The deletion of M4 muscarinic receptors (MRs) changes biological rhythm parameters in females. Here, we searched for the mechanisms responsible for these changes. We performed biological rhythm analysis in two experiments: in experiment 1, the mice [C57Bl/6NTac (WT) and M4 MR -/- mice (KO)] were first exposed to a standard LD regime (12/12-h light/dark cycle) for 8 days and then subsequently exposed to constant darkness (for 24 h/day, DD regime) for another 16 days. In experiment 2, the mice (after the standard LD regime) were exposed to the DD regime and to one light pulse (zeitgeber time 14) on day 9. We also detected M1 MRs in brain areas implicated in locomotor biological rhythm regulation. In experiment 1, the biological rhythm activity curves differed: the period (τ, duration of diurnal cycle) was shorter in the DD regime. Moreover, the day mean, mesor (midline value), night mean and their difference were higher in KO animals. The time in which the maximal slope occurred was lower in the DD regime than in the LD regime in both WT and KO but was lower in KO than in WT mice. In experiment 2, there were no differences in biological rhythm parameters between WT and KO mice. The densities of M1 MRs in the majority of areas implicated in locomotor biological rhythm were low. A significant amount of M1 MR was found in the striatum. These results suggest that although core clock output is changed by M4 MR deletion, the structures involved in biological rhythm regulation in WT and KO animals are likely the same, and the most important areas are the striatum, thalamus and intergeniculate leaflet.

• MeSH
• Actigraphy MeSH
• Locomotion physiology   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout MeSH
• Neostriatum physiology   MeSH
• Periodicity * MeSH
• Receptor, Muscarinic M4 genetics   physiology   MeSH
• Thalamus physiology   MeSH
• Animals MeSH
• Check Tag
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

INTRODUCTION: M4 muscarinic receptor (mAChR) knockout changed the female activity biological rhythm parameters. In this study, we focus on the biological rhythms of mAChRs (total + M1 mAChRs), acetylcholinesterase (AChE), and butyrylcholinesterase (BuChE) in M4 mAChR knockout (M4KO) and wild-type (WT) mice in specific brain areas. METHODS: Female mice were sacrificed every 4 hours, brains were removed, mAChRs were determined by autoradiography, and punching was used for the measurement of acetylcholinesterase and butyrylcholinesterase activity. The density of mAChRs was correlated with locomotor activity. RESULTS: An ultradian rhythm in total mAChRs was found in the suprachiasmatic nucleus (SCN) (both M4KO and WT). M4KO had a positive correlation between the number of mAChRs and locomotor activity. This rhythm was changed to circadian in WT with a peak in the active phase and to circadian rhythm in M4KO with phase shifts to the inactive/active phase in the intergeniculate leaflet (IgL) (positive correlation in KO), subparaventricular zone (SPVZ) (negative correlation in WT), and posterior hypothalamic area (PHA) (positive correlation in WT). The thalamus (TH) reveals circadian rhythms in WT and M4KO, with a peak in the active phase (no correlation). The striatum (Str), i.e., caudate ncl-putamen (CPu) (decrease in M4KO, positive correlation in both WT and KO) and the motor cortex (MCx) (no correlation), showed circadian rhythms (peak in active phase). Caudate ncl-putamen M1 mAChRs rhythm in WT was circadian, while M4KO animals revealed an ultradian rhythm. Cholinesterases revealed ultradian and circadian rhythms in different areas. DISCUSSION: We conclude that muscarinic receptor-directed biological rhythm of activity is determined in the striatum (caudate ncl-putamen) as a key structure mainly by M4 mAChRs with a supportive role of M1 mAChRs.

• Publication type
• Journal Article MeSH

OBJECTIVES: M4 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 METHODS: We 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. RESULTS: We 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. CONCLUSIONS: We 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.

• MeSH
• Behavior, Animal physiology   MeSH
• Models, Animal MeSH
• Brain physiology   MeSH
• Mice, Inbred C57BL MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Periodicity * MeSH
• Motor Activity genetics   physiology   MeSH
• Receptor, Muscarinic M4 deficiency   genetics   MeSH
• Sex Factors MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH