Modelling the neuromechanics of exploration and taxis in larval Drosophiila

The Drosophila larva is emerging as a useful tool in the study of complex behaviours, due to its relatively small size, its genetic tractability, and its varied behavioural repertoire. The larva executes a stereotypical exploratory routine that appears to consist of stochastic alternation between straight peristaltic crawling and reorientation events through lateral bending. The larva performs taxis by biasing this behavioural pattern, allowing it to move up or down attractive and aversive stimulus gradients. Existing explanations of exploration and taxis behaviour often neglect the larva's embodiment, focusing on central pattern generation and decision making circuits within the nervous system. In Chapter 1 of this thesis, I review the current state of knowledge regarding larval peristalsis, exploration, and taxis behaviours, as well as existing theories of their generation. I argue that an understanding of the animal's embodiment should lead to a deeper understanding of its behaviour. In Chapter 2, I present a model of the axial mechanics of the larva, and demonstrate how the animal's body physics can be exploited to produce peristalsis by using segmentally localised, positive feedback of strain rate. The mechanical model includes viscoelastic tissue mechanics, muscular inputs, and substrate interaction while sensory feedback is modelled as a linear feedback control law. In Chapter 3, I extend the mechanical model to study motion in the plane, including both axial and transverse deformations of the body. The feedback law is replaced by a simple model of the larval nervous system. The model includes both a segmentally localised reflex arc as well as long-range, mutual inhibition between segments. The complete model is capable of generating both peristalsis and spontaneous reorientation, leading to emergent exploration behaviour in the form of a deterministic superdiffusion process grounded in the chaotic mechanics of the larva's body. In Chapter 4, I consider taxis behaviour. I introduce a transverse reflex capable of modulating the effective transverse viscosity of the larval body. When the larva is experiencing an increasing attractive (aversive) stimulus, the reflex acts to increase (decrease) the effective transverse viscosity, causing bending to occur less (more) easily. As a result, the model larvae approach attractive stimuli and avoid aversive stumuli. On a population level, I show that the transverse reflex can be thought of as biasing the model animals towards sub- or super-diffusion. I compare the statistics of this behaviour to those of the real larva. In Chapter 5, I shift focus to engineered soft systems. Having successfully deployed an energy-based modelling approach in Chapters 2--4, I argue for the adoption of an energy-focused (specifically, port-Hamiltonian) approach within the field of soft robotics. In Chapter 6, I present some initial theoretical extensions to the models presented in chapter 2--4. I first focus on the mechanics of self-righting and rolling behaviours, before modelling the ventral nerve cord of the larva using a ring attractor architecture. Finally, in Chapter 7, I summarise the results of the previous chapters and discuss directions for future research

Continuous lateral oscillations as a core mechanism for taxis in Drosophila larvae

Taxis behaviour in Drosophila larva is thought to consist of distinct control mechanisms triggering specific actions. Here, we support a simpler hypothesis: that taxis results from direct sensory modulation of continuous lateral oscillations of the anterior body, sparing the need for ‘action selection’. Our analysis of larvae motion reveals a rhythmic, continuous lateral oscillation of the anterior body, encompassing all head-sweeps, small or large, without breaking the oscillatory rhythm. Further, we show that an agent-model that embeds this hypothesis reproduces a surprising number of taxis signatures observed in larvae. Also, by coupling the sensory input to a neural oscillator in continuous time, we show that the mechanism is robust and biologically plausible. The mechanism provides a simple architecture for combining information across modalities, and explaining how learnt associations modulate taxis. We discuss the results in the light of larval neural circuitry and make testable predictions. DOI: http://dx.doi.org/10.7554/eLife.15504.00

An analysis of the locomotory behaviour and functional morphology of errant polychaetes

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Mechanisms of mechanosensation in Drosophila melanogaster proprioceptors

Proprioception is the ability to detect position in space. It is necessary for normal motor control and could share molecular mechanisms with other senses, such as hearing. These mechanisms are poorly understood and clarifying them may reveal novel targets for treatment of muscle spasticity, seizure and hardness of hearing. This research uses Drosophila models to clarify the behavioural role and molecular properties of proprioceptors; the dbd neuron and the chordotonal neurons. I hypothesise that the dbd neuron is both a pain and stretch receptor that requires DmPiezo to respond to both physiological and nociceptive stimuli. In contrast, evidence suggests that chordotonal neurons sense could sound and stretch stimuli through different mechanisms, which depend on nan/ iav/ NompC and DmPiezo respectively. We employed optogenetics, crawling, nociceptive reflex (‘pinch’ response), GCaMP imaging and whole-cell patch-clamp electrophysiology to investigate the role and mechanisms of mechanosensation in the dbd neuron. Similarly, I used crawling, hearing and GCaMP experiments to assess the role and mechanisms of mechanosensation in the chordotonal neurons. I found the dbd neuron difficult to investigate; a ‘nociceptive’ phenotype originally attributed to dbd neuron stimulation disappeared when the related driver, Bd-Gal4, was expressed in the background of a mutant (amos1) that lacks the dbd neuron. Moreover, while electrophysiology gave results like those published previously, my data were limited by issues including low seal values (~40MΩ, significantly lower than the desired 1GΩ) that were exacerbated by stretch. Chordotonal (ch) neurons were easier to study. GCaMP imaging of the larval ventral nerve cord showed that ch neurons respond to both tonal (1024Hz) and muscle contraction stimulation (mean ΔF/ F0 (%) 11.47 ± 2.93 and 7.56 ± 4.38, respectively). I imaged the ch neurons (lch1-5, vch1 and vchAB) directly, and doing so revealed some interesting spatial and temporal differences in response to sound, which implies specific tuning of neurons within the chordotonal neuron population(s)(s). GCaMP imaging also showed that CG17669, a gene with a human orthologue (DNAAF3) associated with primary ciliary dyskinesia, is necessary for ch neuron response to 1024Hz and muscle contraction. In conclusion, the behavioural role and mechanisms of the dbd neuron remain unclear and require further investigation. However, it appears that while the ch neurons can detect stretch (and so act as proprioceptors), this function is not required for normal movement in larvae. The ch neurons appear to be a sense organ with a single mechanism of mechanosensation, that is optimised for detection of tonal stimuli in the hearing range. Finally, this research is the first to: (1) image the response of vch1 and vchAB ch neurons response to sound; (2) provide evidence that subsets of Drosophila ch neurons may be tuned to respond to specific amplitudes and/ or frequencies; (3) use real-time calcium imaging to demonstrate the effect of CG17669 mutation on the function of ch neurons

Étude de la mécanotransduction dans la scoliose idiopathique de l’adolescence (SIA)

À ce jour, la scoliose idiopathique de l’adolescent (SIA) est la déformation rachidienne la plus commune parmi les enfants. Il est bien connu dans le domaine de recherche sur la SIA que les forces mécaniques, en particulier les forces biomécaniques internes dans le système musculosquelettique, pourraient jouer un rôle majeur dans l’initiation et le développement de la maladie. Cependant, les connaissances sur la transformation des forces et des stimulations mécaniques en activité biochimique sont peu abondantes. Cet axe de recherche est très prometteur et peut nous fournir de nouvelles idées dans le dépistage et le traitement de la SIA. Dans le cadre de cette étude, nous visons à caractériser la mécanotransduction chez les patients atteints de la SIA en employant des techniques novatrices aux niveaux in vivo et in vitro. Antérieurement dans notre laboratoire, nous avons démontré que les niveaux d’Ostéopontine (OPN) plasmatique chez l’humain corrèlent avec la progression et la sévérité de la maladie, et que ces changements sont observables avant le début de la scoliose. En plus, selon la littérature, l’OPN est une molécule sensible à la force mécanique, dont l’expression augmente en réponse dans de nombreux types de cellules chez plusieurs espèces. Toutefois, il n’existe aucune preuve que ce résultat soit valide in vivo chez l’humain. L’hétérogénéité physique et biochimique de la SIA pose un gros défi aux chercheurs. Souvent, il est très difficile de trouver des résultats ayant une grande applicabilité. Les études portant sur les facteurs biomécaniques ne font pas exception à cette tendance. En dépit de tout cela, nous croyons qu’une approche basée sur l’observation des contraintes de cisaillement présentes dans le système musculosquelettique pourrait aider à surmonter ces difficultés. Les contraintes de cisaillement physiologique sont générées par des courants de fluide en mouvement à l’intérieur des os. Aussi, elles sont omniprésentes et universelles chez l’humain, peu importe l’âge, le sexe, la condition physique, etc., ce qui veut dire que l’étudier pourrait fort bien avancer nos connaissances en formant une base fondamentale avec laquelle on pourra mieux comprendre les différences quant à la mécanotransduction chez les patients atteints de la SIA par rapport aux sujets sains. Pour ce projet, donc, nous proposons l’hypothèse que les sujets atteints de la SIA se différencient par leurs réponses respectives à la force mécanique au niveau cellulaire (en termes de l’expression génique) ainsi qu’au niveau in vivo (en termes du marqueur OPN et son récepteur, sCD44). Afin de vérifier la partie de notre hypothèse de recherche concernant l’aspect in vivo, nous avons recruté une cohorte de patients âgés de 9-17 ans, y compris i) des cas pré-chirurgicaux (angle de Cobb > 45°), ii) des cas modérément atteints (angle de Cobb 10-44°), iii) des témoins, et iv) des enfants asymptomatiques à risque de développer la scoliose (selon nos dépistages biochimiques et fonctionnels) d’âge et sexe appariés. Une pression pulsatile et dynamique avec une amplitude variant de 0-4 psi à 0.006 Hz a été appliquée à un des bras de chacun de nos sujets pour une durée de 90 minutes. Au tout début et à chaque intervalle de 30 minutes après l’initiation de la pression, un échantillon de sang a été prélevé, pour pouvoir surveiller les niveaux d’OPN et de sCD44 circulants chez les sujets. Nous avons découvert que le changement des niveaux d’OPN plasmatique, mais pas des niveaux de sCD44, corrélaient avec la sévérité de la difformité rachidienne chez les sujets, ceux ayant une courbe plus prononcée démontrant une ampleur de réponse moins élevée. Pour vérifier la partie de notre hypothèse de recherche concernant la réponse mécanotransductive cellulaire, des ostéoblastes prélevées à 12 sujets ont été mis en culture pour utilisation avec notre appareil (le soi-disant « parallel plate flow chamber »), qui sert à fournir aux ostéoblastes le niveau de contraintes de cisaillement désiré, de manière contrôlée et prévisible. Les sujets étaient tous femelles, âgées de 11-17 ans ; les patients ayant déjà une scoliose possédaient une courbe diagnostiquée comme « double courbe majeure ». Une contrainte fluidique de cisaillement à 2 Pa, 0.5 Hz a été appliquée à chaque échantillon ostéoblastique pour une durée de 90 minutes. Les changements apportés à l’expression génique ont été mesurés et quantifiés par micropuce et qRT-PCR. En réponse à notre stimulation, nous avons trouvé qu’il n’y avait que quelques gènes étant soit différentiellement exprimés, soit inchangés statistiquement dans tous les groupes expérimentaux atteints, en exhibant simultanément la condition contraire chez les témoins. Ces résultats mettent en évidence la grande diversité de la réponse mécanotransductive chez les patients comparés aux contrôles, ainsi qu’entre les sous-groupes fonctionnels de la SIA. Globalement, cette œuvre pourrait contribuer au développement d’outils diagnostiques innovateurs pour identifier les enfants asymptomatiques à risque de développer une scoliose, et évaluer le risque de progression des patients en ayant une déjà. Aussi, dans les années à venir, les profils mécanotransductifs des patients pourraient s’avérer un facteur crucial à considérer cliniquement, particulièrement en concevant ou personnalisant des plans de traitements pour des personnes atteintes.Adolescent idiopathic scoliosis (AIS) is the most commonly occurring musculoskeletal deformity among children today. It is generally well accepted in scoliosis research that mechanical forces, especially the internal biomechanical forces of the musculoskeletal system, could well have a major role in the induction and pathogenesis of the disease. However, the process by which mechanical loads or stimuli are converted into biochemical activity (mechanotransduction) has not been explored so deeply. This emerging facet of research in AIS holds much promise for new insights into the disease. Here, we aim to characterize mechanotransduction in scoliosis patients using some novel techniques at both the in vivo and in vitro levels. Previously in our lab, we demonstrated that the level of plasma osteopontin (OPN) and sCD44 in the human body is a strong indicator of disease progression and severity, and that these changes are observable before scoliosis onset. In the literature, OPN in vitro is known to be mechanosensitive, showing upregulation in response to mechanical stress in a variety of cell types across many species. However, to the best of the author’s knowledge, no literature exists as to whether this behaviour carries over in vivo in humans. A major difficulty in AIS research is the heterogeneity of the disease, both physically and biochemically. Because of this, many times it is difficult to find results with wide applicability to patients. Study of biomechanical factors in AIS is no exception. We believe, however, that study of fluid shear stress in the musculoskeletal system may be able to solve this problem for mechanotransduction-related issues in AIS. Native physiological fluid shear stresses in humans are experienced in the musculoskeletal system, caused by fluid movement over cells therein. These fluid shear stresses are omnipresent and universal in all humans, regardless of age, gender, fitness level, etc., which means that studying it could very well go a long way towards establishing a fundamental basis of understanding the differences as to mechanotransduction in scoliosis patients as opposed to normal cases. In this project, then, we advanced the hypothesis that AIS patients are distinguishable in the way they respond to mechanical force at both the cellular level (in terms of gene expression) as well as globally at the in vivo level (in terms of the scoliosis marker OPN and its receptor sCD44). To test the in vivo portion of our hypothesis, we recruited a cohort of patients between the ages of 9-17, each one of which fell into one of 4 subject groups: i) surgical cases (pre-surgery, Cobb angle > 45°), ii) moderately affected cases (Cobb angle 10-44°), iii) controls, or iv) asymptomatic children at risk of developing scoliosis matched for age and gender against healthy controls. A dynamic, pulsatile, compressive pressure of variable amplitude from 0-4 psi at 0.006 Hz was applied to the arm of each subject for a period of 90 minutes. Initially and at intervals of 30 minutes after the start of force application, blood samples were taken in order to monitor circulating plasma OPN and sCD44 levels in subjects. We found that the change of circulating OPN levels, but not sCD44 levels, measured in vivo in response to our mechanical stimulation was statistically significantly correlated to status of spinal deformity severity, with more severely affected subjects demonstrating lower magnitudes of ΔOPN. To test the cellular portion of our hypothesis, osteoblasts from severely affected AIS patients and unaffected controls were cultured for use with our parallel plate flow chamber (PPFC) apparatus setup, which permits application of fluid shear stress patterns to cells in a predictable, controllable manner. Subjects were all females who fell into the 11-17 years age range, with scoliotic patients presenting with double major curves. A dynamic, sinusoidal and oscillatory fluid shear stress pattern was applied to osteoblasts at 2 Pa, 0.5 Hz for 90 minutes. Overall gene expression changes across RNA samples as a result of our stimulation were measured using microarray and qRT-PCR approaches. In response, only a very small number of genes are either mutually differentially expressed or statistically unchanged across all functional scoliotic subgroups while having the opposite condition in the control group, indicating a great degree of difference in terms of mechanotransductive response as compared internally between AIS functional subgroups, as well as between control and AIS patients. Globally, this project’s work may contribute to the development of innovative diagnostic tools to identify asymptomatic children at risk of developing scoliosis, and to assess the risk of curve progression at an early stage in those already affected. Also, in years to come, the mechanotransductive profile of a patient could be another integral factor to weigh, clinically, when considering or designing treatment plans for affected persons