A reduction in voluntary physical activity during pregnancy in mice is mediated by prolactin

As part of the maternal adaptations to pregnancy, mice show a rapid, profound reduction in voluntary running wheel activity (RWA) as soon as pregnancy is achieved. Here, we evaluate the hypothesis that prolactin, one of the first hormones to change secretion pattern following mating, is involved in driving this suppression of physical activity levels during pregnancy. We show that prolactin can acutely suppress RWA in virgin female mice, and that conditional deletion of prolactin receptors (Prlr) from either all forebrain neurons or from GABA neurons prevented the early pregnancy-induced suppression of RWA. Deletion of Prlr specifically from the medial preoptic area, a brain region associated with multiple homeostatic and behavioural roles including parental behaviour, completely abolished the early pregnancy-induced suppression of RWA. Our data demonstrate a key role for prolactin in suppressing voluntary physical activity during early pregnancy, highlighting a novel biological basis for reduced physical activity in pregnancy.

comparisons test: vehicle vs prolactin 12h P=0.03). The measure of "ambulation distance" in 166 these cages is generated by an algorithm that interprets multiple infra-red beam breaks in a 167 consecutive direction as forward movement or 'ambulation'. Interestingly, when total counts 168 of XY beam breaks were examined, prolactin treatment tended to increase, rather than

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To test the hypothesis that prolactin is acting in the brain to mediate the pregnancy-      experiments using a mouse line with a specific deletion of Prlr from GABA neurons (Prlr lox/lox 301 crossed with vGAT-cre mice, as previously described (11)

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Mice with Prlr deleted from kisspeptin neurons were generated by crossing Kiss1-cre mice 344 (25) with Prlr lox/lox mice (Prlr lox/lox /Kiss1-cre) (23). We have previously shown that these mice   with the associated increased risk in pregnancy complications (31). Increasing numbers of 449 women are coming into pregnancy already overweight or obese, and over 50% of women 450 now exceed the Institute of Medicine (IOM) guidelines for optimal weight gain during pregnancy (32-34). Indeed, obesity during pregnancy has been described as the "most 452 common clinical risk factor encountered in obstetric practice" (35). As it is difficult to alter 453 energy intake, increasing energy expenditure through exercise during pregnancy has become 454 highly advocated as a therapeutic intervention that produces healthier gestational weight gain pregnancy, yet as pregnancy progressed, the neuron-specific Prlr knockout mice still showed 484 reduced RWA compared with non-pregnant animals. It is possible that this is simply due to 485 physical constraints, as growth of the fetuses provides an additional burden on the mothers.

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However, the fact that very low RWA persists throughout lactation points more to a hormonal 487 mechanism being involved, in addition to the inhibition provided by prolactin. A possible 488 candidate is progesterone, which is known to decrease RWA in rodents through opposing the 489 positive influence of estradiol (13). In mice, progesterone begins to be increased around 48 490 hours following detection of a copulatory plug and then is high throughout pregnancy until 491 around 24 hours before parturition (47, 48). After birth, progesterone secretion is re-initiated 492 in rodents, due to prolactin-induced rescue of the corpus luteum following a postpartum 493 ovulation. While there is evidence from progesterone receptor knock-out rats that 494 progesterone interactions with this receptor are not required for regulating RWA during the 495 estrous cycle (49), it seems likely that the very high levels of progesterone seen during 496 pregnancy are facilitating the prolactin-induced suppression of RWA as pregnancy advances.

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Overall, the data provide novel and convincing evidence of a prolactin-mediated 499 adaptive change in voluntary physical exercise during early pregnancy in mice. Whether such 500 a response also occurs in women requires attention. Prolactin, and its closely related 501 placental analogue, placental lactogen are both highly regulated hormones during human 502 pregnancy, among the top 1% of proteins increasing in the blood at this time (50, 51). These 503 changes occur progressively throughout human pregnancy, as opposed to being an 504 immediate mating-induced increase as seen in rodents, but are still likely to be sufficiently 505 elevated in the blood to be having effects in the brain by the end of the first trimester. It 506 seems likely that these elevated levels of lactogenic hormones are contributing to the broad 507 range of adaptations that are occurring during pregnancy (2, 52). As discussed above, 508 however, in the current environment some of these changes may have become maladaptive 509 and potentially compromise healthy behaviors. Such a possibility needs to be seriously 510 considered in providing obstetric advice to pregnant women.
Female mice starting at age 8-12 weeks, were housed in a temperature-and lighting-515 controlled environment (22 ± 1 C, 12 h light:12 h dark, lights on at 0700h) and allowed access 516 to food and water ad libitum. When required, mice were handled daily to monitor estrous 517 cyclicity. All experimental protocols were approved by the University of Otago Animal Ethics 518 Committee. Groups of C57BL/6J mice were used for characterization of activity during 519 pregnancy and to investigate the effects of acute prolactin on activity. Prlr lox/lox mice were 520 generated as previously described (11). CamKII-alpha cre (CamK-cre) mice (53), vGat-cre 521 (54)(Jackson lab stock no. 028862) and kiss1-cre (25)

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Our data (Fig 1 and (3)) demonstrate that while there is a wide variation in stable, total daily 567 RWA between different mice, pregnancy induces an approximately 50% decrease in RWA as 568 soon pregnancy is achieved independent of whether a mouse is a 'high' or 'low' runner. Due 569 to the varying range of stable running levels in individual mice, to assess the pregnancy 570 induced change in behaviour we analysed the percentage change in RWA for each individual mouse as opposed to using total distance per day for each mouse (although this data is 572 provided in source data for each transgenic line). Virgin RWA was calculated for the four 573 days prior to mating as a percentage of the average daily RWA over at least a week following 574 running wheel habituation. RWA activity for each day of pregnancy was also calculated in a 575 similar manner, as a percentage of average daily RWA over at least a week.

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Inc., VA) for the next 30 minutes. OF testing was carried out under sodium lighting to allow 596 video recording but perceived as 'dark' to rodents. After completing a full estrous cycle, on 597 the next metestrus mice were similarly injected with vehicle or prolactin (5mg/kg) 598 approximately 4 hours in to the dark phase of the light/dark cycle and then an hour later mice 599 were placed in an elevated plus maze. During the EPM mice were exposed to white light for 600 the duration of the test. The activity of the mouse was recorded using TopScan software and both OF and EPM tests were analyzed for distance traveled during the time period (OF: 30 602 minutes, EPM: 5 minutes) using TopScan software.
handling (removal from cages) and estrous cycle monitoring prior to experiment. On 607 metestrus, mice received an injection of prolactin (5mg/kg) or vehicle approximately 30 608 minutes before the start of the dark phase of the light/dark cycle. On the metestrous of the 609 following estrous cycle, mice received the alternative treatment as described above. Data was 610 collected from Promethion cages using Metascreen software and processed using Expedata 611 software. End points analyzed included total ambulation distance, total infra-red beam breaks 612 and non-ambulatory movements. Vector Biosystems) for control group into the MPOA (coordinates were 0.07 anterior to 626 Bregma, 0.3 mm lateral to midline, and depth from top of the brain was adjusted by body 627 weight: <22g 4.7mm depth, >22g 4.9 mm). Injections were given at a rate of 80nL/min, and 628 the syringes were left in situ for 3 minutes before and 10 minutes following completion of 629 injection. Mice were left to recover for one week, then housed with running wheels for at least induced pSTAT5 and GFP immunohistochemistry was used to demonstrate deletion of Prlr in 645 MPOA AAV-cre injected Prlr lox/lox mice. For pSTAT5 immunohistochemistry, mice received an 646 i.p. injection of prolactin (5mg/kg) 45 minutes before perfusion. Mice were anesthetized with 647 sodium pentobarbital then perfused transcardially with 4% paraformaldehyde. Brains were 648 removed and processed for immunohistochemistry for GFP or pSTAT5, as previously 649 described (11). Briefly, for pSTAT5 immunohistochemistry coronal brain sections (30 micron) 650 underwent an antigen retrieval procedure consisting of incubation in 0.01 mM Tris (pH 10) at 651 90 C for 5 minutes, then were left to cool for 10 minutes. Sections were incubated in rabbit 652 anti-phospho STAT5 antibody (dilution 1:1000, polyclonal rabbit anti-phospho-STAT5, Tyr 653 694, #9351, Cell signaling Technology, Beverly, MA, USA) for 48 hours. Following this, 654 sections were incubated in biotinylated goat anti-rabbit IgG (dilution 1:300, BA-1000, Vector 655 Laboratories, Inc., Burlingame, CA, USA) for 90 minutes and then incubated in Vector Elite 656 avidin-biotin-HRP complex (dilution 1:100) for 60 minutes and labelling was visualized with 657 nickel diaminobenzidine tetrahydrochloride using glucose oxidase to create a black, nuclear 658 precipitate. For GFP immunohistochemistry sections were incubated in anti-GFP antibody vehicle treatment. Two-way ANOVA followed by Sidak's multiple comparison test was used to 675 analyse the change in RWA from virgin (four days prior to mating) to early pregnancy (days 1-676 3 of pregnancy). Repeated measures ANOVA followed by Sidak's multiple comparisons test 677 was used to analyse prolactin effects on RWA, ambulation, XY beam breaks, fine movement 678 in virgin state. One-way ANOVA was used to analyse daily EE and ambulation during the 679 different weeks of pregnancy. Mixed effects analysis followed by Sidak's multiple 680 comparisons test was used to analyse changes in daily RWA, body weight and food intake 681 across pregnancy.Mann-Whitney tests was used to analyse estrous cycle data, pup number 682 during early lactation and GFP positive cell number.