Cephalopod behaviour.

Underlying all animal behaviors, from the simplest reflexive reactions to the more complex cognitive reasoning and social interaction, are nervous systems uniquely adapted to bodies, environments, and challenges of different animal species. Coleoid cephalopods - octopuses, squid, and cuttlefish - are widely recognized as the most behaviorally complex invertebrates and provide exciting opportunities for studying the neural control of behaviour. These unusual molluscs evolved over 400 million years ago from slow-moving armored forms to active predators of coastal and open ocean ecosystems. In this primer we will discuss how, during cephalopod evolution, the relatively simple ganglion-based molluscan nervous system has been extensively transformed to control the complex bodies and process extensive visual, tactile, and chemical sensory inputs, and summarize some recent findings about their fascinating behaviors.

Cephalopod learning and memory.

Cephalopod molluscs are renowned for their unique central nervous system - a donut-shaped brain organised around the oesophagus. This brain supports sophisticated learning and memory abilities. Between the 1950s and 1980s, these cognitive abilities were extensively studied in octopus (Figure 1A) - a now leading model for the study of memory and its neural substrates (approximately 200 papers during this period). The focus on octopus learning and memory was mainly due to their curious nature and the fact that they adapt to laboratory-controlled conditions, making them easy to test and maintain in captivity. Research on cephalopod cognition began to widen in the late 20th century, when scientists started focusing on other coleoid cephalopods (i.e., cuttlefish and squid) (Figure 1B,C), and not just on associative learning and memory per se, but other more complex aspects of cognition such as episodic-like memory (the ability to remember the what, where, and when of a past event), source memory (the retrieval of contextual details from a memory), and self-control (the ability to inhibit an action in the present to gain a more valuable future reward). Attention broadened further over the last two decades to focus on the shelled cephalopods - the nautiloids (Figure 1D). The nautiloids have relatively primitive brains compared to their soft-bodied cousins (octopus, cuttlefish, and squid) but research shows that they are still able to comparatively succeed in some cognitive tasks. In this primer, we will provide a general description of the types of memory studied in cephalopods, and discuss learning and memory experiments that address the main challenges cephalopods face during their daily lives: navigation, timing, and food selection. Determining the type of information cephalopods learn and remember and whether they use such information to overcome ecological challenges will highlight why these invertebrates evolved large and sophisticated brains.

Embodied mechanisms of motor control in the octopus.

Achieving complex behavior in soft-bodied animals is a hard task, because their body morphology is not constrained by a fixed number of jointed elements, as in skeletal animals, and thus the control system has to deal with practically an infinite number of control variables (degrees of freedom). Almost 30 years of research on Octopus vulgaris motor control has revealed that octopuses efficiently control their body with strategies that emerged during the adaptive coevolution of their nervous system and body morphology. In this minireview, we highlight principles of embodied organization that were revealed by studying octopus motor control, and that are used as inspiration for soft robotics. We describe the evolved solutions to the problem, implemented from the lowest level, the muscular system, to the network organization in higher motor control centers of the octopus brain. We show how the higher motor control centers, where the sensory-motor interface lies, can control and coordinate limbs with large degrees of freedom without using body-part maps to represent sensory and motor information, as they do in vertebrates. We demonstrate how this unique control mechanism, which allows efficient control of the body in a large variety of behaviors, is embodied within the animal's body morphology.

Mechanisms of octopus arm search behavior without visual feedback.

The octopus coordinates multiple, highly flexible arms with the support of a complex distributed nervous system. The octopus's suckers, staggered along each arm, are employed in a wide range of behaviors. Many of these behaviors, such as foraging in visually occluded spaces, are executed under conditions of limited or absent visual feedback. In coordinating unseen limbs with seemingly infinite degrees of freedom across a variety of adaptive behaviors, the octopus appears to have solved a significant control problem facing the field of soft-bodied robotics. To study the strategies that the octopus uses to find and capture prey within unseen spaces, we designed and 3D printed visually occluded foraging tasks and tracked arm motion as the octopus attempted to find and retrieve a food reward. By varying the location of the food reward within these tasks, we can characterize how the arms and suckers adapt to their environment to find and capture prey. We compared these results to simulated experimental conditions performed by a model octopus arm to isolate the primary mechanisms driving our experimental observations. We found that the octopus relies on a contact-based search strategy that emerges from local sucker coordination to simplify the control of its soft, highly flexible limbs.

The Inner Lives of Cephalopods.

The minds of cephalopods have captivated scientists for millennia, yet the extent that we can understand their subjective experiences remains contested. In this article, we consider the sum of our scientific progress towards understanding the inner lives of cephalopods. Here, we outline the behavioral responses to specific experimental paradigms that are helping us to reveal their subjective experiences. We consider evidence from three broad research categories, which help to illuminate whether soft-bodied cephalopods (octopus, cuttlefish, and squid) have an awareness of self, awareness of others, and an awareness of time. Where there are current gaps in the literature, we outline cephalopod behaviors that warrant experimental investigation. We argue that investigations, especially framed through the lens of comparative psychology, have the potential to extend our understanding of the inner lives of this extraordinary class of animals.

Connectomics of the Octopus vulgaris vertical lobe provides insight into conserved and novel principles of a memory acquisition network.

Here, we present the first analysis of the connectome of a small volume of the Octopus vulgaris vertical lobe (VL), a brain structure mediating the acquisition of long-term memory in this behaviorally advanced mollusk. Serial section electron microscopy revealed new types of interneurons, cellular components of extensive modulatory systems, and multiple synaptic motifs. The sensory input to the VL is conveyed via~1.8 × 106 axons that sparsely innervate two parallel and interconnected feedforward networks formed by the two types of amacrine interneurons (AM), simple AMs (SAMs) and complex AMs (CAMs). SAMs make up 89.3% of the~25 × 106VL cells, each receiving a synaptic input from only a single input neuron on its non-bifurcating primary neurite, suggesting that each input neuron is represented in only~12 ± 3.4SAMs. This synaptic site is likely a 'memory site' as it is endowed with LTP. The CAMs, a newly described AM type, comprise 1.6% of the VL cells. Their bifurcating neurites integrate multiple inputs from the input axons and SAMs. While the SAM network appears to feedforward sparse 'memorizable' sensory representations to the VL output layer, the CAMs appear to monitor global activity and feedforward a balancing inhibition for 'sharpening' the stimulus-specific VL output. While sharing morphological and wiring features with circuits supporting associative learning in other animals, the VL has evolved a unique circuit that enables associative learning based on feedforward information flow.

Toward an Understanding of Octopus Arm Motor Control.

Octopuses have the extraordinary ability to control eight prehensile arms with hundreds of suckers. With these highly flexible limbs, they engage in a wide variety of tasks, including hunting, grooming, and exploring their environment. The neural circuitry generating these movements engages every division of the octopus nervous system, from the nerve cords of the arms to the supraesophegeal brain. In this review, the current knowledge on the neural control of octopus arm movements is discussed, highlighting open questions and areas for further study.

Wake-like skin patterning and neural activity during octopus sleep.

While sleeping, many vertebrate groups alternate between at least two sleep stages: rapid eye movement and slow wave sleep1-4, in part characterized by wake-like and synchronous brain activity, respectively. Here we delineate neural and behavioural correlates of two stages of sleep in octopuses, marine invertebrates that evolutionarily diverged from vertebrates roughly 550 million years ago (ref. 5) and have independently evolved large brains and behavioural sophistication. 'Quiet' sleep in octopuses is rhythmically interrupted by approximately 60-s bouts of pronounced body movements and rapid changes in skin patterning and texture6. We show that these bouts are homeostatically regulated, rapidly reversible and come with increased arousal threshold, representing a distinct 'active' sleep stage. Computational analysis of active sleep skin patterning reveals diverse dynamics through a set of patterns conserved across octopuses and strongly resembling those seen while awake. High-density electrophysiological recordings from the central brain reveal that the local field potential (LFP) activity during active sleep resembles that of waking. LFP activity differs across brain regions, with the strongest activity during active sleep seen in the superior frontal and vertical lobes, anatomically connected regions associated with learning and memory function7-10. During quiet sleep, these regions are relatively silent but generate LFP oscillations resembling mammalian sleep spindles11,12 in frequency and duration. The range of similarities with vertebrates indicates that aspects of two-stage sleep in octopuses may represent convergent features of complex cognition.

Comparative anatomy and functional implications of variation in the buccal mass in coleoid cephalopods.

In contrast to the well-studied articulated vertebrate jaws, the structure and function of cephalopod jaws remains poorly known. Cephalopod jaws are unique as the two jaw elements do not contact one another, are embedded in a muscular mass and connected through a muscle joint. Previous studies have described the anatomy of the buccal mass muscles in cephalopods and have proposed variation in muscle volume depending on beak shape. However, the general structure of the muscles has been suggested to be similar in octopuses, squids, and cuttlefish. Here we provide a quantitative analysis of the variation in the buccal mass of coleoids using traditional dissections, histological sections and contrast-enhanced computed tomography scans. Our results show that the buccal mass is composed of four main homologous muscles present in both decapodiforms and octopodiforms as suggested previously. However, we also report the presence of a muscle uniquely present in octopodiforms (the postero-lateral mandibular muscle). Our three dimensional reconstructions and quantitative analyses of the buccal mass muscles pave the way for future functional analyses allowing to better model jaw closing in coleoids. Finally, our results suggest differences in beak and muscle function that need to be validated using future in vivo functional analyses.

Biomechanics, motor control and dynamic models of the soft limbs of the octopus and other cephalopods.

Muscular hydrostats are organs composed entirely of packed arrays of incompressible muscles and lacking any skeletal support. Found in both vertebrates and invertebrates, they are of great interest for comparative biomechanics from engineering and evolutionary perspectives. The arms of cephalopods (e.g. octopus and squid) are particularly interesting muscular hydrostats because of their flexibility and ability to generate complex behaviors exploiting elaborate nervous systems. Several lines of evidence from octopus studies point to the use of both brain and arm-embedded motor control strategies that have evolved to simplify the complexities associated with the control of flexible and hyper-redundant limbs and bodies. Here, we review earlier and more recent experimental studies on octopus arm biomechanics and neural motor control. We review several dynamic models used to predict the kinematic characteristics of several basic motion primitives, noting the shortcomings of the current models in accounting for behavioral observations. We also discuss the significance of impedance (stiffness and viscosity) in controlling the octopus's motor behavior. These factors are considered in light of several new models of muscle biomechanics that could be used in future research to gain a better understanding of motor control in the octopus. There is also a need for updated models that encompass stiffness and viscosity for designing and controlling soft robotic arms. The field of soft robotics has boomed over the past 15 years and would benefit significantly from further progress in biomechanical and motor control studies on octopus and other muscular hydrostats.

Sensory specializations drive octopus and squid behaviour.

The evolution of new traits enables expansion into new ecological and behavioural niches. Nonetheless, demonstrated connections between divergence in protein structure, function and lineage-specific behaviours remain rare. Here we show that both octopus and squid use cephalopod-specific chemotactile receptors (CRs) to sense their respective marine environments, but structural adaptations in these receptors support the sensation of specific molecules suited to distinct physiological roles. We find that squid express ancient CRs that more closely resemble related nicotinic acetylcholine receptors, whereas octopuses exhibit a more recent expansion in CRs consistent with their elaborated 'taste by touch' sensory system. Using a combination of genetic profiling, physiology and behavioural analyses, we identify the founding member of squid CRs that detects soluble bitter molecules that are relevant in ambush predation. We present the cryo-electron microscopy structure of a squid CR and compare this with octopus CRs1 and nicotinic receptors2. These analyses demonstrate an evolutionary transition from an ancestral aromatic 'cage' that coordinates soluble neurotransmitters or tastants to a more recent octopus CR hydrophobic binding pocket that traps insoluble molecules to mediate contact-dependent chemosensation. Thus, our study provides a foundation for understanding how adaptation of protein structure drives the diversification of organismal traits and behaviour.

Structural basis of sensory receptor evolution in octopus.

Chemotactile receptors (CRs) are a cephalopod-specific innovation that allow octopuses to explore the seafloor via 'taste by touch'1. CRs diverged from nicotinic acetylcholine receptors to mediate contact-dependent chemosensation of insoluble molecules that do not readily diffuse in marine environments. Here we exploit octopus CRs to probe the structural basis of sensory receptor evolution. We present the cryo-electron microscopy structure of an octopus CR and compare it with nicotinic receptors to determine features that enable environmental sensation versus neurotransmission. Evolutionary, structural and biophysical analyses show that the channel architecture involved in cation permeation and signal transduction is conserved. By contrast, the orthosteric ligand-binding site is subject to diversifying selection, thereby mediating the detection of new molecules. Serendipitous findings in the cryo-electron microscopy structure reveal that the octopus CR ligand-binding pocket is exceptionally hydrophobic, enabling sensation of greasy compounds versus the small polar molecules detected by canonical neurotransmitter receptors. These discoveries provide a structural framework for understanding connections between evolutionary adaptations at the atomic level and the emergence of new organismal behaviour.

Recording electrical activity from the brain of behaving octopus.

Octopuses, which are among the most intelligent invertebrates,1,2,3,4 have no skeleton and eight flexible arms whose sensory and motor activities are at once autonomous and coordinated by a complex central nervous system.5,6,7,8 The octopus brain contains a very large number of neurons, organized into numerous distinct lobes, the functions of which have been proposed based largely on the results of lesioning experiments.9,10,11,12,13 In other species, linking brain activity to behavior is done by implanting electrodes and directly correlating electrical activity with observed animal behavior. However, because the octopus lacks any hard structure to which recording equipment can be anchored, and because it uses its eight flexible arms to remove any foreign object attached to the outside of its body, in vivo recording of electrical activity from untethered, behaving octopuses has thus far not been possible. Here, we describe a novel technique for inserting a portable data logger into the octopus and implanting electrodes into the vertical lobe system, such that brain activity can be recorded for up to 12 h from unanesthetized, untethered octopuses and can be synchronized with simultaneous video recordings of behavior. In the brain activity, we identified several distinct patterns that appeared consistently in all animals. While some resemble activity patterns in mammalian neural tissue, others, such as episodes of 2 Hz, large amplitude oscillations, have not been reported. By providing an experimental platform for recording brain activity in behaving octopuses, our study is a critical step toward understanding how the brain controls behavior in these remarkable animals.

Multiple nerve cords connect the arms of octopuses, providing alternative paths for inter-arm signaling.

Octopuses are remarkable in their ability to use many arms together during behavior (e.g., see Levy et al., 1 Mather,2 Byrne et al.,3 and Hanlon et al.4). Arm responses and multi-arm coordination can occur without engagement of major brain regions,5 which indicates the importance of local proprioceptive responses and peripheral connections. Here, we examine the intramuscular nerve cords (INCs),6,7,8,9 the key proprioceptive anatomy in the arms. INCs are understood to include proprioceptive neurons, multipolar neurons, and motoneurons (reviewed by Graziadei10) and are thought to contribute to structuring whole-arm movement.11 There are four INCs running the full length of each arm (e.g., see Guérin-Ganivet,6 Martoja and May,8 and Graziadei9); we focused on the pair closest to the suckers, called the oral INCs. In tracking the oral INCs, we found that they extend proximally and continue beyond the arm, through the arm's base. Each oral INC bypasses two adjacent arms and is continuous with the nearer oral INC of the third arm over. As a result, an arm connects through oral INC pathways to arms that are two arms away to the right and left of it. In addition to connecting distant arms, nerve fibers project from the central region of the INCs, suggesting function in local tissues. The other two INCs, paired aboral INCs, also extend proximally beyond the arm's base with trajectories suggestive of the oral INC pattern. These data identify previously unknown regions of the INCs that link distant arms, creating anatomical connections. They suggest potential INC proprioceptive function in extra-arm tissues and contribute to an understanding of embodied organization for octopus behavioral control.12,13,14,15.

Octopus bimaculoides' arm recruitment and use during visually evoked prey capture.

Octopus' limb hyper-redundancy complicates traditional motor control system theory due to its extensive sensory inputs, subsequent decision-making, and arm coordination. Octopuses are thought to reduce flexibility control complexity by relying on highly stereotypical motor primitives (e.g., reaching1,2,3,4 and crawling5) and multi-level processes to coordinate movement,6,7 utilizing extensive peripheral nervous system (PNS) processing.2,8,9 Division of labor along the anterior-posterior axis10 and limb specialization of the four anterior arms in T-maze food retrieval11 further simplify control. However, specific arm recruitment and coordination during visually guided reaching behavior remains poorly understood. Here, we investigated visually evoked Octopus bimaculoides' prey capture capabilities12,13 by eliciting and examining prey-specific arm recruitment. When striking crabs, octopuses preferred synchronous arm recruitment, while sequential arm recruitment with a characteristic swaying movement is employed for shrimp. Such behavioral selection aligns with specific prey escape strategies and the octopus' flexible arm biomechanical constraints. Although side bias existed, we found significant bilateral symmetry, with one side being functionally a mirror of the other rather than anterior arm use being functionally equal and differing to posterior arm use. Among arms, the second limb is unequivocally dominant for goal-directed monocularly driven prey capture. Although the eight arms share gross anatomy and are considered equipotential,10,14 such arm use for specific actions could reflect subtle evolutionary adaptations. Finally, we quantitatively show, corroborating earlier observations,10,15 that octopuses employ a dimension reduction strategy by actively deciding to recruit adjacent arms over other available arms during either sequential or synchronous visually evoked prey attack.

Behavioral changes in senescent giant Pacific octopus (Enteroctopus dofleini) are associated with peripheral neural degeneration and loss of epithelial tissue.

Most species of octopus experience extreme physical decline after a single reproductive bout which extends over a period of days, weeks, or months before eventual death. Although outward indicators of senescence are widely recognized, comparatively little is known about physiological and neural changes accompanying terminal decline in octopuses. Here, we measured changes in behavioral response to nociceptive stimuli across the lifespan in giant Pacific octopus (GPO), Enteroctopus dofleini, held in public aquariums in the USA. Post-euthanasia, tissue was collected from arm tips, and neural and epithelial cell degeneration was quantified and compared with biopsies of arm tips from healthy, pre-reproductive GPOs. Behavioral assays showed significant changes both in low threshold mechanosensory responses and nociceptive behavioral responses beginning early in senescence and extending until euthanasia. Histology data showed that while the ratio of apoptotic cells to total cell number stayed constant between healthy and senescent GPOs, overall neural and epithelial cell density was significantly lower in terminally senescent octopuses compared with healthy controls. Our data provide new insight into the time-course and causes of sensory dysfunction in senescent cephalopods and suggest proactive welfare management should begin early in the senescence phase, well before animals enter terminal decline.

Steroid hormones of the octopus self-destruct system.

Among all invertebrates, soft-bodied cephalopods have the largest central nervous systems and the greatest brain-to-body mass ratios, yet unlike other big-brained animals, cephalopods are unusually short lived.1-5 Primates and corvids survive for many decades, but shallow-water octopuses, such as the California two-spot octopus (Octopus bimaculoides), typically live for only 1 year.6,7 Lifespan and reproduction are controlled by the principal neuroendocrine center of the octopus: the optic glands, which are functional analogs to the vertebrate pituitary gland.8-10 After mating, females steadfastly brood their eggs, begin fasting, and undergo rapid physiological decline, featuring repeated self-injury and leading to death.11 Removal of the optic glands completely reverses this life history trajectory,10 but the signaling factors underlying this major life transition are unknown. Here, we characterize the major secretions and steroidogenic pathways of the female optic gland using mass spectrometry techniques. We find that at least three pathways are mobilized to increase synthesis of select sterol hormones after reproduction. One pathway generates pregnane steroids, known in other animals to support reproduction.12-16 Two other pathways produce 7-dehydrocholesterol and bile acid intermediates, neither of which were previously known to be involved in semelparity. Our results provide insight into invertebrate cholesterol pathways and confirm a remarkable unity of steroid hormone biology in life history processes across Bilateria.

Contact chemoreception in multi-modal sensing of prey by Octopus.

Octopuses have keen vision and are generally considered visual predators, yet octopuses predominantly forage blindly in nature, inserting their arms into crevices to search and detect hidden prey. The extent to which octopuses discriminate prey using chemo- versus mechano-tactile sensing is unknown. We developed a whole-animal behavioral assay that takes advantage of octopuses' natural searching behavior to test their ability to discriminate prey from non-prey tastes solely via contact chemoreception. This methodology eliminated vision, mechano-tactile sensing and distance chemoreception while testing the contact chemosensory discriminatory abilities of the octopus arm suckers. Extracts from two types of prey (crab, shrimp) and three types of non-prey (sea star, algae, seawater) were embedded in agarose (to control for mechano-tactile discrimination) and presented to octopuses inside an artificial rock dome; octopuses reached their arms inside to explore its contents - imitating natural prey-searching behavior. Results revealed that octopuses are capable of discriminating between potential prey items using only contact chemoreception, as measured by an increased amount of sucker contact time and arm curls when presented with prey extracts versus non-prey extracts. These results highlight the importance of contact chemoreception in the multi-modal sensing involved in a complex foraging behavior.

Octopus-inspired sucker to absorb soft tissues: stiffness gradient and acetabular protuberance improve the adsorption effect.

Rigid suckers commonly used in surgical procedures often cause absorption damage, while their soft counterparts are difficult to handle due to their weak anchoring. Alternatively, the octopus sucker is both soft and has strong suction power. Further observation revealed that its structure is self-sealing and that the tissues are layered in hardness. Inspired by said structure and the characteristics of associated materials, a bionic soft sucker with stiffness gradient and acetabular roof structure was proposed, made of silicone with varying hardness including structures such as acetabular roof and circle muscles. The automatic tensile force measurement system was used to experimentally analyze the adsorption performance of the suckers to the soft curved contact surface. Both dry and wet conditions were tested, along with practical tests on organisms. The bionic sucker adsorption force was increased by 25.1% and 34.6% on the cylindrical surface, and 45.2% and 7.3% on the spherical surface for dry and wet conditions, respectively. During the experiment, the bionic suckers did not cause notable suction damage to the contact surfaces. Thus, this type of bionic sucker shows good application prospects in the field of surgery.