ADHD animal models with reduced extracellular dopamine

In this article, we compile animal models of ADHD that exhibit reduced extracellular dopamine levels.
We have also provisionally included here animal models in which the only known factor is a reduction in dopamine, without knowing whether it is extracellular or phasic.
If we have inferred a reduced extracellular dopamine level solely on the basis of elevated DAT, this is indicated.

1.1. Spontaneously hypertensive rat (SHR) (increased DAT = decreased extracellular DA, phasically decreased)¶

The SHR is the most important animal model for ADHD research.¹²

The Spontaneous Hypertensive Rat (SHR) was first bred in 1963 as an animal model for hypertension.³ These animals possess genes that cause them (without early-life stress) to develop progressively higher blood pressure with age, reaching the level of hypertension around day 28 and accompanied by hyperactivity.⁴
The SHR strain was developed as a consequence of crossing male Wistar-Kyoto mice with markedly elevated blood pressure with females with slightly elevated blood pressure. Subsequently, brothers were mated with sisters, with continued selection for spontaneous hypertension.⁵
In 1992, it was determined that SHRs also serve as a model for ADHD-HI.⁶ Since then, SHRs have been used in ADHD research as a scientific animal model for ADHD-HI (with hyperactivity).
Although dopamine influences cortical blood flow, high blood pressure is not a symptom of ADHD.⁷ This illustrates that the SHR model represents only one of many different ADHD models and by no means universally represents ADHD.

The significance of the SHR as a model for ADHD should be assessed appropriately. Just as experts barely have any doubt that there are many different pathways leading to ADHD in humans (hundreds, if not thousands, of genes are involved, which can interact in very different combinations in people with ADHD), the SHR is not the only animal model for ADHD, and specifically ADHD-C. The SHR can therefore, at best, represent a possible model for ADHD. If 1,000 genes were indeed involved (most of which can produce multiple alternative gene variants with different expression profiles), there would be, purely mathematically speaking, an almost infinite number of possible combinations. Certainly, not all candidate genes have the same influence or the same frequency, but this line of reasoning shows that the SHR can be only one of many possible genetic constellations of ADHD.

1.1.1. ADHD-related behaviors of the SHR¶

The SHR exhibits (with the exception of sex differences) all major human ADHD-C traits (including hyperactivity).
ADHD symptoms are genetic and not caused by upbringing, as shown by a study in which SHR puppies were raised by WKY mothers.⁹

1.1.1.1. Hyperactivity¶

*¹⁰ Hyperactivity
* Standing upright on the hind legs, usually leaning against the walls. This represents an exploratory orientation toward the environment, which is operationally defined as non-selective attention.¹¹ In SHR and NHE rats, this activity is somewhat “hectic,” with long episodes of very short scanning durations.¹²
* SHR and NHE rats exhibit hyperactivity and altered non-selective attention, along with impaired selective spatial attention, as observed in an eight-arm radial maze.¹¹
* Hyperactivity does not manifest itself in new environments—just as with ADHD in humans. Only once a new environment becomes more familiar do SHR mice exhibit hyperactivity. This also distinguishes SHR hyperactivity from that of DAT-KO mice and rats, which initially exhibit hyperactivity in new environments but see it decrease over time.¹³¹⁴
* Hyperactivity does not develop until the age of 3 to 4 weeks, that is, before puberty¹³¹⁵
* DAT expression in the midbrain of SHR rats is reduced during the first postnatal month, whereas it is increased in adult SHR rats compared with control animals¹⁶¹⁷
* Hyperactivity is alleviated by ADHD medications through
* MPH¹⁸⁷
* AMP⁷
* Guanfacine¹⁹
* Atomoxetine²⁰¹⁸
* administration of dexmedetomidine over several days (25 μg/kg)²¹
* Duloxetine (10 mg/kg, SNRI)¹⁸
* Venlafaxine (10 and 30 mg/kg, SNRI)¹⁸
* Milnacipran (30 mg/kg, SNRI)¹⁸
* Reboxetine (10 mg/kg, NRI)¹⁸
* Desipramine (10 and 30 mg/kg, TZA)¹⁸
* But not by citalopram (30 mg/kg, SSRI)¹⁸
* Hyperactivity present⁷
* Only compared to WKY rats
* WKY rats are hyperactive during the first 15 minutes in an unfamiliar environment; SHR rats remain hyperactive even beyond that time.²²
* In environments such as the eight-armed radial maze
* Hyperactive compared to WKY rats in operant discrimination tests.
* No hyperactivity
* In familiar surroundings
* Less active than Sprague-Dawley rats in open-field tests²³
* Less active in running wheels than WKY rats²³
* Less active than Sprague-Dawley and Wistar rats in both the open-field and home-cage tests²⁴

1.1.1.2. Impulsivity¶

• Impulsivity¹⁰
  • Impaired ability to inhibit responses¹⁰
  • Develops with age
    *⁷, enhanced by MPH and AMP
  • Improved by guanfacine¹⁹
• Choice impulsivity (preferring an immediate small reward over a delayed larger reward)²⁵

1.1.1.3. Inattention¶

• Inattention¹⁰
  • Improved by MPH
  • Improved by guanfacine¹⁹

SHR rats appear to have unimpaired sustained attention, as they performed just as well as WKY and Sprague-Dawley rats on the 5-Choice Serial Reaction Time (5-CSRT) test¹³²⁶²⁷

1.1.1.4. Impaired spatial working memory¶

People with SHR show deficits in spatial working memory²⁸²⁹³⁰

• improved by the administration of choline and uridine³¹
• improved by dexmedetomidine²¹

1.1.1.5. Delay-Dependent Working Memory Deficits¶

SHR exhibited delay-dependent working memory deficits that, although less severe, are similar to those seen in rats with hippocampal lesions.³²

1.1.1.6. Lack of motivation¶

SHR exhibited a significantly lower PCA index compared to Wistar rats³³ as well as compared to Sprague-Dawley rats³⁴, suggesting that SHR have a motivational deficit.

1.1.1.7. Time-Processing Issues¶

Female SHRs showed impairments in processing elapsed time, particularly in distinguishing longer time intervals.³²

1.1.1.8. Impairment of goal-directed behavior¶

• Impairment of goal-directed behavior
  • Restored by MPH³⁵

1.1.1.9. Reduced performance stability¶

• Low performance stability¹⁰

1.1.1.10. Increased sensitivity to stress¶

• Increased Responses to Stress³⁶
• With advancing age and in parallel with rising blood pressure, SHR is associated with increased sensitivity of the HPA axis to stress.³⁷
  High blood pressure is a physical consequence of chronic stress.³⁸

1.1.1.11. Emotional symptoms¶

There is evidence of altered emotional communication and responses in SHR.
Rats communicate their emotional state through ultrasonic vocalizations (USVs). 22 kHz represents aversive reactions, while 50 kHz represents appetitive reactions.
Following fear conditioning, SHRs emitted more short 22-kHz ultrasonic vocalizations (USVs) and fewer 50-kHz USVs overall. In addition, SHR emitted fewer long 22-kHz USVs than Wistar rats. SHR showed no increase in heart rate (HR) in response to a 50-kHz playback, but a sharp decrease in HR in response to a 22-kHz playback. These phenomena observed in SHR may represent deficits in emotional perception and processing, similar to those seen in people with ADHD.³⁹

1.1.1.12. Social interaction is impaired¶

In SHR, social interaction is impaired.⁴⁰

1.1.1.13. Impaired learning ability¶

In an operant conditioning task, SHRs learned to press a lever to receive a reward. They learned this just as quickly as the control group only when the reward was delivered a few seconds after correct behavior. When the reward for correct behavior was sporadically delayed by about 3 minutes, SHRs were unable to learn a new rule. Even after 25 trials, their performance accuracy did not improve.⁴¹ SHRs learned just as quickly as WKYs when the reward was immediate, and learned more slowly when the reinforcement was delayed.⁴²

1.1.1.14. Subtypes corresponding to ADHD-HI and ADHD-I¶

A study was conducted on SHR subgroups that differed significantly in terms of impulsivity. Compared to non-impulsive SHRs and WKYS (used as controls, although the WKYS did not exhibit any behavioral subgroups), impulsive SHRs showed:⁴³

• Decreased norepinephrine levels
  • In the cingulate cortex
  • In the medial frontal cortex
• Reduced serotonin turnover
  • In the medial frontal cortex
• Reduced density of CB1 cannabinoid receptors (CB1R)
  • In the PFC
  • Acute administration of a cannabinoid agonist reduced impulsivity in impulsive SHR, with no change in WKY

Since the SHR strain does not consist of genetically identical, cloned animals, but rather of a strain bred for specific symptoms—and whose individual animals therefore still exhibit certain genetic differences—the subtypes could also have a genetic origin. To date, however, no heritability has been established for stress endophenotypes (typically an externalizing or internalizing stress response, corresponding to the ADHD-HI subtype/ADHD-C and the ADHD-I subtype).

Another study also found evidence of reduced CB1R activity in SHR.⁴⁴

The reduced levels of norepinephrine in the ADHD-HI and ADHD-C subtypes of the SHR appear to contradict Woodman’s findings:

• In Woodman’s study, aggression and outwardly directed anger were correlated with elevated levels of norepinephrine⁴⁵
• In Woodman’s study, however, fear was correlated with elevated adrenaline levels⁴⁵

1.1.2. Effect on SHR Symptoms¶

1.1.2.1. Effect of MPH on SHR¶

SHR responds to MPH:

• Improved attention and memory⁴⁶
• Dose-dependent reduction in impulsivity⁴⁷
• Hyperactivity;
  • Unchanged at low and moderate doses⁴⁶
  • Increases at high doses⁴⁶
  • Decreases at very high doses⁶
• Goal-directed behavior restored by MPH³⁵
• Reduced ADHD symptoms⁴⁸

Contrary to the view of the authors of the meta-analysis by Leffa et al. (2019), we see no reason to question the SHR as a model for ADHD-HI. Since ADHD is multifactorial and SHRs are merely an animal model bred to exhibit specific symptoms, SHRs can represent only one variant of ADHD (which, moreover, corresponds more closely to ADHD-HI than to ADHD-I). It has consequences, therefore, that the effects observed in SHRs cannot be generalized to all people with ADHD; rather, the neurophysiological mechanisms underlying individual symptoms and effects must be considered.

MPH administered before puberty was able to normalize the otherwise elevated DAT density in the striatum in adulthood. The improvement was more pronounced in the SHR/NCrl rat (which serves as a model for the mixed type) than in the WKC/NCrl rat, which serves as a model for the ADHD-I subtype.⁴⁹

MPH did not alter serotonin transporters in the striatum, even with long-term administration.⁵⁰

1.1.2.2. Effects of Amphetamine-Based Medications on SHR¶

Amphetamine-based medications reduced hyperactivity in the SHR.⁶ ADHD symptoms are reduced.⁴⁸

1.1.2.3. Effect of Atomoxetine on SHR¶

Atomoxetine reduced hyperactivity.²⁰

1.1.2.4. Enriched Environment¶

An enriched environment reduced […], in SHR

• Hyperactivity and inattention⁵¹
• cognitive deficits⁵²

1.1.2.5. Carbonic anhydrase inhibitors¶

The carbonic anhydrase inhibitors acetazolamide and metazolamide significantly reduced hyperactivity in SHRs without altering the behavior of Wistar-Kyoto rats. Acetazolamide also reduced impulsivity in SHRs. Low doses of acetazolamide had the strongest inhibitory effect on hyperactivity and impulsivity without impairing the spatial learning ability of SHRs.⁵³

1.1.3. Dopamine system impaired¶

The data suggest hypodopaminergic function in the SHR model.⁷

1.1.3.1. Reduced tyrosine hydroxylase; impaired dopamine synthesis¶

In SHR, the miRNA let-7d is reportedly overexpressed in the PFC, and the expression of galectin-3 is reduced, leading to downregulation of tyrosine hydroxylase, which is a precursor in dopamine synthesis.⁵⁴ The consequences are impaired dopamine synthesis. However, one study found elevated plasma galectin-3 levels in children with ADHD.⁵⁵
Compared to WKY, SHR

• Tyrosine hydroxylase levels are reduced in⁵⁶
  • Neostriatum
  • Nucleus accumbens
  • Lateral septal nucleus
  • Tractus diagonalis
• Calmodulin levels are reduced in⁵⁶
  • Medial neostriatum
• A decrease in serum calcium levels leads, via a calmodulin-dependent mechanism, to a decrease in dopamine levels in the neostriatum and nucleus accumbens⁵⁶

The synthesis of dopamine in the brain occurs in two steps. First, the enzyme tyrosine hydroxylase catalyzes the conversion of the amino acid tyrosine into L-3,4-dihydroxyphenylalanine (L-DOPA); then, L-DOPA is decarboxylated to form dopamine.
On days P5 and P7 (5 and 7 days after birth, respectively), reduced tyrosine hydroxylase gene expression was observed.¹⁶
Reduced tyrosine hydroxylase expression in the neostriatum and nucleus accumbens, with identical levels of dopamine and dopamine metabolites in the striatum of SHR and control rats.⁷

Furthermore, dopamine uptake in the striatum was significantly reduced in SHR during the first month of life.¹⁶

SHR exhibited a reduced release of dopamine and acetylcholine in the striatum in response to glutamate.⁵⁷

1.1.3.2. DAT decreased during the first few weeks, but increased in adult SHRs¶

Reports on DAT expression in SHR mice are inconsistent.

• Decreased during the first month of life, then returns to normal⁵⁸
  *¹⁶ is reduced in the midbrain from day P27 to P49
• Increased over the course of a lifetime¹⁷
• Overexpressed in adult SHR mice¹⁶

Surprisingly, the dopamine reuptake inhibitor nomifensine increases dopamine release to the same extent in SHR and WKY rats in the nucleus accumbens and the caudate-putamen. Given the elevated tonic dopamine levels in SHR rats, one would expect a different outcome.⁷

1.1.3.3. Increased D1 receptor expression¶

The majority of studies confirm increased DRD1 and DRD2 expression in SHR in the nucleus accumbens, striatum, and PFC. Some studies found no differences in DRD1 and DRD2 expression. The higher expression of DRD1 and DRD2 is consistent with reduced dopamine release, which causes upregulation of the receptors.⁷

Consistent with the subsequent decline in stimulus-induced dopamine, postsynaptic DRD1 levels in the caudate-putamen and nucleus accumbens are elevated in SHR, which is consistent with a role for dopamine in ADHD.⁵⁹¹⁷

1.1.3.4. Increased D2 receptor expression¶

The SHR exhibits significantly increased dopamine D2 receptor expression in the PFC, striatum, and hypothalamus. Atomoxetine significantly and dose-dependently reduced dopamine D2 gene expression in the PFC, striatum, and hypothalamus.^20606162
Other studies found no increased D2 expression in SHR¹⁶ compared to WYK rats.⁵⁸

It appears that in SHR, postsynaptic D1/D2-like receptors are less sensitive, while presynaptic dopamine D2-like autoreceptors—which are found primarily in the nucleus accumbens—are likely more sensitive.⁶³

Increased DRD2 expression may serve as a compensatory mechanism for reduced DAT function during early development in SHR. It has been hypothesized that the net effect of such changes leads to elevated extracellular dopamine levels during the preweaning period in SHR (Russell, 2000), which later transitions into dopamine deficiency.

1.1.3.5. D3 receptor expression remains unchanged¶

Previous studies found that the DRD3 receptor was unchanged in SHR.⁷

1.1.3.6. Reduced D4 receptor expression in the PFC (?)¶

SHR exhibited significantly reduced dopamine D4 receptor gene expression and protein synthesis in the PFC. Other dopaminergic genes in the midbrain, PFC, temporal cortex, striatum, or amygdala of SHR were unchanged compared to WKY.⁶⁴
Mill found no change in D4 receptor expression compared to WYK rats.⁵⁸

1.1.3.7. Changes in extracellular (tonic) dopamine?¶

Several studies found reduced basal extracellular dopamine concentrations in the caudate nucleus and nucleus accumbens at 8 to 9 weeks of age.⁶⁰¹⁵⁶⁵
A study found increased extracellular tonic dopamine release in the shell of the nucleus accumbens.³⁶
Some studies found no differences in extracellular dopamine concentrations.⁶⁶⁶⁷

1.1.3.8. Phasic dopamine release is reduced¶

In SHR, the dopaminergic presynapses of the mesocortical, mesolimbic, and nigrostriatal neurons appear to release less phasic dopamine in response to electrical stimulation/depolarization due to high extracellular K+ concentrations.⁶⁸
Compared to WKY/NCrl (an ADHD-I model), SHR/NCrl exhibited reduced KCl-evoked dopamine release in the dorsal striatum.⁶⁹

1.1.3.9. Dopamine uptake in the striatum is accelerated¶

SHR/NCrl mice showed faster dopamine uptake in the ventral striatum and nucleus accumbens than controls, while WKY/NCrl mice (an ADHD-I model) exhibited faster dopamine uptake only in the nucleus accumbens.⁶⁹ This is consistent with increased DAT activity in SHR.

Striatal and mesolimbic dopaminergic neurotransmission is elevated in SHR (which would explain the SHR’s hyperactivity), while basal norepinephrine efflux in the PFC is reduced.⁷⁰

1.1.3.10. Is vesicular dopamine storage impaired?¶

In SHR, vesicular storage of dopamine may be impaired, leading to the release of dopamine into the cytoplasm.⁷¹
* In vitro, methylphenidate and electrical stimulation resulted in lower dopamine release from vesicles than dextroamphetamine
* In vitro, dextroamphetamine released more dopamine from cytoplasmic stores via the DAT

We wonder whether the observed effect might simply describe the increase in DAT efflux known to occur with (high-dose) amphetamine.

1.1.3.11. Mesocortical dopamine system remains unchanged¶

No changes were observed in the mesocortical dopaminergic system in juvenile SHR:⁷²

• no change in the surface density of TH-immunoreactive (TH-ir) dopaminergic neurons in the VTA
• No changes in the volumetric density of TH-ir fibers in layer I of the prelimbic subregion of the mPFC
• no changes in the percentage of dopaminergic TH-ir fibers in layer I of the PrL subregion of the mPFC

The target regions of the mesocorticolimbic dopamine system in the forebrain are hyperinnervated in SHR and undergo pruning (reduction of connections) during puberty.⁷³ The density of D1R and D2R receptors is elevated in SHR, as in WKY, at 42 days of age, primarily in the dorsal striatum (caudate-putamen) and in the ventral striatum (nucleus accumbens, both in the core and in the shell).⁷⁴
Anteriorly, in the rostrocaudal direction, there is a sector with a significant increase in density of D1R and D2R, while posteriorly, the density is markedly reduced.
Signal transduction mechanisms such as Ca2+/calmodulin-dependent kinase II (CaMKII) and the transcription factor c-FOS are downregulated⁷⁵, although here as well, density is increased in the accumbens (NAcc, i.e., ventral striatum) and reduced in the rostral part of the caudate-putamen (i.e., dorsal striatum, dStr).⁷⁶

1.1.3.12. High Blood Pressure as a Consequence of Dopamine Deficiency¶

The high blood pressure that SHRs typically develop as they age could also be a consequence of dopamine deficiency, primarily due to increased peripheral sympathetic nerve activity.⁷⁷

1.1.3.13. Response to MPH / AMP¶

Stimulation of dopamine release by MPH or AMP in the shell of the nucleus accumbens resulted in a greater increase in dopamine in SHR than in WKY. Increasing KCl stimulation in the shell of the nucleus accumbens reversed this difference in the increase (a greater increase in extracellular dopamine release in WKY than in SHR).³⁶ This suggests that in SHR, a higher dopamine tone in the shell of the nucleus accumbens, combined with lower intracellular dopamine reserves, contributes to increased activity compared to WKY.⁷

In vitro, dextroamphetamine released 7 to 17 times more dopamine than methylphenidate from slices of the prefrontal cortex, the nucleus accumbens, and the caudate-putamen. Methylphenidate released significantly less dopamine from nucleus accumbens slices of SHR rats than from normotensive Wistar-Kyoto controls. Dextroamphetamine released more dopamine from prefrontal cortex, nucleus accumbens, and caudate-putamen slices from SHR compared to WKY, unlike methylphenidate. Low-dose methylphenidate inhibited DAT to the same extent in both SHR and WKY tissue. Dextroamphetamine stimulates the release of acetylcholine equally in both SHR and WKY. Methylphenidate induced inhibition of the electrically stimulated release of acetylcholine from nucleus accumbens or caudate-putamen sections in both SHR and WKY rats.⁷¹

1.1.4. Alterations in the adenosine system in SHR¶

The adenosine system interacts with the dopamine system.
In SHR, adenosine is⁷⁸ in blood plasma, and the number of adenosine A2A receptors in frontocortical nerve terminals (presynapses) is increased.⁷⁹ The bioavailability of adenosine in vascular tissues and arteries of SHR appears to be increased, while at the same time the adenosine transporters (ENT) as well as the A1 and A2A receptors are downregulated. In veins, the expression of ARs and ENTs appears unchanged, while the A2A receptor appears to be upregulated and the ENT2 transporter downregulated.⁸⁰

Adenosine receptor antagonists improve various ADHD symptoms in SHR

• Caffeine (non-selective A1 and A2A adenosine receptor antagonist)
  • Object recognition⁸¹
  • social recognition⁸²
  • spatial learning⁸³
  • No effect on high blood pressure⁸³
• DPCPX (8-cyclopenthyl-1,3-dipropylxanthine, A1 antagonist)
  • Object recognition⁸¹
  • No effect on high blood pressure⁸³
• ZM241385 (4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo-[2,3-a][1,3,5]triazin-5-yl-amino]ethyl) phenol, A2A antagonist)
  • Object recognition⁸¹
  • social recognition⁸²
  • No effect on high blood pressure⁸³
• Theobromine⁸⁴
  • Male SHR rats (6 weeks old) that were fed a diet containing 0.05% theobromine for 40 days showed
    • Hyperactivity improved
    • Improved attention span
    • Improves working memory deficits
    • Dopamine levels in the PFC return to normal
    • Normalized expression levels of TH, DRD2, DRD4, SNAP-25, and BDNF in the PFC
    • The expression of DRD1, DRD3, DRD5, DAT, and VMAT-2, which was normal in untreated SHR, was only minimally affected
    • Theobromine binds to adenosine receptors

Chronic Caffeine Intake:⁷⁹

• normalized dopaminergic function
• improved memory and attention deficits
• caused upregulation of A2A receptors in fronto-cortical nerve terminals

Chronic administration of caffeine or MPH before puberty resulted in improved object recognition in adult SHR rats, whereas the same treatment impaired object recognition in adult Wistar rats.⁸⁵

There is evidence of an interaction between the cannabinoid and adenosine systems in relation to impulsive behavior in the SHR:⁸⁶

• WIN55212-2 (cannabinoid receptor agonist) increased impulsive behavior
• Acute pretreatment with caffeine reversed this effect
• Chronic caffeine intake increased impulsivity

In SHR, adenosine-mediated presynaptic inhibition of adrenergic transmission appears to be genetically reduced.⁸⁷
The A1 agonist CPA increased alpha-2 adrenoceptor binding in the nucleus tractus solitarius in SHR by about 10 times more than in WKY.⁸⁸

Adenosine affects blood pressure.⁸⁹ Adenosine lowered blood pressure in SHR even more than in WKY. Adenosine decreased heart rate in SHR and increased it in WKY.⁹⁰

1.1.5. Increased norepinephrine release¶

In the laboratory, PFC brain cells from SHR rats showed increased norepinephrine release in response to glutamate. This effect was not mediated by NMDA receptors, as NMDA did not alter norepinephrine release. It is hypothesized that the noradrenergic system in the PFC of SHR is overactivated⁹¹ or dysregulated, possibly in the form of increased alpha-adrenoceptor sensitivity.²² The A1 agonist CPA increased alpha2-adrenoceptor binding in the nucleus tractus solitarius in SHR approximately 10 times more than in WKY.⁸⁸
In SHR, adenosine-mediated presynaptic inhibition of adrenergic transmission appears to be genetically reduced.⁹²

In SHR, autoreceptor-mediated inhibition of norepinephrine release appears to be further impaired, suggesting poorer regulation of noradrenergic function in the PFC. The behavioral disturbances associated with ADHD may result from an imbalance between the noradrenergic and dopaminergic systems in the PFC, with reduced inhibitory dopaminergic activity and increased noradrenergic activity.⁹³⁶⁸⁴⁸

1.1.6. Serotonin in SHR¶

SHRs show an increased number of serotonin transporters in the striatum in adulthood, a finding that was not altered by MPH.⁵⁰
A 5-HT1A agonist (ipsapiron) or a 5-HT2A antagonist (MDL 100907) improved ADHD symptoms. The improvement correlated (as it did with guanfacine) with:⁹⁴

• the upregulation of 5-HT2A receptors in the prefrontal cortex, striatum, and substantia nigra
• the upregulation of 5-HT1A in the PFC and substantia nigra
• the downregulation of D1R
  • by 5-HT1A agonists (ipsapiron) in the prefrontal cortex, striatum, and substantia nigra
  • via a 5-HT2A antagonist (MDL 100907) in the PFC and striatum
  • via guanfacine in the PFC and substantia nigra

1.1.7. Changes in the endocannabinoid system¶

For information on the cannabinoid system in ADHD, see Cannabinoids In the section “ : Neurotransmitters in ADHD” in the chapter “ : Neurological Aspects”.

1.1.7.1. CB1R is reduced in impulsive SHRs¶

For information on CB1 cannabinoid receptors (CB1R) in SHR, see CB1R is reduced in SHR in the article “Cannabinoids.”

Among other things, reduced CB1R levels in the PFC were characteristic of both impulsive SHR (ADHD-HI/ADHD-C) and distinguished them from non-impulsive SHR (ADHD-I).⁴³

1.1.7.2. Cannabinoids Affect Social Interaction and Hyperactivity in SHR¶

• Rimonabant (an inverse CB1R agonist) did not affect social interaction or locomotion in either Wistar rats or SHR, regardless of the dose.⁴⁰
• WIN55-212,2 (CB1R/CB2R agonist):⁴⁰
  • reduced locomotion in Wistar rats (1 mg/kg)
  • reduced social interaction in Wistar rats (0.1 and 0.3 mg/kg)
  • increased social interaction in SHR (1 mg/kg)
• ACEA (CB1R agonist):⁴⁰
  • Increased locomotion in Wistar and SHR (0.3 mg/kg)
  • reduced locomotion in Wistar and SHR rats (1 mg/kg)
• AM404 (AEA reuptake inhibitor, AEA degradation inhibitor):⁴⁰
  • increased social interaction in SHR (5 mg/kg)
  • reduced locomotion in SHR (5 mg/kg)
  • reduced social interaction in Wistar rats (1 mg/kg)
  • increased locomotion in Wistar rats (1 mg/kg)
• Capsaicin (TRPV1 agonist):⁴⁰
  • increased social interaction in Wistar and SHR rats (2.5 mg/kg)
  • reduced locomotion in SHR (2.5 mg/kg) and Wistar (0.5 mg/kg and 2.5 mg/kg)
• Capsazepine (TRPV1 antagonist):⁴⁰
  • reduced locomotion in both strains (5 mg/kg)
  • increased social interaction in Wistar rats (5 mg/kg)
  • reduced social interaction in Wistar rats (10 mg/kg)

1.1.8. Glutamatergic changes affecting dopamine¶

SHR exhibited a defect in glutamate-stimulated dopamine release in the shell of the nucleus accumbens. This could contribute to a disorder in reinforcement of appropriate behavior.⁹⁵

N-methyl-D-aspartate receptor (NMDAR)-dependent long-term changes in synaptic efficacy in the CNS are fundamental cellular mechanisms underlying certain forms of learning in mammals. Synaptic abnormalities in excitatory, glutamatergic synaptic transmission appear to contribute to behavioral changes in SHRs. SHRs exhibited reduced synaptic transmission between CA3 and CA1 in the hippocampus, whereas short-term forms of synaptic plasticity, such as paired-pulse facilitation, frequency facilitation, and delayed response potentiation, as well as long-term potentiation (LTP) of synaptic transmission, were unchanged compared to WKY rats. However, LTP in SHRs was significantly reduced (by 50%) by the NR2B-specific blocker CP-101,606 (10 microM), whereas the blocker had no effect on LTP in WKY rats. This suggests a functional dominance of NR2B in SHR. A functional predominance of NR2B is characteristic of the early developmental stages of these synapses. Quantitative immunofluorescence and post-embedding immunogold cytochemistry using electron microscopy of the three major NMDAR subunits (NR1, NR2A, and NR2B) in stratum radiatum spine synapses revealed no differences between SHR and WKY.
An enriched environment promotes LTP induction as a consequence of p38-mediated NR2B activation. p38 is an LTD-associated MAP kinase that normally does not contribute to LTP in conventionally h .⁹⁶
Functional impairments in glutamatergic synaptic transmission may be one of the underlying causes of abnormal behavior in SHR (and possibly also in ADHD in humans).⁹⁷

1.1.9. GABA in SHR¶

SHR exhibited reduced [3H]-GABA uptake and release, suggesting a defective striatal GABAergic transport system.
In vitro, caffeine improved the following in the striatum of SHR:

• GABA release (which is reduced in SHR)
• GABA reuptake via the GAT1 transporter (which is reduced in SHR)

whereas this was not the case in Wistar rats (which do not serve as an animal model for ADHD).⁹⁸

A study found evidence suggesting that the extracellular concentration of GABA in the SHR hippocampus may be reduced. An underlying defect in GABA function could be the cause of the catecholamine transmission dysfunction observed in SHRs and could underlie their ADHD-like behavior.⁹⁹

The GABA antagonist Oroxylin A appears to improve ADHD-like behaviors in SHR by enhancing dopaminergic neurotransmission, rather than by modulating the GABA signaling pathway, as previously reported.¹⁰⁰

1.1.10. Altered Vitamin D3 Metabolism in SHR Mice¶

In SHR, the activity of 25-hydroxyvitamin D 1-alpha-hydroxylase appears to be reduced. This could be due to impaired renal metabolism or a reduced responsiveness to cyclic adenosine-3’,5’-monophosphate. In both SHR and WKY rats, a one-week restriction of dietary phosphorus led to an increase in plasma D3 concentration. This did not result in any change in blood pressure.¹⁰¹ Another study found both elevated and reduced D3 levels.¹⁰²

1.1.11. Changes to Stress Systems¶

1.1.11.1. Excessive HPA axis stress response in SHR compared to WKY¶

7-week-old SHRs show a significant difference compared to WKYs of the same age¹⁰³

• Increased corticosterone responses to bleeding and ether stress
  Note: Since SHR is a model for ADHD-HI and not for ADHD-I, we would expect blunted stress corticosterone responses, as have been found in other studies¹⁰⁴
• Elevated basal corticosterone levels
  Note: People with ADHD generally have lower basal cortisol levels, regardless of their subtype.
• Reduced plasma ACTH responses to bleeding and ether-induced stress
• Lower plasma ACTH responses to IV CRH injection
• Identical plasma ACTH responses to vasopressin
• Lower CRH concentrations in the hypothalamus (median eminence), posterior pituitary lobe, and cerebral cortex
• Reduced CRH release from the hypothalamus
• Identical CRH response to 56 mM KCl

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

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

The administration of corticosterone as a substitute restored the elevated blood pressure in SHR.

Dexamethasone, as a glucocorticoid receptor (GR) agonist, improved ADHD-C symptoms in SHR.¹⁰⁵ A GR antagonist (mifepristone) induced ADHD-C symptoms in other rat strains (which do not normally exhibit ADHD symptoms).¹⁰⁶
Dexamethasone (as a GR agonist) increased the previously reduced (compared to WKY) serotonin levels in the PFC of SHR and improved attention deficit and hyperactivity. In contrast, a GR inhibitor (RU486) increased inattention and hyperactivity. Dexamethasone increased the expression of 5-HT and 5-HT2AR in the PFC and decreased the expression of 5-HT1AR. RU486, on the other hand, decreased the expression of 5-HT and 5-HT2AR and increased the expression of 5-HT1AR.¹⁰⁷

These results suggest that:

• HPA Axis Is Overactive in Young SHRs
• Reduced ACTH response to stress and to CRH due to higher plasma corticosterone levels
• Glucocorticoids play a key role in the development of high blood pressure in SHR
• In ADHD-HI / ADHD-C (with hyperactivity), the GR receptor may not be adequately stimulated, either because there are too few GR receptors or their sensitivity is too low, or because there are an excessive number of MR receptors
• In SHR, the glucocorticoid system is closely linked to the serotonin system

Other studies have observed significantly reduced baseline levels of⁸

• Aldosterone at 8 weeks of age
• 18-Hydroxy-11-deoxycorticosterone (18-OH-D0C)1 at 12 weeks of age
• Deoxycorticosterone (DOC) at 20 weeks of age
• Corticosterone at 12 and 20 weeks of age.

1.1.11.2. Increased mineralocorticoid receptor expression as the cause of excessive HPA axis stress responses in SHR¶

SHR mice have genetically determined overexpression of mineralocorticoid receptors (MR) and normal expression of glucocorticoid receptors (GR).¹⁰⁸
A shift in the balance between MR and GR toward increased MR therefore leads to increased basal and stress-induced activity of the HPA axis.
⇒ The Corticosteroid Receptor Hypothesis of Depression

Dexamethasone, as a glucocorticoid receptor (GR) agonist, corrected inattention and hyperactivity in SHR^{107109 110} and also increased dopamine and norepinephrine by reducing DAT expression, whereas a GR antagonist decreased these levels.¹⁰⁹
Conversely, a GR antagonist (mifepristone) induced ADHD-HI symptoms in other rat strains (that do not otherwise exhibit ADHD symptoms).¹⁰⁶

This is consistent with our view that ADHD-HI and ADHD-C (with hyperactivity) are caused or regulated by an impaired response of GR relative to MR.
We wonder whether ADHD-I might, conversely, be characterized by a reduced number of MRs relative to GRs.

MRs regulate the day-to-day functioning of cortisol. GRs, on the other hand, are only activated when cortisol levels are very high, and their function is to shut down the HPA axis. When MR receptors are predominant and the cortisol stress response is reduced (as is typical for ADHD-HI), the unoccupied MR receptors absorb the cortisol, so that the GR receptors are not sufficiently occupied to trigger the shutdown of the HPA axis.
If, on the other hand, MRs are underrepresented or the cortisol stress response (as in ADHD-I) is excessive, the GRs are activated too quickly and the HPA axis is shut down too frequently.

1.1.11.3. MiRNA Expression in SHR Alters the Glucocorticoid Receptor¶

For the miRNA

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

A significantly reduced expression of SHR was found in the ADHD rat model, which was associated with promoter-inhibitory activity of the glucocorticoid receptor No. 3c1.¹¹¹

1.1.12. SHR and the Immune System¶

1.1.12.1. Young SHR¶

Compared to WKYs, young SHRs show:

• Elevated levels of cytokines¹¹⁶
• Elevated levels of chemokines¹¹⁶
• Elevated levels of markers of oxidative stress¹¹⁶
• Reduced PFC volumes¹¹⁶
• Elevated levels of dopamine D2 receptors¹¹⁶
• In the motor cortex and PFC:¹¹⁷
  • Interleukin-1α (IL-1α) is elevated
  • Elevated IL-6
  • The serine/threonine protein Mammalian Target of Rapamycin is elevated
  • RAC-alpha serine/threonine protein kinase is elevated
  • Increased glucocorticoid receptor β
  • Malondialdehyde levels increase
  • Increased sulfhydryl groups
  • Superoxide dismutase levels are elevated
  • Peroxidase levels are elevated
  • Elevated glutathione reductase
  • Elevated glutathione S-transferase
  • Glucose levels are elevated
  • Elevated fructosamine
  • Iron increases
  • Increased lactic acid
  • Alanine is elevated
  • Elevated aspartate transaminase
  • Elevated lactate dehydrogenase
  • Significant reduction in the motor cortex

1.1.12.2. Older SHR¶

Older SHRs, compared to WKYs, show¹¹⁶

• Normalized cytokine levels
• Normalized levels of chemokines
• Normalized levels of oxidative stress markers
• Elevated levels of steroid hormones.

1.1.12.3. Additional changes in immune parameters in SHR mice¶

In the animal model of ADHD-C, the Spontaneously Hypertensive Rat (SHR), the following were found in adult male animals in the brain regions (but not in peripheral blood):¹¹⁸

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

In contrast, another study found the following in untreated brainstem and cerebellar astrocytes from SHR mice compared to Wistar astrocytes:¹¹⁹

• IL-1β significantly elevated
• IL-10 significantly elevated

Another study found in SHR:¹²⁰

• abnormal microglial activation
  -elevated levels of the pro-inflammatory cytokines IL-6 and IL-1β
• pathological changes in the blood-brain barrier

1.1.12.4. The Gut-Brain Axis in SHR¶

Treatment with dexmedetomidine²¹

• altered the composition of the gut microbiota. Dexmedetomidine increased:
  • Ruminiclostridium
  • Jeotgalicoccus
  • Corynebacterium_1
  • Ruminococcaceae_UCG_010
  • Butyricimonas
  • Parasutterella
  • unclassified_, Muribaculaceae
• promoted the growth of beneficial gut bacterial genera that have been linked to anti-inflammatory effects in SHR
• significantly improved intestinal permeability as well as inflammation markers in the gut and brain
• A fecal microbiota transplant from SHR treated with dexmedetomidine to untreated SHR resulted in the latter exhibiting effects mimicking those of DEX administration (improved hyperactivity, improved spatial working memory, improved theta EEG rhythms)

Administration of Bacteroidetes bifidum BD1 to newborn female SHR rats (109 CFU daily via a nasogastric tube for 3 weeks) resulted in the following after 7 weeks:¹²¹

• significantly reduces congenital hyperactivity
• Elevated levels of DAT and tyrosine hydroxylase in the striatum
• significantly reduced the number of activated microglia
• Significantly elevated levels of Treg cells in the spleen
• Improved α-diversity in the gut microbiota
• Reduced Firmicutes/Bacteroidota ratio
• Muribaculaceae increased
• Proliferation of Clostridia_UCG-014 is suppressed

1.1.12.5. Taurine improved inflammatory markers and hyperactivity and reduced DAT in SHR¶

SHRs treated with taurine showed reduced serum levels of C-reactive protein (CRP) and IL-1β.¹²² While low-dose taurine increased motor activity, high-dose taurine decreased it.

A study conducted on rats concluded that taurine may have positive effects on ADHD.¹²³

• Low taurine intake increased
  • DAT in the striatum was significantly elevated (only) in WKY rats
  • Dopamine uptake in the striatum in both SHR and WKY rats.
• High-dose taurine reduces (only) in SHR rats
  • DAT levels in the striatum were significantly
    • DAT levels in the striatum are elevated in ADHD
  • Dopamine uptake in the striatum
    • Dopamine reuptake in the striatum is increased in ADHD
  • Interleukin (IL)-1β and C-reactive protein
  • Horizontal movement
  • The functional connectivity of the hippocampus (including in WKY)
  • The mean amplitude of low-frequency (0.01 to 0.08 Hz) fluctuations (mALFF, mean amplitude of low-frequency fluctuation (mean ALFF)) in the hippocampus bilaterally (including in WKY)
• Increase both low and high levels of taurine intake
  • Significantly reduced BDNF levels in the striatum in both SHR and WKY rats
    BDNF levels are reduced in ADHD

High-dose taurine reduced hyperactivity in SHR rats by lowering levels of inflammatory cytokines and modulating functional brain signals:¹²⁴
WKY with high urea application

• CRP (C-reactive protein) significantly reduced in serum
  SHR with low or high tire pressure
• Interleukin (IL)-1β significantly reduced
• CRP significantly reduced
  WKY and SHR with low tire pressure
• significantly increased horizontal locomotion
  SHR with high tire pressure
• Horizontal locomotion significantly reduced compared to the SHR control group
  WKY, like SHR, with a high tire pressure setting
• functional connectivity (FC) significantly reduced
• Significantly reduced mean amplitude of low-frequency fluctuation (mALFF) in the bilateral hippocampus
  SHR with low or high tire pressure
• mALFF significantly reduced compared to the SHR control group

1.1.13. Blood-Brain Barrier¶

• Monocarboxylate Transporter 1 (MCT1) is elevated¹²⁵
  • According to the energy deficit hypothesis of ADHD, symptoms are triggered by reduced lactate production by astrocytes in the brain. Hyperactivity is thought to be a compensatory mechanism designed to utilize lactate production in the skeletal muscles. Consistent with this hypothesis, increased expression of the lactate transporter monocarboxylate transporter 1 (MCT1) at the blood-brain barrier of the hippocampus was observed in SHR.¹²⁶

Evidence of structural damage to the blood-brain barrier due to neuroinflammation and excessive autophagy is suggested by:¹²⁷

• MMP2 is elevated
• MMP9 is elevated
• ZO1 is reduced (tight junction protein)
• Reduced levels of occludin (a tight junction protein)

1.1.14. Cholesterol metabolism in the PFC of SHR is altered; MPH reverses the change¶

A study found 12 altered metabolites in the PFC in SHR (compared to WKY). The differences in 7 of these were normalized by MPH:¹²⁸

• 3-Hydroxymethylglutaric acid
• 3-phosphoglyceric acid
• Adenosine monophosphate
• Cholesterol
• Lanosterin
• O-phosphoethanolamine
• 3-Hydroxymethylglutaric acid.

The modified metabolites are part of the cholesterol metabolic pathways.
The SHR found the following in the PFC regarding this

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

1.1.15. Blood Pressure, the Sympathetic Nervous System, Cardiac Hypertrophy, and Vitamin D3 in SHR¶

In SHR rats, compared with WKY rats, the following were found:

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

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

• Higher levels of mRNA and protein expression in
  • High Mobility Box 1 (HMGB1)
  • Receptor for Advanced Glycation End Products (RAGE)
  • Toll-like receptor 4 (TLR4)
  • Nuclear factor-kappa B (NF-κB)
  • Pro-inflammatory cytokines
  • NADPH oxidase subunit
• Elevated levels of reactive oxygen species
• Activation of microglia

as well as with

• Elevated levels of norepinephrine in blood plasma.

These symptoms were resolved by administering a daily infusion of 40 ng of calcitriol.¹²⁹

40 ng of calcitriol is equivalent to 0.04 micrograms of vitamin D3. Given a body weight of approximately 200 g per rat, this would correspond to 0.2 micrograms per kilogram of body weight. The recommended daily dose of vitamin D3 for humans is 0.12 to 1 microgram under close medical supervision, which would correspond to a daily dose of 0.0125 micrograms per kilogram of body weight for an 80-kilogram person. The D3 dosage used in the study is therefore 16 times the upper limit of the recommended daily dose for humans. Such a dosage would pose significant health risks to humans.

High blood pressure in SHR could be a risk factor that, in turn and independently of hyperactivity, causes cognitive symptoms. High blood pressure increases the risk of age-related dementia and Alzheimer’s disease.¹³⁰¹³¹
SHRs that are four to five weeks old do not exhibit high blood pressure, but many studies are conducted on SHRs at this age, and ADHD-related symptoms persist when high blood pressure develops. It is unclear whether late-onset high blood pressure poses a problem in this model. High blood pressure does not develop until the animals are 10 to 12 weeks old.¹³
Comparative studies involving the WK/HT suggest that the ADHD-related symptoms in the SHR are not caused by high blood pressure.³⁰
The elevated blood pressure in SHR rats appears to be a consequence of a disorder of the nigrostriatal dopaminergic system. In vitro, SHR rats showed lower levels of phasic dopamine release in the caudate nucleus compared to Wistar-Kyoto (WKY) rats. In vivo, extracellular (“tonic”) dopamine and the metabolite DOPAC were also lower in the caudate nucleus of SHR than in WKY. Bilateral lesions of the pars compacta of the substantia nigra in 4-week-old SHR and WKY rats resulted in a significant attenuation of the development of hypertension in SHR, without affecting heart rate. The DOPAC/dopamine ratio and the HVA/dopamine ratio were lower in unlesioned SHR than in unlesioned WKY, indicating lower dopamine turnover in SHR. Six weeks after the lesion, dopamine concentrations in the caudate nucleus were reduced in both SHR and WKY. At this time, stimulus-evoked (phasic) dopamine release from the remaining terminals in caudate nucleus slices from SHR was significantly increased, but not in WKY. This normalization of dopaminergic activity could be the underlying factor in the attenuation of hypertension development in SHR following bilateral lesion of the pars compacta of the substantia nigra.¹⁵

1.1.16. Brain Regions Shrink¶

The SHR exhibits various reduced brain regions

• The cerebellar vermis is significantly reduced in size⁶⁴
• Caudate nucleus significantly reduced in size⁶⁴
• Putamen significantly reduced in size⁶⁴
• PFC is lower than in WKY rats¹³²
• Hippocampus smaller than in WKY rats¹³²¹³³
• increased ventricular volume at 3 months of age compared to WKY rats¹³²
• fewer neurons than in WKY rats¹³³

In ADHD as well, brain volume is reduced in the PFC and other regions.

1.1.17. Both local and long-range brain connectivity are impaired¶

Using functional ultrasound imaging, which allows for rapid measurement of cerebral blood volume (CBV), the following was found in SHR:¹³⁴

• increased response to visual stimulation in the visual cortex and superior colliculi
• functional connectivity
  • altered over long distances between geographically separate regions
  • Changes in local/regional connectivity
    • regional homogeneity
      • significantly elevated in parts of the motor and visual cortex
      • reduced in the secondary cingulate cortex, the superior colliculi, and the pretectal area

1.1.18. Changes in PFC neurons¶

PFC neurons from SHR mice exhibited fewer neurite branches, a shorter maximum neurite length, and reduced axonal growth compared to PFC neurons from WKY mice.
The adenosine antagonist caffeine restored neurite branching and extension in SHR neurons via PKA and PI3K signaling.
The A2A agonist CGS 21680 enhanced neurite branching via PKA signaling.
The selective A2A antagonist SCH 58261 restored axonal growth in SHR neurons solely via PI3K signaling (not via PKA signaling).¹³⁵

1.1.19. Nicotine receptors desensitized¶

SHR exhibited reduced nicotinic receptor activity and a lower contribution of GABA(A) receptors to nicotinic receptor activity. As a consequence, nicotinic-induced norepinephrine release was reduced.¹³⁶

Activation of the nicotinic acetylcholine receptor nAChR alpha4beta2 improved task-switching difficulties in stroke-prone SHR.²⁹

1.1.20. Monosodium glutamate influences aggression in a vagus nerve-dependent manner¶

SHR were administered monosodium glutamate (glutamate as a flavor enhancer) during the developmental phase (starting on day 25 for 5 weeks). This resulted in reduced aggressive behavior. Anxiety-related behavior remained unchanged. However, when the SHR’s vagus nerve was severed beforehand (vagotomy), monosodium glutamate did not reduce aggression, suggesting that the effect of monosodium glutamate on aggression is mediated by the gut-brain axis.¹³⁷

In wild-type mice, monosodium glutamate can trigger ADHD symptoms.¹³⁸

1.1.21. θ-Rhythms of the electroencephalogram (EEG)¶

Dexmedetomidine significantly reduced the θ/α ratio and the θ/β ratio in SHR.²¹

1.1.22. Ketogenic Diet for SHR¶

One study reported a slight improvement in ADHD symptoms in SHR following a ketogenic diet, likely due to effects on the gut-brain axis. However, the improvement was much less pronounced than that achieved with MPH.¹³⁹

1.1.23. Sphingolipid levels in the PFC are elevated; inhibitor reduces ADHD¶

Sphingolipids are essential lipids derived from the amino alcohol sphingosine.
They serve as structural components of cell membranes, particularly in nervous tissue. They stabilize lipid rafts, act as signaling molecules, and, as components of the skin barrier, strengthen its protective function. The most important classes include ceramides, sphingomyelins, and glycosphingolipids.

SHR exhibited significantly elevated levels of gangliosides and lysophospholipids in the PFC.¹⁴⁰
Intraperitoneal administration of the SPHK inhibitor SKI II (15 mg/kg, 16 days)

• specifically reduced the sphingomyelin content.
• increased the mRNA and protein expression of SPHK1 and SPHK2, in parallel with increased S1P levels
• selectively reduced Sphk2 mRNA expression (without altering protein levels)
• significantly improved hyperactivity, impulsivity, and anxiety-like behavior
• restored dopamine β-hydroxylase expression

1.2. WK/HA¶

By crossing SHR mice with WKY strains, researchers were able to breed a hyperactive and stress-sensitive, but less aggressive and non-hypertensive strain (WK/HA) and a hypertensive, but non-hyperactive strain (WK/HT). Compared to WKY mice, MK/HA mice exhibit changes in monoamine function, particularly in norepinephrine and dopamine uptake in the PFC. In addition, neuroendocrine responses in the HPA axis as well as POMC peptide levels in the anterior and posterior lobes of the pituitary gland are altered.^{141142 143} However, the WK/HA strain is rarely used in studies.

1.3. WK/HT¶

See the section on WK/HA above.

1.4. SLA16 (SHR.LEW-Anxrr16)¶

SLA16 (SHR.LEW-Anxrr16) is an inbred strain, just like the SHR, and differs from the SHR only in genetic variations on chromosome 4 (Anxrr16).¹⁴⁴
We therefore suspect that SLA 16 (like SHR) is also characterized by reduced extracellular dopamine levels.

SLA16 showed:¹⁴⁴

• higher levels of hyperactivity/impulsivity than SHR
• more severe learning and memory deficits than SHR
• a lower resting blood pressure than SHR
• not the single-nucleotide polymorphism (SNP) in the 3’UTR of the Snca gene, which is upregulated in the hippocampus only in SHR mice.
• not an increase in alpha-synuclein in the hippocampus, as seen in SHR

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

In a study, the stroke-prone spontaneously hypertensive rat (SHRSP/Ezo) showed¹⁴⁵

• a reduced D-serine/D-serine + L-serine ratio in the mPFC and hippocampus
  • D-serine binds to NMDA receptors
• D-amino acid oxidase (DAAO, an enzyme that breaks down D-serine) was elevated in the mPFC
• Serine racemase (SR, which converts L-serine to D-serine) was reduced in the hippocampus
• a microinjection of a DAAO inhibitor¹⁴⁶
  • In the mPFC, it increased the DL ratio and reduced ADHD symptoms such as inattention and hyperactivity in the Y-maze test
  • The DL ratio also increased in the hippocampus, but did not alter ADHD symptoms

The authors conclude that NMDA receptor dysfunction in the mPFC is the cause of ADHD symptoms in the SHRSP/Ezo.

Pure norepinephrine reuptake inhibitors (desipramine) and mixed dopamine/norepinephrine reuptake inhibitors (MPH) enhanced LTP in the mPFC of SHRSP/Ezo.
MPH increased dopamine levels in the mPFC more significantly in WKY/Ezo than in SHRSP/Ezo.
Pure dopamine reuptake inhibitors (GBR-12909) increased dopamine levels in the mPFC only in WKY/Ezo, but not in SHRSP/Ezo. This could be due to a preexisting functional impairment of DAT in SHRSP/Ezo, such that DAT inhibition has no further effect. However, this is contradicted by the fact that basal dopamine levels in the mPFC of SHRSP/Ezo are reduced.¹⁴⁷
Inattention was influenced by:¹⁴⁸

• based on
  • GBR-12909 (1 and 3 mg/kg, i.p., selective DAT inhibitor)
  • Desipramine (1, 3, and 10 mg/kg, i.p., NET inhibitor)
  • Milnacipran (30 mg/kg, i.p., NET inhibitor)
• unchanged
  • Fluvoxamine (10 and 30 mg/kg, i.p., SSRI)

Hyperactivity in the open field was influenced by:¹⁴⁸

• improved
  • Desipramine: improves
  • Milnacipran: improves
  • Fluvoxamine (30 mg/kg, i.p., SSRI)
• worsened
  • GBR-12909, high dose (10 mg/kg, i.p.)

Anxiety-related behavior was influenced by:¹⁴⁸

• improved
  • Fluvoxamine (3 mg/kg, i.p., SSRI)

1.6. WKY/NCrl (increased DAT = decreased extracellular DA)¶

WKY/NCrl serves as an animal model for the ADHD-I subtype (attention deficit without hyperactivity).^14915049

1.6.1. Elevated Tyrosine Hydroxylase Levels in WKY/NCrl¶

As adults, WKY/NCrl mice exhibit increased tyrosine hydroxylase gene expression.⁴⁹

1.6.2. Elevated DAT in WKY/NCrl Adults¶

WKY/NCrl, like SHR/NCrl (ADHD-C model), exhibit DAT gene expression on day P25. In the WKY/NCrl group, it was not quite as strongly elevated as in the SHR/NCrl group. A two-week treatment with MPH reduced DAT levels, although the reduction was less pronounced than in the SHR/NCrl group, particularly before puberty.⁴⁹

Based on the elevated DAT levels, we conclude that there is a decrease in extracellular dopamine.

1.6.3. Increased dopamine uptake in the nucleus accumbens¶

While WKY/NCrl (an ADHD-I model) exhibited faster dopamine uptake than controls only in the nucleus accumbens, SHR/NCrl exhibited faster dopamine uptake in both the nucleus accumbens and the ventral striatum.⁶⁹

1.6.4. Dopamine release in the striatum remains unchanged¶

Unlike SHR/NCrl, WKY/NCrl did not show reduced KCl-evoked dopamine release in the dorsal striatum compared with WKY/NHsd controls.⁶⁹

1.7. SNAP-25-KO coloboma mice (CM) (reduced dopamine, increased norepinephrine)¶

The coloboma mouse mutant (Cm) serves as an animal model for research into ADHD.¹²⁴⁸
CM mice carry a mutation in the SNAP-25 gene and are not viable in the homozygous form, but only in the heterozygous form. Heterozygous Coloboma mice have only 50% of the normal SNAP-25 protein concentration. The relationship between SNAP-25 and ADHD remains unclear. SNAP-25 is a presynaptic protein that regulates the exocytotic release of neurotransmitters (fusion of the vesicles with the cell membrane, which releases the neurotransmitter stored in the vesicles into the synaptic cleft).

Cm mice exhibit the following symptoms:

• Hyperactivity¹⁵¹¹⁵²
  • With head bobbing / head circling¹⁵³
  • Amphetamines reduce hyperactivity¹⁵² at low doses¹⁵⁴
  • Methylphenidate increased hyperactivity when administered by subcutaneous injection at doses ranging from 2 mg/kg to 32 mg/kg¹⁵²
  • Norepinephrine depletion reduced hyperactivity¹⁵⁵
• Impulsivity is questionable
  • For that¹⁵⁶¹⁵⁷
  • On the other hand⁴⁸¹⁵⁸
• Inattention is questionable
  *¹⁵⁶
  • On the other hand⁴⁸¹⁵⁸

Compared to control mice, Cm mice show:¹⁵⁹

• Changes in the HPA axis¹⁵⁹
  • No increase in CRH in the hypothalamus due to acetylcholine
  • A more pronounced increase in plasma corticosterone levels due to stress caused by restricted movement
• Changes in the dopamine system
  • Tyrosine hydroxylase:¹⁶⁰¹⁶¹
    • Unchanged in the VTA
    • Unchanged in the substantia nigra
    • Increased in the locus coeruleus
  • Reduced glutamate release due to (K+) depolarization in cortical synaptosomes
  • DRD2 expression¹⁶¹
    • Increased in the VTA
    • Increased in the substantia nigra
      -> which suggests a reduced firing rate of dopamine neurons
    • Unchanged in the striatum
  • DRD1 expression
    • Unchanged in the striatum¹⁶¹
  • Dopamine release
    • Decreased in the striatum
      • Reduced only dorsally, not ventrally¹⁵⁹
    • Reduced in the nucleus accumbens¹⁶¹
    • Reduced levels of the dopamine metabolites DOPAC and HVA in the striatum
      -> consistent with reduced dopamine release and reduced dopamine turnover¹⁶²
      -> hypofunctional dopaminergic system, similar to that seen in SHR⁴⁸
• Changes in the noradrenergic system
  • Expression of α2A-adrenoceptors in the locus coeruleus is increased¹⁶¹¹⁶⁰
  • Noradrenergic function appears to be increased
    • Elevated norepinephrine levels in the striatum and nucleus accumbens¹⁶¹
    • Withdrawal of norepinephrine via DSP-4 reduces hyperactivity but does not completely eliminate it¹⁵⁵
• Changes in the serotonergic system
  • Significantly reduced serotonin levels in the dorsal striatum, but not in the ventral striatum¹⁵⁹

1.8. SNAP-25 null mutant heterozygous (Snap25+/-) mice¶

Snap25+/− mice show:¹⁶³

• moderate hyperactivity, which disappeared in adult animals
• Impaired associative learning and memory
• frequent spikes in the EEG, indicating diffuse hyperexcitability of the network
• increased susceptibility to kainate-induced seizures, which is associated with degeneration of the hilus neurons

When Snap25+/− mice are exposed to nicotine prenatally and postnatally, they exhibit:¹⁶⁴

• increased hyperactivity (similar to coloboma mice)
• Deficits in social interactions
• Deficits in long-term depression (LTD), a measure of synaptic plasticity in corticostriatal circuits
• Changes in the affinity of D2 receptors, the key components of the signaling pathway responsible for corticostriatal LTD

1.9. Mouse/rat with a 6-OH-dopamine lesion (reduced dopamine levels)¶

6-Hydroxydopamine (6-OHDA) has a high affinity for DAT and NET.
¹⁶⁵
6-OHDA inhibits mitochondrial complex I. This leads to the generation of hydrogen peroxide, superoxide, and hydroxyl radicals, resulting in a deficiency of adenosine triphosphate (ATP).

6-OHDA mice and rats are administered 6-hydroxydopamine 5 days after birth, which destroys the dopaminergic and noradrenergic cells (all cells expressing DAT or NET).¹⁶⁶. By administering norepinephrine reuptake inhibitors (e.g., desipramine)¹⁶⁷ together with 6-OHDA, it is possible to destroy only the dopaminergic cells while preserving the noradrenergic cells.¹⁶⁶
6-OHDA-lesioned rodents, in combination with desipramine (to protect noradrenergic neurons), represent the most commonly used neurotoxin-based animal model for studying ADHD. Unless otherwise noted, the following discussion refers to mice treated with 6-OHDA alone, without a norepinephrine reuptake inhibitor.

6-OHDA-treated mice and rats are considered a model for ADHD and exhibit the following symptoms:^48168156

• Hyperactivity (in open space)¹⁶⁹
  • Decreased at first, then increased with repetitions
  • Decreasing after puberty¹⁷⁰ 
    • Motor hyperactivity on postnatal day (PT) 25, but not on PT 37 or PT 60¹⁷¹
      • DRD4 was significantly elevated at PT 25 in the caudate nucleus-putamen and reduced in the nucleus accumbens, but not at PT 37 or 60.
      • Relatively minor changes in DRD1 and DRD2 receptor binding in various forebrain regions
  • Improved by MPH^172170169
  • Enhanced by AMP¹⁷⁰¹⁶⁹
  • Not improved by selective dopamine reuptake inhibitors¹⁷³
  • Improved by selective serotonin reuptake inhibitors (citalopram, fluvoxamine)¹⁷³⁴⁸
  • Enhanced by selective norepinephrine reuptake inhibitors (desipramine, nisoxetine)¹⁷³⁴⁸
    • These also cause reduced dopamine uptake in noradrenergic presynapses, including those in
      * PFC
      * Nucleus accumbens
  • Improved by selective DRD4 antagonists⁴⁸¹⁷¹
    • D4R-KO mice show no hyperactivity and exhibit normal avoidance behavior when treated with 6-hydroxydopamine, in contrast to the lack of inhibition observed in lesioned wild-type animals¹⁷⁰
• Attention Deficit in Older Adults
  • Improved by MPH¹⁶⁹
  • Enhanced by AMP¹⁶⁹
• Impulsivity¹⁷⁰
  • In old age (Five-Choice Serial Reaction Time Task)^48168156
  • Others don’t see any impulsiveness⁷
• Anxiety-like behavior (in the elevated plus maze test)
• Antisocial behavior (in social interactions)
• Impaired cognitive function (difficulty recognizing unfamiliar objects)
• Learning Difficulties in a Spatial Discrimination Task
  • Enhanced by MPH¹⁷² and AMP
• Increased sensitivity to pain
  • Atomoxetine was found to reduce pain sensitivity¹⁷⁴
  • Pain sensitivity is thought to be mediated by α-adrenergic, β-adrenergic, and D2/D3 receptors¹⁷⁴
  • The following are signs of increased baseline nociceptive sensitivity:¹⁷⁵
    • increased licking of the hind legs in response to thermal or mechanical stimuli
    • increased sensitivity to pathological inflammatory stimuli
  • were located in lamina II of the posterior horn of the spinal cord¹⁷⁵
    • the inhibitory synaptic connections remain unchanged
    • the excitatory connections were significantly increased
      • This could promote pain sensitization

Neurophysiological changes in the 6-OHDA mouse/rat:

• Changes in the dopamine system:
  • Dopamine deficiency in the striatum and nucleus accumbens¹⁶⁹¹⁷⁶
  • Increased DRD4 expression in the caudate nucleus and putamen¹⁷⁷
    • A selective D4 antagonist reduced hyperactivity, while a D4 agonist increased it¹⁷⁷¹⁷⁸
    • Motor hyperactivity correlated positively with an increased number of D4R receptors in the striatum¹⁷⁰
  • Reducing hyperactivity by:¹⁷³
    • Selective norepinephrine reuptake inhibitors
    • Methylphenidate
    • Amphetamine
  • No increase in D2 receptor expression¹⁷⁷
  • Decreases in dopamine, as is typical in ADHD¹⁶⁸¹⁵⁶
  • Changes in cortical thickness, as is typical in ADHD¹⁶⁸¹⁵⁶
  • Abnormalities in the neurons of the anterior cingulate cortex, as is typical in ADHD¹⁶⁸¹⁵⁶
• Changes in the serotonin system
  • Serotonergic hyperinnervation as a reactive consequence of dopaminergic denervation of the striatum following a 6-OHDA lesion of the striatum¹⁷⁶
  • Serotonin transporter:¹⁷⁹
    • Increased binding in the striatum
    • Binding remains unchanged in the PFC

1.10. Tal1c knockout mice (reduced dopamine)¶

Most GABAergic neurons in the dopaminergic nuclei of the midbrain depend on the Tal1 transcription factor for their development. Here, Tal1 acts as a cell fate selector gene that promotes GABAergic differentiation at the expense of alternative glutamatergic neuronal identities. The brainstem nuclei that harbor Tal1-dependent neurons are involved in the control of dopamine neurons and in the regulation of movement, motivated behavior, and learning.
Mice carrying the En1Cre16 and Tal1flox11 alleles were crossed to generate En1Cre/+; Tal1flox/flox (Tal1cko) mice. In Tal1c-KO mice, the En1Cre allele drives tissue-specific recombination in both the midbrain and rhombomere 1; however, this leads to a failure of GABAergic neurogenesis in the brainstem only in embryonic rhombomere 1.
Tal1c-KO mice showed:¹⁸⁰

• Hyperactivity
• Increased motor impulsivity
• Altered response to rewards
• Delay discounting (delay aversion)
• Learning disabilities
• The paradoxical calming response—typical of ADHD—to pharmacologically stimulated dopamine release caused by amphetamine and atomoxetine
• Developmental changes in the anterior GABAergic and glutamatergic neurons of the brainstem.
  These are involved in
  • Regulation of dopaminergic pathways
  • Basal Ganglia Output
• Lower body temperature
• A smaller rise in body temperature during stress
• Less nest-building
• Less grooming (brooding/nurturing behavior)
• Reduced levels of dopamine and dopamine metabolites in
  • Nucleus accumbens (most clearly)
  • Dorsal striatum
  • PFC
• Unchanged number of dopaminergic cells in
  • Substantia nigra
  • Ventral tegmentum
• Unchanged serotonin and 5-HIAA levels in
  • Dorsal striatum
  • Nucleus accumbens
  • PFC
• Unchanged norepinephrine levels in
  • PFC
• Unchanged social behavior

1.11. THRSP-OE mice (elevated DAT = reduced extracellular DA)¶

The thyroid hormone-responsive protein gene is also known as the Spot14 gene.

A line of mice with overexpression of the THRSP gene in the striatum (THRSP-OE) showed:¹⁴⁹

• Inattention when recognizing novel objects and during the Y-maze test, however
• no hyperactivity in the field test
• No impulsivity in the task of avoiding cliffs and limiting delays
• increased expression of dopamine genes (genes for dopamine transporters, tyrosine hydroxylase, and dopamine D1 and D2 receptors) in the striatum
• Methylphenidate (5 mg/kg) improved attention and normalized the expression of dopamine-related genes in THRSP-OE mice

In young adult THRSP-OE mice, the following was also found¹⁸¹

• increased SNAP25 expression in the striatum
• reduced dopamine D1 receptor binding concentrations
• markedly low dopamine levels
• Repeated MPH injections over 7 days
  • improved low dopamine levels
  • reduced the EEG theta/beta ratio

The THRSP-OE mice could therefore serve as an animal model for ADHD-I.¹⁸²¹⁸¹

THRSP-OE mice exhibiting ADHD-I-like traits were found to have an altered protein network involved in Wnt signaling. Compared to THRSP knockout (KO) mice, THRSP-OE mice exhibited:¹⁸³

• Attention problems
• Memory problems
• dysregulated Wnt signaling, which impaired cell proliferation in the dentate gyrus of the hippocampus and the expression of markers for neural stem cell (NSC) activity.
• An enriched environment combined with treadmill training improved
  • Behavioral deficits
  • Wnt signaling
  • NSV Activity

SHR/NCrl rats (ADHD-HI, hypertension) and Wistar-Kyoto rats (WKY/NCrl) (inattention) also show increased expression of the THRSP gene.¹⁴⁹

The THRS gene is involved in the regulation of lipogenesis, particularly in the lactating mammary gland. It plays an important role in the biosynthesis of triglycerides with medium-chain fatty acids.

1.12. TARP-γ-8 knockout mice / CACNG8 knockout mice (elevated DAT = reduced extracellular DA)¶

Names:
TARP γ-8: Transmembrane α-Amino-3-Hydroxy-5-Methyl-4-Isoxazolpropionic Acid (AMPA) Receptor Regulatory Protein γ-8
CACNG8: Calcium Voltage-Gated Channel Auxiliary Subunit Gamma 8

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

Young TARP γ-8 knockout (KO) mice exhibited:¹⁸⁴

• ADHD-like behaviors:
  • Hyperactivity
  • Impulsivity
  • Anxiety
  • impaired cognition
  • Memory deficits
• a dysfunction of the AMPA glutamate receptor complex in the hippocampus
• dysregulation of dopaminergic and glutamatergic transmission in the PFC
• MPH improved significantly
  • the most significant behavioral deficits
  • the abnormal synaptosomal proteins, particularly in the PFC
    • a reversal of the upregulation of Grik2
    • a reversal of the upregulation of DAT (Slc6a3)
  • the function of the synaptic AMPAR complex through the upregulation of other AMPAR accessory proteins in hippocampal synaptosomes
• Aniracetam also improved ADHD symptoms¹⁸⁵

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

Given the upregulation of DAT in TARP γ-8 knockout mice, we hypothesize that these mice have reduced extracellular dopamine levels in the PFC.

1.13. D2 autoreceptor knockout mouse (autoDRD2KO mice) (extracellular DA unchanged, phasically elevated)¶

D2 receptors act as heteroreceptors postsynaptically on non-dopamine neurons and as autoreceptors presynaptically at the terminals of dopamine neurons.
In D2 autoreceptor knockout mice (autoDrd2KO mice), only the D2 autoreceptor located on dopamine neurons is inactivated, while the postsynaptic heteroreceptor remains unaffected.
autoDRD2KO mice show:

• Hyperactivity in an open field and when exposed to cocaine, but not in a familiar environment¹⁸⁶
• increased sensitivity to cocaine¹⁸⁷¹⁸⁸
• increased dopamine synthesis and release¹⁸⁷
• increased motivation for food rewards¹⁸⁷

Studies in autoDRD2KO mice showed that not only D2 autoreceptors but also D2 heteroreceptors are involved in dopamine regulation. This D2 heteroreceptor-mediated mechanism is more efficient in the dorsal striatum than in the nucleus accumbens. D2R signaling thus appears to regulate mesolimbic and nigrostriatal functions differently.¹⁸⁶

D2-null mice (D2-/-) exhibited¹⁸⁹

• normal extracellular DA levels
• reduced DA uptake
• uninhibited DA release¹⁹⁰
• Unaltered inhibition of dopamine release through activation of GABA-B receptors¹⁹¹

1.14. FOXP2HUM mice (reduced dopamine)¶

A substitution of two amino acids (T303N, N325S) in the FOXP2 transcription factor in mice resulted in the following:¹⁹²

• reduced dopamine levels in
  • Nucleus accumbens
  • Frontal cortex
  • Cerebellum
  • Putamen and caudate nucleus
  • Globus pallidus
• Glutamate, GABA, and serotonin remain unchanged
• increased dendrite length and increased synaptic plasticity of medium spiny neurons (MSNs) in the striatum
• ultrasonic vocalizations that differ in quality
• reduced exploratory behavior
• increased caution / anxiety (stayed closer to the wall of the test area)
• viable and capable of reproduction
  • unlike FOXP2-KO mice

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

1.15. Mice Fed Monosodium glutamate (Reduced Dopamine)¶

Mice that were fed monosodium glutamate for 8 weeks showed¹³⁸

• Changes in glutamate levels in the brain
• reduced dopamine levels in the brain
• impaired attention
• a marked increase in spontaneous movements
• impaired spatial memory
• increased anxious behavior
• increased compulsive behavior

The flavonoid hesperetin improved ADHD-induced behavioral changes:¹³⁸

• Spontaneous locomotor activity has improved to some extent
• Partial improvement in spatial learning and memory
• partial correction of locomotor hyperkinesia
• Reduces the number of rears, the number of squares crossed, and the activity index in a dose-dependent manner
• Improves repetitive, compulsive behavior
• Improved attention
• improves cognitive function
• Reduced anxiety-related behavior
• The number of correct responses in the Y-maze test increased in a dose-dependent manner

1.16. FRM1 knockout mouse¶

We do not have information on striatal dopamine in FRM1-KO animals ; however, dopamine activity in the PFC is reduced due to the absence of D1R.

FRM1-KO animals serve as animal models for Fragile X syndrome (FXS).
FXS is caused by a CGG triplet expansion in the FMR1 gene, which leads to a deficiency of the encoded Fragile X Mental Retardation Protein (FMRP). This mRNA-binding protein regulates the localization and translation of specific mRNAs in synaptic regions and controls the activity of synaptic membrane proteins independently of translation. FXS causes an excess of long, thin, and “twisted” dendritic spines in the cerebral cortex.¹⁹³
The FMR1 knockout mouse shows

• Abnormalities in the maturation of dendritic spines
• reduced KCNMA1 protein expression¹⁹⁴
  • KCNMA1 is required for associative learning
  • KCNMA1 knockout mice exhibit altered responses to reward outcomes from trial to trial during probabilistic learning tasks¹⁹⁵
• many behavioral characteristics similar to those seen in human FXS:¹⁹⁴¹⁹³
  • changes in social interaction
  • repetitive behaviors
  • Hyperactivity
  • cognitive dysfunction
  • intellectual disability
  • compulsive behavior
  • Seizures
  • Autism
• significant difficulties in learning the TPSD task¹⁹⁶
  • reduced distinctiveness indices
  • atypical leak patterns
  • equivalent performance on purely visual stimuli compared to the multimodal task
  • TPSD: sensory discrimination task (Temporal Pattern Sensory Discrimination Task) for awake mice
    • Stimuli consist of paired audiovisual stimuli that differ in duration

Due to reduced D1R levels in the PFC, FMR1-KO mice lack the long-lasting potentiation of evoked inhibitory postsynaptic currents (IPSCs) via D1R that is normally mediated by dopamine.¹⁹⁴

Without FMRP, signal transmission via group 1 metabotropic glutamate receptors (mGluR 1/5) is disrupted. This dysfunction could be the cause of the excessive synaptic long-term depression observed in FMR1-KO mice or could underlie the insensitivity to mGluR 1/5-mediated stimulation of neuronal/synaptic protein synthesis.¹⁹³

The selective BKCa channel opener BMS-204352 restored dendritic spine function in vitro. A single injection of BMS-204352 (2 mg/kg) restored glutamate homeostasis in the hippocampus in vivo and corrected behavioral abnormalities in social recognition and interaction, non-social anxiety, and spatial memory.¹⁹³

1.17. NCX3+/- mice¶

A deficiency in NCX3 appears to impair dopaminergic neurotransmission in the PFC and trigger ADHD symptoms.
NCX3 is abundantly expressed in dopaminergic VTA neurons. NCX3 knockdown in N27 cells (a dopaminergic neuron cell line frequently used as an in vitro model in Parkinson’s disease research) induces a direct physical interaction between calcium/calmodulin-dependent protein kinase II alpha and the dopamine transporter, which leads to an excess of extracellular dopamine due to a disorder in dopamine clearance in the PFC.
NCX3 heterozygous (NCX3+/-) mice exhibited:¹⁹⁷

• Hyperactivity
• cognitive deficits
• social dysfunction

Methylphenidate reduced the deficits.¹⁹⁷

NCX3+/- mice also showed:¹⁹⁷

• a sustained increase in basal dopamine levels
• reduced phasic extracellular dopamine levels in the PFC in response to social stimuli
• elevated extracellular dopamine levels in the PFC
• Activation of dopamine D1 receptor signaling pathways in the PFC

1.18. Pitx3 homozygous null (aphakia) mouse¶

In the Pitx3 homozygous null (aphakia) mouse, the Pitx3 gene is nonfunctional. Due to their motor and non-motor symptoms, such as depression-like behaviors, these mice serve as a Parkinson’s disease model.
Pitx3 homozygous null (aphakia) mice exhibit:¹⁹⁸

• severe developmental disorders
• Absence of the eye’s lens (aphakia)¹⁹⁹
• postnatal loss of dopamine neurons in the substantia nigra pars compacta of the midbrain²⁰⁰
• reduced dopamine levels in the dorsal striatum
• reduced dopamine signaling
• motor deficits
  • Deficits in motor coordination
  • Movement disorders
  • reduced overall activity²⁰⁰
  • Increased spontaneous activity in the cage during their usual sleep period
  • Fewer transitions, but greater emphasis on certain movement patterns, such as rearing up and horizontal movements
• Learning and memory disorders
• Depression-like behaviors
• increased susceptibility to stress
• Signs of anhedonia (loss of pleasure)
• Impaired serotonin function in the striatum as a consequence of Pitx3 deficiency¹⁶⁶

1.19. NHL Rats¶

Juvenile rats with neonatal habenula lesions (NHL) exhibit some ADHD symptoms. A neonatal habenula lesion (NHL) induced by microinjection of ibotenic acid into the habenula on postnatal day 7 (PND 7) resulted in the following during the juvenile phase (PND 28–35):²⁰¹

• Hyperlocomotion
• Impulsivity
• Attention deficits
• Changes in tissue concentrations of DA and 5-HT only in some mesocorticolimbic regions
• Improvement of ADHD-typical behavioral changes and normalization of the NHL-induced DA-to-5HT imbalance in several brain regions through simultaneous pharmacological manipulation of the DA and 5-HT systems using Astragalus membranaceus and its active ingredient, formononetin

1.20. Prenatal Nicotine Exposure in Mice¶

Presumably reduced extracellular dopamine.²⁰²

Mice exposed to nicotine prenatally show increased ADHD symptoms²⁰³ such as

• Hyperactivity^{204205206207208209210211}
  • in both males and females²⁰²
  • even if raised by other mothers²¹²
  • epigenetically heritable²¹⁰
• Impulsivity²¹³
• Attention problems²¹³²¹⁴
• Aggressiveness²⁰⁸
• Impaired working memory²¹⁴
• Pre-pulse inhibition is impaired²¹⁴
• Learning difficulties²⁰⁹
• Enforcement issues²¹⁵

Methylphenidate improves behavior and neuroplasticity in these mice²¹⁶ when administered orally, but not when administered intraperitoneally²⁰².
Among other factors, this is attributed to the composition of AMPA receptor subunits and the morphology of synaptic spines in the hippocampus in ADHD.²¹⁷

From a neurophysiological perspective, mice exposed to nicotine prenatally show

• reduced dopamine levels in the mPFC²¹⁸
• reduced dopamine turnover in the PFC²⁰²
  • Dopamine turnover increased as a result of MPH
  • while basal dopamine levels were unchanged and remained unchanged by MPH
  • Dopamine levels in the tissue of the frontal cortex were elevated, although it remained unclear whether this represented an increase in vesicular dopamine (intracellular stores) and/or extracellular (i.e., synaptically released) dopamine
  • a significantly reduced DOPAC-to-dopamine ratio in the frontal cortex. Since only extracellular dopamine is broken down into DOPAC, the reduced DOPAC-to-dopamine ratio could indicate a decrease in synaptic dopamine, i.e., a hypodopaminergic state, similar to that seen in ADHD.
• Reduced norepinephrine turnover in the PFC in adulthood²¹⁴.
• increased neuroinflammation²¹⁹
• elevated levels of reactive oxygen species (ROS)²¹⁹
• Impaired blood-brain barrier²²⁰

1.21. Neurokinin-1 (Substance P, tachykinin) receptor knockout mouse (NK1R-KO mouse)¶

The neurokinin-1 receptor is called NK1R in mice and TACR1 in humans.²²¹

The NK1R-KO mouse is another rodent model for ADHD.²²²

NK1R-KO mice (^{NK1R-/– mice}) show:²²³¹⁵⁸

• Hyperactivity
  • likely a direct consequence of the absence of NK1R
  • AMP and MPH eliminate these²²⁴
• Perseveration
  • likely a direct consequence of the absence of NK1R
• Inattention
  • possibly a direct consequence of the absence of NK1R
  • no inattention¹⁵⁸
• Impulsivity
  • was due to an interaction between NK1R dysfunction and other (possibly environmental and/or epigenetic) factors

From a neurophysiological perspective, the following was observed in NK1R-KO mice:²²⁴

• increased norepinephrine release in the PFC
• a deficit in dopaminergic transmission in the PFC (“hypofrontality”)
• a lack of increase in dopamine efflux in the dorsal striatum following systemic administration of d-amphetamine
• the absence of a d-amphetamine- or morphine-induced conditioned place preference.

NK1R antagonists cause hyperactivity in wild-type mice. AMP eliminates the hyperactivity²²⁴
Virtually all dopaminergic neurons in the substantia nigra pars compacta and many in the substantia nigra pars reticulata express neurokinin-1 receptors.²²⁵ Substance P exerts its effects via postsynaptic neurokinin-1 heteroreceptors as well as via presynaptic neurokinin-1 autoreceptors. Substance P has an excitatory effect and modulates the inhibitory action of GABA in the substantia nigra.
Substance P is involved in the regulation of dopamine release in the striatum.²²⁶²²⁷ The effect of substance P on dopaminergic transmission appears to be mediated by a nigro-thalamo-cortico-striatal loop.²²⁸
Substance P increases dopamine levels in the nucleus accumbens, but not in the neostriatum.²²⁹
The NK1R is associated with dopaminergic activity in the striatum.²³⁰ NK1Rs are highly expressed on (cholinergic) interneurons in the striatum, where they mediate the release of acetylcholine and dopamine.²²³

In our view, these data suggest reduced dopamine levels in the striatum of NK1R-KO mice.

NK1R mediates the effect of the protein kinase C pathway in downregulating DAT and NET. The effect appears to be mediated primarily through NET rather than through DAT.²³¹

^{NK1R+/+ mice} treated with an anxiolytic or antidepressant drug exhibit behavior similar to that of NK1R-KO mice.²³²²³³ Antidepressants increase monoamine transmission. Anesthetized ^{NK1R-/– mice exhibited} a 2- to 4-fold increase in extracellular norepinephrine concentration compared to ^{NK1R+/+ mice}. Systemic administration of the α2-adrenoceptor antagonist 2-(2,3-dihydro-2-methoxy-1,4-benzodioxan-2-yl)-4,5-dihydro-1H-imidazoline (RX 821002) increased the extracellular norepinephrine concentration exclusively in ^{NK1R+/+ mice}, both in anesthetized and free-roaming animals.
This suggests that in NK1R-KO mice, the function of α2a autoreceptors, which modulate noradrenergic transmission, is impaired.
This is consistent with the finding that in NK1R-KO mice, [^35S]GTPγS binding to activated α2a-adrenoceptors in the locus coeruleus was 70% lower in NK1R-KO mice compared to ^{NK1R+/+ mice}, while it remained unchanged in the PFC. Administration of RX 821002, followed by the norepinephrine reuptake inhibitor desipramine into the frontal cortex of anesthetized mice, increased extracellular norepinephrine in both NK1R-KO and ^{NK1R+/+ mice}.
^NK1R-/– and ^NK1R+/+ mice exhibited the same amount of NET.
The increased basal norepinephrine efflux in NK1R-/- mice could be explained by enhanced transmitter release combined with desensitization of the somatodendritic α2a-adrenoceptors.²³²²³³

Animal models with reduced striatal extracellular DA but without typical ADHD symptoms¶

1.21. NET-KO mice (elevated norepinephrine; elevated dopamine in the PFC; reduced dopamine in the striatum)¶

Since NET reabsorbs slightly more dopamine than norepinephrine in the PFC and thus represents one of the most important dopamine-clearing mechanisms, we suspect that dopamine levels in the PFC are not reduced but rather increased. In the striatum, however, dopamine is broken down less by NET and more by DAT.

Mice with a genetically inactivated norepinephrine transporter (NET-KO mice) showed

• Norepinephrine levels increased by 55 to 75% in the tissue of the PFC, hippocampus, and cerebellum.
• Dopamine levels in the striatum were reduced by about 20% in the tissue, and extracellular dopamine levels and dopamine metabolites were reduced by 50%.²³⁴

NET-KO mice showed

• reduced anxiety-related behavior
• reduced depressive symptoms
• increased sensitivity to stimulants as an indication of increased vulnerability to addiction
• a greater increase in motor activity caused by D2/D3 agonists, but not by D1 agonists

NET-KO mice showed:²³⁵

• WT mice exhibited bone loss following NET blockade with reboxetine
• NET-KO mice exhibited reduced bone formation and increased bone resorption, resulting in suboptimal peak bone mass and suboptimal mechanical properties, which were associated with low sympathetic outflow and high plasma NE levels.
• Differentiated osteoblasts express NET (similar to neurons), reabsorb norepinephrine via NET, but cannot produce norepinephrine.
• Daily activation of the sympathetic nervous system through mild chronic stress did not cause bone loss, provided that NET activity was not blocked.
  This raises the question of whether ADHD medications that inhibit norepinephrine reuptake could have harmful effects on bone formation.