This study investigates the effects of botulinum neurotoxin on neurotransmitter release in neuronal cultures. By employing electrophysiological techniques, the research aims to elucidate the inhibitory mechanisms of the toxin on synaptic transmission.
Treat neuronal cultures with increasing concentrations of a test toxin.
The toxin enters the cells and is released into the cytosol.
Depending on its potential, the toxin may prevent vesicular fusion, inhibiting neurotransmitter release.
Replace the medium with a buffer containing specific neuropharmacological compounds to block neuronal action potentials.
Place the culture dish on an electrophysiology stage. To start recording, connect a neuron soma with a pipette containing a recording electrode.
Apply steady negative pressure to aspirate a small membrane portion, forming a gigaseal, a tight seal that ensures minimal interference during recording.
Maintain a constant membrane voltage to measure neuronal currents.
Apply negative pressure pulses to rupture the membrane, establishing direct contact between the neuron and the pipette.
Record the currents produced in the neuron due to the uptake of neurotransmitters, termed mEPSCs.
A decrease in mEPSC frequency with increased toxin exposure indicates the inhibition of neurotransmitter release.
Dilute botulinum neurotoxin serotype A to 100 times the desired final concentration in ESN culture medium and warm to 37 degrees Celsius. Then, add an appropriate volume of toxin to DIV 21+ ESN cultures. Swirl the culture dish, and return to the incubator.
At the desired time point, aspirate the ESN culture medium, and wash twice with extracellular recording buffer or ERB. Then, add 4 milliliters of ERB supplemented with 5 micromolar tetrodotoxin to block action potentials and 10 micromolar bicuculline to antagonize GABAA receptor activity. Next, transfer the dish to the electrophysiology rig. Neither perfusion nor temperature control is required for the measured inhibition of synaptic transmission or MIST assay.
After using a micropipette puller to pull borosilicate pipettes with 5 to 10 megaohms of resistance, back-fill a pipette with intracellular recording buffer. Then, gently dip the pipette in siliconizing reagent. Secure the recording pipette onto the electrode holder and attach an air-filled syringe to tubing that is ported into the electrode holder. Provide steady positive pressure via the syringe while lowering the recording pipette into the ERB.
After gently landing the recording pipette on the soma of the neuron to be recorded, seize positive pressure by breaking the seal on the syringe. Reconnect the syringe and apply sustained negative pressure through gentle inspiration to form a gigaohm seal. Once the gigaohm seal is formed, decrease the holding voltage to minus 70 millivolts, then carefully apply short pulses of negative pressure using the syringe to break into wholesale configuration.
Monitor capacitance spikes for 30 seconds to confirm that the patch is stable. Cancel the capacitance spikes in the hacker software, and switch to current clamp mode to monitor and record resting membrane potential. Without adjustment for liquid junction potentials, the resting membrane potential should be between minus 67 and minus 82 millivolts.
Adjust the gain to 2 millivolts per picoamp. Switch to voltage-clamp mode and perform a continuous minus 70 millivolts, recording for 3 to 5 minutes to detect miniature excitatory postsynaptic currents.
Analyze 3 to 5 minutes of recorded data to detect MEPSCs using default settings for AMPA receptor EPSCs in mini-analysis. Collect and save information on detected events. After collecting mEPSC frequencies for 8 to 12 controls and 8 to 12 botulinum neurotoxin-treated samples for each exposure condition, analyze frequency against age and lot-matched controls.
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