Central effects of various ligands on drinking behavior in eels acclimated to seawater
Laboratory of Integrative Physiology, Faculty of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima 739-8521, Japan
* Author for correspondence (e-mail: mando{at}hiroshima-u.ac.jp)
Accepted 18 November 2002
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Summary |
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Key words: seawater eel, Anguilla japonica, drinking behavior, intracranial administration, intravenous administration, angiotensin II, atrial natriuretic peptide, circumventricular organ
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Introduction |
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Among fish, the drinking behavior of the euryhaline eel has been
extensively studied using an esophageal cannulation technique developed by
Hirano (1974). Furthermore,
various regulators that affect water intake are already known in the eel
(Takei et al., 1979
;
Hirano and Hasegawa, 1984
;
Ando et al., 2000a
;
Takei, 2000
). In particular,
endogeneous angiotensin II (eANG II) and atrial natriuretic peptide (eANP) are
considered a potent dipsogen and antidipsogen, respectively
(Takei, 2000
). Application of
these regulators has, however, been restricted to the systemic circulation,
thus limiting our understanding of their effects on central locations such as
brain. It is well known in mammals that circulating ANG II, a most potent
dipsogen, acts on the subfornical organ (SFO), a circumventricular organ (CVO)
that lacks the bloodbrain barrier (BBB)
(Kobayashi and Takei, 1996
;
Fitzsimons, 1998
;
Takei, 2000
), and it is likely
that these systemic regulators may also act on the CVOs in the eel brain. If
these regulators do act on the CVOs, the effects of central administration of
these regulators must be identical to those observed on systemic
application.
The aim of the present study was to identify the ligands affecting drinking
behavior in the brain of the eel. Various ligands were injected intracranially
via a cerebral cannula, and the effects compared with previous
results obtained after intravenous injection
(Ando et al., 2000a).
Similarities and differences between these two applications are discussed in
relation to brain morphology.
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Materials and methods |
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To insert a cerebral cannula, the skin of the occipital region was incised and the muscle removed. After exposing the skull, the surface was flattened with a grinder (No. 28511, Kiso Power Tool, Osaka), then drilled (approx. 1 mm) at the front of the supraoccipital bone (Fig. 1A). A needle (approx. 0.5 mm) was inserted through the dura into the fourth ventricle, and the cerebral cannula (No. 9571, BAS, Tokyo) filled with 0.9% NaCl solution was inserted into the pore, 1.4 mm below the skull (Fig. 1B,C). The cannula was fixed to the skull with dental cement (1-1PKG, GC Corporation, Tokyo).
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After the operation, the incision was closed using silk suture and all cannulae were sutured to the body. Eels were then transferred to a plastic trough of the same size. Well aerated seawater was circulated continuously through the trough at room temperature (20-23°C). Experiments were started on the following day, when the drinking rate was relatively constant. Only eels responsive to intracranial eANG II (5x10-10 mol l-1, 5 µl) but not to 0.9% NaCl (10 µl) were used for the following experiments. To avoid habituation to a ligand, each ligand was injected at intervals of more than 5h, and different ligands were used on the same day. The sequence of ligand application was random. If the response obtained was unexpected, responsiveness to eANG II and 0.9% NaCl as controls was checked again. Under such experimental conditions, eels survived for more than 1 week. After the experiments, 1% Methylene Blue (5 µl) was injected through the cerebral cannula, and the fourth ventricle and a part of the third ventricle were stained with dye.
Reagents were all dissolved in 0.9% NaCl solution (vehicle) and injected intracranially with a syringe pump (MF-9090, Bioanalytical Systems, IN, USA) at a rate of 1.25 µl min-1 for 4 min (total 5 µl). Since the dead volume of the cerebral cannula was 5 µl, a further 5 µl of vehicle was injected into the cannula, making the total volume of injectate 10 µl.
Acetylcholine chloride (ACh), carbamylcholine chloride (CCh), histamine
dihydrochloride (HA), 5-hydroxytriptamine creatine sulphate (5-HT),
phenylephrine HCl, sheep prolactin (PRL) and propranolol HCl were purchased
from Sigma. Arginine vasotocin (AVT), eel angiotensin II
([Asn1]eANG II), cholecystokinin (26-33) (CCK-8), human ghrelin,
substance P (SP), saralasin and vasoactive intestinal polypeptide (VIP) were
purchased from Peptide Institute, Osaka, Japan; dopamine HCl (DA),
-amino butyric acid (GABA), noradrenaline HCl (NA) and heparin sodium
from Katayama Chemical, Osaka, Japan; eel atrial natriuretic peptide (eANP)
from Peninsula Laboratories, CA, USA; PD 123319 ditrifluoroacetate and CGP
42112 from Research Biochemicals International, Natick, USA. Losartan
potassium (Banyu, Tokyo, Japan), Exp 3174 (Dupont, Wilmington, USA) and CV
11974 (Takeda Chemical Industries, Tokyo, Japan) were kind gifts from these
companies.
For intravenous injection, a venous cannula (SP-10, Natume, Tokyo, Japan)
filled with heparinized saline (100i.u. ml-1) was inserted into the
posterior cardinal vein as described previously
(Ando et al., 2000a).
The effects of ligands were evaluated by comparing the drinking rates for a 20 min period before and after application of the ligand. Statistical analyses of the results were performed using a paired t-test. Results are given as means ± S.E.M. and were considered significant at P<0.05.
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Results |
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The drinking rate was also enhanced by acetylcholine (ACh) in a
dose-dependent manner (Fig. 2),
but the effect was smaller than that of eANG II, with a threshold at
5x10-12 mol and a 1.4-fold increase at
5x10-10 mol. An acetylcholine agonist, carbachol (CCh),
enhanced the water intake to 266±71% (N=6) at
5x10-10 mol (not shown). Isoproterenol, a ß-adrenoceptor
agonist that accelerates drinking in mammals upon systemic administration
(Fitzsimons, 1998), also
enhanced water intake in a dose-dependent manner, with a threshold at
5x10-11 mol, and maximal effect at 5x10-10
mol (Fig. 2). Substance P (SP)
increased the drinking rate to 197±51% (N=5) at
5x10-10 mol (data not shown). In some preparations,
cholecystokinin (CCK-8, 5x10-13-5x10-11 mol)
and dopamine (DA, 5x10-12-5x10-8 mol)
increased the drinking rate, but the reproducibility and dose-dependency were
low (not shown).
Substances inhibiting drinking rate
When eel atrial natriuretic peptide (eANP, 5x10-11 mol)
was injected intracranially, basal drinking rate was decreased after a few
minutes and then returned to the initial level after 1 h. The inhibitory
effect of eANP was dose-dependent, with a threshold at
5x10-13 mol. Fig.
3 shows doseresponse curves for various inhibitors,
including ANP, ghrelin, serotonin (5-HT), phenylephrine (an
-adrenoceptor agonist), prolactin (PRL), and
-amino butyric acid
(GABA, a general inhibitory neurotransmitter in the brain). Ghrelin, a
28-amino-acid peptide isolated originally from rat stomach and exhibiting an
orexigenic effect (Kojima et al.,
1999
), was the most potent among these inhibitors examined. Water
intake was also inhibited by arginine vasotocin (AVT, 50±7%,
N=3, at 5x10-12 mol), vasoactive intestinal peptide
(VIP, 80±7%, N=6, at 5x10-11 mol),
noradrenaline (NA, 51±11%, N=5, at 5x10-9
mol) and somatostatin (SS-14, 66±13%, N=5, at
5x10-12 mol) (not shown). Although ß-endorphin
(5x10-11 mol), an opioid peptide that affects drinking in
mammals (Fitzsimons, 1998
),
reduced the drinking rate in some preparations, the reproducibility was low
(not shown).
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When glutamate (a general excitatory neurotransmitter in the brain; 5x10-11-5x10-7 mol), histamine (5x10-11-5x10-8 mol), glycine (an inhibitory neurotransmitter; 5x10-9-5x10-8 mol) and eel intestinal pentapeptide (EIPP; 5x10-13-5x10-12 mol) were applied intracranially, the water intake was not altered (not shown).
Comparison between intracranial and intravenous administrations of
ANG II
Fig. 4 shows the time course
after the intracranial and intravenous administration of eANG II, as well as
that after intracranial administration of eANP. The dose of eANG II was chosen
to induce a comparable effect, i.e. 5x10-11 mol for
intracranial and 10-7 mol for intravenous application. Both
applications of eANG II enhanced the water intake transiently, with similar
peaks after 5 min. However, the effect of intracranial ANG II lasted longer
than that of the intravenous ANG II. eANP (5x10-11 mol),
however, decreased water intake, and the inhibitory effect was maintained for
more than 40 min. The drinking rate 15 min after intravenous administration of
eANG II was almost identical to that after eANP.
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Discussion |
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With the exception of 5-HT, PRL, HA and EIPP, all ligands examined in the
present study had qualitatively similar effects, irrespective of the site of
administration. Recently, intravenous ghrelin (10-10 mol) was
demonstrated to inhibit drinking robustly (Y. Watanabe and M. Ando,
unpublished observation). The most basic explanation for this similarity is
that central and peripheral ligands act on identical sites concerned with
regulating drinking behavior, and these sites are most probably situated in a
central location within the brain. However, as the brain is for the most part
isolated from the systemic circulation by the bloodbrain barrier (BBB),
ligands administrated intravenously would only have access to the specific
control sites where there is no BBB. Such specific regions are called
circumventricular organs (CVOs) (see
Fitzsimons, 1998;
Takei, 2000
). Recently, we
demonstrated that the magnocellular preoptic nucleus (PM), the anterior
tuberal nucleus (ATN) and the area postrema (AP) are analogous CVOs in the
brain of the eel (T. Mukuda and M. Ando, unpublished observation). The PM and
the ATN are situated in the hypothalamus and the AP is in the medulla
oblongata. Since the eel brain possesses CVOs, where neurons can respond
similarly both to the cerebrospinal fluid and to the systemic circulation,
intravenous ligands can act on the CVOs and control drinking behavior
directly.
The intracranial effects of 5-HT, PRL, HA and EIPP were different from the
intravenous effects (Table 1).
Intravenous 5-HT, PRL, HA and EIPP may not directly act on the CVOs. Indeed,
the intravenous effects of 5-HT and HA were completely blocked by intravenous
pretreatment with captopril, an inhibitor of angiotensin converting enzyme,
suggesting that intravenous 5-HT and HA stimulate ANG II synthesis and ANG II
acts directly on the CVOs to enhance the water intake
(Ando et al., 2000a). PRL and
EIPP might act indirectly on the CVOs via other mediators that are
synthesized peripherally but not yet identified.
Although eANG II showed a qualitatively similar effect with both
intracranial and intravenous administration, the time courses differed for the
two treatments (Fig. 4). The
stimulatory effect lasted for 10 min after intracranial administration,
whereas it was reduced to the original level after 10 min following
intravenous application and significantly inhibited after 30 min.
Interestingly, the low level was almost the same as that seen after
intracranial eANP administration. These results could be explained by a dual
effect of intravenous eANG II. Circulating eANG II may act on the CVOs
directly to stimulate drinking, and simultaneously on the atrium to stimulate
ANP (an antidipsogen) secretion. Indeed, a 1.6-fold increase in plasma ANP
levels was induced following intra-arterial administration of
10-10mol ANG II in the Japanese eel
(Tsuchida and Takei, 1999).
Such enhancement of plasma ANP may result in an inhibition of drinking, since
the antidipsogenic effect of systemic eANP was 100 times more potent than the
dipsogenic effect of eANG II (Takei,
2000
).
Although a higher dose of eANG II is required to give the same peak effect when injected intravenously than intracranially, this phenomenon can be explained by difference in the volumes of blood and cerebrospinal fluid (CSF) that flow from the third ventricle to the fourth ventricle. In addition, intravenous ANG II may be inactivated by peptidases before arriving at the CVOs.
Angiotensin receptor in the eel seems to be different from mammalian types,
since mammalian AT1- and AT2-receptor antagonists did
not inhibit eANG II action. Similar lack of effect has been observed in
non-mammalian species (Ji et al.,
1993; Murphy et al.,
1993
; Tierney et al.,
1997
).
Comparing the present results with those obtained in mammals, the effects
of AVT (AVP in the case of mammals) and VIP have opposite effects, whereas
intracranial ANG II, ACh, ANP and GABA show similar effects in both vertebrate
groups (Table 1). In mammals,
it is well known that blood hyperosmolarity stimulates AVP secretion (Brimble
et al., 1977) and drinking rate (Gilman,
1937; Olsson,
1972
; Thrasher et al.,
1980a
,b
;
Kadekaro et al., 1995
). On the
other hand, in the seawater eel, blood hyperosmolarity decreases water intake
(Ando et al., 2000a
;
Takei, 2000
). Therefore, the
different effects of antidiuretic hormone (ADH) seen in mammals and eels may
be attributable to their different drinking behavior responses to blood
hyperosmolarity. The difference in the effect of VIP may be also due to a
different response to ADH, as it is known that intracerebroventricular
administration of VIP releases AVP in rats
(Bardrum et al., 1988
;
Murase et al., 1993
;
Nagai et al., 1996
). Except
for ADH, most ligands act in a similar way in both eel and mammalian brain.
Isoproterenol is known to be a potent thirst stimulus upon systemic
administration (Fitzsimons,
1998
), and to stimulate water intake after injection into the SFO
in the brain of rats (Menani et al.,
1984
). Therefore, the eel may be a suitable model for analyzing
drinking behavior in vertebrates.
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Acknowledgments |
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References |
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