Novel Pulsed Ultrahigh-frequency Spinal Cord Stimulation Inhibits Mechanical Hypersensitivity and Brain Neuronal Activity in Rats after Nerve Injury.

BACKGROUND: Spinal cord stimulation (SCS) is an important pain treatment modality. This study hypothesized that a novel pulsed ultrahigh-frequency spinal cord stimulation (pUHF-SCS) could safely and effectively inhibit spared nerve injury-induced neuropathic pain in rats. METHODS: Epidural pUHF-SCS (± 3V, 2-Hz pulses comprising 500-kHz biphasic sinewaves) was implanted at the thoracic vertebrae (T9 to T11). Local field brain potentials after hind paw stimulation were recorded. Analgesia was evaluated by von Frey-evoked allodynia and acetone-induced cold allodynia. RESULTS: The mechanical withdrawal threshold of the injured paw was 0.91 ± 0.28 g lower than that of the sham surgery (24.9 ± 1.2 g). Applying 5-, 10-, or 20-min pUHF-SCS five times every 2 days significantly increased the paw withdrawal threshold to 13.3 ± 6.5, 18.5 ± 3.6, and 21.0 ± 2.8 g at 5 h post-SCS, respectively (P = 0.0002, < 0.0001, and < 0.0001; n = 6 per group) and to 6.1 ± 2.5, 8.2 ± 2.7, and 14.3 ± 5.9 g on the second day, respectively (P = 0.123, 0.013, and < 0.0001). Acetone-induced paw response numbers decreased from pre-SCS (41 ± 12) to 24 ± 12 and 28 ± 10 (P = 0.006 and 0.027; n = 9) at 1 and 5 h after three rounds of 20-min pUHF-SCS, respectively. The areas under the curve from the C component of the evoked potentials at the left primary somatosensory and anterior cingulate cortices were significantly decreased from pre-SCS (101.3 ± 58.3 and 86.9 ± 25.5, respectively) to 39.7 ± 40.3 and 36.3 ± 20.7 (P = 0.021, and 0.003; n = 5) at 60 min post-SCS, respectively. The intensity thresholds for pUHF-SCS to induce brain and sciatic nerve activations were much higher than the therapeutic intensities and thresholds of conventional low-frequency SCS. CONCLUSIONS: Pulsed ultrahigh-frequency spinal cord stimulation inhibited neuropathic pain-related behavior and paw stimulation evoked brain activation through mechanisms distinct from low-frequency SCS.

Inhibiting the LPS-induced enhancement of mEPSC frequency in superficial dorsal horn neurons may serve as an electrophysiological model for alleviating pain.

Pain is a major primary health care problem. Emerging studies show that inhibition of spinal microglial activation reduces pain. However, the precise mechanisms by which microglial activation contributes to nociceptive synaptic transmission remain unclear. In this study, we measured spontaneous synaptic activity of miniature excitatory postsynaptic currents (mEPSCs) in rat spinal cord superficial dorsal horn (SDH, laminae I and II) neurons. Lipopolysaccharide (LPS) and adenosine triphosphate (ATP) increased the frequency, but not amplitude, of mEPSCs in SDH neurons. Microglial inhibitors minocycline and paeonol, as well as an astrocyte inhibitor, a P2Y1 receptor (P2Y1R) antagonist, and a metabotropic glutamate receptor 5 (mGluR5) antagonist, all prevented LPS-induced enhancement of mEPSC frequency. In mouse behavioral testing, minocycline and paeonol effectively reduced acetic acid-induced writhing and LPS-induced hyperalgesia. These results indicate that LPS-activated microglia release ATP, which stimulates astrocyte P2Y1Rs to release glutamate, triggering presynaptic mGluR5 receptors and increasing presynaptic glutamate release, leading to an increase in mEPSC frequency and enhancement of nociceptive transmission in SDH neurons. We propose that these effects can serve as a new electrophysiological model for evaluating pain. Moreover, we predict that pharmacologic agents capable of inhibiting the LPS-induced enhancement of mEPSC frequency in SDH neurons will have analgesic effects.

Paeonol promotes hippocampal synaptic transmission: The role of the Kv2.1 potassium channel.

Paeonol is a major constituent of the Chinese herb Moutan cortex radices. Recent studies report that paeonol has neuroprotective effects and improves impaired learning and memory. However, its underlying mechanisms by which paeonol contributes to synaptic transmission remain unclear. In this study, we found that paeonol increased the frequency of miniature excitatory postsynaptic currents (mEPSCs) and spontaneous excitatory postsynaptic currents (sEPSCs), but had no effect on the amplitude in rat hippocampal CA1 neurons. Similarly, the acetylcholinesterase (AChE) inhibitor rivastigmine increased the frequency of mEPSCs, but had no effect upon amplitude in rat hippocampal neurons. Rivastigmine also inhibited the delayed outward K+ currents in rat hippocampal CA1 neurons, but had no effect in nucleus ambiguus (NA) neurons. The Kv2 blocker guangxitoxin-1E increased the frequency of both mEPSCs and sEPSCs of rat hippocampal CA1 neurons, without affecting their amplitude. Our results suggest that paeonol and rivastigmine enhance spontaneous presynaptic transmitter release, which may be associated with the inhibition of the hippocampal Kv2 current and with therapeutic potential in neurotransmitter deficits found in Alzheimer's disease (AD). Moreover, our data also show that paeonol protects against Aß25-35-induced impairment of long-term potentiation (LTP) in mouse hippocampal neurons. However, guangxitoxin-1E failed to potentiate the evoked field excitatory postsynaptic potentials (fEPSPs), LTP and Aß25-35-induced impairment of LTP. These results indicate that paeonol may has the potential to improve learning and memory in AD. Interestingly, this effect is not involved in the inhibition of the hippocampal Kv2 current.

A comparison of the delayed outward potassium current between the nucleus ambiguus and hippocampus: sensitivity to paeonol.

Whole-cell patch-clamp recordings investigated the electrophysiological effects of 2'-hydroxy-4'-methoxyacetophenone (paeonol), one of the major components of Moutan Cortex, in hippocampal CA1 neurons and nucleus ambiguus (NA) neurons from neonatal rats as well as in lung epithelial H1355 cells expressing Kv2.1 or Kv1.2. Extracellular application of paeonol at 100µM did not significantly affect the spontaneous action potential frequency, whereas paeonol at 300µM increased the frequency of spontaneous action potentials in hippocampal CA1 neurons. Paeonol (300µM) significantly decreased the tetraethylammonium-sensitive outward current in hippocampal CA1 neurons, but had no effect upon the fast-inactivating potassium current (IA). Extracellular application of paeonol at 300µM did not affect action potentials or the delayed outward currents in NA neurons. Paeonol (100µM) reduced the Kv2.1 current in H1355 cells, but not the Kv1.2 current. The inhibitor of Kv2, guangxitoxin-1E, reduced the delayed outward potassium currents in hippocampal neurons, but had only minimal effects in NA neurons. We demonstrated that paeonol decreased the delayed outward current and increased excitability in hippocampal CA1 neurons, whereas these effects were not observed in NA neurons. These effects may be associated with the inhibitory effects on Kv2.1 currents.

Inhibition of voltage-gated Na+ channels by hinokiol in neuronal cells.

BACKGROUND: Hinokiol is a naturally occurring diterpenoid compound isolated from plants such as Taiwania cryptomerioides. Anti-oxidation, anti-cancer, and anti-inflammation effects of this compound have been reported. It is not yet known if hinokiol affects neurons or neuronal ion channel activities. We reported here that hinokiol inhibited voltage-gated Na(+) channels (VGSC) in neuronal cells and we characterized the mechanisms of block. METHODS: The effects of hinokiol on Na(+) channels were examined using the voltage-clamp (whole-cell mode) technique. RESULTS: VGSC was blocked by hinokiol in a concentration-dependent and state-dependent manner in neuroblastoma N2A cells: IC(50) are 11.3 and 37.4µM in holding potentials of -70 and -100 mV, respectively. In the presence of hinokiol there was a 13-mV left shift in steady-state inactivation curves; however, activation gating was not altered. VGSC inhibition by hinokiol did not require channel opening and was thus considered to be closed-channel block. In the presence of hinokiol, since the degree of block did not enhance with stimulation frequency, block by hinokiol thus did not exhibit use-dependence. Recovery from channel inactivation was not significantly affected in the presence of hinokiol. In addition, hinokiol also inhibited VGSC of differentiated neuronal NG108-15 cells and rat hippocampal CA1 neurons. CONCLUSION: Our results therefore suggest hinokiol inhibited VGSC in a closed-channel block manner and such inhibition involved intensification of channel inactivation.

Inhibitory effects of imperatorin on voltage-gated K(+) channels and ATP-sensitive K(+) channels.

BACKGROUND: Imperatorin is a furocoumarin isolated from Angelica archangelica and Cnidium monnieri. It has multiple neuromodulatory actions such as anticonvulsant effects, anxiolytic effects and anti-nociceptive effects. Although there have been reports demonstrating its effects on voltage-gated Na(+) channels (VGSC) and transient reversal potential (TRP) V1 channels in neurons, there is hitherto no work showing whether this compound affects voltage-gated K(+) (Kv) channels and ATP-sensitive K(+) (KATP) channels. METHODS: We investigated the effects of imperatorin on K(+) channels using whole-cell configuration voltage-clamp technique. RESULTS: Imperatorin inhibited Kv channels in differentiated neuronal NG108-15 cells, and caused a left shift in the steady-state inactivation curve without affecting activation gating. Imperatorin also inhibited heterologously expressed wild type Kv1.2 and Kv2.1 channels, but became much less potent in inhibiting Kv1.2 V370G, a mutant defective in C-type inactivation, implying that drug inhibition depends on C-type inactivation. At a high concentration (100µM), imperatorin also suppressed KATP channels. CONCLUSION: Our results suggest that imperatorin inhibited both Kv and KATP channels.

(±)3,4-methylenedioxyamphetamine inhibits the TEA-sensitive K⁺ current in the hippocampal neuron and the Kv2.1 current expressed in H1355 cells.

The whole-cell patch clamp method was used to study the effects of (±)3,4-methylenedioxyamphetamine (MDA) in hippocampal CA1 neurons from neonatal rats and in lung epithelial H1355 cells expressing Kv2.1. Extracellular application of MDA (30 µM) induced bursts of action potentials in hippocampal CA1 neurons exhibiting single spike action potentials without a bursting firing pattern, and promoted action potential bursts in hippocampal CA1 neurons exhibiting bursting firing of action potentials. Whereas MDA (30 and 100 µM) markedly decreased the delayed outward current in hippocampal CA1 neurons, MDA (100 µM) only slightly decreased the fast-inactivating K(+) current (I(A)) current. Furthermore, MDA (100 µM) substantially decreased the delayed outward current in the presence of 4-aminopyridine (4-AP; 3 mM), but did not significantly decrease the delayed outward current in the presence of tetraethylammonium (TEA; 30 mM). MDA (100 µM) also inhibited the current in H1355 cells expressing Kv2.1. Our results have shown that MDA inhibits the TEA-sensitive K(+) current in the hippocampus and the Kv2.1 current expressed in H1355 cells. These effects may contribute to the pharmacological and toxicological effects of MDA.

Suppression of voltage-gated Na(+) channels and neuronal excitability by imperatorin.

Imperatorin is a naturally occurring furocoumarin compound isolated from plants such as Angelica archangelica and Cnidium monnieri. It has multiple pharmacological effects including anticonvulsant effects. Here we determined the effects of imperatorin on voltage-gated Na(+) channels (VGSC) using whole-cell patch clamp techniques in differentiated neuronal NG108-15 cells. We showed that imperatorin inhibited VGSC; such inhibition did not show state-dependence. Imperatorin caused a left shift in the steady-state inactivation curve without affecting activation gating. The inhibition of VGSC by imperatorin displayed a mild frequency-dependence. Imperatorin was also shown to inhibit VGSC and action potential amplitude without affecting voltage-gated K(+) channels in rat hippocampal CA1 neurons. In conclusion, our results suggest that imperatorin dampens neuronal excitability by inhibiting VGSC.

Coumarsabin hastens C-type inactivation gating of voltage-gated K⁺ channels.

During prolonged depolarization, voltage-gated K(+) (Kv) channels display C-type inactivation, a process which is due to selectivity filter destabilization and serves to limit K(+) flux. Here we reported that coumarsabin, a coumarin derivative isolated from Juniperus Sabina, could hasten C-type inactivation and thus cause block of Kv channels in neuronal NG108-15 cells and Kv1.2 channels heterologously expressed in lung epithelial H1355 cells. In NG108-15 cells, extracellular, but not intracellular, coumarsabin (30 µM) strongly speeded up Kv current decay and caused a left-shift in the steady-state inactivation curve. Coumarsabin inhibited end-of-pulse Kv currents with an IC50 of 13.4 µM. The kinetics and voltage-dependence of activation were not affected by coumarsabin. The degree of block by coumarsabin was not enhanced by a reduction in intracellular K(+) concentration. Data reveal that coumarsabin was a closed channel blocker and it displayed a frequency-independent mode of inhibition. Coumarsabin did not accelerate current decay in a Kv1.2 mutant (V370G) defective in C-type inactivation. Taken together, our data suggest that Kv channel inhibition by coumarsabin did not appear to result from a direct obstruction of the outer pore but relied on C-type inactivation.