Table 8 Effects of subcutaneous injection and prior handling by tail or tunnel on exploratory behaviour in a modified open field test.

PC1 score
After 1^st and 5^th injection
Handling method	F_1,36 = 14.03	P = 0.001
Injection vs control pick up	F_1,36 = 0.05	P = 0.83
Repetition (first vs multiple)	F_1,36 = 27.55	P < 0.001
Method × injection	F_1,36 = 0.60	P = 0.44
Method × repetition	F_1,36 = 0.52	P = 0.48
Injection × repetition	F_1,36 = 0.01	P = 0.93
3 way interaction	F_1,36 = 1.02	P = 0.32
After first injection
Handling method	F_1,36 = 14.20	P = 0.001
Injection vs control pick up	F_1,36 = 0.06	P = 0.81
Method × injection	F_1,36 = 0.05	P = 0.83
After 5^th injection
Handling method	F_1,36 = 8.34	P = 0.007
Injection vs control pick up	F_1,36 = 0.02	P = 0.88
Method × injection	F_1,36 = 1.26	P = 0.27

Mice were picked up briefly by their assigned handling method (tail or tunnel) for 10 days prior to treatment. Mice then experienced either control pick up by their assigned method, or pick up, scruff restraint and subcutaneous injection, repeated 5 times. Anxiety in a modified open field test was assessed immediately after first and fifth experience of injection or control pick up. Principal components analysis derived a single component that explained 43% of variance across all trials. This contrasted the total number of line crosses (weighted 0.68), frequency of visits to urine tile (0.80) and time sniffing stimulus urine (0.61) with latency to first enter the central zone (−0.75) and frequency of stretch attend postures (−0.35), such that positive scores reflected high exploratory behaviour and low anxiety (data shown in Fig. 4C). Repeated measures ANOVA examined consistency of behaviour after single or repeated treatment while univariate ANOVA examined each time point separately. Significant effects (P < 0.05) are in bold.

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