Magnitude Assessment of Adult Neurogenesis in the Octopus vulgaris Brain Using a Flow Cytometry-Based Technique

Abstract

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.

Cognitive Stimulation Induces Differential Gene Expression in Octopus vulgaris: The Key Role of Protocadherins

“…Adult neurogenesis can be considered as an intrinsic factor to the maintenance of a healthy brain and to memory assessment [ 63 ]. O. vulgaris possesses high cognitive capabilities, and just recently, it has never been shown that they have adult neurogenesis [ 10 , 12 ]. We now extend the knowledge to the genes involved in this process.…”

“…The optic and olfactory lobes integrate sensory stimuli detected by sensory organs from the environment while the spine of the peduncle lobe is considered an analog of the vertebrate cerebellum. These lobes are involved in the coding of the visual and chemical inputs as well as the coordination of voluntary movements, such as posture resulting in smooth and balanced muscular activity supporting the neurophysiological bases of octopus’ sophisticated behavior [ 11 , 12 , 59 , 70 , 71 , 72 , 73 , 74 ].…”

“…Octopuses, subjected to problem-solving tasks, revealed an increment of cell proliferation in supraesophageal mass, in particular in the vertical-frontal system and the optic–olfactory lobes, brain areas involved in learning memory, and sensory stimuli integration, respectively [ 10 ]. Moreover, bivariant analysis of flow cytometry using BrdU incorporation allowed assessment of the magnitude of adult neurogenesis in those brain lobes, previously identified, characterized by the presence of adult neurogenesis niches, highlighting the amount of cells exhibiting de novo DNA synthesis [ 12 ]. What remains puzzling is what genes are majorly involved in these intriguing processes in Octopus vulgaris .…”

The Cephalopod Large Brain Enigma: Are Conserved Mechanisms of Stem Cell Expansion the Key?

“… Buresi et al (2013) suggest a prolongation of proliferative capacities of the ganglia in cephalopod hatchlings which implies preservation of quiescent stem cells to allow delayed adult neurogenesis ( Baratte and Bonnaud, 2009 ). Excitotoxic lesion by kainic acid in the vertical lobe of S. officinalis induced proliferation as measured by BrdU incorporation ( Graindorge et al, 2008 ) and recently, Di Cosmo et al (2018) observed active proliferation in the O. vulgaris nervous system after in vitro administration of BrdU. PCNA levels seem to increase in the vertical and frontal lobes of O. vulgaris housed in an enriched environment suggesting active cell division takes place ( Bertapelle et al, 2017 ), however, leaving the reader in the dark on the precise cellular location of the presumptive raise in mitotic activity.…”

OctoPartenopin: Identification and Preliminary Characterization of a Novel Antimicrobial Peptide from the Suckers of Octopus vulgaris

“…Octopus and all cephalopods, as a result of evolutionary selective pressure, have developed a winning strategy for surviving in different environments [8,9]. O. vulgaris is an economically important seafood species [10], and a model for studying complex behavioral, neuronal, and genomic plasticity [11,12]. Moreover, it is well known for its intelligence linked to adult neurogenesis mechanisms, curiosity, and adaptability; recently, transcriptomic and genomic tools were developed to investigate these issues on this species and the congeneric O. bimaculoides [8,[13][14][15].…”