The Effect Of Tetrodotoxin Research Articles

Single cell adhesion measuring apparatus (SCAMA): application to cancer cell lines of different metastatic potential and voltage-gated Na+ channel expression

We have developed a simple yet effective apparatus, based upon negative pressure directed to the tip of a micro-pipette, to measure the adhesiveness of single cells. The "single cell adhesion measuring apparatus" (SCAMA) could differentiate between the adhesion of strongly versus weakly metastatic cancer cells as well as normal cells. Adhesion was quantified as "detachment negative pressure" (DNP) or "DNP relative to cell size" (DNPR) where a noticeable difference in cell size was apparent. Thus, for rat and human prostate and human breast cancer cell lines, adhesiveness (DNPR values) decreased in line with increased metastatic potential. Using the SCAMA, we investigated the effect of tetrodotoxin (TTX), a specific blocker of voltage-gated Na(+) channels (VGSCs), on the adhesion of rat and human prostate cancer cell lines of markedly different metastatic potential. Following pretreatment with TTX (48 h with 1 microM), the adhesion values for the Mat-LyLu cells increased significantly 4.3-fold; there was no effect on the AT-2 cells. For the strongly metastatic PC-3M cells, TTX treatment caused a significant (approximately 30%) increase in adhesion. The adhesion of PNT2-C2 ("normal") cells was not affected by the TTX pretreatment. The TTX-induced increase in the adhesiveness of the strongly metastatic cells was consistent with the functional VGSC expression in these cells and the proposed role of VGSC activity in metastatic cell behaviour. In conclusion, the SCAMA, which can be constructed easily and cheaply, offers a simple and effective method to characterise single-cell adhesion and its modulation.

High concentrations of adrenergic antagonists prolong sciatic nerve blockade by tetrodotoxin.

Millimolar-range concentrations of some adrenergic antagonists have been shown to have local anesthetic-like properties, and to stimulate GTPase activity in vitro. In this report, we investigate whether these agents can potentiate the effect of tetrodotoxin (TTX) and bupivacaine, a conventional local anesthetic, and whether GTPase activation plays a role. Rats received sciatic nerve blockade with tetrodotoxin or bupivacaine co-injected with adrenergic antagonists and/or agonists, or pertussis toxin. Thermal nociceptive blockade was quantified with modified hotplate testing. Nerve block from TTX alone lasted 153 (99-223) min (median and 25th and 75th percentiles). Co-injection with 20 mM phentolamine, propranolol, and yohimbine prolonged TTX block to 856 (765-862), 486 (444-510), and 465 (413-495) min respectively (P<0.005 in all cases, compared to TTX alone). Micromolar concentrations of adrenergic antagonists (which inhibited the prolongation of TTX block by epinephrine) did not prolong TTX block. Injection of adrenergic antagonists alone did not produce specific nerve block. They did not prolong TTX block when injected at a remote subcutaneous site. Prolongation of TTX block by phentolamine was not inhibited by co-injection with pertussis toxin. Adrenergic antagonists did not prolong bupivacaine block. High concentrations of adrenergic antagonists markedly prolonged TTX block, but not bupivacaine block. This locally mediated action does not appear to be adrenergic-receptor-specific, or mediated by GTPase activation.

Tetrodotoxin-sensitive release of adrenaline, noradrenaline and neuropeptide Y-like immunoreactivity in the pithed guinea-pig in the absence of electrical preganglionic nerve stimulation

The effect of tetrodotoxin (TTX) on plasma noradrenaline (NA), adrenaline (A) and neuropeptide Y-like immunoreactivity (NPY-LI) levels was evaluated in the pithed guinea pig, in the absence of electrical stimulation and following preganglionic nerve stimulation (PNS). Blood samples for determination of NA, A and NPY-LI were collected 5 min before and 5 min after injection of saline (control group, n = 6) or TTX (13 μg/kg, i.v., TTX-treated group, n = 8), respectively. In both groups, blood samples were collected 20 s and 40 s after initiation of PNS. In the absence of electrical stimulation, TTX reduced the plasma NA level by 66%, NPY-LI level by 42%, whereas the plasma A level was not significantly altered by the neurotoxin. In the control group, PNS elicited a 27-fold increase of plasma NA, a 60-fold increase of plasma A and enhanced the NPY-LI levels by 13%. Pretreatment with TTX completely blocked this release of sympathetic transmitters. The present results suggest that a certain degree of spontaneous nerve activity is present in the pithed animal model, also during baseline conditions, since TTX significantly reduced NA and NPY-LI in the absence of electrical stimulation. This spontaneous nerve activity is probably a consequence of local mechanical activity of the pithing rod capable of eliciting action potentials at the preganglionic level of the sympathetic nerve terminals.

The effect of tetrodotoxin on the sodium gating current in the squid giant axon

The effect of tetrodotoxin (TTX) on the sodium gating current in the squid giant axon was examined by recording the current that flowed at the pulse potential at which the ionic current fell to zero, first in the absence and then in the presence of TTX. The addition of 1 microM TTX to the bathing solution had no consistent effect on the size of the initial peak of the gating current, but resulted in small changes in the timecourse of its subsequent relaxation which were mainly caused by a reduction of about one quarter in the component that has a delayed onset and may possibly arise from changes in the state of ionization of groups in the channel wall when the lumen fills with water. Our findings suggest that the binding of TTX at the outer face of the sodium channel does not interfere with the mechanisms of activation and inactivation by the voltage sensors, but has an allosteric effect on the access of internal cations to the inside of the channel.

Characterization of dopamine release in the substantia nigra by in vivo microdialysis in freely moving rats

Dopamine (DA) is released not only from the terminals of the nigrostriatal projection, but also from the dendrites of these neurons, which arborize in the substantia nigra pars reticulata (SNR). Although striatal DA release has been extensively studied by in vivo microdialysis, dendritic DA release in the SNR has not been characterized by this technique. Extracellular DA was monitored simultaneously in the ipsilateral striatum and SNR. The nigral probe was implanted at a 50 degree angle, permitting 2.5 mm of SNR to be dialyzed. Delivery of the tracer Fluoro-Gold into the striatal probe retrogradely labeled tyrosine hydroxylase-positive cell bodies and dendrites in the vicinity of the nigral probe. Hence, it could be demonstrated that dopaminergic neurons near the nigral probe projected to the vicinity of the striatal probe. Addition of 50 mM KCl to the SNR perfusion solution produced a 3.5-fold increase in DA and a 50% reduction in dihydroxyphenylacetic acid (DOPAC) in the SNR; in contrast, this manipulation in the SNR caused DA release in the striatum to be decreased by 20%, while striatal DOPAC was increased by 50%. Local administration of nomifensine (10 microM) in the SNR produced a sevenfold increase in SNR DA but had no effect on striatal DA. Systemic injection of d-amphetamine (2 mg/kg, s.c.) elevated DA in the SNR and striatum five- to sevenfold, while DOPAC was decreased in both structures by at least 40%. To determine the effect of tetrodotoxin (TTX), basal concentrations of DA in the SNR were first elevated threefold by including nomifensine (1 microM) in the nigral perfusion solution.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of tetrodotoxin, lidocaine, and quinidine on the transient inward current of sheep Purkinje fibres.

The effect of tetrodotoxin (TTX), lidocaine, and quinidine on the transient inward current (TI) was studied in voltage-clamped sheep cardiac Purkinje fibres. The TI was induced by elevation of extracellular Ca or addition of strophanthidin. Reduction of external Na had a biphasic effect on the steady state TI magnitude; a moderate (less than 50%) reduction of external Na had an enhancing effect on the TI; a further decrease of extracellular Na was accompanied by a decline of TI amplitude. The TI could not be induced in Na-free medium (external Ca less than or equal to 9.0 mM). TTX, lidocaine, and quinidine reduced the magnitude of the TI in a dose-dependent way. The blocking effect of these agents could be compensated for by a moderate (less than 50%) reduction of external Na or an elevation of extracellular Ca. It is suggested that the blocking effect of TTX, lidocaine, and quinidine on the TI is due to a reduction of intracellular Na, which causes a decay of intracellular Ca via the Na-Ca exchange mechanism.

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

(1)Volgate-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.

The postnatal development of the rat primary visual cortex during optic nerve impulse blockade by intraocular tetrodotoxin: a quantitative electron microscopic analysis

The effect of tetrodotoxin (TTX)-induced monocular impulse blockade on various parameters of synaptogenesis during the first 3 postnatal weeks of the developing rat visual cortex was investigated by quantitative electron microscopy. During the injection period, beyond 14 days postnatal (dpn), the effectiveness of TTX in blocking optic nerve impulses was monitored by loss of the pupillary light reflex. Between 5 and 21 dpn, TTX treatment reduced the number of type I axodendritic synapses by approximately 23%, when compared to sham-injected controls. These reductions were found in layers III, IV, and the superficial region of layer V. Layer IV exhibited the greatest decrease (24%) while layers III and V showed reduction of 20% and 18%, respecitvely. At 21 dpn, the number of type II axodendritic synapses decreased by 19% in the same layers, but no reductions were found at earlier ages. TTX also reduced the mean number of synaptic vesicles within type I and type II terminals by 27% and 15%, respectively. At 9 dpn, reductions were first found in layers IV and V, but by 21 dpn significant decreases were found in layers II/III, IV and V. TTX had no effect on the length of the postsynaptic density of both synaptic types or on cortical thickness at any age. These data indicate that optic impulses are important mediators of synaptogenesis in the developing visual cortex, the loss of which induces localized and specific synaptic alterations, possibly due to a change in cortical circuitry.

Effects of intraocular tetrodotoxin on dendritic spines in the developing rat visual cortex: a Golgi analysis

The effect of tetrodotoxin (TTX)-induced monocular impulse blockade on the growth of dendritic spines in the developing rat primary visual cortex was analysed by quantitative Golgi techniques. Between 5 and 21 days postnatal (dpn), rats were injected with TTX every 2 days into the right eye to chronically eliminate optic impulses. Effectiveness of TTX was monitored by loss of the pupillary light reflex. At 21 dpn, the number of spines located on the portion of the apical dendrite within layers III, IV and the superficial region of layer V was reduced by approximately 26%. These decreases were found on the apical dendrites of both large and medium sized pyramidal cells. TTX also reduced the number of spines on the proximal portion of oblique dendrites in layer IV by 16%, yet did not change the number of spines on basilar dendrites. No evidence of transneuronal degeneration was seen following long-term TTX treatment. These data indicate that dendritic spine development in the visual cortex is sensitive to the loss of optic impulses and that the decrease in spine population is principally due to a reduction in spine growth.

Neuronal and glial activity during spreading depression in cerebral cortex of cat.

1. Extra- and intracellular potentials were recorded from neurons and glia during spreading depression (SD) in cerebral cortex of cats. The glial membrane depolarized during SD and the time course of depolarization was concurrent with the surface DC change of SD. The glial depolarization evoked by 20-Hz repetitive cortical stimulation disappeared during the negative DC shift of SD. Simultaneous recording of the extra- and intracellular potentials from a single glial cell with a coaxial microelectrode showed that the extracellular DC potential change was of opposite polarity to the glial intracellular potential, which suggests that the slow glial depolarization concurrent with SD is not the field potential. In contrast to glial cells, the neuronal burst discharges as well as the neuronal membrane depolarization associated with SD did not show a close relationship to SD: the neuronal membrane depolarization and discharge were frequently delayed by 10-3- s from the onset of the SD slow wave. Sometimes SD was observed without accompanying neuronal depolarization. The degree of neuronal depolarization was not always correlated with the amplitude of the negative wave of SD. 2. The effect of tetrodotoxin (TTX) on the negative DC potential of SD was examined. Simultaneous recording of glial membrane potential and the neuronal unit activity as well as extracellular DC potential and surface DC potential during SD was performed and the TTX-treated cortex was compared with the normal state. TTX did not change the DC level of the cerebral cortex. SD could be evoked by KCl when neuronal discharge was completely abolished by TTX application...

Passive electrical properties of squid axon membrane.

The admittance of the resting membrane of squid axon is investigated in natural and artificial seawater with alternating currents using an internal axial electrode. Corrections for the series electrolyte resistance between electrodes and membranes were applied to the measured admittance and the membrane capacitance and conductance are calculated. The membrane capacity was found to decrease from 1 μF/cm2 to about 0.6μF/cm2 between 1 kHz and 50 kHz. The conductivity was found to increase from 1.2 mmho/cm2 to 40 nmho/cm2 in the same frequency range. In addition to the capacitive and conductive components, and inductive element is observed at low frequencies confirming the early work by Cole and co-workers. The effect of tetrodotoxin (TTX) and tetraethylammonium (TEA) on the membrane admittance was investigated. It was found that TTX did not have any effect on the capacitive component but has a profound effect on the inductive element. TEA alters the inductive element even more drastically than TTX.

Tetrodotoxin sensitivity of muscle action potentials in pufferfishes and related fishes

The effect of tetrodotoxin (TTX) was examined on action potentials in various fishes obtained in the waters of the Great Barrier Reef. The action potentials in muscles of all pufferfishes examined (seven species) were found to be insensitive to 3 × 10−6 M TTX. On the other hand, TTX blocked the action potentials in common hardy-head (Pranesus ogilbyi), slender remora (Echeneis naucrates) and parrotfish (Callyodon pyrrhostethus) at a concentration of 3 × 10−7 M. The dose-response curve fits well the curve predicted by the Langmuir adsorption isotherm. The dissociation constants thus determined in the above mentioned fishes were 3 × 10−8 M, 6 × 10−8 M and 5 × 10−8 M respectively. In contrast to the small dissociation constants found in ordinary fishes, some fishes closely related to pufferfishes have large dissociation constants. They are 3 × 10−7 M in porcupinefish (Diodon holacanthus), 1 × 10−7 M in boxfish (Rhynchostracion nasus) and 5 × 10−7 M in triggerfish (Rhinecanthus aculeatus). We speculate that the resistivity of pufferfish action potentials to TTX is a result of evolutionary change and that the selection may have been done by TTX itself. The effect of saxitoxin (STX) on pufferfish action potentials (Arothron hispidus) was examined and it was found that the action potentials are also resistant to 3 × 10−7 M STX whereas those of common hardy-head (Pranesus ogilbyi) muscle were blocked by 3 × 10−8M. Possible molecular mechanisms of the TTX resistivity are discussed.

Effects of tetrodotoxin on impedance in normal and asphyxiated cerebral tissue

The effect of tetrodotoxin (TTX) on cerebral tissue impedance was studied in the cat, as was the effect of TTX on impedance changes induced by calcium and asphyxiation. Impedance measurements were made at 1.0 kHz, in focal volumes of approximately 1.0 mm 3, using a Wheatstone bridge technique. In cats with chronically implanted electrodes, introduction of 1–250 ng of TTX in 0.5 μl CSF into the lateral geniculate body caused no change in cerebral tissue impedance. In acute experiments, topical applications of 0.05–50 μg of TTX in 0.1 ml CSF markedly reduced the amplitude of the cortical EEG without altering cortical tissue impedance. When asphyxiation was preceded by treatment of cortex with TTX, postasphyxial increases in both resistive and reactive impedance were delayed by 4–6 min. A similar delay, together with a marked reduction in the postasphyxial resistance increase, has been shown previously to occur following treatment of the cortex with CaCl 2. The application of CaCl 2 to the cortex prior to asphyxiation did not interfere with the postasphyxial reactance rise. When TTX treatment of the cortex was followed by applications of CaCl 2, the resulting cortical impedance increases were indistinguishable from those following application of CaCl 2 without prior TTX treatment. Postasphyxial resistance changes in calcium treated cortices were inversely related to the resistance changes induced by the calcium prior to asphyxiation. However, when the cortex was treated with TTX followed by CaCl 2 prior to asphyxiation, this inverse relation was lost. The postasphyxial impedance change following cortical pretreatment with TTX and calcium were delayed as with TTX alone. In terms of models of cerebral tissue impedance the results suggest that conductivity changes in hydrated extracellular macromolecules may play an important role in cerebral tissue impedance changes.

Effects of some inhibitors of ionic permeabilities on ventricular action potential and contraction of rat and guinea-pig hearts

The effect of tetrodotoxin (TTX), which blocks Na conductance increase, tetraethylammonium (TEA), which reduces K conductance, and manganese (Mn), which suppresses Ca conductance, on the ventricular intracellular action potential (AP) and contraction was studied in isolated, perfused rat and guinea-pig hearts. In the rat, TTX produces an important decrease of the AP amplitude (initial component) and a shortening of the plateau without marked modification of repolarization. TEA produces resting depolarization and an elevation and lengthening of the plateau. Mn ions diminish the amplitude and duration of the plateau. In the guinea-pig, TTX produces only a very small decrease in AP amplitude, but the first part of the ascending phase decreases markedly, the second part being on the other hand only slightly modified. Under the action of TEA the cells are depolarized, the amplitude of the first part of the plateau decreased, and the repolarization delayed. Mn leads to marked slowing of the second part of the ascending phase and to a lengthening of the plateau. In both species, the contraction is very slightly modified by TTX, reduced or suppressed by TEA and Mn. Initial effects of low-Ca media are similar to Mn effects, but changes in contraction and AP during potentiation are opposite. Differences between rat and guinea-pig AP and the possible participation of Ca ions in the genesis of the ventricular AP are discussed.

Effect of tetrodotoxin on membrane currents in mammalian cardiac fibres.

THE action potential of cardiac fibres differs from that of most other cell types: the fast rising phase is followed by a plateau lasting a few 100 msec. The results of voltage clamp analysis in Purkinje fibres1 show that a depolarizing voltage step elicits a large sodium current, which has a slow component which declines for several hundred milliseconds. The potassium current falls after depolarization with a similar time constant. The slow decline of both currents can explain the plateau of the cardiac action potential. The sodium current was identified by comparing the membrane currents in Tyrode solution with those in a sodium-free solution1. An independent check of this procedure would be useful; we therefore investigated the effect of tetrodotoxin (TTX) on short Purkinje fibres of sheep. TTX at concentrations of 10−8 to 10−7 g/ml. is known to block selectively the excitatory inward sodium current in invertebrate axons2,3. We recorded membrane potentials in fourteen preparations and passed current through a second intracellular glass microelectrode. Details of the preparation and of the electronic apparatus are given elsewhere4.