Elsevier

Journal of Psychiatric Research

Volume 82, November 2016, Pages 109-118
Journal of Psychiatric Research

Transferring the blues: Depression-associated gut microbiota induces neurobehavioural changes in the rat

https://doi.org/10.1016/j.jpsychires.2016.07.019Get rights and content

Abstract

The gut microbiota interacts with the host via neuroimmune, neuroendocrine and neural pathways. These pathways are components of the brain-gut-microbiota axis and preclinical evidence suggests that the microbiota can recruit this bidirectional communication system to modulate brain development, function and behaviour. The pathophysiology of depression involves neuroimmune-neuroendocrine dysregulation. However, the extent to which changes in gut microbiota composition and function mediate the dysregulation of these pathways is unknown. Thirty four patients with major depression and 33 matched healthy controls were recruited. Cytokines, CRP, Salivary Cortisol and plasma Lipopolysaccharide binding protein were determined by ELISA. Plasma tryptophan and kynurenine were determined by HPLC. Fecal samples were collected for 16s rRNA sequencing. A Fecal Microbiota transplantation was prepared from a sub group of depressed patients and controls and transferred by oral gavage to a microbiota-deficient rat model. We demonstrate that depression is associated with decreased gut microbiota richness and diversity. Fecal microbiota transplantation from depressed patients to microbiota-depleted rats can induce behavioural and physiological features characteristic of depression in the recipient animals, including anhedonia and anxiety-like behaviours, as well as alterations in tryptophan metabolism. This suggests that the gut microbiota may play a causal role in the development of features of depression and may provide a tractable target in the treatment and prevention of this disorder.

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.

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