D2 Receptor Knockout Mice Research Articles

Dopamine regulates colonic glial cell-derived neurotrophic factor secretion through cholinergic dependent and independent pathways.

Glial cell-derived neurotrophic factor (GDNF) maintains gut homeostasis. Dopamine promotes GDNF release in astrocytes. We investigated the regulation by dopamine of colonic GDNF secretion. D1 receptor knockout (D1 R-/- ) mice, adeno-associated viral 9-short hairpin RNA carrying D2 receptor (AAV9-shD2 R)-treated mice, 6-hydroxydopamine treated (6-OHDA) rats and primary enteric glial cells (EGCs) culture were used. Incubation fluid from colonic submucosal plexus and longitudinal muscle myenteric plexus were collected for GDNF and ACh measurements. D2 receptor-immunoreactivity (IR), but not D1 receptor-IR, was observed on EGCs. Both D1 receptor-IR and D2 receptor-IR were co-localized on cholinergic neurons. Low concentrations of dopamine induced colonic GDNF secretion in a concentration-dependent manner, which was mimicked by the D1 receptor agonist SKF38393, inhibited by TTX and atropine and eliminated in D1 R-/- mice. SKF38393-induced colonic ACh release was absent in D1 R-/- mice. High concentrations of dopamine suppressed colonic GDNF secretion, which was mimicked by the D2 receptor agonist quinpirole, and absent in AAV-shD2 R-treated mice. Quinpirole decreased GDNF secretion by reducing intracellular Ca2+ levels in primary cultured EGCs. Carbachol ( ACh analogue) promoted the release of GDNF. Quinpirole inhibited colonic ACh release, which was eliminated in the AAV9-shD2 R-treated mice. 6-OHDA treated rats with low ACh and high dopamine content showed decreased GDNF content and increased mucosal permeability in the colon. Low concentrations of dopamine promote colonic GDNF secretion via D1 receptors on cholinergic neurons, whereas high concentrations of dopamine inhibit GDNF secretion via D2 receptors on EGCs and/or cholinergic neurons.

Interaction of Brain-Derived Neurotrophic Factor with the Effects of Chronic Methamphetamine on Prepulse Inhibition in Mice Is Independent of Dopamine D3 Receptors.

The aim of the present study was to gain a better understanding of the role of brain-derived neurotrophic factor (BDNF) and dopamine D3 receptors in the effects of chronic methamphetamine (METH) on prepulse inhibition (PPI), an endophenotype of psychosis. We compared the effect of a three-week adolescent METH treatment protocol on the regulation of PPI in wildtype mice, BDNF heterozygous mice (HET), D3 receptor knockout mice (D3KO), and double-mutant mice (DM) with both BDNF heterozygosity and D3 receptor knockout. Chronic METH induced disruption of PPI regulation in male mice with BDNF haploinsufficiency (HET and DM), independent of D3 receptor knockout. Specifically, these mice showed reduced baseline PPI, as well as attenuated disruption of PPI induced by acute treatment with the dopamine receptor agonist, apomorphine (APO), or the glutamate NMDA receptor antagonist, MK-801. In contrast, there were no effects of BDNF heterozygosity or D3 knockout on PPI regulation in female mice. Chronic METH pretreatment induced the expected locomotor hyperactivity sensitisation, where female HET and DM mice also showed endogenous sensitisation. Differential sex-specific effects of genotype and METH pretreatment were observed on dopamine receptor and dopamine transporter gene expression in the striatum and frontal cortex. Taken together, these results show a significant involvement of BDNF in the long-term effects of METH on PPI, particularly in male mice, but these effects appear independent of D3 receptors. The role of this receptor in psychosis endophenotypes therefore remains unclear.

Regulation of Cdc42 signaling by the dopamine D2 receptor in a mouse model of Parkinson's disease.

Substantial spine loss in striatal medium spiny neurons (MSNs) and abnormal behaviors are common features of Parkinson's disease (PD). The caudate putamen (CPu) mainly contains MSNs expressing dopamine D1 receptor (dMSNs) and dopamine D2 receptor (iMSNs) exerting critical effects on motor and cognition behavior. However, the molecular mechanisms contributing to spine loss and abnormal behaviors in dMSNs and iMSNs under parkinsonian state remain unknown. In the present study, we revealed that Cell division control protein 42 (Cdc42) signaling was significantly decreased in the caudate putamen (CPu) in parkinsonian mice. In addition, overexpression of constitutively active Cdc42 in the CPu reversed spine abnormalities and improved the behavior deficits in parkinsonian mice. Utilizing conditional dopamine D1 receptor (D1R) or D2 receptor (D2R) knockout mice, we found that such a decrease under parkinsonian state was further reduced by conditional knockout of the D2R but not D1R. Moreover, the thin spine loss in iMSNs and deficits in motor coordination and cognition induced by conditional knockout of D2R were reversed by overexpression of constitutively active Cdc42 in the CPu. Additionally, conditional knockout of Cdc42 from D2R‐positive neurons in the CPu was sufficient to induce spine and behavior deficits similar to those observed in parkinsonian mice. Overall, our results indicate that impaired Cdc42 signaling regulated by D2R plays an important role in spine loss and behavioral deficits in PD.

Dopamine D1 receptors mediate methamphetamine-induced dopaminergic damage: involvement of autophagy regulation via the AMPK/FOXO3A pathway.

Clinical studies have revealed that methamphetamine abuse increases risk for developing Parkinson's diseases. It is thus important to elucidate the mechanisms by which methamphetamine damages dopaminergic neurons. The present study was designed to elucidate the role of the dopamine D1 receptor in methamphetamine-mediated dopaminergic neuronal damage and its underlying mechanisms. Mice were treated for 4days with vehicle, methamphetamine, or the D1 agonist SKF38393 and then assessed for locomotion and performance in the pole and rotarod tests. Cellular indices of autophagy, LC3, P62, and Beclin-1, tyrosine hydroxylase, and the AMPK/FOXO3A pathway were analyzed in striatal tissue from treated mice, in PC12 cells, and in D1 receptor mutant mice. Repeated treatment with a relatively high dose of methamphetamine for 4days induced both loss of dopaminergic neurons and activation of autophagy in the striatum as evidenced by increased expression of LC3 and P62. However, such treatment did not induce either loss of dopaminergic neurons or activation of autophagy in D1 receptor knockout mice. D1 receptor-mediated activation of autophagy was also confirmed in vitro using dopaminergic neuronal PC12 cells. Further studies demonstrated that the AMPK/FOXO3A signaling pathway is responsible for D1 receptor-mediated activation of autophagy. The present data indicate a novel mechanism for methamphetamine-induced dopaminergic neuronal damage and reveal an important role for D1 receptors in the neurotoxicity of this drug.

Pramipexole regulates depression-like behavior via dopamine D3 receptor in a mouse model of Parkinson’s disease

Depression is one of the strongest predictors of quality of life in patients with Parkinson’s disease (PD). Despite the high prevalence of depression, there is no clear guidance for its treatment in PD because the evidence for the efficacy of most antidepressants remains insufficient. Pramipexole, a dopamine agonist, is one of the few drugs that has proven to be clinically useful. However, the underlying mechanisms of antidepressive effects of pramipexole are still unknown. A 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model, dopamine D2 receptor (DRD2) and D3 receptor (DRD3) knockout mice were used in our study. Compared with other dopamine D2-like receptor agonists and madopar, pramipexole improved depression-like behavior and alleviate bradykinesia in an MPTP-induced mouse model of PD. Pramipexole significantly improved depression-like behavior in DRD2−/− mice but not in DRD3−/− mice. These results demonstrate that the antidepressive effect of pramipexole is mediated by DRD3 but not DRD2. Our findings highlight the need to develop novel dopamine agonists specifically targeting DRD3 for the treatment of depression in PD in the future.

Knockout or Knock-in? A Truncated D2 Receptor Protein Is Expressed in the Brain of Functional D2 Receptor Knockout Mice

Null mice for the dopamine D2 receptor (D2R) have been instrumental in understanding the function of this protein. For our research, we obtained the functional D2R knockout mouse strain described initially in 1997. Surprisingly, our biochemical characterization showed that this mouse strain is not a true knockout. We determined by sequence analysis of the rapid 3′ amplification of cDNA ends that functional D2R knockout mice express transcripts that lack only the eighth exon. Furthermore, immunofluorescence assays showed a D2R-like protein in the brain of functional D2R knockout mice. We verified by immunofluorescence that the recombinant truncated D2R is expressed in HEK293T cells, showing intracellular localization, colocalizing in the Golgi apparatus and the endoplasmic reticulum, but with less presence in the Golgi apparatus compared to the native D2R. As previously reported, functional D2R knockout mice are hypoactive and insensitive to the D2R agonist quinpirole. Concordantly, microdialysis studies confirmed that functional D2R knockout mice have lower extracellular dopamine levels in the striatum than the native mice. In conclusion, functional D2R knockout mice express transcripts that lead to a truncated D2R protein lacking from the sixth transmembrane domain to the C-terminus. We share these findings to avoid future confusion and the community considers this mouse strain in D2R traffic and protein–protein interaction studies.

Central dopamine D2 receptors regulate plasma glucose levels in mice through autonomic nerves

Recent evidence suggests that the central nervous system (CNS) regulates plasma glucose levels, but the underlying mechanism is unclear. The present study investigated the role of dopaminergic function in the CNS in regulation of plasma glucose levels in mice. I.c.v. injection of neither the dopamine D1 receptor agonist SKF 38393 nor the antagonist SCH 23390 influenced plasma glucose levels. In contrast, i.c.v. injection of both the dopamine D2 receptor agonist quinpirole and the antagonist l-sulpiride increased plasma glucose levels. Hyperglycemia induced by quinpirole and l-sulpiride was absent in dopamine D2 receptor knockout mice. I.c.v. injection of quinpirole and l-sulpiride each increased mRNA levels of hepatic glucose-6-phosphatase and phosphoenolpyruvate carboxykinase, which are the key enzymes for hepatic gluconeogenesis. Systemic injection of the β2 adrenoceptor antagonist ICI 118,551 inhibited hyperglycemia induced by l-sulpiride, but not by quinpirole. In contrast, hyperglycemia induced by quinpirole, but not by l-sulpiride, was inhibited by hepatic vagotomy. These results suggest that stimulation of central dopamine D2 receptors increases plasma glucose level by increasing hepatic glucose production through parasympathetic nerves, whereas inhibition of central dopamine D2 receptors increases plasma glucose level by increasing hepatic glucose production through sympathetic nerves.

Inhibiting MAPK14 showed anti-prolactinoma effect

BackgroundThe specific underlying pathogenesis of prolactinoma has not been clarified yet, to the best of our knowledge. p38 mitogen-activated protein kinase (MAPK) signaling including p38α MAPK (MAPK14), p38β (MAPK11), p38γ (MAPK12) and p38δ (MAPK13) is associated with the development and progression of several types of cancer.MethodsImmunofluorescence analysis was performed on the prolactin (PRL) and MAPK14 expressions of pituitary gland in C57BL/6 mice and human prolactinoma specimen. In the present study, the role of MAPK14 in prolactinoma was determined using estradiol-induced mice and dopamine D2 receptor knockout (DRD2−/−) mice models in C57BL/6 wild-type (WT), MAPK14−/− and DRD2−/−MAPK14+/− mice. GH3 cells were transfected with different sets of MAPK14 small interfering RNA, which to study MAPK14 and PRL expression in GH3 cells.ResultsImmunofluorescence analysis showed that PRL and MAPK14 expression were colocalized and increased in the pituitary gland of mice and human prolactinoma specimen compared with the control specimen. It was shown that PRL and MAPK14 expression was colocalized and increased significantly in the pituitary gland of estradiol-injected prolactinoma mice compared with the control mice. Knockout of MAPK14 significantly inhibited tumor overgrowth, and PRL expression was decreased in estradiol-induced mice. Furthermore, MAPK14 knockout of DRD2−/−MAPK14+/− mice significantly reduced the overgrowth of pituitary gland and PRL production and secretion compared with DRD2−/− mice. MAPK14 knockout using siRNA inhibited PRL production in GH3 cells.ConclusionThese results suggest that MAPK14 serves a promoting role in the formation of prolactinoma, and highlights the potential of MAPK14 as a potential therapeutic target in the treatment of prolactinoma.

Behavioral sensitization and cellular responses to psychostimulants are reduced in D2R knockout mice.

Repeated cocaine exposure causes long-lasting neuroadaptations that involve alterations in cellular signaling and gene expression mediated by dopamine in different brain regions, such as the striatum. Previous studies have pointed out to the dopamine D1 receptor as one major player in psychostimulants-induced behavioral, cellular, and molecular changes. However, the role of other dopamine receptors has not been fully characterized. Here we used dopamine D2 receptor knockout (D2-/- ) mice to explore the role of D2 receptor (D2R) in behavioral sensitization and its associated gene expression after acute and chronic cocaine and amphetamine administration. We also studied the impact of D2R elimination in D1R-mediated responses. We found that cocaine- and amphetamine-induced behavioral sensitization is deficient in D2-/- mice. The expression of dynorphin, primarily regulated by D1R and a marker of direct-pathway striatal neurons, is attenuated in naïve- and in cocaine- or amphetamine-treated D2-/- mice. Moreover, c-Fos expression observed in D2-/- mice was reduced in acutely but not in chronically treated animals. Interestingly, inactivation of D2R increased c-Fos expression in neurons of the striatopallidal pathway. Finally, elimination of D2R blunted the locomotor and striatal c-Fos response to the full D1 agonist SKF81297. In conclusion, D2R is critical for the development of behavioral sensitization and the associated gene expression, after cocaine administration, and it is required for the locomotor responses promoted by D1R activation.

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-1β (IL-1β), 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.

Crucial Role of Dopamine D2 Receptor Signaling in Nicotine-Induced Conditioned Place Preference.

Nicotine in tobacco causes psychological dependence through its rewarding effect in the central nervous system (CNS). Although nicotine dependence is explained by dopamine receptor (DR) signaling together with nicotinic acetylcholine receptors (nAChRs), the synaptic molecular mechanism underlying the interaction between dopamine receptor and nAChRs remains unclear. Since reward signaling is mediated by dopamine receptors, we hypothesized that the dopamine D2 receptor (D2R), in part, mediates the synaptic modulation of nicotine-induced conditioned place preference (CPP) in addition to dopamine D1 receptor. To investigate the involvement of D2R, wild-type (WT) and dopamine D2 receptor knockout (D2RKO) mice were assessed using the CPP task after induction of nicotine-induced CPP. As expected, D2RKO mice failed to induce CPP behaviors after repeated nicotine administration (0.5mg/kg). When kinase signaling was assessed in the nucleus accumbens and hippocampal CA1 region after repeated nicotine administration, both Ca2+/calmodulin-dependent protein kinase (CaMKII) and extracellular signal-regulated kinase (ERK) were upregulated in WT mice but not in D2RKO mice. Likewise, nicotine-induced CPP was associated with elevation of pro- brain-derived neurotropic factor (BDNF) and BDNF protein levels in WT mice, but not in D2RKO mice. Taken together, in addition to dopamine D1 receptor signaling, dopamine D2 receptor signaling is critical for induction of nicotine-induced CPP in mice.

Loss of Function in Dopamine Receptor‐3 (D3R) Attenuates Left Ventricular Cardiac Fibroblast Migration and Proliferation In Vitro

Dopamine is a neurotransmitter heavily involved in neural pathways regulating the reward response, body movement, mood, and cognitive function. As such, much of the research on dopamine and its five receptor subtypes has been conducted to observe the in vivo function of each receptor subtype in neurological disorders. However, recent studies by colleagues from our institution have suggested that D3Rs may play a role in cardiac‐related aging, as 2‐month old D3 receptor knock‐out (D3KO) mice show age‐related changes in cardiac function similar to 2‐year old wild‐type (WT) mice. Thus, it would be interesting to understand the role of D3R expression in LV cardiac fibroblast and its function. We isolated, cultured and characterized cardiac fibroblasts from the left ventricles (LV) of WT (23‐weeks old) and D3KO (5 and 31‐week‐old) mice. Immunohistochemistry was performed to determine the expression of D3R in LV cardiac fibroblasts, and cell culture experiments were performed to examine possible changes in WT and D3KO cell migration and proliferation. Proliferation was measured via total cell count over time points of 6, 12, 24, and 36 hours, while migration in response to scratch wound was examined as percent change in area at 3, 6, 12, 24 hours, and every 12 hours thereafter up to 86 hours. Staining with antibodies for D3R successfully showed expression of this receptor in WT fibroblasts, while cell culture experiments showed reduced proliferation and migration of D3KO fibroblasts compared to WT. Migration of D3KO fibroblasts from 5‐week old mice were significantly different (*p<0.05) in response to an artificial wound when compared to 23‐week‐old mice. Additionally, upon isolation of LV cardiac fibroblasts from 5‐week‐old and 31‐week‐old D3KO and 23‐week‐old WT mice, cell volume from D3KO fibroblast isolation was markedly reduced compared to WT isolation under identical conditions (5‐fold and 10‐fold decrease in 5‐week and 31‐week D3KO, respectively). This data suggests that D3Rs play a role in LV cardiac fibroblast migration and proliferation and support previous observations of age‐related changes in cardiac function of D3KO mice.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of ovarian hormones on emotional behaviors in dopamine D3 receptor knockout mice

Ovarian hormones reportedly have beneficial effects on affective behaviors. However, the functions of ovarian hormones in neurotransmitter signaling must be identified to understand their role in anxiety and depression. Several studies have provided evidence of the relationship between ovarian hormones and the dopaminergic system, but the interaction between ovarian hormones and dopamine D3 receptor (DRD3) is poorly understood. The aim of the present study was to examine the role of DRD3 in the anxiety-like and depression-like behavioral changes induced by estrogen and progesterone. We subjected D3 receptor knockout (D3KO) and wild-type (WT) mice to a series of behavioral tests. Mice were ovariectomized 4 weeks before testing, and we randomly administered 17β-estradiol (E2, 0.2 mg/kg), progesterone (P4, 10 mg/kg), E2 (0.2 mg/kg) plus P4 (10 mg/kg) or vehicle (VEH, corn oil, 0.2 ml) subcutaneously daily for 9 consecutive days, starting 4 days before the testing day. On the testing day, the mice were injected 30 min prior to behavioral testing. Compared with WT mice in the same treatment group, D3KO mice displayed hyperactivity in the light-dark box test (LDBT) but lower activity in the open field test (OFT). In addition, D3KO mice but not their WT littermates showed behavioral changes after E2 treatment compared with those after VEH treatment in the LDBT only. In depression tests, D3KO-VEH mice displayed significantly longer immobility times than did WT-VEH mice. In addition, only D3KO mice exhibited an obvious decrease in immobility time after E2 and P4 administration. These results indicate that the anxiolytic and antidepressant effects of ovarian hormones can be influenced by DRD3 expression and that DRD3 knockout may induce varying sensitivities to ovarian hormones that depend on various factors, including test paradigms and experiences in animal models. Our research provides a novel insight, i.e., DRD3 may play a role in the efficacy of hormone therapy.

Selective dopamine D3 receptor antagonist YQA14 inhibits morphine-induced behavioral sensitization in wild type, but not in dopamine D3 receptor knockout mice.

Increasing preclinical evidence demonstrates that dopamine D3 receptor (D3R) antagonists are a potential option for the treatment of drug addiction. The reinstatement of the addiction can be triggered by environmental stimuli that acquire motivational salience through repeated associations with the drug's effects. YQA14 is a novel D3R antagonist that has exhibited pharmacotherapeutic efficacy in reducing cocaine and amphetamine reward and relapse to drug seeking in mice. In this study we investigated the effects of YQA14 on morphine-induced context-specific locomotor sensitization in mice. We showed that repeated injection of YQA14 (6.25-25 mg/kg every day ip) prior to morphine (10 mg/kg every day sc) not only inhibited the acquisition, but also significantly attenuated the expression of morphine-induced locomotor sensitization. Furthermore, in the expression phase, one single injection of YQA14 (6.25-25 mg/kg, ip) dose-dependently inhibited the expression of morphine-induced behavioral sensitization. Moreover, YQA14 inhibited the expression of morphine-induced behavioral sensitization in wild mice (WT), but not in D3R knockout (D3R-/-) mice in the expression phase. In addition, D3R-/- mice also displayed the reduction in the expression phase compared with WT mice. In summary, this study demonstrates that blockade or knockout of the D3R inhibits morphine-induced behavior sensitization, suggesting that D3R plays an important role in the pathogenesis and etiology of morphine addiction, and it might be a potential target for clinical management of opioid addiction.

Genetic deletion of the dopamine D3 receptor increases vulnerability to heroin in mice

Despite extensive research, the neurobiological risk factors that convey vulnerability to opioid abuse are still unknown. Recent studies suggest that the dopamine D3 receptor (D3R) is involved in opioid self-administration, but it remains unclear whether altered D3R availability is a risk factor for the development of opioid abuse and addiction. Here we used dopamine D3 receptor-knockout (D3-KO) mice to investigate the role of this receptor in the different phases of opioid addiction. D3-KO mice learned to self-administer heroin faster and took more heroin than wild-type mice during acquisition and maintenance of self-administration. D3R-KO mice also displayed higher motivation to work to obtain heroin reward during self-administration under progressive-ratio reinforcement, as well as elevated heroin-seeking during extinction and reinstatement testing. In addition, deletion of the D3R induced higher baseline levels of extracellular dopamine (DA) in the nucleus accumbens (NAc), higher basal levels of locomotion, and reduced NAc DA and locomotor responses to lower doses of heroin. These findings suggest that the D3R is critically involved in regulatory processes that normally limit opioid intake via DA-related mechanisms. Deletion of D3R augments opioid-taking and opioid-seeking behaviors. Therefore, low D3R availability in the brain may represent a risk factor for the development of opioid abuse and addiction.

Arrestin recruitment to dopamine D2 receptor mediates locomotion but not incentive motivation.

SUMMARYThe dopamine (DA) D2 receptor (D2R) is an important target for the treatment of neuropsychiatric disorders such as schizophrenia and Parkinson’s disease. However, the development of improved therapeutic strategies has been hampered by our incomplete understanding of this receptor’s downstream signaling processes in vivo and how these relate to the desired and undesired effects of drugs. D2R is a G protein-coupled receptor (GPCR) that activates G proteindependent as well as non-canonical arrestin-dependent signaling pathways. Whether these effector pathways act alone or in concert to facilitate specific D2R-dependent behaviors is unclear. Here, we report on the development of a D2R mutant that recruits arrestin but is devoid of G protein activity. When expressed virally in “indirect pathway” medium spiny neurons (iMSNs) in the ventral striatum of D2R knockout mice, this mutant restored basal locomotor activity and cocaine-induced locomotor activity in a manner indistinguishable from wildtype D2R, indicating that arrestin recruitment can drive locomotion in the absence of D2R-mediated G protein signaling. In contrast, incentive motivation was enhanced only by wildtype D2R, signifying a dissociation in the mechanisms that underlie distinct D2R-dependent behaviors, and opening the door to more targeted therapeutics.

Ketamine effects on mammalian target of rapamycin signaling in the mouse limbic system depend on functional dopamine D3 receptors.

Ketamine is a noncompetitive glutamate N-methyl-D-aspartic acid receptor antagonist. When acutely administered to rodents, it produces a rapid antidepressant effect. There is evidence that N-methyl-D-aspartic acid receptor blockade enhances glutamatergic transmission preferentially engaging α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors leading to mTOR (mammalian target of rapamycin) pathways activation, thus resulting into downstream neuroadaptive changes in limbic structures. Recent in-vitro data on primary neuronal cultures showed that ketamine activates mTOR also in dopaminergic neurons, and this activation depends on the presence of functional dopamine D3 receptors. The aim of this work was to study the in-vivo relevance of viable D3 receptors in mediating the effects of acute ketamine administration on the mTOR downstream substrate p70 ribosomal S6 kinase (p70S6K), an obligatory substrate for mTOR. We compared the effects of single ketamine 5 mg/kg, 10 mg/kg, or vehicle injection in wild-type and D3 receptor knockout mice. Animals were killed after 60 min, and their brains were processed for p-p70S6K immunohistochemistry. Ketamine increased p70S6K phosphorylation in prefrontal cortex, nucleus accumbens core and shell, ventral tegmental area, substantia nigra, hippocampal CA1, CA2, and CA3, and basolateral amygdala of wild-type mice but not in mutant mice. Our study demonstrates that ketamine-induced p70S6K phosphorylation is dependent on viable D3R expressed in most of limbic structures.

Distinctive binding properties of the negative allosteric modulator, [3H]SB269,652, at recombinant dopamine D3 receptors

Recently, employing radioligand displacement and functional coupling studies, we demonstrated that SB269,652 (N-[(1r,4r)−4-[2-(7-cyano-1,2,3,4-tetrahydroisoquinolin-2-yl)ethyl]cyclohexyl]−1H-indole-2-carboxamide) interacts in an atypical manner with dopamine D3 receptor displaying a unique profile reminiscent of a negative allosteric ligand. Here, we characterized the binding of radiolabelled [3H]SB269,652 to human dopamine D3 receptor stably expressed in Chinese Hamster Ovary cells. Under saturating conditions, SB269,652 showed a KD value of ≈ 1nM. Consistent with high selectivity for human dopamine D3 receptor, [3H]SB269,652 binding was undetectable in cells expressing human dopamine D1, D2L or D4 receptors and absent in synaptosomes from dopamine D3 receptor knockout vs. wild-type mice. In contrast to saturation binding experiments, the dissociation kinetics of [3H]SB269,652 from human dopamine D3 receptors initiated with an excess of unlabelled ligand were best fitted by a bi-exponential binding model. Supporting the kinetic data, competition experiments with haloperidol, S33084 (a dopamine D3 receptor antagonist) or dopamine, were best described by a two-site model. In co-transfection experiments binding of SB269,652 to dopamine D3 receptor was able to influence the functional coupling of dopamine D2 receptor, supporting the notion that SB269,652 is a negative allosteric modulator across receptor dimers. However, because SB269,652 decreases the rate of [3H]nemonapride dissociation, the present data suggest that SB269,652 behaves as a bitopic antagonist at unoccupied dopamine D3 receptor, binding simultaneously to both orthosteric and allosteric sites, and as a pure negative allosteric modulator when receptors are occupied and it can solely bind to the allosteric site.

Cocaine-induced adaptation of dopamine D2S, but not D2L autoreceptors

The dopamine D2 receptor has two splice variants, D2S (Short) and D2L (Long). In dopamine neurons, both variants can act as autoreceptors to regulate neuronal excitability and dopamine release, but the roles of each variant are incompletely characterized. In a previous study we used viral receptor expression in D2 receptor knockout mice to show distinct effects of calcium signaling on D2S and D2L autoreceptor function (Gantz et al., 2015). However, the cocaine-induced plasticity of D2 receptor desensitization observed in wild type mice was not recapitulated with this method of receptor expression. Here we use mice with genetic knockouts of either the D2S or D2L variant to investigate cocaine-induced plasticity in D2 receptor signaling. Following a single in vivo cocaine exposure, the desensitization of D2 receptors from neurons expressing only the D2S variant was reduced. This did not occur in D2L-expressing neurons, indicating differential drug-induced plasticity between the variants.

Role of Dopamine D2 Receptor in Stress-Induced Myelin Loss

Dopaminergic systems play a major role in reward-related behavior and dysregulation of dopamine (DA) systems can cause several mental disorders, including depression. We previously reported that dopamine D2 receptor knockout (D2R−/−) mice display increased anxiety and depression-like behaviors upon chronic stress. Here, we observed that chronic stress caused myelin loss in wild-type (WT) mice, while the myelin level in D2R−/− mice, which was already lower than that in WT mice, was not affected upon stress. Fewer mature oligodendrocytes (OLs) were observed in the corpus callosum of stressed WT mice, while in D2R−/− mice, both the control and stressed group displayed a decrease in the number of mature OLs. We observed a decrease in the number of active β-catenin (ABC)-expressing and TCF4-expressing cells among OL lineage cells in the corpus callosum of stressed WT mice, while such regulation was not found in D2R−/− mice. Administration of lithium normalized the behavioral impairments and myelin damage induced by chronic stress in WT mice, and restored the number of ABC-positive and TCF4-positive OLs, while such effect was not found in D2R−/− mice. Together, our findings indicate that chronic stress induces myelin loss through the Wnt/β-catenin signaling pathway in association with DA signaling through D2R.