ADHD in animal model

There are several rat breeds that represent ADHD-HI, ADHD-I and non-affected as animal models. Among them, SHR and Wystar-Kyoto are mainly the subject of research.
The Spontaneous(ly) hypertensive rat (SHR) represents a form of ADHD-HI (with hyperactivity), while Wistar-Kyoto rats (WKY) usually represent non-affected individuals as an opposite model. In addition, there is an SHR strain, the SHR/NCrl, that shows symptoms of ADHD-C and a strain, the WKY/NCrl, that shows symptoms of ADHD-I (attention deficit without hyperactivity).¹²³⁴ Unless studies differentiate this, it is regularly assumed that Wistar-Kyoto rats (WKY) refers to the non-affected model.
In rats, the predominant glucocorticoid is corticosterone, instead of cortisol, which is predominant in humans.

The respective rat lines were bred for specific symptoms. The rearing of these animals does not involve any stress.⁵
That these animal models express their symptoms based on genetic makeup alone and without the influence of early childhood stress is a strong argument that certain genes alone represent a distinct pathway for the development of mental disorders such as ADHD and that the two developmental pathways of genes alone and genes + environment coexist.
Interestingly, the findings about SHR do not weaken the theory that ADHD (like many other mental disorders) causes its symptoms through a disruption of the HPA axis, but strengthen it enormously - because SHR already show a disrupted HPA axis simply because of their genetic predisposition.

ADHD is also discussed in dogs.⁶

The various animal models show vividly that symptoms such as hyperactivity, impulsivity or attention problems can have very different causes. The mediation of symptoms must be distinguished from the causes (e.g., a specific genetic defect). Thus, very different causes (e.g., gene defects) can cause a dopamine deficit or others can cause a dopamine excess, both of which in turn mediate nearly identical symptoms due to deviation from optimal dopamine levels (inverted-U). To clarify this, we have divided animal models, as far as we know, into those with a dopamine (effect) deficiency and a dopamine (effect) excess. Although dopamine is a particularly important factor in ADHD, the other influences are also relevant.

• 1. Animal models of ADHD with decreased dopamine levels / decreased dopamine action
  • 1.1. Spontaneous(ly) hypertensive rat (SHR)
    • 1.1.1. Dopamine system impaired
      • 1.1.1.1. Dopamine synthesis impaired
      • 1.1.1.2. D2 receptor increased
      • 1.1.1.3. (Only) D4 receptor reduced in the PFC
      • 1.1.1.4. Dopamine release reduced
      • 1.1.1.5. Dopamine uptake in the striatum accelerated
    • 1.1.2. Adenosine system altered in SHR
    • 1.1.3. Norepinephrine release increased
    • 1.1.4. Serotonin in SHR
    • 1.1.5. GABA in SHR
    • 1.1.6. Vitamin D3 metabolism altered in SHR
    • 1.1.7. Stress systems changed
      • 1.1.7.1. Overintense HPA axis stress response in SHR compared with WKY
      • 1.1.7.2. Increased mineralocorticoid receptor expression as a cause of hyperintense HPA axis stress responses in SHR
      • 1.1.7.3. MiRNA expression in SHR alters glucocorticoid receptor
    • 1.1.8. SHR and immune system
      • 1.1.8.1. Young SHR
      • 1.1.8.2. Older SHR
      • 1.1.8.3. Other altered immune values in SHR
      • 1.1.8.4. Taurine improved inflammatory markers in SHR
    • 1.1.9. Cholesterol metabolism altered in PFC of SHR; MPH revises change
    • 1.1.10. Blood pressure, sympathetic nervous system, cardiac hypertrophy and vitamin D3 in SHR
    • 1.1.11. Brain regions reduced in size
    • 1.1.12. Brain connectivity impaired locally as well as over a wide area
    • 1.1.13. PFC neurons altered
    • 1.1.14. Monosodium glutamate affects aggression in a vagus nerve-dependent manner
    • 1.1.15. Drug effect on SHR
      • 1.1.15.1 Effect of MPH on SHR
      • 1.1.15.2. Effect of amphetamine drugs on SHR
      • 1.1.15.3. Effect of atomoxetine on SHR
    • 1.1.16. Altered emotional communication and response of SHR
    • 1.1.17. SHR exhibit behavioral subgroups corresponding to ADHD-HI and ADHD-I
  • 1.2. WKY/NCrl - Rats
    • 1.2.1. Increased tyrosine hydroxylase in WKY/NCrl
    • 1.2.2. Increased DAT in adulthood from WKY/NCrl
    • 1.2.3. Dopamine release in the striatum unchanged
    • 1.2.3. Dopamine uptake increased (only) in the nucleus accumbens
  • 1.3. DAT-KO Mouse / DAT-KO Rat
  • 1.4. DAT (+/-) Mouse
  • 1.5. Coloboma mice (CM)
  • 1.6. 6-OHDA mouse, 6-OH dopamine-lesioned rat
  • 1.7. Tal1cko mice
  • 1.8. THRSP-OE Mice
  • 1.9. SORCS2 -/- Mice
  • 1.10. ICR mice
  • 1.11. FOXP2HUM mice
  • 1.12. TARP γ-8-KO mice / CACNG8-KO mice
  • 1.13. D4R-KO mice
• 2. Animal models with increased dopamine levels
  • 2.1. LPHN3 knockout rat/mouse (ADGRL3-KO mouse)
  • 2.2. P35-KO mouse
  • 2.3. FOXP2wt/ko mice
• 3. Animal models with unknown influence on dopamine levels
  • 3.1. GIT1 KO mouse
  • 3.2. ATXN7 Overexpressed Mouse
  • 3.3. Grin1 mouse
  • 3.4. AGCYAP1-KO mouse
  • 3.5. Other ADHD mouse/rat models
  • 3.6. Drosophila (fruit fly)
• 4. Animal models that inadequately represent ADHD
  • 4.1. Naples high-excitability rat (NHE)
  • 4.2. WKHA advice
  • 4.3. Acallosal mouse
  • 4.4. Hyposexual advice
  • 4.5. PCB-exposed rat
  • 4.6. Lead-exposed mouse
  • 4.7. Rat reared in social isolation
  • 4.8. TAAR-1-KO mouse
  • 4.9. MACROD1 and MACROD2-KO mice
  • 4.10. Stroke-prone spontaneously hypertensive rat (SHRSP/Ezo)

1. Animal models of ADHD with decreased dopamine levels / decreased dopamine action¶

By “decreased dopamine” here we mean decreased phasic dopamine in the striatum.
Decreased phasic dopamine in the striatum is accompanied by increased tonic dopamine in the PFC.

1.1. Spontaneous(ly) hypertensive rat (SHR)¶

SHR exhibits (with the exception of sex differences) all major human ADHD-HI traits (with hyperactivity):

• Hyperactivity⁷
  • Develops with age
  • Reduced by MPH and AMP
• Impulsivity⁷
  • Impaired ability to withhold reactions⁷
  • Develops with age
  • Reduced by MPH
• Inattention⁷
  • Reduced by MPH
• Targeted behavior impaired
  • Restored by MPH⁸
• Low power stability⁷
• Elective impulsivity (preference for immediate smaller rewards over delayed larger rewards)⁹

The SHR serves as an animal model for the study of ADHD.¹⁰¹¹

The Spontaneous(ly) hypertensive rat (SHR) is a strain of rat bred for specific symptoms, beginning in 1963, as an animal model of hypertension.¹² The animals have genes that cause them (without early childhood stress experience in old age) to experience increasing hypertension.
in 1992, SHR were found to be a model of ADHD-HI at the same time.¹³ Since then, SHR have served as a scientific animal model of ADHD-HI (with hyperactivity) in ADHD research.

SHR was developed in 1963 by mating Wistar Kyoto males with marked elevation of blood pressure with females with slightly elevated blood pressure. Subsequently, brothers were mated with sisters under continued selection for spontaneous hypertension.¹⁴
That all specimens exhibit identical behavior that stresses young to exactly the same degree should be impossible. Nevertheless, the pathological behavior patterns are present in all specimens.¹⁵ This indicates that certain genetic constellations can cause psychological disorders even without the addition of stressful environmental influences, i.e. that the formula genes + environment is a frequent but not exclusive etiological model for psychological disorders.
Interestingly, the first generations of SHR had a massive problem of cannibalism on newborns. This problem has since been solved by keeping pregnant rat mothers in isolation until the young reach a certain age. It would be interesting to know if the SHR also shows special behavior towards young animals in other ways.

With increasing age and in parallel with increasing hypertension, SHR is observed to have an increasing sensitivity of the HPA axis to stress.¹⁶
High blood pressure is an organic consequence of chronic stress.¹⁷

The SHR could be further bred to a hyperactive and stress-sensitive but less aggressive and nonhypertensive strain (WK/HA) and a hypertensive but nonhypertensive strain (WK/HT) by crossing with WKY strains. MK/HA exhibit alterations in monoamine function, particularly in norepinephrine and dopamine uptake by the PFC. In addition, neuroendocrine responses in the HPA axis and POMC peptides in the anterior and posterior pituitary lobes are altered.¹⁸¹⁹

The importance of SHR as an ADHD model should be appreciated to the right extent. Just as in humans there should be little doubt that there are very many different ADHD pathways (hundreds, if not thousands, of genes are involved, which may act in very different compositions in affected individuals), the SHR is not the only model animal for ADHD, and here ADHD-C. Therefore, the SHR can at best be a possible model for ADHD. If 1000 genes were actually involved (most of which can form several alternative gene variants with different expression profiles), there would be an almost infinite number of possibilities, in purely mathematical terms, for how they could be composed. Certainly not all candidate genes have the same influence and frequency, but the line of reasoning shows that SHR can be only one of many possible genetic constellations of ADHD.

1.1.1. Dopamine system impaired¶

1.1.1.1. Dopamine synthesis impaired¶

In SHR, the miRNA let-7d is reported to be overexpressed in the PFC and the expression of galectin-3 is decreased, leading to downregulation of tyrosine hydroxylase, which is a precursor of dopamine synthesis.²⁰ This results in impaired dopamine synthesis. One study, however, found excessive galectin-3 blood plasma levels in ADHD-affected children.²¹
The synthesis of dopamine in the brain occurs in two steps. First, the amino acid tyrosine is catalyzed by the enzyme tyrosine hydroxylase and converted into l-3,4-dihydroxyphenylalanine (L-DOPA), then L-DOPA is decarboxylated to produce dopamine.

At day P5 and P7 after birth, decreased tyrosine hydroxylase gene expression was found, and at day P27 to P49, decreased midbrain dopamine transporter (DAT) gene expression was found. In adult SHR, DAT are overexpressed, which decreases dopamine levels in the synaptic cleft.²²

Further, in SHR, dopamine uptake in the striatum was markedly reduced in the first month of life.²²

SHR showed weaker release of dopamine and acetylcholine in the striatum on glutamate.²³

1.1.1.2. D2 receptor increased¶

SHR showed significantly increased dopamine D2 receptor expression in PFC, striatum, and hypothalamus. Atomoxetine significantly decreased dopamine D2 gene expression in PFC, striatum, and hypothalamus in a dose-dependent manner.²⁴
Another study did not find increased D2 expression in SHR.²²

Another study found that in SHR, postsynaptic D1-/D2-like receptors appear to be reduced in sensitivity, whereas presynaptic dopamine D2-like autoreceptors, found primarily in the nucleus accumbens, are arguably increased in sensitivity.²⁵

1.1.1.3. (Only) D4 receptor reduced in the PFC¶

SHR showed significantly decreased dopamine D4 receptor gene expression and protein synthesis in the PFC. Other dopaminergic genes in midbrain, PFC, temporal cortex, striatum, or amygdala of SHR were unchanged compared with WKY.²⁶

1.1.1.4. Dopamine release reduced¶

In SHR, the dopaminergic presynapses of mesocortical, mesolimbic, and nigrostriatal neurons appear to release less dopamine in response to electrical stimulation/depolarization because of high extracellular K+ concentrations.²⁷

SHR/NCrl showed reduced KCl-evoked dopamine release in the dorsal striatum compared with WKY/NCrl (an ADHD-I model).²⁸

1.1.1.5. Dopamine uptake in the striatum accelerated¶

SHR/NCrl showed faster dopamine uptake in the ventral striatum and nucleus accumbens than controls, whereas WKY/NCrl (an ADHD-I model) showed faster dopamine uptake only in the nucleus accumbens.²⁸ This is consistent with increased DAT activity in SHR.

1.1.2. Adenosine system altered in SHR¶

The adenosine system interacts with the dopamine system.
In SHR, adenosine is increased in blood plasma²⁹ and the amount of adenosine A2A receptors in frontocortical nerve terminals (presynapses).³⁰ The bioavailability of adenosine in vascular tissues and in arteries of SHR seems to be increased, whereas at the same time adenosine transporters (ENT) and A1 and A2A receptors are downregulated. In veins, the expression of ARs and ENTs appears unchanged, whereas the A2A receptor appears to be upregulated and the ENT2 transpprter downregulated.³¹

Adenosine receptor antagonists improve various ADHD symptoms in SHR

• Caffeine (non-selective A1 and A2A adenosine receptor antagonist)
  • Object detection³²
  • social recognition³³
  • spatial learning³⁴
  • no influence on high blood pressure³⁴
• DPCPX (8-cyclopenthyl-1,3-dipropylxanthine, A1 antagonist)
  • Object detection³²
  • no influence on high blood pressure³⁴
• ZM241385 (4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-yl-amino]ethyl) phenol, A2A-Antagonist)
  • Object detection³²
  • social recognition³³
  • no influence on high blood pressure³⁴

Chronic Caffeine Input³⁰

• normalized the dopaminergic function
• improved memory and attention deficits
• induced upregulation of A2A receptors in frontocortical nerve terminals

Chronic administration of caffeine or MPH before puberty later improved object recognition in adult SHR, whereas the same treatment worsened it in adult Wistar rats³⁵

There is evidence for an interaction between the cannabinoid and adenosine systems in relation to impulsive SHR behavior:³⁶

• WIN55212-2 (cannabinoid receptor agonist) increased impulsive behavior
• acute pretreatment with caffeine reversed this
• chronic caffeine intake increased impulsivity

In SHR, adenosine-mediated presynaptic inhibition of adrenergic transmission appears to be genetically reduced.³⁷
The A1 agonist CPA increased alpha2-adrenoceptor binding in the nucleus tractus solitarius about 10 times more in SHR than in WKY.³⁸

Adenosine affects blood pressure.³⁹ Adenosine decreased blood pressure in SHR even more than in WKY. Adenosine decreased heart rate in SHR and increased it in WKY.((Ohnishi, Biaggioni, Deray, Branch, Jackson (1986): Hemodynamic effects of adenosine in conscious hypertensive and normotensive rats. Hypertension. 1986 May;8(5):391-8. doi: 10.1161/01.hyp.8.5.391. PMID: 3699881.))

1.1.3. Norepinephrine release increased¶

In the laboratory, PFC brain cells from SHR showed increased norepinephrine release in response to glutamate. This effect was not mediated by NMDA receptors because NMDA did not alter norepinephrine release. It is suggested that the noradrenergic system is overactivated in the PFC of SHR.⁴⁰

The A1 agonist CPA increased alpha2-adrenoceptor binding in the nucleus tractus solitarius in SHR about 10 times more than in WKY.³⁸ In SHR, adenosine-mediated presynaptic inhibition of adrenergic transmission appears to be genetically reduced.⁴¹.

In SHR, autoreceptor-mediated inhibition of norepinephrine release appears to be further impaired, suggesting poorer regulation of noradrenergic function in the PFC. The behavioral disturbances of ADHD may be the result of an imbalance between noradrenergic and dopaminergic systems in the PFC, with decreased inhibitory dopaminergic activity and increased noradrenergic activity.²⁷⁴²

1.1.4. Serotonin in SHR¶

SHR show increased numbers of serotonin transporters in the striatum in adulthood, unchanged by MPH.⁴³

1.1.5. GABA in SHR¶

SHR showed decreased [3H]-GABA uptake and release, suggesting a defective striatal GABA-ergic transport system.
Caffeine improved in vitro in the striatum of SHR the

• GABA release (intrinsically reduced in SHR)
• GABA reuptake via GAT1 transporter (reduced in SHR per se)

whereas this was not the case in Wistar rats (which are not an ADHD animal model).⁴⁴

One study found evidence that extracellular concentrations of GABA may be reduced in the SHR hippocampus. An underlying defect in GABA function could be the cause of the dysfunction of catecholamine transmission found in the SHR and underlie their ADHD-like behavior.⁴⁵

The GABA antagonist oroxylin A appears to ameliorate ADHD-like behaviors in SHR via enhancement of dopaminergic neurotransmission rather than modulation of GABA signaling as previously reported.⁴⁶

1.1.6. Vitamin D3 metabolism altered in SHR¶

In SHR, 25-hydroxyvitamin D-1-alpha-hydroxylase activity appears to be decreased. This could be due to impaired renal metabolism or responsiveness to cyclic adenosine 3’,5’-monophosphate. In SHR as in WKY, a one-week restriction of dietary phosphorus resulted in an increase in plasma D3 concentration. There was no change in blood pressure as a result.⁴⁷ Another study found increased as well as decreased D3 levels.⁴⁸

1.1.7. Stress systems changed¶

1.1.7.1. Overintense HPA axis stress response in SHR compared with WKY¶

7-week-old SHR show significantly compared to WKY of the same age⁴⁹

• Increased corticosterone responses to hemorrhage and ether stress
  Note: Because SHR represent a model for ADHD-HI and not ADHD-I, we would expect flattened stress corticosterone responses as found in other studies⁵⁰
• Elevated basal corticosterone levels
  Note: Decreased basal cortisol levels are usually found in people with ADHD regardless of subtype
• Reduced plasma ACTH responses to hemorrhage and ether stress
• Lower plasma ACTH responses to iv CRH injection
• Identical plasma ACTH responses to vasopressin
• Lower CRH concentrations in hypothalamus (median eminence), posterior pituitary and cerebral cortex
• Decreased CRH release from the hypothalamus
• Identical CRH response to 56 mM KC1

If the adrenal glands, which are the source of glucocorticoids for the HPA axis, were removed in both species, the

• The ACTH response to stress is identical
• The CRH concentrations in hypothalamus (median eminence) identical
• Prevented the development of hypertension in SHR

Corticosterone given as a substitute restored the blood pressure elevation in SHR.

Dexamethasone as a glucocorticoid receptor (GR) agonist improved ADHD-HI symptoms in SHR.⁵¹ A GR antagonist (mifepristone) elicited ADHD-HI symptoms in other rat species (not otherwise exhibiting ADHD symptoms).⁵²
Dexamethasone (as a GR agonist) increased previously (compared with WKY) decreased serotonin levels in the PFC of SHR and improved attention deficit and hyperactivity. In contrast, a GR inhibitor (RU486) increased inattention and hyperactivity. Dexamethasone increased the expression of 5-HT and 5-HT2AR in the PFC and decreased the expression of 5-HT1AR. In contrast, RU486 decreased the expression of 5-HT and 5-HT2AR and increased the expression of 5-HT1AR.⁵³

These results indicate that,

• That the HPA axis is overactivated in young SHR
• That a reduced ACTH response to stress, as to CRH, is due to higher plasma corticosterone levels
• That glucocorticoids are essential for the development of hypertension in SHR
• That in ADHD-HI (with hyperactivity), the GR receptor may be under-addressed, whether due to insufficient number or sensitivity of GRs, or excessive number of MRs
• That in SHR, the glucocorticoid system is closely linked to the serotonin system

Other studies observed significantly decreased basal levels of in SHR¹⁵

• Aldosterone at 8 weeks of age
• 18-hydroxy-lldeoxycorticosterone (18-0H-D0C)1 at 12 weeks of age
• Deoxycorticosterone (DOC) at 20 weeks of age
• Corticosterone at 12 and 20 weeks of age.

1.1.7.2. Increased mineralocorticoid receptor expression as a cause of hyperintense HPA axis stress responses in SHR¶

SHR genetically have excessive expression of mineralocorticoid receptors (MR) and normal expression of glucocorticoid receptors (GR).⁵⁴
Accordingly, a shift in the balance between MR and GR toward increased MR leads to increased basal and stress-responsive activity of the HPA axis.
⇒ Corticosteroid receptor hypothesis of depression

Dexamethasone as a glucocorticoid receptor (GR) agonist improved ADHD-HI symptoms in SHR. In a mirror image, a GR antagonist (mifepristone) elicited ADHD-HI symptoms in other rat species (which otherwise do not exhibit ADHD symptoms).⁵²

This is consistent with our view that ADHD-HI (with hyperactivity) is caused or controlled by a worsened response of GR relative to MR.
We wonder whether ADHD-I might be inversely characterized by a reduced number of MR relative to GR.

MR regulate the day-to-day activities of cortisol. GR, on the other hand, are only addressed when cortisol levels are very high and have the function of shutting down the HPA axis again. In the presence of MR overload and a reduced cortisol stress response (typical of ADHD-HI), the unoccupied MRs soak up cortisol so that the GRs are not sufficiently occupied to trigger HPA axis shutdown.
In contrast, if MRs are underrepresented or if the cortisol stress response is excessive (as in ADHD-I), GRs are addressed too quickly and the HPA axis is shut down too frequently.

1.1.7.3. MiRNA expression in SHR alters glucocorticoid receptor¶

For the miRNA

• MiR-138
• MiR-138*
• MiR-34c*
• MiR-296
• MiR-494

significantly decreased expression was found in the ADHD rat model of SHR, which was related to promoter inhibitory activity of the glucocorticoid receptor Nr3c1.⁵⁵

1.1.8. SHR and immune system¶

1.1.8.1. Young SHR¶

Young SHR show in comparison to WKY⁶⁰

• Increased levels of cytokines
• Increased levels of chemokines
• Increased levels of markers for oxidative stress
• Reduced PFC volumes
• Increased levels of dopamine D2 receptors.

1.1.8.2. Older SHR¶

Older SHR show in comparison to WKY⁶⁰

• Normalized levels of cytokines
• Normalized levels of chemokines
• Normalized levels of markers for oxidative stress
• Increased levels of steroid hormones.

1.1.8.3. Other altered immune values in SHR¶

In the animal model of ADHD-HI (with hyperactivity), the Spontaneous(ly) hypertensive rat (SHR) was found in brain regions (not peripheral blood) in adult male animals:⁶¹

• Increased levels of reactive oxygen species (ROS) in cortex, striatum and hippocampus
• Decreased glutathione peroxidase activity in the PFC and hippocampus
• Decreased TNF-α levels in the PFC, the rest of the cortex, hippocampus and striatum
• Decreased IL-1β levels in the cortex
• Decreased IL-10 levels in the cortex.

1.1.8.4. Taurine improved inflammatory markers in SHR¶

SHR rats treated with taurine showed decreased serum levels of C-reactive protein (CRP) and IL-1β.⁶² While low levels of taurine increased motor activity, high levels of taurine decreased it.

1.1.9. Cholesterol metabolism altered in PFC of SHR; MPH revises change¶

One study found 12 altered metabolites in PFC in SHR (compared with WKY). The deviations of 7 of them were equalized by MPH:⁶³

• 3-Hydroxymethylglutaric acid
• 3-phosphoglyceric acid
• Adenosine monophosphate
• Cholesterol
• Lanosterol
• O-Phosphoethanolamine
• 3-hydroxymethylglutaric acid.

The altered metabolites belong to the metabolic pathways of cholesterol.
In the case of the SHRs, the PFC found for this purpose

• Reduced activity of 3-hydroxy-3-methyl-glutaryl-CoA reductase
  • Unchanged by MPH
• Decreased expression of sterol regulatory element-binding protein-2
  • Increased by MPH
• Decreased expression of the ATP-binding cassette transporter A1
  • Increased by MPH.

1.1.10. Blood pressure, sympathetic nervous system, cardiac hypertrophy and vitamin D3 in SHR¶

In SHR, compared with WKY rats, found to be

• Increased systolic blood pressure
• Increased sympathetic drive
• Cardiac hypertrophy and cardiac remodeling.

These abnormalities correlated in the paraventricular nucleus of the hypothalamus (PVN) with

• Higher mRNA and protein expression levels of
  • High mobility box 1 (HMGB1)
  • Receptor for advanced glycation end products (RAGE)
  • Toll-like receptor 4 (TLR4)
  • Nuclear factor-kappa B (NF-κB)
  • Proinflammatory associated cytokines
  • NADPH oxidase subunit
• Increased level of reactive oxygen species
• Microglia activation

as well as with

• Increased level of norepinephrine in the blood plasma.

These phenomena could be eliminated by an infusion of 40 ng of calcitriol daily.⁶⁴

40 ng calcitriol corresponds to 0.04 micrograms vitamin D3. At a weight of about 200 g / rat, this should correspond to 0.2 micrograms / kg body weight. The recommended daily dose of D3 for humans is 0.12 to 1 microgram with close medical supervision, which would correspond to a daily dose of 0.0125 microgram / kg body weight at 80 kg. The D3 dosage used in the study therefore corresponds to 16 times the upper limit of the daily dose recommended in humans. At such a dosage, considerable health risks would have to be expected in humans.

1.1.11. Brain regions reduced in size¶

An MRI scan showed that the vermis cerebelli, nucleus caudatus, and putamen were significantly reduced in SHR.²⁶

1.1.12. Brain connectivity impaired locally as well as over a wide area¶

With functional ultrasound imaging, which allows rapid measurement of cerebral blood volume (CBV), was found in SHR:⁶⁵

• increased response to visual stimulation in visual cortex and superior colliculi
• functional connectivity
  • changed over long distances between spatially separated regions
  • local / regional connectivity changed
    • regional homogeneity
      • strongly increased in parts of the motor and visual cortex
      • in the secondary cingulate cortex, the colliculi superiores and the area pretectalis reduced

1.1.13. PFC neurons altered¶

PFC neurons of SHR showed fewer neurite branches, shorter maximum neurite length, and lower axonal growth than PFC neurons of WKY.
The adenosine antagonist caffeine restored neurite branching and extension in SHR neurons via PKA and PI3K signaling.
The A2A agonist CGS 21680 enhanced neurite branching via PKA signaling.
The selective A2A antagonist SCH 58261 restored axonal growth of SHR neurons via PI3K- alone (not by PKA signaling)⁶⁶

1.1.14. Monosodium glutamate affects aggression in a vagus nerve-dependent manner¶

SHR were given monosodium glutamate (glutamate as a flavor enhancer) during the developmental phase (from day 25 for 5 weeks). This resulted in reduced aggressive behavior. Fear behavior remained unchanged. However, when the SHR were previously vagus nerve transected (vagotomy), monosodium glutamate did not decrease aggression, suggesting mediation of the effect of monosodium glutamate on aggression by the gut-brain axis.⁶⁷

1.1.15. Drug effect on SHR¶

1.1.15.1 Effect of MPH on SHR¶

SHR respond to MPH:

• Increased attention and memory⁶⁸
• Reduced impulsivity in a dose-dependent manner⁶⁸
• Hyperactivity;
  • Unchanged at low and medium doses⁶⁸
  • Increased at high doses⁶⁸
  • Reduced at very high doses¹³
• Goal-directed behavior restored by MPH⁸

Contrary to the view of the metastudy authors, we see no reason to question SHR as a model of ADHD-HI. Because ADHD is multifactorial and SHRs are merely an animal model bred for specific symptoms, SHRs can only represent one variant of ADHD (which, moreover, corresponds more to ADHD-HI than ADHD-I). At the same time, it follows that the effects in SHR cannot be generalized to all ADHD sufferers, but that the neurophysiological mechanisms mediating individual symptoms and effects must be considered.

MPH before puberty was able to normalize the otherwise increased DAT density in the striatum in adulthood. The improvement was more pronounced in the SHR/NCrl (serving as a model of the mixed type) than in the WKC/NCrl rat, which serves as a model of the ADHD-I subtype.⁴

Serotonin transporters in the striatum were not altered by MPH even with long-term administration.⁴³

1.1.15.2. Effect of amphetamine drugs on SHR¶

Amphetamine medications caused a reduction in hyperactivity in SHR.¹³

1.1.15.3. Effect of atomoxetine on SHR¶

Atomoxetine produced a reduction in hyperactivity.²⁴

1.1.16. Altered emotional communication and response of SHR¶

Rats communicate their emotional state via ultrasonic vocalizations (USV). 22 kHz represents aversive, 50 kHz represents appetitive responses.
SHR emitted more short 22-kHz and fewer 50-kHz USV overall. After fear conditioning. In addition, SHR emitted fewer long 22-kHz USV than did Wistar rats. SHR showed no increase in heart rate (HR) to 50-kHz playback, but a sharp drop in HR to 22-kHz playback. These phenomena of SHR could represent deficits in emotional perception and processing, as also seen in ADHD subjects.⁶⁹

1.1.17. SHR exhibit behavioral subgroups corresponding to ADHD-HI and ADHD-I¶

One study found subgroups on SHR that differed significantly in terms of impulsivity. Impulsive SHR showed no behavioral subgroups compared with nonimpulsive SHR and WKY (as controls, with WKY showing no behavioral subgroups):⁷⁰

• Reduced noradrenaline levels
  • In the cingulate cortex
  • In the medial-frontal cortex
• Reduced serotonin turnover
  • In the medial-frontal cortex
• Reduced density of CB1 cannabinoid receptors
  • In PFC
  • Acute administration of a cannabinoid agonist decreased impulsivity in impulsive SHR, with no change in WKY

Since SHR are not gene-identical, cloned animals, but a strain bred for specific symptoms, whose individual animals thus still contain certain genetic differences, the subtypes could also be of genetic origin. So far, however, no heritability has been established for stress endophenotypes (typically more externalizing or internalizing stress response, corresponding to the ADHD-HI subtype/ADHD-C and the ADHD-I subtype).

The reduced norepinephrine level in ADHD-I subtypes of SHR appears to be consistent with Woodman’s findings:

• Aggression and outward anger correlate with elevated norepinephrine⁷¹
• Anxiety, on the other hand, correlates with increased adrenaline⁷¹

1.2. WKY/NCrl - Rats¶

WKY/NCrl represent an animal model for the ADHD-I subtype (attention deficit without hyperactivity).²³⁴

1.2.1. Increased tyrosine hydroxylase in WKY/NCrl¶

WKY/NCrl show increased tyrosine hydroxylase gene expression as adults.⁴

1.2.2. Increased DAT in adulthood from WKY/NCrl¶

WKY/NCrl show increased DAT gene expression at day P25, but not as much increased as SHR/NCrl. Two weeks of treatment with MPH decreased DAT, with a greater reduction when administered before puberty.⁴

1.2.3. Dopamine release in the striatum unchanged¶

Unlike SHR/NCrl, KY/NCrl did not show reduced KCl-evoked dopamine release in the dorsal striatum compared with WKY/NHsd controls.²⁸

1.2.3. Dopamine uptake increased (only) in the nucleus accumbens¶

Whereas WKY/NCrl (an ADHD-I model) showed faster dopamine uptake than controls in the nucleus accumbens only, SHR/NCrl showed faster dopamine uptake in the nucleus accumbens and ventral striatum.²⁸

1.3. DAT-KO Mouse / DAT-KO Rat¶

DAT-KO mice/rats are often cited as models for increased dopamine levels. This is also correct, but related to the extracellular and thus tonic dopamine level in the striatum. In the interest of comparability of animal models, we take phasic dopamine in the striatum as a reference point, which is significantly decreased in DAT-KO model animals.

The dopamine transport knockout mouse or rat (DAT1 KO) serves as an animal model for ADHD research.¹⁰¹¹

The DAT-KO mouse, whose dopamine transporter is nearly deactivated in monozygous animals and approximately halved in heterozygous animals, shows:⁷²⁷³⁷⁴

• Symptomatology:
  • Hyperactivity, spontaneous in unknown environment⁷⁵
    • However, hyperactivity was only evident in mice that had no DAT at all or 90% less DAT and whose extracellular (tonic) dopamine levels were thus increased 5-fold or at least doubled. Mice that had 50% of the usual number of DAT also had doubled extracellular dopamine levels, but did not exhibit hyperactivity.
      Motor activity is controlled by dopamine changes in the subsecond range, thus by phasic dopamine, which typically originates from the storage vesicles, since it cannot be synthesized so quickly. 50% DAT should be able to replenish the vesicles much better than 10% DAT. This may explain why the two mouse strains differed in terms of hyperactivity despite equally doubled extracellular dopamine levels. Mice with 30% increased DAT showed hypoactivity in novel environments. However, mice with a doubled DAT number showed no variation in hyperactivity or hypoactivity.⁷⁶
    • Hyperactivity in DAT-KO mouse as in DAT-KO rat remediable by
      • AMP and MPH^{7277 78}
        • Indicating that stimulants do not act alone as dopamine reuptake inhibitors:
          • In DAT-KO mice, amphetamine and methylphenidate reduced hyperactivity (occurring only in novel environments), while causing hyperactivity and stereotypy in normal mice. One study suspects that this calming effect is serotonergically mediated. Similarly, stimulants do not reduce the elevated extracellular dopamine levels in DAT-KO mice.⁷⁸
          • In rats whose dopaminergic cells were chemically destroyed (causing ADHD symptoms⁷⁹, serotonin and norepinephrine reuptake inhibitors (but not dopamine reuptake inhibitors) decreased hyperactivity (in novel environments), whereas they did not in normal mice, and dopamine reuptake inhibitors actually increased hyperactivity.⁸⁰
      • A TAAR1 receptor agonist⁷²
      • Haloperidol⁷²
        • Haloperidol increases extracellular DA concentration in the dorsal caudate more effectively than that in the PFC.⁸¹
      • Non-selective serotonin receptor agonist 5CT⁸²
      • Not by dopamine or norepinephrine reuptake inhibitors⁴²
        • Such as atomoxetine⁸²
    • Indifferent locomotor activity in response to cocaine and AMP⁷³
    • Non-focal, conservative movement patterns⁷⁵
  • Impulsivity⁷⁸
  • Increased reactivity and aggression rates after mild social contact⁸³
  • Cognitive impairments
    • Impaired erasure of habit memory⁸⁴ with otherwise unchanged learning behavior
    • Deficits in spatial learning⁷⁸
    • Memory deficits⁷⁸ and impairments in spatial cognitive function in the radial labyrinth
    • Slightly increased long-term potentiation and strongly decreased long-term depression at excitatory hippocampal CA3-CA1 synapses⁸⁵
      • Which can cause learning and memory problems, such as difficulty adapting to changes in the environment
      • The dopamine antagonist haloperidol prevented these effects
    • Increased long-term potentiation in the nucleus accumbens⁸⁶
    • Improving cognitive impairment through⁸²
      • Atomoxetine
      • Stimulants
      • Guanfacine ( alpha2A-adrenoceptor agonist)⁸⁷
        • In contrast, worsening by alpha2A-adrenoceptor antagonist yohimbine
    • No improvement in cognitive impairment by⁸²
      • Non-selective serotonin receptor agonist 5CT
  • Reward motivation deficits
    • Tendency to hedonic positive taste in food⁸⁸
    • Increased resistance to extinction of food-stressed operant behavior⁸⁴
    • Sucrose preference
      • Increases⁸⁹
      • Reduces⁹⁰
    • Increased reward responses to selective norepinephrine and serotonin blockers⁹¹
  • Sleep impaired
    • Reduced sleep⁹¹
      • Non-REM sleep
      • REM sleep
      • Lower total sheep time
    • No wakefulness effect from⁹¹
      • Modafinil
      • Methamphetamine
      • The selective DAT blocker GBR12909
    • Excessive wake-promoting effect of⁹¹
      • Caffeine
    • Normal circadian patterns of inactivity and activity⁹¹
  • Compulsive behavior⁹⁰
    • Rigid pattern behavior
    • Compulsive stereotypies in delay reward tasks
• Neurophysiological changes
  • In the dopamine system:
    • Increased extracellular dopamine levels to 5 to 6 times⁸⁵⁷⁴ in the striatum⁸⁵⁸³
    • Reduction of tissue dopamine levels to below 5%⁷⁴
      • Reduced to 1/20 the amount of dopamine in the storage vesicles usually refilled by the DAT, which reserve dopamine for phasic release, making dopaminergic functions totally dependent on the limitations of dopamine synthesis⁷⁶
    • Reduction of phasic dopamine release to 25%⁷⁴, corresponding to 1/4 reduced amplitude of evoked dopamine release⁷⁶
    • Extended lifetime of dopamine in the synaptic cleft by 300 times⁷⁴
      • Inhibition of serotonin transporters, norepinephrine transporters, MAOA, or COMT did not alter dopamine degradation. This seems to occur more by diffusion in the absence of DAT in the striatum⁷⁴
    • Increased tonic dopamine extracellular = outside the synaptic cleft⁹²
    • Decreased phasic dopamine = in synaptic cleft⁹²
      • Decreased phasic dopamine release to electrical stimulation, equivalent to hypodopaminergic functionality⁹³ as seen in SHR and coloboma mice⁹⁴
    • Medium-sized spike-bearing projection neurons (the most common class of dopamine receptive neurons, such as D1 receptor, D2 receptor, and DARPP-32)⁹⁵ show high-grade localized loss of spines (spikes) on the dendrites of the proximal segment, but no overall morphological change in terms of dendrite length, number, or overlap, or in synapse-to-neuron ratio.⁹⁶
    • Downregulation of D1 receptors by 50%⁷³ in the striatum⁸³
    • Downregulation of postsynaptic D2 receptors in the striatum by 50%.⁴²
    • Downregulation of (presynaptic) D2 autoreceptors⁹⁷ in the striatum⁸³
    • Decreased postsynaptic density of PSD-95 in the striatum and nucleus accumbens, as occurred in other models of increased dopamine levels⁸⁶
  • BDNF
    • In PFC
      • Decreased BDNF gene expression⁹⁸
      • Total BDNF and BDNF exon IV mRNA levels reduced⁹⁹
      • MRNA levels of BDNF exon VI unchanged⁹⁹
      • Decreased mBDNF levels and decreased trkB activation⁹⁹
      • Decreased activation of αCaMKII in the PFC⁹⁹
    • In the dorsolateral striatum
      • MBDNF level in the homogenate increased⁹⁹
      • MBDNF level in the cytosol increased⁹⁹
      • MBDNF levels in the postsynaptic density reduced.⁹⁹
    • TrkB expression in the dorsolateral striatum postsynaptically reduced⁹⁹
      • TrkB is a high-affinity BNDNF receptor
  • PSD-95 expression postsynaptically reduced in the dorsolateral striatum⁹⁹
    • PSD-95 is an index of glutamate spin density and measures the interaction between dopaminergic and glutamatergic systems in the striatum, which is important for cognitive processing
  • Decreased GHRH levels¹⁰⁰
    • Dopamine receptors in the hypothalamus inhibit the release of GHRH in the hypothalamus¹⁰¹
  • Deficient sensorimotor gating as measured by prepulse inhibition (PPI) of startle response⁷⁵
  • Anterior pituitary underdeveloped¹⁰⁰
    • The anterior pituitary (the adenohypophysis) is a part of the HPA axis (stress axis)

Quite a few of these features were found (to a lesser extent) in mice with only reduced DAT and doubled extracellular dopamine levels.⁷⁴

The symptoms of DAT-KO mouse could be explained by:⁷⁶

• A increased tonic dopamine level, which (due to the exhausted salivary vesicles) is accompanied by a decreased phasic dopamine level, so that too little dopamine is available for short-term steering tasks.
  Due to the lack of DAT, the remaining dopamine stores in the vesicles used for phasic release are completely dependent on the re-synthesis of dopamine.
  • This could correspond to the situation after (partial) death of dopaminergic cells, such as after encephalitis, which is also associated with hyperactivity. A (partial) death of dopaminergic cells is accompanied by a significant reduction in the number of dopaminergic presynapses and the corresponding dopamine reuptake sites¹⁰².
  • This may further correspond to the model of mice neonatally treated with the DAT toxin 6-hydroxydopamine (6-OHDA), which show hyperactivity and cognitive impairment for a time thereafter.
• Of indirect regulation of dopaminergic neurotransmission by noradrenergic and serotonergic⁷⁸ mechanisms of AMP and MPH.
• From a reduction in exocytotic dopamine release due to decreased phosphorylation of synapsin¹⁰³

Studies in other mouse strains that have more DAT than DAT-KO mice but have less DAT than wild mice showed that the number of DAT correlates with decreased basal dopamine levels, and as DAT number increases, basal dopamine levels decrease.⁷⁶

Methylphenidate and amphetamine medication remediate hyperactivity in the DAT-KO mouse (= DAT(-/-) mouse). MPH was also able to remedy and normalize the learning impairment in shuttle-box avoidance behavior. Here, the effective dose of MPH increased extracellular dopamine in the PFC but not in the striatum, whereas MPH increased dopamine in the PFC and striatum in the DAT(+/-) and DAT(+/+) mice.¹⁰⁴ The authors discuss that MPH, which also acts as a norepinephrine reuptake inhibitor, may have inhibited NET in the PFC, thereby causing the therapeutically effective dopamine increase in the PFC. NET also degrade dopamine in the PFC. Another option would be that the increased norepinephrine in the PFC due to NET inhibition could have mediated the therapeutic effect.
On the other hand, DAT-KO mice suffer from an extremely high level of dopamine in the striatum, which did not decrease even when the level of dopamine in the PFC was increased.
Guanfacine (single as well as chronic) in DAT-KO rats:¹⁰⁵

• improved spatial working memory
• improved prepulse inhibition (PPI)
• altered power spectra and coherence of brain activity
  The authors see this as confirmation of the importance of the intricate balance of norepinephrine and dopamine in attention regulation

1.4. DAT (+/-) Mouse¶

DAT (+/-) mice, unlike DAT-KO mice, still have dopamine transporter function present but reduced compared with wild type (DAT hypofunction).

DAT (+/-) mice showed¹⁰⁶

• Hyperactivity
  • Starting already before the youth
  • Remediable by amphetamines
• Unchanged reactions to external stimuli
• Unchanged sensorimotor gating abilities
• General cognitive impairment
  • In juvenile males and females
  • Partially improved in adult males
    • Attention deficits remained evident
    • Impulsivity remained evidently increased
  • Unchanged in adult females
  • Remediable by amphetamines
• Reduced expression of Homer1a
  • In PFC
  • Not in other brain regions (striatum)
  • Amphetamines shifted Homer1a expression reduction of PFC in striatum
• ARC and Homer1b unchanged

1.5. Coloboma mice (CM)¶

The Coloboma mouse mutant (Cm) serves as an animal model for ADHD research.¹⁰¹¹⁴²
Cm mice show a mutation in the SNAP-25 gene and are viable only in the heterozygous form. The relationship between SNAP-25 and ADHD is unclear. NAP-25 is a presynaptic protein that regulates the exocytotic release of neurotransmitters; coloboma mice have only 50% of normal protein levels.

Cm mice show the following symptoms:¹⁰⁷

• Impulsivity
  • Different Russell et al⁴²
• Inhibition problems
• Probably also inattention
  • Different Russell et al⁴²

Cm mice show compared to control mice:¹⁰⁸

• Changes in the HPA axis¹⁰⁸
  • No CRH elevation in the hypothalamus due to acetylcholine
  • More elevated plasma corticosterone levels due to exercise restriction stress
• Changes in the dopamine system
  • Reduced release of glutamate by (K+) depolarization in cortical synaptosomes [156]
  • DRD2 Expression¹⁰⁹
    • Increased in VTA
    • Increased in the substantia nigra
      -> suggesting increased inhibition of the firing rate of dopamine neurons
    • Unchanged in striatum
  • DRD1 expression
    • Unchanged in striatum¹⁰⁹
  • Dopamine release
    • Reduced in the striatum[156,158]
      • Reduced only dorsally, not ventrally¹⁰⁸
    • In the nucleus accumbens is reduced¹⁰⁹
    • Dopamine metabolites DOPAC and HVA decreased in the striatum
      -> consistent with decreased dopamine release and decreased dopamine turnover¹¹⁰
      -> hypofunctional dopaminergic system, similar to SHR⁴²
  • Expression of the tyrosine hydroxylase gene¹⁰⁹
    • In the VTA unchanged
    • In the substantia nigra unchanged
    • Increased in the locus coeruleus
• Changes in the noradrenergic system
  • Expression of α2A-adrenoceptors increased in the locus coeruleus¹⁰⁹
  • Noradrenergic function seems to be increased
    • Experimental withdrawal of norepinephrine by DSP-4 reduces hyperactivity but does not completely abolish it¹¹¹
  • Norepinephrine levels increased in striatum and nucleus accumbens¹⁰⁹
• Changes in the serotonergic system
  • Markedly reduced serotonin levels in the dorsal but not in the ventral striatum¹⁰⁸

1.6. 6-OHDA mouse, 6-OH dopamine-lesioned rat¶

6-OHDA mice are mice in which dopaminergic cells are destroyed 5 days after birth using 6-hydroxydopamine. They are considered an ADHD model and show symptoms:^42112107

• Hyperactivity (in the open field)¹¹³
  • Initially reduced, increased with repetitions
  • Improved by MPH and AMP
  • Unchanged by DAT reuptake inhibitors
  • Reduced by⁴²
    • DRD4 antagonists
      • D4R-KO mice show no hyperactivity upon treatment with 6-hydroxydopamine as well as normal avoidance behavior in contrast to the lack of inhibition in lesioned wild-type animals¹¹⁴
    • Serotonin transporter reuptake inhibitor
    • Norepinephrine transporter reuptake inhibitor
      • Also causes reduced dopamine uptake into noradrenergic presynapses in, among other places
        • PFC
        • Nucleus accumbens
• Attention deficit in old age
• Impulsivity in old age (five-choice serial reaction time task)
• Anxiety-like behavior (in the elevated plus maze test)
• Antisocial behavior (in social interaction)
• Decreased cognitive functions (problems with recognition of novel objects)
• Learning difficulties in a spatial discrimination task
  • Improved by MPH and AMP
• Increased sensitivity to pain¹¹⁵
  • Pain sensitivity is thought to be mediated by α-adrenergic, β-adrenergic, and D2/D3 receptors
  • Atomoxetine could reduce pain sensitivity

Neurophysiological changes in the 6-OHDA mouse/rat:

• Changes in the dopamine system:
  • Dopamine deficiency in the striatum and nucleus accumbens¹¹³
  • DRD4 expression increased in caudate nucleus and putamen¹¹⁶
    • A selective D4 antagonist decreased hyperactivity, a D4 agonist increased it¹¹⁶
    • Locomotor hyperactivity correlated positively with increased D4R count in the striatum¹¹⁴
  • Reducing hyperactivity by:¹¹⁷
    • Selective norepinephrine reuptake inhibitors
    • Methylphenidate
    • Amphetamine
  • D2 receptor expression not increased¹¹⁶
  • Dopamine reductions, as typical in ADHD¹¹²¹⁰⁷
  • Changes in cortical thickness typical in ADHD¹¹²¹⁰⁷
  • Abnormalities in the neurons of the anterior cingulate cortex, as is typical in ADHD¹¹²¹⁰⁷
• Changes in the serotonin system
  • Serotonin transporter¹¹⁸
    • Binding increased in the striatum
    • Binding unchanged in PFC

1.7. Tal1cko mice¶

Most GABAergic neurons in midbrain dopaminergic nuclei are dependent on the transcription factor Tal1 for their development. Tal1 functions here as a cell fate selector gene that promotes GABAergic differentiation at the expense of alternative glutamatergic neuron identities. Brainstem nuclei harboring Tal1-dependent neurons have been implicated in the control of dopamine neurons and in the regulation of movement, motivated behavior, and learning.
Mice carrying En1Cre16 and Tal1flox11 alleles were crossed to generate En1Cre/+; Tal1flox/flox (Tal1cko) mice. In the Tal1cko mice, the En1Cre allele drives recombination in a tissue-specific manner in both midbrain and rhombomere 1, but this leads to a failure of GABAergic neurogenesis in the brainstem only in embryonic rhombomere 1.
Tal1cko mice showed:¹¹⁹

• Hyperactivity
• Increased motor impulsivity
• Changed reaction to reward
• Delay discounting (delay aversion)
• Impaired learning
• The ADHD-typical paradoxical calming response to pharmacologically stimulated dopamine release by amphetamine and atomoxetine
• Developmental changes in anterior brainstem GABAergic and glutamatergic neurons.
  These are involved in
  • Regulation of the dopaminergic pathways
  • Basal Ganglia Outpout
• Lower body temperature
• Lower body temperature rise during stress
• Lower nesting
• Lower grooming (brooding/grooming behavior)
• Decreased levels of dopamine and dopamine metabolites in
  • Nucleus accumbens (most prominent)
  • Dorsal striatum
  • PFC
• Unchanged number of dopaminergic cells in
  • Substantia nigra
  • Ventral tegmentum
• Unchanged serotonin and 5-HIAA levels in
  • Dorsal striatum
  • Nucleus accumbens
  • PFC
• Unchanged noradrenaline level in
  • PFC
• Unchanged social behavior

1.8. THRSP-OE Mice¶

A line of mice with overexpressed THRSP gene in the striatum (THRSP-OE) showed inattention in novel object recognition and Y-maze test, but no hyperactivity in the open-air test and no impulsivity in the cliff avoidance and delay restriction task. Expression of dopamine-related genes (genes for dopamine transporters, tyrosine hydroxylase, and dopamine D1 and D2 receptors) in the striatum was increased. Methylphenidate (5 mg/kg) improved attention and normalized the expression of dopamine-related genes in THRSP-OE mice.² Therefore, the THRSP-OE mice may represent an animal model for ADHD-I.¹²⁰

We tentatively infer from increased DAT gene expression a deficiency of phasic dopamine in the striatum.

THRSP-OE mice with ADHD-I traits were found to have an altered protein network involved in Wnt signaling. Compared with THRSP knockout (KO) mice, THRSP-OE mice showed attentional and memory impairments accompanied by dysregulated Wnt signaling that impaired cell proliferation in the hippocampal dentate gyrus and expression of neural stem cell (NSC) activity markers. Combined exposure to an enriched environment and treadmill training was able to improve behavioral deficits in THRSP OE mice as well as Wnt signaling and NSC activity¹²¹

SHR/NCrl - Rats (ADHD-HI, hypertension) as well as Wistar-Kyoto rats (WKY/NCrl) (inattention) also show increased expression of the THRSP gene²

The THRS gene is involved in the regulation of lipogenesis, particularly in the lactating mammary gland. It is significant for the biosynthesis of triglycerides with medium-length fatty acid chains.

1.9. SORCS2 -/- Mice¶

The SORCS2 gene is a candidate gene for ADHD-HI and is also associated with bipolar disorder, schizophrenia, and symptoms of alcohol withdrawal.
SORCS2 influences the outgrowth of neurites in the brain. During embryonic development, SORCS2 is expressed in dopaminergic precursors of the later ventral tegmentum and substantia nigra.

SORCS2-/- mice are severely deficient in Sorcs2. This causes significant changes in the dopaminergic system.
Embryos of SORCS2-/- mice were found to have increased midbrain projections expressing tyrosine hydroxylase. In adult SORCS2-/- mice, the frontal cortex is hyperinnervated (supplied with more nerve fibers), arguing for a critical role of SORCS2 in growth cone shrinkage (the branched tip of an outgrowing axon of a neuron) during dopaminergic innervation.¹²²
SORCS2-/- mice show¹²²

• Hyperactivity in new environment
  • Is reduced by amphetamine administration
• Risk appetite
• Inattention
• Reduced interest in sugar

Neurophysiologically, SORCS2-/- mice showed ¹²²

• D1 receptor sensitivity reduced
• D2 receptor sensitivity increased
• Decreased phasic and increased tonic dopamine signaling in the ventral tegmentum.
  • Tonic dopamine release provides a stable baseline level of extrasynaptic dopamine
  • Phasic (rapid, high-amplitude, intra-synaptic) dopamine release is involved in reward and goal-directed behavior

1.10. ICR mice¶

ICR mice are more motor active than C57BL/6J or CBA/N mice.
ICR mice showed increased levels of L-tyrosine, a dopamine precursor, and decreased dopamine levels in striatum and cerebellum. Administration of L-dopa improved hyperactivity in ICR mice and increased dopamine levels in cerebellum, hippocampus, striatum, and PFC. Administration of BH4 increased dopamine levels in the cerebellum and hippocampus but did not alter behavior. BH4 did not affect serotonin levels.¹²³

1.11. FOXP2HUM mice¶

Substitution of two amino acids (T303N, N325S) in the transcription factor FOXP2 in mice showed:¹²⁴

• reduced dopamine levels in
  • Nucleus accumbens
  • Frontal cortex
  • Cerebellum
  • Putamen caudatus
  • Globus pallidus
• Glutamate, GABA, serotonin unchanged
• increased dendrite length and increased synaptic plasticity of medium spiny neurons (MSN) in the striatum
• qualitatively different ultrasound vocalizations
• reduced exploratory behavior
• higher cautiousness / anxiety (stayed closer to the wall of the test field)
• viable and capable of reproduction
  • in contrast to FOXP2-KO mice

Since FOXP2 is not expressed in dopaminergic cells, it is an indirect effect on dopamine levels.

1.12. TARP γ-8-KO mice / CACNG8-KO mice¶

Names:
TARP γ-8: transmembrane-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor regulatory protein γ-8
CACNG8: Calcium Voltage-Gated Channel Auxiliary Subunit Gamma 8

The TARP γ-8 protein is a subunit of the AMPA receptor (AMPAR).

Juvenile TARP γ-8-knockout (KO) mice showed:¹²⁵

• ADHD-like behaviors:
  • Hyperactivity
  • Impulsivity
  • Anxiety
  • impaired cognition
  • Memory deficits.
• a dysfunction of the AMPA-glutamate receptor complex in the hippocampus
• a dysregulation of dopaminergic and glutamatergic transmissions in the PFC.
• MPH significantly improved
  • the most important behavioral deficits
  • the abnormal synaptosomal proteins, especially in the PFC
    • a reversal of the upregulation of Grik2
    • a reversal of the upregulation of the DAT (Slc6a3)
  • the function of the synaptic AMPAR complex by upregulating other AMPAR auxiliary proteins in hippocampal synaptosomes

In humans, there are also strong associations between SNP of TARP γ-8 genes and susceptibility to ADHD.

Based on DAT upregulation in TARP γ-8-KO mice, we assume that they are deficient in phasic dopamine.

1.13. D4R-KO mice¶

D4R-KO mice have an inactivated dopamine D4 receptor.

Show D4R-KO mice

• Hyperexcitability of frontal cortical P neurons¹²⁶¹²⁷
• The gain of function of the D4 receptor by the D4.7R gene variant, on the other hand, shows a decrease in cortico-striatal glutamatergic transmission¹²⁸

2. Animal models with increased dopamine levels¶

By increased dopamine levels, we mean (phasic) dopamine levels in the striatum.

2.1. LPHN3 knockout rat/mouse (ADGRL3-KO mouse)¶

Latrophilin-3 (LPHN3; ADGRL3), a G protein-coupled receptor, belongs to the adhesion receptor subfamily. LPHN3 regulates synaptic function and serves to maintain in brain regions that mediate locomotor activity, attention, and place and path memory

LPHN3 / ADGRL3 is a candidate gene for ADHD.^129130131132
LPHN3 binds to Gαi1, Gαi2, Gαs, Gαq, and Gα13. In particular, gene variants that cause impaired Gα13 binding appear to be relevant in ADHD.¹³³

LPHN3-KO mice show:¹³²

• no increased anxiety behavior
• greatly reduced maternal caregiving behavior (less than half)
• Hyperactivity
• no habituation to the open field
• decreased ability to distinguish between new and known objects
• Impairment of visual-spatial memory
• increased sociability with simultaneously impaired social memory
• Absence of aggression in the inhabitant-intruder paradigm
• increased impulsivity in the continuous performance test (CPT)
• decreased motivation to eat on the continuous performance test (CPT)
• no elective impulsivity (no preference for immediate smaller versus delayed larger rewards)⁹

Severe alteration of gene expression:¹³²

• PFC: 180 genes with significantly altered expression
  • 115 (63.9%) highly regulated, some of them more than twice as active, e.g.
    • Interleukin 31 (Il31)
    • Starch Binding Domain 1 (Stbd1)
  • 65 genes downregulated
    • 22 thereof by at least 50 %.
    • DAT gene in the PFC most downregulated
      • However, DAT is hardly involved in dopamine degradation in the PFC.
• Hippocampus: 36 genes with significantly altered expression
  • 23 genes (63.9%) upregulated
  • only 2 genes by at least double, here also Stbd1

The Sprague-Dawley LPHN3 knockout rat exhibits learning and memory deficits and increased dopamine release and dopamine reuptake in the striatum,¹³⁴ as well as hyperactivity.¹³⁵
LPHN3-KO rats showed higher DA release with reduced duration compared with wild-type rats.¹³⁴

Based on the reported decrease in expression of the DAT gene, however, we would have rather suspected a decreased phasic dopamine activity.

LPHN3-KO rats show a reduced effect of stimulants on ADHD symptoms.¹³⁶

ADGRL3.1 null zebrafish larvae (ADGRL3.1-/-) exhibit a robust hyperactive phenotype:¹³⁷
Hyperactivity can be remedied by three non-stimulant ADHD medications, but all significantly impaired sleep.
Four other compounds showed comparable effects to atomoxetine:

• Aceclofenac
• Amlodipine
• Doxazosin
• Moxonidine
  • Moxonidine has a high affinity for imidazoline-1 receptors
  • the selective imidazoline-1 agonist LNP599 showed a comparable effect to other non-stimulant ADHD agents
  • Clonidine apparently addresses the imidazoline-1 receptor nonselectively

2.2. P35-KO mouse¶

Mice that cannot produce the P35 protein (P35-KO mouse) show spontaneous hyperactivity that can be reduced by MPH and AMP.¹³⁸ They have increased dopamine levels with decreased dopamine turnover and concomitant decreased CDK5 activity. The number of DAT in the striatum and thus dopamine reuptake is decreased.¹³⁹
In vitro, inhibition of Cdk5 activity in N2a cells caused a significant increase in constitutive DAT endocytosis with a concomitant increase in DAT localization in recycling endosomes. ¹³⁹

2.3. FOXP2wt/ko mice¶

Heterozygous Foxp2wt/ko mice have intermediate levels of Foxp2 protein and thus can be used to assess the consequences of reduced Foxp2 expression:¹²⁴

• increased dopamine levels in
  • Nucleus accumbens
  • Frontal cortex
  • Cerebellum
  • Putamen caudatus
  • Globus pallidus
• slightly increased exploratory behavior
• decreased dendrite length and reduced synaptic plasticity of medium spiny neurons (MSNs) in the striatum

Since FOXP2 is not expressed in dopaminergic cells, it is an indirect effect on dopamine levels.

3. Animal models with unknown influence on dopamine levels¶

In subsequent animal models, we have not yet been able to determine whether (phasic) dopamine levels in the striatum are increased or decreased.

3.1. GIT1 KO mouse¶

The G-protein coupled receptor kinase 1 knockout mouse (GIT1 KO) serves as an animal model for ADHD research.¹⁰¹¹
The GIT1 KO mouse shows hyperactivity, learning disorders and memory loss as ADHD symptoms. The hyperactivity in GIT1 KO mice is remediable by amphetamine and methylphenidate.¹⁴⁰
GIT1 regulates dopamine receptors. Overexpression of GIT1 interferes with the internalization of numerous G protein-coupled receptors, including dopamine receptors.¹⁴¹ The latter suggests a model of reduced dopamine action.

3.2. ATXN7 Overexpressed Mouse¶

The ATXN7 Overexpressed mice (ATXN7-OE) feature

• Hyperactivity
• Impulsivity
• no inattention

Dires corresponds to the ADHD-HI subtype.
Ataxin-7 gene (ATXN7) correlates with hyperactivity. ATXN7-OE mice have overexpression of the Atxn7 gene and protein in the PFC and striatum. Atomoxetine (3 mg/kg, intraperitoneal) decreases ADHD-HI-like behavior and ATXN7 gene expression in the PFC and striatum.¹⁴²

3.3. Grin1 mouse¶

Grin1 mice are a heterozygous mutant strain. Grin1 (glutamate [NMDA] receptor subunit zeta-1) encodes a protein required for NMDA receptor function. Grin2B may be associated with ADHD. Grin1 mice show:

• Hyperactivity¹⁴³
• Novelty seeking¹⁴³
• Reduced social interaction¹⁴³
• Anxiety¹⁴⁴

The attentional abilities of Grun1 mice have not yet been studied.¹⁴³

Hyperactivity improved by high-dose methylphenidate. Whereas in control mice c-FOS was very low in the prelimbic cortex and striatum and increased by MPH, in GRIN1Rgsc174 ⁄ + mice c-FOS was high in the prelimbic cortex and was reduced by MPH (at very high doses). Grin1Rgsc174 ⁄ + mice further showed increased phosphorylation of the protein ERK2 in the nucleus accumbens, which hardly changed even after a very extreme MPH dose (30 mg/kg). The authors concluded that the behavioral symptoms of the GRUN1 mouse were due to NMDA receptor dysfunction in the relevant brain regions, and that the effect of MPH in the GRIN1 mouse was not mediated specifically via the DAT but via other receptors or influences, since the DAT should have already shown effects at much lower doses.¹⁴⁴ The authors further point out that glutamatergic neurotransmission is also altered in SHR. SHR do not respond at all to MPH with respect to hyperactivity, but do respond to AMP (see there).

3.4. AGCYAP1-KO mouse¶

The Adcyap1 gene encodes the pituitary-generated neuropeptide adenylate cyclase activating polypeptide 1. Mice lacking the ADXAP1 gene (Adcyap1(-/-)) show increased novelty seeking and hyperactivity. One study found sensory-motor gating deficits in them in the form of prepulse inhibition (PPI) deficits. Amphetamine was able to normalize PPI and hyperactivity. This occurred via serotonin 1A (5-HT(1A)) receptor signaling. Wild-type mice also developed hyperactivity in response to the 5-HT(1A) agonist 8-hydroxy-2-(di-n-propylamino)tetralin, which could likewise be relieved by AMP. AMP-treated AGCYAP1-KO mice were also found to have increased c-Fos-positive neurons in the PFC, suggesting increased inhibitory control by prefrontal neurons.¹⁰⁴

3.5. Other ADHD mouse/rat models¶

Other rodent models of ADHD that we have not previously described include:¹⁴⁵

• Prenatal Alcohol Exposure Rat
• Prenatal Nicotine Exposure Rat / Mouse
• Neurokinin-1 Receptor Knockout Mouse

3.6. Drosophila (fruit fly)¶

Research on Drosophila showed that gene variants determined the behavior of Drosophila to, for example, unpleasant air blasts.
Drosophila that showed a hyperactive response to air blasts for a particularly long time had a specific mutation of the dopamine transporter gene, which is one of the most important candidate genes in ADHD.¹⁴⁶ When these Drosophila were treated with cocaine, they quieted down more quickly.
The dopamine D1 receptor was essential for learning behavior in Drosophila. Drosophila with an artificially silenced D1 receptor (throughout the brain) could not learn that a particular odor acted as a warning signal for an air blast.¹⁴⁷
If the D1 receptor gene was repaired exclusively in the brain region of the “Central Complex”, the Drosophila were no longer hyperactive, but were still unable to learn. If, on the other hand, the D1 receptor gene was repaired only in the brain region of the “mushroom body”, the ability to learn was restored, while the hyperactivity remained.¹⁴⁶

A Drosophila breeding line that was also bred to have (in)sleep problems simultaneously showed considerable hyperactivity and increased sensitivity to environmental stimuli after 60 generations.¹⁴⁸

4. Animal models that inadequately represent ADHD¶

There are quite a few other animal models that show symptoms of ADHD. However, many of them have only single symptoms or are not suitable to describe the etiology of ADH)S for other reasons:⁴²¹⁴⁹

4.1. Naples high-excitability rat (NHE)¶

• Hyperactive in new environment
• Not hyperactive or impulsive in familiar surroundings
• No permanent attention problems

4.2. WKHA advice¶

• Hyperactive
• Not impulsive
• No problems with sustained attention

4.3. Acallosal mouse¶

• Hyperactivity
  • Becomes hyperactive only with age
• Impulsive
  • Impulsivity decreases with the number of tests on this; this does not correspond to ADHD
• Impairment in conditioned learning tasks

4.4. Hyposexual advice¶

4.5. PCB-exposed rat¶

• Hyperactivity
• No permanent attention problems

4.6. Lead-exposed mouse¶

• Hyperactivity
• Ataxia
• Other symptoms of lead poisoning well distinguishable from ADHD

4.7. Rat reared in social isolation¶

• Hyperactivity in new environment
• Increased omission errors
• Endurance problems
• No impulsivity
• No impairment in the 5-choice serial reaction time (5-CSRT) sustained attention test task acquisition measure

4.8. TAAR-1-KO mouse¶

• Reduced prepulse inhibition¹⁵⁰
• Unchanged:¹⁵⁰
  • Weight, height, body temperature
  • Anxiety Behavior
  • Stress Responses
• Amphetamine administration¹⁵⁰
  • Has a stronger psychomotor stimulating effect
  • Increased increase in dopamine and norepinephrine in the dorsal striatum
  • Correlates with increase in high-affinity D2 receptors (D2-high) in the striatum by 262% (48.5% D2-high receptors in the stratum compared to 18.5% in normal mice)

4.9. MACROD1 and MACROD2-KO mice¶

• (Female only) MACROD1-KO mice showed motor coordination problems.¹⁵¹
• MACROD2-KO mice show hyperactivity, which further increased with age, in combination with a bradykinetic gait pattern (slower and shuffling gait, as in Parkinson’s disease)¹⁵¹

4.10. Stroke-prone spontaneously hypertensive rat (SHRSP/Ezo)¶

The stroke-prone spontaneously hypertensive rat (SHRSP/Ezo) showed in one study¹⁵²

• a reduced D-serine/D-serine + L-serine ratio in the mPFC and the hippocampus
  • D-serine binds to NMDA receptors
• D-amino acid oxidase (DAAO, a D-serine-degrading enzyme) was increased in mPFC
• Serine racemase (SR, D-serine biosynthetic enzyme) was decreased in the hippocampus
• a microinjection of a DAAO inhibitor
  • in the mPFC increased the DL ratio and decreased ADHD symptoms such as inattention and hyperactivity in the Y-maze test
  • into the hippocampus also increased the DL ratio, but did not alter ADHD symptoms

The authors conclude NMDA receptor dysfunction in the mPFC as the cause of ADHD symptoms in SHRSP/zo