Knowledge about the role of neuropeptides in the organization of
behavior has grown tremendously over the past decade and is derived
from diverse sources, including basic studies in various animal species
and studies of human neuro- and psychopathology (e.g.(1, 2, 3, 4) ). These studies
suggest that complex strategies of peptide expression, synthesis, and
action underlie the control of behavior. Bioactive peptides are
commonly synthesized in the form of a larger precursor protein from
which they are liberated by the action of prohormone convertases. Then
the peptides are modified (e.g. amidated, acetylated and
glycosylated) and sorted into dense core vesicles that are targeted to
the appropriate neuronal compartments, where the peptides are released
in response to depolarization. Often, several peptide genes are
co-expressed, frequently together with classical
transmitters(2, 3) . It has recently been suggested (5) that functionally related and closely apposed neurons may
produce overlapping yet distinct sets of peptides. This may be achieved
by one or a combination of various molecular and cellular processes, e.g. differential gene expression(5) , cell-specific
processing(6) , and sorting into distinct types of
granules(7) . As research progresses in this area, it is clear
that the study of the significance of the intrinsic peptide patterns
for behavior will require innovative techniques that allow examination
of the entire peptide profile in defined small brain loci and
even single neurons as well as target tissue. In the present study, we
use the newly developed technique of direct mass spectrometry of
nervous tissue in combination with conventional peptide chemistry, cDNA
cloning data, and neurobiological techniques to examine the intrinsic
patterns of peptide expression and targeting underlying behavior. We
studied the mollusk Lymnaea stagnalis, a model neurobiological
preparation that has been widely used for integrated molecular,
physiological, and behavioral studies (e.g.(8, 9, 10, 11, 12) ).
We examined the penis putative motoneurons which occur as
identifiable clusters in the right cerebral and pedal ganglia and as
scattered neurons in the right pleural and parietal
ganglia(12, 13) . These neurons send their axons into
the penis nerve, the sole nerve to innervate the penis complex
(comprising of the preputium, penis, retractor muscles, and vas
deferens). Several peptide messengers synthesized by the penis
motoneurons in the cerebral ganglion have been elucidated, and their
effects on various muscular systems of the penis complex have been
studied(10, 11, 14, 15) . However,
by using conventional methods of peptide chemistry and molecular
biology it is difficult to gain insight into the whole spectrum of
peptide messengers sent by central neurons to the penis complex.
Equally, peptide contents of the unidentified scattered motoneurons in
the right pleural and parietal ganglia are inaccessible for biochemical
analysis by conventional methodology. To circumvent these problems, we
employ here an alternative strategy that makes use of the newly
developed direct peptide profiling of nervous tissue by mass
spectrometry(5, 16) . The penis nerve was subjected to
direct mass determinations by matrix-assisted laser desorption
ionization mass spectrometry (MALDI-MS), (
)and the measured
masses of all the peptides were scrutinized closely. We detected in the
penis nerve two sets of identified peptides, the tetrapeptides FMRFa
and FLRFa and the heptapeptides GDPFLRFa and SDPFLRFa. Notwithstanding
extensive studies of their site of
synthesis(17, 18, 19) , the heptapeptides in Lymnaea, in contrast to the tetrapeptides (18) , have
never been implicated in the control of the activities of the penis.
In Lymnaea, the FMRFa gene is alternatively spliced into
two transcripts (Fig. 1), the FMRFa transcript encoding the
tetrapeptides and the GDPFLRFa transcript encoding the heptapeptides (20) . In view of our finding that both sets of peptides are
targeted to the penis complex, we hypothesized that in principle
different aspects of the complex copulation behavior may be controlled
by these different sets of peptides through mutually exclusive
expression in different penis motoneurons. Indeed, we could confirm the
presence of the peptides in the penis complex by peptide purification
and characterization. Moreover, we were able to identify the central
neurons that synthesize the peptides by backfilling the penis nerve in
conjunction with immunocytochemistry using two antibodies that
specifically recognize end products of the tetrapeptide and
heptapeptide transcripts, respectively. Finally, we could show by using in vitro bioassays that both sets of peptides have distinctly
different effects on the penis retractor muscle.
Figure 1:
The precursor structures as generated
by alternative splicing of the FMRFa gene of Lymnaea. The
signal sequence expressed by exon I of the FMRFa gene can be spliced to
the sequence expressed by exon II, which encodes the FMRFa precursor
from which the peptides FMRFa, FLRFa, and the SEEPLY peptide with other
putative peptides can be derived. Alternatively, the signal sequence
can also be spliced to the sequence expressed by exon III and exon
IV/V, which encode the GDPFRFa precursor from which the peptides
GDPFLRFa, SDPFLRFa, and the DEILSR peptide together with other putative
peptides can be derived(20) .
MATERIALS AND METHODS
MALDI-MS of the Penis Nerve
Small pieces of
penis nerve were dissected from mature laboratory-bred L. stagnalis under a microscope and transferred to 1-µl drops of matrix
solution (2,5-dihydroxybenzoic acid) on a stainless steel target. Each
sample was ruptured by a pair of tiny hooks to release the peptides in
the matrix. The solution was dried within minutes by a gentle stream of
cool air, and the target was inserted into the mass spectrometer
immediately afterwards. The acidic nature of the matrix (pH 2.0)
inhibits the activities of enzymes that might be released during cell
lysis. MALDI-MS was performed on a Finnigan MAT Vision 2000 laser
desorption time of flight mass spectrometer equipped with a pulse
nitrogen laser. An external standard peptide, renin, was used for
calibration. Usually, 30 individual spectra were accumulated to
increase the signal:noise ratio.
Electrospray Ionization Mass
Spectrometry
Fractions obtained by reversed phase high
performance liquid chromatography (rpHPLC) were dried in a Speed Vac
and redissolved in 100 µl of 7.0 mM trifluoroacetic acid
in 60% acetonitrile. Mass spectrometry was performed as described (21) with minor modifications. 8 µl of each fraction were
injected via a 10-µl loop into a Fisons BioQ triple-quadrupole mass
spectrometer equipped with an electrospray atmospheric pressure
ionization source. The mobile phase was 50% acetonitrile in 7.0
mM trifluoroacetic acid; the flow rate was 10 µl/min.
Peptide Purification
The penis complex (consisting
of the preputium, penis, penis retractor muscles, and vas deferens) was
used as the starting material for purification of tetra- and
heptapeptides. 750 penis complexes were dissected, collected on dry
ice, and stored at -20 °C until used. Extracts were made as
described previously (14, 15) and subjected to high
performance gel permeation chromatography (HPGPC) using an I-125
Protein Pak column (7.8
300 mm) and an I-300 Protein Pak column
(7.5
300 mm; both from Waters Associates) connected in series,
with a liquid chromatograph system (Waters Associates). The running
solvent was 7.0 mM trifluoroacetic acid in 30% acetonitrile,
the flow rate was 1 ml/min, and 1-min fractions were collected and
dried in a Speed Vac. The small peptides were pooled and further
resolved by sequential rpHPLC, using a Gynkotek 480 G HPLC
system(21) . Two different columns were used, a wide-pore
5-µm C18 column (4.6
250 mm; Nucleosil) at a flow-rate of 1
ml/min, and a wide-pore 7-µm C18 column (2.1
220 mm;
Brownlee), at a flow rate of 400 µl/min. The conditions for the
different rpHPLC steps are depicted in Table 1. In the first
rpHPLC purification step, 1-min fractions were collected, and, in the
following rpHPLC steps, 0.5-min fractions were collected. rpHPLC
fractions were dried in a Speed Vac, redissolved in 50 µl of
distilled water, and 1-µl aliquots were used in the
dot-immunobinding assay.
Dot-Immunobinding Assay
The assays were performed
as described previously(14, 15) . In brief, the
samples were dot-blotted onto nitrocellulose paper and conjugated to a
carrier protein precoated on the nitrocellulose paper. After drying,
the nitrocellulose paper was incubated with anti-FMRFa antibodies.
These antibodies recognize both the tetrapeptides and the
heptapeptides(22) . Subsequently, the nitrocellulose was
incubated in the secondary antibodies and developed in diaminobenzidine
solution containing 0.2% H
O
.
Amino Acid Sequencing
Amino acid sequencing was
done on an Applied Biosystems model 473 pulse liquid Sequencer, using
the sequencing program recommended by the company.
Backfilling and Immunocytochemistry
Central
ganglia of the Lymnaea brain were dissected in Hepes-buffered
saline, and, after removal of connective tissue, the penis nerve was
cut and drawn into a glass micropipette using a mild vacuum. The saline
in the pipette was then replaced by nickel-lysine (1.79 g of
NiCl-6H
O, 3.5 g of L-lysine free base in 20 ml of
H
O), and the preparation was left at room temperature for
18 h. The backfilled neurons were visualized by immersing the brain in
2 ml of fresh Hepes-buffered saline to which 5 drops of a saturated
ethanolic rubeanic acid solution had been added for 10-20 min.
Backfilled neurons displayed a blue to gray color. Then, the tissue was
fixed in an ascending series of paraformaldehyde in Tris-buffered
saline (1%, 0.5 h; 2%, 0.5 h; 4%, 3-18 h), dehydrated, and
embedded in paraffin. Alternating sections were placed onto sets of two
slides: one was used for immunocytochemistry, the other was dewaxed in
xylene and mounted with a coverslip. For immunocytochemistry, slides
were dewaxed, rehydrated, and incubated in Tris-buffered saline:gelatin
for 20 min prior to primary antiserum incubation. Sections were
incubated in a 1:300 dilution of antibodies against either the
synthetic peptide KQQVATDDSGELDDEILSR (DEILSR) or the synthetic peptide
SEQPDVDDYLRDVVLQSEEPLY (SEEPLY) in Tris-buffered saline:gelatin at 4
°C for 18 h. Both antibodies were a kind gift of Dr. N. Santama.
After washing, the secondary antiserum (peroxidase-conjugated swine
anti-rabbit; DAKO, 1:100 in Tris-buffered saline:gelatin) was applied
at room temperature for 1 h. Immunoreactivity was visualized as a brown
reaction product by addition of 0.5 mg/ml diaminobenzidine, 0.01%
H
O
in Tris-buffered saline, after which the
slides were dehydrated and mounted with a coverslip. The brain section
with the labeled cells as well as the backfilled counterparts in the
alternate section were photographed on Kodak EPY 64 T slide film.
Bioassay
The bioassays were performed as described
previously(14, 15) . In brief, the penis retractor
muscle was allowed to relax for 1.5 h before application of the
samples. Immediately after the application, the flow of Ringer was
stopped for 5 min. The mixing of sample was ensured by continuously
bubbling the Ringer with a small stream of gas. Muscle contractions
induced by the peptides were measured using an isotonic displacement
transducer and recorded by a chart recorder (Kipp & Zonen). The
following synthetic peptides were assayed: FMRFa, FLRFa, GDPFLRFa, and
SDPFLRFa.
RESULTS
Direct Peptide Profiling of the Penis Nerve by
MALDI-MS
Small pieces (
0.5 mm) of the penis nerve taken
from its origin were ruptured and immediately subjected to MALDI-MS.
The peptide patterns from several preparations were identical, and an
example of a mass spectrum is given in Fig. 2. This spectrum
revealed that a large number of peptides is present in the nerve and
that two of the peptides have masses corresponding to those of FMRFa
(calculated versus measured mass, 598.8 Da versus 598.3 Da) and FLRFa (580.7 Da versus 580.4 Da). The peak
height of the putative FMRFa peak is several times higher than that of
the putative FLRFa peak, suggesting that more FMRFa than FLRFa is
present, which agrees with the number of copies, 9 and 2, respectively,
in the precursor (cf. Fig. 1). Interestingly, two
prominent peaks correspond exactly to the masses of GDPFLRFa
(calculated mass versus measured mass, 850.0 Da versus 850.0 Da) and SDPFLRFa (880.0 Da versus 880.0 Da) that
are represented by 7 and 6 copies, respectively, in the precursor.
Again, the signal intensity reflects the relative number of peptides
contained in the precursor. These results suggest that, in addition to
neurons containing tetrapeptides, also central neurons expressing the
alternative GDPFLRFa transcript encoding the heptapeptides innervate
the penis complex.
Figure 2:
MALDI-MS spectrum of the penis nerve. The
mass spectrum of a piece of the penis nerve at the origin is shown
within the mass range of 550-950 Da. It includes the molecules
with masses corresponding to FMRFa (calculated versus measured
mass, 598.8 Da versus 598.3 Da), FLRFa (580.7 Da versus 580.4 Da), GDPFLRFa (850.0 Da versus 850.0 Da), and
SDPFLRFa (880.0 Da versus 880.0 Da). x axis, m/z, mass to charge ratio; y axis, arbitrary
units.
Peptide Purification and Characterization
FMRFa
and GDPFLRFa and SDPFLRFa have been previously isolated from the
central nervous system of Lymnaea(221) . Based on our
MALDI-MS data, we propose that these peptides are transported from the
central nervous system to the penis complex via the penis nerve. To
further substantiate this suggestion, we decided to demonstrate
unequivocally the presence of the peptides in the penis complex. To
this end, 750 penis complexes were extracted and the supernatant was
size-fractionated by HPGPC. Fractions 26 to 32 containing molecules
<1 kDa were further separated by rpHPLC using trifluoroacetic acid
as a counterion (Step 1; Table 1). All fractions were dotted to
nitrocellulose paper and screened for immunoreactivity by the
anti-FMRFa antibodies. Fig. 3A reveals that four groups of
immunoreactive fractions were present, which were designated A, B, C,
and D. These fractions were resolved separately using HCl as a
counterion (Fig. 3, B-E). The resulting
immunoreactive fractions from the groups B-D (Fig. 3, C-E) appeared to be pure and were subjected to chemical
characterization (Table 2), which revealed the presence of FMRFa
(group B), FLRFa (group C), and GDPFLRFa and SDPFLRFa which are both
contained in the same HPLC fraction (group D). The immunoreactive
material from group A was purified further using a third step of rpHPLC
with trifluoroacetic acid as a counterion (Step 3, Table 1; Fig. 3F). The immunoreactive material was subjected to
Edman degradation, which yielded an identical amino acid sequence as
found in group B (Table 2). However, mass measurement revealed
that this peptide is 16 Da heavier than the intact FMRFa. This suggests
that the peptide is oxidized at the methionine residue, presumably
during the extraction procedure(23, 24) .
Figure 3:
Purification of FMRFarelated peptides from
the penis complex. After HPGPC, FMRFa-related peptides were purified
using three subsequent rpHPLC steps. Fractions were screened using
anti-FMRFa antibodies. The immunoreactivity is indicated by bars. A, HPGPC fractions containing small molecules
were further purified by rpHPLC using a 5-µm C18 column (see Step
1, Table 1). B, the fractions from group A (see A) were purified further by rpHPLC using a 7-µm C18 column
(see Step 2, Table 1). C, the fractions from group B
(see A) were purified further by rpHPLC using a 7-µm C18
column (see Step 2, Table 1). The fractions corresponding to the
peak indicated by the asterisk were used for chemical
characterization. D, the fractions from group C (see A) were purified further by rpHPLC using a 7-µm C18 column
(see Step 2, Table 1). The fractions corresponding to the peak
indicated by the asterisk were used for chemical
characterization. E, the fractions from group D (see A) were purified further by rpHPLC using a 7-µm C18 column
(see Step 2, Table 1). The fractions corresponding to the peak
indicated by the asterisk were used for chemical
characterization. F, the immunoreactive fractions from group A
after a second rpHPLC step (see B) were purified further by
rpHPLC using a 7-µm C18 column (see Step 3, Table 1). The
fractions corresponding to the peak indicated by the asterisk were used for chemical
characterization.
Distribution and Identification of Immunopositive
Neurons
Because the tetrapeptides FMRFa and FLRFa and the
heptapeptides GDPFLRFa and SDPFLRFa are structurally similar, the
antibodies raised against FMRFa also recognize the other peptides (see
above). To be able to selectively detect the presence of products from
each one of the two precursors, antibodies raised against the peptides
SEEPLY and DEILSR were used that are contained by the FMRFa precursor
and the GDPFLRFa precursor, respectively (see Fig. 1). In
previous experiments(18, 19) , the specificity of
these antibodies has been rigorously tested: anti-SEEPLY antibodies
recognize only neurons in the nervous system that contain the peptides
encoded by the FMRFa transcript, whereas anti-DEILSR antibodies
specifically recognize neurons that contain the peptides encoded by the
GDPFLRFa transcript. DEILSR immunoreactive fibers could be detected in
the penis nerve and the penis complex. No immunoreactive cell bodies
could be detected in the penis complex (data not shown). As previous
studies did not give any cue of the identities of the central penis
motoneurons that express the GDPFLRFa transcript, we decided to
re-examine this issue. Backfilling of the penis nerve combined with
immunostaining revealed that there are several neurons in the right
pleural and parietal ganglia that send their axons to the penis complex (Fig. 4, A and C) and that are immunopositive
with the DEILSR antibodies (Fig. 4, B and D).
These cells therefore form the origin of the heptapeptides present in
the penis nerve and penis complex. The immunoreactive penis motoneurons
in the right parietal ganglion appear to be part of the identified
cluster of B cells(8) . The antibodies against SEEPLY
immunostained a number of neurons in the right cerebral ventral lobe
(see below). In accordance with the innervation pattern of these
neurons(13) , immunoreactive fibers were found in the penis
nerve and throughout the penis complex. In addition, backfilling of the
penis nerve with nickel lysine followed by staining with antibodies
against SEEPLY confirmed that the immunopositive anti-SEEPLY neurons of
the right cerebral ventral lobe indeed project down the penis nerve (Fig. 4E).
Figure 4:
Identification of central neurons involved
in male copulation behavior by a combination of backfills and
immunocytochemistry. Neurons backfilled via the penis nerve are
recognized by their blue/gray color; the
immunoreactive neurons are brown, whereas neurons that are
stained by both nickel lysine and antibodies have an intermediate
color. A, three backfilled neurons (arrows) in the
right parietal ganglion are shown. B, on the alternate section
of A, one backfilled neuron (arrowhead) was also
immunoreactive to the anti-DEILSR antibodies, whereas the others were
not (arrows). The location and the immunoreactivity of the
cell suggest that it is a member of the B group cells. C,
here, only one (arrow) of several backfilled neurons present
in the right pleural ganglion is shown. D, on the alternate
section of C, the backfilled neuron appeared immunoreactive
also after staining with anti-DEILRS antibodies (arrow). Other
immunoreactive neurons are also present (arrowheads). E, by applying anti-SEEPLY antibodies to a cross-section of
the ventral lobe of the right cerebral ganglion, it could be confirmed
that a number of the backfilled neurons contain peptides derived from
the FMRFa precursor, which are double-labeled with blue/gray and brown (arrows).
Non-immunoreactive backfilled neurons are also present (arrowheads).
Determination of the Effects of Tetrapeptides and
Heptapeptides on the Penis Retractor Muscle
The effects of
application of various concentrations of the synthetic peptides FMRFa,
FLRFa, GDPFLRFa, and SDPFLRFa were examined using the penis retractor
muscle as an in vitro bioassay preparation. When tetrapeptides
were applied, the muscle exhibited a fast contraction reaching a
plateau within seconds, then returned to basal levels during extensive
washing (Fig. 5, A and B). GDPFLRFa induced a
slow relaxing effect at 3
10
M,
which was often not fully reversible after extensive washing (Fig. 5C). At a high concentration of 10
M, however, GDPFLRFa induced a small tonic contraction,
which was often followed by relaxation (Fig. 5D). The
effect of SDPFLRFa was similar to that of GDPFLRFa. The dose-response
curves of FMRFa and FLRFa were similar (Fig. 6), with a
threshold dose for both peptides at 3
10
M, and a maximum effect at 3
10
M. The EC
values are 1.07
10
M and 1.10
10
M for FMRFa and FLRFa, respectively. At
a high concentration of 10
M, however,
FLRFa induced the muscle to contract at a lower level of about 75% of
the maximum level. The dose-response relationships of GDPFLRFa and
SDPFLRFa were profoundly different from those of FMRFa and FLRFa.
GDPFLRFa showed a biphasic curve with a minimum effective dose of
10
M that induced a relaxing effect. This
relaxing effect increased with higher concentrations, but at
10
M showed a (minor) contractile effect.
The effect of SDPFLRFa was similar to that of GDPFLRFa: at lower
concentrations (3
10
-3
10
M) SDPFLRFa induced a small relaxing
effect, and at higher concentrations
(10
-10
M) a small
tonic contraction (Fig. 6).
Figure 5:
Physiological effects of the tetra- and
heptapeptides on the penis retractor muscle in vitro. At the first arrow, the peptides were applied and the flow of Ringer
was stopped, and, at the second arrow, the flow of Ringer was
started again. A, effect of 3
10
M FMRFa on the penis retractor muscle. B,
effect of 3
10
M FLRFa on the penis
retractor muscle. C, effect of 3
10
M GDPFLRFa on the penis retractor muscle. D,
effect of 10
M GDPFLRFa on the penis
retractor muscle.
Figure 6:
Dose-response relationships of tetra- and
heptapeptides on the penis retractor muscle. In each experiment, the
effects of the peptides are indicated relative to the effect of
10
M FMRFa, which is set at 100%. Each data
point indicates the average of four independent experiments. Closed
squares, FMRFa; open squares, FLRFa; closed
circles, GDPFLRFa; open circles,
SDPFLRFa.
DISCUSSION
By using direct MALDI-MS determinations of peptides in the
penis nerve we were able to gain insight into the diversity of
candidate peptides that may be involved in the control of male
copulation in Lymnaea. We focused on two sets of closely
related peptides that belong to the FMRFa family and that are derived
from the alternatively spliced transcripts of the FMRFa gene. We
hypothesized that these two sets of peptides are synthesized by
different central neurons to coordinate the complex and flexible
copulation behavior of Lymnaea(12, 13) . In
the following we will present arguments that lead to the conclusions
that the sets of tetra- and heptapeptides encoded by the alternative
FMRFa and GDPFLRFa transcripts, respectively, indeed are expressed in a
mutually exclusive way in different penis motoneurons and, in addition,
have different effects on their peripheral targets, i.e. the
penis retractor muscles. The outcome of these experiments demonstrates
a novel and attractive principle of molecular and cellular regulation
of different aspects of a complex behavior by one peptide gene. More
generally, this finding may have important implications for theories
and models of the control of behavior and physiological processes by
bioactive peptides.
Tetrapeptides and Heptapeptides Derived from Two Alternatively
Spliced Transcripts of the FMRFa Gene Are Neuropeptides Involved in the
Control of Penis Functions
Neuropeptides are an important class
of messengers used by the nervous system for cell-to-cell
communication. Generally, their characterization is a first step toward
the understanding of the molecular basis of the functioning of a
neuronal circuit that governs a particular behavior(25) . Here
we focus on the peptides that are contained by the penis nerve. Because
the penis nerve is the sole nerve that relates the neuronal information
from the central ganglia to the penis complex and vice versa, the
peptides present in this nerve must be involved in copulation. As
conventional methodology lacks the ability to envision the whole
peptide profile present in a given tissue, we applied the novel
MALDI-MS method to a single penis nerve and demonstrated that multiple
peptides are contained by the nerve. We then focused on two sets of
neuropeptides, the tetrapeptides and heptapeptides of the Lymnaea FMRFa family of bioactive peptides. Previous peptide chemical (22) and recombinant DNA studies (17, 18, 19, 26) have revealed the
presence of these peptides in the Lymnaea nervous system. Our
mass spectrometric data strongly suggest that the peptides are also
present in the penis nerve because, firstly, the measured masses of the
peptides are in full agreement with the calculated masses of the
peptides, and, secondly, the ratios of the tetrapeptides FMRFa and
FLRFa and the heptapeptides GDPFLRFa and SDPFLRFa as determined in the
mass spectra also reflect the molar ratios that are predicted based on
the cDNA cloning studies. It follows then that the peptides must be
present in the penis complex, which was confirmed by their purification
from this organ.
Tetrapeptides and Heptapeptides Are Each Contained in
Different Central Penis Motoneurons
We postulated that the
tetrapeptides and heptapeptides are synthesized by central neurons and
transported to the penis complex. Indeed, our immunocytochemical
studies detect only immunoreactive fibers but not immunoreactive cells
in the penis complex. By backfilling the penis nerve, we were able to
localize the penis motoneurons, and, in combination with
immunocytochemistry using highly specific antibodies, we could
unequivocally show that tetrapeptides and heptapeptides are synthesized
in a mutually exclusive fashion in different penis motoneurons. The
penis motoneurons containing the tetrapeptides could be mapped to a
subset of neurons in the right cerebral ventral lobe, a cellular
location which has been proposed previously(18) . More
intriguing, however, is the finding that heptapeptides are also present
in the penis nerve and penis complex. The loci of synthesis of these
peptides have as yet not been described. However, we demonstrate here
that the penis motoneurons that contain the heptapeptides are the
``scattered'' neurons in the right pleural and parietal
ganglia. The location and phenotype of the neurons in the right
parietal ganglia strongly suggest that they constitute a subset of the
previously described group of B neurons(8) . Thus, our results
show that the B neurons do not form a homogeneous group, instead they
are neurons with different physiological (and behavioral) effects.
Tetrapeptides and Heptapeptides Have Distinct Effects on
the Penis Retractor Muscle
The tetrapeptides FMRFa and FLRFa
induce very similar fast contractions with typical plateau
characteristics when applied to the penis retractor muscle suspended in vitro. The EC
values obtained in the
dose-response experiments indicate that both peptides bind to the
receptors with very similar affinities. However, the dose-response
studies indicate also that at higher concentrations FLRFa, but not
FMRFa, elicits submaximal contractions. Because FLRFa and the
heptapeptides SDPFLRFa and GDPFLRFa share the same carboxyl-terminal
sequence (i.e. FLRFa), and because the heptapeptides have a
relaxing effect on the penis retractor muscle (see below), this
observation can be explained by assuming that at higher concentrations
FLRFa binds also to the heptapeptide receptors.Interestingly, at
concentrations up to 10
M, the main effects
of GDPFLRFa and SDPFLRFa are opposite to those of FMRFa and FLRFa. At
these concentrations, the heptapeptides induce a similar slow
relaxation of the penis retractor muscle. However, at higher
concentrations, they induce a small contraction, suggesting that at
higher concentrations the heptapeptides may in addition activate the
tetrapeptide receptors. In conclusion, our results show that at
physiological concentrations the tetrapeptides and heptapeptides have
opposite effects on the penis retractor muscle.
Multiple Peptides Are Involved in the Modulation of Penis
Complex Activities
Copulation as a male in Lymnaea involves a series of intricate movements of the penis complex, i.e. extrusion of the preputium and penis, probing for the
vagina, intromission, and transfer of semen followed by retraction. It
is apparent that the activities of the various parts of the penis
complex need to be accurately coordinated. Moreover, in case of
improper intromission, the behavior needs to be (partially)
resumed(12, 13) . These activities therefore call for
extensive fine-tuning of the activities of muscles in order to generate
the appropriate sequence of events underlying copulation. The present
(see e.g.Fig. 2) and previous experiments (10, 14, 15, 16) suggest that a
multitude of neuropeptides is involved in the control of the penis
complex in Lymnaea. A number of these peptides has been
structurally characterized, and their effects on individual parts of
the penis complex have been examined in detail. These studies have
revealed various different molecular and cellular strategies that
underlie the peptidergic control of copulation behavior. Thus,
conopressin and APGWa have opposite effects on the activity of the vas
deferens(14) , whereas different isoforms of the myomodulin
family of peptides have overlapping yet distinct effects on the penis
retractor muscle(15) . Conopressin (27) and APGWa (11) are encoded by distinct genes that are expressed in penis
motoneurons in the right cerebral anterior lobe, whereas the myomodulin
isoforms (15) are encoded by a single gene that is expressed in
motoneurons of the ventral lobe. The present study demonstrates another
important principle of peptidergic control of a complex and flexible
behavior by a single peptide gene: mutually exclusive cellular
expression of distinct sets of peptides encoded by the FMRFa gene that
are all structurally related yet have antagonistic actions. Thus, in
the context of the copulation behavior of Lymnaea, this
ingenious molecular and cellular strategy allows for a finely attuned
control of the same target, the penis retractor muscles, by distinct
penis motoneurons that in principle can operate independently.