Recently, many epilepsy candidate gene have been found and need to be validate by animal experiments. We deliver a set of techniques to study the epilepsy-related neuroactivity, including whole-cell recording, evoked EPSP recording, and the new one that we will introduce here, ex vivo calcium imaging. Since the integrity of the brain and its neural networks cannot be fully replicated in cell culture or brain slices, the main advantage of the current experiment is to obtain intact brain tissue protecting neural networks from damage.
We have established an ex vivo calcium imaging technique along with the bang-sensitive seizure-like behavior assay for efficiently screening the epilepsy-associated genes and exploring the underlying mechanisms of epilepsy at the cellular level. We implemented isolated, intact Drosophila brain tissues for calcium imaging, which can avoid the complex surgical techniques and preserve the integrity of neural networks. And the ex vivo approach can also use the superior signal-to-noise ratio when compared with the in vivo imaging techniques.
To begin, collect the virgin flies of the tub-Gal4 hybrid line and the male flies of the UAS-cac-RNAi line. transfer the virgin and male flies into the same vial to harvest the offspring. After three to five days of eclosion, use a brush to collect the offspring and the tub-Gal4>UAS-cac-RNAi flies.
A day before testing, transfer the flies to new clean vials with food. Next, carefully place four to six carbon-dioxide-anesthetized flies into individual fresh vials. Place a camera on a tripod in front of a whiteboard.
Manually adjust the camera's focus with an empty vial. Now, vortex the vials with the flies at the highest setting for 10 seconds. Immediately place the vial on the whiteboard and observe the flies for any seizure-like behavior.
Measure the recovery time as the time required for the flies to regain the ability to stand upright. The cac knockdown flies showed significantly higher rates of seizure-like behavior than the wild-type flies. The recovery percentage of knockdown flies within one second was significantly lower than the wild-type flies.
To begin, profuse the external solution with oxygenated saline for five minutes. With a pipette, transfer 10 microliters of the dissection solution to a Petri dish. Now, use syringe needles and a microscope to carefully dissect the brains of the anesthetized established wild-type and knockout flies.
With a pipette, transfer the prepared brain into a recording dish with five milliliters of external solution. Immobilize the brains with a C-sharp holder. Next, capture the confocal image of each brain at 20X.
Use the XYT scanning mode and identify the mushroom body neurons at an additional 4.5 times digital amplification. Set the laser excitation to 488 nanometers with 16 microwatt laser power to acquire the whole brain GCaMP6m emission. Then, set the scanning parameters to 1, 400 speed with a pixel size of 256 by 256 pixels.
Set the acquisition rate to 5.3 hertz and record for three minutes. Analyze the fluorescence of five to eight regions of interest. Manually determine the cell body of mushroom body neurons as the region of interest.
Use ImageJ to label the identified neurons and measure their fluorescence intensities. Analyze the fluorescence data of GCaMP6m as shown. Define the intracellular fluorescence increasing between two and 2.5 standard deviations as small spikes, and those increasing more than 2.5 standard deviations as large spikes.
Calcium signals were observed in the mushroom body of flies. The cac knockdown flies showed more large spikes than small spikes.
