Abstract
Cephalopod molluscs are among the most behaviorally and neurologically complex invertebrates. As they are now included in research animal welfare regulations in many countries, humane and effective anesthesia is required during invasive procedures. However, currently there is no evidence that agents believed to act as anesthetics produce effects beyond immobility. In this study we demonstrate, for the first time, that two of the most commonly used agents in cephalopod general anesthesia, magnesium chloride and ethanol, are capable of producing strong and reversible blockade of afferent and efferent neural signal; thus they are genuine anesthetics, rather than simply sedating agents that render animals immobile but not insensible. Additionally, we demonstrate that injected magnesium chloride and lidocaine are effective local anesthetic agents. This represents a considerable advance for cephalopod welfare. Using a reversible, minimally invasive recording procedure, we measured activity in the pallial nerve of cuttlefish (Sepia bandensis) and octopus (Abdopus aculeatus, Octopus bocki), during induction and reversal for five putative general anesthetic and two local anesthetic agents. We describe the temporal relationship between loss of behavioral responses (immobility), loss of efferent neural signal (loss of “consciousness”) and loss of afferent neural signal (anesthesia) for general anesthesia, and loss of afferent signal for local anesthesia. Both ethanol and magnesium chloride were effective as bath-applied general anesthetics, causing immobility, complete loss of behavioral responsiveness and complete loss of afferent and efferent neural signal. Cold seawater, diethyl ether, and MS-222 (tricaine) were ineffective. Subcutaneous injection of either lidocaine or magnesium chloride blocked behavioral and neural responses to pinch in the injected area, and we conclude that both are effective local anesthetic agents for cephalopods. Lastly, we demonstrate that a standard euthanasia protocol—immersion in isotonic magnesium chloride followed by surgical decerebration—produced no behavioral response and no neural activity during surgical euthanasia. Based on these data, we conclude that both magnesium chloride and ethanol can function as general anesthetic agents, and lidocaine and magnesium chloride can function as local anesthetic agents for cephalopod molluscs.
Highlights
There is no direct evidence that cephalopods experience pain or distress as a result of noxious sensory input (Crook and Walters, 2011; Crook, 2013; Crook et al, 2013; Hague et al, 2013; Alupay et al, 2014), in many nations cephalopods are included in regulations that govern the ethical use of vertebrate animals in research, and such legislation [for example, in the UK Scientific Procedures Act (1986), EU Directive 2010/63/EU, and the Canadian Council on Animal Care] requires that anesthesia be provided during potentially harmful procedures
We demonstrate that local anesthesia using either injected lidocaine or high concentrations of magnesium chloride is effective at blocking afferent signal and behavioral responses to noxious stimulation over the injected area; We demonstrate that these agents, not commonly accepted as anesthetics in other taxa, achieve the primary goal of anesthesia—protecting the welfare of the animal when subject to invasive and potentially painful procedures
We have demonstrated that non-invasive monitoring of neural signal during anesthesia is a highly reliable indicator of anesthetic efficacy
Summary
Cephalopods are used frequently in studies of camouflage, motor control, cognition, visual processing, environmental toxicology, and microbiology (Hanlon and Messenger, 1996; Dickel et al, 2001; Sumbre et al, 2001; Darmaillacq et al, 2004; Lee et al, 2009; Mäthger et al, 2009; Allen et al, 2014; Chiao et al, 2015; Zepeda et al, 2017). In the course of such studies, it may be necessary to anesthetize, immobilize or euthanize cephalopods. 17 used high concentrations of magnesium chloride, 23 used some concentration of ethanol in seawater, 3 used a combination of these two agents, and 5 used a different agent. None of these studies used “modern” volatile or injectable anesthetic agents that are standard in the fields of vertebrate animal anesthesia (e.g., volatiles such as Isoflourane or Sevoflourane, or injectables such as Ketamine or Propofol). The field relies overwhelmingly on magnesium chloride and ethanol, which are generally considered effective for immobilization and from which cephalopods recover reliably
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