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Cited by 269Gut microbiota and healthTryptophan and brain disordersGastrointestinal motility and disorders

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Indole, a Signaling Molecule Produced by the Gut Microbiota, Negatively Impacts Emotional Behaviors in Rats

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Jaglin, Mathilde, Rhimi, Moez, Philippe, Catherine, Pons, Nicolas, Bruneau, Aurélia, Goustard, Bénédicte, Daugé, Valérie, Maguin, Emmanuelle, Naudon, Laurent, Rabot, Sylvie·2018·Frontiers in Neuroscience
DOI 10.3389/fnins.2018.00216PMC PMC5900047
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Abstract

Gut microbiota produces a wide and diverse array of metabolites that are an integral part of the host metabolome. The emergence of the gut microbiome-brain axis concept has prompted investigations on the role of gut microbiota dysbioses in the pathophysiology of brain diseases. Specifically, the search for microbe-related metabolomic signatures in human patients and animal models of psychiatric disorders has pointed out the importance of the microbial metabolism of aromatic amino acids. Here, we investigated the effect of indole on brain and behavior in rats. Indole is produced by gut microbiota from tryptophan, through the tryptophanase enzyme encoded by the tnaA gene. First, we mimicked an acute and high overproduction of indole by injecting this compound in the cecum of conventional rats. This treatment led to a dramatic decrease of motor activity. The neurodepressant oxidized derivatives of indole, oxindole and isatin, accumulated in the brain. In addition, increase in eye blinking frequency and in c-Fos protein expression in the dorsal vagal complex denoted a vagus nerve activation. Second, we mimicked a chronic and moderate overproduction of indole by colonizing germ-free rats with the indole-producing bacterial species Escherichia coli. We compared emotional behaviors of these rats with those of germ-free rats colonized with a genetically-engineered counterpart strain unable to produce indole. Rats overproducing indole displayed higher helplessness in the tail suspension test, and enhanced anxiety-like behavior in the novelty, elevated plus maze and open-field tests. Vagus nerve activation was suggested by an increase in eye blinking frequency. However, unlike the conventional rats dosed with a high amount of indole, the motor activity was not altered and neither oxindole nor isatin could be detected in the brain. Further studies are required for a comprehensive understanding of the mechanisms supporting indole effects on emotional behaviors. As our findings suggest that people whose gut microbiota is highly prone to produce indole could be more likely to develop anxiety and mood disorders, we addressed the issue of the inter-individual variability of indole producing potential in humans. An in silico investigation of metagenomic data focused on the tnaA gene products definitively proved this inter-individual variability.

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Figure 1 Flow diagram of the experimental design employed in the conventional rats. (A) Rats received DMSO (controls) or oxindole or isatin at 2 different doses (treated) through an intra-peritoneal (i.p.) injection. (B) Rats received corn oil (controls) or indole (treated) through an intra-cecal (i.c.) injection. Eye blinking frequency and locomotor activity were measured in (A) and (B) animals.
Figure 1 Flow diagram of the experimental design employed in the conventional rats. (A) Rats received DMSO (controls) or oxindole or isatin at 2 different doses (treated) through an intra-peritoneal (i.p.) injection. (B) Rats received corn oil (controls) or indole (treated) through an intra-cecal (i.c.) injection. Eye blinking frequency and locomotor activity were measured in (A) and (B) animals.CC BY · PubMed Central
Figure 2 Flow diagram of the experimental design employed in the gnotobiotic rats. (A) A first series of rats was subjected to an investigation of social behavior (social interaction test) and anxiety-like behavior (elevated plus maze and open-field tests). (B) As differences occurred in the anxiety-like behavior of the (A) rats, further behavioral analyses were carried out in a second series of rats: confirmation of the anxiety-like behavior through a novelty test, exploration of memory (object recognition task) and of despair behavior (tail suspension test). The eye blinking frequency was also measured in these rats. All rats were aged 2.5–3 months when the behavioral tests started (Day 1).
Figure 2 Flow diagram of the experimental design employed in the gnotobiotic rats. (A) A first series of rats was subjected to an investigation of social behavior (social interaction test) and anxiety-like behavior (elevated plus maze and open-field tests). (B) As differences occurred in the anxiety-like behavior of the (A) rats, further behavioral analyses were carried out in a second series of rats: confirmation of the anxiety-like behavior through a novelty test, exploration of memory (object recognition task) and of despair behavior (tail suspension test). The eye blinking frequency was also measured in these rats. All rats were aged 2.5–3 months when the behavioral tests started (Day 1).CC BY · PubMed Central
Figure 3 Oxindole and isatin concentrations in the brain of conventional rats treated with oxindole, isatin or indole. (A) Oxindole and (B) Isatin brain concentrations (nmol/g) after i.p. injection of oxindole or isatin (50 and 100 mg/kg) or of the DMSO vehicle (n = 7–8 rats/group, Kruskal-Wallis test followed by a Dunn's multiple comparison test). (C) Oxindole and isatin brain concentrations (nmol/g) after intra-cecal administration of indole (500 mg/kg) or of the corn oil vehicle (n = 11 rats/group, Mann-Whitney test). Data are medians with interquartile ranges. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group.
Figure 3 Oxindole and isatin concentrations in the brain of conventional rats treated with oxindole, isatin or indole. (A) Oxindole and (B) Isatin brain concentrations (nmol/g) after i.p. injection of oxindole or isatin (50 and 100 mg/kg) or of the DMSO vehicle (n = 7–8 rats/group, Kruskal-Wallis test followed by a Dunn's multiple comparison test). (C) Oxindole and isatin brain concentrations (nmol/g) after intra-cecal administration of indole (500 mg/kg) or of the corn oil vehicle (n = 11 rats/group, Mann-Whitney test). Data are medians with interquartile ranges. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group.CC BY · PubMed Central
Figure 4 Motor activity (locomotion and rearings) measured in an open-field in conventional rats treated with oxindole, isatin or indole. (A) Distance traveled and (B) Number of rearings after i.p. injection of oxindole or isatin (50 and 100 mg/kg) or of the DMSO vehicle (n = 7–8 rats/group, Kruskal-Wallis test followed by a Dunn's multiple comparison test). (C) Distance traveled and number of rearings after intra-cecal administration of indole (500 mg/kg) or of the corn oil vehicle (n = 12 rats/group, Mann-Whitney test). Data are medians with interquartile ranges. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group.
Figure 4 Motor activity (locomotion and rearings) measured in an open-field in conventional rats treated with oxindole, isatin or indole. (A) Distance traveled and (B) Number of rearings after i.p. injection of oxindole or isatin (50 and 100 mg/kg) or of the DMSO vehicle (n = 7–8 rats/group, Kruskal-Wallis test followed by a Dunn's multiple comparison test). (C) Distance traveled and number of rearings after intra-cecal administration of indole (500 mg/kg) or of the corn oil vehicle (n = 12 rats/group, Mann-Whitney test). Data are medians with interquartile ranges. *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control group.CC BY · PubMed Central
Figure 5 Eye blinking frequency in conventional rats treated with oxindole, isatin, or indole. (A) Number of eye blinkings/min during 10 min, from 2 min after i.p. injection of oxindole or isatin (50 and 100 mg/kg) or of the DMSO vehicle (n = 7–8 rats/group, Kruskal-Wallis test followed by a Dunn's multiple comparison test). (B) Number of eye blinkings/min during 10 min, from 2 min after intra-cecal injection of indole (500 mg/kg) or of the corn oil vehicle (n = 12 rats/group, Mann-Whitney test). Data are medians with interquartile ranges. ***P < 0.001.
Figure 5 Eye blinking frequency in conventional rats treated with oxindole, isatin, or indole. (A) Number of eye blinkings/min during 10 min, from 2 min after i.p. injection of oxindole or isatin (50 and 100 mg/kg) or of the DMSO vehicle (n = 7–8 rats/group, Kruskal-Wallis test followed by a Dunn's multiple comparison test). (B) Number of eye blinkings/min during 10 min, from 2 min after intra-cecal injection of indole (500 mg/kg) or of the corn oil vehicle (n = 12 rats/group, Mann-Whitney test). Data are medians with interquartile ranges. ***P < 0.001.CC BY · PubMed Central
Figure 6 c-Fos immunohistochemistry in the brain of conventional rats treated with indole. (A) c-Fos immunolabeled cells were counted in the DVC (area indicated by a dark oval) at two levels, −13.38 and −13.68 mm from the bregma. (B) Number of c-Fos positive cells in the counting areas after intra-cecal administration of indole (500 mg/kg) or of the corn oil vehicle (n = 5–6 rats/group, Mann-Whitney test). Data are medians with interquartile ranges. **P < 0.01. (C,D) Photomicrographs illustrating c-Fos immunoreactivity in the DVC of the corn oil and indole treated rats, respectively. Arrows indicate c-Fos immunolabeled cells.
Figure 6 c-Fos immunohistochemistry in the brain of conventional rats treated with indole. (A) c-Fos immunolabeled cells were counted in the DVC (area indicated by a dark oval) at two levels, −13.38 and −13.68 mm from the bregma. (B) Number of c-Fos positive cells in the counting areas after intra-cecal administration of indole (500 mg/kg) or of the corn oil vehicle (n = 5–6 rats/group, Mann-Whitney test). Data are medians with interquartile ranges. **P < 0.01. (C,D) Photomicrographs illustrating c-Fos immunoreactivity in the DVC of the corn oil and indole treated rats, respectively. Arrows indicate c-Fos immunolabeled cells.CC BY · PubMed Central
Figure 7 Social interactions in gnotobiotic rats mono-associated with the WT E. coli strain (“I+” rats) or with the mutant inactivated for the tnaA gene (“I−” rats). (A) Duration of the total interaction behavior of pairs of “I−” and “I+” rats, (B,C) Duration of the two main behaviors, respectively sniffing and crawling over (n = 12 pairs/group, Mann-Whitney test). Data are medians with interquartile ranges. *P < 0.05, ***P < 0.001.
Figure 7 Social interactions in gnotobiotic rats mono-associated with the WT E. coli strain (“I+” rats) or with the mutant inactivated for the tnaA gene (“I−” rats). (A) Duration of the total interaction behavior of pairs of “I−” and “I+” rats, (B,C) Duration of the two main behaviors, respectively sniffing and crawling over (n = 12 pairs/group, Mann-Whitney test). Data are medians with interquartile ranges. *P < 0.05, ***P < 0.001.CC BY · PubMed Central
Figure 8 Elevated plus maze test in gnotobiotic rats mono-associated with the WT E. coli strain (“I+” rats) or with the mutant inactivated for the tnaA gene (“I−” rats). (A) Ratio of the time spent in open arms divided by the time spent in all arms, (B) Ratio of the number of visits in open arms divided by the number of visits in all arms, (C) Number of visits at the end of open arms, (D) Number of head dippings, (E) Number of self-groomings (n = 5–7 rats/group, Mann-Whitney test). Data are medians with interquartile ranges. *P < 0.05, **P < 0.01.
Figure 8 Elevated plus maze test in gnotobiotic rats mono-associated with the WT E. coli strain (“I+” rats) or with the mutant inactivated for the tnaA gene (“I−” rats). (A) Ratio of the time spent in open arms divided by the time spent in all arms, (B) Ratio of the number of visits in open arms divided by the number of visits in all arms, (C) Number of visits at the end of open arms, (D) Number of head dippings, (E) Number of self-groomings (n = 5–7 rats/group, Mann-Whitney test). Data are medians with interquartile ranges. *P < 0.05, **P < 0.01.CC BY · PubMed Central
Figure 9 Open-field test in gnotobiotic rats mono-associated with the WT E. coli strain (“I+” rats) or with the mutant inactivated for the tnaA gene (“I−” rats). (A) Distance traveled, (B) Number of rearings, (C) Number of self-groomings (n = 12 rats/group, Mann-Whitney test). Data are medians with interquartile ranges. *P < 0.05.
Figure 9 Open-field test in gnotobiotic rats mono-associated with the WT E. coli strain (“I+” rats) or with the mutant inactivated for the tnaA gene (“I−” rats). (A) Distance traveled, (B) Number of rearings, (C) Number of self-groomings (n = 12 rats/group, Mann-Whitney test). Data are medians with interquartile ranges. *P < 0.05.CC BY · PubMed Central
Figure 10 Novelty test and locomotor activity in gnotobiotic rats mono-associated with the WT E. coli strain (“I+” rats) or with the mutant inactivated for the tnaA gene (“I−” rats). (A) Latency to approach the object, (B) Distance traveled (n = 10–12 rats/group, Mann-Whitney test). Data are medians with interquartile ranges. **P < 0.01.
Figure 10 Novelty test and locomotor activity in gnotobiotic rats mono-associated with the WT E. coli strain (“I+” rats) or with the mutant inactivated for the tnaA gene (“I−” rats). (A) Latency to approach the object, (B) Distance traveled (n = 10–12 rats/group, Mann-Whitney test). Data are medians with interquartile ranges. **P < 0.01.CC BY · PubMed Central
Figure 11 Tail suspension test in gnotobiotic rats mono-associated with the WT E. coli strain (“I+” rats) or with the mutant inactivated for the tnaA gene (“I−” rats). Duration of immobility (n = 11 rats/group, Mann-Whitney test). Data are medians with interquartile ranges. *P < 0.05.
Figure 11 Tail suspension test in gnotobiotic rats mono-associated with the WT E. coli strain (“I+” rats) or with the mutant inactivated for the tnaA gene (“I−” rats). Duration of immobility (n = 11 rats/group, Mann-Whitney test). Data are medians with interquartile ranges. *P < 0.05.CC BY · PubMed Central
Figure 12 Phylogenetic analysis of TnaA proteins retrieved from the Human microbiota catalog. Assigned proteins are indicated in color according to the phyla they belong to, while the proteins that cannot be assigned to cultivated species are in black.
Figure 12 Phylogenetic analysis of TnaA proteins retrieved from the Human microbiota catalog. Assigned proteins are indicated in color according to the phyla they belong to, while the proteins that cannot be assigned to cultivated species are in black.CC BY · PubMed Central
Figure 13 Distribution of the non-redundant TnaA proteins from the microbiota among the bacterial phyla. The pie-chart represents the relative percentage of TnaA-proteins retrieved from the Human microbiota catalog in the various bacterial phyla represented in the intestinal tract.
Figure 13 Distribution of the non-redundant TnaA proteins from the microbiota among the bacterial phyla. The pie-chart represents the relative percentage of TnaA-proteins retrieved from the Human microbiota catalog in the various bacterial phyla represented in the intestinal tract.CC BY · PubMed Central
Figure 14 Pie charts showing the family-level distribution of TnaA for the 3 phyla containing the larger number of genes putatively encoding TnaA.
Figure 14 Pie charts showing the family-level distribution of TnaA for the 3 phyla containing the larger number of genes putatively encoding TnaA.CC BY · PubMed Central
Figure 15 Richness of the non-redundant tnaA genes within the microbiota of 203 healthy individuals.
Figure 15 Richness of the non-redundant tnaA genes within the microbiota of 203 healthy individuals.CC BY · PubMed Central

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  1. Paxinos, 2005, The Rat Brain in Stereotaxic Coordinates, 5th Edn.

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Metadata

Authors
Jaglin, M., Rhimi, M., Philippe, C., Pons, N., Bruneau, A., Goustard, B., Daugé, V., Maguin, E., Naudon, L., & Rabot, S.
Affiliations
  • AgroParisTech (FR)
  • Université Paris-Saclay (FR)
  • Centre National de la Recherche Scientifique (FR)
Corresponding author
  • Naudon, Laurent
  • Rabot, Sylvie
ORCID
Venue
Frontiers in Neuroscience
Publisher
Frontiers Media SA
Published
2018-04-09
Volume
12
Pages
216-216
ISSN
1662-453X (ISSN-L)
DOI
10.3389/fnins.2018.00216
PMC
PMC5900047
OpenAlex
W2793407886
Cited by
269
References
66
Citations by year
2026: 17 · 2025: 33 · 2024: 52 · 2023: 37 · 2022: 42 · 2021: 33 · 2020: 34 · 2019: 17 · 2018: 4
FWCI
7.83
Type
journal article
Language
en
Open access
Licence
Locations
  • Frontiers in Neuroscience (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • HAL (submittedVersion, other-oa)
  • HAL (submittedVersion, cc-by)
  • HAL (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Institut National de la Recherche Agronomique
  • Centre National de la Recherche Scientifique
  • Ministère de l'Education Nationale, de l'Enseignement Supérieur et de la Recherche
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Gut microbiota and health
  • Life Sciences › Neuroscience › Biological Psychiatry › Tryptophan and brain disorders
  • Health Sciences › Medicine › Gastroenterology › Gastrointestinal motility and disorders
Keywords
indole, gut microbiota, gut-brain axis, tryptophan metabolism, tryptophanase, germ-free rats, Escherichia coli, anxiety-like behavior, helplessness, dorsal vagal complex, blinking, oxindoles, isatin, interindividual variability, metagenomics, emotional behavior

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