Barroso-Chinea, P. (P.)
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- Expression of the mRNA coding the cannabinoid receptor 2 in the pallidal complex of Macaca fascicularis(Sage Publications, 2010) Lluis, C. (Carmen); Conte-Perales, L. (L.); Lanciego, J.L. (José Luis); Rico, A.J. (Alberto J.); Roda, E. (Elvira); Lopez, I.P. (Iciar P.); Franco, R. (Rafael); Barroso-Chinea, P. (P.); Callen, L. (L.); Labandeira-Garcia, J.L. (José L.); Gomez-Bautista, V. (V.)The putative presence of the cannabinoid receptor type 2 (CB(2)-R) in the central nervous system is still a matter of debate. Although first described in peripheral and immune tissues, evidence suggesting the existence of CB(2)-Rs in glial cells and even neurons has been made available more recently. By taking advantage of newly designed CB(2)-R mRNA riboprobes, we have demonstrated by in situ hybridization and PCR the existence of CB2-R transcripts in a variety of brain areas of the primate Macaca fascicularis, including the cerebral cortex and the hippocampus, as well as in the external and internal divisions of the globus pallidus, both pallidal segments showing the highest abundance of CB(2)-R transcripts. In this regard, the presence of the messenger coding CB(2)-Rs within the pallidal complex highlights their consideration as potential targets for the treatment of movement disorders of basal ganglia origin.
- High-resolution neuroanatomical tract-tracing for the analysis of striatal microcircuits(Elsevier, 2008) Salin, P. (Pascal); Kachidian, P. (P.); Lanciego, J.L. (José Luis); Rico, A.J. (Alberto J.); Castle, M. (M.); Lopez, I.P. (Iciar P.); Kerkerian-Le-Goff, L. (Lydia); Barroso-Chinea, P. (P.); Coulon, P. (Patrice)Although currently available retrograde tracers are useful tools for identifying striatal projection neurons, transported tracers often remained restricted within the neuronal somata and the thickest, main dendrites. Indeed, thin dendrites located far away from the cell soma as well as post-synaptic elements such as dendritic spines cannot be labeled unless performing intracellular injections. In this regard, the subsequent use of anterograde tracers for the labeling of striatal afferents often failed to unequivocally elucidate whether a given afferent makes true contacts with striatal projections neurons. Here we show that such a technical constraint can now be circumvented by retrograde tracing using rabies virus (RV). Immunofluorescence detection with a monoclonal antibody directed against the viral phosphoprotein resulted in a consistent Golgi-like labeling of striatal projection neurons, allowing clear visualization of small-size elements such as thin dendrites as well as dendritic spines. The combination of this retrograde tracing together with dual anterograde tracing of cortical and thalamic afferents has proven to be a useful tool for ascertaining striatal microcircuits. Indeed, by taking advantage of the trans-synaptic spread of RV, different subpopulations of local-circuit neurons modulating striatal efferent neurons can also be identified. At the striatal level, structures displaying labeling were visualized under the confocal laser-scanning microscope at high resolution. Once acquired, confocal stacks of images were firstly deconvoluted and then processed through 3D-volume rendering in order to unequivocally identify true contacts between pre-synaptic elements (axon terminals from cortical or thalamic sources) and post-synaptic elements (projection neurons and/or interneurons labeled with RV).
- Neuroanatomical tracing combined with in situ hybridization: analysis of gene expression patterns within brain circuits of interest(Elsevier, 2010) Wouterlood, F.G. (Floris G.); Conte-Perales, L. (L.); Lanciego, J.L. (José Luis); Rico, A.J. (Alberto J.); Roda, E. (Elvira); Lopez, I.P. (Iciar P.); Barroso-Chinea, P. (P.); Gomez-Bautista, V. (V.)Most of our current understanding of brain circuits is based on hodological studies carried out using neuroanatomical tract-tracing. Our aim is to advance one step further by visualizing the functional correlate in a given circuit. In this regard, we believe it is feasible to combine retrograde tracing with fluorescence, non-radioactive in situ hybridization (ISH) protocols. The subsequent detection at the single-cell level of the expression of a given mRNA within retrograde-labeled neurons provides information regarding cellular function. This may be of particular interest when trying to elucidate the performance of brain circuits of interest in animal models of brain diseases. Several combinations of retrograde tracing with either single- and double-ISH are presented here, together with some criteria that influence the selection of the tracer to be used in conjunction with the strong demands of the ISH.
- Origin of calretinin-containing, vesicular glutamate transporter 2-coexpressing fiber terminals in the entorhinal cortex of the rat(wiley Blackwell, 2008) Zaborszky, L. (Laszlo); Hur, E.E. (E. E.); Wouterlood, F.G. (Floris G.); Aliane, V. (V.); Lanciego, J.L. (José Luis); Boekel, A.J. (A.J.); Barroso-Chinea, P. (P.); Härtig, W. (W.); Witter, M.P. (Menno P.)The entorhinal cortex of the rat (EC) contains a dense fiber plexus that expresses the calcium-binding protein calretinin (CR). Some CR fibers contain vesicular glutamate transporter 2 (VGluT2, associated with glutamatergic neurotransmission). CR-VGluT2 coexpressing fibers may have an extrinsic origin, for instance, the midline thalamic nucleus reuniens. Alternatively, they may belong to cortical interneurons. We studied the first possibility with anterograde and retrograde neuroanatomical tracing methods combined with CR and VGluT2 immunofluorescence and confocal laser scanning. The alternative possibility was studied with in situ hybridization fluorescence histochemistry for VGluT2 mRNA combined with CR immunofluorescence. In the anterograde tracing experiments, we observed many labeled reuniens fibers in EC expressing CR. Some of these labeled fibers contained immunoreactivity for VGluT2 and CR. In the complementary retrograde tracing experiments, we found retrogradely labeled cell bodies in nucleus reuniens of the thalamus that coexpressed CR. We also examined the colocalization of VGluT2 and CR in the entorhinal cortex by using in situ hybridization and CR immunofluorescence. In these experiments, we observed CR-immunopositive cortical neurons that coexpressed VGluT2. For the same sections, with CR as the principal marker and parvalbumin as a control marker, we found that parvalbumin neurons were negative for VGluT2 mRNA. Thus, CR-VGluT2-expressing axon terminals in EC belong to two sources: projection fibers from the thalamus and axon collaterals of local interneurons. VGluT2 expression is linked to the synaptic transmission of the excitatory neurotransmitter glutamate, so these thalamic CR-VGluT2 projection neurons and entorhinal CR-VGluT2 interneurons should be regarded as excitatory.
- Location of prorenin receptors in primate substantia nigra: effects on dopaminergic cell death(Lippincott Williams and Wilkins, 2010) Villar, B. (Begoña); Valenzuela, R. (Rita); Lanciego, J.L. (José Luis); Joglar, B. (B.); Barroso-Chinea, P. (P.); Labandeira-Garcia, J.L. (José L.); Muñoz-Patiño, A.M. (A.M.)Angiotensin II acts via angiotensin type 1 receptors and is a major inducer of inflammation and oxidative stress. Local renin-angiotensin systems play a major role in the development of age-related disorders in several tissues. These processes are delayed, but not totally abolished, by blockade of angiotensin signaling. A specific receptor for renin and its precursor prorenin has recently been identified. We previously showed that neurotoxin-induced dopaminergic (DA) cell loss is decreased by inhibition of angiotensin receptors, but the location and functional effects of prorenin receptor (PRR) in the brain, including the DA system, are unknown. In the substantia nigra of Macaca fascicularis and in rat primary mesencephalic cultures, double immunofluorescence analysis revealed PRR immunoreactivity in neurons (including DA neurons) and microglia, but not in astrocytes. Administration of the PRR blocker, handle region peptide, led to a significant decrease in 6-hydroxydopamine-induced DA cell death in the cultures,whereas administration of renin with simultaneous blockade of angiotensin receptors led to an increase in 6-hydroxydopamine-induced cell death. These results suggest that active agent angiotensin II-independent PRR intracellular signaling may contribute to exacerbation of DA cell death in vivo. Therefore, potential neuroprotective strategies for DA neurons in Parkinson disease should address both angiotensin and PRR signaling.
- A direct projection from the subthalamic nucleus to the ventral thalamus in monkeys(Elsevier, 2010) Conte-Perales, L. (L.); Obeso, J.A. (José A.); Lanciego, J.L. (José Luis); Rico, A.J. (Alberto J.); Roda, E. (Elvira); Barroso-Chinea, P. (P.); Gendive, M. (M.); Gomez-Bautista, V. (V.)The current basal ganglia model considers the internal division of the globus pallidus and the substantia nigra pars reticulata as the sole sources of basal ganglia output to the thalamus. However, following the delivery of retrograde tracers into the ventral anterior/ventral lateral thalamic nuclei, a moderate number of labeled neurons were found within the subthalamic nucleus (STN) in control cases, MPTP-treated monkeys and animals with levodopa-induced dyskinesias. Furthermore, dual tracing experiments showed that subthalamo-thalamic and subthalamo-pallidal projections arise from different subpopulations of STN efferent neurons. Moreover, upregulated expression of the mRNA coding the vesicular glutamate transporter 2 (vGlut2) was found in retrogradely-labeled STN neurons in MPTP-treated monkeys. By contrast, there is a reduction in vGlut2 mRNA expression in subthalamo-thalamic neurons in dyskinetic monkeys. In conclusion, our findings support the presence of a direct projection from the STN to the ventral thalamus that appears to be functionally modulated by dopaminergic activity.
- Changes to interneuron-driven striatal microcircuits in a rat model of Parkinson's disease(Elsevier, 2009) Salin, P. (Pascal); Kachidian, P. (P.); Lanciego, J.L. (José Luis); Rico, A.J. (Alberto J.); Lopez, I.P. (Iciar P.); Kerkerian-Le-Goff, L. (Lydia); Barroso-Chinea, P. (P.); Gomez-Bautista, V. (V.); Coulon, P. (Patrice)Striatal interneurons play key roles in basal ganglia function and related disorders by modulating the activity of striatal projection neurons. Here we have injected rabies virus (RV) into either the rat substantia nigra pars reticulata or the globus pallidus and took advantage of the trans-synaptic spread of RV to unequivocally identify the interneurons connected to striatonigral- or striatopallidal-projecting neurons, respectively. Large numbers of RV-infected parvalbumin (PV+/RV+) and cholinergic (ChAT+/RV+) interneurons were detected in control conditions, and they showed marked changes following intranigral 6-hydroxydopamine injection. The number of ChAT+/RV+ interneurons innervating striatopallidal neurons increased concomitant with a reduction in the number of PV+/RV+ interneurons, while the two interneuron populations connected to striatonigral neurons were clearly reduced. These data provide the first evidence of synaptic reorganization between striatal interneurons and projection neurons, notably a switch of cholinergic innervation onto striatopallidal neurons, which could contribute to imbalanced striatal outflow in parkinsonian state.
- Expression of vesicular glutamate transporters 1 and 2 in the cells of origin of the rat thalamostriatal pathway(Elsevier, 2008) Lanciego, J.L. (José Luis); Castle, M. (M.); Aymerich-Soler, M.S. (María Soledad); Barroso-Chinea, P. (P.)The present study is focused on the analysis of the vesicular glutamate transporters 1 and 2 (VGLUT1 and VGLUT2) used by thalamic neurons giving rise to the thalamostriatal system. Instead of studying the distribution of VGLUT proteins at the level of thalamostriatal terminals, this report is focused on identifying the expression of the VGLUT mRNAs within the parent cell bodies of thalamic neurons innervating the striatum. For this purpose, we have combined dual in situ hybridization to detect both VGLUT1 and VGLUT2 mRNAs together with retrograde tracing with cholera toxin. Our results show that VGLUT2 is the only vesicular glutamate transporter expressed in thalamostriatal-projecting neurons located in the midline and intralaminar nuclei, whereas all neurons from the ventral thalamic nuclei innervating the striatum express both VGLUTs, at least at the mRNA level. Indeed, the mRNAs encoding for VGLUT1 and VGLUT2 displayed a sharp complementary subcellular distribution within neurons from the ventral thalamic nuclei giving rise to thalamostriatal projections. The differential distribution of VGLUT mRNAs lead us to conclude that the thalamostriatal pathway is a dual system, composed by a preponderant projection arising from the midline and intralaminar nuclei using VGLUT2 as the glutamate transporter, together with another important source of striatal afferents arising from neurons in the ventral thalamic relay nuclei containing both kinds of vesicular glutamate transporters.
- Expression of the mRNAs encoding for the vesicular glutamate transporters 1 and 2 in the rat thalamus(Wiley Blackwel, 2007) Tuñon, M.T. (María Teresa); Erro-Aguirre, M.E (María Elena); Lanciego, J.L. (José Luis); Castle, M. (M.); Aymerich-Soler, M.S. (María Soledad); Barroso-Chinea, P. (P.); Perez-Manso, M. (Mónica)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.
- Pallidothalamic-projecting neurons in Macaca fascicularis co-express GABAergic and glutamatergic markers as seen in control, MPTP-treated and dyskinetic monkeys(Springer Verlag, 2011) Sierra, S. (Salvador); Conte-Perales, L. (L.); Lanciego, J.L. (José Luis); Rico, A.J. (Alberto J.); Roda, E. (Elvira); Luquin, N. (Natasha); Barroso-Chinea, P. (P.); Gomez-Bautista, V. (V.)GABAergic neurons within the internal division of the globus pallidus (GPi) are the main source of basal ganglia output reaching the thalamic ventral nuclei in monkeys. Following dopaminergic denervation, pallidothalamic-projecting neurons are known to be hyperactive, whereas a reduction in GPi activity is typically observed in lesioned animals showing levodopa-induced dyskinesia. Besides the mRNAs coding for GABAergic markers (GAD65 and GAD67), we show that all GPi neurons innervating thalamic targets also express transcripts for the isoforms 1 and 2 of the vesicular glutamate transporter (vGlut1 and vGlut2 mRNA). Indeed, dual immunofluorescent detection of GAD67 and vGlut1/2 confirmed the data gathered from in situ hybridization experiments, therefore demonstrating that the detected mRNAs are translated into the related proteins. Furthermore, the dopaminergic lesion resulted in an up-regulation of expression levels for both GAD65 and GAD67 mRNA within identified pallidothalamic-projecting neurons. This was coupled with a down-regulation of GAD65/67 mRNA expression levels in GPi neurons innervating thalamic targets in monkeys showing levodopa-induced dyskinesia. By contrast, the patterns of gene expression for both vGlut1 and vGlut2 mRNAs remained unchanged across GPi projection neurons in control, MPTP-treated and dyskinetic monkeys. In summary, both GABAergic and glutamatergic markers were co-expressed by GPi efferent neurons in primates. Although the status of the dopaminergic system directly modulates the expression levels of GAD65/67 mRNA, the observed expression of vGlut1/2 mRNA is not regulated by either dopaminergic removal or by continuous stimulation with dopaminergic agonists.