The Directive 2010/63/EU on animal experimentation may skew the conclusions of pharmacological and behavioural studies

Abstract

All laboratory animals shall be provided some form of environmental enrichment (EE) in the nearest future (Directive 2010/63/EU). Displacing standard housing with EE entails the possibility that data obtained under traditional housing may be reconsidered. Specifically, while EE often contrasts the abnormalities of consolidated disease models, it also indirectly demonstrates that their validity depends on housing conditions. We mimicked a situation in which the consequences of a novel pharmacological compound were addressed before and after the adoption of the Directive. We sub-chronically exposed standard- or EE-reared adolescent CD1 mice (postnatal days 23-33) to the synthetic compound JWH-018, and evaluated its short- and long-term potential cannabinoid properties on: weight gain, locomotion, analgesia, motor coordination, body temperature, brain metabolism ((1)H MRI/MRS), anxiety- and depressive-related behaviours. While several parameters are modulated by JWH-018 independently of housing, other effects are environmentally mediated. The transition from standard housing to EE shall be carefully monitored.

Figure 1. Experimental timeline (see Methods for details).

Figure 2

(a) Body temperature (°C, mean ± SEM) in adult mice 60 minutes after a single injection of vehicle VEH or JWH-018 (0.3 mg/kg). *p < 0.05 significantly different from baseline conditions; (b) pain perception and drug effects measured as the latency (mean ± SEM) to the first hind-paw licking on the first day of drug administration in the hot-plate test. *p < 0.05 significantly different from VEH in post-hoc tests. (AFR = animal facility rearing; EE = environmental enrichment; VEH = vehicle; JWH-018 = JWH-018).

Figure 3. Spontaneous locomotion in adolescent mice measured in the home-cage, expressed in counts per minute (mean ± SEM), during a single 2-hr session starting 30 minutes after i.p injection with either vehicle (VEH) or JWH-018 (0.3 mg/kg), during the first (P23) (panel a) and last (P33) (panel b) administration day.

*p < 0.05 and **p < 0.01 significantly different from EE-VEH. Data are expressed as average ± SEM. (AFR = animal facility rearing; EE = environmental enrichment; VEH = vehicle; JWH-018 = JWH-018).

Figure 4. Motor coordination measured in drug-free state one month after JWH-018 administration through a rotating rod.

Falling latency (mean ± SEM) was measured during a single 4-min test session. *p < 0.05 significantly different from AFR. (AFR = animal facility rearing; EE = environmental enrichment; VEH = vehicle; JWH-018 = JWH-018).

Figure 5. Anxiety-related responses: (a) Time spent in the open sectors (s) and (b) SAP (stretched attend posture) frequency (mean ± SEM) in the elevated 0-maze.

*p < 0.05 significantly different from corresponding VEH. (AFR = animal facility rearing; EE = environmental enrichment; VEH = vehicle; JWH-018 = JWH-018).

Figure 6. Depressive-like responses: Number of nose pokes (mean ± SEM) in reinforced hole in the progressive ratio operant procedure, during the FR3 stage, conducted in drug-free state in adult mice that received either VEH or JWH-018 for 11 days during adolescence.

*p < 0.05 significantly different from VEH in post-hoc tests (AFR = animal facility rearing; EE = environmental enrichment; VEH = vehicle; JWH-018 = JWH-018).

Figure 7. Principal Component Analysis (PCA) of metabolite levels in hippocampus (a) and prefrontal cortex (b).

Factor 1 in hippocampus was composed of NAA, NAA + NAAG,Glu,Glu + Gln; Factor 2: PCr, Ins, and Tau; Factor 3: Gln, GPC + PCh. In PFC, Factor 1 was composed of: NAA, GPC + PCh, NAA + NAAG, Cr + PCr; Factor 2: Ins and Tau; Factor 3: Glu, Glu + Gln (AFR = animal facility rearing; EE = environmental enrichment; VEH = vehicle; JWH = JWH-018).

Figure 8. MRI panel: Example of in vivo sagittal T2-weighted spin-echo images (TR/TE = 3000/70 ms, slice thickness 0.8 mm, NS = 2, FOV = 20 × 20 mm², matrix 128 × 128).

Voxels localised on PFC and Hip are indicated by the white rectangles. MRS panel – Examples of in vivo ¹H spectra (as a black trace), acquired from the Hip (PRESS, TR/TE = 4000/23 ms, NS = 256). The result of LCModel fit is shown as a red trace overimposed on the spectrum. Metabolite assignments: Ins, inositol; Cr, creatine; PCr, phospho-creatine; Glu, glutamine; Gln, glutamate; Tau, taurine; PCho, phospho-choline; GPC, glicero-phospho-choline; NAA, N-acetyl-aspartate; NAAG, N-acetyl-aspartyl-glutamate; MM, macromolecules.