DOI: 10.5281/zenodo.16950322
title: HPCA translocation in response to LTD-inducing iontophoretic NMDA application

description: Given the neuronal Ca2+ sensor protein hippocalcin (HPCA) involvement in NMDA-dependent long-term depression (LTD) associated Ca2+-dependent endocytosis of AMPA receptors [1], we sought to confirm HPCA translocation from the cytosol into the cell membranes in response to LTD-inducing iontophoretic NMDA application (100 mM NMDA for 60 s). To detect HPCA insertion into the plasma membrane, a Förster resonance energy transfer (FRET) imaging technique was used. We monitored the energy transfer between ECFP-conjugated HPCA (HPCA-ECFP, donor) and membrane-bound EYFP (EYFP-Memb, acceptor), a nonspecific membrane tag [2]. Since FRET required the distance between donor and acceptor fluorophores to be approximately 100 Å, an increase in FRET suggested the insertion of HPCA-ECFP into the cell membranes [3]. In our experiments, we used a three-cube approach to capture fluorescence of the acceptor elicited by the excitation of the donor [4,5] and quantitatively evaluated FRET efficiency (Eapp) to assess HPCA translocation to the membranes.

Data and File Overview

File Naming Convention: Files are named by date and number in experimental day format (YY_MM_DD_num).

Folder & Files Structure:



README.mdREADME file with a comprehensive description.

rawThe folder includes raw image files in TIFF format, along with the corresponding Live Acquisition software metadata files in XML format.

processedThe folder includes processed images, masks, and all intermediate image data obtained during image processing and analysis using the domb-napari plugin for napari. For information on file naming and descriptions of processing steps, please refer to the plugin documentation in the GitHub repository.


Experimental Design & Image Acquisition Protocols

Biological materials: Cultured hippocampal neurons DIV 10–20.

Genetic materials: N1 vector-based plasmids for HPCA-ECFP and EYFP-Mem [2] expression. 

Experimental design: Given that NMDA receptors are saturated with ~1 mM of NMDA, for these experiments, we used a 15 mM NMDA iontophoretic solution to create a saturating concentration gradient across the entire field of view. The iontophoretic electrode (resistanve 100-120 MOhm) was positioned 30–50 μm from the soma and 10 μm above the apical dendrite. 

Acquisition protocols: Images were acquired at a frequency of 0.1 Hz for a total of 300 seconds (30 frames). The combination of two excitation wavelengths (435 nm and 505 nm) and the dual-view system (detection above and below two channels at 505 nm) enabled us to acquire four spectral channels for the next FRET efficiency estimation.

The recording protocol was as follows: - 60 s baseline, the retention current was set to +5 nA to prevent NMDA leakage from iontophoretic electrode.- 60 s iontophoretic application with the current set to -100 nA.- NMDA washout for 180 s with retention current at +5 nA.

Experimental Setup Configuration

Imaging and electrophysiology:- Inverted microscope (Olympus, model: IX71)- 40× oil-immersion high-aperture objective (Olympus, model: UApo/340 40×/1.35)- Optical filters set (suitable for selected fluorophores, in our case, Chroma, model: 69008)- Dual-view splitter (Optical Insights, model: MSMI-DV-FC)- Digital camera (PCO, model: SensiCam QE)- Vibration isolation table (CleanBench, model: TMC)- Imaging control unit (TILL Photonics, model: ICU2)- Monochromator (TILL Photonics, model: Polychrome V)- Patch clamp amplifier (HEKA, model: EPC 10)- Micromanipulators (Sutter Instrument, model: MPC-200)- Ag/AgCl pellets (WPI, catalog number: EP1)- Experimental chamber (Warner Instruments, model: RC-25 or DIY analog)- Microelectrode puller (Sutter Instrument, model: P-97)

Experimental setup control:- PatchMaster (v2x69, HEKA, Germany)- Live Acquisition (v2.6.0.12, FEI, Germany)

Links to Related Publications

Recommended citation for this dataset:

Olifirov, B. et al. (2025). Local Iontophoretic Application for Pharmacological Induction of Long-Term Synaptic Depression. Bio-protocol 15(11): e5338. DOI: 10.21769/BioProtoc.5338

Reference List

1. Palmer, C. L., Lim, W., Hastie, P. G., Toward, M., Korolchuk, V. I., Burbidge, S. A., Banting, G., Collingridge, G. L., Isaac, J. T., Henley, J. M., et al. (2005). Hippocalcin Functions as a Calcium Sensor in Hippocampal LTD. Neuron. 47(4): 487–494. https://doi.org/10.1016/j.neuron.2005.06.0142. Liu, Y., Fisher, D. and Storm, D. (1994). Intracellular sorting of neuromodulin (GAP-43) mutants modified in the membrane targeting domain. J Neurosci. 14(10): 5807–5817. https://doi.org/10.1523/jneurosci.14-10-05807.19943. King, C., Sarabipour, S., Byrne, P., Leahy, D. J. and Hristova, K. (2014). The FRET Signatures of Noninteracting Proteins in Membranes: Simulations and Experiments. Biophys J. 106(6): 1309–1317. https://doi.org/10.1016/j.bpj.2014.01.0394. Zal, T. and Gascoigne, N. R. (2004). Photobleaching-Corrected FRET Efficiency Imaging of Live Cells. Biophys J. 86(6): 3923–3939. https://doi.org/10.1529/biophysj.103.0220875. Chen, H., Puhl, H. L., Koushik, S. V., Vogel, S. S. and Ikeda, S. R. (2006). Measurement of FRET Efficiency and Ratio of Donor to Acceptor Concentration in Living Cells. Biophys J. 91(5): L39–L41. https://doi.org/10.1529/biophysj.106.088773