RESUMEN
The dorsal raphe nucleus (DRN) receives dopaminergic inputs from the ventral tegmental area (VTA). Also, the DRN contains a small population of cells that express dopamine (DRNDA neurons). However, the physiological role of dopamine (DA) in the DRN and its interaction with serotonergic (5-HT) neurons is poorly understood. Several works have reported moderate levels of D1, D2, and D3 DA receptors in the DRN. Furthermore, it was found that the activation of D2 receptors increased the firing of putative 5-HT neurons. Other studies have reported that D1 and D2 dopamine receptors can interact with glutamate NMDA receptors, modulating the excitability of different cell types. In the present work, we used immunocytochemical techniques to determine the kind of DA receptors in the DRN. Additionally, we performed electrophysiological experiments in brainstem slices to study the effect of DA agonists on NMDA-elicited currents recorded from identified 5-HT DRN neurons. We found that D2 and D3 but not D1 receptors are present in this nucleus. Also, we demonstrated that the activation of D2-like receptors increases NMDA-elicited currents in 5-HT neurons through a mechanism involving phospholipase C (PLC) and protein kinase C (PKC) enzymes. Possible physiological implications related to the sleep-wake cycle are discussed.
Asunto(s)
Núcleo Dorsal del Rafe , Receptores de Dopamina D2 , Receptores de N-Metil-D-Aspartato , Neuronas Serotoninérgicas , Animales , Núcleo Dorsal del Rafe/metabolismo , Núcleo Dorsal del Rafe/efectos de los fármacos , Receptores de Dopamina D2/metabolismo , Neuronas Serotoninérgicas/metabolismo , Neuronas Serotoninérgicas/efectos de los fármacos , Neuronas Serotoninérgicas/fisiología , Masculino , Receptores de N-Metil-D-Aspartato/metabolismo , Receptores de Dopamina D3/metabolismo , N-Metilaspartato/farmacología , N-Metilaspartato/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D1/agonistas , Agonistas de Dopamina/farmacología , Ratas , Fosfolipasas de Tipo C/metabolismo , Ratas WistarRESUMEN
Dopamine D2 receptor (D2R) is expressed in striatopallidal neurons and decreases forskolin-stimulated cyclic adenine monophosphate (cAMP) accumulation and gamma-aminobutyric acid (GABA) release. Dopamine D3 receptor (D3R) mRNA is expressed in a population of striatal D2R-expressing neurons. Also, D3R protein and binding have been reported in the neuropil of globus pallidus. We explore whether D2R and D3R colocalize in striatopallidal terminals and whether D3R modulates the D2R effect on forskolin-stimulated [3H]cAMP accumulation in pallidal synaptosomes and high K+ stimulated-[3H]GABA release in pallidal slices. Previous reports in heterologous systems indicate that calmodulin (CaM) and CaMKII modulate D2R and D3R functions; thus, we study whether this system regulates its functional interaction. D2R immunoprecipitates with CaM, and pretreatment with ophiobolin A or depolarization of synaptosomes with 15 mM of K+ decreases it. Both treatments increase the D2R inhibition of forskolin-stimulated [3H]cAMP accumulation when activated with quinpirole, indicating a negative modulation of CaM on D2R function. Quinpirole also activates D3R, potentiating D2R inhibition of cAMP accumulation in the ophiobolin A-treated synaptosomes. D2R and D3R immunoprecipitate in pallidal synaptosomes and decrease after the kainic acid striatal lesion, indicating the striatal origin of the presynaptic receptors. CaM-kinase II alfa (CaMKIIα) immunoprecipitates with D3R and increases after high K+ depolarization. In the presence of KN62, a CaMKIIα blocker, D3R potentiates D2R effects on cAMP accumulation in depolarized synaptosomes and GABA release in pallidal slices, indicating D3R function regulation by CaMKIIα. Our data indicate that D3R potentiates the D2R effect on cAMP accumulation and GABA release at pallidal terminals, an interaction regulated by the CaM-CaMKIIα system.
Asunto(s)
Calmodulina , Receptores de Dopamina D3 , Sesterterpenos , Receptores de Dopamina D3/metabolismo , Quinpirol/farmacología , Calmodulina/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Colforsina , Receptores de Dopamina D2/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Most neurodegenerative diseases are multifactorial, and the discovery of several molecular mechanisms related to their pathogenesis is constantly advancing. Dopamine and dopaminergic receptor subtypes are involved in the pathophysiology of several neurological disorders, such as schizophrenia, depression and drug addiction. For this reason, the dopaminergic system and dopamine receptor ligands play a key role in the treatment of such disorders. In this context, a novel series of conformationally restricted N-arylpiperazine derivatives (5a-f) with a good affinity for D2/D3 dopamine receptors is reported herein. Compounds were designed as interphenylene analogs of the drugs aripiprazole (2) and cariprazine (3), presenting a 1,3-benzodioxolyl subunit as a ligand of the secondary binding site of these receptors. The six new N-arylpiperazine compounds were synthesized in good yields by using classical methodologies, and binding and guanosine triphosphate (GTP)-shift studies were performed. Affinity values below 1 µM for both target receptors and distinct profiles of intrinsic efficacy were found. Docking studies revealed that Compounds 5a-f present a different binding mode with dopamine D2 and D3 receptors, mainly as a consequence of the conformational restriction imposed on the flexible spacer groups of 2 and 3.
Asunto(s)
Enfermedades Neurodegenerativas , Receptores de Dopamina D3 , Dopamina/metabolismo , Humanos , Ligandos , Enfermedades Neurodegenerativas/tratamiento farmacológico , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/metabolismoRESUMEN
Prenatal exposure to amphetamine induces changes in dopamine receptors in mesolimbic areas and alters locomotor response to amphetamine during adulthood. Sex differences have been reported in amphetamine-induced brain activity and stress sensitivity. We evaluated the effects of prenatal amphetamine exposure on locomotor activity, dopamine receptors and tyrosine hydroxylase mRNA expression in nucleus accumbens and caudate-putamen in response to amphetamine challenge in adult female and male rats. The role of estrogen in the response to restraint stress was analyzed in ovariectomized, prenatally amphetamine-exposed rats. Pregnant rats were treated with D-amphetamine during days 15-21 of gestation. Nucleus accumbens and caudate-putamen were processed for mRNA determination by real-time PCR. In nucleus accumbens, higher mRNA dopamine (D3) receptor expression was found in basal and D-amphetamine-challenge conditions in female than male, and prenatal amphetamine increased the difference. No sex differences were observed in caudate-putamen. Basal saline-treated females showed higher locomotor activity than males. Amphetamine challenge in prenatally amphetamine-exposed rats increased locomotor activity in males and reduced it in females. In nucleus accumbens, estrogen diminished mRNA D1, D2 and D3 receptor expression in basal, and D1 and D3 in ovariectomized stressed rats. Estrogen prevented the increase in tyrosine hydroxylase expression induced by stress in ovariectomized prenatally exposed rats. In conclusion, estrogen modulates mRNA levels of D1, D2 and D3 receptors and tyrosine hydroxylase expression in nucleus accumbens; prenatal amphetamine-exposure effects on D3 receptors and behavioral responses were gender dependent.
Asunto(s)
Anfetamina , Dopamina , Anfetamina/farmacología , Animales , Dopamina/metabolismo , Estrógenos/farmacología , Femenino , Masculino , Núcleo Accumbens/metabolismo , Embarazo , Ratas , Receptores Dopaminérgicos , Receptores de Dopamina D3/metabolismoRESUMEN
BACKGROUND: Recent evidence has shown dopamine as a major regulator of inflammation. Accordingly, dopaminergic regulation of immune cells plays an important role in the physiopathology of inflammatory disorders. Multiple sclerosis (MS) is an inflammatory disease involving a CD4+ T-cell-driven autoimmune response to central nervous system (CNS) derived antigens. Evidence from animal models has suggested that B cells play a fundamental role as antigen-presenting cells (APC) re-stimulating CD4+ T cells in the CNS as well as regulating T-cell response by mean of inflammatory or anti-inflammatory cytokines. Here, we addressed the role of the dopamine receptor D3 (DRD3), which displays the highest affinity for dopamine, in B cells in animal models of MS. METHODS: Mice harbouring Drd3-deficient or Drd3-sufficient B cells were generated by bone marrow transplantation into recipient mice devoid of B cells. In these mice, we compared the development of experimental autoimmune encephalomyelitis (EAE) induced by immunization with a myelin oligodendrocyte glycoprotein (MOG)-derived peptide (pMOG), a model that leads to CNS-autoimmunity irrespective of the APC-function of B cells, or by immunization with full-length human MOG protein (huMOG), a model in which antigen-specific activated B cells display a fundamental APC-function in the CNS. APC-function was assessed in vitro by pulsing B cells with huMOG-coated beads and then co-culturing with MOG-specific T cells. RESULTS: Our data show that the selective Drd3 deficiency in B cells abolishes the disease development in the huMOG-induced EAE model. Mechanistic analysis indicates that although DRD3-signalling did not affect the APC-function of B cells, DRD3 favours the CNS-tropism in a subset of pro-inflammatory B cells in the huMOG-induced EAE model, an effect that was associated with higher CXCR3 expression. Conversely, the results show that the selective Drd3 deficiency in B cells exacerbates the disease severity in the pMOG-induced EAE model. Further analysis shows that DRD3-stimulation increased the expression of the CNS-homing molecule CD49d in a B-cell subset with anti-inflammatory features, thus attenuating EAE manifestation in the pMOG-induced EAE model. CONCLUSIONS: Our findings demonstrate that DRD3 in B cells exerts a dual role in CNS-autoimmunity, favouring CNS-tropism of pro-inflammatory B cells with APC-function and promoting CNS-homing of B cells with anti-inflammatory features. Thus, these results show DRD3-signalling in B cells as a critical regulator of CNS-autoimmunity.
Asunto(s)
Autoinmunidad/fisiología , Linfocitos B/metabolismo , Dopamina/metabolismo , Encefalomielitis Autoinmune Experimental/metabolismo , Receptores de Dopamina D3/metabolismo , Secuencia de Aminoácidos , Animales , Linfocitos B/inmunología , Células Cultivadas , Sistema Nervioso Central/inmunología , Sistema Nervioso Central/metabolismo , Dopamina/inmunología , Encefalomielitis Autoinmune Experimental/genética , Encefalomielitis Autoinmune Experimental/inmunología , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptores de Dopamina D3/genética , Receptores de Dopamina D3/inmunologíaRESUMEN
Evidence from inflammatory bowel diseases (IBD) patients and animal models has indicated that gut inflammation is driven by effector CD4+ T-cell, including Th1 and Th17. Conversely, Treg seem to be dysfunctional in IBD. Importantly, dopamine, which is abundant in the gut mucosa under homoeostasis, undergoes a sharp reduction upon intestinal inflammation. Here we analysed the role of the high-affinity dopamine receptor D3 (DRD3) in gut inflammation. Our results show that Drd3 deficiency confers a stronger immunosuppressive potency to Treg, attenuating inflammatory colitis manifestation in mice. Mechanistic analyses indicated that DRD3-signalling attenuates IL-10 production and limits the acquisition of gut-tropism. Accordingly, the ex vivo transduction of wild-type Treg with a siRNA for Drd3 induced a potent therapeutic effect abolishing gut inflammation. Thus, our findings show DRD3-signalling as a major regulator of Treg upon gut inflammation.
Asunto(s)
Colitis/inmunología , Neuronas Dopaminérgicas/inmunología , Inflamación/inmunología , Enfermedades Inflamatorias del Intestino/inmunología , Intestinos/inmunología , Receptores de Dopamina D3/metabolismo , Linfocitos T Reguladores/inmunología , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Humanos , Terapia de Inmunosupresión , Interleucina-10/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuroinmunomodulación , ARN Interferente Pequeño/genética , Receptores de Dopamina D3/genética , Receptores Mensajeros de Linfocitos/metabolismo , Transducción de SeñalRESUMEN
Somatosensory information can be modulated at the spinal cord level by primary afferent depolarization (PAD), known to produce presynaptic inhibition (PSI) by decreasing neurotransmitter release through the activation of presynaptic ionotropic receptors. Descending monoaminergic systems also modulate somatosensory processing. We investigated the role of D1-like and D2-like receptors on pathways mediating PAD in the hemisected spinal cord of neonatal mice. We recorded low-threshold evoked dorsal root potentials (DRPs) and population monosynaptic responses as extracellular field potentials (EFPs). We used a paired-pulse conditioning-test protocol to assess homosynaptic and heterosynaptic depression of evoked EFPs to discriminate between dopaminergic effects on afferent synaptic efficacy and/or on pathways mediating PAD, respectively. DA (10 µM) depressed low-threshold evoked DRPs by 43 %, with no effect on EFPs. These depressant effects on DRPs were mimicked by the D2-like receptor agonist quinpirole (35 %). Moreover, by using selective antagonists at D2-like receptors (encompassing the D2, D3, and D4 subtypes), we found that the D2 and D3 receptor subtypes participate in the quinpirole depressant inhibitory effects of pathways mediating PAD.
Asunto(s)
Inhibición Neural/fisiología , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/metabolismo , Médula Espinal/metabolismo , Transmisión Sináptica/fisiología , Animales , Potenciales Postsinápticos Excitadores , Ratones , Vías Nerviosas/metabolismo , Neuronas Aferentes/metabolismo , Receptores Presinapticos/metabolismoRESUMEN
Dopamine D3 R are widely expressed in basal ganglia where interact with D1 R. D3 R potentiate cAMP accumulation and GABA release stimulated by D1 R in striatonigral neurons through "atypical" signaling. During dopaminergic denervation, D3 R signaling changes to a "typical" in which antagonizes the effects of D1 R, the mechanisms of this switching are unknown. D3 nf splice variant regulates membrane anchorage and function of D3 R and decreases in denervation; thus, it is possible that D3 R signaling switching correlates with changes in D3 nf expression and increases of membranal D3 R that mask D3 R atypical effects. We performed experiments in unilaterally 6-hydroxydopamine lesioned rats and found a decrease in mRNA and protein of D3 nf, but not of D3 R in the denervated striatum. Proximity ligation assay showed that D3 R-D3 nf interaction decreased after denervation, whereas binding revealed an increased Bmax in D3 R. The new D3 R antagonized cAMP accumulation and GABA release stimulated by D1 R; however, in the presence of N-Ethylmaleimide (NEM), to block Gi protein signaling, activation of D3 R produced its atypical signaling stimulating D1 R effects. Finally, we investigated if the typical and atypical effects of D3 R modulating GABA release are capable of influencing motor behavior. Injections of D3 R agonist into denervated nigra decreased D1 R agonist-induced turning behavior but potentiated it in the presence of NEM. Our data indicate the coexistence of D3 R typical and atypical signaling in striatonigral neurons during denervation that correlated with changes in the ratio of expression of D3 nf and D3 R isoforms. The coexistence of both atypical and typical signaling during denervation influences motor behavior.
Asunto(s)
Receptores de Dopamina D3/metabolismo , Transducción de Señal , Sustancia Negra/metabolismo , Animales , AMP Cíclico/metabolismo , Neuronas Dopaminérgicas/metabolismo , Neuronas Dopaminérgicas/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Masculino , Movimiento , Bloqueo Nervioso , Empalme del ARN , Ratas , Ratas Wistar , Receptores de Dopamina D3/genética , Sustancia Negra/citología , Sustancia Negra/fisiología , Ácido gamma-Aminobutírico/metabolismoRESUMEN
BACKGROUND: Neuroinflammation constitutes a pathogenic process leading to neurodegeneration in several disorders, including Alzheimer's disease, Parkinson's disease (PD) and sepsis. Despite microglial cells being the central players in neuroinflammation, astrocytes play a key regulatory role in this process. Our previous results indicated that pharmacologic-antagonism or genetic deficiency of dopamine receptor D3 (DRD3) attenuated neuroinflammation and neurodegeneration in two mouse models of PD. Here, we studied how DRD3-signalling affects the dynamic of activation of microglia and astrocyte in the context of systemic inflammation. METHODS: Neuroinflammation was induced by intraperitoneal administration of LPS. The effect of genetic DRD3-deficiency or pharmacologic DRD3-antagonism in the functional phenotype of astrocytes and microglia was determined by immunohistochemistry and flow cytometry at different time-points. RESULTS: Our results show that DRD3 was expressed in astrocytes, but not in microglial cells. DRD3 deficiency resulted in unresponsiveness of astrocytes and in attenuated microglial activation upon systemic inflammation. Furthermore, similar alterations in the functional phenotypes of glial cells were observed by DRD3 antagonism and genetic deficiency of DRD3 upon LPS challenge. Mechanistic analyses show that DRD3 deficiency resulted in exacerbated expression of the anti-inflammatory protein Fizz1 in glial cells both in vitro and in vivo. CONCLUSIONS: These results suggest that DRD3 signalling regulates the dynamic of the acquisition of pro-inflammatory and anti-inflammatory features by astrocytes and microglia, finally favouring microglial activation and promoting neuroinflammation.
Asunto(s)
Astrocitos/metabolismo , Mediadores de Inflamación/metabolismo , Microglía/metabolismo , Receptores de Dopamina D3/metabolismo , Transducción de Señal/fisiología , Animales , Astrocitos/efectos de los fármacos , Células Cultivadas , Inflamación/inducido químicamente , Inflamación/genética , Inflamación/metabolismo , Lipopolisacáridos/toxicidad , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/efectos de los fármacos , Trastornos Parkinsonianos/inducido químicamente , Trastornos Parkinsonianos/genética , Trastornos Parkinsonianos/metabolismo , Receptores de Dopamina D3/antagonistas & inhibidores , Receptores de Dopamina D3/genética , Transducción de Señal/efectos de los fármacosRESUMEN
Substantia nigra pars reticulata is the output station in basal ganglia; its GABAergic neurons control the activity of thalamo-cortical premotor nuclei, thus controlling motor behavior. D1-like and D2-like presynaptic dopamine receptors on subthalamo-nigral afferents by modulation of glutamate release change the firing rate of nigral neurons; however, their relative contribution to the control of glutamate release and their pharmacological properties have not been studied. This is important since the prevalence of the inhibition or stimulation of release determines the firing rate of nigral neurons, therefore motor activity. Here we used depolarization induced [3H]-glutamate release in slices of rat substantia nigra from reserpinized and non-reserpinized rats to explore the relative contribution of the D1-like and D2-like receptor subtypes to the control of glutamate release. We found a significant control of release by D1-like and D3R, and a modest effect of D2R. D4R exerted no effect. Dopamine showed more potency for D3R than for D1-like receptors; however, these latter enhanced release to a greater degree, as shown by the Emax. We also co-activated these to test their interaction; an antagonist interaction of D1-like with D2 and D3R, and an additive between D2 and D3R were found. Pharmacological receptor antagonist effects in release from reserpinized vs. non-reserpinized slices were similar, suggesting that endogenous dopamine stimulates receptors in the same way. These findings suggest differences in the control of glutamate release by different dopamine receptors in the substantia nigra, which could contribute to explaining the effect of dopamine and its agonists on motor behavior.
Asunto(s)
Ácido Glutámico/metabolismo , Terminales Presinápticos/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D3/metabolismo , Sustancia Negra/metabolismo , Tritio/metabolismo , Animales , Dopamina/farmacología , Relación Dosis-Respuesta a Droga , Masculino , Técnicas de Cultivo de Órganos , Terminales Presinápticos/efectos de los fármacos , Ratas , Ratas Wistar , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D3/agonistas , Sustancia Negra/efectos de los fármacosRESUMEN
ONC201/TIC10 is a first-in-class small molecule inducer of TRAIL that causes early activation of the integrated stress response. Its promising safety profile and broad-spectrum efficacy in vitro have been confirmed in Phase I/II trials in several advanced malignancies. Binding and reporter assays have shown that ONC201 is a selective antagonist of the dopamine D2-like receptors, specifically, DRD2 and DRD3. We hypothesized that ONC201's interaction with DRD2 plays a role in ONC201's anticancer effects. Using cBioportal and quantitative reverse-transcription polymerase chain reaction analyses, we confirmed that DRD2 is expressed in different cancer cell types in a cell type-specific manner. On the other hand, DRD3 was generally not detectable. Overexpressing DRD2 in cells with low DRD2 levels increased ONC201-induced PARP cleavage, which was preceded and correlated with an increase in ONC201-induced CHOP mRNA expression. On the other hand, knocking out DRD2 using CRISPR/Cas9 in three cancer cell lines was not sufficient to abrogate ONC201's anticancer effects. Although ONC201's anticancer activity was not dependent on DRD2 expression in the cancer cell types tested, we assessed the cytotoxic potential of DRD2 blockade. Transient DRD2 knockdown in HCT116 cells activated the integrated stress response and reduced cell number. Pharmacological antagonism of DRD2 significantly reduced cell viability. Thus, we demonstrate in this study that disrupting dopamine receptor expression and activity can have cytotoxic effects that may at least be in part due to the activation of the integrated stress response. On the other hand, ONC201's anticancer activity goes beyond its ability to antagonize DRD2, potentially due to ONC201's ability to activate other pathways that are independent of DRD2. Nevertheless, blocking the dopamine D1-like receptor DRD5 via siRNA or the use of a pharmacological antagonist promoted ONC201-induced anticancer activity.
Asunto(s)
Antineoplásicos/farmacología , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Neoplasias/metabolismo , Receptores Dopaminérgicos/metabolismo , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Resistencia a Antineoplásicos , Expresión Génica , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Técnicas de Inactivación de Genes , Humanos , Imidazoles , Neoplasias/genética , Piridinas , Pirimidinas , ARN Interferente Pequeño/genética , Receptores Dopaminérgicos/genética , Receptores de Dopamina D2/genética , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/genética , Receptores de Dopamina D3/metabolismo , Receptores de Dopamina D5/genética , Receptores de Dopamina D5/metabolismoRESUMEN
Neuroinflammation involves the activation of glial cells, which is associated to the progression of neurodegeneration in Parkinson's disease. Recently, we and other researchers demonstrated that dopamine receptor D3 (D3R)-deficient mice are completely refractory to neuroinflammation and consequent neurodegeneration associated to the acute intoxication with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). In this study we examined the therapeutic potential and underlying mechanism of a D3R-selective antagonist, PG01037, in mice intoxicated with a chronic regime of administration of MPTP and probenecid (MPTPp). Biodistribution analysis indicated that intraperitoneally administered PG01037 crosses the blood-brain barrier and reaches the highest concentration in the brain 40 min after the injection. Furthermore, the drug was preferentially distributed to the brain in comparison to the plasma. Treatment of MPTPp-intoxicated mice with PG01037 (30 mg/kg, administrated twice a week for five weeks) attenuated the loss of dopaminergic neurons in the substantia nigra pars compacta, as evaluated by stereological analysis, and the loss of striatal dopaminergic terminals, as determined by densitometric analyses of tyrosine hydroxylase and dopamine transporter immunoreactivities. Accordingly, the treatment resulted in significant improvement of motor performance of injured animals. Interestingly, the therapeutic dose of PG01037 exacerbated astrogliosis and resulted in increased ramification density of microglial cells in the striatum of MPTPp-intoxicated mice. Further analyses suggested that D3R expressed in astrocytes favours a beneficial astrogliosis with anti-inflammatory consequences on microglia. Our findings indicate that D3R-antagonism exerts a therapeutic effect in parkinsonian animals by reducing the loss of dopaminergic neurons in the nigrostriatal pathway, alleviating motor impairments and modifying the pro-inflammatory phenotype of glial cells.
Asunto(s)
Benzamidas/administración & dosificación , Neuronas Dopaminérgicas/efectos de los fármacos , Encefalitis/prevención & control , Fármacos Neuroprotectores/administración & dosificación , Trastornos Parkinsonianos/prevención & control , Piridinas/administración & dosificación , Receptores de Dopamina D3/antagonistas & inhibidores , Animales , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Benzamidas/sangre , Benzamidas/farmacología , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/patología , Neuronas Dopaminérgicas/patología , Encefalitis/etiología , Masculino , Ratones , Ratones Endogámicos C57BL , Microglía/efectos de los fármacos , Actividad Motora/efectos de los fármacos , Fármacos Neuroprotectores/sangre , Fármacos Neuroprotectores/farmacología , Enfermedad de Parkinson/complicaciones , Enfermedad de Parkinson/prevención & control , Trastornos Parkinsonianos/complicaciones , Porción Compacta de la Sustancia Negra/efectos de los fármacos , Porción Compacta de la Sustancia Negra/patología , Piridinas/sangre , Piridinas/farmacología , Receptores de Dopamina D3/metabolismoRESUMEN
BACKGROUND: Neonatal lesion in the ventral hippocampus (NLVH) is a validated animal model to study schizophrenia from a neurodevelopmental perspective. This animal model is also used to investigate how neonatal lesions may alter the genetic expression of dopaminergic receptors. The present study compares mRNA expression levels of dopamine receptors (drd2 and drd3) in lymphocytes and brain of NLVH animals at two different age stages: young and adult. METHODS: The NLVH procedure was performed on 20 male Wistar rats at postnatal days 5-7. The mRNA expression levels of drd2 and drd3 genes in lymphocytes, nucleus accumbens, hippocampus and prefrontal cortex were measured and analyzed at postnatal days 45 and 90. The results were compared and contrasted with respective sham groups. RESULTS: In lymphocytes, only in NLVH-adult group we observed drd2 mRNA expression, while drd2 mRNA expression was not observed in the NLVH-juvenile rats; on the other hand, the drd3 mRNA expression did not show significant statistical differences. In hippocampus no differences were observed between drd2 mRNA or drd3 mRNA expression when comparing juvenile/adult shams with NLVH groups. In the prefrontal area, a decrease in drd2 mRNA expression levels were observed in the NLVH-adult group (F(1,3) = 52.83, p = 0,005) in comparison to the sham-adult group. Finally, in the nucleus accumbens, a strong decrease of drd3 mRNA expression was observed in the NLVH-adult group in comparison to the sham-adult group (F(1,3) = 123,2, p < 0.001). CONCLUSIONS: Our results show that differences in drd2 and drd3 mRNA levels in NLVH-adults are patent when compared to the sham-adult group or with the NLVH-juvenile group. These findings suggest that the expression levels may be regulated during adulthood, leading to behavioral and neurochemical changes related to schizophrenia. Therefore, more studies are necessary to determine the role of dopamine receptors as possible molecular markers for neurodevelopmental changes associated with schizophrenia.
Asunto(s)
Hipocampo/metabolismo , Núcleo Accumbens/metabolismo , Corteza Prefrontal/metabolismo , Receptores de Dopamina D2/metabolismo , Receptores de Dopamina D3/metabolismo , Esquizofrenia/genética , Animales , Animales Recién Nacidos , Modelos Animales de Enfermedad , Hipocampo/patología , Linfocitos/metabolismo , Masculino , Núcleo Accumbens/patología , Corteza Prefrontal/patología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas Wistar , Receptores de Dopamina D2/genética , Receptores de Dopamina D3/genéticaRESUMEN
Dopamine receptors have been described in T-cells, however their signalling pathways coupled remain unknown. Since cAMP and ERKs play key roles regulating T-cell physiology, we aim to determine whether cAMP and ERK1/2-phosphorylation are modulated by dopamine receptor 3 (D3R) and D5R, and how this modulation affects CD4(+) T-cell activation and differentiation. Our pharmacologic and genetic evidence shows that D3R-stimulation reduced cAMP levels and ERK2-phosphorylation, consequently increasing CD4(+) T-cell activation and Th1-differentiation, respectively. Moreover, D5R expression reinforced TCR-triggered ERK1/2-phosphorylation and T-cell activation. In conclusion, these findings demonstrate how D3R and D5R modulate key signalling pathways affecting CD4(+) T-cell activation and Th1-differentiation.
Asunto(s)
Antígenos CD4/metabolismo , Diferenciación Celular/fisiología , AMP Cíclico/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Receptores de Dopamina D3/metabolismo , Receptores de Dopamina D5/metabolismo , Linfocitos T/fisiología , Animales , Diferenciación Celular/efectos de los fármacos , Diferenciación Celular/genética , Dopaminérgicos/farmacología , Relación Dosis-Respuesta a Droga , Activación Enzimática/efectos de los fármacos , Activación Enzimática/genética , Inhibidores Enzimáticos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Subunidad alfa del Receptor de Interleucina-2/metabolismo , Ionomicina/farmacología , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/genética , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fosforilación/efectos de los fármacos , Fosforilación/genética , Receptores de Dopamina D3/genética , Receptores de Dopamina D5/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Linfocitos T/efectos de los fármacosRESUMEN
Recent studies indicate the involvement of dopamine receptors D1 and D3 in the regulation of locomotor stimulant and conditioned responses to morphine in mice. Moreover, expression of brain-derived neurotrophic factor (BDNF) may be modulated by D1 and D3 receptor activities in the nucleus accumbens (NAc) and prefrontal cortex (PFC). However, the underlying interactions between D1 and D3 receptors and BDNF in the expression of behavioral responses controlled by drug-associated cues have not yet been fully elucidated. In this study, we used dopamine receptor mutant mice to explore the roles of the D1 and D3 receptors in locomotion and morphine-induced place preference; furthermore, we investigated the effects of morphine on BDNF expression in the NAc and PFC of the mouse brain. Our results show that D1 receptor but not D3 receptor mutant mice had decreased sensitivity to acute morphine-induced (10 mg/kg) locomotion (D1: 3814.82 ± 319.9 cm vs D3: 8089.64 ± 967.4 cm). Furthermore, D1 receptor mutant mice did not acquire morphine-conditioned place preference (D1: -18.3 ± 59.9, D3: 217.7 ± 64.1) and showed decreased BDNF expression in the NAc (D1: 0.33 ± 0.07 fold, D3: 2.21 ± 0.18 fold) and PFC (D1: 0.74 ± 0.15 fold, D3: 1.68 ± 0.22 fold) compared with wild-type and D3 receptor mutant mice. These findings suggest that the D1 receptor is necessary for the induction of cue-associated morphine seeking and modulates locomotor habituation processes in response to acute morphine. The dopamine receptor D1 but not the D3 is also critical for morphine-induced BDNF expression in the NAc and PFC.
Asunto(s)
Condicionamiento Psicológico/efectos de los fármacos , Morfina/farmacología , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D3/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Conducta de Elección , Masculino , Ratones Endogámicos C57BL , Ratones Mutantes , Actividad Motora/efectos de los fármacos , Núcleo Accumbens/efectos de los fármacos , Núcleo Accumbens/metabolismo , Corteza Prefrontal/efectos de los fármacos , Corteza Prefrontal/metabolismoRESUMEN
In striatonigral projections activation of dopamine D3 receptors (D3Rs) potentiates the stimulation of GABA release and cAMP production caused by activation of dopamine D1 receptors (D1Rs). Cytoplasmic [Ca(2+)] in the terminals controls this response by modulating CaMKII, an enzyme that depresses D3R action. To examine the effects of dopamine deprivation on D3R signaling we investigated their function in striatonigral terminals of hemiparkinsonian rats. Denervation switched the signaling cascade initiated by D3R activation. In the non-lesioned side activation of D3R potentiated the stimulatory effects of D1R activation on cAMP production and K(+)-depolarization induced [(3)H] GABA release. In contrast, in the denervated side the stimulatory effects of both D1R activation and forskolin administration were blocked by D3R activation. In non-lesioned slices, D3R responses were inhibited by the activation of CaMKII produced by K(+)-depolarization (via increased Ca(2+) entry). The CaMKII-induced inhibition was blocked by the selective inhibitor KN-62. In denervated tissues the response to D3R stimulation was not modified either by K(+) depolarization or by blocking CaMKII with KN-62. Immunoblotting studies showed that depolarization-induced CaMKII binding to the D3 receptor and CaMKII phosphorylation were suppressed in denervated tissues. We also determined calmodulin expression with PCR and immunoblot techniques. Both techniques showed that calmodulin expression was depressed in the lesioned side. In sum, our studies show that dopaminergic denervation switches the D3R signaling cascade and depresses CaMKII signaling through a process that appears to involve reduced calmodulin levels. Since calmodulin is a major cytoplasmic Ca(2+) buffer our findings suggest that abnormal Ca(2+) buffering may be an important component of the abnormalities observed during dopaminergic denervation.
Asunto(s)
Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Calmodulina/metabolismo , Cuerpo Estriado/metabolismo , Neuronas Dopaminérgicas/metabolismo , Receptores de Dopamina D3/metabolismo , Sustancia Negra/metabolismo , Animales , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , Cuerpo Estriado/efectos de los fármacos , AMP Cíclico/metabolismo , Fosfatos de Dinucleósidos/metabolismo , Dopamina/metabolismo , Neuronas Dopaminérgicas/efectos de los fármacos , Masculino , Haz Prosencefálico Medial/fisiopatología , Vías Nerviosas/efectos de los fármacos , Vías Nerviosas/metabolismo , Oxidopamina , Fosforilación/efectos de los fármacos , Ratas Wistar , Transducción de Señal , Sustancia Negra/efectos de los fármacos , Técnicas de Cultivo de Tejidos , Ácido gamma-Aminobutírico/metabolismoRESUMEN
CaMKIIα is expressed at high density in the nucleus accumbens where it binds to postsynaptic D3 receptors inhibiting their effects. In striatonigral projections, activation of presynaptic D3 receptors potentiates D1 receptor-induced stimulation of cAMP production and GABA release. In this study we examined whether the presynaptic effects of D3 receptor stimulation in the substantia nigra reticulata (SNr) are modulated by Ca²âº activation of CaMKIIα. In SNr synaptosomes two procedures that increase cytoplasmic Ca²âº, ionomycin and Kâº-depolarization, blocked the additional stimulation of cAMP accumulation produced by coactivating D3 and D1 dopamine receptors. The selective CaMKIIα inhibitor KN-62 reversed the blockade produced by ionomycin and Kâº-depolarization. Incubation in either Ca²-free solutions or with the selective Ca²âº blocker nifedipine, also reversed the blocking effects of Kâº-depolarization. Immunoblot studies showed that Kâº-depolarization increased CaMKIIα phosphorylation in a KN-62 sensitive manner and promoted CaMKIIα binding to D3 receptors. In Kâº-depolarized tissues, D3 receptors potentiated D1 receptor-induced stimulation of [³H]GABA release only when CaMKIIα was blocked with KN-62. In the presence of this inhibitor, the selective D3 agonist PD 128,907 reduced the ED50 for the D1 agonist SKF 38393 from 56 to 4 nM. KN-62 also enhanced the effects of dopamine on depolarization induced [³H]GABA release. KN-62 changed ED50 for dopamine from 584 to 56 nM. KN-62 did not affect D1 and D4 receptor responses. These experiments show that in striatonigral projections, CaMKIIα inhibits the action of D3 receptors in a Ca²âº dependent manner blocking their modulatory effects on GABA release. These findings suggest a mechanism through which the frequency of action potential discharge in presynaptic terminals regulates dopamine effects.
Asunto(s)
Señalización del Calcio , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Terminales Presinápticos/metabolismo , Receptores de Dopamina D3/metabolismo , Sustancia Negra/metabolismo , Animales , Bloqueadores de los Canales de Calcio , Señalización del Calcio/efectos de los fármacos , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/antagonistas & inhibidores , AMP Cíclico/metabolismo , Agonistas de Dopamina/farmacología , Técnicas In Vitro , Masculino , Potenciales de la Membrana/efectos de los fármacos , Proteínas del Tejido Nervioso/agonistas , Concentración Osmolar , Fosforilación/efectos de los fármacos , Terminales Presinápticos/efectos de los fármacos , Inhibidores de Proteínas Quinasas/farmacología , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Ratas , Ratas Wistar , Receptores de Dopamina D1/agonistas , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D3/agonistas , Sustancia Negra/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacosRESUMEN
Emerging evidence has demonstrated that CD4(+) T cells infiltrate into the substantia nigra (SN) in Parkinson's disease (PD) patients and in animal models of PD. SN-infiltrated CD4(+) T cells bearing inflammatory phenotypes promote microglial activation and strongly contribute to neurodegeneration of dopaminergic neurons. Importantly, altered expression of dopamine receptor D3 (D3R) in PBLs from PD patients has been correlated with disease severity. Moreover, pharmacological evidence has suggested that D3R is involved in IFN-γ production by human CD4(+) T cells. In this study, we examined the role of D3R expressed on CD4(+) T cells in neurodegeneration of dopaminergic neurons in the SN using a mouse model of PD. Our results show that D3R-deficient mice are strongly protected against loss of dopaminergic neurons and microglial activation during 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD. Notably, D3R-deficient mice become susceptible to MPTP-induced neurodegeneration and microglial activation upon transfer of wild-type (WT) CD4(+) T cells. Furthermore, RAG1 knockout mice, which are devoid of T cells and are resistant to MPTP-induced neurodegeneration, become susceptible to MPTP-induced loss of dopaminergic neurons when reconstituted with WT CD4(+) T cells but not when transferred with D3R-deficient CD4(+) T cells. In agreement, experiments analyzing activation and differentiation of CD4(+) T cells revealed that D3R favors both T cell activation and acquisition of the Th1 inflammatory phenotype. These findings indicate that D3R expressed on CD4(+) T cells plays a fundamental role in the physiopathology of MPTP-induced PD in a mouse model.
Asunto(s)
Linfocitos T CD4-Positivos/metabolismo , Neuronas Dopaminérgicas/patología , Proteínas de Homeodominio/genética , Enfermedad de Parkinson/patología , Receptores de Dopamina D3/metabolismo , 1-Metil-4-fenil-1,2,3,6-Tetrahidropiridina , Traslado Adoptivo , Animales , Linfocitos T CD4-Positivos/inmunología , Cuerpo Estriado/metabolismo , Modelos Animales de Enfermedad , Neuronas Dopaminérgicas/inmunología , Inflamación/inmunología , Interferón gamma/biosíntesis , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microglía/metabolismo , Degeneración Nerviosa/metabolismo , Enfermedad de Parkinson/metabolismo , Receptores de Dopamina D3/biosíntesis , Receptores de Dopamina D3/genética , Bazo , Sustancia Negra/inmunología , Sustancia Negra/metabolismo , Factor de Necrosis Tumoral alfa/biosíntesisRESUMEN
The firing rate of substantia nigra reticulata (SNr) neurons is modulated by GABA release from striatonigral and pallidonigral projections. This release is, in turn, modulated by dopamine acting on dopamine D1 receptors at striatonigral terminals and D4 receptors at pallidonigral terminals. In addition, striatal neurons that express D1 receptors also express D3 receptors. In this study we analyzed the possible significance of D3 and D1 receptor colocalization in striatonigral projections. We found that these receptors coprecipitate in SNr synaptosomes suggesting their close association in this structure. D1 agonist SKF 38393 administered alone increased mIPSC frequency in SNr slices and cAMP production in SNr synaptosomes, however, the selective D3 agonist PD 128,907 increased mIPSC frequency and cAMP production only when D1 receptors were concurrently stimulated. The D1 antagonist SCH 23390 blocked completely the effects of the concurrent administration of these agonists while the selective D3 antagonist GR 103691 blocked only the potentiating effects of PD 128,907. These findings further indicate that D1 and D3 receptors are localized in the same structure. The D4 agonist PD 168,077 decreased mIPSCs frequency without changing amplitude, an effect that was blocked by the selective D4 antagonist L 745,870. The effects of D4 receptor stimulation disappeared after lesioning the globus pallidus. D3 agonist PD 128,907 did not reduce mIPSC frequency even in neurons that responded to D4 agonist. In sum, activation of D3 receptors in SNr potentiates the stimulation of transmitter release and cAMP production caused by D1 receptor activation of striatonigral projections while it is without effects in terminals, probably of pallidal origin, that are inhibited by activation of D4 receptors.
Asunto(s)
Neuronas GABAérgicas/metabolismo , Terminales Presinápticos/metabolismo , Receptores de Dopamina D1/metabolismo , Receptores de Dopamina D3/metabolismo , Receptores de Dopamina D4/metabolismo , Sustancia Negra/metabolismo , Animales , Agonistas de Dopamina/metabolismo , Agonistas de Dopamina/farmacología , Antagonistas de Dopamina/metabolismo , Antagonistas de Dopamina/farmacología , Neuronas GABAérgicas/efectos de los fármacos , Masculino , Terminales Presinápticos/efectos de los fármacos , Unión Proteica/fisiología , Ratas , Ratas Wistar , Sustancia Negra/efectos de los fármacosRESUMEN
Amygdaloid dopamine D(2) receptors play an important role in the modulation of fear/anxiety. Their topographical distribution within the amygdala is however unclear, and their role in unconditioned fear/anxiety remains largely unknown. The aim of this paper was to study the intra-amygdaloid distribution of D(2) receptors and to ascertain their role in unconditioned anxiety. Chemical anatomical studies in the rat, using D(2) and D(3)in situ hybridization, quantitative receptor autoradiography with either [(3)H]raclopride or [(125)I]sulpiride, and D(2)-like immunocytochemistry showed that the highest density of dopamine D(2) receptors is present in the central amygdaloid nucleus, particularly within its latero-capsular division, in which a D(2) but not a D(3) mRNA signal was observed. However, although at considerably reduced densities dopamine D(2) receptors were also found in other locations within the amygdala, including the basolateral nucleus. Behaviorally, the infusion of raclopride (0.75-4 µg/side) in the area of the central amygdaloid nucleus resulted at low doses in the appearance of anxiogenic-like effects in the Shock-Probe Burying test, whereas no effects of raclopride treatment were found at any dose in the Elevated Plus-Maze and the Open-Field test. Our results indicate that amygdaloid dopamine D(2)-like receptors have a topographically differentiated distribution within the rat amygdala, the major location being in the central amygdaloid nucleus. D(2)-like receptors play a role in the modulation of anxiety responses involving a potential differential function of D(2)-like receptors in the central amygdaloid nucleus versus the basolateral amygdaloid nucleus.