Morphological and functional polarization of neurons depends on the generation and maintenance of the axon initial segment (AIS). This axonal domain maintains axonal properties but is also the place where the action potential (AP) is generated. All these functions require the AIS, a complex structure that is not fully understood. An integrated structure of voltage-gated ion channels, specific cytoskeleton architecture, as well as, scaffold proteins contributes to these functions. Among them, ankyrinG plays a crucial role to maintain ion channels and membrane proteins. However, it is still elusive how the AIS performs its complex structural and functional regulation. Recent studies reveal that AIS is dynamically regulated in molecular composition, length and location in response to neuronal activity. Some mechanisms acting on AIS plasticity have been uncovered recently, including Ca2+, calpain or calmodulin-mediated modulation, as well as post-translational modifications of cytoskeleton proteins and actin-associated proteins. Neurons are able to respond to different kind of physiological and pathological stimuli from development to maturity by adapting their AIS composition, position and length. This raises the question of which are the neuronal receptors that contribute to the modulation of AIS plasticity. Previous studies have shown that purinergic receptor P2X7 activation is detrimental to AIS maintenance. During initial axonal elongation, P2X7 is coordinated with P2Y1, another purinergic receptor that is essential for proper axon elongation. In this study, we focus on the role of P2Y1 receptor on AIS development and maintenance. Our results show that P2Y1 receptor activity and expression are necessary during AIS initial development, while has no role once AIS maturity is achieved. P2Y1 inhibition or suppression results in a decrease in ankyrinG, βIV-spectrin and voltage-gated sodium channels accumulation that can be rescued by actin stabilization or the modulation of actin-binding proteins at the AIS. Moreover, P2X7 or calpain inhibition also rescues ankyrinG decrease. Hence, a dynamic balance of P2Y1 and P2X7 receptors expression and function during AIS assembly and maturation may represent a fine regulatory mechanism in response to physiological or pathological extracellular purines concentration.

Neuroblasts migrate as directed chains of cells during development and following brain damage. A fuller understanding of the mechanisms driving this will help define its developmental significance and in the refinement of strategies for brain repair using transplanted stem cells. Recently, we reported that in adult mouse there are ionic gradients within the extracellular spaces that create an electrical field (EF) within the rostral migratory stream (RMS), and that this acts as a guidance cue for neuroblast migration. Here, we demonstrate an endogenous EF in brain slices and show that mimicking this by applying an EF of physiological strength, switches on chain migration in mouse neurospheres and in the SH-SY5Y neuroblastoma cell line. Firstly, we detected a substantial endogenous EF of 31.8 ± 4.5 mV/mm using microelectrode recordings from explants of the subventricular zone (SVZ). Pharmacological inhibition of this EF, effectively blocked chain migration in 3D cultures of SVZ explants. To mimic this EF, we applied a physiological EF and found that this increased the expression of N-cadherin and β-catenin, both of which promote cell-cell adhesion. Intriguingly, we found that the EF up-regulated P2Y purinoceptor 1 (P2Y1) to contribute to chain migration of neuroblasts through regulating the expression of N-cadherin, β-catenin and the activation of PKC. Our results indicate that the naturally occurring EF in brain serves as a novel stimulant and directional guidance cue for neuronal chain migration, via up-regulation of P2Y1.

Astrocytes from the hippocampus of chronic epileptic rats exhibit an abnormal pattern of intracellular calcium oscillations, characterized by an augmented frequency of long lasting spontaneous Ca2+ transients, which are sensitive to purinergic receptor antagonists but resistant to tetrodotoxin. The above suggests that alterations in astroglial Ca2+-dependent excitability observed in the epileptic tissue could arise from changes in astrocyte-to-astrocyte signaling, which is mainly mediated by purines in physiological and pathological conditions. In spite of that, how purinergic signaling contributes to astrocyte dysfunction in epilepsy remains unclear. Here, we assessed the possible contribution of P2Y1R as well as pannexin1 and connexin43 hemichannels-both candidates for non-vesicular ATP-release-by performing astroglial Ca2+ imaging and dye uptake experiments in hippocampal slices from control and fully kindled rats. P2Y1R blockade with MRS2179 decreased the mean duration of astroglial Ca2+ oscillations by reducing the frequency of slow Ca2+ transients, and thereby restoring the balance between slow (ST) and fast transients (FT) in the kindled group. The potential contribution of astroglial pannexin1 and connexin43 hemichannels as pathways for purine release (e.g., ATP) was assessed through dye uptake experiments. Astrocytes from kindled hippocampi exhibit three-fold more EtBr uptake than controls, whereby pannexin1 hemichannels (Panx1 HCs) accounts for almost all dye uptake with only a slight contribution from connexin43 hemichannels (Cx43 HCs). Confirming its functional involvement, Panx1 HCs inhibition decreased the mean duration of astroglial Ca2+ transients and the frequency of slow oscillations in kindled slices, but had no noticeable effects on the control group. As expected, Cx43 HCs blockade did not have any effects over the mean duration of astroglial Ca2+ oscillations. These findings suggest that P2Y1R and Panx1 HCs play a pivotal role in astroglial pathophysiology, which would explain the upregulation of glutamatergic neurotransmission in the epileptic brain and thus represents a new potential pharmacological target for the treatment of drug-refractory epilepsy.

Fine processes of astrocytes enwrap synapses and are well positioned to sense neuronal information via synaptic transmission. In rodents, astrocyte processes sense synaptic transmission via Gq-protein coupled receptors (GqPCR), including the P2Y1 receptor (P2Y1R), to generate Ca2+ signals. Astrocytes display numerous spontaneous microdomain Ca2+ signals; however, it is not clear whether such signals are due to local synaptic transmission and/or in what timeframe astrocytes sense local synaptic transmission. To ask whether GqPCRs mediate microdomain Ca2+ signals, we engineered mice (both sexes) to specifically overexpress P2Y1Rs in astrocytes, and we visualized Ca2+ signals via a genetically encoded Ca2+ indicator, GCaMP6f, in astrocytes from adult mice. Astrocytes overexpressing P2Y1Rs showed significantly larger Ca2+ signals in response to exogenously applied ligand and to repetitive electrical stimulation of axons compared with controls. However, we found no evidence of increased microdomain Ca2+ signals. Instead, Ca2+ waves appeared and propagated to occupy areas that were up to 80-fold larger than microdomain Ca2+ signals. These Ca2+ waves accounted for only 2% of total Ca2+ events, but they were 1.9-fold larger and 2.9-fold longer in duration than microdomain Ca2+ signals at processes. Ca2+ waves did not require action potentials for their generation and occurred in a probenecid-sensitive manner, indicating that the endogenous ligand for P2Y1R is elevated independently of synaptic transmission. Our data suggest that spontaneous microdomain Ca2+ signals occur independently of P2Y1R activation and that astrocytes may not encode neuronal information in response to synaptic transmission at a point source of neurotransmitter release.SIGNIFICANCE STATEMENT Astrocytes are thought to enwrap synapses with their processes to receive neuronal information via Gq-protein coupled receptors (GqPCRs). Astrocyte processes display numerous microdomain Ca2+ signals that occur spontaneously. To determine whether GqPCRs play a role in microdomain Ca2+ signals and the timeframe in which astrocytes sense neuronal information, we engineered mice whose astrocytes specifically overexpress the P2Y1 receptor, a major GqPCR in astrocytes. We found that overexpression of P2Y1 receptors in astrocytes did not increase microdomain Ca2+ signals in astrocyte processes but caused Ca2+ wavelike signals. Our data indicate that spontaneous microdomain Ca2+ signals do not require activation of P2Y1 receptors.

The mammalian circadian clock drives daily oscillations in physiology and behavior through an autoregulatory transcription feedback loop present in central and peripheral cells. Ablation of the core clock within the endocrine pancreas of adult animals impairs the transcription and splicing of genes involved in hormone exocytosis and causes hypoinsulinemic diabetes. Here, we developed a genetically sensitized small-molecule screen to identify druggable proteins and mechanistic pathways involved in circadian β-cell failure. Our approach was to generate β-cells expressing a nanoluciferase reporter within the proinsulin polypeptide to screen 2640 pharmacologically active compounds and identify insulinotropic molecules that bypass the secretory defect in CRISPR-Cas9-targeted clock mutant β-cells. We validated hit compounds in primary mouse islets and identified known modulators of ligand-gated ion channels and G-protein-coupled receptors, including the antihelmintic ivermectin. Single-cell electrophysiology in circadian mutant mouse and human cadaveric islets revealed ivermectin as a glucose-dependent secretagogue. Genetic, genomic, and pharmacological analyses established the P2Y1 receptor as a clock-controlled mediator of the insulinotropic activity of ivermectin. These findings identify the P2Y1 purinergic receptor as a diabetes target based upon a genetically sensitized phenotypic screen.

Extracellular purines are important signaling molecules involved in numerous physiological and pathological processes via the activation of P2 receptors. Information about the spatial and temporal P2 receptor (P2R) expression and its regulation remains crucial for the understanding of the role of P2Rs in health and disease. To identify cells carrying P2X2Rs in situ, we have generated BAC transgenic mice that express the P2X2R subunits as fluorescent fusion protein (P2X2-TagRFP). In addition, we generated a BAC P2Y1R TagRFP reporter mouse expressing a TagRFP reporter for the P2RY1 gene expression. We demonstrate expression of the P2X2R in a subset of DRG neurons, the brain stem, the hippocampus, as well as on Purkinje neurons of the cerebellum. However, the weak fluorescence intensity in our P2X2R-TagRFP mouse precluded tracking of living cells. Our P2Y1R reporter mice confirmed the widespread expression of the P2RY1 gene in the CNS and indicate for the first time P2RY1 gene expression in mouse Purkinje cells, which so far has only been described in rats and humans. Our P2R transgenic models have advanced the understanding of purinergic transmission, but BAC transgenic models appeared not always to be straightforward and permanent reliable. We noticed a loss of fluorescence intensity, which depended on the number of progeny generations. These problems are discussed and may help to provide more successful animal models, even if in future more versatile and adaptable nuclease-mediated genome-editing techniques will be the methods of choice.

G protein-coupled receptors (GPCRs) cause various cellular responses through activating heterotrimeric G protein upon the agonist binding. The interaction with G protein has been suggested to stabilize the agonist-bound active conformation of GPCRs. We previously reported the effects of Gq protein on the stabilization of the active conformation of the muscarinic receptor type 1 (M1R), using a fluorescence resonance energy transfer (FRET) technique. In this study, we aimed at examining whether or not the binding of Gq protein affects the agonist-induced active conformation of receptors other than the M1R. For this purpose, functionally intact fluorescent receptors of the metabotropic purinergic receptor type 1 (P2Y1R) and muscarinic receptor type 3 (M3R) were constructed, by inserting junctional linkers between the short intracellular third loops (i3) and yellow fluorescent protein (YFP). The YFP-fused receptors also showed the agonist-induced increases in FRET from the cyan fluorescent protein (CFP) tethered with Gαq subunit, indicating that they interacted with Gq protein. The agonist-induced conformational changes of the receptors were detected as the agonist-induced decrease in FRET between YFP at the i3 and CFP at the C-tail. The FRET decrease of the M3R but not of the P2Y1R was enhanced by coexpression of Gq protein. In addition, coexpression of Gq protein significantly decelerated the FRET recovery of the M3R construct but not of the P2Y1R construct upon the agonist removal. These results suggest that the effects of the Gq binding on the active conformation of the receptor differ depending on the type of GPCRs.

Adenosine 5'-triphosphate (ATP) mediates a variety of biological functions following nerve-evoked release, via activation of either G-protein-coupled P2Y- or ligand-gated P2X receptors. In smooth muscle, ATP, acting via P2Y receptors (P2YR), may act as an inhibitory neurotransmitter. The underlying mechanism(s) remain unclear, but have been proposed to involve the production of inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] by phospholipase C (PLC), to evoke Ca(2+) release from the internal store and stimulation of Ca(2+)-activated potassium (K(Ca)) channels to cause membrane hyperpolarization. This mechanism requires Ca(2+) release from the store. However, in the present study, ATP evoked transient Ca(2+) increases in only ∼10% of voltage-clamped single smooth muscle cells. These results do not support activation of K(Ca) as the major mechanism underlying inhibition of smooth muscle activity. Interestingly, ATP inhibited Ins(1,4,5)P(3)-evoked Ca(2+) release in cells that did not show a Ca(2+) rise in response to purinergic activation. The reduction in Ins(1,4,5)P(3)-evoked Ca(2+) release was not mimicked by adenosine and therefore, cannot be explained by hydrolysis of ATP to adenosine. The reduction in Ins(1,4,5)P(3)-evoked Ca(2+) release was, however, also observed with its primary metabolite, ADP, and blocked by the P2Y(1)R antagonist, MRS2179, and the G protein inhibitor, GDPβS, but not by PLC inhibition. The present study demonstrates a novel inhibitory effect of P2Y(1)R activation on Ins(1,4,5)P(3)-evoked Ca(2+) release, such that purinergic stimulation acts to prevent Ins(1,4,5)P(3)-mediated increases in excitability in smooth muscle and promote relaxation.

1. The presence of ionotropic P2X receptors, targets of ATP in fast synaptic transmission, as well as metabotropic P2Y receptors, known to activate K(+) currents in cultured neostriatal neurones, was investigated in medium-spiny neurones and cholinergic interneurones contained in neostriatal brain slices from 5-26-day-old rats. 2. In these cells, adenosine-5'-triphosphate (ATP) (100-1000 microm), 2-methylthioadenosine-5'-triphosphate (2MeSATP), alpha,beta-methyleneadenosine-5'-triphosphate (alpha,betameATP, 30-300 microm, each) and adenosine-5'-O-(3-thiotriphosphate (ATPgammaS) (100 microm) failed to evoke P2X receptor currents even when 8-cyclopentyl-1,3-dipropylxanthine (DPCPX, 0.1 microm), apyrase (10 U ml(-1)) or intracellular Cs(+) was used to prevent occluding effects of the ATP breakdown product adenosine, desensitisation of P2X receptors by endogenous ATP and an interference with the activation of K(+) channels, respectively. P2X receptor agonists were also ineffective in outside-out patches withdrawn from the brain slice tissue. Muscimol (10 microm) evoked GABA(A) receptor-mediated currents under all these conditions. 3. When used as a control, locus coeruleus neurones responded with P2X receptor-mediated currents to ATP (300 microm), 2MeSATP and alpha,betameATP (100 microm, each). 4. ATP and adenosine-5'-diphosphate (ADP) (100 microm, each) did not activate K(+) currents in the neostriatal neurones. 5. Despite the observed lack of function, P2X(2) and P2Y(1) immunofluorescence was found in roughly 50% of the medium-spiny neurones and cholinergic interneurones. 6. A role of ATP in synaptic transmission to striatal medium-spiny neurones and cholinergic interneurones appears unlikely, however, the otherwise silent P2X and P2Y receptors may gain functionality under certain yet unknown conditions.

Adenosine and ATP, via their specific P1 and P2 receptors, modulate a wide variety of cellular and tissue functions, playing a neuroprotective or neurodegenerative role in brain damage conditions. Although, in general, adenosine inhibits excitability and ATP functions as an excitatory transmitter in the central nervous system, recent data suggest the existence of a heterodimerization and a functional interaction between P1 and P2 receptors in the brain. In particular, interactions of adenosine A1 and P2Y1 receptors may play important roles in the purinergic signalling cascade. In the present work, we investigated the subcellular localization/co-localization of the receptors and their functional cross-talk at the membrane level in Wistar rat hippocampus. This is a particularly vulnerable brain area, which is sensitive to adenosine- and ATP-mediated control of glutamatergic transmission. The postembedding immunogold electron microscopy technique showed that the two receptors are co-localized at the synaptic membranes and surrounding astroglial membranes of glutamatergic synapses. To investigate the functional cross-talk between the two types of purinergic receptors, we evaluated the reciprocal effects of their activation on their G protein coupling. P2Y1 receptor stimulation impaired the potency of A1 receptor coupling to G protein, whereas the stimulation of A1 receptors increased the functional responsiveness of P2Y1 receptors. The results demonstrated an A1-P2Y1 receptor co-localization at glutamatergic synapses and surrounding astrocytes and a functional interaction between these receptors in hippocampus, suggesting ATP and adenosine can interact in purine-mediated signalling. This may be particularly important during pathological conditions, when large amounts of these mediators are released.

Purinergic P2X and P2Y receptors are involved in mediating intercellular signalling via purines such as adenosine triphosphate (ATP). P2X and P2Y receptors have been implicated in numerous body functions including learning, memory and sleep. All of these body functions show time-of-day-dependent variations controlled by the master circadian oscillator located in the suprachiasmatic nucleus (SCN). Evidence exists for a role of purinergic signalling in intercellular coupling within SCN. However, few studies have been performed on the expression of purinergic receptors in SCN. Therefore, we analyse the expression of seven P2X (P2X1-7) and eight P2Y (P2Y1-2, 4, 6, 11-14) receptors in mouse SCN and address their time-of-day-dependent variation by using immunohistochemistry and real-time polymerase chain reaction. At the early light phase, P2X and P2Y receptors show a low to moderate, homogenously distributed immunoreaction throughout SCN. P2Y13 reveals strong immunoreaction in fibres within the core region of SCN. From the fifteen analysed P2 receptors, seven exhibit a time-of-day-dependent variation in SCN. P2X1 immunoreaction is very low in the early light phase with a minor increase at the end of the dark phase. P2X4 immunoreaction strongly increases during the dark phase in soma cells in the core region and in a dense network of fibres in the shell region of SCN. P2X3 immunoreaction is moderately elevated during the dark phase. Conversely, immunoreaction for P2Y2, P2Y12 and P2Y14 moderately increases at the early light phase and P2Y6 immunoreaction displays a moderate increase at the mid-light phase. Thus, this study demonstrates a time-of-day-dependent variation of P2 receptors in mouse SCN.

The dura mater and its vasculature have for decades been central in the hypothesis of migraine and headache pathophysiology. Although recent studies have questioned the role of the vasculature as the primary cause, dural vessel physiology is still relevant in understanding the complex pathophysiology of migraine. The aim of the present study was to isolate the middle meningeal artery (MMA) from rodents and characterize their purinergic receptors using a sensitive wire myograph method and RT-PCR. The data presented herein suggest that blood flow through the MMA is, at least in part, regulated by purinergic receptors. P2X1 and P2Y6 receptors are the strongest contractile receptors and, surprisingly, ADPβS caused contraction most likely via P2Y1 or P2Y13 receptors, which is not observed in other arteries. Adenosine addition, however, caused relaxation of the MMA. The adenosine relaxation could be inhibited by SCH58261 (A2A receptor antagonist) and caffeine (adenosine receptor antagonist). This gives one putative molecular mechanism for the effect of caffeine, often used as an adjuvant remedy of cranial pain. Semi-quantitative RT-PCR expression data for the receptors correlate well with the functional findings. Together these observations could be used as targets for future understanding of the in vivo role of purinergic receptors in the MMA.

ATP-sensitive P2 receptors are suggested to play an important role in the cerebral signal transduction. We examined the expression of the P2Y1 receptor and the possibly downstream-related neuronal nitric oxide synthase (nNOS) in the hypothalamus of rats food-restricted for 3 or 10 days and rats refed after a restriction of 10 days. The restriction caused a reduction of the body weight and plasma triacylglyceride, an increase of non-esterified fatty acid levels correlating with a decrease of leptin levels and an enhancement of plasma corticosterone. All changes returned to basal levels after refeeding. The restriction induced an enhanced intake within 30 min after food presentation and a reduction in the latency. Interestingly, the latter was not abolished by refeeding. The daily food intake induced by refeeding was enhanced at the first day only. The expression of hypothalamic P2Y1 receptor/nNOS mRNA and protein and of leptin receptor mRNA were enhanced after restricted feeding. These changes were abolished after 3 days of refeeding. Immunofluorescence studies indicated that P2Y1 receptor and nNOS immunoreactivities are present in the dorsomedial, ventromedial and lateral hypothalamus and in the nucleus arcuatus. P2Y1 receptor-positive cells were partially also nNOS-positive. The P2Y1 receptor labeling was restricted to cell bodies of obviously non-glial cells, whereas nNOS labeling could be detected also at cellular processes of these cells. In the nucleus arcuatus, astrocytes were identified, expressing P2Y1 receptors at cell bodies and cellular processes. The data suggest that restricted feeding may enhance the sensitivity of the hypothalamus to extracellular ADP/ATP by regulation of the expression of P2Y1 receptors and possibly of their signal transduction pathway via nitric oxide production.

Extracellular agonists linked to inositol-1,4,5-trisphosphate (IP3) formation elicit cytosolic Ca2+ oscillations in many cell types, but despite a common signaling pathway, distinct agonist-specific Ca2+ spike patterns are observed. Using qPCR, we show that rat hepatocytes express multiple purinergic P2Y and P2X receptors (R). ADP acting through P2Y1R elicits narrow Ca2+ oscillations, whereas UTP acting through P2Y2R elicits broad Ca2+ oscillations, with composite patterns observed for ATP. P2XRs do not play a role at physiological agonist levels. The discrete Ca2+ signatures reflect differential effects of protein kinase C (PKC), which selectively modifies the falling phase of the Ca2+ spikes. Negative feedback by PKC limits the duration of P2Y1R-induced Ca2+ spikes in a manner that requires extracellular Ca2+. By contrast, P2Y2R is resistant to PKC negative feedback. Thus, the PKC leg of the bifurcated IP3 signaling pathway shapes unique Ca2+ oscillation patterns that allows for distinct cellular responses to different agonists.

Perisynaptic glial cells respond to neural activity by increasing cytosolic calcium, but the significance of this pathway is unclear. Terminal/perisynaptic Schwann cells (TPSCs) are a perisynaptic glial cell at the neuromuscular junction that respond to nerve-derived substances such as acetylcholine and purines. Here, we provide genetic evidence that activity-induced calcium accumulation in neonatal TPSCs is mediated exclusively by one subtype of metabotropic purinergic receptor. In P2ry1 mutant mice lacking these responses, postsynaptic, rather than presynaptic, function was altered in response to nerve stimulation. This impairment was correlated with a greater susceptibility to activity-induced muscle fatigue. Interestingly, fatigue in P2ry1 mutants was more greatly exacerbated by exposure to high potassium than in control mice. High potassium itself increased cytosolic levels of calcium in TPSCs, a response which was also reduced P2ry1 mutants. These results suggest that activity-induced calcium responses in TPSCs regulate postsynaptic function and muscle fatigue by regulating perisynaptic potassium.

Purinergic P2X and P2Y receptors are broadly expressed on both neurons and glial cells in the central nervous system (CNS), including dentate gyrus (DG). The aim of this research was to determine the synaptic and proliferative response of the DG to severe oxygen and glucose deprivation (OGD) in acute rat hippocampal slices and to investigate the contribution of P2X7 and P2Y1 receptor antagonism to recovery of synaptic activity after OGD. Extracellular field excitatory post-synaptic potentials (fEPSPs) in granule cells of the DG were recorded from rat hippocampal slices. Nine-min OGD elicited an irreversible loss of fEPSP and was invariably followed by the appearance of anoxic depolarization (AD). Application of MRS2179 (selective antagonist of P2Y1 receptor) and BBG (selective antagonist of P2X7 receptor), before and during OGD, prevented AD appearance and allowed a significant recovery of neurotransmission after 9-min OGD. The effects of 9-min OGD on proliferation and maturation of cells localized in the subgranular zone (SGZ) of slices prepared from rats treated with 5-Bromo-2'-deoxyuridine (BrdU) were investigated. Slices were further incubated with an immature neuron marker, doublecortin (DCX). The number of BrdU+ cells in the SGZ was significantly decreased 6 hours after OGD. This effect was antagonized by BBG, but not by MRS2179. Twenty-four hours after 9-min OGD, the number of BrdU+ cells returned to control values and a significant increase of DCX immunofluorescence was observed. This phenomenon was still evident when BBG, but not MRS2179, was applied during OGD. Furthermore, the P2Y1 antagonist reduced the number of BrdU+ cells at this time. The data demonstrate that P2X7 and P2Y1 activation contributes to early damage induced by OGD in the DG. At later stages after the insult, P2Y1 receptors might play an additional and different role in promoting cell proliferation and maturation in the DG.

Calcitonin gene-related peptide (CGRP), an endogenous neuropeptide released from perivascular sensory nerves, exerts a powerful vasodilatation. Interestingly, adenosine triphosphate (ATP) stimulates the release of CGRP by activation of prejunctional P2X2/3 receptors, and adenosine 5'-O-2-thiodiphosphate (ADPβS), a stable adenosine diphosphate (ADP) analogue, produces vasodilator/vasodepressor responses by endothelial P2Y1 receptors. Since the role of ADP in the prejunctional modulation of the vasodepressor sensory CGRPergic drive and the receptors involved remain unknown, this study investigated whether ADPβS inhibits this CGRPergic drive. Accordingly, 132 male Wistar rats were pithed and subsequently divided into two sets. In set 1, ADPβS (5.6 and 10 µg/kg·min) inhibited the vasodepressor CGRPergic responses by electrical stimulation of the spinal T9-T12 segment. This inhibition by ADPβS (5.6 µg/kg·min) was reverted after i.v. administration of the purinergic antagonists MRS2500 (300 µg/kg; P2Y1) or MRS2211 (3000 µg/kg; P2Y13), but not by PSB0739 (300 µg/kg; P2Y12), MRS2211 (1000 µg/kg; P2Y13) or the KATP blocker glibenclamide (20 mg/kg). In set 2, ADPβS (5.6 µg/kg·min) failed to modify the vasodepressor responses to exogenous α-CGRP. These results suggest that ADPβS inhibits CGRP release in perivascular sensory nerves. This inhibition, apparently unrelated to activation of ATP-sensitive K+ channels, involves P2Y1 and probably P2Y13, but not P2Y12 receptors.

Purinergic P2Y1 and P2Y12 receptors (P2Y1-R and P2Y12-R) are G protein-coupled receptors (GPCR) activated by adenosine diphosphate (ADP) to mediate platelet activation, thereby playing a pivotal role in hemostasis and thrombosis. While P2Y12-R is the major target of antiplatelet drugs, no P2Y1-R antagonist has yet been developed for clinical use. However, accumulating data suggest that P2Y1-R inhibition would ensure efficient platelet inhibition with minimal effects on bleeding. In this context, an accurate characterization of P2Y1-R antagonists constitutes an important preliminary step.

Microglia and astrocytes become reactive following traumatic brain injury (TBI). However, the coordination of this reactivity and its relation to pathophysiology are unclear. Here, we show that microglia transform astrocytes into a neuroprotective phenotype via downregulation of the P2Y1 purinergic receptor. TBI initially caused microglial activation in the injury core, followed by reactive astrogliosis in the peri-injured region and formation of a neuroprotective astrocyte scar. Equivalent changes to astrocytes were observed in vitro after injury. This change in astrocyte phenotype resulted from P2Y1 receptor downregulation, mediated by microglia-derived cytokines. In mice, astrocyte-specific P2Y1 receptor overexpression (Astro-P2Y1OE) counteracted scar formation, while astrocyte-specific P2Y1 receptor knockdown (Astro-P2Y1KD) facilitated scar formation, suggesting critical roles of P2Y1 receptors in the transformation. Astro-P2Y1OE and Astro-P2Y1KD mice showed increased and reduced neuronal damage, respectively. Altogether, our findings indicate that microglia-astrocyte interaction, involving a purinergic signal, is essential for the formation of neuroprotective astrocytes.

Microglia are highly sensitive to even small changes in the brain environment, such as invasion of non-hazardous toxicants or the presymptomatic state of diseases. However, the physiological or pathophysiological consequences of their responses remain unknown. Here, we report that cultured microglia sense low concentrations of the neurotoxicant methylmercury (MeHg(low)) and provide neuroprotection against MeHg, for which astrocytes are also required. When exposed to MeHg(low), microglia exocytosed ATP via p38 MAPK- and vesicular nucleotide transporter (VNUT)-dependent mechanisms. Astrocytes responded to the microglia-derived ATP via P2Y1 receptors and released interleukin-6 (IL-6), thereby protecting neurons against MeHg(low). These neuroprotective actions were also observed in organotypic hippocampal slices from wild-type mice, but not in slices prepared from VNUT knockout or P2Y1 receptor knockout mice. These findings suggest that microglia sense and respond to even non-hazardous toxicants such as MeHg(low) and change their phenotype into a neuroprotective one, for which astrocytic support is required.