Autoradiography of 3H-pirenzepine and 3H-AFDX-384 in Mouse Brain Regions: Possible Insights into M1, M2, and M4 Muscarinic Receptors Distribution

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.

Introduction

Muscarinic receptors (MR) are typical members of G protein coupled receptors family (Kruse et al., 2013) and can be divided into 5 subtypes (M₁–M₅) (Eglen, 2012), which activate different G proteins (G_q, G_i). Odd-numbered subtypes activate G_q, even-numbered activate G_i protein (Eglen, 2012; Kow and Nathanson, 2012; Reiner and Nathanson, 2012).

Respective MR subtypes have been assigned to different functions in CNS (Wess et al., 2007; Thomsen et al., 2017). Odd-numbered receptors are considered to be localized primarily post-synaptically, however, both M₂MR and M₄MR are localized both pre- and post-synaptically. As cholinergic autoreceptors, M₂ and M₄ provide feedback control of acetylcholine release (Zhang et al., 2002; Shin et al., 2015).

One of the means to determine MR density in different CNS areas is autoradiography. In vitro autoradiography has high sensitivity allowing to explore brain regions even with few MR. The use of very thin tissue sections in vitro autoradiography provides several advantages over the large tissue blocks used in binding studies in homogenates/membrane fractions. Brain sectioning allows analyzing MR density in virtually all brain areas of a single animal greatly reducing the number of experimental animals. Moreover, sectioning of a single brain generates sufficient number of tissue sections to explore the binding of multiple radioligands in a particular brain area of the same animal. This further reduces the number of experimental animals and allows comparing the effect of treatment on multiple targets (receptors, transporters) in a single animal (Farar and Myslivecek, 2016).

An important issue is the selectivity of radioligand used in experiments. A general problem in identification of MR subtypes present in specific regions of the central nervous system is the lack of highly subtype-selective muscarinic antagonists. The MR subtypes affinities for pirenzepine and AFDX-384, the most commonly used ones for discrimination of M₁ and M₂MR, respectively, are shown in Table 1. It can be deduced from this table that both pirenzepine and AFDX-384 have not only high affinity for M₁, and M₂ MRs, respectively, but also for M₄ MR subtype. In radioligand binding studies, it is therefore necessary to use a combination of various antagonists. However, for autoradiography detection this approach is not suitable because of evaluation limitations of such changed “binding.” Thus, the present protocols for M₁ and M₂ MR subtypes identification should be considered as method for detection of M₁ (or M₂) and also yet unidentified portion of M₄ MRs. Unfortunately, only few papers report these binding sites as M₂/M₄ MRs (e.g., Zavitsanou et al., 2003; Wang et al., 2014).

TABLE 1

Table 1. Muscarinic antagonist affinity constants (log affinity or pKi values) for mammalian muscarinic receptor subtypes.

The lack of MR subtype-selective ligands constitutes a significant obstacle in anatomical localization studies of MR, which are fundamental to our understanding of MR neuronal circuits. Three key strategies were developed to address regional distribution and relative abundance of particular MR subtype in the central nervous system.

First, in situ hybridization studies have identified neurons that synthetize MR (Buckley et al., 1988; Vilaró et al., 1990; Weiner et al., 1990). These and several other studies have shown that all five MR subtypes are expressed in the brain and mRNA for an individual MR subtype is distributed in a brain-region specific manner. However, while in situ hybridization studies provide valuable information about the sites of MR expression they do not address the real distribution of final proteins.

Second, MR subtype selective antibodies have been developed to map and quantify the distribution of individual MR by means of immunocytochemistry and immunoprecipitation (Levey et al., 1991; Yasuda et al., 1993). A body of reports have provided information about the distribution and quantities of individual MR. However, the reliability and selectivity of commonly used antibodies against MR have been questioned by their testing in specimens devoidof particular MR subtypes. This knockout-proof specific labeling has shown that the vast majority of tested antisera have identical labeling patterns in wild-type and knockout mice tissues (Jositsch et al., 2009).

Finally, anatomical localization and relative quantification of MR can be done by means of in vitro radioligand binding studies. This includes direct radioligand binding studies in tissue homogenates or plasma membrane preparation and indirect in vitro autoradiographic assays. In vitro autoradiography offers several advantages over direct binding studies. These includes tissue saving, high precision and reproducibility of results, high sensitivity and high degree of anatomical resolution. Moreover several ligands can be applied on consecutive sections derived from the same animal, further reducing numbers of experimental animals (Farar and Myslivecek, 2016).

Historically, tritiated pirenzepine was used as ligand that binds to MR with distinct binding in specific brain areas (Yamamura et al., 1983). Further, distinct distribution was found in the central nervous system. ³H-pirenzepine labels regions of the cerebral cortex, hippocampus, striatum and dorsal horn of the spinal cord, while sites in the cerebellum, nucleus tractus solitarius, facial nucleus and ventral horn of the spinal cord are labeled with ³H-QNB (non-specific muscarinic ligand) and not by ³H-pirenzepine (Wamsley et al., 1984). These observations indicated binding to different subtypes of MR. This was further expanded to definition of binding sites as M₁ MR (Villiger and Faull, 1985). In the middle of eighties pirenzepine binding sites were considered as M₁ MR (Buckley and Burnstock, 1986; Cortes and Palacios, 1986). On the other hand, ³H-AFDX-384 was considered from the beginning as a M₂ MR specific ligand (Aubert et al., 1992) and some authors were aware of limited selectivity (e.g., Mulugeta et al., 2003). In many cases³H-AFDX-384 and ³H-pirenzepine are still considered as selective ligands (Tien et al., 2004; Wolff et al., 2008).

Here we performed series of in vitro autoradiographies with ³H-AFDX-384 (2 nM, concentration below K_D and the most commonly used one), and ³H-pirenzepine (5 nM, concentration below K_D and the most used one). We took advantage of knockout mice models in the standard autoradiography procedures to compare binding in WT, M₁ KO, M₂ KO and M₄ KO mice. With this approach we addressed the selectivity of in vitro ³H-AFDX-384 autoradiography and provide a guide on the interpretation of results.

Moreover, we tried to block M₄MR using three concentration of MT3 toxin isolated from Dendroaspis angusticeps venom (1, 10, and 100 nmol/l). Another method to disable the binding to M₄MR was co-incubation with specific M₄MR antagonist PD102807 (10, 100, and 1 μmol/l). With an aim to block M₁MR we used MT7 toxin isolated from Dendroaspis angusticeps venom (1, 10, or 100 nmol/l) or pirenzepine (10 and 100 nmol/l).

In another of our experiment, using radiolabeled non-selective anatagonist, which binds all five MR subtypes (³H-QNB), we determined the contribution of M₁, M₂, and M₄ MRs to the total expression of MRs in the mouse brain.

We can conclude that ³H-pirenzepine showed high selectivity toward M₁MR. In contrast, 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 not only in the autoradiographies but also when these antagonists (pirenzepine, AFDX-384, PD102807 or MT3 toxin) are used as a mean to detect M₁MR, M₂MR, or M₄MR effects in functional studies. Experiments with ³H-QNB binding decrease in M₁, ${\text{M}}_{2^{··}}$, and M₄ KO animals showed the highest proportion (usually above 50%) of M₁MR in virtually all studied brain areas. M₂MR take up to 20% in cortical areas and 34% in thalamus. M₄MR were abundant (40% approximately) in thalamus, striatum and ventral striatum (NAc and OT), about 20% of M₄MR can be found in cortical structures.

Methods

Drugs

Atropine sulfate and pirenzepine dihydrochloride were purchased from Sigma-Aldrich (Sigma-Aldrich Co, St. Louis, MO, USA). PD102807 was purchased from Tocris Bioscience (Tocris Bioscience, Bristol, United Kingdom). MT3 toxin and MT7 toxin were purchased from Peptide Institute (Peptide Institute, Inc., Osaka, Japan). Pirenzepine [N-methyl-³H] (83.4 Ci/mmol), and AFDX-384 [2,3-dipropylamino-3H] (100.0 Ci/mmol) were from American Radiolabeled Chemicals (ARC, Inc.), Qinuclidinyl benzilate L-[benzilic-4,4′-³H] (50.5 Ci/mmol) was purchased from Perkin Elmer (Perkin Elmer Inc., USA).

Animals

Mice were treated in accordance with the legislature of the Czech Republic and EU legislature [European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes (Council of Europe No 123, Strasbourg 1985)], and the experimental protocol was approved by the Committee for the Protection of Experimental Animals of the 1st Medical Faculty, Charles University, Prague under N° MSMT-6316/2014-39.

Mice were maintained (3 per cage) under controlled environmental conditions (12 h/12 h light/dark cycle, 22±1°C, light on at 06:00 a.m.). Food and water were available ad libitum. Knockout mice and their WT counterparts of both genders (weighting 20–25 g, 11–13 weeks old), were used in the study. We studied fully backcrossed (at least 10 generations) muscarinic KO and WT littermates.

M₁ KO Mice

Mice lacking M₁ MR subtype were generated in the Wess laboratory (Bymaster et al., 2003) and then bred in our animal facility (Prague, Czech Republic). Their genetic background was C57Bl6/NTac. WT and KO genotypes were confirmed using polymerase chain reaction (PCR) analysis as previously described (Cea-del Rio et al., 2010).

M₂ KO Mice

Mice lacking M₂ MR subtype was generated in the Wess laboratory (Gomeza et al., 1999a) and then bred in our animal facility (Prague, Czech Republic). The genetic background was maintained on C57Bl6/NTac mouse line. WT and KO genotypes were confirmed using PCR analysis as previously described (Cea-del Rio et al., 2010).

M₄ KO Mice

Mice lacking M₄ MR subtype were generated in Wess laboratory (Gomeza et al., 1999b) and then bred in our animal facility (Prague, Czech Republic). Their genetic background was C57Bl6/NTac. WT and KO genotypes were confirmed using PCR analysis as previously described (Cea-del Rio et al., 2010).

Receptor Autoradiography

Tissue Preparation

For receptor determination, autoradiography was performed in several brain areas [motor cortex (MOCx), somatosensory cortex (SSCx), visual cortex (VisCx), striatum (Caudatum-Putamen, CPu), nucleus accumbens (NAc), thalamus (TH), hippocampus (Hipp) and its specific areas CA1, CA3 and dentate gyrus (DG), olfactory tubercle (OT), pons (Pons), and medulla oblongata (MY)] on sagittal brain sections of M₁KO, M₂KO, M₄KO mice and their WT littermates. Brains were rapidly removed (4–6 brains per group), frozen on dry ice, and then stored at −80°C until cryostat sectioning. Sixteen-micrometer thick sagittal sections were cut on a cryostat at −20°C and thaw-mounted on Superfrost^® Plus glass slides (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) and stored in storage boxes at −80°C until use. For autoradiography experiments brain sections were allowed to thaw and dry for 30 min at 22°C.

Autoradiography of Muscarinic Receptors on M₁KO, M₂KO, M₄KO, and WT Mice (³H-Pirenzepine, ³H-AFDX-384, and ³H-QNB Radioligand Binding)

Brain sections were allowed to thaw and dry for 30 min at 22°C and density of receptors was determined as previously described (Farar et al., 2012; Farar and Myslivecek, 2016). Dry M₁KO M₂KO, M₄KO, and WT sagittal brain sections were pre-incubated for 30 min in 50 mM potassium phosphate buffer (pH 7.4) at room temperature (RT). Following pre-incubation, sections were transferred into fresh 50 mM potassium phosphate buffer containing 2 nM ³H-AFDX-384, or 5 nM ³H-pirenzepine, or 2 nM ³H-QNB, and incubated for 60 min at RT. Non-specific binding was assessed on adjacent sections in the presence of 10 μM atropine sulfate. After incubation, sections were washed two times for 5 min each in ice-cold buffer and dipped for 2 s in ice-cold water to remove buffer salts. Wet sections were immediately dried by gentle stream of cold air. Dry sections were opposed to tritium sensitive Fuji BAS imaging plates (GE Healthcare Europe GmbH, Freiburg, Germany) in Kodak Biomax autoradiographic cassettes (Carestream Health, Inc., Rochester, NY).

Autoradiography of M₂ Muscarinic Receptors Labeled with ³H-AFDX-384 and Simultaneous Blocking of M₄ Receptors with PD102807

The procedure was similar as described above. However, the sections were transferred into buffer containing 2 nM ³H-AFDX-384 together with 10; 100 or 1,000 nM PD102807 or without PD102807 but with addition of DMSO which serves as dissolving agent for PD102807 and incubated for 60 min at RT. The final concentration of DMSO in incubation buffer must be less than 1%, in our case it was 0.1%. The non-specific binding was assessed as described above. This specific experiment was conducted on M₂ KO and M₄ KO brain sections only. After incubation, sections were treated as stated above.

Autoradiography of M₂ Muscarinic Receptors Labeled with ³H-AFDX-384 and Simultaneous Blocking of M₄ Receptors with MT3 Toxin Isolated from Dendroaspis Angusticeps Venom

The procedure was similar as described above. However, the sections were transferred into buffer containing 2 nM ³H-AFDX-384 together with 1; 10 or 100 nM MT3 toxin or without MT3 toxin and incubated for 60 min at RT. Non-specific binding was assessed as described above. After incubation, sections were treated as described above.

Autoradiography of M₂ Muscarinic Receptors Labeled with ³H-AFDX-384 and Simultaneous Blocking of M₁ Receptors with MT7 Toxin Isolated from Dendroaspis Angusticeps Venom or with Pirenzepine

The procedure was similar as described above. However, the sections were transferred into buffer containing 2 nM ³H-AFDX-384 together with 10 or 100 nM pirenzepine or without pirenzepine (serves as control) and incubated for 60 min at RT. Blocking of M₁ MR with MT7 toxin was conducted as following: the brain sections were at first pre-incubated with different concentrations of MT7 toxin (1; 10 or 100 nM) for 45 min. After pre-incubation with MT7, ³H-AFDX-384 with final concentration 2 nM was added into the buffer containing MT7 toxin and incubated for another 45 min. Non-specific binding as mentioned earlier. After incubation, sections were processed as described above.

Quantification of Receptor Density

To assure linearity of the signal, autoradiographic standards (American Radiolabeled Chemicals, Inc., St. Louis, MO, USA) were exposed along with the samples to the screens. Imaging plates were processed in Fuji Bioimaging Analyzer BAS-5000 (FUJIFILM corporation, Tokio, Japan) and digitized images analyzed with MCID analysis software (InterFocus GmbH, Mering, Germany). Measurements were taken and averaged from three sections for each animal and brain region.

Statistical Analysis

Statistical significance between groups was determined using 1-way ANOVA with Sidak post-hoc analysis. Student's t-test was used for comparison between two groups (typically WT vs. KO only).

Results

The Selectivity of ³H-Pirenzepine Toward M₁MR

Autoradiography with ³H-pirenzepine showed high selectivity of this ligand toward M₁MR (see Figure 1). In many brain areas, except in CPu and OT the binding in M₁ KO animals decreased by 90–99% (see Table 2). Especially, in hippocampal areas (dorsal hippocampus, CA1, CA3 area, and DG) ³H-pirenzepine binding sites were almost completely abolished. Please note very low binding in TH. The selectivity of pirenzepine was confirmed in M₂ KO animals (see Table 3) where no significant difference was shown in M₂ KO animals when compared to WT animals. Similar results were obtained when using M₄ KO animals (see Table 4). However, in some brain areas, we found small, but significant decrease in ³H-pirenzepine binding in M₄ KO animals (MOCx, SSCx, VisCx, CPu; 7, 13, 9, and 11%, respectively).

FIGURE 1

Figure 1. Changes in ³H-pirenzepine specific binding in M₁KO mice (top) and ³H-AFDX binding in M₂KO (middle) and M₄KO mice (bottom) when compared to WT animals. Non-specific binding was determined in the presence of 10 μM atropine sulfate. Ordinate: brain areas [motor cortex (MOCx), somatosensory cortex (SSCx), visual cortex (VisCx), striatum (Caudatum-Putamen, CPu), nucleus accumbens (NAc), thalamus (TH), hippocampus (Hipp) and its specific areas CA1, CA3, and dentate gyrus (DG), olfactory tubercle (OT)], abscissa: relative density [nCi/mg]. WT, wild type; KO, knockout mice. *p < 0.05, **p < 0.01, ***p < 0.001.

TABLE 2

Table 2. Percentage difference in ³H-pirenzepine binding in M₁KO, and in ³H-AFDX-384 to M₂KO, and M₄KO animals to WT animals.

TABLE 3

Table 3. Binding and percentage difference in ³H-pirenzepine binding in M₂KO animals and WT animals.

TABLE 4

Table 4. Binding and percentage difference in ³H-pirenzepine binding in M₄KO animals and WT animals.

The Selectivity of ³H-AFDX-384 Toward M₂MR

In contrast to the above mentioned experiment, ³H-AFDX-384 did not show selectivity toward M₂MR. If ³H-AFDX-384 was highly selective toward M₂MR, the binding of ³H-AFDX-384 would be barely visible in M₂ KO mice, as they do not express M₂MR. As it can be deduced from Figure 1 and Table 2, the binding reduction in many brain areas varied between 20 and 50%, with exception of pons, medulla oblongata and TH, where uniform population of M₂MR was found. Olfactory tubercle is the only area, in which the binding of ³H-AFDX-384 was completely preserved.

The Selectivity of ³H-AFDX-384 Toward M₄MR

Similar to the experiment in M₂ KO mice, the binding in M₄ KO mice showed only limited selectivity of ³H-AFDX-384 toward this MR subtype. However, ³H-AFDX-384 had similar selectivity to M₄MR and M₂MR (see Figure 1 and Table 2). A decrease in ³H-AFDX-384 binding depends on the brain area analyzed. This experiment also verified that pons and medulla oblongata express only M₂MR which can be deduced from the unchanged binding in M₄ KO mice. However, thalamus was not confirmed as an area with only M₂MR. Representative autoradiograms for M₁, M₂ and M₄ WT and KO mice are shown in Figure 2.

FIGURE 2

Figure 2. Representative autoradiograms in WT and KO mice: ³H-pirenzepine binding in M₁ WT and KO mice (top), ³H-AFDX 384 binding in M₂ WT and KO mice (middle), and ³H-AFDX 384 binding in M₄ WT and KO mice (bottom).

Binding of ³H-AFDX-384 in the Presence of M₄MR Antagonist PD102807

The effort to block M₄MR using antagonist PD102807 was unsuccessful as demonstrated in M₂ KO mice and M₄ KO mice. When the slices were incubated with 1 μmol/l of PD102807, we have recorded 100% inhibition of residual binding in M₂ KO mice (see Table 5). However, PD102807 also dose-dependently reduced ³H-AFDX-384 binding in M₄ KO mice, suggesting limited selectivity toward M₄MR.

TABLE 5

Table 5. Binding and percentage difference in ³H-AFDX 384 binding with PD102807 in M₂KO, and M₄KO animals vs. control (no PD102807).

Binding of ³H-AFDX-384 in the Presence of M₄MR Specific Toxin MT3

Similar to PD102807, MT3 toxin was unable to block M₄MR. This can be seen in competitions binding experiment with increasing concentration of MT3 toxin (Table 6). MT3 toxin was unable to completely block residual ³H-AFDX-384 binding in M₂ KO mice. Moreover, MT3 also dose-dependently reduced ³H-AFDX-384 binding in M₄ KO mice.

TABLE 6

Table 6. Binding and percentage difference in ³H-AFDX 384 binding with MT3 toxin in M₂KO, and M₄KO animals vs. control (no MT3 toxin).

Binding of ³H-AFDX-384 in the Presence of Pirenzepine and M₁MR Specific Toxin MT7 in WT and M₄ KO Mice

Incubation with pirenzepine (Table 7) showed dose-dependent reduction of ³H-AFDX-384 binding both in WT and M₄ KO mice. Irreversible, and very specific toxin MT7 also dose-dependently reduced ³H-AFDX-384 binding in both WT and M₄ KO mice (see Table 8).

TABLE 7

Table 7. Binding and percentage difference in ³H-AFDX 384 binding with pirenzepine in M₄WT and M₄KO animals vs. control (no pirenzepine).

TABLE 8

Table 8. Binding and percentage difference in ³H-AFDX 384 binding with MT7 toxin in M₄WT, and M₄KO animals vs. control (no MT7 toxin).

Binding of ³H-QNB in WT and M₁ KO, M₂ KO, and M₄ KO Mice

M₁ KO mice

Binding decrease in M₁ KO mice (Figure 3, top) showed high densities of M₁ MR in almost all brain areas. In cortical structures, there was approximately 60% of M₁ MR (64, 58, and 61% in MoCx, SSCx, and VisCx, respectively). The highest density of M₁ MR was found in hippocampus (91%) and in hippocampal regions (CA1: 89%, CA3 88%, dentate gyrus 98%, respectively). In striatum, there was 37%. Similar density of M₁ MR was found ventral striatum, i.e., in NAc (46%) and OT (48%).

FIGURE 3

Figure 3. Changes in ³H-QNB specific binding in M₁KO mice (top), M₂KO mice (middle), and M₄KO mice (bottom) when compared to WT animals. Non-specific binding was determined in the presence of 10 μM atropine sulfate. Ordinate: brain areas [motor cortex (MOCx), somatosensory cortex (SSCx), visual cortex (VisCx), medulla oblongate (MY), pons Varoli (pons), striatum (Caudatum-Putamen, CPu), nucleus accumbens (NAc), thalamus (TH), hippocampus (Hipp) and its specific areas CA1, CA3 and dentate gyrus (DG), olfactory tubercle (OT)], abscissa: relative density [nCi/mg]. WT, wild type; KO, knockout mice. *p < 0.05, **p < 0.01, ***p < 0.001.

M₂ KO Mice

Binding decrease in M₂ KO mice (Figure 3, middle) showed relatively low density of M₂ MRs in MoCx, SSCx, and VisCx (20, 22, 21%, respectively). In pons and medulla oblongata, there was 100% decrease suggesting pure M₂ MR population in these regions.

M₄ KO Mice

The decrease in binding in M₄ KO mice (Figure 3, bottom) revealed 21, 26, and 27% of M₄ MRs in MoCx, SSCx, and VisCx, respectively. The population of M₄ MRs is represented by 43% of total number of MRs in the thalamus. However, the muscarinic population in thalamus is low. The highest proportion of M₄ MRs was found in CPu, NAc, OT, where it represents 46, 38, and 49%, respectively.

Discussion

Here we used knockout proof concept to ascertain the selectivity of well-established and commonly used autoradiography protocols for labeling of putative M₂ and M₁MR. Putative M₂ and M₁MR were labeled with ³H-AFDX-384 and ³H-pirenzepine, respectively. We compared the pattern and relative density of ³H-AFDX-384 and ³H-pirenzepine specific binding sites in WT with M₁, M₂, and M₄ KO mice.

Our results demonstrate that ³H-pirenzepine labels predominantly, albeit not exclusively, M₁MR. Thus, according to our results, ³H-pirenzepine can be used as M₁MR selective ligand in brain cortex (MOCx, SSCx, VisCx) and hippocampus, in which more than 94% of ³H-pirenzepine binding sites are attributable to M₁MR. In the striatum and olfactory tubercle 10–13% of ³H-pirenzepine specific binding sites correspond to another MR subtype. Apart from these limitations, ³H-pirenzepine can be considered (in concentration 5 nmol/l and in the protocol described in Methods section) as M₁MR specific ligand.

To test whether ³H-pirenzepine binds also to M₄MR, we performed also ³H-pirenzepine autoradiography in M₄ KO mice, (see Table 1 for affinities). We assumed, that in case that ³H-pirenzepine binds M₄MR, there should be decrease in ³H-pirenzepine binding in M₄ KO mice. Indeed, ³H-pirenzepine binding in M₄KO mice is decreased in cortical areas and in caudate putamen. This result can be explained, however by two ways. Firstly, ³H-pirenzepine binds to M₄MR. Secondly, M₁MR are decreased in M₄ KO mice. M₄MR are enriched in the striatum and expressed at modest level in cortex (Gomeza et al., 1999b). Therefore, the decrease of ³H-pirenzepine binding due to the lack of M₄MR should be mostly seen in striatum and not in the cortex. We showed that there is decrease in ³H-pirenzepine binding by 11% in striatum of M₄ KO mice and that the residual binding of ³H-pirenzepine in M₁ KO mice is 10–13%. This suggest that indeed, in striatum ³H-pirenzepine binds also to M₄MR. In contrast, there was more than 95% reduction of ³H-pirenzepine binding in cortex of M₁ KO mice. The density of M₄MR in the cortex is approximately three-fold lower than in the striatum, but decrease in ³H-pirenzepine binding in cortex of M₄ KO mice was similar to that in striatum. Taken together, decrease in ³H-pirenzepine binding in the cortex of M₄ KO mice suggests rather alteration in the density of M₁ MR than binding to M₄MR. Once again and taking above mentioned results into account, we can consider ³H-pirenzepine as M₁MR highly specific ligand.

In contrast, ³H-AFDX-384 has poor selectivity toward M₂MR and labels mixed population of MR in a brain area dependent manner. Autoradiography in M₂ and M₄KO mice showed that ³H-AFDX-384 binds to multiple MR populations and that this population differs between brain areas. Moreover, ³H-AFDX-384 binds to M₄MR subtype in similar way as to M₂MR subtype what is in contrast to opinion that ³H-AFDX-384 is M₂MR preferential ligand. Our working hypothesis was that in case of ³H-AFDX-384 high selectivity toward M₂MR, gene deletion of M₂MR resulting in absence of M₂MR protein in M₂KO mice should result in complete loss of ³H-AFDX-384-specific binding. Even though ³H-AFDX-384 specific binding was reduced in M₂KO mice throughout the brain, the reduction was far less than expected. Surprisingly ³H-AFDX-384 specific binding was also reduced in M₄KO mice. In brain regions rich in M₄MR, such as striatum the ³H-AFDX-384 specific binding was reduced more than 70%. This suggest that³H-AFDX-384 binds to M₄MR subtype as well. Even though affinity of AFDX-384 is high (see Table 1), the extent of ³H-AFDX-384 binding to M₄MR in vitro autoradiography is surprising. In order to increase the selectivity of ³H-AFDX-384 toward M₂MR, we tried to block ³H-AFDX-384 binding to M₄MR by addition of antagonist PD102807 (with pKi higher by two orders of magnitude for M₄MR than for other MR subtypes: 7.3–7.4 vs. 5.2–6.7, see Table 1). Assuming the high selectivity of PD102807 for M₄MR and binding of ³H-AFDX-384 also to M₄MR, PD102807 should markedly reduce the residual ³H-AFDX-384 binding in M₂KO mice. Indeed, addition of PD102807 into incubation medium dose-dependently reduced the residual ³H-AFDX-384 specific binding in M₂KO mice, suggesting effective blocking of putative M₄MR. At the highest concentration (1,000 nmol/l), there was no residual binding of ³H-AFDX-384 in M₂KO mice. To test the potential binding capacity of PD102807 to other MR subtypes we performed similar experiment in M₄KO mice. In case that PD102807 is selective M₄MR ligand, adding PD102807 to the incubation medium should not interfere with ³H-AFDX-384 with binding capacity in M₄KO mice, since there are no M₄MR. However, PD102807 also dose-dependently reduced ³H-AFDX-384 specific binding in M₄KO mice with no M₄MR, indicating that under our experimental conditions PD102807 has poor selectivity toward M₄MR. Assuming that ³H-AFDX-384 predominantly labels M₂MR and M₄MR and no other MR subtype our results suggest, that PD102807 binds to both M₄MR and M₂MR. We can therefore conclude that co-incubation with PD102807 is not a suitable protocol for M₄MR binding elimination and M₂MR specific binding autoradiography. Thus, we explored another specific binding antagonist—MT3 toxin from Dendroaspis angusticeps venom—with effort to eliminate binding to M₄MR. This toxin is believed to block binding to M₄MR with great order of magnitude difference to other MR subtypes (pKi = 8.7 (M₄MR) vs. < 6 in M₂, M₃, M₅MR and 7.1 in M1MR, see Table 1). Similarly to PD102807, MT3 toxin was unable to effectively block ³H-AFDX-384 binding to M₄MR. There remained ³H-AFDX-384 binding capacity in M₂ KO mice in the highest (100 nmol/l) MT3 toxin concentration which should be able to block all M₄MR. Moreover, similarly to PD102807, MT3 not only dose-dependently decreased residual binding of ³H-AFDX-384 in M₂ KO mice, but also in M₄ KO mice. This suggest that MT3 toxin does not distinguish between M₄ and M₂MR, at least under our experimental conditions. Finally, we tested the hypothesis that ³H-AFDX-384 binds also to M₁MR. We tested our hypotheses in M₄ KO animals using two approaches: binding in the presence of pirenzepine (see Table 1 for affinities) and irreversible, and very specific toxin MT7 (with pKi = 9.8 to M₁MR and pKi <6 to other muscarinic subtypes). We choose M₄ KO mice in order to exclude possible binding of pirenzepine toward M₄MR in striatum as discussed above. Even at 10 nM concentration, pirenzepine significantly reduced ³H-AFDX-384 specific binding in M₄ KO mice, suggesting that ³H-AFDX-384 binds to a certain extent also M₁MR. Moreover, irreversible, and very specific toxin MT7 showed dose-dependent reduction of ³H-AFDX-384 specific binding in M₄ KO mice further supporting binding of ³H-AFDX-384 to M₁MR. It is therefore possible to conclude that ³H-AFDX-384 is not suitable ligand for M₂MR. Moreover, we were not successful in designing any protocol to block M₄ or M₁MR and increase the selectivity of ³H-AFDX-384 autoradiography of M₂MR.

All five MR subtypes are highly expressed in the brain (Levey et al., 1991; Wess et al., 2003; Oki et al., 2005). The M₁ and M₄ MRs represent the most abundant subtypes with the highest expression in the cortex, hippocampus and striatum that can be well illustrated using ³H-QNB autoradiography in our M₁ KO and M₄ KO mice.

In vitro receptor autoradiography has been used for decades for mapping anatomical distribution and quantification of broad range of receptors (Manuel et al., 2015; Farar and Myslivecek, 2016). The standard autoradiography is based on equilibrium binding of radioactively labeled antagonist. The key aspect of autoradiography is thus the selectivity of radioligand toward its target. MR subtypes show often overlapping pattern of expression and most MR ligands have poor selectivity, making the discrimination of individual subtypes difficult. While there are several ligands available, which specifically label MR, they do not distinguish individual MR subtypes. In vitro heterologous expression systems have helped to describe the affinity of a broad range of antagonists toward each of the five MR (Buckley et al., 1989; Dörje et al., 1991; Dong et al., 1995). These studies have identified so-called preferential MR ligands such as AFDX-384 and pirenzepine, which are commonly used to target putative M₂ and M₁MR, respectively. AFDX-384 has the highest affinity toward M₂MR, but also similar affinity toward M₄MR (Dörje et al., 1991).

We have proofed here that autoradiography protocol for ³H-pirenzepine is suitable for M₁MR detection (with limitations in CPu, NAc and OT) where pirenzepine binds also by approximately 10% to another MR subtype, likely M₄MR). Thus, this ligand can be used as M₁MR specific as before (e.g., Wamsley et al., 1984; McCabe et al., 1987; Farar and Myslivecek, 2016).

On the other hand, ³H-AFDX-384 is not suitable to detect M₂MR as believed previously (Entzeroth and Mayer, 1990; Wolff et al., 2008; Grailhe et al., 2009). However, there are some studies that previously correctly defined ³H-AFDX-384 as M₂ partially selective (Mulugeta et al., 2003) or as M₂/M₄ ligand (Nieves-Martinez et al., 2012).

The fact that ³H-AFDX-384 is not selective toward M₂MR was predictable in the light of AFDX-384 affinity to MR subtypes (see Table 1). However, almost identical binding to M₂MR and M₄MR is a new finding. We also tried to find specific protocol for M₂MR specific binding using different antagonists (PD102807, MT3 toxin, pirenzepine, and MT7 toxin). None of these antagonists were able to completely block other receptors. And thus it is necessary to conclude that there is no way to make the binding more specific to M₂MR.

Another aspect of our data is demonstration of M₁MR, M₂MR, and M₄MR distribution. It can be deduced from Figure 1 that M₁MR are not as hugely present in the cortical structures as in some hippocampal areas (dorsal hippocampus, CA1 area, and dentate gyrus). Very low M₁MR density is in the thalamus. Comparing binding in M₂WT and M₂KO mice we can conclude that there is relatively high density of M₂MR in cortical structures, medulla oblongata, pons and thalamus. In contrast, hippocampus and striatum does not have high amount of M₂MR. Striatum is, however, rich in M₄MR similarly to nucleus accumbens and olfactory tubercles. Also, the density of M₄MR in the cortex is relatively high. However, one should take into account that these data represent relative proportions of respective MR subtypes, since the binding was determined in radioligand concentrations around K_D and the receptors were not saturated. ³H-pirenzepine has pK_D around 7.9 (Watson et al., 1984), ³H-AFDX 384 has KD between 3 and 4 nmol.l⁻¹ (Castoldi et al., 1991) what is comparable to ³H-pirenzepine. In order to verify the proportion of respective MR subtypes we have used specific MR knockouts (M₁, M₂, and M₄ KOs) and measured a decrease in non-specific radioligand (³H-QNB) binding. This radioligand has much higher affinity to MR and pK_D ranging between 10.6 and 10.8 (Peralta et al., 1987). These experiments showed the highest proportion (usually above 50%) of M₁MR in virtualy all studied brain areas. M₂MR take up to 20% in cortical areas and 34% in thalamus. M₄MR were abundant (40% approximately) in thalamus, striatum and ventral striatum (NAc and OT), about 20% of M₄MR can be found in cortical structures. This is in general agreement with previously published data (Levey et al., 1991; Wess et al., 2003; Oki et al., 2005), although we have obtained slightly different pattern of MR subtypes distribution what can be caused by the use of different radioligand. Some discrepancies between results could be attributed to the fact that not all antibodies are selective (Pradidarcheep and Michel, 2016). As referenced by Manuel et al. (2015) relatively good correlation exists between the radioligand detected receptor number and the immunolabeled receptor protein for M₁ subtype.

Our results concerning the MR distribution are generally in in agreement with previous study (Oki et al., 2005) which employed different muscarinic knockouts but used non-specific radioligand (³H-NMS). However, in this study less brain areas were investigated and different method (direct radioligand binding) used. We sldo found similar pattern of MR subtype distribution as investigated using antibodies and electron microscopy (Hersch et al., 1994), immunoprecipitation (Levey et al., 1991; Tice et al., 1996) and radioligand binding (Flynn and Mash, 1993). Another study (Ferrari-Dileo et al., 1994) that used selective labeling found similar pattern of M₄MR distribution as here. All five MR subtypes are highly expressed in the brain (Levey, 1996; Oki et al., 2005). The M₁ and M₄ MR represent the most abundant subtypes with the highest expression in the cortex, hippocampus and striatum that can be well illustrated using ³H-QNB autoradiography in our M₁ KO and M₄ KO mice. Previous research has shown that MRs, mostly M₁ and M₄ MRs, might be an interesting pharmacological target for the treatment of neurodegenerative and neuropsychiatric diseases such as Alzheimer's disease, Parkinson's disease, schizophrenia, depression and also drug abuse (Bodick et al., 1997; Scarr et al., 1999; Brady et al., 2008; Langmead et al., 2008; Bradley et al., 2017; Dall et al., 2017). Therefore, in vitro radioligand binding studies, direct and indirect, represent an important pharmacological tool to precisely characterize the involvement of specific MR subtypes in such a disease as well as to study the binding properties, affinity and efficacy of new chemical compounds with the potential to become a selective drug toward the receptor.

Conclusion

We can therefore conclude that ³H-pirenzepine showed high selectivity toward M₁MR and can be used with minor limitations as M₁MR specific ligand. In contrast, ³H-AFDX-384 binding sites represent different populations of MR subtypes which is brain-region-specific. This finding has to be taken into account when interpreting the binding data. Our experiments with ³H-QNB binding decrease in M₁, ${\text{M}}_{2^{··}}$, and M₄ KO animals showed the highest proportion of M₁MR in virtualy all studied brain areas. M₂MR were expressed in cortical areas and in thalamus. M₄MR were abundant in thalamus, striatum and ventral striatum (NAc and OT as well as in cortical structures.

Author Contributions

JM and VF contributed to the conception and design of the reported studies. PV, IK, and VF conducted all of the experiments. SF, VF, and PV analyzed the data. PV and VF contributed to the drafting and revision of the manuscript. JM wrote the manuscript in the final form. All authors approved the final version and agreed to be accountable for all aspects of the work.

Funding

This research was supported by Supported by Grant 328314 from GAUK, CZ, PROGRES Q25/1LF/2 from Charles University, CZ, and by Czech Science Foundation Grant No 17-03847S.

Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The reviewer AB and handling Editor declared their shared affiliation.

Acknowledgments

The authors thank Katerina Janisova for her technical assistance.

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