The goal of this procedure, which combines optogenetics with the freeze-fracture replica immunolabeling technique, is to analyze the density of ionotropic glutamate receptors at synapses in the mouse amygdala with identified pre, and post, synaptic elements. The high reproducibility and versatility of that freeze-fracture replica immunolabeling technique, when combined with optogenetics, offers a very powerful approach for the correlated analysis of the structural and functional properties of synapses. The main advantages of this procedure are:the planar view of the pre and post synaptic elements, and the quantitative analysis of proteins in these specialized micro-domains.

Different technique can be adapted to a variety of different tissues to study the distribution and organization of integral membrane proteins. Generally, this combined optogenetic FRIL approach is challenging for beginners because of the large range of different apparatus and machines required. Beginning the modulation with different method is critical as some other steps are difficult to learn.

Such as fracturing and replicating frozen specimens To begin this procedure glue a coronal block of mouse brain onto the holder of the vibro-slicer. Orient the tissue block so that the neo-cortex faces the vibrating blade. Next, slice the coronal sections containing the amygdala at a thickness of 140 micrometers in zero point one molar, ice-cold PB.And collect them in a six-well dish in the same buffer.

Under a stereo-microscope, trim out the region of interest from the slices, in a petri dish coated with silicone elastomer and filled with zero point one molar PB.Then, transfer the trimmed block into the cryo-protection solution, and keep overnight at six degrees Celsius. In this step, prepare the copper carriers for use in the successive stages of the FRIL procedure, by polishing them with sheet of shammy skin as a tarnish remover. Under the microscope, attach a ring of double-sided tape to the copper carrier, which will serve as the holding well for the trimmed block.

Then, place the trimmed block in the hole of the double-sided tape using a platinum wire loop. Remove excess cryoprotectant solution using a filter paper or a brush. Afterward, cover the holding carrier with another carrier.

So that the tissue block is sandwiched between the two carriers. To freeze the specimen, insert the carrier sandwich into the specimen holder. Subsequently, insert the specimen holder into the high-pressure freezing unit.

Initiate the freezing cycle by pressing the jet-auto button, remove the specimen holder immediately, and submerge the tip into an insulated box with liquid nitrogen. Then, carefully remove the carrier sandwich from the specimen holder, and place it in a pre-chilled cryovial. Store the cryovials containing the carriers in a cryotank until replication.

Before inserting the electron beam gun, remove the shield with the deflector plate. Place the setting gauge, for centering the filament, into the collet chuck through the lower cathode cover. Next, slide the new filament over the gauge, until the pressure laminae clamp the ends of the filament.

Then, remove the setting gauge and insert the carbon rod. Fix it, by tightening the collet chuck of the evaporator rod holder. Ensuring that the height of the end of the rod is at the middle of the second coil from the bottom.

After that, replace the deflector plate, and insert the electron-beam gun into the freeze-fracture unit. In this procedure, adjust the current and voltage for evaporation. Next, insert the frozen carrier sandwich into the double-replica table in liquid nitrogen.

Then, transfer the double-replica table to Dewar vessel, and fix it to the specimen stage receiver at an angle of 45 degrees. Pick up the double-replica table with a table manipulator. Insert it into the freeze-fracture unit onto the cold stage, and wait approximately 20 minutes to allow the temperature of the double-replica table to adjust to negative 115 degrees Celsius.

Afterward, fracture the tissue by rotating the wheel counterclockwise manually, which is connected to the shroud above the double-replica table. When the shroud turns, it forces the double-replica table to open, which results in fracturing the tissue. A layer of carbon from a 90 degree angle is applied to the fractured faces.

Subsequently, remove the replicated specimens from the double-replica table, and transfer them to a ceramic 12-well plate filled with TBS. Using a platinum loop-wire rod, remove the replicated tissue from the specimen carrier. For SDS digestion, transfer a replica to a four milliliter glass vial filled with one milliliter of SDS-digestion buffer.

Allow it to digest for 18 hours at 80 degrees Celsius with shaking. For immunolabeling, wash the replica for 10 minutes in fresh SDS-digestion buffer. Then, incubate it with primary and secondary antibodies, diluted in 2%BSA-TBS, in a humid chamber at 15 degrees Celsius for 24 to 72 hours.

After that, mount the replica on form-far coated, 100-line parallel bar grid. Image the replica with a transmission-electron microscope at 80 or 100 kilo-volts. Then, acquire the digital images through a CCD camera.

When it is offline, find the corresponding regions on the image from the replica, using different landmarks. Four weeks after AAV injection, to express Channelrhodopsin-2 in the posterior thalamic nuclear group, Channelrhodopsin-2 is effectively transported anterogradely along the thalamic axons to reach the amygdala intercalated cell masses. The post-synaptic specialization of glutamatergic synapses can be recognized in a replica as a cluster of intramembrane particles on the E-face of the plasma membrane, and is often accompanied by the P-face of its presynaptic element.

Here, Channelrhodopsin-2 and glutamate receptors were visualized using gold particles of different sizes conjugated to the secondary antibodies. Because of the lack of structural or molecular tools to detect on the same replica whether the postsynaptic membrane belonged to the intercalated neurons. The corresponding replica was labeled for mu opioid receptors, a marker for these neurons.

As an example of the quantitative analysis of glutamate receptor density at these synapses, here are the scatter plots of the number of gold particles for ampa receptors versus the synaptic area on spines and dendrites. Which reveal a positive correlation in both structures. While attempting this procedure, it's important to remember, that the individual steps are highly interdependent.

Therefore, a mistake in one of the steps can jeopardize the whole procedure. Viewing large portion of plasma membrane specializations on a two-dimensional surface of the replica, allows the inspection of the spatial distribution and physical continuity of molecules of interest without laborious and time-consuming reconstruction of serial artrophin sections. This approach can be used by other investigators to gain insights into structure-function relationships of specific synapses in neural circuits.

Where it is untangling the origin of the inputs and the nature of the postsynaptic elements. Is crucial, but problematic.

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