Small Dorsal Root Ganglion Neurons Research Articles

Computational modeling to study the impact of changes in Nav1.8 sodium channel on neuropathic pain.

Nav1.8 expression is restricted to sensory neurons; it was hypothesized that aberrant expression and function of this channel at the site of injury contributed to pathological pain. However, the specific contributions of Nav1.8 to neuropathic pain are not as clear as its role in inflammatory pain. The aim of this study is to understand how Nav1.8 present in peripheral sensory neurons regulate neuronal excitability and induce various electrophysiological features on neuropathic pain. To study the effect of changes in sodium channel Nav1.8 kinetics, Hodgkin-Huxley type conductance-based models of spiking neurons were constructed using the NEURON v8.2 simulation software. We constructed a single-compartment model of neuronal soma that contained Nav1.8 channels with the ionic mechanisms adapted from some existing small DRG neuron models. We then validated and compared the model with our experimental data from in vivo recordings on soma of small dorsal root ganglion (DRG) sensory neurons in animal models of neuropathic pain (NEP). We show that Nav1.8 is an important parameter for the generation and maintenance of abnormal neuronal electrogenesis and hyperexcitability. The typical increased excitability seen is dominated by a left shift in the steady state of activation of this channel and is further modulated by this channel's maximum conductance and steady state of inactivation. Therefore, modified action potential shape, decreased threshold, and increased repetitive firing of sensory neurons in our neuropathic animal models may be orchestrated by these modulations on Nav1.8. Computational modeling is a novel strategy to understand the generation of chronic pain. In this study, we highlight that changes to the channel functions of Nav1.8 within the small DRG neuron may contribute to neuropathic pain.

RdHSV-CA8 non-opioid analgesic gene therapy decreases somatosensory neuronal excitability by activating Kv7 voltage-gated potassium channels.

Chronic pain is common and inadequately treated, making the development of safe and effective analgesics a high priority. Our previous data indicate that carbonic anhydrase-8 (CA8) expression in dorsal root ganglia (DRG) mediates analgesia via inhibition of neuronal ER inositol trisphosphate receptor-1 (ITPR1) via subsequent decrease in ER calcium release and reduction of cytoplasmic free calcium, essential to the regulation of neuronal excitability. This study tested the hypothesis that novel JDNI8 replication-defective herpes simplex-1 viral vectors (rdHSV) carrying a CA8 transgene (vHCA8) reduce primary afferent neuronal excitability. Whole-cell current clamp recordings in small DRG neurons showed that vHCA8 transduction caused prolongation of their afterhyperpolarization (AHP), an essential regulator of neuronal excitability. This AHP prolongation was completely reversed by the specific Kv7 channel inhibitor XE-991. Voltage clamp recordings indicate an effect via Kv7 channels in vHCA8-infected small DRG neurons. These data demonstrate for the first time that vHCA8 produces Kv7 channel activation, which decreases neuronal excitability in nociceptors. This suppression of excitability may translate in vivo as non-opioid dependent behavioral- or clinical analgesia, if proven behaviorally and clinically.

Amyloid β reduces mechanical sensation by attenuating the function of TRPV4 channels in rat small dorsal root ganglion neurons

Amyloid β (Aβ) is well known to be a key trigger for the progression of Alzheimer's disease and dementia. Aβ forms oligomers that develop into amyloid plaques, causing neuronal death and dysfunction. Aβ-induced neuronal dysfunction has been reported in peripheral neurons as well as in the brain. We previously reported that transient receptor potential vanilloid 4 (TRPV4) channels play an important role in mechanical sensation in sensory neurons innervating skeletal muscle. However, to date, it remains unclear whether Aβ modulates mechanosensation or TRPV4 channel activity in peripheral sensory neurons. As such, we hypothesized that Aβ acutely impairs the response of small dorsal root ganglion (DRG) neurons to mechanical stimuli by attenuating the function of TRPV4 channels. Purpose: The aim of this investigation was to determine the effects of Aβ1-40 and 1-42 on mechanosensation in small DRG neurons. Method: In in vitro whole-cell patch clamp recordings from cultured rat DRG neurons (Sprague-Dawley rat, n = 4, body weight: 316 ± 57 g), mechanically-activated (MA) current amplitudes or GSK1016790A (a selective agonist of TRPV4, 1 μM)-induced inward currents were measured before and 5 min after application of either HEPES buffered solution (control), Aβ1-40 (2.5 ng/μL), Aβ1-42 (2.5 ng/μL) or Aβ1-40/42 solution (2.5 ng/μL, respectively). Results: MA current amplitude was significantly decreased 5 min after application of both Aβ1-40 (Before vs. After: -71.7 ± 14.5 vs. -13.5 ± 4.8 pA. P < 0.01) and Aβ1-42 (Before vs. After: -63.7 ± 16.2 vs. -14.5 ± 5.9 pA. P < 0.05) while HEPES application did not alter MA currents (Before vs. After: -69.5 ± 17.9 ± -66.2 ± 13.9 pA. P = 0.36). Likewise, GSK1016790A-induced inward current was significantly suppressed by Aβ1-40/42 solution (Fold change in total charge transfer: HEPES vs. Aβ1-40/42: 0.92 ± 0.47 vs. 0.33 ± 0.06. P < 0.05). Conclusion: The data demonstrate that both Aβ1-40 and 1-42 suppress 1) the cellular response to mechanical stimulation and 2) TRPV4 currents in rat small DRG neurons. These findings suggest that both Aβ1-40 and 1-42 attenuate mechanical sensation in DRG neurons by inhibiting TRPV4 channels. Supported by NIH HL-151632 and JSPS 23KJ2207. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Nav1.8 in small dorsal root ganglion neurons contributes to vincristine-induced mechanical allodynia.

Vincristine-induced peripheral neuropathy (VIPN) is a common side effect of vincristine treatment, which is accompanied by pain and can be dose-limiting. The molecular mechanisms that underlie vincristine-induced pain are not well understood. We have established an animal model to investigate pathophysiological mechanisms of vincristine induced pain. Our previous studies have shown that the tetrodotoxin-sensitive (TTX-S) voltage-gated sodium channel NaV1.6 in medium-diameter dorsal root ganglion (DRG) neurons contributes to the maintenance of vincristine-induced allodynia. In this study, we investigated the effects of vincristine administration on excitability in small-diameter DRG neurons and whether the tetrodotoxin-resistant (TTX-R) NaV1.8 channels contribute to mechanical allodynia. Current-clamp recordings demonstrated that small DRG neurons become hyper-excitable following vincristine treatment, with both reduced current threshold and increased firing frequency. Using voltage-clamp recordings in small DRG neurons we now show an increase in TTX-R current density and a -7.3 mV hyperpolarizing shift in V1/2 of activation of NaV1.8 channels in vincristine-treated animals, which likely contributes to the hyperexcitability that we observed in these neurons. Notably, vincristine treatment did not enhance excitability of small DRG neurons from NaV1.8 knockout mice, and the development of mechanical allodynia was delayed but not abrogated in these mice. Together, our data suggest that sodium channel NaV1.8 in small DRG neurons contributes to the development of vincristine-induced mechanical allodynia.

The transmembrane channel-like 6 (TMC6) in primary sensory neurons involving thermal sensation via modulating M channels.

Introduction: The transmembrane channel-like (TMC) protein family contains eight members, TMC1-TMC8. Among these members, only TMC1 and TMC2 have been intensively studied. They are expressed in cochlear hair cells and are crucial for auditory sensations. TMC6 and TMC8 contribute to epidermodysplasia verruciformis, and predispose individuals to human papilloma virus. However, the impact of TMC on peripheral sensation pain has not been previously investigated. Methods: RNAscope was employed to detect the distribution of TMC6 mRNA in DRG neurons. Electrophysiological recordings were conducted to investigate the effects of TMC6 on neuronal characteristics and M channel activity. Zn2+ indicators were utilized to detect the zinc concentration in DRG tissues and dissociated neurons. A series of behavioural tests were performed to assess thermal and mechanical sensation in mice under both physiological and pathological conditions. Results and Discussion: We demonstrated that TMC6 is mainly expressed in small and medium dorsal root ganglion (DRG) neurons and is involved in peripheral heat nociception. Deletion of TMC6 in DRG neurons hyperpolarizes the resting membrane potential and inhibits neuronal excitability. Additionally, the function of the M channel is enhanced in TMC6 deletion DRG neurons owing to the increased quantity of free zinc in neurons. Indeed, heat and mechanical hyperalgesia in chronic pain are alleviated in TMC6 knockout mice, particularly in the case of heat hyperalgesia. This suggests that TMC6 in the small and medium DRG neurons may be a potential target for chronic pain treatment.

Expression of MHC II in DRG neurons attenuates paclitaxel-induced cold hypersensitivity in male and female mice.

Chemotherapy is often a life-saving treatment, but the development of intractable pain caused by chemotherapy-induced peripheral neuropathy (CIPN) is a major dose-limiting toxicity that restricts cancer survival rates. Recent reports demonstrate that paclitaxel (PTX) robustly increases anti-inflammatory CD4+ T cells in the dorsal root ganglion (DRG), and that T cells and anti-inflammatory cytokines are protective against CIPN. However, the mechanism by which CD4+ T cells are activated, and the extent cytokines released by CD4+ T cells target DRG neurons are unknown. Here, we are the first to detect major histocompatibility complex II (MHCII) protein in mouse DRG neurons and to find CD4+ T cells breaching the satellite glial cell barrier to be in close proximity to neurons, together suggesting CD4+ T cell activation and targeted cytokine release. MHCII protein is primarily expressed in small nociceptive neurons in male and female mouse DRG but increased after PTX in small nociceptive neurons in only female DRG. Reducing one copy of MHCII in small nociceptive neurons decreased anti-inflammatory IL-10 and IL-4 producing CD4+ T cells in naïve male DRG and increased their hypersensitivity to cold. Administration of PTX to male and female mice that lacked one copy of MHCII in nociceptive neurons decreased anti-inflammatory CD4+ T cells in the DRG and increased the severity of PTX-induced cold hypersensitivity. Collectively, our results demonstrate expression of MHCII protein in mouse DRG neurons, which modulates cytokine producing CD4+ T cells in the DRG and attenuates cold hypersensitivity during homeostasis and after PTX treatment.

Differential expression of slow and fast-repriming tetrodotoxin-sensitive sodium currents in dorsal root ganglion neurons.

Sodium channel Nav1.7 triggers the generation of nociceptive action potentials and is important in sending pain signals under physiological and pathological conditions. However, studying endogenous Nav1.7 currents has been confounded by co-expression of multiple sodium channel isoforms in dorsal root ganglion (DRG) neurons. In the current study, slow-repriming (SR) and fast-repriming (FR) tetrodotoxin-sensitive (TTX-S) currents were dissected electrophysiologically in small DRG neurons of both rats and mice. Three subgroups of small DRG neurons were identified based on the expression pattern of SR and FR TTX-S currents. A majority of rat neurons only expressed SR TTX-S currents, while a majority of mouse neurons expressed additional FR TTX-S currents. ProTx-II inhibited SR TTX-S currents with variable efficacy among DRG neurons. The expression of both types of TTX-S currents was higher in Isolectin B4-negative (IB4-) compared to Isolectin B4-positive (IB4+) neurons. Paclitaxel selectively increased SR TTX-S currents in IB4- neurons. In simulation experiments, the Nav1.7-expressing small DRG neuron displayed lower rheobase and higher frequency of action potentials upon threshold current injections compared to Nav1.6. The results suggested a successful dissection of endogenous Nav1.7 currents through electrophysiological manipulation that may provide a useful way to study the functional expression and pharmacology of endogenous Nav1.7 channels in DRG neurons.

Potassium channel modulation in macrophages sensitizes dorsal root ganglion neurons after nerve injury.

Macrophages and satellite glial cells are found between injured and uninjured neurons in the lumbar dorsal root ganglia (DRG). We explored the mechanism of neuro-immune and neuron-glia crosstalk leading to hyperexcitability of DRG neurons. After spared nerve injury (SNI), CX3CR1+ resident macrophages became activated, proliferated, and increased inward-rectifying potassium channel Kir 2.1 currents. Conditioned medium (CM) by macrophages, obtained from DRG of SNI mice, sensitized small DRG neurons from naïve mice. However, treatment with CM from GFAP+ glial cells did not affect neuronal excitability. When subjected to this macrophage-derived CM, DRG neurons had increased spontaneous activity, current-evoked responses and voltage-gated NaV 1.7 and NaV 1.8 currents. Silencing Kir 2.1 in macrophages after SNI prevented the induction of neuronal hyperexcitability from their CM. Blocking vesicular exocytosis or soluble tumor necrosis factor in CM or interfering with the downstream intracellular p38 pathway in neurons, also prevented neuronal hyperexcitability. Blocking protein trafficking in neurons reduced the effect of CM, suggesting that the hyperexcitable state resulted from changes in NaV channel trafficking. These results suggest that DRG macrophages, primed by peripheral nerve injury, contribute to neuron-glia crosstalk, NaV channel dysregulation and neuronal hyperexcitability implicated in the development of neuropathic pain.

Ih current stabilizes excitability in rodent DRG neurons and reverses hyperexcitability in a nociceptive neuron model of inherited neuropathic pain.

We show here that hyperpolarization-activated current (Ih ) unexpectedly acts to inhibit the activity of dorsal root ganglion (DRG) neurons expressing WT Nav1.7, the largest inward current and primary driver of DRG neuronal firing, and hyperexcitable DRG neurons expressing a gain-of-function Nav1.7 mutation that causes inherited erythromelalgia (IEM), a human genetic model of neuropathic pain. In this study we created a kinetic model of Ih and used it, in combination with dynamic-clamp, to study Ih function in DRG neurons. We show, for the first time, that Ih increases rheobase and reduces the firing probability in small DRG neurons, and demonstrate that the amplitude of subthreshold oscillations is reduced by Ih . Our results show that Ih , due to slow gating, is not deactivated during action potentials (APs) and has a striking damping action, which reverses from depolarizing to hyperpolarizing, close to the threshold for AP generation. Moreover, we show that Ih reverses the hyperexcitability of DRG neurons expressing a gain-of-function Nav1.7 mutation that causes IEM. In the aggregate, our results show that Ih unexpectedly has strikingly different effects in DRG neurons as compared to previously- and well-studied cardiac cells. Within DRG neurons where Nav1.7 is present, Ih reduces depolarizing sodium current inflow due to enhancement of Nav1.7 channel fast inactivation and creates additional damping action by reversal of Ih direction from depolarizing to hyperpolarizing close to the threshold for AP generation. These actions of Ih limit the firing of DRG neurons expressing WT Nav1.7 and reverse the hyperexcitability of DRG neurons expressing a gain-of-function Nav1.7 mutation that causes IEM. KEY POINTS: Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels, the molecular determinants of hyperpolarization-activated current (Ih ) have been characterized as a 'pain pacemaker', and thus considered to be a potential molecular target for pain therapeutics. Dorsal root ganglion (DRG) neurons express Nav1.7, a channel that is not present in central neurons or cardiac tissue. Gain-of-function mutations (GOF) of Nav1.7 identified in inherited erythromelalgia (IEM), a human genetic model of neuropathic pain, produce DRG neuron hyperexcitability, which in turn produces severe pain. We found that Ih increases rheobase and reduces firing probability in small DRG neurons expressing WT Nav1.7, and demonstrate that the amplitude of subthreshold oscillations is reduced by Ih . We also demonstrate that Ih reverses the hyperexcitability of DRG neurons expressing a GOF Nav1.7 mutation (L858H) that causes IEM. Our results show that, in contrast to cardiac cells and CNS neurons, Ih acts to stabilize DRG neuron excitability and prevents excessive firing.

CoREST1 in primary sensory neurons regulates neuropathic pain in male mice

AimsEpigenetic regulation is implicated in the neurogenesis of neuropathic pain. The repressor element 1 (RE1) silencing transcription factor (REST) corepressor (CoREST) proteins function as corepressors in the REST complex and/or LSD1 epigenetic complex. In the current study, we aimed to find the expression profile of CoREST1 in the dorsal root ganglion (DRG) and investigate whether it plays a role in neuropathic pain. Main methodsThe evoked pain behaviors in mice were examined by the von Frey test and thermal test in a spinal nerve ligation (SNL)-induced neuropathic pain mice model. CoREST1 siRNA or virus was administered by DRG microinjection or intrathecal injection. The CoREST1 expression in DRGs was examined by immunofluorescence, quantitative PCR, Western blotting, and co-immunoprecipitation. Key findingsCoREST1 was non-selectively expressed in large, medium, and small DRG neurons, and it exclusively colocalized with LSD1. In neuropathic pain models, peripheral nerve injury induced the upregulation of CoREST1 and increased binding of CoREST1 with LSD1 in injured DRGs in male mice. Furthermore, CoREST1 siRNA prevented the development of SNL-induced pain hypersensitivity as well as led to the reduction of established pain hypersensitivity during the maintenance period in SNL mice. Conversely, the overexpression of CoREST1 in DRGs by in vivo transfection of virus-induced pain hypersensitivity in naive mice. SignificanceOur study demonstrated that CoREST1, along with LSD1, was expressed in primary sensory neurons specifically in response to nerve injury, and promoted nociceptive pain hypersensitivity in mice. Thus, CoREST1 might serve as a potential target for treating neuropathic pain.

Antagonism of TRPV4 channels decreases mechanically activated current in rat small dorsal root ganglion neurons

A mechanical stimulus to skeletal muscle during exercise activates the sympathetic nervous system via the skeletal muscle mechanoreflex. However, to date, the receptor mechanisms responsible for mechanotransduction in skeletal muscle afferents have not been fully identified. Transient receptor potential vanilloid 4 (TRPV4), a multimodally activated, nonselective cation channel, is sensitive to mechanical stimuli such as shear stress, osmotic pressure and stretch in various organs. Therefore, it is hypothesized that TRPV4 contributes to mechanotransduction in thin-fiber primary afferents innervating skeletal muscle. Purpose: The aim of this investigation was to determine 1) the extent to which TRPV4 and the afferent marker peripherin are co-localized in dorsal root ganglion (DRG) neurons subserving thin-fiber skeletal muscle afferents using a retrograde tracing technique, and 2) the impact of a selective TRPV4 antagonist, HC067047 on mechanotransduction in small DRG neurons. Methods: To label muscle afferent DRG neurons, dioctadecyl-3,3,3,3-tetramethylindocarbocyanine perchlorate, (Dil, 60 mg/mL, 3 μL total) was injected into the white portion of the gastrocnemius muscle. Five days after the injection, L4-L6 DRGs were harvested from rats (Sprague-Dawley (SD), n = 3, body weight: 318.0 ± 8.7 g). The number of positively stained small-diameter DRG neurons (i.e. less than 30 μm in diameter) were counted and the percentage of peripherin- or DiI-positive neurons in TRPV4-positive neurons was calculated. In vitro whole-cell patch clamp recordings from cultured rat DRG neurons (SD, n = 10, body weight: 352.1 ± 52.4 g), mechanically-activated (MA) current amplitude and mechanical threshold were measured before and 5 min after the application of either HEPES-buffered solution as a control or 1 μM HC067047 solution. Results: Fluorescence immunostaining demonstrated that 20.1 ± 10.1% of TRPV4 positive neurons were expressed with Dil-labeled small DRG neurons. Of these, 9.5 ± 6.1% and 10.6 ± 7.2% were peripherin-positive and -negative neurons, respectively. In in vitro whole-cell patch clamp recordings, MA current amplitude was significantly decreased 5 min after the application of HC067047 ( P < 0.01), while mechanical threshold did not change. Conclusion: The data demonstrates that 1) TRPV4 is expressed in DRGs innervating group IV muscle afferent C-fibers, and 2) HC067047 suppresses the response to mechanical stimulation in DRG neurons. These findings suggest that TRPV4 channels play an important role as mechanoreceptors contributing to mechanotransduction in thin muscle afferents. Supported by NIH HL-151632, JSPS 21J20070 & 22KK0154, and UMF 202131013 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

A sensory neuron-specific long non-coding RNA reduces neuropathic pain by rescuing KCNN1 expression.

Nerve injury to peripheral somatosensory system causes refractory neuropathic pain. Maladaptive changes of gene expression in primary sensory neurons are considered molecular basis of this disorder. Long non-coding RNAs (lncRNAs) are key regulators of gene transcription; however, their significance in neuropathic pain remains largely elusive.Here, we reported a novel lncRNA, named sensory neuron-specific lncRNA (SS-lncRNA), for its expression exclusively in dorsal root ganglion (DRG) and trigeminal ganglion. SS-lncRNA was predominantly expressed in small DRG neurons and significantly downregulated due to a reduction of early B cell transcription factor 1 in injured DRG after nerve injury. Rescuing this downregulation reversed a decrease of the calcium-activated potassium channel subfamily N member 1 (KCNN1) in injured DRG and alleviated nerve injury-induced nociceptive hypersensitivity. Conversely, DRG downregulation of SS-lncRNA reduced the expression of KCNN1, decreased total potassium currents and afterhyperpolarization currents and increased excitability in DRG neurons and produced neuropathic pain symptoms.Mechanistically, downregulated SS-lncRNA resulted in the reductions of its binding to Kcnn1 promoter and heterogeneous nuclear ribonucleoprotein M (hnRNPM), consequent recruitment of less hnRNPM to the Kcnn1 promoter and silence of Kcnn1 gene transcription in injured DRG.These findings indicate that SS-lncRNA may relieve neuropathic pain through hnRNPM-mediated KCNN1 rescue in injured DRG and offer a novel therapeutic strategy specific for this disorder.

Antagonism of TRPV4 channels partially reduces mechanotransduction in rat skeletal muscle afferents.

Mechanical distortion of working skeletal muscle induces sympathoexcitation via thin fibre afferents, a reflex response known as the skeletal muscle mechanoreflex. However, to date, the receptor ion channels responsible for mechanotransduction in skeletal muscle remain largely undetermined. Transient receptor potential vanilloid 4 (TRPV4) is known to sense mechanical stimuli such as shear stress or osmotic pressure in various organs. It is hypothesized that TRPV4 in thin-fibre primary afferents innervating skeletal muscle is involved in mechanotransduction. Fluorescence immunostaining revealed that 20.1 ± 10.1% of TRPV4 positive neurons were small dorsal root ganglion (DRG) neurons that were DiI-labelled, and among them 9.5 ± 6.1% of TRPV4 co-localized with the C-fibre marker peripherin. In vitro whole-cell patch clamp recordings from cultured rat DRG neurons demonstrated that mechanically activated current amplitude was significantly attenuated after the application of the TRPV4 antagonist HC067047 compared to control (P=0.004). Such reductions were also observed in single-fibre recordings from a muscle-nerve ex vivo preparation where HC067047significantly decreased afferent discharge to mechanical stimulation (P=0.007). Likewise, in an in vivo decerebrate rat preparation, the renal sympathetic nerve activity (RSNA) and mean arterial pressure (MAP) responses to passive stretch of hindlimb muscle were significantly reduced by intra-arterial injection of HC067047 (ΔRSNA: P=0.019, ΔMAP: P=0.002). The findings suggest that TRPV4 plays an important role in mechanotransduction contributing to the cardiovascular responses evoked by the skeletal muscle mechanoreflex during exercise. KEY POINTS: Although a mechanical stimulus to skeletal muscle reflexively activates the sympathetic nervous system, the receptors responsible for mechanotransduction in skeletal muscle thin fibre afferents have not been fully identified. Evidence suggests that TRPV4 is a mechanosensitive channel that plays an important role in mechanotransduction within various organs. Immunocytochemical staining demonstrates that TRPV4 is expressed in group IV skeletal muscle afferents. In addition, we show that the TRPV4 antagonist HC067047 decreases the responsiveness of thin fibre afferents to mechanical stimulation at the muscle tissue level as well as at the level of dorsal root ganglion neurons. Moreover, we demonstrate that intra-arterial HC067047 injection attenuates the sympathetic and pressor responses to passive muscle stretch in decerebrate rats. These data suggest that antagonism of TRPV4 attenuates mechanotransduction in skeletal muscle afferents. The present study demonstrates a probable physiological role for TRPV4 in the regulation of mechanical sensation in somatosensory thin fibre muscle afferents.

Capsaicin-Induced Endocytosis of Endogenous Presynaptic CaV2.2 in DRG-Spinal Cord Co-Cultures Inhibits Presynaptic Function.

The N-type calcium channel, CaV2.2 is key to neurotransmission from the primary afferent terminals of dorsal root ganglion (DRG) neurons to their postsynaptic targets in the spinal cord. In this study, we have utilized CaV2.2_HA knock-in mice, because the exofacial epitope tag in CaV2.2_HA enables accurate detection and localization of endogenous CaV2.2. CaV2.2_HA knock-in mice were used as a source of DRGs to exclusively study the presynaptic expression of N-type calcium channels in co-cultures between DRG neurons and wild-type spinal cord neurons. CaV2.2_HA is strongly expressed on the cell surface, particularly in TRPV1-positive small and medium DRG neurons. Super-resolution images of the presynaptic terminals revealed an increase in CaV2.2_HA expression and increased association with the postsynaptic marker Homer over time in vitro. Brief application of the TRPV1 agonist, capsaicin, resulted in a significant down-regulation of cell surface CaV2.2_HA expression in DRG neuron somata. At their presynaptic terminals, capsaicin caused a reduction in CaV2.2_HA proximity to and co-localization with the active zone marker RIM 1/2, as well as a lower contribution of N-type channels to single action potential-mediated Ca2+ influx. The mechanism of this down-regulation of CaV2.2_HA involves a Rab11a-dependent trafficking process, since dominant-negative Rab11a (S25N) occludes the effect of capsaicin on presynaptic CaV2.2_HA expression, and also prevents the effect of capsaicin on action potential-induced Ca2+ influx. Taken together, these data suggest that capsaicin causes a decrease in cell surface CaV2.2_HA expression in DRG terminals via a Rab11a-dependent endosomal trafficking pathway.

Intramuscular insulin administration potentiates sympathetic and pressor responses to capsaicin in rats

Insulin centrally activates the sympathetic nervous system. However, little is known about the effects of insulin on peripheral primary sensory neurons. Transient receptor potential vanilloid 1 (TRPV1), of which capsaicin (CAP) is an agonist, is widely expressed in small dorsal root ganglia (DRG) neurons subserving skeletal muscle thin‐fiber afferents expressing sensory nociceptors and/or metabolically sensitive skeletal muscle receptors. Evidence suggests that insulin increases the sensitivity of these sensory receptors as well as translocation of TRPV1 from cytosol to plasma membrane in DRG neurons. We therefore hypothesized that insulin potentiates the responsiveness of thin fiber muscle afferents to TRPV1 activation.PurposeThe aim of this investigation was 1) to examine whether TRPV1 and insulin receptors (IR) are co‐localized in small‐diameter (< 30 µm) DRG neurons subserving thin‐fiber afferents (C‐fibers or A‐delta fibers), and 2) to determine the effects of insulin on renal sympathetic nerve activity (RSNA) and mean arterial blood pressure (MAP) responses to stimulation of TRPV1 by injection of CAP into the arterial supply of the hindlimb in decerebrated rats.MethodsL4‐L6 DRGs were harvested from Sprague‐Dawley rats (n=3, 19 weeks, body weight: 518 ± 6 g). The percentage of neurons that expressed IR and/or isolectin B4 binding (IB4, a C‐fiber neuronal marker) in TRPV1 positive small DRGs neurons were assessed. RSNA and MAP responses to intra‐arterial administration of CAP (0.3 µg/100 µL) were measured in decerebrate Sprague‐Dawley rats (10 ± 1 weeks, body weight: 330 ± 43 g) before and after intramuscular injection of either insulin (5 U/mL, 30 µL) or saline (vehicle control, 30 µL). To avoid insulin/saline spill over into the systemic circulation, a vascular occluder placed around the abdominal aorta was inflated immediately prior to intramuscular administration of insulin or saline.ResultsImmunohistochemical staining demonstrated that 22 % of TRPV1 positive DRG neurons expressed IR and IB4 binding. Intramuscular insulin administration did not change blood glucose as well as plasma insulin levels assessed from the jugular vein. The RSNA response to CAP after insulin administration was significantly greater than post injection of vehicle (vehicle: 25 ± 24, n=9, vs. insulin: 54 ± 32 %, n=8, P=0.04). While the MAP response to CAP was not significantly changed by vehicle injection (before: 26 ± 13 vs. after: 24 ± 12 mmHg, n=18), insulin administration significantly increased MAP responsiveness (before: 24 ± 1 vs. after: 29 ± 15 mmHg, n=22, P=0.01).ConclusionThe data demonstrate that 1) TRPV1 and IR co‐localize on small DRG neurons and 2) intramuscular insulin administration significantly augments sympathetic and pressor responses to chemical stimulation of skeletal muscle afferents. These findings suggest that insulin potentiates TRPV1 responsiveness at the muscle tissue level, possibly contributing to insulin‐induced sympathoexcitation.

The Slack Channel Deletion Causes Mechanical Pain Hypersensitivity in Mice

The role of the Slack (also known as Slo2.2, KNa1.1, or KCNT1) channel in pain-sensing is still in debate on which kind of pain it regulates. In the present study, we found that the Slack–/– mice exhibited decreased mechanical pain threshold but normal heat and cold pain sensitivity. Subsequently, X-gal staining, in situ hybridization, and immunofluorescence staining revealed high expression of the Slack channel in Isolectin B4 positive (IB4+) neurons in the dorsal root ganglion (DRG) and somatostatin-positive (SOM+) neurons in the spinal cord. Patch-clamp recordings indicated the firing frequency was increased in both small neurons in DRG and spinal SOM+ neurons in the Slack–/– mice whereas no obvious slow afterhyperpolarization was observed in both WT mice and Slack–/– mice. Furthermore, we found Kcnt1 gene expression in spinal SOM+ neurons in Slack–/– mice partially relieved the mechanical pain hypersensitivity of Slack–/– mice and decreased AP firing rates of the spinal SOM+ neurons. Finally, deletion of the Slack channel in spinal SOM+ neurons is sufficient to result in mechanical pain hypersensitivity in mice. In summary, our results suggest the important role of the Slack channel in the regulation of mechanical pain-sensing both in small neurons in DRG and SOM+ neurons in the spinal dorsal horn.

Changes in dorsal root ganglion CGRP expression in mouse pinch nerve injury model: Modulation by Somatostatin type-2 receptor

IntroductionOur previous work has shown that somatostatin effectively inhibits neuropathic pain by activating its type 2 receptor (SSTR2) in the dorsal root ganglion (DRG) and spinal cord of mice. However, the underlying mechanism of this activation has not been elucidated. MethodsTo explore further mechanisms, we examined pain behavior and the expression of neuropeptides such as calcitonin gene-related peptide (CGRP) in dorsal root ganglion neurons(DRGs) as well as the changes of the number of CGRP-IR DRGs in the mouse model of sciatic pinch nerve injury. ResultsIn this model, the number of medium and small DRG neurons in ipsilateral CGRP-IR was slightly increased, but not significantly, compared with sham animals at 3, 7, and 9 days after pinch nerve injury. This correlated with the behavioral readouts of hypersensitivity at the same time points. However, the magnitude of the painful behavior (Autotomy) was observed after application of SSTR2 antagonist (CYN154806, 5 mg/kg) in the injured nerve groups compared to the saline-treated injured group as well as the sham-operated group. Following pinch nerve injury, there was a significant decrease in the number of ipsilateral CGRP-IR small and medium DRG neurons in SSTR2 antagonist (anti-SSTR2)- but not saline-treated mice. These data also correlated with painful behavioral readouts where hypersensitivity was significantly increased by anti-SSTR2 but not saline treatment. Discussion/conclusionIn all, application of the SSTR2 antagonist to the pinched sciatic nerve suppressed CGRP expression and aggravated painful behavior, suggesting that CGRP expression in DRG neurons can be an important component of the pain mechanism and an indicator of pain behavior.

Multitarget nociceptor sensitization by a promiscuous peptide from the venom of the King Baboon spider

The King Baboon spider, Pelinobius muticus, is a burrowing African tarantula. Its impressive size and appealing coloration are tempered by reports describing severe localized pain, swelling, itchiness, and muscle cramping after accidental envenomation. Hyperalgesia is the most prominent symptom after bites from P. muticus, but the molecular basis by which the venom induces pain is unknown. Proteotranscriptomic analysis of P. muticus venom uncovered a cysteine-rich peptide, δ/κ-theraphotoxin-Pm1a (δ/κ-TRTX-Pm1a), that elicited nocifensive behavior when injected into mice. In small dorsal root ganglion neurons, synthetic δ/κ-TRTX-Pm1a (sPm1a) induced hyperexcitability by enhancing tetrodotoxin-resistant sodium currents, impairing repolarization and lowering the threshold of action potential firing, consistent with the severe pain associated with envenomation. The molecular mechanism of nociceptor sensitization by sPm1a involves multimodal actions over several ion channel targets, including NaV1.8, KV2.1, and tetrodotoxin-sensitive NaV channels. The promiscuous targeting of peptides like δ/κ-TRTX-Pm1a may be an evolutionary adaptation in pain-inducing defensive venoms.

Insulin potentiates the response to capsaicin in dorsal root ganglion neurons in vitro and muscle afferents ex vivo in normal healthy rodents.

Systemic insulin administration evokes sympathoexcitatory actions, but the mechanisms underlying these observations are unknown. We reported that insulin sensitizes the response of thin-fibre primary afferents, as well as the dorsal root ganglion (DRG) that subserves them, to mechanical stimuli. However, little is known about the effects of insulin on primary neuronal responses to chemical stimuli. TRPV1, whose agonist is capsaicin (CAP), is widely expressed on chemically sensitive metaboreceptors and/or nociceptors. The aim of this investigation was to determine the effects of insulin on CAP-activated currents in small DRG neurons and CAP-induced action potentials in thin-fibre muscle afferents of normal healthy rodents. Additionally, we investigated whether insulin potentiates sympathetic nerve activity (SNA) responses to CAP. In whole-cell patch-clamp recordings from cultured mice DRG neurons in vitro, the fold change in CAP-activated current from pre- to post-application of insulin (n=13) was significantly (P<0.05) higher than with a vehicle control (n=14). Similar results were observed in single-fibre recording experiments ex vivo as insulin potentiated CAP-induced action potentials compared to vehicle controls (n=9 per group, P<0.05). Furthermore, insulin receptor blockade with GSK1838705 significantly suppressed the insulin-induced augmentation in CAP-activated currents (n=13) as well as the response magnitude of CAP-induced action potentials (n=9). Likewise, the renal SNA response to CAP after intramuscular injection of insulin (n=8) was significantly (P<0.05) greater compared to vehicle (n=9). The findings suggest that insulin potentiates TRPV1 responsiveness to CAP at the DRG and muscle tissue levels, possibly contributing to the augmentation in sympathoexcitation during activities such as physical exercise. KEY POINTS: Evidence suggests insulin centrally activates the sympathetic nervous system, and a chemical stimulus to tissues activates the sympathetic nervous system via thin fibre muscle afferents. Insulin is reported to modulate putative chemical-sensitive channels in the dorsal root ganglion neurons of these afferents. In the present study, it is demonstrated that insulin potentiates the responsiveness of thin fibre afferents to capsaicin at muscle tissue levels as well as at the level of dorsal root ganglion neurons. In addition, it is demonstrated that insulin augments the sympathetic nerve activity response to capsaicin in vivo. These data suggest that sympathoexcitation is peripherally mediated via insulin-induced chemical sensitization. The present study proposes a possible physiological role of insulin in the regulation of chemical sensitivity in somatosensory thin fibre muscle afferents.

Distribution of Large and Small Dorsal Root Ganglion Neurons in Common Marmosets.

The aim of this study was to elucidate the size and distribution of dorsal root ganglion (DRG) neurons in non-human primates and to compare them with those of rodent DRG neurons. By measuring the size of NeuN-, NF200-, and peripherin-positive DRG neurons in the lumbar spinal cord of rats and marmosets, we found that the cell size distribution pattern was comparable in both species, although DRG neurons in marmosets were larger than those of rodents. This is the first demonstration that DRG neurons in marmosets have a bimodal size distribution, which has been well established in rodents and humans.