Sex differences in the sensitivity of CBA mice to convulsions induced by GABA antagonists are age-dependent

The administration of the GABA-blocking agents picrotoxin and bicuculline to adult (2.5–3 months old) CBA/HZgr mice resulted in the appearance of convulsions, the occurrence and/or lethality of which was greater in males than in females. The latency of picrotoxin-induced convulsions was also shorter in male mice. Strchnine, a drug which induces convulsions by blocking glycine receptors was equally effective in producing convulsions in both male and female adult mice. Unlike adult mice, young (20 days old) or old (2 years old) mice fail to display sex differences following the picrotoxin administration. Accordingly, the observed sex differences in the sensitivity of CBA mice to administration of convulsive agents are specific for the GABA system and present only in sexually mature, but not in immature or old animals.     

Effect of daily saline, drug or blank injections on the susceptibility to the convulsant effect of drugs.

The daily intraperitoneal injection to rats of doses of metrazol (30 mg/Kg), strychnin sulfate (1 mg/Kg) or picrotoxin (1.2 mg/Kg) that were initially subconvulsant, caused after a number of days which varied with the drug, clonic convulsions in a high percentage of the animals. However, after 18 daily injections of saline there was a similar increase of seizure susceptibility to the 3 drugs. The daily handling of rats as for injection, either followed or not by actual abdominal pricking (blank injection), had a similar though less pronounced effect. In animals that were housed in the same room where the others were tested, but which were not handled, the above mentioned doses of metrazol, strychnine and picrotoxin had no convulsant effect. These results indicate that the procedure of submitting rats to daily intraperitoneal injections is not as unconsequential as is usually thought to be, and that it may induce neurological changes.     

Differential sensitivity of genetically Fast vs. Slow kindling rats strains to GABAergic convulsive agents

Following selective breeding for seizure-proneness vs. seizure-resistance to amygdala kindling, two strains of rats were developed with non-overlapping kindling rates, i.e. the number of stimulations required to develop fully generalized convulsive seizures (Epilepsy Res. 35 (1999) 183). In the temporal cortices of these two strains, the local seizure thresholds to electrical stimulation have been reported to be approximately two times lower in the seizure-prone (Fast kindling) compared to the seizure-resistant (Slow kindling) strain (McIntyre et al., 1999). In the present experiment, the pharmacological sensitivities of the two strains to three GABAergic antagonists, pentylenetetrazol, bicuculline and picrotoxin, were determined, and compared to the glycine antagonist, strychnine. Paralleling kindling epileptogenesis, na?ve rats of the Fast kindling strain developed convulsive seizures to doses of the GABAergic antagonists that were significantly (approximately 30%) lower than the na?ve Slow kindling strain. In contrast, there were no strain differences in their response to strychnine. These data indicate substantial GABAergic sensitivity differences between the two strains with an emphasis on forebrain mechanisms, which is consistent with other physiological and molecular data related to their differential profiles of epileptogenesis.     

Differential effects of convulsants on visually evoked responses in the albino rat

Visually evoked responses (VERs) were recorded from primary visual cortex in unanesthetized albino rats during pharmacological modulation by one of the following convulsant agents: Physostigmine, picrotoxin, strychnine and Metrazol. The data were analyzed in terms of effects on VER components. The rat VER consists of six distinct waves constituted by three positive peaks(P1-P3) and three negative peaks (N1-N3). Results indicate a differential convulsant action on VER components. The administration of picrotoxin, resulted in a suppression of the peak amplitude of P1-N1 and delayed peak latencies of all components. Strychnine reliably shortened P1, N1 and P2 peak latencies, significantly increased N3 peak latency and only suppressed P3-N3 amplitude. Physostigmine essentially suppressed all component amplitudes but only increased peak latencies for P2 and P3 components. Metrazol, in general, was found to be relatively ineffective in the alteration of any VER component in a systematic manner. The data are discussed in terms of differential modes of convulsant action on the visual system. The results are likewise discussed with respect to their implications for convulsant modulation of photically evoked after-discharges.     

Effects of toluene on seizures induced by convulsants acting at distinct ligand-gated ion channels

Toluene is one of the most widely used solvents. Electrophysiological studies indicated that this solvent directly affects various ligand gated ion channels including NMDA, GABA_A, nicotinic and glycine receptors. The effect of toluene on seizures induced by chemoconvulsants acting on these receptors was compared. Mice were pretreated with toluene (100–1000 mg/kg, i.p.) or corn oil followed by a timed intravenous infusion of NMDA, bicuculline, picrotoxin, nicotine or strychnine to induce seizures. Toluene increased seizure thresholds and lethal doses induced by nicotine, NMDA, picrotoxin and bicuculline, but not strychnine in the used doses. The relative susceptibility to anticonvulsant effect of toluene was in the order: nicotine > NMDA > bicuculline > picrotoxin > strychnine. These findings support a unique anticonvulsant profile of toluene and suggest that nicotinic and NMDA receptors may be more sensitive than GABA_A and glycine receptors to toluene exposure in seizure-related neural circuits.     

Interactions of pentobarbital and phenobarbital with GABAergic drugs against chemoconvulsants in rats

Pentobarbital and phenobarbital exhibited anticonvulsant effects against picrotoxin (10 mg/kg, IP) as well as against strychnine (4 mg/kg, IP). Pentobarbital was also effective against bicuculline whereas only hypnotic doses of phenobarbital provided some protection against bicuculline- (8 mg/kg, IP) induced convulsions. Diazepam as well as THIP, but not baclofen, were also effective against all the three chemoconvulsants. Baclofen or subeffective doses of diazepam or THIP, but not baclofen, were also effective against all of pentobarbital exhibited anticonvulsant activity against all the chemoconvulsants studied. On the other hand, a combination of subeffective doses of these agents with subeffective doses of phenobarbital provided protection only against picrotoxin and strychnine. These observations indicate that pentobarbital is quite effective against convulsions caused by agents acting at picrotoxin site, GABA_A receptor or glycine receptor whereas phenobarbital is effective only against agents acting at picrotoxin site and glycine receptor, and is very weak anticonvulsant against agents causing blockade of GABA_A receptors. Furthermore, activation of GABA_A receptors or benzodiazepine receptors also provide protection against agents acting at GABAergic system or glycine receptors. On the contrary, activation of only GABA_B receptors is inadequate to provide the protective effect. However, the activation of GABA_A as well as GABA_B receptors facilitate the anticonvulsant effect of both the barbiturates. Furthermore, pentobarbital, but not phenobarbital, facilitates the anticonvulsant effect of benzodiazepines against chemoconvulsants acting at GABAergic site or glycine receptors.     

GABA and hypothalamic feeding systems. II. A comparison of GABA,glycine and acetylcholine agonists and their antagonists

Microinjections of various compounds into the paraventricular hypothalamic nucleus (PVH) were made and the effects on feeding observed. During the light phase of the lighting cycle, injections of 0.3 μl of muscimol (100 ng) and flurazepam diHCl (20 μg) increased feeding. Similar injections of glycine (500 ng) did not influence feeding during the light phase. During the dark phase, 0.3 μl injections of bicuculline methiodide (30 ng) and picrotoxin (160 ng) suppressed feeding. Similar injections of carbachol increased drinking during the dark phase. Injections of strychnine during this phase were without effect. Tilt box activity levels were not altered by injection of picrotoxin (160 ng) into the PVH.     

Involvement of central cholinoceptors in metrazol-induced convulsions

Atropine sulphate (10 mg/kg IP) afforded 90% protection against clonic convulsions induced by standard doses of metrazol (80 mg/kg SC) in mice, whereas atropine methonitrate (10 mg/kg IP) did not offer any protection. Furthermore, physostigmine (1 mg/kg IP) caused recurrence of convulsions in atropinzed metrazol-treated mice and converted the subconvulsive dose of metrazol (40 mg/kg SC) into a 100% convulsive dose. However, neostigmine (1 mg/kg IP) did not antagonize the protection afforded by atropine sulphate against metrazol. The results of the study suggest an involvement of central cholinergic mechanisms in metrazol-induced convulsions.     

Relation of rat brain acetylcholine levels to duration of self-stimulation and escape behavior

Total brain acetylcholine (ACh) was assayed in groups of animals after various periods of operant responding maintained by electrical stimulation of the lateral posterior hypothalamus or of escape behavior induced by electrical stimulation of the midbrain tegmentum. Different groups of trained rats were placed in identical Skinner boxes for periods of 1 to 24 hr. The following groups were studied: controls, self-stimulators receiving electrical stimulation, escapers from brain stimulation or peripherally applied aversive stimulation, self-stimulators not receiving electrical stimulation prior to decapitation, tubocurarine-paralyzed respired rats with electrodes in the posterior-lateral hypothalamus not receiving stimulation, and a group of tubocurarine-paralyzed, respired rats receiving electrical stimulation automatically. It was found that brain stimulation decreased total brain ACh, regardless of whether the stimulation was positive, as during self-stimulation behavior, or negative, as during escape behavior. Animals that received positive stimulation while being paralyzed showed similar decreases in total brain ACh, but the change in ACh was smaller. No changes occurred in animals that were paralyzed that received no electrical stimulation. It is concluded that brain usage produced by electrical stimulation of discrete functional pathways causes a reduction of total ACh, but this is unrelated to the specific motivational properties of the electrical stimuli.     

Effect of diazepam on mouse whole brain and brain area acetylcholine and choline levels

A single i.v. dose of diazepam, 5 mg/kg, increased mouse whole brain acetylcholine levels. Choline levels, choline acetyltransferase and acetylcholinesterase activities were not affected, which is consistent with the hypothesis that diazepam blocks release of acetylcholine. Diazepam increased acetylcholine levels in the hemispheres and diencephalon but not in the cerebellum or mesencephalon. The effect lasted for 4 hr in the hemispheres and for 30 min in the diencephalon. This short-lastig biochemical action precludes a correlation with the long-lasting action of diazepam against pentylenetetrazole.     

Effect of central stimulants and depressants on mouse brain acetylcholine and choline levels

Brain concentrations of acetylcholine and choline were measured within 5–30 min after an i.p. injection of central stimulant or depressant drugs. The acetylcholine concentration in whole mouse brain of controls was 2.35 ± 0.06 μg/g wet wt. (16.1 nmol/g wet wt.) while the choline concentration was 6.52 ± 0.21 μg/g wet wt. (62.7 nmol/g). Physostigmine, 0.5 mg/kg, oxotremorine, 2 mg/kg free base, and haloperidol, 4 and 8 mg/kg, increased both acetylcholine and choline 20 min after administration. Pentobarbital, 55 mg/kg, and diazepam, 5–40 mg/kg, increased only acetylcholine without affecting choline. Atropine, 50 mg/kg, lowered acetylcholine without affecting choline. Haloperidol, 1 mg/kg, chlorpromazine, 10 mg/kg, reserpine, 2.5 mg/kg, 20 hr, desmethylimipramine, 20 mg/kg, and amphetamine, 7 and 15 mg/kg, had no effect on either acetylcholine or choline. No drug studied caused a simultaneous decrease in these two quaternary amines. The possible significance of these findings on the mechanism of action of these drugs is discussed.     

Convulsant properties of GABA antagonists and anticonvulsant properties of ethanol in selectively bred Long- and Short-Sleep mice

The convulsant potency of bicuculline, a GABA antagonist, was shown to be greater in Short-Sleep (SS) mice than in Long-Sleep (LS) mice. LS mice, selectively bred for lengthy ethanol-induced narcosis, had longer latencies to myoclonus and clonus following administration of bicuculline and picrotoxin than did ethanol-resistant SS mice. SS mice were also more susceptible to pentylenetetrazol-induced myoclonus, but not clonus. F₁ hybrids showed bicuculline seizure sensitivity intermediate to the two parent lines. Ethanol weakly inhibited bicuculline-induced myoclonus in both LS and SS mice. Clonus was clearly antagonized by ethanol in both lines, but to a similar degree. These data provide evidence for a GABAergic role in geno-type-dependent sensitivity to ethanol.     

Application of high-powered microwave irradiation for acetylcholine analysis in mouse brain

Acetylcholine (ACh) levels in whole brains or discrete areas of mice brains were measured after sacrificing by decapitation, conventional or high-powered microwave irradiation. ACh levels in mice brains varied according to the time required to inactivate the enzymes. ACh levels after high-powered microwave irradiation for a shorter time were significantly higher than those after conventional microwave irradiation.     

Proconvulsant-induced seizures in alpha(4) nicotinic acetylcholine receptor subunit knockout mice

The genetic basis of a number of epilepsy syndromes has been identified but the precise mechanism whereby these mutations produce seizures is unknown. Three mutations of the alpha(4) subunit of the neuronal nicotinic acetylcholine receptor (nAChR) have been identified in autosomal dominant nocturnal frontal lobe epilepsy. In vitro studies of two mutations suggest an alteration of receptor function resulting in decreased ion channel current flow. We investigated the response of alpha(4) nAChR subunit knockout mice to the gamma-aminobutyric acid (GABA) receptor antagonists; pentylenetetrazole (PTZ) and bicuculline (BIC), the glutamate receptor agonist kainic acid (KA), the glycine receptor antagonist strychnine and the K(+) channel blocker 4-aminopyridine (4-AP). Mutant (Mt) mice had a greater sensitivity to PTZ and BIC, with an increase in major motor seizures and seizure-related deaths. Furthermore, Mt mice were more sensitive to KA and strychnine, but the effects were much smaller compared to those seen with the GABA receptor antagonists. Paradoxically, Mt mice appeared to be relatively protected from 4-AP-induced major motor seizures and death.The results show that a functional deletion of the alpha(4) nAChR subunit in vivo is associated with a major increase in sensitivity to GABA receptor blockers.     

Effects of chronic lead exposure on levels of acetylcholine and choline and on acetylcholine turnover rate in rat brain areas in vivo

Rats were exposed to lead acetate from birth, and were killed at the age of 44–51 days for analysis of levels and turnover rates of acetylcholine (ACh). Steady-state levels of ACh were not altered in midbrain, cortex, hippocampus, or striatum of lead-exposed rats. Similarly, no changes in choline (Ch) concentrations were found in cortex, hippocampus, or striatum. In the midbrain, however, a 30% reduction in Ch levels was observed. Changes in specific activity of Ch and ACh were measured as a function of time in selected brain areas of rats infused with a radio-labeled precursor of Ch. Specific activities of ACh were not altered. Ch specific activities were, however, significantly elevated in all brain areas examined, as compared with age-matched control rats. The in vivo ACh turnover rate in cortex, hippocampus, midbrain, and striatum was diminished by 35%, 54%, 51% and 33%, respectively. These findings provide direct evidence for an inhibitory effect of lead exposure from birth on central cholinergic function in vivo. Since a significant reduction of body weight was found in those animals treated with lead acetate, the alteration of central cholinergic function may partially be attributed to malnutrition observed in the lead-exposed animals.Part of this work was presented in abstract form (Fed. Proc. 36. 977 (1977))     

Changes in acetylcholine and noradrenaline sensitivity of chick smooth muscle wholly innervated by sympathetic nerve during development.

Developmental changes in sensitivity of the isolated expansor sedumdariorum muscle of posthatching chicks to noradrenaline (NA), Acetylcholine (ACh) and some other drugs were investigated. This muscle responded to both NA and ACh in early life. The sensitivity to ACh decreased progressively with increasing age and disappeared on day 40 after hatching, however, a corresponding elevation of cholinesterase activity was not observed. The sensitivity to NA remained at the same level during the period of 2-60 days after hatching. The Contractile action of ACh on this muscle was not affected by d-tubocurarine, hexamethonium or phentolamine, but was completely abolished by atropine. These results suggest that there are at least two kinds of responsive sites on the expansor secumdariorum muscle in the new-born chick and that the sites sensitive to ACh degenerate progressively during the developmental processes. The cholinergic sensitive sites of this muscle in the new-born chick may be muscarinic.     

Steady-state concentrations of choline and acetylcholine in rat brain parts during a constant rate infusion of deuterated choline

Summary An intravenous infusion of deuterated choline at constant rate for 6 min (5 or 25 moles kg^–1 min^–1) significantly increases the concentration of choline in plasma, occipital cortex and striatum. Both 5 and 25 moles kg^–1 min^–1 increase the concentration of acetylcholine in cortex but only 25 moles kg^–1 min^–1 increases the acetylcholine content in striatum. In contrast, 1 mole kg^–1 min^–1 does not change the choline or acetylcholine content in cortex or striatum. A single pulse injection of choline (200 moles kg^–1) causes a significant increase in the concentration of choline in striatum 30 sec following injection. The choline content returns to normal values within 2 min. These studies show that when a pulse injection of a non-tracer dose of radioactive choline is used to measure brain acetylcholine turnover rate the maintenance of steady state must be verified within seconds after the pulse injection of radioactive choline. When constant infusion of deuterated choline is used to measure turnover rate of acetylcholine in the brain of rats, a dose of 1 mole kg^–1 min^–1 appears to be a maximal infusion rate.     

Effect of metrazol on brain norepinephrine: a possible factor in amnesia produced by the drug

Metrazol-induced seizures reduced brain norepinephrine (NE) of mice trained in a passive avoidance paradigm. A reduction of NE was observed 5 but not 1440 min following injections. The implications of the results are discussed with respect to amnesia produced by metrazol.     

Convulsions elicited by handling: A sensitive method of measuring CNS excitation in mice treated with reserpine or convulsant drugs

Mice treated with reserpine, picrotoxin or pentylenetetrazol showed convulsions on handling. These seizures closely resembled the previously described convulsions on handling in mice undergoing alcohol withdrawal reactions. The mildest form of the convulsion was tonic, in a characteristic posture, and was evoked by gently spinning the mouse by the tail. More severely affected mice showed clonic seizures when simply lifted by the tail. Reserpine-treated mice had no spontaneous seizures but convulsions could be repeatedly elicited by handling for about 24 h after a single dose. Mice treated with picrotoxin or pentylenetetrazole showed elicited convulsions at doses that were too low to cause spontaneous seizures or at times when the overt convulsant action had ceased. The number of mice showing elicited convulsions was dose-related. Strychnine treatment caused convulsions on handling only at near-lethal doses and then in only a few mice. Convulsions on handling may be useful as a simple empirical sign of CNS hyperactivity in mice.This work was supported by a grant from the U. S. Brewers Association.