The impact of environmental enrichment in laboratory rats—Behavioural and neurochemical aspects

Abstract

The provision of environmental enrichment (EE) for laboratory rats is recommended in European guidelines governing laboratory animal welfare. It is believed the EE implementation can improve animals’ well-being and EE has been used to demonstrate learning and plasticity of the brain in response to the environment. This review suggests that the definition and duration of EE varies considerably across laboratories. Notwithstanding this, some EE protocols have revealed profound effects on brain neurochemistry and resulting behaviour, suggesting that EE can have the potential to significantly modify these parameters in rats. For this review, a literature search was conducted using PubMed and the search terms “Environmental Enrichment” and “rats”. From the results of this search the most important variables for consideration in the implementation of EE are identified and summarised, and include cage size and housing density; rat age, sex and strain; duration of EE; the EE protocol and enrichment items employed; and the use of appropriate controls. The effects of EE in a number of behavioural tests and its effects on neurotransmitters, neurotrophic factors, stress hormones and neurogenesis and proliferation are outlined. The findings summarised in the present review show the range of EE protocols employed and their effects in tests of activity, learning and affect, as well neurochemical effects which mediate enhanced plasticity in the brain. EE, as is provided in many laboratories, may be of benefit to the animals, however it is important that future work aims to provide a better understanding of EE effects on research outcomes.

Introduction

Environmental enrichment (EE) is a term for exposing laboratory animals to physical and/or social stimulation that is greater than they would receive under standard housing conditions [1]. EE can be divided into two types: physical and social enrichment. Physical enrichment strategies involve structural modifications, including increased floor space and the inclusion of features which allow exercise, play, exploration and permit animals some control over their environment [2]; these include (in the case of rats) bedding enhanced with natural materials (paper parchment, fibre-based bedding), plastic tunnels, wooden objects to gnaw, ropes, swings, running wheels, balls, ramps, ladders and other appropriately sized animal toys. Social enrichment on the other hand refers to housing social animals in groups wherever possible. Although social enrichment is probably easier to implement with cagemate(s) being a source of constant dynamic interaction and unpredictability, there are situations where animals must be housed alone and in such cases physical enrichment is particularly useful. Ideally, a combination of both social and physical enrichment elements are thought to be preferable [3].

Animals have been held in captivity for scientific purposes for almost 200 years [4]. However it was many years later before the impact of being held under such conditions on the animal's welfare became a concern. In the 1920s, the primatologist Robert Yerkes was a pioneering force behind improvements to the housing conditions of non-human primates in both zoos and laboratories. His lead was followed by many others as the century progressed. Increasing public pressure in the 1960s called for more naturalistic enclosures for zoo animals, which led to the issuing of guidelines and legislation to improve the welfare of all animals held in captivity, encompassing not just zoos, but also farms and research laboratories [5]. Sustained public concern and pressure has meant that there has been a growing obligation for the widespread implementation of EE policies in animal laboratories. Such concerns led, in 1997, to the EU Commission issuing a resolution on the accommodation and care of laboratory animals, which defined “enrichment” in terms of social interaction, activity-related use of the space and provision of appropriate stimuli and materials (Council of Europe, ETS 123 1997). In 2007, the Commission issued guidelines for the “Accommodation and Care of Animals used for Scientific and other Purposes”, which included a section entitled “Housing, enrichment and care”. As well as recommending the provision of social enrichment for rodents, the guidelines also encouraged that appropriate physical enrichment should be made available, such as wood sticks for gnawing, nesting materials, refuges, tubes, boxes and climbing racks (Council of Europe, 2007/526/EC).

The importance of enrichment for laboratory rodents was first formally recognised by Donald Hebb in the 1940s who found that laboratory rats given freedom to roam in his home as pets had superior problem-solving and learning abilities than rats housed in standard laboratory conditions [6]. In their natural environment, as in captivity, rats engage in nocturnal activity, nest building and burrowing, coprophagia (consumption of faeces), thigmotaxis (preference for the periphery of a novel environment), foraging and gnawing; however in captivity rodents exhibit maladaptive stereotyped behaviours, such as barbering (excessive grooming causing hair and whisker loss) and bar-biting [2]. One reason for a growing interest in EE is that such maladaptive behaviour repertoires can interfere with studies where the research aims to monitor ethologically an animal's natural behaviour; perseverance and stereotypies may affect learning and/or conditioning studies which depend on an animal's adaptability [7]. If one wishes to observe natural activity, inasmuch as possible, in behavioural experiments it is imperative to develop methods of biologically appropriate environmental complexity for animals and try to reduce such stereotypies. Another reason for the steady growth of EE as a discipline since the 1960s is the growing evidence that rodents reared in enriched conditions display a range of plastic responses in the brain including neurogenesis, increased dendritic branching, increased cell size and improvements in learning and memory [8], [9], [10], which have implications for recovery in animal models of neurodegenerative disease, brain injury and psychiatric disorders [11], [12], [13], [14], [15]. In such studies EE is introduced as an experimental variable and can contribute to assessing the external validity and robustness of various models as they are tested across a range of environmental conditions [16]. The use of EE as an experimental variable however requires standardisation of procedures and outcome measures if conclusions are to be valid and not simply an artefact of a particular laboratory [17].

A common theme in the area of EE is its inconsistent nature across scientific literature; furthermore the definitions of EE can vary greatly according to different authors, as some are vague and overly general [18]. The mandatory introduction of EE is generally said to be to improve animal welfare, however this itself is a difficult subject to measure, Wurbel argues that introducing shelters to the cages of male mice can increase stress for the less dominant cagemates [19]. Furthermore, the unintended consequences of EE on behavioural, neurochemical and physiological endpoints [20] may make it difficult for laboratories to compare findings to those seen prior to the implementation of EE. Similarly the lack of consensus surrounding EE makes comparisons both within- and between laboratories challenging. This would suggest the need for standardisation of EE in order to produce replicable results. Wurbel however warns against excessive standardisation as it may actually contribute to spurious results and does not necessarily guarantee reproducible results and that some variability is desirable as representative of a diverse population [21]. A concern for behavioural scientists however is what effect EE introduction will have on experimental outcomes in studies where environment is not a variable of interest, and how this can be comparable within and across laboratories [20]. To protect animal welfare, EE must be incorporated without increasing variability which would increase the number of animals required in studies, and this can only be achieved by standardisation of procedures. The focus of this review is the variability of EE procedures used for rats in scientific literature and whether a consensus toward a standardised protocol is identifiable.

In drafting this review, the search terms “Environmental enrichment” and “rats” were entered into the PubMed search engine and the results yielded 536 articles. After careful review, the article list was filtered to leave only 361 articles addressing the effects of environmental enrichment in rats. The number of articles and decade of publication between 1960 and 2009 are presented in Fig. 1. Immediately the intensity of the research field of EE is evident, particularly over the last decade.

The aim of this review is to outline a number of variables involved in assessing EE for rats, which include; types of EE and types of controls, rat strain and gender, and EE duration. In addition, this review examined the parameters investigated using EE, such as the commonly used behavioural tests, neurochemical and neuroanatomical aspects.

Behavioural studies in EE research are fraught with inconsistencies due to the aforementioned variables such as duration and type of EE, animal age, strain and gender and the use of controls. Some of the behavioural tests commonly employed in EE experiments and referred to in this review are outlined below and summarised in Table 1.

The open field test (OFT) consists of a large, high-walled arena which is often used to monitor rats’ behaviours. This test exploits rats’ natural tendency to display thigmotaxis, which refers to their preference to explore the periphery of a novel environment. The OFT can be used to measure anxiety by quantifying the time spent in the inner arena and/or the outer arena (periphery). This is one of the most common behavioural tests used to investigate the effects of EE on locomotor activity and habituation in rats.

The elevated plus maze (EPM) is a widely-used behavioural measure of anxiety. It consists of a 4-arm maze elevated ∼50 cm from the floor; two of the arms are open and two are enclosed. Anxiety is measured by the preference of rats for the closed arms over the open ones. Rats who have been administered anxiolytic drugs will make more entries to the open arms and spend more time on the open arms than controls [22].

The forced swim test (FST) [23] is test of behavioural despair and is used to assess the anti-depressant activity of compounds. During the test, rats are placed in glass cylinders, filled with water (23–25 °C); the water level in the cylinders must be high enough to prevent the animal from touching the bottom of the cylinder with their paws or tail, and low enough to avoid an escape through the top opening of the cylinder. The animals are thus forced to swim in the cylinders. The test consists of a 15-min pre-test followed 24 h later by a 5-min test. The “non-depressed” rat will try to escape (as evidenced by swimming and climbing behaviours) whereas the “depressed” rat adopts a posture of immobility, which is indicative of despair and hopelessness.

The social interaction test can be used to measure anxiety without the use of food or water deprivation or electric shock and it does not require training of the animal [24]. Pairs of rats (same sex, of similar weight) are placed in a clean cage/monitoring arena for 10 min and their behaviour is observed on a television monitor. The time spent in active social contact are later scored for behaviours such as sniffing, grooming, following, mounting, boxing, wrestling, jumping on, crawling under or over the partner [25]. Increased anxiety is reflected in decreased social interaction.

The holeboard test provides independent measures of motor activity and exploration. The apparatus usually consists of a wooden or Perspex box approximately 3600 cm² with four holes, 3–4 cm in diameter, equally spaced in the floor. Each rat is placed in the centre of the holeboard for a 5–10-min trial and at the end of each trial any faecal boluses are removed and the box is wiped clean with a damp cloth. Behaviours recorded are the number of head dips, the time spent head-dipping, locomotor activity in the internal and external areas of the box, and the number of rears [26]. As a measure of emotionality, reduced head-dips and locomotion in the central arena suggest increased anxiety.

The Morris water maze (MWM) [27] is a behavioural test of spatial learning and memory. The rat is placed into a pool of water and the time it takes to find a hidden escape platform is recorded. Over time most rats learn the location of the platform and the time taken to find it decreases as acquisition trials progress. Following the acquisition phase a probe trial is carried out; here the platform is removed and the time spent in the target quadrant (i.e. where the platform was throughout training) is recorded.

The novel object recognition (NOR) test is also used to assess spatial learning and memory in rats. This is based on evidence that rats spend more time exploring a novel object than one previously explored [28]. In the first phase of the test the rat is placed in a novel arena with the sample (familiar) object. Following this exposure, the rat is returned to the homecage for a retention period. In the second exposure, the rat is placed in the arena with the sample object and a novel object. Object recognition is then defined in the second exposure by more time spent interacting with the novel object [29].

The radial arm maze (RAM) is a test of spatial working and reference memory. The maze is comprised of eight horizontal arms (57 cm × 11 cm) are placed at a 360° angle, forming a circle around an elevated central platform 80 cm above the floor. A Plexiglas wall, ∼25 cm high, surrounds the central platform. Each of the arms are 87 cm long and 10 cm wide and have a 5-mm-deep hole 1 cm from the end, which is used as a food cup. In addition, each arm is separated from the centre platform by a transparent Plexiglas guillotine door which can be raised or lowered to allow or prevent entry. The guillotine doors are connected by individual strings to a pulley system that allows the experimenter to open any door from one location within the testing room. Short walls (2 cm high) along the edge of the maze arms prevent the animal from falling off the maze [30]. The radial maze protocol consists of three consecutive phases: habituation (3 days), the learning task (15 days maximal) and the test task (6 days). Rats are trained every day, once per day. Each rat is placed on the central platform with all doors closed. After 30 s, all doors are opened, and the maze could be visited during 10 min. Each rat's activity was recorded and experimenter scored. Data considered are (i) arm entries and their order; (ii) total trial time; (iii) first entry latency. With those data, the number of working memory (WM) errors was counted. Every entry in an already visited arm was considered as a WM error. A non-visited arm was also considered as an error. Animals were classified according to their performances—the number of visited arms, the number of WM errors, latency to first error and finally, by total trial time [31].