Figure 4. KLB expression in BLA→NAc projection neurons is necessary and sufficient for suppression of alcohol consumption by FGF21 and PF-05231023.

(A) Schematic illustrating placement of a transcriptional blocking sequence (splice acceptor (orange) followed by 3x poly A tail sequence (yellow)) in the intronic region between the 1^st and 2^nd exon of Klb flanked by loxP sites to generate KLB loxTB mice.

(B) Schematic illustrating the dual recombinase strategy used to return endogenous KLB expression specifically to BLA→NAc projecting neurons using an AAVrg-Flp expressing virus injected into the NAc and an AAV virus expressing a Flp-dependent HA-tagged version of Cre (AAV-fDIO-Cre-HA) injected into the BLA.

(C-E) Ethanol (EtOH) consumption (g/day/kg) in wildtype mice (black, WT), KLB loxTB control mice (blue, KLB loxTB^Ctrl, KLB^loxTB/loxTB mice which received AAV-fDIO-Cre-HA in the BLA, but AAVrg-GFP in the NAc instead of AAVrg-Flp), and mice in which endogenous KLB expression was returned to BLA→NAc projecting neurons (green, KLB loxTB^BLA→NAc; KLB^loxTB/loxTB mice which received AAV-fDIO-Cre-HA in the BLA, and AAVrg-Flp in the NAc) during daily vehicle administration followed by daily FGF21 administration (IP, 1 mg/kg) as indicated by the brackets above the plots followed by a washout period.

(F) Percent (%) change in EtOH consumption comparing FGF21 treatment period relative to the vehicle treated period for each group (n = 5 WT mice, 12 KLB loxTB^Ctrl, 9 KLB loxTB^BLA→NAc, one-way ANOVA w/Dunnett’s multiple comparison’s test, *=P<0.05 relative to WT mice).

(G-I) EtOH consumption in WT, KLB loxTB^Ctrl mice, and KLB loxTB^BLA→NAc during daily vehicle administration followed by a single injection of PF-05231023 (IP, 10 mg/kg) as indicated by the brackets and arrow, respectively, above the plots.

(J) Quantification of the percent (%) change in EtOH consumption comparing 1 day following PF-05231023 treatment relative to the vehicle treated period for each group (n = 5 WT mice, 12 KLB loxTB^Ctrl, 9 KLB loxTB^BLA→NAc, one-way ANOVA w/Dunnett’s multiple comparison’s test, *=P<0.05 relative to WT mice).

(K-M) Sucrose consumption (mL/day) in WT, KLB loxTB^Ctrl mice, and KLB loxTB^BLA→NAc during daily vehicle administration followed by daily FGF21 administration (IP, 1mg/kg) as indicated by the brackets above the plots.

(N) Quantification of the percent (%) change in sucrose consumption comparing FGF21 treatment period relative to the vehicle treated period for each group (n = 5 WT mice, 12 KLB loxTB^Ctrl, 9 KLB loxTB^BLA→NAc, one-way ANOVA w/Holm-Sidak’s multiple comparison’s test, *=P<0.05 for KLB loxTB^BLA→NAc mice relative to WT mice).

Data are presented as mean ± S.E.M.

See also Figure S3.