Squid have nociceptors that display widespread long-term sensitization and spontaneous activity after bodily injury

Abstract

Bodily injury in mammals often produces persistent pain that is driven at least in part by long-lasting sensitization and spontaneous activity (SA) in peripheral branches of primary nociceptors near sites of injury. While nociceptors have been described in lower vertebrates and invertebrates, outside of mammals there is limited evidence for peripheral sensitization of primary afferent neurons, and there are no reports of persistent SA being induced in primary afferents by noxious stimulation. Cephalopod molluscs are the most neurally and behaviorally complex invertebrates, with brains rivaling those of some vertebrates in size and complexity. This has fostered the opinion that cephalopods may experience pain, leading some governments to include cephalopods under animal welfare laws. It is not known, however, if cephalopods possess nociceptors, or whether their somatic sensory neurons exhibit nociceptive sensitization. We demonstrate that squid possess nociceptors that selectively encode noxious mechanical but not heat stimuli, and that show long-lasting peripheral sensitization to mechanical stimuli after minor injury to the body. As in mammals, injury in squid can cause persistent SA in peripheral afferents. Unlike mammals, the afferent sensitization and SA are almost as prominent on the contralateral side of the body as they are near an injury. Thus, while squid exhibit peripheral alterations in afferent neurons similar to those that drive persistent pain in mammals, robust changes far from sites of injury in squid suggest that persistently enhanced afferent activity provides much less information about the location of an injury in cephalopods than it does in mammals.

Figure 1.

Squid have mechanosensitive nociceptors in their fins. A, Innervation of the fin by the fin nerve, which provides the only direct communication with the brain. B, Fin nerve branches visualized by injection of air into the main trunk of the fin nerve. C, Excised fin-nerve preparation showing the recording electrode, crush injury site, and medial (M) and caudal (C) test sites on the fin, proximal and distal to the crush site, respectively. D, Extracellularly recorded activity evoked by pressing a glass probe (1.5 mm diameter) with progressively increasing force against the fin. Arrows indicate the first appearance of identified units. E, Activity at the same site evoked by application of von Frey filaments having progressively greater bending forces (indicated). Colored arrows indicate initial spikes of the same units shown in D.

Figure 2.

Squid mechanoafferents show rapid sensitization after noxious stimulation of the excised fin. A, Sensitization produced by punctate noxious stimulation. When the ascending sequence of punctate forces used to identify nociceptors was followed by a descending sequence of the same forces, the second application of all but the 100 g force evoked more spikes than the first application. B, Minor crush injury increased activity evoked by the indicated filaments. Note the large units responding to lower forces 5 min after injury. C, The same injury reduced mechanosensory (left) and electrical (right) thresholds of units that had been activated solely by 100 g before crush. D, Activity evoked by repeated tests (10 g) proximal and distal to the crush (Fig. 1C) showed a rapid, prolonged increase after fin injury (arrow). Activity was measured as the total number of spikes >4 μV during the 1 s period of maximal firing. Compared with corresponding responses in control fins, evoked activity was greater in injured fins during all medial tests, and all but the 0.2 min caudal test. E, Local injection of isotonic MgCl₂ solution blocked immediate activation of afferents by crush injury. Comparisons were to injections with seawater (SW) given before or after the MgCl₂ solution. F, Localized injection of isotonic MgCl₂ solution at the site to be crushed prevented crush-induced enhancement of activity evoked at nearby medial and distant caudal test sites. *p < 0.05, **p < 0.01.

Figure 3.

Fin injury in vivo produces short- and long-term sensitization of mechanoafferents and SA in the fin nerve. A, In vivo fin crush 30 min before excision sensitized mechanoafferents in the injured and contralateral fins. Activity evoked by 10 g tests showed increases at medial and caudal sites. B, In vivo fin crush 24 h before excision caused long-term sensitization of mechanosensory afferents in crushed and contralateral fins. C, In vivo but not ex vivo fin crush increased SA. The injured fin showed significantly higher SA 45 min after in vivo crush, whereas the injured and contralateral fins showed higher rates 24 h after in vivo crush. D, SA and activity evoked by tactile stimulation in fins from animals with natural injuries (received during interactions with conspecifics during the preceding 24 h) showed changes similar to those in experimentally injured animals. *p < 0.05, **p < 0.01.