hrs following BDNF withdrawal in the microarray which was reliably reproducible by qPCR with a small but MGCD516 site significant decrease of 0.3-fold at 3 and 6hrs. Vamp4 was also decreased in both microarray and qPCR although the 3hr timepoint had an opposite response to the treatment for qPCR compared to microarray. For the microarray, Vamp4 increased slightly above baseline by 3hrs then decreased at 612hrs. For qPCR Vamp4 maintained a gradual decrease; starting with a 0.4fold decrease at 1.5hrs which was sustained up to the 12hr timepoint. Since qPCR was a validation for at least 3 independent experiments, the trend for qPCR more likely portrays an accurate and consistent Vamp4 response to BDNF withdrawal. Given the well-established functions of these genes in golgi maintenance, and vesicle trafficking, our results could be suggesting a potential disassembly of the protein trafficking and secretory machinery upon BDNF withdrawal. We utilized a well-established method of TrkB-FC application to sequester BDNF and NT-4, which also binds TrkB. Four early timepoints were selected to capture different Dev Neurobiol. Author manuscript; available in PMC 2016 February 01. Mariga et al. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19850363 Page 7 phases of transcriptional activity. These time points were specifically chosen to identify signaling pathways that are activated prior to the process of cell death initiation based on previous reports that commitment to cell death following NGF deprivation occurs approximately 1620 hours after removal of NGF from sympathetic and sensory neurons. We anticipated identifying individual genes and groups of transcripts in hippocampal neurons as BDNF withdrawal proceeds. Early timepoints were predicated to identify changes in immediate early genes, among others, whereas later time points would likely activate initiation mitochondrial changes associated with programmed cell death. During NGF withdrawal, cell death occurs in sympathetic neurons with increases in c-jun, c-myb mkp-1, cyclin D1 and the pro-apoptotic Bim transcripts. However, due to the time course examined, the microarray screen would not be expected to detect genes involved in cell death. Indeed, instead of proapoptotic genes, we detected significant enrichment in genes involved in synaptic function. Hippocampal neurons were selected for microarray analysis following BDNF withdrawal, as BDNF has profound effects upon long-term potentiation, synaptic plasticity and cell morphology in the hippocampus, where its receptor, TrkB is highly expressed. The functional analysis with DAVID indicated that many relevant pathways were represented although changes in expression levels were within 2030% range. It is worth 
noting that in neuronal populations, relatively small changes can be significant given the nature of neuronal signaling relative to heterologous cell lines or tissue with admixed cell types. Most importantly, BDNF withdrawal resulted in a significant decrease in genes that are associated with vesicular trafficking and synaptic function as well as selective increases in phosphatases and extracellular matrix genes. DUSP5, a stress inducible MAP kinase phosphatase that deactivates Erk1/2 in the MAP kinase pathway, was significantly upregulated upon BDNF withdrawal. Recently, the role of MAP kinase phosphatases in the development of CNS primary neurons was described in which expression of DUSPs is regulated by neurotrophins to modulate structural plasticity. The induction of MKP-1/DUSP1 by BDNF is influenced by
