Cholinergic agents elicit prominent smooth muscle contractions via stimulation of muscarinic receptors that comprise five distinct subtypes (M₁–M₅). Although such contractions are important for autonomic organs, the role of each subtype has not been characterized precisely because of the poor selectivity of the currently available muscarinic ligands. Here, we generated a mutant mouse line (M₂−/−M₃−/− mice) lacking M₂ and M₃ receptors that are implicated in such cholinergic contractions. The relative contributions of M₂ and M₃ receptors in vitrowas ∼5 and 95% for the detrusor muscle contraction and ∼25 and 75% for the ileal longitudinal muscle contraction, respectively. Thus, M₁, M₄, or M₅receptors do not seem to play a role in such contractions. Despite the complete lack of cholinergic contractions in vitro, M₂−/−M₃−/− mice were viable, fertile, and free of apparent intestinal complications. The urinary bladder was distended only in males, which excludes a major contribution by cholinergic mechanisms to the urination in females. Thus, cholinergic mechanisms are dispensable in gastrointestinal motility and female urination. After 10 Hz electrical field stimulation, noncholinergic inputs were found to be increased in the ileum of M₂−/−M₃−/− females, which may account for the lack of apparent functional deficits. Interestingly, the M₂−/−M₃−/− mice had smaller ocular pupils than M₃-deficient mice. The results suggest a novel role of M₂ in the pupillary dilation, contrary to the well known cholinergic constriction. These results collectively suggest that an additional mechanism operates in the control of pupillary constriction–dilatation.

The M₁ and M₃ subtypes are the major muscarinic acetylcholine receptors in the salivary gland and M₃ is reported to be more abundant. However, despite initial reports of salivation abnormalities in M₃‐knockout (M₃KO) mice, it is still unclear which subtype is functionally relevant in physiological salivation. In the present study, salivary secretory function was examined using mice lacking specific subtype(s) of muscarinic receptor. The carbachol‐induced [Ca²⁺]_i increase was markedly impaired in submandibular gland cells from M₃KO mice and completely absent in those from M₁/M₃KO mice. This demonstrates that M₃ and M₁ play major and minor roles, respectively, in the cholinergically induced [Ca²⁺]_i increase. Two‐dimensional Ca²⁺‐imaging analysis revealed the patchy distribution of M₁ in submandibular gland acini, in contrast to the ubiquitous distribution of M₃. In vivo administration of a high dose of pilocarpine (10 mg kg⁻¹, s.c.) to M₃KO mice caused salivation comparable to that in wild‐type mice, while no salivation was induced in M₁/M₃KO mice, indicating that salivation in M₃KO mice is caused by an M₁‐mediated [Ca²⁺]_i increase. In contrast, a lower dose of pilocarpine (1 mg kg⁻¹, s.c.) failed to induce salivation in M₃KO mice, but induced abundant salivation in wild‐type mice, indicating that M₃‐mediated salivation has a lower threshold than M₁‐mediated salivation. In addition, M₃KO mice, but not M₁KO mice, had difficulty in eating dry food, as shown by frequent drinking during feeding, suggesting that salivation during eating is mediated by M₃ and that M₁ plays no practical role in it. These results show that the M₃ subtype is essential for parasympathetic control of salivation and a reasonable target for the drug treatment and gene therapy of xerostomia, including Sjögren's syndrome.

Background: The formation of long-term memory (LTM) and the late phase of long-term potentiation (L-LTP) depend on macromolecule synthesis, translation, and transcription in neurons. vesl-1S (V ASP/E na-related gene upregulated during s eizure and L TP, also known as homer-1a) is an LTP-induced immediate early gene. The short form of Vesl (Vesl-1S) is an alternatively spliced isoform of the vesl-1 gene, which also encodes the long form of the Vesl protein (Vesl-1L). Vesl-1L is a postsynaptic scaffolding protein that binds to and modulates the metabotropic glutamate receptor 1/5 (mGluR1/5), the IP₃ receptor, and the ryanodine receptor. Vesl-1 null mutant mice show abnormal behavior, which includes anxiety- and depression-related behaviors, and an increase in cocaine-induced locomotion; however, the function of the short form of Vesl in behavior is poorly understood because of the lack of short-form-specific knockout mice. Results: In this study, we generated short-form-specific gene targeting (KO) mice by knocking in part of vesl-1L/homer-1c cDNA. Homozygous KO mice exhibited normal spine number and morphology. Using the contextual fear conditioning test, we demonstrated that memory acquisition and short-term memory were normal in homozygous KO mice. In contrast, these mice showed impairment in fear memory consolidation. Furthermore, the process from recent to remote memory was affected in homozygous KO mice. Interestingly, reactivation of previously consolidated fear memory attenuated the conditioning-induced freezing response in homozygous KO mice, which suggests that the short form plays a role in fear memory reconsolidation. General activity, emotional performance, and sensitivity to electrofootshock were normal in homozygous KO mice. Conclusion: These results indicate that the short form of the Vesl family of proteins plays a role in multiple steps of long-term, but not short-term, fear memory formation.

Muscarinic acetylcholine receptors in mammals consist of five subtypes (M₁-M₅) encoded by distinct genes. They are widely expressed throughout the body and play a variety of roles in the peripheral and central nervous systems. Although their pharmacological properties have been studied extensively in vitro, colocalization of the multiple subtypes in each tissue and lack of subtype-specific ligands have hampered characterization of the respective subtypes in vivo. We have mapped mouse genomic loci for all five genes (Chrm1-5) by restriction fragment length variant (RFLV) analyses in interspecific backcross mice. Chrm1, Chrm2, and Chrm3 were mapped to chromosome (Chr) 19, 6, and 13, respectively. Both Chrm4 and Chrm5 were mapped to Chr 2. Although a comparison of their map positions with other mutations in their vicinities suggested a possibility that the El2 (epilepsy 2) allele might be a mutation in Chrm5, sequencing analyses of the Chrm5 gene in the El2 mutant mice did not support such a hypothesis.