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Excitation-inhibition imbalance of GABAergic interneurons is predisposed to develop chronic temporal lobe epilepsy (TLE). We have previously shown that virtually every neuronal nitric oxide synthase (nNOS)-positive cell is a GABAergic inhibitory interneuron in the denate gyrus. The present study was designed to quantify the number of nNOS-containing hilar interneurons using stereology in pilocapine- and kainic acid (KA)-exposed transgenic adult mice that expressed GFP under the nNOS promoter. In addition, we studied the properties of miniature excitatory postsynaptic current (mEPSC) and paired-pulse response ratio (PPR) of evoked EPSC in nNOS interneurons using whole cell recording techniques. Results showed that there were fewer nNOS-immunoreactive interneurons of chronically epileptic animals. Importantly, patch-clamp recordings revealed reduction in mEPSC frequency, indicating diminished global excitatory input. In contrast, PPR of evoked EPSC following the granule cell layer stimulation was increased in epileptic animals suggesting reduced neurotransmitter release from granule cell input. In summary, we propose that impaired excitatory drive onto hippocampal nNOS interneurons may be implicated in the development of refractory epilepsy., (Copyright © 2021. Published by Elsevier B.V.)

The malformative cortical lesions in the cerebral cortex that are characteristic of focal cortical dysplasia type IIb (FCDIIb) and tuberous sclerosis complex (TSC) are well-recognized causes of chronic intractable epilepsy in children. Increasing evidence suggests that extracellular matrix molecules play important roles in epileptogenesis. Matrix metalloproteinase 9 (MMP9), a typical extracellular matrix proteolytic protease, has been shown to participate in the occurrence of seizures in experimental models. In the present study, we used immunoblotting to analyze the levels of MMP9 protein in FCDIIb lesions, TSC tubers and control samples, which included epileptic neocortices from temporal lobe epilepsy and non-epileptic normal cortices (CTX). The cellular distribution of MMP9 was further investigated by immunohistochemical methods. Our findings demonstrated the elevated levels of the inactive and active forms of MMP9 protein in FCDIIb and TSC lesions compared with CTX. Furthermore, the immunohistochemical results showed that MMP9 was characteristically expressed in the following misshapen cells: hypertrophic neurons, dysmorphic neurons, balloon cells and giant cells. Additionally, double immunofluorescent staining revealed that the reactive astrocytes, but not the microglia, expressed high levels of MMP9. Taken together, our findings suggest that the overexpression and spatial distribution patterns of MMP9 may be linked with the intractable epilepsy caused by FCDIIb and TSC., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Nitric oxide (NO) has been implicated in the neuronal hyperexcitability hence its involvement in the pathophysiology of epilepsy is clear. However, some studies indicate that NO has anticonvulsant effects while others present its convulsive effects. In the present study we tested the involvement of NO in pentylenetetrazol (Metrazol) induced Status Epilepticus (SE) rats, using the nonspecific inhibitor, N-omega-nitro-L-arginine methyl ester (L-NAME) and the neuronal-specific inhibitor, 7-nitroindazole (7N1). The effects of NOS (NO synthase) inhibitors were tested, within the seizures and between them, using the Multiparametric Assembly (MPA) which continuously monitored Cerebral Blood Flow (CBF) by Laser Doppler flowmetry, mitochondrial NADH redox state by the fluorometric technique, extracellular K(+) and H(+) levels using selective mini-electrodes and electrical activity (DC potential and ECoG) using special electrodes. Between seizures a trend of increase in CBF with oxidation of NADH was seen, with no change in K(+) and H(+) extracellular levels. Pre-treatment with L-NAME prevented this trend of increase in CBF whereas the injection of 7NI even decreased CBF between seizures. Within seizures, CBF increased and mitochondrial NADH was oxidized at the first seizures, while in the last seizure NADH was reduced. The use of NOS inhibitors significantly increased the degree of NADH oxidation at the latest convulsions. In conclusion our results demonstrated beneficial effect of NOS inhibitors on the brain cortex under SE induced by Metrazol, implying that they may serve as anticonvulsant drugs., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Aromatase is a key enzyme in estrogen biosynthesis that is involved in neuronal plasticity in the rodent hippocampus. Although aromatase mRNA expression has been detected in the human hippocampus, its cellular distribution has yet to be determined. Here, we have examined the immunohistochemical distribution of aromatase in the normal and the epileptic and sclerotic human hippocampus. In both the normal and epileptic hippocampus, aromatase was detected in numerous CA1-CA3 pyramidal neurons, in granule cells of the dentate gyrus and in interneurons that co-expressed the calcium-binding proteins calbindin, calretinin or parvalbumin. However, only a small subpopulation of astrocytes was immunoreactive for aromatase in either the normal and epileptic hippocampus. The widespread expression of aromatase in a large population of neurons in the normal and damaged hippocampus suggests that local estrogen formation may play an important role in human hippocampal function., (2009 Elsevier B.V. All rights reserved.)

Status epilepticus is a life-threatening form of seizure activity that represents a major medical emergency associated with significant morbidity and mortality. Protein Kinase A is an important regulator of synaptic strength that may play an important role in the development of status epilepticus-induced neuronal pathology. This study demonstrated an increase in PKA activity against exogenous and endogenous substrates during later stages of SE. As SE progressed, a significant increase in PKA-mediated phosphorylation of an exogenous peptide substrate was demonstrated in cortical structures. The increased activity was not due to altered expression of either regulatory or catalytic subunits of the enzyme. Through the use of phospho-specific antibodies, this study also investigated the effects of SE on the phosphorylation of the GluR1 subunit of the AMPA subtype of glutamate receptor. After the onset of continuous seizure activity, an increase in phosphorylation of the PKA site on the GluR1 subunit of the AMPA receptor was observed. These data suggest a potential mechanism by which SE may increase neuronal excitability in the cortex, potentially leading to maintenance of seizure activity or long-term neuronal pathology.

The sialic acid in the brain is split from sialoglucoconjugates by sialidases (neuraminidases, EC 3.2.1.18), and is postulated to act as an inhibitor of cellular adhesion and to play a role in various membrane functions. Since epilepsy alters cellular interactions and connectivity, it is reasonable to propose that sialidases can be affected by this pathological state or, alternately, by seizures. Therefore, we studied the activity of total, soluble, and membranal sialidases in various brain regions in normal, kindled epileptic and non-epileptic seizing rats. The results showed that in kindled rats, the total activity of the sialidases significantly decreased in cerebral cortex (11.38%) and cerebellum (28.58%), whereas it increased in brainstem (35.51%), hypothalamus (2.88%) and hippocampus (9.37%). The activity of the membranous sialidases in kindled rats followed the same pattern as the total activity, whereas the activity of soluble sialidase was significantly lower than membranous activity. Interestingly, the activity of total and membranal sialidases in non-epileptic seizing rats paralleled that observed in kindled rats. We suggest that the seizure-induced decrease of sialidasic activity may not modify the number of sialic acid molecules bound to gangliosides in cell membranes, as compared to areas of increased activity, that may decrease them. These changes in sialidases' activity may reflect functional disturbances of membrane polysialylated gangliosides related to the functional and anatomical plastic changes associated to seizures. Our data indicate that these changes are related to the presence of seizures rather than to an established epileptic state.

To identify the roles of pyridoxal kinase (PLK) in epileptogenesis and the recovery mechanisms in spontaneous seizure, a chronological and comparative analysis of PLK expression in the gerbil hippocampus was conducted. PLK immunoreactivity in a pre-seizure group of seizure sensitive (SS) gerbils was more strongly detected than that in a seizure resistant (SR) group. The density of PLK immunoreactivity in a 30-min postictal group was significantly lower than that of a pre-seizure group. In a 12 h postictal group, PLK immunodensity recovered to pre-seizure level. The over-expression of PLK in the hippocampus of pre-seizure SS gerbils suggests that PLP play an important role in the modulation of GAD activity and GABA reuptake as mediated by membrane transporter via neurons.

Testosterone's (T) anti-seizure effects may be mediated in part by actions of its 5alpha-reduced metabolites. To test this hypothesis, T was administered to knockout mice deficient in the 5alpha-reductase type I enzyme and wildtype controls and their ictal activity following pentylenetetrazole (PTZ; 85 mg/kg i.p.) was compared to mice administered vehicle. T to wildtype mice increased latencies to forelimb clonus, tonic clonic seizures, hindlimb extension, and death compared to that seen with vehicle administration. Moreover, incidence of tonic clonic seizures and hindlimb extension were reduced in wildtype mice administered T compared to vehicle-administered mice. T administration to wildtype mice reduced ictal activity compared to T to knockout mice, which were not different than vehicle-administered control mice. T to wildtype mice increased the latencies and decreased the incidence of forelimb clonus compared to T to knockout mice, which were not different from vehicle-administered mice. These data are consistent with T having anti-convulsant effects and that 5alpha-reduced metabolites may mitigate some of T's anti-seizure effects.

Expression of tyrosine hydroxylase (TH) immunostaining in the cerebellum was examined in dilute-lethal mice (DL) prior to and following the onset of ataxia. DL walked normally on postnatal days 7 and 8. Falling over when walking was exhibited by about 20% of DL on day 9 and by all DL by day 10. TH-positive Purkinje cells in lobules IX and X of the vermis of either ataxic or non-ataxic DL were clearly observed on day 9 when compared to control mice, and had drastically increased by day 10. These results revealed that abnormal TH expression occurred in some Purkinje cells of DL cerebella, preceding the onset of ataxia.

Nitric oxide synthase (NOS) activity was examined in forebrain, cerebellum and optic lobes of adult domestic fowl, having a hereditary primary generalized convulsive disorder. NOS was approximately 2-fold higher in only the forebrain of adult epileptic fowl compared to non-epileptic (carrier) hatchmates. A significant increase in NOS was also evident in forebrains of 1-day-old epileptic chicks. Ca(2+)-dependency experiments confirmed that these increments were principally due to type I NOS (NOS-I). Induction of convulsions by intermittent photic stimulation did not affect pre-existing forebrain NOS-I activity. The present data suggest that an enhanced NO signaling may ensue in selected regions of the brain as an adaptive response to hereditary epileptogenesis.

In previous studies we demonstrated that the membrane-associated protein kinase C (PKC) activities in the right and left hippocampus (HIPP) of rats kindled from the left HIPP increased significantly 4 weeks and 4 months after the last seizure compared with those in matched control rats. In this study, we investigated the long-lasting effect of HIPP-kindling on the membrane-associated activities of PKC subspecies in the bilateral HIPP 1 and 4 weeks after the last generalized kindled seizure had occurred. The membrane-associated activities of PKC subspecies were found to be subject to differential regulation. The activity of the alpha-subspecies was unchanged, whereas the respective activities of the beta- and gamma-subspecies in the kindled group increased significantly, compared with the controls, one (21%, P < 0.0001 for the beta-subspecies, and 23%, P < 0.001 for the gamma-subspecies) and 4 weeks (19%, P < 0.02 for the beta-subspecies, and 19%, P < 0.05 for the gamma-subspecies) after the last seizure. There were no significant differences in cytosolic PKC activity between the control and kindled groups for any subspecies examined at either time after the last seizure. These results suggest that activation of the PKC beta- and gamma-subspecies may play an important role in the enduring seizure susceptibility associated with kindling.

N-Acetylated-alpha-linked acidic dipeptidase (NAALADase) is a membrane-bound peptidase which hydrolyzes the endogenous neuropeptide N-acetylaspartylglutamate (NAAG) to N-acetylaspartate (NAA) and the excitatory amino acid, glutamate (Glu). Although there is evidence that NAAG might be a neurotransmitter, this dipeptide could also function as a precursor form of Glu, which is liberated by the dipeptidase. We found that the activity of this NAAG hydrolyzing enzyme in genetically epilepsy-prone rats was 11-26% greater than control in brain regions, including the amygdala, hippocampus and cerebellum, as well as the pyriform, entorhinal and frontal cortices. This is consistent with possible increased availability of Glu in certain CNS synapses in these rats, which are reported to have increased susceptibility to audiogenically, electrically and chemically induced convulsions.

Quinolinic acid (QUI), a structural analogue of neurotransmitters such as L-glutamate and L-aspartate, may act as an 'excitotoxin' when it is abundant in the brain. The compound has been causally related to various neurodegenerative disorders, including epilepsy. We tested the capacity of the brains of epilepsy-prone El mice to synthesize QUI. The activity of 3-hydroxyanthranilate 3,4-dioxygenase in the cerebral cortex of El mice was about 17 times that of ddY mice, the parent strain of El mice. The activity of this enzyme was undetectable in brains of BALB/cA mice and C3H/HeN mice. In El mice the sexes had comparable enzyme activity. The enzyme activity increased gradually as the animals aged. An injection of endotoxin caused a further increase in the enzyme activity. The enzyme activity in the spleen of El mice did not differ from that of ddY mice, and endotoxin did not affect the enzyme activity in the spleen. No strain-difference was observed in the activity of quinolinate phosphoribosyltransferase, a QUI-degrading enzyme, in the cerebral cortex. These results suggest that an increase in the synthesis of QUI in the brain is involved in the pathogenesis of epileptic seizures in El mice.

Protein kinase C (PKC) activity assayed by phosphorylation of exogenous histone, was measured in neocortex obtained from 32 patients following surgery for focal epilepsy and from 6 non-epileptic patients. PKC activity was not significantly different in either the particulate or cytosolic fraction from epileptic foci (n = 17) versus samples from non-spiking regions (n = 22) or neocortex from non-epileptic patients (n = 6). From 67% to 70% of total PKC activity was present in the cytosolic fraction. Phosphorylation of endogenous cytosolic substrate proteins was also not significantly different in epileptic foci.

We investigated the influence of milacemide, a glycinamide derivative with putative antiepileptic activity, on the K(+)-activation of Na+,K(+)-ATPase in bulk isolated glial cells and synaptosomes of control and epileptogenic cortex of cats with a chronic freeze lesion. In the primary and secondary epileptic foci of non-treated animals, glial Na+,K(+)-ATPase lost its physiological K(+)-activation, while the synaptosomal enzyme was unchanged. These data reproduced previous work done on the kinetic measurement of the enzymic activities. In treated animals (500 mg/kg milacemide given orally for 2 weeks after the freeze lesion), the glial enzyme showed a normal K(+)-activation in the epileptic foci. These results confirm the existence of an abnormal glial Na+,K(+)-ATPase in cold-induced focal epilepsy and suggest that the antiepileptic activity of milacemide might be secondary to an activation of glial Na+,K(+)-ATPase, contributing to antagonize ictal transformation and seizure spread.

Epileptic and normal Macaca mulatta monkey cortex was investigated using ligand binding techniques. Subpial injections of aluminum hydroxide gel into the left sensorimotor cortex produced stable seizure frequencies over a two year period and resulted in specific biochemical and receptor abnormalities. Pair matched CSF samples comparing epileptic and non-epileptic hemispheres showed a significant decreased GABA concentration over the epileptic side. The epileptic cortex demonstrated markedly reduced GABA receptor binding and diminished tissue GABA concentration and GAD activity. Two patterns of receptor loss were observed: nonspecific local cellular drop out involving multiple neurotransmitter receptors; and distal receptor loss which was specific for the neurotransmitter intervention pattern of the cortex. GABAergic receptor loss was more marked than receptor losses for the other neurotransmitter and was more widespread. Scatchard plot analysis demonstrated that the diminished GABAergic receptors within the focus were due to receptor loss and not affinity changes. Spearman rank correlations showed a significant correlation only between the degree of GABAergic receptor loss or decrease in GAD activity and the seizure frequency. Epilepsy appears to be a multifactoral disorder with multiple neuroreceptor abnormalities, the most notable of which are the destruction of GABAergic neurons and GABA receptors.

The characteristics of a glial Na+,K+-pump dependent on extracellular K+ within epileptogenic cortex were studied electrophysiologically, biochemically and histochemically in vitro using slices from cobalt-induced epileptogenic cortex of rat. When the extracellular K+ concentration ([K+]o) was varied between 4 and 40 mM, the mean slope of membrane potential plotted against [K+]o was about 57 mV in glia from the normal cortex (tissue A) and about 44 mV in glia from the epileptogenic cortex (tissue B); whereas no significant difference in the resting membrane potential of these tissues was observed. In glia from tissue B, a marked transient hyperpolarization above control level was caused by replacement of elevated [K+]o with the normal medium. Ouabain abolished these phenomena observed in glia from tissue B, but had no effect on the membrane potential during normal [K+]o. Reduction of extracellular Na+, Ca2+ and Cl- did not significantly affect the membrane potential of glia from either tissue. In tissue A, the cells marked by intracellular injection of horseradish peroxidase after intracellular recording were protoplasmic astrocytes; in tissue B, fibrous astrocytes with abnormal processes predominated. K+-dependent stimulation of Na+,K+-ATPase activity of the astrocyte-enriched fraction and its membrane preparation from tissue B was much larger than that from tissue A. A certain amount of the reaction product of K+-pNPPase activity was seen on glial plasma membrane within tissue B but not on that from tissue A. The above findings suggest that a glial Na+,K+-pump within actively firing epileptogenic cortex may be modified to increase in its activity.

The activities of GABA-transaminase (GABA-T) were examined in several brain regions of amygdala-kindled rats, pretreated either with or without gabaculine, an irreversible GABA-T inhibitor. Histochemical and biochemical studies demonstrated that GABA-T activities decreased significantly in some brain regions 16 h after the gabaculine treatment. In contrast, no such alteration was detected in kindled animals after a 48-h survival period either with or without the pharmacological manipulation. The present results suggest that kindling causes retardation of GABA-T resynthesis in neurons, since the GABA-T activities detected 16 h after the drug treatment are due to newly synthetized enzyme in presumptive GABA neurons but not glial cells.