In Vivo NGF Deprivation Reduces SNS Expression and TTX-R Sodium Currents in IB4-Negative DRG Neurons

1999 ◽  
Vol 81 (2) ◽  
pp. 803-810 ◽  
Author(s):  
Jenny Fjell ◽  
Theodore R. Cummins ◽  
Kaj Fried ◽  
Joel A. Black ◽  
Stephen G. Waxman
Keyword(s):  
Sodium Channel ◽  
Current Density ◽  
Sodium Current ◽  
High Antibody ◽  
Sodium Currents ◽  
Drg Neurons ◽  
Adult Rats ◽  
Antibody Titers

In vivo NGF deprivation reduces SNS expression and TTX-R sodium currents in IB4-negative DRG neurons. Recent evidence suggests that changes in sodium channel expression and localization may be involved in some pathological pain syndromes. SNS, a tetrodotoxin-resistant (TTX-R) sodium channel, is preferentially expressed in small dorsal root ganglion (DRG) neurons, many of which are nociceptive. TTX-R sodium currents and SNS mRNA expression have been shown to be modulated by nerve growth factor (NGF) in vitro and in vivo. To determine whether SNS expression and TTX-R currents in DRG neurons are affected by reduced levels of systemic NGF, we immunized adult rats with NGF, which causes thermal hypoalgesia in rats with high antibody titers to NGF. DRG neurons cultured from rats with high antibody titers to NGF, which do not bind the isolectin IB4 (IB4−) but do express TrkA, were studied with whole cell patch-clamp and in situ hybridization. Mean TTX-R sodium current density was decreased from 504 ± 77 pA/pF to 307 ± 61 pA/pF in control versus NGF-deprived neurons, respectively. In comparison, the mean TTX-sensitive sodium current density was not significantly different between control and NGF-deprived neurons. Quantification of SNS mRNA hybridization signal showed a significant decrease in the signal in NGF-deprived neurons compared with the control neurons. The data suggest that NGF has a major role in the maintenance of steady-state levels of TTX-R sodium currents and SNS mRNA in IB4− DRG neurons in adult rats in vivo.

Upregulation of a Silent Sodium Channel After Peripheral, but not Central, Nerve Injury in DRG Neurons

1999 ◽  
Vol 82 (5) ◽  
pp. 2776-2785 ◽  
Author(s):  
J. A. Black ◽  
T. R. Cummins ◽  
C. Plumpton ◽  
Y. H. Chen ◽  
W. Hormuzdiar ◽  
...  
Keyword(s):  
Sciatic Nerve ◽  
Sodium Channel ◽  
Sodium Channels ◽  
Sodium Current ◽  
Sodium Currents ◽  
Drg Neurons ◽  
Adult Rats ◽  
Voltage Range ◽  
Type Iii ◽  
Axonal Projections

After transection of their axons within the sciatic nerve, DRG neurons become hyperexcitable. Recent studies have demonstrated the emergence of a rapidly repriming tetrodotoxin (TTX)-sensitive sodium current that may account for this hyperexcitability in axotomized small (<27 μm diam) DRG neurons, but its molecular basis has remained unexplained. It has been shown previously that sciatic nerve transection leads to an upregulation of sodium channel III transcripts, which normally are present at very low levels in DRG neurons, in adult rats. We show here that TTX-sensitive currents in small DRG neurons, after transection of their peripheral axonal projections, reprime more rapidly than those in control neurons throughout a voltage range of −140 to −60 mV, a finding that suggests that these currents are produced by a different sodium channel. After transection of the central axonal projections (dorsal rhizotomy) of these small DRG neurons, in contrast, the repriming kinetics of TTX-sensitive sodium currents remain similar to those of control (uninjured) neurons. We also demonstrate, with two distinct antibodies directed against different regions of the type III sodium channel, that small DRG neurons display increased brain type III immunostaining when studied 7–12 days after transection of their peripheral, but not central, projections. Type III sodium channel immunoreactivity is present within somata and neurites of peripherally axotomized, but not centrally axotomized, neurons studied after <24 h in vitro. Peripherally axotomized DRG neurons in situ also exhibit enhanced type III staining compared with control neurons, including an accumulation of type III sodium channels in the distal portion of the ligated and transected sciatic nerve, but these changes are not seen in centrally axotomized neurons. These observations are consistent with a contribution of type III sodium channels to the rapidly repriming sodium currents observed in peripherally axotomized DRG neurons and suggest that type III channels may at least partially account for the hyperexcitibility of these neurons after injury.

Rescue of α-SNS Sodium Channel Expression in Small Dorsal Root Ganglion Neurons After Axotomy by Nerve Growth Factor In Vivo

1998 ◽  
Vol 79 (5) ◽  
pp. 2668-2676 ◽  
Author(s):  
S. D. Dib-Hajj ◽  
J. A. Black ◽  
T. R. Cummins ◽  
A. M. Kenney ◽  
J. D. Kocsis ◽  
...  
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Dorsal Root Ganglion ◽  
Sodium Channel ◽  
Dorsal Root ◽  
Sodium Current ◽  
Mrna Levels ◽  
Drg Neurons ◽  
Nerve Growth

Dib-Hajj, S. D., J. A. Black, T. R. Cummins, A. M. Kenney, J. D. Kocsis, and S. G. Waxman. Rescue of α-SNS sodium channel expression in small dorsal root ganglion neurons after axotomy by nerve growth factor in vivo. J. Neurophysiol. 79: 2668–2676, 1998. Small (18–25 μm diam) dorsal root ganglion (DRG) neurons are known to express high levels of tetrodotoxin-resistant (TTX-R) sodium current and the mRNA for the α-SNS sodium channel, which encodes a TTX-R channel when expressed in oocytes. These neurons also preferentially express the high affinity receptor for nerve growth factor (NGF), TrkA. Levels of TTX-R sodium current and of α-SNS mRNA are reduced in these cells after axotomy. To determine whether NGF participates in the regulation of TTX-R current and α-SNS mRNA in small DRG neurons in vivo, we axotomized small lumbar DRG neurons by sciatic nerve transection and administered NGF or Ringer solution to the proximal nerve stump using osmotic pumps. Ten to 12 days after pump implant, whole cell patch-clamp recording demonstrated that TTX-R current density was decreased in Ringer-treated axotomized neurons (154 ± 45 pA/pF; mean ± SE) compared with nonaxotomized control neurons (865 ± 123 pA/pF) and was restored partially toward control levels in NGF-treated axotomized neurons (465 ± 78 pA/pF). The V 1/2 for steady-state activation and inactivation of TTX-R currents were similar in control, Ringer- and NGF-treated axotomized neurons. Reverse transcription polymerase chain reaction revealed an upregulation of α-SNS mRNA levels in NGF-treated compared with Ringer-treated axotomized DRG. In situ hybridization showed that α-SNS mRNA levels were decreased significantly in small Ringer-treated axotomized DRG neurons in vivo and also in small DRG neurons that were dissociated and maintained in vitro, so as to correspond to the patch-clamp conditions. NGF-treated axotomized neurons had a significant increase in α-SNS mRNA expression, compared with Ringer-treated axotomized cells. These results show that the administration of exogenous NGF in vivo, to the proximal nerve stump of the transected sciatic nerve, results in an upregulation of TTX-R sodium current and of α-SNS mRNA levels in small DRG neurons. Retrogradely transported NGF thus appears to participate in the control of excitability in these cells via actions that include the regulation of sodium channel gene expression in vivo.

4966Targeting INaL by a neuronal sodium channel isoform improves survival in a CaMKII-transgenic heart failure mouse model

2019 ◽  
Vol 40 (Supplement_1) ◽  
Author(s):  
P Bengel ◽  
C Krekeler ◽  
S Ahmad ◽  
P Tirilomis ◽  
K Toischer ◽  
...  
Keyword(s):  
Heart Failure ◽  
Mouse Model ◽  
Sodium Channel ◽  
Sodium Current ◽  
Heart Weight ◽  
Knock Out ◽  
In Vivo Experiments ◽  
Antiarrhythmic Effects

Abstract Background Cardiac pathologies like hypertrophy and heart failure are known to be associated with proarrhythmogenic triggers like early- (EADs) and delayed afterdepolarizations (DADs) that can be partly attributed to an augmentation of late sodium current (INaL). Enhanced INaL is closely connected with increased activity of Ca2+/calmodulin dependent-kinase II (CaMKII) in pathology as it is enhanced by CaMKII on the one hand but can also indirectly increase CaMKII-activity on the other. We recently found neuronal sodium channel NaV1.8 to be involved in INaL-augmentation in heart failure and cardiac hypertrophy. Here, we studied possible antiarrhythmic effects of NaV1.8-inhibition in a transgenic mouse model with enhanced CaMKII-expression by selectively knocking out NaV1.8. Methods/Results To investigate antiarrhythmic effects of NaV1.8-depletion in-vivo and in-vitro we crossbred CaMKII-transgenic mice (CaMKII+/T) with NaV1.8-knock-out mice (SCN10A−/−). Surprisingly, CaMKII+/T-mice lacking NaV1.8 (CaMKII+/T & SCN10A−/−) showed a significantly improved survival compared to CaMKII+/T alone (97.5 vs 72.0 days, p<0.05). Heart weight to tibia length ratio was significantly increased in CaMKII+/T-mice compared to wild-type, without any differences between CaMKII+/T and CaMKII+/T & SCN10A−/−. To investigate the underlying mechanisms out of this observation we isolated single cardiomyocytes and performed patch-clamp experiments as well as confocal microscopy to measure Ca2+-transients and diastolic Ca2+-waves. INaL-integral was significantly smaller in cardiomyocytes from CaMKII+/T & SCN10A−/−-mice compared to CaMKII+/T alone. During action potential recordings, significantly less afterdepolarizations occurred in CaMKII+/T & SCN10A−/− compared to cardiomyocytes from CaMKII+/T -mice (16.7/min vs 34.9/min, p<0.05). There was a trend of less cells exhibiting diastolic Ca2+-waves in Ca2+-measurements from CaMKII+/T & SCN10A−/− compared to CaMKII+/T (15% vs 25%, p=0.09). As some cells showed more than one event, we calculated the frequency of Ca2+-waves and found a significant reduction of Ca2+-waves in CaMKII+/T & SCN10A−/− vs. CaMKII+/T (22.8/min vs 43.0/min, p<0.05). Moreover, the time to the first event was significantly longer in CaMKII+/T & SCN10A−/−. Ca2+-transient amplitude (F/F0) was significantly lower in CaMKII+/T compared to CaMKII+/T & SCN10A−/− (4.6 vs. 5.3, p=0.05). Further, Ca2+-extrusion from the cytosol was significantly faster in CaMKII+/T & SCN10A−/−. Conclusion Our data demonstrates, that inhibition of INaL by targeting NaV1.8 has a potent antiarrhythmic potential as we found a reduction of EADs, DADs and diastolic Ca2+-waves in CaMKII+/T & SCN10A−/−-cardiomyocytes. This antiarrhythmic potential appears to be potent enough to improve survival and to rescue the proarrhythmogenic phenotype of CaMKII-overexpression. However, further in-vivo experiments are necessary to investigate NaV1.8-inhibition for a possible therapeutic approach.

scholarly journals Estrogen Elicits Dorsal Root Ganglion Axon Sprouting via a Renin-Angiotensin System

Endocrinology ◽  
2008 ◽  
Vol 149 (7) ◽  
pp. 3452-3460 ◽  
Author(s):  
Anuradha Chakrabarty ◽  
Audrey Blacklock ◽  
Stanislav Svojanovsky ◽  
Peter G. Smith
Keyword(s):  
Estrogen Receptor ◽  
Dorsal Root Ganglion ◽  
Dorsal Root ◽  
Drg Neurons ◽  
Predisposing Factor ◽  
Adult Rats ◽  
Axon Sprouting ◽  
Pain Syndromes

Many painful conditions occur more frequently in women, and estrogen is a predisposing factor. Estrogen may contribute to some pain syndromes by enhancing axon outgrowth by sensory dorsal root ganglion (DRG) neurons. The objective of the present study was to define mechanisms by which estrogen elicits axon sprouting. The estrogen receptor-α agonist propyl pyrazole triol induced neurite outgrowth from cultured neonatal DRG neurons, whereas the estrogen receptor-β agonist diarylpropionitrile was ineffective. 17β-Estradiol (E2) elicited sprouting from peripherin-positive unmyelinated neurons, but not larger NF200-positive myelinated neurons. Microarray analysis showed that E2 up-regulates angiotensin II (ANGII) receptor type 2 (AT2) mRNA in vitro, and studies in adult rats confirmed increased DRG mRNA and protein in vivo. AT2 plays a central role in E2-induced axon sprouting because AT2 blockade by PD123,319 eliminated estrogen-mediated sprouting in vitro. We assessed whether AT2 may be responding to locally synthesized ANGII. DRG from adult rats expressed mRNA for renin, angiotensinogen, and angiotensin converting enzyme (ACE), and protein products were present and occasionally colocalized within neurons and other DRG cells. We determined if locally synthesized ANGII plays a role in estrogen-mediated sprouting by blocking its formation using the ACE inhibitor enalapril. ACE inhibition prevented estrogen-induced neuritogenesis. These findings support the hypothesis that estrogen promotes DRG nociceptor axon sprouting by up-regulating the AT2 receptor, and that locally synthesized ANGII can induce axon formation. Therefore, estrogen may contribute to some pain syndromes by enhancing the pro-neuritogenic effects of AT2 activation by ANGII.

Calmodulin Regulates Current Density and Frequency-Dependent Inhibition of Sodium Channel Nav1.8 in DRG Neurons

2006 ◽  
Vol 96 (1) ◽  
pp. 97-108 ◽  
Author(s):  
Jin-Sung Choi ◽  
Andy Hudmon ◽  
Stephen G. Waxman ◽  
Sulayman D. Dib-Hajj
Keyword(s):  
Sodium Channel ◽  
Current Density ◽  
Low Frequency ◽  
Drg Neurons ◽  
Frequency Dependent ◽  
Iq Motif ◽  
Calmodulin Binding ◽  
C Terminus ◽  
Dependent Inhibition

Sodium channel Nav1.8 produces a slowly inactivating, tetrodotoxin-resistant current, characterized by recovery from inactivation with fast and slow components, and contributes a substantial fraction of the current underlying the depolarizing phase of the action potential of dorsal root ganglion (DRG) neurons. Nav1.8 C-terminus carries a conserved calmodulin-binding isoleucine–glutamine (IQ) motif. We show here that calmodulin coimmunoprecipitates with endogenous Nav1.8 channels from native DRG, suggesting that the two proteins can interact in vivo. Treatment of native DRG neurons with a calmodulin-binding peptide (CBP) reduced the current density of Nav1.8 by nearly 65%, without changing voltage dependency of activation or steady-state inactivation. To investigate the functional role of CaM binding to the IQ motif in the Nav1.8 C-terminus, the IQ dipeptide was substituted by DE; we show that this impairs the binding of CaM to the IQ motif. Mutant Nav1.8IQ/DE channels produce currents with roughly 50% amplitude, but with unchanged voltage dependency of activation and inactivation when expressed in DRG neurons from Nav1.8-null mice. We also show that blocking the interaction of CaM and Nav1.8 using CBP or the IQ/DE substitution causes a buildup of inactivated channels and, in the case of the IQ/DE mutation, stimulation even at a low frequency of 0.1 Hz significantly enhances the frequency-dependent inhibition of the Nav1.8 current. This study presents, for the first time, evidence that calmodulin associates with a sodium channel, Nav1.8, in native neurons, and demonstrates a regulation of Nav1.8 currents that can significantly affect electrogenesis of DRG neurons in which Nav1.8 is normally expressed.

α-SNS Produces the Slow TTX-Resistant Sodium Current in Large Cutaneous Afferent DRG Neurons

2000 ◽  
Vol 84 (2) ◽  
pp. 710-718 ◽  
Author(s):  
M. Renganathan ◽  
T. R. Cummins ◽  
W. N. Hormuzdiar ◽  
S. G. Waxman
Keyword(s):  
Sodium Channel ◽  
Current Density ◽  
Low Voltage ◽  
Gene Knockout ◽  
Sodium Current ◽  
Afferent Neurons ◽  
Wild Type ◽  
Drg Neurons ◽  
Gene Knockout Mice ◽  
Current Densities

In this study, we used sensory neuron specific (SNS) sodium channel gene knockout (−/−) mice to ask whether SNS sodium channel produces the slow Na+current (“slow”) in large (>40 μm diam) cutaneous afferent dorsal root ganglion (DRG) neurons. SNS wild-type (+/+) mice were used as controls. Retrograde Fluoro-Gold labeling permitted the definitive identification of cutaneous afferent neurons. Prepulse inactivation was used to separate the fast and slow Na+ currents. Fifty-two percent of the large cutaneous afferent neurons isolated from SNS (+/+) mice expressed only fast-inactivating Na+ currents (“fast”), and 48% expressed both fast and slow Na+ currents. The fast and slow current densities were 0.90 ± 0.12 and 0.39 ± 0.16 nA/pF, respectively. Fast Na+ currents were blocked completely by 300 nM tetrodotoxin (TTX), while slow Na+ currents were resistant to 300 nM TTX, confirming that the slow Na+ currents observed in large cutaneous DRG neurons are TTX-resistant (TTX-R). Slow Na+ currents could not be detected in large cutaneous afferent neurons from SNS (−/−) mice; these cells expressed only fast Na+ current, and it was blocked by 300 nM TTX. The fast Na+ current density in SNS (−/−) neurons was 1.47 ± 0.14 nA/pF, approximately 60% higher than the current density observed in SNS (+/+) mice ( P< 0.02). A low-voltage–activated TTX-R Na+current (“persistent”) observed in small C-type neurons is not present in large cutaneous afferent neurons from either SNS (+/+) or SNS (−/−) mice. These results show that the slow TTX-R Na+ current in large cutaneous afferent DRG is produced by the SNS sodium channel.

scholarly journals Compensatory mechanisms in resistant Anopheles gambiae AcerKis and KdrKis neurons modulate insecticide-based mosquito control

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Stéphane Perrier ◽  
Eléonore Moreau ◽  
Caroline Deshayes ◽  
Marine El-Adouzi ◽  
Delphine Goven ◽  
...  
Keyword(s):  
Sodium Channel ◽  
Anopheles Gambiae ◽  
Calcium Concentration ◽  
Mosquito Control ◽  
Point Mutations ◽  
Resting Membrane Potential ◽  
Compensatory Mechanisms ◽  
Pyrethroid Insecticides

AbstractIn the malaria vector Anopheles gambiae, two point mutations in the acetylcholinesterase (ace-1R) and the sodium channel (kdrR) genes confer resistance to organophosphate/carbamate and pyrethroid insecticides, respectively. The mechanisms of compensation that recover the functional alterations associated with these mutations and their role in the modulation of insecticide efficacy are unknown. Using multidisciplinary approaches adapted to neurons isolated from resistant Anopheles gambiae AcerKis and KdrKis strains together with larval bioassays, we demonstrate that nAChRs, and the intracellular calcium concentration represent the key components of an adaptation strategy ensuring neuronal functions maintenance. In AcerKis neurons, the increased effect of acetylcholine related to the reduced acetylcholinesterase activity is compensated by expressing higher density of nAChRs permeable to calcium. In KdrKis neurons, changes in the biophysical properties of the L1014F mutant sodium channel, leading to enhance overlap between activation and inactivation relationships, diminish the resting membrane potential and reduce the fraction of calcium channels available involved in acetylcholine release. Together with the lower intracellular basal calcium concentration observed, these factors increase nAChRs sensitivity to maintain the effect of low concentration of acetylcholine. These results explain the opposite effects of the insecticide clothianidin observed in AcerKis and KdrKis neurons in vitro and in vivo.

Sodium Currents in Neurons From the Rostroventrolateral Medulla of the Rat

2003 ◽  
Vol 90 (3) ◽  
pp. 1635-1642 ◽  
Author(s):  
Ilya A. Rybak ◽  
Krzysztof Ptak ◽  
Natalia A. Shevtsova ◽  
Donald R. McCrimmon
Keyword(s):  
Sodium Channels ◽  
Sodium Current ◽  
Cell Voltage ◽  
Kinetic Properties ◽  
Persistent Sodium Current ◽  
Sodium Currents ◽  
Bötzinger Complex ◽  
Window Current ◽  
Bursting Activity

Rapidly inactivating and persistent sodium currents have been characterized in acutely dissociated neurons from the area of rostroventrolateral medulla that included the pre-Bötzinger Complex. As demonstrated in many studies in vitro, this area can generate endogenous rhythmic bursting activity. Experiments were performed on neonate and young rats (P1-15). Neurons were investigated using the whole cell voltage-clamp technique. Standard activation and inactivation protocols were used to characterize the steady-state and kinetic properties of the rapidly inactivating sodium current. Slow depolarizing ramp protocols were used to characterize the noninactivating sodium current. The “window” component of the rapidly inactivating sodium current was calculated using mathematical modeling. The persistent sodium current was revealed by subtraction of the window current from the total noninactivating sodium current. Our results provide evidence of the presence of persistent sodium currents in neurons of the rat rostroventrolateral medulla and determine voltage-gated characteristics of activation and inactivation of rapidly inactivating and persistent sodium channels in these neurons.

scholarly journals In vitro analysis of the origin and maintenance of O-2Aadult progenitor cells.

1992 ◽  
Vol 116 (1) ◽  
pp. 167-176 ◽  
Author(s):  
D Wren ◽  
G Wolswijk ◽  
M Noble
Keyword(s):  
Adult Life ◽  
Cell Types ◽  
Adult Rats ◽  
Optic Nerves ◽  
Limited Life ◽  
Old Rats ◽  
Vitro Analysis

We have been studying the differing characteristics of oligodendrocyte-type-2 astrocyte (O-2A) progenitors isolated from optic nerves of perinatal and adult rats. These two cell types display striking differences in their in vitro phenotypes. In addition, the O-2Aperinatal progenitor population appears to have a limited life-span in vivo, while O-2Aadult progenitors appear to be maintained throughout life. O-2Aperinatal progenitors seem to have largely disappeared from the optic nerve by 1 mo after birth, and are not detectable in cultures derived from optic nerves of adult rats. In contrast, O-2Aadult progenitors can first be isolated from optic nerves of 7-d-old rats and are still present in optic nerves of 1-yr-old rats. These observations raise two questions: (a) From what source do O-2Aadult progenitors originate; and (b) how is the O-2Aadult progenitor population maintained in the nerve throughout life? We now provide in vitro evidence indicating that O-2Aadult progenitors are derived directly from a subpopulation of O-2Aperinatal progenitors. We also provide evidence indicating that O-2Aadult progenitors are capable of prolonged self renewal in vitro. In addition, our data suggests that the in vitro generation of oligodendrocytes from O-2Aadult progenitors occurs primarily through asymmetric division and differentiation, in contrast with the self-extinguishing pattern of symmetric division and differentiation displayed by O-2Aperinatal progenitors in vitro. We suggest that O-2Aadult progenitors express at least some properties of stem cells and thus may be able to support the generation of both differentiated progeny cells as well as their own continued replenishment throughout adult life.

scholarly journals CELL-MEDIATED CYTOTOXICITY DURING REJECTION AND ENHANCEMENT OF ALLOGENEIC SKIN GRAFTS IN RATS

1972 ◽  
Vol 135 (6) ◽  
pp. 1301-1315 ◽  
Author(s):  
Hans-Hartmut Peter ◽  
Joseph D. Feldman
Keyword(s):  
Lymph Nodes ◽  
Thoracic Duct ◽  
Lymphoid Cells ◽  
Target Cells ◽  
Skin Grafts ◽  
Peak Activity ◽  
Adult Rats ◽  
Background Levels

Cell-mediated cytotoxicity (CMC) in spleens and lymph nodes of allografted rats was determined by release of 51Cr from labeled target cells incubated with aggressor lymphoid cells. CMC was first detected in grafted adult rats on day 5, peaked on days 7 and 8, and declined rapidly to background levels by days 9 to 11. In allografted neonates and in cyclophosphamide-treated or neonatally thymectomized adults CMC was a fraction of that observed in normal adult rats. Enhancing antibodies deferred in vivo peak activity of CMC in allografted neonates for 3–4 days, and blocked in vitro the action of aggressor lymphocytes by binding to target cells. Enhancing antibodies had no effect on the cytotoxicity of aggressor cells, but horse antibodies to rat thoracic duct cells inhibited in vitro CMC of aggressor cells.

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