The use of zebrafish reward mutants to search for novel transcripts mediating the behavioral effects of amphetamine

Addiction is a pathological dysregulation of brain reward systems, determined by several complex genetic pathways. The conditioned place preference (CPP) test provides an evaluation of the effects of drugs in animal models, allowing the investigation of substances at a biologically relevant level with respect to reward. Our lab has previously reported the development of a reliable CPP paradigm for zebrafish. Here, this CPP test was used to isolate a dominant ENU-induced mutant, no addiction (naddne3256), which fails to respond to amphetamine, and which we used as an entry point towards identifying the behaviorally- relevant transcriptional response to amphetamine. Using microarray technology, we compare the wildtype with the mutant response to the drug, we compare the transcriptomes of the wildtype and mutants without drug and we also compared the transcriptomes of wildtype fish with the drug and wildtype fish without the drug. Through the combination of microarray experiments comparing the adult brain transcriptome of mutant and wild-type siblings under normal conditions, as well as their response to amphetamine, we identified genes that correlate with the mutants’ altered CPP behavior. In addition to pathways classically involved in reward, this gene set shows a striking enrichment in transcription factor-encoding genes classically involved in brain development, which later appear to be re-used within the adult brain. We selected a subset of them for validation by quantitative PCR and in situ hybridization, revealing that specific brain areas responding to the drug through these transcription factors include domains of ongoing adult neurogenesis. Finally, network construction revealed functional connections between several of these genes. Together, our results identify a new network of coordinated gene regulation that influences or accompanies amphetamine-triggered CPP behavior and that may underlie the susceptibility to addiction. Overall design includes three separate comparisons. Firstly, we took newly identified nad/+ mutants and their wildtype siblings. Both groups have had amphetamine administered. Secondly, we took nad/+ mutants and their wildtype siblings and compared them without drug. In a third experiment, we compared wildtype with drug vs. wildtype fish without drug. We pooled 4-5 brains per biological replicate and had in total 3 biological replicates per treatment. We used one-color arrays and compared the control and reference samples.