Genetic reduction of muscarinic M4 receptor modulates analgesic response and acoustic startle response in a mouse model of fragile X syndrome (FXS)

Abstract

Introduction: The G-protein coupled muscarinic acetylcholine receptors, widely expressed in the CNS, have been implicated in fragile X syndrome (FXS). Recent studies have reported an overactive signaling through the muscarinic receptors in the Fmr1KO mouse model. Hence, it was hypothesized that reducing muscarinic signaling might modulate behavioral phenotypes in the Fmr1KO mice. Pharmacological studies from our lab have provided evidence for this hypothesis, with subtype-preferring muscarinic M1 and M4 receptor antagonists modulating select behaviors in the Fmr1KO mice. Since the pharmacological antagonists were not highly specific, we investigated the specific role of M4 receptors in the Fmr1KO mouse model, using a genetic approach.

Methods: We created a double mutant heterozygous for the M4 receptor gene and hemizygous for the Fmr1 gene and examined the mutants on various behaviors. Each animal was tested on a behavior battery comprising of open-field activity (activity), light-dark (anxiety), marble burying (perseverative behavior), prepulse inhibition (sensorimotor gating), rotarod (motor coordination), passive avoidance (learning and memory) and hotplate (analgesia). Animals were also tested on the audiogenic seizure protocol and testis weights were measured.

Results: Reduction of M4 receptor expression in the heterozygotes completely rescued the analgesic response and partly rescued the acoustic startle response phenotype in the Fmr1KO mice. However, no modulation was observed in a number of behaviors including learning and memory, activity, perseverative behavior and audiogenic seizures.

Conclusion: Reducing M4 receptor signaling altered only select behavioral phenotypes in the Fmr1KO mouse model, suggesting that other targets are involved in the modulation of fragile X behaviors.

Fig 1

Modification of analgesic response by the M₄ receptor in the Fmr1KO mice. Latency to hind limb response in the hot plate assay across different genotypes. # p<0.01 compared to the Fmr1KO and $ p<0.05 compared to the WT (n = 8–14).

Fig 2

Modification of acoustic startle response by the M₄ receptor in the Fmr1KO mice. Bars indicate mean ± SEM for acoustic startle response to the 120dB startle stimulus (a), average percentage inhibition of the startle response (b) across the four genotypes. * p<0.05 compared to the WT (n=8–14).

Fig 3

Effect of reducing M₄ protein levels on activity and perseverative behavior. Bars represent ± SEM for total distance in the open field as a measure of activity (a) and number of marbles buried as a measure of perseverative behavior (b) across the four genotypes. *p<0.01 compared to the WT (n=8–14).

Fig 4

Absence of modification of the learning and memory phenotype in the Fmr1KO mice. Bars indicate mean ± SEM for latency to enter the dark chamber in the passive avoidance assay on day 1 (a) and day 2(b) across four genotypes (n=8–14)

Fig 5

No modulation of anxiety like responses by the M₄ receptor. Bars represent ± SEM for the center: total distance traveled in an open field, an index of anxiety response (a) and number of light-dark transitions in the light-dark assay (b). * p<0.05 compared to the WT (n=8–14)

Fig 6

Effect of reducing M₄ protein levels on motor coordination assessed by rotarod assay. Bars indicate ± SEM for the average time spent on the rotating rod across the four genotypes (n=8–14).

Fig 7

No modulation of AGS by the M₄ receptors. Bars represent value ± SEM for percentage of seizures across genotypes. *p<0.05 compared to the WT

Fig 8

No modulation of the macroorchidism phenotype in the Fmr1KO mice. Bars represent values ± SEM for testis: body weight measurements across four genotypes. *p<0.05 compared to the WT (n=8–14)