Assessing Neural Connection Formation in a Co-Culture using Electrophysiological Recordings

Abstract

Source: Su, C. T. E., et al. An Optogenetic Approach for Assessing Formation of Neuronal Connections in a Co-culture System. J. Vis. Exp. (2015)

The video demonstrates electrophysiological recording to assess neural connections between iPSC-derived and rat cortical neurons, confirming synaptic contacts in the co-culture by activating light-sensitive ion channels and detecting postsynaptic currents.

Protocol

1. Electrophysiological Recordings of iPSC-derived Neurons

1. Confirm whether human iPSCs behave like mature neurons.
   1. Find cells differentiated from human iPSCs by visualization of tdTomato using an upright confocal microscope equipped with a 60X (0.9 NA) water immersion lens.
   2. Pull the recording pipette to a resistance of 4 to 6 M. Perfuse cells at room temperature in an external solution bubbled constantly with 95% O₂/ 5% CO₂. Fill the recording micropipette with the internal solution.
   3. Patch tdTomato+ cell in whole-cell mode and record action potential firing in response to current injection (1 sec duration, 10 pA increments) in the current clamp.
2. Confirm if the action potential of cortical cells expressing ChR2 is reliably evoked by light stimulation.
   1. Find cortical cells expressing ChR2 by visualization of GFP under a confocal microscope. Perform whole-cell patch clamp recording on cortical cells by following steps 1.1.2 to 1.1.3.
   2. Check that the action potential is reliably evoked by light illumination with a mercury arc lamp (100 W). The wavelength of the excitation filter is 480/40 nm.
3. Perform optogenetic stimulation.
   1. Patch tdTomato+ cell in whole-cell mode and set voltage clamp at -70mV. Filter current signals at 2 kHz and digitize at 10 kHz. Monitor series resistances ranging from 10 to 20 M for consistency during recordings.
   2. Stimulate the whole field with 100 W mercury lamp with a 480/40 nm excitation filter for 30 sec.
   3. Record postsynaptic currents (PSCs) of patched cells induced by photostimulation of ChR2-expressing presynaptic cortical neurons. Detect and measure PSCs.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
NaCl	Sigma	S7653
KCl	Sigma	P5405
NaHCO3	Sigma	S5761
NaH2PO4	Fluka	71505
MgCl2	Sigma	M8266
CaCl2	Sigma	C1016
D(+)-Glucose	Sigma	G7520	For electrophysiological studies
K-gluconate	Sigma	P1847
KOH	KANTO Chemical	3234400
HEPES	Sigma	H3375	For electrophysiological studies
EGTA	Sigma	E3889
Na2ATP	Sigma	A7699
Na3GTP	Sigma	G8877
Borosilicate glass capillaries	World precision instruments, Inc	1604323
Mercury short-arc lamp	OSRAM	HBO 103 W/2