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Alternate without alternative: neither preference nor learning explains behaviour of C57BL/6J mice in the T-maze

In: Behaviour
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  • 1 German Federal Institute for Risk Assessment (BfR), German Center for the Protection of Laboratory Animals (Bf3R), Max-Dohrn-Straße 8–10, D-10589 Berlin, Germany
  • | 2 Institute of Animal Welfare, Animal Behavior and Laboratory Animal Science, Freie Universität Berlin, Königsweg 67, D-14163 Berlin, Germany
Open Access

Abstract

In rodents, the T-maze is commonly used to investigate spontaneous alternating behaviour, but it can also be used to investigate preference between goods. However, for T-maze preference tests with mice there is no recommended protocol and researchers frequently report reproduction difficulties. Here, we tried to develop an efficient protocol with female C57BL/6J CrL mice for preference tests. We used two different designs, adapting habituation, cues and trial timing. However, in both experiments mice did not show any preference, although we used goods which we knew mice find rewarding. Instead, they alternated choices indicating that exploratory behaviour overruled preference. We argue that this behavioural strategy has evolved as an adaptive trait in saturated conditions where there is no need to take the reward immediately. Therefore, we deem the T-maze unsuitable for preference testing with the procedures we used here.

Abstract

In rodents, the T-maze is commonly used to investigate spontaneous alternating behaviour, but it can also be used to investigate preference between goods. However, for T-maze preference tests with mice there is no recommended protocol and researchers frequently report reproduction difficulties. Here, we tried to develop an efficient protocol with female C57BL/6J CrL mice for preference tests. We used two different designs, adapting habituation, cues and trial timing. However, in both experiments mice did not show any preference, although we used goods which we knew mice find rewarding. Instead, they alternated choices indicating that exploratory behaviour overruled preference. We argue that this behavioural strategy has evolved as an adaptive trait in saturated conditions where there is no need to take the reward immediately. Therefore, we deem the T-maze unsuitable for preference testing with the procedures we used here.

1. Introduction

The T-maze is a behavioural test using a maze with a start arm (sometimes connected to a start cage) and two choice arms branching off at the same point from the start arm. In the classic design the arms lie exactly opposite each other, so that they form a T together with the starting arm. In the Y-maze variation, the arms branch off from the start arm at a steeper angle so that the overall shape of the apparatus is y-shaped. During a T-maze test, an animal is placed either in the start cage or directly inside the maze at the beginning of the start arm. At the end of the start arm, the animal has then to choose between entering the left or the right arm. Depending on the setup, in addition to the spatial position the arms can provide further cues, e.g., visual (mice: Lione et al., 1999; broilers: Buckley et al., 2011), tactile (compare Cunningham et al., 2006) or olfactory cues (Mayeux-Portas et al., 2000). Also, none, one or both arms can contain a reward, which can be food (Crusio et al., 1990; Deacon & Rawlins, 2006; Deacon, 2006), shelter (Pilz et al., 2020) or a platform (in case of the water T-maze, Granholm et al., 2000; Belzung et al., 2001; Guariglia & Chadman, 2013).

The T-maze is an important behavioural test to assess the effect of drugs (mice: Correa et al., 2015; rats: Lohninger et al., 2001), genetic alterations (mice: Granholm et al., 2000; Mayeux-Portas et al., 2000) or diseases (mice: Belzung et al., 2001; rats: Sánchez-Santed et al., 1997; Wu et al., 2018). It is often used to assess spontaneous alternating behaviour, spatial memory and/or discrimination of stimuli (Dember & Fowler, 1958; Wenk, 1998; Belzung et al., 2001; Dudchenko, 2004; Deacon & Rawlins, 2006; Deacon, 2006; Sharma et al., 2010b). Spontaneous alternating behaviour describes the tendency of rodents to choose the arm they did not visit in the preceding trial. This kind of behaviour occurs spontaneously and is not necessarily related to a resource being exploited in the preceding trial (mice: Gerlai, 1998; gerbils: Dember & Kleinman, 1973; rats: Sánchez-Santed et al., 1997). In position discrimination tests (also: spatial memory tests), only one spatial location, either the left or the right arm, is baited (mice: Lione et al., 1999; Granholm et al., 2000; Belzung et al., 2001; Sharma et al., 2010a; Guariglia & Chadman, 2013; Pioli et al., 2014). Thus, the spontaneous alternating is a way to evaluate the working memory (which location was last visited?), while the position discrimination test evaluates the reference memory (Deacon & Rawlins, 2006), similar to the conditioned place preference test (Wenk, 1998; Sharma et al., 2010b; Shoji et al., 2012; Hieu et al., 2020). In a further modification of the position discrimination, the T-maze can also be used as general discrimination test, using additional cues instead of merely the spatial one to provide information on the baited arm (mice: Lione et al., 1999; Granholm et al., 2000; Mayeux-Portas et al., 2000; broilers: Buckley et al., 2011). Note that with different tasks different memory types are tested: For alternating behaviour, the working memory is important (remembering which arm was last visited). For position or stimulus discrimination behaviour, the working memory is also important (which cue was rewarded?) but between testing days, this information has to be retrieved from the reference memory (Sharma et al., 2010b).

In a modification of the discrimination test, the T-maze is also used as a preference test: The arms are provided with different goods, and the animal is required to choose between them. This form of preference test seems to be easily performed with a variety of animal species (mice: Roder et al., 1996; Correa et al., 2015; Cutuli et al., 2015; wild mice: Nunes et al., 2009; rats: Patterson-Kane et al., 2001; Ras et al., 2002; Denk et al., 2004; van der Plasse et al., 2007; Cunningham et al., 2015; Hernandez-Lallement et al., 2015; Wadhera et al., 2017; Leenaars et al., 2019; pigs: Rooijen & Metz, 1987; hens: Dawkins, 1977; broilers: Buckley et al., 2011; zebrafish: Hieu et al., 2020; fruit flies: Fujita & Tanimura, 2011). Preference is usually assessed by offering the goods in the choice arms of the maze but in some cases, it might be useful to use stimuli which are associated with the to-be-tested goods instead, e.g., in tests for social preference, the real mouse might be replaced by urinary stimuli (Nunes et al., 2009; compare also Fitchett et al., 2006). It also has to be kept in mind that offering the goods itself can lead to saturation and/or influence the choice in the next trial (Kirkden & Pajor, 2006), in the same way as humans might prefer milk after eating something spicy (Nasrawi & Pangborn, 1990).

Preference tests in T-mazes can be performed with discrete or continuous choices: In a discrete measurement task, an animal has to perform multiple trials in which it can choose between the left or the right arm (mice: Tellegen et al., 1969; rats: Patterson-Kane et al., 2001; Ras et al., 2002; van der Plasse et al., 2007; Pioli et al., 2014). In a continuous measurement task, the animal stays in the T-maze for a defined period of time and the time the animal spends in the left or the right arm is used to ascertain preference (mice: Roder et al., 1996; Cutuli et al., 2015; wild mice: Nunes et al., 2009; Correa et al., 2015; compare also Pennycuik & Cowan, 1990; using a U-shaped maze and wild mice).

There are various protocols and recommendations on the conduction of T-maze tests for behavioural measures such as memory and discrimination. However, there is to date no protocol for T-maze preference tests: The protocols focus either on spontaneous (unrewarded) alternation (Wenk, 1998; Deacon & Rawlins, 2006), rewarded alternation (Deacon & Rawlins, 2006; Shoji et al., 2012; Wenk, 1998) or position discrimination (Deacon, 2006; Shoji et al., 2012). A short comparison of different protocols is given in Table 1.

Table 1.
Table 1.

Comparison of T-maze protocols by Deacon & Rawlins, 2006; Deacon, 2006; Shoji et al., 2012 and Wenk, 1998.

Citation: Behaviour 158, 7 (2021) ; 10.1163/1568539X-bja10085

Table 1.
Table 1.

(Continued.)

Citation: Behaviour 158, 7 (2021) ; 10.1163/1568539X-bja10085

In general, for spontaneous alternation, no food restriction or habituation is needed. Animals should just be well-habituated to their environment and the handling, before they are placed into the maze. Protocols for rewarded alternation and position discrimination are more complex and differ in their recommendations. Often, food restriction to 85% of free-feeding weight is recommended, although Deacon & Rawlins (2006) at the same time state that well habituated animals should also perform the T-maze without food restriction (Deacon & Rawlins, 2006). For rewarded alternation, forced trials are recommended, in which animal are only allowed to visit one arm by blocking the other. In the following trial, animals get a free choice with both arms accessible. If the animals visit the previously blocked arm, they made an alternating choice. In position discrimination, on the other hand, no forced trials are conducted, and trials are always free choice. Also, rewarded alternation and position discrimination differ with regard to the recommendations made about cleaning: While for rewarded alternation tasks, cleaning seems to be more common, for position discrimination Deacon (2006) explicitly states that not cleaning maximizes the learning potential (Deacon, 2006). However, protocols for both types of tests differ greatly in their recommendations for habituation procedure (individuals or group, duration, free exploration or trials, reward or no reward) and intertrial interval (immediately or more than 10 min). All protocols recommend at least ten trials per day, but depending on the intertrial interval this leads to differing test durations from 50 min (Shoji et al., 2012) to several hours (Deacon, 2006). None of the protocols gives instructions with regard to testing time, and only one of the protocols (Shoji et al., 2012) provides an example for testing time, but only to emphasise that the tests should be repeated in the same time frame (their example is between 9:00 am and 6:00 pm, with lights 7:00 am–7:00 pm). Searching original studies instead of protocols, the time frame of experiments (if stated) varies, e.g., starting 2 h into the dark phase (Locurto et al., 2002), 3 h before the end of the light phase (Guariglia & Chadman, 2013), 3 h into the light phase (Derenne et al., 2014) or in general ‘during the light phase’ (Moy et al., 2008; Shipton et al., 2014). However, day time might influence motivation to gain food (Acosta et al., 2020; Koch et al., 2020) and should therefore be considered carefully.

Thus, there is not ‘one perfect test design’ with regard to rewarded alternation or position discrimination but various ways to perform it, depending on the research question. However, this makes it difficult to develop a protocol for preference tests. Personal correspondence with other researchers resulted mainly in reports of difficulties in reproduction of the T-maze test, especially when trying to alter the existing protocols for preference tests. In general, varying success rates might be caused by differences in strain performances (Gerlai, 1998; Moy et al., 2008). However, there are various additional factors which might influence results, e.g., differences in handling technique (base of the tail compared to cup or tube handling, Hurst & West, 2010; Gouveia & Hurst, 2017), stress (Mitchell et al., 1985), habituation (Deacon & Rawlins, 2006; Rudeck et al., 2020), level of food restriction (Richman et al., 1986).

One interesting solution for the factor handling is provided by Zhang et al. (2018), who developed an automated T-maze system (Zhang et al., 2018). Here, no handling is involved, and thus, influence of the researcher is reduced. Taking it one step further, Pioli et al. (2014) introduced an automated T-maze which is even home cage based. Here, mice can conduct the test when active and most motivated to work for the reward, which also makes food restriction superfluous (Pioli et al., 2014). However, this automated T-maze is designed for single housing (there is only a companion animal behind a partition), which might not be the desired husbandry condition. In addition, this automated T-maze is meant for spontaneous alternation tasks and it would probably need adjustments for preference tests with regard to, e.g., cue presentation and change of presentation side.

Thus, a working protocol for the conduction of a T-maze preference test is still needed. Here, we performed two experiments in search for such a protocol: In experiment 1, we investigated the preference between two fluids (apple juice vs. almond milk). In experiment 2, we changed the test design and offered one arm containing millet and bedding, and one arm containing only bedding. For both experiments, we used C57BL/6J mice because this is the mouse strain most commonly used; therefore, a working protocol would have the greatest impact for the research community. In addition, we tried to develop a protocol without food or water restriction because this condition itself might change the preference of the mice (see also in the discussion).

2. Material and methods

2.1. Animals

A group of thirteen female C57BL/6J CrL mice was purchased in December 2017 at the age of 3 weeks from Charles River, Sulzfeld. This group was used in experiment 1 (‘group 1’). Another group consisting of twelve female C57BL/6J CrL mice was purchased in June 2019 at the age of 4 weeks from Charles River, Sulzfeld. This group was used for experiment 2 (‘group 2’). We used females because they show less aggression in groups and we needed these large group sizes for other home cage based experiments.

For both groups applies that all mice within a group had different mothers and different nurses to ensure maximal behavioural variability within the inbred strain. At the age of five weeks, transponders were implanted, a procedure performed under anaesthesia and analgesia (for details see the Appendix). Mice were always handled by tube handling. Both groups took part in multiple other experiments, including the development of an home cage based automated tracking system and conditioned place preference tests. By the time the T-maze test was performed, they were around 12 months (group 1, start in November 2018) or 11 months old (group 2, start in April 2020). In the sense of the 3R, we decided to use these groups despite their rather old age. Especially, because the repeatability of activity measures increases with the age of the mice (Brust et al., 2015), and performance levels of C57BL/6J mice in visual detection, pattern discrimination and visual acuity tasks are not decreased with 12 months (Wong & Brown, 2007). It has to be noted that by the start of the experiment 2, eleven of twelve mice in group 2 at least partly lacked their whiskers. This is important as it might influence their tactile-guided behaviour, for example, novel object recognition or open field activity (Haridas et al., 2018; Tur & Belozertseva, 2018). However, this should not have influenced the mice’s ability to perceive visual, olfactory or spatial cues (left or right body turn) and to act on them. In addition, as barbering is a model for a disorder (trichotillomania), it is also important to note that mice which barber show no difference in learning ability itself, with the exception of a extra dimensional shift task (Garner et al., 2011). Here, however, only simple learning was required.

2.2. Housing

One group of mice was kept in two type IV macrolon cages (L × W × H: 598 × 380 × 200 mm, Tecniplast, Buguggiate, Italy) with filter tops. The two cages were connected via a Perspex tube (40 mm in diameter). This cage system was chosen because of other research purposes, and mice had lived in it since they were around 2 months (group 1) or 3 months old (group 2). Food (autoclaved pellet diet, LAS QCDiet, Rod 16, Lasvendi, Soest, Germany) and tap water (two bottles each cage) were available ad libitum in both cages. Cages were equipped each with bedding material (Lignocel FS14, spruce/fir, 2.5–4 mm, JRS, J. Rettenmaier & Söhne, Rosenberg, Germany) of 3–4 cm height, a red house (The MouseHouse, Tecniplast), papers, cotton rolls, strands of additional paper nesting material, and two wooden bars to chew on. Both cages also contained a Perspex tube (40 mm in diameter, 17 cm long), which was used for tube handling.

Room temperature was maintained at 22 ± 3°C, the humidity at 55 ± 15%. Animals were kept at 12 h/12 h dark/light cycle with the light phase starting at 7:00 am (winter time) or 8:00 am (summer time), respectively. Between 6:30 and 7:00 am (winter time) or 7:30 and 8:00 (summer time) a sunrise was simulated using a Wake-up light (HF3510, Philips, Hamburg, Germany). Once per week, the home cages were cleaned and all mice were scored and weighed. In this context, mice also received a colour code on the base of their tails, using Edding 750 paint markers, to facilitate individual recognition.

2.3. T-maze setup

For the T-maze test, a start cage (type III, L × W × H: 425 × 266 × 155 mm, Tecniplast) filled with 1 cm bedding was connected via a tube to the T-maze. The tube contained an automated door. In experiment 1, the connection between the start cage and the T-maze resembled part of the setup used for habituation so mice were already habituated to it (compare Figure 1a and Figure 1b): a 15 cm tube with an radio frequency identification (RFID) antenna between cage and door, and a 6 cm tube with a light barrier between door and maze. If the mouse interrupted the light barrier in front of the door or was detected by the RFID antenna, the door opened for 5 s. For experiment 2 (without automated habituation), the tube connected to the start cage was 14 cm long and contained an RFID antenna, followed by the automated door and a 1 cm long tube (see Figure 1c). Here, the door also opened for 5 s whenever the transponder of a mouse was detected. There was no light barrier on the other side of the door because this time mice were not allowed to return to the start cage by themselves.

Figure 1.
Figure 1.

T-maze setup as a schematic drawing for experiment 1 habituation (a) and test (b), and test of experiment 2 (c). (d) Photo of the experiment 2 setup, the box on the bottom left contains the Arduino, which operates the automatic door, the device to its left (with the hole) is an example of the RFID antenna and light barrier constructions. LB, light barrier; door, automatic door; RFID, radio frequency identification antenna.

Citation: Behaviour 158, 7 (2021) ; 10.1163/1568539X-bja10085

The T-maze itself consisted of grey plastic and had three arms, each 32 cm long and 11 cm wide, with 20 cm high walls (see Figure 1d). On either side of the arms a mark was made outside the T-maze so that a virtual line could be drawn 11 cm from the central arm during video analysis. If a mouse crossed this line with its whole body (but not yet with its tail), this was defined as a choice being made.

For video recording, in both experiments a webcam (C390e, Logitech, Lausanne, Switzerland) was mounted above the maze on a metal beam construction. The connected computer was placed near the T-maze in such a way that the experimenter could observe the mouse in the T-maze via the computer screen.

2.4. T-maze test

In the first experiment, the T-maze test was used to compare the preference for two fluids. Mice performed discrete choices between the two arms, which contained a droplet of either almond milk or apple juice. Because insufficient habituation might slow the performance in the maze (Deacon & Rawlins, 2006) and might be one of the main problems, we conducted a thorough habituation phase: For about two weeks, mice had free access to the T-maze via a connection to the home cage. After one week, fluids were presented for 24 h inside the home cage. (As the mice drank extensively from the almond milk bottle during that time, a longer presentation seemed unnecessary.) After thirteen days, mice were moved to the testing room, to habituate to it before the start of the actual T-maze test.

Figure 2.