ADHD animal models with elevated extracellular dopamine

In this article, we compile animal models of ADHD that exhibit elevated extracellular dopamine levels. To ensure comparability among the animal models, we use extracellular dopamine in the striatum as a reference point.
We have also provisionally included here animal models of ADHD in which we know only that dopamine levels are elevated, without knowing whether they are extracellular or phasic.
If we have inferred an elevated extracellular dopamine level solely on the basis of reduced DAT, this is indicated.

2. Animal models with elevated extracellular dopamine¶

2.1. DAT-KO Mouse / DAT-KO Rat (DA levels are elevated extracellularly but reduced in phases)¶

DAT-KO mice and rats are often cited as models of elevated dopamine levels. However, this refers to the extracellular—and thus tonic—dopamine levels in the striatum. Phasic dopamine in the striatum is significantly reduced in DAT-KO model animals. When less dopamine is reabsorbed, the vesicles that supply the stimulus-evoked phasic release of dopamine can only be filled by newly generated dopamine, so that less dopamine is available for phasic release.

The dopamine transporter knockout mouse or rat (DAT1 KO) serves as an animal model for research on ADHD.¹²

The DAT-KO mouse, in which the dopamine transporter is virtually inactivated in monozygotic animals and reduced by about half in heterozygous animals, exhibits the following symptoms in monozygotic animals:³⁴⁵

2.1.1. Symptoms¶

2.1.1.1. Hyperactivity, occurring spontaneously in unfamiliar surroundings¶

DAT-KO mice exhibit spontaneous hyperactivity only in unfamiliar environments.⁶ To the best of our understanding, this initially corresponds to a fundamentally elevated extracellular dopamine level, which is barely maintained until (due to the new environment) increased arousal or acute stress sets in, further elevating dopamine and norepinephrine to significantly elevated levels.

• In contrast, hyperactivity in children with ADHD does not occur in new situations.^{78 9} To our understanding, this is consistent with the reduced extracellular dopamine levels typical of ADHD, which are elevated to a physiologically appropriate level by the excitement of a new environment, at which point no ADHD symptoms occur for the duration of that excitement. A high level of intrinsic interest has the same effect.

However, according to DAT-KO, exposure to the new environment does not appear to correlate with a corresponding increase in dopamine levels. The behavioral changes therefore do not seem to be driven solely by the dopamine system.¹⁰
However, hyperactivity was observed only in mice that had no DAT at all or 90% less DAT, and whose extracellular (tonic) dopamine levels were consequently increased fivefold (no DAT) or at least doubled (90% less DAT). Mice with 50% of the normal amount of DAT also had extracellular dopamine levels that were doubled, but they did not exhibit hyperactivity.
Motor function is controlled by sub-second changes in dopamine levels—that is, by phasic dopamine, which typically originates from storage vesicles, since it cannot be synthesized that quickly. A 50% DAT level is likely to replenish the vesicles significantly better than a 10% DAT level. This could explain why the two mouse strains differed in terms of hyperactivity despite both having doubled extracellular dopamine levels. Mice with a 30% increase in DAT levels exhibited hypoactivity in a new environment. However, mice with a twofold increase in DAT levels showed no difference in hyperactivity or hypoactivity.¹¹

2.1.1.1.1. Hyperactivity can be treated with stimulants (dopaminergic?)¶

Hyperactivity in DAT-KO mice and DAT-KO rats can be reversed by

• AMP and MPH³¹⁶ and cocaine¹⁷¹⁰ , which indicates that stimulants do not act solely as dopamine reuptake inhibitors:
• In DAT-KO mice, amphetamine, methylphenidate, and cocaine reduced hyperactivity (which occurs only in new environments), whereas in normal mice they caused hyperactivity and stereotypy¹⁰
• Subchronic amphetamine administration (10 days) significantly reduced hyperactivity in female, but not in male, DAT-KO rats¹⁸
• While normal rodents show an increase in dopamine following AMP or MPH injection, dopamine levels remain unchanged in DAT-KO mice following such injections¹⁹
• A study suggests that this calming effect is mediated by serotonin. Similarly, stimulants do not reduce the elevated extracellular dopamine levels in DAT-KO mice.¹⁷
• Another one points to the norepinephrine transporter²⁰
• Haloperidol³
• Haloperidol increases extracellular DA concentrations more effectively in the dorsal caudate than in the PFC.²¹

N-methyl-D-aspartate (NMDA) antagonists block the hyperactivity-normalizing effects of amphetamine, methylphenidate, fluoxetine, quipazine (a 5-HT2 receptor agonist), and L-tryptophan in DAT-KO mice²²

2.1.1.1.2. Hyperactivity Can Be Treated with Serotonergic Medications¶

Hyperactivity in DAT-KO appears to be triggered by an increase in serotonergic tone.

Hyperactivity in DAT-KO can be corrected by

• The non-selective serotonin receptor agonist 5CT²³
• SSRIs, serotonin agonists, serotonin precursors
  • The serotonin reuptake inhibitor fluoxetine drastically reduced hyperactivity¹⁰, as did other serotonergic medications, such as selective serotonin 2A receptor antagonists or serotonin precursors¹⁰²⁴

2.1.1.1.3. Hyperactivity Can Be Treated with TAAR1 Agonists¶

A TAAR1 receptor agonist corrects hyperactivity in DAT-KO.³

2.1.1.1.4. Hyperactivity Cannot Be Treated with Norepinephrine Reuptake Inhibitors¶

Although increased noradrenergic signaling from the locus coeruleus to the PFC resolves the hyperactivity observed in DAT-KO mice²⁵, noradrenergic medications generally do not help.

Norepinephrine reuptake inhibitors do not correct the hyperactivity observed in DAT-KO, such as:

• Atomoxetine²³ — at least not at a dose of 3 mg/kg²⁶²⁷²³
• Nisoxetine (SNRI)¹⁰
• Indifferent locomotor activity in response to cocaine and AMP⁴

In rats whose dopaminergic and noradrenergic cells were chemically destroyed by 6-OHDA, causing ADHD symptoms²⁸, serotonin and norepinephrine reuptake inhibitors (but not dopamine reuptake inhibitors) reduced hyperactivity (in a new environment), whereas they did not do so in normal mice, and dopamine reuptake inhibitors actually increased hyperactivity.²⁹ For more on this, see 6-OH-dopamine-lesioned mice/rats.

2.1.1.1.5. Hyperactivity is slightly inhibited by an α2A agonist and enhanced by an α2A adrenoceptor antagonist¶

• Guanfacine (0.25 mg/kg, α2A-adrenoceptor agonist)³⁰
  • Minimal inhibitory effect on the hyperactivity of DAT-KO rats in the maze
  • Significantly improved the perseverative activity pattern
  • Reduced the time spent in maze error zones
• Yohimbine (1 mg/kg, α2A-adrenoceptor antagonist)³⁰
  • Increased hyperactivity
  • Increased perseverative responses
  • Increased the amount of time spent in the labyrinth’s error zones.

2.1.1.2. Impulsivity¶

Increased impulsivity.¹⁷²⁵
DAT-KO mice show significant cognitive impairments in the eight-arm radial maze test, a standard procedure for assessing spatial cognitive function in rodents. Specifically, the mutant animals make significantly more perseveration errors, suggesting that these mice may suffer from reduced behavioral inhibition.¹⁹

2.1.1.3. Aggressiveness¶

Increased reactivity and aggression following mild social contact.³¹

2.1.1.4. Attention Problems¶

The DAT-KO exhibits attention deficits in auditory prepulse inhibition (PPI).³² This can be corrected with MPH.
In a curiosity test using a black-and-white box, adult DAT-heterozygous rats (DAT-HET) exhibited increased curiosity-driven exploratory activity compared to wild-type rats, while DAT-KO rats failed to recognize novel objects.³³
In a test of novelty preference using a 3-chamber apparatus with different shapes, DAT-KO rats exhibited reduced hyperactivity during the first 10 minutes, which appeared to be related to curiosity and attention toward the new environment.³³

2.1.1.5. Learning and memory problems, cognitive problems¶

Learning and Memory Deficits²⁵

• Deficits in Spatial Learning and Memory³⁴
  • DAT-KO mice exhibit significant cognitive impairments in the eight-arm radial maze test, a standard procedure for assessing spatial cognitive function in rodents. Specifically, the mutant animals make significantly more perseveration errors, which suggests reduced behavioral inhibition.¹⁹
• Impaired extinction of habitual memory³⁵ while learning behavior otherwise remained unchanged
• Long-term potentiation is impaired³⁶
  • Reduced synaptic strength
  • Impairment of associative learning
• Deficits in Spatial Learning¹⁷
• Memory deficits¹⁷ and impairments in spatial cognitive function in the radial labyrinth
• Slightly increased long-term potentiation and markedly reduced long-term depression at excitatory hippocampal CA3-CA1 synapses³⁷
  • 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 accumbens³⁸
• Improvement in cognitive impairment through:²³
  • Atomoxetine
  • Stimulants
  • Guanfacine (alpha-2A-adrenoceptor agonist)³⁹
    • Worsening, however, due to the alpha-2A-adrenoceptor antagonist yohimbine
  • Increased norepinephrine signaling from the locus coeruleus to the PFC improved cognitive abnormalities in spatial learning tasks by increasing persistence and reducing the number of visits to error zones²⁵
• No improvement in cognitive impairment due to:²³
  • Non-selective serotonin receptor agonists 5CT

2.1.1.6. Sleep Disorders¶

• Sleep Disorders⁴⁰
• Reduced sleep:⁴¹
  • Non-REM sleep
  • REM sleep
  • Shorter overall shearing time
    *⁴¹ has no stimulating effect
  • Modafinil
  • Methamphetamine
  • The selective DAT blocker GBR12909
    *⁴¹ ’s excessive wakefulness-promoting effect
  • Caffeine
• Circadian rhythm
  • Normal circadian patterns of inactivity and activity⁴¹
  • Circadian rhythm altered⁴²

2.1.1.7. Deficits in reward-based motivation¶

• A preference for hedonically positive flavors in food⁴³
• Increased resistance to the extinction of food-reinforced operant behavior³⁵
• Preference for sucrose
  • Increased⁴⁴
  • Reduced⁴⁵
• Increased reward responses to selective norepinephrine and serotonin blockers⁴¹

2.1.1.8. Startle reflex altered¶

In DAT-KO, the amplitude of the startle reflex was significantly lower than in WT. ATX reduced the amplitude of the startle reflex in both DAT-KO and WT. Under ATX, the startle response in DAT-KO was still lower compared to WT. Atomoxetine improved pre-pulse inhibition in both DAT-KO and WT.²⁶
Prepulse inhibition in DAT-KO was improved by:⁴⁶

• 60 mg/kg of cocaine
• 60 mg/kg methylphenidate
• Nisoxetine (10 or 30 mg/kg, selective norepinephrine reuptake inhibitor)
• Fluoxetine (30 mg/kg, SSRI)
• but not by citalopram (30 or 100 mg/kg), an SSRI

2.1.1.9. Extinction impairs¶

Impaired extinction in operant tasks.⁴⁷
Extinction: A learning process in which a learned behavior or response is eliminated due to a lack of reinforcement

2.1.1.10. Compulsive behavior and stereotypies (not typical of ADHD)¶

• Compulsive Behavior⁴⁵
  • Alternative interpretation: DAT-KO mice buried fewer marbles, likely due to extreme hyperactivity and the resulting inattention⁴⁸
• Rigid behavioral patterns⁴⁵
• Compulsive stereotypies in delay-reward tasks⁴⁵
• Stereotypical behavior²⁵

Atomoxetine reduced repetitive behavior in DAT-KO rats.²⁶

2.1.1.11. Movement patterns (not typical of ADHD)¶

Non-focal, perseverative movement patterns (inflexible behavioral responses).⁶

2.1.1.12. Growth restrictions (not typical of ADHD)¶

Growth is limited.⁴⁹

2.1.1.13. Reduced anxiety (not typical of ADHD)¶

The DAT-KO exhibits deficits in the cliff avoidance response (CAR).³²

2.1.1.14. Increased mortality (not typical of ADHD in this form)¶

Increased mortality.⁴⁹

2.1.1.15. Social behavior is impaired¶

Subchronic amphetamine administration (10 days) significantly improved social behavior in female, but not in male, DAT-KO rats¹⁸

2.1.2. Neurophysiological changes¶

2.1.2.1. Dopamine¶

• Elevated extracellular dopamine levels
  • To 5 to 6 times³⁷⁵ in the striatum^{3731 36} due to a reduction in dopamine clearance to 1/100⁴⁷ to 1/300, corresponding to a 300-fold increase in the half-life of dopamine in the synaptic cleft⁵⁵⁰
  • To 3.6 times the level in the PFC⁵¹
• Doubling the DA synthesis rate⁵⁰
• Reduction of dopamine levels in tissue to less than 5%⁵⁵⁰
  • A reduction in dopamine levels to 1/20 in the storage vesicles—which are normally refilled by DAT and hold dopamine for phasic releases—making dopaminergic functions entirely dependent on the limitations of dopamine synthesis¹¹
• Elevated tonic dopamine levels extracellularly = outside the synaptic cleft⁵²
• Reduction in phasic dopamine release⁵²⁵³ as also observed in SHR and coloboma mice⁵⁴ to 25%⁵, corresponding to a 1/4 reduction in the amplitude of evoked dopamine release¹¹
  • Inhibition of serotonin transporters, norepinephrine transporters, MAO-A, or COMT did not alter dopamine degradation. In the absence of DAT in the striatum, this appears to occur primarily through diffusion.⁵

• profound dysregulation of dopamine neurotransmission and reduced tissue levels of dopamine in⁵⁵
  • Striatum
  • Midbrain
  • PFC
  • Hippocampus
  • Medulla oblongata
  • Spinal cord
• significant changes in the gene expression of monoamine-degrading genes⁵⁵
  • Striatum
    • MAO-A is reduced (-60%)
    • MAO-B is reduced (-80%)
    • COMT unchanged
  • PFC
    • MAO-A increased (+110%)
    • MAO-B increased (+100%)
    • COMT increased (+20%)
  • Hippocampus
    • MAO-A is reduced (-40%)
    • MAO-B increased (+120%)
    • COMT increased (+100%)
  • Medulla oblongata
    • MAO-A is reduced (-90%)
    • MAO-B is reduced (-90%)
    • COMT is reduced (-80%)
  • Cerebellum
    • MAO-A is reduced (-80%)
    • MAO-B is reduced (-80%)
    • COMT increased (+250%)
  • Spinal cord
    • MAO-A is reduced (-10%)
    • MAO-B increased (+1,200%)
    • COMT increased (+980%)

• Medium-sized spiny projection neurons (the most common class of dopamine-receptor-expressing neurons, such as D1 receptors, D2 receptor, and DARPP-32)⁵⁶ exhibit a high degree of localized loss of spikes on the dendrites of the proximal segment, but no general morphological changes in terms of dendrite length, number, or overlap, or in the synapse-to-neuron ratio.⁵⁷
• A 50% downregulation of D1 receptors in the substantia nigra, VTA⁴, and striatum³¹
• Downregulation of postsynaptic D2 receptors in the striatum²⁷
• 50% downregulation of (presynaptic) D2 autoreceptors⁵⁸ in the striatum³¹
• Reduced postsynaptic density of PSD-95 in the striatum and nucleus accumbens, as has also been observed in other models of elevated dopamine levels³⁸
  *Loss of sensitivity to cocaine and amphetamine⁴⁷

Several of these characteristics were observed (to a lesser extent) in mice with only reduced DAT and doubled extracellular dopamine levels.⁵

The symptoms observed in the DAT-KO mouse could be explained by:¹¹

• , an elevated extracellular (“tonic”) dopamine level , which (due to depleted storage vesicles) is accompanied by a reduced phasic dopamine release , resulting in an insufficient supply of dopamine for short-term control tasks. ****
  Due to the lack of DAT, the remaining dopamine stores in the vesicles—which are used for phasic release—are entirely dependent on the de novo synthesis of dopamine.
  • This could correspond to the situation following a (partial) loss of dopaminergic cells, such as after encephalitis, which is also associated with hyperactivity. A (partial) loss of dopaminergic cells is associated with a significant reduction in the number of dopaminergic presynapses and the corresponding dopamine reuptake sites⁵⁹.
  • This could further correspond to the model of neonatal mice treated with the neurotoxic agent 6-hydroxydopamine (6-OHDA), which subsequently exhibit hyperactivity and cognitive impairments for a period of time.
• An indirect regulation of dopaminergic neurotransmission by noradrenergic and serotonergic¹⁷ mechanisms of AMP and MPH.
• Resulting from a reduction in exocytotic dopamine release due to decreased phosphorylation of synapsin⁶⁰

Studies of other mouse strains that have more DAT than DAT-KO mice, but less DAT than wild-type mice, showed that the number of DAT molecules correlates with reduced basal dopamine levels, and that basal dopamine levels decrease as the number of DAT molecules increases.¹¹

Methylphenidate and amphetamine-based medications reverse hyperactivity in DAT-KO mice (= DAT(-/-) mice). MPH was also able to reverse and normalize the impairment in learning observed in the shuttle-box avoidance test. The effective dose of MPH increased extracellular dopamine in the PFC but not in the striatum, whereas in DAT(+/-) and DAT(+/+) mice, MPH increased dopamine in both the PFC and the striatum.⁶¹ The authors discuss that MPH, which also acts as a norepinephrine reuptake inhibitor, may have inhibited NET in the PFC and thereby caused the therapeutically effective increase in dopamine in the PFC. NET also degrade dopamine in the PFC. Another possibility is that the increased norepinephrine in the PFC resulting from NET inhibition may have mediated the therapeutic effect.
On the other hand, DAT-KO mice have extremely high dopamine levels in the striatum, which were not reduced even by increasing dopamine levels in the PFC.
Guanfacine (single dose and chronic administration) in DAT-KO rats:⁶²

• improved spatial working memory
• improved prepulse inhibition (PPI)
• changes in the spectrum of brain activity and its coherence
  The authors view this as confirmation of the importance of the complex balance between norepinephrine and dopamine in the regulation of attention.

2.1.2.2. BDNF¶

• In the PFC
  • Reduced BDNF gene expression⁶³
  • Total BDNF and BDNF exon IV mRNA levels were reduced⁶⁴
  • MRNA levels of BDNF exon VI remain unchanged⁶⁴
  • Reduced mBDNF levels and reduced trkB activation⁶⁴
  • Reduced activation of αCaMKII in the PFC⁶⁴
• In the dorsolateral striatum
  • Elevated mBDNF levels in the homogenate⁶⁴
  • Elevated mBDNF levels in the cytosol⁶⁴
  • MBDNF levels in the postsynaptic density are reduced.⁶⁴
• TrkB expression in the dorsolateral striatum is reduced postsynaptically⁶⁴
  • TrkB is a high-affinity BNDF receptor

2.1.2.3. Glutamate¶

• PSD-95 expression in the dorsolateral striatum is reduced postsynaptically⁶⁴
  • PSD-95 is an index of glutamate spine density and measures the interaction between dopaminergic and glutamatergic systems in the striatum, which is important for cognitive processing
• Injections of the NMDA antagonist MK-801 (dizocilpin) resulted in:⁶⁵
  • In WT rats
    • A sharp increase in their physical activity
  • In DAT-KO rats
    • A decrease in hyperactivity
      • Signs of chronic stress, including:
        • Elevated basal corticosterone
        • Elevated basal aldosterone
    • Anxiety reduced

Differences were found in the modulation of glutamatergic transmission between males and females, primarily in the infralimbic cortex, but not in the prelimbic prefrontal cortex—the glutamatergic synapse may contribute to differences in behavioral responses to amphetamine between the two sexes.¹⁸

2.1.2.4. Serotonin¶

A notable change in tissue serotonin levels, particularly in⁵⁵

• Cerebellum
  • Serotonin turnover significantly increased (4-fold)
• Spinal cord
  • Serotonin turnover significantly increased (3.5-fold)
• Medulla oblongata
  • Serotonin turnover is no longer detectable
• PFC
  • Increased serotonin turnover (1.5 times)
• Hippocampus
  • Serotonin turnover remains unchanged
• Striatum
  • Serotonin turnover increased by 30%⁴⁸

Elevated serotonin levels in⁴⁸

• PFC
• Hippocampus
• 5-HT1A autoreceptor function is intact

2.1.2.5. Additional Changes¶

• significant changes in the mRNA production of enzymes involved in monoamine metabolism⁵⁵

• Reduced GHRH levels⁶⁶
  • Dopamine receptors in the hypothalamus inhibit the release of GHRH in the hypothalamus⁶⁷
• Impaired sensorimotor gating, as measured by prepulse inhibition (PPI) of the startle response⁶
• Underdeveloped anterior pituitary gland⁶⁶
  • The anterior pituitary gland (adenohypophysis) is part of the HPA axis (stress axis)
• LTP is no longer present in the PFC.⁵¹ This explains the learning and memory problems in the DAT-KO mouse.

2.2. DAT-KD Mouse / DAT (+/-) Mouse (elevated extracellular DA)¶

Unlike DAT-KO mice, DAT (+/-) mice still have dopamine transporter function, although it is reduced compared to the wild type (DAT hypofunction). DAT-KD mice have 90% less DAT.⁶⁸⁶⁹

DAT (+/-) mice showed

• Hyperactivity⁷⁰

  • Starting even before adolescence⁷⁰
  • Can be treated with amphetamines⁷⁰
  • Can be corrected with valproate (resulting in a 90% reduction in DAT)⁶⁸
  • Attenuated by the DRD1/2 agonist apomorphine⁴⁹
  • Attenuated by the DRD2 agonist quinpirole⁴⁹
  • Hyperactivity occurs even with a 33% reduction in DAT in the ventral midbrain, but only 7 days after the reduction in gene expression (wild-type mouse in which DAT levels were reduced surgically)⁷¹

• Impulsivity⁷⁰⁴⁹

• Attention Problems⁷⁰⁴⁹

• Perseverative motor behavior (inflexible behavioral responses)⁶⁸, which is more consistent with Tourette syndrome and obsessive-compulsive disorder:⁷²

  • A complex, serial pattern of grooming behavior that became increasingly rigid and persistent in its sequence²⁷
  • Persistent walking in a straight line
  • Pursued certain incentives excessively
  • Valproate alleviates perseverative behavior⁶⁸

• General cognitive impairments⁷⁰

  • In adolescent males and females
  • Partially improved in adult males
  • Unchanged in adult females
  • Can be treated with amphetamines

• A greater “desire” for sweet rewards⁶⁹

  • But no higher “liking”

• No abnormalities in prepulse inhibition⁶⁸

• Unchanged responses to external stimuli⁷⁰

• Unchanged sensorimotor gating abilities⁷³

• No growth restriction⁴⁹

• No increased mortality⁴⁹

• 70% increase in extracellular dopamine⁶⁹

• Reduced expression of Homer1a

  • In the PFC
  • Not in other regions of the brain (striatum)
  • Amphetamines shifted the reduction in Homer1a expression from the PFC to the striatum

• ARC and Homer1b remain unchanged

2.3. DAT-Val559 Knock-in Mice (Elevated Extracellular DA)¶

Five individuals with the rare, functional coding substitution Ala559Val in DAT exhibited ADHD, ASD, or bipolar disorder.⁷⁴ The DAT Val559 variant does not appear to affect dopamine recognition or reuptake; instead, it promotes DAT-dependent dopamine efflux. This appears to increase extracellular dopamine levels in vivo.⁷⁵

DAT-Val559 knock-in mice show:⁷⁴

• Impulsivity⁷⁶
  • Impulsivity depends on the reward context
  • Impulsivity occurs when the mice have to delay their response in order to receive a reward
  • Impulsivity does not occur when there is a likelihood of a reward for correctly refusing.
  • Impulsivity is likely driven by a heightened motivational phenotype, which also results in faster task comprehension in operant tasks and may trigger an increased maladaptive desire for reward
• increased motivation for rewards (untypical for ADHD)⁷⁷
• conditional hyperactivity⁷⁸⁷⁶
  • a faster escape response to an attack
  • no spontaneous hyperactivity
  • reduced motor activation due to AMP
• Vertical activity (rearing up) is significantly reduced (in heterozygous animals)
• no inattention⁷⁶
• Compulsive behavior (not typical of ADHD)⁷⁷
  • Apomorphine (a DA agonist) induces locomotor stereotypies in DAT Val559 mice, but not in WT mice.

• Compulsive Stereotypies in Delayed-Reward Tasks

• General startle response remains unchanged
• Anxiety remains unchanged
• Increased dendritic spine density in the dorsal medial striatum⁷⁷
• increased responsiveness to upcoming actions⁷⁹
• increased serotonin activity⁷⁹
  • particularly at 5-HT2C receptors
• Cocaine causes⁷⁹
  • no locomotor effects, while the conditioned place preference is maintained
    • presumably due to SERT inhibition
    • independent of striatal DA release
    • The SERT inhibitor fluoxetine reversed the methylphenidate-induced motor activity in DAT Val559 mice, thereby mimicking the effects observed with cocaine
  • no increase in extracellular dopamine
• Changes in psychostimulant responses, social behavior, and cognitive performance are gender-dependent⁸⁰
• The effect of increased DAT efflux on D2 autoreceptor regulation of DAT is specific to both sex and brain region⁸⁰
  • D2AR/DAT coupling in the dorsal striatum occurs only in males
  • D2AR/DAT coupling in the ventral striatum occurs only in females
• Sulpiride (D2R antagonist) administered chronically⁸⁰
  • blocks efflux-controlled DAT traffic
  • prevents the behavioral changes typical of DAT-Val559 in both sexes

2.4. LPHN3 knockout rat/mouse/fish (ADGRL3-KO animals) (DA elevated extracellularly and in phases)¶

Dopamine levels are elevated in a phasic and extracellular manner.
In contrast, one study classified LPHN3-KO rats as hypodopaminergic, just as SHR rats are.⁸¹

Latrophilin-3 (LPHN3; ADGRL3), a G-protein-coupled receptor, belongs to the subfamily of adhesion receptors. LPHN3 regulates synaptic function and helps maintain it in brain regions involved in motor activity, attention, and spatial and route memory.

LPHN3 / ADGRL3 is a candidate gene for ADHD^{8283 84 85} and influences dopamine. For more information, see ADGRL3, LPHN3, Latrophilin-3 (chromosome 4q13.1) as a candidate gene for ADHD.

LPHN3 binds to Gαi1, Gαi2, Gαs, Gαq, and Gα13. In particular, genetic variants that result in impaired Gα13 binding appear to be relevant in ADHD.⁸⁶

2.4.1. Behavior of LPHN3-KO Animals¶

LPHN3-KO mice and rats showed

• no increased anxiety⁸⁵
  • but no acclimatization to the open field⁸⁵
• a significant decrease in maternal caregiving behavior (less than half)⁸⁵
• Hyperactivity⁸⁵⁸⁷
  • in an unfamiliar environment
  • even in familiar surroundings (unlike most ADHD animal models)⁴⁰
  • no reduction in hyperactivity due to amphetamines⁸⁷
• increased (behavioral) impulsivity⁸⁵⁸⁴
  • in the continuous performance test (CPT)
  • Problems with the differential reinforcement of low response rates to contingent reinforcement
  • no choice impulsivity (no preference for an immediate small reward over a delayed larger reward)⁸⁸
• Attention problems⁸⁹
  • According to another source, he reportedly has no attention problems⁷⁶
• reduced ability to distinguish between new and familiar objects⁸⁵
• Learning and memory deficits⁹⁰
• Impaired visual-spatial working memory⁸⁹
• increased sociability accompanied by impaired social memory⁸⁵
• Absence of aggression in the resident-intruder paradigm⁸⁵
• reduced motivation to eat during the continuous performance test (CPT)
• increased reactivity to an acoustic startle stimulus
• cognitive deficits on tests of egocentric learning and memory in the Cincinnati Water Maze
  • Evidence of problems with striatal dopamine⁹¹⁹²⁹³
• Deficits in allocentric (spatial) learning and memory in the Morris water maze
  • Indication of glutamatergic problems⁸⁴
• impaired cognitive flexibility⁸⁴
• Working memory problems
  • Deficits in delayed spatial change⁸⁹
  • Another study found no working memory problems⁹⁴
• Anxiety⁹⁵
  • adgrl3.1-/- fish spent more time at the bottom in the Novel Tank Diving Test and showed a greater preference for the dark zone in the light-dark task
  • Both are signs of increased anxiety-like behavior
• Stress response⁹⁵
  • lower baseline cortisol levels combined with an increased cortisol response to stress signals
• impaired cognitive flexibility⁹⁵
  • increased number of repetitions in the FMP-Y maze
  • Combined with stress, this reflected a shift toward more rigid behavioral strategies and impaired cognitive flexibility

ADGRL3.1-null zebrafish larvae (ADGRL3.1-/-) exhibit a robust phenotype of hyperactivity and impulsivity.⁹⁶⁹⁷⁹⁸

2.4.2. Dopamine and Other Neurotransmitters in LPHN3-KO Animals¶

Unmodified mirrors (after HPLC) from:⁹⁹

• Dopamine / dopamine metabolites (?)
  • Another study found elevated levels of dopamine in the dorsal striatum¹⁰⁰
• Norepinephrine / norepinephrine metabolites
• Serotonin / serotonin metabolites (?)
  • in the brain regions where LPHN3 is most frequently expressed:
    • Hippocampus
    • PFC
  • Another study found elevated levels of serotonin in the dorsal striatum¹⁰⁰

Increased dopamine release and dopamine reuptake in the striatum:⁹⁰

• Increased tyrosine hydroxylase
• DAT increased
• Reduced DRD1 expression
  • possible downregulation due to excessive dopamine synthesis
• DARPP-32 reduces
  • could be a response to a reduction in DRD1
• Dopamine in the striatum increases¹⁰⁰ in vitro:¹⁰¹
  • extracellular
  • Release
  • Resumption
• higher DA release with a shorter duration compared to wild-type rats⁹⁰
  These results suggest that increased dopamine synthesis and release lead to synaptic overflow, which could explain the hyperactivity observed in LPHN3-KO rats.⁴⁰
• increased electrically evoked dopamine release throughout the striatum ex vivo¹⁰²
• Reduced task-induced dopamine signals in the nucleus accumbens during in vivo fixed- e operant task¹⁰²
  • The reduction was not due to impaired dopamine availability, as the amphetamine-induced release remained unchanged

LPHN3-KO rats exhibited⁸¹

• reduced DA release in the nucleus accumbens compared to LPHN-WT rats (controls)
• reduced DA release in the mPFC compared to LPHN-WT rats (controls)
• increased DA half-life in the mPFC compared to LPHN-WT rats (controls)
• Unchanged DA half-life in the nucleus accumbens core compared to LPHN-WT rats serving as controls
• DAT blockade with nomifensine (10 mg/kg, IP) increased DA release in the NAcc of LPHN3-KOs even more than in SHR
  SHR performed “⁸¹ “
• reduced DA release in the nucleus accumbens compared to WKY rats (controls)
• Unchanged DA release in the mPFC compared to WKY rats (controls)

2.4.3. Gene Expression in LPHN3-KO Animals¶

Significant changes in gene expression in LPHN3-KO mice:⁸⁵

• PFC: 180 genes with significantly altered expression
  • 115 (63.9%) were upregulated, some of which were more than twice as active, e.g.,
    • Interleukin 31 (IL-31)
    • Starch-binding domain 1 (Stbd1)
  • 65 genes downregulated
    • 22 of them by at least 50%.
    • The DAT gene is most strongly downregulated in the PFC
      • However, DAT plays barely any role in dopamine breakdown in the PFC
• Hippocampus: 36 genes with significantly altered expression
  • 23 genes (63.9%) were upregulated
  • only 2 genes that are at least twice as large; here, too, it is Stbd1
• increased mRNA expression of:¹⁰⁰
  • Slc6a4
  • 5-HT2a
  • DAT1
  • DRD4
  • Ncam
  • Nurr1
  • Tyrosine hydroxylase

2.4.4. Drug Effects in LPHN3-KO Animals¶

Established ADHD medications reduced hyperactivity in adgrl3.1-/- zebrafish:

• Methylphenidate⁹⁶
• Atomoxetine⁹⁷⁹⁶
• Guanfacine⁹⁷
• Clonidine⁹⁷

In addition, five other compounds showed an effect comparable to that of atomoxetine:⁹⁷

• Aceclofenac
  • nonsteroidal anti-inflammatory drug and nonselective cyclooxygenase inhibitor (COX; prostaglandin endoperoxide synthase). COX cleaves arachidonic acid to form prostaglandin H2, which is then catalyzed by prostaglandin D synthase (PTGDS) to form prostaglandin D2. The PTGDS gene is downregulated in SHR.⁹⁷
• Amlodipine⁹⁷⁹⁸ * (Some of the authors are shareholders or employees of a company conducting research on amlodipine as a treatment for ADHD.)*
• Doxazosin
  • Doxazosin is a quinazoline and a selective α1-adrenoceptor antagonist.¹⁰³ It is used to treat high blood pressure and benign prostatic hyperplasia to improve urine flow.
  • The SHR exhibits an increase in functional α1 receptors, which leads to higher spontaneous neuronal activity in the locus coeruleus (LC) (hyperexcitability, roughly: increased sensitivity), which can be reversed with α1 antagonists¹⁰⁴ ADRA1A, which encodes this receptor, is a candidate gene for ADHD. For more on this, see ADRA2A, ALPHA-2A-ADRENERGIC RECEPTOR (Chromosome 10q25.2)
• Moxonidine
  • Moxonidine is a potent imidazoline-1 receptor agonist and a weak alpha-2 receptor agonist.
  • The selective imidazoline-1 agonist LNP599 demonstrated an effect comparable to that of other non-stimulant ADHD active ingredients
  • Clonidine appears to bind non-selectively to the imidazoline-1 receptor
    LNP599
  • selective imidazoline-1 agonist
  • showed an effect comparable to that of other non-stimulant ADHD active ingredients

2.5. P35-KO mouse (reduced DAT = increased extracellular DA)¶

Mice that cannot produce the P35 protein (P35-KO mice) exhibit spontaneous hyperactivity, which can be reduced by MPH and AMP.¹⁰⁵ They have elevated dopamine levels accompanied by reduced dopamine turnover and, at the same time, reduced CDK5 activity. The number of DAT receptors in the striatum—and thus dopamine reuptake—is reduced.¹⁰⁶
In vitro, inhibition of Cdk5 activity in N2a cells resulted in a significant increase in constitutive DAT endocytosis, accompanied by an increase in DAT localization in recycling endosomes. ¹⁰⁶

Given the reduced DAT, we expect an increase in extracellular dopamine levels.

2.6. FOXP2wt/ko mice (elevated DA)¶

Heterozygous Foxp2wt/ko mice express intermediate levels of Foxp2 protein and can therefore be used to assess the consequences of reduced Foxp2 expression:¹⁰⁷

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

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

2.7. Pregnancy-stress-offspring mouse (DA increased extracellularly, decreased phasically)¶

Stress during phases— —in which brain systems develop particularly rapidly makes them susceptible to developmental abnormalities; maternal stress during pregnancy impairs the development of the offspring. This can be replicated using a mouse model in which the mothers were exposed to immobilization stress during pregnancy.¹⁰⁸ One example of a stress protocol involves immobilizing pregnant rats three times a day for 45 minutes under bright light in a transparent Plexiglas cylinder, starting on the 11th day of gestation and continuing until birth at 21–22 days of gestation.
In adulthood, the offspring exhibit changes in behavior, the HPA axis, and the dopamine system that are similar to those seen in ADHD.
High maternal corticosterone levels could contribute to the long-term effects described in the offspring, in addition to possible internal vasoconstriction, which would impair blood flow to the placenta.¹⁰⁸

2.7.1. Behavioral Changes¶

2.7.1.1. Hyperactivity or Hypoactivity¶

• Hypoactivity
  • Increased immobility in response to acute foot shocks in females¹⁰⁹
  • Reduced activity in females in new environments¹⁰⁹
• Hyperactivity^110111112
  • a greater range of motion¹¹⁰
  • reduced activity caused by dopamine antagonists¹¹⁰

2.7.1.2. Impulsivity¶

Offspring of female rats that were injected with corticosterone during pregnancy exhibited increased impulsivity.¹¹¹

2.7.1.3. Attention Problems¶

Offspring of female rats that were injected with corticosterone during pregnancy exhibited attention problems.¹¹¹

2.7.1.4. Memory problems¶

• Memory problems in old age during hippocampus-dependent tasks (males and females)¹¹³
• Memory performance improves in adult females¹¹³
• reduced hippocampal plasticity in males¹¹³
• increased hippocampal plasticity in females¹¹³

2.7.1.5. Learning difficulties¶

• Learning difficulties in older animals¹¹⁴¹¹⁵

2.7.1.6. Changes in Stress Responses¶

Changes in stress response in females.¹⁰⁹
Difficulty adjusting to new situations.¹¹⁰

2.7.1.7. Sleep Disorders¶

Sleep Disorders in Males.¹¹³
The HPA response (see below) was usually accompanied by changes in the circadian rhythm of corticosterone release.¹¹³

2.7.1.8. Anxiety / Risky Behavior¶

• reduced anxiety symptoms, as measured by increased total time spent in the elevated plus maze test¹¹¹¹¹⁰
• Reduced avoidance of an “aversive” context in females.¹⁰⁹
• increased risk-taking behavior¹¹²
• otherwise: high anxiety levels (adults; females may be slightly less anxious than males).¹¹³¹¹⁶

2.7.1.9. Depression¶

Depression-like behavior (adult males and females).¹¹³¹¹⁷
Can be treated with imipramine.¹¹⁸

2.7.1.10. Increased Risk of Addiction¶

Increased risk of drug abuse correlates with HPA axis hyperactivity in adult rats.^119120121122

2.7.2. Neurophysiological changes¶

2.7.2.1. Changes in the dopamine system¶

• functional hyperdopaminergic state¹¹⁰
• reduced DAT expression¹¹⁰
• increased DA turnover in the striatum¹¹⁰
• an altered response to DA receptor and DA transporter (DAT) blockers¹¹⁰
• Altered expression of DA receptors and striatal DA-regulated neuropeptide genes¹¹⁰
  • D2 receptor binding in the nucleus accumbens significantly increased (+24%)¹²¹
  • D3 receptor binding is significantly reduced in the shell (-16%) and core (-26%) of the nucleus accumbens¹²¹
  • D1 receptor binding in the striatum or nucleus accumbens remains unchanged¹²¹
• severe abnormalities in neural development and brain morphology¹²³¹²⁴
• increased DRD2 dopamine receptors¹²³¹²⁴
  • leads to reduced dopamine release in the PFC following amphetamine stimulation
• Nurr1 expression is increased in the VTA but unchanged in the substantia nigra¹²³¹²⁴
  • Increase on postnatal days 7, 28, and 60
  • possibly a compensatory mechanism to counteract the decrease in dopamine levels following prenatal stress
  • Nurr1 is
    • a specific dopaminergic transcription factor
    • ubiquitous distribution in the cerebral cortex, the hippocampus, the thalamus, the amygdala, and the midbrain
    • is expressed during critical periods of DA neuron differentiation
    • regulates several proteins that are necessary for dopamine synthesis and regulation
• Pitx3 expression in the VTA first decreased, then increased; it remained unchanged in the substantia nigra¹²³¹²⁴
  • Decrease on postnatal day 28 and increase on postnatal day 60
  • possibly a compensatory mechanism to counteract the decrease in dopamine levels following prenatal stress
  • Pitx3 is
    • a specific dopaminergic transcription factor
    • occurs in mesencephalic DA neurons of the substantia nigra and VTA
    • is expressed during critical periods of DA neuron differentiation
    • is specifically involved in the terminal differentiation and maintenance of dopamine neurons
• Tyrosine hydroxylase altered¹²³¹²⁴
  • Decrease on postnatal day 7; no further changes observed on postnatal days 28 and 60
  • Normalization may be a consequence of the increase in Nurr1 and Pitx3

Given the reduced DAT, we expect a decrease in extracellular dopamine levels.
Given the increased dopamine turnover in the striatum, we hypothesize that there is increased phasic dopamine firing.

2.7.2.2. HPA Axis Permanently Altered¶

Stress responses to acute stress were altered by changes in the HPA axis.

• Persistently elevated plasma corticosterone levels¹²⁵¹²⁶
  • could be reversed by adoption by another mother (whether stressed or unstressed)¹²⁷
• Increased negative feedback from the HPA axis in females ¹²⁸
• A persistently attenuated corticosterone stress response in females to unavoidable electric shocks as an acute stressor¹¹³
• HPA axis reactivity has changed
  • increased¹²⁹
  • reduced in males in response to alcohol as an acute stressor¹¹³¹³⁰
    • This could explain the increased susceptibility to alcohol addiction
• Long-lasting hyperactivation of the HPA axis in male infants, young, adult, and elderly animals¹¹³
• The HPA response was usually accompanied by changes in the circadian rhythm of corticosterone release¹¹³
• reduced levels of mineralocorticoid and glucocorticoid receptors in the hippocampus during adolescence and adulthood^129130127129
• exacerbates age-related dysfunction of the HPA axis¹⁰⁸
• The period of hyporesponsiveness of the HPA axis in newborns has been resolved¹²⁹
• Circulating glucocorticoid levels in middle-aged animals are similarly high to those in older, non-stressed animals¹¹⁴
• pro-inflammatory effects on the immune system in adults¹³¹
• Prolonged increase in plasma glucocorticoid levels in males exposed to acute immobilization stress = negative glucocorticoid feedback¹³²
  • in cases of prenatal stress in the mother during the 3rd (but not the 2nd) week of pregnancy
• Increased motor activity in males on amphetamine on the 56th postnatal day, but not on the 35th postnatal day¹³²
  • in cases of prenatal stress in the mother during the 3rd (but not the 2nd) week of pregnancy
• reduced prepulse inhibition of the acoustic startle response in adult males¹³²
• Impaired auditory sensory gating, as measured by the N40 response in adult males¹³²

2.7.2.3. Changes in the serotonin system¶

• Increased 5-HT2 receptors¹³³
• Increased 5HT1A mRNA expression in the PFC¹¹⁸

2.7.2.4. Acetylcholine¶

• Acetylcholine release in the hippocampus increases following mild stress¹³⁴

2.8. SORCS2 -/- mice (DA levels elevated extracellularly, but reduced in phases within the VTA)¶

The SORCS2 gene is a candidate gene for ADHD-HI.¹³⁵ It is also associated with bipolar disorder, schizophrenia, and alcohol withdrawal symptoms.
SORCS2 influences the growth of neurites in the brain. During embryonic development, SORCS2 is expressed in dopaminergic precursors of the future ventral tegmental area and substantia nigra. It is involved in BDNF signaling.

SORCS2-/- mice have a severe deficiency of SORCS2. This causes significant changes in the dopaminergic system.
In SORCS2-/- mouse embryos, an increased number of projections expressing tyrosine hydroxylase were found in the midbrain. In adult SORCS2-/- mice, the frontal cortex is hyperinnervated (supplied with more nerve fibers), suggesting a critical role for SORCS2 in the retraction of the growth cones (the branched tip of an outgrowing axon of a nerve cell) during dopaminergic innervation.¹³⁶
SORCS2-/- mice show¹³⁶

• Hyperactivity in a New Environment
  • Is reduced by the administration of amphetamines
• Risk-taking
• Inattention
• Decreased interest in sugar
• Decreased interest in alcohol¹³⁷

Neurophysiologically, SORCS2-/- mice showed ¹³⁶

• Reduced D1 receptor sensitivity
• Increased D2 receptor sensitivity
• Reduced phasic and increased tonic dopamine signaling in the ventral tegmental area.
  • Tonic dopamine release ensures a stable baseline level of extrasynaptic dopamine
  • Phasic (rapid, high-amplitude, intrasynaptic) dopamine release is involved in reward and goal-directed behavior

2.9. DAT T356M Mouse (ASS Mouse Model) (DA levels elevated extracellularly)¶

The DAT polymorphism observed in ASS (DAT T356M) appears to cause sustained dopamine efflux while DAT levels remain unchanged, without impairing the ability of presynaptic dopaminergic terminals to release stored dopamine from vesicles in a phasic manner. Mice homozygous for this mutation exhibited impaired striatal dopamine neurotransmission and altered dopamine-dependent behaviors corresponding to some ASD behavioral phenotypes (hyperactivity, repetitive behavior, social deficits). DAT blockade abolished the hyperactivity. The reduced DAT-mediated reuptake of released DA from the extracellular space appears to lead to D2R desensitization, The consequences of reduced dopamine synthesis are elevated synaptic dopamine levels, and ultimately a reduced total DA content in the tissue.¹³⁸

2.10. Prenatal/Neonatal Ethanol Mouse (Decreased DAT = Increased Extracellular DA)¶

Alcohol consumption by the mother during pregnancy can cause fetal alcohol spectrum disorder (FASD) in her child, which is associated with increased ADHD symptoms.
For more information, see *Prenatal Stressors as Environmental Causes of ADHD*in the chapter “ : The Origins (of ADHD)”.

The first two weeks of life in rodents correspond, at least in part , to human prenatal development during the third trimester of pregnancy; therefore, neonatal ethanol exposure in rodents is equivalent to prenatal ethanol exposure in humans.¹³⁹

Rats exposed to ethanol before birth or during the first few days of life exhibit ADHD symptoms:

• Hyperactivity¹⁴⁰
  • in cases of alcohol exposure on days 1 through 7¹⁴¹
    • increased by methylphenidate¹⁴²
    • increased by amphetamine only in males¹⁴³
• Impulsivity¹⁴⁰
• Attention deficits¹⁴⁰
  • in the 5-CSRTT⁴⁰
• Learning difficulties
  • in the visuospatial domain in the Morris water maze¹⁴⁴¹⁴⁵
  • Difficulties in inhibiting previously learned responses¹⁴⁶
• Working memory problems
  • in the radial-arm maze¹⁴⁷
  • during the spontaneous change of course in the T-maze¹⁴⁸

Mice and rats that were exposed to ethanol before birth or during the first few days of life showed

• Changes in the dopamine system¹⁴⁹
  which, however, are inconsistent overall and may depend on the rat strain studied

  • Dopamine levels and dopamine turnover
    • unchanged¹⁵⁰
    • All dopamine levels vary by sex¹⁵⁰
    • Dopamine levels decrease in the striatum and hypothalamus,¹⁵¹¹⁵² while remaining unchanged throughout the rest of the brain
  • Increased tyrosine hydroxylase⁴⁰
  • Reduced DAT in the striatum¹⁴⁰
  • DRD1
    • Downregulation of DRD1 receptors in the cortex and hippocampus¹⁵³¹⁵⁴
    • a temporary increase in DRD1 receptor binding, but not in DRD2 receptor binding¹⁵⁰
  • DRD2
    • reduced dopamine D2 receptor expression in the striatum, along with other changes¹⁵⁵
    • DRD2 unchanged¹⁵⁴
  • Altered dopamine modulation of GABAergic transmission in basolateral amygdala pyramidal neurons during periadolescence¹³⁹
  • DRD1-mediated potentiation of spontaneous inhibitory postsynaptic currents (IPSCs) is significantly attenuated¹³⁹
    • accompanied by a compensatory decrease in DRD3-mediated suppression of miniature IPSCs¹³⁹
    • These effects were not attributable to changes in DRD1 or DRD3 levels¹³⁹
    • significantly lower levels of the dopamine precursor L-3,4-dihydroxyphenylalanine in the BLA¹³⁹
    • unchanged dopamine levels in the BLA¹³⁹
      • likely a consequence of reduced dopamine breakdown¹³⁹

• reduced response to food rewards¹⁵⁶

• enhanced effects of amphetamine¹⁵⁶

• Norepinephrine

  • significantly reduced throughout the brain¹⁵¹
  • NET in the PFC is elevated¹⁴⁰

• increased neuronal apoptosis in the cortex and hippocampus¹⁵³

• reduced growth during periadolescence¹⁵⁰

• No change in anxiety-like behavior¹³⁹

• no increased stereotypy¹⁵⁷

2.11. Nicotine transporter knockout mouse¶

There are various nicotine receptors, and we summarize their knockout mouse models here.

• β2-KO mice (all β2-containing nAChRs are inactivated)¹⁵⁸
  • β2-nicotine receptors have a high affinity for nicotine
  • β2-nicotinic receptors are particularly important for the activation of dopaminergic neurons in the reward system
  • β2-KO mice exhibit a marked reduction or absence of dopamine release in response to nicotine and reduced activity of dopaminergic neurons
  • significant deficits in spatial discrimination
• α4-KO mice (All α4-containing nAChRs are inactivated)
  • α4 nicotinic receptors have a high affinity for nicotine
  • Similar to β2-KO mice
• α7-KO mice¹⁵⁸
  • α7 nicotinic receptors have a low affinity for nicotine
  • Changes in dopamine function are usually more subtle and can vary depending on the region of the brain
  • significant deficits in spatial discrimination
  • impaired temporal processing of auditory stimuli¹⁵⁹
  • Slows down procedural learning¹⁶⁰

2.12. Naples high-excitability rat (NHE)¶

The Naples high-excitability rat was selected based on increased exploration in a L-maze (a test environment used to measure arousal in response to novelty).^161
162, developed, validated, and used a model for the mesocortical variant of ADHD that demonstrates hyperactivity, altered attention, and executive functions, but no high blood pressure.¹⁶³ The PFC exhibits elevated dopamine levels with overexpression of mRNA species involved in basal metabolism and reduced dopamine D1 receptors.¹⁶⁴¹⁵ The NHE was also classified as not representative of ADHD.²⁷¹⁶⁵

• Problems with sustained attention¹⁶¹
• Hyperactive and impulsive in new surroundings¹⁶⁶¹⁶⁷
• Not hyperactive or impulsive in familiar surroundings¹⁵¹⁶⁸

• NHE mice exhibit hyperactivity in the open field, but not in their home cage¹⁶⁹(anders als SHR)¹⁶¹
• Impaired working memory and reference memory¹⁵
• NHE become more active as environmental complexity increases¹⁶⁴
• Handling
  • Handling: Young, unweaned animals are removed from their litter and mother for a few minutes each day and placed alone in an empty cage, without any further handling. They are then returned to their mother and siblings.¹⁷⁰
    Barbara
  • Gentle handling affects the activity of NHE rats differently than vigorous handling¹⁷¹
  • Handling during the fifth and sixth weeks of life can reduce hyperactivity in SHR mice and normalize neuronal markers (DRD1, CAMKII, c-fos)¹⁶¹
• Hyperdopaminergic innervation of the PFC with overexpression of mRNA species involved in basal metabolism¹⁶⁴ (overactivated mesocorticolimbic system)
• Downregulation of dopamine D1 receptors¹⁶⁴
• DAT increased¹⁶¹
  • Consequences of excess dopamine during early development
  • NHE have more dopamine-synthesizing terminals, more dopamine, and as a consequence more DAT
  • The DA neurons of the midbrain are hypertrophic and have more terminals that express tyrosine hydroxylase.
• Compared to normally bred rats (NRB), NHE rats show the following in the forebrain:¹⁶³
  • an overactive mesocortical DA pathway¹⁶⁴
  • a higher (+30%) volume of A10 neurons¹⁷²
  • a higher density of tyrosine hydroxylase (TH)-positive fibers
  • higher expression of the TH protein
  • higher expression of a DA-related phosphoprotein (DARPP32) in the VTA, but not in the substantia nigra
  • a lower density of D1 and D2 receptors
  • a higher density of the dopamine transporter (DAT)
  • higher expression of DARPP32 in the PFC)
• Increased tyrosine hydroxylase¹⁶¹
  • Normalization using MPH
  • In SHR, tyrosine hydroxylase levels are reduced, which is also normalized by MPH in this case¹⁶³

2.13. DAT-CI mouse, DAT-cocaine-insensitive mouse (extracellular DA levels elevated)¶

DAT-CI mice (“DAT cocaine-insensitive (DAT-CI) mice”) exhibit a triple point mutation at the cocaine-binding sites L104V, F105C, and A109V¹⁷³ of DAT. DAT-CI mice exhibit:

• no conditioned placepreference For cocaine¹⁷⁴
• a conditioned place-, and aversion To cocaine¹⁷⁵
• Cocaine has no rewarding effect¹⁷⁴
• Hyperactivity¹⁷⁶¹⁷³ pronounced¹⁷⁷
  • treatable with psychostimulants¹⁷⁸¹⁷³
• impaired striatal DA transmission¹⁷⁸
• elevated dopamine levels¹⁷³ in the striatum¹⁷⁹ extracellular¹⁷⁸
• CB1R in the striatum is nonfunctional¹⁷⁷
  • The sensitivity of CB1R, which controls GABA-mediated synaptic currents, had been completely lost in the striatum
  • The CB1Rs, which regulate glutamate transmission and GABA(B) receptors, remained unchanged
  • Blockade of CB1R((GABA)) function remained complete even after cocaine or environmental manipulations that activate the endogenous DA-dependent reward system and are known to sensitize these receptors in control animals.
  • Sugar continued to be viewed in a hedonistically positive light
  • CB1R receptors could be a useful target in the treatment of ADHD

2.14. DAT-CNR2 mice¶

DAT-CNR2 mice lack cannabinoid CB2 receptors (CB2R) on dopaminergic neurons in the midbrain.
DAT-CNR2 mice show:¹⁸⁰

• Hyperactivity (both males and females)
• Impulsivity in decision-making
• reduced anxiety-related behavior
• increased risk-taking behavior

Amphetamine at a therapeutic dose (2 mg/kg) reduces hyperactivity and ADHD symptoms. This is consistent with the typical paradoxical effect of stimulants in ADHD.

2.15. VMAT2-deficient mice¶

VVglut2f/f;CaMKII-Cre mice, in which VGLUT2 was inactivated in the cortex, hippocampus, and amygdala, showed¹⁸¹

• Hyperactivity
• increased risk-taking behavior
• reduced social dominance
• increased sensitivity
• impaired long-term spatial memory

The mice continued to show

• a 17% increase in striatal dopamine
• a 70% increase in striatal binding of a DRD2 agonist, which would suggest a decrease in dopamine levels.
  It is possible that this genetically determined chronic downregulation of VGLUT2 affects other aspects of synaptic function (e.g., the actual density of dopamine receptors).

AMP further increased the hyperactivity.
The partial D2R agonist aripiprazole improved sensory filtering (reduced heightened sensitivity).
Increased sensitivity to amphetamines.

2.16. C57BL/6J mice¶

Methylphenidate, atomoxetine, and amphetamine improved performance only in low-performing C57BL/6J mice, but did not affect the performance of high-performing animals. The drugs reduced premature responses among the more impulsive animals. Methylphenidate, guanfacine, and modafinil increased premature (impulsive) responses in the group with low drive. Modafinil impaired performance in higher-performing mice by increasing the false alarm rate.¹⁸²

C57BL/6J mice showed better impulse control than DBA/2J mice.¹⁸³

A sublineage of C57BL/6J mice, the C57BL/6J-bg(J)/bg(J) (beige-J mice), exhibited:¹⁸⁴

• increased physical activity
• increased dopamine turnover

A splenectomy (removal of the spleen) corrected the hyperlocomotion and reduced dopamine turnover.

2.17. SELENOT-KO Mice¶

The endoplasmic reticulum membrane protein SELENOT is described as neuroprotective.
SELENOT deficiency in dopaminergic neurons or throughout the entire brain of mice (but not a SELENOT deficiency limited to astrocytes):¹⁸⁵

• impairs dopamine signaling in the midbrain
• reduces DAT expression and, consequently, dopamine reuptake
• triggers ADHD-like behaviors such as hyperactivity

Methylphenidates, amphetamine-based medications, or increased expression of SELENOT in dopaminergic neurons eliminated the hyperactivity.

A SELENOT deficiency reduces the expression of the dopamine transporter (DAT), impairs dopamine reuptake, and enhances postsynaptic excitatory input.

SELENOT

• interacts with SERCA2 in the endoplasmic reticulum (ER) to regulate Ca2+ flux between the ER and the cytosol, and subsequently the expression of NURR1 and DAT.
• regulates Ca2+ flux between the endoplasmic reticulum (ER) and the cytosol in dopaminergic neurons through its interaction with SERCA2, thereby modulating NURR1 activity and maintaining DAT expression levels.

2.18. BMAL1 conditional knockout (cKO) mice¶

BMAL1 is a key circadian gene.
Knocking out BMAL1 in dopaminergic neurons leads to ADHD-like symptoms in male mice due to hyperactive dopamine signaling, such as¹⁸⁶

• Hyperactivity
• Attention problems
• Working memory problems

Bmal1-cKO mice exhibited increased dopamine release and increased neuronal excitability in striatal neurons. Amphetamine and the D1 receptor antagonist SCH23390 reduced this hyperactivity.

2.19. Alpha-Syn-/- mice¶

Mice lacking alpha-synuclein exhibit dysfunction in the nigrostriatal dopamine system.¹⁸⁷
They are characterized by elevated extracellular dopamine levels and exhibit¹⁸⁸

• Hyperactivity only in new environments¹⁸⁹
• Memory problems
• a slight lack of attention
• Reduced effect of dAMP compared to WT mice