A novel mechanism for nicotinic potentiation of glutamatergic synapses

Abstract

Selective strengthening of specific glutamatergic synapses in the mammalian hippocampus is critical for encoding new memories. This is most commonly achieved by input-specific Hebbian-type plasticity involving glutamate-dependent coincident presynaptic and postsynaptic depolarization. Our results demonstrate a novel mechanism by which nicotinic signaling, independently of coincident fast glutamatergic transmission, increases synaptic strength in the hippocampus. Electrophysiological recordings from rat hippocampal neurons in culture revealed that 1-3 h of exposure to 1 μm nicotine, even with action potentials being blocked, produced increases in both the frequency and amplitude of miniature EPSCs. Possible mechanisms were analyzed both in mouse organotypic slice culture and in rat cell culture by inducing the cells to express super-ecliptic pHluorin-tagged GluA1-containing AMPA receptors, which fluoresce only on the cell surface. Pharmacological and genetic manipulation of the cells, in combination with fluorescence-recovery-after-photobleaching experiments, revealed that nicotine, acting through α7-containing nicotinic acetylcholine receptors on the postsynaptic neuron, induces the stabilization and accumulation of GluA1-containing AMPA receptors on dendritic spines. The process relies on intracellular calcium signaling, PDZ [postsynaptic density-95 (PSD-95)/Discs large (Dlg)/zona occludens-1 (ZO-1)] interactions with members of the PSD-95 family, and lateral diffusion of the GluA1 receptors on the cell surface. These findings define a new avenue by which nicotinic signaling modulates synaptic mechanisms thought to subserve learning and memory.

Keywords: FRAP; GluA1; hippocampus; nAChR; nicotine; plasticity.

Figure 1.

Nicotine treatment increases mEPSC frequency and amplitude generated by native AMPARs independently of AP-driven activity. A, Representative traces of mEPSCs in cells treated with nicotine for 1–3 h (Nic) versus controls (Ctrl). B, Cumulative histograms of mEPSC interevent intervals showing that the nicotine treatment increased the frequency of events (decreased the interevent intervals; Ctrl vs Nic, n = 2800 events for both, p < 0.0001, KS). C, Amplitude histograms showing that the nicotine treatment increases mEPSC amplitude (Ctrl vs Nic, n = 2800 events for both, p < 0.0001, KS). D, Representative traces of mEPSCs in cells treated with TTX to block APs while incubated with nicotine or control solution. E, Reduced interevent intervals, meaning increased mEPSC frequency, in cells treated with nicotine + TTX for 1–3 h (Ctrl + TTX vs Nic + TTX, n = 2500, 2600 events, p < 0.0001, KS). F, TTX did not prevent nicotine from increasing mEPSC amplitude (Ctrl + TTX vs Nic + TTX, n = 2400, 2600 events, p < 0.0001, KS).

Figure 2.

Nicotine stabilizes GluA1s on dendritic spines in hippocampal cell culture. A, Depiction of an AMPAR containing a transgenic GluA subunit tagged with SEP, a pH-sensitive GFP that only fluoresces while on the cell surface. B, GluA1-SEP fluorescence on spines of neurons treated with 1 μm nicotine for 1–3 h in cell culture recovers less in 16.5 min after photobleaching than do corresponding controls, indicating decreased mobility (Ctrl vs Nic: 96 ± 3 vs 77 ± 2%, n = 24,24; p = 0.00002, WC). C, Control condition images of GluA1-SEP in dissociated culture with accompanying cytosolic-RFP images. Top and bottom arrows indicate bleached spines. Scale bar, 2 μm. D, The nicotine effect on GluA1-SEP FRAP occurs gradually over 2 h. Cultures were transferred into a perfusion chamber with 1 μm nicotine and imaged for 2 h. Data points represent the final recovery point from different FRAP experiments. Values were fit with a linear regression demonstrating a trend in nicotine-treated cultures, but in not control cultures (control: slope −0.03 ± 0.12%, y-intercept 87.18 ± 8.42%, n = 25, r² = 0.0026, p = 0.8087; nicotine: slope −0.22 ± 0.10%, y-intercept 85.85 ± 6.86%, n = 27, r² = 0.1429, p = 0.0519). E, Control condition images of GluA2-SEP FRAP in dissociated culture with accompanying cytosolic-RFP images. F, Nicotine has no effect on GluA2-SEP mobility, yielding a recovery equivalent to control (Ctrl vs Nic: 81 ± 3 vs 84 ± 3%; n = 15,17; p = 0.49).

Figure 3.

In organotypic slice culture, nicotine increases mEPSC frequency and amplitude generated by native AMPARs on CA1 pyramidal neurons and stabilizes GluA1 on dendritic spines. A, Representative traces of mEPSCs from CA1 pyramidal neurons in OT slices treated with nicotine for 1.5–3 h (Nic) versus controls (Ctrl). B, Cumulative histograms of mEPSC interevent intervals showing that the nicotine treatment increased the frequency of events (decreased the interevent intervals; Ctrl vs Nic, n = 2400,2200 events, p = 0.0017, KS). C, Amplitude histograms showing that the nicotine treatment increased mEPSC amplitude (Ctrl vs Nic, n = 2500,2400 events, p < 0.0001, KS). D, Images of CA1 pyramidal neuron basal dendrites depicting GluA1-SEP FRAP under control conditions with accompanying cytosolic-RFP images. Arrows indicate bleached spines. Scale bar, 2 μm. E, GluA1-SEP fluorescence on spines treated with 1 μm nicotine for 2–3 h recovers to a lesser extent in 31 min after photobleaching than do corresponding controls, indicating decreased mobility (Ctrl vs Nic: 98 ± 4 vs 77 ± 5%; n = 12,14; p = 0.0104).

Figure 4.

The nicotine effect on GluA1-SEP FRAP is restricted to spines and represents a change in the rate of exchange between spine and dendrite surface receptors. A, Representative images depicting the bleaching of a spine-sized area of GluA1-SEP on the dendrite. The bleached region and region measured for FRAP were the same, represented by the outlined area. Scale bar, 2 μm. B, FRAP is quite rapid on the dendrite, and there is no difference in mobility of GluA1-SEP on the dendrite between control and nicotine-treated cells (Ctrl vs Nic: 102 ± 3 vs 101 ± 2%; n = 14,15; p = 0.87). C, Representative images depicting bleach of GluA1-SEP signal along 10 μm of the dendrite surrounding a bleached spine. Solid outline indicates the region used to measure FRAP. Dotted outline represents the entire area that was bleached. Scale bar, 3 μm. D, When the reserve pool of surface fluorescent receptors is depleted, GluA1-SEP recovery at the spine is greatly attenuated both in control and nicotine-treated cells, indicating that previously observed recoveries represented exchange between receptor pools on spines and dendrites (Ctrl vs Nic: 55 ± 3 vs 50 ± 3%; n = 12,12; p = 0.28).

Figure 5.

The nicotine-induced decrease in GluA1-SEP mobility on spines correlates with an increase in receptor density on spines. A, Representative prebleach images from FRAP experiments in which spine area was calculated from the RFP signal. B, Representative prebleach images from FRAP experiments demonstrating nicotine-induced enrichment of GluA1-SEP at spines. C, Quantification of GluA1-SEP enrichment on spines by measuring spine/shaft optical density yielded a significant increase in cells exposed to nicotine, suggesting that nicotine leads to the accumulation of receptors on spines. (Ctrl vs Nic: 0.99 ± 0.05 vs 1.67 ± 0.1; n = 29,28; p = 0.00000014, WC). D, The nicotine effect on GluA1-SEP spine enrichment occurs gradually over 2 h. Cultures were transferred into a perfusion chamber with 1 μm nicotine and imaged for 2 h. Data points represent the spine enrichment of GluA1-SEP measured from the baseline images of corresponding FRAP experiments of different spines. Values were fit with a linear regression demonstrating a trend in nicotine-treated cultures, but not control cultures (control: slope −0.003 ± 0.002, y-intercept 1.52 ± 0.15 n = 27, r² = 0.0554, p = 0.2373; nicotine: slope 0.007 ± 0.003, y-intercept 1.446 ± 0.21, n = 27, r² = 0.1572, p = 0.0406). E, Representative images depicting the effect of nicotine on the population distribution of GluA1-SEP spine enrichment on dendritic arbors. Images were taken at 5× zoom to visualize multiple spines from a single dendrite (enrichment images were usually at 15×). Z-stacks were collapsed as a sum of all slices in ImageJ. Arrows indicate enriched spines. Scale bar, 5 μm. F, Frequency histogram of GluA1-SEP enrichment at spines from multiple neuronal dendritic arbors revealing a shift toward greater enrichment in nicotine-treated cells and the appearance of a heavily enriched subpopulation (Ctrl, n = 85 spines from 5 cells; Nic, n = 71 spines from 5 cells, 1 week of plating). G, Cumulative frequency plot of data from B demonstrating the statistically significant difference between the two distributions (Ctrl vs Nic, p = 0.0007, KS).

Figure 6.

Receptor blockade shows that nicotine acts via α7-nAChRs, not α4β2-nAChRs, to stabilize GluA1s on spines independent of AMPA and NMDA receptor activation. A, Time course of GluA1-SEP FRAP after 1–3 h in nicotine with and without 100 nm MLA to block α7-nAChRs (MLA vs MLA + Nic: 97 ± 5 vs 96 ± 5%, n = 12,15; p = 0.86; Ctrl vs Nic: 97 ± 5% vs 78 ± 3%, n = 14,14; p = 0.0038, WC). B, Relative GluA1-SEP enrichment on spines under conditions from A (MLA vs MLA + Nic: 1.23 ± 0.11 vs 1.17 ± 0.09; n = 16,16; p = 0.66; Ctrl vs Nic: 1.09 ± 0.10 vs 1.61 ± 0.11; n = 16,16; p = 0.0013). C, Blockade of α4β2-nAChRs with 1 μm DHβE during the nicotine treatment had no effect on GluA1-SEP mobility (DHβE vs DHβE + Nic: 93 ± 3 vs 79 ± 5%, n = 11,12; p = 0.024, WC; Ctrl vs Nic: 99 ± 3 vs 78 ± 4%, n = 12,11; p = 0.0004). D, DHβE did not prevent nicotine-induced GluA1-SEP enrichment on spines (DHβE vs DHβE + Nic: 1.03 ± 0.11 vs 1.57 ± 0.18; n = 1 2,12; p = 0.0168; Ctrl vs Nic: 0.94 ± 0.08 vs 1.57 ± 0.12; n = 12,12; p = 0.0002). E, Blockade of AMPARs and NMDARs with 50 μm APV and 20 μm NBQX, respectively, during the nicotine treatment had no effect on GluA1-SEP mobility on spines (APV + NBQX vs APV + NBQX + Nic: 98 ± 6 vs 58 ± 7%, n = 10,11; p = 0.0006; Ctrl vs Nic: 94 ± 4 vs 71 ± 5%, n = 10,11; p = 0.0017). F, APV and NBQX failed to block GluA1-SEP enrichment on spines (APV + NBQX vs APV + NBQX + Nic: 1.2 ± 0.11 vs 1.73 ± 0.21; n = 11,11; p = 0.0405, WC; Ctrl vs Nic: 1.18 ± 0.08 vs 1.67 ± 0.1; n = 11,11; p = 0.001).

Figure 7.

Knock-down of α7-nAChRs by shRNA shows that the receptors are required on the postsynaptic cell for the nicotinic effect. A, Representative image of a neuron in dissociated hippocampal culture expressing cytosolic RFP (red) and a SEP-tagged GluA subunit (green). Individual channels are in gray-scale in the images at the top and a merged image is shown on the bottom. GluA-SEP fluorescence, representing surface receptors, is found on the cell body, along the dendritic shaft, and on dendritic spines. It is lacking from putative axons, which only express RFP, indicated by arrows. Scale bar, 5 μm. B, Representative RFP image of an experimentally acceptable scenario free of transfected axons. Scale bar, 2 μm. C, RFP image exemplifying an unacceptable experimental scenario in which a transfected axon runs juxtaposed to a dendritic spine, possibly forming a synapse. The arrow indicates the putative axon. Scale bar, 2 μm. D, E, Nicotine had no effect on GluA1-SEP trafficking on neurons expressing α7-shRNA, although it remained effective on cells expressing the control Scr-shRNA. Nicotine acts directly through α7-nAChRs on the postsynaptic neuron to stabilize and enrich GluA1-SEP at spines (α7-shRNA vs α7-shRNA + Nic, FRAP: 92 ± 5 vs 94 ± 5%, n = 12,12; p = 0.79; enrichment: 1.08 ± 0.08 vs 1.2 ± 0.1; n = 12,12; p = 0.37; Scr-shRNA vs Scr-shRNA + Nic, FRAP: 97 ± 4, vs 76 ± 4%, n = 11,12, p = 0.0007; enrichment: 1.13 ± 0.09 vs 1.91 ± 0.23, n = 11,12, p = 0.0065, WC).

Figure 8.

The nicotine-induced effects on GluA1 trafficking can be blocked by chelating intracellular calcium, disrupting PSD-95 family member PDZ-interactions, or restricting lateral diffusion of surface GluA1s with antibody x-linking. A, B, GluA1-SEP FRAP and enrichment on spines in controls versus nicotine with and without the calcium chelator BAPTA-AM applied 30 min before and throughout the nicotine treatment (BAPTA-AM vs BAPTA-AM + Nic, FRAP: 89 ± 3 vs 88 ± 3%, n = 15,15; p = 0.75; enrichment: 1.14 ± 0.1 vs 1.14 ± 0.08; n = 15,15; p = 0.98; Ctrl vs Nic, FRAP: 99 ± 3, vs 78 ± 3%, n = 14,13, p = 0.000087; enrichment: 1.03 ± 0.07 vs 1.74 ± 0.18, n = 14,13, p = 0.0014, WC). C, D, GluA1-SEP FRAP and enrichment on spines in controls veresus nicotine with and without transfection with CRIPT, a dominant-negative protein that disrupts PDZ-interactions with PSD-95 family members (CRIPT vs CRIPT+Nic, FRAP: 93 ± 3 vs 88 ± 2%, n = 14,13; p = 0.18, WC; Enrichment: 0.94 ± 0.05 vs 1.01 ± 0.06; n = 14,14; p = 0.42; Ctrl vs Nic, FRAP: 91 ± 3, vs 71 ± 4%, n = 12,12, p = 0.0004; enrichment: 1.06 ± 0.07 vs 1.53 ± 0.09, n = 12,12, p = 0.00052). E, GluA1-SEP FRAP on spines in controls vs nicotine after x-linking GluA1-SEP with a GFP-specific antibody (x-link vs x-link+Nic; 33 ± 6 vs 32 ± 4%, n = 10,10; p = 0.88). F, GluA1-SEP enrichment on spines in controls vs nicotine after antibody x-linking of GluA1-SEP (x-link vs x-link + Nic; 1.21 ± 0.16 vs 0.9 ± 0.15, n = 10,10; p = 0.19). G, H, GluA1-SEP FRAP and enrichment on spines in controls vs nicotine with and without BFA treatment 30 min before and during the nicotine incubation to block receptor exocytosis (BFA vs BFA+Nic, FRAP: 90 ± 4 vs 77 ± 4%, n = 13,12, p = 0.0135; enrichment: 0.99 ± 0.06 vs 1.36 ± 0.06, n = 13,13, p = 0.0002; Ctrl vs Nic, FRAP: 93 ± 6, vs 70 ± 4%, n = 10,10, p = 0.0048; enrichment: 0.93 ± 0.08 vs 1.56 ± 0.09, n = 10,10, p = 0.00008). Existing surface GluA1-SEP are sufficient for the calcium- and PDZ-dependent effects of nicotine without requiring exocytosis of additional receptors.

Figure 9.

Preventing nicotine-induced GluA1 enrichment with antibody x-linking blocks the corresponding increase in mEPSC amplitude. A, Representative traces from control and nicotine-treated cells after antibody x-linking of surface GluA1s. B, Cumulative histograms of mEPSC interevent intervals in control versus nicotine-treated cells after antibody x-linking of surface GluA1s (Ctrl vs Nic, p < 0.0001, n = 2700, 2600 events; KS). C, Cumulative histograms of mEPSC amplitudes in controls and nicotine-treated cells after antibody x-linking (Ctrl vs Nic, n = 2600, 2800 events; p < 0.0001, KS, due to more small events in nicotine-treated cells; no difference was seen in larger events, unlike results obtained before x-linking as in Fig. 1).

Figure 10.

Model diagramming the nicotine-dependent changes in GluA1 mobility and stabilization on dendritic spines. Most GluA1s are mobile and not synaptically incorporated. Nicotine activates α7-nAChRs on the postsynaptic cell to trap surface-diffusing GluA1s on spines independently of fast, excitatory glutamatergic transmission. The process requires intracellular calcium and the availability of PDZ-binding scaffold slots. The enrichment and stabilization of GluA1s on spines represents synaptic incorporation of the receptors, strengthening synaptic transmission, as indicated by the influx arrows and by the large depolarizing trace within the spine head.