Modulation of Electrical Activity by 5-Hydroxytryptamine in Crayfish Neurosecretory Cells

Abstract

The effect of 5-hydroxyptryptamine (5-HT) was tested in a population of X organ neurosecretory cells in the eyestalk of the crayfish Procambarus clarkii. Tests were conducted both in situ and on isolated neurones kept in culture.
The application of 5-HT induced action potentials in silent cells. In spontaneously active neurones, 5-HT increased the firing rate and either induced firing or enhanced bursting activity. The effect of 5-HT was dose-dependent within the range 1–100 μmol l−1 in cells of the intact organ. The effect persisted for 20–30 min after 5-HT had been removed from the bathing solution. Successive applications of 5-HT onto the same neurone reduced responsiveness, suggesting that desensitization had occurred. The effects of 5-HT were blocked by prior incubation with the 5-HT antagonist methysergide.
In X organ cells whose axons and branches in the neuropile had been severed, 5-HT induced a depolarisation associated with a slow inward current. In X organ neurones isolated from the eyestalk and kept in culture, 5-HT was capable of evoking bursts of action potentials and elicited a slow inward current. This effect was also blocked by methysergide (10−4 mol l–1).
These results suggest a direct modulatory effect of 5-HT on the pattern of electrical activity in the X organ cells.

Full text

5-Hydroxytryptamine (5-HT) has been proposed as a
transmitter or modulator mediating a variety of physiological
functions in crustaceans. A facilitatory role on neuromuscular
transmission in the lobster (Glusman and Kravitz, 1982) and
in the crayfish (Fischer and Florey, 1983; Dixon and Atwood,
1985) has been described. A specific behavioural pattern of
abdominal muscle flexion is induced by 5-HT injection in
lobster and crayfish (Livingstone et al. 1980), apparently
mediated by a combination of flexor motoneurone excitation
and extensor motoneurone inhibition (Kravitz, 1988). In
addition, 5-HT has been found to modulate the crayfish escape
response (Glanzman and Krasne, 1983; Yeh et al. 1996).
Peripheral sensory mechanisms are also modulated by 5-HT.
A facilitatory action has been described on primary
mechanoreceptor afferents (Pasztor and Bush, 1989) and on
receptor muscles of the lobster abdominal stretch receptor
(Pasztor and Golas, 1993), as well as on crayfish peripheral
mechanoreceptors (El Manira et al. 1991).
An enhancement of retinal responsiveness to light is also
induced by 5-HT in the crayfish Procambarus clarkii mediated
by a dual action: (a) increasing the gain of retinal
photoreceptors, by acting on a light-induced conductance, and
(b) promoting the retraction of intracellular pigment granules
within the photoreceptors, thereby increasing the photon flux
on the photosensitive membrane (Aréchiga et al. 1990).
Another likely target of 5-HT action in crustaceans is the
neurosecretory system. The injection of 5-HT in the crayfish
Orconectes limosus raises blood sugar levels, while this effect
can be prevented by eyestalk ablation (Keller and Beyer,
1968), suggesting that 5-HT acts as a modulator of crustacean
hyperglycaemic hormone (CHH) release in the X organ–sinus
gland system of the eyestalk. However, a direct
hyperglycaemic effect of 5-HT has been described in the shore
crab Carcinus maenas (Bauchau and Mengeot, 1966; Luschen
et al. 1993). It has also been suggested that 5-HT facilitates the
release of red pigment dispersing hormone (RPDH) in the
dwarf crayfish Cambarellus shufeldtii (Rao and Fingerman,
1975), of neurodepressing hormone (NDH) in the crayfish
Procambarus bouvieri (Aréchiga et al. 1985), of moult
inhibiting hormone (MIH) in the crab Cancer antennarius
(Mattson and Spaziani, 1985) and of black pigment dispersing
hormone (BPDH) in Cancer maenas (Bauchau and Mengeot,
1966; Fingerman and Nagabhushanam, 1992). A 5-HT-
induced increase in the number of exocytotic figures in
3079
The Journal of Experimental Biology 200, 000–000 (1997)
Printed in Great Britain © The Company of Biologists Limited 1997
JEB0685
The effect of 5-hydroxyptryptamine (5-HT) was tested in
a population of X organ neurosecretory cells in the eyestalk
of the crayfish Procambarus clarkii. Tests were conducted
both in situ and on isolated neurones kept in culture.
The application of 5-HT induced action potentials in
silent cells. In spontaneously active neurones, 5-HT
increased the firing rate and either induced firing or
enhanced bursting activity. The effect of 5-HT was
dose-dependent within the range 1–100µmoll−1in cells
of the intact organ. The effect persisted for 20–30min
after 5-HT had been removed from the bathing
solution. Successive applications of 5-HT onto the same
neurone reduced responsiveness, suggesting that
desensitization had occurred. The effects of 5-HT were
blocked by prior incubation with the 5-HT antagonist
methysergide.
In X organ cells whose axons and branches in the
neuropile had been severed, 5-HT induced a depolarisation
associated with a slow inward current. In X organ neurones
isolated from the eyestalk and kept in culture, 5-HT was
capable of evoking bursts of action potentials and elicited
a slow inward current. This effect was also blocked by
methysergide (10−4moll–1).
These results suggest a direct modulatory effect of 5-HT
on the pattern of electrical activity in the X organ cells.
Key words: serotonin, neuromodulation, neurosecretory systems,
bursting, neurosecretion, crayfish, Crustacea, Procambarus clarkii.
Summary
Introduction
MODULATION OF ELECTRICAL ACTIVITY BY 5-HYDROXYTRYPTAMINE IN
CRAYFISH NEUROSECRETORY CELLS
FRANCISCO SÁENZ1, UBALDO GARCÍA1AND HUGO ARÉCHIGA2,*
1Departamento de Fisiología, Biofísica y Neurociencias, Centro de Investigación y de Estudios Avanzados, IPN,
México DF and 2División de Estudios de Posgrado e Investigación, Facultad de Medicina,
Universidad Nacional Autónoma de México, México DF
Accepted 14 August 1997
*Author for correspondence (e-mail: arechiga@servidor.unam.mx).

3080
neurosecretory endings of the sinus gland of the crayfish
Astacus leptodactylus has also been documented (Strolemberg
and van Herp, 1977).
5-HT has been identified and quantified in the eyestalk of
various crustacean species (Eloffson et al. 1982; Laxmyr,
1984; Kulkarni and Fingerman, 1992). Neurone somata and
fibres reacting with anti-5-HT antisera have been described in
all eyestalk ganglia, as well as in other central ganglia
(Eloffson, 1983; Beltz and Kravitz, 1983; Bellon-Humbert and
van Herp, 1988; Sandeman et al. 1988). 5-HT-like
immunoreactivity has been located in dense-cored vesicles in
nerve endings in the medulla terminalis (Andrew and
Saleuddin, 1978) and in the retina (Aréchiga et al. 1990). 5-
HT-like immunopositivity has also been detected in the
crayfish, in a bundle of efferent axons running from the
supraoesophageal ganglion to the medulla terminalis, and 5-
HT release by electrical stimulation of the optic nerve has been
documented (Rodríguez-Sosa et al.1997).
This evidence suggests a role for 5-HT in the control of
hormonal secretion from the eyestalk. However, no direct
evidence exists for an effect on the neurosecretory cells
themselves. Although 5-HT has been reported to elicit changes
in the electrical activity of X organ cells in the crabs
Cardisoma carnifex and Podophthalmus vigil (Nagano and
Cooke, 1981), no analysis has been made of its possible effects.
It is the purpose of this paper to present evidence indicating a
direct action of 5-HT on the pattern of electrical activity of X
organ neurones, both in situ and in isolated neurones in culture.
Materials and methods
The experiments were carried out in adult crayfishes
Procambarus clarkii (Girard) or in isolated and cultured
neurosecretory cells removed from the X organ. Animals were
of either sex and in intermoult at the time of the experiment.
The specimens were collected from Rio Conchos, Chihuahua,
México, and adapted to laboratory conditions for 2 weeks,
either under a natural light:dark cycle or with a 12h:12h
light:dark programme. All experiments were conducted at
room temperature (20–22°C) and during day time.
Eyestalks were excised and placed in normal saline solution
containing (in mmoll−1): 205 NaCl, 5.4 KCl, 2.6 MgCl2, 13.5
CaCl2and 10 Hepes (pH7.4). The exoskeleton, muscles and
connective tissue were carefully removed under a microscope
to expose the neurone somata. The somata selected for our
study are in the most external layer of the X organ. Their
electrophysiological features have been reported previously
(Onetti et al. 1990).
Cell location was visualized under a microscope and
confirmed after the experiment by intracellular injection of the
fluorescent dye Lucifer Yellow. As illustrated in Fig. 1, the X
organ neurones send their axons in a well-defined dorso-lateral
direction and are easily recognisable. Intracellular dye
injections were made as previously described (Alvarado-
Álvarez et al. 1993).
The preparations were allowed to stabilize for 1h before
recording and for another 30min after impalements. Only
those cells with stable resting potentials over −45mV and with
spikes with at least 10mV of overshoot were selected for
further testing. Data were collected from 64 neurones. A new
preparation was used for each experiment. 5-HT (Sigma) and
methysergide (Research Biochemicals International) were
freshly prepared in normal saline solution at the time of
experiments and were applied either by perfusion to the
recording chamber or by pressure pulses onto the neurones in
isolated X organs (using 69kPa pressure pulses from 5-HT-
containing pipettes) placed near (100–150µm) the somata.
Intracellular impalements were made using borosilicate
glass microelectrodes pulled with a Sutter P87 pipette puller;
microelectrodes were filled with prefiltered (0.22µm,
Millipore) 3moll−1KCl to tip resistances of 40–50MΩ.
Recordings were made with an M-707 WPI amplifier.
For experiments on isolated neurones, the cells were
removed from the X organ by dissociating them by gentle
suction through fire-polished micropipettes, as previously
described (García et al. 1990). Isolated neurones were plated
F. SAENZ AND OTHERS
AB
Fig. 1. Photomicrographs of
the crayfish optic peduncle
showing an X organ
neurone filled with Lucifer
Yellow after a recording
has been made. (A) ON,
optic nerve; MI, medulla
interna; ME, medulla
externa; MT, medulla
terminalis. (B) Organ cell
during recording. Scale
bars, A, 1mm; B, 50µm.

30815-HT modulates electrical activity in crayfish neurosecretory cells
in a recording chamber previously coated for 2h with
Concanavalin A (Type III, Sigma). Cells were cultured in
modified Leibovitz L-15 medium, containing (in mmoll−1):
205 NaCl; 4.5 KCl; 13.5 CaCl2; 2.5 MgCl2; 10 Hepes; 5.5
glucose; 2 L-glutamine; gentamycin (16µgml−1, Schering
Plough); streptomycin (5µgml−1, Sigma); penicillin (5i.u.,
Sigma). Cultured cells were kept at 20–22°C in darkness.
Voltage-clamp experiments were performed either in the
whole-cell configuration or using a perforated patch
technique. Experiments were performed with an Axoclamp
2A amplifier (Axon Instruments). Voltage and current
recordings were stored on a video code modulator (PCM 400,
Vetter), and selected portions of the data were stored on the
hard disk of an 80486 computer (Acer 433) using
commercially available acquisition hardware and software
(pClamp 5.1, DigiData 1200 and Axotape 2, Axon
Instruments). For the perforated patch technique, a nystatin
(300µg ml−1) patch was used.
Membrane potential and spiking activity were acquired at 10
kHz through the Axotape program. Selected portions of
recordings were exported in ASCII format and analysed with
a computer program developed in the laboratory to determine
interspike intervals and the maximum slopes of depolarisation
and repolarisation during individual spikes (see Fig. 4). From
these data, instantaneous frequency graphs were prepared
using SigmaPlot version 5.0 (Jandel, Inc.).
Results
Effects of 5-HT on the various patterns of discharge in X
organ neurones
In 64 complete experiments, all of the impaled neurones
were responsive to 5-HT in a manner that was dependent on
their previous activity. As described earlier (Onetti et al. 1990),
there are three types of activity in crayfish X organ neurones.
They may be (a) silent, but capable of firing action potentials
by direct depolarisation through the recording electrode, (b)
tonically active, with a regular firing rate, or (c) spontaneously
bursting. As seen in Fig. 2A, topical application of 5-HT elicits
a prolonged depolarisation in a previously silent cell, which
gave rise to a burst of spikes lasting 2–5min. Bursting usually
started approximately 10s after perfusion of 5-HT into the
bathing solution. In tonically active neurones (Fig. 2B), 5-HT
induced a change in the activity pattern, eliciting prolonged
bursting activity lasting for 20–30min. All the bursting
neurones recorded responded to 5-HT with an enhancement of
activity. In these cells, 5-HT increased the burst duration and
shortened the inter-burst interval (Fig. 2C). In most
experiments, the duration of the 5-HT pulse was kept short,
since long exposures to 5-HT led to a prolonged desensitization
(see below). When 5-HT was applied to bursting neurones
shortly before the expected burst onset, the most noticeable
effect was a lengthening of the next burst, as shown in Fig. 3A.
When the application was made shortly after the end of a burst,
the main effect was a shortening of the interval to the next burst
(Fig. 3B), although there was also an increase in the length of
that burst.
To analyze the effects of 5-HT on the firing rate, the
interspike interval was evaluated as the time between two
successive maximum depolarisation slopes (Fig. 4A), and the
instantaneous firing frequency (the reciprocal of the interspike
interval) was determined before, during and after 5-HT
application (Fig. 4C–E). Fig. 4B shows the effect of 5-HT on
a tonically active neurone. The samples of activity used for
interspike interval analysis are marked in Fig. 4B, and the
results are presented in the corresponding graphs (Fig. 4C–E).
B
A
C
0
−50
0
−50
0
−50
Membrane potential (mV)
1min
Fig. 2. Effects of 5-hydroxytryptamine(5-HT)
(50µmoll−1) on three X organ neurones showing
representative patterns of activity. (A) A
previously silent neurone; (B) a neurone firing
tonically; and (C) a spontaneously bursting
neurone. 5-HT applications are indicated by black
bars under the recordings.

3082
As can be seen, the increase in the firing rate induced by 5-HT
(Fig. 4D) lasted over 3min and was followed by a prolonged
hyperpolarisation. The cell was silent for a few seconds, and
electrical activity was reinitiated in a bursting manner
(Fig. 4E). This pattern usually lasted 20–30 min (not shown in
the figure).
Although 5-HT-induced bursting was preceded by a slow
depolarisation, it cannot be explained solely on that basis, since
direct depolarisation of neurones through the microelectrode
elicited sustained spiking activity, but not bursting. However,
when bursting is prevented by hyperpolarising the cell, brief
depolarising pulses may trigger action potentials, but not bursts
(Fig. 5, upper traces), whereas after 5-HT application, the same
depolarising pulse is capable of eliciting a long burst after the
initial brief response (Fig. 5, lower traces).
Repetitive application of 5-HT resulted in a reduction of
responsiveness, as shown in Fig. 6 for the effect of successive
applications of 5-HT at 10min intervals at the same
F. SAENZ AND OTHERS
B
A
0
−50
0
−50
0
−50
0
−50
Membrane potential (mV)
1min
Fig. 3. Effects of 5-HT on a
bursting neurone. (A) 5-HT
applied at the onset of a
burst prolonged the burst
duration (top trace). (B) 5-
HT applied after the end of
a burst shortened the
interburst interval and
prolonged the subsequent
burst (top trace). Both
effects were reversed
after washing (lower
traces). 5-HT (50µmoll−1)
applications are indicated by
black bars.
Fig. 4. Effect of 5-HT on the
instantaneous spike frequency.
(A) The interspike interval was
defined as shown (see text). a,
Maximum depolarization slope;
b, maximum repolarization
slope. (B) Recording of a
tonically active neurone before,
during and after a 1min 5-HT
application (indicated by black
bar below trace) (50µmoll−1).
(C–E) Instantaneous firing rates
for the events indicated on the
recording shown in B. Note the
changes in the firing pattern.
B
A
a
b
0mV
−50mV
CDE
0mV
Inter-spike
interval
CDE
1.5
1.0
0.5
0
Instantaneous
frequency (Hz)
0 10 20 30 40 50 60 70 0 50 100 150 200 250 0 40 80 120 160
Time (s)

30835-HT modulates electrical activity in crayfish neurosecretory cells
0
−50
0
−50
0
−50
0
−50
Membrane potential (mV)
Control
Hyperpolarised
5-HT + hyperpolarised
Wash
0306090
Time (s)
0nA
−0.2nA
0nA
−0.2nA
Fig. 5. Hyperpolarisation prevents
bursting, but brief depolarising pulses
(0.2nA) (lower traces) elicit spiking
activity (upper traces). In the presence
of 5-HT (50µmoll−1), spiking is
evoked in the hyperpolarised neurone.
After washing out 5-HT, the
membrane potential returns to its
resting value and bursting is resumed.
B
A
D
C
0
−50
0
−50
0
−50
0
−50
Membrane potential (mV)
1min
Fig. 6. Desensitization to 5-HT.
Response of an X organ bursting
neurone to successive 5-HT
applications (50µmoll−1) at 10min
intervals. Notice the shortening of the
5-HT-associated burst and the
splitting of successive bursts. 5-HT
pulses are indicated by black bars
under the recordings.

3084
concentration (50µmoll−1) to a bursting neurone. Notice that
the bursts evoked by the 5-HT pulses were progressively
reduced in duration. After 5-HT removal, bursts were also split
into shorter trains of action potentials. These observations
suggest that a desensitization occurs.
Since 5-HT has been reported to affect the duration of
individual spikes in other systems, an analysis of spike
duration was made in our preparations. However, no effects
were found on the waveform of individual spikes, other than
those related to the higher level of depolarisation during 5-HT
application compared with control activity. In samples of 300
spikes in each of three experiments, neither the depolarising
and repolarising phases of individual spikes nor the overshoot
and total duration of the spikes were affected by 5-HT.
Dose-dependency of 5-HT effect
Although the long duration of the effect of 5-HT and the
resulting desensitization hampered the analysis of
dose–response relationships, it was possible in some
preparations to explore a wide range of doses in a single
neurone. Intervals between successive 5-HT applications had
to be longer than 10min (various interval lengths were tested).
As seen in Fig. 7A, for a bursting neurone subjected to pulses
of 5-HT applied over an increasing range of concentrations
from 1 to 100µmoll−1at intervals that also increased (15, 30
and 45min). The number of bursts evoked and the duration of
the effect were proportional to the dose of 5-HT. The interspike
intervals within bursts were also shortened in proportion to 5-
HT concentration. As shown in Fig. 7B, the mean frequency
of spikes within a burst was almost doubled over the dose range
tested. The value shown in the figure is the mean for all the
bursts during the time of 5-HT application. A proportional
relationship can also be seen between inter-burst interval and
the dose applied (Fig. 7C).
Pharmacological blockage
The effect of 5-HT could be blocked by methysergide
topically applied at the same doses (10–100µmoll−1)
previously found to suppress 5-HT modulatory action on the
retina (Aréchiga et al. 1990). In the experiment illustrated in
Fig. 8 (representative of three experiments with the same
dose), the preparation was incubated in 100µmoll−1
methysergide for 10 min prior to the pulse of 50µmoll−15-HT.
Both the depolarisation and the increase in bursting activity
F. SAENZ AND OTHERS
A
0
−50
0
−50
0
−50
0
−50
Membrane potential (mV)
1min
B
1.2
1.0
0.8
0.6
0.4
0.2
0
Mean frequency (Hz)
10−610−510−4
[5-HT] (moll−1)
C
10
8
6
4
2
0
Burst frequency
(burstsmin−1)
10−610−510−4
Fig. 7. Dose–response relationship for
5-HT on a bursting neurone. (A) 5-HT
pulses of increasing concentrations (1,
10, 50 and 100µmoll−1) given at
increasing intervals of 15, 30 and
45min, respectively, from top to
bottom. (B) The response was
evaluated as the mean frequency of
spikes within each burst evoked
during 5-HT perfusion. (C) Effect of
5-HT concentration on burst
frequency, in the 5-HT responses
shown in A. Each point represents the
mean value of four experiments. Error
bars indicate standard deviation.

30855-HT modulates electrical activity in crayfish neurosecretory cells
were blocked. It is interesting to note that no effect of
methysergide was detected on spontaneous synaptic input to X
organ cells.
Site of action of 5-HT
From the experiments described so far, no definite view can
be derived as to the site(s) of action of 5-HT on X organ
neurones. The issue is of interest since it may help to clarify
whether 5-HT acts as a neurotransmitter at specific synaptic
sites or as a modulator on wide areas of the neuronal surface.
To explore this issue, 5-HT was tested on isolated clusters of
X organ cells. Fig. 9C shows the isolated X organ cluster in
which at least five neurone somata can be distinguished. Their
axons were severed before the emergence of the branches in
the neuropile of the medulla terminalis. As seen in Fig. 9A, the
axotomized neurone was no longer capable of generating fast
spikes or spontaneous bursting activity but, in response to
depolarising pulses, did produce slow action potentials, that are
known to be Ca2+-dependent (Onetti et al. 1990). During 5-HT
perfusion (1µmoll−1), a 5mV depolarisation was evoked
(Fig. 9B, upper trace). This effect was reversible after 5-HT
removal. The time course of the depolarisation evoked by 5-
HT corresponds to the slow inward current recorded under
voltage-clamp conditions (Fig. 9B, middle trace). Both traces
0mV
−50mV
1min
Methysergide 5-HT
+
methysergide
Fig. 8. Blockade of the effect of 5-HT by
methysergide. As seen in the control (top trace),
bursting activity was enhanced by 5-HT (black
bar, 50µmoll−1) and this response was
suppressed by continuous perfusion with
methysergide (100µmoll−1), indicated by the
open bar beneath the lower trace (recorded 30 min
after the control).
C
B
A
0
−50
0
−50
10
−10
Em(mV)Em(mV)
Im
(pA)
5s
100ms
5-HT
Fig. 9. Effect of 5-HT (1µmoll−1) on the response of a neurone in the isolated X organ. (A) Control recording; a single spike is evoked by a
brief depolarising pulse. (B) A 5-HT pulse (1µmoll−1, 69kPa, 100ms, shown in the bottom trace) evokes, under current-clamp mode, a 5mV
depolarisation and a single spike (upper trace). In the whole-cell configuration, 5-HT evoked a slow inward current (Im) measured at a holding
potential of −50mV. (C) A photomicrograph of an isolated X organ from the eyestalk. Scale bar, 110µm.

3086
were obtained after applying 5-HT by pressure pulses onto the
somata of the cells.
Effect of 5-HT on isolated X organ neurones
Although the effects so far described for 5-HT on X organ
cells appear to be due to a direct action on the neurones, an
indirect action cannot be ruled out while working on neurones
in intact X organ. These neurones are known to receive
synaptic inputs (Iwasaki and Satow, 1971) mediating the
influence of light (Glantz et al. 1983), and GABA evokes
depolarising responses and trains of action potentials in a dose-
dependent manner (García et al. 1994). Although neither 5-HT
nor methysergide was found to have any effects on the synaptic
activity recorded in our preparations, the only certain way to
explore a direct action of 5-HT on neurones was to test it on
isolated X organ cells.
As illustrated in Fig. 10, in an isolated X organ neurone from
the same population recorded in situ, that had been cultured for
24h, topical application of 50µmoll−15-HT elicited a slow
depolarisation, recorded under current-clamp conditions.
Concurrent with the depolarisation, there was a 33% increase
in the input resistance, as determined by the application of brief
F. SAENZ AND OTHERS
B
A
D
C
0
−50
0
−50
0
−25
−50
−100
E
Em(mV)Em(mV)Im(pA)
Em(mV)
Vh= −30 mV
Vh= −50 mV
Vh= −50 mV
Vh= −70 mV
1min
5-HT
Fig. 10. Responses of a cultured X
organ neurone to 5-HT (50µmoll−1)
recorded under perforated patch-
clamp (A, B and D under current-
clamp mode; C under voltage-clamp
mode). In A and D, input resistance is
probed by brief hyperpolarising
pulses applied through the recording
pipette. Voltage (mV) for ordinates in
A, B and D; current (pA) for ordinate
in C. Black bars indicate 5-HT
applications. (E) Photomicrograph of
an isolated neurone from the X organ.
Vh, holding potential; Em, membrane
potential; Im, membrane current.
Scale bar, 50µm.

30875-HT modulates electrical activity in crayfish neurosecretory cells
(200ms at 0.5Hz) pulses of 10pA (Fig. 10A). The membrane
potential was set to −30mV by depolarising current injection.
Notice the firing of one action potential during the
depolarisation. When the membrane potential was shifted to
−50mV, a similar 5-HT pulse was capable of eliciting a burst
of action potentials (Fig. 10B). Under voltage-clamp, at a
holding potential of −50mV, 5-HT applied under similar
conditions as in the previous tests evoked a slow inward
current with a similar time course to that of the conductance
changes observed in the previous tests (Fig. 10C). After
shifting the membrane potential to −70mV (Fig. 10D), again
under current-clamp conditions, the enhancement of input
resistance was only 25%.
Previous incubation of a cultured neurone in 100µmoll−1
methysergide resulted in a considerable suppression of the
response to 5-HT under perforated patch conditions. As seen
in Fig. 11, 5-HT (50 µmoll−1) elicited a prolonged burst (upper
trace). This response was largely blocked by prior incubation
of the neurone in methysergide (middle trace). A substantial
recovery of the response to 5-HT occurred after the removal
of methysergide (lower trace). The small difference from the
control response might be expected given the desensitization
observed in the in situ experiments.
Discussion
The sensitivity of the X organ neurosecretory cells to 5-HT
supports a role for this amine in stimulating the release of
neurohormones in the eyestalk, as proposed from
pharmacological studies (for a review, see Fingerman and
Nagabhushanam, 1992). It also accords with the abundance of
5-HT-like immunoreactive cell bodies and fibres observed in
the crayfish medulla terminalis neuropile where the X organ
cells receive synaptic inputs (Elofsson et al. 1982; Sandeman
et al. 1988; Rodríguez-Sosa et al. 1997). We presume that the
neurones examined in this study are those containing CHH,
since their location, size and shape coincide with those of cells
in which immunopositivity to antibodies against CHH has been
documented (Jaros and Keller, 1979; van Herp and van
Buggenum, 1979). It is reasonable to suggest that the bursting
activity elicited by the applications of 5-HT on X organ
neurones that we describe in this paper is the mechanism
mediating the release of CHH induced by 5-HT in the crayfish
Orconectes leniusculus (Keller and Beyer, 1968). However,
since no immunocytochemical characterization was made, the
effects of 5-HT could be exerted on X organ cells containing
other hormones.
5-HT could mediate the effects of stress, which is known to
raise blood sugar levels in the lobster Homarus americanus
(Telford, 1986), and the effects of darkness, since both the
blood sugar concentration (Gorles-Kallen and Vooter, 1986)
and the 5-HT content in the eyestalk (Fingerman and
Fingerman, 1977) are higher at night. Since not all eyestalk
neurohormones appear to be released under the influence of 5-
0
−50
0
−50
0
−50
Membrane potential (mV)
5-HT
Methysergide
Fig. 11. Effects of 5-HT (50µmoll−1, 1min) and
methysergide (100µmoll−1) on an X organ neurone
kept in culture and recorded using the perforated patch
technique. The upper trace shows bursting elicited by
a 1min application of 5-HT (indicated by black bar).
After a 10min incubation in methysergide, the effect
was greatly reduced (middle trace). After a 10min
wash, bursting could again be evoked by 5-HT (lower
trace).

3088
HT (Fingerman and Nagabhushanam, 1992), it would be
interesting to test this amine on other groups of neurosecretory
cells in the eyestalk.
The induction of a bursting pattern of activity in X organ
cells by 5-HT would be an effective way of triggering hormone
release. In fact, bursting activity has been proposed as the most
efficient temporal pattern of action potential distribution in
releasing neurosecretory products from vertebrate
hypothalamic neurosecretory cells (Wakerly and Lincoln,
1973; Dutton and Dyball, 1979; Poulain and Wakerly, 1982;
Cazalis et al. 1985), although this relationship has been
questioned for crustacean neurosecretory cells (Keller et al.
1994).
Most features of the effects of 5-HT on X organ neurones
are similar to those described in other systems in which a
facilitatory action has been documented for 5-HT. The
persistence of the effect of 5-HT has also been described in
preparations such as the lobster neuromuscular junction
(Glusman and Kravitz, 1982) and the crayfish neuromuscular
junction (Dixon and Atwood, 1985) and retina (Aréchiga et al.
1990), where it may last for over 30min. The persistence of
the effect long after 5-HT has been removed from the bathing
fluid suggests the participation of an intracellular messenger
stage, as has been described for many other systems involving
5-HT. Desensitization to the effects of 5-HT is another feature
that has been described in invertebrate neurones in the past
(Gerschenfeld and Paupardin-Tritsch, 1974).
The dose–response relationship for the effects of 5-HT on
the X organ cells in situ is similar to that described for the
behavioural responses of the lobster (Livingstone et al. 1980)
and the retinal effects in the crayfish (Aréchiga et al. 1990).
The effective doses are higher than those necessary for
peripheral effects, such as those on the neuromuscular junction,
and also higher than the 5-HT content in the haemolymph
(Livingstone et al. 1980). This has been attributed to
permeability barriers and to active uptake systems which limit
the availability of the amine at the receptor sites (Livingstone
et al. 1980). This view is consistent with our observation that
the sensitivity to 5-HT increases when it is tested on isolated
neurones.
Methysergide has been shown to block 5-HT effects in other
crustacean systems, such as the crayfish retina (Frixione and
Hernández, 1989; Aréchiga et al. 1990) and central neurones
(see Zhang and Harris-Warrick, 1994), as well as in neurones
of other invertebrates, such as the snail Achatina fulica
(Furukawa and Kobayashi, 1988). However, its specificity is
unknown, so the nature of the 5-HT receptors mediating the
excitatory effect of 5-HT is still an open issue.
The sensitivity to 5-HT of the X organ neurone somata after
axotomy is more consistent with a role for 5-HT as a modulator
rather than as a transmitter. In fact, the regional sensitivity of
these neurones to 5-HT is different from that to γ-aminobutyric
acid (GABA), which is ineffective when tested on organ
somata, since sensitivity is confined to the neuropile (García et
al. 1994) where all synaptic connections to X organ neurones
appear to be made (Andrew et al. 1978; Glantz et al. 1983).
This is also consistent with the lack of effect of methysergide
on spontaneous synaptic activity in these neurones. Besides a
local modulatory role for 5-HT released at the neuropile of the
medulla terminalis, it may also have a hormonal action, since
this amine has been identified in crayfish haemolymph
(Livingstone et al. 1980).
Various ionic mechanisms have been described to account
for 5-HT-induced excitatory responses. Since no effects of 5-
HT were detected on the amplitude or the duration of
individual spikes of X organ cells, no action on fast voltage-
dependent currents can be assumed. A host of slow inward
currents have been described when 5-HT is introduced into a
number of preparations, such as invertebrate neurones
(Gerschenfeld and Paupardin-Trisch, 1974; Deterre et al. 1981;
Boyle et al. 1984; Furukawa and Kobayashi, 1988; Levitan and
Levitan, 1988; Baxter and Byrne, 1989; Harris-Warrick and
Marder, 1991; Price and Goldberg, 1993; Pellmar, 1984) and
vertebrate neurones (Andrade and Chaput, 1991; Colino and
Halliwell, 1987; Hounsgaard and Kiehn, 1989; Pape and
McCormick, 1989; Wallén et al. 1989; Stefani et al. 1990;
Anwyl, 1992). 5-HT-induced depolarisations have also been
reported as a result of inhibition of an electrogenic Na+pump
(Catarsi et al. 1993). Of particular interest for the results
reported in this paper is the induction of bursting activity by
5-HT, which has been reported in a variety of systems; 5-HT
increases bursting activity in the AB/PD neurones of the
stomatogastric ganglion of the crab Cancer borealis (Zhang
and Harris-Warrick, 1994). In the same ganglion, 5-HT
induces plateau potentials in the dorsal gastric (DG)
motoneurone; this effect is achieved by the combination of an
enhancement of a hyperpolarisation-activated inward current
and a reduction of a Ca2+-dependent outward current (Kiehn
and Harris-Warrick, 1992).
Another effect, similar to the one reported here, is that 5-
HT augments bursting pacemaker activity in the PON
neurones of the suboesophageal ganglion of the snail Achatina
fulica; both the depolarising and the post-burst
hyperpolarising phases are enhanced by 5-HT. The ionic
mechanism responsible for this effect is the enhancement of a
Na+-dependent negative slope resistance region (NSR) in the
steady-state current–voltage relationship and the induction of
a Ca2+-dependent NSR (Funase et al. 1993). This effect may
be of particular relevance to our results because, as proposed
by Onetti et al. (1990), the bursting activity in X organ cells
is related to an NSR mediated by Na+and modulated by
intracellular Ca2+. Given the voltage-dependence of the
inward current elicited by 5-HT in our experiments, in
conjunction with the evoked increase of input resistance, the
reduction of a K+current appears to be a likely mechanism
for the action of 5-HT. However, to establish this, a thorough
search will be necessary, bearing in mind that more than one
ionic mechanism may underlie the depolarising and the burst-
generating responses in these neurones.
This project was partly supported by CONACyT grant no.
0804-N9110.
F. SAENZ AND OTHERS

30895-HT modulates electrical activity in crayfish neurosecretory cells
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F. SAENZ AND OTHERS