Psychopharmacology (1985) 86 : 137-141 Psychopharmacology 9 Springer-Verlag 1985 Long-lasting anticonvulsant effects of diazepam in different mouse strains: Correlations with brain concentrations and receptor occupancy Sandra E. File 1, D.J. Greenblatt 2, I.L. Martin 3, and Chloe Brown 3 1 Department of Pharmacology, The School of Pharmacy, University of London, Brunswick Square, London WC1N lAX, UK 2 Division of Clinical Pharmacology, Tufts New England Medical Center, Boston, USA 3 MRC Neurochemical Pharmacology Unit, Cambridge, UK Abstract. There were marked strain differences in the dura- tion of the protective effects of diazepam against the con- vulsant actions of penylenetetrazole and picrotoxin in mice. In no case was significant protection found at 12 h or lon- ger, regardless of whether the incidence of or the latencies to myoclonus or tonic-clonic convulsions were considered. These behavioural differences could not be explained simply in terms of strain differences in benzodiazepine metabolism or in percent of receptor occupancy, as determined by the fractional displacement of 3H-flunitrazepam binding in vivo. It is suggested that there might be strain differences in the percent of receptor occupancy neeced in order to produce an anticonvulsant effect. Key words: Benzodiazepines - Anticonvulsant - Strain dif- ferences Diazepam - Desmethyldiazepam Oxazepam - Receptor occupancy - Mouse There is evidence in the literature that diazepam has very long-lasting anticonvulsant effects against seizures induced by pentylenetetrazole, and it is likely that its metabolites desmethyldiazepam and oxazepam make a significant con- tribution to this effect (Garattini et al. 1973). Garattini et al. (1973) reported protective effects persisting for 20 h in mice after diazepam (5 mg/kg IV). Paul et al. (1979) claimed sig- nificant protection in mice for as long as 48 h after diaze- pain (4 mg/kg IP), although the basis for this claim was that one tenth of the mice did not seize to the test dose of pentylenetetrazole. However, they did find that 90% of the mice were protected (i.e. did not have full convulsions) against pentylenetetrazole 12 h after diazepam injection. Significant strain differences have been found in the rate at which mice develop tolerance to the anti-pentylenetetra- zole effects of diazepam with chronic treatment (File 1983). The main purpose of the present paper was to determine whether there were strain differences in the duration of protection induced by diazepam following a single acute dose. This was assessed against two chemical convulsants, picrotoxin and pentylenetetrazole. In order to determine whether any strain differences in protection could be due to differences in the metabolism of diazepam, we measured the plasma and brain concentra- tions of diazepam and its metabolites, desmethyldiazepam Offprint requests to: S.E. File and oxazepam, 6 h after diazepam injection. This was the time at which the strain differences in the incidence of con- vulsions were most marked. We were also interested in whether the strain differences seen behaviourally 6 h after diazepam injection were reflected in strain differences in receptor occupancy at this time. Materials and methods Animals We tested male mice from two inbred strains (C3H/HE from Olac Ltd, Bicester, 30 35 g, and NIH from Hacking and Churchill, Huntingdon, 30-35 g) and two outbred strains (Tuck No. 1 strain from A. Tuck and Sons, South- end, 35-40 g, and CFLP from Hacking and Churchill, 45-50 g). The mice were housed in groups of eight with food and water freely available. Drugs Diazepam (Roche Products Ltd) was suspended by ultra- sonic dispersion in water to which a drop of Tween-20 was added. Pentylenetetrazole and picrotoxin (Sigma) were dis- solved in water. All drugs were given intraperitoneally in a volume of 4 ml/kg. Procedure a) Duration of anticonvulsant effects. Because of variations in pentylenetetrazole (PTZ) threshold with batches of mice, temperature, season etc., pilot experiments were conducted immediately before the main experiment in order to estab- lish the appropriate dose of PTZ that caused seizures in 100% of each strain. The minimum dose of diazepam that totally blocked these seizures was also established for each strain. On the basis of these results, a challenge dose of PTZ (80 mg/kg) and a protective dose of 4 mg/kg diazepam was found suitable for all strains. In each strain mice were randomly allocated (n=8/ group) among the following groups: A) Challenge with PTZ (80 mg/kg) alone, or 0.5, 1.5, 3, 6, 12, 18 or 24 h after injection with diazepam (4 mg/kg); B) Challenge with picrotoxin (8 mg/kg) alone or 0.5, 1.5, 3 or 6 h after injec- tion with diazepam (4 mg/kg). The mice were returned to their home cages after the diazepam injection. The injec- tions were timed so that all the convulsion testing took