2.5. Horizontal locomotor activity (HLA)
During the habituation phase (2 days), mice received an intraperitoneal (i.p.) saline injection and placed into a black Plexiglass open field arena (25x25x40 cm) under low-light conditions and white noise for 60 minutes. On the test day, horizontal locomotor activity (HLA) was measured as described by (Duart-Castells et al., 2019), with minor modifications. Briefly, the animals were given their corresponding i.p. injection (saline (5 ml/kg), pentedrone (3, 10 or 30 mg kg-1), N-ethyl-pentedrone (1, 3 or 10 mg kg-1), N,N-diethyl-pentedrone (3.5, 12.5 or 35 mg kg-1), α-PVP (1, 3 or 10 mg kg-1) or α-PpVP (7.5, 25 or 75 mg kg-1) and immediately placed in the open field arena. HLA was video-monitored for 60 min using a tracking software (Smart 3.0 Panlab, Barcelona, Spain) and their total travelled distance (in cm) was measured. All HLA experiments were performed between 8:00 am and 3:00 pm. On one hand, pentedrone and α-PVP doses were chosen following the results reported by Hwang et al., 2017 and Wojcieszak et al., 2018. On the other hand, there is no data available about the doses of N-ethyl-pentedrone, N,N-diethyl-pentedrone and α-PpVP used in animal research. Therefore, these doses were chosen in pilot experiments and according to the psychostimulant effect induced by pentedrone or α-PVP in our experiments at the medium doses tested in order to have, at least, one dose of N-ethyl-pentedrone, N,N-diethyl-pentedrone and α-PpVP equally effective (similar distance travelled after injection). Moreover, all the highest doses used were always 10-fold higher than the lowest dose tested, a protocol also used by the same authors when injecting pentedrone or α-PVP (Hwang et al., 2017; Wojcieszak et al., 2018).