No abstract

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.

The cephalopod olfactory organ was described for the first time in 1844 by von Kölliker, who was attracted to the pair of small pits of ciliated cells on each side of the head, below the eyes close to the mantle edge, in both octopuses and squids. Several functional studies have been conducted on decapods but very little is known about octopods. The morphology of the octopus olfactory system has been studied, but only to a limited extent on post-hatching specimens, and the only paper on adult octopus gives a minimal description of the olfactory organ. Here, we describe the detailed morphology of young male and female Octopus vulgaris olfactory epithelium, and using a combination of classical morphology and 3D reconstruction techniques, we propose a new classification for O. vulgaris olfactory sensory neurons. Furthermore, using specific markers such as olfactory marker protein (OMP) and proliferating cell nuclear antigen (PCNA) we have been able to identify and differentially localize both mature olfactory sensory neurons and olfactory sensory neurons involved in epithelium turnover. Taken together, our data suggest that the O. vulgaris olfactory organ is extremely plastic, capable of changing its shape and also proliferating its cells in older specimens.

This study focused on the morphological and functional characterization of the haemocytes from Octopus vulgaris as the first agents responsible for innate immunity. Three major haemocytes types were identified by light microscopy based on nucleus/cytoplasm ratio and the presence or absence of cytoplasm granules: haemoblast-like cells, hyalinocytes and granulocytes. The presence of three haemocyte populations was also confirmed by flow cytometry. Cytochemical characterization suggests that they perform different activities during humoral responses. Using the plate radial diffusion method we demonstrated the bactericidal activity of haemocytes in the presence of different bacteria strains. To investigate the presence of soluble biotic compounds responsible for antibacterial activity, using a disc diffusion method and the minimum inhibitory concentration, we have also tested the methanolic acid extract from these cells. Our results pave the way for the development of potent antibacterial drugs that could lead to several applications.

Adult neurogenesis is widespread among metazoans, it occurs in animals with a network nervous system, as cnidarians, and in animals with a complex and centralized brain, such as mammals, non-mammalian vertebrates, ecdysozoans, and a lophotrochozoan, Octopus vulgaris. Nevertheless, there are important differences among taxa, especially in the number of the regions involved and in cell proliferation rate during the life-cycle. The comparative evaluation of adult neurogenesis among different brain regions is an arduous task to achieve with only stereological techniques. However, in Octopus vulgaris we recently confirmed the presence of active proliferation in the learning-memory centers, multisensory integration centers, and the motor centers of the adult brain. Here, using a flow cytometry technique, we provide a method to quantify the active proliferation in octopus nervous system using a BrdU in vitro administration without exposing the animals to stress or painful injections usually used. This method is in line with the current animal welfare regulations regarding cephalopods, and the flow cytometry-based technique enabled us to measure adult neurogenesis more quickly and reliably than histological techniques, with the additional advantage of processing multiple samples in parallel. Flow cytometry is thus an appropriate technique for measuring and comparing adult neurogenesis in animals that are in a different physiological and/or environmental contexts. A BrdU immunoreactivity distribution, to define the neurogenic areas, and the effective penetration in vitro of the BrdU is also provided.