532 Aid et al. Fig. 5. Activity-dependent regulation of BDNF exon-speciﬁc mRNAs in rat hippocampus by kainic acid-induced seizures. The effect of kainate-induced seizures on the expression of different BDNF transcripts in the hippocampus of adult rat brain was examined. Adult rats were injected subcutaneously with kainic acid (8 mg per kg body weight) and sacriﬁced 1, 3, 6, 12, and 24 hr posttreatment. Total RNA was extracted, and semiquantitative RT-PCR was performed. Untreated rat hippocampus RNA was used as a control. 2004) and provide the ﬁrst evidence that the novel BDNF mRNAs are differentially regulated by kainic acid. Our data strongly suggest that as yet unexplored regula- tory elements within BDNF gene contribute to the activ- ity-dependent regulation of BDNF mRNA expression. Antisense-BDNF Transcripts Are Not Expressed in Mouse and Rat It was shown recently (Liu et al., 2005) that protein noncoding antisense transcripts are expressed from human BDNF gene locus. Analyses of mouse and rat BDNF gene loci with the AntiHunter software tool (Lavorgna et al., 2004) did not reveal any BDNF antisense transcripts from EST databases. Moreover, alignment of human antisense BDNF exons sequences with mouse and rat EST databases at NCBI did not reveal any rodent ESTs homologous to human antisense BDNF transcripts. Failure to ﬁnd anti- sense ESTs transcribed from mouse and rat BDNF gene loci could be explained by the fact that, although EST databases are growing rapidly, they are still undersampling the full mammalian transcriptome. Therefore, we aligned the sequences of human antisense BDNF exons with the respective regions of mouse and rat BDNF genomic sequence. Interestingly, sequences with signiﬁcant homol- ogy to human antisense exons, though present in chim- panzee genome, were missing from mouse and rat genomes. RT-PCR analysis with mouse- and rat-speciﬁc primers annealing to the very short regions of homology with human antisense transcripts failed to detect expression of antisense BDNF transcripts in mouse and rat tissues. Therefore, we concluded that antisense BDNF transcripts are human- or primate-speciﬁc, as was proposed earlier by Liu and colleagues (2005). DISCUSSION Since the puriﬁcation of BDNF protein, deﬁnitive evidence has emerged for its central role in mammalian brain development, physiology, and pathology. However, the structural organization of rodent BDNF gene has not been revisited since four 50 exons were ﬁrst discovered and nomenclature of exons established for rat BDNF gene (Timmusk et al., 1993). This numeration of BDNF exons is currently widely used by the scientiﬁc community. In the present work, we show, however, that mouse and rat BDNF gene structure is much more complex than was accepted before. According to our data, mouse and rat BDNF genes consist of a common 30 exon that encodes the pro-BDNF protein and at least eight 50 noncoding exons (exons I–VIII). In each BDNF transcript, one 50 exon is spliced to the protein coding exon. All 50 exons are controlled by distinct promoters as evidenced by our RACE analysis of the 50 ends of these exons, as well as expression analysis data. In addition, we identiﬁed a novel BDNF transcript both in mouse and in rat that contains only exon IXA, the 50 extended protein coding exon. Here we suggest a new numbering system for mouse and rat BDNF exons. With regard to the old nomenclature (Timmusk et al., 1993), former exon III corresponds to exon IV, previous exon IV is now exon VI, and the cod- ing exon previously called exon V is now exon IX. Pro-BDNF, a 32-kDa precursor, undergoes cleavage to release mature 14-kDa BDNF protein as well as a minor truncated form of the precursor (28 kDa). Secreted pro- BDNF activates a heteromeric receptor complex of p75 and sortilin to initiate cell death (Teng et al., 2005) and binds to p75 in hippocampal neurons to enhance long- term depression (Woo et al., 2005). Studies suggest that proneurotrophins account for a signiﬁcant amount of the total neurotrophins secreted extracellularly, particularly in CNS neurons (Farhadi et al., 2000; Mowla et al., 2001). In mouse, rat, and human, exon I transcripts contain an in- frame AUG that can serve as an alternative translation ini- tiation codon, extending the prepro- region of BDNF by eight amino acids (Timmusk et al., 1993). It can be hypothesized that additional amino acids in the N-termi- nus of prepro-BDNF can affect the intracellular trafﬁcking of BDNF and play a role in pro-BDNF secretion. In human, BDNF 50 exons VIB and VII (according to Liu et al., 2005) can contribute to alternative BDNF protein isoforms, because exon VIB can add 15 amino acids to the N-terminus of prepro-BDNF, and exon VII can undergo alternative in-frame splicing leading to the mature BDNF protein isoform that lacks 48 amino acids internally (Liu et al., 2005). None of the novel rodent BDNF exons in- Journal of Neuroscience Research DOI 10.1002/jnr