Abstracts

Elevated levels of extracellular glutamate have been linked to the initiation and maintenance of seizures in patients with pharmaco-resistant temporal lobe epilepsy (TLE). Impairment of the glutamate-glutamine cycle, responsible for the inactivation and recycling of extracellular glutamate, has been implicated.One of the key neuronal components of this cycle is the vesicular glutamate transporter (VGLUT), which mediates the uptake of glutamate into synaptic vesicles. Three types of VGLUTs have been identified so far. VGLUT1 is the major type in the hippocampus. Little is known about VGLUT expression in the human hippocampus. The aim of this study is to investigate changes in VGLUT expression in the hippocampus of patients with TLE., We examined the hippocampal distribution of the VGLUT subtypes by immunohistochemistry, immunofluorescence and in situ hybridisation in 7 [mu]m paraffin sections. Protein levels were assessed by semi-quantitative western blotting. Measurements were performed in hippocampal biopsies obtained during surgical resection as treatment for TLE. Comparisons were made between hippocampi of TLE patients with (HS group) and without hippocampal sclerosis (nonHS group) and autopsy controls without neurological symptoms., VGLUT1 immunoreactivity (IR) showed a diffuse neuropil staining of the subiculum, the pyramidal cell layer of all CA fields, the hilus, and the inner molecular layer of the dentate gyrus (DG). Large punctate structures around neuronal cell bodies and axons were observed in CA4 and CA3. No IR was detected in cell bodies. VGLUT1 mRNA was abundant in granule cells and pyramidal neurons. In nonHS and HS hippocampi VGLUT1 mRNA levels were decreased compared to controls, especially in areas with neuron loss. However, VGLUT1 protein levels were significantly increased in the nonHS hippocampus compared to both the HS and control hippocampus. This upregulation indicates translational regulation within hippocampal neurons. In addition, VGLUT1 mRNA and protein were upregulated in the DG of the HS hippocampus, consistant with the increased number of mossy fiber synapses onto granule cells due to mossy fiber sprouting.
VGLUT2 IR was very weak in controls, but it was upregulated in the molecular layer of the DG in TLE patients. In the nonHS group VGLUT2 IR was only found in the outer molecular layer, whereas in the HS group VGLUT2 was also present in the inner molecular layer. These data indicate an upregulation of VGLUT2 associated with mossy fiber sprouting., Hippocampal mRNA expression of VGLUT1 is decreased and correlates with loss of glutamatergic synapses due to neuron loss. The increase in VGLUT1 and VGLUT2 protein found in TLE patients is most likely due to new glutamatergic synapse formation. This increase in glutamatergic signalling may contribute to the progression and pathology of TLE.,