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The mechanisms of neuronal degeneration following traumatic head injury are not well understood and no adequate treatment is currently available for the prevention of traumatic brain damage in humans. Seven day old rat pups were subjected to mechanical percussion of the head. Cortical damage in infant rats was reduced by pre-treatment with the N-methyl-D-aspartate (NMDA) antagonists dizocilpine (MK-801) or 3-((+/-)-2-carboxypiperazin-4-yl)-propyl-I-phosphonate (CPP). The AMPA antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo (f) quinoxaline (NBQX) did not significantly suppress cortical damage in infant rats. In adult rats, traumatic head injury leads to primary (at impact-cortex) and secondary (distant-hippocampus) damage to the brain. Morphometric analysis demonstrated that both cortical and hippocampal damage was mitigated by pre-treatment with either the NMDA antagonist CPP or the non-NMDA antagonist NBQX. Neither treatment prevented primary damage in the cortex when therapy was started after trauma. Delayed treatment of rats with NBQX, but not with CPP, beginning between 1 and 7 h after trauma prevented the hippocampal damage. No protection was seen when therapy with NBQX was started 10 h after trauma. These data indicate that NMDA antagonists may possess better neuroprotective properties against excitotoxic processes triggered by traumatic brain injury in young individuals whereas AMPA antagonists may be more beneficial in adults.

A superoxide dismutase 1 (SOD-1)genetic defect has been identified in familial amyotrophic lateral sclerosis (ALS) and motor neuron degeneration has been described in SOD-1 transgenic mice. Because an excitotoxic mechanism has been implicated in ALS, we undertook studies to provide a description of excitotoxic degeneration of spinal motor neurons for comparison with the degenerative process observed in SOD-1 transgenic mice. Excitotoxin agonists selective for each of the three major types of inotropic glutamate receptors were applied directly onto the lumbar spinal cord of 21-day old rats following posterior laminectomy. N-methyl-D-aspartate (NMDA) preferentially affected dorsal horn neurons, whereas the non-NMDA agonist, kainic acid, preferentially affected motor neurons. Cytopathological changes in motor neurons closely resembled those described in SOD-1 mice. These changes consist of massively swollen dendritic processes in the presence of well-preserved presynaptic axon terminals; cell bodies of motor neurons filled with vacuoles that originate both from endoplasmic reticulum and mitochondria; pleomorphic changes in mitochondria; axons of motor neuron becoming swollen proximally with accumulation of vacuoles, organelles, filaments, and degeneration products in the swollen segment. The observed changes in motor axons resemble changes described in the spinal cord of ALS patients. These findings are consistent with the proposal that motor neuron degeneration in ALS may be mediated by an excitotoxic process involving hyperactivation with non-NMDA glutamate receptors.

It is well established that glutamate receptors play a major role in mediating acute ischemic neuronal degeneration in the CNS. Cerebral ischemia and head or spinal cord trauma are associated with excessive release and extracellular accumulation of glutamate, which leads to persistent activation of glutamate receptors and acute neurotoxic degeneration of the hyperstimulated neuron. It has been more difficult to link neuronal degeneration that occurs in chronic neurodegenerative disorders to an excitotoxic mechanism. However, accumulating evidence suggests that impairment of intracellular energy metabolism associated with hyperactivation of glutamate receptors may be a common mechanism contributing to neuronal death in such disorders. It is proposed that impaired energy metabolism results in deterioration of membrane function and loss of the voltage-dependent Mg2+ block of N-methyl-D-aspartate receptors, which allows persistent activation of these receptors by glutamate, even if concentrations of glutamate at the receptor are within the normal physiological range. Studies in rodents using mitochondrial respiratory chain toxins, such as aminooxyacetic acid, 1-methyl-4-phenylpyridinium ion, malonic acid, and 3-nitropropionic acid, suggest that these agents do induce CNS degeneration by a process involving an excitotoxic mechanism. Striatal and nigral degeneration induced by mitochondrial toxins in rodents resembles neuropathology seen in humans suffering from Huntington's or Parkinson's disease and can be attenuated by glutamate receptor antagonists and agents that improve energy metabolism. Such experimental observations suggest that disturbed energy metabolism and glutamate may be involved in neuronal death leading to abiotrophic/neurodegenerative disorders in humans. If so, glutamate antagonists or agents that improve energy metabolism may slow the degenerative process and offer a therapeutic approach for temporarily retarding the progression of these disabling disorders.

Structural damage of the human brain (perinatal damage, cerebral trauma, head injury, cerebrovascular and degenerative diseases, intracranial tumor, metabolic diseases, toxins, drug-induced seizures) may lead to chronic epilepsy in survivors. Epidemiologic analyses show that a considerable time-delay occurs between the exposure of the brain to injury and the appearance of seizures. Such seizures are usually partial or mixed, may develop at any age, and are difficult to treat. In rats subjected to structural damage of the brain induced by sustained convulsions triggered by systemic administration of the cholinergic agent pilocarpine, spontaneous seizures may develop after a mean latency of 14-15 days. The mean frequency of spontaneous recurrent convulsions remains constant for several months. Evolution of these convulsions proceeds through several electrographic and behavioral stages resembling kindling. Kindling may be otherwise induced in rodents by repeated systemic administration of convulsants or by repeated electrical stimulation of sensitive brain regions. These observations demonstrate that structural damage of the brain may lead to spontaneously recurrent convulsions (chronic epilepsy) in rats and that kindling may be involved in the evolution of such a condition. This finding suggests that kindling mechanisms underlie the development of epileptic foci from structural brain lesions. Such mechanisms may be involved in the etiology of some forms of epilepsy in humans.

The neuropathological, biochemical, and behavioral effects of intrastriatal injection of aminooxyacetic acid (AOAA), a non-specific transaminase inhibitor, were examined in rats. AOAA, 0.1-1 mumol, produced neuronal damage when injected into the striatum of adult rats but failed to damage the striatum of 6-d-old or decorticated rats. AOAA-induced (0.25 mumol-1 mumol) striatal lesions in adult rats displayed excitotoxic characteristics and could be prevented by the N-methyl-D-aspartate (NMDA) receptor antagonists (-)-2-amino-7-phosphono-heptanoate (AP7; 0.25 mumol) or kynurenate (KYNA; 0.5 mumol), but not by the non-NMDA antagonist 2,3-dihydroxy-6-nitro-7-sulphamoyl-benzo(F)quinoxaline (NBQX; 0.25 mumol). AOAA produced a dose-dependent reduction in striatal L-glutamate decarboxylase activity, as measured 14 d following intrastriatal injection, which could also be prevented by AP7 or KYNA, but not by NBQX. These findings suggest that AOAA-induced lesions are preferentially mediated by activation of the NMDA subtype of excitatory amino acid receptors. Behavioral studies revealed that the cataleptic response to haloperidol, 2 mg/kg, was decreased whereas the cataleptic response to arecoline, 15 mg/kg, and morphine, 15 mg/kg, was potentiated in AOAA lesioned animals 14 d following bilateral intrastriatal injections of AOAA, 0.25 and 1 mumol. In rats which received unilateral intrastriatal injection of AOAA, 0.1-1 mumol, apomorphine, 0.5 mg/kg, induced circling towards the lesioned side. Rats which received AP7, 0.25 mumol, or KYNA, 0.5 mumol, coadministered with AOAA, 0.25 mumol, behaved as vehicle-treated controls, while those which received NBQX, 0.25 mumol, and AOAA, 0.25 mumol, had behavioral patterns similar to those subjected to AOAA alone.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of excitatory amino acid antagonists on convulsions induced by intracerebroventricular (i.c.v.) or systemic (s.c.) administration of the gamma-aminobutyric acidA (GABAA) antagonist bicuculline (BIC) were tested in mice. 3-[+/-)-2-Carboxypiperazin-4-yl)-propyl-1-phosphonate (CPP), 2-amino-7-phosphonoheptanoate (AP7) and (+)-5-methyl-10,11-dihydro-5H-dibenzo(a,d)cycloheptan-5,10-imine maleate (MK-801) were used as representatives of N-methyl-D-aspartate (NMDA) antagonists. gamma-D-Glutamylaminomethylsulphonate (gamma-D-GAMS) typified a preferential kainate (KA) antagonist, 6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX) represented a preferential quisqualate (QA) antagonist, and kynurenic acid (KYNA) was used as a mixed NMDA/KA antagonist. Bicuculline methiodide (BMI) induced clonic convulsions following i.c.v. administration with a CD50 of 0.183 nmol (range 0.164-0.204). The excitatory amino acid antagonists blocked clonic seizures induced by BMI in the dose of 0.224 nmol (approximately CD97) when coinjected into the lateral ventricle. CPP (ED50 0.0075 nmol) was the most potent anticonvulsant and was followed by AP7 (0.182 nmol), MK-801 (0.22 nmol), gamma-D-GAMS (0.4 nmol), KYNA (1.7 nmol) and CNQX (5.17 nmol). Muscimol (MSC), the GABAA agonist, blocked BMI-induced seizures with an ED50 of 0.25 nmol. Systemic (s.c.) administration of BIC induced in mice generalized seizures with a CD50 of 2.2 mg/kg (range 1.9-2.5) for clonus and CD50 of 2.4 mg/kg (range 2.2-2.7) for tonus.2+ the pathogenesis of seizures triggered by bicuculline in mice.

The involvement of dopamine (DA) in human and experimental epilepsy has been discounted as DAergic drugs have little effect on convulsions. This work presents evidence that bilateral microinjection of the DAD1 agonist SKF-38393 into the substantia nigra enhances the susceptibility of rats to seizures, with an ED50 of 20 pmol (range 13-31 pmol), converting subconvulsant doses of the cholinergic agonist pilocarpine (200 mg/kg; i.p.) into convulsant ones. The proconvulsant action of SKF-38393 was reversed by blocking D1-mediated transmission in the substantia nigra with the D1 antagonist SCH-23390. The D2 agonist LY-171555 did not modulate the threshold for limbic seizures when injected into the substantia nigra. In the striatum, the D2 agonist LY-171555 protected rats against limbic seizures induced by systemic administration of pilocarpine (380 mg/kg; i.p.), with an ED50 of 2 pmol (range 1.4-2.8 pmol). The anticonvulsant action of LY-171555 in the striatum was reversed by haloperidol. The D1 agonist SKF-38393 did not affect pilocarpine seizures following administration into the striatum. Systemic administration of DAergic drugs showed that the D1 agonist SKF-38393 decreased the threshold for pilocarpine seizures, with an ED50 of 0.81 mg/kg (range 0.45-1.47 mg/kg), whereas the D2 agonist LY-171555 had no effect on susceptibility of rats to pilocarpine. The proconvulsant action of SKF-38393 was blocked by the D1 antagonist SCH-23390. These results suggest that DA differentially modulates seizure threshold in the forebrain acting via D1 mechanisms in the substantia nigra and D2 mechanisms in the striatum.

Genetically spastic rats were used for studying the effect on muscle tone of beta-carboline-3-carboxylic acid methylester (beta-CCM), an inverse benzodiazepine (BDZ) agonist, and that of Ro 15-1788 and CGS 8216, both putative antagonists of pharmacological actions of BDZs. These animals exhibit pathologically increased muscle tone, which can be recorded and quantified in the electromyogram (EMG) of the gastrocnemius (GS) muscle. beta-CCM, 2.5 and 3.0 mg/kg i.p., augmented the tonic activity in the EMG of GS muscle in spastic rats while it did not modify muscle tone at doses of 1.0 and 2.0 mg/kg. Diazepam, 0.4 mg/kg i.p., and Ro 15-1788, 5 mg/kg i.p., but not CGS 8216, 5 mg/kg i.p., antagonised the effect of the beta-carboline on muscle tone. Ro 15-1788, at doses of 0.1-5 mg/kg i.p., did not affect muscle tone in the genetically spastic rats, whilst at doses of 25-200 mg/kg the imidazodiazepine dose dependently increased the tonic activity in the EMG. The action of Ro 15-1788, 50 mg/kg i.p., was reversed by diazepam, 0.4 mg/kg i.p., and beta-CCM, 2 mg/kg i.p., while CGS 8216, 5 mg/kg i.p., facilitated the effect of Ro 15-1788 on muscle tone. CGS 8216 at doses of 5 and 25 mg/kg i.p. was devoid of any effect on muscle tone, whilst at doses of 50 and 200 mg/kg the pyrazoloquinoline increased the tonic activity recorded in the EMG from the GS muscle of the spastic rats.(ABSTRACT TRUNCATED AT 250 WORDS)

High-dose treatment with pilocarpine hydrochloride, a cholinergic muscarinic agonist, induces seizures in rodents following systemic or intracerebral administration. Pilocarpine seizures are characterized by a sequential development of behavioral patterns and electrographic activity. Hypoactivity, tremor, scratching, head bobbing, and myoclonic movements of the limbs progress to recurrent myoclonic convulsions with rearing, salivation, and falling, and status epilepticus. The sustained convulsions induced by pilocarpine are followed by widespread damage to the forebrain. The amygdala, thalamus, olfactory cortex, hippocampus, neocortex, and substantia nigra are the most sensitive regions to epilepsy-related damage following convulsions produced by pilocarpine. Spontaneous seizures are observed in the long-term period following the administration of convulsant doses of pilocarpine. Developmental studies show age-dependent differences in the response of rats to pilocarpine. Seizures are first noted in 7-12 day-old rats, and the adult pattern of behavioral and electroencephalographic sequelae of pilocarpine is seen in 15-21-day-old rats. During the third week of life the rats show an increased susceptibility to the convulsant action of pilocarpine relative to older and younger animals. The developmental progress of the convulsive response to pilocarpine does not correlate with evolution of the brain damage. The adult pattern of the damage is seen after a delay of 1-2 weeks in comparison with the evolution of seizures and status epilepticus. The susceptibility to seizures induced by pilocarpine increases in rats aged over 4 months. The basal ganglia curtail the generation and spread of seizures induced by pilocarpine. The caudate putamen, the substantia nigra, and the entopeduncular nucleus govern the propagation of pilocarpine-induced seizures. The antiepileptic drugs diazepam, clonazepam, phenobarbital, valproate, and trimethadione protect against pilocarpine-induced convulsions, while diphenylhydantoin and carbamazepine are ineffective. Ethosuximide and acetazolamide increase the susceptibility to convulsant action of pilocarpine. Lithium, morphine, and aminophylline also increase the susceptibility of rats to pilocarpine seizures. The pilocarpine seizure model may be of value in designing new therapeutic approaches to epilepsy.

Accumulating evidence suggests that the N-methyl-D-aspartate (NMDA) subtype of glutamate receptor may play an important role in hypoxic/ischemic (H/I) brain damage. Accordingly, it has been shown that the NMDA antagonist, MK-801, partially protects the infant rat brain against H/I damage. Here we show that reducing the body temperature of the infant rat also confers partial protection against H/I brain damage and that mild hypothermia plus MK-801 treatment provides total protection against such damage. Relevance of these findings to the prevention of perinatal brain damage in humans is discussed.

The increasing amount of data tends to suggest that adenosine-mediated inhibition may play a role in the anticonvulsant activity of a number of antiepileptic drugs. Consequently, we tried to reverse the protective action of acetazolamide [(40 and 80 mg/kg) i.p.; 60 min before the test]; carbamazepine (20 and 30 mg/kg i.p., 60 min); diazepam (5 and 10 mg/kg i.p., 60 min); diphenylhydantoin (8 and 12 mg/kg i.p., 120 min), phenobarbital (20 and 30 mg/kg i.p., 120 min) and valproate (200 and 300 mg/kg i.p., 30 min) with aminophylline (50 and 100 mg/kg i.p., 30 min) against electroconvulsions in mice. Aminophylline markedly decreased the anticonvulsant efficacy of almost all drugs studied, acetazolamide (40 and 80 mg/kg) and carbamazepine (30 mg/kg) being the only exceptions. The ethylenediamine component of aminophylline did not modify the anticonvulsant activity at all. These results seem to support the suggestion that aminophylline-induced blockade of adenosine receptors might be involved in the reversal of the protective action of at least some drugs studied. Regardless of the nature of the aminophylline-induced impairment in the anticonvulsant efficacy of a number of antiepileptic drugs, the use of methylxanthines in epileptic patients for the treatment of obstructive lung diseases should be avoided.

The muscarinic cholinergic agonist bethanechol (0.25-1.0 micrograms) injected bilaterally into various parts of the rat neostriatum induced a tonic electromyogram (EMG) activity in the gastrocnemius muscle which is considered to be a measure of limb rigidity. This tonic EMG activity was found to be dose-dependent and muscarine-specific since it could be blocked by coadministration of the muscarinic antagonist N-methylscopolamine (1.0 micrograms). Tonic EMG activity of comparable amount was observed after injections of bethanechol (1.0 microgram) into all regions of the neostriatum but not into the globus pallidus, thalamus, zona incerta or cortex. The tonic EMG activity induced by intrastriatal injection of bethanechol (1.0 microgram) was abolished by a subsequent injection of the GABAmimetic drug muscimol (25 ng) into the posterior part of the substantia nigra pars reticulata suggesting that bethanechol-induced limb rigidity is mediated via impairment of GABAergic transmission within the substantia nigra pars reticulata.

Aminophylline (50 and 100 mg/kg) and CGS 8216 (20 and 40 mg/kg) decreased the anticonvulsant potency of diazepam (5 and 10 mg/kg) against electroshock-induced seizures. It should be emphasized that aminophylline moderately affected the protective action of the benzodiazepine at a dose of 5 mg/kg, whereas it was equipotent with CGS 8216 with regard to diazepam at a dose of 10 mg/kg. Consequently, participation of a purinergic component in the anticonvulsant action of diazepam is suggested. On the other hand, the use of aminophylline in epileptic patients suffering from asthma seems unjustified.

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.

The effects of 2-chloroadenosine, aminophylline, bicuculline, beta-carboline-3-carboxylic acid methylester and Ro 15-1788 on seizures produced by pilocarpine were examined in rats. In animals pretreated with aminophylline at doses of 25-100 mg/kg, non-convulsant dose of pilocarpine, 100 mg/kg, resulted in severe motor limbic seizures, which rapidly developed into the status epilepticus. Electroencephalographic monitoring showed progressive evolution of seizure activity with initial high-voltage fast activity followed by high-voltage spiking and electrographic seizures. Morphological analysis of frontal forebrain sections with light microscopy demonstrated widespread damage to the hippocampal formation, thalamus, amygdala, olfactory cortex, substantia nigra and neocortex. Bicuculline, 2 mg/kg, beta-carboline-3-carboxylic acid methylester, 5 mg/kg, and Ro 15-1788, 50 mg/kg, did not augment seizures produced by pilocarpine, 100 mg/kg. 2-Chloroadenosine, 5 and 10 mg/kg, blocked the appearance of behavioral and electrographic seizures produced by pilocarpine, 380 mg/kg, and prevented the occurrence of brain damage. The results indicate that purinergic mechanisms are involved in the buildup of pilocarpine-induced convulsions and seizure-related brain damage in rats.