Jakub Otáhal

Gamma Oscillations and Spontaneous Network Activity in the Hippocampus Are Highly Sensitive to Decreases in pO2 and Concomitant Changes in Mitochondrial Redox State

Gamma oscillations have been implicated in higher cognitive processes and might critically depend on proper mitochondrial function. Using electrophysiology, oxygen sensor microelectrode, and imaging techniques, we investigated the interactions of neuronal activity, interstitial pO2, and mitochondrial redox state [NAD(P)H and FAD (flavin adenine dinucleotide) fluorescence] in the CA3 subfield of organotypic hippocampal slice cultures. We find that gamma oscillations and spontaneous network activity decrease significantly at pO2 levels that do not affect neuronal population responses as elicited by moderate electrical stimuli. Moreover, pO2 and mitochondrial redox states are tightly coupled, and electrical stimuli reveal transient alterations of redox responses when pO2 decreases within the normoxic range. Finally, evoked redox responses are distinct in somatic and synaptic neuronal compartments and show different sensitivity to changes in pO2. We conclude that the threshold of interstitial pO2 for robust CA3 network activities and required mitochondrial function is clearly above the “critical” value, which causes spreading depression as a result of generalized energy failure. Our study highlights the importance of a functional understanding of mitochondria and their implications on activities of individual neurons and neuronal networks.

Endogenous Nitric Oxide Is a Key Promoting Factor for Initiation of Seizure-Like Events in Hippocampal and Entorhinal Cortex Slices

Nitric oxide (NO) modulates synaptic transmission, and its level is elevated during epileptic activity in animal models of epilepsy. However, the role of NO for development and maintenance of epileptic activity is controversial. We studied this aspect in rat organotypic hippocampal slice cultures and acute hippocampal–entorhinal cortex slices from wild-type and neuronal NO synthase (nNOS) knock-out mice combining electrophysiological and fluorescence imaging techniques. Slice cultures contained nNOS-positive neurons and an elaborated network of nNOS-positive fibers. Lowering of extracellular Mg2+ concentration led to development of epileptiform activity and increased NO formation as revealed by NO-selective probes, 4-amino-5-methylamino-2′,7′-difluorofluorescein and 1,2-diaminoanthraquinone sulfate. NO deprivation by NOS inhibitors and NO scavengers caused depression of both EPSCs and IPSCs and prevented initiation of seizure-like events (SLEs) in 75% of slice cultures and 100% of hippocampal–entorhinal cortex slices. This effect was independent of the guanylyl cyclase/cGMP pathway. Suppression of SLE initiation in acute slices from mice was achieved by both the broad-spectrum NOS inhibitor N-methyl-l-arginine acetate and the nNOS-selective inhibitor 7-nitroindazole, whereas inhibition of inducible NOS by aminoguanidine was ineffective, suggesting that nNOS activity was crucial for SLE initiation. Additional evidence was obtained from knock-out animals because SLEs developed in a significantly lower percentage of slices from nNOS−/− mice and showed different characteristics, such as prolongation of onset latency and higher variability of SLE intervals. We conclude that enhancement of synaptic transmission by NO under epileptic conditions represents a positive feedback mechanism for the initiation of seizure-like events.

Five percent CO2 is a potent, fast-acting inhalation anticonvulsant

Purpose:  CO2 has been long recognized for its anticonvulsant properties. We aimed to determine whether inhaling 5% CO2 can be used to suppress seizures in epilepsy patients. The effect of CO2 on cortical epileptic activity accompanying behavioral seizures was studied in rats and nonhuman primates, and based on these data, preliminary tests were carried out in humans.

Seizures induced in immature rats by homocysteic acid and the associated brain damage are prevented by group II metabotropic glutamate receptor agonist (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate.

The present study has examined the anticonvulsant and neuroprotective effect of group II metabotropic glutamate receptor (mGluR) agonist (2R,4R)-4-aminopyrrolidine-2,4-dicarboxylate (2R,4R-APDC) in the model of seizures induced in immature 12-day-old rats by bilateral intracerebroventricular infusion of dl-homocysteic acid (DL-HCA, 600 nmol/side). For biochemical analyses, rat pups were sacrificed during generalized clonic-tonic seizures, approximately 45-50 min after infusion. Comparable time intervals were used for sacrificing the pups which had received 2R,4R-APDC. Low doses of 2R,4R-APDC (0.05 nmol/side) provided a pronounced anticonvulsant effect which was abolished by pretreatment with a selective group II mGluR antagonist LY341495. Generalized clonic-tonic seizures were completely suppressed and cortical energy metabolite changes which normally accompany these seizures were either normalized (decrease of glucose and glycogen) or markedly reduced (an accumulation of lactate). EEG recordings support the marked anticonvulsant effect of 2R,4R-APDC, nevertheless, this was only partial. In spite of the absence of obvious motor phenomena, isolated spikes or even short periods of partial ictal activity could be observed. Isolated spikes could also be seen in some animals after application of 2R,4R-APDC alone, reflecting most likely subclinical proconvulsant activity of this agonist. The neuroprotective effect of 2R,4R-APDC was evaluated after 24 h and 6 days of survival following DL-HCA-induced seizures. Massive neuronal degeneration, as revealed by Fluoro-Jade B staining, was observed in a number of brain regions following infusion of DL-HCA alone (seizure group), whereas 2R,4R-APDC pretreatment provided substantial neuroprotection. The present findings support the possibility that group II mGluRs are a promising target for a novel approach to treating epilepsy.

Anticonvulsant and neuroprotective effect of (S)-3,4-dicarboxyphenylglycine against seizures induced in immature rats by homocysteic acid

The present study has examined the anticonvulsant and neuroprotective effect of (S)-3,4-dicarboxyphenylglycine ((S)-3,4-DCPG), a highly selective agonist for subtype 8 of group III metabotropic glutamate receptors (mGluRs), against seizures induced in immature 12-day-old rats by bilateral icv infusion of DL-homocysteic acid (DL-HCA, 600 nmol/side). For biochemical analyses, rat pups were sacrificed during generalized clonic-tonic seizures, approximately 45-50 min after infusion. Comparable time intervals were used for sacrificing the animals which had received (S)-3,4-DCPG (0.25 nmol/each side, 15-20 min prior to infusion of DL-HCA or saline). This agonist provided a pronounced anticonvulsant effect, generalized clonic-tonic seizures were completely suppressed and cortical energy metabolite changes which normally accompany these seizures were either normalized (decrease of glucose and glycogen) or markedly reduced (an accumulation of lactate). Anticonvulsant effect of (S)-3,4-DCPG was also evident from the EEG recordings, nevertheless, it was not complete. In spite of the absence of obvious motor phenomena, sporadic ictal activity could be seen in some animals. Isolated spikes could also be observed in some animals after administration of (S)-3,4-DCPG alone. The neuroprotective effect of (S)-3,4-DCPG was evaluated after 24 h and 6 days of survival following DL-HCA-induced seizures. Massive neuronal degeneration was observed in a number of brain regions following infusion of DL-HCA alone (seizure group), whereas pretreatment with (S)-3,4-DCPG provided substantial neuroprotection. The present findings suggest that receptor subtype 8 of group III mGluRs may be considered a promising target for drug therapy in childhood epilepsies in the future.

Degenerative neuronal changes in the rat thalamus induced by status epilepticus at different developmental stages

SE was induced in Wistar rats at post-natal (P) days 12, 15, 18, 21, and 25 to determine distribution and severity of thalamic damage in relation to time after SE. Six different intervals from 4 h up to 1 week were studied using Fluoro-Jade B (FJB) staining. Severity of damage was semi-quantified for every age-and-interval group. Distribution of neuronal damage within various thalamic nuclei was mapped by a computer-aided digitizing system. A consistent neuronal damage occurred in functionally heterogenous thalamic nuclei. Damage was found in all age groups although its extension and time course as well as the number of involved thalamic nuclei varied. Number of injured thalamic nuclei rapidly increased with age on SE-onset. In P12 group, degenerating neurons were consistently seen in the mediodorsal and lateral dorsal thalamic nuclei. Since P15, neurodegeneration was observed additionally in midline, ventral and caudal thalamic nuclei (visual and auditory thalamic nuclei), in the lateral posterior and in the reticular nucleus. In P21 and P25 animals, the majority of thalamic nuclei exhibited marked neuronal damage. Nuclei with a small number (anterior and intralaminar) or no FJB-positive neurons (the ventral nucleus of the lateral geniculate body) were exceptional. The pattern of thalamic damage is age-specific; its extent and severity increases with age.

Intrahippocampal injection of endothelin-1 in immature rats results in neuronal death, development of epilepsy and behavioral abnormalities later in life

The direct injection of endothelin‐1 (ET‐1) into brain parenchyma was recently suggested as a suitable model of stroke. The present study was designed to assess whether intrahippocampal injection of ET‐1 in immature rats causes neurodegeneration and immediate seizures, and results in impairment of motor development, cognitive decline, epilepsy and chronic hippocampal lesion. ET‐1 was injected unilaterally into the dorsal hippocampus in doses of 20 or 40 pmol at the age of 12 (P12) or 25 (P25) days. Video‐electroencephalographic monitoring performed during 100 min after the injection of ET‐1 demonstrated the development of convulsive epileptic seizures in 75–100% of animals of individual age‐and‐dose groups. Long‐term behavioral follow‐up did not reveal impairment of motor development in any dose‐and‐age group. At 2 months after ET‐1 injection, impairment of spatial memory occurred only in rats with 40 pmol of ET‐1 at P12. At 3 months after ET‐1 injection spontaneous electrographic seizures occurred in 62.5–100% animals of both ages with no relation to the dose used. Seizures were always non‐convulsive. The total seizure duration per 24 h was higher in the P12 than the P25 group, suggesting more severe epilepsy. The extent of the hippocampal lesion increased with the dose of ET‐1 and was significantly higher in the P12 than the P25 group. The severity of the ET‐1‐induced lesion correlated positively with total seizure duration per 24 h at both ages. Our results document that early intrahippocampal injection of ET‐1 results in lesion development and both immediate seizures and chronic epilepsy in either age group. Cognitive impairment occurred only in rats with ET‐1 injection at P12.

Brain superoxide anion formation in immature rats during seizures: Protection by selected compounds

The widely-held assumption was that oxidative stress does not occur during seizures in the immature brain. The major finding of the present study concerns evidence of oxidative stress in the brain of immature rats during seizures induced by DL-homocysteic acid. Seizures were induced in 12-day-old rats by bilateral intracerebroventricular infusion of DL-homocysteic acid (DL-HCA, 600 nmol/side) and oxidative stress was evaluated by in situ detection of superoxide anion (O(2)·(-)). Using hydroethidine (Het) method, the fluorescent signal of the oxidized products of Het (reflecting O(2)·(-) production) significantly increased (by 50%-60%) following 60 min lasting seizures in all the studied structures, namely CA1, CA3 and dentate gyrus of the hippocampus, cerebral cortex and thalamus. The enhanced O(2)·(-) production was substantially attenuated or completely prevented by substances providing an anticonvulsant effect, namely by a competitive NMDA receptor antagonist AP7, a highly selective and potent group II metabotropic glutamate receptor (mGluR) agonist 2R,4R-APDC and highly selective group III mGluR, subtype 8 agonist (S)-3,4-DCPG. Complete protection was achieved by two SOD mimetics Tempol and MnTMPYP which strongly suggest that the increased fluorescent signal reflects O(2)·(-) formation. In addition, both scavengers provided a partial protection against brain damage associated with the present model of seizures. Signs of neuronal degeneration, as evaluated by Fluoro-Jade B staining, were detected at 4h following the onset of seizures. The present findings thus suggest that the increased superoxide generation precedes neuronal degeneration and may thus play a causative role in neuronal injury. Occurrence of oxidative stress in brain of immature rats during seizures, as demonstrated in the present study, can have a clinical relevance for a novel approach to the treatment of epilepsy in children, suggesting that substances with antioxidant properties combined with the conventional therapies might provide a beneficial effect.

Changes in Cytochrome Oxidase in the Piriform Cortex after Status Epilepticus in Adult Rats

Summary:  Purpose: The piriform cortex is involved in genesis and propagation of temporal lobe seizures. Degenerating neurons demonstrated by FluoroJade B staining are visible early after status epilepticus (SE) as well as after longer intervals. Furthermore, the piriform cortex is activated during an early phase of experimental temporal seizures, as described by magnetic resonance imaging (MRI) studies. It indicates that the early activity of the piriform cortex should be accompanied by increased adenosine triphosphate (ATP) production. Cytochrome oxidase activity in the brain may be used as an endogenous metabolic marker for neurons. The present research studied activity of the cytochrome oxidase separately in the rostral and caudal parts of the piriform cortex after lithium chloride–pilocarpine–induced SE in adult rats.

Effect of free radical spin trap N-tert-butyl-α-phenylnitrone (PBN) on seizures induced in immature rats by homocysteic acid

The present study has examined the effect of free radical spin trap N-tert-butyl-alpha-phenylnitrone (PBN) in the model of seizures induced in immature 12-day-old rats by bilateral intracerebroventricular infusion of dl-homocysteic acid (dl-HCA, 600 nmol/side). PBN was given i.p. in two doses (100 mg/kg each), 30 min prior and 30 min after dl-HCA infusion. PBN did not significantly influence the severity of seizures, evident both from the behavioral symptoms and EEG recordings. PBN normalized decreased ATP levels in the hippocampus, occurring during the acute phase of seizures ( approximately 45-50 min after infusion) and persisting until the end of the 24-h recovery period. PBN also led to normalization of decreased glucose levels and to a significant reduction of lactate accumulation in the cerebral cortex and hippocampus. The neuroprotective effect of PBN was evaluated after 24 h and 6 days of survival following dl-HCA-induced seizures (Nissl and Fluoro-Jade B staining). The administration of PBN resulted in a partial amelioration of severe damage observed in many brain regions following infusion of dl-HCA alone. The data suggest that increased free radical production is apparently occurring during seizures induced in immature rats by homocysteic acid. Free radical scavenger PBN had a clear-cut protective effect, evident as the improved recovery of brain energy status and as a partial, but significant, attenuation of neuronal degeneration associated with this model of seizures.