Gut-brain axis and ADHD

With 100 million neurons, the intestinal nervous system contains about as many as the spinal cord. Both are therefore independent nervous systems.
Most intestinal nervous system neurons are located in:¹

• Plexus myentericus Auerbach (in the muscle wall)
• Plexus submucosus Meissner (adjacent to the mucosa).

Specialized neurons of the intestinal nervous system exert an influence within the intestine (promoting or inhibiting depending on the transmitter and receptor):¹

• Motor skills (different movement patterns)
• Secretion (water, electrolytes, hormones)
• Perfusion (vascular tone, stimulating (vasodilation) or inhibiting (vasoconstriction) blood flow)
• Resorption
• Signal substance formation

The gut microbiome also has far-reaching interactive effects on the human body:²

• gastrointestinal
  • Metabolism
  • Nutrient intake
    • Carbohydrates
    • Proteins
    • Bile acid
    • Vitamins
    • other bioactive compounds
• non-gastrointestinal (especially during the child’s developmental period and then irreversibly, already prenatally through the maternal microbiome)
  • Brain development
  • Maturation of the immune system
  • Maturation of the neuroendocrine system

The enteric nervous system is connected to the body and the brain via the vagus nerve.
Influence vagal afferents:³

• anxiety-like and anxiety-related behavior
• Left-right differentiation and reversal learning
• sensorimotor gating (pre-pulse inhibition)
• Attention control
  • for associative learning
  • with conditioned taste aversion
• gene expression in the nucleus accumbens
• the effects of L. reuteri on the social behavior of autism mouse models (improvement is prevented by vagotomy)

The gut-brain axis plays a role in brain development, particularly in infancy, early childhood and childhood.⁴ The mother’s microbiome, the type of birth and the environment influence the child’s microbiome. Breastfeeding and a healthy diet provide the child’s gut with important probiotic elements, while antibiotics can disrupt the intestinal flora. The intestinal flora also influences neurogenesis.⁴ Microbiota are necessary for normal stress response, anxiety-like behaviors, social behavior and cognition and regulate the homeostasis of the central nervous system via immune function and the integrity of the blood-brain barrier⁵
Stress can significantly influence the gut-brain axis.³ The bidirectional communication of the gut-brain axis includes the central nervous system (CNS), the autonomic nervous system (ANS), the enteric nervous system (ENS) and the HPA axis (stress axis) ⁶⁷
In adulthood, the gut microbiome is relatively stable.⁸

Since microbiota can not only improve but also worsen symptoms / behaviors^{910 11 12} , it is strongly recommended not to take microbiota without caution. Anything that works can also do harm. In addition, genetic disposition and diet can differentiate between the advantages and disadvantages of an increased or decreased microbiome component. Sometimes the effect of an intestinal bacterium even depends on the strain used.
We find the fact that there are almost no studies reporting a worsening of symptoms or neurophysiological parameters due to the administration of microbiota disconcerting. Since the administration of microbiota resulting in an increase in the relative abundance of one bacterial species is inevitably accompanied by a relative decrease in the abundance of other bacterial species, we see a risk of biased reporting here or that the changes may in fact not be the result of the administration of certain bacterial species, but of an associated other effect, such as a change in the microbiome itself, possibly due to an increase in diversity¹³

• 1. Gut-brain axis
  • 1.1. Microbiome
  • 1.2. Microbiota / intestinal bacteria
  • 1.3. Intestinal neurons and nerves connecting to the brain
    • 1.3.1. Vagus nerve
    • 1.3.2. Spinal nerves
    • 1.3.3. Spinal cord
• 2. What influences the gut-brain axis
  • 2.1. Vagotomy
  • 2.2. Antibiotics
  • 2.3. Vagus nerve stimulation
  • 2.4. Food
    • 2.4.1. Short-chain fatty acids, SCFA
    • 2.4.2. Probiotics
  • 2.5. Fecal transplantation
  • 2.6. Intestinal flora and stress
• 3. Pathways of the gut microbiome on the brain
  • 3.1. Metabolic / neuroendocrine pathway
    • 3.1.1. Modulation of neurotransmitters by the microbiome
    • 3.1.2. Immune system pathway and microbiome
    • 3.1.3. Inflammation pathway
  • 3.2. HPA axis is influenced by the microbiome
  • 3.3. Nerve pathway of the microbiome
  • 3.4. Epigenetics and the microbiome
  • 3.5. MiRNA (microRNA) and microbiome
• 4. Gut microbiota in ADHD
  • 4.1. Gut bacteria as a possible causal cause of ADHD?
  • 4.2. Microbiome and short-chain fatty acids (SCFA) in ADHD
  • 4.3. Gut microbiota differences in ADHD
    • 4.3.1. Reduced intestinal bacteria in ADHD
    • 4.3.2. Increased intestinal bacteria in ADHD
    • 4.3.3. Alpha diversity
    • 4.3.4. Beta diversity
    • 4.3.5. Gut microbiota similar in ADHD and ASD
    • 4.3.6. Urinary microbiota in ADHD
• 5. Intestinal microbiome and other disorders
  • 5.1. Microbiota against anxiety / depression
    • 5.1.1. Studies on rodents
    • 5.1.2. Studies on humans
  • 5.2. Microbiota against ASS
    • 5.2.1. Studies on rodents
    • 5.2.2. Studies on humans
  • 5.3. Microbiota and multiple sclerosis
  • 5.4. Mirobiota and systemic lupus erythematosus (SLE)

1. Gut-brain axis¶

1.1. Microbiome¶

Trillions of microorganisms (“microbiota”) live in the human body, which together with their genome are called the “microbiome”. The microbiome is the totality of all bacteria, archaea, fungi, parasites, viruses and protozoa as well as their genes and metabolic products.
The microbiota of the digestive tract comprises more than 100 trillion microorganisms from 300-3000 different species. Together, these have over 200 times as many genes as humans.²
The composition of the microbiome is different for every person and is constantly changing.

1.2. Microbiota / intestinal bacteria¶

Intestinal bacteria mainly include the six major phyla:²

• Bacteroidetes (dominant)
• Proteobacteria (dominant)
• Actinomycetes
• Verrucomicrobia
• Fusobacteria

1.3. Intestinal neurons and nerves connecting to the brain¶

There are two types of sensory nerves within the intestine:¹⁴

• extrinsic primary afferent neurons
  The somata of these nerve cells are located outside the intestine
  in humans: 50,000
• intrinsic primary afferent neurons (IPANs)
  The somata of these nerve cells are located within the intestinal wall
  in humans: 100,000,000

Certain bacteria and bacterial components in the intestinal lumen can modulate both the extrinsic and intrinsic intestinal sensory systems and thereby influence peristalsis, nociception, brain chemistry and mood.¹⁴

The means of communication used include¹⁴

• Serotonin
• Substance P
• Somatostatin
• Cholecystokinin (CCK)
• GABA
• ATP
• Glucagon-like peptide-1 (GLP-1)
• Peptide YY (PYY)
• Hormones
  • Leptin
  • Orexin

The following microbiota metabolites increased serotonin synthesis in the intestine in vitro:¹⁵

• α-Tocopherol
• Butyrate
• Cholate
• Deoxycholate
• p-Aminobenzoate
• Propionate
• Tyramine

1.3.1. Vagus nerve¶

The communication of the gut-brain axis is bidirectional. The brain influences the motor, sensory and secretory functions of the gastrointestinal tract top-down via efferent fibers of the vagus nerve. The gut influences the function of the brain bottom-up, in particular the amygdala and hypothalamus, via the afferent vagal fibers.¹⁶

The afferent fibers of the vagus nerve present in all layers of the intestinal wall do not cross the epithelial layer of the intestine, so that the luminal microbiota cannot interact with them directly. SCFA can cross the epithelial barrier and activate the chemoreceptors of the vagus nerve.¹⁷
After stimulation of microbial pattern recognition receptors on their luminal side, enteroendocrine cells also release paracrine factors that can activate the chemoreceptors of the vagus nerve, such as¹⁷

• Serotonin (5-HT)
• Cholecystokinin (CCK)
• Glucagon-like peptide-1 (GLP-1)
• Peptide YY (PYY)

In addition, intrinsic primary afferent neurons (IPAN) of the intestinal nervous system (which make up the majority of sensory fibers innervating the intestinal mucosa) could receive microbial signals and subsequently modulate vagal activity via intramural synaptic transmission.¹⁷

The vagus nerve has a nicotinic intramural sensory synapse that can receive signals from IPAN. The majority of vagal afferent action potentials elicited by L. rhamnosus depend on this intramural synaptic transmission and can be interrupted by nicotinic or complete synaptic blockade.¹⁸

The vagus nerve is the tenth pair of cranial nerves and the longest and most widespread pair. The vagus nerve consists of 20 % efferent and 80 % afferent fibers and includes sensory and motor nerve fibers. The sensory neurons of the vagus nerve project centrally into the brain stem and end at the nucleus tractus solitarius and the nucleus tractus spinalis of the trigeminal nerve. The motor neurons originate from the ambiguous nucleus of the medulla oblongata and the dorsal nucleus of the vagus nerve.¹⁹

1.3.2. Spinal nerves¶

In addition to the vagus nerve, spinal nerves also connect the intestinal nervous system with the brain. Spinal afferents are particularly relevant in relation to the microbial modulation of visceral pain perception.¹⁴

One of the most important pain receptors in the intestine is the transient receptor potential vanilloid 1 (TRPV1). TRPV1 is expressed in the gastrointestinal tract mainly in spinal and vagal primary afferent neurons. L. reuteri DSM 17938 decreased the firing frequency of nociceptive spinal fibers but not the firing frequency of vagal fibers in the mesenteric nerve bundle. This is probably due to a potent specific direct or indirect blockade of TRPV1 ion channels in extrinsic spinal primary sensory fibers and their corresponding DRG cell bodies. In contrast, the antinociceptive effect of the L. rhamousus JB-1 strain was independent of TRPV1 antagonism.¹⁴

1.3.3. Spinal cord¶

In addition to the vagus nerve and the spinal nerves, the spinal cord also serves as an afferent connection from the intestinal nervous system to the brain.¹⁴

2. What influences the gut-brain axis¶

An imbalance in the gut microbiome can be caused by a variety of factors such as caesarean delivery, a diet high in fat and sugar, psychological stress, infections and antibiotics.⁸

2.1. Vagotomy¶

If the vagus nerve, which connects the intestinal nervous system with the brain, is surgically interrupted (vagotomy), this causes behavioral changes:³

• Increase in psychiatric disorders
• neurogenic bowel disorders more common²⁰
• reduced locomotor activity during the dark phase of rodents
• elevated noradrenaline blood plasma levels
  • basal
  • after immobilization stress
• reduced proliferation and survival of newborn cells, reduced number of immature neurons
• Activation of microglia in the dentate gyrus of the hippocampus

In the case of Salmonella typhimurium-induced enteritis, which causes anxiety-like behavior, a unilateral cervical vagotomy²¹

• reduced anxiety-like behavior
• reduced neuronal activation in the nucleus of the tractus solitaire and in the amygdala
• Attenuation of abnormal glial cell activation in the hippocampus and amygdala
• reduced serum endotoxin levels
• Increase in Salmonella concentration in the spleen reduced
• altered expression of inflammatory cytokines (including IL-6, IL-1β and TNF-α) in the gastrointestinal tract and brain
• reduced expression of IL-22 and CXCL1
• increased levels of beneficial intestinal microbiota (including Alistipes and Lactobacillus)
• increased GABA synthesis in the intestine
  • Administration of GABA replicated the positive effects of vagotomy on reducing intestinal inflammation and anxiety-like behavior in infected mice
  • Blockade of GABA receptors by picrotoxin abolished the protective effect of vagotomy against intestinal inflammation without affecting anxiety-like behavior

2.2. Antibiotics¶

Antibiotics change the intestinal microbiota. They influence their effects on the brain not only in children, but also in adults.²²

Oral administration of the antibiotics neomycin and bacitracin together with the antifungal agent primaricin in adult BALB/c mice²³

• temporary change in the composition of the intestinal microbiota
• increased urge to explore
• reduced anxiety
• BDNF mirror
  • reduced in amygdala
  • increased in the hippocampus
• Fecal transplantation into another mouse strain caused similar behavioral changes in this strain

Mice that were continuously treated with an antibiotic cocktail from weaning (postnatal day 21) showed:²⁴

• impoverished and restructured intestinal microbiota
• less anxiety-like behavior
• fewer cognitive deficits
• increased tryptophan and reduced kynurenine levels in serum
• reduced expression of BDNF, oxytocin and vasopressin in the brain

An administration of the antibiotic vancomycin in the early phase of life (4th to 13th postnatal day) in rats showed that²⁵

• fear-like behavior unchanged
• cognitive performance unchanged
• long-term increase in visceral hypersensitivity only in males
• reduced alpha-2-adrenoceptors and TRPV1 in the lumbo-sacral section of the spinal cord in adulthood

Rifaximin prevented chronic stress-induced visceral hypersensitivity, mucosal inflammation and impaired mucosal barrier function in rats²⁶

• this correlated with an increase in Lactobacillus in the ileum

Neomycin, on the other hand, did not prevent visceral hypersensitivity²⁶

2.3. Vagus nerve stimulation¶

Vagus nerve stimulation is approved in Europe for the treatment of drug-resistant epilepsy and refractory depression.
For vagus nerve stimulation, electrodes are surgically implanted under general anesthesia on the left vagus nerve and a generator and cable are implanted under the skin.

Vagus nerve stimulation influences / works for:³

• the regulation of mood
• the perception of pain (chronic pain)
• Crohn’s disease
• certain epilepsies
• increases neurogenesis in the hippocampus of adults
• modulates the release of noradrenaline, 5-HT and dopamine in brain regions associated with anxiety and depression
• increases the expression of BDNF in the hippocampus, which improved depression-like behaviors in animals with chronic immobilization stress
• influences the reward behavior of mice

2.4. Food¶

High-energy food seems to promote reactive aggressive behavior in combination with certain bacterial strains on the one hand and with ADHD on the other.²⁷

2.4.1. Short-chain fatty acids, SCFA¶

The primary functions of the microbiota include²⁸

• Protection against pathogens by increasing mucus production and thus stabilizing the intestinal-blood barrier
• Support for the immune system
• Production of vitamins
• Production of short-chain fatty acids (SCFAs) from indigestible carbohydrates (“dietary fiber”).

A low-fiber diet reduces SCFA levels, as do antibiotics.

SCFA can activate the chemoreceptors of the vagus nerve. The afferent fibers of the vagus nerve present in all layers of the intestinal wall do not cross the epithelial layer of the intestine, so that the luminal microbiota cannot interact with them directly. SCFA can cross the epithelial barrier.¹⁷

Short-chain fatty acids are:

Abbreviation of the fatty acid	Trivial name	Systemic name	Trivial name salt/ester	Systemic name salt/ester	Chemical formula	Typical plasma value29
C1:0 (no SCFA)	Formic acid	Methanoic acid	Formates	Methanoates	HCOOH
C2:0	Acetic acid	Ethanoic acid	Acetates	Ethanoates	CH3COOH	64 μM
C3:0	Propionic acid	Propanoic acid	Propionates	Propanoates	CH3CH2COOH	2.2 μM
C4:0	Butyric acid	Butanoic acid	Butyrate	Butanoate	CH3(CH2)2COOH	0.54 μM
C4:0	Isobutyric acid	2-Methylpropanoic acid	Isobutyrate	2-Methylpropanoate	(CH3)2CHCOOH	0.66 μM
C5:0	Valeric acid	Pentanoic acid	Valerate	Pentanoate	CH3(CH2)3COOH	0.18 μM
C5:0	Isovaleric acid	3-Methylbutanoic acid	Isovalerate	3-Methylbutanoate	(CH3)2CHCH2COOH	0.40 μM
C6:0	Caproic acid	Hexanoic acid	Capronate	Hexanoate	CH3(CH2)4COOH	0.34 μM

In humans, acetates, propionates and butyrates make up 95% of SCFA, in a ratio of 3:1:1.³ A more recent study found other and more detailed differences, which are shown in the table above²⁹

SCFAs corrected the changes that chronic psychosocial stress caused in the gut-brain axis.³⁰ SCFAs

• mitigated the changes in reward behavior triggered by psychosocial stress
• increased the ability to react to an acute stressor
• increased the in vivo intestinal permeability
• had an antidepressant effect
• had an anxiolytic effect
• did not influence the stress-related increase in body weight

2.4.2. Probiotics¶

There is evidence that when prebiotics are administered, an effect (in the brain) only occurs after several weeks, comparable to the effect of antidepressants.³¹ Several weeks after discontinuation, some, but not all, neurotransmitter levels in the brain had returned to their original value.

2.5. Fecal transplantation¶

Rodents raised germ-free have:¹⁴

• an altered brain chemistry
• increased permeability of the blood-brain barrier
• an underdeveloped intestinal nervous system (ENS)
• reduced peripheral 5-HT production
• altered intestinal motility and physiology
• numerous deficits of the immune system.

Nevertheless, they are particularly suitable for investigating the effects of fecal transplants and have shown that the transfer of gut microbiota into germ-free animals can transfer behavioral traits of the donor animals.¹⁴
Fecal transplantation from depressed individuals to rats with depleted microbiota caused anxious behavior and changes in tryptophan metabolism.³²

Fecal transplants of 5 boys with ADHD to rats also triggered ADHD symptoms in them, while this was not the case for 8 boys without ADHD.³³

Fecal transplantation from healthy rats to Lister hooded rats (an ADHD model animal that showed more hyperactivity, impulsivity and inattention than SHR) reduced hyperactivity in transplant recipients.³⁴

2.6. Intestinal flora and stress¶

Microbiota are necessary for normal stress response, anxiety-like behaviors, social behavior and cognition and regulate central nervous system homeostasis via immune function and blood-brain barrier integrity⁵

The vagus nerve and HPA axis influence each other.
Vagal nerve stimulation³

• increased the expression of CRF mRNA in the hypothalamus of rodents
• markedly increased the plasma levels of ACTH and corticosterone

In animal models, psychological stress increased the permeability of the intestine and caused a relocation of intestinal bacteria into the host. The activation of the immune response due to exposure to bacteria and bacterial antigens beyond the epithelial barrier causes proinflammatory cytokine secretion and ultimately activates the HPA axis.³⁵

The gut microbiome is essential for the development and function of the HPA axis (stress axis).³⁶²
Germ-free reared mice show an exaggerated HPA axis response and reduced sensitivity to negative feedback signals. Bifidobacterium infantis given early reversed this response.²

Early childhood stress influences the microbiome.³⁷

3. Pathways of the gut microbiome on the brain¶

Intestinal bacteria (intestinal microbiome, intestinal flora) influence the nervous system via various mechanisms.³⁸

3.1. Metabolic / neuroendocrine pathway¶

Metabolic / neuroendocrine pathway:¹⁶²

3.1.1. Modulation of neurotransmitters by the microbiome¶

The microbiome modulates neurotransmitters³⁸ such as GABA, serotonin, dopamine, noradrenaline¹⁶

• direct synthesis²
• indirectly via biosynthetic pathways of the host organism
  • Synthesis of precursors of neurotransmitters (e.g. for dopamine)²
• by secretion of short-chain fatty acids (SCFAs)¹⁶. These:
• activate microglial cells³⁹
• influence the permeability of the blood-brain barrier⁴⁰
• Suppression of the synthesis of the pro-inflammatory TNF by converting L-histidine into the immunoregulatory histamine (L. reuteri)⁴¹

Bacteria can synthesize neurotransmitters and hormones and react to them:¹⁶

The production of dopamine, noradrenaline and serotonin in intestinal neurons does not mean that the neurotransmitters synthesized in this way reach the brain.

• Blood-brain barrier
  Acetylcholine can cross the blood-brain barrier. However, dopamine, noradrenaline, serotonin and GABA cannot, which means that these latter neurotransmitters produced in the gut do not directly change the levels in the brain.
• extracellular vesicles (EV) can cross the blood-brain barrier
  Intestinal microbiota - like other bacteria - produce extracellular vesicles (EV).⁴⁷ If these originate from gram-negative bacteria, EV are called “outer membrane vesicles (OMV)”.⁴⁸
  These nano-sized EVs (20 to 1000 nm) can penetrate the inner mucus layer, enter the bloodstream and cross the blood-brain barrier to the brain.^495051525354
  95 % of serotonin is produced by enterochromaffin cells in the intestine, 5 % in the brain. Akkermansia muciniphila, which makes up around 5 % of intestinal bacteria and in which a deficiency correlates with various chronic inflammatory bowel diseases such as colitis and Crohn’s disease, influenced serotonin levels in the hippocampus of mice via its EVs.⁴⁹ Another study shows that extracellular vesicles of Escheria coli enter the hippocampus.⁵⁵
• Axonal transport
  We wonder whether neurotransmitters synthesized in the gut could be transported to the brain via the vagus nerve. So far there is no evidence for this. However, there is evidence that nerve fibers of the vagus nerve contain dopamine.⁵⁶ Furthermore, peripheral nerves such as the vagus nerve can transport nanoparticles into the brain.⁵⁷ Synuclein can also be transported from the body into the brain via nerves, which may be of interest with regard to the development of Parkinson’s disease.⁵⁸ In cohort studies, a truncal vagotomy showed a significant protective effect against Parkinson’s disease.⁵⁹⁶⁰
• Influencing the prodrug balance
  Intestinal bacteria influence the blood levels of the precursors of dopamine, noradrenaline, serotonin and GABA, which can cross the blood-brain barrier. Consequences are that the blood level of the precursors could influence the amount of neurotransmitters synthesized from them in the brain. For example, a slight increase in Bifidobacterium in the gut, as found in ADHD, is associated with increased production of cyclohexadienyl dehydratase, which is a precursor to phenylanaline, which is a precursor to dopamine. At the same time, the increase in Bifidobacterium is thought to be associated with reduced reward anticipation, suggesting reduced dopamine levels in the striatum.⁶¹ How these two seemingly contradictory pathways fit together is not yet clear to us.

3.1.2. Immune system pathway and microbiome¶

The microbiome influences the immune system via circulating cytokines.¹⁶²

3.1.3. Inflammation pathway¶

An increased ratio of Phyla Firmicutes to Bacteroidetes is associated with increased levels of inflammation in the intestinal tissue.⁸⁶²⁶³
Reduced abundance of butyrate-producing Faecalibacterium prausnitzii (phylum Firmicutes) correlated with reduced low-grade inflammation in obese individuals and patients with type 2 diabetes.⁶⁴⁶⁵⁶⁶ Obesity is considered an inflammatory condition. Obesity is also a major risk factor for the development of gallstones. Gallstones are associated with an increased Firmicutes to Bacteroidetes ratio. However, gallstones themselves are not associated with increased levels of inflammation. , which in our view raises the question of whether the Firmicutes to Bacteroidetes ratio is the direct pathway for inflammation.⁶⁷⁶⁷

ASA is said to be associated with a significantly higher Firmicutes to Bacteroidetes ratio, among other changes in microbiota diversity.⁸ While two of the sources available to us found an increased Firmicutes to Bacterioides ratio in ASA⁶⁸⁶⁹ and one source reported a strongly increased Clostridium species⁷⁰, which represents at least one - albeit very specific - sub-case of the Firmicutes strain, another of the sources cited⁷¹ did not comment on this topic and a further source cited reported a reduced Firmicutes to Bacteroidetes ratio (fewer Firmicutes, more Bacteroidetes) in ASA.⁷².
Our further searches found a systematic review that found an increased Firmicutes to Bacteroidetes ratio in ASA⁷³ and a meta-analysis that found increased Firmicutes and increased Bacteriodetes in ASA (meta-analysis, k = 18, n = 897)⁷⁴.

3.2. HPA axis is influenced by the microbiome¶

The microbiome influences HPA axis activity. Germ-free (without mirobiota) mice have an overactivated HPA axis with an increased release of stress hormones such as corticosterone and ACTH during restraint stress.⁷⁵

3.3. Nerve pathway of the microbiome¶

The microbiome influences the brain via the neural pathway.²

• by stimulation of the vagus nerve:¹⁶⁷⁶⁴⁶
  • The vagus nerve has 80 % afferent fibers, which transmit sensory stimuli from the body to the brain, and 20 % efferent fibers, which transport motor signals from the brain to the body.¹
• by means of the enteric nervous system²

3.4. Epigenetics and the microbiome¶

Influence on epigenetic processes in the brain (neuroepigenetic)⁷⁷

• e.g. through SCFA (short-chain fatty acids); these influence
  • Acetylation and butyrylation of histone proteins⁷⁸
    • facilitates binding of transcription factors to DNA and thus transcription of genes
  • post-translational modification of histones (crotonylation, butyrylation)⁷⁹⁸⁰⁸¹
  • Neuroplasticity (e.g. in the visual cortex of adult mice)⁸²
  • Gene expression in cortical astrocytes⁸³
• DNA methylation⁷⁷
  • Changes in DNA methylation occur during learning and memory consolidation
  • the one-carbon metabolic pathway⁸⁴ of DNA methylation is regulated by the availability of cofactors. Some of these (such as cobalamin, folate, pyridoxine and riboflavin) are metabolic products of intestinal bacteria
  • The gut microbiome influences DNA methylation in intestinal epithelial cells
  • Influence of the gut microbiome on DNA methylation and transcription in the brain has not yet been proven.

3.5. MiRNA (microRNA) and microbiome¶

Lack of gut microbiota correlates with changes in the expression of various miRNAs, particularly in the amygdala and PFC.⁷⁷

miRNA regulate e.g: ⁷⁷

• the dendritic morphology
• the spine density in the neurons of the hippocampus
• visual cortical plasticity (by influencing spine remodeling)
• cortical plasticity

4. Gut microbiota in ADHD¶

4.1. Gut bacteria as a possible causal cause of ADHD?¶

One study found evidence of a causal relationship between gut bacteria and ADHD.⁸⁵ (Note: Even if causality were confirmed, it should be assumed that this is only one of many different possible ways in which ADHD can develop and would therefore not apply to all people with ADHD)

One study found that mice whose guts were contaminated with gut bacteria from people with ADHD had structural changes in the brain (white matter, gray matter, hippocampus, internal capsule), decreased connectivity between motor and visual cortices right in the resting state, and higher anxiety than mice in which gut bacteria from people without ADHD were used.⁸⁶

A single case study reported an improvement in ADHD symptoms in a young woman with gut bacteria replacement related to a recurrent Clostridioides difficile infection.⁸⁷

Taxonomic differences were found in the microbiota of adolescents and young adults with ADHD compared to healthy controls:⁶¹

• An abundance of actinobacteria was found in persons with ADHD
• the capacity of the intestinal microbiome to produce monoamine precursors (phenylalanine) was increased
• the abundance of cyclohexadienyl dehydratase (CDT) genes in the microbiome involved in phenylalanine production correlated negatively with reward anticipation responses in the ventral striatum (with ventral striatum activation for reward anticipation reduced in ADHD)

A study of urine and fecal samples using 1H nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry found gender-specific patterns in the metabolic phenotype in ADHD⁸⁸

• Urine profile
  • Hippurate (a product of microbial host co-metabolism that can cross the blood-brain barrier)
    • increased (men only)
    • correlated negatively with IQ (in men)
    • correlated with fecal metabolites associated with microbial metabolism in the gut.
• Fecal profile (independent of ADHD medication, age and BMI)
  • Stearoyl-linoleoyl-glycerol increased
  • 3,7-Dimethylurate increased
  • FAD increased
  • Glycerol-3-phosphate reduced
  • Thymine reduced
  • 2(1H)-quinolinone reduced
  • Aspartate reduced
  • Xanthine reduces
  • Hypoxanthine reduces
  • Orotate reduced

Gut microbiome and dopamine

• The gut microbiome influences dopamine levels in the PFC and striatum of rodents²
• A slight increase in Bifidobacterium in the gut, as found in ADHD, is thought to be associated with increased production of cyclohexadienyl dehydratase, which is a precursor to phenylanaline, which is a precursor to dopamine. At the same time, the increase in Bifidobacterium is thought to be associated with reduced reward anticipation, suggesting reduced dopamine levels in the striatum.⁶¹ How these two seemingly contradictory pathways fit together is not yet clear to us.
• Gut microbiome composition correlates with impulsivity, increased striatal D1R and decreased D2R with increasing susceptibility to alcohol dependence⁸⁹
• Intestinal inflammation can impair the dopamine metabolism⁹⁰
  • An infection with Citrobacter rodentium triggers an inflammatory bowel disease in mice. This affected not only the intestinal microbiota but also the brain dopamine metabolism. An additional administration of MPTP (a precursor of the neurotoxin 1-methyl-4-phenylpyridinium (MPP+), which can trigger Parkinson’s symptoms by damaging dopaminergic neurons) compared to the administration of Citrobacter rodentium or MPTP alone:
    • worsened behavioral performance
    • increased dopaminergic degeneration and overactivation of glial cells in the nigrostriatal signaling pathway
    • increased the expression of TLR4 and NF-κB p65 in the colon and striatum
    • increased the expression of pro-inflammatory cytokines.

4.2. Microbiome and short-chain fatty acids (SCFA) in ADHD¶

Studies on short-chain fatty acids found reduced SCFA blood levels in ADHD:⁹¹⁹²

• Adults with ADHD
  • Formic acid reduces
  • Acetic acid reduces
  • Propionic acid reduces
  • Succinic acid reduced (C4H6O, an aliphatic dicarboxylic acid; food additive number E 363)
• Children with ADHD
  • Formic acid lower than in adults
  • Propionic acid lower than in adults
  • Isovaleric acid lower than in adults
• Antibiotic medication in the last 2 years caused
  • Formic acid reduces
  • Propionic acid reduces
  • Succinic acid reduces
• current stimulant use in children caused
  • Acetic acid reduces
  • Propionic acid reduces

4.3. Gut microbiota differences in ADHD¶

Studies have found deviations in the intestinal flora of children with ADHD compared to those not affected.²⁸ In contrast, the intestinal flora differs only slightly between ADHD and ASD.⁹³
ADHD correlated with leaky gut, neuroinflammation and overactivated microglial cells. The colonic microbiota exhibits a pro-inflammatory shift and harbors more gram-negative bacteria that contain immune-triggering lipopolysaccharides in their cell walls.⁹⁴

Disorders of the developing gut microbiota early in life can affect neurological development and potentially lead to adverse mental health outcomes later in life.⁹⁵
75 infants were randomly assigned to receive either Lactobacillus rhamnosus GG or a placebo in the first 6 months of life. After 13 years, ADHD or ASD was found in 17% of the placebo group and none in the probiotic group. Bifidobacteria were significantly reduced in the intestinal microbiome of the children with ADHD in the first 6 months of life.⁹⁶⁹⁷

It is known that caesarean section compared to vaginal birth and bottle feeding compared to breastfeeding (especially in the first three months) changes the composition of the infant’s microbiome and increases the risk of ADHD. More on this at Caesarean section In the chapter Birth circumstances as a cause of ADHD and Bottle feeding increases (up to + 270 %), breastfeeding reduces ADHD risk (- 23 % to -74 %) in the chapter Stressful physical or emotional childhood experiences as a cause of ADHD

4.3.1. Reduced intestinal bacteria in ADHD¶

4.3.2. Increased intestinal bacteria in ADHD¶

Changes in the frequency of Ruminococcaceae_UGC_004 correlated with inattention.¹²³

4.3.3. Alpha diversity¶

Alpha diversity (e.g. Shannon and Simpson index) indicates the degree of heterogeneity of taxa within a sample.⁸¹²⁴

Study results on the alpha diversity of the gut microbiota in ADHD are inconsistent.
We will collect study results on this topic here.

Meta-analyses, reviews:

• Several meta-analyses found no change in alpha diversity in ADHD.
  • (k = 4, n = 619 adults)¹⁰¹
  • (k = 9).⁸
  • also a review¹²⁵

Studies:

• unchanged alpha diversity^{98126 104 105}
  • in mice with ADHD⁸⁶
• significantly lower α-diversity (Shannon index, observed species, Faith PD index)¹²⁷

4.3.4. Beta diversity¶

Beta diversity (e.g. Jaccard distance) indicates the degree of dissimilarity of taxa between samples.⁸¹²⁴

Study results on beta diversity of the gut microbiota in ADHD are inconsistent, with a lack of correlation not as clear as with alpha diversity.
We will collect study results on this topic here.

Meta-analyses, reviews:

• A meta-analysis (k = 4, n = 619 adults) found a significant correlation between beta diversity and ADHD in 3 of the 4 studies.¹⁰¹
• no deviating beta diversity¹²⁵
• A meta-analysis (k = 9) found few differences in beta diversity between people with and without ADHD, mainly in children and adolescents⁸

Studies:

• A study on n = 73 subjects found a trend towards the significance of β-diversity (weighted UniFrac).¹²⁸
• no relevant change in beta diversity¹²⁶
• altered beta diversity in ADHD-C, but not in ADHD-C⁹⁸
• increased beta diversity in mice with ADHD⁸⁶
• differences were found in both taxonomic and functional beta diversity in n = 136 adults¹²⁹
• Children with ADHD (n = 63), had a higher incidence of psychostimulant use (n = 33)¹²⁹
  • significantly different taxonomic beta diversity
  • lower functional and taxonomic uniformity
  • lower frequency of the strain Bacteroides stercoris CL09T03C01
  • lower frequency of bacterial genes coding for an enzyme of vitamin B12 synthesis
  • higher plasma levels of the vascular inflammation markers sICAM-1 and sVCAM-1

4.3.5. Gut microbiota similar in ADHD and ASD¶

The gut microbiota in ADHD and ASD are quite similar in both alpha and beta diversity and differ significantly from non-affected individuals.
In addition, a subgroup of ADHD and ASD cases had increased levels of lipopolysaccharide-binding protein, which positively correlated with interleukin IL-8, IL-12 and IL-13, compared to non-affected children. This suggests an intestinal barrier disorder and immune system dysregulation in a subgroup of children with ADHD or ASD.¹³⁰

Germ-free mice that received a fecal transplant from persons with ADHD subsequently showed¹³¹

• characteristic autistic behaviors
• alternative splicing of ASD-relevant genes in the brain

When ASD model mice received appropriate microbial metabolites, this improved the behavioral abnormalities and modulated neuronal excitability in the brain.¹³¹

4.3.6. Urinary microbiota in ADHD¶

A study of the urinary microbiome in ADHD found:¹³²

• a lower alpha diversity in the urine bacteria of the ADHD group
  • reduced Shannon and Simpson indices (p < 0.05)
• significant differences in beta diversity
• were common with ADHD:
  • Phyla Firmicutes
  • Actinobacteriota
  • Ralstonia (genus)
  • Afipia (genus)
• less frequently with ADHD:
  • Phylum Proteobacteria
  • Corynebacterium (genus)
  • Peptoniphilus (genus)
• Afipia correlated significantly with the Child Behavior Checklist Attention Problems score and the DSM-oriented ADHD subscale

5. Intestinal microbiome and other disorders¶

5.1. Microbiota against anxiety / depression¶

5.1.1. Studies on rodents¶

Lacticaseibacillus rhamnosus JB-1 (Lactobacillus rhamnosus) caused

• only in non-vagotomized mice:⁴⁶
  • GABA(B1b) mRNA
    • increased expression in the ACC and prelimbic cortex
    • reduced expression in the hippocampus, amygdala and locus coeruleus
  • GABA(Aα2) mRNA
    • increased in the hippocampus
    • reduced in the amygdala
  • Corticosterone stress response reduced
  • Reduced anxiety behavior
  • Depressive behavior reduced
• prevented the behavioral changes caused by chronic unpredictable mild stress in rats¹³³
  • reduces anxiety-like behavior
  • depression-like behavior reduced
  • Decrease in glutamine and glutathione levels in the hippocampus avoided
  • Decreases in taurine in the hippocampus.

Acute and chronic administration of the short-chain fatty acid sodium butyrate in addition to fluoxetine significantly reduced immobility values in mice in the tail suspension test by 20-40% compared to administration of only one of the two drugs¹³⁴
Injection of sodium butyrate caused

• short-term histone hyperacetylation in the hippocampus and in the PFC
• a transient, at least 50% increase in the BDNF transcript in the PFC

5.1.2. Studies on humans¶

Daily administration of a combination of L. helveticus and B. longum in a double-blind, randomized, placebo-controlled study in healthy women and men reduced perceived stress, anxiety and depression levels and 24-hour urinary cortisol levels slightly but statistically significantly.¹³⁵

Daily administration of Lactobacillus casei Shirota in a double-blind, randomized, placebo-controlled study in patients with chronic fatigue significantly reduced anxiety symptoms, but not depressive symptoms.¹³⁶

5.2. Microbiota against ASS¶

5.2.1. Studies on rodents¶

Fructo-oligosaccharides and galacto-oligosaccharides reduced chronic stress-induced social avoidance, cognitive dysfunction, anhedonia, HPA axis hyperreactivity, and anxiety- and depression-like behavior in mice.¹³⁷

Limosilactobacillus reuteri administration caused

• only in non-vagotomized mice
  • Change in social behavior in autism mouse model³
  • Improvement of social behavior in different autism mouse models¹³⁸
    • via the vagus nerve and oxytocinergic and dopaminergic signaling in the brain
      • not by restoring the composition of the gut microbiome per se
    • by induction of synaptic plasticity in the VTA of ASA mice
      • by (via L. reuteri) increased oxytocin¹³⁹¹⁴⁰ in various ASA mouse models:
        • genetic ASD mouse models
          • Shank3B-/-¹⁴¹¹³⁸
          • Cntnap2-/-¹⁴²
            • L. reuteri levels are reduced in Cntnap2-/- mice
            • L. reuteri remedied the deficits of the oxytocin-producing neurons
            • L. reuteri corrected the social deficits in both young and adult Cntnap2-/- (KO-I) mice
            • L. reuteri did not eliminate the hyperactivity typical of Cntnap2-/- mice
        • environmental ASS mouse models
          • VPA¹³⁸
          • GF¹³⁸
          • MHFD¹³⁸
          • Lipopolysaccharides (LPS) (unclear whether via oxytocin)¹⁴³ (Lacticaseibacillus rhamnosus also had an effect here)
        • idiopathic ASD mouse model (BTBR)¹³⁸
      • not in mice with missing or blocked oxytocin receptors in dopaminergic neurons

In another study, ASA also correlated with reduced plasticity in the VTA, which was caused by a high-fat diet in the mother. The ASA behavior was transferred from the offspring to germ-free mice.¹³⁹

In a preclinical ASD mouse model, only L. reuteri strain ATCC-PTA-6475, but not strain DSM-17938, improved social deficits.¹⁴⁴

A study on Cntnap2-/- and Cntnap2+/+ mice found that both mouse lines showed the hyperactivity known from this ASD mouse model. In the Cntnap2+/+, however, social behavior was normalized, as was the microbiome. This also indicates that the ASD-typical changes in social behavior are caused by the microbiome ¹⁴²

The study results with regard to L. reuteri in ASD are impressive. The question mentioned by and in the introduction as to which part of the improvement is due to the administration of specific biota (here: L. reuteri) and which is due to the increase in the diversity of the microbiome through the administration of microbiota should have little relevance here. The argument that people with ADHD could have impaired intestinal flora diversity due to restricted food preferences is certainly worth considering, but should only apply to humans and not to mice. In any case, selective feeding habits of ASD model mice have not been reported so far.
The same applies to possible influences on the oral bacterial flora due to sensory stress, which makes tooth cleaning more difficult.¹⁴⁵

B. fragilis improves deficits in communicative, stereotypic, anxiety-like and sensorimotor behavior in an ASD mouse model in which ASD was caused by prenatal virus administration. It also corrected intestinal permeability and altered the microbiome composition.¹⁴⁶

5.2.2. Studies on humans¶

A double-blind, randomized, placebo-controlled study on the effect of L. reuteri in children with ASD found:¹⁴⁴

• significantly improved social functioning across various metrics
• no change from
  • ASA overall severity
  • repetitive behaviors
  • Microbiome composition
  • Immune profile

A study on children with ASD reports a positive effect of an elimination diet on gastrointestinal complaints and a prebiotic on social behavior.¹⁴⁷

5.3. Microbiota and multiple sclerosis¶

In one study, Limosilactobacillus reuteri improved the severity of experimental autoimmune encephalomyelitis in mice, which serves as a model for multiple sclerosis, indicating a deficiency of L. reuteri in MS.¹¹ In contrast, two other studies found an excess of L. reuteri as a risk factor for MS.¹¹¹²
It is possible that the gut microbiota and diet could act synergistically in people genetically susceptible to MS, with diet-dependent gut microbial metabolites serving as a key mechanism for the disease. Due to these gene-gut microbiota interactions, specific microbial taxa could have different effects depending on MS risk alleles or other gene polymorphisms, which are also highly dependent on dietary intake. This could explain why results differ across geographic, dietary and genetic conditions. Prophylactic or therapeutic modulation of the gut microbiome to prevent or treat MS would therefore require careful and personalized consideration of genetic makeup, gut microbiota composition and dietary habits.¹⁴⁸ This is consistent with the ability of L. helveticus to attenuate anxiety-like behavior in mice only as a function of genotype diet.¹⁴⁹

Applied to other disorders, contradictory results of effects of certain microbiota on certain symptoms should be regarded as a warning against careless testing of the effect on individuals.

5.4. Mirobiota and systemic lupus erythematosus (SLE)¶

In the Toll-like receptor 7 (TLR7) mouse model of systemic lupus erythematosus (SLE), L. reuteri increased autoimmune manifestations under specific-pathogenic and gnotobiotic conditions, particularly through an increase in plasmacytoid dendritic cells and interferon signaling. The incidence and translocation of L. reuteri to other organs could be reduced by resistant starch in the diet using SCFA. Resistant starch also reduced plasmacytoid dendritic cells, interferon signaling and mortality.¹⁵⁰