Blockade of synaptic activity induces homeostatic plasticity, in part by stimulating synthesis of all-transretinoic acid (RA), which in turn increases AMPA receptor synthesis. However, the synaptic signal that triggers RA synthesis remained unknown. Using multiple activity-blockade protocols that induce homeostatic synaptic plasticity, here we show that RA synthesis is activated whenever postsynaptic Ca²⁺entry is significantly decreased and that RA is required for upregulation of synaptic strength under these homeostatic plasticity conditions, suggesting that Ca²⁺plays an inhibitory role in RA synthesis. Consistent with this notion, we demonstrate that both transient Ca²⁺depletion by membrane-permeable Ca²⁺chelators and chronic blockage of L-type Ca²⁺-channels induces RA synthesis. Moreover, the source of dendritic Ca²⁺entry that regulates RA synthesis is not specific because mild depolarization with KCl is sufficient to reverse synaptic scaling induced by L-type Ca²⁺-channel blocker. By expression of a dihydropyridine-insensitive L-type Ca²⁺channel, we further show that RA acts cell autonomously to modulate synaptic transmission. Our findings suggest that, in synaptically active neurons, modest “basal” levels of postsynaptic Ca²⁺physiologically suppress RA synthesis, whereas in synaptically inactive neurons, decreases in the resting Ca²⁺levels induce homeostatic plasticity by stimulating synthesis of RA that then acts in a cell-autonomous manner to increase AMPA receptor function.

Members of the transmembrane AMPA receptor-regulatory protein (TARP) family modulate AMPA receptor (AMPA-R) trafficking and function. AMPA-Rs consist of four pore-forming subunits. Previous studies show that TARPs are an integral part of the AMPA-R complex, acting as accessory subunits for mature receptors in vivo. The TARP/AMPA-R stoichiometry was previously measured indirectly and found to be variable and dependent on TARP expression level, with at most four TARPs associated with each AMPA-R complex. Here, we use a single-molecule technique in live cells that selectively images proteins located in the plasma membrane to directly count the number of TARPs associated with each AMPA-R complex. Although individual GFP-tagged TARP subunits are observed as freely diffusing fluorescent spots on the surface of Xenopus laevis oocytes when expressed alone, coexpression with AMPA-R–mCherry immobilizes the stargazin-GFP spots at sites of AMPA-R–mCherry, consistent with complex formation. We determined the number of TARP molecules associated with each AMPA-R by counting bleaching steps for three different TARP family members: γ-2, γ-3, and γ-4. We confirm that the TARP/AMPA-R stoichiometry depends on TARP expression level and discover that the maximum number of TARPs per AMPA-R complex falls into two categories: up to four γ-2 or γ-3 subunits, but rarely above two for γ-4 subunit. This unexpected AMPA-R/TARP stoichiometry difference has important implications for the assembly and function of TARP/AMPA-R complexes.

Inactivation of the Fmr1 gene that is mutated in fragile X syndrome leads to loss of retinoic acid–mediated homeostatic plasticity in human neurons.

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.