Skip to main content
. 2020 Jul 9;9:e53945. doi: 10.7554/eLife.53945

Figure 2. Chemogenetic inhibition of GnRH neurons by intraperitoneal (iP) injection of CNO suppresses both the surge and pulse profiles of LH secretion.

(A) Schematic showing experimental protocol with GnRH-Cre mice injected with Cre-dependent Flex-hM4D(Gi)-mCherry AAVs bilaterally into the region of the median eminence and CNO given by IP injection. MS, medial septum. CNO, Clozapine N-oxide. (B) Fluorescence images of GnRH neurons expressing GnRH (green) and mCherry (red) in GnRH::Flex-hM4D(Gi)-mCherry mice. Scale bar, 20 μm. (C,D) LH profiles for all of the wild-type (WT) OVX+E+P mice given saline control (C, n = 9) or CNO (D, n = 11). (E,F) LH profiles for all of the GnRH-Cre OVX+E+P mice given saline control (E, n = 12) or CNO (F, n = 11). (G) Mean (± SEM) LH levels for the four experimental groups. *p<0.05, ***p<0.001, two-way repeated measures ANOVA with Holm-Sidak test. (H-M) Representative examples of pulsatile LH secretion in OVX GnRH-Cre mice given IP saline (H,I) or CNO (J,K). LH pulses are indicated by asterisks. (L,M) Mean (± SEM) amplitude and frequency of LH pulses in saline (n = 6) and CNO (n = 6). ***p<0.001 Mann-Whitney U-tests.

Figure 2.

Figure 2—figure supplement 1. LH surge profiles of wild-type and un-injected GnRH-Cre mice and expression of hM4D(Gi)-mCherry in GnRH neurons.

Figure 2—figure supplement 1.

LH surge profiles in all individual wild-type (A, n = 7) and GnRH-cre (B, n = 8) OVX+E+P female mice over 6 hr, with tail-tip blood sampling every hour. (C) Mean (± SEM) LH levels in wild-type and GnRH-Cre OVX+E+P female mice with tail-tip blood sampling every hour. Lights off is at 18:00 for all experiments. (D) Photomicrographs showing GnRH immunoreactivity (green), mCherry (red), and overlay in GnRH-Cre mice bilaterally injected with Cre-dependent Flex-hM4D(Gi)-mCherry AAVs into the region of the median eminence. White triangles indicate GnRH neurons expressing mCherry. Scale bar, 50 μm.
Figure 2—figure supplement 2. Inhibition of GnRH neurons expressing hM4D(Gi) by CNO.

Figure 2—figure supplement 2.

(A) Schematic of the acute brain slice and representative cell-attached recording showing the effect of 5 mM clozapine-N-oxide (CNO) on the firing rate of a GnRH neuron expressing hM4D(Gi)-mCherry. (B) Mean firing rate of six GnRH neurons (from 3 mice) before and after applying 5 mM CNO. ** p<0.01, paired t-test.