Thesis (Ph.D.)--University of Rochester. School of Medicine & Dentistry. Dept. of Neuroscience Graduate Program, 2012.
The HVC of the adult songbird forebrain undergoes gonadal steroid-mediated survival of new neurons throughout life. Angiogenesis and neuronal recruitment are serially and causally linked in this process, such that testosterone-induced angiogenesis is necessary for neuronal recruitment. To better understand the contribution of testosterone-induced transcriptional programs to this adult neuronal recruitment, we assessed the effects of androgen exposure on gene expression in the adult canary HVC. We first generated and sequenced a library of cDNAs derived from testosterone-treated adult female and male canary HVCs. We then used these sequences to establish two microarray platforms--a spotted cDNA array and a high-density oligonucleotide microarray. Using the higher quality high-density oligonucleotide array, we profiled the mRNAs of laser capture microdissected HVC sections, derived from testosterone-treated female canaries killed at varying time-points after androgen treatment. This analysis revealed >1000 probe sets that were regulated by testosterone; gene ontology analysis indicated strong differential regulation of genes associated with neuronal survival, while pathway analysis revealed strong differential expression of genes involved in nitric oxide signaling. In situ hybridization (ISH) confirmed that NRGN, RGS4, PPP3CA and CAMK2B mRNA were highly differentially expressed in HVC, whereas PLCB1 mRNA was diminished. Real-time quantitative PCR analysis confirmed that testosterone induced HVC RGS4 mRNA expression, but suppressed HVC NRGN, calcineurin/PPP3CA and CAMK2B. This pattern of regulation suggests that the nitric oxide regulatory pathways are critically involved in the androgenic modulation of cellular plasticity in the adult HVC, since this set of transcripts is highly associated with NO synthesis and signaling. In addition, we noted that the angiogenic modulators CRHBP and ADD3 were selectively enriched in the HVC; ISH then revealed that CRHBP mRNA expression was stimulated by testosterone specifically in HVC interneurons, while RGS4 was expressed in a complementary fashion in HVC excitatory projection neurons. Together, our transcriptional data suggest that HVC interneurons may subserve a regulatory role in testosterone-dependent angiogenesis, which may in turn govern HVC neuronal recruitment through NO-modulatory pathways.
