PICK1 facilitates lasting reduction in GluA2 concentration in the hippocampus during chronic epilepsy.

Overstimulation of glutamate receptors resulting in excessive intracellular calcium concentrations is a major cause of neuronal cell death in epilepsy. The main source of increased calcium concentration during this excitotoxicity is an influx through NMDA subtype of glutamate receptors. The GluR2 (GluA2) hypothesis states that following a neurological insult such as an epileptic seizure, the AMPA receptor subunit GluR2 protein is downregulated. This increases the likelihood of the formation of GluR2-lacking, calcium-permeable AMPA receptor which might further enhance the toxicity of the neurotransmitter, glutamate. The cytosolic protein, PICK1, facilitates the removal of GluA2 subunits from the synaptic plasma membrane. High calcium concentrations may cause PICK1 to bind to the GluA2 subunit of calcium-impermeable AMPARs, leading to an increased internalization of these receptor subunits and a relative increase of GluA2-lacking, calcium-permeable AMPARs. This further escalates the cytosolic calcium concentration. In order to test this hypothesis, we have used kainic acid to induce epilepsy in rats. Using semi-quantitative western blotting combined with univariate and multivariate statistical analyses, we found that both GluA2 and PICK1 were down-regulated in kainate-treated rats for as long as eight weeks after induction of epilepsy. An interesting finding was that statistical analysis indicates that the functional role of PICK1 in our material is to increase GluA2 concentrations in the cells. The observed reduction in PICK1 concentration may thus be an independent contributor to the observed GluA2 reduction. This reduction may possibly be an adaptive mechanism, serving to prevent further loss of GluA2 from the synapses.

Mislocalization of AQP4 precedes chronic seizures in the kainate model of temporal lobe epilepsy.

It has been suggested that loss of the astrocytic water channel aquaporin-4 (AQP4) from perivascular endfeet in sclerotic hippocampi contributes to increased seizure propensity in human mesial temporal lobe epilepsy (MTLE). Whether this loss occurs prior to or as a consequence of epilepsy development remains to be resolved. In the present study, we investigated whether the expression and distribution of AQP4 was altered prior to (i.e., in the latent phase) or after the onset of chronic epileptic seizures (i.e., in the chronic phase) in the kainate (KA) model of MTLE. Immunogold electron microscopic analysis revealed that AQP4 density in adluminal endfoot membranes was reduced in KA treated rats already in the latent phase, while the AQP4 density in the abluminal endfoot membrane was stable or slightly increased. The decrease in adluminal AQP4 immunogold labeling was accompanied by a reduction in the density of AQP4's anchoring protein alpha-syntrophin. The latent and chronic phases were associated with an upregulation of the M1 isoform of AQP4, as judged by semi-quantitative Western blot analysis. Taken together, the findings in this model suggest that a mislocalization of AQP4--reflecting a loss of astrocyte polarization--is an integral part of the epileptogenic process.

Reduced astrocytic contribution to the turnover of glutamate, glutamine, and GABA characterizes the latent phase in the kainate model of temporal lobe epilepsy.

The occurrence of spontaneous seizures in mesial temporal lobe epilepsy (MTLE) is preceded by a latent phase that provides a time window for identifying and treating patients at risk. However, a reliable biomarker of epileptogenesis has not been established and the underlying processes remain unclear. Growing evidence suggests that astrocytes contribute to an imbalance between excitation and inhibition in epilepsy. Here, astrocytic and neuronal neurotransmitter metabolism was analyzed in the latent phase of the kainate model of MTLE in an attempt to identify epileptogenic processes and potential biomarkers. Fourteen days after status epilepticus, [1-(13)C]glucose and [1,2-(13)C]acetate were injected and the hippocampal formation, entorhinal/piriform cortex, and neocortex were analyzed by (1)H and (13)C magnetic resonance spectroscopy. The (13)C enrichment in glutamate, glutamine, and γ-aminobutyric acid (GABA) from [1-(13)C]glucose was decreased in all areas. Decreased GABA content was specific for the hippocampal formation, together with a pronounced decrease in astrocyte-derived [1,2-(13)C]GABA and a decreased transfer of glutamine for the synthesis of GABA. Accumulation of branched-chain amino acids combined with decreased [4,5-(13)C]glutamate in hippocampal formation could signify decreased transamination via branched-chain aminotransferase in astrocytes. The results point to astrocytes as major players in the epileptogenic process, and (13)C enrichment of glutamate and GABA as potential biomarkers.

Expression of glutamine synthetase and glutamate dehydrogenase in the latent phase and chronic phase in the kainate model of temporal lobe epilepsy.

It has been suggested that astrocytic glutamate release or perturbed glutamate metabolism contributes to the proneness to epileptic seizures. Here we investigated whether astrocytic contents of the major glutamate degrading enzymes glutamine synthetase (GS) and glutamate dehydrogenase (GDH) decreases on moving from the latent phase (prior to seizures) to the chronic phase (after onset of seizures) in the kainate (KA) model of temporal lobe epilepsy. Western blotting and immunogold analysis of hippocampal formation indicated similar levels of GDH in the latent and chronic phases of KA injected rats and in corresponding controls. In contrast, the level of GS was increased in the latent phase compared with controls, as assessed by Western blots of whole hippocampal formation and subregions. The increase in GS paralleled that of glial fibrillary acidic protein (GFAP). Compared with the latent phase, the chronic phase revealed a lower level of GS (approaching control levels) but an unchanged GFAP content. The decrease in GS from latent to chronic phase was significant in whole hippocampal formation, dentate gyrus and CA3. It is concluded that kainate treated rats show an initial increase in GS, pari passu with the increase in GFAP, and a secondary decrease in GS that is not accompanied by a similar loss of GFAP. In a situation where glutamate catabolism is in high demand the secondary reduction in GS level may be sufficient to contribute to the seizure proneness that develops between the latent and chronic phases.

Limbic structures show altered glial-neuronal metabolism in the chronic phase of kainate induced epilepsy.

A better understanding is needed of how glutamate metabolism is affected in mesial temporal lobe epilepsy (MTLE). Here we investigated glial-neuronal metabolism in the chronic phase of the kainate (KA) model of MTLE. Thirteen weeks following systemic KA, rats were injected i.p. with [1-(13)C]glucose. Brain extracts from hippocampal formation, entorhinal cortex, and neocortex, were analyzed by (13)C and (1)H magnetic resonance spectroscopy to quantify (13)C labeling and concentrations of metabolites, respectively. The amount and (13)C labeling of glutamate were reduced in the hippocampal formation and entorhinal cortex of epileptic rats. Together with the decreased concentration of NAA, these results indicate neuronal loss. Additionally, mitochondrial dysfunction was detected in surviving glutamatergic neurons in the hippocampal formation. In entorhinal cortex glutamine labeling and concentration were unchanged despite the reduced glutamate content and label, possibly due to decreased oxidative metabolism and conserved flux of glutamate through glutamine synthetase in astrocytes. This mechanism was not operative in the hippocampal formation, where glutamine labeling was decreased. In neocortex labeling and concentration of GABA were increased in epileptic rats, possibly representing a compensatory mechanism. The changes in the hippocampus might be of pathophysiological importance and merit further studies aiming at resolving metabolic causes and consequences of MTLE.

In vivo mapping of temporospatial changes in manganese enhancement in rat brain during epileptogenesis.

Mesial temporal lobe epilepsy is associated with structural and functional abnormalities, such as hippocampal sclerosis and axonal reorganization. The temporal evolution of these changes remains to be determined, and there is a need for in vivo imaging techniques that can uncover the epileptogenic processes at an early stage. Manganese-enhanced magnetic resonance imaging may be useful in this regard. The aim of this study was to analyze the temporospatial changes in manganese enhancement in rat brain during the development of epilepsy subsequent to systemic kainate application (10 mg/kg i.p.). MnCl(2) was given systemically on day 2 (early), day 15 (latent), and 11 weeks (chronic phase) after the initial status epilepticus. Twenty-four hours after MnCl(2) injection T1-weighted 3D MRI was performed followed by analysis of manganese enhancement. In the medial temporal lobes, there was a pronounced decrease in manganese enhancement in CA1, CA3, dentate gyrus, entorhinal cortex and lateral amygdala in the early phase. In the latent and chronic phases, recovery of the manganese enhancement was observed in all these structures except CA1. A significant increase in manganese enhancement was detected in the entorhinal cortex and the amygdala in the chronic phase. In the latter phase, the structurally intact cerebellum showed significantly decreased manganese enhancement. The highly differentiated changes in manganese enhancement are likely to represent the net outcome of a number of pathological and pathophysiological events, including cell loss and changes in neuronal activity. Our findings are not consistent with the idea that manganese enhancement primarily reflects changes in glial cells.

Increased expression of phosphate-activated glutaminase in hippocampal neurons in human mesial temporal lobe epilepsy.

Patients with mesial temporal lobe epilepsy (MTLE) have increased basal concentrations of extracellular glutamate in the epileptogenic versus the non-epileptogenic hippocampus. Such elevated glutamate levels have been proposed to underlie the initiation and maintenance of recurrent seizures, and a key question is what causes the elevation of glutamate in MTLE. Here, we explore the possibility that neurons in the hippocampal formation contain higher levels of the glutamate synthesizing enzyme phosphate-activated glutaminase (PAG) in patients with MTLE versus patients with other forms of temporal lobe epilepsy (non-MTLE). Increased PAG immunoreactivity was recorded in subpopulations of surviving neurons in the MTLE hippocampal formation, particularly in CA1 and CA3 and in the polymorphic layer of the dentate gyrus. Immunogold analysis revealed that PAG was concentrated in mitochondria. Double-labeling experiments indicated a positive correlation between the mitochondrial contents of PAG protein and glutamate, as well as between PAG enzyme activity and PAG protein as determined by Western blots. These data suggest that the antibodies recognize an enzymatically active pool of PAG. Western blots and enzyme activity assays of hippocampal homogenates revealed no change in PAG between MTLE and non-MTLE, despite a greatly (>50%) reduced number of neurons in the MTLE hippocampal formation compared to non-MTLE. Thus, the MTLE hippocampal formation contains an increased concentration and activity of PAG per neuron compared to non-MTLE. This increase suggests an enhanced capacity for glutamate synthesis-a finding that might contribute to the disrupted glutamate homeostasis in MTLE.

The pentylenetetrazole-kindling model of epilepsy in SAMP8 mice: glial-neuronal metabolic interactions.

Recently, a new experimental model of epilepsy was introduced by the authors [Neurochem. Int. 40 (2002) 413]. This model combines pentylenetetrazole (PTZ)-kindling in senescence-accelerated mice P8 (SAMP8), a genetic model of aging. Since imbalance of glutamate and GABA is a major cause of seizures, the study of glial-neuronal interactions is of primary importance. Nuclear magnetic resonance spectroscopy (NMRS) is an excellent tool for metabolic studies. Thus, we examined whether NMRS when combined with administration of [1-13C]glucose and [1,2-13C]acetate might give valuable insights into neurotransmitter metabolism in this new model of epilepsy and aging. The 2- and 8-month-old SAMP8 were kindled with PTZ alone, received PTZ and phenobarbital (PB), or served as controls. In older animals, PTZ-kindling decreased labeling in glutamate C-4 from [1-13C]glucose, whereas, in the younger mice, labeling in glutamine C-4 was decreased both from [1-13C]glucose and [1,2-13C]acetate. It could be concluded that PTZ-kindling affected astrocytes in younger and glutamatergic neurons in older animals. In the presence of PTZ, phenobarbital decreased labeling of most metabolites in all cell types, except GABAergic neurons, from both labeled precursors in the younger animals. However, in older animals only GABAergic neurons were affected by phenobarbital as indicated by an increase in GABA labeling.