Figure 2. Kiss1ARC (KNDy) neuron stimulates LH release in Kiss1Cre/+but not Kiss1Cre/Cre (i.e., Kiss1 KO) mice supplemented with estradiol.
(A) Schematic representation of the site used to inject a pAAV encoding a Cre-dependent hM3Dq DREADD tagged to mCherry (pAAV5/hSyn-DIO-hm3Dq:mCherry; titer 3 × 1012 genome copies per ml; 1 µl per hemisphere) in heterozygous Kiss1Cre/+ (n = 10) and homozygous Kiss1Cre/Cre (i.e., Kiss1 KO) female mice (n = 20). (B) Representative photomicrograph of a coronal brain section stained for green fluorescent protein (GFP; green), mCherry (red) and merged GFP and mCherry immunoreactivity in the ARC of a homozygous Kiss1Cre/Cre (i.e., Kiss1 KO) female mouse >3 weeks after hM3Dq:mCherry injection (Scale bar 50 µm). In this animal model Cre and GFP have been targeted to the Kiss1 locus and are, therefore, expressed solely in Kiss1 neurons. Colocalization of GFP and m-Cherry immunoreactivity represents Kiss1ARC cells that have been infected with the pAAV encoding a Cre-dependent hM3Dq DREADD. (C, D) Mean ± SEM LH concentrations (ng/ml) and area under the curve (AUC) after an i.p. injection of saline (grey line-empty bar) or CNO (10 mg/kg dissolved in saline; green line-green bar) three weeks after the hM3Dq DREADD-injection in (C) heterozygous Kiss1Cre/+ and (D) homozygous Kiss1Cre/Cre (i.e., Kiss1 KO supplemented with estradiol) female mice (n = 5–8/group). Blood samples were collected just before saline or CNO i.p. injection (0) and then every 15 min for 90 min. 3V: third ventricle, ARC: arcuate nucleus. Repeated LH concentrations at multiple time-points and between treatments were compared using a 2-WAY ANOVA and a Fishers posthoc test when appropriate. Area under the curve was compared with 2-tailed student t tests. *p < 0.035, **p < 0.006, ***p < 0.0001.
Figure 2—figure supplement 1. Representative merged images of mCherry (red) and GnRH (green) immunoreactivity in the ARC of Kiss1Cre/+ (A) and Kiss1Cre/Cre (B).
