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1.
Glia ; 71(4): 1081-1098, 2023 04.
Artigo em Inglês | MEDLINE | ID: mdl-36598109

RESUMO

Astrocytes are increasingly shown to operate as an isopotential syncytium in brain function. Protoplasmic astrocytes acquire this ability to functionally go beyond the single-cell level by evolving into a spongiform morphology, cytoplasmically connecting into a syncytium, and expressing a high density of K+ conductance. However, none of these cellular/functional features exist in neonatal newborn astrocytes, which imposes a basic question of when a functional syncytium evolves in the developing brain. Our results show that the spongiform morphology of individual astrocytes and their spatial organization all reach stationary levels by postnatal day (P) 15 in the hippocampal CA1 region. Functionally, astrocytes begin to uniformly express a mature level of passive K+ conductance by P11. We next used syncytial isopotentiality measurement to monitor the maturation of the astrocyte syncytium. In uncoupled P1 astrocytes, the substitution of endogenous K+ by a Na+ -electrode solution ([Na+ ]p ) resulted in the total elimination of the physiological membrane potential (VM ), and outward K+ conductance as predicted by the Goldman-Hodgkin-Katz (GHK) equation. As more astrocytes are coupled to each other through gap junctions during development, the [Na+ ]p -induced loss of physiological VM and the outward K+ conductance is progressively compensated by the neighboring astrocytes. By P15, a stably established syncytial isopotentiality (-73 mV), and a fully compensated outward K+ conductance appeared in all [Na+ ]p -recorded astrocytes. Thus, in view of the developmental timeframe wherein a singular syncytium is anatomically and functionally established for intra-syncytium K+ equilibration, an astrocyte syncytium becomes fully operational at P15 in the mouse hippocampus.


Assuntos
Astrócitos , Hipocampo , Camundongos , Animais , Astrócitos/fisiologia , Potenciais da Membrana/fisiologia , Junções Comunicantes/fisiologia , Região CA1 Hipocampal
2.
Neurochem Res ; 48(4): 1191-1210, 2023 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-35796915

RESUMO

Now astrocytes appear to be the key contributors to the pathophysiology of major depression. Evidence in rodents shows that chronic stress is associated with a decreased expression of astrocytic GFAP-immunoreactivity within the cortex in addition to changes in the complexity and length of astrocyte processes. Furthermore, postmortem brains of individuals with depression have revealed a decrease in astrocyte density. Notably, astrocytes are extensively coupled to one another through gap junctions to form a network, or syncytium, and we have previously demonstrated that syncytial isopotentiality is a mechanism by which astrocytes function as an efficient system with respect to brain homeostasis. Interestingly, the question of how astrocyte network function changes following chronic stress is yet to be elucidated. Here, we sought to examine the effects of chronic stress on network-level astrocyte (dys)function. Using a transgenic aldh1l1-eGFP astrocyte reporter mouse, a six-week unpredictable chronic mild stress (UCMS) paradigm as a rodent model of major depression, and immunohistochemical approaches, we show that the morphology of individual astrocytes is altered by chronic stress exposure. Additionally, in astrocyte syncytial isopotentiality measurement, we found that UCMS impairs the syncytial coupling strength of astrocytes within the hippocampus and prefrontal cortex-two brain regions that have been implicated in the regulation of mood. Together, these findings reveal that chronic stress leads to astrocyte atrophy and impaired gap junction coupling, raising the prospect that both individual and network-level astrocyte functionality are important in the etiology of major depression and other neuropsychiatric disorders.


Assuntos
Depressão , Transtorno Depressivo Maior , Camundongos , Animais , Astrócitos/metabolismo , Encéfalo , Camundongos Transgênicos , Transtorno Depressivo Maior/metabolismo , Modelos Animais de Doenças , Hipocampo
3.
Prog Neurobiol ; 213: 102264, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35283239

RESUMO

The complexity of astrocyte morphology and syncytial coupling through gap junctions are crucial for astrocyte function in the brain. However, the ultrastructural details of astrocyte arborization and interactions between neighboring astrocytes remain unknown. While a prevailing view is that synapses selectively contact peripheral astrocyte processes, the precise spatial-location selectivity of synapses abutting astrocytes is unresolved. Additionally, knowing the location and quantity of vesicles and mitochondria are prerequisites to answer two emerging questions - whether astrocytes have a signaling role within the brain and whether astrocytes are highly metabolically active. Here, we provided structural context for these questions by tracing and 3D reconstructing three neighboring astrocytes using serial block-face scanning electron microscopy. Our reconstructions reveal a spongiform astrocytic morphology resulting from the abundance of reflexive and leaflet processes. At the interfaces, varying sizes of astrocyte-astrocyte contacts were identified. Inside an astrocyte domain, synapses contact the entire astrocyte, and synapse-astrocyte contacts increase from soma to terminal leaflets. In contrast to densely packed vesicles at synaptic boutons, vesicle-like structures were scant within astrocytes. Lastly, astrocytes contain dense mitochondrial networks with a mitochondrial volume ratio similar to that of neurites. Together, these ultrastructural details should expand our understanding of functional astrocyte-astrocyte and astrocyte-neuron interactions.


Assuntos
Astrócitos , Sinapses , Astrócitos/metabolismo , Encéfalo , Humanos , Mitocôndrias , Neurônios/fisiologia , Sinapses/metabolismo
4.
Dev Neurobiol ; 80(7-8): 277-301, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32902152

RESUMO

Axons in the adult mammalian central nervous system (CNS) fail to regenerate inside out due to intrinsic and extrinsic neuronal determinants. During CNS development, axon growth, synapse formation, and function are tightly regulated processes allowing immature neurons to effectively grow an axon, navigate toward target areas, form synaptic contacts and become part of information processing networks that control behavior in adulthood. Not only immature neurons are able to precisely control the expression of a plethora of genes necessary for axon extension and pathfinding, synapse formation and function, but also non-neuronal cells such as astrocytes and microglia actively participate in sculpting the nervous system through refinement, consolidation, and elimination of synaptic contacts. Recent evidence indicates that a balancing act between axon regeneration and synaptic function may be crucial for rebuilding functional neuronal circuits after CNS trauma and disease in adulthood. Here, we review the role of classical and new intrinsic and extrinsic neuronal determinants in the context of CNS development, injury, and disease. Moreover, we discuss strategies targeting neuronal and non-neuronal cell behaviors, either alone or in combination, to promote axon regeneration and neuronal circuit formation in adulthood.


Assuntos
Axônios/fisiologia , Doenças do Sistema Nervoso Central/metabolismo , Sistema Nervoso Central/lesões , Sistema Nervoso Central/metabolismo , Regeneração Nervosa/fisiologia , Neurônios/metabolismo , Animais , Sistema Nervoso Central/crescimento & desenvolvimento , Humanos , Sinapses/metabolismo , Traumatismos do Sistema Nervoso/metabolismo
5.
Brain Sci ; 10(4)2020 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-32252295

RESUMO

Astrocyte syncytial isopotentiality is a physiological mechanism resulting from a strong electrical coupling among astrocytes. We have previously shown that syncytial isopotentiality exists as a system-wide feature that coordinates astrocytes into a system for high efficient regulation of brain homeostasis. Neuronal activity is known to regulate gap junction coupling through alteration of extracellular ions and neurotransmitters. However, the extent to which epileptic neuronal activity impairs the syncytial isopotentiality is unknown. Here, the neuronal epileptiform bursts were induced in acute hippocampal slices by removal of Mg2+ (Mg2+ free) from bath solution and inhibition of γ-aminobutyric acid A (GABAA) receptors by 100 µM picrotoxin (PTX). The change in syncytial coupling was monitored by using a K+ free-Na+-containing electrode solution ([Na+]p) in the electrophysiological recording where the substitution of intracellular K+ by Na+ ions dissipates the physiological membrane potential (VM) to ~0 mV in the recorded astrocyte. However, in a syncytial coupled astrocyte, the [Na+]p induced VM loss can be compensated by the coupled astrocytes to a quasi-physiological membrane potential of ~73 mV. After short-term exposure to this experimental epileptic condition, a significant closure of syncytial coupling was indicated by a shift of the quasi-physiological membrane potential to -60 mV, corresponding to a 90% reduction of syncytial coupling strength. Consequently, the closure of syncytial coupling significantly decreased the ability of the syncytium for spatial redistribution of K+ ions. Altogether, our results show that epileptiform neuronal discharges weaken the strength of syncytial coupling and that in turn impairs the capacity of a syncytium for spatial redistribution of K+ ions.

6.
J Clin Invest ; 130(1): 345-358, 2020 01 02.
Artigo em Inglês | MEDLINE | ID: mdl-31793909

RESUMO

Axon regeneration failure causes neurological deficits and long-term disability after spinal cord injury (SCI). Here, we found that the α2δ2 subunit of voltage-gated calcium channels negatively regulates axon growth and regeneration of corticospinal neurons, the cells that originate the corticospinal tract. Increased α2δ2 expression in corticospinal neurons contributed to loss of corticospinal regrowth ability during postnatal development and after SCI. In contrast, α2δ2 pharmacological blockade through gabapentin administration promoted corticospinal structural plasticity and regeneration in adulthood. Using an optogenetic strategy combined with in vivo electrophysiological recording, we demonstrated that regenerating corticospinal axons functionally integrate into spinal circuits. Mice administered gabapentin recovered upper extremity function after cervical SCI. Importantly, such recovery relies on reorganization of the corticospinal pathway, as chemogenetic silencing of injured corticospinal neurons transiently abrogated recovery. Thus, targeting α2δ2 with a clinically relevant treatment strategy aids repair of motor circuits after SCI.


Assuntos
Axônios/metabolismo , Gabapentina/farmacologia , Regeneração Nervosa/efeitos dos fármacos , Traumatismos da Coluna Vertebral/tratamento farmacológico , Animais , Axônios/patologia , Modelos Animais de Doenças , Feminino , Masculino , Camundongos , Camundongos Transgênicos , Regeneração Nervosa/genética , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Traumatismos da Coluna Vertebral/genética , Traumatismos da Coluna Vertebral/metabolismo , Traumatismos da Coluna Vertebral/patologia
7.
Mol Neurobiol ; 57(3): 1332-1346, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31728930

RESUMO

TREK-1, a two-pore-domain K+ channel, is highly expressed in the central nervous system. Although aberrant expression of TREK-1 is implicated in cognitive impairment, the cellular and functional mechanism underlying this channelopathy is poorly understood. Here we examined TREK-1 contribution to neuronal morphology, excitability, synaptic plasticity, and cognitive function in mice deficient in TREK-1 expression. TREK-1 immunostaining signal mainly appeared in hippocampal pyramidal neurons, but not in astrocytes. TREK-1 gene knockout (TREK-1 KO) increases dendritic sprouting and the number of immature spines in hippocampal CA1 pyramidal neurons. Functionally, TREK-1 KO increases neuronal excitability and enhances excitatory and inhibitory postsynaptic currents (EPSCs and IPSCs). The increased EPSCs appear to be attributed to an increased release probability of presynaptic glutamate and functional expression of postsynaptic AMPA receptors. TREK-1 KO decreased the paired-pulse ratio and severely occluded the long-term potentiation (LTP) in the CA1 region. These altered synaptic transmission and plasticity are associated with recognition memory deficit in TREK-1 KO mice. Although astrocytic expression of TREK-1 has been reported in previous studies, TREK-1 KO does not alter astrocyte membrane K+ conductance or the syncytial network function in terms of syncytial isopotentiality. Altogether, TREK-1 KO profoundly affects the cellular structure and function of hippocampal pyramidal neurons. Thus, the impaired cognitive function in diseases associated with aberrant expression of TREK-1 should be attributed to the failure of this K+ channel in regulating neuronal morphology, excitability, synaptic transmission, and plasticity.


Assuntos
Cognição/fisiologia , Potenciais Pós-Sinápticos Excitadores/fisiologia , Plasticidade Neuronal/genética , Neurônios/fisiologia , Canais de Potássio de Domínios Poros em Tandem/genética , Animais , Astrócitos/metabolismo , Potenciais Pós-Sinápticos Excitadores/genética , Hipocampo/metabolismo , Potenciação de Longa Duração/fisiologia , Camundongos Knockout , Plasticidade Neuronal/fisiologia , Células Piramidais/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia
8.
Neural Regen Res ; 14(4): 595-596, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30632498
9.
Glia ; 66(12): 2756-2769, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30277621

RESUMO

Syncytial isopotentiality, resulting from a strong electrical coupling, emerges as a physiological mechanism that coordinates individual astrocytes to function as a highly efficient system in brain homeostasis. However, whether syncytial isopotentiality occurs selectively to certain brain regions or is universal to astrocytic networks remains unknown. Here, we have explored the correlation of syncytial isopotentiality with different astrocyte subtypes in various brain regions. Using a nonphysiological K+ -free/Na+ electrode solution to depolarize a recorded astrocyte in situ, the existence of syncytial isopotentiality can be revealed: the recorded astrocyte's membrane potential remains at a quasi-physiological level due to strong electrical coupling with neighboring astrocytes. Syncytial isopotentiality appears in Layer I of the motor, sensory, and visual cortical regions, where astrocytes are organized with comparable cell densities, interastrocytic distances, and the quantity of directly coupled neighbors. Second, though astrocytes vary in their cytoarchitecture in association with neuronal circuits from Layers I-VI, the established syncytial isopotentiality remains comparable among different layers in the visual cortex. Third, neurons and astrocytes are uniquely organized as barrels in Layer IV somatosensory cortex; interestingly, astrocytes both inside and outside of the barrels do electrically communicate with each other and also share syncytial isopotentiality. Fourth, syncytial isopotentiality appears in radial-shaped Bergmann glia and velate astrocytes in the cerebellar cortex. Fifth, although fibrous astrocytes in white matter exhibit a distinct morphology, their network syncytial isopotentiality is comparable with protoplasmic astrocytes. Altogether, syncytial isopotentiality appears as a system-wide electrical feature of astrocytic networks in the brain.


Assuntos
Astrócitos/fisiologia , Encéfalo/citologia , Junções Comunicantes/fisiologia , Potenciais da Membrana/fisiologia , Rede Nervosa/fisiologia , Família Aldeído Desidrogenase 1 , Animais , Animais Recém-Nascidos , Células Cultivadas , Conexina 43/metabolismo , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Técnicas In Vitro , Isoenzimas/genética , Isoenzimas/metabolismo , Lisina/análogos & derivados , Lisina/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Técnicas de Patch-Clamp , Fosfopiruvato Hidratase/metabolismo , Retinal Desidrogenase/genética , Retinal Desidrogenase/metabolismo , Sódio/metabolismo , Substância Branca/citologia
10.
Exp Neurol ; 303: 1-11, 2018 05.
Artigo em Inglês | MEDLINE | ID: mdl-29407729

RESUMO

Membrane potential (VM) depolarization occurs immediately following cerebral ischemia and is devastating for the astrocyte homeostasis and neuronal signaling. Previously, an excessive release of extracellular K+ and glutamate has been shown to underlie an ischemia-induced VM depolarization. Ischemic insults should impair membrane ion channels and disrupt the physiological ion gradients. However, their respective contribution to ischemia-induced neuronal and glial depolarization and loss of neuronal excitability are unanswered questions. A short-term oxygen-glucose deprivation (OGD) was used for the purpose of examining the acute effect of ischemic conditions on ion channel activity and physiological K+ gradient in neurons and glial cells. We show that a 30 min OGD treatment exerted no measurable damage to the function of membrane ion channels in neurons, astrocytes, and NG2 glia. As a result of the resilience of membrane ion channels, neuronal spikes last twice as long as our previously reported 15 min time window. In the electrophysiological analysis, a 30 min OGD-induced dissipation of transmembrane K+ gradient contributed differently in brain cell depolarization: severe in astrocytes and neurons, and undetectable in NG2 glia. The discrete cellular responses to OGD corresponded to a total loss of 69% of the intracellular K+ contents in hippocampal slices as measured by Inductively Coupled Plasma Mass Spectrometry (ICP-MS). A major brain cell depolarization mechanism identified here is important for our understanding of cerebral ischemia pathology. Additionally, further understanding of the resilient response of NG2 glia to ischemia-induced intracellular K+ loss and depolarization should facilitate the development of future stroke therapy.


Assuntos
Astrócitos/fisiologia , Fenômenos Biofísicos/fisiologia , Glucose/metabolismo , Hipóxia/fisiopatologia , Potenciais da Membrana/fisiologia , Neurônios/fisiologia , Potássio/metabolismo , Animais , Animais Recém-Nascidos , Antígenos/metabolismo , Fenômenos Biofísicos/efeitos dos fármacos , Condutividade Elétrica , Feminino , Células Gigantes/fisiologia , Hipocampo/citologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oxigênio/farmacologia , Técnicas de Patch-Clamp , Proteoglicanas/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo
11.
Mol Brain ; 9: 34, 2016 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-27004553

RESUMO

BACKGROUND: Neonatal astrocytes are diverse in origin, and undergo dramatic change in gene expression, morphological differentiation and  syncytial networking throughout development. Neonatal astrocytes also play multifaceted roles in neuronal circuitry establishment. However, the extent to which neonatal astrocytes differ from their counterparts in the adult brain remains unknown. RESULTS: Based on ALDH1L1-eGFP expression or sulforhodamine 101 staining, neonatal astrocytes at postnatal day 1-3 can be reliably identified in hippocampal stratum radiatum. They exhibit a more negative resting membrane potential (V M), -85 mV, than mature astrocytes, -80 mV and a variably rectifying whole-cell current profile due to complex expression of voltage-gated outward transient K(+) (IKa), delayed rectifying K(+) (IKd) and inward K(+) (IKin) conductances. Differing from NG2 glia, depolarization-induced inward Na(+) currents (INa) could not be detected in neonatal astrocytes. A quasi-physiological V M of -69 mV was retained when inwardly rectifying Kir4.1 was inhibited by 100 µM Ba(2+) in both wild type and TWIK-1/TREK-1 double gene knockout astrocytes, indicating expression of additional leak K(+) channels yet unknown. In dual patch recording, electrical coupling was detected in 74 % (14/19 pairs) of neonatal astrocytes with largely variable coupling coefficients. The increasing gap junction coupling progressively masked the rectifying K(+) conductances to account for an increasing number of linear voltage-to-current relationship passive astrocytes (PAs). Gap junction inhibition, by 100 µM meclofenamic acid, substantially reduced membrane conductance and converted all the neonatal PAs to variably rectifying astrocytes. The low density expression of leak K(+) conductance in neonatal astrocytes corresponded  to a ~50 % less K(+) uptake capacity compared to adult astrocytes. CONCLUSIONS: Neonatal astrocytes predominantly express a variety of rectifying K(+) conductances, form discrete cell-to-cell gap junction coupling and are deficient in K(+) homeostatic capacity.


Assuntos
Astrócitos/metabolismo , Fenômenos Eletrofisiológicos , Hipocampo/metabolismo , Animais , Bário/metabolismo , Junções Comunicantes/metabolismo , Ativação do Canal Iônico , Cinética , Camundongos Endogâmicos C57BL , Fenótipo , Canais de Potássio Corretores do Fluxo de Internalização/metabolismo
12.
Front Cell Neurosci ; 10: 13, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26869883

RESUMO

We have recently shown that a linear current-to-voltage (I-V) relationship of membrane conductance (passive conductance) reflects the intrinsic property of K(+) channels in mature astrocytes. While passive conductance is known to underpin a highly negative and stable membrane potential (V M) essential for the basic homeostatic function of astrocytes, a complete repertoire of the involved K(+) channels remains elusive. TREK-1 two-pore domain K(+) channel (K2P) is highly expressed in astrocytes, and covalent association of TREK-1 with TWIK-1, another highly expressed astrocytic K2P, has been reported as a mechanism underlying the trafficking of heterodimer TWIK-1/TREK-1 channel to the membrane and contributing to astrocyte passive conductance. To decipher the individual contribution of TREK-1 and address whether the appearance of passive conductance is conditional to the co-expression of TWIK-1/TREK-1 in astrocytes, TREK-1 single and TWIK-1/TREK-1 double gene knockout mice were used in the present study. The relative quantity of mRNA encoding other astrocyte K(+) channels, such as Kir4.1, Kir5.1, and TREK-2, was not altered in these gene knockout mice. Whole-cell recording from hippocampal astrocytes in situ revealed no detectable changes in astrocyte passive conductance, V M, or membrane input resistance (R in) in either kind of gene knockout mouse. Additionally, TREK-1 proteins were mainly located in the intracellular compartments of the hippocampus. Altogether, genetic deletion of TREK-1 alone or together with TWIK-1 produced no obvious alteration in the basic electrophysiological properties of hippocampal astrocytes. Thus, future research focusing on other K(+) channels may shed light on this long-standing and important question in astrocyte physiology.

13.
Mol Neurobiol ; 53(9): 6169-6182, 2016 11.
Artigo em Inglês | MEDLINE | ID: mdl-26553349

RESUMO

TWIK-1 two-pore domain K+ channels are highly expressed in mature hippocampal astrocytes. While the TWIK-1 activity is readily detectable on astrocyte membrane, the majority of channels are retained in the intracellular compartments, which raises an intriguing question of whether the membrane TWIK-1 channels could be dynamically regulated for functions yet unknown. Here, the regulation of TWIK-1 membrane expression by Gi/Go-coupled metabotropic glutamate receptor 3 (mGluR3) and its functional impact on ammonium uptake has been studied. Activation of mGluR3 induced a marked translocation of TWIK-1 channels from the cytoplasm to the membrane surface. Consistent with our early observation that membrane TWIK-1 behaves as nonselective monovalent cation channel, mGluR3-mediated TWIK-1 membrane expression was associated with a depolarizing membrane potential (V M). As TWIK-1 exhibits a discernibly high permeability to ammonium (NH4+), a critical substrate in glutamate-glutamine cycle for neurotransmitter replenishment, regulation of NH4+ uptake capacity by TWIK-1 membrane expression was determined by response of astrocyte V M to bath application of 5 mM NH4Cl. Stimulation of mGluR3 potentiated NH4+-induced V M depolarization by ∼30 % in wild type, but not in TWIK-1 knockout astrocytes. Furthermore, activation of mGluR3 mediated a coordinated translocation of TWIK-1 channels with recycling endosomes toward astrocyte membrane and the mGluR3-mediated potentiation of NH4+ uptake required a functional Rab-mediated trafficking pathway. Altogether, we demonstrate that the activation of mGluR3 up-regulates the membrane expression of TWIK-1 that in turn enhances NH4+ uptake in astrocytes, a mechanism potentially important for functional coupling of astrocyte glutamate-glutamine cycle with the replenishment of neurotransmitters in neurons.


Assuntos
Compostos de Amônio/metabolismo , Astrócitos/metabolismo , Membrana Celular/metabolismo , Citoplasma/metabolismo , Hipocampo/citologia , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Animais , Endocitose , Endossomos/metabolismo , Exocitose , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores de Glutamato Metabotrópico/genética , ATPase Trocadora de Sódio-Potássio/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo
14.
Glia ; 64(2): 214-26, 2016 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-26435164

RESUMO

Astrocytes are extensively coupled through gap junctions into a syncytium. However, the basic role of this major brain network remains largely unknown. Using electrophysiological and computational modeling methods, we demonstrate that the membrane potential (VM) of an individual astrocyte in a hippocampal syncytium, but not in a single, freshly isolated cell preparation, can be well-maintained at quasi-physiological levels when recorded with reduced or K(+) free pipette solutions that alter the K(+) equilibrium potential to non-physiological voltages. We show that an astrocyte's associated syncytium provides powerful electrical coupling, together with ionic coupling at a lesser extent, that equalizes the astrocyte's VM to levels comparable to its neighbors. Functionally, this minimizes VM depolarization attributable to elevated levels of local extracellular K(+) and thereby maintains a sustained driving force for highly efficient K(+) uptake. Thus, gap junction coupling functions to achieve isopotentiality in astrocytic networks, whereby a constant extracellular environment can be powerfully maintained for crucial functions of neural circuits.


Assuntos
Astrócitos/fisiologia , Junções Comunicantes/fisiologia , Potenciais da Membrana/fisiologia , Animais , Cátions Monovalentes/metabolismo , Células Cultivadas , Espaço Extracelular/metabolismo , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Hipocampo/fisiologia , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Vias Neurais/fisiologia , Potássio/metabolismo , Técnicas de Cultura de Tecidos
15.
J Neurophysiol ; 113(10): 3744-50, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25810481

RESUMO

Mature astrocytes exhibit a linear current-to-voltage K(+) membrane conductance (passive conductance) and an extremely low membrane resistance (Rm) in situ. The combination of these electrophysiological characteristics establishes a highly negative and stable membrane potential that is essential for basic functions, such as K(+) spatial buffering and neurotransmitter uptake. However, astrocytes are coupled extensively in situ. It remains to be determined whether the observed passive behavior and low Rm are attributable to the intrinsic properties of membrane ion channels or to gap junction coupling in functionally mature astrocytes. In the present study, freshly dissociated hippocampal tissues were used as a new model to examine this basic question in young adult animals. The morphologically intact single astrocytes could be reliably dissociated from animals postnatal day 21 and older. At this animal age, dissociated single astrocytes exhibit passive conductance and resting membrane potential similar to those exhibited by astrocytes in situ. To precisely measure the Rm from single astrocytes, dual-patch single-astrocyte recording was performed. We show that dissociated single astrocytes exhibit a low Rm similarly to syncytial coupled astrocytes. Functionally, the symmetric expression of high-K(+) conductance enabled rapid change in the intracellular K(+) concentrations in response to changing K(+) drive force. Altogether, we demonstrate that freshly dissociated tissue preparation is a highly useful model for study of the functional expression and regulation of ion channels, receptors, and transporters in astrocytes and that passive behavior and low Rm are the intrinsic properties of mature astrocytes.


Assuntos
Astrócitos/fisiologia , Junções Comunicantes/fisiologia , Hipocampo/citologia , Potenciais da Membrana/fisiologia , Animais , Biofísica , Estimulação Elétrica , Técnicas In Vitro , Camundongos , Microscopia Confocal , Técnicas de Patch-Clamp , Potássio/metabolismo , Rodaminas/metabolismo
16.
PLoS One ; 7(11): e49020, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23152843

RESUMO

Glycobiology research with Caenorhabditis elegans (C. elegans) has benefitted from the numerous genetic and cell biology tools available in this system. However, the lack of a cell line and the relative inaccessibility of C. elegans somatic cells in vivo have limited the biochemical approaches available in this model. Here we report that C. elegans primary embryonic cells in culture incorporate azido-sugar analogs of N-acetylgalactosamine (GalNAc) and N-acetylglucosamine (GlcNAc), and that the labeled glycoproteins can be analyzed by mass spectrometry. By using this metabolic labeling approach, we have identified a set of novel C. elegans glycoprotein candidates, which include several mitochondrially-annotated proteins. This observation was unexpected given that mitochondrial glycoproteins have only rarely been reported, and it suggests that glycosylation of mitochondrially-annotated proteins might occur more frequently than previously thought. Using independent experimental strategies, we validated a subset of our glycoprotein candidates. These include a mitochondrial, atypical glycoprotein (ATP synthase α-subunit), a predicted glycoprotein (aspartyl protease, ASP-4), and a protein family with established glycosylation in other species (actin). Additionally, we observed a glycosylated isoform of ATP synthase α-subunit in bovine heart tissue and a primate cell line (COS-7). Overall, our finding that C. elegans primary embryonic cells are amenable to metabolic labeling demonstrates that biochemical studies in C. elegans are feasible, which opens the door to labeling C. elegans cells with other radioactive or azido-substrates and should enable the identification of additional post-translationally modified targets and analysis of the genes required for their modification using C. elegans mutant libraries.


Assuntos
Caenorhabditis elegans/química , Glicoproteínas/química , Actinas/química , Animais , Azidas/química , Caenorhabditis elegans/embriologia , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/química , Carboidratos/química , Células Cultivadas , Glicoproteínas/metabolismo , Glicosilação , Isoenzimas/química , ATPases Mitocondriais Próton-Translocadoras/química
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