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. 2017 Aug 2;95(3):550–563.e5. doi: 10.1016/j.neuron.2017.07.013

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© 2017 The Author(s)

This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).

PMC Copyright notice

M₁ and M₄ Are Excessively Synthesized and Overexpressed in Fmr1^−/y CA1 Pyramidal Neurons

(A) Chrm4 and Chrm1, but not Chrm3, are enriched in the Fmr1^−/y CA1-TRAP IP (Chrm4: WT = 1.00 ± 0.124, KO = 1.72 ± 0.195, ^∗p = 0.0044, n = 14; Chrm1: WT = 1.00 ± 0.062, KO = 1.47 ± 0.079, ^∗p < 0.0001, n = 14; Chrm3: WT = 1.00 ± 0.085, KO = 1.184 ± 0.073, p = 0.192, n = 10).

(B) Total mRNA levels of Chrm4, Chrm1 and Chrm3 are unchanged in FACS-isolated Fmr1^−/y CA1 pyramidal neurons (Chrm4: WT = 1.00 ± 0.209, KO = 0.98 ± 0.154, p = 0.926, n = 9; Chrm1: WT = 1.00 ± 0.150, KO = 0.87 ± 0.185, p = 0.602, n = 11; Chrm3: WT = 1.00 ± 0.232, KO = 1.14 ± 0.230, p = 0.668, n = 8).

(C) Immunoblotting shows overexpression of M₄ protein in hippocampal slice homogenates (WT = 100% ± 5.7%, KO = 121% ± 5.7%, ^∗p = 0.0186, n = 12) and synaptoneurosomes (WT = 100% ± 7.10%, KO = 121% ± 6.89%, ^∗p = 0.0203, n = 9).

(D and E) Hippocampal slice homogenates show no difference in M₁ (D) (WT = 100% ± 2.5%, KO = 105% ± 2.5%, p = 0.129, n = 12) or M₃ (E) (WT = 100% ± 9.5%, KO = 98% ± 12.9%, p = 0.94, n = 9) expression.

(F) Schematic shows steps for FACS immunostaining.

(G) FACS-immunostaining reveals significant increase in the expression of M₄ and M₁ (M₄: WT = 0.925 ± 0.045, KO = 1.075 ± 0.045, ^∗p = 0.0389, n = 6; M₁: WT = 0.920 ± 0.002, KO = 1.080 ± 0.046, ^∗p = 0.0092, n = 6), but not M₃ (WT = 0.962 ± 0.07, KO = 1.038 ± 0.1011 p = 0.547, n = 7), in Fmr1^−/y CA1 pyramidal neurons. n = number of littermate pairs. Error bars indicate SEM.