Periodic Remodeling in a Neural Circuit Governs Timing of Female Sexual Behavior

Abstract

Behaviors are inextricably linked to internal state. We have identified a neural mechanism that links female sexual behavior with the estrus, the ovulatory phase of the estrous cycle. We find that progesterone-receptor (PR)-expressing neurons in the ventromedial hypothalamus (VMH) are active and required during this behavior. Activating these neurons, however, does not elicit sexual behavior in non-estrus females. We show that projections of PR+ VMH neurons to the anteroventral periventricular (AVPV) nucleus change across the 5-day mouse estrous cycle, with ∼3-fold more termini and functional connections during estrus. This cyclic increase in connectivity is found in adult females, but not males, and regulated by estrogen signaling in PR+ VMH neurons. We further show that these connections are essential for sexual behavior in receptive females. Thus, estrogen-regulated structural plasticity of behaviorally salient connections in the adult female brain links sexual behavior to the estrus phase of the estrous cycle.

Keywords: anteroventral periventricular nucleus; emotional behavior; estrogen, progesterone; estrous cycle; plasticity; sexual behavior; sexually dimorphic behavior; ventromedial hypothalamus.

Figure 1:. Female Pvl neurons are active during mating

(A) Pvl neurons are required for behavioral estrus, but whether Pvl neurons link behavioral estrus to physiological estrus is unknown. (B) Strategy to express virally encoded, Cre-dependent transgenes in Pvl neurons of PR^Cre females. Middle: Coronal section shows that virally delivered mCherry is expressed in VMHvl but not surrounding regions [scale bars = 500μm and 100 μm (inset)]. Right: mCherry is expressed in PR^Cre/PL but not PR^PL/PL neurons (scale bar = 10 μm). (C,D) Vast majority of mCherry+ or nβgal + Pvl neurons express nβgal or mCherry, respectively. Each dot in a bar graph in this and other Figures is data from one mouse. (E-O) Fiber photometry imaging setup and activity of Pvl neurons in PR^Cre females upon insertion into test cage. (G) Cre-dependent GCaMP6s expression in Pvl neurons (scale bar = 100 μm). (H-K) Activation of Pvl neurons in PR^Cre female during interactions with WT male or primed female or wood block. (H,J) Peri-event time plot (PETP) of GCaMP6s fluorescence around entry into test cage (H) and sniffing of resident animal or object (J). In this and Figure S1, dark line and lighter shading in same color indicates Mean and SEM of change in fluorescence for that group of mice. (I,K) Pvl neurons are activated more upon entry into male cage (I) or upon sniffing male (K). (L-O) Activity of Pvl neurons during mating with WT male. Data from interactions with male are from same animals and tests shown in (H-K). (L-N) Top: PETP of GCaMP6s fluorescence around onset of male sniffing of female (L), male mounting (M), and lordosis (N). Bottom: Heat map of PETP, with individual events/row; data from a single female. (O) Increased activation of Pvl neurons upon being mounted and during lordosis compared to being sniffed. Mean ± SEM. n = 3 (C,D); n = 10 (H-O). *p<0.05, **p<0.01. See also Figure S1, Table S1, and Movie S1.

Figure 2:. Activity of Pvl neurons is necessary but not sufficient for female sexual receptivity

(A-E) Chemogenetic inhibition of Pvl neurons with DREADDi. (B-E) Primed females given CNO show diminution in sexual behavior and an increase in time rejecting males, leading to fewer males ejaculating during the test. Lordosis quotient = (# lordosis events)/(# mounts or intromissions); Receptivity index = (# intromissions)/(# mounts). (F-J) Chemogenetic activation of Pvl neurons with DREADDq. (G-J) No increase in sexual behavior of unprimed females 30 or 90 min after CNO. (K-O) Optogenetic activation of Pvl neurons with ChR2. (L-O) No increase in sexual behavior of unprimed females upon laser illumination of ChR2+ Pvl neurons. Mean ± SEM. n = 8 (B-E); n = 5,6 (G-J); n = 4 (L-O). *p<0.05, **p<0.01. See also Figure S2.

Figure 3.. Ovarian sex hormones increase presynaptic termini of Pvl neurons in AVPV

(A-F) Labeling presynaptic termini of female Pvl neurons. (B) Pvl neurons send ~1.2 mm projections to AVPV. (C) More presynaptic termini (mCherry+) of Pvl neurons in AVPV visualized in primed females. Insets are higher magnification images of box outlined in top panels. (D-F) Higher density of mCherry+ Pvl termini in AVPV but not other targets in primed females. Density of mCherry+ termini in this and other Figures has been normalized to number of mCherry+ soma of Pvl neurons to account for subtle variability in infection of these cells. Change in density represented as fold change compared to unprimed female. (G-K) Labeling presynaptic termini of male Pvl neurons. (H) No visible difference in mCherry+ Pvl termini in AVPV in males under various hormonal regimes. (I-K) No difference in density of mCherry+ Pvl presynaptic termini in AVPV or other targets. Change in density represented as fold change compared to intact male. Mean ± SEM. n = 6 (Vehicle), 7 (Primed) (D-F); n = 4 (Intact), 7 (Vehicle Cx), 6 (Primed Cx) (I-K). Scale bars = 50 μm (C, top) and 10 μm (C insets, H). **p<0.01. See also Figure S3.

Figure 4.. More Pvl projection termini in AVPV of naturally cycling estrus females

(A) Strategy to examine presynaptic termini of Pvl neurons in gonadally intact females. (B) More mCherry+ Pvl termini in AVPV visualized in estrus female. (C-E) Higher density of mCherry+ Pvl termini in AVPV, but not other target regions, in estrus female. Change in density represented as fold change compared to diestrus female. Mean ± SEM. n = 11 (Diestrus), 17 (Estrus) (C-E). Scale bar = 10μm. **p<0.01. See also Figure S4.

Figure 5.. Ovarian sex hormones increase excitatory inputs to AVPV.

(A-C) Examining Fos induction in AVPV following activation of Pvl neurons. (B,C) Activating Pvl neurons with CNO induces Fos in AVPV. More Fos+ AVPV neurons in primed than unprimed females. Counts of Fos+ AVPV neurons were normalized to number of DREADDq+ Pvl neurons for the respective experimental condition, and they are represented as fold change compared to unprimed female given saline. (D-G) mEPSC recording with whole-cell voltage-clamp of AVPV neurons. (E) Sample mEPSC traces recorded from AVPV neurons in unprimed and primed female. (F) Shift in cumulative probability distribution of mEPSC inter-event interval (left) and higher mean mEPSC frequency (right) in primed than unprimed females. (G) No difference in cumulative probability plots of mEPSC amplitudes (left) or mean amplitude (right) of mEPSCs between primed and unprimed females. (H-J) Whole-cell voltage-clamp recording of oEPSCs in AVPV neurons elicited by 450nm laser illumination of ChR2+ Pvl axons. (I) Sample oEPSC traces recorded from AVPV neurons primed and unprimed female. (J) Larger peak amplitudes of oEPSCs in AVPV from primed compared to unprimed females. Mean ± SEM. n = 3/condition (C); n = 24 (Unprimed), 25 (Primed) (F, G), 40 (Unprimed), 34 (Primed) (J) cells. Scale bar = 20μm. *p<0.05, **p<0.01, ***p<0.001. See also Figure S5.

Figure 6.. Inhibiting Pvl projection termini in AVPV reduces female sexual receptivity

(A) Experimental strategy to inhibit Pvl projection termini in AVPV in primed females. (B) Coronal sections showing eNpHR3.0+ Pvl neurons in VMHvl and their projection termini in AVPV. For clarity, left VMHvl and left and right AVPV are shown. (C-F) Continuous illumination of Pvl projection termini in AVPV reduces lordosis and increases rejection of male mating attempts, leading to fewer males ejaculating during the test. (G-J) Intermittent illumination of Pvl projection termini in AVPV reduces lordosis and increases rejection of male mating attempts, leading to fewer males ejaculating when the laser is switched on. Mean ± SEM. n = 4 (EYFP), 9 (eNpHR3.0) (C-F), 7/condition (G-J). Scale bar = 100 μm. *p<0.05, **p<0.01, ***p<0.001. See also Figure S6 and Movie S2.

Figure 7.. Estrogen signaling regulates plasticity of Pvl projections to AVPV

(A-C) Labeling presynaptic termini of Pvl neurons in vehicle or estrogen treated Ovx females. (B) More mCherry+ presynaptic termini visualized in estrogen primed female AVPV. (C) More mCherry+ presynaptic termini in estrogen primed female AVPV. Change in density represented as fold change compared to vehicle treated female. (D-F) Labeling presynaptic termini of non-Pvl VMH neurons in female. Only Cre− neurons in VMH express Syp:mCherry. (E) mCherry+ presynaptic termini in AVPV are apparent in PR^PL/PL but not PR^Cre/PL females. (F) Lower density of mCherry+ presynaptic termini in AVPV of PR^Cre/PL compared to PR^PL/PL females. Change in density represented as fold change compared to PR^PL/PL female. (G-I) Examining role of Esr1 in plasticity of female VMH neuron projections to AVPV. AAV encoding constitutively expressed Syp:mCherry was delivered bilaterally whereas lentiviruses encoding EGFP or Cre:EGFP were each delivered unilaterally to the VMHvl of Esr1^Flox/Flox females. (H) Fewer mCherry+ presynaptic termini apparent in AVPV of female on the side that received Cre:EGFP (right) compared to the side that received EGFP (left) (I) Fewer mCherry+ presynaptic termini in AVPV upon deletion of Esr1 in ipsilateral VMH. Change in density represented as fold change compared to control (EGFP) side. Mean ± SEM. n = 4 (Vehicle), 10 (Estrogen) (C), n = 3/genotype (F), n = 6 (I). Scale bar = 10 μm. *p<0.05, **p<0.01. See also Figure S7.