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5-Hydroxytryptamine (5-HT) has been proposed as a
neurotransmitter in crustaceans on several grounds. Levels
have been measured in the nervous system of various
crustacean species (Livingstone et al. 1981; Elofsson et al.
1982; Laxmyr, 1984; Kulkarni and Fingerman, 1992). A
number of immunopositive neurones have been identified in
central ganglia using anti-5-HT antisera (Eloffson, 1983; Beltz
and Kravitz, 1983; Sandeman et al. 1988; Bellon-Humbert and
Van Herp, 1988). 5-HT has also been measured in the
haemolymph (Livingstone et al. 1980; Elofsson et al. 1982),
thus suggesting a possible role as a neurohormone.
Various effects have been reported for 5-HT that implicate
it as a neuroactive agent in crustaceans. Its injection into the
circulation is known to induce a behavioural pattern of flexion
in lobster and crayfish (Livingstone et al. 1980), and neuronal
targets for this action have been identified in the thoracic and
abdominal ganglia (Kravitz, 1988; Ma et al. 1992). A
modulatory role on the crayfish escape response has also been
3067
The Journal of Experimental Biology 200, 3067–3077 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
JEB0864
The content and regional distribution of 5-
hydroxytryptamine (5-HT) in the crayfish eyestalk was
determined by high-performance liquid chromatography.
Levels of the 5-HT precursors L-tryptophan (L-TRP) and
5-hydroxytryptophan (5-OH-TRP), and of three
metabolites, 5-hydroxytryptophol (5-HTPH), N-
acetylserotonin (NA-5-HT) and 5-hydroxy-indole-3-acetic
acid (5-HIAA), were also determined. The
total content of 5-HT in the eyestalk was
95.4±49.3pgmg
−1
wetmass (mean ± S.D., N=55) while the
specific content was 9.6±4.9fmolµg
−1
protein (mean ± S.D.
N=5). 5-HT was present in all four ganglia of the eyestalk.
The highest proportion was found in the medulla
terminalis (40.2%) and the lowest in the retina lamina
ganglionaris (9.9%), which also had the lowest specific
content. Conversely, the highest specific content of L-TRP
was in the retina lamina ganglionaris.
5-HT biosynthesis and metabolism were explored in
isolated eyestalks. The monoamine oxidase blocker pargyline,
at concentrations between 0.8 and 10mmoll
−1
, elicited a dose-
dependent increase in 5-HT content. The biosynthesis of 5-
HT in the crayfish eyestalk is suggested by the presence of its
immediate precursor (5-OH-TRP) and by the suppression of
5-HT synthesis induced by m-hydroxybenzyl-hydrazine (m-
HBH), a blocker of 5-OH-TRP decarboxylase.
The presence of immunopositive cell bodies and axons
was demonstrated using an anti-5-HT antiserum. 5-HT-
like immunopositivity was detected in various regions of
the eyestalk. Efferent immunopositive axons were also
identified in the optic nerve, and these may have
originated in the protocerebral lobe of the
supraoesophageal ganglion. The branchings of these
axons were profusely distributed in the neuropile of the
medulla terminalis.
A basal level release of 5-HT was detected in isolated
eyestalks. The amount recovered was increased two-to
threefold after blocking 5-HT uptake with fluoxetine
(1µmoll
−1
). Incubation of eyestalks in solutions containing
a high K
+
concentration (80mmoll
−1
) released 5-HT.
Electrical stimulation of the optic nerve released 5-HT as
a function of the intensity of stimulation. Both the basal and
evoked release were suppressed by lowering the Ca
2+
concentration in the medium.
These observations support a role for 5-HT as a
neurotransmitter or neuromodulator in the crayfish
eyestalk.
Key words: serotonin, neurosecretion, crayfish, Procambarus clarkii,
crustacean.
Summary
Introduction
LOCALIZATION AND RELEASE OF 5-HYDROXYTRYPTAMINE IN THE CRAYFISH
EYESTALK
LEONARDO RODRÍGUEZ-SOSA
1
, ARTURO PICONES
2,
*, GABINA CALDERÓN ROSETE
1
,
SOCORRO ISLAS
2
AND HUGO ARÉCHIGA
1,
†
1
División de Estudios de Posgrado e Investigación, Facultad de Medicina, Universidad Nacional Autónoma de
México, México DF and
2
Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de
Estudios Avanzados, IPN, México DF
Accepted 14 August 1997
*Present address: Department of Physiology, University of California, San Francisco, CA 94143, USA.
†To whom correspondence should be addressed (e-mail: arechiga @servidor.unam.mx).
3068
proposed (Glanzman and Krasne, 1986; Yeh et al. 1996). 5-
HT is known to exert a facilitatory effect on lobster and
crayfish neuromuscular transmission (Dudel, 1965; Glusman
and Kravitz, 1982; Fischer and Florey, 1983; Dixon and
Atwood, 1985) and to elicit changes in the discharge pattern
of neurones in the lobster stomatogastric ganglion (Marder and
Hooper, 1985; Harris-Warrick and Marder, 1991). 5-HT has
also been shown to enhance sensitivity to light in crayfish
retinal photoreceptors (Aréchiga et al. 1990).
It has been suggested that 5-HT induces the release of
various neurohormones, such as the hyperglycaemic hormone
in the crayfish Orconectes limosus (Keller and Beyer, 1968),
the red and black pigment dispersing hormones in the fiddler
crab Uca pugilator (Fingerman and Nagabhushanam, 1992)
and the gonad-stimulating hormone in the crayfish
Procambarus clarkii (Sarojini et al. 1995a,b). Topical
application of 5-HT to the isolated eyestalk has been reported
to modify electrical activity in neurosecretory cells (Nagano
and Cooke, 1981). However, the possible role of 5-HT as a
neurotransmitter or neuromodulator on neurosecretory cells of
the crayfish eyestalk has not been confirmed (see Fingerman
and Nagabhushanam, 1992; Lüschen et al. 1993). The presence
of 5-HT, its biosynthesis and/or degradation have not been
established in the isolated eyestalk in the specific regions
where it would be expected to exert its physiological actions.
In addition, the release of 5-HT in the eyestalk ganglia has not
been explored.
It is the purpose of this study (a) to examine in the various
regions of the crayfish eyestalk for the presence of 5-HT, its
precursors, L-tryptophan (L-TRP) and 5-hydroxytryptophan
(5-OH-TRP) and its metabolites, 5-hydroxytryptophol (5-
HTPH) and 5-hydroxy-indole-3-acetic acid (5-HIAA),
produced after oxidation of 5-HT by the enzyme monoamine
oxidase, and N-acetyl-5-HT (NA-5-HT), produced after
acetylation by N-acetyltransferase, and to determine their
regional distribution; (b) to determine the location of 5-HT-
immunopositive cell bodies and axons in the eyestalk and optic
nerve, particularly in the areas of efferent input to the
neurosecretory cells in the X organ, and (c) to measure the
release of 5-HT from the isolated eyestalk after stimulation.
Materials and methods
The experiments were carried out using adult crayfishes
Procambarus clarkii (Girard) of either sex and in intermoult
at the time of the experiment. The crayfish were collected from
Rio Conchos, Chihuahua, México, and adapted to laboratory
conditions for 2 weeks, either under natural light:dark cycles
or on a 12h:12h light:dark programme.
Eyestalks were excised and placed in chilled saline solution
(van Harreveld, 1936). The exoskeleton, muscles and
connective tissue sheath were removed, leaving intact the
neural part of the optic peduncle, which was then divided by
microdissection into four segments (see Fig. 1). However, we
shall use the term eyestalk in this paper. Unless otherwise
stated, all experiments were conducted at room temperature
(22–24°C) and at night, when the 5-HT content has been
reported to be at its highest (Fingerman and Fingerman, 1977;
Fingerman et al. 1978).
Amine determinations
The amine determinations were made using high-
performance liquid chromatography (HPLC) following
techniques devised by Eloffson et al. (1982), Kilpatrick et al.
(1986) and Leung and Tsao (1992). Eyestalks were dissected
out, and groups of two eyestalks or eight eyestalk segments
(see Fig. 1) were pooled and their mass recorded. The neural
tissue was homogenized in 500µl of HClO
4
(100mmoll
−1
)
containing variable amounts of 3,4-dihydroxybenzylamine
(DHBA), and centrifuged at 1600g for 20min at 4°C using
a Sorvall centrifuge. The supernatant was filtered through a
nylon sieve with a pore diameter of 0.22µm. The samples
were stored at 0–5°C until analysis, which was conducted on
the same day. The protein content of the homogenized tissue
was determined by a modification (Cerbón and Aréchiga,
1986) of the Lowry method using small samples of nervous
tissue.
The samples were injected into a guard column of C-18,
connected to a reverse-phase analytical column of C-18
(25cm×4.6mm) packed with a particle size of 5µm (LDC-
Analytical). The amines were detected either by fluorescence
or with an electrochemical detector. The HPLC system with
fluorescence detector consisted of a programmable solvent
delivery module (Waters, model 590), a universal LC injector
(model U6K) and a scanning fluorescence detector (model
470). The signals were recorded on a data module (model 730).
Fluorescence was detected at an excitation wavelength of
254nm and an emission wavelength of 338nm.
Electrochemical detection was carried out according to
Kilpatrick et al. (1986). This HPLC system consisted of a
model PM-60 pump (Bioanalytical System) and a Rheodyne
7125 injector. A 200µl sample loop was routinely employed.
The volume eluted was analyzed by an amperometric detector
LC4B (Bioanalytical System). A potential of 0.8V, with
respect to a Ag/AgCl reference electrode, was applied for
oxidation of 5-HT. The current generated was recorded on an
x,y chart recorder (Cole-Palmer).
Three mobile phases were used. One of them (mobile phase
A in Table 1) was described by Leung and Tsao (1992); its
composition was as follows: sodium acetate (40mmoll
−1
),
citric acid (10mmoll
−1
), disodium EDTA (130µmoll
−1
),
octane-1-sulphonic acid (420µmoll
−1
), sodium chloride
(13mmoll
−1
) and methanol 10% (v/v), pH4.68. Mobile phase
B only differed from phase A in the proportion of methanol,
which was 20% (v/v). It was used for the detection of 5-HT
and NA-5-HT. A third mobile phase was used for
electrochemical detection, with the following composition:
sodium acetate (90mmoll
−1
), citric acid (35mmoll
−1
),
disodium EDTA (130µmoll
−1
), octane-1-sulphonic acid
(230µmoll
−1
) and methanol 10.5% (v/v). The pH was adjusted
to 4.35. The mobile phases were degassed before use and
recycled. The flow rate was usually kept at 0.8mlmin
−1
for
L. RODRÍGUEZ-SOSA AND OTHERS
30695-HT localization and release in crayfish eyestalk
electrochemical detection, but was variable for fluorescence
detection (see Table 1).
The amine solutions were freshly prepared for each
experiment and dissolved in perchloric acid (100mmoll
−1
).
With both procedures, the systems were calibrated to define
the ranges of linearity for the following substances (all from
Sigma Chemicals): L-tryptophan (L-TRP), 5-
hydroxytryptophan (5-OH-TRP), 5-hydroxytryptamine (5-
HT), 5-hydroxytryptophol (5-HTPH), N-acetylserotonin (NA-
5-HT), 5-hydroxy-indole-3-acetic acid (5-HIAA) and
3,4-dihydroxybenzylamine (DHBA), which was used as an
internal standard. The amounts of the amines in the samples
were calculated from the peak heights. For fluorescence
detection, the linear range is indicated in Table 1; for
electrochemical detection, the peak height of the current was
linear from a minimum detection value of 0.15pmol up to
1.5pmol of 5-HT. The corresponding peaks in the
chromatogram were confirmed by determining the increase in
amplitude of the peak after adding pure substances to the
column. The loss of substances during chromatography was
estimated as 10–15%, with DHBA as the internal standard.
Release of 5-HT
Eyestalks were incubated in the test solutions for 10min.
The perfusate was collected and HClO
4
(5mmoll
−1
) was
added. Samples were kept at 4°C until chromatographed. In
some experiments, 5-HT uptake was prevented by adding
fluoxetine (Ely Lilly) (1µmoll
−1
) and monoamine oxidase was
inhibited using pargyline (Sigma Chemicals)
(0.8–10mmoll
−1
). When basal or high-[K
+
]-induced 5-HT
release was measured, the eyestalks were incubated in a 400µl
pool in a modified van Harreveld’s saline (1936) (solution A
in Table 2). For release induced by high K
+
concentrations in
the medium, K
+
was substituted for Na
+
in the bathing
solutions at several concentrations. The results reported here
correspond to saline solutions with the compositions indicated
in Table 2.
For electrically evoked release, the eyestalk was mounted in
a 200µl pool of saline solution. Trains of pulses were delivered
through bipolar metal microelectrodes inserted into the optic
nerve (0.6–2µA, 50ms, 2Hz, for 10min). Pulses were
generated with a Grass S-48 stimulator through a PSIU 6
stimulus isolation unit and monitored on a Tektronix 2201
oscilloscope.
Immunocytochemistry
Cellular localization of 5-HT was made by
immunocytochemistry, using a primary antibody against 5-HT
(rabbit anti-5-HT, Immunonuclear Corp.). The crayfish were
anaesthetised in iced water (4°C) and the whole preparation
was removed by microdissection. Eyestalks were excised and
the neural tissue was cleansed as described above. In some
experiments, the eyestalk was removed with the optic nerve
and the supraoesophageal ganglion attached.
The preparations were left for 3h at 4°C in 4%
paraformaldehyde, in phosphate buffer (PBS 0.1moll
−1
), and
then transferred for 30min to a 30% sucrose solution. The
sample was then frozen and 16–18µm thick cryostat sections
prepared. In other experiments, in which the eyestalks were
whole-mounted, the dissection and fixation procedures were
the same, but the tissue was not incubated in the sucrose
solution. Either sections or whole mounts were incubated for
18h at room temperature with the primary antiserum. Various
dilutions of the primary antiserum were tried and the best
results were obtained with a 1:400 dilution in PBS containing
0.3% Triton X-100. After washing, preparations were exposed
to fluorescein-isothiocyanate-conjugated goat anti-rabbit IgG,
which was used as the secondary antibody (see Aréchiga et al.
1990). It was diluted (1:20) in PBS for 1h at 4°C. In some
experiments, preparations were dehydrated by successive
Table 1. Chromatographic conditions for the measurement of 5-HT, its precursors and three metabolites by fluorescence
Retention Linear range
Mobile Flow rate time of detection
Compound phase (mlmin
−1
) (min) (pmol)
L-Tryptophan, L-TRP A 1 15 0.176–2.13
5-Hydroxytryptophan, 5-OH-TRP A 1 7.5 0.336–1.36
5-Hydroxytryptamine, 5-HT B 1.2 34.6 0.34–3.51
5-Hydroxytryptophol, 5-HTPH A 1 21.6 0.417–1.29
N-Acetylserotonin, NA-5-HT B 1.2 31.6 0.338–1.016
5-Hydroxyindole-3-acetic acid, 5-HIAA A 1 11.8 0.183–0.774
3,4-Dihydroxybenzylamine* A 1 10 4.54–20.44
*Internal standard.
Table 2. Composition of solutions used for eyestalk
superfusion and/or incubation
Solution Na+ K
+
Ca
2+
Mg
2+
Cl
−
Hepes
A
205 5.4 13.5 2.6 242.6 2.5
B
205 5.4 0.1 16 242.6 2.5
C
130.4 80.0 13.5 2.6 242.6 2.5
D
130.4 80.0 0.1 16 242.6 2.5
Concentrations are given in moll
−1
.
The solutions were adjusted to pH7.4 using NaOH (0.1moll
−1
).
3070
incubations in ethanol at increasing concentrations from 10%
to 100% and finally cleared in methyl salicylate. Preparations
were observed under a fluorescence microscope (Zeiss).
Results
Regional distribution of 5-HT, its precursors and metabolites
in the eyestalk
To determine the regional distribution of 5-HT, the eyestalks
were divided into four segments, as shown in Fig. 1, which
presents the proportion of 5-HT contained in each segment
(hatched bars) as a percentage of the total content, which was
3.14±1.6pmol per eyestalk (95.4pgmg
−1
of wet tissue). The
specific 5-HT content (fmolµg
−1
of protein) was also
determined (black bars). The most distal segment, comprising
the retina and the lamina ganglionaris (R-LG), had a low total
5-HT content (9.9%), as well as a low specific content
(1.1fmolµg
−1
protein), while the medulla terminalis (MT) and
optic nerve (ON) had the highest proportion in the eyestalk
(40.2%). However, the specific content of this region had only
an intermediate value (13.5fmolµg
−1
protein). The two middle
segments corresponded to the medulla externa (ME) and the
medulla interna (MI); these had intermediate proportions of 5-
HT (27.2% in the ME and 22.8% in the MI) and high specific
contents (17.3fmolµg
−1
protein in the ME and
22.4fmolµg
−1
protein in the MI) (see Table 3).
The regional distribution was also determined for the 5-HT
precursors L-tryptophan (L-TRP) and 5-hydroxytryptophan (5-
OH-TRP) and for three of its metabolites, 5-hydroxytryptophol
(5-HTPH), 5-hydroxy-indole-3-acetic acid (5-HIAA) and N-
acetylserotonin (NA-5-HT). As can be seen in Table 3, all six
substances were present in the various regions of the eyestalk,
and wide differences in regional content were apparent. The
specific content of the precursors was higher than that of either
5-HT or its metabolites, except for 5-OH-TRP in MT, as seen
in Table 3. The highest specific content of L-TRP was found
in the retina lamina ganglionaris segment (see Table 3).
5-HT inactivation by monoamine oxidase in the eyestalk
The inactivation of 5-HT by monoamine oxidase (MAO)
was tested by using pargyline, an inhibitor of MAO activity
(see Neff and Yang, 1974). Groups of five eyestalks each
were incubated for 2h with various concentrations of
pargyline. Experiments were carried around midnight, when
MAO has been found in other nocturnal crustaceans to be
most active (Fingerman et al. 1978). Within the range
0.8–10mmoll
−1
, pargyline induces a dose-dependent
increase of 5-HT content and a simultaneous decrease in the
L. RODRÍGUEZ-SOSA AND OTHERS
60
30
0
5-HT content (%)
[5-HT] (fmolµg
−1
protein)
4
2
0
5
5
60
30
0
40
20
0
5
5
60
30
0
40
20
0
4
4
60
30
0
40
20
0
6
6
R
LG
ME
MI
MT
ON
Fig. 1. Regional distribution of 5-hydroxytryptamine
(5-HT) in the crayfish eyestalk. The diagram
represents the ganglia in the eyestalk. R, retina; LG,
lamina ganglionaris; ME, medulla externa; MI,
medulla interna; MT, medulla terminalis; ON, optic
nerve. The hatched bars represent the 5-HT content
(mean +
S.D.) as the percentage in each structure and
the black bars show the specific content
(fmolµg
−1
protein; mean + S.D.). The number of
experiments is indicated above each bar.
30715-HT localization and release in crayfish eyestalk
content of 5-HTPH (Fig. 2). Control groups of eyestalks
incubated in van Harreveld’s solution (Solution A in Table 2)
did not show any significant changes in either 5-HT or 5-
HTPH within the 2h period.
5-HT biosynthesis in the eyestalk
The biosynthesis of 5-HT from 5-OH-TRP was explored by
using m-hydroxybenzyl-hydrazine (m-HBH), a selective
blocker of 5-OH-TRP decarboxylase (Falck et al. 1966). Basal
5-HT content was determined, and groups of nine eyestalks
were incubated in 80mmoll
−1
K
+
solution for 30min (Solution
C in Table 2). As shown below, this procedure releases 5-HT
in the eyestalk. Samples of the depleted eyestalks (three each)
were taken for 5-HT determination. A pool of six eyestalks was
transferred to 20% Leibovitz L-15 culture medium in normal
saline solution, and samples consisting of three eyestalks were
taken after 60 and 120min intervals to determine 5-HT content.
Another pool of six eyestalks was incubated in the same
culture-medium-enriched saline solution to which m-HBH
(2mgml
−1
) had been added, and samples were also taken at
intervals of 60 and 120min for 5-HT determination. This
protocol was repeated several times (Fig. 3). All experiments
were conducted at room temperature (22–24°C), near dusk. As
seen in Fig. 3, the 5-HT content in the eyestalks incubated in
the inhibitor remained unchanged during the 2h period. This
was in contrast to the increase in 5-HT content seen after a 2h
incubation in culture medium.
Table 3. Content and regional distribution of 5-HT and related substances in the eyestalk
ES R-LG ME MI MT
Protein content 325.1±101 178.7±103.1 26.5±9.2 13.5±12 54.8±19.6
(µg per structure)
L-TRP 353.3±65.6 177.4±79.4 38.2±23.5 40.2±14.6 91.2±60.1
5-OH-TRP 57.6±37.8 25.5±4.2 18.3±9.4 45.7±26.1 7.1±3.5
5-HT 9.6±4.9 1.10±0.22 17.3±13.9 22.4±10.3 13.5±5.03
5-HIAA 7.1±5.9 0.7±0.5 14.1±12.1 2.3±1.9 4.3±2.07
5-HTPH 3.1±0.7 2.0±0.5 17.6±7.8 24.8±10.3 6.7±0.93
NA-5-HT 6.1±3.8 1.5±0.4 17.6±11.1 12.2±2.4 9.1±6.8
Values are expressed as fmolµg
−1
protein and are presented as means ± standard deviation (N=5).
ES, eyestalk; R-LG, retina and lamina ganglionaris; ME, medulla externa; MI, medulla interna; MT, medulla terminalis.
For abbreviations of chemicals, see Table 1.
Fig. 2. Effect of pargyline on the content of 5-hydroxytryptamine (5-
HT, filled circles) and its metabolite 5-hydroxytryptophol (5-HTPH;
open circles). Higher pargyline concentrations result in a greater
reduction of 5-HT degradation into 5-HTPH. All values correspond
to mean and standard deviations of five determinations.
25
20
15
10
5
0
[5-HT] (fmolµg
−1
protein)
[5-HTPH] (fmolµg
−1
protein)
5-HTPH
5-HT
5
4
3
2
1
0
0.8 1.6 4.0 10.0
[Pargyline] (mmoll
−1
)
Fig. 3. Blockage of 5-HT biosynthesis by m-hydroxybenzyl-
hydrazine (m-HBH). Bars indicate the amount of 5-HT present in
controls after 5-HT depletion (control, C) and after incubation in
culture medium for 60 and 120min, both in untreated medium
(hatched bars) and after addition of m-HBH to the medium (black
bars). Values represent mean ±
S.D. The number of experiments is
indicated above the bars; the asterisk indicates a significant difference
(P<0.05, student’s t-test) compared with the content of the depleted
eyestalk.
*
*
15
10
5
0
[5-HT] (fmolµg
−1
protein)
Culture medium
m-HBH
C 60 120
3
3
4
5
4
Time (min)
3072
Immunocytochemical localization of 5-HT-containing
structures
The distribution of 5-HT-immunopositive cell bodies and
fibres was assessed using an antibody against 5-HT. Whole
mounts of eyestalks were used. Immunoreactive cells were
found in all eyestalk regions, confirming results from other
authors. The labelled somata and fibres were widely
distributed. Various types of cell bodies were found in all four
neuropile regions of the eyestalk (see Elofsson, 1983;
Sandeman et al. 1988). The 5-HT-like immunopositivity found
in the lamina ganglionaris was described in detail in an earlier
communication (Aréchiga et al. 1990). Both the medulla
externa and the medulla interna contain immunopositive cell
bodies and fibres. This is consistent with the HPLC assessment,
which indicates the presence of 5-HT in all the eyestalk
ganglia. Since the distribution of 5-HT-containing elements in
the ME and MI was similar to that reported in detail for other
crustacean species, no further analysis was made in our
preparations.
Of particular relevance to the role of 5-HT in the modulation
of neurosecretory activity (see Sáenz et al. 1997) is the
presence of abundant immunopositive fibres in the neuropile
of the medulla terminalis, near the area where the X organ cells
are known to branch. As seen in Fig. 4, a group of
immunopositive cell bodies (10–15 of them) is located in the
area immediately above the hemi-ellipsoid body. The
diameters of the cells range from 15 to 40µm. Profuse
branchings of axons and abundant varicosities of these
neurones in the neuropile of the medulla terminalis were
observed in all preparations.
Interestingly, two separate bundles of 10–15
immunopositive axons each were identified in the optic nerve,
one running along the medial side of the nerve and the other
along the lateral side (Figs 5, 6). All fibres branched in the
neuropile of the medulla terminalis (Fig. 5A). Their number
and location were consistent in eight preparations. Their course
and pattern of endings suggested that they might be efferent
axons running from the supraoesophageal ganglion to the
medulla terminalis. A group of cell bodies in the protocerebral
lobe of the supraoesophageal ganglion, which were
consistently stained, may be the origin of the efferent fibres to
the medulla terminalis (Fig. 5B). However, this point requires
further elucidation.
To test the efferent nature of these fibres, in five animals,
one optic nerve was looped with a fine nylon thread, the
ligature was removed after 72h, and the two eyestalks, still
attached by the optic nerves to the supraoesophageal ganglion,
were excised and processed for immunocytochemistry. As seen
in Fig. 6, the immunopositive fibres in the medial bundle are
no longer present in the segment distal to the ligature, whereas
those in the proximal segment appear dilated. Axons in the
lateral bundle are still present in the distal stump.
L. RODRÍGUEZ-SOSA AND OTHERS
AB
hb
hb
X-o
X-o
Fig. 4. Immunopositive cell bodies and fibres in various regions of the crayfish eyestalk, particulary in the medulla terminalis. (A) Right eyestalk;
(B) left eyestalk. X-o, X organ; hb, hemi-ellipsoid body. Scale bar, 200µm.
30735-HT localization and release in crayfish eyestalk
Release of 5-HT
Both basal and evoked 5-HT release were examined. To
enhance recovery, the active uptake of 5-HT in the eyestalk
was inhibited by fluoxetine (Fuller et al. 1991; Chen and Reith,
1994). Four groups of four eyestalks each were incubated for
10min in 400µl of solution A (see Table 2) to which fluoxetine
(1µmoll
−1
) was added. As shown in Fig. 7, the basal release
of 5-HT in the eyestalk is increased over twofold after
incubation in fluoxetine.
Two methods were used to test the evoked release of 5-HT
from the eyestalk. From a group of 30 eyestalks, 12 were
incubated in a solution in which a high concentration of K
+
(80mmoll
−1
) was substituted for the equivalent amount of Na
+
(Solution C in Table 2). As shown in Fig. 8, the release of 5-
HT, which under control conditions is very low, even after
suppressing 5-HT uptake with fluoxetine, is greatly increased
by incubation for 30min in the high-[K
+
] solution.
To test the possible dependence of 5-HT release on [Ca
2+
]
o
,
Ca
2+
was substituted by Mg
2+
in the bathing fluid, leaving only
0.1mmoll
−1
[Ca
2+
]
o
(solutions B and D in Table 2). Groups of
six eyestalks were incubated in low-[Ca
2+
]
o
solution, three of
them in normal [K
+
]
o
and three in 80mmoll
−1
[K
+
]
o
. This
procedure was repeated several times, as indicated in Fig. 8.
Lowering [Ca
2+
]
o
greatly reduced both basal and the high-
[K
+
]-induced 5-HT release.
The other way of exploring 5-HT release was by selectively
stimulating the optic nerve with brief electric pulses of varying
intensities, as described in Materials and methods. These
experiments were carried out on individual eyestalks,
stimulated with pulses of 50ms at 2Hz for 10min. As shown
in Fig. 9, in 28 preparations, trains of pulses delivered to the
optic nerve are capable of inducing a considerable release of
5-HT to the medium, in proportion to the intensity of
stimulating current applied to the nerve. Again, 5-HT release
is reduced by lowering [Ca
2+
]
o
in the bathing fluid (not shown
in Fig 9). After trains of pulses, a lasting depletion of 5-HTwas
commonly observed for as long as 30min. However, in most
preparations, 5-HT release almost completely recovered after
2–3h.
As can be seen in the chromatograms shown in Figs 8 and
9, both the K
+
-induced and the electrically evoked stimulation
also caused the release of 5-HT-related substances. These
compounds were not identified in the experiments reported
here.
A
B
hb
on
sog
on
mt
on
mt
M
L
Fig. 5. Serial photomicrographs from whole mounts including part of the supraoesophageal ganglion (sog), medulla terminalis (mt) and optic
nerve (on) (see inset). (A) Immunopositive axons in the optic nerve, ending in the medulla terminalis (arrows) and presumably arising from a
group of somata in the supraoesophageal ganglion shown in B (indicated by an arrow). Note the profuse branching of immunopositive fibres in
the medulla terminalis near X organ cells, signalled with a black arrow in A. hb, hemi-ellipsoidal body. Scale bars, 500 µm. M, medial; L, lateral.
3074
Discussion
The total content of 5-HT in the eyestalk of Procambarus
clarkii (95.4pgmg
−1
wettissue) is close to the values reported
for other crayfish; for example, 100pgmg
−1
wettissue
determined by Eloffson et al. (1982) in Pacifastacus leniusculus
and 102pgmg
−1
wettissue by Kulkarni and Fingerman (1992)
in Procambarus clarkii. It is also similar to values found in
other crustacean species (Laxmyr, 1984). There is no other
systematic survey of the regional distribution of 5-HT in
crustaceans, but since 5-HT-like immunopositive cells have
been found in the various eyestalk regions, it is not surprising
to find 5-HT precursors and metabolites as well. The variability
of the values may be related to differences in the manipulation
of the tissue and the time between excision and measurement,
which could result in a lowering of 5-HT content.
The low content of 5-HT in the retina lamina ganglionaris
segment was to be expected, since most of it is non-neural
tissue, including the pigment cells and crystalline elements. A
similar situation may explain the difference between the
proportional and specific contents of 5-HT in the segment
comprising the medulla terminalis and the distal end of the
optic nerve; although the proportion is highest in this part of
the eyestalk, the specific content is lower than that of the ME
and MI. Whereas the mass of this segment (and its protein
content) is more than twice that of ME and four times that of
L. RODRÍGUEZ-SOSA AND OTHERS
Fig. 6. Reconstruction from serial photomicrographs of a crayfish optic nerve, showing the loss of medial immunopositive axons in the distal
stump, 72h after ligating the nerve. The arrow indicates the centripetal direction towards the supraoesophageal ganglion (see inset). Scale bar,
200µm. M, medial; L, lateral.
18
12
6
0
5-HT release (fmolmin
−1
)
C Fluoxetine
Fig. 7. Fluoxetine increases the release of 5-HT from the crayfish
eyestalk. 5-HT release per eyestalk was measured before and after
adding fluoxetine (1µmoll
−1
) to the bathing medium. Black bar,
control (C). Hatched bar, after addition of fluoxetine. Values are
means ±
S.D., N=4.
M
L
30755-HT localization and release in crayfish eyestalk
the MI (see Table 3), a large proportion of its mass corresponds
to optic nerve axons, only a few of which are 5-HT-
immunopositive, and to the neurosecretory system of the X
organ, also devoid of 5-HT. In fact, as seen in Figs 4 and 5, 5-
HT immunopositivity in the MT is located in the neuropile
branches and in a few somata, and it is remarkable that these
elements have such a high content of 5-HT. The high content
of L-TRP in the same region, compared with the low content
in the haemolymph (van Marrewijk and Ravestein, 1974),
suggests an active uptake mechanism for L-TRP, hitherto
unexplored, and presumably active biosynthesis of 5-HT.
Immunocytochemical evidence has identified a profuse
network of 5-HT-like immunopositive cell bodies and fibres in
the R-LG region (Aréchiga et al. 1990). These might be the
sites of 5-HT biosynthesis. This would be in agreement with
the role proposed for 5-HT as a local modulator of retinal
activity (Aréchiga et al. 1990).
The biosynthesis of 5-HT in the crayfish eyestalk is clearly
suggested by the presence of its precursor 5-OH-TRP, with a
similar regional distribution to that of the amine. The
suppression induced by 5-HT synthesis inhibitors on the
restitution of 5-HT levels after its depletion lends support to
this notion. This is in agreement with similar findings in the
crustacean Upogebia littoralis, by Marmaras and Fragoulis
(1970) and Fingerman et al. (1978) in the crab Uca pugilator,
where observations were made on intact animals. Our present
evidence supports the hypothesis of a process of 5-HT
biosynthesis intrinsic to the eyestalk.
The enhancement of 5-HT recovery from the perfusion fluid
after incubation in fluoxetine as well as the rise of 5-HT content
induced by pargyline suggest that two of the common
mechanisms of 5-HT inactivation are present in the crayfish
eyestalk: uptake of 5-HT, which is blocked by fluoxetine
(Fuller et al. 1991; Chen and Reith, 1994), and 5-HT oxidation
by MAO, which is inhibited by pargyline, as demonstrated
earlier in Uca pugilator (Fingerman et al. 1974, 1978) but not
detected by Barker et al. (1972) and Kennedy (1978) in the
lobster Homarus americanus. From the evidence we present
here, it is reasonable to conclude that MAO activity is present
in the crayfish eyestalk.
The presence of NA-5-HT suggests a role for acetylation in
the degradation pathway of 5-HT in the eyestalk. No particular
emphasis was placed in this study on exploring the activity of
the N-acetyltransferase system after finding the metabolite NA-
5-HT, since the activity of this enzyme has already been
30
40
20
10
0
5-HT release (fmolmin
−1
)
5.4
13.5
5.4
0.1
[KCl]
[CaCl
2
]
80
13.5
80
0.1
8
4
12
6
20min
1nA
5-HT
5-HT
Fig. 8. Release of 5-HT induced by incubation of the eyestalk in saline
solutions containing a high K
+
concentration (80mmoll
−1
) and its
dependence on Ca
2+
concentration. The amount of 5-HT released per
eyestalk was measured. Values are means ±
S.D. and the number of
experiments is indicated above each bar. Concentrations of KCl and
CaCl
2
are indicated (in mmoll
−1
) under each bar. HPLC traces in the
inset correspond to the control (black bar) and release of 5-HT after
incubation in a high K
+
concentration (see text).
75
50
25
0
5-HT release (fmolmin
−1
)
14
6
5
3
20min
1nA
5-HT
5-HT
B
C
D
A
012
Stimulation intensity (µA)
Fig. 9. Release of 5-HT evoked by electrical stimulation of the optic
nerve. D shows the amount of 5-HT released per eyestalk as
stimulation intensity increased. Values are means ±
S.D., values of N
are given above each point. Chromatograms: (A) control; (B) after
electrical stimulation at 0.7µA, (C) after stimulation at 1.3µA.
3076
established in the prawn Macrobrachium rosenbergii
(Withyachumnarnkul et al. 1992, 1993). The interaction of this
system with that of MAO in the crayfish remains to be
elucidated.
The location of immunopositive elements is similar to that
described for other crustacean species (Elofsson, 1983;
Sandeman et al. 1988). Of particular interest in this study is
the large 5-HT content and the profuse ramifications of 5-HT-
containing fibres present in the medulla terminalis neuropile,
the probable site of arrival of efferent input to the X organ cells.
The efferent nature of the immunopositive axons identified in
the optic nerve is supported by their disappearance in the distal
stump of the optic nerve after ligation and their continued
presence in the proximal stump. These may be the same axons
in which dense granules have been described, both in the optic
nerve (Larimer and Smith, 1980) and in the neuropile (Andrew
and Saleuddin, 1978). The possible site of origin of the
immunopositive axons in the optic nerve may be the small
immunopositive neurones present in the protocerebral lobe of
the supraoesophageal ganglion; further studies are needed to
confirm this, since other 5-HT-like immunopositive cell
somata have been described in various central ganglia,
although they are not known to send axons to the optic nerve
(Sandeman et al. 1988).
The maximal release of 5-HT evoked by stimulation
corresponds to approximately 2–3% of the total 5-HT content.
It is comparable to values found in other systems, such as the
giant cerebral neurones in Aplysia californica (Gerschenfeld et
al. 1978) and the Retzius cell of the leech (Henderson, 1983).
The dependence of 5-HT release on [Ca
2+
]
o
suggests that there
is an exocytotic mechanism, such as has been amply
documented for other 5-HT-releasing preparations
(Gerschenfeld et al. 1978; Livingstone et al. 1981; Henderson,
1983). The larger amount of 5-HT released by electrical
stimulation compared with incubation in high-[K
+
] solutions
suggests that short, phasic stimuli are more efficient in eliciting
release than a sustained, constant depolarization. The protocol
of stimulation was chosen after testing various combinations
of rate, voltage and train duration. The observation that other
amines are co-released with 5-HT by high [K
+
] and electrical
stimulation is not surprising since the stimulation was not
restricted to serotonergic neurones.
The widespread distribution of 5-HT in the various eyestalk
neuropiles suggests that it may have a multiplicity of functions.
So far, only the effects on the retina (Aréchiga et al. 1990) and
on neurosecretory activity (Fingerman and Nagabhushanam,
1992; Sáenz et al. 1997) have been described. A more detailed
search for other possible functions is necessary.
The results reported here support the concept that 5-HT is a
neurotransmitter or a modulator in the crayfish eyestalk.
Besides its modulatory role in retinal activity, it may act at
other levels of the eyestalk, as suggested by the wide
distribution of neurones immunopositive to the anti-5-HT
antiserum. The presence of a mesh of immunopositive axons
in the neuropile of the medulla terminalis points to a role in
the modulation of neurosecretory activity at that level. This
view is also supported by the demonstration of a direct action
of 5-HT on X organ neurones (see Sáenz et al. 1997).
The authors are indebted to Elizabeth Becerra and Victor
Anaya for valuable assistance in various aspects of the
experimental work. This project was partly supported by
CONACyT grant no. 0804-N9110.
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