First visualization of cholinergic cells and fibers by immunohistochemistry for choline acetyltransferase of the common type in the optic lobe and peduncle complex of Octopus vulgaris

Abstract

This study provides the first immunohistochemical evidence visualizing cholinergic octopus neurons containing choline acetyltransferase (ChAT), the synthetic enzyme of acetylcholine. Because the antiserum applied here was raised against a recombinant protein encoded by exons 7 and 8 of the rat gene for ChAT, and initially used for studies in mammals, to validate antibody specificity for the octopus counterpart enzyme we therefore used three methods. Immunoprecipitation using Pansorbin indicated that immunoreactive octopus brain molecules were capable of synthesizing acetylcholine. Western blot analysis after denatured gel electrophoresis of octopus brain extracts revealed a single band at Ϸ81 kDa. A gel slice containing the 81-kDa protein after native (nondenatured) gel electrophoresis exhibited high ChAT activity. All findings obtained with these three methods clearly indicated that the antiserum effectively recognizes octopus ChAT. The immunohistochemical use of the antiserum in the retina, optic lobe, and its neighboring peduncle complex detected enzyme-containing neuronal cell bodies in only two regions, the cell islands of the optic lobe medulla and the cortical layer of the posterior olfactory lobule. Immunoreactive fibers and probable nerve terminals were also found in the plexiform layer of the deep retina, within the stroma of the optic gland, and the neuropils of the optic lobe, peduncle lobe, and olfactory lobe. These results provide information on the morphology and distribution patterns of cholinergic neurons in the octopus visual system, a useful invertebrate model for learning and memory where the cholinergic system, as in higher vertebrates including mammals, plays an important role.

Immunolocalization of choline acetyltransferase of common type in the central brain mass of Octopus vulgaris

“…72,73 Two different color classes of chromatophore motoneurons, that excite black chromatophores or yellow, orange and red chromatophores, are inhibited by descending cholinergic pathways derived from optic lobes. 20,29,74,75 Our findings agree with previous investigation on AChE presence in the neuropil of all the chromatophore lobes and the optic lobes, 29 and with cChAT immunoreactivity in the optic lobes. 30 In the magnocellular lobes, we found many cChAT-IR cell bodies and fibers.…”

Confocal analysis of nervous system architecture in direct-developing juveniles of Neanthes arenaceodentata (Annelida, Nereididae)

“…Efforts to establish a clearer understanding of polychaete parapodial ganglia and motor-neuronal arrangements for somatic musculature will benefit from the fine-scale resolution and efficiency of CLSM. Retrograde labeling with DiI [85,86] and immunohistochemistry for various myoactive substances such as serotonin [87], Polychaete Excitatory Peptide [88], and acetylcholine [5] (visualized with antibodies to choline acetyltranferase [89]) should prove effective in locating motor-neuronal perikarya and mapping their axonal pathways to muscle.…”

Peripheral type of choline acetyltransferase: Biological and evolutionary implications for novel mechanisms in cholinergic system

“…Late Professor Tindaro Renda and his wife L. D'Este have initiated a series of studies to delineate firstly cholinergic nervous systems in the marine mollusc Octopus vulgaris (D'Este et al, 2008) and terrestrial mollusc Limax maximus (D'Este et al, 2011) by immunohistochemistry using rat cChAT antiserum . They have simultaneously started their studies also by pChAT immunohistochemistry in the terrestrial snails and slugs as well as the octopus.…”

“…Subsequently, several antisera were raised against purified preparation of ChAT from the CNS (Cozzari and Hartman, 1980;Levey et al, 1981;Eckenstein and Thoenen, 1982;Ichikawa et al, 1987;Ostermann et al, 1990;Poethke et al, 1997). All were successfully applied for ChAT immunohistochemistry and became invaluable tools to explore the neuroanatomy of central cholinergic systems in rat (Woolf et al, 1983(Woolf et al, , 1984Phelps and Vaughn, 1986;Satoh and Fibiger, 1986;Woolf and Butcher, 1986;Hallanger et al, 1987) and other mammals (Vincent and Reiner, 1987) including humans (Oda and Nakanishi, 2000), and non-mammal vertebrates including birds (Medina and Reiner, 1994), fishes (Giraldez-Perez et al, 2009), amphibians (Marin et al, 1997) and reptiles (Brauth et al, 1985), and some invertebrates including insects (Gorczyca and Hall, 1987), flatworms (Nishimura et al, 2010), nematodes (Johnson and Stretton, 1985) and molluscs (Yasuyama et al, 1995;D'Este et al, 2008D'Este et al, , 2011. Beside, these antisera are also applicable to visualize ChAT in non-neuronal cells (Parnavelas et al, 1985;Oda et al, 1996) including cancer cells (Song et al, 2003;Cheng et al, 2008;Pettersson et al, 2009).…”