Phenotypic differences between fast and slow methionine sulfoximine-inbred mice: Seizures, anxiety, and glutamine synthetase

Dose–response curve (A), latency as a function of MSO doses (B) and times necessary for inbred mice to reach various stages of MSO-dependent convulsion (C). Mice were administered with various doses of MSO and the development of seizures was observed. Ten males and 10 females were used, MSO-Slow (triangle, broken line) and MSO-Fast mice (square, full line). To observe stages I–V of seizures MSO was administered to MSO-Slow (n=40, 20 males, 20 females) and MSO-Fast (n=40, 20 males, 20 females) mice at a dose of 350 and 75mg/kg, respectively (C). Values are expressed as mean±SEM, and significance between two inbred mice was assumed using Student t-test as *p<0.05, **p<0.01, ***p<0.001. Statistical significance between the two inbred mice for stages I–V of MSO-dependent seizure is estimated using repeated ANOVA test: ***p<0.001.

Typical electroencephalograms (EEG) of inbreed mice to 75mg/kg of methionine sulfoximine (MSO) and 350mg/kg for MSO-Fast and MSO-Slow mice, respectively. EEG from the convulsive period (upper panels A and B) was shown and 2h-EEG after MSO were displayed (lower panels A and B) as the control period. The EEG recording is one out of two performed on two different MSO-Fast mice (A) and two different MSO-Slow mice (B). Surface electrodes were implanted over the cortex through the skull under isoflurane anesthesia, and EEG was recorded 6–8 days later.

Latency (A) and percent of convulsion (B) toward various convulsants of MSO-Slow (full bars) and MSO-Fast (open bars) inbred mice. Latencies toward PC (pilocarpine, 300mg/kg) and KA (kainic acid, 25mg/kg) are given in minutes, while latency to PTZ (pentylenetetrazole, 75mg/kg) is given in seconds. We used 10 males and 10 females of the two inbred mice. Values are expressed as mean±SEM, and significance between two inbred mice was estimated using Student t-test: *p<0.05; ***p<0.001.

Effect of two anticonvulsants on MSO-dependent seizures in MSO-Slow and MSO-Fast mice. We used 10 males and 10 females of MSO-Slow inbred mice (full bars), and 10 males and 10 females of MSO-Fast inbred mice (open bars). MSO was administered at a dose of 350 and 75mg/kg to MSO-Slow and MSO-Fast inbred mice, respectively. Latencies to generalised convulsion are given in minutes, and values are expressed as mean±SEM. Statistical differences were assumed using Student t-test: *p<0.05, **p<0.01, ***p<0.001. (A) Valproic acid (VPA) was administered (ip, 250mg/kg) 30min before MSO (VPA+MSO); MK-801 (ip, 1mg/kg) was administered to mice 30min before MSO (MK-801+MSO). (B) Comparison of latencies to seizures induced by MK-801 administered either alone (MK-801) or 120min after MSO (MSO+MK-801).

Brain cortical glutamine synthetase activity in MSO-Slow and MSO-Fast mice. We used 5 females and 4 males of MSO-Slow inbred mice (full bars, full triangles), and 5 females and 4 males of MSO-Fast inbred mice (open bars, full squares). Values are expressed as mean±SEM and statistical difference was estimated using Student t-test: ***p<0.001, **p<0.01. (A) Glutamine synthetase activity expressed as nmol of glutamate hydroxamate produced per hour per mg of proteins, and is determined as V_max by increasing glutamic acid concentration. V_max is calculated using the linear part of the curve. (B) K_m determination for glutamic acid using various concentrations. Both values in A and B were calculated using plot of Lineweaver–Burk, i.e., 1/V as a function of 1/S. (C) Determination of K_i for MSO using increasing MSO concentrations, and calculated using log [MSO] as function of activity with a variable slope.

Data obtained using the open-field test. Mice from the 7–9 inbreeding procedure, analysed at 10–12 weeks of age, were submitted to the open-field test. Data are expressed as mean±SEM of 40 mice of both sexes corresponding to 20 mice of each sex. First movement, represents the times spend in the centre of open-field before first mice movement and are expressed in second. Total squares visited represent the numbers of lines crossed. Full bars: MSO-Slow inbred mice. Open bars: MSO-Fast inbred mice. Statistical differences were assumed using repeated ANOVA test: *p<0.05, **p<0.01, ***p<0.001.