Ethanol enhancement of GABA-activated chloride current in rat dorsal root ganglion neurons.

The acute effects of ethanol on the gamma-aminobutyric acid (GABA)-activated current were studied with the rat dorsal root ganglion neurons in primary culture using the whole-cell patch-clamp technique. GABA produced an inward chloride current, which was composed of an initial transient and a subsequent sustained phase. Ethanol at concentrations ranging from 30 to 300 mM enhanced the transient current in a concentration-dependent manner without affecting the sustained current.     

Taurine-induced modulation of voltage-sensitive Na+ channels in rat dorsal root ganglion neurons

The physiological role of taurine, an abundant free amino acid in the neural system, is still poorly understood. The aim of this study was to investigate its effect on TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) Na+ currents in enzymatically dissociated neurons from rat dorsal root ganglion (DRG) with conventional whole-cell recording manner under voltage-clamp conditions. A TTX-S Na+ current was recorded preferentially from large DRG neurons and a TTX-R Na+ current preferentially from small ones. For TTX-S Na+ channel, taurine of the concentration > or = 10 mM shifted the activation curve in the depolarizing direction and the inactivation curve in the hyperpolarizing direction. There was no change in the activation curve for TTX-R Na+ channel and the inactivation curve was shifted in the hyperpolarizing direction slightly in the presence of taurine > or = 20 mM. When the recovery kinetics was examined, the presence of taurine resulted in a slower recovery from inactivation of TTX-S currents and no change of TTX-R ones. All the effects of taurine were weakly concentration-dependent and partly recovered quite slowly after washout. Our data indicate that taurine alters the properties of Na+ currents in intact DRG neurons. These may contribute to the understanding of taurine as a natural neuroprotectant and the potential of taurine as a useful medicine for the treatment of sensory neuropathies.     

N-Ethylmaleimide modulation of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglion neurons

The effects of N-ethylmaleimide (NEM), an alkylating reagent to protein sulfhydryl groups, on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in rat dorsal root ganglion (DRG) neurons were studied using the whole cell configuration of patch-clamp technique. When currents were evoked by step depolarizations to 0 mV from a holding potential of -80 mV NEM decreased the amplitude of TTX-S sodium current, but exerted little or no effect on that of TTX-R sodium current. The inhibitory effect of NEM on TTX-S sodium channel was mainly due to the shift of the steady-state inactivation curve in the hyperpolarizing direction. NEM did not affect the voltage-dependence of the activation of TTX-S sodium channel. The steady-state inactivation curve for TTX-R sodium channel was shifted by NEM in the hyperpolarizing direction as that for TTX-S sodium channel. NEM caused a change in the voltage-dependence of the activation of TTX-R sodium channel unlike TTX-S sodium channel. After NEM treatment, the amplitudes of TTX-R sodium currents at test voltages below -10 mV were increased, but those at more positive voltages were not affected. This was explained by the shift in the conductance-voltage curve for TTX-R sodium channels in the hyperpolarizing direction after NEM treatment.     

N-Ethylmaleimide modulation of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglion neurons

The effects of N-ethylmaleimide (NEM), an alkylating reagent to protein sulfhydryl groups, on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in rat dorsal root ganglion (DRG) neurons were studied using the whole cell configuration of patch-clamp technique. When currents were evoked by step depolarizations to 0 mV from a holding potential of −80 mV NEM decreased the amplitude of TTX-S sodium current, but exerted little or no effect on that of TTX-R sodium current. The inhibitory effect of NEM on TTX-S sodium channel was mainly due to the shift of the steady-state inactivation curve in the hyperpolarizing direction. NEM did not affect the voltage-dependence of the activation of TTX-S sodium channel. The steady-state inactivation curve for TTX-R sodium channel was shifted by NEM in the hyperpolarizing direction as that for TTX-S sodium channel. NEM caused a change in the voltage-dependence of the activation of TTX-R sodium channel unlike TTX-S sodium channel. After NEM treatment, the amplitudes of TTX-R sodium currents at test voltages below −10 mV were increased, but those at more positive voltages were not affected. This was explained by the shift in the conductance–voltage curve for TTX-R sodium channels in the hyperpolarizing direction after NEM treatment.     

ATP modulation of sodium currents in rat dorsal root ganglion neurons

The modulation of tetrodotoxin-sensitive (TTX-S) and slow tetrodotoxin-resistant (TTX-R) sodium currents in rat dorsal root ganglion neurons by ATP was studied using the whole-cell patch-clamp method. The effects of ATP on two types of sodium currents were either stimulatory or inhibitory depending on the kinetic parameters tested. At a holding potential of -80 mV ATP suppressed TTX-S sodium currents when the depolarizing potential was positive to -30 mV but it increased them when the depolarizing potential was negative to -30 mV. At the same holding potential slow TTX-R sodium currents were always increased by ATP regardless of the depolarizing potential. In both types of sodium currents ATP shifted both the conductance-voltage relationship curve and the steady-state inactivation curve in the hyperpolarizing direction, and accelerated the time-dependent inactivation. ATP decreased the maximum conductance of TTX-S sodium currents but increased that of slow TTX-R sodium currents. The results suggest that ATP would decrease the excitability of neurons with TTX-S sodium channels but would increase that of neurons with slow TTX-R sodium channels. The effects of ATP on sodium currents were preserved in the presence of a G-protein inhibitor, GDP-beta-S, or purinergic antagonists, suramin and Reactive Blue-2, suggesting that purinergic receptors might not be involved in ATP modulation of sodium currents.     

Three types of sodium channels in adult rat dorsal root ganglion neurons

Several types of Na+ currents have previously been demonstrated in dorsal root ganglion (DRG) neurons isolated from neonatal rats, but their expression in adult neurons has not been studied. Na+ current properties in adult dorsal root ganglion (DRG) neurons of defined size class were investigated in isolated neurons maintained in primary culture using a combination of microelectrode current clamp, patch voltage clamp and immunocytochemical techniques. Intracellular current clamp recordings identified differing relative contributions of TTX-sensitive and -resistant inward currents to action potential waveforms in DRG neuronal populations of defined size. Patch voltage clamp recordings identified three distinct kinetic types of Na+ current differentially distributed among these size classes of DRG neurons. 'Small' DRG neurons co-express two types of Na+ current: (i) a rapidly-inactivating, TTX-sensitive 'fast' current and (ii) a slowly-activating and -inactivating, TTX-resistant 'slow' current. The TTX-sensitive Na+ current in these cells was almost completely inactivated at typical resting potentials. 'Large' cells expressed a single TTX-sensitive Na+ current identified as 'intermediate' by its inactivation rate constants. 'Medium'-sized neurons either co-expressed 'fast' and 'slow' current or expressed only 'intermediate' current. Na+ channel expression in these size classes was also measured by immunocytochemical techniques. An antibody against brain-type Na+ channels (Ab7493)10 labeled small and large neurons with similar intensity. These results demonstrate that three types of Na+ currents can be detected which correlate with electrogenic properties of physiologically and anatomically distinct populations of adult rat DRG neurons.     

Differential properties of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglion neurons.

TTX-sensitive (TTX-S) and TTX-resistant (TTX-R) sodium channel currents were analyzed in acutely dissociated dorsal root ganglion (DRG) neurons isolated from 3-12-d-old and adult rats. Currents were recorded using the whole-cell patch-clamp technique. TTX-R current was more likely to be present in younger animals (3-7 d), whereas TTX-S current was more common in older animals (7-10 d), although TTX-R current was recorded from adult rat DRG neurons. The TTX-R and TTX-S currents differed in their steady-state inactivation, with 50% inactivation voltage at -40 +/- 5 mV (n = 10) for TTX-R currents and -70 +/- 4 mV (n = 10) for TTX-S currents. These current types also differed in their activation kinetics, with 50% activation values of -15 +/- 5 mV (n = 5) for TTX-R currents and -26 +/- 6 mV (n = 5) for TTX-S currents. The interactions of TTX-R and TTX-S channels with various pharmacological agents and divalent cations were studied. The Kd values for TTX-S and TTX-R currents were estimated to be 0.3 nM and 100 microM for TTX, 0.5 nM and 10 microM for saxitoxin, and 50 microM and 200 microM for lidocaine, respectively. TTX-S channels did not exhibit a marked use-dependent block by lidocaine, whereas lidocaine significantly decreased TTX-R current in a use-dependent manner at frequencies ranging from 1 to 33.3 Hz. Several external divalent cations exerted different effects on these current types.(ABSTRACT TRUNCATED AT 250 WORDS)     

Single-channel analysis of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels in rat dorsal root ganglion neurons

Tetrodotoxin-sensitive and tetrodotoxin-resistant single sodium channel currents were recorded from rat dorsal root ganglion neurons. The two types of sodium channel currents could be distinguished by the effects of predepolarization, 10 nM tetrodotoxin, and the inactivation during depolarization. Single-channel conductances were calculated to be 6.3 and 3.4 pS in the tetrodotoxin-sensitive and tetrodotoxin-resistant channels, respectively.     

Substratum-induced modulation of acetylcholinesterase activity in cultured dorsal root ganglion neurons.

Acetylcholinesterase (AChE) has been shown to be transiently expressed in the developing nervous system during periods of neuronal migration and axonal outgrowth. We are investigating the possible interaction of substratum with AChE activity in dorsal root ganglion neurons (DRGN) cultured on substrata with varying degrees of permissiveness for neurite outgrowth: (1) extracellular matrix substrata: reconstituted basal lamina Matrigel (MGEL), laminin (LAM) and type I collagen (COL), and (2) organotypic substrata: unfixed, frozen sections of sciatic nerve (SN) and spinal cord (SC). In group 1, histochemical staining for AChE in DRGN was lowest on MGEL where outgrowth was most vigorous, intermediate on LAM, and highest on COL where neurite outgrowth was reduced by 55% compared to Matrigel and highly fasciculated. A similar trend was seen when the cultures were assayed biochemically, 2.84 +/- 0.14 nmoles ACh hydrolyzed/ganglion/hr (MGEL), 4.42 +/- 0.19 (LAM), 5.79 +/- 0.37 (COL). In group 2, SN supported an expansive outgrowth with lower AChE activity than in DRGN grown on SC where outgrowth was minimal. These studies show that the levels of AChE activity can be modulated by substratum, perhaps in proportion to the permissiveness of the substratum to neuritic outgrowth. These results are discussed in relation to possible non-cholinergic roles of AChE.     

Time course and temperature dependence of allethrin modulation of sodium channels in rat dorsal root ganglion cells

Key effects of the pyrethroid insecticide allethrin, delivered to or washed out from cells at 10 or 100 μM in 0.1% DMSO, on neuronal Na⁺ channel currents were studied in rat dorsal root ganglion (DRG) cells under whole-cell patch clamp. Tetrodotoxin-resistant (TTX-R) Na⁺ channels were more responsive to allethrin than tetrodotoxin-sensitive (TTX-S) Na⁺ channels. On application of 10 or 100 μM allethrin to cells with TTX-R Na⁺ channels, the Na⁺ tail current during repolarization developed a large slowly decaying component within 10 min. This slow tail developed multiphasically, suggesting that allethrin gains access to Na⁺ channels by a multiorder process. On washout (with 0.1% DMSO present), the slow tail current disappeared monophasically (exponential τ=188±44 s). Development and washout rates did not depend systematically on temperature (12°, 18°, or 27°C), but washout was slowed severely if DMSO was absent. As the duration of a depolarizing pulse was increased (range 0.32–10 ms), the amplitude of the slow component of the succeeding tail conductance first increased then decreased. Tail current amplitude had the same dependence on preceding pulse duration (at 18°) at 10 or 100 μM, consistent with allethrin modification of Na⁺ channels at rest before opening. At 10 μM, slow tail conductance was at maximum 40% of the peak conductance during the previous depolarization, independent of temperature; evidently, the fraction of open modified channels did not change. However, at low temperature, the tail is more prolonged, bringing more Na⁺ ions into a cell. In functioning neurons, this Na⁺ influx would cause a larger depolarizing afterpotential, a condition favoring the repetitive discharges, which are signatory of pyrethroid intoxication.     

Chloride current induced by alcohols in rat dorsal root ganglion neurons.

We have recently demonstrated that ethanol and longer-chain alcohols (n-alcohols) enhance gamma-aminobutyric acid (GABA)-induced chloride currents before desensitization takes place. The potencies of n-alcohols increase with lengthening of the carbon chain. We now report that n-alcohols induce chloride currents by themselves in rat dorsal root ganglion neurons in primary culture. The whole cell variation of the patch clamp techniques was used to record currents as induced by external application of alcohols and other test compounds. Ethanol, n-butanol, n-hexanol and n-octanol induced inward currents with their potencies increasing in that order. The potencies were approximately one order of magnitude less than those to augment GABA-induced currents. The maximum amplitudes of currents induced by the alcohols were less than those produced by GABA. The n-octanol-induced currents were carried largely by chloride ions because the reversal potentials were changed according to the Nernst chloride potential as the internal chloride concentration was changed. Bicuculline and picrotoxin suppressed the n-octanol-induced current, and chlordiazepoxide and pentobarbital augmented the n-octanol-induced current. Therefore, the alcohol-induced chloride currents flow through the chloride channels associated with the GABAA receptors. When applied after the GABA-induced current was desensitized to a lower level, n-octanol suppressed rather than augmented the current. Thus, n-alcohols mimic barbiturates in augmenting the GABA-induced currents and in generating chloride currents by themselves. These actions of both agents may play a role in causing anxiolytic, sedative and/or anesthetic effects.     

Tetramethylpyrazine inhibits ATP-activated currents in rat dorsal root ganglion neurons

Tetramethylpyrazine (TMP) is one of the alkaloids contained in Ligustrazine which has been used in traditional Chinese medicine as an analgesic for injury and dysmenorrhea. ATP can elicit the sensation of pain. This study observed the effects of TMP on ATP-activated current (IATP) in rat DRG neurons. TMP (0.1-1 mM) concentration-dependently inhibited ATP (100 microM)-activated current in rat DRG neurons. The inhibitory time of ATP (100 microM)-activated current appeared at 15 s after preapplication of TMP and reached its peak at about 45 s. The dose-response curves for IATP in the absence and presence of 1 mM TMP showed that TMP (1 mM) shifted the concentration-response curve of IATP downward markedly and the two EC50 values were very close (75 vs. 82 microM), while the threshold value remained unchanged. Therefore, the inhibitory effect of TMP on IATP may be noncompetitive. TMP did not alter the reversal potential (0 mV) of ATP-activated current, indicating that the site of TMP action is on or near the exterior surface of channel protein and not within the channel pore. Externally applied TMP (1 mM) increases the inhibitory effect of chelerythrine (PKC inhibitor) contained in pipette solution on IATP. The site of TMP action may be the binding of TMP to an allosteric site on the large extracellular region of ATP receptor-ion channel complex (P2X receptors) or PKC site of the N-terminus of P2X receptors. The mechanism of TMP action may be the allosteric regulation via acting on the large extracellular region of ATP receptor-ion channel complex (P2X receptors) and promoting the phosphorylation of PKC site of the N-terminus of P2X receptors.     

Modulation of tetrodotoxin-resistant sodium channels by dihydropyrazole insecticide RH-3421 in rat dorsal root ganglion neurons

The effects of the dihydropyrazole insecticide RH-3421 on the retrodotoxin-resistant (TTX-R) voltage-gated sodium channels in rat dorsal root ganglion (DRG) neurons were studied using the whole-cell patch clamp technique. RH-3421 at 10 nM to 1 microM completely blocked action potentials. The sodium currents were irreversibly suppressed by 1 microM RH-3421 in a time- and a dose-dependent manner and the IC50 value of RH-3421 was estimated to be 0.7 microM after 10 min of application. RH-3421 blocked the sodium currents to the same extent over the entire range of test potentials. The sodium conductance-voltage curve was not shifted along the voltage axis by 1 microM RH-3421 application In contrast, both fast and slow steady-state sodium channel inactivation curves were shifted in the hyperpolarizing direction in the presence of 1 microM RH-3421. It was concluded that RH-3421 bound to the resting and inactivated sodium channels to cause block with a higher affinity for the latter state.     

G-proteins are involved in riluzole inhibition of high voltage-activated calcium channels in rat dorsal root ganglion neurons

Effects of riluzole on high voltage-activated (HVA) calcium channels of rat dorsal root ganglion neurons were studied using the whole-cell patch-clamp technique. Riluzole at 30 μM inhibited the HVA currents. The onset and offset of riluzole inhibitory effect were slow usually taking more than 3 min. Riluzole inhibition of the HVA currents was abolished and partially reduced by addition of 500 μM GDP-β-S and 1 mM N-ethylmaleimide, respectively, to the pipette solution. Pre-treatment with pertussis toxin or application of depolarizing pre-pulses did not affect riluzole's inhibitory effect on the HVA currents. Riluzole inhibition of the HVA currents was also blocked by internal application of 50 μg/ml protein kinase A inhibitory peptide. It was concluded that pertussis toxin-insensitive G-proteins and protein kinase A may be involved in riluzole inhibition of the HVA currents.     

Hypoxia-induced apoptosis in adult rat dorsal root ganglion neurons in vitro

Following spinal root injury, dorsal root ganglia suffer mechanical trauma and compromised blood supply. Little is known about the consequences for neuronal survival. Here we used cyanide treatment in vitro to examine effects of moderate hypoxia on adult rat dorsal root ganglion cells identified by GAP-43 immunostaining. 400 microM-4 mM cyanide caused sustained increases in intracellular Ca2+. Cyanide at 2 mM led to a significant increase in apoptosis, detected using TUNEL labelling and confirmed by ultrastructural analysis, and a further increase when cultures were left overnight in fresh medium. Our study shows that dorsal root ganglion neurons die by apoptosis following hypoxia and that cell death increases over time. Cyanide response provides a simple assay for testing neuroprotective agents and examining underlying mechanisms.     

Calcium channels in the somatic membrane of the rat dorsal root ganglion neurons, effect of cAMP

Isolated rat dorsal root ganglion neurons have been perfused with potassium-free solutions containing cAMP, ATP and Mg2+ ions. In these conditions stable inward calcium currents can be recorded in the somatic membrane of all investigated cells. The kinetics of these currents can be approximated by a modified Hodgkin-Huxley equation using a square power of the m-variable; its inactivation is extremely slow. The corresponding channels pass Ba2+ ions about twice more effective than Ca2+.     

Differential sensitivity of tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels to the insecticide allethrin in rat dorsal root ganglion neurons

The pyrethroid insecticides are known to modify neuronal sodium channels to cause a prolongation of whole cell current. The sodium channels expressed in the dorsal root ganglion neurons of the rat are of two types, one highly sensitive to tetrodotoxin and the other highly resistant to tetrodotoxin. The pyrethroid allethrin exerted profound effects on tetrodotoxin-resistant sodium channels while causing minimal effects on tetrodotoxin-sensitive sodium channels. Currents derived from tetrodotoxin-resistant sodium channels were greatly prolonged during a step depolarization; the tail currents upon repolarization were also augmented and prolonged. In the tetrodotoxin-sensitive sodium channel currents, these changes caused by allethrin were much smaller or negligible. The activation and inactivation voltages of tetrodotoxin-resistant peak sodium currents were not significantly altered by allethrin. The differential action of allethrin on the two types of sodium channels would be important not only in identifying the target molecular structure but also in interpreting the symptoms of poisoning in mammals.     

Resveratrol inhibits Na+ currents in rat dorsal root ganglion neurons

Resveratrol, a phytoalexin found in grapevines, exerts neuroprotective, cancer chemopreventive, antiinflammatory and cardioprotective activities. Studies have also shown that resveratrol exhibits analgesic effects. Cyclooxygenase inhibition and K+ channel opening have been suggested as underlying mechanisms for the resveratrol-induced analgesia. Here, we investigated the effects of resveratrol on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) Na+ currents in rat dorsal root ganglion (DRG) neurons. Resveratrol suppressed both Na+ currents evoked at 0 mV from -80 mV. TTX-S Na+ current (K(d), 72 microM) was more susceptible to resveratrol than TTX-R Na+ current (K(d), 211 microM). Although the activation voltage of TTX-S Na+ current was shifted in the depolarizing direction by resveratrol, that of TTX-R Na+ current was not. Resveratrol caused a hyperpolarizing shift of the steady-state inactivation voltage and slowed the recovery from inactivation of both Na+ currents. However, no frequency-dependent inhibition of resveratrol on either type of Na+ current was observed. The suppression and the unfavorable effects on the kinetics of Na+ currents in terms of the excitability of DRG neurons may make a great contribution to the analgesia by resveratrol.