Emerging evidence indicates that disruption of the gut microbial community (dysbiosis) impairs mental health. Germ-free mice and antibiotic-induced gut dysbiosis are two approaches to establish causality in gut microbiota-brain relationships. However, both models have limitations, as germ-free mice display alterations in blood-brain barrier and brain ultrastructure and antibiotics may act directly on the brain. We hypothesized that the concerns related to antibiotic-induced gut dysbiosis can only adequately be addressed if the effect of intragastric treatment of adult mice with multiple antibiotics on (i) gut microbial community, (ii) metabolite profile in the colon, (iii) circulating metabolites, (iv) expression of neuronal signaling molecules in distinct brain areas and (v) cognitive behavior is systematically investigated. Of the antibiotics used (ampicillin, bacitracin, meropenem, neomycin, vancomycin), ampicillin had some oral bioavailability but did not enter the brain. 16S rDNA sequencing confirmed antibiotic-induced microbial community disruption, and metabolomics revealed that gut dysbiosis was associated with depletion of bacteria-derived metabolites in the colon and alterations of lipid species and converted microbe-derived molecules in the plasma. Importantly, novel object recognition, but not spatial, memory was impaired in antibiotic-treated mice. This cognitive deficit was associated with brain region-specific changes in the expression of cognition-relevant signaling molecules, notably brain-derived neurotrophic factor, N-methyl-d-aspartate receptor subunit 2B, serotonin transporter and neuropeptide Y system. We conclude that circulating metabolites and the cerebral neuropeptide Y system play an important role in the cognitive impairment and dysregulation of cerebral signaling molecules due to antibiotic-induced gut dysbiosis.

The microbial ecosystem that inhabits the gastrointestinal tract of all mammals—the gut microbiota—has been in a symbiotic relationship with its hosts over many millennia. Thanks to modern technology, the myriad of functions that are controlled or modulated by the gut microbiota are beginning to unfold. One of the systems that is emerging to closely interact with the gut microbiota is the body’s major neuroendocrine system that controls various body processes in response to stress, the hypothalamic–pituitary–adrenal (HPA) axis. This interaction is of pivotal importance; as various disorders of the microbiota–gut–brain axis are associated with dysregulation of the HPA axis. The present contribution describes the bidirectional communication between the gut microbiota and the HPA axis and delineates the potential underlying mechanisms. In this regard, it is important to note that the communication between the gut microbiota and the HPA axis is closely interrelated with other systems, such as the immune system, the intestinal barrier and blood–brain barrier, microbial metabolites, and gut hormones, as well as the sensory and autonomic nervous systems. These communication pathways will be exemplified through preclinical models of early life stress, beneficial roles of probiotics and prebiotics, evidence from germ-free mice, and antibiotic-induced modulation of the gut microbiota.Electronic supplementary materialThe online version of this article (10.1007/s13311-017-0600-5) contains supplementary material, which is available to authorized users.

Toxicity of nanoparticles (NPs) upon oral exposure has been studied in animals using physiological changes, behavior, histology, and blood analysis for evaluation. The effects recorded include the combination of the action on cells of the exposed animal and the reaction of the microorganisms that populate the external and internal surfaces of the body. The importance of these microorganisms, collectively termed as microbiota, for the health of the host has been widely recognized. They may also influence toxicity of NPs but these effects are difficult to differentiate from toxicity on cells of the gastrointestinal tract. To estimate the likelihood of preferential damage of the microbiota by NPs the relative sensitivity of enterocytes and bacteria was compared. For this comparison NPs with antimicrobial action present in consumer products were chosen. The comparison of cytotoxicity with Escherichia coli as representative for intestinal bacteria and on gastrointestinal cells revealed that silver NPs damaged bacteria at lower concentrations than enterocytes, while the opposite was true for zinc oxide NPs. These results indicate that silver NPs may cause adverse effects by selectively affecting the gut microbiota. Fecal transplantation from NP-exposed animals to unexposed ones offers the possibility to verify this hypothesis.

The depression-like behaviour induced by prolonged HFD in mice is associated with distinct alterations of intestinal microbiome, brain metabolome, NPY system, and DPP-4-like activity. Importantly, the HFD-evoked behavioural disturbance remains unaltered by DPP-4 inhibition and antidepressant treatment with imipramine.

Toll-like receptors (TLRs) and nuclear-binding domain (NOD)-like receptors (NLRs) are sensors of bacterial cell wall components to trigger an immune response. The TLR4 agonist lipopolysaccharide (LPS) is a strong immune activator leading to sickness and depressed mood. NOD agonists are less active but can prime immune cells to augment LPS-induced cytokine production. Since the impact of NOD and TLR co-activation in vivo has been little studied, the effects of the NOD1 agonist FK565 and the NOD2 agonist muramyl dipeptide (MDP), alone and in combination with LPS, on immune activation, brain function and sickness behavior were investigated in male C57BL/6N mice.Intraperitoneal injection of FK565 (0.001 or 0.003 mg/kg) or MDP (1 or 3 mg/kg) 4 h before LPS (0.1 or 0.83 mg/kg) significantly aggravated and prolonged the LPS-evoked sickness behavior as deduced from a decrease in locomotion, exploration, food intake and temperature. When given alone, FK565 and MDP had only minor effects. The exacerbation of sickness behavior induced by FK565 or MDP in combination with LPS was paralleled by enhanced plasma protein and cerebral mRNA levels of proinflammatory cytokines (IFN-γ, IL-1β, IL-6, TNF-α) as well as enhanced plasma levels of kynurenine. Immunohistochemical visualization of c-Fos in the brain revealed that NOD2 synergism with TLR4 resulted in increased activation of cerebral nuclei relevant to sickness.These data show that NOD1 or NOD2 synergizes with TLR4 in exacerbating the immune, sickness and brain responses to peripheral immune stimulation.Our findings demonstrate that the known interactions of NLRs and TLRs at the immune cell level extend to interactions affecting brain function and behavior.

Microbial metabolites are known to affect immune system, brain, and behavior via activation of pattern recognition receptors such as Toll-like receptor 4 (TLR4). Unlike the effect of the TLR4 agonist lipopolysaccharide (LPS), the role of other TLR agonists in immune-brain communication is insufficiently understood. We therefore hypothesized that the TLR2 agonist lipoteichoic acid (LTA) causes immune activation in the periphery and brain, stimulates the hypothalamic-pituitary-adrenal (HPA) axis and has an adverse effect on blood-brain barrier (BBB) and emotional behavior. Since LTA preparations may be contaminated by LPS, an extract of LTA (LTA), purified LTA (LTA), and pure LPS (LPS) were compared with each other in their effects on molecular and behavioral parameters 3h after intraperitoneal (i.p.) injection to male C57BL/6N mice. The LTA (20mg/kg) induced anxiety-related behavior in the open field test, enhanced the circulating levels of particular cytokines and the cerebral expression of cytokine mRNA, and blunted the cerebral expression of tight junction protein mRNA. A dose of LPS matching the amount of endotoxin/LPS contaminating the LTA reproduced several of the molecular and behavioral effects of LTA. LTA (20mg/kg) increased plasma levels of tumor necrosis factor-α (TNF-α), interleukin-6 and interferon-γ, and enhanced the transcription of TNF-α, interleukin-1β and other cytokines in the amygdala and prefrontal cortex. These neuroinflammatory effects of LTA were associated with transcriptional down-regulation of tight junction-associated proteins (claudin 5, occludin) in the brain. LTA also enhanced circulating corticosterone, but failed to alter locomotor and anxiety-related behavior in the open field test. These data disclose that TLR2 agonism by LTA causes peripheral immune activation and initiates neuroinflammatory processes in the brain that are associated with down-regulation of BBB components and activation of the HPA axis, although emotional behavior (anxiety) is not affected. The results obtained with an LTA preparation contaminated with LPS hint at a facilitatory interaction between TLR2 and TLR4, the adverse impact of which on long-term neuroinflammation, disruption of the BBB and mental health warrants further analysis.

Altered levels of colonic peptide YY (PYY) have been reported in patients suffering from functional and inflammatory bowel disorders. While the involvement of neuropeptide Y (NPY) and Y receptors in the regulation of nociception is well established, the physiological role of PYY in somatic and visceral pain is poorly understood. In this work, the role of PYY in pain sensitivity was evaluated using PYY knockout (PYY(−/−)) mice and Y2 receptor ligands. PYY(−/−) mice were more sensitive to somatic thermal pain compared to wild type (WT) mice. Visceral pain was assessed by evaluating pain-related behaviors, mouse grimace scale (MGS) and referred hyperalgesia after intrarectal administration of allyl isothiocyanate (AITC, 1 or 2%) or its vehicle, peanut oil. The pain-related behaviors induced by AITC were significantly exaggerated by PYY deletion, whereas the MGS readout and the referred hyperalgesia were not significantly affected. The Y2 receptor antagonist, BII0246, increased pain-related behaviors in response to intrarectal AITC compared to vehicle treatment while the Y2 receptor agonist, PYY(3–36), did not have a significant effect. These results indicate that endogenous PYY has a hypoalgesic effect on somatic thermal and visceral chemical pain. The effect on visceral pain seems to be mediated by peripheral Y2 receptors.

Opioids rank among the most potent analgesic drugs but gastrointestinal side effects, especially constipation, limit their therapeutic utility. The adverse effects of opioids have been attributed to stimulation of opioid receptors, but emerging evidence suggests that opioids interact with the innate immune receptor Toll-like receptor 4 (TLR4) and its signalling pathway. As TLR4 signalling affects gastrointestinal motility, we examined the involvement of TLR4 in morphine-induced depression of peristaltic motility in the guinea-pig intestine in vitro and male C57BL/6N mice in vivo. While the TLR4 antagonist TAK-242 (0.1 μM and 1 μM) did not alter the morphine-induced inhibition of peristalsis in the isolated guinea-pig small intestine, the morphine-induced decrease in pellet propulsion velocity in colonic segments was attenuated by TAK-242 (0.1 μM). The ability of TAK-242 (4 mg/kg) to mitigate the morphine-induced suppression of colonic motility was replicated in mice in vivo by measuring the expulsion time of beads inserted in the distal colon. The inhibition of upper gastrointestinal transit of mice by morphine was not affected by pre-treatment with TAK-242 (4 mg/kg) in vivo. This is the first report that morphine-induced inhibition of colonic peristalsis is alleviated by TLR4 antagonism. We therefore conclude that TLR4 may contribute to opioid-induced constipation.