A chronic model of epilepsy in organotypic brain slice cultures of the rat entorhinal cortex

Epilepsy affects around 50 million people worldwide (60,000 diagnosed cases in the UK). Twenty or 30 out of every 100 do not respond to available drug treatments. Nearly 1000 people a year in the UK die directly or indirectly from their epilepsy. The need for better understanding of the disease is enormously important if we are to develop new and improved drugs. Research into epilepsy and antiepileptic drugs depends heavily on the use of animal models of the disease. The main approach is to use chemicals to provoke a serious convulsion in the animal and this is eventually followed (weeks to months) by the appearance of unprovoked epileptic fits. We can then sacrifice the animal, isolate brain tissue from it and establish what changes have occurred in nerve cell properties and connections. A major drawback is that this does not tell us what the changes are that lead up to the appearance of regular seizures. This cannot be done in a single animal, se we need to sacrifice and study the brains of groups of animals at intervals after the first fit. This leads to a large variation in the data, and requires large animal groups (up to 100) to establish a single change occurring during the development of seizures. Our research aims to establish a model that can provide the same, or more, information from a single animal without the need to subject it to the severe stress of the initial seizure. We will take the brain from one normal animal and prepare multiple samples (essentially thin slices) from it. These slices will then be maintained alive in a culture medium for period of weeks to months. We will use a chemical insult to provoke seizure-like activity in the slices, which will result in changes in the nerve cell properties and connections leading to the appearance of regular, electrically recorded, epileptic-like discharges. Since we will have multiple tissue samples from each animal we can follow the time course of these changes in a single experimental animal throughout the process. We estimate that this will reduce the number of animals needed for each experiment by around 90% and provide a more rapid way of gaining meaningful information from the studies. This should ultimately increase the speed at which we gain understanding of epilepsy and also the rate at which we can design and develop better drug treatments.

Epilepsy is a serious neurological disorder affecting around 50 million people world wide. Research into basic mechanisms of the disorder relies heavily on the use of animals. A favoured approach in this area is to establish chronic epileptic conditions in vivo and then use brain tissue from these animals in in vitro experiments to determine changes in neuronal networks that accompany overt chronic seizures. The in vivo chronic models involve instigating a severe acute seizure in the experimental animal, which is followed after a latent period of weeks to months by establishment of chronic spontaneous recurrent seizures. However, determination of changes underlying the development of the chronic condition is difficult, as the approach dictates that longitudinal changes in synaptic networks over the development phase cannot be conducted in a single animal. Individual animals must be sacrificed at arbitrary time points without knowledge of where that animal is in the chronic development process. This makes for a high degree of variability in the data, and is extremely expensive in terms of the total number of animals required. The current application seeks to development an in vitro model of the whole process in tissue from individual animals. Brain slices will be prepared from normal rats, and maintained in long-term organotypic cultures. After a period of stabilisation they will be subjected to a similar acute insult to that induced in the in vivo models, and thereafter monitored (electrophysiologically and histologically) at regular intervals to establish changes occurring during the period leading to appearance of chronic electrographic seizures activity. Since multiple slices can be prepared from a single animal, these can be used to monitor longitudinal changes in network activity in that animal, with untreated slices from the same animal used a parallel controls. Such an approach has the major advantages of eliminating the need for the severe and stressful acute induction phase in the animals, and dramatically reducing the number of animals needed to establish time-dependent changes in networks leading to chronic seizures. We will develop this model in organotypic slices taken from young adult animals and once the model is established we will use it to investigate changes in glutamate and GABA transmission that underlie the development of chronic seizures.