Fig. 3

UCN1-immunoreactive (ir) neurons of the centrally-projecting Edinger–Westphal nucleus (EWcp) undergo neurodegeneration and show altered functional neuromorphology in the rotenone model of Parkinson’s disease. In rotenone-treated rats (red bars) the number of EWcp/UCN1 neurons (green in B) decreased (C), compared with oil-injected controls (A, black bars). EWcp/UCN1 neurons also showed swollen morphology (D), and contained alpha-synuclein (αSYN, red) immunoreactive (ir) inclusions (see arrows in insets of panel B and histogram in E). Occasionally, ionized calcium binding adaptor molecule 1 (IBA1)-ir phagocytotic microglial cells (red G, H) were observed around UCN1 immunoreactive cell fragments (green) (inset in G, panels H and H’ and see also Additional file 1: Video 1) and rotenone treatment increased the number of interactions between UCN1 neurons and microglia (I). Glial fibrillary acidic protein (GFAP) labeling (white in J, K) revealed astrogliosis (L) upon rotenone treatment in the EWcp, while astrocyte activity score in the adjacent ventrolateral periaqueductal gray (vlPAG) remained unchanged (M). RNAscope in situ hybridization for Ucn1 mRNA (white in N, N’, O, O’) revealed reduced expression (P) in rotenone-treated rats. Immunolabeling in the same preparations revealed increased UCN1 peptide specific signal density (SSD) of the cells (green in N, N”, O, O”) upon rotenone treatment (Q). Cells with very low Ucn1 mRNA and high UCN1 peptide content were occasionally approached by phagocytotic microglial cells (IBA1, red in O, O”). DAPI (4′,6-diamidino-2-phenylindole dihydrochloride) labeling for nuclear staining is shown in blue (N’, N”, O’, O”). A negative correlation between UCN1 SSD and motor performance (R), SNpc/TH cell count and UCN1 SSD (S) as well as UCN1 SSD and Ucn1 mRNA expression (T) was detected. a.u.: arbitrary unit; *p < 0.05, **p < 0.01, ***p < 0.001 in Student’s t test, or in Mann–Whitney U test in L and M). ns not significant; n = 8–12. Bars: 50 µm