Hello, my name's Greg McLeod. My laboratory studies calcium regulation in nerve terminals and its impact on neuro transmission. We do this in Oph Melanogaster.
The fruit fly an attractive model for neuroscientists because of its genetic malleability and simple accessible nervous system. In this nervous system, genetically ENC covid fluorescent indicators are available to detect calcium changes in nerve terminals. However, these indicators have limited sensitivity to calcium and often show a nonlinear response.
Synthetic indicators are better suited for measuring the rapid calcium changes associated with nerve activity. We'll demonstrate a technique for loading dextrin conjugated synthetic calcium indicators into live nerve terminals into oph lava. Dextrin conjugation helps prevent calcium indicators from being sequestered into organelles such as mitochondria.
The loading technique can be applied equally To larvae embryos and adults. We'll dissect a lava in a Small cigar bath on a glass slide. The blue dental wax will support a glass loading the bed.
Take a wandering third in star lava from the side of an uncrowded vial. Place the larva in several drops of chilled Schneider solution and pin it through The head and tail with the dorsal midline utmost. Use a sharp pair of micro scissors To nick.
A small incision between the tra here, large enough to allow the bottom blade of the scissors to penetrate. Cut through the cuticle along the dorsal midline between the traia all the way to the head. Turn the larva 180 degrees and extend the incision all the way to the tail.
It is important not to let the bottom blade penetrate too deep into the viscera, but the larva as this will damage the muscles and nerves in wish to examine. Most of the viscera will push up through the incision, snip the track here close to the tail one track here, and snip through the underlying trache all the way to the head. Repeat on the other side.
Remove most of the viscera by cutting it away and removing it from the bath. Take a moment to put down the scissors and pick up a second pair of forceps. Grab the cut edge of the cuticle and pin it tort to one side.
Repeat for the other side. Proceed four segments towards the head to repeat the procedure.Trim. Any remaining viscera at this stage, Ensure that no damage has been done to muscles 7, 6, 13 or 12 in any of the segments.
If any have been damaged, dissect another lava as this damage inevitably results in genic contractions that will make any subsequent imaging problematic. The brain hemispheres, ventral ganglion and segment nerves are now apparent in the following steps. We'll draw the pressure cut end into the segment nerve into a glass FOP bed, and use a plastic filling filament to apply a calcium indicator to the exposed end of the nerve.
But first, we'll demonstrate How to make the filling filament and filling effect to make the filling filament. Two stage pulling technique will be applied to a Yellow defective, slowly heated halfway along its length until the plastic becomes soft and transparent. Stop heating at this point and immediately pull up the tip to draw it out into a fine strand.
That is about half millimeter in diameter. Cut the filament to a length of 50 millimeters. Now place the filament under a dissection microscope and anchor the cut.
End with a thumbnail and pull the other end to draw out a fine filament one centimeter in length. This must be cut with a new razor Blade on tissue paper to avoid creaming it sharp to make the filling prepared. Start with a thin walled capillary Without any filament.
Draw it to a fine tapered point on a pet puller. Take this prepared to a dissection scope and score the tapered portion with a piece of sand paper. Push the tip distal to the scoring away from the score side such that the tip snaps squarely.
The inner diameter of the break should be greater than the outside diameter. At the end of the filling filament, use a micro forg to heat shrink the broken into the glass per effect until it reaches an internal diameter of about 12 microns. Use a Bumps and burner to polish the blunt end of the effect.
Negative pressure can be applied to the blunt End of the effect using a snug fitting Celtic tube coupled to stiff polyline tubing and a syringe. The same tube and syringe will be used to draw up and expel the calcium indicator from the filament. Calcium indicator should be dissolved in distilled water to a concentration of five milli.
Divide this solution into aliquots of five microliters. Heat these in four degree fish. Do not freeze these quats to keep them unfrozen until needed.
Now, draw up one to two centimeters of the calcium indicator into the filament and you Are ready to proceed to the most critical step of this loading technique. Take your scissors and forceps and cut the most anterior segment nerves individually and close to the ventral ganglia. The most AOR segment nerve mass from the ventral vent.
Cut the off any debris. Place the filling filament on the dental wax ramp with the tip in the center of the larva. Attach the scholastic tube and syringe.
Draw up some Schneider's solution into a perfect tip. Now draw up The end of the nerve into perfect it. The fit must be snowed but not tip tight.
It must block further entry of the Schneiders to the protect. Generally remove the scholastic tube and syringe and attach it to the F filament. Touch the side of the filament to the Schneiders in the bath to discharge any static electricity and immediately insert the filament gum effect within 50 microns at the cutting of the nerve without actually touching.
It.Slowly ejecting up calcium indicator to increase the volume of the Schneider in effect by about one third. This will be the final calcium indicator concentration of just under two milli, both the preparation in a Dark, humid location and room temperature for about 40 minutes. I'd like to make two important points here.
First, a physiological solution must be present in the tip to dilute the calcium indicator. As the calcium indicator was solubilized in water alone. The physiological solution must contain calcium at a concentration of several millimolar as axon ceiling is a calcium dependent process, and axon ceiling is essential to preserve the health of the nerve terminals.
The second point is that no more than five minutes should elapse between cutting the nerves and applying dye to the cut end of the nerve. After five minutes, the ceiling ends of the axons will exclude the dextrin conjugated calcium indicators with molecular weights of 10, 000. Beating this time limit will take some practice.
Don't attempt to retard axon ceiling by reducing the calcium levels as this will only compromise the future physiological Function of the nerve damage. After the 40 minute incubation, the preparation in Schneiders now touch the side of the filling filament to the bath and reinserted into the remove most of the calcium indicator solution from the tip And the starter. Again, using the filament draw up fresh Schneiders from the bath and completely filled the bed with Schneiders.
There must be no bubbles in the bed as the Schneider solution will serve as one of the conductors insulate from the bath while the pet glass stimulated nerve, again place the preparation in a dark location for at least 60 minutes, but no longer than four hours and RINs in fresh Schneiders every 30 minutes during the incubation, the calcium indicator is transported Down. The axon concentrates in the nerve terminal 20 minutes before you wish to examine the preparation. Rinse it with HL six solution.
Contain the desired level of calcium when the calcium level is above about 0.4 millimolar at glutamate or L glutamic acid to the HL six at seven millimolar to stop nerve evoke muscle contraction. Schneider's solution is not appropriate for calcium imaging as it already contains high levels of both glutamate and calcium. These levels you will wish to adjust.
Tate is also present making Schneider's autofluorescent loading as well as imaging can be performed in HL six as long as calcium is present at at least 2 million during loading. When setting up the preparation on a compound microscope for imaging, again, be sure to discharge all static electricity s itself. Then touch both the solution in the bath and the objective with a pair of forceps.
If the objective is an immersion objective, lower its co to the bath, then momentarily earth base stimulating wise, or putting one in the bath and Inserting the other down the filling the bed square DC pulses of less Than three volts and 300 microseconds duration will reliably evoke an action potential in the nerve. Here we see two sets of nerve terminals in the cleft between muscle six and seven have been made visible through driving the cytosolic expression of GFP. They've also been forward filled with the calcium indicator Rod Dextrin, which has different excitation and emission spectra to GFP.
This field of view still contains the nerve terminals, but Rod Rin has a low affinity for calcium and is almost invisible while the nerve is quiescent. It is, however highly responsive to the calcium that enters the terminals during single action potentials or short two second trains of stimuli. As shown here in the fluorescence analysis of the closely spaced 80 hertz strains just shown responses in the type one S terminals are usually greater than those in type one B terminals.
Low affinity calcium indicators as rod dextrin and flu. A four dextrin shown almost linear response to nerve stimulation frequencies in these terminals over a range of 10 to 80 hertz. The example shown here comes from a nerve terminal On muscle number four, loaded with Rod Dextrin.
