Optimising reliability of mouse performance in behavioural testing: the major role of non-aversive handling - PubMed (original) (raw)

Optimising reliability of mouse performance in behavioural testing: the major role of non-aversive handling

Kelly Gouveia et al. Sci Rep. 2017.

Abstract

Handling laboratory animals during test procedures is an important source of stress that may impair reliability of test responses. Picking up mice by the tail is aversive, stimulating stress and anxiety. Responses among anxious animals can be confounded further by neophobia towards novel test environments and avoidance of test stimuli in open areas. However, handling stress can be reduced substantially by using a handling tunnel, or cupping mice without restraint on the open hand. Here we establish whether non-aversive handling, brief prior familiarisation with the test arena and alternative stimulus placement could significantly improve performance of mice in behavioural tests. We use a simple habituation-dishabituation paradigm in which animals must discriminate between two urine stimuli in successive trials, a task that mice can easily perform. Tail handled mice showed little willingness to explore and investigate test stimuli, leading to poor test performance that was only slightly improved by prior familiarisation. By contrast, those handled by tunnel explored readily and showed robust responses to test stimuli regardless of prior familiarisation or stimulus location, though responses were more variable for cup handling. Our study shows that non-aversive tunnel handling can substantially improve mouse performance in behavioural tests compared to traditional tail handling.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1

Figure 1. The habituation-dishabituation task.

(a) Principle underlying the habituation-dishabituation task. Repeated presentation of a novel stimulus for three successive trials (light blue bars) induces a reduction in investigation as the animal recognises an increasingly familiar stimulus (habituation, measured as less investigation in trial 3 compared to trial 1). When an unfamiliar stimulus is presented on the fourth trial (dark blue bar), investigation increases if the animal recognises that the stimulus is novel (dishabituation, measured as more investigation in trial 4 than trial 3). (b) Test arena, showing the location of a urine stimulus either at the centre (cr) or periphery (pr) of the arena. Mice were introduced at the lower edge of the arena (start location marked with X). Animals handled with a tunnel were tipped out backwards at floor level. Urine stimuli used in trials 1–4 came from unrelated male mice.

Figure 2

Figure 2. Handling method and stimulus location influences test performance in a habituation-dishabituation task.

In experiment 1, time sniffing a urine stimulus was recorded in successive trials (1–4), with urine derived from one male in trials 1–3 and from a different male in trial 4. Mice were handled by tail (pink), tunnel familiar from the home cage (blue) or cupped on the hand (orange). Data are medians ± interquartile range. (a) Wilcoxon signed ranks tests assessed habituation (reduced stimulus investigation of the same scent in trial 3 compared to trial 1), and dishabituation (greater investigation of novel scent in trial 4 compared to familiar scent in trial 3). Tests with the stimulus in different locations are pooled. Comparison of (b) habituation responses (trial 1-trial 3 investigation) and (c) dishabituation responses (trial 4-trial 3 investigation) according to handling method and stimulus location (arena centre: cr, solid bars; periphery: pr, hatched bars). P values from likelihood ratio tests comparing mixed effects models (Table 1). N sizes: Tail cr (8), pr (8); Tunnel cr (8), pr (7); Cup cr (6), pr (7).

Figure 3

Figure 3. Effects of handling method and stimulus location on exploratory behaviour in experiment 1.

PCA extracted a single component (PC1) that accounted for 75% of variance in behaviour averaged over four successive trials for each subject. This contrasted high positive weights for active exploration (movement around the arena: 0.94, visits to the stimulus: 0.92, time sniffing stimulus: 0.83) with negative weighting for cautious behaviour (frequency of stretched attend postures: −0.77). (a) Effects of handling method and stimulus location on PC1 scores (means ± sem), P values from likelihood ratio tests comparing mixed effects models (Table 1). Correlation between exploratory behaviour and (b) habituation (stimulus investigation in trial 1-trial 3), or (c) dishabituation (stimulus investigation in trial 4-trial 3). The stimulus was placed in the centre (squares) or periphery (triangles) of the arena and animals were handled by tail (pink), tunnel familiar from the home cage (blue) or cupped on the open hand (orange). N sizes as in Fig. 2.

Figure 4

Figure 4. Familiarisation and handling method influence test performance in the habituation-dishabituation task.

Comparison of (a) habituation (trial 1-trial 3 stimulus investigation) and (b) dishabituation (trial 4-trial 3 investigation) according to handling method (pink: tail; blue: tunnel) and prior familiarisation (familiarised: f, cross hatched; naïve: n, solid bars). Data are medians ± interquartile range, P values from likelihood ratio tests comparing mixed effects models (Table 2). (c) Time sniffing the urine stimulus in successive trials (1–4) for mice handled by tail (pink) or tunnel (blue) when naïve (solid bars) or familiarised (cross hatched bars) with the test arena (medians ± interquartile range). P values from Wilcoxon matched pairs exact tests examining habituation (greater stimulus investigation of same scent in trial 1 than trial 3), and dishabituation (greater investigation of novel scent in trial 4 compared to familiar scent in trial 3). N = 8 mice in each method x familiarisation group.

Figure 5

Figure 5. Effects of familiarisation and handling method on exploratory behaviour in experiment 2.

PCA extracted a single component (PC1) that accounted for 78% of variance in behaviour averaged over four successive trials. This contrasted high positive weights for active exploration (movement around the arena: 0.95, visits to the stimulus: 0.95, time sniffing stimulus: 0.92) with negative weighting for cautious behaviour (frequency of stretched attend postures: −0.67). (a) PC1 scores (means ± s.e.m.) according to handling method and familiarisation (familiarised: f, cross hatched; naïve: n, solid bars). P values from likelihood ratio tests comparing mixed effects models (Table 2). Correlation between exploratory behaviour and (b) habituation (stimulus investigation in trial 1–3), or (c) dishabituation (stimulus investigation in trial 4–3). Mice were either naïve (squares) or familiarised with the test arena (circles) prior to testing, and handled by the tail (pink), or with a tunnel familiar from the home cage (blue). N = 8 in each method x familiarisation group.

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