ADHD animal models with increased extracellular dopamine

In this paper, we collect animal models of ADHD that exhibit elevated extracellular dopamine levels. In the interest of comparability of the animal models, we take the extracellular dopamine in the striatum as a reference point.
ADHD animal models where it is only known that dopamine is elevated, without knowing whether extracellular or phasic, we have also included here for the time being.
If we have concluded an increased extracellular dopamine level only on the basis of reduced DAT, this is characterized.

• 2. Animal models with increased extracellular dopamine
  • 2.1. DAT-KO mouse / DAT-KO rat (DA extracellularly increased, phasically decreased)
    • 2.1.1. Symptomatology
      • 2.1.1.1. Hyperactivity, spontaneous in unfamiliar surroundings
      • 2.1.1.2. Impulsiveness
      • 2.1.1.3. Aggressiveness
      • 2.1.1.4. Attention problems
      • 2.1.1.5. Learning and memory problems, cognitive problems
      • 2.1.1.6. Sleep disorders
      • 2.1.1.7. Reward motivation deficits
      • 2.1.1.8. Startle reflex changes
      • 2.1.1.9. Extinction impaired (not typical of ADHD)
      • 2.1.1.10. Compulsive behavior and stereotypies (not typical of ADHD)
      • 2.1.1.11. Movement patterns (not typical of ADHD)
      • 2.1.1.12. Restricted growth (not typical of ADHD)
      • 2.1.1.13. Reduced anxiety (not typical of ADHD)
      • 2.1.1.14. Increased mortality (not typical of ADHD in this form)
    • 2.1.2. Neurophysiological changes
      • 2.1.2.1. Dopamine
      • 2.1.2.2. BDNF
      • 2.1.2.3. Glutamate
      • 2.1.2.4. Serotonin
      • 2.1.2.5. Further changes
  • 2.2. DAT-KD mouse / DAT (+/-) mouse (DA extracellularly increased)
  • 2.3. DAT-Val559 knock-in mice (DA extracellularly increased)
  • 2.4. LPHN3 knockout rat/mouse (ADGRL3-KO mouse) (DA extracellular and phasic increased)
  • 2.5. P35-KO mouse (DAT decreased = DA extracellular increased)
  • 2.6. FOXP2wt/ko mice (DA increased)
  • 2.7. Pregnancy stress offspring mouse (DA extracellularly increased, phasically decreased)
    • 2.7.1. Behavioral changes
      • 2.7.1.1. Hyperactivity or hypoactivity
      • 2.7.1.2. Impulsiveness
      • 2.7.1.3. Attention problems
      • 2.7.1.4. Memory problems
      • 2.7.1.5. Learning problems
      • 2.7.1.6. Stress reactions changed
      • 2.7.1.7. Sleep disorders
      • 2.7.1.8. Anxiety / risk behavior
      • 2.7.1.9. Depression
      • 2.7.1.10. Increased risk of addiction
    • 2.7.2. Neurophysiological changes
      • 2.7.2.1. Dopamine system changed
      • 2.7.2.2. HPA axis permanently altered
      • 2.7.2.3. Serotonin system changed
      • 2.7.2.4. Acetylcholine
  • 2.8. SORCS2 -/- mice (DA extracellularly increased, phasically decreased in VTA)
  • 2.9. DAT T356M mouse (ASS mouse model) (DA extracellularly increased)
  • 2.10. Prenatal / neonatal ethanol mouse (DAT decreased = DA extracellular increased)
  • 2.11. NET-KO mouse (DA extracellularly increased in PFC)
  • 2.12. Naples high-excitability rat (NHE)

2. Animal models with increased extracellular dopamine¶

2.1. DAT-KO mouse / DAT-KO rat (DA extracellularly increased, phasically decreased)¶

DAT-KO mice/rats are often cited as models for increased dopamine levels. However, this refers to the extracellular and thus tonic dopamine level in the striatum. Phasic dopamine in the striatum is significantly reduced in DAT-KO model animals. When less dopamine is reuptaken, the vesicles that feed 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 transport knockout mouse or rat (DAT1 KO) serves as an animal model for research into ADHD.¹²

The DAT-KO mouse, whose dopamine transporter is almost deactivated in monozygous animals and approximately halved in heterozygous animals, shows the following symptoms in monozygous animals:³⁴⁵

2.1.1. Symptomatology¶

2.1.1.1. Hyperactivity, spontaneous in unfamiliar surroundings¶

Spontaneous hyperactivity, only in unfamiliar surroundings⁶

• However, hyperactivity was only observed in mice that had no DAT or 90% less DAT and whose extracellular (tonic) dopamine level was therefore 5-fold higher (no DAT at all) or at least doubled (90% less DAT). Mice that had 50% of the usual DAT number also had a doubled extracellular dopamine level, but showed no hyperactivity.
  Motor activity is controlled by dopamine changes in the sub-second range, i.e. by phasic dopamine, which typically comes from the storage vesicles as it cannot be synthesized so quickly. 50 % DAT should be able to replenish the vesicles much better than 10 % DAT. This could explain why the two mouse strains differed in terms of hyperactivity despite equally doubled extracellular dopamine levels. Mice with a 30% increase in DAT showed hypoactivity in novel environments. However, mice with doubled DAT levels showed no difference in hyperactivity or hypoactivity.⁷
• Hyperactivity in DAT-KO mice and DAT-KO rats can be remedied by
  • AMP and MPH^{38 9} , indicating that stimulants do not act solely as dopamine reuptake inhibitors:
    • In DAT-KO mice, amphetamine and methylphenidate reduced hyperactivity (occurring only in novel environments), whereas they cause hyperactivity and stereotypy in normal mice.
    • One study suspects that this calming effect is serotonergically mediated. Similarly, stimulants do not reduce the increased extracellular dopamine levels in DAT-KO mice.⁹
    • Another refers to the norepinephrine transporter¹⁰, which is contradicted by studies showing that atomoxetine does not remedy hyperactivity¹¹¹²
    • In rats whose dopaminergic cells were chemically destroyed, causing ADHD symptoms¹³, serotonin and noradrenaline reuptake inhibitors (but not dopamine reuptake inhibitors) reduced hyperactivity (in novel environments), whereas in normal mice they did not and dopamine reuptake inhibitors actually increased hyperactivity.¹⁴ For more information, see 6-OH dopamine-lesioned mouse/rat.
  • A TAAR1 receptor agonist³
  • Haloperidol³
    • Haloperidol increases the extracellular DA concentration in the dorsal caudate more effectively than in the PFC.¹⁵
  • The non-selective serotonin receptor agonist 5CT¹²
  • SSRIs, serotonin agonists, serotonin precursors
    • The serotonin reuptake inhibitor fluoxetine drastically reduced hyperactivity¹⁶ as did other serotonergic drugs such as selective serotonin 2A receptor antagonists or serotonin precursors¹⁶¹⁷
    • Hyperactivity in DAT-KO appears to be triggered by an increase in serotonergic tone
  • Hyperactivity cannot be remedied by
    • Noradrenaline reuptake inhibitors¹¹, e.g:
      • Atomoxetine¹², at least not by 3 mg/kg¹⁸
      • Nisoxetine (SNRI)¹⁶
    • Indifferent locomotor activity in response to cocaine and AMP⁴
    • Guanfacine (0.25 mg/kg, α2A-adrenoceptor agonist)¹⁹
      • Minimal inhibitory effect on the hyperactivity of DAT-KO rats in the labyrinth
      • Significantly improved the perseverative activity pattern
      • Reduced the time spent in maze error zones
    • Yohimbine (1 mg/kg, α2A-adrenoceptor antagonist)¹⁹
      • Increased hyperactivity
      • Increased perseverative reactions
      • Increased the time spent in the maze error zones.

2.1.1.2. Impulsiveness¶

Increased impulsivity.⁹

2.1.1.3. Aggressiveness¶

Increased reactivity and aggression rates after mild social contact.²⁰

2.1.1.4. Attention problems¶

The DAT-KO shows attention deficit in auditory prepulse inhibition (PPI).²¹ This can be remedied by MPH.

2.1.1.5. Learning and memory problems, cognitive problems¶

Learning and memory deficits [15,16].

• Deficits in spatial learning and memory²²
• Impaired erasure of habitual memory²³ with otherwise unchanged learning behavior
• Long-term potentiation impaired²⁴
  • Reduced synaptic strength
  • Impairment of associative learning
• Deficits in spatial learning⁹
• Memory deficits⁹ and impairments of spatial cognitive function in the radial labyrinth
• Slightly increased long-term potentiation and greatly reduced long-term depression at excitatory hippocampal CA3-CA1 synapses²⁵
  • 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 accumbens²⁶
• Improvement of cognitive impairment through¹²
  • Atomoxetine
  • Stimulants
  • Guanfacine (alpha2A-adrenoceptor agonist)²⁷
    • Deterioration due to alpha2A-adrenoceptor antagonist yohimbine
• No improvement in cognitive impairment due to the¹²
  • Non-selective serotonin receptor agonists 5CT

2.1.1.6. Sleep disorders¶

• Sleep disorders²⁸
• Reduced sleep²⁹
  • Non-REM sleep
  • REM sleep
  • Less total sheep time
• No wakefulness-promoting effect of²⁹
  • Modafinil
  • Methamphetamine
  • The selective DAT blocker GBR12909
• Excessive wake-promoting effect of²⁹
  • Caffeine
• Circadian rhythm
  • Normal circadian patterns of inactivity and activity²⁹
  • Circadian rhythm changes³⁰

2.1.1.7. Reward motivation deficits¶

• Tendency to hedonically positive taste in food³¹
• Increased resistance to the extinction of food-strengthened operant behavior²³
• Preference for sucrose
  • Increased³²
  • Reduced³³
• Increased reward reactions to selective noradrenaline and serotonin blockers²⁹

2.1.1.8. Startle reflex changes¶

The amplitude of the startle reflex was significantly smaller in DAT-KO than in WT. ATX reduced the amplitude of the startle reflex in DAT-KO as in WT. Under ATX, the startle reflex was still smaller in DAT-KO compared to WT. Atomoxetine improved the pre-pulse inhibition in DAT-KO as in WT.¹⁸
Prepulse inhibition with DAT-KO has been improved by:³⁴

• 60 mg/kg cocaine
• 60 mg/kg Methylphenidate
• Nisoxetine (10 or 30 mg/kg, selective noradrenaline reuptake inhibitor)
• Fluoxetine (30 mg/kg, SSRI)
• but not by citalopram (30 or 100 mg/kg), SSRIs

2.1.1.9. Extinction impaired (not typical of ADHD)¶

Impaired extinction in operant tasks.³⁵

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

• Compulsive behavior³³
• Rigid pattern behavior
• Compulsive stereotypes with delay reward tasks

Atomoxetine reduced repetitive behavior in DAT-KO rats.¹⁸

2.1.1.11. Movement patterns (not typical of ADHD)¶

Non-focal, preseverative movement patterns (inflexible behavioral reactions).⁶

2.1.1.12. Restricted growth (not typical of ADHD)¶

Growth restricted.³⁶

2.1.1.13. Reduced anxiety (not typical of ADHD)¶

The DAT-KO shows deficits in the cliff avoidance response (CAR)²¹

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

Increased mortality.³⁶

2.1.2. Neurophysiological changes¶

2.1.2.1. Dopamine¶

• Increased extracellular dopamine levels
  • To 5 to 6 times²⁵⁵ in the striatum^{2520 24} due to a reduction in dopamine clearness to 1/100³⁵ to 1/300, corresponding to a 300-fold increase in the lifetime of dopamine in the synaptic cleft⁵³⁷
  • To 3.6 times in the PFC³⁸
• Doubling of the DA synthesis rate³⁷
• Reduction of the dopamine level in the tissue to below 5 %⁵³⁷
  • Reduced to 1/20 the amount of dopamine in the storage vesicles normally refilled by the DAT, which reserve dopamine for phasic release, making dopaminergic functions completely dependent on the limitations of dopamine synthesis⁷
• Increased tonic dopamine extracellular = outside the synaptic cleft³⁹
• Reduction in phasic dopamine release³⁹⁴⁰ as also observed in SHR and coloboma mice⁴¹ to 25 %⁵, corresponding to a 1/4 reduction in the amplitude of evoked dopamine release⁷
  • Inhibition of serotonin transporters, noradrenaline transporters, MAO-A or COMT did not alter dopamine degradation. In the absence of DAT in the striatum, this appears to occur more by diffusion⁵

• profound dysregulation of dopamine neurotransmission and reduced dopamine tissue levels in⁴²
  • Striatum
  • Midbrain
  • PFC
  • Hippocampus
  • Medulla oblongata
  • Spinal cord
• significant changes in the gene expression of monoamine degradation genes⁴²
  • Striatum
    • MAO-A reduced (- 60 %)
    • MAO-B reduced (- 80 %)
    • COMT unchanged
  • PFC
    • MAO-A increased (+ 110 %)
    • MAO-B increased (+ 100 %)
    • COMT increased (+ 20 %)
  • Hippocampus
    • MAO-A reduced (- 40 %)
    • MAO-B increased (+ 120 %)
    • COMT increased (+ 100 %)
  • Medulla oblongata
    • MAO-A reduced (- 90 %)
    • MAO-B reduced (- 90 %)
    • COMT reduced (- 80 %)
  • Cerebellum
    • MAO-A reduced (- 80 %)
    • MAO-B reduced (- 80 %)
    • COMT increased (+ 250 %)
  • Spinal cord
    • MAO-A reduced (- 10 %)
    • MAO-B increased (+ 1,200 %)
    • COMT increased (+ 980 %)

• Medium-sized spine-bearing projection neurons (the most common class of dopamine receptive neurons, such as D1 receptor, D2 receptor and DARPP-32)⁴³ show highly localized loss of spines (spikes) on the dendrites of the proximal segment, but no overall morphological change in dendrite length, number or overlap, or in synapse-to-neuron ratio.⁴⁴
• Downregulation of D1 receptors by 50 % in substantia nigra, VTA⁴ and striatum²⁰
• Downregulation of the postsynaptic D2 receptors in the striatum¹¹
• Downregulation of the (presynaptic) D2 autoreceptors by 50 %⁴⁵ in the striatum²⁰
• Reduced postsynaptic density of PSD-95 in the striatum and nucleus accumbens, as seen in other models of increased dopamine levels²⁶
  *Loss of sensitivity to cocaine and amphetamine³⁵

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

The symptoms of the DAT-KO mouse could be explained by:⁷

• An increased extracellular (“tonic”) dopamine level, which (due to the exhausted salivary vesicles) is accompanied by a reduced phasic dopamine release, so that too little dopamine is available 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 completely dependent on the new synthesis of dopamine.
  • This could correspond to the situation after (partial) death of the 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 sites⁴⁶.
  • This could further correspond to the model of mice treated neonatally with the DAT toxin 6-hydroxydopamine (6-OHDA), which subsequently show hyperactivity and cognitive impairment for a period of time.
• Indirect regulation of dopaminergic neurotransmission by noradrenergic and serotonergic⁹ mechanisms of AMP and MPH.
• From a reduction in exocytotic dopamine release due to reduced phosphorylation of synapsin⁴⁷

Studies in other mouse strains, which have more DAT than DAT-KO mice but less DAT than wild mice, showed that the number of DAT correlates with decreased basal dopamine levels, and as DAT numbers increase, basal dopamine levels decrease.⁷

Methylphenidate and amphetamine medication eliminate hyperactivity in the DAT-KO mouse (= DAT(-/-) mouse). MPH was also able to correct and normalize the impairment of learning in shuttle box avoidance behavior. 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 DAT(+/-) and DAT(+/+) mice.⁴⁸ The authors suggest that MPH, which also acts as a norepinephrine reuptake inhibitor, may have inhibited NETs in the PFC and thereby caused the therapeutically effective increase in dopamine in the PFC. NETs also degrade dopamine in the PFC. Another option would be that the increased noradrenaline 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 dopamine level in the striatum, which was not reduced by increasing the dopamine level in the PFC.
Guanfacine (single and chronic) in DAT-KO rats:⁴⁹

• 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 complicated balance of noradrenaline and dopamine in the regulation of attention.

2.1.2.2. BDNF¶

• In the PFC
  • Reduced BDNF gene expression⁵⁰
  • Total BDNF and BDNF exon IV mRNA levels reduced⁵¹
  • MRNA levels of BDNF exon VI unchanged⁵¹
  • Reduced mBDNF levels and reduced trkB activation⁵¹
  • Reduced activation of αCaMKII in the PFC⁵¹
• In the dorsolateral striatum
  • MBDNF level increased in the homogenate⁵¹
  • MBDNF levels in the cytosol increased⁵¹
  • MBDNF levels in the postsynaptic density are reduced.⁵¹
• TrkB expression in the dorsolateral striatum postsynaptically reduced⁵¹
  • TrkB is a high-affinity BNDF receptor

2.1.2.3. Glutamate¶

• PSD-95 expression in the dorsolateral striatum postsynaptically reduced⁵¹
  • 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 (dizocilpine):⁵²
  • For WT rats
    • A sharp increase in their physical activity
  • For DAT-KO rats
    • A decrease in hyperactivity
      • Signs of chronic stress, including:
        • Corticosterone basally elevated
        • Aldosterone basally elevated
    • Anxiety weakened

2.1.2.4. Serotonin¶

Remarkable change in the tissue level of serotonin, especially in⁴²

• Cerebellum
  • Serotonin turnover significantly increased (4-fold)
• Spinal cord
  • Serotonin turnover significantly increased (3.5-fold)
• Medulla oblongata
  • Serotonin turnover no longer detectable
• PFC
  • Serotonin turnover increased (1.5-fold)
• Hippocampus
  • Serotonin turnover unchanged

2.1.2.5. Further changes¶

• significant changes in the mRNA production of enzymes of the monoamine metabolism⁴²

• Reduced GHRH levels⁵³
  • Dopamine receptors in the hypothalamus inhibit the release of GHRH in the hypothalamus⁵⁴
• Inadequate sensorimotor gating, measured by prepulse inhibition (PPI) of the startle response⁶
• Anterior pituitary lobe underdeveloped⁵³
  • The anterior pituitary gland (the adenohypophysis) is part of the HPA axis (stress axis)
• LTP in the PFC no longer exists.³⁸ This explains the learning and memory problems of the DAT-KO mouse

2.2. DAT-KD mouse / DAT (+/-) mouse (DA extracellularly increased)¶

In contrast to DAT-KO mice, DAT (+/-) mice still have a dopamine transporter function (DAT hypofunction) that is present but reduced compared to the wild type. DAT KD mice have 90 % less DAT.⁵⁵⁵⁶

DAT (+/-) mice showed

• Hyperactivity⁵⁷

  • Starting even before adolescence⁵⁷
  • Can be remedied by amphetamines⁵⁷
  • Recoverable with valproate (with 90 % less DAT)⁵⁵
  • Attenuated by DRD1/2 agonist apomorphine³⁶
  • Attenuated by DRD2 agonists quinpirole³⁶
  • Hyperactivity even with 33% less DAT in the ventral midbrain, but then only occurring after 7 days after reduction of gene expression (wild-type mouse in which DAT was reduced by intervention)⁵⁸

• Impulsiveness⁵⁷³⁶

• Attention problems⁵⁷³⁶

• Perseverative motor behavior (inflexible behavioral reactions)⁵⁵

  • Valproate eliminates perseverative behavior

• General cognitive impairments⁵⁷

  • In juvenile males and females
  • Partially improved in adult males
  • Unchanged in adult females
  • Can be remedied by amphetamines

• Higher “wanting” of sweet rewards⁵⁶

  • But no higher “Liking”

• No deficits in prepulse inhibition⁵⁵

• Unchanged reactions to external stimuli⁵⁷

• Unchanged sensorimotor gating abilities⁵⁹

• No growth restriction³⁶

• No increased mortality³⁶

• 70 % increased extracellular dopamine⁵⁶

• Reduced expression of Homer1a

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

• ARC and Homer1b unchanged

2.3. DAT-Val559 knock-in mice (DA extracellularly increased)¶

5 People with the rare, functional coding substitution Ala559Val in DAT showed ADHD, ASD or bipolar disorder.⁶⁰ DAT-Val559 variant does not appear to affect dopamine recognition or reuptake, but instead promoted DAT-dependent dopamine efflux. This appears to increase dopamine extracellularly in vivo⁶¹

DAT-Val559 knock-in mice show:⁶⁰

• Impulsiveness
  • impulsiveness depends on the reward context
  • Impulsivity occurs when the mice have to delay the reaction for a reward
  • impulsivity does not occur if there is a probability of a reward for a correct refusal.
  • Impulsivity is likely driven by an enhanced motivational phenotype, which also causes faster task acquisition in operant tasks and which may trigger increased maladaptive reward seeking
• increased reward motivation (atypical for ADHD)⁶²
• conditioned hyperactivity⁶³
  • faster escape reaction to attack
  • no spontaneous hyperactivity
  • attenuated motor activation due to AMP
• vertical activity (rearing) significantly reduced (in heterozygous animals)
• Compulsive behavior (atypical for ADHD)⁶²
  • Apomorphine (DA agonist) induces stereotypies of locomotion in DAT Val559 mice, but not in WT mice.

• Compulsive stereotypes with delay reward tasks

• general startle reaction unchanged
• Fear unchanged
• increased dendritic spine density in the dorsal medial striatum⁶²
• increased reactivity to upcoming actions⁶⁴
• increased serotonin activity⁶⁴
  • especially at 5-HT2C receptors
• Cocaine causes⁶⁴
  • no locomotor effects, if the conditioned place preference is maintained
    • probably due to SERT blockade
    • ndependent on the striatal DA release
    • SERT blocker fluoxetine abolished methylphenidate-induced locomotor activity in DAT Val559 mice, mimicking the effects observed with cocaine
  • no increase in extracellular dopamine
• Changes in psychostimulatory reactions, social behavior and cognitive performance are gender-dependent⁶⁵
• Influence of increased DAT efflux on D2 autoreceptor regulation of DAT is both sex and brain region specific⁶⁵
  • D2AR/DAT coupling in the dorsal striatum only in males
  • D2AR/DAT coupling in the ventral striatum only in females
• Sulpiride (D2R antagonist) given chronically⁶⁵
  • stops Efflux-controlled DAT traffic
  • prevents the behavioral changes typical of DAT-Val559 in both sexes

2.4. LPHN3 knockout rat/mouse (ADGRL3-KO mouse) (DA extracellular and phasic increased)¶

Dopamine increased phasically and extrecellularly.

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

LPHN3 / ADGRL3 is a candidate gene for ADHD.^66676869
LPHN3 binds to Gαi1, Gαi2, Gαs, Gαq and Gα13. In particular, gene variants that cause impaired Gα13 binding appear to be relevant in ADHD.⁷⁰

LPHN3-KO mice/rats show:

• no increased anxiety behavior⁶⁹
  • but no habituation to the open field⁶⁹
• greatly reduced maternal care behavior (less than half)⁶⁹
• Hyperactivity⁶⁹
  • in unknown surroundings
  • also in a familiar environment (unlike most ADHD animal models)²⁸
  • no reduction in hyperactivity due to amphetamines⁷¹
• increased (action) impulsiveness⁶⁹⁶⁸
  • in the continuous performance test (CPT)
  • Problems with differential reinforcement of low response rates to contingent reinforcement
  • no choice impulsivity (no preference for immediate small rewards over delayed larger rewards)⁷²
• Attention problems⁷³
• reduced ability to distinguish between new and familiar objects⁶⁹
• Learning and memory deficits⁷⁴
• Impairment of the visual-spatial working memory⁷³
• increased sociability with simultaneously impaired social memory⁶⁹
• Absence of aggression in the inhabitant-intruder paradigm⁶⁹
• reduced motivation to eat in the continuous performance test (CPT)
• increased reactivity to an acoustic startle stimulus
• cognitive deficits in tests of egocentric learning and memory in the Cincinnati water maze
  • Indication of problems with striatal dopamine⁷⁵⁷⁶⁷⁷
• Deficits in allocentric (spatial) learning and memory in the Morris water maze
  • Indication of glutamatergic problems⁶⁸
• impaired cognitive flexibility⁶⁸
• Working memory problems
  • Deficits with delayed spatial change⁷³
  • another study found no working memory problems⁷⁸

Unchanged levels (after HPLC) from⁷⁹

• Dopamine / dopamine metabolites (?)
  • another study found increased dopamine in the dorsal striatum⁸⁰
• Noradrenaline / noradrenaline metabolites
• Serotonin / serotonin metabolites (?)
  • in the brain regions where LPHN3 is most frequently expressed:
    • Hippocampus
    • PFC
  • another study found serotonin increased in the dorsal striatum⁸⁰

Increased dopamine release and dopamine reuptake in the striatum:⁷⁴

• Tyrosine hydroxylase increased
• DAT increased
• DRD1 expression reduced
  • possibly downregulation due to excessive dopamine synthesis
• DARPP-32 reduced
  • could be a reaction to DRD1 reduction
• Dopamine in the striatum increases⁸⁰ in vitro:⁸¹
  • extracellular
  • Release
  • Resumption
• higher DA release with reduced duration compared to wild-type rats⁷⁴
  These results suggest that increased synthesis and release of dopamine leads to synaptic overflow, which could explain the hyperactivity observed in Lphn3-KO rats.²⁸

Strong change in gene expression:⁶⁹

• PFC: 180 genes with significantly altered expression
  • 115 (63.9 %) are highly regulated, some of them more than twice as active, e.g.
    • Interleukin 31 (Il31)
    • Starch binding domain 1 (Stbd1)
  • 65 genes downregulated
    • 22 of which by at least 50 %.
    • DAT gene in the PFC most strongly downregulated
      • However, DAT is barely 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
• increased mRNA expression of:⁸⁰
  • Slc6a4
  • 5-HT2a
  • DAT1
  • DRD4
  • Ncam
  • Nurr1
  • Tyrosine hydroxylase

ADGRL3.1-null zebrafish larvae (ADGRL3.1-/-) show a robust hyperactive phenotype:⁸²
The hyperactivity can be remedied by three non-stimulant ADHD medications, but all of them significantly impaired sleep.
Four other compounds showed a comparable effect 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 active ingredients for ADHD
  • Clonidine apparently addresses the imidazoline-1 receptor non-selectively

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

Mice that cannot produce the P35 protein (P35-KO mice) show spontaneous hyperactivity, which can be reduced by MPH and AMP.⁸³ They have an increased dopamine level with reduced dopamine turnover and simultaneously reduced CDK5 activity. The number of DAT in the striatum and thus dopamine reuptake is reduced.⁸⁴
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. ⁸⁴

Due to the reduced DAT, we assume an increased extracellular dopamine level.

2.6. FOXP2wt/ko mice (DA increased)¶

Heterozygous Foxp2wt/ko mice have intermediate levels of Foxp2 protein and can therefore be used to assess the Consequences of reduced Foxp2 expression:⁸⁵

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

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

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

Stress in phases in which brain systems develop particularly strongly makes them susceptible to maldevelopment, stress of the mother during pregnancy impairs the development of the offspring. This can be reproduced in the model of mice whose mothers were exposed to immobilization stress during pregnancy.⁸⁶ One stress protocol used is to immobilize pregnant rats three times a day for 45 minutes under bright light in a transparent Plexiglas cylinder from the 11th day of gestation until birth at the age of 21-22 days.
In adulthood, the offspring show changes in behavior, the HPA axis and the dopamine system that are similar to those in ADHD.
High maternal corticosterone levels could contribute to the described long-term effects in the offspring, in addition to possible internal vaconstriction, which would impair the blood supply to the placenta.⁸⁶

2.7.1. Behavioral changes¶

2.7.1.1. Hyperactivity or hypoactivity¶

• Hypoactivity
  • Increase in immobility to acute foot shocks in females⁸⁷
  • Hypoactivity in new environment in females⁸⁷
• Hyperactivity⁸⁸⁸⁹⁹⁰
  • a greater movement distance⁸⁸
  • reduced activity due to dopamine antagonists⁸⁸

2.7.1.2. Impulsiveness¶

Offspring of rat mothers injected with corticosterone during pregnancy showed increased impulsivity.⁸⁹

2.7.1.3. Attention problems¶

The offspring of rat mothers injected with corticosterone during pregnancy showed attention problems.⁸⁹

2.7.1.4. Memory problems¶

• Memory problems in old age in hippocampus-dependent tasks (males and females)⁹¹
• Memory performance improved in females in adulthood⁹¹
• reduced hippocampal plasticity in males⁹¹
• increased hippocampal plasticity in females⁹¹

2.7.1.5. Learning problems¶

• Learning impairments in old animals⁹²⁹³

2.7.1.6. Stress reactions changed¶

Stress management changes in females.⁸⁷
Getting used to new things is impaired.⁸⁸

2.7.1.7. Sleep disorders¶

Sleep disorders in males.⁹¹
HPA response (see below) was usually associated with altered circadian rhythm of corticosterone secretion.⁹¹

2.7.1.8. Anxiety / risk behavior¶

• reduced anxiety symptoms, measured by increased total time in the Elevated Plus Maze Test⁸⁹⁸⁸
• reduced avoidance of an “aversive” context in females.⁸⁷
• increased risk behavior⁹⁰
• different: high anxiety levels (adults; females may be slightly lower than males).⁹¹⁹⁴

2.7.1.9. Depression¶

Depression-like behavior (adult males and females).⁹¹⁹⁵
Can be remedied with imipramine.⁹⁶

2.7.1.10. Increased risk of addiction¶

Increased risk of drug abuse correlating with hyperactivity of the HPA axis in adult rats.^979899100

2.7.2. Neurophysiological changes¶

2.7.2.1. Dopamine system changed¶

• functional hyperdopaminergic state⁸⁸
• reduced DAT expression⁸⁸
• increased DA turnover in the striatum⁸⁸
• an altered response to DA receptor and DA transporter (DAT) blockers⁸⁸
• Expression of DA receptors and striatal DA-regulated neuropeptide genes altered⁸⁸
  • D2 receptor binding in the nucleus accumbens significantly increased (+24%)⁹⁹
  • D3 receptor binding significantly reduced in shell (-16%) and nucleus (-26%) of the nucleus accumbens⁹⁹
  • D1 receptor binding in the striatum or nucleus accumbens unchanged⁹⁹
• severe anomalies in neuronal development and brain morphology¹⁰¹¹⁰²
• increased DRD2 dopamine receptors¹⁰¹¹⁰²
  • leads to reduced dopamine release in the PFC after amphetamine stimulation
• Nurr1 expression increased in the VTA, unchanged in substantia nigra¹⁰¹¹⁰²
  • Increase on postnatal days 7, 28 and 60
  • possibly a compensatory mechanism to counteract the reduction 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 at critical moments in the differentiation of DA neurons
    • regulates several proteins that are required for dopamine synthesis and regulation
• Pitx3 expression in the VTA first reduced, then increased, unchanged in substantia nigra¹⁰¹¹⁰²
  • Decrease on postnatal day 28 and increase on postnatal day 60
  • possibly a compensatory mechanism to counteract the reduction in dopamine levels following prenatal stress
  • Pitx3 is
    • a specific dopaminergic transcription factor
    • occurs in mesencephalic DA neurons of substantia nigra and VTA
    • is expressed at critical moments in the differentiation of DA neurons
    • is specifically involved in the terminal differentiation and maintenance of dopamine neurons
• Tyrosine hydroxylase altered¹⁰¹¹⁰²
  • Decrease at postnatal day 7, no longer changed at postnatal day 28 and 60
  • Normalization possibly a consequence of the increase in Nurr1 and Pitx3

Due to the reduced DAT, we assume a reduced extracellular dopamine level.
Due to the increased dopamine turnover in the striatum, we assume an increased phasic dopamine firing.

2.7.2.2. HPA axis permanently altered¶

Stress reactions to acute stress were altered by modifications of the HPA axis.

• Long-term elevated plasma corticosterone¹⁰³¹⁰⁴
  • could be canceled by adoption by another mother (equally by a stressed or an unstressed one)¹⁰⁵
• negative feedback of the HPA axis increases in females ¹⁰⁶
• permanently attenuated corticosterone stress response in females to inescapable electric shocks as an acute stressor⁹¹
• HPA axis reactivity changed
  • increased¹⁰⁷
  • attenuated in males to alcohol as an acute stressor⁹¹¹⁰⁸
    • this could explain the increased susceptibility to alcohol addiction
• long-lasting hyperactivation of the HPA response in males in infants, young, adult and old animals⁹¹
• HPA response was usually associated with altered circadian rhythm of corticosterone secretion⁹¹
• reduced mineralocorticoid and glucocorticoid receptor levels in the hippocampus in adolescence and adulthood ^107108105107
• age-related dysfunctions of the HPA axis intensified⁸⁶
• Period of hyporesponsiveness of the HPA axis in newborns abolished¹⁰⁷
• circulating glucocorticoid levels in middle-aged animals are similar to those in old, non-stressed animals⁹²
• inflammation-increasing effects on the immune system in adults¹⁰⁹
• prolonged increase in plasma glucocorticoid levels in males in response to acute immobilization stress = negative glucocorticoid feedback¹¹⁰
  • in case of prenatal stress of the mother in the 3rd (but not in the 2nd) week of pregnancy
• increased motor response to amphetamine in males on postnatal day 56, but not on postnatal day 35¹¹⁰
  • in case of prenatal stress of the mother in the 3rd (but not in the 2nd) week of pregnancy
• reduced prepulse inhibition of the acoustic startle response in adult males¹¹⁰
• auditory sensory gating impaired as measured by the N40 response in adult males¹¹⁰

2.7.2.3. Serotonin system changed¶

• 5-HT2 receptors increased¹¹¹
• 5HT1A mRNA expression increased in the PFC⁹⁶

2.7.2.4. Acetylcholine¶

• Acetylcholine release in the hippocampus increased after mild stress¹¹²

2.8. SORCS2 -/- mice (DA extracellularly increased, phasically decreased in VTA)¶

The SORCS2 gene is a candidate gene for ADHD-HI. It 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. It is involved in BDNF signaling.

SORCS2-/- mice have a severe deficiency of Sorcs2. This causes significant changes in the dopaminergic system.
In embryos of SORCS2-/- mouse embryos, projections expressing tyrosine hydroxylase were found to be increased in the midbrain. In adult SORCS2-/- mice, the frontal cortex is hyperinnervated (supplied with more nerve fibers), suggesting a critical role of SORCS2 in the shrinkage of the growth cone (the branching tip of a neuron’s outgrowing axon) during dopaminergic innervation.¹¹³
SORCS2-/- mice show¹¹³

• Hyperactivity in a new environment
  • Is reduced by amphetamine administration
• Risk appetite
• Inattention
• Reduced interest in sugar
• Reduced interest in alcohol¹¹⁴

Neurophysiologically, SORCS2-/- mice showed ¹¹³

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

2.9. DAT T356M mouse (ASS mouse model) (DA extracellularly increased)¶

The DAT polymorphism (DAT T356M) occurring in ASD appears to cause sustained dopamine efflux in the presence of unaltered DAT number without impairing the ability of presynaptic dopaminergic terminals to phasically release stored dopamine from vesicles. Mice homozygous for this mutation showed impaired striatal dopamine neurotransmission and altered dopamine-dependent behaviors consistent with some ASD behavioral phenotypes (hyperactivity, repetitive behavior, social deficits). DAT blockade terminated the hyperactivity. Reduced DAT-mediated reuptake of released DA from the extracellular space appears to lead to D2R desensitization, decreased dopamine synthesis as a result of increased synaptic dopamine levels, and ultimately decreased total tissue DA content.¹¹⁵

2.10. Prenatal / neonatal ethanol mouse (DAT decreased = DA extracellular increased)¶

Alcohol consumption by the mother during pregnancy can trigger fetal alcohol spectrum disorder (FASD) in the offspring, which is associated with increased ADHD symptoms.
More on this at *Prenatal stressors as environmental causes of ADHD *in the chapter Development (of ADHD).

The first two weeks of life of rodents correspond at least in part to prenatal development in humans in the third trimester of pregnancy, which is why neonatal ethanol exposure in rodents corresponds to prenatal ethanol exposure in humans.¹¹⁶

Rats exposed to ethanol prenatally or in the first days of life show ADHD symptoms:

• Hyperactivity¹¹⁷
  • for alcohol exposure on day 1 to 7¹¹⁸
    • increased by methylphenidate¹¹⁹
    • increased by amphetamine only in males¹²⁰
• Impulsiveness¹¹⁷
• Attention deficits¹¹⁷
  • in the 5-CSRTT²⁸
• Learning problems
  • in the visuospatial area in the Morris water labyrinth¹²¹¹²²
  • Deficits in the inhibition of previously learned reactions¹²³
• Working memory problems
  • in the radial arm labyrinth¹²⁴
  • during the spontaneous diversion in the T-Labyrinth¹²⁵

Mice and rats that were exposed to ethanol prenatally or in the first days of life showed

• Changes in the dopamine system¹²⁶
  which, however, are inconsistent overall and may depend on the rat strain studied

  • Dopamine levels and dopamine turnover
    • unchanged¹²⁷
    • all dopamine levels differ between the sexes¹²⁷
    • Dopamine reduced in the striatum and hypothalamus,¹²⁸¹²⁹ unchanged in the rest of the brain
  • Tyrosine hydroxylase increased²⁸
  • DAT in the striatum reduced¹¹⁷
  • DRD1
    • Downregulation of DRD1 receptors in the cortex and hippocampus¹³⁰¹³¹
    • transient increase in DRD1, but not DRD2 receptor binding¹²⁷
  • DRD2
    • reduced dopamine D2 receptor expression in the striatum along with other changes¹³²
    • DRD2 unchanged¹³¹
  • altered dopamine modulation of GABAergic transmission in basolateral amygdala pyramidal neurons during periadolescence¹¹⁶
  • DRD1-mediated potentiation of spontaneous inhibitory postsynaptic currents (IPSCs) significantly attenuated¹¹⁶
    • associated with compensatory decrease in DRD3-mediated suppression of miniature IPSCs¹¹⁶
    • these effects were not due to altered DRD1 or DRD3 levels¹¹⁶
    • significantly lower levels of the dopamine precursor L-3,4-dihydroxyphenylalanine in the BLA¹¹⁶
    • unchanged dopamine levels in the BLA¹¹⁶
      • probably a consequence of reduced dopamine degradation¹¹⁶

• reduced reaction to food reward¹³³

• increased effect of amphetamine¹³³

• Noradrenaline

  • significantly reduced throughout the brain¹²⁸
  • NET in the PFC increased¹¹⁷

• increased neuronal apoptosis in the cortex and hippocampus¹³⁰

• reduced periadolescent growth¹²⁷

• fear-like behavior does not change¹¹⁶

• no increased stereotypy¹³⁴

2.11. NET-KO mouse (DA extracellularly increased in PFC)¶

Norepinephrine transporter (NET)-KO mice:¹³⁵

• WT mice showed bone loss in response to NET blockade by reboxetine.
• NET-KO mice showed decreased bone formation and increased bone resorption, resulting in suboptimal peak bone mass and suboptimal mechanical properties associated with low sympathetic outflow and high plasma NE levels.
• Differentiated osteoblasts express the NET (similar to neurons), take up noradrenaline again via the NET, but cannot produce noradrenaline.
• Daily activation of the sympathetic nervous system by mild chronic stress did not trigger bone loss unless NET activity was blocked.
  This leads to the question of whether noradrenaline reuptake inhibiting (ADHD) drugs could have detrimental effects on bone formation.

Since the NET reabsorbs slightly more dopamine than noradrenaline in the PFC, we assume an increased extracellular dopamine level in the PFC in the NET-KO mouse.

2.12. Naples high-excitability rat (NHE)¶

The Naples high-excitability rat was selected on the basis of increased exploration in a Làt-Maze (a test environment for measuring arousal to news).¹³⁶
It represents a model for the mesocortical variant of ADHD developed, validated and used¹³⁷ and shows hyperactivity, altered attentional performance and executive functions, but no hypertension.¹³⁸ The PFC shows hyperdopaminergic innervation with overexpression of mRNA species involved in basal metabolism and downregulation of dopamine D1 receptors¹³⁹

• Permanent attention problems¹³⁶
• Hyperactive and impulsive in a new environment¹⁴⁰¹⁴¹
• Not hyperactive or impulsive in a familiar environment

• NHE show hyperactivity in the field, but not in the home cage¹⁴²(anders als SHR)¹³⁶
• NHE become more active with increasing environmental complexity¹³⁹
• Handling
  • Handling: young, unweaned animals are removed from the litter and mother for a few minutes each day and placed alone in an empty cage without any further manipulation. They are then returned to their mother and siblings¹⁴³
    Barbara
  • light handling influences the activity of NHE rats differently than heavy handling¹⁴⁴
  • Handling in the fifth and sixth week of life can reduce hyperactivity in SHR and normalize neuronal markers (DRD1, CAMKII, c-fos)¹³⁶
• Hyperdopaminergic innervation of the PFC with overexpression of mRNA species involved in basal metabolism¹³⁹ (overactivated mesocorticolimbic system)
• Downregulation of dopamine D1 receptors¹³⁹
• DAT increased¹³⁶
  • Consequences of an excess of dopamine in early development
  • NHE have more dopamine-synthesizing terminals, more dopamine and, as a result, more DAT
  • The midbrain DA neurons are hypertrophic and have more terminals expressing tyrosine hydroxylase.
• In comparison normally bred rats (NRB) show NHE in the forebrain:¹³⁸
  • an overactivated mesocortical DA branch¹³⁹
  • a higher (+30%) volume of A10 neurons¹⁴⁵
  • a higher density of tyrosine hydroxylase (TH)-positive fibers
  • a 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)
• Tyrosine hydroxilase increased¹³⁶
  • Normalization through MPH
  • tyrosine hydroxilase is reduced in SHR, which is also normalized there by MPH¹³⁸