Functional Role of Peptidergic Anterior Lobe Neurons in Male Sexual
Behavior of the Snail Lymnaea stagnalis
PAMELA A.C.M. DE BOER,1 ANDRIES TER MAAT,1 ANTON W. PIENEMAN,1 ROGER P. CROLL,2
MAKOTO KUROKAWA,3 AND RENÉ F. JANSEN1
1
Department of Organismal Neurobiology, Faculty of Biology, Vrije Universiteit, 1081 HV Amsterdam, The
Netherlands; 2Department of Physiology and Biophysics, Dalhousie University, Halifax, Nova Scotia B3H 4H7,
Canada; and 3Department of Biology, Tokyo Metropolitan University, Hachioji-shi, Tokyo 192-03, Japan
INTRODUCTION
Considerable evidence indicates that a large number of
neuropeptides are involved in the control of male sexual
behavior in various species (e.g., Argiolas and Melis 1995;
De Boer et al. 1996; Dornan and Malsbury 1989). Because
sexual behavior is often not a simple cyclic pattern but a
complex behavioral sequence comprising several elements,
it is difficult to elucidate the specific role of peptidergic
neurons and of the neuropeptides they contain. Invertebrates,
such as the hermaphroditic snail Lymnaea stagnalis, can
serve as valuable model systems for the study of peptidergic
control of sexual behavior because their CNS consists of a
limited number of neurons that are often large and identifiable. Moreover, the well-described, restricted behavioral
repertoire of this snail makes it easier to characterize specific
neuronal functions. Still, even in these animals copulation
is not a fixed action pattern but rather a chain of acts that
is variable in duration and is modulated by the motivational
state of the animal (De Boer et al. 1997). Here we describe
a distinct function for a group of peptidergic neurons and
their co-localized neuropeptides in the control of male sexual
behavior of L. stagnalis.
L. stagnalis is a simultaneous hermaphrodite (Holm 1946)
in which each adult possesses the complete neural circuitry
and endocrine centers necessary for ovulation and egg laying
as well as spermiation and copulation in both the female
and male roles. The stereotyped egg laying behavior has
been studied in great detail in Lymnaea, and large parts of
the neural circuitry of egg laying behavior have been described (reviewed in Ter Maat et al. 1992), as well as the
neuropeptides that are involved with the different aspects of
the behavior (Hermann et al. 1994). Also, the considerably
more complex male copulation behavior has now been studied on the behavioral, neural, and molecular levels. The
appetitive male copulatory behavior consists of a number of
elements that have variable durations and do not always
appear in the same sequence (De Boer et al. 1996; Van
Duivenboden and Ter Maat 1988). In addition, motivational
factors have been shown to affect the behavioral sequence
(De Boer et al. 1997; Van Duivenboden and Ter Maat
1985). The complex copulation behavior is thought to be
under the control of at least 10 different neurotransmitters
and neuropeptides (reviewed in De Boer et al. 1996; De
Lange et al. 1997). Although the functions of the different
messenger molecules are not yet clearly defined, there is a
strong indication that it is difficult to deduct specific functions for the peptides on the basis of morphological and
physiological evidence obtained so far. Here we concentrate
on the co-localized neuropeptides, APGWamide (Ala-ProGly-Try-NH2 ) and the vasopressin/oxytocin related conopressin. APGWamide has been shown to inhibit serotoninand dopamine-induced contractions of retractor muscles in
a dose-dependent fashion (Croll et al. 1991; Li et al. 1992).
The male copulatory organ, which comprises the preputium,
penis, and retractor muscles, is everted during copulation
putatively by relaxation of the retractor muscles. APGWamide-containing axons and terminals are located in muscles
of the male copulatory apparatus, the prostate gland, the vas
deferens, and on the body wall surrounding the male gonopore (Croll and Van Minnen 1992; Croll et al. 1991). Conopressin is co-localized with APGWamide in neuronal fibers
on the vas deferens (Van Golen et al. 1995). Application
of conopressin induces contractions in the vas deferens and
this action is inhibited in the presence of APGWamide (Van
Kesteren et al. 1995a,b). These data suggest that APGWam-
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De Boer, Pamela A.C.M., Andries Ter Maat, Anton W. Pieneman, Roger P. Croll, Makoto Kurokawa, and René F. Jansen.
Functional role of peptidergic anterior lobe neurons in male sexual
behavior of the snail Lymnaea stagnalis. J. Neurophysiol. 78:
2823 –2833, 1997. A morphologically defined group of peptidergic neurons in the CNS of the hermaphroditic snail, Lymnaea
stagnalis, is concerned with the control of a very specific element
of male sexual behavior. These neurons are located in the anterior
lobe of the right cerebral ganglion ( rAL). By using chronically
implanted electrodes, we show that the rAL neurons are selectively
active during eversion of the penis-carrying structure, the preputium. The preputium is normally contained inside the body cavity
and is everted during copulation in the male role. Electrical stimulation of the rAL neurons through the implanted electrodes, induced eversion of the preputium in vivo. Injection of APGWamide
(Ala-Pro-Gly-Try-NH2 ) , a small neuropeptide that is present in
all rAL neurons, induced eversion of the preputium. Application
of APGWamide to in vitro preparations of the preputium caused
relaxation of this organ. In contrast, injection of the neuropeptide
conopressin, which is co-localized with APGWamide in 60% of
the rAL neurons, did not induce any behavior associated with
male sexual activities. These results show that the neurons of the
rAL can induce an eversion of the preputium as occurs during
male copulation by release of APGWamide during a period of
electrical activity.
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METHODS
Animals
Adult, laboratory-bred specimens of L. stagnalis (shell heights
28–33 mm) were used. The snails were bred and kept in tanks
with continuous water change at 19–217C (SE) (Van Der Steen
et al. 1969). The animals used for the continuous recording with
fine wire electrodes were housed individually for 8 days before
the implantation. This procedure induces an increase in male sexual
drive in these animals (De Boer et al. 1997; Van Duivenboden
and Ter Maat 1985), thereby causing more animals to copulate
during the recording. A 12:12 h light:dark cycle was maintained
and lettuce leaves were provided ad libitum.
Retrograde and anterograde staining
The central ganglia were dissected and pinned out in saline (pH
7.8). The saline had the following composition (in mM): 4.0
CaCl2 , 1.7 KCl, 1.5 MgCl2 , 30.0 NaCl, 5.0 NaHCO3 , 10.0
NaCH3SO4 , and 10.0 N-2-hydroxyethylpiperazine-N *-2-ethanesulfonic acid (HEPES). The technique for axonal backfilling was
modified after the procedure described by Fredman (1987). The
cut stump of the penis nerve was drawn tightly into the end of a
finely tipped glass pipette of the appropriate diameter. Saline within
the pipette was replaced with a solution of nickel-lysine (1.7 g
NiCl2-6H2O and 3.5 g L-lysine free base in 20 ml H2O). This
preparation was maintained at room temperature for 18–24 h before the pipette was removed and the ganglia were washed in fresh
saline. Nickel was precipitated by adding 5–10 drops of a saturated
alcohol solution of rubeanic acid (dithiooxamide) to the 10 ml
saline bath. After 20–30 min, the ganglia were fixed overnight in
4% paraformaldehyde in saline. After several rinses of saline, the
staining was intensified with silver according to the technique described by Croll (1986). After this procedure the ganglia were
dehydrated through an ascending alcohol series, cleared in methyl
salicylate and mounted in Entellan (Merck).
Morphological details of individual cells were obtained by intracellular staining with Lucifer yellow as described by Stewart
(1978).
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Electrophysiology
Extracellular recordings of the penis nerve were made with a
suction electrode. Intracellular recordings of various cells were
made with glass microelectrodes (resistance 5–50 MV ), which
were filled with a saturated K2SO4 solution. Conventional electrophysiological apparatus was used.
A stainless steel fine wire electrode (25 mm diam, California Fine
Wire Company) was used for extracellular recording of electrical
activity from the intact rAL in vivo. The procedure of implantation
has been described elsewhere (Hermann et al. 1994; Yeoman et
al. 1994). After the electrode was implanted in the animal it was
connected to an amplifier with a high-impedance head stage
(DAM80, WPI). The animal was placed in a glass tank (20 cm
high, 30 cm long, and 6 cm wide), which was continuously perfused with water. Camcorders (Blaupunkt) on both sides of the
tank continuously recorded the animal’s behavior. These recordings together with the electrical activity of the rAL neurons
were stored on super-VHS videotape. A stimulus isolator (Neurolog Nl-800, Digitimer) was used for electrical stimulation via the
fine wire electrode. The electrical recordings were digitized with
a Cambridge Electronic Design A/D converter (model 1401,
12-bit).
Behavioral experiments
Injections of 20 ml of saline, APGWamide (American peptide
company), or conopressin (Saxon Biochemicals, Germany) solutions were made through the sole of the foot into the body cavity.
After injection all snails were placed in individual containers and
held with the foot in contact with the wall of the container until
they attached themselves. When the snails are in this position the
observer has the clearest view of the male aperture. Animals that
did not attach were placed on the bottom of the container in such
a way that any eversion of the preputium would be visible. The
behavior was stored on super-VHS tape. During the experiment the
observer was unaware of the treatment each snail had undergone.
Surgery
The penis nerve together with the penis artery were lesioned in
animals anesthetized with 1.5 ml of 50 mM MgCl2 . The penis
nerve originates from the right cerebral ganglion near the base of
the median lip nerve (Fig. 1). A network of fine branches of the
nerve innervates the penis artery, the preputium, the penis, and the
muscles (for a detailed description of the branching pattern see De
Boer et al. 1997; Elo 1938). The nerve and artery were exposed
after opening the body wall with a 0.5-cm incision behind the right
tentacle and were cut with fine scissors. In one group of animals,
the penis artery was separated from the nerve with fine forceps
and was then cut, leaving the penis nerve intact. Sham operations
consisted of opening the animal and manipulation of the penis
nerve without inducing any damage. After surgery all animals were
left to recover overnight. Within 2 h after surgery the animals were
again crawling about. All operations were verified with dissections
after the behavioral observations.
Bioassay
The preputium was used to test the effects of APGWamide and
conopressin on muscle contraction and relaxation. The preputium
is part of the penial complex that is situated in the head/foot region
of the body and occupies a considerable part of this cavity (Fig.
1). The preputium originates at the male gonopore in the lateral
body wall just caudal to the right tentacle and extends posteriorly
where it is attached by retractor muscles (Holm 1946) to the main
trunk of the columellar muscle. The penis sheath is attached at the
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ide and conopressin are involved in the control of various
elements of male copulation behavior.
Neurons of the anterior lobe of the right cerebral ganglion
(rAL) of the CNS are the major site of expression of the
APGWamide and the conopressin genes (Croll and Van
Minnen 1992; De Lange et al. 1997; Smit et al. 1992; Van
Golen et al. 1995). All rAL neurons express the APGWamide gene, whereas in 60% of the neurons the conopressin
gene is co-expressed (De Lange et al. 1997; Van Golen et
al. 1995). Retrograde stainings have identified those rAL
neurons that send axons in the sole nerve innervating the
male copulatory apparatus, the penis nerve (Smit et al. 1992;
Van Duivenboden 1984). These results suggest that the rAL
neurons have an important role in the control of male copulation in L. stagnalis.
In this paper we analyze the function of the rAL neurons
and the co-localized neuropeptides APGWamide and conopressin in the control of male copulation of L. stagnalis
through the use of in vivo and in situ recordings and injection
experiments. We demonstrate that the neurons of the rAL
can control the eversion of the male sexual organ and that
APGWamide induces such an eversion after injection into
the body cavity.
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tip of the preputium. The penis cannot be seen because it is enveloped by the penis sheath. The preputium was dissected and
attached to a length transducer with fine hooks made out of insect
pins (0.15 mm diam) in a 0.5-ml chamber filled with saline. The
preputium was held under a constant tension of 0.28 g. The preparations were continuously perfused with saline at a flow rate of 0.5
ml/min by using a pump (Minipuls 3, Gilson, France). Data were
stored on a DAT-recorder modified to record DC-signals (JVC,
Japan). Experimental and control solutions were applied in 1-ml
volumes by continuous perfusion. Two minutes after the start of
application, when the volume of the chamber was exchanged twice,
the perfusion was stopped for 1 min. Then the pump was turned
on again to wash out the sample solution with fresh saline.
RESULTS
rAL neurons project into the penis nerve
FIG . 2. Neurons in CNS of Lymnaea that have projections in penis
nerve. A: a nickel lysine backfill of penis nerve. Ventral view of right half
of the CNS with pedal commisure cut and pedal ganglion displaced laterally.
B: localization of neurons in CNS that have an axon in the penis nerve
(PN). Ventral view of the CNS. Ce, cerebral ganglion; Pl, pleural ganglion;
Pa, parietal ganglion; Pe, pedal ganglion; Vi, visceral ganglion; AL, anterior
lobe cluster; VL, ventral lobe cluster; PeIb, pedal Ib cluster. (Modified
after De Boer et al. 1996.)
FIG . 1. Body cavity of Lymnaea. Dorsal view of head/foot with dorsal
body wall removed (modified after De Boer et al. 1997). Only the penis
nerve is shown ( . ); all other nerves are omitted. Preputium is attached to
body wall by muscle bands. Muscle bands near male gonopore are protractor
muscles. Muscle bands near distal end are retractor muscles. –––, part of
vas deferens and a branch of penis nerve that run through body wall. AP,
artery of penis; BM, buccal mass; BW, body wall; P, preputium; PG,
prostate gland; PM, protractor muscle; PN, penis nerve; RM, retractor muscle; VD, vas deferens.
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extracellular recordings of the penis nerve. In an isolated
central ganglia ring the rAL was easily recognized by its
outer layer of relatively large (35–70 mm diam) and uniformly pale orange to yellow cells. Intracellular recordings
showed that the neurons were generally silent, with resting
membrane potentials between 070 and 080 mV. At intracellular stimulation with depolarizing square current pulses, the
neurons generated action potentials and either small or large
spikes could be recorded in the nerve. Occasionally a neuron
was recorded that showed spontaneous activity (Fig. 3).
From the 125 somata impaled in the anterior lobe (26 prepa-
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Both tract tracing experiments and electrophysiology were
used to identify the neurons that project into the penis nerve.
Retrograde staining with nickel-lysine was used to localize
neurons with axons in the penis nerve. Five groups of neurons could be identified, all restricted to the right half of the
CNS (Fig. 2). They were located in the anterior and ventral
lobes of the right cerebral ganglion, the pedal Ib cluster
(PeIb)-cluster of the right pedal ganglion, and in scattered
populations within the right pleural and parietal ganglia. In
the rAL Ç60–70 backfilled somata were identified. Generally about 75% of the neurons at the surface were stained.
The stained neurons were found scattered over the entire
lobe.
To determine the projections of the rAL neurons electrophysiologically, we combined intracellular recordings with
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DE BOER, TER MAAT, PIENEMAN, CROLL, KUROKAWA, AND JANSEN
rations), 115 somata had spikes which were followed one
for one with spikes in the nerve at constant latencies. In one
preparation 18 somata were impaled, 14 of which had a
corresponding element in the penis nerve. Electrical stimulation of the penis nerve induced action potentials in the rAL
neurons. In four preparations a repetitive firing pattern (afterdischarge) occurred as a result of the stimulation. This
feature has been reported earlier by Chase and Li (1994).
Several rAL neurons were also injected with Lucifer yellow (n Å 12). Figure 4 is an example of a staining of a
rAL neuron with axonal projections into the ventral lobe of
the right cerebral ganglion and from there into the penis
nerve and into the cerebropedal connective toward the PeIbcluster and into the superior cervical nerve. In one preparation only one axonal branch extended from the cell body,
projected toward the right ventral lobe, and from there into
the penis nerve. In the other 11 preparations a single axonal
branch extended from the cell body and projected to the
right ventral lobe where the initial branch split into two or
three branches. In eight of the preparations one or two of
these axonal branches projected into the cerebropedal connective and the rest projected into the penis nerve. In the
other preparations all branches projected into the penis
nerve. In four preparations an axonal branch was found in
the PeIb-cluster.
These data indicate that most cells at the outer surface of
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the rAL send axons into the penis nerve. This is in accordance with the results of the nickel-lysine backfills where
only some cells at the surface of the lobe were left unstained.
The Lucifer yellow stainings show that individual rAL neurons do not solely project into the penis nerve. Furthermore,
the branching patterns suggest that rAL neurons communicate with the neurons in the right ventral lobe and PeIbcluster.
rAL neurons are electrically active during eversion of the
preputium
The electrical activity of rAL neurons was studied in
freely behaving animals over 2-day periods by means of
chronically implanted fine wires. During this period all the
animals (n Å 41) appeared healthy and exhibited a variety
of ongoing behaviors (e.g., egg laying, rasping, and lung
ventilation). However, only two recordings were made during successful copulation (i.e., where sperm transfer had
taken place) and 2 were made during sham copulation (preputium eversion without sperm transfer).
The electrical recordings, including those from the animals that did not copulate, showed few and infrequent spikes
not associated with the execution of any (noncopulatory)
behavior. Figure 5A, top, shows a digitized recording of the
rAL made from an animal while it crawled about in the tank,
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FIG . 3. Identification of neurons projecting into penis nerve. A: neurons in the rAL that have a projection in penis nerve
(black somata) or have not (white somata), as revealed by simultaneous recording of neurons and penis nerve (PN). B:
simultaneous intracellular recording of rAL neurons ( bottom) and extracellular recordings of penis nerve ( top). Examples
of a spontaneously active neuron ( right) and of electrically silent neurons that show either a small or large component in
penis nerve after depolarization of the soma (left and middle, respectively). All spikes recorded from somata were followed
1 for 1 with spikes in nerve at constant latencies. DB, dorsal body; rCG: right cerebral ganglion.
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Although normally the preputium is only everted during
male copulatory activities, one animal was recorded while
the preputium was everted in the absence of a female copulation partner. The animal everted its preputium for 5 min
during which the activity in the rAL neurons was increased
(Fig. 6).
These findings show that the neurons of the anterior lobe
are active during preputium eversion. This suggests that the
neurons of the rAL are involved in the control of preputium
eversion. Furthermore, it was demonstrated that the different
units in the recording do not become active simultaneously
during the eversion of the preputium.
Stimulation of the anterior lobe neurons induces eversion
of the preputium in vivo
occasionally rasped and ventilated. In this recording there
was little activity. By contrast, during successful copulation
intense electrical activity is associated with eversion of the
preputium (Fig. 5A, bottom). Electrical activity commenced
when eversion of the preputium started and ceased when the
preputium was retracted into the body cavity 31 min later.
Both recordings, in Fig. 5A, were similarly amplified, filtered, and digitized. All four recordings made during male
copulatory activities showed increased electrical activity
during the eversion of the preputium. The average firing rate
in these recordings was 20.6 { 12.2 (SE) spikes/min during
the 10 min before eversion and 146.9 { 10.2 spikes/min
during the first 10 min of eversion.
In such recordings, the electrode always covered a number
of anterior lobe neurons and probably recorded the electrical
activity from more than one neuron. This can already be inferred from the wide range of amplitudes in the recording of
Fig. 5A. To examine the firing pattern of the different elements
in the compound signal, a template matching algorithm for
waveform recognition was used (Jansen and Ter Maat 1992).
A group of waveforms that is associated with one template is
referred to as one unit. Figure 5B shows that not all units
started firing simultaneously but that more units were recruited
as copulation progressed. Conversely, retraction of the preputium was attended by a gradual decrease of the number of units
firing. However, most units started firing at the moment the
penial complex started to evert. Two of those units (4 and 6)
showed a decrease in firing activity within a couple of minutes.
Other units were active throughout the entire period the preputium was outside (1, 2, and 5). Finally, one-third of the units
(7–9) did not start to increase in firing rate until intromission
was already in progress.
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Injection of APGWamide induces eversion of the
preputium
Almost all rAL neurons contain APGWamide, whereas in
60% of the rAL neurons APGWamide is coexpressed with the
conopressin gene (De Lange et al. 1997). Because little is
known about the release sites, injections of these neuropeptides
provide a first approximation of their function. Injection experiments were performed to investigate the role of APGWamide
and conopressin in the control of preputium eversion.
APGWAMIDE INJECTIONS. Three different concentrations of
APGWamide were tested: 10 05 M (n Å 7), 5 1 10 06 M
(n Å 6), and 10 06 M (n Å 4). Control injections (n Å 4)
consisted of saline only. Saline injected animals attached
to the container wall and remained stationary for 5–10 s.
Thereupon, the animals started moving around the container
in an apparently normal manner. Within the first minute after
all APGWamide injections, animals were distinguishable
from saline-injected animals by their decreased locomotory
activity. All of the snails injected with either 10 05 M or 5 1
10 06 M and two of the four animals injected with 10 06 M
APGWamide showed eversion of the preputium. In five
snails injected with 10 05 M APGWamide the eversion was
full; in the other animals the eversion was partial. Two of
the snails injected with 10 06 M APGWamide did not show
any eversion. A typical full eversion is shown in Fig. 7.
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FIG . 4. Camera lucida drawing of a rAL neuron filled with Lucifer
yellow. Ventral view of anterior right side of central ganglia ring. PeIb,
pedal Ib-cluster; PN, penis nerve; rCG, right cerebral ganglion; rPeG, right
pedal ganglion; rVL, ventral lobe of the right cerebral ganglion; SCN,
superior cervical nerve.
To investigate whether or not the rAL neurons can indeed
control the eversion of the preputium, the rAL neurons were
stimulated via an implanted fine wire in another group of
animals (n Å 24). Initial recording from the fine wire electrodes revealed the same extracellular activity of the rAL
neurons in all animals: generally silent with only occasional
spikes. The implanted fine wires were then used to stimulate
the rAL neurons. Three of the 24 animals responded to the
electrical stimulation by fully everting the preputium, 20
showed partial eversion (ranging from 25 to 50%), and 1
did not exhibit any overt effect. The eversion started within
30–60 s after the onset of the stimulation and the preputium
was retracted again within 10 s after the end of stimulation.
The stimulations lasted 5–10 min.
These results show that stimulation of the rAL neurons
can cause eversion of the preputium, thus further indicating
that these neurons are involved in the control of the penial
complex.
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Within 2 min the partially everted preputium became visible
at the right side of the animal (3). About 1 min later, the
preputium was fully everted (6). This eversion lasted 10–
15 s, after which the preputium was partially retracted into
the body cavity. After 9 min, the preputium is fully retracted
and locomotion started again (9). Generally the eversion
started within 2–7 min after the injection. Within 10 min,
the preputium was retracted back into the body cavity in all
animals and the animals started moving again. Within another 5 min, all animals had resumed normal behaviors.
FIG . 6. Electrical activity of rAL neurons during spontaneous eversion of preputium in vivo. Schematic drawings of the
snail indicate when preputium is everted. –––, start and end of different behavioral acts during recording. During eversion
there is an increase in firing activity. Note that eversion of preputium in absence of a partner is a rare event.
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FIG . 5. Electrical activity of rAL neurons in freely
behaving Lymnaea. A: digitized recording from rAL
neurons. Top: an animal not involved in any male
copulatory activities. Schematic drawings of a snail
illustrate that preputium is not everted. Bottom: same
animal when it was copulating in male role. Schematic
drawings of a snails illustrate 4 different stages of
male copulatory behavior. Snail on top is animal acting as a male. This is the snail from which we recorded. Left: partial eversion, total eversion, and intromission. Right: partial retraction, total retraction.
–––, start and end of different behavioral acts during
recording. Electrical recording shows an increase in
firing activity during eversion of preputium. B: spike
trains of different units in rAL recorded during preputium eversion as resolved from compound signal with
wave recognition analysis. Units are retracted from
same signal shown in A, bottom. Each vertical line
represents 1 spike. Note that more units are recruited
as copulation progresses. Time base: A Å B.
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FIG . 7. Effect of APGWamide injection in vivo. Images of a snail taken after
injection with 20 ml of 5 1 10 05 M
APGWamide. Animal is placed against
transparent side of experimental container.
Within 2 min after injection, preputium is
everted and becomes visible as a white
structure at right side of animal (r , starting
point). Preputium become fully everted (6)
and are retracted again. After Ç9 min, animal is crawling around again (9). Time
interval between images is 1 min for 1st 3
images, 20 s for next 5 images, and 5 min
and 20 s for last image.
Lesion of the penis nerve prevents retraction of the
preputium, not eversion
Eversion of the preputium might be caused either by a
peripheral effect of APGWamide on the male copulatory
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organs or by a central effect of APGWamide on the neurons in the CNS and thereby mediating an eversion. To
investigate whether or not eversion of the preputium can
be induced by peripheral actions of APGWamide, we injected 20 ml of 10 05 M APGWamide into animals that
had their penis nerve cut 3 or 4 days earlier ( n Å 7 ) .
Because the penis artery is attached to the penis nerve, a
lesion of the penis nerve implies also a lesion of the penis
artery which supplies the preputium with blood. To investigate possible effects of a disturbed blood supply on the
ability to evert and retract the preputium, we also injected
APGWamide in animals that had only the penial artery
cut ( n Å 3 ) . Finally, we also injected APGWamide into
sham-operated animals ( n Å 4 ) .
The sham-operated animals behaved similarly to the
nonoperated animals. Two of the animals showed a partial
eversion, whereas the other two showed a total eversion
of the preputium. All lesioned animals, except for one
with only the artery cut, showed a total eversion of the
preputium. Generally, the eversion started within 2 – 4 min
after the injection. Preputium length in the animals with
both penis nerve and artery cut appeared to be increased
as compared with the artery-lesioned, nonoperated, and
sham-operated animals ( not quantified ) . Animals with
both the artery and penis nerve lesioned did not retract
their preputium into the body cavity. By contrast, the artery-lesioned animals did retract the preputium within 10
min after the injection.
These results show that the ability to evert the preputium
after injection with APGWamide persists after a lesion of
the penis nerve and artery. It is concluded that eversion of the
male copulatory apparatus can be induced by the peripheral
action of APGWamide. Retraction of the preputium is abolished by lesions of the penis nerve but not the penis artery.
From these data we conclude that retraction of the penial
complex is an active process requiring the activity of a central circuit.
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There was no effect of the different concentrations on the
temporal pattern of the eversion and retraction of the preputium. From these data it can be concluded that APGWamide
injection induces eversion of the preputium.
CONOPRESSIN INJECTIONS. Three different concentrations of
conopressin were tested: 10 06 M (n Å 4), 10 07 M (n Å 6),
and 10 08 (n Å 2). Conopressin induced a strong contraction
of the foot. During this contraction, the foot was folded
with the tip of the posterior end contacting the anterior side.
Possibly as a result of this the animals did not attach to the
wall of the container after injection. The contraction of the
foot lasted 2–10 min for the animals injected with either
10 07 M or 10 08 M and 8–18 min for the animals injected
with 10 06 M. Within 1–2 min after the relaxation of the
foot, the animals attached to the surface and started to crawl
around again, in an apparently normal manner. Two of the
six animals injected with 10 07 M conopressin, showed minimal eversion of the preputium. Less than Ç20% of the preputium everted. However, none of the other animals showed
any eversion.
COMBINED INJECTIONS. APGWamide and conopressin are colocalized in neurons of the rAL. In previous studies the neuropeptides were found to have opposite effects on muscle tension.
To investigate whether conopressin could alter the effect of
APGWamide described above, combined injections of 10 05 M
APGWamide and 10 07 M conopressin were performed. The
effect on the behavior was a combination of the behaviors seen
after injections of the separate peptides. All animals (n Å 7)
showed eversion of the preputium and simultaneously contraction of the foot, which again prevented attachment. This
suggests that conopressin does not alter the gross effect of
APGWamide when simultaneously injected.
2830
DE BOER, TER MAAT, PIENEMAN, CROLL, KUROKAWA, AND JANSEN
Effect of APGWamide and conopressin on muscles of the
preputium
The effect of APGWamide and conopressin on the relaxation or contraction of the preputium was tested by application of these peptides to the preputium in vitro while the
length of this organ was measured continuously with a length
transducer. Seven different concentrations of APGWamide
were tested, ranging from 10 09 M to 10 03 M (n Å 5 for
10 07 to 10 04 M, n Å 4 for the other concentrations). Bath
application of APGWamide induced a relaxation of the preputium at a concentration of 10 08 M. The relaxation was
maximal at a concentration of 10 04 M (Fig. 8B). An example of a recording made during application of 10 05 M is
given in Fig. 8A, top. Within 30 s after the start of the
application the preputium starts to relax. Sixteen minutes
after the wash out of sample solution, the preputium has
resumed its initial length. The relaxation of the preputium
was dose-dependent (Fig. 8B). The half-maximal effective
concentration (EC50 ) value was 3.63 1 10 06 M.
Conopressin was tested in three different concentrations
ranging from 10 06 to 10 04 M (n Å 3 for each concentration).
Bath application of conopressin had no effect on the relaxation or contraction of the preputium (Fig. 8A, bottom). We
also tested whether or not conopressin could alter the effect
of APGWamide by simultaneous application of APGWamide
(10 05 M) and conopressin (10 04 M) (n Å 4). No effect of
conopressin was found (Fig. 8B, n ). From these data it can
be concluded that the effect of APGWamide on the preputium
is not modulated in the presence of conopressin.
DISCUSSION
This study provided evidence that the neurons of the rAL
can control the eversion of the preputium in the snail L.
stagnalis. Fine wire experiments have revealed electrical
activity of the neurons during eversion. Electrical stimulation
of the neurons induced eversion of the preputium. Finally,
injection of the neuropeptide APGWamide, which is present
in all neurons of the rAL (Croll and Van Minnen 1992;
Croll et al. 1991; Smit et al. 1992), caused eversion of the
preputium.
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Role of the rAL neurons in the eversion of the preputium
The electrophysiological experiments showed that spikes
in rAL somata were followed one for one with spikes in the
penis nerve. Direct projections of rAL neurons in the penis
nerve were not checked by blocking the synaptic activity.
However, both the constant latencies of the nerve spikes
with the somata spikes and the morphological demonstration
of projections of rAL neurons in the penis nerve are consistent with direct projections. The projections of the rAL neurons were studied earlier. With the use of intracellular Lucifer yellow staining, Khennak and McCrohan (1988) demonstrated that at least 45% of the neurons send projections into
the penis nerve. Here we show with retrograde tract tracing,
Lucifer yellow stainings, and electrophysiological recordings that this number must be higher. The results from
intracellular recording indicate that this number is Ç80%.
The electrical activity of the rAL neurons is correlated
with the eversion of the preputium. Both intra- and extracellular recordings showed that the neurons are generally electrically silent. During the eversion of the preputium the electrical activity of Ç10 units was increased. This amount is
very likely the maximum that can be recorded from in this
configuration, because the recording electrode covered about
one-fifth of the total amount of somata at the surface of the
rAL. Furthermore, the number of units that was recognized
was similar in all recordings, although the precise position
of the electrode varied between animals. Therefore, we conclude that the majority of the rAL neurons are probably
active during the eversion of the preputium. Neurons controlling male copulation have been recorded from in some other
gastropods. In Aplysia californica pedal cluster neurons (the
‘‘primary cluster’’) were found to produce one for one excitatory potentials in the penis retractor muscle (Rock et al.
1977). In the terrestrial snail Helix aspersa, neurons in the
right mesocerebrum have projections in the penial nerve
(Li and Chase 1995). Stimulation of these neurons causes
contractions of either the dart sac, the penis, or both (Chase
1986). In this animal, the mesocerebrum is asymmetrical,
the right hand side being appreciably larger than the left side.
Furthermore, neurons in this cluster were found to display
APGWamide-like immunoreactivity (Griffond et al. 1992).
In this respect, the mesocerebrum resembles the anterior lobe
of Lymnaea.
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FIG . 8. Effects of application of
APGWamide and conopressin on preputium. A: APGWamide (10 05 M) induces
a relaxation of preputium (top); whereas
conopressin (10 04 M) has no effect ( bottom). B: dose-response relationship of effect of APGWamide on preputium. Mean
values of response for each concentration
were plotted semilogarithmically vs. concentration of APGWamide that was applied
(n Å 5 for 10 07 –10 04 M; n Å 4 for other
concentrations). n, combined application
of APGWamide and conopressin. There is
no effect of conopressin on effect of
APGWamide. Error bars represent standard
deviations. A sigmoidal function was plotted through the APGWamide data points.
Half-maximal effective concentration Å
3.63 1 10 06 M. Rate of relaxation is normalized to length of preputium at start of
experiment.
CONTROL OF MALE SEXUAL BEHAVIOR IN LYMNAEA
Functional role of APGWamide
In Lymnaea several different neuropeptides have been
demonstrated in the neurons that project into the penis nerve
(reviewed in De Boer et al. 1996). APGWamide is the
neuropeptide that has been studied most extensively. Practically all neurons in the rAL contain APGWamide. The current experiments show that injection of APGWamide induces an eversion of the preputium not only in intact animals
but also in animals with the penis artery lesioned and in
animals with both the penis nerve and artery lesioned. These
data suggest that the injected APGWamide exerts its action
via receptors in the periphery.
The presence of the peptide has been demonstrated in the
periphery. Immunoreactive fibers are located in the penis
retractor muscle, throughout the preputium, along the inner
surface of the penis sheath, and in the body wall surrounding
the male gonopore. (Croll and Van Minnen 1992; De Lange
et al. 1997). The overall action of the APGWamide in Lymnaea seems to be relaxation of muscles. The neuropeptide
inhibits contractions of the retractor muscle that are induced
by serotonin and dopamine (Croll et al. 1991; Li et al. 1992),
it inhibits the spontaneous contractions of the vas deferens
(Van Golen et al. 1995) and relaxes the preputium. Recently,
the presence of cDNA-encoding APGWamide has been
demonstrated in Aplysia (Fan et al. 1997; Nagle et al. 1996).
The cDNA was found almost exclusively in the right cerebral
ganglion and in male reproductive organs. This suggests a
functional role of APGWamide in male copulation of
Aplysia. There are studies on the functional role of
APGWamide in mollusks other than Lymnaea, although
these studies did not relate to male sexual activities. In the
prosobranch snail Fusinus ferrugineus, APGWamide potentiates twitch contractions in the radula retractor muscle
and shows an inhibitory action on the radula protractor muscle (Kuroki et al. 1990). In Mytilus edulis, APGWamide
inhibits the electrically induced contractions of the byssus
muscles (Muneoka and Kobayashi 1992). These reports suggest that APGWamide has a general function in the modulation of muscles in mollusks.
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In addition to peripheral effects of APGWamide, there is
also evidence that it has central actions. Just as in the periphery, immunoreactivity and effects of APGWamide are found
in the CNS. In addition to the rAL neurons, the peptide has
also been demonstrated to occur in some cells of the anterior
lobe of the left cerebral ganglion, the ventral lobe of the
right cerebral ganglion, and in the ring neuron (Croll and
Van Minnen 1992; Croll et al. 1991). This suggests that
APGWamide is not exclusively involved in preputium eversion. This idea is supported by the tract tracing, immunocytochemical, and electrophysiological data. Croll and Van Minnen (1992) and De Lange et al. (1997) describe that
APGWamide positive neurites of the rAL neurons project
in a fascicle toward the right ventral lobe and from there into
both the penis nerve and the right cerebral-pedal connective.
Furthermore, they describe APGWamide-immunoreactive
terminals surrounding somata of the PeIb-cluster of the right
pedal ganglion. These terminals originate from neurons of
the rAL. Our data show that these neurons do indeed project
to the PeIb-cluster. Together, these results suggest that the
rAL neurons have APGWamidergic inputs on the PeIb-cluster neurons of the right pedal ganglion. Central effects of
APGWamide have been found in other gastropods. In the
stylommatophoran snails Achatina fulica and H. aspersa, it
was demonstrated that the peptide has a hyperpolarizing effect on neurons (Chen and Walker 1992; Liu et al. 1991).
In Lymnaea, APGWamide induces an inhibition of the light
green cells (H. Van Tol-Steye and K. S. Kits, personal communication) and also an inhibition of the caudodorsal cells
(Croll et al. 1991) which control ovulation and oviposition
(Ter Maat et al. 1992).
Now that the functional role of the rAL neurons and their
major neuropeptide APGWamide becomes more and more
clear, it is interesting to know what function the co-localized
peptides have in male sexual behavior. APGWamide is colocalized with conopressin in 60% of the rAL neurons (De
Lange et al. 1997; Van Golen et al. 1995). Because conopressin has excitatory effects on muscles of the vas deferens
(Van Golen et al. 1995; Van Kesteren et al. 1995a) Van
Kesteren and colleagues (1995a) suggested that conopressin
may be involved in the control of ejaculation of semen during intromission in Lymnaea. However, this idea cannot yet
be confirmed by the injection experiments described in this
paper. Although the animals were observed carefully, no
ejaculation was observed after injection of conopressin alone
or in combination of APGWamide. Possibly conopressin has
a functional role in the CNS. The receptor for conopressin,
LSCPR, is present on rAL neurons (Van Kesteren et al.
1995b). Recent data show that conopressin excitates the rAL
neurons (Van Soest and Kits 1997). More experiments have
to be done to elucidate the function of conopressin in male
copulation behavior.
Control of preputium eversion
On the basis of the present data we postulate the following
model of preputium eversion as induced by APGWamide.
A discharge of the rAL neurons causes APGWamide to be
released in parts of the penial complex. This release induces
the simultaneous relaxation of the retractor muscles and preputium muscles. The preputium is everted inside out starting
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Although the in vitro studies indicate a possible role for
central neurons, this is the first report of in vivo activities
of neurons controlling male copulation behavior in gastropods. Of the 41 animals with an implanted electrode only 4
showed male copulation activities. The animals that did not
engage in such reproduction were not apparently ill. In other
studies in which in vivo electrical recordings were made
there is no report of such a low percentage of animals showing a specific behavior (Begnoche et al. 1996; Hermann et
al. 1994; Yeoman et al. 1994). Male copulation behavior
of Lymnaea was demonstrated to be under the control of
motivational factors (De Boer et al. 1997). In previous studies in which individually housed animals (without any implanted electrodes) were used, 40% of the animals showed
intromission (De Boer et al. 1997; Van Duivenboden and
Ter Maat 1985), a percentage much higher than reported
here. The performance of the more complex male copulation
behavior is easily disrupted by relative small disturbances.
Chronically implanted electrodes obviously induce enough
disturbance to cease male copulation behavior.
2831
2832
DE BOER, TER MAAT, PIENEMAN, CROLL, KUROKAWA, AND JANSEN
We thank Prof. N. M. van Straalen for valuable comments on earlier
drafts of this manuscript.
This research was supported by the Foundation for Life Sciences (SLW)
Grant 805-25-305, which is subsidized by the Netherlands Organization for
Scientific Research (NWO).
Address for reprint requests: A. Ter Maat, Dept. of Organismal Biology,
Faculty of Biology, Vrije University, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands.
Received 24 June 1997; accepted in final form 8 August 1997.
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