Imaging

These are not results, rather they are only the very beginning of figuring out how to get results. But they’re promising and pretty.

What I am trying to image, is the longitudinal extension of sensory afferents in the neural tube. The cell bodies of these axons live in little clumps all up and down either side of the spinal cord, and they project towards the mid-line (eventually into the grey matter of the cord itself) and towards the periphery (so you can detect sensations like pain, temperature and pressure). In the embryo, chicken, these cells develop in the ganglia and then grow into the cord. But before they grow in, they grow rostrally (north) and caudally (south) in neural tube. How this growth progresses normally, and what effect inhibited or enhanced cAMP (part of cellular messaging systems) has on the growth – these are the things I am trying to understand and visualize.

With that in mind, the little branches at the bottom are what interest me. The big blobs at the top are the dorsal root ganglia, painstakingly labeled with DiI in live tissue, and then preserved. The extensions out of the blobs are the dorsal roots extending into the neural tube, and the branching at the bottom appears to be the longitudinal extension of these axons within the neural tube.

So, the theory goes, if I can get these pictures, I can get better pictures, live pictures (skipping the preservation step), and eventually sort of a movie of the growth in real time using that fancy microscope. Right now, it seems like it is not an impossibility; and in truth, I am looking forward to giving it a shot.

Here is my favorite. The clearest globe-like ganglion with it’s little roots and longitudinal extensions just streaming out, reaching for those other segments. Enjoy!

Microscopes

Dorsal root ganglion, all by its lonesome.

Now that my main job is microdissection, I spend a lot of time using different microscopes. Only in passing will I note that most microscopes in my lab come with notes on them to remind us all of the basics of using anything; “turn off after use”, etc. And yes, I do have a favorite scope – the Leica Wild 10 in the embryology room. It doesn’t slip out of focus, is located in a room where I can turn off all the lights and shut the door, has appropriate levels levels of magnification (because I need to dissect little tiny things!), and nice functioning adjustable lights

Although I love the Leica Wild, it may soon be replaced with a new winning scope. Last week I went down down down into the basement and visited the BioRad MultiPhoton microscope for the first time. This microscope is so cool that it exists in a private room jammed with what I can only describe as tons of complicated stuff and what seems to be one helluva computer. According to the word on the street, this baby cost a ton of money . On top of that, it requires a full time guy (appropriately bearded and outfitted in a seemingly endless series of tucked in flannel shirts) to teach people like me how to get images out of it.  This guy is not a tech (Editing from the future: I soon figure out he does MUCH more than just teach people like me to not break expensive equipment). He’s the director of Imaging and Physiology who helps teach classes with titles like “Three-dimensional Cryo Electron Microscopy of Single Particles”. And he has a wife and a two year old child – obviously amazing.

But as much as I would like to figure out exactly how one becomes expert in operating such machines, and in the process of three-dimensional cryo electron microscopy of single particles, while still finding the time and inclination to procreate – it will be a while until I get to take my little pulsating blobs of living tissue down  to make movies of their insides. Crawling before walking… and really, before I even think about crawling I have got to flip myself over onto my belly and turn on some primal brainstem and spinal cord pattern generators to see if they’re working.

In terms of the project, this means I have to get the live tissue dissected out and properly labeled to see how certain axons grow during the different stages of development. Today was devoted to getting that live tissue dissected out. This involves taking one of those gorgeous embryos, removing it from the egg, eviscerating, decapitating, and “blocking” it so that only the spinal column (with T4 carefully marked) remains. Then begins the rather painstaking task of removing the neural tube, with dorsal root ganglia and (hopefully) dorsal roots attached. Amazingly, after plenty of frustration, and the acquisition of my very own personal pair of very pointy number five forceps, I was able to do it three times. And at five pm, my difficult to please mentor said “That’s nice and clean -but maybe too clean, you’ve probably severed the dorsal roots”.

Ah, science. Frustration, trial and error, and very picky people who notice every little thing. But, in any case, I left the lab floating on a minute cloud of satisfying accomplishment. Ready settle into the evening with a review paper on live imaging, or at least to carry it with me from room to room with good intentions.

Embryos

I called an old friend tonight and blurted out, “I started my job today and I’m experimenting on animals.” And it’s true, I am – but until I said that to her, it had not occurred to me to think of it that way. Today was my first day of my first job in science. I will spend the next few weeks learning to dissect chick embryos, “window” eggs, and use a microscope properly. As someone who has restricted and unrestricted her diet in many ways, “I started my job today and I’m experimenting on animals”, was not a sentence I ever considered as a possible future utterance. I have read the books, joined the organizations, I thought I believed in animal rights. But apparently, some cognitive dissonance needs sorting.

Big girl was my first dissection, a cat, for my anatomy and physiology class at community college last year (when I first returned to school). Half of the A+P sections worked with clay models, the other half did the more traditional cat dissections. Everyone did the sheep brain and cow eye. And luck of the draw had me in a cat section. Faced with an embalmed cat, my beliefs were out the window – I wanted to learn the anatomy, and told myself a convenient lie that the cats were euthanized from shelters. Still can’t figure out what part of my brain made that up. But, however flawed and false my rationalizations, there is no denying that I learned from Big Girl. Named by Ana, one of my Polish lab partners who refused to touch her, I became intimately acquainted with the muscles, nerves, organs and blood vessels of this large female cat. I liked working with my hands, learning with my hands – and I can say confidently that a cadaver would have been better, but the cat taught me all of the foundations.

By saying that a cadaver would have been better, does that somehow excuse me from participating in the death of this cat to teach a bunch of community college kids anatomy? I guess that’s my way of qualifying – sure I would have learned from a human, why don’t you donate your body? But, by calling that class a bunch of community college kids, I am oversimplifying and not giving them or myself any credit. Everyone who finished that class wanted to work in health care. The majority will be LPNs, nurse’s aids or RNs, some will become NPs, PAs or doctors. I can only assume that those cats gave the rest of the people in the class a similar foundation, and what’s that worth? At the risk of dramatizing, what’s that worth when you’re in an ambulance?

Well, this question will not be resolved easily, or through monologue. Back to the embryos. They are small, really tiny. Surrounded by yolk and other membranes that I can not yet name, they are so perfectly perfect when you get up close to them. It is a shame to poke at them with blades “dull as ditch water” according to Betsy. They are also somewhat colorless, aside from the blood, making texture the main distinguishing feature between tissues. I tried and failed to extract three embryos from their yolks. I saved two more eggs for tomorrow, hopefully sleep will refine my motor skills.

First days are first days. And, philospohical concerns aside, hopefully these embryos will teach me as much as Big Girl did.

Chick Embryo

The coolest dissection

This brain is dissected so that the optic radiations are visible. The pretty swooshes are bundles of axons that go from the (lateral geniculate nucleus of the) thalamus to the visual cortex in the occipital lobe. Interestingly, because things get all backwards and upside down in the brain, damage to the most inferior parts of the loop results in an upper quadrant loss of vision in the opposite eye.

Slices

Coronal Slice

Here is a big thick slice. This a coronal slice (think parallel with the front of the body , down through the middle) which illustrates beautifully the grey-white-grey organization of the cortex, tracts, and basal ganglia (gray stuff beneath the surface). The grey areas contain more cell bodies, the white areas are jam packed with myelinated axons (which function sort of like insulated wires) connecting cells to other cells

Slice is the cool-kid word for cross-section of brain. If you’re preparing a slice for the microscope, it’s gonna be thin, really thin, so that the light can shine up through it and you can see cells. Think about the time you spent working at the deli, how some people wanted their cold cuts really thin, and others wanted them weirdly thick. Microscope bound slices of brain are ideally only a few cells thick. Slices bound for gross anatomical identification, to sit out on a table and have undergraduates poke at them, are going to be thicker.

Learning the brain, brain stem and spinal column through slices seems to me to be a little bit like learning to drive. Before I got a learner’s permit, I never paid much attention to where things were in relation to other things. But when I began driving, a series of ephinaies “Ah hah! Making a right at the end of Beekman Ln brings me to the Middle School!” taught me how everything was connected. Rather embarrassingly, in my grandfather’s town, I knew the bagel store, (named, in some stroke of genius, Two if Buy Bagel) and I knew the supermarket, but I did not realize until I had a drivers license that they were actually on the same road, only about a mile or two apart.

This may speak more to a certain kind of detached and spacey childhood than anything else.  But to learn the brain and brain stem in slices is a process of viewing independent, floating segments of a big and interconnected mess (one that really would never naturally be seen in isolated pieces) and trying to reconstruct the roads in and out of the place from you memory of the thing as a whole.

Ah hah, this tract veers laterally because these other tracts appear.  Codes and latin names.

My favortie mnemonic, for structures lining the fourth ventricle at the level of the Rostral Medulla; Has Death Stopped Vice In Central Park? Hypoglossal nucleus, DMX, Solitary nuclues, Vestibulochchlear complex, Inferior Cerebellar Peduncle.

Now, how to pull it together for the next exam.  I always make the stupidest mistakes.