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1.
Purinergic Signal ; 2023 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-37713157

RESUMO

Over the last decades, since the discovery of ATP as a transmitter, accumulating evidence has been reported about the role of this nucleotide and purinergic receptors, in particular P2X7 receptors, in the modulation of synaptic strength and plasticity. Purinergic signaling has emerged as a crucial player in orchestrating the molecular interaction between the components of the tripartite synapse, and much progress has been made in how this neuron-glia interaction impacts neuronal physiology under basal and pathological conditions. On the other hand, pannexin1 hemichannels, which are functionally linked to P2X7 receptors, have appeared more recently as important modulators of excitatory synaptic function and plasticity under diverse contexts. In this review, we will discuss the contribution of ATP, P2X7 receptors, and pannexin hemichannels to the modulation of presynaptic strength and its impact on motor function, sensory processing, synaptic plasticity, and neuroglial communication, with special focus on the P2X7 receptor/pannexin hemichannel interplay. We also address major hypotheses about the role of this interaction in physiological and pathological circumstances.

2.
J Neurochem ; 157(4): 1377-1391, 2021 05.
Artigo em Inglês | MEDLINE | ID: mdl-32974913

RESUMO

Perivascular pericytes are key regulators of the blood-brain barrier, vascular development, and cerebral blood flow. Deciphering pericyte roles in health and disease requires cellular tracking; yet, pericyte identification remains challenging. A previous study reported that the far-red fluorophore TO-PRO-3 (642/661), usually employed as a nuclear dye in fixed tissue, was selectively captured by live pericytes from the subventricular zone. Herein, we validated TO-PRO-3 as a specific pericyte tracer in the nervous system (NS). Living pericytes from ex vivo murine hippocampus, cortex, spinal cord, and retina robustly incorporated TO-PRO-3. Classical pericyte immunomarkers such as chondroitin sulphate proteoglycan neuron-glial antigen 2 (NG2) and platelet-derived growth factor receptor beta antigen (PDGFrß) and the new pericyte dye NeuroTrace 500/525 confirmed cellular specificity of dye uptake. The TO-PRO-3 signal enabled quantification of pericytes density and morphometry; likewise, TO-PRO-3 labeling allowed visualization of pericytes associated with other components of the neurovascular unit. A subset of TO-PRO-3 stained cells expressed the contractile protein α-SMA, indicative of their ability to control the capillary diameter. Uptake of TO-PRO-3 was independent of connexin/pannexin channels but was highly sensitive to temperature and showed saturation, suggesting that a yet unidentified protein-mediated active transport sustained dye incorporation. We conclude that TO-PRO-3 labeling provides a reliable and simple tool for the bioimaging of pericytes in the murine NS microvasculature.


Assuntos
Carbocianinas , Corantes Fluorescentes , Pericitos , Coloração e Rotulagem/métodos , Animais , Camundongos
3.
Glia ; 63(5): 795-811, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25643695

RESUMO

Glia plays an active role in neuronal functions and dysfunctions, some of which depend on the expression of astrocyte connexins, the gap junction channel and hemichannel proteins. Under neuroinflammation triggered by the endotoxin lipopolysacharide (LPS), microglia is primary stimulated and releases proinflammatory agents affecting astrocytes and neurons. Here, we investigate the effects of such microglial activation on astrocyte connexin-based channel functions and their consequences on synaptic activity in an ex vivo model. We found that LPS induces astroglial hemichannel opening in acute hippocampal slices while no change is observed in gap junctional communication. Based on pharmacological and genetic approaches we found that the LPS-induced hemichannel opening is mainly due to Cx43 hemichannel activity. This process primarily requires a microglial stimulation resulting in the release of at least two proinflammatory cytokines, IL-1ß and TNF-α. Consequences of the hemichannel-mediated increase in membrane permeability are a calcium rise in astrocytes and an enhanced glutamate release associated to a reduction in excitatory synaptic activity of pyramidal neurons in response to Schaffer's collateral stimulation. As a whole our findings point out astroglial hemichannels as key determinants of the impairment of synaptic transmission during neuroinflammation.


Assuntos
Astrócitos/metabolismo , Conexina 43/metabolismo , Hipocampo/citologia , Microglia/fisiologia , Neurônios/fisiologia , Animais , Animais Recém-Nascidos , Astrócitos/efeitos dos fármacos , Antígeno CD11b/metabolismo , Carbenoxolona/farmacologia , Conexina 30 , Conexina 43/genética , Conexinas/deficiência , Conexinas/genética , Conexinas/farmacologia , Citocinas/metabolismo , Proteína Glial Fibrilar Ácida/genética , Proteína Glial Fibrilar Ácida/metabolismo , Ácido Glutâmico/metabolismo , Técnicas In Vitro , Potenciais Pós-Sinápticos Inibidores/efeitos dos fármacos , Potenciais Pós-Sinápticos Inibidores/genética , Lipopolissacarídeos/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Minociclina/farmacologia , Proteínas do Tecido Nervoso/farmacologia , Neurônios/efeitos dos fármacos , Peptídeos/farmacologia , Fatores de Tempo
4.
Cell Signal ; 117: 111113, 2024 05.
Artigo em Inglês | MEDLINE | ID: mdl-38395185

RESUMO

The emerging role of glial cells in modulating neuronal excitability and synaptic strength is a growing field in neuroscience. In recent years, a pivotal role of gliotransmission in homeostatic presynaptic plasticity has been highlighted and glial-derived ATP arises as a key contributor. However, very little is known about the glial non-vesicular ATP-release pathway and how ATP participates in the modulation of synaptic strength. Here, we investigated the functional changes occurring in neurons upon chronic inactivity and the role of the purinergic signaling, connexin43 and pannexin1 hemichannels in this process. By using hippocampal dissociated cultures, we showed that blocking connexin43 and pannexin1 hemichannels decreases the amount of extracellular ATP. Moreover, Ca2+ imaging assays using Fluo-4/AM revealed that blocking connexin43, neuronal P2X7Rs and pannexin1 hemichannels decreases the amount of basal Ca2+ in neurons. A significant impairment in synaptic vesicle pool size was also evidenced under these conditions. Interestingly, rescue experiments where Panx1HCs are blocked showed that the compensatory adjustment of cytosolic Ca2+ was recovered after P2X7Rs activation, suggesting that Panx1 acts downstream P2X7Rs. These changes were accompanied by a modulation of neuronal permeability, as revealed by ethidium bromide uptake experiments. In particular, the permeability of neuronal P2X7Rs and pannexin1 hemichannels is increased upon 24 h of inactivity. Taken together, we have uncovered a role for connexin43-dependent ATP release and neuronal P2X7Rs and pannexin1 hemichannels in the adjustment of presynaptic strength by modulating neuronal permeability, the entrance of Ca2+ into neurons and the size of the recycling pool of synaptic vesicles.


Assuntos
Conexina 43 , Conexinas , Receptores Purinérgicos P2X7 , Trifosfato de Adenosina/metabolismo , Conexina 43/metabolismo , Conexinas/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Animais , Camundongos , Ratos , Receptores Purinérgicos P2X7/metabolismo
5.
Proc Natl Acad Sci U S A ; 107(52): 22659-64, 2010 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-21148774

RESUMO

Spinal astrocytes are coupled by connexin (Cx) gap junctions and express pannexin 1 (Px1) and purinergic receptors. Fibroblast growth factor 1 (FGF-1), which is released in spinal cord injury, activated spinal astrocytes in culture, induced secretion of ATP, and permeabilized them to relatively large fluorescent tracers [ethidium (Etd) and lucifer yellow (LY)] through "hemichannels" (HCs). HCs can be formed by connexins or pannexins; they can open to extracellular space or can form gap junction (GJ) channels, one HC from each cell. (Pannexins may not form gap junctions in mammalian tissues, but they do in invertebrates). HC types were differentiated pharmacologically and by Px1 knockdown with siRNA and by use of astrocytes from Cx43 knockout mice. Permeabilization was reduced by apyrase (APY), an ATPase, and by P2X(7) receptor antagonists, implicating secretion of ATP and autocrine and/or paracrine action. Increased permeability of cells exposed to FGF-1 or ATP for 2 h was mediated largely by Px1 HCs activated by P2X(7) receptors. After a 7-h treatment, the permeability was mediated by both Cx43 and Px1 HCs. FGF-1 also caused reduction in gap junctional communication. Botulinum neurotoxin A, a blocker of vesicular release, reduced permeabilization when given 30 min before FGF-1 application, but not when given 1 h after FGF-1. We infer that ATP is initially released from vesicles and then it mediates continued release by action on P2X(7) receptors and opening of HCs. These changes in HCs and gap junction channels may promote inflammation and deprive neurons of astrocyte-mediated protection in spinal cord trauma and neurodegenerative disease.


Assuntos
Trifosfato de Adenosina/metabolismo , Astrócitos/efeitos dos fármacos , Conexinas/metabolismo , Fator 1 de Crescimento de Fibroblastos/farmacologia , Proteínas do Tecido Nervoso/metabolismo , Animais , Animais Recém-Nascidos , Astrócitos/citologia , Astrócitos/metabolismo , Western Blotting , Toxinas Botulínicas Tipo A/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Conexina 43/genética , Conexina 43/metabolismo , Conexinas/genética , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Proteínas do Tecido Nervoso/genética , Neurotoxinas/farmacologia , Interferência de RNA , Ratos , Medula Espinal/citologia , Fatores de Tempo
6.
J Neurosci ; 31(13): 4962-77, 2011 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-21451035

RESUMO

The mechanisms involved in Alzheimer's disease are not completely understood and how glial cells contribute to this neurodegenerative disease remains to be elucidated. Because inflammatory treatments and products released from activated microglia increase glial hemichannel activity, we investigated whether amyloid-ß peptide (Aß) could regulate these channels in glial cells and affect neuronal viability. Microglia, astrocytes, or neuronal cultures as well as acute hippocampal slices made from GFAP-eGFP transgenic mice were treated with the active fragment of Aß. Hemichannel activity was monitored by single-channel recordings and by time-lapse ethidium uptake, whereas neuronal death was assessed by Fluoro-Jade C staining. We report that low concentrations of Aß(25-35) increased hemichannel activity in all three cell types and microglia initiate these effects triggered by Aß. Finally, neuronal damage occurs by activation of neuronal hemichannels induced by ATP and glutamate released from Aß(25-35)-activated glia. These responses were observed in the presence of external calcium and were differently inhibited by hemichannel blockers, whereas the Aß(25-35)-induced neuronal damage was importantly reduced in acute slices made from Cx43 knock-out mice. Thus, Aß leads to a cascade of hemichannel activation in which microglia promote the release of glutamate and ATP through glial (microglia and astrocytes) hemichannels that induces neuronal death by triggering hemichannels in neurons. Consequently, this work opens novel avenues for alternative treatments that target glial cells and neurons to maintain neuronal survival in the presence of Aß.


Assuntos
Peptídeos beta-Amiloides/toxicidade , Morte Celular/fisiologia , Neuroglia/fisiologia , Neurônios/patologia , Fragmentos de Peptídeos/toxicidade , Trifosfato de Adenosina/metabolismo , Animais , Animais Recém-Nascidos , Células Cultivadas , Conexina 43/antagonistas & inibidores , Conexina 43/deficiência , Conexina 43/metabolismo , Ácido Glutâmico/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neuroglia/metabolismo , Neuroglia/patologia , Neurônios/fisiologia
7.
Mol Neurobiol ; 59(6): 3738-3754, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35381889

RESUMO

Iron deficiency anemia is a prevalent health problem among pregnant women and infants, particularly in the developing countries that causes brain development deficits and poor cognitive outcomes. Since tissue iron depletion may impair myelination and trigger cellular hypoxic signaling affecting blood vessels, we studied myelination and the neurovascular unit (NVU) in infant rats born to mothers fed with an iron deficient (ID) or control diet from embryonic day 5 till weaning. Blood samples and brains of rat pups at postnatal day (PND) 14 and 30 were analyzed. PND 14 ID rats had severe microcytic hypochromic anemia that was almost reversed at PND 30 although hypomyelination and astrocyte immature phenotype in the corpus callosum were significant at that age. In CA1 hippocampal region, PND 14 and PND 30 ID rats showed significant reduced expression of the receptor ß of the platelet-derived growth factor localized in pericytes and associated to aquaporin 4 (AQP4) immunopositive capillaries. Shorter AQP4 + capillaries and reduced AQP4 expression were also evidenced in PND 14 and PND 30 ID rats. In addition, pericyte membrane permeability through large-pore channels was transiently increased in ID rats at PND 14 but not at PND 30, while the blood-brain barrier permeability was not affected. Remarkably, transient increased pericyte permeability found in PND 14 ID rats was not directly related to iron depletion, suggesting the involvement of other iron deficiency anemia-induced mechanisms. In summary, severe ID during gestation and lactation produces persistent hypomyelination and significantly affects hippocampal pericytes and astrocytes in the NVU which may trigger impaired neurovascular function.


Assuntos
Anemia Ferropriva , Deficiências de Ferro , Anemia Ferropriva/complicações , Anemia Ferropriva/metabolismo , Animais , Animais Recém-Nascidos , Feminino , Hipocampo/metabolismo , Humanos , Ferro/metabolismo , Lactação , Gravidez , Ratos
8.
J Neurochem ; 118(4): 546-57, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21644995

RESUMO

The source size and density determine the extent of nitric oxide (NO) diffusion which critically influences NO signaling. In the brain, NO released from postsynaptic somas following NMDA-mediated activation of neuronal nitric oxide synthase (nNOS) retrogradely affects smaller presynaptic targets. By contrast, in guinea pig trigeminal motor nucleus (TMN), NO is produced presynaptically by tiny and disperse nNOS-containing terminals that innervate large nNOS-negative motoneurons expressing the soluble guanylyl-cyclase (sGC); consequently, it is uncertain whether endogenous NO supports an anterograde signaling between pre-motor terminals and postsynaptic trigeminal motoneurons. In retrogradely labeled motoneurons, we indirectly monitored NO using triazolofluorescein (DAF-2T) fluorescence, and evaluated sGC activity by confocal cGMP immunofluorescence. Multiple fibers stimulation enhanced NO content and cGMP immunofluorescence into numerous nNOS-negative motoneurons; NOS inhibitors prevented depolarization-induced effects, whereas NO donors mimicked them. Enhance of cGMP immunofluorescence required extracellular Ca(2+), a nNOS-physiological activator, and was prevented by inhibiting sGC, silencing neuronal activity or impeding NO diffusion. In conclusion, NO released presynaptically from multiple cooperative tiny fibers attains concentrations sufficient to activate sGC in many motoneurons despite of the low source/target size ratio and source dispersion; thus, endogenous NO is an effective anterograde neuromodulator. By adjusting nNOS activation, presynaptic Ca(2+) might modulate the NO diffusion field in the TMN.


Assuntos
Sistema Nervoso Central/fisiologia , Óxido Nítrico/fisiologia , Receptores Pré-Sinápticos/fisiologia , Transdução de Sinais/fisiologia , Animais , Tronco Encefálico/fisiologia , Sinalização do Cálcio/fisiologia , Sistema Nervoso Central/citologia , GMP Cíclico/fisiologia , Fenômenos Eletrofisiológicos , Ativação Enzimática/fisiologia , Fluoresceína , Corantes Fluorescentes , Guanilato Ciclase/metabolismo , Cobaias , Processamento de Imagem Assistida por Computador , Imuno-Histoquímica , Técnicas In Vitro , Microscopia Confocal , Neurônios Motores/fisiologia , Fibras Nervosas/fisiologia , Óxido Nítrico Sintase Tipo I/metabolismo , Recrutamento Neurofisiológico/fisiologia , Transmissão Sináptica/fisiologia , Nervo Trigêmeo/fisiologia
9.
Bio Protoc ; 11(22): e4222, 2021 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-34909443

RESUMO

This protocol describes a method for high-resolution confocal imaging of pericytes with the far-red fluorophore TO-PROTM-3 Iodide 642/661 in cerebral slices of murine. Identification of pericytes with TO-PRO-3 is a short time-consuming, high cost-effective and robust technique to label pericytes with no need for immunostaining or generation of reporter mice. Since the TO-PRO-3 stain resists immunofluorescence, and lacks spectral overlap, the probe is well suited for multiple labelling. Our procedures also combine TO-PRO-3-staining of pericytes with fluorescent markers for astrocytes and vessels in brain slices. These approaches should enable the assessment of pericyte biology in gliovascular unit.

10.
Mol Neurobiol ; 57(6): 2856-2869, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32388797

RESUMO

A key feature of neurotransmission is its ability to adapt to changes in neuronal environment, which is essential for many brain functions. Homeostatic synaptic plasticity (HSP) emerges as a compensatory mechanism used by neurons to adjust their excitability in response to changes in synaptic activity. Recently, glial cells emerged as modulators for neurotransmission by releasing gliotransmitters into the synaptic cleft through pathways that include P2X7 receptors (P2X7R), connexons, and pannexons. However, the role of gliotransmission in the activity-dependent adjustment of presynaptic strength is still an open question. Here, we investigated whether glial cells participate in HSP upon chronic inactivity and the role of adenosine triphosphate (ATP), connexin43 hemichannels (Cx43HCs), and pannexin1 (Panx1) channels in this process. We used immunocytochemistry against vesicular glutamate transporter 1 (vGlut1) to estimate changes in synaptic strength in hippocampal dissociated cultures. Pharmacological manipulations indicate that glial-derived ATP and P2X7R are required for HSP. In addition, inhibition of Cx43 and Panx1 channels reveals a pivotal role for these channels in the compensatory adjustment of synaptic strength, emerging as new pathways for ATP release upon inactivity. The involvement of Panx1 channels was confirmed by using Panx1-deficient animals. Lacking Panx1 in neurons is sufficient to prevent the P2X7R-dependent upregulation of presynaptic strength; however, the P2X7R-dependent compensatory adjustment of synapse density requires both neuronal and glial Panx1. Together, our data supports an essential role for glial ATP signaling and Cx43HCs and Panx1 channels in the homeostatic adjustment of synaptic strength in hippocampal cultures upon chronic inactivity.


Assuntos
Trifosfato de Adenosina/metabolismo , Conexinas/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/metabolismo , Neurônios/metabolismo , Sinapses/metabolismo , Transmissão Sináptica/fisiologia , Animais , Conexina 43/metabolismo , Conexinas/genética , Hipocampo/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Ratos , Receptores Purinérgicos P2X7/metabolismo
11.
Mol Neurobiol ; 56(11): 7694-7707, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31104295

RESUMO

Glutaric acidemia I (GA-I) is an inherited neurometabolic childhood disease characterized by bilateral striatal neurodegeneration upon brain accumulation of millimolar concentrations of glutaric acid (GA) and related metabolites. Vascular dysfunction, including abnormal cerebral blood flow and blood-brain barrier damage, is an early pathological feature in GA-I, although the affected cellular targets and underlying mechanisms remain unknown. In the present study, we have assessed the effects of GA on capillary pericyte contractility in cerebral cortical slices and pericyte cultures, as well as on the survival, proliferation, and migration of cultured pericytes. GA induced a significant reduction in capillary diameter at distances up to ~ 10 µm from the center of pericyte somata. However, GA did not affect the contractility of cultured pericytes, suggesting that the response elicited in slices may involve GA evoking pericyte contraction by acting on other cellular components of the neurovascular unit. Moreover, GA indirectly inhibited migration of cultured pericytes, an effect that was dependent on soluble glial factors since it was observed upon application of conditioned media from GA-treated astrocytes (CM-GA), but not upon direct GA addition to the medium. Remarkably, CM-GA showed increased expression of cytokines and growth factors that might mediate the effects of increased GA levels not only on pericyte migration but also on vascular permeability and angiogenesis. These data suggest that some effects elicited by GA might be produced by altering astrocyte-pericyte communication, rather than directly acting on pericytes. Importantly, GA-evoked alteration of capillary pericyte contractility may account for the reduced cerebral blood flow observed in GA-I patients.


Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/patologia , Encefalopatias Metabólicas/patologia , Movimento Celular/efeitos dos fármacos , Glutaratos/farmacologia , Glutaril-CoA Desidrogenase/deficiência , Pericitos/patologia , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Capilares/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/patologia , Meios de Cultivo Condicionados/farmacologia , Citocinas/metabolismo , Pericitos/efeitos dos fármacos , Pericitos/metabolismo , Ratos Sprague-Dawley , Vasoconstrição/efeitos dos fármacos
13.
Respir Physiol Neurobiol ; 161(2): 210-3, 2008 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-18329965

RESUMO

Cultured glomus cells from rat carotid bodies were prepared for optical studies of intracellular calcium using the Fura-2 dye. The baseline calcium had a mean of about 40 nM showing either a relatively steady level or large calcium spikes. Some cells did not show measurable levels of [Ca(2+)](i). Stirring the fluid bathing the cultures induced large increases in [Ca(2+)](i) which were abolished when the bathing medium had zero Ca(2+) and EGTA. It is concluded that glomus cells respond to mechanical stimulation when directly exposed to this stimulus and are not protected by supporting structures. It is unknown if the electrical properties of these cells are also affected by mechanical challenges.


Assuntos
Cálcio/metabolismo , Corpo Carotídeo/metabolismo , Células Quimiorreceptoras/metabolismo , Animais , Corpo Carotídeo/citologia , Células Cultivadas , Células Quimiorreceptoras/citologia , Técnicas In Vitro , Pressão , Ratos , Estresse Mecânico , Tato
14.
Front Mol Neurosci ; 11: 435, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30564096

RESUMO

The classical view of synapses as the functional contact between presynaptic and postsynaptic neurons has been challenged in recent years by the emerging regulatory role of glial cells. Astrocytes, traditionally considered merely supportive elements are now recognized as active modulators of synaptic transmission and plasticity at the now so-called "tripartite synapse." In addition, an increasing body of evidence indicates that beyond immune functions microglia also participate in various processes aimed to shape synaptic plasticity. Release of neuroactive compounds of glial origin, -process known as gliotransmission-, constitute a widespread mechanism through which glial cells can either potentiate or reduce the synaptic strength. The prevailing vision states that gliotransmission depends on an intracellular Ca2+/exocytotic-mediated release; notwithstanding, growing evidence is pointing at hemichannels (connexons) and pannexin channels (pannexons) as alternative non-vesicular routes for gliotransmitters efflux. In concurrence with this novel concept, both hemichannels and pannexons are known to mediate the transfer of ions and signaling molecules -such as ATP and glutamate- between the cytoplasm and the extracellular milieu. Importantly, recent reports show that glial hemichannels and pannexons are capable to perceive synaptic activity and to respond to it through changes in their functional state. In this article, we will review the current information supporting the "double edge sword" role of hemichannels and pannexons in the function of central and peripheral synapses. At one end, available data support the idea that these channels are chief components of a feedback control mechanism through which gliotransmitters adjust the synaptic gain in either resting or stimulated conditions. At the other end, we will discuss how the excitotoxic release of gliotransmitters and [Ca2+]i overload linked to the opening of hemichannels/pannexons might impact cell function and survival in the nervous system.

15.
Front Cell Neurosci ; 8: 306, 2014.
Artigo em Inglês | MEDLINE | ID: mdl-25374505

RESUMO

In the brain, astrocytes represent the cellular population that expresses the highest amount of connexins (Cxs). This family of membrane proteins is the molecular constituent of gap junction channels and hemichannels that provide pathways for direct cytoplasm-to-cytoplasm and inside-out exchange, respectively. Both types of Cx channels are permeable to ions and small signaling molecules allowing astrocytes to establish dynamic interactions with neurons. So far, most pharmacological approaches currently available do not distinguish between these two channel functions, stressing the need to develop new specific molecular tools. In astrocytes two major Cxs are expressed, Cx43 and Cx30, and there is now evidence indicating that at least Cx43 operates as a gap junction channel as well as a hemichannel in these cells. Based on studies in primary cultures as well as in acute hippocampal slices, we report here that Gap19, a nonapeptide derived from the cytoplasmic loop of Cx43, inhibits astroglial Cx43 hemichannels in a dose-dependent manner, without affecting gap junction channels. This peptide, which not only selectively inhibits hemichannels but is also specific for Cx43, can be delivered in vivo in mice as TAT-Gap19, and displays penetration into the brain parenchyma. As a result, Gap19 combined with other tools opens up new avenues to decipher the role of Cx43 hemichannels in interactions between astrocytes and neurons in physiological as well as pathological situations.

16.
Nat Neurosci ; 17(4): 549-58, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24584052

RESUMO

Astrocytes play active roles in brain physiology by dynamic interactions with neurons. Connexin 30, one of the two main astroglial gap-junction subunits, is thought to be involved in behavioral and basic cognitive processes. However, the underlying cellular and molecular mechanisms are unknown. We show here in mice that connexin 30 controls hippocampal excitatory synaptic transmission through modulation of astroglial glutamate transport, which directly alters synaptic glutamate levels. Unexpectedly, we found that connexin 30 regulated cell adhesion and migration and that connexin 30 modulation of glutamate transport, occurring independently of its channel function, was mediated by morphological changes controlling insertion of astroglial processes into synaptic clefts. By setting excitatory synaptic strength, connexin 30 plays an important role in long-term synaptic plasticity and in hippocampus-based contextual memory. Taken together, these results establish connexin 30 as a critical regulator of synaptic strength by controlling the synaptic location of astroglial processes.


Assuntos
Astrócitos/patologia , Movimento Celular/fisiologia , Conexinas/metabolismo , Ácido Glutâmico/metabolismo , Sinapses/fisiologia , Transmissão Sináptica/fisiologia , Animais , Astrócitos/metabolismo , Comportamento Animal , Conexina 30 , Feminino , Hipocampo/citologia , Hipocampo/metabolismo , Hipocampo/patologia , Masculino , Memória/fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mutação/genética , Plasticidade Neuronal/fisiologia
17.
Methods Mol Biol ; 814: 283-303, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22144314

RESUMO

A typical feature of astrocytes is their high level of connexin expression. These membrane proteins constitute the molecular basis of two types of channels: gap junction channels that allow direct cytoplasm-to-cytoplasm communication and hemichannels that provide a pathway for exchanges between the intra- and extracellular media. An unusual property of these channels is their permeability for ions but also for small signaling molecules. They support intercellular communication that contribute to dynamic neuroglial interaction and interplay with neuronal activity and survival. Here, we describe multiple techniques based either on electrophysiological approaches or the monitoring of dye intercellular diffusion and uptake that permits an investigation of the properties of gap junction channels and hemichannels, respectively. These techniques are applied in astrocyte studies using in vitro models, mainly primary cultures and acute brain slices.


Assuntos
Astrócitos/metabolismo , Comunicação Celular/fisiologia , Conexinas/metabolismo , Potenciais da Membrana/fisiologia , Animais , Astrócitos/fisiologia , Conexinas/fisiologia , Eletrofisiologia , Corantes Fluorescentes , HEPES , Técnicas Histológicas , Camundongos , Microscopia de Fluorescência , Técnicas de Patch-Clamp
18.
Science ; 322(5907): 1551-5, 2008 Dec 05.
Artigo em Inglês | MEDLINE | ID: mdl-19056987

RESUMO

Astrocytes provide metabolic substrates to neurons in an activity-dependent manner. However, the molecular mechanisms involved in this function, as well as its role in synaptic transmission, remain unclear. Here, we show that the gap-junction subunit proteins connexin 43 and 30 allow intercellular trafficking of glucose and its metabolites through astroglial networks. This trafficking is regulated by glutamatergic synaptic activity mediated by AMPA receptors. In the absence of extracellular glucose, the delivery of glucose or lactate to astrocytes sustains glutamatergic synaptic transmission and epileptiform activity only when they are connected by gap junctions. These results indicate that astroglial gap junctions provide an activity-dependent intercellular pathway for the delivery of energetic metabolites from blood vessels to distal neurons.


Assuntos
Astrócitos/metabolismo , Conexina 43/metabolismo , Conexinas/metabolismo , Junções Comunicantes/fisiologia , Glucose/metabolismo , Hipocampo/fisiologia , Transmissão Sináptica , Animais , Glicemia/metabolismo , Permeabilidade da Membrana Celular , Conexina 30 , Difusão , Epilepsia/fisiopatologia , Potenciais Pós-Sinápticos Excitadores , Ácido Glutâmico/metabolismo , Hipocampo/irrigação sanguínea , Hipocampo/citologia , Técnicas In Vitro , Ácido Láctico/metabolismo , Redes e Vias Metabólicas , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Neurônios/fisiologia , Técnicas de Patch-Clamp , Receptores de AMPA/metabolismo
19.
J Neurophysiol ; 88(2): 639-49, 2002 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12163517

RESUMO

The activity of gap junction channels between cultured and clustered carotid body glomus cells of the rat was studied with dual voltage clamping during normoxia (PO(2) 300 Torr) and hypoxia induced by sodium dithionite (Na(2)S(2)O(4)) or 100% N(2). Na(2)S(2)O(4) reduced the saline PO(2) to approximately 10 Torr, whereas 100% N(2) reduced ambient O(2) to approximately 60 Torr. The following observations were made. 1) In normoxia, the intercellular macroconductance (G(j) = 3.0 +/- 1.01 ns, mean +/- SE) was changed unevenly (increased and decreased) under hypoxic conditions by either agent, although N(2) produced the largest changes. 2) The intercellular microconductances of the channels (g(j) = 104.44 +/- 10.16 pS under normoxic conditions) significantly decreased in 100% N(2) but showed depressions and enhancements in Na(2)S(2)O(4). 3) The conductance of single-junction channels (SChs), calculated as g(j) variance/mean g(j), yielded a mean of approximately 17.6 pS. Larger values were obtained with manual measurements of the data (approximately 34 pS). Hypoxic hypoxia (induced by 100% N(2)) significantly depressed the conductance of SChs when calculated from digitized records or from manual measurements. Hypoxia induced by Na(2)S(2)O(4) did not significantly change junctional conductance. 4) The number of intercellular channels, calculated as g(j)/SCh g(j), had a mean of approximately 452 (range 1 to 2,471). During N(2)-induced hypoxia, this number significantly decreased to approximately 84 but remained unchanged during Na(2)S(2)O(4) hypoxia. 5) The mean open time of junction channels varied from 4 to 30 ms in different experiments, having an overall mean of mu = 11.33 +/- 0.33 ms. This value was significantly reduced by 100% N(2) but was not changed by Na(2)S(2)O(4). 6) Intracellular calcium ([Ca(2+)](i)), 46.2 +/- 4.84 nM under normoxia, significantly increased to 77.32 +/- 11.27 nM with Na(2)S(2)O(4) and to 66.39 +/- 11.64 nM with 100% N(2). It is concluded that 100% N(2) uncouples glomus cells by significantly reducing intercellular macro- and microconductances. Hypoxia induced by Na(2)S(2)O(4) had variable effects. The coupling effects of hypoxia may depend on, or be aided by, increases in [Ca(2+)](i) and/or intracellular pH changes. However, secreted transmitters and ATP plus the effects of hypoxia on second messengers and other cytoplasmic components may also play an important role in this phenomenon.


Assuntos
Corpo Carotídeo/metabolismo , Hipóxia Celular , Junções Comunicantes/metabolismo , Animais , Cálcio/metabolismo , Corpo Carotídeo/efeitos dos fármacos , Células Cultivadas , Eletrofisiologia , Junções Comunicantes/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Nitrogênio/farmacologia , Técnicas de Patch-Clamp , Ratos , Ratos Wistar , Sulfatos/farmacologia
20.
J Neurophysiol ; 88(1): 497-506, 2002 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-12091571

RESUMO

We demonstrate the presence of nitric oxide synthase containing fibers within the guinea pig trigeminal motor nucleus and describe the effects of nitric oxide (NO) on trigeminal motoneurons. Using immunohistochemical techniques, we observed nitrergic fibers displaying varicosities and giving rise to bouton-like structures in apposition to retrogradely labeled motoneuron processes, most of which were dendrites. NO-donors evoked a membrane depolarization (mean 7.5 mV) and a decrease in rheobase (mean 38%). These substances also evoked an apparent increase in an hyperpolarization-activated cationic current (I(H)). These changes were not accompanied by any modification of the motoneurons' input resistance or time constant. The effects were suppressed by blocking the cytosolic guanlyate cyclase. A membrane-permeant cyclic guanosine 3,5'-monophosphate (cGMP) analogue mimicked the effects of NO. There was a considerable increase in synaptic activity following NO-donors or db-cGMP application. Tetrodotoxin supressed the increase in synaptic activity evoked by NO-donors. The histological and electrophysiological evidence, taken together, indicates the existence of a nitrergic system able to modulate trigeminal motoneurons under yet unknown physiological conditions.


Assuntos
Neurônios Motores/fisiologia , Neurotransmissores/fisiologia , Óxido Nítrico/fisiologia , Espermina/análogos & derivados , Nervo Trigêmeo/fisiologia , Animais , GMP Cíclico/fisiologia , Eletrofisiologia , Guanilato Ciclase/fisiologia , Cobaias , Neurônios Motores/ultraestrutura , Doadores de Óxido Nítrico/farmacologia , Óxidos de Nitrogênio , Espermina/farmacologia , Sinapses/fisiologia , Triazenos/farmacologia , Nervo Trigêmeo/ultraestrutura
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