Article Figures & Data
Figures
- Figure 1.
DREADD expression and function in GnRH neurons. A, Mice homozygous for Cre and GFP were bred to mice expressing either the 3Dq or 4Di under the CAG promoter; mC, mCitrine. B, Dual immunofluorescence for GnRH (green) and HA tag (red) in P14 female GnRH-3Dq (top) and GnRH-4Di (bottom) female mice. C, D, CNO alters GnRH neuron firing rate in vitro in GnRH-GFP-3Dq (C) and GnRH-GFP-4Di (D) mice. Scale bar is the same in C, D; open symbols, control; closed symbols, CNO treatment. Circles show adults, squares prepubertal mice; *p < 0.05 two-tailed Wilcoxon matched-pair signed-rank test (C), two-tailed paired Student’s t test (D).
- Figure 2.
Activation of 4Di-coupled DREADDs targeted to GnRH neurons reduces LH. A, B, LH pulse patterns in OVX GnRH-Cre mice (A) or OVX GnRH-4Di mice (B) during a control period, after intraperitoneal saline injection, after intraperitoneal CNO (1 mg/kg) injection, and after intraperitoneal GnRH (150–200 ng/kg) injection. Cluster detected pulses before GnRH treatment are shown as white symbols, dashed gray lines show times of intraperitoneal injections. C, Individual values and mean ± SEM number of LH pulses during the three treatment periods in A, B, two-way repeated-measures ANOVA (interaction F(2,10) = 6.820, p = 0.0135; genotype F(1,5) = 2.770, p = 0.1569; treatment F(2,10) = 24.86, p = 0.0001; subject F(5,10) = 3.716, p = 0.0368, Sidak post hoc; *p < 0.0001). D, left, Mean LH values in samples before and after intraperitoneal injection of 0.95 mg/ml clozapine to GnRH-Cre mice. Right, Mean GnRH neuron firing rate for 4 min before and minutes 7–10 during 1 μm clozapine treatment.
- Figure 3.
CNO induces LH release in GnRH-3Dq mice in a GnRH-dependent manner. A, LH profiles in three GnRH-Cre (black) and three GnRH-3Dq mice (green) before and after intraperitoneal CNO injection (1 mg/kg; arrow). B, Mean LH in 10 samples before (control) and after CNO injection (left and center) and six samples before and after CNO (right). Two-way repeated-measures ANOVA (interaction F(2,5) = 10, p = 0.0174; genotype F(2,5) = 9.6, p = 0.0192; treatment F(1,5) = 13, p = 0.0165; subject F(5,5) = 1, p = 0.5005, Sidak post hoc; **p < 0.01). C, LH response to CNO injection (0.3 mg/kg) remains elevated for several hours after a single injection; black and gray traces are from two separate animals tested.
- Figure 4.
Reproductive parameters are not altered by prepubertal administration of CNO. A, B, Age at VO (A) and first estrus (B) in GnRH-GFP-4Di (left) and GnRH-GFP-3Dq (right) mice; *p < 0.05 two-way ANOVA/Sidak. C, D, Representative cycles (P, proestrus; D, diestrus; E, estrus) from GnRH-GFP-3Dq (C) and GnRH-GFP-4Di (D) mice. E, Individual values and mean ± SEM of number of days spent in each cycle stage; *p < 0.05, **p < 0.01 three-way repeated-measures ANOVA/Fisher.
- Figure 5.
Reducing GnRH neuron activity in GnRH-GFP-4Di mice from two to three weeks of age changes GnRH neuron firing rate but not GABAergic transmission in adults. A, Representative extracellular recordings from each group. B–F, Individual values and mean ± SEM of GnRH neuron firing rate (B), GABA sPSC frequency (C), GABA mPSC frequency (D), GABA sPSC amplitude (E), and GABA mPSC amplitude (F) in GnRH-GFP-4Di mice; **p < 0.01 two-tailed Mann–Whitney U test. Gray symbols in B indicate mice receiving twice daily intraperitoneal injections of saline or CNO, open and black symbols denote osmopump administration of saline and CNO, respectively. Note that elimination of the cell firing at >2 Hz in B does not alter the observation of a significant difference in firing rate.
- Figure 6.
Prepubertal increase of GnRH neuron activity in GnRH-GFP-3Dq mice does not alter adult GnRH neuron activity when compared with controls, but expression of the 3Dq receptor increases control GnRH neuron activity. A, Representative extracellular recordings from each group. B, Individual values and mean ± SEM GnRH neuron firing rate for GnRH-GFP-3Dq mice. Gray symbols in B indicate mice receiving twice daily intraperitoneal injections of saline or CNO, open symbols denote osmopump administration of saline and black symbols osmopump administration of CNO; all PNA mice received osmopumps. C, Individual values and mean ± SEM comparison of firing rate from diestrous vehicle controls over three different experiments examining firing rate versus that in PNA mice. 1Naive animals were unmanipulated (data from Dulka and Moenter, 2017); 2sham animals received sham OVX surgery (data from Dulka et al., 2020). *Kruskal–Wallis (KW = 8.361, p = 0.0391)/Dunn’s versus naive p = 0.0169.
Tables
Abbreviation Genotype GnRH-Cre GnRH-Cre+/+ or GnRH-Cre+/- GnRH-GFP GnRH-GFP+/+ or GnRH-GFP+/- GnRH-Cre/GnRH-GFP GnRH-Cre+/+/GnRH-GFP+/+ GnRH-4Di GnRH-Cre+/-/CAG-hM4Di+/- GnRH-3Dq GnRH-Cre+/-/CAG-hM3Dq+/- GnRH-GFP-4Di GnRH-Cre+/-/GnRH-GFP+/-/CAG-hM4Di+/- GnRH-GFP-3Dq GnRH-Cre+/-/GnRH-GFP+/-/CAG-hM3Dq+/- Peptide target Antigen sequence Name of antibody Source, catalog number, RRID information Species/type Hemagglutinin (HA) HA peptide sequence (YPYDVPDYA) Anti-HA High Affinity Roche catalog #11867431001, RRID:AB_390919 Rat monoclonal antibody (clone 3F10) GnRH GnRH conjugated to bovine serum albumin EL-14 Dr. Oline Rønnekleiv, Oregon Health and Science University, RRID: AB_2715535 Rabbit/polyclonal n Results GnRH-GFP-4Di (two-tailed unpairedStudent’s t test) VO
(4A, left panel)10 saline
9 CNOp = 0.8931, t = 0.1365, df = 17 Age at first estrus
(4B, left panel)10 saline
9 CNOp = 0.1936, t = 0.1353, df = 17 GnRH-GFP-3Dq (two-way ANOVA) VO
(4A, right panel)16 control saline
20 control CNO
12 PNA saline
13 PNA CNO*Control vs PNA: F(1,57) = 17.11, p = 0.0001
Saline vs CNO: F(1,57) = 0.0897, p = 0.766
Interaction: F(1,57) = 0.0204, p = 0.887Age at first estrus
(4B, right panel)15 control saline
19 control CNO
10 PNA saline
10 PNA CNO*Control vs PNA: F(1,50) = 18.45, p < 0.0001
Saline vs CNO: F(1,50) = 1.349, p = 0.2510
Interaction: F(1,50) = 1.12, p = 0.2950Estrous cycles GnRH-GFP-4Di (two-way ANOVA) 11 saline
6 CNOSaline vs CNO: F(1,45) = 0, p > 0.9999
*Cycle stage: F(2,45) = 151.1, p < 0.0001
*Interaction: F(2,45) = 4.654, p = 0.0145GnRH-GFP-3Dq (three-way ANOVA) 6 control saline
11 control CNO
10 PNA saline
14 PNA CNO*Cycle stage: F(1.084,40.10) = 257.8, p < 0.0001
*Control vs PNA: F(1,37) = 0.0178, p = 0.895
Saline vs CNO: F(1,37) = 1.237, p = 0.2733
*Cycle stage × control vs PNA: F(2,74) = 20.0, p < 0.0001
Cycle stage × saline vs CNO: F(2,74) = 1.085, p = 0.3423
Control vs PNA × saline vs CNO: F(1,37) = 0.0178, p = 0.895
Cycle stage × control vs PNA × saline vs CNO:
F(2,74) = 0.477, p = 0.622- Table 4
Statistical parameters for comparisons of firing rate and GABAergic PSCs in GnRH-GFP-4Di mice (two-tailed Mann–Whitney U test)
Comparison n cells U p value Extracellular recordings Frequency (Hz) 12 saline, 9 CNO 16 p = 0.0056 Spontaneous GABAergic PSCs Frequency (Hz) 10 saline, 8 CNO 29 p = 0.3599 Amplitude (pA) 10 saline, 8 CNO 39 p = 0.3599 Miniature GABAergic PSCs Frequency (Hz) 11 saline, 7 CNO 33 p = 0.6590 Amplitude (pA) 11 saline, 7 CNO 28 p = 0.3749 - Table 5
Passive property values and comparisons for GABAergic sPSC and mPSC recordings in GnRH-GFP-4Di mice (mean ± SEM, p values from two-tailed unpaired Student’s t test with or without Welch correction)
Saline CNO p value* t df Spontaneous GABAergic PSCs n 10 8 Series resistance (MΩ) 12.8 ± 0.9 11.4 ± 0.7 0.260 1.167 16 Input resistance (MΩ) 602.9 ± 41.6 660.4.6 ± 53.6 0.402 0.861 16 Holding current (pA) –54.0 ± 7.3 –49.0 ± 6.9 0.629 0.492 16 Capacitance (pF) 14.4 ± 1.2 13.9 ± 0.5 0.710 0.380 12.4 Miniature GABAergic PSCs n 11 7 Series resistance (MΩ) 13.2 ± 1.1 12.6 ± 1.3 0.760 0.310 16 Input resistance (MΩ) 681.0 ± 62.4 734.4.4 ± 116.9 0.665 0.441 16 Holding current (pA) –49.4 ± 4.3 –53.8 ± 10.5 0.660 0.440 16 Capacitance (pF) 13 ± 1.1 14.7 ± 1.1 0.609 0.522 16 ↵* compares listed passive properties between saline and CNO groups.
- Table 6
Two-way ANOVA and Kruskal–Wallis parameters for extracellular recordings in GnRH-GFP-3Dq and GnRH-GFP mice
Comparison (figure) Control vs PNA Saline vs CNO Interaction Two-way ANOVA for firing rate (Hz) inGnRH-GFP-3Dq mice (6A) F(1,35) = 0.021, p = 0.885 F(1,35) = 1.865, p = 0.181 F(1,35) = 0.104, p = 0.749 Naive vs sham Naive vs 4Di Naive vs 3Dq Kruskal–Wallis (KW = 8.361)/Dunn (6B) p > 0.99 p > 0.99 p = 0.0169
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