Article Figures & Data
Figures
- Figure 1.
Confirmation of PNA phenotype in adults and surviving littermates of 3wk-old mice used for electrophysiology. A, B, D, Individual values (CON open symbols, PNA black symbols) and mean ± SEM for anogenital (AG) distance (A), body mass (B), or percent days in each cycle stage (D). C, Representative estrous cycles over a 3wk period; P, proestrus; D, diestrus; E, estrus. CON are shown on the left in grey, and PNA are on the right in black. Statistical parameters reported in Table 2; *p < 0.05, ****p ≤ 0.0001.
- Figure 2.
Effect of age and PNA treatment on KNDy neuron firing activity. A, Representative long-term firing patterns (1-min bins). CON are shown on the top in gray, PNA on the bottom in black for each age. The time of the traces shown in B is designated by the magenta dashed lines in panel A. B, Examples of raw firing data (60 s) from the areas indicated in A, details as in A. The selected 60-s bins are representative of the mean firing rate of each group. C, Proportion of cells with a firing frequency >0.005 Hz (black bars) versus <0.005 Hz (white bars); numbers are cell counts in each group. D, Individual values (CON open symbols, PNA black symbols) and mean ± SEM and median (magenta line) for firing frequency across the duration of long-term recordings. Statistical parameters reported in Table 3.
- Figure 3.
Senktide activates KNDy neuron firing activity. Individual values and mean ± SEM of firing rate during 5-min control (C) and senktide (SK) periods. CON mice are shown in open symbols, PNA mice in black symbols; **p ≤ 0.01, ***p ≤ 0.001. Statistical parameters in Table 4.
- Figure 4.
Effect of age and PNA treatment on burst parameters. A–F, Individual values (CON open symbols, PNA black symbols) and mean ± SEM and median (magenta lines) for burst frequency (A), burst duration (B), spikes per burst (C), intraburst interval (D), single spike frequency (E), and interevent interval (F). B–D, Burst parameters are only calculated for cells with at least one burst; 3wk-CON n = 6 cells from 5 mice in 4 litters, 3wk-PNA n = 15 cells from 10 mice in 5 litters, adult-CON n = 9 cells from 8 mice in 5 litters, adult-PNA n = 9 cells from 8 mice in 5 litters. F, Calculating interevent interval also requires multiple events, 3wk-CON n = 7 cells from 6 mice in 5 litters, 3wk-PNA n = 17 cells from 10 mice in 5 litters, adult-CON n = 10 cells from 10 mice in 6 litters, adult-PNA n = 12 cells from 9 mice in 5 litters; *p < 0.05. Statistical parameters in Table 5.
- Figure 5.
Effect of age and PNA treatment on arcuate nucleus mRNA transcripts. Individual values (CON open symbols, PNA black symbols) and mean ± SEM for Actb, Syn1, Kiss1, Kiss1r, Tac2, Tacr3, Pdyn, Oprk1, Ar, Esr1, and Pgr mRNA isolated from arcuate nucleus tissue punches. Statistical parameters are reported in Table 6; ***p ≤ 0.001, ****p ≤ 0.0001.
Tables
Transcript Probe 5′—>3′ Forward 5′—>3′ Reverse 5′—>3′ Actb CTG GCC TCA CTG TCC ACC TTC C GAT TAC TGC TCT GGC TCC TAG GAC TCA TCG TAC TCC TGC TTG Syn1 ACG TGT CTA CCC ACA ACT TGT ACC TG CTT GAG CAG ATT GCC ATG TC ACC TCA ATA ATG TGA TCC CTT CC Kiss1 CGG ACT GCT GGC CTG TGG AT CTG CTT CTC CTC TGT GTC G TTC CCA GGC ATT AAC GAG TTC Kiss1r TCA ATC CGC TGC TCT ATG CCT TCC CTC ACT GCA TGT CCT ACA GC GCC TGT CTG AAG TGT GAA CC Tac2 AGC TTT GTC CTT CAG GCA CCA TGA CTG CAC TCT TGT CTC TGT CT ACA GCC GCA AAC AGC AT Tacr3 TCT CTT GAA GCC TGC ACG AAA TCT TTT G AGC TCA ACC ATG TAC AAC CC CTC ATC GTA GCT GGA GAC TTG Pdyn TCA ACC CCC TGA TTT GCT CCC TG GTG CAG TGA GGA TTC AGG ATG CAT GTC TCC CAC TCC TCT GA Opkr1 AGA GAA TTG CCC ACT AAG CCC ACC CAT CAC CGC TGT CTA CTC TG GGT CTT CAT CTT CGT GTA TCG G Ar ACC ACA TGC ACA AGC TGC CTC T CTG CCT TGT TAT CTA GCC TCA ATA CTG AAT GAC CGC CAT CTG Esr1 TGC CTT CCA CAC ATT TAC CTT GAT TCC T CCT GTT TGC TCC TAA CTT GCT GAA CCG ACT TGA CGT AGC C Pgr AGA TTC AGA AGC CAG CCA GAG CC CGC CAT ACC TTA ACT ACC TGA G CCA TAG TGA CAG CCA GAT GC Property Two-tailed unpaired Welch-
corrected Student’s t testMean difference Effect size, Cohen’s d Anogenital distance (mm) t(30.13) = 10.052,
p < 0.0001Diff 2.06 [CI 1.64, 2.48] d = 3.03 Body mass (g) t(32.03) = 2.331,
p = 0.0262Diff 2.26 [CI 0.29, 4.24] d = 0.707 Property χ2 test Estrus Diestrus Proestrus Estrous cycle stage distribution χ2 = 111.392,
n = 992,
df = 2,
p < 0.0001Std resid: −1.79 7.08 10.11 Fisher’s exact test; Bonferroni adjusted: p = 0.226 p < 0.0001 p < 0.0001 Bold indicates p < 0.05. Std resid, standardized residuals of PNA group from χ2 test.
Parameter Breslow–Day test for independence Mantel–Haenszel χ2 Proportion firing χ2 = 1.285, df = 1, p = 0.257 χ2 = 0.069, df = 1, p = 0.7932 Parameter Age PNA treatment Interaction Firing frequency Diff −0.193
[CI −0.537, 0.151]Diff −0.104
[CI −0.448, 0.240]Diff 0.130
[CI −0.558, 0.819]F(1,66) = 1.251; p = 0.267 F(1,66) = 0.362;
p = 0.549F(1,66) = 0.143;
p = 0.707Independence of treatment and age with firing proportion was assessed with Breslow–Day test. This was followed by the Mantel–Haenszel χ2 test with continuity correction to determine the effect of treatment on firing proportion when controlling for age (Simonoff, 2003). A two-way ANOVA was conducted for firing frequency.
- Table 4
Statistical parameters from three-way mixed model ANOVA assessing the effect of age and PNA treatment on the KNDy cell response to senktide (Fig. 3)
Effect Statistic Generalized η2 Senktide F(1,38) = 52.35, p < 0.0001 0.395 Age F(1,38) = 0.7515, p = 0.391 0.010 PNA treatment F(1,38) = 2.235, p = 0.143 0.030 Senktide × age F(1,38) = 0.5459, p = 0.465 0.007 Senktide × PNA treatment F(1,38) = 2.051, p = 0.160 0.025 Age × treatment F(1,38) = 0.2633, p = 0.611 0.004 Senktide × age × PNA treatment F(1,38) = 1.179, p = 0.284 0.015 Bonferroni’s multiple comparisons test Mean diff (Hz), 95% CI of diff Statistic SK 3wk-CON – C 3wk-CON Diff 2.36, [CI 0.59, 4.13] t(38) = 4.055, p = 0.003 SK 3wk-PNA – C 3wk-PNA Diff 1.04, [CI −0.41, 2.49] t(38) = 2.192, p = 0.415 SK adult-CON – C adult-CON Diff 2.178, [CI 0.40, 3.95] t(38) = 3.743, p = 0.007 SK adult-PNA – C adult-PNA Diff 2.00, [CI 0.66, 3.34] t(38) = 4.540, p = 0.001 C 3wk-PNA – C 3wk-CON Diff 0.11, [CI −1.51, 1.72] t(76) = 0.194, p > 0.999 C adult-PNA – C adult-CON Diff −0.18, [CI −1.75, 1.39] t(76) = 0.340, p > 0.999 SK 3wk-PNA – SK 3wk-CON Diff −1.21, [CI −2.82, 0.40] t(76) = 2.223, p = 0.350 SK adult-PNA – SK adult-CON Diff −0.36, [CI −1.93, 1.20] t(76) = 0.682, p > 0.999 C adult-CON – C 3wk-CON Diff 0.19, [CI −1.58, 1.95] t(76) = 0.315, p > 0.999 C adult-PNA – C 3wk-PNA Diff −0.10, [CI −1.49, 1.29] t(76) = 0.207, p > 0.999 SK adult-CON – SK 3wk-CON Diff 0.01, [CI −1.76, 1.77] t(76) = 0.011, p > 0.999 SK adult-PNA – SK 3wk-PNA Diff 0.86, [CI −0.53, 2.25] t(76) = 1.825, p = 0.863 The effect size, generalized η2, is reported for each effect. Bonferroni multiple comparisons for cells that differ by only one factor. Bold indicates p < 0.05.
Property Age PNA treatment Interaction Burst frequency Diff −0.030
[CI −0.086, 0.025]Diff −0.014
[CI −0.070, 0.041]Diff 0.023
[CI −0.088, 0.134]F(1,66) = 1.184; p = 0.281 F(1,66) = 0.267; p = 0.608 F(1,66) = 0.167; p = 0.684 Burst duration Diff 0.116
[CI 0.011, 0.221]Diff 0.063
[CI −0.042, 0.168]Diff 0.116
[CI −0.94, 0.326]F(1,35) = 5.068; p = 0.031 F(1,35) = 1.479; p = 0.232 F(1,35) = 1.263; p = 0.269 Spikes per burst Diff 0.954
[CI −0.178, 2.087]Diff 0.560
[CI −0.572, 1.693]Diff 0.955
[CI −1.310, 3.220]F(1,35) = 2.927; p = 0.096 F(1,35) = 1.009; p = 0.322 F(1,35) = 0.733; p = 0.398 Intraburst interval Diff 0.002
[CI −0.039, 0.044]Diff 0.021
[CI −0.021, 0.062]Diff −0.003
[CI −0.085, 0.079]F(1,35) = 0.013; p = 0.911 F(1,35) = 1.021; p = 0.319 F(1,35) = 0.005; p = 0.942 Single spike frequency Diff −0.073
[CI −0.187, 0.041]Diff −0.015
[CI −0.129, 0.099]Diff −0.122
[CI −0.350, 0.106]F(1,66) = 1.63; p = 0.206 F(1,66) = 0.072; p = 0.790 F(1,66) = 1.147; p = 0.288 Interburst interval Diff −14.87
[CI −84.09, 54.36]Diff 13.72
[CI −55.51, 82.94]Diff 87.73
[CI −50.71, 226.2]F(1,42) = 0.188; p = 0.667 F(1,42) = 0.160; p = 0.691 F(1,42) = 1.635; p = 0.208 Bold indicates p < 0.05.
Transcript Age PNA treatment Interaction Actb Diff −0.018
[CI −0.074, 0.038]Diff −0.019B2
[CI −0.074, 0.037]Diff −0.019
[CI −0.131, 0.092]F(1,28) = 0.436; p = 0.515 F(1,28) = 0.466; p = 0.501 F(1,28) = 0.126; p = 0.725 Syn1 Diff 0.018
[CI −0.038, 0.074]Diff 0.019
[CI −0.037, 0.074]Diff 0.019
[CI −0.092, 0.131]F(1,28) = 0.436; p = 0.515 F(1,28) = 0.466; p = 0.501 F(1,28) = 0.126; p = 0.725 Kiss1 Diff 0.068
[CI −0.358, 0.495]Diff 0.019
[CI −0.407, 0.445]Diff 0.028
[CI −0.824, 0.880]F(1,28) = 0.108; p = 0.744 F(1,28) = 0.009; p = 0.926 F(1,28) = 0.004; p = 0.947 Kiss1r Diff −0.019
[CI −0.170, 0.132]Diff 0.129
[CI −0.022, 0.280]Diff 0.150
[CI −0.152, 0.453]F(1,28) = 0.068; p = 0.796 F(1,28) = 3.062; p = 0.091 F(1,28) = 1.038; p = 0.317 Tac2 Diff 1.075
[CI 0.584, 1.566]Diff 0.009
[CI −0.482, 0.500]Diff 0.264
[CI −0.718, 1.245]F(1,28) = 20.113; p = 0.0001 F(1,28) = 0.001; p = 0.97 F(1,28) = 0.302; p = 0.587 Tacr3 Diff 1.064
[CI 0.707, 1.420]Diff 0.108
[CI −0.249, 0.464]Diff −0.476
[CI −1.189, 0.238]F(1,28) = 37.321; p < 0.0001 F(1,28) = 0.381; p = 0.542 F(1,28) = 1.865; p = 0.183 Pdyn Diff 0.138
[CI −0.224, 0.499]Diff 0.331
[CI −0.030, 0.693]Diff −0.153
[CI −0.876, 0.571]F(1,28) = 0.608; p = 0.442 F(1,28) = 3.520; p = 0.071 F(1,28) = 0.187; p = 0.669 Opkr1 Diff 0.127
[CI −0.044, 0.298]Diff 0.095
[CI −0.076, 0.266]Diff −0.170
[CI −0.512, 0.172]F(1,28) = 2.313; p = 0.139 F(1,28) = 1.294; p = 0.265 F(1,28) = 1.033; p = 0.318 Ar Diff 0.673
[CI 0.493, 0.854]Diff 0.041
[CI −0.140, 0.221]Diff 0.150
[CI −0.212, 0.511]F(1,28) = 58.323; p < 0.0001 F(1,28) = 0.215; p = 0.647 F(1,28) = 0.720; p = 0.403 Esr1 Diff 0.091
[CI −0.195, 0.377]Diff 0.002
[CI −0.284, 0.288]Diff −0.105
[CI −677, 0.467]F(1,28) = 0.428; p = 0.519 F(1,28) = 0.0002; p = 0.989 F(1,28) = 0.141; p = 0.71 Pgr Diff 0.955
[CI 0.585, 1.325]Diff −0.114
[CI −0.484, 0.257]Diff 0.076
[CI −0.664, 0.817]F(1,28) = 27.92; p < 0.0001 F(1,28) = 0.395; p = 0.535 F(1,28) = 0.045; p = 0.834 Bold indicates p < 0.05.
Extended Data 1
Code used to detect and analyze events in Igor Pro (coding-project) and for data analysis in R (PNA-KNDy). Download Extended Data 1, ZIP file.
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