*3.2. Gut-Brain Axis*

The gut-brain axis describes the bidirectional communication between the microbes, enteric nervous system and the CNS [37]. So far, there are three known means of communication between these compartments: Neural, immune, and endocrine [4,38].

The neural pathway describes the hypothalamic-pituitary-adrenal axis (HPA axis), which is the most important efferent stress pathway. It is of great importance to understand to what extent the HPA axis plays a role in the pathogenesis of ADHD, as it influences pathways in the body that are often deviating in ADHD patients [39,40] as for example: Circadian rhythm [41], sleep [42], and emotions [43]. The stimulation of the HPA axis by stress or pro-inflammatory cytokines results in a release of corticotropin-releasing factor (CRF) from the hypothalamus, as well as adrenocorticotropic hormone (ACTH) from the pituitary gland, finally resulting in the secretion of cortisol from the suprarenal (adrenal) glands [38].

One study using 69 healthy children and 123 children with ADHD observed an increase in salivatory cortisol in ADHD patients after waking up in the morning [44]. The effect of stressors was studied in one paper showing that after being exposed to stress children with ADHD of combined type (high levels of hyperactivity and impulsivity) have decreased salivary cortisol levels in comparison to other ADHD patients [45]. In contrast, adult ADHD patients with an inattentive type showed higher levels of cortisol in comparison to the combined types, which showed normal levels of cortisol [46]. Finally, Lackschewitz et al. discovered that adults with ADHD who undergo a stress-inducing exam show a trend towards reduced cortisol levels [47]. These reports portray the association between altered cortisol levels and different types of ADHD. However, the heterogeneity of the results can be explained by various stressors on a differing target group all acting as confounders. Thus, only future studies using the same stressor, examining similar and large patient groups will allow drawing further reliable conclusions.

The neuroimmune communication pathway describes how intestinal microbes influence the function and maturation of immune cells in the CNS, whereby microglia cells play an important role [48]. These cells are activated, as well as produced, by pro-inflammatory cytokines, and are important regulators for autoimmunity, neuroinflammation, and neurogenesis [49]. Germ-free (GF) mice showed defects in microglia activation, which in turn lead to a deficient innate immune response when exposed to pathogenic bacteria [50]. The same study showed the immense effect the microbiome has on microglial cells, as introducing microbiota into GF mice resulted in restored microglial functions. Reversely, eradicating various bacteria in specific pathogen-free (SPF) mice resulted in microglial cells maturing less rapidly [50]. As neuroinflammation plays an important part in the pathophysiology of ADHD, the proper activation and maturation of microglia in ADHD patients have to be thoroughly investigated in order to determine if it has a pathogenic influence.

The enteric nervous system communicates with the brain mainly through the parasympathetic vagus nerve [51], and partially though the sympathetic spinal cord pathway [52]. Furthermore, the vagus nerve predominantly consists of afferent nerve fibers with a ratio of 9:1 to efferent fibers [53]. Even though a definite conclusion cannot be made, various studies have demonstrated that the autonomic nervous system of ADHD patients shows alterations. A study testing 19 children with ADHD showed that the patients had an underactive parasympathetic and an overactive sympathetic nervous system [54]. Another study comparing 32 ADHD patients to 34 healthy controls registered under-aroused parasympathetic nervous systems, while the sympathetic part did not show any difference between the groups [55].

It has become clear that all three ways of communication between the microbiome, gut, and CNS could play an important role in the pathophysiology of ADHD. The neural communication over the HPA axis shows abnormalities in ADHD patients. Additionally, studies detected that microbes influence the function of pro-inflammatory microglia, a key finding, as neuroinflammation in ADHD patients is commonly found. Finally, the autonomic nervous system shows aberrations as the main research results show an under-arousal of the parasympathetic nervous system.

#### *3.3. Etiology of ADHD and the Genetic and Environmental Influences*

As the exact pathophysiology of ADHD is still unclear, its causes are still being researched. Nevertheless, it has been established that there is an interplay between genes and the environment resulting in a complex etiology. Genetic predisposition plays an important part in the pathophysiology of ADHD as children from parents that have been diagnosed with ADHD have a 50% higher chance to be diagnosed with the same disorder [56]. Similarly, twin studies have shown a high heritability, as especially for inattentive and combined types an inheritance of 71–90% could be discovered [56,57]. On the other hand, one study showed, that 20–30% of the risk of developing ADHD is due to environmental factors [58]. These include perinatal maternal smoking, stress, mineral and micronutrient deficiencies and premature birth [59]. Additionally, research showed that 10–40% of the variance inheritance of ADHD could be caused due to the environment highlighting the interplay of genetic and environmental risk factors [60]. Due to these complex interactions, it is believed that ADHD can be manifested with highly heterogenous symptoms depending on the exact pathway and etiology involved [61].

Research shows that the dysfunction of monoaminergic neurotransmitters, including noradrenaline (NE), serotonin (5-HT) and dopamine (DA), plays an important role in the pathophysiology of ADHD [62].

#### 3.3.1. Dopamine

DA is a catecholamine that acts both as a hormone and neurotransmitter (NT). It is a product of the essential amino acid L-phenylalanine, which must be provided in our diet. As seen in Figure 2, this is then turned into L-tyrosine, and finally into DA and NE [63].

**Figure 2.** The synthesis pathway from L-phenylalanine to noradrenaline including all its intermediary products. Dopamine acts as an important metabolite for the emotional response and reward system [64].

The dopamine hypothesis links ADHD to alterations in dopamine metabolism. The hypothesis describes the increased expression of presynaptic dopamine transports (DAT) in ADHD patients leading to an increased dopamine transporter density (DTD), and finally results in a decreased level of the bioavailable NT [65]. The dopamine hypothesis gained attention due to the way methylphenidate (MPH) and amphetamines (AMP), the most commonly used pharmacotherapies to treat ADHD, interact with the DA and NE metabolism. MPH and AMP exert a stimulatory effect in ADHD as they inhibit the reuptake of NE and DA by blocking the metabolizing enzyme, monoamine oxidase (MAO), thereby increasing the concentration of the two monoamines in the synaptic cleft. One differentiates between MAO-A and MAO-B as the former is mostly expressed in the liver and GI-tract and the latter in blood platelets [66]. Nevertheless, both are manifested in the CNS and are able to break down DA [66]. Furthermore, amphetamines have the ability to release NTs from the presynaptic neuron, which additionally increases the monoaminergic concentration in the synapse [67].

Moreover, recent research shows that not only the metabolization, but also the production of DA plays an important role in the pathophysiology of ADHD. One of the influencers on the production of NTs seems to be the microbiome in the GI-tract [68]. Bacteria, such as the genus Bifidobacterium belonging to the phylum Actinobacteria potentially influence the levels of available DA in the body by encoding cyclohexadienyl dehydratase (CDT) [69]. This enzyme is important for the synthesis of the essential amino acid phenylalanine [69], which acts as a precursor of the amino acid tyrosine, which in turn is metabolized into DA and lastly to NE [70]. Aarts et al. found an increase in Bifidobacterium in ADHD patients, and thus, higher levels of CDT. By analyzing BOLD responses of the ventral striatal using fMRI measurements they deduced a negative correlation between the abundance of CDT and reward anticipation [69], a key symptom in ADHD [71], and target of DA [72]. Finally, this study concluded that high levels of phenylalanine might be a risk factor for abnormal dopamine signaling and could lead to a reduced reward response [69]. Although another study supports the findings by Aarts et al. [73], the correlation still appears to be inconsistent as two older studies found a decreased level of phenylalanine in ADHD patients [74,75], even if data of source [74] are not statistically significant. Finally, a more recent study found no correlation between phenylalanine levels and ADHD [76]. A summary of these finfings is given in Table 1.

**Table 1.** Various studies that tested phenylalanine levels in ADHD patients. ↑ represent the increase of phenylalanine found in ADHD patients and ↓ the decrease of the amino acid in comparison to healthy controls (HC). The symbol — describes that the study found no correlation between ADHD and phenylalanine levels. The accumulative data to date do not allow a definite correlation between a change in phenylalanine levels and ADHD. *p* levels less than 0.05 were considered statistically different.

