Seizure-like activity in the entorhinal cortex of organotypic brain slices requires NMDA and GluK1 kainate receptors

PV Massey, A Lench, RSG Jones. University of Bath, Bath, UK

Epilepsy is a highly prevalent disorder and it is crucial to understand its aetiology to inform therapeutic approaches. Much research relies on in vivo animal models of acquired epilepsy in which a single dose of convulsant provokes a severe acute seizure which is followed after a latent period (4-10 weeks) by the appearance of chronic seizures. These models are heavy on animal usage, stressful and have drawbacks and limitations.

We are developing a novel, in vitro model of chronic epilepsy in cultured organotypic hippocampus-entorhinal slices. The initial aim is to develop a reliable method to provoke acute seizure-like events (SLEs) to induce the appearance of chronic spontaneous, epileptic-like discharges. Once this is established treated slices can be studied longitudinally to investigate cellular and molecular changes acquired during the latent period.

Rhythmic discharges such as those seen in temporal lobe epilepsy can be mimicked in vitro (1). We have demonstrated that reducing external [Mg2+]o and raising [K+]o can more reliably provoke regular spontaneous SLEs in brain slices than application of standard convulsants alone. As pre- and postsynaptic NMDA (NMDA) receptors and kainate (KA) receptors are known to play important roles in the modulation of excitability in EC (2), we set out to investigate their involvement in the generation of SLEs triggered by changes in external ionic composition.

Acute brain slices were prepared as described (3) from neonatal (P10-14) rats, and then maintained in organotypic culture using the interface method (4). Spontaneous extracellular field potentials were recorded from entorhinal cortex (EC) of cultured brain slices (14-30 days in vitro) and epileptiform activity induced by perfusion of artificial cerebrospinal fluid (aCSF) containing low (0.5 mM) [Mg2+]o and high (12 mM) [K+]o.

Perfusion of NMDAR antagonist AP5 (100 µM) alone significantly reduced the amplitude, but also increased the frequency of SLEs (P <0.05, n=9). The GluK1 subunit-specific KA antagonist UBP310 (20 µM) alone significantly reduced the amplitude and frequency of the events (P < 0.05, n=7). Applied together AP5 and UBP310 almost completely abolished SLEs (n=7).

These results confirm the importance of GluK1-containing KARs and NMDARs in the control of excitability in EC. Further work is required to understand the precise role of these receptors and their subtypes in generating SLEs triggered by changes in external ionic composition.

Funded by NC3Rs

References

1. Avoli et al (2002) Prog Neurobio 68: 167-207

2. Chamberlain et al (2012) Hippocampus 22 (3): 555-576

3. Jones & Heinemann (1988) J Neurophysiol 59:1476-1497

4. Stoppini et al (1991) J Neurosci Methods 37(2):173-82