ObjectivesM4 muscarinic receptors (MR) presumably play a role in motor coordination. Previous studies have shown different results depending on genetic background and number of backcrosses. However, no attention has been given to biorhythms.Material and MethodsWe therefore analyzed biorhythms under a light/dark cycle obtained telemetrically in intact animals (activity, body temperature) in M4
KO mice growth on the C57Bl6 background using ChronosFit software. Studying pure effects of gene knockout in daily rhythms is especially important knowledge for pharmacological/behavioral studies in which drugs are usually tested in the morning.ResultsWe show that M4
KO mice motor activity does not differ substantially from wild‐type mice during light period while in the dark phase (mice active part of the day), the M4
KO mice reveal biorhythm changes in many parameters. Moreover, these differences are sex‐dependent and are evident in females only. Mesor, night–day difference, and night value were doubled or tripled when comparing female KO versus male KO. Our in vitro autoradiography demonstrates that M4
MR proportion represents 24% in the motor cortex (MOCx), 30% in the somatosensory cortex, 50% in the striatum, 69% in the thalamus, and 48% in the intergeniculate leaflet (IGL). The M4
MR densities were negligible in the subparaventricular zone, the posterior hypothalamic area, and in the suprachiasmatic nuclei.ConclusionsWe conclude that cholinergic signaling at M4
MR in brain structures such as striatum, MOCx, and probably with the important participation of IGL significantly control motor activity biorhythm. Animal activity differs in the light and dark phases, which should be taken into consideration when interpreting the results.