RESUMEN
The brain controls nearly all bodily functions via spinal projecting neurons (SPNs) that carry command signals from the brain to the spinal cord. However, a comprehensive molecular characterization of brain-wide SPNs is still lacking. Here we transcriptionally profiled a total of 65,002 SPNs, identified 76 region-specific SPN types, and mapped these types into a companion atlas of the whole mouse brain1. This taxonomy reveals a three-component organization of SPNs: (1) molecularly homogeneous excitatory SPNs from the cortex, red nucleus and cerebellum with somatotopic spinal terminations suitable for point-to-point communication; (2) heterogeneous populations in the reticular formation with broad spinal termination patterns, suitable for relaying commands related to the activities of the entire spinal cord; and (3) modulatory neurons expressing slow-acting neurotransmitters and/or neuropeptides in the hypothalamus, midbrain and reticular formation for 'gain setting' of brain-spinal signals. In addition, this atlas revealed a LIM homeobox transcription factor code that parcellates the reticulospinal neurons into five molecularly distinct and spatially segregated populations. Finally, we found transcriptional signatures of a subset of SPNs with large soma size and correlated these with fast-firing electrophysiological properties. Together, this study establishes a comprehensive taxonomy of brain-wide SPNs and provides insight into the functional organization of SPNs in mediating brain control of bodily functions.
Asunto(s)
Encéfalo , Perfilación de la Expresión Génica , Vías Nerviosas , Neuronas , Médula Espinal , Animales , Ratones , Hipotálamo , Neuronas/metabolismo , Neuropéptidos , Médula Espinal/citología , Médula Espinal/metabolismo , Encéfalo/citología , Encéfalo/metabolismo , Neurotransmisores , Mesencéfalo/citología , Formación Reticular/citología , Electrofisiología , Cerebelo/citología , Corteza Cerebral/citologíaRESUMEN
The function of the mammalian brain relies upon the specification and spatial positioning of diversely specialized cell types. Yet, the molecular identities of the cell types and their positions within individual anatomical structures remain incompletely known. To construct a comprehensive atlas of cell types in each brain structure, we paired high-throughput single-nucleus RNA sequencing with Slide-seq1,2-a recently developed spatial transcriptomics method with near-cellular resolution-across the entire mouse brain. Integration of these datasets revealed the cell type composition of each neuroanatomical structure. Cell type diversity was found to be remarkably high in the midbrain, hindbrain and hypothalamus, with most clusters requiring a combination of at least three discrete gene expression markers to uniquely define them. Using these data, we developed a framework for genetically accessing each cell type, comprehensively characterized neuropeptide and neurotransmitter signalling, elucidated region-specific specializations in activity-regulated gene expression and ascertained the heritability enrichment of neurological and psychiatric phenotypes. These data, available as an online resource ( www.BrainCellData.org ), should find diverse applications across neuroscience, including the construction of new genetic tools and the prioritization of specific cell types and circuits in the study of brain diseases.
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Encéfalo , Perfilación de la Expresión Génica , Animales , Ratones , Encéfalo/anatomía & histología , Encéfalo/citología , Encéfalo/metabolismo , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento , Hipotálamo/citología , Hipotálamo/metabolismo , Mesencéfalo/citología , Mesencéfalo/metabolismo , Neuropéptidos/metabolismo , Neurotransmisores/metabolismo , Fenotipo , Rombencéfalo/citología , Rombencéfalo/metabolismo , Análisis de Expresión Génica de una Sola Célula , Transcriptoma/genéticaRESUMEN
Single-cell analyses parse the brain's billions of neurons into thousands of 'cell-type' clusters residing in different brain structures1. Many cell types mediate their functions through targeted long-distance projections allowing interactions between specific cell types. Here we used epi-retro-seq2 to link single-cell epigenomes and cell types to long-distance projections for 33,034 neurons dissected from 32 different regions projecting to 24 different targets (225 source-to-target combinations) across the whole mouse brain. We highlight uses of these data for interrogating principles relating projection types to transcriptomics and epigenomics, and for addressing hypotheses about cell types and connections related to genetics. We provide an overall synthesis with 926 statistical comparisons of discriminability of neurons projecting to each target for every source. We integrate this dataset into the larger BRAIN Initiative Cell Census Network atlas, composed of millions of neurons, to link projection cell types to consensus clusters. Integration with spatial transcriptomics further assigns projection-enriched clusters to smaller source regions than the original dissections. We exemplify this by presenting in-depth analyses of projection neurons from the hypothalamus, thalamus, hindbrain, amygdala and midbrain to provide insights into properties of those cell types, including differentially expressed genes, their associated cis-regulatory elements and transcription-factor-binding motifs, and neurotransmitter use.
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Encéfalo , Epigenómica , Vías Nerviosas , Neuronas , Animales , Ratones , Amígdala del Cerebelo , Encéfalo/citología , Encéfalo/metabolismo , Secuencia de Consenso , Conjuntos de Datos como Asunto , Perfilación de la Expresión Génica , Hipotálamo/citología , Mesencéfalo/citología , Vías Nerviosas/citología , Neuronas/metabolismo , Neurotransmisores/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Rombencéfalo/citología , Análisis de la Célula Individual , Tálamo/citología , Factores de Transcripción/metabolismoRESUMEN
Food and water are rewarding in part because they satisfy our internal needs1,2. Dopaminergic neurons in the ventral tegmental area (VTA) are activated by gustatory rewards3-5, but how animals learn to associate these oral cues with the delayed physiological effects of ingestion is unknown. Here we show that individual dopaminergic neurons in the VTA respond to detection of nutrients or water at specific stages of ingestion. A major subset of dopaminergic neurons tracks changes in systemic hydration that occur tens of minutes after thirsty mice drink water, whereas different dopaminergic neurons respond to nutrients in the gastrointestinal tract. We show that information about fluid balance is transmitted to the VTA by a hypothalamic pathway and then re-routed to downstream circuits that track the oral, gastrointestinal and post-absorptive stages of ingestion. To investigate the function of these signals, we used a paradigm in which a fluid's oral and post-absorptive effects can be independently manipulated and temporally separated. We show that mice rapidly learn to prefer one fluid over another based solely on its rehydrating ability and that this post-ingestive learning is prevented if dopaminergic neurons in the VTA are selectively silenced after consumption. These findings reveal that the midbrain dopamine system contains subsystems that track different modalities and stages of ingestion, on timescales from seconds to tens of minutes, and that this information is used to drive learning about the consequences of ingestion.
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Dopamina , Neuronas Dopaminérgicas , Hipotálamo , Vías Nerviosas , Nutrientes , Estado de Hidratación del Organismo , Área Tegmental Ventral , Animales , Señales (Psicología) , Digestión , Dopamina/metabolismo , Neuronas Dopaminérgicas/fisiología , Ingestión de Alimentos , Tracto Gastrointestinal/metabolismo , Hipotálamo/citología , Hipotálamo/fisiología , Mesencéfalo/citología , Mesencéfalo/fisiología , Ratones , Nutrientes/metabolismo , Estado de Hidratación del Organismo/efectos de los fármacos , Recompensa , Factores de Tiempo , Área Tegmental Ventral/citología , Área Tegmental Ventral/fisiología , Agua/metabolismo , Agua/farmacología , Equilibrio HidroelectrolíticoRESUMEN
Parkinson's disease (PD) is a common neurodegenerative disorder characterized by the loss of midbrain dopaminergic (DA) neurons. Neural stem cells (NSCs) are the most promising cells for cell-replacement therapy for PD. However, the poor differentiation and maturation of DA neurons and decreased cell survival after transplantation are a challenge. Tetrahydroxystilbene glucoside (2,3,5,4'-tetrahydroxystilbene-2-O-glucoside; TSG), an active component of the popular traditional Chinese medicinal plant Polygonum multiï¬orum Thunb, possesses multiple pharmacological actions. In this study, we determined whether TSG can induce neural stem cell (NSCs) differentiation into neurons, especially DA neurons, and the possible involvement of Wnt/ß-catenin signaling pathways. Results revealed that NSCs differentiated primarily into astrocytes when cultured in 2 % serum-containing medium. However, TSG treatment during NSC differentiation in vitro increased the number of Tuj-1-positive neurons, as well as the proportion of tyrosine hydroxylase(TH)-positive cells and dopamine- transporter- positive neurons, a late marker of mature DA neurons. We also found that TSG enhanced the expression of nuclear receptor related factor 1, a transcription factor specific for the development and maintenance of midbrain DA neurons in inducing NSC differentiation into TH -immunoreactive DA neurons. Moreover, TSG upregulated the expression of Wnt/ß-catenin signaling molecules (Wnt1, Wnt3a, Wnt5a, and ß-catenin). However, these promoting effects were significantly inhibited by the application of IWR1, a Wnt signaling-specific blocker in culture. Our findings suggested that TSG may have potential in inducing the DA neuronal differentiation of mouse NSCs mediated by triggering the Wnt/ß-catenin signaling pathway. These results indicated the possible role for TSG in the transplantation of NSCs for PD.
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Diferenciación Celular/efectos de los fármacos , Neuronas Dopaminérgicas/efectos de los fármacos , Glucósidos/farmacología , Mesencéfalo/efectos de los fármacos , Células-Madre Neurales/efectos de los fármacos , Estilbenos/farmacología , Animales , Diferenciación Celular/fisiología , Células Cultivadas , Neuronas Dopaminérgicas/fisiología , Femenino , Glucósidos/uso terapéutico , Mesencéfalo/citología , Mesencéfalo/fisiología , Ratones , Ratones Endogámicos BALB C , Células-Madre Neurales/fisiología , Enfermedad de Parkinson/fisiopatología , Enfermedad de Parkinson/terapia , Embarazo , Estilbenos/uso terapéutico , Vía de Señalización Wnt/efectos de los fármacos , Vía de Señalización Wnt/fisiologíaRESUMEN
INTRODUCTION: GPCR share a common structural feature, i.e., the presence of seven trans-membrane helices having three intracellular and three extracellular loops. The carboxyl terminal is intracellular whereas amino terminal is extracellular. Various conformational changes are observed in structure of GPCR during the binding with ligand, coupling with G protein and interaction with other proteins. In Rhodopsin class of GPCR the basic structure of GPCR is resolved by X-ray crystallography. Ligand acts as an extracellular stimulus for GPCRs to bring physiological changes in organisms. GPR139 has been found to have effective physiological role in primary dopaminergic midbrain neurons and in central nervous system. Recent reports suggested that the ligand of GPR139 protein inhibits the growth of primary dopaminergic midbrain neurons in central nervous system. These discoveries indicated the potential involvement and influence of GPR139 protein in central nervous system METHOD: Therefore, we used multi-approach analysis to investigate the role of GPR139 in the molecular mechanisms of central nervous system. In silico screening was performed to study compound 1 binding with GPR139 protein in their predicted three-dimensional structures. Compound 1 was subjected to molecular dynamics (MD) simulation and stability analysis. RESULTS: The results of MD analysis suggested that the loop region in GPR139 protein structure could affect its binding with drugs. Finally, we cross-validated the predicted compound 1 through systems biology approach. Our results suggested that GPR139 might play an important role in primary dopaminergic midbrain neurons therapy.
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Neuronas Dopaminérgicas/citología , Evaluación Preclínica de Medicamentos , Mesencéfalo/citología , Simulación de Dinámica Molecular , Neuroprotección , Receptores Acoplados a Proteínas G/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Biología de Sistemas , Sitios de Unión , Diabetes Mellitus Tipo 2/metabolismo , Neuronas Dopaminérgicas/efectos de los fármacos , Humanos , Simulación del Acoplamiento Molecular , Neuroprotección/efectos de los fármacos , Enfermedad de Parkinson/metabolismo , Dominios Proteicos , Receptores Acoplados a Proteínas G/química , Transducción de Señal/efectos de los fármacos , Bibliotecas de Moléculas Pequeñas/química , Relación Estructura-Actividad , Factores de TiempoAsunto(s)
Supervivencia Celular/efectos de los fármacos , Neuronas Dopaminérgicas/efectos de los fármacos , Ilex paraguariensis , Mesencéfalo/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Extractos Vegetales/farmacología , Animales , Células Cultivadas , Neuronas Dopaminérgicas/citología , Mesencéfalo/citología , RatasRESUMEN
The perioculomotor (pIII) region of the midbrain was postulated as a sleep-regulating center in the 1890s but largely neglected in subsequent studies. Using activity-dependent labeling and gene expression profiling, we identified pIII neurons that promote non-rapid eye movement (NREM) sleep. Optrode recording showed that pIII glutamatergic neurons expressing calcitonin gene-related peptide alpha (CALCA) are NREM-sleep active; optogenetic and chemogenetic activation/inactivation showed that they strongly promote NREM sleep. Within the pIII region, CALCA neurons form reciprocal connections with another population of glutamatergic neurons that express the peptide cholecystokinin (CCK). Activation of CCK neurons also promoted NREM sleep. Both CALCA and CCK neurons project rostrally to the preoptic hypothalamus, whereas CALCA neurons also project caudally to the posterior ventromedial medulla. Activation of each projection increased NREM sleep. Together, these findings point to the pIII region as an excitatory sleep center where different subsets of glutamatergic neurons promote NREM sleep through both local reciprocal connections and long-range projections.
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Hipotálamo/metabolismo , Mesencéfalo/metabolismo , Neuronas/metabolismo , Fases del Sueño/fisiología , Animales , Colecistoquinina/metabolismo , Hipotálamo/citología , Mesencéfalo/citología , Ratones , Ratones Transgénicos , Neuronas/citología , OptogenéticaRESUMEN
Lipopolysaccharide (LPS)-induced neuroinflammation triggers and accelerates the pathogenesis of Parkinson's disease (PD). Carthamus tinctorius L., a traditional Chinese medicine, has been widely used for the treatment of cerebrovascular disease. Hydroxysafflor Yellow A (HSYA) is an active component of C. tinctorius. The purpose of this study was to investigate whether HSYA could attenuate LPS-induced neurotoxicity and neuroinflammation in primary mesencephalic cultures. Cell viability was measured by MTT and LDH assays. The number of tyrosine hydroxylase (TH) positive neuron was observed by immunohistochemistry. NF-κB p65 and iNOS expressions were evaluated with western blotting method. Pro-inflammatory cytokines including IL-1ß and TNF-α were determined by ELISA kits. Nitric oxide (NO) content in the culture medium was assayed. The results showed that HSYA treatment significantly attenuated the LPS-induced dopaminergic neurons damage. HSYA partially inhibited the expressions of NF-κB p65 and iNOS. Furthermore, HSYA decreased the content of IL-1ß, TNF-α and NO in the supernatants. Taken together, these results suggest that HSYA exerts protective effects on LPS-induced neurotoxicity in dopaminergic neurons and the mechanisms may be associated with the inhibition of inflammatory response.
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Chalcona/análogos & derivados , Inflamación/tratamiento farmacológico , Lipopolisacáridos/farmacología , Neuronas/efectos de los fármacos , Quinonas/farmacología , Animales , Carthamus tinctorius/química , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Chalcona/química , Chalcona/farmacología , Citocinas/metabolismo , Humanos , Mesencéfalo/citología , Ratones Endogámicos C57BL , Neuronas/metabolismo , Óxidos de Nitrógeno , Cultivo Primario de Células , Quinonas/química , Transducción de Señal , Distribución Tisular , Factor de Transcripción ReIA/metabolismoRESUMEN
OBJECTIVES: Nuclear factor erythroid 2-related factor (Nrf2) in astrocyte plays important roles in brain homeostasis. Fermented papaya preparation (FPP) has anti-oxidative, anti-inflammatory, immunoregulatory properties. The present study investigated the effects of FPP on activation of Nrf2 and release of Nrf2-regulated neuroprotective antioxidants and detoxifying molecules. METHODS: Primary cultured astrocytes from rat embryos were treated with FPP for 6 or 24 hours. The expression levels of nuclear Nrf2 and cytoplasmic Nrf2-regulated molecules were determined by western blot analysis and immunohistochemistry. Glutathione levels were measured in cells and medium. Dopaminergic neurons were exposed 6-hydroxydopamine (6-OHDA) with/without pre-treatment with FPP astrocytes. Mice were treated orally with FPP for 2 weeks. RESULTS: FPP increased nuclear translocation of Nrf2 in striatal astrocytes, induced up-regulation of NAD(P)H quinine oxidoreductase-1, glutathione-S transferase and hemeoxygenase-1, and increased glutathione level and the percentage of metallothionein-expressing astrocytes. Moreover, FPP suppressed 6-OHDA-induced dopaminergic neuronal loss in not only neuron-astrocyte mixed culture, but also neuron-rich cultures pre-treated with glial conditioned medium. Two-week oral treatment of mice with FPP resulted in Nrf2 activation and increase in glutathione level in striatum. DISCUSSION: The results indicated that FPP enhances the anti-oxidative capacity through activation of Nrf2 in astrocytes, suggesting it may provide neuroprotection in oxidative stress-related neurodegenerative diseases.
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Astrocitos/metabolismo , Carica/química , Suplementos Dietéticos , Frutas/química , Factor 2 Relacionado con NF-E2/agonistas , Fármacos Neuroprotectores/metabolismo , Transducción de Señal , Animales , Antioxidantes/administración & dosificación , Antioxidantes/metabolismo , Astrocitos/citología , Carica/crecimiento & desarrollo , Células Cultivadas , Azúcares de la Dieta/administración & dosificación , Azúcares de la Dieta/metabolismo , Suplementos Dietéticos/microbiología , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/metabolismo , Fermentación , Feto/citología , Frutas/crecimiento & desarrollo , Glucosa/administración & dosificación , Glucosa/metabolismo , Masculino , Mesencéfalo/citología , Mesencéfalo/metabolismo , Ratones Endogámicos ICR , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas del Tejido Nervioso/agonistas , Proteínas del Tejido Nervioso/metabolismo , Fármacos Neuroprotectores/administración & dosificación , Estrés Oxidativo , Ratas Sprague-Dawley , Corteza Visual/citología , Corteza Visual/metabolismoRESUMEN
Neurons in cortical layer 5B (L5B) connect the cortex to numerous subcortical areas. Possibly the best-studied L5B cortico-subcortical connection is that between L5B neurons in the rodent barrel cortex (BC) and the posterior medial nucleus of the thalamus (POm). However, the spatial organization of L5B giant boutons in the POm and other subcortical targets is not known, and therefore it is unclear if this descending pathway retains somatotopy, i.e., body map organization, a hallmark of the ascending somatosensory pathway. We investigated the organization of the descending L5B pathway from the BC by dual-color anterograde labeling. We reconstructed and quantified the bouton clouds originating from adjacent L5B columns in the BC in three dimensions. L5B cells target six nuclei in the anterior midbrain and thalamus, including the posterior thalamus, the zona incerta, and the anterior pretectum. The L5B subcortical innervation is target specific in terms of bouton numbers, density, and projection volume. Common to all target nuclei investigated here is the maintenance of projection topology from different barrel columns in the BC, albeit with target-specific precision. We estimated low cortico-subcortical convergence and divergence, demonstrating that the L5B corticothalamic pathway is sparse and highly parallelized. Finally, the spatial organization of boutons and whisker map organization revealed the subdivision of the posterior group of the thalamus into four subnuclei (anterior, lateral, medial, and posterior). In conclusion, corticofugal L5B neurons establish a widespread cortico-subcortical network via sparse and somatotopically organized parallel pathways.
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Mesencéfalo , Red Nerviosa , Neuronas , Tálamo , Animales , Mesencéfalo/citología , Mesencéfalo/fisiología , Ratones , Red Nerviosa/citología , Red Nerviosa/fisiología , Neuronas/citología , Neuronas/fisiología , Tálamo/citología , Tálamo/fisiologíaRESUMEN
Mitochondria are sensitive targets of environmental chemicals. Dieldrin (DLD) is an organochlorine pesticide that remains a human health concern due to high lipid bioaccumulation, and it has been epidemiologically associated to an increased risk for Parkinson's disease (PD). As mitochondrial dysfunction is involved in the etiology of PD, this study aimed to determine whether DLD impaired mitochondrial bioenergetics in dopaminergic cells. Rat immortalized dopaminergic N27 cells were treated for 24 or 48h with one dose of either a solvent control, 2.5, 25, or 250µM DLD. Dopaminergic cells treated with 250µM DLD showed increased Casp3/7 activity at 24 and 48h. DLD also caused a dose dependent reduction in cell viability of â¼25-30% over 24h. No significant effects on cell viability, apoptosis, nor cytotoxicity were detected at 24 or 48h with 2.5µM DLD. Following a 24h exposure to 2.5 and 25µM DLD, viable cells were subjected to a mitochondrial stress test using the Seahorse XFe24 Extracellular Flux Analyzer. Following three independent experiments conducted for rigor, dopaminergic cells that were treated with 2.5 and 25µM DLD consistently showed a reduction in maximum respiration and spare capacity compared to the control group. Molecular responses were measured to determine mechanisms of DLD-induced mitochondrial dysfunction. There were no changes in transcripts associated with mitochondrial membrane potential and permeability (e.g. Ant, Hk1, Tspo, Vdac), nor PI3 K/Akt/mTor signaling or mitochondrial-associated apoptotic factors (Bax, Bcl2, Casp3). However, transcript levels for Chop/Gadd153 (DNA Damage Inducible Transcript 3), an apoptotic gene activated following endoplasmic reticulum (ER) stress, were 3-fold higher in N27 cells treated with DLD, suggesting that DLD-induced mitochondrial dysfunction is related to ER stress. Dopamine cells were also assessed for changes in tyrosine hydroxylase (TH) protein, which did not differ among treatments. This study demonstrates that DLD impairs oxidative respiration in dopamine cells, and ER stress is hypothesized to be associated with the DLD-induced mitochondrial dysfunction. This is important as ER stress is also linked to PD. This study presents mechanistic insight into pesticide-induced mitochondrial dysfunction using a chemical that is reported to be associated to a higher risk for neurodegenerative disease.
Asunto(s)
Dieldrín/farmacología , Neuronas Dopaminérgicas/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Neurotoxinas/farmacología , Adenosina Trifosfato/metabolismo , Animales , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Neuronas Dopaminérgicas/ultraestructura , Inhibidores Enzimáticos/farmacología , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Mesencéfalo/citología , Oligomicinas/farmacología , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Ratas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
In mice, various instinctive behaviors can be triggered by olfactory input. Despite growing knowledge of the brain regions involved in such behaviors, the organization of the neural circuits that convert olfactory input into stereotyped behavioral output remains poorly understood. Here, we mapped the neural circuit responsible for enhancing sexual receptivity of female mice by a male pheromone, exocrine gland-secreting peptide 1 (ESP1). We revealed specific neural types and pathways by which ESP1 information is conveyed from the peripheral receptive organ to the motor-regulating midbrain via the amygdala-hypothalamus axis. In the medial amygdala, a specific type of projection neurons gated ESP1 signals to the ventromedial hypothalamus (VMH) in a sex-dependent manner. In the dorsal VMH, which has been associated with defensive behaviors, a selective neural subpopulation discriminately mediated ESP1 information from a predator cue. Together, our data illuminate a labeled-line organization for controlling pheromone-mediated sexual behavioral output in female mice.
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Amígdala del Cerebelo/metabolismo , Hipotálamo/metabolismo , Mesencéfalo/metabolismo , Red Nerviosa/metabolismo , Neuronas/metabolismo , Proteínas/metabolismo , Atractivos Sexuales/metabolismo , Conducta Sexual Animal/fisiología , Amígdala del Cerebelo/citología , Amígdala del Cerebelo/fisiología , Animales , Señales (Psicología) , Femenino , Hipotálamo/citología , Hipotálamo/fisiología , Péptidos y Proteínas de Señalización Intercelular , Masculino , Mesencéfalo/citología , Mesencéfalo/fisiología , Ratones , Ratones Transgénicos , Red Nerviosa/fisiología , Neuronas/fisiología , Conducta Predatoria , Caracteres SexualesRESUMEN
Neurotrophic factors are among the most promising treatments aiming at slowing or stopping and even reversing Parkinson's disease (PD). However, in most cases, they cannot readily cross the human blood-brain-barrier (BBB). Herein, we propose as a therapeutic for PD the small molecule 17-beta-spiro-[5-androsten-17,2'-oxiran]-3beta-ol (BNN-20), a synthetic analogue of DHEA, which crosses the BBB and is deprived of endocrine side-effects. Using the "weaver" mouse, a genetic model of PD, which exhibits progressive dopaminergic neurodegeneration in the Substantia Nigra (SN), we have shown that long-term administration (P1-P21) of BNN-20 almost fully protected the dopaminergic neurons and their terminals, via i) a strong anti-apoptotic effect, probably mediated through the Tropomyosin receptor kinase B (TrkB) neurotrophin receptor's PI3K-Akt-NF-κB signaling pathway, ii) by exerting an efficient antioxidant effect, iii) by inducing significant anti-inflammatory activity and iv) by restoring Brain-Derived Neurotrophic Factor (BDNF) levels. By intercrossing "weaver" with NGL mice (dual GFP/luciferase-NF-κΒ reporter mice, NF-κΒ.GFP.Luc), we obtained Weaver/NGL mice that express the NF-κB reporter in all somatic cells. Acute BNN-20 administration to Weaver/NGL mice induced a strong NF-κB-dependent transcriptional response in the brain as detected by bioluminescence imaging, which was abolished by co-administration of the TrkB inhibitor ANA-12. This indicates that BNN-20 exerts its beneficial action (at least in part) through the TrkB-PI3K-Akt-NF-κB signaling pathway. These results could be of clinical relevance, as they suggest BNN-20 as an important neuroprotective agent acting through the TrkB neurotrophin receptor pathway, mimicking the action of the endogenous neurotrophin BDNF. Thus BNN-20 could be proposed for treatment of PD.
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Deshidroepiandrosterona/análogos & derivados , Dopamina/metabolismo , Neuronas Dopaminérgicas/efectos de los fármacos , Mesencéfalo/citología , Receptor trkB/metabolismo , Adyuvantes Inmunológicos/farmacología , Animales , Animales Recién Nacidos , Antígenos CD1/metabolismo , Azepinas/farmacología , Benzamidas/farmacología , Células CHO , Cricetulus , Deshidroepiandrosterona/farmacología , Proteínas de Transporte de Dopamina a través de la Membrana Plasmática/metabolismo , Femenino , Masculino , Mesencéfalo/efectos de los fármacos , Mesencéfalo/metabolismo , Ratones , Ratones Mutantes Neurológicos , Modelos Genéticos , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Tubulina (Proteína)/metabolismo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
In this study, to study the effect of rhynchophylline on TH in midbrain of methamphetamine-induced conditioned place preference (CPP) adult zebrafish, place preference adult zebrafish models were established by methamphetamine (40µg/g) and the expression of TH was observed by immunohistochemistry technique and Western blot. Ketamine (150µg/g), high dose of rhynchophylline (100µg/g) group can significantly reduce the place preference; immunohistochemistry results showed that the number of TH-positive neurons in midbrain was increased in the methamphetamine model group, whereas less TH-positive neurons were found in the ketamine group and high dosage rhynchophylline group. Western blot results showed that the expression of TH protein was significantly increased in the model group, whereas less expression was found in the ketamine group, high dosage rhynchophylline group. Our data pointed out that TH plays an important role in the formation of methamphetamine-induced place preference in adult zebrafish. Rhynchophylline reversed the expression of TH in the midbrain demonstrates the potential effect of mediates methamphetamine induced rewarding effect.
Asunto(s)
Condicionamiento Operante/efectos de los fármacos , Alcaloides Indólicos/farmacología , Metanfetamina/farmacología , Tirosina 3-Monooxigenasa/metabolismo , Pez Cebra , Trastornos Relacionados con Anfetaminas , Animales , Conducta Animal/efectos de los fármacos , Ketamina/farmacología , Masculino , Mesencéfalo/citología , Mesencéfalo/enzimología , Neuronas/enzimología , OxindolesRESUMEN
Previous research has shown that nitric oxide (NO) synthase inhibitors prevent rodents' sensorimotor gating impairments induced by dopamine releasing drugs, such as amphetamine (Amph) and methylphenidate. The mechanisms of this effect have not been entirely understood. In the present work, we investigated some possible mechanisms by which the NO donor, NOC-12 (3-ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene), influence spontaneous and Amph-induced dopamine release, using rat mesencephalic primary cultured neurons preparations. Our results showed that NOC-12 increased dopamine release in a concentration-dependent manner and potentiated the Amph-induced one. Dopamine release induced by NOC-12 was disrupted by N-acetyl-L-cystein (NAC-a free radical scavenger) and MK-801, a NMDA (N-methyl-D-aspartate) non-competitive antagonist, and was concentration dependently affected by oxadiazolo[4,3]quinoxalin-1-one, an inhibitor of the soluble guanylate cyclase (sGC). In contrast, dopamine released by Amph was facilitated by NAC and by MK-801 and not affected by nifedipine (a L-type-Ca(+2) channel blocker), which enhanced NOC-12-induced dopamine release. The present work demonstrates that DA release induced by NOC-12 is partially dependent on sGC and on NMDA activation, and is modulated by L-type Ca(+2) channel and the antioxidant NAC. This mechanism differs from the Amph-induced one, which appears not to depend on L-type Ca(+2) channel and seems to be facilitated by NMDA channel blocking and by NAC. These results suggest that Amph and NOC-12 induce dopamine release through complementary pathways, which may explain the potentiation of Amph-induced dopamine release by NOC-12. These findings contribute to understand the involvement of NO in dopamine-related neuropsychiatric and neurodegenerative diseases.
Asunto(s)
Anfetamina/farmacología , Dopamina/metabolismo , Mesencéfalo/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Donantes de Óxido Nítrico/farmacología , Animales , Bloqueadores de los Canales de Calcio/farmacología , Células Cultivadas , Maleato de Dizocilpina/farmacología , Embrión de Mamíferos , Femenino , Fármacos Neuroprotectores/farmacología , Nifedipino/farmacología , Nitritos/metabolismo , Compuestos Nitrosos/farmacología , Embarazo , Ratas , Ratas Wistar , Transducción de Señal/efectos de los fármacos , Factores de TiempoRESUMEN
Dietary supplements containing plant materials of Annonaceae species (Annona muricata L., A. squamosa L., A. mucosa JACQ., A. squamosa × cherimola Mabb.) were extracted by hot, pressurized ethyl acetate and analyzed for their effect in vitro on Lund human mesencephalic neurons. Cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and cell death was determined by lactate dehydrogenase levels. Three supplements strongly decreased the cell viability at extract concentrations of 1 µg/mL, of which 1 decreased cell viability at 0.1 µg/µL. Also, strong neuronal toxicities of these supplements were found. Cell death was observed at concentrations of 10 µg/mL. The degree of toxicity was comparable to the ones found in Annonaceous fruit extracts. Two fruit pulps of Annonaceae (A. muricata and A. squamosa) showed a reduction in cell viability at lower concentrations. The fruit pulp extract of A. muricata revealed the strongest neurotoxic effect, with 67% cell death at a concentration of 1 µg/mL. A high reduction in cell viability coupled with pronounced cell death was found at 0.1 µg/mL for an Annonaceous seed extract. These results demonstrate that the intake of dietary supplements containing plant material from Annonaceae may be hazardous to health in terms of neurotoxicity.
Asunto(s)
Annonaceae/toxicidad , Suplementos Dietéticos/toxicidad , Síndromes de Neurotoxicidad/patología , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Frutas/química , Frutas/toxicidad , Humanos , L-Lactato Deshidrogenasa/análisis , L-Lactato Deshidrogenasa/metabolismo , Mesencéfalo/citología , Mesencéfalo/efectos de los fármacos , Neuronas/efectos de los fármacos , Extractos Vegetales/toxicidad , Semillas/química , Semillas/toxicidad , Sales de Tetrazolio , TiazolesRESUMEN
Resveratrol is originally extracted from huzhang, a Chinese herbal medicine. Recently, resveratrol has attracted a great of attention due to its antioxidant and antiapoptotic properties. Although the neuroprotection of resveratrol on neural damages in various models has been well characterized, little is known about the role of resveratrol in methamphetamine (MA) induced neurotoxicity in mesencephalic dopaminergic neurons. Dopaminergic neurons were isolated from midbrain of mouse embryos at embryonic day 15 and cultured in the presence of MA and resveratrol. Cell viability was examined by MTT assay and the apoptosis was assessed using Hoechst33342/PI double staining. To evaluate the Oxidative damage, ROS assay was performed. Moreover, the changes of time course of intracellular free calcium concentration ([Ca(2+) ]i) were analyzed with Fluo-3/AM tracing. The data showed that MA induced the neurotoxicity of cultured cells in a dose-dependent manner. Resveratrol significantly increased cellular viability and retarded cell apoptosis. Furthermore, resveratrol also attenuated MA induced ROS production and intracellular free calcium overload. Our results suggest that resveratrol protects dopaminergic neurons from MA-induced neuronal cytotoxicity, which, at least partly, is mediated by inhibition of [Ca(2+) ]i and oxidative stress. © 2015 BioFactors 41(4):252-260, 2015.
Asunto(s)
Antioxidantes/farmacología , Inhibidores de Captación de Dopamina/antagonistas & inhibidores , Neuronas Dopaminérgicas/efectos de los fármacos , Metanfetamina/antagonistas & inhibidores , Fármacos Neuroprotectores/farmacología , Estilbenos/farmacología , Compuestos de Anilina , Animales , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Supervivencia Celular/efectos de los fármacos , Dopamina/metabolismo , Inhibidores de Captación de Dopamina/toxicidad , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/metabolismo , Embrión de Mamíferos , Colorantes Fluorescentes , Mesencéfalo/citología , Mesencéfalo/efectos de los fármacos , Mesencéfalo/metabolismo , Metanfetamina/toxicidad , Ratones , Estrés Oxidativo , Cultivo Primario de Células , Especies Reactivas de Oxígeno/agonistas , Especies Reactivas de Oxígeno/antagonistas & inhibidores , Especies Reactivas de Oxígeno/metabolismo , Resveratrol , XantenosRESUMEN
Glioblastoma multiforme is a highly aggressive brain tumor whose prognosis is very poor. Due to early invasion of brain parenchyma, its complete surgical removal is nearly impossible, and even after aggressive combined treatment (association of surgery and chemo- and radio-therapy) five-year survival is only about 10%. Natural products are sources of novel compounds endowed with therapeutic properties in many human diseases, including cancer. Here, we report that the water extract of Ruta graveolens L., commonly known as rue, induces death in different glioblastoma cell lines (U87MG, C6 and U138) widely used to test novel drugs in preclinical studies. Ruta graveolens' effect was mediated by ERK1/2 and AKT activation, and the inhibition of these pathways, via PD98058 and wortmannin, reverted its antiproliferative activity. Rue extract also affects survival of neural precursor cells (A1) obtained from embryonic mouse CNS. As in the case of glioma cells, rue stimulates the activation of ERK1/2 and AKT in A1 cells, whereas their blockade by pharmacological inhibitors prevents cell death. Interestingly, upon induction of differentiation and cell cycle exit, A1 cells become resistant to rue's noxious effects but not to those of temozolomide and cisplatin, two alkylating agents widely used in glioblastoma therapy. Finally, rutin, a major component of the Ruta graveolens water extract, failed to cause cell death, suggesting that rutin by itself is not responsible for the observed effects. In conclusion, we report that rue extracts induce glioma cell death, discriminating between proliferating/undifferentiated and non-proliferating/differentiated neurons. Thus, it can be a promising tool to isolate novel drugs and also to discover targets for therapeutic intervention.
Asunto(s)
Apoptosis/efectos de los fármacos , Glioblastoma/patología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Células-Madre Neurales/citología , Extractos Vegetales/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ruta/química , Animales , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Activación Enzimática/efectos de los fármacos , Humanos , Mesencéfalo/citología , Mesencéfalo/efectos de los fármacos , Ratones , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Agua/químicaRESUMEN
Parkinson's disease (PD) is an obstinate progressive neurodegenerative disease and characterized by locomotor impairment and dopaminergic neuronal degeneration in the substantia nigra pars compacta (SNc). We examined in here the dietary effect of nucleoprotein (NP) extracted from salmon soft roe on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-injected PD-like mice model to prevent the symptom as an alternative medicine. Male C57/BL6 mice were given either an artificially modified NP-free diet (NF) or NF supplied with 1.2% NP for 1 week. Then, mice were injected intraperitoneally four times with 20 mg/kg MPTP. Seven days later, locomotor activity was examined, and the brains were immunostained with tyrosine hydroxylase (TH) and Iba1 antibodies. Moreover, in situ detection of superoxide anion (O2(-)) and gene expression of mitochondrial electron transfer chain gene, Cox8b was evaluated in midbrains. NP-fed animals showed significantly reduced locomotor impairment and an increased number of TH-positive cells in the SNc compared with NF animals. The NP-fed animals also showed reduced lower levels of O2(-) and up-regulation of Cox8b levels and Iba1 immunoreactivity, suggesting that inflammation and oxidative stress were suppressed and mitochondrial impairment was relieved in these animals. Supplementation of the diet with NP may serve as a useful preventive measure to slow the onset of PD.