In the present review article, we will focus on the role of D3 receptors in schizophrenia and drug addiction, as these are two conditions which have received the most exploration. Indeed, a large number of studies have addressed the preclinical pharmacology of D3‐selective compounds and D3 receptor binding measured using brain imaging in humans. Furthermore, a few clinical trials with D3 receptor‐selective compounds have been reported in patients with schizophrenia or drug addiction.

The high affinity of dopamine agonists for the D3 receptor (Sokoloff et al ., 1990 ) also suggested that this receptor could be involved in Parkinson's disease and its treatment. D3 receptors were found to be regulated in a very peculiar manner in experimental conditions mimicking the dopaminergic denervation that occurs in the disease, and after treatment with levodopa, the gold standard medication. Namely, D3 receptors appear to be downregulated following dopaminergic lesion in rats (Lévesque et al ., 1995 ) and monkeys (Bezard et al ., 2003a ). Recently, this downregulation was confirmed in drug‐naïve patients with Parkinson's disease (Boileau et al ., 2009 ). Repeated treatment with levodopa eventually induces, in most of patients, debilitating and pharmacoresistant involuntary movements (i.e., dyskinesia) likely resulting from an excessive response to dopamine. Repeated treatment with levodopa induces a D1‐mediated ectopic expression of D3 receptors in the dorsal striatum and sensitization to levodopa in rats (Bordet et al ., 1997 ) and drug‐induced dyskinesia and an upregulation of D3 receptors in monkeys (Bezard et al ., 2003a ). The mechanism of this regulation was later found to involve a Brain‐derived Neurotrophic factor (BDNF)‐dependent D3 receptor expression (Guillin et al ., 2001 ). The involvement of D3 receptors in levodopa‐induced dyskinesia has been recently confirmed using D3 receptor knockout mice (Solis et al ., 2016 ) and in a PET study showing increased D3 receptor binding in patients with dyskinesia (Payer et al ., 2016 ). The role of D3 receptors in levodopa‐induced dyskinesia has been reviewed elsewhere (Visanji et al ., 2009 ). BDNF‐dependent D3 receptor expression also has implications for depression. Although antidepressant drugs essentially target the serotonin and noradrenaline systems, these systems converge onto mesolimbic dopamine neurons. The data in the literature are rather consistent with respect to decreased activity of mesolimbocortical dopamine neurons in states of chronic stress and in depression, which normalizes through chronic antidepressant treatments (Grenhoff et al ., 1993 ; Prisco & Esposito, 1995 ). BDNF is expressed by mesolimbic dopamine neurons (Seroogy et al ., 1994 ). Therefore, changes in mesolimbic dopamine neuron activity after stress or chronic antidepressant treatment would alter the expression of BDNF‐regulated genes in target neurons. In agreement, various antidepressant treatments, including tricyclic antidepressants, serotonin‐selective reuptake inhibitors, inhibitors of monoamine oxidase, and electroconvulsive shocks, all selectively increase D3 receptor expression in the shell subdivision of the nucleus accumbens (Maj et al ., 1998 ; Lammers et al ., 2000 ). The potential in depression of drugs acting through D3 receptors has also been previously reviewed (Leggio et al ., 2013 ).

Following the identification of 7‐OH‐DPAT as a D3 receptor‐selective ligand, this compound has been widely used to define D3 receptor‐mediated behavioral responses. However, as extensively discussed (Lévesque, 1996 ), 7‐OH‐DPAT revealed D3 receptor selectivity in binding largely under experimental conditions which are not consistent with in vivo conditions in the brain extracellular space (e.g., high sodium concentrations). Indeed, as 7‐OH‐DPAT has since been shown to demonstrate high potency at the D2 receptor along with the D3 receptor, studies aimed at characterizing D3 receptor‐selective behavioral responses using this compound have been misguided. While the selectivity of dopamine agonists for the D3 receptor were overestimated in these previous binding studies (Sautel et al ., 1995 ), a range of D3 receptor‐preferring agonists with preference over the D2 receptor have now been identified, with PD‐128907 having the highest (54‐fold) and bromocriptine the lowest (0.15‐fold) preference. These dopamine agonists with varying D3 receptor preference have been used to establish D3 receptor‐mediated responses in the absence of D3‐selective antagonist. A D3‐mediated dopaminergic response of particular interest was the decrease in cocaine self‐administration in rats (Caine & Koob, 1993 ), which resulted in a left shift in cocaine dose‐effect function (Caine & Koob, 1995 ). Importantly, the potencies of the various dopamine agonists to decrease cocaine self‐administration were found to correlate with their functional in vitro potencies at D3 receptors, but not D2 receptors (Caine & Koob, 1993 ). These results suggested that dopamine agonists acting at D3 receptors either mimic or enhance the effects of cocaine, an effect consistent with the prototypical agonist substitution therapy for drug dependence. In the second part of this article, the main findings relating D3 receptors to drug addiction will be reviewed.

This seminal article (Sokoloff et al ., 1990 ) also reported a higher affinity of the D3 receptor, relative to the D2 receptor, for dopamine and its agonists, suggesting that the D3 receptor could be a target for drugs used to treat Parkinson's disease. Subsequently, 7‐hydroxy‐ N,N′ ‐di‐ n ‐propyl‐aminotetraline (7‐OH‐DPAT) was identified as a presumably agonist‐selective ligand of the D3 receptor, and thereby used to label native D3 receptors in the brain (Lévesque et al ., 1992 ). Remarkably, the localization studies using [ 3 H]‐7‐OH‐DPAT confirmed an earlier localization study of D3 receptor mRNA (Bouthenet et al ., 1991 ), and also permitted the characterization of the phenotype of D3 receptor‐expressing neurons (Diaz et al ., 1995 ). Taken together, these studies support the restricted distribution of the D3 receptor in the brain, which is potentially reflective of its role in the limbic brain functions. Hence, the hypothesis has been put forward that the D3 receptor could be involved in the pathophysiology of several psychiatric disorders, including schizophrenia (Sokoloff et al ., 2006 ).

In September 1990, the molecular cloning of a rather unexpected novel dopamine receptor subtype was published (Sokoloff et al ., 1990 ), expanding the dopamine receptor family beyond the existing D1 and D2 receptors. The newly identified D3 receptor was characterized as a potential target for neuroleptics (the term antipsychotics will be preferred hereafter) based on the observed in vitro affinity for compounds of this therapeutic class. The implications of this finding were that (i) antipsychotic effects might be mediated by blocking either the D2 or D3 receptor, or both, and (ii) targeting the D3 receptor rather than the D2 may provide a more efficacious and better tolerated treatment of psychosis. The first part of this review article will review the available data supporting a role of D3 receptors in schizophrenia and its treatment, with reference to recent preclinical and clinical data.

D3 receptors and schizophrenia

Dopamine vs. glutamate theories of schizophrenia Most theories of the clinical manifestations of schizophrenia and their treatment postulate an imbalance of dopamine and glutamate/γ‐aminobutyric acid (GABA) neurotransmissions, leading to uncontrolled excitatory activity in the brain, particularly in the prefrontal cortex (Carlsson, 1988; Goff & Coyle, 2001; Javitt, 2004). The dopamine theory of schizophrenia stems from the serendipitous discovery of the antipsychotic activity of chlorpromazine (Delay et al., 1952), which was later found to block dopamine receptors (Carlsson & Lindqvist, 1963; Creese et al., 1976; Seeman et al., 1976). This theory received further support through the observed induction of de novo psychosis in non‐psychotic subjects and worsening of psychotic symptoms in remitted patients following psychostimulant administration known to trigger dopamine release (Angrist et al., 1974). However, the central role of dopamine and its receptors in psychotic symptoms and its treatment has received the strongest support from brain imaging studies. Antipsychotic drugs bind to brain D2‐like receptors, labeled by non‐selective D2/D3 receptor radioligands, in the striatum in proportion to their clinical efficacy (Farde & Nordstrom, 1993). Moreover, the psychostimulant amphetamine produces greater dopamine release in psychotic patients during acute drug administration than in control subjects (Laruelle et al., 1996; Abi‐Dargham et al., 1998). More generally, presynaptic dopaminergic function is elevated in the striatum of schizophrenic patients (Howes et al., 2012), which has led to the concept of the ‘sensitized state’ in schizophrenia, a term analogous to sensitization seen with drugs of abuse (Laruelle, 2000; Featherstone et al., 2007). However, dopamine deficiency in the prefrontal cortex is recognized as a hallmark of the disease (Davis et al., 1991; Carlsson & Carlsson, 2006; Slifstein et al., 2015). Supporting this hypothesis, a prospective resting‐state functional magnetic resonance imaging (rs‐fMRI) study showed that functional connectivity of ventral tegmental area (VTA) neurons, the principal source of mesocorticolimbic dopamine, is impaired in schizophrenia and restored by antipsychotic medication (Hadley et al., 2014). Nevertheless, there is no proof of the direct involvement of dopamine in the etiology of schizophrenia. Since the seminal observations of psychotogenic effects of the NMDA receptor blocker ketamine (Luby et al., 1962; Carlsson, 1988; Goff & Coyle, 2001), the glutamatergic theory of schizophrenia has evolved toward the concept of hyper‐excitability of cortical pyramidal cells. Such hyper‐excitability results from a loss of inhibition from GABAergic interneurons, an effect which may be mimicked by blocking NMDA receptors on these neurons (Moghaddam & Javitt, 2012). This results in enhanced electrical spike activity and disorganization of the firing of cortical neurons, adding ‘noise’ and interrupting the ability of these neurons to process information. This also results in enhanced glutamate release by pyramidal cells that project to various subcortical areas, including the striatum and nucleus accumbens. Imaging studies have consistently given credence to glutamate abnormalities in schizophrenia (Poels et al., 2014). Hence, therapies aimed at increasing NMDA receptor function or limiting glutamate release have been most readily explored for the treatment of schizophrenia (Moghaddam & Javitt, 2012). However, this therapeutic approach has been largely unfruitful, with failures in Phase III clinical trials of pomaglumetad, a glutamate presynaptic receptor mGluR2/3 agonist from Lilly (Downing et al., 2014) and bitopertin, a GlyT1 glycine transporter blocker from Roche (http://www.roche.com/media/store/releases/med-cor-2014-01-21.htm). Thus, although interventions based on modulation of glutamate function are still attractive (Goff, 2015), they lack clinical proof‐of‐concept.

D3 receptor occupancy by antipsychotics More than 60 years after the introduction of chlorpromazine as an antipsychotic drug, pharmacological treatment of schizophrenia has remained essentially unchanged, with the exception of improved motor side effects seen with atypical antipsychotics, mainly based on D2‐like receptor blockade (Kapur & Mamo, 2003). However, whether antipsychotics block both D2 and D3 receptors at clinically relevant dosage has long remained a debated question. Initially, antipsychotics were found to bind in vitro with similar affinities to D2 and D3 receptors, with only a slight (2–5‐fold) preference for D2 receptors (Sokoloff et al., 1992; Malmberg et al., 1993). Based on this, it may be anticipated that a substantial fraction of D3 receptors are occupied in vivo by antipsychotics, if one assumes that dopamine occupancy does not greatly differ at D2 and D3 receptors. This assumption is not evident, because dopamine was found to bind more tightly to D3 than to D2 receptors (Sokoloff et al., 1990). However, a two‐site analysis of inhibition at high‐ and low‐affinity receptor sites revealed that the difference between dopamine affinity at D2 and D3 receptors occurs mainly at the D2 low‐affinity receptor site (Sokoloff et al., 1992), which may not be the relevant sites for antipsychotic action (Seeman et al., 2006). The recent identification of radioligand [11C](+)‐4‐propyl‐9‐hydroxynaphthoxazine ([11C]‐(+)‐PHNO) (Narendran et al., 2006), with higher affinity for D3 receptors relative to D2 receptors [estimated to be around 30–50‐fold in vitro (Freedman et al., 1994; van Vliet et al., 2000; Dijkstra et al., 2002) and 20–48‐fold in vivo (Rabiner et al., 2009; Gallezot et al., 2012)] permitted the direct measurement of D3 receptor occupancy by antipsychotics, through the use of Positron Emission Tomography (PET). Moreover, specific human brain regions were identified whereby D3 receptors represent either the main (globus pallidus) or exclusive (substantia nigra) [11C]‐(+)‐PHNO‐binding sites (Graff‐Guerrero et al., 2010; Searle et al., 2010, 2013; Tziortzi et al., 2011). Hence, [11C]‐(+)‐PHNO has distinct advantages which enables its use for in vivo imaging of brain D3 receptors. Most strikingly, clinical PET studies with [11C]‐(+)‐PHNO did not find evidence for D3 receptor occupancy by antipsychotics in patients with schizophrenia receiving chronic treatment. Thus, patients receiving long‐term treatment (> 4 weeks) with clozapine, risperidone, or olanzapine did show occupancy with [11C]‐(+)‐PHNO in D2‐rich regions such as the striatum, but not in the globus pallidus, a D3‐rich region (Graff‐Guerrero et al., 2009). On the contrary, [11C]‐(+)‐PHNO binding in the globus pallidus seemed to be increased, and this unblocked binding was inhibited by a single‐dose treatment with pramipexole. Notably, this initial study did not use a within‐subject baseline PET scan design, but instead an estimation of basal, drug‐free [11C]‐(+)‐PHNO binding obtained in sex‐ and age‐matched healthy subjects. Nevertheless, similar findings were reported in a longitudinal PET study in patients with a first‐episode of schizophrenia with a within‐subject follow‐up design permitting the measurement of [11C]‐(+)‐PHNO binding in the drug‐free, basal state (Mizrahi et al., 2011). Specifically, patients receiving risperidone or olanzapine for at least 2.5 weeks showed reductions of [11C]‐(+)‐PHNO binding in D2‐rich regions, but, unexpectedly, increases of [11C]‐(+)‐PHNO binding the D3‐rich regions globus pallidus and substantia nigra. On the contrary, D3 receptor occupancy by antipsychotics could be demonstrated after an acute challenge with antipsychotics. Thus, [11C]‐(+)‐PHNO binding was decreased in baboons treated with acute therapeutically active doses of clozapine and haloperidol (Girgis et al., 2011) and in healthy human subjects treated with risperidone (Girgis et al., 2015) and cariprazine (Slifstein et al., 2013), with D2/D3 in vivo selectivity corresponding to their in vitro binding selectivity. In addition, significant D3 receptor occupancy could be demonstrated after an acute challenge with the D3 receptor‐selective partial agonist BP897 in baboons (Narendran et al., 2006) and the D3‐selective antagonist ABT‐925 in humans (Graff‐Guerrero et al., 2010). Thus, it seems that the discrepancies shown in PET studies with respect to occupancy by antipsychotics result from adaptive changes occurring after chronic treatment with antipsychotics, which could have masked reductions in [11C]‐(+)‐PHNO binding due to occupancy by these compounds. Two hypotheses can be put forward to explain these changes: (1) either true upregulation of D3 receptor expression or (2) a decrease in endogenous dopamine in D3‐rich regions, making more receptors available for [11C]‐(+)‐PHNO binding. The former hypothesis contradicts preclinical findings showing that no change in D3 receptor expression occurred after a 2‐week treatment with haloperidol (Lévesque et al., 1995; Damask et al., 1996); however, this expression has not been measured in globus pallidus or substantia nigra and discrepant findings have been reported (D'Souza et al., 1997). The latter hypothesis contradicts results of a preliminary study which found that endogenous dopamine, measured with [11C]‐(+)‐PHNO binding after dopamine depletion, did not differ in healthy subjects and schizophrenic patients receiving long‐term treatment with olanzapine in substantia nigra and globus pallidus, although this result was based on a very limited number of subjects (Caravaggio et al., 2015). It should be noted that [11C]‐(+)‐PHNO binding is highly sensitive to change in endogenous dopamine levels, but these levels seem low to very low in the globus pallidus and substantia nigra (Caravaggio et al., 2014). Clearly, further PET studies with [11C]‐(+)‐PHNO are needed to understand the long‐term effects of chronic treatment with antipsychotics on D3 receptors, although there is now little doubt that these compounds readily occupy the D3 receptor after acute treatment.

D3 receptor localization and interactions with glutamate In the rat brain, the largest receptor densities occur in granule cells of the islands of Calleja and in medium‐sized spiny neurons of the rostral and ventromedial shell of nucleus accumbens (Fig. 1), which co‐express the D1 receptor, substance P, dynorphin and/or neurotensin (Diaz et al., 1995; Le Moine & Bloch, 1996). These output neurons from the nucleus accumbens receive their dopaminergic innervations from the ventral tegmental area and reach the entorhinal and prefrontal cortice after relays in the ventral pallidum and mediodorsal thalamus. In turn, the shell of nucleus accumbens receives projections from the cerebral cortex (infralimbic, ventral, agranular, insular and piriform areas), hippocampus and amygdala and also projects to the ventral tegmental area from which dopaminergic afferents originate (Zahm & Brog, 1992; Pennartz et al., 1994). These various specific connections of the shell of nucleus accumbens, a part of the ‘extended amygdala’ (Heimer et al., 1995), suggest that this area is involved in a series of feedback or feed‐forward loops, involving notably the prefrontal cortex and ventral tegmental area and subserving control of emotions, motivation, and reward. In the human and non‐human primate brains, the phenotype of neurons expressing the D3 receptor are not yet identified, but several studies show their distribution to be rather similar to that in the rat with, however, with higher densities and larger distribution in the ventral part of the caudate putamen and the cerebral cortex (Landwehrmeyer et al., 1993; Hall et al., 1996; Girgis et al., 2011; Gallezot et al., 2012). Figure 1 Open in figure viewer PowerPoint et al., 2000 The D3R is expressed in the shell of the nucleus accumbens (A, B), but is also an autoreceptor in the mesencephalum (C, D). Comparisons of D3 receptor binding (A) and D3 receptor immunoreactivity (B) in brain slices of rat taken at the level of the striatal complex. D3 receptor binding sites were labeled with the D3 receptor‐selective radioligand [125I]7‐trans‐OH‐PIPAT and immunoreactivity was revealed using diaminobenzidine. Note the selective distribution in the island of Calleja and in the shell of nucleus accumbens. C and D show neurons in the VTA. C shows D3 receptor immunofluorescence alone (Cy3, red), D shows double labeling immunofluorescence of D3 receptor (Cy3, red) and TH (FITC, green). All TH‐positive neurons also express the D3 receptor. ac, anterior commisura; Co, nucleus accumbens core; ICj, islands of Calleja; ICjM, island of Calleja major; lv, lateral ventricle; Sh, nucleus accumbens shell. Bar: 0.5 mm. Adapted with permission from (Diaz.,). Electron transmission microscopy and immunostaining was performed with a specific anti‐D 3 receptor antibody, which was thoroughly validated by immunoprecipitation of solubilized D 3 receptor binding, overlapping of immunolabeling with D3 receptor binding and suppression of immunolabeling in D3 receptor knockout mice (Diaz et al., 2000). D3 receptor immunoreactivity was surprisingly detected at the level of asymmetric synapses at the head of dendritic spines (Fig. 2). The synaptic localization of the D3 receptor is in marked contrast with those of D1 and D2 receptors, which are either perisynaptic or spread all over dendrites and dendritic spines in medium‐sized spiny neurons of striatum (Hersch et al., 1995; Delle Donne et al., 1997) and nucleus accumbens (Hara & Pickel, 2005). Synapses of the asymmetric type are typical of glutamatergic synapses (Uchizono, 1965; Kemp & Powell, 1971), whereas the majority of presumed dopamine terminals forms symmetric synapses (Arluison et al., 1984; Bolam et al., 2000), where D3 receptor‐associated immunoreactivity was rarely observed. Figure 2 Open in figure viewer PowerPoint et al., 2013 Localization by electron microscopy of D3 receptors at asymmetrical synapses in the nucleus accumbens of the rat brain. A–D show D3 receptor immunoreactivity at asymmetric synapses in dendritic spines (ed) in medium‐size spiny neurons of the nucleus accumbens. Note the high density of labeling in, or near, the region of the postsynaptic density at the head of dendritic spines (particularly visible in a sagittal section of a dendritic spine in D), which is juxtaposed to presynaptic terminal profiles (t), containing clear vesicles and forming asymmetric synapses. E shows immunoreactivity for D3R in a dendrite profile (d), apposed to an unlabeled axon terminal (t) that forms a symmetric synapse. Scale bar 0.25 μm. Reprinted from (Sokoloff.,), with permission. Considering that asymmetrical synapses bearing the D3 receptor are formed with edges of glutamatergic terminals located at some distance of dopamine terminals, the D3 receptor transmission uses a peculiar and unprecedented variety of volume transmission, in which released dopamine diffuses and dilutes over some distance in the interstitial space, to stimulate post‐synaptic receptors of distal non‐dopaminergic synapses. This mode of transmission is possible owing to the higher affinity of D3 receptors for dopamine, as compared to other dopamine receptor subtypes (Sokoloff et al., 1990). It suggests that D3 receptors would be less sensitive to rapid (i.e., phasic changes on a seconds time scale) than to slower (i.e., tonic changes on a minutes to hour range) changes in synaptic dopamine concentrations. If phasic dopamine release from mesolimbic dopamine neurons mediates behaviorally salient responses, whereas tonic release mediates the amplitude of these responses (Grace, 2016), an enhanced D3 receptor sensitivity would result in a state of aberrant salience, which has been hypothesized in schizophrenia (Kapur, 2003). The peculiar localization of D3 receptors suggested direct interactions at glutamatergic synapses, which bear α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole‐propionic acid (AMPA), and NMDA receptors (Bernard et al., 1997; Bernard & Bolam, 1998) at post‐synaptic sites. Liu et al., (2009) showed that the D3 receptor binds to Ca2+/calmodulin‐dependent protein kinase II (CaMKII) in a Ca2+‐dependent manner in the glutamatergic‐associated post‐synaptic densities (PSD)‐enriched fraction of accumbal neurons. NMDA receptor activation that increases intracellular Ca2+ stimulates D3 receptor‐CaMKII interactions. D3 receptor‐CaMKII interactions further impact downstream effectors of cyclic AMP/protein kinase A, such as other constituents of the glutamatergic synapses, for example, AMPA receptor GluR1 subunit. In turn, D3 receptor–CaMKII interactions downregulate D3 receptor functions. A reciprocal regulation of glutamate and dopamine at synaptic glutamatergic synapses results from this process, by which D3 receptor function is exacerbated in conditions of NMDA receptor blockade. Indeed, this latter situation can be produced by non‐competitive NMDA blockers such as phencyclidine or ketamine, which have dissociative and psychotomimetic effects in humans (see above). They produce schizophrenic‐like symptoms in healthy volunteers, including both positive (hallucinations, delusions) and negative (formal thought disorder, social withdrawal) symptoms, as well as cognitive dysfunction (Luby et al., 1959; Cosgrove & Newell, 1991; Jentsch & Roth, 1999). Therefore, the considerations above support the view that blockade of D3 receptor could reverse the effects of NMDA receptor blockade and elicit antipsychotic‐like actions. In medium‐sized GABAergic neurons of the nucleus accumbens, the D3 receptor is not only expressed on the dendritic spines but also on the terminals of these neurons projecting to the lateral part of the ventral tegmental area and medial part of substantia nigra of the mesencephalon (Diaz et al., 2000). The expression of the D3 receptor at the terminals of descending accumbal GABAergic neurons constitutes the majority of D3 receptor binding in the ventral tegmental area/substantia nigra, which are labeled by [11C]‐(+) PHNO. The D3 receptor may exert a tonic inhibition on dopamine neurons in the ventral tegmental area projecting to the nucleus accumbens by stimulating GABA release at accumbal neuron terminals or by an autoreceptor control. Several lines of evidence support this conclusion. D3 receptors potentiate D1 receptor‐induced stimulation of [³H]GABA release from K⁺‐depolarized synaptosomal preparations of substantia nigra pars reticulate containing terminals of nigrostriatal neurons (Avalos‐Fuentes et al., 2013; Cruz‐Trujillo et al., 2013). In addition, D3 receptors are also expressed by dopamine neurons in the ventral tegmental area and substantia nigra (Diaz et al., 2000). D3 receptors inhibit dopamine release (Tang et al., 1994) and synthesis (O'Hara et al., 1996) in a transfected mesencephalic cell line. Dopamine extracellular levels in the nucleus accumbens (Koeltzow et al., 1998) and striatum (Joseph et al., 2002) are twice as high in D3 receptor‐deficient as in wild‐type mice, and blocking the D3 receptor by selective antagonists increases extracellular levels of dopamine in the prefrontal cortex (Lacroix et al., 2003), a projecting area of mesencephalic dopamine neurons. This suggests a negative control that D3 receptors have on dopamine neurons, directly through its autoreceptor function or indirectly through control of GABA release, which in turn results in a downregulation of dopamine release in the prefrontal cortex, and consequently an excitation of glutamate pyramidal cells. This control is relevant to schizophrenia, in view of the dopamine deficiency in the prefrontal cortex (see above). Regulation of glutamate activity by D3 receptor blockade was studied by using functional neuroimaging based on quantification of c‐fos mRNA expression, a non‐specific marker of neuronal activity, and by electrophysiology in a mouse model of chronic NMDA receptor blockade (Sokoloff et al., 2013). c‐fos mRNA levels were significantly reduced in the medial prefrontal cortex after treatment with MK‐801, a non‐competitive NMDA receptor antagonist, and this effect was reduced after BP 897 administration. At the cellular level, c‐fos mRNA levels in VGluT1‐positive, presumably glutamate neurons, decreased globally following treatment with MK‐801 in a large fraction of the neuronal population, with the appearance, however, of heavily c‐fos‐labeled neurons. Notably, these neurons expressing abnormally high c‐fos mRNA levels were absent after pre‐treatment with BP897. In addition, the treatment by MK‐801 elicited hyper‐responsiveness of cortical efferent glutamatergic neurons, as measured by paired‐pulse facilitation of field potentials after orthodromic stimulation in brain slices, and this effect was reversed by BP897 (Sokoloff et al., 2013). These results demonstrate long‐lasting hyper‐responsiveness of prefrontal cortex efferent glutamatergic neurons after NMDA hypofunction and normalization by D3 receptor blockade.

Behavioral effects of D3 receptor‐selective antagonists in animal models of schizophrenia An important achievement toward the elucidation of the role of D3 receptors in schizophrenia has been the identification of D3 receptor‐selective partial agonists or antagonists. Starting with the phenylpiperazine derivative BP 897 (Pilla et al., 1999), a number of original compounds with D3 receptor selectivity higher than 50‐fold were designed (Micheli & Heidbreder, 2006; Boeckler & Gmeiner, 2007). A patent survey indicates that, since 2005, 110 patents or patent applications have been published, which shows that this is still a very active research area today, in both academic and industrial laboratories (Cao et al., 2016; Capet et al., 2016; Micheli et al., 2016a,b; Sun et al., 2016b). The most representative compounds of this class are SB‐277011A (Reavill et al., 2000; Stemp et al., 2000), S33084 (Millan et al., 2000a,b), ABT‐925 (Gross et al., 1997; Geneste et al., 2006), GSK598809 (Searle et al., 2010), and F17141 (Sokoloff et al., 2013). The psychotic effects of dissociative anesthetics acting by blocking the NMDA receptor in humans (see above) are supportive of not only a conceptual framework for understanding schizophrenia but also a predictive pharmacological model of the disease. In rodents, NMDA blockers elicit hyperactivity and stereotypies that can be reversed by treatment with antipsychotic drugs (Corbett et al., 1993; Jentsch & Roth, 1999; Bradford et al., 2010; Adell et al., 2012). Hyperactivity induced by low acute doses of MK‐801 was largely reduced in D3 receptor‐deficient mice and readily antagonized by the D3 receptor‐selective partial agonist BP 897 (Leriche et al., 2003), suggesting that this behavioral abnormality was largely dependent on D3 receptor hyper‐stimulation. These observations were not confirmed in another laboratory (Yarkov et al., 2010), suggesting that the measurement of MK‐801‐induced hyperactivity could depend on either the mouse strain or other experimental conditions. However, inhibition of MK‐801‐induced hyperactivity has been reproduced by a variety of compounds with varying D3 receptor selectivity (Table 1). Remarkably, most D3 receptor‐selective compounds inhibited MK‐801‐induced hyperactivity at much lower doses than spontaneous locomotor activity, which was also the case of clozapine, but not of the other antipsychotics aripiprazole and haloperidol. A more recent compound, F17464, developed by Pierre Fabre Laboratories, is a highly potent D3 receptor antagonist and 5‐HT1A agonist, with moderate affinity for the D2 receptor (Table 1). This compound showed high potency and selectivity against MK‐801‐induced hyperactivity, with little effects on spontaneous locomotor activity (Table 1). It also inhibited amphetamine‐induced hyperactivity (ID 50 = 0.28 mg/kg i.p.) and increased dopamine extracellular levels in the medial prefrontal cortex at 2.5 mg/kg i.p. (Sokoloff et al., 2014). Table 1. Effects of D3 receptor antagonists and partial agonists, compared to selected antipsychotics in the MK‐801 test in the mouse Compound D3/D2L selectivitya ED 50 for inhibiting spontaneous activityb (mg/kg, i.p.) ED 50 for inhibiting MK‐801‐induced hyperactivityc (mg/kg, i.p.) Ratio ED 50 (spontaneous/MK‐801‐induced) Aripiprazole 0.32 0.48 0.21 2.3 Haloperidol 0.41 0.21 0.08 2.6 Clozapine 0.52 6.3 0.49 13 Cariprazine 5.8 0.11 0.02 5.5 ABT‐925 17.3 9.8 < 0.16 > 61 BP 897d 281 7.7 0.40 19 S33084 324 > 10 1.5 > 6.6 SB‐277011A 871 28 14 2 F17141 151 > 10 0.28 > 36 F17464 71 > 2.5 0.09 > 28 F17141 and F17464 were also tested against MK‐801‐induced social interactions deficits in mice (Sokoloff et al., 2013, 2014) in a ‘resident‐intruder’ paradigm (Dixon et al., 1994; Mohn et al., 1999). In the test, a resident mouse is housed alone for 2 weeks and then confronted to a group‐housed intruder. Typically, the resident actively initiates social investigations of the intruder, sometimes initiating fights and rarely avoiding social interaction by escape. When both residents and intruders were treated by continuous subcutaneous infusion of MK‐801 at a low dose (0.48 mg/kg per day), social interaction deficits developed and consisted of both a reduction in social investigations and an increase in escape behavior displayed by the resident mice. Acute treatment with F17141 of the resident and intruder, 30 min before confrontation, dose‐dependently and completely inhibited the social investigation deficit and escape behavior induced by MK‐801 (Sokoloff et al., 2013). Similar results were obtained with F17464 (Fig. 2). Clozapine, at the dose of 1 mg/kg, i.p. also completely inhibited the social interaction deficits and escape behavior in this same test. Haloperidol, at a dose at which it does not produce catalepsy, was inactive. Taken together, the results obtained with D3 receptor antagonists indicate that blocking the D 3 receptor produces antipsychotic‐like effects in mouse models mimicking both positive and negative symptoms. The putative mechanism of action of this class of compounds is depicted on Fig. 3. D3 receptor‐selective antagonists like F17464 act to restore the glutamate/GABA homeostasis in the prefrontal cortex, and a normal glutamate input in projecting subcortical areas, such as the striatum and nucleus accumbens. They do so by interacting with the glutamate system, either directly at the level of the hyperactive excitatory asymmetrical synapses in the striatum, which co‐express D3 receptors, or indirectly by restoring abated dopamine neuron activity that regulate pyramidal glutamate neurons through D1 receptors in the prefrontal cortex. The action on dopamine neurons is exerted through D3 receptors present in the ventral tegmental area, either as autoreceptors, or presynaptic heteroreceptors on GABA terminals, through which blockade reduces GABA release and inhibition on dopamine neuron activity. Figure 3 Open in figure viewer PowerPoint et al., 2002 2004 2006 et al., 2004 2004 2015 Schematic representation of the putative actions of D3 receptor‐selective antagonists like F17464, compared to current antipsychotics acting at both D2 and D3 receptors. A D3 receptor antagonist may interfere with glutamate at the level of assymetrical synapses in the nucleus accumbens (A), or regulate dopamine neuron activity in the ventral tegmental area (VTA), through regulation of GABA release by striato‐nigral GABA terminals which express D3 receptors (B), thus normalizing dopamine release in the prefrontal cortex (cortex) (C). Current antipsychotics, in addition to blocking D3 receptors, also block D2 receptors, in the striatum and nucleus accumbens, causing motor side‐effects and interfering with the reward system, and in the prefrontal cortex, interfering negatively with GABA neuron activity (Gorelova.,; Seamans & Yang,), which may be deleterious to the normalization of the glutamate/GABA balance and task‐dependent neuronal activity, decision‐making, effort‐based procedures (Floresco & Magyar,; Goldman‐Rakic.,; Seamans & Yang,). Adapted from Cukier‐Meisner () (D). D3 antagonists have also consistently demonstrated an ability to increase cognitive performance or to reverse cognitive deficits in rodents and monkeys (reviewed in Nakajima et al., 2013). F17464 also shares this feature, particularly observable in the passive avoidance paradigm, in which the cognitive deficit was induced by blocking cholinergic neurotransmission with scopolamine (Fig. 3). F17464 dose‐dependently reversed scopolamine‐induced deficits in the 0.16–2.5 mg/kg dose‐range, and its effects seem to be stronger than those of cariprazine, which also showed some efficacy, yet at one dose only. Cariprazine was also found efficacious in other rodent models of negative symptoms and cognitive impairments associated with schizophrenia (Neill et al., 2016). Both F17464 and cariprazine had more effect than the antipsychotics aripiprazole, risperidone, olanzapine, and clozapine (Fig. 4). The improvement of cognitive deficits induced by scopolamine appears to implicate the blockade of dopamine‐induced inhibition of gamma oscillations in the hippocampus (Lemercier et al., 2015). Figure 4 Open in figure viewer PowerPoint 1992 P < 0.05 and **P < 0.01 vs. Vehicle (Veh) by Kruskal–Wallis anova followed by Dunn's post hoc test. F17464 and antipsychotics were given i.p. Adapted from Sokoloff et al. ( 2014 Procognitive effects of F17464 in the passive avoidance test in the rat and comparison with antipsychotics. The experiment assesses in male rats the acquisition/retention of passive avoidance of an inescapable scrambled electric footshock, after deficit induced by scopolamine (Chopin & Briley,). Any increase at the testing trial in step‐through latency to enter the compartment where the shock has been received, means that acquisition/retention has improved. *0.05 and **0.01 vs. Vehicle (Veh) by Kruskal–Wallisfollowed by Dunn's post hoc test. F17464 and antipsychotics were given i.p. Adapted from Sokoloff. (). These preclinical results support the use of D3 receptor‐selective antagonists in the treatment of positive and negative symptoms of schizophrenia, as well as cognitive deficits associated with the disease. They have received some confirmation by a clinical study, showing that high midbrain D3 receptor availability, measured by PET with [11C]‐PHNO, is associated with reduced functional connectivity, measured by resting state‐fMRI, between the orbitofrontal cortex and networks implicated in cognitive control and salience processing (Cole et al., 2012). Hence, blocking selectively the D3 receptor offers a therapeutic option for schizophrenia and its associated cognitive deficits (Gross et al., 2013).