M4 muscarinic receptor knockout mice display abnormal social behavior and decreased prepulse inhibition

Abstract

Background: In the central nervous system (CNS), the muscarinic system plays key roles in learning and memory, as well as in the regulation of many sensory, motor, and autonomic processes, and is thought to be involved in the pathophysiology of several major diseases of the CNS, such as Alzheimer's disease, depression, and schizophrenia. Previous studies reveal that M4 muscarinic receptor knockout (M4R KO) mice displayed an increase in basal locomotor activity, an increase in sensitivity to the prepulse inhibition (PPI)-disrupting effect of psychotomimetics, and normal basal PPI. However, other behaviorally significant roles of M4R remain unclear.

Results: In this study, to further investigate precise functional roles of M4R in the CNS, M4R KO mice were subjected to a battery of behavioral tests. M4R KO mice showed no significant impairments in nociception, neuromuscular strength, or motor coordination/learning. In open field, light/dark transition, and social interaction tests, consistent with previous studies, M4R KO mice displayed enhanced locomotor activity compared to their wild-type littermates. In the open field test, M4R KO mice exhibited novelty-induced locomotor hyperactivity. In the social interaction test, contacts between pairs of M4R KO mice lasted shorter than those of wild-type mice. In the sensorimotor gating test, M4R KO mice showed a decrease in PPI, whereas in the startle response test, in contrast to a previous study, M4R KO mice demonstrated normal startle response. M4R KO mice also displayed normal performance in the Morris water maze test.

Conclusions: These findings indicate that M4R is involved in regulation of locomotor activity, social behavior, and sensorimotor gating in mice. Together with decreased PPI, abnormal social behavior, which was newly identified in the present study, may represent a behavioral abnormality related to psychiatric disorders including schizophrenia.

Figure 1

Normal nociception and motor function of M₄R KO mice. (A) No difference in latency was observed between genotypes in the hot plate test. M₄R KO mice (Mutants), n = 28; wild-type mice (Controls), n = 28. (B) No significant difference in percent falling within 60 s was observed between genotypes in the wire hang test. M₄R KO mice, n = 26; wild-type mice, n = 28. (C) No significant differences in latency to fall were observed between genotypes in the rotarod test. M₄R KO mice, n = 30; wild-type mice, n = 30.

Figure 2

Locomotor hyperactivity of M₄R KO mice. (A-D) M₄R KO mice displayed an increase in novelty-induced locomotor hyperactivity in the open field test. (A) M₄R KO mice traveled significantly longer than wild-type mice during the whole experimental period. Locomotor hyperactivity of M₄R KO was detected in the earlier (1-10 min) part of the experiment but not in the middle (11-20 min) or later (21-30 min) parts of the experimental period. No significant differences were observed between genotypes in vertical activity (B) or margin time (C). (D) M₄R KO mice demonstrated a significant increase in stereotypic behavior counts. M₄R KO, n = 19; wild-type mice, n = 27. (E-H) M₄R KO mice exhibited locomotor hyperactivity in the light/dark transition test. (E) M₄R KO mice traveled significantly longer than wild-type mice in the dark box but not in the light box. No significant difference was observed between genotypes in time spent in the light box (F), the number of transitions between the light and dark sides (G), or latency to entering the light box (H). M₄R KO mice, n = 28; wild-type mice, n = 28.

Figure 3

Abnormal social behavior of M₄R KO mice. (A) No significant difference in total duration of contacts was detected between genotypes. (B) The number of contacts was slightly but not significantly increased in M₄R KO mice. (C) M₄R KO mice displayed a significant decrease in the mean duration of each contact compared to wild-type mice. (D) The distance traveled by M₄R KO mice was significantly increased compared to that of wild-type mice. (E) Scatter plot of distance traveled against mean duration per contact. M₄R KO mice, n = 14 pairs; wild-type mice, n = 14 pairs.

Figure 4

Normal startle response and decreased PPI in M₄R KO mice. (A) M₄R KO mice demonstrated normal acoustic startle response for the 100 and 110 dB startle stimulus. (B) No significant differences in PPI for the 74 and 78 dB prepulse sound level followed by 100 dB startle stimulus were observed between genotypes. (C) M₄R KO mice demonstrated a significant decrease in prepulse inhibition for the 74 and 78 dB prepulse sound level followed by 110 dB startle stimulus compared to wild-type mice. M₄R KO mice, n = 28; wild-type mice, n = 28.

Figure 5

Normal spatial reference, working, and episodic-like memory of M₄R KO mice in the Morris water maze. (A-E) No significant differences in the conventional hidden platform version of the Morris water maze were detected between genotypes. Escape latency (A), swimming speed (B), and time spent in the perimeter of the pool (C) did not differ significantly between the two genotypes during either original or reversal learning. M₄R KO mice, n = 13; wild-type mice, n = 14. In probe trials, both M₄R KO mice and wild-type mice selectively searched the location where the platform had been located. M₄R KO mice, n = 11; wild-type mice, n = 12. (D) Both genotypes spent significantly more time in the training quadrant (black bars) compared with the other quadrants (opposite quadrant, white bars; right quadrant, dark gray bars; left quadrant, light gray bars) in the probe trials conducted after original training. (E) Also, both genotypes crossed the training site significantly more often than the equivalent sites in the other three quadrants in the probe trials. (F) No significant difference in the delayed matching-to-place (DMP) task of the Morris water maze was detected between genotypes. M₄R KO mice showed normal latency in the first five trials of new platform training, averaged over six training sessions. M₄R KO mice n = 13; wild-type mice, n = 14.