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Jul 2018

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Enriched Environment Procedures for Rodents: Creating a Standardized Protocol for Diverse Enrichment to Improve Consistency across Research Studies
啮齿动物的环境丰容程序:创建提高研究一致性的多样化富集标准化协议   

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Abstract

Exposure to environmental enrichment has beneficial effects on learning and memory, diverse neurobiological effects, and promotes recovery of function after brain injury. The effect of enrichment is produced by a combination of increased social interaction, physical activity, spatial complexity, and novelty. Procedures in the literature have, however, been idiosyncratic with poor consistency in the manner or extent to which protocols provide consistent enrichment. We provide an environmental enrichment protocol that can be easily replicated with minor details determined locally so that animals across cohorts and cages all experience a comparable level of enrichment. Procedures are outlined to generate and use a daily pool of suitably varied objects using a standardized format, with objects systematically varied up to a 40-day continuous period. Together with using a large group of rats in a suitably-sized cage, and regular shifting of the position of food and water and cage location, these procedures have produced robust effects in different laboratories and rat strain, thereby improving comparisons within and across laboratories. Non-enriched comparisons can vary, but typically would include grouped animals in standard laboratory housing without objects and with stable food and water locations. Enrichment is a safe non-pharamacological tool to examine behavioral and neurobiological processes in animal models of the lifespan, brain dysfunction and injury.

Keywords: Enrichment (富集), Environmental enrichment (环境丰容), Enriched environment (丰富环境), Recovery of function (功能恢复), Cognitive reserve (认知储备), Novel objects (新奇目标), Social stimulation (社会刺激), Rat (大鼠), Mouse (小鼠)

Background

Enriched environments are a proven therapeutic tool to ameliorate cognitive and affective deficits associated with age-related decline and after many types of brain injury (van Praag et al., 2000; Will et al., 2004; Nithianantharajah and Hannan, 2006; Pang and Hannan, 2013; Alwis and Rajan, 2014; Hannan, 2014). This includes improved cognitive reserve, recovery from relatively diffuse brain injury, such as stroke, traumatic brain injury and various neurodegenerative diseases (Johansson, 2004; Pang and Hannan, 2013; Alwis and Rajan, 2014; Hannan, 2014), as well as from acute localized lesions in cortex, hippocampal formation and thalamus (Will and Kelche, 1992; Will et al., 2004; Harland et al., 2014; Dalrymple-Alford et al., 2015). These effects are likely to be mediated by increased activation of multiple brain networks, including their recruitment during behavioral tasks. Neurobiological effects range from the size and morphology of brain regions to the survival and complexity of neurons, adult neurogenesis, enhanced cell excitability, synaptic plasticity, place cell function, and a wide array of neuroprotective molecular responses that reflect multiple genetic processes, including gene-environment interactions, neuroinflammation and trophic factors (Rampon et al., 2000a and 2000b; Will et al., 2004; Leger et al., 2012; Bonaccorsi et al.,2013; Briones et al., 2013; Eckert and Abraham, 2013; Alwis and Rajan, 2014; Hirase and Shinohara, 2014; Bilkey et al., 2017; Kempermann, 2019; Ohline and Abraham, 2019). These benefits of enrichment are thought to arise from a combination of increased exercise, socialization, and novelty, producing stronger effects than each of these components administered individually (Einon et al., 1980; Will et al., 1986; Olson et al., 2006; Cracchiolo et al., 2007; Sozda et al., 2010). Environmental enrichment is often used as general ‘cognitive treatment’ and produces behavioral and cognitive enhancements in young and old intact or ‘sham-operated’ rats (Gonzalez-Ramirez et al., 2014; Harland et al., 2014). Laboratories that study spatial navigation, memory, or neurogenesis, often raise experimental animals in enriched environments as a cognitive enhancer, for example, groups that study in vivo neuronal electrophysiology (e.g., Moser group, Kavli Institute, Norway; Jeffery group, University College London, United Kingdom).

An enriched environment for rats or mice in a laboratory setting generally includes a moderate (10-12) group of animals housed in a large cage in which they are exposed to stimulation far beyond that afforded by the standard, generally barren laboratory cage that often houses 3 to 6 animals (Simpson and Kelly, 2011). Explicit environmental stimulation is provided by adding multiple objects within the enrichment cage. However, these objects are sometimes infrequently or randomly varied in many studies. More consistent enrichment should regularly introduce new objects and new configurations, preferably on a daily basis. This more systematic approach provides informal, but varied and multisensory cognitive stimulation and learning, together with physical and social experiences. Unlike natural (feral) conditions, laboratory enrichment establishes a safe complex environment that has relatively stable social conditions but in which the inanimate physical conditions are frequently changing. Environmental enrichment procedures, such as the number of rats per cage, cage equipment and space, and especially the type and frequency of objects, have often varied markedly across enrichment studies (Simpson and Kelly, 2011). A range of different exposure periods has been implemented, with the majority of studies focusing on enrichment for 30 or 40 days. Periods as short as 4 days or non-continuous exposures of just several hours a day have also been used (Kolb and Gibb, 1991; Wallace et al., 1992; Bindu et al., 2005). The current protocol outlines a systematic method to produce standardized continuous enrichment paradigms for use with rats (or mice) to allow different cohorts of animals to experience comparable enrichment that can be easily replicated in most laboratories to achieve more consistent enrichment procedures. A rigidly standardized protocol across all labs is not feasible, but our protocol provides a clear guide of how a greater degree of standardized enrichment can be achieved than is currently the case. It describes a systematic approach in the frequency, variation and placement of objects to standardize this aspect of the enrichment procedure across experiments within any given lab and to encourage greater similarity across laboratories with respect to object-based enrichment protocols.

Materials and Reagents

  1. Bedding
    Bedding should be changed at least once per week; for us this was once per week just after the cages were washed. We used Natures Flame pellet fuel (Nature’s Flame Limited, New Zealand 1PSI) for bedding in standard (control) cages, which breaks down into sawdust over a few days. The same bedding, but already in sawdust form, was used in the enrichment cages, to help facilitate the positioning of objects; this was created by soaking and breaking up the Natures Flame pellets in a little water and then leaving them to dry. Presoaking of the bedding material for the standard-housed rats could also be used, but the additional technician time required for the standard cages is not necessary. Any suitable laboratory-standard animal bedding is suitable from your supplier and this does not need to be varied between enriched and standard caging.
  2. Rats
    We have used male hooded PVGc rats (e.g., Harland et al., 2014), as well as female hooded PVGc rats, with this protocol, which were bred in-house in our Facility. Only single-sex housing is used in the enrichment literature. If female rats are used, we have not found it necessary to control for the estrus cycle; this decision may depend on the particular study, especially where hormonal changes are relevant. Our protocols have been shown to be effective in Long-Evans rats (Ali et al., 2017). Any persistently aggressive animal should be removed, and more frequently monitored if a given strain is known in this regard. Our rat strain was originally supplied by an Animal Resources Centre in Australia. A wide variety of different rat (and mouse) strains have been used in the literature; direct comparisons of rat strain for enrichment effects are uncommon. Although it is common in the current literature to provide the weight of rat, for enrichment the more relevant variable is the actual age of the animals used (an estimate of age on arrival can be provided by your supplier if in-house breeding is not employed). The age of rat used would be determined by the specific experimental questions under study. In standard laboratory housing conditions, some strain of rat (e.g., Long-Evans; some albino strains) can show evidence of obesity after young adulthood (Tordoff et al., 2008). Obesity in these strains needs to be countered by advance planning of mild food restriction when young adults and this may require comparisons of weight gain across the two housing conditions. Enriched rats are generally lighter than standard-housed rats, but marked differences in weight should be avoided in our view by mild food restriction in both groups.
  3. Enrichment objects
    A large collection of objects with at least four copies of each–see Step A2 for guide on collecting suitable objects. Only clean objects should be introduced.
  4. Clips
    An assortment of clips to connect enrichment objects to each other or to the walls of the cage. For example, we used: Medium fold-back paper clips (e.g., Medium Binder Clip, Staples, catalog number: 103549 ) and S Hooks (e.g., 1” S Hook, Staples, catalog number: 272145 ).
  5. Detergent
    To clean enrichment objects daily. We used Pyroneg powder (2-3 grams per litre; Thermo Fisher, catalog number: DIVHH13231 ).
  6. Wooden blocks
    Non-toxic small hard-wood blocks from your local supplier (8.5 x 9.4 cm, and 1.5 cm thick or similar), are used for rats to gnaw on after initially sterilized. These blocks minimize any damage to objects. Any contaminant-free wooden blocks would be suitable (e.g., Wood Gnawing Blocks, Bio-Serv, catalog number: K3512 ).

Equipment

  1. Large enrichment cages
    We used locally built wire-mesh hot-dip galvanized cages to house 10-12 rats (maximum of 12 in our case). The cage was 85 cm long by 60 cm wide by 30 cm high, with a solid metal floor and two hinged mesh lids. Figures 1A-1B show our enrichment cage. Figures 1C-1D shows two groups of rats participating in the enriched environment (the environment changes daily). Enrichment cages can be constructed by local manufacturers (as in our case) or custom built by cage suppliers. Another option could be to use suitable commercially available cages for pet rats, hamsters or birds. Multi-level cages may be used, but these are regarded as a modification to standardized enrichment protocols. The size of cage and number of rats should accommodate the strain being used and local conditions and requirements. The floor area per animal for the enrichment cage (height x width of floor divided by number of animals; 425 cm2 in our protocol with 12 male rats) is a little greater than the floor area per animal in the standard laboratory cages (375 cm2 in our protocol with 4 male rats). The minimum floor space per rat based on weight should follow the National Research Council: Guide for the use and Care of Laboratory Animals (2011) (Reference 23) guidelines: e.g., for rats < 100 g floor area per animal > 109.6 cm2, for rats < 200 g floor area per animal > 148.35 cm2, for rats < 300 g floor area per animal > 187.05 cm2, for rats < 400 g floor area per animal > 258 cm2, for rats < 500 g floor area per animal > 387 cm2, for rats > 500 g floor area per animal > 451.5 cm2. The guide also recommends that cages should be < 17.8 cm high, but we recommend < 27 cm high if possible, to allow rats to stand fully upright (Makowska and Weary, 2016). Very large rats (> 900 g) may need even more space, such as 1000 cm2 floor area per animal and 30 cm cage height (see Lawlor, 1990). “Crowding” in our enrichment cages does not occur because rats have the whole area to explore and rats generally socially interact and aggregate together no matter what the size of cage provided. For example, object #13 (Appendix 1 in Supplemental) shows a 15 x 15 x 15 cm clear Perspex box with holes for entry/exit; there may be 2 to 6 rats who self-select to spend periods of time within this box. Within reason, social interaction is more critical than the space afforded per rat within the cage. However, rats in enrichment should be monitored for any overt signs of stress (behavioral changes, vocalization, chromodacryorrhea, unexpected weight loss). Plasma corticosterone response could also be monitored as an analog of stress; other researchers using our enrichment protocol reported relatively low levels of intracardial plasma corticosterone (150 ng/ml) and no differences on this measure between enriched and standard housed rats at the end of a 40-day continuous period of differential housing (see Supplementary Figure 2 from Ali et al., 2017). Small variations in cage size is unlikely to have a substantial effect on the results. Our cages were specifically designed to be able to be housed within racks in our colony rooms, so it is important to consider these kinds of restrictions when ordering cages. Cages need to be high-grade stainless steel and be able to withstand repeated exposure to an industrial (walk-through) cage washer. Alternatively, iron or steel cages can be hot-dip galvanized in zinc to make them rustproof.


    Figure 1. Examples of the enrichment setup used in our laboratory. A and B. The enrichment cage, containing an example of the daily configuration of objects, is shown from (A) the side, and (B) the front. C and D. Rats participating in environmental enrichment in two different object configurations are shown in (E). E. Example of standard cages in the colony room. F. Moveable metal food hoppers that could be attached at different heights and positions within the enrichment cages. G. Glass water bottle mounted in moveable metal holder; the bottle cap (shown in insert) was hard rubber and contained a glass spout angled into the cage.

  2. Standard cages
    We used opaque (autoclavable) plastic cages to house 3-4 rats. These standard housing cages were 50 cm long by 30 cm wide by 22 cm high, with stainless steel grille lids. Figure 1E shows our standard cages on a stainless-steel rack in a colony room. Any kind of regular laboratory cage, which houses 2-6 animals (we usually have 3 or 4), would be suitable for use as the “standard” housing condition. If there is a local preference that one or two objects be placed in this cage and remain unchanged (other than for cleaning) we do not regard this as “enrichment”.
  3. Food hoppers
    Figure 1F shows the food hoppers allowing easy relocation of food around the inner walls of the enrichment cage (e.g., Ancare mouse or rat feeders, catalog number: MF342/RF452 ). Any standard laboratory rat chow is suitable to use as food for both the enriched and standard housing.
  4. Metal bottle holders
    Figure 1G shows glass bottles (600 ml) and metal bottle holders used in our studies allowing easy relocation of water position across days (e.g., Ancare sure lock bottle holders, catalog number: H-100 to H-102). Any bottle compatible with the holder would be suitable. The drinking spout should be rodent compatible (i.e., with one or more metal ball bearings to hold water contents) and long enough to protrude inside the cage wall.
  5. (Optional) Light
    We use a reversed light cycle in the colony room so that behavioral testing is conducted in their dark period, that is, when rats are naturally more often active. The test environment can of course have lighting, because the modern-day natural environment would also include lit areas during the true night-time period. This is a local preference issue.

Note: All equipment was cleaned once a week using an industrial rack-sized cage wash, but any suitable cleaning arrangement can be used.

Procedure

  1. Preparing a systematic enrichment protocol
    Note: This section describes all of the steps for designing and creating an enrichment protocol.
    1. Decide on enrichment parameters
      1. Decide on the length of the continuous enrichment period. It is common to use 30 or 40 days, and positive molecular, cellular and behavioral effects in rodents have been shown at these enrichment durations (see Background). However, the duration of enrichment is based on the particular experimental design and specific questions. Positive effects have been demonstrated after as few as four continuous days of enrichment, or non-continuous exposures of just several hours a day.
      2. Decide on the number of objects per day. We recommend 10-15 objects per day (we used 13). It is best to balance between number of objects and the remaining space within the cage. The objects should be distributed throughout the cage, but not restrict the movement of the rats. Our enrichment templates are in the Supplemental section and provide examples that can be followed.
      3. Designate a number of days (referred to as a “cycle” of days) in which all of the objects will be used only once (i.e., the same object is never repeated for a given cage). To make things easy, this cycle should be divisible by the enrichment period (i.e., use 5 or 6 days for a 30-day period; use 5 or 8 days for a 40-day period). For our 40-day enrichment protocol, we created a cycle of 6 object-days, followed by a 7th day with ‘no objects’, followed by an 8th day that exclusively used 13 PVC tubing objects combined to make a tunnel system (Step A3 contains additional information about these extra “themed” days). This gave an 8-day cycle, repeated 5 times but with explicitly different arrangements of objects per 8-day cycle for the 40-day period. See Supplemental for all of our enrichment day templates. Information about the cycles is listed in the caption below each template.
        Note: Using a cycle of days ensures objects are not re-used too often and that the same arrangement of objects is never used. This is achieved without the need for unique objects every single day of a long enrichment period. Using the cycle of days is also compatible with having multiple enrichment cages running concurrently while using the same overall pool of objects (see Step B1).
      4. Calculate the minimum number of objects required. That is, use the number of objects per day (for us, 13 objects) multiplied by the number of days in a cycle (for us, 6 days). So in our case, we used 13 objects per day x 6-day cycle = 78 different objects.
    2. Create a collection of enrichment objects
      1. A large number of objects need to be collected which are durable enough to survive multiple exposures to rats/mice, including animals that may be on food restriction. Objects need to exhibit as much variation in their size, shape, texture, color, and physical makeup as possible (Figure 2). It is also crucial to have multiple copies of each object to allow easy recycling/simultaneous use of objects in different cages/replacement in the event of any breakages. Objects must be able to endure multiple washes with industrial grade detergents. We generally avoid enrichment objects that have any fragrance and the requirement for multiple washes with industrial-grade detergents would dissipate any object-related odors. The introduction of explicitly odorized objects may be used, but generally only if a specific experimental variation is desired. This section contains suggestions for compiling such a collection of objects as cheaply as possible.


        Figure 2. Examples of enrichment objects. These four example objects consist of different materials and vary in shape and texture. The ‘object list’ in Supplemental shows all objects used in our studies.

      2. Types of enrichment objects: Good materials for objects are: hard plastics, sturdy glass, ceramic, stainless steel metal. Materials that should not be used are: any plastic thin enough to be quickly chewed, metal that can rust, paper or cardboard or any other material that can be easily chewed and consumed. We do not recommend the use of wooden objects, especially if the treatment or paint used is unknown and could be poisonous. In addition, soft wood that can be chewed and untreated hard wood that can soak up urine should not be used. Variation in size and shape is good, but avoid objects with sharp edges, and objects that are too large, heavy, or bulky for the environment size. Some thin plastic objects with no exposed edges, such as table tennis balls, are suitable.
        Examples of suitable object types:
        1. Ceramic statues, figurines, candle holders, bowls, cups
        2. Glass or perspex bottles, jars, paperweights, tubes, tunnels
        3. Hard plastic toys, figurines
        4. Metal chains, large screws, nuts, parts of taps or door handles, oven racks, tongs, candle-holders
        5. PVC (polyvinyl chloride) tubes of different shapes, sizes
        6. Multiple versions of each object type can be used, with variations in size, color, and shape. For further examples of suitable objects see Supplemental which contains photos of all objects that we used in our study. For information on how this object list was made, see Step A3b. The minimum number of objects that is required is “the number of objects per day multiplied by the number of cycled days”.
      3. List of suitable outlets to obtain cheap objects:
        1. Thrift stores: ‘pound stores’ (UK), ‘dollar stores’ (USA, New Zealand). These are particularly good for obtaining multiple copies of cheap ceramic objects, toys, figurines, jars, bottles etc.
        2. Hardware stores: great for obtaining small metal and wooden items, and PVC tubing, metal racks, e.g., ‘Home Depot’ (USA), ‘Bunnings’ (New Zealand).
        3. Discount department stores or stationary stores: items generally more expensive than thrift stores, e.g., ‘Walmart’ (USA), ‘Argos’ (UK), ‘Warehouse’ (New Zealand).
        4. Scrap/recycle/second-hand stores: great to find cheap intact household metal/plastic items, however, harder to find identical multiple copies of objects.
    3. Create Enrichment day templates
      Templates should be created for each enrichment day detailing the set of objects used for that day and their start configuration within the cage. Using templates allows these configurations to be accurately reproduced, even by other experimenters. Templates show the start configuration of objects within the cage before rats are introduced to the cage. Figure 3 shows an example template; additional example templates are shown in Supplemental. Ideally, each enrichment template will include a list, with images, of all objects used for a given day’s configuration. It will also include images of the configuration of objects within the cage preferably from different angles to enable accurate positioning of objects.


      Figure 3. Example of enrichment template. An object list at the top designates the images, ID numbers, and names of all 13 objects used in the configuration (scale bars = 15 cm). Four images below this show the setup position of objects in the cage from different viewpoints.

      1. First, create a database of objects, containing a photo and ID number for each object. Place a ruler or scale bar on a clear horizontal surface and take digital photographs from above each object (Figure 2). Transfer 15-20 images per page into a word document (Microsoft Word or Apache OpenOffice Writer). Assign each object a unique ID number, and unique name (to describe that object in 1-3 words).
        Note: Inclusion of a 15 cm scale bar facilitates a permanent record of the empirical size of each object and their relative size.
      2. Set up and take photos of the configurations for every enrichment day. The easiest way to achieve this is to create templates for each “cycle” (in which each object is used once) and then move on to create templates for the next cycle. First, set-up the empty enrichment cage (no rats, but containing bedding) on the floor or on a low table or bench in a well-lit room. Divide the objects into groups containing the specified ‘number of objects per day’; these initial groups of objects will make up the templates for the first cycle. For each group of objects, set them up inside the enrichment cage. Place objects so that they are arranged in diverse ways. Use suitable clips to attach objects to the walls of the cage and suspend some to incorporate the vertical space within the cage. Once a group of objects is set up, take high quality photographs from several angles so that the arrangements can be easily repeated. For example, we used an overhead photo, and ~45° angle photos of the cage from the front and side (see Figure 3). Now remove those objects, repeat the process with the next group of objects until unique object setups have been created and photographed for each day for the first cycle. An important step is to recombine the objects into new groups containing the specified ‘number of objects per day’ and repeat the process to create the 2nd, 3rd, and so on, cycles, until you have created unique configurations for every day of the enrichment period. For an example of this process, see our enrichment templates in Supplemental; the five cycles we used for our enrichment (templates 1-6, 9-14, 17-22, 25-30, 33-38), in which every object was used once, were made using the method described above.
      3. Optional: Include additional ‘themed’ days at the end of each cycle. For example, every 7th enrichment day we had a ‘no objects’ day in which the animals were introduced to an empty enrichment cage (containing just the bedding and food/water). This was to give the animals a different experience, and to enhance subsequent ‘object days. Every 8th enrichment day, the 13 objects consisted of PVC tubing pieces connected to make unique tunnel systems. A different configuration of the PVC tubes was used across cycles. The tunnels were designed to have entry/exits facing many different directions within the cage to promote spatial learning. These PVC tubing pieces were exclusively used for these configurations, although the general object list included several tubes/tunnels as well. Another possibility would be to use LEGO Duplo bricks (or any other durable plastic brick large enough not to be consumed) to make unique walls and structures.
      4. After the first cycle, ensure no object is reintroduced for at least 2 days. When re-grouping the objects into new unique combinations for the second cycle, make sure that at least 2 groups contain none of the objects from the last 2 days of the previous cycle and use these groups as your 1st and 2nd days for the second cycle. Similarly, when re-grouping objects to create configurations for the third cycle, ensure at least 2 groups contain none of the objects from the last 2 template days of the second cycle and use these groups as your 1st and 2nd days for the third cycle. Repeat this same process for each cycle. Because we included 2 additional themed days at the end of each cycle (‘no object day’ and ‘tunnels only day’) this meant no single enrichment object was reintroduced for at least 4 days.

  2. Running an enrichment protocol
    This section describes all of the steps and procedures necessary to commence and maintain the enrichment over the specified period and beyond.
    1. Assigning rats to enrichment cage groups
      1. Rats should be designated to enrichment cage groups, and maintained in those groups throughout the enrichment period. Combining rats (or mice) from different (previous) standard cages is important, so that both some old and new cage-mates are now housed together to promote new social interactions. It is also important, however, that the standard-housed rodents are rehoused with new mates in a similar fashion to avoid differences between enriched and standard-housed mates in terms of this general manipulation. Note that some effects of enrichment may not be as strong when combining rats that have been raised individually (single-housed; Hellemans et al., 2004). For rats, raising them individually is not preferred, unless there is an explicit experimental question in mind. For example, there can be a relatively permanent increase in open field activity and poor inhibition after isolation rearing (Dalrymple-Alford and Benton, 1984). Our enrichment procedure has used 11-12 rats per enrichment cage. If much smaller or larger rats are being used, a smaller or larger enrichment cage could be used. However, the social interaction is more critical than the space afforded each rat within the cage for environmental enrichment so this is not strictly necessary. Cage size for enrichment can, of course, be adapted for use in mice (or younger rats), although the researcher should check the social interactions. Some male (house) mouse strains can be very aggressive, unlike most rat strains. Do not put rats together that have recently, for whatever reason, been interacting with opposite sex animals, as even male rats can then become aggressive.
      2. It is important to establish method(s) to keep track of individual animals within the enrichment cages. Our rats had earpunch labels, but all animals also had individual tail markings made with a permanent marker and refreshed at least once weekly or sooner. These methods allowed us to keep track of animals for any return to standard housing, cage-cleaning, object changes, and behavioral testing.
      3. If using environmental enrichment as a treatment model, we recommend combining control and experimental rats within each enrichment group and standard housing. This removes any possible unique injury- or treatment-related effect on the enrichment experience.
      4. Multiple enrichment cages/groups can be run simultaneously by ensuring each different cage has a different order of templates within that cycle. This ensures that enrichment objects are only needed in one of the cages per day, and that cages all experience a similar sequence of enrichment days and the same sequence of cycles. Table 1 shows an example of how to run multiple enrichment cages with a different order of enrichment days but within the same cycle.

        Table 1. Method for running concurrent multiple enrichment cages. Enrichment days 1-40 are shown on subsequent rows. The numbers in the columns for Cage 1-Cage 4 specify the enrichment template to use for that cage on the given day.

        Note: Days 8, 16, 24, 32, and 40 were “no object” days for our enrichment paradigm, so all four cages are the same for those days.

    2. Daily maintenance of enrichment novelty
      1. Once per day, enrichment objects are removed and a new configuration is set up within the cages. Rats should first be removed from the enrichment cage and returned to their designated holding cages while the configuration is changed. This would usually be for only 10-30 min, when rats were food-restricted, they would be given their daily food at this time while in their holding cage groups. The used objects are removed from the cage. A new set of clean objects is set up within the cage in the configuration specified by the template for that day. The rats are then returned to the enrichment cage.
      2. Water bottles and food hoppers should be refilled and then replaced in new positions within the cage.
      3. Rats can be returned to the enrichment cage, and then the same process can be repeated with the next enrichment cage.
      4. Once all cages have been changed, all the used objects should be washed in hot water and detergent and then left to dry overnight so that they can be used again as clean objects to be added to an enrichment cage the next day.
      5. This process is repeated every day for the duration of the enrichment. Enrichment cages should be cleaned periodically (at least once a week), during which rats are placed in a holding cage while the sawdust bedding is replaced.
      6. Move the location of the enrichment cage(s) every x days. Our custom-made cages sat on large stainless-steel racks. Every four days we switched the position of the four trolleys in the colony room to give each enrichment cage varied lighting and views out of the cage in each direction. Alternatively, if possible, enrichment cages could be moved between colony rooms every set number of days to enhance the novelty of the surrounding environment.
      7. Optional: Continue enrichment overnight-only after the all-day + all-night enrichment period. In our study, after the 40 days of continuous enrichment, rats were removed daily to their designated standard cages at ~9 am (lights now off; we use reversed lighting) to undergo spatial memory testing. At ~5-6 pm all rats were returned to their enrichment cages overnight. Each enrichment cage repeated the cycle of configurations shown in Table 1. Standard-housed control rats stayed in their standard cages throughout this period. This was done to sustain and/or maximize any cognitive enhancement past the continuous period of environmental enrichment.

Data analysis

This section includes an example of experimental results from Harland et al. (2014), obtained using this enrichment protocol. In this study, rats received acute neurotoxic brain lesions to the anterior thalamic nuclei, a nodal point in the extended hippocampal memory system (Dalrymple-Alford et al., 2015), or a “sham” control surgery in which no neurotoxin was delivered. After a recovery period, roughly half of the animals were placed in an enriched environment for 40 days, and the others were housed in standard caging for this period. This produced four groups: 1. Sham standard-housed rats (SH-Std), 2. Lesion Standard-housed rats (AT-Std), 3. Sham enriched rats (SH-Enr), 4. Lesion enriched rats (AT-Enr). Spatial memory was tested in a spatial memory task in an 8-arm radial maze (Figure 4A), previously shown to be sensitive to anterior thalamic nuclei lesions. The number of errors was assessed in the four groups over 35 days of testing (7 blocks of 5 days each; Figure 4B). There were significant main effects for both Surgery-type and Housing-type (Two-Way ANOVA, Surgery Effect, F(1,41) = 49.94, P < 0.001; d = 1.86, Housing Effect, F(1,41) = 26.52, P < 0.001). Post-hoc Newman-Keuls tests showed that sham enriched rats had significantly reduced errors compared to sham standard-housed rats (P < 0.05). Interestingly, lesion enriched rats had significantly reduced errors compared to lesion standard-housed rats (P < 0.001), returning them to a level of performance that did not differ from the sham standard-housed rats (P > 0.15). Coronal sections from the hippocampus of these animals were processed with a metallic Golgi-Cox stain and the density of dendritic spines per 10 µm on the basal and apical arbors of CA1 pyramidal neurons was assessed (Figure 4C). The hippocampus is a brain region associated with spatial memory in rodents. There were significant main effects for Surgery-type and Housing-type for both basal and apical dendritic spines [see Figure 4D, Two-Way ANOVAs, Basal Spines (Surgery Effect, F(1,41) = 104.50, P < 0.001; Housing Effect, F(1,41) = 69.37, P < 0.001); Apical Spines (Surgery Effect, F(1,41) = 43.87, P < 0.001; Housing Effect, F(1,41) = 22.68, P < 0.001)]. Post-hoc Newman-Keuls tests of basal spine density showed that sham enriched rats had denser spines compared to sham standard-housed rats (P < 0.001). Lesion enriched rats had denser basal spines compared to lesion standard-housed rats (P < 0.001), comparable to the density observed in sham standard-housed rats (P > 0.15; see Figure 4E). Similarly, post hoc Newman-Keuls tests showed higher apical spine density in sham enriched rats compared to sham standard-housed rats (P < 0.001). Lesion enriched rats had denser apical spines compared to lesion standard-housed rats (P < 0.02), again similar to the density observed in sham standard-housed rats (P > 0.15). See Harland et al. (2014) for more details on these experiments.


Figure 4. Examples of enrichment data. Adapted from Harland et al. (2014). A. Enrichment reduced the number of errors in a spatial memory task in a radial maze. B. Enriched rats that received a “sham” control surgery (SH-Enr) outperformed standard housed sham rats (SH-Std) in the radial-maze. Enriched rats with anterior thalamic nuclei lesions (AT-Enr) had reduced errors compared to standard-housed lesion rats performing the task at a comparable level to the standard-housed controls. C. Dendritic spine density was assessed along the basal and apical portions of CA1 pyramidal neurons in the hippocampus. D. Enrichment increased spine density in sham and lesion rats for both basal and apical dendritic spines. E. Representative overlaid projections of basal spine density are shown for each group, scale bar = 2 µm. Enrichment returned spine density in lesion rats to a similar level observed in sham standard-housed rats.

Acknowledgments

These protocols were designed for experiments published in Harland et al. (2014). We would like to thank Neroli Harris and Silvana De Freitas Costa for technical assistance. This work was supported by a Health Research Council of New Zealand Grant 09/051.

Competing interests

The authors declare no conflicts of interest or competing interests.

Ethics

All procedures are in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the University of Canterbury NZ Animal Ethics Committee ref 2015/21R (2015-2017).

References

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简介

[摘要 ] 暴露于环境富集对学习和记忆,神经生物学多样的效果有益效果,和大脑后促进功能恢复injury.The 通过增加社会交往,体力活动,空间复杂性的一个组合产生富集效果,和新颖。然而,文献中的专业方法与协议提供一致的富集方式的一致性或程度不一致,因此我们提供了一种环境富集协议,该协议可以轻松地复制本地确定的次要细节,以便跨队列和笼养动物概述了程序以标准格式生成和使用每天适当变化的对象库的程序,这些对象在长达40天的连续时间内系统地变化,并与大量大鼠一起使用。一个适当大小的笼子,并定期转移食物和水的位置,以及 这些程序在不同的实验室和大鼠品系中产生了稳健的影响,从而改善了实验室内部和实验室之间的比较。非丰富的比较可能会有所不同,但通常包括将成组动物放在标准实验室中,没有物体,食物和水稳定富集是一种安全的非古生物学工具,用于检查寿命,脑功能障碍和损伤的动物模型中的行为和神经生物学过程。

[背景 ] 丰富环境是一个成熟的治疗手段与年龄相关的下降相关改善认知和情感障碍和许多类型的脑损伤(面包车后普拉格等,2000;威尔等人,2004; Nithianantharajah 和汉南,2006; Pang和Hannan ,2013 ; Alwis 和Rajan ,2014; Hannan ,2014)。这包括改善的认知储备,从相对弥漫性脑损伤中恢复,例如中风,脑外伤和各种神经退行性疾病(Johansson,2004; Pang和Hannan , 2013年;Alwis 和Rajan ,2014年;Hannan ,2014年),以及来自皮质,海马形成和丘脑的急性局部病变(Will和Kelche ,1992年; Will 等,2004年; Harland 等,2014年; Dalrymple- Alford 等人,2015)。这些作用可能是由多个大脑网络的激活增强所介导的,包括在行为任务期间对其的募集。神经生物学作用范围从大脑区域的大小和形态到s。神经元的存活和复杂性,成年神经发生,增强的细胞兴奋性,突触可塑性,位置细胞功能以及反映多种遗传过程的多种神经保护性分子反应,包括基因-环境相互作用,神经炎症和营养因子(Rampon 等, 2000a和2000b; Will 等,2004; Leger 等,2012; Bonaccorsi 等,2013; Briones 等,2013; Eckert和Abraham,2013; Alwis 和Rajan ,2014; Hirase 和Shinohara,2014; Bilkey 等人,2017; Kempermann ,2019; Ohline 和亚伯拉罕,2019) 。这些富集的益处被认为从增加运动,社会主义社会发展的组合,以产生ž 通货膨胀,和新颖性,产生比每个这些组分单独给药的效果更强。 (Einon 等人,1980; Will 等人,1986; Olson 等人,2006;Cracchiolo 等人,2007;Sozda 等人,2010)。环境富集通常被用作一般的“ 认知治疗”并产生行为和认知 完整和“假手术”的幼鼠和老鼠的增强作用(Gonzalez-Ramirez 等,2014; Harland 等,2014)。研究空间导航,记忆或神经发生作用的实验室通常会在丰富的环境中饲养实验动物,例如认知增强剂,例如,研究体内神经元电生理的小组(例如,挪威Kavli 研究所的Moser小组;英国伦敦大学学院的Jeffery小组)。

在实验室环境中为大鼠或老鼠提供的丰富环境通常包括中型(10-12)的动物,它们被关在一个大笼子里,在那里它们受到的刺激远远超出了通常为贫瘠的普通笼子所能提供的刺激饲养3至6只动物(Simpson和Kelly,2011)。通过在浓缩笼中添加多个物体来提供明确的环境刺激,然而在许多研究中,这些物体有时很少或随机变化,更一致的浓缩应定期引入新的物体这种更系统的方法可提供非正式的,但多样的,多感觉的认知刺激和学习以及身体和社会经验,与自然(野性)条件不同,实验室浓缩可建立相对安全的复杂环境稳定的社会条件,但是无生命的身体条件经常发生变化。环境富集程序,例如每个笼子中的老鼠数量,笼子设备和空间,尤其是物体的类型和频率,在富集研究中通常有显着差异(Simpson和Kelly,2011)。已实施,大多数研究集中于富集30或40天,也使用了短至4天或每天仅几个小时的非连续暴露的时期(Kolb and Gibb,1991; Wallace et al 。,1992 ; 宾度等..,2005)当前协议概述了系统的方法来产生标准化的不断丰富范式适用于大鼠(或鼠标),让不同的客群组动物来体验相媲美的丰富,可以很容易被复制在大多数实验室获得更一致的富集程序。在所有实验室中使用严格标准化的协议是不可行的,但是我们的协议为如何提供更大程度的标准化富集提供了清晰的指南 被一个chieved比目前case.It d escribes在频率,变化和对象的放置的系统方法任何给定的实验室内横跨实验规范富集方法的这个方面,并鼓励相对于整个实验室更高相似性的对象-基于浓缩协议。

关键字:富集, 环境丰容, 丰富环境, 功能恢复, 认知储备, 新奇目标, 社会刺激, 大鼠, 小鼠

材料和试剂


 


寝具
床上用品应每周至少更换一次;对我们来说,这是在笼子刚洗完后每周一次。我们使用Natures Flame颗粒燃料(新西兰的Nature's Flame Limited 1PSI)在标准(对照)笼子中进行床上用品破损在浓缩笼中使用了相同但已为锯末形式的被褥,以方便物体定位;这是通过将Natures Flame颗粒在少量水中浸泡并破碎后制成的,然后将其晾干。也可以使用标准大鼠的床上用品预先浸泡,但是不需要标准笼子增加技术人员的时间。供应商可以使用任何适合实验室标准的动物床上用品无需在富集和标准笼中进行切换。


老鼠
我们已将雄性带帽PVGc 大鼠(例如Harland 等人,2014)以及雌性带帽PVGc 大鼠用于该实验方案,并在我们的设施中进行了内部繁殖。 。如果使用雌性大鼠,我们尚未发现有必要控制发情周期;这一决定可能取决于具体的研究,尤其是在以下地方:荷尔蒙的变化与我们的方案有关,已证明对长埃文斯族大鼠有效(Ali et al 。,2017)。应移除任何持续侵略性动物,并在此方面已知给定品系的情况下进行更频繁的监测。我们的大鼠品系最初由澳大利亚动物资源中心提供。不同的大鼠(和鼠标)株的HA Ve的蜜蜂N用于文献;.直接比较大鼠品系富集效应是罕见本书虽然是中呃,通常在现有文献,以提供大鼠体重,对于丰富更相关变量精算师 使用的动物l的年龄.The(年龄在抵达时的估计值可以通过您的供应商,如果在内部滋生不使用提供)使用将由study.In根据具体实验的问题来确定鼠的年龄标准实验室的住房条件,某些大鼠品系(例如,Long-Evans;某些白化病品系)可以显示成年后肥胖的迹象(Tordoff 等人,2008)。这些品系的肥胖症需要通过提前计划适度饮食限制来解决成年大鼠,这可能需要比较两种饲养条件下的体重增加。增高的大鼠通常比标准饲养的大鼠轻,但我们认为,两组均应通过适度的食物限制来避免体重明显不同。


富集对象
大量物品至少每件都有四份副本- 有关收集合适物品的指南,请参阅St Ap ,仅应引入干净的物品。


短片
各种各样的夹子,用于将富集对象彼此连接或连接到笼子的壁上。例如,我们使用了:中号折回曲别针(例如,中型装订夹,订书钉,货号:103549)和S型钩(例如,1英寸S形钩,订书钉,货号:272145)。


洗涤剂
每天要清洁浓缩物。我们使用了吡咯烷粉(每升2-3克;Therm o F 是她,目录号:DIVHH13231)。


木积木
非从当地供应商光毒性小硬木块(8.5×9.4厘米和1.5厘米厚的或类似的),被用于大鼠啃上后最初sterilized.These块尽量减少objects.Any任何损坏无污染的木块将是合适的(例如,Wood Gnawing块,Bio- Serv ,目录号:K3512)。


 


设备


 


大型浓缩笼
我们使用本地建造的金属丝网热浸镀锌笼子饲养10-12只老鼠(本例中最多容纳12只)。笼子长85厘米,宽60厘米,高30厘米,高金属地板,图铰链1A-1B显示丰富我们的笼子里。图1C-1D显示了两个群体参与丰富环境(环境每天都在变化).Enrichment笼可以通过大鼠的本土制造商建造(如我们的情况下,或由笼子供应商定制建造。另一种选择是使用适合于宠物大鼠,仓鼠或鸟类的市售笼子。可以使用多层笼子,但这些笼子被视为对标准浓缩协议的修改。富集笼每只动物的建筑面积(高度x地板宽度除以动物数量;在我们的实验方案中,每12只雄性大鼠为425 cm 2),并且每只大鼠的数量应适应所用菌株以及当地条件和要求。略大于每平方米的建筑面积 在标准实验室笼子中饲养动物(在我们的实验方案中为375 cm 2 ,有4只雄性大鼠)。根据体重,每只大鼠的最小占地面积应遵循美国国家研究委员会:《实验动物的使用和护理指南》(2011)(参考)23)准则:例如,对于每只动物< 100 g地板面积> 109.6 cm 2 的大鼠,对于每只< 200 g地板动物> 148.35 cm 2 的老鼠,对于< 300 g每只动物的地板面积> 187.05 cm 2 的老鼠,大鼠< 每只动物400 g的地面面积> 258 cm 2 ,对于老鼠< 每只动物500 g的地面面积> 387 cm 2 ,对于老鼠> 每只动物的500 g地面面积> 451.5 cm 2 ,老鼠。指南还建议笼子的尺寸应< 17.8 高的老鼠,但建议尽可能< 27 厘米,以使大鼠完全直立(Makowska 和Weary,2016年)。超大的老鼠(> 900 g)可能需要更多的空间,例如每只老鼠的地面面积为1000 cm 2 动物和笼高30 厘米(请参阅Lawlor,1990年)。在我们的浓缩笼中不会发生“拥挤”的情况,因为大鼠有待探索的整个区域,而老鼠 enerally社会互动和骨料一起无论什么笼provided.For示例的大小,对象#13 (附录1 中Supplementa 升)示出了15×15×15 厘米明确有机玻璃具有用于进入/退出孔箱;可存在2 6只自选在此框中度过一段时间的老鼠。在一定程度上,社交互动比每只老鼠在笼子里提供的空间更为关键。但是,应监控处于富集状态的老鼠是否有明显的压力迹象(行为CH 安格斯,发声,血泪,意外的重量损失)。血浆皮质酮响应也可以被监测,因为应力的模拟;使用我们的富集方法其他研究人员报道的相对低水平的心脏内血浆皮质酮(150 纳克/毫升)并在此没有差异在连续40天的差异饲养期结束时测量富集和标准饲养的大鼠之间的距离(参见Ali 等人,2017年的补充图2 )。笼尺寸的微小变化不大 我们的笼子经过专门设计,可以容纳在我们殖民地房间的架子中,因此在订购笼子时考虑这些限制非常重要。笼子必须是高级不锈钢钢制的,并且能够承受工业(穿墙式)笼式洗衣机的反复暴露。包括铁制或铁制的笼子,可用锌进行热浸镀锌处理以使其防锈。


 


D:\ Reformatting \ 2020-1-6 \ 1902446 --1300 John C.Dalrymple-Alford 683103 \ Figs jpg \图1.jpg


网络连接ģ 茜1。实施例的丰富安装用于在我们的模式L Aboratory。A和B 的丰富笼,含一个例子的每日配置对象,示出了从(甲)的一侧,(B)的前面。 ç 和D大鼠参与环境富集在两个不同的对象con 音型显示在(ē )。E. Examp 乐标准机箱在殖民地房间。˚F 。这可能是连在不同的高度和位置中移动的金属食品料斗该富集笼摹。玻璃水瓶安装在可移动的金属制支架;瓶盖(显示在插入)的硬质橡胶,含有一个玻璃槽斜角入笼。


 


标准笼
我们使用不透明的(可高压灭菌的)塑料笼子饲养3-4只大鼠,这些标准的笼子长50厘米,宽30厘米,高22厘米,并带有不锈钢格栅盖。图1E显示了我们在不锈钢机架上的标准笼子在殖民地房间中。任何能容纳2-6只动物(我们通常有3或4只)的常规实验室笼子都适合用作“标准”住房条件。如果当地偏爱一到两只将物品放在该笼子中并保持不变(除了清洁外),我们不认为这是“浓缩”。


料斗
图1F显示了供料斗,可轻松在富集笼的内壁周围重新放置食物(例如,Ancare 老鼠或大鼠喂食器,目录号:MF342 / RF452)。任何标准实验室鼠粮都适合用作两种食物丰富的标准住房。


金属瓶架
图1G显示了我们的研究中使用的玻璃瓶(600毫升)和金属瓶架,可在几天之内轻松重新放置水位(例如,Ancare sure Lock瓶架,目录号:H-100至H-102)。持有人将suitable.The饮用口应该是啮齿动物兼容(即,与一个或多个金属球轴承,以保持水分)和足够长的笼壁内侧突出。


(可选)指示灯
我们在集落室中使用了反向的光周期,以便在黑暗的时期(即老鼠自然活跃的时候)进行行为测试。测试环境当然可以有照明,因为现代的自然环境也会包括在真正的夜间时段的照明区域。这是当地的偏爱问题。


注意:每周使用工业机架大小的笼式清洗机清洁所有设备,但可以使用任何合适的清洁方式。


 


程序


 


准备系统的浓缩方案
注意:本节描述了设计和创建扩充协议的所有步骤。


确定浓缩参数
确定连续富集期的长度,通常使用30或40天,并且在这些富集持续时间内已显示出对啮齿动物有积极的分子,细胞和行为效应(参见背景)。根据特定的实验设计和特定的问题,在连续短短四天的浓缩或每天仅几个小时的非连续暴露后,就显示出了积极的效果。
确定每天的对象数。我们建议每天使用10-15个对象(我们使用d 13)。最好在对象数与笼子中的剩余空间之间取得平衡。对象应分布在整个笼子中,但不限制大鼠的活动。我们的浓缩模板在补充部分中,并提供了可以遵循的示例。
指定一个天数(称为“天的周期”),其中所有对象将仅使用一次(即,同一对象永远不会在给定的笼子中重复使用)。为了简化操作,此周期应可被浓缩期整除(即30天使用5或6天; 40 天使用5或8天)。对于40天富集方案,我们创建了一个6个对象的循环-days,随后是7 个天with'no 对象,接着是8 个一天,专门用于13 PVC管材对象相结合,使一个隧道系统(S TEP A3包含有关这些额外的‘主题’天附加信息)。这给了一个8天为一周期,重复5次,但每8天周期对象为40天的明确不同的安排period.See 小号upplemental所有OU的[R富集日templates.Information关于在上市周期每个模板下方的标题。
注意:使用几天的周期可以确保对象不会被频繁地重复使用,并且永远不会使用相同的对象排列方式,而无需在漫长的浓缩周期中的每一天都使用唯一的对象。与使用同一总对象池同时运行多个浓缩笼的情况也兼容(请参阅步骤B1)。


计算所需的最小对象数,即使用每天的对象数(对于我们来说是13个对象)乘以一个周期中的天数(对于我们来说是6天),因此在本例中,我们使用了13每天x 六天的周期= 78个不同的对象。
创建丰富对象的集合
需要大量的对象的被收集,其是耐用的,足以生存多次曝光到大鼠/小鼠,包括动物,其可以是对食品restriction.Objects需要表现出如在吨太多变化嗣的大小,形状,质地,结肠R,尽可能地进行物理化妆(图2),每个对象具有多个副本也很重要,以便在发生任何损坏的情况下可以方便地在不同的笼子中方便地回收/同时使用对象/更换对象。对象必须能够承受多次清洗工业级洗涤剂通常避免浓缩任何有香气的物体,而工业级洗涤剂多次洗涤的要求会消散任何与物体有关的气味。可以使用引入明显气味的物体,但通常仅在经过特殊实验的情况下使用本节包含有关尽可能便宜地编译此类对象集合的建议。
D:\ Reformatting \ 2020-1-6 \ 1902446 --1300 John C.Dalrymple-Alford 683103 \ Figs jpg \图2.jpg


图茜2.例子富集的对象。这四个实例对象由不同材料组成,具体费用在形状和质地。在“对象列表”在小号Upplemental显示所有对象使用在我们的研究。


 


富集对象的类型:可以用作对象的好材料是:硬塑料,坚固的玻璃,陶瓷,不锈钢金属。不应该使用的材料是:任何足以快速咀嚼的薄塑料,会生锈的金属,纸张或纸板等其他不易被咀嚼和消耗的材料。我们不建议使用木制物体,特别是如果不知道所使用的处理方法或油漆是否可能有毒的情况下。此外,可以咀嚼的软木和未经处理的硬木可以浸泡尺寸和形状变化良好,但应避免使用边缘锋利的物体,以及对于环境尺寸而言太大,太重或笨重的物体。不应使用一些没有外露边缘的薄塑料物体,例如桌子上的尿液。网球是合适的。
合适的对象类型的示例:


C 陶瓷雕像,小雕像,烛台,碗,杯
ģ 小姑娘或有机玻璃瓶,罐,纸镇,管,隧道
H Ard塑料玩具,小雕像
金属链,大螺钉,螺母,水龙头或门把手的零件,烤箱架,钳子,烛台
不同形状,尺寸的PVC(聚氯乙烯)管
每个对象类型的多个版本都可以使用,W i个大小,结肠变化r和shape.For合适对象的其他例子,看小号upplemental它包含了我们在使用的所有对象的照片study.For关于如何这个对象名单被提出,见步骤A3B。所需物体的最小号码为“ 在每一天的循环天数堆叠对象的数量”。
获得便宜物品的合适商店列表:
T Hrift商店:。“英镑商店”(英国),“美元商店”(美国,新西兰)这些特别适合于获取廉价陶瓷物品,玩具,小雕像,罐子,瓶子等的多份副本。
H Ardware商店:非常适合获取小型金属和木制物品,以及PVC管,金属架,例如,“ Home Depot”(美国),“ Bunnings”(新西兰)。
d iscount百货公司或专卖店静止:项目普遍比旧货店更贵,比如,“沃尔玛”(美国),“阿尔戈斯”(英国),“仓库”(新西兰)。
小号掷骰子/再循环/二手店:大找到便宜的完整家用金属/塑料物品,但是,很难找到对象的相同多拷贝..
创建丰富日模板
应该在每个浓缩日创建模板,详细说明当天使用的对象及其在笼子中的开始配置,使用模板可以准确地复制这些配置,即使其他实验者也可以使用。模板显示笼子中对象的开始配置。前大鼠介绍d笼子图3显示了一个示例模板;其他实例模板显示在小号Upplemental。理想情况下,每个富集模板将包括一个列表,图像,所有对象用于给定一天的配置会。还包括优选地从不同角度的笼子内物体构型的图像,以便精确定位物体。


D:\ Reformatting \ 2020-1-6 \ 1902446 --1300 John C.Dalrymple-Alford 683103 \ Figs jpg \图3.jpg


图Ure 3.浓缩模板示例E. 顶部的对象列表指定配置中使用的所有13个对象的图像,ID号和名称(比例尺= 15 Cm)。从不同的角度来看笼子里的物体。


 


首先,创建一个对象数据库,其中包含每个对象的照片和ID号;将标尺或比例尺放在清晰的水平表面上,并从每个对象上方进行数字照片拍摄(图2)。每页传输15-20张图像放入Word文档(Microsoft Word或Apache OpenOffice Writer)。为每个对象分配唯一的ID号和唯一的名称(以1-3个字描述该对象)。
注意:包含15厘米的比例尺条有助于永久记录每个对象的经验大小及其相对大小。


为每个浓缩日设置并拍照。实现此目的的最简单方法是为每个“周期”(每个对象使用一次)创建模板,然后继续为下一个周期创建模板。杉木ST,集- 了空富集笼(没有老鼠,但包含床上用品)在地板上或矮桌,在光线充足的房间或替补划分对象成每一天的对象specified'number”组;这些最初的对象组将构成第一个循环的模板。对于每组对象,将它们设置在浓缩笼内。放置对象以使其以不同的方式排列。使用合适的夹子将对象附着到墙上设置好对象组后,可以从多个角度拍摄高质量的照片,以便可以轻松地重复排列。例如,我们使用了头顶照片和笼子的照片,并悬吊了一些照片以在笼子中合并垂直空间。约45 ° 角的照片 现在删除那些对象,对下一组对象重复此过程,直到创建唯一的对象设置并为第一个周期的每一天拍照为止。重要的步骤是从正面和侧面重新组合e(参见图3)。将对象划分为包含指定的“每天对象数量”的新组,并重复该过程以创建第二,第三等循环,直到您为富集期的每一天创建了唯一的配置。有关此过程的信息,请参阅补充中的富集模板;我们用于富集的五个循环(模板1-6、9-14、17-22、25-30、33-38),每个对象都使用一次使用上述方法制成。
可选:在每个周期结束时添加额外的“主题”日。例如,每第7 个浓缩日,我们有一个“无对象”日,将动物引入空的浓缩笼中(仅包含被褥和食物/水)。这是给动物不同的体验,并提高后续的“对象天,每8 钍富集日,13对象包括了PVC管件连接,使独特的隧道系统。不同的配置的PVC管中隧道的设计使入口/出口面向笼子内的许多不同方向,以促进空间学习。这些PVC管件专门用于这些配置,尽管一般对象列表中还包括多个管道/ 隧道。另一种可能性是使用乐高Duplo砖(或其他足够大的耐用塑料砖,不被消耗)制成独特的墙和结构。
在第一个周期之后,请确保至少有2天没有重新引入任何对象。在第二个周期中将这些对象重新分组为新的唯一组合时,请确保至少有2个组在该对象的最后2天中没有包含任何对象。前一个周期以及将这些群组为您的1 日和2 次天为第二个周期。同样,当重新分组对象来创建第三个周期的配置,保证至少2组包含任何对象从过去的2模板天第二个周期的和使用这些组作为1 日和2 次天的第三cycle.Repeat每个cycle.Because我们包括在2个额外的主题的各天周期结束(“没有对象日”和'同一过程仅一天的时间”),这意味着至少4天没有重新引入任何富集对象。
 


运行浓缩协议
本节介绍了在指定时期及以后开始和维持浓缩所需的所有步骤和程序。


将大鼠分配至富集笼组
应将大鼠指定为富集笼组,并在整个富集期间将其维持在这些组中。将不同(先前)标准笼中的大鼠(或小鼠)合并在一起很重要,因此现在将一些新旧笼子同窝在一起促进新的社会互动。然而,同样重要的是,将标准笼养的啮齿动物与新配偶以相同的方式重新安置在一起,以避免在这种一般操作上富人和标准笼养的同伴之间出现差异。结合单独饲养的大鼠时,其强度可能不那么强(单笼 ; Hellemans 等,2004)。对于大鼠,除非考虑到明确的实验性问题,否则不建议单独饲养它们。在隔离饲养后,它可以是一个相对永久性的开放场活动增加和抑制性差的问题(Dalrymple-Alford和Benton,1984)我们的富集程序每个enri使用了11-12只大鼠 Chment笼子:如果使用更大或更小的老鼠,则可以使用更大或更小的浓缩笼子,但是,社会互动比笼子中每只老鼠为环境富集所提供的空间更为关键,因此这并不是绝对必要的。当然,尽管研究人员应该检查社交互动,但是可以使用于饲养的笼子大小适合老鼠(或更年轻的老鼠)。某些雄性(家)小鼠品系可能具有攻击性,这与大多数大鼠品系不同。最近,无论出于何种原因,它们都与异性动物相互作用,因为甚至雄性大鼠也可能变得具有攻击性。
重要的是要建立追踪动物在浓缩笼中的方法,我们的大鼠带有耳穿孔标签,但所有动物的尾巴标记都带有永久性标记,并且至少每周或更快地刷新一次。允许我们跟踪动物以便返回标准住房,清洁笼子,更换对象和进行行为测试。
如果使用环境富集作为治疗模型,我们建议在每个富集组和标准住房中合并对照组和实验大鼠,这样可以消除对富集体验的任何可能的与伤害或治疗相关的独特影响。
通过确保每个不同的笼子在该周期内具有不同顺序的模板,可以同时运行多个浓缩笼子/组,从而确保每天只在其中一个笼子中需要富集对象,并且每个笼子都经历相似的序列。富集天数和相同的循环顺序表1显示了一个示例,说明如何以不同的富集天数顺序但在同一循环中运行多个富集笼。




表1.运行的并发多富集cages.Enrichment法天1-40显示在随后的rows.The 数列中的S代表笼1 - 凯奇4 指定富集模板使用该笼在给定的一天。





笼子1


笼子2


笼子3


笼子4


1个


1个


2


3


四个


2


2


3


四个





3


3


四个





6


四个


四个





6


7








6


7


1个


6


6


7


1个


2


7


7


1个


2


3


8


8


8


8


8


9


9





11


12








11


12


13


11


11


12


13


14


12


12


13


14


15


13


13


14


15


9


14


14


15


9





15


15


9





11


16


16


16


16


16


17


17


18岁


19


20


18岁


18岁


19


20


二十一


19


19


20


二十一


二十二


20


20


二十一


二十二


二十三


二十一


二十一


二十二


二十三


17


二十二


二十二


二十三


17


18岁


二十三


二十三


17


18岁


19


二十四


二十四


二十四


二十四


二十四


二十五


二十五


26


27


28


26


26


27


28


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注:日小号8,16,24,32和40是为了丰富我们的范式“没有对象”的日子,所以所有四个笼子是为那些日子一样。


 


日常维护浓缩新奇
每天一次,富集画线A ..再删除和新的配置设置中的笼大鼠首先应移除了丰富笼,并返回到其指定的控股笼而配置改变这通常将只有10 - 30分钟,当大鼠的食物- 限制,他们将给予他们的日常食物在这个时候,而在红外控股笼groups.The使用的对象从cage.A新一套干净的对象被删除被设置在笼中按照当天模板指定的配置,然后将大鼠放回浓缩笼。
应重新装满水瓶和食物漏斗,然后在笼子中的新位置更换。
可以将大鼠放回浓缩笼,然后在下一个浓缩笼中重复相同的过程。
更换完所有笼子后,所有用过的物体应在热水和洗涤剂中清洗,然后晾干一整夜,以便它们可以再次用作清洁物体,第二天再添加到浓缩笼子中。
每天在富集期间重复此过程。应定期清洁富集笼(至少每周一次),在此过程中,将大鼠放在保持笼中,同时更换木屑垫层。
每隔x 天移动一次浓缩笼的位置。我们定制的笼子坐在大型不锈钢架子上,每隔四天我们将四个手推车的位置切换到菌落室中,以使每个浓缩笼具有不同的照明和尽管有可能,也可以每隔一定的天数在集落室之间移动一个富集笼,以增强周围环境的新颖性。
可选:全天+整夜富集期结束后,仅隔夜继续富集。在我们的研究中,连续富集40天后,每天约上午9 点将大鼠移至指定的标准笼子中(现在熄灯;使用反向照明)进行空间记忆测试。下午5 点至下午6 点,所有大鼠返回其富集笼过夜,每个富集笼重复表1所示的配置周期。这样做是为了维持和/或最大化连续不断丰富环境后的认知能力。
 


数据一nalysis


 


本节包括使用该富集方案获得的Harland 等人(2014)的实验结果示例。在本研究中,大鼠接受了丘脑前核的急性神经毒性脑损伤,这是海马扩展记忆系统的一个结点(Dalrymple -Alford et al 。,2015),或不进行神经毒素递送的“假”对照手术。恢复期后,大约一半的动物被置于丰富的环境中40天,而其他动物则被固定在动物体内。这期间分为四个组:1.假手术标准房大鼠(SH- Std ),2.病损标准房大鼠(AT- Std ),3.假手术丰富大鼠(SH- Enr ),4。病变富集大鼠(AT-恩诺沙星)。空间内存在的空间记忆任务在测试的8臂迷宫(图4A),先前显示为到前塔莱敏感麦克风核病变。n个错误的棕土中进行了评估这四组受试者经过35天的测试(7块,每组5天;图4B)。对于手术类型和住房类型都有显着的主要影响(双向方差分析,手术效果,F (1,41)= 49.94,P <0.001; d = 1.86,住房效果,F (1,41)= 26.52,P <0.001)后。- 特设Newman- 科伊尔斯试验表明,深水富集大鼠具有显着降低错误相比深水标准圈养大鼠(P <0.05)有趣的是,病变富集大鼠具有显着降低错误相比病变标准-饲养的大鼠(P <0.001),使其恢复到与假手术标准饲养的大鼠相同的水平(P > 0.15)。对这些动物海马的冠状切片进行了金属高尔基-柯克斯染色处理,评估了CA1锥体神经元的基础和根尖上每10 µm的树突棘的密度(图4C)。海马是与啮齿类动物的空间记忆相关的大脑区域。键入基础和心尖树突棘[S ee值图 ure 4D,双向方差分析,基底棘(手术效果,F (1,41)= 104.50,P <0.001;住房效应,F (1,41)= 69.37,P <0.001 ); 根尖棘(手术效果,˚F (1,41)= 43.87,P <0.001;房屋的影响,˚F (1,41)= 22.68,P <0.001)] 。后- 特设Newman- 科伊尔斯即深水富集大鼠具有测试基底脊柱密度表现为密集的棘与假手术组相比(P <0.001)。与假手术组相比,富集大鼠的基底棘较密(P <0.001),与假手术组相比(P > 0.15; s 参见图4E),类似地,纽曼Keuls 事后测试显示,与假手术组相比,假手术组大鼠的顶棘密度更高(P <0.001);假手术组比假手术组(P <0.001)的顶棘密度更高。P <0.02),再次类似于假手术标准大鼠的密度(P > 0.15)。有关这些实验的更多详细信息,请参见Harland 等(2014)。


 


D:\ Reformatting \ 2020-1-6 \ 1902446 --1300 John C.Dalrymple-Alford 683103 \ Figs jpg \图4.jpg


图4 富集数据示例。改编自Harland 等人(2014 ).A 。富集减少了径向迷宫中空间记忆任务中的错误数量.B。接受了“假”对照手术的富集大鼠( SH- Enr )在radial骨迷宫中的表现优于标准的假手术大鼠(SH- Std )。与标准的病灶大鼠相比,富集的具有丘脑前核病变的大鼠(AT-Enr )的错误率降低了C.沿海马CA1锥体神经元的基部和根尖评估树突棘密度D.在假和病变大鼠中充实增加的假性和病变大鼠的棘突密度,包括基础和根尖突棘E.代表覆盖每组显示了基础脊柱密度的投影,比例尺= 2 µm。病变大鼠的充实使脊柱密度恢复到假手术标准大鼠中观察到的相似水平。


 


致谢


 


这些协议是为Harland 等人(2014)发表的实验而设计的。我们要感谢Neroli Harris和Silvana De Freitas Costa的技术援助。这项工作得到了新西兰卫生研究理事会Grant 09/051的支持。


 


利益争夺


 


作者声明没有利益冲突或利益冲突。






伦理


 


所有程序均符合《 NIH实验动物的护理和使用指南》,并已获得新西兰坎特伯雷大学动物伦理委员会ref 2015 / 21R(2015-2017)批准。


 


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引用:Harland, B. C. and Dalrymple-Alford, J. C. (2020). Enriched Environment Procedures for Rodents: Creating a Standardized Protocol for Diverse Enrichment to Improve Consistency across Research Studies. Bio-protocol 10(11): e3637. DOI: 10.21769/BioProtoc.3637.
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