Rat Dorsal Root Ganglion Neurons Research Articles

The paracetamol metabolite N-acetylp-benzoquinone imine reduces excitability in first- and second-order neurons of the pain pathway through actions on KV7 channels.

Paracetamol (acetaminophen, APAP) is one of the most frequently used analgesic agents worldwide. It is generally preferred over nonsteroidal anti-inflammatory drugs because it does not cause typical adverse effects resulting from the inhibition of cyclooxygenases, such as gastric ulcers. Nevertheless, inhibitory impact on these enzymes is claimed to contribute to paracetamols mechanisms of action which, therefore, remained controversial. Recently, the APAP metabolites N-arachidonoylaminophenol (AM404) and N-acetyl-p-benzoquinone imine (NAPQI) have been detected in the central nervous system after systemic APAP administration and were reported to mediate paracetamol effects. In contrast to nonsteroidal anti-inflammatory drugs that rather support seizure activity, paracetamol provides anticonvulsant actions, and this dampening of neuronal activity may also form the basis for analgesic effects. Here, we reveal that the APAP metabolite NAPQI, but neither the parent compound nor the metabolite AM404, reduces membrane excitability in rat dorsal root ganglion (DRG) and spinal dorsal horn (SDH) neurons. The observed reduction of spike frequencies is accompanied by hyperpolarization in both sets of neurons. In parallel, NAPQI, but neither APAP nor AM404, increases currents through KV7 channels in DRG and SDH neurons, and the impact on neuronal excitability is absent if KV7 channels are blocked. Furthermore, NAPQI can revert the inhibitory action of the inflammatory mediator bradykinin on KV7 channels but does not affect synaptic transmission between DRG and SDH neurons. These results show that the paracetamol metabolite NAPQI dampens excitability of first- and second-order neurons of the pain pathway through an action on KV7 channels.

Agomelatine modulates calcium signaling through protein kinase C and phospholipase C-mediated mechanisms in rat sensory neurons.

Agomelatine, a novel antidepressant exerting its effects through melatonergic and serotonergic systems, implicated to be effective against pain including neuropathic pain but without any knowledge of mechanism of action. To explore the possible role of agomelatine on nociceptive transmission at the peripheral level, the effects of agomelatine on intracellular calcium ([Ca2+ ]i ) signaling in peripheral neurons were investigated in cultured rat dorsal root ganglion (DRG) neurons. Using the fura-2-based calcium imaging technique, the effects of agomelatine on [Ca2+ ]i and roles of the second messenger-mediated pathways were assessed. Agomelatine caused [Ca2+ ]i signaling in a dose-dependent manner when tested at 10 and 100 μM concentration. Luzindole, a selective melatonin receptor antagonist, almost completely blocked the agomelatine-induced calcium signals. The agomelatine-induced calcium transients were also nearly abolished following pretreatment with the 100 ng/ml pertussis toxin, a Gi/o protein inhibitor. The stimulatory effects of agomelatine on [Ca2+ ]i transients were significantly reduced by applications of phospholipase C (PLC) and protein kinase C (PKC) blockers, 10 μM U73122, and 10 μM chelerythrine chloride, respectively. The obtained results of agomelatine-induced [Ca2+ ]i signals indicates that peripheral mechanisms are involved in analgesic effects of agomelatine. These mechanisms seems to involve G-protein-coupled receptor activation and PLC and PKC mediated mechanisms.

Imaging Sodium Flux during Action Potentials in Neurons with Fluorescent Nanosensors and Transparent Microelectrodes.

Sodium flux plays a pivotal role in neurobiological processes including initiation of action potentials and regulation of neuronal cell excitability. However, unlike the wide range of fluorescent calcium indicators used extensively for cellular studies, the choice of sodium probes remains limited. We have previously demonstrated optode-based nanosensors (OBNs) for detecting sodium ions with advantageous modular properties such as tunable physiological sensing range, full reversibility, and superb selectivity against key physiological interfering ion potassium. (1) Motivated by bridging the gap between the great interest in sodium imaging of neuronal cell activity as an alternative to patch clamp and limited choices of optical sodium indicators, in this Letter we report the application of nanosensors capable of detecting intracellular sodium flux in isolated rat dorsal root ganglion neurons during electrical stimulation using transparent microelectrodes. Taking advantage of the ratiometric detection scheme offered by this fluorescent modular sensing platform, we performed dual color imaging of the sensor to monitor the intracellular sodium currents underlying trains of action potentials in real time. The combination of nanosensors and microelectrodes for monitoring neuronal sodium dynamics is a novel tool for investigating the regulatory role of sodium ions involved during neural activities.

Effect of oxycodone on function of GABAA receptors in dorsal root ganglion neurons of rats with neuropathic pain

Objective To evaluate the effect of oxycodone on function of GABAA receptors in dorsal root ganglion (DRG) neurons of rats with neuropathic pain (NP). Methods Thirty-six adult male Sprague-Dawley rats, weighing 180-220 g, aged 10 weeks, were allocated into 3 groups (n=12 each) using a random number table method: sham operation group (group S), group NP and oxycodone group (group O). The sciatic nerve was only isolated but not ligated in group S. NP was induced by chronic constriction injury.The sciatic nerve was exposed and 4 loose ligatures were placed on the sciatic nerve at 1 mm intervals with 4-0 chromic catgut.Oxycodone 15 μg/kg was intraperitoneally injected once a day for 14 consecutive days from ligating the sciatic nerve to satisfaction in group O. The thermal paw withdrawal latency (TWL) was measured at 1 day before establishing the model (T0) and 3, 5, 7, 10 and 14 days after establishing the model (T1-5). The rats were sacrificed after measurement of pain threshold at T5, and DRG neurons were acutely isolated for recording the amplitude of GABAA receptors-activated currents using whole-cell patch-clamp technique. Results Compared with group S, the TWL was significantly shortened at T1-5, and the amplitude of GABAA receptors-activated currents in DRG neurons was decreased in NP and O groups (P<0.05). Compared with group NP, the TWL was significantly prolonged at T1-5, and the amplitude of GABAA receptors-activated currents in DRG neurons was increased in group O (P<0.05). Conclusion Oxycodone can enhance the function of GABAA receptors-activated currents in DRG neurons and thus enhance GABAA receptors-mediated presynaptic inhibition, which may be related to the mechanism of oxycodone-induced reduction of NP in rats. Key words: Oxycodone; Neuralgia; Ganglia, spinal; Receptors, GABA-A

Granulocyte-macrophage colony-stimulating factor receptor expression in clinical pain disorder tissues and role in neuronal sensitization.

Granulocyte-macrophage colony-stimulating factor receptor (GM-CSFR) is highly expressed in peripheral macrophages and microglia, and is involved in arthritis and cancer pain in animal models. However, there is limited information on GM-CSFR expression in human central nervous system (CNS), peripheral nerves, or dorsal root ganglia (DRG), particularly in chronic pain conditions. Immunohistochemistry was used to quantify GM-CSFR expression levels in human tissues, and functional sensory effects of GM-CSF were studied in cultured DRG neurons. Granulocyte-macrophage colony-stimulating factor receptor was markedly increased in microglia at lesional sites of multiple sclerosis spinal cords (P = 0.01), which co-localised with macrophage marker CD68 (P = 0.009). In human DRG, GM-CSFR was expressed in a subset of small/medium diameter cells (30%) and few large cells (10%), with no significant change in avulsion-injured DRG. In peripheral nerves, there was a marked decrease in axonal GM-CSFR after chronic painful nerve injury (P = 0.004) and in painful neuromas (P = 0.0043); CD-68-positive macrophages were increased (P = 0.017) but did not appear to express GM-CSFR. Although control synovium showed absent GM-CSFR immunostaining, this was markedly increased in macrophages of painful osteoarthritis knee synovium. Granulocyte-macrophage colony-stimulating factor receptor was expressed in 17 ± 1.7% of small-/medium-sized cultured adult rat DRG neurons, and in 27 ± 3.3% of TRPV1-positive neurons. Granulocyte-macrophage colony-stimulating factor treatment sensitized capsaicin responses in vitro, which were diminished by p38 MAPK or TrkA inhibitors. Our findings support GM-CSFR as a therapeutic target for pain and hypersensitivity in clinical CNS and peripheral inflammatory conditions. Although GM-CSFR was decreased in chronic painful injured peripheral nerves, it could mediate CNS neuroinflammatory effects, which deserves study.

Costorage of High Molecular Weight Neurotransmitters in Large Dense Core Vesicles of Mammalian Neurons.

It is today widely accepted that several types of high molecular weight (MW) neurotransmitters produced by neurons are synthesized at the cell body, selectively stored within large dense core vesicles (LDCVs) and anterogradely transported to terminals where they elicit their biological role(s). Among these molecules there are neuropeptides and neurotrophic factors, the main focus of this perspective article. I here first provide a brief resume of the state of art on neuronal secretion, with primary emphasis on the molecular composition and mechanism(s) of filling and release of LDCVs. Then, I discuss the perspectives and future directions of research in the field as regarding the synthesis and storage of multiple high MW transmitters in LDCVs and the possibility that a selective sorting of LDCVs occurs along different neuronal processes and/or their branches. I also consider the ongoing discussion that diverse types of neurons may contain LDCVs with different sets of integral proteins or dial in a different fashion with LDCVs containing the same cargo. In addition, I provide original data on the size of LDCVs in rat dorsal root ganglion neurons and their central terminals in the spinal cord after immunogold labeling for calcitonin gene-related peptide (CGRP), neuropeptide K, substance P, neurokinin A or somatostatin. These data corroborate the idea that, similarly to endocrine cells, LDCVs undergo a process of maturation which involves a homotypic fusion followed by a reduction in size and condensation of cargo. They also give support to the conjecture that release at terminals occurs by cavicapture, a process of partial fusion of the vesicle with the axolemma, accompanied by depletion of cargo and diminution of size.

Constitutive activity of transient receptor potential vanilloid type 1 triggers spontaneous firing in nerve growth factor-treated dorsal root ganglion neurons of rats

Dorsal root ganglion (DRG) neurons cultured in the presence of nerve growth factor (NGF, 100 ng/ml) often show a spontaneous action potential. Underlying mechanisms of this spontaneous firing were examined using the patch clamp technique. The spontaneous firing in the on-cell configuration was abolished by a decrease in the Na+ concentration and by the TRPV1 antagonists capsazepine (10 μM) and BCTC (1 μM). These responses were accompanied by hyperpolarization of the resting potential. The holding current observed in neurons voltage clamped at –60 mV in the whole-cell configuration was significantly larger in the neurons that fired spontaneously, indicating that these neurons had an additional cation conductance that caused depolarization and triggered action potentials. The holding current in the firing neurons was decreased by extracellular Na+ reduction, capsazepine and BCTC. The amplitudes of the capsazepine- or BCTC-sensitive component of the holding current in the spontaneously firing neurons were ten times as large as those recorded in the other neurons showing no spontaneous firing. However, the amplitudes of the current responses to capsaicin (1 μM) were not different regardless of the presence of spontaneous firing or treatment with NGF. These results indicate that chronic NGF treatment of cultured DRG neurons in rats induces a constitutively active cation conductance through TRPV1, which depolarizes the neurons and triggers spontaneous action potentials in the absence of any stimuli. Since NGF in the DRG is reported to increase after nerve injury, this NGF-mediated regulation of TRPV1 may be a cause of the pathogenesis of neuropathic pain.

Dexmedetomidine relieves neuropathic pain by inhibiting hyperpolarization-activated cyclic nucleotide-gated currents in dorsal root ganglia neurons.

This study was designed to examine the effect and mechanism of dexmedetomidine (Dex) on neuropathic pain (NP). The NP model was established by performing chronic sciatic nerve constriction injury (CCI). Seven days after CCI surgery, the rats were injected intraperitoneally with Dex, ZD7288 (an HCN channel inhibitor), and saline, respectively. The paw withdrawal threshold to mechanical stimulation and the thermal withdrawal latency tests were performed. After administration, the L4, L5 dorsal root ganglia (DRG) neurons of rats were isolated. In addition, hyperpolarization-activated cyclic nucleotide-gated (HCN) channels subtype plasmids were transfected into human embryonic kidney (HEK)293 cells. Whole-cell clamp recordings were used to examine the properties of HCN currents (Ih) expressed in HEK293 cells and DRG neurons. After surgery, the paw withdrawal threshold to mechanical stimulation and thermal withdrawal latency were reduced, the HCN currents (Ih) amplitude of DRG neurons was increased, and the semiactivated voltage (V1/2) value was decreased in CCI rats (P<0.05). CCI rats treated with Dex or ZD7288 had reduced mechanical and thermal hyperalgesia. The Ih amplitude was lower and the V1/2 value was increased in DRG neurons in CCI rats treated with Dex or ZD7288 (P<0.05). In addition, Dex inhibited HCN1 and HCN2 currents in HEK293 cells; caused a decrease in maximal currents, an increase in the inhibition rate of Ih, and a negative shift in V1/2 (P<0.05). Taken together, our finding suggested that Dex alleviates NP and the effect is likely because of the inhibition of HCN currents.

Nogo-A promotes inflammatory heat hyperalgesia by maintaining TRPV-1 function in the rat dorsal root ganglion neuron.

Nogo-A is a key inhibitory molecule of axon regeneration in oligodendrocytes. However, little is known about its role in adult neurons. In this study, we showed an important function of Nogo-A on regulation of inflammatory pain in dorsal root ganglion (DRG) neurons. In adult rats with complete Freund's adjuvant (CFA) hind paw inflammation, DRG neurons showed a significant increase in Nogo-A expression. Disruption of Nogo-A signaling with Nogo-66 receptor antagonist peptide, Nogo-A blocking antibody, Nogo-A short hairpin RNA, or Nogo-A gene knockout attenuated CFA-induced inflammatory heat hyperalgesia. Moreover, disruption of Nogo-A signaling suppressed the function and expression in DRG neurons of the transient receptor potential vanilloid subfamily member (TRPV)-1 channel, which is known to be the endogenous transducer of noxious heat during inflammation. These effects were accompanied with a reduction in LIM domain kinase (LIMK)/cofilin phosphorylation and actin polymerization. Similar disruption of actin filament architecture by direct action of Latrunculin A reduced the TRPV-1 activity and up-regulation of TRPV-1 protein caused by CFA. We conclude that Nogo-A plays an essential role in the development of inflammatory heat hyperalgesia, partly through maintaining TRPV-1 function via activation of the LIMK/cofilin pathway, which regulates actin filament dynamics. These findings support a therapeutic potential of modulating Nogo-A signaling in pain management.-Hu, F., Liu, H.-C., Su, D.-Q., Chen, H.-J., Chan, S.-O., Wang, Y., Wang, J. Nogo-A promotes inflammatory heat hyperalgesia by maintaining TRPV-1 function in the rat dorsal root ganglion neuron.

Characterization of temperature-sensitive leak K+ currents and expression of TRAAK, TREK-1, and TREK2 channels in dorsal root ganglion neurons of rats

Leak K+ currents are mediated by two-pore domain K+ (K2P) channels and are involved in controlling neuronal excitability. Of 15 members of K2P channels cloned so far, TRAAK, TREK-1, and TREK-2 are temperature sensitive. In the present study, we show that strong immunoreactivity of TRAAK, TREK-1 and TREK-2 channels was present mainly in small-sized dorsal root ganglion (DRG) neurons of rats. The percentages of neurons with strong immunoreactivity of TRAAK, TREK-1 and TREK-2 channels were 27, 23, and 20%, respectively. Patch-clamp recordings were performed to examine isolated leak K+ currents on acutely dissociated small-sized rat DRG neurons at room temperature of 22 °C, cool temperature of 14 °C and warm temperature of 30 °C. In majority of small-sized DRG neurons recorded (76%), large leak K+ currents were observed at 22 °C and were inhibited at 14 °C and potentiated at 30 °C, suggesting the presence of temperature-sensitive K2P channels in these neurons. In a small population (18%) of small-sized DRG neurons, cool temperature of 14 °C evoked a conductance which was consistent with TRPM8 channel activation in cold-sensing DRG neurons. In these DRG neurons, leak K+ currents were very small at 22 °C and were not potentiated at 30 °C, suggesting that few temperature-sensitive K2P channels was present in cold-sensing DRG neurons. For DRG neurons with temperature-sensitive leak K+ currents, riluzole, norfluoxetine and prostaglandin F2α (PGE2α) inhibited the leak K+ currents at both 30 °C and 22 °C degree, and did not have inhibitory effects at 14 °C. Collectively, the observed temperature-sensitive leak K+ currents are consistent with the expression of temperature-sensitive K2P channels in small-sized DRG neurons.

A SNAP-25 cleaving chimera of botulinum neurotoxin /A and /E prevents TNFα−induced elevation of the activities of native TRP channels on early postnatal rat dorsal root ganglion neurons

Transient receptor potential (TRP) vallinoid 1 (TRPV1) and ankyrin 1 (TRPA1) are two transducing channels expressed on peripheral sensory nerves involved in pain sensation. Upregulation of their expression, stimulated by inflammatory cytokines and growth factors in animal pain models, correlate with the induction of nociceptive hyper-sensitivity. Herein, we firstly demonstrate by immuno-cytochemical labelling that TNFα augments the surface content of these channels on rat cultured dorsal root ganglion (DRG) neurons which, in turn, enhances the electrophysiological and functional responses of the latter to their specific agonists. A molecular basis underlying this TNFα-dependent enhancement was unveiled by pre-treating DRGs with a recently-published chimeric protein, consisting of the protease light chain (LC) of botulinum neurotoxin (BoNT) serotype E fused to full-length BoNT/A (LC/E-BoNT/A). This cleaves synaptosomal-associated protein of Mr 25k (SNAP-25) and reported previously to exhibit anti-nociceptive activity in a rat model of neuropathic pain. Low pM concentrations of this chimera were found to prevent the TNFα-stimulated delivery of TRPV1/A1 to the neuronal plasmalemma and, accordingly, decreased their incremental functional activities relative to those of control cells, an effect accompanied by SNAP-25 cleavage. Advantageously, LC/E-BoNT/A did not reduce the basal surface contents of the two channels or their pharmacological responses. Thus, use of multiple complementary methodologies provides evidence that LC/E-BoNT/A abolishes the TNFα-dependent augmented, but not resting, surface trafficking of TRPV1/A1. As TNFα is known to induce nociceptive hyper-sensitivity in vivo, our observed inhibition by LC/E-BoNT/A of its action in vitro could contribute to its potential alleviation of pain.

Purification and Characterization of a Novel Insecticidal Toxin, μ-sparatoxin-Hv2, from the Venom of the Spider Heteropoda venatoria.

The venom of the spider Heteropoda venatoria produced lethal effect to cockroaches as reported in our previous study, and could be a resource for naturally-occurring insecticides. The present study characterized a novel cockroach voltage-gated sodium channels (NaVs) antagonist, μ-sparatoxin-Hv2 (μ-SPRTX-Hv2 for short), from this venom. μ-SPRTX-Hv2 is composed of 37 amino acids and contains six conserved cysteines. We synthesized the toxin by using the chemical synthesis method. The toxin was lethal to cockroaches when intraperitoneally injected, with a LD50 value of 2.8 nmol/g of body weight. Electrophysiological data showed that the toxin potently blocked NaVs in cockroach dorsal unpaired median (DUM) neurons, with an IC50 of 833.7 ± 132.2 nM, but it hardly affected the DUM voltage-gated potassium channels (KVs) and the DUM high-voltage-activated calcium channels (HVA CaVs). The toxin also did not affect NaVs, HVA CaVs, and Kvs in rat dorsal root ganglion (DRG) neurons, as well as NaV subtypes NaV1.3–1.5, NaV1.7, and NaV1.8. No envenomation symptoms were observed when μ-SPRTX-Hv2 was intraperitoneally injected into mouse at the dose of 7.0 μg/g. In summary, μ-SPRTX-Hv2 is a novel insecticidal toxin from H. venatoria venom. It might exhibit its effect by blocking the insect NaVs and is a candidate for developing bioinsecticide.

CDMP1 overexpression mediates inflammatory cytokine‑induced apoptosis via inhibiting the Wnt/β‑Catenin pathway in rat dorsal root ganglia neurons.

Cartilage-derived morphogenetic protein-1 (CDMP1) is a polypeptide growth factor with specific cartilage inducibility, which is predominantly expressed in the developmental long bone cartilage core and in the pre-cartilage matrix in the embryonic stage. The aim of the present study was to investigate the roles and the mechanisms of CDMP1 overexpression on the apoptosis of rat dorsal root ganglia (DRG) neurons that were induced by inflammatory cytokines. Cell counting Kit-8 assay, flow cytometry and TdT-mediated dUTP nick-end labeling assay were performed to examine cell viability and apoptosis. ELISA, hematoxylin and eosin staining and immunohistochemistry assays were performed to examine the levels of several factors in DRG tissues. Western blot analysis and reverse transcription-quantitative polymerase chain reaction assays were used to determine the mRNA and protein expression levels, respectively. The results demonstrated that CDMP1 expression was downregulated, while inflammatory cytokine expression was upregulated in DRG tissues derived from lumbar disc herniation (LDH) model rats. In addition, DRG cells from LDH rats exhibited increased apoptosis compared with control rats. CDMP1 overexpression enhanced the cell viability of inflammatory cytokine-induced DRG cells, and suppressed the apoptosis of inflammatory cytokine-induced DRG cells via regulating the expression levels of Caspase-3/8/9, BCL2 apoptosis regulator, and BCL2 associated X. Furthermore, CDMP1 overexpression was demonstrated to affect the Wnt/β-Catenin pathway in the inflammatory cytokine-induced DRG cells. In conclusion, the present findings suggested that CDMP1 overexpression mediated inflammatory cytokine-induced apoptosis via Wnt/β-Catenin signaling in rat DRG cells.

Dimethyl fumarate ameliorates chemotherapy agent-induced neurotoxicity in vitro

Chemotherapy agents such as oxaliplatin, cisplatin, paclitaxel, and bortezomib frequently cause severe peripheral neuropathy and there is currently no effective strategy to prevent this. Dimethyl fumarate (DMF) is a new oral drug for the treatment of multiple sclerosis, and has neuroprotective effects via up-regulation of the nuclear factor-erythroid-2-related factor 2 (Nrf2)-dependent antioxidant response. In this study, we investigated the effect of DMF on chemotherapy agent-induced neurodegenerations in cultured cells. We found that DMF and its metabolite monomethyl fumarate (MMF) attenuated oxaliplatin-, cisplatin-, and bortezomib- (but not paclitaxel-) induced inhibition of neurite outgrowth, but had no effect on cell death as a result of these agents in cultured PC12 cells and primary cultured rat dorsal root ganglion (DRG) neurons. Furthermore, Nrf2 DNA binding activity was increased by DMF and MMF in PC12 cells. These findings suggest that DMF, which activates Nrf2 pathway, has a potential protective action against chemotherapy-induced neurotoxicity, particularly neurite impairments.

Enhanced total neurite outgrowth and secondary branching in dorsal root ganglion neurons elicited by low intensity pulsed ultrasound

Objective. Despite the prevalence of peripheral nerve injuries (PNI), challenges remain in restoring full functionality to those afflicted. For recovery to occur, axons must extend across the injury site to connect with distal targets, where injury gap size is a critical factor in the probability of restoration of function. Current clinical therapies often achieve limited neural regeneration, motivating the development of new therapeutic interventions such as biophysical stimulation. Approach. To investigate the potential for low intensity, pulsed ultrasonic simulation (LIPUS) to impact peripheral nerve regeneration, primary neonatal rat dorsal root ganglion neurons were examined in vitro in response to ultrasound (US). Dissociated neurons were stimulated with varied acoustic power (low, medium, high) and their morphometrics, including total outgrowth, branching, and length, were analyzed acutely after 18 h of growth. Main results. Results show US increases total neurite outgrowth by 2.83-fold compared to unstimulated controls at the highest power. Neurite branching at medium and high-power US increased approximately 2-fold compared to controls, while low stimulation exhibited more muted trends. Neurite branching is also impacted by US, with medium and high power eliciting the highest branching, of approximately 2-fold compared to low power and unstimulated controls. Significance. These results demonstrate that US stimulation of DRG neurons in vitro impacts neurite morphology and enhances total extension, indicating the potential for advancing and understanding driving mechanisms of ultrasonic therapies for peripheral nerve regeneration.

BDNF Reduces eEF2 Phosphorylation and Enhances Novel Protein Synthesis in the Growth Cones of Dorsal Root Ganglia Neurons.

The local translation, which is regulated by extracellular stimuli such as guidance molecules, in growth cones of neurons provides a molecular mechanism for axonal development. In this study, we performed immunocytochemistry together with atomic force microscopy to investigate the localization of ribosomal proteins in the growth cones of rat dorsal root ganglion (DRG) neurons. The immunoreactivity of ribosomal protein P0/1/2 and S6, and novel protein synthesis were observed in the central, sterically bulky region of growth cones. Brain derived neurotrophic factor (BDNF) reduced the eEF2 phosphorylation, indicating its activation, and enhanced protein synthesis within 30min. The effects of BDNF were completely inhibited by rapamycin, an inhibitor of mammalian target of rapamycin (mTOR). These results indicated that BDNF rapidly activates translation and enhances novel protein synthesis in growth cones of DRG though the mTOR signaling.

Upregulation of N-type calcium channels in the soma of uninjured dorsal root ganglion neurons contributes to neuropathic pain by increasing neuronal excitability following peripheral nerve injury

N-type voltage-gated calcium (Cav2.2) channels are expressed in the central terminals of dorsal root ganglion (DRG) neurons, and are critical for neurotransmitter release. Cav2.2 channels are also expressed in the soma of DRG neurons, where their function remains largely unknown. Here, we showed that Cav2.2 was upregulated in the soma of uninjured L4 DRG neurons, but downregulated in those of injured L5 DRG neurons following L5 spinal nerve ligation (L5-SNL). Local application of specific Cav2.2 blockers (ω-conotoxin GVIA, 1–100 μM or ZC88, 10–1000 μM) onto L4 and 6 DRGs on the operated side, but not the contralateral side, dose-dependently reversed mechanical allodynia induced by L5-SNL. Patch clamp recordings revealed that both ω-conotoxin GVIA (1 μM) and ZC88 (10 μM) depressed hyperexcitability in L4 but not in L5 DRG neurons of L5-SNL rats. Consistent with this, knockdown of Cav2.2 in L4 DRG neurons with AAV-Cav2.2 shRNA substantially prevented L5-SNL-induced mechanical allodynia and hyperexcitability of L4 DRG neurons. Furthermore, in L5-SNL rats, interleukin-1 beta (IL-1β) and IL-10 were upregulated in L4 DRGs and L5 DRGs, respectively. Intrathecal injection of IL-1β induced mechanical allodynia and Cav2.2 upregulation in bilateral L4–6 DRGs of naïve rats, whereas injection of IL-10 substantially prevented mechanical allodynia and Cav2.2 upregulation in L4 DRGs in L5-SNL rats. Finally, in cultured DRG neurons, Cav2.2 was dose-dependently upregulated by IL-1β and downregulated by IL-10. These data indicate that the upregulation of Cav2.2 in uninjured DRG neurons via IL-1β over-production contributes to neuropathic pain by increasing neuronal excitability following peripheral nerve injury.

Upregulation of Cav3.2 T-type calcium channels in adjacent intact L4 dorsal root ganglion neurons in neuropathic pain rats with L5 spinal nerve ligation

Besides the injured peripheral dorsal root ganglion (DRG) neurons, the adjacent intact DRG neurons also have important roles in neuropathic pain. Ion channels including Cav3.2 T-type calcium channel in the DRG neurons are important in the development of neuropathic pain. In the present study, we aimed to examine the expression of Cav3.2 T-type calcium channels in the intact DRG neurons in neuropathic pain. A neuropathic pain model of rat with lumbar 5 (L5) spinal nerve ligation (SNL) was established, in which the L4 DRG was separated from the axotomized L5 DRG, and the molecular, morphological and electrophysiological changes of Cav3.2 T-type calcium channels in L4 DRG neurons were investigated. Western blotting showed that total and membrane protein levels of Cav3.2 in L4 DRG neurons increased, and voltage-dependent patch clamp recordings revealed an increased T-type current density with a curve shift to the left in steady-state activation in the acutely isolated L4 DRG neurons in neuropathic pain rats. Immunofluorescent staining further showed that the membrane expression of Cav3.2 increased in CGRP-, IB4-positive small neurons and NF200-positive large ones. In conclusion, the membrane expression and the function of Cav3.2 T-type calcium channels are increased in the intact L4 DRG neurons in neuropathic pain rats with peripheral nerve injury like SNL.

(Invited) Wireless Photoelectrochemical Control of Neuronal Activity with Coaxial Silicon Nanowires

Optical methods for modulating cellular behavior are promising for both fundamental and clinical applications. However, most available methods are either mechanically invasive, require genetic manipulation of target cells, or cannot provide sub-cellular specificity. Here, we address all these issues by showing optical neuromodulation with free-standing coaxial p-type/intrinsic/n-type silicon nanowires (PIN-SiNWs). We revealed the presence of atomic gold on the nanowire surfaces, likely due to gold diffusion during the material growth. To evaluate how surface gold impacts the photoelectrochemical properties of single PIN-SiNWs, we used modified quartz pipettes from a patch clamp and recorded sustained cathodic photocurrents from single PIN-SiNWs. We show that these currents can elicit action potentials in primary rat dorsal root ganglion neurons through a primarily atomic gold-enhanced photoelectrochemical process. This work shows that wireless and non-genetic photoelectrochemical neuromodulation with single nanostructures is possible, and has implications for both basic research and photo-responsive therapies for neurodegenerative diseases.

Muscarinic Acetylcholine Type 1 Receptor Constrains Neurite Outgrowth by Inhibiting Microtubule Polymerization and Mitochondrial Trafficking in Adult Sensory Neurons: A Phenotype Rescued by Antagonist Treatment

ObjectiveThe muscarinic acetylcholine type 1 receptor (M1R) is a metabotropic G protein coupled receptor. Knockout of M1R or exposure to selective or specific receptor antagonists elevates neurite outgrowth in adult sensory neurons and blockade of associated cholinergic pathways is therapeutic in diverse models of peripheral neuropathy. We tested the hypothesis that endogenous M1R activation constrained neurite outgrowth via a negative impact on the cytoskeleton and subsequent mitochondrial trafficking and that this could be rescued by antagonist treatment.MethodologyWe overexpressed M1R and M1R DREADD (designer receptors exclusively activated by designer drugs) mutant in primary cultures of adult rat dorsal root ganglion (DRG) neurons and in tumor cell lines and studied the physiological and molecular consequences related to regulation of cytoskeletal/mitochondrial dynamics and neurite outgrowth. The M1R‐DREADD mutant contained two mutations of conserved orthosteric site residues that cause minimal responsiveness to ACh. In addition, we used CRISPR/Cas9 based Gα12/13 knockout HEK293 cells and siRNA mediated knockdown of Gα13 in cultured DRG neurons to study M1R Gα13 protein mediated tubulin cytoskeletal dynamics. No M1R ligand was added to the culture media.ResultsOverexpression of M1R by adult sensory neurons caused dissolution of the polymerized tubulin cytoskeleton, but not the actin network and significantly reduced neurite outgrowth. Conversely, over‐expression of a M1R‐DREADD mutant increased neurite outgrowth. These data suggest that endogenously released M1R ligand interacts with the M1R to suppress neurite outgrowth. M1R‐dependent disruption of the cytoskeleton significantly diminished mitochondrial abundance and trafficking in the distal neurites. Treatment with the specific M1R antagonist – muscarinic toxin 7 (MT7, 100 nM) or the selective M1R antagonist pirenzepine (1 μM) rescued cytoskeletal abnormalities caused by M1R overexpression by sequestering G‐proteins (Gα12/13) to the receptor. In addition, Gα12/13 null HEK293 cells exhibited increased polymerized tubulin and knockdown of Gα13 protected from tubulin cytoskeletal destabilization and restored growth in neurons overexpressing the M1R.ConclusionsOur study indicates that constitutively high expression of M1R destabilizes cytoskeletal dynamics through activation of Gα13 protein that increased tubulin de‐polymerization, impeded mitochondrial transport and reduced neurite outgrowth. This suggests a novel mechanism of M1R control of G protein‐dependent (Gα13) modulation of the tubulin cytoskeleton, mitochondrial trafficking and neurite outgrowth in axons of adult sensory neurons. Manipulation of this novel pathway could be used to treat dying‐back neuropathies since anti‐muscarinic drugs are currently utilized for other clinical conditions.Support or Funding InformationSupported by CIHR grant # MOP‐130282 (PF) and NIH grant NS081082 (NAC)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.