Mice are nocturnal animals. Surprisingly, the majority of physiological/pharmacological studies are performed in the morning, i.e., in the non-active phase of their diurnal cycle. We have shown recently that female (not male) mice lacking the M4 muscarinic receptors (MR, M4KO) did not differ substantially in locomotor activity from their wild-type counterparts (C57Bl/6Tac) during the inactive period. Increased locomotion has been shown in the active phase of their diurnal cycle. We compared the effects of scopolamine, oxotremorine, and cocaine on locomotor response, hypothermia and spontaneous behavior in the open field arena in the morning (9:00 AM) and in the evening (9:00 PM) in WT and in C57Bl/6NTac mice lacking the M4 MR. Furthermore, we also studied morning vs. evening densities of muscarinic, GABAA, D1-like, D2-like, NMDA and kainate receptors using autoradiography in the motor, somatosensory and visual cortex and in the striatum, thalamus, hippocampus, pons, and medulla oblongata. At 9:00 AM, scopolamine induced an increase in motor activity in WT and in M4KO, yet no significant increase was observed at 9:00 PM. Oxotremorine induced hypothermic effects in both WT and M4KO. Hypothermic effects were more evident in WT than in M4KO. Hypothermia in both cases was more pronounced at 9:00 AM than at 9:00 PM. Cocaine increased motor activity when compared to saline. There was no difference in behavior in the open field between WT and M4KO when tested at 9:00 AM; however, at 9:00 PM, activity of M4KO was doubled in comparison to that of WT. Both WT and KO animals spent less time climbing in their active phase. Autoradiography revealed no significant morning vs. evening difference. Altogether, our results indicate the necessity of comparing morning vs. evening drug effects.
- Publikační typ
- časopisecké články 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
- chování zvířat fyziologie MeSH
- modely u zvířat MeSH
- mozek fyziologie MeSH
- myši inbrední C57BL MeSH
- myši knockoutované MeSH
- myši MeSH
- periodicita * MeSH
- pohybová aktivita genetika fyziologie MeSH
- receptor muskarinový M4 nedostatek genetika MeSH
- sexuální faktory MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Autoradiography helps to determine the distribution and density of muscarinic receptor (MR) binding sites in the brain. However, it relies on the selectivity of radioligands toward their target.3H-Pirenzepine is commonly believed to label predominantly M1MR,3H-AFDX-384 is considered as M2MR selective ligand. Here we performed series of autoradiographies with3H-AFDX-384 (2 nM), and3H-pirenzepine (5 nM) in WT, M1KO, M2KO, and M4KO mice to address the ligand selectivity. Labeling with3H-pirenzepine using M1KO, M2KO, and M4KO brain sections showed the high selectivity toward M1MR. Selectivity of3H-AFDX-384 toward M2MR varies among brain regions and depends on individual MR subtype proportion. All binding sites in the medulla oblongata and pons, correspond to M2MR. In caudate putamen, nucleus accumbens and olfactory tubercle, 77.7, 74.2, and 74.6% of3H-AFDX-384 binding sites, respectively, are represented by M4MR and M2MR constitute only a minor portion. In cortex and hippocampus,3H-AFDX-384 labels almost similar amounts of M2MR and M4MR alongside significant amounts of non-M2/non-M4MR. In cortex, the proportion of3H-AFDX-384 binding sites attributable to M2MR can be increased by blocking M4MR with MT3 toxin without affecting non-M4MR. PD102807, which is considered as a highly selective M4MR antagonist failed to improve the discrimination of M2MR. Autoradiography with3H-QNB showed genotype specific loss of binding sites. IN CONCLUSION: while3H-pirenzepine showed the high selectivity toward M1MR,3H-AFDX-384 binding sites represent different populations of MR subtypes in a brain-region-specific manner. This finding has to be taken into account when interpreting the binding data.
- Publikační typ
- časopisecké články MeSH
Brain acetylcholinesterase (AChE) variant AChERexpression increases with acute stress, and this persists for an extended period, although the timing, strain and laterality differences, have not been explored previously. Acute stress transiently increases acetylcholine release, which in turn may increase activity of cholinesterases. Also the AChE gene contains a glucocorticoid response element (GRE), and stress-inducible AChE transcription and activity changes are linked to increased glucocorticoid levels. Corticotropin-releasing hormone knockout (CRH-KO) mice have basal glucocorticoid levels similar to wild type (WT) mice, but much lower levels during stress. Hence we hypothesized that CRH is important for the cholinesterase stress responses, including butyrylcholinesterase (BChE). We used immobilization stress, acute (30 or 120 min) and repeated (120 min daily × 7) in 48 male mice (24 WT and 24 CRH-KO) and determined AChER, AChE and BChE mRNA expression and AChE and BChE activities in left and right brain areas (as cholinergic signaling shows laterality). Immobilization decreased BChE mRNA expression (right amygdala, to 0.5, 0.3 and 0.4, × control respectively) and AChERmRNA expression (to 0.5, 0.4 and 0.4, × control respectively). AChE mRNA expression increased (1.3, 1.4 and 1.8-fold, respectively) in the left striatum (Str). The AChE activity increased in left Str (after 30 min, 1.2-fold), decreased in right parietal cortex with repeated stress (to 0.5 × control). BChE activity decreased after 30 min in the right CA3 region (to 0.4 × control) but increased (3.8-fold) after 120 min in the left CA3 region. The pattern of changes in CRH-KO differed from that in WT mice.
- MeSH
- acetylcholinesterasa metabolismus MeSH
- butyrylcholinesterasa metabolismus MeSH
- funkční lateralita fyziologie MeSH
- fyzické omezení MeSH
- fyziologický stres fyziologie MeSH
- hormon uvolňující kortikotropin genetika metabolismus MeSH
- mozek metabolismus MeSH
- myši knockoutované MeSH
- myši MeSH
- psychický stres metabolismus MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Klíčová slova
- transportéry cholinu,
- MeSH
- acetylcholin analýza fyziologie MeSH
- cholin * analýza fyziologie MeSH
- mikrodialýza MeSH
- mikroelektrody MeSH
- modely u zvířat MeSH
- myši MeSH
- nervový přenos fyziologie MeSH
- prefrontální mozková kůra MeSH
- statistika jako téma MeSH
- Check Tag
- myši MeSH
- Publikační typ
- abstrakt z konference MeSH
- Klíčová slova
- omezení stresu, M2KO, signální kaskáda aktivovaná MR, levá srdeční komora,
- MeSH
- adenylátcyklasy sekrece MeSH
- exprese genu genetika MeSH
- fosfolipasy typu C sekrece MeSH
- funkce levé komory srdeční fyziologie účinky léků MeSH
- lidé MeSH
- myši MeSH
- prospektivní studie MeSH
- proteinkinasa C sekrece MeSH
- psychický stres * patofyziologie MeSH
- receptor muskarinový M2 MeSH
- receptory muskarinové * fyziologie genetika klasifikace MeSH
- synthasa oxidu dusnatého sekrece MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- grafy a diagramy MeSH
