RESUMO
GABAergic projections emitted from the entopeduncular nucleus (ENT) and the substantia nigra pars reticulata (SNr) innervate different thalamic nuclei and they are known to be hyperactive after dopaminergic depletion. Here we show that isoform 2 of the vesicular glutamate transporter (VGLUT2) is expressed by neurons in the ENT nucleus but not in the SNr. Indeed, dual in situ hybridization demonstrated that the ENT nucleus contains two different subpopulations of projection neurons, one single-expressing GAD65/67 mRNAs and another one that co-expresses either of the GAD isoforms together with VGLUT2 mRNA. Unilateral dopaminergic depletion induced marked changes in pallidothalamic-projecting neuron gene expression, resulting in increased expression of GAD65/67 mRNAs together with a clear down-regulation of VGLUT2 mRNA expression. Our results indicate that the increased thalamic inhibition typical of dopamine depletion might be explained by a synergistic effect of increased GABA outflow coupled to decreased glutamate levels, both neurotransmitters coming from ENT neurons.
Assuntos
Globo Pálido/metabolismo , Ácido Glutâmico/metabolismo , Transtornos Parkinsonianos/metabolismo , Tálamo/metabolismo , Proteína Vesicular 2 de Transporte de Glutamato/metabolismo , Ácido gama-Aminobutírico/metabolismo , Animais , Dopamina/deficiência , Regulação para Baixo/fisiologia , Vias Eferentes/metabolismo , Vias Eferentes/fisiopatologia , Núcleo Entopeduncular/metabolismo , Núcleo Entopeduncular/fisiopatologia , Regulação Enzimológica da Expressão Gênica/genética , Globo Pálido/fisiopatologia , Glutamato Descarboxilase/genética , Glutamato Descarboxilase/metabolismo , Masculino , Transtornos Parkinsonianos/fisiopatologia , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Substância Negra/metabolismo , Substância Negra/fisiopatologia , Transmissão Sináptica/fisiologia , Tálamo/fisiopatologia , Regulação para Cima/fisiologia , Proteína Vesicular 2 de Transporte de Glutamato/genéticaRESUMO
Vesicular glutamate transporters (VGLUTs) are responsible for glutamate trafficking and for the subsequent regulated release of this excitatory neurotransmitter at the synapse. Three isoforms of the VGLUT have been identified, now known as VGLUT1, VGLUT2, and VGLUT3. Both VGLUT1 and VGLUT2 have been considered definitive markers of glutamatergic neurons, whereas VGLUT3 is expressed in nonglutamatergic neurons such as cholinergic striatal interneurons. It is widely believed that VGLUT1 and VGLUT2 are expressed in a complementary manner at the cortical and thalamic levels, suggesting that these glutamatergic neurons fulfill different physiological functions. In the present work, we analyzed the pattern of VGLUT1 and VGLUT2 mRNA expression at the thalamic level by using single and dual in situ hybridization. In accordance with current beliefs, we found significant expression of VGLUT2 mRNA in all the thalamic nuclei, while moderate expression of VGLUT1 mRNA was consistently found in both the principal relay and the association thalamic nuclei. Interestingly, individual neurons within these nuclei coexpressed both VGLUT1 and VGLUT2 mRNAs, suggesting that these individual thalamic neurons may have different ways of trafficking glutamate. These results call for a reappraisal of the previously held concept regarding the mutually exclusive distribution of VGLUT transporters in the central nervous system.
Assuntos
RNA Mensageiro/metabolismo , Tálamo/metabolismo , Proteína Vesicular 1 de Transporte de Glutamato/genética , Proteína Vesicular 2 de Transporte de Glutamato/genética , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Ácido Glutâmico/metabolismo , Habenula/anatomia & histologia , Habenula/metabolismo , Hibridização In Situ , Núcleos Intralaminares do Tálamo/anatomia & histologia , Núcleos Intralaminares do Tálamo/metabolismo , Masculino , Vias Neurais/anatomia & histologia , Vias Neurais/metabolismo , Ratos , Ratos Wistar , Transmissão Sináptica/fisiologia , Tálamo/anatomia & histologiaRESUMO
We have compared the performance of two methods designed to simultaneously detect two different mRNAs within a single brain section by dual ISH. Specific mRNA riboprobes labeled with biotin and digoxigenin were simultaneously hybridized and visualized using either brightfield or fluorescence microscopy. For brightfield visualization, the biotin-labeled riboprobe was detected with a peroxidase chromogen, whereas, an alkaline phosphatase substrate was used for the detection of the digoxigenin-labeled riboprobe. Dual fluorescent ISH involved the detection of the biotin-labeled riboprobe with an Alexa((R))488-conjugated streptavidin followed by the visualization of the digoxigenin-labeled riboprobe with the red fluorescent substrate HNPP. The dual ISH protocols presented here offer sensitive methods to detect the expression of two mRNAs of interest, with both colorimetric and fluorescent ISH each having its strengths and limitations. For example, dual colorimetric ISH has proven to be particularly useful to study the distribution of two mRNAs in different brain nuclei, whereas, dual fluorescent ISH has provided better results when studying the co-localization of two different mRNAs in single neurons. The comprehensive step-by-step procedure is presented, together with a troubleshooting section in which the advantages and limitations of these procedures are reviewed in depth. Moreover, alternative protocols for dual ISH were also compared to those presented here.
Assuntos
Encéfalo/fisiologia , RNA Mensageiro/análise , Animais , Biotina , Encéfalo/citologia , Colorimetria/métodos , Amplificação de Genes , Transportador de Glucose Tipo 2/genética , Glutamato Descarboxilase/genética , Hibridização In Situ/métodos , Indicadores e Reagentes , Isoenzimas/genética , Masculino , Plasmídeos , RNA Mensageiro/genética , Ratos , Ratos Wistar , Espectrometria de Fluorescência/métodosRESUMO
Neuroanatomical tracing when considered as an isolated method produces relatively straightforward answers. Although single-, double- or even triple-tracing paradigms produce valuable data on the organization of brain circuits, the final outcome often is too simplistic since it is not possible to elucidate the activity of these circuits. In this regard, emerging technologies contribute with additional information about the status of neuronal circuits. The laser-guided capture microdissection microscope (LCM) allows the accurate dissection of small brain areas under the microscope that could be further analyzed for gene expression or proteomics. In order to elucidate the gene expression of a given circuit of interest, we have developed a combination of methods comprising (i) fluorescent non-radioactive in situ hybridization for the detection of vGLUT2 mRNA expression combined with retrograde tracing with Fluoro-Gold (FG; analysis performed under the confocal microscope) and (ii) laser-guided capture microdissection of brain areas containing neurons retrogradely labeled with FG followed by the measurement of changes in mRNA levels encoding for vGLUT2 by real-time PCR. Our goal was to detect changes in gene expression of the thalamostriatal pathway in unilaterally 6-OHDA lesioned rats. Taking advantage of this procedure, we found a three-fold increase in vGLUT2 mRNA expression within thalamic neurons projecting to the dopamine-depleted striatum when compared with the activity of the thalamic neurons innervating the control striatum.
Assuntos
Encéfalo/anatomia & histologia , Encéfalo/fisiologia , Regulação da Expressão Gênica , Animais , Mapeamento Encefálico , Dissecação/métodos , Masculino , RNA Mensageiro/genética , Ratos , Ratos Wistar , Tálamo/anatomia & histologia , Tálamo/fisiologia , Proteína Vesicular 2 de Transporte de Glutamato/genéticaRESUMO
We have investigated the clinical feasibility of the major urinary metabolite of prostaglandin (PG) E2, tetranor-PGEM, as a biomarker of inflammation in infants with fever. We tested two different and clinically relevant sampling methods, using self-adhesive urinary bags or gauze pads, with respect to stability of tetranor-PGEM and ease of sampling from infants. Liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis was used to quantify tetranor-PGEM in urine, and different normalization parameters, i.e., urinary creatinine and body surface area, were investigated. To study inflammation, infants (1 month-1 year) that were hospitalized with fever of unknown origin at admittance (n=14) were compared to age-matched healthy controls (n=14). Levels of urinary tetranor-PGEM in infants with viral induced fever were increased compared to controls (102.4±56.2 vs. 37.0±21.6pmol/ml/m(2) body surface area, p<0.001). We conclude that urinary tetranor-PGEM is a potential non-invasive biomarker of inflammation in infants.
Assuntos
Biomarcadores/urina , Febre/virologia , Prostaglandinas/urina , Viroses/urina , Cromatografia Líquida , Dinoprostona/metabolismo , Feminino , Febre/urina , Humanos , Lactente , Recém-Nascido , Masculino , Espectrometria de Massas em TandemRESUMO
Mutations in the E3 ubiquitin ligase parkin cause early-onset, autosomal-recessive juvenile parkinsonism (AJRP), presumably as a result of a lack of function that alters the level, activity, aggregation or localization of its substrates. Recently, we have reported that phospholipase Cgamma1 is a substrate for parkin. In this article, we show that parkin mutants and siRNA parkin knockdown cells possess enhanced levels of phospholipase Cgamma1 phosphorylation, basal phosphoinositide hydrolysis and intracellular Ca2+ concentration. The protein levels of Ca2+-regulated protein kinase Calpha were decreased in AJRP parkin mutant cells. Neomycin and dantrolene both decreased the intracellular Ca2+ levels in parkin mutants in comparison with those seen in wild-type parkin cells, suggesting that the differences were a consequence of altered phospholipase C activity. The protection of wild-type parkin against 6-hydroxydopamine (6OHDA) toxicity was also established in ARJP mutants on pretreatment with dantrolene, implying that a balancing Ca2+ release from ryanodine-sensitive stores decreases the toxic effects of 6OHDA. Our findings suggest that parkin is an important factor for maintaining Ca2+ homeostasis and that parkin deficiency leads to a phospholipase C-dependent increase in intracellular Ca2+ levels, which make cells more vulnerable to neurotoxins, such as 6OHDA.
Assuntos
Cálcio/metabolismo , Homeostase , Fosfolipase C gama/fisiologia , Transdução de Sinais/fisiologia , Ubiquitina-Proteína Ligases/fisiologia , Linhagem Celular Tumoral , Dantroleno/farmacologia , Humanos , Oxidopamina/toxicidade , Fosfatidilinositóis/metabolismo , Proteína Quinase C-alfa/fisiologia , Ubiquitina-Proteína Ligases/deficiênciaRESUMO
The situation of the caudal intralaminar thalamic nuclei within basal ganglia circuits has gained increased attention over the past few years. Although initially considered as a "non-specific" thalamic nuclei, tract-tracing studies carried out over the past two decades have demonstrated that the centromedian-parafascicular thalamic complex (CM-Pf) is connected to virtually all basal ganglia components and related nuclei. Although the anatomical basis sustaining the thalamic modulation of basal ganglia circuits has long been characterized, the functional significance of these transverse circuits still remain to be properly accommodated within the basal ganglia model, both under normal conditions as well as in situations of dopaminergic depletion. However, the recent demonstration of primary (e.g., non-dopamine related) neurodegenerative phenomena restricted to the CM-Pf in Parkinson's disease (PD) has renewed interest in the role played by the caudal intralaminar nuclei in the pathophysiology of PD. Concomitantly, evidence has become available of increased metabolic activity in the caudal intralaminar nuclei in rodent models of PD. Finally, CM-Pf neurosurgery in patients suffering from PD has produced contrasting outcomes, indicating that a consensus is still to be reached regarding the potential usefulness of targeting the caudal intralaminar nuclei to treat movement disorders of basal ganglia origin.