Dynamics of pulsatile activities of arcuate kisspeptin neurons in aging female mice
- Laboratory for Comparative Connectomics, RIKEN Center for Biosystems Dynamics Research, Japan
Figures
Figure 1 with 1 supplement
Dynamics of synchronous episodes of ARCkiss (SEskiss) in the regular estrus cycle of the reproductive phase.
(A) Representative 6 h photometry raw data of SEskiss in each stage of the estrus cycle. Red arrowheads indicate SEskiss. (B) Numbers of SEskiss (lower: line plots) along with the estrus cycle stages (upper: color bars) for 7 days (n=4 animals). SEskiss were recorded for 6 h in both light and dark (LD) periods. (C) The ratio of SEskiss in each dark period normalized to those in the preceding light period. Dotted line = 0.5. (D) Numbers of SEskiss per 6 h in 5 days along with the estrus cycle anchoring at proestrus as Day 0. Different letters (a–c) in the upper part of the graph denote significant differences at p<0.05 by one-way repeated measures ANOVA followed by the Tukey–Kramer post hoc test. (E) Intensities of SEskiss as measured by the ΔF/F values in each estrus stage normalized to that in the light period of the proestrus. *p<0.05 by one-way repeated measures ANOVA followed by the Tukey–Kramer post hoc test. (F) Averaged traces of SEkiss waveforms in the light period of each estrus stage. (G) Top, averaged traces magnifying the onset and offset of SEskiss in the proestrus and estrus/metestrus stages in the light period. Data are expressed as mean ± SEM. Bottom, p-value by t-test. Red dots represent p-values <0.05. (H) Quantifications of detailed parameters of SEkiss waveforms in each estrus stage: the normalized LWQM, FWQM, and RWQM. *p<0.05 by one-way repeated measures ANOVA followed by the Tukey–Kramer post hoc test. P: proestrus, E: estrus, M: metestrus, D: diestrus.
Figure 1—figure supplement 1
Inter-pulse intervals and waveforms of synchronous episodes of ARCkiss (SEskiss) in each estrus stage.
(A) Inter-pulse intervals of SEskiss in each estrus stage. Different letters (a–c) in the upper part of the graph denote significant differences at p<0.05 by one-way repeated measures ANOVA followed by the Tukey–Kramer post hoc test. (B) SEkiss waveforms in the light and dark periods of each estrus stage. Data are expressed as mean (black line) ± SEM (colored shadow).
Figure 2
Decrease in estrus cycle frequency during the transition to reproductive senescence.
(A) Number of estrus cycles per 25 days at 3–4 (n=5), 8–9 (n=8), 11–12 (n=6), and 14–15 (n=12) months in old wild-type C57BL/6 mice. Different letters (a, b) in the upper part of the graph denote significant differences at p<0.05 by one-way ANOVA followed by the Tukey–Kramer post hoc test. (B) Chronic monitoring of estrus cycle frequency from the individuals used for Ca2+ imaging of synchronous episodes of ARCkiss (SEskiss) (C57BL/6 Kiss-Cre mice with AAV-mediated GCaMP6s expression). Black: average number of estrus cycles, gray: individual data (n=12 animals in total).
Figure 3 with 2 supplements
Chronic monitoring of synchronous episodes of ARCkiss (SEskiss) from the reproductive to acyclic phase.
(A) Representative photometry raw data of SEskiss in the diestrus stage from the reproductive to acyclic phase (n=2 animals). Red arrowheads indicate SEskiss. (B) Numbers of SEskiss in the estrus stages (color bars) for 7 days from the reproductive to acyclic phase. SEskiss were recorded for 6 h in both light and dark periods. (C) Number of SEskiss in individual estrus stages (shown in different colors) of individual female mice. ns: not significant by one-way ANOVA. (D) Cumulative probability of inter-pulse interval. ns: not significant by the Kolmogorov–Smirnov test. (E) Peak height as assessed by ΔF/F of SEskiss from the reproductive to acyclic phase. Different letters (a–d, w–z) in the lower part of the graph denote significant differences at p<0.05 by the Kruskal–Wallis test followed by the Mann–Whitney U test. Re; reproductive, RC; regular cyclic, IrC; irregular cyclic, Ac; acyclic.
Figure 3—figure supplement 1
Raw data of the estrus cycle, related to Figure 3.
Graphical representation of estrus stage within a 25 day time window from the reproductive to acyclic phase (n=2 animals). Red dots and lines represent the photometric recording timing shown in Figure 3B. Arrows indicate one estrus cycle.
Figure 3—figure supplement 2
Waveforms and intensities of synchronous episodes of ARCkiss (SEskiss), and gene expression analysis.
(A) Averaged traces of SEkiss waveforms of the same female mice in the reproductive (Re), regular cyclic (RC), irregular cyclic (IrC), and acyclic (Ac) phases. (B) Quantifications of detailed parameters of SEkiss waveforms in individual female mice: LWQM, FWQM, and RWQM as defined in Figure 1. Different letters (a–b, x–y) in the upper part of the graph denote significant differences at p<0.05 by the Kruskal–Wallis test followed by the Mann–Whitney U test. In ID#5, the shape of the waveform was changed in the IrC compared with the Re phase, and the shape reverted in the Ac phase. In ID#6, significant differences in the length of LWQM and RWQM were observed between the Re and Ac phases. The origin of these individual differences is unknown, but the bias of estrus cycles in aging female mice might underlay the variations. (C) Coefficient of variances of the intensities of SEskiss in a 6 h window from Re, RC, IrC, and Ac individual mice. *p<0.05 by the Kruskal–Wallis test followed by the Mann–Whitney U test. (D) The gene expression levels of Kiss1, Tac2, and Pdyn relative to a reference gene were compared among mice in Re, RC, IrC, and Ac phases (n=8, 8, 8, and 6, respectively) during the diestrus stage of the estrous cycle. Each data point was normalized to an average of the corresponding gene expression in Re phase. ns: not significant by the Kruskal–Wallis test.
Figure 4 with 1 supplement
Characterization of synchronous episodes of ARCkiss (SEskiss) in aging female mice.
(A) Representative numbers of SEskiss in the estrus stages (color bars) for 7 days from regular cyclic, irregular cyclic, and acyclic female mice. SEskiss were recorded for 6 h in both light and dark periods. (B) The ratio of SEskiss in the dark period normalized to those in the preceding light period of regular cyclic (n=4), irregular cyclic (n=3), and acyclic (n=3) female mice, respectively. Dotted line = 0.5. (C) Average numbers of SEskiss in 6 h in the light (open boxes in the LD cycle) and dark (closed boxes in the LD cycle) periods of reproductive (Re), regular cyclic (RC), irregular cyclic (IrC), and acyclic (Ac) mice. Red horizontal lines indicate the mean. No interaction effect between estrus cyclicity and LD cycle was found by two-way ANOVA. *p<0.05 by paired t-test. Of note, the Re group (age 4–6 months) represents a reanalysis of the 5 days of data reported in Figure 1 (corresponding to one estrus cycle). (D) Cumulative probability of inter-peak intervals among the Re (black), RC (green), IrC (blue), and Ac (orange) groups. ns: not significant by the Kolmogorov–Smirnov test. (E) Intensities of SEskiss during reproductive aging as assessed by the fold change of ΔF/F values in the IrC (#ID8, 9) and Ac (ID#7, 10) phases normalized to those in the RC phase. *p<0.05 by the Mann–Whitney U test. ns: not significant.
Figure 4—figure supplement 1
Histochemical analysis after imaging.
(A) Representative coronal sections of the arcuate nucleus (ARC) showing Tac2 mRNA expression (magenta), a marker of kisspeptin neurons in the hypothalamic arcuate nucleus (ARCkiss) neurons, and GCaMP6s (green) expression, counterstained with DAPI (blue) in control (healthy reproductive phase without potential damage caused by repeated photometry recording and/or prolonged expression of GCaMP6s, see Methods) and the aging recorded mice. The expressions of Tac2 mRNA and GCaMP6s protein were well comparable between the reproductive healthy control and aging recorded samples. Scale bars, 100 µm. (B) Left: Quantification of the GCaMP6s targeting efficiency (Tac2+ GCaMP6s+/Tac2+). Middle: The number of GCaMP6s+ cells. Right: The number of Tac2+ cells in the GCaMP6s targeted site normalized to that of the contralateral non-injected and non-imaged site. ns, no significant difference was found between the control and recorded groups by the Mann–Whitney U test. n=5 and 3 in control and recorded mice, respectively. Error bars, SEM.
Author response image 1
Analysis of RWQM values during the diestrus stage in aging female mice.
(A) Quantifications of RWQM of SEkiss waveforms in two individual aging female mice (those analyzed in Figure 3). A typical decline in RWQM values is observed from reproductive (Re) to regular cyclic (RC) during aging, and then they increase again as the estrus cycle becomes irregular cyclic (IrC) or acyclic (Ac). Different letters (a–c, x–z) denote significant differences at P < 0.05 by the Kruskal–Wallis test followed by the Mann–Whitney U test. (B) The fold-change of RWQM values from RC to either IrC or Ac phase in four aging female mice (those analyzed in Figure 4). Unlike ID#5 and #6, these animals display a further reduction of RWQM values during the reproductive senescence. Although the origin of these individual variations remains unclear, our data show that 5 out of 6 aging animals exhibit significant changes in RWQM values during the diestrus phase associated with the transition from RC to IrC or Ac.
Additional files
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MDAR checklist
- https://cdn.elifesciences.org/articles/82533/elife-82533-mdarchecklist1-v1.docx
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Source code 1
A custom-made code in R for analyzing calcium signals.
- https://cdn.elifesciences.org/articles/82533/elife-82533-code1-v1.zip
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Dynamics of pulsatile activities of arcuate kisspeptin neurons in aging female mice
eLife 12:e82533.
https://doi.org/10.7554/eLife.82533
