The goal of this video is to illustrate how hippocampal slice cultures can be used to decipher the neural immune signaling that modulates susceptibility to spreading depression, the most likely underlying cause of migraine aura and perhaps migraine headache. Spreading depression is easily evoked in hippocampal slice cultures. Since the cultures are maintained in vitro for weeks, the behavior of individual cell types can be measured, mimicked and modulated over extended periods.
This is especially important for the study of neural immune signaling. Since mature hippocampal brain slices attain a quiescent immune state, immune responses of spreading depression can be assessed without the potentially confounding effects of anesthesia using modern molecular biological techniques. Sensitive detection of cytokines signaling from as few as 50 cells to approximately 90, 000 cells in a slice can be determined accordingly.
Hippocampal brain slice cultures are ideally suited for use in the development of novel neural immune therapeutics to alleviate migraine. My name is Yian Berg and this is Aya Puss and Heidi Mitchell from the laboratory of Richard Craig and the Department of Neurology at the University of Chicago Medical Center. Today we will demonstrate the use of rat hippocampus, lice, cultures, and the study of low level immune signaling of spreading depression.
Spreading depression is a paroxysmal neural event that's triggered by a burst of increased synaptic activity that's then quickly followed by a transient loss of all activity. Increasing evidence shows that low-level immune signaling can influence and in return be influenced by synaptic activity within the brain. We hypothesize that cytokines modulate susceptibility to spreading depression and as a result, migraine aura and perhaps migraine headache as well.
The procedures we describe here are designed to minimize potentially confounding immune cell activation and increase our ability to detect low level immune signaling. This lets us detect the widest array of neur immune signaling and its interactions with spreading depression. To begin this procedure, make ground electrodes by cutting glass tubes to 4.5 millimeter lengths.
Then briefly fire polish the cut sections at both ends. Next, bring 100 milliliters of one molar potassium chloride to a boil add 3.5 grams of agar and 0.5 grams of EDTA to it. Stir the mixture continuously to produce a clear yellow solution.
Fit a piece of short flexible tubing over each glass tube. Then draw the warm potassium chloride agri solution into the glass tube and a short distance into the flexible tubing. Release suction and allow the potassium chloride agar to drip slowly out of the open tip of the glass tube.
As the agar drips out, touch a piece of ice with the tip of the open glass tube, which will prompt the agar to gel at the electrode tip and not gel after receding back up into the glass tube tube. Once the agar is jelled, gently remove the flexible tube without altering the agra's position. In the glass tubes, put 0.5 milliliters of one molar potassium chloride into each well of a 1.5 milliliter centrifuge tube rack.
Then place the tip of each potassium chloride afil glass tube in individual wells to keep the tips moist. Next, hold an 80 centimeter length 15 gauge silver wire with a hemostat. Clean each of the four sides by scraping the wire through an emery board held to the countertop.
After that, cut the wire into lengths of four centimeters. Put them into a scintillation vial filled with bleach for 20 to 30 minutes. In order to place a firm silver to silver chloride coat over the cleaned silver surface, bend the wire in half and insert it into the glass tube filled with potassium chloride agar leaving about four to six millimeters exposed.
Then coat the glass tube tip containing the silver wire and a small extent of the wire with goop, a water resistant and flexible glue to prevent the potassium chloride agar from drying, repeat for all electrodes and let the glue dry overnight. After drawing, store the electrodes in scintillation vials with five milliliters of one molar potassium chloride and 25 milligrams of EDTA. About five to six electrodes per vial at four degrees Celsius.
Make recording electrodes with bo silicate glass tubes with filaments. Next, make a stimulating electrode by cutting about a 20 centimeter length of Teflon coated platinum meridium. Then bend the length in half, tie the loose ends into a loose square knot, leaving about two centimeters from the knot to the ends of the wire.
Secure the knot in the chuck of an electric can drill placed on a table while holding the other end of the wire with a hemostat. Briefly turn the drill on and off until the wire is tightly twisted. To attach the platinum meridium twisted wires to lead wires first tape the platinum meridium twisted wire to a bench top and drop a puddle of concentrated hydrochloric acid over one free end of the twisted wire.
Then place the stripped end on a lead wire adjacent to the twisted wire end and apply heat from a fine tip soldering iron then solder until the two wires are firmly attached. Slip a short length of heat shrink tubing over the exposed wire connection and shrink it with heat fire. Polish the tip of a 15 centimeter past pipette and guide the twisted platinum meridian Wire down the pipette.
Use a water repellent epoxy to seal both ends of the electrode. Finally, attach banana plugs to the lead wire ends for the connection to the stimulus isolator under stereoscopic observation. Place the electrode tip in PBS and look for electrolytic produced bubbles coming from only the cut end of the electrode tips if any bubbles come from the sides of the twisted wires.
Cut the electrode transversely using a single edge razor blade to reestablish intact insulation to the long axes of the wires. When troubleshooting to resolve abberant stimulus effects, try to make a freshly cut electrode tip. Another important step is to prepare the recording dishes, which consists of a slice culture insert and a 35 millimeter culture dish that sits on two 1.0 x 12 millimeter glass rods spaced about 1.0 centimeters apart.
Glass rods are attached to the dish base by heating the glass tubes and then pressing them into the base with forceps. For static recording conditions add 1.5 milliliters of media to the dish. Following this soak a piece of sterile cotton that is 10 millimeters wide and three to four centimeters long.
In 1.0 milliliters of media, fold the cotton in half along the long axis and place it along the inner wall on the insert. With sterile forceps, the wet cotton helps maintain dish humidity. Three compressible four millimeter long pieces of tubing are equally spaced around the insert.
Cover the dish tightly with polyvinyl chloride film to allow gas exchange. Then cut around its mid vertical wall with a single edge razor blade to remove excess polyvinyl chloride. Film evoke ca three field potential with 100 microsecond pulses at 0.2 hertz, beginning with a current of about one to five microamps that is sufficient to trigger a response.
Move the recording electrode in increments along the parametal neuron long axis until the field potential is maximal. Next, increase the current with a recording electrode at this position and note the maximal current response of about 10 to 20 micro amps. Finally, use half maximal stimulus intensity for experiments to determine the threshold for trans synaptically evoked spreading depression.
Switch the stimulation paradigm to manually evoke single bursts of 100 microseconds per pulse at 10 hertz. Progressively increase the current intensity per stimulus burst until spreading depression is triggered. Report spreading depression threshold in lums trigger spreading depression every nine minutes for an hour.
Record ca three area fast potential and slow potential changes via separate digital signal processing systems. After the last spreading depression, return the culture insert to normal incubation conditions and mark the culture dish lid. To note the culture that experienced spreading depression change media every three to four days until culture harvest.
Begin this procedure by diluting ISO selectin to 20 micrograms per milliliter in growth media. Then incubate slice cultures at normal incubation conditions four to 10 hours before imaging turn on the microscope camera UV light temperature control and gases at least one hour before imaging. Next, set up the metamorph software for imaging following this place the insert in a 35 millimeter culture dish with two one millimeter glass rods in the bottom.
Then fit three two millimeter pieces of rubber tubing between the walls of the insert and the culture dish. To further stabilize the insert place a 10 millimeter wide piece of cotton soaked in an additional one milliliter of media inside the insert. To maintain a humidified environment, cover the top of the culture dish tightly with polyvinyl chloride film.
To prevent fluid loss, make sure that the focus of the imaging is on the CA three parametal cell layer. Note the slices orientation by taking phase pictures at five x 10 x and 20 x in the fine focus window set range current to be eight and accuracy to be one click find focus to ensure an in-focus image in the acquire time-lapse window. Select time interval to be one minute and duration to be six hours to accurately capture microglial movements.
Imaging should be no less frequent than once per minute at the end of the movie. Check for cell injury by CY talk screening for PCR experiments. Submerge slice culture inserts in two to three milliliters of RNA later and store them at four degrees Celsius for up to three days until further processing.
To harvest, remove extraneous tissue using a glass knife, put the slices into individual RNAs. DNAs free 1.5 milliliter centrifuge tubes containing 0.5 milliliters of sterile phosphate buffered saline. Using a fine tipped paintbrush spin samples, the 10, 000 RPM for one minute in a stereotypic fashion so that all slices adhere to the same side of their tubes.
Remove the PBS supernatant and resuspend the sample in 500 microliters of triol. Store the samples at minus 80 degrees Celsius or keep on ice until RNA extraction Protein expression changes from SI RNA knockdown can be sensitively assessed by enhancing traditional DAB immuno staining via silver intensification and subsequent densi metric quantification. Shown here is the slice culture electrophysiological recording paradigm in which a recording micro electrode is placed in the CA three parametal neuron layer and a bipolar stimulating electrode is placed in the dentate gyrus.
Here is an example of the evoked CA three field potentials by dentate gyrus stimulation, which is used to establish the half maximal response here shows the CA three field potential responses to 10 hertz bursts left that were progressively increased in an intensity to eventually trigger spreading depression. In the lower right. Shown here is an exemplary vital immune cell.
This imaging demonstrates for the first time that a fraction of microglia move long distances in an optimal search pattern as indicated in red known as levy flights that are inversely proportional to synaptic activity. Preem amplification of CDNA allows the detection of IFN gamma in hippocampal slice cultures. Initially, the CT for a reference gene RPL 13 A was 20.5 and IFN gamma was undetectable on the left.
However, after Preem amplification indicated on the right, IFN gamma was easily detected at 29.0. With RPL 13 A registering at 14.1 to indicate a 12 fold increase of signal amplification immunohistochemical confirmation of a efficiency is shown here. Differentiated silver intensification of peroxidase diamine benzine immuno staining is used to quantify the significant reduction of cyclophilin B production in siRNA treated hippocampal slice cultures One once mastered.
The techniques outlined in this video can be performed over several days. We wish to emphasize that our hippocampal slice cultures closely approximate their in vivo counterpart and are ideal for studying immune signaling and spreading depression. However, it is essential to maintain quiescent immune environment under control and sham conditions.
To minimize confounding immune activation, cultures must be maintained under sterile condition. They should not be exposed to excessive chemical, mechanical, or thermo perturbations. Since physiological cytokine signaling occurs at the femto molar level, highly sensitive and reproducible measurement techniques must be applied.
We use PCRA technology and sensitive proteomic techniques. Finally, cytokines are both pleiotropic and redundant.Accordingly. Multiplex cytokine targets measured over time are useful for studying how immune signaling modulates susceptibility to sprain depression and by extension migraine.
