Electroacupuncture at different intensities inhibits nociceptive discharges of wide dynamic range neurons in spinal dorsal horn of rats. / ä¸åŒå¼ºåº¦ç”µé’ˆå¯¹å¤§é¼ è„Šé«“èƒŒè§’å¹¿åŠ¨åŠ›ç¥žç»å ƒä¼¤å®³æ€§æ”¾ç”µçš„æŠ‘åˆ¶ä½œç”¨.

OBJECTIVES: To observe the effect of electroacupuncture (EA) at different intensities on nociceptive discharges of wide dynamic range (WDR) neurons in the spinal dorsal horns (DHs) of rats, so as to explore its regulatory characteristics on nociceptive signals at the spinal level. METHODS: A total of 25 male SD rats were used in the present study. A microelectrode array was used to record the discharge activity of WDR neurons in the lumbar spinal DHs of normal rats. After finding the WDR neuron, electrical stimulation (pulse width of 2 ms) was administered to the plantar receptive field (RF) for determining its response component of discharges according to the latency of action potential generation (Aß ï¼»0 to 20 msï¼½, Aδ ï¼»20 to 90 msï¼½, C ï¼»90 to 500 msï¼½ and post-discharge ï¼»500 to 800 msï¼½). High-intensity electrical stimulation was continuously applied to the RF at the paw's plantar surface to induce DHs neuronal windup response. Subsequently, EA stimulation at different intensities (1 mA and 2 mA) was applied to the left "Zusanli"(ST36) at a frequency of 2 Hz/15 Hz for 10 min. The induction of WDR neuronal windup was then repeated under the same conditions. The quantity of nociceptive discharge components and the windup response of WDR neurons before and after EA stimulations at different intensities were compared. RESULTS: Compared to pre-EA, both EA1 mA and EA2 mA significantly reduced the number of Aδ and C component discharges of WDR neurons during stimulation, as well as post-discharge (P<0.01, P<0.001). The inhibitory rate of C component by EA2 mA was significantly higher than that by EA1 mA (P<0.05). Meanwhile, both EA1 mA and EA2 mA attenuated the windup response of WDR neurons (P<0.05, P<0.01), and the effect of EA2 mA was stronger than that of EA1 mA (P<0.05). Further analysis showed that when EA1 mA and EA2 mA respectively applied to both non-receptive field (non-RF) and RF, a significant reduction in the number of Aδ component, C component and post-discharge was observed (P<0.05, P<0.01). EA2 mA at the non-RF and RF demonstrated a significant inhibitory effect on the windup response of WDR neurons (P<0.01, P<0.05), but EA1 mA only at the non-RF showed a significant inhibitory effect on the windup response (P<0.01). CONCLUSIONS: EA can suppress nociceptive discharges of spinal DHs WDR neurons in rats. The inhibitory impact of EA is strongly correlated with the location and intensity of EA stimulation, and EA2 mA has a stronger inhibitory effect than EA1 mA.

Selective inhibition of A-fiber-mediated excitatory transmission underlies the analgesic effects of KCNQ channel opening in the spinal dorsal horn.

Neuronal voltage-gated KCNQ (Kv7) channels, expressed centrally and peripherally, mediate low-threshold and non-inactivating M-currents responsible for the control of tonic excitability of mammalian neurons. Pharmacological opening of KCNQ channels has been reported to generate analgesic effects in animal models of neuropathic pain. Here, we examined the possible involvement of central KCNQ channels in the analgesic effects of retigabine, a KCNQ channel opener. Behaviorally, intraperitoneally applied retigabine exerted analgesic effects on thermal and mechanical hypersensitivity in male mice developing neuropathic pain after partial sciatic nerve ligation, which was antagonized by the KCNQ channel blocker XE991 preadministered intraperitoneally and intrathecally. Intrathecally applied retigabine also exerted analgesic effects that were inhibited by intrathecally injected XE991. We then explored the synaptic mechanisms underlying the analgesic effects of retigabine in the spinal dorsal horn. Whole-cell recordings were made from dorsal horn neurons in spinal slices with attached dorsal roots from adult male mice developing neuropathic pain, and the effects of retigabine on miniature and afferent-evoked postsynaptic currents were examined. Retigabine reduced the amplitude of A-fiber-mediated EPSCs without affecting C-fiber-mediated excitatory synaptic transmission. A-fiber-mediated EPSCs remained unaltered by retigabine in the presence of XE991, consistently with the behavioral findings. The frequency and amplitude of mEPSCs were not affected by retigabine. Thus, opening of KCNQ channels in the central terminals of primary afferent A-fibers inhibits excitatory synaptic transmission in the spinal dorsal horn, most likely contributing to the analgesic effect of retigabine.

Tonic excitation by astrocytic GABA causes neuropathic pain by augmenting neuronal activity and glucose metabolism.

Neuropathic pain is a debilitating condition caused by the hyperexcitability of spinal dorsal horn neurons and is often characterized by allodynia. Although neuron-independent mechanisms of hyperexcitability have been investigated, the contribution of astrocyte-neuron interactions remains unclear. Here, we show evidence of reactive astrocytes and their excessive GABA release in the spinal dorsal horn, which paradoxically leads to the tonic excitation of neighboring neurons in a neuropathic pain model. Using multiple electrophysiological methods, we demonstrated that neuronal hyperexcitability is attributed to both increased astrocytic GABA synthesis via monoamine oxidase B (MAOB) and the depolarized reversal potential of GABA-mediated currents (EGABA) via the downregulation of the neuronal K+/Cl- cotransporter KCC2. Furthermore, longitudinal 2-deoxy-2-[18F]-fluoro-D-glucose microPET imaging demonstrated increased regional glucose metabolism in the ipsilateral dorsal horn, reflecting neuronal hyperexcitability. Importantly, inhibiting MAOB restored the entire astrocytic GABA-mediated cascade and abrogated the increased glucose metabolism and mechanical allodynia. Overall, astrocytic GABA-mediated tonic excitation is critical for neuronal hyperexcitability, leading to mechanical allodynia and neuropathic pain.

Actions of remimazolam on inhibitory transmission of rat spinal dorsal horn neurons.

Remimazolam is an ultra-short benzodiazepine that acts on the benzodiazepine site of γ-aminobutyric acid (GABA) receptors in the brain and induces sedation. Although GABA receptors are found localized in the spinal dorsal horn, no previous studies have reported the analgesic effects or investigated the cellular mechanisms of remimazolam on the spinal dorsal horn. Behavioral measures, immunohistochemistry, and in vitro whole-cell patch-clamp recordings of dorsal horn neurons were used to assess synaptic transmission. Intrathecal injection of remimazolam induced behavioral analgesia in inflammatory pain-induced mechanical allodynia (six rats/dose; p < 0.05). Immunohistochemical staining revealed that remimazolam suppressed spinal phosphorylated extracellular signal-regulated kinase activation (five rats/group, p < 0.05). In vitro whole-cell patch-clamp analysis demonstrated that remimazolam increased the frequency of GABAergic miniature inhibitory post-synaptic currents, prolonged the decay time (six rats; p < 0.05), and enhanced GABA currents induced by exogenous GABA (seven rats; p < 0.01). However, remimazolam did not affect miniature excitatory post-synaptic currents or amplitude of monosynaptic excitatory post-synaptic currents evoked by Aδ- and C-fiber stimulation (seven rats; p > 0.05). This study suggests that remimazolam induces analgesia by enhancing GABAergic inhibitory transmission in the spinal dorsal horn, suggesting its potential utility as a spinal analgesic for inflammatory pain.

Development and characterization of a Gucy2d-cre mouse to selectively manipulate a subset of inhibitory spinal dorsal horn interneurons.

Recent transcriptomic studies identified Gucy2d (encoding guanylate cyclase D) as a highly enriched gene within inhibitory dynorphin interneurons in the mouse spinal dorsal horn. To facilitate investigations into the role of the Gucy2d+ population in somatosensation, Gucy2d-cre transgenic mice were created to permit chemogenetic or optogenetic manipulation of this subset of spinal neurons. Gucy2d-cre mice created via CRISPR/Cas9 genomic knock-in were bred to mice expressing a cre-dependent reporter (either tdTomato or Sun1.GFP fusion protein), and the resulting offspring were characterized. Surprisingly, a much wider population of spinal neurons was labeled by cre-dependent reporter expression than previous mRNA-based studies would suggest. Although the cre-dependent reporter expression faithfully labeled ~75% of cells expressing Gucy2d mRNA in the adult dorsal horn, it also labeled a substantial number of additional inhibitory neurons in which no Gucy2d or Pdyn mRNA was detected. Moreover, cre-dependent reporter was also expressed in various regions of the brain, including the spinal trigeminal nucleus, cerebellum, thalamus, somatosensory cortex, and anterior cingulate cortex. Injection of AAV-CAG-FLEX-tdTomato viral vector into adult Gucy2d-cre mice produced a similar pattern of cre-dependent reporter expression in the spinal cord and brain, which excludes the possibility that the unexpected reporter-labeling of cells in the deep dorsal horn and brain was due to transient Gucy2d expression during early stages of development. Collectively, these results suggest that Gucy2d is expressed in a wider population of cells than previously thought, albeit at levels low enough to avoid detection with commonly used mRNA-based assays. Therefore, it is unlikely that these Gucy2d-cre mice will permit selective manipulation of inhibitory signaling mediated by spinal dynorphin interneurons, but this novel cre driver line may nevertheless be useful to target a broader population of inhibitory spinal dorsal horn neurons.

Multimodal sensory control of motor performance by glycinergic interneurons of the mouse spinal cord deep dorsal horn.

Sensory feedback is integral for contextually appropriate motor output, yet the neural circuits responsible remain elusive. Here, we pinpoint the medial deep dorsal horn of the mouse spinal cord as a convergence point for proprioceptive and cutaneous input. Within this region, we identify a population of tonically active glycinergic inhibitory neurons expressing parvalbumin. Using anatomy and electrophysiology, we demonstrate that deep dorsal horn parvalbumin-expressing interneuron (dPV) activity is shaped by convergent proprioceptive, cutaneous, and descending input. Selectively targeting spinal dPVs, we reveal their widespread ipsilateral inhibition onto pre-motor and motor networks and demonstrate their role in gating sensory-evoked muscle activity using electromyography (EMG) recordings. dPV ablation altered limb kinematics and step-cycle timing during treadmill locomotion and reduced the transitions between sub-movements during spontaneous behavior. These findings reveal a circuit basis by which sensory convergence onto dorsal horn inhibitory neurons modulates motor output to facilitate smooth movement and context-appropriate transitions.

Neural Circuitry Polarization in the Spinal Dorsal Horn (SDH): A Novel Form of Dysregulated Circuitry Plasticity during Pain Pathogenesis.

Pathological pain emerges from nociceptive system dysfunction, resulting in heightened pain circuit activity. Various forms of circuitry plasticity, such as central sensitization, synaptic plasticity, homeostatic plasticity, and excitation/inhibition balance, contribute to the malfunction of neural circuits during pain pathogenesis. Recently, a new form of plasticity in the spinal dorsal horn (SDH), named neural circuit polarization (NCP), was discovered in pain models induced by HIV-1 gp120 and chronic morphine administration. NCP manifests as an increase in excitatory postsynaptic currents (EPSCs) in excitatory neurons and a decrease in EPSCs in inhibitory neurons, presumably facilitating hyperactivation of pain circuits. The expression of NCP is associated with astrogliosis. Ablation of reactive astrocytes or suppression of astrogliosis blocks NCP and, concomitantly, the development of gp120- or morphine-induced pain. In this review, we aim to compare and integrate NCP with other forms of plasticity in pain circuits to improve the understanding of the pathogenic contribution of NCP and its cooperation with other forms of circuitry plasticity during the development of pathological pain.

Methylglyoxal activates transient receptor potential A1/V1 via reactive oxygen species in the spinal dorsal horn.

Methylglyoxal (MGO), a highly reactive dicarbonyl metabolite of glucose primarily formed during the glycolytic pathway, is a precursor of advanced glycation end-products (AGEs). Recently, numerous studies have shown that MGO accumulation can cause pain and hyperalgesia. However, the mechanism through which MGO induces pain in the spinal dorsal horn remains unclear. The present study investigated the effect of MGO on spontaneous excitatory postsynaptic currents (sEPSC) in rat spinal dorsal horn neurons using blind whole-cell patch-clamp recording. Perfusion of MGO increased the frequency and amplitude of sEPSC in spinal horn neurons in a concentration-dependent manner. Additionally, MGO administration increased the number of miniature EPSC (mEPSC) in the presence of tetrodotoxin, a sodium channel blocker. However, 6-cyano-7-nitroqiunocaline-2,3-dione (CNQX), an AMPA/kainate receptor antagonist, blocked the enhancement of sEPSC by MGO. HC-030031, a TRP ankyrin-1 (TRPA1) antagonist, and capsazepine, a TRP vanilloid-1 (TRPV1) antagonist, inhibited the action of MGO. Notably, the effects of MGO were completely inhibited by HC-030031 and capsazepine. MGO generates reactive oxygen species (ROS) via AGEs. ROS also potentially induce pain via TRPA1 and TRPV1 in the spinal dorsal horn. Furthermore, we examined the effect of MGO in the presence of N-tert-butyl-α-phenylnitrone (PBN), a non-selective ROS scavenger, and found that the effect of MGO was completely inhibited. These results suggest that MGO increases spontaneous glutamate release from the presynaptic terminal to spinal dorsal horn neurons through TRPA1, TRPV1, and ROS and could enhance excitatory synaptic transmission.

BDNF in Neuropathic Pain; the Culprit that Cannot be Apprehended.

In males but not in females, brain derived neurotrophic factor (BDNF) plays an obligatory role in the onset and maintenance of neuropathic pain. Afferent terminals of injured peripheral nerves release colony stimulating factor (CSF-1) and other mediators into the dorsal horn. These transform the phenotype of dorsal horn microglia such that they express P2X4 purinoceptors. Activation of these receptors by neuron-derived ATP promotes BDNF release. This microglial-derived BDNF increases synaptic activation of excitatory dorsal horn neurons and decreases that of inhibitory neurons. It also alters the neuronal chloride gradient such the normal inhibitory effect of GABA is converted to excitation. By as yet undefined processes, this attenuated inhibition increases NMDA receptor function. BDNF also promotes the release of pro-inflammatory cytokines from astrocytes. All of these actions culminate in the increase dorsal horn excitability that underlies many forms of neuropathic pain. Peripheral nerve injury also alters excitability of structures in the thalamus, cortex and mesolimbic system that are responsible for pain perception and for the generation of co-morbidities such as anxiety and depression. The weight of evidence from male rodents suggests that this preferential modulation of excitably of supra-spinal pain processing structures also involves the action of microglial-derived BDNF. Possible mechanisms promoting the preferential release of BDNF in pain signaling structures are discussed. In females, invading T-lymphocytes increase dorsal horn excitability but it remains to be determined whether similar processes operate in supra-spinal structures. Despite its ubiquitous role in pain aetiology neither BDNF nor TrkB receptors represent potential therapeutic targets.

Novel aspects of signal processing in lamina I.

The most superficial layer of the spinal dorsal horn, lamina I, is a key element of the nociceptive processing system. It contains different types of projection neurons (PNs) and local-circuit neurons (LCNs) whose functional roles in the signal processing are poorly understood. This article reviews recent progress in elucidating novel anatomical features and physiological properties of lamina I PNs and LCNs revealed by whole-cell recordings in ex vivo spinal cord. This article is part of the Special Issue on "Ukrainian Neuroscience".

Nerve injury disrupts temporal processing in the spinal cord dorsal horn through alterations in PV+ interneurons.

How mechanical allodynia following nerve injury is encoded in patterns of neural activity in the spinal cord dorsal horn (DH) remains incompletely understood. We address this in mice using the spared nerve injury model of neuropathic pain and in vivo electrophysiological recordings. Surprisingly, despite dramatic behavioral over-reactivity to mechanical stimuli following nerve injury, an overall increase in sensitivity or reactivity of DH neurons is not observed. We do, however, observe a marked decrease in correlated neural firing patterns, including the synchrony of mechanical stimulus-evoked firing, across the DH. Alterations in DH temporal firing patterns are recapitulated by silencing DH parvalbumin+ (PV+) interneurons, previously implicated in mechanical allodynia, as are allodynic pain-like behaviors. These findings reveal decorrelated DH network activity, driven by alterations in PV+ interneurons, as a prominent feature of neuropathic pain and suggest restoration of proper temporal activity as a potential therapeutic strategy to treat chronic neuropathic pain.

DNA N6-methyladenine methylase N6AMT1 controls neuropathic pain through epigenetically modifying Kcnj16 in dorsal horn neurons.

ABSTRACT: Nerve injury-induced aberrant changes in gene expression in spinal dorsal horn neurons are critical for the genesis of neuropathic pain. N6-methyladenine (m 6 A) modification of DNA represents an additional layer of gene regulation. Here, we report that peripheral nerve injury significantly decreased the level of m 6 A-specific DNA methyltransferase 1 ( N6amt1 ) in dorsal horn neurons. This decrease was attributed, at least partly, to a reduction in transcription factor Nr2f6 . Rescuing the decrease in N6amt1 reversed the loss of m 6 A at the promoter for inwardly rectifying potassium channel subfamily J member 16 ( Kcnj16 ), mitigating the nerve injury-induced upregulation of Kcnj16 expression in the dorsal horn and alleviating neuropathic pain hypersensitivities. Conversely, mimicking the downregulation of N6amt1 in naive mice erased DNA m 6 A at the Kcnj16 promoter, elevated Kcnj16 expression, and led to neuropathic pain-like behaviors. Therefore, decreased N6amt1 caused by NR2F6 is required for neuropathic pain, likely through its regulation of m 6 A-controlled KCNJ16 in dorsal horn neurons, suggesting that DNA m 6 A modification may be a potential new target for analgesic and treatment strategies.

Upregulation of Spinal MDGA1 in Rats After Nerve Injury Alters Interactions Between Neuroligin-2 and Postsynaptic Scaffolding Proteins and Increases GluR1 Subunit Surface Delivery in the Spinal Cord Dorsal Horn.

Previous studies suggested that postsynaptic neuroligin-2 may shift from inhibitory toward excitatory function under pathological pain conditions. We hypothesize that nerve injury may increase the expression of spinal MAM-domain GPI-anchored molecule 1 (MDGA1), which can bind to neuroligin-2 and thereby, alter its interactions with postsynaptic scaffolding proteins and increase spinal excitatory synaptic transmission, leading to neuropathic pain. Western blot, immunofluorescence staining, and co-immunoprecipitation studies were conducted to examine the critical role of MDGA1 in the lumbar spinal cord dorsal horn in rats after spinal nerve ligation (SNL). Small interfering ribonucleic acids (siRNAs) targeting MDGA1 were used to examine the functional roles of MDGA1 in neuropathic pain. Protein levels of MDGA1 in the ipsilateral dorsal horn were significantly upregulated at day 7 post-SNL, as compared to that in naïve or sham rats. The increased levels of GluR1 in the synaptosomal membrane fraction of the ipsilateral dorsal horn tissues at day 7 post-SNL was normalized to near sham level by pretreatment with intrathecal MDGA1 siRNA2308, but not scrambled siRNA or vehicle. Notably, knocking down MDGA1 with siRNAs reduced the mechanical and thermal pain hypersensitivities, and inhibited the increased excitatory synaptic interaction between neuroligin-2 with PSD-95, and prevented the decreased inhibitory postsynaptic interactions between neuroligin-2 and Gephyrin. Our findings suggest that SNL upregulated MDGA1 expression in the dorsal horn, which contributes to the pain hypersensitivity through increasing the net excitatory interaction mediated by neuroligin-2 and surface delivery of GluR1 subunit in dorsal horn neurons.

Microglial Refinement of A-Fiber Projections in the Postnatal Spinal Cord Dorsal Horn Is Required for Normal Maturation of Dynamic Touch.

Sensory systems are shaped in postnatal life by the refinement of synaptic connectivity. In the dorsal horn of the spinal cord, somatosensory circuits undergo postnatal activity-dependent reorganization, including the refinement of primary afferent A-fiber terminals from superficial to deeper spinal dorsal horn laminae which is accompanied by decreases in cutaneous sensitivity. Here, we show in the mouse that microglia, the resident immune cells in the CNS, phagocytose A-fiber terminals in superficial laminae in the first weeks of life. Genetic perturbation of microglial engulfment during the initial postnatal period in either sex prevents the normal process of A-fiber refinement and elimination, resulting in an altered sensitivity of dorsal horn cells to dynamic tactile cutaneous stimulation, and behavioral hypersensitivity to dynamic touch. Thus, functional microglia are necessary for the normal postnatal development of dorsal horn sensory circuits. In the absence of microglial engulfment, superfluous A-fiber projections remain in the dorsal horn, and the balance of sensory connectivity is disrupted, leading to lifelong hypersensitivity to dynamic touch.

Constitutive KCC2 Cell- and Synapse-Specifically Regulates NMDA Receptor Activity in the Spinal Cord.

K+-Cl- cotransporter-2 (KCC2) critically controls neuronal chloride homeostasis and maintains normal synaptic inhibition by GABA and glycine. Nerve injury diminishes synaptic inhibition in the spinal cord via KCC2 impairment. However, how KCC2 regulates nociceptive input to spinal excitatory and inhibitory neurons remains elusive. Here, we show that basal GABA reversal potentials were significantly more depolarized in vesicular GABA transporter (VGAT)-expressing inhibitory neurons than those in vesicular glutamate transporter-2 (VGluT2)-expressing excitatory neurons in spinal cords of male and female mice. Strikingly, inhibiting KCC2 with VU0463271 increased currents elicited by puff NMDA and the NMDAR-mediated frequency of mEPSCs in VGluT2, but not in VGAT, dorsal horn neurons. Notably, VU0463271 had no effect on EPSCs monosynaptically evoked from the dorsal root in VGluT2 neurons. Furthermore, VU0463271 augmented α2δ-1-NMDAR interactions and their protein levels in spinal cord synaptosomes. In Cacna2d1 KO mice, VU0463271 had no effect on puff NMDA currents or the mEPSC frequency in dorsal horn neurons. Disrupting α2δ-1-NMDAR interactions with α2δ-1 C-terminus mimicking peptide diminished VU0463271-induced potentiation in the mEPSC frequency and puff NMDA currents in VGluT2 neurons. Additionally, intrathecal injection of VU0463271 reduced mechanical and thermal thresholds in wild-type mice, but not in Cacna2d1 KO mice. VU0463271-induced pain hypersensitivity in mice was abrogated by co-treatment with the NMDAR antagonist, pregabalin (an α2δ-1 inhibitory ligand), or α2δ-1 C-terminus mimicking peptide. Our findings suggest that KCC2 controls presynaptic and postsynaptic NMDAR activity specifically in excitatory dorsal horn neurons. KCC2 impairment preferentially potentiates nociceptive transmission between spinal excitatory interneurons via α2δ-1-bound NMDARs.Significance statementImpaired function of potassium-chloride cotransporter-2 (KCC2), a key regulator of neuronal inhibition, in the spinal cord plays a major role in neuropathic pain. This study unveils that KCC2 controls spinal nociceptive synaptic strength via NMDA receptors in a cell type- and synapse type-specific manner. KCC2 inhibition preferentially augments presynaptic and postsynaptic NMDA receptor activity in spinal excitatory interneurons via α2δ-1 (previously known as a calcium channel subunit). Importantly, spinal KCC2 impairment triggers pain hypersensitivity through α2δ-1-coupled NMDA receptors. These findings pinpoint the cell and molecular substrates for the reciprocal relationship between spinal synaptic inhibition and excitation in chronic neuropathic pain. Targeting both KCC2 and α2δ-1­NMDA receptor complexes could be an effective strategy in managing neuropathic pain conditions.

Characterization of synaptic structural plasticity in mouse spinal dorsal horn neurons.

Here, we present a pipeline for the characterization of synaptic structural plasticity in mouse spinal dorsal horn (SDH) neurons. We describe steps for the intra-SDH microinjection of the EGFP virus to sparsely label L4 SDH neurons without laminectomy, wide dynamic range neuron imaging, dendritic spine morphometric analysis, and F-actin to G-actin ratio measurement. This protocol can be applied to investigate the synaptic structural plasticity mechanisms in the SDH as well as in the brain. For complete details on the use and execution of this protocol, please refer to Li et al. (2023).1.

Acute inhibition of acid sensing ion channel 1a after spinal cord injury selectively affects excitatory synaptic transmission, but not intrinsic membrane properties, in deep dorsal horn interneurons.

Following a spinal cord injury (SCI), secondary damage mechanisms are triggered that cause inflammation and cell death. A key component of this secondary damage is a reduction in local blood flow that initiates a well-characterised ischemic cascade. Downstream hypoxia and acidosis activate acid sensing ion channel 1a (ASIC1a) to trigger cell death. We recently showed that administration of a potent venom-derived inhibitor of ASIC1a, Hi1a, leads to tissue sparing and improved functional recovery when delivered up to 8 h after ischemic stroke. Here, we use whole-cell patch-clamp electrophysiology in a spinal cord slice preparation to assess the effect of acute ASIC1a inhibition, via a single dose of Hi1a, on intrinsic membrane properties and excitatory synaptic transmission long-term after a spinal cord hemisection injury. We focus on a population of interneurons (INs) in the deep dorsal horn (DDH) that play a key role in relaying sensory information to downstream motoneurons. DDH INs in mice treated with Hi1a 1 h after a spinal cord hemisection showed no change in active or passive intrinsic membrane properties measured 4 weeks after SCI. DDH INs, however, exhibit significant changes in the kinetics of spontaneous excitatory postsynaptic currents after a single dose of Hi1a, when compared to naive animals (unlike SCI mice). Our data suggest that acute ASIC1a inhibition exerts selective effects on excitatory synaptic transmission in DDH INs after SCI via specific ligand-gated receptor channels, and has no effect on other voltage-activated channels long-term after SCI.

miR-199a-3p mediates bone cancer pain through upregulation of dnmt3a expression in spinal dorsal horn neurons.

Due to its complex pathological mechanisms, bone cancer pain (BCP) has become an increasingly challenging clinical issue, there is an urgent need to identify the underlying mechanisms of BCP. In our present study, we found that decreased expression of miR-199a-3p in spinal dorsal horn (SDH) neurons contributed to BCP hypersensitivity. Intrathecal administration of miR-199a-3p agomir alleviated the initiation of tumor inoculation induced pain hypersensitivity and suppressed the expression of DNMT3A. Subsequently, luciferase assays confirmed direct binding between miR-199a-3p and Dnmt3a mRNA. AAV-DNMT3A-shRNA microinjection relieved mechanical hyperalgesia and upregulated the expression of Nrf2 levels in BCP. In naïve rats, Overexpression of DNMT3A yielded the opposite effects. Finally, increase of DNMT3A by lentiviral vector abolished miR-199a-3p-mediated alleviation hypersensitivity effects on BCP progression. Taken these together, our findings highlighted a novel contribution of miR-199a-3p to BCP and provided a fresh outlook on potential mechanism research for BCP.

Network model of nociceptive processing in the superficial spinal dorsal horn reveals mechanisms of hyperalgesia, allodynia, and spinal cord stimulation.

The spinal dorsal horn (DH) processes sensory information and plays a key role in transmitting nociception to supraspinal centers. Loss of DH inhibition during neuropathic pain unmasks a pathway from nonnociceptive Aß-afferent inputs to superficial dorsal horn (SDH) nociceptive-specific (NS) projection neurons, and this change may contribute to hyperalgesia and allodynia. We developed and validated a computational model of SDH neuronal circuitry that links nonnociceptive Aß-afferent inputs in lamina II/III to a NS projection neuron in lamina I via a network of excitatory interneurons. The excitatory pathway and the NS projection neuron were in turn gated by inhibitory interneurons with connections based on prior patch-clamp recordings. Changing synaptic weights in the computational model to replicate neuropathic pain states unmasked a low-threshold excitatory pathway to NS neurons similar to experimental recordings. Spinal cord stimulation (SCS) is an effective therapy for neuropathic pain, and accumulating experimental evidence indicates that NS neurons in the SDH also respond to SCS. Accounting for these responses may inform therapeutic improvements, and we quantified responses to SCS in the SDH network model and examined the role of different modes of inhibitory control in modulating NS neuron responses to SCS. We combined the SDH network model with a previously published model of the deep dorsal horn (DDH) and identified optimal stimulation frequencies across different neuropathic pain conditions. Finally, we found that SCS-generated inhibition did not completely suppress model NS activity during simulated pinch inputs, providing an explanation of why SCS does not eliminate acute pain.NEW & NOTEWORTHY Chronic pain is a severe public health problem that reduces the quality of life for those affected and exacts an enormous socio-economic burden worldwide. Spinal cord stimulation (SCS) is an effective treatment for chronic pain, but SCS efficacy has not significantly improved over time, in part because the mechanisms of action remain unclear. Most preclinical studies investigating pain and SCS mechanisms have focused on the responses of deep dorsal horn (DDH) neurons, but neural networks in the superficial dorsal horn (SDH) are also important for processing nociceptive information. This work synthesizes heterogeneous experimental recordings from the SDH into a computational model that replicates experimental responses and that can be used to quantify neuronal responses to SCS under neuropathic pain conditions.

Brn3a controls the soma localization and axonal extension patterns of developing spinal dorsal horn neurons.

The spinal dorsal horn comprises heterogeneous neuronal populations, that interconnect with one another to form neural circuits modulating various types of sensory information. Decades of evidence has revealed that transcription factors expressed in each neuronal progenitor subclass play pivotal roles in the cell fate specification of spinal dorsal horn neurons. However, the development of subtypes of these neurons is not fully understood in more detail as yet and warrants the investigation of additional transcription factors. In the present study, we examined the involvement of the POU domain-containing transcription factor Brn3a in the development of spinal dorsal horn neurons. Analyses of Brn3a expression in the developing spinal dorsal horn neurons in mice demonstrated that the majority of the Brn3a-lineage neurons ceased Brn3a expression during embryonic stages (Brn3a-transient neurons), whereas a limited population of them continued to express Brn3a at high levels after E18.5 (Brn3a-persistent neurons). Loss of Brn3a disrupted the localization pattern of Brn3a-persistent neurons, indicating a critical role of this transcription factor in the development of these neurons. In contrast, Brn3a overexpression in Brn3a-transient neurons directed their localization in a manner similar to that in Brn3a-persistent neurons. Moreover, Brn3a-overexpressing neurons exhibited increased axonal extension to the ventral and ventrolateral funiculi, where the axonal tracts of Brn3a-persistent neurons reside. These results suggest that Brn3a controls the soma localization and axonal extension patterns of Brn3a-persistent spinal dorsal horn neurons.