Alan M. Kuzirian

Bryostatin Enhancement of Memory in Hermissenda

Bryostatin, a potent agonist of protein kinase C (PKC), when administered to Hermissenda was found to affect acquisition of an associative learning paradigm. Low bryostatin concentrations (0.1 to 0.5 ng/ml) enhanced memory acquisition, while concentrations higher than 1.0 ng/ml down-regulated the pathway and no recall of the associative training was exhibited. The extent of enhancement depended upon the conditioning regime used and the memory stage normally fostered by that regime. The effects of two training events (TEs) with paired conditioned and unconditioned stimuli, which standardly evoked only short-term memory (STM) lasting 7 min, were—when bryostatin was added concurrently—enhanced to a long-term memory (LTM) that lasted about 20 h. The effects of both 4- and 6-paired TEs (which by themselves did not generate LTM), were also enhanced by bryostatin to induce a consolidated memory (CM) that lasted at least 5 days. The standard positive 9-TE regime typically produced a CM lasting at least 6 days. Low concentrations of bryostatin (<0.5 ng/ml) elicited no demonstrable enhancement of CM from 9-TEs. However, animals exposed to bryostatin concentrations higher than 1.0 ng/ml exhibited no behavioral learning. Sharp-electrode intracellular recordings of type-B photoreceptors in the eyes from animals conditioned in vivo with bryostatin revealed changes in input resistance and an enhanced long-lasting depolarization (LLD) in response to light. Likewise, quantitative immunocytochemical measurements using an antibody specific for the PKC-activated Ca2+/GTP-binding protein calexcitin showed enhanced antibody labeling with bryostatin. Animals exposed to the PKC inhibitor bisindolylmaleimide-XI (Ro-32-0432) administered by immersion prior to 9-TE conditioning showed no training-induced changes with or without bryostatin exposure. However, if animals received bryostatin before Ro-32, the enhanced acquisition and demonstrated recall still occurred. Therefore, pathways responsible for the enhancement effects induced by bryostatin were putatively mediated by PKC. Overall, the data indicated that PKC activation occurred and calexcitin levels were raised during the acquisition phases of associative conditioning and memory initiation, and subsequently returned to baseline levels within 24 and 48 h, respectively. Therefore, the protracted recall measured by the testing regime used was probably due to bryostatin-induced changes during the acquisition and facilitated storage of memory, and not necessarily to enhanced recall of the stored memory when tested many days after training.

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.

Hydrogen Peroxide: An Effective Treatment for Ballast Water

polyphemus size and age. The analysis of cohorts demonstrated that C. fornicata in Pleasant Bay can be divided into 5 size cohorts (Fig. 1A), with C. fornicata of approximately 4–6 mm in length appearing to represent the most recent spatfall. The cohorts differed in abundance, reflecting different magnitudes of recruitment from year to year. Growth rates in this study did not decrease with increased size and age (Fig. 1B). This may be due to low numbers of larger (50 mm 1) and older C. fornicata. Published data of sizes and ages (1, 2) match those found in this study, and thus confirm the conversion from size to age of the C. fornicata. The largest C. fornicata found resident on a horseshoe crab was 58 mm. This size C. fornicata could be from 8–11 years old (2). There was no evident relationship between maximum length and age of C. fornicata and size of the host horseshoe crabs (Fig. 1C). Male horseshoe crabs were consistently smaller than females, but in both sexes the length and presumed age of C. fornicata varied greatly, and was independent of the size of the crab. It is not possible to establish a strong relationship between true horseshoe crab length and the length of the C. fornicata upon it. At most the data of Figure 1C support that a minimum age can be calculated by adding the maximum C. fornicata length on a given horseshoe crab to the minimum age of horseshoe crabs at maturity. Using 9 years as the age at maturity (4), the crabs in this study were from 12 to 17 years old. This study was funded by the Woods Hole Marine Science Consortium and a grant from the Friends of Pleasant Bay.

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.

Induction of metamorphosis in Hermissenda crassicornis larvae (Molluscs: Nudibranchia) by GABA, choline and serotonin

Summary The nudibranch mollusc Hermissenda crassicornis is currently used as a biomedical model in neurobiological studies. It possesses planktotrophic larvae which metamorphose in the laboratory in the presence of the hydroid Tubularia crocea in yields of about 2–5% (Tamse et al., 1990). This study presents evidence that artificial metamorphic inducers such as GABA (γ-aminobutyric acid), choline, and serotonin (alone or combined with the natural inducer T. crocea), can be used to improve the metamorphic success in H. crassicornis larvae. GABA at 10−5M and 10−4M, choline at 10−3M and 10−4M, and serotonin at 10−5 M and 10−4M, were most effective, while serotonin at 10−3 M was toxic. Larvae 47 and 63 days old metamorphosed at different rates when exposed to the same concentrations of chemicals, but these had no positive effect on young larvae, i.e., 28 and 39 days old. On the other hand, when young larvae were exposed to the natural inducer, an habituation phenomenon was observed, i.e., the larvae did not m...

Visual phototransduction components in cephalopod chromatophores suggest dermal photoreception

ABSTRACT 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. Highlighted Article: Squid and cuttlefish skin chromatophores contain rhodopsin, Gqα and sTRP channels, which are necessary components for photoreception.

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

SUMMARY The blue-ringed octopus (Hapalochlaena lunulata), one of the worlds 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.

Pavlovian conditioning-specific increases of the Ca2+- and GTP-binding protein, calexcitin in identified Hermissenda visual cells.

Hermissenda CNS, immunolabeled for the memory protein calexcitin showed significant immunostaining over background in the B-photoreceptor cells of the eye. The degree of staining correlated positively with the number of Pavlovian training events experienced by the animals and the degree of Pavlovian conditioning induced. The training regime consisted of exposing animals to light (conditioned stimulus, CS) paired with orbital rotation (unconditioned stimulus, US). In animals that exhibited the conditioned response, calexcitin immunolabeling was more intense than was found for naive (unconditioned) animals or animals given the CS and US in random sequence. Animals exposed to lead (maintained in 1.2 ppm lead acetate) at a dosage known to impair learning in children, showed reduced learning and less intense calexcitin staining whether the CS and US were paired or given randomly. However, the levels were still higher than that of naive animals. Immuno-electron microscopy indicated that the labeling was predominantly within calcium sequestering organelles such as the endoplasmic reticulum, and to lesser extent within mitochondria, and photopigments. The calexcitin density after a short-term memory (STM) regime was the same whether measured 5 minutes after conditioning (when STM was evidenced by foot contraction) or 90 minutes later when no recall was detected. The staining density was also similar to the levels found 5 minutes after long-term memory (LTM) conditioning. However, the LTM regime produced a greater calexcitin intensity at 90 minutes when the memory had been consolidated. This learning-specific increase in calexcitin is consistent with the previously implicated sequence of molecular events that are associated with progressively longer time domains of memory storage.

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 papillas 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 papillas base contract and push the overlying tissue upward and away from the mantle surface, while horizontally arranged dermal erector muscles pull the papillas 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 papillas muscular core and the skin layer that contains the pigmented chromatophores. In extended papillae, the connective tissue layer appeared thinner above the papillas 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. J. Morphol., 2013.

Strontium is Required in Artificial Seawater for Embryonic Shell Formation in Two Species of Bivalve Molluscs

Strontium is required in artificial seawater for the formation of the calcareous embryonic shell of the bivalves Mercenaria mercenaria (Linne, 1758) and Bankia gouldi (Bartsch, 1908). Embryos reared in defined media without strontium chloride become swimming larvae that appear normal except that the mineralized portion of the prodissoconch I shell is absent. Calcification is sensitive to small differences in strontium concentrations and a level of 6–8 ppm is required for normal development. Structural defects also result at high concentrations of this element, 3 to 10 times that of natural seawater, or when barium is substituted for strontium. Exposure to strontium is required only between hours 15–30 of the 40–45 hour embryonic period. Shell mineralization is initiated at hour 20 while the organic layer is observed with SEM at hour 18 and appears complete by hour 26. Larval shell growth (prodissoconch II) and statolith formation in M. mercenaria do not require the presence of strontium. These results are compared with those obtained earlier for the gastropod Aplysia californica (Cooper, 1863) and the implications are discussed.