DZF-Project: Scott M. Thompson
1994
| Hippocampal slice cultures: an in vitro alternative for studying anti-convulsant withdrawal seizures |
| Scott M. Thompson Current Institute and Address: Dept. of Physiology, University of Maryland School of Medicine, 655 W. Baltimore St., Baltimore, Maryland 21201 USA sthom003(at)umaryland.edu Keywords: epilepsy, benzodiazepine, barbiturates, anti-convulsant Begin and End of the Project: 1994 – 1996 Background and Aim Background: Barbiturates and benzodiazepines are widely used as anti-convulsant drugs in epilepsy treatment, and also in numerous other pathologies such as anxiety and insomnia. Such treatment usually extends for periods ranging from months to years. When epileptic patients stop taking their anti-convulsant medication, seizures very commonly occur within a very few days. Little is known, however, about the mechanisms underlying these withdrawal seizures. Aim: We intended to address the following questions in vitro: 1) Does chronic exposure to BZs lead to a decrease in the number of postsynaptic GABAA receptors? 2) Is there any change in the kinetic properties of the receptors that reflects a change in their subunit composition? 3) What are the mechanisms underlying the long-term BZ effects on GABA receptors? Methods and Results We used electrophysiological techniques to investigate benzodiazepine function in organotypic slice cultures of the rat hippocampus. These cultures maintain the appropriate cellular phenotypes and connectivity characteristic of the hippocampus, thus facilitating the use of electrophysiological techniques to study synaptic transmission between defined cell types. Because the slices survive in culture for up to 4 weeks, drugs could be applied under carefully controlled conditions and at well defined concentrations for relatively long periods of time. Inhibitory synaptic transmission was then investigated using whole-cell voltage-clamp recordings from hippocampal pyramidal cells. We described several novel aspects of acute benzodiazepine action on GABAA receptor-mediated synaptic inhibition in area CA3 of the hippocampus. The benzodiazepine agonist midazolam increased the decay time constant of miniature inhibitory postsynaptic currents (mIPSCs) recorded from pyramidal cells by ~40%, but did not significantly affect their activation rate or amplitude, consistent with saturation of postsynaptic GABAA receptors by a quantum of GABA. Nonstationary variance analysis of mIPSCs revealed that the unitary conductance of synaptic GABAA channels (~31 pS) was unaffected by midazolam. Midazolam increased not only the decay time constant (51%), but also the amplitude (23%) of unitary IPSPs, recorded from pairs of monosynaptically connected inhibitory and pyramidal cells. Simulation of unitary IPSPs indicated that the increased amplitude was due primarily to the slow time constant of pyramidal cells. Finally, the mean amplitude, decay time constant, and single channel conductance of mIPSCs recorded in cultures chronically exposed to midazolam (0.1 - 10 µM) for 2 weeks were not different from control mIPSCs, nor was their response to midazolam. Conclusions We concluded that benzodiazepines increase synaptic GABAA channel open time and that this results in an increase in both the amplitude and duration of IPSPs in pyramidal cells, but we were unable to induce and alterations in GABA receptor function with chronic benzodiazepine exposure. These results were published: 1) Poncer, J.-C., R. Dürr, B.H. Gähwiler and S.M. Thompson. Modulation of synaptic GABAA receptor function by benzodiazepines in area CA3 of rat hippocampal slice cultures. Neuropharmacol., 35: 1169-1179, 1996. 2) Thompson, S.M., J.-C. Poncer, M. Capogna, and B.H. Gähwiler. Properties of spontaneous miniature GABAA receptor mediated synaptic currents in area CA3 of rat hippocampal slice cultures. Can. J. Physiol. Pharmacol., 75: 495-499, 1997. Relevance for 3R Our ultimate goal was to develop the use of hippocampal slice cultures as an in vitro alternative to currently used whole animal models with which to examine the long-term effects of chronically-applied substances on brain tissue. Current methods for the screening of potential anti-epileptic and neuroprotective compounds involve the use of enormous numbers of animals, mostly rats and mice. In order to reduce, refine and replace current animal models with alternative in vitro methods, we suggested that screening of anti-epileptic and neuroprotective compounds can be accomplished in an efficient and cost-effective manner using the slice culture technique. Although such are cultures are in fact derived from animals, 25-35 slice cultures can be produced routinely from one rat pup. The use of slice cultures for these purposes will lead ultimately to a reduction in the number of animals required in the development of these essential medications. |