ADHD in Animal Models - Introduction

Animal models used to study clinical conditions are evaluated based on three key validity criteria:¹

• Observational validity (face validity)
  • Similarity of behaviors or symptoms to the core symptoms of the Disorder in humans
• Construct validity
  • Consistency with a theoretically sound pathophysiological basis
• Predictive validity
  • The transferability of results from animal models to humans for predicting the therapeutic efficacy of new forms of treatment

Rodents are very popular as animal models because they are very similar to humans in their physiology, are easy to care for, and have very rapid development cycles and metabolisms. One day in the life of a rat is equivalent to approximately 27 human days.² However, due to their accelerated metabolism, the required dose of stimulants is also higher, meaning that rats require significantly higher doses of an active ingredient than humans, a factor that must be taken into account when evaluating drug studies.

1. Model Animals¶

There are various rat strains that serve as animal models representing ADHD-HI, ADHD-C, ADHD-I, and unaffected individuals. These models are used to investigate which neurophysiological changes are associated with which symptoms. Animal models are generated in various ways:

• Breeding or selection for specific traits (e.g., Spontaneously Hypertensive Rat, SHR)
• Manipulation of a single gene, usually a knockout (KO) (e.g., dopamine transporter knockout, DAT-KO)
• Treatment of the parents before conception or pregnancy (nicotine, ethanol)
• Exposure to chemical substances, stressors, or pathogens during pregnancy or early childhood (e.g., hypoxia; 6-hydroxydopamine, 6-OHDA).
• surgical procedures (e.g., lesions of the right frontal cortex, transection of the corpus callosum)
• Exposure to social factors (e.g., isolation in early childhood)

1.1. How Knockout Animals Are Created¶

Adapted from Gainetdinov & Caron, 2001³

Normally, to breed animals with a specific gene knockout, a mutation is first induced in the relevant gene in the laboratory, which (as a rule) causes the gene product to become inactive. This inactivated gene is usually introduced into the animal by microinjecting the mutated gene into embryonic stem cells (ES cells). ES cells can be cultured and manipulated quite easily in Petri dishes in the laboratory. During homologous recombination, the mutated gene injected into the ES cells can integrate into the chromosomal region containing the normal copy. Homologous recombination is the natural process that occurs when both copies of a chromosome align side by side in germ cells and exchange genetic material between the paternal and maternal chromosomes.
Following the process of homologous recombination, the chromosomal region can be exchanged.

ES cells in which this rare chromosomal rearrangement has occurred can be selected so that they grow preferentially in the culture dish. To do this, a second gene is added to the injected DNA; this gene encodes a protein that makes the cells resistant to certain antibiotics. While the unmodified ES cells are killed by the antibiotic neomycin, only the cultured lines with the rare recombination survive and are able to grow. As a consequence, DNA is extracted from some of these ES cells for verification to confirm the presence of the mutated gene. These verified cells are then implanted into the uterus of animals (usually mice or rats). The first generation of offspring now carries one normal and one inactive copy of the manipulated gene. If these animals are bred with one another, according to the general rules of genetics, about one-quarter of the next generation will carry two copies of the inactivated gene. These are selected and subsequently serve as a research strain.

1.2. Animal Models for ADHD¶

Both the SHR and the DAT-KO represent a form of ADHD-HI, while Wistar-Kyoto rats (WKY) typically serve as controls. In addition, there is the SHR/NCrl strain, which exhibits symptoms of ADHD-C, and the WKY/NCrl strain, which exhibits symptoms of ADHD-I (attention deficit without hyperactivity).^{45 6 7} Unless studies specify otherwise, it is generally assumed that Wistar-Kyoto (WKY) rats refer to the non-affected model.
The fact that these animal models exhibit their symptoms solely on the basis of their genetic makeup and without the influence of early childhood stress⁸ shows that certain gene variants alone can represent a distinct pathway to the development of mental disorders such as ADHD, and that the two pathways—genes alone and genes plus environment—coexist.
Interestingly, SHRs already have a dysfunctional HPA axis (stress axis) simply because of their genetic predisposition.

In rodents, the predominant glucocorticoid is corticosterone, rather than cortisol, which is predominant in humans. Otherwise, the stress systems of rodents and hominids are surprisingly similar, despite the fact that their evolutionary lineages diverged some 90 million years ago.
ADHD is also being discussed in dogs.⁹¹⁰ An ADHD rating scale for dogs yielded reliable results.¹¹ Another scale for measuring ADHD in dogs is currently under development.¹²

2. Drug Dosage in Animal Models¶

Drug doses cannot be adjusted linearly based on an animal’s weight. Dosage conversion is usually based on body surface area or other pharmacokinetic scaling methods.¹³¹⁴
Pharmacokinetics in rodents are significantly faster than in humans.

2.1. Human Equivalent Dose (HED)¶

Human Equivalent Dose (HED): Conversion Based on Body Surface Area¹⁵

The standard method is the Human Equivalent Dose (HED), based on body surface area:

HED (mg/kg) = Animal dose (mg/kg) × (𝐾𝑚 (animal) / 𝐾𝑚 (human))

Km values according to FDA guidelines (based on body surface area):

• Mouse: 3
• Rat: 6
• Adult (70 kg): 37

Sample Calculation

• Mouse:
  • 1 mg/kg of MPH corresponds to the following for a 70-kg person: HED = 1 × (3 / 37) = 0.081 mg/kg = 5.7 mg
  • 10 mg/kg of MPH corresponds to the following for a 70-kg person: HED = 10 × (3 / 37) = 0.81 mg/kg = 57 mg
  • 15 mg/kg MPH corresponds to the following for a 70-kg person: HED = 15 × (3 / 37) = 1.21 mg/kg = 85.5 mg
• Rat
  • 1 mg/kg MPH; for a 70-kg person, this corresponds to: HED = 1 × (6 / 37) = 0.16 mg/kg = 11.4 mg
  • 5 mg/kg MPH; for a 70-kg person, this corresponds to: HED = 5 × (6 / 37) = 0.81 mg/kg = 57 mg
  • 10 mg/kg MPH; for a 70-kg person, this corresponds to: HED = 10 × (6 / 37) = 1.62 mg/kg = 114 mg

Only doses administered by the same route are considered equivalent. An injection is metabolized more quickly than an oral dose. Therefore, 15 mg/kg of MPH administered orally to a mouse corresponds to a very high drug dose, whereas 15 mg/kg of MPH administered subcutaneously corresponds to a therapeutic dose.

2.2. Typical oral doses of methylphenidate in animal studies¶

These doses are commonly used in animal studies to investigate therapeutic or subtoxic effects.

• Mice (oral, commonly used in the literature):
  • Low: 1–2 mg/kg
  • Average: 5–10 mg/kg
  • High: 20–30 mg/kg
• Rats (oral):
  • Low: 0.5–2 mg/kg
  • Average: 2–10 mg/kg
  • High: 10–20 mg/kg

Typical therapeutic doses in humans range from 0.3–1 mg/kg orally (e.g., 18–72 mg per day for ADHD treatment). It follows that animal doses of 5–10 mg/kg in mice or 2–10 mg/kg in rats fall within the range of human equivalence when the HED conversion is applied.

3. Animal models allow for the analysis of the causes of various symptoms¶

The various animal models clearly demonstrate that symptoms such as hyperactivity, impulsivity, or attention problems can each be triggered by very different causes. A distinction must be made between the causes (e.g., a specific genetic defect) and the manifestation of symptoms. For example, very different causes (e.g., genetic defects) can result in a dopamine deficiency, while others can lead to a dopamine excess; both, in turn, produce nearly identical symptoms due to the deviation from optimal dopamine levels (inverted-U curve).¹⁶ To illustrate this, we have categorized the animal models, to the best of our knowledge, into those with a dopamine deficiency and those with a dopamine excess. Although dopamine is the most important factor in ADHD, the other influences also play a role.
As far as we understand, whether an extracellular hypo- or hyperdopaminergic state prevails is of little relevance—both are associated with ADHD symptoms. Extracellular dopamine levels are too low when DAT is overactive, and the dopamine released in bursts is reabsorbed by the overactive DAT before it can transmit its signal to the receptors. With underactive DATs, extracellular dopamine levels are too high, causing “noise” that interferes with signal transmission. Above all, however, there is a lack of reabsorbed dopamine to replenish the vesicles for phasic dopamine release.
Both extracellular hyperdopaminergic and hypodopaminergic states impair signaling via phasic dopamine.

The most commonly used criteria for model validity include face validity, construct validity, and predictive validity.
Face validity: The reliability with which the model reproduces certain state characteristics (e.g., the behavioral symptoms of a psychiatric disorder).
Construct validity: The reliability with which the model measures what it is intended to measure (the etiology and pathogenesis of a Disorder)
Predictive validity: The reliability with which the model predicts the relationship between a causative factor and an observable effect of the disease, on the one hand, and the observable effects of a therapeutic agent on the disease, on the other.

Interestingly, mouse models in which dopamine receptors have been deactivated show barely any ADHD symptoms, further underscoring the importance of DAT and its influence on extracellular and phasic dopamine.

4. ADxS: Animal Models Based on Extracellular Dopamine Levels¶

On the following pages, we have categorized the animal models based on their extracellular dopamine levels:

1. ADHD animal models with reduced extracellular dopamine
2. ADHD animal models with elevated extracellular dopamine
3. ADHD animal models with dopamine changes that are unknown to us
4. Animal models that do not adequately represent ADHD

Cabana-Domínguez et al. have compiled a list of nearly 200 mouse models in which the manipulation of individual genes triggered hyperactivity, hyper- and hypoactivity, impulsivity, or inattention.¹⁷ For more on this, see Monogenic Animal Models for ADHD Symptoms in the article Monogenic Causes of ADHD