scholarly journals Determining of Apoptosis at in vitro Conditioned DRG Neurons with TUNEL Method

2017 ◽  
Vol 7 (2) ◽  
pp. 80-84
Author(s):  
Elif KAVAL OĞUZ
Keyword(s):  
Tunel Method ◽  
Drg Neurons

scholarly journals Exendin-4 Promotes Schwann Cell Survival/Migration and Myelination In Vitro

2021 ◽  
Vol 22 (6) ◽  
pp. 2971
Author(s):  
Shizuka Takaku ◽  
Masami Tsukamoto ◽  
Naoko Niimi ◽  
Hideji Yako ◽  
Kazunori Sango
Keyword(s):  
Phosphatidyl Inositol ◽  
Specific Protein ◽  
Myelin Protein ◽  
Drg Neurons ◽  
Antibody Treatment ◽  
Pi3k Inhibitor Ly294002 ◽  
Adult Rat ◽  
Drg Neuron ◽  
Peripheral Nerve Lesions

Besides its insulinotropic actions on pancreatic β cells, neuroprotective activities of glucagon-like peptide-1 (GLP-1) have attracted attention. The efficacy of a GLP-1 receptor (GLP-1R) agonist exendin-4 (Ex-4) for functional repair after sciatic nerve injury and amelioration of diabetic peripheral neuropathy (DPN) has been reported; however, the underlying mechanisms remain unclear. In this study, the bioactivities of Ex-4 on immortalized adult rat Schwann cells IFRS1 and adult rat dorsal root ganglion (DRG) neuron–IFRS1 co-culture system were investigated. Localization of GLP-1R in both DRG neurons and IFRS1 cells were confirmed using knockout-validated monoclonal Mab7F38 antibody. Treatment with 100 nM Ex-4 significantly enhanced survival/proliferation and migration of IFRS1 cells, as well as stimulated the movement of IFRS1 cells toward neurites emerging from DRG neuron cell bodies in the co-culture with the upregulation of myelin protein 22 and myelin protein zero. Because Ex-4 induced phosphorylation of serine/threonine-specific protein kinase AKT in these cells and its effects on DRG neurons and IFRS1 cells were attenuated by phosphatidyl inositol-3′-phosphate-kinase (PI3K) inhibitor LY294002, Ex-4 might act on both cells to activate PI3K/AKT signaling pathway, thereby promoting myelination in the co-culture. These findings imply the potential efficacy of Ex-4 toward DPN and other peripheral nerve lesions.

Contribution of Nav1.8 Sodium Channels to Action Potential Electrogenesis in DRG Neurons

2001 ◽  
Vol 86 (2) ◽  
pp. 629-640 ◽  
Author(s):  
Muthukrishnan Renganathan ◽  
Theodore R. Cummins ◽  
Stephen G. Waxman
Keyword(s):  
Action Potential ◽  
Sodium Channels ◽  
Action Potentials ◽  
Input Resistance ◽  
Resting Membrane Potential ◽  
Drg Neurons ◽  
Significant Difference ◽  
Steady State Inactivation ◽  
Current Threshold

C-type dorsal root ganglion (DRG) neurons can generate tetrodotoxin-resistant (TTX-R) sodium-dependent action potentials. However, multiple sodium channels are expressed in these neurons, and the molecular identity of the TTX-R sodium channels that contribute to action potential production in these neurons has not been established. In this study, we used current-clamp recordings to compare action potential electrogenesis in Nav1.8 (+/+) and (−/−) small DRG neurons maintained for 2–8 h in vitro to examine the role of sodium channel Nav1.8 (α-SNS) in action potential electrogenesis. Although there was no significant difference in resting membrane potential, input resistance, current threshold, or voltage threshold in Nav1.8 (+/+) and (−/−) DRG neurons, there were significant differences in action potential electrogenesis. Most Nav1.8 (+/+) neurons generate all-or-none action potentials, whereas most of Nav1.8 (−/−) neurons produce smaller graded responses. The peak of the response was significantly reduced in Nav1.8 (−/−) neurons [31.5 ± 2.2 (SE) mV] compared with Nav1.8 (+/+) neurons (55.0 ± 4.3 mV). The maximum rise slope was 84.7 ± 11.2 mV/ms in Nav1.8 (+/+) neurons, significantly faster than in Nav1.8 (−/−) neurons where it was 47.2 ± 1.3 mV/ms. Calculations based on the action potential overshoot in Nav1.8 (+/+) and (−/−) neurons, following blockade of Ca2+ currents, indicate that Nav1.8 contributes a substantial fraction (80–90%) of the inward membrane current that flows during the rising phase of the action potential. We found that fast TTX-sensitive Na+ channels can produce all-or-none action potentials in some Nav1.8 (−/−) neurons but, presumably as a result of steady-state inactivation of these channels, electrogenesis in Nav1.8 (−/−) neurons is more sensitive to membrane depolarization than in Nav1.8 (+/+) neurons, and, in the absence of Nav1.8, is attenuated with even modest depolarization. These observations indicate that Nav1.8 contributes substantially to action potential electrogenesis in C-type DRG neurons.

scholarly journals Phosphoinositide 3-Kinase Binds to TRPV1 and Mediates NGF-stimulated TRPV1 Trafficking to the Plasma Membrane

2006 ◽  
Vol 128 (5) ◽  
pp. 509-522 ◽  
Author(s):  
Alexander T. Stein ◽  
Carmen A. Ufret-Vincenty ◽  
Li Hua ◽  
Luis F. Santana ◽  
Sharona E. Gordon
Keyword(s):  
Plasma Membrane ◽  
Total Internal Reflection ◽  
Specific Inhibitor ◽  
Thermal Hyperalgesia ◽  
Hek293 Cells ◽  
Drg Neurons ◽  
Excised Patches ◽  
Phosphoinositide 3 Kinase ◽  
Inside Out

Sensitization of the pain-transducing ion channel TRPV1 underlies thermal hyperalgesia by proalgesic agents such as nerve growth factor (NGF). The currently accepted model is that the NGF-mediated increase in TRPV1 function during hyperalgesia utilizes activation of phospholipase C (PLC) to cleave PIP2, proposed to tonically inhibit TRPV1. In this study, we tested the PLC model and found two lines of evidence that directly challenge its validity: (1) polylysine, a cationic phosphoinositide sequestering agent, inhibited TRPV1 instead of potentiating it, and (2) direct application of PIP2 to inside-out excised patches dramatically potentiated TRPV1. Furthermore, we show four types of experiments indicating that PI3K is physically and functionally coupled to TRPV1: (1) the p85β subunit of PI3K interacted with the N-terminal region of TRPV1 in yeast 2-hybrid experiments, (2) PI3K-p85β coimmunoprecipitated with TRPV1 from both HEK293 cells and dorsal root ganglia (DRG) neurons, (3) TRPV1 interacted with recombinant PI3K-p85 in vitro, and (4) wortmannin, a specific inhibitor of PI3K, completely abolished NGF-mediated sensitization in acutely dissociated DRG neurons. Finally, simultaneous electrophysiological and total internal reflection fluorescence (TIRF) microscopy recordings demonstrate that NGF increased the number of channels in the plasma membrane. We propose a new model for NGF-mediated hyperalgesia in which physical coupling of TRPV1 and PI3K in a signal transduction complex facilitates trafficking of TRPV1 to the plasma membrane.

Neurotrophins affect the pattern of DRG neurite growth in a bioassay that presents a choice of CNS and PNS substrates

Development ◽  
1995 ◽  
Vol 121 (5) ◽  
pp. 1301-1309 ◽  
Author(s):  
R. Tuttle ◽  
W.D. Matthew
Keyword(s):  
Spinal Cord ◽  
Central Nervous System ◽  
Nervous System ◽  
Sciatic Nerve ◽  
Sympathetic Neurons ◽  
Neurite Growth ◽  
Drg Neurons ◽  
Substrate Preferences

Neurons can be categorized in terms of where their axons project: within the central nervous system, within the peripheral nervous system, or through both central and peripheral environments. Examples of these categories are cerebellar neurons, sympathetic neurons, and dorsal root ganglion (DRG) neurons, respectively. When explants containing one type of neuron were placed between cryosections of neonatal or adult sciatic nerve and neonatal spinal cord, the neurites exhibited a strong preference for the substrates that they would normally encounter in vivo: cerebellar neurites generally extended only on spinal cord, sympathetic neurites on sciatic nerve, and DRG neurites on both. Neurite growth from DRG neurons has been shown to be stimulated by neurotrophins. To determine whether neurotrophins might also affect the substrate preferences of neurites, DRG were placed between cryosections of neonatal spinal cord and adult sciatic nerve and cultured for 36 to 48 hours in the presence of various neurotrophins. While DRG cultured in NGF-containing media exhibited neurite growth over both spinal cord and sciatic nerve substrates, in the absence of neurotrophins DRG neurites were found almost exclusively on the CNS cryosection. To determine whether these neurotrophin-dependent neurite patterns resulted from the selective survival of subpopulations of DRG neurons with distinct neurite growth characteristics, a type of rescue experiment was performed: DRG cultured in neurotrophin-free medium were fed with NGF-containing medium after 36 hours in vitro and neurite growth examined 24 hours later; most DRG exhibited extensive neurite growth on both peripheral and central nervous system substrates.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Apoptosis in chicken ovarian follicles following in vitro exposure to TCDD, PCB 126 and PCB 153

2017 ◽  
Vol 17 (3) ◽  
pp. 787-798
Author(s):  
Piotr A. Antos ◽  
Anna Hrabia ◽  
Anna Gdula ◽  
Andrzej Sechman
Keyword(s):  
Caspase 3 ◽  
Suppressive Effect ◽  
Ovarian Follicles ◽  
Apoptotic Cells ◽  
Tunel Method ◽  
Ovarian Stroma ◽  
Pcb 126 ◽  
Preovulatory Follicles ◽  
Vitro Effect

Abstract The study was conducted in order to compare the in vitro effect of 2,3,7,8-tetrachlorodibenzo-pdioxin (TCDD), 3,3′,4,4′,5-pentachlorobiphenyl (PCB 126) and 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB 153) on the number of apoptotic cells and the activity of caspase-3 in chicken ovarian follicles. The ovarian stroma, white (WF) and yellowish (YF) prehierarchical follicles and fragments of the theca and granulosa layers of the 3 largest preovulatory follicles (F3-F1) were in vitro exposed to TCDD (10 nM), PCB 126 (10 nM) and PCB 153 (10 μM) for 24 h. After incubation the number of apoptotic cells and caspase-3 activity were determined by TUNEL method and fluorometric assay, respectively. PCB 126 and PCB 153 increased the number of apoptotic cells in the ovarian stroma while TCDD and PCB 126 elevated it in the WF follicles. Under the control conditions, caspase-3 activity steadily increased along with maturation of the follicles, reaching the highest level in the theca layer of the F1 follicle. The activity of this enzyme in the granulosa layer of F3-F1 follicles was on average 60% lower in comparison to the stroma. Exposure to TCDD elevated caspase-3 activity in prehierarchical follicles and in the granulosa layer of F2 and F1 preovulatory follicles. On the contrary, PCB 126 exerted a suppressive effect on caspase-3 activity in the WF follicles and the granulosa layer of the F2 follicle, and PCB 153 in the theca layer of F2 and F1 and the granulosa layer of the F3 follicle. In conclusion, the results indicate that TCDD and PCBs affect apoptosis in chicken ovarian follicles and in consequence may disrupt follicle development.

scholarly journals Nicotine suppresses hyperexcitability of colonic sensory neurons and visceral hypersensivity in mouse model of colonic inflammation

2012 ◽  
Vol 302 (7) ◽  
pp. G740-G747 ◽  
Author(s):  
Galya R. Abdrakhmanova ◽  
Minho Kang ◽  
M. Imad Damaj ◽  
Hamid I. Akbarali
Keyword(s):  
Mouse Model ◽  
Action Potential ◽  
Action Potentials ◽  
Drg Neurons ◽  
Action Potential Firing ◽  
Colonic Inflammation ◽  
Single Action ◽  
Nicotine Administration ◽  
Potential Firing

Recently, we reported that nicotine in vitro at a low 1-μM concentration suppresses hyperexcitability of colonic dorsal root ganglia (DRG; L1-L2) neurons in the dextran sodium sulfate (DSS)-induced mouse model of acute colonic inflammation ( 1 ). Here we show that multiple action potential firing in colonic DRG neurons persisted at least for 3 wk post-DSS administration while the inflammatory signs were diminished. Similar to that in DSS-induced acute colitis, bath-applied nicotine (1 μM) gradually reduced regenerative multiple-spike action potentials in colonic DRG neurons to a single action potential in 3 wk post-DSS neurons. Nicotine (1 μM) shifted the activation curve for tetrodotoxin (TTX)-resistant sodium currents in inflamed colonic DRG neurons (voltage of half-activation changed from −37 to −32 mV) but did not affect TTX-sensitive currents in control colonic DRG neurons. Further, subcutaneous nicotine administration (2 mg/kg b.i.d.) in DSS-treated C57Bl/J6 male mice resulted in suppression of hyperexcitability of colonic DRG (L1-L2) neurons and the number of abdominal constrictions in response to intraperitoneal injection of 0.6% acetic acid. Collectively, the data suggest that neuronal nicotinic acetylcholine receptor-mediated suppression of hyperexcitability of colonic DRG neurons attenuates reduction of visceral hypersensitivity in DSS mouse model of colonic inflammation.

Pronociceptive effects of a TRPA1 channel agonist methylglyoxal in healthy control and diabetic animals

2013 ◽  
Vol 4 (4) ◽  
pp. 260-260
Author(s):  
Hanna Viisanen ◽  
Maria Lasierra ◽  
Hong Wei ◽  
Ari Koivisto ◽  
Karl E. Åkerman ◽  
...  
Keyword(s):  
Diabetes Mellitus ◽  
Pain Behavior ◽  
Drg Neurons ◽  
Mechanical Hypersensitivity ◽  
Conditioned Place Avoidance ◽  
Healthy Control ◽  
Place Avoidance ◽  
Ongoing Pain ◽  
Endogenous Release

Abstract Aims Methylglyoxal (MG), a reactive carbonyl compound generated in diabetes mellitus (DM), activates the TRPA1 ion channel. Here we studied whether MG induces mechanical hypersensitivity or ongoing pain and whether the pronociceptive effect of MG is changed following its sustained endogenous release in DM. Methods DM was induced by streptozotocin (50-60 mg/kg i.p.) in the rat. MG and Chembridge-5861528 (CHEM), a selective TRPA1 channel antagonist, were administered intraplantarly (i.pl.) in control and diabetic animals. Limb withdrawal to monofilaments was used as an index of hypersensitivity, and observation of sustained pain-like behavior and conditioned place-avoidance test were used to assess ongoing pain. In vitro calcium imaging was used to study whether MG induces sustained activation of dorsal root ganglion (DRG) neurons of diabetic as well as control animals. Results MG produced mechanical hypersensitivity and ongoing pain behavior in control animals, which effects were reduced in diabetic animals. CHEM treatment at a dose suppressing the MG-induced mechanical hypersensitivity failed to suppress the MG-induced ongoing pain behavior. MG was able to produce sustained calcium inflow in DRG neurons of DM as well as control animals. Conclusions The results suggest that MG induces hypersensitivity and ongoing pain that are reduced in diabetes mellitus, possibly due to changes caused by the DM-induced sustained endogenous release of MG. Moreover, the MG-induced mechanical hypersensitivity can be more effectively reversed by a TRPA1 antagonist than the MG-induced ongoing pain behavior.

scholarly journals Injury-free priming: induction of Primary Afferent Collateral Sprouting in uninjured sensory neurons in vivo primes them for enhanced axon outgrowth in vitro

2018 ◽  
Author(s):  
Sara Soleman ◽  
Jeffrey C. Petruska ◽  
Lawrence D.F. Moon
Keyword(s):  
Axon Regeneration ◽  
Axonal Injury ◽  
Axon Growth ◽  
Drg Neurons ◽  
Primary Afferent ◽  
Collateral Sprouting ◽  
Axon Elongation ◽  
Defined Media

AbstractPrior “conditioning” nerve lesions can prime DRG neurons for enhanced axon regeneration. Here, we tested the hypothesis that adult DRG neurons can be primed for axon elongation in vitro without axonal injury by prior induction of Primary Afferent Collateral Sprouting (PACS) in vivo. Thoracic cutaneous nerves (T9, T10, T12, T13 but not T11) were transected to create zones of denervated skin. Neurons from the uninjured T11 DRG underwent PACS within the skin, as demonstrated by the expansion of its zones responsive to pinch up to 14 days. At 7 or 14 days after induction of collateral sprouting, DRG neurons were dissociated and cultured for 18 hours in defined media lacking neurotrophins and growth factors. Neurons from the uninjured T11 DRG had longer mean neurite lengths than neurons from naïve DRG. A larger proportion of neurons from the uninjured T11 DRG showed an elongating or arborizing phenotype than neurons from naïve DRG. Transcriptomic analysis of the uninjured T11 DRG and denervated/reinnervated skin reveal regulation of receptor/ligand systems and regulators of growth during collateral sprouting. For example, the glial cell-derived neurotrophic family ligands Artemin and Persephin were upregulated in denervated skin after 7 and/or 14 days. We suggest that extracellular cues in denervated skin modify the intrinsic growth program of uninjured DRG neurons that enhances their ability to elongate or arborize even after explantation. Collectively, these data confirm that induction of collateral sprouting does not induce an injury response yet primes many of these uninjured neurons for in vitro axon growth.

scholarly journals Role of CaMK II α in the Injury of Dorsal Root Ganglion Neurons Induced by Ropivacaine Hydrochloride in the Dorsal Root Ganglion of Rats

2021 ◽  
pp. 1-7
Author(s):  
Wu Zhaoxia ◽  
Chen Meixin ◽  
Li Yiqun ◽  
Yang Shuxuan ◽  
Wen Xianjie
Keyword(s):  
Dorsal Root Ganglion ◽  
Cell Viability ◽  
Intracellular Calcium ◽  
Dorsal Root ◽  
Dorsal Root Ganglion Neurons ◽  
Drg Neurons ◽  
Apoptosis Rate ◽  
Ganglion Neurons ◽  
Ropivacaine Hydrochloride

Objective: To investigate whether CaMKⅡα participates in the dorsal root ganglion neurotoxicity induced by ropivacaine hydrochloride. Methods: DRG neurons were isolated from 1-day-old SD rats and cultured in vitro. pAd-shRNA-CaMKⅡα-DRG cells were constructed by RNA interference technique to inhibit the expression of CaMKⅡα. The experiment was divided into six groups: DRG group (DRG group), vector DRG group (vector group), pAd-shRNA- CaMKIIα-DRG group (pAd-shRNA group), DRG + ropivacaine group (DRG + R group), vector DRG + ropivacaine group (vector + R group), pAd-shRNA-CaMKII α - DRG + ropivacaine group (pAd-shRNA + R group), and the last three groups were treated with 3 mM ropivacaine hydrochloride for 4 hours. MTT assay was used to detect cell viability, flow cytometry was used to detect cell apoptosis rate, laser confocal microscopy was used to detect intracellular calcium level, and real-time PCR was used to detect the mRNA expression of CaMKⅡα, Cav3.2 and Cav3.3. Results: The cell viability of DRG+R group, vector+R group and pAd-shRNA+R group decreased significantly after 3 mM ropivacaine hydrochloride treatment for 4 h. Compared with DRG+R group, the cell viability of pAd-shRNA+R group was significantly higher. After 3 mM ropivacaine hydrochloride treatment for 4 h, the apoptosis rate of DRG + R group, vector + R group and pAd-shRNA + R group increased significantly. Compared with DRG+R group, the apoptosis rate in pAd shRNA+R group was significantly lower. After 3 mM ropivacaine hydrochloride treatment for 4 h, the intracellular calcium levels in DRG + R group, vector + R group and pAd-shRNA + R group were significantly increased, and the intracellular calcium levels in pAd-shRNA + R group were significantly lower than those in DRG + R group. The mRNA expressions of CaMKⅡα, Cav3.2 and Cav3.3 were significantly decreased in pAd- shRNA group. The mRNA expressions of CaMK Ⅱ α, Cav3.2 and Cav3.3 were up-regulated in DRG + R group, vector + R group and pAd-shRNA + R group after 3 mm ropivacaine treatment for 4 h. The mRNA expressions of CaMKⅡα, Cav3.2 and Cav3.3 in pAd-shRNA + R group were significantly lower than those in DRG + R group. Conclusion: Inhibition of CaMKⅡα expression can down regulate the expression of Cav3.2 and Cav3.3 mRNA, increase cell viability of DRG neurons, reduce the apoptosis rate, and improve the dorsal root ganglion neurotoxicity induced by ropivacaine hydrochloride.

scholarly journals Regulation of TRPM8 channels by PKCβ mediates morphine-induced cold hypersensitivity.

2020 ◽  
Author(s):  
Mircea Iftinca ◽  
Lilian Basso ◽  
Robyn Flynn ◽  
Charlie Kwok ◽  
Corinne Roland ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Chronic Administration ◽  
Repeated Administration ◽  
Drg Neurons ◽  
Trpm8 Channel ◽  
Directed Mutation ◽  
Cold Sensitive ◽  
Cold Hyperalgesia ◽  
Cold Hypersensitivity

Abstract Postoperative shivering and cold hypersensitivity are major side effects of acute and chronic opioid treatments respectively. TRPM8 is a cold and menthol-sensitive channel found in a subset of dorsal root ganglion (DRG) nociceptors. Deletion or inhibition of the TRPM8 channel was found to prevent the cold hyperalgesia induced by chronic administration of morphine. Here, we examined the mechanisms by which morphine was able to promote cold hypersensitivity in DRG neurons and transfected HEK cells. Mice daily injected with morphine for five days developed cold hyperalgesia. Treatment with morphine did not alter the expressions of cold sensitive TREK-1, TRAAK and TRPM8 in DRGs. However, TRPM8-expressing DRG neurons isolated from morphine-treated mice exhibited hyperexcitability. Sustained morphine treatment in vitro sensitized TRPM8 responsiveness to cold or menthol and reduced activation-evoked desensitization of the channel. Blocking the phospholipase C (PLC) as well as protein kinase C beta (PKCβ), but not protein kinase A (PKA) or Rho-associated protein kinase (ROCK), restored channel desensitization. Identification of two PKC phosphorylation consensus sites, S1040 and S1041, in the TRPM8 and their site-directed mutation were able to prevent the MOR-induced reduction in TRPM8 desensitization. Our results show that activation of MOR by morphine 1) promotes hyperexcitability of TRPM8-expressing neurons and 2) induces a PKCβ-mediated reduction of TRPM8 desensitization. This MOR-PKCβ dependent modulation of TRPM8 may underlie the onset of cold hyperalgesia caused by repeated administration of morphine. Our findings point to TRPM8 channel and PKCβ as important targets for opioid-induced cold hypersensitivity.

Sign in / Sign up

Export Citation Format

Share Document