The overall goal of this procedure is to prepare healthy brain slices from mature adult animals that are suitable for patch clamp electrophysiology experiments. So this optimized NMDG Protective Recovery Method for brain slice preparation allows researchers to explore the intrinsic and synaptic properties of brain cell types across many different brain regions and for animals of virtually any age. So the main advantage of this particular technique is that it allows one to prepare brain slices that have a higher degree of neuronal preservation compared to prior methodologies.

And also that these brain slices are more suitable for patch clamp recording. This method was optimized for adult mouse brain tissue. It can be utilized for a variety of other species including neurosurgically resected human brain tissue.

To begin this procedure setup the slicing station with the tissue slicer machine and surgical instruments. Attach a zirconium ceramic injector blade to the blade arm using fast adhesive glue. Then insert the specimen holder and align the leading edge of the blade to the specimen holder rim.

Leaving a tiny gap to ensure the blade does not scrape the metal. Fill a 250 milliliter beaker with 200 milliliters of NMDG HEPES aCSF. And pre-chill it on ice with constant carbogenation for more than 10 minutes.

Then setup the initial brain slice recovery chamber by filling it with 150 milliliters of NMDG HEPES aCSF. And place the chamber in a water bath at 32 to 34 degrees Celsius. To setup a brain slice holding chamber fill the reservoir with 450 milliliters of HEPES aCSF and let it warm to room temperature under constant carbogenation until use.

Next, prepare molten agarose for tissue embedding by cutting out a block of 2%agarose from previously prepared dish using the open end of a 50 milliliter conical vial. Loosely cap the conical vial then microwave it for 10 to 30 seconds until the agarose starts melting. Do not overheat.

Pour the molten agarose into 1.5 milliliter tubes. Maintain the agarose in the molten state using a thermomixer set to 42 degrees Celsius with a vigorous shaking. And carefully ensure that the molten agarose does not solidify prematurely.

Pre-cool the accessory chilling block for the slicer on ice at this time. In this procedure, make a cut on the skin of the head to expose the skullcap. Then, use fine super cut scissors to cut away the skin over the skullcap.

And make small incisions laterally on both sides of the caudal ventral base of the skull. Make additional shallow cuts starting at the caudal dorsal aspect of the skull moving in the rostral direction up the dorsal midline. Taking care not to damage the underlying brain.

After that, make a final T cut perpendicular to the midline at the level of the olfactory bulbs. Subsequently, use the round tip forceps to grasp the skull starting at the rostral medial aspect and peel towards the caudal lateral direction at one side. Repeat this procedure for the other side to crack open.

And remove the dorsal halves of the skull cap to expose the brain. Gently scoop out the intact brain into the beaker of pre-chilled NMDG HEPES aCSF. And allow the brain to uniformly cool for about one minute.

Now, use a large spatula to transfer the brain from the beaker onto the Petri dish covered with filter paper. Trim and mount the brain according to the preferred angle of slicing and desired brain region of interest. Work quickly to avoid prolonged oxygen deprivation during handling.

Afterward, affix the brain block to the specimen holder using adhesive glue. Retract the inner piece of the specimen holder to withdraw the brain block fully inside. Then pour the molten agarose directly into the holder until the brain block is fully covered in agarose.

Subsequently, clamp the pre-cooled accessory chilling block around the specimen holder for ten seconds until the agarose has solidified. Insert the specimen holder into the receptacle on the slicer machine and verify proper alignment. Following that, fill the reservoir with the remaining pre-chilled oxygenated NMDG HEPES aCSF from the 250 milliliter beaker and place the bubble stone into the reservoir for the duration of slicing, to ensure adequate oxygenation.

Next, adjust the micrometer to begin advancing the agarose embedded brain specimen. Start the slicer and empirically adjust the advance speed and oscillation frequency to the desired level. Continue advancing and slicing the tissue in 300 micrometer increments until the brain region of interest is fully sectioned.

The total time for the slicing procedure should be less than 15 minutes. For initial NMDG recovery. Upon completion of the sectioning procedure collect all of the slices using a cutoff plastic pasture pipette and transfer them into a 34 degree Celsius recovery chamber filled with 150 milliliters of NMDG HEPES aCSF.

The timing of the acute brain slice recovery step is very important to be able to strike a balance between morphological preservation and the functional recovery of the electrophysiological properties of each neuron. After determining the optimal sodium spike-in schedule, according to mouse age carry out the Step Y sodium spike-in procedure by adding the indicated volumes of sodium spike-in solution at the indicated times. Add the sodium spike-in solution directly into the bubbler chimney of the initial recovery chamber to facilitate rapid mixing.

The sodium spike-in schedule that was provided with a variety of different age ranges is a really good starting point. But it should be optimized and utilized for your own specific experimental design. Afterward, transfer all the slices to the HEPES aCSF long-term holding chamber maintained at room temperature.

Allow the slices to recover for another hour in the HEPES holding chamber prior to patch clamp recording experiments. Shown here are the representative IR DIC images that were acquired from diverse brain regions and acute slices from a three month old mouse for the evaluation of the morphological preservation of neurons. The results shown here are from the use of the control NMDG protective cutting method without a protective recovery step.

Whereas these results are from the optimized NMDG protective recovery method. Overall, improved neuronal preservation is observed with the optimized NMDG protective recovery method. The optimized NMDG protective recovery method with a gradual sodium spike-in procedure was compared with the original NMDG protective recovery method.

The average time for GigaOhm seal formation and patch clamp recording was dramatically and significantly reduced when the gradual sodium spike-in procedure was applied together with the NMDG protective recovery step. The improved neuron preservation achieved with this brain slice method facilitates challenging experimental applications including but not limited to multi-electrode patch clamp electrophysiology to probe local synaptic connectivity. As demonstrated here, using adult human ex vevo neocortical brain slices derived from neuro surgery.

Scientists should have a good understanding from watching this video about how to implement our optimized NMDG Protective recovery method including the new sodium spike-in procedure that we've described, for preparing brain slices that are suitable for patch clamp recordings. So after mastering this protocol it should be possible to complete the brain slice procedure in less than one hour each day. And should yield high-quality reproducible brain slices and will work for animals of virtually any age.

So this procedure, this method can help to prepare brain slices that are suitable for addressing questions about brain function using techniques such as electrophysiology, live optical imaging and optogenetics, receptor trafficking assays and other kinds of functional assays for exploring the function of brain cell types. So this technique is also useful because it will help researchers to be able to functionally probe different brain regions including brain regions that are not traditionally amenable for patch clamp recording from adult animals in brain slices.

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