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. Author manuscript; available in PMC: 2008 Nov 21.
Published in final edited form as: Neuron. 2007 Nov 21;56(4):670–688. doi: 10.1016/j.neuron.2007.09.016

Figure 3. Effects of cGKII activation on GluR1 trafficking.

Figure 3

(Ai) Hippocampal neurons expressing HAGluR1 and treated for 5 min with saline (control), 8-Br-cGMP, or 8-Br-cGMP following pretreatment with the cGK inhibitor KT5823. Neurons were stained live for surface HA (red), followed by fixation, permeabilization and staining for total HA (green). Scale bar, 20 μm. (Aii) Quantitation of surface/total ratio of HAGluR1 on neurons from experiments as the one shown in (Ai). Bar graph shows mean±SEM; n=35 for control, 30 for 8-Br-cGMP and 20 for 8-Br-cGMP+KT5823; **p<0.01.

(Bi) Representative images of hippocampal neurons expressing HAGluR1 or HAGluR1-S845A and treated for 5 min with saline (control) or 8-Br-cGMP. Staining was done as in (Ai). Scale bar, 20 μm. (Bii) Quantitation of surface/total ratio of HAGluR1 and HAGluR1-S845A on neurons from experiments as the one shown in (Bi). Bar graph shows mean±SEM; n=33 for HAGluR1 wt, 20 for HAGluR1-S845A and 20 for HAGluR1-S845A+8-Br-cGMP; NS = non-significant.

(C) Quantitation of surface/total ratio of HAGluR1 wt and HAGluR1-S845A on neurons treated for 10 min with saline (control) or NOR-3. Bar graph shows mean±SEM; n=20 for HAGluR1 wt control, 18 for HAGluR1 wt + NOR-3, 22 for HAGluR1-S845A control and 24 for HAGluR1-S845A + NOR-3; **p<0.01.

(D) Images of surface GluR1 (red) and SV2 (green) staining in hippocampal neurons treated with saline (control), glycine for chemLTP, 8-Br-cAMP or 8-Br-cGMP. Neurons were stained live for surface GluR1, and after fixation and permeabilization, for SV2. Scale bar, 2 μm.

(E) Quantitation of surface levels of GluR1 on neurons from experiments as the one shown in (D). Bar graph shows mean±SEM; n=18 for control, 15 for glycine, 16 for 8-Br-cAMP and 19 for 8-Br-cGMP; ***p<0.001.

(F) Quantitation of synaptic GluR1 on neurons from experiments similar to those of (D). Synaptic GluR1 was expressed as the intensity of the GluR1 signal that overlapped with SV2 divided by the total intensity of the GluR1 signal. Bar graph shows mean±SEM; n = same as in (B); ***p<0.001.

(G) Examples of mEPSCs before (-5 min), and 5 and 25 min after the application of glycine.

(H) Application of glycine (n=6, filled red circles), but neither 8-Br-cGMP (n=6, open green squares), nor 8-Br-cAMP (n=6, open black squares), enhanced mEPSC amplitude in cultured hippocampal neurons. Controls were performed with vehicle (n=5, open red circles). The thick line represents the duration of glycine, 8-Br-cGMP or 8-Br-cAMP application and error bars represent SEM.