Research ReportRodent BDNF genes, novel promoters, novel splice variants, and regulation by cocaine☆
Introduction
The brain-derived neurotrophic factor (BDNF) gene encodes peptides that are important for the function of a number of brain circuits, including those involved in the behavioral and physiological effects of abused drugs (Bolanos et al., 2004). The evidence that supports such roles include BDNF's effects on morphology and function of reward-associated dopaminergic neurons in vitro (Hyman et al., 1991, Kontkanen et al., 1999) and in vivo (Shen et al., 1994). BDNF is packaged in the large dense-core neuronal vesicles and is released from hippocampal and striatal slices upon neuronal stimulation (Balkowiec et al., 2002, Goggi et al., 2002, Hartmann et al., 2001, Mowla et al., 1999). BDNF infusions into the nucleus accumbens or the ventral tegmental area enhance cocaine-induced locomotor activity, cocaine-induced potentiation of conditioned reward, and drug seeking after cocaine withdrawal (Horger et al., 1999, Lu et al., 2004). Inhibition of BDNF in the nucleus accumbens reduces amphetamine-induced dopamine release (Narita et al., 2003). Cocaine self-administration and subsequent withdrawal result in time-dependent increases in BDNF protein levels in the ventral tegmental area, nucleus accumbens, and amygdala that persist for 90 days after the last exposure to cocaine (Grimm et al., 2003). Heterozygous BDNF knockout mice with about half of normal levels of BDNF expression display about half wild-type levels of striatal dopamine (Dluzen et al., 1999) and are less sensitive to the locomotor and rewarding effects of cocaine than wild-type mice (Hall et al., 2003). We and others have linked genomic markers at the human BDNF locus to individual differences in vulnerability to polysubstance abuse (Liu et al., 2005b, Uhl et al., 2001).
These previous findings make the understanding of the BDNF gene structure, function, and regulation of interest. The previous work on BDNF's gene structure in rodent was not complete, and, therefore, the annotation of BDNF exons was unclear (Aoyama et al., 2001, Bishop et al., 1994, Timmusk et al., 1993). We have recently reported detailed studies of the human BDNF gene that support a complex pattern of regulation, the use of a variety of promoters, and the involvement of a number of cis- and trans-acting transcriptional control processes to produce a variety of differentially spliced sense and antisense transcripts. The human BDNF gene thus contains seven 5′ exons, each of which is spliced independently to a major coding exon. We renamed human BDNF exons in accordance with 5′ to 3′ order, exon I and II are homologous to Timmusk's rat exon I and II (Timmusk et al., 1993), respectively. Exon III is homologous to Bishop's rat exon 1a (Bishop et al., 1994). Exons IV and V are homologous to Timmusk's rat exon III and IV, respectively. Exons VIA and VIB are homologous to Aoyama's human exon 4I, and the exon VII has the same transcription initiation site of Aoyama's human exon 5U. However, exon VII is spliced to the exon VIII instead of directly connected to the major coding exon as found for Aoyama's exon 5U (Aoyama et al., 2001). The major coding exons from different laboratories are the same, the exon VIII in our study, exon V in Timmusk's, and exon 5 in Aoyama's work (Aoyama et al., 2001, Timmusk et al., 1993). The opposite strand of human BDNF gene encodes an eleven-exon gene that we have termed BDNFOS (AF411339). This gene's eleven exons are alternatively spliced to produce natural antisense transcripts (NATs) that produce 225 bp regions that are complementary to sequences found in the major protein coding exon VIII of the BDNF gene itself (Liu et al., 2005b).
Studies of BDNF's roles in drug addiction and other mental disorders and more generally in brain function would be facilitated by improved understanding of the gene and its structure/function relationships in rodents. However, previous studies lack the details that proved essential for improved understanding of the human BDNF gene. Thus, we now report work that characterizes the rat and mouse BNDF genes' exon–intron structures, identifies novel BDNF exons, and defines two- and three-part BDNF transcripts. These observations reveal striking differences between rodent and primate BDNF genes, including the apparent absence of rodent antisense transcripts that correspond to the human BDNFOS. Studies on the effect of cocaine exposure reveal promoter-specific upregulation of specific BDNF's transcripts in striatum and frontal cortex by acute, but not by repeated drug exposures.
Section snippets
Genomic structures of mouse and rat BDNF genes
Aligning human chromosome 11 BDNF exon sequences with those from mouse (Mus musculus) chromosome 2 and rat (Rattus norvegicus) chromosome 3 genomic contigs AY057907 and NW_047673, respectively, yielded good alignments for rat and mouse exons that corresponded to each human sense BDNF exon except exon VII. Previously identified rat exons I–V (Timmusk et al., 1993) and 1a exon (Bishop et al., 1994) were readily apparent; gaps in previous sequence assemblies appear to have hidden several novel
Discussion
The rodent BDNF gene structures and gene regulation that emerges from these studies and the comparison of these sequences with data from the BDNF genes in humans and other species indicate a significant diversity in the gene and in its likely regulatory mechanisms between species, within species, and after cocaine exposures.
Mouse and rat BDNF genes display similar structures, sequences, and homologies to other vertebrate BDNF genes. BDNF genes with multiple transcriptional initiation sites and
Acute and chronic cocaine administration
Rats and mice (Charles River, Raleigh, NC, USA) were kept under 12-h light/dark cycle with free access to water and food. The experimental procedures followed the “Principles of Laboratory Animal Care” (NIH publication no. 86-23, 1996) and were approved by the local Animal Care and Use Committee. Tissues were obtained from male 20–30g C57/BL6 mice sacrificed by cervical dislocation. For acute cocaine treatments, male Sprague–Dawley or Long–Evans rats (350–400 g, n = 6) were injected with
Acknowledgments
We acknowledge financial support from NIDA-IRP, NIH/DHSS. Some of these data were generated through the use of the Celera Discovery System and Celera's associated databases.
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NCBI GenBank accession numbers. Mouse BDNF gene sequence: AY057907. Mouse BDNF cDNA sequences: mBDNF1, AY057908; mBDNF2A, AY057909; mBDNF2B, AY057910; mBDNF2C, AY057911; mBDNF3, AY057912; mBDNF4, AY057913; mBDNF5, AY057914; mBDNF6A, AY231131; and mBDNF6B, AY231132. Rat cDNA sequences: rBDNF3, AY559248; rBDNF6A, AY559249; rBDNF6B, AY559250.