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 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 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.

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.

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 Expression of Cholinergic Markers and Characterization of Splice Variants during Ontogenesis of Rat Dorsal Root Ganglia Neurons

2021 ◽  
Vol 22 (11) ◽  
pp. 5499
Author(s):  
Veronica Corsetti ◽  
Carla Perrone-Capano ◽  
Michael Sebastian Salazar Intriago ◽  
Elisabetta Botticelli ◽  
Giancarlo Poiana ◽  
...  
Keyword(s):  
Dorsal Root Ganglia ◽  
Dorsal Root ◽  
Splice Variants ◽  
Multiple Roles ◽  
Pcr Analysis ◽  
Drg Neurons ◽  
Embryonic Developmental Stage ◽  
Embryo Stage ◽  
Dorsal Root Ganglia Neurons ◽  
Cholinergic Markers

Dorsal root ganglia (DRG) neurons synthesize acetylcholine (ACh), in addition to their peptidergic nature. They also release ACh and are cholinoceptive, as they express cholinergic receptors. During gangliogenesis, ACh plays an important role in neuronal differentiation, modulating neuritic outgrowth and neurospecific gene expression. Starting from these data, we studied the expression of choline acetyltransferase (ChAT) and vesicular ACh transporter (VAChT) expression in rat DRG neurons. ChAT and VAChT genes are arranged in a “cholinergic locus”, and several splice variants have been described. Using selective primers, we characterized splice variants of these cholinergic markers, demonstrating that rat DRGs express R1, R2, M, and N variants for ChAT and V1, V2, R1, and R2 splice variants for VAChT. Moreover, by RT-PCR analysis, we observed a progressive decrease in ChAT and VAChT transcripts from the late embryonic developmental stage (E18) to postnatal P2 and P15 and in the adult DRG. Interestingly, Western blot analyses and activity assays demonstrated that ChAT levels significantly increased during DRG ontogenesis. The modulated expression of different ChAT and VAChT splice variants during development suggests a possible differential regulation of cholinergic marker expression in sensory neurons and confirms multiple roles for ACh in DRG neurons, both in the embryo stage and postnatally.

Neurotoxicity of Seven MEIC Chemicals Evaluated in Organotypic Cultures of Chick Embryonic Dorsal Root Ganglia

1997 ◽  
Vol 25 (3) ◽  
pp. 303-309
Author(s):  
Václav Mandys ◽  
Katerina Jirsová ◽  
Jirí Vrana
Keyword(s):  
Toxic Effect ◽  
Neurite Outgrowth ◽  
Dorsal Root Ganglia ◽  
Dorsal Root ◽  
Growth Parameters ◽  
In Vitro Cytotoxicity ◽  
Organotypic Cultures ◽  
Response Curves ◽  
Agar Culture

The neurotoxic effects of seven selected Multicenter Evaluation of In Vitro Cytotoxicity programme chemicals (methanol, ethanol, isopropanol, sodium chloride, potassium chloride, iron [II] sulphate and chloroform) were evaluated in organotypic cultures of chick embryonic dorsal root ganglia (DRG), maintained in a soft agar culture medium. Two growth parameters of neurite outgrowth from the ganglia — the mean radial length of neurites and the area of neurite outgrowth — were used to evaluate the toxicities of the chemicals. Dose-dependent decreases of both parameters were observed in all experiments. IC50 values (the concentration causing 50% inhibition of growth) were calculated from the dose-response curves established at three time-points during culture, i.e. 24, 48 and 72 hours. The lowest toxic effect was observed in cultures exposed to methanol (the IC50 ranging from 580mM to 1020mM). The highest toxic effect was observed in cultures exposed to iron (II) sulphate (the IC50 ranging from 1.2mM to 1.7mM). The results of other recent experiments suggest that organotypic cultures of DRG can be used during in vitro studies on target organ toxicity within the peripheral nervous system. Moreover, these cultures preserve the internal organisation of the tissue, maintain intercellular contacts, and thus reflect the in vitro situation, more precisely than other cell cultures.

Ty1 in vitro integration: effects of mutations in cis and in trans

1994 ◽  
Vol 14 (9) ◽  
pp. 5731-5740
Author(s):  
L T Braiterman ◽  
J D Boeke
Keyword(s):  
Missense Mutation ◽  
Frameshift Mutations ◽  
Amino Terminal ◽  
Dna Molecule ◽  
Conserved Domain ◽  
In Trans ◽  
In Cis ◽  
Obvious Similarity ◽  
Insertion Mutations

Mutations within the TYB gene of Ty1 encoding integrase (IN) as well as alterations in its substrate, a linear DNA molecule, were examined for their effects on in vitro IN activity, using a recently developed physical assay. Five different codon-insertion mutations, two frameshift mutations, and one missense mutation, previously identified as transposition-deficient mutations, were tested. Virus-like particles, the source of IN, from two different protease mutants and a reverse transcriptase mutant exhibited near-normal to normal IN activity. Two frameshift mutations mapping within the phylogenetically variable C-terminal domain of IN resulted in significant in vitro IN activity. In contrast, three mutations within the amino-terminal conserved domain of IN completely abolished IN activity. When the substrate termini were mutated, we found that substrates with as few as 4 bp of Ty1 termini were capable of efficiently generating integration products. Surprisingly, certain substrates that lacked obvious similarity to Ty1 termini were also readily integrated into both linear and circular targets, whereas others were not used as substrates at all. Termini rich in adenosine residues were among the more active substrates; however, certain substrates lacking terminal adenosine residues can form small quantities of integration products, including complete integration reactions.

Dorsal root ganglia cocultured with macrophages: an in vitro model to study experimental demyelination

1994 ◽  
Vol 88 (5) ◽  
pp. 459-464 ◽  
Author(s):  
W. Br�ck ◽  
Y. Br�ck ◽  
U. Diederich ◽  
R. L. Friede
Keyword(s):  
Dorsal Root Ganglia ◽  
Dorsal Root ◽  
In Vitro Model ◽  
Experimental Demyelination ◽  
Vitro Model
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