Exploring the emotional brain: Neural correlates of homeostatic and sensory-evoked emotions and their interaction in emotional rivalry

Human emotions are essential for survival. They are vital for the satisfaction of basic needs, the regulation of personal life and successful integration into social structures. Depending on which aspect of an emotion is used in its definition, many different theories offer possible answers to the questions of what emotions are and how they can be distinguished. The systematic investigation of emotions in cognitive neuroscience is relatively new, and neuroimaging studies specifically focussing on the neural correlates of different categories of emotions are still lacking. Therefore, the current thesis aimed at investigating the behavioural and neurophysiological correlates of different human emotional levels and their interaction in healthy subjects. We differentiated between emotions according to their cerebral entry site and neural processing pathways: homeostatic emotions, which are elicited by metabolic changes and processed by the interoceptive system (such as thirst, hunger, and need for air), and sensory-evoked emotions, which are evoked by external inputs via the eyes, ears or nose, or their corresponding mental representations and processed in the brain as sensory perception (e.g. fear, disgust, or pride). Using functional magnetic resonance imaging (fMRI) and behavioural parameters, we examined both the specific neural underpinnings of a homeostatic emotion (thirst) and a sensory-evoked emotion (disgust), and their interaction in a situation of emotional rivalry when both emotions were perceived simultaneously. This thesis comprises three research articles reporting the results of this research. The first paper presents disgust-related brain imaging data in a thirsty and a satiated condition. We found that disgust mainly activated the anterior insular cortex. In the thirsty condition, however, we observed an interaction effect between disgust and thirst: when thirsty, the subjects rated the disgusting stimulus as less repulsive. On the neurobiological level, this reduction of subjective disgust was accompanied by significantly reduced neural activity in the insular cortex. These results provide new neurophysiological evidence for a hierarchical organization among homeostatic and sensory-evoked emotions, revealing that in a situation of emotional conflict, homeostatic emotions are prioritized over sensory-evoked emotions. In the second paper, findings on brain perfusion over four different thirst stages are reported, with a special focus on the parametric progression of thirst. Cerebral perfusion differences over all thirst stages were found in the posterior insular cortex. Taking this result together with the findings of the first paper, the insular cortex seems to be a key player in human emotional processing, since it comprises specific representations of homeostatic and sensory-evoked emotions and also represents the site of cortical interaction between the two levels of emotions. Finally, although this thesis focussed on the homeostatic modulation of disgust, we were also interested in whether dehydration modulates taste perception. The results of this behavioural experiment are described in the third paper, where we show that dehydration alters the perception of neutral taste stimuli.

Serotonin versus catecholamine deficiency: behavioral and neural effects of experimental depletion in remitted depression

Despite immense efforts into development of new antidepressant drugs, the increases of serotoninergic and catecholaminergic neurotransmission have remained the two major pharmacodynamic principles of current drug treatments for depression. Consequently, psychopathological or biological markers that predict response to drugs that selectively increase serotonin and/or catecholamine neurotransmission hold the potential to optimize the prescriber's selection among currently available treatment options. The aim of this study was to elucidate the differential symptomatology and neurophysiology in response to reductions in serotonergic versus catecholaminergic neurotransmission in subjects at high risk of depression recurrence. Using identical neuroimaging procedures with [(18)F] fluorodeoxyglucose positron emission tomography after tryptophan depletion (TD) and catecholamine depletion (CD), subjects with remitted depression were compared with healthy controls in a double-blind, randomized, crossover design. Although TD induced significantly more depressed mood, sadness and hopelessness than CD, CD induced more inactivity, concentration difficulties, lassitude and somatic anxiety than TD. CD specifically increased glucose metabolism in the bilateral ventral striatum and decreased glucose metabolism in the bilateral orbitofrontal cortex, whereas TD specifically increased metabolism in the right prefrontal cortex and the posterior cingulate cortex. Although we found direct associations between changes in brain metabolism and induced depressive symptoms following CD, the relationship between neural activity and symptoms was less clear after TD. In conclusion, this study showed that serotonin and catecholamines have common and differential roles in the pathophysiology of depression.

Neuroanatomical correlates of tube dependency and impaired oral intake after hemispheric stroke.

BACKGROUND AND PURPOSE Acute stroke patients with severely impaired oral intake are at risk of malnutrition and dehydration. Rapid identification of these patients is necessary to establish early enteral tube feeding. Whether specific lesion location predicts early tube dependency was analysed, and the neural correlates of impaired oral intake after hemispheric ischaemic stroke were assessed. METHODS Tube dependency and functional oral intake were evaluated with a standardized comprehensive swallowing assessment within the first 48 h after magnetic resonance imaging proven first-time acute supratentorial ischaemic stroke. Voxel-based lesion symptom mapping (VLSM) was performed to compare lesion location between tube-dependent patients versus patients without tube feeding and impaired versus unimpaired oral intake. RESULTS Out of 119 included patients 43 (36%) had impaired oral intake and 12 (10%) were tube dependent. Both tube dependency and impaired oral intake were significantly associated with a higher National Institutes of Health Stroke Scale score and larger infarct volume and these patients had worse clinical outcome at discharge. Clinical characteristics did not differ between left and right hemispheric strokes. In the VLSM analysis, mildly impaired oral intake correlated with lesions of the Rolandic operculum, the insular cortex, the superior corona radiata and to a lesser extent of the putamen, the external capsule and the superior longitudinal fascicle. Tube dependency was significantly associated with affection of the anterior insular cortex. CONCLUSIONS Mild impairment of oral intake correlates with damage to a widespread operculo-insular swallowing network. However, specific lesions of the anterior insula lead to severe impairment and tube dependency and clinicians might consider early enteral tube feeding in these patients.

Correlation of regional cerebral blood flow and change of plasma sodium concentration during genesis and satiation of thirst

Positron emission tomography studies were conducted during genesis of moderate thirst by rapid i.v. infusion of hypertonic saline (0.51 M) and after satiation of thirst by drinking water. The correlation of regional cerebral blood flow with the change in the plasma Na concentration showed a significant group of cerebral activations in the anterior cingulate region and also a site in the middle temporal gyrus and in the periaqueductal gray. Strongest deactivations occurred in the parahippocampal and frontal gyri. The data are consistent with an important role of the anterior cingulate in the genesis of thirst.

Functional neuroimaging studies of encoding, priming, and explicit memory retrieval

Human functional neuroimaging techniques provide a powerful means of linking neural level descriptions of brain function and cognition. The exploration of the functional anatomy underlying human memory comprises a prime example. Three highly reliable findings linking memory-related cognitive processes to brain activity are discussed. First, priming is accompanied by reductions in the amount of neural activation relative to naive or unprimed task performance. These reductions can be shown to be both anatomically and functionally specific and are found for both perceptual and conceptual task components. Second, verbal encoding, allowing subsequent conscious retrieval, is associated with activation of higher order brain regions including areas within the left inferior and dorsal prefrontal cortex. These areas also are activated by working memory and effortful word generation tasks, suggesting that these tasks, often discussed as separable, might rely on interdependent processes. Finally, explicit (intentional) retrieval shares much of the same functional anatomy as the encoding and word generation tasks but is associated with the recruitment of additional brain areas, including the anterior prefrontal cortex (right > left). These findings illustrate how neuroimaging techniques can be used to study memory processes and can both complement and extend data derived through other means. More recently developed methods, such as event-related functional MRI, will continue this progress and may provide additional new directions for research.

General and specific brain regions involved in encoding and retrieval of events: what, where, and when.

Remembering an event involves not only what happened, but also where and when it occurred. We measured regional cerebral blood flow by positron emission tomography during initial encoding and subsequent retrieval of item, location, and time information. Multivariate image analysis showed that left frontal brain regions were always activated during encoding, and right superior frontal regions were always activated at retrieval. Pairwise image subtraction analyses revealed information-specific activations at (i) encoding, item information in left hippocampal, location information in right parietal, and time information in left fusiform regions; and (ii) retrieval, item in right inferior frontal and temporal, location in left frontal, and time in anterior cingulate cortices. These results point to the existence of general encoding and retrieval networks of episodic memory whose operations are augmented by unique brain areas recruited for processing specific aspects of remembered events.

Le rôle de la méthylation de l’ADN dans les fonctions cérébrales fronto-limbiques et la réactivité au stress quotidien

La sérotonine (5-HT) joue un rôle crucial dans l'étiologie des troubles mentaux comme la dépression majeure, les troubles de comportement et les troubles anxieux. Des études ont montré que des altérations précoces du système 5-HT peuvent potentiellement influencer le développement du cerveau et le fonctionnement du système fronto-limbique, engendrant des conséquences pour la régulation émotionnelle. Il existe aussi des évidences que le stress précoce peut affecter la méthylation de l'ADN résultant d'une altération de l'expression génique. Toutefois, le lien entre la méthylation de l'ADN et la réactivité comportementale à des facteurs de stress de la vie quotidienne est inconnu. La méthylation du gène transporteur 5-HT (SLC6A4) est d'un intérêt particulier, étant donné le rôle de SLC6A4 dans le développement du cerveau, les troubles mentaux et la régulation du stress. L'objectif de cette thèse est d'étudier l'association entre (1) les niveaux périphériques de méthylation de l'ADN dans le gène SLC6A4 et les réponses neurales aux stimuli émotionnels dans les circuits fronto-limbiques du cerveau, ainsi qu’entre (2) la méthylation périphérique de SLC6A4 et la réactivité comportementale au stress de la vie quotidienne. Nous explorons également l'association entre les réponses neuronales fronto-limbique à des stimuli émotionnels et la réactivité comportementale au stress de la vie quotidienne (3). À cette fin, vingt-deux personnes (11 femmes) d’âge moyen de 34,0 ans (SD : 1,5) avec différents niveaux de méthylation au gène SLC6A4 ont été recrutés à partir de deux études longitudinales. Les participants ont subi une analyse IRMf qui comprenait une tâche de traitement émotionnel. Un questionnaire en ligne sur la réactivité au stress quotidien de la vie a été réalisé pendant 5 jours consécutifs. Des analyses corrélationnelles et de régression ont été effectuées pour examiner les associations entre les variables primaires. Les résultats préliminaires de cette étude ont montré que la méthylation de l'ADN est associée à la désactivation significative du gyrus précentral et gyrus fusiforme respectivement face à des stimuli de peur et de tristesse. Aucune association significative n'a été observée entre les niveaux de méthylation et l'activation de l'amygdale. En outre, les scores obtenus aux variables de stress de la vie quotidienne tels que la détresse chronique ont été associées à la désactivation du précuneus et du cortex cingulaire postérieur face à la tristesse. Ces résultats suggèrent l'implication potentielle des processus épigénétiques dans l'activation cérébrale spécifique et la sensibilité au stress de la vie courante.

Corrélats neuronaux du circuit des récompenses chez les jeunes à risque parental de troubles de l'humeur

Cette thèse a pour objectif l’investigation du circuit des récompenses, sur les plans comportementaux et neuronaux, chez des adolescents à risque parental élevé de dépression majeure et de trouble bipolaire, en comparaison à des jeunes à risque parental peu élevé. Plus précisément, le but est d’identifier des marqueurs comportementaux et neuronaux du risque de développer une dépression majeure ou un trouble bipolaire, afin d’être en mesure de détecter et de prévenir ces troubles le plus tôt possible pour éviter, ou du moins retarder, leur émergence. Pour ce faire, nous avons réalisé deux études, présentées ici dans deux articles empiriques. Dans le premier article, le fonctionnement comportemental et neuronal du circuit des récompenses a été investigué au moyen d’une tâche d’anticipation et d’obtention de gains et de pertes monétaires, chez des adolescents à risque parental de dépression majeure (i.e., jeunes asymptomatiques dont un des parents souffre de dépression majeure), des adolescents à risque parental de trouble bipolaire (i.e., jeunes asymptomatiques dont un des parents souffre de trouble bipolaire) et des adolescents contrôles (i.e., jeunes asymptomatiques dont les deux parents sont en bonne santé mentale). Au niveau comportemental, les résultats ont révélé une meilleure performance chez les jeunes à risque de dépression majeure lorsqu’ils devaient éviter d’obtenir des pertes monétaires de magnitude variée (0,20$, 1$ ou 5$), ainsi qu’une meilleure performance chez les jeunes à risque de trouble bipolaire sur les essais impliquant d’éviter des pertes monétaires de magnitude nulle (0$). Au niveau neuronal, les jeunes à risque de dépression majeure démontraient une diminution de l’activation du cortex préfrontal dorsolatéral lors de l’anticipation de potentielles pertes monétaires de magnitude variée, tandis que les jeunes à risque de trouble bipolaire démontraient une diminution de l’activation du cortex préfrontal dorsolatéral lors de l’anticipation de potentielles pertes monétaires de magnitude nulle. De plus, les jeunes à risque de dépression majeure tendaient à démontrer une augmentation de l’activité du cortex orbitofrontal durant l’évitement réussi de pertes monétaires, tandis que les jeunes à risque de trouble bipolaire tendaient à démontrer une augmentation de l’activité du cortex orbitofrontal lors de l’obtention de pertes monétaires. Dans le deuxième article, l’intégrité structurelle des régions fronto-limbiques a été investiguée, au moyen de mesures du volume, de l’épaisseur corticale et de la superficie corticale. Les résultats ont mis en évidence, chez les jeunes à risque de trouble bipolaire, un volume plus élevé du cortex préfrontal dorsolatéral, par rapport aux jeunes à risque de dépression majeure et contrôles. De plus, les jeunes à risque de trouble bipolaire présentaient un volume plus élevé du cortex cingulaire postérieur, en comparaison aux jeunes à risque de dépression majeure. Enfin, une diminution de l’épaisseur corticale du cortex orbitofrontal et du gyrus frontal moyen a été observée chez les adolescents à risque de trouble bipolaire, en comparaison au groupe contrôle. L’ensemble de ces résultats démontre ainsi l’existence de particularités comportementales et d’altérations neuronales sur les plans fonctionnel et structurel, chez des jeunes à risque élevé de troubles de l’humeur, et ce, avant même l’émergence des premiers symptômes thymiques. Plus particulièrement, ces caractéristiques pourraient constituer des marqueurs du risque de développer un trouble de l’humeur. Par conséquent, ces marqueurs pourraient aider à mieux identifier les jeunes qui sont le plus à risque de développer un trouble de l’humeur, et ainsi permettre la mise en place précoce de stratégies préventives adaptées, afin d’éviter des trajectoires développementales psychopathologiques.

Corrélats neuronaux du circuit des récompenses chez les jeunes à risque parental de troubles de l'humeur

Cette thèse a pour objectif l’investigation du circuit des récompenses, sur les plans comportementaux et neuronaux, chez des adolescents à risque parental élevé de dépression majeure et de trouble bipolaire, en comparaison à des jeunes à risque parental peu élevé. Plus précisément, le but est d’identifier des marqueurs comportementaux et neuronaux du risque de développer une dépression majeure ou un trouble bipolaire, afin d’être en mesure de détecter et de prévenir ces troubles le plus tôt possible pour éviter, ou du moins retarder, leur émergence. Pour ce faire, nous avons réalisé deux études, présentées ici dans deux articles empiriques. Dans le premier article, le fonctionnement comportemental et neuronal du circuit des récompenses a été investigué au moyen d’une tâche d’anticipation et d’obtention de gains et de pertes monétaires, chez des adolescents à risque parental de dépression majeure (i.e., jeunes asymptomatiques dont un des parents souffre de dépression majeure), des adolescents à risque parental de trouble bipolaire (i.e., jeunes asymptomatiques dont un des parents souffre de trouble bipolaire) et des adolescents contrôles (i.e., jeunes asymptomatiques dont les deux parents sont en bonne santé mentale). Au niveau comportemental, les résultats ont révélé une meilleure performance chez les jeunes à risque de dépression majeure lorsqu’ils devaient éviter d’obtenir des pertes monétaires de magnitude variée (0,20$, 1$ ou 5$), ainsi qu’une meilleure performance chez les jeunes à risque de trouble bipolaire sur les essais impliquant d’éviter des pertes monétaires de magnitude nulle (0$). Au niveau neuronal, les jeunes à risque de dépression majeure démontraient une diminution de l’activation du cortex préfrontal dorsolatéral lors de l’anticipation de potentielles pertes monétaires de magnitude variée, tandis que les jeunes à risque de trouble bipolaire démontraient une diminution de l’activation du cortex préfrontal dorsolatéral lors de l’anticipation de potentielles pertes monétaires de magnitude nulle. De plus, les jeunes à risque de dépression majeure tendaient à démontrer une augmentation de l’activité du cortex orbitofrontal durant l’évitement réussi de pertes monétaires, tandis que les jeunes à risque de trouble bipolaire tendaient à démontrer une augmentation de l’activité du cortex orbitofrontal lors de l’obtention de pertes monétaires. Dans le deuxième article, l’intégrité structurelle des régions fronto-limbiques a été investiguée, au moyen de mesures du volume, de l’épaisseur corticale et de la superficie corticale. Les résultats ont mis en évidence, chez les jeunes à risque de trouble bipolaire, un volume plus élevé du cortex préfrontal dorsolatéral, par rapport aux jeunes à risque de dépression majeure et contrôles. De plus, les jeunes à risque de trouble bipolaire présentaient un volume plus élevé du cortex cingulaire postérieur, en comparaison aux jeunes à risque de dépression majeure. Enfin, une diminution de l’épaisseur corticale du cortex orbitofrontal et du gyrus frontal moyen a été observée chez les adolescents à risque de trouble bipolaire, en comparaison au groupe contrôle. L’ensemble de ces résultats démontre ainsi l’existence de particularités comportementales et d’altérations neuronales sur les plans fonctionnel et structurel, chez des jeunes à risque élevé de troubles de l’humeur, et ce, avant même l’émergence des premiers symptômes thymiques. Plus particulièrement, ces caractéristiques pourraient constituer des marqueurs du risque de développer un trouble de l’humeur. Par conséquent, ces marqueurs pourraient aider à mieux identifier les jeunes qui sont le plus à risque de développer un trouble de l’humeur, et ainsi permettre la mise en place précoce de stratégies préventives adaptées, afin d’éviter des trajectoires développementales psychopathologiques.

Identification of candidate genes at the corticoseptal boundary during development

Cortical midline glia are critical to the formation of the corpus callosum during development. The glial wedge is a Population of midline glia that is located at the corticoseptal boundary and expresses repulsive/growth-inhibitory molecules that guide callosal axons as they cross the midline. The glial wedge are the first cells within the cortex to express GFAP and thus may express molecules specific for glial maturation. The corticoseptal boundary is a genetically defined boundary between the cingulate cortex (dorsal telencephalon) and the septum (ventral telencephalon). The correct dorso-ventral position of this boundary is vital to the formation of both the glial wedge and the corpus callosum. Our aim was to identify genes expressed specifically within the glial wedge that might be involved in either glial differentiation, formation of the corticoseptal boundary or development of the corpus callosum. To identify such genes we have performed a differential display PCR screen comparing RNA isolated from the glial wedge with RNA isolated from control tissues such as the neocortex and septum, of embryonic day 17 mouse brains. Using 200 different combinations of primers, we identified and cloned 67 distinct gene fragments. In situ hybridization analysis confirmed the differential expression of many of the genes, and showed that clones G24F3, G39F8 and transcription factor LZIP have specific expression patterns in the telencephalon of embryonic and postnatal brains. An RNase Protection Assay (RPA) revealed that the expression of G39F8, G24173 and LZIP increase markedly in the telencephalon at E16 and continue to be expressed until at least PO, during the period when the corpus callosum is forming. (c) 2005 Elsevier B.V. All rights reserved.

Visual evoked electrical and magnetic response to half-field stimulation using pattern reversal stimulation

The visual evoked magnetic response to half-field stimulation using pattern reversal was studied using a d.c. SQUID coupled to a second order gradiometer. The main component of the magnetic response consisted of a positive wave at around 100 ms (P100M). At the time this component was present the response to half-field stimulation consisted of an outgoing magnetic field contralateral and extending to the midline. When the left half field was stimulated the outgoing field was over the posterior right visual cortex and when the right half field was stimulated it was over the left anterior visual cortex. These findings would correctly identify a source located in the contralateral visual cortex. The orientation of the dipoles was not that previously assumed to explain the paradoxical lateralization of the visual evoked potential. The results are discussed in terms of both electrical and magnetic models of the calcarine fissure. © 1992.

Visual evoked electrical and magnetic response to half-field stimulation using pattern reversal stimulation

The visual evoked magnetic response to half-field stimulation using pattern reversal was studied using a dc-SQUID coupled to a second-order gradiometer. The main component of the magnetic response consisted of a positive wave at around 100ms (P100M). At the same time this component was present the reponse to half-field stimulation consisted of an outgoing field contralateral and extending to the midline. When the left half-field was stimulates the outgoing field was over the posterior right visual cortex and when the right half field was stimulated it was over the left anterior visual cortex. These findings would correltly identify a source located in the contralateral visual cortex. The orientation of the dipoles was not that previously assumed to explain the paradoxical lateralization of the visual evoked potential. The results are discussed in terms of both electrical and magnetic models of the calcarine fissure.

Contradiction in universal and particular reasoning

A wide range of essential reasoning tasks rely on contradiction identification, a cornerstone of human rationality, communication and debate founded on the inversion of the logical operators "Every" and "Some." A high-density electroencephalographic (EEG) study was performed in 11 normal young adults. The cerebral network involved in the identification of contradiction included the orbito-frontal and anterior-cingulate cortices and the temporo-polar cortices. The event-related dynamic of this network showed an early negative deflection lasting 500 ms after sentence presentation. This was followed by a positive deflection lasting 1.5 s, which was different for the two logical operators. A lesser degree of network activation (either in neuron number or their level of phase locking or both) occurred while processing statements with "Some," suggesting that this was a relatively simpler scenario with one example to be figured out, instead of the many examples or the absence of a counterexample searched for while processing statements with "Every." A self-generated reward system seemed to resonate the recruited circuitry when the contradictory task is successfully completed.

Cataract, macular characteristics and assessing lens opacities

Age-related macular degeneration and cataract are very common causes of visual impairment in the elderly. Macular pigment optical density is known to be a factor affecting the risk of developing age-related macular degeneration but its behaviour due to light exposure to the retina and the effect of macular physiology on this measurement are not fully understood. Cataract is difficult to grade in a way which reflects accurately the visual status of the patient. A new technology, optical coherence tomography, which allows a cross sectional slice of the crystalline lens to be imaged has the potential to be able to provide objective measurements of cataract which could be used for grading purposes. This thesis set out to investigate the effect of cataract removal on macular pigment optical density, the relationship between macular pigment optical density and macular thickness and the relationship between cortical cataract density as measured by optical coherence tomography and other measures of cataract severity. These investigations found: 1) Macular pigment optical density in a pseudophakic eye is reduced when compared to a fellow eye with age related cataract, probably due to differences in light exposure between the eyes. 2) Lower macular pigment optical density is correlated with thinning of the entire macular area, but not with thinning of the fovea or central macula. 3) Central macular thickness decreases with age. 4) Spectral domain optical coherence tomography can be used to successfully acquire images of the anterior lens cortex which relate well to slit lamp lens sections. 5) Grading of cortical cataract with spectral domain optical coherence tomography instruments using a wavelength of 840nm is not well correlated with other established metrics of cataract severity and is therefore not useful as presented as a grading method for this type of cataract.

Dissociable patterns of neural activity during response inhibition in depressed adolescents with and without suicidal behavior

Objectives - Impaired attentional control and behavioral control are implicated in adult suicidal behavior. Little is known about the functional integrity of neural circuitry supporting these processes in suicidal behavior in adolescence. Method - Functional magnetic resonance imaging was used in 15 adolescent suicide attempters with a history of major depressive disorder (ATTs), 15 adolescents with a history of depressive disorder but no suicide attempt (NATs), and 14 healthy controls (HCs) during the performance of a well-validated go-no-go response inhibition and motor control task that measures attentional and behavioral control and has been shown to activate prefrontal, anterior cingulate, and parietal cortical circuitries. Questionnaires assessed symptoms and standardized interviews characterized suicide attempts. Results - A 3 group by 2 condition (go-no-go response inhibition versus go motor control blocks) block-design whole-brain analysis (p < .05, corrected) showed that NATs showed greater activity than ATTs in the right anterior cingulate gyrus (p = .008), and that NATs, but not ATTs, showed significantly greater activity than HCs in the left insula (p = .004) to go-no-go response inhibition blocks. Conclusions - Although ATTs did not show differential patterns of neural activity from HCs during the go-no-go response inhibition blocks, ATTs and NATs showed differential activation of the right anterior cingulate gyrus during response inhibition. These findings indicate that suicide attempts during adolescence are not associated with abnormal activity in response inhibition neural circuitry. The differential patterns of activity in response inhibition neural circuitry in ATTs and NATs, however, suggest different neural mechanisms for suicide attempt versus major depressive disorder in general in adolescence that should be a focus of further study.