socio-emotional development evaluated by output … · page 1 of 8 study protoco icensee o...

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Study protocol

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For citation purposes: Koshiba M, Nakamura S, Mimura K, Senoo A, Karino G, Amemiya S, et al. Socio-emotional development evaluated by Behaviour Output analysis for Quantitative Emotional State Translation (BOUQUET): towards early diagnosis of individuals with developmental disorders. OA Autism 2013 Jul 21;1(2):18. Co

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ts Socio-emotional development evaluated by Behaviour Output analysis for Quantitative Emotional State

Translation: towards early diagnosis of individuals with developmental disorders

M Koshiba1*, S Nakamura2, K Mimura2, A Senoo1,2, G Karino1,2, S Amemiya1, T Miyaji1, T Kunikata1, H Yamanouchi1

AbstractIntroductionThe main symptoms of autism spec-trum disorders are difficulties in so-cial communication. The complexity in defining the quality and extent of these social communication difficul-ties in terms of behaviour hampers the precision of diagnostic compre-hension and early treatment inter-vention. This research attempts to understand the emotional altera-tion through social communication behaviours. This is done through an approach using active and inter-ventional observation. The study analyses how animal and human participants produce social-context dependent behaviours allowing researchers to interpret the socio-emotional valence of behaviours. The comprehension algorithms are explored through the visuali-sation of behaviour features in a statistically constructed space us-ing a method called the ‘Behaviour Output analysis for Quantitative Emotional State Translation (BOU-QUET)’. This is based on principal component analysis in two model animals: domestic fowl chicks (Gal-lus gallus domesticus) and common marmosets (Callithrix jacchus). This is also a preliminary study of the BOUQUET application with a pre-mature infant inside an incubator of

Neonatal Intensive Care Unit at high risk for developmental disorders.Study ProtocolThe models used were chick models, common marmoset models, preterm infant study and a BOUQUET analysis was performed.DiscussionBOUQUET is a graphical presentation of a time-dependent multivariate cor-relation. It visualised a trajectory of socio-emotional development, brain region-specific molecular dynamics and nutritive effects in peripheral molecules and physiology on socio-emotional behaviour. Further ap-plication of BOUQUET on a preterm infant preliminarily suggested the behavioural diversification different from other primitive reflexes. The aim of the research is to make BOU-QUET contributory towards quanti-tative early diagnosis.ConclusionStructuralising of multivariate corre-lation between behaviour and physi-ology, potentially contributes objec-tive diagnosis and care design in the developmental psychiatry.

IntroductionThe diagnosis of persons with au-tism spectrum disorder (ASD)1 has been solely based on behaviours ac-cording to the 10th revision of the International Statistical Classifica-tion of Diseases and Related Health Problems (ICD-10) and Diagnostic and Statistical Manual of Mental Dis-orders, Fifth Edition (DSM-V). This situation is based on clinical history and also from the neurobiological

complexity of the socio-emotional symptom2–5. Children with ASD often have hyper-sensitivity and difficul-ties in vision, auditory and somato-sensory information processing, in addition to motor control difficulties. The uniqueness of early developed sensory-motor systems may lead to different sensory attention towards one’s environment, difficulty of interpretation of others’ goal-direct-ed behaviour and a social communi-cation development different from the ‘norms’ of a society. In parallel and correlated sensory-motor infor-mation processing, the emotional limbic system may take different de-velopmental pathways in attachment formation, empathy, reward systems and theory of mind in later stages. The outcome of these differences would cause a highly significant vari-ance in a complex brain system6,7. Whatever the reason, the objective quantification of complex behaviours is necessary stating points to under-stand the neurobiological bases of the disorders. In this study, a devel-oped multivariate behaviour analysis method is used to evaluate a particu-lar participant’s behaviour among the affected group’s behaviour. There is difficulty in defining and attempting to reintegrate the extracted behav-iour parameters into a statistically defined space through video-record-ed data of the core features of a com-plicated symptom8. To address this issue, a visualised method called the Behaviour Output analysis for Quan-titative Emotional State Translation’ (BOUQUET) (Figure 1) was devel-

* Corresponding author Email: [email protected] Saitama Medical University, Saitama, Japan2 Tokyo University of Agriculture and Technol-

ogy, Tokyo, Japan

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For citation purposes: Koshiba M, Nakamura S, Mimura K, Senoo A, Karino G, Amemiya S, et al. Socio-emotional development evaluated by Behaviour Output analysis for Quantitative Emotional State Translation (BOUQUET): towards early diagnosis of individuals with developmental disorders. OA Autism 2013 Jul 21;1(2):18. Com

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conditions: socially isolated (Iso) or grouped (Grp), six chicks without car-egivers’ handling. In the behavioural test, a subject chick was transferred to a novel meeting cage to face the un-familiar reference chicks presented with both visual and acoustic cues for 2 min (Figure 2). All behaviour was recorded using a top video camera (SONY, Panasonic or Victor, Japan). Monoamines were measured by high-performance liquid chromatography (HPLC) (EiCOM, Kyoto) with each of 13 divided brain parts (Figure 3c) of six females per group prepared at P14 around 2 pm after leaving under the dark for 30 min in the home cage. The subject’s behaviour was tested 1 day before brain sampling.

Common marmoset modelsThe experimental protocol was ap-proved by the Ethics Review Com-mittee for Animal Experiments of Tokyo University of Agriculture and Technology, TUAT (20-21) that fol-lows the animal care and experimen-tal guidelines of Japan Neuroscience Society and National Institutes of Health in USA. Two subjects, a 1.5- to 2-year-old female (A) and male (B) were kept in each cage and their behaviours were recorded by an USB camera (Logicool, Tokyo) and the rel-ative body surface temperature by in-frared camera (TP-L, Chino, Tokyo). The reference peers for behavioural test included another female and two other males in unfamiliar condi-tion. Four mg/kg weight of ubiqui-nol (QH, reduced form of coenzyme Q10, Kaneka) was administered to either subject mixed with milk (1 ml through syringe) at 9–10 am every morning for 2 months followed by cross-over control administration for 2 months after 1-month-interval. Blood was sampled from the tail vein before ubiquinol administration and the concentrations of ubiquinol and ubiquinone in plasma were de-termined by HPLC (Alliance 2695 separations module, Waters, USA). Blood glucose was also measured

Figure 1: The BOUQUET concept for objective analysis of emotion, supposedly constructed by multimodal nervous functions. Considering many emotion-relevant factors as possible to comprehend a psychological state, especially in the context of psychological or physical environment should be experimentally considered as a window for active/interventional observation. BOUQUET (Behaviour Output analysis for Quantitative Emotional state Translation) is the generic term for translation between subjective and objective information.

oped based on principal component analysis (PCA)9–19. One drawback of the quantification is the tendency to ignore qualitative difference among patients. To link both quantita-tive and qualitative information, an experimental animal model of psy-chological development was studied with multivariate analysis. Consider-ing comparative simulation of emo-tional behaviour, we chose two diur-nal, rich-vocalised species with full of homologous priorities for develop-mental neurobiological psychiatry18 domestic fowl chicks (Gallus gallus domesticus)8–13,18–21 from avian22 and common marmosets (Callithrix jac-chus)8,14–18. This article presents the result of two lines of experimental animal studies. The first one is the con-specific social context-depend-ent motor activities such as vocalisa-tion, social facing and distance. These were extracted from video-record-ing and their integrated statistical correlation patterns were challenged

to interpret psych-cognitive mean-ings. Second, their physiological and biomolecular dynamics in vivo were quantified and the information was again statistically correlated with the behaviour data by BOUQUET to integrate over hetero modal informa-tion into a brain mapping. Finally, the approach was applied to preterm in-fants in an effort to realise early diag-nosis and environmental support for persons with ASD.

Study ProtocolChick modelsThis protocol was approved by the Ethics Review Committee for Animal Experiments of Tokyo University of Agriculture and Technology, TUAT (19-19). Fertilised eggs from domes-tic chicks (G. g. domesticus), white Leghorn, Maria strain, were hatched (the day of hatching was labelled as postnatal day 1: P1) and reared un-til the day for behavioural tests in home incubator under two different

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structuralisation and ellipse ap-proximation whose centre was the average of the plots, the long and short axes were derived from the first and second component eigen-vectors multiplied by the square root of the eigenvalue, using vari-ance–covariance matrix. The statis-tical analysis was performed with Wilks’ lambda distribution and two-sampled t-test using free software R and Excel (Microsoft). The 3D graph of two PCA components and time was plotted and lined by OriginPro, 7.5 ( OriginLab, Northampton). The factor loading vector reflecting the correlation of the eigenvector with the two component axes was illus-trated only with the plus direction and the minus vector was omit-ted. The most significantly differ-ent components for discrimination were used depending on each pur-pose of group comparison.

Age-dependent modulation and a flexion period of peer social behaviour revealed by PCA multimodal processingIso and Grp chicks were tested once on P3.7.13.17.2110. Their behaviour was analysed and the developmen-tal trajectory is shown in Figure 2a. The flexion point of the develop-mental trajectory in Iso appeared most clearly in a 3D time space at P7 (black star, Figure 2b,d), where the ellipse of Iso chicks promi-nently shifted to a ‘negative’ direc-tion on the factor loading vectors’ clustering (Figure 2c) that could be translated as ‘immobile’ at P3 (in the third quadrant), while they emitted negative calls in response to unfamiliar con-specific chicks at P7 but less immobility (in the fourth quadrant), however, returned to im-mobile state until P21. Grp chicks exhibited gradual modulation on the first component axis from posi-tive to negative over developmental time from P3 to P21, which means becoming less active. The Grp

Figure 2: A flexion period for peer-social response at P7 was revealed by behavioural development analysis using multivariate correlation processing. (a) A subject chick (left) of either socially isolated (Iso) or grouped (Grp) at home met a few unfamiliar reference chicks in a behavioural test. The head and beak coordinate, call type and frequency from spectrogram were extracted from video-recorded image and subjected to PCA-based BOUQUET analysis. (b) The developmental trajectories of Grp and Iso chicks in the 3D-graph consisted of the first and second PCA component as X, Y and age as Z axes. (c) The factor loading vectors of 14 behavioural factors calculated by PCA at all ages. Consequently, the correlation vectors highlighted four clusters denoted as active, immobile, positive and negative. The same vectors multiplied arbitrarily are shown at the bottom of the 3D-graph (b). (d) PCA scores for the Grp/Iso data at each stage as top view of (b) were plotted with variance ellipses. Statistical significance by Wilks’ lambda to compare between Iso and Grp chicks are represented asterisks (**P < 0.01, *P < 0.05).

by the glucose sensor (GLUCOCARD-ARKRAY. Factory Inc., Shiga).

Preterm infant studyAll the protocol was approved by the Institute Review Board for Clinical Analysis of Saitama Medical Universi-ty (12-073-1). On the code of the ap-proval, written informed consent was provided to the parents of the patient. The participant was a typically devel-oping preterm male infant, corrected gestational age 39 weeks at our ob-servation, who was born at 30 weeks, weighing 728 g with apgar score 7 at 1 min, and 7 at 5 min, had been cared in an incubator of Neonatal Intensive Care Unit (NICU). A top view video camera (Logicool) recorded the in-fant through the incubator’s plastic

cover for a few minutes while the in-fant was turned over from prone to supine position by the examiner.

BOUQUET analysisFrom the recorded WAVE and JPEG files per second, the parameters (animals: the x, y coordinate of head centre, forehead direction, the hori-zontal or rotation velocity of head, local preference, pecking and vo-cal call behaviours that had previ-ously identified four spectrogram patterns depending on socio-emo-tional contexts; human infant; the X, Y specific points of arthrometry: head, trunk, arms and legs). Mul-tiple parameters were integrated by BOUQUET9–19 with PCA based on a correlation matrix for general

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chicks’ data plots were allocated in the first quadrant at P3, which was featured as socially affiliated by the factor loading vectors, such as ap-proaching to peers, facing peers and positive calls (not a distress-type call but a pleasure call)9.

Chick peer-social brain map characterised by correlation between brain monoamines in darkness and behaviour features one-day-before monoamines samplingNeuro-molecular studies of peer-so-cial development were sequentially explored in these chick models (so-cially isolated and grouped chicks) first with the behaviour test and the subsequent brain monoamine analysis13. In the results of screen-ing any differences between Iso and Grp (P < 0.1; two sampled t-test) in ten kinds of monoamines, focus was

on serotonergic metabolism, 5-hy-droxyindoleacetic acid (5-HIAA) and four kinds of catecholamines, which are dopamine (DA), dihydroxypheny-laceticacid (DOPAC), norepinephrine (NE) and epinephrine (Epi). To find any correlation between behaviour feature and monoamines, behavioural parameters together with one spe-cies of monoamines in each of 13 brain sub-regions were analysed us-ing BOUQUET and visualised (Figure 3b). The correlation coefficient (R) of Pearson product-moment between monoamine concentration and ei-ther the first or second PCA scores of behaviour data was defined as the monoamine vector (Figure 3b, a red arrow). The monoamine vector was colour-coded according to the quadrant valence and colour-graded by each R value (Figure 3b,c). Three kinds of monoamines were signifi-cantly expressed more in left caudal

forebrain of Iso chicks. The coloured brain map expressed that the corre-lated behavioural features of 5-HIAA, DA and DOPAC were ‘immobile’, ‘neg-ative’ and ‘immobile’, respectively. Meanwhile, the right thalamus-mid-brain region showed its correlation of NE and Epi with immobile behaviour.

Chronic administration effect of antioxidant nutrition on primate social behaviour revealed by BOUQUET reduced-oxidised axisTwo isolated adult marmosets, a fe-male and a male, were observed with 4 mg/kgwt/day of the ubiquinol or vehicle (corn oil) dietary adminis-tration in milk for two terms of 3 months16. After a 2-month interval followed the first term, we swapped the condition in the second term (Figure 4b, vertical time axis from bottom to top). The socio-emotional effect of ubiquinol dietary supple-mentation was recorded by our be-havioural test8,14–18 per second with relative body surface temperature versus room temperature by infrared cameras. The metabolic effect of di-etary ubiquinol was measured with plasma ubiquinol and ubiquinone that are reduced and oxidised form of coenzyme Q10 and blood glucose ac-quired by a stress-repressed sampler and assay kits. BOUQUET derived from all the parameters consequent-ly revealed a projected plane featur-ing negatively correlated ubiquinol–ubiquinone axis by the factor loading vectors (Figure 4c). In these screened multiple parameters, the factors cor-related with plasma ubiquinol were ‘approach’ positively and ‘relative body surface temperature’ negative-ly that was weakly correlated with ubiquinone positively (Figure 4c). It is consistently confirmed in Figure 4b that any distribution of ubiquinol-administrated animals (blue ellip-ses) emerged at the ubiquinol factor loading vector direction than vehicle administered ones (grey ellipses) but simple active mass axis (the longest green vector) appeared not much

Figure 3: The whole-brain maps representing the correlation of peer-social environment dependent monoamines and behavioural features. (a) Brain sampled at home in darkness expressed more monoamines, 5-HIAA, DA, DOPAC, NE, and Epi in Iso than Grp chicks. (b) The schematic representation of the correlation of a single species of monoamine in a sub-brain region with behaviour parameters by BOUQUET analysis (see more in text). The colour density indicates as ‘0.3 < R < 0.6 (shorter length vector, light)’ or ‘R ≥ 0.6 (longer length vector, dark)’ and this rule is used for colour of (c). (c) BOUQUET revealed 5-HIAA and DOPAC in the left caudal forebrain, or NE and Epi in the right thalamus-midbrain expression, which correlated with ‘immobile’ behaviour more in Iso chicks. DA in the left caudal forebrain weakly correlated with negative behaviour features.

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Figure 4: BOUQUET for dietary supplementary effect in adult common marmoset’s social emotions by visible/infrared cameras, plasma Redox forms of CoQ10 and blood glucose. (a) A scene of social behaviour test detected by USB (upper) and infrared (lower) cameras. (b, c) BOUQUET 3-D analytic space (b) is constructed by the plane explained by the factor loading vectors (c) and vertically age axis in month. The results of five times behaviour tests were traced for either subject A or B by each line dotted or solid, respectively. Note that the subject behaviour administered with ubiquinol (blue variance ellipse, marked by star) shifted towards the increment of first component.

correlated with the chronic nutrition appeared as plasma glucose level (the shortest red vector).

Preliminary trial of BOUQUET application to a preterm infant BOUQUET was applied to a pre-term infant inside the NICU incuba-tor for preventive intervention of developmental disorders. The multi-ple points of the infant’s extremities and trunk were tracked when he was turned over from the prone to supine position as shown in Figure 5a, and both relative velocity of the points

and the vector angles between two lines linking two adjacent points along the body part were assessed for BOUQUET coordinate of components x and y (Figure 5b, c). These plots demarcated four kinds of groups in order of time, depending on the sub-jective discrimination as ‘Prone’, two kinds of primitive reflexes23,24 ‘Moro’ and ‘Palmar’ and ‘Dorsal Flexion’. The term Dorsal Flexion is arbitrar-ily defined as a kind of endeavouring movement of whole body posture leading to dorsal arching (Figure 5a, top). To explore any features quanti-

tatively, it was projected on both the points’ velocity and tilt angle shown as the factor loading vectors in Figure 5d. As a result, referring to the first points of prone in the third quadrant, the ‘Dorsal Flexion’ rather scattered around negative component y, ap-peared highly discriminative from the two primitive reflexes, Moro and Palmar around the second quadrant. In comparison of each relative area of approximated ellipse, Dorsal Flex-ion (3.7) appeared certainly the most diversified than either Moro (1.7) or Palmer (1.0).

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social communication difficulties such as ASD. First, the multivariate statistic method was developed to in-terpret the behaviour of experimen-tal animals based on the correlation structure of behaviour parameters

stage-specific, species-specific, but has impacts among cross-species as well. This study explored the pos-sibility to translate behaviour into emotional states, which should be relevant for objective diagnosis of

Discussion Socio-emotional development is a complex process through interac-tion and integration of sensory-motor, limbic and cognitive sys-tems5–15,17–21,25–30. This process is

Figure 5: Preliminary trial of BOUQUET application for a preterm immature infant. (a) A male infant was video recorded for a few minutes of the given posture modulation from prone to turned-up. Molor reflex (red), Palmar reflex (green), and dorsal flexion (blue) sequentially emerged (arrow). The coordinates of multiple points (circle) of body were analysed. (b–d) The BOUQUET 3D space (b) is explained by the factor loading vectors (d). The top view of 3D space is presented in (c) with colour plots matched to the colour code of reflexes and flexion behaviours (a).

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as ‘Dorsal Flexion’ that must give the infant more chances to interact with environment and learn. Sub-ject number was small and further expansion is certainly required. The development of BOUQUET applica-tion for infants’ behavioural diag-nosis may discriminate prospective problems quantitatively and suggest a personalised intervention such as rehabilitation and supplemental nu-trition16,19 from early life. With fur-ther comprehension of molecular or any other studies of paediatric neu-rology29,30, BOUQUET would possibly open the next stage of developmental psychiatry.

The study presented the develop-ment of the comprehensive method-ology, ‘BOUQUET’ for the analysis of social interaction development with two experimental animals, domestic chick and small New World primate, common marmoset. This method may contribute to visualise complex, higher-order neuronal functions and their time-dependent modulations, thus can be applicable to early di-agnosis support of person with ASD and neurobiologically relevant inter-vention to improve their quality of lives.

ConclusionFor the coming generation of objec-tive diagnosis and care design, in de-velopmental psychiatry, our model animal and human infant studies suggested that imaging of multivari-ate correlation between behaviours, molecules and physiology, named as BOUQUET, quantitatively charac-terised development of emotional functions.

AbbreviationsASD, autism spectrum disorder; DA, dopamine; DOPAC, dihydroxypheny-laceticacid; Epi, epinephrine; HPLC, high-performance liquid chroma-tography; NE, norepinephrine; PCA, principal component analysis; Pn, postnatal day n (P1 = first day after hatching); TUAT, Tokyo University of

which might summarise a set of brain socialisation-specific network system between right thalamus-midbrain in-cluding ventral tegmental area, raphe nuclei and locus coeruleus, and left caudal forebrain including amygdale homolog although additional studies must be needed for confirmation.

In the BOUQUET following veri-fication to visualise adult primate social emotion (Figure 4), the plot distribution in the coordinate struc-turalised some antioxidant dietary effects as multimodal correlation of social behaviour, physiology of body temperature and plasma redox mol-ecules, quantitatively. The repeated appearances of ubiquinol uptake el-lipses confirmed by its concentration in plasma could be translated as high-er social affinity higher with relative lower body surface temperature.

Finally, BOUQUET can be applied to quantitative behavioural diagnosis for human early development (Figure 5) because of ASD relevance to low birth weight and small for gestation-al age25. We observed ‘Dorsal Flexion’ besides primitive reflexes of Moro and Palmar. ‘Dorsal Flexion’ contin-ued for more than a minute after be-ing turned-over from prone position and the infant seemed to integrate activity by multiple muscles with making the body lift against the grav-ity, though its uneconomical usage of energy, implying some primitive voluntary movement involving mid-brain function such as righting reflex seen at 2–6 months after birth23,24. The postural adjustment movement is led by vestibular, visual and soma-tosensory functions whose problems are well-known as the cause of ASD5. Kuniyoshi26, Asada27 and Pfeifer28 et al. suggested initialisation and self-organised optimisation of recurrent neural network connectivity using mathematical models and simulation by robotics and computation. Using the synthetic approach, it would be interesting to explore the neuronal basis of the diversified motor activ-ity other than primitive reflex such

that can be captured by video-re-cording. The principal component analysis was used for extracting the multivariate correlation since it con-stitutes a vector space where each behavioural parameter is represent-ed as a factor loading vector in the space and the inner product of the factor loading vectors shows the cor-relation of the behaviour parameters without any prior-statistical mod-els. Another concern was a design of social context to explore the socio-emotional interpretation. Through prior design, the psycho-cognitive contexts can interpret the emotional valence of behaviour change over the contexts. This led to the behav-iour analysis approach being named BOUQUET9–19. BOUQUET has four current applications: (1) chick social behaviour development depending on social environment during early developmental stages, (2) the effect of reduced form CoQ10 on marmo-set social behaviour and peripheral physiology, (3) construction of a functional brain map based on corre-lation analysis of monoamines con-tent in brain sub-regions and social behaviours and (4) evaluation of be-haviours of a human preterm infant in an incubator of NICU to objectively characterise future preventive diag-nosis as early as possible. The fol-lowing briefly summarises the main findings and their interpretations in the research field of social commu-nication, affective neuroscience and developmental disorders.

Considering even simpler develop-mental function in chicks as prelimi-nary studies, BOUQUET with multi-ple factors contributed to visualise complex and refined developmental trajectory lines depending on social environment as shown in Figure 2. By this coordinate translation, a unique flexion point during this ani-mal’s development could be discrim-inated. The BOUQUET availability of multimodal parameters allowed this animal’s brain monoaminergic cor-relation maps as shown in Figure 3,

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Agriculture and Technology; 5-HIAA, 5-hydroxyindoleacetic acid.

AcknowledgementsWe thank Professor Masakazu Koni-shi at Caltech for encouragement. This work was partially supported by MEXT/JSPS Kakenhi (21200017, 25119509 and 25282221), and by MHLW (Intramural Research Grant (22–6)), JST CREST (2005–2006), JST-A-step, ALCA, JSPS Research Fel-low, DC2, Japan.

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