Figure 4.
M4 Synthesis Downstream of mGlu5 Is Mimicked and Occluded in the Fmr1−/y Hippocampus
(A) Time course for DHPG stimulation experiments.
(B) Analysis of transcripts encoding hippocampal mAChR subunits reveals a striking upregulation of Chrm4 mRNA in CA1-TRAP IP after mGlu1/5 stimulation (Veh = 1.00 ± 0.12, DHPG = 1.72 ± 0.23, ∗p = 0.0047, n = 15), with no changes seen in Chrm1 or Chrm3 (Chrm1: Veh = 1.00 ± 0.07, DHPG = 1.03 ± 0.06, p = 0.75, n = 16; Chrm3: Veh = 1.00 ± 0.12, DHPG = 0.82 ± 0.09, p = 0.209, n = 14).
(C) DHPG stimulation of WT slices shows dramatic increase in Chrm4 mRNA in the TRAP IP fraction. In Fmr1−/y slices, Chrm4 mRNA is already significantly elevated in the TRAP IP and does not increase further with mGlu1/5 activation (WT vehicle = 1.00 ± 0.24, WT DHPG = 2.13 ± 0.41, KO vehicle = 2.28 ± 0.43, KO DHPG = 2.34 ± 0.44, ANOVA genotype ∗p = 0.03, treatment ∗p = 0.048, WT versus KO veh ∗p = 0.03, KO veh versus DHPG p > 0.999, n = 7). Chrm1 mRNA is significantly elevated in the Fmr1−/y CA1-TRAP IP, but DHPG does not increase Chrm1 in either WT or Fmr1−/y CA1-TRAP IPs (WT vehicle = 1.00 ± 0.09, WT DHPG = 1.13 ± 0.08, KO vehicle = 1.50 ± 0.13, KO DHPG = 1.47 ± 0.16, ANOVA genotype ∗p = 0.005, treatment p = 0.753, WT versus KO veh ∗p = 0.04, WT veh versus DHPG p = 0.944, n = 7). Chrm3 mRNA is neither increased in the Fmr1−/y CA1-TRAP IP nor elevated with DHPG (WT veh = 1.00 ± 0.14, WT DHPG, = 1.029 ± 0.18, KO veh = 0.946 ± 0.19, KO DHPG = 1.09 ± 0.19, ANOVA genotype p = 0.97, treatment p = 0.55, n = 7).
(D) Immunoblotting shows a robust increase in M4 expression in WT slices after 5 min of DHPG stimulation, which is maintained at 30 min and 60 min post-stimulation. In contrast, the elevated expression of M4 in Fmr1−/y slices is not further increased with DHPG stimulation (WT vehicle = 100% ± 6.63%, WT DHPG 5 min = 159.59% ± 9.37%, WT DHPG 30 min = 131.09% ± 13.23%, WT DHPG 60 min = 141.66% ± 12.08%, KO vehicle = 132.70% ± 7.31%, KO DHPG 5 min = 146.95% ± 7.94% KO DHPG 30 min = 131.04% ± 13.01%, KO DHPG 60 min = 155.75% ± 10.28%, ANOVA treatment × genotype ∗p = 0.037, n = 7).
(E) Time course for MTEP slice experiments.
(F) Incubation with 10 μM MTEP reduces Chrm4 in the Fmr1−/y CA1-TRAP to WT levels (WT vehicle = 1.00 ± 0.112, WT MTEP = 1.48 ± 0.230, KO vehicle = 2.63 ± 0.352, KO MTEP = 1.63 ± 0.161, ANOVA genotype ∗p = 0.0014, treatment p = 0.1923, genotype × treatment ∗p = 0.0119, WT veh versus KO veh ∗p = 0.0024, WT veh versus WT MTEP p = 0.306, KO veh versus KO MTEP ∗p = 0.0289, WT MTEP versus KO MTEP p = 0.880, n = 8).
(G) Immunoblotting shows a significant reduction in M4 expression in MTEP-treated Fmr1−/y slices (WT vehicle = 100% ± 9.7%, WT MTEP = 97% ± 5.4%, KO vehicle = 181% ± 32.6%, KO MTEP = 103% ± 14.1%, ANOVA genotype ∗p = 0.034, WT veh versus KO veh ∗p = 0.0181, KO veh versus KO MTEP ∗p = 0.0145, n = 6). n = number of littermate pairs. Error bars indicate SEM.