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.     

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)     

Tetrodotoxin-resistant sodium channels Na(v)1.8/SNS and Na(v)1.9/NaN in afferent neurons innervating urinary bladder in control and spinal cord injured rats

Tetrodotoxin-resistant (TTX-R) sodium channels Na(v)1.8/SNS and Na(v)1.9/NaN are preferentially expressed in small diameter dorsal root ganglia (DRG) neurons. The urinary bladder is innervated by small afferent neurons from L6/S1 DRG, of which approximately 75% exhibit high-threshold action potentials that are mediated by TTX-R sodium channels. Following transection of the spinal cord at T8, the bladder becomes areflexic and then gradually hyper-reflexic, and there is an attenuation of the TTX-R sodium currents in bladder afferent neurons. In the present study, we demonstrate that Na(v)1.8 is expressed in both bladder and non-bladder afferent neurons, while Na(v)1.9 is expressed in non-bladder afferent neurons but is rarely observed in bladder afferent neurons. In spinal cord transected rats 28-32 days following transection, there is a decreased expression of Na(v)1.8 sodium channels in bladder afferents, but no change in the expression of Na(v)1.8 in non-bladder afferent neurons. Both bladder and non-bladder afferent neurons exhibit limited increases in Na(v)1.9 expression following spinal cord transection. These results demonstrate that the expression of TTX-R channels in bladder afferent neurons changes after spinal cord transection, and these changes may contribute to the increased excitability of these neurons following spinal cord injury.     

Modulation of sodium currents in rat dorsal root ganglion neurons by sulfur dioxide derivatives

The effect of sulfur dioxide (SO2) derivatives, a common air pollutant and exists in vivo as an equilibrium between bisulfate and sulfite, on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium channels in cultured post-natal dorsal root ganglion (DRG) neurons were studied using the whole cell configuration of patch-clamp technique. SO2 derivatives on two types of sodium currents were either inhibitory or stimulatory depending on the kinetic parameters tested. At a holding potential of -80 mV, SO2 derivatives suppressed TTX-S sodium currents when depolarizing potential was negative to -30 mV and TTX-R sodium currents when negative to -10 mV but they increased them when the depolarizing potential was positive to -30 or -10 mV. SO2 derivatives shifted the conductance-voltage curve for TTX-R sodium currents in the depolarizing direction but had little effect on that for TTX-S sodium currents. The steady-state inactivation curve for TTX-R sodium channel was shifted by SO2 derivatives in the depolarizing direction as that for TTX-S sodium channel. SO2 derivatives changed the reversal potential and increased the maximum conductance of two types of sodium channels. SO2 derivatives postponed the activating time and delayed the inactivation of sodium currents. The results suggest that SO2 derivatives would increase the excitability of neurons and alter the ion selectivity for two types of sodium currents.     

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.     

Differential effects of the pyrethroid tetramethrin on tetrodotoxin-sensitive and tetrodotoxin-resistant single sodium channels

The differential effects of the pyrethroid tetramethrin on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) single sodium channel currents in rat dorsal root ganglion (DRG) neurons were investigated using the outside-out configuration of patch-clamp technique. Channel conductances were 10.7 and 6.3 pS for TTX-S and TTX-R sodium channels, respectively, at a room temperature of 24–26°C. The single-channel current of TTX-S sodium channels at the test potential of −30 mV was −1.27 ± 0.25 pA, and was not changed after exposure to 10 μM tetramethrin (−1.28 ± 0.23 pA). The open time histogram of TTX-S single-channel currents could be fitted by a single exponential function with a time constant of 1.27 ms. After exposure to 10 μM tetramethrin, the open time histogram could be fitted by the sum of two exponential functions with time constants of 1.36 ms (τ_fast) and 5.73 ms (τ_low). The percentage of contribution of each component to the population was 62% for the fast component representing the normal channels and 38% for the slow component representing the tetramethrin modified channels. The amplitudc of TTX-R single-channel currents was slightly changed from −0.72 ± 0.14 to −0.83 ± 0.07 pA by 10 μM tetramethrin. The open time histogram of TTX-R single-channel currents could be fitted by a single exponential function with a time constant of 1.92 ms. In the presence of 10 μM tetramethrin, the open time histogram could be fitted by the sum of two exponential functions with time constants of 2.07 ms (τ_fast) and 9.75 ms (τ_slow). The percentage of contribution of each component was 15% for the fast, unmodified component and 85% for the slow, modified component. Differential effects of tetramethrin on the open time distribution of single sodium channel currents explains the differential sensitivity of TTX-S and TTX-R sodium channels.     

Effects of lysophosphatidic acid on sodium currents in rat dorsal root ganglion neurons

Lysophosphatidic acid (LPA), a simple phospholipid, induces pain. To elucidate an involvement of ion channel mechanism in the LPA-induced pain, its effects on sodium currents in rat dorsal root ganglion (DRG) neurons were investigated. LPA suppressed tetrodotoxin-sensitive (TTX-S) sodium current, but increased tetrodotoxin-resistant (TTX-R) sodium current, when currents were evoked by step depolarizations to 0 mV from a holding potential of -80 mV. In both types of currents, LPA produced a hyperpolarizing shift of both activation and inactivation voltages. LPA had a negligible effect on the maximal conductance of TTX-S current, but increased that of TTX-R current. The results suggest that the enhancement of TTX-R current may contribute to the LPA-induced pain.     

Block of sodium currents in rat dorsal root ganglion neurons by diphenhydramine

To elucidate the local anesthetic mechanism of diphenhydramine, its effects on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium currents in rat dorsal root ganglion (DRG) neurons were examined by the whole-cell voltage clamp method. Diphenhydramine blocked TTX-S and TTX-R sodium currents with K(d) values of 48 and 86 microM, respectively, at a holding potential of -80 mV. It shifted the conductance-voltage curve for TTX-S sodium currents in the depolarizing direction but had little effect on that for TTX-R sodium currents. Diphenhydramine caused a shift of the steady-state inactivation curve for both types of sodium currents in the hyperpolarizing direction. The time-dependent inactivation became faster and the recovery from the inactivation was slowed by diphenhydramine in both types of sodium currents. Diphenhydramine produced a profound use-dependent block when the cells were repeatedly stimulated with high-frequency depolarizing pulses. The use-dependent block was more pronounced in TTX-R sodium currents. The results show that diphenhydramine blocks sodium channels of sensory neurons similarly to local anesthetics.     

Different voltage-gated sodium currents are expressed by human neuroblastoma NB69 cells when cultured in defined serum-free and in astroglial-conditioned media

Voltage-gated Na⁺ currents (I_Na) were analysed with the whole-cell patch-clamp technique in human neuroblastoma NB69 cells plated in serum-free “defined” medium (DM) or in “astroglial-conditioned” medium (CM). Cells survived in both media and expressed the microtubule associated protein 1A, indicating neuron-like differentiation. Two I_Na types with different time-, voltage-dependent properties and tetrodotoxin (TTX) sensitivities were expressed in DM and CM. The I_Na in DM-plated cells was present from day 4 and its surface density increased from 11 pA/pF (days 5–7) to 68 pA/pF (days 15–30). The underlying conductance (G_Na) half-activated (V_0A) at −24 mV. I_Na inactivation was fitted by single exponentials with 7.5 ms time constant (t_h) at the −35 mV half-inactivation voltage (V_0I). I_Na was not affected by 10 nM, was reduced (65%) by 100 nM, and not completely abolished (92%) by 300 nM tetrodotoxin (TTX). The I_Na of CM-plated cells appeared at day 3–4 and its surface density increased from 14 pA/pF (days 3–6) to 28 pA/pF (days 11–14). The G_Na V_0A was −29 mV and inactivation was fitted by single exponentials with 2.6 ms t_h at the −58 mV V_0I. This I_Na was reduced (55%) by 10 nM and totally abolished by 100 nM tetrodotoxin (TTX). In conclusion, NB69 cells displayed a slow, “TTX-resistant,” or a fast, “TTX-sensitive” I_Na in DM and CM, respectively, suggesting that the CM contained diffusible trophic factors of astroglial origin that induced the expression of a different Na⁺ channel type. About half of the CM- and DM-plated cells also displayed a persistent Na⁺ current (I_NaP). © 1997 Wiley-Liss Inc.     

Effects of ATP on TTX-sensitive and TTX-resistant sodium currents in rat sensory neurons.

Differential effects of ATP on tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium currents in rat dorsal root ganglion neurons were studied using the whole-cell variation of path-clamp technique. Currents were evoked by step depolarizations to 0 mV from a holding potential of -80 mV. ATP suppressed TTX-S sodium currents while it increased TTX-R sodium currents. The effects were concentration-dependent and were reversible upon washout with ATP-free external solution. ATP-gamma-S, a hydrolysis-resistant ATP analog, also affected two types of sodium currents similarly to ATP, excluding the possibility that the effects were caused by the products of ATP hydrolysis, namely adenosine. ATP by modulating sodium currents may exert profound effects on the transmission of sensory information such as nociception.     

Expression and kinetic properties of Na(+) currents in rat cardiac dorsal root ganglion neurons

The expression and properties of voltage-gated Na(+) currents in cardiac dorsal root ganglion (DRG) neurons were assessed in this study. Cardiac DRG neurons were labelled by injecting the Fast Blue fluorescent tracer into the pericardium. Recordings were performed from 138 cells. Voltage-dependent Na(+) currents were found in 115 neurons. There were 109 neurons in which both tetrodotoxin-sensitive (TTX-S, blocked by 1 microM of TTX) and tetrodotoxin-resistant (TTX-R, insensitive to 1 microM of TTX) Na(+) currents were present. Five cells expressed TTX-R current only and one cell only the TTX-S current. The kinetic properties of Na(+) currents and action potential waveform parameters were measured in neurons with cell membrane capacitance ranging from 15 to 75 pF. The densities of TTX-R (110.0 pA/pF) and TTX-S (126.1 pA/pF) currents were not significantly different. Current threshold was significantly higher for TTX-R (-34 mV) than for TTX-S (-40.4 mV) currents. V(1/2) of activation for TTX-S current (-19.6 mV) was significantly more negative than for TTX-R current (-9.2 mV), but k factors did not differ significantly. V(1/2) and the k constant for inactivation for TTX-S currents were -35.6 and -5.7 mV, respectively. These values were significantly lower than those recorded for TTX-R current for which V(1/2) and k were -62.3 and -7.7 mV, respectively. The action potential threshold was lower, the 10-90% rise time and potential width were shorter before than after the application of TTX. Based on this we drew the conclusion that action potential recorded before adding tetrodotoxin was mainly TTX-S current dependent, while the action potential recorded after the application of toxin was TTX-R current dependent. We also found 23 cells with mean membrane capacitance ranging from 12 to 35 pF (the smallest labelled DRG cells found in this study) that did not express the Na(+) current. The function of these cells is unclear. We conclude that the overwhelming majority of cardiac dorsal root ganglion neurons in which voltage-dependent Na(+) currents were present, exhibited both TTX-S and TTX-R Na(+) currents with remarkably similar expression and kinetic properties.     

The voltage‐dependent conductances of rat neocortical layer I neurons

Whole cell patch-clamp techniques were used to study voltage-dependent sodium (Na⁺), calcium (Ca²⁺), and potassium (K⁺) conductances in acutely isolated neurons from cortical layer I of adult rats. Layer I cells were identified by means of γ-aminobutyric acid (GABA) immunocytochemistry. Positive stainings for the Ca²⁺-binding protein calretinin in a subset of cells, indicated the presence of Cajal–Retzius (C-R) cells. All investigated cells displayed a rather homogeneous profile of voltage-dependent membrane currents. A fast Na⁺ current activated at about −45 mV, was half-maximal steady-state inactivated at −66.6 mV, and recovery from inactivation followed a two-exponential process (τ₁ = 8.4 ms and τ₂ = 858.8 ms). Na⁺ currents declined rapidly with two voltage-dependent time constants, reaching baseline current after some tens of milliseconds. In a subset of cells (< 50%) a constant current level of < 65 pA remained at the end of a 90 ms step. A transient outward current (I_fast) activated ≈–40 mV, declined rapidly with a voltage-insensitive time constant (τ≈ 350 ms) and was relatively insensitive to tetraethylammonium (TEA, 20 mm ). I_fast was separated into two components based on their sensitivity to 4-aminopyridine (4-AP): one was blocked by low concentrations (40 μm ) and a second by high concentrations (6 mm ). After elimination of I_fast by a conditioning prepulse (50 ms to −50 mV), a slow K⁺ current (I_KV) could be studied in isolation. I_KV was only moderately affected by 4-AP (6 mm ), while TEA (20 mm ) blocked most (> 80%) of the current. I_KV activated at about −40 mV, declined monoexponentially in a voltage-dependent manner (τ≈ 850 ms at −30 mV), and revealed an incomplete steady-state inactivation. In addition to I_fast and I_KV, indications of a Ca²⁺-dependent outward current component were found. When Na⁺ currents, I_fast, and I_KV were blocked by tetrodotoxin (TTX, 1 μm ), 4-AP (6 mm ) and TEA (20 mm ) an inward current carried by Ca²⁺ was found. Ca²⁺ currents activated at depolarized potentials at about −30 mV, were completely blocked by 50 μm cadmium (Cd²⁺), were sensitive to verapamil (≈ 40% block by 10 μm ), and were not affected by nickel (50 μm ). During current clamp recordings, isolated layer I neurons displayed fast spiking behaviour with short action potentials (≈ 2 ms, measured at half maximal amplitude) of relative small amplitude (≈ 83 mV, measured from the action potential threshold).     

ATP evokes different currents in TTX-sensitive and TTX-resistant cells of dorsal root ganglia

The relationship between the level of expression of tetrodotoxin-sensitive (TTX-S) and tetrodotoxin-resistant (TTX-R) sodium currents and the occurrence of two kinetically different ATP-induced currents in rat dorsal root ganglion neurons was studied. ATP evoked two distinct types of currents, one with fast activation and desensitization (I-fast) and the other with slow and persistent development (I-slow). In all TTX-S cells which expressed solely TTX-S sodium currents ATP evoked I-fast. The other cells expressed a variable proportion of TTX-S and TTX-R sodium currents. Only 15% of these TTX-R+S cells responded to ATP with I-fast. I-slow was evoked in both cell types but the magnitude of response was much greater in TTX-R+S cells. This result suggests that a different array of ion channels is equipped in different types of sensory neurons.     

Activation of Tetrodotoxin-Resistant Sodium Channel NaV1.9 in Rat Primary Sensory Neurons Contributes to Melittin-Induced Pain Behavior

Tetrodotoxin-resistant (TTX-R) sodium channels Na_V1.8 and Na_V1.9 in dorsal root ganglion (DRG) neurons play important roles in pathological pain. We recently reported that melittin, the major toxin of whole bee venom, induced action potential firings in DRG neurons even in the presence of a high concentration (500 nM) of TTX, indicating the contribution of TTX-R sodium channels. This hypothesis is fully investigated in the present study. After subcutaneous injection of melittin, Na_V1.8 and Na_V1.9 significantly upregulate mRNA and protein expressions, and related sodium currents also increase. Double immunohistochemical results show that Na_V1.8-positive neurons are mainly medium- and small-sized, whereas Na_V1.9-positive ones are only small-sized. Antisense oligodeoxynucleotides (AS ODNs) targeting Na_V1.8 and Na_V1.9 are used to evaluate functional significance of the increased expressions of TTX-R sodium channels. Behavioral tests demonstrate that AS ODN targeting Na_V1.9, but not Na_V1.8, reverses melittin-induced heat hypersensitivity. Neither Na_V1.8 AS ODN nor Na_V1.9 AS ODN affects melittin-induced mechanical hypersensitivity. These results provide previously unknown evidence that upregulation of Na_V1.9, but not Na_V1.8, in small-sized DRG neurons contributes to melittin-induced heat hypersensitivity. Furthermore, melittin-induced biological effect indicates a potential strategy to study properties of TTX-R sodium channels.     

Comparative study of lacosamide and classical sodium channel blocking antiepileptic drugs on sodium channel slow inactivation

Many antiepileptic drugs (AEDs) exert their therapeutic activity by modifying the inactivation properties of voltage‐gated sodium (Na_v) channels. Lacosamide is unique among AEDs in that it selectively enhances the slow inactivation component. Although numerous studies have investigated the effects of AEDs on Na_v channel inactivation, a direct comparison of results cannot be made because of varying experimental conditions. In this study, the effects of different AEDs on Na_v channel steady‐state slow inactivation were investigated under identical experimental conditions using whole‐cell patch‐clamp in N1E‐115 mouse neuroblastoma cells. All drugs were tested at 100 μM, and results were compared with those from time‐matched control groups. Lacosamide significantly shifted the voltage dependence of Na_v current (I_Na) slow inactivation toward more hyperpolarized potentials (by ?33 ± 7 mV), whereas the maximal fraction of slow inactivated channels and the curve slope did not differ significantly. Neither SPM6953 (lacosamide inactive enantiomer), nor carbamazepine, nor zonisamide affected the voltage dependence of I_Na slow inactivation, the maximal fraction of slow inactivated channels, or the curve slope. Phenytoin significantly increased the maximal fraction of slow inactivated channels (by 28% ± 9%) in a voltage‐independent manner but did not affect the curve slope. Lamotrigine slightly increased the fraction of inactivated currents (by 15% ± 4%) and widened the range of the slow inactivation voltage dependence. Lamotrigine and rufinamide induced weak, but significant, shifts of I_Na slow inactivation toward more depolarized potentials. The effects of lacosamide on Na_v channel slow inactivation corroborate previous observations that lacosamide has a unique mode of action among AEDs that act on Na_v channels. © 2012 Wiley Periodicals, Inc.     

Slow calcium current is not reduced in malignant hyperthermic porcine myotubes

Malignant hyperthermia-susceptible (MHS) pigs express a sarcoplasmic reticulum (SR) Ca²⁺-release channel mutation that results in lower than normal contractile thresholds in skeletal muscles. In adult MHS pig muscles the L-type calcium current (I_s) is also reduced. We tested the hypothesis that there is a causal relationship between I_s and the lower contractile threshold by recording I_s from MHs and normal porcine myotubes using the whole cell patch-clamp technique. Current voltage relationships for both MHS and normal myotubes were similar, with peak I_s between +20 and +30 mV. Maximum I_s amplitudes were not different (normal: 4976 ± 566 pA; MHS: 6516 ± 1088 pA) nor was I_s specific density (normal: 9.0 ± 0.8; MHS: 8.8 ± 1.1 pA/pF). In both MHS and normal myotubes, both the dihydropyridine antagonist PN200 –110 (200 nmol/L) and holding the membrane potential at −10 mV for 5 min decreased I_s significantly (by more than 50%). There was no apparent direct relationship between the mutation in the SR Ca²⁺-release channel and lowered I_s. We concluded that reduced I_s in adult MHS pig muscles may be a secondary effect of the SR Ca²⁺-release channel mutation on muscle development. © 1996 John Wiley & Sons, Inc.     

Changes in the properties of developing glycine receptors in cultured mouse spinal neurons

We studied several neurophysiological properties of in vitro maturing glycine receptors in mouse spinal cord neurons cultured for various times: 3–7 days (early), 10–12 days (intermediate), and 17–24 days (mature), using whole-cell and gramicidin-perforated techniques. The glycine-activated Cl⁻ conductance increased about 6-fold during in vitro development, and the current density increased from 177 ± 42 pA/pF in early to 504 ± 74 pA/pF in mature neurons. The sensitivity to glycine increased transiently from 39 ± 2.8 μM in early neurons to 29 ± 1 μM in intermediate neurons. Using whole-cell recordings, we found that E_Cl did not change during development. With the gramicidin-perforated technique, on the other hand, E_Cl shifted from −27 to −52 mV with development. Thus, immature neurons were depolarized by the activation of glycine receptors, whereas mature neurons were hyperpolarized. The current decayed (desensitized) during the application of 500 μM glycine. The decay was single exponential and the time constant increased from 2,212 ± 139 msec in early neurons to 4,580 ± 1,071 msec in mature neurons. Picrotoxin (10 μM) inhibited the current to a larger extent in early neurons (46 ± 6% of control), and the sensitivity of these receptors to strychnine (IC₅₀) increased from 23 ± 3 nM to 9 ± 1 nM in mature neurons. In conclusion, several properties of spinal glycine receptors changed during in vitro neuronal maturation. This indicates that, similar to GABA_A receptors, the functions of these receptors are developmentally regulated. These changes should affect the excitability of spinal neurons as well as other maturation processes. Synapse 28:185–194, 1998. © 1998 Wiley-Liss, Inc.     

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.