Neuronal and neurohormonal control of the heart in the stomatopod crustacean, Squilla oratoria
1 Department of Oral Physiology, Matsumoto Dental University School of
Dentistry, Shiojiri 399-0781, Japan
2 Neurobiology Laboratory, Faculty of Science, Okayama University of
Science, Ridai-cho 1-1, Okayama 700-0005, Japan
* Author for correspondence (e-mail: kuwasawa-kiyoaki{at}das.ous.ac.jp)
Accepted 6 September 2004
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Summary |
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The heartbeat was activated by application of glutamate, serotonin, dopamine, octopamine or acetylcholine, which were applied to the heart by perfusion into an organ bath. Joro-spider toxin (JSTX) blocked myocardial excitatory junctional potentials evoked by the cardiac ganglion. Neuronal cell bodies and processes in the heart were examined using immunocytochemical techniques. All 15 neurons of the cardiac ganglion showed glutamate-like immunoreactivity. Glutamate may be a neurotransmitter of the cardiac ganglion neurons.
JSTX also blocked cardiac acceleration by activation of CA1 and CA2 axons. CA1 and CA2 axons showed glutamate-like immunoreactivity. It is likely that glutamate is a neurotransmitter for the cardio-acceleratory neurons.
The heartbeat was inhibited by application of -amino-butyric acid
(GABA). Cardiac inhibition induced by activation of CI axons was blocked by
picrotoxin. CI axons showed GABA-like immunoreactivity. These results may
support the identification of GABA as an extrinsic inhibitory
neurotransmitter.
Key words: cardioregulatory neuron, cardiac ganglion neuron, neurotransmitter, stomatopod, Squilla oratoria, immunocytochemistry, GABA, glutamate
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Introduction |
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The neurotransmitter candidates for the intrinsic and extrinsic heart
neurons have been identified in a few species within the sub-class
Malacostraca from the results of pharmacological and immunocytochemical
studies. Dealing with the decapods, Yazawa and Kuwasawa
(1994) proposed that
-amino-butyric acid (GABA) and dopamine (DA) are the extrinsic
neurotransmitters of the cardio-inhibitory and cardio-acceleratory nerves,
respectively, in the hermit crab Aniculus aniculus. They also
proposed that acetylcholine (ACh) and DA are the intrinsic neurotransmitters
of the small and large neurons, respectively, in the cardiac ganglion of that
species (Yazawa and Kuwasawa,
1992
). On the other hand, glutamate (Glu) has been proposed for
the neurotransmitter of both of the small and large neurons in the cardiac
ganglion of the lobster Panulirus argus
(Delgado et al., 2000
). In
isopods, it has been proposed that Glu is the intrinsic excitatory
neurotransmitter released by the motoneurons of the cardiac ganglion of
Bathynomus doederleini (Yazawa et
al., 1998
) and Ligia exotica
(Sakurai et al., 1998
).
Furthermore, GABA has been proposed as the extrinsic cardio-inhibitory
neurotransmitter, while ACh has been proposed as the extrinsic
cardio-acceleratory neurotransmitter in B. doederleini
(Tanaka et al., 1992
).
In stomatopods, epinephrine (E), norepinephrine (NE), and ACh have
cardioexcitatory effects on the heart of S. mantis
(Alexandrowicz and Carlisle,
1953; Florey and Rathmayer,
1990
). Additionally, pharmacological experiments indicate that
GABA may be a neurotransmitter of the extrinsic cardio-inhibitor nerves of
S. oratoria (Watanabe et al.,
1968
). However, candidate neurotransmitters of the cardiac
ganglion neurons and cardio-accelerator nerves have not yet been proposed for
the stomatopod (recently reviewed by
Cooke, 2002
).
In this study, we describe the central ganglia and the nerve roots from
which the cardioregulatory axons emerge, and identify their cell bodies in the
ganglia. Furthermore, we identify neurotransmitter candidates for all the
cardioregulatory neurons and neurons of the cardiac ganglion, from the results
of electrophysiological, pharmacological and immunocytochemical studies.
Preliminary reports have appeared elsewhere in abstract form
(Ando and Kuwasawa, 1993;
Ando and Kuwasawa, 1994
;
Ando et al., 1995
).
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Materials and methods |
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Neuroanatomy
Before dissection, the animals were anesthetized by injection of an
isotonic MgCl2 (0.36 mol l1) solution, through a
syringe inserted into the thorax. After anesthetization, they were pinned to
the Silpot (Dow Corning, Kanagawa, Japan)-lined bottom of a chamber filled
with cold seawater, kept in a refrigerator. The carapace in the cephalo-thorax
was carefully peeled off to expose the terminal region of the cardioregulatory
nerves on the heart inside the pericardium. The peripheral cardioregulatory
nerves were stained by means of a vital staining technique, in Methylene Blue
filtered seawater (SW). The stained preparations were fixed in a 4% ammonium
molybdateSW solution overnight and washed in running tapwater for
several hours (Alexandrowicz,
1932). Then, the fixed preparations were stained in a Methylene
Bluetapwater solution (Kihara and
Kuwasawa, 1984
), and dissection was continued to trace the
cardioregulatory nerves up to the central ganglia.
According to their functions, as elucidated by physiological experiments
(see Electrophysiology section below), we refer to the nerves named ,
ß and
by Alexandrowicz
(1934
) as, respectively, the
cardio-inhibitor (CI) nerve, and the 1st and 2nd cardio-accelerator (CA1 and
CA2) nerves. Those are terms that have been commonly used in a variety of
crustacean species: in the decapods
(Maynard, 1953
;
Yazawa and Kuwasawa, 1984
) and
in the isopods (Kihara and Kuwasawa,
1984
; Sakurai and Yamagishi,
1998
).
Electrophysiology
After the heart and cardioregulatory nerves were exposed, the nerves were
cut in the pericardial cavity. The isolated heart was pinned dorsal side up to
a Silpot-lined experimental bath (3.5 ml). The distal cut-stumps of the
nerves, extending to the heart, were introduced one by one into a glass
capillary suction electrode containing a AgAgCl wire, used to apply
stimulus pulses to the nerve. Heartbeat of the whole heart was monitored using
either a mechanogram, with a strain gauge transducer (TB-611T Nihon Kohden,
Tokyo, Japan) or an electrocardiogram (ECG), which was recorded from the outer
surface of the heart with a glass capillary suction electrode.
Myocardial intracellular potentials were recorded from the isolated heart,
which was opened by longitudinal incision of the ventral wall of the heart.
After incision, the heart was spread out and then pinned dorsal side up to a
Silpot-lined experimental bath (1.5 ml). Intracellular potentials were
recorded with a conventional glass capillary microelectrode filled with 3 mol
l1 KCl (tip resistance, 2035 M). Excitatory
junctional potentials (EJPs) in myocardial cells were evoked by electrical
stimuli applied to the cardiac ganglion at the cut stump of the trunk of the
ganglion. Preparations were routinely perfused with Squilla saline
(in mmol l1: NaCl, 450; KCl, 15; CaCl2, 10;
MgCl2, 20; Hepes, 5; pH 7.8) by gravity-feeding through a cannula
at the rate of 34 ml min1. Saline was suctioned for
removal. The saline was modified from the saline of Watanabe et al.
(1967
). The perfusate was
maintained at 1720°C by a cooling device (EC-201, Scinics, Tokyo,
Japan).
Application of agents
The following agents were used: acetylcholine chloride (ACh), atropine
sulfate monohydrate, dopamine hydrochloride (DA),
-amino-N-butyric acid (GABA), sodium L-glutamate
monohydrate, histamine dihydrochloride, joro spider toxin 3 (JSTX),
L-noradrenaline bitartrate, picrotoxin (PTX),
serotonincreatinine sulfate, serotonin (5-HT),
D-tubocurarine hydrochloride (Wako Pure Chemical Industries, Osaka,
Japan), adrenaline bitartrate, chlorpromazine hydrochloride, octopamine
hydrochloride (Sigma Chemicals, St Louis, MO, USA), phentolamine mesylate,
Regitin (Ciba-Geigy, Basle, Switzerland).
The salines containing the agents were applied to preparations when the cock of a three-way valve was turned in the perfusion line, except for JSTX, when saline containing the agent was added to the experimental bath by a pipette.
Resin sections for photomicroscopic observation
Pieces of nerve, isolated from the CI, CA1 and CA2, were pre-fixed in a 3%
(w/v) glutaraldehyde solution and post-fixed in a 0.1% (w/v) osmium solution.
After washing in the buffer solution and dehydrating in a graded ethanol
series, they were embedded in Quetol-812 (Nissin EM, Tokyo, Japan). Sections
were cut at 1 µm and stained with the mixed 1% (w/v) Methylene Blue and
0.1% (w/v) Azur II solutions. They were observed under a microscope and
photographed (BX-51, Olympus, Tokyo, Japan).
Back-filling of the cardioregulatory neurons with Co2+ and Ni2+ ions
In order to locate the cell bodies of the CI, CA1 and CA2 neurons in the
central nervous system (CNS), a proximal cut-stump of the cardioregulatory
nerve was introduced into a glass capillary filled with a mixed solution of 1
mol l1 CoCl2 and 1 mol l1
NiCl2. The preparation was incubated for 48 days at 4°C,
and then rinsed with SW. Some drops of a saturated Rubeanic Acidethanol
solution were added to precipitate a sulfate compound
(Quicke and Brace, 1979). The
stained preparation was fixed with 4% (w/v) formaldehyde, dehydrated with a
graded ethanol series, and cleared with methyl salicylate. The preparation was
observed under a microscope and photographed (BX-51, Olympus).
Immunocytochemistry
We examined immunoreactivity of neural processes in the heart and extrinsic
nerves against four kinds of antibodies: anti-GABA, anti-glutamate,
anti-serotonin and anti-histamine. In immunocytochemistry using anti-GABA and
anti-glutamate, preparations were processed as described for the isopod by
F.-Tsukamoto and Kuwasawa
(2003). For controls,
preparations were processed in the same manner but without treatment with the
primary antibodies. For each antibody, no immunoreactivity was detected in the
control preparations. The preparations were observed under a microscope and
photographed (BX-51, Olympus).
1. Treatment with anti-GABA antibodies
To locate GABA-like immunoreactive neural processes, specimens were fixed
with a solution of 4% (w/v) paraformaldehyde0.1% glutaraldehyde in 0.1
mol l1 phosphate buffer (pH 7.2) containing 15% (w/v)
sucrose for 23 h at 4°C. After fixation they were rinsed in a 0.1
mol l1 phosphate buffer solution containing 15% (w/v)
sucrose over 1 day at 4°C. The specimens were embedded in paraffin and
sectioned serially at 10 µm. The sections were dried on the slides,
deparaffinized, rehydrated and immersed in distilled water (DW). In order to
eliminate intrinsic peroxidase activity in the sectioned tissues, the slides
were treated with 0.3% (w/v) H2O2 in DW for 30 min at
room temperature. They were incubated with a 0.1 mol l1
phosphate buffer containing 0.1% (w/v) Triton X-100 (pH 7.2) (0.1% PBT) for 15
min, then with the primary antibody (rabbit anti-GABA; cat. no. 20094,
Incstar, Stillwater, MN, USA), diluted 1:2000 in a 0.1% PBT for 24 h and
rinsed in a 0.1% PBT for 1 h. The specimens were treated with the secondary
antibody (goat anti-rabbit IgG; Sigma), diluted 1:200 in a 0.1% PBT for
1.52 h at room temperature and rinsed in a 0.1% PBT for 1 h. They were
treated with the third antibody [rabbit peroxidaseantiperoxidase (PAP)
complex; Sigma], diluted 1: 200 in a 0.1% PBT for 2 h and rinsed in a 0.1% PBT
for 1 h. 0.03% (w/v) 3,3'-diaminobenzidine-tetrahydrochloride (DAB) in
0.05 mol l1 Tris buffer (pH 7.6) containing 0.006% (w/v)
H2O2 was applied for about 10 min at room temperature in
dark. The peroxidase reaction was stopped by transferring the slides to DW.
The sections were counterstained with Methyl Green, dehydrated in a graded
ethanol series, cleared in xylene and mounted in Bioleit (Oken Shoji, Tokyo,
Japan).
For whole-mount preparations, fixed specimens were immersed in 0.2% (w/v) collagenase in SW, rinsed in 0.3% PBT, and immersed in 1% PBT overnight. They were incubated with the primary antibody diluted 1:1000 in a 0.3% PBT for 48 h, with the secondary antibody for 24 h andfurther with PAP complex for 24 h. After each incubation they were rinsed in 0.3% PBT for 1 h. The specimens were stained with DAB solutions, dehydrated with a graded ethanol series, cleared in methyl salicylate and then in xylene and mounted on glass slides in Bioleit.
2. Treatment with anti-glutamate antibodies
Specimens were treated as described for GABA immunocytochemistry before
treatment with the antibodies. Specimens were treated with the primary
antibody (mouse anti-glutamate; cat. no. 22523, Incstar or cat. no. G9282,
Sigma) diluted 1:1000 (Incstar) or 1:20000 (Sigma) in a 0.1% PBT. The
specimens were treated with the secondary antibody (goat anti-mouse IgG;
Sigma), diluted 1:200 in a 0.1% PBT and with the third antibody (mouse PAP
complex; Sigma), diluted 1:200 in a 0.1% PBT. Then the specimens were
processed and examined as described above.
3. Treatment with anti-serotonin antibodies
To locate serotonin-like immunoreactive neural cells and processes,
specimens were fixed with a solution of 4% (w/v) paraformaldehyde in 0.1 mol
l1 phosphate buffer containing 15% (w/v) sucrose (pH 6.5)
for 30 min at room temperature, followed by a 4% (w/v) paraformaldehyde in 0.1
mol l1 sodium tetraborate buffer containing 15% (w/v)
sucrose (pH 9.0) for 2.53 h at 4°C. The specimens were treated with
the primary antibody (rabbit anti-serotonin; cat. no. 2008, Incstar), diluted
1:2000 in a 0.1% PBT for 24 h and rinsed in a 0.1% PBT. Then, the specimens
were processed and examined as described above.
4. Treatment with anti-histamine antibodies
To locate histamine-like immunoreactive neural cells and processes,
specimens were fixed with a solution of 4% (w/v) ethyl-dimethylcarbodiimide in
0.1 mol l1 phosphate buffer (pH 7.3) for 60 min at 4°C,
followed by a 4% (w/v) paraformaldehyde in 0.1 mol l1
phosphate buffer containing 15% sucrose (pH 7.2) overnight at 4°C. The
specimens were treated with the primary antibody (rabbit anti-histamine; cat.
no. 22939, Incstar), diluted 1:1000 in a 0.1% PBT for 24 h and rinsed in a
0.1% PBT. Then, the specimens were processed and examined as described
above.
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Results |
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The cardiac ganglion (CG), which lies on the outer surface of the heart,
consists of 1316 ganglion cells in stomatopods
(Alexandrowicz, 1934;
Irisawa and Irisawa, 1957
;
Brown, 1964a
;
Watanabe et al., 1967
;
Florey and Rathmayer, 1990
).
We used Methylene Blue staining techniques to determine the number of neurons
in the CG of the present material. We counted 15 cell bodies in the area of
the CG between the 2nd and 14th lateral arteries for more than 50
preparations, which were well stained with the dye. Three of the 15 cell
bodies formed a cluster at the base of the 2nd arteries, and the other 12 cell
bodies were individually located at each base of the lateral arteries. The
number of cell bodies coincides with that of pairs of the lateral arteries.
Fig. 2 shows the cardiac
ganglion cells in the CG in the anterior part of the heart. Three anterior
cell bodies of the ganglion lie forming a cluster at the 2nd lateral
artery.
|
Three pairs of cardioregulatory nerves join the CG at the level of the origins of the 2nd, 3rd and 4th lateral arteries in the cephalo-thorax, for CI, CA1 and CA2 nerves, respectively (Fig. 2AC).
Anatomically, we traced the nerves all the way to the central nervous system from which they arose, and located their origins in the SEG. We decided that the CI, CA1 and CA2 nerves emerged from, respectively, the 10th the 16th and 19th nerve roots (10th NR, 16th NR and 19th NR) of the SEG (Fig. 3A). In order to verify these findings, we observed the effects of stimulation of their roots on heart beat.
|
When the 10th NR was electrically stimulated at a distal cut-stump of the nerve, heart rate decreased (Fig. 3B). The effect was intensified with increased stimulus frequency (Fig. 3C). By contrast, when either the 16th or 19th NR was electrically stimulated at a distal cut-stump of each nerve, heart rate and amplitude of action potentials increased (Fig. 3B). The acceleratory effect was intensified with increased stimulus frequency (Fig. 3C). Fig. 3C shows the effects of stimulation of the nerve roots on heart rate. Heart rate in controls was 33.2±8.9 for the 10th NR, 30.0±7.7 for the 16th NR and 26.6±6.1 for the 19th NR (mean ± S.D.; N=7 for the 10th NR, N=9 for the 16th NR, N=10 for the 19th NR). The increased amplitude of action potential, during the stimulations, may indicate an increase of contraction force of the heart. Thus, the anatomical results described above were confirmed by these results.
In order to examine the axonal composition of the cardioregulatory nerves, extracellular impulses were recorded from the nerves. Only one kind of orthodromic (Fig. 4A orth) or antidromic unit impulse (Fig. 4A ant) was recorded for CI, CA1 and CA2. Additional impulse units never appeared, even when stimulated at higher intensity. These results were confirmed in seven preparations. Resin cross sections obtained from the CI, CA1 and CA2 nerves were observed under a microscope (Fig. 4B), and showed that each of the CI, CA1 and CA2 nerves contains one axon (68 µm in diameter) wrapped in the heavily stained perineurium (about 1 µm in thickness), which is surrounded by epineurium (1020 µm in thickness), thus forming the nerves.
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The axons were back-filled with Ni2+ and Co2+ ions at their proximal cut-stumps. The cell body (about 30 µm in diameter) of the CI axon was found stained at a site near the midline on the side contralateral to the nerve root through which the CI axon emerged in the 1st SEG segment (Fig. 5). The cell body (about 20 µm in diameter) of the CA1 axon was found stained at a site near the midline on the side ipsilateral to the nerve root through which the CA1 axon emerged in the 3rd SEG segment. The cell body (20 µm in diameter) of the CA2 axon was found stained at a site near the midline on the side ipsilateral to the nerve root through which the CA2 axon emerged in the 4th SEG segment.
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Effects of putative neurotransmitters and neurohormones
In order to examine the effects on the heart of putative neurotransmitter
substances known in crustaceans (Cooke and
Sullivan, 1982), mechanograms of heartbeat were recorded from
isolated hearts treated with the substances.
Effects of GABA and histamine on the heart are shown in
Fig. 6. GABA
(>106 mol l1) decreased heart rate and
contraction force depending on the dose
(Fig. 6A). Similar results were
reported by Watanabe et al.
(1968) for GABA application to
the heart of S. oratoria. Histamine (HA) (>106
mol l1) also exerted inhibitory effects on the heart rate
and contraction force (Fig.
6B). Picrotoxin, a GABAergic antagonist, at 104
mol l1, completely blocked the cardiac inhibition induced by
stimulation of the CI (Fig.
6C).
|
5-HT, DA, OA, ACh and Glu were applied to the isolated heart, and doseresponse curves are shown in Fig. 7. 5-HT initially transiently decreased both heart rate and contraction force, and then increased both heart rate and contraction force (Fig. 7A). The effects of 5-HT on heart rate and contraction force increased in a dose-dependent manner. Threshold concentrations for 5-HT in both the first and second phases were between 109 and 108 mol l1. DA (Fig. 7B), OA (Fig. 7C), ACh (Fig. 7D) and Glu (Fig. 7E) increased heart rate and force. Threshold concentrations for both DA and OA were approximately 108 mol l1, and those for both ACh and Glu around 106 mol l1. Epinephrine (E) and norepinephrine (NE) showed weak excitatory effects compared to the effects induced by stimuli of 5-HT, DA and OA (data not shown).
|
Antagonists for various putative neurotransmitter substances were tested in order to predict the neurotransmitters of the extrinsic CA axons and cardiac ganglion neurons. The catecholaminergic blockers, chlorpromazine 105 mol l1 and phentolamine 105 mol l1, did not antagonize the cardio-acceleratory effects of CA nerves and force of the myocardium beats induced by the cardiac ganglion. The cholinergic blockers, atropine 104 mol l1 and d-tubocurarine 104 mol l1, were also ineffective on the acceleratory effects produced by stimulation of the CA nerves (data not shown). Three preparations were used for each of the agents.
JSTX (105 mol l1), known as a glutamate
antagonist (Kawai et al.,
1982; Chiba and Tazaki,
1992
; Sakurai et al.,
1998
; F.-Tsukamoto and
Kuwasawa, 2003
), blocked cardio-acceleratory effects of
stimulation of the CA1 and CA2 axon (Fig.
8). Acceleratory effects of stimulation of CA1 or CA2 decreased to
38.5±12.7% of control value for CA1 and 24.5±17.5% for CA2
(means ± S.D., N=3).
|
5x106 mol l1 JSTX blocked EJPs in cardiac muscle, evoked by stimulation of the cardiac ganglion trunk (Fig. 9). The amplitude of EJPs decreased to 29.1±14.6% of control values (mean ± S.D., N=9). EJPs partially recovered at 3 h after washout of JSTX.
|
Immunocytochemistry
No immunoreactivity was detected in a negative control preparation of
wholemount preparations or paraffin sections incubated without the primary
anti-GABA and anti-glutamate antibodies.
GABA-like immunoreactivity
The CI axon showed GABA-like immunoreactivity to anti-GABA antibodies
(Fig. 10A). An axon showing
GABA-like immunoreactivity runs in the CG
(Fig. 10B). Although many
histamine-like immunoreactive neuronal processes, as well as GABA-like
immunoreactive ones, were observed in the SEG, the CI nerves showed no
histamine-like immunoreactivity to anti-histamine antibodies (data not
shown).
|
Glutamate-like immunoreactivity
Fig. 11 shows
glutamate-like immunoreactive neuronal processes, evoked in response to
anti-glutamate antibodies. Both the CA1 and CA2 axons and the CG showed
glutamate-like immunoreactivity (Fig.
11A). A glutamate-like immunoreactive cell body was observed in
Fig. 10B. Glutamate-like
immunoreactivity was shown in a major motor branch extending from the CG
(Fig. 11C). Glutamate-like
imunoreactivity was observed in all cells in the CG (data not shown). The
immunoreactivity was not observed in the cardioregulatory nerves and in the
heart (data not shown), but it was observed that serotonin-like immunoreactive
neuronal processes ran out of the SEG and extended on the ventral skeletal
muscles.
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Discussion |
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A common neurotransmitter of CI neurons
In the present material, GABA and histamine produced inhibitory effects on
the heartbeat (Fig. 6).
Picrotoxin blocked neurally induced cardiac inhibition induced by impulses of
the CI nerve (Fig. 6C). Though
picrotoxin is not a GABA-specific antagonist but a chloride channel blocker,
the CI nerves showed immunoreactivity to anti-GABA antibodies
(Fig. 10) not to
anti-histamine antibodies (data not shown). These results show that GABA may
be a neurotransmitter for the CI neurons in S. oratoria.
Neurotransmitters for cardioregulatory neurons and cardiac ganglion neurons
have been proposed for some crustaceans. It has been proposed that GABA may be
a neurotransmitter of the CI neuron in the isopod, B. doederleini
(Tanaka et al., 1992;
F.-Tsukamoto and Kuwasawa,
2003
), and in the decapods, A. aniculus
(Yazawa and Kuwasawa, 1994
)
and Panulirus argus (Delgado et
al., 2000
). It is concluded that GABA may be commonly a
cardio-inhibitory neurotransmitter in all those crustacean taxa.
Neurotransmitters for CA neurons
The heart of S. mantis is activated by application of E, NE
(Alexandrowicz and Carlisle,
1953) and ACh (Florey and
Rathmayer, 1990
). While NA and DA are reported to produce cardio
inhibitory activity on the heart of an isopod, Bathynomus
doederleini, 5-TH, OA and Glu are reported to produce cardio-acceleratory
activity (Tanaka et al., 1992
;
Yazawa et al., 1998
;
F.-Tsukamoto and Kuwasawa,
2003
). ACh has been proposed to be a neurotransmitter for
cardio-accelerator nerves in B. doederleini
(Tanaka et al., 1992
), and DA
to be a neurotransmitter for cardio-accelerator nerves in the decapod species,
A. aniculus (Yazawa and Kuwasawa,
1994
) and the crab (Fort and
Miller, 2001
; reviewed by
Cooke, 2002
). In the present
study the heart was activated by 5-HT, DA, OA and Glu, as well as E, NE and
ACh (Fig. 7). No serotonin-like
immunoreactivity was observed in CA axons and cardiac ganglion neurons (data
not shown), although 5-HT activated heartbeat
(Fig. 7A). Cardiac acceleration
induced by stimulation of the CA nerves was not antagonized by cholinergic
blockers, atropine and d-tubocurarine, or by catecholaminergic blockers,
chlorpromazine and phentolamine (data not shown). Thus, ACh, 5-HT and
catecholamines may be excluded from the list of transmitter candidates for the
CA neurons.
Glu increased not only contraction force but also heart rate
(Fig. 7E). JSTX blocked cardiac
acceleration induced by the CA1 and CA2 nerves
(Fig. 8). Glutamate-like
immunoreactivity was observed in both the CA1 and CA2 axons
(Fig. 11). JSTX is known to
antagonize glutamatergic actions on the skeletal muscle of the lobster
Palinurus japonicus (Kawai et
al., 1982), on the stomach muscle of S. oratoria
(Chiba and Tazaki, 1992
), the
heart of L. exotica (Sakurai et
al., 1998
) and on the arterial valve in B. doederleini
(F.-Tsukamoto and Kuwasawa,
2003
). These results may indicate that, in S. oratoria,
both the CA1 and CA2 neurons are glutamatergic. It has been suggested that DA
may be a neurotransmitter of the CA nerves in the hermit crab, A.
aniculus (Yazawa and Kuwasawa,
1994
) and the crab (Fort and
Miller, 2001
; reviewed by
Cooke, 2002
), while Glu had
little effect on the cardiac ganglion (Yazawa and Kuwasawa,
1992
,
1994
). In the isopod, B
doederleini, ACh may be a neurotransmitter of the CA neurons
(Tanaka et al., 1992
).
Thus, it seems that the transmitters of cardio-accelerator nerves may be varied among the crustacean taxa.
Neurotransmitters for cardiac ganglion neurons
EJPs in the myocardium induced by motor impulses of the cardiac ganglion
were blocked by JSTX (Fig. 9).
Glutamate-like immunoreactivity was observed in all of the 15 cardiac ganglion
neurons (Fig. 11). On the
other hand, heartbeat triggered by the cardiac ganglion neurons was not
antagonized by the cholinergic blockers and catecholaminergic blockers (data
not shown). These results may indicate that, in S. oratoria, the
cardiac ganglion neurons are glutamatergic. Pharmacological experiments
suggested that ACh and DA may be neurotransmitters of small (pacemaker) and
large (motor) neurons of the cardiac ganglion, respectively, although Glu
exerted little effect on the cardiac ganglion and cardiac muscle in the hermit
crab A. aniculus (Yazawa and Kuwasawa,
1992,
1994
). On the other hand,
glutamate-like immunoreactivity has been observed in the small and large
neurons of the cardiac ganglion in the lobster P. argus
(Delgado et al., 2000
). A
neurotransmitter of the cardiac ganglion neurons appears to differ in the
anomuran (A. aniculus) and palinuran (P. argus) decapod
species examined. Since Glu increased contraction force but not heart rate in
the isopod, B. doederleini, Glu was proposed to be a neurotransmitter
of the cardiac ganglion neurons (Yazawa et
al., 1998
). Glutamate-like immunoreactivity has been observed in
the motor neurons of the cardiac ganglion in the isopod, L. exotica
(Sakurai et al., 1998
).
Glutamate may be a neurotransmitter of the cardiac ganglion neurons of S.
oratoria as of the case in a decapod, P. argus, and in isopods,
B. doederleini and L. exotica.
Neurohormones for control of the heart
Cardiac excitation
Alexandrowicz (1952)
observed that, in S. mantis, unpaired nerves ran in the connectives
between the subesophageal, thoracic and abdominal ganglia, and extended toward
the ventral body muscles. He called this nervous system `the system of the
median connectives' and suggested that it had a neurosecretory function. He
(Alexandrowicz, 1953
) also
observed neuropile-like networks on the inner surface of the pericardium in
S. mantis and referred to it as the pericardial organ. The
pericardial organ contained neuronal cell bodies and endings of nerves
extending from the CNS. Pericardial organ extracts increased heart rate and
contraction force in S. mantis
(Alexandrowicz and Carlisle,
1953
; Brown,
1964b
). Substances contained in the pericardial organ and in the
system of the median connectives have not yet been examined in any stomatopod
species. The decapod pericardial organ contains 5-HT, DA, OA and several
neuropeptides (Cooke and Sullivan,
1982
; Cooke, 1988
;
Christie et al., 1995
;
reviewed by Cooke, 2002
). In
the present species, 5-HT, DA and OA increased heart rate and contraction
force (Fig. 7AC). Since
we observed serotonin-like immunoreactivity in the system of the median
connectives as well as the CNS (data not shown), 5-HT may be one of the
cardioexcitatory neurohormones. ACh may be a neurotransmitter for small
neurons of the pacemaker in the cardiac ganglion of the hermit crabs
(Yazawa and Kuwasawa, 1992
)
and for the CA axons of the isopod (Tanaka
et al., 1992
). It is suggested that in the stomatopod a
transmitter of CA neurons is Glu but not ACh, although ACh increased both
heart rate and contraction force (Fig.
7D). It is of interest for further study to determine whether ACh
is contained as a neurohormonal substance in the pericardial organ of S.
oratoria.
Cardiac inhibition
Cardio-inhibitory substances other than GABA have not previously been
reported in any stomatopod species. This study showed that HA induced
inhibitory effects on the heart (Fig.
6B). However, no histamine-like immunoreactivity was observed in
the CI neurons. Since many histamine-like immunoreactive neurons were located
throughout the CNS in the present material (data not shown), it cannot be
excluded that HA is possibly a cardio-inhibitory neurohormone, as proposed for
Homarus americanus
(Hashemzadeh-Gargari and Freschi,
1992).
List of abbreviations
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Acknowledgments |
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References |
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