Methods for measuring dopamine release

HPLC with electrochemical detection

Biological samples are mixtures of many compounds. The assay of biological samples usually begins with separation of the mixture into its components. Classically, dopamine and its metabolites have been separated from tissue samples by extraction into organic solvents or by ion exchange. During the past 25 years, the preferred method of separation of dopamine and its metabolites has been HPLC. Using this technique, dopamine, its acidic metabolites, and its amino acid precursors can be separated from extracts of brain tissue, or in samples of extracellular fluid acquired by cerebral microdialysis. Once this separation has been obtained, the separate analytes must be detected by some means, either due to the presence of a radioactive label, or on the basis of some other inherent property.

The chemical structure of dopamine is based upon catechol, an aromatic ring in which two adjacent ring protons are replaced with hydroxyl groups. The catechol structure imparts critical properties related to the interactions between dopamine and its receptors, but also reduces the chemical stability of molecules bearing it. The catechol group is highly oxidizable, meaning that electrons are readily withdrawn by other molecules or by catalytic surfaces. Exposure of a catechol to an electric field with a potential difference of one volt causes an oxidation reaction in which four electrons are transferred to the surface of the electrode, with the production of an oxidized quinone molecule.

This book is timely and will prove useful for many researchers interested not only in the specific topic, “Imaging Dopamine,” but also in more general aspects of dopamine. In neurotransmitter research, dopamine has served a spearhead function ever since its discovery in the brain half a century ago. Dopamine has also played a key role in molecular imaging research; the imaging of dopamine receptors started very early in the history of positron emission tomography.

Although this book has its focus on imaging, the full utilization of imaging techniques depends on the background knowledge gained from other methodologies, a theme that has been duly considered by the author. Thus, the various aspects of dopamine, dealing, for example, with its synthesis, storage, release, and metabolism, as well as with the enzymes and transporter proteins involved in these processes, are treated in sufficient detail to provide a well-integrated and reasonably complete picture of the very complex dopamine transmission machinery.

It should go without saying that the growth of knowledge regarding the various aspects of neurotransmission has not taken place without intervals of considerable disagreement and controversy. In the course of the past half century's intense research, many issues have been resolved, whereas others are still being debated. I am pleased to find that the author has devoted some space to historical aspects, starting out with a scheme of the dopamine nerve terminal published by me in 1966.

This book is a biography of dopamine, as illuminated by classical neurochemical methods and especially by molecular imaging with positron emission tomography (PET), or single photon emission tomography (SPET), a closely related technology.

Since the early 1980s, molecular imaging has become indispensable for the study of normal physiology, disease processes, and novel therapeutics. Using external detection with PET or SPET, the uptake and metabolism or binding of radioligands is monitored and quantified in living brain. This book summarizes the state of knowledge of the half-dozen molecular targets in the dopamine system, which have been investigated by imaging techniques. A key advantage of molecular imaging is that aspects of the life of dopamine can be studied in living brain, both in preclinical studies and in humans afflicted with neurodegenerative or psychiatric disorders in which dopamine is implicated. A key disadvantage is presented by the type of knowledge obtained by molecular imaging, which can be only indirectly informative of the step of the pathway for dopamine neurotransmission under investigation. Thus, the interpretation of molecular imaging results must always be grounded in basic aspects of the biochemistry of dopamine and the pharmacology of its binding sites.

Although several groups of dopamine neurons are found in the brain, the entire emphasis here is to be placed on the mesencephalic dopamine systems, which innervate the extended striatum and specific limbic structures of the forebrain.

Molecular biology and enzymology

Basic aspects of the biochemistry of TH have been investigated in considerable detail. Alternative post-transcriptional splicing of the cloned cDNA for human TH gives rise to four distinct messages (Kaneda et al. 1987). Of these, the TH type I resembles in sequence most closely the single transcript expressed in rat (Grima et al. 1987). When purified from tissue, the TH enzyme is associated with dopamine and possesses a bright blue-green color due to the complex between iron(III) and the catecholamine. Purified in the absence of catecholamines from transgenic bacteria, the enzyme is light green due to the complex between iron(III) and several histidine residues close to the active site (Ramsey, Hillas, & Fitzpatrick 1996). When purified to homogeneity, human TH type I expressed in Escherichia coli is a tetrameric protein of subunits with molecular mass of about 60 kDa (Nakashima et al. 1999), consistent with the native tetramer from rat, human, and bovine sources (Oka et al. 1982; Rosenberg & Lovenberg 1983).

The catalytic cycle for TH requires the reduction of iron(III), which has a half-life of 1 s in the presence of reduced biopterin (Ramsey, Hillas, & Fitzpatrick 1996), also known as tetrahydrobiopterin (BH4). Tyrosine hydroxylation requires the reaction between equimolar amounts of molecular oxygen and BH4, or certain synthetic reducers such as 2-amino-4-hydroxy-6,7-dimethyltetrahydropterin (DMPH4) and 6-methyltetrahydropterin (6MPH4). Indeed, the reaction rate is faster for 6MPH4 than for the endogenous compound, BH4 (Lazar et al. 1982; Lazar, Mefford, & Barchas 1982).

Accumulation of DOPA after treatment with NSD 1015

General aspects

In the previous chapter, the kinetics of TH in vitro has been discussed in detail. It is clear that the net activity of TH under physiological conditions must be determined by the multiplicity of regulatory factors prevailing in the living brain. This chapter reviews and contrasts the several methods developed for the assay of TH in living brain. In vitro assays of TH activity are based on the formation of product in a reaction vessel under controlled conditions. However, there is no known mechanism for sequestering or concentration of DOPA in living brain. Basal concentrations of DOPA in brain are normally low because of its unimpeded diffusion back into circulation, and because of the high activity of AAADC relative to that of TH. The normally prodigious activity of cerebral AAADC in the brain of living rat is substantially blocked by treatment with the irreversible enzyme inhibitor m-hydroxybenzyl-hydrazine (NSD 1015), which forms a hydrazone derivative with the pyridoxal phosphate co-factor of AAADC. This blockade is essentially complete in rat brain when the drug is administered at a dose of at least 50 mg kg−1 (Watanabe 1985). In the absence of AAADC activity, DOPA formed from tyrosine then accumulates in the brain tissue (Carlsson et al. 1972). This accumulation appears to be linear with time, from which the rate of DOPA synthesis can be calculated by linear regression.

Enzymology

The oxidative deamination of dopamine and other monoamines is catalyzed by two distinct forms of MAO with high sequence homology (MAO-A, MAO-B), the genes of which have both been localized to nearby bands on the X-chromosome (Kochersperger et al. 1986). The deduced amino acid sequences for the enzymes expressed in human liver predict molecular weights close to 59 000 for both forms, and contain a C-terminus alpha helix which is the likely site of fixation in membranes (Bach et al. 1988). The two genes share considerable homology and have the same intron–exon structure, suggesting an ancient duplication event of the ancestral gene (Grimsby et al. 1991). Consistent with their ancient origin, the amino acid sequences have considerable homology across species, especially in the domain containing the cystein residue which binds the prosthetic flavin adenine dinucleotide (FAD) (Kwan, Bergeron, & Abell 1992). The pure enzyme is devoid of iron or other transition metal ions. The catalytic cycle for MAO involves the successive binding of the amine substrate and oxygen, followed by liberation of the aldehyde product and equimolar amounts of ammonia and hydrogen peroxide. In general, the aldehyde produced by MAO is immediately oxidized further by non-specific enzymes, yielding a carboxylic acid, hence the acidic metabolites of dopamine. The production of (toxic) hydrogen peroxide may be problematic under certain circumstances, especially given the location of MAO in the outer mitochondrial membrane.

Most molecular imaging studies make use of a single injection of the radioligand at very high specific activity, such that the mass of substance is negligible. Consequently, the index of receptor availability (pB) is formally ambiguous, being a function of the number of receptors (Bmax), divided by the apparent affinity of the ligand in vivo (Kdapp). Separate determination of the saturation binding parameters requires multiple tracer injections, such that a range of receptor occupancies are obtained. This chapter summarizes the relationship between PET estimates of dopamine receptor abundance, and estimates obtained using preparations in vitro. The various findings in the cases of dopamine D1 receptors (Table 13.1) and D2 receptors (Table 14.1) reveal the extent of agreement between the several quantitative methods.

The absolute density of dopamine receptors in the brain can be quantified using a variety of methods in vitro (Seeman 1987). The saturation binding parameters for a radioligand are measured under controlled conditions, using either washed membranes or brain cryostat sections. The experimentalist exposes the tissue samples to a range of radioliogand concentrations, ideally extending at least one order of magnitude to each side of the half-saturation concentration, Kd. The ionic composition of the incubation buffer, the incubation temperature, and the duration of incubation are determined entirely by laboratory procedures; the specific binding is measured relative to the binding in the presence of a non-radioactive competitor for the same site.

Presenting the life history of dopamine has been a matter of assigning quantities to the arrows depicted in the schematic diagram of Carlsson, introduced over 40 years ago (Figure 1.2). This endeavor has made use of a range of methods, extending from classical enzymology and receptor pharmacology, to modern molecular imaging techniques. All of these methods have been brought to bear on the implicit task of breathing life into a model, with the expectation that garnering enough specific information would allow the construction of an explicit model of dopamine neurotransmission. Needless to say, completion of this task remains to be fulfilled. But after 50 years of dopamine research, enough information has been gathered such that a biological model of considerable complexity can now be defined and constrained, encompassing the main elements of metabolic regulation, compartmentation, and anatomical connectivity of dopamine in the basal ganglia.

The neurochemical anatomy of the basal ganglia has a fine structure; many of its components in human brain are simply too small to be seen with PET. The spatial resolution of PET has dramatically increased in the past quarter century. Thus, it was nearly impossible to resolve the human caudate and putamen in the earliest PET images, which had a spatial resolution of 6–8 mm. Newer tomographs with a resolution of 4 mm allowed the defining of a region of interest encompassing the ventral striatum, which has attracted much attention due to its key importance in the rewarding properties of psychostimulants.

General aspects of the quantitation of FDOPA utilization

PET recordings consist only of the total radioactivity concentration in brain volumes as a function of circulation time. How then is one to deduce the rate of decarboxylation of AAADC substrates in living brain, knowing (without the luxury of dissection and HPLC analysis of brain extracts) only the total amount of radioactivity in a volume of brain tissue and the concentration of the tracer in blood? The exogenous AAADC substrate FDOPA has been used in hundreds of PET studies. The quantitation of FDOPA uptake in brain is formally similar to the cases of fluorodeoxyglucose (FDG) and [11C]tyrosine. Thus, FDOPA passes reversibly across the blood–brain barrier by a process of facilitated diffusion. In the brain, FDOPA can be trapped as non-diffusible [18F]fluorodopamine at a rate defined by the local AAADC activity, k3D. In most PET studies, substantial amounts of the inert plasma metabolite O-methyl-FDOPA (OMFD) also enter the brain, creating a diffuse and non-specific background radioactivity on which the specific signal is superimposed. The model for FDOPA kinetics is schematically represented inFigure 5.1, which is essentially a simplification of the more detailed compartmental model, presented in the preceding chapter. The objective of this chapter is to review the various methods that have been brought to bear on the quantification of PET studies with FDOPA and other substrates for AAADC (Hoshi et al. 1993) and to show how these different methods illuminate different aspects of the life history of dopamine in different clinical conditions.

Kinetic properties of AAADC in vitro

DOPA and other substrates are decarboxylated by aromatic amino acid decarboxylase (AAADC), some biochemical properties of which are summarized inTable 4.1. AAADC purified from pig kidney occurs as a homodimer, with two catalytical sites, each of which binds a single pyridoxal phosphate (Vitamin B6) (Dominici et al. 1990). The pyridoxal phosphate co-factor is essential for catalytic activity, and the majority (80%) of AAADC in rat brain normally occurs as the holozyme (Kawasaki et al. 1992), endowed with an equimolar amount of pyridoxal phosphate. Pyridoxine is transferred across the blood–brain barrier by a saturable process and is phosphorylated in the brain by a specific kinase; brain pyridoxal phosphate concentrations can be increased by peripheral loading with pyridoxine (Spector & Shikuma 1978).

AAADC substrates form reversibly a Schiff base with the pyridoxal phosphate group. The consequent withdrawal of electrons from the amino acid moiety weakens the carbonyl bond, encouraging the irreversible loss of carbon dioxide, which is followed by release of the decarboxylated amine and recycling of the co-factor for the next catalytic cycle. Electrophilic substituents on the α-carbon decrease the reaction rate for AAADC substrates. In the case of the “suicide substrate” α-fluoromethyl-DOPA, an irreversible covalent bond is formed with the holozyme, resulting in a permanent loss of catalytic activity of the enzyme (Maycock, Aster, & Patchett 1980). Another suicide inhibitor, NSD 1015 (the α-hydrazine derivative of DOPA), is commonly used for the assay of DOPA synthesis, as reviewed in Chapter 5.

Molecular biology of DAT and regulation of expression

The DAT gene cloned from a rat brain expression library codes for 620 amino acids, with a sequence predicting 12 transmembrane domains (Giros et al. 1991). Its expression in various cell lines confers vulnerability to the toxin MPP+ (Pifl, Giros, & Caron 1993), in addition to conferring the ability to transport dopamine. The uptake of dopamine in striatal slices and synaptosomes has a saturable component with high affinity, and apparently also a high-capacity, low-affinity site (Mireylees, Brammer, & Buckley 1986). It is the saturable component that is characterized by sensitivity to cocaine and related compounds. Thus, the potency of many drugs displacing [3H]cocaine from striatal membranes is highly correlated with the inhibition of [3H]dopamine uptake in slices or synaptosome preparations (Madras et al. 1989).

The intracellular domain of DAT presents several potential sites for phosphorylation by protein kinases C and A (Giros et al. 1992). Treatment with phorbol esters, which activate PKC directly, reduced the maximal velocity of [3H]dopamine uptake by synaptosomes from rat striatum (Copeland et al. 1996), and also reduced the velocity of dopamine uptake in frog oocytes expressing human DAT (Zhu et al. 1997). This latter reduction in velocity was associated with reduced [3H]mazindol binding to intact cells, without any concomitant change in the ligand binding to homogenates, which indicates that the treatment altered the association of functional transporters with the plasma membrane.

Pharmacological modulation

Dopamine antagonists

Just as the abundance of striatal dopamine D2 receptors increases after prolonged dopamine depletion, chronic pharmacological blockade can also result in receptor upregulation. For example, chronic neuroleptic treatment increased dopamine D2/3 receptor binding site density in rat striatum by 19%, whereas the specific binding to D4 receptors increased two-fold (Schoots et al. 1995). In another study, chronic haloperidol treatment increased [3H]spiperone binding (in the presence of a 5HT2 antagonist) in rat striatum membranes by 40%, but the antipsychotic treatment was without effect on the apparent fraction of those receptors which could be displaced by agonists, i.e. D2High (MacKenzie & Zigmond 1984). Similar increases in D2 antagonist binding have also been seen in the striatum of monkeys after prolonged pharmacological blockade with receptor antagonists (Huang et al. 1997).

Pharmacologically evoked changes in dopamine receptor availability can be extremely long-lasting. In a primate PET study, daily treatment with raclopride (10 μg k g− 1 × 30 days) increased the striatal binding of the D2-selective antagonist [18F]fluoroclebopride by 12–20%, an increase which persisted for 1 year in two of the three monkeys investigated (Czoty, Gage, & Nader 2005). In a case report of two patients with schizophrenia who had undergone treatment with haloperidol for many years, a non-smoker had a 98% increase in [11C]raclopride pB and suffered from tardive dyskinesia, whereas a smoker, treated at a much higher haloperidol dose, had somewhat lower elevation in pB, and no tardive symptoms (Silvestri et al. 2004).

A brief overview of the dopamine pathway

The life history of a dopamine molecule begins in the liver, with the synthesis of the precursor tyrosine, and ends in the kidney, with the elimination of the conjugated dopamine metabolites to the urine. Only during a brief and specific interval in its life can a dopamine molecule engage in its proper function, which is the mediation of signaling via activation of dopamine receptors. The chemical structures of molecules in the dopamine biosynthesis and catabolic pathway are illustrated in Figure 1.1. This scheme does not include the catecholamines noradrenaline and adrenaline, for which dopamine is a precursor, since these substances might properly serve as the topic of another book.

As presented in Figure 1.1, all the reactants and enzymes in the dopamine pathway seem to be present in the same space. However, in the living organism, molecules and chemical reactions normally occur within strictly segregated spaces, known as metabolic compartments. Transfer of a molecule in the dopamine pathway from one compartment to another may be strictly impeded by diffusion barriers, or may occur via carrier-mediated facilitated diffusion or by ATP-driven active transport. Thus, the schematic pathway for dopamine synthesis in the living organism should be projected onto a model containing cellular compartments. The model proposed by Carlsson (1966) illustrates the blood, extracellular space, intracellular space, and vesicles as distinct compartments (Figure 1.2). Enzymes and transporters conduct the transfer of mass from one compartment to another, here represented as arrows.

Biochemistry of COMT

COMT catalyzes the transfer of active methyl from S-adenosylmethionine (SAM) to dopamine and other catechols (Axelrod & Tomchick1958). The methylation is preferentially directed to the p-hydroxyl group of most substrates. In the brain, the enzyme exists in two distinct molecular forms: a soluble form with low affinity for catecholamine substrates, and a membrane-bound form with μM affinity with respect to dopamine (Jeffery & Roth 1984). The membrane-bound form can be solubilized with strong detergent, suggesting that it is an integral membrane protein. The activity of both forms is dependent on the presence of Mg2+, and is maximal at pH greater than 7. Mechanistic studies of the membrane-bound form suggest that catalysis is initiated with binding of the co-substrate SAM, followed by the formation of a ternary complex with dopamine. The reaction is inhibited by low concentrations of the end-product S-adenosylhomocysteine (Rivett & Roth 1982). Some biochemical properties of COMT are summarized in Table 6.1.

The membrane-bound form of COMT differs from the soluble form in having an extra 50-residue hydrophobic sequence at the N-terminus (Ulmanen et al. 1997). When expressed in transfected mammalian cells, the membrane-bound COMT is associated with the endoplasmic reticulum and nuclear membranes, but not in the plasma membrane – an indication that its substrates must be present in the cytosol rather than on the external plasma membrane, as was once believed.

Biochemistry of vesicular monoamine transporters

Two vesicular monoamine transporters of adrenal chromaffin cells (VMAT1) and catecholamine neurons (VMAT2) belong to a family of vaculolar-type proton-ATPase proteins, which are related to the bacterial antibiotic resistance transporters. Both vesicular monoamine transporters accumulate monoamines within vesicles, employing an intrinsic ATPase activity to generate the proton gradient that establishes and maintains the accumulation of monoamine substrates; in a sense, vesicles behave as inside-out mitochondria. The genetic sequence for VMAT1 was isolated on the basis of its ability to and impart resistance to MPP+, the neurotoxic metabolite of MPTP (Liu et al. 1992), apparently by sequestering the toxic metabolite out of harm's way. Indeed, the reserpine-sensitive uptake of MPP+ by chromaffin and brain synaptic vesicles could produce 100-fold concentration gradients in the presence of ATP (Moriyama, Amakatsu, & Futai 1993).

Around the same time as the cloning of VMAT1, a sequence encoding VMAT2 was cloned from the human cDNA library on the basis of its ability to impart serotonin uptake to organelles in permeabilized cells (Erickson, Eiden, & Hoffman 1992). The sequence of the VMAT2 gene predicts a 512 amino acid protein with 12α-helical membrane spanning domains, both termini and several potential phosphorylation sites being present on the cytoplasmic side. In situ hybridization signal for VMAT2 is present in the dopamine, serotonin, noradrenaline, and adrenaline neurons of the brain, all of which biogenic amines are good substrates for vesicular uptake.

General properties of D2 ligands

Irreversible ligands

N-[3H]methylspiperone ([3H]NMSP) is a butyrophenone compound binding to at least two pharmacologically distinct sites in homogenates from the mammalian brain. Although the bindings had similar kinetics in the caudate and in the cortex, the preponderance of binding in the caudate could be displaced with dopamine D2 antagonists, whereas most of the cortical binding was displaced with serotonin 5HT2 antagonists (Lyon et al. 1986). The in vitro association kinetics for both sites was rapid, while dissociation kinetics was so slow as to be nearly irreversible in the time course of a PET study. Displacement studies in living mice likewise revealed that whereas 90% of the [3H]spiperone binding in the frontal cortex was to serotonin receptors, 80% of the binding in the striatum was to dopamine D2 receptors (Frost et al. 1987). With the caveat that functional selectivity is mainly imparted by the differing distributions of the two main binding sites, [11C]NMSP and related compounds can potentially be used to measure dopamine and serotonin receptors in the same PET session (Borbely et al. 1999).

Displacement studies of [3H]spiperone from rat brain membranes indicated the presence of considerable amounts of non-dopaminergic, non-serotonergic sites, although these were of ten-fold lower apparent affinity than were the identified components of the binding (List & Seeman 1981).