RESUMO
Signaling through GABA(A) receptors controls neural progenitor cell (NPC) development in vitro and is altered in schizophrenic and autistic individuals. However, the in vivo function of GABA(A) signaling on neural stem cell proliferation, and ultimately neurogenesis, remains unknown. To examine GABA(A) function in vivo, we electroporated plasmids encoding short-hairpin (sh) RNA against the Na-K-2Cl cotransporter NKCC1 (shNKCC1) in NPCs of the neonatal subventricular zone in mice to reduce GABA(A)-induced depolarization. Reduced GABA(A) depolarization identified by a loss of GABA(A)-induced calcium responses in most electroporated NPCs led to a 70% decrease in the number of proliferative Ki67(+) NPCs and a 60% reduction in newborn neuron density. Premature loss of GABA(A) depolarization in newborn neurons resulted in truncated dendritic arborization at the time of synaptic integration. However, by 6 weeks the dendritic tree had partially recovered and displayed a small, albeit significant, decrease in dendritic complexity but not total dendritic length. To further examine GABA(A) function on NPCs, we treated animals with a GABA(A) allosteric agonist, pentobarbital. Enhancement of GABA(A) activity in NPCs increased the number of proliferative NPCs by 60%. Combining shNKCC1 and pentobarbital prevented the shNKCC1 and the pentobarbital effects on NPC proliferation, suggesting that these manipulations affected NPCs through GABA(A) receptors. Thus, dysregulation in GABA(A) depolarizing activity delayed dendritic development and reduced NPC proliferation resulting in decreased neuronal density.
Assuntos
Diferenciação Celular/fisiologia , Ventrículos Cerebrais/citologia , Dendritos/fisiologia , Neurônios/citologia , Bulbo Olfatório/citologia , Receptores de GABA-A/metabolismo , Simportadores de Cloreto de Sódio-Potássio/deficiência , Fatores Etários , Análise de Variância , Animais , Animais Recém-Nascidos , Cálcio/metabolismo , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ventrículos Cerebrais/crescimento & desenvolvimento , Dendritos/efeitos dos fármacos , Ácido Egtázico/análogos & derivados , Ácido Egtázico/metabolismo , Eletroporação , Feminino , Moduladores GABAérgicos/farmacologia , Agonistas de Receptores de GABA-A/farmacologia , Proteínas de Fluorescência Verde/genética , Técnicas In Vitro , Antígeno Ki-67/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Masculino , Camundongos , Muscimol/farmacologia , Células-Tronco Neurais , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Técnicas de Patch-Clamp , Pentobarbital/farmacologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Membro 2 da Família 12 de Carreador de Soluto , Transfecção , Proteína Vermelha FluorescenteRESUMO
In the postnatal subventricular zone (SVZ), S phase entry of neural progenitor cells (NPCs) correlates with a local increase in blood flow. However, the cellular mechanism controlling this hemodynamic response remains unknown. We show that a subpopulation of SVZ cells, astrocyte-like cells or B-cells, sends projections ensheathing pericytes on SVZ capillaries in young mice. We examined whether calcium increases in pericytes or B-cells led to a vascular response in acute slices using the P2Y(2/4) receptor (P2Y(2/4)R) agonist UTP, electrical stimulation, or transgenic mice expressing exogenous Gq-coupled receptors (MrgA1) in B-cells. UTP increased calcium in pericytes leading to capillary constrictions. Electrical stimulation induced calcium propagation in SVZ cells followed by capillary constrictions involving purinergic receptors. In transgenic mice, selective calcium increases in B-cells induced P2Y(2/4)R-dependent capillary constrictions, suggesting that B-cells release ATP activating purinergic receptors on pericytes. Interestingly, in the presence of a P2Y(2/4)R blocker, dilation was observed. Intraventricular UTP injection transiently decreased blood flow monitored in vivo using laser Doppler flowmetry. Using neonatal electroporation, we expressed MrgA1 in slow cycling radial glia-derived B1 cells, i.e., NPCs. Intraventricular injection of an MrgA1 ligand increased blood flow in the SVZ. Thus, upon intracellular calcium increases B-cells/NPCs release ATP and vasodilating factors that activate purinergic receptors on pericytes triggering a vascular response and blood flow increase in vivo. Considering that NPCs receive signals from other SVZ cells, these findings further suggest that NPCs act as transducers of neurometabolic coupling in the SVZ.
Assuntos
Capilares/fisiologia , Ventrículos Cerebrais/fisiologia , Circulação Cerebrovascular/fisiologia , Células-Tronco Neurais/fisiologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/fisiologia , Animais , Animais Recém-Nascidos , Astrócitos/fisiologia , Sinalização do Cálcio/fisiologia , Ventrículos Cerebrais/irrigação sanguínea , Estimulação Elétrica , Eletroporação , Feminino , Imunofluorescência , Processamento de Imagem Assistida por Computador , Fluxometria por Laser-Doppler , Masculino , Camundongos , Tono Muscular/fisiologia , Músculo Liso Vascular/fisiologia , Pericitos/fisiologia , Vasoconstrição/fisiologia , Vasodilatação/fisiologiaRESUMO
The role of putrescine, spermidine and spermine in phorbol 12-myristate-13-acetate (PMA)-induced macrophage differentiation was examined in human HL-60 and U-937 myeloid leukemia cells. Unlike other polyamines, spermine affected this differentiation by acting as a negative regulator. This negative regulation was established by showing that the PMA-induced macrophage phenotype, but not PMA-associated replication arrest, was abrogated (a) by replenishing the PMA-evoked decrease in cellular spermine levels with this polyamine from an exogenous source and (b) by blocking PMA-induced expression of the polyamine catabolic enzyme N(1)-spermidine/spermine acetyltransferase (SSAT) with antisense oligonucleotides in the presence of low substrate level. The PMA-evoked reduction in cellular spermine appears to result from an increase in the activity of SSAT and a decrease in the activity of ornithine decarboxylase, the polyamine biosynthetic enzyme. To a degree, these changes are due to corresponding changes in the expression of the genes that code for these enzymes. When cell differentiation is initiated, SSAT expression is increased after PMA-evoked activation of protein kinase C-beta. The present studies raise the possibility that agents able to reduce spermine levels in patients' myeloid leukemia cells may enhance the activity of differentiation therapy drugs for this type of leukemia.
Assuntos
Macrófagos/patologia , Espermina/fisiologia , Acetiltransferases/biossíntese , Acetiltransferases/genética , Acetiltransferases/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Poliaminas Biogênicas/biossíntese , Diferenciação Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Ativação Enzimática , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Guanidinas/farmacologia , Células HL-60 , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Fator 2 Relacionado a NF-E2 , Proteína Quinase C/metabolismo , Proteína Quinase C beta , Espermina/antagonistas & inibidores , Espermina/metabolismo , Espermina/farmacologia , Acetato de Tetradecanoilforbol/antagonistas & inibidores , Acetato de Tetradecanoilforbol/farmacologia , Transativadores/fisiologia , Células U937RESUMO
BACKGROUND: Numerous gaseous microemboli (GME) are delivered into the arterial circulation during cardiopulmonary bypass (CPB). These emboli damage end organs through multiple mechanisms that are thought to contribute to neurocognitive deficits after cardiac surgery. Here, we use hypobaric oxygenation to reduce dissolved gases in blood and greatly reduce GME delivery during CPB. METHODS: Variable subatmospheric pressures were applied to 100% oxygen sweep gas in standard hollow fiber microporous membrane oxygenators to oxygenate and denitrogenate blood. GME were quantified using ultrasound while air embolism from the surgical field was simulated experimentally. We assessed end-organ tissues in swine postoperatively using light microscopy. RESULTS: Variable sweep gas pressures allowed reliable oxygenation independent of carbon dioxide removal while denitrogenating arterial blood. Hypobaric oxygenation produced dose-dependent reductions of Doppler signals produced by bolus and continuous GME loads in vitro. Swine were maintained using hypobaric oxygenation for 4 hours on CPB with no apparent adverse events. Compared with current practice standards of oxygen/air sweep gas, hypobaric oxygenation reduced GME volumes exiting the oxygenator (by 80%), exiting the arterial filter (95%), and arriving at the aortic cannula (â¼100%), indicating progressive reabsorption of emboli throughout the CPB circuit in vivo. Analysis of brain tissue suggested decreased microvascular injury under hypobaric conditions. CONCLUSIONS: Hypobaric oxygenation is an effective, low-cost, common sense approach that capitalizes on the simple physical makeup of GME to achieve their near-total elimination during CPB. This technique holds great potential for limiting end-organ damage and improving outcomes in a variety of patients undergoing extracorporeal circulation.
Assuntos
Ponte Cardiopulmonar/efeitos adversos , Embolia Aérea/etiologia , Embolia Aérea/terapia , Oxigenoterapia/métodos , Animais , Embolia Aérea/patologia , SuínosRESUMO
Tuberous sclerosis complex (TSC) is an autosomal dominant monogenetic disorder that is characterized by the formation of benign tumors in several organs as well as brain malformations and neuronal defects. TSC is caused by inactivating mutations in one of two genes, TSC1 and TSC2, resulting in increased activity of the mammalian Target of Rapamycin (mTOR). Here, we explore the cytoarchitectural and functional CNS aberrations that may account for the neurological presentations of TSC, notably seizures, hydrocephalus, and cognitive and psychological impairments. In particular, recent mouse models of brain lesions are presented with an emphasis on using electroporation to allow the generation of discrete lesions resulting from loss of heterozygosity during perinatal development. Cortical lesions are thought to contribute to epileptogenesis and worsening of cognitive defects. However, it has recently been suggested that being born with a mutant allele without loss of heterozygosity and associated cortical lesions is sufficient to generate cognitive and neuropsychiatric problems. We will thus discuss the function of mTOR hyperactivity on neuronal circuit formation and the potential consequences of being born heterozygous on neuronal function and the biochemistry of synaptic plasticity, the cellular substrate of learning and memory. Ultimately, a major goal of TSC research is to identify the cellular and molecular mechanisms downstream of mTOR underlying the neurological manifestations observed in TSC patients and identify novel therapeutic targets to prevent the formation of brain lesions and restore neuronal function.
Assuntos
Sistema Nervoso Central/metabolismo , Transtornos Cognitivos/etiologia , Epilepsia/etiologia , Esclerose Tuberosa , Animais , Sistema Nervoso Central/patologia , Transtornos Cognitivos/genética , Modelos Animais de Doenças , Epilepsia/genética , Humanos , Camundongos , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Esclerose Tuberosa/complicações , Esclerose Tuberosa/genética , Esclerose Tuberosa/patologiaRESUMO
To investigate the role of astrocytes in regulating synaptic transmission, we generated inducible transgenic mice that express a dominant-negative SNARE domain selectively in astrocytes to block the release of transmitters from these glial cells. By releasing adenosine triphosphate, which accumulates as adenosine, astrocytes tonically suppressed synaptic transmission, thereby enhancing the dynamic range for long-term potentiation and mediated activity-dependent, heterosynaptic depression. These results indicate that astrocytes are intricately linked in the regulation of synaptic strength and plasticity and provide a pathway for synaptic cross-talk.