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Depolarization drives neuronal plasticity. However, whether depolarization drives sensitization of peripheral nociceptive neurons remains elusive. By high-content screening (HCS) microscopy, we revealed that depolarization of cultured sensory neurons rapidly activates protein kinase A type II (PKA-II) in nociceptors by calcium influx through Ca(V)1.2 channels. This effect was modulated by calpains but insensitive to inhibitors of cAMP formation, including opioids. In turn, PKA-II phosphorylated Ser1928 in the distal C terminus of Ca(V)1.2, thereby increasing channel gating, whereas dephosphorylation of Ser1928 involved the phosphatase calcineurin. Patch-clamp and behavioral experiments confirmed that depolarization leads to calcium-and PKA-dependent sensitization of calcium currents ex vivo and local peripheral hyperalgesia in the skin in vivo. Our data suggest a local activity-driven feed-forward mechanism that selectively translates strong depolarization into further activity and thereby facilitates hypersensitivity of nociceptor terminals by a mechanism inaccessible to opioids.

Genetic loss of the voltage-gated sodium channel Nav1.7 (Na(v)1.7(-/-)) results in lifelong insensitivity to pain inmice and humans. One underlying cause is an increase in the production of endogenous opioids in sensory neurons. We analyzed whether Na(v)1.7 deficiency altered nociceptive heterotrimeric guanine nucleotide-binding protein-coupled receptor (GPCR) signaling, such as initiated by GPCRs that respond to serotonin (pronociceptive) or opioids (antinociceptive), in sensory neurons. We found that the nociceptive neurons of Na(v)1.7 knockout (Na(v)1.7(-/-)) mice, but not those of Na(v)1.8 knockout (Na(v)1.8(-/-)) mice, exhibited decreased pronociceptive serotonergic signaling through the 5-HT4 receptors, which are G alpha(s)-coupled GPCRs that stimulate the production of cyclic adenosinemonophosphate resulting in protein kinase A (PKA) activity, as well as reduced abundance of the RIIb regulatory subunit of PKA. Simultaneously, the efficacy of antinociceptive opioid signaling mediated by the G alpha(i)-coupled mu opioid receptors was increased. Consequently, opioids inhibited more efficiently tetrodotoxin-resistant sodium currents, which are important for pain-initiating neuronal activity in nociceptive neurons. Thus, Na(v)1.7 controls the efficacy and balance of GPCR-mediated pro-and antinociceptive intracellular signaling, such that without Na(v)1.7, the balance is shifted toward antinociception, resulting in lifelong endogenous analgesia.

A recent digital public archaeology project (HeritageTogether) sought to build a series of 3D ditigal models using photogrammetry from crowd-sourced images. The project saw over 13000 digital images being donated, and resulted in models of some 78 sites, providing resources for researchers, and condition surveys. The project demonstrated that digital public archaeology does not stop at the 'trowel's edge', and that collaborative post-excavation analysis and generation of research processes are as important as time in the field. We emphasise in this contribution that our methodologies, as much as our research outputs, can be fruitfully co-produced in public archaeology projects.

Archaeologists are increasingly working with crowd-sourced digital data. Using evidence from other disciplines about the nature of crowd-sourcing in academic research, we suggest that archaeological projects using donated data can usefully be differentiated between generative projects (which rely on data collected by citizen scientists), and analytical projects (which make use of volunteers to classify, or otherwise analyse data that are provided by the project). We conclude that projects which privilege hyper-local research (such as surveying specific sites) might experience tension if the audience they are appealing to are 'cyber local'. In turn, for more 'traditional' archaeological audiences (when the primary motivating interests may be the tangible, physical nature of portable material culture or the archaeological site itself), then intangible, digital simulacra may not provide an effective medium through which to undertake digital public archaeology

Background: The translocator protein (18 kDa) (TSPO) is a mitochondrial protein expressed on reactive glial cells and a biomarker for gliosis in the brain. TSPO ligands have been shown to reduce neuroinflammation in several mouse models of neurodegeneration. Here, we analyzed TSPO expression in mouse and human retinal microglia and studied the effects of the TSPO ligand XBD173 on microglial functions. Methods: TSPO protein analyses were performed in retinoschisin-deficient mouse retinas and human retinas. Lipopolysaccharide (LPS)-challenged BV-2 microglial cells were treated with XBD173 and TSPO shRNAs in vitro and pro-inflammatory markers were determined by qRT-PCR. The migration potential of microglia was determined with wound healing assays and the proliferation was studied with Fluorescence Activated Cell Sorting (FACS) analysis. Microglial neurotoxicity was estimated by nitrite measurement and quantification of caspase 3/7 levels in 661 W photoreceptors cultured in the presence of microglia-conditioned medium. The effects of XBD173 on filopodia formation and phagocytosis were analyzed in BV-2 cells and human induced pluripotent stem (iPS) cell-derived microglia (iPSdM). The morphology of microglia was quantified in mouse retinal explants treated with XBD173. Results: TSPO was strongly up-regulated in microglial cells of the dystrophic mouse retina and also co-localized with microglia in human retinas. Constitutive TSPO expression was high in the early postnatal Day 3 mouse retina and declined to low levels in the adult tissue. TSPO mRNA and protein were also strongly induced in LPS-challenged BV-2 microglia while the TSPO ligand XBD173 efficiently suppressed transcription of the pro-inflammatory marker genes chemokine (C-C motif) ligand 2 (CCL2), interleukin 6 (IL6) and inducible nitric oxide (NO)-synthase (iNOS). Moreover, treatment with XBD173 significantly reduced the migratory capacity and proliferation of microglia, their level of NO secretion and their ne

Hyperalgesic priming is characterized by enhanced nociceptor sensitization by pronociceptive mediators, prototypically PGE(2). Priming has gained interest as a mechanism underlying the transition to chronic pain. Which stimuli induce priming and what cellular mechanisms are employed remains incompletely understood. In adult male rats, we present the cytokine OncostatinM(OSM), a member of the IL-6 family, as an inducer of priming by a novel mechanism. We used a high content microscopy based approach to quantify the activation of endogenous PKA-II and ERK of thousands sensory neurons in culture. Incubation with OSM increased and prolonged ERK activation by agents that increase cAMP production such as PGE(2), forskolin, and cAMP analogs. These changes were specific to IB4/CaMKII alpha positive neurons, required protein translation, and increased cAMP-to-ERK signaling. In both, control and OSM-treated neurons, cAMP/ERK signaling involved RapGEF2 and PKA but not Epac. Similar enhancement of cAMP-to-ERK signaling could be induced by GDNF, which acts mostly on IB4/CaMKII alpha-positive neurons, but not by NGF, which acts mostly on IB4/CaMKII alpha-negative neurons. In vitro, OSM pretreatment rendered baseline TTX-R currents ERK-dependent and switched forskolin-increased currents from partial to full ERK-dependence in small/medium sized neurons. In summary, priming induced by OSM uses a novel mechanism to enhance and prolong coupling of cAMP/PKA to ERK1/2 signaling without changing the overall pathway structure.

Hyperalgesic priming is characterized by enhanced nociceptor sensitization by pronociceptive mediators, prototypically PGE(2). Priming has gained interest as a mechanism underlying the transition to chronic pain. Which stimuli induce priming and what cellular mechanisms are employed remains incompletely understood. In adult male rats, we present the cytokine OncostatinM(OSM), a member of the IL-6 family, as an inducer of priming by a novel mechanism. We used a high content microscopy based approach to quantify the activation of endogenous PKA-II and ERK of thousands sensory neurons in culture. Incubation with OSM increased and prolonged ERK activation by agents that increase cAMP production such as PGE(2), forskolin, and cAMP analogs. These changes were specific to IB4/CaMKII alpha positive neurons, required protein translation, and increased cAMP-to-ERK signaling. In both, control and OSM-treated neurons, cAMP/ERK signaling involved RapGEF2 and PKA but not Epac. Similar enhancement of cAMP-to-ERK signaling could be induced by GDNF, which acts mostly on IB4/CaMKII alpha-positive neurons, but not by NGF, which acts mostly on IB4/CaMKII alpha-negative neurons. In vitro, OSM pretreatment rendered baseline TTX-R currents ERK-dependent and switched forskolin-increased currents from partial to full ERK-dependence in small/medium sized neurons. In summary, priming induced by OSM uses a novel mechanism to enhance and prolong coupling of cAMP/PKA to ERK1/2 signaling without changing the overall pathway structure.