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…
開示
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 |
