI of the menthol vs. implicit baseline condition in ADHD vs. HC revealed a cluster in the right ACC only (Fig. 3B). Here, in the peak voxel, children with ADHD showed positive coupling with fPIR compared to negative coupling in HC. Again, contrasting HC vs. ADHD revealed no significantly activated clusters. The additional direct PPI-comparison between PEA and menthol contrasting ADHD vs. HC revealed clusters in bilateral posterior as well as left anterior insula, ACC and putamen. In all these regions, the PPI-contrast estimates were shown to be positive in the patients and negative in HC. 4. Discussion As expected, increased sensitivity was found in children with ADHD only in the olfactory but not in trigeminal threshold performance when compared to healthy controls (HC). Furthermore, an increased volume of the OB was observed in ADHD. There were no group differences in neuronal activation towards olfactory and trigeminal stimuli (PEA and menthol). However, analysis of psychophysiological interactions (PPI) during olfactory stimulus processing revealed group differences in the functional connectivity
Table 2 Descriptive statistics of subjective ratings of chemosensory stimuli, rated on a visual analogue scale ranging from 0 (not at all) to 5 (maximum). PEA [Mean ± SD] ADHD Intensity Pleasantness Unpleasantness Familiarity
2.69 2.65 0.98 1.6
± ± ± ±
0.63 1.23 1.07 1.53
HC 2.81 2.52 1.76 1.54
± ± ± ±
0.46 1.26 1.46 1.28
Menthol [Mean ± SD] Total 2.75 2.58 1.38 1.57
± ± ± ±
0.54 1.22 1.33 1.38
ADHD 3.03 3.12 1.31 3.93
± ± ± ±
.63 1.26 1.61 1.17
Note: ADHD ¼ Attention-deficit/hyperactivity disorder, HC ¼ healthy controls, PEA ¼ phenylethyl alcohol, SD ¼ standard deviation.
HC 3.0 3.14 1.45 4.1
± ± ± ±
0.68 1.33 1.44 1.22
Total 3.02 3.13 1.38 4.0
± ± ± ±
0.65 1.27 1.5 1.18
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Fig. 3. Functional connectivity (PPI) in ADHD vs. HC. Whole brain analysis projected on DARTEL-template in neurological covention (p < .001, minimum cluster size ¼ 10 voxels, uncorrected). A: PEA B: Menthol. ACC ¼ anterior cingulate, SMG ¼ supramarginal gyrus, SN ¼ substantia nigra.
Skinner, 1988). Thus, cortical processes in terms of olfactory learning and attention (Huart et al., 2013) but also altered salience attribution could possibly influence the chemosensory circuits of the OB. The piriform cortex exerts a net inhibitory effect on bulbar excitation (Boyd et al., 2012). In the extreme case of disrupted centrifugal fibers, OB excitation could be enhanced by dampening of these inhibitory influences (Gray and Skinner, 1988). The OBvolume is shaped through mechanisms of synaptic plasticity as well as neurogenesis as a function of olfactory sensitivity (Huart et al., 2013). A higher olfactory performance due to increased bulbar excitability could thus lead to increased OB volume in ADHD. Whether or not dysregulations in dopamine-sensitive networks cause decreased neurogenesis of dopaminergic inhibitory interneurons leading to increased excitability of OB (Romanos et al., 2008; Schecklmann et al., 2011a,b) remains speculative. There is also the possibility of a peripheral mechanism since Hegg and Lucero (2004) have observed dopaminergic suppression through D2-receptors in olfactory receptor neurons. However, our fMRI results indicate changes in central mechanisms rather than pure peripheral effects. But we cannot rule out that peripheral mechanisms might play an additional role. Consequently, PETstudies (e.g. tracing dopaminergic signaling in the OB and olfactory/salient network) as well as the application of MPH in further fMRI-studies are required to elucidate the exact dopaminergic mechanisms. Processing of trigeminal menthol also involves connectivity changes between the fPIR and the salience network, albeit to a lesser extent than seen in olfactory processing. This indicates a global sensory alteration and not limited to the olfactory domain. Superior sensitivity in the olfactory domain in ADHD may be unique due to direct cortical feedback loops to the OB (bypassing thalamic relay). A possible confounding influence of IQ (marginal significant group difference) was controlled by adopting intelligence as a covariate in behavioral and volumetric analysis. Lower IQ scores in ADHD (but distinctly above cut-off of 85) may reflect the observed inability to sustain attention during the test procedure. Considerable overlap of olfactory and trigeminal stimulation in the olfactory cortex was observed. This is not surprising, considering that CO2 is the only chemo-stimulus which does not stimulate the olfactory system (Hummel and Livermore, 2002). In the present study, menthol was used as a trigeminal stimulus because this substance is more child-oriented than pungent CO2. Lack of additional somatosensory activation in the contrast menthol vs. clean air may be due to stimulus intensity not strong enough to elicit an effect of pungency. In addition, we already had to choose a liberal significance level because olfactory compared to e.g. visual stimuli are known to produce low intensity activations, high variability and low contrast-to-noise ratio (Morrot et al., 2013). Also, PPI-analyses tend to lack power and bear the risk for beta errors (O'Reilly et al., 2012). Since PEA and menthol were presented intermixed in an
event-related design, there might be a concern of possible interactions between the olfactory and trigeminal stimulus processing. Although the ITI in the fMRI measurement was considerably long (20.5 s on average), the ITI itself was variable (jitter: 18.8e26.2 s), and the order of chemosensory stimuli was pseudorandomized, we still cannot exclude such possible interactions. For example, an events-in-blocks design (Albrecht et al., 2009) with longer ITIs might help not only to increase the signal power but also to reduce possible interactions between different kinds of chemosensory stimuli. Taken together, children with ADHD displayed a selective superior olfactory sensitivity, accompanied by an increased OB volume. Functional connectivity between the primary olfactory cortex and regions of the dopamine-sensitive salience network were altered in ADHD, suggesting a hyper-sensitive olfactory processing that might trigger processes of OB neuroplasticity. Conflicts of interest None Contributions Anna Lorenzen: Data collection, analysis of the data, article preparation. Deborah Scholz-Hehn: Data collection, analysis of the data, article preparation. Christian D. Wiesner: Contributed in data analysis and interpretation. Author has approved final article Stephan Wolff: Contributed in data acquisition. Author has approved final article Til O. Bergmann: Contributed in data analysis and interpretation. Author has approved final article Thilo van Eimeren: Contributed in data analysis and interpretation. Author has approved final article Luisa Lentfer: Diagnostics. Author has approved final article Lioba Baving: Contributed in data analysis and interpretation. Author has approved final article Alexander Prehn-Kristensen: Supervision of data acquisition, analysis and article preparation. Acknowledgements We would like to thank Oliver Granert (Department of Neurology, University Hospital Schleswig-Holstein, Kiel, Germany) for his valuable advice concerning the fMRI design as well as Susanne Kell and Petra Schneckenburger (Department of Child and
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