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ORIGINAL ARTICLE
Immunohistochemical localization of two types of cholineacetyltransferase in neurons and sensory cells of the octopus arm
Yuko Sakaue • Jean-Pierre Bellier •
Shin Kimura • Loredana D’Este • Yoshihiro Takeuchi •
Hiroshi Kimura
Received: 21 July 2012 / Accepted: 28 December 2012
� Springer-Verlag Berlin Heidelberg 2013
Abstract Cholinergic structures in the arm of the ceph-
alopod Octopus vulgaris were studied by immunohisto-
chemistry using specific antisera for two types (common
and peripheral) of acetylcholine synthetic enzyme choline
acetyltransferase (ChAT): antiserum raised against the rat
common type ChAT (cChAT), which is cross-reactive with
molluscan cChAT, and antiserum raised against the rat
peripheral type ChAT (pChAT), which has been used to
delineate peripheral cholinergic structures in vertebrates,
but not previously in invertebrates. Western blot analysis of
octopus extracts revealed a single pChAT-positive band,
suggesting that pChAT antiserum is cross-reactive with an
octopus counterpart of rat pChAT. In immunohistochem-
istry, only neuronal structures of the octopus arm were
stained by cChAT and pChAT antisera, although the pat-
tern of distribution clearly differed between the two anti-
sera. cChAT-positive varicose nerve fibers were observed
in both the cerebrobrachial tract and neuropil of the axial
nerve cord, while pChAT-positive varicose fibers were
detected only in the neuropil of the axial nerve cord. After
epitope retrieval, pChAT-positive neuronal cells and their
processes became visible in all ganglia of the arm,
including the axial and intramuscular nerve cords, and in
ganglia of suckers. Moreover, pChAT-positive structures
also became detectable in nerve fibers connecting the dif-
ferent ganglia, in smooth nerve fibers among muscle layers
and dermal connective tissues, and in sensory cells of the
suckers. These results suggest that the octopus arm has two
types of cholinergic nerves: cChAT-positive nerves from
brain ganglia and pChAT-positive nerves that are intrinsic
to the arm.
Keywords Choline acetyltransferase � cChAT � pChAT �Cephalopod � Sensory � Motor
Abbreviations
ACh Acetylcholine
AChE Acetylcholinesterase
ChAT Choline acetyltransferase
cChAT Common type of choline acetyltransferase
pChAT Peripheral type of choline acetyltransferase
Introduction
Acetylcholine (ACh) was the first neurotransmitter to be
identified, but no reliable method has been established for
direct visualization of the molecule (Anglade and Larabi-
Godinot 2010). Identification of morphologic features of
cholinergic structures has been achieved by immunohisto-
chemistry, in various species including vertebrates and
invertebrates (for references see D’Este et al. 2008),
using antiserum against choline acetyltransferase (ChAT)
(Kimura et al. 1980), the enzyme responsible for ACh
biosynthesis (Nachmansohn and Machado 1943).
Y. Sakaue � J.-P. Bellier (&) � S. Kimura � H. Kimura
Molecular Neuroscience Research Center,
Shiga University of Medical Science, Seta Tsukinowa-cho,
Otsu, Shiga 520-2192, Japan
e-mail: [email protected]
Y. Sakaue � Y. Takeuchi
Department of Pediatrics, Shiga University of Medical Science,
Seta Tsukinowa-cho, Otsu, Shiga 520-2192, Japan
L. D’Este
Laboratory of Immunohistochemistry Tindaro G. Renda,
Department of Anatomic, Histologic, Forensic and Locomotor
Apparatus Sciences, Sapienza University of Rome,
00161 Rome, Italy
123
Brain Struct Funct
DOI 10.1007/s00429-012-0502-6
A novel form of ChAT has been identified in the rat
(Tooyama and Kimura 2000) and is referred to as
peripheral type ChAT (pChAT) because it is yielded by
alternative splicing occurring mainly in the peripheral
nervous system. The classical form of ChAT was then
renamed as common type ChAT (cChAT) due to its
presence in both the central and peripheral nervous sys-
tems. Antisera to cChAT and pChAT have been raised
against a recombinant peptide specific for each protein. A
peptide encoded by exons 7 and 8 was used as a cChAT-
specific sequence because it is absent from pChAT
(Kimura et al. 2007). Because pChAT is a splice variant of
cChAT, with pChAT mRNA lacking exons 6–9, a pChAT-
specific peptide encoded by alternative splicing between
exons 5 and 10 was used to raise pChAT antiserum
(Tooyama and Kimura 2000). In mammals so far studied,
pChAT is expressed in all known peripheral, but only a
few central, cholinergic neurons (for review, see Bellier
and Kimura 2011). In addition, pChAT also exists in
sensory, reportedly non-cholinergic, neurons of mammals.
Thus, the pChAT antiserum has enabled the immunohis-
tochemical labeling of peripheral cholinergic and sensory
neurons in mammals, which have often been difficult to
stain using cChAT antisera. Beside the above differences
in molecular structure and neuronal localization, pChAT
further varies from cChAT in ChAT activity (Bellier and
Kimura 2007) and nuclear-cytoplasmic trafficking (Matsuo
et al. 2005).
The octopus and other cephalopods such as cuttlefish
and squids have the most advanced nervous networks
among invertebrates. The networks consist of a central
system of nerve cords surrounding the esophagus and a
peripheral system of neural ganglia in the arm, viscera and
mantle (Fraser Rowell 1966; Graziadei 1971; Hochner
et al. 2006; Young 1963a). In octopus, many neurons are
packed into the encephalized brain ganglia, but the eight
arms together contain many more neurons (350 million
cells) that account for 70 % of the total neurons (500
million cells) in the entire nervous system (Young 1963a).
Many indices of ACh neurotransmission seen in verte-
brates have also been detected in cephalopods. These
include ACh content, ChAT activity, acetylcholinesterase
activity, transporter mechanisms, and receptor-mediated
responses (for reviews, see Messenger 1996; Bellier et al.
2012). In octopuses and squids, ACh is present in both the
central brain (Loe and Florey 1966; Welsch and Dettbarn
1972) and the peripheral ganglia (Loe and Florey 1966).
Physiologic studies have shown that the systemic injection
of ACh induces miosis and paling in Sepia (Chichery and
Chanelet 1972; Chichery and Chichery 1985), while in
octopus, it produces mydriasis, paling, respiratory arrest,
and the muscle paralysis of mantle, arm and skin (Andrews
et al. 1981, 1983). Involvement of central cholinergic
control has been proposed in octopus visual transduction
(Bacq and Mazza 1935; Lam et al. 1974; Piscopo et al.
2007), octopus visual learning and memory (Fiorito et al.
1998), cuttlefish brain memory formation (Bellanger et al.
2003, 2005), and cuttlefish predatory behavior (Halm et al.
2002). Peripheral cholinergic innervation has been shown
to occur in octopus arm muscles (Matzner et al. 2000),
squid muscles of the arm, funnel, tentacle retractors and
head retractors (Bone et al. 1982), squid muscles control-
ling the color change of skin (Smotherman 2002) and eye
(Wardill et al. 2012; Hanlon et al. 1990; Mathger et al.
2004), octopus sensory epithelia of the statocyst (Auerbach
and Budelmann 1986; Williamson 1989), octopus digestive
tract (Andrews and Tansey 1983), nautilus pyriform
appendage (Spintzik et al. 2009), and cuttlefish heart
(Gebauer et al. 1999). Our understanding of octopus cho-
linergic nerves has, however, been hampered by the lack of
reliable methods for morphological observation. Several
neuronal cells and fibers in the octopus arm contain ace-
tylcholinesterase (Talesa et al. 1995), but the enzyme is not
a specific cholinergic marker (Eckenstein and Sofroniew
1983).
Recent evidence indicates that the rat cChAT antiserum
described above (Kimura et al. 2007) also binds to a ChAT
protein from octopus (D’Este et al. 2008), thus allowing the
first immunohistochemical description of central choliner-
gic neurons in the octopus optic lobe. The antiserum has
been used to study cChAT-containing neurons and their
processes in other central ganglia of octopus (Casini et al.
2012) and in central ganglia of the terrestrial mollusc Li-
max (D’Este et al. 2011). Using this cChAT antiserum,
here we report the presence of cChAT-positive nerve fibers
in the axial nerve cord of the octopus arm. We also used
pChAT antiserum (Tooyama and Kimura 2000) to allow
the first mapping of the wide distribution of pChAT-posi-
tive neuronal structures and sensory cells in various por-
tions of the arm.
Materials and methods
Animals
Nine live octopus (Octopus vulgaris) specimens weighing
about 150–200 g and collected by fishermen from the
Island Sea of Japan near Akashi (Hyogo Prefecture, Japan)
were delivered within 24 h to our laboratory in individual
plastic bags filled with ice-cold seawater and oxygen gas.
All experimental procedures were designed to minimize
the number of animals and their suffering in accordance
with ‘‘The UFAW handbook on the care and management
of cephalopods in the laboratory’’ (Boyle 1991) and also
with a recent review (Moltschaniwskyj et al. 2007). The
Brain Struct Funct
123
animals were killed after being anesthetized by placement
on crushed ice.
Reagents and antisera
Unless otherwise indicated, all chemical reagents were
purchased from Nacalai Tesque (Kyoto, Japan). Primary
antisera were home-made rabbit anti-rat cChAT (Kimura
et al. 2007) and anti-rat pChAT (Tooyama and Kimura
2000). The specificity of each antiserum has been well
established in rat tissues. In molluscs, the specificity of the
cChAT antiserum has been described previously in octopus
(D’Este et al. 2008) and slug (D’Este et al. 2011).
Western blot
Axial nerve cord tissues were dissected out of the entire
length of the arm, and suckers were collected randomly
from the tip to the base of the arm. Central lobes were also
dissected out of the brain capsule as a cChAT-rich tissue
specimen. These specimens were quickly frozen in liquid
nitrogen and stored at -80 �C until use. The frozen tissues
of either the arm or the sucker were homogenized (10 %
w/v) in 50 mM Tris–HCl (pH 7.4) containing protease-
inhibitor cocktail (P-2714, Sigma, St. Louis, MO) using a
Polytron device. After centrifugation at 12,000g for
20 min, the supernatant was collected and its protein
concentration was determined. Aliquots containing
approximately 20 lg protein and protein molecular weight
marker (BenchMark Pre-stained Protein Ladder, Invitro-
gen, Carlsbad, CA, USA) were processed for 5–20 %
gradient sodium dodecyl sulfate polyacrylamide gel elec-
trophoresis (SDS-PAGE) (Wako Chemicals, Osaka, Japan)
under reducing conditions, as described previously (Bellier
and Kimura 2007). Protein concentration was measured
using a Protein assay kit based on the method of Bradford
(Bio-Rad Laboratories, Tokyo, Japan). Bovine serum
albumin was used as a standard.
In our pilot experiments, we found that a number of
octopus tissue molecules reacted with the secondary anti-
bodies against anti-rabbit IgG on Western blot after SDS-
PAGE. To solve this problem, we labeled primary anti-
bodies with peroxidase using a Peroxidase Labeling Kit-
NH2 (Dojindo Molecular Technologies, Kumamoto,
Japan) according to the manufacturer’s instructions.
Fractions from electrophoresis were transferred to a
polyvinylidene fluoride membrane (Immobilon-P, Milli-
pore Corp., Billerica MA), followed by fixation with a
solution containing 4 % paraformaldehyde in 0.1 M
phosphate buffer (PB, pH 7.4) for 20 min at room tem-
perature. After extensive washing in 0.1 M Tris–HCl buf-
fered saline (pH 7.4, TBS), strips of the membrane were
incubated for 1 h with 10 % skimmed milk in TBS,
followed by incubation overnight at room temperature with
the peroxidase-labeled primary antiserum against pChAT
(diluted 1:2,500) with immunoreaction enhancer solution
(Toyobo, Osaka, Japan). The peroxidase activity was ren-
dered visible by reacting the membrane with the enhanced
chemiluminescence reagent (Chemi-Lumi One Super,
Nacalai Tesque, Kyoto, Japan) and the emitted signals
were counted for 15 min using a Lumino-Image Analyzer
LAS-4000 (Fujifilm, Tokyo, Japan). As a control, a per-
oxidase-labeled pre-immune serum of the rabbit that pro-
duced pChAT antiserum was used.
Immunohistochemistry
To examine the cerebrobrachial tracts and interbrachial
tracts, two neighboring arms were dissected around the
beak between the bases of the arms and the brain. The
tissues were fixed for 24 h by dipping in a fixative con-
taining 4 % paraformaldehyde, 0.2 % picric acid, and
0.1 M phosphate buffer (PB, pH 7.4) at 4 �C. After
washing with PB, the tissues were immersed for at least
24 h in PB containing 15 % sucrose, either embedded or
not embedded in PB containing 10 % gelatin, frozen and
cut into 20-lm thick coronal sections in a cryostat. Free
floating sections were collected and stored to improve tis-
sue permeability for at least 3 days in ice-cold 10 mM
phosphate buffer (pH 7.4) containing 0.9 % NaCl and
0.3 % Triton X-100 (PBST). Prior to cryostat sectioning,
the gelatin-embedded tissues were fixed again with the
same fixative for 24 h and then immersed in PB containing
15 % sucrose for at least 24 h at 4 �C. Immediately before
immunohistochemical processing, glass-mounted sections
of non-gelatin-embedded or gelatin-embedded tissues were
incubated for 30 min at room temperature with PBST
containing 0.1 % sodium azide and 0.5 % hydrogen per-
oxide to inactivate endogenous peroxidase activity (Li
et al. 1987). Non-gelatin-embedded sections treated with or
without proteolytic-induced epitope retrieval and gelatin-
embedded sections without epitope retrieval were both
incubated for 3–4 days at 4 �C with cChAT antiserum
(diluted 1:10,000) or overnight at room temperature with
pChAT antiserum (diluted 1:10,000). These sections were
then incubated at room temperature for 90 min with bio-
tinylated goat anti-rabbit IgG (BA-1000, Vector Labora-
tories, Burlingame, CA; diluted 1:4,000) and for 90 min
with avidin–biotin–peroxidase complex (PK-6100, Vector
Laboratories; diluted 1:4,000). PBST was used for diluting
all the reagents and for washing of sections after each step.
The localization of peroxidase activity was visualized by
treating the sections for 20 min at room temperature with a
solution containing 0.04 % 3,30-diaminobenzidine-4HCl,
0.4 % nickel ammonium sulfate, and 0.003 % H2O2 in
50 mM Tris–HCl buffer (pH 7.6) to yield a dark blue
Brain Struct Funct
123
precipitate. The stained sections were washed with tap
water, counterstained with or without nuclear fast red
(Kernechtrot; Merck, Darmstadt, Germany), dehydrated,
cleared, and coverslipped with Entellan (Merck). For
immunohistochemical controls, we used each pre-immune
serum of the rabbit that produced the cChAT or pChAT
antiserum. For histological verification, sections neigh-
boring immunolabeled sections were stained with crystal
violet (Merck). Digital images obtained with a camera
(Nikon-D90, Tokyo, Japan) attached to a microscope
(BX50, Olympus, Tokyo, Japan) were handled with an
open-source computer software program (Paint.net-3.5.10)
to adjust only for contrast and brightness. All artworks
were created using Inkscape-0.48, an open-source vector
graphics editor.
For proteolytic-induced epitope retrieval, non-gelatin-
embedded cryostat sections were mounted on glass slides,
dried, and then incubated for 5 h with the protease papain
(30 K IU/ml, Merck, Darmstadt, Germany) dissolved in
10 mM phosphate-buffered saline containing 0.3 % Triton
X-100 (pH 7.6) at 40 �C. The enzyme was inactivated by
soaking the mounted sections for 20 min with PBST con-
taining 0.2 N NaOH, followed by extensive washing with
PBST.
Results
Western blot after SDS-PAGE using pChAT antiserum
The pChAT antiserum gave a specific, clearly stained
single band for octopus arm tissues composed mainly of
axial nerve cords (Fig. 1). This band was not detected by
pre-immune serum. Other octopus arm tissues mainly
consisting of the rim and infundibulum of suckers gave
essentially identical results (Fig. 1). The band had a
molecular weight of approximately 62 kDa, which was
significantly smaller than that (approximately 81 kDa) on
octopus central lobes labeled by cChAT antiserum (Fig. 1).
The molecular size of octopus cChAT matched well with
that reported for cChAT from octopus optic lobe (D’Este
et al. 2008). The result supports the idea that the octopus
pChAT may be a short splice variant of octopus cChAT, as
their rat counterparts have been proved to be so.
cChAT immunohistochemistry
We used the descriptions given by Graziadei (1971) and
Kier and Stella (2007) for the anatomical organization and
terminology of the nervous system of the octopus arm. In
most cases, we examined stained arm sections in the
transverse, sagittal, or horizontal plane. Transverse and
sagittal section planes are, respectively, defined as planes
perpendicular and parallel to the longitudinal axis of the
arm, while horizontal sections are cut parallel to the sucker
surface. The inner and outer sides in the axial nerve cord
are referred to as the directions toward and opposite to the
sucker, respectively. Because our results indicated that the
pattern of staining obtained with cChAT antiserum differed
from that with pChAT antiserum, we describe the findings
with each antiserum separately. This description is also
convenient because our epitope retrieval method gave a
dramatic improvement in staining results using pChAT
antiserum, whereas the same method had no effect on
cChAT staining. For this reason, we describe positive
staining for cChAT seen in tissues that were not treated
with protease. Non-neuronal structures or neuronal cell
bodies did not stain for cChAT. When the primary serum
was omitted or substituted with pre-immune serum, no
specific staining was found (data not shown). Such control
studies were particularly important for identifying cChAT-
negative chromatophores that were stained intensely due to
binding of the secondary antibody or the avidin–biotin
complex.
Positive staining for cChAT immunoreactivity was
confined to nerve fibers and terminal-like dots lying in the
Fig. 1 Left panel Western blot using pre-immune control serum (left)or pChAT antiserum (right) on crude extracts of the octopus axial
nerve cord and sucker. Right panel Western blot using cChAT
antiserum on crude extracts of the octopus central lobes. Bars on theleft indicate the position of molecular marker proteins in kDa. pChAT
antiserum recognizes a single band at about 62 kDa, while cChAT
antiserum detects a single band at about 81 kDa. ANC axial nerve
cord
Brain Struct Funct
123
axial nerve cord (cerebrobrachial tracts plus brachial gan-
glia), interbrachial commissure, and brachial nerves.
The cerebrobrachial tract
The axial nerve cord, extending along the center of each
arm to its tip, consists of two dorsal cerebrobrachial tracts
and a chain of ventral brachial ganglia. The pair of
cerebrobrachial tracts is composed of axons projecting to
and from the brain (Graziadei 1971; Gutfreund et al.
2006; Fraser Rowell 1966). cChAT-positive dots were
observed in the cerebrobrachial tract in transverse sections
of the axial nerve cord. Figure 2b shows that relatively
dense clusters of such dots with either large (black
arrowheads) or small (white arrowheads) diameters were
intermingled with unstained small caliber nerve fibers
located near the brachial artery. Such clusters of cChAT-
positive dots were almost always observed near the artery
in different transverse sections of the cerebrobrachial tract
at the level of an interganglionic region or a sucker. In
serial transverse sections, we found that cChAT-positive
fibers run obliquely from the proximal-outer to distal-
inner parts of lateral regions in the cerebrobrachial tract at
the level of a sucker, but run in the midline of the tract
almost vertically at the level of an interganglionic region
between two neighboring suckers. A schematic diagram
deduced from these observations is presented later
(Fig. 13).
In sagittal sections of the cerebrobrachial tract, a number
of cChAT-positive fine fibers with small varicosities ran
longitudinally among many unstained small caliber fibers
in the outer bundles (white arrowheads in Fig. 3b). These
positive fibers occasionally extended branches (arrow in
Fig. 3b) to the neuropil of brachial ganglion (arrowhead in
Fig. 3b). In horizontal sections of the cerebrobrachial tract,
cChAT-positive fine fibers were observed running longi-
tudinally, as expected, most richly at section planes con-
sisting largely of the outer bundles (white arrowheads in
Fig. 4b).
Collectively, the observations in these three planes
indicate that the major bundle of cChAT-positive nerve
fibers runs longitudinally along the brachial artery in the
outer layer of the cerebrobrachial tract. From this major
bundle, a few cChAT-positive fibers appear to bifurcate to
innervate the neuropil of each brachial ganglion. The route
of such a bifurcation within the cerebrobrachial tract differs
depending on the level of the arm with or without the
sucker. Although most of the ramified cChAT-positive
fibers appear to terminate directly within their corre-
sponding ganglion, a few are likely to be involved in short
interconnecting systems between two (or more) neighbor-
ing ganglia. A schematic diagram deduced from these
observations is presented later (Fig. 13).
The brachial ganglion
The brachial ganglia display typical invertebrate organi-
zation, with an internal neuropil surrounded by an outer
cellular layer. The neuropil contains few cell bodies but a
dense network of nerve fibers, while the cellular layer
comprises of some large and many small nerve cells. Many
nerves spread from each ganglion in different directions.
Graziadei (1971) divided them into two groups, termed
recently the ventral and dorsal roots (Gutfreund et al.
2006). The ventral roots project ventrally from the ganglia
to innervate the suckers. The dorsal roots, arising laterally
from the ganglia, are thought to carry motor fibers to the
Fig. 2 cChAT immunohistochemical staining in a transverse section
of the axial nerve cord, counterstained with nuclear fast red. a Low
magnification view. b High magnification view of the cerebrobrachial
tract (boxed area) showing clusters of cChAT-positive large (blackarrowheads) and small (white arrowheads) dots. c High magnification
view of a lateral part of the ganglion neuropil (boxed area) showing
positive dots and fibers aligned vertically. d High magnification view
of a basal part of the ganglion neuropil (boxed area), showing positive
large (black arrowheads) and small (white arrowheads) dots depos-
ited somehow regularly. BA brachial artery, CBT cerebrobrachial
tract, CL cellular layer, NP neuropil. Scale bars: a 100 lm, b 50 lm,
c 25 lm, d 37 lm
Brain Struct Funct
123
intrinsic musculature and to the chromatophores, and sen-
sory fibers from peripheral regions of the arm. In transverse
sections of the axial nerve cord, cChAT-positive dots were
observed in the neuropil of each ganglion, whereas the
cellular layer was completely devoid of staining (Fig. 2a).
These positive dots varied greatly in size and were dis-
tributed richly in lateral sides of the neuropil along the
medial side of the cell layer (Fig. 2c). Clusters of such
positive dots were also situated in the neuropil near the
sucker (Fig. 2d).
In sagittal sections through the lateral sides of the gan-
glion neuropil (Fig. 3a), cChAT-positive dots lying in the
neuropil appeared to align, implying that they were vari-
cose terminals of nerve fibers (Fig. 3c). High power mag-
nification indicated that the varicose nerves (for which
intervaricose fibers were not clearly visible) ran mainly
along the longitudinal axis of the arm and partly in the
lateral-vertical plane of the neuropil (Fig. 3d).
In horizontal sections cut at different vertical levels of the
ganglion neuropil (Fig. 4a), cChAT-positive varicose fibers
were always found in the neuropil with different patterns of
distribution (Figs. 4c–g). As might be anticipated, positive
fibers occurred most densely in sections through the inner
part of the ganglion near the sucker (Fig. 4g). Here, the
varicose fibers tended to form a nerve network, implying the
existence of intense neural transmission. In contrast, in sec-
tions of other vertical planes, positive fibers generally ran
along the longitudinal axis of the ganglion following its
curvature (Fig. 4c–e). Occasionally, such varicose fibers
lying in a ganglion appeared to extend their branches into the
neighboring ganglion, suggesting a role of cChAT-positive
fibers in linkage between ganglia (Fig. 4e, f).
Fig. 3 cChAT
immunohistochemical staining
in a sagittal section of the axial
nerve cord, counterstained with
nuclear fast red. a The dottedline in the transverse section of
a shows the approximate level
of the sagittal sections of b–
d. b cChAT-positive fine fibers
with small varicosities running
longitudinally in the
cerebrobrachial tract (whitearrowheads) and appearing to
extend branches (arrow) toward
the ganglion neuropil (blackarrowheads). c Low
magnification view. d High
magnification view of the boxedarea in c. cChAT-positive dots
and fibers (black arrowheads)
are distributed preferentially in
the neuropil outside the islets
encircled by dotted lines, most
richly in the ventral (inner) part
(white arrowheads). CBTcerebrobrachial tract, CLcellular layer, NP neuropil.
Scale bars: a 195 lm, b 35 lm,
c 160 lm, d 25 lm
Brain Struct Funct
123
Interbrachial commissure and brachial nerves
The interbrachial commissure is a ring of nerves located
at the base of the eight arms. It consists of two roots. One
root, which arises from the cerebrobrachial tract of each
arm, carries axons extending to and from its right and left
neighbor. The second root, which arises from the neuropil
of the first brachial ganglion, contains fibers from each
arm to join the more distant arms (Graziadei 1971). In
horizontal sections of two neighboring arms at the level
of the interbrachial commissure, many cChAT-positive
varicose fibers were seen in both the interbrachial com-
missure (Fig. 5b) and the brachial nerves between the
commissure and central brain ganglia (Fig. 6b–d). Within
the commissure, cChAT-positive fibers were found almost
exclusively in upper commissure bundles forming a
complete ring of nerve fibers near the brain, while very
few in lower commissure bundles (Fig. 5b). In contrast,
within the brachial nerves, cChAT-positive fibers exten-
ded from the brain toward the cerebrobrachial tract and
the first ganglion neuropil, respectively, and tended to run
in the outer (black arrowheads in Fig. 5c, d) and inner
(white arrowheads in Fig. 5c, d) parts of the brachial
nerve.
pChAT immunohistochemistry
As described above, positive staining with pChAT antise-
rum observed differed greatly in sections with or without
papain treatment, we will describe the results separately.
Fig. 4 cChAT
immunohistochemical staining
in a horizontal section of the
axial nerve cord, counterstained
with nuclear fast red. a The
dotted lines in the transverse
section of a show the
approximate level of the
horizontal sections of b–g.
b cChAT-positive fine fibers
with small varicosities (whitearrowheads) are seen to run
longitudinally in outer parts of
the cerebrobrachial tract. c At
the outermost level of the
ganglion neuropil, cChAT-
positive fibers and varicosities
(white arrowheads) are less
dense than those at the inner
level. d At the middle level of
the ganglion neuropil, cChAT-
positive tiny dots (arrowheads)
are seen along the longitudinal
axis of the arm. e Low
magnification of a horizontal
section containing a series of
brachial ganglia. f High
magnification view of the boxedarea in e, showing cChAT-
positive tiny dots in short
interconnecting bundles
between two neighboring
ganglia. g At the ventral most
(innermost) level of the
ganglion neuropil, cChAT-
positive fibers and varicosities
are clustered densely (blackarrowheads) or sprinkled
irregularly (white arrowheads).
CBT cerebrobrachial tract, CLcellular layer, NP neuropil.
Scale bars: a 195 lm, b 45 lm,
c 190 lm, d 40 lm, e 30 lm,
f 70 lm, g 40 lm
Brain Struct Funct
123
pChAT immunohistochemistry without epitope retrieval
Positive staining for pChAT occurred in nerve fibers and
terminal-like dots. Nerve cells and non-neuronal structures
including chromatophores did not stain for pChAT.
Immunohistochemical controls performed similarly to
those described for cChAT gave no specific staining (data
not shown).
The cerebrobrachial tract No positive staining for
pChAT was observed in the cerebrobrachial tract.
Fig. 6 cChAT immunohistochemical staining in the brachial nerve
between the arm and central brain ganglia. a The same diagram as
that shown in Fig. 5. The staining result in the boxed area (B) is
shown in b. b Low magnification view of the brachial nerve. Staining
results in the two boxed areas (C and D) are shown at high
magnification in c and d. c cChAT-positive varicose fibers are seen in
the outer (black arrowheads) and inner (white arrowheads) parts of
the brachial nerve. d At the merger of the brachial nerve, interbrachial
commissure and the first ganglion of the axial nerve cord, positive
fibers are seen in the outer part of the cerebrobrachial tract (blackarrowheads), the inner part of the brachial nerve (white arrowheads),
and the ganglionic neuropil (arrows). BrN brachial nerve, CBTcerebrobrachial tract, NP neuropil. Scale bars: b 450 lm, c 110 lm,
d 70 lm
Fig. 5 cChAT and pChAT
immunohistochemical staining
at the junction between the
brachial nerve and the
interbrachial commissure. a A
schematic diagram showing the
location of the octopus brain,
brachial nerves, brachial ganglia
and interbrachial commissure.
Staining results in the boxedarea are shown at high
magnification in b and c,
respectively. b cChAT-positive
varicose fibers (arrowheads) are
seen in an upper part of the
interbrachial commissure. c No
pChAT staining is seen in the
interbrachial commissure. BrNbrachial nerve, CL cellular
layer, Com Int. interbrachial
commissure. Scale bars: b,
c 50 lm
Brain Struct Funct
123
The brachial ganglion In transverse sections of the axial
nerve cord, pChAT-positive dots were deposited in the
neuropil of each ganglion (Fig. 7a). In the lateral part of the
neuropil, some of these dots were arranged in succession,
implying that they were part of varicose nerve fibers
(arrowheads in Fig. 7b). In the inner part of the neuropil
near the sucker, pChAT-positive dots were sprinkled
unevenly in a pattern similar to that described for cChAT
(arrowheads in Fig. 7c). In sagittal sections of the lateral
part of the neuropil, pChAT-positive varicose fibers ran
longitudinally along both the outer (near the cerebrobra-
chial tract) and inner (near the sucker) parts of the neuropil
(Fig. 8b). In horizontal sections of the outer part of the
neuropil, pChAT-positive varicose fibers running longitu-
dinally connected neighboring ganglia, presumably as part
of the interbrachial inter-connecting system (Fig. 8c).
Interbrachial commissure and brachial nerves No
positive staining for pChAT was observed in the interbra-
chial commissure. However, a few pChAT-positive vari-
cose fibers extended from the first ganglion neuropil toward
the brain in the inner part of the brachial nerve (white
arrowheads in Fig. 9c, e).
pChAT immunohistochemistry after epitope retrieval
Pretreatment of tissue sections with papain digestion
revealed pChAT-positive staining in a surprising number of
structures, including all six nerve centers of the arm and in
presumed sensory cells of the arm. Labeled neuronal cells
were detected in the axial nerve cord, four intramuscular
nerve cords, and ganglia of suckers. Well-stained nerve
fibers were also found in cerebrobrachial tracts, anastomotic
tracts between intramuscular nerve cords, and nerve bun-
dles connecting the brachial ganglion with intrinsic mus-
cles, intramuscular nerve cords, and the sucker. Probable
primary receptor cells containing pChAT were densely
distributed in the sucker and were scattered widely in the
epithelium covering the surface of the arm. Immunohisto-
chemical controls carried out similarly to those described
for cChAT gave no positive staining (data not shown).
The cerebrobrachial tract In transverse arm sections,
pChAT-positive fine dots were found in the dorsal-most
part of the cerebrobrachial tract (Fig. 10a). These dots,
which became detectable after treating tissues with papain,
were too tiny for morphologic analysis (Fig. 10b). How-
ever, in sagittal sections, long pChAT-positive fibers with
tiny varicosities were seen to run longitudinally along the
dorsal-most part of the cerebrobrachial tract (Fig. 10c).
Positive fibers running in the dorsal part of the cerebro-
brachial tract were also found in horizontal sections (data
not shown).
The brachial ganglion In transverse sections of the cell
layer, pChAT immunoreactivity was found in almost all
neuronal cell bodies, in which the reaction products for
pChAT were condensed near the cell surface (Fig. 10a, d).
In contrast, in transverse sections of the neuropil, reaction
products were precipitated densely and diffusely and with
no clearly distinguishable structure, either neural or non-
neural (Fig. 10a, d). A control study using pre-immune
serum suggested that the diffuse-staining products were
artifacts due to non-specific binding of immunoglobulin to
tissue molecules produced by papain digestion (data not
shown).
Interbrachial commissure and brachial nerves A few
pChAT-positive nerve fibers became visible in the inter-
brachial commissure (Fig. 10e) and in the brachial nerve.
Fig. 7 pChAT immunohistochemical staining without papain treat-
ment in a transverse section of the axial nerve cord, counterstained
with nuclear fast red. a Low magnification view. b High magnifica-
tion view of the boxed area in a at a lateral part of the neuropil,
showing that pChAT-positive dots and fibers (arrowheads) are
aligned vertically. c High magnification view of the boxed area in
a at a basal part of the neuropil, showing that positive dots
(arrowheads) are scattered irregularly. CBT cerebrobrachial tract,
CL cellular layer, NP neuropil. Scale bars: a 80 lm, b 35 lm,
c 30 lm
Brain Struct Funct
123
Fig. 9 pChAT immunohistochemical staining without papain treat-
ment in the brachial nerve between the arm and brain ganglia, with no
counterstaining. a The same diagram as that shown in Fig. 5. The
staining result in the boxed area (B) is shown in b. b Low
magnification view of the brachial nerve. Staining results in the twoboxed areas (C and E) are shown at high magnification in c and
e. c pChAT-positive varicose fibers (white arrowheads) are seen in
the inner, but not the outer, part of the brachial nerve. d pChAT-
positive fibers in the inner part of the brachial nerve. e At the merger
of the brachial nerve, interbrachial commissure and first ganglion of
the axial nerve cord, pChAT-positive fibers are present in the
ganglionic neuropil (black arrowheads) and the inner part of the
brachial nerve (white arrowheads). BrN brachial nerve, NP neuropil.
Scale bars: b 340 lm, c 80 lm, d 14 lm, e 60 lm
Fig. 8 pChAT immunohistochemical staining without papain treat-
ment in sagittal and horizontal sections of the axial nerve cord,
counterstained with nuclear fast red. a The dotted lines in the
transverse section of a show the approximate levels of the sagittal
section of b and horizontal section of c. b pChAT-positive fibers with
small varicosities in the outer part (black arrowheads) and inner part
(white arrowheads) of the ganglion neuropil. c pChAT-positive fibers
in the short interconnecting bundle extending longitudinally from a
ganglion neuropil (demarcated by a dotted line) to the neighboring
ganglion. These fibers occur in both the midline (black arrowheads)
and lateral (white arrowheads) parts of the interconnecting bundle.
CBT cerebrobrachial tract, CL cellular layer, NP neuropil. Scale bars:
b and c 60 lm
Brain Struct Funct
123
Intramuscular nerve cords and communication bundles
Four intramuscular nerve cords are situated among the
intrinsic muscles in small canals extending the entire length
of the arm. The structure of these cords resembles that of
the brachial ganglia, with an external cellular layer envel-
oping a neuropil core. The cellular layer is assumed to
involve motor, sensory, and interneurons. The anastomotic
tracts, which connect intramuscular nerves cords together,
are made up of nerve fiber bundles around which multi-
polar, presumably of muscle receptor, neurons lie. The
intramuscular nerve cords are connected to the axial nerve
cord via segmental thick bundles of the dorsal roots
(Graziadei 1971; Gutfreund et al. 2006).
In transverse sections of the arm (Fig. 11a), pChAT-
positive nerve bundles extended from each ganglion of the
axial nerve cord towards the four intramuscular nerve cords
lying in the dorsal (i.e. to the outer side) and lateral edges
of arm muscles. We refer to these bundles as the dorsal
roots (Fig. 11b, d). Within the intramuscular nerve cord,
almost all ganglionic cells were positive for pChAT (white
arrowheads in Fig. 11c). In these ganglia, we often
observed pChAT-positive small roots extending into the
longitudinal and transverse muscles (black arrowheads in
Fig. 11c). Although pChAT-positive smooth fibers also
extended from ganglia of the intramuscular nerve cord
towards the oblique muscle array (black arrowhead in
Fig. 11b), we could trace these only for a short distance,
and the exact termination sites remain uncertain. pChAT-
positive nerve fibers were seen in the communication
bundles between neighboring intramuscular nerve cords
(Fig. 11d, e), which probably correspond to the anasto-
motic tracts identified by Graziadei (1965). Observations in
horizontal sections helped to reveal the patterns of the
pChAT-positive structures of the intramuscular nerve cord
and their associated communication bundles.
Ganglion of the sucker and ventral roots of the axial
nerve cord The ganglion of the sucker is a small cluster of
nerve cells embedded among connective tissue and
peduncular muscles below the acetabular cup of each
sucker. This ganglion exhibits a unique structure. A cel-
lular layer of neurons surrounds the neuropil in the core.
This complex is then encircled by a crown-like bundle of
nerve fibers distributed like one of the planetary rings of
Saturn. Although the significance of such a complicated
structure is unknown, morphological studies indicate that
the ganglion contain motor neurons for muscles of the
peduncle and sucker, and receives axons from sensory
receptors (Graziadei 1971).
Figure 11a also shows pChAT-positive nerve bundles
that extended ventrally (i.e., to the inner sucker side) from
the axial nerve cord. We refer to these bundles as the
ventral roots. In sagittal sections, ventral roots containing
many pChAT-positive fibers were clearly seen to extend
toward the suckers and ganglia (Fig. 11f). Examination of
horizontal sections of the peduncle of the sucker indicated
that the sucker ganglia contain many pChAT-positive cell
bodies (white arrowhead in Fig. 11g). These cell bodies
were surrounded by a crown-like peripheral ring consisting
of pChAT-positive ventral root fibers (Fig. 11g).
Epidermis, dermal connective tissue and chromato-
phores In transverse, sagittal or horizontal sections, bun-
dles of pChAT-positive fibers were distributed irregularly
throughout the dermal connective tissue beneath the epi-
dermis. An example of these positive fibers is shown in a
transverse section, where pChAT-positive fine and smooth
fibers seemed to terminate on pChAT-negative epidermal
epithelial cells (white arrowhead in Fig. 12a). Such
pChAT-positive fibers often ran spirally (Fig. 12b).
Rim, muscle and superficial epithelium of the sucker In
the sucker and its associated structures, pChAT immuno-
reactivity occurred in cell bodies and fibers. These positive
structures were best seen in sagittal sections (Fig. 12c, d).
Most, if not all, fusiform cells in the rim and infundibulum
Fig. 10 pChAT immunohistochemical staining in a papain-pretreated
transverse section of the axial nerve cord. a Low magnification view.
b High magnification view of a transverse section of the cerebrobra-
chial tract, showing abundant pChAT-positive dots. c High magni-
fication view of a sagittal section of the cerebrobrachial tract, showing
that pChAT-positive fibers (white arrowheads) run longitudinally
within the tract. d High magnification view of a transverse section at
the boundary between the neuropil and the cellular layer. In the
cellular layer, pChAT-positive cells are recognized by deposition of
reaction products in the cytoplasm near the cell surface (whitearrowheads). e pChAT-positive fibers (arrowheads) are seen in the
interbrachial commissure. CBT cerebrobrachial tract, CL cellular
layer, Com Int. interbrachial commissure, NP neuropil. Scale bars:
a 80 lm, b 50 lm, c 65 lm, d 25 lm, e 50 lm
Brain Struct Funct
123
of the sucker were stained intensely for pChAT (arrow in
Fig. 12d and white arrowhead in Fig. 12f). In connective
tissues near the intrinsic sucker muscle and in muscles of
the sucker, many cells appeared to contain pChAT (white
arrowheads in Fig. 12c, d). These positive cells were often
seen to emit bipolar processes: one directed to join the
pChAT-positive fiber bundles of the ventral root (black
arrowheads in Fig. 12c) and the other targeted toward
pChAT-positive fusiform cells in the rim of the sucker
(black arrowheads in Fig. 12d, f).
Summary of immunohistochemical results
Distribution of neuronal cells and fibers immunoreactive
for pChAT (with or without epitope retrieval using papain)
and cChAT in the arm is presented in Table 1.
c
Brain Struct Funct
123
Discussion
Presence of two cholinergic marker candidates, cChAT
and pChAT, in octopus arm
ACh was first isolated from the optic lobe of the octopus
brain and identified chemically (Bacq and Mazza 1935).
Despite its long history, knowledge of the anatomy and
morphology of octopus cholinergic structures has been
limited to histochemical studies of acetylcholinesterase, the
enzyme that degrades ACh (Loe and Florey 1966; Talesa
et al. 1995, 1998). However, this enzyme is not a reliable
cholinergic marker, as mentioned above. The recent appli-
cation of rabbit antiserum against rat cChAT (Kimura et al.
2007) has enabled immunohistochemical analysis of the
octopus optic lobe (D’Este et al. 2008) and central brain
lobes (Casini et al. 2012). The cross-reactive specificity of
rat cChAT antiserum for octopus cChAT has been verified
by Western blot and by blue-native PAGE combined with
an enzyme activity assay for ChAT (D’Este et al. 2008).
The cChAT antiserum has further been proved to cross-
react with cChAT of the terrestrial slug Limax (D’Este et al.
2011). Since the cChAT antiserum was raised against a
peptide sequence encoded by exons 7–8 of the rat cChAT
gene, it is likely that these marine and terrestrial molluscs
possess a gene structure that is at least partly similar to that
of rat cChAT. This assumption may be useful in searching
for molluscan cChAT genes and transcripts, for there is
currently no published data. Regardless, the present study
shows that the cChAT antiserum is useful for investigation
of cChAT-containing cholinergic nerves in the octopus arm.
In this study, we also provided the first evidence of
pChAT-containing structures in octopus tissues. The rat
pChAT antiserum used (Tooyama and Kimura 2000) has
been extensively characterized in rodents (Chiocchetti
Fig. 11 pChAT-immunohistochemical staining in papain-pretreated
sections of the arm. a Low magnification view of a transverse section
of the whole arm. The two dotted lines (F and G) show the
approximate angles of arm sections cut as shown in f and g,
respectively. b High magnification view of a transverse section in a
dorsal (outer) part of the arm, showing that pChAT-positive fibers
form a part of the dorsal root (black arrowhead) that spans between
two pChAT-positive structures: the axial nerve cord and the
intramuscular nerve cord. c High magnification view of a transverse
arm section showing pChAT-positive cells (white arrowhead) in the
intramuscular nerve cord, from which pChAT-positive fine and
smooth fibers (black arrowhead) appear to extend towards the
transverse and longitudinal muscles. d A transverse arm section in a
ventrolateral part of the arm, showing that pChAT-positive fibers
extend from the axial nerve cord to join the dorsal root (blackarrowheads) and the ventral root (arrow). The pChAT-positive fibers
in the dorsal root appear to form a neural circuit with pChAT-positive
fibers in the anastomotic tract via pChAT-positive cells in the
intramuscular nerve cord. e High magnification view of a transverse
arm section showing pChAT-positive staining in the anastomotic tract
(white arrowhead). A pChAT-positive thin and smooth fiber (blackarrowhead) is seen toward the muscle layer. f A sagittal arm section
showing the projection pathway of pChAT-positive fibers in the
ventral root, spanning between the axial nerve cord and the subdermal
connective tissue of the sucker. g Horizontal section (cut parallel to
the surface of suckers) of the peduncle of the sucker in the arm,
showing pChAT-positive staining in ganglion cell bodies of the
sucker (white arrowhead), fibers of the ventral root (arrow), and
connecting fibers (black arrowhead) between the ganglion of the
sucker and the ventral root. ANC axial nerve cord, AT anastomotic
tract, DR dorsal root, INC intramuscular nerve cord, GS ganglion of
the sucker, Su sucker, VR ventral root. Scale bars: a 340 lm,
b 125 lm, c 50 lm, d 125 lm, e 50 lm, f 100 lm, g 200 lm
b
Fig. 12 pChAT-immunohistochemical staining in papain-pretreated
sections of the skin and sucker of the arm. a A transverse arm section
showing a thick bundle of pChAT-positive fibers (black arrowhead)
in the connective tissue beneath the skin slightly distant from the
sucker. A thin bundle of pChAT-positive fibers running spirally
(white arrowhead) is also seen. The dotted line indicates the boundary
between the dermis and subdermis. b High magnification view of the
thin bundle of pChAT-positive fibers terminating in the boundary
between the dermis and subdermis, as shown in a. c A transverse
section of the sucker showing pChAT-positive cells (white arrow-heads) and their probable processes (black arrowheads) in subdermal
connective tissue of the sucker. d A transverse section of the sucker at
the level of radial muscles, showing a pChAT-positive cell (whitearrowhead) and its bipolar processes that extend centrifugally to
pChAT-positive epithelia of the infundibulum of the sucker (blackarrowheads) or centripetally to pChAT-positive fibers in the ventral
root. Arrows indicate pChAT-positive epithelial cells, likely with
receptors. e High magnification view of a pChAT-positive bipolar cell
in radial muscles of the sucker. f High magnification view of pChAT-
positive epithelial cells (white arrowheads) in the sucker rim. Fine
pChAT-positive fibers (black arrowhead) are seen close to the base of
the pChAT-positive cells, although the origin of these fibers is
unclear. Scale bars: a 40 lm, b 10 lm, c 80 lm, d 60 lm, e 12 lm,
f 8 lm
Brain Struct Funct
123
et al. 2003; Kimura et al. 2007; Matsuo et al. 2005;
Yasuhara et al. 2003), but not in octopus. Since the pChAT
antiserum gave intense immunohistochemical staining in
octopus tissues in a pilot study, we tested the immuno-
chemical specificity of this antiserum. The single protein
band (62 kDa) detected by Western blot using octopus
axial nerve cord and sucker extracts was smaller than that
(81 kDa) reported for octopus cChAT (D’Este et al. 2008)
and confirmed in the present study. The enzymatic prop-
erties of the pChAT-immunoreactive protein band remain
to be determined.
As shown in the schematic diagram in Fig. 13, the
pattern of immunohistochemical staining for cChAT
reported in this study differs clearly from that for pChAT,
suggesting that the octopus arm receives two distinct
cholinergic innervations: cChAT is likely to be involved in
the cholinergic supply from the brain, while pChAT may
play roles in the intrinsic cholinergic network of the arm.
All cChAT-positive fibers in the arm are likely to be
derived from brain
We found no cChAT-positive cell bodies in the octopus
arm. However, cChAT-positive varicose nerve fibers
occurred in the brachial commissure, cerebrobrachial tract,
ganglion neuropil of the axial nerve cord, and connections
between neighboring ganglia of the axial nerve cord. In the
brachial commissure, the cChAT-positive fibers were
located exclusively in an upper zone of the commissure
close to the brain. Since nerve fibers of the brachial com-
missure are known to run in both directions between the
brain and the arm, the cChAT-positive fibers we observed
may represent either afferent or efferent fibers with respect
to the brain. Because no cChAT-positive cells seem to exist
within the arm, and because there are abundant cChAT-
positive cells in various brain regions (Casini et al. 2012),
these results strongly suggest that cChAT-positive fibers
extend from the brain to the arm. The lack of cChAT-
containing cells in the arm is supported by the finding that
few or no cChAT-positive fibers occur in the lower zone of
the brachial commissure, which is composed of cerebro-
brachial nerve fibers from one arm to a neighboring arm
(Graziadei 1971).
Previous studies using tracing methods indicate that
neurons in various lobes of the brain project axons to the
arm, including the superior buccal, posterior buccal, sub-
vertical, brachial, chromatophore, and magnocellular lobes
(Budelmann and Young 1985, 1987; Young 1963b).
Interestingly, all of these ganglia contain cChAT-positive
neuronal cells (Casini et al. 2012). To date, however, the
brain origins of cChAT-positive nerves to the arm have not
been determined. However, centrifugal cChAT-positive
fibers in brachial nerves appear to join the brachial com-
missure, run through the cerebrobrachial tract, and fre-
quently extend branches to terminate in the neuropil of one
or more ganglia of the axial nerve cord.
pChAT in octopus arm
pChAT immunohistochemistry with or without epitope
retrieval
To improve cChAT and pChAT immunostaining, we tested
several methods for epitope retrieval, including use of heat
(using a water-bath or autoclave) and proteases (proteinase
K, trypsin, chymotrypsin, and papain). None of these
methods affected the results for cChAT immunohisto-
chemistry. In contrast, all of the techniques improved
Table 1 Distribution of structures immunoreactive for cChAT and
pChAT in the arm of octopus
Octopus regions
examined
Structures, if
stained positively
cChAT pChAT
No
papain
After
papain
Cerebrobrachial
tract
Nerve fibers ? - ?
Brachial ganglion
Cellular layer Nerve cells - - ?
Neuropil Nerve fibers ? ? n.d.
Dorsal roots Nerve fibers - - ?
Ventral roots Nerve fibers - - ?
Interbrachial
commissure
Nerve fibers ? - ?
Brachial nerve Nerve fibers ? ? ?
Intramuscular nerve cords
Cellular layer Nerve cells - - ?
Neuropil Nerve fibers - - ?
Anastomic tracts
Cellular layer Nerve cells - - ?
Fiber bundle Nerve fibers - - ?
Muscular layer Nerve fibers - - ?
Ganglion of the sucker
Ganglion Nerve cells - - ?
Fiber bundle Nerve fibers - - ?
Connective tissue
of the skin
Nerve fibers - - ?
Epithelium of the
skin
Epithelial
(sensory)
cells
- - ?
Chromatophore Pigment cells n.d. n.d. -
Epithelium of the
sucker
Epithelial
(sensory)
cells
- - ?
No papain, without epitope retrieval; after papain, following epitope
retrieval; ?, present; -, absent
n.d. not determinated due to non-specific staining
Brain Struct Funct
123
quality and quantity of staining for pChAT immunohisto-
chemistry, and treatment with papain gave the best and
most stable outcome. We did not examine the reason for
the efficacy of papain, but it has been reported that prote-
ases may cleave peptides that mask the antibody epitope
(Leong and Leong 2007). Following papain treatment,
pChAT-positive nerve fibers that were already detectable
without the treatment were stained more intensely. In
addition, papain treatment permitted observation of many
pChAT-positive structures that were invisible without the
treatment in selective regions in the octopus arm. For
reasons yet unknown, however, there was a disadvantage in
that the papain treatment brought about non-specific
staining in the neuropil of the brachial ganglion as
mentioned earlier. We were therefore unable to trace which
pChAT-positive neurons in the arm project to the neuropil.
Although other epitope retrieval methods tested did not
give such a non-specific staining, they often failed to
clearly label the pChAT-positive cells and fibers that
became detectable by the papain treatment.
pChAT-positive cells and fibers are essentially intrinsic
to the arm
The most prominent effect of papain treatment was the
ability to visualize pChAT-positive cells and some asso-
ciated fibers. These included neuronal cells in all of the six
main nerve centers containing motor neurons for the arm,
Fig. 13 Schematic diagrams showing the patterns of cChAT- and
pChAT-positive immunoreactivity in the nervous system of the
octopus arm. a cChAT-immunoreactivity is mainly located in nerve
fibers running longitudinally through the cerebrobrachial tract,
particularly in its outer layer (arrows). These cChAT-positive fibers
give off branches ventrally to terminate largely in lateral parts of the
neuropil of the ganglion at the level of the sucker (black arrowhead),
while at the level of the interganglionic region only a few cChAT-
positive collaterals are bifurcated toward the midline of the neuropil
(white arrowhead). b Distribution and projection patterns of cChAT-
positive fibers compared with those of pChAT-positive fibers in the
longitudinal aspect of the axial nerve cord. The ganglion neuropil
contains both cChAT- and pChAT-positive nerve fibers and terminals.
In the cerebrobrachial tract, cChAT-positive fibers run along the outer
layer, whereas pChAT fibers run along the inner part. c A schematic
drawing showing pChAT-positive neuronal cells, fibers and terminals
(labeled in red) in a transverse section of the octopus arm. 1 Cells and
fibers in the intramuscular nerve cord. 2 Cells and fibers in the
anastomotic tract. 3 Cells in the ganglion of the sucker. 4 Cells in the
connective tissue of the sucker. 5 Cells in muscles of the sucker. 6Cells in epithelia of the sucker. 7 Fibers in the dorsal root. 8 Fibers in
the ventral root. 9 Fibers in connective tissues of the mantle skin. 10Terminals in the transverse muscles. 11 Terminals in the longitudinal
muscles. 12 Terminals in the dermis. The solid gray area represents
the median oblique muscle array, which lacks innervation by pChAT
terminals. CBT cerebrobrachial tract, CL cellular layer, NP neuropil
Brain Struct Funct
123
and sensory cells densely packed in the epithelium of the
sucker. Both the pChAT-positive neuronal and sensory
cells are therefore likely to be involved in sensory-motor
control of the arm. Although papain treatment increased
pChAT-positive fibers in both number and staining inten-
sity, only a few were found in arm nerve tracts to and from
the brain. This finding indicates that pChAT-immunore-
active molecules are probably intrinsic to the arm.
pChAT-positive neuronal cells in nerve centers
Every ganglion in the axial nerve cord, the four intra-
muscular nerve cords, and the sucker contained pChAT-
positive neuronal cells. As most, if not all, cells of various
sizes stained positive, pChAT-labeled cells appear to par-
ticipate in a variety of functions performed by these cen-
ters. Among these, large cells in the cellular layer of the
axial nerve cord are thought to be motor neurons for
muscles of the sucker and the arm itself (Graziadei 1971).
Therefore, it is possible that the large pChAT-positive cells
are involved in a motor control mechanism. Some pChAT-
positive cells may also be motor neurons in the four
intramuscular nerve cords and ganglia of the suckers.
Indeed, axons emitted from pChAT-positive cells can
occasionally be traced to innervate muscles of the sucker
and some of the intrinsic musculature of the arm. The
distribution pattern of these pChAT-positive fibers partly
resembles that reported for acetylcholinesterase (Talesa
et al. 1995).
At present, the roles of pChAT-immunoreactive octopus
molecules are unknown, while rat pChAT has been proved
to possess ChAT activity. Given that pChAT-immunore-
active octopus molecules also have ChAT activity, ACh
transmission may occur in pChAT-positive nerves. How-
ever, whether ACh is a neuromuscular neurotransmitter in
invertebrates is still a matter of debate. Glutamate has been
shown to be such a neurotransmitter in insect (Usherwood
et al. 1968; Clements and May 1974) and crustacea
(Thieffry and Bruner 1978), while ACh has been proposed
to have a similar role in crustacea (Futamachi 1972). In
molluscs, glutamate is accepted as a nerve-muscle trans-
mitter (Kerkut et al. 1965), and Bone et al. (1982) have
provided evidence that ACh and glutamate act separately to
contract muscles of different types. The data reported here
support the latter view, in that pChAT-positive fibers are
present in the longitudinal and transversal muscle groups,
but absent from the oblique muscle group (except for
pChAT-positive fiber bundles cutting across this group).
This observation is important for future studies of the
pharmacologic responses of ACh and glutamate in these
muscle groups. The presence of two transmitters used
separately in each muscle group of the arm may ensure
controlled movements that are performed by coordination
of two neighboring groups of muscles without a bony or
cartilaginous skeleton (Kier 1988; Messenger 1996).
Because most cells are stained positive for pChAT in the
intramuscular nerve cord, and because the cord is known to
contain not only motor neurons but also sensory neurons
acting as muscle receptors, we cannot rule out the possi-
bility that some pChAT neurons in the cord reflect the
presence of such receptors. The presence of stained pro-
cesses extending from pChAT-positive cells in the intra-
muscular nerve cord supports this possibility. These
processes run together to form a part of the anastomotic
tract towards the oblique muscles, and the morphologic
feature closely resembles that described for muscle recep-
tors (Alexandrowicz 1960; Graziadei 1971).
The presence of pChAT-positive smooth fibers scattered
throughout the connective tissue beneath the skin outside
the suckers suggests two possible roles for these fibers. One
possibility is that they are sensory-free nerve endings, since
their fine processes are often apposed to the epithelium
covering the surface of the arm. Because no pChAT-posi-
tive cells were found in the epithelium of the skin, except
the suckers, the pChAT-positive fibers are not the cen-
tripetal processes of epidermal cells, but are likely to be
afferents derived via the dorsal roots from the ganglia of
the axial nerve cord. The exact function of such sensory
endings is unknown. Another possibility is that the pChAT-
positive fibers may represent efferent motor nerves to
chromatophores. Such motor nerves are thought to be
supplied from the ganglia of the axial nerve cord (Graz-
iadei 1971; Gutfreund et al. 2006). Activation of chro-
matophores is induced by intravascular injection of ACh
(Andrews et al. 1981), while L-glutamate (Florey et al.
1985) and both L-glutamate and serotonin (Messenger et al.
1997) are neurotransmitters of motor efferents to chro-
matophore muscles. The contribution of pChAT-positive
nerves in these motor neurons remains to be determined.
pChAT-positive sensory cells in the arm
Numerous pChAT-positive epithelial cells were observed
in the rim and infundibulum of the suckers. Because these
cells have only short processes, they appear to correspond
to type T2a and T4 receptor cells (Graziadei and Gagne
1976). These two types of cells relay sensory signals via
axons of encapsulated nerve cells to the axial nerve cord
(Graziadei 1964, 1965). Whether such encapsulated nerve
cells contain pChAT remains to be elucidated.
Possible presence of two types of ChAT in the octopus arm
Physiological and biochemical evidence clearly indicates
that cholinergic innervation occurs in the octopus brain and
arm, but little is known about the precise distribution of
Brain Struct Funct
123
cholinergic structures and there is no good histochemical
technique for detecting ACh. In vertebrates, two molecules
have been widely used as markers of cholinergic nerves:
ChAT and the vesicular acetylcholine transporter, which is
responsible for loading ACh into synaptic vesicles. The
antibody against this transporter has been used in many
studies, but these are limited to a few mammals due to little
cross-reactivity with its counterpart in other animals,
including invertebrates.
As mentioned above, recent applications of cChAT
antiserum for immunohistochemical labeling of choliner-
gic elements in the optic lobe (D’Este et al. 2008) and
various brain ganglia of octopus (Casini et al. 2012)
prompted us to conduct a similar study in the octopus arm.
Again, it should be noted that the octopus 81-kDa protein
recognized by the cChAT antiserum is capable of pro-
ducing ACh. To our surprise, cChAT-positive nerve fibers
had a far lower density and distribution than we expected
from previous physiologic data showing an association of
cholinergic roles in sensory-motor nerve regulation in
regions of the arm. Therefore, we used the antiserum
against rat pChAT for immunohistochemistry to screen the
octopus arm. The results in this study are interesting in
that pChAT-positive reaction products in both neuronal
and sensory cells and fibers were distributed abundantly
throughout various regions of the arm. Such wide and
dense distributions of pChAT-positive staining are in
accordance with the physiological results mentioned
above. However, it is unclear whether octopus proteins
recognized by pChAT antiserum possess enzyme activity
to synthesize ACh. Studies in cephalopods have described
the properties of ChAT purified from squid ganglia (Prince
1967; Husain and Mautner 1973), but nothing is known
about the structure of cChAT and pChAT at the protein
and mRNA levels. To address this question, the suckers of
octopus arm may be promising material for molecular
biology analysis, since they contain intense immunoreac-
tivity for pChAT but not for cChAT.
Conclusion
The present study first shows the localization in the octopus
arm of nerve fibers containing octopus cChAT, the bio-
synthetic enzyme for ACh. Since no cChAT-positive cells
were found in the arm, cChAT-positive fibers appear to be
supplied from the brain. The pattern of distribution sug-
gests that cChAT-positive nerves serve as a part of the
brain control limited to nerve centers within the arm. We
also showed the presence of neuronal or sensory cells and
fibers immunoreactive for antiserum against rat pChAT.
These positive structures appear to be largely intrinsic to
the arm with few fibers connecting to and from the brain.
Whether pChAT-immunoreactive molecules possess ACh
biosynthetic activity remains to be determined.
Acknowledgments The authors thank the late Professor Tindaro G.
Renda (University la Sapienza, Roma) for performance of preliminary
immunohistochemical staining using pChAT antiserum in the octopus
arm. J-P. B was supported by a Grant-in-Aid for KAKENHI from the
Japan Society for the Promotion of Science (No. 24592334).
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