1 2 3 Behavioral responses to emotional challenges in female rats living in a seminatural 4 5 6 environment: The role of estrogen receptors 7 8 9 10 11 Olivia Le Moëne and Anders Ågmo 12 13 14 15 16 Department of Psychology, University of Tromsø, Norway 17 18 19 Corresponding author: [email protected] 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 2 61 62 Abstract: 63 64 65 Estrogen receptors (ERs) are involved in sexual as well as non-sexual behaviors. In the 66 67 present study we assessed the effects of stimuli inducing positive or negative affect on 68 69 sociosexual, exploratory and fear-related behaviors of female rats housed in groups (4 70 71 females, 3 males) in a seminatural environment. Ovariectomized females were treated with 72 73 74 oil, 17β-estradiol benzoate (EB, 18 μg/kg), the ERα agonist propylpyrazoletriol (PPT), or the 75 76 ERβ agonist diarylpropionitrile (DPN) (both 2 x 10 mg/rat). On the test day, the females were 77 78 exposed to a sequence of events consisting of lavender odor, Mozart’s Sonata for Two Pianos 79 80 K448, chocolate pellets, white noise and fox odor (2,3,5-Trimethyl-3-thiazoline, TMT). All 81 82 these events are known to induce positive or negative affect. Behavior was carefully observed 83 84 from the video record. White noise suppressed sexual behaviors and reduced the time spent in 85 86 the open area of the environment. TMT had no consistent effect whereas exposure to music 87 88 caused avoidance of the open area. Exposure to chocolate increased exploratory and social 89 90 91 behavior. Lavender odor enhanced exploratory behavior. PPT and EB stimulated sexual 92 93 behaviors, whereas DPN was ineffective. Co-occurrence analyses of the sequence of 94 95 behavioral patterns revealed that PPT and EB consistently belonged to clusters different from 96 97 oil and DPN, whereas DPN was separate from oil only under fear-inducing experimental 98 99 conditions. These data, from a procedure with external validity, confirm that the ERα is 100 101 crucial for sexual behaviors, that these behaviors are reduced under stressful conditions, and 102 103 that the ERβ may have some role in fear-related behaviors. 104 105 106 107 108 Key words: seminatural environment, sexual behavior, social behavior, fear, positive emotion, 109 110 co-occurrence analysis, propylpyrazoletriol, diarylpropionitrile 111 112 113 114 115 116 117 118 119 3 120 121 1. Introduction 122 123 Female rodents only express sexual behavior when their brain is exposed to 124 125 appropriate concentrations of ovarian hormones. Estrogens and progesterone normally act 126 127 synergistically, but high doses of estrogens can activate all aspects of female sexual behavior 128 129 130 in the absence of progesterone, whereas progesterone is ineffective in the absence of 131 132 estrogens regardless of dose (e.g. Södersten & Eneroth, 1982). It is known that all female 133 134 sexual behaviors, including being attractive for males and being attracted to males (reviewed 135 136 in Le Moëne & Ågmo, 2017) as well as the display of paracopulatory behaviors and lordosis 137 138 (e.g. Ogawa et al., 1998; Rissman et al., 1997), are dependent on the estrogen receptor α 139 140 (ERα). The estrogen receptor β (ERβ) does not contribute to these behaviors, since female 141 142 mice lacking this receptor show perfectly normal sexual behaviors (Ogawa et al., 1999; Walf 143 144 et al., 2008; Antal et al., 2012) and since ERβ agonists are unable to activate these behaviors 145 146 147 in ovariectomized female rats (Mazzucco et al., 2008). 148 149 In addition to being necessary for the display of sexual behaviors, estrogens have several 150 151 behavioral effects, some of which may be relevant for the sex behaviors. Among those, 152 153 modifications of fear and anxiety responses might be particularly important. There are also 154 155 data suggesting that estrogens may alter the response to events inducing positive affect, for 156 157 example the ingestion of tasty foods like sucrose or chocolate (e.g. Clarke & Ossenkopp, 158 159 1998; Reynaert et al., 2016). However, the effects of fear-inducing stimuli or situations or of 160 161 stimuli causing positive affect on sexual behavior in female rats have not been studied. In 162 163 fact, available data are limited to studies of the effects of stress on subsequent sexual 164 165 166 behavior. It can be argued that fear stimuli activate stress responses, and studies of stress 167 168 could therefore provide some information about the potential effects of fear on sexual 169 170 behavior. Acute stress in the form of short restraint reduces the display of female copulatory 171 172 behavior in females rendered sexually receptive with estradiol alone, whereas no effect was 173 174 observed in females given estradiol + progesterone (Truitt et al., 2003). When females can 175 176 177 178 4 179 180 pace sexual interaction, restraint reduces the time spent with the male as well as the number 181 182 183 of mounts received regardless of the presence or absence of progesterone. Receptivity was not 184 185 modified, though (Uphouse et al., 2005). Interestingly, restraint stress had no effect in a test 186 187 for sexual incentive motivation (Uphouse et al., 2008). It appears, then, that acute stress has 188 189 minor or no consequences for female sexual behavior. Chronic stress, however, has 190 191 consistently been found to facilitate the display of lordosis and paracopulatory behavior and 192 193 to reduce rejections (Brotto et al., 1999; Williams et al., 1992). 194 195 A common feature of all these studies is that they have evaluated the effects of prior, 196 197 but not present, stress. Thus, the immediate consequences of fear-inducing stimuli for sexual 198 199 behavior remains unknown. There is, however, abundant evidence for a role of estrogens in 200 201 202 non-sexual, anxiety-like responses. Treatment of ovariectomized mice and rats with estradiol 203 204 has been reported to enhance the time spent on the open arms of an elevated plus maze (e.g. 205 206 Nomikos & Spyraki, 1988), or in the center of an open field (e.g. Walf & Frye, 2007), and to 207 208 reduce a passive avoidance response (Díaz-Véliz et al., 1997). All these effects are usually 209 210 interpreted as suggesting reduced fear or anxiety. Other studies have failed to find an effect of 211 212 estradiol in anxiety tests (e.g. Walf & Frye, 2008; Martínez-Mota et al., 2000), and still others 213 214 found anxiogenic effects (Mora et al., 1996). One hypothesis proposed to account for these 215 216 contradictory observations is that estrogens are anxiogenic in threatening environments and 217 218 219 anxiolytic in safe environments (Morgan & Pfaff, 2001). Such an effect could conceivably be 220 221 useful for assuring that another ERα dependent behavior, copulation, occurs more easily in 222 223 safe than in dangerous environments (see, e.g. Frye et al., 2006, for an elaboration of this 224 225 argument). Direct experimental evidence for this conjecture is lacking, though. 226 227 The anxiolytic-like effects of estrogens are often attributed to actions at the ERβ. Mice 228 229 without a functional ERβ are more fearful than the wildtype (Krezel et al., 2001), and 230 231 treatment with a selective ERβ agonist reduces fear in female rats (Kudwa et al., 2014) and 232 233 234 235 236 237 5 238 239 mice (Krezel et al., 2001; Oyola et al., 2012; Walf et al., 2008b), whereas selective ERα 240 241 242 agonists are ineffective. It has also been reported that ERα knock-out mice are not different 243 244 from the wildtype in several of the anxiety procedures (Krezel et al., 2001). However, 245 246 anxiogenic effects of a selective ERα agonist in fear-inducing environments (elevated plus 247 248 maze and novel open field) have been reported (Lund et al., 2005). It has also been found that 249 250 the ERα is anxiogenic in the light/dark box and in a brightly lit open field (Spiteri et al., 251 252 2010b; Spiteri et al., 2012). Thus, it can be proposed that ERα and ERβ agonists might have 253 254 opposite effects in fear-inducing contexts. 255 256 One of the purposes of the present study was to determine whether fear-inducing 257 258 stimuli actually inhibit female sexual behavior, and if agonists selective for the ERα and ERβ 259 260 261 would have different effects on the nonsexual responses to these stimuli. To that end, 262 263 ovariectomized females were given either estradiol or selective ER agonists. Fear was induced 264 265 by exposing the females to a 90 dB white noise or to synthetic fox odor. Loud noise as well as 266 267 2,5-dihydro-2,4,5-trimethylthiazoline (TMT) produce strong fear responses in rats (e.g. 268 269 Endres et al., 2005; Fendt et al, 2005; Homiack et al., 2017; Weyers et al., 1994). 270 271 Another purpose of this study was to evaluate the effects of stimuli inducing positive 272 273 affect rather than fear, and the potential role of the ERs for responses to such stimuli. 274 275 Estrogen-modulation of responses to attractive, non-sexual stimuli have only been 276 277 278 systematically studied with regard to food ingestion. It is well known that estrogens reduce 279 280 food intake (e.g. Butera, 2010). It appears that the ERα is responsible for the effects of 281 282 estrogens, since ERα knockout mice do not reduce food intake in response to hormone 283 284 treatment (Geary et al., 2001). Moreover, a specific ERα agonist does reduce food intake 285 286 whereas an ERβ agonist is ineffective (Shen et al., 2017). It has been suggested that post- 287 288 ingestive factors rather than reduced hedonic impact of tastants underlie the reduced food 289 290 intake (Hrupka et al., 1997; Flanagan-Cato et al., 2001). This proposal is reinforced by the 291 292 293 294 295 296 6 297 298 fact that estrogens enhance the hedonic response to and consumption of tasty foods, such as 299 300 301 sucrose (Clarke & Ossenkopp, 1998) or chocolate (Reynaert et al., 2016; Boswell et al., 2006; 302 303 Lampert et al., 2013). 304 305 A less known effect of estrogens is to increase the fear-reducing effect of music. It has 306 307 repeatedly been reported that music has anxiolytic activity in several of the standard 308 309 procedures (Li et al., 2010; Cruz et al., 2015). When the sonata for 2 pianos (Köchel number 310 311 448) by W.A. Mozart is played to ovariectomized female rats, the anxiolytic effect is reduced 312 313 compared to that found in proestrus females, but it can be restored by treatment with estradiol 314 315 (Escribano et al., 2014). In the same study, it was found that the anxiogenic effect of loud 316 317 white noise was not altered by ovariectomy or estrogen treatment. In mice, the effects of 318 319 320 music seem less dependent on ovarian hormones, although both estradiol and progesterone 321 322 had some effects (Chikahisa et al., 2007). The potential role of the different ERs has not been 323 324 explored. 325 326 Another stimulus with anxiolytic effects in several rodent procedures is the odor of 327 328 lavender oil (Umezu et al., 2006; Shaw et al., 2007; Tsang & Ho, 2010; Tsang et al., 2013; 329 330 Linck et al., 2010). There are also observations suggesting that this oil activates positive 331 332 affect in rodents and humans (Frasnelli et al., 2015). To our knowledge, there are no data 333 334 concerning possible modifications of the effects of lavender oil by ovarian hormones. 335 336 337 An additional purpose of the present study was to evaluate the effects of the positive 338 339 stimuli lavender oil, music, and chocolate on estradiol + progesterone-activated sexual 340 341 behavior in ovariectomized rats and to determine if and how non-sexual responses were 342 343 modified by ER ligands. 344 345 We have previously argued that an understanding of the behavioral consequences of 346 347 the central nervous actions of the ovarian hormones is best achieved in experimental setups 348 349 with external validity (Chu & Ågmo, 2014; Chu & Ågmo, 2015b; Chu & Ågmo, 2016). This 350 351 352 353 354 355 7 356 357 means that the setup should include as many as possible of the elements found in the natural 358 359 360 context in which the behavior normally is shown. In the case of sexual behaviors in rats, an 361 362 important feature is that it occurs in multi-male, multi-female groups, and in a physical 363 364 environment making it possible for the rats to temporarily escape from other group members. 365 366 In view of these considerations, we administered estradiol as well as the selective ERα 367 368 agonist propylpyrazoletriol (PPT) and the selective ERβ agonist diarylpropionitrile (DPN) to 369 370 ovariectomized female rats housed in a seminatural environment in groups consisting of 4 371 372 females and 3 males. During the period in which the agonists could be expected to have their 373 374 maximal effect, we introduced the events mentioned earlier into the environment. This made 375 376 it possible not only to determine the role of the ERs in social and sexual interactions in a 377 378 379 group of rats, but also how they affected the response to these events, and how the positive 380 381 and negative events themselves affected behavior. These data would provide us with a better 382 383 understanding of how the ERs control sexual behavior and responses to emotion-inducing 384 385 events in a procedure with external validity. 386 387 388 389 390 391 2. Material and Methods 392 393 2.1 Subjects 394 395 396 Wistar rats (females, 250 g and males 300 g upon arrival) were obtained from Charles River 397 ® 398 WIGA (Sulzfeld, Germany). The rats were housed in same-sex pairs in standard Macrolon 399 400 IV cages prior to the beginning of the experiment. Commercial rat pellets (RM1, Special 401 402 Diets Services, Witham, UK) and tap water were available ad libitum. The animal rooms were 403 404 maintained at 21 ± 1°C and humidity was 55 ± 10%. Lights were set on a reversed 12:12 h 405 406 cycle, being on between 23:00 and 11:00 h. Females were ovariectomized 14 days prior to the 407 408 409 410 411 412 413 414 8 415 416 introduction into the seminatural environment under isofluorane anesthesia. For a detailed 417 418 419 description of the surgical procedure see Ågmo (1997). 420 421 All experimental procedures employed in the present experiment were approved by 422 423 the Norwegian Food Safety Authority and were in agreement with the European Union 424 425 council directive 2010/63/EU. 426 427 428 429 2.2 Apparatus 430 431 The seminatural environment has previously been used in a number of studies and has 432 433 been described in detail elsewhere (Chu & Ågmo, 2014; Chu & Ågmo, 2015b). Briefly, it 434 435 consisted of a complex burrow system and a large open area (Fig. 1). The burrow system 436 437 438 included four nest boxes provided with nest material and was maintained in complete 439 440 darkness for the whole experiment. Infrared (850 nm) lamps provided the light necessary for 441 442 video recording. The open area (1.2 * 2 m) contained 12 wooden sticks and three small 443 444 shelters made of transparent red plastic. The open field was submitted to a reversed light/dark 445 446 cycle (12D:12L) with a 180 lx light from 23:00 to 11:00 and a 30 lx light from 11:00 to 23:00. 447 448 Artificial dusks and dawns were provided by slowly changing light intensity from night to day 449 450 and day to night during a 30 min period preceding and following the light period. Video 451 452 cameras were fixed to the ceiling about 2 m above the burrow and the open area, respectively. 453 454 455 They were connected to digital video recorders. 456 457 The ventilation system in the animal facility produced an ambient noise of about 40 458 459 dB. 460 461 462 463 2.3 Hormones and selective estrogen receptor ligands 464 465 Estradiol benzoate (EB) and progesterone (P) (both from Sigma Aldrich, St Louis, 466 467 MO) were dissolved in peanut oil (Den norske Eterfabrikk, Norway) and administered 468 469 470 471 472 473 9 474 475 subcutaneously (SC) in a dose of 18 μg/kg and 1 mg/rat, respectively. The injection volume 476 477 478 was 1 ml/kg for EB and 0.2 ml/rat for progesterone. EB was administered 48 h before P. 479 480 The estrogen receptor agonists propylpyrazoletriol (PPT) and diarylpropionitrile 481 482 (DPN) were obtained from Tocris Bioscience, Bristol, UK. Both PPT and DPN were 483 484 dissolved in undiluted dimethylsulfoxide (Sigma Aldrich) right before SC injection. Both 485 486 were administered at a dose of 2 * 10 mg/kg body weight in a volume of 1 ml/kg. There was 487 488 an interval of 24 h between injections. PPT is selective to ERα with a 410-fold preference 489 490 compared to ERβ, and with a relative binding affinity of 50% compared to estradiol (Stauffer 491 492 et al., 2000). DPN is selective to the ERβ with a 72-fold preference compared to ERα with a 493 494 relative binding affinity of 18% (Meyers et al., 2001). PPT and DPN reach their maximum 495 496 497 serum concentration about 30 min after SC injection and have a half-life of 6.0 ± 0.03 h and 498 499 8.2 ± 1.7 h, respectively (Sepehr et al., 2012). 500 501 The doses of EB and P employed here have been used successfully in several earlier 502 503 studies (e.g. Chu et al., 2017; Snoeren et al., 2015). They produce close to maximal 504 505 receptivity and high intensity of paracopulatory behaviors (Spiteri & Ågmo, 2006). The dose 506 507 of PPT was based on earlier studies. One showed that 2 * 10 mg/kg of PPT given 48 and 24 h 508 509 before test produced a high lordosis quotient, similar to that of 2 * 2 μg/rat of EB. A dose of 2 510 511 mg/kg was inactive (Miller et al., 2005). Another study showed that 2 * 5 mg/rat, 48 h and 512 513 514 24h before test, produced a lordosis quotient of about 0.8 when combined with progesterone 515 516 0.5 mg/rat (Mazzucco et al., 2008). Thus, a dose of 2 * 10 mg/kg of PPT was used in order to 517 518 assure clear behavioral effects. Concerning DPN, the dose was chosen somewhat arbitrarily. 519 520 The ERβ does not participate in the activation of sexual behavior, so we needed to find 521 522 another basis for determining the appropriate dose. Since many studies comparing the effects 523 524 of PPT and DPN employ the same dose of both compounds (e.g. Pisani et al., 2016; Walf & 525 526 Frye, 2005) we simply decided to do the same. 527 528 529 530 531 532 10 533 534 535 536 537 2.4 Experimental conditions 538 539 Since the aims of this study include an analysis of behavior in situations producing 540 541 positive and negative affect, it was essential to introduce events into the seminatural 542 543 environment that reasonably could be expected to produce diverse emotional reactions. Five 544 545 such events, all mentioned in the Introduction, were used. 546 547 1. Lavender odor stemmed from an essential oil extracted from Lavandula angustifolia 548 549 (AromaBio, Lyon, France). Immediately before the beginning of the experimental session, 1.5 550 551 ml of this oil was put on a cotton pad in an airtight jar. An air stream could be made to flow 552 553 through the jar whenever desired with the help of an air pump and a system of solenoid valves 554 555 556 (Olfactory Stimulus Package, Medical associates, Georgia, Vt). Odorless plastic tubing (I.D. 557 ® 558 3/16” , O.D. 5/16” , wall thickness 1/16”; TYGON Inert, Saint-Gobain Verneret, Charny, 559 560 France) connected the pump with the valves and eventually with a nozzle in the wall of one of 561 562 the tunnels in the burrow section of the seminatural environment, as well as with another 563 564 nozzle in the wall of the open area. The airflow (around 3 l/min) could be directed through 565 566 one of two jars, or made to bypass the jars and consist of unscented room air instead. Room 567 568 air was blown through the nozzles during the entire experimental session, except when it was 569 570 replaced by an olfactory stimulus. Lavender odor was introduced into the seminatural 571 572 573 environment for 30 min with the help of this system. The amount of lavender oil used here 574 575 has been found to alter behavior even with short exposure times (e.g. Shaw et al., 2007). 576 577 2. Classical music (Mozart’s sonata for two pianos K448), played by Murray Perahia and 578 579 Radu Lupu, recorded at Snape Maltings Concert Hall, Suffolk, England (CD from Sony 580 581 Music Entertainment). The piece lasts 24 min and 18 s. The music file was uploaded on a 582 583 computer and played through A60 stereo speakers from Creative (Clas Ohlson, Norway) at a 584 585 sound intensity of 55-60 dB as measured at floor level. 586 587 588 589 590 591 11 592 593 3. Thirty-five one g chocolate pellets (Bioserve, Flemington, NJ) were placed on a Petri dish 594 595 596 (diameter 100 mm) which was put in the middle of the open area. After 30 min, the dish was 597 598 removed. At that time, most pellets had been removed by the rats. 599 600 4. A 90 dB white noise was produced by a noise generator (Lafayette instruments, Lafayette, 601 602 IN) connected to two loudspeakers (Scan-Speak Discovery 10F/8414G10, Hifi Kit Electronic, 603 604 Stockholm), one suspended about 2 m above the burrow, and another at the same height 605 606 above the open area. The noise was turned on for a period of 15 min. 607 608 5. Fox odor was produced by making air flow through a jar containing 35 μl of 2,5-dihydro- 609 610 2,4,5-trimethylthiazoline (TMT; Contech, Delta, BC, Canada) on a cotton pad. The odor 611 612 system described above was used. Short exposure to odor from this amount of TMT has been 613 614 615 found to alter behavior in a large open field (Morrow et al., 2002). The odor exposure lasted 616 617 for 30 min. 618 619 Music and chocolate may not be standard parts of rats’ natural habitat, and these 620 621 stimuli might therefore reduce external validity. We used them because they are among the 622 623 few, non-sexual stimuli, known to cause positive affect in non-deprived rats. Sucrose might 624 625 have been used instead of chocolate pellets, but the latter have the advantage of allowing for 626 627 for quantification of each individual’s consumption. This would not have been possible with a 628 629 sucrose solution. 630 631 632 633 634 2.5 Procedure 635 636 Prior to introducing each experimental group into the seminatural environment, the 637 638 floor of the entire environment was covered with a three cm thick layer of aspen wood chips 639 640 (Tapvei, Harjumaa, Estonia). Four 0.5 l water bottles and about 5 kg of standard food pellets 641 642 were located in a corner of the open area. After each experimental session, the bedding was 643 644 removed and the entire environment was cleaned and disinfected. 645 646 647 648 649 650 12 651 652 The rats were released in the seminatural environment on day 0 at 13:00. About four 653 654 655 hours before, they had been weighed and marked in order to be identifiable on the video 656 657 record. 658 659 The rats were left undisturbed for the first 5 days in the seminatural environment. On 660 661 days five and six the females were captured and injected with the appropriate compound. On 662 663 day 7, all females received P. Four hours later, the sequence of experimental conditions 664 665 started. All experimental conditions were separated by a 50-minutes rest period (Table 1). The 666 667 experiment was terminated after the last experimental condition, and the females were again 668 669 weighed. 670 671 [Insert table 1 here] 672 673 674 The order of presentation of the experimental conditions was fixed throughout the 675 676 whole experiment. Four hours after the P injection, lavender odor was presented, followed by 677 678 music, chocolate, white noise and the fox odor. 679 680 This order of events was based on several considerations. Predator odor has been 681 682 reported to alter behavior for several hours (Fendt et al., 2005). Consequently, exposure to fox 683 684 odor needed to be the last condition. The duration of potential effects of lavender oil is not 685 686 known, but anxiolytic effects are normally obtained immediately or within few minutes after 687 688 the end of exposure (e.g. Tsang et al., 2013). The ventilation system in the room housing the 689 690 691 seminatural environment assures 15 air changes per hour, leading to a fast decline in the 692 693 concentration of odorant. We found it reasonable to suppose that the 50 min interval before 694 695 the next condition would be sufficient both for the odor and for its possible effects to 696 697 dissipate. In fact, the data confirmed that supposition. Concerning music, the reported effects 698 699 were usually obtained during exposure (e.g. Escribano et al., 2014). There are no data as to 700 701 the duration of effect, but again we supposed that it should be less than 50 min after the end of 702 703 the piece. The duration of the positive affect produced by chocolate eating or of the fear 704 705 706 707 708 709 13 710 711 reaction produced by white noise has not been determined. It may be pointed out, though, that 712 713 714 the chocolate-induced positive affect is present already during consumption (La Mela et al., 715 716 2010; Reynaert et al., 2016). With regard to white noise, pilot data revealed that the 717 718 behavioral effects of the noise were most evident at the onset, and that behavior began to 719 720 normalize already during the last few minutes of noise exposure. Therefore, the 50 min 721 722 interval was probably sufficient for any noise effects to dissipate. 723 724 725 726 2.6 Design 727 728 Each group in the seminatural environment consisted of seven rats, four females and 729 730 three males. The group members came from different cages, meaning that they were unknown 731 732 733 to each other before the introduction into the environment. Ten such groups were used in this 734 735 experiment. 736 737 [insert Figure 1 here] 738 739 In all groups, each of the four females received a different treatment. 1. Oil on days 5 740 741 and 6, P on day 7. 2. EB on day 5, oil on day 6, and P on day 7. 3. PPT on days 5 and 6, P on 742 743 day 7. 4. DPN on days 5 and 6, P on day 7. This means that all treatments were present in all 744 745 housing groups. 746 747 748 749 750 2.7 Behavioral observations 751 752 Based on extensive observation of the video record, we established an ethogram for 753 754 the scoring of the rats’ behavior (Table 2). Scoring was made with the Observer XT 12.5 755 756 software (Noldus, Wageningen, the Netherlands). Pilot data showed that a 15 min observation 757 758 period was sufficient to detect behavioral differences between conditions and treatments. 759 760 Thus, the last 15 min of the lavender, music, and fox odor exposure were observed, whereas 761 762 we recorded behaviors for the first 15 min of chocolate availability. This allowed us to 763 764 765 766 767 768 14 769 770 determine the latency to approach and grab the chocolate. The entire 15 min period of 771 772 773 exposure to white noise was observed. Behavior during the 15 min preceding the lavender 774 775 odor was recorded as a baseline. The frequency and/or duration of the occurrences of the 776 777 behavior patterns were recorded, specifying the individual initiating the behavior, the 778 779 individual to whom it was directed, and the location of the behavior. We also calculated the 780 781 number of transitions between zones (see Figure 1), as well as the number of visits to and the 782 783 time spent in the open area. In the case of latencies, subjects not displaying the behavior were 784 785 assigned a latency of 900 s, the duration of the observation. The lordosis quotient (LQ, 786 787 number of lordoses / number of mounts) was also calculated. Please note that some females 788 789 displayed one or more lordoses in the absence of male mounting, usually in response to tactile 790 791 792 stimulation of posterior body parts. The LQ may, consequently, be larger than 1. 793 794 [insert Table 2 here] 795 796 797 798 2.8 Statistical analysis 799 800 Whenever possible, data were analyzed with two-factor ANOVA for repeated 801 802 measures on one factor. The between-groups factor was treatment and the within-groups 803 804 factor was experimental condition. After significant main effect, the Tukey HSD test was 805 806 used for a posteriori comparisons. We calculated the effect size η2 for the effect of treatment 807 808 2 809 and the partial effect size η for effect of experimental condition and for the interaction. The 810 811 effect size for Tukey’s HSD was expressed as Cohen’s d (d = (x1-x2)/σ). 812 813 When the data deviated from the normal distribution according to Shapiro-Wilk’s test, 814 815 or the error variances were non-homogenous according to Hartley’s Fmax test, we used 816 817 nonparametric tests. The effect of treatment was evaluated with the Kruskal-Wallis test 818 819 whereas the effect of experimental condition was analyzed with Friedman’s ANOVA. In case 820 821 822 of significance, post hoc analyses were made as recommended by Conover (Conover, 1999). 823 824 825 826 827 15 828 829 The effect size was calculated as eta squared (η2 ) for the Kruskal-Wallis tests and as 830 831 832 Kendall’s W for the Friedman test (Tomczak & Tomczak, 2014). Cliff’s δ was used for the 833 834 non-parametric post hoc comparisons (Cliff, 1996). Some data were analyzed with the χ2 test, 835 836 and/or the Fisher exact test. Effects sizes for these tests were calculated with Cramer’s V and 837 838 Cohen’s d, respectively. 839 840 Significance level was p < 0.05. Data in text and figures are expressed as mean + 841 842 843 SEM. The IBM SPSS Statistics, version 23 was used for parametric tests and the free 844 845 software R, version 3.4.3 with base, PMCMRplus, effsize and lsr packages for non-parametric 846 847 tests. 848 849 850 851 2.9 Co-occurrence analysis 852 853 The seminatural environment allows the subjects to express a substantial part of their 854 855 natural behavioral repertoire. In fact, the continuous flow of behavior patterns is recorded. 856 857 This makes it possible to determine treatment- or condition-induced modifications of that 858 859 860 flow. In other words, how the experimental manipulations might have altered the structure of 861 862 behavior. Analyses of the frequency or duration of particular behavioral items cannot reveal 863 864 this kind of effects. Thus, in order to fully exploit the data obtained, we subjected the 865 866 behavioral record to an analysis of co-occurrence. Since the behavior patterns were recorded 867 868 in chronological order, this is easily made. We used a moving window of four behavior 869 870 patterns, and determined how often one behavior pattern occurred together with another in the 871 872 same window. This is defined as a co-occurrence. The window moved, by steps of one 873 874 behavior pattern, over the entire individual record. The frequency of co-occurrences was 875 876 877 entered in a matrix with the behavior patterns in rows and columns, the co-occurrence 878 879 frequency appearing at the intersections. Treatments and experimental conditions were also 880 881 included in the matrix. These were the raw data for the analysis. Descending hierarchical 882 883 884 885 886 16 887 888 classification was used in order to find clusters of related behavior (Reinert, 1983; Reinert, 889 890 891 1990; Valax et al., 1990, see also LePape et al., 1997). The descending hierarchical 892 893 classification is based on the probability for an item to be proportionally more present in a 894 895 cluster than it is in the entire data set, as evaluated by χ2 analysis. Each item is permutated 896 897 from one cluster to the other to test the robustness of the classification, until statistically 898 899 independent profiles of items appear (Marchand & Ratinaud, 2012). Communities can 900 901 therefore be interpreted as groups of individuals and behaviors significantly more co- 902 903 occurring together than with items of another community. 904 905 906 The criterion for including elements in their respective classes is a higher frequency 907 2 908 compared to the average occurrence, as well as an association with the class determined by χ 909 910 values equal to or higher than 3.84. This gives an error margin of 0.05 when df = 1 (de 911 912 Oliveira Andrade Jr. & de Oliveira Andrade, 2016). 913 914 Finally, co-occurrence networks were established and visualized using the 915 916 Fruchterman-Reingold algorithm. Calculations were performed with the free software 917 918 IRAMUTEQ (Interface de R pour les Analyses Multidimensionnelles de Textes et de 919 920 921 Questionnaires; available at http://www.iramuteq.org/). 922 923 924 925 3. Results 926 927 The pattern of effects of experimental condition and of treatment were similar for the 928 929 frequency of recorded behaviors and the total duration as well as the mean duration of each 930 931 behavioral episode, whenever these could be calculated. Therefore, we only present frequency 932 933 data. These have the advantage of being available for all behaviors. 934 935 936 937 3.1 Effects of experimental conditions 938 939 940 3.1.1 Female sexual behaviors 941 942 943 944 945 17 946 947 The sex behavior data did not satisfy the criteria for ANOVA. The distribution greatly 948 949 950 deviated from normality according to the Shapiro-Wilk test and Hartley’s Fmax test showed 951 952 error variances to be non-homogenous. Therefore, these data were analyzed using non- 953 954 parametric statistics. Moreover, since the aim of these analyses was to determine how 955 956 experimental conditions affected sexual behaviors, we limited the analyses to females treated 957 958 with EB or PPT. The females treated with oil or DPN expressed an extremely low level, or 959 960 none at all, of these behaviors (see 3.2.1). Thus, these females could not contribute with any 961 962 useful data to an analysis of the effects of experimental condition on sexual behaviors, since 963 964 no such behaviors were displayed in any condition. 965 966 The lordosis frequency in the collapsed EB and PPT groups differed between 967 968 969 conditions (χ² DF=5 = 12.67, p = 0.027, W = 0.12). It was lower during exposure to white noise 970 971 (p = 0.023, δ = 0.30) than at baseline (Fig. 2 A). The LQ also differed between experimental 972 973 conditions (χ² DF=5 = 15.56, p = 0.008, W = 0.16), being lower during exposure to white noise 974 975 (p = 0.002, δ = 0.45) than at baseline (Fig. 2 B). Likewise, the frequency of paracopulatory 976

977 behaviors differed between conditions (χ² DF=5 = 15.57, p = 0.008, W = 0.16). It was lower 978 979 during exposure to white noise (p = 0.004, δ = 0.37) than at baseline (Fig. 2 C). The frequency 980 981 of rejection did not vary between the experimental conditions (χ² = 1.72, p = 0.887, W = 982 DF=5 983 0.02; Fig. 2 D), 984 985 986 987 988 3.1.2 Female attractivity to males 989 990 Behaviors indicative of female attractiveness were analyzed using non-parametric 991 992 statistics due to lack of normality. Here, all treatments were included since also the females 993 994 treated with Oil or DPN were somewhat attractive to the males. The number of mounts 995 996 2 received by the females was affected by the experimental conditions (χ DF=5 = 12.20, p = 997 998 0.032, W = 0.32), but none of the conditions differed from baseline (ps > 0.092; data not 999 1000 1001 1002 1003 1004 18 1005 1006 shown). The frequency of male pursuit of the females also differed between the experimental 1007

1008 2 1009 conditions (χ DF=5 = 19.07, p = 0.002, W = 0.50). The pursuit frequency was lower during 1010 1011 exposure to fox odor than at baseline (p = 0.043, δ = 0.17). The other conditions had no effect 1012 1013 on the frequency of pursuit (all ps > 0.270; Fig. 3 A). 1014 1015 The frequency of male anogenital sniffing of the females did not vary between the 1016 2 1017 experimental conditions (χ DF=5 = 8.27, p = 0.142, W = 0.05) and there was no meaningful 1018 1019 effect on male resting with females (data not shown). To the contrary, the frequency of male 1020 1021 2 sniffing of the females differed between the experimental conditions (χ DF=5 = 16.85, p = 1022 1023 0.005, W = 0.09). Males sniffed females more often during exposure to chocolate than at 1024 1025 baseline (p = 0.019, δ = 0.34; Fig. 3 B). 1026 1027 1028 1029 1030 3.1.3 Exploratory behavior 1031 1032 Ambulatory activity, expressed as the frequency of transition between zones in the 1033 1034 entire seminatural environment, differed between the experimental conditions (F5,170 = 10.59, 1035 1036 2 p < 0.001, ηp = 0.24). Lavender (p < 0.05, d = 0.62), chocolate (p < 0.05, d = 0.92) and white 1037 1038 noise (p < 0.001, d = 0.80) enhanced activity, whereas exposure to music and TMT had no 1039 1040 1041 effect (ps > 0.06; Fig. 4 A). 1042 1043 The transitions between zones in the open area also differed between experimental 1044 2 1045 conditions (F5,170 = 10.17, p < 0.001, ηp =0.23). Exposure to lavender odor (p < 0.05, d = 1046 1047 0.67) and to chocolate (p < 0.05, d = 0.63) increased activity. The other conditions had no 1048 1049 effect (ps > 0.166; Fig. 4 B). The number of transitions between zones in the burrow was also 1050 1051 2 affected by experimental condition (F5,170 = 17.34, p < 0.001, η =0.34). Exposure to 1052 p 1053 1054 chocolate (p < 0.05, d = 1.04) and white noise (p < 0.05, d = 1.13) increased ambulatory 1055 1056 activity in the burrow compared to baseline (Fig. 4 C). 1057 1058 1059 1060 1061 1062 1063 19 1064 1065 There was an effect of experimental condition on the time spent in the open area (F 1066 5,170 1067 2 1068 = 7.73, p < 0.001, ηp = 0.19). Music (p < 0.05, d = 0.65) and white noise (p < 0.05, d = 0.62; 1069 1070 Fig. 4 D) reduced the time spent in the open area compared to baseline, whereas the other 1071 1072 conditions had no effect. Logically, the time spent in the burrow was also affected by 1073 2 1074 experimental condition (F5,170 = 7.73, p < 0.001, ηp = 0.19) in a way opposite to the open area. 1075 1076 Music (p > 0.05, d = 0.65) and white noise (p <0.05, d = 0.62) enhanced the time spent in the 1077 1078 burrow (Fig. 4 E). 1079 1080 1081 The frequency of rearing was also modified by the experimental condition (F5,170 = 1082 2 1083 5.81, p < 0.001, ηp = 0.15). More rearing episodes were observed during exposure to white 1084 1085 noise than at baseline (p < 0.05, d = 0.70; Fig. 4 F). The experimental conditions also altered 1086 1087 2 the frequency of sniffing the floor (F5,170 = 26.57, p < 0.001, ηp = 0.44). Exposure to chocolate 1088 1089 (p < 0.05, d = 1.00) and white noise (p < 0.05, d = 1.42) enhanced the frequency relative to 1090 1091 1092 baseline (Fig. 4 G). 1093 1094 1095 1096 3.1.4 Female prosocial behaviors. 1097 1098 These behaviors could, obviously, be directed towards the other females in the group 1099 1100 or to the males. We found it useful to analyze female-female and female-male interactions 1101 1102 separately. The frequency of resting with another female did not change between 1103 1104 2 experimental conditions (F(5,170) = 2.19, p = 0.058, ηp =0.06). To the contrary, the time resting 1105 1106 2 1107 with males differed between experimental conditions (F(5,170) = 23.88, p < 0.001, ηp =0.41) 1108 1109 The females rested more with males during exposure to chocolate than at baseline (p < 0.05, d 1110 1111 = 0.70). These data are illustrated in Fig. 5 A. We also found main effects of experimental 1112

1113 condition both on the frequency of female sniffing another female (F5,170 = 12.66, p < 0.001, 1114 1115 2 2 ηp=0.27) and a male (F5,170 = 10.37, p < 0.001, ηp = 0.23). The frequency was always higher 1116 1117 1118 1119 1120 1121 1122 20 1123 1124 during exposure to chocolate than at baseline (female-female, p < 0.05, d = 0.56; female- 1125 1126 1127 male, p < 0.05, d = 0.53). Data are shown in Fig. 5 B. 1128 1129 1130 1131 3.1.5 Female antisocial behavior towards males and females 1132 1133 Only the female nose-off behavior satisfied the criteria for parametric analysis. All the 1134 1135 other antisocial behaviors were analyzed using non-parametric statistics. 1136 1137 The nose-off frequency varied between the experimental conditions regardless of the 1138 1139 2 sex of the other party (males, F5,170 = 3.95, p = 0.002, ηp = 0.10; females, F5,170 = 7.32, p < 1140 1141 2 1142 0.001, ηp = 0.18). More nose-off episodes occurred during exposure to chocolate than at 1143 1144 baseline in both cases (males, p < 0.05, d = 0.35; females, p < 0.05, d = 0.61). White noise 1145 1146 increased nose-off of other females (p < 0.05, d = 0.97) but not of males (p > 0.05, d = 0.24). 1147 1148 Data are found in Fig. 6 A. As can be seen in Fig. 6 B, the frequency of fleeing from the 1149 1150 males as well as from other females differed between conditions (males, χ² DF=5 = 29.89, p < 1151 1152 0.001, W = 0.16; females, χ² DF=5 = 48.90, p < 0.001, W = 0.26). The fleeing frequency was 1153 1154 higher during exposure to chocolate and white noise than at baseline (males, chocolate- 1155 1156 1157 baseline: p = 0.001, δ = 0.48; white noise-baseline: p < 0.001, δ = 0.41; females, chocolate- 1158 1159 baseline: p = 0.001, δ = 0.52; white noise-baseline: p < 0.001, δ = 0.58). 1160 1161 1162 1163 3.1.6 Non-social behaviors. There was no systematic effect of experimental condition on 1164 1165 drinking, self-grooming or resting alone (data not shown). 1166 1167 1168 1169 3.1.7 Summary of the effects of experimental condition 1170 1171 The only experimental condition with an effect on the display of female sexual 1172 1173 1174 behaviors was white noise. The stimulus reduced these behaviors. The other conditions were 1175 1176 ineffective with regard to sex behaviors, and none of the conditions modified female 1177 1178 1179 1180 1181 21 1182 1183 attractivity. The availability of chocolate pellets stimulated ambulatory activity throughout the 1184 1185 1186 seminatural environment and enhanced both prosocial and antisocial interactions with both 1187 1188 sexes. White noise was associated with avoidance of the open area and increased ambulatory 1189 1190 activity in the burrow only. Antisocial behaviors were also enhanced in this condition. The 1191 1192 other purportedly fear-inducing stimulus, fox odor, had no consistent effect. This was also the 1193 1194 case for lavender odor and music. 1195 1196 1197 1198 3.2 Effects of hormone treatment 1199 1200 3.2.1 Female sexual behaviors 1201 1202 When the treatment effect was collapsed over all experimental conditions, it was 1203 1204 1205 found that the frequency of lordosis differed between treatments (H3, N = 38 =22.71, p < 0.001, 1206 2 1207 η = 0.60). The Oil group displayed less lordoses than the EB group (p < 0.001, δ = 0.90) and 1208 1209 the PPT group (p = 0.001, δ = 0.60). There was no difference between the Oil group and the 1210 1211 DPN group (p = 1, δ = 0.00). When evaluating treatment effects within each of the 1212 1213 experimental conditions it was found that the number of lordoses differed between treatments 1214 1215 at baseline (H , = 10.34, p = 0.016, η2 = 0.27), during exposure to lavender odor (H , 1216 3 N = 38 3 N = 1217 2 2 1218 38 = 10.42, p = 0.015, η = 0.27), music (H3, N = 38 = 12.14, p = 0.007, η = 0.32) and chocolate 1219 2 1220 (H3, N = 38 = 19.25, p < 0.001, η = 0.51). No difference between the treatments was observed 1221 1222 2 during exposure to the negative conditions, white noise (H3, N = 38 = 5.76, p = 0.124, η = 1223 1224 2 1225 0.15) and TMT odor (H3, N = 38 = 6.30, p = 0.098, η = 0.17). The EB group showed more 1226 1227 lordoses than the Oil group at baseline (p = 0.006, δ = 0.50), during exposure to lavender odor 1228 1229 (p = 0.038, δ = 0.40), music (p = 0.003, δ = 0.40) and chocolate (p < 0.001, δ = 0.60). White 1230 1231 noise and TMT odor suppressed responding, since the EB and Oil groups did no longer differ. 1232 1233 The PPT group displayed more lordoses than the Oil group only during exposure to lavender 1234 1235 1236 1237 1238 1239 1240 22 1241 1242 odor (p = 0.009, δ = 0.500). DPN failed to stimulate lordosis in all conditions. Results are 1243 1244 1245 illustrated in Fig. 7 A. 1246 2 1247 There was a treatment effect on the LQ (H3, N = 38 = 13.50, p = 0.004, η = 0.36) when 1248 1249 all experimental conditions were collapsed. Only the EB group differed significantly from Oil 1250 1251 (p = 0.001, δ = 0.60). Treatment with PPT failed to significantly enhance the LQ (p = 0.053, δ 1252 1253 = 0.40), whereas DPN was completely inactive (p = 1, δ = 0). The treatment effect was absent 1254 1255 2 1256 during exposure to white noise (H3, N = 38 = 2.80, p = 0.424, η = 0.07) or fox odor (H3, N = 38 = 1257 2 1258 6.30, p = 0.098, η = 0.17), but present in all other conditions (baseline, H3, N = 38 = 10.36, p = 1259 1260 2 2 0.016, η = 0.27; lavender odor, H3, N = 38 = 10.81, p = 0.013, η = 0.29; music, H3, N = 38 = 1261 1262 12.14, p = 0.007, η2 = 0.32; chocolate, H , = 19.43, p < 0.001, η2 = 0.51). During these 1263 3 N = 38 1264 1265 conditions, EB-treated females systematically had a higher LQ than oil-treated females 1266 1267 (baseline: p = 0.006, δ = 0.50; lavender: p = 0.006, δ = 0.50; music: p = 0.003, δ = 0.40; 1268 1269 chocolate: p < 0.001, δ = 0.60). Exclusively during exposure to lavender, the PPT group 1270 1271 displayed a higher LQ than the Oil group (p = 0.050, δ = 0.40). Data are illustrated in Fig. 7 1272 1273 1274 B. 1275 1276 There was also an effect of treatment on the frequency of paracopulatory behaviors 1277 1278 when all experimental conditions were included in the analysis (H3, N = 38 = 21.06, p < 0.001, 1279 2 1280 η = 0.55). The Oil group displayed less paracopulatory behaviors than the EB group (p < 1281 1282 0.001, δ = 0.90) and the PPT group (p < 0.001, δ = 0.80). There was no difference between 1283 1284 the Oil group and the DPN group (p = 0.273, δ = 0.30). 1285 1286 1287 We then proceeded to evaluate treatment effects on paracopulatory behavior under 1288 1289 each of the experimental conditions. There was a difference between treatments at baseline 1290 2 1291 (H3, N = 38 = 10.34, p = 0.016, η = 0.27), during exposure to lavender odor (H3, N = 38 = 12.68, 1292 1293 2 2 p = 0.005, η = 0.33) and chocolate (H3, N = 38 = 11.84, p = 0.021, η = 0.26. No difference 1294 1295 1296 1297 1298 1299 23 1300 1301 was found between the treatments in the frequency of paracopulatory behaviors during the 1302

1303 2 1304 exposure to music (H3, N = 38 = 6.95, p = 0.074, η = 0.18), white noise (H3, N = 38 = 5.75, p = 1305 2 2 1306 0.124, η = 0.15) and fox odor (H3, N = 38 = 7.46, p = 0.059, η = 0.20). The EB group showed 1307 1308 more paracopulatory behaviors than the Oil group at baseline (p = 0.003, δ=0.60), during 1309 1310 exposure to lavender odor (p = 0.002, δ = 0.60) and chocolate (p = 0.003, δ = 0.60). The PPT 1311 1312 group displayed more paracopulatory behaviors than the Oil group during exposure to 1313 1314 lavender odor (p = 0.038, δ = 0.40) but not during the other conditions. The DPN group did 1315 1316 1317 not differ from the Oil group in any condition. These data are found in Fig. 7 C. The 1318 2 1319 frequency of rejections was not modified by the treatments (H3, N = 38 = 3.30, p = 0.347, η = 1320 1321 0.09; data not shown). 1322 1323 1324 1325 3.2.2 Female attractivity to males 1326 1327 All experimental conditions collapsed, there was a main effect of treatment on the 1328 1329 number of mounts received H , = 20.03, p < 0.001, η2 = 0.53). The females in the EB 1330 3 N = 38 1331 1332 group were mounted more than the females in the Oil group (p < 0.001, δ = 0.90) and so were 1333 1334 females treated with PPT (p = 0.024, δ = 0.40). The DPN group (p = 0.678, δ = 0.10) did not 1335 1336 differ from the Oil group. 1337 1338 The number of mounts received differed between treatments during exposure to 1339 1340 2 2 lavender odor (H3, N = 38 = 10.25, p = 0.017, η = 0.27), music (H3, N = 38 = 8.86, p = 0.031, η 1341 1342 = 0.23) and chocolate (H , = 15.60, p = 0.001, η2 = 0.41). No difference was found at 1343 3 N = 38 1344 1345 baseline, during exposure to white noise or fox odor (all ps > 0.103). The EB group received 1346 1347 more mounts than the Oil group during exposure to lavender odor (p = 0.012; δ = 0.50), music 1348 1349 (p = 0.016; δ = 0.30) and chocolate (p < 0.001, δ = 0.50). Only during exposure to lavender 1350 1351 odor, females treated with PPT were more mounted than those treated with Oil (p = 0.031; 1352 1353 1354 1355 1356 1357 1358 24 1359 1360 δ=0.50). The DPN group was never different from the Oil group (all ps = 1). Data are 1361 1362 1363 summarized in Fig. 8 A. 1364 1365 All experimental conditions collapsed, there was an effect of the treatment on the 1366 2 1367 frequency of male pursuit of the females (H3, N = 38 = 13.73, p = 0.003, η = 0.36). The males 1368 1369 pursued females given EB more than those given oil (p = 0.002, δ = 0.78). Neither PPT- nor 1370 1371 DPN-treated females differed from Oil (ps > 0.120). 1372 1373 The number of pursuit episodes differed between the treatments during exposure to 1374 1375 lavender odor (H , = 8.24, p = 0.041, η2 = 0.22). There was no difference between the 1376 3 N = 38 1377 1378 treatments in the frequency of male pursuit at baseline, during exposure to music, chocolate, 1379 1380 white noise and fox odor (all p’s > 0.053). During exposure to lavender odor the EB group 1381 1382 was more pursued by the males than the Oil group (p = 0.017, δ = 0.55). The other treatment 1383 1384 groups were not different from the Oil group (all ps > 0.129; Fig. 8 B). 1385 1386 The frequency of male anogenital sniffing of the females was unaffected by the 1387 1388 2 treatments (H3, N = 38 = 4.67, p = 0.198, η = 0.12) when all experimental conditions were 1389 1390 1391 considered. We then proceeded with an analysis of the effects of EB in each of the 1392 1393 experimental conditions. It turned out that the frequency of male anogenital sniffing did not 1394 1395 differ between treatments at any of the experimental conditions (ps > 0.058). Neither the 1396 1397 frequency of male resting with females nor the frequency of sniffing the females differed 1398 1399 between treatments at any of the experimental conditions (all ps > 0.100; data not shown). 1400 1401 1402 1403 3.2.3 Exploratory behaviors 1404 1405 There was no main effect of treatment for any of the exploratory behaviors (all ps > 1406 1407 1408 0.071), and no interaction treatment * condition (ps > 0.286). 1409 1410 1411 1412 3.2.4 Female prosocial behavior 1413 1414 1415 1416 1417 25 1418 1419 There was no main effect of treatment on the frequency of resting with another female 1420

1421 2 2 1422 (F3,34 = 1.33, p = 0.281, η = 0.11) nor with a male (F3,34 = 1.60, p = 0.207, η = 0.12). The 1423 1424 interaction treatment * experimental condition was, however, significant with regard to 1425 2 1426 resting with a male (F15,170 = 2.99, p < 0.001, = 0.21). This prompted tests for simple main 1427 1428 effects of treatment within each of the experimental conditions. It turned out that there was a 1429 1430 treatment effect during exposure to chocolate. The Tukey HSD test revealed that the females 1431 1432 treated with PPT rested less with males than the females treated with oil (p = 0.006, d = 2.13). 1433 1434 There was no effect at any other experimental condition. Data are shown in Fig. 9. 1435 1436 1437 None of the treatments affected the frequency of sniffing another female or of the 1438 1439 males. Likewise, the interactions between treatment and experimental condition was 1440 1441 nonsignificant (all ps > 0.605; data not shown). 1442 1443 1444 1445 3.2.5 Female antisocial behavior towards males and females 1446 1447 The frequency of nose-off or of fleeing involving other females or males was not 1448 1449 affected by treatment and there was no interaction between treatment and experimental 1450 1451 condition (ps > 0.075; data not shown). 1452 1453 1454 1455 1456 3.2.6 Female non-social behavior 1457 1458 There was no main effect of treatment on drinking, resting alone and self-grooming 1459 1460 and there was no interaction between treatment and experimental condition (all ps > 0.230; 1461 1462 data not shown). 1463 1464 1465 1466 3.2.7 Treatment effects on white-noise specific behaviors 1467 1468 Data for all the condition specific behaviors, except “hide alone” and “hide with 1469 1470 1471 another rat”, greatly deviated from the normal distribution and were analyzed with non- 1472 1473 1474 1475 1476 26 1477 1478 parametric statistics. There was no treatment effect on the frequency of hiding alone, hiding 1479 1480 1481 with another rat, freezing, or startle (all ps > 0.662). The proportion of females fleeing from 1482 2 1483 the noise differed between the treatments (χ 3 = 8.43, p = 0.038; V = 0.47) and so did the 1484 2 1485 latency to flee (H3, N=38 = 8.23, p = 0.041, ηH = 0.22). More females in the PPT group fled 1486 1487 from the noise (Fisher exact test, p = 0.038, d = 0.66; Fig. 10 A) and they had a shorter 1488 1489 latency to flee than the Oil group (p = 0.008, δ = 0.50; Fig. 10 B). None of the other 1490 1491 treatments differed from oil (ps > 0.512). 1492 1493 1494 1495 1496 3.2.8 Treatment effects on chocolate-specific behaviors 1497 1498 The treatments did not influence the frequency of grabbing a chocolate pellet, of 1499 1500 eating or sniffing the chocolate or the latency to approach the chocolate (all ps > 0.210; data 1501 1502 not shown). 1503 1504 1505 1506 3.2.9 Weight gain 1507 1508 We calculated the weight difference between the moment the females were introduced 1509 1510 1511 into the seminatural environment and the moment they were removed. The weight change was 1512 1513 expressed as a proportion of initial weight. After the 8 days spent in the environment, the 1514 2 1515 weight gain was not evenly distributed among the females (F3,34 = 9.62, p < 0.001 ηp = 0.46). 1516 1517 The EB group gained less weight than the Oil group (p < 0.001, d = 2.36). The PPT and the 1518 1519 DPN groups did not differ from the Oil group (PPT-Oil: p = 0.068, d = 1.22; DPN-Oil: p = 1520 1521 0.852, d = 0.36). Data are shown in Fig. 11. 1522 1523 1524 1525 1526 3.2.10 Summary of the treatment effects 1527 1528 EB, and to some extent PPT, stimulated the sexual behaviors. These treatments also 1529 1530 enhanced some aspects of females’ attractivity. DPN had no effect. Pro- or antisocial 1531 1532 1533 1534 1535 27 1536 1537 behaviors were not modified by any of the treatments. This was also the case for exploratory 1538 1539 1540 behaviors. 1541 1542 EB stimulated sexual behaviors at baseline, during exposure to lavender odor and 1543 1544 music, and when chocolate was available. During white noise or fox odor, EB-treated females 1545 1546 did not differ from those treated with oil. PPT enhanced these behaviors only during exposure 1547 1548 to lavender odor. During exposure to lavender odor and music, as well as when chocolate was 1549 1550 available, EB enhanced female attractivity. PPT did so only during exposure to lavender odor. 1551 1552 When chocolate was available, PPT reduced the time spent resting with males, and during 1553 1554 white noise this compound facilitated the flight reaction. DPN did not affect any behavior in 1555 1556 any condition. 1557 1558 1559 1560 1561 4. Analysis of co-occurrences 1562 1563 Co-occurrence analysis identified the behavioral associations typical of the 1564 1565 experimental condition without considering treatment. The baseline condition showed modest 1566 1567 associations between sniffing another rat, nose off and fleeing. In a common cluster, we find 1568 1569 white noise and chocolate availability. Associated to the white noise we find rearing. The 1570 1571 main behavior in this cluster is sniffing the floor, probably a result of the enhanced 1572 1573 exploratory behavior observed during these two conditions. Lavender odor is associated with 1574 1575 1576 the sexual behaviors as well as with male pursuit of the female, anogenital sniffing, and 1577 1578 rejection. Finally, there is a cluster containing the conditions of fox odor and music, and the 1579 1580 behaviors of resting alone and resting with another rat. Perhaps this illustrates that these 1581 1582 conditions somewhat reduced social behaviors, making the non-social activity of resting more 1583 1584 preeminent. 1585 1586 Analysis of treatment effects, ignoring experimental conditions, revealed that EB and 1587 1588 PPT each belonged to a different cluster whereas Oil and DPN were found in the same 1589 1590 1591 1592 1593 1594 28 1595 1596 cluster. EB is associated with the sexual behaviors and drinking, whereas PPT is associated 1597 1598 1599 with exploratory behaviors, grooming, chocolate related behaviors and behavior patterns 1600 1601 indicative of fear like freezing, hiding and startle. Oil and DPN are mainly related to sniffing 1602 1603 other rats and nose off. 1604 1605 We then evaluated the treatments in each experimental condition. During baseline, Oil 1606 1607 and DPN were found in the same cluster, whereas EB and PPT were in separate clusters (Fig. 1608 1609 12 A). During exposure to lavender odor, Oil and DPN were found in the same cluster, while 1610 1611 EB was clearly associated with sexual behaviors, and PPT was related to exploratory 1612 1613 behaviors and grooming. Data are found in Fig. 12 B. During music, a separate cluster 1614 1615 containing EB and sexual behavior was found. PPT was found in another cluster, together 1616 1617 1618 with nose-off and fleeing. DPN formed a cluster together with rejection and drinking, whereas 1619 1620 Oil was associated with exploratory and social behaviors (Fig. 12 C). Chocolate exposure 1621 1622 again made Oil and DPN appear in the same cluster, with EB and PPT clearly separated. EB 1623 1624 was associated with sexual behaviors, whereas PPT had a strong association with chocolate- 1625 1626 related and exploratory behaviors (Fig. 12 D). White noise seemed to have altered behavior. 1627 1628 All treatments now belonged to different clusters, and some behavior patterns formed clusters 1629 1630 unrelated to the treatments. However, behaviors associated with white noise avoidance were 1631 1632 found in the same cluster as DPN (Fig. 12 E). Fox odor also caused each of the treatments to 1633 1634 1635 belong to different clusters, with EB having a minor association with lordosis, and PPT with 1636 1637 paracopulatory behavior (Fig. 12 F). DPN appeared in a separate cluster during exposure to 1638 1639 TMT. 1640 1641 1642 1643 4. Discussion 1644 1645 4.1 General considerations 1646 1647 1648 1649 1650 1651 1652 1653 29 1654 1655 The data obtained in this study are meaningful only if two requirements are met: That 1656 1657 1658 the emotion-inducing procedures actually induced the intended emotion and that the 1659 1660 experimental treatments were active with about the same intensity during the 5.83 h between 1661 1662 the beginning of the baseline observation and the end of the observation during fox odor 1663 1664 exposure. If one or both of the requirements fail, then we cannot determine the effects of 1665 1666 negative and positive emotions on female sexual behavior. Likewise, it would be impossible 1667 1668 to determine the role of the estrogen receptors in the responses to the experimental conditions. 1669 1670 Concerning the effectiveness of the experimental conditions, it is evident that some of 1671 1672 them indeed altered behavior in the expected way. This is particularly the case for chocolate 1673 1674 availability and white noise. The data show that the availability of chocolate had clear 1675 1676 1677 behavioral effects. Both prosocial, antisocial, and exploratory behaviors were stimulated. All 1678 1679 these responses may be interpreted as manifestations of increased arousal. It is unlikely that 1680 1681 the enhanced arousal was caused by a fear reaction, since the females made more visits to the 1682 1683 open area than at baseline. It is known that food reward causes increased arousal (Killeen et 1684 1685 al., 1978), often called food arousal (Tuersley & McCrohan, 1987), in addition to positive 1686 1687 affect. The response to chocolate observed here is, then, what would be expected if this 1688 1689 stimulus indeed induced positive affect. White noise clearly caused a fear reaction, manifested 1690 1691 as avoidance of the open area and enhanced antisocial behavior. The effect of lavender odor 1692 1693 1694 on non-sexual behaviors was limited to enhanced activity in the open area. Whether this is a 1695 1696 manifestation of positive affect, as we expected during lavender exposure, is an open 1697 1698 question. However, the fact that lavender odor altered sexual behavior reinforces the notion 1699 1700 that this stimulus might have had the desired effect. To the contrary, music did not produce 1701 1702 any behavioral manifestation of positive affect. It rather appeared to cause a fear reaction, 1703 1704 since the time in the open area was reduced. Finally, fox odor had very slight behavioral 1705 1706 effects, and it cannot be concluded that the females responded with fear to this odor. It is 1707 1708 1709 1710 1711 1712 30 1713 1714 worth noting, though, that the co-occurrence analysis localized music and fox odor to the 1715 1716 1717 same cluster. Perhaps recent data showing that TMT is inferior to fox feces for producing fear 1718 1719 responses in rats (Rampin et al., 2018) could explain the modest effect of this compound. This 1720 1721 proposal is, of course, at variance to a substantial number of reports on the effectiveness of 1722 1723 TMT (reviewed in Rosen et al., 2015). 1724 1725 In sum, the data allow us to suggest that chocolate availability, white noise, and 1726 1727 perhaps lavender odor, had the intended effects, whereas music rather seemed to have an 1728 1729 effect opposite to what we expected. Fox odor had slight effects, especially when compared to 1730 1731 the other aversive stimulus, white noise. Nevertheless, we conclude that the first requirement 1732 1733 mentioned above is, at least partially, satisfied. 1734 1735 1736 The second requirement, the constant effect of the treatments during the entire 1737 1738 observation period, is only possible to answer for oil and EB. The no-effect of oil is obviously 1739 1740 constant, and there are good reasons to believe that the effects of EB outlast the observation 1741 1742 period. We have earlier reported that female sociosexual behaviors are remarkably stable 1743 1744 during the entire period of behavioral estrus in intact, cycling females in a seminatural 1745 1746 environment. There is no significant change in lordosis frequency or LQ from the moment the 1747 1748 first lordosis of the estrus period is displayed until the last lordosis (Chu & Ågmo, 2014). The 1749 1750 change from non-receptivity to full receptivity and vice versa is almost instantaneous (Chu & 1751 1752 1753 Ågmo, 2015a). Similar data have been obtained in ovariectomized females, given the same 1754 1755 doses of EB and P as used here. The duration of behavioral estrus in those females was 6.35 ± 1756 1757 0.42 h (mean ± SEM) (Le Moëne et al., 2015). This is longer than the duration of the present 1758 1759 observation. Thus, the intrinsic effects of EB should have remained constant under all 1760 1761 experimental conditions. The duration of the effects of PPT and DPN is unknown, but there is 1762 1763 no compelling reason to assume that it is much different from that of EB. The same molecular 1764 1765 events underlying sexual behavior are probably activated both by EB and PPT, and once 1766 1767 1768 1769 1770 1771 31 1772 1773 activated these events will have a similar time course (see Pfaff, 2017 for an extensive review 1774 1775 1776 of the molecular events underlying estrogen-induction of lordosis). Other actions, as well as 1777 1778 those of DPN, can have a different time course, so present data need to be interpreted with 1779 1780 some caution. Nevertheless, we propose that the effects of the treatments remained reasonably 1781 1782 stable throughout the observation period. 1783 1784 In addition to the two requirements discussed above, there are to caveats to the 1785 1786 meaningfulness of the data obtained in this experiment. The first is the possibility that one or 1787 1788 several of the experimental conditions influenced the subsequent condition or conditions. We 1789 1790 have no data to refute this possibility. However, it can also be argued that a sequence of 1791 1792 events probably is part of rats’ nightly experience in their natural habitat. Consequently, our 1793 1794 1795 design would contribute to enhance external validity compared to an experiment consisting of 1796 1797 a single event. Nevertheless, it cannot be excluded that the specific sequence used here 1798 1799 somewhat affected the results. 1800 1801 A second caveat concerns the confounding effects of potential circadian rhythms 1802 1803 causing variations in behavior during the rather long observation. This, however, is highly 1804 1805 unlikely. There is no change in receptivity from the beginning to the end of estrus (Chu and 1806 1807 Ågmo, 2014). Locomotor activity shows peaks at both ends of the dark period, but it remains 1808 1809 at a stable, high level during the middle part (Spiteri et al., 2012). Also food intake remains 1810 1811 1812 stable during that period (e.g. Kersten et al., 1980). Thus, circadian variations cannot explain 1813 1814 the differences between the experimental conditions. Finally we would like to point out that 1815 1816 the males’ sexual activity was as high at the end of the observation period as at baseline 1817 1818 (mean ± SEM number of mounts was 0.38 ± 0.18 at baseline vs. 0.31 ± 0.22 during exposure 1819 1820 to TMT, V = 36, p = 0.412). 1821 1822 4.2 Negative and positive emotions and sexual behavior 1823 1824 1825 1826 1827 1828 1829 1830 32 1831 1832 One aim of the present study was to determine the effects of fear on female sexual 1833 1834 1835 behavior. White noise strongly inhibited sexual behaviors. In fact, females treated with EB 1836 1837 did not show more of these behaviors in the presence of the noise than females treated with 1838 1839 oil. It is interesting to note that fox odor also eliminated the difference between oil- and EB- 1840 1841 treated females, even though this odor had no observable effects on behaviors indicating fear 1842 1843 or anxiety. Perhaps female sexual behaviors are more sensitive to potential threats than other 1844 1845 behaviors, at least in the seminatural environment. Little is known about the differential effect 1846 1847 of stress on the entire behavioral repertoire, and the relative sensitivity of each behavior to the 1848 1849 environmental context. It could even be proposed that observations of sex behavior in the 1850 1851 seminatural environment are a potential, externally valid procedure for evaluating anxiolytic 1852 1853 1854 and anxiogenic drugs. This possibility should be further explored. We also want to point out 1855 1856 that this is the first study in which the effects of aversive or fearful stimulation present in the 1857 1858 test situation on sexual behavior have been evaluated. 1859 1860 While fear-inducing situations inhibited female sexual behavior, it appears that 1861 1862 situations putatively leading to positive affect enhance these behaviors. This is evident for 1863 1864 lavender odor, in which both EB- and PPT-treated females showed a non-significant tendency 1865 1866 to display more sexual behaviors than in at baseline. These treatments also enhanced female 1867 1868 attractivity to the males during the presence of lavender odor. Considering that lavender odor 1869 1870 1871 might induce a state of positive affect, it could be suggested that such affect facilitates female 1872 1873 sexual behaviors, and makes the female more attractive to males. Whether the enhanced 1874 1875 female attractiveness was due to factors intrinsic to the female or to lavender-induced, 1876 1877 enhanced male responsivity to the females is not known. However, since female receptivity 1878 1879 also was increased during exposure to lavender odor, it is likely that lavender-induced 1880 1881 changes in the females was the main factor behind the observed behavioral changes. 1882 1883 Chocolate availability did not have any particular effect on the sexual behaviors, despite the 1884 1885 1886 1887 1888 1889 33 1890 1891 fact that its consumption should have caused positive affect, just as lavender odor. The fact 1892 1893 1894 that our observations were limited to the moment when chocolate was available may, 1895 1896 however, obscure any possible effect. The behavioral consequences of the positive affect 1897 1898 caused by chocolate availability might have been counteracted by the urge to collect and 1899 1900 consume the pellets. 1901 1902 Too little is known about the actions of lavender odor and chocolate to make any 1903 1904 informed speculation about the causes of differences in effects on female behavior. 1905 1906 Furthermore, as was the case with aversive stimuli, the influence of positive affect on sexual 1907 1908 behavior has not been studied before, rendering any effort to propose explanations for these 1909 1910 discrepancies still more difficult. 1911 1912 1913 The many studies of the effects on female sexual behavior of drugs producing positive 1914 1915 or negative affect (e.g. Guarraci & Bolton, 2014; Ågmo, 2014) are not directly relevant for 1916 1917 the issue of how emotional state might alter sexual behaviors. The drugs have many effects in 1918 1919 addition to altering the emotional state (e.g. Paredes & Ågmo, 2004; López, 2010), making 1920 1921 such studies difficult to interpret. In fact, drug effects are usually explained in terms of altered 1922 1923 neurotransmission rather than in terms of altered emotional states. 1924 1925 1926 1927 4.3 Anxiogenic and anxiolytic effects of estrogen receptor activation 1928 1929 1930 We did not obtain much evidence for estrogen effects on fear behavior. EB and DPN 1931 1932 had no effect whatsoever on the frequency of individual behavioral items, whereas PPT 1933 1934 showed two signs of having produced or enhanced fear reactions. It reduced female resting 1935 1936 with males in the presence of chocolate, and it enhanced the flight reaction in the presence of 1937 1938 white noise. Both these effects can be interpreted as manifestations of fear or anxiety. PPT 1939 1940 would then be anxiogenic in the chocolate and noise conditions. These conditions were 1941 1942 associated with heightened arousal, and it has been shown that PPT indeed is anxiogenic in 1943 1944 1945 1946 1947 1948 34 1949 1950 such situations (Lund et al., 2005; Morgan et al., 2004; Spiteri et al., 2010a; Spiteri et al, 1951 1952 1953 2010b; see also Borrow & Handa, 2017, for a review). It is also noteworthy that PPT 1954 1955 belonged to a cluster separate from the other treatment clusters at all experimental conditions. 1956 1957 This shows that the females treated with this compound had a behavioral structure different 1958 1959 from all other treatments. This fact can probably be attributed to the fact that PPT stimulated 1960 1961 sexual behaviors under some conditions and enhanced anxiety-like behaviors under others. 1962 1963 The complete lack of effect of DPN on the frequency and duration of the behaviors 1964 1965 recorded here would indicate that the ERβ receptor is of little or no importance for sexual 1966 1967 activity as well as for fear and anxiety in test procedures with external validity. However, the 1968 1969 co-occurrence analysis showed that DPN belonged to a separate cluster in the situations that 1970 1971 1972 might be considered aversive, i.e. during exposure to music, white noise and fox odor. In the 1973 1974 neutral or positive conditions, i.e. at baseline, during exposure to lavender odor and chocolate, 1975 1976 DPN and oil belonged to the same cluster. This important observation suggests that actions at 1977 1978 the ERβ only becomes apparent in contexts being aversive or even inducing fear. It appears, 1979 1980 then, that present data confirm the lack of a role for the ERβ in sexual behavior as well as its 1981 1982 importance for anxiety-related behaviors. 1983 1984 The potential role of the membrane receptor GPER1 has not been mentioned. This 1985 1986 receptor is obviously activated in the EB-treated females, and perhaps also in the females 1987 1988 1989 treated with PPT. High concentrations of this ERα agonist bind to the GPER1, and DPN 1990 1991 might be still less active (Petrie et al., 2013). Since the GPER1 has been implicated in fear 1992 1993 responses as well as in female sexual behavior (reviewed in Hadjimarkou and Vasudevan, 1994 1995 2018), and since there is evidence for crosstalk between GPER1 and the ERα, it is possible 1996 1997 that the GPER1 may have contributed to the effects observed in the present study. This issue 1998 1999 is, however, too complex for being analyzed here. 2000 2001 2002 2003 2004 2005 2006 2007 35 2008 2009 Another issue not addressed here is the possible contribution of local synthesis of 2010 2011 2012 estrogens. Although such synthesis has been suggested to affect some of the behaviors studied 2013 2014 here (reviewed in Cornil, 2018), present data have no relevance for this question. 2015 2016 2017 2018 4.4 On the utility of a seminatural environment and the problem of opposing effects of ERα 2019 2020 and ERβ 2021 2022 When the experimental subjects are given the opportunity to express a substantial 2023 2024 proportion of their natural behavioral repertoire, the multitude of data generated needs to be 2025 2026 made comprehensible in some way or another. Moreover, the frequency or duration of 2027 2028 behavioral items give only a rudimentary description of behavior. Behavior patterns are 2029 2030 2031 displayed in a continuous flow, and the sequence of behavior is completely ignored in 2032 2033 frequency and duration analysis. The co-occurrence analysis and the clustering and 2034 2035 visualization techniques employed here makes the patterning of behavior intelligible, and 2036 2037 subtle modifications can be discovered. This, for example, made it possible to see that DPN 2038 2039 affected behavior in aversive situations, even though the frequency and duration of none of 2040 2041 the behaviors was altered. 2042 2043 At the time of the experimental manipulations, the rats had lived in the seminatural 2044 2045 environment for 7 days. Consequently, they have had plenty of time to familiarize themselves 2046 2047 2048 to the environment and to the other rats. One manifestation of this is the almost complete 2049 2050 absence of aggressive interactions. It is reasonable to assume that the subjects considered the 2051 2052 environment as a safe place. We introduced the experimental conditions upon this baseline. A 2053 2054 similar approach was employed in the studies of fear and aggression in the visual burrow 2055 2056 system (Blanchard et al., 1995; Blanchard et al., 2001; Blanchard & Blanchard, 1989), a 2057 2058 procedure not entirely different from the one used here. The present results may be most 2059 2060 2061 2062 2063 2064 2065 2066 36 2067 2068 illustrative with regard to the behavioral consequences of the activation of the estrogen 2069 2070 2071 receptors. 2072 2073 In nature, rats live and copulate in groups, and most of their activities are localized 2074 2075 within the well-known home range. These characteristics are preserved in the seminatural 2076 2077 environment but entirely absent in most other tests. It can be maintained that our procedure 2078 2079 satisfies the requirements for a representative design in brunswikian terms (see Brunswik, 2080 2081 1955; Petrinovich, 1980, also Chu & Ågmo, 2016). Data from such designs have external 2082 2083 validity in the sense that they might be applicable to situations other than the same in which 2084 2085 the data were obtained. Therefore, we propose that activation of the ERα in female rats leads 2086 2087 to the display of sexual behaviors and enhanced fear in unsafe or novel situations, even 2088 2089 2090 outside the laboratory setting. The ERβ does not modify the sexual behaviors, but it may be 2091 2092 important for reducing fear in fear-inducing contexts, even outside of the laboratory setting. 2093 2094 Unfortunately, opposing actions of the ERα and ERβ would complicate the 2095 2096 understanding of the behavioral actions of estrogens. In the intact animal, both receptors 2097 2098 would be stimulated simultaneously, and opposing actions would then be nulled out. It is 2099 2100 difficult to find a situation in which circulating estradiol would stimulate one receptors and 2101 2102 not the other, meaning that opposing actions would not be physiologically relevant. The 2103 2104 solution to this conundrum is not immediately apparent. 2105 2106 2107 2108 2109 Acknowledgments 2110 2111 Financial support was received from the faculty of Health Sciences, University of Tromsø. It 2112 2113 is a pleasure to acknowledge generous and patient help from Gilles Le Pape regarding the use 2114 2115 of IraMuTeQ. 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Physiology and Behavior, 52, 755-760. 2847 2848 2849 2850 2851 2852 2853 2854 2855 2856 2857 2858 2859 2860 2861 2862 2863 2864 2865 2866 2867 2868 2869 2870 2871 2872 2873 2874 2875 2876 2877 2878 2879 2880 2881 2882 2883 2884 2885 2886 2887 2888 2889 2890 2891 2892 50 2893 2894 Table 1. Summary of the experimental procedure on the test day. 2895 2896 2897 Experimental condition 2898 2899 Baseline Lavender odor Music Chocolate White noise Fox odor 2900 2901 Time of the day 2902 12:30-13:00 13:00-13:30 14:20-14:44 15:35-16:05 16:55-17:10 18:00-18:30 2903 2904 Observation time 2905 2906 12:45-13:00 13:15-13:30 14:29-14:44 15:35-15:50 16:54-17:09 18:15-18:30 2907 2908 Duration 2909 30 min 30 min 24 min 18 s 30 min 15 min 30 min 2910 2911 2912 2913 2914 2915 2916 2917 2918 2919 2920 2921 2922 2923 2924 2925 2926 2927 2928 2929 2930 2931 2932 2933 2934 2935 2936 2937 2938 2939 2940 2941 2942 2943 2944 2945 2946 2947 2948 2949 2950 2951 51 2952 2953 Table 2. Definition of recorded behaviors. 2954 2955 f = frequency; d = duration; l = latency; o = occurrence. 2956 2957 Category Behavior pattern Definition 2958 2959 Female sexual Lordosis; f Posture of the female arching her back, exposing her vagina. 2960 2961 behaviors 2962 Paracopulatory behaviors; f,d Approach to a male followed by runaway, often associated with hops, darts, and ear 2963 wiggling. 2964 2965 2966 Rejection; f Female kicks, boxes or assumes a belly up posture. 2967 Female 2968 Mounts received; f Male catches the female by her waist and puts his belly over her back, with pelvic 2969 attractivity 2970 thrusting. 2971 2972 Male pursuit; f,d Male runs after a female with his snout in close to the anogenital zone of the female. 2973 2974 2975 Male sniffing female; f,d Snout close to a female, sniffing the fur 2976 2977 Male sniffing anogenital area, f,d Male sniffs the anogenital zone of a female by putting his snout under her tail. 2978 Prosocial 2979 2980 behaviors Resting with other females; f,d Rests immobilized in relaxed position at a distance shorter than one rat to one or 2981 several females. 2982 2983 Rests immobilized in relaxed position at a distance shorter than one rat to one or 2984 Resting with males,f,d 2985 several males. 2986 2987 2988 2989 2990 2991 2992 52 2993 2994 Snout close to a female, sniffing the fur 2995 2996 2997 Sniffing other females,f,d Snout close to a male, sniffing the fur 2998 2999 Sniffing males; f,d 3000 Antisocial 3001 b; behaviors Hiding with another rat f,d Immobilized in a corner or in a nest box within one body length of the other rat. 3002 3003 Nose-off male; f,d The female faces a male, nose to nose, heads up, with or without boxing. 3004 3005 Nose-off female; f,d The female faces another female, nose to nose, heads up, with or without boxing. 3006 3007 3008 3009 3010 Flee from male; f Escapes from agonistic interaction by running away or simply turning head away 3011 from a male. 3012 Solitary 3013 Escapes from agonistic interaction by running away or simply turning head away 3014 behaviors Flee from another female; f 3015 from a female. 3016 3017 Resting alone; f,d Rests immobilized in relaxed position at a distance longer than one rat to a 3018 3019 conspecific. 3020 3021 Drinking; f,d Self explanatory. 3022 3023 Selfgrooming and scratching; f,d Self explanatory. 3024 3025 3026 3027 3028 3029 3030 3031 3032 3033 53 3034 3035 Hide aloneb; f,d Immobilized in a corner or nest box at a distance longer than one body length to 3036 3037 another rat. 3038 3039 Approach to chocolatea; f,l Coming close enough for making snout or paw contact with the chocolate pellets. The 3040 3041 latency is the time between putting the petri dish on the floor of the open area and the 3042 first approach. 3043 3044 3045 Grabbing chocolate with paws or mouth. 3046 Grabbinga; f 3047 3048 Eatinga; f,d Chew on chocolate. 3049 3050 Freezingb; f,d Immobilized in rigid position without any movement including those of vibrissa. 3051 3052 Startleb; o Sudden reflex contractions of the major muscles of the body, leading to a little jump 3053 3054 on the spot. Only observed in response to onset of the white noise. 3055 3056 Flee from noiseb; o,l Rush into the burrows at the onset of the white noise. The latency is the time from 3057 Exploratory 3058 onset of the noise until the rat escapes from the open field into the burrow. behaviors and 3059 3060 ambulatory Sniffing the floor, f,d Sniffs the floor material with all four paws on the floor 3061 activity 3062 Rearing; f,d Sniffs the air while standing on the hind legs. 3063 3064 Transitions; f Displays a behavior in a zone different from the one in which the previous behavior 3065 3066 was displayed. 3067 3068 3069 3070 3071 3072 3073 3074 54 3075 3076 a, behavior observed only in the presence of chocolate. b, behavior only observed during exposure to white noise. 3077 3078 3079 3080 3081 3082 3083 3084 3085 3086 3087 3088 3089 3090 3091 3092 3093 3094 3095 3096 3097 3098 3099 3100 3101 3102 3103 3104 3105 3106 3107 3108 3109 3110 3111 3112 3113 3114 3115 55 3116 3117 Figure legends: 3118 3119 Figure 1. A. Picture of the seminatural environment. B. The division in zones. 3120 3121 3122 Figure 2. Effect of the experimental conditions on female sexual behavior, for EB- and PPT- 3123 3124 treated groups only, both treatments collapsed. A. Lordosis frequency. B. Lordosis quotient. 3125 3126 C. Frequency of paracopulatory behaviors. D. Rejection frequency. Data are mean ± SEM. 3127 3128 Friedman test, post hoc: Conover-Iman test. *, different from baseline. N=20. 3129 3130 3131 Figure 3. Effect of the experimental conditions on female attractivity to males, all treatments 3132 3133 collapsed. A. Pursuit frequency. B. Frequency of male sniffing of a female. Data are mean ± 3134 3135 SEM. Friedman test, post hoc: Conover-Iman test. *, different from baseline. N=38. 3136 3137 3138 Figure 4. Effect of the experimental conditions on female exploratory behavior, all treatments 3139 3140 collapsed. A. Frequency of transition between the zones of the seminatural environment. B. 3141 3142 Frequency of transition between the zones of the open area. C. Frequency of transition 3143 3144 between the zones of the burrow system. D. Time spent in the open area. E. Time spent in the 3145 3146 burrow system. F. Rearing frequency. G. Frequency of sniffing the floor. Data are mean ± 3147 3148 3149 SEM. Repeated measures two-way ANOVA, post hoc: Tukey HSD. *, different from 3150 3151 baseline. N=38. 3152 3153 Figure 5. Effect of the experimental conditions on female social behavior directed to males 3154 3155 3156 (black) and to other females (white), all treatments collapsed. A. Frequency of resting with 3157 3158 another rat. B. Frequency of sniffing another rat. Data are mean ± SEM. Repeated measures 3159 3160 two-way ANOVA, post hoc: Tukey HSD. *, different from baseline. N=38. 3161 3162 3163 Figure 6. Effect of the experimental conditions on female anti-social behavior directed to 3164 3165 males (black) and to other females (white), all treatments collapsed. A. Nose-off frequency; 3166 3167 repeated measures two-way ANOVA, post hoc: Tukey HSD. B. Frequency of fleeing from 3168 3169 3170 3171 3172 3173 3174 56 3175 3176 another rat; Friedman test, post hoc: Conover-Iman test. Data are mean ± SEM. *, different 3177 3178 3179 from baseline; N=38. 3180 3181 Figure 7. Effect of the treatment on female sexual behavior. A. Lordosis frequency. B. 3182 3183 Lordosis quotient. C. Frequency of paracopulatory behaviors. Kruskal-Wallis test, post hoc: 3184 3185 Conover test. Data are mean ± SEM. *, different from the Oil group. Oil: n=8, EB: n=10, 3186 3187 3188 PPT: n=10, DPN: n=10. 3189 3190 Figure 8. Effect of the treatment on female attractivity to males. A. Mount frequency. B. 3191 3192 Pursuit frequency. Kruskal-Wallis test, post hoc: Conover test. Data are mean ± SEM. *, 3193 3194 3195 different from the Oil group. Oil: n=8, EB: n=10, PPT: n=10, DPN: n=10. 3196 3197 Figure 9. Effect of the interaction between the treatment and the experimental condition on 3198 3199 the frequency of female resting with a male. Repeated measures two-way ANOVA, post hoc: 3200 3201 3202 Tukey HSD. *, different from the Oil group. Oil: n=8, EB: n=10, PPT: n=10, DPN: n=10. 3203 3204 Figure 10. Effect of the treatment on the response to the onset of white noise. A. Probability 3205 3206 for an individual to flee from the white noise; Fisher exact test; statistical significance: *, 3207 3208 3209 different from the Oil group. Latency to flee the noise at its onset (B); Kruskal-Wallis test, 3210 3211 post hoc: Conover test; statistical significance: * different from the Oil group. Oil: n=8, EB: 3212 3213 n=10, PPT: n=10, DPN: n=10. 3214 3215 3216 Figure 11. Effect of the treatment on weight gain. Repeated measures two-way ANOVA, post 3217 3218 hoc: Tukey HSD. Data are mean ± SEM. *, different from the Oil group. Oil: n=8, EB: n=10, 3219 3220 PPT: n=10, DPN: n=10. 3221 3222 3223 Figure 12. Co-occurrence analysis showing main behavioral associations typical of each of 3224 3225 the treatments. A. Baseline. B. Exposure to lavender odor. C. Music. D. Chocolate. E. White 3226 3227 noise. F. Fox (TMT) odor. Clusters of behavioral association are represented in halos of 3228 3229 different colors. 3230 3231 3232