Persistent reduction of hippocampal glutamine synthetase expression after status epilepticus in immature rats.

Mesiotemporal sclerosis (MTS), the most frequent form of drug-resistant temporal lobe epilepsy, often develops after an initial precipitating injury affecting the immature brain. To analyse early processes in epileptogenesis we used the juvenile pilocarpine model to study status epilepticus (SE)-induced changes in expression of key components in the glutamate-glutamine cycle, known to be affected in MTS patients. SE was induced by Li(+) /pilocarpine injection in 21-day-old rats. At 2-19 weeks after SE hippocampal protein expression was analysed by immunohistochemistry and neuron damage by FluoroJade staining. Spontaneous seizures occurred in at least 44% of animals 15-18 weeks after SE. As expected in this model, we did not observe loss of principal hippocampal neurons. Neuron damage was most pronounced in the hilus, where we also detected progressive loss of parvalbumin-positive GABAergic interneurons. Hilar neuron loss (or end-folium sclerosis), a common feature in patients with MTS, was accompanied by a progressively decreased glutamine synthetase (GS)-immunoreactivity from 2 (-15%) to 19 weeks (-33.5%) after SE. Immunoreactivity for excitatory amino-acid transporters, vesicular glutamate transporter 1 and glial fibrillary acidic protein was unaffected. Our data show that SE elicited in 21-day-old rats induces a progressive reduction in hilar GS expression without affecting other key components of the glutamate-glutamine cycle. Reduced expression of glial enzyme GS was first detected 2 weeks after SE, and thus clearly before spontaneous recurrent seizures occurred. These results support the hypothesis that reduced GS expression is an early event in the development of hippocampal sclerosis in MTS patients and emphasize the importance of astrocytes in early epileptogenesis.

Characterization of functional and structural integrity in experimental focal epilepsy: reduced network efficiency coincides with white matter changes.

BACKGROUND: Although focal epilepsies are increasingly recognized to affect multiple and remote neural systems, the underlying spatiotemporal pattern and the relationships between recurrent spontaneous seizures, global functional connectivity, and structural integrity remain largely unknown. METHODOLOGY/PRINCIPAL FINDINGS: Here we utilized serial resting-state functional MRI, graph-theoretical analysis of complex brain networks and diffusion tensor imaging to characterize the evolution of global network topology, functional connectivity and structural changes in the interictal brain in relation to focal epilepsy in a rat model. Epileptic networks exhibited a more regular functional topology than controls, indicated by a significant increase in shortest path length and clustering coefficient. Interhemispheric functional connectivity in epileptic brains decreased, while intrahemispheric functional connectivity increased. Widespread reductions of fractional anisotropy were found in white matter regions not restricted to the vicinity of the epileptic focus, including the corpus callosum. CONCLUSIONS/SIGNIFICANCE: Our longitudinal study on the pathogenesis of network dynamics in epileptic brains reveals that, despite the locality of the epileptogenic area, epileptic brains differ in their global network topology, connectivity and structural integrity from healthy brains.

Prolonged increase in rat hippocampal chemokine signalling after status epilepticus.

Temporal lobe epilepsy (TLE) is one of the most common focal epilepsy syndromes. In a genome-wide expression study of the human TLE hippocampus we previously showed up-regulation of genes involved in chemokine signalling. Here we investigate in the rat pilocarpine model for TLE, whether changes in chemokine signalling occur during epileptogenesis and are persistent. Therefore we analysed hippocampal protein expression and cellular localisation of CCL2, CCL4, CCR1 and CCR5 after status epilepticus. We found increased CCL4 (but not CCL2) expression in specific populations of hilar astrocytes at 2 and 19 weeks after SE concomitant with a persistent up-regulation of its receptor CCR5. Our results show an early and persistent up-regulation of CCL4/CCR5 signalling during epileptogenesis and suggest that CCL4 signalling, rather than CCL2 signalling, could have a role in the epileptogenic process.

Hippocampal distribution of vesicular glutamate transporter 1 in patients with temporal lobe epilepsy.

PURPOSE: Vesicular glutamate transporters (VGLUTs) are responsible for loading synaptic vesicles with glutamate, determining the phenotype of glutamatergic neurons, and have been implicated in the regulation of quantal size and presynaptic plasticity. We analyzed VGLUT subtype expression in normal human hippocampus and tested the hypothesis that alterations in VGLUT expression may contribute to long-term changes in glutamatergic transmission reported in patients with temporal lobe epilepsy (TLE). METHODS: VGLUT immunohistochemistry, immunofluorescence, in situ hybridization, Western blotting, and quantitative polymerase chain reaction (qPCR) were performed on biopsies from TLE patients without (non-HS) and with hippocampal sclerosis (HS) and compared to autopsy controls and rat hippocampus. VGLUT1 expression was compared with synaptophysin, neuropeptide Y (NPY), and Timm's staining. RESULTS: VGLUT1 was the predominant VGLUT in human hippocampus and appeared to be localized to presynaptic glutamatergic terminals. In non-HS hippocampi, VGLUT1 protein levels were increased compared to control and HS hippocampi in all subfields. In HS hippocampi VGLUT1 expression was decreased in subfields with severe neuronal loss, but strongly up-regulated in the dentate gyrus, characterized by mossy fiber sprouting. DISCUSSION: VGLUT1 is used as marker for glutamatergic synapses in the human hippocampus. In HS hippocampi VGLUT1 up-regulation in the dentate gyrus probably marks new glutamatergic synapses formed by mossy fiber sprouting. Our data indicate that non-HS patients have an increased capacity to store glutamate in vesicles, most likely due to an increase in translational processes or upregulation of VGLUT1 in synapses from afferent neurons outside the hippocampus. This up-regulation may increase glutamatergic transmission, and thus contribute to increased extracellular glutamate levels and hyperexcitability.