In Vivo Recording of Neural and Behavioral Correlates of Anesthesia Induction, Reversal, and Euthanasia in Cephalopod Molluscs

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.

Keywords: analgesia; cephalopoda; general anesthesia; immobilization; local anesthesia; neurophysiology; welfare impact.

Figure 1

We studied effects of anesthesia on three tropical, commercially available cephalopod species. (A) Sepia bandensis. (B) Abdopus aculeatus. (C) Octopus bocki. Recordings were conducted in vivo using a minimally-invasive hook electrode, attached to one pallial nerve. Twenty-four hours after experiments, animals showed normal camouflage and signaling behavior (D,E) the same specimen of S. bandensis shown in (A); (F,G) the same specimen of A. aculaeatus shown in (B), demonstrating that neither the anesthesia nor the experimental procedure itself caused any long-term damage to the animal's nervous systems or behaviors. Scale bar: 15 mm.

Figure 2

Background neural activity during quiescent, awake periods of cuttlefish (A) and Octopus (O. bocki, B), showing rhythmic bursts associated with mantle contractions during respiration. Respiration rates during anesthesia can be computed from electrophysiological traces, and respiration signal was a good indicator that the electrode was well-positioned, the nerve was healthy, and that animal was physiologically stable. (C) Respiration rates of animals in various procedure stages did not differ significantly. Awake animals showed slightly elevated rates of respiration which was likely due to handling stress. Respiration rates from cuttlefish in the EtoH and MgCl₂ treatment groups were measured during periods in which no afferent neural signal was detected during mantle pinch. Breathing rates were not significantly slower than during awake periods, and were within the range of normal respiration rates of unrestrained animals observed in home tanks.

Figure 3

Effective doses and the time to anesthesia (the loss of neural signal at both the medial and distal stimulation sites). (A) Effective doses of ethanol ranges from 1 to 4% in ASW. There was a weakly positive correlation between does and time-to-induction for cuttlefish (Pearson r² = 0.89, p = 0.02), line shows best fit from linear regression, dotted lines are 95% CI), but no relationship for octopus. (B) Nearly all subjects were effectively anesthetized in a solution of 1:3 MgCl₂: ASW, but times to anesthesia varied considerably. There was insufficient variation in dose to test dose/time relationships.

Figure 4

Latencies to behavioral and neural indicators of tri-partite anesthesia induction for cuttlefish and octopus (O. bocki and A. aculeatus pooled). (A) Ethanol anesthesia (effective doses pooled) showed tight temporal coupling of immobility, loss of evoked behavioral responses and loss of evoked neural responses. While magnesium chloride achieved the three requirements of anesthesia—immobility, loss of afferent signal and loss of efferent signal (not shown on figures, see Results for details)—there was a significant delay between the loss of behavioral response and the loss of neural response at the distal stimulation site (denoted by ^*). Latencies to medial and distal anesthesia were significantly delayed compared with the same measures for ethanol anesthesia (denoted by +). (B) The same variables shown for octopuses. Octopus sample sizes were smaller, and octopuses were inherently more variable, thus although patterns are similar to those of cuttlefish there are no significant differences. Within-treatment comparisons: paired t-tests. Between treatment comparisons: unpaired t-tests. All p-values are two-tailed. ^*p < 0.05.

Figure 5

Examples of electrophysiological traces recorded during pinch on the distal stimulations site, for cuttlefish in ethanol (A) and MgCl₂ (B). (A) A fully-awake cuttlefish has a strong response to pinch prior to anesthesia induction. This trace shows motor efferent signal associated with escape jetting (this animal makes 7 jets in rapid succession, coinciding with peaks in the signal). After 2 min in 3% ethanol, the animal ceases spontaneous behavior, and there is no neural signal in response to pinch on the contralateral side to the electrode (trace not shown). Evoked behavior (which may be reflexive or intentional) ceases after 4 min, at the same time that neural afferent signal ceases. Reversal in ASW is rapid, with simultaneous return of evoked behavioral and neural signals. (B) Cuttlefish undergoing MgCl₂ anesthesia showed a different pattern. While spontaneous and evoked behavior ceased at a similar time to the animal in ethanol in these examples, afferent and efferent neural signal persisted for much longer, and their loss was temporally more separated than for ethanol. Evoked afferent signal in response to pinch was present for more than 10 min after the cessation of evoked behavioral responses; this period is of great concern for welfare. Upon reversal, neural signal returned rapidly, but there was a length period in which behavioral responses remained absent; again, the existence of this extended period is likely to be problematic for researchers using behavioral measures to ascertain anesthesia. Fully awake behavior usually lagged behind return of evoked behavioral responses by more than 5 min.

Figure 6

Examples of electrophysiological traces recorded during pinch on the distal stimulation site, for one octopus undergoing ethanol (A) or MgCl₂ (B) anesthesia. (A) A fully awake octopus shows strong neural response to pinch. Within 2 min of induction in ethanol, spontaneous behaviors have ceased and neural signal is almost completely abolished. By 5 min there is no neural and no behavioral responses to pinch. Similar to cuttlefish under ethanol anesthesia, reversal is rapid with simultaneous return of neural and behavioral responses to pinch. (B) In this example of an octopus in MgCl₂, anesthesia induction is uncharacteristically rapid (compare with means shown in Figure 4), with minimal delay between loss of behavioral responses and loss of neural responses. We selected this example to show the very prolonged recovery process that was typical of octopuses anesthetized in MgCl₂. By 4 min after reversal begins, neural signal is apparent, and it remains strong and mostly unchanging for the next 20 min. There is a slight increase in signal amplitude and duration at 24 min, and the first behavioral response to pinch is recorded at 25 min after reversal begins. During this period there is no behavioral indication that the animal's nervous system is functional. Behaviors indicating that animal is fully awake typically returned 5–10 min after evoked behaviors.

Figure 7

Latencies to behavioral and neural indicators of anesthesia reversal for cuttlefish and octopus (O. bocki and A. aculeatus pooled). (A) Cuttlefish undergoing reversal of ethanol anesthesia (A, bottom) showed tight temporal coupling of return of spontaneous behaviors, return of evoked behavioral responses and return of evoked neural responses. Return of neural signal in cuttlefish under magnesium chloride anesthesia (A, top) occurred on a similar timescale to that for ethanol, but return of behavioral responses was significantly delayed, both with respect to neural signal in the animals (denoted by ^*), and with respect to behavioral recovery for ethanol (denoted by +). (B) The same variables shown for octopuses. (B), bottom: Octopuses undergoing reversal from ethanol anesthesia showed tight temporal coupling of all reversal indicators. Octopuses undergoing reversal of MgCl₂ anesthesia showed significant delays of neural responses at the medial stimulation site, behavioral responses at both sites, and latency to full recovery compared with ethanol-treated octopuses, but within-group, data were highly variable and not significantly different. Within-treatment comparisons: paired t-tests, marked with ^*. Between treatment comparisons: unpaired t-tests, marked with +. All p-values are two-tailed. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 8

Lag of neural signal loss (compared with behavior) upon induction, and advance of its return (compared with behavior) upon reversal. For each experimental trial, we used the following formula to compute “neural lag” (for induction) and “neural advance” (for recovery). Lag or Advance = (latency to loss/return of neural signal—latency to loss/return of behavioral signal). Thus, a positive value upon induction shows that neural signal persisted longer than behavioral responses did, and a negative value upon reversal shows that neural signal returned sooner than behavioral responses returned. Each outcome is tested with a one-sample t-test against an expected value of zero (i.e., neural and behavioral responses are lost or return simultaneously) (A) Cuttlefish induced with ethanol had very little very lag, while for those in magnesium chloride group, there was a significant lag at the distal site. Upon reversal (B), cuttlefish in the ethanol group had advances of zero, while both stimulation sites showed significant neural advances for cuttlefish in the magnesium chloride group. We also compared the advance of neural recovery compared with the return of spontaneous behavior (C), here also there was effectively zero advance for ethanol but a significant advance for magnesium chloride. Patterns were largely similar for octopuses. (D) Both the medial and distal stimulation sites showed significant neural lags for octopuses in the magnesium chloride group, and although there was some slight lags for the ethanol group, neither was significantly different from zero. (E) Upon reversal, there were noticeable but non-significant advances (medial; p = 0.08, distal, p = 0.13) at both stimulation sites for the magnesium chloride group, but effectively zero advances for the ethanol group. (F) Advance of neural recovery compared with the return of spontaneous behavior for octopuses was significant (p = 0.002, note that this is in the positive direction, i.e., spontaneous behavior occurred sooner than neural recovery, see results for explanation). There was a long advance for octopuses in the magnesium chloride group, but this was not significantly different from zero (p = 0.12), most likely due to the highly variable advances across trials. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001.

Figure 9

Behavioral signifiers of anesthesia induction and reversal. (A) Cuttlefish undergoing ethanol induction most frequently showed an all-pale (E) body color as their last spontaneous behavior before loss of neural signal. All-yellow (F) was also common for ethanol, and chromtophore pulsing, flickering, or waving occurred in some animals in the MgCl₂ group. Upon reversal of anesthesia (B), cuttlefish in the ethanol groups showed all-yellow most frequently as the behavior occurring closest to re-emergence of neural responses to pinch. Other behaviors included increased respiration rate (see Figure 2 for example), and other color changes (G). In the MgCl₂ group, behavioral indicators were more varied, with all-yellow, other color change, movement, and increased respiration noted as the behavior occurring closest to return of neural signal. For octopus inductions (C), all-pale was the most common behavioral sing of anesthesia in both ethanol and magnesium chloride groups. Respiration rate change in the ethanol group and loss of righting response (H) in the magnesium chloride group were also frequent indicators. Reversal for octopuses (D) was reliably signaled in both groups by a sudden coiling or curling of the arms (I). In the ethanol group, respiration rate changes and color change were also noted.

Figure 10

Representative images of the distal mantle of a cuttlefish injected with (A). 0.1 mL of 0.5% lidocaine in ASW, and (B). 0.1 mL of isotonic (330 mM) magnesium chloride. Images taken 20 min after injection. Scale bars 10 mm.

Figure 11

Euthanasia. Immersion in isotonic (330 mM) magnesium chloride resulted in rapid loss of afferent and efferent signal and respiratory arrest. No neural signal was recorded upon skin and cranial incision 5 min after respiratory arrest. This interval is shown as the “decerebration window” where the animal is deeply anesthetized and insensible during destruction of the CNS.