ArticlePDF Available

Abstract and Figures

Cephalopod molluscs are the most neurally and behaviorally complex invertebrates, with brains rivaling those of some vertebrates in size and complexity. This has fostered the opinion that cephalopods, particularly octopuses, may experience vertebrate-like pain when injured. However, it is not known whether octopuses possess nociceptors or if their somatic sensory neurons exhibit sensitization after injury. Here we show that the octopus Abdopus aculeatus expresses nocifensive behaviors including arm autotomy, and displays marked neural hyperexcitability both in injured and uninjured arms for at least 24h after injury. These findings do not demonstrate that octopuses experience pain-like states; instead they add to the minimal existing literature on how cephalopods receive, process, and integrate noxious sensory information, potentially informing and refining regulations governing use of cephalopods in scientific research.
Content may be subject to copyright.
Neuroscience
Letters
558 (2014) 137–
142
Contents
lists
available
at
ScienceDirect
Neuroscience
Letters
jou
rn
al
hom
epage:
www.elsevier.com/locate/neulet
Arm
injury
produces
long-term
behavioral
and
neural
hypersensitivity
in
octopus
Jean
S.
Alupaya,
Stavros
P.
Hadjisolomoub,
Robyn
J.
Crookc,d,
aDepartment
of
Integrative
Biology,
University
of
California,
Berkeley,
CA,
United
States
bDepartment
of
Psychology,
Brooklyn
College
of
the
City
University
of
New
York,
Brooklyn,
NY,
United
States
cDepartment
of
Integrative
Biology
and
Pharmacology,
University
of
Texas
Medical
School,
Houston,
TX,
United
States
dProgram
in
Sensory
Physiology
and
Behavior,
Marine
Biological
Laboratory,
Woods
Hole,
MA,
United
States
h
i
g
h
l
i
g
h
t
s
Arm
injury
evokes
hypersensitivity
to
touch
and
wound-directed
protective
behavior.
Low-threshold
and
nociceptive
mechanosensory
neurons
sensitize
after
injury.
Pattern
of
acute
(5
min)
and
persistent
(24
h)
neuronal
hyperexcitability
is
similar.
Distinction
between
pain
and
nociception
in
octopus
is
more
complex
than
in
squid.
a
r
t
i
c
l
e
i
n
f
o
Article
history:
Received
7
September
2013
Received
in
revised
form
29
October
2013
Accepted
1
November
2013
Keywords:
Autotomy
Cephalopod
Nociception
Octopus
Pain
Sensitization
a
b
s
t
r
a
c
t
Cephalopod
molluscs
are
the
most
neurally
and
behaviorally
complex
invertebrates,
with
brains
rivaling
those
of
some
vertebrates
in
size
and
complexity.
This
has
fostered
the
opinion
that
cephalopods,
partic-
ularly
octopuses,
may
experience
vertebrate-like
pain
when
injured.
However,
it
is
not
known
whether
octopuses
possess
nociceptors
or
if
their
somatic
sensory
neurons
exhibit
sensitization
after
injury.
Here
we
show
that
the
octopus
Abdopus
aculeatus
expresses
nocifensive
behaviors
including
arm
autotomy,
and
displays
marked
neural
hyperexcitability
both
in
injured
and
uninjured
arms
for
at
least
24
h
after
injury.
These
findings
do
not
demonstrate
that
octopuses
experience
pain-like
states;
instead
they
add
to
the
minimal
existing
literature
on
how
cephalopods
receive,
process,
and
integrate
noxious
sensory
information,
potentially
informing
and
refining
regulations
governing
use
of
cephalopods
in
scientific
research.
Published by Elsevier Ireland Ltd.
1.
Introduction
Most
animals
are
capable
of
detection
and
reflexive
avoidance
of
noxious
or
damaging
stimuli
(‘nociception’),
but
historically
only
vertebrate
animals
have
been
considered
to
experience
pain,
defined
by
the
International
Association
for
the
Study
of
Pain
as
‘the
unpleasant
sensory
and
emotional
experience
associated
with
actual
or
potential
tissue
damage’
[1].
While
nociceptors
have
been
described
in
many
invertebrates
[2],
and
there
have
been
some
attempts
to
demonstrate
pain-like
states
in
crustaceans
[3],
ques-
tions
remain
over
the
analogy
of
putative
affective
states
in
animals
with
vastly
different
brains
and
behaviors
[4],
and
the
validity
Corresponding
author
at:
Department
of
Integrative
Biology
and
Pharmacology,
University
of
Texas
Medical
School,
6431
Fannin
Street,
Houston,
TX
77030,
United
States.
Tel.:
+1
2812540588.
E-mail
address:
Robyn.crook@uth.tmc.edu
(R.J.
Crook).
of
anthropocentric
measures
of
pain
and
distress
in
invertebrates
[5,6].
Octopuses
have
the
most
complex
nervous
system
among
invertebrates
[7].
Speculative
connections
drawn
between
octopus
‘intelligence’
and
pain
[8],
have
been
used
to
support
increased
reg-
ulation
of
cephalopod-based
research
in
multiple
countries
[9,10].
However,
until
recently
there
has
been
no
concerted
effort
to
examine
nociception
or
pain
in
any
cephalopod.
Nociceptors
and
injury-induced
sensitization
have
been
described
in
squid
(Dory-
teuthis
pealeii)
[11,12],
but
there
is
no
comparable
evidence
for
octopuses
and
none
for
affective
states
resembling
pain
experience
in
any
cephalopod
[5,10].
Here
we
address
several
basic
questions
about
how
octopuses
(Abdopus
aculeatus)
respond
to
noxious
stimulation.
We
asked
whether
injury
to
one
arm
induces
behavioral
or
neural
hyper-
sensitivity,
whether
changes
were
spatially
restricted
to
injury
or
generalized
across
the
body,
and
if
octopuses
express
ongoing
attention
to
wounded
areas
or
long-term
changes
to
motivational
or
spontaneous
behaviors.
0304-3940/$
see
front
matter.
Published by Elsevier Ireland Ltd.
http://dx.doi.org/10.1016/j.neulet.2013.11.002
138 J.S.
Alupay
et
al.
/
Neuroscience
Letters
558 (2014) 137–
142
Fig.
1.
Abdopus
aculeatus.
(A)
Arms
are
numbered
L
(left),
R
(right),
1–4
from
front
(anterior)
to
rear
(posterior).
Zigzag,
location
of
arm
crush;
white
line,
plane
of
autotomy.
Numbered
circles
show
order
of
stimulation
for
behavior
testing.
(B)
Arm
section
showing
axial
nerve
cord.
AT,
axonal
tract;
AG,
axial
ganglion;
CM,
circular
muscle;
HM,
helical
muscle;
LM,
longitudinal
muscle;
S,
sucker.
(C)
Mantle
section
showing
peripheral
stellar
nerves
(SN)
converging
on
the
stellate
ganglion
(SG).
MM,
mantle
muscle;
BV,
blood
vessel.
Unlike
the
axial
nerve
cord
that
contains
peripheral
ganglia,
SNs
in
the
mantle
contain
only
axons.
Arrows
show
aboral
(AbO)
and
oral
(Or)
surfaces,
orientation
for
both
sections.
Schematics
show
section
location
and
plane.
Mallory’s
triple
stain:
muscle,
deep
pink;
connective
tissue,
blue;
nervous
tissue,
light
pink/violet.
Scale
bars
1000
m.
(For
interpretation
of
the
references
to
color
in
figure
legend,
the
reader
is
referred
to
the
web
version
of
the
article.)
A.
aculeatus
autotomizes
arms
as
a
defensive
tactic
[13],
and
many
octopus
species
regenerate
arms
lost
due
to
prey
defenses
or
predatory
attacks
[14,15].
Arm
amputation
was
a
standard
method
of
tissue
harvest
in
earlier
studies
of
physiology
[16,17].
We
chose
to
utilize
a
species
that
readily
autotomizes
arms
to
examine
whether
arm
loss,
either
via
autotomy
or
amputation,
induces
changes
to
behavior
or
physiology.
2.
Methods
Animals.
Nine
adult
A.
aculeatus
(4
F,
5
M,
mantle
length
220–480
mm)
from
a
commercial
vendor
(LiveAquaria)
were
housed
for
up
to
three
weeks
in
individual
20
L
tanks
in
flow-
through
seawater
(SW)
at
24–26 C,
and
fed
daily
on
grass
shrimp
(Palaemon
spp.).
Ethical
considerations.
IACUC
protocols
are
not
issued
for
inver-
tebrate
research
in
the
USA.
Anesthesia
was
given
where
it
did
not
conflict
with
study
aims,
and
animals
that
received
noxious
stim-
ulation
were
monitored
for
signs
of
ill
health
such
as
autophagy,
poor
surface
suction
and
persistently
elevated
respiration,
none
of
which
were
observed.
Two
animals
showing
evidence
for
incom-
plete
decerebration
(see
below)
were
euthanized.
Sample
size
was
limited
to
that
needed
to
demonstrate
large
effects
and
experi-
ments
terminated
at
24
h
after
injury,
precluding
assessment
of
a
longer
time-course
of
effects.
Behavioral
tests
of
sensory
threshold.
An
octopus
was
moved
to
a
15
L
experimental
tank
and
acclimated
for
10
min.
An
ascend-
ing
series
of
Semmes–Weinstein
filaments
(Stoelting,
hair
numbers
1.65,
3.61,
4.31
and
5.07,
producing
maximum
tip
pressures
of
0.008,
0.4,
2
and
10
g,
respectively)
were
applied
to
the
mantle,
the
base
of
the
treated
arm
(L1
or
R1),
adjacent
arm,
and
fourth
arm
on
the
contralateral
side
(Fig.
1A).
Filaments
were
held
for
1
s
after
bending
(filaments
reach
maximum
tip
pressure
upon
bending).
We
considered
the
2
and
10
g
filaments
likely
to
be
noxious
as
they
produced
tissue
indentation
and
puncture,
respec-
tively,
when
applied
for
5–10
s
on
excised
arms.
Local
response
thresholds
(movement
of
the
suckers
or
local
muscle
contraction),
and
thresholds
for
inducing
coordinated
whole-body
avoidance
behavior
were
recorded
prior
to,
and
at
10
min,
6
h
and
24
h
after
injury
or
sham
treatment.
Arm
crush.
The
mid-proximal
area
of
one
arm
was
crushed
in
serrated
forceps
for
up
to
20
s.
Control-treated
animals
received
light
arm
touch.
Terminal
anesthesia,
decerebration
and
arm
removal.
Octopus
tis-
sue
has
limited
viability
post-mortem
[18].
To
permit
sequential
harvest
of
each
arm
without
distress,
octopuses
were
surgically
decerebrated
following
[19],
before
arms
were
removed.
General
anesthesia
was
induced
by
progressive
exposure
to
ethanol
in
natural
seawater
(0.1–1.5%
over
20
min).
Skin
and
mus-
cle
overlying
the
cranium
were
injected
with
0.1
ml,
0.5%
(w/v)
lidocaine
hydrochloride
in
a
mix
of
3:1
SW
and
ethanol.
The
supraoesophageal
ganglion
(SOG)
was
exposed
and
excised
via
a
dorsal
midline
incision,
which
was
closed
with
3-0
Ethicon
sutures.
Importantly,
we
observed
in
two
animals
that
incomplete
removal
of
the
SOG,
leaving
the
OL
stalks
and
some
basal
lobe
tissue
intact,
did
not
prevent
grooming
and
guarding
of
the
surgery
site
after
recovery
from
anesthesia.
Complete
removal
of
the
SOG
abolished
wound
attention.
Recovery
in
fresh,
aerated
SW
occurred
in
all
cases
within
5
min.
Decerebrate
animals
respire
normally,
exhibit
regular
locomotion
patterns,
and
respond
reflexively
to
touch,
maintaining
good
tissue
health
[18].
To
block
activity-dependent
sensitizing
effects
of
arm
removal,
each
arm
was
injected
intramuscularly
in
its
base
with
0.5
mL
isotonic
(330
mM)
MgCl2solution
prior
to
removal
distal
to
the
injection
site.
Arms
were
removed
in
random
order
then
the
animal
J.S.
Alupay
et
al.
/
Neuroscience
Letters
558 (2014) 137–
142 139
2
3
4
5
Trea
ted arm
Control, local movement
Inju red, local movement
Threshold (log bending force)
Control, general movement
Inju red, general movement
Adjacent arm
Distant arm Mantle
Pre10 min6 h 24 h
2
3
4
5
Pre10 mi
n6
h24 h
Threshold (log bending force)
** ***
++ ++ +*
++
++
*
+
+
A
B
CD
A
D
M
T
Fig.
2.
Thresholds
for
local
reflex
and
whole-body
escape
responses
decrease
for
at
least
24
h
after
arm
injury.
(A)
Touch
on
the
treated
arm
(T
on
schematic),
(B)
the
adjacent
arm
(A
on
schematic),
(C)
on
a
distant
arm
(D
on
schematic),
and
(D)
on
the
mantle
(M
on
schematic).
+,
difference
between
thresholds
for
general
movement
in
control
and
injured
octopuses;
*,
differences
between
thresholds
for
local
movement.
Mann–Whitney
U
tests;
+
or
*,
p
<
0.05.
++
or
**,
p
<
0.01.
was
killed
by
deep
ethanol
anesthesia
and
removal
of
the
remaining
CNS.
The
mantle
was
cut
along
the
ventral
midline
and
the
viscera
removed
to
permit
recordings
from
mantle
nerves.
Neural
responses
to
mechanical
stimulation.
The
cut
end
of
each
arm
was
pinned
to
a
Sylgard-coated
recording
chamber
filled
with
seawater,
and
a
suction
electrode
applied
to
the
axial
nerve
cord
(see
Fig.
1B).
The
same
series
of
Semmes–Weinstein
filaments
used
for
sensory
threshold
testing
was
applied
to
two
positions
on
the
arm–one
proximal
(within
20
mm)
to
the
cut
end,
and
one
within
30
mm
of
the
tip.
For
injured
arms
that
were
autotomized,
we
recorded
from
the
remaining
arm
stump
and
stimulated
the
prox-
imal
site
only.
Mantle
nerves
were
cut
and
recorded
distal
to
the
stellate
ganglion
(see
Fig.
1C).
In
control
animals
we
also
tested
short-term
sensitization
of
sensory
responses
by
applying
arm
crush
distal
to
the
second
stim-
ulation
site
after
pre-tests,
then
repeating
the
stimulation
sequence
after
five
minutes.
Data
analysis
and
statistical
procedures.
Behavioral
sensory
thresholds
were
compared
between
injured
and
control
animals
with
Mann–Whitney-U
tests.
Activity
from
arm
and
mantle
nerves
was
sampled
at
10
kHz
and
digitized
using
a
Powerlab
with
LabChart
software
(AD
Instruments).
Counts
of
spikes
evoked
dur-
ing
the
1
s
maximal-pressure
application
of
each
filament
from
injured,
contralateral,
and
sham-treated
arms
from
control
ani-
mals
were
analyzed
with
nested
ANOVA.
Short-term
sensitization
to
ex
vivo
crush
was
analyzed
using
a
2-factor
ANOVA.
Sponta-
neous
firing
rates
were
compared
between
a
20
s
interval
prior
to
any
test
stimulation
in
injured
and
control
animals,
and
before
and
after
ex
vivo
crush.
If
significant
effects
were
detected,
post
hoc
independent-samples
t-tests
compared
between
groups
and
paired
t-tests
compared
before
and
after
treatment.
The
critical
alpha
was
Bonferroni-corrected
and
set
at
0.05.
All
p
values
were
two-tailed.
Statistical
analysis
was
conducted
using
Prism
6.0
(GraphPad).
3.
Results
3.1.
Noxious
stimulation
induces
arm
autotomy
and
evokes
sustained
wound-directed
behavior
In
four
of
five
injured
animals,
arm
crush
induced
autotomy
at
a
consistent
site,
around
suckers
five
to
seven
on
the
injured
arm.
All
animals
inked
and
jetted
at
the
onset
of
stimulation
and
showed
immediate
wound-grooming
behavior,
where
the
arm
stump
or
crushed
site
was
held
in
the
beak,
until
the
10-min
behavior
test
and
in
two
individuals
for
at
least
20
min.
By
the
6
h
behavior
test
no
animals
expressed
ongoing
grooming,
and
mechanical
stimulation
did
not
re-induce
it.
Instead
the
injured
area
was
contracted
and
held
close
to
the
body,
and
some
animals
(n
=
3)
used
adjacent
arms
to
curl
around
the
injured
site.
This
guarding
behavior
was
pro-
voked
further
by
stimulation
of
the
wounded
arm.
At
24
h
injured
sites
were
no
longer
contracted
prior
to
stimulation,
but
light
touch
was
sufficient
to
induce
contraction
that
persisted
throughout
the
behavioral
test
sequence.
Sham-treated
animals
(n
=
4)
never
groomed
or
guarded
arms.
3.2.
Noxious
stimulation
reduces
sensory
thresholds
for
local
reflex
and
full-body
escape
behaviors
Sensory
thresholds
for
both
local
and
full-body
movement
were
significantly
decreased
at
each
post-test
for
stimulation
on
the
injured
arm
(Fig.
2A),
but
local
thresholds
were
only
transiently
decreased
in
response
to
touch
on
other
arms
(Fig.
2B–D).
Thresh-
olds
for
evoking
general
movement
were
decreased
across
the
body.
Sham-treated
animals
showed
consistent
sensory
thresholds.
3.3.
Sensory
units
encoding
noxious
stimulation
are
present
in
arms
and
mantle
Mechanical
stimulation
of
excised
arms
produced
afferent
activ-
ity
of
one
or
two
units
for
light
(<1
g)
pressure
and
typically
four
or
more
for
heavier
pressures
(2
g)
(Fig.
3A,
S1),
but
the
identity
and
specificity
of
the
units
were
not
easily
determined;
increasing
spike
rates
to
noxious
stimulation
may
have
been
from
primary
sensory
units
or
interneurons
conveying
mixed
sensory
information.
Supplementary
material
related
to
this
article
can
be
found,
in
the
online
version,
at
http://dx.doi.org/10.1016/j.neulet.2013.
11.002.
Both
light
and
firm
touches
induced
local,
reflexive
sucker
tur-
ning
toward
the
stimulus
site
[20]
and
sometimes
also
induced
coordinated
sucker
‘stalking’
[21],
whipping,
or
coiling
motions
of
the
arm.
Injection
of
isotonic
MgCl2blocked
all
afferent
activity
in
140 J.S.
Alupay
et
al.
/
Neuroscience
Letters
558 (2014) 137–
142
Fig.
3.
Arm
injury
sensitizes
mechanosensory
afferents.
(A)
Complex
neural
responses
to
touch
of
varying
force
on
the
arm.
Traces
show
two
identified
wide
dynamic
range
mechanoreceptors
responding
to
increasing
force
(2.0
g
not
shown).
Boxed
units
in
each
trace
expanded,
below.
More
examples
are
shown
in
Fig.
S1.
(B)
Injured
arms
produce
significantly
more
spikes
compared
with
sham-treated
arms
when
stimulated
with
filaments
delivering
2
and
10
g
of
tip
pressure,
but
not
by
lighter
filaments.
(C)
Untreated
arms
ipsilateral
to
the
injured
arm
show
similar
increases
in
response
to
both
light
and
heavy
force
compared
with
arms
from
matched
sides
of
sham-treated
control
animals.
Bars
show
mean
±
SEM
of
spike
count
during
the
1
s
maximal-pressure
application
of
each
filament.
Schematics
show
arms
included
in
each
group
for
analysis.
Independent
samples
t-tests
with
Bonferroni
correction;
*p
<
0.05;
**p
<
0.01.
the
injected
region
and
conduction
of
impulses
from
distal
to
the
injection
site.
Distinct
primary
sensory
afferents
in
the
mantle
nerves,
typ-
ically
with
larger
spikes,
responded
to
only
the
two
firmest
filaments,
suggesting
a
nociceptor-activation
threshold
of
<2
g
of
pressure
(Fig.
S1).
Similar
signal
was
obtained
from
recordings
made
from
the
pallial
nerve
(not
shown).
3.4.
Arm
injury
has
long-term
sensitizing
effects
in
injured
and
uninjured
arms
At
24
h
after
injury,
injured
arms
produced
more
spikes
in
response
to
application
of
2
and
10
g
filaments
compared
with
sham-treated
arms
from
controls
(Fig.
3B),
but
not
to
the
0.008
and
0.4
g
filaments.
Ipsilateral
arms
to
injury
showed
stronger
responses
to
each
filament
application
compared
to
sham-treated
animals.
Responses
from
contralateral
arms
were
not
different
(Fig.
3C).
Spontaneous
activity
(Hz)
was
not
significantly
higher
in
injured
arms
(10.3
±
2.2)
compared
with
intact
arms
from
injured
animals
(6.4
±
0.9,
p
=
0.08),
but
intact
arms
had
higher
rates
of
spontaneous
activity
compared
with
arms
from
uninjured
control
animals
(3.6
±
0.7,
p
=
0.03).
3.5.
Ex
vivo
arm
injury
has
short-term
sensitizing
effects
that
mirror
long-term
in
vivo
changes
Crush
on
arm
tips
produced
robust
short-term
sensitization
of
mechanosensory
units
5
min
after
crush,
with
significantly
more
spikes
produced
in
response
to
both
light
and
heavy
mechanical
stimulation
compared
both
to
pre-crush
and
to
post-tests
for
sham-
treated
arms
(Fig.
4).
Spontaneous
activity
at
5
min
post-crush
was
also
significantly
higher
than
in
sham-treated
arms
(10.1
±
0.5
vs.
2.3
±
1.2
Hz,
p
=
0.04)
4.
Discussion
Arm
injury
in
A.
aculeatus
induced
various
nocifensive
behavioral
responses
including
autotomy,
long-term
site-specific
sensitization,
directed
wound
attention
and
long-term
decreased
thresholds
for
initiating
escape
responses.
Whether
these
changes
imply
cognitive
or
emotional
responses
to
injury
is
uncertain;
local
hyperreflexia
and
autotomy
are
generally
considered
evidence
for
nociception
but
insufficient
to
indicate
pain
[5,22].
Directed
wound
attention
and
increased
motivation
to
escape
from
noxious
stim-
ulation
are
accepted
as
indicative
of
pain
in
mammals
[23],
but
remain
controversial
for
invertebrates
[24].
The
attention
to
injuries
we
observed
after
arm
crush
is
qualita-
tively
similar
to
those
observed
in
mammals
that
lick,
rub
or
conceal
a
painful
site
[25],
but
unlike
responses
in
squid,
which
never
dis-
played
wound-directed
attention
[11].
It
has
been
suggested
that
the
vertical
and
frontal
lobes
would
generate
hypothesized
emo-
tional
responses
in
octopus
[26],
but
removal
of
these
areas
alone
was
insufficient
to
abolish
wound-directed
behavior;
either
central
representations
of
injury
form
outside
these
lobes,
or
wound-
directed
attention
in
octopuses
is
not
a
robust
indicator
of
pain,
as
is
presumed
for
mammals.
It
is
therefore
not
yet
clear
whether
arm
injury
or
autotomy
is
distressing
to
octopuses,
or
whether
autotomy
and
amputation
are
represented
similarly
in
the
central
brain.
Patterns
of
neural
sensitization
after
injury
were
similar
to
site-
specific
sensitization
in
the
gastropod
Aplysia
[27]
and
in
mammals
[28],
but
somewhat
different
to
those
described
in
squid
[12],
where
J.S.
Alupay
et
al.
/
Neuroscience
Letters
558 (2014) 137–
142 141
Fig.
4.
Ex
vivo
arm
crush
produces
short-term
sensitization
that
mirrors
long-term
sensitization
in
arms
injured
in
vivo.
The
zigzag
line
shows
position
of
arm
crush,
large
dot
indicates
stimulation
site.
Stimulation
at
medial
and
distal
sites
reveals
increased
firing
after
crush,
compared
against
both
pre-test
counts
for
the
same
arm
and
post-test
counts
from
arms
receiving
light
touch
(sham
treatment).
+,
comparisons
to
sham-treated
post-tests
with
unpaired,
Bonferroni-corrected
t-tests;
*,
pre-test
counts
of
the
same
arm
made
with
paired,
corrected
t-tests.
*p
<
0.05;
**p
<
0.01.
nociceptors
close
to
injury
and
on
the
opposite
side
of
the
body
were
sensitized
and
behavioral
response
thresholds
were
reduced
all
over
the
body
after
arm
injury
[11].
Here
we
found
generally
decreased
behavioral
thresholds
only
at
10
min
after
injury,
but
site-specific
behavioral
and
neural
effects
at
longer
intervals.
How-
ever,
similar
spontaneous
activity
among
crushed
and
intact
arms
on
injured
animals,
but
generally
higher
rates
in
arms
from
injured
animals
compared
with
controls,
matches
the
pattern
in
squid
[12].
Likewise,
spontaneous
firing
rates
are
too
low
to
account
for
increased
spike
counts
to
mechanical
stimulation.
Elevated
spon-
taneous
activity
is
present
in
all
arms
after
one
is
injured,
thus
it
is
not
clear
if
it
is
implicated
in
wound-directed
attention,
as
occurs
in
mammals
[29].
Noxious
mechanosensation
appears
to
be
coded
by
increased
firing
of
multiple
distinct
units
in
arms,
probably
a
mixed
popu-
lation
of
primary
sensory
neurons
and
interneurons
[19];
further
work
is
needed
to
identify
true
nociceptors
and
circuitry
driving
peripherally-mediated
reflexive
avoidance
[30,31].
In
the
mantle
we
identified
putative
primary
afferents,
as
the
mantle
does
not
contain
peripheral
cell
bodies
outside
stellate
ganglia
[32].
Similar
to
squid
[12],
nociceptive
mechanical
stimulation
may
be
encoded
by
specific
units
not
activated
by
innocuous
touch,
although
activa-
tion
thresholds
are
somewhat
lower
in
A.
aculeatus
than
D.
pealei.
Similar
units
recorded
from
pallial
nerves
indicate
that
not
all
noci-
ceptive
inputs
terminate
in
local
reflex
connections
in
the
stellate
ganglion.
A
few
studies
have
examined
centrally
projecting
sensory
fibers
[33,34]
but
CNS
circuitry
that
might
convey
sensory
inputs
to
higher
processing
centers
analogous
to
those
involved
in
pain
experience
in
mammals
has
not
been
described
in
any
cephalopod.
5.
Conclusions
Octopuses
respond
to
noxious
stimulation
with
reflex
avoid-
ance
that
probably
does
not
require
higher
cognitive
processing,
but
unlike
squid,
also
engage
in
directed,
protective
behaviors
that
persist
for
considerable
periods.
Arms
and
mantle
contain
sen-
sory
units
that
conduct
noxious
stimulation
to
higher
processing
centers,
although
whether
there
is
pain
associated
with
noxious
sensory
input
is
unclear.
Both
low-threshold
mechanoreceptors
and
putative
nociceptors
are
hyperexcitable
for
at
least
24
h
after
142 J.S.
Alupay
et
al.
/
Neuroscience
Letters
558 (2014) 137–
142
injury,
and
ex
vivo
arm
crush
produces
similar
short-term
sensi-
tization
of
mechanosensory
afferents.
Acknowledgements
We
thank
Edgar
T.
Walters
and
Roger
T.
Hanlon
for
providing
funding
and
laboratory
space
to
RJC
from
NSF
IOS-1146987
and
IOS-1145478,
and
for
insightful
discussion
of
results.
JSA
and
SPH
were
partially
supported
by
The
University
of
California,
Berke-
ley
Department
of
Integrative
Biology
and
Graduate
Division
and
a
Doctoral
Student
Research
Grant
from
the
Graduate
Center
of
the
City
University
of
New
York,
respectively.
Justine
Allen
prepared
tissue
sections
and
photographed
animals
and
tissue,
Julia
Carroll
assisted
with
data
collection
and
George
Bell
built
animal
housing.
References
[1]
E.T.
Walters,
Injury-related
behavior
and
neuronal
plasticity:
an
evolutionary
perspective
on
sensitization,
hyperalgesia,
and
analgesia,
Int.
Rev.
Neurobiol.
(1994)
325.
[2]
E.S.J.
Smith,
G.R.
Lewin,
Nociceptors:
a
phylogenetic
view,
J.
Comp.
Physiol.
A:
Neuroethol.
Sens.
Neural
Behav.
Physiol.
195
(2009)
1089–1106.
[3]
R.W.
Elwood,
Pain
and
suffering
in
invertebrates?
ILAR
J.
52
(2011).
[4]
V.
Braithwaite,
Do
Fish
Feel
Pain?
OUP,
Oxford,
2010.
[5]
R.J.
Crook,
E.T.
Walters,
Nociceptive
behavior
and
physiology
of
molluscs:
ani-
mal
welfare
implications
ILAR
J.
52
(2011)
185–195.
[6]
P.L.R.
Andrews,
Laboratory
invertebrates:
only
spineless,
or
spineless
and
pain-
less?
ILAR
J.
52
(2011)
121.
[7]
B.
Hochner,
T.
Shomrat,
G.
Fiorito,
The
octopus:
a
model
for
a
comparative
analysis
of
the
evolution
of
learning
and
memory
mechanisms,
Biol.
Bull.
210
(2006)
308–317.
[8]
J.A.
Mather,
R.C.
Anderson,
Ethics
and
invertebrates:
a
cephalopod
perspective,
Dis.
Aquat.
Organ.
75
(2007)
119–129.
[9]
J.A.
Smith,
P.L.R.
Andrews,
P.
Hawkins,
S.
Louhimies,
G.
Ponte,
L.
Dickel,
Cephalo-
pod
research
and
EU
directive
2010/63/EU:
requirements,
impacts
and
ethical
review,
J.
Exp.
Mar.
Biol.
Ecol.
447
(2013)
31–45.
[10]
P.L.R.
Andrews,
A.-S.
Darmaillacq,
N.
Dennison,
I.G.
Gleadall,
P.
Hawkins,
J.B.
Messenger,
D.
Osorio,
V.J.
Smith,
J.A.
Smith,
The
identification
and
management
of
pain,
suffering
and
distress
in
cephalopods,
including
anaesthesia,
analgesia
and
humane
killing,
J.
Exp.
Mar.
Biol.
Ecol.
447
(2013)
46–64.
[11]
R.J.
Crook,
T.
Lewis,
R.T.
Hanlon,
E.T.
Walters,
Peripheral
injury
induces
long-
term
sensitization
of
defensive
responses
to
visual
and
tactile
stimuli
in
the
squid
Loligo
pealeii,
Lesueur
1821,
J.
Exp.
Biol.
214
(2011)
3173–3185.
[12]
R.J.
Crook,
R.T.
Hanlon,
E.T.
Walters,
Squid
have
nociceptors
that
display
widespread
long-term
sensitization
and
spontaneous
activity
after
bodily
injury,
J.
Neurosci.
33
(2013)
10021–10026.
[13]
C.L.
Huffard,
Ethogram
of
Abdopus
aculeatus
(d’Orbigny,
1834)
(Cephalopoda:
Octopodidae):
can
behavioural
characters
inform
octopodid
taxomony
and
sys-
tematics?
J.
Molluscan
Stud.
73
(2007)
185–193.
[14]
M.M.
Lange,
On
the
regeneration
and
finer
structure
of
the
arms
of
the
cephalopods,
J.
Exp.
Zool.
31
(1920)
1–57.
[15]
J.R.
Voight,
Movement,
injuries
and
growth
of
members
of
a
natural
pop-
ulation
of
the
Pacific
pygmy
octopus,
Octopus
digueti,
J.
Zool.
228
(1992)
247–264.
[16]
H.
Matzner,
Neuromuscular
system
of
the
flexible
arm
of
the
octopus:
physio-
logical
characterization,
J.
Neurophysiol.
83
(2000)
1315–1328.
[17]
Y.
Gutfreund,
H.
Matzner,
T.
Flash,
B.
Hochner,
Patterns
of
motor
activity
in
the
isolated
nerve
cord
of
the
octopus
arm,
Biol.
Bull.
211
(2006)
212–222.
[18]
C.H.F.
Rowell,
Excitatory
and
inhibitory
pathways
in
the
arm
of
octopus,
J.
Exp.
Biol.
40
(1963)
257–270.
[19]
C.H.F.
Rowell,
Activity
of
interneurones
in
the
arm
of
octopus
in
response
to
tactile
stimulation,
J.
Exp.
Biol.
44
(1966)
589–605.
[20]
J.
Altman,
Control
of
accept
and
reject
reflexes
in
the
octopus,
Nature
229
(1971)
204–206.
[21]
W.M.
Kier,
A.M.
Smith,
The
structure
and
adhesive
mechanism
of
octopus
suck-
ers,
Integr.
Comp.
Biol.
42
(2002)
1146–1153.
[22]
D.
Broom,
Animal
welfare:
concepts
and
measurement,
J.
Anim.
Sci.
(1991)
4167–4175.
[23]
C.
Allen,
Animal
pain,
Noûs
38
(2004)
617–643.
[24]
B.
Magee,
R.W.
Elwood,
Shock
avoidance
by
discrimination
learning
in
the
shore
crab
(Carcinus
maenas)
is
consistent
with
a
key
criterion
for
pain,
J.
Exp.
Biol.
216
(2013)
353–358.
[25]
Q.
Hogan,
Animal
pain
models,
Reg.
Anesth.
Pain
Med.
27
(2002)
385–401.
[26]
J.A.
Mather,
To
boldly
go
where
no
mollusc
has
gone
before:
personality,
play,
thinking,
and
consciousness
in
cephalopods,
Am.
Malacol.
Bull.
24
(2008)
51–58.
[27]
E.T.
Walters,
Site-specific
sensitization
of
defensive
reflexes
in
aplysia:
a
simple
model
of
long-term
hyperalgesia,
J.
Neurosci.
7
(1987)
400–407.
[28]
C.J.
Woolf,
Q.
Ma,
Nociceptors
noxious
stimulus
detectors,
Neuron
55
(2007)
353.
[29]
J.
Xu,
T.J.
Brennan,
Guarding
pain
and
spontaneous
activity
of
nociceptors
after
skin
versus
skin
plus
deep
tissue
incision,
Anesthesiology
112
(2010)
153–164.
[30]
T.
Hague,
M.
Florini,
P.L.R.
Andrews,
Preliminary
in
vitro
functional
evidence
for
reflex
responses
to
noxious
stimuli
in
the
arms
of
Octopus
vulgaris,
J.
Exp.
Mar.
Biol.
Ecol.
447
(2013)
100–105.
[31]
J.
Ten
Cate,
Contribution
a
L’innervation
des
ventouses
chez
Octopus
vulgaris,
Arch.
Neerl.
Physiol.
Homme.
Anim.
13
(1928)
541–551.
[32]
F.B.
Hoffmann,
Gibt
es
in
der
Muskulatur
der
Mollusken
periphere,
kontinuier-
lich
leitende
Nervennetze
bei
Abwesenheit
von
Ganglienzellen?
II:
Weitere
leitende
Nervennetze
bei
Abwesenheit
von
Ganglienzellen?
II:
Weitere
Unter-
suchungen
an
den
Chromatophoren
der
Kephalopoden,
Pflug.
Arch.
Gesamte
Physiol.
Menschen
Tiere
132
(1910)
43–81.
[33]
E.
Monsell,
Cobalt
and
horseradish
peroxidate
tracer
stdies
in
the
stellate
gan-
glion
of
octopus,
Brain
Res.
184
(1980)
1–9.
[34]
B.
Budelmann,
J.
Young,
Central
pathways
of
the
nerves
of
the
arms
and
mantle
of
octopus,
Phil.
Trans.
R.
Soc.
Lond.
B:
Biol.
Sci.
310
(1985)
109–122.
... In multiple studies already reviewed under criterion 1, electrophysiological measurements were taken at the nerve cords linking peripheral nerves to the central brain and found to show increased activity in response to noxious stimuli (Crook et al., 2013;Alupay et al., 2014, Perez et al., 2017Bazarini & Crook, 2020;Crook, 2021). This shows compellingly that signals from nociceptors are reaching the brain, but it does not show that they are reaching the vertical lobe system. ...
... There is also behavioural evidence that suggests information about noxious stimuli must be processed within central brain regions. For example, as a result of sophisticated behavioural responses to noxious stimuli in their tests, Alupay et al. (2014) infer that perception of noxious stimuli in the arms and mantle was conveyed to "higher processing centres". However, this evidence is discussed under other headings, and (as in Section 2.1) we want to focus on neurophysiological evidence in this section. ...
... Full review of evidence: What we are looking for here is robust evidence of self-protective behaviours that go beyond reflexes: to meet this criterion, the animal should be able to vary its response in a targeted way, according to where on the body the noxious stimulus is administered. Alupay et al. (2014) provides strong evidence to support criterion 6 in octopods, demonstrating that algae octopus, Abdopus aculeatus, (n = 9) exhibit flexible self-protective behaviours to an injured site. Injured octopuses received a crush to one arm with serrated forceps (n = 5) and sham-treated octopuses (n = 4) received a light arm touch. ...
Technical Report
Full-text available
Sentience is the capacity to have feelings, such as feelings of pain, pleasure, hunger, thirst, warmth, joy, comfort and excitement. It is not simply the capacity to feel pain, but feelings of pain, distress or harm, broadly understood, have a special significance for animal welfare law. Drawing on over 300 scientific studies, we have evaluated the evidence of sentience in two groups of invertebrate animals: the cephalopod molluscs or, for short, cephalopods (including octopods, squid and cuttlefish) and the decapod crustaceans or, for short, decapods (including crabs, lobsters and crayfish). We have also evaluated the potential welfare implications of current commercial practices involving these animals.
... Supporting this, there is a range of evidence for pain experience in cephalopods. Octopus and squid have been found to possess nociceptors, and these connect to the central nervous system (Alupay et al., 2014;Bazarini & Crook, 2020;Crook, 2021;Crook et al., 2013;Howard et al., 2019;Perez et al., 2017), though connection to the substantial integrative brain region of the vertical lobe is still to be established. Octopus and squid show behavioural changes after exposure to noxious stimuli, including defencive behaviour (Bazarini & Crook, 2020;Crook et al., 2011;Howard et al., 2019), increased responsiveness to threats Oshima et al., 2016), and grooming injured arms (Alupay et al., 2014;Crook, 2021). ...
... Octopus and squid have been found to possess nociceptors, and these connect to the central nervous system (Alupay et al., 2014;Bazarini & Crook, 2020;Crook, 2021;Crook et al., 2013;Howard et al., 2019;Perez et al., 2017), though connection to the substantial integrative brain region of the vertical lobe is still to be established. Octopus and squid show behavioural changes after exposure to noxious stimuli, including defencive behaviour (Bazarini & Crook, 2020;Crook et al., 2011;Howard et al., 2019), increased responsiveness to threats Oshima et al., 2016), and grooming injured arms (Alupay et al., 2014;Crook, 2021). Injured octopus have also been found to show a preference for a chamber containing analgesia (Crook, 2021). ...
Article
Full-text available