Given the current knowledge on the association between the gut microbiota and neurodevelopmental disorders via the gut-brain axis [29,30,31], we hypothesized that the microbiota of individuals with ADHD, when transplanted into young GF mice, would affect brain structure and/or function as well as behavior. We show that miceADHD had an impaired structural integrity of both white and gray matter, and showed decreased functional connectivity between the right visual cortex and right motor cortex.

In the current study, we investigated if there were any structural brain differences in miceADHD compared to micecontrol using diffusion tensor imaging (DTI). One of the brain regions selected was the hippocampus. The hippocampus is highly sensitive to aging, life experiences such as stress, and environmental factors like malnutrition and altered microbial composition [44,45,46]. The hippocampus is important for learning and memory, mood regulation, and neural plasticity [44]. It is therefore not surprising that impaired hippocampal function has been implicated in many neurodevelopmental and psychiatric disorders, including ASD, ADHD, and MDD [16, 47,48,49,50]. The DTI data revealed a decreased FA and increased MD in the right hippocampus, accompanied by increased RD and unchanged AD in miceADHD. This is indicative of decreased myelination rather than axonal damage or degeneration [42]. A decreased FA and increased MD have been reported in children and adults with ADHD in several brain regions, including the internal capsule, but not in the hippocampus [51, 52]. However, a smaller hippocampal volume has been reported in ADHD [15, 16].

We also observed white matter alterations in the right internal capsule. The internal capsule is composed of afferent and efferent myelinated fibers of the cerebral cortex [53]. Abnormalities in the structure of the internal capsule have been implicated in neurodevelopmental and psychiatric disorders like schizophrenia, ADHD, and bipolar disorder [52, 54, 55]. We report decreased FA and increased RD in miceADHD in the internal capsule, indicative of decreased myelination in this region. This finding may be related to the observed decreased functional connectivity between the right primary motor cortex (M1) and the right primary visual cortex (V1) of miceADHD because the fibers of these regions course through the internal capsule [56]. Both the primary motor cortex and visual cortex are part of the visuomotor network [57]. Reduced visuomotor adaptation has also been reported in schizophrenia and in individuals with ADHD [58, 59].

We used various behavioral tests to investigate diverse aspects of mouse behavior that might be influenced by microbiota from individuals with ADHD. One of the tests that the mice performed was the OFT. In this test, we observed that miceADHD showed more anxiety than micecontrol, which was measured as time spent in the corners of the open field. Although this behavior is not specific to ADHD, many psychiatric disorders, including ADHD, MDD, and ASD, are highly comorbid with anxiety [60,61,62]. Altered anxiety-related behavior is often found after microbiota manipulations, and this behavior can also be caused by fecal transplantation [19]. This suggests that components of the gut microbiota are able to regulate anxiety. We found no differences in motor activity between groups in either the OFT or in home cage activity during active and inactive phase of the mice. Additionally, no apparent effects were seen in the MBT assessing compulsivity/impulsivity nor in the memory assessing NOR task.

Previous studies have examined and discovered some differences in the microbiota composition between individuals with ADHD and controls [34, 35], although transplantation of the microbiota of individuals with ADHD into mice is novel. Therefore we investigated the microbial composition within and between samples and on different levels in the current study. The overall microbial composition, after transplantation, revealed a clear separation (beta-diversity) between microbiota from micecontrol and miceADHD (Fig. 1b). We did not observe, however, any significant differences in the alpha-diversity indices. Furthermore, in the global microbial composition, we found a difference in microbial community composition, with 31 genera showing different relative abundance between the animal groups. Most of these genera (23 of the 31) are also present in our human donors (ADHD and/or control), though we cannot be sure about the uncultured or unknown genera (6 out of 31). This means that our study has a translational value, especially as the microbial composition of both the original human samples and the mouse samples cluster per experimental group (ADHD and control).

One of the genera we found to differ between our groups was Lachnoclostridium. It appeared to be decreased in relative abundance in miceADHD. The relative abundance of Lachnoclostridium was also found decreased in children with ASD [63] and in ADHD patients compared to controls [34, 35]. Going up to the phylum level, the differentially abundant genera in our study belonged predominantly (24 out of 31) to the phylum Firmicutes, and families Lachnospiraceae and Ruminococcaceae, which have been associated with stress and social avoidance behavior [64, 65]. Ten genera of the Lachnospiraceae family were more abundant in miceADHD compared to micecontrol; in the latter group, three other Lachnospiraceae genera were enriched. Genera of the Lachnospiraceae family have been associated with immune responses and are able to control regulatory T cells [66].

Our correlation analysis showed that there was a positive correlation between white matter integrity and the relative abundance of Holdemania and Eubacterium. These bacteria are both known to be changed in abundances in other disorders: Holdemania is increased in individuals with MDD [67,68,69], and Eubacterium is decreased in children with ASD [70]. However, to our knowledge, this is the first study that found significant correlations between these bacteria and brain structural and functional changes. Based on our finding, it would therefore be of interest to investigate this further in the clinical populations of the mentioned disorders.

Changes in the relative abundance of the family Ruminococcaceae have been observed in multiple psychiatric and neurodevelopmental diseases including ADHD, ASD, and MDD [71, 72]. Our results show that three genera, Anaerostipes, Roseburia (both from the Lachnospiraceae family), and Ruminococcaceae UGC-004 (Family Ruminococcaceae), positively correlated with anxiety levels, while Eubacterium appeared to be negatively correlated to the severity of anxiety and positively with anxiety reduction. Importantly, these genera were also found to be more abundant in persons with ADHD, compared to controls, as part of our ongoing study of ADHD patients from the NeuroIMAGE study [73] (n = 56) and healthy participants (n = 49) [74].

This work should be considered in the light of its strengths and limitations. First, we only used six donor samples (three of individuals with ADHD and three of controls) in our study. While this low number of samples may possibly reduce the general distribution of taxonomic groups observable in adult individuals with ADHD, the samples used were carefully selected based only on the disease status (not on behavioral or MRI-based variables in these individuals). To reduce individual differences, samples were pooled before colonization (ADHD patients or controls). Additionally, the choice of collecting the fecal samples from adult male donors was based on the fact that the adult form of ADHD is thought to be the most severe outcome of this disorder [75] and to avoid sex effects and non-controlled hormonal effects, like the use of contraceptives and the menstrual cycle. These hormonal effects are likely to play a role in the shaping of the gut microbial composition, both in humans and mice [76,77,78,79], and therefore only male mice were colonized. Besides the hormonal effects, it is possible that unmeasured factors of our donors, e.g., body mass index, also influenced the microbial composition. Though, based on the inclusion criteria of the subjects in the NeuroImage cohort [73], we are confident that the subjects in this study were physically healthy.

Second, we used germ-free mice, housed in isolators, as opposed to antibiotic-treated mice. While this approach limited our behavioral testing options (in terms of equipment), the sterile isolators used in this study provided a contamination-free environment at the time of behavioral testing. Additionally, this allowed us to make sure that the animals were only exposed to the microorganisms from the donor samples, and competition between the native and newly transplanted micro-organisms in germ-free animals was absent, which allows for the study of a defined microbial composition [80]. However, germ-free mice show various developmental and physiological differences when compared to conventionally raised animals. For example, germ-free mice show decreased anxiety and decreased expression of genes that are connected to synaptogenesis [12, 81]. Nevertheless, germ-free mice, housed in isolators, still seem to be the best controlled animal model to study the impact of microbial transplantation [80]. Moreover, by including a group of germ-free mice that received the transplantation from humans without ADHD, a positive control was present. However, it might be interesting to add a third group of germ-free mice that is not colonized (negative control or mock) in future studies, to be able to investigate baseline behavioral (i.e., anxiety) and brain structural and functional indices.

Third, we analyzed the relative microbial composition using 16S rRNA sequencing data. This approach is preferred for comparison across different samples (i.e., different strains of mice), treatments, or timepoints. Additionally, 16S rRNA sequencing techniques are more cost-efficient than metagenomics sequencing; it shows lower signal distortion due to host contamination [82] and has many well-developed analytical tools available [83]. Moreover, we (i) used mock communities in the sequencing experiment in order to check for the “true” bacterial distribution (which validated our taxonomic distribution), (ii) focused on common taxa, and (iii) applied a stringent QC for the statistical analysis to reduce the chance of false positives. However, the choice of sequencing primers (V1-V2 variable region) includes a bias in the sensitivity to identify specific bacterial taxa, for example, the Bifidobacteria and Faecalibacterium that were observed to differ between ADHD patients and controls [34, 35], could not be measured properly in our samples. Therefore, sequencing the same region of the 16S rRNA gene and the use of similar analysis pipelines is needed in order to properly compare studies in the best possible way.