J Neurophysiol 94: 4002– 4010, 2005.
First published August 24, 2005; doi:10.1152/jn.00432.2005.
Serotonin Mediates Learning-Induced Potentiation of Excitability
Brian D. Burrell1 and Christie L. Sahley2
1
Neuroscience Group, Division of Basic Biomedical Sciences, University of South Dakota School of Medicine, Vermillion, South Dakota;
and 2Department of Biological Sciences and Graduate Neuroscience Program, Purdue University, West Lafayette, Indiana
Submitted 28 April 2005; accepted in final form 19 August 2005
Burrell, Brian D. and Christie L. Sahley. Serotonin mediates learning-
induced potentiation of excitability. J Neurophysiol 94: 4002– 4010,
2005. First published August 24, 2005; doi:10.1152/jn.00432.2005.
Sensitization potentiates excitability in an interneuron, the S-cell, that
is critical for this form of learning in the whole-body shortening reflex
of the medicinal leech. Serotonin (5-HT) also increases S-cell excit-
ability, and serotonergic modulation is known to be critical for
sensitization of whole-body shortening, suggesting that 5-HT medi-
ates learning-induced enhancement of S-cell excitability. In this pa-
per, the role of 5-HT in mediating sensitization-induced potentiation
of S-cell excitability was examined. Potentiation of S-cell excitability
by 5-HT was blocked by the 5-HT receptor antagonist methysergide
and by intracellular injection of the G-protein inhibitor GDP-␤-S,
indicating that a metabotropic 5-HT receptor was involved. Bath
application of Rp-cAMP, an inhibitor of protein kinase A (PKA),
blocked 5-HT-induced potentiation of excitability, whereas db-cAMP,
a cAMP analogue that activates PKA, mimicked the potentiating
effects of 5-HT on the S-cell. During sensitization of the shortening
reflex in semi-intact preparations, methysergide and Rp-cAMP pre-
vented learning-induced potentiation of S-cell excitability, as well as
the increase in S-cell activity that normally occurs during sensitiza-
tion. Furthermore, sensitization-induced increases in the shortening
reflex did not occur in preparations treated with methysergide or
Rp-cAMP. These results demonstrate that sensitization-induced en-
hancement of S-cell excitability is mediated by 5-HT and suggests
that these changes may contribute to this form of learning.
INTRODUCTION
There has been increased appreciation for the role that
modulation of neuronal excitability plays during learning and
memory (see reviews by Daoudal and Debanne 2003; Wu et al.
2002; Zhang and Linden 2003). Increases in excitability during
learning have been observed in a number of different regions of
the mammalian brain including CA1 and CA3 pyramidal cells
in the hippocampus (Moyer et al. 1996, 2000; Oh et al. 2003;
Stackman et al. 2002; Thompson et al. 1996), cerebellar
Purkinje neurons (Shreurs et al. 1997, 1998), and pyramidal
cells in the pyriform cortex (Saar et al. 1998). Learning-
induced increases in excitability also have been observed in a
number of invertebrate species, including sensory cells in
Hermissenda and Aplysia (Alkon et al. 1985; Antonov et al.
2001; Cleary et al. 1998), motor neurons in Lymnaea (Straub
and Benjamin 2001), and interneurons in Helix and the medic-
inal leech (Burrell et al. 2001; Gainutdinov et al. 1998).
The medicinal leech is particularly useful for studying the
mechanisms that underlie learning-related neuroplasticity. Like
many invertebrates, it has a well-characterized CNS in which it
Address for reprint requests and other correspondence: B. D. Burrell, Asst.
Professor, Neuroscience Group, Div. of Basic Biomedical Sciences, University
of South Dakota School of Medicine, 414 E. Clark St. Vermillion, SD 57069
(E-mail: bburrell@usd.edu).
4002 0022-3077/05 $8.00 Copyright © 2005 The American Physiological Society
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is possible to record from single, identifiable neurons and to
monitor simultaneously learning-induced changes in behavior
and the response properties of individual neurons. An addi-
tional advantage of the leech is that it is possible to focus on a
single interneuron type, the S-cell, which not only integrates a
variety of afferent inputs and disseminates this information
throughout the CNS but also appears to be critical to certain
forms of learning-related behavioral plasticity.
The S-cells form a linear network of electrically coupled
interneurons that extends throughout the leech CNS (Fig. 1A).
Each S-cell receives afferent input from touch, pressure, and
nociceptive mechanosensory neurons as well as photoreceptive
neurons (Baccus et al. 2000; Muller and Scott 1981; Peterson
1984), and action potentials that are elicited by this afferent
input propagate reliably throughout the entire S-cell network.
The S-cell contributes to learning-related plasticity of the leech
whole-body shortening reflex, a defensive withdrawal reflex
that can be initiated by either mechanical or photo stimulation
and involves the near-simultaneous contraction of the entire
body. Although the S-cell receives input from afferents that
elicit shortening, is active during this behavior, and has direct
input to at least one type of shortening motor neuron (the L
motor neurons; see Fig. 1B), its activity alone is not sufficient
to trigger shortening, and lesions of the S-cell network do not
affect the animal’s capacity to shorten (Gardner-Medwin et al.
1973; Shaw and Kristan 1995, 1999). However, lesions of the
S-cell chain completely disrupt sensitization and partially dis-
rupt dishabituation of the leech shortening response (Burrell et
al. 2003; Modney et al. 1997; Sahley et al. 1994). In addition,
S-cell activity during elicited shortening is enhanced after
sensitization, which may be mediated, in part, by increases in
the excitability of the S-cell that also occur during sensitization
(Burrell et al. 2001). This increase in excitability may due to
the effects of serotonin (5-HT), a modulatory neurotransmitter
that is critical for sensitization, full dishabituation, and asso-
ciative learning of leech shortening (Ehrlich et al. 1992; Sahley
1994). Bath application of 5-HT directly increases S-cell ex-
citability (Burrell et al. 2001) and the interneuron’s response to
mechanosensory stimuli (Belardetti et al. 1982; Burrell et al.
2002). S-cell excitability is also enhanced by stimulation of the
5-HT-containing Retzius cells (Fig. 1B) (Burrell et al. 2001).
Although these results suggest that 5-HT contributes to
sensitization-induced increases in S-cell excitability, a direct
link between 5-HT- and sensitization-induced changes in S-cell
excitability has not been established. Therefore the major goal
of this paper is to determine the role of 5-HT in sensitization-
mediated increases in S-cell excitability by blocking the effects
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 MODULATION OF EXCITABILITY DURING LEARNING
of 5-HT on the S-cell during sensitization training in a behav-
iorally intact preparation. The results demonstrate that 5-HT-
induced potentiation of S-cell excitability is due to activation
of a metabotropic 5-HT receptor coupled to a cAMP/protein
kinase A (PKA) second-messenger pathway and that blocking
5-HT’s effect on the S-cell, either at the receptor or cAMP/
PKA level, prevents sensitization-induced increases in excit-
ability. These experiments represent the first time, to our
knowledge, that learning-induced changes in excitability have
been directly monitored and blocked at the same time that
learning was taking place.
METHODS
Electrophysiology
Medicinal leeches (Hirudo medicinalis) weighing 3 g were ob-
tained from a commercial supplier (Leeches USA) and maintained in
pond water (0.5 g/1 l H2O Hirudo salt (Leeches USA) at 18°C. Single
ganglia were dissected in leech saline [which contained (in mM) 110
NaCl, 5 NaOH, 4 KCl, 1.8 CaCl2, 1.5 MgCl2, and 10 HEPES] and
placed in a recording chamber (chamber volume ⬇ 0.75 ml) under
constant perfusion (⬇1 ml/min). Intracellular recordings were made
by impaling the cell with a sharp microelectrode made from borosili-
cate glass (1.0 mm OD, 0.78 mm ID; FHC) and pulled to a tip
resistance of 20 –30 M⍀ using a Sutter P-97 puller (Sutter Instru-
ments). Electrodes were filled with 3 M potassium acetate (KAc).
Recordings were made using a BA-1S amplifier (NPI). Either a Grass
S88 stimulator with SIU5 stimulus isolation units (Astromed) or an
STG 1004 programmable stimulator (Multichannel Systems) was
used to deliver current pulses. Electrophysiological data were viewed
on a digital oscilloscope and stored on a computer using a Digidata
1322A analog/digital interface and Axoscope 9.0 acquisition software
(Axon Instruments).
S-cells were identified by their position in the ganglion (variable,
but always in the central glial packet on the ventral side of the
ganglion), size (5–10 ␮m), and shape of their action potential (50 to
60 mV amplitude, ⬇2-ms spike width). Measurements of intrinsic
electrical properties of the S-cell were made as follows. Input resis-
tance was measured by injecting a hyperpolarizing current pulse (1
nA, 500 ms) and measuring the resulting change in membrane
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FIG. 1. The S-cell network and organization of the whole-
body shortening neural circuit. A: leech CNS consists of a
chain of 21 ganglia (1 per body segment) plus fused ganglia
in the head and tail regions (not shown) that are linked by a
connective nerve, and each ganglion contains a single S
interneuron that projects an axon into the anterior and pos-
terior connective nerves. At the midpoint of each connective,
these axons form an S-to-S electrical synapse, resulting in a
chain or network that extends throughout the leech CNS. B:
whole-body shortening can be initiated by mechanosensory
stimuli that activate touch (T), pressure (P), and sometimes
nociceptive (N) neurons, which have synaptic input onto
local motor neurons (Nicholls and Purves 1970), i.e., those
that are in either the same segment or in the segment adjacent
to where the stimuli are applied (L, longitudinal motorneu-
ron; DE and VE, dorsal and ventral excitor motor neurons).
More distant motorneurons must be activated by an unknown
neuron or neurons that are responsible for triggering whole-
body shortening (these triggering elements presumably re-
ceive input from the T-, P-, and N-cells). The S-cell is also
activated during shortening by the mechanosensory neurons
and has synaptic output onto the L-motor neurons and the
serotonin (5-HT)-containing Retzius cell (Wang 1999). The
Retzius cell, in turn, has an excitatory (⫹) modulatory input
onto the S-cell (Burrell et al. 2001). Connections marked (*)
are polysynaptic.
potential. Excitability was measured by recording the number of
action potentials initiated during a 500 ms, 1 nA depolarizing current
pulse (AP/pulse). Additional excitability measurements were made
from the S-cell’s response to this stimulus. The membrane potential at
which action potential initiation occurred (membrane potential thresh-
old) was measured and defined as the point at which the velocity of
the change in membrane potential equaled 20 mV/s (Bekkers and
Delaney 2001). The rate of the increase in membrane potential (slope)
prior to the first action potential initiation was also measured by taking
the slope of the membrane potential depolarization (dV/dt) during a
5-ms window, 10 ms prior to action potential initiation (Cudmore and
Turrigiano 2004). Finally, the interspike interval of the first pair of
action potentials initiated during the stimulus pulse was measured (1st
ISI). The resting potential of the S-cell was kept constant for both pre-
and post-5-HT treatment measures of excitability.
Changes in S-cell excitability were tested prior to (pretest) a 5-min
perfusion with 10 ␮M 5-HT (n ⫽ 9). This was followed by a 5-min
wash in normal saline and then a second test of excitability (posttest).
This treatment has been shown to produce robust increases in excit-
ability that have reached a plateau by this time, although enhancement
of excitability lasts ⱖ1 h (Burrell et al. 2001). The 5-HT-induced
changes in excitability were compared with groups of S-cells in which
the 5-HT treatment was replaced with normal saline (n ⫽ 9), 5-HT
plus 100 ␮M methysergide (a nonspecific antagonist of metabotropic
5-HT receptors; n ⫽ 7), methysergide alone (n ⫽ 3), 5-HT plus 1 mM
GDP-␤-S (a membrane impermeable competitive G-protein antago-
nist; n ⫽ 4), GDP-␤-S alone (n ⫽ 4), 5-HT plus 50 ␮M Rp-cAMP (a
membrane permeable inhibitor of PKA; n ⫽ 5), Rp-cAMP alone (n ⫽
5), or 50 ␮M dibutyryl cAMP (db-cAMP; a membrane permeable
analogue of cAMP; n ⫽ 3). GDP-␤-S was dissolved in filtered 3 M
KAc and applied intracellularly by using the GDP-␤-S solution to fill
the recording electrode and then allowing it to diffuse into the
interneuron. The remaining drugs were dissolved in leech saline and
applied via the perfusion through the recording chamber. All drugs
were obtained from Sigma (St. Louis).
Behavioral training and recordings from
semi-intact preparations
Dissections were carried out in an ice-lined dissecting dish with
ice-cold leech saline. Segments 1–2 were left intact and pinned dorsal
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4004 B. D. BURRELL AND C. L. SAHLEY
side up in a silicone elastomer (Sylgard)-lined dish (Fig. 2). A
longitudinal incision was made down the dorsal midline of segments
3– 8 so that the cylinder of the leech body could be opened up, and the
resulting flat sheet of skin was rotated and pinned ventral side up (this
will be referred to as the body-wall portion of the preparation). A
small longitudinal incision was made on the ventral side of segment 4,
exposing the segmental ganglion for S-cell intracellular recordings.
Segments 9 –11 were left intact and pinned dorsal side up with the
posterior end connected to an isometric tension transducer (model
FIG. 2. Diagram of the semi-intact preparation used for sensitization train-
ing. The ganglion in the 4th segment was exposed so that intracellular
recordings could be made from the S-cell before and after training. This
portion of the preparation (segments 3– 8) was rotated so that it was ventral
side up during training to accommodate S-cell recordings. The rest of the
preparation was pinned dorsal side up. Changes in the whole-body shortening
reflex were monitored using a tension transducer attached to segments 9 –11.
S-cell activity during shortening was monitored using a suction electrode
applied to the connective nerve posterior of the segment 14 ganglion.
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72– 4481; Harvard Apparatus) using a nylon monofilament so that
whole-body shortening could be recorded and quantified. The remain-
ing portion of the preparation, segments 12–14, consisted of the CNS
only (ganglia and connective nerve), and the connective nerve poste-
rior of the segment 14 ganglion was pulled into a suction electrode for
extracellular recordings of S-cell activity. The S-cell action potential
produces the largest signal in such recordings (Frank et al. 1975), so
S-cell activity during elicited shortening responses could be easily
monitored and recorded. The preparation was allowed to rest for 30
min in saline with 10 mM glucose before undergoing sensitization
training. All training was conducted at room temperature (22–24°C).
Whole-body shortening was elicited by delivering controlled elec-
troshocks to the skin using implanted Teflon-coated silver wire
electrodes (75-␮m bare diameter; A-M Systems). Each electrode
consisted of a pair of silver wires in which the Teflon was removed
from the portions of the wire that were in contact with the skin, and
two pairs of wire electrodes were implanted in the skin. One pair was
implanted at segment 4 and elicited whole-body shortening (test site)
using stimulation parameters from Shaw and Kristan (1995): five
stimulus pulses, 1 ms pulse duration, 10 Hz frequency. The stimulus
intensity at the test site was set at just above threshold for eliciting
shortening. The second set was implanted between segments 7 and 8
and was used to deliver the sensitizing stimulus (sensitizing site; 10
stimulus pulses, 1 ms pulse duration, 10 Hz frequency, intensity set at
⬇25% above threshold).
Immediately before training, intracellular recordings were made
from the segment 4 S-cell, and its excitability properties were mea-
sured in the same manner used for the 5-HT experiments described
previously. In preparations that would undergo sensitization in normal
saline (n ⫽ 7), training began by making two presensitization (pre-
test), see Fig 4A measurements of the shortening response and the
accompanying S-cell activity at an intertrial interval (ITI) of 2.5 min.
This was followed by the delivery of two trains of sensitizing stimuli,
again with a 2.5 min ITI. Five minutes after the last sensitizing trial,
two postsensitization (posttest) measurements of the shortening re-
flex/S-cell activity were made (2.5 min ITI). Postsensitization mea-
surements of S-cell excitability were made immediately after the last
behavioral posttest.
To test whether sensitization-induced potentiation of S-cell excit-
ability was mediated by 5-HT, sensitization training was carried in the
presence of 100 ␮M methysergide (n ⫽ 6) or 50 ␮M Rp-cAMP (n ⫽
5). Control experiments, in which delivery of the sensitizing stimuli
was omitted, were carried out in normal saline (n ⫽ 6), 100 ␮M
methysergide (n ⫽ 6) or 50 ␮M Rp-cAMP (n ⫽ 6). All drugs were
bath-applied 5 min before the first pretest. Injection of membrane
impermeable agents into the S-cell that might antagonize 5-HT-
induced changes in excitability (e.g., GDP-␤-S or PKA inhibitory
peptides) was not possible using these semi-intact preparations. Mech-
anosensory stimuli that elicit shortening cause multiple S-cells to fire
(2–5 adjacent interneurons) and increases in the S-cell network’s
response due to increased stimulus intensity, 5-HT, or sensitization
are distributed across multiple S-cells (Baccus et al. 2001; Burrell et
al. 2002; Cruz et al. 2003). Therefore using injectable drugs to block
potential serotonergic modulation of the S-cell would require finding
and injecting a minimum of three separate S-cells, by which time there
would have been a substantial reduction in the viability of the
semi-intact preparation.
Data analysis
Changes in S-cell excitability properties from both the 5-HT and
sensitization training experiments were analyzed as follows. For each
neuron, multiple measurements of a given excitability property (AP/
pulse, slope, input resistance, etc.) were made and an average calcu-
lated for both the pre- and posttest excitability measurements. The
averaged value from each neuron was then used for statistical analysis
and graphical representation. One-way ANOVA in which the post-
94 • DECEMBER 2005 • www.jn.org
 MODULATION OF EXCITABILITY DURING LEARNING
treatment levels had been normalized relative to the pretreatment level
was used to detect differences between the various treatment groups.
Behavioral and S-cell activity data from the sensitization training
experiments were analyzed using a one-way ANOVA with repeated
measures (presensitization trial 2 and postsensitization trials 1 and 2,
all normalized to the 1st presensitization trial).
RESULTS
5-HT activates a metabotropic receptor coupled to a cAMP/
PKA pathway
Application of 10 ␮M 5-HT induced a significant increase in
the number of action potentials fired by the S-cell during
current injection [Fig. 3A; AP/pulse 1-way ANOVA, F(8,36) ⫽
5.96, P ⬍ 0.0001]. This was accompanied by a significant
decrease in the interval between the first pair of action poten-
tials [Fig. 3B; 1st ISI 1-way ANOVA, F(8,34) ⫽ 3.15, P ⬍
0.01] and an increase in the rate of depolarization prior to the
first action potential [Fig. 3C; slope 1-way ANOVA, F(8,36) ⫽
3.53, P ⬍ 0.005]. The 5-HT-induced changes in these excit-
ability properties were significantly different when compared
with S-cells in which 5-HT application was omitted (saline
group; Fig. 3D; post hoc test: AP/pulse, P ⬍ 0.0005; 1st ISI,
P ⬍ 0.05; slope, P ⬍ 0.01). No significant change was
observed in the membrane potential threshold [1-way ANOVA,
F(8,35) ⫽ 0.48] or in input resistance [1-way ANOVA,
F(8,35) ⫽ 0.50]. 5HT-induced increases of S-cell excitability
were blocked by co-application of 100 ␮M methysergide (Fig.
3D; 5-HT⫹methysergide). Excitability measures from the
5-HT⫹methysergide group were significantly different com-
pared with the 5-HT group (post hoc test: AP/pulse, P ⬍ 0.001;
1st ISI, P ⬍ 0.01; slope, P ⬍ 0.01) but were not significantly
different from the saline group. S-cells treated with methyser-
gide alone were not significantly different from the saline or
5-HT⫹methysergide groups but were significantly different
from the 5-HT-treated group in terms of AP/pulse (post hoc
P ⬍ 0.001), first ISI (P ⬍ 0.01), and slope (P ⬍ 0.01).
There was no significant difference in membrane potential
threshold or input resistance in S-cells from the saline-,
5-HT⫹methysergide-, methysergide-, and the 5-HT-treated
groups.
Although methysergide was effective at blocking 5-HT-
mediated increases in S-cell excitability, it is a relatively
nonspecific 5-HT receptor antagonist and does not help in
identifying the 5-HT receptor subtype(s) involved in this mod-
ulation. It is difficult to identify 5-HT receptor subtypes in
invertebrates using vertebrate pharmacological tools (Tierney
2001). Therefore components of the signal transduction path-
ways activated by the 5-HT receptors were examined instead.
This approach was used not only to characterize the cellular
mechanisms mediating 5-HT modulation of the S-cell but also
to provide tools with which to disrupt this process during
sensitization training.
The first step was to identify whether a G-protein-coupled
receptor mediated 5-HT-induced changes in S-cell excitability
because there are both iono- and metabotropic 5-HT receptors
(Hoyer et al. 2002). Therefore 1 mM GDP-␤-S was included in
the recording electrode used to monitor S-cell excitability
while the interneuron was perfused with 10 ␮M 5-HT
(5HT⫹GDP-␤-S). GDP-␤-S prevented 5-HT-induced in-
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4005
creases in S-cell excitability (Fig. 3E). Changes in AP/pulse
(post hoc P ⬍ 0.001), first ISI (P ⬍ 0.05), and slope (P ⬍ 0.05)
in the 5-HT⫹GDP-␤-S group were significantly different from
those treated with 5-HT alone but not significantly different
from the saline group. The same result was observed in S-cells
treated with GDP-␤-S alone (Fig. 3E; post hoc results: AP/
pulse, P ⬍ 0.05; 1st ISI, P ⬍ 0.01; slope, P ⬍ 0.01). No
significant difference in S-cell membrane potential threshold or
input resistance was observed among the 5-HT⫹GDP-␤-S,
GDP-␤-S, 5-HT, or saline groups (Fig. 3E).
Next, the involvement of a cAMP/PKA second-messenger
cascade was examined. 5-HT-induced increases in S-cell ex-
citability properties were blocked when 50 ␮M Rp-cAMP was
included in the perfusate (Fig. 3F; 5HT⫹Rp-cAMP). Changes
in AP/pulse (post hoc P ⬍ 0.005), first ISI (P ⬍ 0.05), and
slope (P ⬍ 0.01) in the 5-HT⫹Rp-cAMP group were signifi-
cantly different from those treated with 5-HT alone but not
significantly different from the saline group. The same result
was observed in S-cells treated with Rp-cAMP alone (Fig. 3F),
indicating that the drug did not affect S-cell excitability di-
rectly (post hoc results: AP/pulse, P ⬍ 0.0005; 1st ISI, P ⬍
0.05; slope, P ⬍ 0.01). No significant difference in S-cell
membrane potential threshold or input resistance was observed
among the 5-HT⫹Rp-cAMP, Rp-cAMP, 5-HT, or saline
groups (Fig. 3F). If 5-HT modulation of excitability does
require the activity of a cAMP/PKA second-messenger system,
then increasing the intracellular level of cAMP should mimic
the effects of 5-HT on this interneuron. A 5-min perfusion of
the S-cell with 50 ␮M db-cAMP produced increases in AP/
pulse and slope and decreases in first ISI that were statistically
indistinguishable from those of 5-HT (Fig. 3F) but statistically
different from the saline group (post hoc results: AP/pulse, P ⬍
0.001; 1st ISI, P ⬍ 0.05; slope, P ⬍ 0.05). No significant
difference in S-cell membrane potential threshold or input
resistance was observed between the db-cAMP, 5-HT, or saline
groups (Fig. 3F). Together, the results from the Rp-cAMP and
db-cAMP experiments demonstrate that 5-HT-induced in-
creases in S-cell excitability require activation of a cAMP/PKA
second-messenger cascade.
Sensitization is blocked by treatments that inhibit 5-HT
modulation of excitability
To examine whether sensitization-induced increases in S-
cell excitability are the result of serotonergic modulation,
sensitization training was carried out under conditions that
block potentiation of excitability by 5-HT. Specifically, semi-
intact preparations were treated with either 100 ␮M methyser-
gide or 50 ␮M Rp-cAMP during sensitization training. In
addition, the effects of these treatments on sensitization of
whole-body shortening and the accompanying increase in S-
cell activity during shortening were examined.
The intensity of the whole-body shortening reflex more than
doubled after delivery of the sensitizing stimuli (Fig. 4, B and
C) in preparations that underwent sensitization training in
normal saline (sens/saline group). S-cell activity during short-
ening also increased relative to presensitization levels (Fig. 4,
B and D). No change in either the shortening reflex or S-cell
activity was observed in preparations from sensitization control
experiments in which delivery of the sensitizing stimuli was
94 • DECEMBER 2005 • www.jn.org
4006 B. D. BURRELL AND C. L. SAHLEY

omitted (Fig. 4, C and D; con/saline group). Sensitization-

induced increases in both the shortening reflex and S-cell
activity were blocked in preparations treated with either me-
thysergide (sens/methy group) or Rp-cAMP (sens/Rp-cAMP
group; Fig. 4, C and D).
Sensitization-induced changes in whole-body shortening
were confirmed using two-way ANOVA with repeated mea-
sures [treatment effect, F(5,29) ⫽ 4.00, P ⬍ 0.01; trial effect,
F(2,58) ⫽ 0.11; interaction effect, F(10,58) ⫽ 3.20, P ⬍
0.005]. Post hoc analysis confirmed that the postsensitization
shortening in the sens/saline group was significantly greater
compared with shortening in all the sensitization control
groups (con/saline, P ⬍ 0.001; con/methy, P ⬍ 0.005; con/
Rp-cAMP, P ⬍ 0.0005). Furthermore, shortening in the sens/
saline group was significantly greater than in preparations that
underwent sensitization training in the presence of either me-
thysergide (sens/methy P ⬍ 0.01) or Rp-cAMP (sens/Rp-
cAMP, P ⬍ 0.01). The level of shortening in the sens/methy
and sens/Rp-cAMP groups was not significantly different from
any of the sensitization control groups (con/saline, con/methy,
and con/Rp-cAMP). Similarly, sensitization-induced increases
in S-cell activity during the shortening response were con-
firmed statistically [treatment effect, F(5,29) ⫽ 3.02, P ⬍ 0.05;
trial effect, F(2,58) ⫽ 7.91, P ⬍ 0.001; interaction effect,
F(10,58) ⫽ 2.25, P ⬍ 0.05]. Post hoc analysis showed that
postsensitization S-cell activity in the sens/saline group was
significantly greater than activity in the preparations sensitized
in the presence of methysergide (sens/methy, P ⬍ 0.01) or
Rp-cAMP (sens/Rp-cAMP, P ⬍ 0.05) or any of the sensitiza-
tion control groups (con/saline, P ⬍ 0.001; con/methy, P ⬍
0.01; con/Rp-cAMP, P ⬍ 0.05). Postsensitization S-cell activ-
ity was not significantly different between any of the sensiti-
zation control groups or between these groups and the sens/
methy or sens/Rp-cAMP groups.
The lack of sensitization in methysergide- or Rp-cAMP-
treated groups was not due to any detrimental effect of these
drugs on the preparations during training. Shortening and
S-cell responses were stable during sensitization control exper-
iments in these preparations and were statistically indistin-
guishable from the con/saline group (Fig. 4, C and D). In
addition, the lack of sensitization in the sens/methy or sens/
Rp-cAMP groups was not due to any differences in the level of
the whole-body shortening response at the start of training.
There was no significant difference in the presensitization trial
1 shortening reflex from the sens/saline, sens/methy, and sens/
Rp-cAMP groups [1-way ANOVA, F(2,16) ⫽ 1.20]. Further-
more, the lack of sensitization was not due to any deficits in the

FIG. 3. 5-HT increases excitability via a metabotropic 5-HT receptor that

activates a cAMP/protein kinase A (PKA) second-messenger pathway.
Changes in excitability properties before and after application of 10 ␮M 5-HT:
action potentials (AP)/pulse (A), 1st interspike interval (ISI) in the train (B),
and slope of depolarization (dV/dT) prior to the 1st AP in the train (C). D:
average percent change (⫾SE) in excitability properties in 5-HT-treated (black
bars) and saline control (white bars) S-cells (same data used in E and F).
Co-application of methysergide blocked 5-HT’s effects on excitability (dark
gray-bars) but did not alter excitability itself (light gray-filled bars). E:
co-application of GDP-␤-S blocked 5-HT’s effects (fine right-hatched bars)
but had no effect by itself (coarse right-hatched bars). F: co-application of
Rp-cAMP block 5-HT’s effects (fine left-hatched bars) but had no effect by
itself (coarse left-hatched bars). Application of db-cAMP (cross-hatched bars)
mimicked the effects of 5-HT.

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 MODULATION OF EXCITABILITY DURING LEARNING 4007

the sens/saline group, 21 ⫾ 2.43 and 18.8 ⫾ 3.68 for the

sens/methy group, and 21 ⫾ 4.13 and 15.3 ⫾ 3.78 for the
sens/Rp-cAMP group (average presensitization S-cell response
during shortening was 7.71 ⫾ 1.54 action potentials). One-way
ANOVA with repeated measures showed no significant effect
of treatment group [F(2,13) ⫽ 0.06] and no significant inter-
action effect [F(2,16) ⫽ 1.97]. There was a significant effect
for the repeated measures [F(1,13) ⫽ 8.73, P ⬍ 0.05] that was
the result of the first sensitizing stimulus consistently eliciting
more S-cell action potentials than the second sensitizing stim-
ulus in all three treatment groups.

Sensitization alters S-cell frequency distribution

Changes in the frequency distribution of S-cell activity as a
result of sensitization were also analyzed. The average fre-
quency from the two pre- and postsensitization trials was
calculated at 100 ms intervals following onset of the test
stimulus and then averaged across all preparations in the
sens/saline group. The frequency of S-cell activity from the
postsensitization trials was enhanced relative to the presensi-
tization trials with differences between the average activity
levels becoming greater at later poststimulus intervals (Fig. 5).
This sensitization effect was statistically significant based on
ANOVA [F(1,36) ⫽ 10.94, P ⬍ 0.005]. Not surprisingly, there
was also a significant effect of the poststimulus interval as a
result of the frequency decreasing at each time interval
[F(1,36) ⫽ 5.35, P ⬍ 0.001]. There was no interaction effect
[F(5,36) ⫽ 0.27]. The pre- versus postsensitization change in
overall S-cell frequency elicited during shortening is shown in
the Fig. 5, inset.

Sensitization-induced enhancement of S-cell excitability

is 5-HT dependent
In agreement with earlier observations (Burrell et al. 2001),
S-cell excitability increased after sensitization training, and the
pattern of sensitization-induced increases in excitability prop-
erties was identical to those produced by 5-HT in this study.
Sensitization produced an increase in AP/pulse [Fig. 6, A and
D; 1-way ANOVA, F(5,15) ⫽ 5.52, P ⬍ 0.005], a decrease in

FIG. 4. Methysergide and Rp-cAMP block sensitization-induced increases

the shortening reflex and S-cell activity. A: time line of training protocol. B:
sensitization training increased the intensity of the elicited shortening response
(top traces) and the S-cell activity during shortening (bottom traces). Average
(⫾SE) increase in the whole-body shortening reflex (C) and S-cell activity (D)
in the sensitized (F) vs. control (E) preparations trained in normal saline. Both
methysergide (
) and Rp-cAMP (■) blocked sensitization-induced increases in
behavior and S-cell activity. Neither methysergide nor Rp-cAMP had any
effect on shortening or S-cell activity when applied to preparations that did not
undergo sensitization training (ƒ and 䊐, respectively).
FIG. 5. Sensitization increases the rate of S-cell activity. Frequency distri-
ability of the sensitizing stimuli to activate the S-cell, which is bution of S-cell activity prior to (䊐) and after (■) sensitization. For each
preparation, the average S-cell frequency from both pre- and postsensitization
critical for the induction of sensitization (Burrell et al. 2003). tests was calculated for each interval and then those numbers were used to
The number of S-cell action potentials elicited during the two calculate the average and SE shown in the graph. Inset: average (⫾SE) overall
sensitizing stimulation trials was 20.4 ⫾ 1.5 and 19 ⫾ 3.44 for S-cell frequency during elicited shortening.

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4008 B. D. BURRELL AND C. L. SAHLEY
FIG. 6. Sensitization increases S-cell excitability and those changes are
blocked by treatments that antagonized 5-HT’s effect on this interneuron.
Changes in excitability properties before and after application of sensitization
training: AP/pulse (A), 1st ISI in the train (B), slope of depolarization (dV/dT)
prior to the first action potential in the train (C). D: as with 5-HT, S-cells from
preparations that had undergone sensitization training in normal saline (sens/
saline; black bars) exhibited an increase in AP/pulse and slope, a decrease in
1st ISI, and no change in membrane potential threshold or input resistance. No
significant change in these excitability properties was observed in S-cells from
the control (no sensitizing stimuli delivered) preparations in normal saline
(con/saline; white bars). Sensitization-induced increases in excitability were
not observed in preparations trained in methysergide (sens/methys; fine
hatched bars) or Rp-cAMP (Sens/Rp-cAMP; fine cross-hatched bars). No
changes in excitability were observed in control preparations treated with
either methysergide (con/methys; coarse hatched bars) or Rp-cAMP (con/Rp-
cAMP; coarse cross-hatched bars).
the first ISI [Fig. 6, B and D; F(5,15) ⫽ 4.48, P ⬍ 0.05], and
an increase in slope [Fig. 6, C and D; F(5,15) ⫽ 4.72, P ⬍
0.01] relative to S-cells from nonsensitized preparations, but
there was no change in membrane potential threshold
[F(5,15) ⫽ 0.29] or input resistance [F(5,15) ⫽ 1.56]. Further-
more, sensitization-induced potentiation of excitability was
blocked by treatments that prevented 5-HT-induced enhance-
ment of excitability, namely methysergide and Rp-cAMP (Fig.
6D), demonstrating that enhanced S-cell excitability during
sensitization was mediated by 5-HT. Post hoc analyses con-
firmed that sensitization-induced changes in excitability prop-
erties were significantly different in the S-cells in the sens/
saline group compared with those in the sens/methy (AP/pulse,
P ⬍ 0.001; 1st ISI, P ⬍ 0.005; slope, P ⬍ 0.005) or the
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sens/Rp-cAMP (AP/pulse, P ⬍ 0.001; 1st ISI, P ⬍ 0.005;
slope, P ⬍ 0.01) groups. Excitability changes in the sens/saline
group were also significantly different from the con/saline
(AP/pulse, P ⬍ 0.005; 1st ISI, P ⬍ 0.005; slope, P ⬍ 0.001),
con/methy (AP/pulse, P ⬍ 0.005; 1st ISI, P ⬍ 0.01; slope, P ⬍
0.005), and con/Rp-cAMP (AP/pulse, P ⬍ 0.01; 1st ISI, P ⬍
0.05; slope, P ⬍ 0.05) groups. There were no significant
differences in the excitability properties between any of the
sensitization control groups or between the sens/methy, sens/
Rp-cAMP and any of the control groups (Fig. 6D).
DISCUSSION
These results confirm previous work (Burrell et al. 2001) in
which both sensitization training and 5-HT induced similar
increases in excitability in the S-cell and extends those findings
by demonstrating that 5-HT mediates sensitization-induced
potentiation of S-cell excitability. 5-HT-induced potentiation
of S-cell excitability is mediated by a metabotropic 5-HT
receptor that upregulates cAMP production and activates PKA,
although additional signaling pathways may be involved as
well. The fact that serotonergic modulation of the S-cell
requires a cAMP/PKA second-messenger cascade is consistent
with earlier findings showing that 5-HT-mediated increases in
the S-cell response to mechanosensory stimuli utilize a cAMP/
PKA pathway (Belardetti et al. 1982). Sensitization-induced
enhancement of S-cell excitability was blocked by treatments
that either antagonized 5-HT binding (methysergide) or pre-
vented cAMP activation of PKA (Rp-cAMP), demonstrating
that learning-induced potentiation of S-cell excitability is a
5-HT-dependent process.
Interestingly, treatments that blocked 5-HT-mediated poten-
tiation of S-cell excitability during sensitization training pre-
vented sensitization-induced increases in S-cell activity and
whole-body shortening. These results suggest that increased
S-cell excitability contributes to sensitization of the shortening
reflex but cannot be considered conclusive because the drugs
used to block serotonergic modulation of the S-cell were
bath-applied (see METHODS) and may have prevented 5-HT- or
cAMP/PKA-dependent changes in other cells that contribute to
sensitization. For example, 5-HT decreases afterhyperpolariza-
tion (AHP) in T and P mechanosensory neurons through a
cAMP-dependent process (Catarsi and Brunelli 1991; Catarsi
et al. 1995). Decreased AHP in these cells could enhance
afferent input to the S-cell (or other neurons that contribute to
shortening) by reversing branch point propagation failure of
action potentials (Mar and Drapeau 1996) or inducing action
potential reflection at these branch points (Baccus et al. 2000,
2001). In addition, 5-HT modulation of the S-to-S electrical
synapses may increase the instantaneous frequency of action
potentials as they propagate through the S-cell network (Moss
et al. 2005), which could also contribute to increases in the rate
of S-cell activity. It is not known if 5-HT modulation of the
S-to-S electrical synapses is also a PKA-dependent process.
The S-cell appears to have two roles during sensitization.
First, an intact S-cell network is necessary during the delivery
of the sensitizing stimuli based on experiments in which
sensitization was prevented when the S-cell chain had been
lesioned (Burrell et al. 2003; Modney et al. 1997; Sahley et al.
1994). The precise role of the S-cell during this induction stage
of sensitization is not known, but as the present results show,
94 • DECEMBER 2005 • www.jn.org
 MODULATION OF EXCITABILITY DURING LEARNING
sensitizing stimuli strongly activate the S-cell. This may lead to
the stimulation of 5-HT-containing Retzius cells throughout
the leech CNS because these cells receive synaptic input from
the S-cell (Wang 1999). Second, the S-cell itself may contrib-
ute to producing the sensitized response, i.e., increased short-
ening. Sahley et al. (1994) observed a strong correlation
between the level of S-cell activity and the intensity of the
sensitized shortening response, whereas no such correlation
was detected in the nonsensitized shortening reflex. In addition,
results presented here and from previous experiments (Burrell
et al. 2001; Sahley et al. 1994) show that S-cell excitability and
activity during shortening are enhanced during sensitization.
The S-cell may also be incorporated into other behaviors as a
result of sensitization. Debski and Friesen (1986) observed an
increase in S-cell activity during sensitization of leech swim-
ming behavior, which is also 5-HT- and cAMP-dependent
(Zaccardi et al. 2004), and suggested that increased S-cell
activity might contribute to this behavior during sensitization
even though this interneuron is not part of the swimming neural
circuit. However, the necessity of the S-cell in the “expression”
of the sensitization for shortening or swimming has not been
conclusively demonstrated.
The biophysical basis for 5-HT-induced increases in S-cell
excitability is not known, but it likely involves the modulation
of multiple membrane conductances (see reviews by Frick and
Johnston 2005; Zhang and Linden 2003). The observed in-
crease in slope suggests an enhancement of voltage-gated Na⫹
currents or a reduction of voltage-gated K⫹ currents. The
increase in the AP’s/pulse (and possibly the decrease in 1st ISI)
suggests modulation of currents that regulate the repeated
firing of action potentials by a neuron, such as those mediated
by Ca2⫹-activated K⫹ channels or persistent Na⫹ channels
(Sah and Faber 2002; Wu et al. 2004).
Potentiation of excitability is an important mechanism for
encoding and storing information during learning and memory.
Increased excitability has been observed after learning in both
vertebrates and invertebrates (Alkon et al. 1985; Antonov et al.
2001; Burrell et al. 2001; Cleary et al. 1998; Gainutdinov et al.
1998; Moyer et al. 1996, 2000; Oh et al. 2003; Saar et al. 1998;
Shreurs et al. 1997, 1998; Stackman et al. 2002; Straub and
Benjamin 2001; Thompson et al. 1996) and dysfunctions in the
modulation of excitability may contribute to age-related defi-
cits in learning and memory (Moyer et al. 2000; Wu et al.
2002). In addition, modulation of excitability interacts with
activity-dependent forms of neuroplasticity that may contribute
to learning and memory, such as long-term potentiation (LTP)
or long-term depression (LTD) (Andersen et al. 1980; Bliss
and Lomo 1973; Daoudal et al. 2002; Frick et al. 2004).
Modulation of excitability may also act to change the threshold
for induction of LTP or LTD (Cohen et al. 1999; Le Ray et al.
2004; Morozov et al. 2003; Nolan et al. 2004), a process often
referred to as metaplasticity (Abraham and Bear 1996). LTP
and LTD are observed in synapses formed by the mechanosen-
sory T- and P-cells onto the S-cell (Burrell and Sahley 2004),
but it is not known if 5-HT modulation of S-cell excitability
interacts with either form of synaptic plasticity.
The results presented here show a relationship between
learning- and 5-HT-induced potentiation of excitability in the
S-cell. To our knowledge, these experiments represent the first
time that learning-induced modulation of excitability has been
directly monitored and blocked at the same time that learning-
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4009
induced changes in behavior were taking place. These results
suggest, albeit not conclusively, that increased S-cell excitabil-
ity contributes to sensitization of the shortening reflex. It is
hypothesized that potentiation of S-cell excitability during
learning contributes to increases in the rate of S-cell activity
during the shortening response and that this increase in activity
allows the interneuron to contribute to the reflex in a way that
is does not under basal conditions (Lisman 1997). The S-cell
would be effectively “recruited” into the shortening neural
circuit as a result of learning, a process that has been observed
in both vertebrates and invertebrates (Daly et al. 2004; Moita et
al. 2003). Such recruitment of a cell into a neural circuit may
represent a general mechanism by which modulation of excit-
ability re-configures neural networks during learning.
ACKNOWLEDGMENTS
The authors thank Drs. Brenda L. Moss and Kevin M. Crisp for helpful
discussions during the preparation of this paper and Rodney McPhail at the
Purdue University Department of Biological Sciences for the Fig. 2 drawing.
GRANTS
This work was supported by National Institute of Neurological Disorders
and Stroke Grant R01-NS-34927 to C. L. Sahley with Kenneth J. Muller,
National Science Foundation Grant IBN-0432683 to B. D. Burrell, and a
subproject of National Institute of Research Resources Grant P20 RR-015567
to B. D. Burrell that is designated as a Center of Biomedical Research
Excellence.
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