Neocortical Layer-5 tLTD Relies on Non-Ionotropic Presynaptic NMDA Receptor Signaling

Thomazeau, Aurore 1 ; Rannio, Sabine2; Brock, Jennifer A.2; Wong, Hovy Ho-Wai3; Sjöström, P. Jesper 2

Research facility: McGill University Health Centre

Published Jul 14, 2025 on Dryad. https://doi.org/10.5061/dryad.3n5tb2rwb

Abstract

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 suggest 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 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.

Dataset DOI: 10.5061/dryad.3n5tb2rwb

Data description

We induced spike-timing dependent depression or potentiation in synaptically coupled primary visual cortex layer-5 pyramidal cells using in vitro whole-cell patch clamp electrophysiology in mouse tissue under various genetic or pharmacological conditions. This dataset was acquired with custom software MultiPatch (https://github.com/pj-sjostrom/MultiPatch), running in Igor Pro (Wavemetrics Inc).

In current clamp mode, synaptically coupled PC→PC connections were identified by evoking five spikes at 30 Hz by 5-ms-long current injections of ~1.3 nA magnitude, repeated every 20 seconds, and averaged across 10–20 repetitions. We induced:

tLTD: Postsynaptic firing 25 ms before presynaptic, either at:
- 20 Hz: High-frequency (HF) tLTD
- 1 Hz: Low-frequency (LF) tLTD
tLTP: Presynaptic firing 10 ms before postsynaptic at 50 Hz

Unless otherwise specified, experiments were carried out in C57BL/6 (WT) mice.

Data Structure and Usage

Data are organized by figure number. Data for each figure is found in separate ZIP archive files (called Figure_1.zip, Sup_Figure_1.zip, etc.), which contain subfolders for each panel.
File formats:
- .pxp for analysis files (open with Igor Pro)
- .txt for exports (open using scripts below running in Igor Pro)
- .xslx for overview (open with Excel)
Required Igor Pro procedure files (JespersTools_v03.ipf, MP_DatAn 20.ipf, and MP_CompileExperiments v28.ipf) are available at https://github.com/pj-sjostrom/. See the Code/software section below for additional details. Briefly, MP_DatAn 20.ipf (MultiPatch Data Analysis) is analysis code used to view sample experiments, whereas MP_CompileExperiments v28.ipf (MultiPatch Experiment Compiler) is used to view ensemble data as analyzed and extracted with MP_DatAn 20.ipf. Both MP_DatAn 20.ipf and MP_CompileExperiments v28.ipf rely on a library of function calls that are available in JespersTools_v03.ipf (Jesper's Tools).
See the Excel file Plasticity_overview_data.xlsx for a summary.

Note: In all likelihood, the vast majority of users inspecting this data set will be satisfied with the Plasticity_overview_data.xlsx Excel file and will not need to used the above-mentioned Igor Pro procedure files (JespersTools_v03.ipf, MP_DatAn 20.ipf, and MP_CompileExperiments v28.ipf).

Figure Summary

Figure 1: HF tLTD with or without preNMDARs

Figure_1.zip: ZIP-archived data for Figure 1, organized as described below.
Panel A: Presynaptic NMDAR WT sample — HF tLTD Presynaptic NMDAR WT sample experiment.pxp. When you load this sample file with Igor, you will be prompted to locate the path to the Igor Stuff folder, which is where the .ipf procedure files should be located. You will also be prompted to locate the raw data, which is found in /raw data/SR20230116_Ephys/SR20230116_Ephys Folder/. The specific experiment date is shown in several places, e.g., in the title bar of the MultiPatch Data Analysis panel.
Panel B: Presynaptic NMDAR KO sample — HF tLTD Presynaptic NMDAR KO sample experiment.pxp. You will need to locate Igor Stuff and the raw data folders, like above.
Panels C–E: Ensemble — folder: Panel C-E HF tLTD NMDAR KO Ensemble

Figure 2: tLTP with MK-801

Figure_2.zip: ZIP-archived data for Figure 2, organized as described below.
Panel A: Control + MK-801 — tLTP MK-801 sample experiment.pxp. You will need to locate Igor Stuff and the raw data folders, like for Figure 1 sample experiments.
Panel A: Control no drug — tLTP no drug sample experiment.pxp. You will need to locate Igor Stuff and the raw data folders, like for Figure 1 sample experiments.
Panel B: MK-801 — tLTP MK-801 sample experiment.pxp
Panels C–E: Ensemble — folder: Panel C-E tLTP MK-801 Ensemble

Figure 3: HF or LF tLTD with MK-801

Figure_3.zip: ZIP-archived data for Figure 3, organized as described below.
Panel A: HF — HF tLTD MK-801 sample experiment.pxp. You will need to locate Igor Stuff and the raw data folders, like for Figure 1 sample experiments.
Panel B: LF — LF tLTD MK-801 sample experiment.pxp. You will need to locate Igor Stuff and the raw data folders, like for Figure 1 sample experiments.
Panels C–E: Ensemble — folder: Panel C-E tLTD MK-801 Ensemble

Figure 4: LF tLTD with 7-CK

Figure_4.zip: ZIP-archived data for Figure 4, organized as described below.
Panel A: Sample — LF tLTD 7CK sample experiment.pxp. You will need to locate Igor Stuff and the raw data folders, like for Figure 1 sample experiments.
Panels B–D: Ensemble — folder: Panel B-D LF tLTD 7CK Ensemble

Figure 5: HF tLTD with RIM1αβ KO or SP600125

Figure_5.zip: ZIP-archived data for Figure 5, organized as described below.
Panel A: RIM1αβ KO — HF tLTD Rim1KO sample experiment.pxp. You will need to locate Igor Stuff and the raw data folders, like for Figure 1 sample experiments.
Panel B: SP600125 — HF tLTD SP600125 sample experiment.pxp. You will need to locate Igor Stuff and the raw data folders, like for Figure 1 sample experiments.
Panels C–F: Ensemble — folder: Panel C-F HF tLTD Rim1KO vs SP600125 Ensemble

Figure 6: Peptide experiments disrupting JNK2/STX1a

Figure_6.zip: ZIP-archived data for Figure 6, organized as described below.
Panel A: Presynaptic JGRi1 — HF tLTD presynaptic peptide sample experiment.pxp. You will need to locate Igor Stuff and the raw data folders, like for Figure 1 sample experiments.
Panel B: Postsynaptic JGRi1 — HF tLTD postsynaptic peptide sample experiment.pxp. You will need to locate Igor Stuff and the raw data folders, like for Figure 1 sample experiments.
Panels C–F: HF Ensemble — folder: Panel C-F HF tLTD Peptide Ensemble
Panels G–J: LF Ensemble — folder: Panel G-J LF tLTD Peptide Ensemble

Supplementary Figures

Supplementary Figure 1: MK-801 wash-in with LF evoked release

Sup_Figure_1.zip: ZIP-archived data for Supplemental Figure 1, organized as described below.
File: LF evoked release under MK-801.pxp
Folder: Panel A-B LF evoked release under MK-801

Supplementary Figure 2: 7-CK wash-in with LF evoked release

Sup_Figure_2.zip: ZIP-archived data for Supplemental Figure 2, organized as described below.
File: LF evoked release under 7-CK.pxp
Folder: Panel A-B LF evoked release under 7-CK

Excel Summary File

File: Plasticity_overview_data.xlsx

Includes ensemble data per condition, organized by figure. The Excel file represents the ensemble average EPSP amplitude for each experimental category, normalized to the initial baseline period, expressed in percent.

Code/software

Required Igor Pro procedure files:
- JespersTools_v03.ipf (Jesper's Tools) — a library of functions that the below two procedure files rely on.
- MP_DatAn 20.ipf (MultiPatch Data Analysis) — used to analyze individual sample experiments. This code is likely not necessary for most circumstances, but is necessary for viewing the individual sample experiments provided in the paper.
- MP_CompileExperiments v28.ipf (MultiPatch Experiment Compiler) — used to analyze ensemble data sets. You would use this code to get details on the ensemble data sets. However, if you just need individual data points from data presented in the figures of the paper, you might be satisfied with what is provided in the Plasticity_overview_data.xlsx file.
- Available at:
  - https://github.com/pj-sjostrom/qMorph
  - https://github.com/pj-sjostrom/MultiPatch

How to View Files

Download a figure ZIP archive.
Decompress the ZIP archive.
Place GitHub procedure files MP_DatAn 20.ipf and MP_CompileExperiments v28.ipf in a folder named Igor Stuff (folder must be manually created).
Place the GitHub procedure file JespersTools_v03.ipf in the folder Igor procedures (which was automatically created when Igor Pro was first installed).
Launch Igor Pro and load a .pxp file; select the Igor Stuff folder when prompted.
For sample experiment files, also select the correct raw data folder when prompted.