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. 2015 Jul 15;29(14):1535–1551. doi: 10.1101/gad.260869.115

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© 2015 Wang et al.; Published by Cold Spring Harbor Laboratory Press

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Figure 3. — Ca_V3.2 channels regulate synaptic expression of glutamatergic receptors. (A) GluA2/3 immunogold labeling at synapses in the control nonexpressing TRN and the TRN expressing hCa_V3.2(WT)-GFP, hCa_V3.2(C456S)-GFP, and Ca_V3.2ct-GFP. Arrows point to silver-enhanced gold particles associated with PSDs postsynaptic to L (pink arrows) or D (cyan arrows) terminals. (B, left) Relative distributions of GluA2/3 silver–gold particles at thalamic synapses contacted by L terminals in the TRN expressing hCa_V3.2(WT)-GFP, hCa_V3.2(C456S)-GFP, or Ca_V3.2ct-GFP (wild type: n = 178 particles from 147 synapses; C456S: n = 227 particles from 207 synapses; Ca_V3.2ct: n = 523 particles from 443 synapses) compared with that in the control nonexpressing TRN (Ctrl: n = 232 particles from 202 synapses) as well as cortical synapses contacted by D terminals in the TRN expressing hCa_V3.2(WT)-GFP, hCa_V3.2(C456S)-GFP, and Ca_V3.2ct-GFP (wild type: n = 298 particles from 232 synapses; C456S: n = 545 particles from 502 synapses; Ca_V3.2ct: n = 993 particles from 851 synapses) terminals compared with that in the control nonexpressing TRN (Ctrl: n = 563 from 469 synapses). (Right) Average percentages of GluA2/3 silver–gold particles in synaptic pools of thalamic synapses in the TRN expressing hCa_V3.2(WT)-GFP, hCa_V3.2(C456S)-GFP, and Ca_V3.2ct-GFP (wild type: 8.5% ± 2.1%, n = 12 animals, U = 75.0, P = 0.89; C456S: 16.6% ± 1.2%, n = 12 animals, U = 126.5, P < 0.001; Ca_V3.2ct: 2.2% ± 0.7%, n = 12 animals, U = 34.0, P < 0.05) compared with that in the control nonexpressing TRN (Ctrl: 7.7% ± 1.7%, n = 12 animals); in deliverable pools of thalamic synapses in the TRN expressing hCa_V3.2(WT)-GFP, hCa_V3.2(C456S)-GFP, and Ca_V3.2ct-GFP (wild type: 18.4% ± 1.9%, n = 12 animals, U = 81.0, P = 0.63; C456S: 10.0% ± 2.2%, n = 12 animals, U = 118.0, P < 0.01; Ca_V3.2ct: 24.0% ± 1.7%, n = 12 animals, U = 117.0, P < 0.05) compared with that in the control nonexpressing TRN (Ctrl: 18.3% ± 2.5%, n = 12 animals); in synaptic pools of cortical synapses in the TRN expressing hCa_V3.2(WT)-GFP, hCa_V3.2(C456S)-GFP, and Ca_V3.2ct-GFP (wild type: 10.1% ± 1.5%, n = 12 animals, U = 78.5, P = 0.71; C456S: 17.3% ± 1.7%, n = 12 animals, U = 130.0, P < 0.0005; Ca_V3.2ct: 3.3% ± 0.8%, n = 12 animals, U = 10.0, P < 0.005) compared with that in control nonexpressing TRN (Ctrl: 9.4% ± 0.7%, n = 12 animals); and in deliverable pools of cortical synapses in the TRN expressing hCa_V3.2(WT)-GFP, hCa_V3.2(C456S)-GFP, and Ca_V3.2ct-GFP (wild type: 16.8% ± 3.1%, n = 12 animals, U = 71.0, P = 0.98; C456S: 8.4% ± 1.2%, n = 12 animals, U = 16.0, P < 0.001; Ca_V3.2ct: 23.7% ± 1.7%, n = 12 animals, U = 122.0, P < 0.005) compared with that in the control nonexpressing TRN (Ctrl: 15.6% ± 1.3%, n = 12 animals). (C) GluN1 immunogold labeling at synapses in the control nonexpressing TRN and the TRN expressing hCa_V3.2(WT)-GFP, hCa_V3.2(C456S)-GFP, and Ca_V3.2ct-GFP. Arrows point to silver-enhanced gold particles associated with the PSDs postsynaptic to L (pink arrows) or D (cyan arrows) terminals. (D, left) Relative distributions of GluN1 silver–gold particles at thalamic synapses contacted by L terminals in the TRN expressing hCa_V3.2(WT)-GFP, hCa_V3.2(C456S)-GFP, and Ca_V3.2ct-GFP (wild type: n = 262 particles from 230 synapses; C456S: n = 257 particles from 196 synapses; Ca_V3.2ct: n=301 particles from 238 synapses) compared with that in the control nonexpressing TRN (Ctrl: n = 197 particles from 175 synapses) as well as cortical synapses contacted by D terminals in the TRN expressing hCa_V3.2(WT)-GFP, hCa_V3.2(C456S)-GFP, and Ca_V3.2ct-GFP (wild type: n = 399 particles from 343 synapses; C456S: n = 444 particles from 339 synapses; Ca_V3.2ct: n = 576 particles from 456 synapses) compared with that in the control nonexpressing TRN (Ctrl: n = 334 particles from 300 synapses). (Right) Average percentages of GluN1 silver–gold particles in synaptic pools of thalamic synapses in the TRN expressing hCa_V3.2(WT)-GFP, hCa_V3.2(C456S)-GFP, and Ca_V3.2ct-GFP (wild type: 9.3% ± 1.5%, n = 12 animals, U = 70.5, P = 0.95; C456S: 17.4% ± 1.8%, n = 14 animals, U = 127.5, P < 0.05; Ca_V3.2ct: 3.4% ± 0.8%, n = 12 animals, U = 32.5, P < 0.05) compared with that in the control nonexpressing TRN (Ctrl: 10.0% ± 2.3%, n = 12 animals); in deliverable pools of thalamic synapses in the TRN expressing hCa_V3.2(WT)-GFP, hCa_V3.2(C456S)-GFP, and Ca_V3.2ct-GFP (wild type: 17.7% ± 2.1%, n = 12 animals, U = 77.0, P = 0.80; C456S: 10.0% ± 1.9%, n = 14 animals, U = 36.0, P < 0.05; Ca_V3.2ct: 23.2% ± 1.7%, n = 12 animals, U = 106.5, P < 0.005) compared with that in control nonexpressing TRN (Ctrl: 18.1% ± 2.5%, n = 12 animals); in synaptic pools of cortical synapses in the TRN expressing hCa_V3.2(WT)-GFP, hCa_V3.2(C456S)-GFP, and Ca_V3.2ct-GFP (wild type: 9.8% ± 1.7%, n = 12 animals, U = 59.0, P = 0.47; C456S: 17.6% ± 2.4%, n = 14 animals, U = 129.0, P < 0.05; Ca_V3.2ct: 3.2% ± 0.8%, n = 12, U = 7.5, P < 0.005) compared with that in the control nonexpressing TRN (Ctrl: 10.0% ± 0.9%, n = 12 animals); and in deliverable pools of cortical synapses in the TRN expressing hCa_V3.2(WT)-GFP, hCa_V3.2(C456S)-GFP, and Ca_V3.2ct-GFP (wild type: 16.8% ± 0.7%, n = 12 animals, U = 67.0, P = 0.80; C456S: 10.4% ± 1.7%, n = 14 animals, U = 29.0, P < 0.01; Ca_V3.2ct: 23.1% ± 1.4%, n = 12 animals, U = 109.0, P < 0.05) compared with that in the control nonexpressing TRN (Ctrl: 17.8% ± 2.0%, n = 12 animals). Note the complementary changes in the amounts of GluA2/3 and GluN1 silver–gold particles in the PSD and the nearby deliverable pool of both thalamic and cortical synapses in the TRN in all experiments. Asterisks indicate P < 0.05 (Mann-Whitney test).