Article Figures & Data
Figures
- Figure 1.
Confirmation of PNA phenotype. A–C, Individual values and mean ± SEM for age of vaginal opening (VO; A), body mass at VO (B), and adult anogenital distance (AGD, mm; C). D, Representative estrous cycles over 14 d. P, proestrus; D, diestrus; E, estrus. E, Individual values and mean ± SEM days in each cycle stage over 14 d. Statistical parameters are in Table 1; ***p < 0.005, ****p < 0.0001.
- Figure 2.
Recording quality parameters. A–D, Individual values and mean ± SEM for compensated series resistance (A), capacitance (B), input resistance (C), holding current (D). Statistical parameters are in Table 2.
- Figure 3.
GnRH neuron excitability is increased, and action potential properties altered, in adult versus three-week-old mice. A, Representative membrane voltage responses (top) to depolarizing current injections (bottom); only three current steps are shown for clarity. B, Mean ± SEM # of action potentials (APs) fired as a function of current injection in age-combined groups. C, representative traces of the rheobase AP for each experimental group. D–K, Individual values and mean ± SEM for AP threshold (D), latency (E), rate of rise (F), AP amplitude (G), full width at half-maximum (FWHM; H), rheobase (I), afterhyperpolarization potential (AHP) time (J), and AHP amplitude (K). Statistical parameters are in Tables 3 and 4.
- Figure 4.
Neither development nor PNA treatment alter the response of GnRH neurons to hyperpolarizing current. A, Representative membrane voltage (top) responses to hyperpolarizing current injections (bottom); only three steps are shown for clarity. Individual values ± SEM for sag (B), and GnRH neuron rebound following the hyperpolarizing current relative to baseline membrane potential (C). Statistical parameters shown in Table 5.
Tables
- Table 1
Descriptive statistics and statistical parameters characterizing the PNA phenotype (Fig. 1)
Mean ± SEM for age at VO, body mass at VO and AGD Property VEH PNA Age at vaginal opening (d) 31.7 ± 0.5 27.6 ± 0.7 Body mass (g) at vaginal opening 13.8 ± 0.3 12.1 ± 0.4 AGD (mm) 4.9 ± 0.1 5.7 ± 0.1 Property Unpaired, two-tailed Student’s t test Mean difference (PNA-VEH) Effect size r2 Age at vaginal opening (d) t(4.986), df = 34; p < 0.0001 Diff [Cl, −5.824, −2.451]
−4.138 ± 0.8299r2 = 0.4223 AGD (mm) t(5.124), df = 23; p < 0.0001 Diff [Cl, 0.8206, 0.1601]
0.8206 ± 0.160r2 = 0.5331 Property Mann–Whitney U test Two-tailed p-value Body mass (g) at vaginal opening U = 55 p = 0.0006 Mean ± SEM days per estrous cycle stage Cycle stage VEH PNA Estrus 5.2 ± 0.3 4.5 ± 0.7 Diestrus 6.4 ± 0.3 9.2 ± 0.8 Proestrus 2.4 ± 0.2 0.1 ± 0.1 Property χ2 test Estrous cycle stage distribution χ2 = 36.1672, n = 425, df= 2, p = 0.000000014 Estrus Diestrus Proestrus Std. residual 0.3578012 3.771665 5.7238930 Fisher’s exact test; Bonferroni adjusted p = 1.0 p = 0.000573 p < 0.00005 Bold indicates p < 0.05.
- Table 2
Descriptive statistics and statistical parameters from two-way ANOVA for recording quality parameters and passive properties (Fig. 2)
Descriptive statistics (mean ± SEM) Property 3-week controls 3-week PNA Adult controls Adult PNA Series resistance (MΩ) 12.2 ± 0.8 12.259 ± 0.8 13.0 ± 0.8 12.841 ± 0.729 Capacitance (pF) 11.9 ± 0.9 11.8 ± 0.6 12.5 ± 0.9 13.654 ± 0.651 Input resistance (MΩ) 648.6 ± 30.2 710.3 ± 39.3 858.6 ± 53.4 775.946 ± 41.81 Holding current (pA) −63.9 ± 5.8 −61.6 ± 4.6 −48.2 ± 5.3 −48.456 ± 3.820 Two-way ANOVA Property Age Treatment Interaction Series resistance (MΩ) Diff, −0.7058
[Cl, −2.250, 0.8387]
F(1,60) = 0.8356; p = 0.3643Diff, 0.04112
[Cl, −1.503, 1.586]
F(1,60) = 0.002837; p = 0.9577Diff, −0.2489
[Cl, −2.840, 3.338]
F(1,60) = 0.02597; p = 0.8725Capacitance (pF) Diff, −1.220
[Cl, −2.750, 0.3089]
F(1,60) = 2.548; p = 0.3906Diff, −0.5158,
[Cl, −2.045, 1.014]
F(1,60) = 0.4551; p = 0.5025Diff, 1.322
[Cl, −4.381, 1.736]
F(1,60) = 0.7478; p = 0.5025Input resistance (MΩ) Diff, −137.8
[Cl, −221.8, −53.87]
F(1,60) = 10.78; p = 0.0017Diff, 10.43
[Cl, −73.53, 94.39]
F(1,60) = 0.06174, p = 0.8046Diff, −144.4
[Cl, −312.3, 23.56]
F(1,60) = 2.957; p = 0.0907Bonferroni 3-week VEH
vs 3-week PNA3-week VEH
vs adult VEH3-week PNA
vs adult PNAAdult VEH
vs adult PNAp > 0.9999 p = 0.0065 p ≥ 0.9999 p > 0.9999 Holding current (pA) Diff, −14.42
[Cl, −24.17, −4.658]
F(1,60) = 8.732; p = 0.0045Diff, −0.9966
[Cl, −10.75, 8.761]
F(1,60) = 0.04174; p = 0.8388Diff, −2.608
[Cl, −22.12, 16.91]
F(1,60) = 0.07146; p = 0.7901Bonferroni 3-week VEH
vs 3-week PNA3-week VEH
vs adult VEH3-week PNA
vs adult PNAAdult VEH
vs adult PNAp > 0.9999 p = 0.1846 p = 0.3271 p > 0.9999 Bold indicates p < 0.05.
- Table 3
Descriptive statistics and statistical parameters from three-way repeated-measures ANOVA for GnRH neuron excitability (Fig. 3)
Descriptive statistics number of APs (mean ± SEM) Current (pA) 3-week controls 3-week PNA Adult controls Adult PNA 0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 5 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 0.0 ± 0.0 10 0.0 ± 0.0 0.0 ± 0.0 0.2 ± 0.12 0.1 ± 013 15 0.1 ± 0.14 0.2 ± 0.15 1.1 ± 0.50 0.7 ± 0.30 20 0.8 ± 0.35 0.8 ± 0.40 2.3 ± 0.75 2.0 ± 0.56 25 2.3 ± 0.70 1.8 ± 0.56 4.3 ± 1.09 3.8 ± 0.71 30 3.6 ± 0.84 4.0 ± 0.57 6.2 ± 1.36 5.6 ± 0.88 35 5.9 ± 1.10 6.1 ± 0.63 8.0 ± 1.45 7.2 ± 0.90 40 7.7 ± 1.00 7.8 ± 0.68 9.7 ± 1.51 8.8 ± 0.94 Three-way repeated-measures ANOVA Two-way (age) Two-way (treatment) Current (pA) F(8,408) = 176.9; p < 0.0001 F(1.341,71.06) = 183.1; p < 0.0001 F(1.348,71.46) = 173.9; p < 0.0001 Age F(1,51) = 4.515; p = 0.0385 F(1,53) = 4.478; p = 0.0391 Treatment F(1,51) = 0.1273; p = 0.7227 F(1,53) = 0.02802; p = 0.8677 Current × age F(8,408) = 2.977; p = 0.0030 F(8,424) = 2.974; p = 0.0030 Current × treatment F(8,408) = 0.120; p = 0.9984 F(8,424) = 0.06764; p = 0.9998 Age × treatment F(1,51) = 0.1738; p = 0.1738 Current × age × treatment F(8,408) = 0.228; p = 0.9857 Bonferroni of age-consolidated data Three weeks (CON and PNA) vs adult (CON and PNA) 0, 5, 10, 15 pA p > 0.9999 20 pA p = 0.3820 25 pA p = 0.0302 30 pA p = 0.0169 35 pA p = 0.1481 40 pA p = 0.3349 Bold indicates p < 0.05.
Descriptive statistics (mean ± SEM) Property 3-week controls 3-week PNA Adult controls Adult PNA Threshold (mV) −43.8 ± 1.31 −44.8 ± 1.24 −42.2 ± 1.17 −43.2 ± 0.95 Latency (s) 0.2 ± 0.11 0.2 ± 0.10 0.3 ± 0.12 0.3 ± 0.10 Amplitude (mV) 87.5 ± 0.84 88.4 ± 1.28 85.7 ± 1.46 86.7 ± 1.05 FWHM (ms) 0.8 ± 0.04 0.8 ± 0.02 0.8 ± 0.04 0.8 ± 0.03 Rate of rise (mV/ms) 0.4 ± 0.01 0.4 ± 0.02 0.4 ± 0.02 0.5 ± 0.01 Rheobase (mV) 27.5 ± 2.15 25.0 ± 1.60 21.7 ± 2.41 21.9 ± 1.57 AHP amplitude (mV) −22.6 ± 0.85 −24.3 ± 1.02 −24.8 ± 1.15 −25.8 ± 0.68 AHP time (ms) 3.6 ± 0.31 4.1 ± 0.26 4.5 ± 0.52 4.8 ± 0.36 Property Age Treatment Interaction Threshold (mV) Diff, 1.170
[Cl, −3.918, 0.7768]
F(1,52) = 1.803; p = 0.1852Diff, 0.9859
[Cl, −1.361, 3.333]
F(1,52) = 0.7103; p = 0.4032Diff, −0.01028
[Cl, −4.705, 4.684]
F(1,52) = 1.932e-005; p = 0.9965Latency (s) Diff, −0.05101
[Cl, −0.1089, 0.006901]
F(1,51) = 0.3.127; p = 0.0.0830Diff, 0.003030
[Cl, −0.05489, 0.06095]
F(1,51) = 0.0.01103; p = 0.9168Diff, −0.05693
[Cl, −0.1728, 0.05890]
F(1,51) = 0.9735; p = 0.3285Amplitude (mV) Diff, 1.731
[Cl, −0.5921, 4.055]
F(1,51) = 2.238; p = 0.1408Diff, −0.9231
[Cl, −3.247, 1.400]
F(1,51) = 0.0.6361; p = 0.4288Diff, 0.002851
[Cl, −4.650,4.644]
F(1,51) = 1.517e006; p = 0.9990FWHM (ms) Diff, 0.02731
[Cl, −0.03989, 0.09454]
F(1,51) = 0.6660; p = 0.4183Diff, −0.01879
[Cl, −0.8601, 0.04843]
F(1,51) = 0.3149; p = 0.5771Diff, 0.05826
[Cl, −0.07618, 0.1927]
F(1,51) = 0.7568; p = 0.3884Rate of rise (mV/ms) Diff, −0.01261
[Cl, −0.04178, 0.01657]
F(1,51) = 0.7524; p = 0.3898Diff, 0.01453
[Cl, −0.04740, 0.01095]
F(1,51) = 0.2155; p = 0.2155Diff, −0.0002991
[Cl, −0.05865, 0.05805]
F(1,51) = 0.0001059; p = 0.9918Rheobase (pA) Diff, 4.479
[Cl, 0.5934, 8.365]
F(1,51) = 5.355; p = 0.0247Diff, 1.146
[Cl, −2.740, 5.032]
F(1,51) = 0.3505; p = 0.5565Diff, 2.708
[Cl, −5.063, 10.48]
F(1,51) = 0.4895; p = 0.4873Bonferroni 3-week VEH
vs 3-week PNA3-week VEH
vs adult VEH3-week PNA
vs adult PNAAdult VEH
vs adult PNAp > 0.9999 p = 0.2568 p > 0.9999 p > 0.9999 AHP amplitude (mV) Diff, 1.827
[Cl, −0.01161, 3.666]
F(1,51) = 3.980; p = 0.0514Diff, 1.369
[Cl, −0.4701, 3.208]
F(1,51) = 2.233; p = 0.1412Diff, 0.7832
[Cl, −2.895, 4.461]
F(1,51) = 0.1827; p = 0.6708Bonferroni 3-week VEH
vs 3-week PNA3-week VEH
vs adult VEH3-week PNA
vs adult PNAAdult VEH
vs adult PNAp > 0.9999 p = 0.6064 p > 0.9999 p > 0.9999 AHP time (ms) Diff, −0.7990
[Cl, −1.538, −0.05992]
F(1,51) = 4.710; p = 0.0347Diff, −0.3644
[Cl, −1.103, 0.3747]
F(1,51) = 0.9797; p = 0.3270Diff, −0.1663
[Cl, −1.644, 1.312]
F(1,51) = 0.05100; p = 0.8222Bonferroni 3-week VEH
vs 3-week PNA3-week VEH
vs adult VEH3-week PNA
vs adult PNAAdult VEH
vs adult PNAp > 0.9999 p = 0.6283 p = 0.9836 p > 0.9999 Bold indicates p < 0.05.
- Table 6
Mean ± SEM and two-way ANOVA parameters for GnRH neurons hyperpolarized between −90 and −95 mV (Fig. 4)
Current (pA) needed to hyperpolarize the cell to −90 to −95 mV VEH 3 weeks PNA 3 weeks VEH adult PNA adult −37.3 ± 1.95 −35.0 ± 1.78 −31.0 ± 2.67 −31.5 ± 1.73 Steady-state to sag-peak membrane potential difference VEH 3 weeks PNA 3 weeks VEH adult PNA adult 0.2 ± 0.12 0.04 ± 0.20 −0.01 ± 0.15 0.3 ± 0.26 Repolarization to basal membrane potential difference VEH 3 weeks PNA 3 weeks VEH adult PNA adult 0.3 ± 0.26 0.3 ± 0.48 −0.1 ± 0.26 0.5 ± 0.32 Two-way ANOVA Property Age Treatment Interaction Current (pA) needed to
hyperpolarizethe cell to
−90 to −95 mVDiff, −4.867
[Cl, −8.960, −0.7745]
F(1,41) = 5.768; p = 0.0209Diff, −0.8671
[Cl, −4.960, 3.225]
F(1,41) = 0.1831;
p = 0.6710Diff, −2.811
[Cl, −11.0, 21.915.374
F(1,41) = 0.4811; p = 0.4918Bonferroni 3-week VEH
vs 3-week PNA3-week VEH
vs adult VEH3-week PNA
vs adult PNAAdult VEH
vs adult PNAp > 0.9999 p = 0.2398 p > 0.9999 p > 0.9999 Steady-state to sag-peak
potential differenceDiff, −0.04333
[Cl, −0.4615,
0.3748]
F(1,41) = 0.04380;
p = 0.8353Diff, −0.09561
[Cl, −0.5138, 0.00928503226]
F(1,41) = 0.2132;
p = 0.6467Diff, 0.4902
[Cl, −0.3461,
1.327]
F(1,41) = 1.401;
p = 0.2433Repolarization to basal
potential differenceDiff, 0.09892
[Cl, −0.5909, 0.7887]
F(1,41) = 0.08386;
p = 0.7736Diff, −0.3164
[Cl, −1.006, 0.3735]
F(1,41) = 0.8579;
p = 0.3597Diff, 0.7403
[Cl, −0.6393, 2.120]
F(1,41) = 1.174;
p = 0.2848Bold indicates p < 0.05.
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