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. 2013 Apr;18(4):471-84.
doi: 10.1038/mp.2012.80. Epub 2012 Jun 26.

Regulation of AMPA receptor surface trafficking and synaptic plasticity by a cognitive enhancer and antidepressant molecule

H Zhang  1 , L-A Etherington, A-S Hafner, D Belelli, F Coussen, P Delagrange, F Chaouloff, M Spedding, J J Lambert, D Choquet, L Groc
Affiliations
Free PMC article

Regulation of AMPA receptor surface trafficking and synaptic plasticity by a cognitive enhancer and antidepressant molecule

H Zhang et al. Mol Psychiatry. 2013 Apr.
Free PMC article

Abstract

The plasticity of excitatory synapses is an essential brain process involved in cognitive functions, and dysfunctions of such adaptations have been linked to psychiatric disorders such as depression. Although the intracellular cascades that are altered in models of depression and stress-related disorders have been under considerable scrutiny, the molecular interplay between antidepressants and glutamatergic signaling remains elusive. Using a combination of electrophysiological and single nanoparticle tracking approaches, we here report that the cognitive enhancer and antidepressant tianeptine (S 1574, [3-chloro-6-methyl-5,5-dioxo-6,11-dihydro-(c,f)-dibenzo-(1,2-thiazepine)-11-yl) amino]-7 heptanoic acid, sodium salt) favors synaptic plasticity in hippocampal neurons both under basal conditions and after acute stress. Strikingly, tianeptine rapidly reduces the surface diffusion of AMPA receptor (AMPAR) through a Ca(2+)/calmodulin-dependent protein kinase II (CaMKII)-dependent mechanism that enhances the binding of AMPAR auxiliary subunit stargazin with PSD-95. This prevents corticosterone-induced AMPAR surface dispersal and restores long-term potentiation of acutely stressed mice. Collectively, these data provide the first evidence that a therapeutically used drug targets the surface diffusion of AMPAR through a CaMKII-stargazin-PSD-95 pathway, to promote long-term synaptic plasticity.

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Figures

Figure 1
Figure 1
GluA2-AMPAR surface diffusion is rapidly decreased by tianeptine (TIA) application. (a) Experimental scheme showing that 12 min after the incubation of TIA, surface GluA2-AMPARs were tracked for 20 min in the extrasynaptic and synaptic compartments of days in vitro (DIV) 11–12 cultured hippocampal neurons using quantum dot (QD) coupled to the antibody specific for the extracellular domain of endogenous GluA2 subunit. (b) Typical QD trajectory (yellow) in neurons treated with vehicle (left panel), TIA (10 μM) (middle panel) and TIA (100 μM) (right panel). (Lower panels) Enlarged single GluA2-AMPAR-QD trajectories. The arrows represent homer1c cluster, that is, synapses. Scale bar=10 μm. (c) Diffusion coefficients (median±25–75% interquartile range (IQR)) of extrasynaptic GluA2-AMPARs was decreased in neurons incubated with TIA (10 and 100 μM) compared to vehicle-treated neurons (vehicle=0.11±0.022–0.24 μm2 s−1, n=753; TIA (10 μM)=0.087±0.025–0.20 μm2 s−1, n=446; TIA (100 μM)=0.055±0.007–0.142 μm2 s−1, n=255; ***P<0.001). (d) Synaptic GluA2-AMPAR diffusion coefficient was also decreased after TIA incubation (10 and 100 μM) (vehicle=0.08±0.03–0.18 μm2 s−1, n=281; TIA (10 μM)=0.04±0.01–0.11 μm2 s−1, n=245; TIA (100 μM)=0.03±0.007–0.09 μm2 s−1, n=214; ***P<0.001). (Right panel) Cumulative distributions of diffusion coefficients of neurons treated with vehicle or TIA (10 and 100 μM). Note the shift toward the left in the presence of TIA, indicating a reduced GluA2-AMPAR surface diffusion immobilized at synapses.
Figure 2
Figure 2
Tianeptine (TIA) reduces the surface diffusion of GluA1-AMPAR. (a) Typical time-lapse images during fluorescence recovery after photobleaching (FRAP) in hippocampal neurons co-transfected with homer1c∷DsRed and GluA1∷SEP. Homer1c∷DsRed image (very left panel) was recorded 5 s before photobleaching. The rest were GluA1∷SEP images 5 s before photobleaching (t−5 s), at photobleaching (t0 s) and 40, 80, 120 s after photobleaching (t+40 s, t+80 s, t+120 s). White arrows indicate a typical synaptic area. (b) Quantified relative fluorescence intensity recovery during 150 s at extrasynaptic (left panel) and synaptic (right panel) compartments. Compared to vehicle, TIA (10 and 100 μM) slowed down GluA1-SEP fluorescence recovery in the extrasynaptic and synaptic compartments. (c) Quantified GluA1-AMPARs mobile fraction at extrasynaptic (left panel) and synaptic (right panel) compartments. The mobile fraction was considered as the percentage of fluorescence recovery at the end of FRAP (elapse time 150 s) (vehicle=78±3.5%, n=24; TIA (10 μM)=66±4.4%, n=21; TIA (100 μM): 55±4.9%, n=21; right panel, vehicle=60±2.5%, n=44; TIA (10 μM)=40±2.8%, n=30; TIA (100 μM)=38±1.9%, n=20; **P<0.01; ***P<0.001).
Figure 3
Figure 3
Tianeptine (TIA)-induced GluA-AMPA receptor (AMPAR) surface diffusion decrease is a Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent mechanism. (a) Surface GluA2-AMPARs were tracked in the synaptic compartments of days in vitro (DIV) 11–12 cultured hippocampal neurons in the presence/absence of TIA (10 μM) (left panel) and of 100 μM TIA (right panel) with or without CaMKII inhibitor, KN93 (10 μM) (median±25–75% interquartile range (IQR); vehicle=0.06±0.01–0.15 μm2 s−1, n=276; TIA (10 μM)+DMSO=0.04±0.01–0.1 μm2 s−1, n=210; TIA (10 μM)+KN93=0.08±0.02–0.17 μm2 s−1, n=244; ***P<0.001; right panel: vehicle=0.09±0.03–0.19 μm2 s−1, n=198; TIA (100 μM)+DMSO=0.04±0.01–0.13 μm2 s−1, n=155; TIA (100 μM)+KN93=0.08±0.03–0.19 μm2 s−1, n=173; ***P<0.001). (b) Ensemble AMPAR mobility was assessed by fluorescence after photobleaching (FRAP) at synaptic compartments in the presence/absence of TIA (10 μM) (left panel) and of TIA (100 μM) (right panel) with or without CaMKII inhibitor, KN93 (10 μM). The decreased GluA1-AMPAR fluorescence recovery induced by TIA (10 or 100 μM) was fully restored in the presence of KN93. (c) Quantified GluA1-AMPARs mobile fraction at synaptic compartments in vehicle, TIA and TIA+KN93 conditions. The mobile fraction was considered as the percentage of fluorescence recovery at the end of FRAP (elapse time 150 s) (left panel: vehicle=64±4.7%, n=26; TIA (10 μM)=40±3.2%, n=21; TIA (10 μM)+KN93=57±3%, n=32; right panel: vehicle=64±4.7%, n=26; TIA (100 μM)=31±3.6%, n=24; TIA (100 μM)+KN93=55±4.4%, n=18; **P<0.01; ***P<0.001). DMSO, dimethylsulfoxide.
Figure 4
Figure 4
The phosphorylation of stargazin and its PDZ binding site are required to mediate tianeptine's (TIA's) effects on AMPA receptor (AMPAR) surface diffusion. (a) Global (extrasynaptic and synaptic) surface GluA2-AMPAR were tracked in days in vitro (DIV) 11–12 cultured hippocampal neurons transfected with wild-type stargazin (WT Stg) and dephosphorylated stargazin S9A mutant (StA) from which nine serines at the putative Ca2+/calmodulin-dependent protein kinase II/protein kinase C (CaMKII/PKC) phosphorylation sites were mutated to alanines. In WT Stg-expressing cells, TIA (10 μM) significantly decreased the GluA2-AMPAR lateral diffusion, while in StA-expressing cells this effect was fully blocked (right panel) (median±25–75% interquartile range (IQR); WT Stg-vehicle=0.10±0.04–0.21 μm2 s−1, n=454; WT Stg-TIA (10 μM)=0.05±0.01–0.15 μm2 s−1, n=365; StA-vehicle=0.11±0.05–0.20 μm2 s−1, n=424; StA-TIA (10 μM) median=0.11±0.04–0.20 μm2 s−1, n=563; ***P<0.001). The left panel shows typical quantum dot (QD) trajectories in the four groups. (b) Global surface GluA2-AMPAR were tracked in DIV 11–12 cultured hippocampal neurons transfected with WT Stg or a mutant stargazin (ΔC Stg) in which the last four C terminus amino acids corresponding to the PDZ binding site of PSD-95 were deleted. In ΔC Stg- but not WT Stg-expressing neurons, TIA failed to reduce GluA2-AMPAR surface diffusion (median±25–75% IQR; WT Stg-vehicle=0.186±0.08–0.32 μm2 s−1, n=189; WT Stg-TIA (10 μM)=0.09±0.02–0.21 μm2 s−1, n=241; ΔC Stg-vehicle=0.22±0.10–0.39 μm2 s−1, n=173; ΔC Stg-TIA (10 μM)=0.20±0.11–0.32 μm2 s−1, n=132; ***P<0.001).
Figure 5
Figure 5
Tianeptine (TIA) enhances stargazin to PSD-95 binding in a Ca2+/calmodulin-dependent protein kinase II (CaMKII)-dependent process. The interaction between stargazin and PSD-95 was studied by Förster resonance energy transfer (FRET)- fluorescence lifetime imaging microscopy (FLIM) in synaptic and extrasynaptic compartments in days in vitro (DIV) 9 cultured hippocampal neurons with or without TIA treatments. (a) Experimental schemes showing that neurons were transfected with PSD-95∷GFP and stargazin∷tetracysteine (Stg∷4cys). The site that ReAsH binds to 4cys tags is on the C terminus domain of stargazin 17 amino acids away from the PDZ binding domain. The interaction between stargazin and PSD-95 was monitored by FRET/FLIM, indicated by a decrease in GFP lifetime. (b) Sample images of neurons expressing Stg∷4cys and PSD-95∷GFP with vehicle (left panels) or with TIA treatment (right panels). ReAsH labeling was diffused (top panels). PSD-95∷GFP was present in soma, dendrites and clustered in synapses (middle panels). Short lifetime was indicated by blue areas where PSD-95∷GFP binds to ReAsH-labeled Stg∷4cys (bottom panels). Scale bar=10 μm. (c) Synaptic measurements of PSD-95∷GFP lifetime in the presence of ReAsH. GFP lifetime was significantly decreased only in neurons co-expressing Stg∷4cys and PSD-95, but not in neurons expressing PSD-95 alone or with HA∷stargazin (HA∷Stg) (PSD-95∷GFP=2.182±0.013 ns, n=14; PSD-95∷GFP/HA∷Stg=2.191±0.015 ns, n=14; PSD-95∷GFP/Stg∷4cys=1.839±0.011 ns, n=24; ***P<0.0001). (d) Synaptic analysis of PSD-95∷GFP lifetime after ReAsH labeling in PSD-95∷GFP/Stg∷4cys expressing neurons with vehicle or TIA treatment. TIA (10 μM) significantly decreased PSD-95∷GFP lifetime (vehicle=1.839±0.011 ns, n=25; +TIA=1.607±0.012 ns, n=18; ***P<0.0001). Its synaptic effect was partially blocked by KN93 (10 μM) (TIA+KN93=1.767±0.013 ns, n=20; ***P<0.0001). And 10 μM of KN93 alone increased PSD-95∷GFP lifetime (KN93=2.084±0.021 ns, n=4; ***P<0.0001). (e) Extrasynaptic analysis of PSD-95∷GFP lifetime after ReAsH labeling in PSD-95∷GFP/Stg∷4cys expressing neurons with vehicle or TIA treatment. TIA (10 μM) significantly decreased PSD-95∷GFP lifetime (vehicle=2.224±0.013 ns, n=25; +TIA=2.062±0.021 ns, n=18; ***P<0.0001). Its extrasynaptic effect was fully block by KN93 (10 μM) (TIA+KN93=2.200±0.020 ns, n=20; ***P<0.0001). And 10 μM of KN93 had no effect on PSD-95∷GFP lifetime (KN93=2.251±0.027 ns, n=4; ***P<0.0001). GFP, green fluorescent protein.
Figure 6
Figure 6
Tianeptine increases excitatory transmission and AMPA receptor (AMPAR) synaptic content. (a) Control field excitatory postsynaptic potentials (fEPSPs) were generated by stimulation (0.033 Hz) of the afferent Schaffer collateral–commissural pathway from the CA3 area to the CA1 region. Illustrated is a representative trace of an fEPSP before (black) and after (red) tianeptine (10 μM; 30 min). (b) A histogram demonstrating the increase of the slope of the fEPSP (% of the control fEPSP) produced by 10 and by 50 μM tianeptine. Note that the increase of the fEPSP produced by tianeptine (10 μM) is completely prevented by pre-treatment with the Ca2+/calmodulin-dependent protein kinase II (CaMKII) inhibitor KN93 (10 μM). Each bar represents the mean±s.e.m. of 3–4 independent experiments. (c) Sample live fluorescence images of neurites coexpressing homer1c∷DsRed (top panels) as postsynaptic density marker of glutamatergic synapses and GluA1∷SEP (middle panels) indicating surface AMPARs before (left panels) and 30 min after (right panels) tianeptine application. The bottom panels were GluA1∷SEP images in pseudocolor. Scale bar, 10 μm. (d) Quantified GluA1∷SEP intensity at synaptic (colocalization with the postsynaptic protein, homer1c) and extrasynaptic compartments from paired experiments. Each point represents the mean of GluA1∷SEP intensity from 10 to 12 regions before (‘0 min' left points) or 30 min after (right points) tianeptine/vehicle application. The 30-min incubation with vehicle (H2O) (top panels) did not significantly affect GluA1∷SEP intensity at synapses (0 min: 44±5.9 arbitrary unit (a.u.), n=5; 30 min=36±5 a.u., n=5) or in the extrasynaptic compartment (0 min: 89±10.3 a.u., n=5; 30 min=89±10 a.u., n=5). Incubation of neurons with tianeptine (10 μM, 30 min) (bottom panels) significantly increased GluA1∷SEP intensity at synapses (0 min: 41±5.7 .u., n=5; 30 min=48±6.5 a.u., n=5; **P<0.01). The extrasynaptic content was not significantly altered, but a tendency toward an increase content was clearly noted (0 min=101±10 a.u., n=5; 30 min=123±13 a.u., n=5; P=0.06). a.u., arbitrary unit.
Figure 7
Figure 7
Tianeptine enhances hippocampal CA1 long-term potentiation (LTP) and rescues the suppression of LTP induced by acute stress, or by corticosterone. (a) The slope of the field excitatory postsynaptic potentials (fEPSP) (% of the control fEPSP) is plotted as a function of time before and after delivery of a 3-pulse theta burst stimulation (TBS; at t=10 min). The stimulus is designed to induce a submaximal form of LTP (black). Tianeptine (10 μM) greatly enhanced this submaximal LTP (red). Each time point (one every 30 s) represents the mean±s.e.m. of 6–12 independent experiments. (b) The histogram compares the magnitude of LTP (% increase from control of the slope of the fEPSP) determined at 50 min post-TBS and illustrates that a 3-pulse TBS protocol (black) is far less effective in producing LTP than a 4-pulse TBS (white). However, tianeptine (10 μM) greatly increased the magnitude of LTP induced by a 3-pulse TBS to a level similar to that produced by the 4-pulse TBS protocol (red). In contrast to the effect of tianeptine (10 μM) on 3-pulse TBS, this concentration of the drug has no effect on maximal LTP induced by the 4-pulse TBS protocol (gray). Each bar represents the mean±s.e.m. of six independent experiments (*P<0.05). (c) The slope of the fEPSP (% of the control fEPSP) is plotted as a function of time after delivery of a 4-pulse TBS (at t=10 min) for control (black squares), ‘stressed' (open squares) and stressed plus 10 μM tianeptine (gray triangles). Note that pre-exposure to acute stress greatly decreased the magnitude of LTP, when compared to that achieved with control neurons. Tianeptine (10 μM) reversed this stress-induced deficit of LTP. Each time point (one every 30 s) represents the mean±s.e.m. of 6–12 independent experiments. (d) The histogram compares the magnitude of LTP determined at 50 min post 4-pulse TBS. Acute stress and corticosterone (10 μM) result in a clear reduction in the extent of LTP when compared to control, which are fully rescued by (10 and 3μM) tianeptine. Each bar represents the mean±s.e.m. of 6–12 independent experiments (*P<0.05).
Figure 8
Figure 8
Tianeptine (TIA) prevents corticosterone-induced GluA2- AMPA receptor (AMPAR) surface diffusion increase. Surface GluA2-AMPARs were tracked in the extrasynaptic and synaptic compartments of days in vitro (DIV) 11–12 cultured hippocampal neurons after vehicle/vehicle, corticosterone (Cort)/vehicle or Cort/TIA application. (a) Experimental scheme showing that neurons were incubated with Cort/vehicle (phosphate-buffered saline (PBS)) for 20 min, washed, incubated with TIA (10 μM)/vehicle (H2O) for 90 min, washed and then tracked for 15–30 min. (b) Representative trajectories (20–40 s) of single surface GluA2-AMPAR in vehicle/vehicle, Cort/vehicle and Cort/TIA conditions. The first point of the trajectory is represented by an arrowhead and the last point by a filled circle. (c) The diffusion coefficients (median±25–75% interquartile range (IQR)) of extrasynaptic and synaptic GluA2-AMPAR were increased in neurons incubated with Cort compared to vehicle-treated neurons. These increases were fully prevented by 10 μM TIA (extrasynaptic: vehicle+vehicle=0.16±0.08–0.29 μm2 s−1, n=153; Cort+vehicle=0.24±0.11–0.44 μm2 s−1, n=350; Cort+TIA (10 μM)=0.11±0.05–0.26 μm2 s−1, n=214; P<0.001; synaptic: vehicle+vehicle=0.05±0.025–0.16 μm2 s−1, n=101; Cort+vehicle=0.09±0.05–0.17 μm2 s−1, n=210; Cort+TIA (10 μM)=0.04±0.02–0.08 μm2 s−1, n=165; ***P<0.001). (d) Schematic representation of TIA's effects on AMPAR surface diffusion and synaptic long-term potentiation (LTP).
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