Transferring the blues: Depression-associated gut microbiota induces neurobehavioural changes in the rat
Introduction
Accumulating evidence from preclinical studies suggests that the gut microbiota can modulate brain activity and behaviour via neuroendocrine, neuroimmune, neural and humoral pathways (Cryan and Dinan, 2012, Dinan and Cryan, 2013). This emerging link between the gut microbiota and the central nervous system suggests that gut microbiota modification may have translational applications in the treatment of neuropsychiatric disorders (Cryan and Dinan, 2015, Desbonnet et al., 2014, Hsiao et al., 2013).
Depression is a common, often recurrent (Eaton et al., 2008) heterogeneous disorder responsible for significant disability worldwide (WHO, 2008). The complex aetiology, involves dysregulated neuroendocrine (Stetler and Miller, 2011) neuroimmune (Dowlati et al., 2010), metabolic (Jokela et al., 2014) and neurotransmitter systems (Berton and Nestler, 2006). Current pharmacological interventions are suboptimal (Fava, 2003) and there has been little progress in the identification of biomarkers.
Data from animal studies provides evidence that the gut microbiota may impact on the neurobiological features of depression (Park et al., 2013), such as low-grade immune activation (Bailey et al., 2011), hypothalamic-pituitary-adrenal axis (HPA) activity (Sudo et al., 2004), altered tryptophan metabolism (Clarke et al., 2013, El Aidy et al., 2012, O'Mahony et al., 2015, Yano et al., 2015), neurotrophic factors (Bercik et al., 2011), and neurogenesis (Möhle et al., 2016, Ogbonnaya et al., 2015).
A number of studies have shown that when the microbiome is transplanted from one animal (either stressed or obese) to another control animal it can significantly alter anxiety-like behaviours, a common comorbidity of depression (Bercik et al., 2011, Bruce-Keller et al., 2015).
Different lactobacillus and bifidobacteria species have been shown to modulate depression and stress-related behaviours in animal models (Bravo et al., 2011, Desbonnet et al., 2010, Savignac et al., 2015). Furthermore, a growing number of small studies in healthy individuals suggest pre- and probiotic consumption can positively affect aspects of mood and anxiety (Messaoudi et al., 2011, Steenbergen et al., 2015), modulate HPA function (Messaoudi et al., 2011, Schmidt et al., 2015) and alter brain activity (Tillisch et al., 2013). However, there are a paucity of studies in relevant clinical populations (Jiang et al., 2015, Naseribafrouei et al., 2014, Zheng et al., 2016).
We investigated alterations in the gut microbiota composition in patients with depression with respect to signature physiological alterations in HPA axis function, immune activation and altered tryptophan metabolism. Next, we aimed to identify the functional consequences of the gut microbiota alterations in depression by determining levels of fecal short chain fatty acids. We then assessed gut permeability as a potential mechanism by which gut bacteria may influence brain function (Julio-Pieper et al., 2014, Kelly et al., 2015).
Finally, to confirm that an altered gut microbiota specifically influences aspects of depressive symptomatology, we carried out a fecal microbiota transplantation from depressed patients to a microbiota depleted antibiotic rat model and assessed if a depressive-like phenotype emerged in the treated animals.
Approval of the study protocol was granted by the Cork University Hospital (CUH) ethics committee and written informed consent was obtained from all subjects. The study was carried out in accordance with the Declaration of Helsinki. Thirty four depressed patients were recruited from outpatient and inpatient psychiatric clinics. Thirty three healthy subjects matched for gender, age and ethnicity were recruited (See Supplementary Information).
Extracted Fecal DNA was prepared for sequencing on the Illumina Miseq platform. The concentration of SCFA was measured using a Varian 3800 GC flame ionization system, fitted with a ZB-FFAP column (30 m × 0.32 mm × 0.25 mm; Sigma) (See SI).
Participants were instructed to collect three saliva samples using Salivettes (Sarstedt AG and Co, Numbrecht, Germany) at the following time points: (t0) upon wakening, 30 min post wakening (t+30), and 150 min post wakening (t+150) (See SI).
Plasma Tryptophan and kynurenine pathway metabolites were determined as previously described (Clarke et al., 2009). (See SI).
Plasma levels of IL-6, IL-8, TNF-α, and CRP were assayed in duplicate using high sensitivity commercially available electrochemiluminescence MULTI-SPOT® Meso Scale Discovery kits (MSD, Rockville, MD, 75USA) (See SI).
LBP concentrations were determined using the Enzyme Immunoassay Kit for free human LBP (Enzo®, Life Sciences).
All experiments were in full accordance with the European Community Council Directive (86/609/EEC). Adult male Sprague-Dawley rats (n = 28) were used and maintained as described in SI. They were divided into control (n = 15) and depressed groups (n = 13) matched for average body weight. Rats were then given a cocktail of ampicillin and metronidazole (all at 1 g/L), vancomycin (500 mg/L), ciprofloxacin HCl (200 mg/L), imipenem (250 mg/L) once daily for 28 consecutive days in drinking water. Seventy-two hours later, animals were colonized via daily oral gavage of donor microbiota (300 μL) for 3 days. Donor microbiota was acquired from pooled fecal samples from 3 of the most severely depressed male patients and 3 age and sex matched healthy controls. To offset potential confounder and/or cage effects and to reinforce the donor microbiota phenotype, booster inoculations were given twice per week throughout the study.
Sucrose preference; SP, Open field; OF, Elevated plus maze; EPM, Forced swim test FST were carried out as detailed in Supplementary Materials.
Rats were given 200 μl of 6% carmin red in 0.5% methylcellulose (in PBS) given by oral gavage. The cages were inspected every 10 min post gavage and the appearance of the first red fecal pellet recorded.
Plasma corticosterone levels were assayed using a commercially-available ELISA kit (Corticosterone EIA Kit, Enzo®, Life Sciences).
Plasma CRP was determined using commercially available RayBio® Rat CRP ELISA Kit. Cytokines were analyzed using a commercially available electrochemiluminescence multiplex system (MSD, Gaithersburg, MD, USA).
Plasma LBP concentrations were determined using the Enzyme Immunoassay Kit (Enzo®, Life Sciences).
The concentrations of SCFA were measured using a Varian 3800 GC flame-ionization system, fitted with a ZB-FFAP column (30 m × 0.32 mm x 0.25 μm; Phenomenex, Macclesfield, Cheshire, UK).
Data that were normally distributed according to Shapiro-Wilk test were analyzed using unpaired t tests. Outliers were removed by Grubbs' test. Data that were not normally distributed were transformed by square root transformation. Microbiota data were analyzed using non parametric tests. Benjamini-Hochberg procedure was used to correct for multiple comparisons with a FDR-adjusted p-value ≤0.1 considered significant. Statistical procedures were carried out using IBM SPSS 20.0. Graphs were generated using GraphPad Prism 5. Macronutrient data was generated using Diet Plan 6.
Section snippets
Demographic data and health indicators
Other than education level, employment status, smoking and alcohol consumption, there were no differences between the groups (Table 1). Clinical characteristics of the depressed patients are presented in (Table 2).
Daily Macronutrient Consumption similar in depressed patients and controls
We assessed Daily Macronutrient Consumption using a food frequency questionnaire (Table S1). Apart from Trans fats (t (61) = 2.06, p = 0.05) there were no significant differences in diet between the groups.
Proinflammatory profile in depression
The data in this study confirm that the depressed group had increased levels of
Discussion
The present findings represent definitive evidence that depression-associated alterations in the gut microbiome are sufficient to disrupt behavioural and physiological homeostasis. Specifically, transplantation of the perturbed microbiota signature from depressed patients to microbiota-depleted rats induced the development of some of the behavioural and physiological features of the depressive phenotype. Furthermore, this data indicates that a gut microbiota transfer from depressed patients
Conclusions
We show that depression is characterised by alterations in the gut microbiota. We have demonstrated that it is possible to reproduce aspects of depressed behaviour and physiology via a gut microbiota transfer. This suggests that the gut microbiota could play a causal role in the complex mechanisms underlying the development of depression. The profile of depression-like behaviours and physiological alterations noted following FMT suggests that this represents a novel paradigm in behavioural
Funding source
The APC Microbiome institute is funded by Science Foundation Ireland (SFI). This publication has emanated from research conducted with the financial support of Science Foundation Ireland (SFI) under Grant Number SFI/12/RC/2273.
Contributors
Dr Kelly and Dr Borre contributed to study design, data processing, data collection, data analysis, and manuscript preparation. Ciaran O’ Brien, Dr Patterson, Dr El Aidy, Dr Kennedy, Jennifer Deane, Sasja Beers, Dr Karen Scott, Dr Gerard Moloney and Alan E. Hoban contributed to data processing and data analysis. Dr Lucinda Scott and Patrick Fitzgerald contributed to data collection. Prof Ross, Prof Stanton, Dr Clarke, Prof Cryan and Prof Dinan contributed to study design, data analysis, and
Declaration of interest
TGD and JFC are also supported by the Irish Health Research Board (HRA_POR/2011/23) and (HRA_POR/2012/32), the Department of Agriculture, Food & the Marine and Enterprise Ireland. GC is supported by a NARSAD Young Investigator Grant from the Brian and Behaviour Research Foundation (Grant Number 20771). TGD and JFC are principal investigators in the APC Microbiome Institute, University College Cork. GC is a faculty member of the APC Microbiome Institute. The APC Microbiome Institute has
Acknowledgements
None.
References (66)
- et al.
Biological and psychological markers of stress in humans: focus on the trier social stress test
Neurosci. Biobehav. Rev.
(2014) - et al.
Human microbiota-associated mice: a model with challenges
Cell Host Microbe
(2016) - et al.
The effects of lofepramine and desmethylimipramine on tryptophan metabolism and disposition in the rat
Biochem. Pharmacol.
(1991) - et al.
Exposure to a social stressor alters the structure of the intestinal microbiota: implications for stressor-induced immunomodulation
Brain, Behav. Immun.
(2011) - et al.
Obese-type gut microbiota induce neurobehavioral changes in the absence of obesity
Biol. Psychiatry
(2015) - et al.
Characterization and functional significance of glucocorticoid receptors in patients with major depression: modulation by antidepressant treatment
Psychoneuroendocrinology
(2003) - et al.
Effects of the probiotic Bifidobacterium infantis in the maternal separation model of depression
Neuroscience
(2010) - et al.
A meta-analysis of cytokines in major depression
Biol. Psychiatry
(2010) Diagnosis and definition of treatment-resistant depression
Biol. Psychiatry
(2003)- et al.
Microbiota modulate behavioral and physiological abnormalities associated with neurodevelopmental disorders
Cell
(2013)
Altered fecal microbiota composition in patients with major depressive disorder
Brain, Behav. Immun.
Brain-gut-microbiota axis: challenges for translation in psychiatry
Ann. Epidemiol
Expression of toll-like receptors in peripheral blood mononuclear cells and response to cognitive-behavioral therapy in major depressive disorder
Brain, Behav. Immun.
Ly6Chi monocytes provide a link between antibiotic-induced changes in gut microbiota and adult hippocampal neurogenesis
Cell Rep.
Serotonin, tryptophan metabolism and the brain-gut-microbiome axis
Behav. Brain Res.
Early life stress alters behavior, immunity, and microbiota in rats: implications for irritable bowel syndrome and psychiatric illnesses
Biol. Psychiatry
Glucocorticoid receptors in major depression: relevance to pathophysiology and treatment
Biol. Psychiatry
Bifidobacteria modulate cognitive processes in an anxious mouse strain
Behav. Brain Res.
Antidepressant-like effects of the histone deacetylase inhibitor, sodium butyrate, in the mouse
Biol. Psychiatry
A randomized controlled trial to test the effect of multispecies probiotics on cognitive reactivity to sad mood
Brain, Behav. Immun.
Microbes & neurodevelopment–Absence of microbiota during early life increases activity-related transcriptional pathways in the amygdala
Brain, Behav. Immun.
Indigenous bacteria from the gut microbiota regulate host serotonin biosynthesis
Cell
Effects of acute paroxetine administration on tryptophan metabolism and disposition in the rat
Br. J. Pharmacol.
The intestinal microbiota affect central levels of brain-derived neurotropic factor and behavior in mice
Gastroenterology
New approaches to antidepressant drug discovery: beyond monoamines
Nat. Rev. Neurosci.
Ingestion of lactobacillus strain regulates emotional behavior and central GABA receptor expression in a mouse via the vagus nerve
Proc. Natl. Acad. Sci. U. S. A.
Tryptophan degradation in irritable bowel syndrome: evidence of indoleamine 2,3-dioxygenase activation in a male cohort
BMC Gastroenterol.
The microbiome-gut-brain axis during early life regulates the hippocampal serotonergic system in a sex-dependent manner
Mol. Psychiatry
Mind-altering microorganisms: the impact of the gut microbiota on brain and behaviour
Nat. Rev. Neurosci.
Gut microbiota: microbiota and neuroimmune signalling-Metchnikoff to microglia
Nat. Rev. Gastroenterol. Hepatol.
High-level adherence to a mediterranean diet beneficially impacts the gut microbiota and associated metabolome
Gut
Microbiota and host determinants of behavioural phenotype in maternally separated mice
Nat. Commun.
Microbiota is essential for social development in the mouse
Mol. Psychiatry
Cited by (1060)
The bidirectional relationship of depression and disturbances in B cell homeostasis: Double trouble
2024, Progress in Neuro-Psychopharmacology and Biological PsychiatryRole of soluble epoxide hydrolase in pain and depression comorbidity
2024, Neurobiology of DiseaseQuality of Life and Bidirectional Gut-Brain Interactions in Irritable Bowel Syndrome From Adolescence to Adulthood
2024, Clinical Gastroenterology and HepatologyCeliac disease and depressive disorders as nutritional implications related to common factors – A comprehensive review
2024, Behavioural Brain ResearchProbiotic, prebiotic, synbiotic and fermented food supplementation in psychiatric disorders: A systematic review of clinical trials
2024, Neuroscience and Biobehavioral Reviews