Roda, E. (Elvira)
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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.
- 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.
- Preferential expression of SCN1A in GABAergic neurons improves survival and epileptic phenotype in a mouse model of Dravet syndrome(2023) Rubinstein, M. (Moran); Honrubia, A. (Adriana); Vides-Urrestarazu, I. (Irene); Ricobaraza, A. (Ana); Lanciego, J.L. (José Luis); Fadila, S. (Saja); Sanchez-Carpintero, R. (Rocío); Roda, E. (Elvira); Arnaiz, P. (Patricia); Banderas, J. (Julliana); Sola-Sevilla, N. (Noemi); Hernandez-Alcoceba, R. (Rubén); Buñuales, M. (María); Gonzalez-Aparicio, M. (Manuela)The SCN1A gene encodes the alpha subunit of a voltage-gated sodium channel (Nav1.1), which is essential for the function of inhibitory neurons in the brain. Mutations in this gene cause severe encephalopathies such as Dravet syndrome (DS). Upregulation of SCN1A expression by different approaches has demonstrated promising therapeutic effects in preclinical models of DS. Limiting the effect to inhibitory neurons may contribute to the restoration of brain homeostasis, increasing the safety and efficacy of the treatment. In this work, we have evaluated different approaches to obtain preferential expression of the full SCN1A cDNA (6 Kb) in GABAergic neurons, using high-capacity adenoviral vectors (HC-AdV). In order to favour infection of these cells, we considered ErbB4 as a surface target. Incorporation of the EGF-like domain from neuregulin 1 alpha (NRG1α) in the fiber of adenovirus capsid allowed preferential infection in cells lines expressing ErbB4. However, it had no impact on the infectivity of the vector in primary cultures or in vivo. For transcriptional control of transgene expression, we developed a regulatory sequence (DP3V) based on the Distal-less homolog enhancer (Dlx), the vesicular GABA transporter (VGAT) promoter, and a portion of the SCN1A gene. The hybrid DP3V promoter allowed preferential expression of transgenes in GABAergic neurons both in vitro and in vivo. A new HC-AdV expressing SCN1A under the control of this promoter showed improved survival and amelioration of the epileptic phenotype in a DS mouse model. These results increase the repertoire of gene therapy vectors for the treatment of DS and indicate a new avenue for the refinement of gene supplementation in this disease. KEY MESSAGES: Adenoviral vectors can deliver the SCN1A cDNA and are amenable for targeting. An adenoviral vector displaying an ErbB4 ligand in the capsid does not target GABAergic neurons. A hybrid promoter allows preferential expression of transgenes in GABAergic neurons. Preferential expression of SCN1A in GABAergic cells is therapeutic in a Dravet syndrome model.
- 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.
- 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.
- Detection of cannabinoid receptors CB1 and CB2 within basal ganglia output neurons in macaques: changes following experimental parkinsonism(Springer, 2014) Sierra, S. (Salvador); Lanciego, J.L. (José Luis); Rico, A.J. (Alberto J.); Vazquez, A. (Alfonso); Roda, E. (Elvira); Franco, R. (Rafael); Dopeso-Reyes, I.G. (Iria G.); Luquin, N. (Natasha); Martinez-Pinilla, E. (Eva); Labandeira-Garcia, J.L. (José L.); Gomez-Bautista, V. (V.)Abstract Although type 1 cannabinoid receptors (CB1- Rs) are expressed abundantly throughout the brain, the presence of type 2 cannabinoid receptors (CB2Rs) in neurons is still somewhat controversial. Taking advantage of newly designed CB1R and CB2R mRNA riboprobes, we demonstrate by PCR and in situ hybridization that transcripts for both cannabinoid receptors are present within labeled pallidothalamic-projecting neurons of control and MPTP-treated macaques, whereas the expression is markedly reduced in dyskinetic animals. Moreover, an in situ proximity ligation assay was used to qualitatively assess the presence of CB1Rs and CB2Rs, as well as CB1R–CB2R heteromers within basal ganglia output neurons in all animal groups (control, parkinsonian and dyskinetic macaques). A marked reduction in the number of CB1Rs, CB2Rs and CB1R–CB2R heteromers was found in dyskinetic animals, mimicking the observed reduction in CB1R and CB2R mRNA expression levels. The fact that chronic levodopa treatment disrupted CB1R–CB2R heteromeric complexes should be taken into consideration when designing new drugs acting on cannabinoid receptor heteromers.