Figure 6. Reproductive-state dependent responses of female VMHvll^Cckar cells during male interaction.

(A) Fiber photometry recording and histology. Shade indicates optic fiber track.

(B) Recording timeline.

(C) Parameters analyzed in the figure.

(D) Representative traces of Ca²⁺ signal of VMHvll^Cckar cells in a solitary females during different reproductive states. Red: transient peaks. Yellow: transient troughs.

(E-F) Frequency (E) and magnitude (F) of spontaneous Ca²⁺ events during estrus diestrus and lactation.

(G) Heatmap of Ca²⁺ traces from individual females (G1) and the average of all females under a specific reproductive state (G2) during male-female interaction. For each animal, signals were normalized to 0–1 by the maximum Ca²⁺ signal across all recording sessions of that animal.

(H) Male entry aligned PETHs of Ca²⁺ signals from all animals under a specified reproductive state.

(I) Peak ΔF/F within the first 60 s after male entry.

(J) The time for the normalized ΔF/F to reach 0.2 after peaking during the male entry.

(K) Mean ΔF/F from 1–10 minutes after male entry.

(L) The accumulated male-female interaction time over 10 minutes when the females were under different reproductive states.

(M1-M3) PETHs of normalized ΔF/F aligned to the offset of male-initiated approach towards the female (M1), value at the offset of approach (M2), change in normalized ΔF/F during the last second of approach (M3).

(N1-N3) Same as (M1-M3) but for female-initiated approach.

(O1-O3) PETHs of normalized ΔF/F aligned to the onset of male investigation of the recording female (O1), Peak ΔF/F during male investigation (O2), and the difference between the peak response and the mean ΔF/F at the preceding baseline (−3 to −1 s) (O3).

(P-Q) Same as (O). Signal aligned to the onset of female investigation of the male (P1-P3) and onset of male mounting attempts (Q1-Q3).

(R) Trace of normalized ΔF/F from an estrous female during copulation. Color shades indicate male behaviors.

(S1-S3) PETHs of normalized ΔF/F aligned to the onset of male mounting (S1), intromission (S2) and ejaculation (S3).

(S4) Mean signal during various male sexual behaviors.

(T1) PETHs of normalized ΔF/F aligned to the onset of female lordosis (red) and wiggling (blue).

(T2) Peak signal during wiggling and lordosis.

(U) The time windows for spontaneous Ca²⁺ transient analysis.

(U) Example traces of Ca²⁺ signals before male introduction (left) and after male removal (right). Top: no ejaculation. Bottom: with ejaculation.

(W and X) Frequency (W) and magnitude (X) of spontaneous Ca²⁺ transients.

Data are mean ± s.e.m. (E, F, I-K, M2-Q2, M3-Q3, S4) One-way ANOVA with repeated measures, followed by Tukey’s multiple comparisons test. (L) Two-way ANOVA, followed by Tukey’s multiple comparisons test, red and blue dots indicate periods when accumulated time differed significantly (p<0.05) between E and D vs. L and D, respectively. (T2) Two-tailed paired t test. (W, X) Two-way ANOVA, followed by Sidak’s multiple comparisons test. *p<0.05; **p<0.01; ***p<0.001.

n = number of animals. (D-Q3) n=9 estrous, 9 diestrous and 7 lactating females. (S) n=9 estrous females. (T2) n=5 females. (W-X) n=7 estrous females.

See also Figures S12–S15.