Sex dependent effects of post-natal penicillin on brain, behavior and immune regulation are prevented by concurrent probiotic treatment

Abstract

There is increasing awareness of the need to consider potential long-term effects of antibiotics on the health of children. In addition to being associated with immune and metabolic diseases, there is evidence that early-life antibiotic exposure can affect neurodevelopment. Here we investigated the effect of low dose of penicillin V on mice when administered for 1 week immediately prior to weaning. We demonstrated that exposure to the antibiotic during the pre-weaning period led to long-term changes in social behaviour, but not anxiety-like traits, in male mice only. The change in behaviour of males was associated with decreased hippocampal expression of AVPR1A and AVPR1B while expression of both receptors was increased in females. Spleens of male mice also showed an increase in the proportion of activated dendritic cells and a corresponding decrease in regulatory T cells with penicillin exposure. All changes in brain, behaviour and immune cell populations, associated with penicillin exposure, were absent in mice that received L. rhamnosus JB-1 supplementation concurrent with the antibiotic. Our study indicates that post-natal exposure to a clinically relevant dose of antibiotic has long-term, sex dependent effects on the CNS and may have implications for the development of neuropsychiatric disorders. Importantly, we also provide further evidence that probiotic based strategies may be of use in counteracting detrimental effects of early-life antibiotics on neurodevelopment.

Figure 1

Experimental protocol. PND, post-natal day; OFT, open field test; EPM, elevated plus maze; 3CS, 3 chamber sociability.

Figure 2

Effect of post-natal penicillin treatment on behavior. Male and female mice were treated with penicillin (PenV), penicillin and concurrent L. rhamnosus JB-1 (PenV + JB-1) or vehicle and tested for locomotor activity (A,B) and anxiety-like behavior in the open field (C,D) and elevated plus maze (E–H). Social behavior was assessed in the 3 chamber test (I,J). Data presented as mean ± standard deviation. (Males, n = 15 vehicle, 15 PenV, 14 PenV+JB1: Females n = 15 vehicle, 15 PenV, 15 PenV+JB-1: *p < 0.05, **p < 0.01).

Figure 3

Effect of post-natal penicillin treatment on gene expression in the brain. Male and female mice were treated with penicillin (PenV), penicillin and concurrent L. rhamnosus JB-1 (PenV + JB-1) or vehicle and expression of Arginine Vasopressin Receptors 1a (AVPR1a) and 1b (AVPR1b), oxytocin receptor (OxtR) and brain derived neurotrophic factor (BDNF) was assessed in the hippocampus (A,B), hypothalamus (C,D) and frontal cortex (E&F). (n = 9, *p < 0.05, **p < 0.01).

Figure 4

Effect of post-natal penicillin treatment brain expression of Claudin V and Occludin. Male and female mice were treated with penicillin (PenV), penicillin and concurrent L.rhamnosus JB-1 (PenV + JB-1) or vehicle and expression of the tight junction proteins Claudin V (Claudin) and Occludin assessed in the hypothalamus (A,B), hippocampus (C,D) and frontal cortex (E,F). (n = 9, *p < 0.05, **p < 0.01).

Figure 5

Effect of post-natal penicillin treatment on immune cell populations. Male and female mice were treated with penicillin (PenV), penicillin and concurrent L.rhamnosus JB-1 (PenV + JB-1) or vehicle and flowcytometry used to determine proportion of CD4⁺CD25⁺ splenocytes expressing Foxp3 (A,B), and CD4 + CD25 + Foxp3+ cells expressing IL-10 (C,D). The proportion of dendritic cells (CD11c+) expressing CD86 (E,F), MHCII (G,H) and CD80 (I,J) were also assessed (n = 12, *p < 0.05, **p < 0.01).

Figure 6

Fecal Microbiome composition at 3 weeks. Alpha diversity calculated using Faith’s PD (A,B), beta-Diversity calculated using unweighted UniFrac matrix (C,D) and relative abundance of bacteria phyla, expressed in percentage (E,F) in male and female mice immediately following treatment with penicillin, penicillin and concurrent L.rhamnosus JB-1 (PenV + JB-1) or vehicle from postnatal day 14–21 (Males, n = 11 vehicle, 12 PenV, 12 PenV + JB1: Females n = 15 vehicle, 15 PenV, 15 PenV + JB-1).

Figure 7

Fecal Microbiome composition at 6 weeks. Alpha diversity calculated using Faith’s PD (A,B), beta-Diversity calculated using unweighted UniFrac matrix (C,D) and relative abundance of bacteria phyla, expressed in percentage (E,F) in male and female mice 3 weeks following treatment with penicillin (PenV), penicillin and concurrent L.r hamnosus JB-1 (PenV + JB-1) or vehicle from postnatal day 14–21 (Males, n = 15 vehicle, 14 PenV, 12 PenV + JB1: Females n = 15 vehicle, 15 PenV, 15 PenV + JB-1).