成長円錐の高分解能イメージングのためのアメフラシから神経細胞の培養

In the pseudo lab, we culture back cell neurons from the Marine Mollusk Plegia California car. These neurons project very large growth cons, which make them a very good model system for high resolution studies of growth con motility and guidance, especially for analyzing cytoskeletal dynamics. In this video, we demonstrate how to perform an plegia dissection and back cell culture.

Hi, I'm Daniel Suter from the Department of Biological Sciences at Purdue University. Today we are gonna show you procedure for dissection culture and imaging of plegia back cell neurons. Neurons from the C, her apia CAA have been extensively used for studies of learning and memory.

Since Apia has a simple nervous system and large neurons that are suitable for electrophysiology. In our lab we use these neurons because their growth cones are large and very suitable for high resolution imaging of protein dynamics related to growth con motility and guidance. The complete procedure involves the following steps.

On day one, the animal is sacrificed and dissected to obtain the abdominal ganglia. On day two, culture plates are prepared. Back cell clusters are dissected and individual neurons are plated.

On day three, an imaging chamber for time-lapse imaging of the cells with DIC differential interference contrast microscopy is constructed, demonstrating and narrating. The procedure today will be iTune Lee, a graduate student in my laboratory. She will now show you how to prepare back cell neurons.

Let's begin. The back cell neurons are part of the abdominal ganglion of the plegia nervous system, so we will be dissecting out the entire abdominal ganglion for enzymatic digestion. Then we'll separate the two bag cell clusters from the rest of the ganglion.

The abdominal ganglion will be digested in this phase two solution for 15 to 16 hours to loosen the connective tissues. Now we need to prepare a large 60 milliliter syringe filled with 0.5 molar of magnesium chloride to sacrifice the animal and clean dissection tools, one forcep dissection, scissors, and large scissors. We are wild plegia California car from the Californian coast, which are about 150 grams of weight.

We keep the animals in a lobster tank for a maximum of three to four weeks. The animal is injected near the head into the body cavity with the magnesium chloride solution. Rubbing the animal helps spreading the solution and prevents it from contracting.

Pin the animal at the head and tail down onto a styrofoam board using syringe needles. Lift the body wall muscle with one forcep and cut open the body cavity with a large scissors. Then cut the body wall open along the side from head to tail and pin the muscle to the board.

Next, the abdominal ganglion must be found beneath the ovary, which is a brush like yellowish organ. The abdominal ganglion can be found. Disconnect the ganglion by cutting the nerves with the dissection scissors on the anterior and the posterior side of the ganglion.

Then transfer the ganglion into a test tube with one milliliter of the displaced solution and place it into a 22 degree water bath for 15.5 hours. During this incubation, the connective tissue will loosen to a point where the back cell clusters can easily be removed. Before beginning the back cell culture, we will prepare the cover slips on which the neurons are cultured.

We culture back cell neurons on lyin coated cover slips in 35 millimeter Petri dishes. They are then submerged in L 15 A SW medium, which is a high salt culture medium. For this type of cells, we typically prepare eight to 10 Petri dishes with cover slips per animal.

Now let's discuss the tools used for plating the bag cell neurons. For dissection of bag cell clusters, we would need three peachy dishes without cover slips filled with L 15 A SW, fine forceps and scissors sterilized with alcohol and a bent yellow tip attached to a P 20 pipe. The yellow tip is burned by quickly passing it over a benson burner flame and using forceps to bend it.

Be careful not to crush the bend part. Otherwise, the bag cell cluster might get stuck there later. Dissection and plating of plegia bag cells is done in room temperature under the dissecting scope.

Now the 15.5 hour enzyme incubation has ended and we can transfer the abdominal ganglion from the disc space to one of the working dishes with L 15 A SW medium. Before showing how bag cells are isolated, let's first look at schematic. To visualize how the abdominal ganglion is dissected, we first cut the abdominal ganglion through the center.

As you can see by this line. We then make two cuts in front and back of the back. So cluster as you can see here.

Now looking through the stereoscope, you can see that the abdominal ganglion is cut through the center and now you can see that the two cuts are made in the front and back of the back cell cluster. Then we use forceps to find an opening in the connective tissue and gently push down the connective tissue sheath surrounding the bag cell cluster. Remove the connective tissue from the cluster as one piece if possible using the dissecting scissors.

But be careful not to damage the bag cell cluster. With a medium taken into the bent tip, gently transfer the bag cell cluster to another working dish. Gently ter the bag cell cluster with a yellow tip to remove individual bag cells from the cluster.

To do this, take the cluster into the pipette tip and push it out without causing any air bubbles. Transfer live cells from the cluster to dishes containing poly coated cover slips. Dead cells appear black in the center while live cells are white.

It's important to place the cells at the center of the cover slip for microscopy and to remove any cell debris and dead cells as soon as possible. By picking up the unwanted material or blowing it away, healthy cells will attach quickly to the polylysine plate. About 15 to 25 cells on each cover slip.

When all the cells from the first cluster have been positioned, the connective tissue of the second cluster is removed and the same cell plating procedure continues. After all plates are finished, let them sit at room temperature in the floor bench for at least two hours to allow cells to attach properly while avoiding any vibrations. Then place the dishes on a styrofoam or a plastic tray and transfer them to a 14 degree incubator before further use.

We typically image the plegia growth cones one or two days after plating. For lifestyle imaging of neuronal growth cone dynamics, we make a small imaging chamber of two cover slips separated by plastic spaces. The lower cover glass has the neurons attached to them.

The upper cover slip is cut shorter with a diamond pen to allow easy medium exchange. On the microscope stage, the cover slip sandwich is then placed into a custom made holder for mounting onto the microscope. Stage high vacuum grease is used to hold the chamber elements together without medium leaking out Using a small syringe, high vacuum grease is applied to the top cover glass and then we use forceps to position the spaces and then apply some vacuum grease on top.

Then we pick up the whole assembly, turn it around and gently press it onto the cover Slip with cells still immersed in the dish. Use Q-tips to align the cover slips and press down the top cover slip without breaking it. Then we remove the sandwich carefully from the dish, wipe off any extra medium and put it onto the base of the holder.

Place the sandwich in the holder without cracking the cover glass by pressure. Clean both cover glasses with sparkle glass cleaner on a Q-tip and we prefer to use sparkle because it leaves fewer streaks on the glass. Clean the covered glass with circular motion from the center and immediately follow with a dry Q-tip to avoid streaks.

Clean covered glass is very important for high quality imaging, especially with DIC optics. Plegia back cell neuronal cultures are low density cultures, so it'll be very helpful if your microscope has stage positioning functions for easy switching between different cells. Now we're ready to observe growth core morphology and motility.

We visualize growth on morphology and motility with high resolution differential interference contrast, DIC time-lapse imaging. DIC imaging is useful for this application because it provides a surface view of the growth con structure in this 60 XDIC time-lapse movie. One can clearly see the three domains of a typical growth cone.

The peripheral domain that is made up of affect enrich la mela podia, and op podia. The transition zone that is characterized by ruffling activity and the central domain that mainly contains micro tubals and organelles. To study the mechanisms of growth con steering, we have developed the restrained bead interaction RBI assay in this assay.

Microbeads coated with artesian proteins are used to mimic cellular target substrates. The bead is placed onto the peripheral domain of the growth con with a microneedle in the DIC time-lapse movie. One can see retrograde effect and flow by retrograde moving waves in the central domain.

Organelles move on micro tubals. After an initial period during which no significant changes happen, one can see a sudden forward movement of the central domain boundary towards the restrained bead. A companion by leading edge growth and tension increase in addition effect in flow attenuation and micro tubal extension can be observed specifically along the bead interaction axis.

To analyze cytoskeletal dynamics in growth cones, we use fluorescent speckle microscopy. We micro inject fluorescently labeled tubulin and acting probes into the neuronal cell body and take time-lapse movies. Of the dynamic movements of the cytoskeleton, the affecting shown in red is assembled along the leading edge retrograde lead translocated by retrograde flow and recycled in the transition zone.

Dynamic micro tubal shown in green explored the growth con periphery by a combination of assembly and translocation behavior. The low amount of labeling results in the speckled pattern, allowing us to quantitatively analyze the various parameters of cytoskeletal dynamics. We have shown You how to do plegia back cell dissection, culture and imaging if you would like to do high resolution imaging of neuronal growth cones.

This is really the system to use because of the large size of the growth cones, the define cytoplasmic regions and the well-described cytoskeleton. So that's it. Thanks for watching and good luck with your experiments.