Search

El parlamentarismo describe tanto la teoría como la práctica de la forma parlamentaria de Gobierno, una de las múltiples opciones de la democracia representativa. Resultado de un proceso acumulativo, el parlamentarismo ha conocido diversas expresiones políticas e institucionales. A pesar de que el tipo ideal de parlamentarismo se ha ligado tradicionalmente a la vía inglesa y británica, resulta indiscutible que esta forma de gobierno ha superado el alcance de los territorios que encuentran su origen democrático bajo la influencia de la cultura británica. De este modo, en la Europa continental se encuentran tanto un amplísimo abanico de sistemas parlamentarios con sus diversas especificidades como el espacio de referencia geopolítica de la integración supranacional europea. En este sentido, entre los casos en los que se ha materializado y consolidado este esquema institucional destaca el Parlamento Europeo, siendo el único Parlamento supraestatal del mundo. A lo largo del presente artículo se repasan no solamente los pilares de la democracia representativa y el parlamentarismo, así como diversas nociones imprescindibles para el entendimiento de estos, sino que también se analizan y exponen las peculiaridades del Parlamento Europeo, órgano particular que ha ido evolucionando y adaptándose a la naturaleza única del proceso y de la experiencia de la integración europea.

The integrity of the perirhinal cortex (PRh) is essential for object recognition memory (ORM) function, and damage to this brain area in animals and humans induces irreversible ORM deficits. Here, we show that activation of area V2, a brain area interconnected with brain circuits of ventral stream and medial temporal lobe that sustain ORM, by expression of regulator of G-protein signaling 14 of 414 amino acids (RGS14414) restored ORM in memory-deficient PRh-lesioned rats and nonhuman primates. Furthermore, this treatment was sufficient for full recovery of ORM in rodent models of aging and Alzheimer's disease, conditions thought to affect multiple brain areas. Thus, RGS14414-mediated activation of area V2 has therapeutic relevance in the recovery of recognition memory, a type of memory that is primarily affected in patients or individuals with symptoms of memory dysfunction. These findings suggest that area V2 modulates the processing of memory-related information through activation of interconnected brain circuits formed by the participation of distinct brain areas.

The inhibition of capping enzymes such as guanine-N7-methyltransferase (GMT) is an attractive target for regulating viral replication, transcription, virulence, and pathogenesis. Thus, compounds that target the Severe Acute Respiratory Syndrome Corona Virus 2 GMT (S2GMT) will enhance drug development against COVID-19. In this study, an in-house library of 249 phytochemicals from African medicinal plants was screened using computational approaches including homology modeling, molecular docking, molecular dynamic simulations, binding free energy calculations based on molecular mechanics/Poisson-Boltzmann surface area (MMPBSA) and Absorption-Distribution-Metabolism-Excretion-Toxicity (ADMET) analysis for inhibitors of S2GMT. The top-ten ranked phytochemicals (TTRP) obtained from the docking analysis to S2GMT were further docked to SARS-COV N7-MTase. Among the TTRP, the top-four ranked phytocompounds (TFRP) viz: 3 alkaloids (Isocryptolepine, 10’–Hydroxyusambarensine and Isostrychnopentamine) and a flavonoid (Mulberrofuran F) interacted strongly with critical catalytic residues whose interference either reduce or completely abolish N7-MTase activity, indicating their potential as capping machinery disruptors. The interactions of TFRP with the catalytic residues of S2GMT were preserved in a 100 ns simulated dynamic environment, thereby, demonstrating high degree of structural stability. The MMPBSA binding free energy calculations corroborated the docking scores with biscryptolepine having the highest binding free energy to S2GMT. The TFRP showed favourable drug-likeness and ADMET properties over a wide range of molecular descriptors. Therefore, the TFRP can be further explored as potential S2GMT inhibitors in in vitro and in vivo experiments. Communicated by Ramaswamy H. Sarma

Despite the ongoing vaccination against the life-threatening COVID-19, there is need for viable therapeutic interventions. The S-adenosyl-l-Methionine (SAM) dependent 2-O'-ribose methyltransferase (2'-O-MTase) of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) presents a therapeutic target against COVID-19 infection. In a bid to profile bioactive principles from natural sources, a custom-made library of 226 phytochemicals from African medicinal plants with especially anti-malarial activity was screened for direct interactions with SARS-CoV-2 2'-O-MTase (S2RMT) using molecular docking and molecular dynamics (MD) simulations as well as binding free energies methods. Based on minimal binding energy lower than sinefungin (a reference methyl-transferase inhibitor) and binding mode analysis at the catalytic site of S2RMT, a list of 26 hit phytocompounds was defined. The interaction of these phytocompounds was compared with the 2'-O-MTase of SARS-CoV and MERS-CoV. Among these compounds, the lead phytocompounds (LPs) viz: mulberrofuran F, 24-methylene cycloartenol, ferulate, 3-benzoylhosloppone and 10-hydroxyusambarensine interacted strongly with the conserved KDKE tetrad within the substrate binding pocket of the 2'-O-MTase of the coronavirus strains which is critical for substrate binding. The thermodynamic parameters analyzed from the MD simulation trajectories of the LPs-S2RMT complexes presented an eminent structural stability and compactness. These LPs demonstrated favorable druggability and

BACKGROUND: In children, the complications of severe acute respiratory syndrome coronavirus 2 infection occur less frequently than in adults but the characteristics of this disease in oncology patients are not well characterized. METHODS: This was a retrospective study in patients younger than 18 years of age with coronavirus disease 2019 (COVID-19) and cancer diagnoses between April and September 2020. Demographic variables, laboratory, and radiologic findings and complications of each case were identified. A descriptive analysis was performed. RESULTS: A total of 33 patients were identified; the median age was 10 years. Fifteen patients (42%) were in chemotherapy at the time of the infection diagnosis, in two patients the chemotherapy protocol was permanently suspended. The most common symptom was fever in 20 patients (60%). Seven patients (21.2%) showed mild pneumonia, four patients (12.1%) severe pneumonia, and three cases (9.0%) were classified as critical. In the evaluated cohort, five patients (15.1%) died, and in two of those, death was caused by COVID-19 infection. CONCLUSIONS: Children with an oncologic disease, the search for COVID cases should be oriented to patients with fever, including febrile neutropenia, the presence of respiratory symptoms, and the search for epidemiologic contact. A higher frequency of complications and mortality attributed to COVID-19, two in pediatric oncohematologic patients was found. Institutional strategies to detect the infection early and lower institutional infection are indicated.

En nuestro país, por lo general, las aulas han sido construidas sin considerar criterios acústicos, generando ambientes inadecuados para el proceso de enseñanza-aprendizaje. Ésta situación, interfiere tanto en el desempeño académico de los alumnos, como en la salud de los docentes. En este trabajo se analizan los principales factores acústicos intervinientes en dicho proceso, tales como: geometría y materiales del recinto, ruido de fondo, tiempos de reverberación y relación señal/ruido. Los resultados evidencian que una cantidad significativa de aulas utilizadas en el dictado de carreras de grado no presentan condiciones adecuadas para el desarrollo del proceso de enseñanza-aprendizaje. Los resultados presentados se obtuvieron en el marco del PID UTN 1658: “Estudio de características principales del campo sonoro en aulas y auditorios.

Antipsychotic drugs remain the current standard for schizophrenia treatment. Although they directly recognize the orthosteric binding site of numerous monoaminergic G protein-coupled receptors (GPCRs), these drugs, and particularly second-generation antipsychotics such as clozapine, all have in common a very high affinity for the serotonin 5-HT2A receptor (5-HT2AR). Using classical pharmacology and targeted signaling pathway assays, previous findings suggest that clozapine and other atypical antipsychotics behave principally as 5-HT2AR neutral antagonists and/or inverse agonists. However, more recent findings showed that antipsychotics may also behave as pathway-specific agonists. Reversible phosphorylation is a common element in multiple signaling networks. Combining a quantitative phosphoproteomic method with signaling network analysis, we tested the effect of clozapine treatment on the overall level of protein phosphorylation and signal transduction cascades in vitro in mammalian cell lines induced to express either the human 5-HT2AR or the H452Y variant of the gene encoding the 5-HT2AR receptor. This naturally occurring variation within the 5-HT2AR gene was selected because it has been repeatedly associated with schizophrenia patients who do not respond to clozapine treatment. Our data show that short time exposure (5 or 10 min) to clozapine (10−5 M) led to phosphorylation of numerous signaling components of pathways involved in processes such as endocytosis, ErbB signaling, insulin signaling or estrogen signaling. Cells induced to express the H452Y variant showed a different basal phosphoproteome, with increases in the phosphorylation of mTOR signaling components as a translationally relevant example. However, the effect of clozapine on the functional landscape of the phosphoproteome was significantly reduced in cells expressing the 5-HT2AR-H452Y construct. Together, these findings suggest that clozapine behaves as an agonist inducing phosphorylation of numerous pathways downstream of the 5-HT2AR, and that the single nucleotide polymorphism encoding 5-HT2AR-H452Y affects these clozapine-induced phosphorylation-dependent signaling networks.

Antipsychotic drugs remain the current standard for schizophrenia treatment. Although they directly recognize the orthosteric binding site of numerous monoaminergic G protein-coupled receptors (GPCRs), these drugs, and particularly second-generation antipsychotics such as clozapine, all have in common a very high affinity for the serotonin 5-HT

Sp8 and Sp6 are two closely related Sp genes expressed in the limb ectoderm where they regulate proximo-distal and dorso-ventral patterning. Mouse genetics revealed that they act together in a dose-dependent manner but with Sp8 making a much greater contribution. Here, we combine ChIP-seq and RNA-seq genome-wide analyses to investigate the Sp8 regulatory network and mechanism of action. We find that Sp8 predominantly binds to putative distal enhancers to activate crucial limb patterning genes, includingFgf8, En1, Sp6andRspo2. Sp8 exerts its regulatory function by directly binding DNA at Sp consensus sequences or indirectly through Dlx5 interaction. Overall, our work underscores Sp8 master regulatory functions and supports a model in which it cooperates with other Dlx and Sp cofactors to regulate target genes. We believe that this model could help to properly understand the molecular basis of congenital malformations.Impact SentenceIn the limb ectoderm, Sp8 regulates master genes through a dual mechanism: directly binding DNA at Sp consensus sequences and indirectly engaging through Dlx5 interaction.

Membrane trafficking processes regulate G protein-coupled receptor (GPCR) activity. Although class A GPCRs are capable of activating G proteins in a monomeric form, they can also potentially assemble into functional GPCR heteromers. Here, we showed that the class A serotonin 5-HT(2A) receptors (5-HT(2A)R) affected the localization and trafficking of class C metabotropic glutamate receptor 2 (mGluR2) through a mechanism that required their assembly as heteromers in mammalian cells. In the absence of agonists, 5-HT(2A)R was primarily localized within intracellular compartments, and coexpression of 5-HT(2A)R with mGluR2 increased the intracellular distribution of the otherwise plasma membrane-localized mGluR2. Agonists for either 5-HT(2A)R or mGluR2 differentially affected trafficking through Rab5-positive endosomes in cells expressing each component of the 5-HT(2A)R-mGluR2 heterocomplex alone, or together. Additionally, overnight pharmacological 5-HT(2A)R blockade with clozapine, but not with M100907, decreased mGluR2 density through a mechanism that involved heteromerization between 5-HT(2A)R and mGluR2. Using TAT-tagged peptides and chimeric constructs that are unable to form the interclass 5-HT(2A)R-mGluR2 complex, we demonstrated that heteromerization was necessary for the 5-HT(2A)R-dependent effects on mGluR2 subcellular distribution. Expression of 5-HT(2A)R also augmented intracellular localization of mGluR2 in mouse frontal cortex pyramidal neurons. Together, our data suggest that GPCR heteromerization may itself represent a mechanism of receptor trafficking and sorting.

The 5-HT receptor is a homodimeric G protein-coupled receptor implied in multiple diseases, including schizophrenia. Recently, its co-crystallisation with the antipsychotic drugs zotepine and risperidone has revealed the importance of its extracellular domains in its pharmacology. Previous studies have shown that the non-specific disruption of extracellular disulphide bridges in the 5-HT receptor decreases ligand binding and receptor activation. There is enough evidence to hypothesize that this decrease may be due to a reduction of the disulphide bridge that links transmembrane domain 3 (TM-3) and extracellular loop 2 (ECL-2) of the 5-HT receptor via cysteine 148 (C148) and C227. Thus, to study the influence of the C148–C227 disulphide bridge on 5-HT receptor pharmacology, we substituted C148 and C227 in the human 5-HT receptor (WT) with alanines, to obtain two single mutants (C148A and C227A) and a double mutant (C148A/C227A), and the resultant DNA constructs were used to generate four stable cell lines. These substitutions reduced the binding of the 5-HT receptor to [H]lysergic acid diethylamide ([H]LSD) and impeded the 5-HT receptor-mediated activation of phospholipase C (PLC). Furthermore, bioluminescence resonance energy transfer (BRET) and western blotting analysis revealed that these mutations did not alter the homodimeric nature of the 5-HT receptor. However, fluorescence microscopy showed that these mutations hindered receptor trafficking to the cell membrane. These results illustrate the importance of the disulphide bridge between TM-3 and ECL-2 in maintaining the correct 5-HT receptor conformation to allow ligand binding and migration of the homodimeric receptor to the cell membrane., This work was supported by the Spanish Ministry of Economy and Competitiveness (SAF2014-57138-C2-1-R and SAF2017-85225-C3-1-R) and the European Regional Development Fund (ERDF). MC and LGG were supported by a grant from the Consellería de Cultura, Educación y Ordenación Universitaria, partially co-funded by the European Social Fund (ESF) program.

Exposure and infections by Trypanosoma cruzi are the fourth cause of loss of potential life years between parasitic and infectious diseases. We describe the case of a 11-year-old patient with intestinal occlusion, surgically treated with intestinal volvulus, the surgical specimen is sent to histopathology reporting Chagasic megacolon. The age range of presentation is a challenge in the absence of nonspecific symptoms. There is no pediatric statistical data that define trypanosomiasis in a latent or chronic state and will be diagnosed in adult stages due to the physiopathological alterations that they will present.La exposición y las infecciones por Trypanosoma cruzi ocupan el cuarto lugar entre las causas de pérdida de años de vida potenciales por enfermedades parasitarias e infecciosas. Se describe el caso de un niño de 11 años, con cuadro de oclusión intestinal, intervenido quirúrgicamente con datos de vólvulo intestinal. La pieza quirúrgica se envió a histopatología, que reportó megacolon chagásico. El rango de edad de presentación es un reto ante la falta de síntomas inespecíficos. No se cuenta con datos estadísticos pediátricos que definan la tripanosomiasis en estado latente o crónico, y estos niños serán diagnosticados en la etapa adulta por las alteraciones fisiopatológicas que presentarán.

Exposure and infections by Trypanosoma cruzi are the fourth cause of loss of potential life years between parasitic and infectious diseases. We describe the case of a 11-year-old patient with intestinal occlusion, surgically treated with intestinal volvulus, the surgical specimen is sent to histopathology reporting Chagasic megacolon. The age range of presentation is a challenge in the absence of nonspecific symptoms. There is no pediatric statistical data that define trypanosomiasis in a latent or chronic state and will be diagnosed in adult stages due to the physiopathological alterations that they will present.

Memory deficits affect a large proportion of the human population and are associated with aging and many neurologic, neurodegenerative, and psychiatric diseases. Treatment of this mental disorder has been disappointing because all potential candidates studied thus far have failed to produce consistent effects across various types of memory and have shown limited to no effects on memory deficits. Here, we show that the promotion of neuronal arborization through the expression of the regulator of G-protein signaling 14 of 414 amino acids (RGS14

Memory deficits affect a large proportion of the human population and are associated with aging and many neurologic, neurodegenerative, and psychiatric diseases. Treatment of this mental disorder has been disappointing because all potential candidates studied thus far have failed to produce consistent effects across various types of memory and have shown limited to no effects on memory deficits. Here, we show that the promotion of neuronal arborization through the expression of the regulator of G-protein signaling 14 of 414 amino acids (RGS14414) not only induced robust enhancement of multiple types of memory but was also sufficient for the recovery of recognition, spatial, and temporal memory, which are kinds of episodic memory that are primarily affected in patients or individuals with memory dysfunction. We observed that a surge in neuronal arborization was mediated by up-regulation of brain-derived neurotrophic factor (BDNF) signaling and that the deletion of BDNF abrogated both neuronal arborization activation and memory enhancement. The activation of BDNF-dependent neuronal arborization generated almost 2-fold increases in synapse numbers in dendrites of pyramidal neurons and in neurites of nonpyramidal neurons. This increase in synaptic connections might have evoked reorganization within neuronal circuits and eventually supported an increase in the activity of such circuits. Thus, in addition to showing the potential of RGS14414 for rescuing memory deficits, our results suggest that a boost in circuit activity could facilitate memory enhancement and the reversal of memory deficits.-Masmudi-Martin, M., Navarro-Lobato, I., Lopez-Aranda, M. F., Delgado, G., Martin-Montanez, E., Quiros-Ortega, M. E., Carretero-Rey, M., Narvaez, L., Garcia-Garrido, M. F., Posadas, S., Lopez-Tellez, J. F., Blanco, E., Jimenez-Recuerda, I., Granados-Duran, P., Paez-Rueda, J., Lopez, J. C., Khan, Z. U. RGS14414 treatment induces memory enhancement and rescues episodic memory deficits.

Oxidative stress (OxS) is involved in the development of cell injures occurring in retinal diseases while Poly(ADP-ribose) Polymerase-1 (PARP-1) is a key protein involved in the repair of the DNA damage caused by OxS. Inhibition of PARP-1 activity with the pharmacological inhibitor PJ34 in mouse retinal explants subjected to H2O2-induced oxidative damage resulted in an increase of apoptotic cells. Reduction of cell growth was also observed in the mouse cone like cell line 661 W in the presence of PJ34 under OxS conditions. Mass spectrometry-based phosphoproteomics analysis performed in 661 W cells determined that OxS induced significant changes in the phosphorylation in 1807 of the 8131 peptides initially detected. Blockade of PARP-1 activity after the oxidative treatment additionally increased the phosphorylation of multiple proteins, many of them at SQ motifs and related to the DNA-damage response (DDR). These motifs are substrates of the kinases ATM/ATR, which play a central role in DDR. Western blot analysis confirmed that the ATM/ATR activity measured and the phosphorylation at SQ motifs of ATM/ATR substrates was augmented when PARP-1 activity was inhibited under OxS conditions, in 661 W cells. Phosphorylation of ATM/ATR substrates, including the phosphorylation of the histone H2AX were also induced in organotypic cultures of retinal explants subjected to PARP-1 inhibition during exposure to OxS. In conclusion, inhibition of PARP-1 increased the phosphorylation and hence the activation of several proteins involved in the response to DNA damage, like the ATM protein kinase. This finally resulted in an augmented injury in mouse retinal cells suffering from OxS. Therefore, the inhibition of PARP-1 activity may have a negative outcome in the treatment of retinal diseases in which OxS is involved.

Current drug discovery procedures require fast and effective quantification of the pharmacological response evoked in living cells by agonist compounds. In the case of G-protein coupled receptors (GPCRs), the efficacy of a particular drug to initiate the endocytosis process is related to the formation of endocytic vesicles or endosomes and their subsequent internalisation within intracellular compartments that can be observed with high spatial and temporal resolution by fluorescence microscopy techniques. Recently, an algorithm has been proposed to evaluate the pharmacological response by estimating the number of endosomes per cell on time series of images. However, the algorithm was limited by the dependence on some manually set parameters and in some cases the quality of the image does not allow a reliable detection of the endosomes. Here we propose a simple, fast and automated image analysis method-the Delta m algorithm- to quantify a pharmacological response with data obtained from fluorescence microscopy experiments. This algorithm does not require individual object detection and computes the relative increment of the third order moment in fluorescence microscopy images after filtering with the Laplacian of Gaussian function. It was tested on simulations demonstrating its ability to discriminate different experimental situations according to the number and the fluorescence signal intensity of the simulated endosomes. Finally and in order to validate this methodology with real data, the algorithm was applied to several time-course experiments based on the endocytosis of the mu opioid receptor (MOP) initiated by different agonist compounds. Each drug displayed a different Am sigmoid time-response curve and statistically significant differences were observed among drugs in terms of efficacy and kinetic parameters., The authors acknowledge financial support from the Spanish Project AYA2015-66357-R 288 (MINECO/FEDER).

© The Author(s) 2019., A better understanding of the molecular mechanisms that participate in the development and clinical manifestations of schizophrenia can lead to improve our ability to diagnose and treat this disease. Previous data strongly associated the levels of deregulated ADAMTS2 expression in peripheral blood mononuclear cells (PBMCs) from patients at first episode of psychosis (up) as well as in clinical responders to treatment with antipsychotic drugs (down). In this current work, we performed an independent validation of such data and studied the mechanisms implicated in the control of ADAMTS2 gene expression. Using a new cohort of drug-naïve schizophrenia patients with clinical follow-up, we confirmed that the expression of ADAMTS2 was highly upregulated in PBMCs at the onset (drug-naïve patients) and downregulated, in clinical responders, after treatment with antipsychotics. Mechanistically, ADAMTS2 expression was activated by dopaminergic signalling (D1-class receptors) and downstream by cAMP/CREB and mitogen-activated protein kinase (MAPK)/ERK signalling. Incubation with antipsychotic drugs and selective PKA and MEK inhibitors abrogated D1-mediated activation of ADAMTS2 in neuronal-like cells. Thus, D1 receptors signalling towards CREB activation might participate in the onset and clinical responses to therapy in schizophrenia patients, by controlling ADAMTS2 expression and activity. The unbiased investigation of molecular mechanisms triggered by antipsychotic drugs may provide a new landscape of novel targets potentially associated with clinical efficacy., This work was supported by: SAF2016-76046-R and SAF2013-46292-R (MINECO and FEDER) to B.C.F., PI16/00156 (isciii and FEDER) to J.P.V., LUCHAMOS POR LA VIDA project to F.R.J. and J.P.V., SAF2017-83702-R (MINECO and FEDER), Red TERCEL RD12/0019/0024 (ISCIII) and GVA-PROMETEO 2018/041 (Generalitat Valenciana) to S.M. J.P.V. is supported by the RyC research programme (RYC-2013-14097) and F.R.J. by the predoctoral research programme (BES-2014-070615), from MINECO and FEDER.

The neuropsychological effects of naturally occurring psychoactive chemicals have been recognized for millennia. Hallucinogens, which include naturally occurring chemicals such as mescaline and psilocybin, as well as synthetic compounds, such as lysergic acid diethylamide (LSD), induce profound alterations of human consciousness, emotion, and cognition. The discovery of the hallucinogenic effects of LSD and the observations that LSD and the endogenous ligand serotonin share chemical and pharmacological profiles led to the suggestion that biogenic amines like serotonin were involved in the psychosis of mental disorders such as schizophrenia. Although they bind other G protein-coupled receptor (GPCR) subtypes, studies indicate that several effects of hallucinogens involve agonist activity at the serotonin 5-HT2A receptor. In this chapter, we review recent advances in understanding hallucinogen drug action through characterization of structure, neuroanatomical location, and function of the 5-HT2A receptor.

Morphine and related opioids are the mainstay of analgesic treatment, especially in patients suffering chronic pain. Besides their antinociceptive effects they may also exhibit anxiolytic-like properties that could contribute to pain relief. The pharmacological manipulation of the serotonergic system may not only modulate pain transmission and processing but also other behavioral effects of opioids. The present study aimed to analyze the effect of the concurrent treatment with citalopram, a selective serotonin reuptake inhibitor, on the antinociceptive, locomotor and anxiety-related effects induced by acute and subchronic administration of morphine in mice. Citalopram (15 mg/kg) enhanced the acute antinociceptive effects of morphine when concurrently administered as evidenced by a two-fold increase in the ED for the antinociceptive effect of morphine in the hot-plate test. Chronic studies also revealed that concurrent citalopram treatment (15 mg/kg) delayed the development of tolerance to the thermal antinociceptive effects of morphine. Additionally, morphine-induced hyperlocomotion was potentiated by citalopram as assessed in the open-field test and in the spontaneous activity recording in the home cage, a behavioral outcome to which tolerance or desensitization was not developed. Interestingly, chronic administration of both drugs promoted an anxiolytic effect as evidenced by the increased central activity in the open field test. Future investigations on this pharmacological interaction, such as the possible translational research in clinics, might have consequences in future strategies for the therapeutic management of pain., This research was supported by the grant SAF2010-15663 from the Spanish Government (MINECO).

Ibi, Daisuke et al., Antipsychotic drugs remain the standard for schizophrenia treatment. Despite their effectiveness in treating hallucinations and delusions, prolonged exposure to antipsychotic medications leads to cognitive deficits in both schizophrenia patients and animal models. The molecular mechanisms underlying these negative effects on cognition remain to be elucidated. Here we demonstrate that chronic antipsychotic drug exposure increases nuclear translocation of NF-κB in both mouse and human frontal cortex, a trafficking event triggered via 5-HT-receptor-dependent downregulation of the NF-κB repressor IκBα. This upregulation of NF-κB activity led to its increased binding at the Hdac2 promoter, thereby augmenting Hdac2 transcription. Deletion of HDAC2 in forebrain pyramidal neurons prevented the negative effects of antipsychotic treatment on synaptic remodeling and cognition. Conversely, virally mediated activation of NF-κB signaling decreased cortical synaptic plasticity via HDAC2. Together, these observations may aid in developing therapeutic strategies to improve the outcome of schizophrenia treatment., NIH R01 MH084894 (J.G.M.), NIH R01 MH111940 (J.G.M.), Dainippon Sumitomo Pharma (J.G.M.), NARSAD (J.G.M.), the Japan Society for the Promotion of Science (JSPS) 15H06719 and 16K19786 (D.I.), NIH R01 MH104491 (G.W.H.), NIH R01 MH086509 (S.A.), NIH P50 MH096890 (S.A.), MINECO/ERDF SAF2009-08460 (J.J.M. and L.F.C.), SAF2013-45084R (J.J.M. and L.F.C.), Basque Government IT616-13 (J.J.M.), NIH R21 MH103877 (S.D.) and NIH R01 MH090264 (S.J.R.) participated in the funding of this study. RNA-seq analysis was supported in part through the computational resources and staff expertise provided by Scientific Computing at the Icahn School of Medicine at Mount Sinai and the NIH infrastructure grant S10OD018522. C.M. and A.G.B. were recipients of a postdoctoral and a predoctoral fellowship from the Basque Government, respectively. D.I. was a recipient of postdoctoral fellowships from JSPS (Young Scientists JSPS 23-3454) and the Uehara Memorial Foundation.

Animal models of schizophrenia constitute at this moment fundamental experimentation tools to further explore on the etiological basis of this pathology, leading to the discovery of new biological targets and innovative therapeutic strategies for its treatment., This study was funded by NIH R01 MH084894 and R01 MH111940 grants and SAF2010-20840-C02-01/02 and SAF2013-46292-R from MINECO (Spain

The neuropsychological effects of naturally occurring psychoactive chemicals have been recognized for millennia. Hallucinogens, which include naturally occurring chemicals such as mescaline and psilocybin, as well as synthetic compounds, such as lysergic acid diethylamide (LSD), induce profound alterations of human consciousness, emotion, and cognition. The discovery of the hallucinogenic effects of LSD and the observations that LSD and the endogenous ligand serotonin share chemical and pharmacological profiles led to the suggestion that biogenic amines like serotonin were involved in the psychosis of mental disorders such as schizophrenia. Although they bind other G protein-coupled receptor (GPCR) subtypes, studies indicate that several effects of hallucinogens involve agonist activity at the serotonin 5-HT2A receptor. In this chapter, we review recent advances in understanding hallucinogen drug action through characterization of structure, neuroanatomical location, and function of the 5-HT2A receptor.

El pdf del artículo es la versión post-print.-- et al., Serotonin and glutamate G protein-coupled receptor (GPCR) neurotransmission affects cognition and perception in humans and rodents. GPCRs are capable of forming heteromeric complexes that differentially alter cell signaling, but the role of this structural arrangement in modulating behavior remains unknown. Here, we identified three residues located at the intracellular end of transmembrane domain four that are necessary for the metabotropic glutamate 2 (mGlu2) receptor to be assembled as a GPCR heteromer with the serotonin 5-hydroxytryptamine 2A (5-HT2A) receptor in the mouse frontal cortex. Substitution of these residues (Ala-6774.40, Ala-6814.44, and Ala-6854.48) leads to absence of 5-HT2A·mGlu2 receptor complex formation, an effect that is associated with a decrease in their heteromeric ligand binding interaction. Disruption of heteromeric expression with mGlu2 attenuates the psychosis-like effects induced in mice by hallucinogenic 5-HT2A agonists. Furthermore, the ligand binding interaction between the components of the 5-HT2A·mGlu2 receptor heterocomplex is up-regulated in the frontal cortex of schizophrenic subjects as compared with controls. Together, these findings provide structural evidence for the unique behavioral function of a GPCR heteromer., This work was supported, in whole or in part, by National Institutes of Health Grants R01 MH084894 (to J.G.M.), R01 NS37731 (to D.L.B.), and P01 DA12923 (to S.C.S.). This work was also supported by Dainippon Sumi-tomo Pharma (to J.G.M.), National Alliance for Research on Schizophrenia and Depression (to J.G.M.), The Mortimer D. Sackler Foundation (to J.G.M.), Ministerio de Ciencia e Innovación Grant SAF2009-084609 (to J.J.M.), Basque Government Grant IT-199-07 (to J.J. M.), Consejo Superior de Investigaciones Científicas Grants PA1003176 and 200980I110 (to J.F.L.-G.), and Medical Research Council United Kingdom Grant G0900050 (to G.M.). Recipient of a predoctoral fellowship from University of the Basque Country, Spain. Recipient of a postdoctoral fellowship from Fundacion Alicia Koplowitz, Spain. Recipient of a predoctoral fellowship from Consejo Superior de Investigaciones Científicas, Spain.

PMCID: PMC4819166.-- Moreno et al., Heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs) can form multiprotein complexes (heteromers), which can alter the pharmacology and functions of the constituent receptors. Previous findings demonstrated that the G-coupled serotonin 5-HTA receptor and the G-coupled metabotropic glutamate 2 (mGlu2) receptor-GPCRs that are involved in signaling alterations associated with psychosis-assemble into a heteromeric complex in the mammalian brain. In single-cell experiments with various mutant versions of the mGlu2 receptor, we showed that stimulation of cells expressing mGlu2-5-HT heteromers with an mGlu2 agonist led to activation of G proteins by the 5-HTA receptors. For this crosstalk to occur, one of the mGlu2 subunits had to couple to G proteins, and we determined the relative location of the G-contacting subunit within the mGlu2 homodimer of the heteromeric complex. Additionally, mGlu2-dependent activation of G, but not G, was reduced in the frontal cortex of 5-HT knockout mice and was reduced in the frontal cortex of postmortem brains from schizophrenic patients. These findings offer structural insights into this important target in molecular psychiatry., This work was supported, in whole or in part, by the NIH grants R01MH084894 and R56MH084894 (to J.G.-M.), R01HL59949 (to D.E.L.), R37AG017926 and R01AG008200 (to N.K.R.), R01DA025036 and R01DA027460 (to J.A.M.), R01NS047229 and P50AG05138 (to A.G.), and S10RR027411 (to M.C.). This work was also supported by Dainippon Sumitomo Pharma (to J.G.-M.), Spanish MINECO/EDR Funds SAF2009-68460 and SAF2013-48586R (to J.J.M.), the Basque Government (to J.J.M.), the Spanish Government SAF2010-15663 grant (MICINN) (to J.F.L.G.), and Medical Research Council (UK) grants MR/L023806/1 and G0900050 (to G.M.). P.M.-A. and A.G.-B. were recipients of predoctoral fellowships from UPV/EHU and the Basque Government in Spain, respectively.

A common way to experimentally characterize a linear time-invariant acoustic system is by measuring its impulse response for each location of interest. Currently several methods exist for such purpose, signal deconvolution being the one which exhibits best performance. Moreover, measurement systems are usually aimed to particular applications, working with expensive platforms and proprietary software. This paper describes design and development of specific software, which manages an automatic measurement system for acoustic devices. SAMSoft presents a modular, scalable and easy to upgrade design developed in MATLab, based on the model-view-control system pattern which enables source code reuse and facilitates further development. This software allows impulse response measurement with different excitation signals, user-selected processing intervals, time and frequency domain visualization, and spectral analysis in octave and one-third octave bands. It runs on hardware consisting of a control unit and a mobile platform, and is capable of measuring 360° in the horizontal plane with an angular resolution of up to 0.06°. Impulse responses measured from different acoustic transducers showed a signal noise ratio of up to 40 dB in frequency bands under 100 Hz. In power measurements for octave and one-third octave bands a maximum error of 0.12 dB was obtained.A common way to experimentally characterize a linear time-invariant acoustic system is by measuring its impulse response for each location of interest. Currently several methods exist for such purpose, signal deconvolution being the one which exhibits best performance. Moreover, measurement systems are usually aimed to particular applications, working with expensive platforms and proprietary software. This paper describes design and development of specific software, which manages an automatic measurement system for acoustic devices. SAMSoft presents a modular, scalable and easy to upgrade design developed in MATLab, based on the model-view-control system pattern which enables source code reuse and facilitates further development. This software allows impulse response measurement with different excitation signals, user-selected processing intervals, time and frequency domain visualization, and spectral analysis in octave and one-third octave bands. It runs on hardware consisting of a control unit and a mob...

Facial emotional expression constitutes a basic guide in the social interaction and, thus, the alterations in its expression or recognition imply an important limitation for the communication. On the other hand, cognitive impairment and the presence of depressive symptoms, which are commonly found in patients with multiple sclerosis, it is unknown how they influence cognitive function and depression on emotional recognition.To consider the evaluation of time reaction and response accuracy of facial expression recognition in people affected by multiple sclerosis, and to assess the possible variables that may be modulating the emotion recognition, such as depression and cognitive functions.The study has a cross-sectional non-experimental design with a single measurement. The sample is compound by 85 participants, 45 diagnosed as multiple sclerosis and 40 control subjects.Multiple sclerosis subjects reveal significant differences in both reaction time and response accuracy in neuropsychological tests in comparison to the control group. Explanatory models were identified in the emotional recognition.Multiple sclerosis subjects face difficulties at recognising facial emotions; and differences at attention memory, processing speed and depressive symptomatology were observed in regard to the control group.Precision y tiempo de reaccion en el reconocimiento de emociones faciales en personas con esclerosis multiple.Introduccion. La expresion facial emocional constituye una guia basica en la interaccion social y, por lo tanto, las alteraciones en su expresion o reconocimiento implican una limitacion importante para la comunicacion. Por otro lado, el deterioro cognitivo y la presencia de sintomas depresivos, que se encuentran comunmente en los pacientes con esclerosis multiple, no se sabe como influyen en el reconocimiento emocional. Objetivo. Considerar la evaluacion del tiempo de reaccion y precision en la respuesta de reconocimiento de expresiones faciales de las personas afectadas por esclerosis multiple y valorar las posibles variables que pueden modular el reconocimiento de emociones, como la depresion y las funciones cognitivas. Sujetos y metodos. El estudio tiene un diseño no experimental transversal con una sola medicion. La muestra esta compuesta por 85 participantes, 45 con diagnostico de esclerosis multiple y 40 sujetos control. Resultados. Los sujetos con esclerosis multiple revelaban diferencias significativas tanto en el tiempo de reaccion y la precision de respuesta en pruebas neuropsicologicas en comparacion con el grupo control. Se identificaron modelos explicativos en el reconocimiento emocional. Conclusion. Los sujetos con esclerosis multiple se enfrentan a dificultades en el reconocimiento de emociones faciales, y se observaron diferencias en la memoria, atencion, velocidad de procesamiento y sintomatologia depresiva en relacion con el grupo control.

Calcium flux through L-type voltage-activated calcium (Cav1) channels is crucial for regulating brain functions including memory formation and behavior. Alterations in Ca²+ homeostasis have been linked to many cognitive disorders, and understanding the regulation of this process is crucial for their remedy. Therefore, here, we have evaluated the effect of a multifunctional protein known to be involved in memory functions called regulator of G-protein signaling 14 (RGS-14) on Cav1 channel activity in neuronal cell lines NG108-15 and SH-SY5Y. RGS-14 protein produced significant reduction in Ca²+ influx in both cell lines and this effect was dependent on nifedipine-sensitive Cav1 channels. Thus, our results provide evidence supporting the idea that RGS-14 may facilitate the cognitive processing by modulating Cav1 channel-mediated intracellular Ca²+ transients.

Treatment with dopamine and other dopamine D2 receptor agonists has been shown to induce cell death through activation of caspase-3 pathway. However, initial step that leads to the activation of caspase-3 in D2 receptor-mediated apoptotic pathway remains unclear. Recently, it was shown that a spliced variant of Galphai2 protein (sGalphai2) forms intracellular complex with D2 receptors by protein-protein interaction and that D2 drugs treatment causes the liberation of sGalphai2 protein from complex. Now, we show that the unbound form of sGalphai2 protein is able to activate caspase-3 pathway in baby hamster kidney (BHK) cells. Expression of sGalphai2 protein in BHK cells led to the production of active form of caspase-3 and activation of p38 mitogen-activated protein kinase (p38 MAPK) and extracellular regulated kinase 1/2 (ERK1/2). Co-expression of sGalphai2 with either D2 short (D2S) or D2 long (D2L) isoforms of dopamine D2 receptors blocked the activation of caspase-3 pathway. Thus, our results demonstrate that high level of unbound sGalphai2 protein can affect the cell survival and engagement of this protein with D2 receptors can block this process. It is suggested that this process may be a crucial step in the initiation of D2 receptor-mediated cellular apoptosis through this pathway.

The function of sGalphai2 protein in central nervous system is not well understood. Therefore to explore the possible role of this protein in postnatal brain development, we have analyzed the protein expression pattern of brain obtained from rats of postnatal day 0 (P0) to P90 by dot-blots and immunocytochemistry techniques. In dot-blots, both nuclear and membrane fractions showed a gradual decrease from P0 to P60. Highest protein level was observed at the age of P0. There was also a trend of decline in the sGalphai2 protein from P0 to P90 in brain sections stained by immunocytochemistry method. At P0, the protein labeling was highest in cerebral cortex, hippocampus, cerebellum and mitral cell layer. In cerebral cortex, a drop in the immunolabeling of sGalphai2 protein was observed at P3, which was significantly increased at the age of P5. However, in striatum and olfactory tubercle, it was maintained through P0-P10 and P0-P5, respectively. Thalamus was one of the areas where labeling was not as strong as cortex, hippocampus or striatum. In contrary to other areas, immunostaining of sGalphai2 in corpus-callosum and lacunosum-molecular was not seen at P0 and appeared in advanced postnatal ages. A detectable level of sGalphai2 protein was observed at P5 in carpus-callosum and at P20 in lacunosum-molecular. A high level of sGalphai2 protein in the period when cellular layer organization and synaptic innervations, synaptic connections and maturation take place, suggests for a potential role of this protein in the early postnatal brain development.

Approaches to identify G protein-coupled receptor oligomers rather than dimers have been lacking. Using concatamers of fluorescent proteins, we established conditions to monitor sequential three-color fluorescence resonance energy transfer (3-FRET) and used these to detect oligomeric complexes of the alpha(1b)-adrenoceptor in single living cells. Mutation of putative key hydrophobic residues in transmembrane domains I and IV resulted in substantial reduction of sequential 3-FRET and was associated with lack of protein maturation, prevention of plasma membrane delivery, and elimination of signaling function. Although these mutations prevented cell surface delivery, bimolecular fluorescence complementation studies indicated that they did not ablate protein-protein interactions and confirmed endoplasmic reticulum/Golgi retention of the transmembrane domain I plus transmembrane domain IV mutated receptor. The transmembrane domain I plus transmembrane domain IV mutated receptor was a "dominant-negative" in blocking cell surface delivery of the wild-type receptor. Mutations only in transmembrane domain I did not result in a reduction in 3-FRET, whereas restricting mutation to transmembrane domain IV did result in reduced 3-FRET. Mutations in either transmembrane domain I or transmembrane domain IV, however, were sufficient to eliminate cell surface delivery. Terminal N-glycosylation is insufficient to determine cell surface delivery because both transmembrane domain I and transmembrane domain IV mutants matured as effectively as the wild-type receptor. These data indicate that the alpha(1b)-adrenoceptor is able to form oligomeric rather than only simple dimeric complexes and that disruption of effective oligomerization by introducing mutations into transmembrane domain IV has profound consequences for cell surface delivery and function.

This is an open access article distributed under the terms of the Creative Commons Attribution License., The activation of G-protein coupled receptors by agonist compounds results in diverse biological responses in cells, such as the endocytosis process consisting in the translocation of receptors from the plasma membrane to the cytoplasm within internalizing vesicles or endosomes. In order to functionally evaluate endocytosis events resulted from pharmacological responses, we have developed an image analysis method -the Q-Endosomes algorithm- that specifically discriminates the fluorescent signal originated at endosomes from that one observed at the plasma membrane in images obtained from living cells by fluorescence microscopy. Mu opioid (MOP) receptor tagged at the carboxy-terminus with yellow fluorescent protein (YFP) and permanently expressed in HEK293 cells was used as experimental model to validate this methodology. Time-course experiments performed with several agonists resulted in different sigmoid curves depending on the drug used to initiate MOP receptor endocytosis. Thus, endocytosis resulting from the simultaneous activation of co-expressed MOP and serotonin 5-HT2C receptors by morphine plus serotonin was significantly different, in kinetics as well as in maximal response parameters, from the one caused by DAMGO, sufentanyl or methadone. Therefore, this analytical tool permits the pharmacological characterization of receptor endocytosis in living cells with functional and temporal resolution., This work was supported by the SAF2010-15663 grant from the Spanish Government (MINECO).

The structural basis of the quaternary organization of rhodopsin has recently been explored and modeled. Because information obtained from studying rhodopsin has frequently been directly applicable to other G protein-coupled receptors we wished to ascertain if dimeric and/or oligomeric forms of the alpha(1b)-adrenoceptor could be observed and if so whether rhodopsin might provide insights into the quaternary structure of this receptor. Co-immunoprecipitation and both conventional and time-resolved fluorescence resonance energy transfer studies demonstrated quaternary structure of the alpha(1b)-adrenoceptor and, in concert with the reconstitution of fragments of this receptor, provided information on the molecular basis of these interactions. Development of three color fluorescence resonance energy transfer (FRET) allowed the imaging of alpha(1b)-adrenoceptor oligomers in single living cells. Mutation of hydrophobic residues in transmembrane domains I and IV of the receptor resulted in marked reduction in three color FRET suggesting an alteration in oligomeric organization and potential similarities with rhodopsin. The mutated alpha(1b)-adrenoceptor was unable to reach the cell surface, did not become terminally N-glycosylated and was unable to signal.

At advanced stages, Alzheimer’s disease (AD) is characterized by an extensive neuronal loss. However, the early neurodegenerative deficiencies have not been yet identified. Here we report an extensive, selective and early neurodegeneration of the dendritic inhibitory interneurons (oriens-lacunosum moleculare, O-LM, and hilar perforant path-associated, HIPP, cells) in the hippocampus of a transgenic PS1 × APP AD model. At 6 months of age, from 22 different pre- and postsynaptic mRNA markers tested (including GABAergic, glutamatergic and cholinergic markers), only the expression of somatostatin (SOM) and NPY neuropeptides (O-LM and HIPP markers) displayed a significant decrease. Stereological cell counting demonstrated a profound diminution (50–60%) of SOM-immunopositive neurons, preceding the pyramidal cell loss in this AD model. SOM population co-expressing NPY was the most damaged cell subset. Furthermore, a linear correlation between SOM and/or NPY deficiency and Abeta content was also observed. Though the molecular mechanism of SOM neuronal loss remains to be determined, these findings might represent an early hippocampal neuropathology. Therefore, SOM and NPY neuropeptides could constitute important biomarkers to assess the efficacy of potential early AD treatments.

Three distinct genes encode α1-adrenoceptors. Although homodimers of each subtype have been reported, certain but not all combinations of heterodimers of the α1-adrenoceptors appear to form. Key studies in this field are reviewed and the approaches that have been applied to monitoring the selectivity and the basis of α1-adrenoceptor dimerization are discussed.

Combinations of coimmunoprecipitation, single-cell fluorescence resonance energy transfer, and cell-surface time-resolved fluorescence resonance energy transfer demonstrated protein-protein interactions and quaternary structure for the alpha(1b)-adrenoceptor. Self-association of transmembrane domain 1 and its interaction with the full-length receptor indicated a symmetrical interface provided by this domain. Lack of effect of mutation of the glycophorin-A dimerization-like region within this helix demonstrated that this did not provide the molecular mechanism. Multiple interactions were observed between the alpha(1b)-adrenoceptor and fragments derived from its sequence. Fragments comprising transmembrane domains 3 and 4 and transmembrane domains 5 and 6, but not transmembrane domain 7, were also able to interact with the full-length receptor. Transmembrane domain 7 failed to interact significantly with any element of the receptor and was not transported to the cell surface after coexpression with the full-length receptor. Symmetrical interactions were also noted between fragments incorporating transmembrane domain 4, but this segment of the receptor failed to interact with transmembrane domains 1 and 2 or transmembrane domains 5 and 6. Time-resolved fluorescence resonance energy transfer studies were also consistent with contributions of transmembrane domains 1 and/or 2 and transmembrane domains 3 and/or 4 to protein-protein interactions within the quaternary structure of the alpha(1b)-adrenoceptor, and with a contribution of transmembrane domains 5 and/or 6. These data are consistent with a complex oligomeric quaternary structure of the alpha(1b)-adrenoceptor in which major, symmetrical interactions may define intradimeric contacts with other contributions, providing interdimer contacts to generate oligomeric complexes akin to those observed for murine rhodopsin. A model derived from this was developed.

Using combinations of bioluminescence resonance energy transfer, time-resolved fluorescence resonance energy transfer and the functional complementation of pairs of inactive receptor-G protein fusion proteins, the human alpha(1A-1)-adrenoceptor was shown to form homodimeric/oligomeric complexes when expressed in human embryonic kidney (HEK) 293 cells. Saturation bioluminescence resonance energy transfer studies indicated the alpha(1A-1)-adrenoceptor homodimer interactions to be high affinity and some 75 times greater than interactions between the alpha(1A-1)-adrenoceptor and the delta opioid peptide receptor. Only a fraction of the alpha(1A-1)-adrenoceptors was at the plasma membrane of HEK293 cells at steady state. However, dimers of alpha(1A-1)-adrenoceptors were also present in intracellular membranes, and the dimer status of those delivered to the cell surface was unaffected by the presence of agonist. Splice variation can generate at least three forms of the human alpha(1A-1)-adrenoceptor with differences limited to the C-terminal tail. Each of the alpha(1A-1), alpha(1A-2a), and alpha(1A-3a)-adrenoceptor splice variants formed homodimers/oligomers, and all combinations of these splice variants were able to generate heterodimeric/oligomeric interactions. Despite the coexpression of these splice variants in human tissues that possess the pharmacologically defined alpha(1L)-adrenoceptor binding site, coexpression of any pair in HEK293 cells failed to generate ligand binding characteristic of the alpha(1L)-adrenoceptor.

Despite the importance of glutamatergic signalling in the co-ordination of hormone secretion, the identity of the enzyme for the production of glutamate in β-cells is still unresolved. We have found that the endocrine pancreas co-expresses two isoforms of GA (glutaminase), denoted as kidney-type (KGA) and liver-type (LGA), with a complementary cellular pattern of expression. Whereas KGA was mainly present in α-cells, LGA was very abundant in β-cells. This spatial segregation may have important functional implications, facilitating a differential regulation of glutamate production in insulin- and glucagon-secreting cells.

G-protein-coupled receptors (GPCRs) represent the largest family of receptors involved in transmembrane signaling. Although these receptors were generally believed to be monomeric entities, accumulating evidence supports the presence of GPCRs in multimeric forms. Here, using immunoprecipitation as well as time-resolved fluorescence resonance energy transfer to assess protein-protein interactions in living cells, we unambiguously demonstrate the occurrence of dimerization of the human histamine H(1) receptor. We also show the presence of domain-swapped H(1) receptor dimers in which there is the reciprocal exchange of transmembrane domain TM domains 6 and 7 between the receptors present in the dimer. Mutation of aspartate(107) in transmembrane (TM) 3 or phenylalanine(432) in TM6 to alanine results in two radioligand-binding-deficient mutant H(1) receptors. Coexpression of H(1)D(107) A and H(1)F(432)A, however, results in a reconstituted radioligand binding site that exhibits a pharmacological profile that corresponds to the wild-type H(1) receptor. Interestingly, the H(1) receptor radioligands [(3)H]mepyramine and [(3)H]-(-)-trans-1-phenyl-3-N,N-dimethylamino-1,2,3,4-tetrahydronaphthalene show differential saturation binding values (B(max)) for wild-type H(1) receptors but not for the radioligand binding site that is formed upon coexpression of H(1) D(107)A and H(1) F(432)A receptors, suggesting the presence of different H(1) receptor populations.

Human D2Long (D2L) and D2Short (D2S) dopamine receptor isoforms were modified at their N-terminus by the addition of a human immunodeficiency virus (HIV) or a FLAG epitope tag. The receptors were then expressed in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus system, and their oligomerization was investigated by means of co-immunoprecipitation and time-resolved fluorescence resonance energy transfer (FRET). [3H]Spiperone labelled D2 receptors in membranes prepared from Sf9 cells expressing epitope-tagged D2L or D2S receptors, with a pKd value of approximately 10. Co-immunoprecipitation using antibodies specific for the tags showed constitutive homo-oligomerization of D2L and D2S receptors in Sf9 cells. When the FLAG-tagged D2S and HIV-tagged D2L receptors were co-expressed, co-immunoprecipitation showed that the two isoforms can also form hetero-oligomers in Sf9 cells. Time-resolved FRET with europium and XL665-labelled antibodies was applied to whole Sf9 cells and to membranes from Sf9 cells expressing epitope-tagged D2 receptors. In both cases, constitutive homo-oligomers were revealed for D2L and D2S isoforms. Time-resolved FRET also revealed constitutive homo-oligomers in HEK293 cells expressing FLAG-tagged D2S receptors. The D2 receptor ligands dopamine, R-(-)propylnorapomorphine, and raclopride did not affect oligomerization of D2L and D2S in Sf9 and HEK293 cells. Human D2 dopamine receptors can therefore form constitutive oligomers in Sf9 cells and in HEK293 cells that can be detected by different approaches, and D2 oligomerization in these cells is not regulated by ligands.