RESUMO
BACKGROUND: Basolateral amygdala (BLA) excitatory projections to medial prefrontal cortex (PFC) play a key role controlling stress behavior, pain, and fear. Indeed, stressful events block synaptic plasticity at the BLA-PFC circuit. The stress responses involve the action of corticotrophin releasing factor (CRF) through type 1 and type 2 CRF receptors (CRF1 and CRF2). Interestingly, it has been described that dopamine receptor 1 (D1R) and CRF peptide have a modulatory role of BLA-PFC transmission. However, the participation of CRF1 and CRF2 receptors in BLA-PFC synaptic transmission still is unclear. METHODS: We used in vivo microdialysis to determine dopamine and glutamate (GLU) extracellular levels in PFC after BLA stimulation. Immunofluorescence anatomical studies in rat PFC synaptosomes devoid of postsynaptic elements were performed to determine the presence of D1R and CRF2 receptors in synaptical nerve endings. RESULTS: Here, we provide direct evidence of the opposite role that CRF receptors exert over dopamine extracellular levels in the PFC. We also show that D1R colocalizes with CRF2 receptors in PFC nerve terminals. Intra-PFC infusion of antisauvagine-30, a CRF2 receptor antagonist, increased PFC GLU extracellular levels induced by BLA activation. Interestingly, the increase in GLU release observed in the presence of antisauvagine-30 was significantly reduced by incubation with SCH23390, a D1R antagonist. CONCLUSION: PFC CRF2 receptor unmasks D1R effect over glutamatergic transmission of the BLA-PFC circuit. Overall, CRF2 receptor emerges as a new modulator of BLA to PFC glutamatergic transmission, thus playing a potential role in emotional disorders.
Assuntos
Complexo Nuclear Basolateral da Amígdala/metabolismo , Dopamina/metabolismo , Ácido Glutâmico/metabolismo , Córtex Pré-Frontal/metabolismo , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Receptores de Dopamina D1/metabolismo , Animais , Masculino , Microdiálise , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND AND PURPOSE: Dopamine and corticotrophin-releasing hormone (CRH; also known as corticotrophin-releasing factor) are key neurotransmitters in the interaction between stress and addiction. Repeated treatment with cocaine potentiates glutamatergic transmission in the rat basolateral amygdala/cortex pathway through a synergistic action of D1 -like dopamine receptors and CRH type-2α receptors (CRF2 α receptors). We hypothesized that this observed synergism could be instrumented by heteromers containing the dopamine D1 receptor and CRF2 α receptor. EXPERIMENTAL APPROACH: D1 /CRF2 α receptor heteromerization was demonstrated in HEK293T cells using co-immunoprecipitation, BRET and FRET assays, and by using the heteromer mobilization strategy. The ability of D1 receptors to signal through calcium, when singly expressed or co-expressed with CRF2 α receptors, was evaluated by the calcium mobilization assay. KEY RESULTS: D1 /CRF2 α receptor heteromers were observed in HEK293T cells. When singly expressed, D1 receptors were mostly located at the cell surface whereas CRF2 α receptors accumulated intracellularly. Interestingly, co-expression of both receptors promoted D1 receptor intracellular and CRF2 α receptor cell surface targeting. The heteromerization of D1 /CRF2 α receptors maintained the signalling through cAMP of both receptors but switched D1 receptor signalling properties, as the heteromeric D1 receptor was able to mobilize intracellular calcium upon stimulation with a D1 receptor agonist. CONCLUSIONS AND IMPLICATIONS: D1 and CRF2 α receptors are capable of heterodimerization in living cells. D1 /CRF2 α receptor heteromerization might account, at least in part, for the complex physiological interactions established between dopamine and CRH in normal and pathological conditions such as addiction, representing a new potential pharmacological target.
Assuntos
Membrana Celular/metabolismo , Receptores de Hormônio Liberador da Corticotropina/metabolismo , Receptores de Dopamina D1/metabolismo , Cálcio/metabolismo , Hormônio Liberador da Corticotropina/metabolismo , Dopamina/metabolismo , Células HEK293 , Humanos , Imunoprecipitação , Transdução de SinaisRESUMO
Urocortin 1, highly conserved metazoan gene of the corticotropin-releasing hormone family, is a simple gene structured in two exons and the corresponding intron. The urocortin 1 prepropeptide is entirely coded in the second exon. Preliminary non-isotopic in situ hybridization experiments with an oligonucleotide complementary to an intron sequence of the urocortin 1 gene showed a significant cytoplasmic-like staining, suggesting the occurrence of an intron-retained urocortin 1 transcript. This observation prompted us to study whether the urocortin 1 gene presents alternative splicing by intron retention event. Confocal fluorescent in situ hybridization for urocortin 1 RNA and the use of the specific DNA dye TOPRO-3 allowed us to show significant expression of the intron-retained urocortin 1 transcript that did not colocalize with TOPRO-3 staining indicating a cytoplasmic localization for the intron-retained urocortin 1 transcript. The natural occurrence of a polyadenylated intron-retained urocortin 1 RNA was further documented by reverse transcriptase polymerase chain reaction (PCR), primed with oligo(dT), of total RNA extracted from three brain regions, a midbrain region containing the Edinger-Westphal nucleus, cerebellum and prefrontal cortex. In the three brain regions studied, it was possible to amplify both intron-less as well as intron-retained urocortin 1 transcripts. The use of PCR primers that simultaneously amplify both urocortin 1 transcripts allowed us to show that the expression of both urocortin 1 transcripts differs among the brain regions analyzed, suggesting a tissue specific regulation of this alternative splicing. In silico analysis of the five known mammalian urocortin 1 genomic sequences showed high conservation of the urocortin 1 intron sequence. Further studies should investigate the regulation of this intron retention event and its consequence for the functionality of the urocortin 1 gene.
Assuntos
Processamento Alternativo/genética , Hormônio Liberador da Corticotropina/genética , Variação Genética , Íntrons/genética , Animais , Cerebelo/metabolismo , Hormônio Liberador da Corticotropina/fisiologia , Masculino , Mesencéfalo/metabolismo , Córtex Pré-Frontal/metabolismo , RNA/biossíntese , RNA/genética , Ratos , Ratos Sprague-Dawley , UrocortinasRESUMO
Extracellular levels of norepinephrine (NE) and glutamate (Glu) in the ventral bed nucleus of the stria terminalis (vBNST) of saline- and chronic morphine-treated rats, with or without withdrawal, were studied by means of the in vivo microdialysis technique in anesthetized rats. In addition, the tissue concentration of NE was studied at different rostrocaudal levels of the vBNST. Chronic morphine treatment significantly increased extracellular levels of NE, but not Glu, in vBNST. At 48 h after naloxone-induced morphine withdrawal there was a further significant increase in the extracellular levels of NE, but not Glu, in vBNST. The presence of UK 14304, an alpha(2)-adrenergic agonist, induced a significant decrease in NE extracellular levels in all experimental groups. In contrast, UK 14304 induced a significant decrease in Glu extracellular levels only in saline-treated rats. The results also show that the vBNST presents a rostrocaudal gradient of NE and contains 9.4% of total brain NE. The increase in NE extracellular levels in vBNST induced by chronic morphine treatment and the further increase in NE levels 48 h after naloxone-induced morphine withdrawal suggest that NE in vBNST may be involved in the pharmacological effects of chronic morphine and withdrawal.
Assuntos
Encéfalo/metabolismo , Ácido Glutâmico/metabolismo , Dependência de Morfina/metabolismo , Norepinefrina/metabolismo , Núcleos Septais/metabolismo , Síndrome de Abstinência a Substâncias/metabolismo , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Tartarato de Brimonidina , Espaço Extracelular/efeitos dos fármacos , Espaço Extracelular/metabolismo , Técnicas In Vitro , Masculino , Microdiálise , Morfina/farmacologia , Quinoxalinas/farmacologia , Ratos , Ratos Sprague-Dawley , Núcleos Septais/efeitos dos fármacosRESUMO
The bed nucleus of the stria terminalis pars ventralis (vBNST) receives dense noradrenergic terminals and contains the highest concentration of noradrenaline (NA) in the brain. We used autoradiography following retrograde axonal transport of [(3)H]-NA to identify selectively whether noradrenergic neurons innervating the vBNST originate in the medulla oblongata and/or the locus coeruleus. In combination with this technique, non-isotopic in situ hybridization for the NMDA-NR1 receptor subunit mRNA was used to examine, on the same brain sections, its expression in noradrenergic neurons that innervate the vBNST. The results showed that 60 +/- 6% and 35 +/- 7% of the total number of radiolabeled cells detected after injection of [(3)H]-NA in the vBNST were located in brainstems A1 and A2 noradrenergic cell groups, respectively. In addition, 18.5 +/- 4.2% of radiolabeled cells in A1 and 15.7 +/- 5% in A2 also expressed the mRNA for the NMDA-NR1 receptor subunit. In contrast, only 4 +/- 3% of the radiolabeled cells were present in the locus coeruleus, and none of these cells was positive to NMDA-NR1 receptor subunit mRNA. The present results provide evidence that BNST noradrenergic fibers and terminals originate predominantly from A1 and A2 noradrenergic cell groups, and that a significant number of these noradrenergic neurons also express the mRNA for the NMDA-NR1 receptor subunit. The observation that brainstem noradrenergic neurons innervating the vBNST express NMDA receptor mRNA gives anatomical support to the regulation of NA release by NMDA presynaptic receptors.
Assuntos
Neurônios/metabolismo , Norepinefrina/metabolismo , Receptores de N-Metil-D-Aspartato/genética , Núcleos Septais/citologia , Simpatomiméticos/metabolismo , Animais , Autorradiografia , Expressão Gênica/fisiologia , Hibridização In Situ , Masculino , Neurônios/química , Norepinefrina/farmacologia , RNA Mensageiro/análise , Ensaio Radioligante , Núcleos da Rafe/química , Núcleos da Rafe/citologia , Ratos , Ratos Sprague-Dawley , Receptores de N-Metil-D-Aspartato/análise , Núcleos Septais/química , Simpatomiméticos/farmacologia , TrítioRESUMO
The participation of N-methyl-d-aspartate (NMDA) receptors on dopamine (DA) efflux in the striatum of anaesthetized rats, which had their DA nigrostriatal pathway previously lesioned with different doses of 6-hydroxydopamine (6-OH-DA), was assessed by in vivo microdialysis methodology. In addition, the in vivo basal DA and dihydroxy-phenyl-acetic acid (DOPAC) effluxes and the effect of local K+-depolarization on DA release were also evaluated in the striatum of these 6-OH-DA treated rats. Lesioned rats were divided in three groups corresponding to animals with 25-75%, 75-95% and >95% of striatum tissue DA depletion, respectively. Striatal DA tissue depletion between 25-75% occurred in parallel with a 30% reduction in DA extracellular levels, with a moderate 10% increase in basal fractional DA efflux, and with no statistical changes in the fractional DA efflux induced by NMDA (500 microM) receptor stimulation by reverse dialysis. Rats with higher DA tissue depletion (between 75-95%) exhibited a 60% reduction in DA extracellular levels in the striatum and this reduction occurred in parallel with a modest rise in basal fractional DA efflux, but with a striking decrease in the NMDA-induced fractional DA efflux. In rats with extreme or >95% of striatal DA tissue depletion, basal fractional DA efflux in the striatum increased quite substantially along with a recovery in the ability of NMDA receptor stimulation to induce fractional DA release. The >95% striatal DA-depleted rats also exhibited a significant decrease in tissue and extracellular DOPAC/DA ratio when compared to sham and partially DA-depleted rats. In contrast to the previous results, fractional DA efflux induced by reverse dialysis with K+ (40 mM) remained the same in the striatum of sham and all groups of DA-tissue depleted rats. The present findings suggest the existence of at least three features associated to the regulation of basal and NMDA-induced extracellular levels of DA in the striatum of rats as a function of striatal tissue DA depletion produced by 6-OH-DA. They also support the view that a differential regulation of basal and NMDA-induced DA extracellular levels occur in partial and extreme DA-depleted striatum after 6-OH-DA treatment. Such findings may have implications as regard to the participation of the NMDA receptor in the compensatory mechanisms associated to the progress of Parkinson's disease, as well as in the therapeutic treatment of this neurological disorder.
Assuntos
Corpo Estriado/citologia , Corpo Estriado/metabolismo , Dopamina/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Substância Negra/citologia , Ácido 3,4-Di-Hidroxifenilacético/metabolismo , Animais , Química Encefálica/efeitos dos fármacos , Corpo Estriado/química , Denervação , Maleato de Dizocilpina/farmacologia , Antagonistas de Aminoácidos Excitatórios/farmacologia , Espaço Extracelular/química , Espaço Extracelular/metabolismo , Masculino , Microdiálise , Vias Neurais , Oxidopamina , Potássio/farmacologia , Ratos , Ratos Sprague-Dawley , SimpatolíticosRESUMO
Antisense digoxigenin-labeled deoxyoligonucleotides probes and non-isotopic in situ hybridization (HIS) techniques have been used to explore the NMDA-NR1 receptor subunit mRNA distribution in different brain areas of rats which had their dopaminergic nigrostriatal pathway previously lesioned with intracerebral administration of 6-OH-dopamine (6-OH-DA). Intense and significant hybridization signals for NR1 mRNA were found in dentate gyrus and regions CA1-CA2-CA3 of the hippocampus, in layers II-III and V-VI of the cerebral cortex, and in the cerebellum of sham-treated rats. Basal ganglia structures such as the striatum exhibited few NR1 mRNA hybridization signals as compared to the hippocampus and cerebral cortex. In contrast, both zona compacta and reticulata of substantia nigra (SN) showed a reduced number of cells with nevertheless intense NR1 mRNA HIS signals. The NR1 mRNA distribution in the brain was affected in a brain regional selective manner by 6-OH-DA induced lesions of DA neuronal systems. A striking increase in NR1 mRNA HIS signals was observed in both striata after unilateral lesioning with 6-OH-DA. Instead, in SN compacta but not in reticulata, a moderate but significant bilateral reduction of NR1 mRNA was observed after unilateral 6-OH-DA injection. No significant changes in NR1 mRNA were detected in cerebral cortex and other brain regions after 6-OH-DA treatment. These studies, and others reported in the literature, support the view that extensive lesions of nigrostriatal DA-containing neurons in the brain may trigger compensatory or adaptative responses in basal ganglia structures such as striatum and substantia nigra which involve glutamateric neurons and the genic expression of NMDA receptors.
Assuntos
Encéfalo/efeitos dos fármacos , Fragmentos de Peptídeos/genética , RNA Mensageiro/biossíntese , Receptores de N-Metil-D-Aspartato/química , Animais , Encéfalo/metabolismo , Hibridização In Situ/métodos , Técnicas In Vitro , Masculino , Neurotoxinas , Oxidopamina , Ratos , Ratos Sprague-DawleyRESUMO
After 5 weeks of haloperidol, positive symptoms in drug-naive schizophrenic patients substantially subsided. Negative symptoms, although with a different temporal pattern, decreased after the fifth week of haloperidol treatment; specifically, a decrease was seen in anhedonia and affective flattening, whereas avolition-apathy and attentional impairment presented no changes. Alogia showed a decrease during the third week and a trend to return to placebo scores during weeks 4 and 5. Changes in affective flattening, alogia and attentional impairment correlated with changes in positive symptoms. During placebo, plasma homovanillic acid (HVA) correlated with negative symptoms and with changes presented by negative symptoms between the first and the fifth treatment week. These data show that negative symptoms respond differentially to neuroleptics and suggest that avolition-apathy may represent a different behavioral component of the schizophrenia process.
Assuntos
Haloperidol/uso terapêutico , Ácido Homovanílico/sangue , Receptores Dopaminérgicos/efeitos dos fármacos , Esquizofrenia/sangue , Psicologia do Esquizofrênico , Adolescente , Adulto , Afeto/efeitos dos fármacos , Nível de Alerta/efeitos dos fármacos , Atenção/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Relação Dose-Resposta a Droga , Método Duplo-Cego , Feminino , Humanos , Masculino , Motivação , Escalas de Graduação PsiquiátricaRESUMO
The effect of a lack of dopamine (DA) in the striatum upon the K(+)-evoked release of cholecystokinin (CCK) from superfused rat striatal slices has been studied. Two pharmacological tools were used to deplete the nigrostriatal DA system: administration of alpha-methyl-p-tyrosine which competitively inhibits DA synthesis and lesions with 6-hydroxydopamine of the medial forebrain bundle. In both cases there was a significant inhibition of the K(+)-evoked release of CCK. The observed effects might be relevant on pathological conditions implying depletion of the DA system.