This study examined the acute effects of ethanol (EtOH) on the firing patterns of Purkinje cells (PCs) using an intracellular recording in slice preparation of rat cerebellum. The experiments were performed in sagittal cerebellar slices (400 microm) of adult Sprague-Dawley rats (80-100g). Ethanol was applied by a bath superfusion with a known concentration expressed as the percentage of solution by volume (v/v) at 0.1, 0.5, 1, 2, and 4%. The result of the Chi-square test illustrated that the firing patterns were altered significantly after EtOH (p=0.007). However, the firing patterns that were altered by EtOH application were not affected by EtOH concentration (p= 0.1296). Among the 54 PCs tested, 30 PCs did not display any spontaneous firing activity and 24 PCs displayed spontaneous spike activity, either spiking in the simple manner (n=14) or cyclicly oscillating (n=10). In the presence of EtOH, 31 PCs were quiet, 22 PCs exhibited simple spiking activity and 1 PC continued to oscillate. Most PCs that displayed spontaneous activity before EtOH application progressively slowed their spike activity after EtOH superfusion. Especially, it was evident that 9 out of 10 oscillating PCs stopped their regular cyclic activity. In addition, 9 out of 14 PCs that displayed simple spike activity ceased to fire after EtOH application. Eleven out of 30 quiet PCs began to fire irregularly after EtOH application and this phenomenon usually occurred with membrane depolarization. EtOH induced spontaneous activity in 36.7% (11/30) of the quiescent PCs. In conclusion, there was differential EtOH sensitivity in the vitro slice preparation. EtOH depressed the endogenously generated spontaneous activity, especially the oscillatory firing activity. In contrast, the silent PCs were excited after EtOH application. Since this differential sensitivity persists in the presence of tetrodotoxin (TTX), it is suggested that this differential sensitivity is peculiar to the PCs.
Animal ; Ethanol/*toxicity ; In Vitro ; Purkinje Cells/*drug effects/physiology ; Rats ; Rats, Sprague-Dawley ; Tetrodotoxin/pharmacology

In the present study, we used in vitro whole-cell patch-clamp technique to record and analyze oscillatory activity of neurons in the optic tectum of Xenopus. Two patterns of subthreshold oscillations were induced by long-term depolarizing current pulses. One of the oscillating patterns occurred without a slow inward current (SIC); the other was superimposed on the SIC. The subthreshold oscillations were induced by depolarization in 48% of the recorded neurons. Both the oscillations and the SIC were tetrodotoxin (TTX)-resistant, but neither occurred when the slices were immersed in Ca(2+) free solutions. The evocation of the oscillations was voltage-sensitive: only when the initial membrane potentials of the neurons were held at -40 mV or -50 mV, 10 mV depolarization could induce the subthreshold oscillations. The amplitude and duration of the SIC depended on the level of the initial membrane potential. The subthreshold oscillations might play an important role in the physiological and behavioral functions of frogs, e.g. pattern discrimination, prey recognition, avoiding behavior etc., furthermore, these oscillations might play roles in the integration of neural activity in both mammals and non-mammalian vertebrates.
Animals ; Cell Polarity ; Membrane Potentials ; Neurons ; cytology ; Patch-Clamp Techniques ; Tetrodotoxin ; pharmacology ; Xenopus

Animals ; GAP-43 Protein ; metabolism ; Ganglia, Spinal ; drug effects ; metabolism ; Nerve Block ; Rats ; Rats, Wistar ; Sciatic Nerve ; drug effects ; injuries ; Tetrodotoxin ; pharmacology

Hainantoxin-IV (HNTX-IV) purified from the venom of the spider Selenocosmia hainana is a potent antagonist that acts on tetrodotoxin-sensitive (TrX-S) sodium channels. It is a 35-residue polypeptide and includes three disulfide bridges. In order to investigate the structure-function relationship of HNTX-IV, two mutants (S12A-HNTX-IV and R29A-HNTX-IV) of HNTX-TV in which Ser12 and Arg29 were replaced by Ala respectively, were synthesized by solid-phase Fmoc chemistry, followed by oxidative refolding of purified peptides under the optimal conditions. The synthetic mutants were analyzed by MALDI-TOF mass spectrometry, nuclear magnetic resonance spectroscopy (NMR) and electrophysiological experiments for molecular weight, conformation and physiological activity, respectively. The results show that the mutants and native HNTX-IV (nHNTX-IV) have almost identical three-dimensional structures. The bioactivity level of S12A-HNTX-IV is also about the same as that of nHNTX-IV, suggesting that Ser12 does not play any important role for the bioactivity of this toxin. The bioactivity of R29A-HNTX-IV is reduced by at last 155 times, indicating that Arg29 is a key residue relative to the bioactivity of HNTX-IV. It is presumed that the decrease in activity of R29A-HNTX-IV is due to the changes of the property in the binding site rather than the change in the basic conformation of the molecule.
Amino Acid Substitution ; Animals ; Mutation ; Sodium Channel Blockers ; Sodium Channels ; drug effects ; physiology ; Spider Venoms ; chemical synthesis ; genetics ; Structure-Activity Relationship ; Tetrodotoxin ; pharmacology

Growth differentiation factor 15 (GDF-15) is a member of the transforming growth factor-β superfamily. It is widely distributed in the central and peripheral nervous systems. Whether and how GDF-15 modulates nociceptive signaling remains unclear. Behaviorally, we found that peripheral GDF-15 significantly elevated nociceptive response thresholds to mechanical and thermal stimuli in naïve and arthritic rats. Electrophysiologically, we demonstrated that GDF-15 decreased the excitability of small-diameter dorsal root ganglia (DRG) neurons. Furthermore, GDF-15 concentration-dependently suppressed tetrodotoxin-resistant sodium channel Nav1.8 currents, and shifted the steady-state inactivation curves of Nav1.8 in a hyperpolarizing direction. GDF-15 also reduced window currents and slowed down the recovery rate of Nav1.8 channels, suggesting that GDF-15 accelerated inactivation and slowed recovery of the channel. Immunohistochemistry results showed that activin receptor-like kinase-2 (ALK2) was widely expressed in DRG medium- and small-diameter neurons, and some of them were Nav1.8-positive. Blockade of ALK2 prevented the GDF-15-induced inhibition of Nav1.8 currents and nociceptive behaviors. Inhibition of PKA and ERK, but not PKC, blocked the inhibitory effect of GDF-15 on Nav1.8 currents. These results suggest a functional link between GDF-15 and Nav1.8 in DRG neurons via ALK2 receptors and PKA associated with MEK/ERK, which mediate the peripheral analgesia of GDF-15.
Analgesia ; Animals ; Ganglia, Spinal ; Growth Differentiation Factor 15 ; NAV1.8 Voltage-Gated Sodium Channel ; Rats ; Sensory Receptor Cells ; Sodium Channels ; Tetrodotoxin/pharmacology*

OBJECTIVEConcentration of extracellular calcium ([Ca(2+)](o)) in the central nervous system decreases substantially in different conditions. It results in facilitating neuronal excitability. The goal of this study is to examine the mechanisms of enhanced neuronal excitation in low [Ca(2+)](o) in order to provide new clues to treat the hyperexcitability diseases in clinic.

METHODSWhole-cell patch-clamp technique and neuron culture were used in the study.

RESULTSThe firing threshold of cultured hippocampal neurons decreased markedly in low [Ca(2+)](o) saline. Unexpectedly, apamine and isoprenaline, antagonists of medium afterhyperpolarization (mAHP) and slow AHP (sAHP) respectively, had no statistic significant effect on excitability of neurons. TTX at a low concentration was sufficient to inhibit I(NaP), which blocked the increase of firing frequency in low [Ca(2+)](o). It also reduced the number of spikes in normal [Ca(2+)](o).

CONCLUSIONThese results suggest that in cultured hippocampal neurons, modulation of spiking threshold but not AHP may cause the increased excitability in low [Ca(2+)](o).

Action Potentials ; drug effects ; Animals ; Apamin ; pharmacology ; Calcium ; pharmacology ; Cells, Cultured ; Dose-Response Relationship, Drug ; Electric Stimulation ; Embryo, Mammalian ; Hippocampus ; cytology ; Neurons ; drug effects ; Patch-Clamp Techniques ; Rats ; Sodium Channel Blockers ; pharmacology ; Tetrodotoxin ; pharmacology

Effective drug to manage constipation has been unsatisfactory. We sought to determine whether methionine has effect on the human colon. Human colon tissues were obtained from the specimens of colon resection. Microelectrode recording was performed and contractile activity of muscle strips and the propagation of the contractions in the colon segment were measured. At 10 microM, methionine depolarized the resting membrane potential (RMP) of circular muscle (CM) cells. In the CM strip, methionine increased the amplitude and area under the curve (AUC) of contractions. In the whole segment of colon, methionine increased the amplitude and AUC of the high amplitude contractions in the CM. These effects on contraction were maximal at 10 microM and were not observed in longitudinal muscles in both the strip and the colon segment. Methionine reversed the effects of pretreatment with sodium nitroprusside, tetrodotoxin and Nw-oxide-L-arginine, resulting in depolarization of the RMP, and increased amplitude and AUC of contractions in the muscle strip. Methionine treatment affected the wave pattern of the colon segment by evoking small sized amplitude contractions superimposed on preexisting wave patterns. Our results indicate that a compound mimicking methionine may provide prokinetic functions in the human colon.
Area Under Curve ; Arginine/pharmacology ; Colon/drug effects/physiology ; Humans ; Membrane Potentials/drug effects ; Methionine/*pharmacology ; Microelectrodes ; Muscle Contraction/*drug effects ; Muscle, Smooth/drug effects/*physiology ; Nitroprusside/pharmacology ; Tetrodotoxin/pharmacology

The objectives of this study were to investigate the effects of veratridine (VER) on persistent sodium current (I(Na.P)), Na(+)/Ca(2+) exchange current (I(NCX)), calcium transients and the action potential (AP) in rabbit ventricular myocytes, and to explore the mechanism in intracellular calcium overload and myocardial contraction enhancement by using whole-cell patch clamp recording technique, visual motion edge detection system, intracellular calcium measurement system and multi-channel physiological signal acquisition and processing system. The results showed that I(Na.P) and reverse I(NCX) in ventricular myocytes were obviously increased after giving 10, 20 μmol/L VER, with the current density of I(Na.P) increasing from (-0.22 ± 0.12) to (-0.61 ± 0.13) and (-2.15 ± 0.14) pA/pF (P < 0.01, n = 10) at -20 mV, and that of reverse I(NCX) increasing from (1.62 ± 0.12) to (2.19 ± 0.09) and (2.58 ± 0.11) pA/pF (P < 0.05, n = 10) at +50 mV. After adding 4 μmol/L tetrodotoxin (TTX), current density of I(Na.P) and reverse I(NCX) returned to (-0.07 ± 0.14) and (1.69 ± 0.15) pA/pF (P < 0.05, n = 10). Another specific blocker of I(Na.P), ranolazine (RAN), could obviously inhibit VER-increased I(Na.P) and reverse I(NCX). After giving 2.5 μmol/L VER, the maximal contraction rate of ventricular myocytes increased from (-0.91 ± 0.29) to (-1.53 ± 0.29) μm/s (P < 0.01, n = 7), the amplitude of contraction increased from (0.10 ± 0.04) to (0.16 ± 0.04) μm (P < 0.05, n = 7), and the baseline of calcium transients (diastolic calcium concentration) increased from (1.21 ± 0.08) to (1.37 ± 0.12) (P < 0.05, n = 7). After adding 2 μmol/L TTX, the maximal contraction rate and amplitude of ventricular myocytes decreased to (-0.86 ± 0.24) μm/s and (0.09 ± 0.03) μm (P < 0.01, n = 7) respectively. And the baseline of calcium transients reduced to (1.17 ± 0.09) (P < 0.05, n = 7). VER (20 μmol/L) could extend action potential duration at 50% repolarization (APD(50)) and at 90% repolarization (APD(90)) in ventricular myocytes from (123.18 ± 23.70) to (271.90 ± 32.81) and from (146.94 ± 24.15) to (429.79 ± 32.04) ms (P < 0.01, n = 6) respectively. Early afterdepolarizations (EADs) appeared in 3 out of the 6 cases. After adding 4 μmol/L TTX, APD(50) and APD(90) were reduced to (99.07 ± 22.81) and (163.84 ± 26.06) ms (P < 0.01, n = 6) respectively, and EADs disappeared accordingly in 3 cases. It could be suggested that: (1) As a specific agonist of the I(Na.P), VER could result in I(Na.P) increase and intracellular Na(+) overload, and subsequently intracellular Ca(2+) overload with the increase of reverse I(NCX). (2) The VER-increased I(Na.P) could further extend the action potential duration (APD) and induce EADs. (3) TTX could restrain the abnormal VER-induced changes of the above-mentioned indexes, indicating that these abnormal changes were caused by the increase of I(Na.P). Based on this study, it is concluded that as the I(Na.P) agonist, VER can enhance reverse I(NCX) by increasing I(Na.P), leading to intracellular Ca(2+) overload and APD abnormal extension.
Acetanilides ; pharmacology ; Action Potentials ; Animals ; Calcium ; metabolism ; Myocardial Contraction ; Myocytes, Cardiac ; cytology ; drug effects ; Patch-Clamp Techniques ; Piperazines ; pharmacology ; Rabbits ; Ranolazine ; Sodium-Calcium Exchanger ; metabolism ; Tetrodotoxin ; pharmacology ; Veratridine ; pharmacology

Using patch clamp technique the effects of dragon's blood and its component loureirin B on tetrodotoxin-sensitive sodium channel currents in dorsal root ganglion cells were observed. The experimental data were simulated with Hodgkin-Huxley model and the corresponding parameters were estimated. In addition, computer-simulated neuron action potentials in the absence and presence of drugs were produced using Hodgkin-Huxley model. The results show that the conductance of tetrodotoxin-sensitive sodium channel was fitted with m3h model well, the half-activated potentials of the sodium channel in the presence of drugs were shifted to the depolarizing direction and the threshold intensity of the cells in the presence of drugs was increased. These results demonstrate that dragon's blood and loureirin B did not resemble the tetrodotoxin which inhibited tetrodotoxin-sensitive sodium channel currents completely. Perhaps the analgesic effects of dragon's blood were partly caused by loureirin B affecting the activation, blocking the action potential generation and interfering with the transmission of painful signals into the central nervous system.
Action Potentials ; drug effects ; Analgesics, Non-Narcotic ; pharmacology ; Animals ; Computer Simulation ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Ganglia, Spinal ; cytology ; Models, Biological ; Neurons ; drug effects ; Rats ; Resins, Plant ; pharmacology ; Sodium Channels ; drug effects ; Tetrodotoxin ; pharmacology

The effects of scorpion venom heat resistant protein (SVHRP) on sodium channel were studied in freshly isolated hippocampal neurons in rat using the whole-cell patch-clamp technique. The results indicated that tetrodotoxin-sensitive voltage-dependent sodium current in hippocampal neurons was inhibited by SVHRP in a dose-dependent manner. The half-inhibition concentration (IC(50)) was (0.0034+/-0.0004) microg/mL, Hill constant (n) was 0.4361+/-0.0318. After SVHRP application, a clear shift of the activation curve of Na(+) channel was shown towards more depolarized potential, resulting in channel opening at more positive membrane potentials. In the presence of 0.1 mug/mL SVHRP, the voltage for half-activation (V(1/2)) and the slope factor of the activation curve were (-23.96+/-0.41) mV and 3.73+/-0.08 (n=8, P<0.05) compared with the control recordings of (-34.38+/-0.62) mV and 4.52+/-0.52 (n=16), respectively. Averaged and normalized curve of steady-state inactivation of Na(+) channel was shifted towards negative potential after treatment of 0.1 and 0.01 mug/mL SVHRP. In the presence of 0.1 mug/mL SVHRP, the voltage for half-inactivation (V(1/2)) and the slope factor determined by a sigmoid fit of the inactivation curve were (-50.69+/-2.55) mV (n=8, P<0.01) and 5.49+/-0.72 (n=8, P<0.05) compared with the control recordings of (-32.60+/-1.52) mV and 6.73+/-0.51 (n=16), respectively. These results suggest that SVHRP blocks the voltage-dependent sodium currents and alters the sodium channel kinetics to decrease the excitability of neurons. This might be an interpretation for the antiepileptic effects of SVHRP.
Animals ; Dose-Response Relationship, Drug ; Female ; Hippocampus ; drug effects ; physiology ; In Vitro Techniques ; Male ; Patch-Clamp Techniques ; Rats ; Rats, Sprague-Dawley ; Scorpion Venoms ; pharmacology ; Sodium Channel Blockers ; pharmacology ; Tetrodotoxin ; pharmacology