Anesthetic Efficacy of Magnesium Chloride and Ethyl Alcohol in Temperate Octopus and Cuttlefish Species

Abstract

Cephalopods are important in biologic and biomedical research, yet relatively little objective information is available to guide researchers and veterinarians regarding the best methods for anesthetizing these animals for various experimental procedures. Recent studies demonstrate that ethyl alcohol and magnesium chloride are effective at depressing efferent and afferent neural signals in some tropical cephalopod species when measured via the pallial nerve. Here we used similar methods to test 2 temperate species (Octopus bimaculoides and Sepia officinalis) and demonstrate that (1) ethyl alcohol and magnesium chloride were effective at reversibly depressing evoked activity in the pallial nerve, (2) ethyl alcohol generally had shorter induction and recovery times compared with magnesium chloride, (3) both agents were associated with a latency between the behavioral and neural effects, and it was longer with magnesium chloride, and (4) senescent animals generally had longer induction or recovery times than young animals. Both agents successfully anesthetized both life stages; however, our data show that assessing anesthesia based solely on behavior may lead to premature commencement of invasive procedures. We conclude that temperate cephalopods can be humanely, effectively, and completely anesthetized by using these 2 agents and that the loss of neural signal we show here is consistent with true anesthesia and not merely paralysis. This relatively simple, nondestructive nerve recording technique can be applied to the study of other prospective anesthetic agents in cephalopods.

Figure 1.

Electrode placement in temperate cephalopods. (A) Juvenile Octopus bimaculoides. (B) Senescent adult Octopus bimaculoides. (C) Illustration of where the U-shaped electrode is hooked gently around the large pallial nerve in Octopus bimaculoides, (D) subadult Sepia officinalis, and (E) senescent adult Sepia officinalis. (F) Illustration of where the U-shaped electrode is hooked gently around the large pallial nerve in Sepia officinalis.

Figure 2.

Responses of O. bimaculoides to anesthesia via EtOH and MgCl₂ (8 adult, 6 juvenile). (A) Juvenile and adult octopuses show no significant differences in time to loss of behavioral response to pinch (paralysis) or in time to loss of afferent neural signal (anesthesia). (B) Extended induction times were apparent for adults when exposed to magnesium chloride, compared with juveniles. (C) Recovery times from EtOH anesthesia were short and showed little variation in either adults or juveniles. (D) Recovery times from magnesium chloride were highly variable, but no significant differences were apparent. Data are given as mean ± SEM; the cartoons show the groups included in the figure in gray shading. Significant differences between age classes are indicated (unpaired t test, §, P < 0.0001).

Figure 3.

Responses of cuttlefish (S. officinalis) to anesthesia via EtOH and (adults: 5 EtOH, 3 MgCl₂; juveniles: 5 EtOH, 5 MgCl₂). (A) Juvenile and adult cuttlefish show no significant differences in time to loss of behavioral response to pinch (paralysis), but juveniles had significantly longer latency to loss of afferent neural signal (anesthesia) compared with adults. (B) There were no differences in induction times when MgCl₂ was used. (C) Recovery times after EtOH anesthesia were more variable for juveniles than for adults but did not differ significantly between groups. (D) Recovery of neural signal after MgCl₂ anesthesia was significantly longer for adults, but behavioral recovery did not differ significantly between age groups. Data are given as mean ± SEM; cartoons show the groups included in the figure in gray shading. Significant difference between age classes are indicated (unpaired t test, †, P < 0.01).

Figure 4.

Comparisons of the 2 anesthetic substances plotted separately for juvenile (n = 6) and adult (n = 8) octopuses. (A) In juvenile octopuses, latency from application of the anesthetic agent until loss of behavioral response (paralysis) was significantly (P = 0.0003) longer for MgCl₂ compared with EtOH. There was also a significant (P = 0.011) lag in the EtOH group between the loss of behavioral response (paralysis) and the loss of neural signal (anesthesia). (B) In adult octopuses, both paralysis and anesthesia took longer (paralysis, 0.008, anesthesia, P = 0.001) in the MgCl₂ group compared with EtOH. There was also a significant lag between paralysis and anesthesia with MgCl₂ (P = 0.02) (C) Recovery of juveniles from paralysis (that is, return of behavioral responses to the stimulus) took longer for animals exposed to MgCl₂ compared with EtOH (P = 0.04). In adults, recovery of neural signal (reversal of anesthesia) took longer for MgCl₂ (P = 0.02). Data are given as mean ± SEM. Comparisons within groups according to agent made by using paired t tests; comparisons of paralysis or anesthesia times between substances were made by using unpaired t tests, *, P < 0.05. †, P < 0.01 ‡, P < 0.001

Figure 5.

Comparisons of the 2 anesthetic substances plotted separately for juvenile and adult cuttlefish. (A) In juvenile cuttlefish, both anesthetic substances produced significant lag of anesthesia onset compared with the onset of paralysis (EtOH, P = 0.002, MgCl_2, P = 0.02). Time to anesthesia was also longer in the EtOH group compared with the MgCl₂ group (n = 5 per group, P = 0.003). (B) In adults, onset of anesthesia lagged significantly behind the onset of paralysis for MgCl₂ (n = 3, P = 0.045) but not for EtOH (n = 5). (C) Recovery from anesthesia (i.e., return of afferent neural signal) did not differ in juveniles in the MgCl₂ group compared with EtOH. (D) Recovery of both behavioral (P < 0.0001) and neural responses (P < 0.0001) were delayed significantly in adults treated with MgCl₂ compared with those anesthetized with EtOH . Data are shown as Comparisons within groups according to agent made by using paired t tests; comparisons of paralysis or anesthesia times between substances were made by using unpaired t tests. *, P < 0.05. †, P < 0.01. ‡, P < 0.001