Estrous Cycle Mediates Midbrain Neuron Excitability Altering Social Behavior upon Stress

Abstract

The estrous cycle is a potent modulator of neuron physiology. In rodents, in vivo ventral tegmental area (VTA) dopamine (DA) activity has been shown to fluctuate across the estrous cycle. Although the behavioral effect of fluctuating sex steroids on the reward circuit is well studied in response to drugs of abuse, few studies have focused on the molecular adaptations in the context of stress and motivated social behaviors. We hypothesized that estradiol fluctuations across the estrous cycle acts on the dopaminergic activity of the VTA to alter excitability and stress response. We used whole-cell slice electrophysiology of VTA DA neurons in naturally cycling, adult female C57BL/6J mice to characterize the effects of the estrous cycle and the role of 17β-estradiol on neuronal activity. We show that the estrous phase alters the effect of 17β-estradiol on excitability in the VTA. Behaviorally, the estrous phase during a series of acute variable social stressors modulates subsequent reward-related behaviors. Pharmacological inhibition of estrogen receptors in the VTA before stress during diestrus mimics the stress susceptibility found during estrus, whereas increased potassium channel activity in the VTA before stress reverses stress susceptibility found during estrus as assessed by social interaction behavior. This study identifies one possible potassium channel mechanism underlying the increased DA activity during estrus and reveals estrogen-dependent changes in neuronal function. Our findings demonstrate that the estrous cycle and estrogen signaling changes the physiology of DA neurons resulting in behavioral differences when the reward circuit is challenged with stress.SIGNIFICANCE STATEMENT The activity of the ventral tegmental area encodes signals of stress and reward. Dopaminergic activity has been found to be regulated by both local synaptic inputs as well as inputs from other brain regions. Here, we provide evidence that cycling sex steroids also play a role in modulating stress sensitivity of dopaminergic reward behavior. Specifically, we reveal a correlation of ionic activity with estrous phase, which influences the behavioral response to stress. These findings shed new light on how estrous cycle may influence dopaminergic activity primarily during times of stress perturbation.

Keywords: dopamine; estrogen; estrous cycle; potassium channel; stress; ventral tegmental area.

Figure 1.

The estrous cycle modulates electrophysiological properties of VTA DA neurons. A three-pronged approach to determining estrous cycle stage was used in terminal experiments. a, A representation of the estrous cycle and relative estrogen and progesterone levels at each stage of estrous. b, Measured serum concentrations of estrogen (E2), progesterone (P4), and uterine weights at each estrous cycle stage determined by vaginal cell cytology. c, Uterine weight was significantly higher in animals during proestrus (F_(3,32) = 23.52, p < 0.0001, n = 7–12 mice per group). Scale bar, 1 cm. d, Vaginal cytology showing leucocytes, nucleated epithelial cells, and cornified epithelial cells at each stage of the estrous cycle. Scale bars: 100 μm. Proestrus is typified by the presence of small, round, nucleated epithelial cells, which are often observed in clusters. Estrus was typified by the presence of anucleated keratinized epithelial cells and the absence of neutrophils. Metestrus is typified by the presence of anucleated keratinized epithelial cells and neutrophils. Diestrus is typified by the presence of primarily neutrophils. Arrows indicate time points used for electrophysiology studies. e, Spontaneous firing rate does not change across the estrous cycle (1-way ANOVA, F_(3,128) = 1.88, p = 0.14, n = 28–39 cells per estrous stage, 7–12 mice per group; nested ANOVA, F_(3,33) = 1.23, p = 0.32, 2–7 cells per mouse, 7–12 mice per group). f, Excitability is increased during estrus compared with proestrus and metestrus (2-way ANOVA, current injection × estrous cycle stage interaction, p < 0.05, F_(12,360) = 3.28, p = 0.0002, n = 10–33 = cells per group, 7–12 mice per group; nested ANOVA, +100 pA, F_(3,32) = 4.44, p = 0.006; +75 pA, F_(3,32) = 4.37, p = 0.01, 2–7 cells per mouse, 7–12 mice per group). g, I_h across the estrous cycle does not change (2-way ANOVA, voltage step × cycle interaction, F_(24,719) = 0.368, p = 0.99, n = 15–33 cells, 7–12 mice per group). h, Action potential width across the estrous cycle (2-way ANOVA, estrous stage vs AP width, F_(3,94) = 5.044, p = 0.002, n = 28–39 cells per estrous stage, 7–12 mice per group; nested ANOVA, F_(3,133)= 6.17, p = 0.0006, 2–7 cells per mouse, 7–12 mice per group). i, I–V curves obtained from neurons across the estrous cycle (2-way ANOVA, +50 pA current injection × estrous cycle stage interaction, F_(3,16) = 1.427, p = 0.271, n = 5–12 = cells per group, 3 mice per group; nested ANOVA, +50 pA, F_(3,8) = 1.29, p = 0.34, n = 2–4 cells per mouse, 3 mice per group). j, Peak potassium current across the estrous cycle is increased in diestrus (2-way ANOVA, voltage step × cycle interaction, F_(33,594) = 4.835, p < 0.0001; multiple comparisons, *p < 0.05 **p < 0.01 between estrus and diestrus; +p < 0.05 between proestrus and diestrus; nested two-way ANOVA, significant voltage × cycle interaction, F_(33,429) = 4.88, p < 0.001, 2–3 cells per mouse, 7–12 mice per group). Sustained potassium current is increased during metestrus (2-way ANOVA significant voltage × cycle interaction, F_(33,550) = 2.913, p < 0.0001; multiple comparisons, #p < 0.01 between estrus and metestrus, n = 8–22 cells per phase, 7–12 mice per group; nested 2-way ANOVA, significant voltage × cycle interaction, F_(33,429) = 3.35, p < 0.001, 2–3 cells per mouse, 7–12 mice per group). Error bars indicate mean ± SEM.

Figure 2.

Effect of estradiol incubation on I_h across the estrous cycle. a, During proestrus there was no change in I_h during estradiol incubation (2-way ANOVA, voltage step × treatment interaction F_(9,531) = 0.7959, p = 0.6202, n = 27–34 cells, n = 6 mice). b, During estrus there was no change in I_h during estradiol incubation (2-way ANOVA, voltage step × treatment interaction, F_(9,261) = 0.460, p > 0.9999, n = 13–19 cells, n = 6 mice). c, During metestrus there was no change in I_h during estradiol incubation (2-way ANOVA, voltage step × treatment interaction, F_(9,234) = 0.50, p = 0.86, 13–15 cells, n = 6 mice). d, During diestrus there was no change in I_h during estradiol incubation (voltage step × treatment, F_(9,385) = 1.021, p = 0.422, n = 14-15 cells, n = 6 mice).

Figure 3.

Effects of estradiol incubation and constant perfusion on VTA DA neuron excitability across the estrous cycle. a, Proestrus, estradiol incubation significantly reduces the excitability of DA neurons during proestrus (2-way ANOVA, current step × treatment interaction, F_(7,280) = 3.49, p = 0.001, n = 18–24 cells, n = 6 mice). b, Estrus, estradiol incubation significantly reduces the excitability of DA neurons during estrus (2-way ANOVA, current step × treatment interaction, F_(7,168) = 3.602, p = 0.001, n = 10–16 cells, n = 6 mice). c, Metestrus, estradiol treatment significantly increases excitability during metestrus (2-way ANOVA current step × treatment interaction, F_(7,182) = 3.30, p = 0.003, n = 13–15 cells, n = 6 mice). d, Diestrus, estradiol incubation does not affect excitability during diestrus (2-way ANOVA, current step × treatment interaction F_(7,189) = 0.363, p = 0.923, n = 18–25 cells, n = 6 mice). (*p < 0.05, **p < 0.005) Error bars indicate mean ± SEM.

Figure 4.

Estrous cycle stage during AVSS influences social interaction behaviors. a, Timeline of acute variable social stress and behavioral testing. b, AVSS increases serum corticosterone (Cort) levels in female mice (t₍₁₅₎ = 8.74, p< 0.0001). c, Social interaction time 24 h after AVSS (t₍₇₀₎ = 1.48, p = 0.14, n = 34–38 mice per group). d, Corner zone time is increased 24 h after AVSS (t₍₇₀₎ = 2.61, p = 0.01, n = 34–38). e, There is no significant effect of AVSS on average velocity with no target present (t₍₇₀₎ = 1.37, p = 0.17, n = 34–38). f, Distance traveled with no target present (t₍₇₀₎ = 2.37, p = 0.02, n = 34–38). g, Reduced social interaction time is driven by females in estrus on the day of AVSS stress acquisition (2-way ANOVA, cycle × stress interaction, F_(3,64) = 2.87, p = 0.04, n = 7–12 mice per group). h, Corner zone time is significantly higher in mice that undergo AVSS during estrus compared with unstressed control (2-way ANOVA, cycle × stress interaction, F_(3,64) = 3.47, p = 0.02). i, There is no significant effect of estrous phase on velocity following AVSS (2-way ANOVA, cycle × stress interaction, F_(3,64) = 0.79, p = 0.51). (****p < 0.0001, *p < 0.05, n.s. = not significant) Error bars indicate mean ± SEM.

Figure 5.

Estrous cycle stage during AVSS influences time spent grooming in the the splash test. a, Timeline. b, AVSS does not significantly alter time spent grooming (t₍₄₁₎ = 1.9, p = 0.06, n =21–22). c, AVSS increases the latency to groom (t₍₄₁₎ = 3.1, p = 0.003, n = 21–22). d, Estrous phase has no significant effect on time spent grooming during the splash test in nonstressed mice (open circle) (F_(3,17) = 2.04, p = 0.15, n = 5–6 per cycle phase). AVSS during the estrus phase decreases the time spent grooming (black circles) (2-way ANOVA, cycle × stress interaction, F_(3,35) = 4.20, p = 0.01, n = 5–6 mice per cycle phase). e, There is no significant interaction between cycle and stress in latency to groom (2-way ANOVA, cycle × stress interaction, F_(3,35) = 1.65, p = 0.20, n = 5–6 mice per cycle phase). (*p < 0.05, **p < 0.01) Error bars indicate mean ± SEM.

Figure 6.

VTA infusion of ICI 182,780 before AVSS in female mice during diestrus increases behavioral stress susceptibility. a, In vitro ICI 182,780 bath perfusion increases excitability in VTA brain slices of diestrus mice (2-way ANOVA, F_(7,104) = 2.30, p = 0.032, n = 14 cells, 5 mice). b, Timeline of behavioral assessment following infusion and AVSS in diestrus mice. c, Sample traces of animal location and velocity in the absence of a social target. d, ICI 182,780 in vivo infusion and stress has no significant interaction on time spent in social interaction zone (2-way ANOVA, F_(3,23) = 0.96, p = 0.42, n = 8–12 mice per group). e, Time spent in corner zone (F_(3,23) = 0.43, p = 0.73). f, velocity with no target present (F_(3,23) = 0.53, p = 0.67). g, Sample traces of animal location and velocity with novel female social target. h, ICI 182,780 infusion before AVSS significantly decreases time spent in the interaction zone in the presence of a social target (ICI × stress interaction (F_(3,23) = 6.68, p = 0.002). i, ICI 182,780 infusion before AVSS significantly increases time spent in the corner zone in the presence of a social target (F_(3,23) = 12.2, p <0.0001). j, There is no significant effect of ICI or AVSS on average velocity F_(3,23) = 1.184, p = 0.34). (*p < 0.05, **p < 0.01, n.s. = not significant) Error bars indicate mean ± SEM.

Figure 7.

Retigabine infusion to the VTA decreases behavioral susceptibility to AVSS in mice during estrus. a, In vitro retigabine bath perfusion decreases excitability in VTA brain slices of estrus mice (paired t test, 60 pA step, t₍₁₁₎ = 2.65, *p < 0.05 12 cells, 6 mice). b, Timeline of behavioral assessment following infusion and AVSS in estrus mice. c, Sample traces of animal location and velocity during social interaction test. d, In vivo infusion of retigabine to the VTA before stress increases the time spent in social interaction zone with novel social target (ANOVA, F_(3,28) = 10.32, p < 0.0001, n = 8 mice per group) and e, reduces time spent in corner zone (F_(3,28) = 2.40, p = 0.08). f, There is no significant effect of retigabine on average velocity with the target present (paired t tests, t₍₁₄₎ = 0.763, p = 0.46). Error bars indicate mean ± SEM.