Here we demonstrate how to employ the ATP-sensitive FRET sensor ATeam 1.03 YEMK for dynamic measurements of changes in neuronal and astrocytic ATP levels in organotypically cultured mouse hippocampal slices. The main advantage of this technique is that the one can study energy metabolism in living brain tissue in a controlled setting, an environment with high sensor expression levels. This technique may help in gaining insights into pathomechanisms, underlying diseases, or brain damage, for example, caused by energy deprivation, like ischemic stroke.
It can, in principle, be used to study ATP levels, not only in brain tissue, but in other organs as well. To successfully run this experiment, it is essential to perform all steps involved in culturing the tissue extremely careful and to maintain sterility. Moreover, some knowledge of cellular fluorescent imaging is required.
Tissue handling in most sophisticated approach and a visualization is vital to understand the proper procedures. The same is true for imaging experiments. Demonstrating the procedure will be Rodrigo Lerchundi, a postdoc, and Na Huang, a Master student from the laboratory.
In an ice-cold Petri dish filled with ACSF, place the brain on a filter membrane. Separate the hemispheres and perform a parasagittal cut at an angle of 45 degrees. Fix one hemisphere at the Vibratome tissue stage with superglue, and immediately transfer the tissue block to the Vibratome bath containing ice-cold ACSF bubbled with 5%carbon dioxide and 95%oxygen.
Align the tissue in the Vibratome bath. Keep the second hemisphere in ice-cold ACSF until slicing. Adjust the Vibratome to cut slices at 250 to 400 micrometers.
After cutting the slice, identify the hippocampal formation based on its typical morphological appearance and isolate it using hypodermic needles, keeping the part of the cerebral cortex adjacent to the hippocampus. Place the slice on a mesh in ACSF, warmed to 34 degrees Celsius and bubbled with 5%carbon dioxide and 95%oxygen until all the slices are collected. Transfer the slices to the laminar flow cabinet to continue under sterile conditions.
With an inverted, sterile, glass Pasteur pipette, gently transfer the slices from the ACSF into one of the pre-warmed Petri dishes filled with sterile Hanks'salt solution. Change the pipette and transfer the slices to the second culture dish. Repeat the process five times overall.
Transfer as little HBSS as possible to the following culture plates. Using a pipette, gently place one slice at a time on the top of the culture insert. Avoid turbulences in the pipette, and wait until the slice descends to the tip of the Pasteur pipette.
Repeat the process for each slice. Place two slices on a membrane. Carefully remove any excess Hank solution from the top of the insert by using a fine tip.
Keep the cultures in a humidified incubator at 5%carbon dioxide and 37 degrees Celsius until the day of the experiment. Replace the medium every two to three days. Without touching the tissue, apply 0.5 microliters of the diluted vector directly to the top of each slice.
Finally, place the slices back into the incubator and maintain them there for at least six more days. Just before starting an experiment, transfer an insert containing cultured slices into the sterile hood, and place it into a 30 millimeter dish containing one milliliter of organotypic slice culture medium, or minimal essential medium. Place the dish under the stereoscope and focus onto the surface of the slice.
Use two sterile hypodermic needles to make a short cross-cut right on the narrow edges of a chosen slice, and in the upper layer without damaging the tissue underneath. To remove the prepared slice from the insert, hold the edges of the membrane with tweezers and use a sterile scalpel to make straight, parallel cuts to the membrane, forming a square or a triangle with the slice in the center. If the insert hosts additional slices, transfer it back to the original plate and into the incubator.
The surface tension of the medium will prevent its leakage onto the surface of the membrane. Prepare experimental ACSF and obtain a pH of 7.4 by bubbling it with 95%oxygen and 5%carbon dioxide through an inserted tubing connected to the carbogen supply for at least thirty minutes. Keep the saline bubbled during the entire experiment.
Then switch on the fluorescent light source of the monochrometer. Transfer the organotypic slice culture into the experimental chamber. Place a grid on top of the organotypic slice culture with the frame down, not touching the culture, and the threads up, touching the membrane.
Place the chamber on the microscope stage and connect it to the perfusion system. Switch on the peristaltic pump at a flow rate of 1.5 to 2.5 milliliters per minute. Make sure there is no leaking of the perfusion system.
Using transmission light, bring the cultured slice into focus and identify the area where experiments shall be performed. Before starting imaging experiments, wait at least 15 minutes to allow slices to adapt to the saline conditions, then switch on the camera and the imaging software. Excite the donor fluorescent protein at 435 nanometers.
Set the exposure time to between 40 to 90 milliseconds. Then insert the dichroic mirror and the filters into the beam splitter unit. Split the fluorescence emission at 500 nanometers with an emission image splitter, and employ band pass filters at 482 plus or minus 16 and 542 plus or minus 13.5 nanometers to further isolate donor and acceptor fluorescence.
Select a region of interest apparently devoid of cellular fluorescence for background subtraction. Circle single structures of labeled tissue in the image on the screen to create ROIs. Set the frequency of image acquisition and the overall recording time.
For experiments longer than 30 minutes, an acquisition frequency of 0.2 to 0.5 Hertz is recommended to prevent phototoxicity. Subsequently, start the recording. To induce changes in intracellular ATP, switch the perfusion tube from standard ACSF to a saline containing metabolic inhibitors, for example, chemical ischemia solution.
Alternatively, use a saline with elevated potassium concentration at eight millimolar to mimic release of potassium ion from active neurons. Directly after the recordings, exchange the experimental ACSF with heaps-buffered ACSF. Then transfer the recording chamber containing the slice culture to the confocal laser scan microscope.
Take Z stack images at the highest Z resolution possible at the given optical configuration. In this protocol, ten days after a transduction, neurons expressing ATeam 1.03 YEMK were found at high density in the neocortex of cultured tissue slices at depths of up to 50 micrometers below the slice surface. Comparable results were achieved in the hippocampus.
For astrocytes, ATeam 1.03 YEMK was expressed under the control of the human glial fibrolary acidic protein promoter, and organotypic slices expressing ATeam 1.03 YEMK in the hippocampal neurons selected ROIs represent the somata of pyramidal cells. After exposing the slice to 5 millimolar sodium azide in the absence of extracellular glucose for one minute, opposite changes were induced in the emission intensity of the FRET pair. A reversible decrease in the ATeam FRET ratio was also observed.
Long-term ATeam FRET ratio in 14 different cells under baseline conditions in neurons and astrocytes show that the ATeam is a reliable and stable sensor. Please note that the chemical ischemia resulted in the expected strong drop in the ATeam ratio in both cell types at the end of this experiment. An increase in the extracellular potassium concentration from three to eight millimolar for three minutes did not result in a detectable change in the neurons expressing ATeam 1.03 YEMK.
In contrast, astrocytes reacted to the increase in extracellular potassium by a reversible increase in the ATeam FRET ratio, indicating an increase in intracellular ATP levels. Again, chemical ischemia caused an immediate decrease in the ATeam ratio, demonstrating that the sensor can detect changes in ATP. When attempting FRET-based cellular imaging as described here, it's important to keep in mind that the production of high-quality slices in organotypic cultures is a key step.
Moreover, careful removal of the glial scar and expert-level imaging are required. Following this procedure, one should also be able to perform experiment incorporating different other FRET-based nanosensor for cellular metabolites. For example, those for glucose or lactate.
Last, but not least, it needs to be emphasized that relevant acts for protection of animals must always be observed. This is also true for the effective laws governing the handling of genetically modified organisms.
