DOI: 10.5061/dryad.3n5tb2rwb
title: Neocortical Layer-5 tLTD Relies on Non-Ionotropic Presynaptic NMDA Receptor Signaling

description: 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.
description: # Data for Thomazeau *et al.*, 'Neocortical Layer-5 tLTD Relies on
 Non-Ionotropic Presynaptic NMDA Receptor Signaling', eLife 2025
 Dataset DOI: [10.5061/dryad.3n5tb2rwb](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](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/](https://github.com/pj-sjostrom/MultiPatch). 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/qMorph) * [https://github.com/pj-sjostrom/MultiPatch](https://github.com/pj-sjostrom/MultiPatch) ### How to View Files 1. Download a figure ZIP archive. 2. Decompress the ZIP archive. 3. Place GitHub procedure files `MP_DatAn 20.ipf` and `MP_CompileExperiments v28.ipf` in a folder named `Igor Stuff` (folder must be manually created). 4. Place the GitHub procedure file `JespersTools_v03.ipf` in the folder `Igor procedures` (which was automatically created when Igor Pro was first installed). 5. Launch Igor Pro and load a `.pxp` file; select the `Igor Stuff` folder when prompted. 6. For sample experiment files, also select the correct raw data folder when prompted.