Introduction

A rapidly growing body of evidence points to a role of the microbiota in the regulation of brain and behaviour. The microbiota–gut–brain axis represents a bidirectional network of communication between the intestinal microbiota and the brain (Rhee et al., 2009; Bercik et al., 2012; Cryan & Dinan, 2012; Dinan & Cryan, 2012; Sampson & Mazmanian, 2015). The gut microbiota is required for development of the hypothalamic–pituitary–adrenal (HPA) axis, optimal stress responsivity and social cognition (Neufeld et al., 2011; Clarke et al., 2013; Desbonnet et al., 2014). Dysregulation of the microbiota–gut–brain axis may contribute to the development of psychiatric and gastrointestinal diseases, a link supported by the comorbidity found between anxiety disorders and irritable bowel syndrome (Fond et al., 2014) as well as the abnormal composition of gut microbiota in patients with autism (Mayer et al., 2014).

Germ‐free mice (GF; microbiota‐deficient from birth) have provided critical insights into the role of the microbiota in regulating brain function (Luczynski et al., 2016). We and others have previously shown that GF mice exhibit exaggerated HPA axis responses to acute stressors, reduced anxiety‐like behaviours and deficits in social cognition (Sudo et al., 2004; Heijtz et al., 2011; Neufeld et al., 2011; Clarke et al., 2013; Desbonnet et al., 2014; Arentsen et al., 2015), effects which are influenced by the amygdala and hippocampus (Sah et al., 2003; Ledoux, 2007; Tovote et al., 2015). Indeed, signalling between the basolateral amygdala (BLA) and the ventral hippocampus modulates both anxiety and social behaviours (Felix‐Ortiz et al., 2013; Allsop et al., 2014; Felix‐Ortiz & Tye, 2014).

Studies have documented altered amygdalar and hippocampal structure in both humans and animals with exaggerated stress responses. Changes in amygdalar and hippocampal volume have been associated with anxiety disorders in humans and with early‐life stress in rodents (De Bellis et al., 2000; Isgor et al., 2004; Salm et al., 2004; Salzer & Weniger, 2010; Machado‐de‐Sousa et al., 2014). There is also evidence showing long‐lasting dendritic hypertrophy of excitatory neurons in the BLA of rodents repeatedly exposed to stressors (Vyas et al., 2002, 2004, 2006). In contrast, repeated stress induces dendritic atrophy in hippocampal neurons (Magariños & McEwen, 1995a; Vyas et al., 2002). Thus, although the mechanisms underpinning the ability of the gut microbiota to influence stress responsivity and behaviour remain to be elucidated, many lines of evidence point to the amygdala and hippocampus as likely targets.

This study is based on the hypothesis that the GF phenotype could be due to morphological changes in amygdalar and hippocampal subregions with implications for neural output. To this end we determined if the presence of a gut microbiota is necessary for normal structure of the amygdala and hippocampus. We firstly utilized design‐based stereology to compare the volumes of well‐defined brain regions between adult GF and conventionally colonized (CC) animals. Secondly, we evaluated the dendritic morphology of single neurons in the BLA and hippocampus, regions known to undergo neuronal remodelling and to regulate both anxiety and social behaviours (Vyas et al., 2002; Allsop et al., 2014).