GCaMP expression in RP3VKISS neurons.

Confocal images of (A) GFP (GCaMP6s, green) and (B) kisspeptin (red) and immunofluorescence in the RP3V, and (C) an overlay of both in the periventricular nucleus of a proestrous female mouse. Scale bar = 100 μm.

GCaMP signals in RP3VKISS neurons across the estrous cycle.

(A) Coronal section showing location of tapered optic fibre (white outline) in relation to GCaMP-expressing cells (green) lining the third ventricle (3V). Scale bar = 100 μm. (B&C) Two representative examples of 22-hour GCaMP fibre photometry recordings across the complete estrous cycle in two mice. Light-off period is represented by grey shaded area. (D) Mean area under the curve (AUC) calculated from 22-hour recordings across the estrous cycle: metestrus (M), diestrus (D), proestrus (P), and estrus (E). Each dot represents an animal (N=8). Friedman test followed by Dunn’s multiple comparisons tests. * p<0.05, *** p<0.001. (E&F) Examples of 22-hour GCaMP fibre photometry recordings from two proestrous female mice with misplaced optic fibers in which signals remain < 3% of baseline.

Deconvolution of RP3VKISS neuron GCaMP signals and relation to the LH surge on proestrus.

(A,B) Two representative examples showing the relationship between the increase in GCaMP activity (black) with luteinizing hormone (LH) surge (red). Light-off period is represented by grey shaded area. (C,D) Representative examples of 22-hour photometry recordings in proestrus from the same two female mice in Figure 2 showing (i) the original recording, (ii) a 30-minute moving average highlighting the baseline oscillations (purple) with identified oscillations labelled with dots, (iii) high frequency transients (green) after the moving average is subtracted from the original recording, (iv) expanded views of the traces showing moving average and high frequency transients. Light-off period is represented by grey shaded area.

Baseline oscillations of RP3VKISS neurons during proestrus.

(A-D) Individual baseline oscillation traces from 22-hour recordings for each animal across each of the estrous cycle stages (n=8). (E) Mean number of oscillations identified across each stage of the estrous cycle. Each dot represents an animal (Friedman Test: χ²=18.75, p=0.0003; Dunn’s multiple comparisons tests versus proestrus: p=0.0402 (metestrus), p=0.0029 (diestrus), p=0.0117 (estrus). (F) Mean baseline oscillations showing variations across the estrous cycle: metestrus (M, red), diestrus (D, blue), proestrus (P, green), and estrus (E, purple). Shaded regions around the traces indicate ± SEMs of corresponding colours (N=8). Triangle marks the oscillation occurring before lights-off. Light-off period is represented by grey shaded area.

Changes in RP3VKISS neuron high frequency transients across the cycle.

(A) An example of high frequency transient signals across the estrous cycle in one mouse. Identified significant transients are highlighted in yellow and expanded views given below. Note different y-axes for diestrus and proestrous graphs. (B) Mean frequency (Hz), (C) amplitude (dF/F, %) and (D) duration (seconds) of transients in metestrus (M), diestrus (D), proestrus (P), and estrus (E). Each dot represents an animal (N=8). Repeated measures one-way ANOVA followed by Tukey’s multiple comparisons test; * p<0.05, ** p<0.01, *** p<0.001 (see text for exact p values).

Variable oscillations in RP3VKISS neuron activity across proestrous surges in the same mice.

(A&B) Two representative examples showing code-extracted baseline profiles of RP3VKISS neuron GCaMP activity across three proestrous surges in the same mouse. Light-off period is represented by grey shaded area. Heat maps below show calcium signal dynamics (dF/F) with each row representing one proestrus. Colour intensity indicates dF/F values, with yellow and green colours representing higher signal amplitudes. Legends of heat maps show colour codes for dF/F (%).

Effects of ovariectomy and estrogen replacement on RP3VKISS neuron activity.

Experimental plan is shown at the top with times of photometry recordings highlighted in blue. Representative examples of 22-hour photometry recordings from two female mice (right and left) in (A) diestrus, (B) one week after OVX, (C) five days after the estradiol implant (OVX+E2), (D) on the day of estradiol benzoate injection (OVX+E2+EB), (E) at the time of the expected estradiol-induced LH surge (OVX+E2+EB surge), and for comparison (F) in proestrus (before ovariectomy). Light-off period is represented by grey shaded area.

Changing RP3VKISS neuron basal and transient activity following OVX + estrogen treatment regimens.

(A) Mean area under the curve (AUC) of 22-hour recordings for mice in diestrus (D) and following their ovariectomy (OVX), paired t-test: p=0.002 (B) Mean AUC calculated in OVX, OVX+E2, OVX+E2+EB, and OVX+E2+EB surge conditions. Friedman test: χ²=10.68, p=0.0055, Dunn’s multiple comparisons test p=0.021. (C) Mean AUC from intact proestrous mice (P) compared to the same mice under OVX+E2+EB surge conditions, paired t-test: p=0.014. Each dot represents one animal (N=5). (D) Mean moving average of baseline recordings over 22-hour recordings showing variations after OVX (yellow), five days after estradiol implant (OVX+E2, orange), on the day of EB injection (OVX+E2+EB, aqua), and on the day of OVX+E2+EB surge (plum). Shaded regions around the traces indicate SEMs of corresponding colours (n=5). Triangle marks the pre-lights-off oscillation. Light-off period is represented by grey shaded area. (E) Example of high frequency transient activity from a representative mouse 7 days after OVX and then continuously for three days encompassing the fifth day after E2 implant (OVX+E2), on the day of EB injection (OVX+E2+EB), and the day of the expected OVX+E2+EB LH surge. Triangle indicates time of EB injection. Identified significant transients are highlighted in yellow. Expanded views of the traces are shown below.

High frequency transients parameters in different treatment groups (Mean ± SEM)

Comparison between RP3VKISS and GnRH neuron activity patterns on proestrus.

Mean 30-minute moving average of 22-hour GCaMP recordings across proestrus from RP3VKISS neurons (pink; taken from Fig.4F; N=8) and GnRH neuron dendron activity (green; raw data obtained from (Han, Yeo et al. 2025); n=7) processed in the same manner. Shaded regions around the traces indicate ± SEMs of corresponding colours. Text provides mean ± SEM duration of total elevated activity, duration of each oscillation, and variation in onset of oscillations between animals.