Rico, A.J. (Alberto J.)
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- 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.
- Glutamatergic pallidothalamic projections and their implications in the pathophysiology of Parkinson's disease(Elsevier, 2008) Lanciego, J.L. (José Luis); Rico, A.J. (Alberto J.); Luis-Ravelo, D. (Diego); Roda, E. (Elvira); Lopez, I.P. (Iciar P.); Barroso-Chinea, P. (P.); Perez-Manso, M. (Mónica)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.
- Adeno-associated viral vectors serotype 8 for cell-specific delivery of therapeutic genes in the central nervous system(Frontiers Media, 2017) Lopez-Franco, E. (Esperanza); González-Aseguinolaza, G. (Gloria); Sucunza, D. (Diego); Vales, A. (África); Lanciego, J.L. (José Luis); Rico, A.J. (Alberto J.); Hommel, M. (Mirja); Dopeso-Reyes, I.G. (Iria G.); Vanrell, L. (Lucía); Pignataro, D. (Diego)Adeno-associated viruses (AAVs) have become highly promising tools for research and clinical applications in the central nervous system (CNS). However, specific delivery of genes to the cell type of interest is essential for the success of gene therapy and therefore a correct selection of the promoter plays a very important role. Here, AAV8 vectors carrying enhanced green fluorescent protein (eGFP) as reporter gene under the transcriptional control of different CNS-specific promoters were used and compared with a strong ubiquitous promoter. Since one of the main limitations of AAV-mediated gene delivery lies in its restricted cloning capacity, we focused our work on small-sized promoters. We tested the transduction efficacy and specificity of each vector after stereotactic injection into the mouse striatum. Three glia-specific AAV vectors were generated using two truncated forms of the human promoter for glial fibrillar acidic protein (GFAP) as well as a truncated form of the murine GFAP promoter. All three vectors resulted in predominantly glial expression; however we also observed eGFP expression in other cell-types such as oligodendrocytes, but never in neurons. In addition, robust and neuron-specific eGFP expression was observed using the minimal promoters for the neural protein BM88 and the neuronal nicotinic receptor β2 (CHRNB2). In summary, we developed a set of AAV vectors designed for specific expression in cells of the CNS using minimal promoters to drive gene expression when the size of the therapeutic gene matters.
- The added value of rabies virus as a retrograde tracer when combined with dual anterograde tract-tracing(Elsevier, 2010) Salin, P. (Pascal); Kachidian, P. (P.); Conte-Perales, L. (L.); 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)Rabies virus (RV) has widely been used as a trans-synaptic retrograde tracer to analyze chains of connected neurons. The use of antibodies directed against the viral nucleoprotein enables viral nucleocapsids to be visualized within the cell soma, as well as within the thickest main dendrites. However, through this approach it is often difficult to accurately define post-synaptic elements (thin dendrites and/or dendritic spines). This limitation can now easily been circumvented by taking advantage of antibodies directed against a soluble viral phosphoprotein that spreads throughout the cytoplasm of the infected neuron, thereby producing Golgi-like immunofluorescent labeling of first-order projection neurons that are infected with RV. Furthermore, when combined with anterograde tracers such as Phaseolus vulgaris-leucoagglutinin (PHA-L) and biotinylated dextran amine (BDA), this procedure to detect RV facilitates the accurate visualization of both the pre- and post-synaptic elements. Finally, this method of viral detection is sufficiently sensitive to detect weakly labeled second-order neurons, which can then be further characterized neurochemically. Several examples are provided to illustrate why retrograde trans-synaptic tracing using RV can be regarded as an important breakthrough in the analysis of brain circuits, providing an unprecedented level of resolution.
- Calbindin content and differential vulnerability of midbrain efferent dopaminergic neurons in macaques(Frontiers, 2014) Sucunza, D. (Diego); Sierra, S. (Salvador); Lanciego, J.L. (José Luis); Rico, A.J. (Alberto J.); Chang-Azancot, L. (Luis); Roda, E. (Elvira); Dopeso-Reyes, I.G. (Iria G.); Lanz, M. (María); Pignataro, D. (Diego)Calbindin (CB) is a calcium binding protein reported to protect dopaminergic neurons from degeneration. Although a direct link between CB content and differential vulnerability of dopaminergic neurons has long been accepted, factors other than CB have also been suggested, particularly those related to the dopamine transporter. Indeed, several studies have reported that CB levels are not causally related to the differential vulnerability of dopaminergic neurons against neurotoxins. Here we have used dual stains for tyrosine hydroxylase (TH) and CB in 3 control and 3 MPTP-treated monkeys to visualize dopaminergic neurons in the ventral tegmental area (VTA) and in the dorsal and ventral tiers of the substantia nigra pars compacta (SNcd and SNcv) co-expressing TH and CB. In control animals, the highest percentages of co-localization were found in VTA (58.2%), followed by neurons located in the SNcd (34.7%). As expected, SNcv neurons lacked CB expression. In MPTP-treated animals, the percentage of CB-ir/TH-ir neurons in the VTA was similar to control monkeys (62.1%), whereas most of the few surviving neurons in the SNcd were CB-ir/TH-ir (88.6%). Next, we have elucidated the presence of CB within identified nigrostriatal and nigroextrastriatal midbrain dopaminergic projection neurons. For this purpose, two control monkeys received one injection of Fluoro-Gold into the caudate nucleus and one injection of cholera toxin (CTB) into the postcommissural putamen, whereas two more monkeys were injected with CTB into the internal division of the globus pallidus (GPi). As expected, all the nigrocaudate- and nigroputamen-projecting neurons were TH-ir, although surprisingly, all of these nigrostriatal-projecting neurons were negative for CB. Furthermore, all the nigropallidal-projecting neurons co-expressed both TH and CB. In summary, although CB-ir dopaminergic neurons seem to be less prone to MPTPinduced degeneration, our data clearly demonstrated that these neurons are not giving rise to nigrostriatal projections and indeed CB-ir/TH-ir neurons only originate nigroextrastriatal projections.
- Glutamatergic and cholinergic pedunculopontine neurons innervate the thalamic parafascicular nucleus in rats: changes following experimental parkinsonism(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.)The tegmental pedunculopontine nucleus (PPN) is a basal ganglia-related structure that has recently gained renewed interest as a potential surgical target for the treatment of several aspects of Parkinson's disease. However, the underlying anatomical substrates sustaining the choice of the PPN nucleus as a surgical candidate remain poorly understood. Here, we characterized the chemical phenotypes of different subtypes of PPN efferent neurons innervating the rat parafascicular (PF) nucleus. Emphasis was placed on elucidating the impact of unilateral nigrostriatal denervation on the expression patterns of the mRNA coding the vesicular glutamate transporter type 2 (vGlut2 mRNA). We found a bilateral projection from the PPN nucleus to the PF nucleus arising from cholinergic and glutamatergic efferent neurons, with a small fraction of projection neurons co-expressing both cholinergic and glutamatergic markers. Furthermore, the unilateral nigrostriatal depletion induced a bilateral twofold increase in the expression levels of vGlut2 mRNA within the PPN nucleus. Our results support the view that heterogeneous chemical profiles account for PPN efferent neurons innervating thalamic targets. Moreover, a bilateral enhancement of glutamatergic transmission arising from the PPN nucleus occurs following unilateral dopaminergic denervation, therefore sustaining the well-known hyperactivity of the PF nucleus in parkinsonian-like conditions. In conclusion, our data suggest that the ascending projections from the PPN that reach basal ganglia-related targets could play an important role in the pathophysiology of Parkinson's disease.
- The search for a role of the caudal intralaminar nuclei in the pathophysiology of Parkinson's disease(Elsevier, 2009) Tuñon, M.T. (María Teresa); Erro-Aguirre, M.E (María Elena); Gonzalo, N. (Nancy); Conte-Perales, L. (L.); Lanciego, J.L. (José Luis); Rico, A.J. (Alberto J.); Castle, M. (M.); Combarro, C. (C.); Aymerich-Soler, M.S. (María Soledad); Roda, E. (Elvira); Lopez, I.P. (Iciar P.); Molina, C. (Carmeen); Barroso-Chinea, P. (P.); Perez-Manso, M. (Mónica)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.
- Glucocerebrosidase expression patterns in the non-human primate brain(Springer, 2018) Rodriguez-Perez, A.I. (Ana I.); Sucunza, D. (Diego); Lanciego, J.L. (José Luis); Rico, A.J. (Alberto J.); Roda, E. (Elvira); Dopeso-Reyes, I.G. (Iria G.); Marín-Ramos, D. (David); Labandeira-Garcia, J.L. (José L.); Pignataro, D. (Diego)Glucocerebrosidase (GCase) is a lysosomal enzyme encoded by the GBA1 gene. Mutations in GBA1 gene lead to Gaucher’s disease, the most prevalent lysosomal storage disorder. GBA1 mutations reduce GCase activity, therefore promoting the aggregation of alphasynuclein, a common neuropathological finding underlying Parkinson’s disease (PD) and dementia with Lewy bodies. However, it is also worth noting that a direct link between GBA1 mutations and alpha-synuclein aggregation indicating cause and effect is still lacking, with limited experimental evidence to date. Bearing in mind that a number of strategies increasing GCase expression for the treatment of PD are currently under development, here we sought to analyze the baseline expression of GCase in the brain of Macaca fascicularis, which has often been considered as the gold-standard animal model of PD. Although as with other lysosomal enzymes, GCase is expected to be ubiquitously expressed, here a number of regional variations have been consistently found, together with several specific neurochemical phenotypes expressing very high levels of GCase. In this regard, the most enriched expression of GCase was constantly found in cholinergic neurons from the nucleus basalis of Meynert, dopaminergic cells in the substantia nigra pars compacta, serotoninergic neurons from the raphe nuclei, as well as in noradrenergic neurons located in the locus ceruleus. Moreover, it is also worth noting that moderate levels of expression were also found in a number of areas within the paleocortex and archicortex, such as the entorhinal cortex and the hippocampal formation, respectively.
- 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).