Citric acid water as an alternative to food restriction to motivate task performance in mice during touchscreen testing

Citric acid water as an alternative to food restriction to motivate task performance in mice during touchscreen testing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Citric acid water as an alternative to food restriction to motivate task performance in mice during touchscreen testing Leila Dzinic, Olivia Ghosh-Swaby, Joel Antolin, Julie Dumont, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5575045/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 06 Apr, 2026 Read the published version in Lab Animal → Version 1 posted You are reading this latest preprint version Abstract Rodent behavioural testing paradigms in touchscreen operant chambers have successfully provided insight into the neural mechanisms underlying various cognitive domains in healthy and disease models. Touchscreen testing has previously required food restriction to sufficiently motivate rodents to complete behavioural tests, limiting the use of interventions, for example diet-based interventions, that alter animals’ motivation for food in experimental design. Here, we explored the safety and efficacy of water manipulation via addition of citric acid in motivating behavioural performance in touchscreen operant chambers 1) in comparison with food restriction and 2) when mice are fed an obesogenic high-fat, high-sugar (HFHS) diet. Water manipulation and food restriction produced similar performance on the progressive ratio task in non-obesogenic, standard-fed mice. However, when water-manipulated mice were fed a HFHS diet they showed deficits in this motivation-sensitive task. Critically, all groups, regardless of restriction type or diet, showed similar learning curves during a pairwise visual discrimination task. Together, these findings demonstrate that water manipulation can safely and effectively motivate mice to perform touchscreen tasks for reward, even when fed a highly satiating HFHS diet, which opens the possibility of using interventions, especially diet-based, in conjunction with touchscreen cognitive testing batteries. Biological sciences/Neuroscience/Cognitive neuroscience Biological sciences/Neuroscience/Learning and memory Biological sciences/Neuroscience/Motivation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Introduction Advancements in the understanding and treatment of human diseases are largely due to the use of pre-clinical experimental models. Pre-clinical models allow direct manipulation and observation of factors underlying putative disease states. 1,2 In neuroscience research, studies using rodent models have provided significant insight into the mechanisms underlying many neurological conditions; 2 however, many of these study methods involve tests of cognition that are not directly translatable to human populations. 3–6 Automated touchscreen operant chambers offer several advantages to traditional hand-testing tasks by minimizing experimenter interference, using standardized operating procedures, and enabling cross-species investigations through a visual-based modality. Tasks specifically developed for touchscreens allow for a greater level of control and translatability by administering virtually identical and visual-based paradigms to both rodent and human subjects. 4–7 Studies with human participants increasingly use computerized test batteries, including CogState, Mindstreams, and the Cambridge Neuropsychological Test Automated Battery (CANTAB), amongst others. 8 Rodent touchscreen operant chambers use similar principles, bridging the gap between rodent research and behavioural assessments in humans. 9 Rodent touchscreen paradigms, like many other behavioural paradigms, require the use of an appetitive reinforcer (reward), such as strawberry milkshake. 7,9,10 Food or liquid rewards avoid the use of aversive stimuli, removing stress or pain-induced conditioned responding in mice. 11 This approach motivates spontaneous animal behaviour and supports animal welfare. 11 Motivation to complete tasks is usually enhanced through food restriction – limiting food access by duration or quantity. 12–14 The need for food restriction is a significant limitation in studies that may alter an animal’s motivation for food or liquid rewards as part of an experimental manipulation, such as the cuprizone diet in studies of demyelination or high-fat, high-sugar (HFHS) diet in studies of diet-induced obesity. 15,16 Specialized diets often require ad libitum availability to produce the effects of the diet in a way that are translatable to humans. 17,18 An alternative protocol is thus required to maintain the desirability of an appetitive reinforcer so that rodents on diet manipulations can be motivated to perform touchscreen tests of cognition. Water restriction is one such alternative to food restriction. Water restriction has been applied in two different ways: by limiting the duration of access to water 19 or by limiting the amount of water that is accessible to the rodent. 20 Previous studies have demonstrated that water restriction protocols in animals not undergoing food restriction effectively increase motivation in touchscreen and operant paradigms that use liquid reinforcers. 12,21 Despite its efficacy, water restriction presents greater health risks than food restriction and requires rigorous monitoring to ensure the health of the rodents. 12 Water manipulation differs from water restriction in that it does not limit the availability or quantity of water. 22 Water manipulation occurs through the addition of a small amount of citric acid (CA) to ad libitum drinking water, which creates a solution with a mild sour taste 23 and in turn reduces the quantity of water that mice consume. 22,23 This reduction of water consumption is sufficient to motivate rodents to successfully complete behavioural conditioning paradigms when presented with water rewards with reduced risk of dehydration in comparison to other water restriction protocols. 22,23 Water manipulation using CA has previously demonstrated efficacy in motivating rodents to complete a test of learning; however, these studies did not include and explicit test of motivation. This study aimed to investigate 1) whether mice undergoing water manipulation perform similarly to food restricted mice on touchscreen tasks of motivation and discrimination learning, and 2) whether mice consuming an ad libitum HFHS diet are as motivated as mice consuming standard chow to complete touchscreen tasks while undergoing the water manipulation protocol. Mice were evaluated in terms of motivation (using touchscreen fixed ratio (FR) and progressive ratio (PR) tasks), learning (using a touchscreen pairwise visual discrimination (PVD) task), and health outcomes. Additionally, sex is considered in this study as male and female mice respond to changes in diet differently, including body weight and consumption. 24 Results Three experimental conditions were used (Fig. 1 ). All three conditions included male and female C57/Bl6J mice. The mice in Condition A were food restricted on standard chow to 85–90% of their free-feeding body weight with no water manipulation (i.e., unrestricted access to tap water). Mice in Condition B were fed ad libitum standard chow diet while on the water manipulation protocol (consuming CA water). Finally, mice in Condition C were fed ad libitum HFHS diet while on the water manipulation protocol (consuming CA water). A group with food restriction and ad libitum HFHS diet was of course not possible. Previously, Yang et al. 25 established the importance of food restriction in motivating mice to complete touchscreen tasks by demonstrating decreased response rates and interactions with touchscreens when mice were returned to ad libitum feeding following testing while on food restriction. As such, free-feeding mice that were not undergoing water manipulation were not used in the current study. Our analyses thus are based on two different comparisons of these Conditions: 1) the Standard Diet Comparison , comparing Conditions A and B, and 2) the Water Manipulation Comparison , which compare Conditions B and C. The aim of Comparison 1 is to establish whether water manipulation motivates mice as well as food restriction, whereas the aim of Comparison 2 is to establish whether water manipulation is sufficient to motivate touchscreen task performance in mice fed a highly appetitive HFHS diet. In consideration of the 3Rs (Replacement, Reduction, and Refinement), mice in Condition B were the same in both comparisons. Standard Diet Comparison: Does restriction type affect motivation, discrimination learning, or health outcomes? Mice undergoing water manipulation complete both the fixed-ratio and progressive-ratio task with similar levels of motivation to food-restricted mice Mice were habituated to the touchscreen chambers and strawberry milkshake reward over three days before undergoing a series of pretraining stages to interact with the touchscreens. To train and progress to PR, mice moved through FR reward schedules, each requiring a higher number of responses to earn rewards (see Methods and Materials below and full standard operating protocol on TouchscreenCognition.org). To address a sharp loss in weight in water-manipulated groups, prior to touchscreen testing, the CA concentration was reduced from 2–1.5% (Fig. 1 ). This change in CA concentration effectively stabilized body weight at 85% or greater, allowing a safe return to the 2% CA concentration without further significant weight decline. Consequently, all mice performed FR while on 1.5% CA water only. In the groups fed a standard diet, there were no effects of restriction type or sex on sessions to criterion within the FR task (ps > 0.275, Fig. 2 A) for advancement to PR. Similarly, the rates of responses (i.e. responses per minute) during the FR task were not affected by sex or restriction type (ps > 0.275; Fig. 2 B) but increased in all groups with the number of responses required to receive reward (main effect of reward schedule: F (2.33, 80.383) = 374.44, η 2 = 0.513, p < 0.001; Fig. 2 B). Therefore, mice in all groups progressed at a similar rate from FR to PR. After completing the FR task, mice performed the PR test of motivation, where the number of responses required for a reward increased progressively within a reward schedule by either four, eight, or 12 responses (i.e., PR4, PR8, PR12, respectively; see Methods and Materials). To address the change in CA water concentrations from 1.5–2% (Fig. 1 ), mice performed PR once while water-manipulated mice were on 1.5% CA water, and then again when they returned to 2% CA water. These two runs were analyzed separately. Breakpoint, an important measure of motivation, was defined as the maximum number of responses made to receive reward. While water-manipulated mice received 1.5% CA, there were no significant differences in breakpoint between restriction types or sexes (ps > 0.096; Fig. 2 C). As expected, there was a main effect of PR reward schedule (F (1.695, 61.023) = 17.630, η 2 = 0.059, p < 0.001, Fig. 2 C) where breakpoints progressively increased from PR4 to PR8 to PR12 as demands to respond were higher. To assess non-specific or impulsive behavior, the number of blank touches (i.e. responses to non-target windows) made during image presentation were recorded. The number of blank touches made significantly decreased from PR4 to PR8 to PR12 (p < 0.001; main effect of PR reward schedule: F (2, 72) = 8.316, η 2 = 0.036, p 0.061). Following testing on 1.5% CA, water-manipulated mice were returned to 2% CA, and all mice were tested again on PR. There were again no effects of sex or restriction type (ps > 0.051, Fig. 2 C) on breakpoint. Overall, mice had higher breakpoints at PR8 than PR4 and PR12, an effect driven by the males (PR8 > PR4, p PR12, p = 0.004; reward schedule by sex interaction: F (1.374, 49.464) = 4.091, η 2 = 0.011, p = 0.036; main effect of PR reward schedule: F (1.374, 49.464) = 9.082, η 2= 0.066, p = 0.002; Fig. 2 C). There was a main effect of PR reward schedule in the number of blank touches (F (1.713, 61.67) = 9.865, η 2 = 0.066, p 0.117; Fig. 2 D). Overall, there is no evidence that water-manipulated mice showed lower motivation to receive reward than food restricted mice in a task highly sensitive to changes in motivation. Both restriction types produce high yield responding during the touchscreen task, indicating the mice are motivated to seek reward regardless of restriction type. Standard chow diet fed mice on CA water mice perform similarly to food-restricted mice in a pairwise visual discrimination task After completing the PR task, mice performed the PVD task, in which they learned to discriminate between and respond to one of two different images presented simultaneously to receive a reward. Accuracy and the number of correction trials were recorded over 10 sessions of 30 trials each. There was a main effect of session (F (5.708, 205.49) = 46.643, η 2 = 0.425, p < 0.001; Fig. 3 A), indicating that groups learned progressively over the 10 sessions. There was also an interaction between session, sex, and restriction type (F (5.708, 205.49) = 2.417, η 2 = 0.022, p = 0.011; no statistically significant and relevant pairwise comparisons), but no main effects of sex or restriction type (ps > 0.160). When evaluating correction trials – which occurred following an incorrect response and were repeated until the animal made a correct response but did not count towards total trial count during the session – there were main effects of session (F (5.052, 181.882) = 40.579, η 2 = 0.411, p 0.420). This suggests that mice with CA manipulation are overall no different to food restricted mice in the number of correction trials required. Significant session by sex (F (5.052, 181.882) = 3.080, η 2 = 0.031, p = 0.001) and session by restriction type (F (5.052, 181.882) = 1.963, η 2 = 0.020, p = 0.043) interactions were observed, but without relevant and statistically significant post hoc comparisons. To further contextualize these findings, perseveration indices (PI) were calculated. The PI is a ratio of the number of correction trials to incorrect non-correction trials, representing the number of corrections required for the mouse to complete the trial correctly. Only a main effect of session (F (5.537,199.333) = 14.582, η 2 = 0.180, p 0.852; Fig. 3 C). Collectively, these data show that restriction type did not affect pairwise visual discrimination learning. Mice on CA water maintained near-baseline weight and scored positively on health scores of activity levels, hydration, and grooming Mice were monitored for health using a scoring rubric for categories of posture and grooming, activity levels, signs of dehydration, and eating and drinking (see Materials & Methods, Table 1 ). Each category has a set of behaviors or conditions assigned a score from 0 (normal) to 3 (severe), with higher scores indicating signs of distress or poor health. Low scores indicated no significant health changes within a single category (< 2 out 3) or across categories (< 5 out of 11). Overall health concerns were minimal with no mice on CA water scoring values of greater than 1 in any single health category or cumulatively. Reported scores of 1 in a single health category or cumulatively were predominantly for activity levels and signs of dehydration (i.e., skin turgor test, lack of urine and feces) in both males (n = 2) and females (n = 4) during the first four days of CA water administration (Table 1 ). Body weight was monitored throughout the experiment (Fig. 4 ) and analyzed following onset of restriction. Overall, water-manipulated mice weighed significantly more than food restricted mice (main effect of restriction type: F (1,36) = 16.565, η²=0.027, p < 0.001) and male mice were heavier than female mice (main effect of sex: F (1,36) = 516.522, η²=0.827, p < 0.001). There was a significant sex by restriction type interaction (F (1,36) = 5.236, η²= 0.008, p = 0.028) revealing that the effect of restriction type was significant in the male mice (Cohen’s d = 1.678, p < 0.001) but not in female mice (p = 0.216). Over the course of the experiment, mice on water manipulation gained weight following restriction whereas food restricted mice did not (week by restriction type interaction: F (5.571, 200.546) = 22.889, η²=0.018, p < 0.001) with this being predominantly driven by the male mice (sex by week interaction: F (5.571, 200.546) = 2.801, η²=0.002, p = 0.014). There was also a three-way sex by restriction type by week interaction (F (5.571, 200.546) = 3.146, η²=0.002, p = 0.007; Fig. 4 ). A main effect of week was observed (F (5.571, 200.546) = 36.327, η²=0.029, p = 0.001). These results indicate that although CA water-manipulated male mice weighed more compared with the food restricted mice. Water Manipulation Comparison: Does diet affect motivation, discrimination learning, or health outcomes? Mice on HFHS diet are less motivated to perform FR and PR tasks for reward than mice on standard chow diet In the water manipulation groups, mice on HFHS diet required more sessions to reach advancement criteria on the FR task than mice on standard chow diet (F (1, 51) = 7.412, η 2 = 0.119, p = 0.009; Fig. 5 A). Sessions to reach criterion were not significantly affected by sex (p = 0.31) and there was no sex by diet interaction (p = 0.105; Fig. 5 A). When comparing rate of responses across FR reward schedules and diet groups, as the number of responses required to obtain reward increased so too did the rate of response (F (2.107, 107.482) = 123.241, η 2 = 0.448, p < 0.001; Fig. 5 B). However, HFHS mice had overall lower response rates in FR compared to standard fed mice (F (1, 51) = 7.648, η²=0.040, p = 0.008; Fig. 5 B) particularly during FR5 (Cohen’s d = 1.125, p < 0.001; reward schedule by diet interaction: F (2.107,107.482) = 5.187, η²=0.019, p = 0.006; Fig. 5 D). Males on HFHS diet responded at a lower rate than male mice on standard chow diet (Cohen’s d = 1.074, p = 0.004; sex by diet interaction (F (1, 51) = 5.603, η²=0.030, p = 0.022). Notably, female mice did not vary by diet (p = 0.778), suggesting that the effects of diet observed are driven by the male mice. The main effect of sex was not significant (p = 0.988). Overall, mice on a HFHS diet took longer to progress through the FR task and responded at lower rates than standard diet fed mice, particularly in male mice and when greater responses for reward were required. This demonstrates that diet significantly affects training speed and response rates across the FR task. Following the FR task, mice were run on the PR task. While on 1.5% CA, chow-fed mice had higher breakpoints than HFHS mice (main effect of diet: F (1,51) = 22.204, η 2 = 0.245, p < 0.001; Fig. 5 C), with no effect of sex (p = 0.914). Mice of both diets had lower breakpoints at PR4 than at PR8 or PR12 (ps < 0.016; main effect of reward schedule: F (1.660, 84.652) = 9.259, η 2 = 0.025, p < 0.001; Fig. 5 C). Number of blank touches differed by diet (F (1,51) = 11.162, η 2 = 0.141, p = 0.002, Fig. 5 D), with no effect of sex. A main effect of reward contingency was observed (F (2,102) = 11.942, η 2 = 0.033, p < 0.001; Fig. 5 D), where blank touches decreased from PR4 to PR8 to PR12. After returning to 2% CA water, and like performance on 1.5%, a main effect of diet on breakpoint was present (F (1, 51) = 5.108, η 2 = 0.071, p = 0.028, Fig. 5 C) with standard chow mice responding more for reward than HFHS diet mice. Once again, there was also a main effect of PR reward schedule (F (2, 102) = 4.603, η 2 = 0.010, p = 0.012) but no main effect of sex (p = 0.160). A diet by sex interaction was present (F (1, 51) = 4.780, η 2 = 0.067, p = 0.033), with a significant drop in performance in HFHS-fed male mice compared to standard chow-fed male mice (Cohen’s d = 1.129, p = 0.016), but no differences observed in female mice (ps > 0.063). Blank touches differed across PR reward schedule (F (2, 102) = 13.712, η 2 = 0.063, p 0.390). Finally, there was a sex by diet interaction for blank touches (F (1,51) = 8.550, η 2 = 0.098, p = 0.005) with a trend for fewer blank touches in male mice on HFHS diet than male mice on standard chow diet; however, this (p = 0.056) and other pairwise comparisons did not reach statistical significance (all others, ps > 0.209). Collectively, these results demonstrate that water-manipulated mice fed a HFHS diet were significantly less motivated to obtain reward than chow-fed mice in touchscreen PR. Male mice on HFHS diet were impaired compared to their chow-fed counterparts. Water-manipulated mice consuming HFHS diet perform similarly to standard chow mice in the pairwise visual discrimination Following PR, water-manipulated mice performed the PVD task. All mice improved in performance across the 10 sessions (F (6.039, 301.929) = 77.071, η 2 = 0.484, p < 0.001; Fig. 6 A). Male mice made fewer correct responses on average compared with female mice (main effect of sex: F (1, 50) = 4.210, η 2 = 0.013, p = 0.045; Fig. 6 ), but no significant difference was found between diet groups and no interaction (ps > 0.235). Overall, mice performed fewer correction trials across sessions (F (5.187, 259.375) = 83.072, η 2 = 0.534 p < 0.001; Fig. 6 B). HFHS diet mice made fewer correction trials than standard fed mice (main effect of diet: F (1, 50) = 4.443, η 2 = 0.003 p = 0.040), but there was no effect of or interaction with sex or sessions (ps > 0.067). Lastly, perseveration index (PI) decreased across sessions (F (4.541, 227.033) = 16.376, η 2 = 0.199 p 0.192). HFHS diet induces significant weight gain in water-manipulated mice with few other health concerns Mice were monitored for health using a scoring rubric for categories of posture and grooming, activity levels, signs of dehydration, and eating and drinking (see Materials & Methods, Table 1 ). Low scores indicated no significant health changes within a single category (< 2 out 3) or across categories (< 5 out of 11). All standard-chow, water-manipulated mice reported scores of one or less in activity levels or sign of dehydration, indicating no major health concerns. Only four (2 male, 2 female) HFHS diet mice were reported to have cumulative scores of either 2 or 3 within the activity level or posture categories that prompted monitoring by veterinary care. These scores later decreased as mice acclimatized to the combination of CA water manipulation and HFHS diet. Body weight was monitored throughout the experiment (Fig. 7 ). Overall, HFHS diet fed mice weighed significantly more than standard diet fed mice (main effect of diet: F (1,51) = 42.98, η²=0.149, p < 0.001) and male mice were heavier than female mice (main effect of sex: F (1,51) = 162.695, η²=0.565, p < 0.001). There was a significant sex by restriction type interaction (F (1,51) = 7.755, η²= 0.027, p = 0.008) revealing that the effect of diet was greater in male mice compared with female mice but significant in both (males: Cohen’s d = 2.318, p < 0.001; females: Cohen’s d = 0.936, p = 0.011). As weeks elapsed, mice on HFHS diet gained more weight compared with mice on standard diet (week by diet interaction: F (3.242, 165.325) = 7.083, η²=0.005, p < 0.001) once started on CA water manipulation. Male mice gained more weight than female mice (week by sex interaction: F (3.242, 165.325) = 3.121, η²=0.002, p = 0.024; no significant three-way, sex by diet by week, interaction: p = 0.132; Fig. 7 ). Discussion Specialized diets, such as demyelinating and HFHS diets, are used as experimental manipulations in many studies but their use in food-motivated tasks is problematic due to the need in such tasks for adequate motivation for food reward. The present findings show that water manipulation using CA water supports behavioural performance on strawberry milkshake-rewarded touchscreen-based tasks in both standard-fed and HFHS-fed mice. Notably, differences in touchscreen performance were observed between diet groups in the PR task, which is highly sensitive to changes in motivation, but this decrease in motivation was not sufficient to affect performance on the PVD task used to assess visual discrimination learning. Thus, CA water manipulation allows testing of mice on diets such as HFHS on appetitively motivated touchscreen tests of cognition. Comparison 1: Water manipulation is sufficient to motivate standard chow-fed mice on touchscreen tasks Previous work by Urai et al. 22,23 and Reinagel et al. 23 confirmed the efficacy of CA water manipulation as a motivator in an apparatus that paired a computer display with either a steering wheel or licking ports for responses, respectively. In line with those publications, the present study also found CA water manipulation to be sufficient to elicit behavioural performance on touchscreen tests of motivation and visual discrimination. Under standard diet conditions, both CA water manipulation and food restriction were equally effective in motivating standard chow-fed mice to respond for strawberry milkshake reward without changes in latency to respond (Supplementary Figure 1) on a touchscreen-based motivation task. This motivated performance further extended to learning in a pairwise visual discrimination task on which there were no differences between water-manipulated and food-restricted mice. Thus, CA water manipulation is a sufficient motivator of behaviour in touchscreen chambers in standard fed mice, extending the utility of both water manipulation as a motivator and touchscreens tasks in animal research. Comparison 2: HFHS diet reduces motivation in the PR task but does not restrict performance in pairwise visual discrimination learning motivated by CA water manipulation. As the need for food restriction was previously recommended for touchscreen testing, rendering the use of specialized diets in this testing modality problematic, we sought to determine whether CA water manipulation – which enables animals to both drink and eat ad libitum – is sufficient to drive responding in touchscreens when mice were fed a HFHS diet, perhaps the most satiating of all specialized diets. It is documented that a HFHS diet typically results in impaired motivation. 26,27 Particularly, it is both the composition of the food such as increased sugar and fats, and caloric intake that affects motivation. 28,29 HFHS diet often leads to metabolic dysfunction, dysregulated dopamine signaling, 18,30 reduced reward sensitivity, 21,29 and lowered activity levels, 31 all of which would be expected to significantly demotivate animals and/or reduce task engagement. Even with these well-known demotivating effects and reductions in task engagement, we observed that CA water manipulation was sufficient to motivate mice on an HFHS diet to complete the PR task, a task highly sensitive to changes in motivation. While HFHS fed mice on water manipulation took longer to train on the task and showed decreased motivation compared to ad libitum standard chow-fed mice (also motivated with CA water manipulation), they could perform the task. Consequently, we were able to robustly quantify differences in motivation for an appetitive reward in mice on HFHS diet versus standard chow diet. This is the first study to investigate the effects of HFHS diet on discrimination learning as measured by the PVD task, which may be due to limitations in restriction protocols to motivate behaviour. Rather, most studies observe HFHS diet-induced impairments in tests of spatial memory, working memory, and object recognition that depend on spontaneous, non-rewarded testing paradigms in rodents 34 and humans. 28,29,35 While HFHS diet led to lower breakpoints on a motivation-sensitive PR task, this did not translate into learning impairments on the PVD task. This has similarly been seen in rats that were food restricted on standard chow diet and provided 70ml/day of high sucrose water on the same PVD task. 36 The PVD task, like the FR1 task, requires only a single response to the correct stimulus to obtain a reward, leading to consistent reinforcement with each correct response. This simple response requirement likely protected performance from motivational deficits observed in tasks with higher effort demands such as PR, where multiple responses revealed motivational impairments. Thus, it is likely that CA water manipulation will similarly elicit performance in touchscreen tasks that assess other cognitive domains, such as attention, even in mice on specialized diets. In this study, we were able to conclude that HFHS diet does not impact the rate of learning on a PVD task compared to standard diet. Sex-specific differences between restriction types and diets Beyond the expected and normal differences in body weight between male and female mice, there were notable sex differences observed in this study, especially following HFHS diet consumption. In Comparison 1 (Standard Diet Comparison), few sex differences were observed in PR and PVD task performance. For example, during PR, male mice exhibited higher breakpoints on PR8 compared to PR4 and P12, irrespective of restriction type. In Comparison 2 (Water Manipulation Comparison), males on a HFHS diet exhibited lower response rates during the FR task, showed reduced breakpoints in the PR task, and tended to make fewer non-specific responses when compared with males on a standard chow diet. Interestingly, water-manipulated female mice made more correct responses in PVD than water-manipulated male mice, irrespective of diet. Females, conversely, showed no differences in weight between restriction types, and a more moderate increase in weight on HFHS diet when compared to the substantial weight gains seen in HFHS-fed males. Further investigation into how body weight, adiposity, and circulating nutrients resulting from different diets (including drinking water) affect motivation and learning in males and females is needed. CA water manipulation is practical to implement and promotes positive health outcomes in mice The use of water manipulation resulted in no significant health changes and allowed for mice to maintain or gain weight throughout the study while maintaining task performance. Mice given CA water stabilized or regained their weight, and presented with few signs of dehydration, lowered activity levels, and abnormal posture or grooming. Water manipulation allowed for changes in weight during development and diet consumption in contrast with the food restriction group which, by design, is maintained at 85-90% of baseline weight throughout testing. Therefore, CA water manipulation may be a useful alternative in studies using adolescent mice to allow for normal growth and development. Additionally, water manipulation requires minimal experimenter labour when compared to food restriction or other types of water restriction. CA water preparations were completed at the start of each week and animals only required daily monitoring and weighing. Unlike food restriction, there are also no concerns regarding time of feeding (especially relative to behavioural experiments), missed feedings, or competition over food in grouped housed animals. CA may also offer health benefits in animals. Urai et al. highlight that CA increases the acidity of water, which may benefit gut health in animals. 22 Previous literature has also suggested that CA is an antioxidant and limits excessive generation of reactive oxygen species and free radicals, which benefits liver health. 37 Considerations for future use Water manipulation affected neither motivation nor learning in standard fed mice. In contrast, HFHS diet reduced motivation in water-manipulated mice compared with standard fed mice on water manipulation. It is therefore critical for researchers to consider their control groups and confounds that may exist with future use of water manipulation. If diet is the dependent variable in an experiment, it will be important to consider how diet may affect motivation in testing and how that can impact the dependent variables collected. Even in experiments where diet is consistent across experimental groups, caution should be used to ensure accurate interpretation of the data, including how the results may generalize to standard and other non-standard diets. Conclusion In conclusion, water manipulation using CA water is an effective alternative to food restriction in motivating mice across both standard and HFHS diet conditions. While motivation was reduced in mice on CA water when consuming a HFHS diet, mice were still able to perform the tasks. Importantly, these motivational changes did not significantly impact learning performance, highlighting the potential utility of water manipulation using CA in future studies where food restriction may not be feasible or desirable. Materials and Methods All experiments were conducted in compliance with the standards set by the Canadian Council of Animal Care and under direct veterinary supervision at the Western University. Experimental design Seventy-two C57BI/6J mice (36 males and 36 females, Jackson Laboratory, US) arrived at 8–12 weeks of age. Mice were group-housed by sex with 4 mice per cage in a temperature (23 ± 1°C) and humidity (50 ± 1%) controlled room under a reverse 12h light/dark cycle (lights off at 9:00). The complete study design is illustrated in Fig. 1 . Mice were acclimated to their cages for 5 days with ad libitum access to standard Teklad diet (Envigo) and untreated water. Mice were randomly assigned to standard pre-weighed diet (Bioserv F0078, 3.6 kcal/g, 5.6% fat, 59.1% carbohydrates, 18% protein; n = 20 per sex) or HFHS chow (Bioserv F6724, 4.57 kcal/g, 21.2% fat, 48.5% carbohydrates, 17.3% protein; n = 16 per sex). Mice were maintained on their respective diets for three weeks after which baseline weights were calculated as mean bodyweight over three days. Following baseline calculations, food restriction and water manipulation protocols were implemented. Sixteen mice (n = 8/sex) in the standard diet group were randomly assigned to undergo food restriction to 85–90% of their baseline weight. Twenty-four mice (n = 12/sex) received ad libitum standard diet, and thirty-two mice (n = 16/sex) received ad libitum HFHS diet (see Fig. 1 ). All mice with ad libitum access to diet also received 2% citric acid water manipulation ad libitum . Two grams of CA (citric acid anhydrous, Thermofisher Scientific, USA) were dissolved in 100mL of tap water to produce 2% CA water. Mice were weighed 3-6x per week in accordance with the animal use protocol. Mice undergoing food restriction had a healthy weight defined as 85–90% of their baseline weight whereas mice consuming 2% CA water had no upper boundary of permissible body weight. 2% CA water-manipulated mice had their weight recorded between 10:00AM and 12:00PM each day and were administered CA water with a reduced concentration of CA if they experienced abrupt weight loss to an amount less than 85% of their baseline weight. Food restricted mice found to be underweight were provided additional diet (minimum of 0.5g increase); water-manipulated mice found to be underweight were provided one hour of access to 0.5% CA water to encourage increased water consumption. Health scores were provided for each mouse based on their posture and grooming, dehydration levels, activity, and eating or drinking using a health scoring rubric (Table 1 ). Table 1 Health scoring rubric used to assess activity levels and hydration of mice on citric acid water. Mice undergoing water manipulation treatment were monitored using this table 6 times per week. Activity Score Moves around the cage 0 Moves slowly around the cage 1 Moves only when touched 2 Does not move 3 Posture and Grooming Normal posture and smooth fur 0 Hunched posture or ruffled fur 1 Hunched posture and slightly ruffled fur 2 Hunched posture and all fur ruffled 3 Signs of Eating and Drinking Feces and urine observed 0 Minimal fecal and/or urine 1 No signs or feces and/or urine 2 Signs of Dehydration Skin does not tent when scuffed 0 Skin tents briefly but returns to normal 1 Skin tents and takes more than 2 seconds 2 Skin tents and stays tented 3 Total Scores Any animal that has a score: • ≤ 4 cumulatively or ≤ 1in any one category should be monitored but no action required • ≥ 2 in any one category or cumulatively to ≥ 5 required veterinary support to monitor the animal Touchscreen Behavioural Testing All touchscreen behavioural testing was completed using standard Bussey-Saksida mouse touchscreen chambers (model 80614, Lafayette Instrument Company, Lafayette IN) as described in detail elsewhere 7 and using task specific standard operating procedures published to TouchscreenCognition.org. Briefly, the apparatus contains a trapezoidal chamber with a touchscreen at one end and a reward magazine on the other end. A space in front of the touchscreen allows the insertion of removable masks (black plastic sheets with apertures that allow access to the touchscreen) specialized to each cognitive test. The reward magazine illuminates and dispenses liquid milkshake reward (Nielson’s Strawberry Milkshake) upon successful trial completion following interaction with the touchscreen. Therefore, the number of trials completed is equivalent to the number of rewards obtained. Mice were habituated to the chambers and milkshake rewards over three days and were given several pretraining stages in order that the mice learned to interact with the touchscreens, and successfully retrieve the reward. 9,10,38 Touchscreen Tests of Motivation: Fixed and Progressive Ratio Tasks Training using the Fixed-Ratio (FR) Touchscreen Task Following habituation and pretraining, mice were trained for PR using the FR task. The FR task was comprised of several reward schedules with each reward schedule requiring a different number of nose pokes to the stimulus (white square) to obtain a reward. For example, mice were trained to collect a reward (~ 24ul dispensed via 800ms pulse of a peristaltic pump) by completing one touchscreen response to the stimulus in the FR1 reward schedule. 9 Following each reward collection, there was a 20s intertrial interval (ITI) before the next trial can be initiated and a stimulus (white square) appears in the centre of a 5-window mask. After achieving 30 rewards in 60 minutes, mice were advanced to the subsequent reward schedule. 9 The following reward schedules FR2, FR3, and FR5 required 2, 3 or 5 responses to the stimulus, respectively. 38 Therefore, FR5 required the greatest effort, requiring five responses to the stimulus for a single reward. The number of responses, time of session completion, and rewards collected were recorded. To examine the rate of responding, the number of responses per minute were calculated. Additionally, the number of sessions to complete all of FR was totaled. Following successful completion of FR5, the PR task began. One male HFHS diet mouse failed to reach criterion and so did not progress to PR from FR. PR task parameters were identical to those in the FR task with the exception that the number of responses to the touchscreen required to obtain a reward differed: The number of touches required increased incrementally within a session, such that the responses required for reward in PR4 increases by four for each subsequent reward (i.e., Trial 1 required one response, Trial 2: five responses, Trial 3: nine responses, etc.). After completing PR4, mice completed PR8and PR12, where the number of required responses for a reward increased by 8 or 12 on each trial, respectively. Mice were only given one PR session (either PR4, PR8, or PR12) per day. To examine motivation, the breakpoint for each mouse was calculated; breakpoint is the number of responses made in the final successfully completed trial. For example, in PR4, if a mouse successfully completed the first four trials with reward requirements of 1, 5, 9, and 13 responses, respectively, but failed to complete the next (17 responses for reward), it thus reached a breakpoint of 13. Additionally, reward latency and number of non-stimulus touches (blank touches) were recorded. Pairwise Visual Discrimination (PVD) Task Following FR/PR, mice performed the pairwise visual discrimination (PVD) task. 7 Mice were first pretrained for PVD on punish incorrect, where correct touches were rewarded, and blank touches triggered a 5-second timeout with the house light illuminated. Mice needed to complete ≥ 24/30 trials correct (i.e., minimum 24 rewards) within 60 min for 2 consecutive sessions before moving on to PVD. One water-manipulated female HFHS diet mouse did not progress to PVD due to health complications not related to diet or CA water. The PVD task required mice to discriminate between 2 images (diagonal lines 45° clockwise or anticlockwise from vertical) and respond to the correct (rewarded) stimulus image (S+) and withhold responding to the incorrect (unrewarded) stimulus image (S-) when they were both displayed simultaneously on either side of the touchscreen (Horner et al., 2013). The relative left and right presentation of the stimuli varied pseudo-randomly across each trial and stimuli were not displayed in the same location for more than three consecutive trials. Next, the mouse could initiate the following trial. In contrast, an incorrect response to the S- resulted in a 5s illumination of the chamber by the house light. Following the 5s penalty, a 20s ITI is initiated. Following the ITI, a correction trial began in which the stimuli were presented in the same configuration as the previous trial and this trial configuration was repeated until the mouse selected the S + and collected a reward. Mice completed a total of 10 sessions of 30 successful trials each. The percent correct responses out of 30 trials (i.e., accuracy) and the number of correction trials were recorded for each session. When a mouse did not reach 30 trials in a single session, it was tested the next day on the trials remaining to reach 30 trials; therefore, each session analyzed consisted of a total of 30 trials. Due to running errors, one mouse (male, HFHS diet, water-manipulated) completed 33 trials during session 1 and another (male, HFHS diet, water-manipulated) completed only 20 trials during session 3. Formulae for perseveration indices were adapted in these cases. In cases where a mouse completed only nine sessions, data from the ninth session was used for both Sessions 9 and 10 (1 female, standard diet, water-manipulated; 4 male, HFHS diet, water-manipulated). Data Analysis The dependent variables were examined using two separate analyses to address the main questions of this study: 1) Comparison of the restriction protocol (food restriction, CA water manipulation) and sex (male, female) within the standard diet groups (i.e., mice given standard chow), and 2) a comparison of diet (standard, HFHS) and sex (male, female) within water-manipulated groups (i.e., mice given ad libitum CA water). The comparison between standard diet with food restriction and HFHS with water manipulation mice was considered irrelevant to the research aims, and therefore were not directly compared statistically, which reduced potential type 2 errors. Moreover, using the same standard-diet mice consuming CA water in both analyses aligns with the principles of the 'Three Rs' of animal research, reducing animal use while maximizing data collection from the same cohort. This design addresses 1) whether CA water and food restriction could sufficiently motivate mice consuming standard chow to a similar degree during touchscreen testing, and 2) whether CA water can motivate mice consuming HFHS diet to a similar degree as standard chow diet mice. Data were analyzed using JASP (Version 0.16.3 Intel). Three-way mixed model analyses of variance (ANOVA) were used, with restriction protocol (food restriction, CA water manipulation) or diet (standard chow, HFHS) and sex (male, female) as between-subject factors and day, week, or session as the within-subject repeated factors. In instances where sphericity assumptions were violated (Mauchly’s test), Greenhouse-Geisser corrections were applied. When interaction effect was detected, Tukey or Holm post-hoc analyses were performed where appropriate. Data are presented as mean ± standard error of the mean (SEM). Significance was set at α < 0.05. Declarations All experiments were conducted in compliance with the standards set by the Canadian Council of Animal Care and under approval and direct veterinary supervision by the animal care committee at the Western University. Acknowledgements This research was supported by through the Canada First Research Excellence Fund (CFREF), Brain Canada, the Canadian Institutes of Health Research (CIHR PJT 426966; Vanier Canada Graduate Scholarship to ORG-S), the Natural Sciences and Engineering Research Council (NSERC RGPIN-2019-06102, RGPIN-2019-06087), the Canada Foundation for Innovation, and the Ontario Research Fund. LMS is the Canada Research Chair in Translational Cognitive Neuroscience and TJB is the Western Research Chair in Behavioural Neuroscience.The authors also thank Claire Lipton for her invaluable assistance with animal handling and monitoring. Competing Interest Statement TJB and LMS have established a series of targeted cognitive tests for animals, administered via touchscreen within a custom environment known as the “Bussey-Saksida touchscreen chamber”. Cambridge Enterprise, the technology transfer office of the University of Cambridge, supported commercialization of the Bussey-Saksida chamber, culminating in a license to Campden Instruments. Any financial compensation received from commercialization of the technology is fully invested in further touchscreen development and/or maintenance. Campden Instruments play no role in the conceptualization, design, data collection, analysis, decision to publish, or preparation of the manuscript. References Moran, P. et al. Gene × Environment Interactions in Schizophrenia: Evidence from Genetic Mouse Models. Neural Plast 2016 , 1–23 (2016). Jucker, M. & Ingram, D. K. Murine models of brain aging and age-related neurodegenerative diseases. Behavioural Brain Research 85 , 1–25 (1997). 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Obesity (Silver Spring) 18 , 463–469 (2010). Yang, J.-H. et al. Pharmacological studies of effort-related decision making using mouse touchscreen procedures: effects of dopamine antagonism do not resemble reinforcer devaluation by removal of food restriction. Psychopharmacology (Berl) 237 , 33–43 (2020). Arcego, D. M. et al. Chronic high-fat diet affects food-motivated behavior and hedonic systems in the nucleus accumbens of male rats. Appetite 153 , 104739 (2020). Rossi, H. L. et al. Effects of diet-induced obesity on motivation and pain behavior in an operant assay. Neuroscience 235 , 87–95 (2013). Attuquayefio, T., Stevenson, R. J., Oaten, M. J. & Francis, H. M. A four-day Western-style dietary intervention causes reductions in hippocampal-dependent learning and memory and interoceptive sensitivity. PLoS One 12 , e0172645 (2017). Attuquayefio, T. et al. A high-fat high-sugar diet predicts poorer hippocampal-related memory and a reduced ability to suppress wanting under satiety. J Exp Psychol Anim Learn Cogn 42 , 415–428 (2016). Blaisdell, A. P. et al. Food quality and motivation: A refined low-fat diet induces obesity and impairs performance on a progressive ratio schedule of instrumental lever pressing in rats. Physiol Behav 128 , 220–225 (2014). Vellers, H. L., Letsinger, A. C., Walker, N. R., Granados, J. Z. & Lightfoot, J. T. High Fat High Sugar Diet Reduces Voluntary Wheel Running in Mice Independent of Sex Hormone Involvement. Front Physiol 8 , (2017). Gladding, J. M., Bradfield, L. A. & Kendig, M. D. Diet and obesity effects on cue-driven food-seeking: insights from studies of Pavlovian-instrumental transfer in rodents and humans. Front Behav Neurosci 17 , (2023). Ducrocq, F. et al. Decrease in Operant Responding Under Obesogenic Diet Exposure is not Related to Deficits in Incentive or Hedonic Processes. Obesity 27 , 255–263 (2019). Abbott, K. N., Arnott, C. K., Westbrook, R. F. & Tran, D. M. D. The effect of high fat, high sugar, and combined high fat-high sugar diets on spatial learning and memory in rodents: A meta-analysis. Neurosci Biobehav Rev 107 , 399–421 (2019). Francis, H. M. & Stevenson, R. J. Higher Reported Saturated Fat and Refined Sugar Intake Is Associated With Reduced Hippocampal-Dependent Memory and Sensitivity to Interoceptive Signals. Behavioral Neuroscience 125 , 943–955 (2011). Miles, B. et al. High sucrose diet does not impact spatial cognition in rats using advanced touchscreen technology. Behavioural Brain Research 418 , 113665 (2022). Abdel-Salam, O. M. E., Shaffie, N. M., Omara, E. A. & Yassen, N. N. Citric Acid an Antioxidant in Liver. in The Liver 183–198 (Elsevier, 2018). doi:10.1016/B978-0-12-803951-9.00016-1. Heath, C. J. et al. A Touchscreen Motivation Assessment Evaluated in Huntington’s Disease Patients and R6/1 Model Mice. Front Neurol 10 , (2019). Additional Declarations Yes there is potential Competing Interest. (Non-financial interest) Tim Bussey and Lisa Saksida have established a series of targeted cognitive tests for animals, administered via touchscreen within a custom environment known as the “Bussey-Saksida touchscreen chamber”. Cambridge Enterprise, the technology transfer office of the University of Cambridge, supported commercialization of the Bussey-Saksida chamber, culminating in a license to Campden Instruments. Any financial compensation received from commercialization of the technology is fully invested in further touchscreen development and/or maintenance. Campden Instruments play no role in the conceptualization, design, data collection, analysis, decision to publish, or preparation of the manuscript. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5575045","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":387235432,"identity":"e518f44d-e70a-4791-be79-fc34078ab3a3","order_by":0,"name":"Leila Dzinic","email":"","orcid":"","institution":"Graduate Program in Neuroscience, Western University, London, ON, Canada","correspondingAuthor":false,"prefix":"","firstName":"Leila","middleName":"","lastName":"Dzinic","suffix":""},{"id":387235433,"identity":"00e6431a-e698-412b-b67b-f00d523f4a3c","order_by":1,"name":"Olivia Ghosh-Swaby","email":"","orcid":"https://orcid.org/0000-0002-4603-8560","institution":"Graduate Program in Neuroscience, Western University, London, ON, Canada","correspondingAuthor":false,"prefix":"","firstName":"Olivia","middleName":"","lastName":"Ghosh-Swaby","suffix":""},{"id":387235434,"identity":"ccab5cbb-ecf1-48ad-9e0a-707f6399ad1f","order_by":2,"name":"Joel Antolin","email":"","orcid":"","institution":"Undergraduate Program in Physiology and Pharmacology, Western University, London, ON, Canada","correspondingAuthor":false,"prefix":"","firstName":"Joel","middleName":"","lastName":"Antolin","suffix":""},{"id":387235435,"identity":"6e7367a2-d0dd-4bbd-beca-abca28ffa26e","order_by":3,"name":"Julie Dumont","email":"","orcid":"","institution":"BrainsCAN, Western University, London, ON, Canada","correspondingAuthor":false,"prefix":"","firstName":"Julie","middleName":"","lastName":"Dumont","suffix":""},{"id":387235431,"identity":"252a1199-a792-4b91-9532-9e23b9e333ca","order_by":4,"name":"Paul Sheppard","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuElEQVRIiWNgGAWjYFACHjBZz0+ylgTJBpK1GBwgVoM5/9mDjwt32OUZ38gxfMBQY0dYi+WMvGTjmWeSi81u5BgbMBxLJqzF4AaPmTRvGzPjthu52yQYG5iJ0HL+jPlv3rZ6xs0zwFrqidByIMeMmbftcOIGCbCWw8T4JcdYeuaZ48YSZ95/Nkg4dpywFnP+M4afC3dUy/G3pyU++FBTTYTDgJiZsQHKSyCsAV3LKBgFo2AUjAJsAADtzDgDu3hAjwAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0001-7432-1446","institution":"Robarts Research Institute, Schulich School of Medicine \u0026 Dentistry, Western University, London, ON, Canada","correspondingAuthor":true,"prefix":"","firstName":"Paul","middleName":"","lastName":"Sheppard","suffix":""},{"id":387235436,"identity":"8c14f4f3-d46c-4976-b7f0-6e2f1cbff45c","order_by":5,"name":"Timothy Bussey","email":"","orcid":"https://orcid.org/0000-0002-3180-3709","institution":"BrainsCAN, Robarts Research Institute, Department of Physiology and Pharmacology, Schulich School of Medicine \u0026 Dentistry, Western University, London, ON, Canada","correspondingAuthor":false,"prefix":"","firstName":"Timothy","middleName":"","lastName":"Bussey","suffix":""},{"id":387235437,"identity":"4391492c-396c-4cca-bb4b-44d61a9898d7","order_by":6,"name":"Lisa Saksida","email":"","orcid":"https://orcid.org/0000-0002-8416-8171","institution":"BrainsCAN, Robarts Research Institute, Department of Physiology and Pharmacology, Schulich School of Medicine \u0026 Dentistry, Western University, London, ON, Canada","correspondingAuthor":false,"prefix":"","firstName":"Lisa","middleName":"","lastName":"Saksida","suffix":""}],"badges":[],"createdAt":"2024-12-03 21:35:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5575045/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5575045/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1038/s41684-026-01711-y","type":"published","date":"2026-04-06T04:00:00+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":71850229,"identity":"fc26626c-2083-4800-9305-313cdf358bf4","added_by":"auto","created_at":"2024-12-19 07:20:34","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":842692,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eStudy timeline, conditions, and groups\u003c/strong\u003e. Mice were fed either a standard laboratory chow or HFHS diet and then placed on either food restriction or water manipulation with CA water following week 3. This produced 3 experimental conditions. All mice were then habituated to and pretrained on touchscreen testing. Following this, mice performed the FR and PR tasks of motivation and the PVD learning task. Weight and health were monitored throughout. Created with Biorender.com. HFHS = high-fat, high-sugar diet\u003c/p\u003e","description":"","filename":"Picture1.png","url":"https://assets-eu.researchsquare.com/files/rs-5575045/v1/cdd161434e76679936476050.png"},{"id":71848608,"identity":"85807598-aa9e-4c0e-bfe8-5632825f7e5b","added_by":"auto","created_at":"2024-12-19 07:04:34","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1589277,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eFood restriction and water manipulation produce similar levels of motivation in mice. \u003c/strong\u003e\u003cbr\u003e\n Mice were trained on FR prior to advancing to PR testing. A) The groups did not differ significantly in the sessions required to reach criterion for advancement to PR. B) As expected, mice made more responses as the task demands required additional responses to earn milkshake reward. C) Restriction type did not affect breakpoint. D) Restriction type did not affect responses made to non-active touchscreen windows. Food restricted (n=8 per sex), Water-manipulated (n=12 per sex). Data presented as mean±SEM, \u003cem\u003e***p\u0026lt;0.001. \u003c/em\u003eCA = citric acid,\u003cem\u003e \u003c/em\u003eFR = fixed ratio;\u003cem\u003e \u003c/em\u003ePR = progressive ratio.\u003c/p\u003e","description":"","filename":"Picture2.png","url":"https://assets-eu.researchsquare.com/files/rs-5575045/v1/0e7ec4217d19736c726ba5c2.png"},{"id":71848610,"identity":"76611186-d20a-4ef8-a637-542f6c57b664","added_by":"auto","created_at":"2024-12-19 07:04:34","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":838584,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eRestriction type does not affect learning in the PVD task.\u003c/strong\u003e \u003cbr\u003e\n Restriction type did not affect A) percent correct responding, B) number of correction trials, or C) perseverative responding across the 10 sessions of PVD. Food restricted (n=8 per sex), Water-manipulated (n=12 per sex). Data presented as mean ± SEM, *\u003cem\u003ep\u0026lt;0.05. \u003c/em\u003ePVD = pairwise visual discrimination.\u003c/p\u003e","description":"","filename":"Picture3.png","url":"https://assets-eu.researchsquare.com/files/rs-5575045/v1/51792b481b784cebc90b85ea.png"},{"id":71849618,"identity":"ba57b073-b8b0-4b54-b1cd-9c7fe345c475","added_by":"auto","created_at":"2024-12-19 07:12:34","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":989402,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAverage weekly body weight of mice fed a standard diet. \u003c/strong\u003eMice were fed standard chow \u003cem\u003ead libitum\u003c/em\u003e from week 0 (baseline) to week three. Mice were weighed three times per week. Following week 3, mice started receiving either 2% CA water (n=12 per sex) or began food restriction to 85-90% of their week 3 weight (n=8 per sex). Mice began consuming 2% CA water on week 4 but were reduced to 1.5% CA water from week 5 to 8 to combat body weight loss. Following weight stabilization at week 8, mice were returned to 2% CA water until the end of the study. Following week 3, mice were weighed and had their health monitored 6 times per week. Data presented as mean±SEM. CA = citric acid.\u003c/p\u003e","description":"","filename":"Picture4.png","url":"https://assets-eu.researchsquare.com/files/rs-5575045/v1/279bcb8c8b76e3a79abd81c3.png"},{"id":71848620,"identity":"65f64eef-80c5-4f7d-bc20-88ee996c65b0","added_by":"auto","created_at":"2024-12-19 07:04:34","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1684905,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eHFHS fed mice are less motivated than standard chow-fed mice in FR and PR tasks. \u003cbr\u003e\n \u003c/strong\u003eMice were trained on FR prior to advancing to PR testing. A) HFHS diet fed mice required significantly more sessions to reach criterion for advancement to PR. B) HFHS diet fed mice made significantly fewer responses per minute than standard diet fed mice. This effect was driven predominantly by the males. Overall, mice made more responses per minute with increasing number of responses require to obtain reward. C) HFHS diet fed mice had lower breakpoints than standard diet fed mice both on 1.5% CA water and 2% CA water. During the second run (2% CA water), this main effect of diet was driven by differences in the male mice. D) When on 1.5% CA water, HFHS diet fed mice made fewer blank touches than standard diet fed mice, particularly the males. Standard chow diet (n=12 per sex), HFHS diet (n=15 male, n=16 female). Data presented as mean±SEM, \u003cem\u003e*p\u0026lt;0.05, **p\u0026lt;0.01, ***p\u0026lt;0.001, ## p\u0026lt;0.01 for diet comparison, %p\u0026lt;0.05 for diet comparison within a sex. \u003c/em\u003eCA = citric acid, FR = fixed ratio;\u003cem\u003e \u003c/em\u003ePR = progressive ratio.\u003c/p\u003e","description":"","filename":"Picture5.png","url":"https://assets-eu.researchsquare.com/files/rs-5575045/v1/35dfa71d5b95e55e3921ee4f.png"},{"id":71915569,"identity":"3debb794-0653-471a-b6fc-776f3dc9cef0","added_by":"auto","created_at":"2024-12-19 16:24:39","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":285971,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDiet does not affect percent correct responding, but mice fed HFHS diet required fewer correction trials during PVD learning.\u003c/strong\u003eA) Sex, but not diet, affected percent correct responding across the 10 sessions of PVD with females responding with higher percent correct. B) Male mice and mice fed HFHS diet required more correction trials than female mice and standard diet fed mice, respectively, during PVD. There was no interaction between sex and diet. C) Perseverative responding was not affected by diet or sex. Standard chow diet (n=12 per sex), HFHS diet (n=16 male, n=15 female). Data presented as mean ± SEM, *\u003cem\u003ep\u0026lt;0.05, ***p\u0026lt;0.001. \u003c/em\u003eHFHS = high-fat, high-sugar,\u003cem\u003e \u003c/em\u003ePVD = pairwise visual discrimination.\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-5575045/v1/c1d2de03c837b8386c1b1140.jpg"},{"id":71849620,"identity":"04cae8ce-cf12-4738-b095-fa9537ef5815","added_by":"auto","created_at":"2024-12-19 07:12:34","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":1021112,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAverage weekly body weight of mice on water manipulation.\u003cbr\u003e\n\u003c/strong\u003eMice were fed standard chow or HFHS diet \u003cem\u003ead libitum\u003c/em\u003e from week 0 (baseline) until the end of the experiment. Mice were weighed three times per week. Starting week 3, mice received 2% CA water (standard diet: n=12 per sex, HFHS diet: n=16 per sex). Mice began consuming 2% CA water on week 4 but were reduced to 1.5% CA water from week 5 to 8 to combat rapid body weight loss. Following weight stabilization at week 8, mice were returned to 2% CA water until the end of the study. Following week 3, mice were weighed and had their health monitored 6 times per week. Data presented as mean±SEM. CA = citric acid, HFHS = high-fat, high-sugar.\u003c/p\u003e","description":"","filename":"Picture7.png","url":"https://assets-eu.researchsquare.com/files/rs-5575045/v1/988c8d967c6e85965c903efb.png"},{"id":106287159,"identity":"3e80aff5-b7af-4187-8262-93d9be2e72f0","added_by":"auto","created_at":"2026-04-07 07:14:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8718691,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5575045/v1/dee8cbc1-07e6-4647-b812-e88bc7a3619f.pdf"},{"id":71848615,"identity":"5efc13a5-2339-4505-b0e8-6bbef7222e73","added_by":"auto","created_at":"2024-12-19 07:04:34","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":392366,"visible":true,"origin":"","legend":"Supplementary Information","description":"","filename":"SupplementaryInformation.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5575045/v1/3e4931f4029760e8698fea67.pdf"}],"financialInterests":"\u003cb\u003eYes\u003c/b\u003e there is potential Competing Interest.\n(Non-financial interest)\r\nTim Bussey and Lisa Saksida have established a series of targeted cognitive tests for animals, administered via touchscreen within a custom environment known as the “Bussey-Saksida touchscreen chamber”. Cambridge Enterprise, the technology transfer office of the University of Cambridge, supported commercialization of the Bussey-Saksida chamber, culminating in a license to Campden Instruments. Any financial compensation received from commercialization of the technology is fully invested in further touchscreen development and/or maintenance. Campden Instruments play no role in the conceptualization, design, data collection, analysis, decision to publish, or preparation of the manuscript.","formattedTitle":"Citric acid water as an alternative to food restriction to motivate task performance in mice during touchscreen testing","fulltext":[{"header":"Introduction","content":"\u003cp\u003eAdvancements in the understanding and treatment of human diseases are largely due to the use of pre-clinical experimental models. Pre-clinical models allow direct manipulation and observation of factors underlying putative disease states.\u003csup\u003e1,2\u003c/sup\u003e In neuroscience research, studies using rodent models have provided significant insight into the mechanisms underlying many neurological conditions;\u003csup\u003e2\u003c/sup\u003e however, many of these study methods involve tests of cognition that are not directly translatable to human populations.\u003csup\u003e3\u0026ndash;6\u003c/sup\u003e Automated touchscreen operant chambers offer several advantages to traditional hand-testing tasks by minimizing experimenter interference, using standardized operating procedures, and enabling cross-species investigations through a visual-based modality. Tasks specifically developed for touchscreens allow for a greater level of control and translatability by administering virtually identical and visual-based paradigms to both rodent and human subjects.\u003csup\u003e4\u0026ndash;7\u003c/sup\u003e Studies with human participants increasingly use computerized test batteries, including CogState, Mindstreams, and the Cambridge Neuropsychological Test Automated Battery (CANTAB), amongst others.\u003csup\u003e8\u003c/sup\u003e Rodent touchscreen operant chambers use similar principles, bridging the gap between rodent research and behavioural assessments in humans.\u003csup\u003e9\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eRodent touchscreen paradigms, like many other behavioural paradigms, require the use of an appetitive reinforcer (reward), such as strawberry milkshake.\u003csup\u003e7,9,10\u003c/sup\u003e Food or liquid rewards avoid the use of aversive stimuli, removing stress or pain-induced conditioned responding in mice. \u003csup\u003e11\u003c/sup\u003e This approach motivates spontaneous animal behaviour and supports animal welfare.\u003csup\u003e11\u003c/sup\u003e Motivation to complete tasks is usually enhanced through food restriction \u0026ndash; limiting food access by duration or quantity.\u003csup\u003e12\u0026ndash;14\u003c/sup\u003e The need for food restriction is a significant limitation in studies that may alter an animal\u0026rsquo;s motivation for food or liquid rewards as part of an experimental manipulation, such as the cuprizone diet in studies of demyelination or high-fat, high-sugar (HFHS) diet in studies of diet-induced obesity.\u003csup\u003e15,16\u003c/sup\u003e Specialized diets often require \u003cem\u003ead libitum\u003c/em\u003e availability to produce the effects of the diet in a way that are translatable to humans.\u003csup\u003e17,18\u003c/sup\u003e An alternative protocol is thus required to maintain the desirability of an appetitive reinforcer so that rodents on diet manipulations can be motivated to perform touchscreen tests of cognition.\u003c/p\u003e \u003cp\u003eWater restriction is one such alternative to food restriction. Water restriction has been applied in two different ways: by limiting the duration of access to water\u003csup\u003e19\u003c/sup\u003e or by limiting the amount of water that is accessible to the rodent.\u003csup\u003e20\u003c/sup\u003e Previous studies have demonstrated that water restriction protocols in animals not undergoing food restriction effectively increase motivation in touchscreen and operant paradigms that use liquid reinforcers.\u003csup\u003e12,21\u003c/sup\u003e Despite its efficacy, water restriction presents greater health risks than food restriction and requires rigorous monitoring to ensure the health of the rodents.\u003csup\u003e12\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eWater \u003cem\u003emanipulation\u003c/em\u003e differs from water restriction in that it does not limit the availability or quantity of water.\u003csup\u003e22\u003c/sup\u003e Water manipulation occurs through the addition of a small amount of citric acid (CA) to \u003cem\u003ead libitum\u003c/em\u003e drinking water, which creates a solution with a mild sour taste\u003csup\u003e23\u003c/sup\u003e and in turn reduces the quantity of water that mice consume.\u003csup\u003e22,23\u003c/sup\u003e This reduction of water consumption is sufficient to motivate rodents to successfully complete behavioural conditioning paradigms when presented with water rewards with reduced risk of dehydration in comparison to other water restriction protocols.\u003csup\u003e22,23\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eWater manipulation using CA has previously demonstrated efficacy in motivating rodents to complete a test of learning; however, these studies did not include and explicit test of motivation. This study aimed to investigate 1) whether mice undergoing water manipulation perform similarly to food restricted mice on touchscreen tasks of motivation and discrimination learning, and 2) whether mice consuming an \u003cem\u003ead libitum\u003c/em\u003e HFHS diet are as motivated as mice consuming standard chow to complete touchscreen tasks while undergoing the water manipulation protocol. Mice were evaluated in terms of motivation (using touchscreen fixed ratio (FR) and progressive ratio (PR) tasks), learning (using a touchscreen pairwise visual discrimination (PVD) task), and health outcomes. Additionally, sex is considered in this study as male and female mice respond to changes in diet differently, including body weight and consumption.\u003csup\u003e24\u003c/sup\u003e\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003eThree experimental conditions were used (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). All three conditions included male and female C57/Bl6J mice. The mice in Condition A were food restricted on standard chow to 85\u0026ndash;90% of their free-feeding body weight with no water manipulation (i.e., unrestricted access to tap water). Mice in Condition B were fed \u003cem\u003ead libitum\u003c/em\u003e standard chow diet while on the water manipulation protocol (consuming CA water). Finally, mice in Condition C were fed \u003cem\u003ead libitum\u003c/em\u003e HFHS diet while on the water manipulation protocol (consuming CA water). A group with food restriction and \u003cem\u003ead libitum\u003c/em\u003e HFHS diet was of course not possible. Previously, Yang et al.\u003csup\u003e25\u003c/sup\u003e established the importance of food restriction in motivating mice to complete touchscreen tasks by demonstrating decreased response rates and interactions with touchscreens when mice were returned to \u003cem\u003ead libitum\u003c/em\u003e feeding following testing while on food restriction. As such, free-feeding mice that were not undergoing water manipulation were not used in the current study. Our analyses thus are based on two different comparisons of these Conditions: 1) \u003cem\u003ethe Standard Diet Comparison\u003c/em\u003e, comparing Conditions A and B, and 2) \u003cem\u003ethe Water Manipulation Comparison\u003c/em\u003e, which compare Conditions B and C. The aim of Comparison 1 is to establish whether water manipulation motivates mice as well as food restriction, whereas the aim of Comparison 2 is to establish whether water manipulation is sufficient to motivate touchscreen task performance in mice fed a highly appetitive HFHS diet. In consideration of the 3Rs (Replacement, Reduction, and Refinement), mice in Condition B were the same in both comparisons.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n \u003ch2\u003eStandard Diet Comparison: Does restriction type affect motivation, discrimination learning, or health outcomes?\u003c/h2\u003e\n \u003cp\u003e\u003cem\u003eMice undergoing water manipulation complete both the fixed-ratio and progressive-ratio task with similar levels of motivation to food-restricted mice\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eMice were habituated to the touchscreen chambers and strawberry milkshake reward over three days before undergoing a series of pretraining stages to interact with the touchscreens. To train and progress to PR, mice moved through FR reward schedules, each requiring a higher number of responses to earn rewards (see Methods and Materials below and full standard operating protocol on TouchscreenCognition.org). To address a sharp loss in weight in water-manipulated groups, prior to touchscreen testing, the CA concentration was reduced from 2\u0026ndash;1.5% (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). This change in CA concentration effectively stabilized body weight at 85% or greater, allowing a safe return to the 2% CA concentration without further significant weight decline. Consequently, all mice performed FR while on 1.5% CA water only.\u003c/p\u003e\n \u003cp\u003eIn the groups fed a standard diet, there were no effects of restriction type or sex on sessions to criterion within the FR task (ps\u0026thinsp;\u0026gt;\u0026thinsp;0.275, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eA) for advancement to PR. Similarly, the rates of responses (i.e. responses per minute) during the FR task were not affected by sex or restriction type (ps\u0026thinsp;\u0026gt;\u0026thinsp;0.275; Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB) but increased in all groups with the number of responses required to receive reward (main effect of reward schedule: F\u003csub\u003e(2.33, 80.383)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;374.44, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.513, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eB). Therefore, mice in all groups progressed at a similar rate from FR to PR.\u003c/p\u003e\n \u003cp\u003eAfter completing the FR task, mice performed the PR test of motivation, where the number of responses required for a reward increased progressively \u003cem\u003ewithin\u003c/em\u003e a reward schedule by either four, eight, or 12 responses (i.e., PR4, PR8, PR12, respectively; see Methods and Materials). To address the change in CA water concentrations from 1.5\u0026ndash;2% (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e), mice performed PR once while water-manipulated mice were on 1.5% CA water, and then again when they returned to 2% CA water. These two runs were analyzed separately. Breakpoint, an important measure of motivation, was defined as the maximum number of responses made to receive reward. While water-manipulated mice received 1.5% CA, there were no significant differences in breakpoint between restriction types or sexes (ps\u0026thinsp;\u0026gt;\u0026thinsp;0.096; Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). As expected, there was a main effect of PR reward schedule (F\u003csub\u003e(1.695, 61.023)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;17.630, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.059, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC) where breakpoints progressively increased from PR4 to PR8 to PR12 as demands to respond were higher. To assess non-specific or impulsive behavior, the number of blank touches (i.e. responses to non-target windows) made during image presentation were recorded. The number of blank touches made significantly decreased from PR4 to PR8 to PR12 (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; main effect of PR reward schedule: F \u003csub\u003e(2, 72)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;8.316, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.036, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD). There were no interaction effects or no main effects of sex or restriction type on the number of blank touches (ps\u0026thinsp;\u0026gt;\u0026thinsp;0.061).\u003c/p\u003e\n \u003cp\u003eFollowing testing on 1.5% CA, water-manipulated mice were returned to 2% CA, and all mice were tested again on PR. There were again no effects of sex or restriction type (ps\u0026thinsp;\u0026gt;\u0026thinsp;0.051, Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC) on breakpoint. Overall, mice had higher breakpoints at PR8 than PR4 and PR12, an effect driven by the males (PR8\u0026thinsp;\u0026gt;\u0026thinsp;PR4, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; PR8\u0026thinsp;\u0026gt;\u0026thinsp;PR12, p\u0026thinsp;=\u0026thinsp;0.004; reward schedule by sex interaction: F\u003csub\u003e(1.374, 49.464)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4.091, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.011, p\u0026thinsp;=\u0026thinsp;0.036; main effect of PR reward schedule: F\u003csub\u003e(1.374, 49.464)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;9.082, \u0026eta;\u003csup\u003e2=\u003c/sup\u003e0.066, p\u0026thinsp;=\u0026thinsp;0.002; Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eC). There was a main effect of PR reward schedule in the number of blank touches (F \u003csub\u003e(1.713, 61.67)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;9.865, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.066, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eE), but no effects of sex, restriction type, or interaction (ps\u0026thinsp;\u0026gt;\u0026thinsp;0.117; Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eD).\u003c/p\u003e\n \u003cp\u003eOverall, there is no evidence that water-manipulated mice showed lower motivation to receive reward than food restricted mice in a task highly sensitive to changes in motivation. Both restriction types produce high yield responding during the touchscreen task, indicating the mice are motivated to seek reward regardless of restriction type.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eStandard chow diet fed mice on CA water mice perform similarly to food-restricted mice in a pairwise visual discrimination task\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eAfter completing the PR task, mice performed the PVD task, in which they learned to discriminate between and respond to one of two different images presented simultaneously to receive a reward. Accuracy and the number of correction trials were recorded over 10 sessions of 30 trials each. There was a main effect of session (F\u003csub\u003e(5.708, 205.49)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;46.643, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.425, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA), indicating that groups learned progressively over the 10 sessions. There was also an interaction between session, sex, and restriction type (F\u003csub\u003e(5.708, 205.49)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.417, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.022, p\u0026thinsp;=\u0026thinsp;0.011; no statistically significant and relevant pairwise comparisons), but no main effects of sex or restriction type (ps\u0026thinsp;\u0026gt;\u0026thinsp;0.160). When evaluating correction trials \u0026ndash; which occurred following an incorrect response and were repeated until the animal made a correct response but did not count towards total trial count during the session \u0026ndash; there were main effects of session (F\u003csub\u003e(5.052, 181.882)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;40.579, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.411, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB) but no main effect of sex or restriction type (ps\u0026thinsp;\u0026gt;\u0026thinsp;0.420). This suggests that mice with CA manipulation are overall no different to food restricted mice in the number of correction trials required. Significant session by sex (F\u003csub\u003e(5.052, 181.882)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.080, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.031, p\u0026thinsp;=\u0026thinsp;0.001) and session by restriction type (F\u003csub\u003e(5.052, 181.882)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;1.963, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.020, p\u0026thinsp;=\u0026thinsp;0.043) interactions were observed, but without relevant and statistically significant post hoc comparisons. To further contextualize these findings, perseveration indices (PI) were calculated. The PI is a ratio of the number of correction trials to incorrect non-correction trials, representing the number of corrections required for the mouse to complete the trial correctly. Only a main effect of session (F\u003csub\u003e(5.537,199.333)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;14.582, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.180, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), but no effect of sex or restriction type nor any of the interactions were observed (ps\u0026thinsp;\u0026gt;\u0026thinsp;0.852; Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). Collectively, these data show that restriction type did not affect pairwise visual discrimination learning.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eMice on CA water maintained near-baseline weight and scored positively on health scores of activity levels, hydration, and grooming\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eMice were monitored for health using a scoring rubric for categories of posture and grooming, activity levels, signs of dehydration, and eating and drinking (see Materials \u0026amp; Methods, Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Each category has a set of behaviors or conditions assigned a score from 0 (normal) to 3 (severe), with higher scores indicating signs of distress or poor health. Low scores indicated no significant health changes within a single category (\u0026lt;\u0026thinsp;2 out 3) or across categories (\u0026lt;\u0026thinsp;5 out of 11). Overall health concerns were minimal with no mice on CA water scoring values of greater than 1 in any single health category or cumulatively. Reported scores of 1 in a single health category or cumulatively were predominantly for activity levels and signs of dehydration (i.e., skin turgor test, lack of urine and feces) in both males (n\u0026thinsp;=\u0026thinsp;2) and females (n\u0026thinsp;=\u0026thinsp;4) during the first four days of CA water administration (Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eBody weight was monitored throughout the experiment (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e) and analyzed following onset of restriction. Overall, water-manipulated mice weighed significantly more than food restricted mice (main effect of restriction type: F \u003csub\u003e(1,36)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;16.565, \u0026eta;\u0026sup2;=0.027, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and male mice were heavier than female mice (main effect of sex: F\u003csub\u003e(1,36)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;516.522, \u0026eta;\u0026sup2;=0.827, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). There was a significant sex by restriction type interaction (F\u003csub\u003e(1,36)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;5.236, \u0026eta;\u0026sup2;= 0.008, p\u0026thinsp;=\u0026thinsp;0.028) revealing that the effect of restriction type was significant in the male mice (Cohen\u0026rsquo;s d\u0026thinsp;=\u0026thinsp;1.678, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) but not in female mice (p\u0026thinsp;=\u0026thinsp;0.216). Over the course of the experiment, mice on water manipulation gained weight following restriction whereas food restricted mice did not (week by restriction type interaction: F \u003csub\u003e(5.571, 200.546)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;22.889, \u0026eta;\u0026sup2;=0.018, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) with this being predominantly driven by the male mice (sex by week interaction: F\u003csub\u003e(5.571, 200.546)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;2.801, \u0026eta;\u0026sup2;=0.002, p\u0026thinsp;=\u0026thinsp;0.014). There was also a three-way sex by restriction type by week interaction (F\u003csub\u003e(5.571, 200.546)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.146, \u0026eta;\u0026sup2;=0.002, p\u0026thinsp;=\u0026thinsp;0.007; Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e). A main effect of week was observed (F\u003csub\u003e(5.571, 200.546)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;36.327, \u0026eta;\u0026sup2;=0.029, p\u0026thinsp;=\u0026thinsp;0.001). These results indicate that although CA water-manipulated male mice weighed more compared with the food restricted mice.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eWater Manipulation Comparison: Does diet affect motivation, discrimination learning, or health outcomes?\u003c/h3\u003e\n\u003cp\u003e\u003cem\u003eMice on HFHS diet are less motivated to perform FR and PR tasks for reward than mice on standard chow diet\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eIn the water manipulation groups, mice on HFHS diet required more sessions to reach advancement criteria on the FR task than mice on standard chow diet (F\u003csub\u003e(1, 51)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;7.412, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.119, p\u0026thinsp;=\u0026thinsp;0.009; Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). Sessions to reach criterion were not significantly affected by sex (p\u0026thinsp;=\u0026thinsp;0.31) and there was no sex by diet interaction (p\u0026thinsp;=\u0026thinsp;0.105; Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eA). When comparing rate of responses across FR reward schedules and diet groups, as the number of responses required to obtain reward increased so too did the rate of response (F\u003csub\u003e(2.107, 107.482)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;123.241, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.448, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB). However, HFHS mice had overall lower response rates in FR compared to standard fed mice (F\u003csub\u003e(1, 51)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;7.648, \u0026eta;\u0026sup2;=0.040, p\u0026thinsp;=\u0026thinsp;0.008; Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eB) particularly during FR5 (Cohen\u0026rsquo;s d\u0026thinsp;=\u0026thinsp;1.125, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; reward schedule by diet interaction: F\u003csub\u003e(2.107,107.482)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;5.187, \u0026eta;\u0026sup2;=0.019, p\u0026thinsp;=\u0026thinsp;0.006; Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD). Males on HFHS diet responded at a lower rate than male mice on standard chow diet (Cohen\u0026rsquo;s d\u0026thinsp;=\u0026thinsp;1.074, p\u0026thinsp;=\u0026thinsp;0.004; sex by diet interaction (F\u003csub\u003e(1, 51)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;5.603, \u0026eta;\u0026sup2;=0.030, p\u0026thinsp;=\u0026thinsp;0.022). Notably, female mice did not vary by diet (p\u0026thinsp;=\u0026thinsp;0.778), suggesting that the effects of diet observed are driven by the male mice. The main effect of sex was not significant (p\u0026thinsp;=\u0026thinsp;0.988). Overall, mice on a HFHS diet took longer to progress through the FR task and responded at lower rates than standard diet fed mice, particularly in male mice and when greater responses for reward were required. This demonstrates that diet significantly affects training speed and response rates across the FR task.\u003c/p\u003e\n\u003cp\u003eFollowing the FR task, mice were run on the PR task. While on 1.5% CA, chow-fed mice had higher breakpoints than HFHS mice (main effect of diet: F\u003csub\u003e(1,51)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;22.204, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.245, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC), with no effect of sex (p\u0026thinsp;=\u0026thinsp;0.914). Mice of both diets had lower breakpoints at PR4 than at PR8 or PR12 (ps\u0026thinsp;\u0026lt;\u0026thinsp;0.016; main effect of reward schedule: F\u003csub\u003e(1.660, 84.652)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;9.259, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.025, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC). Number of blank touches differed by diet (F\u003csub\u003e(1,51)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;11.162, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.141, p\u0026thinsp;=\u0026thinsp;0.002, Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD), with no effect of sex. A main effect of reward contingency was observed (F\u003csub\u003e(2,102)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;11.942, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.033, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD), where blank touches decreased from PR4 to PR8 to PR12.\u003c/p\u003e\n\u003cp\u003eAfter returning to 2% CA water, and like performance on 1.5%, a main effect of diet on breakpoint was present (F\u003csub\u003e(1, 51)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;5.108, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.071, p\u0026thinsp;=\u0026thinsp;0.028, Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eC) with standard chow mice responding more for reward than HFHS diet mice. Once again, there was also a main effect of PR reward schedule (F\u003csub\u003e(2, 102)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4.603, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.010, p\u0026thinsp;=\u0026thinsp;0.012) but no main effect of sex (p\u0026thinsp;=\u0026thinsp;0.160). A diet by sex interaction was present (F\u003csub\u003e(1, 51)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4.780, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.067, p\u0026thinsp;=\u0026thinsp;0.033), with a significant drop in performance in HFHS-fed male mice compared to standard chow-fed male mice (Cohen\u0026rsquo;s d\u0026thinsp;=\u0026thinsp;1.129, p\u0026thinsp;=\u0026thinsp;0.016), but no differences observed in female mice (ps\u0026thinsp;\u0026gt;\u0026thinsp;0.063). Blank touches differed across PR reward schedule (F\u003csub\u003e(2, 102)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;13.712, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.063, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eD), with no further main effects (ps\u0026thinsp;\u0026gt;\u0026thinsp;0.390). Finally, there was a sex by diet interaction for blank touches (F\u003csub\u003e(1,51)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;8.550, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.098, p\u0026thinsp;=\u0026thinsp;0.005) with a trend for fewer blank touches in male mice on HFHS diet than male mice on standard chow diet; however, this (p\u0026thinsp;=\u0026thinsp;0.056) and other pairwise comparisons did not reach statistical significance (all others, ps\u0026thinsp;\u0026gt;\u0026thinsp;0.209).\u003c/p\u003e\n\u003cp\u003eCollectively, these results demonstrate that water-manipulated mice fed a HFHS diet were significantly less motivated to obtain reward than chow-fed mice in touchscreen PR. Male mice on HFHS diet were impaired compared to their chow-fed counterparts.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eWater-manipulated mice consuming HFHS diet perform similarly to standard chow mice in the pairwise visual discrimination\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eFollowing PR, water-manipulated mice performed the PVD task. All mice improved in performance across the 10 sessions (F\u003csub\u003e(6.039, 301.929)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;77.071, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.484, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eA). Male mice made fewer correct responses on average compared with female mice (main effect of sex: F\u003csub\u003e(1, 50)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4.210, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.013, p\u0026thinsp;=\u0026thinsp;0.045; Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e), but no significant difference was found between diet groups and no interaction (ps\u0026thinsp;\u0026gt;\u0026thinsp;0.235). Overall, mice performed fewer correction trials across sessions (F\u003csub\u003e(5.187, 259.375)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;83.072, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.534 p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eB). HFHS diet mice made fewer correction trials than standard fed mice (main effect of diet: F\u003csub\u003e(1, 50)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;4.443, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.003 p\u0026thinsp;=\u0026thinsp;0.040), but there was no effect of or interaction with sex or sessions (ps\u0026thinsp;\u0026gt;\u0026thinsp;0.067). Lastly, perseveration index (PI) decreased across sessions (F\u003csub\u003e(4.541, 227.033)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;16.376, \u0026eta;\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.199 p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003eC), but did not differ between diet groups or sexes (ps\u0026thinsp;\u0026gt;\u0026thinsp;0.192).\u003c/p\u003e\n\u003ch3\u003eHFHS diet induces significant weight gain in water-manipulated mice with few other health concerns\u003c/h3\u003e\n\u003cp\u003eMice were monitored for health using a scoring rubric for categories of posture and grooming, activity levels, signs of dehydration, and eating and drinking (see Materials \u0026amp; Methods, Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). Low scores indicated no significant health changes within a single category (\u0026lt;\u0026thinsp;2 out 3) or across categories (\u0026lt;\u0026thinsp;5 out of 11). All standard-chow, water-manipulated mice reported scores of one or less in activity levels or sign of dehydration, indicating no major health concerns. Only four (2 male, 2 female) HFHS diet mice were reported to have cumulative scores of either 2 or 3 within the activity level or posture categories that prompted monitoring by veterinary care. These scores later decreased as mice acclimatized to the combination of CA water manipulation and HFHS diet.\u003c/p\u003e\n\u003cp\u003eBody weight was monitored throughout the experiment (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). Overall, HFHS diet fed mice weighed significantly more than standard diet fed mice (main effect of diet: F\u003csub\u003e(1,51)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;42.98, \u0026eta;\u0026sup2;=0.149, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) and male mice were heavier than female mice (main effect of sex: F\u003csub\u003e(1,51)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;162.695, \u0026eta;\u0026sup2;=0.565, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001). There was a significant sex by restriction type interaction (F\u003csub\u003e(1,51)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;7.755, \u0026eta;\u0026sup2;= 0.027, p\u0026thinsp;=\u0026thinsp;0.008) revealing that the effect of diet was greater in male mice compared with female mice but significant in both (males: Cohen\u0026rsquo;s d\u0026thinsp;=\u0026thinsp;2.318, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; females: Cohen\u0026rsquo;s d\u0026thinsp;=\u0026thinsp;0.936, p\u0026thinsp;=\u0026thinsp;0.011). As weeks elapsed, mice on HFHS diet gained more weight compared with mice on standard diet (week by diet interaction: F\u003csub\u003e(3.242, 165.325)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;7.083, \u0026eta;\u0026sup2;=0.005, p\u0026thinsp;\u0026lt;\u0026thinsp;0.001) once started on CA water manipulation. Male mice gained more weight than female mice (week by sex interaction: F\u003csub\u003e(3.242, 165.325)\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;3.121, \u0026eta;\u0026sup2;=0.002, p\u0026thinsp;=\u0026thinsp;0.024; no significant three-way, sex by diet by week, interaction: p\u0026thinsp;=\u0026thinsp;0.132; Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eSpecialized diets, such as demyelinating and HFHS diets, are used as experimental manipulations in many studies but their use in food-motivated tasks is problematic due to the need in such tasks for adequate motivation for food reward. The present findings show that water manipulation using CA water supports behavioural performance on strawberry milkshake-rewarded touchscreen-based tasks in both standard-fed and HFHS-fed mice. Notably, differences in touchscreen performance were observed between diet groups in the PR task, which is highly sensitive to changes in motivation, but this decrease in motivation was not sufficient to affect performance on the PVD task used to assess visual discrimination learning. Thus, CA water manipulation allows testing of mice on diets such as HFHS on appetitively motivated touchscreen tests of cognition.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eComparison 1: Water manipulation is sufficient to motivate standard chow-fed mice on touchscreen tasks\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003ePrevious work by Urai et al.\u003csup\u003e22,23\u003c/sup\u003e and Reinagel et al.\u003csup\u003e23\u003c/sup\u003e confirmed the efficacy of CA water manipulation as a motivator in an apparatus that paired a computer display with either a steering wheel or licking ports for responses, respectively. In line with those publications, the present study also found CA water manipulation to be sufficient to elicit behavioural performance on touchscreen tests of motivation and visual discrimination.\u0026nbsp;Under standard diet conditions, both CA water manipulation and food restriction were equally effective in motivating standard chow-fed mice to respond for strawberry milkshake reward without changes in latency to respond (Supplementary Figure 1) on a touchscreen-based motivation task. This motivated performance further extended to learning in a pairwise visual discrimination task on which there were no differences between water-manipulated and food-restricted mice. Thus, CA water manipulation is a sufficient motivator of behaviour in touchscreen chambers in standard fed mice, extending the utility of both water manipulation as a motivator and touchscreens tasks in animal research.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eComparison 2: HFHS diet reduces motivation in the PR task but does not restrict performance in pairwise visual discrimination learning motivated by CA water manipulation.\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eAs the need for food restriction was previously recommended for touchscreen testing, rendering the use of specialized diets in this testing modality problematic, we sought to determine whether CA water manipulation – which enables animals to both drink and eat \u003cem\u003ead libitum\u003c/em\u003e – is sufficient to drive responding in touchscreens when mice were fed a HFHS diet, perhaps the most satiating of all specialized diets. It is documented that a HFHS diet typically results in impaired motivation.\u003csup\u003e26,27\u003c/sup\u003e Particularly, it is both the composition of the food such as increased sugar and fats, and caloric intake that affects motivation.\u003csup\u003e28,29\u003c/sup\u003e HFHS diet often leads to metabolic dysfunction, dysregulated dopamine signaling,\u003csup\u003e\u0026nbsp;18,30\u003c/sup\u003e reduced reward sensitivity,\u003csup\u003e21,29\u003c/sup\u003e and lowered activity levels,\u003csup\u003e31\u003c/sup\u003e all of which would be expected to significantly demotivate animals and/or reduce task engagement. Even with these well-known demotivating effects and reductions in task engagement, we observed that CA water manipulation was sufficient to motivate mice on an HFHS diet to complete the PR task, a task highly sensitive to changes in motivation. While HFHS fed mice on water manipulation took longer to train on the task and showed decreased motivation compared to \u003cem\u003ead libitum\u003c/em\u003e standard chow-fed mice (also motivated with CA water manipulation), they could perform the task. Consequently, we were able to robustly quantify differences in motivation for an appetitive reward in mice on HFHS diet versus standard chow diet.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis is the first study to investigate the effects of HFHS diet on discrimination learning as measured by the PVD task, which may be due to limitations in restriction protocols to motivate behaviour. Rather, most studies observe HFHS diet-induced impairments in tests of spatial memory, working memory, and object recognition that depend on spontaneous, non-rewarded testing paradigms in rodents\u003csup\u003e34\u003c/sup\u003e and humans.\u003csup\u003e\u0026nbsp;28,29,35\u003c/sup\u003e While HFHS diet led to lower breakpoints on a motivation-sensitive PR task, this did not translate into learning impairments on the PVD task. This has similarly been seen in rats that were food restricted on standard chow diet and provided 70ml/day of high sucrose water on the same PVD task.\u003csup\u003e36\u003c/sup\u003e The PVD task, like the FR1 task, requires only a single response to the correct stimulus to obtain a reward, leading to consistent reinforcement with each correct response. This simple response requirement likely protected performance from motivational deficits observed in tasks with higher effort demands such as PR, where multiple responses revealed motivational impairments. Thus, it is likely that CA water manipulation will similarly elicit performance in touchscreen tasks that assess other cognitive domains, such as attention, even in mice on specialized diets. In this study, we were able to conclude that HFHS diet does not impact the rate of learning on a PVD task compared to standard diet.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eSex-specific differences between restriction types and diets\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003eBeyond the expected and normal differences in body weight between male and female mice, there were notable sex differences observed in this study, especially following HFHS diet consumption. In Comparison 1 (Standard Diet Comparison), few sex differences were observed in PR and PVD task performance. For example, during PR, male mice exhibited higher breakpoints on PR8 compared to PR4 and P12, irrespective of restriction type. In Comparison 2 (Water Manipulation Comparison), males on a HFHS diet exhibited lower response rates during the FR task, showed reduced breakpoints in the PR task, and tended to make fewer non-specific responses when compared with males on a standard chow diet. Interestingly, water-manipulated female mice made more correct responses in PVD than water-manipulated male mice, irrespective of diet. Females, conversely, showed no differences in weight between restriction types, and a more moderate increase in weight on HFHS diet when compared to the substantial weight gains seen in HFHS-fed males. Further investigation into how body weight, adiposity, and circulating nutrients resulting from different diets (including drinking water) affect motivation and learning in males and females is needed.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eCA water manipulation is practical to implement and promotes positive health outcomes in mice\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;The use of water manipulation resulted in no significant health changes and allowed for mice to maintain or gain weight throughout the study while maintaining task performance. Mice given CA water stabilized or regained their weight, and presented with few signs of dehydration, lowered activity levels, and abnormal posture or grooming. Water manipulation allowed for changes in weight during development and diet consumption in contrast with the food restriction group which, by design, is maintained at 85-90% of baseline weight throughout testing. Therefore, CA water manipulation may be a useful alternative in studies using adolescent mice to allow for normal growth and development. Additionally, water manipulation requires minimal experimenter labour when compared to food restriction or other types of water restriction. CA water preparations were completed at the start of each week and animals only required daily monitoring and weighing. Unlike food restriction, there are also no concerns regarding time of feeding (especially relative to behavioural experiments), missed feedings, or competition over food in grouped housed animals. CA may also offer health benefits in animals. Urai et al. highlight that CA increases the acidity of water, which may benefit gut health in animals.\u003csup\u003e22\u003c/sup\u003e Previous literature has also suggested that CA is an antioxidant and limits excessive generation of reactive oxygen species and free radicals, which benefits liver health.\u003csup\u003e37\u003c/sup\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConsiderations for future use\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; Water manipulation affected neither motivation nor learning in standard fed mice. In contrast, HFHS diet reduced motivation in water-manipulated mice compared with standard fed mice on water manipulation. It is therefore critical for researchers to consider their control groups and confounds that may exist with future use of water manipulation. If diet is the dependent variable in an experiment, it will be important to consider how diet may affect motivation in testing and how that can impact the dependent variables collected. \u0026nbsp;Even in experiments where diet is consistent across experimental groups, caution should be used to ensure accurate interpretation of the data, including how the results may generalize to standard and other non-standard diets.\u0026nbsp;\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eIn conclusion, water manipulation using CA water is an effective alternative to food restriction in motivating mice across both standard and HFHS diet conditions. While motivation was reduced in mice on CA water when consuming a HFHS diet, mice were still able to perform the tasks. Importantly, these motivational changes did not significantly impact learning performance, highlighting the potential utility of water manipulation using CA in future studies where food restriction may not be feasible or desirable.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cp\u003e All experiments were conducted in compliance with the standards set by the Canadian Council of Animal Care and under direct veterinary supervision at the Western University.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eExperimental design\u003c/h2\u003e \u003cp\u003eSeventy-two C57BI/6J mice (36 males and 36 females, Jackson Laboratory, US) arrived at 8\u0026ndash;12 weeks of age. Mice were group-housed by sex with 4 mice per cage in a temperature (23\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C) and humidity (50\u0026thinsp;\u0026plusmn;\u0026thinsp;1%) controlled room under a reverse 12h light/dark cycle (lights off at 9:00). The complete study design is illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003eMice were acclimated to their cages for 5 days with \u003cem\u003ead libitum\u003c/em\u003e access to standard Teklad diet (Envigo) and untreated water. Mice were randomly assigned to standard pre-weighed diet (Bioserv F0078, 3.6 kcal/g, 5.6% fat, 59.1% carbohydrates, 18% protein; n\u0026thinsp;=\u0026thinsp;20 per sex) or HFHS chow (Bioserv F6724, 4.57 kcal/g, 21.2% fat, 48.5% carbohydrates, 17.3% protein; n\u0026thinsp;=\u0026thinsp;16 per sex). Mice were maintained on their respective diets for three weeks after which baseline weights were calculated as mean bodyweight over three days. Following baseline calculations, food restriction and water manipulation protocols were implemented. Sixteen mice (n\u0026thinsp;=\u0026thinsp;8/sex) in the standard diet group were randomly assigned to undergo food restriction to 85\u0026ndash;90% of their baseline weight. Twenty-four mice (n\u0026thinsp;=\u0026thinsp;12/sex) received \u003cem\u003ead libitum\u003c/em\u003e standard diet, and thirty-two mice (n\u0026thinsp;=\u0026thinsp;16/sex) received \u003cem\u003ead libitum\u003c/em\u003e HFHS diet (see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). All mice with \u003cem\u003ead libitum\u003c/em\u003e access to diet also received 2% citric acid water manipulation \u003cem\u003ead libitum\u003c/em\u003e. Two grams of CA (citric acid anhydrous, Thermofisher Scientific, USA) were dissolved in 100mL of tap water to produce 2% CA water.\u003c/p\u003e \u003cp\u003e Mice were weighed 3-6x per week in accordance with the animal use protocol. Mice undergoing food restriction had a healthy weight defined as 85\u0026ndash;90% of their baseline weight whereas mice consuming 2% CA water had no upper boundary of permissible body weight. 2% CA water-manipulated mice had their weight recorded between 10:00AM and 12:00PM each day and were administered CA water with a reduced concentration of CA if they experienced abrupt weight loss to an amount less than 85% of their baseline weight. Food restricted mice found to be underweight were provided additional diet (minimum of 0.5g increase); water-manipulated mice found to be underweight were provided one hour of access to 0.5% CA water to encourage increased water consumption. Health scores were provided for each mouse based on their posture and grooming, dehydration levels, activity, and eating or drinking using a health scoring rubric (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003e\u003cb\u003eHealth scoring rubric used to assess activity levels and hydration of mice on citric acid water.\u003c/b\u003e Mice undergoing water manipulation treatment were monitored using this table 6 times per week.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"2\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eActivity\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eScore\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMoves around the cage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMoves slowly around the cage\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMoves only when touched\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDoes not move\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePosture and Grooming\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNormal posture and smooth fur\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHunched posture \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eor\u003c/span\u003e ruffled fur\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHunched posture \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eand\u003c/span\u003e slightly ruffled fur\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eHunched posture \u003cspan type=\"Underline\" class=\"Underline\" name=\"Emphasis\"\u003eand\u003c/span\u003e all fur ruffled\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSigns of Eating and Drinking\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eFeces and urine observed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMinimal fecal and/or urine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eNo signs or feces and/or urine\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSigns of Dehydration\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSkin does not tent when scuffed\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSkin tents briefly but returns to normal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSkin tents and takes more than 2 seconds\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSkin tents and stays tented\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal Scores\u003c/b\u003e\u003c/p\u003e \u003cp\u003eAny animal that has a score:\u003c/p\u003e \u003cp\u003e\u0026bull; \u0026le;\u0026thinsp;4 cumulatively or \u0026le;\u0026thinsp;1in any one category should be monitored but no action required\u003c/p\u003e \u003cp\u003e\u0026bull; \u0026ge;\u0026thinsp;2 in any one category or cumulatively to \u0026ge;\u0026thinsp;5 required veterinary support to monitor the animal\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eTouchscreen Behavioural Testing\u003c/h2\u003e \u003cp\u003eAll touchscreen behavioural testing was completed using standard Bussey-Saksida mouse touchscreen chambers (model 80614, Lafayette Instrument Company, Lafayette IN) as described in detail elsewhere \u003csup\u003e7\u003c/sup\u003e and using task specific standard operating procedures published to TouchscreenCognition.org. Briefly, the apparatus contains a trapezoidal chamber with a touchscreen at one end and a reward magazine on the other end. A space in front of the touchscreen allows the insertion of removable masks (black plastic sheets with apertures that allow access to the touchscreen) specialized to each cognitive test. The reward magazine illuminates and dispenses liquid milkshake reward (Nielson\u0026rsquo;s Strawberry Milkshake) upon successful trial completion following interaction with the touchscreen. Therefore, the number of trials completed is equivalent to the number of rewards obtained. Mice were habituated to the chambers and milkshake rewards over three days and were given several pretraining stages in order that the mice learned to interact with the touchscreens, and successfully retrieve the reward.\u003csup\u003e9,10,38\u003c/sup\u003e\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTouchscreen Tests of Motivation: Fixed and Progressive Ratio Tasks\u003c/h2\u003e \u003cdiv id=\"Sec16\" class=\"Section3\"\u003e \u003ch2\u003eTraining using the Fixed-Ratio (FR) Touchscreen Task\u003c/h2\u003e \u003cp\u003eFollowing habituation and pretraining, mice were trained for PR using the FR task. The FR task was comprised of several reward schedules with each reward schedule requiring a different number of nose pokes to the stimulus (white square) to obtain a reward. For example, mice were trained to collect a reward (~\u0026thinsp;24ul dispensed via 800ms pulse of a peristaltic pump) by completing one touchscreen response to the stimulus in the FR1 reward schedule.\u003csup\u003e9\u003c/sup\u003e Following each reward collection, there was a 20s intertrial interval (ITI) before the next trial can be initiated and a stimulus (white square) appears in the centre of a 5-window mask. After achieving 30 rewards in 60 minutes, mice were advanced to the subsequent reward schedule.\u003csup\u003e9\u003c/sup\u003e The following reward schedules FR2, FR3, and FR5 required 2, 3 or 5 responses to the stimulus, respectively.\u003csup\u003e38\u003c/sup\u003e Therefore, FR5 required the greatest effort, requiring five responses to the stimulus for a single reward. The number of responses, time of session completion, and rewards collected were recorded. To examine the rate of responding, the number of responses per minute were calculated. Additionally, the number of sessions to complete all of FR was totaled.\u003c/p\u003e \u003cp\u003eFollowing successful completion of FR5, the PR task began. One male HFHS diet mouse failed to reach criterion and so did not progress to PR from FR. PR task parameters were identical to those in the FR task with the exception that the number of responses to the touchscreen required to obtain a reward differed: The number of touches required increased incrementally within a session, such that the responses required for reward in PR4 increases by four for each subsequent reward (i.e., Trial 1 required one response, Trial 2: five responses, Trial 3: nine responses, etc.). After completing PR4, mice completed PR8and PR12, where the number of required responses for a reward increased by 8 or 12 on each trial, respectively. Mice were only given one PR session (either PR4, PR8, or PR12) per day. To examine motivation, the breakpoint for each mouse was calculated; breakpoint is the number of responses made in the final successfully completed trial. For example, in PR4, if a mouse successfully completed the first four trials with reward requirements of 1, 5, 9, and 13 responses, respectively, but failed to complete the next (17 responses for reward), it thus reached a breakpoint of 13. Additionally, reward latency and number of non-stimulus touches (blank touches) were recorded.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003ePairwise Visual Discrimination (PVD) Task\u003c/h2\u003e \u003cp\u003eFollowing FR/PR, mice performed the pairwise visual discrimination (PVD) task.\u003csup\u003e7\u003c/sup\u003e Mice were first pretrained for PVD on punish incorrect, where correct touches were rewarded, and blank touches triggered a 5-second timeout with the house light illuminated. Mice needed to complete\u0026thinsp;\u0026ge;\u0026thinsp;24/30 trials correct (i.e., minimum 24 rewards) within 60 min for 2 consecutive sessions before moving on to PVD. One water-manipulated female HFHS diet mouse did not progress to PVD due to health complications not related to diet or CA water. The PVD task required mice to discriminate between 2 images (diagonal lines 45\u0026deg; clockwise or anticlockwise from vertical) and respond to the correct (rewarded) stimulus image (S+) and withhold responding to the incorrect (unrewarded) stimulus image (S-) when they were both displayed simultaneously on either side of the touchscreen (Horner et al., 2013). The relative left and right presentation of the stimuli varied pseudo-randomly across each trial and stimuli were not displayed in the same location for more than three consecutive trials. Next, the mouse could initiate the following trial. In contrast, an incorrect response to the S- resulted in a 5s illumination of the chamber by the house light. Following the 5s penalty, a 20s ITI is initiated. Following the ITI, a correction trial began in which the stimuli were presented in the same configuration as the previous trial and this trial configuration was repeated until the mouse selected the S\u0026thinsp;+\u0026thinsp;and collected a reward. Mice completed a total of 10 sessions of 30 successful trials each. The percent correct responses out of 30 trials (i.e., accuracy) and the number of correction trials were recorded for each session. When a mouse did not reach 30 trials in a single session, it was tested the next day on the trials remaining to reach 30 trials; therefore, each session analyzed consisted of a total of 30 trials. Due to running errors, one mouse (male, HFHS diet, water-manipulated) completed 33 trials during session 1 and another (male, HFHS diet, water-manipulated) completed only 20 trials during session 3. Formulae for perseveration indices were adapted in these cases. In cases where a mouse completed only nine sessions, data from the ninth session was used for both Sessions 9 and 10 (1 female, standard diet, water-manipulated; 4 male, HFHS diet, water-manipulated).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eData Analysis\u003c/h2\u003e \u003cp\u003eThe dependent variables were examined using two separate analyses to address the main questions of this study: 1) Comparison of the restriction protocol (food restriction, CA water manipulation) and sex (male, female) within the standard diet groups (i.e., mice given standard chow), and 2) a comparison of diet (standard, HFHS) and sex (male, female) within water-manipulated groups (i.e., mice given ad libitum CA water). The comparison between standard diet with food restriction and HFHS with water manipulation mice was considered irrelevant to the research aims, and therefore were not directly compared statistically, which reduced potential type 2 errors. Moreover, using the same standard-diet mice consuming CA water in both analyses aligns with the principles of the 'Three Rs' of animal research, reducing animal use while maximizing data collection from the same cohort. This design addresses 1) whether CA water and food restriction could sufficiently motivate mice consuming standard chow to a similar degree during touchscreen testing, and 2) whether CA water can motivate mice consuming HFHS diet to a similar degree as standard chow diet mice.\u003c/p\u003e \u003cp\u003eData were analyzed using JASP (Version 0.16.3 Intel). Three-way mixed model analyses of variance (ANOVA) were used, with restriction protocol (food restriction, CA water manipulation) or diet (standard chow, HFHS) and sex (male, female) as between-subject factors and day, week, or session as the within-subject repeated factors. In instances where sphericity assumptions were violated (Mauchly\u0026rsquo;s test), Greenhouse-Geisser corrections were applied. When interaction effect was detected, Tukey or Holm post-hoc analyses were performed where appropriate. Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Significance was set at α\u0026thinsp;\u0026lt;\u0026thinsp;0.05.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003eAll experiments were conducted in compliance with the standards set by the Canadian Council of Animal Care and under approval and direct veterinary supervision by the animal care committee at the Western University.\u003c/p\u003e\n\u003cp\u003eAcknowledgements\u003c/p\u003e\n\u003cp\u003eThis research was supported by through the Canada First Research Excellence Fund (CFREF), Brain Canada, the Canadian Institutes of Health Research (CIHR PJT 426966; Vanier Canada Graduate Scholarship to ORG-S), the Natural Sciences and Engineering Research Council (NSERC RGPIN-2019-06102, RGPIN-2019-06087), the Canada Foundation for Innovation, and the Ontario Research Fund. LMS is the Canada Research Chair in Translational Cognitive Neuroscience and TJB is the Western Research Chair in Behavioural Neuroscience.The authors also thank Claire Lipton for her invaluable assistance with animal handling and monitoring.\u003c/p\u003e\n\u003cp\u003eCompeting Interest Statement\u003c/p\u003e\n\u003cp\u003eTJB and LMS have established a series of targeted cognitive tests for animals, administered via touchscreen within a custom environment known as the \u0026ldquo;Bussey-Saksida touchscreen chamber\u0026rdquo;. Cambridge Enterprise, the technology transfer office of the University of Cambridge, supported commercialization of the Bussey-Saksida chamber, culminating in a license to Campden Instruments. Any financial compensation received from commercialization of the technology is fully invested in further touchscreen development and/or maintenance. Campden Instruments play no role in the conceptualization, design, data collection, analysis, decision to publish, or preparation of the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eMoran, P. \u003cem\u003eet al.\u003c/em\u003e Gene \u0026times; Environment Interactions in Schizophrenia: Evidence from Genetic Mouse Models. \u003cem\u003eNeural Plast\u003c/em\u003e \u003cstrong\u003e2016\u003c/strong\u003e, 1\u0026ndash;23 (2016).\u003c/li\u003e\n \u003cli\u003eJucker, M. \u0026amp; Ingram, D. K. 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N. Citric Acid an Antioxidant in Liver. in \u003cem\u003eThe Liver\u003c/em\u003e 183\u0026ndash;198 (Elsevier, 2018). doi:10.1016/B978-0-12-803951-9.00016-1.\u003c/li\u003e\n \u003cli\u003eHeath, C. J. \u003cem\u003eet al.\u003c/em\u003e A Touchscreen Motivation Assessment Evaluated in Huntington\u0026rsquo;s Disease Patients and R6/1 Model Mice. \u003cem\u003eFront Neurol\u003c/em\u003e \u003cstrong\u003e10\u003c/strong\u003e, (2019).\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"nature-portfolio","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"","title":"Nature Portfolio","twitterHandle":"","acdcEnabled":false,"dfaEnabled":false,"editorialSystem":"ejp","reportingPortfolio":"","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5575045/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5575045/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRodent behavioural testing paradigms in touchscreen operant chambers have successfully provided insight into the neural mechanisms underlying various cognitive domains in healthy and disease models. Touchscreen testing has previously required food restriction to sufficiently motivate rodents to complete behavioural tests, limiting the use of interventions, for example diet-based interventions, that alter animals\u0026rsquo; motivation for food in experimental design. Here, we explored the safety and efficacy of water manipulation via addition of citric acid in motivating behavioural performance in touchscreen operant chambers 1) in comparison with food restriction and 2) when mice are fed an obesogenic high-fat, high-sugar (HFHS) diet. Water manipulation and food restriction produced similar performance on the progressive ratio task in non-obesogenic, standard-fed mice. However, when water-manipulated mice were fed a HFHS diet they showed deficits in this motivation-sensitive task. Critically, all groups, regardless of restriction type or diet, showed similar learning curves during a pairwise visual discrimination task. Together, these findings demonstrate that water manipulation can safely and effectively motivate mice to perform touchscreen tasks for reward, even when fed a highly satiating HFHS diet, which opens the possibility of using interventions, especially diet-based, in conjunction with touchscreen cognitive testing batteries.\u003c/p\u003e","manuscriptTitle":"Citric acid water as an alternative to food restriction to motivate task performance in mice during touchscreen testing","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-12-19 07:04:28","doi":"10.21203/rs.3.rs-5575045/v1","editorialEvents":[],"status":"published","journal":{"display":false,"email":"[email protected]","identity":"lab-animal","isNatureJournal":true,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"laban","sideBox":"Learn more about [Lab Animal](http://www.nature.com/laban/)","snPcode":"","submissionUrl":"","title":"Lab Animal","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"Nature Research","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"4e7ade14-97c6-4c7b-84b8-8beb868e8cd0","owner":[],"postedDate":"December 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":41270165,"name":"Biological sciences/Neuroscience/Cognitive neuroscience"},{"id":41270166,"name":"Biological sciences/Neuroscience/Learning and memory"},{"id":41270167,"name":"Biological sciences/Neuroscience/Motivation"}],"tags":[],"updatedAt":"2026-04-07T07:13:47+00:00","versionOfRecord":{"articleIdentity":"rs-5575045","link":"https://doi.org/10.1038/s41684-026-01711-y","journal":{"identity":"lab-animal","isVorOnly":false,"title":"Lab Animal"},"publishedOn":"2026-04-06 04:00:00","publishedOnDateReadable":"April 6th, 2026"},"versionCreatedAt":"2024-12-19 07:04:28","video":"","vorDoi":"10.1038/s41684-026-01711-y","vorDoiUrl":"https://doi.org/10.1038/s41684-026-01711-y","workflowStages":[]},"version":"v1","identity":"rs-5575045","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5575045","identity":"rs-5575045","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}