CCK2 receptor nullification attenuates lipopolysaccharide-induced sickness behavior

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CCK2 Receptor Nullification Attenuates Lipopolysaccharide-induced Sickness Behavior

Tracey J. Weiland, Nicholas J. Voudouris, and Stephen Kent

School of Psychological Science, La Trobe University, Bundoora, VIC, 3086, AUSTRALIA

Running Head: CCK2 Receptors and LPS-induced Sickness Behavior

Address correspondence to:

Dr. Tracey WeilandEmergency Practice Innovation CentreDept. Emergency MedicineSt Vincent's Health41 Victoria Pde, Fitzroy, VIC 3065AUSTRALIA

Tel: +61 39288 2734Fax: +61 39479 1956Email: [email protected]

of 53Articles in PresS. Am J Physiol Regul Integr Comp Physiol (July 20, 2006). doi:10.1152/ajpregu.00156.2006

Copyright © 2006 by the American Physiological Society.

mailto:[email protected]

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Abstract

Systemic infection produces a highly regulated set of responses such as fever,

anorexia, adipsia, inactivity and cachexia, collectively referred to as sickness behavior.

Although the expression of sickness behavior requires immune-brain communication, the

mechanisms by which peripheral cytokines signal the brain are unclear. Several mechanisms

have been proposed for neuroimmune communication, including the interaction of cytokines

with peripheral nerves. A critical role has been ascribed to the vagus nerve in mediating

sickness behaviour following intraperitoneally-delivered immune activation, and converging

evidence suggests that this communication may involve neurochemical intermediaries afferent

and/or efferent to this nerve. Mice lacking functional CCK2/gastrin receptors (CCK2KO) and

wild-type (WT) controls were administered LPS (50, 500, or 2500 µg/kg; serotype 0111:B4;

ip). Results indicate a role for CCK2 receptor activation in the initiation and maintenance of

LPS-induced sickness behavior. In comparison to WT controls, CCK2KO mice were

significantly less affected by LPS on measures of Tb, activity, body weight, and food intake,

with the magnitude of effects increasing with increasing LPS dose. Although activation of

CCK2 receptors at the level of the vagus nerve cannot be excluded, a possible role for these

receptors in non-vagal routes of immune-brain communication is suggested.

Keywords: cholecystokinin, LPS, infection, vagus, fever

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The gut-brain peptide, cholecystokinin (CCK), exhibits great potential as a

neurochemical intermediary of vagus-to-brain signalling. CCK binds to CCK1 and CCK2

receptors; the former are primarily localized in peripheral organs and some discrete areas of

the brain, whereas CCK2 receptors are the predominant subtype in the brain (55). Both

subtypes are present on the vagus nerve and vagal afferent connections, including the

brainstem and hypothalamus (46, 51). Thus, the peripheral and central positioning of CCK

receptors provides an anatomical basis for CCK to play a role in vagally-mediated

neuroimmune communication.

A constellation of findings points to a role for CCK in immune regulation, including a

protective function in septic shock induced by lipopolysaccharide (LPS)(41,42), a compound

of the outer cell wall of Gram-negative bacteria (60). CCK induces monocyte chemotaxis

both in vitro and in vivo (16, 65), and stimulates production of proinflammatory cytokines,

tumor necrosis factor (TNF), interleukin (IL)-1β, IL-6, and IL-8 (16). Peripheral

administration of CCK induces a pattern of c-fos expression that is similar to that seen

following administration of the immune activators, LPS, or IL-1β (19). Increases in plasma

CCK have been reported in the rat following intraperitoneal (ip) IL-1α (18) or intravenous

(iv) IL-1β (40). However, limitations in assay sensitivity and specificity, render these findings

questionable, and contradictory evidence has recently been presented (84).

Studies examining the involvement of CCK receptor activation in sickness behavior

(anorexia, inactivity, cachexia) have focussed primarily on the role of the CCK1 receptor.

Antagonism of the CCK1 receptor blocked the decrease in feeding following ip IL-1α in rats

(18), an effect that could not be reproduced for food-motivated behavior in mice with low

doses of LPS or IL-1β administered ip (8). The role of CCK receptor activation in fever

genesis and maintenance is also equivocal. The febrile effects of LPS and IL-1β administered

to rats in low doses were not attenuated by CCK1 receptor antagonism (34,44), although

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naturally occurring mutant rats deficient in CCK1 receptors exhibited an exaggerated febrile

response, a result attributed to a secondary trait of the strain (34).

Although several studies have examined the role of the CCK1 receptor in sickness

behavior, relatively little attention has been paid to CCK2 receptors. Blockade of CCK2

receptors, either peripherally (subcutaneously) or centrally (icv), attenuated fever in rats

administered a low dose of LPS iv (77). The role for CCK2 receptors in fever, however, has

yet to be confirmed when immune activators are administered ip, or with other pyrogens and

species.

Since the effectiveness of vagotomy (VGX) in attenuating behavioral depression (17,

71) and fever (25, 63) induced by LPS or IL-1 is typically dose-dependent, studies of the role

of CCK in sickness behavior have employed low doses of the immune activators. However,

given the diverse peptide and receptor distribution of CCK, and its possible involvement in

thermoregulatory effector mechanisms (24,86), it is important to acknowledge that CCK may

have a role in sickness behavior that is independent of vagal activation, thus underscoring the

importance of examining a broad range of doses of immune activators.

The purpose of this study was to examine the role of CCK2 receptors in the

expression of LPS-induced sickness behavior. Changes in body temperature (Tb), locomotor

activity, body weight, and food and water intake were monitored in response to a 50-fold

range of LPS doses. The responses of mice that lack functional CCK2 receptors (CCK2KO;

53) were compared with responses of the background strain, or wild-type (WT).

Investigations of the mechanisms underlying LPS-induced fever and anorexia, are typically

restricted to observations of a few hours (e.g., 70, 79). However, recent evidence suggests that

the sickness response follows a protracted timecourse (20). Given this, and large range of

doses used in the present study, all parameters were monitored for 4 days post-injection.

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Materials and Methods

Animals

Thirty-two male homozygous CCK2KO mice (La Trobe University, Melbourne,

Australia), and 32 male C57BL/6J mice (mus musculus) as WT mice (La Trobe University)

were used for the present study. All CCK2KO mice were homozygous for the disrupted CCK2

receptor gene and were developed through gene targeting as described previously (53,58).

Briefly, the CCK2 gene was isolated from a 129sv genomic library and the clone inserted in a

vector. Clones exhibiting homologous recombination on the disrupted CCK2 gene were

microinjected into blastocytes of C57BL/6J females. Chimerics were bred with C57BL/6J

mice to enable germ line transmission. The first generation of heterozygous animals were then

bred for 10 generations, selecting for the mutant CCK2 gene at each generation. Fifth

generation heterozygous mice were then bred to generate CCK2KO mice. When backcrossed

with C57BL/6J for several generations, the WT strain or genetic background for such

transgenic strains becomes the C57BL/6J (54). Confirmation that the CCK2KO strain is in

fact deficient in a functional CCK2 receptor has been provided previously (53). Every effort

was made to age-match the animals. At the commencement of the recording period, CCK2KO

mice were aged between 84 and 120 days (mean=98 ± 2 SEM, median= 96) and their average

body weight was 29.5 ± 0.4 and 26.8 ± 0.2 g (median=28.2 g; range: 24.0 to 35.1 g).

Drugs

An immune response was triggered through the ip administration of LPS (serotype:

0111:B4; Sigma, Sydney) in doses of 50 µg/kg, 500 µg/kg, and 2500 µg/kg. Lyophilized LPS

was dissolved in sterile, pyrogen free, isotonic saline solution (Astra Pharmaceuticals, North

Ryde, Australia), aliquotted, and stored at -20° C in Eppendorf tubes until required. Before

injection, aliquots were thawed at 4° C and an appropriate volume of saline was added to

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bring the diluent to the correct concentration for injection. Solutions were then raised to room

temperature. All LPS injections were administered 1 hr following the onset of the photophase.

Procedure

Throughout experimentation mice were housed individually under a 12:12 light:dark

cycle with food and water available ad libitum, and ambient temperature set at 30 ± 1° C,

which is within the thermoneutral zone for this species. Sawdust and tissue were provided as

bedding. Following a 2-week acclimation period, a biotelemetry device (22 mm x 8 mm; 1.83

g; E-4000, Mini Mitter Co., Inc., Bend, OR) was surgically implanted into the peritoneal

cavity of each animal as described previously (83).

A 7-10 day recovery period followed surgery, after which time mice were housed singly

in polypropylene enclosures. This apparatus which enables the determination of Tb, and the

quantification of general locomotor activity, and food and water intake has been described in

detail previously (83). Data for Tb, locomotor activity and food intake were decoded by

VitalView software (Mini Mitter Co., Inc., Bend, OR) at 1min intervals and stored

electronically.

Uneaten pellets were removed from the enclosure each morning and the number

subtracted from the total number delivered each day for the final data set. Body weight and

water consumption were manually recorded each morning. Body weight was measured to 10-2

g using top-loading scales. Water was dispensed following contact with a water nipple fed

from a 25 ml vertical, polypropylene tube with 0.2 ml graduated markings.

All dependent variables (Tb, activity, body weight, food hopper entries, and water

intake) were monitored during an acclimation period of at least 1 week. Mice were considered

to be adapted to the housing environment when: (a) clear and consistent circadian rhythms in

Tb, activity, and food hopper entries were present for at least 3 consecutive days; (b) daily

water intake and body weight varied by less than 1 ml and 1.5 g, respectively, during this

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period; and (c) body weight was greater than pre-surgical weight. Following successful

acclimation, a 4-day baseline period commenced during which time all dependent measures

were monitored.

Following the baseline period, each animal was administered one of three treatments:

50, 500, or 2500 µg/kg LPS, or the LPS vehicle, saline, 1 hr following the onset of the

photophase. Allocation of treatments was random and treatments were administered ip in a

maximum volume of 100 µl/mouse. Dependent measures were then monitored for a further 4

days post-injection.

All procedures were conducted in accordance with the American Physiological Society's

Guiding Prinicples in the Care and Use of Animals, and were approved by the La Trobe

University Animal Ethics Committee.

Data Analyses

Data were averaged and means ± SEM were calculated for each group and all

variables for the 8-day testing period. Data for Tb were converted to 15 min, and 2- and 12-

hourly means. Activity data and food hopper entries were converted to 12-hourly means and

totals respectively, and expressed as percentage difference from the 4-day average baseline.

Raw data for body weight and water consumption were collated and daily means for these

variables were derived.

Data for each dose were analysed separately, using univariate and two-way (time by

strain or time by pretreatment) repeated measures ANOVAs or ANCOVAs. This permitted

the levels of time in the analyses performed to vary as a function of dose, an important

consideration given that the duration of the effects of LPS is dose-dependent.

Identification of potential covariates was made using partial correlation. Missing data

were replaced with the group mean when using food or water intake as covariates. To

overcome violations of sphericity in the Tb data, the Greenhouse-Geisser statistic is reported

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for within-subjects effects. Where required, simple main effects were analyzed using Fisher’s

Least Significant Difference. This statistic is suitable when the number of comparisons is

small (32).

Basal strain differences in Tb, activity, body weight, and water intake from this data set

have been reported previously (83). To control for pre-existing individual and group

differences, results for LPS-induced differences in Tb were analysed on change from 4-day

average baseline. LPS-induced group differences for all other dependent variables were tested

on percentage difference from average baseline and are presented as such. For Tb, activity,

and food intake, data obtained for photophase and scotophase were analysed separately.

For 15 min Tb expressed as difference from baseline, analyses were conducted on

the initial 4 hr period post injection regardless of treatment. For 2 hr Tb analyses were

conducted on the initial 24 hrs post-injection. For 12-hourly Tb, the first 24 hrs post-injection

were analysed for mice treated with saline or 50 µg/kg LPS, and the first 48 hrs were analysed

for mice administered either 500 or 2500 µg/kg LPS. For activity data (percentage difference

from baseline), the number of post-injection days analysed was 1 for mice administered

saline, 2 for mice administered 50 µg/kg LPS, and 3 for mice treated with 500 or 2500 µg/kg

LPS. Regardless of LPS dose, body weight analyses were conducted on data for the entire

post-injection period since bodyweight typically failed to return to baseline. For saline-treated

mice, however, body weight data were examined using data collected during the first day

post-injection. Food pellet consumption, expressed as percentage difference from baseline,

was analysed using separate 2-way repeated measures ANOVAs on data for the first 48 hrs

for mice administered saline or 50 µg/kg LPS, and 96 hrs for mice administered 500 or 2500

µg/kg LPS. Water consumption analyses were conducted on data obtained during the first day

post-injection, for all treatments except 2500 µg/kg LPS, which was tested for a 48 hr period.

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Results

Body temperature. The febrile response to 50 µg/kg LPS was less prominent in

CCK2KO mice than WT mice, with the latter exhibiting a more rapid onset. ANOVA

conducted on 15 min mean difference from average baseline Tb revealed significant effects

for time, strain, and their interaction. The strain difference became significant from 90-105

min post-injection (F(1,14)=5.26, p=0.038 η2=.19) when a change from baseline of 0.48 ± 0.18

and -0.06 ± 0.08 °C occurred for WT and CCK2KO, mice respectively (Fig 1a). For 15 min

data, this difference remained significant for the remainder of the first 4 hr period following

injection. The strain difference was greatest 2-3 hrs post-injection (difference=0.65 °C), and

for both strains fever peaked at 5 hrs post-injection when an increase from baseline of 1.32 ±0.26 and 0.65 ± 0.17 °C occurred for WT and CCK2KO, mice respectively (Fig. 1b). ANOVA

conducted on 2-hourly means indicated a significant strain difference throughout the

photophase (F(4, 56)=3.34, p=.016 η2=.19). The mean 12-hourly increase from baseline during

the initial photophase was significantly greater in WT mice (WT: 0.93 ± 0.23 °C; CCK2KO:

0.30 ± 0.28 °C; F(1, 14)=6.33, p=.025 η2=.31; Fig. 1c). In both strains, circadian patterns of Tb

were re-established by day 2 post-injection.

In response to 500 µg/kg LPS, WT mice mounted a greater febrile response compared

to CCK2KO mice; this difference began to emerge 105-120 min post-LPS (Fig 1d) and

persisted until 8 hrs following injection (Fig. 1e). For 15 min means, significant time, strain,

and interaction effects were detected. The strain difference first became significant 105m

post-injection (F(1, 14)=17.95, p<.001 η2=.58) and remained so for all 15 min periods during

the first 4 hrs. For 2hr means, ANOVA revealed significant effects for time, strain, and their

interaction. The Tb of WT mice was significantly higher than that of CCK2KO mice from 2-8

hrs post-injection (2-3: F(1, 14)=37.51, p<.001 η2=.73; 4-5: F(1, 14)=26.25, p<.001 η2=.65; 6-7:

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F(1, 14)=45.71, p<.001, η2=.77; 8-9: F(1, 14)=38.48, p<.001 η2=.73). The effects of 500 µg/kg

LPS on Tb persisted in both strains for 48 hrs. For both strains fever peaked during the middle

of the photophase periods, with the Tb of WT and CCK2KO mice increasing from baseline by

a maximum of 2.25 ± 0.16 and 0.82 ± 0.23 °C, respectively (Fig. 1e). The first two scotophase

periods were marked by a decrease from baseline Tb of up to 1.01 ± 0.19 (WT) and 1.57 ±0.35 °C (CCK2KO; Fig. 1e). This hypothermic response, was observed from 12-17 hrs and

35-43 hrs for WT mice, and 12-18 hrs and 36-40 hrs for CCK2KO mice. The mean difference

between strains in the first 12 hrs post-injection was 0.35 °C (Fig. 1f). ANOVA indicated

significant strain differences during the first and second photophases (F(1, 14)=30.22, p<.001

η2=.68).

The strain difference induced by 2500 µg/kg was detected throughout the first 4hrs

post injection (Fig 1d). For 15m means ANOVA indicated significant main effects for strain

(F(1, 14)=4.84, p=.047 η2=.27) and time, but not their interaction. During the first 4hrs post-

injection, the difference was greatest 60-75m post-LPS when the change from baseline for

WT and KO was -0.71 ± 0.22 and 0.20 ± 0.14 °C, respectively. Examination of hourly and

12-hourly data revealed that this strain difference was not restricted to photophase periods

(Figs. 1e & 1f); similar to lower doses, the fever observed was followed by a decrease in Tb

during the scotophases. When pre-existing strain differences are taken into account, the

strains differed in their response to LPS from 3-9 hrs and 12-25 hrs following injection, with a

maximum difference of 1.42 °C occurring 23 hrs post-injection (Fig. 1e). Between 26 and 35

hrs post-injection, the Tb of WT mice remained 0.5 °C or more greater than at baseline. The

strain differences throughout the initial photophase and scotophase were confirmed by

ANOVA on 2-hourly means (photophase: F(1, 14)=5.39, p=.036 η2=.28; scotophase: F(1,

14)=23.54, p<.001 η2=.40). Inspection of 12-hourly means indicated substantial strain

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differences in Tb during the first 35 hrs following injection (Fig. 1f). ANOVA on scotophase

data revealed significant effects for time, strain, and their interaction; the Tb of CCK2KO

mice was significantly higher than that of WT mice during the initial post-injection

scotophase (F(1, 14)=22.84, p<.001 η2=.62).

In response to saline, the Tb of WT mice initially decreased in the photophase by 0.39

± 0.12, °C (Fig. 1a) but increased by up to 0.55 ± 0.12°C by the end of this period (Fig. 1a &

1b). The Tb of CCK2KO mice remained similar to baseline during the start of the photophase

but increased by 0.72 ± 0.21 °C at the end of the first photophase. Although CCK2KO mice

did not differ greatly from baseline during the initial scotophase period following injection, a

decrease from baseline of up to 0.80 ± 0.17 °C was found for WT mice (Fig. 1b).

Examination of 15m mean difference from baseline data for the first 4 hrs post-injection

revealed a significant main effect for strain (F(1, 14)=6.05, p=.027 η2=.30) but not time or their

interaction. The mean Tb of saline-treated WT mice was lower than that of CCK2KO mice 5-

9 hrs post-injection (Fig. 1b). For 2 hourly photophase data, ANOVA indicated significant

effects for strain (F(1, 14)=5.89, p=.029 η2=.30) and time, but not their interaction. Analyses of

scotophase data indicated a significant interaction between time and strain. Simple main

effects analyses revealed a significantly higher mean Tb for WTs compared to CCK2KO mice

from 20-23 hrs post-saline (F(1, 14)=12.94, p=.003, η2=.48; F(1, 14)=4.95, p=.043, η2=.26).

ANOVA indicated a significant difference between strains in 12-hourly mean Tb following

saline during the first post-injection photophase (F(1, 14)=5.92, p=.029 η2=.30) and scotophase

(F(1, 14)=5.68, p=.032 η2=.29) (Fig. 1c).

Locomotor activity. Both strains exhibited a decrease in locomotor activity during

the first 2 days following 50 µg/kg LPS (Fig. 2a). These were most prominent during, but not

limited to, the scotophase periods, and were greatest during the post-injection scotophase.

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Compared to WT mice, the activity levels of CCK2KO mice appeared less affected by LPS

(Fig. 2a), however the difference failed to reach significance.

Following 500 µg/kg LPS, locomotor activity levels of both strains decreased

substantially (Fig. 2b). A clear strain difference occurred in response to LPS, with CCK2KO

mice being less affected than WT mice for the entire 4-day period (Fig. 2b). This difference

was most pronounced during the second scotophase when WT mice were 33 ± 11 % less

active, and CCK2KO mice were 12 ± 28 % more active, than at baseline. For scotophase data

ANOVA indicated significant effects for time, and strain (F(1, 11)=5.94, p=.033, η2=.35), but

not their interaction.

Both strains exhibited a profound reduction in activity following 2500 µg/kg LPS

(Fig. 2b). Although activity during the photophase periods remained less than baseline until

day 4 post-injection, the decrease in activity was greatest for both strains during the initial

scotophase period following LPS. During this period, WT and CCK2KO mice were 73 ± 7

and 59 ± 11 % less active than at baseline, with activity falling below photophase levels (Fig.

2b). Although slight strain differences were present for this dose, they were not as distinct as

those seen in mice treated with 500 µg/kg LPS (Fig. 2b), and they failed to reach siginficance.

Following saline, both WT and CCK2KO mice produced a slight decrease from

baseline activity during the initial photophase (Fig. 2a). In the ensuing scotophase, WT mice

approached baseline whereas the activity of CCK2KO mice decreased to 26 ± 6 % less than

baseline. A strain difference in activity was evident in the first scotophase and second

photophase post-injection. However, only the scotophase difference reached significance (F(1,

13)=9.79, p=.008, η2=.43).

Body weight. The cachexic effects of 50 µg/kg LPS were greatest for both strains on

day 1 post-injection; WT and CCK2KO mice lost 4.7 ± 0.9 and 2.0 ± 0.6 % body weight,

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respectively (Fig. 3a). The percentage weight loss of WT mice exceeded that of CCK2KO

mice throughout the post-injection period with the strain difference peaking on day 2 post-

injection (WT: -2.6 ± 0.9 %; CCK2KO: 0.5 ± 0.4 %, relative to baseline). ANOVA indicated

significant main effects for strain (F(1, 14)=13.08, p=.003, η2=.48) and time, but not their

interaction.

When challenged with 500 µg/kg LPS WT and CCK2KO mice lost a maximum of 9.4

± 0.2 and 3.5 ± 0.5 % respectively, with cachexia greatest on day 1 (Fig. 3b). CCK2KO mice

were less affected by LPS with the strain difference being greatest on day 2 post-injection. At

this point, WT mice had lost only 8.3 ± 0.8 %, whereas CCK2KO mice had lost 2.7 ± 0.7 % of

their baseline weights. By the end of the testing period, CCK2KO mice had returned to within

0.3 ± 0.6 % of baseline body weight. In contrast, WT mice had returned to within 3.7 ± 1.2 %

of their initial weights. ANOVA indicated that the difference between strains was significant

across the entire testing period (F(1, 14)=25.02, p<.001, η2=.64).

When treated with the largest dose of LPS, WT and CCK2KO mice lost 12.1 ± 0.6 and

6.4 ± 1.4 % of body weight, respectively. In contrast to lower doses of LPS, the cachexic

effects were greatest on day 2 post-injection, but weight loss was once again greatest in WT

mice (Fig. 3b). The strain difference was largest on day 3 post-injection when CCK2KO mice

lost 2.3 ± 1.1 % and WT mice lost 9.6 ± 0.9 % body weight (Fig. 3b). ANOVA indicated

significant main effects for strain (F(1, 14)=19.23, p<.001, η2=.58), and time, but not their

interaction.

The body weight of saline-treated mice was stable throughout testing with no

significant strain difference following injection (Fig. 3a).

Food intake. In response to 50 µg/kg LPS, both WT and CCK2KO mice exhibited a

decrease in food intake compared to baseline (Fig. 4a). In percentage terms, the anorexic

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effects of LPS were most profound during the initial photophase following injection, when

consumption decreased by approximately 60% for both strains. Compared to WT mice, the

food intake of CCK2KO mice was slightly less affected and by the second photophase,

CCK2KO mice they had returned to within 6 ± 7 % of baseline, whereas WT mice consumed

29 ± 6 % less than baseline. Analyses of scotophase data revealed food consumption to be

significantly less during the first post-injection scotophase regardless of strain. The main

effect for strain was significant (F(1, 14)=4.88, p=.040, η2=.26), but the time by strain

interaction was not.

In response to 500 µg LPS, both WT and CCK2KO mice decreased food consumption

during the 4-day post-injection period (Fig. 4b). The decrease in consumption was greatest for

both strains during the first scotophase period following injection. During this period, the

number of pellets consumed by WT and CCK2KO mice decreased by 39.3 ± 2.6 and 28.9 ±4.2, respectively. Strain differences were observed in mice treated with 500 µg/kg LPS; most

notably during the first scotophase following LPS, when WT mice consumed 94 ± 1 % and

CCK2KO mice consumed 58 ± 10 % less than baseline. ANOVA indicated that scotophase

food intake varied significantly as a function of time, strain (F(1, 12)=8.01, p=.015, η2=.40),

and their interaction (F(1, 10)=5.90, p=.002, η2=.33). Simple main effects analyses revealed that

CCK2KO mice consumed significantly more than their WT counterparts on the first 2 days

post-injection only (F(1, 12)=9.98, p=.008, η2=.45; F(1, 12)=10.17, p=.008, η2=.46).

Consistent with other doses of LPS, the anorexic effects of 2500 µg/kg LPS were most

pronounced during the post-injection scotophase periods (Fig. 4b). For WT mice, the effects

persisted for the entire post-injection period, whereas CCK2KO mice had recovered to

baseline by the third post-injection photophase. Again, for both strains the effects of LPS on

food consumption were maximal during the first scotophase post-injection when food intake

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by WT and CCK2KO mice decreased by 36.2 ± 1.8 and 42.6 ± 4.5 pellets, respectively.

Although strain differences were observed during each scotophase, the greatest differences

between strains occurred during the third photophase, at which time CCK2KO mice had

returned to baseline and WT mice ate 51 ± 13 % less compared to the pre-injection period

(Fig. 4b). ANOVA on photophase data revealed significant effects for time, strain (F(1,

14)=10.71, p=.006, η2=.43), and their interaction (F(3, 42)=6.08, p=.002, η2=.30). Simple main

effects analyses indicated significant differences between strains on the first 3 days post-LPS

with WT mice consuming more than CCK2KO mice during the initial photophase (F(1,

14)=5.82, p=.030, η2=.29), and CCK2KO mice consuming more than controls during

photophases 2 and 3 (F(1, 14)=4.69, p=.048, η2=.25; F(1, 14)=7.56, p=.016, η2=.35). ANCOVA

performed on scotophase data revealed significant main effects for time, strain (F(1, 13)=16.47,

p<.001, η2=.56), and their interaction (F(3, 42)=5.33, p=.003, η2=.28). Simple main effects

analyses revealed that during the scotophase, CCK2KO mice consumed significantly more

that WT mice on days 1-3 post-injection (F(1, 14)=5.28, p=.038, η2=.27; F(1, 14)=14.31, p=.002,

η2=.51; F(1, 14)=10.51, p=.006, η2=.43).

Following saline, food pellet consumption by WT mice increased by 2.2 ± 1.3 and 5.6

± 1.3 (6 ± 3 % and 14 ± 2 %) during the first and second scotophases (Fig. 4a). In contrast,

consumption by CCK2KO mice decreased 8 ± 1 % during the first scotophase but no

difference was found during the second scotophase. A minimal but statistically significant

strain difference was observed during the first 2 scotophase periods post-injection (Fig. 4a).

For scotophase data ANOVA revealed a significant main effect for time and strain (F(1,

11)=9.85, p=.012, η2=.45), but not their interaction.

Water consumption. The decrease in water intake for mice administered 50 µg/kg LPS

was restricted to the first 24 hrs post-injection (Fig. 5a). For WT and CCK2KO mice the

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differences from baseline were 37 ± 9 and 30 ± 4 %, respectively, a difference which failed to

reach significance.

When treated with 500 µg/kg LPS, both strains exhibited a decrease in water

consumption which persisted for 48 hrs post-injection, but was most pronounced in the first

24 hrs following LPS (Fig. 5b). At this time, CCK2KO mice consumed 48 ± 11 % less than

baseline and WT mice consumed 84 ± 6 % less than at baseline. A clear strain difference was

apparent, with CCK2KO mice consuming more than WT mice (relative to baseline) on the

first day post-injection, but not on subsequent days (F(1, 14)=8.62, p=.011, η2=.38) (Fig. 5b).

Similar to the 500 µg/kg dose of LPS, water consumption by mice treated with 2500

µg/kg LPS was affected for the first 48 hrs following injection, with bothn strains decreasing

consumption by approximately 85% (Fig. 5b). CCK2KO mice, however, recovered quicker

and a strain difference was apparent on day 2 of testing. ANOVA revealed a significant time

by strain interaction (F(1, 13)=10.62, p=.006, η2=.45) and simple main effects analyses revealed

a significant increase in water consumption within each strain (WT: F(1, 13)=41.91, p<.001,

η2=.76; CCK2KO: F(1, 13)=110.63, p<.001, η2=.90) during post-injection day 2 compared to

day 1.

In response to saline, water consumption by CCK2KO mice decreased from baseline

by 8 ± 2 % (Fig. 5a). In contrast, the difference from baseline by WT mice was minimal,

though somewhat variable (-1 ± 5 %). After accounting for variance due to bodyweight

(44%), ANCOVA indicated a no significant effects of strain.

Discussion

Compared to controls, CCK2KO mice were significantly less affected by LPS on

measures of Tb, activity, body weight, and food intake. That the effects observed were

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apparent for a wide range of doses of LPS and dependent variables has important implications

for the involvement of CCK2 receptors in pathways of immune-brain signalling following ip

LPS. Studies concerned with elucidating pathways of neuroimmune communication have

ascribed differing levels of importance to vagal activation required for LPS or cytokine-

induced fever compared to behavioral depression (2, 25). A decrease in social interaction has

been shown to depend upon the integrity of vagal afferents regardless of dose (10, 11, 36, 43).

Subdiaphragmatic VGX attenuated the fever and anorexia induced by a low, but not a high,

dose of LPS (2, 71, 12, 43, 63). Thus, the mechanisms of communication from the periphery

to the brain, and their dose-dependence, differ for different components of the immune

response. Consequently, the following discussion will consider the dependent measures of

sickness separately.

There are several reasons to consider CCK as a neurochemical regulator of Tb under

pathological conditions. In addition to the activation of gastric vagal afferents (6), CCK is

capable of activating the common hepatic branch of the vagus (15), thought to be critical in

the genesis of LPS-induced fever (73). The expression of Fos protein in central autonomic

nuclei by CCK and LPS exhibits a similar pattern (23, 49). Both activate the central nucleus

of the amygdala and hypothalamic structures including the paraventricular nucleus (26, 80,

82), which has a role in temperature regulation (1, 26). Furthermore, in the rat, CCK is found

abundantly in the POA/anterior hypothalamus (47), an area long known for thermoregulatory

integration (5), and which has connections with the NTS (68).

CCK2KO mice were less affected than WT mice across a 50-fold range of LPS doses

ranging from modest (50 µg/kg) to high (2500 µg/kg). This is interesting with respect to a

possible mechanism of action since subdiaphragmatic VGX attenuates or abolishes LPS- and

IL-1β-induced fever when they are administered in low doses only (25, 63). Following high

doses VGX failed to prevent the induction of fever (2, 12, 43, 63). This raises the possibility

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that, in response to high doses of LPS, CCK may act on CCK2 receptors to induce the

expression of fever via a central, non-vagal route. This does not, however, preclude an

involvement of CCK2 receptors at the level of the vagus nerve. Rather, it points to the

possibility that activation of CCK2 receptors at this level may be sufficient, but not necessary,

for immune-brain communication required for fever when doses of LPS are high. As

highlighted by recent studies (36, 37), vagal activation may be of lesser importance for fever

genesis and HPA activation than for behavioral depression.

Our findings are in agreement with the reported inhibition of LPS-induced (sc, iv, 10

µg/kg) fever by a CCK2 receptor antagonist capable of crossing the BBB (79). Since fever

suppression persisted for longer when the antagonist was administered icv as opposed to

subcutaneously, the LPS-induced febrile response may occur primarily via the activation of

central CCK2 receptors rather than peripherally located CCK2 receptors. This hypothesis is

consistent with the largely central distribution of CCK2 receptors, including areas implicated

in thermoregulation (67).

In contrast to pharmacological studies examining the role of CCK2 receptors in fever

(77,79), we monitored Tb for several days following LPS. CCK2 receptor nullification

attenuated both the early and late changes in Tb induced by LPS with fever suppressing

persisting longer than 24 hrs with larger doses. This indicates a role for CCK2 receptor

activation in both the induction and maintenance of thermoregulatory changes that occur in

response to LPS.

The eicosanoid, PGE, is well accepted as one of the final steps in the fever process

(69, 76, 81), but cannot fully account for all thermoregulatory changes of the fever course

(79). PGE has been shown to act independently of CCK since the “fever-like” effects of icv

administered PGE1 could not be attenuated by CCK2 receptor antagonism (77). The reverse is

also true; the “fever-like” hyperthermic state induced by icv CCK was attenuated by CCK2

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receptor antagonism, but not the PG synthesis inhibitor, indomethacin (77). Thus, an

exclusive role for either CCK or PGE in the mediation of fever is unlikely.

Cholecystokinin interacts with a variety of neurohormones and peptides including

endogenous opioids (48). This interplay is especially interesting with respect to the present

study since opioids have been implicated in thermoregulation associated with systemic

inflammation. When administered to guinea pigs iv, the opioid antagonist, naloxone,

decreased the first and suppressed the second febrile phase elicited by LPS, and abolished the

febrile changes evoked by interferon-α2, TNF-α, and IL-6 (9). Although centrally

administered µ-opioid receptor antagonism attenuated LPS-induced fever in rats (4) but not

guinea pigs (62), peripheral administration attenuated fever in both species, suggesting that

circulating opioids may play a role in fever production (9,62). Given that the opioid system is

upregulated in CCK2KO mice (58), one may have hypothesised an increased febrile response

to LPS in this strain. This clearly did not occur except in response to the largest dose of LPS.

Pharmacological modulation of the opioid and CCK systems may provide further insight

regarding the coupling of these systems in thermoregulation.

The decrease in scotophase Tb compared to baseline may reflect a “true hypothermic”

reaction to LPS, that is, involving a decrease in set-point. The adaptive value of hypothermia

has been discussed previously (64). More recently, data were presented suggesting that

hypothermia and fever may be two different thermoregulatory strategies of the APR with

differing underlying mechanisms (21). Data from the present study indicate that, following

2500 µg/kg LPS WT mice produced a substantial decrease in scotophase Tb compared to

baseline, whereas no such decrease was observed for CCK2KO mice. CCK has been

implicated in the regulation of sympathetic vasomotor function (24). Further, CCK-8

modulates the firing rate of sympathetic nerves innervating interscapular brown adipose tissue

(85), induces skin vasodilation and a shock-like state, and a subsequent inhibition of heat-

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production and increase in heat-loss (35). Interestingly, however, the hypothermia induced by

CCK receptor activation has been demonstrated to depend on a CCK1, not a CCK2, receptor

mechanism (59, 77).

Prolonged hypothermia accompanied by a fall in mean arterial blood pressure is a

hallmark of septic shock (61). Although a brief decrease from baseline was present for WT

mice soon after injection with 2500 µg/kg LPS, this was rapidly followed by a prolonged

fever, a pattern that contrasts with previous studies employing this dose (39). Given the lack

of prolonged decreases in Tb during the photophase it is unlikely that any of the doses used in

the present study were sufficient to induce septic shock. Interestingly, CCK has been

demonstrated to reverse septic shock induced by large doses of LPS (8 mg/kg, iv, serotype

0111:B4; 41,42). It is unlikely that this is due to the activation of CCK2 receptors since KO

mice of the present study did not have an exacerbated shock response to the largest dose of

LPS. Moreover, it is unlikely that the present findings resulted from strain differences in

circulating CCK as a result of genetic compensation or an altered feedback loop. No

differences were observed between CCK2KO and their background strain in basal

concentrations of plasma, brainstem, or hypothalamic CCK (T.J. Weiland, unpublished

thesis). Furthermore, we have recently presented evidence of a decrease in murine plasma

CCK following low and high doses of LPS (84).

Evidence regarding the respective roles of CCK receptors in satiety is mixed. Since

vagal activation has been demonstrated to be critical for satiety (74), and since CCK1

receptors are present on the vagus nerve and vagal afferent terminals including the NTS, the

CCK1 receptor has been favoured for the regulation of food intake. However, satiety induced

by endogenous CCK has been demonstrated to require activation of the CCK2 receptor, and

although these receptors are located on the vagus nerve, they are also strongly represented in

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the hypothalamus. Indeed, it has been suggested that CCK may act not only at peripheral

sites, but also centrally to regulate food intake (22).

Results of the present study indicated that the anorexic effects of LPS were more

profound in WT compared to CCK2KO mice. In contrast, Bret-Dibat and Dantzer (8) found

that antagonism of the CCK2 receptor using a compound capable of crossing the blood brain

barrier failed to attenuate the suppression of food-motivated behavior in mice induced by a

low dose of ip LPS (serotype 0127:B8; 100 µg/kg) or IL-1β (20 µg/kg). The interpretation of

studies involving pharmacological manipulations is complicated by issues such as incomplete

antagonism, limited half-life, and bioavailability. While CCK2 receptor nullification through

genetic means is permanent, pharmacological receptor antagonism is not. However, since the

receptor antagonist employed by Bret-Dibat and Dantzer (8) is reasonably long-lasting, the

reason for the observed discrepancy is not clear. It should be noted, however, that they did not

verify the biological activity of the CCK2 antagonist employed. In addition, a variety of

procedural differences may account for these discrepant findings. The present study differed

from that of Bret-Dibat and Dantzer (8) in terms of ambient temperature (23 ± 2 °C vs. 30 ±1°C for the present study), timing of immune activation (scotophase vs. the photophase for the

present study), and food schedules (90 % food restricted vs. ad libitum in the present study).

The latter may be particularly relevant since a 10% food restriction increased plasma CCK

levels and reduced pancreatic function in the rat (14).

Assuming that CCK2 receptors are involved in LPS-induced anorexia as the present

study suggests, the potential levels at which CCK could become involved in immune-brain

communication are multiple. Receptors for CCK2 are present on the vagus nerve and at

several central sites. Thus, CCK may act centrally, peripherally, or at both levels. Studies

involving subdiaphragmatic VGX have indicated a critical role for the vagus nerve in food-

motivated behavior (7). Although this has not always been the case for LPS-induced changes

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in food intake itself (70), it is generally accepted that vagal afferents are required for the

expression of LPS-induced anorexia. More recently it has been shown that subdiaphragmatic

VGX blocks the anorexia induced by a “low” (5 µg/kg), but not “high” (125 µg/kg), dose of

ip LPS (71). However, since the serotype of LPS was not reported, it is difficult to compare

information regarding the precise dose with previous studies. Nonetheless, similar to Tb (12,

43, 63), these data suggest that vagal activation is necessary for the expression of anorexia

following a low dose of LPS, and that extra-vagal mechanisms are required following a high

dose. With this in mind, central CCK2 receptors are likely to mediate the anorexic effects of

large doses of LPS, whereas smaller doses may activate either peripheral CCK2 receptors of

the vagus, and/or central CCK2 receptors following vagal activation. However, that the most

robust strain differences occurred with the two large doses is suggestive of an exclusively

central role for CCK. The use of a CCK2 antagonist incapable of crossing the BBB or icv

injections may clarify this issue. Furthermore, since CCK exerts its effect on food intake via

both afferent and efferent vagal fibres (50), future studies may seek to determine the

contribution of CCK from vagal efferent fibres in moderating the response to illness.

The cachexic effects of LPS were more pronounced in WT mice compared to

CCK2KO mice across all doses of LPS. CCK acts synergistically with leptin (45), an

important circulating signal important for body weight regulation (31), and a recently

recognised mediator of LPS-induced anorexia and fever (66). Leptin receptors are also critical

in the recovery from LPS-induced hypothermia (75). In contrast to CCK, however, leptin is

thought to be devoid of a true thermoregulatory action; it acts via metabolic rate but does not

directly affect heat loss mechanisms (78). This synergy between CCK and leptin is

particularly interesting with respect to our results for Tb and body weight since circulating

leptin is decreased in CCK2KO mice compared to WTs (H. Chen, S. Kent & M. Morris,

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unpublished observations). It remains to be determined whether this strain difference remains

following LPS administration, and whether it impacts on thermoregulation and metabolism.

The close relationship of water consumption to food intake, makes it plausible that the

expression of LPS-induced adipsia and anorexia depend on a common pathway. Although

CCK2KO mice were significantly less anorexic compared to WT mice in response to all three

LPS doses, a strain difference in adipsia was observed only in response to the 500 µg/kg dose.

While this may be attributable to the larger variance in water consumption, it may also

indicate that the role, if any, of CCK2 receptors in mediating LPS-induced adipsia is indirect

or not robust.

In addition to food-motivated behavior and social exploration, decreased general

locomotor activity has also been demonstrated to be vagally-mediated (11, 43). Despite this,

relatively little is known regarding the mechanisms that support illness-associated changes in

locomotor activity. Under non-pathological conditions, CCK decreases locomotion in the rat

via a vagally-mediated pathway; conversely, CCK2 receptor antagonism increased the activity

of mice (33). LPS typically results in decreased locomotor activity, but this was minimal in

the CCK2KO mice. Consequently, it is possible that CCK may participate in LPS-induced

hypoactivity by acting on CCK2 receptors of the vagus nerve.

Further “downstream”, the locomotor effects observed may be due to the interaction of

CCK with opioid or dopaminergic systems. In several areas of the CNS, CCK is colocalised

with opioid peptides and/or dopamine (29, 57), and it antagonises opioid and dopamine-

mediated behavior (52, 56). Interestingly, opioid agonists increase locomotor activity in

rodents, and it is well established that this involves dopamine (3). Furthermore, the opioid

antagonist, naltrexone attenuates the LPS-induced decrease in locomotor activity (38). As

discussed earlier, CCK2KO mice differ from WTs in that they exhibit an increase in opioid

agonist tone, as well as a positive coupling of type 2 dopamine receptors to the cAMP

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pathway (58). Thus, it is unlikely that CCK acts alone to affect LPS-induced locomotor

activity.

The LPS-induced strain differences observed in the present study may be also be

related to the role of CCK2 receptors in depression. There is considerable overlap between the

APR and depressive episodes (85), and many features of the APR are prevented or attenuated

by chronic antidepressants. For example, fluoxetine attenuates LPS-induced anorexia and

cachexia, and abolishes its hypothermic effect (85). Similarly, tricyclic antidepressants

prevent LPS-induced anorexia, cachexia, adipsia, and decreased locomotor activity in rats

(72), and an atypical antidepressant attenuates the decreased social exploration and locomotor

effects of peripheral but not central LPS and IL-1β (13). Interestingly, mice treated with

CCK2 antagonists exhibit antidepressant-like responses in tests of reactitvity to stress (27,28).

This antidepressant effect is prevented by a selective dopamine D1 receptor agonist (27), and

by a δ-opioid antagonist (28). Thus, it is conceivable that CCK2 receptor invalidation may

attenuate the sickness syndrome by exerting an antidepressant effect involving stimulation of

δ-opioid and/or D1 receptor stimulation. This hypothesis is in agreement with the enhanced

opioid tone in CCK2KO mice.

Summary and Future Directions

Elucidating the role of CCK receptors in LPS-induced sickness behavior is far from

straightforward. Interpretation of the literature is complicated by species differences,

differences in immune activators, routes and timing of administration, and methods of

quantifying changes in behavior.

Using a genetic approach, this study has explored the roles of CCK2 receptors in LPS-

induced changes in Tb, activity, body weight, food intake, and water consumption. Our

findings strongly indicate an involvement of CCK2 receptors in LPS-induced sickness

behavior. Given the doses used and the fact that most CCK2 receptors are located centrally,

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this has important implications regarding the possible mechanisms of action of immune-to-

brain communication. While these data cannot exclude activation of CCK2 receptors at the

level of the vagus nerve, a possible role for these receptors in non-vagal routes of

communication is suggested.

CCK is known to alter the firing rate of sympathetic nerves innervating interscapular

brown adipose tissue (86), and has a documented involvement in blood pressure regulation

(24) and increased daytime Tb (83). Consequently, it would be invaluable to examine

thermoregulatory effector mechanisms in CCK2KO mice and mice pretreated with a CCK2

antagonist prior to LPS.

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Acknowledgements

We are grateful to Toshimitsu Matsui (Kobe University, Japan) for the kind donation of

homozygous CCK2KO breeding pairs, to Marika Gorrisen, Deb Anthony-Harvey-Beavis, and

Dominic Dwyer for practical assistance, and Ian Moore and Russell Beaton for technical

assistance.

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Figure Legends

Figure 1

a) Mean (± SEM) 15 min Tb (°C) of CCK2KO and WT mice challenged with 50 µg/kg LPS or

vehicle expressed as difference from baseline (n=8 all groups). Mean 15 min Tb 2h prior to

injection and at injection: WT vehicle-treated mice = 37.20 ± .16 and 36.98 ± 0.14; KO

vehicle treated mice = 36.92 ± 0.11 and 37.22 ± 0.15; WT 50 µg/kg treated mice = 37.08 ±0.10 and 37.18 ± 0.22; KO 50 µg/kg treated mice = 37.17 ± 0.13 and 37.23 ± 0.13.

(b) Mean (± SEM) hourly Tb (°C) of CCK2KO and WT mice challenged with 50 µg/kg LPS

or vehicle expressed as difference from baseline (n=8 all groups). Mean hourly Tb 2h prior

to injection and at injection: WT vehicle-treated mice = 37.28 ±.12 and 37.02 ± 0.14; KO

vehicle treated mice = 36.85 ± 0.14 and 37.19 ± 0.14; WT 50 µg/kg treated mice = 37.04 ±0.07 and 37.22 ± 0.21; KO 50 µg/kg treated mice = 37.15 ± 0.13 and 37.20 ± 0.11.

(c) 12-hourly mean (± SEM) Tb (°C) of CCK2KO and WT mice challenged with 50 µg/kg

LPS or vehicle (n=8 all groups).

(d) Mean (± SEM) 15 min Tb (°C) of CCK2KO and WT mice challenged with 500 or 2500

µg/kg LPS expressed as difference from baseline (n=8 all groups). Mean 15 min Tb 2h prior

to injection and at injection: WT 500 µg/kg treated mice = 37.28 ± 0.14 and 37.34 ± 0.14; KO

500 µg/kg treated mice = 37.10 ± 0.15 and 37.71 ± 0.14; WT 2500 µg/kg treated mice = 37.23

± 0.13 and 37.20 ± 0.15; KO 2500 µg/kg treated mice = 37.01 ± 0.20 and 37.67 ± 0.15.

(e) Mean (± SEM) hourly Tb (°C) of CCK2KO and WT mice challenged with 500 or 2500

µg/kg LPS expressed as difference from baseline (n=8 all groups). Mean hourly Tb 2h prior

to injection and at injection: WT 500 µg/kg treated mice = 37.30 ± 0.12 and 37.49 ± 0.14; KO

500 µg/kg treated mice = 37.00 ± 0.09 and 37.75 ± 0.11; WT 2500 µg/kg treated mice = 37.23

± 0.09 and 37.22 ± 0.13; KO 2500 µg/kg treated mice = 36.95 ± 0.20 and 37.75 ± 0.12.

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(f) 12-hourly mean (± SEM) Tb (°C) of CCK2KO and WT mice challenged with 500 or 2500

µg/kg LPS (n=8 all groups).

Figure 2

(a) 12-hourly mean (± SEM) percentage change in locomotor activity of CCK2KO and WT

mice challenged with 50 µg/kg LPS or vehicle (n=8 all groups).

(b) 12-hourly mean (± SEM) percentage change in locomotor activity of CCK2KO and WT

mice challenged with 500 µg/kg LPS (n=7 and 6 respectively) or 2500 µg/kg LPS (n=8 both

groups).

Figure 3

(a) Mean (± SEM) percentage change in body weight of CCK2KO and WT mice challenged

with 50 (n=7 both groups) µg/kg LPS or vehicle (n=8 both groups).

(b) Mean (± SEM) percentage change in body weight of CCK2KO and WT mice challenged

with 500 µg/kg LPS or 2500 µg/kg LPS (n=8 all groups).

Figure 4

(a) 12-hourly mean (± SEM) percentage change in food intake by CCK2KO and WT mice

challenged with 50 µg/kg LPS (n=8 both groups) or vehicle (n=6 and 7 respectively).

(b) 12-hourly mean (± SEM) percentage change in food intake by CCK2KO and WT mice

challenged with 500 µg/kg LPS (n=8 and 6 respectively) or 2500 µg/kg LPS (n=8 both

groups) or vehicle.

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Figure 5

(a) Mean (± SEM) percentage change in average water consumption of CCK2KO and WT

mice administered 50 µg/kg LPS or vehicle (n=8 all groups).

(b) Mean (± SEM) percentage change in average water consumption of CCK2KO and WT

mice administered 500 or 2500 µg/kg LPS or vehicle (n=8 all groups).

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Figure 1(a)

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Figure 1(b)

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Figure 1(c)

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Figure 1(d)

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Figure 1(e)

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Figure 1(f)

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Figure 2(a)

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Figure 2(b)

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Figure 3(a)

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Figure 3(b)

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Figure 4(a)

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Figure 4(b)

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Figure 5(a)

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Figure 5(b)

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CCK2 receptor nullification attenuates lipopolysaccharide-induced sickness behavior

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