Learning and memory impairments in a congenic C57BL/6 strain of mice that lacks the M2 muscarinic acetylcholine receptor subtype

Abstract

The neurotransmitter acetylcholine is an important modulator of cognitive functions including attention, learning, and memory. The actions of acetylcholine are mediated by five distinct muscarinic acetylcholine receptor subtypes (M(1)-M(5)). The lack of drugs with a high degree of selectivity for these subtypes has impeded the determination of which subtypes mediate which components of cholinergic neurotransmission relevant to cognitive abilities. The present study examined the behavioral functions of the M(2) muscarinic receptor subtype by utilizing congenic C57BL/6 mice possessing a null-mutation in the M(2) muscarinic receptor gene (M(2)(-/-) mice). Comprehensive assessment of general health and the neurological function found no major differences between M(2)(-/-) and wild-type (M(2)(+/+)) mice. In the tests of learning and memory, M(2)(-/-) mice were impaired in the acquisition (trials to criterion), but not the retention (72h) of a passive avoidance task. In a novel open field, M(2)(-/-) mice were impaired in between-sessions, but not within-session habituation. In a holeboard test of spatial memory, M(2)(-/-) mice committed more errors in working memory than M(2)(+/+) mice. Reference memory did not differ between the genotypes. M(2)(-/-) mice showed no impairments in either cued or contextual fear conditioning. These findings replicate and extend earlier findings in a hybrid strain and solidify the interpretation that the M(2) receptor plays a critical role in specific components of cognitive abilities.

Figure 1

Spontaneous Motor Activity and Within Session Habituation. (a) There was no difference between M₂^+/+ (n = 23) and M₂^−/− (n = 21) mice in spontaneous motor activity in a novel environment. The genotypes also did not differ in their habituation to the novel environment as indicated by a decrease in motor activity with time. (b) There was no difference between M₂^+/+ and M₂^−/− mice in vertical activity (rearing) in a novel environment.

Figure 2

Passive Avoidance Trials to Criterion. (a) There was no difference between the genotypes in latency to enter the dark chamber on the first training trial. (b) As indicated by the asterisk, on the second training trial, M2^−/− mice (n = 20) had significantly lower latencies (t = 2.29, df = 41, p = 0.03) than M2^+/+ mice (n = 23) to enter the dark chamber. (c) As indicated by the asterisk, M2^−/− mice required significantly more trials (t = −2.77, df = 41, p =0.008) than M2^+/+ mice to reach the criterion of remaining in the lit chamber for 300 sec. (d) There was no effect of genotype on latency to enter the dark chamber in the 72 hr retention trial.

Figure 3

Between Sessions Habituation. As indicated by the asterisk, M₂^−/− (n = 20) mice exhibited a significantly smaller (t = 2.46, df = 38, p = 0.02) degree of habituation (as measured by a decrease in motor activity) than M₂^+/+ (n = 20) mice during the second exposure (4 hr interval) to a novel environment.

Figure 4

Cued and Contextual Fear Conditioning. (a) During training freezing behavior did not differ by genotype (M₂^+/+, n = 23; M₂^−/−, n = 21) prior or subsequent to cue-shock pairings. (b) During testing for contextual conditioning, there was no effect of genotype on freezing. (c) During testing for cued conditioning, freezing was significantly greater during the first minute of “cue on” than during the last minute of “cue off” (p < 0.01), but there was no effect of genotype on freezing.

Figure 5

Holeboard Task of Spatial Memory. (a) During autoshaping trials, the M₂^−/− mice (n = 15) and M₂^+/+ mice (n = 16) did not differ in their propensity to explore the holes. (b) As indicated by the asterisk, during training in the holeboard task, M₂^−/− mice made a significantly greater number of working memory errors than M₂^+/+ mice (F_{(1, 29)} = 8.04, p =0.008). (c) During training in the holeboard task, M₂^−/− and M₂^+/+ made a similar number of reference memory errors.