scholarly journals Adult mouse dorsal root ganglia neurons form aberrant glutamatergic connections in dissociated cultures

PLoS ONE ◽  
2021 ◽  
Vol 16 (3) ◽  
pp. e0246924
Author(s):  
F. Kemal Bayat ◽  
Betul Polat Budak ◽  
Esra Nur Yiğit ◽  
Gürkan Öztürk ◽  
Halil Özcan Gülçür ◽  
...  
Keyword(s):  
Sensory Neurons ◽  
Dorsal Root Ganglia ◽  
Dorsal Root ◽  
Network Formation ◽  
Activity Patterns ◽  
Synaptic Coupling ◽  
Drg Neurons ◽  
Complex Activity ◽  
Evoked Activity

Cultured sensory neurons can exhibit complex activity patterns following stimulation in terms of increased excitability and interconnected responses of multiple neurons. Although these complex activity patterns suggest a network-like configuration, research so far had little interest in synaptic network formation ability of the sensory neurons. To identify interaction profiles of Dorsal Root Ganglia (DRG) neurons and explore their putative connectivity, we developed an in vitro experimental approach. A double transgenic mouse model, expressing genetically encoded calcium indicator (GECI) in their glutamatergic neurons, was produced. Dissociated DRG cultures from adult mice were prepared with a serum-free protocol and no additional growth factors or cytokines were utilized for neuronal sensitization. DRG neurons were grown on microelectrode arrays (MEA) to induce stimulus-evoked activity with a modality-free stimulation strategy. With an almost single-cell level electrical stimulation, spontaneous and evoked activity of GCaMP6s expressing neurons were detected under confocal microscope. Typical responses were analyzed, and correlated calcium events were detected across individual DRG neurons. Next, correlated responses were successfully blocked by glutamatergic receptor antagonists, which indicated functional synaptic coupling. Immunostaining confirmed the presence of synapses mainly in the axonal terminals, axon-soma junctions and axon-axon intersection sites. Concisely, the results presented here illustrate a new type of neuron-to-neuron interaction in cultured DRG neurons conducted through synapses. The developed assay can be a valuable tool to analyze individual and collective responses of the cultured sensory neurons.

scholarly journals Adult mouse sensory neurons on microelectrode arrays exhibit increased spontaneous and stimulus-evoked activity in the presence of interleukin-6

2018 ◽  
Vol 120 (3) ◽  
pp. 1374-1385 ◽  
Author(s):  
Bryan J. Black ◽  
Rahul Atmaramani ◽  
Rajeshwari Kumaraju ◽  
Sarah Plagens ◽  
Mario Romero-Ortega ◽  
...  
Keyword(s):  
Chronic Pain ◽  
Sensory Neurons ◽  
Dorsal Root ◽  
Inflammatory Cytokine ◽  
Adult Mouse ◽  
Microelectrode Arrays ◽  
Drg Neurons ◽  
Lead Discovery ◽  
Evoked Activity

Following inflammation or injury, sensory neurons located in the dorsal root ganglia (DRG) may exhibit increased spontaneous and/or stimulus-evoked activity, contributing to chronic pain. Current treatment options for peripherally mediated chronic pain are highly limited, driving the development of cell- or tissue-based phenotypic (function-based) screening assays for peripheral analgesic and mechanistic lead discovery. Extant assays are often limited by throughput, content, use of tumorigenic cell lines, or tissue sources from immature developmental stages (i.e., embryonic or postnatal). Here, we describe a protocol for culturing adult mouse DRG neurons on substrate-integrated multiwell microelectrode arrays (MEAs). This approach enables multiplexed measurements of spontaneous as well as stimulus-evoked extracellular action potentials from large populations of cells. The DRG cultures exhibit stable spontaneous activity from 9 to 21 days in vitro. Activity is readily evoked by known chemical and physical agonists of sensory neuron activity such as capsaicin, bradykinin, PGE2, heat, and electrical field stimulation. Most importantly, we demonstrate that both spontaneous and stimulus-evoked activity may be potentiated by incubation with the inflammatory cytokine interleukin-6 (IL-6). Acute responsiveness to IL-6 is inhibited by treatment with a MAPK-interacting kinase 1/2 inhibitor, cercosporamide. In total, these findings suggest that adult mouse DRG neurons on multiwell MEAs are applicable to ongoing efforts to discover peripheral analgesic and their mechanisms of action. NEW & NOTEWORTHY This work describes methodologies for culturing spontaneously active adult mouse dorsal root ganglia (DRG) sensory neurons on microelectrode arrays. We characterize spontaneous and stimulus-evoked adult DRG activity over durations consistent with pharmacological interventions. Furthermore, persistent hyperexcitability could be induced by incubation with inflammatory cytokine IL-6 and attenuated with cercosporamide, an inhibitor of the IL-6 sensitization pathway. This constitutes a more physiologically relevant, moderate-throughput in vitro model for peripheral analgesic screening as well as mechanistic lead discovery.

Neuronal intermediate filament expression in rat dorsal root ganglia sensory neurons: An in vivo and in vitro study

Neuroscience ◽  
2008 ◽  
Vol 153 (4) ◽  
pp. 1153-1163 ◽  
Author(s):  
M. Fornaro ◽  
J.M. Lee ◽  
S. Raimondo ◽  
S. Nicolino ◽  
S. Geuna ◽  
...  
Keyword(s):  
Sensory Neurons ◽  
Dorsal Root Ganglia ◽  
Intermediate Filament ◽  
Dorsal Root ◽  
In Vitro Study ◽  
Vitro Study

scholarly journals Despite Differences in Cytosolic Calcium Regulation, Lidocaine Toxicity Is Similar in Adult and Neonatal Rat Dorsal Root Ganglia In Vitro

2014 ◽  
Vol 120 (1) ◽  
pp. 50-61 ◽  
Author(s):  
Lisa V. Doan ◽  
Olga Eydlin ◽  
Boris Piskoun ◽  
Richard P. Kline ◽  
Esperanza Recio-Pinto ◽  
...  
Keyword(s):  
Dorsal Root Ganglia ◽  
Local Anesthetics ◽  
Dorsal Root ◽  
Caspase 3 ◽  
Cytosolic Calcium ◽  
Neonatal Rat ◽  
Dependent Manner ◽  
Drg Neurons ◽  
Induced Changes

Abstract Background: Neuraxial local anesthetics may have neurological complications thought to be due to neurotoxicity. A primary site of action of local anesthetics is the dorsal root ganglia (DRG) neuron. Physiologic differences have been noted between young and adult DRG neurons; hence, the authors examined whether there were any differences in lidocaine-induced changes in calcium and lidocaine toxicity in neonatal and adult rat DRG neurons. Methods: DRG neurons were cultured from postnatal day 7 (P7) and adult rats. Lidocaine-induced changes in cytosolic calcium were examined with the calcium indicator Fluo-4. Cells were incubated with varying concentrations of lidocaine and examined for viability using calcein AM and ethidium homodimer-1 staining. Live imaging of caspase-3/7 activation was performed after incubation with lidocaine. Results: The mean KCl-induced calcium transient was greater in P7 neurons (P < 0.05), and lidocaine significantly inhibited KCl-induced calcium responses in both ages (P < 0.05). Frequency distribution histograms of KCl-evoked calcium increases were more heterogeneous in P7 than in adult neurons. With lidocaine, KCl-induced calcium transients in both ages became more homogeneous but remained different between the groups. Interestingly, cell viability was decreased by lidocaine in a dose-dependent manner similarly in both ages. Lidocaine treatment also activated caspase-3/7 in a dose- and time-dependent manner similarly in both ages. Conclusions: Despite physiological differences in P7 and adult DRG neurons, lidocaine cytotoxicity is similar in P7 and adult DRG neurons in vitro. Differences in lidocaine- and KCl-evoked calcium responses suggest the similarity in lidocaine cytotoxicity involves other actions in addition to lidocaine-evoked effects on cytosolic calcium responses.

scholarly journals Cis and trans RET signaling control the survival and central projection growth of rapidly adapting mechanoreceptors

eLife ◽  
2015 ◽  
Vol 4 ◽  
Author(s):  
Michael S Fleming ◽  
Anna Vysochan ◽  
Sόnia Paixão ◽  
Jingwen Niu ◽  
Rüdiger Klein ◽  
...  
Keyword(s):  
Cell Survival ◽  
Dorsal Root Ganglia ◽  
Dorsal Root ◽  
Developmental Process ◽  
Central Projection ◽  
Drg Neurons ◽  
In Trans ◽  
Cis And Trans ◽  
In Cis

RET can be activated in cis or trans by its co-receptors and ligands in vitro, but the physiological roles of trans signaling are unclear. Rapidly adapting (RA) mechanoreceptors in dorsal root ganglia (DRGs) express Ret and the co-receptor Gfrα2 and depend on Ret for survival and central projection growth. Here, we show that Ret and Gfrα2 null mice display comparable early central projection deficits, but Gfrα2 null RA mechanoreceptors recover later. Loss of Gfrα1, the co-receptor implicated in activating RET in trans, causes no significant central projection or cell survival deficit, but Gfrα1;Gfrα2 double nulls phenocopy Ret nulls. Finally, we demonstrate that GFRα1 produced by neighboring DRG neurons activates RET in RA mechanoreceptors. Taken together, our results suggest that trans and cis RET signaling could function in the same developmental process and that the availability of both forms of activation likely enhances but not diversifies outcomes of RET signaling.

scholarly journals Mineralocorticoid Receptor Blocker Eplerenone Reduces Pain Behaviors In Vivo  and Decreases Excitability in Small-diameter Sensory Neurons from Local Inflamed Dorsal Root Ganglia In Vitro 

2012 ◽  
Vol 117 (5) ◽  
pp. 1102-1112 ◽  
Author(s):  
Fei Dong ◽  
Wenrui Xie ◽  
Judith A. Strong ◽  
Jun-Ming Zhang
Keyword(s):  
Sensory Neurons ◽  
Dorsal Root Ganglia ◽  
Dorsal Root ◽  
Mineralocorticoid Receptor ◽  
Small Diameter ◽  
Mechanical Hypersensitivity ◽  
Proinflammatory Role ◽  
Immunohistochemical Studies

Background Inflammation of the dorsal root ganglia (DRG) may contribute to low back pain, postherpetic neuralgia, and neuropathic pain. The mineralocorticoid receptor (MR) plays a proinflammatory role in many nonrenal tissues, but its role in peripheral pain at the DRG level is not well studied. Methods Local inflammation of the L5 DRG with the immune activator zymosan rapidly leads to mechanical hypersensitivity and increased excitability of sensory neurons. Using this pain model, the authors applied the MR antagonist eplerenone locally to the inflamed DRG. Excitability of small-diameter sensory neurons was examined in acute primary culture by using patch clamp techniques. Results Local eplerenone significantly reduced the mechanical hypersensitivity and shortened its duration. The same dose was ineffective systemically. Immunohistochemical studies showed the MR was present in most neurons and rapidly translocated to the nucleus 1 day after local DRG inflammation. Activation of satellite glia (defined by expression of glial fibrillary acidic protein) in the inflamed DRG was also reduced by local eplerenone. Increased excitability of small-diameter sensory neurons 1 day after inflammation could be observed in vitro. Eplerenone applied in vitro (8-12 h) could reverse this increased excitability. Eplerenone had no effect in neurons isolated from normal, uninflamed DRG. The MR agonist aldosterone (10 nM) applied in vitro increased excitability of neurons isolated from normal DRG. Conclusions The MR may have a pronociceptive role in the DRG. Some of its effects may be mediated by neuronal MR. The MR may represent a novel therapeutic target in some pain syndromes.

scholarly journals Oncomodulin derived from regeneration-associated macrophages in dorsal root ganglia promotes axon regeneration in the spinal cord

2021 ◽  
Author(s):  
Min Kwon ◽  
Yeojin Seo ◽  
Hana Cho ◽  
Jihye Choi ◽  
Hyung Soon Kim ◽  
...  
Keyword(s):  
Spinal Cord ◽  
Neurite Outgrowth ◽  
Dorsal Root Ganglia ◽  
Axonal Regeneration ◽  
Dorsal Root ◽  
Axon Regeneration ◽  
Signal Sequence ◽  
Drg Neurons ◽  
Cord Injury

Preconditioning peripheral nerve injury enhances axonal regeneration of dorsal root ganglia (DRG) neurons in part by driving pro-regenerative perineuronal macrophage activation. How these regeneration-associated macrophages influence the neuronal capacity of axon regeneration remains elusive. The present study reports that oncomodulin (ONCM) is an effector molecule derived from the regeneration-associated macrophages. ONCM was highly upregulated in DRG macrophages following preconditioning injury and necessary for the preconditioning-induced neurite outgrowth. ONCM-deficient macrophages failed to generate neurite outgrowth activity of the conditioned medium in the in vitro model of neuron-macrophage interaction. CCL2/CCR2 signaling is an upstream regulator of ONCM since the ONCM upregulation was dependent on CCR2 and CCL2 overexpression-mediated conditioning effects were attenuated in ONCM-deficient mice. Direct application of ONCM potently increased neurite outgrowth in cultured DRG neurons by activating a distinct gene set, particularly neuropeptide-related genes. AAV-mediated overexpression of ONCM construct with the signal sequence increased neuronal secretion of ONCM and enhanced neurite outgrowth in an autocrine manner. For a clinically relevant approach, we developed a nanogel-mediated system for localized delivery of recombinant ONCM to DRG tissue. Electrostatic encapsulation of ONCM by a reducible epsilon-poly(L-lysine)-nanogel (REPL-NG) resulted in a slow release of ONCM allowing sustained bioactivity. Intraganglionic injection of REPL-NG/ONCM complex achieved a remarkable long-range axonal regeneration beyond spinal cord lesion, surpassing the extent expected from the preconditioning effects. The NG-mediated ONCM delivery could be exploited as a therapeutic strategy for promoting sensory axon regeneration following spinal cord injury.

Two TTX-resistant Na+ currents in mouse colonic dorsal root ganglia neurons and their role in colitis-induced hyperexcitability

2004 ◽  
Vol 287 (4) ◽  
pp. G845-G855 ◽  
Author(s):  
Michael J. Beyak ◽  
Noor Ramji ◽  
Karmen M. Krol ◽  
Michael D. Kawaja ◽  
Stephen J. Vanner
Keyword(s):  
Sensory Neurons ◽  
Dorsal Root Ganglia ◽  
Dorsal Root ◽  
Neuronal Excitability ◽  
Distal Colon ◽  
Inactivation Kinetics ◽  
Drg Neurons ◽  
Trinitrobenzenesulfonic Acid ◽  
Na Currents ◽  
Dorsal Root Ganglia Neurons

The composition of Na+ currents in dorsal root ganglia (DRG) neurons depends on their neuronal phenotype and innervation target. Two TTX-resistant (TTX-R) Na+ currents [voltage-gated Na channels (Na v)] have been described in small DRG neurons; one with slow inactivation kinetics (Na v1.8) and the other with persistent kinetics (Na v1.9), and their modulation has been implicated in inflammatory pain. This has not been studied in neurons projecting to the colon. This study examined the relative importance of these currents in inflammation-induced changes in a mouse model of inflammatory bowel disease. Colonic sensory neurons were retrogradely labeled, and colitis was induced by instillation of trinitrobenzenesulfonic acid (TNBS) into the lumen of the distal colon. Seven to ten days later, immunohistochemical properties were characterized in controls, and whole cell recordings were obtained from small (<40 pF) labeled DRG neurons from control and TNBS animals. Most neurons exhibited both fast TTX-sensitive (TTX-S)- and slow TTX-R-inactivating Na+ currents, but persistent TTX-R currents were uncommon (<15%). Most labeled neurons were CGRP (79%), tyrosine kinase A (trkA) (84%) immunoreactive, but only a small minority bind IB4 (14%). TNBS-colitis caused ulceration, thickening of the colon and significantly increased neuronal excitability. The slow TTX-R-inactivating Na current density (Na v1.8) was significantly increased, but other Na currents were unaffected. Most small mouse colonic sensory neurons are CGRP, trkA immunoreactive, but not isolectin B4 reactive and exhibit fast TTX-S, slow TTX-R, but not persistent TTX-R Na+ currents. Colitis-induced hyperexcitability is associated with increased slow TTX-R (Na v1.8) Na+ current. Together, these findings suggest that colitis alters trkA-positive neurons to preferentially increase slow TTX-R Na+ (Na v1.8) currents.

scholarly journals α7-nAChR-mediated suppression of hyperexcitability of colonic dorsal root ganglia neurons in experimental colitis

2010 ◽  
Vol 299 (3) ◽  
pp. G761-G768 ◽  
Author(s):  
Galya R. Abdrakhmanova ◽  
Shakir AlSharari ◽  
Minho Kang ◽  
M. Imad Damaj ◽  
Hamid I. Akbarali
Keyword(s):  
Mouse Model ◽  
Action Potential ◽  
Dorsal Root Ganglia ◽  
Knockout Mice ◽  
Dorsal Root ◽  
Drg Neurons ◽  
Colonic Inflammation ◽  
Single Action ◽  
Α7 Nachr

Controlled clinical trials of nicotine transdermal patch for treatment of ulcerative colitis have been shown to improve histological and global clinical scores of colitis. Here we report that nicotine (1 μM) suppresses in vitro hyperexcitability of colonic dorsal root ganglia (DRG) (L1–L2) neurons in the dextran sodium sulfate (DSS)-induced mouse model of acute colonic inflammation. Nicotine gradually reduced regenerative multiple-spike action potentials in colitis mice to a single action potential. Nicotine's effect on hyperexcitability of inflamed neurons was blocked in the presence of an α7-nicotinic acetylcholine receptor (nAChR) antagonist, methyllicaconitine, while choline, the α7-nAChR agonist, induced a similar effect to that of nicotine. Consistent with these findings, nicotine failed to suppress hyperexcitability in colonic DRG neurons from DSS-treated α7 knockout mice. Furthermore, colonic DRG neurons from DSS-treated α7 knockout mice were characterized by lower rheobase (10 ± 5 vs. 77 ± 13 pA, respectively) and current threshold (28 ± 4 vs. 103 ± 8 pA, respectively) levels than DSS-treated C57BL/J6 mice. An interesting observation of this study is that 8 of 12 colonic DRG (L1–L2) neurons from control α7 knockout mice exhibited multiple-spike action potential firing while no wild-type neurons did. Overall, our findings suggest that nicotine at low 1 μM concentration suppresses in vitro hyperexcitability of inflamed colonic DRG neurons in a mouse model of acute colonic inflammation via activation of α7-nAChRs.

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