Neocortical layer-5 tLTD relies on non-ionotropic presynaptic NMDA receptor signaling

In the textbook view, NMDA receptors (NMDARs) act as coincidence detectors in Hebbian plasticity by fluxing Ca²⁺ when simultaneously depolarized and glutamate bound. Hebbian coincidence detection requires that NMDARs be located postsynaptically, but enigmatic presynaptic NMDARs (preNMDARs) also exist. It is known that preNMDARs regulate neurotransmitter release, but precisely how remains poorly understood. Emerging evidence suggests that NMDARs can also signal non-ionotropically, without the need for Ca²⁺ flux. At synapses between developing visual cortex layer-5 (L5) pyramidal cells (PCs), preNMDARs rely on Mg²⁺ and Rab3-interacting molecule 1αβ (RIM1αβ) to regulate evoked release during periods of high-frequency firing, but they signal non-ionotropically via c-Jun N-terminal kinase 2 (JNK2) to regulate spontaneous release regardless of frequency. At the same synapses, timing-dependent long-term depression (tLTD) depends on preNMDARs but not on frequency. We, therefore, tested in juvenile mouse visual cortex if tLTD relies on non-ionotropic preNMDAR signaling. We found that tLTD at L5 PC→PC synapses was abolished by pre- but not postsynaptic NMDAR deletion, cementing the view that tLTD requires preNMDARs. In agreement with non-ionotropic NMDAR signaling, tLTD prevailed after channel pore blockade with MK-801, unlike tLTP. Homozygous RIM1αβ deletion did not affect tLTD, but wash-in of the JNK2 blocker SP600125 abolished tLTD. Consistent with a presynaptic need for JNK2, a peptide blocking the interaction between JNK2 and Syntaxin-1a (STX1a) abolished tLTD if loaded pre- but not postsynaptically, regardless of frequency. Finally, low-frequency tLTD was not blocked by the channel pore blocker MK-801, nor by 7-CK, a non-competitive NMDAR antagonist at the co-agonist site. We conclude that neocortical L5 PC→PC tLTD relies on non-ionotropic preNMDAR signaling via JNK2/STX1a. Our study brings closure to long-standing controversy surrounding preNMDARs and highlights how the textbook view of NMDARs as ionotropic coincidence detectors in plasticity needs to be reassessed.