Spinal cord GluR2-lacking AMPA receptors (AMPARs) contribute to nociceptive hypersensitivity in persistent pain, but the molecular mechanisms underlying this event are not completely understood. We report that complete Freund's adjuvant (CFA)-induced peripheral inflammation induces synaptic GluR2 internalization in dorsal horn neurons during the maintenance of CFA-evoked nociceptive hypersensitivity. This internalization is initiated by GluR2 phosphorylation at Ser880and subsequent disruption of GluR2 binding to its synaptic anchoring protein (GRIP), resulting in a switch of GluR2-containing AMPARs to GluR2-lacking AMPARs and an increase of AMPAR Ca2+permeability at the synapses in dorsal horn neurons. Spinal cord NMDA receptor-mediated triggering of protein kinase C (PKC) activation is required for the induction and maintenance of CFA-induced dorsal horn GluR2 internalization. Moreover, preventing CFA-induced spinal GluR2 internalization through targeted mutation of the GluR2 PKC phosphorylation site impairs CFA-evoked nociceptive hypersensitivity during the maintenance period. These results suggest that dorsal horn GluR2 internalization might participate in the maintenance of NMDA receptor/PKC-dependent nociceptive hypersensitivity in persistent inflammatory pain.

Highlights d Induction of synaptic LTP prompts withdrawal of perisynaptic astroglia d The underlying mechanisms involve NKCC1 transporter and cofilin d Reduced synaptic astroglial coverage boosts extrasynaptic glutamate escape d LTP induction thus enhances NMDAR-dependent intersynaptic cross-talk

Peripheral inflammation alters AMPA receptor (AMPAR) subunit trafficking and increases AMPAR Ca 2+ permeability at synapses of spinal dorsal horn neurons. However, it is unclear whether AMPAR trafficking at extrasynaptic sites of these neurons also changes under persistent inflammatory pain conditions. Using patch-clamp recording combined with Ca 2+ imaging and cobalt staining, we found that, under normal conditions, an extrasynaptic pool of AMPARs in rat substantia gelatinosa (SG) neurons of spinal dorsal horn predominantly consists of GluR2-containing Ca 2+ -impermeable receptors. Maintenance of complete Freund's adjuvant (CFA)-induced inflammation was associated with a marked enhancement of AMPA-induced currents and [Ca 2+ ] i transients in SG neurons, while, as we previously showed, the amplitude of synaptically evoked AMPAR-mediated currents was not changed 24 h after CFA. These findings indicate that extrasynaptic AMPARs are upregulated and their Ca 2+ permeability increases dramatically. This increase occurred in SG neurons characterized by intrinsic tonic firing properties, but not in those exhibited strong adaptation. This increase was also accompanied by an inward rectification of AMPA-induced currents and enhancement of sensitivity to a highly selective Ca 2+ -permeable AMPAR blocker, IEM-1460. Electron microcopy and biochemical assays additionally showed an increase in the amount of GluR1 at extrasynaptic membranes in dorsal horn neurons 24 h post-CFA. Taken together, our findings suggest that CFA-induced inflammation increases functional expression and proportion of extrasynaptic GluR1-containing Ca 2+ -permeable AMPARs in tonically firing excitatory dorsal horn neurons. We suggest that the altered extrasynaptic AMPAR trafficking might participate in the maintenance of persistent inflammatory pain.

HighlightsGb3 and Lyso-Gb3, plasma lipids accumulating in Fabry disease, cause mechanical allodynia in mice.Lyso-Gb3 elevates intracellular calcium level in sensory neurons.Lyso-Gb3 enhances voltage-dependent calcium currents in small-diameter DRG neurons.Direct effects of lyso-Gb3 on sensory neurons may contribute to the pain of Fabry disease.

Brain cells continuously produce and release protons into the extracellular space, with the rate of acid production corresponding to the levels of neuronal activity and metabolism. Efficient buffering and removal of excess H+ is essential for brain function, not least because all the electrogenic and biochemical machinery of synaptic transmission is highly sensitive to changes in pH. Here, we describe an astroglial mechanism that contributes to the protection of the brain milieu from acidification. In vivo and in vitro experiments conducted in rodent models show that at least one third of all astrocytes release bicarbonate to buffer extracellular H+ loads associated with increases in neuronal activity. The underlying signalling mechanism involves activity-dependent release of ATP triggering bicarbonate secretion by astrocytes via activation of metabotropic P2Y1 receptors, recruitment of phospholipase C, release of Ca2+ from the internal stores, and facilitated outward HCO3− transport by the electrogenic sodium bicarbonate cotransporter 1, NBCe1. These results show that astrocytes maintain local brain extracellular pH homeostasis via a neuronal activity-dependent release of bicarbonate. The data provide evidence of another important metabolic housekeeping function of these glial cells.

Persistent inflammation promotes internalization of synaptic GluR2-containing Ca2+-impermeable AMPA receptors (AMPARs) and insertion of GluR1-containing Ca2+-permeable AMPARs at extrasynaptic sites in dorsal horn neurons. Previously we have shown that internalization of synaptic GluR2-containing AMPARs requires an activation of spinal cord protein kinase C alpha (PKCα), but molecular mechanisms that underlie altered trafficking of extrasynaptic AMPARs are still unclear. By utilizing the antisence oligodeoxynucleotides that specifically knockdown PKCα, we have found that a decrease in dorsal horn PKCα expression prevents complete Freund’s adjuvant (CFA)-induced increase in a functional expression of extrasynaptic Ca2+-permeable AMPARs in substantia gelatinosa (SG) neurons of the rat spinal cord. This was manifested as an abolishment of augmented AMPA-induced currents and associated [Ca2+]i transients, and as a reverse of the current rectification 1 d post-CFA. These changes were observed specifically in SG neurons characterized by intrinsic tonic firing properties, but not in those exhibiting strong adaptation. Finally, dorsal horn PKCα knockdown produced anti-nociceptive effect on CFA-induced thermal and mechanical hypersensitivity during the maintenance period of inflammatory pain, indicating a role for PKCα in persistent inflammatory pain maintenance. Altogether, our results indicate that inflammation-induced trafficking of extrasynaptic Ca2+-permeable AMPARs in tonically firing SG neurons depends on PKCα, and suggest that this PKCα-dependent trafficking may contribute to the persistent inflammatory pain maintenance.

Frontotemporal dementia and parkinsonism (FTDP-17) caused by the 10+16 splice-site mutation in the gene encoding microtubuleassociated protein tau (MAPT) provides an established platform to model tau-related dementia in vitro. Neurons derived from human induced pluripotent stem cells (iPSCs) have been shown to recapitulate the neurodevelopmental profile of tau pathology during in vitro corticogenesis, as in the adult human brain. However, the neurophysiological phenotype of these cells has remained unknown, leaving unanswered questions regarding the functional relevance and the gnostic power of this disease model. In this study, we used electrophysiology to explore the membrane properties and intrinsic excitability of the generated neurons and found that human cells mature by ∼150 days of neurogenesis to become compatible with matured cortical neurons. In earlier FTDP-17, however, neurons exhibited a depolarized resting membrane potential associated with increased resistance and reduced voltage-gated Na +-and K +channel-mediated conductance. Expression of the Na v 1.6 protein was reduced in FTDP-17. These effects led to reduced cell capability of induced firing and changed the action potential waveform in FTDP-17. The revealed neuropathology might thus contribute to the clinicopathological profile of the disease. This sheds new light on the significance of human in vitro models of dementia.

Upregulation of Ca2+-permeable AMPA receptors (CP-AMPARs) in the dorsal horn (DH) neurons of the spinal cord has been causally linked to the maintenance of persistent inflammatory pain. Therefore, inhibition of CP-AMPARs could potentially alleviate an, otherwise, poorly treatable chronic pain. However, a loss of CP-AMPARs could produce considerable side effects because of the crucial role of CP-AMPARs in synaptic plasticity. Here we have tested whether the inhibition of spinal CP-AMPARs with dicationic compounds, the open-channel antagonists acting in an activity-dependent manner, can relieve inflammatory pain without adverse effects being developed. Dicationic compounds, N1-(1-phenylcyclohexyl)pentane-1,5-diaminium bromide (IEM-1925) and 1-trimethylammonio-5-1-adamantane-methyl-ammoniopentane dibromide (IEM-1460) were applied intrathecally (i.t.) as a post-treatment for inflammatory pain in the model of complete Freund’s adjuvant (CFA)-induced long-lasting peripheral inflammation. The capability of dicationic compounds to ameliorate inflammatory pain was tested in rats in vivo using the Hargreaves, the von Frey and the open-field tests. Treatment with IEM-1460 or IEM-1925 resulted in profound alleviation of inflammatory pain. The pain relief appeared shortly after compound administration. The effects were concentration-dependent, displaying a high potency of dicationic compounds for alleviation of inflammatory hyperalgesia in the micromolar range, for both acute and long-lasting responses. The period of pain maintenance was shortened following treatment. Treatment with IEM-1460 or IEM-1925 changed neither thermal and mechanical basal sensitivities nor animal locomotion, suggesting that inhibition of CP-AMPARs with dicationic compounds does not give rise to detectable side effects. Thus, the ability of dicationic compounds to alleviate persistent inflammatory pain may provide new routes in the treatment of chronic pain.