Table 7 Effects of subcutaneous injection and prior handling by tail or tunnel on voluntary interaction with the handler.

Voluntary interaction	Before 1^st injection		After 1^st injection		After 5^th injection
Handling method	z = 5.41	P < 0.0001	z = 5.34	P < 0.0001	z = 5.41	P < 0.0001
Injection vs control pick up	z = 1.27	P = 0.21	z = 0.23	P = 0.84	z = 0.53	P = 0.53
Comparison between time points
Tail, control pick up	χ² = 1.42	P = 0.49
Tail, injection	χ² = 8.60	P = 0.014
Tunnel, control pick up	χ² = 2.60	P = 0.27
Tunnel, injection	χ² = 0.67	P = 0.72

Mice were picked up briefly by their assigned handling method (tail or tunnel) for 10 days prior to treatment. Half the mice were then assigned to either control pick up by their assigned method or pick up, scruff restraint and subcutaneous injection, repeated on 5 days. Voluntary interaction was assessed immediately before and after handling in the 10^th handling session, 1 week after first subcutaneous injection or control pick up, and one week after 5^th subcutaneous injection or control pick up. The proportion of test time interacting voluntarily with the handler was averaged for tests conducted immediately before and after handling by the assigned handling method at each time point (data shown in Fig. 4A). Mann-Whitney tests assessed differences in averaged voluntary interaction between handling methods and between injection versus control pick up at each time point. Repeated measures nonparametric Friedman tests compared response across the 3 time points for each treatment group. Only mice picked up by tail showed significantly reduced interaction following experience of injection. Significant effects (P < 0.05) are in bold.

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