RESUMO
The angiomotin (Amot)-Yes-associated protein 1 (Yap1) complex plays a major role in regulating the inhibition of cell contact, cellular polarity, and cell growth in many cell types. However, the function of Amot and the Hippo pathway transcription coactivator Yap1 in the central nervous system remains unclear. We found that Amot is a critical mediator of dendritic morphogenesis in cultured hippocampal cells and Purkinje cells in the brain. Amot function in developing neurons depends on interactions with Yap1, which is also indispensable for dendrite growth and arborization in vitro. The conditional deletion of Amot and Yap1 in neurons led to a decrease in the complexity of Purkinje cell dendritic trees, abnormal cerebellar morphology, and impairments in motor coordination. Our results indicate that the function of Amot and Yap1 in dendrite growth does not rely on interactions with TEA domain (TEAD) transcription factors or the expression of Hippo pathway-dependent genes. Instead, Amot and Yap1 regulate dendrite development by affecting the phosphorylation of S6 kinase and its target S6 ribosomal protein.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Ciclo Celular/metabolismo , Dendritos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Locomoção/fisiologia , Proteínas dos Microfilamentos/metabolismo , Angiomotinas , Animais , Hipocampo/citologia , Integrases/metabolismo , Camundongos Endogâmicos C57BL , Morfogênese , Atividade Motora , Fosforilação , Ligação Proteica , Células de Purkinje/metabolismo , Ratos Wistar , Proteína S6 Ribossômica/metabolismo , Proteínas de Sinalização YAPRESUMO
More than two decades after the discovery of adult neurogenesis in humans, researchers still struggle to elucidate the underlying transcriptional and post-transcriptional mechanisms. RNA interference is a crucially important process in the central nervous system, and its role in adult neurogenesis is poorly understood. In this work, we address the role of Dicer-dependent microRNA biogenesis in neuronal differentiation of adult neural stem cells within the subventricular zone of the mouse brain. Loss of the Dicer1 gene in the tailless (Tlx)-positive cells did not cause the decline in their numbers, but severely affected differentiation. Thus, our findings identify yet another phenomenon associated with microRNA pathway deregulation in adult neural stem cells which might be of relevance both for neuroscience and clinical practice.
Assuntos
Proliferação de Células , MicroRNAs/genética , Células-Tronco Neurais/citologia , Neurogênese , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Células Cultivadas , RNA Helicases DEAD-box/genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Ventrículos Laterais/citologia , Ventrículos Laterais/metabolismo , Masculino , Camundongos , Células-Tronco Neurais/metabolismo , Ribonuclease III/genética , TranscriptomaRESUMO
TAR DNA-binding protein 43 (TDP-43) is a hallmark of some neurodegenerative disorders, such as frontotemporal lobar degeneration and amyotrophic lateral sclerosis. TDP-43-related pathology is characterized by its abnormally phosphorylated and ubiquitinated aggregates. It is involved in many aspects of RNA processing, including mRNA splicing, transport, and translation. However, its exact physiological function and role in mechanisms that lead to neuronal degeneration remain elusive. Transgenic rats that were characterized by TDP-43 depletion in neurons exhibited enhancement of the acquisition of fear memory. At the cellular level, TDP-43-depleted neurons exhibited a decrease in the short-term plasticity of intrinsic neuronal excitability. The induction of long-term potentiation in the CA3-CA1 areas of the hippocampus resulted in more stable synaptic enhancement. At the molecular level, the protein levels of an unedited (R) FLOP variant of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) GluR1 and GluR2/3 subunits decreased in the hippocampus. Alterations of FLOP/FLIP subunit composition affected AMPAR kinetics, reflected by cyclothiazide-dependent slowing of the decay time of AMPAR-mediated miniature excitatory postsynaptic currents. These findings suggest that TDP-43 may regulate activity-dependent neuronal plasticity, possibly by regulating the splicing of genes that are responsible for fast synaptic transmission and membrane potential.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Hipocampo/metabolismo , Memória/fisiologia , Plasticidade Neuronal/fisiologia , Neurônios/metabolismo , Animais , Proteínas de Ligação a DNA/genética , Espinhas Dendríticas/metabolismo , Ratos , Ratos Transgênicos , Receptores de AMPA/metabolismo , Transmissão Sináptica/fisiologiaRESUMO
The role of neuronal noncoding RNAs in energy control of the body is not fully understood. The arcuate nucleus (ARC) of the hypothalamus comprises neurons regulating food intake and body weight. Here we show that Dicer-dependent loss of microRNAs in these neurons of adult (DicerCKO) mice causes chronic overactivation of the signaling pathways involving phosphatidylinositol-3-kinase (PI3K), Akt, and mammalian target of rapamycin (mTOR) and an imbalance in the levels of neuropeptides, resulting in severe hyperphagic obesity. Similarly, the activation of PI3K-Akt-mTOR pathway due to Pten deletion in the adult forebrain leads to comparable weight increase. Conversely, the mTORC1 inhibitor rapamycin normalizes obesity in mice with an inactivated Dicer1 or Pten gene. Importantly, the continuous delivery of oligonucleotides mimicking microRNAs, which are predicted to target PI3K-Akt-mTOR pathway components, to the hypothalamus attenuates adiposity in DicerCKO mice. Furthermore, loss of miR-103 causes strong upregulation of the PI3K-Akt-mTOR pathway in vitro and its application into the ARC of the Dicer-deficient mice both reverses upregulation of Pik3cg, the mRNA encoding the catalytic subunit p110γ of the PI3K complex, and attenuates the hyperphagic obesity. Our data demonstrate in vivo the crucial role of neuronal microRNAs in the control of energy homeostasis.
Assuntos
Hiperfagia/complicações , Hipotálamo/metabolismo , MicroRNAs/metabolismo , Obesidade/etiologia , Obesidade/patologia , Absorciometria de Fóton , Proteína Relacionada com Agouti/genética , Proteína Relacionada com Agouti/metabolismo , Animais , RNA Helicases DEAD-box/deficiência , RNA Helicases DEAD-box/genética , Células HeLa , Humanos , Proteínas Luminescentes/genética , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Neuropeptídeo Y/genética , Neuropeptídeo Y/metabolismo , Proteína Oncogênica v-akt/metabolismo , PTEN Fosfo-Hidrolase/deficiência , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/metabolismo , Ribonuclease III/deficiência , Ribonuclease III/genética , Serina-Treonina Quinases TOR/metabolismo , Transdução GenéticaRESUMO
Dopamine plays an important role in synaptic plasticity and learning and is involved in the pathogenesis of various neurological and psychiatric disorders. Here, we reveal staining of dopaminergic fibers in stratum oriens of the mouse hippocampal CA1 region, a finding that is consistent with earlier reports. Furthermore, we examined the effect of dopamine agonists on NMDAR-dependent early long-term potentiation (LTP) (40 min) during γ-aminobutyric acid (GABA)(A)-mediated blockade. LTP of the AMPA component was strongly reduced in stratum oriens but barely affected in stratum radiatum. This layer-specific effect was caused by D4 receptor activation, which augmented the inactivation of synaptic NMDAR-mediated currents (NMDA EPSCs) during LTP induction through a Ca(2+)-dependent G-protein-independent mechanism. A similar dopaminergic modulation of both NMDA EPSCs and LTP was also observed in mice constitutively lacking NR2A but was absent in mice lacking NR2B in principal forebrain neurons. Together, these experiments strongly indicate that dopaminergic modulation of early LTP in stratum oriens occurs through NMDARs containing NR2B subunits via D4Rs. Thus, a dopamine hyperfunction in stratum oriens may result in NMDAR hypofunction that could affect both normal and pathological conditions.
Assuntos
Região CA1 Hipocampal/fisiologia , Potenciação de Longa Duração/fisiologia , Receptores de Dopamina D4/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Técnicas de Patch-ClampRESUMO
Mass spectrometry-based proteomics aims to study the proteome both qualitatively and quantitatively. A key step in proteomic analysis is sample preparation, which is crucial for reliable results. We investigated the effect of the composition of the homogenization buffer used to extract proteins from brain tissue on the yield of protein extraction and the number and type of extracted proteins. Three different types of buffers were compared-detergent-based buffer (DB), chaotropic agent-based buffer (CAB) and buffer without detergent and chaotropic agent (DFB). Based on label-free quantitative protein analysis, detergent buffer was identified as the most suitable for global proteomic profiling of brain tissue. It allows the most efficient extraction of membrane proteins, synaptic and synaptic membrane proteins along with ribosomal, mitochondrial and myelin sheath proteins, which are of particular interest in the field of neurodegenerative disorders research.
RESUMO
Obesity is a growing medical and social problem worldwide. The control of energy homeostasis in the brain is achieved by various regions including the arcuate hypothalamic nucleus (ARH). The latter comprises a number of neuronal populations including the first order metabolic neurons, appetite-stimulating agouti-related peptide (AgRP) neurons and appetite-suppressing proopiomelanocortin (POMC) neurons. Using an in vivo reductionist approach and POMCCre-dependent CRISPR-Cas9, we demonstrate that miR-15a-5p protects from obesity. Moreover, we have identified Bace1, a gene previously linked to energy metabolism imbalance, as a direct target of miR-15a-5p. This work warrants further investigations of non-coding RNA-mediated regulation of energy homeostasis and might contribute to the development of novel therapeutic approaches to treat metabolic diseases.
Assuntos
MicroRNAs , Pró-Opiomelanocortina , Proteína Relacionada com Agouti/genética , Proteína Relacionada com Agouti/metabolismo , Secretases da Proteína Precursora do Amiloide , Animais , Ácido Aspártico Endopeptidases , Camundongos , MicroRNAs/genética , Obesidade/genética , Obesidade/metabolismo , Pró-Opiomelanocortina/genéticaRESUMO
OBJECTIVE: Obesity, a growing threat to the modern society, represents an imbalance of metabolic queues that normally signal to the arcuate hypothalamic nucleus, a critical brain region sensing and regulating energy homeostasis. This is achieved by various neurons many of which developmentally originate from the proopiomelanocortin (POMC)-expressing lineage. Within the mature neurons originating from this lineage, we aimed to identify non-coding genes in control of metabolic function in the adulthood. METHODS: In this work, we used microRNA mimic delivery and POMCCre-dependent CRISPR-Cas9 knock-out strategies in young or aged mice. Importantly, we also used CRISPR guides directing suicide cleavage of Cas9 to limit the off-target effects. RESULTS: Here we found that mature neurons originating from the POMC lineage employ miR-29a to protect against insulin resistance obesity, hyperphagia, decreased energy expenditure and obesity. Moreover, we validated the miR-29 family as a prominent regulator of the PI3K-Akt-mTOR pathway. Within the latter, we identified a direct target of miR-29a-3p, Nras, which was up-regulated in those and only those mature POMCCreCas9 neurons that were effectively transduced by anti-miR-29 CRISPR-equipped construct. Moreover, POMCCre-dependent co-deletion of Nras in mature neurons attenuated miR-29 depletion-induced obesity. CONCLUSIONS: Thus, the first to our knowledge case of in situ Cre-dependent CRISPR-Cas9-mediated knock-out of microRNAs in a specific hypothalamic neuronal population helped us to decipher a critical metabolic circuit in adult mice. This work significantly extends our understanding about the involvement of neuronal microRNAs in homeostatic regulation.
Assuntos
MicroRNAs , Pró-Opiomelanocortina , Animais , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Neurônios/metabolismo , Obesidade/genética , Obesidade/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Pró-Opiomelanocortina/metabolismoRESUMO
Dicer-dependent noncoding RNAs, including microRNAs (miRNAs), play an important role in a modulation of translation of mRNA transcripts necessary for differentiation in many cell types. In vivo experiments using cell type-specific Dicer1 gene inactivation in neurons showed its essential role for neuronal development and survival. However, little is known about the consequences of a loss of miRNAs in adult, fully differentiated neurons. To address this question, we used an inducible variant of the Cre recombinase (tamoxifen-inducible CreERT2) under control of Camk2a gene regulatory elements. After induction of Dicer1 gene deletion in adult mouse forebrain, we observed a progressive loss of a whole set of brain-specific miRNAs. Animals were tested in a battery of both aversively and appetitively motivated cognitive tasks, such as Morris water maze, IntelliCage system, or trace fear conditioning. Compatible with rather long half-life of miRNAs in hippocampal neurons, we observed an enhancement of memory strength of mutant mice 12 weeks after the Dicer1 gene mutation, before the onset of neurodegenerative process. In acute brain slices, immediately after high-frequency stimulation of the Schaffer collaterals, the efficacy at CA3-to-CA1 synapses was higher in mutant than in control mice, whereas long-term potentiation was comparable between genotypes. This phenotype was reflected at the subcellular and molecular level by the elongated filopodia-like shaped dendritic spines and an increased translation of synaptic plasticity-related proteins, such as BDNF and MMP-9 in mutant animals. The presented work shows miRNAs as key players in the learning and memory process of mammals.
Assuntos
RNA Helicases DEAD-box/deficiência , Endorribonucleases/deficiência , Deleção de Genes , Hipocampo/metabolismo , Aprendizagem/fisiologia , Memória/fisiologia , MicroRNAs/genética , Animais , RNA Helicases DEAD-box/biossíntese , RNA Helicases DEAD-box/genética , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/ultraestrutura , Estimulação Elétrica/métodos , Endorribonucleases/biossíntese , Endorribonucleases/genética , Hipocampo/ultraestrutura , Potenciação de Longa Duração/genética , Camundongos , Camundongos Knockout , Camundongos Transgênicos , MicroRNAs/antagonistas & inibidores , MicroRNAs/biossíntese , Técnicas de Cultura de Órgãos , Ribonuclease III , Sinapses/metabolismo , Sinapses/ultraestruturaRESUMO
c-Jun NH(2)-terminal kinases (JNKs) are essential during brain development, when they regulate morphogenic changes involving cell movement and migration. In the adult, JNK determines neuronal cytoarchitecture. To help uncover the molecular effectors for JNKs in these events, we affinity purified JNK-interacting proteins from brain. This revealed that the stathmin family microtubule-destabilizing proteins SCG10, SCLIP, RB3, and RB3' interact tightly with JNK. Furthermore, SCG10 is also phosphorylated by JNK in vivo on sites that regulate its microtubule depolymerizing activity, serines 62 and 73. SCG10-S73 phosphorylation is significantly decreased in JNK1-/- cortex, indicating that JNK1 phosphorylates SCG10 in developing forebrain. JNK phosphorylation of SCG10 determines axodendritic length in cerebrocortical cultures, and JNK site-phosphorylated SCG10 colocalizes with active JNK in embryonic brain regions undergoing neurite elongation and migration. We demonstrate that inhibition of cytoplasmic JNK and expression of SCG10-62A/73A both inhibited fluorescent tubulin recovery after photobleaching. These data suggest that JNK1 is responsible for regulation of SCG10 depolymerizing activity and neurite elongation during brain development.
Assuntos
Axônios/fisiologia , Dendritos/fisiologia , Microtúbulos/metabolismo , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Fatores de Crescimento Neural/metabolismo , Animais , Encéfalo/crescimento & desenvolvimento , Proteínas de Ligação ao Cálcio , Proteínas de Transporte , Linhagem Celular , Células Cultivadas , Embrião de Mamíferos/citologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana , Camundongos , Proteínas dos Microtúbulos , Proteína Quinase 8 Ativada por Mitógeno/análise , Proteína Quinase 8 Ativada por Mitógeno/antagonistas & inibidores , Fosforilação , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , EstatminaRESUMO
The persistent nature of addiction has been associated with activity-induced plasticity of neurons within the striatum and nucleus accumbens (NAc). To identify the molecular processes leading to these adaptations, we performed Cre/loxP-mediated genetic ablations of two key regulators of gene expression in response to activity, the Ca(2+)/calmodulin-dependent protein kinase IV (CaMKIV) and its postulated main target, the cAMP-responsive element binding protein (CREB). We found that acute cocaine-induced gene expression in the striatum was largely unaffected by the loss of CaMKIV. On the behavioral level, mice lacking CaMKIV in dopaminoceptive neurons displayed increased sensitivity to cocaine as evidenced by augmented expression of locomotor sensitization and enhanced conditioned place preference and reinstatement after extinction. However, the loss of CREB in the forebrain had no effect on either of these behaviors, even though it robustly blunted acute cocaine-induced transcription. To test the relevance of these observations for addiction in humans, we performed an association study of CAMK4 and CREB promoter polymorphisms with cocaine addiction in a large sample of addicts. We found that a single nucleotide polymorphism in the CAMK4 promoter was significantly associated with cocaine addiction, whereas variations in the CREB promoter regions did not correlate with drug abuse. These findings reveal a critical role for CaMKIV in the development and persistence of cocaine-induced behaviors, through mechanisms dissociated from acute effects on gene expression and CREB-dependent transcription.
Assuntos
Proteína Quinase Tipo 4 Dependente de Cálcio-Calmodulina/genética , Transtornos Relacionados ao Uso de Cocaína/genética , Transtornos Relacionados ao Uso de Cocaína/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Regulação da Expressão Gênica/genética , Adulto , Análise de Variância , Animais , Brasil , Corpo Estriado/metabolismo , Feminino , Deleção de Genes , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Transgênicos , Plasticidade Neuronal/genética , Neurônios/metabolismo , Polimorfismo de Nucleotídeo Único/genéticaRESUMO
Animals display a rich repertoire of defensive responses adequate to the threat proximity. In social species, these reactions can be additionally influenced by the behavior of fearful conspecifics. However, the majority of neuroscientific studies on socially triggered defensive responses focuses on one type of behavior, freezing. To study a broader range of socially triggered reactions and underlying mechanisms, we directly compared two experimental paradigms, mimicking occurrence of the imminent versus remote threat. Observation of a partner currently experiencing aversive stimulation evokes passive defensive responses in the observer rats. Similar interaction with a partner that has just undergone the aversive stimulation prompts animals to increase active exploration. Although the observers display behaviors similar to those of the aversively stimulated demonstrators, their reactions are not synchronized in time, suggesting that observers' responses are caused by the change in their affective state rather than mimicry. Using opsins targeted to behaviorally activated neurons, we tagged central amygdala (CeA) cells implicated in observers' responses to either imminent or remote threat and reactivated them during the exploration of a novel environment. The manipulation revealed that the two populations of CeA cells promote passive or active defensive responses, respectively. Further experiments confirmed that the two populations of cells at least partially differ in expression of molecular markers (protein kinase C-δ [PKC-δ] and corticotropin-releasing factor [CRF]) and connectivity patterns (receiving input from the basolateral amygdala or from the anterior insula). The results are consistent with the literature on single subjects' fear conditioning, suggesting that similar neuronal circuits control defensive responses in social and non-social contexts.
Assuntos
Complexo Nuclear Basolateral da Amígdala , Núcleo Central da Amígdala , Animais , Antígeno Carcinoembrionário , Hormônio Liberador da Corticotropina , Medo , RatosRESUMO
The retrosplenial cortex (RSC) belongs to the spatial memory circuit, but the precise timeline of its involvement and the relation to hippocampal activation have not been sufficiently described. We trained rats in a modified version of the T maze with transparent walls and distant visual cues to induce the formation of allocentric spatial memory. We used two distinct salient contexts associated with opposite sequences of turns. Switching between contexts allowed us to test the ability of animals to utilize spatial information. We then applied a CatFISH approach with a probe directed against the Arc immediate early gene in order to visualize the associated memory engrams in the RSC and the hippocampus. After training, rats displayed two strategies to solve the maze, with half of the animals relying on distant spatial cues (allocentric) and the other half using egocentric strategy. Rats that did not utilize the spatial cues showed higher Arc levels in the RSC compared to the allocentric group. The overlap between the two context engrams in the RSC was similar in both groups. These results show differential involvement of the RSC and hippocampus during spatial memory acquisition and point toward their distinct roles in forming the cognitive maps.
RESUMO
Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is an invaluable tool for gene function analysis in living organisms. The standard transgenesis method is based on microinjecting foreign DNA fragments into a pronucleus of a fertilized oocyte. This technique is widely used in mice but remains relatively inefficient and technically demanding in other animal species. The transgene can also be introduced into one-cell-stage embryos via lentiviral infection, providing an effective alternative to standard pronuclear injections, especially in species or strains with a more challenging embryo structure. In this approach, a suspension that contains lentiviral vectors is injected into the perivitelline space of a fertilized rat embryo, which is technically less demanding and has a higher success rate. Lentiviral vectors were shown to efficiently incorporate the transgene into the genome to determine the generation of stable transgenic lines. Despite some limitations (e.g., Biosafety Level 2 requirements, DNA fragment size limits), lentiviral transgenesis is a rapid and efficient transgenesis method. Additionally, using female rats that are mated with a fertile male strain with a different dominant fur color is presented as an alternative to generate pseudopregnant foster mothers.
Assuntos
Vetores Genéticos/genética , Lentivirus/genética , Animais , Camundongos , Ratos , Ratos TransgênicosRESUMO
Since the 1980s, we have witnessed the rapid development of genetically modified mouse models of human diseases. A large number of transgenic and knockout mice have been utilized in basic and applied research, including models of neurodegenerative and neuropsychiatric disorders. To assess the biological function of mutated genes, modern techniques are critical to detect changes in behavioral phenotypes. We review the IntelliCage, a high-throughput system that is used for behavioral screening and detailed analyses of complex behaviors in mice. The IntelliCage was introduced almost two decades ago and has been used in over 150 studies to assess both spontaneous and cognitive behaviors. We present a critical analysis of experimental data that have been generated using this device.
Assuntos
Técnicas de Observação do Comportamento/instrumentação , Técnicas de Observação do Comportamento/métodos , Comportamento Animal , Animais , Escala de Avaliação Comportamental , Feminino , Aprendizagem , Masculino , Camundongos , Camundongos TransgênicosRESUMO
Induction of mitotic catastrophe through the disruption of microtubules is an established target in cancer therapy. However, the molecular mechanisms determining the mitotic catastrophe and the following apoptotic or non-apoptotic cell death remain poorly understood. Moreover, many existing drugs targeting tubulin, such as vincristine, have reduced efficacy, resulting from poor solubility in physiological conditions. Here, we introduce a novel small molecule 2-aminoimidazoline derivative-OAT-449, a synthetic water-soluble tubulin inhibitor. OAT-449 in a concentration range from 6 to 30 nM causes cell death of eight different cancer cell lines in vitro, and significantly inhibits tumor development in such xenograft models as HT-29 (colorectal adenocarcinoma) and SK-N-MC (neuroepithelioma) in vivo. Mechanistic studies showed that OAT-449, like vincristine, inhibited tubulin polymerization and induced profound multi-nucleation and mitotic catastrophe in cancer cells. HeLa and HT-29 cells within 24 h of treatment arrested in G2/M cell cycle phase, presenting mitotic catastrophe features, and 24 h later died by non-apoptotic cell death. In HT-29 cells, both agents altered phosphorylation status of Cdk1 and of spindle assembly checkpoint proteins NuMa and Aurora B, while G2/M arrest and apoptosis blocking was consistent with p53-independent accumulation in the nucleus and largely in the cytoplasm of p21/waf1/cip1, a key determinant of cell fate programs. This is the first common mechanism for the two microtubule-dissociating agents, vincristine and OAT-449, determining the cell death pathway following mitotic catastrophe demonstrated in HT-29 cells.
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Glucose is the essential energy source for the brain, whose deficit, triggered by energy deprivation or therapeutic agents, can be fatal. Increased appetite is the key behavioral defense against hypoglycemia; however, the central pathways involved are not well understood. Here, we describe a glucoprivic feeding pathway by tyrosine hydroxylase (TH)-expressing neurons from nucleus of solitary tract (NTS), which project densely to the hypothalamus and elicit feeding through bidirectional adrenergic modulation of agouti-related peptide (AgRP)- and proopiomelanocortin (POMC)-expressing neurons. Acute chemogenetic inhibition of arcuate nucleus (ARC)-projecting NTSTH neurons or their target, AgRP neurons, impaired glucoprivic feeding induced by 2-Deoxy-D-glucose (2DG) injection. Neuroanatomical tracing results suggested that ARC-projecting orexigenic NTSTH neurons are largely distinct from neighboring catecholamine neurons projecting to parabrachial nucleus (PBN) that promotes satiety. Collectively, we describe a circuit organization in which an ascending pathway from brainstem stimulates appetite through key hunger neurons in the hypothalamus in response to hypoglycemia.
Assuntos
Proteína Relacionada com Agouti/metabolismo , Regulação do Apetite , Hipoglicemia/metabolismo , Hipotálamo/metabolismo , Neurônios/metabolismo , Núcleo Solitário/metabolismo , Animais , Feminino , Hipotálamo/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/citologia , Núcleo Solitário/citologiaRESUMO
Local and regulated expression of exogenous genes in the central nervous system is one of the major challenges of modern neuroscience. We have approached this issue by applying the inducible tetracycline system to regulate the expression of EGFP reporter gene in double transgenic rats. We have obtained a strong induction of EGFP only in male testes, which correlated with a high level of rtTA expression only in this organ. To overcome the problem of lack of rtTA protein in the transgenic rat brain, we have delivered this Tet system activator with lentiviral vectors into the dentate gyrus of hippocampus of transgenic EGFP rats. As a result, after systemic application of doxycycline we have obtained inducible, stable and restricted to the desired brain region expression of EGFP. An advantage of this strategy is that the transgene is located in the same genetic milieu in every cell of the transgenic organism. This is crucial to obtain uniform expression of the regulated gene within the target brain structure. Combination of rat transgenesis and lentiviral vectors is a novel approach enabling precise spatiotemporal regulation of genes of interest strictly in the brain structure of choice or in other tissues.
Assuntos
Encéfalo/metabolismo , Vetores Genéticos/genética , Proteínas de Fluorescência Verde/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Giro Denteado/metabolismo , Doxiciclina/farmacologia , Feminino , Imunofluorescência , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Fluorescência Verde/genética , Lentivirus/genética , Masculino , Ratos , Ratos Transgênicos , Ratos Wistar , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Elementos de Resposta/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tetraciclina/farmacologia , Transativadores/genética , Transativadores/metabolismoRESUMO
The Morris Water Maze is commonly used in behavioural neuroscience for the study of spatial learning with rodents. Over the years, various methods of analysing rodent data collected during this task have been proposed. These methods span from classical performance measurements to more sophisticated categorisation techniques which classify the animal swimming path into behavioural classes known as exploration strategies. Classification techniques provide additional insight into the different types of animal behaviours but still only a limited number of studies utilise them. This is primarily because they depend highly on machine learning knowledge. We have previously demonstrated that the animals implement various strategies and that classifying entire trajectories can lead to the loss of important information. In this work, we have developed a generalised and robust classification methodology to boost classification performance and nullify the need for manual tuning. We have also made available an open-source software based on this methodology.