4. Dopamine formation and storage

Dopamine is synthesized in several steps. First, L-phenylalanine from food is converted to L-tyrosine. L-tyrosine is then converted to L-dopa and finally to dopamine. Alternatively, dopamine can also be synthesized directly from L-phenylalanine. Another alternative synthesis pathway is via tyramine, which is converted to dopamine. Noradrenaline is synthesized by converting dopamine, while adrenaline is synthesized from noradrenaline.

The formation of dopamine takes place in dopaminergic cells in various brain regions, primarily in the VTA (A10) and substantia nigra (A9) as well as in the retrorubral nucleus (A8) and the arcuate nucleus.
Dopamine from the VTA influences motivated behavior and reinforcement learning. Different types of VTA neurons are distinguished, including purely dopaminergic, purely GABAergic and VGLUT2-positive neurons. These each have different functions and project to different regions of the brain.

The activity of dopamine synthesis is regulated by various enzymes and factors. These include tyrosine hydroxylase (TH), tetrahydrobiopterin (BH4), GTP cyclohydrolase 1 (GTPCH), aromatic L-amino acid decarboxylase (AADC), pyridoxal phosphate (vitamin B6) and oxygen concentration.
Mechanisms such as phosphorylation, dephosphorylation and feedback inhibition influence the activity of the enzymes. Binding to protein complexes and interaction with other proteins also play a role.

The synthesis and storage of dopamine in vesicles is carried out by the enzyme VMAT2.
Blockade of VMAT function leads to parkinsonian-like behavioral effects that can be remedied by L-DOPA. VMAT2 inhibitors inhibit dopamine formation and reduce dopamine release. The storage of dopamine in vesicles depends on the cytosolic dopamine concentration. VMAT2 can be regulated by several mechanisms, including cytosolic chloride concentration, acidification by V-ATPase, post-translational modifications and the amount of dopamine in the cytoplasm. Various drugs can increase or inhibit VMAT2 activity. Increased VMAT2 expression leads to an increase in the amount of dopamine in vesicles and dopamine release.

• 4. Synthesis and storage of dopamine, noradrenaline, adrenaline
  • 4.1. Synthesis of dopamine, noradrenaline, adrenaline
  • 4.2. Individual elements of dopamine synthesis
    • 4.2.1. L-phenylalanine
    • 4.2.2. Tyrosine
      • 4.2.2.1. Tyrosine increases dopamine and noradrenaline
      • 4.2.2.2. Tyrosine depletion reduces dopamine and noradrenaline
      • 4.2.2.3. Tyrosine excess causes inattention
    • 4.2.3. Tyrosine hydroxylase (TH)
    • 4.2.4. Tetrahydrobiopterin (BH4, sapropterin, INN)
    • 4.2.5. GTP cyclohydrolase 1 (GTPCH)
    • 4.2.6. Aromatic L-amino acid decarboxylase (AADC, Dopa decarboxylase)
    • 4.2.7. Pyridoxal phosphate (PLP, PALP, P5P, active vitamin B6)
    • 4.2.8. Oxygen concentration and dopamine synthesis
    • 4.2.9. Sports and thinking influence dopamine synthesis
  • 4.3. Site of dopamine synthesis
    • 4.3.1. Dopamine synthesis within the neurons
    • 4.3.2. Brain regions in which dopamine is produced
      • 4.3.2.1. Substantia nigra pars compacta
      • 4.3.2.2. VTA: dopamine, GABA, VGLUT2
  • 4.4. Storage of dopamine in the vesicles (VMAT)
    • 4.4.1. VMAT isoforms
    • 4.4.2. Regulation of VMAT
    • 4.4.3. Effect of VMAT2

4. Synthesis and storage of dopamine, noradrenaline, adrenaline¶

4.1. Synthesis of dopamine, noradrenaline, adrenaline¶

The formation of dopamine takes place in several steps:¹²

• L-phenylalanine from food
  is synthesized by phenylalanine hydroxylase using tetrahydrobiopterin (BH4), folic acid and oxygen
• To L-Tyrosine
  this is achieved by tyrosine hydroxylase (TH) using BH4 and calcium citrate
• To L-Dopa (dihydroxyphenylalanine)
  this is achieved by aromatic L-amino acid decarboxylase (AADC, dopa decarboxylase) with the consumption of pyridoxal phosphate (vitamin B6)
• To dopamine and CO2 (carbon dioxide)

Alternative ways of synthesizing dopamine are ²

• L-phenylalanine from food
  is synthesized by aromatic L-amino acid decarboxylase (AADC, dopa decarboxylase) using pyridoxal phosphate (vitamin B6)
• To phenylethylalamine
  this becomes
• To tyramine

or

• L-tyrosine
  is synthesized by aromatic L-amino acid decarboxylase (AADC, dopa decarboxylase) using pyridoxal phosphate (vitamin B6)
• To tyramine
  in humans, this is mediated by CYP2D6³, in rats by CYP2D2, CYP2D4 and CYP2D18⁴
• To dopamine

This alternative synthesis pathway appears to be quantitatively modest under normal physiological conditions in rats, but may be more efficient in the human brain and may be particularly important when the main synthesis pathway is impaired (e.g. tyrosine hydroxylase deficiency or aromatic amino acid decarboxylase deficiency). Furthermore, alternative CYP2D6-mediated dopamine synthesis could be relatively important in individuals who have more than one CYP2D6 gene (e.g. in Mediterranean populations)⁵

In addition, dopamine synthesis occurs - particularly in the blood - through the conversion of

• Dopamine sulfate
  through sulfatases⁶
• To dopamine

This appears to be influenced in particular by steroid sulphatase (STS, arylsulphatase C). An STS deficiency or STS gene defect is associated with a significantly increased risk of ADHD.

This remains the case in dopaminergic cells.
Dopamine is converted to noradrenaline in noradrenergic cells:

• Dopamine
  is produced by the enzyme dopamine β-hydroxylase (DHB) using oxidized vitamin C
• To noradrenaline

This remains the case in noradrenergic cells.
Noradrenaline is converted to adrenaline in adrenergic cells:

• Noradrenaline
  is synthesized by the enzyme phenylethanolamine N-methyltransferase (PNMTase) using S-adenosylmethionine (SAM), pyridoxal phosphate (vitamin B6) and vitamin B12
• To adrenaline.

Dopamine is also a precursor (precursor) for

• Isoquinoline alkaloids
  • E.g. Berber
• Morphine

4.2. Individual elements of dopamine synthesis¶

4.2.1. L-phenylalanine¶

Phenylalanine inhibits the hydroxylation of tyrosine.⁹

While the administration of tyrosine increases dopa and dopamine in the retina of rats, the administration of phenylalanine does not lead directly to an increase in dopa in the retina, but only indirectly via the increase in tyrosine through phenylalanine hydroxylation in the liver.¹⁰
Phenylalanine inhibited the conversion of labeled tyrosine to DOPA less effectively than tyrosine inhibited the conversion of labeled phenylalanine to DOPA. This indicates that the tyrosine hydroxylase affinity of tyrosine is higher than that of phenylalanine.¹⁰
Studies in PC-12 cells indicate that almost all DOPA synthesized in the retina results from tyrosine at normal retinal tyrosine concentrations, regardless of retinal phenylalanine concentration. Fluctuations in retinal phenylalanine have little effect on total DOPA synthesis in the retina.¹⁰

Using PC-12 cells exposed to different concentrations of phenylalanine and tyrosine, it was found that

• High extracellular phenylalanine levels lead to a depletion of intracellular tyrosine and dopamine.
• Unlike physiological phenylalanine concentrations (75 μM):¹¹
  • low (35 μM) phenylalanine concentrations as well as
  • high tyrosine concentrations (275 or 835 μM)
    reduced levels of
    • cellular dopamine
    • Tyrosine hydroxylase (TH)
    • TH phosphorylation values

In the liver, phenylalanine is immediately metabolized to tyrosine.⁹

Phenylketonuria (PKU) is an inborn error of amino acid metabolism in which phenylalanine cannot be metabolized to tyrosine due to a lack of phenylalanine hydroxylases. PKU is associated with increased phenylalanine levels, reduced tyrosine levels and ADHD symptoms.

4.2.2. Tyrosine¶

Tyrosine is a precursor in the synthesis of dopamine. Although the rate-limiting enzyme of catecholamine synthesis is not tyrosine but tyrosine hydroxylase, an increase in tyrosine levels in the brain stimulates catecholamine production, but only in actively firing neurons ⁹¹²

Tyrosine is oxidized by tyrosinase.

4.2.2.1. Tyrosine increases dopamine and noradrenaline¶

Tyrosine administration increased extracellular dopamine in the striatum and nucleus accumbens in rats.¹³ Another source reported this for the striatum only if the animals had previously received a dopamine receptor antagonist (halperidol).¹⁴ Tyrosine administration also significantly increased (in light only) the conversion of tyrosine to L-dopa (part of the dopamine synthesis pathway) in the retina of rats¹⁵ and subsequently increased dopamine in the retina.¹⁶ Since dopamine suppresses melatonin, this pathway could influence the circadian rhythm. More on this at Melatonin and dopamine and the circadian rhythm in the article Melatonin in ADHD
In rats, the tyrosine level in the hypothalamus varies linearly with a range of protein content in the diet between 2 and 10 %. At 20 %, the tyrosine level in the hypothalamus barely increased further.⁹

Protein-rich food increases the tyrosine level in the brain. Theoretically, a protein-rich diet could therefore help to alleviate a dopamine deficiency and the resulting ADHD symptoms. For people with ADHD whose dopamine synthesis functions normally, the effect is likely to be small. If a dopamine deficiency is explicitly caused by dopamine synthesis problems due to tyrosine deficiency, the effect could be stronger. Although this is only very rarely the case, we believe it is conceivable that this could be one of the many pathways leading to ADHD.

This opens up the possibility of treatment with tyrosine for ADHD.
More on this under Tyrosine for ADHD.

4.2.2.2. Tyrosine depletion reduces dopamine and noradrenaline¶

The amount of tyrosine available in the brain is also influenced by other large neutral amino acids that compete with tyrosine for the same transporter across the blood-brain barrier, e.g:⁹¹⁷

• Tryptophan
• Phenylalanine
• L-methionine
• Histidine
• Threonine
• L-glycine
• L-lysine
• L-arginine
• L-leucine
• L-isoleucine
• L-valine
• but not alanine¹⁸

Therefore, administration of these large neutral amino acids (without tyrosine and without phenylalanine, as phenylalanine is immediately metabolized to tyrosine in the liver) can reduce brain tyrosine levels in the hypothalamus and retina within 2 hours.¹⁷ Consequences are that the dopamine level in the brain also decreases, but not in the PFC ⁹¹⁹

This opens up treatment options for disorders characterized by excessive dopamine or noradrenaline levels.
More on this under Tyrosine for ADHD.

4.2.2.3. Tyrosine excess causes inattention¶

Tyrosinemia (here: hereditary tyrosinemia type 1, HT-1 or TT-1), a rare (1:100,000 to 120,000 live births) disorder of tyrosine degradation, which thus leads to increased tyrosine levels, is associated with increased inattention.²⁰²¹

4.2.3. Tyrosine hydroxylase (TH)¶

TH is the rate-limiting enzyme of dopamine synthesis. TH uses BH4 and molecular oxygen to convert tyrosine into DOPA.²²

What influences the activity of TH thus influences dopamine synthesis. Some mechanisms alter the activity of TyrH, others simply bring the enzyme into the vicinity of other related proteins. Post-translational mechanisms of TH regulation include:²²

• Phosphorylation by kinases
  • The activity and stability of tyrosine hydroxylase is influenced by various kinases such as PKA, PKC, CaMPKII, PKG, MPK and ERK, which phosphorylate it at serine sites 8 (rat only), 19, 31 and 40. Phosphorylation of the tyrosine hydroxylase²³
    • at Ser40 strongly increases TH activity (up to 10-fold)
    • to Ser31 slightly increases TH activity
    • at Ser19 or Ser8 (rat only) does not affect TH activity
    • at Ser19 increases the rate of Ser40 phosphorylation, which increases TH activity.
  • Furthermore, the arginine sites 37 and 38 appear to be able to regulate TH. Deletion or replacement of arginine 37 and 38 by glycine or glutamate resulted in improved BH4 affinity and thus increased TH activity.²
• Dephosphorylation by phosphatases
  • The phosphatases PP2A and PP2C reverse the phosphorylation of TH and can thus deactivate TH²
• Feedback inhibition
  • Catecholamines regulate TH. Catecholamines compete with BH4 for the binding of the iron(III) ion at the catalytic site of tyrosine hydroxylase. As a result, high catecholamine levels inhibit tyrosine hydroxylase and thus its own synthesis in the form of a feedback loop.²
  • Dopamine inhibits TH (also in the presence of GTPCH) as a negative feedback loop²
• Oxidation by nitrites
  • TH can be inactivated by nitration using reactive nitrogen species (peroxynitrite) or by S-thiolation on cysteine residues.²
• Integration into protein complexes
  • The stability, activity and probably also the intracellular localization of TH is also regulated by interactions with other proteins, such as²
    • 14-3-3
      • activated TH²⁴
    • α-Synuclein
      • inhibits TH²⁴
    • VMAT-2
    • AADC
    • GTPCH
    • DJ-1 (protein deglycase DJ-1, also known as Parkinson’s disease protein 7, PARK7) regulates TH.
      • DJ-1 regulates TH transcription by altering the acetylation status of the TH promoter. DJ-1 increases TH expression by inhibiting the sumoylation of PSF and preventing its sumoylation-dependent recruitment of histone deacetylase 1. Silencing of DJ-1 decreases the acetylation of TH promoter-bound histones. Histone deacetylase inhibitors restore DJ-1 siRNA-induced repression of TH. DJ-1 silencing thus results in reduced TH expression and reduced dopamine synthesis.²⁵
      • The oxidation state of DJ-1 regulates its own activity and thus also TH expression²²
        • DJ-1 is a transcriptional regulator
        • DJ-1 plays a role in bypassing oxidative stress
        • Certain DJ-1 variants have been identified in Parkinson’s disease²⁶
        • DJ-1 is a TH repressor and binds to the promoter of the TyrH gene
        • DJ-1 binds directly to TyrH itself (as well as to AADC) binds and activates TH like AADC
• Salsolinol, a tetrahydroisoquinoline alkaloid, inhibits TH²⁷
  • is found at up to 25 µg/g in chocolate, cocoa and bananas, but cannot cross the blood-brain barrier.
  • is also formed endogenously in the organism
• Cortisol inhibits TH.²⁸
• Alpha-methyl-p-tyrosine (AMPT, α-methyl-DL-tyrosine, αMPT) inhibits TH so strongly that catecholamine synthesis is blocked and the dopamine tissue level in DAT-KO mice drops to 0.2% of the normal level. This extreme dopamine deficiency after AMPT causes Parkinson’s symptoms such as severe akinesia, rigidity, tremor and ptosis.²⁹
• Aspirin at low doses (2 mg / kg / day) increased TH and subsequently dopamine in the striatum in mice.³⁰
• Prolactin is said to increase TH synthesis and TH activity and thus dopamine synthesis within 12 to 16 hours³¹³²

Tyrosine hydroxylase prefers to bind to tyrosine rather than phenylalanine.¹⁰

4.2.4. Tetrahydrobiopterin (BH4, sapropterin, INN)¶

Tetrahydrobiopterin (BH4) is produced from guanosine triphosphate by GTP cyclohydrolase 1 (GTPCH). GTPCH is the limiting factor in BH4 synthesis.³³

• excessive BH4 levels block GTPCH
  • this prevents GTPCH-TH interaction
  • therefore too much BH4 impairs the TH
• bH4 levels that are too low inhibit TH
  • BH4 is a cofactor for TH
• BH4 only promotes tyrosine hydroxylase from 10 to 25 and up to 100 microM. Lower or higher quantities inhibit it²
  BH4 deficiency or BH4 excess can therefore hinder dopamine synthesis. BH4 administration can improve ADHD symptoms resulting from phenylketunorie.³⁴

Genetically caused BH4 disorders such as

• autosomal recessive (AR) guanosine triphosphate cyclohydrolase deficiency (GTPCH deficiency)
• 6-Pyruvoyl tetrahydropterin synthase deficiency (PTPS)

seem to contribute to ADHD and other mental disorders such as anxiety, depression, aggression or oppositional defiant behavior.³⁵

4.2.5. GTP cyclohydrolase 1 (GTPCH)¶

GTPCH interacts with tyrosine hydroxylase²

• cAMP administration increases GTPCH gene expression
• Phenylalanine induces GTPCH activity
• Phosphorylation of Ser81 increases GTPCH activity.
• BH4 inhibits GTPCH via the GTPCH feedback regulatory protein (GFRP) in the form of a feedback loop

GTPCH interacts with tyrosine hydroxylase²

• The interaction depends on the phosphorylation of TH and GTPCH.
• Interaction with TH prevented BH4-mediated inhibition of GTPCH
  • this increased the GTPCH and TH activity
• GTPCH activity is stimulated by phosphorylated TH
• Dopamine synthesis is dependent on GTPCH
• BH4 does not increase TH activity in Drosophila with inactive GTPCH gene versions
• TH activity depends on an interaction between TH and GTPCH

4.2.6. Aromatic L-amino acid decarboxylase (AADC, Dopa decarboxylase)¶

AADC is involved in the synthesis of catecholamines, indoleamines and trace amines.³⁶

• Decarboxylation of aromatic amino acids occurs in different cell types, so that trace amines can theoretically be formed in these:³⁷
  • Nerve cells
  • Glial cells
  • Blood vessels
  • Cells of the gastrointestinal tract
  • Kidney
  • Liver
  • Lung
  • Stomach
    • Serotonergic AADC, especially in the pylorus³⁸

Cells containing AADC were found in the following areas of the brain:³⁹

• Raphe nuclei (serotonergic, very strong AADC)
• Pons ventral (serotonergic, very strong AADC)
• Medulla ventral (serotonergic, very strong AADC)
• Mesencephalic reticular formation (dopaminergic, moderate to strong AADC)
• Substantia nigra (dopaminergic, moderate to strong AADC)
• VTA (domapinergic, moderate to strong AADC)
• Locus coeruleus (noradrenergic, moderate to strong AADC)
• Subcoeruleus nuclei (noradrenergic, moderate to strong AADC)
• Median and ventrolateral parts of the intermediate reticular nucleus in the medulla oblongata (noradrenergic / adrenergic, moderate AADC)
• Forebrain: few non-aminergic AADC-positive neurons (D-neurons); these were not detectable in other parts of the human brain.

AADC catalyzes the decarboxylation of various aromatic l-amino acids such as:

• L-DOPA
• L-tyrosine
• L-tryptophan
• L-histidine

AADC is itself subject to regulatory influences:

• Transcriptional (slower, longer-lasting regulation through adaptation of gene expression)²
  • Expression is influenced by
    • Different use of promoters
    • Different splicing
• Post-translational (fast-acting, short-term mechanism)
  • Phosphorylation³⁶
    • By PKA in the striatum and midbrain
    • AADC increased by 20 to 70
  • PH value changes⁴⁰
  • Denaturation⁴⁰
  • Neurotransmitter²
    • Antagonists enhance, agonists reduce AADC activity⁴¹
    • Dopamine via
      • Dopamine D1 to D4 receptors⁴¹
      • Dopamine antagonists increase AADC, e.g.
        • Cis-flupenthixol
        • Haloperidol
      • Dopamine increase reduces AADC²
        • MOA inhibitors
      • The destruction of dopaminergic cells only reduced the dopaminergic 3,4-dihydroxyphenylalanine decarboxylase, not the serotonergic 5-hydroxytryptophan decarboxylase activity, which actually increased⁴²
      • In contrast, another study found that the destruction of both dopaminergic and serotonergic neurons reduced both dopaminergic and serotonergic AADC equally.⁴³
    • Serotonin via
      • 5-HT 1A receptor⁴¹
      • 5-HT 2A receptor⁴¹
    • Glutamate via
      • NMDA receptor⁴¹
    • Nicotinic acetylcholine receptors⁴¹

Different forms of AADC induce dopaminergic and serotonergic decarboxylase. The two forms showed different activity maxima depending on pH, temperature and substrate concentrations:⁴⁴

• Dopaminergic: 3,4-dihydroxyphenylalanine decarboxylase
  * Dopaminergic AADC activity:
  * Only in soluble cell fractions⁴⁴
  * Distribution corresponded to lactic acid dehydrogenase⁴⁴
  * Pyridoxal-5-phosphate increases this by a factor of 20⁴⁵
  * Carboxyl scavengers inhibit them completely⁴⁴

• Serotonerg: 5-hydroxytryptophan decarboxylase
  * In soluble and particulate cell fractions⁴⁴
  * Pyridoxal-5-phosphate doubles this⁴⁴
  * Pyridoxal-P antagonists barely affect them⁴⁴
  * Serotonergic AADC is potentiated by 3-isobutyl-1-methylxanthine.³⁸

In ADHD, AADC activity was found to be reduced by 50% in the medial and left PFC, but not in the striatum or midbrain.⁴⁶ Another study by the same research group with a smaller number of subjects found increased AADC activity in ADHD⁴⁷

AADC is also relevant in the synthesis of melatonin. There could be a connection here with the increased sleep problems in ADHD.

4.2.7. Pyridoxal phosphate (PLP, PALP, P5P, active vitamin B6)¶

Pyridoxal phosphate (PLP, PALP, P5P) is one of the most important cofactors in the animal organism. Pyridoxal phosphate is the metabolically active form of vitamin B6.
PLP is involved in various amino acid reactions:

• Transamination
  catalyzed e.g. by
  • GABA transaminase
• Decarboxylation
  catalyzed e.g. by
  • AADC
  • Histidine decarboxylase
  • Ornithine decarboxylase
• Dehydration
• Breakdown of glycogen

4.2.8. Oxygen concentration and dopamine synthesis¶

The intracellular O2 concentration in brain tissue influences the synthesis and stability of dopamine. The O2 concentration in brain tissue is normally 1-5 %, which is significantly lower than the 20 % in the atmosphere. Increased oxygen levels induce dopamine oxidation and thus the formation of ROS.⁴⁸ Hypoxia increases TH activity and thus dopamine synthesis.⁴⁹

4.2.9. Sports and thinking influence dopamine synthesis¶

Dopamine production is increased by

• Exercise and sports⁵⁵
• (Strategic) thinking (context: workaholism)

Some people with ADHD play sports excessively. Sports are “needed”. Sports increase stress resistance.

4.3. Site of dopamine synthesis¶

4.3.1. Dopamine synthesis within the neurons¶

Neurotransmitters are formed in the cell body (soma) and, enclosed in vesicles, are transported to the synapses mainly through the microtubules via the axons in the nerve fibers at up to approx. 25 to 40 cm/day.⁵⁶⁵⁷⁵⁸

4.3.2. Brain regions in which dopamine is produced¶

The formation of dopamine in the brain takes place in nerve cells from:⁵⁹⁶⁰ A8 to A15, as well as in the small cell groups of the Aaq (rostral in the central gray, midbrain) and the telencephalic group (A16, A17).⁶¹

• in the ventral midbrain (around 70 % of DA neurons)
  • VTA (in A10)
    • Mesolimbic system; target: limbic system
    • Mesocortical system; target: cortex
  • Substantia nigra (in A9)
    • Pars reticulata (SNpr) and Pars compacta (SNpc)
    • Mesostriatal system (nigrostriatal system); target: basal ganglia (including striatum)
  • Nucleus retrorubralis (nucleus retrorubricus, A8, in the thalamus).
    • Mesostriatal system (nigrostriatal system); target: basal ganglia (including striatum)
• in the diencephalon: A11 to A15
  • subparafascicular thalamic nucleus, A11
    • from here, dopamine innervates the superior olivary complex and the inferior colliculus in the brainstem (A13)
    • where it is supposed to regulate auditory processing
  • Nucleus arcuatus (nucleus infundibularis, core area at the lower end of the infundibulum of the hypothalamus, A12)
    • Tuberoinfundibular system; target: anterior pituitary gland
    • Inhibition of prolactin
  • Dopaminergic neurons of the preoptic area (A14)
    • release DA into the pituitary portal vein system and thereby regulate the secretion of prolactin and growth hormone⁶²
  • The A15 neurons are located in the periventricular area of the rostral hypothalamus⁶²
• in the telencephalon (end brain)
  • glomerular layer of the olfactory bulb (A16)
  • Retina (A17)
    • Dopamine is formed in amacrine and interplexiform cells in the retina⁶³
    • Lack of bright daylight in childhood promotes short-sightedness due to dopamine deficiency in the eye
• in the brain stem
  • Locus coeruleus^64656667
  • dopaminergic projection in dorsal hippocampus (73 % of dopaminergic tone in dorsal hippocampus; remainder from VTA)
  • dopaminergic projection in PFC

After synthesis in the nerve cell, the dopamine is packaged in vesicles and transported through the axons to the nerve endings. This axonal transport of vesicles takes place at 25 to 40 cm / day.
Dopamine is synthesized in the cytosol. The cytosol is located in the soma, but also in the axon, so that in dopaminergic cells, dopamine is also synthesized in the axon and its endings (terminals).⁵⁸

4.3.2.1. Substantia nigra pars compacta¶

The substantia nigra pars compacta contains more than 70 % of all dopaminergic neurons in humans (in young people there are around 400,000 to 600,000 in total). It is located in Brodman area A9 in the ventral midbrain and bears its name (“black substance”) due to its high iron and neuromelanin levels, which make it appear darker than neighboring areas.⁶¹

A dense network of axons emanates from the dopaminergic neurons of the substantia nigra, via which the target regions in the dorsal striatum in particular are supplied with dopamine.⁶⁸

4.3.2.2. VTA: dopamine, GABA, VGLUT2¶

Dopamine from the VTA modulates motivated behavior and reinforcement learning.
The other neurotransmitters released by VTA neurons also influence motivation.

There are 6 types of VTA neurons:

• purely dopaminergic⁶⁹
  • Activation through positive stimuli such as food, sugar, water or addictive substances
  • Projection according to: Nucleus accumbens
    • NAc D1-type MSN
      • activated when dopamine is released, by strengthening the PKA pathway
      • encode reward/positive stimuli
      • directly inhibit the ventral mesencephalon, which in turn inhibits the thalamus (direct pathway).
    • NAc D2 type MSN
      • activated when dopamine is low by activation of the adenosine A2A receptor (A2AR), which increases intracellular calcium levels
      • encode aversive/negative stimuli
      • disinhibit the ventral mesencephalon by suppressing the ventral pallidum (indirect pathway)
  • half of the dopaminergic VTA neurons are active and fire spontaneously
    • the other half is inactive and does not fire spontaneously⁷⁰ because they are constantly hyperpolarized by an inhibitory GABAergic influence from the ventral pallidum and thus kept inactive. By suppressing the pallidal afferents, the neurons are freed from the GABAergic inhibition and fire spontaneously again.⁷¹⁷²
• purely GABAergic⁶⁹
  • GABAergic VTA neurons project to nucleus accumbens, PFC, central amygdala, lateral habenula and the dorsal raphe nuclei.
  • GABA-A receptors cause hyperpolarization of postneurons through the influx of chloride ions
  • GABA-B receptors further induce hyperpolarization by suppressing adenylyl cyclase and voltage-gated calcium channels
  • Tasks:
    • Inhibition of dopaminergic neurons in the VTA and inhibition of distal brain regions
    • Aversion and interruption of rewards
    • Response to cues and reward-associated learning
    • VTA GABAergic neurons become active and suppress VTA DA neurons when mice anticipate a reward such as sucrose or cocaine after being exposed to a stimulus unrelated to the reward. GABAergic neurons in the VTA thus control VTA dopaminergic activity by interrupting reward consumption and Disorder responsiveness
    • regulate place preference when the projection of caudal GABAergic neurons of the VTA to serotonergic neurons of the DRN is activated
    • GABAergic VTA neurons predict the absence of a reward⁷³
• purely VGLUT2 (type 2 vesicular glutamate transporter)⁶⁹
  • Knockout of glutamatergic VTA neurons reduces motivated behavior
  • VGLUT2-VTA neurons become active during drug self-administration, drug-seeking behavior and reinforcement
  • project into the medial shell of the nucleus accumbens, ventral palladium and lateral habenula
  • directly excite the nucleus accumbens and ventral palladium
  • inhibit lateral habenula
  • Glutamate in the axon terminal binds to N-methyl-D-aspartate receptors (NMDA-Rs), leading to calcium influx that activates the pERK signaling pathway
  • become active in classical conditioning when exposed to a reward or electric shock
    • active firing in response to the conditioned stimulus associated with the reward
  • glutamatergic VTA neurons predict a reward⁷³
• VGLUT2/dopamine neurons⁶⁹
  • release glutamate and dopamine together at the axon terminals
    • Glutamate via asymmetric synapses
    • Dopamine via symmetrical synapses
  • VGLUT2-DA neurons increase survivability and axonal arborization of VTA dopamine neurons
  • project to the cholinergic neurons in the medial shell of the nucleus accumbens
  • contribute to switching the behavioral response in tasks with cognitive reinforcement
• VGLUT2/GABA neurons⁶⁹
  • differ from pure VGLUT neurons at the circuit level
  • are strongly associated with lateral habenula and ventral palladium
  • become active in classical conditioning when exposed to a reward or electric shock
    • no active firing in response to the conditioned stimulus associated with the reward
  • VGLUT2-GABA neurons
    • encode the valence itself
    • signal rewarding and aversive outcomes without signaling learned cues related to these outcomes⁷³
• GABA dopamine neurons⁶⁹
  • are strongly oriented towards the nucleus accumbens
  • Release of neurotransmitters is controlled by uptake mechanism, not by the classic GAD1/2 pathway
    • VTA-DA neurons synthesize GABA via aldehyde dehydrogenase 1A1
    • GABA vesicles are filled by vesicular monoamine transporters, not by vesicular GABA transporters

Overall, VTA neurons are⁶⁹

• dopaminergic: > 70 %
• GABAergic: approx. 30 %
• VGLUT2erg: approx. 30 %

Dopamine release from the VTA follows a 12-hour rhythm. VTA neurons fire highest early in the light cycle and early in the dark cycle. In particular, a small subgroup of VTA neurons appears to be active at night.⁷⁴
Neurons of the substantia nigra, on the other hand, showed no changes across the circadian time.

4.4. Storage of dopamine in the vesicles (VMAT)¶

Dopamine and other catecholamines are stored in vesicles after their formation by VMAT2, where they are stored until they are released:⁷⁵

• small synaptic vesicles in neurons
• small and large dense nuclear vesicles in neurons and neuroendocrine cells.

With a slightly acidic pH value, the vesicles protect the dopamine from oxidation in the cytosol.²⁷⁶

VMAT2-KO mice have reduced dopamine levels in the neurons. Dopamine cannot be released by electrical signals in these mice, but can be released by amphetamine.⁷⁷ Missing or excessively inhibited VMAT2 causes excessive cellular dopamine levels, which have a neurotoxic effect after oxidation. VMAT-Full-KO mice usually only survive for a few days.

While it was previously assumed that vesicles harbor uniformly large quantities (a “quantum”) of neurotransmitters, a number of factors are now known to influence the amount of neurotransmitters in vesicles and the amount released from vesicles.⁷⁸

4.4.1. VMAT isoforms¶

2 VMAT isoforms⁷⁹

Both belong to the SLC18 family.

• VMAT1
  • Is only expressed in endocrine and paracrine cells associated with the stomach, intestine and sympathetic nervous system
• VMAT2
  • Is expressed by monoaminergic neurons throughout the CNS (rat), particularly in⁸⁰
    • Midbrain
    • Striatum
    • Olfactory tubercle
    • The dopaminergic paraventricular nuclei
    • Nucleus tuberomammillaris
    • Nucleus raphe
    • Locus coeruleus.
  • In dopamine neurons on both types of secretory vesicles
    • Small synaptic vesicles
    • Large dense core vesicles

4.4.2. Regulation of VMAT¶

Inhibition of the general VMAT function with reserpine led to clear parkinsonian-like behavioral effects in rats. These could be remedied by the dopamine prodrug L-DOPA⁷⁹
Since L-dopa is only helpful in ADHD in rare cases, VMAT might be correspondingly rarely involved in ADHD.
The selective VMAT2 antagonist tetrabenazine also inhibits dopamine formation.

VMAT2 inhibitors inhibit methamphetamine and amphetamine self-administration in rats⁷⁹

• (+)eCYY477, VMAT2 inhibitor (a dihydrotetrabenazine derivative)
• Lobelan
• GZ-793A (Lobalan analog)
  • also reduces the release of dopamine, especially in limbic end fields (e.g. nucleus accumbens shell).

VMAT2 are dependent on V-ATPase (vacuolar-type H+ ATPase) for their dopaminergic function. Disorders in the function of V-ATPase or the proton gradient it creates impair the storage of dopamine in the vesicles by VMAT2.

The storage of dopamine in vesicles via VMAT2 also depends on the amount of cytosolic dopamine concentration. L-dopa increases dopamine within the cell. Increased L-DOPA causes an increase in vesicle size without a change in vesicle number. The increased vesicle size represents a larger releasable dopamine pool⁷⁹

VMAT2 can be regulated by⁷⁹

• the cytosolic chloride concentration
  • up to a certain maximum, a higher cytosolic chloride concentration promotes dopamine storage by VMAT2
• acidification by V-ATPase is a critical mediator for the increase in dopamine levels by VMAT2
• post-translational modifications
• Amount of dopamine in the cytoplasm appears to directly influence VMAT2
• Changes in the expression level due to
  • Transcription factors
  • Protein kinases
    • Protein Kinase A (PKA)
      • influences VMAT2 expression indirectly:
        • regulates N-terminal glycosylation
        • directly phosphorylates cAMP response element-binding protein (CREB) for trafficking and transcription activation
  • heterotrimeric G proteins
    • depending on an intralumenal loop
  • Bonding partner interactions
  • Early childhood (stress) experiences⁸¹
  • Activity
    • VMAT2 expression can be up- or downregulated depending on activity
  • medicinal
    • Dopamine reuptake inhibitors increase VMAT2
      • Methylphenidate
      • Cocaine
    • Drugs that promote dopamine release reduce VMAT2
      • Amphetamine
        • causes emptying of the vesicular dopamine pool into the cytoplasm
        • causes deacidification of the secretory vesicles
        • typical for DAT-reversing drugs
        • different from DAT inhibitors such as methylphenidate
      • Methamphetamine
    • Apomorphine
      • D2 agonist
        • D2 agonists increase VMAT2 activity, D2 antagonists appear to inhibit VMAT2
      • D1 partial agonist
    • Reserpine
      • inhibits VMAT2

4.4.3. Effect of VMAT2¶

Increased VMAT2 expression increases⁷⁹

• Amount of dopamine in vesicles
• Amount of dopamine released from vesicles
• Frequency of release events from vesicles

VMAT2 forms complexes with TH and AADC enzymes and thus also directly influences dopamine synthesis.
Summary
Dopamine is synthesized in several steps. First, L-phenylalanine from food is converted to L-tyrosine. L-tyrosine is then converted to L-dopa and finally to dopamine. Alternatively, dopamine can also be synthesized directly from L-phenylalanine. Another alternative synthesis pathway is via tyramine, which is converted to dopamine. Noradrenaline is synthesized by converting dopamine, while adrenaline is synthesized from noradrenaline.

The formation of dopamine takes place in various regions of the brain, including the VTA, the substantia nigra, the retrorubral nucleus and the arcuate nucleus. Dopamine from the VTA influences motivated behavior and reinforcement learning. Different types of VTA neurons are distinguished, including purely dopaminergic, purely GABAergic and VGLUT2-positive neurons. These each have different functions and project to different regions of the brain.

The activity of dopamine synthesis is regulated by various enzymes and factors. These include tyrosine hydroxylase (TH), tetrahydrobiopterin (BH4), GTP cyclohydrolase 1 (GTPCH), aromatic L-amino acid decarboxylase (AADC), pyridoxal phosphate (vitamin B6) and oxygen concentration. Different mechanisms, such as phosphorylation, dephosphorylation and feedback inhibition, influence the activity of the enzymes. Binding to protein complexes and interaction with other proteins also play a role.

The synthesis and storage of dopamine in vesicles is carried out by the enzyme VMAT2. There are two isoforms of VMAT, VMAT1 and VMAT2, whereby VMAT2 is mainly expressed in the monoaminergic neurons of the central nervous system.

In summary, the synthesis and storage of dopamine, noradrenaline and adrenaline is regulated by various enzymes, factors and brain regions. The activity of these processes is important for the function of the dopaminergic system and has an impact on behavior, motivation and mental disorders such as ADHD.

The regulation of VMAT2 function can be influenced by various factors. Blocking VMAT function leads to parkinsonian-like behavioral effects, which can be remedied by L-DOPA. VMAT2 inhibitors inhibit dopamine formation and reduce dopamine release. The storage of dopamine in vesicles depends on the cytosolic dopamine concentration. VMAT2 can be regulated by several mechanisms, including cytosolic chloride concentration, acidification by V-ATPase, post-translational modifications and the amount of dopamine in the cytoplasm. Various drugs can increase or inhibit VMAT2 activity. Increased VMAT2 expression leads to an increase in the amount of dopamine in vesicles and dopamine release.