Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA262

Poster Communications

Differences in drug response and spontaneous activity of M4 muscarinic receptor knockout mice during day and night time

P. Valuskova1, V. Farar1, J. Myslivecek1

1. Institute of Physiology, Charles University, 1st Faculty of Medicine, Prague, Czechia.

Scopolamine and oxotremorine are widely used as drugs for testing the motor activity and temperature in animal models. Cocaine is usually applied with aim to study addiction and effects on motor activity. Mice are nocturnal animals. Surprisingly, the majority of pharmacological studies are performed in the morning, i.e. in the non-active phase of their diurnal cycle. Therefore, we have compared the effects of scopolamine (0.5 mg/kg s.c.), oxotremorine (0.2 mg/kg s.c.) and cocaine (20 mg/kg s.c.) in inactive (at 9:00) and in active (at 21:00) period of the day on motor activity and temperature using telemetric system. We have used two mouse strains: wild types (C57BL/6NTac) and mice lacking M4 muscarinic receptors (same genetic background, 12th backcrossed generation, M4 KO), in light/dark 12h/12h conditions, lights on at 6:00. All experiments using drug application were conducted on females (n=74) where only differences in biorhythm parameters between M4 KO and WT were revealed. The telemetric probes were implanted into peritoneal cavity under anaesthesia and mice were left at least for 10 days to recover. The spontaneous motor activity and temperature were analyzed using Chronos-Fit and GraphPad software. The activity in novelty environment was tested using Open field test. We have also compared receptor density (muscarinic, GABAA, D1-like, D2-like dopamine, NMDA and kainite receptors) using autoradiography in several brain areas (motor, somatosensory, visual cortex, striatum, nucleus accumbens, thalamus, hippocampus and CA1, CA3 areas, dentate gyrus, olfactory tubercle, pons and medulla oblongata) in M4KO mice and their WT littermates. For comparison of multiple groups we have used one-way ANOVA with post-hoc Tukey's corrections. Values of p<0.05 were considered as significant. The effects of drugs depended on the time of application, i.e. if applied in active or inactive phase. After scopolamine and cocaine treatment, M4KO mice increased motor activity more profoundly during light (251 % and 348%, respectively) than in dark phase (186.0 % and 198%, respectively) when compared with saline control group. Oxotremorine reduced the temperature more in WT (to 27.3°C in light and 28.4°C in dark phase) than in M4KO (29.8 °C and 29.3°C, respectively). Oxotremorine also caused increase of the area under curve of this parameter at 9:00 than at 21:00. There was no difference in the open field between WT and KO when tested at 9:00 AM; however, at 9:00PM the KO activity was doubled in comparison to WT. At the same time, autoradiography revealed no significant morning vs. evening difference in the number of receptors in the tested brain areas. Altogether our results indicate the necessity of comparing morning vs. evening drug effects.

Where applicable, experiments conform with Society ethical requirements