Action Of Picrotoxin Research Articles

Comparative Analysis of the Effects of Blockade of Striatal Metabotropic and Ionotropic Glutamate Receptors on Motor Behavior in Rats

Chronic experiments on rats addressed the effects of blockade of NMDA and metabotropic glutamate receptors in the neostriatum on conditioned reflex avoidance (in a shuttle box) and spontaneous (in an open field) behavior. Glutamate receptor antagonists were given bilaterally into the neostriatum, in some experiments with the GABAA receptor antagonist picrotoxin (2 μg), which impairs execution of conditioned reflex skills and produces choreomyoclonic hyperkinesia. The most effective agent in preventing the adverse effects of picrotoxin on behavior was the type 5 metabotropic receptor antagonist MTEP (3 μg), which when given into the neostriatum without picrotoxin had no effect on measures of avoidance behavior and did not alter the level of spontaneous motor activity. In contrast, the type 1 metabotropic receptor antagonist EMQMCM (3 μg) degraded measures of normal motor behavior (indicative of a sedative effect) but did not prevent the actions of picrotoxin. The NMDA receptor antagonist MK-801 (dizocilpine, 1 and 5 μg) decreased picrotoxin-induced hyperkinesia but had only mild effects on its adverse influence on conditioned reflex activity; given alone into the striatum, it decreased normal motor activity. Considering the distributions of the study receptors on neostriatal neuron membranes, it is suggested that the most effective action, on type 5 glutamate receptors, may be linked with their involvement in supporting the activity of the “indirect” efferent pathway, the activity of which is impaired in the hyperkinetic type of dysfunction of the extrapyramidal motor system in Huntington’s chorea in humans.

Mechanisms for Picrotoxinin and Picrotin Blocks of α2 Homomeric Glycine Receptors

Contrary to its effect on the gamma-aminobutyric acid type A and C receptors, picrotoxin antagonism of the alpha1 homomeric glycine receptors (GlyRs) has been shown to be non-use-dependent and nonselective between the picrotoxin components picrotoxinin and picrotin. Picrotoxin antagonism of the embryonic alpha2 homomeric GlyR is known to be use-dependent and reflects a channel-blocking mechanism, but the selectivity of picrotoxin antagonism of the embryonic alpha2 homomeric GlyRs between picrotoxinin and picrotin is unknown. Hence, we used the patch clamp recording technique in the outside-out configuration to investigate, at the single channel level, the mechanism of picrotin- and picrotoxinin-induced inhibition of currents, which were evoked by the activation of alpha2 homomeric GlyRs stably transfected into Chinese hamster ovary cells. Although both picrotoxinin and picrotin inhibited glycine-evoked outside-out currents, picrotin had a 30 times higher IC50 than picrotoxinin. Picrotin-evoked inhibition displayed voltage dependence, whereas picrotoxinin did not. Picrotoxinin and picrotin decreased the mean open time of the channel in a concentration-dependent manner, indicating that these picrotoxin components can bind to the receptor in its open state. When picrotin and glycine were co-applied, a large rebound current was observed at the end of the application. This rebound current was considerably smaller when picrotoxinin and glycine were co-applied. Both picrotin and picrotoxinin were unable to bind to the unbound conformation of the receptor, but both could be trapped at their binding site when the channel closed during glycine dissociation. Our data indicate that picrotoxinin and picrotin are not equivalent in blocking alpha2 homomeric GlyR.

Mechanisms for Picrotoxin Block of α2 Homomeric Glycine Receptors

It is well known that the convulsant alkaloid picrotoxin (PTX) can inhibit neuronal gamma-aminobutyric acid (GABA) and homomeric glycine receptors (GlyR). However, the mechanism for PTX block of alpha(2) homomeric GlyR is still unclear compared with that of alpha(1) homomeric GlyR, GABA(A), and GABA(C) receptors. Furthermore, PTX effects on GlyR kinetics have been poorly explored at the single-channel level. Hence, we used the patch-clamp technique in the outside-out configuration to investigate the mechanism of PTX suppression of currents carried by alpha(2) homomeric GlyRs stably transfected into Chinese hamster ovary cells. PTX inhibited the alpha(2) homomeric GlyR current elicited by glycine in a concentration-dependent and voltage-independent manner. Both competitive and noncompetitive mechanisms were observed. PTX decreased the mean open time of the GlyR channel in a concentration-dependent manner, suggesting that PTX can block channel openings and bind to the receptor in the open channel conformation. When PTX and glycine were co-applied, a small rebound current was observed during drug washout. Application of PTX during the deactivation phase of glycine-induced currents eliminated the rebound current and accelerated the deactivation time course in a concentration-dependent manner. PTX could not bind to the unbound conformation of GlyR, but could be trapped at its binding site when the channel closed during glycine dissociation. Based on these observations, we propose a kinetic Markov model in which PTX binds to the alpha(2) homomeric GlyR in both the open channel state and the fully liganded closed state. Our data suggest a new allosteric mechanism for PTX inhibition of wild-type homomeric alpha(2) GlyR.

Pharmacological Evaluation of the Central Nervous System Activity of Aframomum melegueta Seed Extract in Mice

Objective: To study the effects of intraperitoneal injection of aqueous seed extract of Aframomum melegueta (AM) on the central nervous system (CNS) in mice. Materials and methods: The study sought to evaluate the effects of the extract on the general behaviour of the animals (Irwin test), on pentobarbitone-induced sleeping time, on methamphetamine-induced stereotyped behaviour, on motor coordination, and on convulsive seizures induced by isoniazid and picrotoxin. AM was tested at a dose range of 5-400 mg/kg. Results: AM (50-200 mg/kg) produced a significant decrease in spontaneous motor activity and also caused a dose-related prolongation of pentobarbital-induced sleeping time. At a dose range of 100-400 mg/kg, a significant inhibition of methamphetamine (35 mg/kg, i. p) induced stereotyped behaviour was observed. Furthermore, it offered a significant protection against convulsions induced by isoniazid (200 mg/kg, i.p). However, it failed to modify the convulsive action of picrotoxin (10 mg/kg, i.p) and did not cause any significant effect in the motor coordination of animals on the rota-rod machine. Conclusion: The results of this study suggest that A. melegueta seed extract possesses central nervous system depressant activity.

Evidence for an involvement of the ammonia-decreasing action of L-arginine in suppressing picrotoxin-induced convulsions in rats and its additive action with diazepam

The effects of pre- (30 min before challenge) and post-treatment (5 min after challenge) of L-arginine (840 mg kg-1) were tested on picrotoxin-induced increase in ammonia concentrations in brain regions (cerebral cortex, brain stem and cerebellum) and the accompanying convulsive responses in adult male rats. The effect of pre- and post-treatment of L-arginine was tested on the action of diazepam against picrotoxin-induced convulsions. Picrotoxin-induced increase in ammonia concentrations in the brain regions was reverted partially by L-arginine pre-treatment. However, L-arginine pre-treatment failed to inhibit convulsions independently and concurrently with diazepam. On the other hand, L-arginine posttreatment reverted ammonia to control level in all brain regions. A partial but significant inhibition of convulsions was found in these animals. The effect produced concurrently by L-arginine and diazepam post-treatment was much greater than that produced by these agents independently. These results suggest that brain ammonia has a partial but significant participation in the convulsant action of picrotoxin. L-arginine has produced a partial protection of picrotoxin-induced convulsions by reverting brain ammonia to control level. The data further suggest that the duration of action of L-arginine is considerably short and that L-arginine has an additive anticonvulsant action with diazepam. [Neurol Res 2001; 23: 622-626]

Use-dependent and Use-independent blocking actions of picrotoxin and zinc at the GABA C receptor in retinal horizontal cells

The inhibitory actions of picrotoxin (PTX) and zinc on the GABA receptor in acutely isolated catfish cone horizontal cells were studied and compared using the whole-cell patch clamp technique. PTX blocked the GABAcurrent elicited by 30 μM GABA with IC 50 = 0.64 μM. Over a PTX concentration range of 1–100 μ,M, simultaneous application of PTX with GABA (30 uM) produced current transients at both the onset and offset of the drug pulse. When the PTX concentration was maintained before, during, and after GABA application, the current transients at the onset and offset of GABA application disappeared. Thus, these transients seem to reflect a slower initial action of PTX at, and faster washout of PTX from, the GABA receptor than GABA when they were co-applied. The full recovery from PTX inhibition required a second GABA application. Recovery could not be achieved by a prolonged wash in the absence of GABA. These results suggest that PTX effect its use-dependent. Zinc also potently blocked the GABA current elicited by 30 μM GABA with an IC 50 about an order of magnitude higher than that of PTX (IC 50 = 8.2 uM). However, only the onset, but not the offset current transient was observed when zinc was simultaneously applied with GABA. The full recovery of the GABA current from zinc inhibition was obtained after washing for 20 sec and did not require a subsequent GABA application. This indicates that the zinc effect is use-independent. Our findings suggest that: (1) the zinc binding site is on the surface of the GABA receptor molecule; (2) there is a PTX binding site that is probably inside the receptor and its access requires GABA binding to the receptor.

Effect of muscimol on the picrotoxinand bicuculline-induced delay in the desensitization of the GABA receptor/Cl− channel complex

Effects of picrotoxin and bicuculline on the muscimol-dependent36Cl− entry into synaptoneurosomes of the rat cerebral cortex are examined as well as desensitization of36Cl− entry at muscimol concentrations of 5 and 50 μM. At the 5 μM concentration (which is close to the muscimol IC50), picrotoxin and bicuculline inhibited Cl− entry into synaptoneurosomes and decreased the desensitization. At the 50 μM concentration, muscimol completely abolishes the bicuculline effects both on Cl− entry and desensitization. Inhibition of Cl− entry by picrotoxin is also abolished by 50 μM muscimol, whereas the picrotoxin-induced decrease in the desensitization rate is not. It is shown that both bicuculline effects result from inhibition of the GABA receptor, but the action of picrotoxin on the desensitization of Cl− entry into synaptoneurosomes is not closely related to the functional activity of the GABA receptor/Cl− channel complex.

The enhancement of retention performance induced by picrotoxin in mice may be mediated through a release of endogenous vasopressin

Male Swiss mice were tested 48h after training in a one-trial step-through inhibitory avoidance task. Immediately post-training i.p. injection of the GABA antagonist picrotoxin (0.3-3.0mg/kg), at nonconvulsive doses, induced a dose-dependent modification of retention performance. The lower doses of picrotoxin (0.1-1.0mg/kg) enhanced retention, whereas the highest dose (3.0mg/kg) impaired retention. Picrotoxin did not affect response latencies in mice not given the footshock on the training trial, indicating that the actions of picrotoxin on retention performance were not due to nonspecific proactive effects on response latencies. The enhancing effects of picrotoxin (1.0mg/kg) on retention were time-dependent, which suggests that picrotoxin enhanced storage of recently acquired information. The enhancement of retention induced by picrotoxin (1.0mg/kg) was prevented by the vasopressin receptor antagonist, AAVP (0.01µg/kg, s.c.) administered immediately after training, but prior to picrotoxin treatment. This dose of AAVP did not affect retention by itself, either under the standard experimental conditions, or in mice trained with a high footshock. Low subeffective doses of picrotoxin (0.1mg/kg, s.c.) administered immediately after training, and hypertonic saline (1ml of 0.5M NaCl, i.p.), given 10min after training, interacted to improve retention. Considered together, these findings suggest that the better retention performance induced by post-training administration of picrotoxin could result, at least in part, from an endogenous release of vasopressin.

The effect of chronic cyclodiene insecticide treatment on some pharmacological actions of diazepam in rats

The sedative and hypnotic effects of diazepam were tested in rats following chronic treatment with subconvulsive doses of the cyclodiene insecticides, endosulfan and aldrin. The anticonvulsant effect of diazepam was determined on picrotoxin-induced convulsions in insecticide-treated and control animals. Both endosulfan and aldrin promoted the convulsant action of picrotoxin indicating that the insecticides were able to show proconvulsant action following chronic administration. Interestingly, the protective effect of diazepam against picrotoxin-induced convulsions was greater in endosulfan- and aldrin-treated animals than in control animals. The sedative and hypnotic effects of diazepam were also increased in insecticide-treated animals. The mechanisms involved in the interaction were discussed with the support of the findings reported by the present and other investigators on the central and hepatic effects of chronic endosulfan and aldrin treatment.

Effect of perfluorodecalin on picrotoxin toxicity and some detoxication systems

Preliminary administration (at least 7 days before the experiment) of perfluorodecalin boosts resistance to the toxic action of picrotoxin in rodents. The antidotal activity of diazepam rises under these conditions. Perfluorodecalin induces the activation of monooxygenase, conjugation, and antioxidant detoxication systems; the protein content increases in the blood serum and carboxylesterase activity is enchanged.

Changes in benzodiazepine/GABA receptor complex function in benzodiazepine-tolerant mice.

Mice were given flurazepam, 40 mg k-1, IP, once daily for 7 consecutive days. Twenty-four and forty-eight hours after the last injection measurements were made of the effects on convulsion threshold, body temperature and locomotor activity, of drugs acting on the GABA receptor complex. Significant decreases were seen in the hypothermic and hypomobility effects of progabide at 48 h, but no significant changes were seen in the effects of pentylenetetrazol or pentobarbitone. The actions of picrotoxin in all three types of test and the convulsant action of bicuculline (IP) were significantly decreased at 24 h but not at 48 h. The convulsive, but not the hypothermic, effects of picrotoxin were increased at the 48 h interval. These results may suggest that the chronic benzodiazepine treatment decreased some aspects of GABA receptor function at 48 h after the last dose; however, such an effect probably does not explain the previously reported increases in the effects of inverse agonists following chronic agonist treatment.

Antagonist actions of bicuculline methiodide and picrotoxin on extrasynaptic gamma-aminobutyric acid receptors.

The effects of picrotoxin and bicuculline methiodide to block depolarizing responses of extrasynaptic receptors for gamma-aminobutyric acid (GABA) are compared using excitability testing of myelinated axons in amphibian peripheral nerve. The actions of the antagonists appear both complex and dissimilar. Picrotoxin (10-1000 microM) produces large reversible depressions of the maximal response to GABA (0.01-10mM) and increases the EC50 from 0.33 to 12.6 mM. With high concentrations of agonist and antagonist an insensitive component is apparent. The action of picrotoxin is not classically noncompetitive: it may represent a mixed antagonism (competitive and noncompetitive) or a noncompetitive one, masked by the presence of receptor reserve and (or) secondary depolarizing influences (e.g., GABA-evoked [K+] o accumulation). Bicuculline methiodide (10-200 microM) shifts the GABA concentration-response curve to the right; maximal responses persist and are even enhanced. The impression that bicuculline methiodide has a competitive action is supported by analysis of its inhibition of responses to low concentrations of the agonist. It is suggested that the enhancement of GABA responses by bicuculline methiodide and their apparent resistance to block by picrotoxin may be due to a common secondary effect of the antagonists such as a decrease in membrane conductance to K+ and (or) block of transmitter uptake.

The actions of picrotoxin, strychnine, bicuculline and other convulsants and antagonists on the responses to acetylcholine glutamic acid and gamma-aminobutyric acid on Helix neurones

1. 1. Intracellular recordings were made from 3 identifiable neurones in the brain of the snail, Helix aspersa; cell E4 was inhibited by acetylcholine, GABA and glutamate; cell E13 was excited by GABA; and cell F30 was excited by glutamate. These 3 compounds were applied iontophoretically. A range of potential antagonists were tested for blocking actions against acetylcholine, GABA and glutamate. 2. 2. Crotethamide, 800 nmoles, was a specific and reversible antagonist at the inhibitory GABA receptor of cell E4. While pentylenetetrazol, 800 nmoles, was a specific and reversible antagonist at the inhibitory glutamate receptor of the same cell. 3. 3. Strychnine, 8 nmoles, was a specific and reversible antagonist for inhibitory acetylcholine receptors on cell E4. Higher doses, 80 nmoles, will in addition block GABA receptors without any effect on glutamate receptors. 4. 4. HA966, 800 nmoles, had no effect on glutamate receptors but did reduce reversibly the responses to GABA and acetylcholine. 5. 5. Picrotoxin, 80–800 nmoles, reduced all 3 inhibitory responses and GABA excitation but had no effect on glutamate excitation. 6. 6. Bicuculline methochloride, 800 nmoles, reduced reversibly both GABA responses and the response to acetylcholine. It had no effect on either glutamate responses. 7. 7. Bemegride, 800 nmoles, reduced reversibly the inhibitory actions of glutamate and acetylcholine but potentiated irreversibly both responses to GABA. 8. 8. Atropine, 80 nmoles, specifically and reversibly reduced the response to acetylcholine but higher doses were non-specific. Tubocurarine, 8 nmoles, altered all the responses and at higher doses had direct excitatory effects on the cells. It is of little use as a specific antagonist on these neurones.

Increase in striatal acetylcholine by picrotoxin in the rat: Evidence for a gabergic-dopaminergic-cholinergic link

Picrotoxin, 2 mg/kg i.p., a GABA receptor blocking agent, increased rat striatal acetylcholine content by approximately 70% without altering the levels of this amine in the cerebral hemispheres, mesencephalon, diencephalon, hippocampus and cerebellum. Striatal choline levels were concomitantly decreased by about 25%. This dose of picrotoxin also increased striatal homovanillic acid levels by about 30%, an effect which was not antagonized by pretreatment with the dopamine receptor stimulating agent, piribedil. Picrotoxin did not affect striatal choline-O-acetyltransferase or cholinesterase activity after in vitro incubation. The action of picrotoxin on striatal acetylcholine levels was partially antagonized by pimozide and completely blocked by alpha-methyl-para-tyrosine pretreatment while the intraventricular injection of 6-hydroxydopamine was without effect. Convulsions were not prevented by any of these treatments. The results are interpreted as follows: picrotoxin released dopamine through disinhibition of the dopaminergic neurons as a result of blockade of gabergic receptors. The increased dopaminergic activity inhibited cholinergic neurons and lead to an increase in acetylcholine content. The data thus provide evidence for a possible gabergic (inhibitory)—dopaminergic (inhibitory)-cholinergic link terminating in the striatum.

The action of picrotoxin and bicuculline on rat caudate neurons inhibited by GABA

The action of picrotoxin and bicuculline on rat caudate neurons inhibited by GABA

Habituation of the motion detectors of the crayfish optic nerve: their relationship to the visually evoked defense reflex.

AbstractExamination of sustaining units, dimming units, and motion detectors under stimulus conditions associated with defense reflex habituation revealed that only the motion detectors exhibit a response decrement with stimulus repetition. Motion detector habituation was specific to the retinal locus of the repetitive stimulus and thus can provide a basis for the spatially specific component of defense reflex habituation. When stimulated at low repetition rates (2‐‐4 min) the motion detectors exhibited a time course of habituation and spontaneous recovery similar to that observed behaviorally. At high repetition rates (20/mm) the motion detectors exhibited a more rapid rate of habituation than was observed for the defense reflex. Simultaneous recordings of motion detector and cheliped levatory muscle activity revealed consistently significant and frequently substantial positive correlations. Both defense reflex and motion detector habituation were transiently reversed by treatment with the GABA antagonist, picrotoxin (1.8 2.8 mg kg body weight). The action of picrotoxin is likely to be mediated via mechanisms controlling the general level of arousal. It was concluded that the response variations of motion detectors subjected to repetitive stimuli contribute to the habituation of the visually evoked defense refle.

The spontaneous and electrically evoked release of [ 3H]-GABA from the isolated hemisected frog spinal cord

A study has been made of the spontaneous and electrically evoked release of [ 3H]GABA from the isolated, hemisected spinal cord of the frog. The spontaneous efflux of radioactive GABA was exponential with time and was significantly increased by ouabain, p-hydroxy-mercuribenzoate, l-2,4-diaminobutyric acid and also by nonradioactive GABA; of the convulsants investigated, only strychnine affected (reduced) release. Electrically evoked release could only be achieved by stimulation of descending spinal tracts; release was calcium-dependent and was a function of the stimulation frequency, duration and applied current employed. The evoked release was inhibited by ouabain, p-hydroxy-mercuribenzoate and picrotoxin, significantly potentiated by bicuculline whereas strychnine was without effect. The actions of these drugs could not be explained on the basis of their effects on the uptake of [ 3H]GABA by spinal cord tissue. The results suggest that GABA may act as a neurotransmitter in the spinal cord. Furthermore, it is concluded that the actions of picrotoxin and bicuculline on [ 3H]GABA release are not solely the result of GABA receptor blockade.

Pharmacological properties of cat's collicular auditory neurons.

Effects of various drugs on the collicular auditory neurons were examined by means of pressure microinjection and microelectrophoresis methods.1. Acetylcholine had stimulating action on 67% of the nuerons tested. Most of them showed a relatively fast onset and a slow recovery.2. Monosodium-L-glutamate excited 85% of the neurons tested. The action was exerted with a relatively fast time course and by smaller doses than those required for acetylcholine.3. L-aspartic acid also had an excitatory action. It was clearly much weaker than L-glutamate.4. Eserine sulphate stimulated 60% of the neurons tested.5.γ-Aminobutyric acid exerted a potent inhibitory action on both spontaneous discharges and tone-induced responses. The recovery required a much longer time than that needed for the onset of the effect.6. Glycine caused a powerful depression comparable to that of GABA. The neurons tested were of a fast time course of action.7. Atropine sulphate, dihydro-β-erythroidine hydrobromide and d-tubocurarine blocked tone-induced excitation. Atropine had the strongest action among these cholinergic antagonists.8. Disinhibitory effect by picrotoxin was observed with 43% of the “on” type neurons examined, that is, “on” responses were changed to sustained responses. The action of picrotoxin was time-limited in duration to 40-80 msec and mainly affected the discharges during the beginning of the stimulation period.9. Strychnine sulphate was found to have no effect upon “on” type neurons.10. 5-Hydroxytryptamine was also found to have no effect.11. Irreversible blocking effect was observed with tetrodotoxin.12. It is suggested that the receptor sites of the collicular auditory neurons are cholinergic in nature for mediating the excitatory process. GABA may be a principal candidate as an inhibitory transmitter which may produces “on” type responses in the inferior colliculus.

Action of picrotoxin on presynaptic inhibition of various origins in the cat's spinal cord

Action of picrotoxin on presynaptic inhibition of various origins in the cat's spinal cord

A study of the action of picrotoxin on the inhibitory neuromuscular junction of the crayfish.

1. The effect of picrotoxin on the neuromuscular junction of the crayfish (Cambarus clarkii) was investigated. The potential changes were recorded intracellularly and extracellularly with micro-electrodes. The membrane conductance of the muscle fibre was also measured.2. Picrotoxin depressed the amplitudes of the inhibitory junctional potentials and the potential changes produced by iontophoretically applied gamma-aminobutyric acid (GABA), but had no appreciable effect on the excitatory junctional potentials and the potential changes produced by L-glutamate.3. The presynaptic action of GABA and the neural transmitter was depressed by picrotoxin. The presynaptic action of beta-guanidinopropionic acid was also depressed by picrotoxin.4. The increase in the membrane conductance produced by the addition of GABA in the bath fluid was depressed by picrotoxin. The dose-response relation showed that picrotoxin depressed the conductance increase produced by GABA in a non-competitive manner. The action of picrotoxin on the conductance increase produced by GABA was more effective in low Cl- solution.5. The analysis of the dose-response curves showed that the action of picrotoxin was well expressed by the Michaelis-Menten equation, but the slope of the dose-response curve of GABA was steeper than this relation. It is proposed that the conductance of the junctional membrane was increased by the combination of two molecules of GABA with a receptor, and the attachment of one molecule of picrotoxin to a specific site depressed the conductance increase.