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.

The Role of Voltage-Gated Calcium Channels in Basal Ganglia Neurodegenerative Disorders.

Calcium (Ca2+) plays a central role in regulating many cellular processes and influences cell survival. Several mechanisms can disrupt Ca2+ homeostasis to trigger cell death, including oxidative stress, mitochondrial damage, excitotoxicity, neuroinflammation, autophagy, and apoptosis. Voltage-gated Ca2+ channels (VGCCs) act as the main source of Ca2+ entry into electrically excitable cells, such as neurons, and they are also expressed in glial cells such as astrocytes and oligodendrocytes. The dysregulation of VGCC activity has been reported in both Parkinson's disease (PD) and Huntington's (HD). PD and HD are progressive neurodegenerative disorders (NDs) of the basal ganglia characterized by motor impairment as well as cognitive and psychiatric dysfunctions. This review will examine the putative role of neuronal VGCCs in the pathogenesis and treatment of central movement disorders, focusing on PD and HD. The link between basal ganglia disorders and VGCC physiology will provide a framework for understanding the neurodegenerative processes that occur in PD and HD, as well as a possible path towards identifying new therapeutic targets for the treatment of these debilitating disorders.

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.

South-to-south mentoring as a vehicle for implementing sustainable health security in Africa.

BACKGROUND: While sustainability has become a universal precept in the development of global health security systems, supporting policies often lack mechanisms to drive policies into regular practice. 'On-paper' norms and regulations are to a great extent upheld by frontline workers who often lack the opportunity to communicate their first-hand experiences to decisionmakers; their role is an often overlooked, yet crucial, aspect of a sustainable global health security landscape. Initiatives and programs developing transdisciplinary professional skills support the increased bidirectional dialogue between these frontline workers and key policy- and decisionmakers which may sustainably narrow the gap between global health security policy design and implementation. METHODS: The International Federation of Biosafety Associations' (IFBA) Global Mentorship Program recruits biosafety and biosecurity champions across Africa to provide local peer mentorship to developing professionals in their geographic region. Mentors and mentees complete structured one year program cycles, where they are provided with written overviews of monthly discussion topics, and attend optional virtual interactive activities. Feedback from African participants of the 2019-2020 program cycle was collected using a virtual Exit Survey, where aspects of program impact and structure were assessed. RESULTS: Following its initial call for applications, the IFBA Global Mentorship Program received considerable interest from professionals across the African continent, particularly in East and North Africa. The pilot program cycle matched a total of 62 individuals from an array of professional disciplines across several regions, 40 of which were located on the African continent. The resulting mentorship pairs shared knowledge, skills, and experiences towards translating policy objectives to action on the front lines. Mentorship pairs embraced multidisciplinary approaches to harmonize health security strategies across the human and animal health sectors. South-to-South mentorship therefore provided mentees with locally relevant support critical to translation of best technical practices to local capacity and work. CONCLUSION: The IFBA's South-to-South Global Mentorship Program has demonstrated its ability to form crucial links between frontline biosafety professionals, laboratory workers, and policy- and decision-makers across several implicated sectors. By supporting regionally relevant peer mentorship programs, the gap between health security policy development and implementation may be narrowed.

Ion Channels and Electrophysiological Properties of Astrocytes: Implications for Emergent Stimulation Technologies.

Astrocytes comprise a heterogeneous cell population characterized by distinct morphologies, protein expression and function. Unlike neurons, astrocytes do not generate action potentials, however, they are electrically dynamic cells with extensive electrophysiological heterogeneity and diversity. Astrocytes are hyperpolarized cells with low membrane resistance. They are heavily involved in the modulation of K+ and express an array of different voltage-dependent and voltage-independent channels to help with this ion regulation. In addition to these K+ channels, astrocytes also express several different types of Na+ channels; intracellular Na+ signaling in astrocytes has been linked to some of their functional properties. The physiological hallmark of astrocytes is their extensive intracellular Ca2+ signaling cascades, which vary at the regional, subregional, and cellular levels. In this review article, we highlight the physiological properties of astrocytes and the implications for their function and influence of network and synaptic activity. Furthermore, we discuss the implications of these differences in the context of optogenetic and DREADD experiments and consider whether these tools represent physiologically relevant techniques for the interrogation of astrocyte function.

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.

Differential expression of GluN2 NMDA receptor subunits in the dorsal horn of male and female rats.

N-methyl-D-aspartate receptors (NMDARs) are excitatory ionotropic glutamate receptors expressed throughout the CNS, including in the spinal dorsal horn. The GluN2 subtypes of NMDAR subunit, which include GluN2A, GluN2B, and GluN2D in the dorsal horn, confer NMDARs with structural and functional variability, enabling heterogeneity in synaptic transmission and plasticity. Despite essential roles for NMDARs in physiological and pathological pain processing, the distribution and function of these specific GluN2 isoforms across dorsal horn laminae remain poorly understood. Surprisingly, there is a complete lack of knowledge of GluN2 expression in female rodents. We, therefore, investigated the relative expression of specific GluN2 variants in the dorsal horn of lumbar (L4/L5) spinal cord from both male and female rats. In order to detect synaptic GluN2 isoforms, we used pepsin antigen-retrieval to unmask these highly cross-linked protein complexes. We found that GluN2B and GluN2D are preferentially localized to the pain-processing superficial regions of the dorsal horn in males, while only GluN2B is predominantly localized to the superficial dorsal horn of female rats. The GluN2A subunit is diffusely localized to neuropil throughout the dorsal horn of both males and females, while GluN2B and GluN2D immunolabelling are found both in the neuropil and on the soma of dorsal horn neurons. Finally, we identified an unexpected enhanced expression of GluN2B in the medial division of the superficial dorsal horn, but in males only. These sex-specific localization patterns of GluN2-NMDAR subunits across dorsal horn laminae have significant implications for the understanding of divergent spinal mechanisms of pain processing.

Conserved contributions of NMDA receptor subtypes to synaptic responses in lamina II spinal neurons across early postnatal development.

NMDA receptors are heteromeric complexes that contribute to excitatory synaptic transmission and plasticity. The presence of specific variants of GluN2 subunits in these complexes enables diversity in NMDA receptor function and regulation. At brain synapses, there is a switch from slow GluN2B-mediated NMDA receptors to faster GluN2A-dominated NMDA receptors as well as an increase in the ratio of AMPA to NMDA receptors during early postnatal development. This glutamate receptor switch is observed across brain regions and is critical for synaptic maturation, circuit development, and associative learning. However, whether a similar receptor subunit switch occurs within pain processing neurons in the developing spinal cord remains untested. To investigate this, we performed whole-cell patch clamp recordings of excitatory synaptic responses from lamina II dorsal horn neurons of one to three week-old rats. We found that GluN2B and GluN2A both prominently contribute to NMDA receptor responses at neonatal lamina II synapses, with a small contribution from GluN2D as well. Surprisingly, we found that this molecular identity of NMDA receptor responses as well as the relative contribution of AMPA receptors versus NMDA receptors did not change at lamina II synapses across early postnatal development (P7 to P21). The lack of a developmental switch and persistence of slow-decaying GluN2B- and GluN2D-mediated synaptic responses throughout neuronal maturation in the dorsal horn has implications for understanding both the regulation of synaptic glutamatergic receptors as well as spinal mechanisms of pain processing.

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.

GluN2B and GluN2D NMDARs dominate synaptic responses in the adult spinal cord.

The composition of the postsynaptic ionotropic receptors that receive presynaptically released transmitter is critical not only for transducing and integrating electrical signals but also for coordinating downstream biochemical signaling pathways. At glutamatergic synapses in the adult CNS an overwhelming body of evidence indicates that the NMDA receptor (NMDAR) component of synaptic responses is dominated by NMDARs containing the GluN2A subunit, while NMDARs containing GluN2B, GluN2C, or GluN2D play minor roles in synaptic transmission. Here, we discovered NMDAR-mediated synaptic responses with characteristics not described elsewhere in the adult CNS. We found that GluN2A-containing receptors contribute little to synaptic NMDAR responses while GluN2B dominates at synapses of lamina I neurons in the adult spinal cord. In addition, we provide evidence for a GluN2D-mediated synaptic NMDAR component in adult lamina I neurons. Strikingly, the charge transfer mediated by GluN2D far exceeds that of GluN2A and is comparable to that of GluN2B. Lamina I forms a distinct output pathway from the spinal pain processing network to the pain networks in the brain. The GluN2D-mediated synaptic responses we have discovered in lamina I neurons provide the molecular underpinning for slow, prolonged and feedforward amplification that is a fundamental characteristic of pain.

Calcium-permeable ion channels in pain signaling.

The detection and processing of painful stimuli in afferent sensory neurons is critically dependent on a wide range of different types of voltage- and ligand-gated ion channels, including sodium, calcium, and TRP channels, to name a few. The functions of these channels include the detection of mechanical and chemical insults, the generation of action potentials and regulation of neuronal firing patterns, the initiation of neurotransmitter release at dorsal horn synapses, and the ensuing activation of spinal cord neurons that project to pain centers in the brain. Long-term changes in ion channel expression and function are thought to contribute to chronic pain states. Many of the channels involved in the afferent pain pathway are permeable to calcium ions, suggesting a role in cell signaling beyond the mere generation of electrical activity. In this article, we provide a broad overview of different calcium-permeable ion channels in the afferent pain pathway and their role in pain pathophysiology.

Contributions of T-type voltage-gated calcium channels to postsynaptic calcium signaling within Purkinje neurons.

Low threshold voltage-gated T-type calcium channels have long been implicated in the electrical excitability and calcium signaling of cerebellar Purkinje neurons although the molecular composition, localization, and modulation of T-type channels within Purkinje cells have only recently been addressed. The specific functional roles that T-type channels play in local synaptic integration within Purkinje spines are also currently being unraveled. Overall, Purkinje neurons represent a powerful model system to explore the potential roles of postsynaptic T-type channels throughout the nervous system. In this review, we present an overview of T-type calcium channel biophysical, pharmacological, and physiological characteristics that provides a foundation for understanding T-type channels within Purkinje neurons. We also describe the biophysical properties of T-type channels in context of other voltage-gated calcium channel currents found within Purkinje cells. The data thus far suggest that one specific T-type isoform, Ca(v)3.1, is highly expressed within Purkinje spines and both physically and functionally couples to mGluR1 and other effectors within putative signaling microdomains. Finally, we discuss how the selective potentiation of Ca(v)3.1 channels via activation of mGluR1 by parallel fiber inputs affects local synaptic integration and how this interaction may relate to the overall excitability of Purkinje neuron dendrites.

Identification of sodium channel isoforms that mediate action potential firing in lamina I/II spinal cord neurons.

BACKGROUND: Voltage-gated sodium channels play key roles in acute and chronic pain processing. The molecular, biophysical, and pharmacological properties of sodium channel currents have been extensively studied for peripheral nociceptors while the properties of sodium channel currents in dorsal horn spinal cord neurons remain incompletely understood. Thus far, investigations into the roles of sodium channel function in nociceptive signaling have primarily focused on recombinant channels or peripheral nociceptors. Here, we utilize recordings from lamina I/II neurons withdrawn from the surface of spinal cord slices to systematically determine the functional properties of sodium channels expressed within the superficial dorsal horn. RESULTS: Sodium channel currents within lamina I/II neurons exhibited relatively hyperpolarized voltage-dependent properties and fast kinetics of both inactivation and recovery from inactivation, enabling small changes in neuronal membrane potentials to have large effects on intrinsic excitability. By combining biophysical and pharmacological channel properties with quantitative real-time PCR results, we demonstrate that functional sodium channel currents within lamina I/II neurons are predominantly composed of the NaV1.2 and NaV1.3 isoforms. CONCLUSIONS: Overall, lamina I/II neurons express a unique combination of functional sodium channels that are highly divergent from the sodium channel isoforms found within peripheral nociceptors, creating potentially complementary or distinct ion channel targets for future pain therapeutics.

A novel slow-inactivation-specific ion channel modulator attenuates neuropathic pain.

Voltage-gated ion channels are implicated in pain sensation and transmission signaling mechanisms within both peripheral nociceptors and the spinal cord. Genetic knockdown and knockout experiments have shown that specific channel isoforms, including Na(V)1.7 and Na(V)1.8 sodium channels and Ca(V)3.2 T-type calcium channels, play distinct pronociceptive roles. We have rationally designed and synthesized a novel small organic compound (Z123212) that modulates both recombinant and native sodium and calcium channel currents by selectively stabilizing channels in their slow-inactivated state. Slow inactivation of voltage-gated channels can function as a brake during periods of neuronal hyperexcitability, and Z123212 was found to reduce the excitability of both peripheral nociceptors and lamina I/II spinal cord neurons in a state-dependent manner. In vivo experiments demonstrate that oral administration of Z123212 is efficacious in reversing thermal hyperalgesia and tactile allodynia in the rat spinal nerve ligation model of neuropathic pain and also produces acute antinociception in the hot-plate test. At therapeutically relevant concentrations, Z123212 did not cause significant motor or cardiovascular adverse effects. Taken together, the state-dependent inhibition of sodium and calcium channels in both the peripheral and central pain signaling pathways may provide a synergistic mechanism toward the development of a novel class of pain therapeutics.

Functional coupling between mGluR1 and Cav3.1 T-type calcium channels contributes to parallel fiber-induced fast calcium signaling within Purkinje cell dendritic spines.

T-type voltage-gated calcium channels are expressed in the dendrites of many neurons, although their functional interactions with postsynaptic receptors and contributions to synaptic signaling are not well understood. We combine electrophysiological and ultrafast two-photon calcium imaging to demonstrate that mGluR1 activation potentiates cerebellar Purkinje cell Ca(v)3.1 T-type currents via a G-protein- and tyrosine-phosphatase-dependent pathway. Immunohistochemical and electron microscopic investigations on wild-type and Ca(v)3.1 gene knock-out animals show that Ca(v)3.1 T-type channels are preferentially expressed in Purkinje cell dendritic spines and colocalize with mGluR1s. We further demonstrate that parallel fiber stimulation induces fast subthreshold calcium signaling in dendritic spines and that the synaptic Ca(v)3.1-mediated calcium transients are potentiated by mGluR1 selectively during bursts of excitatory parallel fiber inputs. Our data identify a new fast calcium signaling pathway in Purkinje cell dendritic spines triggered by short burst of parallel fiber inputs and mediated by T-type calcium channels and mGluR1s.