ISSN 00220930, Journal of Evolutionary Biochemistry and Physiology, 2022, Vol. 58, No. 5, pp. 1401–1412.

© Pleiades Publishing, Ltd., 2022.

EXPERIMENTAL PAPERS

Characterization of the Brain of the Red Mayan Octopus

(Octopus maya Voss and Solis, 1966)
F. VergaraOvallea, A. GonzalezNavarretea, and H. SánchezCastilloa,b,*
a
Neuropsychopharmacology Laboratory, School of Psychology, National Autonomous University
of Mexico, Mexico City, Mexico
b
Research Unit of Psychobiology and Neurosciences (UIPyN), School of Psychology, National
Autonomous University of Mexico, Mexico City, Mexico
*email: [email protected]
Received June 26, 2022
Revised July 26, 2022
Accepted August 10, 2022

Abstract—Octopuses show a wide behavioral range that allows them great adaptability to their
environment. This makes them a target of great interest for biophysics, cytology, and neurobiology
studies. To adequately execute these investigations, it is essential to have a thorough understanding
of the neuroanatomy of the species that function as good laboratory models, for example, O. maya.
In this work, we undertake the task of extracting and characterizing various structures of the
nervous system of O. maya at the level of lobes and connectivity between them. O. maya has great
advantages as a research model for the neurobiology of cephalopods, such as being reproduced in
captivity, having holobenthic larvae, an adequate size for analysis and performing its behavior out
of hiding places and not burrowing. All this allows behavioral studies to be carried out more easily.
In this study we have characterized the morphology of 30 lobes in the brain of O. maya from young
and adult specimens and the cells that constitute the connectivity pathways between these regions.
We also observed that the brains of O. maya at four weeks after hatching do not show a fully mature
structure, but rather that some lobes related to somatosensory memory have low cell density and a
simpler morphology than that of adult specimens. It is indicative of a maturation process that
should be considered for future experimental designs. We hope that this nervous system mapping
will increase the basis for more detailed investigations into the neurophysiology of O. maya and
other octopuses.
DOI: 10.1134/S0022093022050118
Keywords: Octopus, brain, nervous system, cephalopod

INTRODUCTION allows them great adaptability to their environ

ment. For this reason, they have been the target of
Cephalopods are a group of mollusks that great interest by scientists and are particularly rel
include nautilus, cuttlefish, squid, and octopus. evant in biophysics, cytology, and neurobiology
This group shows a wide behavioral range that studies [1–2]. This diversity of behavior is possible

1401
1402 VERGARAOVALLE et al.

in a highly complex nervous system, the most the case of neurobiology, the anatomy of the ner
complex within invertebrates [3]. Particularly in vous system. Our knowledge of octopuses’ neuro
the case of octopuses, it has been suggested that anatomy arose in the 60’s [23–24]. However,
they possess cognitive abilities and capacities such these descriptions were just detailed throughout
as memory [4–6], play [7–8], problemsolving their works but not organized in the form of a map
[9], recognition of individuals [10], and emotion or an atlas, making their understanding and com
like behaviors [11]. All these functions are com parison difficult for new researchers. Since then,
mon in vertebrates. However, the last common some cephalopod brain atlases have been pub
ancestor between vertebrates and mollusks existed lished, e.g., two for the squids Idiosepius para
approximately 550 million years ago, which is why doxus Ortmann 1888 [25] and Todarodes pacificus
a model of convergent evolution is proposed with Steenstrup 1880 [26], one for the longarm octo
a single nervous system but comparable to that of pus, O. minor [27], as well as a recent comparative
some vertebrates [12]. To take advantage of this analysis between the brains of diurnal and noctur
model, it is imperative to have a thorough under nal octopuses and the vampire squid [28]. These
standing of the nervous system of the cephalopods previous works provide good foundations to
to study how they accomplish these tasks and investigate the nervous system of a particularly
compare them with the structures of other groups useful species as a laboratory model, such as
such as arthropods and vertebrates [13]. O. maya [29]. That is why in this work we took on
In addition, these groups, mainly squid and the task of extracting and characterizing the vari
octopuses, are of great commercial importance, ous structures of the nervous system of O. maya,
which is why in recent decades efforts have been at the level of lobes and connectivity between
increased to breed them in captivity [14]. This them. We hope that this mapping of the nervous
offers many opportunities for physiological and system will increase the basis for more detailed
behavioral studies in the laboratory and under investigations into the neurophysiology of
much more controlled conditions. An example of O. maya, as well as help to correlate future inves
the latter is the case of O. maya, a species of octo tigations about behavior with the nervous system
pus endemic to the Yucatan Peninsula, Mexico, of this species.
whose maintenance conditions, from the egg
stage to the adult stage, have been widely MATERIALS AND METHODS
described [14–16]. O. maya is also known as red
octopus or foureyed octopus because of its char Ethical statement
acteristic color and large doubleringed ocelli All the experiments and handling of animals
under each eye. This distinguishes it from the were carried out under the approval of the bioeth
other species that are distributed in the Yucatan ics committee of the Faculty of Psychology,
peninsula, O. vulgaris Cuvier 1797. In addition, UNAM, and following the directive 2010/63/EU
O. maya has a larger egg size (average 17 mm) and (European parliament) [30].
fewer eggs (1500–2000 eggs per clutch) [17]. The
larvae that hatch from these eggs are holobenthic, Animals
resembling an adult, and much more developed The specimens were obtained from the Applied
than those of O. vulgaris. This characteristic has Ecophysiology Laboratory of the Multidisci
been very convenient to achieve its reproduction plinary Unit for Teaching and Research (UMDI,
in captivity [14–18]. All mentioned above allow Unidad Multidisciplinaria para la Docencia e
O. maya, along with other species such as O. insu Investigación), Port of Sisal, Yucatan, México. A
laris Leite & Haimovici 2008 [19], O. minor total of three adult specimens of O. maya were
Sasaki 1920 [20] and O. vulgaris [21] to be consid captured off the coast of Sisal, Yucatan. The ani
ered laboratory animals for physiological, behav mals were sacrificed using anesthesia (3.5% etha
ioral, and evolutionary studies [22]. As part of nol in artificial seawater) for 15 min and
these studies, it is crucial to know as much as pos subsequently, the brain was removed, immedi
sible about general anatomy and, particularly in ately embedding it in 4% formaldehyde for

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 CHARACTERIZATION OF THE BRAIN OF THE RED MAYAN OCTOPUS 1403

12 hours. Eggs were obtained and these were To compare the number of nervous cells
cared for and kept in the laboratory until hatching between adults and four week old specimens,
so that we could obtain three young specimens somas were counted in the superior frontal, infe
which were overanesthetized with ethanol. rior frontal, and buccal lobes of three adult speci
mens and three fourweekold specimens, using
Histology the ImageJ program. For this, the six closest slides
Cartilage tissue was removed from the brain as to the midline corresponding to L –70, L –140,
described below. The mantle and arms are first L –210, R +70, R +140, R +210 were used (L =
separated, leaving only the segment containing left, R = right, ± X μm).
the eyes and skull. Then the skull was separated
from the muscle tissue and removed. It had a tri Diaphanization
angular shape, two of the sides corresponding to To visualize the macrostructure of the nervous
the basins where the optic ganglia are located, and system, the diaphanization of the young speci
the third side corresponding to the dorsal region. mens was carried out, according to a modified
The top of the skull is slightly rounded. The vertex protocol described in [31], in which the specimen
between the two sides with the eye sockets was cut was kept in PFA for at least 2h and then embed
and from there, cuts were made to the orifice of ded in a 0.1% alizarin red solution for 2h under
the optic tract, in the center of each side of the constant stirring, at room temperature and in the
skull. These cuts allowed the top of the skull to be dark. Later washes were given in decreasing con
opened, thereby exposing the brain. The vertical centrations of ethanol (100% → 50%). To lighten
lobe was quickly identifiable by its characteristic the pigmentation, the specimens were immersed
grooves. Finally, the brain was carefully sepa in a solution of 0.5% KOH and 10% H2O2 for
rated, cutting the nerves of the inferior region and approximately 48 hours. Finally, 3 washes of
cleaning of the remains of cartilage. This tech 20 min each were given in 25% glycerol / 0.1%
nique allows the brain to be removed quickly. KOH and 50% glycerol / 0.1% KOH. To finish,
Following fixation with 4% paraformaldehyde the diaphanized specimens were preserved in
(PFA) in sea water, the brain was embedded in glycerin.
paraffin. Through the use of a KD2258 micro
tome (KEDEE, Zhejiang, China), tissue sections Statistical analysis
of 10 μm thickness were obtained, at a distance of All cell count results were compared using the
every 70 or 80 μm. Hematoxylin and eosin (H&E) Sigma Plot Software (Systat Software Inc, San
and cresyl violet (Nissl) staining were performed Jose California, USA) with the Mann–Whitney
following a standard protocol using the 10 μm sec Utest. p < 0.05 was considered statistically signif
tions. 37–42 sagittal slices were obtained from icant.
each of the 3 specimens and 51–56 from sequen
tial coronal slices. In addition, 51 H&Estained RESULTS
transverse sectional slides were obtained from the
brain of an adult specimen. These were presented Morphology of the brain of O. maya
in the Supplementary material. The size of the tissue located within the carti
laginous cavity (central ganglion) was 6 ± 1.5 mm
Image analysis wide and 9 ± 1.7 mm long (Figs. 1e, 1f). Sections
The stained slides were photographed at 2x, 4x, of both hematoxylineosin (H&E) and Nissl
10x, and 40x magnification using a stereoscopic staining were used to characterize the different
and light microscope. The images obtained were regions of the O. maya brain. In the tissues, it was
analyzed and used to create a threedimensional observed that they maintain a typical arrangement
map, sequentially arranging the photographs using of ganglia in invertebrates with a cluster of somas
an image processing program (Microsoft Paint in the periphery or perikaryon and axonal projec
3D). This was done to allow a better analysis of the tions forming the center of the soma or neuropil.
connectivity and location of the different lobes. However, they showed great heterogeneity in

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Fig. 1. Brain structures of O. maya. Sagittal sections (a, b) of the central ganglion of O. maya with abbreviations on each struc
ture, accordingly to Table 1. Coronal slices (c, d) and photograph of fresh tissue in ventral (e) and dorsal (f) perspective. Bars
represent 1000 μm. P: Posterior, V: Ventral, D: Dorsal (To review the nomenclature see Table 1).

terms of the type of cells observed, their arrange characterization of the tissues, observing the peri
ment, and density. Through the morphological karyon and neuropil sites, it was possible to distin

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 CHARACTERIZATION OF THE BRAIN OF THE RED MAYAN OCTOPUS 1405

Table 1. Names and abbreviations of the structures that make up the brain of O. maya
Lobe Abbrevi Cell types
ation
Vertical LV Glial cells, small amacrine cells; very few large neurons
Vertical medial LVm Glial cells, small amacrine cells; very few large neurons
Vertical lateral LVl Glial cells, small amacrine cells; very few large neurons
Subvertical Anterior SubVa Large neurons, glial cells, pyramidal neurons; axons from the SFL
Subvertical Posterior SubVp Large neurons, glial cells, pyramidal neurons; cell bodies that connect with the
VL entries
Superior Frontal SF Large neurons, glial cells pyramidal neurons and big amacrine cells
Inferior Frontal IF Large neurons, big and small cells
SubFrontal/Posterior SubF Large neurons, big and small cells
Frontal
Buccal Bu Large and small neurons, glia and functional syncytia
Post Buccal BuP Large neurons, big and small cells, glial cells
Dorsal Basal Anterior BaDa Long, large and small neurons with fused axons, small multipolar neurons, glia
Dorsal Basal Posterior BaDp Long, large and small neurons with fused axons, small multipolar neurons, glia
Basal Anterior BaA Long, large and small neurons with fused axons, small multipolar neurons, glia
Basal Medial BaM Long, large and small neurons with fused axons, small multipolar neurons, glia
Basal Lateral BaL Long, large and small neurons with fused axons, small multipolar neurons, glia
Precommissural Precom Long, large and small neurons, glia
PreBraquial PreBra Long, large and small neurons with fused axons, small multipolar neurons,
glia, functional syncytium
PosBraquial PosBra Long, large and small neurons with fused axons, small multipolar neurons, glia
Pedal Anterior PeA Large, pyramidal, and large amacrine cells
Pedal Posterior PeP Large, pyramidal, and large amacrine cells
Palio Visceral PV Large, pyramidal, and glial cells
Siphon Sif Neurons with very large cell bodies, low cell density, “honeycomb” arrange
ment and some small cells at base
Ventral Vasomotor VenVas Neurons with very large cell bodies, low cell density, “honeycomb” arrange
ment and some small cells at base
Medial Vasomotor VasM Neurons with very large cell bodies, low cell density, “honeycomb” arrange
ment and some small cells at base
Interbasal Iba Large neurons with axons intertwined as a “bouquet”, high cell density
Chromatophore Pos ChP Large neurons with axons intertwined as a “bouquet”, high cell density
terior
Magnocellular dorsal Mgd Large neurons with axons intertwined as a “bouquet”, high cell density
Magnocellular poste Mgp Large neurons with axons intertwined as a “bouquet”, high cell density
rior
Olfactory Olf Neurons with ample cytoplasm, cuboidal shape, low cell density

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1406 VERGARAOVALLE et al.

Table 1. (Contd.)
Lobe Abbrevi Cell types
ation
Peduncular Ped Small neurons, high cell density
Optic Opt Amacrine neurons, it shows a layer of long neurons with axonal projections
between perykarion and neuropil, high density of somas in neuropil
Optic Gland Gopt High cell density, no neuropil, high density of interconnected axonal projec
tions
Optic Tract TrOpt
Optic nerves nOpt
Brachial nerve 1–8 nBra 1–8
Siphon nerve anterior nSifa
Siphon nerve poste nSifp
rior
Palial Nerve nPa
Visceral nerve nVis
Buccal nerve nBu
Orbital antor nerve nAntorb
Brain–brachial con Con
nection Cer–Bra
Anterior pedal–basal Con
connection Pe–Baa
Posterior pedal–basal Con
connection Pe–Bap
Palliovisceral–basal Con
connection PV–Ba
Brachial–Paliovis Con
ceral Connection Bra–PV

guish 30 different lobes that make up the central (Figs. 2a–2b). Regarding the nerves and tracts
ganglion and at least two for each of the axial gan coming from the rest of the animal’s body, the
glia, with the identification of at least 10 different eight brachial nerves coming from each brachial
neuronal types (Table 1; Figs. 1a–1d). Since each ganglion stand out, which merge in the prebra
cut was sequential, and knowing the distance chial lobe, together with the anteroorbital nerves.
between each cut, it was possible to calculate the This initiates a complex network of tracts and
distance at which each cut is located to the center nerves connecting the posterior brachial lobe with
of the tissue. the buccal and posterior buccal lobes on one side
Once all the sections were photographed with and with the paliovisceral lobe on the other.
the stereoscope, these photographs were used to Regarding the optic tract, it crosses the posterior
create a threedimensional model that would help region of the supraesophageal mass, connecting
to analyze all the images simultaneously (Fig. 2c). the two optic lobes with the peduncular lobes,
By doing this, existing anatomical connections olfactory lobes, and optic glands. Furthermore,
between the different lobes were determined this tract bifurcates to join the precommissural

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 CHARACTERIZATION OF THE BRAIN OF THE RED MAYAN OCTOPUS 1407

Fig. 2. Scheme of connectivity in the brain of O. maya. The geometric shapes represent each of the lobes in the O. maya brain.
A—Anterior, P—posterior. The blue lines represent adjacent connectivity and gray lines represent distant connectivity
through neuronal tracts or nerves (a, b). Below is a representative image of the threedimensional arrangement of the digi
talized slides, corresponding to the total obtained from a complete octopus’ brain, used for the brain connectivity analysis (c).
This arrangement in three dimensions was made with the Paint 3D, Microsoft, tool. The photographs were arranged as flat
labels in sequential order, with constant spaces between each of them. This allows the user to zoom in and out as well as view
from various perspectives to facilitate interpretation of tissue connectivity and morphology. (To review the nomenclature, see
Table 1).

lobe in the supraesophageal mass and the paliovis Brain of a young octopus continues its development
ceral lobe in the subesophageal mass. The sub 4 weeks after hatching
esophageal and supraesophageal masses form a Visualization by diaphanization allows for
ring via a wide anatomical communication made observing the cartilaginous tissues and clarifying
up of axons and cells (possibly glial) that make up the epithelial and muscular tissues. This tech
the prebrachialbuccal connections on one side nique, together with the histological sections,
and the pedalbasal/paliovisceralbasal connec allowed us to observe that the brain of O. maya
tions on the other. We observed that there is continued its development, even 4 weeks after
extended connectivity between practically all the hatching. This is observed because the brain of
lobes of the brain and wide distribution of nerves young specimens is in a more posterior position
and tracts that propagate sensory information to compared to adults, towards the optic lobes
both brain masses, supra and subesophageal. (Fig. 3). This is corroborated in the histological

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1408 VERGARAOVALLE et al.

Fig. 3. The brain of young specimens of O. maya. Diaphanization of a 4weekold specimen to observe the cartilage covering
the central ganglion, around the esophagus (Arrows) (a). Sagittal section with H&E staining at 4x (b) and 100x magnitude of a
young specimen (Cc) vs. adult specimen (d). (e) Graph showing a lower number of somas in the buccal (Bu), inferior frontal
(IF) and superior frontal (SF) lobes of four weeks old specimens vs adult specimens, Mann–Whitney Utest, p < 0.05.

sections in which the optic lobe can be seen well already has the complexity of the adult brain,
below the rest of the brain. composed of the same number of lobes and the
In addition to the above, it was observed that anatomical connections between them, it still did
although the brain of a young octopus in general not have the final morphology characteristic of

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 CHARACTERIZATION OF THE BRAIN OF THE RED MAYAN OCTOPUS 1409

the adult stage, in the inferior frontal, sub frontal, O. maya, as well as the behavioral and cognitive
buccal, and posterior buccal lobes, which have differences that they could present at different
been proposed to participate in somatosensory stages of their life. This delayed maturation of the
memory. To support these suggestions, a manual nervous system has been described in other ceph
count of the somas of adults and fourweekold alopods such as the cuttlefish Sepia officinalis
specimens was carried out. This count suggests Linnaeus 1758 [32], S. pharaonis Ehrenberg,
that there is a lower cell density in young individ 1831 [33], the squids I. paradoxus [34], and
uals, compared to fully grown ones, at least in the S. lessoniana d’Orbigny, 1826 [35], the octopuses
superior frontal (SF), inferior frontal (IF), and O. ocellatus Gray, 1849 [36] and O. vulgaris [37].
buccal (Bu) lobes (Fig. 3e). This may indicate that it is a conserved feature in
the Cephalopoda class. It is important to men
DISCUSSION tion that although the results suggest a difference
between the number of neurons of adult and
The structure of 30 lobes in the brain of young specimens, the connectivity between the
O. maya from young and adult specimens was different lobes seems to be already formed from
determined, as well as the number of cells that the first stages of life. However, it will be interest
make up the connectivity pathways between these ing to determine if this connectivity modifies
regions. This complex network of lobes and con their electrical and neurotransmitter activity as
nectivity present in the octopus brain underlies individuals mature.
the diversity of behaviors and cognitive abilities These lobes are very similar to those previously
that have been proposed in cephalopods and par described by Young in 1971 [24] and Jung and
ticularly in O. maya. However, it is important to colleagues in 2018 [27] for O. vulgaris and
clarify that the methods used in this work do not O. minor, respectively, although this was expected
allow us to determine the polarity in this connec as they are closely related species [38]. However,
tivity, nor the cell types that make it possible. it is also important to determine the precise neu
Future research is needed that uses various obser roanatomy of O. maya since it is the species that is
vation methods, as well as physiological activity ideal for carrying out physiological and behavioral
analysis, to characterize in more detail the rela studies thanks to its developmental characteristics
tionship between the different structures and their (hatches as holobenthic larvae) and behavioral
participation in the various neurophysiological habits (it does not usually bury itself, or escape
processes. These investigations will probably help from its enclosure, when given adequate food and
us to better define brain structures, considering refugees), as well as the achievements in standard
the types of cells, as well as their function. How izing their laboratory care.
ever, visualization of threedimensional models of Provided that detailed information on the fun
the brain, as achieved in this article, allows us to damental aspects of the species becomes more
give a better idea of the structure and communi available, it promises to be a great model for neu
cation within the brain. robiology research. In addition, the determina
We observed that at four weeks after hatching tion of the morphological characteristics of
the brains of O. maya do not show all character various species allows the comparison between
istics of a fully mature brain. It is the case that them and relates it to their behaviors and cogni
some lobes related to somatosensory memory tive abilities, just as recently discussed by Gutnick
had lower cell density and simpler morphology and colleagues [39] and Chung and colleagues
than that of the adult specimens. These observa [28].
tions could be correlated with behavioral dif We hope that this work will help new research
ferences since at this age the octopuses show a on the nervous system of cephalopods, particu
decreased capacity for memory and inhibition larly to increase neurobiological knowledge about
response [29]. However, more studies are needed O. maya, which, together with the advantage of
to fully understand the process of development being captivebred, increases the justification for
and maturation of the nervous system of using it for studies on cognition in cephalopods.

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AUTHORS’ CONTRIBUTION sequential slides obtained from one of the brains

of an adult specimen of O. maya is shown. Num
Conceptualization: VergaraOvalle, Sánchez bers indicate μm to the left (L –) from the midline
Castillo; Methodology: VergaraOvalle; Formal of the brain. The right side of each photograph
analysis and investigation: VergaraOvalle; Writ corresponds to the ventral region, while the left
ing: VergaraOvalle; Writing: review and editing: side of the photograph corresponds to the dorsal
Sánchez Castillo; Funding acquisition, resources, region. Bars represent 1000 μm. Cresyl violet
and supervision: Sánchez Castillo. staining.
Supplementary 3. Transversal cuts. The totality
FUNDING of sequential slides obtained from one of the
brains of an adult specimen of O. maya is shown.
José Fabián VergaraOvalle would like to thank The lower side of each photograph corresponds to
to the program Posgrade in Biological Sciences, the anterior region, while the upper side of the
(UNAM) the aquarium of the sciences school and photograph corresponds to the posterior region.
“Consejo Nacional de Ciencia y Tecnología” Numbers indicate μm vertical (V +) or Dorsal
(CONACYT) for the fellowship granted. The (D –) from the midline of the brain. Bars repre
research leading to these results has received sent 1000 μm. H&E staining.
funding from the projects PAPIIT IN 208722 and
PAPIME PE300918 granted to HSC. REFERENCES

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