A low dose of LPS of 0.1 mg/kg has no significant behavioural effects in naïve mice

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).

Stress-induced depressive-like behaviours tend to be exacerbated by systemic inflammation

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).

Fig. 3 The effect of low-dose LPS on depressive-like behaviours in stressed mice. Naïve and stressed animals were subjected to either a single dose of LPS (0.1 mg/kg) or vehicle injection and tested 24 h thereafter in a two-bottle sucrose preference test investigating a overall preference for sucrose, b total sucrose consumption, c water intake in a sucrose test, d the period of immobility in the tail suspension test, and in the forced swim test for e latency to floating and f total time spent floating. All animals showed >65 % preference for sucrose at baseline and similar sucrose preference prior to bolus injection of LPS or vehicle (Additional file 1: Figure S2). Data are mean ± SEM; *p < 0.05, **p < 0.01 and ***p < 0.001 when compared to controls; +p < 0.05 and +++p < 0.001 compared to stressed animals Full size image

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.

Inflammation decreases aggression and impulsivity in stressed animals

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).

Fig. 4 The effect of low-dose LPS on anxiety and aggression-like behaviours in stressed mice. Naïve and stressed animals were challenged with a single dose of LPS (0.1 mg/kg) or vehicle (saline) and tested 24 h thereafter in the elevated O-maze for the a latency to exit to the open arms and b number of exits to the open arms; in the resident-intruder paradigm for c duration of social interaction and d latency to attack conspecific, e total number of attacks and f duration of crawl over behaviour. Data are mean ± SEM; *p < 0.05, **p < 0.01 and ***p < 0.001 compared to control animals; +++p < 0.001 and ++++p < 0.0001 compared to stressed animals Full size image

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).

Inflammation and stress cumulatively increase hepatic IL-1β, but not corticosterone

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).

Fig. 5 Cytokine mRNA in the liver and blood corticosterone levels in control, stressed and LPS-treated animals. mRNA levels of a TNFα and b IL-1β were measured by qPCR in the liver of animals after either 10 days of chronic stress, an acute LPS challenge (0.1 mg/kg) or a combination of both. Corticosterone levels in blood (c) were measured by HPLC. qPCR data are expressed as relative-fold expression normalized to GAPDH and naïve mice. Bars are mean ± SEM, (n = 5 in each group), **p < 0.01 and ***p < 0.001 compared to control animals Full size image

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.

Low-dose LPS-induced inflammation does not exacerbate chronic stress-induced changes in 5-HT 2A and SERT expression or CNS cytokine expression

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.

Fig. 6 IL-1β, TNFα, 5-HT 2A receptor and SERT mRNA expression in the brain structures of animals challenged with chronic stress, LPS or a combination of both. mRNA levels of a IL-1β, b TNFα, c 5-HT 2A and d SERT were measured by qPCR in the pre-frontal cortex, striatum, hippocampus and raphe of animals after either 10 days of chronic stress, an acute LPS challenge (0.1 mg/kg) or a combination of both. Values are expressed as relative-fold expression normalized to housekeeping gene GAPDH and to control values within each region. Data are mean ± SEM; n = 5 in each group; *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.0001 compared to control animals Full size image

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).