Behavioral and neurophysiological evidence suggests affective pain experience in octopus

Abstract

Pain is a negative affective state arising from tissue damage or inflammation. Because pain is aversive and its relief is innately rewarding, animals may learn to avoid a context in which pain is experienced and prefer one where pain relief occurs. It is generally accepted that vertebrate animals experience pain; however, there is currently inconclusive evidence that the affective component of pain occurs in any invertebrate. Here, we show that octopuses, the most neurologically complex invertebrates, exhibit cognitive and spontaneous behaviors indicative of affective pain experience. In conditioned place preference assays, octopuses avoided contexts in which pain was experienced, preferred a location in which they experienced relief from pain, and showed no conditioned preference in the absence of pain. Injection site grooming occurred in all animals receiving acetic acid injections, but this was abolished by local anesthesia. Thus, octopuses are likely to experience the affective component of pain.

Keywords: Biological Sciences; Ethology; Neuroscience.

Graphical abstract

Figure 1

CPP design and timeline (A) Octopus bocki in the start chamber of the CPP box. (B) Diagram of the apparatus, with pattern shown on the back and sides only for clarity. In experimental trials, visual cues covered all four walls. (C) Timeline of an experiment showing sequences for CPA and CPP procedures. In this example, an octopus showed an initial preference in session 1 for the dot chamber and is thus trained against initial preference (i.e., the octopus is given AA injection prior to confinement in the dot chamber or lidocaine prior to the stripe chamber). Different animals were used to test each of the four conditions (see Figure 2). Control sequences (saline/saline and saline/lidocaine) not shown.

Figure 2

Conditioned place avoidance (CPA) and conditioned place preference (CPP) assays reveal the affective component of pain in octopus In trials where initially preferred chambers were paired with 0.5% acetic acid (AA) injection, octopuses spent less time in their initially preferred chamber in a post-training period of free exploration, compared with octopuses receiving saline. In trials where octopuses received lidocaine over an area of prior injection (either saline or AA), octopuses preferred the chamber paired with lidocaine only if they had previously been given AA injection. Points show individual change in time values for each subject. Different animals were used to test each condition. Central, wide bars show the mean and whiskers show standard error of the mean. Asterisks indicate significant between-group differences (Bonferroni post-hoc test, ∗∗p < 0.01).

Figure 3

Precise and specific wound-directed grooming behaviors show discriminative pain experience in octopus Top panel shows examples of wound-directed behaviors, and colors surrounding each image correspond to shaded frequencies in stacked bars, below. Arrowheads indicate the location of AA injection on the arm. Behaviors were observed during training trials and 24 h later. AA-injected octopuses showed sustained wound attention and concealment that persisted for at least 24 hr after AA injection. Skin removal behavior was observed in all AA-injected animals, suggesting a specific representation of acid-induced pain that elicits a highly specific behavioral response. Bar acronyms: AA, acetic acid injection; S, saline injection; L, lidocaine injection after earlier AA injection; LC, lidocaine control (lidocaine injected after earlier saline injection). Numbers inside bars in first panel (5 min) are group sample sizes, which are the same for each subsequent interval. Asterisks indicate significant difference in proportions of animals performing each behavior, relative to saline-injected controls (fishers exact tests, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.

Figure 4

Examples of electrophysiology recordings and summary data showing that nociceptive signal from the arms is available to the octopus CNS (A) Examples of spontaneous (ongoing) and evoked activity in the brachial connective before and after injection of acetic acid (AA, shown as a black circle at arm stimulation position 3) and at the point where lidocaine is injected locally over the region of prior AA injection (shown as a red overlay of the black circle on the arm at position 3). Note the almost immediate cessation of ongoing activity after lidocaine injection, and the complete suppression of evoked activity in the region where lidocaine was injected at position 3 on the arm of the octopus. (B) Ongoing, spontaneous firing in the brachial connective is increased after AA injection and blocked by injection of lidocaine into the same position on the arm. (C) Summary data showing responses to touch on the arm at four locations (indicated by shaded blue circles on the octopus body outline). There is clear enhancement of evoked activity after injection that is suppressed by injection of a local anesthetic. Points show individual values for each subject. Central, wide bars show the mean and whiskers show S.E.M. Asterisks above linking bars indicate significant differences between groups (paired, Holm-Bonferroni corrected t-tests, ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001.