Fig. 1. Volcano plot demonstrate differential regulation of PACAP-targeted genes. Sympathetic transcripts identified on the Affymetrix U34 microarray to be regulated by 9 h acute (A) or 96 h chronic (B) peptide treatments were represented as correlation between robust multichip average expression index (M, log₂ fold change) and p-value (−log₁₀ p) for each of the 8799 genes. Criteria boundaries are shown for each plot. Transcripts upregulated following PACAP treatment are designated by open circles in the upper right quadrants; transcript expression diminished by PACAP are shown in upper left quadrants. Compared to panel A, the decrease in number of gene targets in the upper right quadrant of panel B reflects the transient nature of many induced transcripts after chronic PACAP treatment. The transcript in the extreme upper right of each plot is VIP.

Fig. 2. PCR studies corroborate PACAP-regulated transcript response magnitude and temporal dynamics shown by microarray analyses. Sympathetic cultures were treated with 100 nM PACAP27 and prepared for semi-quantitative (A) and quantitative (B) PCR analyses as described in Section 2. The peptide treatment times corresponded to those submitted for the microarray experiments to facilitate comparisons; the transcripts analyzed were representative of peptide, growth factor and receptor transcripts that demonstrated temporal differences in expression. The expression patterns of select transcripts after PACAP treatment followed those described in the gene chip studies; from quantitative PCR, the magnitude of the responses was frequently underestimated in the array data. Data from the quantitative PCR studies represent fold changes compared to untreated control cultures (n = 3) ± S.E.M.

PCR oligonucleotide primers

PACAP-regulated sympathetic transcripts after acute and chronic treatments

Fold change (fold Δ) in the acute (9 h) and chronic (96 h) transcript sets, and the corresponding p-values represent data compared to untreated control cultures (n = 3); transcripts in italics are downregulated after PACAP treatment. nc, not changed from control cultures.

Sympathetic transcripts uniquely regulated by PACAP after chronic treatment

Fold change (fold Δ) for the chronic (96 h) transcript set and the corresponding p-values represent data compared to untreated control cultures (n = 3); transcripts in italics are downregulated after PACAP treatment. nc, not changed from control cultures.