Discrete electrolytic lesion of the preoptic area prevents LPS-induced behavioral fever in toads
Department of Physiology, Dental School of Ribeirao Preto and Department of Physiology, Faculty of Medicine of Ribeirao Preto, University of Sao Paulo, Ribeirao Preto, Sao Paulo, Brazil
* Author for correspondence (e-mail: branco{at}forp.usp.br)
Accepted 5 August 2002
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Summary |
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Key words: behavioral thermoregulation, amphibian, Bufo paracnemis, thermoregulatory set point
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Introduction |
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Although evidence has shown that the POA is an important thermoregulatory
site in some ectotherm species, the neural control of fever in these animals
has received no attention. Fever is a regulated increase in
Tb that is often described as a rise in the
thermoregulatory set point (Kluger,
1991). In mammals, fever is produced by the coordinated actions of
many central nervous system (CNS) regions as an adaptive response to
infection. Some preoptic neurons not only sense changes in deep body
temperature but are also affected by pyrogens that act on these neurons to
cause a number of physiological and behavioral responses that elevate
Tb (Boulant,
1998
).
With few exceptions, both endothermic and ectothermic vertebrates (as well
as invertebrates) develop fever in response to injections of exogenous
pyrogens such as lipopolysaccharide (LPS; endotoxin), viruses, Gram-positive
bacteria and yeast. LPS, which is the most purified form of a compound from
the cell wall of Gram-negative bacteria, usually Escherichia coli,
has been extensively used to induce fever in experimental animals (for a
review, see Kluger, 1991).
Recently, we demonstrated that the toad Bufo paracnemis develops
fever after systemic injection of LPS
(Bicego-Nahas et al., 2000
),
but the possible sites in the CNS involved in this response have not been
assessed.
In the present study, we tested the hypothesis that the POA is important for the development of behavioral fever in B. paracnemis. To this end, we evaluated the effect of electrolytic lesions in the POA on preferred Tb and LPS-induced behavioral fever in B. paracnemis.
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Materials and methods |
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Surgical methods
Animals were anesthetized by submergence in an aqueous 0.3% solution of
3-aminobenzoic acid ethyl ester (MS-222, Sigma, St Louis, MO, USA). The
animal's head was then fixed to a David Kopf stereotaxic apparatus (Model 900
Small Animal Stereotaxic, Tujunga, CA, USA) and the skin covering the skull
was removed with the aid of a bone scraper. An opening was made in the skull
above the diencephalon region using a small drill (Model FM 3545, Foredom
Electric, Bethel, CT, USA). A 0.4 mm tungsten electrode (A-M Systems, Everett,
WA, USA) fixed to the electrode holder was positioned into the region of the
POA (0.5 mm caudal to the telencephalon, 0.1 mm left and right from the
midline, and 0.5 mm above the cranial base) according to the coordinates of
the stereotaxic atlas for B. paracnemis (Hoffman, 1973). The
electrode was connected to the anode of a source of continuous current (Ugo
Basile, Comerio-VA, Italy, model 3500 Lesion Making Device) and the cathode
was connected with a toothed clamp to the animal's paw, previously wrapped in
cotton moistened with physiological saline. The bilateral lesions were made
with a 0.3 mA current applied for a period of 8 s. After lesion, the orifice
was filled with bone wax and acrylic cement. Sham-operated toads were
similarly prepared, but no current was passed through the electrode. The
experiments were performed 4 days after brain surgery. All animals looked
perfectly healthy after experimentation, even the lesioned ones.
Measurement of preferred Tb
Preferred Tb was determined in a thermal gradient
chamber (1.50 m long x 0.15 m high x 0.20 m wide) with an aluminum
floor. One end of the floor was cooled to 10°C by a copper pad connected
to a refrigerated water bath (VWR Scientific, 1160A, Niles, IL, USA). The
other end was heated to 38°C by another copper pad connected to another
water bath (Barnstead/Thermolyne, 310A, Dubuque, IA, USA). Petri dishes filled
with tap water throughout the chamber provided access to water at all
temperatures. An animal with a temperature probe, which was secured 2 cm into
the cloaca with skin sutures, was placed in the center of the thermal
gradient, and the thermistor output was continuously displayed on a chart
recorder (Barnstead/Thermolyne, LR93125, Dubuque, IA, USA). Cold and warm
water was used to calibrate the temperature probes before each experiment.
Experimental procedure
Experiments were performed on unanesthetized and unrestrained toads
previously prepared as described. One toad was placed in the middle of the
temperature gradient and left there for about 24h. After this period, saline
or LPS (from Escherichia coli, serotype 0111:B4, Sigma), was injected
into the dorsal lymph sac of the animals, and Tb was
monitored for 15h after injections. The dose used, 200µg LPS
kg-1 body mass, was based on a previous study on toads
(Bicego-Nahas et al., 2000).
The gradient chamber was continuously flushed with humidified room air at a
rate of 1.5 1 min-1.
Histology
At the end of each experiment the animals were anesthetized by submergence
in 0.3% MS-222 and perfused through the heart with saline followed by 10%
formalin solution. Immediately after, their heads were placed in 10% formalin
for at least 2 days. The brains were then removed from the skull,
histologically processed and immersed in paraffin, and serial coronal sections
(17 µm) were cut and stained by the Nissl method for light microscopy
determination of the electrolytic lesions.
Calculations and statistical analysis
Mean preferred Tb was determined every hour in all
experiments from individual chart paper recordings by manual calculation based
on a previous calibration. Two-way analysis of variance (ANOVA) was performed
followed by TukeyKramer multiple comparisons test. All values are
reported as means ± S.E.M. Values of P<0.05 were considered
to be significant.
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Results |
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Effects of electrolytic lesion of the POA on preferred Tb
of toads in euthermic condition
Toads selected a Tb of approximately 26°C, and no
significant difference was observed among control, sham-operated and lesioned
groups during a period of 24h (Fig.
2).
|
Effects of electrolytic lesions of the POA on behavioral fever of
toads induced by LPS
LPS caused a significant increase in preferred Tb
(P<0.05) of control and sham-operated toads, a response that was
abolished by electrolytic lesion of the POA. Lesions outside the POA did not
alter the LPS-induced fever of toads (Fig.
3).
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Discussion |
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Role of the POA in thermoregulation
In mammals, the POA is considered to be the thermointegrative and
thermosensitive site of the CNS, containing warm-sensitive and
temperature-insensitive neurons, a balance of which determines the
thermoregulatory set point (Boulant and
Dean, 1986; Boulant,
1998
,
2000
). In the tested species,
preoptic warming elicits heat-loss responses, including panting, sweating and
increased skin blood flow, as well as behavioral responses. By comparison,
preoptic cooling evokes heat-production responses, including shivering and
nonshivering thermogenesis, and heat-retention responses, including cutaneous
vasoconstriction and behavioral responses.
In amphibians, the neural control of thermoregulation has received limited
attention over the years. Although experimental verification is scarce for
amphibians, the rostral brain stem has been suggested to be an important site
for the regulation of Tb based on thermal stimulation of
this area in other ectotherm species such as lizards
(Hammel et al., 1967), teleost
fish (Crawshaw and Hammel,
1971
; Hammel et al.,
1969
; Nelson and Prosser,
1979
) and sharks (Crawshaw and
Hammel, 1973
). In a shuttle box set up, brain stem warming leads
these animals to exit from a warm environment earlier and at a lower
Tb than they would normally do. Conversely, brain-stem
cooling produces the opposite response. Moreover, heating the rostral brain
stem at high ambient temperatures can transiently increase the rate of
evaporative water loss in turtles
(Morgareidge and Hammel,
1975
).
Temperature-sensitive neurons have also been reported to exist in the POA
of some ectotherm species such as fish (Nelson and Prosser,
1981a,
b
) and lizards
(Cabanac et al., 1967
), and
also of birds (Nakashima et al.,
1987
). From these findings, it has been inferred that centrally
located temperature-sensitive neurons are components of a thermoregulatory
system in all vertebrates. However, more studies are needed to verify the
existence of this sort of neuron in amphibians.
The role of the hypothalamus in the behavioral thermoregulation of
ectotherms is also supported by studies in which specific brain nuclei were
lesioned in fish (Nelson and Prosser,
1979) and lizards (Berk and
Heath, 1975
). Nelson and Prosser
(1979
) reported that sunfish
(Lepomis cyanellus) and goldfish (Carassius auratus) show a
well-defined preferred Tb, but lesions placed in the
medial preoptic region disrupt this behavior and the fish can then be found
evenly distributed along the thermal gradient. In the lizard Dipsosaurus
dorsalis, Berk and Heath
(1975
) found that specific
hypothalamic areas when lesioned had pronounced effects on both high and low
Tb during shuttling.
For the amphibians, to our knowledge only one study has reported a role of
the hypothalamus in behavioral thermoregulation. After extensive hypothalamic
lesions by electric cautery, juvenile bullfrogs (Rana catesbeiana) do
not move at all when introduced within a thermal gradient. Lesioned bullfrogs
appear alert and jump when prodded but are quiescent when left undisturbed,
even when placed at 35°C (Lillywhite,
1971).
In the present study, we observed no effect of electrolytic lesion in the
POA on thermoregulation of B. paracnemis under euthermic conditions
(Fig. 2). Toads were still
actively moving (Fig. 2),
indicating that thermoregulation was not disrupted after POA lesions.
Conversely, Lillywhite (1971)
observed that thermoregulation of euthermic frogs was abolished after
hypothalamic lesions. This difference might reside in the fact that Lillywhite
(1971
) performed extensive
hypothalamic lesions, whereas in our experiments the lesions were discrete and
restricted to the POA (Fig. 1).
In agreement with our observation, as reviewed by Boulant
(2000
), the compilation of all
studies about lesions in the rostral hypothalamus of mammals suggests that no
single neural area acts as the center for thermoregulation. Rather, there
appears to be a hierarchy of structures extending through the hypothalamus,
brain stem and spinal cord. When the nervous system is intact, the role of the
higher structures (i.e. the preoptic region) becomes apparent. At least in
mammals, the strongest evidence of the importance of the preoptic region in
thermoregulation comes from studies involving direct thermal stimulation of
this area (Boulant, 2000
). This
type of investigation has never been carried out in amphibians.
Role of POA in the behavioral fever induced by LPS
It is interesting to note that although lesion in the POA of toads did not
result in a loss of thermoregulatory capabilities
(Fig. 2). LPS-induced
behavioral fever was completely abolished
(Fig. 3). Moreover, lesions
outside the POA did not change the febrile response of the animals. These
results are in agreement with data about the important role of the POA in
mammalian fever (Boulant, 1998,
2000
). In these animals, which
use autonomic and behavioral mechanisms to regulate Tb,
fever is functionally expressed as an increase in metabolic heat production
and a decrease in heat loss, besides behavior
(Cooper, 1995
). It is thought
that pyrogens and their mediators elevate the thermoregulatory set point by
inhibiting the firing rate of preoptic warm-sensitive neurons
(Eisenman, 1969
;
Matsuda et al., 1992
;
Shibata and Blatteis, 1991
;
cf. Boulant, 1998
,
2000
). Therefore, this
suppresses heat loss and enhances heat production and heat-retention
responses, and so fever occurs (cf.
Boulant, 2000
). Our results
concerning febrile toads, which thermoregulate primarily by behavior,
represent new data favoring the notion that the POA is a site involved in the
thermoregulatory set point rise during fever, regardless of the sort of
thermoeffector mechanisms present in the tested species.
Behavioral fever in ectotherms was first observed in lizards
(Kluger, 1991;
Vaughn et al., 1974
) and has
since been reported in amphibians
(Bicego-Nahas et al., 2000
;
Kluger, 1977
;
Myhre et al., 1977
), fish and
even some invertebrates (cf.
Kluger, 1991
), indicating that
fever has an ancient phylogenetic history. As to the mechanisms of fever, we
now report for the first time an important role of the POA in the development
of fever in toads, indicating a considerable degree of phylogenetic
conservation of this site in the CNS involved in fever among vertebrates.
Perspectives
Since the classic 1938 paper by Magoun et al. (cf.
Boulant, 2000) suggesting that
the POA is a thermosensitive site in the CNS, a growing body of evidence has
confirmed this notion in a wide variety of vertebrate species ranging from
fish to mammals and birds. It is currently accepted that, in mammals, a
balance between warm-sensitive and temperature-insensitive neurons in the POA
determines the thermoregulatory set point. Accordingly, endogenous pyrogens
such as interleukin 1 (Shibata and
Blatteis, 1991
) and prostaglandin E2
(Matsuda et al., 1992
) have
been shown to inhibit the sensitivity of warm-sensitive neurons to
temperature, a response that is in agreement with the increased
thermoregulatory set point during fever
(Boulant, 1998
).
However, even though fever has been reported to be a response that is
extremely widespread among taxa (Kluger,
1991), the role of the POA in the febrile response of ectotherm
species has received no attention. We now add data showing that the POA is
essential for fever development in an ectotherm species that thermoregulates
primarily behaviorally, supporting the notion that the POA is a site involved
in the thermoregulatory set-point rise during fever. This evidence favors the
view that the mechanisms responsible for fever may have an ancient
phylogenetic history. In fact, fever in ectotherms has already been suggested
to involve endogenous pyrogens (Myhre et
al., 1977
) and prostaglandin
(Bicego et al., 2002
;
Hutchison and Erskine, 1981
)
as pyretic mediators, and the vasopressin analogue arginine vasotocin as an
antipyretic molecule (Bicego-Nahas et al.,
2000
). Nevertheless, data are still lacking regarding the
mechanisms involved in the febrile response of ectotherm species to support
this assumption. Further experiments are needed to determine how exogenous
pyrogens are sensed by ectotherms, how endogenously produced pyrogens signal
the brain to produce fever and, once the brain is signalled, what neural
pathways are responsible for the effector response.
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Acknowledgments |
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