Tensile properties and associated cellular composition of axon bundles in the desert locust and other orthopteroid insects

Axons are known to be under tension during growth and development for guidance of the growth cone, synapse formation and stretch growth. Tension is also present during adulthood, but its role is unclear. Here we investigate whether axon bundles in the desert locust are under tension. We then determine whether this tension is reflected in the composition of the axon bundle and/or individual axons. We used microdissection and nerve cutting to observe tension in axon bundles connected to the metathoracic ganglion of adult gregarious desert locusts (Schistocerca gregaria). We show variable levels of tension in three axon bundles, with the highest level in the connectives between the meso- and metathoracic ganglia. Transmission electron microscopy (TEM) allowed us to compare these bundles at the gross anatomical, cellular and sub-cellular levels. Gross anatomical differences in bundle size do not account for the results from the microdissections. Instead, the number of microtubules per axon and the relationship between microtubule number and axon size are the main factors that correlate with results from microdissection. This suggests that the tension of the entire axon bundle is mediated by the cellular cytoskeleton. We use ex vivo uniaxial loading to link microdissection and TEM findings to mechanical properties. We find that high-tension bundles withstand the highest forces prior to failure. We repeated our microdissection nerve cutting across growth stages (instars) immediately prior to and after moulting. This showed that tension does not change during periods of growth. We performed microsurgery on third instar S. gregaria, cutting one of the high-tension connectives to expose the second to greater force. We then resealed the cuticle and allowed the locusts to grow to adulthood. Despite morphological changes, there was no change in in vivo tension in surgery locusts. TEM revealed that the remaining connective had a higher density and count of microtubules than shams or controls, suggesting these had been actively upregulated in response to changing forces. Finally, we repeated the microdissections on solitary phase S. gregaria and Locusta migratoria. We found no differences in tension held in the connectives between the meso- and metathoracic ganglia and those of gregarious desert locusts. We expanded to other orthopteroids (Gryllus bimaculatus, Periplaneta americana) that differ in the extent to which their metathoracic ganglion is fused. In both species the connectives between the meso- and metathoracic ganglia are under less tension than in locusts. This was reflected in TEM results: connectives in locusts had a higher number of microtubules per axon than other species. However uniaxial loading experiments did not reflect microdissection results, which suggests mechanical properties of axons do not necessarily reflect in vivo forces experienced. We suggest in this thesis that axonal tension is a variable property of axons that is correlated with the density of microtubules in the longitudinal orientation. We propose that individual axons have a ‘set-point’ of tension that is actively maintained throughout growth and in response to injury. Combined these results suggest that axonal tension is likely functional, rather than universal, property of axons that is capable of responding to selective pressures