Hyperexcitability induced by GABA withdrawal facilitates hippocampal long-term potentiation.

In some mammals, epileptic seizures have been induced in the cerebral cortex, hippocampus and other limbic structures after the sudden suppression of chronically infused GABA. This hyperexcitability state induced by the endogenous neurotransmitter resembles the withdrawal seizure-responses to other GABA(A) receptor agonists such as benzodiazepines, barbiturates and alcohol. Hyperexcitability induced by GABA withdrawal also persists in in vitro preparation. Hippocampal slices, obtained from rats with seizures induced by GABA-withdrawal showed field potential oscillations and paroxysmal activity in the Ammon's horn region 1. During GABA-withdrawal hyperexcitability the threshold of hippocampal long-term potentiation (LTP) decreased to a point in which a brief frequency stimulation that normally failed to produce long lasting changes in synaptic strength, was now able to induce LTP. Facilitation of the LTP induction was associated with a decreased GABA(A)-mediated inhibitory activity, because the effect of the GABA(A) receptor antagonist, bicuculline, was occluded during hyperexcitability and the dose-response curve for bicuculline showed a 50% efficacy reduction with a shift in the effective concentration required for half-maximal activation from 4.5-1.1 microM relative to controls. Nevertheless, the dissociation constant of the antagonist did not change significantly. Our results support the idea that changes in hippocampal plasticity under altered inhibitory neurotransmission states, like those induced by withdrawal syndromes to anxiolytic, sedative or anticonvulsant drugs may be engaged during seizures.

Hippocampal hyperexcitability induced by GABA withdrawal is due to down-regulation of GABA(A) receptors.

The sudden interruption of an intracortical instillation of exogenous gamma-aminobutyric acid (GABA) generates an epileptic focus in mammals. Seizures elicited by GABA withdrawal (GW) last for weeks. A similar withdrawal-induced hyperexcitability is also produced by several GABA(A) receptor agonists. This work reports a quantitative analysis of GW-induced hyperexcitability produced in the hippocampus in vitro. GW produced a left-ward displacement of the input/output (I/O) function, suggesting that the postsynaptic component is predominant to explain the hyperexcitability. A decrease in the inhibitory efficacy of the GABA(A) receptor agonist, muscimol, confirmed that inhibition was impaired. Binding saturation experiments demonstrated a decrease in [(3)H]-muscimol binding after GABA withdrawal showing a close correlation with the development of hyperexcitability. All these modifications coursed without changes in receptor affinity (K(D)) for muscimol or bicuculline as demonstrated by both binding studies and Schild analysis. It is concluded that, in the CA1 region of the hippocampus, it is the number of functional GABA(A) receptors, and not the affinity of the receptor, what is decreased during GW-induced hyperexcitability.

Long-lasting effects of GABA infusion into the cerebral cortex of the rat.

In electrophysiological terms, experimental models of durable information storage in the brain include long-term potentiation (LTP), long-term depression, and kindling. Protein synthesis correlates with these enduring processes. We propose a fourth example of long-lasting information storage in the brain, which we call the GABA-withdrawal syndrome (GWS). In rats, withdrawal of a chronic intracortical infusion of GABA, a ubiquitous inhibitory neurotransmitter, induced epileptogenesis at the infusion site. This overt GWS lasted for days. Anisomycin, a protein synthesis inhibitor, prevented the appearance of GWS in vivo. Hippocampal and neocortical slices showed a similar post-GABA hyperexcitability in vitro and an enhanced susceptibility to LTP induction. One to four months after the epileptic behavior disappeared, systemic administration of a subconvulsant dose of pentylenetetrazol produced the reappearance of paroxysmal activity. The long-lasting effects of tonic GABAA receptor stimulation may be involved in long-term information storage processes at the cortical level, whereas the cessation of GABAA receptor stimulation may be involved in chronic pathological conditions, such as epilepsy. Furthermore, we propose that GWS may represent a common key factor in the addiction to GABAergic agents (for example, barbiturates, benzodiazepines, and ethanol). GWS represents a novel form of neurono-glial plasticity. The mechanisms of this phenomenon remain to be understood.

Neocortical hyperexcitability after GABA withdrawal in vitro.

The sharp interruption of the intracortical instillation of exogenous gamma-aminobutyric acid (GABA), generates an epileptic focus in mammals. Seizures elicited by GABA withdrawal last several days or weeks. The present work reports that GABA withdrawal-induced hyperexcitability can be produced in vitro: a sudden withdrawal of GABA (5 mM; 120 min) or benzodiazepine (60 microM flunitrazepam) from the superfusion, induced a gradual increase in the amplitude of the evoked population spike (PS) recorded on neocortical slices. PS enhancement reached 150% above the control value 2.5 h after GABA withdrawal. GABA withdrawal-induced hyperexcitability was facilitated by progesterone. PS enhancement induced by GABA withdrawal was associated with an impairment of GABA transmission occurring before epileptiform discharges were fully established. Paired pulse inhibition and evoked [3H]-GABA release appear decreased; suggesting that cortical hyperexcitability as a result of GABA withdrawal involves pre-synaptic changes. Specific muscimol binding decreased during GABA superfusion but recovered after GABA withdrawal. However, the sensitivity of the post-synaptic response to 3alpha-OH-5alpha-pregnan-20-one or allopregnanolone (alloP) was enhanced after GABA withdrawal, suggesting a functional change in the GABA(A) receptors. The changes described may be the cellular correlates of the withdrawal syndromes appearing after interruption of the administration of GABA(A) receptor agonists.

Susceptibility to focal and generalized seizures in Wistar rats with genetic absence-like epilepsy.

The susceptibility to develop cortically induced focal and generalized seizures was examined in Genetic Absence Epilepsy Rats from Strasbourg (GAERS), an inbred strain of Wistar rats with absence epilepsy. A GABA-withdrawal syndrome induced after suppression of a 2-h intracortical GABA infusion was used as a model of focal epileptogenesis: localized cortical discharges appear at the infusion site within 1 h. GAERS were more prone to develop a GABA-withdrawal syndrome than non-epileptic inbred controls and non-selected Wistar rats. After a transient suppression of absence seizures following GABA infusion in GAERS, generalized spike-and-wave discharges and focal spikes were recorded simultaneously in the cortex. GAERS also showed a higher incidence of systemic pentylenetetrazol-induced convulsions at the dose of 25 mg/kg. Higher doses had similar convulsant effects in all groups. In conclusion, the results confirm a genetic susceptibility in GAERS and/or resistance in inbred non-epileptic rats to focal and generalized seizures involving the cortex. Rats with absence epilepsy appear to be more prone to seizures elicited by cortical GABA deficiency.

Functional recovery from cortical hemiplegia in the rat: effects of a callosotomy.

The present work aimed at studying the participation of the homologous contralateral zone to a unilateral somatomotor cortex lesion, once the animals had showed a significant functional recovery. We studied recovery of coordinated walking after unilateral motor cortex aspiration in rats. A callosotomy was performed 20 days after the initial lesion, without significant effects. We conclude that after this time period, the intact hemisphere plays no role in the recovery process, suggesting that at this time point recovery does not depend on the integrity of corpus callosal fibers at this rostral-caudal level.

Ethanol, GABA and epilepsy.

Ethanol exerts its behavioral effects largely by interacting with receptors to brain neurotransmitters. The molecular mechanisms involving these interactions are still not well known since an ideal model for their study is currently unavailable. In addition, responses to alcohol may vary due to factors such as genetic predisposition, ethanol concentration consumed, and stimuli such as stress, socialization, etc. The chronic consumption of alcohol, similar to that of other drugs such as benzodiazepines and barbiturates, is linked to GABAergic neurotransmission. GABA is the predominant inhibitory neurotransmitter in the brain. In a context of substance abuse, these three drugs first cause a gratifying effect, later tolerance and finally, physical and psychological dependence. If consumption is interrupted abruptly, a withdrawal syndrome occurs. The Alcohol Withdrawal Syndrome (AWS) is a state of hyperexcitability characterized by anxiety, fear, muscular rigidity and tonic-clonic seizures with epileptiform-type characteristics. The epileptic seizures seen during AWS are often similar to those seen in experimental epilepsy models such as "kindling" or GABA Withdrawal Syndrome (GWS) models. A possible correlation between these models and AWS will allow for a better understanding of the cellular and molecular effects that alcohol exerts on the brain.

Absence seizures decrease steroid modulation of t-[35S]butylbicyclophosphorothionate binding in thalamic relay nuclei.

Interaction of gamma-aminobutyric acid (GABA), pentobarbital and two neuroactive steroids on t-butylbicyclophosphorothionate ([35S]TBPS) binding to GABAA receptors in thalamus was studied during absence seizures. In control brain sections, the steroids alphaxalone and tetrahydrodeoxycorticosterone (at low 0.1-1 microM concentrations) increased [35S]TBPS binding in thalamic relay nuclei. Both GABA and pentobarbital dose-dependently decreased [35S]TBPS binding in these nuclei. A significant decrease in the ability of steroids to increase [35S]TBPS binding in thalamic relay nuclei was observed during absence seizures induced by gamma-hydroxybutyric acid (GHB). This loss of steroid effect on binding was 1) selective to steroids only as GABA and pentobarbital modulation of [35S]TBPS binding in these nuclei did not change significantly and 2) not causally related to the generation of GHB-induced absence seizures as it was not observed at the onset of GHB-seizures but developed 30 min after the seizure-onset. We tested whether absence seizures were critical for the development of this loss of steroid effect on [35S]TBPS binding in thalamic relay nuclei. The ability of the steroids to increase [35S]TBPS binding in relay nuclei was preserved when GHB-seizures were blocked. When the duration of GHB-seizures was prolonged, the loss of steroid effect on [35S]TBPS binding in thalamus persisted throughout the seizure-duration. These findings suggest that absence seizures cause a rapid loss of steroid effect on [35S]TBPS binding to GABAA receptors in thalamic relay nuclei.

[Neurosteroids. Neuromodulators of cerebral excitability]. / Neuroesteroides. Neuromoduladores de la excitabilidad cerebral.

Steroids which are produced by the brain are called neurosteroids, and they are able to modulate neurotransmissions: GABAergic; glutamatergic; glycinergic, and cholinergic (nicotine receptor). These effects are of short latency and duration, and do not implicate the cellular genome. The interaction of these neurosteroids with membrane receptors contribute to the regulation of neuronal excitability, and their study has allowed a better understanding of cognitive, hormonal, and epileptic phenomena as well as the development of new drugs with anxiolytic, antidepressive, anesthetic and anti-epileptic effects.

Alterations in GABAA receptor alpha 1 and alpha 4 subunit mRNA levels in thalamic relay nuclei following absence-like seizures in rats.

Modification of GABAA receptor mRNA levels by seizure activity can regulate general neuronal excitability. The possibility of absence seizure-induced alteration in GABAA receptor alpha 1, alpha 4, beta 2, and gamma 2 subunit gene expression in thalamic relay nuclei was studied in a rat model of absence seizures induced by gamma-hydroxybutyric acid (GHB). We observed a marked increase in alpha 1 mRNA and a corresponding decrease in alpha 4 mRNA in thalamic relay nuclei 2-4 h after the onset of GHB-induced absence seizures (when the seizures were terminating). These changes were selective to these alpha isoforms as neither beta 2 nor gamma 2 mRNA changed following seizures and occurred only in thalamic relay nuclei but not in hippocampus, a structure from which absence seizures do not evolve. The alterations in alpha 1 and alpha 4 mRNA persisted until about 12 h, and by 24 h after the seizure-onset the mRNA levels normalized. Blocking GHB-seizures produced no change in the levels of alpha 1 and alpha 4 mRNA in thalamic relay nuclei, suggesting that seizures themselves were responsible for mRNA alterations. In order to determine if absence seizure-induced changes in alpha 1 and alpha 4 mRNA had any physiological significance, GHB was readministered in rats 6 and 24 h after the onset of seizures. The total duration of GHB-seizures was found to be significantly decreased when GHB was readministered at 6 h but not 24 h after the seizure-onset. These results suggest that absence seizures regulate GABAA receptor alpha 1 and alpha 4 gene expression in thalamic relay nuclei as a compensatory mechanism by which absence seizures are terminated.

Motor function in young and aged hemiplegic rats: effects of a Ginkgo biloba extract.

We have previously shown beneficial effects of a Ginkgo biloba extract (EGb761-IPSEN) in accelerating functional recovery from hemiplegia induced by unilateral motor cortex ablation. Here, we report the behavioral and histological effects of various dose regimes of EGb761. In young rats (3 months), 10 mg/kg/day for 7 days produced an improvement in motor performance, relative to untreated controls, on the last day of treatment. Applying a priming (P)-maintenance (M) dose regime (P-7 = 7 days, M-21 = 21 days), a P-7 of 50 (all doses expressed in mg/kg/day) and a M-21 of 10 promoted recovery from the second day after surgery. However, in aged rats (26-28 months old) this treatment ameliorated motor performance only after the 10th day of treatment. A P-7 of 100 or 200 and a M-21 of 50 or 100 produced an acceleration of behavioral recovery in aged animals. Improvement was evident by the fifth day of treatment and was maintained after the treatment regimen. These two groups also demonstrated reduced glial fibrillary acid protein (GFAP) immunostaining and ex vacuo hydrocephalus. Thus, the confirmed efficacy of EGb in hemiplegic rats can be enhanced by an appropriate posology.

Effects of GABAB receptor antagonists on two models of focal epileptogenesis.

The acute effects of two GABAB receptor antagonists (phaclofen and CGP-35348) were studied in two types of epileptogenic activity: that produced by intracortical injections of baclofen and that appearing after withdrawal of chronic intracerebral GABA infusion (the GABA-withdrawal syndrome, GWS). Intracortical baclofen induced two types of electrographic paroxysmal discharges: one consisting of single spike-and-wave (pattern I) and another of polyspike-and-wave patterns (pattern II). Both patterns showed similar latencies and temporal evolution of spike frequency discharges. Phaclofen, applied directly into the baclofen-induced epileptogenic focus, suppressed pattern II but was ineffective in modifying both pattern I and the GWS. CGP-35348, administered systemically, inhibited both patterns I and II. Intracortical microinjection of baclofen or phaclofen in rats showing a GWS had no effect, nor the systematically given CGP 35348. These results indicate a differential participation of GABAB receptors in GABA-related epileptic syndromes of cortical origin.

Allopregnanolone potentiates a GABA-withdrawal syndrome in the rat cerebral cortex.

We have studied the neuromodulatory effect of the neurosteroid 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone-3 alpha-5 alpha P-) in the GABA-withdrawal syndrome (GWS). This is a model of partial epilepsy consisting of an enduring paraoxysmal activity recorded at the site of GABA infusion that depends, for its induction, on GABA receptor activation. Rats were chronically implanted for frontal and occipital EEG recording with infusion cannulae fixed on the somatomotor cortical region. When the neurosteroid was infused after or concurrently with GABA, a potentiation of the GWS (i.e. shorter latency and prolonged duration) was observed. No modifications in EEG activity were detected when allopregnanolone was administered alone or prior to GABA administration. These results indicate a neuromodulatory effect of allopregnanolone, dependent on the presence of GABA at the receptor site.

Acceleration of functional recovery from motor cortex ablation by two Ginkgo biloba extracts in rats.

We studied the effects of two extracts of Ginkgo biloba, with and without terpenes, on motor recovery from cortical hemiplegia. Both extracts of the reference product (EGb761-IPSEN) produced a dose-dependent acceleration of behavioral recovery and diminished ventricular dilation in lesion rats. These results indicate that the active substance(s) participating in the beneficial effect of EGb761 is (are) contained in the non-terpenic fraction of the extract.

Decrease of glutamate decarboxylase activity after in vivo cortical infusion of gamma-aminobutyric acid.

gamma-Aminobutyric acid (GABA) levels and the activity of glutamate decarboxylase were measured in homogenates of rat brain cortical tissue, at different times after chronic intracortical infusion of GABA in vivo during 2, 6 or 24 h. Cortical electrical activity was also recorded. As previously described, about 1 h after cessation of the infusion epileptic discharges were observed (GABA-withdrawal syndrome), which lasted for several days. At zero time after cessation of the infusion, before the appearance of seizures, GABA levels were increased 3-6-fold and glutamate decarboxylase activity was decreased 27-48% in the infused cortex, as compared to the contralateral cortex or to tissue from control intact rats. During epileptic discharges GABA levels gradually returned to normal values. In contrast, glutamate decarboxylase activity remained decreased during seizures and returned to normal only after recovery from the GABA-withdrawal syndrome. These results suggest that the persistent decrease in the activity of the decarboxylase is due probably to a lowered amount of the enzymatic protein, occurring as a consequence of a temporarily elevated intracellular GABA concentration. The decreased rate of GABA synthesis might be involved in the pathophysiology of the GABA-withdrawal syndrome.

Chronic infusions of GABA into the medial prefrontal cortex induce spatial alternation deficits in aged rats.

It has been proposed that functions associated with the prefrontal cortex could change as a consequence of aging. Previous experiments in young rats have demonstrated that anatomical lesions or chronic GABA infusions into this area produce deficits in spatial delayed alternation tasks. The present study examines the effect of chronic (7 days) GABA or saline infusion into the prefrontal cortex on the performance of delayed alternation task in old rats (24 months). The results suggested that aged rats needed more sessions to acquire the delayed alternation task. GABA infusions into the prefrontal cortex produced deficits in spatial alternation tasks similar to those previously observed in young rats. Performance rapidly recovered after the infusion period. Histological analysis showed similar lesion size in both groups. The results suggest that aged prefrontal cortex and/or related areas participating in the acquisition of the delayed alternation task are more sensitive to aging processes. Furthermore, the prefrontal cortex is important for the retention of a previously learned spatial delayed alternation task. The structures involved in functional recovery from these deficits appear to be fully functional in aged rats.

Hyperexcitability of hippocampal CA1 region in brain slices after GABA withdrawal.

The interruption of GABA infusion in the cerebral cortex and in the hippocampus produces electrographic seizures in rats. Here, we have used the hippocampal slice preparation to induce a 'GABA withdrawal syndrome (GWS)'. With the stimulation parameters used (0.2 Hz, 200 microseconds), activation of the Schaffer afferents produced one population spike in the CA1 subfield, while multiple population spikes were observed in the slices previously incubated in GABA. Also, we recorded an increase in the amplitude of the population spike when compared to its control value. Paired pulse test showed absence of recurrent inhibition in these slices. These results suggest a dysfunction in GABAergic neurotransmission.

Effects of 3-hydroxy,3-ethyl,3-phenylpropionamide (HEPP) on rat models of generalized and focal epilepsy.

The GABA withdrawal syndrome (GWS) is a new model of focal epilepsy in which paroxysmal activity is induced through the interruption of a chronic, intracortical infusion of GABA. Preliminary studies have shown extraordinary resistance of this epileptogenic activity to classic anticonvulsants including diazepam, the most effective agent for treating status epilepticus. However, GWS can be inhibited by GABA itself. The rat with petit mal-like seizures is a genetic model of generalized non-convulsive epilepsy (GNCE), with behavioral characteristics and electrical (spike-and-wave discharges) signs resembling absences. Moreover, GABAmimetics aggravate this type of seizure. Rats with GWS induced by cessation of a localized GABA infusion (50 micrograms/microliters/h for 24 h), and the rat model of GNCE, were treated with HEPP, a new anticonvulsant agent. In the case of GWS, the drug produced a significant decrease of focal spike activity in animals which started discharging at low frequencies while in rats with higher frequency discharge, HEPP was without effect. HEPP administered on the second day of the GWS in naive rats had no effect. In rats with GNCE, doses of 50 and 100 mg/kg i.p. blocked the spike-and-wave discharges. The higher dose produced sedation in this absence seizures model. Although the mechanism of action of HEPP is still unknown, its unique antiepileptic profile deserves further studies.