Assessment of Drug Proarrhythmic Potential in Electrically Paced Human Induced Pluripotent Stem Cell-Derived Ventricular Cardiomyocytes Using Multielectrode Array.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) have been widely used for the assessment of drug proarrhythmic potential through multielectrode array (MEA). HiPSC-CM cultures beat spontaneously with a wide range of frequencies, however, which could affect drug-induced changes in repolarization. Pacing hiPSC-CMs at a physiological heart rate more closely resembles the state of in vivo ventricular myocytes and permits the standardization of test conditions to improve consistency. In this study, we systematically investigated the time window of stable ion currents in high-purity hiPSC-derived ventricular cardiomyocytes (hiPSC-vCMs) and confirmed that these cells could be used to correctly predict the proarrhythmic risk of Comprehensive In Vitro Proarrhythmia Assay (CiPA) reference compounds. To evaluate drug proarrhythmic potentials at a physiological beating rate, we used a MEA to electrically pace hiPSC-vCMs, and we recorded regular field potential waveforms in hiPSC-vCMs treated with DMSO and 10 CiPA reference drugs. Prolongation of field potential duration was detected in cells after exposure to high- and intermediate-risk drugs; in addition, drug-induced arrhythmia-like events were observed. The results of this study provide a simple and feasible method to investigate drug proarrhythmic potentials in hiPSC-CMs at a physiological beating rate.

VIP-mediated increase in cAMP prevents tetrodotoxin-induced retinal ganglion cell death in vitro.

Afferent influences on natural cell death were modeled in retinal cultures derived from neonatal rats. Tetrodotoxin (TTX) blockade of electrical activity produced a significant reduction in surviving retinal ganglion cell (RGC) neurons during a critical period of development, similar in magnitude to the reduction observed during natural cell death in the intact retina at a similar developmental stage. The addition of vasoactive intestinal peptide (VIP) protected the RGCs from the lethal action of TTX. This effect was specific, since the related peptides PHI-27 and secretin produced no significant increase in RGC survival. Radioimmunoassay of cyclic nucleotides showed that TTX decreased culture levels of cAMP and that this trend was reversed by VIP. Decreases in RGC survival associated with TTX electrical blockade were prevented by 8-bromo:cAMP or forskolin. Furthermore, VIP10-28, the C-terminal fragment that inhibits VIP stimulation of adenylate cyclase, reduced the number of surviving RGCs. Thus, our results suggest that VIP, acting by increasing cAMP, has a neurotrophic effect on electrically blocked RGCs and may be an endogenous factor modulating normal cell death in the retina.

Periodate treatment reduces the tetrodotoxin-sensitivity of voltage-gated Na+ channels.

(1) Voltage-clamped nerve fibres of the frog Rana esculenta were treated with periodate in the extracellular solution. (2) Periodate treatment irreversibly reduced the effect of tetrodotoxin (TTX) on the Na+ currents. (3) The effect of saxitoxin (STX) was also reduced but less than that of TTX. (4) The presence of STX during the application of periodate to the nerve fibre almost completely prevented the effect of the chemical reagent on the TTX sensitivity of the Na+ channels. (5) The reduction of the TTX effect is not due to the reaction of small amounts of periodate with the diol group of this toxin, because the effect was seen after prolonged washing with reagent-free Ringer solution with or without high amounts of ribose. (6) Carboxyl groups present in the Na+ channel seem to be quite important for the binding of TTX and STX. Periodate modifies several amino acid side chains, however, it does not attack carboxyl groups in a peptide chain. Thus, these results suggest that periodate modifies a further group critically involved in the binding of TTX and STX.

Toxin-neutralizing effect and activity-quality relationship for mice tetrodotoxin-specific polyclonal antibodies.

The polyclonal antibodies specific for tetrodotoxin (TTX) were prepared from mice and their capacity of neutralizing TTX was investigated so as to explore the possibility of developing TTX antitoxin. Haptenic TTX was conjugated to Tachypleus tridentatus hemocyanin (TTH) chemically to form artificial antigen TTX-TTH. BALB/c mice were immunized with TTX-TTH and ascites were induced by intraperitoneal administration of Freund's adjuvant. Twenty strains of TTX-specific ascites antibody with apparent affinity varying from 10(-4) to 10(-7)M were obtained. KM mice were challenged with lethal doses (1LD = 14.0 microg/kg, i.p.) of TTX neutralized by antibodies to evaluate the power of antitoxin. The potential of TTX-neutralizing of the antibodies was approved by the increase in survival animal challenged by lethal doses of TTX pre-incubated in vitro or neutralized in vivo with TTX specific antibodies. The highest protection was observed with all animals survived challenge of 1.5 x LD TTX neutralized in vitro, and antibody administration 4 days prior to 1.3 x LD TTX challenge in vivo neutralization. The protective efficiency was antibody quality factor dependent and with the highest detoxifying immunological equivalent as high as 1 300 microg (TTX)/L(ascites) approximately, while the antibody apparent affinity being at the order of 10(-6) to 10(-7)M. These results suggested that chemical vaccine for haptenic TTX could successfully raise high humoral immune response and the antibodies could neutralize TTX effectively both in vitro and in vivo, antibody therapy would be the hopeful means for detoxification of TTX.

Stimulated release of exogenous GABA and glutamate from cerebral cortical synaptosomes and brain slices by bis(acetato)tetrakis(imidazole) copper(II) complex.

In these experiments we have tested the effect of bis(acetato)tetrakis (imidazole) copper(II) on the release and uptake of 14C-GABA and 3H-glutamate from brain slices and brain cortical synaptosomes. Cu(OAc)2(Im)4 in concentrations ranging from 1 to 100 microM has increased the release of GABA and glutamate from brain slices and synaptosomal preparations in a dose-related manner when the effect on GABA release is two-fold greater than glutamate and 10-fold greater than alanine. Pretreatment with a GABA uptake inhibitor such as 1-2 mM nipecotic acid has no effect on 14C-GABA release, whereas hydroxy aspartate, the glutamate uptake inhibitor, has elevated the stimulated release of glutamate. Copper(II) chloride, the inorganic form of copper, had no significant effect either on GABA release or on glutamate release. The stimulated release of exogenous GABA and glutamate was Ca2+-dependent, because it was inhibited by EGTA, and neuronal, because it was blocked by tetrodotoxin. The recent results can explain the anticonvulsant activity of Cu(OAc)2(Im)4 against strychnine-induced seizures by increasing the net release of GABA from cortical neurons.

A novel selective and orally bioavailable Nav 1.8 channel blocker, PF-01247324, attenuates nociception and sensory neuron excitability.

BACKGROUND AND PURPOSE: NaV 1.8 ion channels have been highlighted as important molecular targets for the design of low MW blockers for the treatment of chronic pain. Here, we describe the effects of PF-01247324, a new generation, selective, orally bioavailable Nav 1.8 channel blocker of novel chemotype. EXPERIMENTAL APPROACH: The inhibition of Nav 1.8 channels by PF-01247324 was studied using in vitro patch-clamp electrophysiology and the oral bioavailability and antinociceptive effects demonstrated using in vivo rodent models of inflammatory and neuropathic pain. KEY RESULTS: PF-01247324 inhibited native tetrodotoxin-resistant (TTX-R) currents in human dorsal root ganglion (DRG) neurons (IC50 : 331 nM) and in recombinantly expressed h Nav 1.8 channels (IC50 : 196 nM), with 50-fold selectivity over recombinantly expressed TTX-R hNav 1.5 channels (IC50 : ∼10 µM) and 65-100-fold selectivity over TTX-sensitive (TTX-S) channels (IC50 : ∼10-18 µM). Native TTX-R currents in small-diameter rodent DRG neurons were inhibited with an IC50 448 nM, and the block of both human recombinant Nav 1.8 channels and TTX-R from rat DRG neurons was both frequency and state dependent. In vitro current clamp showed that PF-01247324 reduced excitability in both rat and human DRG neurons and also altered the waveform of the action potential. In vivo experiments n rodents demonstrated efficacy in both inflammatory and neuropathic pain models. CONCLUSIONS AND IMPLICATIONS: Using PF-01247324, we have confirmed a role for Nav 1.8 channels in both inflammatory and neuropathic pain. We have also demonstrated a key role for Nav 1.8 channels in action potential upstroke and repetitive firing of rat and human DRG neurons.

Antagonistic action of uranyl nitrate on presynaptic neurotoxins from snake venoms.

Uranyl ion (UO2+2) antagonized the neuromuscular blocking action and phospholipase A2 activity of neurotoxins which act presynaptically [beta-bungarotoxin (beta-BuTX) and crotoxin] but did not affect the action of alpha-bungarotoxin and tetrodotoxin. On the basis of the kinetic analysis of the UO2+2 and strontium ion (Sr2+) antagonism of muscle paralysis induced by beta-bungarotoxin, it was found that they inhibited both the binding of the toxin and the steps following binding that brought about the neuromuscular blocking action of beta-bungarotoxin. Uranyl ion was about 50 times more potent than Sr2+ in antagonizing beta-bungarotoxin. High Ca2+ (10 mM) abolished but low Ca2+ (0.25-1.25 mM) medium enhanced the antagonizing action of UO2+2 and Sr2+. In low Ca2+ medium, UO2+2 markedly potentiated the amplitude of the twitch, subsequent addition of beta-bungarotoxin produced three phases of effects on the twitches, e.g. an initial depression, followed by the second facilitation and finally a rapid depression of twitches; however, approx. 70 min after beta-bungarotoxin the small twitches reached a steady state which persisted for more than 350 min. Therefore, it is evident that UO2+2 is the most potent antagonist of beta-bungarotoxin so far tested.

Secondary excitation of intestinal smooth muscle.

1. A period of stimulation of intrinsic or extrinsic nerves to intestinal muscle is often followed by a secondary contraction. In the present work, the basis for such secondary contractions in the longitudinal muscle of the large bowel of guinea-pigs and rabbits was examined.2. In general, the action of cholinergic nerves did not contribute significantly to the secondary contractions. Concentrations of atropine or hyoscine, which completely blocked primary cholinergic contractions, potentiated or did not significantly reduce secondary contractions.3. Atropine resistant, nerve mediated primary contractions of the guinea-pig ileum were inhibited by anticholinesterases, although these drugs potentiated secondary contractions in other segments of the gut.4. The occurrence of a secondary contraction following inhibition of smooth muscle activity did not depend on the nature of the initial inhibition. Thus, secondary contractions were observed following the responses to both nonadrenergic and adrenergic inhibitory nerves, adenosine triphosphate, noradrenaline and brief periods of anoxia. The secondary contractions following hyperpolarizing drugs or anoxia were not prevented by tetrodotoxin in a concentration sufficient to paralyse all nerves.5. It is concluded that cholinergic nerves do not contribute significantly to secondary contractions except at frequencies of stimulation higher than about 50 Hz or after the inhibition of cholinesterases. In the segments of gut examined, the secondary contractions were principally myogenic.

Antagonism of tetrodotoxin- and procaine-induced axonal blockade by adenine nucleotides in the frog sciatic nerve.

The effects of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine on compound action potentials were investigated in de-sheathed frog-sciatic nerve preparations. ATP and ADP but not adenosine antagonized the inhibitory action of tetrodotoxin (TTX) on nerve conduction. AMP had little or no antagonistic effect on TTX-induced axonal block. ATP was more effective than ADP. The effects of the nucleotides were related to the degree of the TTX-induced inhibition and were more evident where the blockade was more intense. ATP and ADP but not adenosine antagonized the procaine-induced axonal blockade which, in some experiments, was completely reversed by these nucleotides. ATP and ADP were of similar potency. The axonal blockade induced by pentobarbitone was not antagonized by ATP, ADP, AMP or adenosine. The possibility that ATP stimulates a TTX-sensitive sodium channel is discussed.

Pharmacological profile of a tachykinin antagonist, spantide, as examined on rat spinal motoneurones.

1. The pharmacological profile of a tachykinin antagonist, [D-Arg1, D-Trp7,9, Leu11] substance P (spantide), was studied on motoneurones of the isolated spinal cord of the newborn rat. For this purpose, potentials were recorded from a lumbar ventral root extracellularly and drugs were bath-applied in the presence of tetrodotoxin (TTX). 2. Neurokinin A (NKA), a NK2-receptor selective agonist, induced concentration-dependent depolarizations, which were antagonized by spantide. Analyses of concentration-response curves suggested a competitive type antagonism with a pA2 of 6.5. 3. Depolarizations induced by acetyl-Arg6-septide, a NK1-receptor selective agonist, were also antagonized by spantide with a pA2 of 6.5. 4. Spantide (0.5-16 microM) had no depolarizing action on the ventral root in the presence of TTX. 5. Spantide antagonized the depolarizing action of substance P (SP) when SP was applied at low concentrations (0.1-0.3 microM) or by short duration pulses in artificial cerebrospinal fluid containing TTX, but much higher concentrations of spantide (4-10 microM) were needed to exert an antagonistic action against SP than against acetyl-Arg6-septide or NKA. 6. Thyrotrophin-releasing hormone, L-glutamate, GABA, and noradrenaline, also induced depolarizations of the ventral root in the presence of TTX but the responses to these agonists were not depressed by spantide (16 microM). 7. These results suggest that there is a subtype of tachykinin receptors on neonatal rat spinal motoneurones to which NKA, acetyl-Arg6-septide and spantide bind competitively with high affinity. The present results also suggest the existence on rat motoneurones of another class or other classes of tachykinin receptors that are less sensitive to the antagonistic action of spantide.

Cannabinoid CB1 receptor-mediated modulation of evoked dopamine release and of adenylyl cyclase activity in the human neocortex.

1. The present study investigated the binding characteristics of various ligands to cannabinoid CB(1) receptors in human neocortex and amygdala. In addition, the functionality of CB(1) receptors in the human neocortex was assessed by examining the effects of CB(1) receptor ligands on evoked [(3)H]-dopamine (DA) release in superfused brain slices and on synaptosomal cAMP accumulation. 2. Saturation-binding assays in human neocortical and amygdala synaptosomes using a radiolabelled cannabinoid receptor agonist ([(3)H]-CP55.940) revealed pK(d) values of 8.96 and 8.63, respectively. The numbers of binding sites (B(max)) were 3.99 and 2.67 pmol (mg protein)(-1), respectively. 3. Various cannabinoid receptor ligands inhibited [(3)H]-CP55.940 binding with rank order potencies corresponding to those of previous studies in animal tissues. 4. Electrically evoked [(3)H]-DA release from human neocortical slices was inhibited by CP55.940 (IC(50) 6.76 nm, I(max) 65%) and strongly enhanced by the cannabinoid receptor antagonist AM251. However, [(3)H]-DA release was not influenced in rat neocortex. In human tissue, the estimated endocannabinoid concentration in the biophase of the release-modulating CB(1) receptors was 1.07 nm, expressed in CP55.940 units. 5. K(+)-evoked [(3)H]-DA release in the presence of tetrodotoxin (TTX) was strongly inhibited by CP55.940 in humans, but not in rats. 6. In human tissue, CP55.940 inhibited forskolin-stimulated cAMP accumulation (IC(50) 20.89 nm, I(max) 35%). AM251 blocked this effect and per se increased forskolin-stimulated cAMP accumulation by approximately 20%. 7. In conclusion, cannabinoids modulate [(3)H]-DA release and adenylyl cyclase activity in the human neocortex. CB(1) receptors are located on dopaminergic nerve terminals and seem to be tonically activated by endocannabinoids.

Gangliosides restore the specificity of afferent projection patterns in spinal cord explants chronically exposed to tetrodotoxin.

Sensory afferent projection patterns within organotypic explants of fetal mouse spinal cord-dorsal root ganglia (SC-DRG) were mapped out histologically using an HRP-staining method. Cultures grown in tetrodotoxin-containing medium with added gangliosides, in contrast to those grown without the addition of these compounds, showed preferential DRG innervation of the dorsal half of the cord. It therefore appears that gangliosides can compensate for the absence of functional activity during the development of selective innervation patterns.

Capsaicin blocks tetrodotoxin-resistant sodium potentials and calcium potentials in unmyelinated C fibres of biopsied human sural nerve in vitro.

Topical application of capsaicin has been tested recently for treatment of painful peripheral neuropathy. In the present study, effects of capsaicin were explored on compound action potentials of isolated fascicles from human sural nerve biopsies. Capsaicin reduced the C fibre component by 30-60%; the remaining C fibres were not sensitive to the drug. A good correlation was found between the sensitivity of C fibres to capsaicin and their resistance to tetrodotoxin (TTX), i.e. C fibre action potentials recorded in the presence of TTX were completely blocked by capsaicin. Calcium action potentials seen after inhibition of axonal potassium conductances were also completely suppressed. The data indicate that application of capsaicin nearby human peripheral nerves might prevent action potential conduction in specific subtypes of C fibres.

Sensitization of the smooth muscle by prostaglandin E1 contributes to reversal of drug-induced inhibition of the guinea-pig ileum.

Prostaglandin (PG)E1 reverses drug-induced inhibition of the electrically stimulated guinea-pig ileum. The effect of PGE1 is in part related to a sensitizing action on the smooth muscle and in part to an increased acetylcholine release from the myenteric plexus.

In vivo neutralization of tetrodotoxin by a monoclonal antibody.

This study examined the ability of monoclonal antibody against TTX to neutralize tetrodotoxin (TTX) in vivo. The mice were injected i.p. with 1.5 mouse units of TTX, followed 3 min later by administering graded doses of antibody immunoglobulin G (IgG). Animals injected with 100 micrograms IgG through the tail vein showed 100% survival. The 50% protective dose was c. 2 mg/kg. The present results indicate that monoclonal antibody can neutralize TTX in vivo.

Delayed haemolytic activity by the freshwater puffer Tetraodon sp. toxin.

In order to elucidate the toxin composition of the freshwater puffer in Bangladesh, about 230 specimens of Tetraodon sp. were collected from 1997 to 1999 and extracted. After partitioning the toxins between an aqueous layer and a 1-butanol layer, the toxin in the aqueous layer was characterized as paralytic shellfish poison (PSP) (data not shown), while the toxin in the 1-butanol layer was identified as palytoxin (PTX) or PTX-like substance based on the delayed haemolytic activity which was inhibited by an anti-PTX antibody and ouabain (g-strophanthin). This is the first report on the occurrence of PTX or PTX-like substance(s) in puffer fish.

A tetrodotoxin-binding protein in the hemolymph of shore crab Hemigrapsus sanguineus: purification and properties.

The shore crab Hemigrapsus sanguineus hemolymph contains soluble proteins that bind tetrodotoxin (TTX) and are responsible for high resistance of the crab to TTX. The TTX-binding protein was purified from the hemolymph by ultrafiltration, lectin affinity chromatography and gel filtration HPLC. The purified protein gave only one band in native-polyacrylamide gel electrophoresis (PAGE), confirming its homogeneity. Its molecular weight was estimated to be about 400k by gel filtration HPLC, while it was estimated to be about 82k under non-reducing conditions and about 72 and 82k under reducing conditions by SDS-PAGE, indicating that the TTX-binding protein was composed of at least two distinct subunits. The TTX-binding protein was an acidic glycoprotein with pI 3.5, abundant in Asp and Glu but absent in Trp, and contained 6% reducing sugar and 12% amino sugar. The protein selectively bound to TTX, with a neutralizing ability of 6.7 mouse unit TTX/mg protein, but not to paralytic shellfish poisoning toxins. However, its neutralizing activity was almost lost by treatments with enzymes (protease XIV, thermolysin, trypsin, amyloglucosidase and alpha-amylase) and denaturing agents (1% SDS, 1% dithiothreitol, 8 M urea and 6 M guanidine hydrochloride), suggesting the involvement of both proteinaceous and sugar moieties in the binding to TTX and the importance of the steric conformation of the TTX-binding protein.

Role of the cholinergic system and of apamin-sensitive Ca2+-activated K+ channels on rabbit jejunum spontaneous activity and on the inhibitory effects of adrenoceptor agonists.

1. One reason why rabbit jejunum is suitable for studying the mechanisms underlying the actions of the various neurotransmitters and their interactions is its spontaneous motility. The main regulator of spontaneous motility is the cholinergic system. How the cholinergic system regulates the spontaneous activity in the rabbit jejunum and how it affects the inhibitory action of alpha- and beta-adrenoceptor agonists remains unclear. 2. We studied the influence of the cholinergic system and apamin-sensitive Ca2+-activated K+ channels on spontaneous contractions in the rabbit jejunum and on the inhibitory effects of alpha1- and beta-adrenoceptor agonists. 3. In naïve tissues, atropine (ATR, 7.4 x 10(-8) m) and tetrodotoxin (8 x 10(-8) m) almost completely inhibited - to a similar extent - the amplitude of spontaneous activity. Despite the presence of ATR or TDX, tissue contraction gradually recovered to about 50% of the baseline amplitude within 5-10 min. When ATR or TDX, respectively, were added to the TDX- or ATR-treated tissues, the recovered activity decreased weakly but significantly. After washout and a 45-min rest the contraction amplitude returned to baseline values. A further exposure to ATR or TDX reduced the contraction to a level significantly lower than the one obtained after TDX or ATR added 5 min after ATR or TDX, respectively. In preparations prestimulated for 10 min with acetylcholine (ACh), ATR abolished the TDX-resistant recovered spontaneous activity. 4. Adrenaline (ADR, 0.5-5 x 10(-7) m) and phenylephrine (PHE, 1-10 x 10(-7) m) inhibited tissue motility in naïve and in ATR- and in TDX-exposed preparations. But whereas in naïve preparations the alpha1-adrenoceptor antagonists completely antagonized inhibition induced by both drugs, in ATR- and TDX-exposed tissues they did so only partially for ADR. Agonist-induced inhibition had a rapid onset but rapidly faded; pendular movements took significantly longer to recover in ATR- and TDX-treated tissues than in naïve tissues. In tissues exposed for 2 min to ADR (0.5-5 x 10(-7) m) or PHE (1-10 x 10(-7) m), washout or addition of alpha1-adrenoceptor antagonists caused an immediate short-lasting increase in contraction amplitude. 5. Apamin (APAM, 5 x 10(-9) m) caused a rapid and persistent increase in the amplitude of contractions. It also blocked the inhibitory responses to ADR and PHE, and removed washout-induced contractions. The APAM-induced increase in the contraction amplitude correlated with the increase obtained by washing out ADR or PHE. 6. Isoprenaline (at concentrations up to 2.8 x 10(-7) m) produced no inhibitory response in naïve tissues, but it invariably blocked (at a concentration of 0.7 x 10(-7) m) the recovered spontaneous activity (and sometimes depressed muscletone) in tissues exposed to ATR or TDX. Neither propranolol (3.4 x 10(-7) m) nor APAM (5 x 10(-9) m) counteracted these inhibitory effects. 7. These results indicate that spontaneous motility in the rabbit jejunum is predominantly mediated by neuronal release of ACh and by some other unidentified neuronal activity. Released ACh inhibits myogenic activity and strongly antagonizes beta-adrenoceptor-induced APAM-insensitive inhibition but leaves alpha1 agonist-induced APAM-sensitive inhibition unchanged.

Membrane properties of complex spike firing neurons of the mouse dorsal cochlear nucleus in vitro.

Intracellular recordings were made from complex spike firing neurons of the mouse dorsal cochlear nucleus (DCN) in vitro. The whole cochlear nucleus was dissected out and maintained submerged in rapidly flowing artificial cerebrospinal fluid (CSF). Recordings were made with current clamp techniques in the presence or absence of ion channel blocking drugs tetrodotoxin (TTX, 1 microM), tetraethylammonium (TEA, 20 mM), 4-aminopyridine (4-AP, 5 mM) or verapamil (50, 100, 150, 250 microM). The cells showed both spontaneous firing and responses to injections of depolarising current consisting of a mixture of a tall single action potential and complexes of 2 to 3 smaller wider action potentials superimposed on a plateau depolarisation. The membrane properties were: resting membrane potential -68.8 +/- 8.5 mV, cell resistance 54.1 +/- 26.5 M omega, time constant 9.6 +/- 5.4 ms and capacitance 0.25 +/- 0.5 nF; the first three variables had bimodel distributions. The current/voltage (I/V) relationship at membrane below resting was non-linear. Previously published histological evidence from the mouse DCN has shown that both cartwheel cells and Purkinje-like neurons are present. Both DCN cartwheel cells and cerebellar Purkinje cells are known to fire both tall single action potentials and complexes of smaller wider action potentials. It is therefore possible that the recordings shown here were made from these neuron types. TTX (1 microM) abolished both the tall single and the complexes of smaller action potentials, suggesting that the tall single action potentials are sodium dependent and possibly that a TTX sensitive sodium channel is responsible for the plateau as is suggested for Purkinje cells in the cerebellum. Verapamil (100 microM) abolished only the complex action potentials and the plateau leaving the tall narrow action potentials intact, which is consistent with the smaller complexes being calcium dependent. Higher concentrations abolished all spiking activity. TEA and 4-AP used separately both caused marked depolarisation to around -20 mV, suggesting that there is a large potassium current active at and near resting.

[A study on the gustatory effects of tetrodotoxin in rat (author's transl)].

Effects of tetrodotoxin (TTX) on neural responses of the chorda tympani to four basic taste stimuli were investigated electrophysiologically in rats. When the TTX (10 mg/ml) was applied directly to the tongue surface for 3 minutes, magnitude of the integrated responses of the chorda tympani was diminished to about 60% of that of the control response. This diminution of response was recovered within 30 minutes by degrees and the effect of the TTX was antagonized by guanylate. This result gives a suggestion that guanidyl group in the TTX may play an important role for the inhibitory actions to the responses of the chorda tympani. On the other hand, when the TTX (0.25 mg/100 g b. wt.) was applied intravenously, magnitude of the responses of the chorda tympani to four basic taste stimuli decreased gradually to 20 approximately 30% of that of the control responses within 60 minutes and did not recover more than 10 hours. This is assumed due to the blocking of the sodium pump of nerve fibers in the chorda tympani by the TTX.