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Journal of Molecular Neuroscience Copyright © 2006 Humana Press Inc. All rights of any nature whatsoever are reserved. ISSN0895-8696/06/30:157–160/$30.00 JMN (Online)ISSN 1559-1166 DOI 10.1385/JMN/30:1-2:157
ORIGINAL ARTICLE
M1–M3 Muscarinic Acetylcholine Receptor-Deficient Mice Novel Phenotypes
Dinesh Gautam,1 Alokesh Duttaroy,1 Yinghong Cui,1 Sung-Jun Han,1 Chuxia Deng,2 Thomas Seeger,3 Christian Alzheimer,4 and Jürgen Wess*,1 1Laboratory
of Bioorganic Chemistry; 2Genetics of Development and Diseases Branch, NIH-NIDDK, Bethesda, Maryland 20892; 3Bundeswehr Institute of Pharmacology and Toxicology, D-80937 Munich, Germany; and 4Department of Physiology, University of Kiel, D-24098 Kiel, Germany
Introduction The five muscarinic acetylcholine receptors (M1–M5 mAChRs) mediate a very large number of important physiological functions (Caulfield, 1993; Caulfield and Birdsall, 1998; Wess, 2004). Because of the lack of small molecule ligands endowed with a high degree of receptor subtype selectivity and the fact that most tissues or cell types express two or more mAChR subtypes, identification of the physiological and pathophysiological roles of the individual mAChR subtypes has proved to be a challenging task. To overcome these difficulties, we recently generated mutant mouse lines deficient in each of the five mAChR genes (M1R–/– mice, M2R–/– mice, M3R–/– mice, etc. [Wess, 2004]). Phenotyping studies showed that each of the five mutant mouse lines displayed characteristic physiological, pharmacological, behavioral, biochemical, or neurochemical deficits (Wess, 2004). This chapter summarizes recent findings dealing with the importance of the M2 mAChR for cognitive processes and the roles of the M1 and M3 mAChRs in mediating stimulation of glandular secretion.
Role of M2 mAChRs in Cognition mAChRs are known to play central roles in facilitating cognitive functions (Coyle et al., 1983; Bartus, 2000). Like M1 receptors, M2 receptors are expressed,
both pre- and postsynaptically, in the hippocampus and most other brain regions implicated in learning and memory processes (Levey et al., 1995; Rouse et al., 1997). Using M2R–/– mice as novel experimental tools, we recently tested the hypothesis that the M2 receptor subtype is involved in spatial learning and memory and hippocampal plasticity (Seeger et al., 2004). We found that M2R/ mice showed significant deficits in behavioral flexibility and working memory in the Barnes circular maze and the T-maze delayed alternation test, respectively. To examine whether the behavioral deficits observed with M2R–/ mice correlated with changes in neuronal plasticity, we studied short-term potentiation (STP) and longterm potentiation (LTP) at the Schaffer-CA1 synapse using hippocampal slices from wild-type (WT [M2R+/+]) and M2R–/– mice (Seeger et al., 2004). Strikingly, we found that STP was abolished and LTP was greatly reduced following theta burst electrical stimulation of MR2–/ hippocampi (Fig. 1A). Treatment of M2R –/ hippocampi with the GABA A receptor antagonist, bicuculline (10 RM), fully restored STP and significantly increased LTP (Seeger et al., 2004). A likely explanation for this finding is that presynaptic M2 receptors mediate suppression of GABAergic (GABAA receptor-mediated) inhibition of CA1 neurons in the WT hippocampus, suggesting that
*Author to whom all correspondence and reprint requests should be addressed. E-mail:
[email protected]
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Fig. 1. Hippocampal synaptic plasticity is greatly impaired in M2R–/– mice. (A) STP is absent, and LTP is greatly reduced in hippocampal slices from M2–/– mice. Field excitatory postsynaptic potentials (fEPSPs) were recorded in CA1 stratum radiatum of WT (M2R+/+) and M2R–/– hippocampi before and after theta burst stimulation (TBS). fEPSP slopes were normalized to 100% prior to TBS (n = 8 or 9 per group). (B) Carbachol (CCh)-induced LTPm is absent in hippocampal slices from M2R–/– mice. Superfusion of WT hippocampal slices with a low concentration (0.5 RM) of CCh for 20 min induced a pronounced enhancement of fEPSPs (LTPm). In contrast, the same protocol failed to evoke LTPm in M2R–/– hippocampi (n = 9 per group). Data were taken from Seeger et al. (2004).
induction of STP and LTP requires the activity-dependent stimulation of presynaptic M2 heteroreceptors. This concept was further corroborated by whole-cell recordings from CA1 pyramidal neurons of WT and M2R–/– hippocampi, in which we studied the behavior of pharmacologically isolated inhibitory postsynaptic currents (IPSCs) and excitatory postsynaptic currents (EPSCs) during single and repetitive electrical stimulation (Seeger et al., 2004). Another muscarinic mechanism leading to enhanced hippocampal synaptic plasticity is referred to as muscarinic LTP (LTPm), which is characterized by a long-term enhancement of excitatory transmission in CA1 pyramidal cells following mAChR activation (Segal and Auerbach, 1997). Muscarinic LTP (LTPm) is most probably mediated by mAChRs located postsynaptically on CA1 pyramidal cells (Segal and Auerbach, 1997). Consistent with previous findings, we demonstrated that a low concentration of the cholinergic agonist carbachol (0.5 RM) was able to induce LTPm in WT hippocampal slices. Strikingly, this activity was abolished in M2R–/– mice (Fig. 1b), providing unambiguous evidence that LTPm is mediated by (postsynaptic) M2 receptors expressed by CA1 hippocampal pyramidal cells. In conclusion, our data show that M2 receptors are required for intact working memory, behavioral flexibility, and hippocampal plasticity (Schaffer-CA1 synapse). It is likely that the behavioral deficits are,
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at least partially, due to the observed impairments in hippocampal synaptic plasticity (Chen and Tonegawa, 1997; Silva et al., 1997). Because reduced muscarinic cholinergic neurotransmission is thought to represent a key factor leading to impaired cognition associated with Alzheimer’s disease and old age (Coyle et al., 1983; Bartus, 2000), these results should be of considerable clinical importance.
Role of M1 and M3 mAChRs in Endocrine and Exocrine Secretion Stimulation of glandular mAChRs leads to secretion from virtually all exocrine and endocrine glands. A vast body of pharmacological evidence suggests that the M3 receptor plays a key role in muscarinic regulation of glandular function (Caulfield, 1993). However, several studies suggest that stimulation of other mAChRs, such as the M1 receptor, can also trigger glandular secretion. To address this issue, we used several mAChR mutant mouse strains to define the contribution of M1 and M3 mAChRs to muscarinic stimulation of salivary and pancreatic (both exocrine and endocrine) secretion.
Muscarinic Stimulation of Salivary Secretion The M3 mAChR is thought to play a key role in mAChR-mediated salivary secretion (Caulfield, 1993). Consistent with this concept, injection of a low
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Fig. 2. Carbachol-induced increases in amylase secretion from dispersed pancreatic acini prepared from M1R–/– and M3R–/– single-KO (A) and M1R–/–/M3R–/– double-KO (B) mice, and their corresponding WT controls. Dispersed pancreatic acini were incubated with the indicated concentrations of carbachol for 30 min at 37oC. Results are expressed as the percentage of total cellular amylase activity (before incubation) that was released into the extracellular medium during the incubation. Data are given as means ± S.E.M. of three independent experiments (sets of mice), each carried out in duplicate. Data were taken from Gautam et al. (2005).
dose of pilocarpine (1 mg/kg, sc) resulted in a greatly reduced salivation response in M3R–/– mice (Matsui et al., 2000; Gautam et al., 2004). However, we demonstrated that higher doses of pilocarpine (5 or 15 mg/kg, sc) led to pronounced salivation responses even in the absence of M3 receptors (Gautam et al., 2004). Strikingly, pilocarpine-induced salivation responses were completely abolished in M1R–/–/ M3R–/– double-knockout (KO) mice (Gautam et al., 2004). These observations support the concept that cholinergic stimulation of salivary flow is mediated by a mixture of M1 and M3 receptors and that other mAChRs do not contribute to this activity to a significant extent.
Muscarinic Potentiation of Glucose-Dependent Insulin Secretion from Pancreatic Islets Pancreatic mAChRs play an important role in stimulating insulin secretion from pancreatic G-cells (Gilon and Henquin, 2001). By using isolated pancreatic islets prepared from WT and M3R–/– mice, we recently showed that muscarinic agonist-mediated enhancement of glucose-dependent insulin release was abolished in M3R–/– mice (Duttaroy et al., 2004). We also demonstrated that muscarinic agonist-induced glucagon release (glucagon is synthesized and stored by pancreatic F-cells) was also greatly diminished in islets from M3R–/– mice (Duttaroy et al., 2004). These
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findings suggest that strategies aimed at enhancing signaling through G-cell M3 mAChRs might represent a useful novel approach to promote insulin release in type-2 diabetes.
Muscarinic Stimulation of Digestive Enzyme Secretion from Pancreatic Acinar Cells The extrinsic parasympathetic (vagal) innervation of the pancreas plays a major role in stimulating enzyme secretion from the exocrine pancreas (Holst, 1993). Recent RT-PCR studies demonstrated that rat pancreatic acini express both M3 and M1 mAChRs (Schmid et al., 1998; Turner et al., 2001). To examine the role of acinar M1 and M3 receptors in muscarinic stimulation of exocrine pancreas secretion, we studied carbachol-induced amylase secretion in vitro, using dispersed pancreatic acini prepared from WT and M1 and M3 mAChR mutant mice (Gautam et al., 2005). Quantitative real-time RT-PCR experiments (TaqMan) demonstrated that genetic inactivation of M1 or M3 mAChR genes had no significant effect on mRNA levels of the remaining mAChR subtypes in mouse pancreatic acini. As shown in Fig. 2A, carbachol-mediated maximum secretory responses (E max values) were decreased by ~40% in both M1R/ and M3R/ singleKO mice. Carbachol showed a ~10-fold reduction in potency in acinar preparations from M3R/ mice
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160 (EC50 ~3 × 10–6 M) but similar potencies in preparations from M1R/ and WT mice (EC50 ~3 × 10–7 M). Strikingly, carbachol-induced amylase secretion was completely abolished in pancreatic acinar preparations from M1/M3 receptor double-KO mice (Fig. 2b). Immunoprecipitation studies showed that mouse pancreatic acinar cells express significantly more M3 than M1 receptors (Gautam et al., 2005), providing a possible explanation for the observation that the lack of M3 receptors led to a more pronounced inhibition of carbachol-induced amylase secretion (reduction in both Emax and carbachol potency) than the lack of M1 receptors. Taken together, these data indicate that cholinergic stimulation of pancreatic amylase release is mediated by a mixture of M1 and M3 mAChRs and that other mAChR subtypes do not play a significant role in regulating this activity. Given the vital importance of exocrine pancreatic output, our findings should be of considerable clinical interest.
Conclusions The analysis of mAChR mutant mouse strains has led to a wealth of novel information about the physiological and pathophysiological roles of individual mAChR subtypes. It is likely that this new body of data will facilitate the development of novel muscarinic drugs endowed with increased efficacy and reduced side effects.
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