A Neural Circuit Underlying the Generation of Hot Flushes

Abstract

Hot flushes are a sudden feeling of warmth commonly associated with the decline of gonadal hormones at menopause. Neurons in the arcuate nucleus of the hypothalamus that express kisspeptin and neurokinin B (Kiss1^ARH neurons) are candidates for mediating hot flushes because they are negatively regulated by sex hormones. We used a combination of genetic and viral technologies in mice to demonstrate that artificial activation of Kiss1^ARH neurons evokes a heat-dissipation response resulting in vasodilation (flushing) and a corresponding reduction of core-body temperature in both females and males. This response is sensitized by ovariectomy. Brief activation of Kiss1^ARH axon terminals in the preoptic area of the hypothalamus recapitulates this response, while pharmacological blockade of neurokinin B (NkB) receptors in the same brain region abolishes it. We conclude that transient activation of Kiss1^ARH neurons following sex-hormone withdrawal contributes to the occurrence of hot flushes via NkB release in the rostral preoptic area.

Keywords: chemogenetics; estrogen; hot flashes; kisspeptin; menopause; neurokinin B; optogenetics; temperature regulation.

Figure 1.. Artificial Activation of Kiss1^ARH Neurons Is Sufficient to Drive Heat Dissipation in Both Female and Male Mice

(A) Schematic representation of the viral vectors and injection targets. A unilateral fiber-optic ferrule was implanted above the ARH. (B) DAPI staining of a coronal brain section indicates the fiber-optic terminal in the ARH. Scale bar, 500 μm. (C—E) Telemetry recordings of core body temperature (T-b) show that Kiss1^ARH neuron stimulation (CNO, 1 mg/kg) was sufficient to transiently decrease T-b. (C) 5-day T-b recording of a single mouse, with CNO injected on day 2 at ZT= 13:00 (arrow). The animals were housed on a 12-hr:12-hr lightdark cycle (horizontal bars); Zeitgeber time (ZT) = 12:00 refers to the onset of the dark. (D) Average T-b comparing baseline versus CNO treatment on a scale of 24 hr. (E) Average T-b comparing baseline vs. CNO-treatment on a scale of 2 hr; n = 9; F(1, 144) = 7.50, p = 0.0146. (F) Thermal imaging of tail-skin temperature (T-sk), saline versus CNO-treated females; n = 9; F(1, 128) = 17.07, p < 0.00001. (G) Telemetry recordings of locomotor activity; n = 9; F(1,2178) = 46.48, p < 0.00001. (H) Optogenetic stimulation (2 Hz) of Kiss1^ARH neurons was sufficient to increase T-sk in females; n = 6; F(1,40) = 11.23, p < 0.00001. (I) T-sk in saline versus CNO-treated males; n = 7; F(1,48) = 26.35, p < 0.00001. (J) Optogenetic stimulation (2 Hz) of Kiss1^ARH neurons was sufficient to increase T-sk in males; n = 9; F(1,64) (male) = 12.97, p = 0.0024. F statistics represent the main effect of treatment by two-way repeated measures (RMs)-ANOVA. Error bars represent ± SEM. Crossover design with randomized starts.

Figure 2.. Activation of Kiss1^ARH Neurons Induces Fos Expression in the Rostral POA

(A) Schematic diagram of Kiss1^ARH projections to the POA (numbers indicate distance from Bregma in mm). (B) Serial, coronal sections of hM3Dq:mCherry-transduced Kiss1^Cre neurons with immunohistochemistry for mCherry. (C-E) Representative Fos staining in POA from rostral to caudal (top to bottom). 0.55 (C), 0.25 (D), and 0.15 (E) from Bregma. Left panels: DAPI-stained coronal sections, with pink highlights indicating the POA. Middle and right panels: Fos immunoreactivity in the POA comparing control versus stimulated mice, respectively. (F) There were more Fos-positive cells in the rostral POA of stimulated mice; for Cntl, M = 19.57, SD = 7.56; for Stim, M = 28.73, SD = 13.56; t(83) = 3.06; **p = 0.003. All scale bars, 500 μm.

Figure 3.. Optogenetic Stimulation of Kiss1^ARH Axons in the Rostral POA Promotes a Heat-Dissipation Response in Female Mice

(A) Schematic representation of the targeted viral injection into the ARH and fiber-optic placement 0.5 mm above the POA. (B-D) Optogenetic stimulation (2 Hz) of Kiss1^ARH neuron fibers in the POA was sufficient to transiently decrease T-b. Within-subject comparison of unstimulated baseline (black trace) vs. stimulated (blue trace). (B) 6-day T-b recording of a single mouse, subjected to blue-light stimulation from ZT = 13:00–15:00 on day 2 (blue). (C) Average T-b comparing baseline versus stimulation on a scale of 24 hr. (D) Average T-b comparing baseline vs. stimulation on a scale of 2 hr; n = 5; F(1,72) = 9.589, p = 0.0147. (E) T-sk in control versus terminal-stimulated females; n = 5; F(1,64) = 17.83, p = 0.0029. (F) Telemetry recording of locomotor activity, n = 5, F(1,960) (activity) = 185.8, p < 0.00001. (G) T-sk following a brief (2-min, 2 Hz) stimulation; n = 5; F(1,48) = 9.41, p = 0.0035. F statistics represent the main effect of treatment by two-way RM-ANOVA. Error bars represent ± SEM. Crossover design with randomized starts.

Figure 4.. Evoked Hot Flushes Are Sensitive to Ovarian Estrogens and Are Blocked by Neurokinin Receptor Antagonists Delivered to the POA

(A) Dose response of T-sk to CNO. CNO was injected at ZT = 13:00, and T-sk was monitored for 2 hr; data are presented as the average area under the curve (AOC; hours x °C). mCherry-expressing controls were treated with CNO (0.05 to 1 mg/kg) and averaged. One-way ANOVA: for mCh, n = 8; for hM3Dq, n = 6; **p = 0.0006, asterisks indicate Bonferroni post-test results. Following ovariectomy, females were significantly more sensitive to 0.3 mg/kg CNO. Paired t test, n = 6. Intact: M = 285.6, SD = 22.74; ovari- ectomized, M = 393.1, SD = 29.31. t(5) = 2.605; *p = 0.0480. (B) Schematic representation of targeted viral injection into the ARH and placement of infusion cannula in the POA. (C) Infusion of the cocktail of antagonists was sufficient to block CNO-induced flushing. 30 min prior to recording, either saline or antagonists were infused into the POA of h M3Dq-expressi ng females. T-sk was recorded every 15 min starting immediately prior to CNO delivery at ZT = 13:00. Two-way RM-ANOVA, n = 6; F(1, 45) = 84.05, p < 0.00001. (D) T-sk from ZT = 13:00–15:00 represented as AOC. hM3Dq data were extracted from (B) and compared to mCherry-expressing controls that were treated in the same manner. Two-way RM-ANOVA main effect of antagonists, n = 6 or 8 per group (hM3Dq or mCh); F(1, 12) = 16.49, **p = 0.0016; Bonferroni post-tests compare artificial cerebral spinal fluid (ACSF) with antagonists for paired animals in each group. ns, not significant. Experiments were conducted using a crossover design with randomized start times. All error bars represent SEM.