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The existence of both calcium-binding proteins (CBPs) and neuropeptides in the retina and brain of various species of vertebrates and invertebrates is well documented. Octopus retina is particularly interesting because it represents a case of convergent evolution. The aim of this study was to characterize the distribution of two CBPs, calretinin and calbindin, in Octopus retina using morphology, in situ hybridization, immunocytochemistry and Western blot. Calretinin-like immunoreactivity was found in the photoreceptor cells, but unexpectedly also in the supporting cells. In situ hybridization and Western blot analysis confirmed these results. No immunoreactivity was found for calbindin. Two neuropeptides, Substance P and calcitonin gene-related peptide (CGRP), as well as neurofilament protein and glial fibrillary acidic protein were also localized in the Octopus retina by immunocytochemistry. Our work provides new insights about calcium-binding proteins and neuropeptide distribution in Octopus retina and suggests a functional role for calretinin, a highly conserved protein, in visual signal transduction of cephalopods., (© 2018 Wiley Periodicals, Inc.)

Organisms showing a complex and centralized nervous system, such as teleosts, amphibians, reptiles, birds and mammals, and among invertebrates, crustaceans and insects, can adjust their behavior according to the environmental challenges. Proliferation, differentiation, migration, and axonal and dendritic development of newborn neurons take place in brain areas where structural plasticity, involved in learning, memory, and sensory stimuli integration, occurs. Octopus vulgaris has a complex and centralized nervous system, located between the eyes, with a hierarchical organization. It is considered the most "intelligent" invertebrate for its advanced cognitive capabilities, as learning and memory, and its sophisticated behaviors. The experimental data obtained by immunohistochemistry and western blot assay using proliferating cell nuclear antigen and poli (ADP-ribose) polymerase 1 as marker of cell proliferation and synaptogenesis, respectively, reviled cell proliferation in areas of brain involved in learning, memory, and sensory stimuli integration. Furthermore, we showed how enriched environmental conditions affect adult neurogenesis., (© 2017 Wiley Periodicals, Inc.)

The cephalopod vertical lobe is the largest learning and memory structure known in invertebrate nervous systems. It is part of the visual learning circuit of the central brain, which also includes the superior frontal and subvertical lobes. Despite the well-established functional importance of this system, little is known about neuropil organization of these structures and there is to date no evidence that the five longitudinal gyri of the vertical lobe, perhaps the most distinctive morphological feature of the octopus brain, differ in their connections or molecular identities. We studied the histochemical organization of these structures in hatchling and adult Octopus bimaculoides brains with immunostaining for serotonin, octopus gonadotropin-releasing hormone (oGNRH), and octopressin-neurophysin (OP-NP). Our major finding is that the five lobules forming the vertical lobe gyri have distinct neurochemical signatures. This is most prominent in the hatchling brain, where the median and mediolateral lobules are enriched in OP-NP fibers, the lateral lobule is marked by oGNRH innervation, and serotonin immunostaining heavily labels the median and lateral lobules. A major source of input to the vertical lobe is the superior frontal lobe, which is dominated by a neuropil of interweaving fiber bundles. We have found that this neuropil also has an intrinsic neurochemical organization: it is partitioned into territories alternately enriched or impoverished in oGNRH-containing fascicles. Our findings establish that the constituent lobes of the octopus superior frontal-vertical system have an intricate internal anatomy, one likely to reflect the presence of functional subsystems within cephalopod learning circuitry., (© 2015 Wiley Periodicals, Inc.)

The concentrations of D- and L-lactate, methylglyoxal and pyruvate were measured in tissues of normal and starved Octopus ocellatus. D-Lactate was always more abundant than L-lactate in the tissues. D-Lactate, pyruvate and methylglyoxal were present in 320, 94 and 43 times higher concentrations in tentacle of O. ocellatus of control group than those in normal rat skeletal muscle. The D-lactate concentration in the tentacle of O. ocellatus was 17-fold higher than that in Octopus vulgars. The activities of enzymes involved with D-lactate metabolism such as pyruvate kinase, octopine dehydrogenase, glyoxalase I and II and lactate dehydrogenase were measured in those tissues. The activities of glyoxalase I and II, and D-lactate dehydrogenase were increased in mantle and tentacle of starved octopus, while the levels of D-lactate and related metabolites were lowered in these tissues. The experimental results presented in this report and up to the present indicate that D-lactate is actively used for energy production in the tentacle and mantle of the starved animals. In octopus, especially starved octopus D-lactate was actively produced from methylglyoxal, which is formed via aminoacetone from threonine and glycine., (Copyright 2005 Wiley-Liss, Inc.)

In this study, we have identified a 28-kDa protein resembling the linker H1 in the testis and prostate of the reproductive system of Octopus vulgaris. This protein, OvH1, was partially purified by reverse phase high-pressure liquid chromatography (HPLC) of the perchloric acid extract from testis nuclei. It showed electrophoretic mobility, CD spectrum and amino acid composition highly comparable with those of the mammalian histone. Moreover, it was microheterogeneous, as resulted from prostate and testis HPLC and mass spectrometry analyses. Such analysis showed that in testis there are two H1 subfractions, which do not appear in the prostate. Amino acid composition of the major testis specific variant (OvH1t) showed high similarity with rat testis specific H1t. The histone-like nature of OvH1 was confirmed by its ability to bind DNA as tested both by circular dichroism and protection of the nucleic acid toward deoxyribonuclease I activity. The circular dichroism spectra of Octopus DNA in the absence and presence of increasing amounts of the protein showed a dose-dependent effect, leading to a progressive compactness of the polynucleotide. OvH1/DNA complexes were also resistant to nuclease digestion. The presence of H1 in the testis and prostate of the reproductive system of Octopus is discussed in light of the fact that there is a similarity between its behavior and that of vertebrates., (Copyright 2002 Wiley-Liss, Inc.)

In this study, for the first time we have identified an estradiol-17beta receptor (ER) in the reproductive system of the female of Octopus vulgaris. Scatchard analysis revealed that one binding component with high affinity and low capacity for the ligand was present in the cytosol, but not in the nuclear extract of the ovary and the oviduct. A steroid specificity competition assay showed that 3H-estradiol-17beta binding activity showed a preference for estradiol-17beta. DNA-cellulose chromatography confirmed the presence of one 3H-estradiol-17beta binding component. By using antibodies anti ER (578-595), we have localized by Western blotting one band of about 70 kDa. ER immunoreactivity has been localized in the nuclei of the follicle cells of the ovary, in the nuclei of the epithelium lining the proximal portion of the oviduct and in the nuclei, and in the cytoplasm of the inner region of the oviducal gland and in the cytoplasm of the outer region of the oviducal gland. These data, taken together, provide evidence that in Octopus vulgaris the ER has biochemical and immunohistochemical characteristics resembling those of ER in vertebrates., (Copyright 2002 Wiley-Liss, Inc.)

In cephalopods, the complex rhodopsin-retinochrome system serves to regenerate metarhodopsin and metaretinochrome after illumination. In the dark, a soluble protein, retinal-binding protein (RALBP), shuttles 11-cis retinal released from metaretinochrome located in the photoreceptor inner segments to metarhodopsin present in the rhabdoms. While in the rhabdoms, RALBP delivers 11-cis retinal to regenerate rhodopsin and in turn binds the all-trans isomer released by metarhodopsin. RALBP then returns all-trans retinal to the inner segments to restore retinochrome. The conventional interpretation of retinoid cycling is contradicted by immunocytochemical studies showing that, in addition to rhodopsin, retinochrome is present in the rhabdomal compartment, making possible the direct exchange of chromophores between the metapigments with the potential exclusion of RALBP. By using immunofluorescence and laser scanning confocal microscopy, we have precisely located opsin, aporetinochrome, and RALBP in light-/dark-adapted octopus retinas. We found differences in the distribution of all three proteins throughout the retina. Most significantly, comparison of cross sections though light- and dark-adapted rhabdoms showed a dramatic shift in position of the proteins. In the dark, opsin and retinochrome colocalized at the base of the rhabdomal microvilli. In the light, opsin redistributed along the length of the microvillar membranes, and retinochrome retreated to a location that is perhaps extracellular. RALBP was present in the core cytoplasm of the photoreceptor outer segments in the dark, and RALBP moved to the periphery in the light. Because of the colocalization of opsin and retinochrome in the dark, we believe that the two metapigments participate directly in chromophore exchange. RALBP may serve to transport additional chromophore from the inner segments to the rhabdoms and may not be immediately involved in the exchange process.

Glial fibrillary acidic protein (GFAP) and vimentin proteins are known to be component proteins of glial filaments in the CNS of many vertebrates. The nature of the filaments present in the glial cells of the goldfish optic tectum and in the CNS of two members of the Mollusca (Helix pomatia and Octopus vulgaris) were investigated using immunocytochemical localization of monoclonal antibodies to GFAP and vimentin. Immunoblots visualized by the alkaline phosphatase method showed cross-reactive protein bands to GFAP and vimentin antibodies in total brain homogenates of the goldfish, octopus, and snail. Immunofluorescence staining of the goldfish optic tectum showed GFAP immunoreactivity, primarily in the ependymal cell processes. Immunogold labelling at the ultrastructural level verified that GFAP antibodies were bound to glial filaments. Immunolabelling of the optic lobe of Octopus vulgaris and the cerebral ganglia of Helix pomatia suggests that a protein exhibiting antigenic properties similar to GFAP is a component protein in the filaments of the protoplasmic and filamentous glia randomly distributed throughout the CNS. Unlike anti-GFAP antibodies, which stained relatively specific to filaments, vimentin staining in the CNS tissues of the three organisms studied did not appear to be exclusive to filamentous structures. As vimentin protein has been shown, in previous studies as well as our own, to exist in many tissue types, this suggests that it does not appear to be confined to glial filaments but is shared with other subcellular components. The proteins GFAP and vimentin which are thought to be well conserved in vertebrate evolution also appear to be expressed in the nervous system of some lower organisms.

Cytophotometric analyses of Feulgen-stained nuclei present in homogenates of vertical and subesophageal lobes of octopus brain have shown that the latter region contains larger nuclei with up to several times the amount of DNA present in vertical nuclei. No obvious relationship was found between DNA content and nuclear size. Except for a rather small minority, nuclei of the vertical lobe have a uniform size and the expected diploid amount of DNA. These parameters are not substantially dependent on body weight. In contrast, the DNA content of subesophageal nuclei increases progressively with body weight. The amount of DNA found in subesophageal nuclei does not seem to be a simple multiple of the diploid or haploid value.

Using light and electron microscope cytochemistry and lectin blotting techniques, we have shown that the lectins concanavalin A (Con A), Ricinus communis agglutinin (RCA), and peanut agglutinin (PNA) bind to specific glycoconjugants in the adult cephalopod retina. For light microscope lectin cytochemistry, aldehyde-fixed, frozen, or Araldite-embedded, etched sections of cephalopod retinas were incubated with FITC- or TRITC-conjugated lectins and examined by using epifluorescence microscopy. Con A labeled structures in the entire retina including the inner limiting membrane (ILM), rhabdomeric membranes, interphotoreceptor matrix (IPM), and structures in the photoreceptor inner segments. RCA labeling was similar to that of Con A except that there was a decrease in the staining of the rhabdom tips near the ILM. PNA labeled only the interphotoreceptor matrix between apposing rhabdomeres. The intensity of staining of the IPM by PNA also decreased or was absent toward the rhabdom tips. None of the lectins labeled the myeloid bodies located in the photoreceptor inner segments. Electron microscope (EM) lectin cytochemistry was performed on aldehyde-fixed, LR White-embedded tissue or on Araldite-embedded, periodate-etched sections by using gold-conjugated lectins. EM results confirmed the observations made by light microscopy. Lectin blots with a retinal extract or light-sensitive membrane fraction revealed a variety of protein bands labeled by all three lectins. Con A and RCA labeled opsin and its aggregates whereas PNA did not. None of the lectins labeled retinochrome. The labeling of the cephalopod IPM by PNA suggests a structural similarity between the IPM of vertebrates and invertebrates. In other studies, we have demonstrated the presence of a retinoid binding protein in the IPM of cephalopods.(ABSTRACT TRUNCATED AT 250 WORDS)