Habituation of LG-mediated tailflip in the crayfish

Invert Neurosci (2015) 15:2
DOI 10.1007/s10158-015-0178-8
Habituation of LG-mediated tailflip in the crayfish
Toshiki Nagayama1 • Makoto Araki2
Received: 23 January 2015 / Accepted: 11 March 2015
Ó Springer-Verlag Berlin Heidelberg 2015
Abstract Crayfish escape from threatening stimuli by
tailflipping. If a stimulus is applied to the rear, crayfish
escape up and forwards in a summersault maneuver that is
mediated by the activation of lateral giant (LG) interneurons. The occurrence probability of LG-mediated tailflip,
however, diminishes and habituates if a stimulus is repeatedly applied. Since crayfish have a relatively simple
CNS with many identifiable neurons, crayfish represent a
good animal to analyze the cellular basis of habituation. A
reduction in the amplitude of the EPSP in the LGs, caused
by direct chemical synaptic connection from sensory afferents by repetitive stimulations, is essential to bring about
an inactivation of the LGs. The spike response of the LGs
recovers within several minutes of habituation, but the LGs
subsequently fail to spike when an additional stimulus is
applied after specific periods following habituation. These
results indicate that a decline in synaptic efficacy from the
mechanosensory afferents recovers readily after a short
delay, but then the excitability of the LGs themselves decreases. Furthermore, the processes underlying habituation
are modulated depending on a social status. When two
crayfish encounter each other, a winner–loser relationship
is established. With a short interstimulus interval of 5 s, the
rate of habituation of the LG in both socially dominant and
subordinate crayfish becomes lower than in socially isolated animals. Serotonin and octopamine affect this social
status-dependent modulation of habituation by means of
& Toshiki Nagayama
[email protected]
Department of Biology, Faculty of Science, Yamagata
University, Yamagata 990-8560, Japan
Division of Biological Sciences, Graduate School of Science,
Hokkaido University, Sapporo 060-0810, Japan
activation of downstream second messenger system of
cAMP and IP3 cascades, respectively.
Keywords Chemical synapse Modulation Biogenic
amines Second messengers
Habituation is a well known form of non-associative
learning (Thompson and Spencer 1966) in which reflexive
behavioral responses gradually reduce upon repeated
stimulation. Habituation is subject to plasticity in invertebrates, as has been shown in the siphon withdrawal reflex
in Aplysia (Kandel 2001, 2009), the proboscis extension
response of honeybees (Braun and Bicker 1992; Hammer
and Menzel 1998) and the lateral giant (LG)-mediated
tailflip of crayfish (Krasne 1969; Krasne and Woodsmall
1969; Zucker 1972b; Wine et al. 1975; Bryan and Krasne
1977; Fricke 1984; Marchand and Barnes 1992; Edwards
et al. 1994; Krasne and Teshiba 1995; Edwards et al. 1999;
Araki and Nagayama 2003; Edwards 2009; Nagayama and
Newland 2011). Since the LG-mediated tailflip is a
stereotyped behavior (Wiersma 1947) and crayfish have a
relatively simple central nervous system with relatively
few and often identifiable neurons (Wine 1984; Nagayama
et al. 1993a, b, 1994), crayfish represent a good animal to
analyze the cellular basis of habituation. If strong tactile
stimuli are applied to the abdomen or tailfan, crayfish
produce a rapid flexion of the abdominal musculature that
leads to an escape response directed up and forwards in a
summersault maneuver (Fig. 1a; also Wine and Krasne
1972). The lateral giant interneuron (LG; Fig. 1b) with a
large diameter of ascending axon (Fig. 1c) acts as a command neuron by receiving sensory inputs directly and
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Fig. 1 LG-mediated tailflip of
the crayfish. a Tracing of LG
tailflip. b Intracellular staining
of the LG in the terminal
abdominal ganglion.
c Transverse section of
abdominal third–fourth
connective. These illustrations
were modified from Nagayama
indirectly from extero- and proprioceptive afferents (Wine
and Krasne 1972; Newland et al. 1997; Araki and Nagayama 2003) and by making excitatory outputs with
motor giant (MoG) motor neurons in anterior abdominal
segments (Wine 1984). The rapid flexion of the abdomen is
triggered within 10 ms following spikes in LG.
The probability of occurrence of LG-mediated tailflip
diminishes and habituates if a tapping stimulus to tailfan is
repeatedly applied. Krasne and his colleagues show that
tonic descending inhibition from higher center of brain via
GABAergic pathway plays a role in controlling LG habituation that LGs show subthreshold response to stimulus
(Krasne and Bryan 1973; Krasne and Wine 1975; Vu and
Krasne 1992, 1993; Vu et al. 1993; Krasne and Teshiba
1995; Shirinyan et al. 2006). LGs also show habituation
without descending inputs by severance of the nerve cord
between thorax and abdomen or isolation of abdominal
ganglia from the rest of body (Krasne 1969; Zucker 1972a,
b; Wine et al. 1975). Krasne and Teshiba (1995) show that
removing the influence of higher center on LG circuit reduces the tendency of the LG threshold. Thus, descending
inputs from higher center control and/or modulate the activity of local regulation within the terminal abdominal
ganglion. When a constant stimulus that sets just above the
initial LG threshold is applied, the differences between
intact and abdominal isolated animals are small (Wine
et al. 1975). Here, we will focus on the local regulation
underlying habituation of LG neurones (=LG habituation)
within the terminal abdominal ganglion using isolated
nerve cord preparation with the results from our own
laboratory to describe the synaptic mechanism of LG habituation, recovery from habituation, and social status-dependent modulation of habituation.
Crayfish LG-flips and habituation
To prepare crayfish for electrophysiological analysis
(Fig. 2a), the nerve chain from the second to sixth (terminal) abdominal ganglia with relevant nerve roots is
isolated from the rest of the body and pinned, dorsal side
up, in a Sylgard-lined perfusion chamber, containing
cooled physiological solution (van Harreveld 1936). The
chamber is constantly perfused with fresh saline, and the
bathing solution can be changed with a saline containing a
specific drug. The dorsal ganglionic sheath of the terminal
ganglion is surgically removed with fine forceps to facilitate the penetration of intracellular electrode and drug
perfusion. The spike activity of LG is monitored extracellularly from the third–fourth abdominal connective using a suction electrode. Nerve roots 2, 3 and 4 of the
terminal abdominal ganglion that contain the
mechanosensory afferents innervating the uropods and
telson are electrically stimulated simultaneously using a
Invert Neurosci (2015) 15:2
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Fig. 2 LG habituation. a Experimental setup. b Responses of the LG
to repeated sensory stimulation. When the stimulus is repeated with a
5-s interstimulus interval, the LG elicits spikes to the first three
stimuli. From the fourth stimulus, the LG shows subthreshold
responses. c Habituation curves. LG firing probability is plotted as
the percentage of animals in which LG fired on a given trial
single oil hook electrode, since the spike threshold of the
LGs is relatively high. Square stimulus pulses
(0.01–0.05 ms duration; 1–20 V intensity) are delivered
through the stimulating electrode. Intracellular recordings
from the LG, or relevant ascending interneurons, are made
from their dendritic branches in the left half of the terminal
ganglion neuropil with glass microelectrodes. After 15 min
of rest following dissection, an intracellular microelectrode
is driven into the neuropil of the terminal ganglion. The
spike threshold of LG is then determined by applying a
gradual increase in the intensity of stimulation of the sensory nerves. When LG produces an action potential, an
extracellular spike with very large amplitude is recorded
following the intracellular LG spike from the A3–A4 abdominal connective (Fig. 2b). After the LG spike threshold
is determined, the intensity of stimulation is set so that the
stimulus is just suprathreshold. The preparation is rested
for a further 5 min before repeated sensory stimulation
with the intended interstimulus interval. We have judged
LG habituation to have occurred when LG failed to give
rise to spikes following five continuous stimulus trials. In
the recordings shown in Fig. 2b, the LG habituates from
the fourth trial of the stimulus. In some preparations, the
stimulus intensity is set just below suprathreshold to elicit
LG spikes in order to analyze the synaptic response of the
LGs more quantitatively.
As sensory stimulation was repeated, the rate of firing
probability of LGs in the tested populations declined depending on the stimulus intervals. When the stimulus is
repeated with a 1-s interstimulus interval, the LG response
immediately shows a rapid habituation, decreasing by
80 % of tested animals within four trials of stimulation, and
by 95 % after the twentieth trial (filled circles in Fig. 2c).
As the interstimulus interval is increased, the decrease in
the LG response becomes slower. For example, at a 5-s
interstimulus interval, the response of LG declines by 80 %
after 20 trials (open circles in Fig. 2c) and at interstimulus
intervals of 20 or 60 s, the response of LG decreases by
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50 % after 20 stimuli (Fig. 2c: filled triangles for 20-s intervals and open triangles for 60-s intervals). When a stimulus is repeated with a 300-s interstimulus interval,
however, habituation occurs more readily (filled squares in
Fig. 2c). The response of LG declines by 80 % after only
10 trials, and the decline of habituation curve is similar to
that at a 5-s interstimulus interval. Thus, the number of
stimuli necessary to cause habituation increases when the
interstimulus interval is increased, with the exception of a
300-s interstimulus interval (Araki and Nagayama 2005).
For example, the number of stimuli needed to cause habituation at a 5-s interstimulus interval is significantly
greater than at a 1-s interstimulus interval (p \ 0.001; logrank test). Furthermore, with 20- and 60-s interstimulus
intervals, the number of stimuli significantly increases
compared to that with a 5-s interval (p \ 0.005 and
p \ 0.001, respectively; log-rank test). The reason why the
LGs habituated rapidly in response to stimuli at a 300-s
interstimulus interval will be discussed later in the section
referring to change in LG excitability.
Synaptic mechanism of habituation
The LGs receive sensory inputs through sensory neurons
via direct synapses and by ascending interneurons via
indirect synapses. Low intensities of sensory stimulation
(=9 V, 10-ls duration) evoke compound potentials in the
LG with two components (Fig. 3a top trace). As Krasne
(1969) reported, the first EPSP, termed the a component,
and a later EPSP, the b component, can be temporally
discriminated. With further increases in stimulus intensity
(=10 V), the stimulation evokes a spike rising from the
beginning of b component (Fig. 3a 2nd trace). When the
stimulus is repeated at the same intensity at 0.5 Hz, LG
fails to give rise to a spike from the second stimulus
(Fig. 3a third trace) and continues to respond with subthreshold EPSPs to later stimuli (Fig. 3a bottom trace). A
comparison of the potentials elicited in LG evoked by
stimulus pulses of 9-V intensity and 10-V intensity; the
later part of a component increases following a spike from
the b component (Fig. 3b top trace). A comparison of the
potentials before and after LG habituation to the stimulus
of 10-V intensity shows that the later part of the a component decreases in amplitude after habituation, while the
amplitude of the early part of the a component remains
constant (Fig. 3b middle trace). With repeated stimulation,
the amplitude of the early part of the a component remains
constant in amplitude, while that of the b component decreases gradually. The superimposed potentials of LG
evoked in response to a subthreshold stimulus (9 V in intensity), and the response to the second stimulus at 10 V
shows that the time course and amplitude of the a
Invert Neurosci (2015) 15:2
component vary little (Fig. 3b bottom trace). These results
suggest that the potentials in LG could consist of three
components when LG responds with a spike to sensory
stimulation. The third component of EPSPs, termed as a0
component, is distinguishable at the boundary between the
late a and early b component when the LG produces an
action potential, but is difficult to discriminate after LG
habituation (Araki and Nagayama 2003).
Interneurons A and C, also known as NE-1 and RC-8
(Nagayama et al. 1993a), contribute to the b component of
the LG response to sensory stimulation (Kennedy and
Takeda 1965; Zucker et al. 1971; Zucker 1972a, b). Interneuron C responds to sensory stimulation with a train of
spikes superimposed on a sustained membrane depolarization at stimulus intensities just suprathreshold for LG
spikes (Fig. 3c left). When sensory stimulation is repeated,
LG fails to give rise to a spike, which is indicated by the
disappearance of the largest extracellular spike recorded
from the third–fourth abdominal nerve cord (lower trace in
Fig. 3c right). The spikes of interneuron C reduce in
number following repeated stimulation (upper trace in
Fig. 3c right), but the time course and amplitude of the
initial spike of the interneuron do not change significantly
before and after LG habituation. Since interneuron A has
the next largest axon diameter to the giant interneurons
(LG and MG), spikes of interneuron A are readily distinguishable by means of extracellular recording from the
abdominal nerve cord (arrowheads in Fig. 3c lower trace).
Interneuron A elicits spikes with rather constant latencies
before and after LG habituation. Thus, these interneurons
only show a slight reduction of activity in response to repeated stimulation, even though LG fails to give rise to a
spike upon repeated sensory stimulation (Araki and Nagayama 2003).
If EPSPs are mediated through chemical synapses, they
are reduced in amplitude by the passage of depolarizing
current and increased by hyperpolarizing current. By contrast, current injection usually has little effect on the size of
potentials if they are mediated by electrical transmission
(e.g., Nagayama et al. 1997b; Newland et al. 1997).
Stimulation with an intensity set just subthreshold to elicit
LG spikes evokes compound potentials in LG (upper trace
in Fig. 4a). The passage of 1 nA hyperpolarizing current
injected into LG causes an increase in amplitude of the
sensory-evoked potentials. Superimposed sweeps to compare the EPSPs in LG at resting potential, and during hyperpolarizing current injection, show that the EPSP in the
later part of the a component, that is the a0 component
(asterisks in Fig. 4a), is increased in amplitude, while that
of the early a component or the b component shows no
change in amplitude (Fig. 4a). Since the a and b components of EPSPs are mediated through electrical synapses
from sensory afferents and specific ascending interneurons,
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Fig. 3 Synaptic response of LG and interneuron C to sensory
stimulation. a Subthreshold (top trace) and suprathreshold (second–
fourth traces) stimuli. LG shows habituation from the second
suprathreshold stimulus. Interstimulus interval is 1 s. b Superimposed
sweeps of a. The a0 component of the EPSP is only observed when
the LG gives rise to a spike. c Response of interneuron C before and
after LG habituation. Arrowheads in bottom trace are extracellular
spikes of interneuron A
respectively (Zucker 1972a), this study suggests that LG
receives chemically mediated inputs that are consistent
with the a0 component directly from mechanosensory afferents innervating hairs on the surface of the tailfan (Araki
and Nagayama 2003).
Since mechanosensory afferents are known to release
acetylcholine as an excitatory neurotransmitter (Miller
et al. 1992; Ushizawa et al. 1996), the change in response
of the LGs to sensory stimulation under bath application of
d-tubocurarine, a nicotinic antagonist, is analyzed
(Fig. 4b). The compound potentials in LG elicited by
subthreshold stimulation decrease in amplitude about
6 min after bath application of 50 lL d-tubocurarine. The
decrement in the amplitude of LG continues gradually
(after 13 and 19 min) and recovers partially after about
30 min of washing with normal saline. This decrement in
potentials could be resolved into two temporal phases. The
later part of the a component, that is the a0 component,
quickly reduces in amplitude (shown with asterisk in
Fig. 4b), while the b component decreases gradually. This
different time course of effects on the compound potentials
suggests that two distinct chemically mediated inputs
contribute to the compound potentials in LG. (Araki and
Nagayama 2003).
We further analyze the responses of LG to exteroceptive
mechanical stimulation. Small numbers of mechanosensory
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b Fig. 4 Direct chemically mediated inputs from sensory afferents.
a Injection of 1 nA hyperpolarizing current into LG increases the
amplitude of the EPSP in the later part of a component, that is a0
component. Upper superimposed sweeps of the response of the LG
are adjusted to the base level of each record. Lower superimposed
sweeps are adjusted to the peak level of the EPSP. Asterisks indicate
EPSP in a0 component The EPSP amplitudes of both the early a
component and the b component are constant after injection of
hyperpolarizing current. b Effect of bath application of d-tubocurarine
on the response of LG to sensory stimulation. Superimposed sweeps
of the LG response show that the a0 component of the EPSP (asterisk)
disappears quickly and that of the b component diminishes gradually
in amplitude. c Response of LG to hair stimulation. Superimposed
sweeps triggered from each of two different afferents (c1 and c2)
show that EPSPs consistently follow the sensory spike with a constant
short latency. Superimposed sweeps triggered from the third afferent
spikes (c3) show that the EPSP followed consistently with a constant
long latency, and this EPSP decreased in amplitude under bath
application of d-tubocurarine. d Schematic diagram of circuitry for
LG tailflip
hairs are deflected locally, and the response of LG was
analyzed (Fig. 4c). Superimposed sweeps triggered from
the spikes of a single afferent show that EPSPs in LG
follow consistently with a constant short latency (Fig. 4c1).
The EPSPs are about 0.9 mV in amplitude, have a rise time
of about 0.8 ms, and a decay time to half amplitude of
about 0.9 ms. There is a delay of about 0.8 ms between the
afferent spike recorded in nerve root 3 and the start of the
EPSP in LG. A second sensory afferent with a smaller
amplitude spike in the extracellular recording is sampled
sequentially (Fig. 4c2), and superimposed sweeps triggered
from the spikes of this afferent show that EPSPs in LG
follow consistently with a short constant latency of about
0.9 ms. The EPSPs are about 0.7 mV in amplitude and
have a fast rise time of about 0.8 ms and a decay time to
half amplitude of about 1.0 ms. Superimposed sweeps
triggered from the spikes of the third afferent show that
EPSPs in LG follow consistently (left trace in Fig. 4c3).
There is, however, a rather long delay of about 1.5 ms
between the afferent spike and the start of the EPSP in the
LG. The EPSPs are about 0.8 mV in amplitude and have a
rise time of about 2.4 ms. The falling phase declines
gradually having a decay time to half amplitude of about
3.4 ms. They are, furthermore, decreased in amplitude
under bath application of d-tubocurarine (right trace in
Fig. 4c3). These results strongly indicate that the first two
afferents make convergent electrical connections with the
LG, while the third afferent makes chemically mediated
synaptic transmission. Thus, LGs receive direct excitatory
inputs from the mechanosensory afferents mediated
through both electrical (that is a component) and chemical
synapses (that is a0 component) with indirect electrical
input via sensory interneurons (that is b component) as
shown in Fig. 4d. The decrease in synaptic efficacy of the
direct chemical synapses contributes, at least in part, to
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elicit LG spikes and LG habituation (Araki and Nagayama
Recovery from habituation then change in LG
The spike response of the LGs usually recovers quickly
within minutes after habituation, but the LGs become less
excitable when additional sensory stimuli are applied after
a longer period following habituation (Araki and Nagayama 2005). For example, following a 1-min pause from
habituation, additional sensory stimulation (=test stimulus)
with the same intensity again elicits spikes in LG. By
contrast, in animals rested for 30 min following habituation
with no additional stimulation, the test stimulus fails to
elicit a LG spike (Araki and Nagayama 2005). These observations suggest that habituation is caused by a decline in
transmitter release from mechanosensory afferents (Zucker
1972b), but the synaptic efficacy of these sensory afferents
could recover readily after a short delay that is sufficient to
elicit a LG spike. Following recovery of the LG response,
however, LG soon exhibits a reduction in excitability for
spike generation after certain periods of delay. The a0
component of the EPSP disappears after habituation, while
the a0 component of the EPSP is observed when the test
stimulus is applied (Araki personal observation). At the
moment, the neural mechanisms underlying this decrease
in excitability of LGs remain unclear, but the observation
that the threshold of LG following the change in LG excitability becomes significantly higher than the threshold
just after habituation (Araki and Nagayama 2005) suggests
some physiological change could occur in the LGs during
the period of the delay following habituation.
As the interstimulus interval becomes shorter, the LG
habituation occurs rapidly, but a longer delay is necessary
to decrease excitability of LGs. As the interstimulus interval is increased, the delay needed for decrease in excitability becomes shorter (Araki and Nagayama 2005).
The observation that LG habituates rapidly to repetitive
stimulation with a 300-s interstimulus interval (Fig. 2c)
would explain that the LG response shifts directly from
habituation to reduction of excitability during the course of
longer periods of stimulation.
Status-dependent modulation of habituation
Animal behaviors are modulated according to external and
internal conditions. For example, the crayfish LG response
habituates more slowly when animals are exposed in high
temperatures (Nagayama and Newland 2011). Furthermore, the establishment of social hierarchies also affects
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crayfish behaviors. For example, a gentle mechanical
stimulation to the tailfan evokes an avoidance reaction
when a crayfish shows a stationary resting posture (Nagayama et al. 1986). Small crayfish show an escape-like
dart response, while larger animals show a defensive-like
turn response. When two small crayfish encounter each
other, a winner–loser relationship is established following
several combats, with the winner changing its response to
show a turn response (Fujimoto et al. 2011). The establishment of social status also affects excitability of LGs
(Krasne et al. 1997). Subordinate animals exhibit an increased threshold for LG activation. LG habituation is also
modulated by state-dependent manner (Araki et al. 2013).
When a sensory stimulus is applied repeatedly with a 5-s
interstimulus interval, LG from socially isolated crayfish
rapidly habituates, with a decrease in firing probability by
50 % within four trials of stimulation, and by 75 % after 25
trials (Fig. 5a, filled triangles). By contrast, LG’s responses
from socially subordinate crayfish show a slower rate of
habituation (Fig. 5a, open circles). After the twentieth trial
of stimulation, only 50 % of subordinate animals show LG
habituation, while 35 % still respond with a spike after 40
trials. Dominant crayfish are also found to show a slow
decline in the rate of LG habituation in the isolated abdominal nerve cord (Fig. 5a, filled circles). Approximately
20 trials are needed to decrease the LG firing probability by
50 %, while more than 40 % still give rise to a spike after
40 trials. The stimulus number required to habituate LG in
both the dominant and subordinate animals increases significantly in comparison with socially isolated crayfish
(p \ 0.01; log-rank test). This status-dependent change in
LG habituation is maintained for at least a week (Fig. 5b).
The decline in the habituation curve is very similar for
dominant animals of both first day and seventh day. On the
seventh day, about 40 % of animals do not show LG habituation within 40 trials of stimulation.
Fig. 5 Status-dependent modulation and the effect of biogenic
amines on habituation. a Habituation curves of the response of LG
to repeated sensory stimulation with a 5-s interstimulus interval. The
LG firing probabilities of control (filled triangles), dominant (filled
circles), and subordinate (open circles) animals are plotted. b Longterm memory of status-dependent modulation of habituation. Habituation curve of the response of LG in dominant crayfish on the
seventh day to repeated sensory stimulation with a 5-s interstimulus
interval. c Habituation curves of the response of LG to repeated
sensory stimulation with a 5-s interstimulus interval under bath
application of biogenic amines. The LG firing probabilities of control
(gray squares), 5 lM serotonin (open circles) application and 10 lM
octopamine (filled circles) application are plotted. d Effect of
serotonin (d1) and octopamine (d2) on synaptic response of the LG
to the sensory stimulation. Bath application of 5 lM serotonin (d1) or
10 lM octopamine (d2) enhances both the a ? a0 components and
the b component of the EPSPs. Asterisk indicates spike of LG
Invert Neurosci (2015) 15:2
Since subordinate animals show mainly submissive acts
such as retreats and tailflips in response to the attacks of
dominant animals (Sato and Nagayama 2012; Ueno and
Nagayama 2012), it would be reasonable that subordinate
animals show a slow decline in spike activity of the LGs to
repeated sensory stimulation, since a decrease in the rate of
habituation would be necessary to evade repeated attacks
of dominant animals. By contrast, the result that the rate of
habituation of dominant crayfish is also less than control
animals is contradictory since dominant animals perform
more aggressively and appeared not to need to evade encounters from subordinates. Herberholz et al. (2001) have
reported that crayfish frequently show offensive tailflips
during agonistic encounters. Offensive tailflip begins with
an abdominal extension followed by abdominal flexions
and re-extensions. The abdominal extensions are accompanied by a spread of the tailfan that is maintained during
the abdominal flexion, which occurred primarily around the
anterior abdominal segmental joints, while the posterior
segments remained extended. This configuration helps to
throw the animal up into the water column above the opponent and could act to allow a crayfish to abruptly change
orientation in a short period, i.e., during the LG-mediated
tailflip. Offensive tailflip occurs with a short interval of less
than 5 s before the dominance order is determined.
Dominant animals show a decrease in the rate of habituation in response to sensory stimulation with a 5-s interstimulus interval. It would be advantageous therefore to
prevent habituation of tailflip with short stimulus intervals
during agonistic bouts. Sensory reception between LG and
offensive tailflip might be linked causally, and a common
neural mechanism could occur to prevent habituation. In
fact, with a long interstimulus interval of 60 s, the rate of
habituation of dominant animals is similar to that of socially isolated animals, although subordinate animals still
show a slow rate of the habituation (Araki et al. 2013).
The neuromodulators, serotonin, and octopamine play a
key role in dominance hierarchy formation (Huber et al.
1997; Huber and Delago 1998). Direct injection of serotonin or octopamine into the systemic circulation of crayfish induces dominant-like or subordinate-like status and
motivation, respectively (Momohara et al. 2013). Serotonin
and octopamine also affect the responsiveness of LG to
sensory stimulation (Glanzman and Krasne 1983; Yeh et al.
1997; Edwards et al. 2002; Krasne and Edwards 2002;
Antonsen and Edwards 2007; Lee et al. 2008) and increase
the number of stimuli required to habituate the LG response to sensory stimulation with a 5-s interstimulus interval (Araki et al. 2005). Under bath application of 5 lM
serotonin (open circles in Fig. 5c) and 10 lM octopamine
(filled circles in Fig. 5c), the rate of decease in the response
of LG to sensory stimulation is slower. Only 20–35 % of
animals fail to respond with a spike within 5 trials of
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stimulation. The response of LG decreases by 50 % just
after 15 stimuli, and approximately 30 % of animals still
respond with a spike after 40 stimuli. Thus, both serotonin
and octopamine decrease the rate of habituation, and the
numbers of stimuli needed to habituate LG increase significantly (p \ 0.05; log-rank test), as is the case of both
dominant and subordinate animals. The sensory-evoked
EPSP of LG is significantly increased in amplitude, including both the early a ? a0 component and the later b
component under bath application of 5 lM serotonin
(Fig. 5d1) or 10 lM octopamine (Fig. 5d2). If the stimulus
intensity of sensory stimulation is set subthreshold for LG
spikes during serotonin or octopamine application, sensory-evoked EPSPs of the LGs frequently induce spikes as
shown in Fig. 5d2. These findings suggest that the increment in the number of stimuli required to habituate the LG
response to sensory stimulation is possibly linked to serotonin and octopamine levels of dominant and subordinate
crayfish. Serotonin and octopamine enhance the LG responsiveness to sensory stimulation (Araki et al. 2005;
Araki and Nagayama 2012).
The majority of serotonin and octopamine receptors
belong to a superfamily of G-protein-coupled receptors,
and their effects are mediated by second messengers
(Hoyer et al. 1994; Gerhardt et al. 1997; Roeder 1999). We
therefore confirmed the effects of second messengers upon
LG response to the sensory stimulation. When a cAMP
analogue, sp-cAMPS, is iontophoretically injected into LG,
the amplitude of EPSPs in LG to sensory stimulation is
increased significantly (Fig. 6a). Similarly, intracellular
injection of IP3 agonist, adenophostin A, into the LG also
enhances the LG response to stimulation (Fig. 6b). Bath
application of a cGMP analogue has no obvious effect
upon the process of LG habituation (Araki et al. 2005).
Following activation of adenylate cyclase, cAMP level is
increased, while the level of IP3 increases following activation of phospholipase C. The enhanced effect of bath
application of serotonin (Fig. 5d1) is not detected under
bath application of the mixture of serotonin and adenylate
cyclase inhibitor, SQ22536 (Fig. 6c1). On the other hand,
an enhancing effect of octopamine (Fig. 5d2) is unchanged
under SQ22536 (Fig. 6c2). Thus, the effect of serotonin
appears to be mediated by an increase in the level of
cAMP. Intracellular injection of U-73122, a phospholipase
C inhibitor, into the LG has no obvious effect on the effect
of serotonin on the LG response to sensory stimulation
(Fig. 6d1). Enhancement of LG response to sensory
stimulation, especially that of early a and a0 components
mediated by octopamine, is canceled by intracellular injection of U-73122 into LG (Fig. 6d2). b component of the
EPSP is not affected significantly since responses of sensory interneurons to sensory stimulation also increase
during bath application of octopamine. Thus, effect of
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Fig. 6 Modulatory effects of second messengers on LG response to
sensory stimulation. a Iontophoretically injected 50 lM Sp-cAMPS
into LG enhances the sensory-evoked EPSP of both the a ? a0
components and the b component. b Iontophoretic injection of
100 lM adenophostin A into LG enhances the sensory-evoked EPSP
of both the a ? a0 components and the b component. c Effect of bath
application of the adenylate cyclase inhibitor SQ22536 of 100 lM in
concentration upon the enhancing effect of serotonin (c1) and
octopamine (c2). The serotonin-induced enhancement of the
sensory-evoked EPSP in LG is canceled, while the octopamineinduced enhancement is not affected. d Effect of iontophoretic
injection of the phospholipase C inhibitor U-73122 of 40 lM in
concentration into the LG upon the enhancing effect of serotonin (d1)
and octopamine (d2). Serotonin-induced enhancement of the sensoryevoked EPSP of the LG is not affected, but the octopamine-induced
enhancement of sensory-evoked EPSP of the a ? a0 components is
canceled. Asterisks indicates spikes of LG
octopamine is mediated by an increase in the level of IP3
(Araki and Nagayama 2012). The second messenger,
cAMP system, is linked to the serotonin-induced synaptic
enhancement of the LG response, while IP3 system is
linked to the octopamine-induced synaptic enhancement of
physiologically and morphologically (Wine 1984; Nagayama et al. 1993a, b, 1994), their neurotransmitters have
been characterized (Ushizawa et al. 1996; Nagayama et al.
1997a, 2004; Aonuma and Nagayama 1999), and the
pharmacological profiles of receptors have been clarified
(Miyata et al. 1997; Nagayama 2005; Sosa et al. 2004;
Spitzer et al. 2005, 2008). Thus, the cellular mechanisms
for habituation are accessible behaviorally and neurophysiologically. Furthermore, the process of habituation
shows status-dependent plasticity depending on the effects
of biologic amines, e.g., serotonin and octopamine. Habituation of the LG escape reaction is known to be retained
for several hours in intact animals (Krasne and Woodsmall
1969; Wine et al. 1975). Furthermore, dominant animals
The LG tailflip of the crayfish is a good system to elucidate
the neural basis of habituation, since the LG tailflip is a
stereotyped behavioral act and that many neurons contributing to the LG system are identifiable both
Invert Neurosci (2015) 15:2
show a slow rate of habituation a week after they became
dominants. Descending tonic inputs are thought necessary
to maintain habituation for long periods (Krasne and
Teshiba 1995). The action of serotonin and octopamine is
related to the activation of cAMP and IP3s messenger
systems (Araki et al. 2005; Araki and Nagayama 2012).
Since descending inputs from brain exert moment-to-moment control over LG’s excitability and habituation
(Krasne and Teshiba 1995; Shirinyan et al. 2006), further
studies to clarify the interactions between descending inputs and the local regulation of LG habituation within the
terminal abdominal ganglion will provide the neurophysiological basis for the long-term memory of
The neural circuitry that produces the LG tailflip is
similar to that underlying the fast startle response, the
C-start, of teleost fishes. Mauthner neurons in the hindbrain
have descending large diameter axons that are responsible
for triggering this response (Eaton et al. 1981). Like
crayfish LGs, the Mauthner neurons also receive sensory
inputs directly from sensory afferents via electrical synapses and indirectly from sensory interneurons via chemical synapses. Although the Mauthner neuron is not
command neuron, it belongs to a class of neurons called
reticulospinal neurons. The C-start response is also known
to show habituation (Roberts et al. 2011). To compare the
homology of neural mechanisms for habituation of both
animals would be interesting to better understand the
convergent evolution of neural circuitry.
Acknowledgments This work was supported by grants from the
Ministry of Education, Science, Sport, Culture and Technology to
T.N. We are grateful to Dr. H. Aonuma for his assistance of this work.
Conflict of interest
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