How octopus arm muscle contractile properties and anatomical organization contribute to arm functional specialization.

Octopus arms are highly flexible structures capable of complex motions and are used in a wide repertoire of behaviors. Movements are generated by the coordinated summation of innervation signals to packed arrays of muscles oriented in different directions and moving based on their anatomical relationships. In this study, we investigated the interplay between muscle biomechanics and anatomical organization in the Octopus vulgaris arm to elucidate their role in different arm movements. We performed isometric and isotonic force measurements on isolated longitudinal and transverse arm muscles and showed that longitudinal muscles have a higher rate of activation and relaxation, lower twitch-to-tetanus ratio and lower passive tension than transverse muscles, thus prompting their use as faster and slower muscles, respectively. This points to the use of longitudinal muscles in more graded responses, such as those involved in precise actions, and transverse muscles in intense and sustained actions, such as motion stabilization and posture maintenance. Once activated, the arm muscles exert forces that cause deformations of the entire arm, which are determined by the amount, location, properties and orientation of their fibers. Here, we show that, although continuous, the arm manifests a certain degree of morphological specialization, where the arm muscles have a different aspect ratio along the arm. This possibly supports the functional specialization of arm portions observed in various motions, such as fetching and crawling. Hence, the octopus arm as a whole can be seen as a 'reservoir' of possibilities where different types of motion may emerge at the limb level through the co-option of the muscle contractile properties and structural arrangement.

Beyond muscles: role of intramuscular connective tissue elasticity and passive stiffness in octopus arm muscle function.

The octopus arm is a 'one of a kind' muscular hydrostat, as demonstrated by its high maneuverability and complexity of motions. It is composed of a complex array of muscles and intramuscular connective tissue, allowing force and shape production. In this study, we investigated the organization of the intramuscular elastic fibers in two main muscles composing the arm bulk: the longitudinal (L) and the transverse (T) muscles. We assessed their contribution to the muscles' passive elasticity and stiffness and inferred their possible roles in limb deformation. First, we performed confocal imaging of whole-arm samples and provided evidence of a muscle-specific organization of elastic fibers (more chaotic and less coiled in T than in L). We next showed that in an arm at rest, L muscles are maintained under 20% compression and T muscles under 30% stretching. Hence, tensional stresses are inherently present in the arm and affect the strain of elastic fibers. Because connective tissue in muscles is used to transmit stress and store elastic energy, we investigated the contribution of elastic fibers to passive forces using step-stretch and sinusoidal length-change protocols. We observed a higher viscoelasticity of L and a higher stiffness of T muscles, in line with their elastic fiber configurations. This suggests that L might be involved in energy storage and damping, whereas T is involved in posture maintenance and resistance to deformation. The elastic fiber configuration thus supports the specific role of muscles during movement and may contribute to the mechanics, energetics and control of arm motion.

Synapsins are expressed at neuronal and non-neuronal locations in Octopus vulgaris.

Synapsins are a family of phosphoproteins fundamental to the regulation of neurotransmitter release. They are typically neuron-specific, although recent evidence pointed to their expression in non-neuronal cells where they play a role in exocytosis and vesicle trafficking. In this work, we characterized synapsin transcripts in the invertebrate mollusk Octopus vulgaris and present evidence of their expression not only in the brain but also in male and female reproductive organs. We identified three synapsin isoforms phylogenetically correlated to that of other invertebrates and with a modular structure characteristic of mammalian synapsins with a central, highly conserved C domain, important for the protein functions, and less conserved A, B and E domains. Our molecular modeling analysis further provided a solid background for predicting synapsin functional binding to ATP, actin filaments and secretory vesicles. Interestingly, we found that synapsin expression in ovary and testis increased during sexual maturation in cells with a known secretory role, potentially matching the occurrence of a secretion process. This might indicate that its secretory role has evolved across animals according to cell activity in spite of cell identity. We believe that this study may yield insights into the convergent evolution of ubiquitously expressed proteins between vertebrates and invertebrates.

mTOR as a Marker of Exercise and Fatigue in Octopus vulgaris Arm.

Cephalopods are highly evolved marine invertebrates that colonized almost all the oceans of the world at all depths. This imposed the occurrence of several modifications of their brain and body whose muscle component represents the major constituent. Hence, studying their muscle physiology may give important hints in the context of animal biology and environmental adaptability. One major pathway involved in muscle metabolism in vertebrates is the evolutionary conserved mTOR-signaling cascade; however, its role in cephalopods has never been elucidated. mTOR is regulating cell growth and homeostasis in response to a wide range of cues such as nutrient availability, body temperature and locomotion. It forms two functionally heteromeric complexes, mTORC1 and mTORC2. mTORC1 regulates protein synthesis and degradation and, in skeletal muscles, its activation upon exercise induces muscle growth. In this work, we characterized Octopus vulgaris mTOR full sequence and functional domains; we found a high level of homology with vertebrates' mTOR and the conservation of Ser2448 phosphorylation site required for mTORC1 activation. We then designed and tested an in vitro protocol of resistance exercise (RE) inducing fatigue in arm samples. We showed that, upon the establishment of fatigue, a transient increase in mTORC1 phosphorylation reaching a pick 30 min after exercise was induced. Our data indicate the activation of mTORC1 pathway in exercise paradigm and possibly in the regulation of energy homeostasis in octopus and suggest that mTORC1 activity can be used to monitor animal response to changes in physiological and ecological conditions and, more in general, the animal welfare.

From synaptic input to muscle contraction: arm muscle cells of Octopus vulgaris show unique neuromuscular junction and excitation-contraction coupling properties.

The muscular-hydrostat configuration of octopus arms allows high manoeuvrability together with the efficient motor performance necessary for its multitasking abilities. To control this flexible and hyper-redundant system the octopus has evolved unique strategies at the various levels of its brain-to-body organization. We focus here on the arm neuromuscular junction (NMJ) and excitation-contraction (E-C) properties of the arm muscle cells. We show that muscle cells are cholinergically innervated at single eye-shaped locations where acetylcholine receptors (AChR) are concentrated, resembling the vertebrate neuromuscular endplates. Na+ and K+ contribute nearly equally to the ACh-activated synaptic current mediating membrane depolarization, thereby activating voltage-dependent L-type Ca2+ channels. We show that cell contraction can be mediated directly by the inward Ca2+ current and also indirectly by calcium-induced calcium release (CICR) from internal stores. Indeed, caffeine-induced cell contraction and immunohistochemical staining revealed the presence and close association of dihydropyridine (DHPR) and ryanodine (RyR) receptor complexes, which probably mediate the CICR. We suggest that the dynamics of octopus arm contraction can be controlled in two ways; motoneurons with large synaptic inputs activate vigorous contraction via activation of the two routs of Ca2+ induced contraction, while motoneurons with lower-amplitude inputs may regulate a graded contraction through frequency-dependent summation of EPSP trains that recruit the CICR. Our results thus suggest that these motoneuronal pools are likely to be involved in the activation of different E-C coupling modes, thus enabling a dynamics of muscles activation appropriate for various tasks such as stiffening versus motion generation.

Motor control pathways in the nervous system of Octopus vulgaris arm.

The octopus's arms have virtually infinite degrees of freedom, providing a unique opportunity for studying movement control in a redundant motor system. Here, we investigated the organization of the connections between the brain and arms through the cerebrobrachial tracts (CBT). To do this, we analyzed the neuronal activity associated with the contraction of a small muscle strand left connected at the middle of a long isolated CBT. Both electrical activity in the CBT and muscle contraction could be induced at low threshold values irrespective of stimulus direction and distance from the muscle strand. This suggests that axons associated with transmitting motor commands run along the CBT and innervate a large pool of motor neurons en passant. This type of innervation implies that central and peripheral motor commands involve the simultaneous recruitment of large groups of motor neurons along the arm as required, for example, in arm stiffening, and that the site of movement initiation along the arm may be determined through a unique interplay between global central commands and local sensory signals.