Cephalopods in neuroscience: regulations, research and the 3Rs.

Cephalopods have been utilised in neuroscience research for more than 100 years particularly because of their phenotypic plasticity, complex and centralised nervous system, tractability for studies of learning and cellular mechanisms of memory (e.g. long-term potentiation) and anatomical features facilitating physiological studies (e.g. squid giant axon and synapse). On 1 January 2013, research using any of the about 700 extant species of "live cephalopods" became regulated within the European Union by Directive 2010/63/EU on the "Protection of Animals used for Scientific Purposes", giving cephalopods the same EU legal protection as previously afforded only to vertebrates. The Directive has a number of implications, particularly for neuroscience research. These include: (1) projects will need justification, authorisation from local competent authorities, and be subject to review including a harm-benefit assessment and adherence to the 3Rs principles (Replacement, Refinement and Reduction). (2) To support project evaluation and compliance with the new EU law, guidelines specific to cephalopods will need to be developed, covering capture, transport, handling, housing, care, maintenance, health monitoring, humane anaesthesia, analgesia and euthanasia. (3) Objective criteria need to be developed to identify signs of pain, suffering, distress and lasting harm particularly in the context of their induction by an experimental procedure. Despite diversity of views existing on some of these topics, this paper reviews the above topics and describes the approaches being taken by the cephalopod research community (represented by the authorship) to produce "guidelines" and the potential contribution of neuroscience research to cephalopod welfare.

Arm regeneration in two species of cuttlefish Sepia officinalis and Sepia pharaonis.

To provide quantitative information on arm regeneration in cuttlefish, the regenerating arms of two cuttlefish species, Sepia officinalis and Sepia pharaonis, were observed at regular intervals after surgical amputation. The third right arm of each individual was amputated to ~10-20 % starting length. Arm length, suction cup number, presence of chromatophores, and behavioral measures were collected every 2-3 days over a 39-day period and compared to the contralateral control arm. By day 39, the regenerating arm reached a mean 95.5 ± 0.3 % of the control for S. officinalis and 94.9 ± 1.3 % for S. pharaonis. The process of regeneration was divided into five separate stages based on macroscopic morphological events: Stage I (days 0-3 was marked by a frayed leading edge; Stage II (days 4-15) by a smooth hemispherical leading edge; Stage III (days 16-20) by the appearance of a growth bud; Stage IV (days 21-24) by the emergence of an elongated tip; and Stage V (days 25-39) by a tapering of the elongated tip matching the other intact arms. Behavioral deficiencies in swimming, body postures during social communication, and food manipulation were observed immediately after arm amputation and throughout Stages I and II, returning to normal by Stage III. New chromatophores and suction cups in the regenerating arm were observed as early as Stage II and by Stage IV suction cup number equaled that of control arms. New chromatophores were used in the generation of complex body patterns by Stage V. These results show that both species of cuttlefish are capable of fully regenerating lost arms, that the regeneration process is predictable and consistent within and across species, and provide the first quantified data on the rate of arm lengthening and suction cup addition during regeneration.

Expression of the SOFaRP2 gene in the central nervous system of the adult cuttlefish Sepia officinalis.

FMRFamide-related Peptides (FaRPs) are involved in a variety of physiological processes, including reproduction, feeding, development, body patterning and osmoregulation in vertebrates and invertebrates. Here we investigate the expression pattern of cuttlefish Sepia officinalis FaRP2 gene in the brain by in situ hybridization. The SOFaRP2 gene was found to be expressed most intensively in the posterior chromatophore lobe, vasomotor lobe and subvertical lobe. In addition, positive staining was also found in the fin lobe, brachial lobe, anterior chromatophore lobe, anterior, dorsal and lateral basal lobes, inferior and superior frontal lobes, and optic lobe. The expression pattern of SOFaRP2 suggests its involvement in chromatophore regulation, feeding behavior, and learning and memory.

Molecular analysis of a novel FMRFamide-related peptide gene (SOFaRP(2)) and its expression pattern in the brain of the European cuttlefish Sepia officinalis.

FMRFamide-related peptides (FaRPs) are among several neurotransmitters known to regulate the chromatophore function in the European cuttlefish Sepia officinalis. Here we report the cloning and sequencing of a novel S. officinalis FaRP gene (SOFaRP(2)). The complete 835-base pair cDNA sequence of the SOFaRP(2) gene contains an open reading frame of 567 base pairs encoding 188 amino acids and four putative FaRPs, NSLFRFamide, GNLFRFamide, TIFRFamide and PHTPFRFamide. All except TIFRFamide cause chromatophore expansion when assayed in an in vitro chromatophore bioassay. To investigate the expression pattern of SOFaRP(2) gene in the cuttlefish brain, in situ hybridization was performed using a full length RNA probe. The SOFaRP(2) gene was expressed primarily in the posterior chromatophore, anterior chromatophore, lateral basal and optic lobes among other brain locations. The SOFaRP(2) gene appears to be expressed in all brain regions involved in chromatophore regulation. These data suggests that some or all of the four FaRPs encoded by SOFaRP(2) might be involved in controlling chromatophore activity in cuttlefish.

Principles underlying chromatophore addition during maturation in the European cuttlefish, Sepia officinalis.

The goal of this work was to identify some of the principles underlying chromatophore growth and development in the European cuttlefish, Sepia officinalis. One set of experiments used a regeneration model to follow the re-growth of black chromatophores for 30 days following excision of a small piece of fin tissue. A separate set of experiments tracked and analyzed the addition of new fin chromatophores during a month of normal, undisturbed growth. We also followed the development of individual chromatophores from their initial appearance to full maturation to determine whether their color type was fixed. Based on the results of these studies, we propose five guiding principles for chromatophore growth and maturation. (1) The three chromatophore cell types--black, reddish-brown and yellow--are present at different spatial frequencies in the cuttlefish fin. (2) During normal growth, new chromatophores are inserted at a higher spatial frequency than existing (control) chromatophores of the same color type. (3) In regenerating tissue, new black chromatophores are initially added at low spatial frequencies. As regeneration continues, new black chromatophores appear at increasing spatial frequencies until they are inserted at a spatial frequency higher than that observed in control tissue, similar to the way in which chromatophores were observed to be added in normally growing tissue. (4) All chromatophores first appear as pale orange cells and slowly darken into their respective color types without passing through intermediate color stages. (5) New black chromatophores undergo a doubling in size as they mature, while reddish-brown and yellow chromatophores do not grow at all after they are inserted in the dermis.

Central distribution and three-dimensional arrangement of fin chromatophore motoneurons in the cuttlefish Sepia officinalis.

Cephalopod body patterning is a most complex invertebrate behavior. Generated primarily by pigment-containing chromatophore organs, this behavior enables rapid alteration of body coloration as a result of direct innervation of chromatophores by motoneurons. This study focuses on location and arrangement of fin chromatophore motoneurons in the cuttlefish Sepia and investigates the possibility of central topography. Retrograde labeling of topographically arranged fin nerve branches in the periphery revealed the posterior subesophageal mass (PSEM) of the brain as the primary location of fin chromatophore motoneurons; within this region, most cells were located in the posterior chromatophore and fin lobes. Additionally, a small percentage of labeled motoneurons occurred in the anterior subesophageal mass and the stellate ganglia. Data from three-dimensional reconstructions of PSEMs showed the arrangement of labeled motoneurons within individual lobes; these data suggest no obvious topographic arrangement. Further, electrical stimulation of the PSEM generated chromatophore activity on the fin and mantle. These stimulation results, coupled with the retrograde labeling, suggest that chromatophore motoneurons are located across multiple PSEM lobes.

Neural regulation of a complex behavior: body patterning in cephalopod molluscs.

Unshelled cephalopods have a remarkable ability to alter their appearance, using textural, postural, and chromatic elements to generate a myriad of body patterns. Of the unshelled cephalopods, it is generally acknowledged that cuttlefish express the most detailed and widest range of body patterns, including static and dynamic patterns. In this paper we present data on the neuronal mechanisms underlying this amazing behavior, focusing on the neuroregulation of the chromatic elements, the chromatophore organs, in the European cuttlefish Sepia officinalis. Cephalopod chromatophore organs, including those in Sepia, are unlike those in any other animal taxa; each consists of a pigment-containing chromatophore cell that expands in response to the coordinated activation of a set of radial muscles which are directly attached to the chromatophore cell. We show that the chromatophore muscles are regulated by 2 different excitatory transmitters, glutamate and the family of FMRFamide-related peptides (FaRPs). Glutamate mediates rapid and transient chromatophore cell expansion whereas the FaRPs are responsible for slower, more sustained responses. Using retrograde dye filling, immunocytochemical and in situ hybridization techniques, we demonstrate that the cell bodies of the glutamatergic and FaRPs-containing motoneurons innervating the fin chromatophore muscles are primarily localized to the posterior chromatophore and fin lobes in the posterior subesophageal mass of the Sepia brain. Data are also presented showing that some fin chromatophore motoneurons have multiple axons in different nerve branches, which accounts for overlapping chromatophore motor fields by adjacent peripheral nerves.

Vasoactive intestinal peptide fragment VIP10-28 and active vasodilation in human skin.

A recent study reported the vasoactive intestinal peptide (VIP) fragment VIP(10-28) inhibited the rise in skin blood flow during heat stress. Our laboratory has reported that the nitric oxide (NO) pathway and histamine receptor-1 (H1)-receptor activation is common to both exogenous VIP-mediated dilation and active vasodilation (AVD). The present study aimed to further examine the specific role for VIP in AVD by using VIP(10-28) to antagonize VIP-mediated dilation in the presence of NO synthase (NOS) inhibition and an H1 antagonist. Study 1 (n = 12) examined whether VIP(10-28) antagonizes vasodilation to exogenous VIP via inhibition of NO-dependent mechanisms. Study 2 (n = 6) investigated AVD in skin sites receiving VIP(10-28) alone and in combination with NOS inhibition. Study 3 (n = 6) examined AVD in sites receiving VIP(10-28) alone and combined VIP(10-28) and H1 antagonism. Due to differences in our findings and those previously published, study 4 (n = 6) investigated whether an increase in baseline skin blood flow could result in a diminished rise in AVD. Red blood cell flux was measured using laser Doppler flowmetry, and cutaneous vascular conductance (flux/mean arterial pressure) was normalized to maximal vasodilation (28 mM sodium nitroprusside). VIP(10-28) augmented vasodilation to exogenous VIP (P < 0.05 vs. control) and hyperthermia (P < 0.05 vs. control). NOS inhibition had no effect on the augmented dilation during exogenous VIP or hyperthermia (P > 0.05). Similarly, H1-receptor antagonists had no effect on the augmented dilation during hyperthermia (P > 0.05 vs. VIP(10-28)). In study 4, percentage of maximal cutaneous vascular conductance was attenuated when baseline skin blood flow was elevated before whole body heating. Our results suggest that VIP(10-28) may be an unsuitable antagonist for examining a role for VIP-mediated dilation in human skin.

Neurokinin-1 receptor desensitization to consecutive microdialysis infusions of substance P in human skin.

The neuropeptide substance P is known to be localized in nerve terminals in human skin and substance P-induced vasodilatation is believed to be partially dependent on nitric oxide (NO) and H1 histamine receptor activation. Unlike other neuropeptides investigated in human skin, substance P-induced vasodilatation has been shown to decline during continuous infusion, possibly suggestive of an internalization of neurokinin-1 (NK1) receptors, which are highly specific to substance P. However, questions remain regarding these mechanisms in human skin. Fifteen subjects participated in this series of studies designed to investigate the effect of consecutive infusions and possible mechanisms of substance P-induced vasodilatation in human skin. Two concentrations of substance P (10 microm and 20 microm) were tested via intradermal microdialysis in two groups of subjects. Site 1 served as a control and received substance P only. Site 2 received substance P combined with 10 mm L-NAME to inhibit NO synthase. Site 3 received substance P combined with 500 microm pyrilamine, an H1 receptor antagonist. Site 4 received substance P combined with 10 mm L-NAME plus 500 microm pyrilamine. Red blood cell (RBC) flux was measured via laser-Doppler flowmetry to provide an index of skin blood flow. Cutaneous vascular conductance was calculated as RBC flux/mean arterial pressure and was normalized to maximal vasodilatation via 28 mm sodium nitroprusside. Substance P was perfused through each microdialysis fibre at a rate of 4 microl min(-1) for 15 min. The subsequent increase in skin blood flow was allowed to return to baseline (approximately 45-60 min) and a stable 5 min plateau was used as a new baseline (post-infusion baseline). A second dose of substance P was then delivered to the skin and skin blood flow was monitored for 45-60 min. Substance P produced a dose-dependent increase in skin blood flow with the concentrations of substance P tested, which was significantly attenuated in the presence of L-NAME and the combination of L-NAME plus pyrilamine. However, substance P-induced vasodilatation was unaffected in the presence of pyrilamine. There was no significant difference between the L-NAME-only sites and the L-NAME plus pyrilamine sites. Importantly, the second dose of substance P did not produce a significant increase in skin blood flow compared to the initial baseline or the post-infusion baseline. These data suggest substance P-induced vasodilatation delivered via microdialysis contains an NO component but does not contain an H1 receptor activation component at the doses tested. Additionally, these data provide evidence for NK1 receptor desensitization as there was no observable increase in skin blood flow following a second administration of substance P. This may provide a useful model for studying the role of substance P in the control of skin blood flow in humans.

Sequence and expression of the CAPA/CAP2b gene in the tobacco hawkmoth, Manduca sexta.

The gene coding for cardioacceleratory peptide 2b (CAP2b; pELYAFPRV) has been isolated and sequenced from the moth Manduca sexta (GenBank accession #AY649544). Because of its significant homology to the CAPA gene in Drosophila melanogaster, this gene is called the Manduca CAPA gene. The Manduca CAPA gene is 958 nucleotides long with 29 untranslated nucleotides from the beginning of the sequence to the putative start initiation site. The CAPA gene has a single open reading frame, 441 nucleotides long, that codes for a predicted precursor protein of 147 amino acids. The predicted prepropeptide encodes a single copy of each of three deduced propeptides, a CAP2b propeptide, with a Q substituted for an E at the N-terminus (QLYAFPRVa), and two novel CAP2b-related propeptides (DGVLNLYPFPRVa and TEGPGMWFGPRLa). To reduce confusion and to adopt a more standardized nomenclature, we rename pELYAFPRVa as Mas-CAPA-1 and assign the names of Mas-CAPA-2 to DGVLNLYPFPRVa and Mas-PK-1 (Pyrokinin-1) to TEGPGMWFGPRLa. The spatial and temporal expression pattern of the CAPA gene in the Manduca central nervous system (CNS) was determined in all major post-embryonic stages using in situ hybridization techniques. The CAPA gene is expressed in a total of 27 pairs of neurons in the post-embryonic Manduca CNS. A total of 16 pairs of cells is observed in the brain, two pairs in the sub-esophageal ganglion (SEG), one pair in the third thoracic ganglion (T3), one pair in each unfused abdominal ganglion (A1-A6) and two pairs in the fused terminal ganglion. The mRNA from the CAPA gene is present in nearly every ganglion in each post-embryonic stage. The number of cells expressing the CAPA gene varies during post-embryonic life, starting at 54 cells in first-instar larvae and declining to a minimum of 14 cells midway through adult development.

Peripheral innervation patterns and central distribution of fin chromatophore motoneurons in the cuttlefish Sepia officinalis.

Body patterning behavior in unshelled cephalopod molluscs such as squid, octopuses, and cuttlefish is the ability of these animals to create complex patterns on their skin. This behavior is generated primarily by chromatophores, pigment-containing organs that are directly innervated by central motoneurons. The present study focuses on innervation patterns and location of chromatophore motoneurons in the European cuttlefish Sepia officinalis, specifically those motoneurons that control chromatophores of the fin. The fin is known to be innervated by the large, branching fin nerve. This study further characterizes the innervation of fin chromatophores by the fin nerve, generates a reference system for the location of fin nerve branches across individuals, and localizes the neurons whose axons innervate fin chromatophores through the fin nerve. Data from extracellular stimulation of fin nerve branches in intact animals demonstrate topographic innervation of fin chromatophores, while retrograde labeling data reveal the posterior subesophageal mass of the brain as the primary location of fin chromatophore motoneurons.

Mechanisms of vasoactive intestinal peptide-mediated vasodilation in human skin.

Vasoactive intestinal peptide (VIP) is known to induce histamine release in human skin and to include a nitric oxide (NO)-dependent dilation in several other vascular beds. However, the relative contribution of histamine and NO to VIP-mediated vasodilation in human skin is unknown. Forty-three subjects volunteered to participate in two studies designed to examine the mechanism of VIP-mediated vasodilation in human skin. Study 1 examined the contribution of NO in the skin blood flow response to eight doses of VIP ranging from 25 to 800 pmol. In addition, study 1 examined a specific role for NO in VIP-mediated dilation. Study 2 examined the relative contribution of NO and histamine to VIP-mediated dilation via H1 and H2 histamine receptors. Infusions were administered to skin sites via intradermal microdialysis. Red blood cell flux was measured by using laser-Doppler flowmetry (LDF), and cutaneous vascular conductance (CVC; LDF/mean arterial pressure) was calculated and normalized to maximal vasodilation. VIP-mediated vasodilation includes a NO-dependent component at doses above 100 pmol, where NO synthase inhibition significantly attenuates CVC (P < 0.05). Inhibition of H1 receptors attenuates the rise in CVC to exogenous VIP (P < 0.05); however, combined H1-receptor inhibition and NO synthase inhibition further reduced VIP-mediated vasodilation compared with either H1 inhibition or NO synthase inhibition alone (P < 0.05). In contrast to H1-receptor inhibition, H2-receptor inhibition did not affect vasodilation to exogenous VIP. Thus, in human skin, VIP-mediated vasodilation includes a NO-dependent component that could not be explained by H1- and H2-receptor activation.

Quantitative staging of embryonic development of the tobacco hawkmoth,Manduca sexta.

A complete timetable of embryonic development of the tobacco hawkmoth,Manduca sexta (Lepidoptera: Sphingidae), is presented. Using living embryos, 20 developmental stages from oviposition to hatching are described with respect to their morphological and physiological maturation. This staging series provides a simple method to identify the stage ofManduca development during all phases of embryogenesis.