Schizophrenia: Possible alterations in dopaminergic neurotransmission

Kirstine Callø, Mette Hesselager, Heidi Holst & Søren Tomra

Abstract

This review is concerned with the role of the dopaminergic pathway in schizophrenia. Possible molecular mechanisms resulting in a hypo- and hyperactivity of the dopaminergic system are investigated. The synthesis of dopamine, re-uptake and expression of the dopamine receptors are given special attention, as well as the receptor polymorphisms’ involvement in schizophrenia. Some alterations in the dopamine metabolism have been found, as well as a changed expression of the dopamine receptors. Genetic studies have implicated several minor susceptibility loci; however the clinical impact of these loci on the disease is still uncertain. There is some evidence for the involvement of the dopaminergic system in schizophrenia, but it cannot be concluded if the described changes are the main causes or just consequences of the disease.

COMT	Catechol-O-methyl transferase
CSF	Cerebrospinal fluid
DAT	Dopamine transporter
DBH	Dopamine-b-hydroxylase
DDC	Dopa decarboxylase
Dn	Dopaminergic n
DOPA	Dihydroxyphenylalanine
DOPAC	Dihydroxyphenylacetic acid
HVA	Homovanillic acid
KO	Knock-out
MAO	Monoamine oxidase
PET	Positron emmission technique
PNMT	Phenylethanolamine N-methyl transferase
SSCP	Single stranded conformational polymorphism
TH	Tyrosine hydroxylase
UTR	Untranslated region
VNTR	Variable number of tandem repeats
VTA	Ventral tegmental area

Introduction

Schizophrenia is a complex neurological disease affecting about 1 % of the world’s population [Rang et al., 1999]. It is characterised by various positive and negative symptoms. Positive symptoms include hyperactive behaviour, hallucinations, paranoia, and delusions. Negative symptoms involve loss of motivation, withdrawal from social life, cognitive deficits, depression, slowed and impaired speech, and blunting of emotions [WHO icd 10, 1998]. As not all of the symptoms are necessarily present in one patient, the disease is difficult to define. Therefore the diagnosis is made in agreement with guidelines from the American Psychiatric Association.

The causes of schizophrenia are largely unknown. It has not been possible to find a single reason for the disease and therefore it is now regarded as being a multifactorial disorder.

A role for dopamine has been suggested, which has led to the dopamine hypothesis of schizophrenia. In its original form it was assumed that hyperactivity of the brain’s dopaminergic tracts was responsible for schizophrenia. The theory was based on the discovery that some of the symptoms can be relieved by treatment with antagonists of the dopamine receptors [Farde et al., 1992] and that the clinical outcome of the treatment is correlated to the drugs affinity for the receptors [Creese et al., 1976]. This was combined with the findings that amphetamine and cocaine increases the amount of dopamine in the synaptic cleft and creates schizophrenia-like symptoms.

The theory has later been disputed and modified. Five different isoforms of the dopamine receptors have been discovered (D₁ - D₅) [reviewed by Sibley & Monsma, 1992]. This has revealed that the dopaminergic pathways are much more complex than it was first believed. Furthermore, hypo- as well as hyperactivity have been detected in the dopaminergic system in schizophrenia [reviewed by Willner, 1997]. Hypoactivity may be related to negative symptoms whereas hyperactivity may be related to positive symptoms [Hietala et al., 1999].

In order to understand the consequences of a change in activity in the dopaminergic system, the dopaminergic tracts and their functions have to be considered in details.

Figure 1a. A coronal section of the brain showing the sites of origin and the targets of the four dopaminergic tracts.

1. The nigrostriatal system, from the substantia nigra to the putamen and caudate nucleus; 2. The tuberoinfundibular system, from the arcute nucleus to the infundibulum; 3. The mesolimbic system, from the ventral tegmental area to the limbic system; 4. The mesocortical system, from the ventral tegmental area to the neocotex.

There are four major dopaminergic projections in the brain (figure 1a & 1b). The major dopaminergic neuronal projection extents from the pars compacta, a part of the substantia nigra. It consists of neurones synthesising dopamine whereby neuromelanin is produced as a by-product. It projects to the striatum encompassing the caudate nucleus and the putamen of the basal ganglia in the prosencephalon. The nigrostriatal projection has both excitatory and inhibitory effect on striatal neurones. This projection is implicated in controlled movements. The pathological outcome of degeneration of pars compacta is Parkinson’s disease.

A projection known as the mesolimbic tract conveys signals from the ventral tegmental area (VTA) medial of the substantia nigra to the most rostral and ventral part of the striatum, the nucleus accumbens. It is also known as the mesostriatal tract. The system is thought to influence motivated behaviors.

A third projection, the mesocortical tract, runs from VTA to neocortex. It is implicated in learning and memory.

A minor projection, the tuberoinfundibular tract runs from the arcute nucleus of the hypothalamus to the infundibulum. It regulates prolactin secretion and it seems to be of minor importance in respect to schizophrenia. [Crossman & Neary, 1995; Kandel et al. 1995]

Schizophrenia is probably not caused only by abnormalities in the dopaminergic system but may include malfunction of other neurotransmitter systems as well.

Theories about the cellular mechanisms, which might contribute to an abnormal activity in the dopaminergic system in schizophrenia, are still controversial and ambiguous.

Figure 2 The key steps in the synthesis and degradation of dopamine.

Tyrosine is transported across the cell membrane by the L-transporter, tyrosine is converted to dopamine (DA) via Dopa. It is stored in vesicles before its release to the synaptic cleft. It can bind to five different postsynaptic receptors (D1-5) as well as to presynaptic autoreceptors. Dopamine is cleared from the synaptic cleft either by degradation by catechol-O-methyltransferase (COMT) or by reuptake by a dopamine transporter (DAT) into the presynapse where it is recycled or degraded by monoamine oxidase (MAO).

Dopamine neurotransmission is regulated in the synapse where a number of factors can be involved in abnormal functioning of the dopaminergic system (figure 2). Tyrosine is transported into the neurones by the L-transporter. Dopamine is synthesised from tyrosine via dihydroxyphenylalanine (DOPA) in the presynaptic neurone and is either stored intracellularly, degraded by monoamine oxidase (MAO) or released to the synaptic cleft. In the synaptic cleft dopamine has three possible fates. It can bind to the dopamine receptors. These are localised both pre- and postsynaptically, the former known as autoreceptors. Another possibility is re-uptake of dopamine into the presynapse by the dopamine transporter (DAT) and finally it can be broken down by catechol-O-methyl transferase (COMT) in the postsynaptic membrane.

Changes in the level of dopamine, which is regulated presynaptically and changes of expression or functioning of the dopamine receptors are two major cellular mechanisms taken into consideration in this review.

Metabolism of dopamine

The metabolic precursor for dopamine is tyrosine. Tyrosine is an aromatic amino acid synthesised in the liver. It is present in all body fluids and is transferred by the L-transporter system into the neurones. The function of the L-transporter is to transport neutral amino acids, which compete for the transport across the cell membrane. Tyrosine hydroxylase (TH) is the rate-limiting enzyme and catalyses the conversion of tyrosine to DOPA. TH is found only in the catecholamine-containing cells. DOPA is converted to dopamine by DOPA decarboxylase (DDC). DDC is a relatively non-specific enzyme and it catalyses the decarboxylation of various aromatic amino acids. Dopamine-b-hydroxylase (DBH) converts dopamine to noradrenaline, which can be further metabolised to adrenaline by phenylethanolamine N-methyl transferase (PNMT). DBH is also relatively non-specific and its distribution is restricted to catecholamine-synthesising cells. Dopaminergic neurones lack DBH and, thus, do not produce noradrenaline or adrenaline.

Dopamine is metabolised by MAO and COMT, the main product being dihydroxyphenylacetic acid (DOPAC), which is further metabolised to homovanillic acid (HVA). The metabolism of dopamine is illustrated in figure 3.

Figure 3. The dopamine metabolism.

Left: Biosynthesis of catecholamines. Right: The main pathways for dopamine degradation in the brain.

Availability of tyrosine for dopamine synthesis

Tyrosine is the precursor of dopamine; therefore the amount of dopamine produced is dependent on the amount of tyrosine available. In order to keep dopamine in the right concentration, the intracellular level of tyrosine is strictly regulated. This is possible by regulation of the transport across the cell membrane by the L-transporter. Another way to control the availability of tyrosine in the brain is to keep the concentration of tyrosine in the blood correct by controlling the metabolism of tyrosine. Finally, restraining the amount of tyrosine crossing the blood-brain barrier could control the tyrosine concentration in neurones. Wiesel & Bjerkenstedt (1996) discovered that the level of HVA in the cerebrospinal fluid (CSF) was lower in schizophrenics compared to healthy individuals. They suggested that the concentration of HVA was dependent on the concentration of tyrosine in CSF. Furthermore, the plasma concentration of neutral amino acids except tyrosine was relatively higher in the schizophrenic patients compared to the control group. These results indicate that there might be a lower level of tyrosine in the CSF in schizophrenics and this influences the level of HVA. This potential reduction in tyrosine concentration in CSF could be caused by a reduced transport of tyrosine across the blood-brain barrier or be due to the lower plasma levels of tyrosine, or both.

It was examined whether the low levels of HVA in the CSF were due to a reduction of tyrosine transport across the cell membrane. There was a slower rate (V_max) of tyrosine transport into fibroblasts taken from schizophrenics compared to controls [Wiesel & Bjerkenstedt, 1996; Ramchand et al., 1996]. The L-transporter was intact since the uptake of tyrosine at different concentrations was changed as expected. Therefore a more general alteration of the cell membrane in schizophrenics was suggested. This alteration would affect the whole body and not only the brain, in agreement with the impaired tyrosine transport in fibroblasts.

The transport of tyrosine across the blood-brain barrier was examined by positron emission technique (PET). L-[1-¹¹C]tyrosine was used as a tracer [Wiesel & Bjerkenstedt, 1996]. As expected, a decreased concentration of tyrosine inside the blood-brain barrier was found.

Thus, an impaired uptake of tyrosine both into the fibroblasts and across the blood-brain barrier may take place in schizophrenia.

Tyrosine hydroxylase

Four different isoforms of tyrosine hydroxylase (TH) with different functional characteristics are produced from alternative splicing. The TH gene contains 14 exons spanning approximately 8 kb, it is localised to 11p15. Genetic studies have demonstrated variations in the TH gene and correlated them to schizophrenia [Wei et al., 1997; Burgert et al., 1998; Ishiguro et al., 1998]. Four different regions of the gene have been found to contain polymorphisms, one of these in a VNTR region in the first intron. This region is interesting because it is believed to regulate the expression of the gene [Burgert et al., 1998]. Studies of Caucasian groups have revealed an unequal allelic distribution of the VNTR polymorphism when comparing schizophrenics and controls [Wei et al., 1997; Burgert et al., 1998]. Some of these alleles could contribute to the development of schizophrenia or be a marker for a still not described susceptibility gene. Ishiguro et al. (1998) systematically searched for variations in the whole TH gene, and they found no difference in the distribution of the four alleles between Japanese schizophrenic individuals (208 unrelated individuals) and a Japanese control group (266 unrelated individuals). These results do not support the idea of existence of a widespread causal relationship between TH polymorphisms and schizophrenia. It cannot be ruled out that allelic variations of TH play a role in the Caucasian population, as the genetic composition of this population is different from the Japanese population.

The results with TH polymorphisms have not so far been convincing. An altered expression of TH can influence the levels of dopamine, but whether an altered expression is found in schizophrenics has not been elucidated yet.

DOPA decarboxylase

Uptake of labelled DOPA is commonly used to estimate DDC activity as this enzyme catalyses the formation of dopamine from DOPA (figure 3). An increase in the activity of DDC could be an indication for an increased activity in the dopaminergic system.

Reith et al. (1994) used PET-scanning to study the uptake of ¹⁸FDOPA in the striatum of patients suffering from schizophrenia, but free of medication. The uptake of ¹⁸FDOPA was found to be higher in schizophrenics compared to the controls. This observation was confirmed in neuroleptic-naive patients [Hietala et al., 1999].

Lindström et al. (1999) found elevations in the uptake of ¹¹C-labeled DOPA in the caudate nucleus, putamen and in the limbic part of the medial frontal cortex of schizophrenics, which is consistent with the former results.

DDC is involved in the synthesis of dopamine, noradrenaline and adrenaline and thus, an increased activity of DDC does not necessarily reflect an increase in the activity of dopaminergic neurones. To avoid this problem the measurements were made during the first 45 minutes after adding ¹¹C-labeled DOPA when the synthesis of dopamine takes place [Lindström et al., 1999]. This does not exclude measurement of DOPA uptake in noradrenergic or adrenergic neurones, though, as these will also be synthesising dopamine.

There is no evidence that an elevation in the uptake of labelled DOPA reflects a natural requirement for DOPA or an increased activity of DDC. High amounts of DOPA might lead to an artificially high production of dopamine, and the uptake of labelled DOPA is therefore not necessarily representative for the real requirement of DOPA.

There is a difference between the two DOPA tracers. The ¹⁸F-labelling of DOPA is done by the ¹⁸F modification of the molecule, whereas the radioactive carbon atom substitutes a carbon atom within the DOPA molecule. This means that ¹⁸FDOPA is extensively metabolised outside the brain compared to ¹¹C-labeled DOPA, and radio-labelled ¹⁸F-metabolites cross the blood-brain barrier in disturbing amounts [Lindström et al., 1999]. Comparably less ¹¹C-labeled DOPA is metabolised outside the brain, as the dopaminergic neurones metabolising DOPA primarily exist in the brain.

Depressive symptoms in the schizophrenic patients were found to correlate to a reduction in the ¹⁸FDOPA uptake in the striatum, especially the putamen and the caudate nucleus. Furthermore, the paranoid symptomatology correlated positively with the right putamen ¹⁸FDOPA uptake [Hietala et al., 1999]. This can indicate that changes in DOPA uptake is somehow correlated to the disease.

In healthy individuals a pronounced asymmetric uptake of ¹⁸FDOPA in caudate nucleus with the highest uptake in the right half of the caudate has been observed [Hietala et al., 1999]. This asymmetry was diminished in schizophrenic patients, so that ¹⁸FDOPA uptake was equal in both halves of the caudate nucleus. No asymmetry in the ¹⁸FDOPA uptake in the putamen was seen either in the patient group or in the control group. Lindström et al. (1999) confirmed these results by comparing left and right hemispheres of a healthy and a schizophrenic group. However, how this asymmetry is involved in the disease is unknown.

The degree of DDC activity or degree of expression might be changed in schizophrenia leading to the higher DOPA uptake. There might be different isoforms of or mutations in DDC resulting in different activities. So far, two fairly frequent variations of the DDC gene are known; a 1-bp deletion in the promoter and a 4-bp deletion in the untranslated exon 1 [Børglum et al., 1999]. Both deletion sites affect putative binding sites for known transcription factors so both deletion sites might affect the transcription rate of DDC. The 1-bp deletion is found to be associated with bipolar disease, but whether the same is true for schizophrenia is not known [Børglum et al., 1999].

The dopamine transporter

The dopamine transporter (DAT) belongs to a large family of Na⁺/Cl^- dependent transporters containing 12 transmembrane domains. DAT is responsible for the termination of neurotransmission by a rapid re-uptake of dopamine into the presynaptic terminals (figure 2). It is believed to control the intensity and duration of dopaminergic neurotransmission by resetting the dopamine concentration in the extracellular space. DAT is therefore an important element in regulation of the actions of dopamine on locomotion, cognition, affection and neuroendocrine functions. DAT is also a target for psychoactive drugs such as antidepressants and drugs of abuse including amphetamine and cocaine. Amphetamine and cocaine act by blocking DAT and thus inhibit dopamine re-uptake, which ultimately leads to an increase in extracellular dopamine levels [reviewed by Hitri et al., 1994]. Cocaine has a remarkable psychomotor stimulating effect, causing euphoria, garrulousness, increased motor activity, and a magnification of pleasure, similar to the effects of amphetamine. Both drugs have some negative side effects but cocaine has a less tendency than amphetamine to produce stereotyped behaviour, delusions, hallucinations, and paranoia [Rang et al., 1999]. Amphetamine can produce a behavioural syndrome indistinguishable from an acute schizophrenic episode. These are some of the reasons for the attention being turned towards the dopamine transporter.

DAT mRNA is present only in dopamine synthesising neurones and the corresponding protein coincides with dopaminergic innervation of several regions [Ciliax et al., 1995].

To investigate how high extracellular concentration of neural dopamine can affect the functional state of dopamine receptors, as well as other parameters DAT knock-out (KO) mice were constructed. Striatal synaptosomal preparations from DAT -/- mice showed no significant dopamine re-uptake, suggesting that no other monoamine transporters contribute to dopamine re-uptake [Giros et al., 1996].

The DAT KO mice did not effectively clear extracellular dopamine and exhibited a generally hyperactive dopaminergic state. They revealed a remarkable increase in spontaneous locomotor activity during both night and day. This increase was of the same magnitude as in normal mice treated with very high doses of amphetamine and cocaine. Other physiological changes appeared; the KO mice gained weight more slowly than wild type mice did. Females lacking DAT showed an inability to lactate probably due to the inhibition of prolactin secretion and they showed an impaired capability to care for their offspring. The KO mice demonstrated premature death compared to wild type mice [Giros et al., 1996, reviewed by Gainetdinov et al., 1999].

Giros et al. (1996) measured the extracellular dopamine levels in striatal slices from KO and wildtype mice. There was at least a 100-fold increase in the time dopamine persisted in the extracellular space in DAT-KO mice. The prolonged dopamine clearance rate was also associated with a ~ 75% decrease of dopamine molecules released in response to an electrical pulse. These results suggest that the releasable pools of dopamine are markedly decreased in DAT KO mice [Giros et al., 1996]. The basal extracellular dopamine concentration of dopamine in the striatum was measured by microdialysis and showed a 5-fold elevation in KO mice compared to wildtype [Jones et al., 1998]. The intracellular content of dopamine in the DAT KO mice striatum was found to be less than 5% of wildtype levels. However, dopamine terminals in the striatum were intact, ruling out an anatomical abnormality as a reason for the decrease. The extremely low intracellular content of dopamine was unexpected considering the high basal extracellular levels of dopamine. These results support that a decrease in the number of DAT can result in a large decrease in the intracellular stores of dopamine, implying that the reserve pools of dopamine are acutely dependent on recycled extracellular dopamine [Jones et al., 1998]. The DAT KO mice were also found to have a doubled rate of dopamine synthesis compared to wildtype mice. The level of striatal TH protein in the KO mice was found to be less than 10% of wildtype levels. Thus, a few TH molecules present in DAT KO mice were very efficient in converting tyrosine to DOPA. This can partly be due to the absence of re-uptaken dopamine that normally inhibits TH activity [Jones et al., 1998].

In situ hybridisation revealed that mice lacking DAT had down-regulated the number of dopamine receptors. The coding mRNA for D1 and D2 was found to be down-regulated by 55% and 45%, respectively [Giros et al. 1996].

In the absence of DAT, adaptive changes are concurrently induced in the striatal system. These broad compensatory changes illustrate the importance of DAT not only in regulating dopaminergic transmission but also in maintaining normal dopaminergic homeostasis and function.

There are some similarities between symptoms of schizophrenic patients and the hyperdopaminergic phenotype of DAT KO mice. However, several features of the DAT KO mice do not correlate with those of schizophrenia, which complicates drawing conclusions about the involvement of DAT in the disease.

Because DAT is a key element in controlling the extracellular dopamine levels in the brain, various groups have tried to find evidence for an association between the DAT genes and schizophrenia.

Silverman et al. (1996) found evidence for genetic association of the marker D5S111 with schizophrenia in a large Hispanic family. The D5S111 locus is located centromerically to the DAT gene, which is found in the human chromosome 5p15.3 [King et al., 1997]. However, other groups have not been able to find any association between the DAT gene markers and schizophrenia [King et al., 1997; Persico & Macciardi 1997]. These results do not support the existence of a widespread and significant causal relationship between genetic mutations at the DAT locus and the disease phenotype.

The dopamine autoreceptors

Dopamine autoreceptors provide an important mechanism whereby dopaminergic neurones can regulate cellular functions such as neurotransmitter release, synthesis, and impulse flow.

The dopamine autoreceptors that are found on somadendrites of the midbrain dopaminergic neurones regulate impulse activity, whereas the nerve terminals of these neurones express autoreceptors that modulate both dopamine synthesis and release [Koeltzow et al 1998]. A lot of work has been done to establish the nature of these autoreceptors; especially D₂ and D₃ dopamine receptors have been examined. D₂ receptor KO mice synaptosomes were used to study the autoreceptors in the dopaminergic pathways. It was found that D₂ receptor serves as an inhibitory autoreceptor both on somadendrites and on the nerve terminals, and thus were involved both in the control of synthesis, release and the excitability of the nerve terminals, whereas inhibitory D₃ receptors were not found [L’hirondel et al., 1998]. Studies of D₃ receptor KO mice confirmed that D₃ receptor was not significantly involved in dopamine autoreceptor function, but there was evidence that they participate in postsynaptically activated short loop feedback modulation of dopamine release. [Koeltzow et al., 1998]. Short loops are mono- or multisynaptic paths from the postsynapse terminating on the presynapse.

Presynaptic D₂ autoreceptors have recently been shown to regulate DAT activity. D₂ receptor KO mice were found to have a decreased DAT function. It is likely that local dopamine binds to presynaptic D₂ receptors and thereby increases the DAT activity [Dickinson et al., 1999].

It is evident that the autoreceptors are important regulators of the dopamine homeostasis, but whether an alteration caused by a change in the function of the autoreceptors are involved in schizophrenia is not known.

Loss of dopamine axons

A recent study indicates that the density of TH-immunoreactive axons is decreased by approximately 30 % in the deep layers of the prefrontal cortex of schizophrenic patients [Akil et al., 1999]. To determine the consequence of a partial loss of prefrontal dopamine axons on the activity of the remaining dopamine axons Venator et al. (1999) examined the effect of 6-hydroxydopamine lesions in the medial prefrontal cortex on local extracellular dopamine concentration in the rat. They found that moderate loss of TH-immunoreactive axons in prefrontal cortex was sufficient to reduce extracellular dopamine concentration in this brain region. These findings support the hypothesis that a loss of dopamine axons in schizophrenia subjects may result in decreased amounts of extracellular dopamine contributing to the expression of some of the symptoms [Venator et al., 1999].

The oxidation of dopamine results in the formation of neurotoxic o-semiquionones together with free oxygen radicals. A number of mechanisms are involved to protect cells against these o-semiquionones including o-methylation by COMT [reviewed by Smythies, 1997]. Smythies (1997) suggested that the neurochemical basis of some types of schizophrenia might be a combination of low antioxidant defenses and other defects. These defects could increase the neural level of toxic o-semiquinone metabolites of catecholamines. This could lead to neural damage at critical stages of brain development. There is increasing evidence that a changed free radical metabolism and oxidative injury both contribute to the patophysiology of schizophrenia. This is indicated by the increased levels of lipid peroxidation products in plasma and CSF, and altered levels of both enzymatic and non-enzymatic antioxidants in chronic and drug naïve first-episode schizophrenics [reviewed by Reddy & Yao, 1996].

The dopamine receptors

A well-established hypothesis regarding hyperactivity in the dopaminergic system in schizophrenia is concerned with involvement of the dopamine receptors in the disease. This could be caused by a dysexpression or a malfunction of the receptors.

The high interest in the dopamine receptors originates in the observation that typical neuroleptics antagonize the dopamine receptors [Farde et al., 1992] and this is generally believed to be their mode of action. Furthermore, the clinical outcome of the treatment has been shown to be correlated with the drugs affinity for a subtype of the receptors [Creese et al., 1976]. This is why it has long been speculated that the dopamine receptors might be implicated in schizophrenia.

So far, five distinct neuronal dopamine receptor genes have been identified. The proteins encoded by these genes all belong to the superfamily of G-protein coupled 7 transmembrane domain receptors. Based on pharmacological profiles as well as the molecular structure they have been divided into two groups [reviewed by Sibley & Monsma, 1992]. The “D₁-like” family includes the dopamine D₁ and D₅ receptors. The “D₂-like” family includes the dopamine D₂, D₃ and D₄ receptors. The chromosomal distributions of the genes are as follows: D₁: 5q35.1; D₂: 11q22.5; D₃: 3q13.3; D₄: 11p15.5; D₅: 4p15.2 [reviewed by Portin & Alanen, 1997].

Typical neuroleptics bind mainly to the D₂ receptor and it was therefore suggested that this receptor could be implicated in the development of schizophrenia. However, with the discovery of an atypical neuroleptic, clozapine, which binds to the dopamine D₄ receptor with high affinity [Van Tol et al., 1991], focus has been concentrated on this receptor. Treatment with clozapine does not cause the extra pyramidal side effects (disorganised movements) observed when treating with typical D₂ receptor antagonizers and the D₄ receptor is now thought to play a more central role in schizophrenia than it was first believed. As only the D₂-like receptors seem to be involved in the treatment of the disease by dopamine receptor antagonists only this subgroup will be considered here.

Measurement of expression levels of the dopamine receptors

The elucidation of which part the dopamine receptors may play in schizophrenia is rendered difficult. The methods used to determine the amount of receptors have not led to unambiguous results. One approach used is determining the mRNA expression level by RT-PCR, northern blotting or ribonuclease protection assay. Others are based on measurement of the density of putative receptor binding sites by using radioactive labelled ligands with different affinities for the various receptors.

Measurement of the density of putative D_2, D_3, and D₄ receptor binding sites has typically been done using [³H]raclopride and [³H]nemonapride. [³H]raclopride binds to dopamine D₂ and D₃ receptors with high affinity whereby the combined density of these two receptors can be estimated. [³H]nemonapride binds to dopamine D₂, D₃, and D₄ receptors. Thereby, labelling of brain homogenates with these two different ligands allows an estimation of the putative D₄ receptor sites. This technique is not ideal as different affinities of the [³H] ligands for the receptor subtypes make interpretation of the results more difficult. However, the lack of specific ligands complicated the direct measurements for a long time. With the discovery of the D₄ receptor specific ligand, [³H]NGD-94-1, the direct determination of D₄ receptors has been attempted [Lahti et al., 1998].

However, there is still some inconsistency between the results obtained by different methods as well as by the same method.

Expression of the dopamine receptors in the brains of normal subjects

In brains from normal individuals, all three types of receptor mRNA have been found in moderate levels in the medial temporal lobe structures (e.g. hippocampus, amygdala and entorhinal cortex) as well as in the neocortex. The expression of D₂ and D₃ receptor mRNA is found to be high to moderate in the basal ganglia (striatum and nucleus accumbens) [reviewed by Meador-Woodruff, 1995]. The level of expression of D₄ receptor mRNA in the basal ganglia has not been clarified. D₄ receptor mRNA has been detected in caudate nucleus as well as in nucleus accumbens [Matsumoto et al., 1996; Stefanis et al., 1998] while others have been unable to detect the transcript in this region [reviewed by Meador-Woodruff, 1995]. D₄ receptor mRNA has also been found in frontal cortex [Mulcrone & Kerwin, 1996; Stefanis et al., 1998]. D₂ receptor mRNA expression was high and D₃ and D₄ receptor mRNA levels were undetectable or low in the substantia nigra [Matsumoto et al., 1996; reviewed by Meador-Woodruff 1995].

The results obtained with the D₄ receptor specific ligand [³H]NGD-94-1 indicate that the highest density of D₄ receptor in normal brain is in the hippocampus, entorhinal cortex, insula and collateral sulcus. Lower amounts of binding sites were also detected in the basal ganglia (striatum and nucleus accumbens) [Lahti et al., 1998]. This is in agreement with Murray et al. (1995) who found putative D₄ receptor binding sites to be homogenously distributed in the basal ganglia using [³H]nemonapride and [³H]raclopride labelling.

The reason for the discrepancy between D₄ receptor mRNA distribution and binding sites the basal ganglia is still not clear. It is possible that one or more of the [³H] labelled ligands also bind to undefined sites, which are different from D₄ receptors. Another possibility is that the only D₄ receptors present in the striatum are located presynaptically on afferents from other brain areas. If this is the case, the receptors will be synthesised in distally located cell bodies, explaining the lack of mRNA in striatum. The major corticostriatal afferents to the striatum are glutamatergic [Crossman & Neary, 1995]. The frontal cortex is a major source of glutamatergic afferents in this region, and so the D₄ receptor binding sites detected in striatum may be synthesised in frontal cortex. This is in accordance with the existence of D₄ receptor mRNA in frontal cortex [Stefanis et al., 1998].

So far, there seems to be a number of indications for the existence of the dopamine D₄ receptors in the limbic system as well as in the basal ganglia although their relative level in this latter area may be rather low.

The distribution of the D₂-like receptor mRNA is shown in table 1. The binding sites of the D₄ receptor are shown in table 2.

Table 1: Normal dopamine receptor mRNA distribution

	D2	D3	D4
Basal ganglia: Caudate nuc. Putamen N. accumbens	+++ +++ +++	+ +/++ +++	-/+ -/+ -/+
Amygdala Hippocampus Entorhinal cortex	+ ++ ++	? ++ +	++ ++ ++
Neocortex	+++	+	+
Substantia nigra	+++	-/+	-/+

[Matsumoto et al., 1996; Stefanis et al., 1998; reviewed by Meador-Woodruff 1995]

Table 2: Normal D₄ receptor binding sites

	D4
Basal ganglia: Caudate nuc. Putamen N. accumbens	++ ++ ++
Hippocampus Entorhinal cortex	+++ +++
Neocortex	++
Substantia nigra	0

[Murray et al., 1995; Lahti et al., 1998; Seeman et al., 1993]

Expression of the dopamine receptors in the brains of schizophrenics

A difficulty in determining the role of the dopamine receptors in schizophrenia is the limited availability of human brain material, as well as the fact that the use of neuroleptics has been shown to increase the number of dopamine receptors. One month treatment of healthy rats with a widely used drug haloperidol results in a 2-fold increase of striatal D₄ receptor density and a 19% increase in the combined density of striatal D₂ and D₃ receptor sites, using untreated rats as controls [Schoots et al., 1995]. The result is consistent with an 88% increase in receptor mRNA levels in the striatum of the same rats. No increase in the receptor density in the frontal cortex was found. Therefore, medical records need to be available and considered when estimating the role of the dopamine receptors in schizophrenia.

Comparing dopamine D₄ receptor densities between schizophrenics and control groups has led to several interesting discoveries. Seeman et al. (1993) labelled D₂, D₃ and D₄ receptor binding sites with [³H]raclopride and [³H]nemonapride in brain homogenates from the striatum of postmortem schizophrenics and controls, and estimated the density of of the receptors. The density of D₄ receptors was six times elevated in the schizophrenic group and the density of D₂ and D₃ receptors together was increased by 10%. This group of patients had received neuroleptic treatment prior to their death, though. Another control group consisting of patients with Alzheimers or Huntingtons chorea, who had also received neuroleptic treatment, showed an 1.9 elevation in the density of striatal D₄ receptors. This number is consistent with the results obtained from experiments with rats treated with a neuroleptic drug for one month [Schoots et al., 1995]. Therefore, there are indications that the elevation found by Seeman et al. (1993) is not due entirely to the use of neuroleptics.

The existence of increased density of D₄ dopamine receptor sites in striatum from schizophrenics has been confirmed by others using the same method [Murray et al., 1995]. Stefanis et al. (1998) found increases in the level of D₄ receptor mRNA in frontal cortex of schizophrenics, but this study used brain material from patients receiving neuroleptics just before their death, and it cannot be excluded that this was the actual reason for the observed increase. However, the elevation was found to be restricted to the frontal cortex and did not involve the caudate nucleus, which is just the opposite to the results obtained when treating rats with a neuroleptic [Schoots et al., 1995]. These rats had a regional up-regulation of mainly D₄ receptors in striatum but no effect was seen in the frontal cortex, suggesting that the up-regulation discovered by Stefanis et al. (1998) may be disease-related. However, no elevation in D₄ receptor mRNA levels was found in frontal cortex of schizophrenics who had received neuroleptic treatment prior to death in another study [Mulcrone & Kerwin, 1996].

Using the D₄ receptor specific ligand [³H]NGD-94-1, increases in D₄ receptor density were found in the hippocampus of schizophrenics treated with antipsycotic drugs at the time of death as well as in schizophrenics who did not receive drugs for at least 3 months prior to their death [Lahti et al., 1998]. No changes in the density of D₄ receptors were found in striatum (caudate nucleus and putamen) which is in agreement with the D₄ mRNA measurements made by Stefanis et al. (1998). These results are in contrast to the findings of Murray et al. (1995) and Seeman et al. (1993) as well as to the suggestion that neuroleptics cause an up-regulation in this area.

Using [¹²⁵I]epidepride to label D₂ and D₃ receptors, instead of [³H]raclopride, and [³H]nemonapride to label D₂, D₃, and D₄ receptors_, no striatal D₄ dopamine receptors was detected in schizophrenics or normal controls [Reynolds & Mason, 1995]. The D₂ and D₃ receptor density was increased by two-fold in the schizophrenic group, which was suggested to be due to the neuroleptic treatment of these patients. The absence of D₄ receptor in striatum is consistent with the measurements of D₄ receptor mRNA in this region [reviewed by Meador-Woodruff, 1995]. In another study using the same ligands, an elevation of D₄ receptor binding sites in putamen of schizophrenics could be detected [Seeman et al., 1995]. Conclusion about whether an up-regulation of particularly D₄ receptors takes place in the course of schizophrenia or at least in a subgroup of patients is still controversial. There seems to be some indications for an increase in the expression levels in striatum, but the exact significance of these findings remains to be discovered.

Table 3 summarises D₄ dopamine receptor up-regulation in various brain regions in schizophrenia.

Table 3: D₄ dopamine receptor up-regulation in schizophrenia

	D4
Basal ganglia: Caudate nuc. Putamen N. accumbens	-/ -/ -/
Hippocampus Entorhinal cortex
Neocortex	-/

[Seeman et al., 1993; Murray et al., 1995; Reynolds & Mason, 1995;

Seeman et al., 1993; Seeman et al., 1995; Lahti et al., 1998; Stefanis et al., 1998]

The density of D₃ receptors has been found to be elevated in striatum in a group of postmortem schizophrenics using the D₃-specific radioactive labelled ligand [¹²⁵I]-7-OH-DPAT [Gurevich et al., 1994]. However, the authors do not mention the patients’ history of neuroleptic treatment and others have not confirmed this finding.

It is still not clear whether an up-regulation of the dopamine receptors, in particular the D₄ receptor, do in fact play a role in the development and the cause of schizophrenia.

Moreover, down-regulation of the dopamine receptors has been detected in schizophrenia as well. RT-PCR showed a selective loss of the D₃ transcript in the parietal and motor cortices in post mortem schizophrenics compared to normal controls [Schmauss et al., 1993]. This has been suggested to be due to an up-regulation of an alternative splice variant of D₃, D_3nf, at the expense of normal D₃ mRNA [Schmauss 1996]. The existence of this splice variant does not seem to be due to a splicing error as the transcript was found in the same amounts as normal D₃ mRNA in normal human cortical tissue [Liu et al.,1994]. However, the ratio of D_3nf/ D₃ mRNA has been demonstrated to be increased in the cingulate cortex of schizophrenics compared to normal controls [Schmauss 1996], suggesting an abnormal post-transcriptional processing of D₃ pre-mRNA in schizophrenia. It is possible that unique splicing factors are required for the alternative D_3nf mRNA and that altered expression of such factors underlies the observed abnormality in mRNA processing. The splice variant has an unusual splice site and has a deletion of 98 nucleotides within exon 1. The translated protein is truncated in the putative third cytoplasmic domain, which may affect signaling through the coupled G-protein [Liu et al., 1994]. An important point is whether the altered splicing is initiated due to neuroleptic treatment as all schizophrenics in this study had received treatment.

So far, no conclusive evidence exists for the role of the dopamine receptors in schizophrenia.

Altered signalling through the dopamine receptors

When measuring the levels of dopamine receptor binding sites the labelling of D₂ and D₃ receptors with [³H]raclopride was done using a stable GTP analog (Gpp(NH)p), which elevates the number of raclopride binding sites [Seeman et al., 1993]. This has been suggested to be caused by the GTP analog converting the high affinity binding state of G-protein-linked receptors into the low affinity state, leading to a decrease in the binding of endogenous dopamine [Hall et al., 1992]. Thereby, the binding of antagonists such as [³H]raclopride can be enhanced. The mechanism whereby this happens is believed to depend upon the interaction of Gpp(NH)p with the a-subunit of G-proteins associated with the receptor. The rise in [³H]raclopride binding sites in the presence of the GTP analog has not been observed in brains of schizophrenics [Seeman et al., 1993], thereby indicating a possible abnormality in G-proteins coupled to D₂ and D₃ receptors in schizophrenia. Consistent with this is that the amount of G_i and G_o is decreased in the putamen and the hippocampus of schizophrenics [Okada et al., 1991]. Altered signalling through the receptors may therefore be present some schizophrenics at least, leading to a changed response to dopamine in these patients.

Polymorphisms in the dopamine receptors

The methodology for investigating nucleotide variants and polymorphisms involves a range of studies of promoters, 5’ untranslated regions (UTR), exons, introns and 3’ UTR’s. Deviations in these regions can lead to altered transcription, translation, further processing and sorting of the receptors as well as to a change in function of the proteins. The nucleotide variation may not be pathogenic by itself but could be an association marker for schizophrenia or be in linkage with a pathogenic variation.

The most recent studies on different polymorphisms of the D₂ receptor resulted in controversial conclusions about their influence on the development of schizophrenia and their importance as association markers. Arinami et al. (1997) identified a polymorphism by single stranded conformational polymorphism (SSCP) followed by sequencing nucleotide variants using blood samples from 20 randomly selected Japanese schizophrenic patients and 20 Japanese control subjects. The polymorphism was located in the promoter of the D₂ receptor gene and was a –141 insertion/deletion variant. The most frequent polymorphism found is the insertion variant (two cytosines in a row) compared to the deletion variant (one cytosine deleted) [Arinami et al., 1997].

After such findings two questions have arisen: Is the –141 ins/del polymorphism functional and/or can the polymorphism be used as an association marker? Making an expression system in human cells and thereby measuring the influence of the polymorphisms on the strength of the promoter answered the first question. This showed that the deletion variant was a less strong as a promoter. To answer the second question blood samples from 260 schizophrenics and 312 control subjects were analysed. The deletion variant was underrepresented in the schizophrenic patients suggesting a negative association between the –141del variant and schizophrenia. These results could indicate that individuals carrying the –141del polymorphism are less susceptible to develop schizophrenia.

Several groups have later repeated the association study. Tallerico et al. (1999) were not able to confirm the findings. They performed an association study in a North American schizophrenic group analysing the frequency of the –141 ins/del polymorphism in 50 schizophrenics and 51 control subjects. They did not identify any significant differences in distribution of the alleles between the two groups. Similar studies carried out on German and British subjects did not confirm any association [Stöber et al., 1998; Arranz et al., 1998].

There could be several reasons for these discrepancies. First, the investigations were performed on different populations, Caucasians and Asiatic people. It is well known that allelic frequencies can vary among geographical populations, which could affect the susceptibility for developing schizophrenia. Second, the overall statistical material might be too small. Tallerico et al. (1999) suggested that at least 1500 schizophrenia patients and 1500 control subjects have to be analysed to get reliable results. Despite the fact that the analysed patients are diagnosed on the basis of guidelines from the American Psychiatric Association, the types of schizophrenia may differ due to general difficulties establishing correct diagnosis.

In recent years several reports of an association between schizophrenia and the glycine-9-serine polymorphism of D₃ have emerged. Crocq et al. (1992) described a polymorphism within exon 1 of D₃ that creates a Msc1 restriction site and a change of glycine to serine in the extracellular domain of the receptor. The Msc1 restriction site was found to be associated with schizophrenia in a group of patients [Crocq et al. (1992)]. Asherson et al. (1996) have further investigated the Msc1 polymorphism and proposed two possibilities for the role of this site. The Msc1 allele might be pathogenic, bringing about a small increase in the susceptibility for developing schizophrenia or the Msc1 polymorphism is a marker for a significant polymorphism within the gene. Using SSCP they found that the Msc1 substitution polymorphism was the only important polymorphism in the D₃ receptor in relation to schizophrenia. There was detected a significantly increased homozygosity for Msc1 among the patients, particularly among males.

Segman et al (1999) found a negative association between the Msc1 site and dyskinesia, a disease characterised by involuntary movements. It is a well-known adverse effect of long-term administration of antipsychotic drugs in schizophrenics. In brief, the association was manifested as an increased likelihood of dyskinesia in neuroleptic treated schizophrenic patients with the glycine allele.

Further investigations should elucidate whether a dysfunction of the receptor is related to the amino acid substitution.

The D₄ receptor encompasses a hypervariable segment in the coding region of exon 3 characterized by a varying number of up to 10 times of a 48 bp repeat [Lichter et al., 1993]. The D₄ receptor polymorphism of the 48 bp repeats is not associated with schizophrenia but it was suggested that the receptor allele variants may be related to the neuroleptic response [Tanaka et al., 1995; Hwu et al., 1998].

Hwu et al. (1998) investigated a possible relationship between neuroleptic treatment response in schizophrenia and polymorphisms of the D₄ gene. Two groups of schizophrenic patients were described: The genotypes of group I consisted of one allele carrying 2 times 48 bp repeats and one allele carrying 2,4 or 6 repeats. Group II was a 4 times 48 bp repeat homozygous. DNA from the patients’ lymphocytes were analysed by PCR, and Hwu et al. (1998) concluded that the group with a homozygous 4 times 48 bp repeat in exon 3 of D₄ exhibited a more rapid neuroleptic response. The biological function of the 48 bp repeat polymorphisms is not yet known so the 48 bp might be in linkage with other putative functional polymorphisms.

Discussion

The hypothesis that schizophrenia is caused by hyperactivity in the dopaminergic system has been shown to be far too simple. The dopaminergic system does seem to be involved in the disease but it is still not clear how the dopamine homeostasis is altered and why. Several different mechanisms involved in the dopaminergic pathways have been proposed to play a major role in the development of schizophrenia but so far all of these has been rejected as being a major cause. Nevertheless, many cellular aspects of the dopamine system are changed in subgroups of schizophrenics and some of them may be of importance for the development and symptoms of the disease.

Low activity of the dopaminergic system has been correlated to the negative symptoms of schizophrenia. This low activity can be the result of a decrease in the synthesis of dopamine caused by a low tyrosine uptake in the brain [Ramchand et al., 1996; Wiesel & Bjerkenstedt, 1996] or a decreased activity of DDC [Hietala et al., 1999]. A recent study showed a significant reduction in the number of dopaminergic axons in the prefrontal cortex [Akil et al., 1999], which could be a consequence of a low antioxidant defence in the schizophrenic patients. Although down-regulation of the D₃ receptor has been found, a general down-regulation of the dopamine receptors cannot itself explain a decrease in dopamine transmission in schizophrenia.

Presence of hyperactivity in the dopaminergic system has been extensively searched for. Several groups have found an elevation in the uptake of DOPA in various brain parts. This was interpreted as an increase in the activity of DDC, possibly reflecting a higher dopamine synthesis [Reith et al., 1994; Hietala et al., 1999; Lindström et al., 1999]. An increased density of the dopamine receptors has been found to be associated with schizophrenia. D₄ has been found to be profoundly elevated in the striatum of different patient groups [Seeman et al., 1993; Murray et al., 1995]. Other groups were not able to confirm these results, so up-regulation of D₄ receptors is not a general mechanism contributing to dopaminergic hyperactivity in schizophrenia. However, it is possible that one mechanism whereby hyperactivity arises is an up-regulation of the expression of the dopamine receptors at least in a subgroup of patients.

A major problem interpreting the various results is the existence of many compensatory mechanisms in the dopaminergic pathway. The study on DAT KO mice emphasised this point [Giros et al., 1996; Jones et al., 1998]. Thus, it can be difficult to determine whether an observed change in the dopamine pathway is the primary cause of the disease or just a compensatory mechanism. For instance, the up-regulation of the postsynaptic dopamine receptors can be a mechanism to compensate for a low presynaptic release of dopamine and thus, indicate a general hypodopaminergic state of the system instead of a hyperdopaminergic state. Another complication concerning the receptors is the presence of D₂ autoreceptors. In many studies it is not possible to determine whether the receptors are located on the presynapse or on the postsynapse. This makes it difficult to draw any conclusion about the consequences of an up-regulation of the receptors.

Family, twin, and adoption studies indicate a genetic predisposition to schizophrenia. Genetic studies have implicated several minor susceptibility loci; however, the clinical impact of these loci on the neurobiology of schizophrenia is still unclear [reviewed by Portin & Alanen, 1997].

Studies on different allele variants of TH have shown a weak correlation to schizophrenia in a certain ethnic group [Wei et al., 1997; Burgert et al., 1998]. D5S111, which is a marker for DAT, has been found to be associated with schizophrenia in one family [Silverman et al., 1996] but this has not been generally confirmed [King et al., 1996; Persico & Macciardi, 1997]. Studies on D₃ and D₄ receptors have shown that allele variants encoding structurally slightly different receptors can be associated to schizophrenia [Hwu et al., 1998; Segman et al., 1999]. These receptors might have a different affinity for dopamine and may therefore increase the susceptibility for developing schizophrenia.

These findings support the theory that schizophrenia has multiple genetic causes; mutations in various genes, which affect the dopamine pathway, might cause the same symptoms.

While it is generally acknowledged that genetic factors do play a role in determining an individual’s susceptibility to develop schizophrenia it is becoming increasingly clear that with equal genetic loading, expression of the disease can be influenced by external factors. In rats anoxia during birth and repeated stress contribute to the development of dopaminergic hyperfunction, and thus support the view that birth complications may contribute to the pathophysiology of psychiatric disorders [Brake et al., 1997]. Others believe that failures in early brain development and structural abnormalities are associated to the pathophysiology of schizophrenia. The frequency of in utero insults and/or obstetric complications has been found to be higher in mothers of schizophrenics than in mothers of non-schizophrenics. [reviewed by Egan & Weinberger, 1997]

The failures in brain development may affect the brain areas differently; the activity may be up-regulated in some parts and down-regulated in other parts. This could account for the changed asymmetry and loss of prefrontal axons and might explain that both negative and positive symptoms can be found in the same patient.

Interneural connections in the brain are very complex and the different neurotransmitter systems interact closely. Changes in the activity of the dopaminergic system could be caused by abnormalities elsewhere in the brain. One of the systems that recently has received a lot of attention is the glutamatergic system, which is known to interact with the dopaminergic neurones. Phencyclidine (PCP) blocks a glutamate receptor in some parts of the brain and induces symptoms that closely resemble the mental state in schizophrenia [Rang et al., 1999].

This is just some of many theories concerning the aetiology of schizophrenia. Despite many years of study the dopaminergic systems role in schizophrenia still remains controversial. Changes in the dopamine system are found in many studies, and dopamine is somehow involved in the disease. It seems reasonable to suggest that a disease as complex as schizophrenia might involve both disturbances in different neurotransmitter systems as well as developmental abnormalities. Thus, knowledge from other fields of neurobiology has to be taken into account when unravelling the aetiology of schizophrenia.

References

Akil, M.; Pierri, J.N.; Whitehead, R.E.; Edgar, C.L.; Mohila, C.; Sampson, A. R.& Lewis, D.A. (1999) Lamina-specific alterations in the dopamine innervation of the prefrontal cortex in schizophrenic subjects. Am J Psychiatry 156:1580-1589

Arinami, T.; Gao M, Hamaguchi, H.; Toru, M. (1997) A functional polymorphism in the promotor region of the dopamine D2 receptor gene is associated with schizophrenia. Hum Mol Genet 6: 577-582

Arranz, M .J.; Munro, J., Li, T.; Collier, D.A.; Kirov, G.; Kerwin, R.W. (1998) A polymorphism in the promotor region of the dopamine D2 receptor gene (DRD2) and drug response: Association studies. Schizophrenia res 29:127

Asherson, P.; Mant, R.; Holmans, P.; Willams, J.; Cardno, A.; Murphy, K.; Jones, L.; Collier, D.; Mcguffin, P.; Owen, M.J. (1996) Linkage, association and mutational analysis of the dopamine D3 receptor gene in schizophrenia. Mol Psychiatry 1: 125-132

Brake, W.G.; Noel, M.B.; Boksa, P. & Gratton, A. (1997) Influence of preinatal factors on the nucleus accumbens dopamine response to repeated stress during adulthood: an electrochemical study in the rat. Neuroscience 77: 1067-1076

Burgert, E.; Crocq, M.-A.; Bausch, E.; Macher, J.-P. & Morris-Rosendahl, D.-J. (1997) No association between the tyrosine hydroxylase microsatellite marker HUMTH01 and schizophrenia or bipolar I disorder. Psychiatr Genet. 8: 45-48

Børglum, A. D.; Bruun, T. G.; Kjeldsen, T. E.; Ewald, H.; Mors, O.; Kirov, G.; Russ. C.; Freeman, B.; Collier, D. A. & Kruse, T. A. (1999) Two novel variants in the DOPA decarboxylase gene: association with bipolar affective disorder. Molecular Psychiatry 4: 545-551

Ciliax, B.J.; Heilman, C.; Demchyshyn, L.L.; Pristupa, Z.B.; Ince, E.; Hersch, S.M.; Niznik, H.B. & Levey, A.I. (1995) The dopamine transporter: Immunochemical characterization and localization in brain. J Neurosci 15: 1714-1723

Creese, I.; Burt, D.R. & Snyder, S.H. (1976) Dopamine receptor binding predicts clinical and pharmacological potencies of antischizophrenic drugs. Science 192: 481-483

Crocq, M.A.; Mant, R.; Asherson, P.; Williams, J.; Hode, Y.; Mayerova, A.; Collier, D.; Lannfelt, L.; Sokoloff, P.; Schwarts, J-C.; Gill, M.; Macher, J-P.; McGuffin, P.; Owen, M.J. (1992) Association between schizophrenia and homozygosity at the dopamine D3 receptor gene. J Med Genet 29: 858-860

Dickinson, S. D.; Sabeti, J.; Larson, G. A.; Giardina, K.; Rubinsttein, M.; Kelly, M. A.; Grandy, D. K.; Low, M. J.; Gerhardt, G. A. & Zahniser, N. R. (1999) Dopamine D₂ receptor-deficient mice exhibit decreased dopamine transporter function but no changes in dopamine release in dorsal striatum. J. Neurochem. 72: 148-156

Egan, M. F. & Weinberger, D. R. (1997) Neurobiology of schizophrenia. Curr. Op. Neurobiology 7: 701-707

Farde, L.; Nordström, A.L.; Wiesel, F.-A.; Pauli, S.; Halldin, C. & Sedvall, G. (1992) Positron emission tomographic analysis of central D₁ and D₂ receptor occupancy in patients treated with classical neuroleptics and clozapine. Arch Gen Psychiatry 49: 538 - 544

Gainetdinov. R.R.; Jones, S.R. & Caron, M.G. (1999) Functionl hyperdopaminergia in dopamine transporter knock-out mice. Biol Psychiatry 46: 303-311

Giros, B.; Jaber, M.; Jones, S.R.; Wightman, R.M. & Caron, M.G. (1996) Hyperlocomotion and indifference to cocaine and amphetamine in mice lacking the dopamine transporter. Nature 379: 606-612

Glenthøj, B. Y.; Mackersprang, T.; Bille, A. E. H. & Hemmingsen, R. P. (1999) Transmitterforstyrrelser hos scizofrene. Ugeskr Læger 161: 1391-1398

Gruender, G.; Davies, E.; Heydari, N.; Benz, P.; Schlosser, R.; Schiegel, S.; Andreas, J.; Stoeter, P.; Wong, D. F. & Benkert, O. (1998) DOPA decarboxylase activity in schizophrenia: A PET study with 6-[F-18]fluorodopa. Journal of Nuclear Medicine 39 (5 suppl.): 83P

Gurevich, E.; Kung, M.P.; Kung, H. & Joyce, J.N. (1994) Dopamine D₃ receptors are elevated in schizophrenia and Alzheimer’s (AD) cases. Soc Neurosci Abstracts 20: 1169

Hall, H.; Halldin, C. & Sedvall, G. (1992) Gpp(NH)p stimulates [³H]raclopride binding to homogenates from human putamen and accumbens. Neurosci Lett 136: 79 - 82

Hietala, J.; Syvälahti, E.; Vilkman, H.; Vuorio, K,; Räkköläinen, V.; Bergman, J.; Haaparanta, M.; Solin, O.; Kuoppamäki, M.; Eronen, E.; Ruotsalainen, U. & Salokangas, R. K. R. (1999) Depressive symptoms and presynaptic dopamine function in neuroleptic-naive schizophrenia. Schizophrenia Research 35: 41-50

Hitri, A.; Hurd, Y.L.; Wyatt R.J. & Deutsch, S. I. (1994) Molecular, functional and biochemichal charateristics of the dopamine transporter: Regional differences and clinical relevance. Clinical Neuropharmacology 17: 1-22

Hwu, H-G.; Hong, C-J.; Lee, Y-L.; Lee, P-C.; Lee, S.F.C. (1998) Dopamine D4 receptor gene polymorphism and neuroleptic response in schizophrenia. Biol Psychiatry 44: 483-487

Ishiguro, H.; Arinami, T.; Saito, T.; Akazawa, S.; Enomoto, M.; Mitushio, H.; Fujishiro, H.; Tada, K.; Akimoto, Y.; Mifune, H.; Shiozuka, S.; Hamaguchi, H.; Toru, M. & Shibuya, H. (1998) Systematic Search for Variations in the Tyrosine Hydroxylase Gene and Their Associations With Schizophrenia, Affective Disorders, and Alcoholism. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 81: 388-396

Jones, S. R.; Gainetdinov, R. R.; Jaber, M.; Giros, B.; Wightman, R. M.& Caron, M. G. (1998) Profound neuronal plasticity in response to inactivation of the dopamine transporter. Neurobiology 95: 4029-4034

King, N.; Bassett, A.S.; Honer, W.G.; Marsellis, M. & Kennedy, J.L. (1997) Absence of linkage for schizophrenia on the short arm of chromosome 5 in multiplex canadian families. Am J Med Genet 74: 472-472

Koeltzow, T. E.; Xu, M.; Cooper, D. C.; Hu, X.-T.; Tonegawa, S.; Wolf, M. E. & White, F. J. (1998) Alterations in dopamine release but not dopamine autoreceptor function in dopamine D₃ receptor mutant mice. J. Neurosci. 18: 2231-2238

Lahti, R.A.; Roberts, R.C.; Cochrane, E.V.; Primus, R.J.; Gallager, D.W.; Conley, R.R. & Tamminga, C.A. (1998) Direct determination of dopamine D₄ receptors in schizophrenic postmortem brain tissue: a [³H]NGD-94-1 study. Mol Psychiatry 3: 528 - 533

L’hirondel, M.; Chéramy, A.; Godeheu, G.; Saiardi, A.; Borelli, E. & Glowinski, J. (1998) Lack of autoreceptor-mediated inhibitory control of dopamine release in striatal synaptosomes of D2 receptor-deficient mice. Braain Research 792: 253-262

Lichter, B.L.; Barr, C.L.; Kennedy, J.L.; Van Tol, H.H.M.; Kidd, K.K.; Livak, K.J. (1993) A hypervariable segment in the human dopamine receptor D4 (DRD4) gene. Hum Mol Genet 2:767-773

Lindström, L. H.; Gefvert, O.; Hagberg, G.; Lundberg, T.; Bergström, M.; Hartvig, P. & Långström, B. (1999) Increased Dopamine Synthesis Rate in Medial Prefrontal Cortex and Striatum in Schizophrenia Indicated by L-(b-11C) DOPA and PET. Biol Psychiatry 46: 681-688

Liu, K.; Bergson, C.; Levenson, R. & Schmauss, C. (1994) On the origin of mRNA encoding the truncated dopamine D₃ receptor D_3nf and detection of D_3nf –like immunoreactivity in human brain. J Biol Chem 269: 29220 – 29226

Matsumoto, M.; Hidaka, K.; Tada, S.; Tasaki, Y. & Yamaguchi, T. (1996) Low levels of mRNA for dopamine D₄ receptor in human cerebral cortex and striatum. J Neurochem 66: 915 - 919

Meador-Woodruff, J.H. (1995) Neuroanatomy of dopamine receptor gene expression: potential substrates for neuropsychiatric illness. Clin Neuropharmacol 18: s14 – s24

Mulcrone, J. & Kerwin, R.W. (1996) No difference in the expression of the D₄ gene in postmortem frontal cortex from controls and schizophrenics. Neurosci Lett 219: 163 - 166

Murray, A.M.; Hyde, T.H.; Knable, M.B.; Herman, M.M.; Bigelow, L.B.; Carter, J.M.; Weinberger, D.R. & Kleinman, J.E. (1995) Distribution of putative D₄ dopamine receptors in postmortem striatum from patients with schizophrenia. J Neurosci 15: 2186 - 2191

Okada, F.; Crow, T.J. & Roberts, G.W. (1991) G proteins (G_i, G₀ ) in the medial temporal lobe in schizophrenia: prelimenary report of a neurochemical correlate of structural change. J Neural Transm 84: 147 - 153

Persico, A. & Macciardi, F. (1997) Genotypic association between dopamine transporter gene polymorphisms and schizophrenia. Am J Med Gen 74: 53-57

Portin, P.; Alanen, Y.O. (1997) A critical review of genetic studies of schizophrenia. II. Molecular genetic studies. Acta Psychiatr Scand 95: 73-80

Rang, H. P.; Dale, M. M. & Ritter, J. M. (1999) Pharmacology 4^th ed. Churchilll Livingstone.

Ramchand, C. N.; Peet, M.; Clark, A. E. & Hemmings, G. P. (1996) Decreased tyrosine transport in fibroblasts from schizophrenics: Implications for membrane pathology. Prostaglandins, Leukotrienes and Essential Fatty Acids. 55: 59-64

Reddy, R.D. & Yao, J.K. (1996) Free radical pathology in schizophrenia: a review. Prostaglandins, Leukotrienes and Essential Fatty Acids. 55: 33-43

Reith, J.; Benkelfat, C.; Sherwin, A.; Yashuhara, Y.; Kuwabara, H.; Andermenn, A.; Bachneff, S.; Cumming, P.; Diksic, M.; Dyve, S. E.; Etienne, P.; Evans, A. C.; Lal, S.; Shevell, M.; Savard, G.; Wong, D. F.; Chouinard, G. E. & Gjedde, A. (1994) Elevated dopa decarboxylase activity in living brain of patients with psychosis. Proc. Natl. Acad. Sci. USA. 91: 11651-11654

Reynolds, G.P. & Mason, S.L. (1995) Absence of detectable striatal dopamine D₄ receptors in drug-treated schizophrenia. Eur J Pharmacol 281: R5 – R6

Schmauss, C. (1996) Enhanced cleavage of an atypical intron of dopamine D₃ receptor pre-mRNA in chronic schizophrenia. J Neurosci 16: 7902 – 7909

Schmauss, C.; Haroutunian, V.; Davis, K.L. & Davidson, M. (1993) Selective loss of dopamine D₃-type receptor mRNA expression in parietal and motor cortices of patients with chronic schizophrenia. Proc Natl Acad Sci USA 90: 8942-8946

Schoots, O.; Seeman. P.; Guan, H.C.; Paterson, A.D. & Van Tol, H.H.M. (1995) Long-term haloperidol elevates dopamine D₄ receptors by 2-fold in rats. Eur J Pharmacol 289: 67 – 72

Seeman, P.; Guan, H.C. & Van Tol, H.H.M. (1993) Dopamine D₄ receptors elevated in schizophrenia. Nature 365: 441 – 445

Seeman, P.; Guan, H.C. & Van Tol, H.H.M. (1995) Schizophrenia: elevation of dopamine D₄-like sites, using [³H]nemonapride and [¹²⁵I]epidepride. Eur J Pharmacol 286: R3 – R4

Segman, R.; Neeman, T.; Heresco-Levy, U.; Finkel, B.; Karagichev, L.; Schlafman, M.; Dorevitch, A.; Yakir, A.; Lerner, A.; Shelevoy, A.; Lerer, B. (1999) Genotypic association between the dopamine D3 receptor and tardive dyskinesia in chronic schizophrenia. Molecular Psychiatry 4: 247-253

Sibley, D.R. & Monsma, F.J. (1992) Molecular biology of dopamine receptors. Trends Pharmacol Sci 13: 61 - 69

Silverman, J. M.; Greenberg, D.A.; Altstiel, L.D.; Siever, L.J.; Mohs, R.C.; Smith, C.J.; Zhou, G.; Hollanders, T. E.; Yang, X-P.; Kedache, M.; Li, G. Zaccario, M.L. & Davis, K.L. ( 1996) Evidence of a locus for schizophrenia and related disorders on the short arm of chromosome 5 in a large pedigree. Am J Med Gen 67: 162-171

Smythies, J.R. ( 1997) Oxidative reactions and schizophrenia: a review-discussion. Schizophrenia Res 24: 357-364

Stefanis, N.C.; Bresnick, J.N.; Kerwin, R.W.; Schofield, W.N. & McAllister, G. (1998) Elevation of D₄ dopamine receptor mRNA in postmortem schizophrenic brain. Mol Brain Res 53: 112 – 119

Stöber, G.; Jatzke, S., Heils, A.; Jungkunz, G.; Knapp, M.; Mössner, R.; Riederer, P.; Lesch, K.P. (1998) Insertion/deletion variant (-141C Ins/Del) in the 5’ regulatory region of dopamine D2 receptor gene: Lack of association with schizophrenia and bipolar affective disorder. J Neural Transm 105: 101-109

Tallerico, T.; Ulpian, C.; Liu, I.S.C. (1999) Dopamine D2 receptor promotor polymorphism: no association with schizophrenia. Psychiatry Research 85: 215-219

Tanaka, T.; Igarashi, S.; Onodera, O.; Tanaka, H.; Kameda, K.; Takahashi, K.; Tsuji, S.; Ihda, S. (1995) Lack of association between dopamine D4 receptor gene and schizophrenia. Am J of Med Gen 60: 580-582

Van Tol, H. H. M.; Bunzow, J. R.; Guan, H.-C.; Sunahara, R. K.; Seeman, P.; Niznik, H. B. & Civelli, O. (1991) Cloning of the gene for a human dopamine D₄ receptor with high affinity for the antipsycotic clozapine. Nature 350: 610-614

Venator, D.K.; Lewis, D.A. & Finlay, J.M. ( 1999) Effect of partial dopamine loss in the medial prefrontal cortex on local baseline and stress- evoked extracellular dopamine concentrations Neuroscience 93: 497-505

Wei, J.; Ramchand, C. N. & Hemmings, G. P. (1997) Possible association of catecholamine turnover with the polymorphic (TCAT)_N repeat in the first intron of the human tyrosine hydroxylase gene. Life Sciences 61: 1341-1347

WHO icd 10 (1998) Psykiske lidelser og adfærdsmæssige forstyrrelser. 5. oplag. Munksgaard.

Wiesel, F.-A. & Bjerkenstedt, L. (1996) Tyrosine transport as an indicator of cell membrane dysfunction in schizophrenia. Prostaglandins, Leukotrienes and Essential Fatty Acids 55: 55-58

Willner, P. (1997) The dopamine hypothesis of schizophrenia: current status, future prospects. International Clinical Psycopharmacoloy 12: 297-308