Dorsal Root Ganglion Cell Cultures Research Articles

Calcitonin receptor, calcitonin gene-related peptide and amylin distribution in C1/2 dorsal root ganglia

BackgroundThe upper cervical dorsal root ganglia (DRG) are important for the transmission of sensory information associated with the back of the head and neck, contributing to head pain. Calcitonin receptor (CTR)-based receptors, such as the amylin 1 (AMY1) receptor, and ligands, calcitonin gene-related peptide (CGRP) and amylin, have been linked to migraine and pain. However, the contribution of this system to nociception involving the cervical DRG is unclear. Therefore, this study aimed to determine the relative distribution of the CTR, CGRP, and amylin in upper cervical DRG.MethodsCTR, CGRP, and amylin immunofluorescence was examined relative to neural markers in C1/2 DRG from male and female mice, rats, and human cases. Immunofluorescence was supported by RNA-fluorescence in situ hybridization examining amylin mRNA distribution in rat DRG.ResultsAmylin immunofluorescence was observed in neuronal soma and fibres. Amylin mRNA (Iapp) was also detected. Amylin and CGRP co-expression was observed in 19% (mouse), 17% (rat), and 36% (human) of DRG neurons in distinct vesicle-like neuronal puncta from one another. CTR immunoreactivity was present in DRG neurons, and both peptides produced receptor signalling in primary DRG cell cultures. CTR-positive neurons frequently co-expressed amylin and/or CGRP (66% rat; 84% human), with some sex differences.ConclusionsAmylin and CGRP could both be local peptide agonists for CTR-based receptors in upper cervical DRG, potentially acting through autocrine and/or paracrine signalling mechanisms to modulate neuron function. Amylin and its receptors could represent novel pain targets.

Celecoxib alleviates nociceptor sensitization mediated by interleukin-1beta-primed annulus fibrosus cells

PurposeThis study aims to analyze the effect of pro-inflammatory cytokine-stimulated human annulus fibrosus cells (hAFCs) on the sensitization of dorsal root ganglion (DRG) cells. We further hypothesized that celecoxib (cxb) could inhibit hAFCs-induced DRG sensitization.MethodshAFCs from spinal trauma patients were stimulated with TNF-α or IL-1β. Cxb was added on day 2. On day 4, the expression of pro-inflammatory and neurotrophic genes was evaluated using RT-qPCR. Levels of prostaglandin E2 (PGE-2), IL-8, and IL-6 were measured in the conditioned medium (CM) using ELISA. hAFCs CM was then applied to stimulate the DRG cell line (ND7/23) for 6 days. Then, calcium imaging (Fluo4) was performed to evaluate DRG cell sensitization. Both spontaneous and bradykinin-stimulated (0.5 μM) calcium responses were analyzed. The effects on primary bovine DRG cell culture were performed in parallel to the DRG cell line model.ResultsIL-1ß stimulation significantly enhanced the release of PGE-2 in hAFCs CM, while this increase was completely suppressed by 10 µM cxb. hAFCs revealed elevated IL-6 and IL-8 release following TNF-α and IL-1β treatment, though cxb did not alter this. The effect of hAFCs CM on DRG cell sensitization was influenced by adding cxb to hAFCs; both the DRG cell line and primary bovine DRG nociceptors showed a lower sensitivity to bradykinin stimulation.ConclusionCxb can inhibit PGE-2 production in hAFCs in an IL-1β-induced pro-inflammatory in vitro environment. The cxb applied to the hAFCs also reduces the sensitization of DRG nociceptors that are stimulated by the hAFCs CM.

Notch signaling is activated in knee-innervating dorsal root ganglia in experimental models of osteoarthritis joint pain

BackgroundWe aimed to explore activation of the Notch signaling pathway in knee-innervating lumbar dorsal root ganglia (DRG) in the course of experimental osteoarthritis (OA) in mice, and its role in knee hyperalgesia.MethodsCultured DRG cells were stimulated with the TLR4 agonist, lipopolysaccharide (LPS). Notch signaling in the cells was either inhibited with the γ-secretase inhibitor, DAPT, or with soluble Jagged1, or activated through immobilized Jagged1. CCL2 production was analyzed at mRNA and protein levels. In in vivo experiments, knee hyperalgesia was induced in naïve mice through intra-articular (IA) injection of LPS. The effect of inhibiting Notch signaling was examined by pre-injecting DAPT one hour before LPS. OA was induced through surgical destabilization of the medial meniscus (DMM) in male C57BL/6 mice. Gene expression in DRG was analyzed by qRT-PCR and RNAscope in situ hybridization. Activated Notch protein (NICD) expression in DRG was evaluated by ELISA and immunofluorescence staining. DAPT was injected IA 12 weeks post DMM to inhibit Notch signaling, followed by assessing knee hyperalgesia and CCL2 expression in the DRG.ResultsIn DRG cell cultures, LPS increased NICD in neuronal cells. Inhibition of Notch signaling with either DAPT or soluble Jagged1 attenuated LPS-induced increases of Ccl2 mRNA and CCL2 protein. Conversely, activating Notch signaling with immobilized Jagged1 enhanced these LPS effects. In vivo, IA injection of LPS increased expression of Notch genes and NICD in the DRG. Pre-injection of DAPT prior to LPS alleviated LPS-induced knee hyperalgesia, and decreased LPS-induced CCL2 expression in the DRG. Notch signaling genes were differentially expressed in the DRG from late-stage experimental OA. Notch1, Hes1, and NICD were increased in the neuronal cell bodies in DRG after DMM surgery. IA administration of DAPT alleviated knee hyperalgesia post DMM, and decreased CCL2 expression in the DRG.ConclusionsThese findings suggest a synergistic effect of Notch signaling with TLR4 in promoting CCL2 production and mediating knee hyperalgesia. Notch signaling is activated in knee-innervating lumbar DRG in mice with experimental OA, and is involved in mediating knee hyperalgesia. The pathway may therefore be explored as a target for alleviating OA pain.

Hedgehog signaling plays a crucial role in hyperalgesia associated with neuropathic pain in mice.

Neuropathic pain is a debilitating chronic syndrome of the nervous system caused by nerve injury. In Drosophila, the Hedgehog (Hh) signaling pathway is related to increased pain sensitivity (hyperalgesia) but does not affect the baseline nociceptive threshold. In general, the contribution of the Hh signaling pathway to neuropathic pain in vertebrates is a highly debated issue. Alternatively, we investigated the potential role of Hh signaling in mechanical allodynia using a mouse model of neuropathic pain. Seven days after spinal nerve-transection (SNT) surgery, microglial activation increased in the ipsilateral spinal dorsal horn compared with that in the sham group; however, 21 days after surgery, microglial activation decreased. Contrastingly, astrocyte activation in the spinal cord did not differ between the groups. On day 21 of postsurgery, the SNT group showed marked upregulation of sonic hedgehog expression in peripheral glial cells but not in dorsal root ganglion (DRG) neurons. Intrathecal administration of the Hh signaling inhibitor vismodegib attenuated the mechanical allodynia observed on day 21 postsurgery. Conversely, intrathecal treatment with the Hh signaling activator smoothened agonist in naive mice induced mechanical allodynia, which was abolished by the ATP transporter inhibitor clodronate. Moreover, inhibition of Hh signaling by pretreatment with vismodegib significantly reduced ATP secretion and the frequency/number of spontaneous elevations of intracellular calcium ion levels in cultured DRG cells. Thus, the Hh signaling pathway appears to modulate the neural activity of DRG neurons via ATP release, and it plays an important role in sustaining mechanical allodynia and hypersensitivity in a mouse model of neuropathic pain.

NOTCH SIGNALING CONTRIBUTES TO THE DEVELOPMENT OF KNEE HYPERALGESIA IN MICE

Purpose: Notch signaling plays a fundamental role in neurogenesis and neuroplasticity. Aberrant Notch signaling also contributes to cartilage and bone damage in osteoarthritis (OA) joints. Our previous data showed that Notch signaling is activated in knee-innervating dorsal root ganglia (DRG) after surgical destabilization of the medial meniscus (DMM) in mice, and in vitro studies using DRG cell culture suggested that Notch signaling may interact with toll-like receptor (TLR) signaling to promote synthesis of the pro-algesic chemokine, C-C motif chemokine ligand 2 (CCL2).

In Vitro Model to Investigate Communication between Dorsal Root Ganglion and Spinal Cord Glia.

Chronic discogenic back pain is associated with increased inflammatory cytokine levels that can influence the proximal peripheral nervous system, namely the dorsal root ganglion (DRG). However, transition to chronic pain is widely thought to involve glial activation in the spinal cord. In this study, an in vitro model was used to evaluate the communication between DRG and spinal cord glia. Primary neonatal rat DRG cells were treated with/without inflammatory cytokines (TNF-α, IL-1β, and IL-6). The conditioned media were collected at two time points (12 and 24 h) and applied to spinal cord mixed glial culture (MGC) for 24 h. Adult bovine DRG and spinal cord cell cultures were also tested, as an alternative large animal model, and results were compared with the neonatal rat findings. Compared with untreated DRG-conditioned medium, the second cytokine-treated DRG-conditioned medium (following medium change, thus containing solely DRG-derived molecules) elevated CD11b expression and calcium signal in neonatal rat microglia and enhanced Iba1 expression in adult bovine microglia. Cytokine treatment induced a DRG-mediated microgliosis. The described in vitro model allows the use of cells from large species and may represent an alternative to animal pain models (3R principles).

Leptin Contributes to Neuropathic Pain via Extrasynaptic NMDAR-nNOS Activation

Leptin is an adipocytokine that is primarily secreted by white adipose tissue, and it contributes to the pathogenesis of neuropathic pain in collaboration with N-methyl-D-aspartate receptors (NMDARs). Functional NMDARs are a heteromeric complex that primarily comprise two NR1 subunits and two NR2 subunits. NR2A is preferentially located at synaptic sites, and NR2B is enriched at extrasynaptic sites. The roles of synaptic and extrasynaptic NMDARs in the contribution of leptin to neuropathic pain are not clear. The present study examined whether the important role of leptin in neuropathic pain was related to synaptic or extrasynaptic NMDARs. We used a rat model of spared nerve injury (SNI) and demonstrated that the intrathecal administration of the NR2A-selective antagonist NVP-AAM077 and the NR2B-selective antagonist Ro25-6981 prevented and reversed mechanical allodynia following SNI. Administration of exogenous leptin mimicked SNI-induced behavioral allodynia, which was also prevented by NVP-AAM077 and Ro25-6981. Mechanistic studies showed that leptin enhanced NR2B- but not NR2A-mediated currents in spinal lamina II neurons of naïve rats. Leptin also upregulated the expression of NR2B, which was blocked by the NR2B-selective antagonist Ro25-6981, in cultured dorsal root ganglion (DRG) neurons. Leptin enhanced neuronal nitric oxide synthase (nNOS) expression, which was also blocked by Ro25-6981, in cultured DRG cells. However, leptin did not change NR2A expression, and the NR2A-selective antagonist NVP-AAM077 had no effect on leptin-enhanced nNOS expression. Our data suggest an important cellular link between the spinal effects of leptin and the extrasynaptic NMDAR-nNOS-mediated cellular mechanism of neuropathic pain.

Кріоконсервування культури клітин, отриманої зі спінальних гангліїв неонатальних поросят

The effect of cryopreservation with various concentrations of dimethyl sulfoxide (DMSO) on morphofunctional properties of the dorsal root ganglia cell culture (DRGCC) was investigated in this research. Cells were obtained from the dorsal root ganglia of neonatal piglets and cultured for 7 days in -MEM with 10% fetal calf serum (FCS). These conditions promote a predominant growth of satellite glial cells (SGCs). The resulting culture was cryopreserved at a rate of 0.5 deg / min to –20°C at stage 1, and at 1 deg / min to -80°C at stage 2, afterwards the samples were immersed into liquid nitrogen. Cryoprotective solutions based on α-MEM, 25% FBS, and DMSO at final concentrations of 5, 7.5, and 10% were used. After warming on day 10 of subculturing, the cell viability, relative monolayer area, and glutamine synthetase expression as a marker of SGCs were evaluated. It has been established that cryopreservation of DRGCC using 7.5% DMSO provided 87.7% of viable cells after warming and 85% relative monolayer area in respect of an intact control. The amount of SGCs was about 95%. The obtained results allow us to recommend the chosen regimen for low temperature storage of cell cultures enriched with MG. Probl Cryobiol Cryomed 2020; 30(2): 158–168

Direct and Intervertebral Disc-Mediated Sensitization of Dorsal Root Ganglion Neurons by Hypoxia and Low pH.

ObjectiveIschemia-related risk factors are consistently correlated with discogenic pain, but it remains unclear how the ischemia-associated hypoxia and acidosis influence the peripheral sensory nervous system, namely the dorsal root ganglion (DRG), either directly or indirectly via intervertebral disc (IVD) mediation. MethodsBovine tail IVD organ cultures were preconditioned in different hypoxic and/or acidic conditions for 3 days to collect the conditioned medium (CM). The DRG-derived ND7/23 cells were either treated by the IVD CM or directly stimulated by hypoxic and/or acidic conditions. Neuronal sensitization was evaluated using calcium imaging (Fluo-4) after 3 days. ResultsWe found that direct exposure of DRG cell line to hypoxia and acidosis increased both spontaneous and bradykinin-stimulated calcium response compared to normoxia-neutral pH cultures. Hypoxia and low pH in combination showed stronger effect than either parameter on its own. Indirect exposure of DRG to hypoxia-acidosis-stressed IVD CM also increased spontaneous and bradykinin-stimulated response, but to a lower extent than direct exposure. The impact of direct hypoxia and acidosis on DRG was validated in a primary sheep DRG cell culture, showing the same trend. ConclusionOur data suggest that targeting hypoxia and acidosis stresses both in IVD and DRG could be a relevant objective in discogenic pain treatment.

Activation of EphB receptors contributes to primary sensory neuron excitability by facilitating Ca2+ influx directly or through Src kinase-mediated N-methyl-D-aspartate receptor phosphorylation.

EphrinB-EphB receptor tyrosine kinases have been demonstrated to play important roles in pain processing after peripheral nerve injury. We have previously reported that ephrinB-EphB receptor signaling can regulate excitability and plasticity of neurons in spinal dorsal horn, and thus contribute to spinal central sensitization in neuropathic pain. How EphB receptor activation influences excitability of primary neurons in dorsal root ganglion (DRG), however, remains unknown. Here, we report that EphB receptor activation facilitates calcium influx through N-methyl-D-aspartate receptor (NMDAR) dependent and independent manners. In cultured DRG cells from adult rats, EphB1 and EphB2 receptors were expressed in neurons, but not the glial cells. Bath application of EphB receptor agonist ephrinB2-Fc induced NMDAR-independent Ca influx, which was from the extracellular space rather than endoplasmic reticulum. EphB receptor activation also greatly enhanced NMDAR-dependent Ca influx and NR2B phosphorylation, which was prevented by pretreatment of Src kinase inhibitor PP2. In nerve-injured DRG neurons, elevated expression and activation of EphB1 and EphB2 receptors contributed to the increased intracellular Ca concentration and NMDA-induced Ca influx. Repetitive intrathecal administration of EphB2-Fc inhibited the increased phosphorylation of NR2B and Ca-dependent subsequent signals Src, ERK, and CaMKII as well as behaviorally expressed pain after nerve injury. These findings demonstrate that activation of EphB receptors can modulate DRG neuron excitability by facilitating Ca influx directly or through Src kinase activation-mediated NMDA receptor phosphorylation and that EphB receptor activation is critical to DRG neuron hyperexcitability, which has been considered critical to the subsequent spinal central sensitization and neuropathic pain.

Calcium imaging of primary canine sensory neurons: Small-diameter neurons responsive to pruritogens and algogens.

IntroductionRodent primary sensory neurons are commonly used for studying itch and pain neurophysiology, but translation from rodents to larger mammals and humans is not direct and requires further validation to make correlations.MethodsThis study developed a primary canine sensory neuron culture from dorsal root ganglia (DRG) excised from cadaver dogs. Additionally, the canine DRG cell cultures developed were used for single‐cell ratiometric calcium imaging, with the activation of neurons to the following pruritogenic and algogenic substances: histamine, chloroquine, canine protease‐activated receptor 2 (PAR2) activating peptide (SLIGKT), compound 48/80, 5‐hydroxytryptamine receptor agonist (5‐HT), bovine adrenal medulla peptide (BAM8‐22), substance P, allyl isothiocyanate (AITC), and capsaicin.ResultsThis study demonstrates a simple dissection and rapid processing of DRG collected from canine cadavers used to create viable primary sensory neuron cultures to measure responses to pruritogens and algogens.ConclusionRatiometric calcium imaging demonstrated that small‐diameter canine sensory neurons can be activated by multiple stimuli, and a single neuron can react to both a pruritogenic stimulation and an algogenic stimulation.

Bone marrow mesenchymal stem cell-derived exosome uptake and retrograde transport can occur at peripheral nerve endings

We investigated the occurrence of mesenchymal stem cell (MSC)-derived exosome uptake and retrograde transport at peripheral nerve endings using bone marrow MSCs (bMSCs) transduced with recombinant CD63-green fluorescent protein (GFP) lentiviral plasmid. GFP was used to track the release of bMSC-derived exosomes and the uptake and transport at peripheral nerve terminals, the dorsal root ganglion (DRG), and the spinal cord. In vitro cell culture and injection of a CD63-GFP exosome suspension into the right gastrocnemius muscle of an in vivo rat model were also performed. Fluorescence microscopy of co-cultured CD63-GFP exosomes and SH-SY5Y or BV2 cell lines and primary cultured DRG cells in a separate experiment demonstrated exosome uptake into DRG neurons and glia. Moreover, we observed both retrograde axoplasmic transport and hematogenous transport of exosomes injected into rat models at the DRG and the ipsilateral side of the anterior horn of the spinal cord using fluorescence microscopy, immunohistochemistry, and Western blot analyses. In conclusion, we showed that exosome uptake at peripheral nerve endings and retrograde transport of exosomes to DRG neurons and spinal cord motor neurons in the anterior horn can occur. In addition, our findings propose a novel drug delivery approach for treating neuronal diseases.

Formation of aligned core/sheath microfiber scaffolds with a poly-L-lactic acid (PLLA) sheath and a conductive poly(3,4-ethylenedioxythiophene) (PEDOT) core

Abstract

Sympathoexcitation in response to cardiac and pulmonary afferent stimulation of TRPA1 channels is attenuated in rats with chronic heart failure.

Excessive sympathoexcitation characterizes the chronic heart failure (CHF) state. An exaggerated cardiac sympathetic afferent reflex (CSAR) contributes to this sympathoexcitation. Prior studies have demonstrated that the CSAR to capsaicin [transient receptor potential (TRP) vanilloid 1 agonist] is exaggerated in CHF animal models. We recently discovered that capsaicin application to the lung visceral pleura in anesthetized, vagotomized, open-chested rats increases mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA). We named this response the pulmonary spinal afferent reflex (PSAR). Due to the similarities between TRP vanilloid 1 and TRP ankyrin 1 (TRPA1) channels as well as the excessive sympathoexcitation of CHF, we hypothesized that stimulation of the CSAR and PSAR with a specific TRPA1 agonist would result in an augmented response in CHF rats (coronary ligation model) compared with sham control rats. In response to a TRPA1 agonist, both CSAR and PSAR in sham rats resulted in biphasic changes in MAP and increases in HR and RSNA 10-12 wk postmyocardial infarction (post-MI). These effects were blunted in CHF rats. Assessment of TRPA1 expression levels in cardiopulmonary spinal afferents by immunofluorescence, quantitative RT-PCR, and Western blot analysis 10-12 wk post-MI all indicates reduced expression in CHF rats but no reduction at earlier time points. TRPA1 protein was reduced in a dorsal root ganglia cell culture model of inflammation and simulated tissue ischemia, raising the possibility that the in vivo reduction of TRPA1 expression was, in part, caused by CHF-related tissue ischemia and inflammation. These data provide evidence that reflex responses to cardiopulmonary spinal afferent TRPA1 stimulation may be attenuated in CHF rather than enhanced. NEW & NOTEWORTHY Excessive sympathoexcitation characterizes chronic heart failure (CHF). The contribution of transient receptor potential ankyrin 1 (TRPA1) channel-mediated reflexes to this sympathoexcitation is unknown. We found that application of TRPA1 agonist to the heart and lung surface resulted in increased heart rate and sympathetic output and a biphasic change in mean arterial pressure in control rats. These effects were attenuated in CHF rats, decreasing the likelihood that TRPA1 channels contribute to cardiopulmonary afferent sensitization in CHF.

Effect of Cryopreservation on Different Passages of Porcine Dorsal Root Ganglion Cell Culture

The cell culture obtained from dorsal root ganglia (DRG) is a valuable model used in biology or medical research. However, the effect of cryopreservation on the properties of DRG-derived cell culture of different passages remains unclear up to date. The objective of the study is to assess the effect of cryopreservation with various concentrations of cryoprotectant dimethyl sulfoxide (DMSO) on the viability and morphological features of porcine neonatal DRG cell culture of different passages. Cell suspension was obtained from DRG of neonatal piglets and cultured in an α-MEM nutritional medium supplemented with 10% fetal calf serum (0, 1st, and 2nd passages). Cells of different passages were cryopreserved at a cooling rate of 0.5°C/min to – 20°C (step 1) and 1°C/min to – 80°C (step 2) followed by immersion into liquid nitrogen. Cryoprotective solutions based on α-MEM nutrient medium and 25% fetal calf serum (FCS) containing 5%, 7.5%, and 10% DMSO were used. It was established that the primary culture (passage 0) consisted of four cell morphological types: large rounded cell bodies of sensory neurons (SN) and three types of non-neuronal cells, namely, polygonal cells with pronounced elongated processes (type 1), spindle-shaped cells (type 2), and multipolar flattened fibroblastoid cells (type 3). As the number of passages increases, an elimination of SN from the culture, a decrease in the relative number of 1st and 2nd cell types, and expansion of 3rd cell type were observed. DRG cell culture had sufficiently high resistance to cryopreservation as cell viability was in the range from 83% to 90% using different concentrations of DMSO. The cells of passage 1 were more resistant to cryopreservation in comparison with primary culture cells (passage 0). The best result was achieved by freezing the culture of passage 1 in the cryoprotective medium with 7.5% DMSO, where 90.6% of viable cells were observed after thawing.

Effects of RSC96 Schwann Cell-Derived Exosomes on Proliferation, Senescence, and Apoptosis of Dorsal Root Ganglion Cells In Vitro.

BackgroundStress urinary incontinence is a common condition in women and can be associated with peripheral nerve injury. Exosomes. derived from Schwann cells, can enhance the regeneration of axons of the peripheral nervous system. This study aimed to investigate the effects of RSC96 Schwann cell-derived exosomes in a novel in vitro model of dorsal root ganglion (DRG) cell injury induced by cyclic mechanical strain (CMS).Material/MethodsRSC96 Schwann cells and DRG cells were cultured in vitro. CMS in DRG cells involved mechanical stretch trauma with 5333 μ strain. ExoQuick-TC polymer was used to precipitate exosomes from RSC96 Schwann cell culture medium and identified by nanoparticle tracking analysis, electron microscopy, and Western blot for detection of CD9 and tumor susceptibility gene 101 (Tsg101) protein. Cultured DRG cells were treated with RSC96 Schwann cell-derived exosomes, followed by measurement of cell viability, proliferation, senescence, and apoptosis using the cell counting kit-8 (CCK-8) assay, senescence-associated beta-galactosidase (SA-β-gal) staining, and Hoechst 33258 (blue) fluorescence nucleic acid staining using flow cytometry.ResultsMechanical stretch with 5333 μ strain for 8 hours at 1 Hz decreased the activity of cultured DRG cells. RSC96 Schwann cell-derived exosomes promoted cell proliferation and significantly inhibited apoptosis and senescence of DRG cells following injury induced by CMS.ConclusionsAn in vitro model of DRG cell injury induced by CMS, showed that RSC96 Schwann cell-derived exosomes had a protective effect. The effects of Schwann cell-derived exosomes on peripheral nerve injury, including in stress urinary incontinence, require future in vivo studies.

Dorsal Root Ganglia Isolation and Primary Culture to Study Neurotransmitter Release.

Dorsal root ganglia (DRG) contain cell bodies of sensory neurons. This type of neuron is pseudo-unipolar, with two axons that innervate peripheral tissues, such as skin, muscle and visceral organs, as well as the spinal dorsal horn of the central nervous system. Sensory neurons transmit somatic sensation, including touch, pain, thermal, and proprioceptive sensations. Therefore, DRG primary cultures are widely used to study the cellular mechanisms of nociception, physiological functions of sensory neurons, and neural development. The cultured neurons can be applied in studies involving electrophysiology, signal transduction, neurotransmitter release, or calcium imaging. With DRG primary cultures, scientists may culture dissociated DRG neurons to monitor biochemical changes in single or multiple cells, overcoming many of the limitations associated with in vivo experiments. Compared to commercially available DRG-hybridoma cell lines or immortalized DRG neuronal cell lines, the composition and properties of the primary cells are much more similar to sensory neurons in tissue. However, due to the limited number of cultured DRG primary cells that can be isolated from a single animal, it is difficult to perform high-throughput screens for drug targeting studies. In the current article, procedures for DRG collection and culture are described. In addition, we demonstrate the treatment of cultured DRG cells with an agonist of neuropeptide FF receptor type 2 (NPFFR2) to induce the release of peptide neurotransmitters (calcitonin gene-related peptide (CRGP) and substance P (SP)).

Insulin Potentiates Neuronal Responses to Chemical Stimulation in Thin Muscle Afferents and Dorsal Root Ganglia

The cardiovascular response to physical activity is abnormally exaggerated in patients with type 2 diabetes mellitus (T2D). Recent studies in patients with T2D have suggested that this exaggerated responsiveness is mediated, in part, by the skeletal muscle metaboreflex. However, the mechanisms causing augmentations in muscle metaboreflex function in this disease remain to be elucidated. Chronic hyperinsulinemia associated with peripheral insulin resistance is one of the pathophysiological characteristics of T2D. Evidence suggests that transient receptor potential vanilloid receptors, which contribute to metaboreflex activation, are more responsive to stimuli in the presence of insulin. Given that metaboreflex afferent fibers reside in skeletal muscle, it is suggested that hyperinsulinemia may underlie the skeletal muscle metaboreflex overactivity manifest in T2D. PURPOSE: To examine the impact of insulin on neuronal responses to chemical stimulation in thin muscle afferents and dorsal root ganglia (DRG) of normal healthy rodents. It was hypothesized that insulin potentiates the activity of metabolically sensitive afferent neurons. METHODS: Chemically activated neurons were assessed by single-fiber recordings from rat muscle-nerve preparations in vitro and by whole cell patch-clamp recordings from cultured mice DRG neurons. The magnitude of responses to capsaicin stimulation and the capsaicin activated current were recorded, respectively. RESULTS: Compared to control conditions, thin muscle afferent response magnitude was significantly increased by insulin exposure (0.03±0.03 vs. 0.25±0.02 imp, n=3, P<0.05). In DRG cell culture, total charge transfer by capsaicin activated current was largely augmented by insulin administration (403±159 % changes from control conditions, n=3, P=0.11). CONCLUSIONS: These data demonstrate that thin muscle afferent as well as DRG neuronal responses to capsaicin are augmented by insulin exposure in normal healthy animals. The data support the concept that chronic hyperinsulinemia may potentiate skeletal muscle metaboreflex function in T2D contributing to the abnormal cardiovascular response to exercise characteristic of this disease. Supported by the Lawson & Rogers Lacy Research Fund in Cardiovascular Disease and JSPS KAKENHI JP17K01769

Neural Responses to Mechanical Stimulation in Thin Muscle Afferents and Dorsal Root Ganglia are Sensitized by Insulin

In patients with type 2 diabetes mellitus (T2D), the circulatory responses to physical activity are abnormally exaggerated. Recent studies in patients with T2D have suggested that this exaggerated cardiovascular response is mediated, in part, by the exercise pressor reflex (EPR) which is a primary contributor to autonomic cardiovascular control during exercise. However, the mechanisms underlying this abnormal EPR function in T2D remain unknown. The pathophysiology of T2D is characterized by insulin resistance induced hyperinsulinemia. Further, evidence suggests that insulin increases the sensitivity of transient receptor potential vanilloid receptors localized to muscle afferent neurons that contribute to EPR activation. Importantly, sensitization of muscle receptors associated with the mechanically sensitive component of the EPR (i.e. the muscle mechanoreflex) has been shown to mediate reflex overactivity in other cardiovascular disease states such as heart failure and hypertension. Given this information, it is logical to suggest that excess levels of insulin may potentiate mechanoreflex function in T2D.PURPOSETo examine the effect of insulin on neuronal responses to mechanical stimulation in thin muscle afferents and dorsal root ganglia (DRG) of normal healthy rodents. We hypothesized that insulin sensitizes the activity of mechanically sensitive afferent neurons.METHODSMechanically activated neurons were assessed before (control) and after exposure to insulin 1) by single‐fiber recordings from rat muscle‐nerve preparations in vitro and 2) by whole cell patch‐clamp recordings from cultured mice DRG neurons. The magnitude of the response to mechanical stimulation as well as the mechanical threshold were assessed.RESULTSCompared to control, the mechanical threshold of thin muscle afferents was significantly decreased after insulin administration (58±16 vs. 15±17 mN, n=8, P&lt;0.05) while the response magnitude was not changed (66±22 vs. 68±16 spikes, n=8). In DRG cell culture, mechanically activated current was significantly augmented by insulin (−93 ±12 vs. −190±43 pA, n=16, P&lt;0.05) whereas the mechanical threshold was significantly decreased (4.6±0.4 vs. 2.6±0.3 steps, n=17, P&lt;0.05). Further, after pretreatment of DRG insulin receptors with the inhibitor GSK1838705, insulin failed to augment the mechanically activated current (−91±19 vs. −92±19 pA, n=14) or to reduce the mechanical threshold (3.5 ± 0.4 vs. 3.4 ± 0.5 steps, n=11).CONCLUSIONSThese data demonstrate that thin muscle afferent and DRG neuronal responses to mechanical stimulation are sensitized by the presence of insulin in normal healthy animals. Importantly, the findings provide support for the concept that chronic hyperinsulinemia enhances skeletal muscle mechanoreflex function in T2D contributing to the abnormal cardiovascular response during exercise in this disease.Support or Funding InformationSupported by the Lawson &amp; Rogers Lacy Research Fund in Cardiovascular Disease and JSPS KAKENHI JP17K01769This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Influence of nutrient medium composition on the morphological characteristics of culture of dorsal root ganglion cells of neonatal piglets

Dorsal root ganglion (DRG) is a potential source of neural stem cells because it contains neural crest derived cells that are capable to differentiate into neurons and glial cells. Cell cultures obtained from animals that are close to humans by physiological characteristics can be regarded as an adequate modern model for in vitro studies. In this respect, DRG cell culture obtained from the domestic pig (Sus scrofa domesticus) is a convenient model. The aim of the work was to obtain a primary cell culture of DRG of neonatal piglets and to study its morphological and proliferative properties depending on culture medium composition. The composition of the media prepared on the basis of α-MEM varied depending on the presence of fetal calf serum (FCS) or its modern supplements B-27 and NeuroMax. It is established that morphological differences of primary DRG cell cultures of neonatal pigs depend on the composition of the nutrient medium. When cultured in the presence of 10% FCS, the formation of monolayer which includes satellite glial cells (SGC) and fibroblast-like cells was observed. Small colonies of neurons producing long processes were on the monolayer. When cultured in the presence of NeuroMax and B-27 supplements, the bulk of the cells is not attached, but organized into floating multicellular spheroids (MS). With the passage of culture obtained in the presence of 10% FCS, rapid attachment and proliferation of cells was observed. When MS obtained in the presence of NeuroMax and B-27 were transferred to the medium with 10% FCS, the attachment of MS to the substrate and cell migration were observed. The cells retain the ability to actively proliferate, because the monolayer achieves confluence by 5–7 days of subculture. Regardless of the composition of the primary culture medium, there were 3 morphologically different types of cells in the subcultures: SGC, neuron-like and fibroblast-like cells. The type of cells prevailing in the subculture depends on the composition of the nutrient medium. When MS is transferred from a B-27-containing medium, a significant growth of fibroblast-like cells is observed, whereas when MS is transferred from NeuroMax-containing medium MG and neuron-like cells were abundant.