2 research outputs found
Neuroglial ATP release through innexin channels controls microglial cell movement to a nerve injury
Microglia, the immune cells of the central nervous system, are attracted to sites of injury. The injury releases adenosine triphosphate (ATP) into the extracellular space, activating the microglia, but the full mechanism of release is not known. In glial cells, a family of physiologically regulated unpaired gap junction channels called innexons (invertebrates) or pannexons (vertebrates) located in the cell membrane is permeable to ATP. Innexons, but not pannexons, also pair to make gap junctions. Glial calcium waves, triggered by injury or mechanical stimulation, open pannexon/innexon channels and cause the release of ATP. It has been hypothesized that a glial calcium wave that triggers the release of ATP causes rapid microglial migration to distant lesions. In the present study in the leech, in which a single giant glial cell ensheathes each connective, hydrolysis of ATP with 10 U/ml apyrase or block of innexons with 10 µM carbenoxolone (CBX), which decreased injury-induced ATP release, reduced both movement of microglia and their accumulation at lesions. Directed movement and accumulation were restored in CBX by adding ATP, consistent with separate actions of ATP and nitric oxide, which is required for directed movement but does not activate glia. Injection of glia with innexin2 (Hminx2) RNAi inhibited release of carboxyfluorescein dye and microglial migration, whereas injection of innexin1 (Hminx1) RNAi did not when measured 2 days after injection, indicating that glial cells’ ATP release through innexons was required for microglial migration after nerve injury. Focal stimulation either mechanically or with ATP generated a calcium wave in the glial cell; injury caused a large, persistent intracellular calcium response. Neither the calcium wave nor the persistent response required ATP or its release. Thus, in the leech, innexin membrane channels releasing ATP from glia are required for migration and accumulation of microglia after nerve injury
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Cannabinoid Control of Microglial Migration
In both vertebrates and invertebrates, including leeches, microglia are rapidly activated by central nervous system (CNS) damage and migrate to the lesions. Adenosine triphosphate (ATP), nitric oxide (NO) and endocannabinoids have been implicated in controlling activation and migration, but details of the mechanisms are uncertain. This dissertation tests the hypothesis that endocannabinoids coordinate and influence the microglial response to nerve cord crushing. Chapter 1 reports that application of endocannabinoids to nerve cords at concentrations as low as 100 nM for arachidonylethanolamide (anandamide, or AEA) reduced the number of migrating microglia, but not when cords were pretreated with 10 µM of the CB2 cannabinoid receptor (CB2R) antagonist SR144528. In addition, immunoblots confirmed the expression of CB1-like and CB2-like receptors and immunohistochemistry showed that they were concentrated at lesions, where microglia accumulated. Benzoyl ATP (BzATP) also reduced microglia accumulation, an effect blocked by pretreatment of nerve cords with SR144528, whereas the G-protein coupled P2YR agonists uridine triphosphate (UTP) and methylthio-ATP (MeSATP) at 100 µM did not reduce accumulation. This result suggested that P2X7R activation elicited production and release of a CB2R agonist that influenced microglia movement. Chapter 2 reports that extracellular ATP levels were highest in the CNS within the first 30 min of injury and remained above unharmed controls for at least 2 hours. Application of 10 units (U) of the ATPase apyrase to nerve cords reduced accumulation of microglia at lesions, another indication that microglia require extracellular ATP to accumulate. Chapter 3 reports that AEA stopped ATP-induced movement of microglia, and that this effect was blocked by pretreatment of nerve cords with the CB1R antagonist SR141716A (10µM), the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester (L NAME, 2 mM) or the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-teramethylimidazoline-oxyl-3-oxide (carboxy-PTIO or cPTIO, 1 mM). Thus the migration of microglia to lesions is regulated not only by ATP acting on P2YR and by NO acting on soluble guanylate cyclase (sGC), but also potentially by ATP binding to P2X7-like receptors to increase the production of cannabinoids. Cannabinoids, binding to the CB1R and CB2R cause production of NO, which suppresses microglia movement