The Gut-Brain Axis and ADHD

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

• Auerbach’s myenteric plexus (in the muscle wall)
• Meissner’s submucosal plexus (adjacent to the mucosa).

Specialized neurons in the enteric nervous system exert effects within the intestine (either excitatory or inhibitory, depending on the neurotransmitter and receptor):¹

• Motor skills (various movement patterns)
• Secretion (water, electrolytes, hormones)
• Perfusion (vascular tone, which either stimulates (vasodilation) or inhibits (vasoconstriction) blood flow)
• Resorption
• Signal molecule production

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

• gastrointestinal
  • Metabolism
  • Nutrient absorption
    • Carbohydrates
    • Proteins
    • Bile acid
    • Vitamins
    • other bioactive compounds
• non-gastrointestinal (particularly during childhood development, at which point it becomes irreversible; influenced even before birth by 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.
Vagal afferents influence:³

• anxiety-like and anxiety-related behavior
• Distinguishing Between Left and Right and Learning to Reverse Directions
• sensorimotor gating (pre-pulse inhibition)
• Attention control
  • in associative learning
  • in cases of conditioned taste aversion
• gene expression in the nucleus accumbens
• the effects of L. reuteri on the social behavior of mouse models of autism (the improvement is blocked by vagotomy)

The gut-brain axis plays a role in brain development, particularly during infancy, early childhood, and childhood.⁴⁵ The mother’s microbiome, the mode of delivery, and the environment influence the child’s microbiome. Breastfeeding and a healthy diet supply the child’s gut with important probiotic elements, while antibiotics can disrupt or destroy the gut flora. Furthermore, the gut flora influences neurogenesis.⁵ Through vaginal birth, infants acquire a microbiota rich in Lactobacillus spp. that resembles the mother’s vaginal and fecal flora. With a cesarean section, infants develop a microbiota dominated by Staphylococcus, Corynebacterium, and Propionibacterium spp. and resembling the skin flora. Thereafter, the diversification of the microbiome is primarily shaped by diet. Breastfeeding leads to an increased prevalence of certain Bifidobacterium species in infants, which diversifies after weaning. In contrast, infants fed formula typically show a lower prevalence of Bifidobacteria and a higher abundance of coliforms, Bacteroides, and Clostridium difficile.⁶

Microbiota are essential for a normal stress response, anxiety-like behaviors, social behavior, and cognition, and they regulate the homeostasis of the central nervous system through immune function and the integrity of the blood-brain barrier.⁷
Stress can significantly affect the gut-brain axis.³ The bidirectional communication of the gut-brain axis involves the central nervous system (CNS), the autonomic nervous system (ANS), the enteric nervous system (ENS), and the HPA axis (stress axis).⁸⁹
In adulthood, the gut microbiome is relatively stable.¹⁰

Since the microbiota can not only improve but also worsen symptoms and behaviors^{1112 13 14} , it is imperative to warn against taking microbiota supplements without due consideration. Anything that works can also cause harm. In addition, genetic predisposition and diet can determine whether an increase or decrease in a specific microbiome component results in benefits or drawbacks. Sometimes, the effect of a gut bacterium even depends on the specific strain used.
We find it puzzling that there are virtually no studies reporting a worsening of symptoms or neurophysiological parameters following the administration of microbiota. Since the administration of microbiota, which has consequences for the relative abundance of one bacterial species, inevitably leads to a relative decrease in the abundance of other bacterial species, we see a risk here of biased reporting, or that the changes may in reality not be the consequence of the administration of specific bacterial species, but rather of another associated effect, such as a change in the microbiome itself, possibly due to an increase in diversity.¹⁵

1. Gut-Brain Axis¶

1.1. Microbiome¶

The human body is home to more than 100 billion⁶to trillions of microorganisms (“microbiota”), which, together with their genomes, are called the “microbiome.”¹⁶ The microbiome is the totality of all bacteria, archaea, fungi, parasites, viruses, and protozoa, as well as their genes and metabolic products.¹⁷
The microbiota of the digestive tract comprises more than 100 quadrillion² to 100 quintillion ¹⁸ microorganisms from 300 to 3,000 different species. Together, these have 100¹⁸ to 200² times as many genes as humans.
The composition of the microbiome is different for every person and is constantly changing.

1.2. Microbiota / Gut Bacteria¶

Intestinal bacteria mainly belong to the following six major phyla:²

• Bacteroidetes (dominant)
• Proteobacteria (dominant)
• Actinomycetes (actinobacteria)
• Verrucomicrobia
• Fusobacteria
• as well as Firmicutes⁶

1.3. Intestinal neurons and nerves connecting to the brain¶

There are two types of sensory nerves within the intestine:¹⁹

• extrinsic primary afferent neurons
  The cell bodies of these nerve cells are located outside the intestine
  in humans: 50,000
• intrinsic primary afferent neurons (IPANs)
  The cell bodies 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, thereby influencing peristalsis, nociception, brain chemistry, and mood.¹⁹

The following are among the means of communication used:¹⁹

• 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 gut in vitro:²⁰

• α-Tocopherol
• Butyrate
• Cholat
• Deoxycholate
• p-aminobenzoate
• propionate
• Tyramine

1.3.1. Vagus nerve¶

Communication along the gut-brain axis is bidirectional. The brain exerts a top-down influence on the motor, sensory, and secretory functions of the gastrointestinal tract via efferent fibers of the vagus nerve. The gut exerts a bottom-up influence on brain function—particularly that of the amygdala and the hypothalamus—via afferent vagal fibers.²¹

The afferent fibers of the vagus nerve, which are present in all layers of the intestinal wall, do not cross the intestinal epithelial layer, so the luminal microbiota cannot interact directly with them. SCFAs can cross the epithelial barrier and activate the chemoreceptors of the vagus nerve.²²
Furthermore, upon stimulation of microbial pattern recognition receptors, enteroendocrine cells release paracrine factors on their luminal side that can activate the chemoreceptors of the vagus nerve, such as:²²

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

In addition, intrinsic primary afferent neurons (IPANs) of the enteric nervous system (which account for the majority of the sensory fibers innervating the intestinal mucosa) could receive microbial signals and, as a result, modulate vagal activity via intramural synaptic transmission.²²

The vagus nerve possesses a nicotinic intramural sensory synapse capable of receiving signals from the IPAN. The majority of vagal afferent action potentials evoked by L. rhamnosus depend on this intramural synaptic transmission and can be blocked by nicotinic or complete synaptic blockade.²³

The vagus nerve is the tenth pair of cranial nerves and is also the longest and most widely distributed pair. The vagus nerve consists of 20% efferent and 80% afferent fibers and includes both sensory and motor nerve fibers. The sensory neurons of the vagus nerve project centrally into the brainstem and terminate at the nucleus tractus solitarius and the nucleus tractus spinalis of the trigeminal nerve. The motor neurons originate from the nucleus ambiguus of the medulla oblongata and the dorsal nucleus of the vagus nerve.²⁴

1.3.2. Spinal nerves¶

In addition to the vagus nerve, spinal nerves also connect the enteric nervous system to the brain. Spinal afferents are particularly relevant in terms of the microbial modulation of visceral pain perception.¹⁹

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 primarily in spinal and vagal primary afferent neurons. L. reuteri DSM 17938 reduced the firing rate of nociceptive spinal fibers, but not that of vagal fibers in the mesenteric nerve plexus. This is likely 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.¹⁹

1.3.3. Spinal cord¶

In addition to the vagus nerve and the spinal nerves, the spinal cord also serves as an afferent connection between the enteric nervous system and the brain.¹⁹

2. Factors That Influence the Gut-Brain Axis¶

An imbalance in the gut microbiome can be caused by a variety of factors, such as cesarean delivery, a diet high in fat and sugar, psychological stress, infections, and antibiotics.¹⁰

2.1. Vagotomy¶

If the vagus nerve, which connects the enteric nervous system to the brain, is surgically severed (vagotomy), this causes behavioral changes:³

• Increase in psychiatric disorders
• Neurogenic bowel disorders are more common²⁵
• reduced locomotor activity during the dark phase in rodents
• elevated plasma norepinephrine 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 cases of intestinal inflammation caused by Salmonella typhimurium, which results in anxiety-like behavior, a unilateral cervical vagotomy resulted in:²⁶

• reduced anxiety-like behavior
• reduced neuronal activation in the nucleus of the solitary tract and in the amygdala
• Attenuation of abnormal glial cell activation in the hippocampus and amygdala
• reduced serum endotoxin levels
• Reduced increase in salmonella concentration in the spleen
• altered expression of inflammatory cytokines (including IL-6, IL-1β, and TNF-α) in the gastrointestinal tract and the brain
• reduced expression of IL-22 and CXCL1
• increased levels of beneficial gut microbiota (including Alistipes and Lactobacillus)
• increased GABA synthesis in the gut
  • 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 reversed the protective effect of vagotomy against intestinal inflammation without affecting anxiety-like behavior

2.2. Antibiotics¶

Antibiotics alter the gut microbiota. They affect how the gut microbiota influences the brain, not only in children but also in adults.²⁷

Oral administration of the antibiotics neomycin and bacitracin together with the antifungal agent primaricin resulted in the following effects in adult BALB/c mice:²⁸

• temporary change in the composition of the gut microbiota
• increased curiosity
• reduced anxiety
• BDNF levels
  • reduced in the amygdala
  • increased in the hippocampus
• Fecal transplantation into another mouse strain caused similar behavioral changes in that strain

Mice that were continuously treated with a cocktail of antibiotics starting at weaning (day 21 postnatal) showed:²⁹

• depleted and altered gut microbiota
• less anxiety-like behavior
• fewer cognitive deficits
• elevated serum tryptophan levels and reduced serum kynurenine levels
• reduced expression of BDNF, oxytocin, and vasopressin in the brain

Administration of the antibiotic vancomycin to rats during the early postnatal period (days 4 through 13) showed that:³⁰

• anxiety-like behavior remains unchanged
• cognitive function remains unchanged
• Long-term increase in visceral hypersensitivity observed only in males
• Reduced levels of alpha-2 adrenoreceptors and TRPV1 in the lumbosacral segment of the spinal cord in adulthood

Rifaximin prevented chronic stress-induced visceral hypersensitivity, mucosal inflammation, and impaired mucosal barrier function in rats³¹

• This correlated with an increase in Lactobacillus in the ileum

Neomycin, on the other hand, did not prevent visceral hypersensitivity³¹

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 on the left vagus nerve, along with a generator and cables, under the skin while the patient is under general anesthesia.

Vagus nerve stimulation affects / is effective for:³

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

2.4. Food¶

High-energy foods, when combined with certain strains of bacteria on the one hand and ADHD on the other, appear to promote reactive aggressive behavior.³²

2.4.1. Short-chain fatty acids, SCFA¶

The primary functions of the microbiota include:³³

• Protection against pathogens by increasing mucus production and thereby stabilizing the intestinal blood-mucosa barrier
• Supports 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.

SCFAs can activate the chemoreceptors of the vagus nerve. The afferent fibers of the vagus nerve, which are present in all layers of the intestinal wall, do not cross the intestinal epithelial layer, so the luminal microbiota cannot interact directly with them. SCFAs can cross the epithelial barrier.²²

Short-chain fatty acids are:

Abbreviation for the fatty acid	Common name	Systematic name	Common name (salt/ester)	Systematic name (salt/ester)	Chemical formula	Typical plasma level34
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	Butyrates	Butanoates	CH3(CH2)2COOH	0.54 μM
C4:0	Isobutyric acid	2-Methylpropanoic acid	Isobutyrates	2-Methylpropanoates	(CH3)2CHCOOH	0.66 μM
C5:0	Valeric acid	Pentanoic acid	Valerates	Pentanoates	CH3(CH2)3COOH	0.18 μM
C5:0	Isovaleric acid	3-Methylbutanoic acid	Isovalerates	3-Methylbutanoates	(CH3)2CHCH2COOH	0.40 μM
C6:0	caproic acid	hexanoic acid	capronates	hexanoates	CH3(CH2)4COOH	0.34 μM

In humans, acetate, propionate, and butyrate account for 95% of SCFAs, in a ratio of 3:1:1.³ A recent study found other, more detailed differences, which are shown in the table above.³⁴

SCFAs reversed the changes caused by chronic psychosocial stress in the gut-brain axis.³⁵ SCFAs

• mitigated the changes in reward-seeking behavior triggered by psychosocial stress
• increased responsiveness to an acute stressor
• increased in vivo intestinal permeability
• had an antidepressant effect
• had an anxiolytic effect
• did not affect the stress-induced increase in body weight

2.4.2. Probiotics¶

There is evidence that when prebiotics are administered, an effect (in the brain) does not occur until several weeks later, comparable to the latency period of antidepressants.³⁶ Several weeks after discontinuation, some—but not all—neurotransmitter levels in the brain had returned to their original levels.

2.5. Fecal Transplantation¶

Although rodents raised in a germ-free environment do have:¹⁹

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

Nevertheless, they are particularly well-suited for studying the effects of fecal transplants and have shown that transferring gut microbiota to germ-free animals can confer behavioral traits from the donor animals.¹⁹
A fecal transplant from depressed individuals into rats with depleted microbiota resulted in anxious behavior and changes in tryptophan metabolism.³⁷

Fecal transplants from 5 boys with ADHD into rats also triggered ADHD symptoms in the rats, whereas this was not the case for 8 boys without ADHD.³⁸

A fecal transplant from healthy rats to Lister hooded rats (an ADHD animal model that exhibited more hyperactivity, impulsivity, and inattention than SHR) reduced hyperactivity in the transplant recipients.³⁹

2.6. Gut Flora and Stress¶

Microbiota are essential for a normal stress response, anxiety-like behaviors, social behavior, and cognition, and they regulate the homeostasis of the central nervous system through immune function and the integrity of the blood-brain barrier.⁷

The vagus nerve and the HPA axis influence each other.
Vagus Nerve Stimulation³

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

In animal models, psychological stress increased intestinal permeability and caused a shift of gut bacteria into the host. The activation of the immune response due to exposure to bacteria and bacterial antigens beyond the epithelial barrier triggers pro-inflammatory cytokine secretion and ultimately activates the HPA axis.⁴⁰

The gut microbiome is essential for the development and function of the HPA axis (stress axis).⁴¹²
Mice raised in a germ-free environment exhibit an exaggerated HPA axis response and reduced sensitivity to negative feedback signals. Early administration of Bifidobacterium infantis reversed this response.²

Early-life stress affects the microbiome.⁴²

3. Mechanisms by Which the Gut Microbiome Affects the Brain¶

Gut bacteria (gut microbiome, gut flora) influence the nervous system through various mechanisms.⁴³

3.1. Metabolic / Neuroendocrine Pathway¶

Metabolic/neuroendocrine pathway:²¹²

3.1.1. Modulation of Neurotransmitters by the Microbiome¶

The microbiome modulates neurotransmitters⁴³ such as GABA, serotonin, dopamine, and norepinephrine²¹

• direct synthesis²
• indirectly through the host organism’s biosynthetic pathways
  • Synthesis of neurotransmitter precursors (e.g., for dopamine)²
• through the secretion of short-chain fatty acids (SCFAs)²¹. These:
• activate microglia cells⁴⁴
• affect the permeability of the blood-brain barrier⁴⁵
• Inhibition of the synthesis of the proinflammatory TNF through the conversion of L-histidine into the immunoregulatory histamine (L. reuteri)⁴⁶

Bacteria can synthesize neurotransmitters and hormones and respond to them:²¹

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

• Blood-brain barrier
  Acetylcholine can cross the blood-brain barrier. Dopamine, norepinephrine, serotonin, and GABA, however, cannot, so these neurotransmitters—which are produced in the gut—do not directly affect levels in the brain.
• Extracellular vesicles (EVs) can cross the blood-brain barrier
  Like other bacteria, the gut microbiota produce extracellular vesicles (EVs).⁵² When these originate from Gram-negative bacteria, EVs are called “outer membrane vesicles (OMVs).”⁵³
  These nano-sized EVs (20 to 1,000 nm) can penetrate the inner mucous layer, enter the bloodstream, and cross the blood-brain barrier to reach the brain.^545556575859
  95% of serotonin is produced by enterochromaffin cells in the gut, and 5% in the brain. Akkermansia muciniphila, which accounts for about 5% of gut bacteria and whose deficiency is associated with various chronic inflammatory bowel diseases such as colitis and Crohn’s disease, influenced serotonin levels in the hippocampus of mice via its EVs.⁵⁴ Another study demonstrates that extracellular vesicles from Escherichia coli reach the hippocampus.⁶⁰
• 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 to support this. However, there are indications that nerve fibers of the vagus nerve contain dopamine.⁶¹ Furthermore, peripheral nerves such as the vagus nerve can transport nanoparticles into the brain.⁶² Similarly, synuclein can be transported from the body to the brain via nerves, which may be of interest with regard to the development of Parkinson’s disease.⁶³ In cohort studies, a truncal vagotomy has shown a significant protective effect against Parkinson’s disease.⁶⁴⁶⁵
• Effects on prodrug metabolism
  Gut bacteria influence blood levels of the precursors of dopamine, norepinephrine, serotonin, and GABA, which can cross the blood-brain barrier. As a consequence, blood levels of these precursors—through the amount synthesized—could influence the amount of the neurotransmitters synthesized from them in the brain. For example, 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 phenylalanine—itself a precursor to dopamine. At the same time, the increase in Bifidobacterium is thought to be associated with reduced reward anticipation, which may suggest lower dopamine levels in the striatum.⁶⁶ We do not yet understand how these two seemingly contradictory pathways fit together.

3.1.2. Immune System Pathway and Microbiome¶

The microbiome influences the immune system through circulating cytokines.²¹²

3.1.3. Inflammatory pathway¶

An increased ratio of Firmicutes to Bacteroidetes is associated with elevated levels of inflammation in the intestinal tissue.¹⁰⁶⁷⁶⁸
A reduced abundance of the butyrate-producing bacterium Faecalibacterium prausnitzii (phylum Firmicutes) correlated with reduced low-grade inflammation in obese individuals and patients with type 2 diabetes.^{6970 71} Obesity is considered an inflammatory condition. Obesity is also a major risk factor for the development of gallstones. Gallstones are associated with an elevated Firmicutes-to-Bacteroidetes ratio.⁷² However, gallstones themselves are not associated with elevated inflammatory markers.⁷², which, in our view, raises the question of whether the Firmicutes-to-Bacteroidetes ratio is the direct pathway for inflammation.

Elevated ADHD symptoms correlated with elevated levels of the inflammatory marker GlycA after meals. In addition, elevated peripheral tryptophan levels were found in boys.⁷³

In addition to other changes in microbiota diversity, ASD is reportedly associated with a significantly higher Firmicutes-to-Bacteroidetes ratio.¹⁰ While two of the cited sources available to us found an elevated Firmicutes-to-Bacteroidetes ratio in IBD⁷⁴⁷⁵ and one source reported a sharply increased Clostridium species⁷⁶ —which represents at least one, albeit very specific, —subtype of the Firmicutes phylum, another of the cited sources⁷⁷ did not address this topic, and yet another cited source reported a reduced Firmicutes-to-Bacteroidetes ratio (fewer Firmicutes, more Bacteroidetes) in SSI.⁷⁸.
Our further research identified a systematic review that found an elevated Firmicutes-to-Bacteroidetes ratio in SSA⁷⁹, as well as a meta-analysis that found elevated levels of both Firmicutes and Bacteroidetes in SSA (meta-analysis, k = 18, n = 897)⁸⁰.

3.2. The HPA axis is influenced by the microbiome¶

The microbiome influences HPA axis activity. Germ-free (microbiota-deficient) mice have an overactive HPA axis, with increased release of stress hormones such as corticosterone and ACTH in response to restraint stress.⁸¹

3.3. The Microbiome’s Neural Pathway¶

The microbiome influences the brain via the nervous system.²

• by stimulating the vagus nerve:²¹⁸²⁵¹
  • The vagus nerve consists of 80% afferent fibers, which carry sensory stimuli from the body to the brain, and 20% efferent fibers, which carry motor signals from the brain to the body.¹
• via the enteric nervous system²

3.4. Epigenetics and the Microbiome¶

Influence on epigenetic processes in the brain (neuroepigenetic)⁸³

• e.g., through SCFAs (short-chain fatty acids); these influence
  • Acetylation and butyrylation of histone proteins⁸⁴
    • facilitates the binding of transcription factors to DNA and, consequently, gene transcription
  • post-translational modification of histones (crotonylation, butyrylation)⁸⁵⁸⁶⁸⁷
  • Neuroplasticity (e.g., in the visual cortex of adult mice)⁸⁸
  • Gene expression in cortical astrocytes⁸⁹
• DNA methylation⁸³
  • Changes in DNA methylation occur during learning and memory consolidation
  • The one-carbon pathway⁹⁰ involved in DNA methylation is regulated by the availability of cofactors. Some of these (such as cobalamin, folate, pyridoxine, and riboflavin) are metabolites produced by gut bacteria
  • The gut microbiome influences DNA methylation in intestinal epithelial cells
  • There is currently no evidence that the gut microbiome influences DNA methylation and transcription in the brain.

3.5. MiRNA (microRNA) and the Microbiome¶

Deficiencies in the gut microbiota correlate with changes in the expression of various miRNAs, particularly in the amygdala and PFC.⁸³

miRNAs regulate, for example: ⁸³

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

4. Gut Microbiota in ADHD¶

4.1. Could Gut Bacteria Be a Possible Cause of ADHD?¶

A study found evidence suggesting a causal link between gut bacteria and ADHD.⁹¹ (Note: Even if a causal relationship were confirmed, it should be assumed that this represents only one of many possible mechanisms by which ADHD can develop and therefore would not apply to all people with ADHD.)

A study found that mice whose guts were colonized with gut bacteria from people with ADHD exhibited structural changes in the brain (white matter, gray matter, hippocampus, internal capsule), reduced connectivity between the right motor and visual cortices during the resting state, and higher levels of anxiety than mice that were exposed to gut bacteria from people without ADHD.⁹²

A case study reports an improvement in a young woman’s ADHD symptoms following a fecal microbiota transplant performed in connection with a recurrent Clostridioides difficile infection.⁹³

Taxonomic differences were found in the microbiota of adolescents and young adults with ADHD compared to healthy control subjects:⁶⁶

• A large number of actinobacteria were found in people with ADHD
• the gut microbiome’s capacity to produce monoamine precursors (phenylalanine) was increased
• The abundance of cyclohexadienyl dehydratase (CDT) genes in the microbiome, which are involved in phenylalanine production, was negatively correlated with responses to reward anticipation in the ventral striatum (where activation of the ventral striatum in response to reward anticipation was reduced in ADHD)

A study of urine and fecal samples using 1H nuclear magnetic resonance spectroscopy and liquid chromatography-mass spectrometry identified gender-specific patterns in the metabolic phenotype of ADHD:⁹⁴

• Urine Profile
  • Hippurate (a product of microbial host co-metabolism that can cross the blood-brain barrier)
    • elevated (men only)
    • was negatively correlated 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 glycerin increases
  • Increased 3,7-dimethylurate
  • FAD increased
  • Glycerol-3-phosphate decreases
  • Thyme reduces
  • 2(1H)-quinolinone reduced
  • Aspartate is reduced
  • Xanthine reduces
  • Hypoxanthine decreases
  • Orotate is reduced

Gut Microbiome and Dopamine

• The gut microbiome influences dopamine levels in the PFC and striatum of rodents.²
• A slight increase in Bifidobacterium in the gut, as observed in ADHD, is thought to be associated with increased production of cyclohexadienyl dehydratase, which is a precursor to phenylalanine—itself a precursor to dopamine. At the same time, the increase in Bifidobacterium is thought to be associated with reduced reward anticipation, which may suggest lower dopamine levels in the striatum.⁶⁶ We do not yet understand how these two seemingly contradictory pathways fit together.
• The composition of the gut microbiome correlates with impulsivity, increased striatal D1R, and decreased D2R, along with increased vulnerability to alcohol dependence⁹⁵
• Inflammation of the intestines can affect dopamine metabolism⁹⁶
  • Infection with Citrobacter rodentium triggers inflammatory bowel disease in mice. This affected not only the intestinal microbiota but also dopamine metabolism in the brain. 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:
    • impaired behavioral performance
    • increased dopaminergic degeneration and overactivation of glial cells in the nigrostriatal pathway
    • increased the expression of TLR4 and NF-κB p65 in the colon and the striatum
    • increased the expression of proinflammatory cytokines.

4.2. The Microbiome and Short-Chain Fatty Acids (SCFAs) in ADHD¶

Studies on short-chain fatty acids have found reduced blood levels of SCFAs in ADHD:⁹⁷⁹⁸

• Adults with ADHD
  • Formic acid reduces
  • Acetic acid reduces
  • Propionic acid reduces
  • Reduced succinic acid (C4H6O, an aliphatic dicarboxylic acid; food additive number E 363)
• Children with ADHD
  • Formic acid levels are lower than in adults
  • Propionic acid levels lower than in adults
  • Isovaleric acid levels are lower than in adults
• Antibiotics taken in the past 2 years caused
  • Formic acid reduces
  • Propionic acid reduces
  • Succinic acid reduces
• The current use of stimulants by children resulted in
  • Acetic acid reduces
  • Propionic acid reduces

4.3. Differences in Gut Microbiota in ADHD¶

Studies have found differences in gut microbiota between children with ADHD and those without the condition.³³ In contrast, there is little difference in gut microbiota between ADHD and ASD.⁹⁹
ADHD was associated with a leaky gut, neuroinflammation, and overactivated microglial cells. The colon microbiota exhibits a pro-inflammatory shift and harbors more Gram-negative bacteria, which contain immune-triggering lipopolysaccharides in their cell walls.¹⁰⁰

Early disorders of the developing gut microbiota can affect neurological development and may lead to adverse mental health outcomes later in life.¹⁰¹
75 infants were randomly assigned to receive either Lactobacillus rhamnosus GG or a placebo during the first 6 months of life. After 13 years, 17% of the placebo group were diagnosed with ADHD or ASD, while none in the probiotic group were. Among the people with ADHD, levels of bifidobacteria in the gut microbiome were significantly reduced during the first 6 months of life.¹⁰²¹⁰³

It is known that a cesarean section, compared to a vaginal birth, and bottle-feeding, compared to breastfeeding (especially during the first three months), alter the composition of the infant’s microbiome and increase the risk of ADHD. For more information, see Cesarean section in the chapter Circumstances of Birth as a Cause of ADHD and Bottle-feeding increases (up to +270%), breastfeeding reduces the risk of ADHD (-23% to -74%) in the chapter Traumatic Physical or Emotional Childhood Experiences as a Cause of ADHD

Autism has been linked to high levels of Clostridium species as well as low levels of Bifidobacterium species.¹⁰⁴

Translated with DeepL.com (free version)

4.3.1. Reduced Gut Bacteria in ADHD¶

4.3.2. Elevated Levels of Gut Bacteria in ADHD¶

Changes in the abundance of Ruminococcaceae_UGC_004 correlated with inattention.¹¹⁹

4.3.3. Alpha Diversity¶

Alpha diversity (e.g., the Shannon and Simpson indices) indicates the degree of heterogeneity among taxa within a sample.¹⁰¹³¹

The results of studies on the alpha diversity of the gut microbiota in ADHD are inconsistent.
We will compile research findings on this topic here.

Meta-analyses, Reviews:

• Several meta-analyses found no change in alpha diversity in ADHD.
  • (k = 4, n = 619 adults)¹⁰⁹
  • (k = 9).¹⁰
  • a review as well¹³²

Studies:

• Unchanged alpha diversity^106133112113
  • in mice with ADHD⁹²
• significantly lower α-diversity (Shannon index, observed species, Faith-PD index)¹³⁴

4.3.4. Beta Diversity¶

Beta diversity (e.g., Jaccard distance) indicates the degree of dissimilarity among taxa across samples.¹⁰¹³¹

Study results on the beta diversity of the gut microbiota in ADHD are inconsistent, although the lack of a correlation is not as clear-cut as it is for alpha diversity.
We will compile research findings 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.¹⁰⁹
• no variation in beta diversity¹³²
• A meta-analysis (k = 9) found only a few differences in beta diversity between people with and without ADHD, primarily among children and adolescents.¹⁰

Studies:

• A study of n = 73 participants found a trend toward significance for β-diversity (weighted UniFrac).¹³⁵
• no significant change in beta diversity¹³³
• altered beta diversity in ADHD-C, but not in ADHD-C¹⁰⁶
• Increased beta diversity in mice with ADHD⁹²
• Differences were found in both taxonomic and functional beta diversity among n = 136 adults¹³⁶
• Children with ADHD (n = 63) who were taking psychostimulants (n = 33)¹³⁶
  • significantly different taxonomic beta diversity
  • lower functional and taxonomic uniformity
  • lower prevalence of the Bacteroides stercoris CL09T03C01 strain
  • lower frequency of bacterial genes that encode an enzyme involved in vitamin B12 synthesis
  • higher plasma levels of the vascular inflammatory 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 terms of both alpha and beta diversity and differ significantly from those of unaffected individuals.
Furthermore, a subgroup of children with ADHD and ASD showed elevated levels of lipopolysaccharide-binding protein compared to unaffected children, which correlated positively with interleukin IL-8, IL-12, and IL-13. This suggests a disorder of the intestinal barrier and a dysregulation of the immune system in a subgroup of children with ADHD or ASD.¹³⁷

Germ-free mice that received a fecal transplant from people with ADHD subsequently showed:¹³⁸

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

When ASS model mice were administered appropriate microbial metabolites, this improved their behavioral abnormalities and modulated neuronal excitability in the brain.¹³⁸

4.3.6. Urinary Microbiota in ADHD¶

A study of the urinary microbiome in ADHD found that:¹³⁹

• lower alpha diversity in the urinary bacteria of the ADHD group
  • reduced Shannon and Simpson indices (p < 0.05)
• significant differences in beta diversity
• Common symptoms of ADHD included:
  • Phylum Firmicutes
  • Actinobacteriota
  • Ralstonia (genus)
  • Afipia (genus)
• Less common in ADHD:
  • Phylum Proteobacteria
  • Corynebacterium (genus)
  • Peptoniphilus (genus)
• Afipia correlated significantly with the scores on the Child Behavior Checklist Attention Problems and the DSM-based ADHD subscale

4.3.7. Probiotic Supplementation for ADHD¶

A study reports benefits of administering Xiaoer Huanglong pellets (a traditional Chinese remedy) for ADHD symptoms, which are thought to be mediated by the microbiome.¹⁴⁰

Newborn SHR mice were administered microbiota.¹⁴¹

• 2’-Fucosyllactose (2’-FL)
  • impaired spatial orientation in male mice in the Morris water maze
  • increased mRNA expression in males associated with the γ-aminobutyric acid, serotonin, and norepinephrine systems in the hippocampus
  • An increase in Romboutsia, Clostridium_, sensu_stricto_1, and a decrease in Muribaculaceae correlated with improvements in learning ability and spatial memory
• 2’-FL plus Bifidobacterium bifidum TMC3115
  • reduced hyperactivity in females in the open-field test
  • increased the expression of tyrosine hydroxylase and the dopamine transporter in the striatum of females
  • promoted the diversity of the gut microbiota
  • reduced levels of Bacillus and Turicibacter

5. The Gut Microbiome and Other Clinical Presentations¶

5.1. Probiotic Supplementation for Anxiety and Depression¶

5.1.1. Studies in rodents¶

Lacticaseibacillus rhamnosus JB-1 (Lactobacillus rhamnosus) caused

• only in mice that have not undergone vagotomy:⁵¹
  • 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
  • Reduced corticosterone stress response
  • Reduced anxiety-related behavior
  • Reduced depressive symptoms
• prevented behavioral changes in rats caused by chronic, unpredictable, mild stress¹⁴²
  • Reduces anxiety-like behavior
  • Reduces depression-like behavior
  • Decrease in glutamine and glutathione levels in the hippocampus prevented
  • Reduced levels of taurine in the hippocampus.

Both acute and chronic administration of the short-chain fatty acid sodium butyrate in combination with fluoxetine resulted in a significant 20–40% reduction in immobility scores in the tail suspension test in mice, compared with administration of either agent alone.¹⁴³
An injection of sodium butyrate caused

• transient histone hyperacetylation in the hippocampus and the PFC
• a temporary increase of at least 50% in BDNF transcript levels in the PFC

5.1.2. Human studies¶

A daily dose of a combination of L. helveticus and B. longum in a double-blind, randomized, placebo-controlled study of healthy women and men resulted in a slight, but statistically significant, reductions in levels of perceived stress, anxiety, and depression, as well as in 24-hour urinary cortisol levels.¹⁴⁴

In a double-blind, randomized, placebo-controlled study of patients with chronic fatigue, daily administration of Lactobacillus casei Shirota significantly reduced anxiety symptoms but not depressive symptoms.¹⁴⁵

5.2. Microbiota Supplementation with ASA¶

5.2.1. Studies in 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.¹⁴⁶

Administration of Limosilactobacillus reuteri resulted in

• only in mice that have not undergone vagotomy
  • Changes in Social Behavior in a Mouse Model of Autism³
  • Improvement in social behavior in various mouse models of autism¹⁴⁷
    • via the vagus nerve and oxytocinergic and dopaminergic signaling in the brain
      • not through the restoration of the gut microbiome’s composition per se
    • by inducing synaptic plasticity in the VTA of ASS mice
      • Increased oxytocin levels (via L. reuteri)¹⁴⁸¹⁴⁹ in various ASS mouse models:
        • Genetic ASS mouse models
          • Shank3B-/-¹⁵⁰¹⁴⁷
          • Cntnap2−/−¹⁵¹
            • L. reuteri levels are reduced in Cntnap2-/- mice
            • L. reuteri corrected the deficits in oxytocin-producing neurons
            • L. reuteri corrected the social deficits in both young and adult Cntnap2-/- (KO-I) mice
            • L. reuteri did not correct the hyperactivity typical of Cntnap2-/- mice
        • Environmentally induced ASS mouse models
          • VPA¹⁴⁷
          • GF¹⁴⁷
          • MHFD¹⁴⁷
          • Lipopolysaccharides (LPS) (it is unclear whether this is via oxytocin)¹⁵² (Lacticaseibacillus rhamnosus was also effective here)
        • idiopathic ASS mouse model (BTBR)¹⁴⁷
      • not in mice with missing or blocked oxytocin receptors in dopaminergic neurons

In another study, ASS was also correlated with reduced plasticity in the VTA, which was caused there by a high-fat diet in the mother. The ASS-related behavior was then transmitted from the offspring to mice raised in a germ-free environment.¹⁴⁸

In a preclinical ASS mouse model, only the L. reuteri strain ATCC-PTA-6475—but not the strain DSM-17938—improved social deficits.¹⁵³

A study of Cntnap2−/− and Cntnap2+/+ mice found that both mouse lines exhibited the hyperactivity characteristic of this ASD mouse model. In the Cntnap2+/+ mice, however, social behavior was normalized, as was the microbiome. This, too, suggests that the ASD-typical changes in social behavior are caused by the microbiome.¹⁵¹

The study results regarding L. reuteri in ASS are impressive. The question raised in the introduction—namely, to what extent the administration of a specific microbiota (in this case, L. reuteri) contributes to the improvement, and to what extent the increase in microbiome diversity resulting from the administration of microbiota in general contributes—is likely of little relevance here. The argument that people with ADHD may have impaired gut microbiota diversity due to limited dietary preferences is certainly worth noting, but it likely applies only to humans and not to mice. In any case, selective dietary habits in ASD model mice have not been reported to date.
The same applies to potential effects on the oral bacterial flora resulting from sensory stressors that make teeth cleaning more difficult.¹⁵⁴

B. fragilis improves deficits in communicative, stereotypical, anxiety-like, and sensorimotor behavior in a mouse model of ASD in which ASD was induced by prenatal viral infection. It also corrected intestinal permeability and altered the microbiome composition.¹⁵⁵

5.2.2. Human studies¶

A double-blind, randomized, placebo-controlled study on the effects of L. reuteri in children with ASD found that:¹⁵³

• significantly improved social functioning across various measures
• no change in
  • Overall Severity of AS
  • repetitive behaviors
  • Microbiome composition
  • Immune profile

A study of children with ASD reports that an elimination diet has a positive effect on gastrointestinal symptoms and that a prebiotic has a positive effect on social behavior.¹⁵⁶

5.3. Probiotic Supplementation for Multiple Sclerosis¶

In a study, Limosilactobacillus reuteri reduced the severity of experimental autoimmune encephalomyelitis in mice—a model for multiple sclerosis—suggesting a deficiency of L. reuteri in MS.¹³ In contrast, two other studies found that an excess of L. reuteri is a risk factor for MS.¹³¹⁴
It is possible that, in individuals genetically susceptible to MS, the gut microbiota and diet could act synergistically, with diet-dependent microbial metabolites in the gut serving as a key mechanism for the disease. Due to these gene-gut microbiota interactions, specific microbial taxa may have different effects depending on MS risk alleles or other genetic polymorphisms, and these effects may also depend heavily on dietary intake. This could explain why results vary across different geographic, dietary, and genetic contexts. Prophylactic or therapeutic modulation of the gut microbiome to prevent or treat MS would therefore require careful and personalized consideration of genetic predispositions, gut microbiota composition, and dietary habits.¹⁵⁷ This is consistent with the ability of L. helveticus to attenuate anxiety-like behavior in mice only in a genotype- and diet-dependent manner.¹⁵⁸

When extrapolated to other clinical presentations, conflicting findings regarding the effects of certain microbiota on specific symptoms should be viewed as a warning against carelessly testing these effects on individuals.

5.4. Microbiota 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 pathogen-specific and gnotobiotic conditions, particularly through an increase in plasmacytoid dendritic cells and interferon signaling. The colonization and spread of L. reuteri to other organs could be reduced by resistant starch in the diet via SCFAs. Resistant starch also reduced plasmacytoid dendritic cells, interferon signaling, and mortality.¹⁵⁹