Enhanced Dopamine Transmission and Hyperactivity in the Dopamine Transporter Heterozygous Mice Lacking the D3 Dopamine Receptor.

Dopamine transporter knockout (DATk) mice are known to demonstrate profound hyperactivity concurrent with elevated (5-fold) extracellular dopamine in the basal ganglia. At the same time, heterozygous DAT mice (DATh) demonstrate a 2-fold increase in dopamine levels yet only a marginal elevation in locomotor activity level. Another model of dopaminergic hyperactivity is the D3 dopamine receptor knockout (D3k) mice, which present only a modest hyperactivity phenotype, predominately manifested as stereotypical behaviors. In the D3k mice, the hyperactivity is also correlated with elevated extracellular dopamine levels (2-fold) in the basal ganglia. Cross-breeding was used to evaluate the functional consequences of the deletion of both genes. In the heterozygous DAT mice, inactivation of the D3R gene (DATh/D3k) resulted in significant hyperactivity and further elevation of striatal extracellular dopamine above levels observed in respective single mutant mice. The decreased weight of DATk mice was evident regardless of the D3 dopamine receptor genotype. In contrast, measures of thermoregulation revealed that the marked hypothermia of DATk mice (-2 °C) was reversed in double knockout mice. Thus, the extracellular dopamine levels elevated by prolonging uptake could be elevated even further by eliminating the D3 receptor. These data also suggest that the hypothermia observed in DATk mice may be mediated through D3 receptors.

Microglial activation contributes to depressive-like behavior in dopamine D3 receptor knockout mice.

We previously demonstrated that the dopamine D3 receptor (D3R) inhibitor, NGB2904, increases susceptibility to depressive-like symptoms, elevates pro-inflammatory cytokine expression, and alters brain-derived neurotrophic factor (BDNF) levels in mesolimbic dopaminergic regions, including the medial prefrontal cortex (mPFC), nucleus accumbens (NAc), and ventral tegmental area (VTA) in mice. The mechanisms by which D3R inhibition affects neuroinflammation and onset of depression remain unclear. Here, using D3R-knockout (D3RKO) and congenic wild-type C56BL/6 (WT) mice, we demonstrated that D3RKO mice displayed depressive-like behaviors, increased tumornecrosisfactor-α (TNF-α), interleukin-1ß (IL-1ß), and IL-6 levels, and altered BDNF expression in selected mesolimbic dopaminergic regions. D3R expression was localized to astrocytes or microglia in the mPFC, NAc, and VTA in WT mice. D3RKO mice exhibited a large number of Iba1-labelled microglia in the absence of glial fibrillary acidic protein (GFAP)-labelled astrocytes in mesolimbic dopaminergic brain areas. Inhibition or ablation of microglia by minocycline (25 mg/kg and 50 mg/kg) or PLX3397 (40 mg/kg) treatment ameliorated depressive-like symptoms, alterations in pro-inflammatory cytokine levels, and BDNF expression in the indicated brain regions in D3RKO mice. Minocycline therapy alleviated the increase in synaptic density in the NAc in D3RKO mice. These findings suggest that microglial activation in selected mesolimbic reward regions affects depressive-like behaviors induced by D3R deficiency.

Dopamine D3 receptor blockade rescues hyper-dopamine activity-induced deficit in novel object recognition memory.

Patients afflicted with bipolar disorder demonstrate significant impairments in recognition and episodic memory during acute depressive and manic episodes. These impairments and the related pathophysiology may result from over-activation of the brain dopamine (DA) system. In order to model overactive DA transmission in a well-established novel object recognition (NOR) memory test, we used DA transporter knockdown (DAT-KD) mice, which exhibit reduced DAT expression and display hyper-dopaminergic phenotypes. DAT-KD mice exhibited impaired NOR memory compared to wild-type (WT) mice. This impairment was prevented by administration of FAUC365, a DA D3 receptor (D3R) selective antagonist, prior to object learning. Similarly, D3R knockout (KO)/DAT-KD double mutant mice displayed performance in the NOR test that was comparable to WT mice, suggesting that deficiencies in NOR performance in DAT-KD mice can be compensated by diminishing D3R signaling. GBR12909, a DAT blocker, also impaired NOR performance in WT mice, but not in D3R KO mice. Impaired NOR performance in GBR12909-treated WT mice was also prevented by pretreatment with FAUC365. Together, these findings indicate that reduced DAT activity can impair recognition memory in the NOR test, and D3R appears to be necessary to mediate this effect.

Cariprazine Exhibits Anxiolytic and Dopamine D3 Receptor-Dependent Antidepressant Effects in the Chronic Stress Model.

Background: Cariprazine, a D3-preferring dopamine D2/D3 receptor partial agonist, is a new antipsychotic drug recently approved in the United States for the treatment of schizophrenia and bipolar mania. We recently demonstrated that cariprazine also has significant antianhedonic-like effects in rats subjected to chronic stress; however, the exact mechanism of action for cariprazine's antidepressant-like properties is not known. Thus, in this study we examined whether the effects of cariprazine are mediated by dopamine D3 receptors. Methods: Wild-type and D3-knockout mice were exposed to chronic unpredictable stress for up to 26 days, treated daily with vehicle, imipramine (20 mg/kg), aripiprazole (1 and 5 mg/kg), or cariprazine (0.03, 0.1, 0.2, and 0.4 mg/kg), and tested in behavioral assays measuring anhedonia and anxiety-like behaviors. Results: Results showed that cariprazine significantly attenuated chronic unpredictable stress-induced anhedonic-like behavior in wild-type mice, demonstrating potent antidepressant-like effects comparable with aripiprazole and the tricyclic antidepressant imipramine. This antianhedonic-like effect of cariprazine was not observed in D3-knockout mice, suggesting that the cariprazine antidepressant-like activity is mediated by dopamine D3 receptors. Moreover, cariprazine significantly reduced drinking latency in the novelty-induced hypophagia test in wild-type mice, further confirming its antianhedonic-like effect and showing that it also has anxiolytic-like activity. Conclusions: In combination with previous studies, these results suggest that cariprazine has a unique pharmacological profile and distinct dopamine D3 receptor-dependent mechanism of action that may be beneficial in the treatment of schizophrenia, bipolar disorder, and major depressive disorder.

The Role of Dopamine D1 and D3 Receptors in N-Methyl-D-Aspartate (NMDA)/GlycineB Site-Regulated Complex Cognitive Behaviors following Repeated Morphine Administration.

Background: Opiate addiction is associated with complex cognitive impairment, which contributes to the development of compulsive drug use and relapses. Dopamine and N-methyl-D-aspartate receptors play critical roles in opiate-induced cognitive deficits. However, the roles of D1 and D3 receptors in the N-methyl-D-aspartate/glycineB receptor-regulated cognitive behaviors induced by morphine remain unknown. Methods: The 5-choice serial reaction time task was used to investigate the cognitive profiles associated with repeated morphine administration in D1 (D1-/-)- and D3 (D3-/-)-receptor knockout mice. The expression of phosphorylated NR1, Ca2+/calmodulin-dependent protein kinase II (CaMKII), and cAMP response element-binding protein (CREB) in the brain was examined by western blotting. D1-/- and D3-/- mice were treated with the N-methyl-D-aspartate/glycineB site agonist l-aminocyclopropanecarboxylic acid and the antagonist L-701,324 to chronically disrupt N-methyl-D-aspartate receptor function and investigate their effects on morphine-induced cognitive changes. Results: Repeated morphine administration impaired attentional function and caused impulsive and compulsive behaviors. D1-/- mice exhibited hardly any premature nosepokes. D3-/- mice showed robustly increased morphine-induced impulsive behavior. The numbers of premature responses were decreased by L-701,324 administration and increased by ACPC administration; these effects were completely abolished in D1-/- mice due to their inability to perform reward-based tasks. In contrast, the inhibitory effects of L-701,324 on impulsive behavior were significantly augmented in D3-/- mice. Conclusions: N-methyl-D-aspartate/glycineB site functions may contribute to morphine-induced cognitive deficits, especially those related to impulsive behavior. D1 and D3 receptors may have contrasting effects with respect to modulating impulsive behavior. D3 receptors have inhibitory effects on impulsive behaviors, and these effects are clearly mediated by N-methyl-D-aspartate/glycineB receptor and µ-opioid receptor interactions.

Dopamine D3 and acetylcholine nicotinic receptor heteromerization in midbrain dopamine neurons: Relevance for neuroplasticity.

Activation of nicotinic acetylcholine receptors (nAChR) promotes the morphological remodeling of cultured dopamine (DA) neurons, an effect requiring functional DA D3 receptors (D3R). The aim of this study was to investigate the mechanisms mediating D3R-nAChR cross-talk in the modulation of DA neuron structural plasticity. By using bioluminescence resonance energy transfer2 (BRET2) and proximity ligation assay (PLA), evidence for the existence of D3R-nAChR heteromers has been obtained. In particular, BRET2 showed that the D3R directly and specifically interacts with the ß2 subunit of the nAChR. The D3R-nAChR complex was also identified in cultured DA neurons and in mouse Substantia Nigra/Ventral Tegmental Area by PLA. Cell permeable interfering peptides, containing highly charged amino acid sequences from the third intracellular loop of D3R (TAT-D3R) or the second intracellular loop of the ß2 subunit (TAT-ß2), were developed. Both peptides, but not their scrambled counterparts, significantly reduced the BRET2 signal generated by D3R-GFP2 and ß2-Rluc. Similarly, the PLA signal was undetectable in DA neurons exposed to the interfering peptides. Moreover, interfering peptides abolished the neurotrophic effects of nicotine on DA neurons. Taken together these data first demonstrate that a D3R-nAChR heteromer is present in DA neurons and represents the functional unit mediating the neurotrophic effects of nicotine.

Inhibition of dopamine receptor D3 signaling in dendritic cells increases antigen cross-presentation to CD8+ T-cells favoring anti-tumor immunity.

Dendritic cells (DCs) display the unique ability for cross-presenting antigens to CD8+ T-cells, promoting their differentiation into cytotoxic T-lymphocytes (CTLs), which play a pivotal role in anti-tumor immunity. Emerging evidence points to dopamine receptor D3 (D3R) as a key regulator of immunity. Accordingly, we studied how D3R regulates DCs function in anti-tumor immunity. The results show that D3R-deficiency in DCs enhanced expansion of CTLs in vivo and induced stronger anti-tumor immunity. Co-culture experiments indicated that D3R-inhibition in DCs potentiated antigen cross-presentation and CTLs activation. Our findings suggest that D3R in DCs constitutes a new therapeutic target to strengthen anti-tumor immunity.

Dopamine D3 receptor knockout mice exhibit abnormal nociception in a sex-different manner.

Pain is a complex and subjective experience. Previous studies have shown that mice lacking the dopamine D3 receptor (D3RKO) exhibit hypoalgesia, indicating a role of the D3 receptor in modulation of nociception. Given that there are sex differences in pain perception, there may be differences in responses to nociceptive stimuli between male and female D3RKO mice. In the current study, we examined the role of the D3 receptor in modulating nociception in male and female D3RKO mice. Acute thermal pain was modeled by hot-plate test. This test was performed at different temperatures including 52°C, 55°C, and 58°C. The von Frey hair test was applied to evaluate mechanical pain. And persistent pain produced by peripheral tissue injury and inflammation was modeled by formalin test. In the hot-plate test, compared with wild-type (WT) mice, D3RKO mice generally exhibited longer latencies at each of the three temperatures. Specially, male D3RKO mice showed hypoalgesia compared with male WT mice when the temperature was 55°C, while for the female mice, there was a statistical difference between genotypes when the test condition was 52°C. In the von Frey hair test, both male and female D3RKO mice exhibited hypoalgesia. In the formalin test, the male D3RKO mice displayed a similar nociceptive behavior as their sex-matched WT littermates, whereas significantly depressed late-phase formalin-induced nociceptive behaviors were observed in the female mutants. These findings indicated that the D3 receptor affects nociceptive behaviors in a sex-specific manner and that its absence induces more analgesic behavior in the female knockout mice. © 2016 Wiley Periodicals, Inc.

Genetic blockade of the dopamine D3 receptor enhances hippocampal expression of PACAP and receptors and alters their cortical distribution.

Dopamine D3 receptors (D3Rs) are implicated in several aspects of cognition, but their role in aversive conditioning has only been marginally uncovered. Investigations have reported that blockade of D3Rs enhances the acquisition of fear memories, a phenomenon tightly linked to the neuropeptide pituitary adenylate cyclase-activating peptide (PACAP). However, the impact of D3R ablation on the PACAPergic system in regions critical for the formation of new memories remains unexplored. To address this issue, levels of PACAP and its receptors were compared in the hippocampus and cerebral cortex (CX) of mice devoid of functional D3Rs (D3R(-/-)) and wild-types (WTs) using a series of comparative immunohistochemical and biochemical analyses. Morphometric and stereological data revealed increased hippocampal area and volume in D3R(-/-) mice, and augmented neuronal density in CA1 and CA2/3 subfields. PACAP levels were increased in the hippocampus of D3R(-/-) mice. Expression of PACAP receptors was also heightened in mutant mice. In the CX, PACAP immunoreactivity (IR), was restricted to cortical layer V in WTs, but was distributed throughout layers IV-VI in D3R(-/-) mice, along with increased mRNAs, protein concentration and staining scores. Consistently, PAC1, VPAC1 and VPAC2 IRs were variably redistributed in CX, with a general upregulation in cortical layers II-IV in knockout animals. Our interpretation of these findings is that disturbed dopamine neurotransmission due to genetic D3R blockade may enhance the PACAP/PAC1-VPAC axis, a key endogenous system for the processing of fear memories. This could explain, at least in part, the facilitated acquisition and consolidation of aversive memories in D3R(-/-) mice.

Low dose pramipexole causes D3 receptor-independent reduction of locomotion and responding for a conditioned reinforcer.

Pramipexole is a clinically important dopamine receptor agonist with reported selectivity for dopamine D3 receptors over other dopaminergic and non-dopaminergic sites. Many of its behavioural effects are therefore attributed to D3 receptor activity. Here we relate pramipexole's ex vivo D2 and D3 receptor binding (measured using [(3)H]-(+)-PHNO binding experiments) to its effects on locomotion and operant responding for primary and conditioned reinforcers. We show that pramipexole has inhibitory behavioural effects on all three behaviours at doses that occupy D3 but not D2 receptor. However, these effects are 1) not inhibited by a D3 selective dose of the antagonist SB-277011-A, and 2) present in D3 receptor knockout mice. These results suggest that a pharmacological mechanism other than D3 receptor activity must be responsible for these behavioural effects. Finally, our receptor binding results also suggest that these behavioural effects are independent of D2 receptor activity. However, firmer conclusions regarding D2 involvement would be aided by further pharmacological or receptor knock-out experiments. The implications of our findings for the understanding of pramipexole's behavioural and clinical effects are discussed.

Dopamine receptor D3 deficiency results in chronic depression and anxiety.

Over the last decade accumulating evidence suggests that brain dopamine (DA) has a role in depression, particularly given the high comorbidity of depression with Parkinson's Disease (PD) and the antidepressant effects of the DA receptor subtype 3 (D3R) agonist pramipexole. The present study assesses the role of D3R in depression. Here we hypothesized that D3R mediates the antidepressant effects of DA. Thus, genetic deficiency of D3R in D3R knockout (D3RKO) mice would yield animals with chronic depressive symptoms. Whereas D3R deficient mice did not show significant alterations in locomotion when tested in the openfield, these animals showed anxiety-like symptoms measured as a significant increase in thigmotaxis at the openfield and a significantly lower time spent in the lit compartment at the light/dark exploration test. D3RKO animals also showed depressive-like symptoms as measured by increased immobility time in the Porsolt forced swim test and the tail suspension test, as well as anhedonia measured in the non-motor dependent sucrose test. In conclusion, D3R deficiency results in anxiety-like and depressive-like symptoms that cannot be attributed to motor dysfunction.

The dopamine D3 receptor knockout mouse mimics aging-related changes in autonomic function and cardiac fibrosis.

Blood pressure increases with age, and dysfunction of the dopamine D3 receptor has been implicated in the pathogenesis of hypertension. To evaluate the role of the D3 receptor in aging-related hypertension, we assessed cardiac structure and function in differently aged (2 mo, 1 yr, 2 yr) wild type (WT) and young (2 mo) D3 receptor knockout mice (D3KO). In WT, systolic and diastolic blood pressures and rate-pressure product (RPP) significantly increased with age, while heart rate significantly decreased. Blood pressure values, heart rate and RPP of young D3KO were significantly elevated over age-matched WT, but similar to those of the 2 yr old WT. Echocardiography revealed that the functional measurements of ejection fraction and fractional shortening decreased significantly with age in WT and that they were significantly smaller in D3KO compared to young WT. Despite this functional change however, cardiac morphology remained similar between the age-matched WT and D3KO. Additional morphometric analyses confirmed an aging-related increase in left ventricle (LV) and myocyte cross-sectional areas in WT, but found no difference between age-matched young WT and D3KO. In contrast, interstitial fibrosis, which increased with age in WT, was significantly elevated in the D3KO over age-matched WT, and similar to 2 yr old WT. Western analyses of myocardial homogenates revealed significantly increased levels of pro- and mature collagen type I in young D3KO. Column zymography revealed that activities of myocardial MMP-2 and MMP-9 increased with age in WTs, but in D3KO, only MMP-9 activity was significantly increased over age-matched WTs. Our data provide evidence that the dopamine D3 receptor has a critical role in the emergence of aging-related cardiac fibrosis, remodeling, and dysfunction.

Dopamine receptor 3 might be an essential molecule in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity.

BACKGROUND: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) induces Parkinson's disease (PD)-like neurodegeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc) via its oxidized product, 1-methyl-4-phenylpyridinium (MPP+), which is transported by the dopamine (DA) transporter into DA nerve terminals. DA receptor subtype 3 (D3 receptor) participates in neurotransmitter transport, gene regulation in the DA system, physiological accommodation via G protein-coupled superfamily receptors and other physiological processes in the nervous system. This study investigated the possible correlation between D3 receptors and MPTP-induced neurotoxicity. A series of behavioral experiments and histological analyses were conducted in D3 receptor-deficient mice, using an MPTP-induced model of PD. RESULTS: After the fourth MPTP injection, wild-type animals that received 15 mg/kg per day displayed significant neurotoxin-related bradykinesia. D3 receptor-deficient mice displayed attenuated MPTP-induced locomotor activity changes. Consistent with the behavioral observations, further neurohistological assessment showed that MPTP-induced neuronal damage in the SNpc was reduced in D3 receptor-deficient mice. CONCLUSIONS: Our study indicates that the D3 receptor might be an essential molecule in MPTP-induced PD and provides a new molecular mechanism for MPTP neurotoxicity.

Dopamine D(3) receptor deletion or blockade attenuates cocaine-induced conditioned place preference in mice.

The dopamine (DA) D3 receptor (D3R) has received much attention in medication development for treatment of addiction. However, the functional role of the D3R in drug reward and addiction has been a matter of debate. We recently reported that D3 receptor-knockout (D3(-/-)) mice display increased vulnerability to cocaine self-administration, which we interpret as a compensatory response to attenuated cocaine reward after D3R deletion. Here we report that D3(-/-) mice displayed attenuated cocaine-induced conditioned place response (CPP) compared to wild-type mice. Similarly, blockade of brain D3Rs by YQA-14, a novel DA D3 receptor antagonist, significantly and dose-dependently inhibits acquisition and expression of cocaine-induced CPP in WT mice, but not in D3(-/-) mice. These findings suggest that: 1) D3Rs play an important role in mediating cocaine's rewarding effects; and 2) YQA-14 is a highly potent and selective D3R antagonist in vivo, which deserves further study as a candidate for treatment of cocaine addiction.

Effects of immobilization stress on emotional behaviors in dopamine D3 receptor knockout mice.

A central problem in understanding the dopamine system in anxiety and depression is to specify functions of different members of the dopamine receptor family. Recent studies have reported that the dopamine D2/D3 receptor agonist pramipexole exerts an antidepressant-like effect in the chronic mild stress model and in the behavioral despair model, suggesting dopamine D3 receptor may be an important target for antidepressant actions. The aim of the present study was to examine the role of dopamine D3 receptor on the anxiety-like and depression-like behaviors induced by immobilization stress. We subjected D3 receptor knockout (D3KO) mice to a series of behavioral paradigms after acute (1 h) or chronic (1 h a day for 14 days) immobilization stress. The results showed that immobilization stress significantly altered the anxiety-like behaviors (open field test and elevated plus maze) and depression-like behaviors (tail suspension test) in both D3KO mice and their wild-type littermates. Moreover, further analysis of the data indicated that the D3KO mice, but not their littermates, failed to show a change in immobility time in the tail suspension test after the acute and chronic stress as compared to intact controls, suggesting an increased resistance to the immobilization stress given before behavioral tests. Although our study did not suggest a significant role of D3 receptor in regulating basal anxiety-like and depression-like behaviors, it demonstrated the mice lacking D3 receptor might be more resistant to stressful procedure than their WT littermates.

Dopamine D3 receptor activation promotes neural stem/progenitor cell proliferation through AKT and ERK1/2 pathways and expands type-B and -C cells in adult subventricular zone.

The neurotransmitter dopamine acts on the subventricular zone (SVZ) to regulate both prenatal and postnatal neurogenesis, in particular through D(3) receptor (D(3) R) subtype. In this study, we explored the cellular mechanism(s) underlying D(3) R-mediated cell proliferation and tested if systemic delivery of a D(3) R agonist would induce SVZ multipotent neural stem/precursor cell (NSC/NPC) proliferation in vivo. We found that treatment with the D(3) R agonist, 7-OH-DPAT, enhances cell proliferation in a dose-dependent manner in cultured SVZ neurospheres from wild-type, but not D(3) R knock-out mice. Furthermore, D(3) R activation also stimulates S-phase and enhances mRNA and protein levels of cyclin D1 in wild-type neurospheres, a process which requires cellular Akt and ERK1/2 signaling. Moreover, chronic treatment with low dose 7-OH-DAPT in vivo increases BrdU(+) cell numbers in the adult SVZ, but this effect was not seen in D(3) R KO mice. Additionally, we probed the cell type specificity of D(3) R agonist-mediated cell proliferation. We found that in adult SVZ, GFAP(+) astrocytes, type-B GFAP(+) /nestin(+) and type-C EGF receptor (EGFR(+) )/nestin(+) cells express D(3) R mRNA, but type-A Doublecortin (Dcx)(+) neuroblasts do not. Using flow cytometry and immunofluorescence, we demonstrated that D(3) R activation increases GFAP(+) type-B and EGFR(+) type-C cell numbers, and the newly divided Dcx(+) type-A cells. However, BrdU(+) /Dcx(+) cell numbers were decreased in D(3) R KO mice compared to wildtype, suggesting that D(3) R maintains constitutive NSC/NPCs population in the adult SVZ. Overall, we demonstrate that D(3) R activation induces NSC/NPC proliferation through Akt and ERK1/2 signaling and increases the numbers of type-B and -C NSC/NPCs in the adult SVZ.

Hippocampal neurofibromin and amyloid precursor protein expression in dopamine D3 receptor knock-out mice following passive avoidance conditioning.

Passive avoidance (PA) conditioning is a fear motivated task able to initiate a cascade of altered gene expression within the hippocampus, a structure critical to learning and memory. We have previously shown that neurofibromin (NF1) and amyloid precursor protein (APP), two genes implicated in cognitive function, are differentially expressed in brain of dopamine D3 receptor knock-out mice (D(3)R(-/-)), suggesting that the receptor might have a role in their trascriptional regulation. Here in this study, we hypothesized that during acquisition of PA conditioning the expression of NF1 and APP genes could be influenced by D(3)Rs. To address this issue, we analyzed the expression of NF1 and APP in the hippocampus of both wild-type (WT) and D(3)R(-/-) mice subjected to the single trial step-through PA paradigm. Our finding demonstrated that (1) D(3)R(-/-) mice exhibit increased cognitive performance as compared to WT mice in the step-through PA trial; (2) acquisition of PA increased D(3)R and NF1, but not APP expression in WT mice hippocampus; (3) PA-driven NF1 induction in WT was abrogated in D(3)R(-/-) mice and finally that (4) the heightened basal APP expression observed in naive D(3)R(-/-) mice was totally reversed by acquisition of PA. In conclusion, the present finding show for the first time that both D(3)R and NF1 genes are upregulated following PA conditioning and suggest that hippocampal D(3)Rs might be relevant to NF1 transcriptional regulation in the hippocampus.

Increased excitability of spinal pain reflexes and altered frequency-dependent modulation in the dopamine D3-receptor knockout mouse.

Frequency-dependent modulation and dopamine (DA) receptors strongly modulate neural circuits in the spinal cord. Of the five known DA receptor subtypes, the D3 receptor has the highest affinity to DA, and D3-mediated actions are mainly inhibitory. Using an animal model of spinal sensorimotor dysfunction, the D3 receptor knockout mouse (D3KO), we investigated the physiological consequences of D3 receptor dysfunction on pain-associated signaling pathways in the spinal cord, the initial integration site for the processing of pain signaling. In the D3KO spinal cord, inhibitory actions of DA on the proprioceptive monosynaptic stretch reflex are converted from depression to facilitation, but its effects on longer-latency and pain-associated reflex responses and the effects of FM have not been studied. Using behavioral approaches in vivo, we found that D3KO animals exhibit reduced paw withdrawal latencies to thermal pain stimulation (Hargreaves' test) over wild type (WT) controls. Electrophysiological and pharmacological approaches in the isolated spinal cord in vitro showed that constant current stimulation of dorsal roots at a pain-associated frequency was associated with a significant reduction in the frequency-dependent modulation of longer-latency reflex (LLRs) responses but not monosynaptic stretch reflexes (MSRs) in D3KO. Application of the D1 and D2 receptor agonists and the voltage-gated calcium-channel ligand, pregabalin, but not DA, was able to restore the frequency-dependent modulation of the LLR in D3KO to WT levels. Thus we demonstrate that nociception-associated LLRs and proprioceptive MSRs are differentially modulated by frequency, dopaminergics and the Ca(2+) channel ligand, pregabalin. Our data suggest a role for the DA D3 receptor in pain modulation and identify the D3KO as a possible model for increased nociception.

Distinct roles of dopamine D3 receptors in modulating methamphetamine-induced behavioral sensitization and ultrastructural plasticity in the shell of the nucleus accumbens.

Persistent changes in behavior and psychological function that occur as a consequence of exposure to drugs of abuse are thought to be mediated by the structural plasticity of specific neural circuits such as the brain's dopamine (DA) system. Changes in dendritic morphology in the nucleus accumbens (NAc) accompany drug-induced enduring behavioral and molecular changes, yet ultrastructural changes in synapses following repeated exposure to drugs have not been well studied. The current study examines the role of DA D3 receptors in modulating locomotor activity induced by both acute and repeated methamphetamine (METH) administration and accompanying ultrastructural plasticity in the shell of NAc in mice. We found that D3 receptor mutant (D3−/−) mice exhibited attenuated acute locomotor responses as well as the development of behavioral sensitization to METH compared with wild-type mice. In the absence of obvious neurotoxic effects, METH induced similar increases in synaptic density in the shell of NAc in both wild-type and D3−/− mice. These results suggest that D3 receptors modulate locomotor responses to both acute and repeated METH treatment. In contrast, the D3 receptor is not obviously involved in modulating baseline or METH-induced ultrastructural changes in the NAc shell.

Role of dopamine D3 receptors in basal nociception regulation and in morphine-induced tolerance and withdrawal.

Repeated administration of opioids such as morphine leads to the development of tolerance to their pain-relieving effects as well as to physical dependence. Although the association between the dopamine system and the molecular mechanisms of morphine-induced antinociceptive tolerance has been studied, the possible interaction between morphine-induced tolerance and D3 receptors has not been investigated. In the present study, male mice lacking the dopamine D3 receptor gene were used to investigate the function of D3 receptors in the development of morphine-induced tolerance and withdrawal. Compared with wild-type (WT) mice, the dopamine D3 receptor knockout (D3R KO) mice showed pronounced hypoalgesia. The D3R KO mice clearly developed lower morphine-induced tolerance and showed attenuated withdrawal signs compared with the WT mice. These results suggest that D3 receptors regulate basal nociception and are involved in the development of morphine-induced tolerance and withdrawal.