A Technique to Assess the Inhibitory Effect of Toxin Exposure on Miniature Excitatory Postsynaptic Currents (mEPSCs)

Abstract

Source: Hubbard, K. et. al., Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons. J. Vis. Exp. (2015)

The video demonstrates the electrophysiological analysis of embryonic stem cell-derived neurons after exposure to neurotoxins. Treating these neurons with botulinum neurotoxins reduces the frequency of spontaneous miniature excitatory post-synaptic currents, confirming inhibition of synaptic transmission.

Protocol

1. Adaptation of embryonic stem cells (ESCs) to Feeder Cell-free Suspension Culture Thaw ESCs at 37 °C until a sliver of ice remains. Using a P1000 pipet, gently transfer 1 – 2.5 x 106 dissociated ESCs to a non-tissue culture-treated dish containing 10 ml of pre-warmed ESC medium. Incubate in a humidified tissue culture incubator for 20 hr at 37 °C and 5% carbon dioxide (CO2). After 20 hr, wash cells by gently transferring the suspensio…

Representative Results

Figure 1. Suspension-adapted ESNs remain mitotically stable and express markers of pluripotency. (A) Schematic of ESC maintenance and differentiation. The presence or absence of retinoic acid (RA) or leukemia inhibitory factor (LIF) is marked by a + or –. A comparison between days in vitro (DIV) and classical developmental stages (DS) for primary neu…

Materials

Table 1. ESC and ESN
Knockout DMEM	Life Technologies	10829-018	Media: ESC culture medium
			Formulation and notes: 500 mL
100x MEM NEAA	Life Technologies	11140-050	Media: store at 4 °C for up to 1 month
			Formulation and notes: 6 mL
200 mM L-Alanyl-L-Glutamine	ATCC	30-2115	6 mL
ES qualified FBS	Applied Stem Cell	ASM-5007	90 mL
100x antibiotics	Sigma-Aldrich	A5955	3 mL
55 mM 2-mercaptoethanol	Life Technologies	21985-023	1.1 mL
107 Units/mL LIF	Millipore	ESG1107	60 μL
Knockout DMEM	Life Technologies	10829-018	Media: ESC differentiation medium
			Formulation and notes: 436.6 mL
100x MEM NEAA	Life Technologies	11140-050	Media: store at 4 °C for up to 1 month
			Formulation and notes: 5 mL
200 mM L-Alanyl-L-Glutamine	ATCC	30-2115	5 mL
ESC-qualified serum	Applied Stem Cell	ASM-5007	50 mL
100x antibiotics	Sigma-Aldrich	A5955	2.5 mL
55 mM 2-mercaptoethanol	Life Technologies	21985-023	0.9 mL
Dulbecco's PBS	Sigma-Aldrich	D8537	Media: NPC trypsinization medium
			Formulation and notes: 100 mL
0.5 M EDTA (18.3%)	Sigma-Aldrich	3690	Media: freeze in 5 mL aliquote
			Formulation and notes: 0.266 mL
Trypsin	Sigma-Aldrich	T8802	50 mg
Polyethyleneimine (PEI)	Sigma-Aldrich	P3143	Media: Surface coating solutions
			Formulation and notes: 2.5 µg/mL in H20
Poly-D-lysine (PDL)	Sigma-Aldrich	P7280	Media: use within 1 wk
			Formulation and notes: 100 µg/mL in H20
Laminin	Sigma-Aldrich	L2020	5 µg/mL in H20
DMEM/F12 + GlutaMAX	Life Technologies	10565-018	Media: NPC culture medium
			Formulation and notes: 492.5 mL
100x N2 vitamins	Life Technologies	17502-048	Media: store at 4 °C for up to 1 month
			Formulation and notes: 5 mL
100x antibiotics	Sigma-Aldrich	A5955	2.5 mL
Neurobasal A	Life Technologies	10888-022	Media: ESN culture medium
			Formulation and notes: 482.5 mL
50x B27 vitamins	Life Technologies	17504-044	Media: store at 4 °C for up 1 month
			Formulation and notes: 10 mL
200 mM L-Alanyl-L-Glutamine	ATCC	30-2115	5 mL
100x antibiotics	Sigma-Aldrich	A5955	2.5 mL
5-fluoro-2'-deoxyuridine (5FDU)	Sigma-Aldrich	F0503	Media: 2000x Mitotic inhibitors aliquot and store at -20 °C
			Formulation and notes: dd 150 mg 5FDU and 350 mg uridine to 10 mL Neurobasal A. Sterile filter, aliquot and freeze. Use 2000x in ESN culture medium.
Uridine	Sigma-Aldrich	U3003
TrypLE Express Trypsin	Life Technologies	12605-010	Media: Miscellaneous
			Formulation and notes: store at RT
DMSO	Sigma-Aldrich	D8418	store at RT
Soybean trypsin inhibitor (STI)	Sigma-Aldrich	T6414	store at -20 °C
Ethanol	Sigma-Aldrich	E7023	store at RT
Ascorbic acid	Sigma-Aldrich	A4403	Prepare 100 mM stock by dissolving 100 mg ascorbic acid in 5.7 mL of 50:50 DMSO/ethanol mix.
Retinoic acid (RA)	Sigma-Aldrich	R2625	Resuspend 15 mg RA in 9 mL 50:50 DMSO/ethanol. Supplement with 1 mL of 100 mM ascorbic acid stock. Aliquot and stored at -80 °C. Stable for 6 mos.
Table 2. MIST
NaCl	Sigma-Aldrich	71386	Media: Extracellular Recording Buffer
			Final Concentration (mM): 58.44
			MW: 140
KCl	Sigma-Aldrich	60135	Media: (ERB; pH = 7.3, 315 mO)
			Final Concentration (mM): 74.56
			MW: 3.5
NaH2PO4	Sigma-Aldrich	S3139	Media: Stable at 4 °C for at least 6 mo.
			Final Concentration (mM): 119.98
			MW: 1.25
CaCl2	Sigma-Aldrich	21115	Final Concentration (mM): 110.98
			MW: 2
MgCl2	Sigma-Aldrich	63069	Final Concentration (mM): 95.21
			MW: 1
D-glucose	Sigma-Aldrich	G8644	Final Concentration (mM): 180.16
			MW: 10
HEPES	Sigma-Aldrich	H0887	Final Concentration (mM): 238.3
			MW: 10
K-gluconate	Sigma-Aldrich	P1847	Media: Intracellular Recording Buffer
			Final Concentration (mM): 234.5
			MW: 140
NaCl	Sigma-Aldrich	71386	Media: (IRB; pH = 7.3, 320 mO)
			Final Concentration (mM): 58.44
			MW: 5
Mg-ATP	Sigma-Aldrich	A9187	Media: Stable at 4 °C for at least 6 mo.
			Final Concentration (mM): 507.18
			MW: 2
Li-GTP	Sigma-Aldrich	G5884	Final Concentration (mM): 523.18
			MW: 0.5
CaCl2	Sigma-Aldrich	21115	Media: Stable at 4 °C for at least 6 mo.
			Final Concentration (mM): 110.98
			MW: 0.1
MgCl2	Sigma-Aldrich	63069	Media: Stable at 4 °C for at least 6 mo.
			Final Concentration (mM): 95.21
			MW: 1
EGTA	Sigma-Aldrich	E3889	380.35 Media: Stable at 4 °C for at least 6 mo. Final Concentration (mM): 380.35 MW: 1
HEPES	Sigma-Aldrich	H0887	Media: Stable at 4 °C for at least 6 mo.
			Final Concentration (mM): 238.3
			MW: 10
Bicuculline	Tocris	131	Media: Miscellaneous
			Final Concentration (mM): 0.01
			MW: 417.85
CNQX	Sigma-Aldrich	C239	Final Concentration (mM): 0.01
			MW: 276.12
Tetrodotoxin	Sigma-Aldrich	A8001	Final Concentration (mM): 0.005
			MW: 645.74
BoNT/A-/G	Metabiologics	N/A	Media:
			Final Concentration (mM): TBD
			MW: 150,000
Tetanus toxin	Sigma-Aldrich	T3194	Final Concentration (mM): TBD
			MW: 150,000
Sigmacote	Sigma-Aldrich	SL-2	Final Concentration (mM): N/A
			MW: N/A
Table 3. Equipment and Software
MiniAnalysis (version 6.0.7)	Synaptosoft, Inc.	N/A	Event detection software
Igor Pro (version 6.22A)	WaveMetrics	N/A	Software for visualization of recordings
Patchmaster	Heka	N/A	Data acquisition software
Olympus IX51 inverted microscope	Olympus	N/A
Micromanipulator	Sutter Instrument	MPC-365
Patch-clamp amplifier	Heka	ECB10USB
Micropipette puller	Sutter Instrument	P-1000
Borosilicate capillary tubes	Sutter Instrument	B150-86-10	Corning 7740
18 mm glass coverslips	Fisher	12-545-84
Plasma cleaner	Harrick Plasma	PDC-32G
Cell strainer (40 µm)	Fisher	08-771-1
Stovall Belly Dancer Shaker	Fisher	15-453-211
Low adhesion dishes	Fisher	05-539-101	Corning 3262
Bacterial dishes	VWR	25384-302	100 x 15 mm