Multicellular organisms have co-evolved with complex consortia of viruses, bacteria, fungi and parasites, collectively referred to as the microbiota1. In mammals, changes in the composition of the microbiota can influence many physiologic processes (including development, metabolism and immune cell function) and are associated with susceptibility to multiple diseases2. Alterations in the microbiota can also modulate host behaviours—such as social activity, stress, and anxiety-related responses—that are linked to diverse neuropsychiatric disorders3. However, the mechanisms by which the microbiota influence neuronal activity and host behaviour remain poorly defined. Here we show that manipulation of the microbiota in antibiotic-treated or germ-free adult mice results in significant deficits in fear extinction learning. Single-nucleus RNA sequencing of the medial prefrontal cortex of the brain revealed significant alterations in gene expression in excitatory neurons, glia and other cell types. Transcranial two-photon imaging showed that deficits in extinction learning after manipulation of the microbiota in adult mice were associated with defective learning-related remodelling of postsynaptic dendritic spines and reduced activity in cue-encoding neurons in the medial prefrontal cortex. In addition, selective re-establishment of the microbiota revealed a limited neonatal developmental window in which microbiota-derived signals can restore normal extinction learning in adulthood. Finally, unbiased metabolomic analysis identified four metabolites that were significantly downregulated in germ-free mice and have been reported to be related to neuropsychiatric disorders in humans and mouse models, suggesting that microbiota-derived compounds may directly affect brain function and behaviour. Together, these data indicate that fear extinction learning requires microbiota-derived signals both during early postnatal neurodevelopment and in adult mice, with implications for our understanding of how diet, infection, and lifestyle influence brain health and subsequent susceptibility to neuropsychiatric disorders.
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The algorithm used for automated scoring of freezing behaviour is available at https://www.seas.upenn.edu/~molneuro/software.html. The algorithm used for motion artefact correction in 2P calcium imaging data is available at http://www.cs.cmu.edu/~kangli/code/Image_Stabilizer.html. All other analysis code is available from the corresponding author upon reasonable request.
RNA-seq data, 16S rRNA-seq data and snRNA-seq data are available at Gene Expression Omnibus and BioProject under accession numbers GSE134808, PRJNA556230 and GSE135326, respectively. All datasets generated and/or analysed during the current study are presented in this published article, the accompanying Source Data or Supplementary Information, or are available from the corresponding author upon reasonable request.
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We thank the members of the Artis and Liston laboratories for discussion and reading of the manuscript; G. Eraslan, S. Simmons, and C. Smillie for discussions about the snRNA-seq analyses; and the Metabolic Phenotyping Center of Weill Cornell Medicine for technical advice and support. The summary cartoon (Extended Data Fig. 10f) was created with BioRender.com. This work was supported by the Jill Roberts Institute (to G.G.P.), JSPS Overseas Research Fellowships (to S.M.), the National Institute of General Medical Sciences (1R35GM118182-01, to K.J.T.), the National Institute of Allergy and Infectious Diseases (1P01AI102852-01A1, to K.J.T. and S.S.C.), the National Institutes of Health (NS052819, to F.S.L), the Rita Allen Foundation, the One Mind Institute, the Klingenstein-Simons Foundations, the Brain and Behavior Research Foundation, and the National Institutes of Mental Health (R01 MH109685, R01 MH118451) (all to C.L.), the National Institutes of Health (AI074878, AI095466, AI095608 and AI102942), the Burroughs Wellcome Fund, the Crohn’s and Colitis Foundation and the Rosanne H. Silbermann Foundation (all to D.A.). F.C.S. is a Faculty Scholar of the Howard Hughes Medical Institute. This work was supported by the Klarman Cell Observatory at the Broad Institute. A.R. is a Howard Hughes Medical Institute Investigator.
A.R. is an SAB member of ThermoFisher Scientific and Syros Pharmaceuticals and a co-founder and equity holder of Celsius Therapeutics. D.A. has contributed to scientific advisory boards at MedImmune, Pfizer, FARE, and the KRF. The other authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Peer review information Nature thanks Drew Kiraly and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.
Extended data figures and tables
a–c, Food intake (a), water intake (b) and weight gain (c) of the mice measured using the Promethion Metabolic Cage System. Antibiotic treatment was started two weeks before the experiment and continued for the duration of the experiment. For food (a) and water intake (b), the mice were acclimated to the system for the first four days followed by one day of data collection. Body mass (c) of the mice was measured at the beginning (Start) and the end (End) of the 5-day experiment. n = 4 mice per group. Mean ± s.e.m. Total, full day. Light and Dark denote the light and dark periods of the 12-h cycle. d, 16S rDNA gene copies as quantified by real-time PCR with reverse transcription (RT–PCR) from stool pellets collected from control or ABX mice. Data pooled from two independent experiments. n = 7 mice per group. Mean ± s.e.m.; unpaired two-sided t-test. e–g, PCoA (e), alpha-diversity Shannon index (f) and taxonomic classification (g) of 16S rDNA in stool pellets collected from control or ABX mice. Control n = 4, ABX n = 5. For PCoA plot PERMANOVA: F = 33.579, Df = 1, P = 0.00804. For phylogenetic classification ‘f_’, ‘g_’, ‘uncl_c_’, ‘uncl_d_’ and ‘uncl_o_’ stand for ‘family_’, ‘genus_’, ‘unclassified_class_’, ‘unclassified_domain_’ and ‘unclassified_order_’, respectively. ‘uncl_d_Bacteria’ matches exactly to mitochondria or chloroplasts, probably from the food. Mean ± s.e.m. in f.
Fear extinction in sham-operated control (Ctrl_Sham) or ABX mice (ABX_Sham) and in vagotomised ABX mice (ABX_Vx) mice over the course of 3 days or sessions. Ctrl_Sham n = 10, ABX_Sham n = 10, ABX_Vx n = 12. Mean ± s.e.m.; AUC was calculated for each mouse within each group, followed by unpaired two-sided t-test between groups. P values are as follows: i, 2.57 × 10−7; ii, 9.21 × 10−8.
Extended Data Fig. 3 Comparable percentages and numbers of CD45high leukocytes in the brains of control and ABX or GF mice.
a, Gating strategy for T cells, B cells, dendritic cells (DCs) and macrophages (Mφ) in the brain. b, Population frequencies and numbers of brain-resident CD45high leukocytes in control and ABX mice. c, d, Population frequencies of CD4+ T cells, CD8+ T cells, CD19+ B cells (c), CD11c+ DCs and F4/80+ macrophages (d) gated on brain-resident CD45high leukocytes in control and ABX mice. e, Population frequencies and numbers of brain-resident CD45high leukocytes in control and GF mice. f, g, Population frequencies of CD4+ T cells, CD8+ T cells, CD19+ B cells (f), CD11c+ DCs and F4/80+ macrophages (g) gated on brain-resident CD45high leukocytes in control and GF mice. h, Gating strategy of total myeloid cells and Ly6Chigh monocytes in the brain. i, j, Population frequencies of total myeloid cells and Ly6Chigh monocytes gated on brain-resident CD45high leukocytes in control and ABX (i) or GF (j) mice. Data in b, c, g, j are representative of three independent experiments. n = 4 mice per group. Data in d, i are pooled from two independent experiments. n = 8 mice per group. Data in e, f are pooled from two independent experiments. n = 6 mice group. Data are mean ± s.e.m.; unpaired two-sided t-tests were used. P values are indicated on the figures. k, Fear extinction in control, GF and Rag1−/− mice in the single-session 30-tone fear extinction assay. Data are pooled from two independent experiments. Control n = 18, GF n = 16, Rag1−/− n = 18. Mean ± s.e.m.; AUC was calculated for each mouse within each group followed by one-way ANOVA with Tukey’s multiple comparisons test. F(2,49) = 8.558, P = 0.0006. Adjusted P values are as follows: i = 0.0343, ii = 0.0004. l, Fear extinction of SPF-Rag1−/− and GF-Rag1−/− mice in the single-session 30-tone fear extinction assay. n = 7 mice per group. Mean ± s.e.m.; AUC was calculated for each mouse within each group followed by unpaired two-sided t-test between groups. P value is shown.
Extended Data Fig. 4 Comparable transcriptomes of mPFCs dissected from control and ABX mice in the absence of fear conditioning and extinction.
a, PCA of genome-wide transcriptional profiles of mouse mPFC in the absence of fear conditioning and extinction. Control n = 3, ABX n = 4. PERMANOVA test was used: F = 2.52, Df = 1, P = 0.17. b, Volcano plot of differential expression between control (negative log2FC) and ABX (positive log2FC) groups. DEGs (defined as FDR < 0.1, DESeq2 Wald test) are shown in red. c–f, Immunofluorescence staining of c-FOS (red) (c, e) and the density of c-FOS+ neurons (d, f) in the BLA (c, d) or IL (e, f) of control and GF mice 90 min after classical fear extinction session 3. Data pooled from two independent experiments. n = 6 mice per group. Mean ± s.e.m.; unpaired two-sided t-tests. P values are shown. Scale bar, 200 μm.
a, Proportion of expressing cells (dot size) and mean normalized expression of representative marker genes (columns) associated with the cell clusters shown in Fig. 2a (rows). Clusters are labelled with post facto annotation based on known marker genes. Ambiguous clusters expressing multiple canonical markers across cell types are annotated with both (for example, exPFC/astrocyte), and are likely to represent doublets. b, Number of significantly differentially expressed genes (z-test calculated on coefficients of mixed linear model, Bonferroni-corrected P < 10−7) by cluster after downsampling each cluster to 500 nuclei, ranked from highest to lowest (clusters of doublets and undetermined annotations not included). exPFC, glutamatergic excitatory neurons from the PFC; GABA, γ-aminobutyric acid (GABA)ergic interneurons; OPC, oligodendrocyte progenitor cells; MO, myelinating oligodendrocytes.
Extended Data Fig. 6 Differential gene expression between control and ABX mice in individual clusters of mPFC cells.
Differential expression of ABX versus control (log2FC) in each cluster in Fig. 2a and the associated significance. Blue, genes that are significantly differentially expressed (z-test calculated on coefficients of mixed linear model, Bonferroni-corrected P < 10−7).
Extended Data Fig. 7 Differentially expressed genes in ABX versus control mPFC samples shared by all excitatory neuronal subsets.
Mean fold change in expression in excitatory neurons (columns) from Fig. 2a of genes (rows) that were significantly differentially expressed (z-test calculated on coefficients of mixed linear model, Bonferroni-corrected P < 10−7) in at least 2 of these clusters, and with absolute log2FC ≥ 0.31 in at least 1 cluster.
Extended Data Fig. 8 Differentially expressed genes in ABX versus control mPFC samples shared by multiple cell types.
Mean fold change in expression across all cell clusters (columns) from Fig. 2a of genes (rows) that were significantly differentially expressed (z-test calculated on coefficients of mixed linear model, Bonferroni-corrected P < 10−7) in at least 4 clusters, and with absolute log2FC ≥ 0.31 in at least 1 cluster.
a, Population frequencies and numbers of microglia in control and GF mice. b, Representative flow cytometry histogram and mean fluorescence intensity (MFI) of F4/80 staining on microglia from control and GF mice. c, Representative flow cytometry plots and population frequencies of CSF1R+ microglia in control and GF mice. d, Representative flow cytometry histogram and MFI of CSF1R expression gated on CSF1R+ microglia from control and GF mice. Data in a–d are representative of three independent experiments, n = 4 mice per group. e, Population frequencies and numbers of microglia in control and ABX mice. f, Representative flow cytometry histogram and MFI of F4/80 staining on microglia from control and ABX mice. g, Representative flow cytometry plots and population frequencies of CSF1R+ microglia in control and ABX mice. h, Representative flow cytometry histogram and MFI of CSF1R expression gated on CSF1R+ microglia from control and ABX mice. Data in e are pooled from two independent experiments, n = 8 mice per group. Data in f–h are representative of two independent experiments, n = 4 mice per group. Mean ± s.e.m.; unpaired two-sided t-test. P values shown.
a, Enzyme-linked immunosorbent assay (ELISA) quantification of plasma corticosterone in control and ABX mice. Data pooled from three independent experiments. Control n = 12; ABX n = 11. b, ELISA quantification of plasma corticosterone in control and GF mice. Data pooled from three independent experiments. Control n = 12; GF n = 11. Mean ± s.e.m. c, Structures of phenyl sulfate, pyrocatechol sulfate, 3-(3-sulfooxyphenyl)propanoic acid and indoxyl sulfate. d, Relative abundances of phenyl sulfate, pyrocatechol sulfate, 3-(3-sulfooxyphenyl)propanoic acid and indoxyl sulfate in faecal samples from Ctrl_fostered, GF and ex-GF_fostered mice as determined by LC–MS. n = 3 mice per group. e, Relative abundances of phenyl sulfate, pyrocatechol sulfate, 3-(3-sulfooxyphenyl)propanoic acid and indoxyl sulfate in CSF samples from control and GF mice as determined by LC–MS. Data are representative of two independent experiments, n = 8 mice per group. Mean ± s.e.m.; unpaired two-sided t-test. P values shown. f, Schematic of the microbiota–gut–brain axis in fear extinction learning. Our data inform a model in which alterations in the microbiota and their metabolites influence neuronal function and learning-related plasticity, which may be due to altered microglia-mediated synaptic pruning, and subsequently regulate fear extinction behaviour.
Gene ontology (GO) enrichment analyses of the differentially expressed genes between ABX and control mPFC samples in Fig. 1f.
Gene ontology (GO) enrichment analyses of the differentially expressed genes shared by excitatory neuronal subsets in Fig. 2a.
Gene ontology (GO) enrichment analyses of the differentially expressed genes shared by multiple cell types in Fig. 2a.
Gene ontology (GO) enrichment analyses of the differentially expressed genes in microglia in Fig. 2c.
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Chu, C., Murdock, M.H., Jing, D. et al. The microbiota regulate neuronal function and fear extinction learning. Nature 574, 543–548 (2019). https://doi.org/10.1038/s41586-019-1644-y
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