Despite being best known for their involvement in signal transduction at neuromuscular junctions, these receptors are also expressed throughout the central nervous system (CNS). As mentioned above, nicotinic ACh receptors are ionotropic, i.e., ligand-gated cation channels, whose activation by an agonist evokes a flux of K⁺, Ca²⁺, and Na⁺ ions (however not all subtypes of nAChRs are permeable for Na⁺), which in turn triggers mechanisms of Ca²⁺ signaling (1). These receptors typically comprise five subunits: either a homomeric combination of α subunits (for example α7) or a heteromeric combination of α(1–10), β(1–4), δ, and ε subunits. The specific combination of these subunits results in different pharmacological properties of the individual subtypes, such as ion selectivity and ligand affinity (14). The most common nAChR subtypes found in the brain are α7 and α4β2 receptors. Located at both pre- and postsynaptic sites, they play a pivotal role in various processes, such as learning and cognition (15), decision-making (9), and regulation of the postnatal development of the visual cortex (16). Thus, nicotinic ACh receptors constituted of specific subunits appear to be suitable pharmacological target for cognitive enhancement.

Muscarinic receptors are abundantly expressed throughout the brain; however, they are also found in various other tissues in the body, such as the heart (17, 18), the bladder and pulmonary system (19), and the intestine (20). As mentioned above, mAChRs do not serve as cation channels like nicotinic receptors, but instead are coupled with G-proteins, which transmit signals into the cell by affecting the activity of certain enzymes (such as the adenylyl cyclase, phospholipase C, etc.) (14, 21).

Five subtypes of mAChRs have been described, M1–M5. They differ in their level of expression in various parts of the body and the signal cascades they trigger after binding an agonist. Located mostly postsynaptically, the M1, M3, and M5 receptors (sometimes referred to as “M1-like” receptors) activate phospholipase C via G_q/11 protein, thus inducing a calcium influx into the cell. M2 and M4 on the other hand (the “M2-like” group), when activated lower the level of cyclic adenosine mono-phosphate in the cell by G_o/i protein-mediated inhibition of adenylyl cyclase. They are found both pre- and postsynaptically (8, 21, 22).

The outputs of signaling through specific cholinergic receptor subtypes may vary tremendously depending on the subtype of the receptors and their pre- or postsynaptic localization. The specific tissue and the type of the cell that expresses the receptors is also of major importance, as well as the metabolic state of the neuron at the precise time of receiving the signal, i.e., a cell with high intracellular levels of calcium may react differently to a signal than one with low intracellular concentrations of calcium. To further complicate any predictions of outcomes of cholinergic signaling and behavioral analysis, many neurons corelease ACh and glutamate, or ACh and gamma-aminobutyric acid (4, 21).

One of the major characteristics of the molecular structure of mAChRs is the evolutionarily highly conserved orthosteric ACh binding site, with a key asparagine residue (Asn105). This results in great difficulty when developing direct agonists and antagonists selective for a specific receptor subtype, and non-selective agents such as scopolamine, an antiemetic drug, and 3-iodothyronamine are widely used in research on memory impairment (23–25). Researchers have rather focused on developing compounds acting as allosteric ant/agonists and positive allosteric modulators (8, 26).

As mentioned above, the ACh binding site is evolutionarily highly conserved across all five mAChR subtypes, which in turn complicates the search for subtype-selective ligands. However, there is an abundance of allosteric sites that facilitate receptor activity modulation and are specific for each receptor subtype. These have enabled the development of highly selective compounds (8).

Orthosteric subtype-selective agents are scarce, though some may be found; for example, a recent study reported a novel compound PCS1055 that exhibits high selectivity for the M4 receptor (35). Also, some ligands have been shown to bind at the orthosteric site as well as one of the allosteric sites, thus achieving relatively high selectivity for a specific mAChR subtype. An example may be seen in the work of Jakubík et al. (36), where the mechanism of action of the M2-selective antagonist methoctramine was put under scrutiny. The authors reported that methoctramine binds with high affinity to the orthosteric site and at the same time interacts with lower affinity with an allosteric site at the second and third extracellular loops. Interestingly, in the presence of another orthosteric-binding ligand (such as N-methyl-scopolamine), methoctramine may still bind to the allosteric site, thus preventing the other ligand from dissociating from the receptor. This antagonist occasionally binds the M3 receptor as well, but with a much lower affinity due to the lack of the allosteric site found on M2 (36). Also, the time that antagonists take to bind to the receptor has been shown to be of crucial importance for the efficacy of receptor blockage. For example, due to its relatively slow binding, tiotropium seems less effective at blocking the M3AChR (37).

As to the effects of antimuscarinic drugs on the organism, these naturally depend on the means and site of administration (which determines where the agent exerts its influence, such as the brain following an intracerebroventricular injection or the heart after a systemic application of a drug unable to cross the blood–brain barrier). Thus, as the M1 and M4 receptors are abundantly expressed in parts of the brain affected in neurodegenerative diseases such as AD, it seems probable—and has been repeatedly reported—that stimulating cholinergic transmission via these receptors should enhance cognitive abilities, learning and memory, whereas blocking it would result in cognitive impairment (26).

In spite of the risk of various undesirable side-effects such as cognitive impairment, dry mouth, or even psychosis and addiction, if dosed with care, antimuscarinic drugs provide therapeutic effects in a number of conditions. For illustration, aclidinium and tiotropium are often prescribed in the treatment of chronic pulmonary disease, as well as asthma, overactive bladder, and irritable bowel syndrome (38–41).

Quite recently, scopolamine, a non-selective antagonist capable of crossing the blood–brain barrier, has been found to exhibit antidepressant properties (mediated probably by its binding to M1 and M2 receptors), even in patients unresponsive to standard therapy (11). This has proven beneficial not only to patients with major depressive disorder but also to those suffering from bipolar disorder (42). In addition, scopolamine is also used as an antiemetic, for example in treating postoperational nausea (23).

As mentioned previously, mAChR antagonists (e.g., biperiden, trihexyphenidyl) are also employed as prophylaxis and/or for the treatment of side-effects of antipsychotics prescribed in diseases such as schizophrenia. However, this method is currently on the decline due to the multitude of unwanted side-effects of the anticholinergic treatment (30, 33).

Biperiden, amongst other antimuscarinics, also acts as an antiparkinsonian agent and is thus sometimes prescribed to patients with Parkinson’s disease, as well as other diseases manifesting with parkinsonian symptoms. However, even here the risks of addiction and detrimental side-effects still remain (43, 44).

Quite surprisingly, given the amount of criticism regarding the cognitive side-effects of muscarinic antagonists, a recent study investigating the properties of a new potential treatment for AD reported M1-antagonism for these agents. The tested drug candidate was developed using a newly proposed approach to treating multifactorial diseases such as AD, which aims to hit multiple therapeutic targets with a single drug comprising a series of compounds, in this case combining 7-methoxytacrine and memantine. As the results of other tests (such as successful prevention of β-amyloid fibrillization, AChE inhibition, etc.) looked rather promising, the authors recommended the novel compound as a potential treatment, claiming that the observed M1-antagonism did not seem to exhibit noticeable effects (45). It is conceivable that muscarinic antagonism can act beneficially when it is a part of a broader spectrum of mechanisms of action.

Biperiden hydrochloride (or lactate) is an established M1-receptor selective antagonist. Approved for human usage and sold under the brand name of Akineton, it is prescribed for Parkinsonism (to improve motor abilities such as gait and tremor) and occasionally to suppress the side-effects of neuroleptics.

Apart from clinical practice, biperiden is also used in research as a cognitive impairer (46, 47). Biperiden has been shown to cross the blood–brain barrier without difficulties, thus enabling a simple administration of the drug, such as using intraperitoneal or subcutaneous injections (s.c.). The tissue distribution (V_d) for biperiden has been reported to be relatively high: with a brain to plasma ratio of up to 7–12 (44). The uptake of the drug by the tissues is quite rapid, possibly also due to its substantial transport into lysosomes (48). This makes biperiden a useful candidate as a specific drug, contrarily to scopolamine or atropine.

The Radial arm maze presents another task used to test spatial cognition, namely working and reference memory, but the procedure may also be adjusted to assess acquisition and memory retrieval (91, 92). This task was used for example in the study of Kay et al. (93), which showed that scopolamine elicits a stronger effect on working memory, while 3,4-methylendioxy-metamphtamine administration affects reference memory more prominently (93). Similar results regarding scopolamine administration had also been reported by Pilcher et al. (91), who compared the effects of scopolamine on working memory, acquisition and memory retrieval, concluding that there was stronger impairment in working memory relative to the other types (91).

This task may also be used for investigating differences in the consequences of acute vs. chronic drug administration, as shown for example by Ortega-Alvaro et al. (94). In their study, the authors found a significant impairment in rats’ performance in the radial arm maze following an acute injection of atypical antipsychotics (olanzapine and clozapine, used in the treatment of schizophrenia) and scopolamine, marked among others by a lower speed of movement. However, when following a chronic drug treatment, the observed deficits were absent, hence hinting at the ability to build a tolerance. The authors also concluded that chronic muscarinic antagonism may exert little or no influence over working memory (94).

One possible drawback of this task was raised in a study of Hodges et al. (95). The authors pointed out that the peripheral effects of scopolamine administration include “dry mouth,” which can lead to disruption of a rat’s ability to eat multiple food pellets and thus decrease their reward value.

The natural tendency of rodents to alternate between two choices in successive trials is exploited in a variety of simple T-shaped or Y-shaped mazes. Due to the simplicity of the task, alternation has been employed in the bulk of pharmacological studies using the scopolamine-induced amnesia model. Numerous studies [reviewed in Ref. (96)] have consistently shown that scopolamine treatment disrupts working memory both in discrete (97) and continuous versions of the alternation paradigm (98, 99). A article by Givens and Olton (100) demonstrated that intraseptal injections of scopolamine mimicked the detrimental dose-dependent effect of systemic scopolamine injections, indicating a critical contribution of the medial septal area. Further studies supported the central position of the septohippocampal pathway and revealed a more distributed network including a few other limbic and non-limbic structures (101).

Intraventricular administration of the M1 antagonist pirenzepine exerts similar effects as scopolamine, suggesting that M1 receptors may dominate in mediating spontaneous spatial alternation (102). On the other hand, M2 knockout mice were found to perform worse only under longer (20 s) but not short (5 s) delays in reinforced alternation in a T-maze compared to wild-type controls (103), suggesting a more complex contribution of particular mAChR types. M5 receptors seem to play a role in alternation as well, but the mechanism of action is likely indirect. As M5 receptors are expressed by endothelial cells and control cerebral vasodilatation, M5R−/− mice were found to exhibit a significantly reduced cerebral blood flow in the cerebral cortex, hippocampus, basal ganglia, and thalamus. In consequence, the low blood supply led to impaired long-term potentiation and consequently to a deterioration of spatial alternation (104).

Despite being almost ubiquitous in pharmacological research, the spatial alternation paradigm has some drawbacks. Investigators do not usually configure the maze to enforce animals to use praxis, taxon, or mapping strategies, or any combination of these. Therefore authors cannot report, in contrast to the MWM, whether effects are due to impairment of a particular mode of place navigation. Furthermore, the variability and consistency of results have been disputed, particularly in the spontaneous alternation paradigm. However, this drawback can be counterbalanced by the fact that under some circumstances, spatial alternation has been found to be superb at detecting hippocampal dysfunction (105).

Active place avoidance [(106–117), for review see Ref. (110, 111)] is a behavioral test specifically focusing on a rat’s ability to coordinate two conflicting frames of reference. An animal is placed into a slowly rotating arena where it needs to learn to locate a “to-be-avoided sector,” upon which stepping into it receives a foot-shock. The position of this sector does not change relative to the room frame; i.e., the animal has to actively move to another place in the arena so as not to be carried into the sector. The arena’s surroundings ought to contain distinct extramaze cues for the rats to navigate (110, 112–116).

The first study with scopolamine in this task (117) showed that a deficit induced by scopolamine at doses 1 and 2 mg/kg was not alleviated by intact spatial pretraining. A follow-up study (67) compared the performance of two rat strains obtained from the breeding colony of Institute of Physiology, CAS, Prague (Long-Evans and Wistar) in the MWM and active place avoidance following scopolamine treatment. As already mentioned, whereas in the MWM the disruption in learning and memory was similar, in active place avoidance the Wistar rats exhibited a higher sensitivity to scopolamine than the Long-Evans group (67). In general, active place avoidance tasks are sensitive to antimuscarinic action elicited by scopolamine, yet the effects are strain-specific and also present at relatively higher doses that can also affect procedural aspects. Unfortunately, no active place avoidance results on more selective antagonists, mAChR knockouts or other specific manipulations with the mAChR system are available, indicating the need for future research.

In the Barnes maze, a rat is placed in the center of a circular platform with holes at the edges. An escape cylinder is placed under one of these holes; the animals are trained to locate the position of this cylinder based on distal external cues. The use of odor trails is eliminated by rotating the platform in between trials, and animals presumably use a mapping strategy to locate the target (118).

Evaluations of antimuscarinic agents employing this paradigm are scarce. Consistent with other cognitive mapping taxing tasks, scopolamine was found to impair performance (119). Seeger et al. (103) used this task for investigating changes in cognition and behavior in M2 knock-out mice, reporting a severe impairment in learning, accompanied with decreased short-term and long-term potentiation (103). Another example of the usage of this test is the study by Gawel et al. (120), in which the authors examined the potential of cholinesterase inhibitors (donepezil and rivastigmine) to alleviate ethanol-induced cognitive impairment. The results showed an improvement in both memory retention and cognitive flexibility, the latter being more pronounced for rivastigmine (120).

The cone-field task represents another experimental paradigm for testing spatial learning and memory. It consists of a dodecagonal field with a number of cones topped with un/baited food cups in the middle and four starting boxes on the borders, from which the animal is released into the field. The ability of the rat to learn and remember the position of the baited cones is assessed. A suggested advantage of this test over tasks like the MWM is that it is based on positive reward learning (whereas the MWM relies on aversive learning). This task was used for example by Van der Staay et al. (121) to investigate the effects of AChE inhibitors (donepezil and metrifonate) on scopolamine-induced learning deficits in rats. The results showed that metrifonate, but not donepezil, was able to alleviate the working memory disruption produced by scopolamine (121). Specific conclusions on the role of mAChRs in this task are impossible due to the limited data.

In the hole-board task, an animal is placed in a rectangular box with a number of holes in the floor. Some of these are baited with a food reward. An animal is evaluated in its ability to learn and remember the position (using a mapping strategy) of the baited holes as well as the holes it has already visited. Different variations and adaptions of this task have been used. For example, Post et al. (122) published a article on a hole-board paradigm specially designed for mice (COGITAT) and presented its validation as a tool for testing spatial learning and memory via a scopolamine-induced performance deficit and its alleviation by metrifonate (122). Regarding the involvement of particular types of receptors, M1 receptors were shown to be important for reference memory (for non-baited holes) in a study evaluating biperiden in pigs (47). On the other hand, M2 receptors were shown to be important for working memory (memory for already-visited holes) in a study using transgenic mice (123).