Enriched environment exposure accelerates rodent driving skills

Journal Pre-proof Enriched Environment Exposure Accelerates Rodent Driving Skills L.E. Crawford, L.E. Knouse, M. Kent, D. Vavra, O. Harding, D. LeServe, N. Fox, X. Hu, P. Li, C. Glory, K.G. Lambert

PII:

S0166-4328(19)31176-3

DOI:

https://doi.org/10.1016/j.bbr.2019.112309

Reference:

BBR 112309

To appear in:

Behavioural Brain Research

Received Date:

29 July 2019

Revised Date:

14 October 2019

Accepted Date:

15 October 2019

Please cite this article as: Crawford LE, Knouse LE, Kent M, Vavra D, Harding O, LeServe D, Fox N, Hu X, Li P, Glory C, Lambert KG, Enriched Environment Exposure Accelerates Rodent Driving Skills, Behavioural Brain Research (2019), doi: https://doi.org/10.1016/j.bbr.2019.112309

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Enriched Environment Exposure Accelerates Rodent Driving Skills

L.E. Crawford, L.E. Knouse, M. Kent, D. Vavra, O. Harding, D. LeServe, N. Fox, X. Hu, P. Li, C. Glory, & K.G. Lambert * [email protected]

Dept. of Psychology, University of Richmond, VA USA, 23173

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*Correspondence and requests for materials should be addressed to Kelly Lambert at

Rats can learn the complex task of navigating a car to a desired goal area. Enriched environments enhance competency in a rodent driving task. Driving rats maintained an interest in the car through extinction. Tasks incorporating complex skill mastery are important for translational research.

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ABSTRACT: Although rarely used, long-term behavioral training protocols provide opportunities to shape complex skills in rodent laboratory investigations that incorporate cognitive, motor, visuospatial and temporal functions to achieve desired goals. In the current study, following preliminary research establishing that rats could be taught to drive a rodent operated vehicle (ROV) in a forward direction, as well as steer in more complex navigational patterns, male rats housed in an enriched environment were exposed to the rodent driving regime. Compared to standard-housed rats, enriched-housed rats demonstrated more robust learning in driving performance and their interest in the ROV persisted through extinction trials. Dehydroepiandrosterone/corticosterone (DHEA/CORT) metabolite ratios in fecal samples increased in accordance with training in all animals, suggesting that driving training, regardless of housing group, enhanced markers of emotional resilience. These results confirm the importance of enriched environments in preparing animals to engage in complex behavioral tasks. Further, behavioral models that include trained motor skills enable researchers to assess subtle alterations in motivation and behavioral response patterns that are relevant for translational research related to neurodegenerative disease and psychiatric illness. The most common behavioral tasks used to assess cognitive processes in neuroscience research include operant conditioning, passive avoidance tasks, spatial tasks such as the Morris Water Maze and the Barnes Maze, and object recognition tasks [1]. Although informative, these 1

tasks are rather simple, capturing a narrow window of the animals’ potential behavioral response repertoire that is characteristic in their natural habitats. [2]. The unpredictability and response complexity of the wild rat was previously elucidated when a rat fitted with a tracking device was released on a New Zealand island to monitor the navigation of the animal around the island. To the researchers’ surprise, the rat evaded capture by a seasoned research team for 18 weeks and ultimately swam 400 meters across open waters to another island. Laboratory observations of rodent responses failed to reveal the rats’ diverse arsenal of sophisticated maneuvers to avoid being captured by a team of experienced field researchers [3]. These observations emphasize the

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value of assessing goal-oriented behaviors that provide opportunities to observe varied responses

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in the animals’ pursuit of desired outcomes.

Although advanced cognitive abilities have been demonstrated in rodents [4], it is

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important to evaluate trained motor skills (generally defined as an acquired ability to successfully perform a task) incorporating integrative neural functions in laboratory investigations.

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example, previous rodent research utilizing the trained skill of reaching for food through a

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challenging barrier has been successfully used to determine neural functions and effective therapeutic strategies for recovery from brain damage [5]. According to human fMRI data, as an

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individual shifts from a naïve to expert responder, neural patterns underlying the same behavioral outcome shift in interesting ways that require the recruitment of varying brain areas to continue to execute the skilled response [6]. The utilization of additional animal models of skilled goal-

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oriented responses is necessary to further elucidate the neurobiological correlates of skill mastery. The likelihood of observing complex, self-initiated behavioral responses in laboratory animals is enhanced by enriched environments that, although not entirely comparable, provide a closer match to the animals’ natural environment than more traditional laboratory habitats [7, 8,

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9]. Environmental enrichment has been shown to affect rat learning performance in spatial tasks, as well as enhance hippocampal complexity and emotional resilience [10, 11]. In humans, a large-scale study of mental health in Denmark indicated that childhood exposure to natural green space affected mental health with an associated 55% lower rate of mental illness [12]. Thus, various forms of enriched environments provide opportunities for frequent spontaneous movements leading to mastery over environmental challenges and heightened resilience against the onset of psychiatric illnesses [13, 14] and cognitive decline [15]. 2

In the current study, animals trained in the complex task of driving a specially-designed rat-operated vehicle (ROV), a task that offers opportunities to investigate causal and therapeutic agents in translational models of learning and mental health, exhibited evidence of advanced skill acquisition. Initially, the ROV was designed so that rats could be systematically shaped to drive to a desired food reward in a forward path (See Figure 1a for depiction of ROV). Additionally, we established that rats could be trained to use varying movement sequences to steer the ROV in different directions to arrive at the reward station from different start positions. Thus, our preliminary work with both forward driving and complex steering navigation confirmed that the

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driving response could be shaped and maintained in laboratory animals. Consequently, rats

were housed long-term in a complex, enriched environment and subsequently exposed to the

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forward driving regime to determine the influence of the animals’ habitat on the acquisition of a complex skill. Drawing from research consistently confirming enhanced neuroplasticity in

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animals exposed to enriched environments [10, 16], it was hypothesized that the enriched animals would acquire robust driving skills (defined as the completion of four consecutive

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driving trials following initial fundamental training in the ROV) in comparison to rats housed in standard laboratory cages.

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Young adult male Long-Evans rats that had lived in an enriched environment for four months (n = 5) and age-matched control animals raised in standard laboratory housing (n = 6) were used in the current study. The animals were bred in our laboratory from animals sourced

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from Envigo (Dublin VA USA). All animal procedures were approved by the University of Richmond Institutional Animal Care and Use Committee. During the environmental enrichment exposure (which began at approximately 3.5 weeks of age), animals were group-housed (n=5) in a single 60 cm L x 90 cm W x 60 cm H cage containing multiple levels of living surface; additionally, approximately six natural (e.g., piece of wood) and artificial (e.g., plastic ball)

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objects were changed weekly. The control animals were pair-housed in standard housing environments (34 cm L x 28 cm W x 14cm H single-level cage). Driving training began when the animals were approximately 5 months of age. The driver

compartment of the ROV was a plastic container with an aluminum floor plate and cut out windows spanned by copper bars. The ROV was designed so that the rat could move the car by touching or grabbing a bar and stop movement by releasing contact. Specifically, the car was built from a modified ELEGOO EL-KIT-012 UNO Project Smart Robot Car Kit V 3.0. A one3

gallon clear plastic food container with a white plastic lid served as the cab of the ROV. The cab was adhered to the ELEGOO chassis such that the bottom of the container served as the front face of the car, and the opening of the container served as an entry for the rats. An aluminum plate was placed on the inside of the bottom of the cab and connected to the ground lead from the battery. A rectangular 8.5 cm x 7.5 cm hole was cut into the bottom of the plastic container to create a window at the front of the car. Copper wire was threaded horizontally across this window to form three bars, and the wire was connected to the power lead from the battery. When a rat positioned itself on the aluminum plate and touched the copper wire, a circuit was

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completed that sent power to a micro-controller, which then caused the motors to turn and the car to move forward (as shown in Figure 1A).

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We used operant conditioning (shaping) to train the rats to touch the bars and move the car forward after they voluntarily entered the ROV. Training took place in a 150 cm L x 60 cm

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W x 50 cm arena composed of black acrylic plexiglass. A black and white checkerboard pattern stimulus (56 cm W x 43 cm H) was placed at the target end of the arena to provide a visual

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discrimination for the destination target area. A wire hook apparatus with the end covered in a nitrile exam glove served as the reward dispenser (similar to the gloved hands of the

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experimenters). Miniature marshmallows were attached to the end of the hook as food safe adhesives for the reinforcer (0.25 of a Froot Loops® cereal piece). Importantly, the reward dispenser was only placed at the end of the arena during the final stages of training and animals

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were never permitted access to the reinforcer outside of the ROV. Prior to the actual training trials, a plastic container similar to the one used as the car shell was placed in the animals’ home cages for approximately one week to allow the rats to habituate to the novel stimulus. Additionally, froot loop treats were presented to the rats during the week prior to training to habituate them to the novel food reward.

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As training progressed, increasing distances of driving were required to receive a food

reward from the hand-guided reward dispenser. In pilot work, two young adult male rats (approximately 65 days old) learned to drive forward a distance of about 110 cms after about a month of training sessions (consisting of at least four 15 minute sessions per week). In the current study, animals were trained three times weekly for eight weeks (individual trials were approximately 5 minutes in this study), during which time the rats’ latency to enter the car, contact the driving bars to move the car, and completion of a full drive to the reward station were 4

recorded. Following the last training week, the animals were trained for three additional weeks and were subsequently assessed in the training arena during four extinction trials (on four separate days, with one-two days between trials); specifically, the rats were observed in the driving arena with no opportunity for acquiring a reward to determine the robustness of the response during extinction conditions. To investigate the effect of an enriched environment and training on emotional/endocrine responses, fecal samples were collected prior to training (baseline), mid-way through training

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and during the extinction phase so that corticosterone (CORT) and dehydroepiandrosterone (DHEA) metabolites could be quantified. For fecal sample collection, each animal was

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individually placed in a clean cage until the animal emitted a fecal bolus (typically occurring within one-two minutes). Upon collection, the animal was returned to its home cage and each

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bolus was placed in a centrifuge tube and stored at -80 until the assays were conducted. Hormones were extracted from fecal samples by homogenizing the sample in 100% methanol as we have previously reported [17]. Subsequently, a sample dilution of 1:20 was made

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using an assay buffer (Enzo Scientific, Farmingdale, NY). The resulting samples had a sensitivity of 27 pg/ml within a range of 32-20,000 pg/ml (CORT) and 12.21-50,000 pg/ml

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(DHEA). Next, the standard protocol of an ELISA kit (Enzo) was followed. Density readings were obtained from an automated microplate reader (BioTek, Winooski, VT) and read with the Gen5 software (v. 3.02.1) (BioTek). Readings were made at a wavelength of 405λ with

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correction at 490 λ. The % cross-reactivity of the assay was 100% with CORT and 100% with DHEA. In the case of CORT, the assayed standards generated a line with a correlation coefficient of .99. The intra-assay % CV for corticosterone was 6.6 (high) and 8.0 (low), while the inter-assay % CV was 7.8 (high) and 13.1(low). In the case of DHEA, the assay standards generated a line with a correlation coefficient of 0.98. The intra-assay % CV for DHEA was

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4.8(high) and 6.4(low) while the inter-assay % CV was 6.5 (high) and 8.4 (low). The crossreactivity for other steroids determined by Enzo was 0.28% for tetrahydrocorticosterone and less than 0.03% for 11-dehydrocorticosterone acetate, two steroids specifically found in fecal matter. As hypothesized, the enriched animals exhibited more robust driving performance. Independent samples t-tests indicated a significant difference between environment groups in trials to criterion for the first car entrance, t(9) = 5.793, p = .001, d = 3.665. Due to a ceiling effect, Mann-Whitney U tests indicated a significant difference between housing groups in trials 5

to criterion for both 8 bar touches, U = 4, p = .035, and 4 full drives, U = 3, p = .011 (See Figure 2 A for driving training results). In the fourth extinction trial, the enriched animals continued to exhibit more interest in the ROV by exhibiting shorter latencies to enter the car in the absence of food rewards (U=0, p=.006; See Figure 2B). Thus, considering all of the driving results, the data indicated a poorer performance in the standard-housed animals that extended through extinction. No environment effects were observed in the endocrine data; however, a repeated2

measures ANOVA indicated a significant time effect (F(2,18)=9.126, p=0.002, pn =0.503) with

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baseline values observed to be lower than the driving and extinction samples (p=.023 and .005, respectively); additionally, a nonsignificant trend indicated that the extinction values were also higher than the driving phase values (p=.058). Thus, regardless of the environment the animals

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were housed in, the effects of training seemed to alter the DHEA/CORT metabolite ratio in a

pattern consistent with emotional resilience [i.e., higher DHEA levels in comparison to lower

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CORT levels; 11, 18]; See Figure 2C.

Although rodents have been shown to navigate virtual environments by running on

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moving platforms [19] and intermittently driving a car on a circular track [20], the current findings demonstrate that rats can learn to control a machine for locomotion and spatial

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navigation in complex ways. Further, in the preliminary steering trials, the rats’ self-correcting responses while steering the ROV in right, left, or forward directions toward the goal (located 140 cm from the startpoint in a larger arena), regardless of their starting position, indicated fine-

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tuned responsiveness to the changing demands of the spatial context (see Figure 3 for steering patterns when the car and reward station were changed from the consistent positions utilized during training; essentially, the rats grabbed the bars in the same position they needed to steer). Consequently, these preliminary findings from six female rats housed in standard housing conditions suggest that rats are capable of highly complex skill acquisition when given extensive

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training (i.e., approximately nine months in this case) with protocols requiring self-correction for reinforcement.

Considering the progress demonsrated by the enriched trained animals in the driving

task, the use of long-term highly skilled reponses may be informative in behavioral neuroscience research. Interestingly, past research indicates that even simple motor tasks (i.e., finger tapping) in humans recruit large portions of the cerebral cortex in order to incorporate the planning and execution of the task [21, ]; consequently, motor tasks that extend beyond simple stimulus6

response tasks are necessary to provide meaningful assessments of sophisticated actions in changing contexts [22]. Additionally, it is important to consider the effect of mastery over physical tasks; for example, formula one racecar drivers develop stronger neural connections and enhanced information integration than control participants who do not possess highly skilled driving abilities [23]. Accordingly, the use of behavioral training protocols such as the ROV driving regime offer opportunities for dissecting various aspects of decision-making and task performance in predictable and unpredictable conditions. Across an animal’s lifespan, the accumulation of informative experiences, whether they

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are driving skills or another skill set, likely contribute to emotional resilience, providing buffers against subsequent neural threats and challenges [24]. Interestingly, the enriched rats’ persistent

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interest in the car, evidenced by increased entries during the extinction phase of training in the absence of food rewards, suggests that the enriched animals may have developed a more engaged

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reward system throughout training. Further, because cognition and motoricity are functionally interconnected, incorporating several brain areas including, but not limited to, the frontal cortex,

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basal ganglia and cerebellum [25], motor tasks such as the driving task provide opportunities to explore precise mechanisms and developmental trajectories of specific neural conditions such as

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neurodevelopmental and neurodegenerative diseases. More broadly, investigations of complex skill acquisition in the controlled laboratory environment provide opportunities to investigate changing adaptive responses critical for survival in dynamic natural environmental contexts

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such as rapidly changing urban landscapes.

As previously established, an animal’s housing environment has a significant impact on neural functions [10,11]. In the present study, the positive impact of an enriched environment was observed to extend to the acquired driving response. In the past, our laboratory has focused on behavioral responses considered to be ecologically relevant for rodents; however, in this case,

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the complex driving task was viewed as a model for human-machine interactions such as driving a car or operating other technological devices. Because humans in some cultures spend considerable time engaged in these types of responses, such models are necessary for translational research focused on learning and skill acquisition. Although it was hypothesized that the standard housed animals would not be as efficient in the learning process, we were surprised at their lack of interest and underachieved mastery of the driving task. It will be interesting to determine if future investigations with this task, preferably with increased numbers 7

of animals, will reveal similar results. Unique to this study was the extended duration of environmental enrichment exposure as the animals were housed in their respective environments for almost four months prior to their driving training. These results suggest that rats exposed to rich and diverse environments with an opportunity for more physical interactions demonstrate enhanced achievement of complex behaviors, likely due to a diverse portfolio of potential behavioral responses. However, because the driving training altered the DHEA/CORT ratio in a positive fashion, it appears that the training sessions may have served as an enriched environment of sorts for all the animals, regardless of their assigned laboratory environment

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condition. This finding is consistent with previous research indicating positive effects of

behavioral training on emotional resilience [17, 26]. Because behavior in natural contexts is

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influenced by the accumulation of varied experiences encountered throughout one’s life, both the extended enriched environment and training protocol seemed to provide animals with an

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opportunity to accrue more extensive experiential and cognitive reserves than typically gained in laboratory environments. This skill acquisition model provides preclinical translational

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opportunitites for the investigation of experience-based functional topologies of neural networks necessary for adaptive responses—both natural and acquired. Further, the complex skill set

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associated with the ROV driving model provides a novel approach for the assessment of relevant questions in translational neuropsychiatric research such as the roles of self-efficacy,

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emotional resilience, and individual variation in uncertain and challenging contexts [17, 27, 28].

End Notes:

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Acknowledgments: The authors acknowledge the contributions of Thad Martin and Matthew B. Crawford for technical and engineering support, as well as support from the University of Richmond School of Arts and Sciences, University of Richmond Department of Psychology, and the MacEldin Trawick Endowed Professorship of Psychology (awarded to LEC). Conflict of Interest: The authors report no conflict of interest. References

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Figure 1. Environment, Rodent-Operated Vehicle (ROV) and Training Protocol. (A) The key elements of the ROV are shown, (B) prior to and during driving training, rats were either housed in an enriched environment as shown, or standard rodent laboratory cages, (C) Following habituation to the ROV shell, rats were shaped to engage in behaviors to drive the ROV.

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Figure 2. Driving and Endocrine Data. (A) Training trials to criterion for each experimental phase by animal housing status. Dashed lines represent data from individual animals and solid lines represent the group average. Note that not all animals achieved all criteria; individual data lines truncate to the final criterion met by each animal. Phase labels with an asterisk (*) designate statistically significant differences (B) Enriched animals exhibited a shorter latency to th enter the car at the end of the extinction trials (i.e., 4 trial) and (C) DHEA:CORT ratio across all study animals at each of three time points; baseline values were significantly lower than driving and extinction values and a nonsignificant trend was observed between driving and extinction values.

Figure 3. Preliminary Steering Investigation. (A) The large driving

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arena (140cm x 280 cm x 51 cm) is depicted with the checkered grid indicating the location of the reward station and the solid car outline indicating the typical orientation of the car during training. During training, animals steered straight to the target (B); however when the ROV was positioned away from the reward station (dotted line ROV in diagram), the animals exhibited a strategic turn by grasping bars to the right or left to orient to the reward (C) and, in the most challenging task, when the reward station was repositioned to the opposite side of the driving arena (dashed B circle in diagram on left), the animals initially drove to the previous location of the reward station before eventually orienting and driving to the new location (D). Path lines depict representative paths of all six female rats used in this preliminary study.

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