Retinoic Acid and LTP Recruit Postsynaptic AMPA Receptors Using Distinct SNARE-Dependent Mechanisms

Abstract

Retinoic acid (RA)-dependent homeostatic plasticity and NMDA receptor-dependent long-term potentiation (LTP), a form of Hebbian plasticity, both enhance synaptic strength by increasing the abundance of postsynaptic AMPA receptors (AMPARs). However, it is unclear whether the molecular mechanisms mediating AMPAR trafficking during homeostatic and Hebbian plasticity differ, and it is unknown how RA signaling impacts Hebbian plasticity. Here, we show that RA increases postsynaptic AMPAR abundance using an activity-dependent mechanism that requires a unique SNARE (soluble NSF-attachment protein receptor)-dependent fusion machinery different from that mediating LTP. Specifically, RA-induced AMPAR trafficking did not involve complexin, which activates SNARE complexes containing syntaxin-1 or -3, but not complexes containing syntaxin-4, whereas LTP required complexin. Moreover, RA-induced AMPAR trafficking utilized the Q-SNARE syntaxin-4, whereas LTP utilized syntaxin-3; both additionally required the Q-SNARE SNAP-47 and the R-SNARE synatobrevin-2. Finally, acute RA treatment blocked subsequent LTP expression, probably by increasing AMPAR trafficking. Thus, RA-induced homeostatic plasticity involves a novel, activity-dependent postsynaptic AMPAR-trafficking pathway mediated by a unique SNARE-dependent fusion machinery.

Figure 1. Acute RA treatment impairs hippocampal LTP

(A) Example traces (left) and summary graph (right) of LTP in DMSO- or RA-treated hippocampal CA1 pyramidal neurons from cultured slices (p < 0.0001). (B) Example traces and summary graph of LTP in CA1 pyramidal neurons with RARα deletion. Lentivirus expressing wild-type or mutant inactive Cre recombinase (Cre or mCre) were injected into the CA1 regions of the hippocampal slices obtained from RARα conditional KO mice. LTP was examined 7–10 days after viral injection (mCre/DMSO vs. mCre/RA: p < 0.005; Cre/DMSO vs. Cre/RA: p > 0.5; mCre/RA vs. Cre/RA: p < 0.0001). (C) Example traces and summary graph of LTP in CA1 pyramidal neurons treated with anisomycin, RA or both (RA vs. aniso: p < 0.005; aniso vs. RA+aniso: p > 0.9). Scale bars in(A)–(C): 20 pA, 20 ms. Black bars in all summary graphs indicate the time window for LTP magnitude quantification. All graphs represent average values ± s.e.m.

Figure 2. RA increases AMPAR-mediated excitatory synaptic transmission through an activity-dependent mechanism

(A) Trace examples (left), amplitude (middle) and frequency (right) quantification of mEPSCs recordings from DMSO- or RA-treated CA1 pyramidal neurons (***, p < 0.0005). Scale bars: 10 pA, 1s. (B) Dual component mEPSC recordings in CA1 pyramidal neurons treated with DMSO or RA. Left: traces examples of AMPA only and dual component mEPSCs. Right: quantification of AMPA and NMDA mEPSC amplitude (*, p < 0.05). NMDA mEPSC component was calculated by subtracting the average AMPA mEPSC component from the average dual mEPSC component. Scale bars: 4 pA, 10 ms. (C) Ratio of AMPAR- to NMDAR-mediated EPSCs in CA1 pyramidal neurons treated with DMSO or RA (*, p < 0.05). Representative EPSCs recorded at −60 mV and +40 mV are shown to the left. Scale bars: 50 pA, 20 ms. (D) Trace examples (left) and quantification of mEPSC amplitude and frequency obtained from CA1 pyramidal neurons treated with four hours of DMSO, RA, RA+TTX, or RA+APV (***, p < 0.0001). Scale bars: 10 pA, 1 s. All graphs represent average values ± s.e.m.

Figure 3. RA treatment activates postsynaptic silent synapses by promoting AMPAR insertion into the synaptic membrane

(A) Schematic diagrams of various treatment protocols used in the experiments for this figure. (B–D) Trace examples and scatter plots of eEPSCs recorded from CA1 pyramidal neurons treated with DMSO (B), TTX (60 hours) (C), and RA (4 hr) (D) at −60 mV and +40 mV. Scale bars: 10 pA, 10 ms. (E) Failure rate of eEPSCs recorded at −60 mV and +40 mV from DMSO-, TTX- and RA- treated neurons (***, p < 1 x 10⁻⁹). (F) Failure rate of eEPSCs recorded at −60 mV and +40 mV from neurons treated with TTX wash/RA, TTX no wash/RA, TTX wash, and TTX wash/RA+APV (***, p < 0.0005). (G) Top: example traces of mEPSC recordings. Bottom: quantification of amplitude and frequency of mEPSCs recorded from CA1 pyramidal neurons treated with DMSO, TTX and TTX wash/RA (**, p < 0.01; ***, p < 0.001). Scale bars: 15 pA, 1s. (H) Top: example traces of mEPSC recordings. Bottom: quantification of amplitude and frequency of mEPSCs recorded from CA1 pyramidal neurons treated with DMSO, TTX and TTX no wash/RA (**, p < 0.01; ***, p < 1 x 10⁻⁶). Scale bars: 15 pA, 1s. All graphs represent average values ± s.e.m.

Figure 4. Synaptobrevin-2 and SNAP-47, but not complexin, are required for RA-induced increase in excitatory synaptic transmission

(A) Amplitude and frequency analysis of mEPSCs recorded from CA1 pyramidal neurons infected with lentivirus expressing Cpx KD constructs and treated with DMSO or RA. Neighboring uninfected neurons were recorded as controls (*, p < 0.05; ***, p < 0.001). (B) Amplitude and frequency analysis of mEPSCs recorded from CA1 pyramidal neurons expressing tetanus toxin light chain (tetTox) treated with DMSO or RA. Neighboring uninfected neurons were recorded as controls (*, p < 0.05; ***, p < 1 x 10⁻⁵). (C) Amplitude and frequency analysis of mEPSCs recorded from CA1 pyramidal neurons expressing SNAP-47 KD construct treated with DMSO or RA. Neighboring uninfected neurons were recorded as controls (***, p < 1 x 10⁻⁴). (D) Amplitude and frequency analysis of mEPSCs recorded from CA1 pyramidal neurons infected with lentivirus expressing both SNAP-47 KD and wild-type SNAP-47 rescue constructs (SNAP-47 Rep) treated with DMSO or RA. Neighboring uninfected neurons were recorded as controls (***, p < 1 x 10⁻⁴). All graphs represent average values ± s.e.m.

Figure 5. Syntaxin-4, but not Syntaxin-1 or Syntaxin-3, is required for RA-induced increase in excitatory synaptic transmission

(A) Analysis of amplitude and frequency of mEPSCs recorded from control and Stx-1 KD CA1 pyramidal neurons in cultured hippocampal slices treated with DMSO or RA (**, p < 0.01). Top: schematic of Stx-1 showing its functional domains (Habc, Habc domains; SNARE, soluble NSF-attachment protein receptor motif; TMR, transmembrane domain; green indicates complexin-binding sequence). (B) Summary of mEPSC amplitude and frequency analysis from control and Stx-3 KD CA1 pyramidal neurons in cultured hippocampal slices treated with DMSO or RA (**, p < 0.01; ***, p < 0.0005). Top: schematic of Stx-3 showing its functional domains. (C) Summary of mEPSC amplitude and frequency analysis from control and Stx-4 KD CA1 pyramidal neurons in cultured hippocampal slices treated with DMSO or RA (*, p < 0.05; ***, p < 1 x 10⁻⁶). Top: schematic of Stx-4 showing its functional domains (red indicates non-complexin-binding sequence). (D) Summary graph (left) and trace examples (right) of LTP recorded from CA1 pyramidal neuron expressing Stx-3 KD or Stx-4 KD constructs. Neighboring uninfected neurons were recorded as controls. Black bar in the summary graph indicates the time window for LTP magnitude quantification. Scale bars: 20 pA, 10 ms. (E) Scatter plots of LTP obtained from individual experiments summarized in (D) with bar graphs representing mean ± s.e.m (**, p < 0.01).

Figure 6. Non-complexin-binding sequence of Stx-4 is required for normal Stx-4 function in RA-induced AMPAR-containing vesicle exocytosis

(A) Analysis of amplitude and frequency of mEPSCs obtained from CA1 pyramidal neurons expressing both Stx-4 KD and wildtype Stx-4 rescue constructs (Stx-4 Rep) (*, p < 0.05; ***, p < 0.001). Top: schematic of Stx-4 showing its functional domains (red indicates non-complexin-binding sequence). (B) Analysis of amplitude and frequency of mEPSCs obtained from CA1 pyramidal neurons expressing both Stx-4 KD and Stx-4/3 rescue constructs (Stx-4/3 Rep) (***, p < 1 x 10⁻⁴). Top: schematic of Stx-4/3 showing the non-complexin-binding domain of Stx-4 is replaced by the complexin-binding domain of Stx-3 (green). (C) Analysis of amplitude and frequency of mEPSCs obtained from CA1 pyramidal neurons expressing both Stx-4 KD and Stx-3/4 rescue constructs (Stx-3/4 Rep) (***, p < 0.001). Top: schematic of Stx-3/4 showing the complexin-binding domain of Stx-3 is replaced by the non-complexin-binding domain of Stx-4 (red).

Figure 7. Stx-4 KD rescues LTP in RA-treated hippocampal slices

(A) Summary graph (left) and trace examples (right) of LTP recorded from Stx-4 KD CA1 pyramidal neuron treated with DMSO or RA. Black bar in the summary graph represents the time window for LTP magnitude quantification. Scale bars: 50 pA, 10 ms. (B) Scatter plots of LTP obtained from individual experiments summarized in (A) with bar graphs representing mean ± s.e.m (p > 0.05).

Figure 8. Model of the shared and distinct vesicle fusion machinery for AMPAR-containing vesicle exocytosis during LTP and synaptic RA signaling

Top panel: R-SNARE Synaptobrevin-2 is present in the membrane of AMPAR-containing vesicles. Syntaxin-3 and Syntaxin-4 may form distinct vesicle fusion micro-domains on the synaptic or perisynaptic membranes and define AMPAR-containing vesicle exocytosis locations for LTP and RA pathways, respectively. Q-SNARE SNAP-47 is present and is required for both pathways. Bottom panel: AMPAR-containing vesicle fusion occurs at distinct surface membrane locations during LTP or RA signaling. RA-induced vesicle exocytosis occludes LTP by either depleting the AMPAR-containing vesicle pool or by occupying postsynaptic slots necessary for anchoring AMPARs.