Low-dose lipopolysaccharide (LPS) inhibits aggressive and augments depressive behaviours in a chronic mild stress model in mice

Studies were performed using 3.5-month-old male C57BL/6J mice; 3.5-month-old male CD1 mice were used as intruders for social stress, and 2–5-month-old Wistar rats were used for predator stress. All animals were supplied by the Gulbenkian Institute of Science, Oeiras, Portugal. C57BL/6J mice were housed individually for 14 days before the start of the experiments; CD1 mice and rats were housed in groups of five before the experiment and then individually thereafter. All animals were kept under a reversed 12-h light-dark cycle (lights on: 21:00 h) with food and water ad libitum, under controlled laboratory conditions (22 ± 1 °C, 55 % humidity). A minimum of six animals were used in all the behavioural experiments, and a minimum of five animals per group were used in the molecular biology experiments. All studies were carried out in accordance with the European Communities Council Directive for the care and use of laboratory animals upon approval by the ethics committee of Maastricht University for animal research (CPV, DEC-UM 2009-109) and permission 0421/000/000/2013 issued by the General Directory of Ethical Committee of the New University of Lisbon.

The study design is outlined in the schematic in Fig. 1, and the animals were randomly assigned to test groups. The behavioural responses were studied in three separate cohorts. The first cohort was used to establish a subthreshold working dose of LPS that would not induce altered behaviour in the elevated O-maze and resident-intruder test in naïve mice, so that we would be able to explore the interaction of stress and LPS (Fig. 1a). A lack of immediate behavioural effects with 0.1 mg/kg LPS and a suppression of social and locomotor behaviour with 0.5 mg/kg LPS has been previously reported [38, 39]. The animals were subjected to either an acute LPS challenge, 0.5 or 0.1 mg/kg, or treated with vehicle alone and tested 24 h post-injection or, in order to explore the delayed behavioural response to LPS [40], 48 h post-injection, in separate subgroups. The second cohort (Fig. 1b) was subjected to chronic mild stress or no stress (minimal handling). Separate subgroups of mice were subjected to the novel cage, O-maze, forced swim test and a resident-intruder test or sucrose test and tail suspension or were killed for analysis of central and peripheral changes in gene expression and blood corticosterone. The stressed and non-stressed animals were then subsequently treated with either LPS at 0.1 mg/kg or vehicle 24 h prior to testing. The number of animals per group is indicated in the figure legends. A third cohort, duplicating the first, was employed to establish the effects of 0.1 and 0.5 mg/kg LPS on behaviour in the open field at 24 or 48 h post-injection using TruScan apparatus. Resting time and average speed were recorded.

In the second (stressed) cohort, the animals underwent a previously validated 10-day chronic stress procedure [41]. The stress procedure consisted of rat exposure between the hours of 18:00 and 09:00 h (light phase of dark-light cycle) concomitant with a combination of restraint stress for 2 h and tail suspension for 40 min, applied in a semi-random manner with an inter-session interval of at least 4 h [29]. Briefly, during predation stress, mice were introduced to a transparent glass cylinder (15 cm high × ⌀ 8 cm) and placed into the rat cage for 15 h as described and validated previously [39, 40, 42]. For a restraint stress, mice were placed into a small container (50-ml Falcon tube) with space for breathing but no space for free movement, for 2 h, and for tail suspension, they were hung by their tails during the dark phase of the animals’ light cycle, as described previously [29]. Body weight, sucrose preference and previously defined social behaviour parameters were determined 1 week before the chronic stress procedure [38, 39, 41]. A further cohort of animals were killed, and tissue was collected for messenger RNA (mRNA) analysis.

Behaviour was tested after 24 h because at this point, LPS-induced behavioural changes in stress-naïve mice had returned to baseline for the low-dose LPS challenge (Fig. 2). All behavioural testing was carried out during the dark phase of the animals’ light-dark cycle. Tests were recorded on film and analysis carried out post hoc and blinded, unless otherwise stated in the text.

The apparatus (Technosmart, Rome, Italy) consisted of a circular path (runway width 5.5 cm, diameter 46 cm) that was placed 45 cm above the floor. Two opposing arms were protected by walls (closed area, height 10 cm), and the illumination strength was 25 lx. The apparatus was placed on a dark surface in order to maintain control over lighting conditions during testing. Mice were placed in one of the closed-arm area of the apparatus. Behaviour was assessed using previously validated parameters during a 5-min observation period [36, 43]. The latency of the first exit to the ‘open’ compartments of the maze, the number of exits to the open arms and the percentage of time spent in the open arms were recorded [28].

The resident-intruder test procedure was adapted from previously described protocols [36, 44]. In this paradigm, the C57BL/6J mice were placed individually in an observation cage (30 × 60 × 30 cm) for 30 min to acclimatize. Thereafter, a previously group-housed male CD1 mouse was introduced as an intruder to the same cage and left with the resident mouse for 8 min. During the observation period, both resident mice were scored for the latency and duration of social interaction (nose-nose interactions) as well as the latency to attack, the number of attacks and the duration of crawl over behaviour. Crawl over time was recorded when the resident mouse positioned itself on top of the intruder mouse and often was usually associated with paw pressure on the head of the intruder [45]. During these periods, the intruder mouse showed little, if any, resistance to this mounting and displayed a submission by closing the eyes and not moving.

The novel cage test was performed to assess exploration in a new environment as described elsewhere [44, 46]. Mice were introduced into a standard plastic cage (21 × 21 × 15 cm) filled with fresh sawdust. The number of exploratory rears was counted under red light during a 5-min period.

Mice were placed into TruScan activity boxes (26 × 26 × 39 cm; Coulbourn) for 10 min. The boxes were evenly illuminated with white light at 25 lx. Horizontal movements (speed) and resting time were scored automatically by red beam cells using TruScan software (Coulbourn), as described elsewhere [47]. Mean speed and total duration of resting behaviour, defined by a lack of crossing of more than three beams over 60 s, were evaluated.

Mice were given 24 h of free choice between two bottles of either 1 % sucrose or standard drinking water, as described elsewhere [44]. The bottles were weighed before and after conducting the sucrose preference and consumption calculated accordingly. The beginning of the test started with the onset of the dark (active) phase of the animals’ cycle. To prevent the possible effects of side preference in drinking behaviour, the position of the bottles in the cage was switched at 12 h, halfway through testing. No prior food or water deprivation was applied before the test. Other conditions of the test were applied as described elsewhere [48]. Both baseline and post-stress paradigm sucrose preference tests utilized a 1 % sucrose solution. Percentage preference for sucrose was calculated at the end of the test using the following formula: Sucrose Preference = Volume_{Sucrose solution}/(Volume_{Sucrose solution} + Volume_Water) × 100.

The protocol used in this study was adapted from a previously validated procedure [43, 47]. Mice were suspended by the tails to a rod 50 cm above the floor using adhesive tape. Animals were left on this apparatus for 6 min in a dark room. The apparatus was illuminated with a single spotlight (5 lx at animal height). The trials were recorded by a video camera positioned directly in front of the mice while the experimenter observed the session from a distance in a dark area of the experimental room. The total duration of this behaviour, a putative measure of ‘behavioural despair’, were scored using protocols that were previously validated with automated tools [43, 47]. In accordance with the commonly accepted criteria, immobility was defined as the absence of any movements of the animals’ head and body.

The protocol used for the Porsolt forced swim test was modified to prevent behavioural artefacts caused by stress-induced hyperlocomotion [41]. Mice were placed into a transparent pool (20 × 35 × 15 cm) lit with red light and filled with warm water (30 °C, to a depth of 9.5 cm) for 2 min. Floating behaviour, commonly interpreted as ‘behavioural despair’ in mice [49], was defined as the absence of directed movements of the animals’ head and body. Floating was measured by visual observation which was validated previously in comparison to automated scoring with specialized software [43, 47]. The latency to begin floating was scored as the time between introduction of the animal into the pool and the first moment of complete immobility of the entire body for a duration of >3 s. The total time spent floating was scored for the entire duration of the test using video footage.

Mice were terminally anaesthetized with an intraperitoneal injection of sodium pentobarbitone. The left ventricle was perfused in situ with 10 ml ice-cold saline; the brain and liver of each mouse were dissected. The pre-frontal cortex and striatum were collected by placing the brain, on its ventral side, on a metal plate. The olfactory bulbs were removed, and a 1-mm-thick coronal section of the most anterior cortical tissue was collected. The left and right cortical sections were further dissected to take the medial pre-frontal cortex while avoiding the motor cortex and anterior forceps of the corpus callosum. The left and right striatum was collected by generating a coronal section at bregma 0 and bregma +1. The cortex and corpus callosum were carefully removed and the left and right striatum collected. The hippocampus was removed by generating a coronal slice at bregma −1 and bregma −3. The overlying cortex was carefully removed, and the left and right hippocampus was removed. The dorsal raphe nucleus was collected from a 1-mm-thick section from bregma −4 to bregma −5 by collecting a diamond-shaped piece of tissue under the fourth ventricle. Small segments of liver tissue were isolated and stored at −80 °C.

RNA extraction was performed as previously described from specifically microdissected snap-frozen brain regions and liver biopsies using the RNeasy Mini Kit (Qiagen, UK) [29]. The serotonergic genes 5-HT_2A and SERT were selected for analysis based on prior observation that their expression levels change in response to systemic inflammation [35] and chronic stress paradigms [29]. Primers were custom designed and synthesized, taking basic secondary structure into account during the design process (PrimerDesign Ltd., UK). All primers were validated against a standard complementary DNA (cDNA) biobank (PrimerDesign) to ensure adequate amplification and single melt-curve products. Five hundred nanograms of whole mRNA was converted to cDNA using random primers supplied with the High Capacity Reverse Transcription cDNA Kit (Applied Biosystems, UK), and final samples were diluted to 5 ng/μl. Standard curves were generated from a mixed cohort of cDNA, and analysis was performed using SYBR green (PrimerDesign Ltd., Southampton, UK) and a LightCycler 480 (Roche, UK). Cycle conditions were 8-min enzyme activation (95 °C), 15-s denaturation (95 °C), followed by 40 cycles of denaturation (15 s at 95 °C) and data collection (45 s at 60 °C). Total cDNA was used to enable normalization to expression of the housekeeping gene glyceraldehyde-3-phosphate (GAPDH) using the Pfaffl method [50]. Details of primers are listed in (Additional file 1: Table S1). Results are expressed as relative-fold compared to control animals.

Blood was taken via cardiac puncture immediately before perfusion and stored in heparinized vials prior to centrifugation (10 k rpm, 10 min, 4 °C), and plasma was removed and immediately stored at −20 °C. Corticosterone was analysed using HPLC coupled with mass spectroscopy, based on the principles from Marwah et al. [51]. Briefly, plasma samples were diluted 1:1 with distilled water and applied to 2 ng of internal standard (5-pregnen-3b-ol-20-one-16a-carbonitrile). Diethyl ether was added to separate organic compounds into a water-free layer. Samples were vortexed and centrifuged (5 min, 1500×g) to fully separate solvent and aqueous layers. Solvent layers were removed and dried using a heated vacuum centrifuge. Organic residues were dissolved in 100 μl eluent A (see below) and applied to columns. Separations were carried out using a Waters 2695 separations module (Waters, Elstree, UK) with an ACE C18 3 μm, 100 × 2.1 mm column (Hichrom, Reading) maintained at 35 °C. The specific eluents were 2 mM acetic acid (A) and acetonitrile (B), with a linear gradient of 30–75 % of B over 8 min. The flow rate was 0.25 ml/min. The eluent was monitored using a Waters Micromass ZQ mass detector using positive electrospray ionization in single ion mode and Waters Empower 2 software. Mass spectrometry was performed under the following conditions: capillary voltage, 2.7 kV; source temperature, 125 °C; desolvation temperature, 475 °C; desolvation gas flow, 575 l/h; and cone gas flow, 80 l/h. Corticosterone was monitored at m/z 347.1 (M + H), cone voltage 20 V. The internal standard CA4 was monitored at m/z 302.1, cone voltage 35 V.

Naive mice were challenged with either 0.1 or 0.5 mg/kg LPS to determine the behavioural effects of each dose 24 or 48 h thereafter [35]. In the elevated O-maze, there was a main effect of LPS dose, but not time post-challenge, on the overall latency to exit to the open arms (Fig. 2a; two-way ANOVA dose p < 0.01 F _2,34 = 7.89; time post-challenge p = 0.06 F _1,34 = 0.55; dose:time post-challenge p = 0.78 F _2,34 = 0.25). Post hoc analysis demonstrates that at 24 h post-challenge, 0.5 mg/kg LPS animals have a significantly increased latency to exit to the open arms compared to controls (Fig. 2a; Bonferroni p < 0.05). The total number of exits from the closed area of the O-maze was not affected by either LPS dose or time (Fig. 2b; two-way ANOVA dose p = 0.93 F _2,34 = 0.07; time post-challenge p = 0.28 F _1,34 = 1.18; dose:time post-challenge p = 0.07 F _2,34 = 2.90). There was a non-significant tendency for the higher dose of LPS to affect the total number of exits at 24 h post-challenge; this was not significant (Fig. 2b; p = 0.07). The proportion of time spent in the open arms of the elevated O-maze was also unaffected by treatment (Fig. 2c; two-way ANOVA dose p = 0.60 F _2,34 = 0.5.14; time post-challenge p = 0.25 F _1,34 = 1.343; dose:time post-challenge p = 0.63 F _2,34 = 0.455).

In the resident-intruder test, there was no overall effect of dose or time on social interaction (Fig. 2d; two-way ANOVA dose p = 0.31 F _2,34 = 1.18; time post-challenge p = 0.16 F _1,34 = 2.06; dose:time post-challenge p = 0.13 F _2,34 = 2.14), but post hoc testing showed a significantly increased latency of social interaction in animals receiving 0.5 mg/kg and tested at 24 h when compared to vehicle-treated controls (Fig. 2d; Bonferroni p < 0.05). The total time spent interacting with the intruder was also not affected by LPS at either 24 or 48 h (Fig. 2e; two-way ANOVA dose p = 0.07 F _2,34 = 2.89; time post-challenge p = 0.26 F _1,34 = 1.27; dose:time post-challenge p = 0.57 F _2,34 = 0.57). Using open field and novel cage tests, we observed no effect of either dose on locomotor activity on mean speed (Fig. 2f; two-way ANOVA dose p = 0.77 F _2,34 = 0.257; time post-challenge p = 0.47 F _1,34 = 0.53; dose:time post-challenge p = 0.90 F _2,34 = 0.10) or on resting time or the number of rears (Additional file 1: Figure S1).

Since the lower dose of LPS did not affect the behaviour of naïve mice at 24 h, it was used for the chronic stress study. We first assessed body weight (experimental groups were balanced at baseline) and showed that stress reduced body weight as expected (Additional file 1: Figure S2). Low-dose LPS (0.1 mg/kg) given 24 h prior to testing does not significantly alter parameters of sucrose preference test (Additional file 1: Figure S2) [41]. However, it was hypothesized that if stress increases pro-inflammatory cytokines, stimulation of the system with an inflammatory challenge may significantly alter this behaviour. All animals showed a preference of >65 % for a 1 % sucrose solution prior to testing and a consistent sucrose and water intake (Additional file 1: Figure S2 C–E). Control animals, and animals injected 24 h prior to testing with 0.1 mg/kg LPS, maintained a sucrose preference of >65 % and were not significantly different from each other (Fig. 3a). After 10 days of chronic stress, there was a significant main effect of stress on sucrose consumption but not of LPS, and there was no interaction between stress and LPS (Fig. 3a; two-way ANOVA; stress p < 0.001 F _1,54 = 16.62; LPS p = 0.28 F _1,54 = 1.182; stress:LPS p = 0.41 F _1,54 = 0.689). Consistent with the main effects, post hoc tests showed that after 10 days of chronic stress and a single i.p. dose of saline, animals displayed a significant decrease (<65 %) preference for a sucrose solution (Fig. 3a; Bonferroni post hoc p < 0.05). Post hoc analysis revealed that animals undergoing 10 days of chronic stress combined with a single i.p. dose of LPS (0.1 mg/kg) 24 h prior to testing also showed a decrease in sucrose preference compared to controls (p < 0.001). While there appears to be a decrease in sucrose preference for stressed animals receiving LPS compared to those without LPS, this difference is not significant (p = 0.192). Since sample sizes are unequal across groups, post hoc tests should be considered with caution. Total sucrose intake somewhat reflects this, here showing a main effect of both stress and LPS but no interaction (Fig. 3b; two-way ANOVA; stress p < 0.001 F _1,54 = 25.36; LPS p < 0.01 F _1,54 = 10.27; stress:LPS p = 0.28 F _1,54 = 1.15). Post hoc testing revealed a significant decrease in sucrose consumption in stressed animals when compared to non-stressed controls (Fig. 3b; Bonferroni post hoc p < 0.01). Post hoc testing also reveals a decreased sucrose intake in stressed mice treated with LPS when compared to those treated with vehicle, suggesting a higher degree of anhedonia in these animals (Fig. 3b; Bonferroni post hoc p < 0.0001). Finally, stressed mice show some degree of hyperdipsia, with water consumption being affected by stress, but not by any other factors (Fig. 3c; two-way ANOVA; stress p < 0.05 F _1,54 = 5.38; LPS p = 0.31 F _1,54 = 1.04; stress:LPS p = 0.35 F _1,54 = 0.85).

Tail suspension is used to measure helpless behaviour, which is associated with a depressive-like state in mice [52, 53]. Analysis showed a significant effect of stress on the total time spent immobile in the test (Fig. 3d; two-way ANOVA; stress p < 0.001 F _1,40 = 24.89; LPS p = 0.16 F _1,40 = 1.97; stress:LPS p = 0.35 F _1,40 = 0.89) but no other main effects and no interactions. Post hoc testing showed that all stressed animals, irrespective of treatment, were immobile for significantly longer periods than controls (Fig. 3d; Bonferroni post hoc; stress p < 0.05; stress and LPS p < 0.0001).

In the forced swim test, another test for helpless behaviour, control and LPS-alone animals showed similar values in both the latency to float and total time spent floating. Analysis showed that both stress and LPS had a main effect on the latency to floating behaviour but that there was no interaction between factors and therefore, all results should be considered with caution (Fig. 3e; two-way ANOVA; stress p < 0.001 F _1,43 = 21.46; LPS p < 0.05 F _1,43 = 5.495; stress:LPS p = 0.19 F _1,43 = 1.76). In post hoc tests, chronic stress significantly decreased the latency to float compared to controls (Fig. 3e; Bonferroni post hoc; p < 0.05), as did chronic stress combined with LPS (p < 0.001). Using multiple pairwise comparisons (Bonferroni post hoc), LPS combined with stress is significantly different from stress alone (p < 0.05); however, as there is no interaction between these factors, this result should be interpreted with caution.

There was a main effect of stress, not LPS, on the total duration of floating behaviour, and there was no interaction between factors (two-way ANOVA; stress F _1,43 = 9.654, p < 0.01; LPS F _1,43 = 1.922, p = 0.17; stress: LPS F _1,43 = 0.99, p = 0.32; Fig. 3f). In post hoc tests, the combination of chronic stress and LPS significantly increased the total time spent floating compared to the control group in the forced swim test (Fig. 3f; Bonferroni post hoc; p < 0.05). While this suggests that LPS combined with stress significantly affects floating behaviour in the forced swim test, the lack of interaction makes these results difficult to interpret.

In the O-maze, stress and LPS significantly affected the latency to exit into the open arms independently and through interaction (Fig. 4a; two-way ANOVA; stress F _1,39 = 4.41, p < 0.05; LPS F _1,39 = 9.84, p < 0.01; stress: LPS F _1,39 = 4.87, p < 0.05). In stressed animals, LPS reversed the stress-induced decrease in the latency to exit to the open arms, ameliorating this parameter which is an assumed sign of impulsivity (Fig. 4a; Bonferroni post hoc; p < 0.001). Similarly, the total number of exits to the open arms of the maze was significantly affected by stress and LPS independently and in terms of interaction (Fig. 4b; two-way ANOVA; stress F _1,39 = 4.55, p < 0.05; LPS F _1,39 = 4.58, p < 0.05; stress: LPS F _1,39 = 5.01, p < 0.05). Post hoc testing also demonstrated that the presence of LPS significantly diminished the number of exits to the open arms of the O-maze in stressed animals, thus abolishing the impulsivity/hyperlocomotion in these mice (Fig. 4b; Bonferroni post hoc; p < 0.01).

The resident-intruder test can be used to assess both social and aggressive behaviours [54]. Resident-intruder testing was performed on all animals before undertaking the chronic stress and/or dosing procedure, and all groups were shown to be balanced at baseline (Additional file 1: Figure S3). We found that the duration of social exploration was significantly decreased by LPS and there was also an interaction between stress and LPS (Fig. 4d; two-way ANOVA; stress F _1,35 = 1.17, p = 0.28; LPS F _1,35 = 18.81, p < 0.0001; stress: LPS F _1,35 = 10.15, p < 0.01). Post hoc testing found that stressed animals challenged with LPS interacted with their intruders for significantly less time than those not challenged with LPS (Fig. 4d; Bonferroni post hoc; p < 0.0001).

When aggressive behaviour was examined, we found that the 10 days of chronic stress increased crawl over behaviour and the number of attacks and this was significantly inhibited by LPS treatment (Fig. 4). LPS treatment affected the number of the total number of attacks compared to control animals in an independent fashion, and analysis revealed a further interaction with stress (Fig. 4e; two-way ANOVA; stress F _1,35 = 1.89, p = 0.17; LPS F _1,35 = 7.16, p < 0.01; stress: LPS F _1,35 = 4.39, p < 0.05). LPS significantly reduced the stress-induced rise in the number of attacks analysed with post hoc testing (Fig. 4e; Bonferroni post hoc; p < 0.001). Crawl over behaviour, a measure of a dominant-like interaction [45], was found to be increased in the animals exposed to stress, and this was once more significantly reduced in the stressed animals that were challenged with LPS (Fig. 4f; RM-ANOVA; stress/LPS treatment F _3,35 = 3.59, p < 0.05; before/after F _1,35 = 2.85, p = 0.1; stress/LPS: before/after F _3,35 = 6.78, p < 0.01). Stressed animals showed an increased amount of crawl over behaviour when compared to controls (Fig. 4f; Bonferroni post hoc p < 0.01). Furthermore, stressed animals treated with LPS showed significantly less crawl over behaviour when compared to animals that had undergone stress alone (Fig. 4f; Bonferroni post hoc p < 0.0001).

Behaviour in a novel cage was also examined in all animals. Those animals undergoing 10 days of chronic stress followed by either an LPS challenge or a vehicle challenge showed no significant change in rearing behaviour in this test (two-way ANOVA; stress F _1,32 = 1.29, p = 0.26; LPS F _1,32 = 0.01, p = 0.9; stress: LPS F _1,32 = 0.17, p = 0.67). This suggests that the changes observed in behavioural tests for aggression or social interaction above were unlikely to be a result of confounding alterations in general locomotor activity (Additional file 1: Figure S2).

Systemic inflammation has been shown to increase circulating cytokines, and stress is known to decrease pro-inflammatory cytokine expression via glucocorticoid induction [55]. As mentioned above, the levels of pro-inflammatory cytokines present 24 h after injection of 0.1 mg/kg endotoxin should be relatively low [56].

In this experiment, both LPS and stress had a significant effect on TNFα gene expression; furthermore, there was a significant interaction between the factors (Fig. 5a; two-way ANOVA; stress p < 0.01 F _1,18 = 9.259; LPS p < 0.001 F _1,18 = 22.07; stress:LPS p < 0.05 F _1,18 = 6.472). At 24 h after LPS injection in non-stressed mice, the fivefold increase in hepatic Tnf compared to vehicle-treated controls was statistically significant (Fig. 5a; Bonferroni post hoc; p < 0.001). Chronic stress and LPS, combined, appeared to the levels of TNFα mRNA compared to vehicle controls, but this change was not significant (Fig. 5a).

IL-1β mRNA expression was affected by stress and LPS, but there was no significant interaction between the two factors (Fig. 5b; two-way ANOVA; stress p < 0.001 F _1,18 = 15.56; LPS p < 0.01 F _1,18 = 12.61; stress:LPS p = 0.07 F _1,18 = 3.711). IL-1β mRNA expression was slightly higher in animals treated with stress and LPS alone but in neither case are they significantly different from non-stressed, vehicle-treated controls (Fig. 5b). The combination of 10 days of chronic stress and a low-dose LPS challenge resulted in a significant sixfold increase in hepatic IL-1β mRNA expression (Fig. 5b; Bonferroni post hoc; p < 0.001).

Control animals had an average of 10-nM baseline corticosterone (Fig. 5c). Both stress and LPS had a significant effect on corticosterone levels, and there was a significant interaction between these factors (Fig. 5c; two-way ANOVA; stress p < 0.05 F _1,25 = 4.605; LPS p < 0.01 F _1,25 = 9.355; stress:LPS p < 0.05 F _1,25 = 6.659). More specifically, analysis showed that administration of 0.1 mg/kg LPS significantly increased circulating corticosterone when compared to controls, to an average of 90 nM (Fig. 5c; Bonferroni post hoc; p < 0.01). Following 10 days of stress and 10 days of stress in combination with an LPS challenge, elevated circulating corticosterone levels (100 nM) were also found and were significantly higher than controls (Fig. 5c; Bonferroni post hoc; stress alone p < 0.01, stress and LPS p < 0.01). At no point were stressed or LPS-treated animals different from each other, and stress combined with LPS did not result in an additive increase in corticosterone concentration.

Previous work from our laboratory has demonstrated that both LPS and chronic stress are independently capable of changing the expression of the 5-HT_2A receptor and SERT mRNA expression [29, 35]. The data above demonstrate that LPS is capable of exacerbating certain behaviours induced by the chronic stress. Therefore, it is important to determine whether receptor expression was also cumulatively increased or whether, like corticosterone, low-level inflammation in stressed animals did not affect receptor expression. The addition of both stress and LPS into the model requires a more complex analysis with stress, LPS and brain regions as repeated factors. The general linear model applied to the earlier data remains with unstructured co-variance but with the added capacity of determining whether stress and LPS interact with each other. The number of possible interactions makes reporting this data rather excessive; therefore, only significant values are reported below.

IL-1β mRNA levels were significantly affected by both stressors, either stress or LPS alone or combined and by brain region (Fig. 6a; RM-ANOVA brain region p < 0.001 F _4,48 = 16.91; stress:LPS:brain region p < 0.001 F _4,48 = 13.69). These factors also showed a significant interaction, suggesting that stress/LPS had a differential effect on IL-1β mRNA levels in different brain regions (Fig. 6a; brain region:stressor p < 0.001 F _4,48 = 8.58). Post hoc testing revealed significant effects of LPS alone, and stress combined with LPS, in the dorsal raphe nucleus (Fig. 6a; Bonferroni post hoc; p < 0.001 stress vs LPS; p < 0.05 control vs stress and LPS), and these differences continued in the raphe when comparing animals that were only stressed for 10 days to animals that were stressed but also challenged with LPS (Fig. 6a; Bonferroni post hoc p < 0.001). Other brain regions only showed minor increases in IL-1β receptor mRNA expression after either stress or LPS, and these did not reach significance (Fig. 6a). However, it should be cautioned that large changes in any individual brain region, such as the raphe, are likely to mask smaller changes in other brain regions.

In the brain, TNFα mRNA expression was affected in a similar manner to IL-1β mRNA expression, with significant main effects of both brain region and stressor and a significant interaction (Fig. 6b; RM-ANOVA brain region p < 0.05 F _4,48 = 15.64; stressor p < 0.01 F _4,48 = 7.72; stress:LPS:brain region p < 0.05 F _4,48 = 2.89). Post hoc testing suggests that stress alone does not exacerbate TNFα mRNA expression but as with the IL-1β results, larger changes in other regions may mask specific effects. LPS administration induced a significant increase in TNFα mRNA expression in all regions, with the exception of the hippocampus (Fig. 6b; Bonferroni post hoc; pre-frontal cortex p < 0.0001; striatum p < 0.01; raphe p < 0.0001). Stress combined with an inflammatory challenge results in a significant increase in TNFα expression in similar regions compared to control (Fig. 6b; Bonferroni post hoc; pre-frontal cortex p < 0.001; striatum p < 0.01; raphe p < 0.05). Finally, there were significant differences between stress-alone animals and animals stressed and challenged with LPS but only in the pre-frontal cortex (Fig. 6b; Bonferroni post hoc p < 0.01). However, there was no overt synergy between stress with LPS and LPS alone.

Analysis shows that there was only a significant main effect of brain region on 5-HT_2A mRNA expression, as well as a significant interaction between brain region, stress and LPS challenge (Fig. 6c; RM-ANOVA brain region p < 0.001 F _4,48 = 16.20; stress:LPS:brain region p < 0.01 F _4,48 = 4.96). Post hoc analysis shows 5-HT_2A receptor mRNA expression appeared to increase after a single LPS injection in the pre-frontal cortex, striatum and hippocampus, compared to controls, but was only significantly different in the hippocampus (Fig. 6c; Bonferroni post hoc; p < 0.01). There was no difference, significant or otherwise, in 5-HT_2A mRNA levels in the raphe compared to controls (Fig. 6c). In a similar manner, after 10 days of chronic stress, 5-HT_2A mRNA appeared to be elevated in the pre-frontal cortex as well as the hippocampus but again, only reached significance in the latter when compared to control animals (Fig. 6c; Bonferroni post hoc; p < 0.01). Chronic stress did not change receptor expression in either the striatum or the raphe. In the CNS of animals challenged with 10 days of chronic stress and LPS, 5-HT_2A receptor mRNA expression was not different from controls in any region except the hippocampus, where it showed an increase of a similar magnitude to stress and LPS alone (Fig. 6c; Bonferroni post hoc; p < 0.05).

SERT mRNA expression showed the same main effects as for the 5-HT_2A receptor, but significant interactions were noted. Specifically, there was a main effect of brain region and interactions between brain region and stress, brain region and LPS challenge and all three factors (Fig. 6d; RM-ANOVA; brain region p < 0.001 F _4,48 = 22.23; stress:brain region p < 0.001 F _4,48 = 15.46; LPS:brain region p < 0.001 F _4,48 = 6.42; stress:LPS:brain region p < 0.01 F _4,48 = 12.32). Further analysis showed that SERT expression in the pre-frontal cortex after a single LPS challenge appeared to be higher than controls but did not reach significance (Fig. 6d; Bonferroni post hoc pre-frontal cortex p = 0.081). No other brain regions studied showed any change in SERT mRNA compared to controls after a single dose of LPS. Ten days of chronic stress did not change SERT expression in the pre-frontal cortex or the raphe compared to controls but led to significantly higher expression in the striatum and hippocampus (Fig. 6d; Bonferroni post hoc; striatum p < 0.001, hippocampus p < 0.01). Compared to stress alone, the combination of stress and LPS did give rise to any significant increase SERT mRNA expression in any region studied (Fig. 6d).