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Exp Neurobiol 2018; 27(4): 257-266
Published online August 30, 2018
https://doi.org/10.5607/en.2018.27.4.257
© The Korean Society for Brain and Neural Sciences
Seung-Hyun Jung†, Ha Yeun Song†, Young Se Hyun, Yu-Cheol Kim, Ilson Whang, Tae-Young Choi, and Seonmi Jo*
Department of Genetic Resources Research, National Marine Biodiversity Institute of Korea (MABIK), Seocheon 33662, Korea.
Correspondence to: *To whom correspondence should be addressed.
TEL: 82-41-950-0756, FAX: 82-41-750-0765
e-mail: joseonmi@mabik.re.kr
†These authors contributed equally to this work.
Cephalopods have the most advanced nervous systems and intelligent behavior among all invertebrates. Their brains provide comparative insights for understanding the molecular and functional origins of the human brain. Although brain maps that contain information on the organization of each subregion are necessary for a study on the brain, no whole brain atlas for adult cephalopods has been constructed to date. Here, we obtained sagittal and coronal sections covering the entire brain of adult
Keywords: Cephalopoda, Octopodiformes, Brain, Anatomy, Comparative histology
With the exception of
Within the Octopodiformes, the genus
For the purposes of H&E staining, we used two sexually mature female
For H&E staining, the animals were anesthetized by immersion in artificial seawater containing 2% ethanol for 8 min. When they became relaxed and immobile, cartilaginous tissues containing the brain were excised and fixed with 10% formalin in artificial seawater at 4℃ for 2 days. Paraffin-embedded serial sagittal or coronal sections at 10 µm thickness were obtained from each brain (Histore Inc., Korea). Sections were stained with H&E using a staining kit (ScyTek Laboratories, USA). Briefly, Mayer's Hematoxylin (Lillie's modification) was applied to completely cover the tissue section and incubated for 5 min. After rinsing twice with distilled water to remove any excess stain, a Bluing Reagent was applied for 10~15 s. Following two additional washes in distilled water, slides were dipped in absolute alcohol. Thereafter, the slides were incubated in adequate Eosin Y solution (modified alcoholic) for 2~3 min, rinsed, and dehydrated four times in absolute alcohol. The slides were then cleared and mounted with Histomount (National Diagnostics, USA).
Stereoscopic images of the fixed intact brain were taken after removing the cartilage surrounding the brain as completely as possible using forceps. Images of the intact brain and H&E slide preparations were obtained under a LEICA S6D stereoscope (Leica Microsystems, Germany) using an OPTINITY KCS-2000SS CMOS camera (Korea Lab Tech, Korea). White balance was adjusted using OptiView 3.7 software (Korea Lab Tech, Korea). To establish the coordinates of each image, the distance from the midline point of the posterior end was calculated according to the thickness of the sections. Illustrations of brain gross anatomy were sketched by tracing a stereoscopic image of the intact brain using PowerPoint software (Microsoft, USA). For delineations, 40 representative H&E images for each sagittal and coronal section were selected based on the appearance of distinguishable changes in structural morphology. The delineations were hand-drawn on a transparent film placed over each of the paper-printed H&E images. Outlines of the brain were sketched with thick black lines, the brain internal structures with thin black lines, and Hematoxylin-stained layers with cyan lines or planes. All sketches were scanned and used as templates for tracing (Penodegr Inc., Korea) using Adobe Illustrator software (Adobe, USA).
On the tracings, we indicated the name of each subregion with abbreviations, using Adobe Illustrator software (Adobe, USA). For efficient conveyance of information among cephalopod researchers, we adopted the terms used in previous studies. All nomenclatures and abbreviations used are based on those presented in the book “The anatomy of the nervous system of
For immunofluorescence staining in the supraesophageal and subesophageal masses, 10-µm-thick sagittal paraffin sections were prepared using the same procedure as described for H&E staining. In case of the optic lobe, 30-µm-thick free-floating sagittal sections were prepared using a VT-1200S vibratome (Leica, Germany), after fixation with 4% paraformaldehyde overnight at 4℃, followed by dehydration and rehydration. Immunofluorescence staining of neurons was carried out as previously described using an anti-acetylated α-tubulin antibody (1:500; Sigma-Aldrich, USA) [17]. For fluorescent detection of antibody labeling, we used an Alexa Fluor 568 goat anti-mouse conjugate (1:1000; Molecular Probes, USA). DAPI (ThermoFisher, USA) counterstaining was performed for visualizing cell nuclei. Fluorescence images were obtained using a BX53 digital fluorescence microscope (Olympus, Japan).
We initially isolated the brain with surrounding cranial cartilage, which is located inside the head of
Prior to preparing tissue for construction of the
We subsequently obtained serial sagittal sections covering the entire brain of the sequence-confirmed, sexually mature adult
In the constructed atlas, the
Some of the known lobes in the brains of
From the H&E images of the supraesophageal and subesophageal mass, we found that the thickness and cell density of Hematoxylin-positive layers markedly varied depending upon the region (Fig. 4A). The optic lobe showed a distinctive structure, comprising islands of cells and a thin double Hematoxylin-positive layer near the surface (Fig. 4A). In the magnified images, the size of cellular nuclei in Hematoxylin-positive layer seemed to be diverse (image not shown). Therefore, we questioned the cellular identity of the dense Hematoxylin-positive cell layers of the
Although individual differences in brain size according to body weight and tissue shrinkage after formalin fixation are factors that should be taken into consideration, this study, nevertheless, represents an initial step towards characterizing the brain of
Abbreviation | Structure |
---|---|
b.a. | Anterior basal lobe |
b.d.a. | Anterior dorsal basal lobe |
b.d.p. | Posterior dorsal basal lobe |
b.int. | Interbasal lobe |
b.l. | Lateral basal lobe |
b.med. | Median basal lobe |
b.sul. | Basal sulcus |
br. | Brachial lobe |
br.po. | Postbrachial lobe |
br.pr. | Prebrachial lobe |
br.-pv.con. | Brachio-palliovisceral connective |
buc.p. | Posterior buccal lobe |
buc.s. | Superior buccal lobe |
c.br.sup. | Suprabrachial commissure |
c.opt.ven. | Ventral optic commissure |
cer.-br.con. | Cerebro-brachial connective |
cer.con.p. | Posterior cerebral connective |
ch.p. | Posterior chromatophore lobe |
fr.i.l. | Lateral inferior frontal lobe |
fr.i.med. | Median inferior frontal lobe |
fr.s.l. | Lateral superior frontal lobe |
fr.s.med. | Median superior frontal lobe |
fun.p. | Posterior funnel lobe |
g.opt. | Optic gland |
mag.d. | Dorsal magnocellular lobe |
mag.p. | Posterior magnocellular lobe |
n.antorb.s. | Superior antorbital nerve |
n.antorb.i. | Inferior antorbital nerve |
n.br.1,l.r. | Lateral root of the first brachial nerve |
n.br.1,med.r. | Medial root of the first brachial nerve |
n.br.1. | First brachial nerve |
n.br.2,l.r. | Lateral root of the second brachial nerve |
n.br.2,med.r. | Medial root of the second brachial nerve |
n.br.2. | Second brachial nerve |
n.br.3,l.r. | Lateral root of the third brachial nerve |
n.br.3,med.r. | Medial root of the third brachial nerve |
n.br.3. | Third brachial nerve |
n.br.4,l.r. | Lateral root of the fourth brachial nerve |
n.br.4,med.r. | Medial root of the fourth brachial nerve |
n.br.4. | Fourth brachial nerve |
n.col. | Collar nerve |
n.fun.a. | Anterior funnel nerve |
n.fun.p. | Posterior funnel nerve |
n.lab. | Labial nerve |
n.pal. | Pallial nerve |
n.sal.p. | Posterior salivary nerve |
n.st. | Static nerve |
n.vc.a. | Anterior vena cava nerve |
n.visc. | Visceral nerve |
ol. | Olfactory lobe |
opt. | Optic lobe |
pe.a. | Anterior pedal lobe |
pe.p. | Posterior pedal lobe |
ped. | Peduncle lobe |
ped.sub. | Subpedunculate lobe |
prec. | Precommissural lobe |
pv. | Palliovisceral lobe |
subfr. | Subfrontal lobe |
subv.a. | Anterior subvertical lobe |
subv.p. | Posterior subvertical lobe |
tr.br.-opt. | Brachial-optic lobe tract |
tr.br.-pv. | Brachial-palliovisceral lobe tract |
tr.cer. | Cerebral tract |
tr.opt. | Optic tract |
v. | Vertical lobe |
v.l. | Lateral vertical lobe gyrus |
v.med. | Median vertical lobe gyrus |
v.med.l. | Medio-lateral vertical lobe gyrus |
vas.d.l. | Lateral dorsal vasomotor lobe |
vas.d.med. | Median dorsal vasomotor lobe |
vas.ven. | Ventral vasomotor lobe |