Characterizing Functional Contributions of Specific GluN2 Subunits to Individual Postsynaptic NMDAR Responses Using Biophysical Parameters.

The NMDAR is a heterotetramer composed of two GluN1 subunits and two GluN2 and/or GluN3 subunits, with the GluN2 subunits exhibiting significant diversity in their structure and function. Recent studies have highlighted the importance of characterizing the specific roles of each GluN2 subunit across central nervous system regions and developmental stages, as well as their unique contributions to NMDAR-mediated signaling and plasticity. Understanding the distinct functions of GluN2 subunits is critical for the development of targeted therapeutic strategies for NMDAR-related disorders. However, measuring the functional contribution of individual GluN2 subtypes in ex vivo slices is challenging. Conventionally, pharmacological or genetic approaches are used, but, in many cases, this is not possible or is restricted to population-level NMDAR responses. Here, we describe a technique for using biophysical properties of miniature synaptic NMDAR responses as a proxy to measure the functional contribution of specific GluN2-NMDAR subunits to individual synapses within a neuron.

A cellular taxonomy of the adult human spinal cord.

The mammalian spinal cord functions as a community of cell types for sensory processing, autonomic control, and movement. While animal models have advanced our understanding of spinal cellular diversity, characterizing human biology directly is important to uncover specialized features of basic function and human pathology. Here, we present a cellular taxonomy of the adult human spinal cord using single-nucleus RNA sequencing with spatial transcriptomics and antibody validation. We identified 29 glial clusters and 35 neuronal clusters, organized principally by anatomical location. To demonstrate the relevance of this resource to human disease, we analyzed spinal motoneurons, which degenerate in amyotrophic lateral sclerosis (ALS) and other diseases. We found that compared with other spinal neurons, human motoneurons are defined by genes related to cell size, cytoskeletal structure, and ALS, suggesting a specialized molecular repertoire underlying their selective vulnerability. We include a web resource to facilitate further investigations into human spinal cord biology.

Cannabinoid CB1 Receptor Expression and Localization in the Dorsal Horn of Male and Female Rat and Human Spinal Cord.

Background: Preclinical and clinical evidence suggests that cannabis has potential analgesic properties. However, cannabinoid receptor expression and localization within spinal cord pain processing circuits remain to be characterized across sex and species. Aims: We aimed to investigate the differential expression of the cannabinoid type 1 (CB1) receptor across dorsal horn laminae and cell populations in male and female adult rats and humans. Methods: To investigate and quantify CB1 receptor expression in the spinal dorsal horn across species, we refined immunohistochemical procedures for successful rat and human fixed tissue staining and confocal imaging. Immunohistochemical results were complemented with analysis of CB1 gene (CNR1) expression within rodent and human dorsal horn using single-cell/nuclei RNA sequencing data sets. Results: We found that CB1 was preferentially localized to the neuropil within the superficial dorsal horn of both rats and humans, with CB1 somatic staining across dorsal horn laminae. CB1 receptor immunoreactivity was significantly higher in the superficial dorsal horn compared to the deeper dorsal horn laminae for both rats and humans, which was conserved across sex. Interestingly, we found that CB1 immunoreactivity was not primarily localized to peptidergic afferents in rats and humans and that CNR1 (CB1) but not CNR2 (CB2) was robustly expressed in dorsal horn neuron subpopulations of both rodents and humans. Conclusions: The conserved preferential expression of CB1 receptors in the superficial dorsal horn in male and female rodents and humans has significant implications for understanding the roles of this cannabinoid receptor in spinal mechanisms of nociception and analgesia.

Contexte: Les données probantes précliniques et cliniques indiquent que le cannabis possède des propriétés analgésiques potentielles. Cependant, l'expression et la localisation des récepteurs cannabinoïdes au sein des circuits de traitement de la douleur de la moelle épinière restent à caractériser selon le sexe et les espèces.Objectifs: Nous avons cherché à étudier l'expression différenciée du récepteur cannabinoïde de type 1 (CB1) dans les différentes couches de la corne dorsale et les populations cellulaires chez des rats et des êtres humains adultes de sexe masculin et féminin.Méthodes: Pour étudier et quantifier l'expression des récepteurs CB1 dans la corne dorsale de la moelle épinière chez différentes espèces, nous avons perfectionné les procédures d'immunohistochimie pour obtenir des résultats de coloration réussis sur des échantillons de tissus provenant de rats et d'êtres humains, ainsi que des images confocales. Les résultats immunohistochimiques ont été complétés par l'analyse de l'expression du gène CB1 (CNR1) dans la corne dorsale des rongeurs et des humains en utilisant des ensembles de données de séquençage d'ARN au niveau des cellules uniques et des noyaux.Résultats: Nous avons constaté que le CB1 était principalement localisé dans le neuropile au sein de la corne dorsale superficielle chez les rats et les humains, avec une coloration somatique du CB1 dans les différentes couches de la corne dorsale. Chez les deux espèces, l'immunoréactivité du récepteur CB1 était significativement plus élevée dans la couche superficielle de la corne dorsale par rapport aux couches plus profondes, indépendamment du sexe. De manière intéressante, nous avons constaté que l'immunoréactivité du CB1 n'était pas principalement localisée dans les afférences peptidergiques chez les rats et les humains. De plus, nous avons observé une forte expression du gène CNR1 (CB1), mais pas du CNR2 (CB2), au sein de sous-populations de neurones de la corne dorsale chez les rongeurs et les êtres humains.Conclusions: La localisation privilégiée et constante des récepteurs CB1 dans la couche superficielle de la corne dorsale chez les rongeurs et les humains, quel que soit leur sexe, revêt une importance majeure pour la compréhension des fonctions de ce récepteur des cannabinoïdes dans les mécanismes médullaires de la nociception et de l'analgésie.

Advances and Barriers in Understanding Presynaptic N-Methyl-D-Aspartate Receptors in Spinal Pain Processing.

For decades, N-methyl-D-aspartate (NMDA) receptors have been known to play a critical role in the modulation of both acute and chronic pain. Of particular interest are NMDA receptors expressed in the superficial dorsal horn (SDH) of the spinal cord, which houses the nociceptive processing circuits of the spinal cord. In the SDH, NMDA receptors undergo potentiation and increases in the trafficking of receptors to the synapse, both of which contribute to increases in excitability and plastic increases in nociceptive output from the SDH to the brain. Research efforts have primarily focused on postsynaptic NMDA receptors, despite findings that presynaptic NMDA receptors can undergo similar plastic changes to their postsynaptic counterparts. Recent technological advances have been pivotal in the discovery of mechanisms of plastic changes in presynaptic NMDA receptors within the SDH. Here, we highlight these recent advances in the understanding of presynaptic NMDA receptor physiology and their modulation in models of chronic pain. We discuss the role of specific NMDA receptor subunits in presynaptic membranes of nociceptive afferents and local SDH interneurons, including their modulation across pain modalities. Furthermore, we discuss how barriers such as lack of sex-inclusive research and differences in neurodevelopmental timepoints have complicated investigations into the roles of NMDA receptors in pathological pain states. A more complete understanding of presynaptic NMDA receptor function and modulation across pain states is needed to shed light on potential new therapeutic treatments for chronic pain.

Sexual dimorphism in a neuronal mechanism of spinal hyperexcitability across rodent and human models of pathological pain.

The prevalence and severity of many chronic pain syndromes differ across sex, and recent studies have identified differences in immune signalling within spinal nociceptive circuits as a potential mediator. Although it has been proposed that sex-specific pain mechanisms converge once they reach neurons within the superficial dorsal horn, direct investigations using rodent and human preclinical pain models have been lacking. Here, we discovered that in the Freund's adjuvant in vivo model of inflammatory pain, where both male and female rats display tactile allodynia, a pathological coupling between KCC2-dependent disinhibition and N-methyl-D-aspartate receptor (NMDAR) potentiation within superficial dorsal horn neurons was observed in male but not female rats. Unlike males, the neuroimmune mediator brain-derived neurotrophic factor (BDNF) failed to downregulate inhibitory signalling elements (KCC2 and STEP61) and upregulate excitatory elements (pFyn, GluN2B and pGluN2B) in female rats, resulting in no effect of ex vivo brain-derived neurotrophic factor on synaptic NMDAR responses in female lamina I neurons. Importantly, this sex difference in spinal pain processing was conserved from rodents to humans. As in rodents, ex vivo spinal treatment with BDNF downregulated markers of disinhibition and upregulated markers of facilitated excitation in superficial dorsal horn neurons from male but not female human organ donors. Ovariectomy in female rats recapitulated the male pathological pain neuronal phenotype, with BDNF driving a coupling between disinhibition and NMDAR potentiation in adult lamina I neurons following the prepubescent elimination of sex hormones in females. This discovery of sexual dimorphism in a central neuronal mechanism of chronic pain across species provides a foundational step towards a better understanding and treatment for pain in both sexes.

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.

Loss of STEP61 couples disinhibition to N-methyl-d-aspartate receptor potentiation in rodent and human spinal pain processing.

Dysregulated excitability within the spinal dorsal horn is a critical mediator of chronic pain. In the rodent nerve injury model of neuropathic pain, BDNF-mediated loss of inhibition (disinhibition) gates the potentiation of excitatory GluN2B N-methyl-d-aspartate receptor (NMDAR) responses at lamina I dorsal horn synapses. However, the centrality of this mechanism across pain states and species, as well as the molecular linker involved, remain unknown. Here, we show that KCC2-dependent disinhibition is coupled to increased GluN2B-mediated synaptic NMDAR responses in a rodent model of inflammatory pain, with an associated downregulation of the tyrosine phosphatase STEP61. The decreased activity of STEP61 is both necessary and sufficient to prime subsequent phosphorylation and potentiation of GluN2B NMDAR by BDNF at lamina I synapses. Blocking disinhibition reversed the downregulation of STEP61 as well as inflammation-mediated behavioural hypersensitivity. For the first time, we characterize GluN2B-mediated NMDAR responses at human lamina I synapses and show that a human ex vivo BDNF model of pathological pain processing downregulates KCC2 and STEP61 and upregulates phosphorylated GluN2B at dorsal horn synapses. Our results demonstrate that STEP61 is the molecular brake that is lost following KCC2-dependent disinhibition and that the decrease in STEP61 activity drives the potentiation of excitatory GluN2B NMDAR responses in rodent and human models of pathological pain. The ex vivo human BDNF model may thus form a translational bridge between rodents and humans for identification and validation of novel molecular pain targets.

Potentiation of Synaptic GluN2B NMDAR Currents by Fyn Kinase Is Gated through BDNF-Mediated Disinhibition in Spinal Pain Processing.

In chronic pain states, the neurotrophin brain-derived neurotrophic factor (BDNF) transforms the output of lamina I spinal neurons by decreasing synaptic inhibition. Pain hypersensitivity also depends on N-methyl-D-aspartate receptors (NMDARs) and Src-family kinases, but the locus of NMDAR dysregulation remains unknown. Here, we show that NMDAR-mediated currents at lamina I synapses are potentiated in a peripheral nerve injury model of neuropathic pain. We find that BDNF mediates NMDAR potentiation through activation of TrkB and phosphorylation of the GluN2B subunit by the Src-family kinase Fyn. Surprisingly, we find that Cl--dependent disinhibition is necessary and sufficient to prime potentiation of synaptic NMDARs by BDNF. Thus, we propose that spinal pain amplification is mediated by a feedforward mechanism whereby loss of inhibition gates the increase in synaptic excitation within individual lamina I neurons. Given that neither disinhibition alone nor BDNF-TrkB signaling is sufficient to potentiate NMDARs, we have discovered a form of molecular coincidence detection in lamina I neurons.