To begin this Neurocircuit assay flies are mounted to dental wax. On a slide four electrodes are inserted into the fly. One ground electrode in the abdomen, one recording electrode in the dorsal longitudinal flight, muscles in the thorax, and two stimulating electrodes in the head.
Electrode placement is confirmed by finding the giant fiber threshold and noting a visible response. The fly is delivered a high frequency wave train of the desired voltage, and if no seizure occurs, the trace on the oscilloscope will be flat. If a seizure does occur, the trace will show uncontrolled activity as a response to the high frequency stimulus.
Hi, I'm Mark Toay, professor in the Department of Environmental Science Policy and Management at the University of California Berkeley. Today we'll show you a procedure for assaying electrophysiological seizures in drosophila. We use this procedure in our laboratory to study seizures in drosophila mutants and determine seizure thresholds for these mutants.
So let's get started. The night before the experiment, male flies of the required genotype are anesthetized with carbon dioxide and placed in a fresh food vial capped with a soft nylon plug or cotton ball. Following carbon dioxide anesthetization, the seizure threshold is altered, so the flies are best left for at least six hours, though overnight is ideal.
On the day of the experiment, the flies are mounted one at a time to glass slides with a layer of dental wax on one side. Flies are manipulated without anesthetization by being picked up by the head, using a blunted 23 gauge needle attached to a vacuum line. They are then mounted to the slides by pushing down the legs and wings into the dental wax.
Once the fly is immobilized, wax is pushed up around the neck and eyes to fully immobilize the head. The fly is now mounted and ready for electrophysiology. The electrophysiology setup consists of three sets of micro manipulators, each with one to two sharpened tungsten electrodes attached.
The tungsten electrodes are used bare and require no saline. Voltage is provided by a stimulator that is connected to an isolation unit attached to the stimulating electrodes. The recording electrode is connected to a preamp, which in turn is attached to a digital storage oscilloscope.
While it is not essential, it is useful to have a speaker hooked up to the oscilloscope so that responses can be made audible. The immobilized fly is now placed on the microscope stage and a ground electrode is inserted into the abdomen of the fly. The recording electrode is inserted into one of the dorsal longitudinal muscles in the thorax of the fly.
The sharpened electrodes are strong enough to puncture the exoskeleton. If care is used, occasionally, electrodes will bend and need to be replaced. The stimulating electrodes are inserted through the cuticle of the head in the space between the eye and the antenna.
The fly is now set up for making recordings. Next, prepare the instruments for recording. The stimulator should have the following settings at set up a continuous pulse interval of two seconds between each pulse, zero pulse delay, and a pulse width no larger than 0.2 milliseconds.
The oscilloscope should have the storage feature turned on and have the following settings. Time per division should be set to one millisecond per division. The vaults per division should be set to 100 millivolts per division later.
If the response is not ideally strong, this setting can be changed to 50 millivolts per division. The giant fiber threshold is determined by delivering single electrical pulses of incremental voltage and noting the minimal voltage required to register our response in the recording electrode. This measurement is typically around two volts.
Once the giant fiber threshold has been to determined, stop pulsing the fly and change the settings on the Oslo scope to 0.2 seconds per division. Now, change settings on the stimulator. Set the pulse interval to a single pulse and activate the pulse train.
The waveform parameters are set to 0.5 millisecond pulses at 200 hertz For 300 milliseconds, the voltage is changed to the desired setting and a high frequency wave train of pulses as delivered to the fly's brain to induce a seizure. After the pulse train, the oscilloscope trace will show a flat component. If a seizure is not induced in this case, if the settings are quickly returned to those when recording began, the single pulses will cause a response in the giant fiber system.
If a seizure is induced, the oscilloscope will show the seizure-like activity after the train. Now, if the settings are quickly switched back to the original settings, the single pulses will fail to activate a response. In the giant fiber system.
The flies have a refractory period after a high frequency stimulus wave train during which seizure-like activity cannot be induced. If no seizure occurs, the flies are refractory for approximately seven minutes. And if a seizure is induced, the flies are refractory for approximately 17 minutes after the refractory period.
Additional high frequency stimuli can be delivered to the same fly at voltages, either higher or lower than the previous train until the seizure threshold is determined or until the giant fiber response becomes too weak to see, in which case the fly must be discarded and a new experiment started. After the experiment is completed, the fly is removed from the electrophysiology setup, released from the wax and disposed of improperly mounted flies with correct electrode placement. High frequency stimulation at a chosen voltage can lead to seizure-like activity where no seizure occurs.
The trace immediately after the stimulation is flat. Where a seizure does occur, the trace will show uncontrolled activity. We've just shown you how to assay for seizures and drosophila using electrophysiology.
When using this assay, it is important to have the fly mounted properly so that the electrodes may be inserted easily. Improperly placed. Electrodes are the most difficult cause of problems for this assay.
So that's it. Thanks for watching and good luck with your experiments.
