The brain is a complex neuronal network in which the subtle balance between different neurotransmitter systems is essential for normal functioning. Disease states are often caused by inbalances in these neurotransmitter systems and therefore therapies aim to restore the equilibrium. Epileptic convulsions are the expression of a synchrone burst of neuronal populations in brain regions characterised by excitatory interconnections between the principal nerve cells, such as in hippocampus. Animal models are used in this context for the fundamental study of pathophysiological neurotransmitter changes and the pharmacodynamic neurotransmitter-receptor interaction. We have developed and characterised in a previous project the pilocarpine model for focal epilepsy in freely moving rats. During the pilocarpine-induced epileptic seizures we observed a long-lasting increase in extracellular glutamate concentrations in the hippocampus. Glutamate is the principal excitatory neurotransmitter in the central nervous system. Abnormally high extracellular glutamate can lead to excitotoxic brain damage, morphological alterations and even cell death. Glutamate interacts with both ionotropic glutamate receptors (iGluR) and metabotropic glutamate receptors (mGluR). The iGluRs can be subdivided into AMPA, NMDA and kainate receptors, all present in the central nervous system.
1. Study of subtype-selective glutamate receptor antagonists.
We have previously demonstrated that blockade of NMDA receptors prevents pilocarpine-induced convulsions in freely moving rats. Most NMDA antagonists possess however important motoric and cognitive side effects and are as a result not clinically used in the treatment of epilepsy. Therefore we search for alternative strategies to suppress the glutamate-mediated processes. In this project, we aim to investigate which glutamate receptor subtypes are actually involved in epileptogenesis by using subtype-selective kainate- and AMPA receptor antagonists. These results could contribute to the development of possible new anti-epileptic drugs. Testing the effectiveness of several mGluR-ligands in the pilocarpine model is also an interesting challenge. Development of selective and allosterically modulating molecules for the eight mGluR receptors is a fast expanding domain of excitatory amino acid research. mGluR ligands have a modest influence on fast excitatory synaptic transmission, but possess important modulatory properties, which is an advantage for chronic drug therapies.
2. 'On-line' determination of alterations in the extracellular glutamate concentrations.
Almost all neurochemical and neuropharmacological studies performed in our laboratory have used microdialysis as sampling technique to collect the extracellular neurotransmitters from the conscious animals. Samples are subsequently analysed by microbore liquid chomatography (LC). Microdialysis is a quite unique technique for the neuropharmacologist because neuronal activity can be studied under physiological conditions and the biochemical changes can be correlated with behavioural alterations. Despite a tremendous amount of advantages, such as excellent sensitivity and selectivity, this technique has also some disadvantages: 1) Time resolution exceeds 5 min. 2) The concentrations of the transmitters in the extracellular space is low, resulting in a large analytical challenge. We have already developed several sensitive and robust LC analytical methods for quantitative determination of transmitters in dialysates. The only alternative method for in vivo microdialysis, is in vivo voltammetry. The latter technique is faster and possesses a better time resolution (ms to min), but is not as sensitive and selective as microdialysis. Both methods will therefore deliver complementary information. We would like to develop an implantable glutamate biosensor in collaboration with an engineer with experience in this domain and who is at the moment a postdoc in our laboratory. Because voltammetry should be able to register fast responses, we hope to determine 'on-line' the physiological glutamate release in the freely moving rat. We think that this method would also be able to provide us in a later stage with interesting information on the glutamate release during the ictal and interictal periods of an epileptic attack. In parallel and to validate the biosensor, it remains interesting to compare with microdialysis and thus to increase the sensitivity of our current analytical method for glutamate. This implies that we have to decrease sampling time, resulting in smaller sample volumes. Glutamate in the dialysates is now routinely analysed after precolumn derivatisation with microbore LC and fluorescence detection. It is possible to ameliorate the sensitivity of this method by using a performant fluorescence detector containing a micro 'flow cell'.