Examining persistence of acute environmental enrichment-induced anti-sucrose craving effects in rats

Abstract

A single, overnight (acute) environmental enrichment (EE; a large environment with conspecifics and novel objects) experience robustly decreases sucrose consumption (taking) and responsiveness to sucrose-paired cues (seeking) in rats. Persisting effects of acute EE on sucrose seeking and taking have not yet been identified. In the present study, rats were trained to self-administer a 10% sucrose solution paired with a compound tone+light stimulus for 10 days in 2-h sessions. We then examined the persistence of acute EE effects at reducing sucrose seeking and taking in a 12-h test immediately following acute EE (Exp. 1), or for 7 days with daily 1-h tests immediately following acute EE, or after a 24-h delay (Exp. 2). We also examined the persistence of acute EE effects on sucrose taking in rats responding on a PR schedule in 7 daily sessions following acute EE (Exp. 3). We found that acute EE was effective at reducing responding for both sucrose and a sucrose-paired cue, persisting throughout the 12-h test (Exp. 1). A reduction in sucrose seeking persisted for 24 h and a reduction in sucrose taking persisted for 72 h following acute EE plus a 24-h delay prior to testing (Exp. 2). Decreased PR responding for sucrose was observed following acute EE; this reduction persisted for 48 h (Exp. 3). These findings indicate that acute exposure to EE has persisting effects at reducing sucrose seeking and taking in rats. Acute EE may have translational value as a non-pharmacological intervention to curb sucrose craving.

1. Introduction

Excessive sugar consumption increases the likelihood and severity of several negative health outcomes including diabetes, heart disease, cancers, and obesity (CDC, 2018; Yang et al., 2014). Despite the threat of these negative consequences (Wiss, Avena, & Rada, 2018), many individuals find it difficult to reduce consumption. Factors contributing to excessive consumption include the reinforcing qualities of sucrose including its sweetness and energy content, and the power of sucrose-rich food-paired cues to induce craving (Wolz et al., 2017; Konova, Louie, & Glimcher, 2018). To better understand how sugar comes to serve as such a potent reinforcer, researchers have examined sucrose craving in non-human models that include dietary and environmental controls not achievable in clinical studies. For example, rats will readily and reliably make an operant response for sucrose, providing a measure of consumption (taking). They will also respond for cues previously associated with sucrose delivery (seeking), providing a measure of “conditioned craving” (Grimm, 2011).

Behavioral pharmacological approaches incorporating seeking and taking models have revealed potential pharmacotherapeutics to reduce sugar-directed behaviors. However, nonpharmacological approaches could provide the benefit of reducing seeking and/or taking without drug side effects. For example, addiction treatments include behavioral-based therapies such as contingency management. This approach reduces drug seeking and taking (McPherson et al., 2018) and improves pro-health outcomes such as healthy eating (Kurti et al., 2016). Unfortunately, non-pharmacological approaches to reduce excessive appetite for sweets are not well-represented in the research literature. Our laboratory found environmental enrichment (EE) to be effective at reducing both sucrose seeking and taking in a rat model of craving. The EE effects at reducing sucrose seeking and taking are generally similar to what has been found in rats trained to self-administer drugs of abuse (Grimm et al., 2013).

Thus far, most findings in this area of research are from studies with an EE duration of several weeks or more. In contrast, we found that a one-time, 22 h EE experience (acute EE), resulted in a robust decrease in both sucrose seeking and taking (Grimm et al., 2013). This brief and effective manipulation in a pre-clinical model should be examined for its potential utility as a non-pharmacological treatment approach to reduce sucrose-directed behavior. In humans, EE could be conceived as opportunities to engage in novel reinforcing activities (Clemenson & Stark, 2015).

We have reported that the acute EE effects are observed in both very early and late abstinence from sucrose self-administration (Grimm et al., 2013; Glueck, Ginder, Hyde, North, & Grimm, 2017; Grimm et al., 2018). However, we have not examined the duration of the sucrose appetite reducing effect in detail. Therefore, we evaluated the persistence of the acute EE effects on responding for the 12 h immediately following acute EE and also over several days following acute EE.

2. Materials and methods

2.1. Subjects

A total of 203 male Long-Evans rats bred in the Western Washington University vivarium served as subjects. Rats were at least 3 months old at the start of the study. Rats were individually housed (Micro-Isolator chambers (20 × 32 × 20 cm; Lab Products, Inc., Seaford, DE) under a 12-h reverse day/night cycle. Lights went off at 0700 h. Food (Purina Mills Inc. nutritionally complete Mazuri Rodent Pellets, Saint Louis, MO) and water were available ad libitum. Both were available throughout the study, except for water deprivation 17 h prior to the first Training session. Operant Training and Testing procedures occurred between 0900–1200 h. Rats were weighed every Monday, Wednesday, and Friday. Our procedures followed NIH guidelines (PHS, 2015) and were approved by the Western Washington University IACUC.

2.2. Operant conditioning apparatus

Operant conditioning chambers were from Med Associates (30 × 20 × 24 cm; St. Albans, VT). Each was equipped with one active, retractable lever to the left of the sucrose reward receptacle. An inactive lever to measure non-directed responding was on the opposite wall. To record locomotor activity, each chamber had four infrared photobeam emitters and detectors positioned in a tic-tac-toe pattern (front beams 10.5 cm from wall; side beams 6 cm from wall; all beams 4.5 cm above bar floor). Chambers also included a red house light, a white stimulus light above the active lever, and a tone generator (2 kHz, 15 dB over ambient noise). Chambers were enclosed in sound-attenuated boxes and were equipped with fans to provide white noise and airflow.

2.3. Procedures

2.3.1. Training

Each session began with illumination of the house light and insertion of the active lever. Rats trained in 10 daily 2-h sessions; active lever presses were reinforced with 0.2 mL delivery of 10% sucrose on a fixed-ratio 1 schedule of reinforcement (FR1). Sucrose delivery was accompanied with a 5 s presentation of the white stimulus light and the 2 kHz tone. For this 5 s and the following 35 s, responses were not reinforced but were recorded (40 s “timeout”). Rats in the progressive ratio (PR) experiment followed the 10 days of FR training with 7 days of responding on a PR schedule of reinforcement following the PR escalation of Roberts, Loh, & Vickers (1989). The active lever was retracted during sucrose delivery on this schedule. A PR session lasted a maximum of 3 h.

2.3.2. Environmental enrichment

In all experiments, rats were pseudo-randomly assigned to treatment conditions immediately following the 10^th day of Training in order to balance training behaviors across treatments. Acute EE included three rats housed together in a multi-level cage with toys, a PVC tunnel, and paper towel sheets (Grimm et al., 2013). Rats were in acute EE 22 h. Control (CON) rats remained single-housed. Acute EE rats were placed into EE soon after completing the final day of Training. Testing was either with active responses producing the tone+light cue only (Seeking) or with sucrose available (Taking). The first day of Testing and duration of Testing varied by Experiment, described below.

2.3.3. Experiment 1.

12-h Testing (Seeking or Taking). Rats were first trained to respond for sucrose on a FR and were then pseudo-randomly assigned to CON or EE housing overnight. Rats were tested the next day in a 12-h session. Twelve h was chosen to provide an extended period of observation of operant responding, beyond the typical 2 or 3-h period in our previous studies. Some rats responded for the tone+light cue only (Seeking, Experiment 1A) and some responded for sucrose as during Training (Taking, Experiment 1B).

2.3.4. Experiment 2.

Daily FR testing for one week (Seeking or Taking). Rats were trained as in Experiment 1 and then pseudo-randomly assigned to CON or EE housing overnight. Some rats began Testing the next day, while others did not begin Testing for another 24 h (DELAY). Rats that had been in acute EE were back in single housing during and subsequent to the delay. Testing consisted of 7 daily 1-h sessions where rats were allowed to respond for just the tone+light cue only (Seeking, Experiment 2A) or for sucrose as during Training (Taking, Experiment 2B).

2.3.5. Experiment 3.

PR Testing for one week (Taking only). Rats were trained on a PR following 10 days of FR training. Following the 7^th day of PR Training, rats were pseudo-randomly assigned to remain single-housed (CON) or were placed into EE overnight. Rats responded for sucrose on the PR for the next 7 days.

2.4. Statistical analyses

For all 3 Experiments, Training data were retrospectively compared across groups to verify that groups did not differ just prior to experimental manipulations. For Experiments 1 and 2, rats that responded in Testing for sucrose or only for the tone+light cue were analyzed separately. For analyses, there were two independent variables for Experiments 1 and 3: TIME and HOUSING. For Experiment 2 there was the additional variable DELAY. DELAY was whether rats started testing the day after Training was completed, or 24 h later. TIME was 12, 5-min bins for Experiment 1 or seven days of Testing (Experiments 2 and 3). HOUSING was whether rats were in CON or EE housing immediately after the final Training session. Data were analyzed using repeated measures analysis of variance (RMANOVA with TIME as the repeated variable). Dependent measures were analyzed separately. For seeking rats, cue deliveries were response-contingent deliveries of the tone+light cue, on the same schedule of reinforcement as during Training. For Experiments 1A and B, 12-h totals for all dependent measures were compared using one-tailed t-tests (CON vs. EE). First h active lever data were collapsed into five-min bins and analyzed using RMANOVA with TIME and HOUSING as independent variables. All 12 h of active lever data were then graphed using 1-min bins. We analyzed the trajectory of responding in the 12^th h of Testing for CON and EE groups using linear curve estimation. These analyses were done to assess whether, despite a large overall difference in accumulated reinforcers, rate of responding necessarily differed between HOUSING conditions after 11 h of Testing. Slopes for each subject were calculated with the 60, 1-min bins. Using RMANOVA we compared slopes of zero (no change in responding over the 12^th h) vs. slopes calculated for each subject including the variable HOUSING. For Experiment 3, data were analyzed similar to Experiment 2 but with no DELAY variable. Finally, for each Experiment initial (pre-experiment) and final (end of experiment) body weights were compared with RMANOVA including the HOUSING variable, REINFORCER (Seeking vs. Taking; Experiments 1 and 2), and DELAY (Experiment 2). Family-wise error for post hoc tests following a statistically significant RMANOVA interaction was reduced by using a Šidák-corrected p value calculated incorporating the total number of comparisons. For all other statistical comparisons, p < .05 was used. Effect sizes are indicated in the Results, with Cohen’s d for t-tests and partial eta squared (η_p²) for RMANOVA. IBM SPSS Statistics 25 was used for all statistical calculations, except t-tests were calculated in Excel 2016 and Cohen’s d values were calculated by hand. Figures were created in Excel 2016. Means ± SEMs are indicated in the text and Figures. The datasets generated and/or analyzed during the current study are available from the corresponding author on reasonable request.

3. Results

Body weights increased over the course of each experiment. There were no pre-existing group differences, nor were there any effects of HOUSING, REINFORCER (Experiments 1 and 2), or DELAY (Experiment 2). Body weights pre/post-experiment were (in grams): Experiment 1, 371.3 ± 5.0/ 386.7 ± 6.5, Experiment 2, 383.1 ± 3.1/ 407.6 ± 3.1, and Experiment 3, 356.6 ± 7.6/ 384.8 ± 5.1. Of the 203 rats that started the study, 21 were removed before final data analyses. Six were removed due to a programming error resulting in lost Testing data and 15 for not meeting a Training criterion of an average of 20 sucrose reinforcers over the final 3 days of Training. Final group sizes are indicated in Supplemental Tables 1–4.

Training data (active and inactive lever, reinforcers, and photobeam breaks) are provided for each Experiment (see below). For Testing data, in Experiments 1 and 2 only active lever responses are indicated in Figures. Experiment 3 also includes reinforcers due to the non-linear relationship between active lever responses and reinforcers on the PR schedule. For brevity and clarity, the data presented in Figures are primarily active lever responses. Active lever responding highlights how acute EE alters behavior directed to produce either a sucrose-paired cue (seeking) or sucrose (taking). To facilitate presentation of main findings, statistical results for Testing (other than slope analyses below, Experiment 1) are presented in Tables 1–4. All Testing data dependent measures are presented in Supplemental Tables 1–4.

Table 1.

Experiment 1. Statistically significant T values for 12 h Test data CON vs. EE.

Experiment	Active Lever	Reinforcers	Inactive Lever	Photobeam
1A. Seeking	4.1 (d = 1.80)	4.2 (d = 1.83)	n.s.	2.5 (d = 1.09)
1B. Taking	5.9 (d = 2.50)	7.4 (d = 3.14)	5.0 (d = 2.13)	1.9 (d = 79)

Note: df = 19; not significant (n.s.), otherwise p’s < .05; Cohen’s d indicated for t-tests.

Table 4.

Experiment 3. RMANOVA results for dependent measures over the 7 days of Testing.

Active Lever	TIME F(6,168) = 9.9, ηp2 = .26 TIME X HOUSING F(6,168) = 4.0, ηp2 = .12
Reinforcers	TIME F(6,168) = 16.1, ηp2 = .37 HOUSING F(1,28) = 5.1, ηp2 =.15 TIME X HOUSING F(6,168) =13.2, ηp2 =.25
Inactive Lever	HOUSING F(1,28) = 4.8, ηp2 = .15 TIME X HOUSING F(6,168) = 2.2, ηp2 = .07
Photobeam	TIME X HOUSING F(6,168) = 3.0, ηp2 = .10

Note: partial eta squared (η_p²) indicated for RMANOVA effects and interactions; all p’s < .05.

3.1. Experiment 1.

The experimental design for Experiment 1 is presented in Figure 1A. There were no differences in Training between groups. Training data are depicted in Figure 1B. For this Experiment, and Experiments 2 and 3, rats selectively responded on the active lever. Inactive lever responding and locomotor activity decreased over Training.

Fig 1.

1A depicts the Experiment 1 timeline. 1B is Experiment 1 Training. 1C is active lever responding in 5-min bins over the first h of Testing in Experiment 1A (Seeking). 1D is active lever responding over the 12 h of Testing Experiment 1A. 1E is active lever responding in 5-min bins over the first h of Testing in Experiment 1B (Taking). 1F is active lever responding over the 12 h of Testing in Experiment 1B. Means ± SEMs indicated. * statistically significant difference from CON, p < .05.

In Experiment 1A (Seeking), CON rats responded significantly more across all measures except for inactive lever responding (Supplemental Table 1). In Experiment 1B (Taking), CON rats responded significantly more across all measures (Supplemental Table 1). Active lever time course Testing data are indicated in Figures 1C, D, E, and F. For Experiment 1A (Seeking), first h cumulative responding (Figure 1C) was reduced by EE. Post-hoc t-tests were calculated comparing cumulative responding between conditions at the 20, 40, and 60 min time points. While p values were all below .05 for 15 min and subsequent time points, only responding at the 40 and 60 min time points were significant at the Šidák-corrected p value cutoff of .017. Over 12 h, cumulative active lever responding was lower in EE-exposed rats early in testing, and for the 12-h duration of testing (Figure 1D). Twelfth h slopes were shallow (CON 0.05 ± 0.14; EE 0.02 ± 0.01), but significantly greater than zero in the 12^th h of responding F(1,19) = 19.0, p < .05, η_p² = .50. Slopes did not differ by HOUSING. These results could be interpreted to mean that by the 12^th h, the EE effect on rate responding was no longer apparent. CON and EE rats were responding at similar, albeit modest, rates. As with Seeking, for Experiment 1B Taking first h cumulative responding (Figure 1E) was also noticeably reduced by EE. P values were all below .05 for 5 min and subsequent time points and post-hoc t-tests were significant at the 20, 40, and 60-min time points using the Šidák corrected p value. Over the 12 h of Testing in Experiment 1B, five rats went to a 270 reinforcer maximum (maximum syringe volume available) after an average of 6.6 h of responding (4.9, 5.8, 6.1, 6.8, 9.6 h). All were in the CON condition. Figure 1F, depicting responding across 12 h, indicates data from the experiment with these rats removed. From this Figure, as in the seeking experiment, cumulative active lever responding was noticeably lower in EE-exposed rats early in testing and for the 12-h duration of testing. Twelfth h slopes of these rats responding for sucrose (not including the five rats noted above) were about eight times steeper than in the first experiment where rats responded for a sucrose-paired cue (CON slope average was 0.05, indicated above) with slopes of CON 0.18 ± 0.06, and EE 0.30 ± 0.12. Slopes were significantly greater than zero in the 12^th h of responding F(1,16) = 9.0, p < .05, η_p² = .36, but did not differ by HOUSING. As with the Seeking results, Taking results could be interpreted to mean that, by the 12^th h of Testing, the EE effect on rate of responding was no longer apparent. CON and EE rats were responding at similar rates.

3.2. Experiment 2.

The experimental design for Experiment 2 is presented in Figure 2A There were no differences in Training between groups. Training data are depicted in Figure 2B. Testing data (active lever) are indicated in Figures 2C, D, E, and F. Data in Figures are organized according to RMANOVA main effects or interactions. Supplemental Tables 2 and 3 indicate all dependent measures across the 7 days of Testing.

Fig 2.

2A depicts the Experiment 2 timeline. 2B is Experiment 2 Training. 2C is Experiment 2A (Seeking) Testing. Figure indicates active lever responding over the 7 days of Testing. 2D is Experiment 2B (Taking) Testing. The Figure indicates active lever responding over the 7 days of Testing. 2E is Experiment 2B (Taking) Testing collapsed across DELAY conditions. 2F is Experiment 2B (Taking) collapsed across HOUSING conditions. Means ± SEMs indicated. For Figure 2C, * indicates statistically significant difference from all other groups on that day. Otherwise, * indicates statistically significant difference from CON on that day (Figure 2E) or between DELAY and NO DELAY (Figure 2F), p < .05.

Acute EE reduced sucrose seeking compared to controls in both rats responding immediately following EE and following a 24 h return to the home cage (DELAY) (Figure 2C). The effect did not persist into the second day of Testing. Therefore, the acute EE effect of decreasing sucrose seeking persists for at least 24 h. The effect of acute EE on reducing sucrose taking was more persistent (Figure 2D). Collapsed across DELAY conditions, responding was reduced for 3 testing days (72 h) (Figure 2E). Inspection of Figure 2D reveals this persistence trended toward being greater in the DELAY condition, however the interaction terms in the RMANOVA including DELAY did not reach statistical significance. The DELAY manipulation itself resulted in greater responding for sucrose (Figure 2F).

3.3. Experiment 3.

The experimental design for Experiment 3 is presented in Figure 3A. There were no differences in Training between groups. Training data are depicted in Figures 3B (FR training) and 3C (PR training). Testing data (active lever and reinforcers) are indicated in Figure 3D. Supplemental Table 4 indicates all dependent measures across the 7 days of Testing. Taking sucrose on a PR schedule of reinforcement was reduced by acute EE for 2 days (48 h). This was indicated by significantly decreased active lever responding and total number of sucrose reinforcers (Figure 3D).

Fig 3.

3A depicts the Experiment 3 timeline. 3B is FR Training; 3C is PR Training. 3D is Testing. The Figure indicates active lever (top) and reinforcers (bottom) over the 7 days of Testing. Means ± SEMs indicated. * statistically significant difference from CON on that day of Testing, p < .05.

3.4. Non-directed measures.

As noted above, dependent measures for all Experiments are indicated in Supplemental Tables 1–4. In all 3 Experiments, locomotor activity was reduced following exposure to acute EE. There was also a locomotor effect of DELAY in Experiment 2, but with delayed testing resulting in a persistent increase in locomotion in rats responding for sucrose and a transient increase in locomotion in rats responding for a sucrose-paired cue. Inactive lever responding during Testing across all Experiments was low. Represented by CON subjects, inactive as a percent of active responding was 24% in Experiment 1A, 8% in Experiment 1B, 7% in Experiment 2A, 5% in Experiment 2B, and 7% in Experiment 3. Even so, acute EE had a general effect of reducing inactive lever responding.

4. Discussion

As we have reported previously, a single 22-h EE experience resulted in decreased responding for sucrose cues and for sucrose itself. In the present study we further established the persistence of these effects: decreased responding was observed for several h when assessed in a single Test session and for several days when tested daily on either FR (seeking or taking) or PR (taking) schedules of reinforcement.

4.1. Experiment 1.

The 12-h Test allowed us to examine if the acute EE effect was limited to the time immediately following the rats entering the testing environment. In our previous studies, we tested rats for no more than 2 h. While we considered the possibility that the acute EE effect would dissipate with prolonged habituation to the testing environment, we instead observed an enduring decreased responding for sucrose cues or sucrose. Absolute seeking and taking was much less over the 12 h following acute EE. The effect appears to persist at least 8 h (Figures 1D and F), but any uptick in responding was minor in the EE-exposed rats by the 12^th h.

4.2. Experiment 2.

We were confident we would observe some persistence following a 24-h delay, as we previously reported that sucrose intake was reduced in a test session the day after a cue-reactivity test just following acute (or chronic) EE (Grimm et al., 2013). However, those results were based on repeated exposure to the operant conditioning chamber. In the present study, the delay manipulation allowed us to examine, without a potential exposure confound, whether immediate contrast between EE and the operant conditioning chamber was necessary for EE to reduce seeking and taking. It was not, as the effects of EE decreasing responding (seeking and taking) were observed in the delay conditions. However, the delay did appear to reduce the effectiveness of EE. In addition, the delay itself increased subsequent responding for sucrose (Figure 2F) and may be indicative of incubation of craving (Grimm, Fyall, & Osincup, 2005). Incubation may actually counter the persisting effects of acute EE. Finally, regarding interpretation of the effects of acute EE on Seeking vs. Taking, the present results likely understate the persistence of acute EE at reducing sucrose seeking. This is because each seeking test is a test of responding in extinction conditions that would be expected to decrease over repeated tests even in CON-housed subjects. Further evaluation of the persistence of acute EE on sucrose seeking would require the use of groups with increasing delays before Testing (e.g. 48 h, 72 h,…).

4.3. Experiment 3.

In Experiment 3, acute EE decreased PR responding for sucrose. The effect lasted for 2 Test sessions (2 days). Responding on the PR schedule of reinforcement has been argued to provide a measure of motivation to acquire a particular reinforcer (Arnold & Roberts, 1997). Therefore, the present findings support an effect of EE reducing motivation to work for sucrose. EE has previously been reported to reduce motivation to respond for psychostimulants. For example, EE-housed rats respond for fewer deliveries of intravenous amphetamine on a PR at a low unit dose (Green, Gehrke, & Bardo, 2002). There was a more persistent effect of EE on FR compared to PR responding for sucrose (Figure 2E vs. 3D). This occurred despite the fact that the FR schedule was much richer compared to the PR. For example, rats in Experiment 3 training on the FR earned almost 9 times as many sucrose deliveries as during PR training (Figures 3B and 3C). The fact that the FR responding decrease outlasted that of PR supports a hypothesis that the EE effect is not simply interrupting the ability of rats to respond. Specifically, they are responding near CON levels on the PR by 3 days post EE (Figure 3D) whereas on the FR, responding did not overlap with CON until 4 days post EE (Figure 2E).

4.4. General Discussion

Only a handful of studies have examined EE as an intervention in adult animals, as was done in the present study. Salient examples include addiction model studies (Chauvet, Lardeux, Goldberg, Jaber, & Solinas, 2009; Thiel, Sanabria, Pentkowski, & Neisewander, 2009) where the EE intervention was chronic, lasting several weeks. Few studies have examined effects of acute (single occurrence) EE on physiological and/or behavioral measures.

For example, Thiel, Engelhardt, Hood, Peartree, & Neisewander (2011) and Theil et al. (2012) included measures of rats responding for a cocaine-paired cue the day following placement into EE. In both studies, cocaine cue-reactivity was significantly reduced following EE. We have examined the acute EE effect somewhat more extensively, including the manipulation in studies examining acute EE effects in early or late abstinence from sucrose, and the related effects on brain regional expression of c-Fos (Grimm et al., 2016), perineuronal nets (Slaker, Barnes, Sorg, & Grimm, 2016), and DARPP32 (Grimm et al., 2018). In a parametric study, we evaluated the effectiveness of acute EE components at reducing sucrose seeking (Grimm et al., 2013). We reported that an acute novelty manipulation (placement in an unfamiliar cage) reduced sucrose seeking vs. controls, but rats placed into EE instead (or in the EE cage without social companions) responded significantly less. While not as effective as “full” EE, novelty is likely a key component of the acute EE effect. For instance, it was reported that placement of rats in a novel environment with enriching objects for 15 min prior to self-administration sessions decreased acquisition of amphetamine self-administration (Klebaur, Phillips, Kelly, & Bardo, 2001). The acute EE effects reported here complement findings from decades ago where 48 h EE exposure increased performance in a food-seeking task (Henderson, 1976) and more recent findings of enhanced cognitive performance following 24 h EE exposure in a traumatic brain injury model (de la Tremblaye et al., 2017).

Persistence of the effects of EE have been examined previously, again primarily in studies using EE as a chronic condition. For example, brains of EE-exposed rats are heavier than controls for weeks following cessation of EE (Bennett, Rosenzweig, Diamond, Morimoto, & Hebert, 1974). Many studies of persistence following chronic EE provided EE early in development as a treatment to possibly counter manifestation of a genetic predisposition (i.e. Alzheimer’s disease model mice; Torres-Lista & Gimenez-Llort, 2015) or negative effects of an experimentally created brain lesion (e.g. Cheng et al., 2012). In those studies, chronic EE tended to have persisting effects at reducing physiological and behavioral indicators of disease or trauma, in some instances persisting for months (Davenport, Gonzalez, Carey, Bishop, & Hagquist, 1976). Even relatively brief EE was found to have some enduring effects in disease models. For example, the improved cognitive outcomes following a 24 h EE manipulation of de la Tremblaye et al. (2017), noted above persisted for 2 weeks. Persistence of behavioral changes following EE in other models has also been examined, with no lasting effects or effects persisting for up to several months. For example, Thiel et al. (2011) found no persisting effects of chronic EE on cocaine seeking in a one week follow-up test. Klippel (1978) reported minimal persistence of EE effects on exploratory and passive avoidance behaviors following chronic EE over a 2-week post-EE period. In contrast, Passig et al., (1996) reported enhanced short and long-term memory abilities assessed in the radial 8-arm and Lashley mazes for 6 months in rats with a history of chronic EE. In addition, Amaral, Vargas, Hansel, Izquierdo, & Souza (2008) observed decreased open field behavior (interpreted as enhanced habituation) for 6 months following chronic EE. The limited evidence for persistence of EE effects following acute EE (e.g. de la Tremblaye et al. and the present study) so far complements previous findings that chronic EE can result in enduring behavioral changes. More research is required to determine what aspects of EE, acute or chronic, best result in persistent changes in behavior.

Variabilities in the effectiveness of brief vs. chronic effects of EE, and the persistence of EE effects on behavior, likely arise from the fact that there are many inconsistencies across laboratories in the operational definitions of EE, outcome measures, and related testing procedures. Species and strain differences also likely contribute to variability. Further research is needed to address these issues and, arguably, adoption of a standard EE methodology is warranted. That being said, the fact that EE affects behavior regardless of the wide variety of approaches supports the robustness of the manipulation.

The key mechanism(s) underlying the EE effect on motivated behavior has been considered in several studies. One theory is that EE has anti-stress effects; these effects are hypothesized to reduce behaviors typically increased by stress, such as drug seeking (Chauvet et al., 2009; Solinas, Thiriet, Chauvet, & Jaber, 2010). While intuitive, the theory has not been consistently supported. For example, one measure of stress responsivity, plasma corticosterone, has not reliably been affected by acute EE in rats (increased: Konkle, Kentner, Baker, Stewart, & Bielajew, 2010; decreased: Thiel et al., 2012; no change: Grimm et al., 2016). In addition, chronic EE has also been found to increase anxiety-related behaviors measured as reduced time sent in open arms of an elevated plus maze (Marianno, Abrahao, & Camarini, 2017). Another theory is that motivated behavior is altered by EE due to a behavioral contrast between EE and the reinforcer and/or the environment where reinforcement occurs (Grimm et al., 2013). This theory aligns with a behavioral economics perspective where EE alters reinforcer valuation such that the value of the target reinforcer (e.g. drug) is reduced (Hofford, Beckmann, & Bardo, 2016). Taken as evidence for such a contrast effect, the present results provide new evidence for an enduring contrast. Future studies are needed to better elucidate whether contrast is a main factor in anti-seeking and taking effects of acute EE, and to identify whether chronic EE has similar effects.

In addition to these potential stress and contrast effects, EE (acute or chronic) has other effects on behavior. For example, as noted above, previous studies described boosts in cognitive performance following EE. Future research is required to clarify the multiple affectual and motivational effects of EE. For example, ultrasonic vocalizations could reveal the valence of emotional state following EE. In addition, site-specific manipulations of brain structures involved in reinforcer valuation (e.g. orbitofrontal cortex) following EE, but prior to seeking or taking, could reveal the contribution of these structures to the anti-seeking or taking effects of EE.

Fitting with a multivariate understanding of EE effects, as reported in the present study and in our previous reports, acute or chronic EE typically reduces not only behavior directed at the lever that produced or produces sucrose but also non-directed behaviors of inactive lever responding and locomotor activity (Supplemental Tables 1–4). These changes in behavior following EE are difficult to explain. If these effects are stress-related, they could indicate either less stress behavior (fast habituation) or more (freezing). In addition, from one perspective the decrease in sucrose intake following EE mirrors behavior observed during stress-induced anhedonia (Strekalova et al., 2011). Further research is required to identify the scope of affectual, cognitive, and behavioral changes necessary for acute EE to reduce sucrose appetite. Regardless, the decreased responding and activity reflects less motivation to explore the sucrose-paired environment and respond for a sucrose-paired cue (seeking) or sucrose (taking).

4.5. Summary and Conclusions

Acute EE reduced rate of sucrose seeking and taking for at least 8 h measured within-session, and for days when assessed daily in 1-h sessions. Motivation to consume sucrose, assessed using the PR schedule of reinforcement, was decreased for 2 days and did not completely recover until after 3 days. Overall, these findings support the need for further investigation of acute EE as an intervention to curb sucrose appetite.

Table 2.

Experiment 1. RMANOVA results for first hour active lever test data.

Experiment
1A. Seeking	TIME F(l1,209) = 39.1, ηp2 = .67 HOUSING F(1,19) = 6.9, ηp2 = .27 TIME X HOUSING F(11,209) = 30.4, ηp2 = .34
1B. Taking	TIME F(l1,231) = 39.2, ηp2 = .65 HOUSING F(1,21) = 30.9, ηp2 = .60 TIME X HOUSING F(11,231) = 28.4, ηp2 = .58

Note: partial eta squared (η_p²) indicated for RMANOVA effects and interactions; all p’s < .05.

Table 3.

Experiment 2. RMANOVA results for dependent measures over the 7 days of Testing.

Experiment	Dependent Measure
	Active Lever
2A. Seeking	TIME F(6,294) = 51.6, ηp2 = .51 HOUSING F(1,49) = 5.2, ηp2 = .10 TIME X HOUSING F(6,294) =15.0, ηp2 = .23 TIME X HOUSING X DELAY F(6,294) = 2.3, ηp2 = .05
2B. Taking	TIME F(6,306) = 9.5, ηp2 = .16 HOUSING F(1,51) = 9.1, ηp2 = .15 DELAY F(1,51) = 9.0, ηp2 = .15 TIME X HOUSING F(6,306) = 9.7, ηp2 = .16
	Reinforcers
2A. Seeking	TIME F(6,294) = 32.9, ηp2 = .40 TIME X HOUSING F(6,294) = 9.6, ηp2 = .16 TIME X DELAY F(6,294) = 3.1, ηp2 = .06
2B. Taking	TIME F(6,306) = 14.8, ηp2 = .23 HOUSING F(1,51) = 11.0, ηp2 = .18 DELAY F(1,51) = 10.8, ηp2 =.18 TIME X HOUSING F(6,306) = 11.3, ηp2 = . 18
	Inactive Lever
2A. Seeking	HOUSING F(1,49) = 5.5, ηp2 = .10
2B. Taking	TIME F(6,306) = 2.3, ηp2 = .04 HOUSING F(1,51) = 4.5, ηp2 = .08 TIME X HOUSING F(6,306) = 2.3, ηp2 = .04
	Photobeam
2A. Seeking	HOUSING F(1,49) = 5.8, ηp2 = .07 TIME X HOUSING F(6,294) = 3.7, ηp2 = .1 TIME X DELAY F(6,294) = 2.5, ηp2 = .05
2B. Taking	TIME F(6,300) = 9.7, ηp2 = .16 HOUSING F(1,50) = 13.7, ηp2 = .21 DELAY F(1,50) = 4.6, ηp2 = .08 TIME X HOUSING F(6,300) = 3.7, ηp2 = .07

Note: partial eta squared (η_p²) indicated for RMANOVA effects and interactions; all p’s < .05.