This paper is the result of an international initiative and is a first attempt to develop guidelines for the care and welfare of cephalopods (i.e. nautilus, cuttlefish, squid and octopus) following the inclusion of this Class of ∼700 known living invertebrate species in Directive 2010/63/EU. It aims to provide information for investigators, animal care committees, facility managers and animal care staff which will assist in improving both the care given to cephalopods, and the manner in which experimental procedures are carried out. Topics covered include: implications of the Directive for cephalopod research; project application requirements and the authorisation process; the application of the 3Rs principles; the need for harm-benefit assessment and severity classification. Guidelines and species-specific requirements are provided on: i. supply, capture and transport; ii. environmental characteristics and design of facilities (e.g. water quality control, lighting requirements, vibration/noise sensitivity); iii. accommodation and care (including tank design), animal handling, feeding and environmental enrichment; iv. assessment of health and welfare (e.g. monitoring biomarkers, physical and behavioural signs); v. approaches to severity assessment; vi. disease (causes, prevention and treatment); vii. scientific procedures, general anaesthesia and analgesia, methods of humane killing and confirmation of death. Sections covering risk assessment for operators and education and training requirements for carers, researchers and veterinarians are also included. Detailed aspects of care and welfare requirements for the main laboratory species currently used are summarised in Appendices. Knowledge gaps are highlighted to prompt research to enhance the evidence base for future revision of these guidelines.

Cephalopods have been utilised in neuroscience research for more than 100 years particularly because of their phenotypic plasticity, complex and centralised nervous system, tractability for studies of learning and cellular mechanisms of memory (e.g. long-term potentiation) and anatomical features facilitating physiological studies (e.g. squid giant axon and synapse). On 1 January 2013, research using any of the about 700 extant species of “live cephalopods” became regulated within the European Union by Directive 2010/63/EU on the “Protection of Animals used for Scientific Purposes”, giving cephalopods the same EU legal protection as previously afforded only to vertebrates. The Directive has a number of implications, particularly for neuroscience research. These include: (1) projects will need justification, authorisation from local competent authorities, and be subject to review including a harm-benefit assessment and adherence to the 3Rs principles (Replacement, Refinement and Reduction). (2) To support project evaluation and compliance with the new EU law, guidelines specific to cephalopods will need to be developed, covering capture, transport, handling, housing, care, maintenance, health monitoring, humane anaesthesia, analgesia and euthanasia. (3) Objective criteria need to be developed to identify signs of pain, suffering, distress and lasting harm particularly in the context of their induction by an experimental procedure. Despite diversity of views existing on some of these topics, this paper reviews the above topics and describes the approaches being taken by the cephalopod research community (represented by the authorship) to produce “guidelines” and the potential contribution of neuroscience research to cephalopod welfare.

The hippocampal complex (hippocampus and parahippocampalis) is known to play a role in spatial memory in birds and is known to be larger in food-storing versus non-storing birds. In the present study, we investigated the relative volume of the hippocampal complex in four food-storing corvids: gray-breasted jays (Aphelocoma ultramarina), scrub jays (Aphelocoma coerulescens), pinyon jays (Gymnorhinus cyanocephalus), and Clark's nutcrackers (Nucifraga columbiana). The results show that Clark's nutcrackers have a larger hippocampal complex, relative to both body and total brain size, than the other three species. Clark's nutcrackers rely more extensively on stored food in the wild than the other three species. Clark's nutcrackers also perform better during cache recovery and operant tests of spatial memory than scrub jays. Thus, greater hippocampal volume is associated with better performance in laboratory tests of spatial memory and with stronger dependence on food stores in the wild.

When placed in a rectangular aquarium (arena) containing no objects, blindfolded freshwater crayfish (Cherax destructor) explore by walking along the walls of the arena. Animals taken from their home tanks and placed in the arena for a 40‐min trial each day habituate and exhibit a reduction in their exploratory activity over 4 trials, despite their lack of continuous exposure to the arena. Dishabituation (i.e. an immediate increase in exploratory activity) occurs when animals were placed in the arena after the introduction of short partitions projecting at right angles from the walls. The dishabituation was interpreted as indicating that the animal can detect differences in the spatial configuration of the arena topography. Using dishabituation as a measure, we found that animals responded not only to the presence or absence of the partitions but also to changes in the position of the partitions. Animals with immobilized or lesioned second antennae no longer responded to configurational changes in the spatial arrangement of the partitions in the arena. We conclude that Cherax destructor relies upon the tactile input from its second antennae to detect topographical changes in the environment and that such topographical changes can be retained for at least 24 h. For an organism that forages in and defends a home territory on a daily basis, this seems to be an ecologically relevant time scale.

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In this review we show that the cephalopod vertical lobe (VL) provides a good system for assessing the level of evolutionary convergence of the function and organization of neuronal circuitry for mediating learning and memory in animals with complex behavior. The pioneering work of JZ Young described the morphological convergence of the VL with the mammalian hippocampus, cerebellum and the insect mushroom body. Studies in octopus and cuttlefish VL networks suggest evolutionary convergence into a universal organization of connectivity as a divergence-convergence ('fan-out fan-in') network with activity-dependent long-term plasticity mechanisms. Yet, these studies also show that the properties of the neurons, neurotransmitters, neuromodulators and mechanisms of long-term potentiation (LTP) induction and maintenance are highly variable among different species. This suggests that complex networks may have evolved independently multiple times and that even though memory and learning networks share similar organization and cellular processes, there are many molecular ways of constructing them.

Cephalopods are a large and ancient group of marine animals with complex brains. Forms extant today are equipped with brains, sensors, and effectors that allow them not to just exist beside modern vertebrates as predators and prey; they compete fiercely with marine vertebrates at every scale from small crustaceans to sperm whales. We review the evolution of this group’s brains, learning ability and complex behavior. We outline evidence that although competition with vertebrates has left a deep impression on the brains and behavior of cephalopods, the original reorganization of their complex brains from their molluscan ancestors might have been forged in ancient seas millions of years before the advent of bony fishes.

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