Waldemar A. Turski

Seizures produced by pilocarpine in mice: A behavioral, electroencephalographic and morphological analysis

Increasing doses of pilocarpine, 100-400 mg/kg, were given intraperitoneally to mice and the resulting behavioral, electroencephalographic and neuropathological alterations were studied. No behavioral phenomena were observed in mice treated with the lowest dose of pilocarpine. Occasional tremor and myoclonus of hindlimbs were found in animals which received pilocarpine in a dose of 200 mg/kg. At doses of 300, 325 and 350 mg/kg, pilocarpine produced a sequence of behavioral alterations including staring spells, limbic gustatory automatisms and motor limbic seizures that developed over 15-30 min and built up progressively into a limbic status epilepticus lasting for several hours. The highest dose of pilocarpine, 400 mg/kg, was generally lethal to mice. Pilocarpine produced both interictal and ictal epileptiform activity in the electroencephalogram (EEG). The earliest EEG alterations appeared in the hippocampus and then spread to cortical areas. EEG seizures started 10-15 min after injection of large doses of pilocarpine, 300-350 mg/kg. Ictal periods lasted for 1-2 min, recurred every 5-10 min and were followed by periods of depression of the EEG activity. By 30-45 min paroxysmal activity resulted in a status epilepticus. Examination of frontal forebrain sections with light microscopy revealed a widespread damage to several brain regions including the hippocampus, amygdala, thalamus, olfactory cortex, neocortex and substantia nigra. Scopolamine, 10 mg/kg, and diazepam, 10 mg/kg, prevented the development of convulsive activity and brain damage produced by pilocarpine. The results emphasize that excessive and sustained stimulation of cholinergic receptors can lead to seizures and seizure-related brain damage in mice. It is proposed that systemic pilocarpine in mice provides a useful animal model for studying mechanisms of and therapeutic approaches to temporal lobe epilepsy.

Muscle Relaxant Action of Excitatory Amino Acid Antagonists

Antagonists of neuronal excitation induced by dicarboxylic amino acids were tested in genetically spastic rats of the Han-Wistar strain. These animals exhibit an increased muscle tone which can be measured as a spontaneous tonic activity in the electromyogram of the gastrocnemius-soleus muscle. Compounds that block excitation due to N-methyl-D-aspartic acid reduced the spontaneous activity measured in the electromyogram in a dose-related manner. The most potent compounds, 2-amino-7-phosphonoheptanoic and kynurenic acids were effective muscle relaxants when given either intraperitoneally or intracerebroventricularly. 2-Amino-5-phosphonopentanoic acid possessed much weaker muscle relaxant activity, while L-glutamic acid diethylester was inactive by either route. The results suggest that blockade of N-methyl-D-aspartic acid receptors results in a myorelaxant effect. Specific antagonists of excitation at N-methyl-D-aspartic acid receptors may provide a new class of muscle relaxants.

Susceptibility to seizures produced by pilocarpine in rats after microinjection of isonnazid or γ-vinyl-GABA into the substantia nigra

Pilocarpine, given intraperitoneally to rats, reproduces the neuropathological sequelae of temporal lobe epilepsy and provides a relevant animal model for studying mechanisms of buildup of convulsive activity and pathways operative in the generalization and propagation of seizures within the forebrain. In the present study, the effects of manipulating the activity of the gamma-aminobutyric acid (GABA)-mediated synaptic inhibition within the substantia nigra on seizures produced by pilocarpine in rats, were investigated. In animals pretreated with microinjections of isoniazid, 150 micrograms, an inhibitor of activity of the GABA-synthesizing enzyme, L-glutamic acid decarboxylase, into the substantia nigra pars reticulata (SNR), bilaterally, non-convulsant doses of pilocarpine, 100 and 200 mg/kg, resulted in severe motor limbic seizures and status epilepticus. Electroencephalographic and behavioral monitoring revealed a profound reduction of the threshold for pilocarpine-induced convulsions. Morphological analysis of frontal forebrain sections with light microscopy revealed seizure-related damage to the hippocampal formation, thalamus, amygdala, olfactory cortex, substantia nigra and neocortex, which is typically observed with pilocarpine in doses exceeding 350 mg/kg. Bilateral intrastriatal injections of isoniazid did not augment seizures produced by pilocarpine, 200 mg/kg. Application of an irreversible inhibitor of GABA transaminase, gamma-vinyl-GABA (D,L-4-amino-hex-5-enoic acid), 5 micrograms, into the SNR, bilaterally, suppressed the appearance of electrographic and behavioral seizures produced by pilocarpine, 380 mg/kg. This treatment was also sufficient to protect animals from the occurrence of brain damage. Microinjections of gamma-vinyl-GABA, 5 micrograms, into the dorsal striatum, bilaterally, failed to prevent the development of convulsions produced by pilocarpine, 380 mg/kg. The results demonstrate that the threshold for pilocarpine-induced seizures in rats is subjected to the regulation of the GABA-mediated synaptic inhibition within the substantia nigra.

Blockade of excitatory neurotransmission in the globus pallidus induces rigidity and akinesia in the rat: implications for excitatory neurotransmission in pathogenesis of Parkinson's diseases

Bilateral microinjections of the selective N-methyl-D-aspartate (NMDA) antagonist, (-)-2-amino-7-phosphonoheptanoate (AP7), 0.02-0.5 nmol, into the globus pallidus and ventral-posterior portions of the caudate-putamen result in an increase in the muscle tone (rigidity) and catalepsy (akinesia) in rats. NMDA blocked the actions of AP7 on motility in sensitive regions of the globus pallidus and caudate-putamen. Topographical differences in the action of AP7 in the striatum were detected in the dorsal-ventral and rostral-caudal direction. Microinjections of AP7 into the nucleus accumbens induced neither an increase in the muscle tone nor catalepsy in rats, while ventral regions of the caudate-putamen were sensitive to both actions of AP7. Microinjections of AP7 into the dorsal caudate-putamen resulted in a moderate or no increase in the muscle tone. AP7 failed to induce catalepsy from dorsal regions of the caudate-putamen. These data identify the globus pallidus and a defined subregion of the caudate-putamen as crucial sites where excitatory neurotransmission acts to regulate the final set-point of the respective output neurons providing modulation of the passage of motor information through the basal ganglia.

Differential effects of non-steroidal anti-inflammatory drugs on seizures produced by pilocarpine in rats

The muscarinic cholinergic agonist pilocarpine induces in rats seizures and status epilepticus followed by widespread damage to the forebrain. The present study was designed to investigate the effect of 5 non-steroidal anti-inflammatory drugs, sodium salicylate, phenylbutazone, indomethacin, ibuprofen and mefenamic acid, on seizures produced by pilocarpine. Pretreatment of rats with sodium salicylate, ED50 103 mg/kg (60-174), and phenylbutazone, 59 mg/kg (50-70) converted the non-convulsant dose of pilocarpine, 200 mg/kg, to a convulsant one. Indomethacin, 1-10 mg/kg, and ibuprofen, 10-100 mg/kg, failed to modulate seizures produced by pilocarpine. Mefenamic acid, 26 (22-30) mg/kg, prevented seizures and protected rats from seizure-related brain damage induced by pilocarpine, 380 mg/kg. These results indicate that non-steroidal anti-inflammatory drugs differentially modulate the threshold for pilocarpine-induced seizures.

Substantia nigra and motor control in the rat: effect of intranigral α-kainate and γ-d-glutamylaminomethylsulphonate on motility

Bilateral microinjections of an excitatory amino acid, alpha-kainate (KA), 5-50 ng, into the substantia nigra pars reticulata (SNR) result in an increase in the muscle tone and catalepsy in rats. The preferential KA/quisqualate antagonist, gamma-D-glutamylaminomethylsulphonate (gamma-D-GAMS), 10 micrograms, blocks the actions of KA, 25 ng, when coadministered into the SNR. The chemical lesion of the caudate-putamen with 6-hydroxydopamine (6-OHDA) does not affect either increases in the muscle tone or catalepsy produced by KA, 25 ng, from the SNR. The lesion of the caudate-putamen with ibotenate moderately enhances the effect of KA, 25 ng, on the muscle tone. Microinjections of KA, 25 ng, into the substantia nigra pars compacta (SNC) do not increase the muscle tone and lead to significantly less pronounced catalepsy relative to that observed following the injections of KA into the SNR. Unilateral microinjections of KA, 10-50 ng, into the SNR elicit ipsilateral turning in rats in a dose- and time-dependent manner. Unilateral application of gamma-D-GAMS, 1-10 micrograms, into the SNR produces contralateral turning. The turning evoked by KA, 25 ng, or gamma-D-GAMS, 10 micrograms, is affected neither by 6-OHDA nor by ibotenate lesion of the caudate-putamen. These results demonstrate that excitatory neurotransmission in the substantia nigra participates in the regulation of the muscle tone and posture in rats.

Effect of aminophylline on muscle relaxant action of diazepam and phenobarbitone in genetically spastic rats: Further evidence for a purinergic mechanism in the action of diazepam

The effect of aminophylline on the muscle relaxant action of both diazepam and phenobarbitone was studied in genetically spastic rats of the Han-Wistar strain which exhibit spontaneous tonic activity in the electromyogram of the gastrocnemius-soleus muscle. Both diazepam (0.8 and 4.0 mg/kg i.p.) and phenobarbitone (20 and 30 mg/kg i.p.) reduced the spontaneous activity measured in the electromyogram in a dose-related manner. Aminophylline (50 mg/kg i.p.), a methylxanthine with potent antagonistic activity of adenosine-mediated inhibition, partially reversed the muscle relaxant action of diazepam (4 mg/kg) but not that produced by phenobarbitone. The muscle relaxant effect of phenobarbitone (30 mg/kg) was antagonised by beta-carboline-3-carboxylic acid methylester (beta-CCM), 2 mg/kg i.p. The reversal of the muscle relaxant effect of phenobarbitone produced by beta-CCM was abolished by CGS 8216 (2-phenylpyrazolo-(4,3c)quinolin-3(5H)-one), 5 mg/kg i.p. Aminophylline altered neither the muscle relaxant effect of a low dose of diazepam (0.8 mg/kg) nor the reversal of the muscle relaxant effect of phenobarbitone produced by beta-CCM. These findings indicate that the interaction between diazepam and aminophylline does not involve competition for the benzodiazepine receptor and add further support to the suggestion that purinergic mechanisms may be engaged in the muscle relaxant action of diazepam.

A specific benzodiazepine antagonist CGS 8216 reverses the muscle relaxant effect of diazepam but not that of phenobarbitone

The muscle relaxant effects of diazepam and phenobarbitone were studied in mutant Han-Wistar rats which exhibit spontaneous tonic activity in the electromyogram of the gastrocnemius-soleus muscle. Diazepam and phenobarbitone significantly and dose- relatedly reduced the spontaneous activity measured in the electromyogram. The benzodiazepine receptor antagonist CGS 8216 reversed the depressant effect of diazepam but not that produced by phenobarbitone. The results add further support to the suggestion that CGS 8216 may be useful to investigate the effects modified by benzodiazepine receptors in vivo.

Neurotransmitters in the Basal Ganglia and Motor Thalamus: Their Role for the Regulation of Muscle Tone

Stimulated by Ehringer and Hornykiewicz’s (1960) fundamental finding of a highly decreased dopamine content in the neostriata of Parkinsonian patients there has been increasing interest in neurotransmitter mechanisms in the basal ganglia and their role in physiological and pathological processes. Aided by the advent of new neuroanatomical and neurochemical technigues and the use of modern electrophysiological and pharmacological methods, new concepts of basal ganglia function have emerged which, although still inadaeguate, allow a better integration of the experimental and clinical data of different disciplines involved in basal ganglia research.

The entopeduncular nucleus regulates muscle tone in genetically spastic rats: role of substance P and γ-aminobutyric acid

Microinjections of the substance P (SP) antagonist (D-pro2,D-phe7,D-trp9)-SP, or the gamma-aminobutyric acid (GABA) agonist, muscimol, into the entopeduncular nucleus reduced muscle tone in genetically spastic rats in a dose- and time-dependent manner. Similar injections into the ventral thalamus, zona incerta or amygdala had no effect on muscle tone. The muscle relaxant effect of (D-pro2,D-phe7,D-trp9)-SP injected into the entopeduncular nucleus was blocked by co-injections of SP, and that of muscimol by the GABAA antagonist, bicuculline methiodide. These results suggest that SP- and GABA-dependent mechanisms in the entopeduncular nucleus mediate regulation of the muscle tone.