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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.
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¶
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.
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.
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
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
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
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.
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)
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
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
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
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
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
1.1.7.1. 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
SHR, corticosterone and stress sensitivity
Castrated or sterilized SHR showed decreased blood pressure and increased basal corticosterone levels,56 which, in our opinion, contrary to the authors’ conclusion, may suggest that insufficient basal corticosterone levels (and insufficient response intensity of the HPA axis) may cause the hypertension. In addition, the relationship between stress, sex hormones, and mental disorders is clarified.
The decreased basal corticosterone level in SHR or cortisol level in people with ADHD-HI may result from increased glucocorticoid 6-beta hydroxylation (increased family 3A cytochrome P-450 activity). SHR respond to injected [3H] corticosterone with urinary excretion of 6β- [3H] OH-corticosterone four to five times greater than control Wistar-Kyoto rats, consistently before as well as after the development of hypertension.
Hypertension as well as 6-beta hydroxylation could be inhibited by selective 3A P-450 - cytochrome inhibitors.5758
SHR are much more sensitive to heat or other stressors,59 which correlates with the increased sensitivity present in ADHD.
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.
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
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
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
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
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
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
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 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
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
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
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.
Dopamine metabolites DOPAC and HVA decreased in the striatum
-> consistent with decreased dopamine release and decreased dopamine turnover110
-> hypofunctional dopaminergic system, similar to SHR42
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
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
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
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.
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
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
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
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
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
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
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.
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
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:
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).
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
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
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)
(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
