Animals
WTC and zitter rat used in the present study were supplied by Dr.T.Serikawa (Kyoto University) and bred in our laboratory. All animals were housed in shoe-box type cages with continuous access to food and water, and maintained on a 12-hour/12-hour light/dark cycle. All procedures in the present study were approved by the Animal Welfare Committee of Tottori University School of Medicine.

Northern blot and Slot blot analysis
Total RNA was isolated from three regions (cerebrum, cerebellum and brainstem) of postnatal rat (aged 3,10,30 and 80 days) brain as described by Chomcznski and Sacchi. In Northern blot analysis, 10ug of total RNA were electrophoresed in 1% agarose-formaldehyde gels and transferred to nylon membrane. In slot blot hybridization, 1,3,5 and 6ug of total RNA were denatured and transferred to nylon membrane. Blots were hybridized with [32P]?labeled rat BDNF cDNA as a probes followed by rehybridized wit 18S rRNA.

Zitter rats (3weeks old) showed conspicuous trembling in both head and hund body enough to cause unsteady gait, and the size of brain in zitter rat was became smaller than WTC. The vacuoles at 30 days of age were appeared in the deep cortex, hippocampus, cervical spinal gray matter, and at later stages they were recognized apparently by their symptoms. Hence, we noted BDNF mRNA levels in zitter rat brain at 30 days of age. Northern blot analysis of total RNA revealed that the BDNF gene produced 1.6 and 4.2 kb BDNF mRNA transcripts (Fig. 1). The 1.6 and 4.2 kb BDNF mRNAs were derived from two alternative 3' end polyadenylation signals in the 3' untranslated region of the fifth exon. Total RNA was prepared from cerebrum, cerebellum and brainstem of both WTC and zitter rats. 18S ribosomal RNA bands were reprobed to compensate for variations in samples applied. In cerebrum, the relative expression of BDNF mRNA in zitter rat brain was observed at a half level of that in the control rats. In cerebellum and brainstem, the BDNF mRNA level was also reduced significantly in zitter rat.

Fig. 1. BDNF expression at 30 days of age in WTC and zitter rat brains. Northern blot analysis was performed with total RNA isolated from brain cells of zitter rats (z/z) and age-matched WTC (w/w). Total RNA (10ug) from cerebrum and brainstem + cerebellum was electrophoresed on an 1% agarose-formaldehyde gel, transferred to nylon membrane, and hybridized with BDNF cDNA probe. The membrane was stripped off the BDNF probe, and rehybridized with the 18S rRNA probe. Blots were representative of four independent experiments. The BDNF were estimated to be 1.6 and 4.2 kb, respectively.

Our finding that the BDNF difference in cerebrum of 80 days rat was significantly large compared with that of 3-30 days of age, was suggested that BDNF-producing cells were degenerated by pathological effect of zitter genotype. Because vacuoles became noticeable and many cells of zitter rat brain degenerated after 30 days, the differences in BDNF might be remarkable in progressed age. The constant deviations in BDNF expression in cerebellum and brainstem were associated with the development of brain at early stage in fetal. These results suggested that BDNF expression of zitter rat brain decreased compared with WTC, consequently related to vacuolation in zitter rat brain.