Dana & Richelle: Week 8

Dana's Update:

This week, in our biweekly meeting for The Bridge, Richelle and I talked about how neuroscientists slice brains for physiology experiments in labs.  Richelle had a lot of great questions about how we slice brains, what the equipment looks like, how we keep the neurons alive, and how long the process takes.  After chatting with Richelle, I decided to take some photos of the dissecting and slicing equipment and put together some captions.  My hope is that these images will be useful for Richelle when she creates images to go with my text for our collaborative project.  Here are some of the photos and captions that I sent Richelle:

In this photo, I’m placing the middle of the cerebellum (the tiny bit of brain tissue between the two dissection tools) onto a spot of superglue in the black box. Once the tissue is glued down, I fill the black box with a solution and use the blade (to the right of the tissue) to make 200 micrometer-thick live brain slices.

Here, you can see the cerebellum glued onto the platform just before getting sliced. The chunk of tissue is about the size of an eraser on a pencil.

Here, you can see the blade going through the tissue and cutting a very thin slice, which ends up on top of the blade. It’s a little blurry because the slicer is vibrating side to side as it gradually moves forward through the tissue. It takes about 20 seconds for the blade to make it all the way through the tissue for each slice. The dissection and all the slices have to be completed in about 12-15 minutes in order for the slices to be healthy enough to last through an experiment. We make slices in the mornings, and use the slices for experiments in the afternoons/evenings.

In this photo, I’m using a small suction tube to suck up a brain slice after the blade has completely disconnected it from the rest of the tissue. The blade starts at the top, and then moves down 200 micrometers for each slice to make sure they’re all the same thickness.

This is the chamber that the brain slices recover in for about 1.5 hours after getting sliced. If you look carefully in the bottom right corner of the photo, you can see three slices lined up on top of the silver netting. The slices incubate here inside this box with a solution that has nutrients to keep them alive. In order to stay alive, the neurons on these slices also require oxygen, so we have gas tanks that bubble oxygen into the chamber.

​In this video, you can see the blade coming across the brain tissue and generating a 200 µm slice of the cerebellum. 
 
In addition to chatting about how to slice brains, Richelle and I also talked about the pipettes I use.  These pipettes are special, glass micropipettes that we make each morning.  We attach these micropipettes to the membrane of a neuron in order to measure the electrical activity going on inside that neuron.  We also use these micropipettes to fill some neurons with dye for experiments that generate the colorful images I’ve been posting each week.  Here’s some of the pipettes info I send Richelle:

Here’s my pipette box. These pipettes (five here) are the little glass tubes that I attach to the membrane of a neuron to “patch” the cell and fill it with dye while recording the cell’s electrical activity. In a good day, I’ll use three or four pipettes – one or two to patch neurons, and two to send electrical stimulation into the neurons. In a bad day, when these break (they’re really small and fragile), I’ll go through ten or more pipettes. We make new pipettes each morning. Old ones are used to send electrical stimulation into neurons, and new ones are used to patch cells and fill them with dye.

Here’s an even closer view of one pipette. The pointy tip is open on the end, and the opening is about 2-5 micrometers in diameter. The portion of a neuron that I patch with this is about 17-20 micrometers in diameter. I actually held my iPhone up to the eyepiece on a microscope to take this photo. ☺ The black bar on the bottom is platinum wire that heats up and to fire polish the sharp glass edges of the pipette so they’re not so sharp. This helps ensure that the pipettes won’t tear the cell membrane.

This photo is sideways….tilt your head 90º to the left. Here, I placed a pipette over the tiny platinum wire to fire polish it. The large lenses are attached to a microscope and help us see how the shape of the pipette tip changes as we fire polish it.

​Check back next week for another update.  Cheers!