The distribution of transgenic fyn mRNA in the brain. (A) Transgenic fyn mRNA was detected in horizontal brain sections prepared from F₁ offspring of fyn transgenic mice using a digoxigenin-labeled transgene-specific cRNA probe. No signal was detected in nontransgenic littermates (non-Tg). The regional pattern restricting to the forebrain was essentially similar among the individual lines. (B) In the forebrain of line N8, the transgenic fyn mRNA was expressed highly in neurons of the neocortex (Cx), hippocampal pyramidal layer (Py), dentate granular layer (DG), and lateral amygdala nucleus (LA). Scale bar, 500 μm. (C) In the cerebellum of line N8, the transgenic fyn mRNA was also detected in Purkinje cells (Pu). (ML) Cerebellar molecular layer; (GL) cerebellar granular layer; (WM) white matter. Scale bar, 100 μm.

Seizure susceptibility in fyn transgenic mice induced by penthylenetetrazole and electrical stimulation of the amygdala. (A) Threshold for penthylenetetrazole (PTZ)-induced seizure in the group of mice was determined by a continuous infusion of 1.0% of PTZ (0.1 ml min⁻¹) intravenously. The time required for first twitch and tonic extension was monitored. Data were corrected for body weight and converted into the corresponding PTZ dose (mg/kg body weight). Each bar represents the mean ± s.e.m. (Solid area) Twitch; (open area) extension. The PTZ dose required for tonic extension was reduced significantly in mice expressing mutant Fyn (M27 and 78). (*) P < 0.05 (two-tailed, unpaired t-test). (B) Representative electroencephalograms recorded from the amygdala of a wild-type (wt, upper trace) and a mutant fyn transgenic mice (M78, lower trace) after the electrical stimulation. Thick bar represents the duration of the stimulation. Prolonged electrical response associated by behavioral seizure was often elicited in mice expressing mutant Fyn. (C) The AD duration and convulsion stage of the individual animals after the initial stimulation were plotted. Convulsion stage after the stimulation was determined according to the modified Racine’s classification (see Materials and Methods).

Change in tyrosine phosphorylation of the brain proteins in fyn transgenic mice. (A) PY-containing proteins and Fyn were detected by immunoblotting of RIPA extract (25 μg protein) prepared from wild-type mice (wt) and line N8 (tg) using anti-PY antibody (αPY) and anti-Fyn antibody (αFyn), respectively. Tyrosine phosphorylation of PY180, 120, 100, and Fyn (arrowheads) was enhanced in some brain regions where Fyn was overexpressed. The highest level of the enhancement was observed in the telencephalon, including the cerebral cortex, hippocampus, and amygdala/pyriform cortex (amygdala), where Fyn was overexpressed. Weak but significant enhancement of tyrosine phosphorylation was observed in the olfactory bulb, diencephalon and midbrain. By contrast, the enhancement of tyrosine phosphorylation was not observed in the medulla oblongata and pons (medulla/pons) and cerebellum where transgenic Fyn was expressed at a low level. Molecular weight markers (in kilodaltons) are indicated at left. (B) PY-containing proteins were detected by immunoblotting of the forebrain extracts (25 μg protein) prepared from wild-type (wt), fyn knockout (fyn⁻) and several lines of fyn transgenic mice. Tyrosine phosphorylation of PY180, 120, and 100 (arrowheads) was enhanced in most of fyn transgenic lines and conversely reduced in fyn knockout mice. Molecular mass markers (in kilodaltons) are indicated at left. (C) The level of PY180 (open bars) and 100 (hatched bars) was determined by densitometry of the fluorographic image. Each bar represents the mean value normalized with the level in wild-type mice ± s.e.m. from seven independent experiments. Significant change from the value in wild-type mice is indicated by asterisks; (*) P < 0.05, (**) P < 0.01, (***) P < 0.005 (two-tailed, one group t-test). (D) The relationship between relative level of PY proteins and relative amount of Fyn normalized with that in wild-type mice is plotted. The values shown are obtained from the (□) endogenous or native Fyn and (█) mutant Fyn, respectively. The phosphorylation of each PY protein was enhanced in parallel to the relative level of Fyn protein, and the level of enhancement was higher in mutant Fyn than that in native Fyn.

Effect of MK-801 on kindling development in fyn transgenic mice. MK-801 was systemically administered prior to once-daily stimulation of the amygdala in wild-type mice (wt +, █, n = 9) and fyn transgenic line N8 (N8 +, •, n = 8) from days 2–11. The period of the drug treatment is represented by thick bars. Values represent the means of the AD duration and convulsion stage ± s.e.m. Values in control animals which were not treated with the drug are also represented (wt −, □, n = 7; N8−, ○, n = 7). MK-801 reversibly suppressed the development of kindling in the line N8, as well as that in wild-type mice.

The levels of Fyn protein in fyn transgenic mice. Transgenic Fyn protein was detected by Western blotting of RIPA extract (20 μg of protein) prepared from the forebrain of each transgenic mouse line generated on fyn knockout genetic background (Tg⁺/fyn^−/−). Relative amount of Fyn protein was determined by densitometry of the fluorographic image and compared to that in wild-type mice (wt). Each bar represents the mean ± s.e.m. from seven independent experiments.

Amygdaloid kindling in fyn transgenic mice. (A) Once-daily stimulation of the amygdala was delivered to wild-type mice (wt, □, n = 9) and fyn transgenic mice which did not show convulsion greater than stage 1 after the initial stimulation (N8, █, n = 7; M27 and 78, ▵, n = 9). AD was recorded from the ipsilateral amygdala. Values represent the mean ± s.e.m. The thin line in the upper graph indicates the mean of AD duration at the nineteenth stimulation in wild-type mice (31.7 sec). The AD duration and convulsion stage progressed more rapidly in a line expressing native Fyn N8 and mice expressing mutant Fyn M27 and 78, as compared with wild-type mice. (B) Each bar represents the mean of the AD number required for full kindling ± s.e.m. A decrease in the AD number was significant in the lines N8 and M27 and 78. (*) P < 0.01 (Fisher’s PLSD test). (C) The severity of kindling was compared among the group of mice which were fully kindled. The white bar represents the mean of AD duration ± s.e.m.; the hatched bar represents the mean of convulsion stage ± s.e.m. Whereas the AD duration was not different significantly from that in wild-type mice, convulsion stage was significantly higher in kindled mice expressing mutant Fyn. (*) P < 0.01 (Fisher’s PLSD test).

Change in tyrosine phosphorylation of NR2 subunits in fyn transgenic mice. (A) The NR2B was immunoprecipitated from SDS-solubilized P2 membrane (500 μg of protein) prepared from each group of mouse brain by anti-NR2B antibody, then subjected to SDS-PAGE/immunoblotting with ether anti-PY antibody (αPY, top) or anti-NR2B antibody (αNR2B, bottom). The intensity of the band was determined by densitometric analysis of the fluorographic image. Relative ratio of the level of phosphorylated NR2B to the total level of NR2B was increased significantly in all the fyn transgenic lines except with line M58 and decreased in fyn knockout mice (fyn⁻), as compared to that in wild-type mice (wt). (*) P < 0.05, (**) P < 0.01, (***) P < 0.005 (two-tailed, one-group t-test). (B) The NR2A was immunoprecipitated from SDS-solubilized P2 membrane (500 μg protein) prepared from each group of mouse brain by anti-NR2A antibody and subjected to SDS-PAGE/immunoblotting with ether anti-PY antibody (αPY, top) or anti-NR2A antibody (αNR2A, bottom). The intensity of the band was determined as described above. Relative ratio of the level of phosphorylated NR2A to the total level of NR2A was significantly increased in lines N8 and 92, as compared to that in wild-type mice (wt). *P < 0.05 (two-tailed, one-group t-test).