| Previously in neuroscience, the neuronal network was considered as the only important one, and astrocyte was looked upon as a passive bystanders. More recently, the function of astrocytes has been reconsidered [1,2]. Now they were thought to play a number of active roles in brain, including modulation of synaptic transmission,“tripartite synapse”formation, re-uptake of neurotransmitters and vasomodulation (neurovascular unit) [3,4,5,6,7].Furthermore, astrocytes respond to the extracellular stimulus actively. Upon stimulations, astrocytes release gliotransmitters including glutamate and ATP [1,8,9], which is necessary for formation of long-term potentiation (LTP), a synaptic model of learning and memory and participate in many physiological and pathological processes, such as vasodilatation, trauma and neuropathic pain [10,11].Regarding mechanisms of evoked gliotransmitter release, glutamate is released via Calcium dependent and“kiss-and-run”vesicular exocytosis [12,13]. Currently, a major question in gliotransmission is whether ATP release is via vesicular or non-vesicular pathway following physiological stimulations [3,14], although recent works based on indirect measurements favor the exocytosis hypothesis [15,16,17]. So we investigated the release mechanism of astrocytic ATP in the first part of present study.ATP has been regard as an activity-dependent signaling molecule in communication between neurons and glial cells [18], it is also a nociceptive messenger in nerve system [19,20,21],and is involved in pain sensing[19] and glial activation [21,22]. Despite the framework of sensory neuronal system contribute to neuropathic pain[23], a new conception that glial cells are a driving force for creating and maintaining pain following peripheral injury and inflammation is build up [24]. Nerve injury induced microglial recruitment and activation in the dorsal horn was quick and robust, the same alternations of astrocytes were also detected but relatively late and progresses slowly. Activation of different glial cells in complex temporal patterns requires well organized reciprocal communication between neurons and glial cells, and among glial cells themselves [25], but the organization signal(s) and details are still little known. Different subtypes of purinergic receptors have been found in CNS. Many evidences suggested that the P2X4 and P2X7 receptors play important role on neuropathic pain [26,27,28], but many questions should be further addressed, such as, are these receptors conditionally or continuously expressed on microglia, astrocytes and neurons? What role do the activated P2X4 and P2X7 receptors localized in microglia or astrocytes play at different phases of neuropathic pain development?We further investigated the dynamic alternations of astrocytes, microglia and their P2XRs in never injury induced neuropathic pain and provided a system perspective of the relationships among these receptors and glia in the second part of this study.Part I. Mechanisms of ATP Release in Hippocampal Astrocytes[Objectives]To investigate the characters of evoked astrocytic ATP and glutamate release and determine the evoked astrocytic ATP release pathway.[Methods](1) Astrocytes were primary cultured from the hippocampi of neonatal Sprague-Dawley (SD) rat pups (0-day old) and were used from 14 to 20 days in vitro (DIV) after purification; (3) DNA constructs of AMPA receptor subtypes GluR1/2, Purinergic receptor subtypes P2X4R and P2X7R were transfected into HEK293A cells to express the specific receptor as sniffers; (3) The kinetics of ATP /glutamate release from astrocytes as well as the kinetics of ATP release from astrocytes and rat adrenal chromaffin cells (RACC) were recorded by the“sniff-patch”assay; (4) Vesicular catecholamine release from RACC and dopamine preloaded astrocytes were recorded by the amperometry method using micro-carbon fiber electrode (proCFE); (5) Real-time astrocytic vesicular release were visualized on Two-photon excitation microscopy by detecting the fluorescence discharge of FM dye puncta; (6) Intracellular [Ca2+] changes were monitored on the Calcium Image system (Fura-2 AM) or on confocal microscope system (Fluo-4 AM, Rhod-2 AM); (7) The mRNA level of P2X7R and some stretch-sensitive channels in astrocyte were determined by RT-PCR; (8) Pharmacological assay was used to screen the possible candidate molecules for ATP release; (9) P2X7R shRNA lentiviral particles were used to knock down the P2X7R expression in astrocyte; (10) Proteins expression level were measured by western blot, and the distributions were identified by immunofluorescence assay.[Results]Here we show, at single-vesicle spatiotemporal resolution by using patch-clamped“sniffer cells”expressing receptor channels of ATP or glutamate, the physiology-like stretch-induced ATP and glutamate releases are through fundamentally different mechanistic pathways in astrocytes. Distinct from the evoked quantal glutamate release, the evoked ATP release is exclusively via a non quantal pathway. (1) The evoked glutamate release is Ca2+-dependent, while the evoked ATP release is Ca2+-independent; (2) Block of lysosome eliminates the Ca2+-dependent exocytosis, but has no effect on the evoked ATP release; (3) Instead, block of mitochondria eliminates the evoked ATP release; (4) Finally, the astrocytic P2X7 channel is responsible for the ATP release, because inhibition of P2X7 either by silencing shRNA or by its selective antagonists substantially attenuated the release.[Conclusion]Thus, following a physiology-like stimulation, both ATP and glutamate released from astrocyte in different pathways. Glutamate is released through the Ca2+-dependent exocytosis pathway, while ATP is released through the non vesicular P2X7 channels in astrocytes.Part II. Temporospatial Changes of P2X4 and P2X7 Receptors in Activated Glia in Nerve Injury Induced Neuropathic Pain[Objectives] To investigated the temporospatial pattern of microglia and astrocyte activation and the temporospatial changes of the endogenous purinergic receptors (P2X4 and P2X7) in neurons and glial cells in the lumbar dorsal horn, and determined the dynamic interactions among them.[Methods](1) To simulate clinical peripheral nerve injury induced neuropathic pain, spared nerve injury (SNI) surgery was performed on the adult SD rats; (2) To evaluate the degree of pain (allodynia), paw withdrawal mechanical threshold (PWMT) was measured at each time point (Day0, Day 10, Day 20 and Day 30); (3) Western blot was used to evaluate the P2X4R and P2X7R expression level at each time point; (4) Immunofluorescence assay was used to analyze the expression and localization of P2X4R and P2X7R and activation of glial cells in the lumbar dorsal horn; (5) P2X4R and P2X7R ultrastructural localizations were analyzed with immuno-electron microscopic method; (6) Pharmacological methods were used to evaluate the functions of P2XRs and roles of activated glial cells in allodynia.[Results](1) We showed that SNI induced microglial activation was earlier than astrocytic activation; at the beginning, activated microglia and astrocytes mainly distributed at the superficial layer and more activated glia appeared at the deep layer in the late phase; (2) Microglia responded to nerve injury with the upregulation of both P2X7R and P2X4R; but displayed a different responding pattern: the expression level of P2X7R peaked earlier (at Day 10) than P2X4R (at Day 20); (3) Differently, in astrocyte, only P2X7R expression level markedly upregulated after injury and displayed a continuous elevation which peaked at Day 30; (4) Immuno-electron microscopic analysis showed that both neurons and glia expressed P2X4R and P2X7R; P2X4R expressed in postsynaptic elements and the outer surface of mitochondria, while P2X7R mainly located at presynaptic elements and the inner surface or cristae of mitochondria; (5) Pharmacologically blocked the functions of homologue P2X receptors at the different time point would attenuate the tactile allodynia but the analgesia effects were different. Blockage of the activation of glial cells also blocked tactile allodynia.[Conclusion]These results suggest a prominent cross-talk exists between P2X4R and P2X7R and between astrocytes and microglia, and indicated different roles of each type of receptors and cells in different phases of neuropathic pain. We showed that P2X4R- positive microglia responded to the initiation phase, and P2X7R-positive astrocytes mainly responded to the maintenance phase of neuropathic pain, while the P2X4R- positive microglia also participated. The blockage of each of these two cells also attenuated the allodynia. These results implied that both activated astrocytes and microglia were required for the maintenance of pain, their teamwork made the mechanism of neuropathic pain more complicated. So we should choose the specific therapeutic targets and strategies at different phases of neuropathic pain. |
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