Activation of inflammatory signaling by lipopolysaccharide produces a prolonged increase of voluntary alcohol intake in mice

Abstract

Previous studies showed that mice with genetic predisposition for high alcohol consumption as well as human alcoholics show changes in brain expression of genes related to immune signaling. In addition, mutant mice lacking genes related to immune function show decreased alcohol consumption (Blednov et al., 2011), suggesting that immune signaling promotes alcohol consumption. To test the possibility that activation of immune signaling will increase alcohol consumption, we treated mice with lipopolysaccaride (LPS; 1mg/kg, i.p.) and tested alcohol consumption in the continuous two-bottle choice test. To take advantage of the long-lasting activation of brain immune signaling by LPS, we measured drinking beginning one week or one month after LPS treatment and continued the studies for several months. LPS produced persistent increases in alcohol consumption in C57BL/6J (B6) inbred mice, FVBxB6F1 and B6xNZBF1 hybrid mice, but not in FVB inbred mice. To determine if this effect of LPS is mediated through binding to TLR4, we tested mice lacking CD14, a key component of TLR4 signaling. These null mutants showed no increase of alcohol intake after treatment with LPS. LPS treatment decreased ethanol-conditioned taste aversion but did not alter ethanol-conditioned place preference (B6xNZBF1 mice). Electrophysiological studies of dopamine neurons in the ventral tegmental area showed that pretreatment of mice with LPS decreased the neuronal firing rate. These results suggest that activation of immune signaling promotes alcohol consumption and alters certain aspects of alcohol reward/aversion.

Fig. 1

Schedule for injection of LPS and monitoring of recovery from acute LPS effects. (A) Schedule for injection of LPS and alcohol consumption. (B and C) Monitoring of recovery from acute LPS effects in B6 male mice. (B) Daily changes in body weight. (C) Daily changes in water intake. (D and E) Monitoring of recovery from acute LPS effects (two injections) in B6 female mice. (D) Daily changes in body weight. (E) Daily changes in water intake.

Fig. 2

LPS pretreatment produces long-lasting increase of ethanol intake in C57Bl/6 J male mice. (A–C) Ethanol intake (g/kg/24 h) after single injection of LPS (1 mg/kg). n = 13 per group. (D–F) Ethanol intake (g/kg/24 h) after two injections of LPS. n = 10–14 per group. (A and D) Initial ethanol intake (g/kg/24 h). (B and E) Ethanol intake (g/kg/24 h) after first (one week) ethanol deprivation. (C and F) Ethanol intake (g/kg/24 h) after second (one-month) ethanol-deprivation period.

Fig. 3

LPS pretreatment produces long-lasting increase of ethanol intake in C57Bl/6 J female mice. (A–C) Ethanol intake (g/kg/24 h) after single injection of LPS (1 mg/kg). n = 10–13 per group. (D–F) Ethanol intake (g/kg/24 h) after two injections of LPS. n = 11–13 per group. (A and D) Initial ethanol intake (g/kg/24 h). (B and E) Ethanol intake (g/kg/24 h) after first (one-week) ethanol-deprivation period. (C and F) Ethanol intake (g/kg/24 h) after second (one-month) ethanol-deprivation period.

Fig. 4

CD14 null mutant mice do not show increased ethanol consumption after pretreatment with LPS. (A and B) CD14 null male mice. n = 7–10 per group. (C and D) CD14 null female mice. n = 10 per group. (A and C) Initial ethanol intake (g/kg/24 h). (B and D) Ethanol intake (g/kg/24 h) after first (one-week) ethanol deprivation.

Fig. 5

C57Bl/6 J male mice shows the increase of ethanol intake even one month after LPS injection. (A and B) B6 male mice. n = 10–13 per group. (C and D) CD14 null female mice. n = 10–12 per group. (A and C) Initial ethanol intake (g/kg/24 h). (B and D) Ethanol intake (g/kg/24 h) after first (one-week) ethanol deprivation.

Fig. 6

Ability of LPS to increase the alcohol intake depends on genetic background in female mice. (A–C) FVBxB6F1 female mice. n = 10–12 per group. (D–F) FVB inbred female mice. n = 10–14 per group. (G–I) B6xNZBF1 female mice. n = 8–17 per group. (A, D and G) Initial ethanol intake (g/kg/24 h). (B, E and H) Ethanol intake (g/kg/24 h) after first (one-week) ethanol deprivation. (C, F and I) Ethanol intake (g/kg/24 h) after second (one-month) ethanol deprivation.

Fig. 7

LPS pretreatment produces no changes in olfactory recognition of ethanol in C57Bl/6 J mice. (A–C) B6 male mice. (D–F) B6 female mice. (A and D) Latency to uncover the buried or visible target (odor of 20% of ethanol). (B and E) Latency to uncover the buried target (odor of different concentrations of ethanol). (C and F) Time spent exploring the target in the “block” test. *p < .05, statistically significant difference between LPS- and saline-pretreated groups (Bonferroni’s post-hoc analyses).

Fig. 8

LPS pretreatment reduces the aversive effect of ethanol in B6xNZB F1 female mice. (A) Consumption of saccharin, the conditioned stimulus, is presented as percent of saccharin intake at day 0 (before injection) for all conditioning days. (B) Consumption of saccharin, the conditioned stimulus, is presented as differences in saccharin intake between day 1 (consumption after first injection) and day 0 (consumption at day before injection). n = 10 for both groups of saline-treated mice and n = 15 for both groups of ethanol-treated mice. *p < .05, statistically significant difference between LPS- and saline-pretreated groups (Bonferroni’s post-hoc analyses).

Fig. 9

LPS pretreatment does not change the rewarding properties of ethanol in a conditioned place preference (CPP) test in NZBxB6 F1 female mice. (A) Ethanol-induced CPP. Time (s) spent on Grid + floor (circles) during 30-min test session in ethanol-conditioned (Grid + and Grid−) groups. (B) Motor activity of mice (Grid + and Grid− groups) reported as total distance traveled (cm) during 5-min conditioning trials for CS + and CS− trials. n = 10 per each group.

Fig. 10

LPS pretreatment significantly reduced the basal firing of DA neurons in VTA of C57Bl/6 male mice. Upper panel: Example traces of action potential firing in dopamine neurons from saline- and LPS-treated mice. (A) Average firing frequencies in saline- and LPS-treated groups. (B and C) Time graphs of firing frequency during 5-min recording periods for the neurons shown in the upper panel. These graphs illustrate the stability of firing recording in individual neurons. *p < 0.05, Student’s t-test. n = 9 and 15 in saline and LPS groups, respectively.

Fig. 11

Lack of correlation between ethanol intake and severity of sickness developed after LPS injection. Y-axis: The severity of sickness was calculated as an area under the curve for daily monitoring of body weight after the first injection of LPS. Body weight the day before LPS injection was taken as 100% and body weight for each of the next seven days were expressed as a percentage of the initial body weight. This parameter reflects not only the peak severity of sickness but also the course of recovery. X-axis: Ethanol intake was calculated as an area under the curve for ethanol intake from 3% of ethanol solution up to 18% of ethanol (using amount of ethanol consumed, g/kg/d).