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Research ArticleResearch Article: New Research, Neuronal Excitability

Chemogenetic Suppression of GnRH Neurons during Pubertal Development Can Alter Adult GnRH Neuron Firing Rate and Reproductive Parameters in Female Mice

Eden A. Dulka, R. Anthony DeFazio and Suzanne M. Moenter
eNeuro 8 June 2020, 7 (3) ENEURO.0223-20.2020; https://doi.org/10.1523/ENEURO.0223-20.2020
Eden A. Dulka
1Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109
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R. Anthony DeFazio
1Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109
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Suzanne M. Moenter
1Department of Molecular and Integrative Physiology, University of Michigan, Ann Arbor, MI 48109
2Department of Internal Medicine, University of Michigan, Ann Arbor, MI 48109
3Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI 48109
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  • Figure 1.
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    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).

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    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.

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    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.

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    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.

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    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.

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    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

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    Table 1

    Genotypes of mice and in text abbreviation used

    AbbreviationGenotype
    GnRH-CreGnRH-Cre+/+ or GnRH-Cre+/-
    GnRH-GFPGnRH-GFP+/+ or GnRH-GFP+/-
    GnRH-Cre/GnRH-GFPGnRH-Cre+/+/GnRH-GFP+/+
    GnRH-4DiGnRH-Cre+/-/CAG-hM4Di+/-
    GnRH-3DqGnRH-Cre+/-/CAG-hM3Dq+/-
    GnRH-GFP-4DiGnRH-Cre+/-/GnRH-GFP+/-/CAG-hM4Di+/-
    GnRH-GFP-3DqGnRH-Cre+/-/GnRH-GFP+/-/CAG-hM3Dq+/-
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    Table 2

    Antibodies used for detection of DREADD expression in GnRH neurons

    Peptide targetAntigen sequenceName of antibodySource, catalog number, RRID informationSpecies/type
    Hemagglutinin (HA)HA peptide sequence (YPYDVPDYA)Anti-HA High AffinityRoche catalog #11867431001, RRID:AB_390919Rat monoclonal antibody (clone 3F10)
    GnRHGnRH conjugated to bovine serum albuminEL-14Dr. Oline Rønnekleiv, Oregon Health and Science University, RRID: AB_2715535Rabbit/polyclonal
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    Table 3

    Statistical parameters for reproductive measures

    nResults
    GnRH-GFP-4Di (two-tailed unpairedStudent’s t test)
     VO
     (4A, left panel)
    10 saline
    9 CNO
    p = 0.8931, t = 0.1365, df = 17
     Age at first estrus
     (4B, left panel)
    10 saline
    9 CNO
    p = 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 = 0.887
     Age 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 = 0.2950
    Estrous cycles
     GnRH-GFP-4Di (two-way ANOVA)11 saline
    6 CNO
    Saline 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 = 0.0145
     GnRH-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
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    Table 4

    Statistical parameters for comparisons of firing rate and GABAergic PSCs in GnRH-GFP-4Di mice (two-tailed Mann–Whitney U test)

    Comparisonn cellsUp value
    Extracellular recordings
     Frequency (Hz)12 saline, 9 CNO16p = 0.0056
    Spontaneous GABAergic PSCs
     Frequency (Hz)10 saline, 8 CNO29p = 0.3599
     Amplitude (pA)10 saline, 8 CNO39p = 0.3599
    Miniature GABAergic PSCs
     Frequency (Hz)11 saline, 7 CNO33p = 0.6590
     Amplitude (pA)11 saline, 7 CNO28p = 0.3749
    • View popup
    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)

    SalineCNOp value*tdf
    Spontaneous GABAergic PSCs
     n108
     Series resistance (MΩ)12.8 ± 0.911.4 ± 0.70.2601.16716
     Input resistance (MΩ)602.9 ± 41.6660.4.6 ± 53.60.4020.86116
     Holding current (pA)–54.0 ± 7.3–49.0 ± 6.90.6290.49216
     Capacitance (pF)14.4 ± 1.213.9 ± 0.50.7100.38012.4
     Miniature GABAergic PSCs
     n117
     Series resistance (MΩ)13.2 ± 1.112.6 ± 1.30.7600.31016
     Input resistance (MΩ)681.0 ± 62.4734.4.4 ± 116.90.6650.44116
     Holding current (pA)–49.4 ± 4.3–53.8 ± 10.50.6600.44016
     Capacitance (pF)13 ± 1.114.7 ± 1.10.6090.52216
    • ↵* compares listed passive properties between saline and CNO groups.

    • View popup
    Table 6

    Two-way ANOVA and Kruskal–Wallis parameters for extracellular recordings in GnRH-GFP-3Dq and GnRH-GFP mice

    Comparison (figure)Control vs PNASaline vs CNOInteraction
    Two-way ANOVA for firing rate (Hz) inGnRH-GFP-3Dq mice (6A)F(1,35) = 0.021, p = 0.885F(1,35) = 1.865, p = 0.181F(1,35) = 0.104, p = 0.749
    Naive vs shamNaive vs 4DiNaive vs 3Dq
    Kruskal–Wallis (KW = 8.361)/Dunn (6B)p > 0.99p > 0.99p = 0.0169
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Chemogenetic Suppression of GnRH Neurons during Pubertal Development Can Alter Adult GnRH Neuron Firing Rate and Reproductive Parameters in Female Mice
Eden A. Dulka, R. Anthony DeFazio, Suzanne M. Moenter
eNeuro 8 June 2020, 7 (3) ENEURO.0223-20.2020; DOI: 10.1523/ENEURO.0223-20.2020

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Chemogenetic Suppression of GnRH Neurons during Pubertal Development Can Alter Adult GnRH Neuron Firing Rate and Reproductive Parameters in Female Mice
Eden A. Dulka, R. Anthony DeFazio, Suzanne M. Moenter
eNeuro 8 June 2020, 7 (3) ENEURO.0223-20.2020; DOI: 10.1523/ENEURO.0223-20.2020
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