Submitted as a Research Report to Biology of Sex Differences


 Background: There are sex differences in addiction behaviors. To develop a pre-clinical animal model to investigate this, the present study examined sex differences in sucrose taking and seeking using LongEvans rats. Methods: Five experiments were conducted using separate groups of subjects. The first two examined sucrose or saccharin preference in two-bottle home cage choice tests. Experiment three assessed sucrose intake in a binge model with sucrose available in home cage bottles. Experiments four and five utilized operant-based procedures. In Experiment four rats responded for sucrose on fixed and progressive ratio (FR, PR) schedules of reinforcement over a range of concentrations of sucrose. A final component of experiment four was measuring seeking in the absence of sucrose challenged with the dopamine D1 receptor antagonist SCH23390. Experiment five assessed responding for water on FR and PR schedules of reinforcement. Results: When accounting for body weight, female rats consumed more sucrose than water; but there was no sex difference in saccharin preference over a range of saccharin concentrations. When accounting for body weight, females consumed more sucrose than males in the binge model, and only females increased binge intake over the 14 days of the study. Females responded at higher rates for sucrose under both FR and PR schedules of reinforcement. Females responded at higher rates in extinction (seeking); SCH23390 reduced sucrose seeking of both females and males. Females responded at higher rates for water on FR and PR schedules than males, although rates of responding were low and decreased over sessions. Conclusions: Across bottle-choice, binge intake, and operant procedures, female Long-Evans rats consumed more sucrose and responded at higher rates for sucrose. Although females also responded more for water, the vigor of responding did not explain the consistent sex difference in sucrose taking and seeking. The sex difference in sucrose taking was also not explained by sweet preference, as there was no sex difference in saccharin preference. These data corroborate with findings of sex differences in addiction behaviors in humans, providing a pre-clinical model to further evaluate sex differences in these behaviors and manipulations designed to reduce them.


Background 134
There are sex differences in various aspects of drug addiction behaviors including relapse [1], 135 [2],[3], [4]. A general summary of sex-difference findings for cocaine, for example, is that women not 136 only have higher rates of relapse, but also crave more and take more upon relapse [2]. Furthermore, 137 women are more vulnerable to addiction behaviors related to food. Women are also more likely to 138 crave sweet vs. savory foods and have more difficulty regulating those cravings compared to men [5]. In 139 addition, psychiatric eating disorders are more prevalent in women and women are also more likely to 140 be diagnosed with severe and morbid obesity [6]. Although a number of economic and societal factors 141 influence sex differences in drug-focused and eating behaviors, findings from animal models can provide 142 critical insight into these differences as well. 143 Sugars are high-value reinforcers for humans and other species, including rats. Like humans, 144 rats prefer sucrose-sweetened foods and will consume sucrose beyond caloric need [7], [8]. These and 145 other behaviors including food binging [9] indicate that examination of the reinforcing effects of sucrose 146 and other sugars provides insight into the profound effects of sugar on behavior and neurobiology, 147 especially in the context of food and drugs of abuse [7], [8]. 148 Sex differences in sweet preference by rats have been reported previously. Initial findings using 149 a choice procedure identified females preferred saccharin more than males did [10]. Subsequent 150 studies with sucrose have generally supported a sex difference with females preferring sucrose solutions 151 more than males, however caveats exist depending on methodology and stage of estrus cycle [11], [12]. 152 An extension of the choice procedure is the binge intake model where rats are provided access 153 to a sweet solution for either 12 or 24h per day. Rats in the 12h condition drink more sweet solution 154 during the first hour of daily access [13]. Sex differences in binging of fat or a palatable mixture (fat + 155 sugar) have been described, with females binging more than males [14], [15]. With sucrose alone as the 156 5 reinforcer, females binged slightly more than males [13] and females binged more than males but this 157 effect was observed only in Wistar-Kyoto, but not Wistar rats [16]. 158 Finally, operant reinforcement models are quintessential for developing a deeper understanding 159 of reinforcer-directed behavior. For example, depending on the reinforcement history and schedule of 160 reinforcement, rate of responding on a lever for sucrose could be indicative of motivation to acquire the 161 reinforcer, not just interest or preference. The fixed ratio (FR) schedule of reinforcement is often used 162 to assess interest or preference while the progressive ratio (PR) schedule of reinforcement is argued to 163 provide a measure of motivation [17]. Responding in the absence of reinforcement, such as in 164 extinction or for a cue paired previously with reinforcement, can be used to assess conditioned 165 reinforcing properties of stimuli [18]. The latter approach is an established preclinical model of craving 166 [19]. Currently, three studies have reported no significant sex differences in operant sucrose self-167 administration using the FR schedule of reinforcement; all used sucrose pellets as the reinforcer 168 [20], [21], [22]. PR results with sucrose alone have not yet been reported, however in a study where 169 sucrose plus a preferred flavor was used as the reinforcer, females responded to higher break points, an 170 index of motivation, than males [23]. Operant procedures also allow examination of the reinforcing 171 efficacy of conditioned stimuli including conditioned reinforcers. Thus far, sex differences have been 172 identified but findings both within and between laboratories are inconsistent. Specifically, females 173 9 tone+white light cue. There was a 40 s timeout before availability of the next reinforcer. Rats were 253 then randomly assorted to two different groups to be tested with either 0, 3.75, 7.5 or 7.5, 15, 30% 254 sucrose. Concentrations were tested in counterbalanced order with 3 days of 10% sucrose available in 255 between each test. Following a subsequent FR training day, rats trained with 10% sucrose on a 256 progressive ratio (PR) schedule for 7 days. Sessions began with extension of the active lever and 257 illumination of the red house light. The reinforcement contingency for responding on the active lever 258 then escalated according to [33]. Following a reinforced response, the active lever retracted for the 259 duration of the tone+white light cue (5 s) and .4 mL sucrose delivery (total of 6. preference was calculated over 1 g rat mass. In Experiment 3 intake was calculated over 1 g rat mass. In 279 some instances, in the literature the denominator for preference is 100 g but we chose to present data 280 for Experiments 1-3 to be internally consistent with data presentation. Where warranted, post-hoc 281 analyses were made using t-tests with Šidák-corrected p values calculated incorporating the total 282 number of comparisons made. As an indication of effect size, partial eta squared (η 2 ) is provided for 283 significant F tests and interactions and Cohen's d is provided for significant t-tests. The threshold for 284 statistical significance was p < .05. 285 Experiment 1. Sucrose preference. The final 48 h preference data are reported as (sucrose mL-286 water mL)/weight 1 g. Water mL was the intake from the water bottle on that test day. Calculation of 287 preference using this weight-adjusted formula has been used over the past several decades to 288 characterize saccharin preference, especially to identify low vs. high saccharin-preferring phenotypes in 289 rats [34]. We made one modification of the typical calculation: we used water intake during the choice 290 period instead of water intake during the initial habituation to bottle access. This was implemented to 291 be consistent with Experiment 2. In that experiment, the time between habituation to bottle access and 292 the final choice measurements was over two weeks. We deemed it more reasonable to use water 293 intake measures taken at the same time as saccharin measures. Preference scores were compared 294 between males and females using a t-test. Water intakes measured in mL/weight 1 g were compared 295 between males and females using a t-test. 296 Experiment 2. Saccharin preference. Preference data are reported as (saccharin mL-water 297 mL)/weight 1 g. Water mL was the intake from the water bottle on that test day. Preference scores 298 across the 5 concentrations were compared between males and females using two-way repeated 299 11 measures analysis of variance (2-way RMANOVA). Water intake in mL/weight 1 g was compared 300 between males and females across the five measures using 2-way RMANOVA. 301 Experiment 3. Sucrose binge. Sucrose intake is reported as sucrose mL/weight 1 g. Intake 302 across days of the study was compared between males and females and between sucrose access 303 conditions using 3-way RMANOVA. Separate analyses were conducted for overall daily intake (12 or 24 304 h) and binge test intake (1 h intake). Chow intake is reported as g/weight 1 g. Chow intake across days 305 of the study was compared between males and females and between sucrose access conditions using 3-306 way RMANOVA. 307 Experiment 4. Operant sucrose. Operant conditioning data are reported as the number of active 308 lever presses in each session. Training data for the two dose-response cohorts were combined after 309 initial 3-way RMANOVA of the six dependent measures (days 1 and 10 FR, FR day between FR and PR 310 training, days 1 and 7 PR, and day 3 of FR training prior to FR extinction testing) revealed no cohort 311 effect. Training FR (10 days) and PR (7 days) responses were compared between males and females 312 using 2-way RMANOVA. Testing FR and PR responses were compared between males and females using 313 2-way RMANOVA with the two dose-response cohorts analyzed separately. FR extinction testing data 314 (SCH23390 challenge) were compared between males and females using 2-way RMANOVA with the 315 previously tested dose-response cohorts combined. We did not normalize responding to body weight as 316 the FR with timeout and PR responding only indirectly relate to sucrose intake. FR extinction testing 317 data were analyzed a second time using day 10 of FR training active lever responding as a covariate 318 (ANCOVA). 319 Experiment 5. Operant water. Operant conditioning data are reported as the number of active 320 lever presses in each session. FR training, PR training, and the final FR re-training plus extinction test 321 responses were compared between males and females using 2-way RMANOVA. 322

Results 323
In the text and Figures, group averages are presented as mean ± standard error of the mean 324 (SEM). N sizes and male and female body weights at the start of each experiment are provided below. 325 Experiment 1. Sucrose preference. 10 males and 10 females served as subjects. Weights at the 326 start of the experiment were: males 392.7 ± 6.1 g, females 234.2 ± 4.4 g. Over a 48-h period, females 327 preferred sucrose more than males as measured by consumption of sucrose solution vs. water 328 accounting for body weight t(18) = -2.4, p < .05 (d = 1.2) ( Figure 1). Females also consumed more water 329 during this period, by body weight t(18) = -2.7, p < .01 (d = 1.2). Water intake (intake mL/body weight 1 330 g) during this period was: females .03 ± .003 and males .02 ± .004. 331 Experiment 2. Saccharin preference. 10 males and 10 females served as subjects. Weights at 332 the start of the experiment were: males 411.3 ± 9.6 g, females 244.1 ± 3.7 g. There was no sex 333 difference in saccharin preference across a range of saccharin concentrations (0, .075, .15, .3, .6%) with 334 preference measured as consumption of saccharin solution vs. water accounting for body weight. Both 335 males and females preferred saccharin vs. water across the range of concentrations examined F(4,72) = 336 9.2, p < .001 (η 2 = .3). Figure 2 indicates saccharin preference by females vs. males by body weight. 337 There was a sex difference in water intake across preference tests with females consuming more water. 338 This was not apparent at the 0% concentration ( Figure 2) but when comparing water intake by body 339 weight across all five, 72 h test sessions using 2-way RMANOVA (data not shown). There was a main 340 effect for sex F(1,18) = 9.2, p < .01 (η 2 = .9). Average test intake for males was .07 ± .008 mL/1 g and for 84.8, p < .001 (η 2 = .7), and for 1 h access F(1,44) = 69.2, p < .001 (η 2 = .6). Intake during 12 or 24 h 347 13 access increased across the 14 days of the experiment for both males and females F(13,572) = 6.2, p < 348 .001 (η 2 = .1). Intake during 1 h access increased across the 14 days for females, time x sex F(13,572) = 349 1.9, p < .05 (η 2 = .04). Chow intake (data not shown) decreased by 20.7% across the 14 days of the 350 experiment F(13,572) = 14.4, p < .001 (η 2 = .2) but also varied according to sex or sucrose intake 351 condition. There was a time x sex interaction F(13,572) = 2.9, p < .001 (η 2 = .06), and a time x condition 352 interaction F(13,572) = 2.9, p < .001 (η 2 = .06). For time x sex, females consumed an average of 13.9% 353 more chow over the first three days of the experiment with similar consumption between males and 354 females thereafter. For time x condition, consumption was 17.4% greater on day 1 of the experiment 355 for 24 h sucrose access rats, but for the rest of the experiment consumption was similar between access 356 conditions. 357 Experiment 4. Operant sucrose. 24 males and 24 females served as subjects. Weights at the 358 start of the experiment were: males 363.4 ± 4.5 g, females 219.7 ± 3.6 g. Active lever responses are 359 reported here. Other dependent measures (liquid reinforcer deliveries, inactive lever responses, 360 photobeam breaks) are presented in Supplementary results. As noted in the Methods, the training and 361 extinction data of the two dose-response cohorts were combined after initial 3-way RMANOVA of the six 362 dependent measures (days 1 and 10 FR, FR day between FR and PR training, days 1 and 7 PR, and day 3 363 of FR training prior to FR extinction testing) revealed no cohort effect. 364 FR training. Females responded at a higher rate than males F(1,46) = 33.6, p < .001 (η 2 = .4) and 365 this effect was consistent after the second day of training (time x sex interaction) F(9,414) = 3.3, p < .01 366 (η 2 = .07). Responding of both males and females increased over the 10 days of training F(9,414) = 7.9, p 367 < .001 (η 2 = .1). Figure 4  .01 (η 2 = .1) with a significant post-hoc test comparing 0 vs. the 10 μg/kg dose ( Figure 6). The fact that 390 females responded at a higher rate in extinction is confounded by their higher rate of responding when 391 responding for sucrose. ANCOVA analysis revealed that the extinction sex difference was not accounted 392 for by the higher rate of responding during training; the significant sex difference in extinction 393 responding remained F(1,45) = 45.5, p < .001 (η 2 = .5). ANCOVA results support a sex difference in 394 extinction responding despite higher reinforced responding during training. To address the question of 395 a sex difference in extinction or, arguably, "cue-reinforced" responding further, we calculated ratios of 396 active lever responding to sucrose deliveries on day 10 of training and ratio of active lever responding to 397 cue deliveries during the vehicle pretreatment extinction test day for all rats. We then compared male 398 and female rats with these ratios using 2-way RMANOVA. Females had larger ratios overall F(1,46) = 399 25.5, p < .001 (η 2 = .4). The ratios for male rats did not differ between training (responding for sucrose; 400 2.8 ± .3) and extinction testing (responding for sucrose-paired cue; 3.3 ± .4), however ratios for females 401 increased comparing training to extinction testing (from 4.6 ± .3 to 6.5 ± .6) with a significant post-hoc 402 comparison following a significant interaction F(1,44) = 5.3, p < .05 (η 2 = .1). In summary, males 403 responded with a similar ratio of responses to reinforcers for sucrose itself or a sucrose-paired cue. 404 Females overall responded at a higher rate for both sucrose and a sucrose-paired cue than males. 405 Females also responded approximately 1.4 times as much for a sucrose-paired cue compared to sucrose 406 itself, demonstrating that females are more cue-reactive than males 407 Experiment 5. Operant water. 13 males and 13 females served as subjects. Weights at the start 408 of the experiment were: males 414.0 ± 14.2 g, females 222.6 ± 3.5 g. Active lever responses are 409 reported here. Other dependent measures (liquid reinforcer deliveries, inactive lever responses, 410 photobeam breaks) are presented in Supplementary results. Female rats responded at a higher rate 411 than males for water on the FR schedule of reinforcement F(1,24) = 5.5, p < .05 (η 2 = .2). There was an 412 effect of days of training F(9,216) = 7.3, p < .001 (η 2 = .2) and a significant time x sex interaction F(9,216) 413 = 2.3, p < .05 (η 2 = .1), illustrated in Figure 7 as females responding more than males over the first days 414 of training, but the difference being negligible by day 10 of training ( Figure 7). PR training was similar in 415 this profile (Figure 7) with a main effect of sex F(1,24) = 4.4, p < .05 (η 2 = .2) and time F(6,144) = 3.6, p < 416 .01 (η 2 = .1) but there was no significant interaction. In summary for PR, females responded at a higher 417 rate than males and overall responding decreased over the 7 days of training for both males and 418 females. Reinforcers, inactive lever responses, and locomotor activity followed trajectories similar to 419 active lever responding (Supplementary results). 420 For the FR re-baseline and subsequent extinction test after completing the 7 days of PR training, 421 there were no significant effects. At this point in the study, response rates for water were low and did 422 not differ between males and females. In addition, when responding for the water-paired cue alone 423 (extinction test), rate of responding did not differ between males and females (Supplementary results). 424

Discussion 425
In summary, we observed sex differences in sucrose reinforcement in three assays. Females 426 preferred sucrose over water more than males, females consumed more sucrose than males when 427 available via bottles or after a lever response, and females responded at a higher rate for sucrose-paired 428 cues in extinction conditions. There were no sex differences in saccharin preference, nor in extinction 429 responding following a challenge with the dopamine D1 receptor antagonist SCH23390. 430 Sweet preference. Female rats preferred 10% sucrose over water to a greater degree than male 431 rats. This preference was observed using simple bottle choice considering body weight (Experiment 1) 432 ( Figure 1) and with greater sucrose intake in the binge study (Experiment 3) (Figure 3). Greater 433 preference of sucrose by female vs. male rats has only inconsistently been reported previously, with sex 434 differences more likely to be observed in binge access studies (see below). In a comprehensive 435 examination of tastant preference across 14 rat strains, sex differences in sucrose preference were 436 negligible including for Long-Evans rats [35]. These comparisons were made using sucrose intake as a 437 percentage of total fluid (sucrose + water). Body weight was not considered in these comparisons, 438 despite the substantial sexual dimorphism in body mass observed in many strains of adult rats. In 439 contrast, many studies describing saccharin preference consider body weight. For example, [34] 440 describes a calculation of saccharin preference score similar to that used in the present study, in their 441 case to differentiate low vs. high saccharin preferring phenotypes. Incorporating body weight into the 442 preference calculation emphasizes the importance of the absolute intake of the sweet substance for a 443 specific individual; in effect the measure is a mixture of preference and avidity. This approach to 444 quantifying preference in some studies, but not others, may also explain a lack of sex difference in 445 sucrose preference in other studies (e.g. [36], [37], [38]). For the present study, incorporating body 446 weight also allows within-study qualitative comparison across Experiments 1,2,3. 447 We did not find a sex difference in saccharin preference ( Figure 2)  sucrose intake over the 14 days of the experiment, perhaps suggestive of an escalation in sucrose 480 consumption although this is confounded with an expected accelerating acquisition curve. Binge intake 481 in this model is indicated when rats that have had 12 h access to sucrose consume significantly more 482 sucrose in a 1 h consumption test than rats with 24 h access to sucrose. In the present study, binge 483 intake was apparent by both male and female rats. While there was no main effect of sex for binge 484 intake during the 1h consumption measure, there was a sex difference in binge intake "escalation" in 485 the 1 h access measure where only females slightly increased consumption over days if they were in the 486 12 h but not 24 h access condition (Figure 3). 487 There are few studies in the literature to compare the present results, as sex differences in 488 binge intake with rats has largely been with rats consuming fat or fat+sugar combinations. Of those with 489 sucrose only binge intake, [13] reported a moderate sex effect with 1 h intake in 12 h rats with females 490 19 with greater intake than males on some days of the experiment. As noted above, we observed an 491 increase in 1 h intake across days for females only; [13] observed an increase for both sexes. This lab 492 [50] also found, in a study with only females, binging was more pronounced in proestrus indicating that 493 even if a sex difference in binge intake is moderate, the behavior is related to estrus cycle. However, clinical prevalence rates may be distorted by reporting constrained by ethnic/racial disparities 504 in individuals seeking support for eating disorders [52]. 505 Operant self-administration. As noted above, operant paradigms may provide more robust 506 models of sucrose consumption behavior. This includes studies of responding for reinforcement (taking) 507 and responding in the absence of reinforcement (seeking). The present study evaluated sucrose taking 508 behavior using two schedules of reinforcement, the FR and PR, with the latter argued to be a reasonable 509 measure of motivation to acquire a reinforcer. Using the PR, reinforcing efficacy can be determined 510 [17]. Active lever responding, indirectly tied to reinforcement, is a common measure of operant 511 response behavior. For the present study, all response data in the main manuscript are reported as 512 active lever responses. Active lever responses are not conducive to body weight adjustment, although 513 the number of earned reinforcers would be (see [23]). We did not make this adjustment in the present 514 20 study as females responded at higher rates both in terms of active lever and reinforcers earned. 515 Normalization by body weight would have exaggerated the already apparent sex differences. 516 Our robust sex differences across FR ( Figure 4) and PR ( Figure 5) schedules of reinforcement, 517 and across some concentrations of sucrose, contrast with previous findings reporting no sex differences 518 [20],[21], [22]. These previous studies all used sucrose pellets as reinforcers, compared to sucrose 519 solution used in the present study. A side-by-side comparison of responding for pellets vs. sucrose 520 solution could reveal whether this is a pellet vs. liquid solution issue, or an issue of differences in 521 laboratory procedures. As the sex differences observed thus far are with liquid (present study) but not 522 pellets, it is likely some aspect of the dry vs. liquid reinforcement is most important. 523 As with binge research, sex differences in palatable food taking and seeking has thus far 524 included studies with sucrose, sucrose+flavor, fat, or fat+carbohydrate combinations.
[53] examined sex 525 differences in an operant paradigm with within-session increases in response requirements designed to 526 assess intake depending on demand. Demand at zero (null) cost was estimated. Female rats were 527 estimated to have a higher demand for sucrose and sucrose+fat reinforcement at null cost, but not at 528 higher costs. It is notable that active lever responding on a FR 1 was not different between males and 529 females, but the null estimate that incorporates body weight revealed the significant sex difference at 530 the estimated null cost. Finally, in a study that incorporated PR intake, females had greater intake vs. 531 males of a sucrose+flavor reinforcer [23]. This study, referred to above, also required normalization by 532 body weight (reported as kcals/g^⅔) to reveal the sex difference. 533 Extinction responding (cue-reactivity) was more pronounced in females vs. males in the present 534 study. In Figure 6, females are shown to respond over 3 times as much as males in the vehicle dose 535 condition of the SCH23390 study. Overall, these values (males and females) are larger than what we 536 typically see in sucrose cue-reactivity studies. This is likely due to the fact that the rats had just been 537 responding on the response demanding PR schedule of reinforcement as part of Experiment 4. The 538 21 present results somewhat concur with previous studies of sex differences in conditioned responding to a 539 sucrose-paired cue.
[24] reported females develop more Pavlovian approach behavior compared to 540 males in response to a sucrose-associated cue. [21] found that females respond more for a sucrose-541 paired cue compared to males in an operant conditioning procedure similar to ours. Interestingly, that 542 same group did not observe a sex difference in a subsequent constructive replication [22]. In addition, 543 there was no sex difference in discriminative stimulus effects (CS+) of a palatable pellet (fat,carb,protein 544 combo) [25]. As with other inconsistencies reported above, the differences in procedures, strains, and 545 possibly age of subjects could account for the lack of consistent sex differences in sucrose seeking across 546 studies. What is most salient with the present results are the robust sex differences observed. 547 we are aware of where both male and female rats were challenged with SCH23390 under sucrose self-551 administration extinction conditions. These findings indicate that the sex difference in responding is not 552 specifically tied to D1 receptors, but furthermore that D1 antagonism is equally effective in males and 553 females at reducing sucrose seeking in a preclinical model of craving. 554 The higher response rate by females during sucrose FR and PR training, and the higher, but 555 diminished difference over training, response rate for water by females (Figure 7) introduces a potential 556 confound for interpreting both the sex differences in sucrose training but also cue-reactivity. Response 557 rate during sucrose training was accounted for with the ANCOVA described in the Results. That is, using 558 training response rate as a covariate did not affect the ANOVA outcome of a sex difference in extinction 559 responding. Furthermore, our analysis of the ratio between active lever and reinforcer delivery 560 (sucrose-paired cue delivery for extinction testing) as a means to normalize responding between sexes 561 indicated females responded more for the sucrose-paired cue than males (Results). As the water 562 22 responding was a separate experiment with a new group of subjects, we cautiously make comparisons 563 between experiments here to explore how much the rate of responding for water might account for rate 564 of responding for sucrose. It is not clear just what rats are responding for in our procedure: water is 565 provided with reinforced responses, but a bottle of water is also available in the operant conditioning 566 chamber and the rats are not food or water restricted. It is likely that rats are responding for the 567 novelty of water and/or cue presentations, as described previously with rats responding for a 568 presentation of a light stimulus [54]. However, the magnitude of responding for water does not appear 569 to account for the sex differences in operant responding for sucrose. First, the sex difference in 570 response rate for water (FR, then PR) disappeared over the 10 (FR) or 7 (PR) days of training. Second, 571 we subtracted the grand means of all training days for sucrose for males from those for females and 572 then the same was done for water. Grand means for training were: FR sucrose females -males = 65.6 573 active lever presses, FR water females -males = 21.8, PR sucrose females -males = 122.8, and PR water 574 females -males = 16.7. A relative comparison was then made between males and females in terms of 575 the magnitude of the difference between males and females responding for sucrose or water. The FR 576 difference between males and females was 300% more for sugar vs. for water, while the PR difference 577 between males and females was 736% more for sugar vs. for water. In summary, females responded for 578 water at higher rates than males but the effect was transient and rates were orders of magnitude below 579 that of responding for sucrose. 580 Nonetheless, in nearly every component of the present set of experiments where water intake 581 was measured, water intake varied by sex. Female rats in Experiment 1 (sucrose preference), 582 Experiment 2 (saccharin preference), and Experiment 5 (water self-administration) drank or responded 583 for more water than males. More intake by female rats has been reported in preference studies, for 584 studies that report water intake (e.g., [55]). Further study on this effect is required as water 585 23 homeostasis could affect a number of important variables key to interpreting sex differences in animal 586 models including food intake and drug pharmacokinetics. 587 Other potential confounds. Inactive lever and locomotor (photobeam breaks) data ANOVA 588 results for Experiments 4 (sucrose self-administration) and 5 (water self-administration) are provided in 589 the Supplemental results. A general finding was that female rats were more "active", reflected as 590 increased inactive lever presses and photobeam breaks. It is not clear how this background of increased 591 activity could account for the sex differences in sucrose taking and seeking observed in the present 592 study. Female rats have been noted to move about more than male rats in previous studies. For 593 example, female rats have a greater locomotor response to a novel environment [22]. It is important to 594 note that these measures were higher for females in both the sucrose and water self-administration 595 experiments in the present study. That is, elevated inactive lever and photobeam breaks may be 596 indicative of background activity not necessarily connected to motivated, or even impulsive behavior. 597 That being said, general increased basal activity and response rate is important to consider for future 598 studies. 599 Perspectives and significance 600 The present study provides a baseline for further investigation of sex differences in sucrose 601 taking and seeking in rats. Extending the generality across species, these results mostly parallel a very 602 recent study with mice as subjects [56]. Further studies are required to evaluate a potential role of 603 gonadal hormones in these sex differences, and to explore the neurobiology of sucrose taking and 604 seeking in males and females. For example, mesolimbic estrogen/dopamine interactions [ the Low range of concentrations, females responded more than males and male and females responded 856 more for 3.75 and 7.5% concentrations of sucrose compared to 0% (#vs. 0% concentration, p < .05). 857 Across the High range of concentrations, males and females differed at 7.5% (*p < .05) and only females 858 decreased responding at the 15 and 30% concentrations vs. 7.5% (#vs. 7.5%, p < .05). 859 860 Figure 5. Operant PR training and testing females vs. males. Females responded for sucrose more than 861 males during training and testing. For training (Top), responding by males and females increased over 862 days of training. For testing (Bottom), the only concentration-dependent effect was a decrease in 863 responding at the 30 vs. 7.5% concentration only, for both females and males (#vs. 7.5%, p < .05). 864 865 Figure 6. Operant extinction and D1 antagonist females vs. males. Females responded more than 866 males. SCH23390 reduced responding at the high dose (#vs. 0 dose, p < .05 females and males 867 combined). 868 869 Figure 7. Operant water females vs. males. For FR training (Top), females responded more than males 870 on days 5 and 6 (*p < .05). Overall, responding decreased over days for both females and males. For PR 871 training (Bottom), females responded more than males. Both females and males decreased responding 872 over the 7 days. 873 874 Figure 1 Sucrose preference females vs. males by body weight. Females consumed more sucrose vs. water than males, by body weight. *females vs. males, p < .05.

Figure 2
Saccharin preference females vs. males by body weight. There was no sex difference in preference for saccharin across a range of concentrations. Regardless of sex, rats preferred all concentrations of saccharin over the 0% concentration. #vs. 0% concentration, p < .05 (females and males combined). Binge data 12 vs. 24 h and 1 h intake by access condition females vs. males. Females consumed more sucrose than males across days of the study. Top: Overall intake (12 or 24 h) increased over days for both males and females. Bottom: 1h intake was greater by rats with a history of 12 vs. 24 h sucrose access, indicative of binge intake. 1 h intake increased over days for females only. Operant FR training and testing females vs. males. Top: Females responded more for sucrose than males and responding for both females and males increased over days of training. Bottom: Across the Low range of concentrations, females responded more than males and male and females responded more for 3.75 and 7.5% concentrations of sucrose compared to 0% (#vs. 0% concentration, p < .05). Across the High range of concentrations, males and females differed at 7.5% (*p < .05) and only females decreased responding at the 15 and 30% concentrations vs. 7.5% (#vs. 7.5%, p < .05).

Figure 5
Operant PR training and testing females vs. males. Females responded for sucrose more than males during training and testing. For training (Top), responding by males and females increased over days of training. For testing (Bottom), the only concentration-dependent effect was a decrease in responding at the 30 vs. 7.5% concentration only, for both females and males (#vs. 7.5%, p < .05).

Figure 6
Operant extinction and D1 antagonist females vs. males. Females responded more than males. SCH23390 reduced responding at the high dose (#vs. 0 dose, p < .05 females and males combined). Operant water females vs. males. For FR training (Top), females responded more than males on days 5 and 6 (*p < .05). Overall, responding decreased over days for both females and males. For PR training (Bottom), females responded more than males. Both females and males decreased responding over the 7 days.