RESUMEN
The cell-intrinsic mechanisms underlying the decision of a stem/progenitor cell to either proliferate or differentiate remain incompletely understood. Here, we identify the transmembrane protein Lrig1 as a physiological homeostatic regulator of FGF2-driven proliferation and self-renewal of neural progenitors at early-to-mid embryonic stages of cortical development. We show that Lrig1 is expressed in cortical progenitors (CPs), and its ablation caused expansion and increased proliferation of radial/apical progenitors and of neurogenic transit-amplifying Tbr2+ intermediate progenitors. Notably, our findings identify a previously unreported EGF-independent mechanism through which Lrig1 negatively regulates neural progenitor proliferation by modulating the FGF2-induced IL6/Jak2/Stat3 pathway, a molecular cascade that plays a pivotal role in the generation and maintenance of CPs. Consistently, Lrig1 knockout mice showed a significant increase in the density of pyramidal glutamatergic neurons placed in superficial layers 2 and 3 of the postnatal neocortex. Together, these results support a model in which Lrig1 regulates cortical neurogenesis by influencing the cycling activity of a set of progenitors that are temporally specified to produce upper layer glutamatergic neurons.
Asunto(s)
Janus Quinasa 2 , Glicoproteínas de Membrana , Ratones Noqueados , Células-Madre Neurales , Neurogénesis , Neuronas , Factor de Transcripción STAT3 , Transducción de Señal , Animales , Factor de Transcripción STAT3/metabolismo , Factor de Transcripción STAT3/genética , Janus Quinasa 2/metabolismo , Células-Madre Neurales/metabolismo , Células-Madre Neurales/citología , Ratones , Neurogénesis/genética , Neuronas/metabolismo , Neuronas/citología , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Proliferación Celular , Corteza Cerebral/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/embriología , Diferenciación Celular , Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas del Tejido NerviosoRESUMEN
The symptoms of fragile X syndrome (FXS), caused by a single gene mutation to Fmr1, have been increasingly linked to disordered astrocyte signalling within the cerebral cortex. We have recently demonstrated that the purinergic signalling pathway, which utilizes nucleoside triphosphates and their metabolites to facilitate bidirectional glial and glial-neuronal interactions, is upregulated in cortical astrocytes derived from the Fmr1 knockout (KO) mouse model of FXS. Heightened Fmr1 KO P2Y purinergic receptor levels were correlated with prolonged intracellular calcium release, elevated synaptogenic protein secretion, and hyperactivity of developing circuits. However, due to the relative lack of sensitive and reproducible quantification methods available for measuring purines and pyrimidines, determining the abundance of these factors in Fmr1 KO astrocytes was limited. We therefore developed a hydrophilic interaction liquid chromatography protocol coupled with mass spectrometry to compare the abundance of intracellular and extracellular purinergic molecules between wildtype and Fmr1 KO mouse astrocytes. Significant differences in the concentrations of UDP, ATP, AMP, and adenosine intracellular stores were found within Fmr1 KO astrocytes relative to WT. The extracellular level of adenosine was also significantly elevated in Fmr1 KO astrocyte-conditioned media in comparison to media collected from WT astrocytes. Glycosylation of the astrocyte membrane-bound CD39 ectonucleotidase, which facilitates ligand breakdown following synaptic release, was also elevated in Fmr1 KO astrocyte cultures. Together, these differences demonstrated further dysregulation of the purinergic signalling system within Fmr1 KO cortical astrocytes, potentially leading to significant alterations in FXS purinergic receptor activation and cellular pathology.
Asunto(s)
Astrocitos , Corteza Cerebral , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Síndrome del Cromosoma X Frágil , Ratones Noqueados , Transducción de Señal , Animales , Astrocitos/metabolismo , Ratones , Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Corteza Cerebral/metabolismo , Corteza Cerebral/citología , Apirasa/genética , Apirasa/metabolismo , Células Cultivadas , Adenosina Trifosfato/metabolismo , Medios de Cultivo Condicionados , Adenosina/metabolismo , Adenosina/análogos & derivados , Receptores Purinérgicos P2Y/metabolismo , Receptores Purinérgicos P2Y/genética , Ratones Endogámicos C57BL , Antígenos CDRESUMEN
Cortical neurons exhibit temporally irregular spiking patterns and heterogeneous firing rates. These features arise in model circuits operating in a 'fluctuation-driven regime', in which fluctuations in membrane potentials emerge from the network dynamics. However, it is still debated whether the cortex operates in such a regime. We evaluated the fluctuation-driven hypothesis by analyzing spiking and sub-threshold membrane potentials of neurons in the frontal cortex of mice performing a decision-making task. We showed that while standard fluctuation-driven models successfully account for spiking statistics, they fall short in capturing the heterogeneity in sub-threshold activity. This limitation is an inevitable outcome of bombarding single-compartment neurons with a large number of pre-synaptic inputs, thereby clamping the voltage of all neurons to more or less the same average voltage. To address this, we effectively incorporated dendritic morphology into the standard models. Inclusion of dendritic morphology in the neuronal models increased neuronal selectivity and reduced error trials, suggesting a functional role for dendrites during decision-making. Our work suggests that, during decision-making, cortical neurons in high-order cortical areas operate in a fluctuation-driven regime.
Asunto(s)
Potenciales de Acción , Modelos Neurológicos , Neuronas , Animales , Neuronas/fisiología , Ratones , Potenciales de Acción/fisiología , Corteza Cerebral/fisiología , Corteza Cerebral/citología , Toma de Decisiones/fisiología , Potenciales de la Membrana/fisiología , Dendritas/fisiología , Masculino , Ratones Endogámicos C57BL , Lóbulo Frontal/fisiología , Lóbulo Frontal/citologíaRESUMEN
The emergence of myelinating oligodendrocytes represents a pivotal developmental milestone in vertebrates, given their capacity to ensheath axons and facilitate the swift conduction of action potentials. It is widely accepted that cortical oligodendrocyte progenitor cells (OPCs) arise from medial ganglionic eminence (MGE), lateral/caudal ganglionic eminence (LGE/CGE), and cortical radial glial cells (RGCs). Here, we used two different fate mapping strategies to challenge the established notion that the LGE generates cortical OPCs. Furthermore, we used a Cre/loxP-dependent exclusion strategy to reveal that the LGE/CGE does not give rise to cortical OPCs. Additionally, we showed that specifically eliminating MGE-derived OPCs leads to a significant reduction of cortical OPCs. Together, our findings indicate that the LGE does not generate cortical OPCs, contrary to previous beliefs. These findings provide a new view of the developmental origins of cortical OPCs and a valuable foundation for future research on both normal development and oligodendrocyte-related disease.
Asunto(s)
Corteza Cerebral , Oligodendroglía , Animales , Oligodendroglía/fisiología , Oligodendroglía/citología , Ratones , Corteza Cerebral/embriología , Corteza Cerebral/fisiología , Corteza Cerebral/citología , Células Precursoras de Oligodendrocitos/fisiología , Células Precursoras de Oligodendrocitos/citología , Diferenciación Celular , Eminencia GanglionarRESUMEN
Complex sensory information arrives in the brain from an animal's first-person ('egocentric') perspective. However, animals can efficiently navigate as if referencing map-like ('allocentric') representations. The postrhinal (POR) and retrosplenial (RSC) cortices are thought to mediate between sensory input and internal maps, combining egocentric representations of physical cues with allocentric head direction (HD) information. Here we show that neurons in the POR and RSC of female Long-Evans rats are tuned to distinct but complementary aspects of local space. Egocentric bearing (EB) cells recorded in square and L-shaped environments reveal that RSC cells encode local geometric features, while POR cells encode a more global account of boundary geometry. Additionally, POR HD cells can incorporate egocentric information to fire in two opposite directions with two oppositely placed identical visual landmarks, while only a subset of RSC HD cells possess this property. Entorhinal grid and HD cells exhibit consistently allocentric spatial firing properties. These results reveal significant regional differences in the neural encoding of spatial reference frames.
Asunto(s)
Neuronas , Ratas Long-Evans , Percepción Espacial , Animales , Femenino , Neuronas/fisiología , Ratas , Percepción Espacial/fisiología , Señales (Psicología) , Corteza Entorrinal/fisiología , Corteza Entorrinal/citología , Ambiente , Corteza Cerebral/fisiología , Corteza Cerebral/citologíaRESUMEN
Amyloid-beta peptide (Aß) is a neurotoxic constituent of senile plaques in the brains of Alzheimer's disease (AD) patients. The detailed mechanisms by which protein kinase C-delta (PKCδ) contributes to Aß toxicity is not yet entirely understood. Using fully differentiated primary rat cortical neurons, we found that inhibition of Aß25-35-induced PKCδ increased cell viability with restoration of neuronal morphology. Using cyclin D1, proliferating cell nuclear antigen (PCNA), and histone H3 phosphorylated at Ser-10 (p-Histone H3) as the respective markers for the G1-, S-, and G2/M-phases, PKCδ inhibition mitigated cell cycle reentry (CCR) and subsequent caspase-3 cleavage induced by both Aß25-35 and Aß1-42 in the post-mitotic cortical neurons. Upstream of PKCδ, signal transducers and activators of transcription (STAT)-3 mediated PKCδ induction, CCR, and caspase-3 cleavage upon Aß exposure. Downstream of PKCδ, aberrant neuronal CCR was triggered by overactivating cyclin-dependent kinase-5 (CDK5) via calpain2-dependent p35 cleavage into p25. Finally, PKCδ and CDK5 also contributed to Aß25-35 induction of p53-upregulated modulator of apoptosis (PUMA) in cortical neurons. Together, we demonstrated that, in the post-mitotic neurons exposed to Aßs, STAT3-dependent PKCδ expression triggers calpain2-mediated p35 cleavage into p25 to overactivate CDK5, thus leading to aberrant CCR, PUMA induction, caspase-3 cleavage, and ultimately apoptosis.
Asunto(s)
Péptidos beta-Amiloides , Apoptosis , Ciclo Celular , Corteza Cerebral , Neuronas , Proteína Quinasa C-delta , Péptidos beta-Amiloides/metabolismo , Animales , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Apoptosis/efectos de los fármacos , Ratas , Proteína Quinasa C-delta/metabolismo , Corteza Cerebral/metabolismo , Corteza Cerebral/citología , Ciclo Celular/efectos de los fármacos , Quinasa 5 Dependiente de la Ciclina/metabolismo , Fragmentos de Péptidos/farmacología , Fragmentos de Péptidos/metabolismo , Caspasa 3/metabolismo , Ratas Sprague-Dawley , Células Cultivadas , Transducción de Señal/efectos de los fármacosRESUMEN
53BP1 is a well-established DNA damage repair factor that has recently emerged to critically regulate gene expression for tumor suppression and neural development. However, its precise function and regulatory mechanisms remain unclear. Here, we showed that phosphorylation of 53BP1 at serine 25 by ATM is required for neural progenitor cell proliferation and neuronal differentiation in cortical brain organoids. Dynamic phosphorylation of 53BP1-serine 25 controls 53BP1 target genes governing neuronal differentiation and function, cellular response to stress, and apoptosis. Mechanistically, ATM and RNF168 govern 53BP1's binding to gene loci to directly affect gene regulation, especially at genes for neuronal differentiation and maturation. 53BP1 serine 25 phosphorylation effectively impedes its binding to bivalent or H3K27me3-occupied promoters, especially at genes regulating H3K4 methylation, neuronal functions, and cell proliferation. Beyond 53BP1, ATM-dependent phosphorylation displays wide-ranging effects, regulating factors in neuronal differentiation, cytoskeleton, p53 regulation, as well as key signaling pathways such as ATM, BDNF, and WNT during cortical organoid differentiation. Together, our data suggest that the interplay between 53BP1 and ATM orchestrates essential genetic programs for cell morphogenesis, tissue organization, and developmental pathways crucial for human cortical development.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada , Organoides , Proteína 1 de Unión al Supresor Tumoral P53 , Proteína 1 de Unión al Supresor Tumoral P53/metabolismo , Proteína 1 de Unión al Supresor Tumoral P53/genética , Organoides/metabolismo , Humanos , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Proteínas de la Ataxia Telangiectasia Mutada/genética , Fosforilación , Daño del ADN , Corteza Cerebral/metabolismo , Corteza Cerebral/citología , Células-Madre Neurales/metabolismo , Diferenciación Celular/genética , Proliferación Celular , Reparación del ADN , Neurogénesis/genética , Neuronas/metabolismo , Transducción de SeñalRESUMEN
Alzheimer's disease, a prevalent neurodegenerative condition primarily affecting older adults, remains incurable. Its principle pathological hallmark is the accelerated accumulation of amyloid ß (Aß) protein. This study investigates the potential of photobiomodulation using near infrared light to counteract Aß1-42-induced synaptic degeneration and neurotoxicity. We focused on the effect of 808 nm near-infrared laser diode (LD) on Aß1-42 cytotoxicity in primary cultured cortical neurons. We assessed cell survival using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, observing substantial benefits from LD irradiation with a power of 10 mW and a dose of 30 J. Cells exposed to Aß1-42 exhibited morphological changes indicative of synaptic damage and a significant decrease in the number of postsynaptic density protein-95 (PSD-95) contacts, which were significantly improved with near-infrared LD therapy. Furthermore, this therapy reduced Aß and phosphorylated tau (P-tau) protein accumulation. Additionally, near-infrared LD irradiation substantially lessened the Aß1-42-induced rise in glial fibrillary acid protein (GFAP) and ionized calcium-binding adaptor molecule 1 (IBA1) in astrocytes and microglia. Remarkably, near-infrared LD irradiation effectively inhibited phosphorylation of key proteins involved in Aß1-42-induced necroptosis, namely Receptor-interacting protein kinase-3 (RIP3) and Mixed Lineage Kinase domain-Like protein (MLKL). Our findings suggest that near-infrared LD treatment significantly reduces neurodegeneration by reducing glial overactivation and neuronal necroptosis triggered by Aß1-42. Thus, near-infrared LD treatment emerges as a promising approach for slowing or treating Alzheimer's disease, offering new avenues in its management.
Asunto(s)
Péptidos beta-Amiloides , Supervivencia Celular , Rayos Infrarrojos , Neuronas , Fragmentos de Péptidos , Péptidos beta-Amiloides/metabolismo , Péptidos beta-Amiloides/toxicidad , Animales , Neuronas/efectos de la radiación , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Fragmentos de Péptidos/metabolismo , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/efectos de la radiación , Ratas , Láseres de Semiconductores , Proteínas tau/metabolismo , Terapia por Luz de Baja Intensidad , Células Cultivadas , Homólogo 4 de la Proteína Discs Large/metabolismo , Proteína Ácida Fibrilar de la Glía/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/efectos de la radiación , Astrocitos/metabolismo , Astrocitos/efectos de los fármacos , Astrocitos/efectos de la radiaciónRESUMEN
TRPM4 is a non-selective cation channel activated by intracellular Ca2+ but only permeable to monovalent cations, its activation regulates membrane potential and intracellular calcium. This channel participates in the migration and adhesion of non-excitable cells and forms an integral part of the focal adhesion complex. In neurons, TRPM4 expression starts before birth and its function at this stage is not clear, but it may function in processes such as neurite development. Here we investigate the role of TRPM4 in neuritogenesis. We found that neurons at DIV 0 express TRPM4, the inhibition of TRPM4 using 9-Ph reduces neurite number and slows the progression of neurite development, keeping neurons in stage 1. The genetic suppression of TRPM4 using an shRNA at later stages (DIV2) reduces neurite length. Conversely, at DIV 0, TRPM4 inhibition augments the Cch-induced Ca2 + i increase, altering the calcium homeostasis. Together, these results show that TRPM4 participates in progression of neurite development and suggest a critical role of the calcium modulation during this stage of neuronal development.
Asunto(s)
Calcio , Corteza Cerebral , Neuritas , Neurogénesis , Canales Catiónicos TRPM , Canales Catiónicos TRPM/metabolismo , Canales Catiónicos TRPM/antagonistas & inhibidores , Animales , Neuritas/metabolismo , Neuritas/efectos de los fármacos , Calcio/metabolismo , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Neuronas/metabolismoRESUMEN
The invention of Light Emitting Diode (LED) revolutionized energy-efficient illumination, but concerns persist regarding the potential harm of blue light to our eyes. In this study, we scrutinized the impact of LED light characteristics on eyes using two cell types: M-1 (rich in mitochondria) and CD-1 (neuronal). Variations in color rendering index (CRI) and correlated color temperature (CCT) were investigated, alongside exposure durations ranging from 0 to 24 hours. The findings illuminated the potential benefits of high-quality LED lighting, characterized by a high CRI and low CCT, which emits a greater proportion of red light. This form of lighting was associated with enhanced cell proliferation, elevated ATP levels, and reduced oxidative stress. In contrast, LEDs with low CRI and high CCT exhibited adverse effects, diminishing cell viability and increasing oxidative stress. These results suggest that high-quality LED lighting may have neuroprotective potential as a treatment option, such as for retinal ganglion cells.
Asunto(s)
Supervivencia Celular , Luz , Mitocondrias , Neuronas , Estrés Oxidativo , Animales , Ratones , Mitocondrias/metabolismo , Mitocondrias/efectos de la radiación , Supervivencia Celular/efectos de la radiación , Neuronas/metabolismo , Neuronas/efectos de la radiación , Estrés Oxidativo/efectos de la radiación , Corteza Cerebral/efectos de la radiación , Corteza Cerebral/metabolismo , Corteza Cerebral/citología , Línea Celular , Proliferación Celular/efectos de la radiación , Adenosina Trifosfato/metabolismo , IluminaciónRESUMEN
Cortical neurons store information across different timescales, from seconds to years. Although information stability is variable across regions, it can vary within a region as well. Association areas are known to multiplex behaviorally relevant variables, but the stability of their representations is not well understood. Here, we longitudinally recorded the activity of neuronal populations in the mouse retrosplenial cortex (RSC) during the performance of a context-choice association task. We found that the activity of neurons exhibits different levels of stability across days. Using linear classifiers, we quantified the stability of three task-relevant variables. We find that RSC representations of context and trial outcome display higher stability than motor choice, both at the single cell and population levels. Together, our findings show an important characteristic of association areas, where diverse streams of information are stored with varying levels of stability, which may balance representational reliability and flexibility according to behavioral demands.
Asunto(s)
Neuronas , Animales , Neuronas/fisiología , Ratones , Masculino , Ratones Endogámicos C57BL , Conducta de Elección/fisiología , Corteza Cerebral/fisiología , Corteza Cerebral/citología , Giro del Cíngulo/fisiología , Giro del Cíngulo/citología , Conducta Animal/fisiologíaRESUMEN
Oligodendrocyte precursor cells (OPCs) give rise to myelinating oligodendrocytes of the brain. This process persists throughout life and is essential for recovery from neurodegeneration. To better understand the cellular checkpoints that occur during oligodendrogenesis, we determined the mitochondrial distribution and morphometrics across the oligodendrocyte lineage in mouse and human cerebral cortex. During oligodendrocyte generation, mitochondrial content expands concurrently with a change in subcellular partitioning towards the distal processes. These changes are followed by an abrupt loss of mitochondria in the oligodendrocyte processes and myelin, coinciding with sheath compaction. This reorganization and extensive expansion and depletion take 3 days. Oligodendrocyte mitochondria are stationary over days while OPC mitochondrial motility is modulated by animal arousal state within minutes. Aged OPCs also display decreased mitochondrial size, volume fraction, and motility. Thus, mitochondrial dynamics are linked to oligodendrocyte generation, dynamically modified by their local microenvironment, and altered in the aging brain.
Asunto(s)
Mitocondrias , Vaina de Mielina , Oligodendroglía , Animales , Mitocondrias/metabolismo , Humanos , Oligodendroglía/metabolismo , Oligodendroglía/citología , Ratones , Vaina de Mielina/metabolismo , Células Precursoras de Oligodendrocitos/metabolismo , Células Precursoras de Oligodendrocitos/citología , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Ratones Endogámicos C57BL , Masculino , Dinámicas Mitocondriales , Diferenciación Celular , FemeninoRESUMEN
While guided human cortical organoid (hCO) protocols reproducibly generate cortical cell types at one site, variability in hCO phenotypes across sites using a harmonized protocol has not yet been evaluated. To determine the cross-site reproducibility of hCO differentiation, three independent research groups assayed hCOs in multiple differentiation replicates from one induced pluripotent stem cell (iPSC) line using a harmonized miniaturized spinning bioreactor protocol across 3 months. hCOs were mostly cortical progenitor and neuronal cell types in reproducible proportions that were consistently organized in cortical wall-like buds. Cross-site differences were detected in hCO size and expression of metabolism and cellular stress genes. Variability in hCO phenotypes correlated with stem cell gene expression prior to differentiation and technical factors associated with seeding, suggesting iPSC quality and treatment are important for differentiation outcomes. Cross-site reproducibility of hCO cell type proportions and organization encourages future prospective meta-analytic studies modeling neurodevelopmental disorders in hCOs.
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Diferenciación Celular , Corteza Cerebral , Células Madre Pluripotentes Inducidas , Organoides , Humanos , Organoides/citología , Organoides/metabolismo , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Reproducibilidad de los Resultados , Corteza Cerebral/citología , Corteza Cerebral/metabolismo , Neuronas/metabolismo , Neuronas/citología , Técnicas de Cultivo de Célula/métodos , FenotipoRESUMEN
BACKGROUND: - Alcohol-induced pro-inflammatory activation might influence cellular and synaptic pathology, thus contributing to the behavioral phenotypes associated with alcohol use disorders. In the present study, the possible anti-inflammatory properties of N-[(4-trifluoromethyl)-benzyl]4-methoxybutyramide (GET73), a promising therapeutic agent for alcohol use disorder treatment, were evaluated in primary cultures of rat cortical microglia. METHODS: - Primary cultures of cerebral cortex microglial cells were treated with 100 ng/ml lipopolysaccharide (LPS; 8 h, 37 °C) or 75 mM ethanol (EtOH; 4 days, 37 °C) alone or in the presence of GET73 (1-30 µM). At the end of the incubation period, multiparametric quantification of cytokines/chemokines was performed by using the xMAP technology and Luminex platform. Furthermore, cultured microglial cell viability following the treatment with EtOH and GET73, alone or in combination, has been measured by a colorimetric assay (i.e. MTT assay). RESULTS: - GET73 (10 and 30 µM) partially or fully prevented the LPS-induced increase of IL-6, IL-1ß, RANTES/CCL5 protein and MCP-1/CCL2 levels. On the contrary, GET73 failed to attenuate the TNF-α level increase induced by LPS. Furthermore, GET73 treatment (10-30 µM) significantly attenuated or prevented the EtOH-induced increase of TNF-α, IL-6, IL-1ß and MCP-1/CCL2 levels. Finally, at all the concentrations tested (1-30 µM), the GET73 treatment did not alter the EtOH-induced reduction of microglial cell viability. CONCLUSIONS: - The current results provide the first in vitro evidence of GET73 protective properties against EtOH-induced neuroinflammation. These data add more information on the complex and multifactorial profile of action of the compound, further supporting the significance of developing GET73 as a therapeutic tool for the treatment of individuals with alcohol use disorders.
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Supervivencia Celular , Corteza Cerebral , Citocinas , Etanol , Lipopolisacáridos , Microglía , Animales , Microglía/efectos de los fármacos , Microglía/metabolismo , Etanol/farmacología , Lipopolisacáridos/farmacología , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Corteza Cerebral/citología , Ratas , Citocinas/metabolismo , Células Cultivadas , Supervivencia Celular/efectos de los fármacos , Quimiocinas/metabolismo , Ratas Wistar , Antiinflamatorios/farmacologíaRESUMEN
Primary neuronal cultures allow for in vitro analysis of early developmental processes such as axon pathfinding and growth dynamics. When coupled with methods to visualize and measure microtubule dynamics, this methodology enables an inside look at how the cytoskeleton changes in response to extracellular signaling cues. Here, we describe the culturing conditions and tools required to extract primary cortical neurons from postnatal mouse brains and visualize cytoskeletal components.
Asunto(s)
Corteza Cerebral , Neuronas , Animales , Ratones , Neuronas/citología , Neuronas/metabolismo , Corteza Cerebral/citología , Células Cultivadas , Microtúbulos/metabolismo , Cultivo Primario de Células/métodos , Técnicas de Cultivo de Célula/métodos , Citoesqueleto/metabolismoRESUMEN
During the development of mammalian brains, pyramidal neurons in the cerebral cortex form highly organized six layers with different functions. These neurons undergo developmental processes such as axon extension, dendrite outgrowth, and synapse formation. A proper integration of the neuronal connectivity through dynamic changes of dendritic branches and spines is required for learning and memory. Disruption of these crucial developmental processes is associated with many neurodevelopmental and neurodegenerative disorders. To investigate the complex dendritic architecture, several useful staining tools and genetic methods to label neurons have been well established. Monitoring the dynamics of dendritic spine in a single neuron is still a challenging task. Here, we provide a methodology that combines in vivo two-photon brain imaging and in utero electroporation, which sparsely labels cortical neurons with fluorescent proteins. This protocol may help elucidate the dynamics of microstructure and neural complexity in living rodents under normal and disease conditions.
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Neuronas , Animales , Ratones , Neuronas/citología , Neuronas/metabolismo , Electroporación/métodos , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Espinas Dendríticas/metabolismo , Espinas Dendríticas/ultraestructura , Células Piramidales/metabolismo , Células Piramidales/citología , Femenino , Corteza Cerebral/citología , Dendritas/metabolismoRESUMEN
Neuronal development is characterized by the unidirectional flow of signal from the axon to the dendrites via synapses. Neuronal polarization is a critical step during development that allows the specification of the different neuronal processes as a single axon and multiple dendrites both structurally and functionally, allowing the unidirectional flow of information. Along with extrinsic and intrinsic signaling, a whole network of molecular complexes involved in positive and negative feedback loops play a major role in this critical distinction of neuronal processes. As a result, neuronal morphology is drastically altered during establishment of polarity. In this chapter, we discuss how we can analyze the morphological alterations of neurons in vitro in culture to assess the development and polarity status of the neuron. We also discuss how these studies can be conducted in vivo, where polarity studies pose a greater challenge with promising results for addressing multiple pathological conditions. Our experimental model is limited to rodent hippocampal/cortical neurons in culture and cortical neurons in brain tissues, which are well-characterized model systems for understanding neuronal polarization.
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Polaridad Celular , Hipocampo , Neuronas , Animales , Neuronas/citología , Neuronas/fisiología , Neuronas/metabolismo , Ratones , Hipocampo/citología , Células Cultivadas , Ratas , Axones/fisiología , Axones/metabolismo , Dendritas/fisiología , Dendritas/metabolismo , Corteza Cerebral/citologíaRESUMEN
Egocentric neural representations of environmental features, such as edges and vertices, are important for constructing a geometrically detailed egocentric cognitive map for goal-directed navigation and episodic memory. While egocentric neural representations of edges like egocentric boundary/border cells exist, those that selectively represent vertices egocentrically are yet unknown. Here we report that granular retrosplenial cortex (RSC) neurons in male mice generate spatial receptive fields exclusively near the vertices of environmental geometries during free exploration, termed vertex cells. Their spatial receptive fields occurred at a specific orientation and distance relative to the heading direction of mice, indicating egocentric vector coding of vertex. Removing physical boundaries defining the environmental geometry abolished the egocentric vector coding of vertex, and goal-directed navigation strengthened the egocentric vector coding at the goal-located vertex. Our findings suggest that egocentric vector coding of vertex by granular RSC neurons helps construct an egocentric cognitive map that guides goal-directed navigation.
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Neuronas , Animales , Masculino , Neuronas/fisiología , Ratones , Ratones Endogámicos C57BL , Percepción Espacial/fisiología , Corteza Cerebral/fisiología , Corteza Cerebral/citología , Giro del Cíngulo/fisiología , Giro del Cíngulo/citología , Orientación/fisiología , Navegación Espacial/fisiologíaRESUMEN
Microglia are tissue-resident macrophages of the central nervous system (CNS), performing numerous functions that support neuronal health and CNS homeostasis. They are a major population of immune cells associated with CNS disease activity, adopting reactive phenotypes that potentially contribute to neuronal injury during chronic neurodegenerative diseases such as multiple sclerosis (MS). The distinct mechanisms by which microglia regulate neuronal function and survival during health and disease remain limited due to challenges in resolving the complex in vivo interactions between microglia, neurons, and other CNS environmental factors. Thus, the in vitro approach of co-culturing microglia and neurons remains a valuable tool for studying microglia-neuronal interactions. Here, we present a protocol to generate and co-culture primary microglia and neurons from mice. Specifically, microglia were isolated after 9-10 days in vitro from a mixed glia culture established from brain homogenates derived from neonatal mice between post-natal days 0-2. Neuronal cells were isolated from brain cortices of mouse embryos between embryonic days 16-18. After 4-5 days in vitro, neuronal cells were seeded in 96-well plates, followed by the addition of microglia to form the co-culture. Careful timing is critical for this protocol as both cell types need to reach experimental maturity to establish the co-culture. Overall, this co-culture can be useful for studying microglia-neuron interactions and can provide multiple readouts, including immunofluorescence microscopy, live imaging, as well as RNA and protein assays.
Asunto(s)
Corteza Cerebral , Técnicas de Cocultivo , Microglía , Neuronas , Animales , Técnicas de Cocultivo/métodos , Microglía/citología , Ratones , Neuronas/citología , Corteza Cerebral/citología , Técnicas Citológicas/métodosRESUMEN
Synaptic plasticities, such as long-term potentiation (LTP) and depression (LTD), tune synaptic efficacy and are essential for learning and memory. Current studies of synaptic plasticity in humans are limited by a lack of adequate human models. Here, we modeled the thalamocortical system by fusing human induced pluripotent stem cell-derived thalamic and cortical organoids. Single-nucleus RNA sequencing revealed that >80% of cells in thalamic organoids were glutamatergic neurons. When fused to form thalamocortical assembloids, thalamic and cortical organoids formed reciprocal long-range axonal projections and reciprocal synapses detectable by light and electron microscopy, respectively. Using whole-cell patch-clamp electrophysiology and two-photon imaging, we characterized glutamatergic synaptic transmission. Thalamocortical and corticothalamic synapses displayed short-term plasticity analogous to that in animal models. LTP and LTD were reliably induced at both synapses; however, their mechanisms differed from those previously described in rodents. Thus, thalamocortical assembloids provide a model system for exploring synaptic plasticity in human circuits.