Purinergic signaling regulates important physiological processes and the homeostatic response to stress in the cochlea via extracellular nucleosides (adenosine) and nucleotides (ATP, UTP). Using a previously established organotypic culture model, the current study investigated the effect of purinergic P1 (adenosine) and P2 (ATP) receptor activation on the survival of the sensory hair cell population in the cochlea exposed to the ototoxic aminoglycoside neomycin. Organ of Corti explants were obtained from C57BL/6 mice at postnatal day 3 (P3) and maintained in normal culture medium (with or without purine receptor agonists or analogs) for 19.5 h prior to neomycin exposure (1 mM, 3 h) followed by a further incubation for 19.5 h in culture medium. The cochlear explants were then fixed in 4% paraformaldehyde (PFA) and sensory hair cells labeled with Alexa 488-phalloidin. Neomycin induced a substantial loss of the sensory hair cells, mostly in the middle segment of the cochlea. This neomycin-induced ototoxicity was unaffected by the addition of P2 receptor agonists (ATP and UTP) in the culture medium, whilst the addition of their slowly-hydrolyzable analogs (ATPγS, UTPγS) aggravated neomycin-induced sensory hair cell loss. In contrast, the activation of P1 receptors by adenosine or adenosine amine congener (ADAC) conferred partial protection from neomycin ototoxicity. This study demonstrates a pro-survival effect of P1 receptor stimulation, whilst prolonged activation of P2 receptors has an opposite effect. Based on these findings, we postulate that P1 and P2 receptors orchestrate differential responses to cochlear injury and that the balance of these receptors is important for maintaining cochlear homeostasis following ototoxic injury.

Previous studies suggested that the thrombospondin-1/transforming growth factor-β1 (TSP-1/TGF-β1) pathway might be critical in synaptogenesis during development and that the purinergic P2 receptor family could regulate synaptogenesis by modulating TSP-1 signaling. However, it is unclear whether this pathway plays a role in synaptogenesis during epileptic progression. This study was designed to investigate this question by analyzing the dynamic changes and effects of TSP-1 levels on the density of synaptic markers that are related to epileptic seizure activity. In addition, we evaluated whether P2-type receptors could regulate these effects. We generated a rat seizure model via amygdala kindling and inhibited TSP-1 activity using small interfering RNA (siRNA) interference and pharmacological inhibition. We treated the rats with antagonists of P2 or P2Y receptors, pyridoxalphosphate-6-azophenyl-2',4'-disulfonic (PPADS) or Reactive Blue 2. Following this, we quantified TSP-1 and TGF-β1 immunoreactivity (IR), the density of synaptic markers, and seizure activity. There were significantly more synapses/excitatory synapses in several brain regions, such as the hippocampus, which were associated with progressing epileptic discharges after kindling. These were associated with increased TSP-1 and TGF-β1-IR. Genetic or pharmacologic inhibition of TSP-1 significantly reduced the density of synaptic/excitatory synaptic markers and inhibited the generalization of focal epilepsy. The administration of PPADS or Reactive Blue 2 attenuated the increase in TSP-1-IR and the increase in the density of synaptic markers that follows kindling and abolished most of the epileptic seizure activity. Altogether, our results indicate that the TSP-1/TGF-β1 pathway and its regulation by P2, particularly P2Y-type receptors, may be a critical promoter of synaptogenesis during the progression of epilepsy. Therefore, components of this pathway may be targets for novel antiepileptic drug development.

The present study explores tissue and cellular distribution of ectonucleoside triphosphate diphosphohydrolase 2 (NTPDase2) and the gene and protein expression in rat spinal cord during the course of experimental autoimmune encephalomyelitis (EAE). Given that NTPDase2 hydrolyzes ATP with a transient accumulation of ADP, the expression of ADP-sensitive P2 purinoceptors was analyzed as well. The autoimmune disease was actively induced in Dark Agouti female rats and the changes were analyzed 10, 15 and 29 days after the induction. These selected time points correspond to the onset ( Eo ), peak ( Ep ) and recovery ( Er ) from EAE. In control animals, NTPDase2 was confined in the white matter, in most of the glial fibrillary acidic protein (GFAP)-immunoreactive (ir) astrocytes and in a considerable number of nestin-ir cells, while the other cell types were immunonegative. Immunoreactivity corresponding to NTPDase2 decreased significantly at Eo and Ep and then returned to the baseline levels at Er . The preservation of the proportion of GFAP single-labeled and GFAP/NTPDase2 double-labeled elements along the course of EAE indicated that changes in NTPDase2-ir occurred at fibrous astrocytes that typically express NTPDase2 in normal conditions. Significant downregulation of P2Y1 and P2Y12 receptor proteins at Eo and several-fold induction of P2Y12 and P2Y13 receptor proteins at Ep and/or Er were observed implying that the pathophysiological process in EAE may be linked to ADP signaling. Cell-surface expression of NTPDase2, NTPDase1/CD39 and ecto-5'-nucleotidase (eN/CD73) was analyzed in CD4+ T cells of a draining lymph node by fluorescence-activated cell sorting. The induction of EAE was associated with a transient decrease in a number of CD4+ NTPDase2+ T cells in a draining lymph node, whereas the recovery was characterized by an increase in NTPDase2+ cells in both CD4+ and CD4- cell populations. The opposite was found for NTPDase1/CD39+ and eN/CD73+ cells, which slightly increased in number with progression of the disease, particularly in CD4- cells, and then decreased in the recovery. Finally, CD4+ NTPDase2+ cells were never observed in the spinal cord parenchyma. Taken together, our results suggest that the process of neuroinflammation in EAE may be associated with altered ADP signaling.

The pannexin family of channels consists of three members-pannexin-1 (Panx1), pannexin-2 (Panx2), and pannexin-3 (Panx3) that enable the exchange of metabolites and signaling molecules between intracellular and extracellular compartments. Pannexin-mediated release of intracellular ATP into the extracellular space has been tied to a number of cellular activities, primarily through the activity of type P2 purinergic receptors. Previous work indicates that the opening of Panx1 channels and activation of purinergic receptors by extracellular ATP may cause inflammation and apoptosis. In the CNS (central nervous system) and PNS (peripheral nervous system), coupled pannexin, and P2 functions have been linked to peripheral sensitization (pain) pathways. Purinergic pathways are also essential for other critical processes in the PNS, including myelination and neurite outgrowth. However, whether such pathways are pannexin-dependent remains to be determined. In this study, we use a Panx1 knockout mouse model and pharmacological inhibitors of the Panx1 and the ATP-mediated signaling pathway to fill gaps in our understanding of Panx1 localization in peripheral nerves, roles for Panx1 in axonal outgrowth and myelination, and neurite extension. Our data show that Panx1 is localized to axonal, myelin, and vascular compartments of the peripheral nerves. Knockout of Panx1 gene significantly increased axonal caliber in vivo and axonal growth rate in cultured dorsal root ganglia (DRG) neurons. Furthermore, genetic knockout of Panx1 or inhibition of components of purinergic signaling, by treatment with probenecid and apyrase, resulted in denser axonal outgrowth from cultured DRG explants compared to untreated wild-types. Our findings suggest that Panx1 regulates axonal growth in the peripheral nervous system.

Vesicular nucleotide transporter (VNUT) is required for active accumulation of adenosine tri-phosphate (ATP) into vesicles for purinergic neurotransmission, however, the cell types that express VNUT in the central nervous system remain unknown. This study characterized VNUT expression within the mammalian retina and brain and assessed a possible functional role in purinergic signaling. Two native isoforms of VNUT were detected in mouse retina and brain based on RNA transcript and protein analysis. Using immunohistochemistry, VNUT was found to co-localize with tyrosine hydroxylase (TH) positive, dopaminergic (DA) neurons of the substantia nigra and ventral tegmental area, however, VNUT expression in extranigral non-DA neurons was also observed. In the retina, VNUT labeling was found to co-localize solely with TH-positive DA-cells. In the outer retina, VNUT-positive interplexiform cell processes were in close contact with horizontal cells and cone photoreceptor terminals, which are known to express P2 purinergic-receptors. In order to assess function, dissociated retinal neurons were loaded with fluorescent ATP markers (Quinacrine or Mant-ATP) and the DA marker FFN102, co-labeled with a VNUT antibody and imaged in real time. Fluorescent ATP markers and FFN102 puncta were found to co-localize in VNUT positive neurons and upon stimulation with high potassium, ATP marker fluorescence at the cell membrane was reduced. This response was blocked in the presence of cadmium. These data suggest DA neurons co-release ATP via calcium dependent exocytosis and in the retina this may modulate the visual response by activating purine receptors on closely associated neurons.

There is growing recognition of the important role of interaction between neurons and glial cells for brain longevity. The extracellular ATP have been shown to bring significant contribution into bi-directional glia-neuron communications, in particular into astrocyte-driven modulation of synaptic plasticity. To elucidate a putative impact of brain aging on neuron-glia networks, we explored the aging-related plasticity of the purinoreceptors-mediated signaling in cortical neurons and astrocytes. We investigated the age- and experience-related alterations in purinergic components of neuronal synaptic currents and astroglial calcium signaling in the layer2/3 of neocortex of mice exposed to the mild caloric restriction (CR) and environmental enrichment (EE) which included ad libitum physical exercise. We observed the considerable age-related decline in the neuronal P2X receptor-mediated miniature spontaneous currents which originated from the release of ATP from both synapses and astrocytes. We also found out that purinergic astrocytic Ca2+-signaling underwent the substantial age-related decline but EE and CR rescued astroglial signaling, in particular mediated by P2X1, P2X1/5, and P2Y1 receptors. Our data showed that age-related attenuation in the astroglial calcium signaling caused a substantial decrease in the exocytosis of ATP leading to impairment of astroglia-derived purinergic modulation of excitatory synaptic currents and GABAergic tonic inhibitory currents. On a contrary, exposure to EE and CR, which enhanced purinergic astrocytic calcium signaling, up-regulated the excitatory and down-regulated the inhibitory currents in neurons of old mice, thus counterbalancing the impact of aging on synaptic signaling. Combined, our results strongly support the physiological importance of ATP-mediated signaling for glia-neuron interactions and brain function. Our data also show that P2 purinoreceptor-mediated communication between astrocytes and neurons in the neocortex undergoes remodeling during brain aging and decrease in the ATP release may contribute to the age-related impairment of synaptic transmission.

Merkel cells (MCs) have been proposed to form a part of the MC-neurite complex with sensory neurons through synaptic contact. However, the detailed mechanisms for intercellular communication between MCs and neurons have yet to be clarified. The present study examined the increases in intracellular free Ca2+ concentration ([Ca2+]i) induced by direct mechanical stimulation of MCs. We also measured [Ca2+]i in the trigeminal ganglion neurons (TGs) following direct mechanical stimulation to the MCs in an MC-TGs coculture. The MCs were isolated from hamster buccal mucosa, while TGs were isolated from neonatal Wistar rats. Both cell populations showed depolarization-induced [Ca2+]i. Direct mechanical stimulation to MCs increased [Ca2+]i, showing stimulation strength dependence. In the MC-TGs coculture, the application of direct mechanical stimulation to MCs resulted in increased [Ca2+]i in the TGs. These changes were significantly suppressed by antagonists of glutamate-permeable anion channels (4,4'-diisothiocyanato-2,2'-stilbenedisulfonic acid; DIDS), and non-competitive antagonist of the N-methyl-D-aspartate (NMDA) receptors (MK801). Apyrase, an ATP-degrading enzyme, and suramin, a non-selective P2 purinergic receptor antagonist, did not exert inhibitory effects on these [Ca2+]i increases in the TGs following MC stimulation. These results indicated that MCs are capable of releasing glutamate, but not ATP, in response to cellular deformation by direct mechanical stimulation. The released glutamate activates the NMDA receptors on TGs. We suggest that MCs act as mechanoelectrical transducers and establish synaptic transmission with neurons, through the MC-neurite complex, to mediate mechanosensory transduction.

Adenine nucleotides, such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), as well as the nucleoside adenosine are important modulators of neuronal function by engaging P1 and P2 purinergic receptors. In mitral cells, signaling of the G protein-coupled P1 receptor adenosine 1 receptor (A1R) affects the olfactory sensory pathway by regulating high voltage-activated calcium channels and two-pore domain potassium (K2P) channels. The inflammation of the central nervous system (CNS) impairs the olfactory function and gives rise to large amounts of extracellular ATP and adenosine, which act as pro-inflammatory and anti-inflammatory mediators, respectively. However, it is unclear whether neuronal A1R in the olfactory bulb modulates the sensory function and how this is impacted by inflammation. Here, we show that signaling via neuronal A1R is important for the physiological olfactory function, while it cannot counteract inflammation-induced hyperexcitability and olfactory deficit. Using neuron-specific A1R-deficient mice in patch-clamp recordings, we found that adenosine modulates spontaneous dendro-dendritic signaling in mitral and granule cells via A1R. Furthermore, neuronal A1R deficiency resulted in olfactory dysfunction in two separate olfactory tests. In mice with experimental autoimmune encephalomyelitis (EAE), we detected immune cell infiltration and microglia activation in the olfactory bulb as well as hyperexcitability of mitral cells and olfactory dysfunction. However, neuron-specific A1R activity was unable to attenuate glutamate excitotoxicity in the primary olfactory bulb neurons in vitro or EAE-induced olfactory dysfunction and disease severity in vivo. Together, we demonstrate that A1R modulates the dendro-dendritic inhibition (DDI) at the site of mitral and granule cells and impacts the processing of the olfactory sensory information, while A1R activity was unable to counteract inflammation-induced hyperexcitability.