Gut microbes and the developing brain

The discovery that commensal gut microbiota can influence host development and physiology beyond the gastrointestinal (GI) tract has triggered a paradigm shift in our conceptualization of the origin of human diseases. A growing body of preclinical research has demonstrated that gut microbiota exert a modulatory role on the development and function of brain circuits involved in motor control, emotion and cognition. These findings have lent support to the hypothesis that gut bacteria may play a role in the etiology and/or pathophysiology of human brain disorders. The current challenge is to understand the precise mechanisms mediating the communication between the microbiota and the brain. In the present thesis, we used a combination of mouse models (e.g., germ-free; GF, antibiotic treated, and transgenic mice), molecular, biochemical, and behavioral approaches to gain a deeper insight into the role of gut microbiota on brain development and behavior. A major goal was to explore whether microbial products from the commensal gut microbiota can be translocated into the developing brain and be sensed by pattern recognition receptors (PRRs) of the innate immune system. In Paper I, we took advantage of the GF mouse model (mice raised throughout development in an environment devoid of bacteria) to study the influence of gut microbiota on social behavior. Using the three-chamber social approach task, we demonstrated significant differences in social approach behavior between GF and conventionally raised mice (specific pathogen-free, SPF). Adult GF Swiss-Webster mice displayed higher levels of sociability than SPF mice, as indicated by a stronger preference for time spent close to the unfamiliar stimulus mouse versus the novel object. In addition, they showed reduced expression levels of total BDNF and BDNF exon-containing transcripts I-, IV-, VI-, and IX in the amygdala, a brain region involved in the processing of social stimuli. These findings suggest that alterations in the expression of specific BDNF exon transcripts within the amygdala may contribute to the abnormal development of social behavior in GF mice. In Paper II, we investigated whether antibiotic-induced perturbation of the maternal gut microbiota during pregnancy influences brain development and behavior of the offspring. The juvenile offspring of antibiotic-treated dams showed hyperactivity and sex-specific changes in social behavior (similar to that observed in GF mice), without changes in body weight. In addition, the male juvenile offspring had reduced BDNF mRNA and protein expression in the amygdala. Interestingly, we found a negative correlation between time spent interacting with an unfamiliar stimulus mouse and levels of BDNF protein. These findings in mice indicate that antibiotic-induced perturbation of the maternal gut microbiota during pregnancy has profound effects on brain development leading to abnormal motor and social development of the offspring. In Paper III, we examined the possibility that fragments of bacterial peptidoglycan (PGN), a major component of the bacterial cell wall, derived from commensal gut microbiota can cross the blood brain barrier under normal conditions and influence the developing brain via activation of PRRs. Using various expression-profiling techniques (i.e., qRT-PCR, Western Blot and immunohistochemistry), we showed that two families of PRRs that specifically detect PGN and its derivates (PGN recognition proteins and NOD-like receptors), and the PGN transporter PepT1 are highly expressed in the developing brain during critical windows of postnatal development. In addition, we demonstrated that the expression of several of these PGN-sensing molecules are sensitive to manipulation of the gut microbiota (i.e., GF conditions and antibiotic exposure in early life). Finally, we demonstrated that the absence of PGN recognition protein 2 (Pglyrp2; which is an N-acetylmuramyl-L-alanine amidase that hydrolyzes bacterial PGN between the sugar backbone and the peptide chain) leads to sexspecific changes in social behavior in the prepubertal period. However, we did not observe changes in motor or anxiety-like behavior at this age. These novel findings support the notion that central activation of PRRs by bacterial PGN fragments could be one of the signaling pathways mediating the communication between the gut microbiota and the developing brain. In Paper IV, we tested the hypothesis that the modulatory role of PGN recognition proteins (PGRPs) on behavior changes with age, by using Pglyrp2 knockout (KO) mice. Using a battery of behavioral tests, we demonstrated sex-dependent alterations in motor and anxiety-like behavior in 15-month-old Pglyrp2 KO mice, as well as mild changes in the expression of synaptophysin (a presynaptic marker) and gephyrin (a protein associated with inhibitory synapses) in key brain regions implicated in the processing of emotional stimuli. These observations indicate that the mammalian Pglyrp2 plays an important role in the modulation of brain circuits involved in motor control and anxiety in later life. In summary, this thesis provides conceptually novel evidence that the central activation of PRRs by bacterial PGN fragments could be one of the signaling pathways mediating the communication between the gut microbiota and the developing brain. This new signaling pathway may be a new entry point for the exploration of the role of gut microbiota on brain development, function and behavior. Finally, we propose that alterations within different components of this signaling pathway could lead to deviations in brain developmental trajectories, thus increasing risk for neurodevelopmental and psychiatric disorders

Fluoxetine Exerts Age-Dependent Effects on Behavior and Amygdala Neuroplasticity in the Rat

The selective serotonin reuptake inhibitor (SSRI) Prozac® (fluoxetine) is the only registered antidepressant to treat depression in children and adolescents. Yet, while the safety of SSRIs has been well established in adults, serotonin exerts neurotrophic actions in the developing brain and thereby may have harmful effects in adolescents. Here we treated adolescent and adult rats chronically with fluoxetine (12 mg/kg) at postnatal day (PND) 25 to 46 and from PND 67 to 88, respectively, and tested the animals 7–14 days after the last injection when (nor)fluoxetine in blood plasma had been washed out, as determined by HPLC. Plasma (nor)fluoxetine levels were also measured 5 hrs after the last fluoxetine injection, and matched clinical levels. Adolescent rats displayed increased behavioral despair in the forced swim test, which was not seen in adult fluoxetine treated rats. In addition, beneficial effects of fluoxetine on wakefulness as measured by electroencephalography in adults was not seen in adolescent rats, and age-dependent effects on the acoustic startle response and prepulse inhibition were observed. On the other hand, adolescent rats showed resilience to the anorexic effects of fluoxetine. Exploratory behavior in the open field test was not affected by fluoxetine treatment, but anxiety levels in the elevated plus maze test were increased in both adolescent and adult fluoxetine treated rats. Finally, in the amygdala, but not the dorsal raphe nucleus and medial prefrontal cortex, the number of PSA-NCAM (marker for synaptic remodeling) immunoreactive neurons was increased in adolescent rats, and decreased in adult rats, as a consequence of chronic fluoxetine treatment. No fluoxetine-induced changes in 5-HT1A receptor immunoreactivity were observed. In conclusion, we show that fluoxetine exerts both harmful and beneficial age-dependent effects on depressive behavior, body weight and wakefulness, which may relate, in part, to differential fluoxetine-induced neuroplasticity in the amygdala

Host microbiota modulates development of social preference in mice

Background: Mounting evidence indicates that the indigenous gut microbiota exerts long-lasting programming effects on brain function and behaviour. Objective: In this study, we used the germ-free (GF) mouse model, devoid of any microbiota throughout development, to assess the influence of the indigenous microbiota on social preference and repetitive behaviours (e.g. self-grooming). Methods and results: Using the three-chambered social approach task, we demonstrate that when adult GF mice were given a choice to spend time with a novel mouse or object, they spent significantly more time sniffing and interacting with the stimulus mouse compared to conventionally raised mice (specific pathogen-free, SPF). Time spent in repetitive self-grooming behaviour, however, did not differ between GF and SPF mice. Real-time PCR–based gene expression analysis of the amygdala, a key region that is part of the social brain network, revealed a significant reduction in the mRNA levels of total brain-derived neurotrophic factor (BDNF), BDNF exon I-, IV-, VI-, IX-containing transcripts, and NGFI-A (a signalling molecule downstream of BDNF) in GF mice compared to SPF mice. Conclusion: These results suggest that differential regulation of BDNF exon transcripts in the amygdala by the indigenous microbes may contribute to the altered social development of GF mice

Effect of fluoxetine on body weight in adolescent and adult rats.

<p>Data are presented as mean ± S.E.M. body weight (g) in adolescent rats (A) and adult (B) rats (n = 10). 21 Days of fluoxetine treatment had no effect on bodyweight of adolescent rats, but reduced bodyweight in adult rats. ^*p<0.05 fluoxetine <i>versus</i> methylcellulose in age group.</p

Effect of fluoxetine on exploratory behavior in adolescent and adult rats.

<p>Data are presented as mean ± S.E.M. of distance moved within 60 min (n = 10). 7 Days following chronic fluoxetine treatment (12 mg/kg) exploratory behavior in the open field was not affected in adolescent, nor adult, rats. ^ap<0.05 main age effect.</p

Effect of fluoxetine treatment on anxiety in adolescent and adult rats.

<p>Data are presented as mean ± S.E.M. of time spent in the open of the elevated plus maze (n = 10). 10 Days following chronic fluoxetine treatment (12 mg/kg) anxiety in the elevated plus maze test was increased in both adolescent and adult rats. ^#p<0.05 main treatment effect.</p

PSA-NCAM immunoreactivity in the dorsal raphe nucleus, mPFC, and amygdala of fluoxetine and methylcellulose treated adolescent and adult rats.

<p>Data are presented as mean ± S.E.M. of the numer of immunoreactive neurons in the dorsal raphe nucleus (A), mPFC (B), and amygdala (C) (n = 4–5) per 100×100 µm. 14–17 Days following chronic fluoxetine (12 mg/kg) treatment the number of PSA-NCAM immunoreactivity was lower in adult compared to adolescent rats, but only in the dorsal raphe nucleus. In addition, we obtained a significant age x treatment interaction for PSA-NCAM immunoreactivity in the amygdala, which tended to be increased in adolescent, and decreased in adult rats. ^ap<0.05 main age effect; ^∧p<0.05 age x treatment interaction.</p

Time schedule of experiments.

<p>Each group consisted of adolescent and adult rats treated with either fluoxetine or methylcellulose.</p

Effect of fluoxetine treatment on behavioral despair in adolescent and adult rats.

<p>Data are presented as mean ± S.E.M. of time spent on immobility (n = 10). 10 Days following chronic fluoxetine treatment (12 mg/kg) behavioral despair, expressed as immobility in the forced swim test, was increased in adolescent, but unaffected in adult rats. ^ap<0.05 main age effect; ^∧p<0.05 age x treatment interaction; ^*p<0.05 fluoxetine <i>versus</i> methylcellulose in age group.</p

Fluoxetine (ng/ml) and norfluoxetine (ng/ml) levels in blood plasma.

<p>Fluoxetine (ng/ml) and norfluoxetine (ng/ml) levels in blood plasma.</p