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. 2023 Jan 28;12(2):201.
doi: 10.3390/biology12020201.

Evaluation of Candidates for Systemic Analgesia and General Anesthesia in the Emerging Model Cephalopod, Euprymna berryi

Skyler Deutsch  1 , Rachel Parsons  1 , Jonathan Shia  2 , Sarah Detmering  1 , Christopher Seng  1 , Alyssa Ng  1 , Jacqueline Uribe  1 , Megan Manahan  1 , Amanda Friedman  1 , Gabrielle Winters-Bostwick  1 , Robyn J Crook  1
Affiliations

Evaluation of Candidates for Systemic Analgesia and General Anesthesia in the Emerging Model Cephalopod, Euprymna berryi

Skyler Deutsch et al. Biology (Basel). .

Abstract

Cephalopods' remarkable behavior and complex neurobiology make them valuable comparative model organisms, but studies aimed at enhancing welfare of captive cephalopods remain uncommon. Increasing regulation of cephalopods in research laboratories has resulted in growing interest in welfare-oriented refinements, including analgesia and anesthesia. Although general and local anesthesia in cephalopods have received limited prior study, there have been no studies of systemic analgesics in cephalopods to date. Here we show that analgesics from several different drug classes may be effective in E. berryi. Buprenorphine, ketorolac and dexmedetomidine, at doses similar to those used in fish, showed promising effects on baseline nociceptive thresholds, excitability of peripheral sensory nerves, and on behavioral responses to transient noxious stimulation. We found no evidence of positive effects of acetaminophen or ketamine administered at doses that are effective in vertebrates. Bioinformatic analyses suggested conserved candidate receptors for dexmedetomidine and ketorolac, but not buprenorphine. We also show that rapid general immersion anesthesia using a mix of MgCl2 and ethanol was successful in E. berryi at multiple age classes, similar to findings in other cephalopods. These data indicate that systemic analgesia and general anesthesia in Euprymna berryi are achievable welfare enhancing interventions, but further study and refinement is warranted.

Keywords: Euprymna; analgesia; anesthesia; cephalopod; mollusc; squid; welfare.

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Conflict of interest statement

R.J.C. is a co-editor of the Special Issue in which this paper appears, but was not involved in the editorial process for this paper. The authors declare no other conflicts.

Figures

Figure 1
Figure 1
Euprymna berryi, (A) Hatchling (B) Pre-reproductive adult (C) Senescent adult. All squid were captive bred in the laboratory for this study. Scale bars are 500 µm (A), and 5 mm (B,C).
Figure 2
Figure 2
Responses of uninjured, healthy squid to applications of an ascending series of von Frey filaments after receiving candidate analgesics. (A) Response thresholds (color change, movement of individual body parts, or slow avoidance movements) were tested at one and three hours after drug dosing. Responses were typically variable and no significant effects were found compared with control squid injected with sterile seawater (unpaired t-tests, all NS). Refer to Table 2 for sample sizes. (B) Nociceptive thresholds were determined by recording instances of either inking, reflexive, high-speed escape jetting, or stimulus-directed arm grooming. At one hour nociceptive thresholds were elevated in squid given dexmedetomidine (p = 0.0071) and high-dose buprenorphine (p = 0.0098), compared with control squid (unpaired, Bonferroni-corrected t-tests). At three hours only squid given high dose buprenorphine showed continued elevation of nociceptive thresholds (p = 0.0071). Points show mean and error bars show standard error of the mean (SEM). * p < 0.05. ** p < 0.01).
Figure 3
Figure 3
Peripheral nervous system excitability is suppressed by systemic analgesics. (A) A schematic of the nerve/tissue prep. The mantle was split down the midline and left and right sides were paired drug/control replicates. Drug-treated preparations were incubated in the same drug concentrations as shown in Figure 2, and controls were incubated in filtered artificial seawater. The large pallial nerve carries information to the central brain from the mantle nerves, which converge into the stellate ganglion. A suction electrode on the pallial nerve measures activity in response to stimulation on the mantle and fin tissue with von Frey filaments. (B) Spontaneous afferent firing was recorded for one minute prior to any stimulation being delivered. Only high-dose buprenorphine suppressed spontaneous activity (unpaired t-test vs control). (C) Buprenorphine suppressed evoked firing in response to touch with a light von Frey filament, which activates mechanoreceptors, and with a stiff von Frey filament, which activates nociceptors (paired t-tests vs. control-side prep). (D) Ketorolac suppressed nociceptor firing but had no effect on low-threshold mechanoreceptors. (E) Dexmedetomidine had no effect on pallial nerve activity. Bars show mean and error bars show standard error of the mean (SEM).
Figure 4
Figure 4
Pain-like behavior is suppressed by systemic analgesics. (A) Number of individual ink plumes released in response to fin pinch, and at one, five and ten minute intervals thereafter. Only dexmedetomidine significantly reduced inking (p = 0.04, unpaired t-test), although overall the instances of inking were low and these data show likely floor effects. (B) Site-directed grooming with the arms occurred repeatedly in control squid, but was significantly suppressed by all three drugs at one minute post-injury (Ket, p = 0.01, Dex, p = 0.003, Bup, p = 0.003, Bonferroni-corrected, unpaired t-tests). (C) Jetting showed no significant effects at any time point. (D) Ventilation rate, which is used as an indicator of pain in fish, was significantly higher in control squid at one and five minutes post-pinch compared with all three drug-treated groups (control vs. ketorolac, p = 0.03. Control vs. dexmedetomidine, p = 0.02, control vs. buprenorphine, p = 0.005, (D)). The suppression of respiratory rate was still present at five minutes for all drugs (control vs. ketorolac, p = 0.001. Control vs. dexmedetomidine, p = 0.04, control vs. buprenorphine, 0.004), but only for dexmedetomidine at 10 min post-pinch (vs. control, p = 0.008, (D)). Points show mean and error bars are standard error of the mean (SEM). * p, 0.05. ** p, 0.01. Colors of asterisks show comparisons of each color matched dataset to control.
Figure 5
Figure 5
Multiple sequence alignments of putative receptors for Ketamine (A), Ketorolac (B), and Dexmedetomidine (C). Residues with described functions in vertebrates are highlighted in yellow if they are conserved in cephalopod sequences or highlighted in blue and green if they diverge. (A) Homologs for NMDAR receptors 1 and 2 are conserved across mammals and cephalopod species. Some residues surrounding the central vestibule containing the ketamine binding pocket [41] of NMDAR 1 (Hs_NMDAR1 T648-dark yellow, V644-dark yellow) and NMDAR 2 (Hs_NMDAR2 T646-bright yellow, L642-pale yellow) are conserved across species for which we have sequence data (Eb_NMDAR1 sequence quality was too low to confirm conservation of T648). NMDAR 1 exhibits conservation of N616 (bright yellow), but the conservation of the homologous site (N615) in NMDAR 2 was limited to vertebrates (H. sapiens and D. rerio), with a cephalopod glycine (bright blue) replacing the vertebrate asparagine (bright green). (B) The amino acid sequences for cephalopod cyclooxygenase-2/prostaglandin synthase (COX/PGS) are conserved with those of vertebrates (H. sapiens and D. rerio). Amino acid residues that line the cyclooxygenase channel in H. sapiens prostaglandin synthase (H90, R120, Y355, Y387, R513, E524, S530, and L531) or make contact with the NSAID Ibuprofen [42] (V359, L359, M522, V523, G526, and A527) were assessed for conservation. Complete conservation across cephalopods for which sequences were available) and vertebrates was observed in ten of these fifteen residues (bright yellow), while L359, R513, M522, V523, and L531 were not conserved in all cephalopods (bright blue and green residues). (C) The amino acid sequences for cephalopod (Ob and Es) alpha 2 Adrenergic (α2A) receptors are highly conserved with those of vertebrates (Hs and Dr). Amino acid residues known to line the binding pocket and play some role in ligand or activity [43] were assessed for conservation. Complete conservation across cephalopods (for which sequences were available) and vertebrates was observed in eleven of the twelve residues (yellow), and one residue (C201) was replaced by a Serine in cephalopods. Abbreviations: Cephalopods—Ob: Octopus bimaculoides; Es: Euprymna scolopes; and Eb: Euprymna berryi. Vertebrates—Hs: Homo sapiens; Dr: Danio rerio. NMDAR: N-methyl-D-aspartate receptor, PGS: prostaglandin synthase, COX: Cyclooxygenase, α2A_Rec: Alpha 2 Adrenergic Receptor.
Figure 6
Figure 6
General anesthesia in multiple age-classes of E. berryi with a combination of 1% EtoH (v/v) and 1:3 ratio of isotonic MgCl2:SW. (A) Photographic sequence showing progressive outward signs of anesthesia induction. In all age classes paling of the arms progressed from tip to base (Black arrowheads), which was followed by all-over mantle paling (white arrowhead, 4th panel) and finally relaxation of chromatophores across the head-bar, between the eyes (5th panel). Once squid were completely pale, we tested for complete anesthesia by recording absence of response to visual stimulation, vibratory stimulation, or light touch on the body surface. Latency between complete paling and full anesthesia was variable. (B) Comparisons among the three age classes tested showed no significant differences in latency to paling of arms, mantle or head bars. (C) Reversal times were counted from the point where solutions were changed to fresh seawater. Return of chromatophore tone followed the reverse order of induction. In hatchlings we were only able to reliably identify whole-body darkening on recovery. No significant differences between age classes were found. (D) Latency to complete anesthesia and complete reversal wee compared among the age classes. No differences in induction times were found, but hatchlings recovered significantly faster than juvenile and senescent squid (one-way ANOVA followed by post-hoc Bonferroni corrected t-tests, critical alpha p < 0.05). Points show mean and error bars are standard error of the mean (SEM). * p < 0.05. *** p < 0.01).
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