About the Lab
Our research is primarily focused on the functions of muscarinic receptors. Muscarinic receptors (mAChRs) are typical members of the G protein-coupled receptor (GPCR) family and exist in five subtypes, M1 to M5. Muscarinic receptor subtypes do not sufficiently differ in their affinity for orthosteric antagonists or agonists; therefore, subtype-specific analyses are complicated and prone to misinterpretation.
Researchers mainly specialized in central nervous system (CNS) and peripheral functions and investigating mAChR involvement in behavior, learning, spinal locomotor networks, biological rhythms, cardiovascular physiology, bronchoconstriction, gastrointestinal functions, schizophrenia, and Parkinson’s disease usually use orthosteric ligands, while typically neglecting allosteric ligands. Moreover, they often rely on manufacturers’ claims regarding ligand selectivity, which can be misleading. However, the orthosteric binding site of mACChRs is very similar in all subtypes and thus the ligands declared as selective target to mutiple mAChR subtypes (Myslivecek, 2022). Importantly, limited subtype-selective binding is not unique to mAChRs but represents a general characteristic of most neurotransmitter receptors. Despite this limitation, the multitarget nature of drugs acting on mAChRs may be advantageous in the treatment of complex disorders such as schizophrenia.
To overcome the limited selectivity of orthosteric ligands, we recommend a systematic approach that includes: (i) verification of the presence of specific receptor subtypes in given tissues using knockout models, (ii) careful application of “subtype-selective” agonists and antagonists at appropriate concentrations, and (iii) calculation of the probability of individual subtype involvement in specific functions. This strategy may assist researchers studying CNS functions mediated by muscarinic receptors (Myslivecek, 2022).
Previous research
We have previously studied the functions of M2 muscarinic receptors (mAChRs), their signaling pathways (Tomankova et al., 2015), their role in stress responses (Valuskova et al., 2017), and their interactions with functionally antagonistic β-adrenoceptors (Tomankova et al., 2015). In addition, we have investigated the effects of acetylcholinesterase deficiency in the CNS (zero acetlycholine in CNS) on locomotor activity, selected G protein–coupled receptors, and ligand-gated ion channels (Farar et al., 2012, Farar et al., 2013).
Current Research Focus
Now we are focused on mAChRs functions (specifically on M4 and M1 mAChR).
Discrepancies observed in M4 mAChR–dependent locomotor activity led us to investigate the biological rhythm of locomotor behavior. The results were unexpected: we demonstrated increased locomotor activity during the dark (active) phase in female mice lacking M4 mAChRs, whereas no such difference was observed in M4 KO males (Valuskova et al., 2018a). These changes were reflected by an increase in the mesor, elevated nighttime activity, an increased night–day difference, and alterations in other circadian rhythm parameters.
Because changes in locomotor biological rhythms involve multiple brain regions, we employed in vitro autoradiography to identify brain areas potentially involved in rhythm regulation (Valuskova et al., 2019). This study also revealed marked differences in morning versus evening effects of muscarinic drugs (scopolamine, oxotremorine) in both wild-type (WT) and M4 KO animals. Subsequently, using a constant darkness paradigm to distinguish light responsiveness from endogenous circadian effects (Riljak et al., 2020), we demonstrated that although core clock output is altered by M4 mAChRs deletion, the brain structures involved in rhythm regulation are likely the same in WT and KO animals, namely the striatum, thalamus, and intergeniculate leaflet. Furthermore, we identified the involvement of M1 mAChRs in the striatum (caudate–putamen) in the regulation of locomotor activity biological rhythms.In the next paper, we focused on the biological rhythms of mAChRs (total, M1, and M4 mAcChRs) in different brain areas (Janisova et al., 2025) showing the striatum as a key structure for the M4 mAChRs directed locomotor activity biological rhythm. In detail, we observed the ultradian rhythm of total mAChR density in the suprachiamatic nucleus and there was a positive correlation between the number of mAChRs and locomotor activity. This ultradian rhythm changed to circadian in WT with a peak in the active phase, to circadian rhythm in M4KO with phase shifts to the inactive/active phase in the intergeniculate leaflet (positive correlation in KO), subparaventricular zone (negative correlation in WT), and posterior hypothalamic area (positive correlation in WT). The thalamus reveals circadian rhythms in WT and M4KO, with a peak in the active phase (no correlation). The striatum, i.e., caudate ncl.-putamen (decrease in M4KO, positive correlation both in WT and KO) and the motor cortex (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 with no correlation with locomotor activity.
Pharmacological and Autoradiography Studies
In a pharmacologically oriented study (Valuskova et al., 2018b), we performed a series of autoradiographic experiments using 3H-AFDX-384 (2 nM), and 3H-pirenzepine (5 nM) in WT mice and mAChR knockout models (M1 KO, M2 KO, and M4 KO) to assess ligand selectivity. Labeling with ³H-pirenzepine in M1 KO, M2 KO, and M4 KO brain sections demonstrated high selectivity toward M1 mAChRs.We also quantified the relative abundance of mAChR subtypes in the medulla oblongata and pons, caudate–putamen, nucleus accumbens and olfactory tubercle, cortex, and hippocampus. While ³H-pirenzepine showed high selectivity for M1 mAChRs, ³H-AFDX-384 binding sites represented heterogeneous populations of muscarinic receptor subtypes in a brain region–specific manner.
Lab Members
Jaromir Myslivecek, Head of the Lab
Katerina Janisova, Research Worker
Ph.D. students
Lenka Kleinova
Monika Uhlirova
Tereza Chrbolkova
Recent publications
Janisova, K., Uhlirova, M., Forczek, S. & Myslivecek, J.: Striatal M4 muscarinic receptors determine the biological rhythm of activity, with a supportive role of M1 muscarinic receptors. Frontiers in Pharmacology (2025), 1691118, DOI: 10.3389/fphar.2025.1691118
Myslivecek, J.: Multitargeting nature of muscarinic orthosteric agonists and antagonists. Front Physiol (2022), 974160, PMID: 36148314, DOI: 10.3389/fphys.2022.974160
Riljak, V., Janisova, K., and Myslivecek, J. (2020). Lack of M(4) muscarinic receptors in the striatum, thalamus and intergeniculate leaflet alters the biological rhythm of locomotor activity in mice. Brain Struct Funct225, 1615–1629. PMID: 32409918, DOI: 10.1007/s00429-020-02082-x
Valuskova, P., Riljak, V., Forczek, S.T., Farar, V., and Myslivecek, J. (2019). Variability in the Drug Response of M4 Muscarinic Receptor Knockout Mice During Day and Night Time. Frontiers in Pharmacology 10. PMID: 30936831, DOI: 10.3389/fphar.2019.00237
Valuskova, P., Farar, V., Forczek, S., Krizova, I., and Myslivecek, J. (2018b). Autoradiography of (3)H-pirenzepine and (3)H-AFDX-384 in Mouse Brain Regions: Possible Insights into M(1), M(2), and M(4) Muscarinic Receptors Distribution. Front Pharmacol 9, 124. PMID: 29515448, DOI: 10.3389/fphar.2018.00124
Valuskova, P., Forczek, S.T., Farar, V., and Myslivecek, J. (2018a). The deletion of M(4) muscarinic receptors increases motor activity in females in the dark phase. Brain Behav 8, e01057. PMID: 29978954, DOI: 10.1002/brb3.1057
Other important publications
Farar, V., Mohr F., Legrand M., Lamotte dIncamps B., Cendelin J., Leroy J., Abitbol M., Bernard V., Baud F., Fournet V., Houze P., Klein J., Plaud B., Tuma J., Zimmermann M., Ascher P., Hrabovska A., Myslivecek J and Krejci E.: Near-Complete Adaptation of the Prima Knockout to the Lack of Central Acetylcholinesterase. J Neurochem 122, no. 5 (2012): 1065-80. PMID: 22747514, DOI: 10.1111/j.1471-4159.2012.07856.x
Farar, V., A. Hrabovska, E. Krejci and J. Myslivecek: Developmental Adaptation of Central Nervous System to Extremely High Acetylcholine Levels. PLoS One 8, no. 7 (2013): e68265. PMID: 23861875, DOI: 10.1371/journal.pone.0068265
Myslivecek, J.: Two Players in the Field: Hierarchical Model of Interaction between the Dopamine and Acetylcholine Signaling Systems in the Striatum. Biomedicines (2021), 1, 25, PMID: 33401461, DOI: 10.3390/biomedicines9010025
Tomankova, H., Valuskova, P., Varejkova, E., Rotkova, J., Benes, J. & Myslivecek, J.: The M2 muscarinic receptors are essential for signaling in the heart left ventricle during restraint stress in mice. Stress (2015),2, 208-20, PMID: 25586419, DOI: 10.3109/10253890.2015.1007345
Edited books
Myslivecek J., Jakubik J. (Eds).: Muscarinic receptors: From structure to animal models. Springer USA, 2016 (first edition).
Myslivecek J., Jakubik J. (Eds).: Muscarinic receptors: From structure to animal models. Springer Nature, 2024 (second edition).