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Pharmacological validation of a semi - automated hippocampal brain slice assay for seizure liability assessment in vitro Safety pharmacology studies assessing seizure potential traditionally use low throughput in vivo models associated with high compound and animal usage. A higher throughput in vitro assay would enable compounds to be tested earlier in the drug discovery process. The hippocampal brain slice assay is reported to be able to detect convulsant compounds but conventionally involves recording from one slice at a time (Fountain et al., 1992). This study aimed to validate a semi-automated system that allows concurrent recording from 4 or 8 slices (Slicemaster, Scientifica UK Ltd; Stopps et al., 2004). Hippocampal brain slices (350 μm thick) were prepared from Han Wistar rats (6-12 weeks, male or female, 170 to 350 g). Population spikes (PS) were evoked at 30 s intervals by electrical stimulation of the Schaffer collateral pathway and were recorded using extracellular electrodes positioned in the CA1 cell body layer. Responses were quantified as PS areas (the area above and below the 0 mV line). It was first established in time control experiments that, when applied alone for 80 minutes (the average experimental time course), the vehicle (0.1% DMSO) had no significant effect. Seven validation compounds known to cause seizures in vivo were then tested: 4-aminopyridine (4-AP; 1-100 μM), picrotoxin (0.1–100 μM), pentylenetetrazole (PTZ; 0.1–10 mM), aminophylline (1-100 μM), chlorpromazine (0.3-100 μM), SNC80 (0.1–30 μM) and penicillin (100–2000 μM). Cumulative concentration-effect curves were generated over these ranges. PS data (mean ± sem; n=4-15) were expressed as a percentage of that in vehicle. All compounds evoked a concentration-dependent increase in PS area that was statistically significant at higher concentrations (p<0.05; ANOVA) and comparable to literature data. The percentage effects at the top test concentration were: Picrotoxin 212.9 ± 28.8, PTZ 181.4 ± 24.7, 4-AP 328.9 ± 48.6, aminophylline 124.5 ± 5.9, chlorpromazine 122.1 ± 9.8, SNC80 132.1 ± 12.6 and penicillin 174.7 ± 14.1. The data show this higher throughput (4-8 times) in vitro brain slice assay can detect convulsant compounds and compare them using a consistent experimental format. This may facilitate earlier testing of convulsant liability than is currently possible with in vivo models and also be preferable from an animal welfare perspective.
Fountain S.B. et al. (1992). Toxicology in vitro. 6: 77-87 |
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