Bacterial lipopolysaccharide (LPS) modulates corticotropin-releasing hormone (CRH) content and release in the brain of juvenile and adult tilapia (Oreochromis mossambicus; Teleostei)
Department of Animal Physiology, Faculty of Sciences, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands
* Author for correspondence (e-mail: p.pepels{at}science.ru.nl)
Accepted 29 September 2004
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Summary |
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Key words: immune system, immune-endocrine communication, endotoxin, corticotropin-releasing factor, CRF, -MSH, cortisol, plasma CRF, HPI-axis, pituitary, stress, teleost, telencephalon, NE, norepinephrine, 5-HT, serotonin
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Introduction |
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The question whether LPS treatment also affects the amount of CRH peptide
present in the brain of teleost fish has not been clarified and is
investigated in the present study. Immuneendocrine communication also
occurs in fish, as LPS administration modifies the activity of the
pituitaryinterrenal (PI) axis (Balm
et al., 1995; Haukenes and
Barton 2004
; Holland et al.,
2002
; Wedemeyer,
1969
; White and Fletcher,
1985
). At present, it is unknown whether the in vivo
modulation of the PI axis is an indirect effect caused by modulation of
hypothalamic CRH or a direct effect of LPS on pituitary or head kidney tissue.
In vitro treatment of tilapia (Oreochromis mossambicus
Peters 1852) with LPS from Escherichia coli (E. coli)
resulted in modulation of the activity of the HPI-axis at the level of the
cortisol producing tissue, which became markedly less responsive to
adrenocorticotropin hormone (ACTH; Balm et al., 1997).
In tilapia as in other fish, hypophysiotropic CRH-ir neurons directly
innervate the pituitary gland and, under certain stressful conditions,
stimulate the release of pro-opiomelanocortin (POMC)-derived peptides, such as
ACTH and -melanocyte-stimulating hormone (
-MSH), which in turn
stimulate the synthesis and release of cortisol from the interrenal cells
located in the head kidney (Balm et al.,
1994
; Wendelaar Bonga,
1997
). Besides its corticotropic role and its role in body
coloration,
-MSH may be involved in immuneendocrine interactions
in fish as well as in mammals (see review by
Balm, 1997
;
Lipton and Catania, 1997
).
In the brain of tilapia the largest CRH-ir cell population is found in the
lateral part of the ventral telencephalon Vl (Pepels et al.,
2002a,b
).
These CRH-ir cells in the Vl region are not directly involved in regulation of
the pituitary because these Vl cells massively innervate the anterior part of
the lateral dorsal telencephalon (Dla; see
Fig. 1, original data in
Pepels et al., 2002a
). This
Vl-Dla projection contains the highest amount of CRH-ir measured within the
tilapia brain (Pepels et al.,
2002a
). We recently reported that during the acute stress
associated with capture, CRH is secreted into the blood, probably from these
telencephalic centres (Pepels et al.,
2004
; Fig. 1).
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Fish are markedly tolerant to high doses of LPS compared with mammals (up
to a 1000-fold; Berczi et al.,
1966) and effects of LPS or Gram-negative bacterial infections
become deleterious in fish after chronic infection, but are less severe after
acute infection. Previously, we showed that chronic treatment with LPS
(Balm et al., 1995
) or with
murine Interleukin-1 (IL-1; Balm et al.,
1993
) altered the activity of the PI axis. For these reasons we
treated tilapia chronically. The present study compared the effects of LPS on
hypophysiotropic and non-hypophysiotropic CRH-ir neurons with particular
emphasis on telencephalic CRH. Initially, juveniles were treated stress-free
with E. coli LPS via immersion. Fish larvae immersed in
LPS-containing water have been shown to take up LPS
(Dalmo et al., 2000
). We
recently have shown that rapid sampling and processing of juveniles can be
achieved leading to whole brain CRH,
-MSH and tissue cortisol levels
characteristic for unstressed fish (Pepels
et al., 2002c
; Pepels and
Balm, 2004
). Next, adult fish were used to obtain specific
information on which of the CRH-immunoreactive (ir) brain regions are
modulated by LPS treatment. LPS treatment was combined with confinement
stress. In fish, the acute-phase response and the susceptibility to bacterial
infections are modulated by stress (Maule
et al., 1989
). LPS is the bacterial constituent that triggers the
acute-phase response and, thus, we anticipated that actions of LPS could be
modulated by stress. Finally, to investigate whether LPS may directly act at
the level of the CNS, the telencephalon of tilapia was superfused in
vitro with LPS. Our previous results indicate that the tilapia
telencephalon can be used in vitro to study regulation of CRH release
(Pepels et al., 2002b
).
In mammals there is still debate on the most likely route via which LPS
in vivo stimulates cytokine release, and how these cytokines in turn
stimulate CRH synthesis in the brain (reviewed by
Turnbull and Rivier, 1999).
Lipopolysaccharide tested on hypothalamic explants of rats, inhibited the
in vitro CRH release (Pozzoli et
al., 1994
), although others reported no effects of LPS. In
contrast to the CNS of mammals the CNS of fish contains extremely high numbers
of sessile macrophages (Dowding and
Scholes, 1993
). These can act as intermediates for LPS actions as
fish macrophages produce cytokines such as interleukin-1ß (IL-1ß;
Zou et al., 1999
) and tumour
necrosis factor-
(TNF-
;
MacKenzie et al., 2003
), when
encountering LPS or bacteria. Interleukin-1ß is the common messenger that
mediates LPS signalling to CRH neurons in mammals
(Berkenbosch et al., 1987
;
Mirtella et al., 1994
;
Turnbull and Rivier, 1999
),
and the fish Il-1ß gene has recently been sequenced
(Zou et al., 1999
).
Holland et al., 2002
showed
that homologues IL-1ß stimulates PI-axis activity in trout and serves as
an intermediate for LPS effects on the PI-axis.
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Materials and methods |
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Exposure of juvenile tilapia to LPS
Tilapia (Orechromis mossambicus Peters 1852) breeding couples from
our laboratory stock were kept in glass tanks of 500 l supplied with Nijmegen
tapwater of 24°C under a 12 h photoperiod.
The developmental stage and corresponding age of the larvae was determined
according to the classification criteria of Bauerle and Voss
(1993). Tilapia life stages
younger than 4 weeks post hatching (wph) are here described as larvae, and
life stages older than 4 wph as juveniles. Larvae obtained from the mouth of
breeding females were classified at stage 7 corresponding with 7 days post
fertilisation (7 dpf) and 2 days post hatching (2 dph). Larvae were fed daily
with Micro-min® (Tetramin GmbH, Melle, Germany) at a ration of 2.5% (w/w)
of their total body mass. During the first week after hatching, larvae were
maintained in a 120 l freshwater aquarium, after which period 64 larvae were
equally divided among four transparent plastic tanks (height x depth
x length = 25x20x30 cm) each containing 4 l of freshwater
(Nijmegen tapwater). Water was continuously refreshed (4 l
day1 tank1) and aerated. After an
adaptation period of 3.5 weeks in the 4 l tanks, flow-through was stopped and
two tanks received either 8 ml of water (mixed by the air supply), or 8 ml of
water containing LPS (E. coli, serotype 0111:B4, Sigma L-2630, lot
no. 42K4120). Thereafter, the juveniles were observed for 10 min to see
whether there was a behavioural reaction to the addition of water or LPS. At
this moment the juveniles were approximately 4.5 wph. The LPS concentration in
the fish water was 12.5 mg l1. Starting at exposure day 7,
flow-through was resumed (1 l day1 tank1)
with water (control tanks) or with water containing LPS (12.5 mg
l1). After 10 days of LPS exposure the two control and LPS
tanks were sampled. Tanks were decanted above a fish net and larvae were
immediately frozen by dipping the net into a mixture of dry ice and methanol.
This sample procedure was completed within 30 s per tank. Larvae were removed
from the dry ice methanol mixture, weighed individually and kept in Eppendorf
cups at 20°C. Eight larvae from each group were processed further.
After measuring body length, the head region and body were separated according
to the procedure described previously
(Pepels et al., 2002c
).
Briefly, the fish was put on its lateral side on an ice-cold plate under a
binocular and the head was dissected with a scalpel. The plane of dissection
ran from the rostral tip of the dorsal fin to the most caudal part of the
operculum. Heads, including CNS, pituitary and head kidneys, were homogenised
individually (Pepels et al.,
2002b
,c
)
in a mixture of methanol and 0.01 N HCl (3:1 volume) containing ascorbic acid
(6 mmol l1) and aprotinin (Bayer: 250.000 KIU
l1). CRH and cortisol levels were determined by
radioimmunoassays (RIA) validated for tilapia larvae (Pepels et al.,
2002b
,c
).
-MSH was determined by RIA (Balm et
al., 1993
) in which serial dilutions of juvenile head homogenates
displaced radiolabelled
-MSH from the antibody in a parallel fashion
with dilutions of the
-MSH standard (not shown).
The CRH values measured in head homogenates represent the sum of central
nervous system (CNS), pituitary and head kidney CRH. Levels of head kidney CRH
in comparison to the CRH content of the CNS are less than 1% in tilapia and
thus negligible (Pepels and Balm,
2004). The
-MSH levels measured mainly represent pituitary
MSH, as immunohistochemical
-MSH staining of the brain in young life
stages did not reveal
-MSH-ir cells
(Pepels and Balm, 2004
).
Exposure of adult tilapia to LPS
Thirty-two tilapia (Oreochromis mossambicus) of mixed sex from our
laboratory stock were equally distributed between two 120 l aquaria of neutral
background. Water was continuously aerated, filtered (Eheim Pumps, GmbH,
Deizisan, Germany) and refreshed (10 l h1). Water
temperature was 24°C, the photoperiod was 12 h, and fish were fed twice
daily with Tetramin tropical fish food (Tetramin GmbH) at a daily rate of 1.5%
(w/w) of their body mass.
Fish were left undisturbed in their aquaria for 4 weeks. To obtain samples
from undisturbed fish, on four alternate days one fish from each aquarium was
quickly netted and sampled for blood and brain tissue (pre-injection sample).
On each of these 4 days, aquaria were sampled in random order and sampling of
two fish was completed within 2 min. Blood was collected in tubes containing
EDTA (1.5 mg ml1) and aprotinin (3000 KIU
ml1; Trasylol, Bayer AG, Leverkusen, Germany). Collected
blood was centrifuged, and plasma was separated and stored at 20°C
until analyses. Following spinal dissection, fish were weighed and sexed, fork
length was measured, whole brains were removed including pituitary, and the
tissue samples were frozen at 20°C until analyses. Starting 2 days
after taking the final pre-injection sample, the remaining 12 fish per
aquarium were injected intraperitoneally either with saline, or with 3 mg
kg1 body mass E. coli LPS (Sigma, serotype 0111:B4,
lot no. 110K4060). Fish were injected on three alternate days (day 1, 3, 5)
and were exposed to LPS for 6 days. 1 day following the final injection, six
fish were sequentially sampled from each aquarium (pre-confinement). As the
sampling protocol influences plasma cortisol
(Balm et al., 1994) and plasma
CRH (Pepels et al., 2004
),
strict care was taken to synchronise sampling of the two groups. Fish were
sequentially sampled in alternating order (every other; 1st LPS fish, 1st
saline fish, 2nd LPS fish, 2nd saline fish and so on until fish 6) at 2 min
intervals. Sampling of the two groups was completed within 24 min. The
remaining six fish per aquarium were confined in a net (approximately 1 l
volume) in their home aquarium, and were sampled 24 h later. The same sampling
protocol was used and within 24 min all fish were sampled.
Pituitary and brain samples were treated as described by Pepels et al.
(2002b) with slight
alterations of the dissection into various brain parts (see left upper panel
in Fig. 2). Briefly, brains
were dissected under a binocular on an ice-cold petridish into: telencephalon,
diencephalons, rhombencephalon and pituitary. The tectummidbrain and
spinal cord parts were discarded (grey parts in the
Fig. 2). Brains from
pre-injected fish were not further dissected. CRH levels in the tissue
extracts were measured by RIA (Pepels et
al., 2002b
), and expressed as pg CRH tissue1.
Plasma cortisol was assayed by RIA (Balm et
al., 1994
), plasma glucose was measured using a commercial kit
(Boehringer Mannheim, Germany), and chloride was quantified by flame
photometry. Plasma CRH,
-MSH (in pituitary and plasma) was assayed by
RIA (Balm et al., 1993
;
Pepels et al., 2002b
).
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In vitro basal and stimulated CRH release during LPS incubation
Superfusion of telencephalic tissues was performed as described previously
(Pepels et al., 2002b).
Telencephalic tissues were derived from unstressed tilapia (N=60,
bodymass 31±2 g), and cut into four pieces. In the first in
vitro experiment four superfusion chambers, each containing telencephalic
tissue from three tilapia, were superfused with artificial cerebrospinal fluid
(acf-medium) containing 200 µmol l1 ascorbic acid
(Pepels et al., 2002b
). After
reaching a steady CRH release, tissues were during the time period
210240 min pulsed with 5x106 mol
l1 NE (norepinephrinehydrogentartaat; Centrafarm
Company, RVG 50833 UR, Etten Leur, Netherlands), and during 270300 min
were pulsed with 5x106 mol l1
serotonin (5-hydroxytryptamine-hydrochloride, 5-HT; Sigma H9523). Superfusion
fractions were collected and stored at 20°C. CRH was analysed by
RIA (Pepels et al.,
2002b
).
In the second in vitro experiment 16 superfusion chambers, each
containing telencephalic tissue from three fish, were superfused for 225 min
with acf-medium. Thereafter, eight control chambers were perfused with
acf-medium and the remaining eight chambers were perfused with acf-medium
containing 50 µg LPS ml1. The Escherichia coli
LPS used (serotype 0111:B4) had been chromatographically purified by
gelfiltration (Sigma L3012). This purified LPS was used because standard LPS
preparations contain glutamate and adenosine contaminants
(Hardy and White, 2001), which
have been shown to stimulate the release of NE by cortex tissue in rats
(Hardy and White, 2001
). After
210 min of LPS treatment, tissues were stimulated for 30 min with
5x106 mol l1 norepinephrine.
Finally, to test whether the tissues were still viable and in a
physiologically reactive state, all tissues received acf-medium containing a
high potassium concentration (56 mmol l1 KCl) between 500
and 515 min. Superfusion fractions were collected and stored at
20°C until use for CRH analysis by RIA. In the CRH RIA no
interference of LPS was found when analysing acf-medium containing 50 or 500
µg LPS ml1.
Presentation of data and statistics
Values presented are means ± S.E.M. (N1).
In the experiment using juveniles, the data were subjected to analysis of
variance (two-way independent ANOVA; SPSS statistical package 11.5 version for
windows; SPSS Inc., Chicago, USA) with tank and LPS treatment as independent
variable factors. Since the variables CRH and -MSH content were not
influenced by the tank factor (P>0.533 and P>0.880,
respectively; two-way independent ANOVA, SPSS), we pooled the values from
duplicate tanks to yield N=16. Student's t-test (two-sided)
was used as post-hoc test, and P<0.05 was accepted as
indicative of significant differences.
In the experiment using adult fish the condition (K) factor was calculated
from 100 w l-3 (w = body wet mass; l = fork length). As observed
previously (Balm et al., 1994)
capture sequences were observed in plasma cortisol. Plateau plasma cortisol
levels were calculated by taking the average of cortisol levels of fish 4, 5
and 6 from each group (Balm et al.,
1994
). Hormone or plasma parameters were subjected to analysis of
variance (two-way independent ANOVA, SPSS) with LPS treatment and confinement
stress as independent variable factors. Student's t-test (two-sided)
was used as post-hoc test, and P<0.05 was accepted as
indicative of significant differences. In the figures and tables, levels of
significance are indicated as follows P<0.05*,
P<0.01** and P<0.001***.
Maximally stimulated in vitro CRH release was calculated after determining the peak value of stimulation for each superfusion chamber. Differences between maximally stimulated and prepulse values were tested with the paired Student's t-test (two-sided).
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Results |
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LPS treatment adult tilapia
Throughout the experiment, the feeding response as well as the body
colouration remained unaltered, and none of the fish died as a result of the
experimental treatments. Plasma cortisol levels of fish sampled pre-injection
did not differ between the tanks (4.3±0.3 and 6.0±0.6 ng
ml1 in control and E. coli designated fish,
respectively; N=4). Also whole brain CRH content (including tectum
and pituitary) of fish sampled pre-injection did not differ between the 2
tanks (N=4/tank: 6119±419 and 6461±512 pg/fish in
control and E. coli LPS designated groups, respectively).
In fish sampled 1 day following the final injection with saline or LPS no
differences in body mass or K factor was observed between saline and LPS
treatment groups (not shown, ANOVA: P>0.226 and
P>0.215, respectively). The dissection of the brains was carried
out according to the dotted lines in the diagram of
Fig. 2 (left upper panel).
Overall, confinement stress significantly affected diencephalic and
telencephalic CRH, plasma CRH, plasma -MSH, plasma cortisol and plasma
chloride levels (ANOVA P<0.02, P<0.01,
P<0.01, P<0.001, P<0.02 and
P<0.01, respectively). In saline-treated fish confinement stress
increased telencephalic CRH (P<0.05) content, plasma cortisol
(P<0.02), plasma glucose (P<0.01) and decreased plasma
CRH (P<0.05) and plasma
-MSH (P<0.02) levels
(Fig. 2,
Table 2). In LPS treated fish
confinement increased plasma glucose (P<0.001), diencephalic CRH
(P<0.02), and decreased plasma chloride levels
(P<0.05; Fig. 2,
Table 2).
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LPS treatment alone did not affect the parameters studied, but interactions
were observed between LPS treatment and confinement stress regarding
diencephalic CRH, pituitary CRH, pituitary -MSH and plasma glucose
levels (ANOVA P<0.05, P<0.02, P<0.05,
P<0.02, respectively).
Effects of in vitro LPS treatment on CRH release from telencephalic tissue
In the first 3 h of superfusion CRH release decreased after which period a
steady state was reached with a basal release of approximately 550 fg
min1 tissue1
(Fig. 3A). Both
neurotransmitters stimulated the CRH release
(Fig. 3A). The maximally
stimulated CRH release induced by NE was 1281±105 fg
min1 tissue1 (P<0.001 compared
to prepulse values) and by serotonin was 1004±67 fg
min1 tissue1 (P<0.02). These
rates corresponded to 223±66% and 187±36% of pre-pulse values,
respectively.
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Next, telencephalic tissues were superfused with or without LPS-containing medium. Superfusion with LPS did not alter the basal CRH release compared with controls (Fig. 3B). Prepulse release values were 629±223 and 647±248 fg min1 tissue1 for control and LPS-treated tissues, respectively (Fig. 3B) However, LPS pre-treatment abolished the CRH response of the tissue to NE (Fig. 3B). Control tissues displayed a maximally stimulated CRH release of 1512±295 fg min1 tissue1, when receiving NE, which corresponded to 240±47% of pre-pulse values. In reaction to the 56 mmol l1 K+ pulse, control tissues displayed a maximally stimulated CRH release of 997±186 fg min1 tissue1 (P<0.05; Fig. 3B) and LPS superfused tissues displayed a maximal stimulated CRH release of 908±186 fg min1 tissue1 (P<0.01; Fig. 3B). These rates corresponded to 142±26% and 219±46% of prepulse values, respectively.
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Discussion |
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Our results on the effectiveness of LPS treatment via the ambient water are
consistent with previous findings in Atlantic halibut (Hippoglossus
hippoglossus) larvae. Dalmo et al.
(2000) reported that after 10
days of bath exposure to LPS (Aeromonas salmonicida) immunoreactive
LPS was found in the gut lumen, intestinal epithelial cells, kidney ducts and
epidermis, and fluorescein-labelled LPS was found in endothelial cells in
veins adjacent to the intestinal tissue. This strongly indicates that LPS in
yolk sac larvae is absorbed via the gut and the integument, and subsequently
transferred via the circulation to the kidneys. We have not studied the uptake
of LPS in our fish, but our juveniles had absorbed their yolk sac, and had
already developed a skin with scales. As juvenile tilapia only feed in the
short period during which the flake food floats on the water surface, the
uptake of LPS precipitated on the feed may be neglected. Therefore, LPS must
have been taken up predominantly by drinking. Indeed, using the freshwater
drinking rate described for tilapia larvae by Lin et al.
(2000
), it can be calculated
that during the 10 day experimental period our juvenile tilapia have ingested
22 mg LPS kg1 body mass1 via the water.
This dose is in the same order of magnitude as that administered to the adult
tilapia in this study.
Our juvenile fish were 5.5 weeks (4.5 wph) of age at the start of the
experiment and we propose that the response of their immune system to the LPS
antigen triggered the neuroendocrine effects observed. The first
appearance of lymphocytes in thymus, head kidney, and in gut-associated
lymphoid tissues of tilapia occurs already 1 wph
(Dogget and Harris, 1987). In
one-week-old carp (Cyprinus carpio), monocyte/macrophage-like cells
are already present in thymus, head kidney, spleen, and importantly in blood
and gut-epithelium (Romano et al.,
1997
). B cells appear in the carp head kidney in the second week
post fertilisation (Romano et al.,
1997
). In fish the capacity to produce antibodies, which evidences
the presence of mature immune cells and functional cytokine pathways,
generally starts at an age between 38 weeks (see review
Tatner, 1996
).
The increases in CRH and -MSH contents after LPS exposure were not
related to differences in growth or to enhanced development, as mass and
length of juveniles did not differ between LPS-treated and control groups.
Growth stimulation by LPS has been reported in Atlantic salmon (Salmo
salar) juveniles but only after long term (64 days)feeding of LPS coated
feed (Guttvik et al.,
2002
).
Since cortisol levels did not differ between groups we conclude that LPS
treatment effects were not secondary to HPI activation. The lack in cortisol
response after LPS exposure can not be attributed to immaturity of the
juvenile HPI-axis. In the first week post hatching the hypothalamus,
telencephalon and pituitary gland of tilapia are CRH-immunoreactive (CRH-ir),
POMC-derived peptides are expressed by the pituitary gland
(Pepels and Balm, 2004), and
the cortisol stress response is already present
(Pepels and Balm, 2004
).
Results therefore suggest that LPS directly affected the CRH system after 10
days of treatment.
Similarly, the increase of -MSH content after LPS exposure may
represent a direct effect of LPS at the level of the pituitary. Previously we
showed that in vitro LPS treatment alters the
-MSH release
from pituitary melanotropes in tilapia (Balm et al.,
1993
,
1995
). Since there was no
HPI-axis activation or difference in body colour between experimental groups,
the
-MSH increases after LPS exposure may subserve immune related
actions. As early as 1938 an immunoregulatory role for melanotropins was
reported as fishes' susceptibility to infectious diseases was influenced by
tank colour (Sumner and Douderoff
1938
). In mammals
-MSH acts as a potent immunomodulator,
which inhibits fever and all types of inflammation
(Lipton and Catania, 1997
). In
the periphery,
-MSH inhibits the production, release and actions of
pro-inflammatory cytokines, such as IL-1, interferon (IFN) and TNF-
(Lipton and Catania, 1997
).
Interestingly, in fish the peptide has been reported to have predominantly
stimulatory effects on immune responses in vitro, where it stimulates
phagocytosis of leucocytes (see review Harris and Bird, 2000).
LPS effects on adult tilapia
To obtain more information concerning the specific CRH-ir brain regions
influenced by LPS treatment further dissection of the brain was performed
using adult specimens. The pre-injection results demonstrated that at the
start of the experiment no differences in the HPI-axis activity existed
between tanks as plasma cortisol and brain CRH levels were similar between
groups. These CRH levels corresponded to previously measured CRH levels in
adult unstressed tilapia (Pepels et al.,
2002b). Overall, 6 days pre-treatment with E. coli LPS
modified the reaction of tilapia to the additional stressor of 24 h
confinement, as interactions between LPS treatment and confinement were
observed at the level of the hypothalamus (diencephalic CRH), the pituitary
(CRH and
-MSH contents) and in plasma glucose levels. Our results on
the modulatory effect of LPS on the stress response appear to corroborate the
results of Haukenes and Barton
(2004
), who found that
crowding stress affected the HPI response to a single injection with LPS.
Confinement elevated telencephalic CRH levels in controls, but not in
LPS-treated fish. Whether the small population of hypophysiotropic CRH-ir
cells located in the nucleus preopticus (npo; CRH in these cells accounts for
approximately 5% of total telencephalic CRH contents
(Pepels et al., 2002a), or the
large non-hypophysiotropic population of CRH-ir cells located in the lateral
part of the ventral telencephalon (Vl) was affected by confinement cannot be
ascertained from the present results. The stress-induced elevation of plasma
cortisol levels observed in the controls only could point to activation of the
npo cells in these animals. Similarly, Ando et al.
(1999
) observed an increase in
npo CRH mRNA expression associated with confinement in rainbow trout
(Oncorhynchus mykiss). Activation of the non-hypophysiotropic Vl CRH
cells on the other hand would resemble the stress-induced activation observed
in CRH cells located in the amygdala of rats
(Pich et al., 1995
) and sheep
(Cook, 2002
). We previously
discussed that the lateral part of the ventral telencephalon (Vl) in tilapia
might be comparable with the mammalian amygdaloid complex since in both
situations these regions contain the largest extra hypothalamic CRH-ir cell
population (Pepels et al.,
2002a
).
The most striking effect of the 6 days E. coli endotoxin
pre-treatment was the modulation of the CRH response to confinement at the
level of the diencephalon. The diencephalon of teleost fish including tilapia
contains hypophysiotropic CRH-ir cells located in hypothalamic nuclei (lateral
tuberal nucleus and recessus lateralis nucleus), which directly innervate the
melanotrope and corticotrope cells in the pituitary gland
(Pepels et al., 2002a).
Notably, at the level of the pituitary combined effects of LPS and confinement
were also found on CRH and on
-MSH. In fish the melanotropes are
targets for the CRH-ir nerve endings in the pituitary.
Previously, we reported that CRH appears rapidly, within 5 min after the
onset of capture, in plasma in tilapia
(Pepels et al., 2004). This
rise in plasma CRH was abolished when fish were confined for 48 h before
capture. In the present study this inhibition in plasma CRH response was also
observed in the control fish after 24 h confinement, but not in the LPS
treated groups, which suggests that these compounds in some way interfere with
CRH release. During stress circulating CRH in tilapia may regulate circulating
monocytes (Pepels et al.,
2004
), as in rats where CRH modulates IL-1 release by
LPS-activated blood monocytes (Pereda et
al., 1995
).
In contrast with confined saline-treated fish, confined LPS-treated fish
had difficulties maintaining their osmoregulation as plasma chloride levels
were decreased in comparison to their unstressed LPS-treated counterparts.
During stress, catecholamines increase the permeability of gills to water and
ions, and cortisol counteracts this effect, partly by mobilisation of glucose
(Wendelaar Bonga, 1997). In
view of the combined effects of LPS and confinement found on plasma glucose
levels, effects of LPS on energy reallocation may underlie the inability of
the LPS treated fish to maintain ionic homeostasis.
Regulation of in vitro CRH release
We investigated whether in vivo effects of LPS could be caused by
direct actions of LPS on brain tissue. Regarding mammals, there is still
debate on the most likely route via which LPS in vivo stimulates
cytokine release, and how these cytokines in turn stimulate CRH synthesis in
the brain (reviewed by Turnbull and
Rivier, 1999). One theory is that following IP administration, LPS
is transported via the portal veins to the blood circulation
(Lenczowski et al., 1997
), and
that LPS or LPS-induced blood-borne factors reach the brain to stimulate
IL-1ß production in brain regions where the bloodbrain barrier is
poorly developed (van Dam et al.,
2000
). Among the areas containing CRH-ir neurons in tilapia, the
ventral telencephalon is most richly innervated by blood capillaries
(Pepels et al., 2004
). This
vascular bed provides an opportunity for LPS to gain excess to the brain
in vivo, and we therefore investigated CRH release from this tissue
in vitro (Pepels et al.,
2002b
). We anticipated that stimulation of the CRH neurons by
norepinephrine (NE) or serotonin (5-HT) would be required as monoamine
utilisation in the CNS of mammals is altered by peripheral administration of
LPS (Dunn, 1992
). There is
also substantial evidence for the critical and permissive role of medullary
catacholaminergic innervation of the hypothalamus in the activation of the HPA
axis in response to systemic LPS (Ericsson
et al., 1994
).
The stimulatory role of NE and 5-HT on in vitro CRH release is
reported for the first time in lower vertebrates. Stress stimulates NE release
in the fish brain. Hoglund et al.
(2000) have demonstrated that
turnover rates of 5-HT in the telencephalon, and turnover rates of NE in the
brain stem and optic tectum, were positively correlated with plasma ACTH
levels in socially chronic stressed (subordinate) Arctic charr (Salvelinus
alpinus). Also in goldfish (Carassius auratus) there is support
for a stimulatory role of 5-HT on CRH release, in relation to the regulation
of feeding behaviour (de Pedro et al.,
1998
). The ventral telencephalon as well as the preoptic region of
teleost fish are innervated by NE-ir or dopamine ß-hydroxylase
ir
fibres (see review by Meek,
1994
) and by 5-HT-ir fibres
(Meek and Joosten, 1989
).
Similar to mammals, the NE-ir and DBH-ir cell bodies are located in the locus
coeruleus and the brainstem and the 5-HT-ir cell bodies are mainly located in
the raphe nuclei (Meek, 1994
;
Meek and Joosten, 1989
). In
mammals NE stimulates the in vitro CRH release from telencephalic
tissues containing the amygdala (Raber et
al., 1995
). Previously, we demonstrated in vitro that the
telencephalon synthesised and released CRH for at least 4.5 h of superfusion
(Pepels et al., 2002b
). The
present stimulation of the in vitro CRH release by NE and 5-HT
further confirms that in vitro CRH release is not caused by leakage
but represents regulated release.
Between in vitro experiments the initial and basal CRH release
rates in tilapia appeared remarkably comparable (see also
Pepels et al., 2002b). Basal
in vitro CRH release by the telencephalon was not affected following
3 h of LPS treatment/superfusion. Previously, we showed in tilapia that
concentrations between 150 µg ml1 of LPS in
vitro applied in a similar superfusion set-up dose-dependently inhibited
the basal release of
-MSH by the pituitary gland
(Balm et al., 1993
).
The major in vitro finding in our study was that LPS treatment
inhibited the NE-induced CRH release. This effect was not due to non-specific
effects, such as tissue damage by LPS, as the CRH neurons in the LPS-treated
telencephalon reacted to the high concentration of potassium by increasing the
CRH release in a similar way as the controls. Also, tilapia pituitary tissue
superfused with equally high E. coli LPS concentrations remained
viable (Balm et al., 1993).
There are several possible mechanisms via which LPS may have affected the
NE-induced CRH release, such as a LPS-induced nitric-oxide production, which
inhibited CRH production in mammals
(Kostoglou-Athanassiou et al.,
1998
). Alternatively, preincubation with LPS (or IL-1) could have
desensitised adrenergic receptors because LPS stimulates the NE turnover in
brain tissues of mammals (Ericsson et al.,
1994
; Francis et al.,
2001
). Neurons, microglia cells, sessile macrophages or
endothelial cells may be among the cell types involved in these regulations
(Kostoglou-Athanassiou et al.,
1998
; Turnbull and Rivier
1999
). The observed in vitro inhibition of the CRH
response to NE by LPS may explain why in vivo the confinement
treatment in LPS pre-treated fish, in contrast to controls, was unable to
increase telencephalic CRH.
In summary the present study is the first study in lower vertebrates to demonstrate changes in brain CRH levels after a challenge with the bacterial endotoxin LPS. Whereas the in vivo results were obtained following more-prolonged treatment, the in vitro results demonstrate that LPS in fish also can act within several hours. Our results together with those of Volkhoff and Peter (2004) establish that the role of CRH in immuneendocrine interactions is a phylogenetically old mechanism. A single in vivo LPS (ip or icv) treatment of goldfish elevated mRNA CRH levels in hypophysiotropic and non-hypophysiotropic brain regions (Volkhoff and Peter 2004). Since LPS treatment affects non-hypophysiotropic as well as hypophysiotropic CRH-ir cells, the role of the peptide in immuneendocrine interactions in fish is not limited to regulation of HPI-axis activity. Results indicate that, in particular, the non-hypophysiotropic CRH system located in the ventral telencephalon of tilapia can be modulated directly by LPS.
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