The M₃-muscarinic receptor regulates learning and memory in a receptor phosphorylation/arrestin-dependent manner

Abstract

Degeneration of the cholinergic system is considered to be the underlying pathology that results in the cognitive deficit in Alzheimer's disease. This pathology is thought to be linked to a loss of signaling through the cholinergic M₁-muscarinic receptor subtype. However, recent studies have cast doubt on whether this is the primary receptor mediating cholinergic-hippocampal learning and memory. The current study offers an alternative mechanism involving the M₃-muscarinic receptor that is expressed in numerous brain regions including the hippocampus. We demonstrate here that M₃-muscarinic receptor knockout mice show a deficit in fear conditioning learning and memory. The mechanism used by the M₃-muscarinic receptor in this process involves receptor phosphorylation because a knockin mouse strain expressing a phosphorylation-deficient receptor mutant also shows a deficit in fear conditioning. Consistent with a role for receptor phosphorylation, we demonstrate that the M₃-muscarinic receptor is phosphorylated in the hippocampus following agonist treatment and following fear conditioning training. Importantly, the phosphorylation-deficient M₃-muscarinic receptor was coupled normally to G_q/11-signaling but was uncoupled from phosphorylation-dependent processes such as receptor internalization and arrestin recruitment. It can, therefore, be concluded that M₃-muscarinic receptor–dependent learning and memory depends, at least in part, on receptor phosphorylation/arrestin signaling. This study opens the potential for biased M₃-muscarinic receptor ligands that direct phosphorylation/arrestin-dependent (non-G protein) signaling as being beneficial in cognitive disorders.

Among the multitude of physiological responses regulated by G protein-coupled receptors (GPCRs), one of the most intriguing is the ability of this superfamily of cell-surface receptors to regulate neurological and behavioral processes such as learning and memory (1–4). The members of the muscarinic acetylcholine receptor family are prominent among the GPCR subtypes associated with cognitive function because lesions in cholinergic innervations to the hippocampus and other brain areas are widely thought to underlie the cognitive deficit observed in Alzheimer's disease (5). Whereas the M₁-muscarinic receptor subtype has been proposed to be the subtype associated with acetylcholine-mediated cognition (6, 7), recent gene-knockout experiments have cast doubt on the direct role of this receptor subtype in learning and memory (1, 8). This has been reinforced by the discovery of a novel selective M₁-muscarinic receptor antagonist that was effective in blocking M₁-muscarinic receptor–mediated seizures in vivo but had no effect on hippocampal-based contextual fear conditioning (9). In addition, recent studies using an M₁-muscarinic receptor–positive allosteric modulator, BQCA, have suggested that M₁-muscarinic receptors can mediate learning and memory through an indirect mechanism by stimulating the prefrontal cortex (10). There is some controversy, however, because the same compound has been used in studies that suggest that M₁-muscarinic receptors can directly act on hippocampal-based memory (11).

In light of this uncertainty, we investigate here the possibility that the M₃-muscarinic receptor, which is expressed widely in the central nervous system including the hippocampus (12), might play a role in Pavlovian fear conditioning learning and memory. We also probe the contribution that receptor phosphorylation might play by using a knockin mouse strain that expresses a phosphorylation-deficient mutant of the M₃-muscarinic receptor. Our data point to an important role for the M₃-muscarinic receptor in hippocampal-based learning and memory and to the fact that this process depends on the phosphorylation status of the receptor.

Results

Involvement of the M₃-Muscarinic Receptor in Fear Conditioning.

M₃-muscarinic receptor knock out mice (M3R-KO) (13) showed a profound deficit in contextual [hippocampal based (14)] fear conditioning response (Fig. 1 A and B). In contrast, the tone fear conditioning response [associated with amygdala based learning and memory (15)] showed a trend toward a deficit, but the overall response was not significantly different from control mice (Fig. 1A). Importantly, there was no significant difference in nociception, as determined by pain thresholds, nor innate anxiety as measured in the elevated plus maze, between wild-type and M3R-KO mice (Fig. 1 C and D). These data indicate an important role for the M₃-muscarinic receptor in fear conditioning learning and memory.

Fig. 1.

Fear conditioning response in wild-type and M3R-KO mice. Wild-type (WT) or M₃-muscarinic receptor knockout mice (KO) were subjected to fear conditioning training. 24 h after training mice were analyzed for contextual fear conditioning and 24 h after this the animals were analyzed for cued fear conditioning. (A) Cumulative fear conditioning responses in WT (n = 10) and KO (n = 10) mice. (B) Example of a single movement trace from WT and a KO mouse undergoing a test for contextual fear conditioning. Freezing is defined when movement amplitude falls below the white line. Length and frequency of freezing is also represented by the yellow bars on the bar graph. (C) Measure for anxiety was conducted on WT (n = 10) and KO (n = 10) mice using the elevated plus maze. (D) Pain thresholds determined in WT (n = 10) and KO (n = 10) mice. Data represents the means ± SE. *, P < 0.01 (t test).

Generation of a Knockin Mouse Strain Expressing a Phosphorylation-Deficient M₃-Muscarinic Receptor.

Nearly all GPCRs are rapidly phosphorylated in response to agonist stimulation at multiple sites on the intracellular loops and C-terminal tail (16). The M₃-muscarinic receptor is no exception, being phosphorylated by a number of protein kinases at serine clusters contained within the third intracellular loop (17, 18). We tested the possibility that M₃-muscarinic receptor phosphorylation was important in the mechanism of action of this receptor in physiological responses. Toward this goal, we generated mutant mice in which the wild-type M₃-muscarinic receptor coding sequence had been replaced (by homologous recombination) with a mutant version of the receptor containing 15 point mutations in serine phospho-acceptor sites within the third intracellular loop of the receptor (Fig. S1 A–C). Mice homozygous for the knockin gene targeting event (designated M3R-KI) developed into adult animals with no gross physiological defects and showed normal Mendelian breeding.

Ligand binding analysis of cerebellar granule cell (CG) neurons using the hydrophilic antagonist [H³]-N-methyl scopolamine that labels cell surface receptors demonstrated no significance difference in the expression of the mutant receptor in neurons derived from M3R-KI mice compared with wild-type controls [wild-type B_max= 167 ± 1 fmol/mg protein, M3R-KI B_max = 143 ± 3 fmol/mg protein (n = 3 ± SE)]. Furthermore, immunoprecipitation of the M₃-muscarinic receptor from extracts of CG neurons or from adult hippocampal extracts followed by Western blotting for the M₃-muscarinic receptor revealed that the receptor was expressed similarly in both M3R-KI and wild-type neurons (Fig. S1 D and E).

As anticipated, the mutant M₃-muscarinic receptor showed reduced levels of agonist-mediated phosphorylation as determined by [³²P]orthophosphate labeling of CG neurons derived from wild-type and M3R-KI mice. The mutant M₃-muscarinic receptor showed a 63 ± 1% reduction in agonist-mediated phosphorylation compared with wild-type receptor controls (Fig. 2 A and B).

Fig. 2.

Characterization of the phosphorylation-deficient M₃-muscarinic receptor. (A) Phosphorylation of the M₃-muscarinic receptor was determined in CG neurons derived from wild-type (WT) or M3R-KI (KI) mice metabolically labeled with [³²P]orthophosphate. CG neurons were stimulated with or without methacholine (Meth, 100 μM) for 5 min before solubilization and immunoprecipitation of the M₃-muscarinic receptor. Shown is an example autoradiograph (showing long and short exposures) and the Western blot loading control (M₃-Receptor Western). (B) Quantification of the receptor phosphorylation data shown in A. The data represents the mean ± SE from four independent experiments. (C) Coupling of the M₃-muscarinic receptor to the phosphoinositide pathway was determined in CG neurons derived from wild-type (WT) or M3R-KI (KI) mice transfected with the phosphoinositide biosensor (eGFP-PH_PLCδ1). CG neurons were stimulated with methacholine (0.1 mM; downward arrow) for the indicated time (the bar represents 30 s). Agonist was then removed (upward arrow). The translocation of the biosensor from the plasma membrane to the cytoplasm was monitored on an inverted epifluorescence microscope. Shown are images of a single neuron before and during stimulation with methacholine. On the left is a representative time course of fluorescence change in the cytoplasm expressed as a self ratio (F/F₀). (D) Internalization of M₃-muscarinic receptors expressed in CG neurons derived from wild-type (WT) and M3R-KI (KI) mice determined by stimulating cultures at 37 °C in the absence or presence of methacholine (0.1 mM) for 30 min. Shown is the mean percentage receptor internalization ± SE (n = 5). (E and F) β-Arrestin recruitment to the M₃-muscarinic receptor determined using PathHunter CHO-K1 cells expressing either the wild-type human M₃-muscarinic receptor (WT) or the mutated human M₃-muscarinic receptor lacking phospho-acceptor sites (H-M3_phos-neg) (18). Shown are concentration response curves to the full muscarinic receptor agonists methacholine and acetylcholine. The data are the means ± SE of three independent experiments.

Phosphorylation-Deficient M₃-Muscarinic Receptor Shows “G Protein Bias” in Neurons.

To determine whether the phosphorylation-deficient M₃-muscarinic receptor was functionally coupled to G_q/11 proteins in CG neurons, a biosensor for phosphoinositide signaling was used. This biosensor consisted of the pleckstrin homology (PH) domain of phospholipase Cδ₁ fused to eGFP (eGFP-PH_PLCδ1), which binds phosphoinositide 4,5-bisphosphate (PIP₂) in the membrane under basal conditions but translocates to the cytoplasm following receptor-mediated PIP₂ hydrolysis (19). Using this biosensor, we found that the coupling of the mutant receptor expressed in M3R-KI mice to the phosphoinositide pathway was not significantly different from that observed for the wild-type receptor (Fig. 2C and Fig. S2). These data were confirmed in recombinant CHO cells expressing the phosphorylation-deficient M₃-muscarinic receptor where stimulation of inositol phosphate-mediated calcium mobilization was not significantly different from the wild-type receptor (Fig. S3 A–C).

In contrast, there was a significant deficit in the coupling of the phosphorylation-deficient M₃-muscarinic receptor to phosphorylation/arrestin-dependent processes. GPCR internalization is well known to depend on the phosphorylation of the receptor and the subsequent recruitment of arrestin (20). Here, we show that internalization of the M₃-muscarinic receptor in CG neurons was significantly reduced in neurons derived from the M3R-KI mice, indicating that there was a deficit in phosphorylation/arrestin signaling (Fig. 2D). We next tested whether removal of the phospho-acceptor sites on the M₃-muscarinic receptor affected the ability of the receptor to recruit arrestin. These experiments were conducted on the human M₃-muscarinic receptor where we have shown previously that mutation of the phospho-acceptor sites (comparable to those of the mouse receptor) resulted in a reduction in both agonist-mediated receptor phosphorylation and receptor internalization (18). In an assay for the recruitment of β-arrestin, the phosphorylation-deficient M₃-muscarinic receptor showed a significant reduction in both the potency and efficacy of arrestin-recruitment in response to the full agonists methacholine and acetylcholine (Fig. 2 E and F) [EC₅₀ for arrestin recruitment to the wild-type receptor with methacholine and acetylcholine was −5.35 ± 0.1 M and −5.57 ± 0.1 M (log₁₀), respectively, compared with −4.95 ± 0.1 M and −5.14 ± 0.1 M (log₁₀) for the phosphorylation-deficient receptor (n = 5 ± SE)]. Consistent with the data from the mouse M₃-muscarinic receptor, mutation of the phosphorylation sites in the human receptor did not affect coupling to the G_q/11/calcium mobilization pathway (Fig. S4 A and B).

Involvement of M₃-Muscarinic Receptor Phosphorylation in Fear Conditioning.

The M3R-KI mice were tested for fear conditioning learning and memory. In these experiments, the wild-type mice spent 49.2 ± 8.9% of the time frozen in the contextual fear conditioning test and 63.9 ± 7.2% of the time frozen in the tone-associated fear conditioning test (Fig. 3 A and B). These responses were significantly reduced in M3R-KI animals, where the contextual fear conditioning response was 28.6 ± 3.2% and the tone-associated fear conditioning response was 47.5 ± 3.4% (Fig. 3 A and B). No significant difference was observed in controls for nociception or innate anxiety (Fig. 3 C and D).

Fig. 3.

Fear conditioning response in wild-type and M3R-KI mice. Wild-type (WT) or M3R-KI (KI) mice were subjected to fear conditioning training. Twenty-four hours after training, mice were analyzed for contextual fear conditioning, and 24 h later the animals were analyzed for cued fear conditioning. (A) Cumulative fear conditioning responses in WT (n = 10) and M3R-KI (n = 10) mice. (B) Example of a single movement trace from a WT and a M3R-KI (KI) mouse undergoing a test for contextual fear conditioning. Freezing is defined when movement amplitude falls below the white line. Length and frequency of freezing are also represented by the yellow bars on the bar graph. (C) Measure for anxiety was conducted on WT (n = 10) and M3R-KI (n = 10) mice using the elevated plus maze. (D) Pain thresholds were determined in WT (n = 10) and M3R-KI (n = 10) mice. Data represents means ± SE. *, P < 0.05 (t test).

These data firstly point to an important role for receptor phosphorylation in the mechanism of M₃-muscarinic receptor–mediated fear conditioning learning and memory. Furthermore, due to the G protein bias of the receptor, the data indicate that coupling of the receptor to heterotrimeric G proteins is not the primary mechanism of mediating the fear conditioning response but that rather it is via a G protein-independent process that involves receptor phosphorylation.

The importance of receptor phosphorylation in the fear conditioning response was further confirmed in studies of neuronal activity as measured by changes in the expression of the early/intermediate gene c-Fos. Here, c-Fos expression was rapidly induced in the hippocampus and amygdala of wild-type mice following fear conditioning (Fig. 4). An observation consistent with other studies (21). However, the c-Fos expression after fear conditioning in M3R-KI mice was significantly reduced in the hippocampus when compared with controls (Fig. 4 A and B). Hence, in wild-type animals c-Fos expression in the dentate gyrus increased by 359% and 219%, 30 and 120 min after fear conditioning, respectively. In contrast, c-Fos immunostaining in the dentate gyrus of the M3R-KI mice increased by only 167% and 68%, 30 and 120 min after fear conditioning, respectively (Fig. 4A). Similarly, the induction of c-Fos expression was significantly attenuated in the CA3 region of the hippocampus in M3R-KI animals when compared with wild-type controls (Fig. 4B). Importantly, despite the fact that c-Fos expression in amygdala was substantially increased following fear conditioning, there was no significant difference in c-Fos expression in this brain region observed following fear conditioning in the wild-type and M3R-KI mice (Fig. 4C).

Fig. 4.

c-Fos induction in the hippocampus and amygdala following fear conditioning depends on M₃-muscarinic receptor phosphorylation. Wild-type (WT) or M3R-KI (KI) mice were subjected to fear conditioning training, and the animals were killed by cardiac perfusion of fixative solution 30 or 120 min after training. The brain was then dissected and sectioned before staining for c-Fos expression. Basal conditions represent animals that had not undergone fear conditioning training. (A) Immunofluorescent staining for c-Fos expression in the dentate gyrus of the hippocampus. (B) Immunofluorescent staining for c-Fos expression in the CA3 region of the hippocampus. (C) Immunofluorescent staining for c-Fos expression in the amygdala. Graphs represent quantification of the immunofluorescent staining showing means ± SE (n = 4). *, P < 0.05 (t test). c-Fos appears as the red label and nuclei are stained blue using DAPI.

Interestingly, previous studies had determined that the M₃-muscarinic receptor can directly up-regulate c-Fos expression (22). We investigated here whether this process depended on β-arrestin. siRNA knockdown of β-arrerstin1/2 in CHO cells stably expressing the M₃-muscarinic receptor resulted in a marked reduction in M₃-muscarinic receptor–mediated c-Fos up-regulation (Fig. S5). These data raise the possibility that those neurons in the hippocampus that express the M₃-muscarinic receptor may show reduced c-Fos up-regulation in M3R-KI mice where receptor signaling via β-arrestin is disrupted.

M₃-Muscarinic Receptor Phosphorylation in the Hippocampus.

Because our data pointed to a role for M₃-muscarinic receptor phosphorylation in hippocampal-based fear conditioning learning and memory, we investigated directly the phosphorylation status of the receptor. Mass spectrometry of the recombinant M₃-muscarinic receptor expressed in Chinese hamster ovary cells demonstrated that the receptor was phosphorylated on serine³⁸⁴ in the third intracellular loop (Fig. S6A). A phospho-specific antibody against this site was generated and confirmed to be phospho-specific on the basis that it did not recognize the nonphosphorylated epitope present in a bacterial fusion protein containing the third intracellular loop of the receptor (Fig. S6B). Furthermore, the antibody did identify the intact M₃-muscarinic receptor immunoprecipitated from transfected CHO cells (Fig. S6C) but not following treatment with calf intestinal phosphatase which de-phosphorylated the receptor (Fig. S6C).

Although it was clear from our previous studies that the M₃-muscarinic receptor was phosphorylated at multiple sites (18), not just serine³⁸⁴, we nevertheless investigated the phosphorylation at this single site as an exemplar of receptor phosphorylation. Immunoprecipitation of the M₃-muscarinic receptor from wild-type hippocampus followed by Western blotting using the anti-phosphoserine³⁸⁴ antibody revealed that the receptor was phosphorylated at serine³⁸⁴ in the basal state in the mouse hippocampus (Fig. 5A). The phosphorylation status of the receptor at serine³⁸⁴ increased by 75% following the incubation of the dissected hippocampus with the muscarinic receptor agonist methacholine (Fig. 5A). In these experiments, the specificity of the anti-phosphoserine³⁸⁴ antibody was further confirmed by the lack of a band associated with the M₃-muscarinic receptor in hippocampal preparations from M3R-KI mice expressing the phosphorylation-deficient M₃-muscarinic receptor mutant, which includes the Ser³⁸⁴ to Ala mutation (Fig. 5A).

Fig. 5.

Phosphorylation of serine³⁸⁴ on the M₃-muscarinic receptor in the hippocampus. (A) Hippocampi dissected from wild-type (WT) or M3R-KI (KI) mice were exposed to vehicle or methacholine (Meth, 1 mM) for 10 min. Membranes were then prepared and solubilized. The M₃-muscarinic receptor was then immunoprecipitated using an anti-M₃-muscarinic receptor monoclonal antibody and the immunoprecipitate probed in a Western blot using anti-phosphoserine³⁸⁴ antibody. The blot was then stripped and reprobed with a rabbit polyclonal anti-receptor antibody as a loading control. A typical experiment is shown together with the quantification from three independent experiments. (B) Wild-type mice were subjected to fear conditioning training, and the animals were killed either immediately after training (time point zero) or 5 min after training. The hippocampi were then dissected and membranes were prepared. Phosphorylation of the M₃-muscarinic receptor using anti-phosphoserine³⁸⁴ antibody was then determined as in A. As a control for the stress response to the footshock, animals were subjected to immediate footshock with no fear conditioning training. Under these conditions there was no significant change in the phosphorylation status of serine 384 (Right). Typical experiments are shown together with the quantification from three independent experiments. The graphical data represent the means ± SE of three independent experiments. *, P < 0.05 (t test).

These data established that M₃-muscarinic receptor phosphorylation at serine³⁸⁴ is regulated by agonist in the hippocampus. We next examined whether physiological stimuli, such as fear conditioning, could mediate changes in the phosphorylation status of the receptor. To test this possibility, mice were killed either immediately after fear conditioning training (time point zero) or 30 min after training. The receptor was then immunoprecipitated from hippocampal lysates and probed with the anti-phosphoserine³⁸⁴ antibody. The phosphorylation status of the receptor increased by 30% and 39% immediately after training (time point zero) and 30 min after training, respectively (Fig. 5B), demonstrating that the phosphorylation status of the M₃-muscarinic receptor did change in response to fear conditioning training. Importantly, applying an immediate footshock with no fear conditioning training did not result in a significant change in the phosphorylation status of serine³⁸⁴ (Fig. 5B).

Discussion

We show here that M3R-KO mice displayed a deficit in fear conditioning learning and memory, indicating that this receptor subtype is important in the mechanism of cholinergic-mediated learning and memory. This finding may have significant clinical implications because stimulation of cholinergic transmission by the use of acetyl cholinesterase inhibitors is the front line treatment for the cognitive deficit in Alzheimer's disease (23). The target for the enhanced cognition in response to this treatment was thought to be M₁-muscarinic receptor signaling. This notion is based on the fact that the M₁-muscarinic receptor is the most highly expressed muscarinic receptor subtype in the cortex and hippocampus (12) and that pharmacological studies point to this receptor subtype as being the primary effector of cholinergic-mediated cognition (6, 24, 25). However, recent studies using M₁-muscarinic receptor knockout mice (1) together with studies using more selective pharmacological tools (9, 10) have suggested that the M₁-muscarinic receptor does not mediate hippocampal cognition directly. Hence, our finding that the M₃-muscarinic receptor is important in hippocampal-based learning and memory offers an alternative explanation for the positive effects of promoting cholinergic transmission in patients with a cognitive deficiency.

Interestingly, earlier studies on mice where the M₃-muscarinic receptor had been deleted had not identified a deficit in fear conditioning learning and memory (13). These studies did, however, use mice on a different genetic background (C57BL/J6 × 129SvEv) as well as a different experimental setup (13). Furthermore, the primary focus of these earlier studies was not learning and memory.

The mechanism by which the M₃-muscarinic receptor mediates fear conditioning depends on the phosphorylation status of the receptor. This was revealed by using a knockin mouse strain that expressed a phosphorylation-deficient mutant of the M₃-muscarinic receptor. This mutant mouse was shown to have a deficit in fear conditioning learning and memory similar to that observed in the receptor knockout mouse. Furthermore, the M₃-muscarinic receptor was phosphorylated (at least on serine³⁸⁴) in the hippocampus in an agonist-dependent fashion and phosphorylation at serine³⁸⁴ was also up-regulated in the hippocampus following fear conditioning. These data point to the possibility that agonist-mediated phosphorylation of the M₃-muscarinic receptor at the hippocampus is a regulatory step in fear conditioning learning and memory.

Importantly, mutation of the phospho-acceptor sites on the M₃-muscarinic receptor did not affect cell surface or tissue expression of the receptor. The mutations also did not significantly affect the ability of the receptor to couple to G_q/11/calcium signaling. Interestingly, signaling of the phosphorylation-deficient receptor to G_q/11 in neurons and transfected cells did not show signs of altered desensitization. Because previous studies had demonstrated that M₃-muscarinic receptor G_q/11-responses can be subject to phosphorylation-dependent desensitization (26, 27), it is possible that the phosphorylation-deficient mutant receptor, in vivo, might have different rates of desensitization that might impact on learning and memory.

The phosphorylation-deficient receptor was, however, defective in phosphorylation-dependent signaling such as receptor internalization and the recruitment of arrestin. In this way the mutant M₃-muscarinic receptor could be described as showing G protein bias (28) where signaling is biased toward G_q/11-signaling pathways as opposed to phosphorylation/arrestin-dependent signaling pathways. Because mice expressing this receptor mutant showed a defect in hippocampal learning and memory, it would seem that the mechanism used by the M₃-muscarinic receptor in mediating learning and memory is, at least in part, via phosphorylation and arrestin-dependent signaling. This conclusion is important in the context of recently described “biased ligands” that are able to direct signaling of GPCRs selectively through phosphorylation/arrestin-dependent pathways (28). Such biased ligands have been described for a number of GPCRs (e.g., ref. 29) and raises the possibility that a biased ligand to the M₃-muscarinic receptor that directed the receptor toward phosphorylation and arrestin-dependent (non-G protein) signaling would promote cholinergic-mediated learning and memory.

The M₃-muscarinic receptor can be phosphorylated by a number of protein kinases including members of the GRK family (26, 27) as well as protein kinase CK2 (18) and CK1α (17). In this respect M₃-muscarinic receptors are similar to many other GPCRs where phosphorylation at multiple sites by more than one protein kinase has been observed (30). This has led to the notion that tissue specific GPCR signaling could be mediated, in part, by the employment of a subset of the receptor kinases resulting in a tissue specific phosphorylation profile (16). Whereas it was not possible in the current study to define the receptor kinases used in the phosphorylation of the M₃-muscarinic receptor in the hippocampus, it would seem plausible that one or more of the protein kinases identified from in vitro studies are used to impart a phosphorylation profile on the receptor that allows for the regulation of learning and memory.

In conclusion, the deficit in fear conditioning shown by the M3R-KO mice implicates this receptor in cholinergic-mediated learning and memory. The fact that the mutant mice expressing a phosphorylation-deficient M₃-muscarinic receptor showed a similar fear conditioning deficit to the M3R-KO mice demonstrates the importance of receptor phosphorylation, and potentially arrestin recruitment, in the mechanism of action of this receptor in learning and memory. In light of these findings it would seem plausible that biased ligands that direct the signaling of the M₃-muscarinic receptor through phosphorylation/arrestin pathways might be of clinical benefit in the treatment of cognitive disorders.

Materials and Methods

Generation of the Muscarinic Receptor Mutant Strain M3R-KI Mice.

The mouse M₃-muscarinic receptor coding sequence was subjected to consecutive rounds of mutagenesis (QuikChange; Stratagene) to generate a receptor, termed M3R_phos-neg with serine to alanine mutations at the following positions: 285, 286, 288, 290, 291, 302, 303, 331, 332, 333, 335, 374, 379, 384, and 385 (Fig. S1A). This receptor was used to replace (knock in) the coding sequence of the M₃-muscarinic receptor using homologous recombination. A detailed description can be found in SI Materials and Methods.

Fear Conditioning.

For all behavioral tests, 8- to 15-week-old mice were used. For fear conditioning, mice were placed in the conditioning chamber (Coulbourn Instruments) and after a 2-min adaptation period received three tone/footshock pairings where the footshock [unconditioned stimulus (US); 2 s, 0.4 mA) always co-terminated with a tone [conditioned stimulus (CS); 30 s, 2.8 kHz, 85 dB]. The next day mice were placed back in the conditioning chamber and percent of time spent freezing was recorded for 3 min to assess context-dependent learning. Cued-conditioning was evaluated 48 h after training. For immunohistochemistry, mice were killed either 30 min or 2 h after the training session.

See SI Materials and Methods for a detailed explanation of the methods used.