TITLE: Protocol for neurophysiological imaging in human iPSC-derived neurons
AUTHORS: Natalia Garcia Perez, Yi-Ning Kang, Emma Moles-Garcia, Tom Venneman, Pieter Vanden Berghe

[DESCRIPTION]
The protocol describes how to perform neurophysiological imaging experiments on different types of human iPSC-derived neurons.

[STEPS]
SECTION: iPSC-derived neurons generation
2. Cells were differentiated from human iPSCs (WTC11, KOLF2.1 cell lines) according to published protocols, with some modifications to allow for easier live-cell imaging.
SECTION: iPSC culture and maintenance
1. The iPSC cell lines (WTC11 and KOLF2.1J) are cultured in Essential 8 medium on Geltrex-coated plates. The cultures are passaged every 3 days with 0.5mM EDTA.
SECTION: iPSC-derived neurons generation
2.1. To generate midbrain dopaminergic-like neurons, we followed the Kriks et al. (2011) protocol, with the following modifications:

Simplified basal medium: 
Days 1-20: Neurobasal-A + 1% GlutaMAX + 2% B27 Supplement + 1% N2 Supplement + 1% PenStrep
Days 20+: Neurobasal-A + 1% GlutaMAX + 2% B27 Supplement + 1% PenStrep

Additional 1:2 replating step at Day 8 of differentiation to avoid cell detachment due to overgrowth.

For the replating step at Day 20, the cells are replated under low-density conditions (30 x103 - 100 x103 cells per cm2) as opposed to the recommended high-density conditions (300 x103 - 400 x103 cells per cm2)

If there are non-neuronal cells present in the culture after Day 20, a treatment with 2 µM Ara-C for 24-48h is carried out.
SECTION: iPSC-derived neurons generation
2.2. To generate enteric nervous system-like neurons, we followed the Barber et al. (2019) protocol, with the following modifications:

Additional replating step at Day 20-22. We plate 60 x103 - 100 x103 cells per cm2.

If there are non-neuronal cells present in the culture after Day 20, a treatment with 2 µM Ara-C for 24-48h is carried out.
SECTION: Live-cell imaging
3. Using calcium as a proxy for neuronal activity, we assess calcium dynamics during basal conditions, as well as during stimulations. 

SECTION: Live-cell imaging
3.1. For midbrain dopaminergic-like neurons:
The cells are loaded with the calcium dye Fluo-4-AM at a concentration of 50-100 nanomolar (nM)  for 10 min at 37 °C. 
The cells are washed 3x with HEPES and left to incubate at37 °C for 5 min.
The coverslips are mounted into an imaging holder and the cells covered with HEPES. As an easy alternative, glass bottom culture dishes can be used.
The cells can be imaged for up to 60 min.

SECTION: Live-cell imaging
3.2. For ENS-like neurons:
The cells are loaded with the calcium dye Fluo-4-AM at a concentration of 1 micromolar (µM) for 10 min at Room temperature, on a shaker (<50 rpm). 
The cells are washed 1x with HEPES and mounted into an imaging holder. The cells are covered with HEPES. As an easy alternative, glass bottom dishes can be used.
The cells can be imaged for up to 60 min.

SECTION: Live-cell imaging
4. Using mitochondrial dyes such as MitoTracker (MitoTracker Green/Red/Red CMXRos), we investigate neuritic mitochondrial transport in these cells (Van Steenbergen et al. 2022).
The cells are loaded in5-7.5 nanomolar (nM)  (for dopaminergic-like neurons) or 20 nanomolar (nM) (for ENS-like neurons) MitoTracker for 10 min at 37 °C. 
The cells are washed 3x with HEPES and left to incubate at 37 °C for 5 min.
The coverslips are mounted onto an imaging holder and covered with HEPES. As an easy alternative, glass bottom culture dishes can be used.
The cells can be imaged for up to60 min.
5.2. Local perfusion (for ENS-like cells, as in Boesmans et al. 2019)
High-K+ (75 millimolar (mM))
Substance P (1 micromolar (µM)) 
DMPP (10 micromolar (µM))


5.1. Electrical stimulation
Electrodes (custom field electrodes and focal electrodes; see Materials for details)
Stimulator: Master8 (A.M.P.I) driving the A385 Stimulus Isolator (World Precision Instruments)
Stimulation protocols:
        ‣ Field electrode 
            • 3 - 100 pulses at 20 Hz - 100 Hz
            • Current: 5 - 10 mA
        ‣ Focal electrode
            • Train duration: 2x1000 ms
            • Rate: 2x10 pps
            • Delay: 1x1ms
            • Duration: 3x0.1 ms
            • Volts: 3x10 volts
SECTION: Stimulation
5. Depending on the experiment, we use a combination of different stimuli to assess neuronal physiology.
SECTION: Microscopy
6. A variety of microscopes can be used. We use wide-field systems (Zeiss Axiovert, Nikon coupled to Till Vision image acquisition hard/software) and confocal systems (Zeiss LSM 780-NLO, Zeiss LSM 880 AiryScan). The image acquisition speed should be at least 1-2 Hz in order to capture the relevant physiological events.

Laser powers and exposure times (generally ~ 2 mWatt for widefield, 20-80ms exposure per 500 ms) should be adjusted to avoid bleaching and to preserve neuronal health.