INTEG. AND COMP. BIOL.

, 42:705–715 (2002)

Modulation of the Crayfish Escape Reflex—Physiology and Neuroethology1

FRANKLIN B. KRASNE2 AND DONALD H. EDWARDS

Department of Psychology, UCLA, Los Angeles, California 90095-1563 and
Department of Biology, Georgia State University, Atlanta, Georgia 30302-4010

SYNOPSIS. We review here factors that control the excitability of the giant neuron-mediated tail-flip escape
behavior in crayfish, focusing especially on recent findings concerning serotonergic modulation. Serotonin
can either facilitate or inhibit escape depending on concentration and pattern of application. Low concen-
trations facilitate while high ones inhibit; however, if high concentrations arise gradually they facilitate
instead of inhibiting. The effects of serotonin can also be altered by social experience, with application
regimens that cause facilitation in social isolates coming to produce inhibition after an extended period of
living as a subordinate. Attempts to understand both the possible physiological basis of some of these com-

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plexities and their possible function are discussed. Neuroethological investigations indicate that giant neuron-
mediated escape is inhibited during the initial fights that establish social relationships and is facilitated in
their immediate aftermath. Once the relationship of a pair is well-established, the presence of the dominant
tends to suppress giant neuron-mediated escape (but not tail-flip escape mediated by non-giant circuitry) in
the subordinate, but the presence of the subordinate has relatively little effect on the dominant. These
patterns of modulation can be seen as consistent with the known variations in serotonin’s effect as a function
of concentration and social experience and may provide a biological reason for these variations.

For most neurobiologists it is an article of faith that unique opportunities: (1) The connection between cel-
the behavior which emerges from nervous systems is lular phenomena and behavior is often much clearer in
the product of a neural machine. But the machine is invertebrates due to the relative simplicity of some of
one in which a given neural circuit does not always their behavior-producing neural circuitry; thus, we can
work the same way. Operational properties of a circuit discover the natural uses made of instances of plastic-
can change due to learning and due to modulation by ity and modulation. (2) The very diversity of inverte-
other circuits, imparting to the behavior of a given brates inevitably exposes us to a wider array of phe-
individual the great variety and irregularity that makes nomena than we would see from studying mammals
the behavior of animals and ourselves interesting and alone; thus it helps us distinguish what is general from
a challenge to our understanding. To a great degree, what is not.
though not entirely, changes in the properties of neural We review here work on plasticity seen in the neural
circuits are due to changes in the functional properties circuitry which mediates escape behavior in crayfish,
of their synapses. with a focus on recent surprising findings on seroto-
The last several decades have seen remarkable pro- nergic modulation, and their possible functional sig-
gress in uncovering various forms of synaptic plastic- nificance.
ity induced either by activity or by chemical modula-
tors. Some of the first forms of synaptic plasticity to THE NEURAL CIRCUITRY UNDERLYING
be described and related to behavioral plasticity were ESCAPE BEHAVIOR
in invertebrates—specifically in Aplysia (see articles Escape can be mediated in two rather different ways
by Sutton and Carew, 2002, and Sherff and Carew, as indicated separately on the left and right of Figure
2002 [this issue]; Kandel, 1976) and also in crayfish 1A (for a review see Edwards et al., 1999). The cir-
(Krasne, 1969; Zucker, 1972; Zucker et al., 1971). In cuitry on the left produces either of two distinct types
parallel with these studies on invertebrates were dis-
of response, depending on locus of stimulation. Each
coveries beginning at roughly the same time on mam-
response type is associated with a giant command neu-
malian hippocampal LTP (Bliss and Collingridge,
ron. The medial giants (MGs) sum input from anterior
1993), which is now widely regarded as a possible
sensory channels and make output connections with
mechanism of associative learning. This latter line of
giant flexor motor neurons (motor ‘‘giants’’—MoGs in
work has led to a veritable frenzy of activity directed
both at working out cellular and molecular mecha- Fig. 1) that cause a dart backwards when the excitation
nisms of long-term potentiation (LTP) and at trying to produces even one spike in the MGs. The lateral gi-
fathom what might be its actual roles visa vi behavior. ants (LGs) sum input from posterior channels and
Research on LTP has somewhat eclipsed invertebrate cause an upward rotation that distances the hind end
research, but invertebrate systems continue to provide of the animal from the disturbing stimulus. MG and
LG responses are often referred to as ‘‘reflex’’ respons-
1 From the Symposium Recent Advances in Neurobiology pre-
es or as ‘‘giant fiber (GF)’’ responses for the giant
sented at the Annual Meeting of the Society for Integrative and
axons of the MGs and LGs, which run the length of
Comparative Biology, 2–6 January 2002, at Anaheim, California. the nerve cord. GF responses are very prompt (muscle
2 E-mail: [email protected] potentials can begin within 3 msec) and are good at

705
706 F. B. KRASNE AND D. H. EDWARDS

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FIG. 1. Escape tail-flip circuitry of the crayfish. A. Schematic circuit. Primary mechanosensory afferents of the abdomen (black neurons in
top row) excite LG neurons directly and via an intervening layer of interneurons (black neurons in second row). The LGs in turn excite giant
motor neurons (MoGs) to muscles (marked by solid black squares) whose contraction causes an upward, forward trajectory of movement due
to bending at joints indicated by solid black circles over the crayfish depicted at the bottom of the figure. The overall movement produced is
indicated by the black silhouette at the lower right of the figure. Stippled neurons and marks show a corresponding arrangement for the
production of backward-directed tail-flips commanded by MGs in response by to stimulation of anterior sensory neurons, and the resultant
response is indicated by the stippled drawing at the lower left of the figure. Non-giant tailflip circuitry activates an independent set of flexor
muscles (FFs) that innervate the same flexor muscles. GFs also recruit FFs via the segmental giant neurons (SGs), which excite motor and
pre-motor neurons within the non-G circuitry (modified from Edwards et al. [1999] which should be consulted for further explanation). The
efficacy of synapses at levels I and II are particularly subject to alteration by past activity and by neuromodulators, as discussed in this review
and summarized in Figure 2. B. Form of EPSP evoked in LGs by a volley of sensory root activity. Explanation in text.

getting the crayfish moving away from the source of sophisticated responses of the non-G circuitry come at
stimulation rapidly. a price: They are far from prompt (latencies are about
The circuitry on the right has no giant neurons (non- 100 msec).
G circuitry), is much more complex, and is far from Before beginning to discuss forms of synaptic mod-
fully charted. Whereas the giant-containing circuitry ulation that have been studied in the GF circuitry it
produces only two very stereotyped forms of response should be noted that only the synapses between pri-
(‘‘back’’ and ‘‘upward rotation’’) and always single mary afferents and the sensory interneurons of the GF
flexions, the responses generated by the non-giant cir- circuitry are conventional chemical synapses. The re-
cuitry have a seemingly infinite variety of possible mainder are voltage-gated electrical synapses, which
forms and can occur in repetitive strings (‘‘swims’’). pass current effectively only when the presynaptic side
Using this circuitry crayfish can move directly away of the synapse is made positive relative to the post-
from an oblique stimulus, avoid obstacles, and move synaptic side by the arrival of a presynaptic spike (Ed-
toward specific locations. Unlike GF responses, which wards et al., 1991; Furshpan and Potter, 1959; Giaume
ordinarily occur only in response to abrupt and fairly et al., 1987; Jaslove and Brink, 1986). Qualitatively,
vigorous stimulation, non-G responses are often these voltage-gated electrical synapses have many of
prompted by gradually developing threats. The more the same properties as chemical synapses. These in-
 MODULATION OF THE CRAYFISH ESCAPE REFLEX 707

thresholds when, for example an animal has found a

food source and is avidly feeding (Krasne and Lee,
1988) or when an animal is firmly clutched by a fish-
erman and reflex tailflips would be useless (Krasne and
Wine, 1975).
Recently, work done by former students from each
of our laboratories has begun to suggest that transmis-
sion at the voltage-gated electrical synapses on the
GFs is also subject to intrinsic, activity-dependent
change. In both their experiments, as in other experi-
ments to be discussed here, brief electrical test shocks
to sensory nerves were delivered every few minutes
and responses recorded in the LGs with intracellular
microelectrodes. The shocks produce compound excit-

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atory postsynaptic potentials (EPSPs) (Fig. 1B), which
have a first elevation (the a component) resulting from
monosynaptic input from the primary afferents and a
second (the b component), which is due to input ar-
riving via the sensory interneurons. Shi-Rung Yeh
(personal communication) has found that several sec-
ond long trains of 4 Hz stimuli to the afferents cause
both the monosynaptic and disynaptic components of
FIG. 2. Factors regulating the excitability of GF escape. The levels
(I and II) at which synaptic transmission is affected are those indi- LG EPSPs to grow and stay high for many hours (Fig.
cated in Figure 1. At the right are listed known forms of behavioral 3, top; Fig. 2, line 3). The augmentation is specific to
modulation. Arrows indicate possible causality of forms of behav- input pathways that were given the 4 Hz stimulation
ioral modulation with dashed lines marked by ??? indicating entirely and seems to be entirely prevented if the calcium ion
speculative causality. Details in text.
chelators BAPTA or EGTA were previously injected
into the LGs. Thus, this phenomenon is similar to LTP
clude polarized transmission, temperature sensitive at glutamatergic synapses in that its induction is de-
synaptic delay, and modulation of EPSP size by post- pendent on a transient elevation of calcium ions in the
synaptic membrane potential level. Because of these postsynaptic neuron. Since these are voltage-depen-
similarities it can be very difficult to distinguish volt- dent electrical synapses and not glutamatergic ones,
age-gated electrical synapses from chemical ones, and this is an interesting parallelism. Sun Hee Lee has
as we shall see, EPSPs produced by these electrical found that similar stimulation (5 Hz) given for the
synapses are also subject to modulation of kinds that much longer time of 5 min causes a depression that
one normally associates with chemical synapses. also lasts a long time and is blocked by BAPTA in the
LGs (Lee, 1996; Lee and Park, 1997). This long-term
MODULATION AND PLASTICITY IN GF CIRCUIT depression (LTD)-Iike phenomenon seems to occur
The first form of plasticity discovered in GF cir- mainly in the disynaptic (b) EPSP but presumably still
cuitry circuit was intrinsic depression of transmitter involves depression at synapses directly on the LGs,
release as the result of repetitive presynaptic activity since it is blocked by preventing calcium ion elevation
at the cholinergic synapses made onto sensory inter- in the LGs. The functional consequences of LTP are
neurons that innervate the LGs (Fig. 2, line 1; Krasne, quite unknown, but it seems possible that the LTD may
1976; Miller et al., 1992; Zucker, 1972). Like probably play some role in habituation (Fig. 2, line 4).
all escape behavior, GF responses habituate to repeated
stimulation, and this presynaptic activity-dependent Aminergic neuromodulation of transmission to the
depression is at least in part responsible. Its discovery LGs
in the late 60s was exciting, because it was one of the The story that is the major focus of this review be-
first times that a simple kind of learning had been gan about 20 yr ago when Ed Kavitz and his students
traced to a specific neural mechanism. (Livingstone et al., 1980; Kravitz, 1988) discovered
It was soon after found that a second influence af- that injection of serotonin into lobster or crayfish al-
fecting whether crayfish will escape to threats is a tered synaptic transmission at certain neuromuscular
GABA-ergic inhibitory input directly to the GF den- synapses and caused animals to adopt a posture that
drites (Fig. 2, line 2; Krasne and Wine, 1975; Vu and was seen as similar to that adopted by socially domi-
Krasne, 1993; Vu et al., 1993). This inhibition, which nant animals, whereas octopamine, the arthropod an-
is controlled by higher centers, is turned on under a alog of adrenaline, caused a subordinate-like posture.
variety of circumstances and greatly elevates the stim- It seemed likely that since these agents affected pos-
ulus threshold for producing GF escape. This ‘‘tonic tures associated with fight and flight, they might also
inhibition’’ contributes to habituation (Krasne and affect escape. In particular, it was conjectured that oc-
Teshiba, 1995) and also serves to elevate GF escape topamine might facilitate GF escape while serotonin
708 F. B. KRASNE AND D. H. EDWARDS

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FIG. 3. LTP and LTD-like changes at synapses on the LGs. Left: Changes in amplitude of sensory root shock-evoked test EPSPs in LGs
resulting from short (above) and long (below) trains of 4–5 Hz stimulation via the electrodes used to produce test EPSPs (but set higher than
test voltages to recruit more afferents fibers during trains of LTP and LTD inducing stimulation). Right: Comparison of the effects of trains
under normal conditions and when BAPTA had been infused into LGs prior to the experiment. LTP data from Shi-Rung Yeh, personal
communication, with permission. LTD data from Sun Lee (Lee, 1996; Lee and Park, 1997, and personal communication, with permission).

might suppress it. This conjecture was soon verified. mission at the first synapse that leads to behavioral
Octopamine enhances transmission at the first synapse sensitization of GF escape (Krasne and Glanzman,
(Fig. 2, line 5; Bustamante and Krasne, 1991; Glanz- 1986); the possibility that this is mediated by octopa-
man and Krasne, 1983), and serotonin was initially mine is obvious, but it has not been established. Lim-
found to inhibit transmission (but see further below), ited evidence also suggests that serotonin might under
at least in part by an action at the level of the LGs some circumstances share with GABA a role in the
(Fig. 2, line 6; Glanzman and Krasne, 1983; Vu and tonic inhibition mentioned above (Glanzman and Kras-
Krasne, 1993). It was found soon thereafter that trau- ne, 1986, but see Vu and Krasne, 1993).
matic stimulation also causes a facilitation of trans-
Serotonergic modulation
Though the discovery of serotonergic inhibition was
consistent with the conjectures that had prompted the
first test of serotonin’s effects, it has recently become
clear that these effects are much more complex than
originally believed. Experiments done in the Edwards
lab by Shi-Rung Yeh, over a decade after the first ex-
periments showing an inhibitory effect, consistently
found serotonin to have a facilitatory effect on trans-
mission to the LGs (Yeh et al., 1996, 1997). After a
period of some confusion it eventually became clear
that the difference lay in the regimen of serotonin ap-
plication (Teshiba et al., 2001). When serotonin is in-
troduced as rapidly as possible (FAST in Fig. 4) and
left in place for only 10–15 min (SHORT in Fig. 4),
FIG. 4. Mean time course of b component EPSP amplitude during as was done in the original experiments, inhibition de-
and following 5-HT exposure. Beta component amplitudes were nor-
malized by their values on the last stimulation before the start of 5- velops over 5–10 min and washes out at the same rate
HT exposure. The EPSP amplitude on the last trial in 5-HT is taken (Fig. 4, solid triangles). However, when serotonin lev-
as the 0 time point for the washout graphs (Teshiba et al., 2001). els are allowed to increase only gradually (SLOW in
 MODULATION OF THE CRAYFISH ESCAPE REFLEX 709

Fig. 4), reaching full concentration over some 20–30

min, and are allowed to remain in place for 30–45 min
(Fig. 4, solid circles), facilitation rather than inhibition
is seen, and this facilitation persists for as much as 5
hr or more even during washout (Yeh et al., 1997).
The persistence of facilitation during wash requires ex-
posures of longer than 10 to 15 min; the longevity of
serotonin-induced facilitation in Aplysia is also well
known to depend on duration of exposure (see Sutton
and Carew, 2002 and Sherff and Carw, 2002 [this is-
sue]). One gets persistent facilitation even with fast
application if one uses a low dose of 5-HT (i.e., 1028–
1026 M as opposed to 1024 and above—Fig. 4, open
circles).

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It is not yet known whether sertotonin alters the
properties of the electrical junctions on the LGs. How-
ever, one can in part understand these modulations as
reflections of altered ionic conductances in the LGs.
The inhibition is associated with an increased conduc-
tance and small depolarization postsynaptically (Vu
and Krasne, 1993), which given that the equilibrium FIG. 5. Hypothesis to explain effects of concentration and rate of
application of serotonin on its effect. Fl, F2, and I are unidentified
potential for chloride is about 8 mV above the resting signaling molecules conjectured to mediate modulatory effects of
level, is probably due to increased chloride conduc- serotonin on EPSP amplitude. gK and gCl, respectively, are potassium
tance. The facilitation that is caused at low 5-HT con- ion and chloride ion conductances, changes of which may be re-
centrations (Fig. 2, line 7) is associated with a de- sponsible for alterations of EPSP amplitude. Conjectured 5-HT
creased conductance and also a depolarization (Yeh et threshold and onset characteristics needed to cause production of
signaling molecules are specified at the left. The predictions made
al., 1997), which seems most likely to be due to a by the model, explained in the text, are indicated at the bottom of
decreased potassium ion conductance. Thus inhibition the figure.
and facilitation are due to non-mutually exclusive
causes that could co-exist.
Another way in which the facilitation and inhibition F2, so facilitation then develops. With SLOW-HIGH
are independent is that that their underlying intracel- exposures F2 builds up before 5-HT concentration
lular progenitors can apparently co-exist. Thus, the reaches a level that can activate the inhibitory path-
precursors of the facilitation appear to develop even at way, and F2 has reached a level where it can suppress
high doses that cause inhibition; however the inhibi- the formation of I by the time serotonin is concentrated
tion prevents or masks expression of the facilitation. enough to exceed the threshold of the inhibitory path-
This can be seen when one washes out a high dose of way. These ideas have been developed into a compu-
serotonin that has been in place long enough for per- tational model that correctly predicts (qualitatively) all
sistent facilitation to develop. Inhibition is seen for as of the types of modulation observed (Teshiba et al.,
long as the 5-HT is present, but when it is washed 2001).
away, the inhibition gives way to facilitation (Fig. 4, The various delivery regimens used in our experi-
open triangles, FAST, LONG, HIGH). ments may correspond to different modes of serotonin
Although the signaling molecules that mediate 5- delivery that occur naturally. Serotonin is released
HT’s effects in the LGs are not yet known, Figure 5 both synaptically within abdominal ganglia neuropile,
proposes the logic of an intracellular signaling scheme which could provide natural FAST, HIGH exposures
that could account for the complex effects of 5-HT and is also released into the blood as a hormone from
exposure regimen on modulatory effect (see Teshiba a variety of sites, presumably providing a SLOW,
et al., 2001). A pathway with a low 5-HT threshold LOW form of delivery (Beltz and Kravitz, 1983; Yen
and relatively slow onset produces facilitation, while et al., 1997).
a pathway with a high 5-HT threshold and faster onset
produces inhibition. The buildup of the final stage sig- Social dependence of 5-HT effects
naling molecule of each pathway suppresses the for- There is yet another, and rather extraordinary layer
mation of buildup of the final stage molecule of the of complexity to this story. Crayfish have long been
other pathway. In this scheme FAST-LOW exposures known to form social hierarchies (Bovbjerg, 1953;
cause facilitation because only the facilitatory pathway Lowe, 1956). When two crayfish are brought together,
gets activated. FAST-HIGH exposures cause inhibition one generally becomes dominant and the other sub-
because signaling molecule I builds up first and pre- ordinate after a short period of interaction (see below).
vents formation of F2; however F1 still builds despite Quite remarkably, social experience alters the effects
the manifest inhibition, and when 5-HT is washed out, of serotonin on transmission to the LGs: Whereas in
persisting F1 is allowed to promote the formation of social isolates low concentrations of serotonin facili-
710 F. B. KRASNE AND D. H. EDWARDS

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FIG. 6. Effects of 5-HT depend on animal’s social history. Center: Change in EPSP amplitude (measured at disynaptic b peak of EPSP)
produced by 5-HT in social isolates kept for varying periods with a dominant partner. Left and right: Examples of EPSPs before (CNT) and
during 5-HT exposure (5-HT) in an isolate (left) and after 12 days of subordinance (right). (Data from Yeh et al., 1997).

tate transmission to the LGs (as discussed above), after chloride conductance-increasing type of inhibition pro-
a crayfish has lived for 1–2 wk as a subordinate, se- duced by high 5-HT, but a hyperpolarizing, presum-
rotonin comes to inhibit transmission to the LGs (Fig. ably potassium conductance-increasing inhibition.
6; Yeh et al., 1996, 1997). This is not the depolarizing, Thus, whereas in isolates low concentrations of 5-HT
decrease potassium ion conductance, in subordinates
serotonin increases potassium ion conductance—a di-
rectly opposite effect (Fig. 2, line 8). Living as a dom-
inant causes a more subtle change: Whereas in isolates
prolonged exposure to serotonin causes facilitation that
persists after washout of serotonin, dominants do not
show this persistence of facilitation.
Preliminary pharmacological experiments suggest
that exposure to a vertebrate 5-HT2 receptor agonist
[a-methyl 5-HT] mimics the faciltatory effects of se-
rotonin, while a 5-HT1 agonist [1-(3-chlorophenyl) pi-
perazine] has little effect in isolates but has a large
inhibitory effect in subordinates (Fig. 7; Yeh et al.,
1997). This interesting observation suggests the pos-
sibility that the transformation of serotonin’s net effect
from facilitatory to inhibitory might be due to the in-
sertion of a species of 5-HT1 receptor into LG mem-
FIG. 7. Effects of serotonin and agonists on isolated and subordi- brane or to some alteration in such a receptor’s down-
nate crayfish (Data from Yeh et al., 1997). stream signaling effects. However, the effects of the
 MODULATION OF THE CRAYFISH ESCAPE REFLEX 711

agonists in dominants belie this straight-forward inter- provide any obvious reason for the changes in sero-
pretation, since 5-HT1 agonists also produce an inhib- tonin’s effect as a function of social status. Indeed, if
itory effect in dominants, while 5-HT2 agonist effects one assumes that serotonin is released during agonistic
are not altered. The cloning of crayfish 5-HT receptors encounters, one is faced with the conundrum that se-
is in progress, and soon receptor antibodies will be rotonergic modulation should make subordinates less
used to look for changes in the amount of specific likely to escape during social interactions than are
receptor types on the LGs as the result of social ex- dominants!! Obviously some information on whether
perience (Spitzer et al., 2001). and how escape is in fact modulated during social in-
These are fascinating observations. The finding that teractions would help.
experience can alter the qualitative effect of a neuro-
modulator seems both new and remarkable. Moreover, NEUROETHOLOGY OF GF MODULATION DURING
when one considers that mental illnesses often seem SOCIAL INTERACTIONS
to involve abnormalities in neuromodulation and that When a pair of crayfish previously unknown to each
mental illness seems to have important experiential de- other is placed in the same living space, three phases

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terminants, these observations provide a possible mod- of interaction ensue (Herberholz et al., 2001; Issa et
el for why life experiences might be so important. Es- al., 1999):
pecially interesting from this point of view is the ob-
I. The animals engage in a fight during which one
servation that serotonin’s effect in a subordinate can
animal often grasps the other and engages in ‘‘of-
again take on the typical isolate profile if the subor-
fensive’’ tailflips that appear to be demonstrations
dinate is re-isolated, or can take on the profile typical
of dominance and strength; in lobsters seemingly
of a dominant if the subordinate is paired with an an-
similar behavior can result in the target animal’s
imal over which it becomes dominant. However, once
dismemberment (Huber and Kravitz, 1995). After
an animal has been dominant long enough for its re-
a few minutes phase I ends and phase II is ushered
sponse to serotonin to take on the typical dominant
in when the animal that is getting the worst of it
profile, this profile is retained even if the animal later
gives up and flips away.
becomes subordinate (Yeh et al., 1997). Thus, some
II. The new dominant frequently harasses the new
experience-caused changes in neuromodulation are
subordinate, which backs or flips away.
readily reversible, but others are not.
III. After some days the relationship becomes firmly
We now have a rather long list of ways in which
established. The subordinate cowers at the margin
the GF circuitry is modifiable, and we have some plau-
of the living space and appears to try to keep as
sible connections to behavior (Fig. 2). It is interesting
low a profile as possible, resting close as possible
to note that each known form of behavioral control
to the substrate, while the dominant moves about
may be due to a multiplicity of modulatory mecha-
freely, maintains a tall posture, and very occa-
nisms. Thus, the roles played by each and the possi-
sionally harasses the subordinate, which slinks or
bility of their interaction becomes an interesting topic
flips away. The transition to phase III is illustrated
for future investigation. That there are interactions
in Figure 8 (Issa et al., 1999), which shows that
seems clear. For example, if GABA is infused along
encounters greatly decrease over days and that re-
with 5-HT, the persistent effects of 5-HT usually pro-
treats replace escape as the most common subor-
duced by prolonged application do not develop (Ghiu-
dinate evasion response.
seli and Krasne, unpublished). And experiments in
progress seem to show that in the presence of picro- Edwards’ group recently developed a noninvasive
toxin, which blocks the chloride channel normally procedure to record tailflips from freely behaving
opened by GABA, LTD may not be produced (Lee, animals during their initial encounter in a 30 min
Shirinyan, and Krasne, unpublished). This raises in- session (Herberholz et al., 2001). They found (Fig.
teresting questions for the future. 9) that during phase I there were almost no GF-me-
We turn next to the role of serotonergic modulation diated flips; the frequent offensive tail flips are me-
in the natural economy of the crayfish. It seems plau- diated by non-giant circuitry. Since the animals are
sible to assume that serotonergic modulation of escape fighting vigorously and providing many of the sorts
reflexes plays an important role during social interac- of stimuli that would ordinarily evoke GF escape,
tions, since otherwise its change of effect as a function the lack of responses suggests that GF escape is in-
of social status makes little sense. This belief is sup- hibited. Subsequently, during phase II, the dominant
ported by serotonin’s promotion of a dominant stance harasses the subordinate a great deal and the sub-
(Livingstone et al., 1980) and by the more recent find- ordinate commonly escapes either with GF or non-
ing that injection of serotonin reduces the extent to G responses. It appears that during this period GF
which animals will retreat during fights (Huber et al., excitability is elevated in both subordinates and
1997). However, the general notion that serotonergic dominants, since both animals typically make a few
modulation of the GFs might be used during social GF escape responses per observation period that do
interactions does not help to explain either the reason not appear to be caused by the sorts of sudden stim-
for the complex mix of facilitatory and inhibitory ef- uli that are ordinarily needed to trigger GF escape.
fects as a function of application regimen nor does it Additional GF responses are made during this period
712 F. B. KRASNE AND D. H. EDWARDS

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FIG. 8. Decline in frequency of aggressive interactions following formation of a dominance hierarchy in groups of 5 juvenile crayfish. Top:
Average frequencies (and SD) of encounters between any two animals and of each of four behaviors. Measurements were made over 15 min
perods during the first hour of group formation and over one hour periods during the following days. Bottom: Relative proportions of each
behavior (Issa et al., 1999). There is a gradual decline in the number of encounters and a gradual increase in the extent to which the subordinate
reacts to threats by walking retreats rather than tail-flip escape reactions.

when the dominant attacks the subordinate or when that could be used to directly test the excitability of
the subordinate bumps into the dominant while ex- the LG (Figure 10; Krasne et al., 1997). When the
ecuting a non-G escape response. sensory excitability of the animals tested apart and to-
Animals that have had considerable experience with gether was compared, it was found that, while the an-
one another (phase III) have been studied with chron- imals are together, the sensory threshold of subordi-
ically implanted stimulating and recording electrodes nates is about triple its value when they are apart; in
 MODULATION OF THE CRAYFISH ESCAPE REFLEX 713

FIG. 10. Effect of encounter of a subordinate with its dominant

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partner on LG threshold. The ability of a 0.25 msec voltage pulse
to sensory nerves via chronically implanted electrodes to fire LGs
(and also a large sensory interneuron, A) was tested every few min-
utes with the level of shock being varied so as to continuously eval-
uate the threshold voltage for firing (from Krasne et al., 1997).
FIG. 9. Ethogram of a pair of juvenile crayfish at their first meeting.
The abscissa is the ordinal number of the observed behavioral ac-
tion; this, rather than time, is used so that actions occurring very
close together in time are somewhat spread out along the axis. Giant
neuron-mediated (GF) and non-G mediated (NG) tail flips were dis-
tinguished with the aid of bath recording electrodes. ‘‘Attack’’ refers
to rapid approaches leading to contact. ‘‘Approach’’ refers to slower
approach. ‘‘Retreat’’ refers to movements away not involving tail
flips. ‘‘Swims’’ are strings of tail-flips with about a 100 msec inter-
flip interval (Herberholz et al., 2001).

contrast, the threshold of the dominant is elevated just

slightly while the animals are together.
It may seem odd that the subordinates should inhibit
GF escape in the presence of their dominant partner.
However, inhibition of GF escape does not mean in-
hibition of all escape. Subordinates do in fact execute
escape responses when harassed by the dominant;
however, these are virtually always non-G rather than
GF responses (Krasne et al., 1997). Our interpretation
of the suppression of GF responses by subordinates
depends on the differing capacities of the giant and
non-giant circuitry. GF responses are useful for a sur- FIG. 11. Summary of GF excitability changes during social en-
prise attack when an animal is taken unawares, but the counters. Top row: Estimates of GF reflex excitability during each
more sophisticated non-G responses are more adaptive phase of a developing social relationship are indicated by circles.
if an animal has a chance to see its attacker coming Estimates of excitability when the conspecifics are separated (the
‘‘apart’’ level) are indicated as a dashed line, and the direction of
and time to prepare. GF and non-G escape are in-effect change from this level is emphasized by the arrows. For phase I the
incompatible strategies. An animal that is watching a measure of excitability used is the number of GF response per abrupt
possible attacker approach and is preparing to execute stimulus, which is virtually zero. Although we do not know how
an optimal non-G response at an optimal moment common GF responses would be to the same physical stimulus if
should avoid the production of a stereotyped GF tail- animals were apart, we believe from experience with crayfish that
they would be fairly common. We have therefore placed a dashed
flip, and crayfish do generally inhibit GF escape cir- line with question marks to indicate the ‘‘apart’’ level of responses;
cuitry when preparing to make a non-G responses the placement of this line at 1 is arbitrary, but its placement above
(Krasne and Wine, 1975, 1984). We suggest that sub- zero is consistent with the known sensory sensitivity of GF respons-
ordinates are in a continual state of vigilance and that es. For phase II we have graphed the number of GF responses per
animal that occurred during the approximately 25 min observation
the dominant probably never takes them by surprise. period without any abrupt stimulus of the kind that would normally
If so, it is probably adaptive for subordinates to sup- be needed to trigger GF escape (based on study of the high speed
press GF-mediated responding. It might also make video data of Herberholz et al., 2001). Normally, in animals outside
sense for excitability of non-G responses to increase a social situation, GF responses would not occur without clear abrupt
in subordinates when animals are together, but this has stimuli; therefore, we have placed the dashed ‘‘apart’’ level line at
zero. For phase III, we have used the increase of the directly mea-
not been tested. sured sensory threshold for LG escape, when animals that were sep-
These neuroethological observations are summa- arated were brought together, as a measure of the excitability in-
rized at the top of Figure 11. GF escape appears to be crease of GF escape.
714 F. B. KRASNE AND D. H. EDWARDS

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