Blocking Src-PSD-95 interaction rescues glutamatergic signaling dysregulation in schizophrenia.

The complex and heterogeneous genetic architecture of schizophrenia inspires us to look beyond individual risk genes for therapeutic strategies and target their interactive dynamics and convergence. Postsynaptic NMDA receptor (NMDAR) complexes are a site of such convergence. Src kinase is a molecular hub of NMDAR function, and its protein interaction subnetwork is enriched for risk-genes and altered protein associations in schizophrenia. Previously, Src activity was found to be decreased in post-mortem studies of schizophrenia, contributing to NMDAR hypofunction. PSD-95 suppresses Src via interacting with its SH2 domain. Here, we devised a strategy to suppress the inhibition of Src by PSD-95 via employing a cell penetrating and Src activating PSD-95 inhibitory peptide (TAT-SAPIP). TAT-SAPIP selectively increased post-synaptic Src activity in humans and mice, and enhanced synaptic NMDAR currents in mice. Chronic ICV injection of TAT-SAPIP rescued deficits in trace fear conditioning in Src hypomorphic mice. We propose blockade of the Src-PSD-95 interaction as a proof of concept for the use of interfering peptides as a therapeutic strategy to reverse NMDAR hypofunction in schizophrenia and other illnesses.

Calcium-Dependent Hyperexcitability in Human Stem Cell-Derived Rett Syndrome Neuronal Networks.

Background: Mutations in MECP2 predominantly cause Rett syndrome and can be modeled in vitro using human stem cell-derived neurons. Patients with Rett syndrome have signs of cortical hyperexcitability, such as seizures. Human stem cell-derived MECP2 null excitatory neurons have smaller soma size and reduced synaptic connectivity but are also hyperexcitable due to higher input resistance. Paradoxically, networks of MECP2 null neurons show a decrease in the frequency of network bursts consistent with a hypoconnectivity phenotype. Here, we examine this issue. Methods: We reanalyzed multielectrode array data from 3 isogenic MECP2 cell line pairs recorded over 6 weeks (n = 144). We used a custom burst detection algorithm to analyze network events and isolated a phenomenon that we termed reverberating super bursts (RSBs). To probe potential mechanisms of RSBs, we conducted pharmacological manipulations using bicuculline, EGTA-AM, and DMSO on 1 cell line (n = 34). Results: RSBs, often misidentified as single long-duration bursts, consisted of a large-amplitude initial burst followed by several high-frequency, low-amplitude minibursts. Our analysis revealed that MECP2 null networks exhibited increased frequency of RSBs, which produced increased bursts compared with isogenic controls. Bicuculline or DMSO treatment did not affect RSBs. EGTA-AM selectively eliminated RSBs and rescued network burst dynamics. Conclusions: During early development, MECP2 null neurons are hyperexcitable and produce hyperexcitable networks. This may predispose them to the emergence of hypersynchronic states that potentially translate into seizures. Network hyperexcitability depends on asynchronous neurotransmitter release that is likely driven by presynaptic Ca2+ and can be rescued by EGTA-AM to restore typical network dynamics.

Synapse-specific diversity of distinct postsynaptic GluN2 subtypes defines transmission strength in spinal lamina I.

The unitary postsynaptic response to presynaptic quantal glutamate release is the fundamental basis of excitatory information transfer between neurons. The view, however, of individual glutamatergic synaptic connections in a population as homogenous, fixed-strength units of neural communication is becoming increasingly scrutinized. Here, we used minimal stimulation of individual glutamatergic afferent axons to evoke single synapse resolution postsynaptic responses from central sensory lamina I neurons in an ex vivo adult rat spinal slice preparation. We detected unitary events exhibiting a NMDA receptor component with distinct kinetic properties across synapses conferred by specific GluN2 subunit composition, indicative of GluN2 subtype-based postsynaptic heterogeneity. GluN2A, 2A and 2B, or 2B and 2D synaptic predominance functioned on distinct lamina I neuron types to narrowly, intermediately, or widely tune, respectively, the duration of evoked unitary depolarization events from resting membrane potential, which enabled individual synapses to grade differentially depolarizing steps during temporally patterned afferent input. Our results lead to a model wherein a core locus of proteomic complexity prevails at this central glutamatergic sensory synapse that involves distinct GluN2 subtype configurations. These findings have major implications for subthreshold integrative capacity and transmission strength in spinal lamina I and other CNS regions.

Conserved transcriptional programming across sex and species after peripheral nerve injury predicts treatments for neuropathic pain.

BACKGROUND AND PURPOSE: Chronic pain is a devastating problem affecting one in five individuals around the globe, with neuropathic pain the most debilitating and poorly treated type of chronic pain. Advances in transcriptomics have contributed to cataloguing diverse cellular pathways and transcriptomic alterations in response to peripheral nerve injury but have focused on phenomenology and classifying transcriptomic responses. EXPERIMENTAL APPROACH: To identifying new types of pain-relieving agents, we compared transcriptional reprogramming changes in the dorsal spinal cord after peripheral nerve injury cross-sex and cross-species, and imputed commonalities, as well as differences in cellular pathways and gene regulation. KEY RESULTS: We identified 93 transcripts in the dorsal horn that were increased by peripheral nerve injury in male and female mice and rats. Following gene ontology and transcription factor analyses, we constructed a pain interactome for the proteins encoded by the differentially expressed genes, discovering new, conserved signalling nodes. We investigated the interactome with the Drug-Gene database to predict FDA-approved medications that may modulate key nodes within the network. The top hit from the analysis was fostamatinib, the molecular target of which is the non-receptor spleen associated tyrosine kinase (Syk), which our analysis had identified as a key node in the interactome. We found that intrathecally administrating the active metabolite of fostamatinib, R406 and another Syk inhibitor P505-15, significantly reversed pain hypersensitivity in both sexes. CONCLUSIONS AND IMPLICATIONS: Thus, we have identified and shown the efficacy of an agent that could not have been previously predicted to have analgesic properties.

Neuropathic Pain: Mechanisms, Sex Differences, and Potential Therapies for a Global Problem.

The study of chronic pain continues to generate ever-increasing numbers of publications, but safe and efficacious treatments for chronic pain remain elusive. Recognition of sex-specific mechanisms underlying chronic pain has resulted in a surge of studies that include both sexes. A predominant focus has been on identifying sex differences, yet many newly identified cellular mechanisms and alterations in gene expression are conserved between the sexes. Here we review sex differences and similarities in cellular and molecular signals that drive the generation and resolution of neuropathic pain. The mix of differences and similarities reflects degeneracy in peripheral and central signaling processes by which neurons, immune cells, and glia codependently drive pain hypersensitivity. Recent findings identifying critical signaling nodes foreshadow the development of rationally designed, broadly applicable analgesic strategies. However, the paucity of effective, safe pain treatments compels targeted therapies as well to increase therapeutic options that help reduce the global burden of suffering.

Regulation of neuropathic pain by microglial Orai1 channels.

Microglia are important mediators of neuroinflammation, which underlies neuropathic pain. However, the molecular checkpoints controlling microglial reactivity are not well-understood. Here, we investigated the role of Orai1 channels for microglia-mediated neuroinflammation following nerve injury and find that deletion of Orai1 in microglia attenuates Ca2+ signaling and the production of inflammatory cytokines by proalgesic agonists. Conditional deletion of Orai1 attenuated microglial proliferation in the dorsal horn, spinal cytokine levels, and potentiation of excitatory neurotransmission following peripheral nerve injury. These cellular effects were accompanied by mitigation of pain hyperalgesia in microglial Orai1 knockout mice. A small-molecule Orai1 inhibitor, CM4620, similarly mitigated allodynia in male mice. Unexpectedly, these protective effects were not seen in female mice, revealing sexual dimorphism in Orai1 regulation of microglial reactivity and hyperalgesia. Together, these findings indicate that Orai1 channels are key regulators of the sexually dimorphic role of microglia for the neuroinflammation that underlies neuropathic pain.

Wide spectrum of neuronal and network phenotypes in human stem cell-derived excitatory neurons with Rett syndrome-associated MECP2 mutations.

Rett syndrome (RTT) is a severe neurodevelopmental disorder primarily caused by heterozygous loss-of-function mutations in the X-linked gene MECP2 that is a global transcriptional regulator. Mutations in the methyl-CpG binding domain (MBD) of MECP2 disrupt its interaction with methylated DNA. Here, we investigate the effect of a novel MECP2 L124W missense mutation in the MBD of an atypical RTT patient with preserved speech in comparison to severe MECP2 null mutations. L124W protein had a limited ability to disrupt heterochromatic chromocenters due to decreased binding dynamics. We isolated two pairs of isogenic WT and L124W induced pluripotent stem cells. L124W induced excitatory neurons expressed stable protein, exhibited increased input resistance and decreased voltage-gated Na+ and K+ currents, and their neuronal dysmorphology was limited to decreased dendritic complexity. Three isogenic pairs of MECP2 null neurons had the expected more extreme morphological and electrophysiological phenotypes. We examined development and maturation of L124W and MECP2 null excitatory neural network activity using micro-electrode arrays. Relative to isogenic controls, L124W neurons had an increase in synchronous network burst frequency, in contrast to MECP2 null neurons that suffered a significant decrease in synchronous network burst frequency and a transient extension of network burst duration. A biologically motivated computational neural network model shows the observed changes in network dynamics are explained by changes in intrinsic Na+ and K+ currents in individual neurons. Our multilevel results demonstrate that RTT excitatory neurons show a wide spectrum of morphological, electrophysiological and circuitry phenotypes that are dependent on the severity of the MECP2 mutation.

Methacrylic acid-based biomaterials promote peripheral innervation in the subcutaneous space of mice.

Peripheral nerve innervation is essential for regulating tissue repair and regeneration. MAA-based biomaterials have been previously shown to promote angiogenesis. Here we show a new role for MAA-based biomaterials in promoting terminal axon nerve growth. Our results demonstrate that MAA-based biomaterials promote peripheral nerve growth in an Igf-1 and Shh dependent manner. The resulting nerves increased the sensitivity of treated mice paws to nociception. iDISCO clearing showed that MAA increased the presence of peripheral nerve structures in whole explants. MAA was also able to increase the expression of key neuronal markers and growth factors in a peripheral neuropathy model, the diabetic db/db mouse, suggesting that MAA-based biomaterials may be relevant to treatment of peripheral neuropathy. Moreover, in a peripheral neuropathy model, MAA was able to up-regulate the expression of growth factors for an extended duration suggesting MAA may prevent degeneration through an effect on factors that promote survival. As all tissues are innervated, MAA-based biomaterials could have broad applications in the promoting regeneration and preventing degeneration of peripheral nerves.

Targeting NMDA receptors in neuropsychiatric disorders by drug screening on human neurons derived from pluripotent stem cells.

NMDA receptors (NMDARs), a prominent subtype of glutamatergic receptors, are implicated in the pathogenesis and development of neuropsychiatric disorders such as epilepsy, intellectual disability, autism spectrum disorder, and schizophrenia, and are therefore a potential therapeutic target in treating these disorders. Neurons derived from induced pluripotent stem cells (iPSCs) have provided the opportunity to investigate human NMDARs in their native environment. In this review, we describe the expression, function, and regulation of NMDARs in human iPSC-derived neurons and discuss approaches for utilizing human neurons for identifying potential drugs that target NMDARs in the treatment of neuropsychiatric disorders. A challenge in studying NMDARs in human iPSC-derived neurons is a predominance of those receptors containing the GluN2B subunit and low synaptic expression, suggesting a relatively immature phenotype of these neurons and delayed development of functional NMDARs. We outline potential approaches for improving neuronal maturation of human iPSC-derived neurons and accelerating the functional expression of NMDARs. Acceleration of functional expression of NMDARs in human iPSC-derived neurons will improve the modeling of neuropsychiatric disorders and facilitate the discovery and development of novel therapeutics targeting NMDARs for the treatment of these disorders.

Microglia-independent peripheral neuropathic pain in male and female mice.

ABSTRACT: The dominant view in the field of pain is that peripheral neuropathic pain is driven by microglia in the somatosensory processing region of the spinal dorsal horn. Here, to the contrary, we discovered a form of neuropathic pain that is independent of microglia. Mice in which the nucleus pulposus (NP) of the intervertebral disc was apposed to the sciatic nerve developed a constellation of neuropathic pain behaviours: hypersensitivity to mechanical, cold, and heat stimuli. However, NP application caused no activation of spinal microglia nor was pain hypersensitivity reversed by microglial inhibition. Rather, NP-induced pain hypersensitivity was dependent on cells within the NP which recruited macrophages to the adjacent nerve. Eliminating macrophages systemically or locally prevented NP-induced pain hypersensitivity. Pain hypersensitivity was also prevented by genetically disrupting the neurotrophin brain-derived neurotrophic factor selectively in macrophages. Moreover, the behavioural phenotypes as well as the molecular mechanisms of NP-induced pain hypersensitivity were not different between males and females. Our findings reveal a previously unappreciated mechanism for by which a discrete peripheral nerve lesion may produce pain hypersensitivity, which may help to explain the limited success of microglial inhibitors on neuropathic pain in human clinical trials.

Glial-modulating agents for the treatment of pain: protocol for a systematic review.

INTRODUCTION: Evidence suggests a role for Central nervous system glia in pain transmission and in augmenting maladaptive opioid effects. Identification of drugs that modulate glia has guided the evaluation of glial suppression as a pain management strategy. This planned systematic review will describe evidence of the efficacy and adverse effects of glial-modulating drugs in pain management. METHODS AND ANALYSIS: A detailed search will be conducted on the Cochrane Central Register of Controlled Trials, Medline, and Embase from their inception until the date the final searches are run to identify relevant randomised controlled trials. The reference lists of retrieved studies, as well as online trial registries, will also be searched. English language, randomised, double-blind trials comparing various glial-modulating drugs with placebo and/or other comparators, with participant-reported pain assessment, will be included. Two reviewers will independently evaluate studies for eligibility, extract data and assess trial quality and potential bias. Risk of bias will be assessed using criteria outlined in the Cochrane Handbook for Systematic Review of Interventions. Primary outcomes for this review will include any validated measure of pain intensity and/or pain relief. Dichotomous data will be used to calculate risk ratio and number needed to treat or harm. The quality of evidence will be assessed using Grading of Recommendations Assessment, Development and Evaluation. ETHICS AND DISSEMINATION: This systematic review does not require formal ethics approval. The findings will be disseminated through peer-reviewed publications and conference presentations. PROSPERO REGISTRATION NUMBER: CRD42021262074.

Cholinergic modulation is independent of T lymphocytes in a mouse model of neuropathic pain.

T lymphocytes are increasingly implicated in pain signaling. A subset of T lymphocytes, termed TChAT, express the rate-limiting enzyme for acetylcholine (ACh) production, choline acetyltransferase (ChAT), and mediate numerous physiological functions. Given that cholinergic signaling has long been known to modulate pain processing and is the basis for several analgesics used clinically, we asked whether TChAT could be the intersection between T lymphocyte and cholinergic mediation of pain signaling. In this study, we used a mouse gene knockout strategy to ablate ChAT specifically from T lymphocytes and examined the development and expression of mechanical and thermal hypersensitivity in a spared nerve injury (SNI) mouse model of neuropathic pain. We found that mice with ChAT knockout in T cells (floxed Chat plus CD4-Cre recombinase) did not differ from control mice with intact ChAT (floxed Chat, but no Cre recombinase) in their expression of mechanical sensitivity before or after injury. Similarly, thermal sensitivity was unaffected after injury, with control mice expressing similar patterns of thermal preference to mice whose T cells do not express ChAT. Our experiments demonstrate that cholinergic signaling initiated by T lymphocytes neither dampens nor exacerbates the expression of mechanical or thermal sensitivity in neuropathic mice. Thus, while both cholinergic signaling and T lymphocytes have established roles in modulating pain phenotypes, it is not cholinergic signaling initiated by T lymphocytes that drive this. Our findings will help to narrow in on which aspects of T-cell modulation may prove useful as therapies.

Single-cell RNA sequencing reveals time- and sex-specific responses of mouse spinal cord microglia to peripheral nerve injury and links ApoE to chronic pain.

Activation of microglia in the spinal cord following peripheral nerve injury is critical for the development of long-lasting pain hypersensitivity. However, it remains unclear whether distinct microglia subpopulations or states contribute to different stages of pain development and maintenance. Using single-cell RNA-sequencing, we show that peripheral nerve injury induces the generation of a male-specific inflammatory microglia subtype, and demonstrate increased proliferation of microglia in male as compared to female mice. We also show time- and sex-specific transcriptional changes in different microglial subpopulations following peripheral nerve injury. Apolipoprotein E (Apoe) is the top upregulated gene in spinal cord microglia at chronic time points after peripheral nerve injury in mice. Furthermore, polymorphisms in the APOE gene in humans are associated with chronic pain. Single-cell RNA sequencing analysis of human spinal cord microglia reveals a subpopulation with a disease-related transcriptional signature. Our data provide a detailed analysis of transcriptional states of mouse and human spinal cord microglia, and identify a link between ApoE and chronic pain in humans.

Alternative splicing of GluN1 gates glycine site-dependent nonionotropic signaling by NMDAR receptors.

N-methyl-D-aspartate (NMDA) receptors (NMDARs), a principal subtype of excitatory neurotransmitter receptor, are composed as tetrameric assemblies of two glycine-binding GluN1 subunits and two glutamate-binding GluN2 subunits. NMDARs can signal nonionotropically through binding of glycine alone to its cognate site on GluN1. A consequence of this signaling by glycine is that NMDARs are primed such that subsequent gating, produced by glycine and glutamate, drives receptor internalization. The GluN1 subunit contains eight alternatively spliced isoforms produced by including or excluding the N1 and the C1, C2, or C2' polypeptide cassettes. Whether GluN1 alternative splicing affects nonionotropic signaling by NMDARs is a major outstanding question. Here, we discovered that glycine priming of recombinant NMDARs critically depends on GluN1 isoforms lacking the N1 cassette; glycine priming is blocked in splice variants containing N1. On the other hand, the C-terminal cassettes-C1, C2, or C2'-each permit glycine signaling. In wild-type mice, we found glycine-induced nonionotropic signaling at synaptic NMDARs in CA1 hippocampal pyramidal neurons. This nonionotropic signaling by glycine to synaptic NMDARs was prevented in mice we engineered, such that GluN1 obligatorily contained N1. We discovered in wild-type mice that, in contrast to pyramidal neurons, synaptic NMDARs in CA1 inhibitory interneurons were resistant to glycine priming. But we recapitulated glycine priming in inhibitory interneurons in mice engineered such that GluN1 obligatorily lacked the N1 cassette. Our findings reveal a previously unsuspected molecular function for alternative splicing of GluN1 in controlling nonionotropic signaling of NMDARs by activating the glycine site.

Src and Fyn regulation of NMDA receptors in health and disease.

The Src family kinases (SFKs) are cytoplasmic non-receptor tyrosine kinases involved in multiple signalling pathways. In the central nervous system (CNS), SFKs are key regulators of N-methyl-d-aspartate receptor (NMDAR) function and major points of convergence for neuronal transduction pathways. Physiological upregulation of NMDAR activity by members of the SFKs, namely Src and Fyn, is crucial for induction of plasticity at Schaffer collateral-CA1 synapses of the hippocampus. Aberrant SFK regulation of NMDARs is implicated in several pathological conditions in the CNS including schizophrenia and pain hypersensitivity. Here, evidence is presented to highlight the current understanding of the intermolecular interactions of SFKs within the NMDAR macromolecular complex, the upstream regulators of SFK activity on NMDAR function and the role Src and Fyn have in synaptic plasticity and metaplasticity. The targeting of SFK protein-protein interactions is discussed as a potential therapeutic strategy to restore signalling activity underlying glutamatergic dysregulation in CNS disease pathophysiology.

The T-type calcium channel antagonist, Z944, reduces spinal excitability and pain hypersensitivity.

BACKGROUND AND PURPOSE: T-type voltage-gated calcium channels are an emerging therapeutic target for neurological disorders including epilepsy and pain. Inhibition of T-type channels reduces the excitability of peripheral nociceptive sensory neurons and reverses pain hypersensitivity in male rodent pain models. However, administration of peripherally restricted T-type antagonists failed to show efficacy in multiple clinical and preclinical pain trials, suggesting that inhibition of peripheral T-type channels alone may be insufficient for pain relief. EXPERIMENTAL APPROACH: We utilized the selective and CNS-penetrant T-type channel antagonist, Z944, in electrophysiological, calcium imaging and behavioural paradigms to determine its effect on lamina I neuron excitability and inflammatory pain behaviours. KEY RESULTS: Voltage-clamp recordings from lamina I spinal neurons of adult rats revealed that approximately 80% of neurons possess a low threshold T-type current, which was blocked by Z944. Due to this highly prevalent T-type current, Z944 potently blocked action-potential evoked somatic and dendritic calcium transients in lamina I neurons. Moreover, application of Z944 to spinal cord slices attenuated action potential firing rates in over half of laminae I/II neurons. Finally, we found that intraperitoneal injection of Z944 (1-10 mg·kg-1 ) dose-dependently reversed mechanical allodynia in the complete Freund's adjuvant model of persistent inflammatory pain, with a similar magnitude and time course of analgesic effects between male and female rats. CONCLUSION AND IMPLICATIONS: T-type calcium channels critically shape the excitability of lamina I pain processing neurons and inhibition of these channels by the clinical stage antagonist Z944 potently reverses pain hypersensitivity across sexes.

Purinergic signalling in spinal pain processing.

Purinergic signalling plays important roles in somatosensory and nociceptive transmission in the dorsal horn of the spinal cord under physiological and pathophysiological conditions. Physiologically, ATP mediates excitatory postsynaptic responses in nociceptive transmission in the superficial dorsal horn, and in transmission of innocuous primary afferent inputs in the deep dorsal horn. Additionally, extracellular conversion of ATP to adenosine mediates inhibitory postsynaptic responses from Pacinian corpuscle afferents, and is implicated in analgesia caused by transcutaneous electrical nerve stimulation in humans. In terms of pathological pain, P2X4 receptors de novo expressed on dorsal horn microglia are implicated in pain hypersensitivity following peripheral nerve injury. There is evidence that involvement of such P2X4 receptors is sexually dimorphic, occurring in males but not in females. Thus, the roles of purinergic signalling in physiological and pathological pain processing are complex and remain an ever-expanding field of research.

Intracellular Calcium Responses Encode Action Potential Firing in Spinal Cord Lamina I Neurons.

Maladaptive plasticity of neurons in lamina I of the spinal cord is a lynchpin for the development of chronic pain, and is critically dependent on intracellular calcium signaling. However, the relationship between neuronal activity and intracellular calcium in these neurons is unknown. Here we combined two-photon calcium imaging with whole-cell electrophysiology to determine how action potential firing drives calcium responses within subcellular compartments of male rat spinal cord lamina I neurons. We found that single action potentials generated at the soma increase calcium concentration in the somatic cytosol and nucleus, and these calcium responses invade dendrites and dendritic spines by active backpropagation. Calcium responses in each compartment were dependent on voltage-gated calcium channels, and somatic and nuclear calcium responses were amplified by release of calcium from ryanodine-sensitive intracellular stores. Grouping single action potential-evoked calcium responses by neuron type demonstrated their presence in all defined types, as well as a high degree of similarity in calcium responses between neuron types. With bursts of action potentials, we found that calcium responses have the capacity to encode action potential frequency and number in all compartments, with action potential number being preferentially encoded. Together, these findings indicate that intracellular calcium serves as a readout of neuronal activity within lamina I neurons, providing a unifying mechanism through which activity may regulate plasticity, including that seen in chronic pain.SIGNIFICANCE STATEMENT Despite their critical role in both acute pain sensation and chronic pain, little is known of the fundamental physiology of spinal cord lamina I neurons. This is especially the case with respect to calcium dynamics within these neurons, which could regulate maladaptive plasticity observed in chronic pain. By combining two-photon calcium imaging and patch-clamp electrophysiological recordings from lamina I neurons, we found that action potential firing induces calcium responses within the somatic cytosol, nucleus, dendrites, and dendritic spines of lamina I neurons. Our findings demonstrate the presence of actively backpropagating action potentials, shifting our understanding of how these neurons process information, such that calcium provides a mechanism for lamina I neurons to track their own activity.

Sex-Dependent Mechanisms of Chronic Pain: A Focus on Microglia and P2X4R.

For over two decades, purinergic signaling in microglia has persisted in the spotlight as a major pathomechanism of chronic pain. Of the many purinoreceptors, the P2X4R of the ionotropic family, has a well-described causal role underlying chronic neuropathic pain. This review will briefly examine microglial P2X4R signaling in the spinal cord as it relates to chronic pain through a historical lens, followed by a more in-depth examination of recent work, which has revealed major sex differences. We also discuss the generalizability of sex differences in microglial and P2X4R signaling in other pain conditions as well as in nonspinal regions. Finally, we speculate on remaining gaps in the literature as well as what can be done to address them with the ultimate goal of using our collective knowledge to treat chronic pain effectively and in both sexes. SIGNIFICANCE STATEMENT: Effective treatments are lacking for chronic pain sufferers, and this may be explained by the vast sex differences underlying chronic pain mechanisms. In this minireview, we focus on the roles of microglia and P2X4R in chronic pain, with specific attention to the circumstances under which these pathomechanisms differ between males and females. By delineating the ways in which pain occurs differently between the sexes, we can start developing successful therapies for all.

Tripartite signalling by NMDA receptors.

N-methyl-D-aspartate receptors (NMDARs) are excitatory glutamatergic receptors that are fundamental for many neuronal processes, including synaptic plasticity. NMDARs are comprised of four subunits derived from heterogeneous subunit families, yielding a complex diversity in NMDAR form and function. The quadruply-liganded state of binding of two glutamate and two glycine molecules to the receptor drives channel gating, allowing for monovalent cation flux, Ca2+ entry and the initiation of Ca2+-dependent signalling. In addition to this ionotropic function, non-ionotropic signalling can be initiated through the exclusive binding of glycine or of glutamate to the NMDAR. This binding may trigger a transmembrane conformational change of the receptor, inducing intracellular protein-protein signalling between the cytoplasmic domain and secondary messengers. In this review, we outline signalling cascades that can be activated by NMDARs and propose that the receptor transduces signalling through three parallel streams: (i) signalling via both glycine and glutamate binding, (ii) signalling via glycine binding, and (iii) signalling via glutamate binding. This variety in signal transduction mechanisms and downstream signalling cascades complements the widespread prevalence and rich diversity of NMDAR activity throughout the central nervous system and in disease pathology.