LipopolysaccharideInduced Sickness Behaviour ... - Wiley Online Library

Basic & Clinical Pharmacology & Toxicology, 2012, 110, 359–369

Doi: 10.1111/j.1742-7843.2011.00824.x

Lipopolysaccharide-Induced Sickness Behaviour Evaluated in Different Models of Anxiety and Innate Fear in Rats Gabriel S. Bassi1,2, Alexandre Kanashiro3,4, Francele M. Santin1,2, Glria E. P. de Souza4, Manoel J. Nobre1,2 and Norberto C. Coimbra1,5 1

Institute for Neuroscience and Behaviour (INeC), Campus Universitarius of Ribeir¼o Preto of the University of S¼o Paulo (USP), Ribeir¼o Preto, S¼o Paulo, Brazil, 2Laboratory of Psychobiology, School of Philosophy, Sciences and Literature of Ribeir¼o Preto of the University of S¼o Paulo (FFCLRP-USP), Ribeir¼o Preto, S¼o Paulo, Brazil, 3Department of Pharmacology, Ribeir¼o Preto School of Medicine of the University of S¼o Paulo (FMRP-USP), Ribeir¼o Preto, S¼o Paulo, Brazil, 4Department of Physics and Chemistry, School of Pharmaceutical Sciences of Ribeir¼o Preto of the University of S¼o Paulo (FCFRP-USP), Ribeir¼o Preto, S¼o Paulo, Brazil, and 5Laboratory of Neuroanatomy and Neuropsychobiology, Department of Pharmacology, Ribeir¼o Preto School of Medicine of the University of S¼o Paulo (FMRP-USP), Ribeir¼o Preto, S¼o Paulo, Brazil (Received 18 August 2011; Accepted 10 October 2011)

Abstract: The fact that there is a complex and bidirectional communication between the immune and nervous systems has been well demonstrated. Lipopolysaccharide (LPS), a component of gram-negative bacteria, is widely used to systematically stimulate the immune system and generate profound physiological and behavioural changes, also known as ‘sickness behaviour’ (e.g. anhedonia, lethargy, loss of appetite, anxiety, sleepiness). Different ethological tools have been used to analyse the behavioural modifications induced by LPS; however, many researchers analysed only individual tests, a single LPS dose or a unique ethological parameter, thus leading to disagreements regarding the data. In the present study, we investigated the effects of different doses of LPS (10, 50, 200 and 500 lg ⁄ kg, i.p.) in young male Wistar rats (weighing 180–200 g; 8–9 weeks old) on the ethological and spatiotemporal parameters of the elevated plus maze, light-dark box, elevated T maze, open-field tests and emission of ultrasound vocalizations. There was a dose-dependent increase in anxiety-like behaviours caused by LPS, forming an inverted U curve peaked at LPS 200 lg ⁄ kg dose. However, these anxiety-like behaviours were detected only by complementary ethological analysis (stretching, grooming, immobility responses and alarm calls), and these reactions seem to be a very sensitive tool in assessing the first signs of sickness behaviour. In summary, the present work clearly showed that there are resting and alertness reactions induced by opposite neuroimmune mechanisms (neuroimmune bias) that could lead to anxiety behaviours, suggesting that misunderstanding data could occur when only few ethological variables or single doses of LPS are analysed. Finally, it is hypothesized that this bias is an evolutionary tool that increases animals’ security while the body recovers from a systemic infection.

Several researches have yielded evidence that both humoral and neural pathways communicate the central nervous system about the presence of an invader [1]. During a systemic infection, the immune system conveys a message to the brain via vagus nerve and the circunventricular organs, and then neuronal cells respond producing cytokines that help the body to coordinate an adaptive behavioural response [1–4]. In fact, this behavioural response (or sickness behaviour – [5]) occurs because the organism needs to preserve energy during the response of the immune system to microorganism invasion [6]. Physiologically, these complex mechanisms are important to maintain homeostasis during an invasion of microorganisms. However, an excessive and prolonged production of proinflammatory cytokines in the body can damage tissues including the brain (producing severe behavioural deficits,

Author for correspondence: Norberto C. Coimbra, Laboratory of Neuroanatomy and Neuropsychobiology, Department of Pharmacology, School of Medicine of Ribeir¼o Preto of the University of S¼o Paulo (FMRP-USP), Av. dos Bandeirantes, 3900, Ribeir¼o Preto, SP, 14049-900, Brazil (fax +55 (016) 3633 0619, e-mail [email protected]).

such as depression and anxiety) [7]. For instance, recent studies have demonstrated anxiety- and depression-like behaviours in different animal models of inflammatory chronic diseases, such as systemic lupus erythematosus and rheumatoid arthritis respectively [8,9]. Curiously, in these studies, the sickness behaviour appeared before the beginning of the diseases suggesting that these changes occurred as a result of early inflammatory signals affecting the central nervous system but not as a response to organ damage or even pain. Behavioural alterations during some psychiatric dysfunctions are usually studied in animals using different tests, such as the elevated plus maze, light-dark box, elevated T maze, open field and emission of ultrasound vocalization recordings [10–15]. These assays are based, for example, on the conflict between the innate fear of rodents to open and wide areas versus their desire to explore new environments, while other behavioural strategies depend on the context offered by each additional behavioural test [13,16,17]. In first attempts studying emotionality in ill animals, many authors performed their experiments using only the few and classical parameters of ethological tools [18,19] or only one lipopolysaccharide (LPS) dose [20,21].

2011 The Authors Basic & Clinical Pharmacology & Toxicology 2011 Nordic Pharmacological Society

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Sickness behaviour could be compared to an acute ‘depression-like’ behaviour [22,23], and classical behavioural analysis are useful for assessing anxiety-like behaviours [11,13,15,24,25], then some data could be misinterpreted if only few ethological parameters and tools are used. LPS is a cell membrane component of gram-negative bacteria that induces global hyperalgesia and increases plasma corticosterone [3,26]. High plasma costicosterone increases risk assessment (complementary) behaviours with no effects on classical behavioural parameters [27,28], and the presence of more than one emotional variable (e.g. anxiety and hyperalgesia) decreases the sensitivity of the behavioural analysis if one simple ethological tool is used for a complete ethological event, leading to wrong data interpretation [15]. Considering that complementary ethological categories among the ethological tools are necessary to avoid superimposed and mismatched behavioural analysis, and different ethological tools allow the recording of different types of behaviours [13,29,30], we investigated the influence of LPSinduced immune-system alterations on the affective dimensions of Wistar rats expressing sickness behaviour in several ethological categories in elevated plus maze, dark-light box, open field and elevated T-maze tests (ETM), which have not yet been analysed, as well as on the emission of ultrasonic vocalizations (USV). It was hypothesized that rats under sickness behaviour will be more sensitized to some behavioural parameters than control animals submitted to different ethological tools that commonly evoke fear and anxiety in rodents.

Material and Methods Animals. One hundred and sixty-five nave male Wistar rats, weighing 180–200 g (7–8 weeks old), from the animal house of the University of S¼o Paulo were used. This age period was used because rats become sexually mature at age 6 weeks [31]. Six-week-old rats are more anxious and more exploratory than standard 16-week-old rats (300–320 g) [32], and emit 22 kHz USVs in many environmental conditions, for example, when facing the presence of an imminent danger of death [33,34], as well as during the exposure to stressful experiences [15,35]. The experiments were conducted between 9:00 AM and 5:00 PM. After arriving in the sectorial animal facility of the laboratory, the animals were housed in plastic cages under a 12-hr light:dark cycle (lights on at 7:00 AM) at 20 € 1C and maintained in groups of five per cage (40 · 33 · 18 cm). The animals had free access to water and food. The experiments reported herein were performed in compliance with the recommendations of the SBNeC (Brazilian Society of Neuroscience and Behaviour), and agree with the Ethic Principles of the Brazilian College of Animal Experimentation (COBEA), also based on the US National Institutes of Health Guide for The Care and Use of Laboratory Animals. The number of animal used was the minimum required to ensure reliability of the results, and every effort was made to minimize animal suffering. LPS administration. Lipopolyssacharide (Escherichia coli 0111:B4; Sigma, St. Louis, Missouri, USA; 1 mg ⁄ mL) previously dissolved in physiological saline and stored at )20C was sonicated for 15 min., diluted in sterile saline and administered by intraperitoneal injection (10, 50, 100, 200 and 500 lg ⁄ kg in a final volume of 1 mL) [36,37]. The control group received only sterile saline. After 3–4 hr postinjection, the animals were exposed to one of the tests used, as described below [1,36,38,39].

Ethological analysis procedures. The experiments were conducted between 9:00 AM and 5:00 PM. The rats were maintained grouped in their own cages during all procedure of LPS treatment in a room aside of the ethological test room. They were transported to and from the test room by a small acrylic box (30 · 18 · 13 cm) with sawdust provided from their original cages. The ethological analysis procedures were conducted as below:

Group 1 Group 2 Group 3

Nave rats fi elevated plus maze fi black-white box fi discarded Nave rats fi open field fi elevated T maze fi discarded Nave rats fi emission of ultrasonic vocalizations fi discarded

Experimental apparatus. The elevated plus-maze (EPM). The EPM test is the prototypic anxiety-model. It is based on the aversion of rodents to open spaces, consisting of a bidirectional test and one of the most used ethological tools for measuring and manipulating anxiety in rodents [13,40]. The EPM was made of wood and had two open arms (50 · 10 cm), perpendicular to two enclosed arms of the same size, with 50-cm-high walls, and elevated 50 cm from the floor. To avoid falls, a 3-mm wooden rim surrounded the open arms. The apparatus was located inside a room with a constant noise caused by an air conditioning system (50 dB). The behaviour of the animals was recorded using a video camera (Everfocus Electronics Corporation, Duarte, California, USA) positioned above the maze, allowing for the discrimination of all behaviours. The signal was relayed to a monitor in another room via an enclosed-circuit television camera. Luminosity at the level of the open arms of the plus-maze was 20 lux from a fluorescent light. The maze was cleaned thoroughly after each test using cotton embedded with 70% alcoholic solution. Procedure: Each animal was placed at the centre of the EPM facing one of the enclosed arms, and 5 min. of free exploration was recorded. A trained observer measured all the ethological parameters from a video camera. An arm entry or exit was counted when all four paws of the animal entered or exited the arm, respectively (see [13,41,42] for a complete description of the use of the EPM). The classic parameters analysed were the percentage of entries and time spent in the open-arms and the number of enclosed-arms entries. For complementary analysis, the number of head dipping, end-arm explorations (EAE), rearing, stretch attend positions (SAP), scanning, grooming and rat immobility (no movement observed during 6 sec.) were recorded [13]. The black-white box (BWB) test. In the BWB, a natural conflict situation occurs when the animal, initially in a dark, smaller area, is biased between a tendency to explore or avoid new environments. Such conflict is the result of an innate aversion of rodents to larger and brightly illuminated areas and the spontaneous exploratory behaviour, having been suggested as an index of anxiogenic activity (for a review see [11]). As the EPM, the BWB has been widely used with mice [18,22] and rats [43,44] for the evaluation of anxiety. All experiments were carried out in an acrylic box (light–dark test) composed of two different compartments: a light (45 · 40 · 21 cm; white walls with transparent roof) and a dark side (45 · 40 · 21 cm; opaque black walls and roof), with a 10 · 9 cm opening between the two compartments. The floor of both sides was made by stainless steel bars (2 mm thick) 15 mm apart. The test box was situated inside a wooden chamber with external acoustic and light isolation. An incandescent white bulb (20 lx) was placed inside the chamber, 50 cm above the experimental box, and a micro camera, located 50 cm above the light side, was linked to an external video camera system to monitor the behaviour of the animals during the sessions [45].


LIPOPOLYSACCHARIDE-INDUCED SICKNESS BEHAVIOUR Procedure: Immediately after the EPM test, each animal was placed at the centre of the dark compartment of the BWB facing the opening to the white compartment, and a 10-min. period of free exploration was analysed. A video camera was used to analyse the time each animal spent in either the black or white compartments, as well as the number of crossings, number of entries into the white compartment and the number of rearing movements. The open-field (OF) test. The OF test was first described by Hall in 1934 [10], and is currently a useful tool to assess behaviour. The procedure involves forced confrontation of a rodent with a situation in which there is direct exposure of the animal to a potentially dangerous situation in the environment [46]. The OF test is very sensitive in recording the effects of anxiolytic drugs (for a review, see [29]). Many authors have described the use of the OF test to assess LPSinduced behaviours in rats and mice [18,20,22,36,47,48]. The OF test consists of a large squared arena (45 · 45 · 34 cm) made of Plexiglas. The apparatus was situated inside an external sound attenuation chamber, located in an experimental room with a 50-dB background noise and a white light bulb (15 W) located 60 cm above the centre of the open field. The apparatus has four parallels bars (45 · 4.5 cm), each with 16 infrared sensors that detect the relative position of the animal inside the arena. Procedure: The animal was put in the centre of the OF, and 10 min. of free exploration was allowed. Specific software (Activity Monitor; Insight, Ribeir¼o Preto, S¼o Paulo, Brazil) was used to monitor the animal’s position throughout the 10 min. of free exploration, and a skilled observer analysed the number of centre and outer crossings, time spent in the centre, number of rearing movements, SAPs, grooming and immobility via a video camera. The elevated T-maze test. In 1991, Deakin and Graeff [12] aimed at developing a model to separately study conditioned fear related with generalised anxiety disorders, and unconditioned fear related to panic disorders. Hence, a new model was designed based on the EPM, named ETM [17,24], which blocked the entrance to one of the enclosed arms. The ETM results from shutting the entrance to one of the enclosed arms of the EPM, and has three arms of equal dimensions (50 · 10 cm) [24,49]. From this variation, two measures can be obtained: inhibitory avoidance and escape behaviour. For the inhibitory avoidance task, the rat is placed at the end of the remaining enclosed arm, and the latency to withdraw from this arm with the four paws is recorded in three successive trials made with a 30-sec. interval. Learning is indicated by increased withdrawal latency along trials. For the escape task, which initiates 30 sec. after the completion of the avoidance training, the rat is placed at the end of one of the open arms and the withdrawal latency from this arm is similarly recorded [24]. Procedure: For the inhibitory avoidance analysis, the animals were first placed at the distal end of the enclosed arm of the ETM facing the intersection of the arms. The time taken by the rat to leave this arm with its four paws was recorded (baseline latency). The same measurement was repeated in two subsequent trials (avoidance 1 and 2) at 30 sec. inter-trial intervals, during which animals were placed in a Plexiglas cage where they had been previously habituated. Following avoidance training (30 sec.), rats were placed at the end of one of the open arms and the latency to leave this arm with its four paws was recorded for three consecutive times (escape 1, 2 and 3) with 30 sec. inter-trial intervals (escape behaviour). A cut-off time of 300 sec. was established for the avoidance and escape latencies [24,49]. Ultrasonic vocalization recordings. Rats commonly evoke 22–24 kHz USVs (also known as alarm calls) among their defence repertoire in response to the presence of a potential dangerous stimulus. Therefore, when confronted with aversive situations such as the

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presence of a predator, during defeated or on anticipatory of punishment, rats evoke USVs, mainly at approximately 22 kHz [33,50,51]. This suggests that 22 kHz USVs reflect a negative state [52]. Corroborating this hypothesis, the administration of anxiolytic drugs decreases the emission of alarm calls during aversive encounters [53]. The apparatus used for recording and analysing USVs consisted of an acrylic testing box (25 · 15 · 12 cm). This experimental chamber was situated inside a large, padded, echo-free (sound-attenuated), ventilated box (60 · 40 · 45 cm) with a 28-W red light bulb on the top of the chamber. The USVs were recorded using an electric ultrasound microphone (Emkay FG-3629; Avisoft Bioacoustics, Berlin, Germany) sensitive to frequencies from 1 to 100 kHz with a flat frequency response. The microphone was connected via an Avisoft UltraSoundGate 116 USB audio device (Avisoft Bioacoustics) to a computer, where acoustic data were displayed in real time by Avisoft Recorder (version 2.7; Avisoft Bioacoustics) and recorded at a sampling rate of 214,285 Hz in 16-bit format. For acoustical analysis, the recordings were transferred to SASLab Pro (version 4.38; Avisoft Bioacoustics), and a fast Fourier transformation was automatically performed (512 faster Fourier transformation (FFT)-length, 100% frame, Hamming window, 75% time window overlap). Spectrograms were produced at a frequency resolution of 488 Hz and a time resolution of 0.512 ms. Call detection was provided by an automatic threshold-based algorithm (threshold, )10 dB; start ⁄ end threshold, )20 dB) and a hold-time mechanism (hold time, 20 ms). A lower cut-off frequency of 1 kHz was used for the analysis of the USV parameters. The number of calls emitted at each frequency served as the statistical unit in each subject [15]. Procedure: The sessions consisted of placing the animals individually inside the experimental chamber for 15 min. During each testing session, the microphone was placed through a hole in the middle of the roof of the chamber, 40 cm above the floor, to record the entire spectrum of USVs. A video camera linked to a television was used to monitor behaviour throughout the 15-min. recording period. The USVs obtained from this experiment were stored on a hard disk and subsequently transferred to Tables in the Microsoft Excel spreadsheet program (Microsoft, Redmond, WA, USA) for off-line analyses. Statistical analysis. Elevated plus maze test. The data obtained in the EPM test were analysed using one-way analysis of variance (oneway ANOVA) for each variable in the study, followed by the Dunnet post-hoc test (saline representing control data in comparison with each of the LPS-treated groups). Values of p < 0.05 were considered statistically significant. Black-white box test. Data were reported as mean + S.E.M. Data for the saline group scored for the baseline and treatment group was subjected to one-way ANOVA (saline · treatment). Dunnett’s post-hoc comparisons were carried out (saline as the control data) and a probability level of p < 0.05 was considered significant. Open field test. The data were submitted to a one-way ANOVA followed by Dunnet’s post-hoc test (saline representing control data in comparison with the LPS treatment groups), factor groups refer to saline i.p. injected animals, and the factor condition refers to treatment sessions. Bonferroni’s post-hoc test was performed when appropriated (row factor as the time period and column factor as treatment groups). A value of p < 0.05 was considered significant. Elevated T maze test. Data from the ETM were submitted to a two-way ANOVA for repeated measures (treatment as the independent factor and trials as the repeated measure). When appropriate, posthoc comparisons were performed by Bonferroni’s test (row factor as treatments and column factor as number of trials). A value of p < 0.05 was considered significant.



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Ultrasonic vocalizations recording. Data from ultrasound vocalization recordings were analysed by two-way ANOVA for repeated measures. The group factor refers to treatments, and the condition factor refers to frequencies recorded. If values of F were significant, Bonferroni’s post-hoc test was performed. Values of p < 0.05 were considered statistically significant.

Results Elevated plus-maze. The first experiment was delineated to evaluate the influence of LPS in the anxiety-related responses evoked by rats tested in the EPM. One-way ANOVA revealed a significant effect of the LPS treatment upon the frequency of entries to open arms (F4,35 = 9.59, p < 0.001), percentage of time spent in open arms (F4,35 = 5.87, p < 0.001) and number of entries in enclosed arms (F4,35 = 7.03; p < 0.001). Dunnett’s post-hoc test showed a significant decrease for LPS at 500 lg treatment for entries to open arms (q = 4.27, p < 0.001), time spent in open arms (q = 4.59, p < 0.001) and entries in enclosed arms (q = 3.52, p < 0.01) as shown in fig. 1. One-way ANOVA showed a significant effect in EAE (F4,35 = 8.68, p < 0.001), SAP (F4,35 = 6.65, p < 0.001), head dipping (F4,35 = 4.21, p < 0.01), rearing (F4,35 = 5.25, p < 0.01), grooming (F4,35 = 6.04, p < 0.001) and immobility response (F4,35 = 5.46, p < 0.01). Scanning frequencies did not show any significant difference (F4,35 = 1.49, p > 0.05). Dunnett’s post-hoc test showed a decrease in EAE frequencies for LPS at 50 (q = 3.10, p < 0.05), 200 (q = 3.31, p < 0.01) and 500 (q = 4.14, p < 0.001) lg ⁄ kg-treated groups. For head-dipping, post-hoc test showed decreased head-dipping frequencies for 50 (q = 3.35, p < 0.01), 200 (q = 3.22, p < 0.01) and 500 (q = 3.35, p < 0.01) lg ⁄ kg-treated groups. Decreased rearing and increased time of immobility occurred only at LPS 500 lg ⁄ kg (q = 3.67 and q = 4.19, p < 0.05 and p < 0.01, respectively). Ten, 50, 200 and 500 lg ⁄ kg caused a significant decrease on grooming behaviour (q = 3.24; p < 0.01; q = 3.74; p < 0.01; q = 3.49;

p < 0.05; and q = 4.49; p < 0.001 respectively) compared with the control group, as shown in fig. 2. Black-white box. One-way ANOVA showed that LPS treatment has significant effects upon white compartment entries (F4,35 = 6.62, p < 0.01) and crossings (F4,35 = 8.51, p < 0.001) and black compartment crossings (F4,35 = 9.61, p < 0.001). Dunnett’s post-hoc analyses showed that these parameters were decreased by LPS at 200 and at 500 lg ⁄ kg (4.21 £ q ‡ 5.08). One-way ANOVA demonstrated a significant effect for time spent in white compartments (F4,35 = 5.06; p < 0.01). Dunnett’s post-hoc analyses showed that these effects were caused by LPS at 50, 200 and 500 lg ⁄ kg (q = 2.79; 2.95; and 4.39, respectively). One-way ANOVA also demonstrated a significant effect on the number of rearing movements (F4,35 = 14.89, p < 0.001), and Dunnett’s post-hoc test demonstrated that this effect was decreased by LPS at 200 and at 500 lg ⁄ kg (q = 5.73 and 6.13, respectively) (these data are shown in fig. 3). The q values that resulted from the comparisons between the LPS at 10 lg ⁄ kg-treated group and the physiological saline-treated group (q = 1.31) were far from significant. For this reason, the dose of 10 lg ⁄ kg of LPS was not used in the OF and T-maze tests. Open field test. In this experiment, it was evaluated whether the LPS treatment would affect the motor and emotional behaviour of rats in the OF test. One-way ANOVA showed significant differences in the number of centre crossings (F4,35 = 3,47, p < 0.05), time spent in the centre of the arena (F4,35 = 3.19, p < 0.05), number of outer crossing (F4,35 = 12.41, p < 0.001), rearing (F4,35 = 10.78, p < 0.001), grooming (F4,35 = 25.07, p < 0.001), immobility (F4,35 = 48.55, p < 0.001) and SAP (F4,35 = 4.54, p < 0.05). Dunnett’s post-hoc test showed a significant decrease of crossings in the centre of the arena only

Fig. 1. Effects of lipopolysaccharide (LPS) treatment on the exploratory behaviour of rats submitted to the elevated plus-maze. Each animal was treated with either physiological saline or LPS at 10, 50, 200 or 500 lg ⁄ kg 3 hr before testing (n = 8 in each group). Data were presented as mean € S.E.M. *p < 0.01, **p < 0.001. 2011 The Authors Basic & Clinical Pharmacology & Toxicology 2011 Nordic Pharmacological Society

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Fig. 2. Effects of lipopolysaccharide (LPS) treatment on complementary ethological categories of rats submitted to the elevated plus-maze. Each animal was treated with either physiological saline or LPS at 10, 50, 200 or 500 lg ⁄ kg 3 hr before testing (n = 8 in each group). Data were presented as mean € S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001. EAE, end-arm explorations; SAP, stretch-attend position.

for LPS at higher doses (q = 2.65, p < 0.05). LPS at 200 lg ⁄ kg alone increased statistically the number of SAPs (q = 4.08, p < 0.05). LPS at 200 lg ⁄ kg and at 500 lg ⁄ kg showed a decrease in the number of outer crossings (q = 5.38, p < 0.001), and in the number of rearing movements (q = 5.38, p < 0.001). LPS at 50, 200 and 500 lg ⁄ kg-treated groups decreased grooming (q = 6.23, 8.02 and 12.03; p < 0.001) and time of immobility (q = 3.24, 8.69, 15.93; p < 0.001) compared with the control group (fig. 4). Elevated T maze test. Two-way ANOVA showed that all groups tested acquired inhibitory avoidance as measured by trial effect (F2,47 = 76.44, p < 0.001), and there was a significant treatment effect (F2,47 = 6.32, p < 0.001); however, no significant difference was seen for treatment X trial interaction effect (F2,47 = 0.96, p > 0.05). The LPS treatment has significant effects upon escapes trials (F2,40 = 3.86, p < 0.01) and on the treatment (F2,47 = 3.86, p < 0.01). However, there were no significant effects on interaction (F2,47 = 0.79, p > 0.05). Bonferroni’s post-hoc test demonstrated significant effects

for Avoidance 1 and Avoidance 2 between LPS-treated animals and the control group, and no significant difference on Escape 1 and Escape 2 trials compared with baseline (fig. 5). Ultrasound vocalization. Only 1 of 10 saline-treated rats emitted USVs and three of seven LPS 200 lg ⁄ kg emitted USVs. Two-way ANOVA demonstrated a significant effect of LPS treatment (F4,30 = 5.6, p < 0.001), USV frequencies emitted (F4,30 = 2.33, p < 0.05), and interactions between treatment and USV frequencies (F16,30 = 2.03, p < 0.05). Bonferroni’s post-hoc comparisons revealed that LPS treatment caused an increase in the number of USVs recorded at 22 and 24 kHz for LPS at 200 lg ⁄ kg-treated rats compared with the control group, as shown in fig. 6. Discussion Elevated plus maze. Previous studies postulate that LPS administration in mice and rats induces an anxiety-like behaviour on EPM


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Fig. 3. Effects of lipopolysaccharide (LPS) treatment on the exploratory behaviour of rats submitted to the black-white box. Each animal was treated with either physiological saline or LPS at 10, 50, 200 or 500 lg ⁄ kg 3 hr before testing (n = 8 in each group). Data were presented as mean € S.E.M. *p < 0.05, **p < 0.01, ***p < 0.001.

[18,20,22]. Nevertheless, these authors have only evaluated the open- and enclosed-arm entries and the time spent in each part of the maze. Other variables of the EPM besides time and number of entrances in the arms were not analysed, such as head-dipping, raising, EAE, SAP and grooming [13,54]. If only the classical parameters (open- and enclosedarm entries and time spent) are analysed, misleading data will occur due superposition among other ethological parameters [13,54]. For example, Rodgers et al. [28] reported that, in mice, anxiogenic compounds as mCPP and TFMPP enhance risk-assessment behaviour on the EPM without affecting the time spent in and entries to open arms. In addition, Bassi et al. [15] reported that hyperalgesic rats entered more in the open and enclosed arms of the EPM, data that could not be interpreted as an anxiolytic-like behaviour because there was an increase on emission of 22–24 kHz calls and a non-statistical difference in the time spent in the open arms compared to controls. In the present study, LPS at 10, 50 and 200 lg ⁄ kg did not induce an anxiogenic-like behaviour on the EPM test for the classical parameters (number of entrances and time spent on open arms). However, LPS at 200 lg ⁄ kg-treated rats showed fewer head-dipping movements and EAE, indicating an anxiogenic-like behaviour, despite the less SAP. LPS at 500 lg ⁄ kg induced a motor deficit (as seen for a significant reduction

on enclosed-arm entries compared to the saline-treated group) probably due to its toxic effects on the nervous system. Curiously, grooming behaviour significantly decreased in all LPS-treated groups; data corroborated by earlier studies in which animals evoking LPS-induced sickness behaviour decreased activities that lead to heat loss (grooming) and favour others that increase heat production (shivering) or minimize heat loss (piloerection) [6,55,56]. Hence, grooming is a very sensitive parameter to assess the initial signals of sickness behaviour induced by LPS in rats, as seen for the mouse [57]. Black-white box. Curiously, no statistically significant difference in the number of entries in the illuminated compartment and quadrants crossed in the white and dark compartments was recorded, although Lacosta et al. [18] found less time spent on the illuminated compartment at doses higher than LPS at 1 lg ⁄ kg in mice. This could be explained by (i) rats begin the BWB test in the dark compartment and, due to sickness, prefer to stay in the dark compartment instead of going out to explore a potentially dangerous place like the white compartment, as darker places are innately safer than illuminated ones. For this reason, the BWB test does not offer a higher challenging ⁄ aversive environment in ill rats because the rat has the



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Fig. 4. Effects of lipopolysaccharide (LPS) treatment on the exploratory behaviour evoked in the open-field test. Each animal was treated with either physiological saline or LPS at 50, 200 or 500 lg ⁄ kg 3 hr before testing (n = 8 in each group). Data were presented as mean € S.E.M. *p < 0.05; **p < 0.001, ***p < 0.001. SAP, stretch-attend position.

choice to go out from the dark compartment or stay in it (different from EPM or OF tests, in which the rat is directly exposed to the environment); (ii) There is a superposition of behavioural factors in the BWB test (i.e. locomotion in the dark compartment is highly correlated with both the anxiety and locomotor factor together) [58], and as LPS has effects on various emotional variables (e.g. hyperalgesia and anxiety), some data could be misinterpreted. Despite being a reliable and useful test to assess anxiogenic- or anxiolytic-like behaviour induced by drug treatments [11], due to the difficulties listed above as to assessing the behaviour analysis of ill animals, it is proposed that the BWB test should be used with other ethological tools (such as EPM or OF). Open field. No differences were found for crossings in the centre and outer parts of the open field, demonstrating that LPS treatments up to 200 lg ⁄ kg do not induce motor deficits. The same was observed for time spent in the centre of the arena, indicating a non-anxiogenic-like behaviour. Besides these

data indicating non-anxiogenic-like behaviours, complementary ethological analysis showed an inverse bias. As seen for Labyrinth in Cross Elevated (LCE) data, grooming and immobility responses were significantly decreased compared with control, demonstrating that these parameters are very sensitive to the low doses of LPS used in the present study. The frequency of SAPs was significantly high only for the LPS at 200 lg ⁄ kg, probably due to the high anxiety state produced, reaching a peak of conflict in the rats’ emotional drive. Hence, the animal has to choose between ‘rest-andrecover’ or ‘move-and-search’ for a place to rest in a moment when both options are immediately necessary, but not at the same time. Elevated T-maze test. Our findings have shown that the treatment with LPS did not induce significant changes in the intra and inter treatment groups (fig. 5). These results demonstrate that low doses of LPS do not induce motor deficits and ⁄ or learning impairments in the ETM test because (i) all groups tested acquired inhibitory avoidance, indicating that there is no


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Fig. 5. Effects of lipopolysaccharide (LPS) treatment on T maze test. Each animal was treated with either physiological saline or LPS at 10, 50, 200 or 500 lg ⁄ kg 3 hr before testing (n = 10 in each group). Data were presented as mean € S.E.M. *p < 0.05, **p < 0.001.

impairment on learning and memory (see [24] for review); (ii) there is a statistically significant difference in the treatment effect in all avoidance trials, indicating that there is no motor deficit and ⁄ or the production of a panic-like behaviour observed at the ETM test. Despite the fact that other authors have found impairments on learning and memory in acute and chronic LPS injection [59,60], ETM data corroborate findings from the study of Sanderson et al. [21] in which LPS-treated mice do not have impairments on continuous reinforcement, as at the end of each avoidance and escape trial, the mouse is put back to its cage, and this condition could be assessed as a continuous reward sensitive to doses of LPS used in the present work. Ultrasound vocalization. Ultrasonic vocalizations at frequencies of 20–24 kHz occur only when there is a moderate stressful condition (not as

intense as proximal danger) as a potentially dangerous situation [33,34], demonstrating that 22 kHz USVs are only emitted when associated with anxiety and not fear [53,61]. Bassi et al. [15] reported that hyperalgesic rats emit USVs at 22 and 24 kHz (anxiogenic-like behaviour); however, they explore more the open-arms at LCE (anxiolytic-like behaviour), suggesting the involvement of an emotional-affective state during this model of central pain. Rats treated with LPS at 200 lg ⁄ kg emitted 22 and 24 kHz USVs. Lower doses did not induce an aversive state that could be measured by USVs. The same is seen for the behavioural data on EPM, OF and ETM tests where there is no statistical difference for LPS at 10 and 50 lg ⁄ kg compared with the control group. As USVs are emitted only in the presence of potential or distal threat, LPS at 200 lg ⁄ kg could be a ‘boundary’ dose producing a state of conflict inside the cost and benefit bias, dividing the rat between exploring a new environment (it feels the sickness, but can still search for a better place to rest) and remaining in its current place (the sickness becomes too strong for it to try to search for a better place to rest), generating anxiety and, then, USVs. However, caution must be exercised as three of seven rats emitted USVs. Alarm calls are related to risk assessment behaviours as a sentinel activity, and alpha males are more likely to proceed with these parameters of risk assessment behaviours than subordinates, as dominant animals are more likely to be the first to detect potential threats [62,63]. The occurrence of emission of USVs in three of seven rats could be due to the presence of alpha male rats in the cages, and further studies on alpha males’ sickness behaviour should be performed. General According to our data, LPS produces a dose-dependent conflict inside the rats’ emotional drive, reaching an emotional peak at 200 lg ⁄ kg of LPS, generating anxiety-like behaviours. Despite the production of anxiogenic-like behaviours, some ethological parameters (e.g. time spent and number of entries in aversive areas) are not as sensitive as other complementary parameters (e.g. grooming and SAPs). The use of complementary ethological parameters is necessary due to

Fig. 6. Effects of lipopolysaccharide (LPS) treatment on the number of vocalizations emitted at frequencies of 18–26 kHz during 15 min. of a novelty exposure (n = 7 in each group). Data are expressed as mean € S.E.M. *p < 0.01, **p < 0.001. 2011 The Authors Basic & Clinical Pharmacology & Toxicology 2011 Nordic Pharmacological Society


the sickness behaviour itself, as the rat, trying to resolve its immunological threat, has to choose between ‘stay-and-rest’ and ‘move-and-search’ for a better and safer place, then rest, as the exploratory behaviour optimizes the rat’s security [64]. This biased behaviour (known as cost and benefit behaviour, according to Aubert et al. [65]) is necessary when a given illness is present. However, it can generate some unusual behaviours on ethological tools. Although behavioural tools have been validated in neurobiological research, there are several points concerning the real validity of the ethological parameters analysed on these tools in ill rodents: 1 In the presence of illness, the animal needs to analyse which from two evaluative priorities are possible at the moment, depending on the severity of the illness and the potentiality of threats of the current place: rest and recover or move and search for a better place to rest to assure its security [64]. LPS at 200 lg ⁄ kg induces such a high conflict inside the cost and benefit bias that both these options are immediately necessary, but only one is evoked, generating anxiety-like behaviours that are better observed analysing complementary ethological analysis, as SAPs, groomings and emission of USVs. 2 The increased anxiety observed in some experiments performed in the present work could be a way that sick animals search for a favourable environmental temperature where it would be possible to develop an efficient febrile response. When animals experience fever, they do not seek out cooler environmental temperatures to counteract the changes in equilibrium; rather, they facilitate their fever development by moving to a warmer environment if given the opportunity, increasing their body temperature [56,66]. 3 Different ethological tools have different sensibilities and are complementary to behavioural analysis when an illness is present. Grooming, SAPs and time of immobility are useful recordings to analyse the initial signs of sickness-behaviour; their sensibilities, however, vary depending on which ethological tool was used. In conclusion, although there are several methodological differences between the current study and those from cited reports (dose of LPS, route of administration, animal species, strain of rats, period of behaviour evaluation), the principal key contribution of the present study was using several tests (as well as multi-parameters evaluated) and different doses of LPS to investigate sickness behaviour after activation of the immune system during a given infection. In fact, when the cost and benefit bias is working under the presence of an immunological threat, individual ethological tools, single ethological parameters or classical ethological analysis are not enough to investigate the changes of the behavioural performances (e.g. anxiolytic or anxiogenic-like behaviour). Acknowledgements This study received financial support from the National Council for Scientific and Technological Development (CNPq) (133713 ⁄ 2010-5; 483763 ⁄ 2010-1) and by The State of S¼o Paulo Research Foundation (FAPESP) (Grants

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