VIIIRACE, CCATTTTCAGCAATCGTTTGTTCAGC; rBDNFVIIINested, GAGACACACACCACAGCCTTTCTC; rBDNFIXARACE, GAGTAAACGGTTTCTAAGCAA- GTG; and rBDNFIXANested, CTTCCTCCCACTTTAG- CAGCTCTG. Cell Culture and Animal Experiments Rat glioma C6 and mouse neuroblastoma Neuro2A cells were plated 16 hr before treatment in DMEM (Invitrogen) con- taining 10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin). Trichostatin A (TSA) and 5-aza-20-deoxy- cytidine (5AzadC) were purchased from Sigma-Aldrich (St. Louis, MO). Neuro-2A and C6 cells were treated for 48 hr with 5AzadC (1 mM) or with TSA (333 nM) to analyze the effects of 5AzadC and TSA on the expression of BDNF. Adult male Sprague-Dawley rats were injected with the glutamate analog kainic acid as previously described (Metsis et al., 1993). Animals were sacriﬁced 1, 3, 6, 12, and 24 hr posttreatment. Total RNA from hippocampi was extracted by using RNAwiz RNA Isolation Reagent (Ambion) according to the manufacturer’s recommendations. All animal experi- ments were performed according to the norms of the local Eth- ical Committee of Animal Experimentation. RESULTS New Nomenclature for Mouse and Rat BDNF Gene BDNF gene is transcribed from multiple promoters located upstream of distinct 50 noncoding exons to pro- duce a heterogeneous population of BDNF mRNAs. Although this conserved feature of BDNF has been described for several species, including human (Liu et al., 2005), mouse (Hayes et al., 1997), rat (Timmusk et al., 1993), and zebraﬁsh (Heinrich and Pagtakhan, 2004), detailed analyses of rodent BDNF gene structure have not been performed. In rat, four 50 noncoding exons (I–IV) that are spliced to the common 30 coding exon (Fig. 1A) have previously been identiﬁed (Timmusk et al., 1993). For mouse, only homologues of rat BDNF exons I and II have been reported (Hayes et al., 1997). In silico analysis of mouse and rat BDNF gene structure performed in the present study showed that BDNF exons III and IV are present and expressed in mouse as well. Moreover, a number of EST and mRNA sequences aligned to the locations of potential novel BDNF exons and the respec- tive sequences turned out to be highly conserved in rat and mouse genome. Furthermore, analysis of BDNF 50 RACE products from human hippocampal RNA re- vealed additional novel exons (Kazantseva et al., unpub- lished), the sequences of which were also conserved in mouse and rat genomes. Identiﬁcation of rodent BDNF transcripts containing novel exons by RT-PCR and sub- sequent cloning and sequencing conﬁrmed the bioinfor- matic analyses data. Together our results show that both rat and mouse BDNF gene contains eight 50 noncoding exons and one 30 protein coding exon. All exon–intron junctions display conventional splice-donor and -acceptor sites. A new nomenclature was assigned to mouse and rat Journal of Neuroscience Research DOI 10.1002/jnr BDNF Gene Structure in Rodents 527 BDNF exons (Fig. 1B). In both mouse and rat genomes, the locations of novel BDNF exons are as following: exon III (corresponding to rat exon Ia described by Bishop et al., 1994) is located 0.6 kb downstream of previously described exon II, exon V is 0.25 kb downstream of exon IV (exon IV is the former exon III according to Timmusk et al., 1993), exon VII is located 0.6 kb downstream of exon VI (exon VI corresponds to exon IV in Timmusk et al. 1993), exon VIII is 13.5 kb upstream of the protein coding exon, and exon IXA is a 50 extended variant of the protein coding exon (Fig. 1B). Homology of human and rodent BDNF 50 exons ranges from 95% to 45%, reaching 95% for exon I, 93% for exon II, 62% for exon III, 91% for exon IV, 86% for exon VI (corresponds to exon V in human according to Liu et al., 2005), and 45% for exon VII (corresponds to exon VIA in human according to Liu et al., 2005). All exons that have been deﬁned in human (Liu et al., 2005) are also expressed in mouse and rat, except for human exons VIIB and VIII. Rodent exons V, VIII, and IXA have not been previously described in human (Liu et al., 2005), but according to our data these exons are expressed in human as well (Kazantseva et al., unpublished). Rat BDNF gene has been suggested to undergo cryptic splicing within exon II (Timmusk et al., 1995). In agreement with the recently updated version of GenBank’s submission (AY057907), our results show that usage of alternative splice donor sites (A, B, and C in Fig. 1B) within BDNF exon II leads to three different exon II transcript variants in both in mouse and rat. Expression Analysis of Mouse and Rat BDNF Transcripts Rat BDNF transcripts containing exons I, II, IV (former III), and VI (former IV) and their tissue-speciﬁc expression proﬁles have previously been described (Tim- musk et al., 1993), whereas there are no data on the expression of the novel rat BDNF exons V, VII, VIII, and IXA, and only limited data are available on the expression patterns of rat exon III (Bishop et al., 1994). Furthermore, although promoter regions upstream of mouse BDNF exons I and II have been described (Hayes et al., 1997), no data are available for the expression of mouse BDNF transcripts containing exons I–IXA. In the present work, RT-PCR analysis of the expression proﬁles of all BDNF transcripts was carried out in developing and adult brain as well as in peripheral tissues of mouse and rat (Fig. 2). Expression of rat exon I BDNF mRNA, which was previously described as a brain-speciﬁc transcript (Tim- musk et al., 1993), was also observed at low levels in several nonneural tissues, including testis, lung, thymus, liver, and spleen (Fig. 2B). Expression of mouse exon I transcripts was detected in addition to brain only in thy- mus (Fig. 2A). In adult mouse and rat brain, BDNF exon I mRNAs were expressed in all regions studied, with the lowest levels in cerebellum. In developing mouse and rat brain, low levels of BDNF exon I transcripts were ex- pressed at embryonic (E) days 13 and 15, the expression