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

Published: March 29, 2024

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…

Açıklamalar

The authors have nothing to disclose.

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

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Bu Makaleden Alıntı Yapın
A Technique to Assess the Inhibitory Effect of Toxin Exposure on Miniature Excitatory Postsynaptic Currents (mEPSCs). J. Vis. Exp. (Pending Publication), e22071, doi: (2024).

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