cludes an in-frame ATG, predicting that for these tran- scripts translation is initiated from the BDNF coding exon. BDNF is the most abundant and widely distributed neurotrophin in the mammalian CNS. In addition to re- ﬁning expression patterns of BDNF transcripts that have been identiﬁed earlier, results of this study also show that mouse and rat BDNF novel exons III, V, VII, VIII, and IXA are differentially expressed in adult brain and in pe- ripheral tissues. In general, exons that are closely located in the genome are expressed in a similar manner: exons I, II, and III have brain-enriched expression patterns and exons IV, V, and VI are widely expressed also in nonneu- ral tissues. However, 50 RACE analysis of transcription initiation sites of rat and mouse BDNF new exons and in silico analysis of the regions upstream of these exons (data not shown) suggest that their expression is driven by dis- tinct novel tissue-speciﬁc and development- and activity- regulated promoters. It has been established earlier by using different cellu- lar and animal models that BDNF gene is regulated by neural activity through calcium-mediated pathways (Shieh and Ghosh, 1999; West et al., 2001; Mellstrom et al., 2004) and that BDNF transcripts containing exons I, II, and IV are differentially regulated. BDNF exon I and exon IV transcripts (exons I and III according to Timmusk et al., 1993) have previously been characterized as the most highly induced BDNF mRNAs in response to kainate treatment and KCl-mediated membrane depolarization in embryonic cortical neuron cultures (Tao et al., 1998). Sev- eral calcium-responsive elements and transcription factors binding to these elements have been characterized in the promoter regions upstream of these exons (Timmusk et al., 1999; Tabuchi et al., 2002; Tao et al., 2002; Chen et al., 2003b). Here we show that BDNF exon V, exon VII, exon VIII, and exon IXA transcripts are also regulated by kainic acid and that the induction magnitude is comparable to that of BDNF exon I and IV transcripts. In light of our ﬁndings, it is attractive to speculate that differential regula- tion of nine BDNF exon mRNAs would become apparent in different neurodegenerative diseases in which BDNF levels are altered (Phillips et al., 1991; Mogi et al., 1999; Parain et al., 1999; Zuccato et al., 2001). Also, differential regulation of BDNF mRNAs can take place for example in depression, stress, exercise, and learning (Cotman and Berchtold, 2002; Tyler et al., 2002; Hashimoto et al., 2004; Russo-Neustadt and Chen, 2005). Future characterization of the regulatory sequences and transcription factors media- ting regulation of novel BDNF transcripts in different dis- ease models is important for understanding BDNF gene regulation and its contribution to pathology. The role of chromatin remodeling in the activity of different BDNF promoters has been investigated in several recent studies. Neuronal activity-dependent activation of BDNF gene is mediated by decreased CpG methylation of BDNF promoter IV and release of a repressor complex containing methyl-cytosine binding protein MeCP2, his- tone deacetylases HDAC1 and HDAC2, and corepressor mSin3A (Chen et al., 2003a; Martinowich et al., 2003). It has also been shown that histone modiﬁcations at speciﬁc Journal of Neuroscience Research DOI 10.1002/jnr BDNF Gene Structure in Rodents 533 BDNF promoters are involved in chromatin remodeling during electroconvulsive seizures (Tsankova et al., 2004) and cocaine-induced plasticity (Kumar et al., 2005) in rat and in a mouse model of depression and antidepressant treatment (Tsankova et al., 2006). In addition, zinc ﬁnger transcription factor REST/NRSF (Chong et al., 1995; Schoenherr and Anderson, 1995), which recruits multiple cofactors including HDAC1, HDAC2, and mSin3A (for review see Ballas and Mandel, 2005) to repress its target genes, negatively regulates BDNF gene expression by binding to NRSE/RE1 element in BDNF promoter II (Palm et al., 1998; Timmusk et al., 1999; Bruce et al., 2004; Ballas et al., 2005). The present study showed that the DNA demethylating agent 5AzadC evoked robust activation of BDNF gene expression in C6 rat glioma cells and more moderate activation in Neuro2A mouse neuro- blastoma cells in a transcript-speciﬁc manner: induction of exon I, III, IV, V, VIII, and IXA mRNAs was observed in C6 cells, whereas only exon I and exon III mRNA levels increased in Neuro2A cells. Furthermore, in C6 cells, inhi- bition of histone deacetylation by TSA up-regulated the levels of BDNF exon III, exon VII, and exon IX tran- scripts. The results presented in this study suggest the con- tribution of histone modiﬁcations and methylation of BDNF promoters to the regulation of BDNF gene tran- scription and open up possibilities for addressing these phe- nomena in more detail. Finally, we report that, in contrast with the human BDNF gene locus (Liu et al., 2005), mouse and rat BDNF gene loci do not encode antisense mRNA tran- scripts. These ﬁndings demonstrate that regulation of BDNF gene expression by antisense-BDNF transcripts clearly is a human- or primate-speciﬁc phenomenon and suggest that regulation of rodent and human BDNF gene differs substantially. Human-speciﬁc antisense transcripts have been reported for the tumor suppressor gene ret ﬁn- ger protein 2 (RFP2; Baranova et al., 2003) and for the human protocadherin (PCDH) locus (Lipovich et al., 2006). BDNF has important roles in development, partic- ularly of the nervous system, and plays a central role in brain plasticity-related processes, underscoring the possi- ble role of antisense BDNF gene in regulation of BDNF expression across primates manifesting in speciﬁc behav- ioral phenotypes. During the preparation of this paper, an article by Liu and colleagues examining the gene structure and ex- pression of BDNF in rodents was published (Liu et al., 2006). However, our study increases the understanding of rodent BDNF gene loci, in that we present several novel data that are complementary to the results of Liu and col- leagues. 1) We identiﬁed an additional 50 exon, exon V that was not been reported by Liu et al. Thus, both mouse and rat BDNF genes consist of at least eight 50 exons spliced to the 30 coding exon. In addition, we identiﬁed a novel BDNF transcript, exon IXA mRNA, consisting of only the 50 extended protein coding exon. 2) We deter- mined the transcription initiation sites for novel exons (III, V, VII, VIII, and IXA), showing that these exons are tran- scribed from distinct promoters. 3) Our data show that