Because the high-affinity state of the dopamine D2 receptor, D2High, is the functional state of the receptor, has a role in demarcating typical from atypical antipsychotics, and is markedly elevated in amphetamine-sensitized rats, it is important to have a method for the convenient detection of this state by a ligand. The present data show that, in contrast to [(3)H]spiperone or [(3)H]raclopride, [(3)H]domperidone labels D2High sites in the presence of isotonic NaCl in either striatum or cloned D2Long receptors, yielding a dopamine dissociation constant (1.75 nM) in agreement with that found with [(3)H]dopamine. Increased labeling of D2High sites occurred with [(3)H]domperidone after severe disruption of the cells, suggesting that [(3)H]domperidone has better access to the D2 receptor from the cytoplasmic aspect of the cell membrane. The density of the [(3)H]domperidone-labeled D2 receptors was the same as that of the [(3)H]raclopride-labeled D2 receptors, but twice the density of [(3)H]spiperone sites for human cloned D2Long receptors, compatible with the monomer-dimer concept of the D2 receptor. [(3)H]domperidone readily labels the D2High sites in postmortem human brain homogenates. Although [(3)H]spiperone or [(3)H]raclopride can occupy D2High sites, the inability of 1-10 nM dopamine to displace these ligands under isotonic conditions suggests that these ligands may not be suitable for monitoring the physiological high-affinity state of the dopamine D2 receptor by means of [(11)C]methylspiperone or [(11)C]raclopride in humans.

Although phencyclidine and ketamine are used to model a hypoglutamate theory of schizophrenia, their selectivity for NMDA receptors has been questioned. To determine the affinities of phencyclidine, ketamine, dizocilpine and LSD for the functional high-affinity state of the dopamine D2 receptor, D2 High , their dissociation constants (K i ) were obtained on [ 3 H]domperidone binding to human cloned dopamine D2 receptors. Phencyclidine had a high affinity for D2 High with a K i of 2.7 nM, in contrast to its low affinity for the NMDA receptor, with a K i of 313 nM, as labeled by [ 3 H]dizocilpine on rat striatal tissue. Ketamine also had a high affinity for D2 High with a K i of 55 nM, an affinity higher than its 3100 nM K i for the NMDA sites. Dizocilpine had a K i of 0.3 nM at D2 High , but a K d of 1.8 nM at the NMDA receptor. LSD had a K i of 2 nM at D2 High . Because the psychotomimetics had higher potency at D2 High than at the NMDA site, the psychotomimetic action of these drugs must have a major contribution from D2 agonism. Because these drugs have a combined action on both dopamine receptors and NMDA receptors, these drugs, when given in vivo, test a combined hyperdopamine and hypoglutamate theory of psychosis. Keywords: dopamine receptor; phencyclidine; domperidone; ketamine; NMDA receptors; psychotomimetics While the clinical anti-dopaminergic actions of antipsychotic drugs are compatible with the hyperdopamine hypothesis of psychosis and schizophrenia, 1,2 the psychoses caused by glutamate antagonists such as phencyclidine or ketamine suggest a hypo-glutamate component in psychosis. [3][4][5] However, phencyclidine also lowers plasma prolactin 6 and elicits rotation, 7 suggesting a direct or indirect dopaminemimetic action of phencyclidine.Although phencyclidine and ketamine are not selective for glutamate NMDA receptors, 8 the precise affinities of these drugs for dopamine D2 receptors need to be clarified in order to determine their dopaminergic and non-dopaminergic components of action. More specifically, although phencyclidine had a dissociation constant of 37 000 nM at the D2 receptor in rat striatal homogenate, 8 phencyclidine had a dissociation constant of 1.3 nM for the functional high-affinity site of the cloned D2 receptor, or the D2 High receptor. 9 This apparent discrepancy may be resolved by the recent finding that dopamine itself has a dissociation constant of 3000 nM when competing vs [ 3 H]raclopride at striatal D2 receptors, but has a dissociation constant of 1.5 nM at the D2 High receptor when the link between dopamine D1 and D2 receptors is blocked by the D1 antagonist SCH23390. 10 The link between D1 and D2 receptors arises from several sources, including the colocalization of dopamine D1 and D2 receptors in at least 50% of the medium spiny neurons in the striatum, the cooperation and mutual potentiation of D1 and D2 agonists on various behaviors, and the biochemical conversion of D2 receptors from their functional high-affinity state, D2 High , into their low-affinity state, D2 Lo...

While a range of dopamine D(2)-related behaviors are exaggerated in amphetamine-sensitized animals, studies of the dopamine D(2) receptor have reported either no change or a decrease in dopamine D(2) receptor density--especially when measured using radioraclopride. We hypothesized that a decrease in D(2) receptors may actually be "apparent" and that these receptors may still be present, but are noncompetitively "occupied" by endogenous dopamine. Animals sensitized to amphetamine (and their saline controls) were examined 4 weeks after their last injection. We first measured the [(3)H]raclopride binding in vivo, and observed that sensitized animals showed a 29% lower level of raclopride binding in vivo, suggesting an apparently lower level of dopamine D(2) receptors. To assess the reason for this we examined the density of receptors (using Scatchard analysis in vitro) measured by [(3)H]raclopride in the presence and absence of guanilylimidodiphosphate. This guanine nucleotide converts the dopamine-bound high-affinity state of D(2) receptors into low-affinity states, thereby making measurable the absolute density of the sites. As previously reported, the amphetamine-sensitized animals showed a 31% lower number of D(2) receptors in conventional binding (B(max) 15.6 vs. 22.7 pmol/g). However, with the addition of guanilylimidodiphosphate there was an equalization of both groups (B(max) 25.9 vs. 25.6 pmol/g), revealing an additional 10.3 pmol/g in the sensitized animals, but only 2.9 pmol/g in saline controls. There were no changes in the dissociation constant of [(3)H]raclopride for the receptors. The nearly four-fold increase of dopamine D(2) receptors in the high-affinity state occupied by dopamine may explain why amphetamine-sensitized animals show almost an order of magnitude greater response to dopamine-releasing challenges or dopamine agonists, even though the absolute receptor number is unchanged and the "apparent" receptor number is decreased.

Competition with endogenous dopamine (DA) is usually invoked to explain changes in [11 C]raclopride binding observed after amphetamine administration in animals and humans. This account has recently been questioned because a number of inconsistencies have been reported that contradict it. In the present study, we investigated whether the decrease in [3 H]raclopride binding observed in the rat striatum after an amphetamine challenge reflects true competition with endogenous DA or agonist-mediated internalization of D 2 -receptors. We found that the amphetamine-induced decrease in The addition of Gpp(NH)p had no effect on B max , suggesting that these receptors were not just noncompetitively bound with dopamine at the cell-surface. Subcellular fractionation studies showed that amphetamine treatment led to a decrease in radioligand binding in the cell-surface fraction for both In vivo imaging techniques such as positron emission tomography (PET) and single-photon emission computed tomography (SPECT) have been applied to assess the endogenous levels of dopamine (DA) in both basic and clinical investigations. These techniques have been used to show that behavioral tasks, such as playing video games or writing, induce an increased release of striatal DA (Koepp et al., 1998;de la Fuente-Fernandez et al., 2001) and that patients with schizophrenia show an abnormally high release of DA when challenged with amphetamine (Laruelle et al., 1996;Breier et al., 1997). Although these techniques are being extensively used, PET and SPECT do not provide a direct measurement of endogenous DA levels. Changes in endogenous DA levels are inferred from changes in the binding of (Innis et al., 1992;Volkow et al., 1994;Laruelle et al., 1995;Smith et al., 1997), whereas the opposite effect is observed with drugs that decrease synaptic DA, such as reserpine and ␣-methyl-paratyrosine (Ginovart et al., 1997;Laruelle et al., 1997a

It is known that RGS9-2 gene knockout mice show supersensitivity to DA and have a marked elevation in the proportion of DA D2 receptors in the high-affinity state for DA (D2(High) receptors). As this is a similar profile to that observed in the CNS from subjects with schizophrenia, we examined whether postmortem CNS tissue from subjects with the disorder and brain striata from an animal model of psychosis or schizophrenia (the amphetamine-sensitized rat) had altered levels of RGS9-2. The mRNA for RGS9-2 in 29 control hippocampi was 0.185 +/- 0.015 fg per fg of beta-glucuronidase mRNA (average +/- SE), while that in 29 schizophrenia hippocampi was 0.145 +/- 0.015 fg per fg of beta-glucuronidase mRNA (average +/- SE), a reduction of 22%. Of the many receptor-regulating genes related to G proteins, and of 11 RGS genes, RGS9-2 was the most reduced in the amphetamine-sensitized rat striatum. The reduced levels of RGS9-2 expression in both an animal model of schizophrenia and a postmortem schizophrenia brain provide further evidence implicating RGS9-2 as a candidate gene in schizophrenia.