The microbiota–gut–brain axis is an emerging concept in modern medicine informed by
the ability of gut microbiota to alter brain and behaviour.
1
Although some clinical studies have revealed altered gut microbiota composition in
patients with neurodevelopmental disorders such as autism,
2, 3
the specific contributions of microbiota in early life to the development and programming
of the various facets of social behaviour has not been investigated.
Germ-free (GF) mice have been critical in assessing the role of microbiota in all
aspects of physiology. Indeed, recent studies in GF mice report increases in neuroendocrine
responses to stress,
4, 5
altered neurotrophin levels in the hippocampus and amygdala,
5, 6, 7
reduced anxiety
5, 6, 7
and non-spatial memory,
8
and altered monoamine neurotransmitter levels in the brain.
5, 6
Interestingly, many of the deficits are specific to males
4
in which there are higher incidence rates of neurodevelopmental disorders relative
to females. Here, we examined the effects of GF rearing conditions through early life
and adolescence on social behaviour in adulthood.
Mice, like humans, are a social species and have a natural propensity to seek out
the security and pleasure afforded by stable social scenarios. Social motivation and
preference for social novelty in mice can be assessed in the three-chambered sociability
test.
9
Our initial findings in this test revealed significant social impairments in GF mice,
particularly in males, as indicated by a lack of the normal preference for time spent
in a chamber containing a mouse versus the alternative empty chamber (GF × chamber
interaction: F(1,57)=5.35, P<0.05; Supplementary Figures 1a–c). This was accompanied
by reduced preference for novel social situations, where GF mice did not demonstrate
the normal increase in time spent investigating a novel over a familiar mouse, which
resembles social cognition deficits observed in patients with neurodevelopmental disorders
(GF × chamber interaction: F(1,57)=5.86, P<0.01; Supplementary Figures 1d–f).
To substantiate these results and to assess the capacity for post-weaning bacterial
colonisation of the GF gut (GFC) to reverse the observed social deficits, we repeated
the test in a different male cohort. As expected, GF mice exhibited robust deficits
including social avoidance (GF × chamber interaction: F(1,20)=12.41, P<0.001; Figures
1a–c), and diminished preference for social novelty relative to conventionally colonised
(CC) mice (GF × chamber interaction: F(1,20)=4.45, P<0.05; Figures 1d–f). These effects
were not influenced by changes in general locomotor activity, as any decrease in chamber
entries was specific to the social chamber (Supplementary Figure 2). Intriguingly,
whereas GFC reversed the observed social avoidance, it had no effect on social cognition
impairments. This indicates that although the effects of GF rearing on the latter
behaviour are permanently established in the pre-weaning period, the development of
social avoidance in GF mice is more amenable to microbial-based interventions in later
life.
In addition to symptoms of reduced social motivation, children with autism exhibit
poor social and communication skills and repetitive behaviours. To establish whether
the degree of information transfer during social interaction is disrupted in GF mice,
performance in the social transmission of food preference test was assessed. GF mice
spent a decreased proportion of time engaged in social investigation (F(2,20)=7.51,
P<0.005; Figure 1g) and substantially greater proportion of time engaged in repetitive
self-grooming behaviour (F(2,20)=11.91, P<0.001; Figure 1h) during social interaction.
These behaviours were normalised following GF bacterial colonisation, confirming the
involvement of microbiota in modulation of these behaviours. However, despite the
reduction in social investigation times, the quality of information transfer during
the interaction was not affected in GF mice, as they displayed normal preference for
the novel food (food to which cage-mate was exposed prior to social interaction) in
the subsequent food choice test conducted immediately after the social interaction
and 24 h later (Figures 1i and j), indicating that the ability to process information
per se during social interaction is not affected in GF mice.
This study shows for, what is to our knowledge, the first time that microbiota are
crucial for the programming and presentation of distinct normal social behaviours,
including social motivation and preference for social novelty, while also being important
regulators of repetitive behaviours. Given that these facets of behaviour are impaired
in neurodevelopmental disorders such as schizophrenia and autism
10
and with a similar male preponderance, these data may have implications for our understanding
of the genesis of neurodevelopmental disorders of altered sociability. A better understanding
of the mechanisms underlying these social deficits, which may include modulation of
immune cell cytokines release, changes in vagal nerve activity and neuroendocrine
function, could potentially lead to the emergence of novel and more effective therapies
to combat symptoms in the social domain.