The overall goal of this procedure is to obtain isolated reticulospinal axons with functional presynaptic terminals to permit the recording of calcium currents directly from the release face membrane. This is achieved by first extracting the spinal cord from the lampe body cavity and removing the dorsal column of the spinal cord as a second step. The slice tissue is treated with a mixture of protease and collagenase enzymes to loosen the connective tissue and facilitate the dissociation process.
Next, lateral tracts of the enzyme treated spinal cord are cut at the midpoint in order to localize the dissociation force. The spinal cord is acutely dissociated by gently separating it until the reticular spinal reticulospinal axons are isolated. Results are obtained that demonstrate the viability of the isolated reticulospinal axons and the recording of calcium currents directly from the release face membrane of the individual presynaptic terminals.
The main advantage of this technique over the imaging of presynaptic calcium transient is that the currents can be resolved directly at the presynaptic release face membrane. This cannot be achieved using conventional imaging techniques. The unrestricted access to the release face membrane at individual presynaptic terminals allows us to apply numerous approaches to study aspects of neurotransmitter at these processes.
Single iCal imaging with quantum dots measuring membrane capacitance changes and simultaneous measurement of calcium currents enables a precise measurement of synaptic delay. Begin this procedure by anesthetizing an amite or adult lamp ray with trica methane sulfonate at 100 milligrams per liter. Next, decapitate the lamp ray in four degrees Celsius ringer solution.
Then remove the body wall muscles to expose the dorsal surface of the spinal cord. Subsequently, remove the meanings prima tiva. Do not remove the ventral meaning priva at this stage.
After that, cut the spinal cord into one centimeter long pieces. Next pin a spinal cord piece with the dorsal side facing up on A-P-D-M-S lined slicing base plate in a vibram chamber containing ice cold ringer solution. Then remove a central section of the spinal cord dorsal column and leave behind the intact dorsal column.
Sections on the rostral and coddle ends to serve as handles during the dissociation process. Use the slowest speed setting in slicing and a depth setting that removes only the dorsal column, leaving the underlying reticular spinal axon column undamaged. Afterward, incubate the sliced spinal cord pieces at room temperature for 45 minutes in a cocktail of one milligram per milliliter protease and one milligram per milliliter collagenase prepared in ringer's solution.
Then put the treated spinal cord pieces in A-P-D-M-S lined Petri dish containing four degrees Celsius ringer solution. Next, remove the ventral meanings priva with fine forceps. Cut the lateral tracks of the spinal cord at the midpoint of the sliced dorsal section with a scalpel blade.
After that, place a drop of immersion oil on the lens. Then place the polylysine coated cover slip in the slot of the recording chamber and seal the edges with vacuum grease. Screw the top in place and place the chamber in the inset.
On the recording rig add ringer solution to the recording chamber using a pasture pipette. Next, connect the outflow tubing of the pressure bottle containing antifreeze solution to the input tubing of the thermoelectric cooling device. Then connect the output tubing of the thermoelectric cooling device to the input end of the outer cooling jacket and the output end to the reservoir R.Place the spinal cord pieces in the recording chamber one at a time.
Gently separate them while keeping them along the cover slip at all times until the axons are isolated. Bring the recording solution temperature to 10 degrees Celsius by passing the pressurized antifreeze solution through the outer cooling jacket of the recording chamber. Allow the axons to recover at 10 degrees Celsius for an hour after dissociation.
In this procedure, perfuse the sample with five micromolar FM 1 43 in ringer solution containing 30 millimolar potassium chloride. Afterward, perfuse the sample with one milligram per milliliter ADV seven in ringer solution. To remove excess FM dye before perfusing it with ringer solution for 15 minutes.
Then image the sample with a 100 x oil immersion lens, a digital CCD camera and the image acquisition software micromanager. Identify individual fluorescently labeled pre-synaptic terminals for recording. In this procedure, fabricate the Illumina silicate glass patch pipettes in a P 87 micro pipette puller.
Design the patch pipette such that the pipette tip encompasses the entire presynaptic terminal diameter S guard coat the patch pipettes by dipping them into PDMS under 50 to 60 PSI pressure and dry them using a heat gun. After that fire, polish the patch pipettes using a micro forge under the fluorescence microscope. Identify the isolated axons with labeled presynaptic terminals.
Then fill the patch pipette with the recording solution. Using a syringe, insert a patch pipette into the pipette holder and position it above the bath. Using a motorized manipulator, gently lower the patch pipette into the bath and position it against the face of a fluorescently identified presynaptic terminal.
Advance the patch pipette slowly until it makes contact with the membrane. At this point, form a giga ohm seal by gentle mouth, suction through a tube attached to the pipette holder. For data acquisition, use a standard step protocol in increments of 10 millivolts as stimulus.
The acutely dissociated reticulospinal axon preparation can be utilized to carry out immunohistochemical characterizations. As a first step, fill the dish inset with divalent ion free ringer solution. Fabricate the patch pipettes in a P 87 micro pipette puller.
Suck a small amount of suture glue from an einor tube into the pipette tip by mouth.Suction. Dispense the suture glue at one end of the polylysine coated inset by blowing gently into micro pipette tip. Then place one end of the spinal cord on the suture glue.
Gently press and adhere it to the surface. Next, place another drop of suture glue at the other end of the inset. Perform the dissociations as demonstrated earlier.
Gently stretch the spinal cord until the axons are dissociated. Then gently drag the free spinal cord end over the suture glue and fix in place. Afterward, replace the divalent free ringer solution with the regular ringer solution by perfusion.
Allow the axons to recover at 10 degrees Celsius for 20 minutes after dissociation before proceeding with the immunohistochemistry protocol. These two images show an isolated reticulospinal axon with punctate labeling of pre-synaptic terminals with FM 1 43 and the targeting of an individual pre-synaptic terminal with patch pipette for cell attached patch recording. This is a representative example showing the cell attached recording of calcium currents from the presynaptic release face membrane.
The solid line indicates the closed state of the channel while the dashed lines indicate the opening of one, two, and three channels. Shown here is the immunohistochemical characterization of an R type calcium channel at the individual presynaptic terminals. A polyclonal primary antibody was used to label an epitope in the intracellular loop between domains two and three of the R type calcium channel and the secondary antibody used was LOR 6 33 Hydrocyte conjugated goat anti rabbit, IGG.
The pre-synaptic terminals were identified by BYOR 4 88 Phin labeling of pre-synaptic actin. This image shows the colocalization between R type antibody labeling and Alexa 4 88 phin. The pre incubation of anti R type calcium channel antibody with control antigen did not yield any specific labeling of calcium channels, but Exor 4 88 falo in labeling of pre-synaptic terminals for the same axon showed discreet punctate labeling After its development.
This technique paved the way for direct measurement of calcium currents at the release face membrane of individual presynaptic terminals previously not possible in central synapses. It also provided a way to determine the number of calcium channels that contributes to the calcium domain gating release. After watching this video, you should have a good understanding of recording directly from the release phase membrane of individual presynaptic terminals.
