D5 Dopamine Receptor Knockout Mice and Hypertension

Abnormalities in dopamine production and receptor function have been described in human essential hypertension and rodent models of genetic hypertension. All of the five dopamine receptor genes (D₁, D₂, D₃, D₄, and D₅) expressed in mammals and some of their regulators are in loci linked to hypertension in humans and in rodents. Under normal conditions, D₁-like receptors (D₁ and D₅) inhibit sodium transport in the kidney and the intestine. However, in the Dahl salt-sensitive and spontaneously hypertensive rats, and humans with essential hypertension, the D₁-like receptor-mediated inhibition of sodium transport is impaired because of an uncoupling of the D₁-like receptor from its G protein/effector complex. The uncoupling is genetic, and receptor-, organ-, and nephron segment-specific. In human essential hypertension, the uncoupling of the D₁ receptor from its G protein/effector complex is caused by an agonist-independent serine phosphorylation/desensitization by constitutively active variants of the G protein-coupled receptor kinase type 4. The D₅ receptor is also important in blood pressure regulation. Disruption of the D₅ or the D₁ receptor gene in mice increases blood pressure. However, unlike the D₁ receptor, the hypertension in D₅ receptor null mice is caused by increased activity of the sympathetic nervous system, apparently due to activation of oxytocin, V₁ vasopressin, and non-N-methyl D-aspartate receptors in the central nervous system. The cause of the activation of these receptors remains to be determined.     

Parkin Expression Profile in Dopamine D3 Receptor Knock-Out Mice Brains

Patients affected by autosomic recessive juvenile parkinsonism (ARJP) exhibit parkin gene mutations with brain decrease in dopamine D2/D3 binding sites. To date, there are no data indicating whether the reduction in dopamine D3 receptors (DRD3) may be associated with the expression of specific parkin variants. In the present study we investigated parkin expression profile in DRD3 knock-out mice brains. RT-PCR analysis was performed to assess qualitative changes in parkin isoforms’ distribution pattern and in exons’ expression both in wild type controls and dopamine D3 receptor’s knock-out mice. Real-time PCR was performed to quantify single exons mRNA. Results demonstrated that exons 1, 2, 4, 6, 7, 8, were more expressed in wild type compared to dopamine D3 receptor KO mice brains while some other (3, 9, 10) were lower expressed. The expression levels of exons 5, 11 and 12 did not change in both animal groups. Our analysis was confirmed by western blot, which showed that parkin protein levels were influenced by the absence of DRD3.     

Histamine Receptor Expression, Hippocampal Plasticity and Ammonia in Histidine Decarboxylase Knockout Mice

Genetic ablation of the histamine producing enzyme histidine decarboxylase (HDC) leads to alteration in exploratory behaviour and hippocampus-dependent learning. We investigated how brain histamine deficiency in HDC knockout mice (HDC KO) affects hippocampal excitability, synaptic plasticity, and the expression of histamine receptors. No significant alterations in: basal synaptic transmission, long-term potentiation (LTP) in the Schaffer collateral synapses, histamine-induced transient changes in the CA1 pyramidal cell excitability, and the expression of H1 and H2 receptor mRNAs were found in hippocampal slices from HDC KO mice. However, when compared to WT mice, HDC KO mice demonstrated: 1. a stronger enhancement of LTP by histamine, 2. a stronger impairment of LTP by ammonia, 3. no long-lasting potentiation of population spikes by histamine, 4. a decreased expression of H3 receptor mRNA, and 5. less potentiation of population spikes by H3 receptor agonism. Parallel measurements in the hypothalamic tuberomamillary nucleus, the origin of neuronal histamine, demonstrated an increased expression of H3 receptors in HDC KO mice without any changes in the spontaneous firing of “histaminergic” neurons without histamine and their responses to the H3 receptor agonist (R)-α-methylhistamine. We conclude that the absence of neuronal histamine results in subtle changes in hippocampal synaptic transmission and plasticity associated with alteration in the expression of H3 receptors.     

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.     

多巴胺D5受体转基因小鼠的建立

目的建立人多巴胺D5受体突变基因F173 L（D5F173L）,S390G（D5S390G）及正常人D5基因（hD5 WT）的转基因小鼠,利用该转基因动物模型来研究D5受体在原发性高血压中的发病机制。方法利用显微注射的技术将插入CMV启动子下游的D5F173L,D5S390G及hD5 WT基因的转基因载体注射入C57BL/6小鼠体内,建立多巴胺D5F173L,D5S390G及hD5 WT的转基因小鼠。通过PCR鉴定转基因小鼠的基因型。利用Western Blotting方法鉴定该受体蛋白在肾脏的表达情况。使用智能无创血压测量仪测量转基因小鼠的血压值。结果分别建立了多巴胺D5F173L,D5S390G及hD5 WT转基因C57BL/6小鼠,Western Blotting方法鉴定结果显示,与非转基因C57BL/6小鼠比较,D5转基因小鼠D5受体在肾脏有较高的表达。3-6月龄D5 F173L转基因小鼠的收缩压、舒张压和平均动脉血压均明显高于多巴胺D5S390G及hD5 WT转基因小鼠（n=6-8,P〈0.05）。结论多巴胺D5受体在原发性高血压发病中具有重要作用,但作用机理还有待于进一步研究。     

Pregnenolone Rescues Schizophrenia-Like Behavior in Dopamine Transporter Knockout Mice

Pregnenolone belongs to a class of endogenous neurosteroids in the central nervous system (CNS), which has been suggested to enhance cognitive functions through GABA_A receptor signaling by its metabolites. It has been shown that the level of pregnenolone is altered in certain brain areas of schizophrenic patients, and clozapine enhances pregnenolone in the CNS in rats, suggesting that pregnenolone could be used to treat certain symptoms of schizophrenia. In addition, early phase proof-of-concept clinical trials have indicated that pregnenolone is effective in reducing the negative symptoms and cognitive deficits of schizophrenia patients. Here, we evaluate the actions of pregnenolone on a mouse model for schizophrenia, the dopamine transporter knockout mouse (DAT KO). DAT KO mice mirror certain symptoms evident in patients with schizophrenia, such as the psychomotor agitation, stereotypy, deficits of prepulse inhibition and cognitive impairments. Following acute treatment, pregnenolone was found to reduce the hyperlocomotion, stereotypic bouts and pre-pulse inhibition (PPI) deficits in DAT KO mice in a dose-dependent manner. At 60 mg/kg of pregnenolone, there were no significant differences in locomotor activities and stereotypy between wild-type and DAT KO mice. Similarly, acute treatment of 60 mg/kg of pregnenolone fully rescued PPI deficits of DAT KO mice. Following chronic treatment with pregnenolone at 60 mg/kg, the cognitive deficits of DAT KO mice were rescued in the paradigms of novel object recognition test and social transmission of food preference test. Pregnenolone thus holds promise as a therapeutic candidate in schizophrenia.     

G Protein-coupled Receptor Kinase 4 (GRK4) Regulates the Phosphorylation and Function of the Dopamine D3 Receptor

During conditions of moderate sodium excess, the dopaminergic system regulates blood pressure and water and electrolyte balance by engendering natriuresis. Dopamine exerts its effects on dopamine receptors, including the dopamine D₃ receptor. G protein-coupled receptor kinase 4 (GRK4), whose gene locus (4p16.3) is linked to essential hypertension, desensitizes the D₁ receptor, another dopamine receptor. This study evaluated the role of GRK4 on D₃ receptor function in human proximal tubule cells. D₃ receptor co-segregated in lipid rafts and co-immunoprecipitated and co-localized in human proximal tubule cells and in proximal and distal tubules and glomeruli of kidneys of Wistar Kyoto rats. Bimolecular fluorescence complementation and confocal microscopy revealed that agonist activation of the receptor initiated the interaction between D₃ receptor and GRK4 at the cell membrane and promoted it intracellularly, presumably en route to endosomal trafficking. Of the four GRK4 splice variants, GRK4-γ and GRK4-α mediated a 3- and 2-fold increase in the phosphorylation of agonist-activated D₃ receptor, respectively. Inhibition of GRK activity with heparin or knockdown of GRK4 expression via RNA interference completely abolished p44/42 phosphorylation and mitogenesis induced by D₃ receptor stimulation. These data demonstrate that GRK4, specifically the GRK4-γ and GRK4-α isoforms, phosphorylates the D₃ receptor and is crucial for its signaling in human proximal tubule cells.During conditions of moderate sodium excess, the dopaminergic system sits at the fulcrum of homeostatic control of water and electrolyte balance and blood pressure (1, 2). Dopamine promotes natriuresis by inhibiting sodium chloride reabsorption in specific segments of the nephron. Dopamine exerts its action on dopamine receptors, which belong to the family of G protein-coupled receptors (GPCRs).² The dopamine receptors are classified into two subtypes based on their ability to increase cAMP levels, sequence similarity, G protein coupling, and pharmacological profiles (3, 4). The D₁-like dopamine receptors activate adenylyl cyclase by coupling to stimulatory Gα_s/Gα_olf and include the D₁ (D₁R) and D₅ receptors (D₅R). The D₂-like dopamine receptors inhibit adenylyl cyclase by coupling to Gα_i/Gα_o and consist of the D₂ (D₂R), D₃ (D₃R), and D₄ (D₄R) receptors. The D₃R has also been shown to couple to Gα_o, Gβγ, and to the stimulatory Gα_s (5, 6).The signal transduction that follows ligand occupation of a GPCR is tightly regulated to limit the specificity and extent of cellular response. GPCR-mediated signal transduction is rapidly dampened via receptor desensitization or the waning of the responsiveness of the receptor to agonist with time. Desensitization involves receptor phosphorylation and is carried out by either GPCR kinases (GRKs) or second messenger-activated kinases such as protein kinase A and protein kinase C. Homologous desensitization involves GRKs that selectively phosphorylate only agonist-activated receptors, whereas heterologous desensitization is carried out by second messenger-dependent kinases that indiscriminately phosphorylate agonist-activated receptors and those that have not been exposed to the agonist (7).The GRKs are serine/threonine protein kinases comprising seven isoforms that are grouped into three subfamilies. GRK1 and GRK7 belong to the rhodopsin kinase subfamily and are expressed exclusively in the retina (8–10). GRK2 and GRK3 phosphorylate the β-adrenergic receptor and belong to the β-adrenergic receptor kinase subfamily (11), and GRK4, GRK5, and GRK6 belong to the GRK4 subfamily. GRK4 is highly enriched in the testis and, to a lesser degree, in the kidneys (12, 13). Four splice variants of human GRK4 result from the alternative splicing of exons 2 and 15 (11). GRK4-α is considered the full-length version, whereas GRK4-β, -γ, and -δ are shortened versions of GRK4-α (14). The coding region of the GRK4 gene, whose 4p16.3 locus has been linked to essential hypertension (15, 16), contains several single nucleotide polymorphisms, including R65L, A142V, and A486V, which have been linked to essential hypertension and/or salt sensitivity in various ethnic groups (17).The D₃R gene is found at 3q13.3 (18), a locus that is also linked to essential hypertension (19, 20). Sequence analysis of the D₃R gene shows the presence of several single nucleotide polymorphisms, which do not correlate with either essential hypertension among Japanese (21) or with blood pressure levels and diabetic nephropathy among Finns (22). However, D₃R knock-out mice develop a renin-dependent form of hypertension and fail to excrete a sodium load (23).The D₃R has a long third intracellular loop that contains several putative GRK phosphorylation sites (24). A previous study evaluated the ability of GRK2 and GRK3 to phosphorylate D₃R and showed that co-transfection of GRK3, but not GRK2, resulted in a weak phosphorylation of the heterologously expressed, dopamine-stimulated D₃R in HEK-293 (25), a human embryonic kidney cell line. We tested the hypothesis that GRK4 is required in D₃R signaling in terminally differentiated human renal proximal tubule cells (hPTCs) by determining the spatiotemporal dynamics of the interaction of D₃R and GRK4 through their subfractionation in membrane microdomains and subcellular co-localization via confocal microscopy and bimolecular fluorescence complementation assay (BiFC). We also identified which of the GRK4 splice variants are involved in D₃R phosphorylation and evaluated the physiological roles of GRK4 in D₃R signaling in the hPTCs. We now report that D₃R and GRK4 co-fractionate in lipid rafts and co-localize in both hPTCs and WYK kidneys. Moreover, D₃R is phosphorylated by GRK4-γ and GRK4-α isoforms, and the absence of GRK4 impairs D₃R-mediated mitogenesis and activation of p44/42 in hPTCs.     

G Protein-coupled Receptor Kinase-2 Constitutively Regulates D2 Dopamine Receptor Expression and Signaling Independently of Receptor Phosphorylation

We investigated the regulatory effects of GRK2 on D₂ dopamine receptor signaling and found that this kinase inhibits both receptor expression and functional signaling in a phosphorylation-independent manner, apparently through different mechanisms. Overexpression of GRK2 was found to suppress receptor expression at the cell surface and enhance agonist-induced internalization, whereas short interfering RNA knockdown of endogenous GRK2 led to an increase in cell surface receptor expression and decreased agonist-mediated endocytosis. These effects were not due to GRK2-mediated phosphorylation of the D₂ receptor as a phosphorylation-null receptor mutant was regulated similarly, and overexpression of a catalytically inactive mutant of GRK2 produced the same effects. The suppression of receptor expression is correlated with constitutive association of GRK2 with the receptor complex as we found that GRK2 and several of its mutants were able to co-immunoprecipitate with the D₂ receptor. Agonist pretreatment did not enhance the ability of GRK2 to co-immunoprecipitate with the receptor. We also found that overexpression of GRK2 attenuated the functional coupling of the D₂ receptor and that this activity required the kinase activity of GRK2 but did not involve receptor phosphorylation, thus suggesting the involvement of an additional GRK2 substrate. Interestingly, we found that the suppression of functional signaling also required the Gβγ binding activity of GRK2 but did not involve the GRK2 N-terminal RH domain. Our results suggest a novel mechanism by which GRK2 negatively regulates G protein-coupled receptor signaling in a manner that is independent of receptor phosphorylation.     

Signaling Mechanisms of the D3 Dopamine Receptor

A substantial body of evidence shows the capacity of the dopamine D₃ receptor to couple functionally to G proteins when expressed in an appropriate milieu in heterologous expression systems. In these systems, activation of D₃ receptors inhibits adenylate cyclase, modulates ion flow through potassium and calcium channels, and activates kinases, most notably mitogen-activated protein kinase. Coupling to G_i/G_o is implicated in many of these effects, but other G proteins may contribute. Studies with chimeric receptors implicate the third intracellular loop in the mediation of agonist-induced signal transduction. Finally, D₃-preferring drugs modulate expression of c-fos in neuronal cultures and brain. Signaling mechanisms of the D₃ receptor in brain, however, remain to be definitively determined.     

Analysis of Human Dopamine D3 Receptor Quaternary Structure

The dopamine D₃ receptor is a class A, rhodopsin-like G protein-coupled receptor that can form dimers and/or higher order oligomers. However, the molecular basis for production of these complexes is not well defined. Using combinations of molecular modeling, site-directed mutagenesis, and homogenous time-resolved FRET, the interfaces that allow dopamine D₃ receptor monomers to interact were defined and used to describe likely quaternary arrangements of the receptor. These were then compared with published crystal structures of dimeric β₁-adrenoreceptor, μ-opioid, and CXCR4 receptors. The data indicate important contributions of residues from within each of transmembrane domains I, II, IV, V, VI, and VII as well as the intracellular helix VIII in the formation of D₃-D₃ receptor interfaces within homo-oligomers and are consistent with the D₃ receptor adopting a β₁-adrenoreceptor-like quaternary arrangement. Specifically, results suggest that D₃ protomers can interact with each other via at least two distinct interfaces: the first one comprising residues from transmembrane domains I and II along with those from helix VIII and a second one involving transmembrane domains IV and V. Moreover, rather than existing only as distinct dimeric species, the results are consistent with the D₃ receptor also assuming a quaternary structure in which two transmembrane domain I-II-helix VIII dimers interact to form a ”rhombic” tetramer via an interface involving residues from transmembrane domains VI and VII. In addition, the results also provide insights into the potential contribution of molecules of cholesterol to the overall organization and potential stability of the D₃ receptor and possibly other GPCR quaternary structures.     

Exposure to Far Infrared Ray Protects Methamphetamine-Induced Behavioral Sensitization in Glutathione Peroxidase-1 Knockout Mice via Attenuating Mitochondrial Burdens and Dopamine D1 Receptor Activation

Evidence indicates that stress conditions might lead to drug dependence. Recently, we have demonstrated that exposure to far infrared ray (FIR) attenuates acute restraint stress via induction of glutathione peroxidase-1 (GPx-1) gene. We investigated whether FIR affects methamphetamine (MA)-induced behavioral sensitization and whether FIR-mediated pharmacological activity requires interaction between dopamine receptor and GPx-1 gene. We observed that MA treatment significantly increased GPx-1 expression in the striatum of wild-type (WT) mice. Interestingly, exposure to FIR potentiated MA-induced increase in GPx-1 expression. This phenomenon was also observed in animals receiving MA with dopamine D1 receptor antagonist SCH23390. However, dopamine D2 receptor antagonist sulpiride did not affect MA-induced GPx-1 expression. FIR exposure or SCH23390, but not sulpiride, significantly attenuated MA-induced behavioral sensitization. Exposure to FIR significantly attenuated MA-induced dopamine D1 receptor expression, c-Fos induction and oxidative burdens. FIR-mediated antioxidant effects were also more pronounced in mitochondrial- than cytosolic-fraction. In addition, FIR significantly attenuated against MA-induced changes in mitochondrial superoxide dismutase and mitochondrial GPx activities, mitochondrial transmembrane potential, intramitochondrial Ca²⁺ level, mitochondrial complex-I activity, and mitochondrial oxidative burdens. The attenuation by FIR was paralleled that by SCH23390. Effects of FIR or SCH23390 were more sensitive to GPx-1 KO than WT mice, while SCH23390 treatment did not exhibit any additive effects on the protective activity mediated by FIR, indicating that dopamine D1 receptor constitutes a molecular target of FIR. Our result suggests that exposure to FIR ameliorates MA-induced behavioral sensitization via possible interaction between dopamine D1 receptor and GPx-1 gene.     

Dopamine D3 Receptor Mediates Preadolescent Stress-Induced Adult Psychiatric Disorders

Several studies have shown that repeated stressful experiences during childhood increases the likelihood of developing depression- and anxiety-related disorders in adulthood; however, the underlying mechanisms are not well understood. We subjected drd3-EGFP and drd3-null mice to daily, two hour restraint stress episodes over a five day period during preadolescence (postnatal day 35 to 39), followed by social isolation. When these mice reached adulthood (post-natal day > 90), we assessed locomotor behavior in a novel environment, and assessed depression-related behavior in the Porsolt Forced Swim test. We also measured the expression and function of dopamine D3 receptor in limbic brain areas such as hippocampus, nucleus accumbens and amygdala in control and stressed drd3-EGFP mice in adulthood. Adult male mice subjected to restraint stress during preadolescence exhibited both anxiety- and depression-related behaviors; however, adult female mice subjected to preadolescent restraint stress exhibited only depression-related behaviors. The development of preadolescent stress-derived psychiatric disorders was blocked by D3 receptor selective antagonist, SB 277011-A, and absent in D3 receptor null mice. Adult male mice that experienced stress during preadolescence exhibited a loss of D3 receptor expression and function in the amygdala but not in hippocampus or nucleus accumbens. In contrast, adult female mice that experienced preadolescent stress exhibited increased D3 receptor expression in the nucleus accumbens but not in amygdala or hippocampus. Our results suggest that the dopamine D3 receptor is centrally involved in the etiology of adult anxiety- and depression-related behaviors that arise from repeated stressful experiences during childhood.     

B-50/GAP-43 Phosphorylation and PKC Activity are Increased in Rat Hippocampal Synaptosomal Membranes After an Inhibitory Avoidance Training

Several lines of evidence indicate that protein kinase C (PKC) is involved in long-term potentiation (LTP) and in certain forms of learning. Recently, we found a learning-specific, time-dependent increase in [³H]phorbol dibutyrate binding to membrane-associated PKC in the hippocampus of rats subjected to an inhibitory avoidance task. Here we confirm and extend this observation, describing that a one trial inhibitory avoidance learning was associated with rapid and specific increases in B-50/GAP-43 phosphorylation in vitro and in PKC activity in hippocampal synaptosomal membranes. The increased phosphorylation of B-50/GAP-43 was seen at 30 min (+35% relative to naive or shocked control groups), but not at 10 or 60 min after training. This learning-associated increase in the phosphorylation of B-50/GAP-43 is mainly due to an increase in the activity of PKC. This is based on three different sets of data: 1) PKC activity increased by 24% in hippocampal synaptosomal membranes of rats sacrificed 30 min after training; 2) B-50/GAP-43 immunoblots revealed no changes in the amount of this protein among the different experimental groups; 3) phosphorylation assays, performed in the presence of bovine purified PKC or in the presence of the selective PKC inhibitor CGP 41231, exhibited no differences in B-50/GAP-43 phosphorylation between naive and trained animals. In conclusion, these results support the contention that hippocampal PKC participates in the early neural events of memory formation of an aversively-motivated learning task.     

High-Fat Diet Exposure Increases Dopamine D2 Receptor and Decreases Dopamine Transporter Receptor Binding Density in the Nucleus Accumbens and Caudate Putamen of Mice

This experiment examined dopamine D2 receptor and its transporter (DAT) density in mice fed a high-fat or low-fat diet for twenty days as well as fed twenty days of high-fat diet then changed to low-fat diet for one and seven days. Quantitative autoradiography revealed that twenty days of high-fat diet consumption significantly increased D2 receptor and decreased DAT density in the dorsal and ventral parts of the caudal caudate putamen (D2: 32% and 35% respectively, DAT: 33.3% and 28.8% respectively) compared with low-fat diet. High-fat feeding also increased D2 binding in the nucleus accumbens shell (36%). D2 receptor and DAT density remained unchanged following reversal of the diets from high-fat to low-fat diet. The high-fat diet induced increase of D2 receptor and decrease of DAT binding may have occurred due to defensive control over dopaminergic activity in response to a positive energy balance.     

Dopamine D-1 Receptor and Cyclic AMP-Dependent Phosphorylation in Parkinson''s Disease

D-1 and D-2 receptor densities, evaluated respectively by [3H]SCH 23390 and [3H]spiperone binding, and DARPP-32 (dopamine and adenosine 3':5'-monophosphate-regulated phosphoprotein-32K) concentrations, were studied in the brains of control and parkinsonian subjects postmortem. D-2 receptor density was unchanged in the putamen of parkinsonian patients. D-1 receptor density was unchanged in the putamen and substantia nigra pars reticulata (SNR) of parkinsonian patients, but decreased by 28% in the substantia nigra pars compacta (SNC). DARPP-32, which is localized in the same structures as D-1 receptors of which it is thought to represent the intracellular messenger, decreased by 45% in the putamen, 66% in the SNR, and 79% in the SNC. The decrease in D-1 receptors in the SNC may be due to degeneration of pallidonigral GABAergic neurons, but some of the D-1 receptors may be on the nigrostriatal dopaminergic neurons themselves. The dissociation between the alteration of D-1 receptor densities and DARPP-32 concentrations in both the striatum and substantia nigra, which are of the same order in the two structures, may be an index of functional hypoactivity of D-1 neurotransmission.     

Leukocyte ABCA1 Remains Atheroprotective in Splenectomized LDL Receptor Knockout Mice

Aim

ATP-binding cassette transporter A1 (ABCA1) is an important mediator of macrophage cholesterol efflux. It mediates the efflux of cellular cholesterol to lipid-poor apolipoprotein A-I. LDL receptor (LDLr) knockout (KO) mice deficient for leukocyte ABCA1 (ABCA1 KO→LDLr KO) show increased atherosclerosis and splenic lipid accumulation despite largely attenuated serum cholesterol levels. In the present study, we aimed to explore the importance of the spleen for the atheroprotective effects of leukocyte ABCA1.

Methods

LDLr KO mice were transplanted with bone marrow from ABCA1 KO mice or wild-type (WT) controls. After 8 weeks recovery, mice were either splenectomized (SP-x) or underwent a sham operation, and were subsequently challenged with a Western-type diet (WTD).

Results

In agreement with previous studies, the atherosclerotic lesion area in ABCA1 KO→LDLr KO sham animals (655±82×10³ µm²) was 1.4-fold (p = 0.03) larger compared to sham WT→LDLr KO mice (459±33×10³ µm²) after 8 weeks WTD feeding, despite 1.7-fold (p<0.001) lower serum cholesterol levels. Interestingly, deletion of ABCA1 in leukocytes led to 1.6-fold higher neutrophil content in the spleen in absence of differences in circulating neutrophils. Levels of KC, an important chemoattractant for neutrophils, in serum, however, were increased 2.9-fold (p = 0.07) in ABCA1 KO→LDLr KO mice. SP-x induced blood neutrophilia as compared to WT→LDLr KO mice (1.9-fold; p<0.05), but did not evoke differences in serum cholesterol and anti-oxLDL antibody levels. Atherosclerotic lesion development, however, was 1.3-fold induced both in the presence and absence of leukocyte ABCA1 (WT: 614±106×10³ µm², ABCA1 KO: 786±44×10³ µm²). Two-way ANOVA revealed independent effects on atherosclerosis for both leukocyte ABCA1 deficiency and SP-x (p<0.05).

Conclusions

The observed splenic alterations induced by leukocyte ABCA1 deficiency do not play a significant role in the anti-atherogenic effects of leukocyte ABCA1 on lesion development.     

Evaluation of D1 and D2 Dopamine Receptor Segregation in the Developing Striatum Using BAC Transgenic Mice

The striatum is predominantly composed of medium spiny neurons (MSNs) that send their axons along two parallel pathways known as the direct and indirect pathways. MSNs from the direct pathway express high levels of D1 dopamine receptors, while MSNs from the indirect pathway express high levels of D2 dopamine receptors. There has been much debate over the extent of colocalization of these two major dopamine receptors in MSNs of adult animals. In addition, the ontogeny of the segregation process has never been investigated. In this paper, we crossed bacterial artificial chromosome drd1a-tdTomato and drd2-GFP reporter transgenic mice to characterize these models and estimate D1-D2 co-expression in the developing striatum as well as in striatal primary cultures. We show that segregation is already extensive at E18 and that the degree of co-expression further decreases at P0 and P14. Finally, we also demonstrate that cultured MSNs maintain their very high degree of D1-D2 reporter protein segregation, thus validating them as a relevant in vitro model.