Networks of linked radial muscles could influence dynamic skin patterning of squid chromatophores.

The visibility of cephalopod chromatophore organs is regulated dynamically by rosettes of obliquely striated radial muscles that dilate or relax the diameter of a central pigmented sacculus in 100-300 ms. Each of the several dozen muscles has a flared proximal end that adheres tightly to its pigmented sacculus and an extremely elongated distal end which branches into single fibrils that anchor into the dermis. This geometry provides ample opportunity for overlap of the many muscles from neighboring chromatophores. The temporal activity of these muscles has been believed to be patterned exclusively by monosynaptic projections from sets of efferent motor axons originating in the chromatophore lobes of the suboesophageal brain. Based on historical observations that distal radial muscles from some chromatophores appear to extend closely to muscles from other chromatophores, we asked whether radial muscles actually make specialized contacts. Using 3D electron microscopy of Doryteuthis pealeii mantle skin, we discovered tight putatively functional muscle-to-muscle contacts between radial muscles from different chromatophores, including elaborate sets of axonal processes located adjacent to those myo-myo junctions. These detailed ultrastructural findings demonstrate auxiliary anatomical routes for radial muscle activation and suggest plausible mechanisms whereby local physical synchronization and axo-axonic processing in the periphery can contribute to chromatophore pattern dynamics such as "passing cloud."

New Species Can Broaden Myelin Research: Suitability of Little Skate, Leucoraja erinacea.

Although myelinated nervous systems are shared among 60,000 jawed vertebrates, studies aimed at understanding myelination have focused more and more on mice and zebrafish. To obtain a broader understanding of the myelination process, we examined the little skate, Leucoraja erinacea. The reasons behind initiating studies at this time include: the desire to study a species belonging to an out group of other jawed vertebrates; using a species with embryos accessible throughout development; the availability of genome sequences; and the likelihood that mammalian antibodies recognize homologs in the chosen species. We report that the morphological features of myelination in a skate hatchling, a stage that supports complex behavioral repertoires needed for survival, are highly similar in terms of: appearances of myelinating oligodendrocytes (CNS) and Schwann cells (PNS); the way their levels of myelination conform to axon caliber; and their identity in terms of nodal and paranodal specializations. These features provide a core for further studies to determine: axon-myelinating cell communication; the structures of the proteins and lipids upon which myelinated fibers are formed; the pathways used to transport these molecules to sites of myelin assembly and maintenance; and the gene regulatory networks that control their expressions.

Dynamic pigmentary and structural coloration within cephalopod chromatophore organs.

Chromatophore organs in cephalopod skin are known to produce ultra-fast changes in appearance for camouflage and communication. Light-scattering pigment granules within chromatocytes have been presumed to be the sole source of coloration in these complex organs. We report the discovery of structural coloration emanating in precise register with expanded pigmented chromatocytes. Concurrently, using an annotated squid chromatophore proteome together with microscopy, we identify a likely biochemical component of this reflective coloration as reflectin proteins distributed in sheath cells that envelop each chromatocyte. Additionally, within the chromatocytes, where the pigment resides in nanostructured granules, we find the lens protein Ω- crystallin interfacing tightly with pigment molecules. These findings offer fresh perspectives on the intricate biophotonic interplay between pigmentary and structural coloration elements tightly co-located within the same dynamic flexible organ - a feature that may help inspire the development of new classes of engineered materials that change color and pattern.

White reflection from cuttlefish skin leucophores.

The highly diverse and changeable body patterns of cephalopods require the production of whiteness of varying degrees of brightness for their large repertoire of communication and camouflage behaviors. Leucophores are structural reflectors that produce whiteness in cephalopods; they are dermal aggregates of numerous leucocytes containing spherical leucosomes ranging in diameter from 200-2000 nm. In Sepia officinalis leucophores, leucocytes always occur in various combinations with iridocytes, cells containing plates that function as Bragg stacks to reflect light of particular wavelengths. Both spheres and plates contain the high-refractive-index protein reflectin. Four leucophore skin-patterning components were investigated morphologically and with spectrometry. In descending order of brightness they are: white fin spots, White zebra bands, White square, and White head bar. Different densities, thicknesses and proportions of leucocytes and iridocytes were correlated with the relative brightness measurements of the skin. That is, White fin spots and White zebra bands had leucocytes of the highest density, the greatest number of reflective cell layers, and the highest proportion of leucocytes to iridocytes. In contrast, the White square and White head bar had the lowest density of reflective cells, fewer cell layers and the lowest ratios of leucocytes to iridocytes. Leucophores are white in white light, yet reflect whatever colors are in the available light field: e.g. red in red light, green in green light, etc. Leucophores are physiologically passive, thus their ultrastructure alone is capable of diffusing all ambient wavelengths in all directions, regardless of the angle of incident light. However, the specific optical contributions of spherical leucosomes versus the associated plate-like iridosomes in producing whiteness versus brightness are yet to be determined. This study reveals complex morphological arrangements that produce white structural coloration for different brightnesses of skin by differentially combining spheres and plates.

Visual phototransduction components in cephalopod chromatophores suggest dermal photoreception.

Cephalopod mollusks are renowned for their colorful and dynamic body patterns, produced by an assemblage of skin components that interact with light. These may include iridophores, leucophores, chromatophores and (in some species) photophores. Here, we present molecular evidence suggesting that cephalopod chromatophores - small dermal pigmentary organs that reflect various colors of light - are photosensitive. RT-PCR revealed the presence of transcripts encoding rhodopsin and retinochrome within the retinas and skin of the squid Doryteuthis pealeii, and the cuttlefish Sepia officinalis and Sepia latimanus. In D. pealeii, Gqα and squid TRP channel transcripts were present in the retina and in all dermal samples. Rhodopsin, retinochrome and Gqα transcripts were also found in RNA extracts from dissociated chromatophores isolated from D. pealeii dermal tissues. Immunohistochemical staining labeled rhodopsin, retinochrome and Gqα proteins in several chromatophore components, including pigment cell membranes, radial muscle fibers, and sheath cells. This is the first evidence that cephalopod dermal tissues, and specifically chromatophores, may possess the requisite combination of molecules required to respond to light.

Comparative morphology of changeable skin papillae in octopus and cuttlefish.

A major component of cephalopod adaptive camouflage behavior has rarely been studied: their ability to change the three-dimensionality of their skin by morphing their malleable dermal papillae. Recent work has established that simple, conical papillae in cuttlefish (Sepia officinalis) function as muscular hydrostats; that is, the muscles that extend a papilla also provide its structural support. We used brightfield and scanning electron microscopy to investigate and compare the functional morphology of nine types of papillae of different shapes, sizes and complexity in six species: S. officinalis small dorsal papillae, Octopus vulgaris small dorsal and ventral eye papillae, Macrotritopus defilippi dorsal eye papillae, Abdopus aculeatus major mantle papillae, O. bimaculoides arm, minor mantle, and dorsal eye papillae, and S. apama face ridge papillae. Most papillae have two sets of muscles responsible for extension: circular dermal erector muscles arranged in a concentric pattern to lift the papilla away from the body surface and horizontal dermal erector muscles to pull the papilla's perimeter toward its core and determine shape. A third set of muscles, retractors, appears to be responsible for pulling a papilla's apex down toward the body surface while stretching out its base. Connective tissue infiltrated with mucopolysaccharides assists with structural support. S. apama face ridge papillae are different: the contraction of erector muscles perpendicular to the ridge causes overlying tissues to buckle. In this case, mucopolysaccharide-rich connective tissue provides structural support. These six species possess changeable papillae that are diverse in size and shape, yet with one exception they share somewhat similar functional morphologies. Future research on papilla morphology, biomechanics and neural control in the many unexamined species of octopus and cuttlefish may uncover new principles of actuation in soft, flexible tissue.

Protein kinase C mediates memory consolidation of taste avoidance conditioning in Lymnaea stagnalis.

In Lymnaea stagnalis, in order to obtain a 10 min short-term memory (STM) of taste avoidance conditioning (TAC) at least 10 paired presentations of a conditioned stimulus (CS), sucrose, and an unconditioned stimulus (US), tactile stimulation to the animal's head, are required. Pre-exposure of snails to the protein kinase C (PKC) α and ε activator bryostatin (Bryo) facilitated STM formation in that only 5 paired CS-US trials were required. Typically 20 paired presentations of the CS-US are required for formation of STM and LTM. However, 20 paired presentations do not result in STM or LTM if snails are pre-incubated with a PKC inhibitor, Ro-32-0432. We also found that LTM lasting longer than 48 h was acquired with Bryo incubation for 45 min even after termination of the conditioning paradigm. These data suggest that activation of the α and ε isozymes of PKC is crucially involved in the formation of LTM and provide further support for a mechanism that has been conserved across the evolution of species ranging from invertebrate molluscs to higher mammals.

The structure-function relationships of a natural nanoscale photonic device in cuttlefish chromatophores.

Cuttlefish, Sepia officinalis, possess neurally controlled, pigmented chromatophore organs that allow rapid changes in skin patterning and coloration in response to visual cues. This process of adaptive coloration is enabled by the 500% change in chromatophore surface area during actuation. We report two adaptations that help to explain how colour intensity is maintained in a fully expanded chromatophore when the pigment granules are distributed maximally: (i) pigment layers as thin as three granules that maintain optical effectiveness and (ii) the presence of high-refractive-index proteins-reflectin and crystallin-in granules. The latter discovery, combined with our finding that isolated chromatophore pigment granules fluoresce between 650 and 720 nm, refutes the prevailing hypothesis that cephalopod chromatophores are exclusively pigmentary organs composed solely of ommochromes. Perturbations to granular architecture alter optical properties, illustrating a role for nanostructure in the agile, optical responses of chromatophores. Our results suggest that cephalopod chromatophore pigment granules are more complex than homogeneous clusters of chromogenic pigments. They are luminescent protein nanostructures that facilitate the rapid and sophisticated changes exhibited in dermal pigmentation.

Cuttlefish skin papilla morphology suggests a muscular hydrostatic function for rapid changeability.

Coleoid cephalopods adaptively change their body patterns (color, contrast, locomotion, posture, and texture) for camouflage and signaling. Benthic octopuses and cuttlefish possess the capability, unique in the animal kingdom, to dramatically and quickly change their skin from smooth and flat to rugose and three-dimensional. The organs responsible for this physical change are the skin papillae, whose biomechanics have not been investigated. In this study, small dorsal papillae from cuttlefish (Sepia officinalis) were preserved in their retracted or extended state, and examined with a variety of histological techniques including brightfield, confocal, and scanning electron microscopy. Analyses revealed that papillae are composed of an extensive network of dermal erector muscles, some of which are arranged in concentric rings while others extend across each papilla's diameter. Like cephalopod arms, tentacles, and suckers, skin papillae appear to function as muscular hydrostats. The collective action of dermal erector muscles provides both movement and structural support in the absence of rigid supporting elements. Specifically, concentric circular dermal erector muscles near the papilla's base contract and push the overlying tissue upward and away from the mantle surface, while horizontally arranged dermal erector muscles pull the papilla's perimeter toward its center and determine its shape. Each papilla has a white tip, which is produced by structural light reflectors (leucophores and iridophores) that lie between the papilla's muscular core and the skin layer that contains the pigmented chromatophores. In extended papillae, the connective tissue layer appeared thinner above the papilla's apex than in surrounding areas. This result suggests that papilla extension might create tension in the overlying connective tissue and chromatophore layers, storing energy for elastic retraction. Numerous, thin subepidermal muscles form a meshwork between the chromatophore layer and the epidermis and putatively provide active papillary retraction.

How does the blue-ringed octopus (Hapalochlaena lunulata) flash its blue rings?

The blue-ringed octopus (Hapalochlaena lunulata), one of the world's most venomous animals, has long captivated and endangered a large audience: children playing at the beach, divers turning over rocks, and biologists researching neurotoxins. These small animals spend much of their time in hiding, showing effective camouflage patterns. When disturbed, the octopus will flash around 60 iridescent blue rings and, when strongly harassed, bite and deliver a neurotoxin that can kill a human. Here, we describe the flashing mechanism and optical properties of these rings. The rings contain physiologically inert multilayer reflectors, arranged to reflect blue-green light in a broad viewing direction. Dark pigmented chromatophores are found beneath and around each ring to enhance contrast. No chromatophores are above the ring; this is unusual for cephalopods, which typically use chromatophores to cover or spectrally modify iridescence. The fast flashes are achieved using muscles under direct neural control. The ring is hidden by contraction of muscles above the iridophores; relaxation of these muscles and contraction of muscles outside the ring expose the iridescence. This mechanism of producing iridescent signals has not previously been reported in cephalopods and we suggest that it is an exceptionally effective way to create a fast and conspicuous warning display.

Catalytic activities of recA protein are dependent on the lattice length of the single-strand DNA ligand.

recA protein (RecA) performs diverse catalytic activities that require a complex with single-stranded DNA and an NTP. A subset of these functions shows optimal activity at a high DNA/protein ratio and requires NTP hydrolysis, whereas other catalytic activities are optimal in RecA-saturated complexes that require NTP, but do not hydrolyze it. To analyze the mechanism of catalytic discrimination, we investigated the properties of RecA bound to small oligonucleotides (oligos) of defined sizes. We show that RecA bound to (dT)(16) is optimal for co-protease activity and not active as ATPase whereas the complex with (dT)(24) is competent in ATP hydrolysis but impaired as a co-protease. Thermodynamic measurements of the equilibrium-binding properties of these complexes showed that (dT)(24) promoted a more salt sensitive complex than the one formed with (dT)(16), indicating more ionic interactions between RecA and DNA in the former. X-ray pictures show that the oligo complexes form helixes. We propose that RecA may change its conformation as a function of the number of phosphates available to the monomer in the interacting DNA lattice, thus promoting an allosteric change in catalytic activities. This model offers explanations for the observed inhibition of co-protease activity by excess ssDNA.

Protein synthesis required for long-term memory is induced by PKC activation on days before associative learning.

Protein synthesis has long been known to be required for associative learning to consolidate into long-term memory. Here we demonstrate that PKC isozyme activation on days before training can induce the synthesis of proteins necessary and sufficient for subsequent long-term memory consolidation. Bryostatin (Bryo), a macrolide lactone with efficacy in subnanomolar concentrations and a potential therapeutic for Alzheimer's disease, is a potent activator of PKC, some of whose isozymes undergo prolonged activation after associative learning. Under normal conditions, two training events with paired visual and vestibular stimuli cause short-term memory of the mollusc Hermissenda that lasts approximately 7 min. However, after 4-h exposures to Bryo (0.25 ng/ml) on two preceding days, the same two training events produced long-term conditioning that lasted >1 week and that was not blocked by anisomycin (1 mug/ml). Anisomycin, however, eliminated long-term memory lasting at least 1 week after nine training events. Both the nine training events alone and two Bryo exposures plus two training event regimens caused comparably increased levels of the PKC alpha-isozyme substrate calexcitin in identified type B neurons and enhanced PKC activity in the membrane fractions. Furthermore, Bryo increased overall protein synthesis in cultured mammalian neurons by up to 60% for >3 days. The specific PKC antagonist Ro-32-0432 blocked much of this Bryo-induced protein synthesis as well as the Bryo-induced enhancement of the behavioral conditioning. Thus, Bryo-induced PKC activation produces those proteins necessary and sufficient for long-term memory on days in advance of the training events themselves.

Two different biological configurations for long-term memory.

Long-term memory (LTM) in Hermissenda can be distinguished from consolidated long-term memory (CLTM) by determining how long recall is retained. LTM is retained for approximately 1 day, while CLTM is retained for at least 3 days. During the transition from LTM to CLTM, the extent of retention appears to depend partially on how much consolidation has been completed. Several models are discussed that may be related to the two different manifestations of recall.

Original molluscan radula: comparisons among Aplacophora, Polyplacophora, Gastropoda, and the Cambrian fossil Wiwaxia corrugata.

As the original molluscan radula is not known from direct observation, we consider what the form of the original radula may have been from evidence provided by neomenioid Aplacophora (Solenogastres), Gastropoda, Polyplacophora, and the Cambrian fossil Wiwaxia corrugata (Matthews). Conclusions are based on direct observation of radula morphology and its accessory structures (salivary gland ducts, radular sac, anteroventral radular pocket) in 25 species and 16 genera of Aplacophora; radula morphogenesis in Aplacophora; earliest tooth formation in Gastropoda (14 species among Prosobranchia, Opisthobranchia, and Pulmonata); earliest tooth formation in four species of Polyplacophora; and the morphology of the feeding apparatus in W. corrugata. The existence of a true radula membrane and of membranoblasts and odontoblasts in neomenioids indicates that morphogenesis of the aplacophoran radula is homologous to that in other radulate Mollusca. We conclude from p redness of salivary gland ducts, a divided radular sac, and a pair of anteroventral pockets that the plesiomorphic state in neomenioids is bipartite, formed of denticulate bars that are distichous (two teeth per row) on a partially divided or fused radula membrane with the largest denticles lateral, as occurs in the genus Helicoradomenia. The tooth morphology in Helicoradomenia is similar to the feeding apparatus in W. corrugata. We show that distichy also occurs during early development in several species of gastropods and polyplacophorans. Through the rejection of the null hypothesis that the earliest radula was unipartite and had no radula membrane, we conclude that the original molluscan radula was similar to the radula found in Helicoradomena species.

Time windows for effects of protein synthesis inhibitors on Pavlovian conditioning in Hermissenda: behavioral aspects.

Inhibitors of protein and mRNA syntheses inhibit long-term memory (LTM), but we lack information about the time windows during which those inhibitors are effective. Anisomycin (a protein synthesis translation inhibitor) was given to Hermissenda crassicornis which had received sufficient Pavlovian conditioning to produce LTM. When tested after 90 min, LTM recall was blocked when anisomycin was administered until 13 min after conditioning and from 65 to 75 min. There was good recall from 16 to 60 min. When tested at 240 min, two periods of sensitivity to anisomycin were revealed: one finished before 20 min, while the other lasted from 60 to 220 min.A brief study of the transcription inhibitor actinomycin-D found its action similar to that of anisomycin, except not inhibiting recall from about 160 to 220 min.

Trafficking of axonal K+ channels: potential role of Hsc70.

Voltage-gated potassium ion channels in axons underlie the repolarization phase of the membrane action potential and help to set the resting potential. In addition to being present in the axolemma, they are also found in axoplasm in small vesicles, 30-50 nm in diameter, which may serve as a reserve pool of K+ channel protein (Clay and Kuzirian [2000] J Neurobiol 45:172-184). We have developed a novel technique for extracting these vesicles from axoplasm, which relies on the ability of Texas red to bind to them, thereby reducing their buoyancy so that they are amenable to pelleting by ultracentrifugation (Clay and Kuzirian [2000] J Neurobiol 45:172-184). The mechanism underlying this process may be binding of Texas red to Hsc70, which is primarily a cytosolic protein. However, a small portion of it is located on the surface of vesicles. Kinesin is also on the vesicle surface. This protein is membrane bound in our in vitro vesicle preparation when solutions that do not contain MgATP are added to extruded axoplasm. The addition of MgATP to the solution appears to release a significant amount of kinesin from the vesicles, possibly by the Hsc70-MgATP catalysis mechanism recently proposed by Tsai et al.