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/metabolismoRESUMO
Gamma-Aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the brain. It is produced by interneurons and recycled by astrocytes. In neurons, GABA activates the influx of Cl- via the GABAA receptor or efflux or K+ via the GABAB receptor, inducing hyperpolarization and synaptic inhibition. In astrocytes, the activation of both GABAA and GABAB receptors induces an increase in intracellular Ca2+ and the release of glutamate and ATP. Connexin 43 (Cx43) hemichannels are among the main Ca2+-dependent cellular mechanisms for the astroglial release of glutamate and ATP. However, no study has evaluated the effect of GABA on astroglial Cx43 hemichannel activity and Cx43 hemichannel-mediated gliotransmission. Here we assessed the effects of GABA on Cx43 hemichannel activity in DI NCT1 rat astrocytes and hippocampal brain slices. We found that GABA induces a Ca2+-dependent increase in Cx43 hemichannel activity in astrocytes mediated by the GABAA receptor, as it was blunted by the GABAA receptor antagonist bicuculline but unaffected by GABAB receptor antagonist CGP55845. Moreover, GABA induced the Cx43 hemichannel-dependent release of glutamate and ATP, which was also prevented by bicuculline, but unaffected by CGP. Gliotransmission in response to GABA was also unaffected by pannexin 1 channel blockade. These results are discussed in terms of the possible role of astroglial Cx43 hemichannel-mediated glutamate and ATP release in regulating the excitatory/inhibitory balance in the brain and their possible contribution to psychiatric disorders.
Assuntos
Astrócitos , Conexina 43 , Ratos , Animais , Conexina 43/metabolismo , Astrócitos/metabolismo , Receptores de GABA-A , Bicuculina/farmacologia , Animais Recém-Nascidos , Células Cultivadas , Ácido Glutâmico/farmacologia , Ácido gama-Aminobutírico/farmacologia , Trifosfato de Adenosina/farmacologiaRESUMO
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/metabolismoRESUMO
At least half of human immunodeficiency virus (HIV)-infected individuals suffer from a wide range of cognitive, behavioral and motor deficits, collectively known as HIV-associated neurocognitive disorders (HAND). The molecular mechanisms that amplify damage within the brain of HIV-infected individuals are unknown. Recently, we described that HIV augments the opening of connexin-43 (Cx43) hemichannels in cultured human astrocytes, which result in the collapse of neuronal processes. Whether HIV soluble viral proteins such as gp120, can regulate hemichannel opening in astrocytes is still ignored. These channels communicate the cytosol with the extracellular space during pathological conditions. We found that gp120 enhances the function of both Cx43 hemichannels and pannexin-1 channels in mouse cortical astrocytes. These effects depended on the activation of IL-1ß/TNF-α, p38 MAP kinase, iNOS, cytoplasmic Ca2+ and purinergic signaling. The gp120-induced channel opening resulted in alterations in Ca2+ dynamics, nitric oxide production and ATP release. Although the channel opening evoked by gp120 in astrocytes was reproduced in ex vivo brain preparations, these responses were heterogeneous depending on the CA1 region analyzed. We speculate that soluble gp120-induced activation of astroglial Cx43 hemichannels and pannexin-1 channels could be crucial for the pathogenesis of HAND.
Assuntos
Astrócitos/citologia , Conexina 43/metabolismo , Conexinas/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Astrócitos/metabolismo , Cálcio/metabolismo , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/metabolismo , Camundongos , Óxido Nítrico/metabolismo , Transdução de Sinais , Imagem com Lapso de Tempo , Regulação para CimaRESUMO
Bisphosphonates (BPs), potent inhibitors of bone resorption which inhibit osteoclasts, have also been shown to act on osteocytes and osteoblasts preventing apoptosis via connexin (Cx) 43 hemichannels and activating the extracellular signal regulated kinases ERKs. We previously demonstrated the presence of a saturable, specific and high affinity binding site for alendronate (ALN) in osteoblastic cells which express Cx43. However, cells lacking Cx43 also bound BPs. Herein we show that bound [(3)H]-alendronate is displaced by phosphatase substrates. Moreover, similar to Na3VO4, ALN inhibited the activity of transmembrane and cytoplasmic PTPs, pointing out the catalytic domain of phosphatases as a putative BP target. In addition, anti-phospho-tyrosine immunoblot analysis revealed that ALN stimulates tyrosine phosphorylation of several proteins of whole cell lysates, among which the major targets of the BP could be immunochemically identified as Cx43. Additionally, the transmembrane receptor-like PTPs, RPTPµ and RPTPα, as well as the cytoplasmic PTP1B, are highly expressed in ROS 17/2.8 cells. Furthermore, we evidenced that Cx43 interacts with RPTPµ in ROS 17/2.8 and ALN decreases their association. These results support the hypothesis that BPs bind and inhibit PTPs associated to Cx43 or not, which would lead to the activation of signaling pathways in osteoblasts.