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Muscarinic receptors (mAChRs) are typical members of the G protein-coupled receptor (GPCR) family and exist in five subtypes from M1 to M5. Muscarinic receptor subtypes do not sufficiently differ in affinity to orthosteric antagonists or agonists; therefore, the analysis of receptor subtypes is complicated, and misinterpretations can occur. Usually, when researchers mainly specialized in CNS and peripheral functions aim to study mAChR involvement in behavior, learning, spinal locomotor networks, biological rhythms, cardiovascular physiology, bronchoconstriction, gastrointestinal tract functions, schizophrenia, and Parkinson’s disease, they use orthosteric ligands and they do not use allosteric ligands. Moreover, they usually rely on manufacturers’ claims that could be misleading. This review aimed to call the attention of researchers not deeply focused on mAChR pharmacology to this fact. Importantly, limited selective binding is not only a property of mAChRs but is a general attribute of most neurotransmitter receptors. In this review, we want to give an overview of the most common off-targets for established mAChR ligands. In this context, an important point is a mention the tremendous knowledge gap on off-targets for novel compounds compared to very well-established ligands. Therefore, we will summarize reported affinities and give an outline of strategies to investigate the subtype’s function, thereby avoiding ambiguous results. Despite that, the multitargeting nature of drugs acting also on mAChR could be an advantage when treating such diseases as schizophrenia. Antipsychotics are a perfect example of a multitargeting advantage in treatment. A promising strategy is the use of allosteric ligands, although some of these ligands have also been shown to exhibit limited selectivity. Another new direction in the development of muscarinic selective ligands is functionally selective and biased agonists. The possible selective ligands, usually allosteric, will also be listed. To overcome the limited selectivity of orthosteric ligands, the recommended process is to carefully examine the presence of respective subtypes in specific tissues via knockout studies, carefully apply “specific” agonists/antagonists at appropriate concentrations and then calculate the probability of a specific subtype involvement in specific functions. This could help interested researchers aiming to study the central nervous system functions mediated by the muscarinic receptor.

Social species form organizations that support individuals because the consequent social behaviors help these organisms survive. The isolation of these individuals may be a stressor. We reviewed the potential mechanisms of the effects of social isolation on cholinergic signaling and vice versa how changes in cholinergic signaling affect changes due to social isolation.There are two important problems regarding this topic. First, isolation schemes differ in their duration (1–165 days) and initiation (immediately after birth to adulthood). Second, there is an important problem that is generally not considered when studying the role of the cholinergic system in neurobehavioral correlates: muscarinic and nicotinic receptor subtypes do not differ sufficiently in their affinity for orthosteric site agonists and antagonists. Some potential cholinesterase inhibitors also affect other targets, such as receptors or other neurotransmitter systems. Therefore, the role of the cholinergic system in social isolation should be carefully considered, and multiple receptor systems may be involved in the central nervous system response, although some subtypes are involved in specific functions. To determine the role of a specific receptor subtype, the presence of a specific subtype in the central nervous system should be determined using search in knockout studies with the careful application of specific agonists/antagonists.

The dopaminergic system is one of the most important neurotransmitter systems in the central nervous system (CNS). It acts mainly by activation of the D1-like receptor family at the target cell. Additionally, fine-tuning of the signal is achieved via pre-synaptic modulation by the D2-like receptor family. Some dopamine drugs (both agonists and antagonists) bind in addition to DRs also to α2-ARs and 5-HT receptors. Unfortunately, these compounds are often considered subtype(s) specific. Thus, it is important to consider the presence of these receptor subtypes in specific CNS areas as the function virtually elicited by one receptor type could be an effect of other—or the co-effect of multiple receptors. However, there are enough molecules with adequate specificity. In this review, we want to give an overview of the most common off-targets for established dopamine receptor ligands. To give an overall picture, we included a discussion on subtype selectivity. Molecules used as antipsychotic drugs are reviewed too. Therefore, we will summarize reported affinities and give an outline of molecules sufficiently specific for one or more subtypes (i.e., for subfamily), the presence of DR, α2-ARs, and 5-HT receptors in CNS areas, which could help avoid ambiguous results.

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

Tight interactions exist between dopamine and acetylcholine signaling in the striatum. Dopaminergic neurons express muscarinic and nicotinic receptors, and cholinergic interneurons express dopamine receptors. All neurons in the striatum are pacemakers. An increase in dopamine release is activated by stopping acetylcholine release. The coordinated timing or synchrony of the direct and indirect pathways is critical for refined movements. Changes in neurotransmitter ratios are considered a prominent factor in Parkinson’s disease. In general, drugs increase striatal dopamine release, and others can potentiate both dopamine and acetylcholine release. Both neurotransmitters and their receptors show diurnal variations. Recently, it was observed that reward function is modulated by the circadian system, and behavioral changes (hyperactivity and hypoactivity during the light and dark phases, respectively) are present in an animal model of Parkinson’s disease. The striatum is one of the key structures responsible for increased locomotion in the active (dark) period in mice lacking M4 muscarinic receptors. Thus, we propose here a hierarchical model of the interaction between dopamine and acetylcholine signaling systems in the striatum. The basis of this model is their functional morphology. The next highest mode of interaction between these two neurotransmitter systems is their interaction at the neurotransmitter/receptor/signaling level. Furthermore, these interactions contribute to locomotor activity regulation and reward behavior, and the topmost level of interaction represents their biological rhythmicity.

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 with 3H-AFDX-384 (2 nM), and 3H-pirenzepine (5 nM) in WT, M1KO, M2KO, and M4KO mice to address the ligand selectivity. Labeling with 3H-pirenzepine using M1KO, M2KO, and M4KO brain sections showed the high selectivity toward M1MR. Selectivity of 3H-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% of 3H-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 of 3H-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 with 3H-QNB showed genotype specific loss of binding sites. In conclusion: while 3H-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.

Acetylcholinesterase (AChE) is a key enzyme in termination of fast cholinergic transmission. In brain, acetylcholine (ACh) is produced by cholinergic neurons and released in extracellular space where it is cleaved by AChE anchored by protein PRiMA. Recently, we showed that the lack of AChE in brain of PRiMA knock-out (KO) mouse increased ACh levels 200-300 times. The PRiMA KO mice adapt nearly completely by the reduction of muscarinic receptor (MR) density. Here we investigated changes in MR density, AChE, butyrylcholinesterase (BChE) activity in brain in order to determine developmental period responsible for such adaptation. Brains were studied at embryonal day 18.5 and postnatal days (pd) 0, 9, 30, 120, and 425. We found that the AChE activity in PRiMA KO mice remained very low at all studied ages while in wild type (WT) mice it gradually increased till pd120. BChE activity in WT mice gradually decreased until pd9 and then increased by pd120, it continually decreased in KO mice till pd30 and remained unchanged thereafter. MR number increased in WT mice till pd120 and then became stable. Similarly, MR increased in PRiMA KO mice till pd30 and then remained stable, but the maximal level reached is approximately 50% of WT mice. Therefore, we provide the evidence that adaptive changes in MR happen up to pd30. This is new phenomenon that could contribute to the explanation of survival and nearly unchanged phenotype of PRiMA KO mice.