Figure 3
Ucn1 knockdown in the EWcp selectively reduces long-term excessive alcohol drinking. (a) shUcn1-GFP lentivirus was expressed primarily in Ucn1-positive neurons at multiple bregma levels throughout the rostral-caudal axis of EWcp, indicated by co-localization of GFP in neurons immunostained for Ucn1 (white arrows). (b) Lentiviral vector driving GFP-tagged Ucn1 shRNA sequence. (c) Three weeks following surgery, number of Ucn1-positive neurons was reduced throughout the medial and posterior subregions of EWcp. (d) Eight weeks following surgery, density of Ucn1 immunoreactivity was significantly lower overall in the EWcp of shUcn1 vs Control mice (main effect of virus; F1,30=4.99, *P<0.05). (e) Images demonstrating that shUcn1 virus reduced the number of Ucn1-positive neurons as assessed by diaminobenzidine (DAB) staining (left) and the density of Ucn1 immunoreactivity as assessed by immunfluorescence staining (right). (f) EWcp-shUcn1 KD significantly reduced alcohol intake across the long-term 20% alcohol drinking phase (main effect of virus; F1,228=4.52, *P<0.05), and reduction in alcohol preference was strongly trending toward significance (P=0.055, Inset). Groups did not differ in (g) body weight, (h) total fluid consumption, (i) average food intake, (j) caloric intake nor (k) percent calories consumed from alcohol. EWcp, Edinger-Westphal nucleus; GFP, green florescent protein.
