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First published online July 14, 2008
Journal of Experimental Biology 211, 2388-2396 (2008)
Published by The Company of Biologists 2008
doi: 10.1242/jeb.015040
Nitric oxide induces aspects of egg-laying behavior in Aplysia
1 Leslie and Susan Gonda (Goldschmied) Multidisciplinary Brain Research Center,
Bar Ilan University, Ramat Gan, 52900, Israel
2 Mina and Everard Goodman Faculty of Life Sciences, Bar Ilan University, Ramat
Gan, 52900, Israel
* Author for correspondence (e-mail: avy{at}mail.biu.ac.il)
Accepted 28 May 2008
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: egg-laying, NO, nitric oxide, Aplysia, feed-forward, feed-back, motor coordination
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Sigvardt et al.,
1986; Stuart et al.,
1980), are a central component coordinating egg laying. There have
been conflicting reports on how different aspects of egg laying are
coordinated. It has been suggested that the various peptides secreted by the
bag cells have different functions, and egg laying is coordinated by the
simultaneous release of the different peptides
(Scheller and Axel, 1984;
Scheller et al., 1983). This
view arises from the supposition that egg laying is a fixed act
(Kandel, 1976) in which all of
the components occur in a fixed sequence. A group of peptides released from
the site at which a decision is made to initiate the behavior could coordinate
the sequencing of the behavior. The direct effects of a variety of bag cell
peptides on a number of tissues has been shown, supporting this idea. For
example, ELH directly affects the gonads and causes egg release
(Choate et al., 1993;
Coggeshall, 1970;
Rothman et al., 1983b), and
directly affects central neurons in the buccal and cerebral ganglia and
thereby inhibits feeding (Ram,
1982; Ram, 1983;
Stuart and Strumwasser, 1980;
Teyke et al., 1991), whereas
additional peptides cause depolarization of the bag cells as part of a
positive feedback loop (Brown and Mayeri,
1989; Kauer et al.,
1987; Rothman et al.,
1983a). It has also been suggested that egg laying is coordinated
by feed-forward and feedback loops (Cobbs
and Pinsker, 1982b; Ferguson
et al., 1989b; Ter Maat and
Ferguson, 1996). The performance of certain aspects of egg-laying
behavior act as stimuli that in turn affect other aspects of the behavior
(Cobbs and Pinsker, 1982b;
Ter Maat and Ferguson, 1996).
Thus, injection of only one of the bag cell peptides into the animals, ELH,
induces fully-fledged egg-laying behavior
(Bernheim and Mayeri, 1995),
presumably because it directly activates some aspects of egg laying, and the
aspects directly triggered by ELH in turn act as stimuli that trigger the
additional aspects of egg laying. This report focuses on the effects of the unconventional neurotransmitter nitric oxide (NO) on egg-laying behavior. We have found that NO mediates some aspects of egg-laying behavior. Treating Aplysia with an NO donor induces parts of the coordinated behavior, while leaving out a crucial aspects of egg laying. The finding that an NO donor induces abnormal egg laying provides insight into how some of the different aspects of egg laying may be coordinated.
Egg-laying is thought to be initiated by the activity of neurons in the
cerebral and pleural ganglia (Brown et al.,
1989; Ferguson et al.,
1989b; Shope et al.,
1991) which have axons that reach the neuroendocrine bag cells
via the pleural-abdominal connectives. The bag cells are generally
silent, except preceding egg laying (Dudek
et al., 1979; Pinsker and
Dudek, 1977). Electrical or chemical stimulation of cerebral and
pleural ganglion neurons can trigger a bag cell discharge
(Brown et al., 1989;
Painter et al., 1988). Brief
electrical stimulation of the pleural-abdominal connectives also initiates a
bag cells discharge (Ferguson et al.,
1989a; Kupfermann and Kandel,
1970; Pinsker and Dudek,
1977; Wayne and Wong,
1994), presumably because the stimulus excites axons from the head
ganglia that activate the bag cells. Bag cell activity is characterized by
sustained low-frequency synchronous firing of the cells, which are
electrically coupled to one another
(Kupfermann and Kandel, 1970).
Synchronous bag cell activity is readily recorded via an
extracellular electrode on the plural-abdominal connective, close to the bag
cells (Ferguson et al., 1989a;
Kupfermann and Kandel, 1970;
Wayne and Wong, 1994). Bag
cell firing releases a number of peptide hormones into the hemolymph
(Stuart et al., 1980). The
peptides are synthesized on a single precursor protein, which is then cleaved
(Nagle et al., 1989;
Scheller et al., 1983).
Homologues of the bag-cell peptides are also synthesized in and released from
the atrial gland of the genital tract (Arch
et al., 1978; Heller et al.,
1980; Nagle et al.,
1989; Painter et al.,
1988; Scheller et al.,
1982). In some Aplysia species, including in A.
fasciata that was used in this study, a distinct atrial gland is not
present, and tissue homologous to the atrial gland is present in the anterior
portion of the large hermaphroditic duct
(Painter et al., 1985).
Homogenates of the bag cells or of the atrial gland elicit egg-laying when
injected into Aplysia (Arch et
al., 1978; Bernheim and Mayeri,
1995; Heller et al.,
1980; Kupfermann,
1967), in part because some peptides act on the gonad and cause
the release of yolk-containing eggs. Eggs are then transported via
the small hermaphroditic duct from the gonad to the to the fertilization
chamber, where they are fertilized by previously stored sperm cells. Eggs are
then packaged into capsules and cordons. The packaging is dependent on the
albumen and winding glands (the accessory genital mass), which secrete the
packaging material (Cummins and Nagle,
2005; Thompson and Bebbington,
1969). The egg cordons are transported via the large
hermaphroditic duct to the exterior. Eggs then travel down the genital groove
toward the head. Egg cordons are then deposited onto the substrate
via weaving and tamping movements of the head and lips
(Bernheim and Mayeri, 1995;
Cobbs and Pinsker, 1982a;
Ferguson et al., 1989a).
Appetitive behaviors called waves and undulations precede the extrusion of the
egg cordon. The appetitive behaviors are thought to be produced partially by
the effects of bag cell peptides on neural circuits in the head ganglia that
control the neck (Ferguson et al.,
1986) and partially by feed-forward excitation elicited by egg
cordons stimulating parts of the reproductive system
(Cobbs and Pinsker, 1982b).
The consummatory behaviors are thought to be elicited via
feed-forward excitation elicited by egg cordons stimulating parts of the
reproductive system (Cobbs and Pinsker,
1982b; Ferguson et al.,
1986).
Our data indicate that treating Aplysia with a NO donor elicits the formation of egg cordons containing capsules that usually lack eggs. NO is likely to act on the accessory genital mass which packages eggs. In addition, NO activates appetitive behaviors that normally precede egg laying, even in Aplysia that are unable to lay eggs. This effect is likely to be on pedal ganglion neurons organizing the appetitive behaviors, and are independent of the effect on the accessory genital mass.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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All of the A. fasciata used were sexually mature, as evidenced by
previous egg laying. One experiment examined aspects of egg-laying behavior in
presumably sexually immature animals. Because A. fasciata mature very
quickly, it is difficult to have a large sample of immature animals for
experiments. This experiment was performed on A. californica Copper
1863 (Santa Barbara Marine Bio, Santa Barbara, CA, USA) during the winter
months (February to March), when they are often sexually immature
(Audesirk, 1979;
Strumwasser et al., 1969).
Experimental treatments and observations
Twenty-four hours before an experiment, animals were placed one to a
container in aerated 5 l containers of Mediterranean seawater kept at
21°C. Animals that laid eggs during this 24 h period were not used. To
induce egg laying, animals were injected through the foot into the hemocoel
with a variety of substances. Bag cell (BC) and large hermaphroditic duct
(LHD) preparations from a single donor animal were homogenized in 1 ml
artificial seawater (ASW), and this volume was injected into a single animal.
The NO donor S-nitroso-N-acetyl-penicillamine (SNAP; Sigma,
Rehovot, Israel) was prepared to reach a concentration within the animal of 45
µmol l–1. This concentration depolarizes the giant
metacerebral cell (MCC) of the Aplysia cerebral ganglion
(Jacklet and Tieman, 2004),
and also affects aspects of Aplysia feeding behavior
(Katzoff et al., 2006). The
effect of 45 µmol l–1 N-acetylpenicillamine (NAP;
Sigma, Israel), which lacks the NO donating S-nitroso group, was also
examined. ELH (Peninsula Laboratories, Belmont, CA, USA) was prepared to reach
a concentration of 0.15 µmol l–1 within the animal. The
NOS (nitric oxide synthase) inhibitor
N
-nitro-L-arginine methyl ester (L-NAME; Sigma,
Israel) was prepared to reach a concentration of 1.85 mmol
l–1 within the animal. The dosage used (1.85 mmol
l–1) was five times larger than that used in previous
experiments (Katzoff et al.,
2002; Katzoff et al.,
2006) that examined the effects of L-NAME on feeding behavior.
Methylene Blue, an inhibitor of guanylyl cyclase, was prepared to reach a
concentration of 100 µmoll–1 within the animal. This
concentration affects Aplysia feeding behavior
(Katzoff et al., 2006), as
well as persistent sensitization of nociceprors as a result of a noxious
stimulus (Lewin and Walters,
1999). Animals were observed for 2 h following treatment, and the
start and duration of egg-laying behavior was noted: the person observing the
animals was not informed about the nature of the preceding treatment.
Recording
Extracellular recordings were used to monitor bag cell after-discharge.
Recordings were performed from an isolated abdominal ganglion with a suction
electrode placed on a pleural-abdominal connective which was cut just anterior
to the bag cells. A bag cell after-discharge was initiated by brief electrical
stimulation via the suction electrode. The recordings were made with
a Model 1700 AC differential amplifier (A-M Systems, Carlsborg, WA, USA).
Surgery
Animals were anesthetized and relaxed with isotonic MgCl2 (30%
of their body mass). After the animals were completely flaccid, one of two
surgical procedures was performed. In one procedure, after an initial
incision, the pleural-abdominal connectives were severed bilaterally. In the
other procedure the small hermaphroditic duct was tied with silk braided
thread from a 6.0 suture (Assut Sutures, CE 0482, Pully-Lausanne,
Switzerland). The duct was tied in two places, close to its origin at the
gonad, and close to its terminus at the winding gland. The incision was then
sewn with silk thread. The surgical procedures were performed at 4°C.
After the surgery, animals were returned to 10 l containers of Mediterranean
seawater kept at 21°C. The water was changed twice before the start of an
experiment, 24 h after the surgery.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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To exclude the possibility that SNAP induces egg laying via
effects that do not depend on NO release, we tested the effects of SNAP after
it had already released most of its NO. SNAP releases NO with a half time of 5
h (Ignarro et al., 1981). We
allowed the SNAP solution to sit for 36 h, and then treated the animals with
this solution. Egg-laying behavior was elicited in only one of five tested
animals. To test whether egg laying might be caused by a much lower
concentration of NO, such as that which might be released by SNAP after 36 h
of exposure, we injected into one animal a 10 µmol l–1
concentration of SNAP (less than a quarter of the concentration used
previously). This treatment induced egg laying, suggesting that the small
quantities of NO released by SNAP after 36 h could cause egg laying in the
single animal that laid eggs in response to the stimulus.
We also tested the possible effect on egg-laying of NAP, an analogue of SNAP that lacks the S-nitroso group and therefore does not release NO. Egg-laying was not seen in any of the six animals that were tested.
The physiological effects of NO often occur through its activation of
soluble guanylyl cyclase, which in turn causes an increase in cyclic GMP
(cGMP) (Ahern et al., 2002;
Davis et al., 2001). However,
NO can also act through other mechanisms
(Davis et al., 2001), such as
S-nitrosylation (Ahern et al.,
2002). We tested whether blocking guanylyl cyclase by treatment
with Methylene Blue blocked the effects of the NO donor in inducing
egg-laying. Methylene Blue was injected into five animals 20 min before
treatment with the NO donor. All five animals laid eggs, similar to those
given the NO donor alone, indicating that the effect of NO on egg-laying is
not via the cGMP second messenger pathway.
Appearance of egg cordons
Egg-laying behavior in response to the NO donor was compared with that in
response to treatment with purified ELH (N=3) a BC homogenate
(N=10) or an LHD homogenate (N=4), which in a number of
Aplysia species contains tissue homologous to the atrial gland in
A. californica (Painter et al.,
1985). Such is the case in A. fasciata
(Susswein and Benny, 1985).
The NO donor produced changes in behavior that were similar or identical to
those elicited by the other stimuli. Egg deposition was preceded by appetitive
behaviors, waves and undulations. Eggs were deposited by weaves and tamps,
consummatory behaviors that distribute and attach the egg cordon to the
substrate. The similarities in egg-laying behavior during bouts elicited by
the NO donor and by ELH or bag cell homogenates led to a similarly raveled
appearance of the deposited egg cordons (see
Fig. 1A). Other aspects of
egg-laying behavior, such as mouth puckering, genital groove swelling,
crawling preceding egg deposition and its cessation, and weight of the cordon,
were not examined.
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Latency and duration of egg laying
The latency to egg laying (defined as the first observation of an egg
cordon) in response to the NO donor was compared to the latency in animals
treated with purified ELH, as well with BC and LHD homogenates
(Fig. 2A). There were no
significant differences in latency between animals treated with ELH, or with
BC or LHD homogenates (F2,14=0.83, P=0.46;
one-way analysis of variance), and therefore data from these three treatments
were combined. There was a small but significant decrease in the latency to
the deposition of egg cordons in animals treated with the NO donor, with
respect to the latency in response to other stimuli (P=0.03,
t29=2.28; two-tailed t-test). The duration of egg
laying was also measured (Fig.
2B). Data from animals treated with ELH and with LHD and BC
homogenates were combined, as there were no significant differences between
them (F2,14=2.58, P=0.12; one-way analysis of
variance). There was a significant decrease in the duration of egg laying in
response to the NO donor (P=0.001, t26=3.66;
two-tailed t-test), with respect to the duration in response to the
other three treatments.
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Blocking NO transmission does not block egg laying
Since a NO donor elicits aspects of egg laying, we reasoned that blocking
NO transmission might block aspects of egg-laying behavior. To test this
possibility, animals that were treated with BC or LHD homogenates, or with
ELH, were also treated with L-NAME, a competitive inhibitor of
L-arginine for nitric oxide synthase. The L-NAME was injected 5 min
before treatment with the stimuli eliciting egg laying, since treatment with
L-NAME elicits changes in feeding behavior within 5 min (N.M. and A.J.S.,
unpublished). In 11 of 11 animals normal egg cordons were seen, indicating
that NO release is not necessary for egg laying. Latency to egg laying was not
affected by L-NAME for either animals treated with BC or LHD homogenates, or
for animals treated with ELH. Treatment with L-NAME did not cause significant
effects on the duration of egg laying in animals treated with a bag cell
homogenate or with ELH. However, L-NAME caused a significant increase in the
duration of egg laying in response to the LHD homogenate (P=0.02,
t5=3.19; two-tailed t-test)
(Fig. 2C). These data suggests
that NO release may shorten the duration of egg laying in response to the LHD
homogenate, and treatment with a blocker of NO transmission uncovers this
regulation.
It is possible that L-NAME blocks the effects of ELH, or of BC or LHD homogenates, but itself elicits egg laying. As a control for the possible independent effects of 1.85 mmol l–1 L-NAME, we examined the effect of treatment with this concentration of L-NAME alone (N=5). The treatment did not elicit egg laying.
Sites of NO action
We examined the possible sites (Fig.
3A) of NO action in initiating egg-laying behavior.
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Egg-laying behavior similar to that described above was elicited by the NO donor in five of seven animals in which the pleural-abdominal connectives had been bilaterally severed. The mean latency from treatment with the NO donor to egg-laying behavior was 20.7±0.5 min (mean ± s.e.m.), which is comparable to the latency with the NO donor in unoperated animals (see Fig. 2A). The lack of egg-laying behavior in two of the animals treated with the NO donor is probably due to the effects of the surgery. These data indicate that NO is unlikely to act on neurons in the head ganglia that activate the bag cells and thereby induce egg laying.
Possible action on bag cells
We tested the possibility that NO induces egg-laying behavior by acting
directly on the bag cells and causing them to discharge and release peptides.
Bag cell activity was monitored in an isolated abdominal ganglion preparation
using a suction electrode placed on the cut pleural-abdominal connective,
close to the bag cells. Application of the NO donor (N=5) did not
cause a bag cell after-discharge (Fig.
3Bi). To be certain that the bag cells were capable of responding
to stimuli with an after-discharge, the connective was electrically stimulated
after treatment with the NO donor. In five of five cases, electrical
stimulation of the connective elicited a bag cell after-discharge
(Fig. 3Bii). These data
indicate that NO does not cause egg-laying behavior via possible
effects on the bag cells. NO is likely to act downstream from the bag
cells.
Possible action on gonads and on packaging glands
The ovotestis is one site of action of ELH. NO could act on the gonad,
causing it to release material into the small hermaphroditic duct, and the
presence of material in the duct and distal to it could act as a stimulus for
packaging the contents into capsules and cordons. A second possibility is that
NO acts directly on the accessory genital mass, and elicits the production of
capsules and cordons without the presence of material secreted from the gonad.
We tested these two possibilities by examining the effects of the NO donor on
Aplysia in which the small hermaphroditic duct had been ligated,
thereby preventing material secreted by the gonad from reaching the accessory
genital mass. If this treatment blocks egg laying in response to the NO donor,
one could conclude that NO acts on the gonad. By contrast, if NO still induces
egg laying one could conclude that NO acts directly on the packaging
machinery.
In six of six animals with a ligated small hermaphroditic duct, treatment with the NO donor elicited egg-laying behavior and the deposition of egg cordons. The latency to the start of egg laying was 21.0±0.44 min (± s.e.m.), which is comparable to the latency in intact animals that were treated with NO. Egg cordons in all 6 animals contained empty capsules, similar to those observed in most cases in which intact animals were treated with the NO donor. These data indicate that NO probably works directly on the accessory genital mass that package the eggs, and thereby produces egg cordons and capsules.
The data above suggest that NO may be released during natural egg laying, and acts on the accessory genital mass, inducing packaging of eggs. NO could be released as a result of a direct effect of bag cell peptides acting on the accessory genital mass. An alternative possibility is that transport of eggs via the small hermaphroditic duct, or perhaps a neurally mediated stimulus activated by egg transport, causes the release of NO and the recruitment of packaging by the accessory genital mass. To test between these two possibilities, we examined the effects of a bag cell homogenate on egg-laying behavior in animals in which the small hermaphroditic duct was ligated. If bag cell peptides cause NO release and subsequent egg-laying behavior, this treatment should cause animals to deposit egg cordons. By contrast, if transport of eggs or other neural signals elicit NO release and the deposition of egg cordons, the ligature should prevent the deposition. In five of five ligated animals no egg cordons were deposited in response to the bag cell homogenate.
Direct action on neurons organizing appetitive behavior
We examined whether NO initiates either appetitive or consummatory
behaviors in the absence of egg deposition. The effects of the NO donor were
examined in Aplysia in which egg-laying generally does not occur,
probably because they are sexually immature. Because most A. fasciata
are sexually mature throughout the season in which they are found, this
experiment was performed on A. californica during the winter months,
when many animals do not respond to bag cell homogenates
(Strumwasser et al., 1969). We
reasoned that treatment with the NO donor might not cause winter animals to
deposit an egg cordon, but might still elicit aspects of appetitive or
consummatory behaviors.
A. californica were treated with either ASW or with the NO donor and egg-laying behaviors were observed (Fig. 4). In five of nine A. californica treated with the NO donor, no egg cordons were produced, whereas the NO donor led to the production of egg cordons with empty capsules in the other four animals. Behavior during the hour following the treatment was compared in the five animals that did not produce egg cordons and in the animals treated with ASW. The total time devoted to appetitive behaviors (combined head waves and undulations) was significantly greater in animals treated with the NO donor than in animals treated with ASW (P=0.01, t9=3.03; two-tailed t-test), indicating that NO is able to induce appetitive behaviors even in animals that do not deposit egg cordons. The appetitive behaviors gradually decreased over the hour of observation, as shown by a significant difference in appetitive behaviors between the first and second halves of the observation in immature animals treated with the NO donor (P=0.04, t4=3.75; two-tailed paired t-test with Bonferroni correction). These data indicate that appetitive behaviors elicited by the NO donor are not dependent on the deposition of egg cordons.
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In animals that laid eggs the cordons began to appear 22–25 min after the treatment with the NO donor. Consummatory behaviors (tamps and head weaves) also began to appear at this time. Two of the five animals in which the NO donor did not elicit egg-laying displayed a brief increase in consummatory behaviors from 23–27 min after being treated, indicating that NO may initiate consummatory behaviors, but their maintenance requires stimuli provided by egg cordons.
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DISCUSSION |
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|
TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Hermann et al., 1997;
Jansen and Ter Maat, 1985) is
a complex behavioral sequence under partial neuroendocrine control. It has
been widely used as a model system for examining mechanisms underlying the
sequencing of behavior, and the interactions between behaviors. Eggs are
released from the gonad and then packaged into capsules within cordons, which
are eventually transported externally to the head
(Ter Maat and Ferguson, 1996;
Thompson and Bebbington,
1969), and then deposited by consummatory behaviors affecting body
wall muscles, as well as the mouth and lips. Egg laying has widespread
interactions with other behaviors, such as feeding and withdrawal reflexes,
which are inhibited during egg laying
(Cobbs and Pinsker, 1982b;
Goldsmith and Byrne, 1993;
Ram, 1982;
Stuart and Strumwasser, 1980).
It has been suggested that the sequencing of egg laying, and its interactions
with other behaviors, arises through the actions of a number of neuroendocrine
bag cell peptides (Scheller et al.,
1983; Scheller and Axel,
1984). Support for this hypothesis comes from the finding that
different bag cell peptides directly affect a variety of neural targets
(Branton et al., 1978;
Sigvardt et al., 1986;
Stuart and Strumwasser, 1980),
as well as directly affecting the gonad and causing the release of eggs
(Choate et al., 1993;
Coggeshall, 1970;
Rothman et al., 1983b).
However, other experiments also showed that aspects of egg-laying behavior are
initiated or regulated neurally, as feed-forward or feedback responses to
stimuli originating in other portions of the sequence
(Cobbs and Pinsker, 1982b;
Ter Maat and Ferguson,
1996). We have found that treating Aplysia with an NO donor induces aspects of egg-laying behavior. Exogenous NO presumably calls into play aspects of egg laying that are normally elicited either by neurally mediated signals or by bag cell hormones, which in turn cause the release of NO as an intermediate signal in the context of normal egg laying. Our data indicate that NO acts at specific sites in the egg laying cascade. However, it may also activate aspects of egg laying that it does not directly control, since the sites directly affected may act as stimuli that control other downstream and upstream sites.
Sites of NO action
Our findings rule out a number of sites at which NO could affect egg-laying
behavior. In principle, NO could be activated by peptides or by electrical
signals affecting neurons in the head ganglia. Bag cell or atrial gland
peptides, as well as electrical stimulation, have direct effects on neurons in
the head ganglia that excite the neuroendocrine bag cells and thereby cause
egg laying (Brown et al., 1989;
Ferguson et al., 1989b;
Shope et al., 1991). NO is
unlikely to be an intermediary for these effects, since cutting the
pleural-abdominal connectives by which neurons in the head ganglia communicate
with the bag cells did not block the effect of the NO donor. Bag cell and
atrial gland peptides also directly excite the bag cells
(Heller et al., 1980;
Kauer et al., 1987). NO is
unlikely to be an intermediary for these effects, since stimulating the bag
cells with the NO donor did not cause a bag cell after-discharge
(Fig. 3A).
One of the hormones secreted by the bag cells, ELH, acts directly on the gonad to effect egg release. NO is also unlikely mediate this effect, since most cordons lacked eggs or yolk, which are released from the gonad. In addition, egg cordons were elicited by the NO donor even after the small hermaphroditic duct was ligated, thereby preventing egg transport from the gonad to the accessory genital mass. In unoperated animals treated with the NO donor, egg-filled capsules were sometimes present late in a bout of egg laying, suggesting that feedback to the gonad from a downstream site directly affected by NO can cause egg release. However, we cannot eliminate the possibility that NO acts directly on the gonad, although with weaker, less consistent and longer latency effects than at downstream sites.
Our data provide support for three separate sites at which NO affects egg laying.
Egg packaging
One site of action is likely to be the accessory genital mass, composed of
the albumen and winding glands, which package the eggs into capsules and
cordons. A direct effect on the accessory genital mass would account for the
release of cordons without eggs, which are released from the gonad, upstream
from the accessory genital mass. Empty cordons were also laid in response to
NO when the small hermaphroditic duct connecting the gonad to the accessory
genital mass was ligated, indicating that the formation of egg cordons is not
dependent on stimuli provided by eggs released from the gonad and transported
to the accessory genital mass.
Under natural conditions, the accessory genital mass could be activated by
bag cell peptides acting directly on these tissues, which then release NO. In
our experiments, application of exogenous NO would bypass the hormones that
cause the NO release. A second possibility is that the release of eggs from
the gonad and their transport to the packaging machinery is a feed-forward
stimulus that causes the release of NO and the packaging of the eggs. Our data
provide strong support for the latter hypothesis. First, we found that bag
cell homogenates did not induce egg deposition after the small hermaphroditic
duct was ligated, indicating that the peptides cannot elicit the release of
cordons in the absence of egg transport. This finding is consistent with
previous results (Cobbs and Pinsker,
1982b) showing that ligation of the small hermaphroditic duct
blocks egg laying in response to a bag cells homogenate. Second, the latency
to deposition of a cordon in response to NO was shorter than in response to
other stimuli (Fig. 2),
suggesting that NO bypasses upstream sites whose activation takes time that
adds to the latency. In response to ELH, or BC or LHD homogenates, eggs would
be released from the gonads, and then transported to the accessory genital
mass, where NO would be released. In response to exogenous NO, release of eggs
is not necessary to activate the accessory genital mass, accounting for the
shorter latency.
Although NO is likely to act on the accessory genital mass and cause
packaging of eggs, it is not necessary for this action, since blocking
nitrergic transmission did not block egg laying. Many physiological systems
use multiple transmitters as messengers, with each transmitter sufficient but
not necessary to elicit an effect. NO is likely to act in tandem with another
transmitter in activating the packaging system. This is consistent with the
finding that neuron C2 in the feeding circuit, which uses NO as a transmitter,
also releases histamine. NO and histamine together depolarize follower cells
of C2 (Jacklet and Tieman,
2004).
Appetitive behaviors
A second site of NO action is the neural circuitry that organizes
appetitive behaviors preceding egg deposition. Thus, application of the NO
donor elicited appetitive behaviors even in Aplysia that were unable
to produce egg cordons (Fig.
4A). Previous data on gonadectomised animals that are unable to
lay eggs showed that appetitive behaviors are still initiated by bag cell
hormones (Ferguson et al.,
1986), indicating that these behaviors can be directly induced by
the hormones. Our data suggest that the effects of bag cell peptides on
appetitive behaviors are mediated in part by NO release. Appetitive behaviors
are effected by neurons in the pedal ganglia that innervate body wall muscles
in the neck (Ferguson et al.,
1989b). Moroz (Moroz,
2006) found nitrergic neurons in the pedal ganglia, suggesting
that these might be activated by stimuli initiating the appetitive behaviors
that then release NO, which would activate adjacent neurons that organize
appetitive behaviors.
Previous data (Ferguson et al.,
1986; Ferguson et al.,
1989a; Ter Maat and Ferguson,
1996) also indicated that the consummatory behaviors arise by
feed-forward excitation as a result of egg transport along the genital groove.
Activation of consummatory behaviors causes feedback inhibition of the
preceding appetitive behaviors (Ter Maat
and Ferguson, 1996). Our data are consistent with these findings.
First, consummatory behaviors in response to the NO donor were normal, in
spite of the anomalies in the contents of the egg cordon. Second, consummatory
behaviors were not maintained in non egg-laying animals treated with the NO
donor, although they were briefly initiated in some animals
(Fig. 4B). The lack of
consummatory behaviors in the presumably immature Aplysia led to a
prolongation of the appetitive behaviors that was not seen in animals that
laid eggs (Fig. 4A), in which
the consummatory behaviors apparently inhibited the appetitive behaviors.
Feeding behavior in Aplysia is inhibited during egg laying
(Ram, 1982;
Stuart and Strumwasser, 1980).
This inhibition is partially caused by feed-forward inhibition mediated by the
direct effects of bag cell peptides on neurons controlling feeding
(Ram, 1982;
Ram, 1983;
Sossin et al., 1987;
Teyke et al., 1991), as well
as by lateral inhibition, in which the performance of consummatory egg-laying
behaviors inhibit feeding (Cobbs and
Pinsker, 1982b; Ter Maat and
Ferguson, 1996). Previous data from our laboratory indicated that
the application of inhibitors of NO transmission, or of a NO donor, can
modulate aspects of feeding behavior
(Katzoff et al., 2006).
Increase in NO makes animals less interested in food (N.M., R. Saada, I.
Hurwitz and A.J.S., unpublished), and treatment with L-NAME induces food
arousal (Hurwitz et al.,
2006), suggesting that NO may be involved in the control of
feeding during egg laying.
Bout duration
NO may also act at a third site, and action at this site affects aspects of
bout duration. The duration of egg laying is related to length of the egg
cordon, and presumably to the number of eggs laid. Animals that have a larger
number of mature eggs within the gonads would lay more eggs, and the duration
of egg laying would therefore be longer. Egg laying duration in response to
the NO donor is shorter than in response to the other stimuli
(Fig. 2B), presumably because
there are few or no eggs released, and egg laying is therefore presumably
terminated prematurely. However, our data suggest that the duration of egg
laying may be regulated in a complex manner. Although there were no
differences in the duration of egg laying in response to ELH, or to BC or LHD
homogenates, treatment with the NO blocker L-NAME produced a large increase in
the duration of egg laying in response to the LHD homogenate. These data
suggest that application of different exogenous substances may result in
similar durations of egg laying by calling into play different transmitter
systems. The LHD homogenate may specifically call into play NO release, which
shortens the duration of egg laying. Additional studies will be needed to
determine the factors governing the duration of egg laying.
Latency of egg laying
The latency of egg laying in our experiments was generally shorter than
previously reported in response to stimuli such as shocks to the
pleural-abdominal connectives (Ferguson et
al., 1989a; Pinsker and Dudek,
1977), which in A. californica produced egg laying with
latencies of 30 min or longer. For A. fasciata it has been reported
that latency to egg laying is 27–36 min after the start of the bag cells
discharge (Ter Maat and Ferguson,
1996). Part of the latency in response to these stimuli is
presumably the time needed to produce an after-discharge in the bag cells, and
for the bag cells to release peptides, which in turn then cause egg release
and transport, which also take time. Application of a NO donor would shorten
the latency by bypassing many of these processes. Our data suggest that a
major portion (approximately 20 min) of the latency to egg laying stems from
the time needed for NO and other transmitters to act on the accessory genital
mass to produce capsules and cordons.
Multiple effects of NO
Previous data have suggested that a number of different sites in the
Aplysia nervous system affecting a single behavior may use a common
transmitter. Thus, neurons in the cerebral and pleural ganglia containing
peptides of the bag cells family apparently function in egg laying
(Brown et al., 1989;
Painter et al., 1988;
Pulst et al., 1988). In
addition, peptides related to those expressed in identified neuron R15 are
also expressed in other neurons active in autonomic control
(Romanova et al., 2007).
Serotonin release from a number of loci may also contribute to the
sensitization of defensive behaviors
(Marinesco et al., 2004). The
use of NO at a number of sites affecting egg laying may be an additional
example of this phenomenon. Moroz (Moroz,
2006) demonstrated that nitrergic neurons are present in all of
the central ganglia, as well as in many peripheral sites. It is likely that NO
has other functions unrelated to egg laying. However, many of the nitrergic
neurons may be activated during the control of different aspects of egg
laying.
Feed-forward and feedback control
Complex behavioral sequences in higher animals are controlled by both
feed-forward and feedback mechanisms (Ghez
and Krakauer, 2000). The finding that Aplysia egg laying
is also controlled in this manner indicates that this is a general principle
organizing behavior, even in relatively simple systems. The Aplysia
egg laying system is somewhat atypical in that the feed-forward components of
the system are partially hormonal and partially neural. The hormonally induced
components are relatively stereotyped: once an after-discharge in the bag
cells is elicited, and the hormones are released, there will be little ability
to regulate the subsequent components of egg laying behavior. However, many
aspects of egg laying are variable. The latency to egg deposition as well as
its duration are variable, as are the substrates on which the eggs are
deposited. Coordination of the egg laying sequence will have to be modulated
to take into account the variable aspects of the behavior. Our data, as well
as those of others, indicate that activity initiated by various aspects of egg
laying coordinate the sequence, and probably allow a particular phase of the
sequence to be properly timed. At many levels of the sequence, a neural signal
will act on targets that have already been primed by a preceding hormonal
effect. Our data indicate that specific neural and hormonal signals may be
partially mediated by NO.
LIST OF ABBREVIATIONS:
-nitro-L-arginine methyl ester
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Acknowledgments |
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References |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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