Search

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.

Most species of octopus experience extreme physical decline after a single reproductive bout which extends over a period of days, weeks, or months before eventual death. Although outward indicators of senescence are widely recognized, comparatively little is known about physiological and neural changes accompanying terminal decline in octopuses. Here, we measured changes in behavioral response to nociceptive stimuli across the lifespan in giant Pacific octopus (GPO), Enteroctopus dofleini, held in public aquariums in the USA. Post-euthanasia, tissue was collected from arm tips, and neural and epithelial cell degeneration was quantified and compared with biopsies of arm tips from healthy, pre-reproductive GPOs. Behavioral assays showed significant changes both in low threshold mechanosensory responses and nociceptive behavioral responses beginning early in senescence and extending until euthanasia. Histology data showed that while the ratio of apoptotic cells to total cell number stayed constant between healthy and senescent GPOs, overall neural and epithelial cell density was significantly lower in terminally senescent octopuses compared with healthy controls. Our data provide new insight into the time-course and causes of sensory dysfunction in senescent cephalopods and suggest proactive welfare management should begin early in the senescence phase, well before animals enter terminal decline.

104We study learning in the ancient cephalopod Nautilus pompilius as a way of understanding how behavioral and neural complexity may have evolved in its more derived relatives, the coleoid cephalopods (octopuses, cuttlefish, and squid), whose brains are heavily invested in learning and memory. Nautilus retains a relatively primitive brain whereas coleoid brains are among the most complex of all the invertebrates, containing several discrete lobes dedicated to learning and memory storage—the vertical and frontal lobe complexes. The nautilus CNS lacks known homologues or analogues of these regions, and instead the brain more likely approximates those of the ancestors of the two extant subclasses. Nautilus is therefore uniquely placed in this lineage for studies of the evolution of complex behavior, including learning and memory. We identify and characterize learning, memory formation, composition, and retrieval in Nautilus through controlled classical conditioning studies and also behavioral assays with free-moving animals. In studies of classical conditioning where a light predicted the reward of food, nautiluses learned rapidly, expressing temporally separated short-term and long-term memory (STM and LTM). STM expression in Nautilus was similar to results from coleoids, but LTM was of shorter duration, perhaps due the lack of dedicated learning and memory centers found in more derived cephalopods. Spatial memory studies in complex mazes have revealed far more complex information processing than the “primitive brain” of nautiluses might predict. By providing increasingly complex cue arrays to be learned for spatial navigation to a goal, we investigated how nautiluses perform in complex cognitive tasks similar to those tested in coleoids. Nautiluses were trained to escape from a shallow-water, brightly lit arena (aversive) into deep, dark water (reward) via a hole in the arena—with the aid of either a beacon proximate to the goal or multiple visual cues in the arena located proximate and distant from the escape point. We found evidence for rapid learning, memory of long duration, and dynamic switching among learned navigational tactics when the cue arrays were altered. Current studies focusing on what information is retained, processed, and used to navigate reveal that nautiluses can simultaneously learn and remember net displacement (dead reckoning of turns and distance traveled) and beacon information (both associative and spatial) proximate to the escape point as they navigate toward the goal location.

Endogenous estrogens affect multiple sensory systems, including those involved in processing noxious and painful stimuli. Extensive evidence demonstrates that estrogenic environmental pollutants have profound, negative effects on growth and reproductive physiology, but there is limited information about how estrogenic pollutants might affect sensory systems known to be modulated by endogenous estrogens. Here, we show that ethinyl estradiol, the most common artificial estrogen found in coastal marine environments, disrupts normal behavioral and neural responses to tissue injury in the sepiolid, Euprymna scolopes (Hawaiian bobtail squid), which inhabits shallow tropical waters close to dense human habitation. Behavioral hypersensitivity and neural plasticity that occur normally after tissue injury were impaired both under chronic estrogen exposure beginning during embryogenesis, or after a single, high dose co-incident with injury. This suggests that these naturally selected responses to injury, which function to protect animals from predation and infection risk, may be impaired by anthropogenic pollution.

Endogenous estrogens affect multiple sensory systems, including those involved in processing noxious and painful stimuli. Extensive evidence demonstrates that estrogenic environmental pollutants have profound, negative effects on growth and reproductive physiology, but there is limited information about how estrogenic pollutants might affect sensory systems known to be modulated by endogenous estrogens. Here, we show that ethinyl estradiol, the most common artificial estrogen found in coastal marine environments, disrupts normal behavioral and neural responses to tissue injury in the sepiolid

Injury occurring in the neonatal period in mammals is known to induce plasticity in pain pathways that may lead to pain dysfunction in later life. Whether these effects are unique to the mammalian nervous system is not well understood. Here, we investigate whether similar effects of early-life injury are found in a large-brained comparative model, the cephalopod Euprymna scolopes. We show that the peripheral nervous system of E. scolopes undergoes profound and permanent plasticity after injury of peripheral tissue in the early post-hatching period, but not after the same injury given in the later juvenile period. Additionally, both innate defensive behaviour and learning are impaired by injury in early life. We suggest that these similar patterns of nervous system and behavioural remodelling that occur in squid and in mammals indicate an adaptive value for long-lasting plasticity arising from early-life injury, and suggest that injuries inflicted in very early life may signal to the nervous system that the environment is highly dangerous. Thus, neonatal pain plasticity may be a conserved pattern whose purpose is to set the developing nervous system's baseline responsiveness to threat. This article is part of the Theo Murphy meeting issue ‘Evolution of mechanisms and behaviour important for pain’.

Cephalopod molluscs are known for their extensive behavioral repertoire and their impressive learning abilities. Their primary defensive behaviors, such as camouflage, have received detailed study, but knowledge is limited to intensive study of relatively few species. A considerable challenge facing cephalopod research is the need to establish new models that can be captive bred, are tractable for range of different experimental procedures, and that will address broad questions in biological research. The Hawaiian Bobtail Squid (Euprymna scolopes) is a small, tropical cephalopod that has a long history of research in the field of microbial symbiosis, but offers great promise as a novel behavioral and neurobiological model. It can be bred in the laboratory through multiple generations, one of the few species of cephalopod that can meet this requirement (which is incorporated in regulations such as EU directive 2010/63/EU). Additionally, laboratory culture makes E. scolopes an ideal model for studying ontogeny- and experience-dependent behaviors. In this study, we show that captive bred juvenile and adult E. scolopes produce robust, repeatable defensive behaviors when placed in an exposed environment and presented with a visual threat. Further, adult and juvenile squid employ different innate defensive behaviors when presented with a size-matched model predator. When a 10-minute training procedure was repeated over three consecutive days, defensive behaviors habituated in juvenile squid for at least 5 days after training, but memory did not appear to persist for 14 days. In contrast, adult squid did not show any evidence of long-term habituation memory. Thus we conclude that this species produces a range of quantifiable, modifiable behaviors even in a laboratory environment where ecologically-relevant, complex behavioral sequences may not reliably occur. We suggest that the lack of long-term memory in adult squid may be related to their less escalated initial response to the mimic, and thus indicates less motivation to retain memory and not necessary inability to form memory. This is the first demonstration of age-related differences in defensive behaviors in Euprymna, and the first record of habituation in this experimentally tractable genus of squid.

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. □

Many efforts to improve science teaching in higher education focus on a few faculty members at an institution at a time, with limited published evidence on attempts to engage faculty across entire departments. We created a long-term, department-wide collaborative professional development program, Biology Faculty Explorations in Scientific Teaching (Biology FEST). Across 3 years of Biology FEST, 89% of the department’s faculty completed a weeklong scientific teaching institute, and 83% of eligible instructors participated in additional semester-long follow-up programs. A semester after institute completion, the majority of Biology FEST alumni reported adding active learning to their courses. These instructor self-reports were corroborated by audio analysis of classroom noise and surveys of students in biology courses on the frequency of active-learning techniques used in classes taught by Biology FEST alumni and nonalumni. Three years after Biology FEST launched, faculty participants overwhelmingly reported that their teaching was positively affected. Unexpectedly, most respondents also believed that they had improved relationships with departmental colleagues and felt a greater sense of belonging to the department. Overall, our results indicate that biology department–wide collaborative efforts to develop scientific teaching skills can indeed attract large numbers of faculty, spark widespread change in teaching practices, and improve departmental relations.

Serotonin is a widely studied modulator of neural plasticity. Here we investigate the effect of fluoxetine, a selective serotonin reuptake inhibitor, on short-term, peripheral nociceptive plasticity in the neurologically complex invertebrate, octopus. After crush injury to isolated mantle (body wall) tissue, application of 10 nM fluoxetine increased spontaneous firing in crushed preparations, but had a minimal effect on mechanosensory sensitization. Effects largely did not persist after washout. We suggest that transiently elevated, endogenous serotonin may help promote initiation of longer-term plasticity of nociceptive afferents and drive immediate and spontaneous behaviors aimed at protecting wounds and escaping dangerous situations.

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, particularly octopuses, may experience vertebrate-like pain when injured. However, it is not known whether octopuses possess nociceptors or if their somatic sensory neurons exhibit sensitization after injury. Here we show that the octopus Abdopus aculeatus expresses nocifensive behaviors including arm autotomy, and displays marked neural hyperexcitability both in injured and uninjured arms for at least 24 h after injury. These findings do not demonstrate that octopuses experience pain-like states; instead they add to the minimal existing literature on how cephalopods receive, process, and integrate noxious sensory information, potentially informing and refining regulations governing use of cephalopods in scientific research.

Injury occurring in the neonatal period in mammals is known to induce plasticity in pain pathways that may lead to pain dysfunction in later life. Whether these effects are unique to the mammalian nervous system is not well understood. Here, we investigate whether similar effects of early-life injury are found in a large-brained comparative model, the cephalopod Euprymna scolopes . We show that the peripheral nervous system of E. scolopes undergoes profound and permanent plasticity after injury of peripheral tissue in the early post-hatching period, but not after the same injury given in the later juvenile period. Additionally, both innate defensive behaviour and learning are impaired by injury in early life. We suggest that these similar patterns of nervous system and behavioural remodelling that occur in squid and in mammals indicate an adaptive value for long-lasting plasticity arising from early-life injury, and suggest that injuries inflicted in very early life may signal to the nervous system that the environment is highly dangerous. Thus, neonatal pain plasticity may be a conserved pattern whose purpose is to set the developing nervous system's baseline responsiveness to threat. This article is part of the Theo Murphy meeting issue ‘Evolution of mechanisms and behaviour important for pain’.

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.

Fast dynamic control of skin coloration is rare in the animal kingdom, whether it be pigmentary or structural. Iridescent structural coloration results when nanoscale structures disrupt incident light and selectively reflect specific colours. Unlike animals with fixed iridescent coloration (e.g. butterflies), squid iridophores (i.e. aggregations of iridescent cells in the skin) producedynamically tuneable structural coloration, as exogenous application of acetylcholine (ACh) changes the colour and brightness output. Previous efforts to stimulate iridophores neurally or to identify the source of endogenous ACh were unsuccessful, leaving researchers to question the activation mechanism. We developed a novel neurophysiological preparation in the squidDoryteuthis pealeiiand demonstrated that electrical stimulation of neurons in the skin shifts the spectral peak of the reflected light to shorter wavelengths (greater than 145 nm) and increases the peak reflectance (greater than 245%) of innervated iridophores. We show ACh is released within the iridophore layer and that extensive nerve branching is seen within the iridophore. The dynamic colour shift is significantly faster (17 s) than the peak reflectance increase (32 s), revealing two distinct mechanisms. Responses from a structurally altered preparation indicate that the reflectin protein condensation mechanism explains peak reflectance change, while an undiscovered mechanism causes the fast colour shift.

Animals with detectable injuries are at escalated threat of predation. The anti-predation tactic of schooling reduces individual predation risk overall, but it is not known how schooling behavior affects injured animals, or whether risks are reduced equally for injured animals versus other school members. In this laboratory study we examined the effects of minor fin injury on schooling decisions made by squid. Schooling behavior of groups of squid, in which one member was injured, was monitored over 24h. Injured squid were more likely to be members of a school shortly after injury (0.5-2h), but there were no differences compared with sham-injured squid at longer time points (6-24h). Overall, the presence of an injured conspecific increased the probability that a school would form, irrespective of whether the injured squid was a member of the school. When groups containing one injured squid were exposed to a predator cue, injured squid were more likely to join the school, but their position depended on whether the threat was a proximate visual cue or olfactory cue. We found no evidence that injured squid oriented themselves to conceal their injury from salient threats. Overall we conclude that nociceptive sensitization after injury changes grouping behaviors in ways that are likely to be adaptive.

Previous studies demonstrate that soft-bodied (coleoid) cephalopods are adept at learning and remembering features of their environment, but little is known about their primitive relative, nautilus. Nautilus makes nightly migrations from deep to shallow water along coral reef slopes, covering large areas of varied substrate. Memory of its surroundings may be advantageous, but the nautilus brain is the simplest among extant cephalopods, lacking dedicated neural regions that support learning and memory in other cephalopods. The authors hypothesize that the absence of these regions in nautilus may affect memory storage. Here the authors report the first evidence for spatial memory in 2- and 3-dimensional arenas. In a small open-field maze, nautiluses learned the location of a goal within 3 trials, and memory was stable for at least 2 weeks. In 3-dimensional environments, animals habituated within and across trials when their surroundings were unchanged, but activity increased when the environment changed topographically, although not when the change was visual only. These results are comparable to performances of coleoids in similar tasks and are surprising given the far simpler neuroanatomy of nautilus.

SUMMARYCephalopods are an exceptional taxon for examining the competing influences of ecology and evolutionary history on brain and behaviour. Coleoid cephalopods (octopuses, cuttlefishes and squids) have evolved specialised brains containing dedicated learning and memory centres, and rely on plastic behaviours to hunt prey effectively and communicate intricate visual displays. Their closest living relative, the primitive nautilus, is the sole remnant of an ancient lineage that has persisted since the Cambrian. Nautilus brains are the simplest among the extant cephalopods, and the absence of dedicated learning and memory regions may represent an ancestral condition. It is assumed that the absence of these regions should limit memory storage and recall in nautilus, but this assumption has never been tested. Here we describe the first evidence of learning and memory in chambered nautilus(Nautilus pompilius). Using a Pavlovian conditioning paradigm, we demonstrate that chambered nautilus exhibits temporally separated short- and long-term memory stores, producing a characteristic biphasic memory curve similar to that of cuttlefishes. Short-term memory persisted for less than 1 h post-training, whereas long-term memory was expressed between 6 and 24 h after training. Despite lacking the dedicated neural regions that support learning and memory in all other extant cephalopods, nautilus expressed a similar memory profile to coleoids. Thus the absence of these regions in the nautilus brain does not appear to limit memory expression, as hypothesised. Our results provide valuable insights into the evolution of neural structures supporting memory.

There is some evidence that macrurans recognize each other as individuals. In freshwater crayfish there are conflicting reports and there is limited information about the sensory mechanisms involved. To determine the extent to which the crayfish Cherax destructor is capable of individual recognition, we performed experiments that familiarized animals with each other and then manipulated their recent success in dominance contests. Crayfish were more likely to win an encounter when paired against a familiar opponent than an unfamiliar one after the manipulation stage. In other experiments, animals were attracted to familiar conspecifics when only visual or chemical cues were present. This demonstrates that C. destructor is able to discriminate between a familiar and an unfamiliar opponent. The results highlight the complex nature of intraspecific communication in crayfish and suggest elements likely to be of importance in the social interactions of groups in the wild state.

Chronic pain caused by insults to the CNS (central neuropathic pain) is widely assumed to be maintained exclusively by central mechanisms. However, chronic hyperexcitablility occurs in primary nociceptors after spinal cord injury (SCI), suggesting that SCI pain also depends upon continuing activity of peripheral sensory neurons. The present study in rats (Rattus norvegicus) found persistent upregulation after SCI of protein, but not mRNA, for a voltage-gated Na(+) channel, Nav1.8, that is expressed almost exclusively in primary afferent neurons. Selectively knocking down Nav1.8 after SCI suppressed spontaneous activity in dissociated dorsal root ganglion neurons, reversed hypersensitivity of hindlimb withdrawal reflexes, and reduced ongoing pain assessed by a conditioned place preference test. These results show that activity in primary afferent neurons contributes to ongoing SCI pain.

Summary How an octopus performs complex movements of its eight sucker-studded arms without entanglement has been a mystery. A new study has found that self-recognition of the octopus's skin by its suckers inhibits reflexive grasping of its own arms, simplifying the mechanisms needed to generate intricate arm behavior.

Summary Sublethal injury triggers long-lasting sensitization of defensive responses in most species examined, suggesting the involvement of powerful evolutionary selection pressures [1]. In humans, this persistent nociceptive sensitization is often accompanied by heightened sensations of pain and anxiety [2]. While experimental [3] and clinical [4] evidence support the adaptive value of immediate nociception during injury, no direct evidence exists for adaptive benefits of long-lasting sensitization after injury. Recently, we showed that minor injury produces long-term sensitization of behavioral and neuronal responses in squid, Doryteuthis pealei [5, 6]. Here we tested the adaptive value of this sensitization during encounters between squid and a natural fish predator. Locomotion and other spontaneous behaviors of squid that received distal injury to a single arm (with or without transient anesthesia) showed no measurable impairment 6 hr after the injury. However, black sea bass given access to freely swimming squid oriented toward and pursued injured squid at greater distances than uninjured squid, regardless of previous anesthetic treatment. Once targeted, injured squid began defensive behavioral sequences [7, 8] earlier than uninjured squid. This effect was blocked by brief anesthetic treatment that prevented development of nociceptive sensitization [6, 9]. Importantly, the early anesthetic treatment also reduced the subsequent escape and survival of injured, but not uninjured, squid. Thus, while minor injury increases the risk of predatory attack, it also triggers a sensitized state that promotes enhanced responsiveness to threats, increasing the survival (Darwinian fitness) of injured animals during subsequent predatory encounters.

Chronic neuropathic pain is often a severe and inadequately treated consequence of spinal cord injury (SCI). Recent findings suggest that SCI pain is promoted by spontaneous activity (SA) generated chronically in cell bodies of primary nociceptors in dorsal root ganglia (DRG). Many nociceptors express transient receptor potential V1 (TRPV1) channels, and in a preceding study most dissociated DRG neurons exhibiting SA were excited by the TRPV1 activator, capsaicin. The present study investigated roles of TRPV1 channels in behavioral hypersensitivity and nociceptor SA after SCI. Contusive SCI at thoracic segment T10 increased expression of TRPV1 protein in lumbar DRG 1 month after injury and enhanced capsaicin-evoked ion currents and Ca2+ responses in dissociated small DRG neurons. A major role for TRPV1 channels in pain-related behavior was indicated by the ability of a specific TRPV1 antagonist, AMG9810, to reverse SCI-induced hypersensitivity of hind limb withdrawal responses to mechanical and thermal stimuli at a dose that did not block detection of noxious heat. Similar reversal of behavioral hypersensitivity was induced by intrathecal oligodeoxynucleotides antisense to TRPV1, which knocked down TRPV1 protein and reduced capsaicin-evoked currents. TRPV1 knockdown also decreased the incidence of SA in dissociated nociceptors after SCI. Prolonged application of very low concentrations of capsaicin produced nondesensitizing firing similar to SA, and this effect was enhanced by prior SCI. These results show that TRPV1 makes important contributions to pain-related hypersensitivity long after SCI, and suggest a role for TRPV1-dependent enhancement of nociceptor SA that offers a promising target for treating chronic pain after SCI.

Although most investigations of the mechanisms underlying chronic pain after spinal cord injury (SCI) have examined the central nervous system (CNS), recent studies have shown that nociceptive primary afferent neurons display persistent hyperexcitability and spontaneous activity in their peripheral branches and somata in dorsal root ganglia (DRG) after SCI. This suggests that SCI-induced alterations of primary nociceptors contribute to central sensitization and chronic pain after SCI. Does SCI also promote growth of these neurons' fibers, as has been suggested in some reports? The present study tests the hypothesis that SCI induces an intrinsic growth-promoting state in DRG neurons. This was tested by dissociating DRG neurons 3 days or 1 month after spinal contusion injury at thoracic level T10 and measuring neuritic growth 1 day later. Neurons cultured 3 days after SCI exhibited longer neurites without increases in branching (“elongating growth”), compared to neurons from sham-treated or untreated (naïve) rats. Robust promotion of elongating growth was found in small and medium-sized neurons (but not large neurons) from lumbar (L3–L5) and thoracic ganglia immediately above (T9) and below (T10–T11) the contusion site, but not from cervical DRG. Elongating growth was also found in neurons immunoreactive to calcitonin gene-related peptide (CGRP), suggesting that some of the neurons exhibiting enhanced neuritic growth were nociceptors. The same measurements made on neurons dissociated 1 month after SCI revealed no evidence of elongating growth, although evidence for accelerated initiation of neurite outgrowth was found. Under certain conditions this transient growth-promoting state in nociceptors might be important for the development of chronic pain and hyperreflexia after SCI.

SUMMARY Survivable injuries are a common yet costly experience. The ability to sense and respond to noxious stimuli is an almost universal trait, and prolonged behavioral alterations, including sensitization to touch and other stimuli, may function to ameliorate fitness costs associated with injury. Cephalopods can modify their behavior by learned association with noxious electric shock, but non-associative alterations of behavioral responses after tissue injury have not been studied. The aim of this study was to make the first systematic investigations in any cephalopod of behavioral responses and alterations elicited by explicit, minor injury. By testing responsiveness in the longfin squid, Loligo pealeii, to the approach and contact of an innocuous filament applied to different parts of the body both before and after injury to the distal third of one arm, we show that a cephalopod expresses behavioral alterations persisting for at least 2 days after injury. These alterations parallel forms of nociceptive plasticity in other animals, including general and site-specific sensitization to tactile stimuli. A novel finding is that hyper-responsiveness after injury extends to visual stimuli. Injured squid are more likely to employ crypsis than escape in response to an approaching visual stimulus shortly after injury, but initiate escape earlier and continue escape behaviors for longer when tested from 1 to 48 h after injury. Injury failed to elicit overt wound-directed behavior (e.g. grooming) or change hunting success. Our results show that long-lasting nociceptive sensitization occurs in cephalopods, and suggest that it may function to reduce predation risk after injury.

Molluscs have proven to be invaluable models for basic neuroscience research, yielding fundamental insights into a range of biological processes involved in action potential generation, synaptic transmission, learning, memory, and, more recently, nociceptive biology. Evidence suggests that nociceptive processes in primary nociceptors are highly conserved across diverse taxa, making molluscs attractive models for biomedical studies of mechanisms that may contribute to pain in humans but also exposing them to procedures that might produce painlike sensations. We review the physiology of nociceptors and behavioral responses to noxious stimulation in several molluscan taxa, and discuss the possibility that nociception may result in painlike states in at least some molluscs that possess more complex nervous systems. Few studies have directly addressed possible emotionlike concomitants of nociceptive responses in molluscs. Because the defi nition of pain includes a subjective component that may be impossible to gauge in animals quite different from humans, firm conclusions about the possible existence of pain in molluscs may be unattainable. Evolutionary divergence and differences in lifestyle, physiology, and neuroanatomy suggest that painlike experiences in molluscs, if they exist, should differ from those in mammals. But reports indicate that some molluscs exhibit motivational states and cognitive capabilities that may be consistent with a capacity for states with functional parallels to pain. We therefore recommend that investigators attempt to minimize the potential for nociceptor activation and painlike sensations in experimental invertebrates by reducing the number of animals subjected to stressful manipulations and by administering appropriate anesthetic agents whenever practicable, welfare practices similar to those for vertebrate subjects.

Mechanisms underlying chronic pain that develops after spinal cord injury (SCI) are incompletely understood. Most research on SCI pain mechanisms has focused on neuronal alterations within pain pathways at spinal and supraspinal levels associated with inflammation and glial activation. These events might also impact central processes of primary sensory neurons, triggering in nociceptors a hyperexcitable state and spontaneous activity (SA) that drive behavioral hypersensitivity and pain. SCI can sensitize peripheral fibers of nociceptors and promote peripheral SA, but whether these effects are driven by extrinsic alterations in surrounding tissue or are intrinsic to the nociceptor, and whether similar SA occurs in nociceptorsin vivoare unknown. We show that small DRG neurons from rats (Rattus norvegicus) receiving thoracic spinal injury 3 d to 8 months earlier and recorded 1 d after dissociation exhibit an elevated incidence of SA coupled with soma hyperexcitability compared with untreated and sham-treated groups. SA incidence was greatest in lumbar DRG neurons (57%) and least in cervical neurons (28%), and failed to decline over 8 months. Many sampled SA neurons were capsaicin sensitive and/or bound the nociceptive marker, isolectin B4. This intrinsic SA state was correlated with increased behavioral responsiveness to mechanical and thermal stimulation of sites below and above the injury level. Recordings from C- and Aδ-fibers revealed SCI-induced SA generated in or near the somata of the neuronsin vivo. SCI promotes the entry of primary nociceptors into a chronic hyperexcitable-SA state that may provide a useful therapeutic target in some forms of persistent pain.

Nautilus is an ancient remnant of a largely extinct cephalopod lineage.1 Its status within its clade is the subject of ongoing debate-its morphology, behavior and neuroanatomy may or may not be representative of an ancestral condition, and therefore its value as a model for ancestral cephalopods is uncertain. While the nautilus brain is simpler than that of more derived cephalopods2 (coleoids), it is plausible that this is a secondary simplification related to ecology, and not a precursor to the vertebrate-like CNS of modern cephalopods. However, the absence of the vertical lobe complex, implicated in learning and memory in coleoids, makes studies of cognition in nautilus particularly interesting from a comparative perspective. Our research on the behavior and sensory biology of Nautilus pompilius gives the first indications of learning and memory in this ancient genus,3 and suggests that even with a far simpler brain containing no clearly defined "memory" center, nautilus performs simple cognitive tasks comparably to its more derived relatives.