MPH Part 2 - Mechanism of Action

• (E 2b): Increase in dopamine and norepinephrine in the PFC (textbook, E 4)³ * (animal study, rats, E 2b)⁴ * (animal study, rats, E 2b)⁵
  ** (animal study, rats, full text not available to us, E 2b) (animal study, adolescent rats, 4 weeks, E 2b)⁶ **⁷
  • 1.0 mg/kg in rats
    • increase extracellular norepinephrine in the hippocampus by 100%
    • do not significantly alter dopamine levels in the nucleus accumbens (striatum)
  • 2.5 mg/kg in rats
    • increase extracellular norepinephrine in the hippocampus by 150–175%
    • do not significantly alter dopamine levels in the nucleus accumbens (striatum).
• In healthy adult rats, MPH increases
  • Dopamine in the PFC, striatum, and nucleus accumbens (peak values of 253%, 209%, and 267% of baseline at 3 mg/kg i.p.) and (animal study, rats, author(s) employed by the manufacturer, E 2b.)⁸
    • Highly concentrated ex vivo (100 µM): dopamine efflux in the PFC, partly from noradrenergic terminals (animal study, N = 27, rats, E 2b.)⁹
    • ex vivo at low concentration (10 µM): no dopamine efflux (animal study, N = 27, rats, E 2b.)⁹
    • In mice, chronic administration of 3 mg/kg (21 days) of extracellular dopamine and norepinephrine in the PFC increased (animal study, mice, 21 days, E 2b.)¹⁰
  • Norepinephrine in the PFC (peak value 199% of baseline at 3 mg/kg i.p.; not measured in the striatum or nucleus accumbens) (animal study, rats; author(s) employed by the manufacturer; E 2b.)⁸
  • Dopamine and norepinephrine in the PFC, but barely in other brain regions (animal study, rats, E 2b)¹¹
    • even at low doses of 0.25 mg i.p. per kg
• In healthy monkeys, MPH caused: (animal study, rhesus monkeys, E 2b)¹²
  • Striatum: low and high doses: consistent increase in dopamine
  • PFC: Dopamine levels rise only at high doses
• In mice, a single dose of 3 mg/kg MPH had the same effect as chronic administration (21 days)
  • Striatum: no change in extracellular levels of norepinephrine, dopamine, or serotonin (animal study, mice, 21 days, E 2b.)¹⁰

Stimulants: (animal study, rats, E 2b.)¹⁴

• increased the firing rate of postsynaptic neurons in the globus pallidus and decreased the firing rate of dopaminergic neurons in the substantia nigra pars compacta, without a preferential effect on presynaptic autoreceptors
• shortened the duration of postsynaptic multisecond oscillations in the globus pallidus from about 30 seconds to 5 to 10 seconds. Even 1.0 mg/kg of MPH or 0.2 mg/kg of D-Amp was effective in this regard.
• However, 0.2 mg/kg of D-Amp showed only a slight presynaptic effect. D-Amp had a stronger postsynaptic effect.

The significant reduction in phasic (synaptic) dopamine release is likely due to a decrease in synapsin phosphorylation (animal study, mice, E 2b)¹⁵
The inhibition of phasic dopamine is not a consequence of presynaptic D2 autoreceptor activation, since this (animal study, mice, E 2b)¹⁵

• a. even in the presence of the D2 antagonist sulpiride, and
• b. also occurs in DAT-CI striatal slices, in which increased extracellular DA levels cannot activate D2 autoreceptors
  The inhibition of phasic dopamine could—as with cocaine—possibly be due to a reduction in synaptic vesicular neurotransmitter. (Animal study, mice, E 2b)¹⁵ As a lipophilic weak base, cocaine could cause the vesicular pH gradient to collapse, similar to the “weak base effects” reported for amphetamines, or it could act on the vesicles. There is evidence that synaptic vesicles may be quite “leaky” and constantly lose dopamine into the cytosol. This impression is supported by the effect of reserpine, which can rapidly deplete synaptic vesicles of dopamine.

Stimulants: (Animal study, rats, E 2b.)¹⁴

• increased the firing rate of (non-dopaminergic) neurons in the globus pallidus in a dose-dependent manner
  • Maximum MPH at 3.2 mg/kg
  • Maximum D-AMP at a cumulative dose of 0.8 mg/kg
• reduced the firing rate of dopaminergic neurons in the substantia nigra pars compacta
  • MPH by up to 96%
  • D-AMP by up to 99%

(E 2b): MPH inhibits the firing rate of dopaminergic midbrain neurons and, at the same time, reveals a non-dopaminergic excitation mediated by alpha-1 receptors, which becomes apparent after administration of a D2 antagonist. Electrophysiological animal studies found:

• Inhibition of the firing rate in VTA dopamine neurons, which was reversed by the D2 antagonist raclopride and shifted to alpha-1-mediated (prazosin-sensitive) burst activity that was elevated above baseline. At the same time, MPH prolonged the PFC UP state and shifted the functional coupling between the PFC and VTA from negative to positive (D2-mediated). (Animal study, rats, dual-channel recording PFC/VTA, E 2b.)³⁷
• in dopamine neurons, a DA-mediated inhibition of firing rate accompanied by non-DA-mediated excitation with bursting and a 0.5- to 1.5-Hz oscillation, which required alpha-1 receptors and intact forebrain afferents. (Animal study, rats, single-cell recording from the VTA, E 2b.)³⁸
• in VTA dopamine neurons (2 mg/kg i.v.), reduced firing and burst activity, which was reversed by the D2 antagonist eticloprid. Combined, the activity was above baseline and depended on alpha-1 and D1 receptors as well as NMDA transmission. Chronic MPH administration during adolescence attenuated this effect in adulthood. (Animal study, rats, in vivo single-cell recording, full text not available to us, E 2b.)³⁹
• inhibition of spontaneous firing in midbrain dopamine neurons accompanied by hyperpolarization, which was reversed by sulpiride (D2) and decreased following dopamine depletion. The inhibition was thus due to newly synthesized dopamine that had accumulated extracellularly as a result of reuptake inhibition. (Animal study, rats, midbrain sections, E 2b.)⁴⁰
• a biphasic, dose-dependent effect in the neostriatum. 10 mg/kg MPH decreased the firing rate, while 20 to 25 mg/kg increased it; meanwhile, the substantia nigra pars compacta remained consistently inhibited. All effects were reversible with haloperidol. (Animal study, rats, single-cell recording, E 2b.)⁴¹

(E 2b): The DAT blockade caused by MPH is dose-dependent. PET and SPECT studies found:

• in humans (120 minutes after oral administration), a blockade of 12% (SD 4) at 5 mg, 40% (SD 12) at 10 mg, 54% (SD 5) at 20 mg, 72% (SD 3) at 40 mg, and 74% (SD 2) at 60 mg, with a half-maximal blockade at 0.25 mg/kg. (PET, [11C]cocaine, n = 7, healthy subjects, E 2b.)⁵⁴
• in rats, a decrease in the striatal binding index from 3.5 (± 0.9) to 2.4 (± 0.8) after 3 mg/kg (p = 0.026) and to 1.7 (± 0.6) after 10 mg/kg (p < 0.001). (Animal study, rats, SPECT [123I]FP-CIT, manufacturer-funded, E 2b.)⁵⁵
  The peak brain concentration did not occur until 60 minutes after administration, consistent with the time course of the behavioral effect. This time point was measured in baboons. (PET, [11C]cocaine, n = 7, healthy subjects, E 2b.)⁵⁴ A review added that therapeutic doses block over 50% of the DAT, only d-threo-MPH binds to the DAT, the peak in the brain is reached after 60 to 90 minutes, and the increase in dopamine depends on the dopamine release rate and age (Older adults showed smaller effects). (Narrative review, E 4.)⁵⁶
  A dose-binding relationship for MPH and cocaine was indistinguishable in animal and human comparisons (approximately 50% DAT binding at 0.25 mg/kg, approximately 80% at 1 mg/kg). At the same occupancy level, MPH increased locomotion to a greater extent. (comparison of animal and human data, mice and PET data, E 2b.)⁵⁷
  In untreated adults with ADHD, striatal DAT binding was elevated and decreased significantly with MPH, accompanied by clinical improvement: (E 2b)
• DAT binding 1.43 (± 0.18) compared to controls 1.22 (± 0.06, p < 0.001); under MPH (3 × 5 mg/day) 1.00 (± 0.14, p < 0.001) (SPECT [99mTc]TRODAT-1, n = 17, E 2b.)⁵⁸
• DAT binding 1.43 (± 0.18) compared to controls 1.22 (± 0.05, p < 0.001); under MPH (3 × 5 mg/day, 4 weeks) 1.02 (± 0.23, p < 0.001) (SPECT [99mTc]TRODAT-1, n = 10, 4 weeks, E 2b.)⁵⁹
  On-MPH reduced striatal DAT binding by 29.9% in boys with ADHD (t = −4.12, p = 0.002). A greater reduction and higher baseline binding were associated with higher symptom ratings without MPH (r = 0.68, p = 0.01, and r = 0.56, p = 0.04, respectively). (Within-subject SPECT [123I]Beta-CIT, n = 13, E 2b.)⁶⁰ For more on this aspect of responding, see section 5.6.
  A longitudinal study using dual-label PET found that, under sustained release MPH administration, there was a reduction in DAT binding in the caudate and putamen and in NET binding in the thalamus and pons. Higher baseline DAT levels in the putamen predicted an improvement in sustained attention. (Longitudinal PET, DAT [18F]FEPE2I and NET [18F]FMeNER-D2, n = 21 pre-treatment, n = 12 post-treatment, E 2b.)⁶¹ See sections 5.6 and 2.1.2.2 for more information.
  According to an in vitro study, the interaction of MPH with DAT differs from that of cocaine, GBR12909, and amphetamine. MPH and benztropine exhibited reduced affinity for a DAT mutant (S356,359A) in ligand displacement assays, but not in dopamine reuptake inhibition. (In vitro study, COS-7 cells with wild-type and mutant DAT, E 2b.)⁶²
  The duration of the psychostimulant effect depended on the duration of binding to the DAT. Slow dissociation from the DAT (slow dissociation) predicted a more prolonged effect in vivo. MPH and cocaine had a shorter duration of action than the S-enantiomers of related compounds. (In vitro and computational study using animal data, mice, E 2b.)⁶³
  The reward-enhancing effect of MPH (5 and 10 mg/kg) was abolished in DAT-knockout mice, whereas it occurred in wild-type mice. A very high dose (60 mg/kg) reduced the effect in both groups. This demonstrates the dependence on DAT. (Animal study, DAT knockout and wild-type mice, E 2b.)⁶⁴
  A book chapter review presented DAT as the primary target of MPH and noted that chronic MPH increases DAT expression in animals and humans, suggesting that elevated DAT levels could be a consequence of treatment rather than solely an expression of the pathology. The effects of MPH extend beyond DAT. (Review, book chapter, full text not accessible to us, E 4.)⁶⁵ For time-dependent increases in DAT, see 2.1.1.10.
  An increased DAT density (due to overexpression or intravenous self-administration of MPH in the abuse model) increased the maximum transport rate and correlated with the inhibitory potency of amphetamine and MPH, but not of cocaine (a doubling of the transport velocity tripled the potency). In terms of uptake inhibition, MPH behaved similarly to a releaser. (animal study, rats, fast-scan cyclic voltammetry, E 2b.)⁶⁶ For more on differences in effects compared to amphetamine, see sections 3 and 2.1.4.

Intravenous MPH was shown to increase dopamine transporter levels in healthy individuals in a dose-dependent manner (secondary citation from Volkow (2002)): (Review, E 4.)²⁵

• 78% (± 11) after 0.5 mg/kg
• 67% (± 3) after 0.25 mg/kg
• 62% (± 13) after 0.1 mg/kg
• 35% (± 5) after 0.025 mg/kg

The dose required to block 50% of the DAT (ED50) was 0.075 mg/kg i.v. Oral MPH at therapeutic doses (0.3 to 0.6 mg/kg) likely occupied more than 50% of the DAT without inducing a “high.” (Review, E 4.)²⁵

• Unlike amphetamine, methylphenidate is not considered a substrate for transport into the cytoplasm, which is why it causes, at most, a slight presynaptic release of dopamine. (Review, E 4)⁷²

• Dopamine efflux in rats (ex vivo at 100 µM MPH, but not at 10 µM)

  • in the PFC from both dopamine and norepinephrine terminals (animal study, N = 27, rats, E 2b.)⁹
  • in the striatum through the reversal of DAT, which is dependent on sodium and, to some extent, on vesicular integrity (reserpine-sensitive)
  • As a result, MPH increases extracellular dopamine levels

• Increase the firing rate of PFC pyramidal neurons

  • following chronic administration (4 mg/kg/day orally for 15 days, measured after a 28-day washout period) in the PFC of rats (measured in vivo) (animal study, N = 27, rats, Category 2b)⁹
  • measured using extracellular electrophysiological single-cell recordings
  • Responses to locally applied NMDA remain unchanged

• Desensitization to both dopamine and MPH in striatal regions

  • following chronic administration (4 mg/kg/day orally for 15 days, measured after a 28-day washout period) in the striatum of rats: (animal study, N = 27, rats, E 2b.)⁹
  • Reduced efficacy of extracellular dopamine in modulating NMDA-induced firing activity of medium spiny neurons in the striatum (in vivo, preliminary data)
  • reduced MPH-induced dopamine release (ex vivo, 100 µM)
  • is consistent with the empirical observation that, when starting treatment, a one-time adjustment of the MPH dose is necessary after a few weeks

(E 2b): MPH increases dopamine release via reuptake-mediated mechanisms and, at high doses, via vesicular mechanisms. A microdialysis study found that after 50 mg/kg MPH, dialysate dopamine levels rose to 430% of baseline. The release was Ca²⁺-dependent and could be inhibited by TTX and reserpine, indicating a vesicular and reuptake-mediated mechanism. (Animal study, rats, microdialysis, E 2b.)⁷³
The release triggered by MPH (1 mg/kg) was dependent on alpha-synuclein. MPH reduced the evoked dopamine overflow only in alpha-synuclein knockout mice, while extracellular dopamine levels increased equally in all strains. Release per pulse decreased in knockout mice and increased in wild-type mice. Reuptake inhibition was alpha-synuclein-independent. (Animal study, wild-type and alpha-synuclein knockout mice, voltammetry and microdialysis, E 2b.)⁷⁴ This also applies to the modulation of phasic dopamine (2.1.1.1.2).

Methylphenidate in combination with fluoxetine significantly reduced D2 receptors in rats in: (animal study, rats, n = 8 to 9 per group, 4 weeks, E 2b.)⁴⁹

• dorsal caudate putamen (51.5%)
• dorsolateral caudate-putamen (50.4%)
• ventromedial caudate-putamen (49.1%)
• ventrolateral caudate-putamen (47.7%)
• Nucleus accumbens core (44.8%)

D1R binding remained unchanged. Neither MPH nor fluoxetine alone altered D1R or D2R binding.

(E 2b): Several animal studies demonstrate the redistribution and kinetic upregulation of VMAT2 by MPH:

• MPH increased dopamine transport into membrane-associated vesicles despite a decrease in VMAT-2 immunoreactivity (kinetic upregulation), as well as dopamine content and K+-stimulated dopamine release. (Animal study, rats, subcellular fractionation, E 2b.)⁸⁴
• A single dose of MPH shifted VMAT-2 and vesicles from the synaptosomal membranes into the cytoplasm and increased dopamine transport in both fractions, resulting in higher dopamine content and faster, greater K+-stimulated release. (Animal study, rats, review of authors’ own data, E 2b.)⁸⁵
• Even low doses of MPH and amphetamine shifted VMAT-2 in the same way as high doses, while vesicular glutamate, acetylcholine, and GABA transporters remained unaffected (selectivity for monoaminergic neurons). (Animal study, rats, subcellular fractionation, E 2b.)⁸⁶
• MPH increased vesicular dopamine uptake and VMAT-2 binding in a dose- and time-dependent manner. In vitro, MPH (at concentrations equal to or below 1 µM) had no effect, suggesting an in vivo-mediated effect. D1 (SCH23390) and D2 antagonists (Eticloprid) reversed this effect. (Animal study, rats, purified striatal vesicles, E 2b.)⁸⁷
• MPH increased dopamine transport into membrane-associated vesicles (mimicked by quinpirole, blocked by eticlopride) and K+-stimulated release. Both D2 and muscarinic receptors were required. (Animal study, rats, E 2b.)⁸⁸
• MPH pretreatment increased vesicular dopamine uptake and VMAT-2 binding and prevented persistent, methamphetamine-induced dopaminergic deficits. (Animal study, rats, E 2b.)⁸⁹

In vitro, d-MPH at concentrations of 100 nmol/l or higher (protein homogenates from rat PC12 cells) tyrosine hydroxylase activity significantly and in a dose-dependent manner up to 100 µmol/l (by 50 to 145% compared to the control). L-MPH or racemic MPH did not increase TH. As the proportion of L-MPH increased, the TH activity induced by d-MPH decreased slightly. (In vitro study, rat PC12 cell homogenates, E 2b.)⁹²
This concentration was higher than the mean plasma levels of 42.5 nmol/L observed in children receiving less than 0.7 mg/kg of OROS-MPH. It is unclear whether the increase occurs only peripherally or also in the brain. (Narrative review, E 4.)⁹³ TH gene variants appear to influence the response to MPH. To date, only one TH polymorphism (rs2070762) has been studied. Carriers of the C/C genotype responded less well to MPH (OR 4.34, CI 1.54 to 12.2, secondary citation from Stevens 2019). (Narrative review, E 4.)⁹³

There is no consensus on whether MPH actually increases dopamine synthesis. An [18F]FDOPA-PET study found a 22% reduction in dopamine turnover (kloss) in the putamen following MPH administration (p = 0.003), while at the same time, following a single dose, an 18% increase in the dopamine synthesis rate was observed at the follow-up measurement, indicating a sustained effect (K). The change observed by kloss correlated with the change in cognition. ([18F]FDOPA PET, n = 20, healthy women, 0.5 mg/kg orally, E 2b.)⁹⁴ For long-term effects, see 2.1.1.10.
In contrast, older animal studies found no increase or a decrease in dopamine synthesis due to MPH:

• Cocaine and MPH reduced DOPAC efflux and the rate of dopamine synthesis in the striatum (pure reuptake inhibition), unlike d-amphetamine (biphasic, release). (Animal study, rats, DOPA accumulation and DOPAC efflux, E 2b.)⁹⁵
• Systemic amphetamine reduced dopamine synthesis in a dose-dependent manner (ED50: d-amphetamine 1.1, l-amphetamine 3.5 mg/kg), whereas MPH did not alter dopamine synthesis despite producing comparable stereotypies. In vitro, MPH partially activated synthesis but did not reverse the inhibition caused by reserpine. (Animal study, rats, synaptosomes, E 2b.)⁹⁶
  In a voltammetry study, an increase in tyrosine hydroxylase occurred only after intravenous self-administration of MPH (abuse model), along with elevated DAT levels, increased dopamine uptake, and dopamine release. Cocaine had the opposite effect. (Animal study, rats, voltammetry, nucleus accumbens, E 2b.)⁹⁷

High-dose MPH (administered orally over 3 months) increased D1R binding in several regions of the basal ganglia in rats, particularly in the caudal portions of the caudate-putamen and in the tuberculum olfactorium. D2R binding remained unchanged at both doses tested. After one month of abstinence, no differences remained. (Animal study, rats, n = 8 to 9 per group, 3 months, E 2b.)⁴⁵

The effect of MPH on cognitive performance depends on the individual baseline level of striatal dopamine synthesis, although two [18F]FDOPA-PET studies by the same research group found opposing relationships:

• MPH (and sulpiride) most strongly increased the willingness to engage in cognitive effort when striatal dopamine synthesis rates were low. MPH enhanced the balance of benefits versus costs of cognitive effort. (Placebo-controlled, [18F]DOPA-PET, healthy subjects, E 2b.)¹⁰⁶
• MPH increased the preference for cognitive tasks over leisure activities most significantly at high dopamine synthesis rates; sulpiride had no significant effect. (placebo-controlled, crossover, [18F]DOPA-PET, n = 50, healthy subjects, E 2b.)¹⁰⁷
  MPH improved accuracy and speed in reversal learning. Reward signals were stronger than punishment signals at high dopamine synthesis rates, while surprise signals were stronger at low rates. (Placebo-controlled, fMRI and [18F]DOPA-PET, n = 100, healthy subjects, E 2b.)¹⁰⁸
  In the animal model, the narrow, dose-dependent therapeutic window was confirmed:
• MPH (1.5 mg/kg) improved working memory in half of the animals and impaired it in the other half. Both effects were reversed by the D1 antagonist SCH39266 administered intra-PFC; a D1 agonist was more effective. (Animal study, SHR, behavior and PFC single-cell recording, E 2b.)¹⁰⁹
• MPH improved working memory in a narrow inverted U-shaped curve, while sustained attention improved across a broader dose range. PFC alpha-2 and D1 receptors were involved in both tasks, while alpha-1 receptors were involved only in sustained attention (facilitating). (animal study, rats, PFC infusions, E 2b.)¹¹⁰ For more on norepinephrine receptors, see 2.1.2.5.
  The baseline levels of GABA and glutamate (Glx) modulated the dose-dependent effect on reaction control:
• Striatal GABA+/Glx levels predicted better response inhibition at a low dose of MPH, while (prae-)SMA GABA+/Glx levels predicted a deterioration at a high dose. (Human MRS, healthy adults, E 2b.)¹¹¹
• A moderate dose of MPH reduced subliminal interference the most. Higher striatal GABA+ levels were associated with better control at low doses, while low ACC GABA+ levels were associated with better control at moderate doses. (Human MRS and EEG, RCT, n = 71, healthy adults, MPH 0.25 vs. 0.50 mg/kg, E 1b.)¹¹² See sections 2.1.7 and 2.11 for more information.
  In children with ADHD, MPH improved behavior in a dose-dependent manner, but academic performance improved regardless of dose or task complexity. In the afternoon, a behavioral effect—but no effect on performance—occurred only at a dose of 1.0 mg/kg. (RCT, double-blind, crossover, n = 12, 0.3 vs. 1.0 mg/kg, full text not available to us, E 1b.)¹¹³ In 10 responders, a significant linear dose effect (5, 10, 15 mg) was observed on teacher ratings, on-task behavior, accuracy, and work volume, both individually and task-specifically. (Triple-blind, crossover, n = 12, boys, E 1b.)¹¹⁴ Both findings are also included in 4.2.

• In adolescent rats, a low single dose of MPH (0.5 mg/kg) NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) in the PFC via adrenergic receptors and improved temporal order memory and attention. A high dose (10 mg/kg) suppressed NMDA- and AMPA-receptor-mediated EPSCs and increased locomotion. Both effects were accompanied by opposing changes in glutamate receptors on the cell surface. (Animal study, rats, E 2b.)¹¹⁵

• In rats, the effect of higher doses of MPH on the choice of larger, delayed rewards depended on the baseline level of impulsivity. Up to 10 mg/kg, higher doses increased this choice in impulsive Lewis rats and decreased it in Wistar rats. (Animal study, n = 24, Lewis and Wistar rats, E 2b.)¹¹⁶

Acute administration of MPH increased the firing activity of PFC pyramidal neurons in rats and potentiated NMDA-induced neurotransmission.
Chronic MPH administration (2 × 2 mg/kg/day) resulted in the following changes in pyramidal neurons of the PFC 28 days after the end of MPH administration: (animal study, rats, 15 days, E 2b)⁹

• a long-term increase in combustion activity
• Unchanged burst activity
• unchanged total number of spontaneously firing neurons
• unchanged glutamatergic neurotransmission

Subchronic administration (14 days, 1 mg/kg) of MPH to prepubertal Naples High-Excitability (NHE) rats resulted in the following in adulthood: (animal study, NHE rats, 14 days, E 2b.)¹¹⁷

• Reduced dopamine levels in the PFC and striatum

• Increased norepinephrine in the ventral striatum

• Atomoxetine, on the other hand, increased dopamine levels in the PFC and decreased them in the dorsal striatum

• (E 2b): In adolescent rats, (animal study, adolescent rats, 15 days, E 2b)⁸² * (animal study, young rats, 2 weeks, E 2b)*⁸³

  • a single dose of high-dose MPH (2 mg/kg, which is roughly twice the maximum therapeutic dose typically used in humans)
    • a reduction in the number of vesicular monoamine transporters (VMAT2) in the cerebellum
    • No increase in dopamine turnover in the cerebellum (as measured by the DA metabolite DOPAC)
    • No change in the protein levels of tyrosine hydroxylase (TH) and the dopamine D1 receptor
    • Unchanged levels of dopamine and homovanillic acid (HVA)
  • continuous administration of high-dose MPH (2 mg/kg twice daily) for 14 to 15 days
    • increased number of vesicular monoamine transporters (VMAT2) in the cerebellum
    • significantly increased dopamine turnover in the cerebellum (as measured by the metabolite DOPAC) (animal study, adolescent rats, 15 days, E 2b)⁸²
    • left the protein levels of tyrosine hydroxylase (TH) and the dopamine D1 receptor unchanged (animal study, adolescent rats, 15 days, E 2b)⁸² (elsewhere, the same authors: (animal study, young rats, 2 weeks, E 2b)⁸³ )
    • increased the number of DAT (animal study, young rats, 2 weeks, E 2b)⁸³
      • in the left dorsal striatum
    • did not modify the DAT
      • in the right dorsal striatum
      • in the nucleus accumbens (ventral striatum) (animal study, young rats, 2 weeks, E 2b.)⁸³
    • increased the expression of the
      • Norepinephrine transporter (NET)
      • (E 2b): Monoamine transporter 2 (VMAT2) (animal study, adolescent rats, 15 days, E 2b)⁸² * (animal study, young rats, 2 weeks, E 2b)*⁸³
        • In contrast, amphetamine reduces VMAT2 function (Narrative review, E 4.)¹¹⁸
      • Tyrosine hydroxylase
      • Dopamine D1 receptors
      • more pronounced in the nucleus accumbens (ventral striatum) than in the dorsal striatum
      • more pronounced in the parietal cortex than in the frontal cortex
      • (E 2b): This effect of chronic MPH on increasing DAT, NET, and VMAT2 transporters could suggest that, in the long term, the drug might lose some of its acute effect of raising dopamine and norepinephrine levels. (Animal study, young rats, 2 weeks, E 2b)⁸³ * (Controlled cohort study, N = 29, n = 18, 12 months, E 2b)*⁵¹
        This is consistent with our experience that, for some users, the dosage needs to be adjusted (slightly increased) once after half a year to a year. However, a general habituation effect has not been reported in studies (RCT follow-up, N = 433, n = 256, 1.5 years, E 1b)¹¹⁹ or in clinical practice.
      • Elevated levels of vanillinmandelic acid in the urine of Wistar rats. This was prevented by the adjunctive administration of buspirone. (animal study, Wistar rats, E 2b)¹²⁰
        Vanillinmandelic acid is produced during the breakdown of epinephrine and norepinephrine by MAO-A and COMT, making vanillinmandelic acid an indicator of the activity of the autonomic nervous system (sympathetic nervous system).
    • A single dose of very high-dose MPH (5 mg/kg, which is about 5 to 20 times the standard treatment dose for humans) results in
      • similar changes in metabolites in the cerebellum as with 2 mg
      • Metabolites in the cerebellum that are associated with energy consumption and excitatory neurotransmission—specifically, glutamate, glutamine, N-acetyl aspartate, and inosine—tended to be reduced
      • Furthermore, the levels of certain metabolites associated with inhibitory neurotransmission—in this case, GABA, glycine, acetate, aspartate, and hypoxanthine—were reduced

MPH modulated the activity of the locus coeruleus. An acute dose (2.5 mg/kg) altered the firing rate in 87% of the recorded LC units, with 80% of these showing an increase. Chronic administration led to sensitization (41%) or tolerance (59%). (animal study, rats, free-moving LC recording, E 2b.)¹³² This also applies to the firing rate (2.1.1.3).

Low-dose MPH reduced the tonic and phasic discharge of the locus coeruleus in a dose-dependent (moderate) manner. It decreased the signal-to-noise ratio of the excitatory phasic discharge and increased the inhibitory component. Oral and intraperitoneal administration had comparable effects. (Animal study, rats, LC recording, E 2b.)¹³³

Doses of 10, 20, and 30 mg/kg MPH (well above a therapeutic dose) resulted in: (animal study, rats, E 2b)¹³⁴

• stereotypical behavior (a sign of a sharp increase in extracellular dopamine). 20 mg/kg is just as potent as 2.5 mg/kg of AMP
• Increased extracellular dopamine
• Increased extracellular norepinephrine
• Extracellular serotonin remains unchanged (unlike with AMP, where it is elevated)

NET function (norepinephrine uptake rate) in the orbitofrontal cortex was elevated in the SHR (ADHD model). MPH administered during adolescence normalized NET function in the OFC in adulthood. (Animal study, SHR, WKY, and Wistar; in vivo voltammetry, E 2b.)¹³⁶ This also applies to the long-term effects (2.1.1.10). For information on the reduction of NET in the thalamus under MPH in humans, see Oya (2026) under 2.1.1.2.

MPH (1 and 5 mg/kg) increased norepinephrine levels in the primary somatosensory cortex by 64% and 101%, respectively, and suppressed post-excitatory inhibition as well as the long-latency excitation of sensory responses. (Animal study, rats, microdialysis and SI cortex recording, E 2b.)¹³⁷

In vivo, MPH increased extracellular dopamine and norepinephrine levels in the medial PFC (microdialysis) in rats by: (animal study, rats, E 2b.)¹³⁸

• 286% dopamine and 127% norepinephrine after 5 mg/kg i.p.
• 129% dopamine and 30% norepinephrine after 10 mg/kg administered intragastrically

A 15-minute handling stress altered only the dopamine response, not the norepinephrine response. Stress administered 2 hours before MPH (5 mg/kg i.p.) reduced the dopamine increase to 203%; stress administered 15 minutes after MPH (10 mg/kg intragastric) increased it to 273%. (Animal study, rats, E 2b.)¹³⁸

(E 2b): In animal studies, the effects of MPH were dependent on noradrenergic β-receptors in:

• MPH-induced impulsivity (premature responses in the 5-choice serial reaction time task, 2 mg/kg MPH). The centrally acting propranolol completely reversed this effect, whereas the peripherally acting nadolol did not. The α1 antagonist prazosin partially attenuated them, as did the D4 antagonist L-745,870. Cortical norepinephrine depletion did not alter them. (Animal study, rats, E 2b.)¹³⁹
• Enhanced long-term potentiation (LTP) and long-term depression (LTD) in the hippocampus (CA1, preadolescent rats, 10 to 50 ng/ml MPH) without presynaptic changes (animal study, brain sections, rats, E 2b.)¹⁴⁰
• dose-dependent enhancement of LTP in the hippocampus (CA3-CA1, young rats, EC50 73.44 nM), further mediated by D1/D5 dopamine receptors and PKA, with increased phosphorylation of the AMPA receptor subunit GluA1 (Ser845) and more functional AMPA receptors on the cell surface (animal study, brain sections, rats, E 2b.)¹⁴¹
• Increased phosphorylation of GluA1 (Ser845) in Bergmann glial cells of the cerebellum following administration of MPH (15 mg/kg) and d-amphetamine (10 mg/kg). For d-amphetamine, this effect depended on intact norepinephrine release and β1-adrenoceptors, but not on D1 receptors. (Animal study, mice, E 2b.)¹⁴²

(E 2b): Animal studies have shown that α2-adrenoceptors are involved in the effects of MPH in:

• Increased excitability of pyramidal cells in the deep layers of the infralimbic and prelimbic PFC (juvenile rats, 10 µM MPH). This effect was abolished following catecholamine depletion by reserpine and was mediated via α2 receptors. D1 receptors did not play a discernible role. (animal study, brain sections, rats, E 2b.)¹⁴⁶
• enhanced noradrenergic inhibition in the locus coeruleus of juvenile rats. MPH hyperpolarized the neurons and reduced their spontaneous firing rate (at concentrations above 3 µM), enhanced the norepinephrine-mediated inhibitory postsynaptic current (IPSC), and triggered a barium-sensitive, inward-rectifying potassium current. The α2 antagonist yohimbine blocked the potentiation of the IPSC. (Animal study, brain sections, rats, E 2b.)¹⁴⁷
• the psychomotor-stimulating effect of MPH (1 mg/kg s.c.), which was attenuated by the α2 antagonist RX821002 in the infralimbic cortex. RX821002 did not prevent the suppression of social play by MPH, neither in the anterior cingulate cortex nor in the infralimbic cortex, in the basolateral amygdala, or in the habenula, which the authors interpreted as parallel mediation via prefrontal and limbic regions. (Animal study, rats, E 2b.)¹⁴⁸
• Activity and discrimination accuracy in an operant test. MPH (0.3 to 3 mg/kg s.c.) dose-dependently reduced the number of responses and rewards received in control mice and increased them in α2C-adrenoceptor knockout mice. (Animal study, mice, E 2b.)¹⁴⁹
• the activation of PFC neurons by MPH (1 or 3 mg/kg i.v.). The α2 antagonist yohimbine potentiated this effect and rendered an otherwise ineffective dose (0.3 mg/kg) effective. The D1 antagonist SCH 23390 partially reduced or prevented this response. Locally administered MPH produced a D1-dependent effect; dopamine (but not norepinephrine) enhanced the response. (Animal study, rats, E 2b.)¹⁵⁰

Methylphenidate (3, 10, and 30 mg/kg orally) did not alter extracellular serotonin levels in the PFC of rats and did so only sporadically in the striatum (a single increase to 319% 45 minutes after a 30 mg/kg dose, followed by a decrease 3 to 4 hours after all doses), while it increased norepinephrine and dopamine in the PFC as well as dopamine in the striatum. In the same experiment, lisdexamfetamine additionally increased serotonin in the PFC and striatum, though to a lesser extent and for a shorter duration than norepinephrine and dopamine. (Animal study, rats, manufacturer-funded, author(s) employed by the manufacturer, E 2b.)¹⁵²

(E 2b): MPH did not alter extracellular serotonin levels in animal studies:

• in the PFC and the ventral hippocampus (2.5 mg/kg s.c.; citalopram, by contrast, significantly increased these levels). When combined with citalopram, MPH enhanced the citalopram-induced increase in serotonin in the hippocampus and attenuated it in the cortex. The combination increased dopamine in the PFC, nucleus accumbens, and hippocampus to a greater extent than MPH alone, but not in the striatum. (Animal study, microdialysis, rats, E 2b.)¹⁵⁵
• in the nucleus accumbens (10 mg/kg i.p.) with elevated dopamine levels, unlike methamphetamine (animal study, microdialysis, rats, E 2b.)¹⁵⁶

(E 2b): Chronic MPH altered the density of the serotonin transporter (SERT) in animal studies:

• increased in the medial frontal cortex of adult rats (20 mg/kg orally twice daily for 11 days), 2 and 12 weeks after discontinuation (animal study, n = 30, rats, 11 days, E 2b.)¹⁵⁷
• not in the striatum in adulthood (day 90 of life) following administration of 2 mg/kg/day on days 25 through 39 or 50 through 64 of life, neither in SHR (ADHD animal model) nor in WKY. Older SHR rats had a higher striatal SERT density than WKY rats. (animal study, SHR, WKY, rats, 2 weeks, E 2b.)¹⁵⁸

In children with ADHD, sustained-release MPH did not significantly alter the morning blood serotonin levels, which are typically reduced in ADHD. A circadian rhythm characterized by higher morning melatonin levels disappeared with MPH, and nocturnal excretion of 6-sulfatoxymelatonin decreased. (Controlled, quasi-experimental, open-label study, N = 179, n = 136, 4.6 months, E 2b.)¹⁵⁹

Repeated administration of MPH reduced 5-HT-1A-R expression

• in the nucleus accumbens
  • significant at 2.5 mg/kg (animal study, N = 16, rats, 12 days, E 2b.)¹⁶¹
  • which is relevant to the reinforcing effects of MPH (animal study, N = 16, rats, 12 days, E 2b)¹⁶¹
  • Buspirone
    • Doses of 0.1 and 0.3 mg/kg prevented downregulation, but doses of 1.0 and 2.0 mg/kg of buspirone did not (animal study, rats, E 2b)¹⁶²
    • 10 mg/kg/day prevented the development of tolerance following long-term administration of 2 mg/kg/day MPH (animal study, rats, E 2b)¹⁶³
    • Buspirone is a partial 5-HT1A agonist and a D2R antagonist
• in the PFC
  • Not significant at 2.5 mg/kg (animal study, N = 16, rats, 12 days, E 2b.)¹⁶¹ * (animal study, rats, E 2b.)*¹⁶⁴
  • significant at 5 mg/kg (animal study, rats, E 2b)¹⁶⁴

Low doses (2.5 mg/kg) of MPH improved memory performance in the water maze test, while higher doses (5 mg/kg) impaired it. Improved performance correlated with high 5-HT metabolism in the PFC. (animal study, rats, E 2b)¹⁶⁴

• GABAA receptor mRNA expression is reduced at low doses of MPH (2.5 mg/kg)
• GluN2A expression is reduced at high doses of MPH (5 mg/kg)
• According to the authors, elevated 5-HT levels and reduced GABAA receptor mRNA expression in the PFC appeared to free excitatory glutamate neurons from the inhibitory influence of GABA
• Reduced 5-HT1A receptor mRNA expression enhances the inhibitory effect of GABA on glutamate neurons and thereby impairs cognitive performance.

Repeated administration of MPH (1 mg/kg, 14 days) and tryptophan (100 mg/kg) each increased locomotor activity in rats. Pretreatment or concurrent treatment with tryptophan significantly reduced MPH-induced behavioral sensitization. The authors attributed this to desensitization of somatodendritic 5-HT1A receptors due to increased serotonin levels. (Animal study, rats, 2 × 14 days, full text not available to us, E 2b.)¹⁶⁵

A 5-HT1B receptor agonist (CP 94253, 1 to 10 mg/kg) significantly and dose-dependently potentiated MPH-induced locomotor activation in rats. Pretreatment with methamphetamine or fluoxetine, which increased extracellular serotonin, also potentiated this effect. This effect was blocked by a 5-HT1B antagonist (GR 55562), but not by a 5-HT2 antagonist (ritanserin). MPH itself (10 mg/kg i.p.) increased dopamine in the nucleus accumbens, but not serotonin. (Animal study, rats, E 2b.)¹⁵⁶

Doses of MPH (30 mg/kg) that induce stereotypy and amphetamine (3 mg/kg) increased levels of the serotonin metabolite 5-hydroxyindoleacetic acid (5-HIAA) in the striatum and frontal cortex of rats, which receive serotonergic fibers from the dorsal raphe. In the hippocampus, which is innervated by the median raphe, these doses either decreased 5-HIAA or had no effect on it. The authors interpreted this as evidence of enhanced serotonergic function of the dorsal raphe. Pretreatment with α-methyltyrosine did not prevent the amphetamine-induced increase in 5-HIAA. (Animal study, rats, E 2b.)¹⁶⁸

The response of dorsal raphe neurons to acute and chronic MPH (0.6, 2.5, and 10 mg/kg on 6 days, a 3-day washout period, and re-administration on day 10) differed significantly between young and adult rats (504 and 356 recorded neurons, respectively). Neurons from behaviorally sensitized animals responded differently to the repeat administration than neurons from behaviorally tolerant animals. The proportion of animals exhibiting sensitization or tolerance at the same dose differed between young and adult animals. (Animal study, 860 neurons, rats, 10 days, E 2b.)¹⁷¹

(E 2b): Other studies found inhibition constants (Ki) and half-maximal inhibitory concentrations (IC50) for MPH

• on the date of:
  • Ki 60 nM (human DAT, murine DAT 260 nM, amphetamine at human DAT 640 nM) (in vitro study, human and murine transporters, E 2b.)¹⁷³
  • Ki 210 nM (caudate-putamen brain slices, mice, voltammetry, cocaine 350 nM, amphetamine 420 nM) (in vitro study, mice, E 2b.)¹⁷⁴
  • Ki 341.2 nM for synaptosomal uptake (striatum, rats; vesicular uptake 39,300 nM; d-amphetamine 206 and 565.2 nM, respectively) (In vitro study, rats, manufacturer-funded, E 2b.)¹⁷⁵
• on the NET from:
  • Ki 100 nM (human NET, murine NET 170 nM, amphetamine at human NET 70 nM) (in vitro study, human and murine transporters, E 2b.)¹⁷³
  • Ki 238 nM for synaptosomal uptake (frontal cortex, rats; vesicular uptake 11,400 nM; d-amphetamine 54.8 and 261 nM, respectively; atomoxetine synaptosomal 20.6 nM) (In vitro study, rats, manufacturer-funded, E 2b.)¹⁷⁵
• at the SERT by:
  • Ki 19 µM (brain slices of the substantia nigra, mice, cocaine 10 µM, amphetamine 23 µM) (in vitro study, mice, E 2b.)¹⁷⁴
  • IC50 118 µM (synaptosomes, rats; cocaine 70 µM, d-amphetamine 570 µM) (in vitro study, rats; full text not available to us, E 2b.)¹⁷⁶
  • Ki 132 µM (human SERT, approximately 2,200 times weaker than at human DAT; amphetamine 38 µM) (in vitro study, human and murine transporters, E 2b.)¹⁷³
• at the organic cation transporter hOCT1 (low-affinity, high-capacity monoamine transporter) with an IC50 of 0.36 µM (95% CI 0.27 to 0.46). MPH did not completely inhibit hOCT2, hOCT3, and PMAT. (In vitro study, HEK293 cells, E 2b.)¹⁷⁷

Intravenous MPH blocked DAT in the striatum in vivo in a dose-dependent manner with an ED50 (dose that blocks 50% of DAT) of 0.07 mg/kg; cocaine had an ED50 of 0.13 mg/kg. This was consistent with the reported in vitro affinities (Ki for MPH 390 nM, cocaine 640 nM). Heart rate and blood pressure increased similarly with both substances, but the increase was significantly more prolonged with MPH. (Comparison of two PET studies, N = 25, n = 8 healthy subjects with MPH, n = 17 cocaine users with cocaine, E 2b.)¹⁷⁸

(E 2b): Changes in proton MR spectroscopy following MPH treatment in children and adolescents with ADHD:

• Glutamate/creatine in the right DLPFC (decrease after 8 weeks), plus a decrease in choline/creatine in the left crus 1 of the cerebellum (Open-label pre-post study, n = 15, 8 weeks, full text not available to us, E 2b.)¹⁸²
• Glutamate/creatine in the PFC (decrease of 15% on the right and 14.1% on the left after 12 weeks of 20 mg sustained-release MPH). N-acetylaspartate/creatine increased by 18.8% on the right and 10% on the left; choline/creatine decreased by 12.4% on the right and 16% on the left; myo-inositol/creatine decreased by 14.7% on the left. (Open-label pre-post study, n = 21, 12 weeks, E 2b.)¹⁸³
• Glutamate plus glutamine (absolute and relative to creatine) in the left white matter behind the DLPFC (increase after 2 months of OROS-MPH in responders). In contrast, following atomoxetine treatment, N-acetylaspartate/creatine levels decreased on the left side of the DLPFC, while choline/creatine levels increased on the right side. (Open-label pre-post study, n = 21, including 10 treated with MPH, 2 months, E 2b.)¹⁸⁴
• Glutamate, glutamine, and glutamate-plus-glutamine-to-myo-inositol ratios in the ACC (higher in untreated ADHD than in controls and than after 6 to 8 weeks of OROS-MPH at an average dose of 54 mg/day, not significant) (Open-label study with a control group, N = 22, n = 10, 6 to 8 weeks, manufacturer-funded, E 2b.)¹⁸⁵

(E 2b): In animal studies, MPH altered glutamatergic transmission

• at NMDA receptors via:
  • At a low dose (0.5 mg/kg), NMDA receptor-mediated EPSCs in the PFC of adolescent rats were enhanced via adrenergic receptors and SNAP-25, resulting in improved temporal sequence memory and attention. A high dose (10 mg/kg) suppressed NMDA- and AMPA-receptor-mediated EPSCs and increased locomotion. (Animal study, rats, E 2b.)¹¹⁵
  • Enhancement of NMDA receptor-mediated transmission in pyramidal cells (layers V and VI) of the medial PFC at higher concentrations, in a catecholamine-independent manner via sigma-1 receptors (blocked by sigma-1 antagonists, but not by D1/5 or α2 antagonists) and via PLC, IP3 receptors, and PKC. Locomotor activation by higher doses of MPH also depended on sigma-1 receptors. (Animal study, rats, mice, E 2b.)¹⁸⁶
  • Potentiation of NMDA-induced, but not AMPA-induced, excitation of PFC neurons (1 to 3 mg/kg i.v., as well as d-amphetamine and atomoxetine). The increase in firing rate induced by MPH was D1-dependent. (Animal study, rats, E 2b.)¹⁸⁷
  • A reduction in the NMDA receptor subunits NR1 and NR2B (but not NR2A) in the PFC of juvenile rats following a single dose (1 mg/kg i.p.), accompanied by smaller NMDA- and larger AMPA-receptor-mediated EPSCs, as well as more frequent and stronger LTP (animal study, rats, E 2b.)¹⁸⁸
  • Dose-dependent changes in NMDA receptor binding ([3H]MK-801) in numerous regions (including the prelimbic, insular, and motor cortex; dorsolateral striatum; nucleus accumbens; hippocampus; amygdala; thalamus) after 13 weeks of oral administration in adolescent rats (4/10 and 30/60 mg/kg, respectively); this effect was no longer observed after 4 weeks of abstinence (animal study, rats, 13 weeks, E 2b.)¹⁸⁹
  • Effect on NMDA-receptor-mediated EPSCs in dopaminergic VTA neurons only at high doses; no effect on AMPA/kainate-receptor-mediated EPSCs (amphetamine enhanced both, particularly AMPA/kainate) (Animal study, brain sections, rats, E 2b.)¹⁹⁰
• at AMPA receptors via:
  • A reduction in stop-signal reaction time (SSRT, 1 mg/kg s.c.) was observed only in rats with a long baseline SSRT. An AMPA receptor antagonist (NASPM) in the PFC or a Src kinase inhibitor (PP2) reversed this effect. (Animal study, rats, E 2b.)¹⁹¹
• on extracellular glutamate through:
  • An increase in tonic glutamate in the prelimbic cortex (2 mg/kg s.c.) was observed after an intermediate treatment duration only in SHR, and after 11 days in both SHR and WKY. The amplitude of phasic glutamate signals decreased only in SHR. (Animal study, SHR, WKY, rats, 11 days, E 2b.)¹⁹²
  • Increased glutamate levels in the cerebrospinal fluid and reduced glutamate uptake in the PFC (GLAST and GLT-1 unchanged) with decreased Na+/K+-ATPase activity following 2 mg/kg i.p. daily from day 15 to 45 of life, interpreted by the authors as excitotoxicity (animal study, rats, 30 days, E 2b.)¹⁹³

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 regulator element-binding protein 2
  • increased by MPH
• reduced expression of the ATP-binding cassette transporter A1
  • increased by MPH

(E 2a): The effect of MPH on the parasympathetic nervous system (vagal tone, measured via heart rate variability (HRV) or respiratory sinus arrhythmia (RSA)) was inconsistent. A meta-analysis found no significant changes in the time- and frequency-domain indices of resting HRV due to MPH in children and adolescents with ADHD, with very low certainty of evidence for all endpoints and a high risk of bias. (Systematic review with meta-analysis; full text not available to us; E 2a.)²⁰⁹ Individual studies found

• reduced vagal tone in untreated children with ADHD compared to controls, which approached control levels with MPH (24-hour ECG): (Pilot study with a control group, N = 42, n = 23, E 3.)²¹⁰ * (Pilot study with a control group, N = 50, n = 31, E 3.)*²¹¹
  • rMSSD without MPH: 26.1 ms; with MPH: 36.7 ms; controls: 44.5 ms
  • pNN50: 6.5% without MPH, 14.2% with MPH, controls 21.5%
  • Reduction primarily between 5 p.m. and 6 a.m.; levels under MPH treatment approached those of the control group (pilot study with a control group, N = 42, n = 23, E 3.)²¹⁰
• a shift in autonomic balance toward the parasympathetic system after more than one year of treatment with sustained-release MPH (mean 37.64 mg/day) compared with controls (higher rMSSD and HF, lower LF/HF ratio, p = 0.02, 0.001 and 0.01, respectively), particularly during sleep. Microvolt T-wave alternans (a marker of susceptibility to ventricular arrhythmias) was not significantly elevated. (Case-control study, N = 95, n = 40; full text not available to us; E 3.)²¹²
• reduced vagal tone after 12 weeks of MPH (mean 0.70 mg/kg) in children with ADHD (RMSSD from 47.65 to 38.88 ms, p = 0.018, HF from 6.14 to 5.70 ln ms², p = 0.043), accompanied by an increased heart rate (from 86.38 to 93.54/min, p = 0.007). The authors interpreted this as a shift away from parasympathetic dominance in ADHD. (Open-label before-and-after study, n = 37, 12 weeks, E 2b.)²¹³
• No change in RSA with OROS-MPH compared with placebo (6.20 vs. 6.26). Under placebo, the RSA was reduced compared to controls (6.26 vs. 6.85, ηp² 0.07); under MPH, there was no longer a significant difference compared to controls. (Double-blind, placebo-controlled crossover study with a control group, N = 256, n = 157, E 1b.)²¹⁴

(E 1b): In children with ADHD, MPH normalized markers of reduced sympathetic activation (hypoarousal):

• Resting skin conductance (EDA) was lower in the placebo group compared with the control group (3.80 vs. 5.03, p < 0.04); under OROS-MPH, 4.78 and no longer different from controls (MPH vs. placebo, p = 0.058) (Double-blind, placebo-controlled crossover study with a control group, N = 256, n = 157, E 1b.)²¹⁴
• Resting skin conductance and skin conductance responses to neutral and emotional images were reduced in boys with ADHD not taking MPH; in those taking MPH (0.27 mg/kg one hour beforehand), these responses did not differ from those of controls (case-control study, N = 102, n = 80, E 3.)²¹⁵
• Pupil diameter (a marker of noradrenergic arousal) was reduced during a visuospatial working memory task without MPH, increased under MPH (p = 0.003), and was no longer different from that of controls. Pupil size correlated with performance (rho = 0.63) and inversely with reaction time variability (rho = −0.68). Performance and reaction time variability did not change significantly under MPH. (Case-control study comparing conditions with and without MPH, N = 50, n = 17, E 3.)²¹⁶

The deceleration in heart rate (evoked heart rate) that occurs during error checks was absent in untreated children with ADHD across all feedback conditions. Under MPH, it normalized in the absence of feedback and during punishment, but not completely during reward. (Study with randomized assignment to MPH or medication withdrawal and a control group, N = 50, n = 32, E 2a.)²¹⁷

Single dose of MPH: (animal study, rats, E 2b)²²⁴

• increased superoxide (ROS) levels in the cerebellum of young rats at both low and high doses, and in the hippocampus only at high doses (10 mg/kg)
• did not affect superoxide in adult rats

Chronic administration (as is common with ADHD medications): (animal study, rats, E 2b)²²⁴

• did not affect superoxide in young rats
• Reduced superoxide levels in the cerebellum of adult rats at lower doses

MPH (0.1 mM) in vitro on retinal cells (In vitro and animal study, retinal cell cultures, rats, SHR, E 2b.)²²⁵

• protected the viability of retinal cells
• induced oxidative stress through the activation of the NOX2 and PI3K/AKT/DRP1 signaling pathways
• induced mitochondrial dysfunction
  • Reduced mitochondria
  • Increased mitochondrial fragmentation
  • Impaired membrane potential
  • Reduced oxygen consumption
  • A shift in metabolism toward a glycolytic metabolic profile
• in inflammatory conditions
  • boosted antioxidant defense
  • reduced oxidative stress
  • reduced intracellular calcium levels
  • improved the structure and function of the mitochondria

MPH (1.5 mg/kg/day, oral) caused * In rats (in vitro and animal study, retinal cell cultures, rats, SHR, E 2b.)*²²⁵

• at SHR
  • Reduces oxidative stress
  • Improved mitochondrial function
• for WKY (control tree)
  • adverse effects

MPH (0.01 to 2 mM) promoted astrogliosis, oxidative/nitrosative stress, and the release of TNF in astrocyte cultures, consistent with findings in the hippocampus of WKY rats receiving chronic doses of 1.5 mg/kg/day. MPH had no significant effect on microglial cells other than impaired migration. (In vitro and animal study, N9 microglial cell line, astrocytes, rats (WKY); full text not available to us; E 2b.)²²⁶

Changes in interhemispheric connectivity have been reported in ADHD: (case-control study, E 3.)²³⁸

• reduced interhemispheric coherence in the delta band in frontal brain regions
• Increased coherence in the delta band in posterior regions (only with eyes open)
• increased coherence in the theta band in central regions

In one study, methylphenidate normalized the reduced global connectivity observed in ADHD 400 to 700 ms after a stimulus and prevented the increase in network segregation (segregation) of networks that occurred 100 to 700 ms after the stimulus. These global changes occurred primarily in the task-relevant frontal and parietal regions. The changes in network connectivity relative to baseline were more pronounced and sustained in people with ADHD taking methylphenidate and in controls than in people with ADHD not taking methylphenidate. The study’s results suggest that methylphenidate corrects the impaired network flexibility observed in ADHD. (Double-blind randomized crossover study, N = 37, n = 19, E 1b.)²⁴⁰

(E 1b): MPH altered functional connectivity

• in children and adolescents with ADHD, following a single dose, by:
  • Reduction in resting-state connectivity within a large occipital, temporal, and cerebellar network (visual and executive networks, DMN, 20 mg). This connectivity was elevated in the placebo group compared with controls but no longer differed under MPH. (Randomized, placebo-controlled crossover study, N = 31, n = 16, single dose, E 1b.)²⁴¹
  • Increased regional homogeneity (ReHo) in the bilateral ventral PFC and the vermis, as well as decreased ReHo in the right parietal and visual regions (10 mg). Under MPH, there were no longer any ReHo differences compared to controls. (Randomized, placebo-controlled crossover study, N = 55, n = 23, single dose, E 1b.)²⁴²
  • Normalization of fronto-striatal and fronto-cerebellar connectivity, as well as parieto-temporal activation during sustained attention, and the excessive orbitofrontal activation during reward (0.3 mg/kg, medication-naive children) (Randomized, double-blind crossover study, N = 26, n = 13, single dose, E 1b.)²⁴³
  • Resolution of hyperconnectivity between the right superior parietal gyrus and the left operculum/insula during the n-back test (as well as under atomoxetine). Only under MPH (0.3 mg/kg) did fronto-temporo-parietal and fronto-striato-thalamic connectivity increase, without any effects on performance. (Randomized, double-blind crossover study, N = 39, n = 19, single dose, E 1b.)²⁴⁴
  • Alignment of the average controllability (which was elevated under placebo) and modal controllability (which was reduced under placebo) (Network Control Theory) of attention, reward, and somatomotor networks with those of controls (0.3 mg/kg), interpreted by the authors as stabilization of network dynamics (Randomized, double-blind, crossover study, N = 46, n = 19, single dose, E 1b.)²⁴⁵
  • Reduced connectivity and centrality of the striatum and thalamus in stimulant-naive boys (ages 10 to 12), but increased connectivity and centrality in stimulant-naive men (ages 23 to 40, 0.5 mg/kg), with no significant effects in the dACC and PFC (pre-post study with a control group, N = 122, n = 99, single dose, E 2b.)²⁴⁶
• in cases of ADHD following prolonged treatment with:
  • increased connectivity between the left frontoparietal network and the left insula, as well as between the right frontoparietal network and the brainstem, lower clustering coefficients globally and, among other areas, in the cerebellum; bilaterally increased ALFF in the superior parietal region and decreased ALFF in the right inferior fronto-temporal region after 12 weeks in medication-naive children. These changes explained 27.1% of the variance in improvement on the ADHD Rating Scale. (Open-label pre-post study, n = 20, 12 weeks, Phase 2b.)²⁴⁷
  • sustained increase in whole-brain efficiency (p = 0.01) and DMN whole-brain connectivity strength (p = 0.03) after 4 months of MPH (not placebo) in adults, with no effects observed in children (p > 0.17). The acute connectivity response to MPH remained stable in adults, with no evidence of neurobiological tolerance. (Preprint, secondary analysis of an RCT, n = 99, 4 months, E 1b.)²⁴⁸
  • Increase in resting-state connectivity between the right nucleus accumbens and the somatomotor network after 2 years of real-world stimulant exposure (ABCD cohort), similar to the development observed in children without ADHD, with no association with reliable symptom reduction (Observational study, n = 179, of whom 81 were on stimulants, 2 years, E 2b.)²⁴⁹
• in healthy individuals after a single dose, due to:
  • Increased connectivity between the dorsal attention network and the thalamus (40 mg, men). Frontoparietal and executive networks showed greater connectivity to sensorimotor and visual areas and less to cortico-striato-thalamo-cortical loops; sensorimotor networks showed the opposite pattern. (Randomized, double-blind, placebo-controlled study, n = 54, single dose, E 1b.)²⁵⁰
  • Reduced connectivity of the lateral PFC with motor and somatosensory subregions of the thalamus, with no change in connectivity to premotor, prefrontal, and temporal subregions (20 mg) (Randomized, double-blind, parallel-group study, N = 60, n = 29, single dose, E 1b.)²⁵¹
  • Enhanced connectivity from the thalamus and dorsal striatum to the primary motor cortex and memory circuits (amygdala, hippocampus) with attenuated prefrontal connectivity (45 mg), interpreted by the authors as exceeding the optimal level (Controlled, non-randomized study, N = 48, n = 24, single dose, E 2a.)²⁵²
  • a stronger high-attention network and a weaker low-attention network (connectome-based predictive modeling) compared with unmedicated controls (45 mg) (Controlled, non-randomized study, n = 24 with MPH, single dose, E 2a.)²⁵³
  • Opposing patterns of global connectivity during rapid (intravenous) and slow (60 mg oral) dopamine increases (rho = −0.54). The “fast versus slow” patterns spatially corresponded to D1 receptor distribution (rho = 0.22). Hippocampal connectivity during the rapid rise correlated negatively with the perceived “high” (r = −0.68). (Randomized, double-blind, placebo-controlled study, PET-fMRI, n = 20, single dose, E 1b.)²⁵⁴
  • An increase in global connectivity density (gFCD) and a decrease in the amplitude of low-frequency fluctuations (ALFF) were observed with reduced D2/3 receptor availability in the putamen; this effect was pronounced following intravenous administration (0.25 mg/kg, Cohen’s d > 1.6), and weak after oral administration (60 mg, d < 0.6). The gFCD depended on the magnitude and rate of the dopamine increase. (Randomized, double-blind, placebo-controlled crossover study, PET-fMRI, n = 20, single dose, E 1b.)²⁵⁵
• in children taking stimulants (ABCD cohort), validated in healthy subjects (40 mg MPH), by:
  • Changes in connectivity in sensorimotor regions that corresponded to patterns of arousal, longer nighttime sleep, and NET expression, as well as changes in the salience and parietal memory networks, but not in attention networks (such as the dorsal attention network). Stimulants reversed the effects of sleep deprivation on connectivity and school grades. (cohort analysis, n = 11,875 children, and precision imaging study, n = 5 adults, E 2b.)²⁵⁶

(E 1b): MPH altered the connectivity and synchronization of the DMN

• According to a systematic review, 11 out of 12 studies showed an improvement in DMN regions (ADHD and other neuropsychiatric disorders) (Systematic review, k = 12, E 2b.)²⁶¹
• for ADHD:
  • Children (6 months): Increased connectivity between the left putamen and the DMN (p < 0.001), correlated with symptom reduction (rho = −0.65, p = 0.017), no significant change in connectivity within the DMN (open-label before-and-after study, n = 23, 6 months, E 2b.)²⁶²
  • Children (1 month): In the absence of medication, there is a lack of anti-phase synchronization between the DMN and the task-positive network (TPN); posterior DMN activity correlates with reaction time variability. MPH initiated partial anti-phase synchronization, reduced variability, and eliminated its correlation with the DMN. (Case-control study with before-and-after comparison, N = 22, n = 11, 1 month, E 2b.)²⁶³
  • Adult males (MPH-IR, mean 0.37 mg/kg/day): stronger connectivity between the posterior cingulate cortex and the left lateral parietal cortex (Open-label before-and-after study, n = 18, E 2b.)²⁶⁴
• In healthy individuals (single dose of 20 mg): overall reduced DMN connectivity (particularly with the auditory, cingulo-opercular, and somatomotor networks) and increased connectivity with the parietomedial network. The DMN was divided into two subnetworks with opposing effects of MPH. Reduced connectivity of the DMN subnetwork with the cingulo-opercular network was associated with higher impulsivity. (RCT, crossover, n = 55, single dose, E 1b.)²⁶⁵

In healthy individuals, a single dose of MPH (40 mg) reduced resting-state functional connectivity between the nucleus accumbens and the basal ganglia (subthalamic nucleus, ventral pallidum) as well as between the nucleus accumbens and the medial prefrontal cortex (PFC) and temporal cortex. Connectivity between the mediodorsal thalamic nucleus and the limbic circuit remained unchanged. (RCT, crossover, n = 20, single dose, E 1b.)²⁶⁷

(E 1b): MPH increased the P3 amplitude in ADHD. In contrast, early sensory ECP components, motor planning potentials, and error monitoring potentials showed barely any change. (Systematic review, k = 44, E 2b.)²⁷⁰ * (Systematic review, k = 43, E 2b.)*²⁷¹ The P3 amplitude under MPH was found to

• According to review articles:
  • Pre-/post-MPH (k = 17): increased central and posterior activity; greatest effect observed in Go/No-Go tasks (meta-analysis, k = 17; full text not available to us; E 2b.)²⁷²
  • No overall difference between people with ADHD who have been treated with MPH and controls (k = 13), but a difference in the frontal lobe (meta-analysis, k = 13, full text not available to us, E 2b.)²⁷²
  • In 11 of 20 P3 studies, * Was increased (systematic review, k = 44, E 2b.)*²⁷⁰
  • tends to normalize, accompanied by behavioral improvement (Systematic review, k = 43, E 2b.)²⁷¹
• in individual studies involving MPH responders:
  • greater with MPH than with placebo and not different from controls (auditory oddball task); earlier EEG components (mismatch negativity, N100, N200, P200) remained unchanged (RCT, crossover, N = 28, n = 14, E 1b.)²⁷³
  • only the early central No-Go-P3 subcomponent (mean latency 378 ms) was amplified, but not the late centrofrontal subcomponent (428 ms) (within-subject study on/off MPH, n = 57, single dose, E 2b.)²⁷⁴

(E 1b): Spectral EEG and other EEG measures changed under the influence of MPH or D-MPH in patients with ADHD for:

• Theta Power:
  • reduced during rest (eyes open) compared to physical activity as the control condition (ηp² = 0.12), comparable to neurofeedback (ηp² = 0.08) (RCT, N = 112, n = 81 in the EEG analysis, of whom 25 received MPH, 10 weeks, E 1b.)²⁷⁵
  • reduced during a task compared to physical activity (ηp² = 0.10), unlike during neurofeedback (RCT, N = 112, n = 81 in the EEG analysis, of whom 25 received MPH, 10 weeks, E 1b.)²⁷⁵
  • Absence of task-related theta increases in the frontal and parietal regions in adults normalized (rest-to-task pattern) (case-control study with open-label MPH treatment, N = 89, n = 21 on MPH, E 2b.)²⁷⁶
  • D-MPH: Increased midoccipital theta during retrieval in a spatial working memory task (RCT, n = 172, 8 weeks, E 1b.)²⁷⁷
• Alpha-Power:
  • reduced during a task compared to physical activity (ηp² = 0.12) (RCT, N = 112, n = 81 in the EEG analysis, of whom 25 received MPH, 10 weeks, E 1b.)²⁷⁵
  • the reactivity of high alpha activity (11 to 12 Hz) from rest to focused attention, which is present in control subjects and untreated children with ADHD, is eliminated (case-control study comparing subjects with and without MPH, N = 37, n = 19, Table 3.)²⁷⁸
• Beta power: D-MPH increased centro-parietal beta power (13 to 21 Hz) at rest (guanfacine reduced alpha power; the combination reduced theta power) (RCT, n = 179, 8 weeks, E 1b.)²⁷⁹
• Very low frequencies (VLF-EEG, 0.02 to 0.2 Hz): In adults, increased frontal and parietal VLF activity reduced omission errors to the level of controls and decreased inattention (longitudinal case-control study, N = 88, n = 21 with MPH, mean 9.4 months, E 2b.)²⁸⁰
• lateralized action potential (LRP): reduced differences compared to controls (case-control study comparing groups with and without MPH, N = 33, n = 17, E 3.)²⁸¹

(E 2b): Stimulants altered the EEG in children with ADHD by:

• Increased alpha activity in the central and parietal regions at rest and during cognitive activation; increased frontal beta activity in responders and decreased beta activity in nonresponders (MPH, ages 8 to 12) (Double-blind, placebo-controlled study, n = 36, E 2b.)²⁸³
• Increase in intrahemispheric coherence over short- to medium-range electrode distances (significant in the delta band, within the normal range) without normalization of lateralization abnormalities (MPH or dexamfetamine, 6 months, girls with a good response) (Longitudinal study with a control group, N = 40, n = 20, 6 months, E 2b.)²⁸⁴
• Unchanged theta/beta ratio despite improvement in symptoms (MPH, approximately 8 weeks) (Case-control study, N = 63, n = 32, approximately 8 weeks, author(s) employed by pharmaceutical companies, full text not available to us, E 3.)²⁸⁵

The EEG effects of a single dose of MPH (10 mg) in children with ADHD depended on the DAT1 genotype. Children with the 10R/10R genotype showed more central and parietal beta activity, less right frontal theta activity, and a lower theta/beta ratio while on MPH; carriers of the 9R allele showed the opposite pattern. (Double-blind, placebo-controlled single-dose study, n = 27, Phase 2b.)²⁸⁶

A study in rats using single and repeated doses of 0, 0.6, 2.5, and 10.0 mg MPH/kg found that MPH affected the PFC and the caudate nucleus. The same dose of MPH induced behavioral sensitization in some animals and tolerance in others, with activity in the PFC and caudate nucleus correlating with the animals’ behavioral responses to MPH. A larger proportion of neurons in the caudate nucleus than in the PFC responded to MPH, both following single and repeated administration. In addition, dose-dependent differences in responses were observed between the PFC and the caudate nucleus: Some PFC and caudate nucleus cell units responded to the same dose of MPH with an increase in neuronal firing rate, while others responded with a decrease. (Animal study, rats, E 2b.)²⁹⁶

(E 1b): MPH altered the activation of brain regions

• based on reviews by:
  • Children with ADHD (single dose): Increased activation in the middle and inferior frontal gyri (especially during inhibition tasks), in the basal ganglia, and in the cerebellum, usually with at least some areas returning to levels seen in healthy individuals (Systematic review, k = 9, E 2b.)²⁹⁸
  • ADHD (stimulants and non-stimulants combined): Increased activation in the left supplementary motor area (Z = 1.21, p < 0.0001, central executive network) (Meta-analysis, k = 28, N = 855, n = 396 on medication, E 2b.)²⁹⁹
  • ADHD (stimulants vs. non-stimulants): greater activation in the left amygdala (Z = 1.30, p = 0.0006), superior frontal gyrus (Z = 1.27, p = 0.0006), and middle cingulate gyrus (Z = 1.22, p = 0.0008), regions of the ventral attentional network (meta-analysis, k = 28, N = 855, n = 396 on medication, E 2b.)²⁹⁹
  • ADHD (long-term stimulant use): more normal activation of the right caudate nucleus during attention tasks (meta-regression, preliminary) (meta-analysis, k = 21 and 13 datasets, E 3.)³⁰⁰
• in individual studies on ADHD (single dose) for:
  • Interference inhibition (Simon task, medication-naive boys): Increased activation in the right inferior prefrontal and premotor cortices; underactivation in the right inferior PFC and striato-thalamic region normalized; medial frontal and temporal dysfunction unaffected (RCT, crossover, with healthy control group, N = 25, n = 12, single dose, E 1b.)³⁰¹
  • Sustained attention (adolescents): fronto-parietal and striato-thalamic underactivation normalized (as well as with atomoxetine); only MPH improved performance. MPH-specific increased activation in the left ventrolateral PFC and superior temporal cortex. (RCT, crossover, with healthy control group, N = 41, n = 14, single dose, E 1b.)³⁰²
  • Go/No-Go task (fNIRS, children aged 6 to 13): reduced activation in the right inferior and middle frontal gyri normalized; not observed with the placebo * E (RCT, crossover, with a healthy control group, N = 32, n = 16, single dose, E 1b.)*³⁰³
  • Processing of angry and fearful faces (0.5 mg/kg): Reduced reactivity of the right amygdala in children and adults; reduced reactivity of the left amygdala only in adults (pre-treatment: children −31%, adults +31% compared to controls) (Pharmacological fMRI with an open-label single dose and a control group, N = 104, n = 81, Phase 2b.)³⁰⁴
• in individual studies on ADHD (long-term treatment) for:
  • Go/No-Go task (6 to 8 weeks, comparison with atomoxetine): comparable improvement in symptoms (MPH 55%, atomoxetine 57%), along with reduced activation of the bilateral motor cortex. Under MPH, the improvement was accompanied by reduced activation, while under atomoxetine, it was accompanied by increased activation in the right inferior frontal gyrus, left ACC/supplementary motor area, and bilateral posterior cingulate cortex. (fMRI before-and-after study, parallel groups, N = 36, n = 18, 6 to 8 weeks, partially manufacturer-funded, E 2b.)³⁰⁵
  • Go/No-Go task (fNIRS, children, 6 months): Increased oxygenated hemoglobin in the right medial PFC, improved Go/No-Go accuracy and SNAP-IV scores (Before-and-after study with a healthy control group, N = 48, n = 36, 6 months, E 2b.)³⁰⁶
  • emotional face processing (16 weeks, boys aged 10 to 12 and men): no effect on amygdala reactivity, amygdala connectivity, or internalizing symptoms, unlike after acute administration. ADHD symptoms improved. (RCT, n = 99, 16 weeks, E 1b.)³⁰⁷
  • Magnetic resonance spectroscopy of the ACC (adults, 5 to 6 weeks): Choline-containing compounds decreased, N-acetylaspartate increased, creatine unchanged, with marked clinical improvement (Open-label before-and-after study, n = 7, 5 to 6 weeks, E 2b.)³⁰⁸
• In healthy individuals (single dose) for:
  • Rewarded conflict task: higher accuracy with lower activity in the attention and reward systems (particularly the insula during reward trials), interpreted as an increase in efficiency (RCT, crossover, n = 16, single dose, E 1b.)³⁰⁹
  • Rewarded working memory task (30 mg MPH, 60 mg atomoxetine): In unrewarded trials, only MPH was distinguishable from placebo, showing a reward-like activation pattern during encoding. During the retention period of rewarded trials, MPH increased the activity of working memory networks to a greater extent, while atomoxetine increased that of the DMN to a greater extent. (placebo-controlled crossover study, n = 15, single dose, E 1b.)³¹⁰
• In macaques: improved task performance at certain doses without changes in single-neuron activity, noise correlations, or ensemble activity in the caudal lateral PFC (2,811 neuronal recordings) (animal study, macaques, n = 2, 55 sessions, E 2b.)³¹¹

A review of animal studies found that MPH can trigger inflammatory processes in the brain and alter the apoptosis pathway, depending on age, duration of treatment, route of administration, and dose, with young animals being more susceptible. (Narrative review of animal studies; full text not available to us; E 4.)²³¹

Cranial nerves transmit information electrically. At the point where one nerve connects to another (synapse), the signal is transmitted across the synaptic cleft to another cranial nerve. This transmission of information typically occurs chemically via neurotransmitters (dopamine, norepinephrine, serotonin, and others). The electrical signal triggers the release of neurotransmitters (in this case, dopamine) into the synaptic cleft at the end of the nerve (presynaptic). At the receiving nerve on the other side of the synaptic cleft (postsynaptic), the neurotransmitter (in this case, dopamine) is taken up by (dopamine) receptors and, once a threshold number of activated receptors is reached, triggers the transmission of an (electrical) signal. The neurotransmitter is then released back into the synaptic cleft by the receiving neuron, from where the sending neuron reabsorbs it via specialized reuptake transporters (in the case of dopamine, the dopamine reuptake transporter, DAT) to be stored back into the vesicles for the next signal transmission.
In ADHD, the DAT reuptake transporters (located primarily in the striatum) are overactive. When dopamine is released from the presynaptic neuron into the synaptic cleft, the DATs of the presynaptic neuron reabsorb the dopamine before it can be taken up by the postsynaptic receptors of the target neuron. The signaling pathway is thus disrupted; in terms of dopamine, this is comparable to static in a radio signal (“neural noise”). (Case-control study, n = 61, E 3)³³⁴ Stimulants such as methylphenidate inhibit DAT activity, allowing dopamine to remain in the synaptic cleft long enough for the signal to be transmitted clearly. Thus, MPH reduced neural noise in people with ADHD to the level seen in people without the condition. (case-control study, n = 61, E 3)³³⁴
What is unique about dopaminergic synapses is that, according to the latest findings (2019), there are no dopamine receptors at all on the receiving end of the dopaminergic synapse—instead, there are GABA receptors. Rather, the dopamine receptors are spatially arranged around the synapse and respond to dopamine that diffuses out of the synapse or is released in some other way.

Early-life stress, when combined with a corresponding genetic predisposition, leads to long-term damage to stress regulation systems. Such the establishment of a stress response could potentially be prevented through timely pharmacological treatment. In mice exposed to stress, the serotonin reuptake inhibitor fluoxetine normalized the risk-assessment behavior (an anxiety-related evaluation of dangers) that had been heightened by chronic mild stress, whereas the GABA-A receptor modulator diazepam did not produce this effect. (Animal study, mice, 5 weeks, E 2b.)³³⁶

Chronic administration of caffeine or MPH before puberty resulted in improved object recognition in adult SHR rats (a rat strain that represents a genetic form of ADHD-HI), whereas the same treatment impaired object recognition in adult Wistar rats. (Animal study, rats (SHR, Wistar), 14 days, E 2b)³³⁷

(E 2b): Administration of 2 mg/kg MPH via intraperitoneal injection twice daily before puberty (days 20 through 35) into the abdominal cavity of NON-ADHD model rats resulted in the following effects in adulthood (which could be normalized by fluoxetine or the kappa-opioid antagonist nor-binaltorphimine): (Animal study, rats (Sprague-Dawley), 16 days, E 2b.)³³⁸ * (Animal study, rats (Sprague-Dawley), 16 days, E 2b.)*³³⁹

• increased anxiety
• increased symptoms of depression
• reduced ability to be motivated
  • due to drugs (morphine or cocaine place preference)
  • lower sucrose preference

In contrast, oral administration to non-ADHD model rats beginning on day 27 for 4 to 7 weeks, 5 days a week,

• Did not increase anxiety (animal study, Wistar rats, 4 or 7 weeks, E 2b)³⁴⁰
• temporarily altered hippocampus-sensitive learning but did not cause any cognitive impairments (animal study, Wistar rats, 7 weeks, E 2b)³⁴¹

Wistar rats (a non-ADHD model) that received MPH at a therapeutic dose—but administered via injection—for 14 days during adolescence (days 30 through 44) exhibited reduced impulsivity as adult animals. In addition, total creatine and taurine levels were elevated in the dorsal striatum and reduced in the nucleus accumbens. (Animal study, n = 16, rats (Wistar), 14 days, E 2b.)³⁴²

(E 2b): One study examined choice preferences between frequent small rewards and infrequent large rewards (nudging a lever to obtain food). Adult rats that had received MPH during adolescence showed a shift in preference that can be interpreted as increased efficiency and/or flexibility, leading to a “better” (i.e., more optimal) decision-making process. (Animal study, rats (Wistar), 16 days, E 2b)³⁴³ * (Animal study, n = 16, rats (Wistar), 14 days, E 2b)³⁴² * (Narrative review, E 4)⁷⁹

Sprague-Dawley rats (a non-ADHD model) that had received intraperitoneal injections of MPH (2 mg/kg) or cocaine during adolescence showed the following results as adults in place conditioning tests: (Animal study, rats (Sprague-Dawley), 16 days, E 2b)³⁴⁴

• an aversive reaction to moderate doses of cocaine
• a reduced rewarding effect of high doses of cocaine
• Early exposure to MPH produces depression-like effects in the forced swim test
• early MPH exposure is associated with reduced habituation to the activity chambers (a sign of greater stress sensitivity)

Sprague-Dawley rats (a non-ADHD model) that had received intraperitoneal injections of MPH (2 mg/kg) twice daily during adolescence (days 20 through 35) exhibited the following as adults: (Animal study, rats (Sprague-Dawley), 16 days, E 2b)³⁴⁵

• lower reward reactance to sucrose, novelty-induced activity, and sex
• significantly more sensitive reactions to stressful situations and elevated plasma levels of corticosterone
• more frequent anxiety-like behavior

In adult rats that received MPH during adolescence, changes in taurine levels were observed that could lead to hyperactivity in the dorsal striatum and the associated hypofunction of the nucleus accumbens. Taurine may influence the extent to which a given brain region is capable of processing neural information and regulating behavior. The taurine level in a brain region could serve as an index of regional reactivity to synaptic input signals and thus of the overall function of that brain region. (Narrative Review, E 4)⁷⁹
Furthermore, adult rats that received MPH during adolescence showed an increase in the phosphocreatine-to-creatine ratio in the PFC, which could be a consequence of improved energy metabolism. The authors hypothesize that the enhanced function of the PFC is responsible for downregulating the functional activity of the nucleus accumbens and increasing the activity of the dorsal striatum. Studies suggest distinct functional roles for PFC projections to these two target regions. While the circuit from the PFC to the striatum serves attention, which influences executive planning and the selection of actions, the pathway from the PFC to the nucleus accumbens serves to integrate information about the motivational consequences of an action. This could explain how PFC regions modulate subcortical activity. (Narrative Review, E 4)⁷⁹

While treatment of rats with MPH over 16 days during adolescence resulted in changes in the expression of approximately 700 genes in the striatum at the end of treatment, only 2 genes (Grik2 and Htr7) were found to still have elevated expression in adulthood. (Narrative review, E 4.)⁷⁹

Slight changes were also observed in the average levels of N-acetyl aspartate and glutamine in the nucleus accumbens, as well as in the total choline content in the dorsal striatum.
N-acetylaspartate (Narrative Review, E 4)³⁴⁶ Aging and neurodegenerative processes reduce N-acetylaspartate levels. (Narrative review, E 4)³⁴⁷ Rats exposed to MPH also showed reduced N-acetylaspartate levels as adults. (Narrative review, E 4)⁷⁹

To the extent that the tests listed above were conducted using non-ADHD model animals, we believe they have only very limited relevance to the effects of MPH in rats with ADHD. When administering medication to organisms that do not exhibit the deficit the medication is intended to correct, adverse effects of the medication are virtually to be expected.
However, the tests are valuable in that, in theory, stimulants administered early on have the potential to produce a curative effect.
Tests on ADHD animal models would be valuable.

Adolescents who took psychostimulants showed a slower rate of cortical thinning in the right motor strip, the left middle/inferior frontal gyrus, and the right parieto-occipital region, compared to the elevated rates observed in adolescents with ADHD who were not taking medication (0.03 mm/year vs. 0.16 mm/year = less than one-fifth). (cohort study, n = 43, E 2b)³⁴⁸

Administration of MPH before age 12 was associated with morphological changes in the brain, unlike administration starting at age 12. (Longitudinal study, n = 180, 4.9 years, E 2b.)³⁴⁹ An increased cumulative methylphenidate dose before age 12 was associated with:

• increased gray matter volume in several frontal regions
  • right:
    • paracentral cortex
    • caudal medial frontal cortex
    • superior frontal cortex
    • lateral orbitofrontal cortex
    • rostral medial frontal cortex
    • precentral cortex
  • left:
    • Pars opercularis
    • paracentral cortex
    • superior frontal cortex
• a greater improvement in symptoms of opposition with a larger increase in volume in:
  • right rostral-medial frontal cortex
  • right paracentral cortex
  • right superior frontal cortex
  • left paracentral cortex

Methylphenidate and amphetamine increased the power of the alpha band in rats, while atomoxetine and guanfacine did not. (Animal study, rats; author(s) employed by the manufacturer (of ASP2905); E 2b.)³⁵⁰

It is unclear what concentrations of methylphenidate reach the synaptic cleft. (Animal and in vitro study, mice, E 2b)¹⁵
In mice, the brain concentration of methylphenidate was 4 to 8 times higher than the plasma concentration 15 to 30 minutes after oral administration of a therapeutically relevant dose (0.75 mg/kg). The authors cited passive diffusion with a saturable transport component as the mechanism. (Animal study, mice, E 2b.)³⁵² For cocaine, concentrations in brain regions of rats following intravenous self-administration were 3.3 to 6.2 times higher than in plasma, with the highest levels in the striatum. (Animal study, N = 26, rats (Wistar), E 2b.)³⁵³