This study demonstrates an optogenetic approach to induce ictal events in cortical slices from transgenic mice. By utilizing light-activated cation channels, researchers can manipulate neuronal excitability and observe seizure-like activities.
Take a cortical slice from a transgenic mouse brain that expresses light-activated cation channels in excitatory pyramidal neurons of the cortex.
Secure the slice in a recording chamber and perfuse it with artificial cerebrospinal fluid to maintain tissue viability.
Insert a recording electrode into the superficial cortical layer and record baseline neuronal activity.
Introduce a seizure-inducing drug that blocks voltage-gated potassium channels, preventing potassium outflow and maintaining the neurons in a hyperexcitable state.
The hyperexcitable neurons spontaneously generate bursts of action potentials, termed ictal events, for a prolonged duration.
To generate ictal events on-demand, apply a brief light pulse to activate the cation channels on the excitatory pyramidal neurons.
The resulting cation influx generates action potentials, triggering the release of excitatory neurotransmitters.
These neurotransmitters bind to receptors on postsynaptic neurons, causing an influx of cations.
The influx results in bursts of action potential, generating ictal events for a short duration.
In this procedure, cut out lens paper that is slightly larger than the brain slice. Use a wide bore pipette or a detailing brush to transfer a brain slice onto the cut out lens paper that is held in place using a dental tweezer. Then, transfer the lens paper with a brain slice to the recording chamber and secure it in position with a harp screen.
Subsequently, perfuse the brain slice in the recording chamber with carbogenated ACSF at 35 degrees Celsius, at a rate of three milliliters per minute. Use a digital thermometer to ensure the recording chamber is 33 to 36 degrees Celsius. Then, backfill the glass electrodes with 10 microliters of ACSF, using a Hamilton syringe.
Under the 20 times stereomicroscope, guide the recording glass electrode into the superficial cortical layer 2/3, using manual manipulators. View the electrical activity of the brain slice with standard software. To induce electrographic seizure-like activities, perfuse the brain slice with ACSF containing 4-AP at 100 micromolar. View the electrical activity of the brain slice with standard software.
To generate electrographic seizure-like events using optogenetic strategy on the brain slices from optogenetic mice, use a manual manipulator to position a 1,000 micron core diameter optical fiber directly above the recording region. Apply a brief pulse of blue light to initiate an ictal event.
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