ADHD animal models with unknown dopamine alteration

In this paper, we collect animal models of ADHD in which the way in which dopamine levels change has not yet been revealed to us.

• 3. Animal models with unchanged or (to us) unknown dopamine changes
  • 3.1. 39.XY*O–Mouse (DHEA deficiency) (dopamine unchanged)
  • 3.2. GIT1-KO mouse (dopamine effect reduced?)
  • 3.3. ATXN7 Overexpressed mouse
  • 3.4. Grin1 mouse
  • 3.5. AGCYAP1-KO mouse
  • 3.6. TACR1-KO mouse
  • 3.7. Guanylylcyclase-C-KO mouse (GC-C-KO mouse)
  • 3.8. GAT1-KO mouse
  • 3.9. TR-beta 1 transgenic mouse
  • 3.10. FEZ1-KO mouse
  • 3.11. STA3GAL5(-/-) - Mouse
  • 3.12. PACAP(-/-) - Mouse
  • 3.13. Wheelrunning mouse
  • 3.14. Naples high-excitability rat (NHE)
  • 3.15. PTCHD1-KO mouse
  • 3.16. Neonatal anoxia mouse
  • 3.17. Cry1Δ11 mice
  • 3.18. Pln-/-KO mice
  • 3.19. Dogs with ADHD
  • 3.20. Drosophila (fruit fly)

3. Animal models with unchanged or (to us) unknown dopamine changes¶

3.1. 39.XY*O–Mouse (DHEA deficiency) (dopamine unchanged)¶

39,XY*O mice are genetically unable to produce steroid sulfatase (STS). Among other things, STS breaks down the steroid DHEAS.

39,XY*O mice showed compared to 40,XY mice:¹

• increased reactivity to a new environment¹
• Hyperactivity in the active phase¹
• Inattention²
• increased emotional reactivity¹
• increased water consumption (but not food)¹
• increased motivation²
• no difference in social dominance¹
• significantly lower DHEA serum levels¹
• equivalent corticosterone levels¹
• Increased serotonin²
  • in the striatum
  • in the hippocampus
• Reduced serotonin turnover²
  • in the striatum
  • in the hippocampus
• Noradrenaline turnover reduced²
  • in the striatum
• MOPEG decreases in the striatum²
• Dopamine unchanged in PFC, striatum, hippocampus, thalamus, cerebellum²

The STS gene is a gene candidate for ADHD.

3.2. GIT1-KO mouse (dopamine effect reduced?)¶

The G-protein coupled receptor kinase 1 knockout mouse (GIT1-KO) serves as an animal model for research into ADHD.³⁴
The GIT1 KO mouse shows as ADHD symptoms:⁵

• Hyperactivity
  • remediable with amphetamine and methylphenidate
• Learning disorders
• Memory loss

GIT1 regulates dopamine receptors. Overexpression of GIT1 interferes with the internalization of numerous G-protein-coupled receptors, including dopamine receptors.⁶ The latter suggests a model of reduced dopamine action.
GIT1 is a candidate gene for ADHD.

3.3. ATXN7 Overexpressed mouse¶

The ATXN7 Overexpressed mice (ATXN7-OE) show

• Hyperactivity
• Impulsiveness
• no inattention

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

3.4. Grin1 mouse¶

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

• Hyperactivity⁸
• Novelty seeking⁸
• Reduced social interaction⁸
• Anxiety⁹

The attentional abilities of Grin1 mice have not yet been investigated.⁸

Hyperactivity improved with high-dose methylphenidate. While c-FOS was very low in the prelimbic cortex and striatum of control mice and increased by MPH, c-FOS was high in the prelimbic cortex of GRIN1Rgsc174 ⁄ + mice and was reduced by MPH (at very high doses). Grin1Rgsc174 ⁄ + mice further showed increased phosphorylation of the protein ERK2 in the nucleus accumbens, which barely changed even after a very extreme MPH dose (30 mg/ kg). The authors concluded that the behavioral symptoms of the GRIN1 mouse were due to NMDA receptor dysfunction in the relevant brain regions, and that the effect of MPH in the GRIN1 mouse was not specifically mediated via the DAT but via other receptors or influences, since the DAT should have shown an effect even at much lower doses.⁹ The authors also point to the altered glutamatergic neurotransmission in SHR. SHR do not react at all to MPH with regard to hyperactivity, but do respond to AMP (see there).

3.5. AGCYAP1-KO mouse¶

The Adcyap1 gene encodes the neuropeptide Adenylate Cyclase Activating Polypeptide 1 generated in the pituitary gland. Mice lacking the ADXAP1 gene (Adcyap1(-/-)) show increased novelty seeking and hyperactivity. One study found sensory-motor gating deficits in them in the form of prepulse inhibition (PPI) deficits. Amphetamine was able to normalize PPI and hyperactivity. This occurred via serotonin 1A (5-HT(1A)) receptor signaling. Wild-type mice also developed hyperactivity in response to the 5-HT(1A) agonist 8-hydroxy-2-(di-n-propylamino)tetralin, which was also reversed by AMP. In addition, increased c-Fos-positive neurons were found in the PFC of AGCYAP1-KO mice treated with AMP, indicating increased inhibitory control by prefrontal neurons.¹⁰

3.6. TACR1-KO mouse¶

The Neurokinin-1 (Substance P, Tachikinin) Receptor Knockout Mouse (TACR1-KO mouse) is another rodent model for ADHD.¹¹

Virtually all dopaminergic neurons of the substantia nigra pars compacta and many of the substantia nigra pars reticulata contained neurokinin-1 receptors.¹² Substance P mediates its effect via postsynaptic heteroreceptors as well as via presynaptic autoreceptors. Substance P has an excitatory effect and modulates the inhibitory effect of GABA in the substantia nigra.
Substance P is involved in the regulation of dopamine release in the striatum.¹³¹⁴ The effect of substance P on dopaminergic transmission appears to be mediated by a nigro-thalamo-cortico-striatal loop.¹⁵
Substance P increases dopamine in the nucleus accumbens, but not in the neostriatum.¹⁶
TACR1 is associated with dopaminergic action in the striatum.¹⁷ TACR1 mediates the action of protein kinase C pathway in the downregulation of DAT and NET. The effect appears to be mediated via NET rather than DAT.¹⁸

We believe that these data may be indicative of reduced dopamine levels in TACR1-KO mice.

3.7. Guanylylcyclase-C-KO mouse (GC-C-KO mouse)¶

Guanylyl cyclase-C (GC-C) is a membrane receptor and is found together with tyrosine hydroxylase in the VTA and the substantia nigra pars compacta. GC-C can modulate dopamine signaling. Activation of GC-C by GC-C ligands, such as guanylin or uroguanylin, potentiates the excitatory responses mediated by glutamate and acetylcholine receptors via the activity of guanosine 3’,5’-monophosphate-dependent protein kinase (PKG), which affects dopaminergic cells.

GC-C-KO mice show:

• Hyperactivity (?)
  • in familiar and new environments¹⁹
  • is canceled by
    • systemic AMP injection¹⁹
    • GMP-dependent protein kinase agonist infusions into the VTA¹⁹
  • Another study found no hyperactivity²⁰
• Attention deficit in the Go/No-Go test.¹⁹
  • is canceled by
    • systemic AMP injection¹⁹
    • GMP-dependent protein kinase agonist infusions into the VTA¹⁹
• extensive investigation of new odors¹⁹
• Recognition of new objects impairs²⁰
• tactile shock reduces²⁰
• acoustic shock unchanged²⁰
• increased latency during training trials in the Morris water maze only in females²⁰
  • not for spatial learning attempts with a hidden platform

3.8. GAT1-KO mouse¶

Tonic GABA inhibits the release of dopamine in the striatum (of the mouse) via GABA-A and GABA-B receptors. Only a few GABA-ergic synapses are present at the dopamine axons. Therefore, the tonic inhibition of dopamine release by striatal GABA is probably mediated by extrasynaptic effects of extracellular GABA on receptors presumably located on dopamine axons. GABA therefore shows extrasynaptic effects on other neurons.
The gamma-aminobutyric acid transporter subtype 1 and subtype 3 reabsorbs GABA. If the GABA transporters GAT1 or GAT3 are reduced or switched off, this increases the extracellular GABA and thus reduces the release of dopamine in the dorsal striatum, but not in the nucleus accumbens.²¹²²

The two isoforms of the GAT in the striatum are:

• GAT-1 (Slc6a1)
  • common in axons of GABA-ergic neurons
  • in striatal astrocytes
  • in DA midbrain neurons
  • on striatal DA axons
• GAT-3 (Slc6a11)
  • moderately expressed
  • especially occurring on (striatal) astrocytes
    • Dysregulation of GAT-3 on striatal astrocytes causes profound changes in SPN activity and striatal-driven behavior through decreased extralellular dopamine²³
  • in DA midbrain neurons
  • on striatal DA axons

(GAT1)-KO mice (GAT-1-/- mice) show typical ADHD symptoms:²⁴²⁵

• Hyperactivity
  • AMP and MPH reduce these
• motor problems
  • Ataxia, characterized by deficiencies in motor coordination and balance
• Attention problems
  • Impairment of attentional focus in an “incentive runway test”
• Impulsiveness in an incentive test for passive avoidance
• Memory problems
  • Deficits in spatial reference memory

3.9. TR-beta 1 transgenic mouse¶

Carries a mutated human thyroid receptor TRb 1 gene.

TRbeta-transgenic mice²⁶

• are euthyroid except for a short period during postnatal development (= normal thyroid hormone levels of triiodothyronine (T3) and thyroxine (T4)).
• show until adulthood
  • Changes in the dopaminergic system (increased dopamine turnover)
  • ADHD symptoms
  • paradoxical reaction to MPH

Thus, like the vast majority of children with ADHD, the TRbeta transgenic mice show ADHD symptoms without measurable thyroid abnormalities. It is possible that even transient disorders of developmental thyroid homeostasis cause long-lasting behavioral and cognitive consequences, including the development of the full spectrum of ADHD symptoms.

Symptoms:

• Hyperactivity
• Impulsiveness
• Inattention
• All symptoms
  • Are reduced by methylphenidate
  • As with ADHD, are dynamic and react sensitively to changing environmental conditions, stress and reinforcement

3.10. FEZ1-KO mouse¶

The Fez1 gene (fasciculation and elongation protein zeta-1) is specifically expressed in the nervous system and is involved in neurodevelopment.
FEZ1 knockout mice show:²⁷

• Hyperactivity²⁸
• Impulsiveness
• Tyrosine hydroxilase expression reduced in midbrain and brainstem
• Dopamine and noradrenaline levels and their metabolites in the nucleus accumbens and PFC reduced
• MPH and guanfacine caused
  • Hyperactivity and impulsivity improved
  • Dopamine and noradrenaline levels restored in the nucleus accumbens and PFC
  • Tyrosine hydroxilase expression increased

The FEZ1 gene is specifically expressed in the nervous system and is most strongly expressed during neurodevelopment [21]. FEZ1 is involved in various processes of neurodevelopment, such as:²⁷

• Neurite extension
• dendritic arborization
• axonal transport
• neuronal migration

3.11. STA3GAL5(-/-) - Mouse¶

St3gal5-/- mice. In contrast to B4galnt1-/- mice, the clinical abnormalities only partially regress in these mutants, which could be due to the compensatory synthesis of the 0-series gangliosides GD1α and GM1b. However, the
St3gal5-/- mice lack the most important CNS gangliosides GM3, GM1, GD1a, GD3, GT1b and GQ1b
St3gal5-/- mice show:²⁹

• motor hyperactivity
• Impulsiveness
• Inattention³⁰
• increased insulin sensitivity³¹
• autistic behavior³²
  • conditioned taste aversion impaired in an inhibitory learning task
  • fear-like behavior in the field
  • motor deficits (moderate)
  • abnormal social interactions
  • excessive grooming and rearing behavior
• Platelet activation and neuronal damage after traumatic brain injury
• Proteolipid protein-1 (Plp1) gene and protein expression reduced³²
• proinflammatory cytokine expression increased³²
  • Interleukin1β is upregulated
• Lipopolysaccharides induce sex-dependent abnormalities in inflammatory response and social behavior³²
• Signs of hypomyelination³²

The STA3GAL5-KO mouse is a mild form of the STA3GAL5 disorder.³³ More severe forms are associated with developmental disorders, severe hearing, visual, motor and cognitive impairments and respiratory chain disorders. More on this at STA3GAL5 In the article Gene candidates without a plausible pathway in relation to ADHD

St3gal5-/-/B4galnt1-/- mice with double knockout lack any ganglioside derivative of LacCer.
Soon after birth, they develop severe neurodegeneration with impaired axon-glia interactions, weakness of the hind limbs, ataxia, tremor and increased inflammatory reactions. They die before the age of two months.³⁴

3.12. PACAP(-/-) - Mouse¶

Mice with pituitary adenylate cyclase-activating polypeptide (PACAP) deficiency (PACAP(-/-)):³⁵

• Hyperactivity
• Memory for new objects impaired
• Pre-pulse inhibition impaired

Atomoxetine improved all 3 symptoms and increased extracellular norepinephrine and dopamine in the PFC of PACAP(-/-) mice more than in wild-type mice.

3.13. Wheelrunning mouse¶

Mice bred by selection of those animals with a higher voluntary use of the running wheel showed:³⁶³⁷

• Hyperactivity in a new environment

• more wheel utilization in the form of shorter and faster runs

• Hyperactivity even after 24-hour acclimatization in cages without wheels

• D1/D5 receptors with reduced function

• D2/D3/D4 receptors unchanged

• Cocaine (dopamine reuptake blocker)³⁷

  • reduces average speed, but not duration of wheel use with wheel running mice
  • unchanged impeller use with wild type

• GBR 12909 (dopamine reuptake blocker)³⁷

  • reduces average speed, but not duration of wheel use with wheel running mice
  • unchanged impeller use with wild type

• Ritalin (15 mg/kg and 30 mg/kg)³⁶

  • reduced wheel usage with wheel running mice
  • increased wheel utilization with wild type

• Apomorphine (non-selective D2 agonist)³⁶

  • 0.125 mg/kg reduced wheel use in wheelrunning mice and wild-type mice alike
  • 0.25 mg/kg and 0.5 mg/kg reduced spawning wheel utilization in the wild type to a greater extent

• SCH 23390 (selective D1/D5 antagonist)³⁶

  • 0.025, 0.05 and 0.1 mg/kg reduced running wheel use in wild type more than in running mice

• Racloprid (selective D2 antagonist)³⁶

  • 0.5, 1 and 2 mg/kg reduced wheel use in wheelrunning mice and wild type to a similar extent

• Fluoxetine (SSRI)³⁷

  • reduced running speed and duration of wheel use in wheel-running mice and wild type proportional to baseline activity

3.14. Naples high-excitability rat (NHE)¶

The Naples high-excitability rat was selected based on increased exploration in a Làt-Maze.³⁸

• Permanent attention problems³⁸
• Hyperactive and impulsive in a new environment
• Not hyperactive or impulsive in a familiar environment

• NHE show hyperactivity in the field, but not in the home cage³⁹(anders als SHR)³⁸
• NHE become more active with increasing environmental complexity⁴⁰
• 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 siblings⁴¹
    Barbara
  • light handling influences the activity of NHE rats differently than heavy handling⁴²
  • Handling in the fifth and sixth week of life can reduce hyperactivity in SHR and normalize neuronal markers (DRD1, CAMKII, c-fos)³⁸
• Hyperdopaminergic innervation of the PFC with overexpression of mRNA species involved in basal metabolism⁴⁰ (overactivated mesocorticolimbic system)
• Downregulation of dopamine D1 receptors⁴⁰
• DAT increased³⁸
  • 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.

3.15. PTCHD1-KO mouse¶

In the PTCHD1-KO mouse, the PTCHD1 receptors in the thalamus are deactivated.

Male mice with deactivated PTCHD1 showed:

• Distractibility⁴³
• Problems with recognition memory⁴⁴
  • Atomoxetine eliminated this change.⁴⁴
• Hyperactivity⁴³⁴⁴
  • Atomoxetine eliminated this change.⁴⁴
• Impulsiveness⁴⁴
  • Atomoxetine eliminated this change.⁴⁴
• Learning disorders⁴³
• Hypotension⁴³
• Aggression⁴³
• Sleep fragmentation⁴³

There were also changes in the kynurenine metabolism.⁴⁴

When PTCHD1 was only deactivated in the reticular nucleus of the thalamus, only increased levels of⁴³

• Distractibility
• Hyperactivity
• Sleep problems

3.16. Neonatal anoxia mouse¶

Neonatal anoxia (postnatal lack of oxygen) increases the risk of ADHD.⁴⁵⁴⁶

Symptoms:
Males were more severely affected than females.⁴⁷

• Hyperactivity
  • for males in the open field⁴⁸⁴⁹
  • from day P20 to P45⁵⁰
  • not increased⁵¹
• Permanent deficits in spatial memory⁵⁰⁵²
• cognitive impairments during task acquisition⁵¹
• Deficits in sustained attention⁵¹
• Increased impulsivity⁵¹
• Compulsiveness increased⁵¹
• increased sensation of pain⁵³
  • Gender-specific reactions depending on the nociceptive stimulus
• Fear behavior in adult males⁴⁷

Neurophysiological changes:

• Anomalies with monoamines⁵⁴
• Changed cell density in the CA1 hippocampus⁵⁰
• Cell loss in substantia nigra⁵⁵
• Loss of brain volume, especially ipsilateral, in⁵¹
  • entire hemisphere
  • Cerebral cortex
  • white substance
  • Hippocampus
  • Striatum

Acute anoxia (acute lack of oxygen):⁵⁴

• Within 20 minutes:
  • Noradrenaline reduced in the cerebellum
  • Dopamine reduced in the striatum
  • 5-Hydroxyindoleacetic acid (5-HIAA) increased in the cortex and cerebellum
• P7 (7th day)
  • Noradrenaline increase in the cerebellum
  • Serotonin (5-HT) and 5-HIAA reduced in the cortex and cerebellum
• P21
  • Noradrenaline increase in the hippocampus
  • Increase in homovanillic acid (HVA) in the striatum
  • Serotonin decreases in the striatum
  • 5-HIAA increase in striatum and hippocampus
• P60
  • 3,4-Dihydroxyphenylacetic acid (DOPAC) increased in striatum
  • and 5-HIAA levels increased in the striatum

15 minutes caused perinatal asphyxia (lack of oxygen during birth):⁵⁶

• Tyrosine hydroxylase mRNA levels increased in VTA and substantia nigra
• DRD1 and DRD2 mRNA increased in the striatum

A similar mouse model, which simulates the damage of oxygen deprivation caused by extreme premature birth through repeated hypoxia, showed⁵⁷

• Hyperactivity and impulsivity as a reaction to a delayed reward
• no hyperactivity in an unfamiliar environment
• no inattention
• significant specific loss of dopaminergic neurons (only) in the right VTA

3.17. Cry1Δ11 mice¶

Cry1Δ11 (c. 1717 + 3A > C) mice show ADHD-like symptoms:⁵⁸

• Hyperactivity
• Impulsiveness
• Learning deficits
• Memory deficits
• hyperactive cAMP signaling pathway in the nucleus accumbens
• upregulated c-Fos, mainly localized in dopamine D1 receptor-expressing medium spiny neurons (DRD1-MSNs) in the NAc
• increased neuronal excitability of DRD1-MSNs in the nucleus accumbens
• the CRY1Δ11 protein, unlike the WT CRY1 protein, could not mechanistically interact with the Gαs protein and inhibit DRD1 signaling
• the DRD1 antagonist SCH23390 normalized most ADHD-like symptoms

3.18. Pln-/-KO mice¶

Phospholamban is found at the protein level in the reticular nucleus of the thalamus. This has a major influence on vital neurological processes, including executive functions and the generation of sleep rhythms.

Pln-/- mice exhibit a higher number of littermates compared to their Pln+/+ littermates:⁵⁹

• Hyperactivity
• reduced anxiety behavior
• Deficits in spatial working memory
• unchanged object localization memory
• impaired object recognition memory
• social exploration behavior / sociability / preference for new social territory unchanged

In contrast, ablation of phospholamban, which was limited to the reticular nucleus of the thalamus and did not affect peripheral PLN synthesis in cardiac muscles, skeletal muscles and smooth muscles, leads to:⁵⁹

• Hyperactivity
• Increased impulsivity
• unchanged anxiety behavior
• unchanged spatial working memory
• Waking phases shortened
• REM sleep prolonged, especially in females
• non-REM sleep extended
• Attention unchanged

3.19. Dogs with ADHD¶

A study of dogs with ADHD behaviors found relatively decreased blood dopamine and serotonin levels.⁶⁰

3.20. Drosophila (fruit fly)¶

Research on Drosophila has shown that gene variants determine the behavior of Drosophila in response to unpleasant air blasts, for example.
Drosophila that showed a particularly prolonged hyperactive reaction to bursts of air had a certain mutation in the dopamine transporter gene, which is one of the most important candidate genes in ADHD.⁶¹ When these Drosophila were treated with cocaine, they calmed down more quickly.
The dopamine D1 receptor was essential for the learning behavior of Drosophila. Drosophila with an artificially silenced D1 receptor (throughout the brain) were unable to learn that a particular odor acted as a warning signal for a puff of air.⁶²
If the D1 receptor gene was repaired exclusively in the brain region of the central complex, the Drosophila were no longer hyperactive, but were still unable to learn. If, on the other hand, the D1 receptor gene was only repaired in the brain region of the “mushroom body”, the ability to learn was restored, while the hyperactivity remained.⁶¹

After 60 generations, a Drosophila breeding line that was bred for sleep problems also showed considerable hyperactivity and increased sensitivity to environmental stimuli.⁶³

77 % of human disease genes have homologs in Drosophila.⁶⁴ Although the evolutionary tree of Drosophila and humans diverged 700 million years ago, the Drosophila brain is organized in three parts (forebrain, midbrain and hindbrain) like that of vertebrates and uses the same important neurotransmitters (dopamine, glutamate, GABA, homologues of adrenaline, noradrenaline and serotonin) as well as the corresponding enzymes, transporters and receptors.⁶⁵⁶⁶