I want to diverge for a moment before I get to the titular issue of the piece.
I have begun to see what may be a key part of science writing, and it is a key part of life. If Shimer’s ethos has taught me anything, it is that admitting when you have no idea what is going is not only okay; more often than not, it is necessary.
This has proven true in more personal situations than would make sense to number; it has done me well in the lab, like a little daemon hissing in my ear to ask and not mess something up. What I am about to go into I would like to begin with a disclaimer:
Here, I have no idea what I am talking about.
But Shimer has also given me the confidence to take graduate classes, read publications meant for medical and academic professionals, and speak my mind. So I want to give you all my best guess.
We have been getting some weird results with the NeuN stain. Not bad, as in the procedure is wrong, or being done wrong. They are just weird.
NeuN looks for a protein found in the nucleus of neurons, sometimes found in their cytoplasm as well. In these progranulin-mutant cases, we can see neurons (mostly their nuclei). But there is a crazy amount of background…and a lot of stain-aggregates that shouldn’t be nuclei.
At first I thought it was the embedding, or an indicator of neuronal loss. I still think the latter will be a major factor to consider, but the former I believe is null after reading papers which cite successfully stained paraffin embedded sections; also our control sections (which worked as expected) were paraffin embedded.
I compared these NeuN stains to another, Cresyl Violent (CV), which also stains neurons. Looking at CV-slides from the same tissue, the weirdly stained areas do contain neurons. But the CV didn’t stain the little dye-aggregates we’re seeing in these PPA with progranulin mutant (PRGN) cases.
Up to this week, we had seen them in all locales and both hemispheres…
But this week, we got to a case from 2003 where this laterality of weirdness didn’t hold up - the left side looked the same as all of the other PPA-PRGN NeuN stains. The right, however, did not.
The right looked like controls. This difference, generally, holds for all I have seen of the thirty sections we have done so far in this 2003 case. The inferior frontal and superior temporal (both areas implicated in language) hold this pattern to a “T”. The hippocampal and enthorhinal cortices displayed a bit more control-like look in general.
This would be consistent with a profile like PPA’s where language deteriorates while general mnemonic function is spared.
There has to be a lot more looked into, but preliminarily, I can guess that there may be that there is some sort of proteinopathy related to NeuN, or that NeuN’s expression is being affected by another, already identified, FTLD pathology.
If some gross pathology (large-scale) was present in the left hemisphere in this case (i.e. the right was mostly spared) or the patient exhibited a shorter disease course, the hemispheric difference in NeuN staining may indicate… something. But it is exciting.
This is what is so important about being hands-on, and involved. I don’t know what is going to happen with this difference, but I am sure there will be much to discuss with my supervisor come Monday.
There hasn’t been much change. I have learn how to do the immuno stains, and even done one by myself (and didn’t completely screw it up).
It goes something like this (for the NeuN stain I have been working with - it stains for neuronal nuclei, hence the abbreviated name) :
-Dry the tissue
-“Retrieve” the antigen (in a very expensive pressure cooker)
-Cool and wash (washing is done in biologically neutral solutions and de-ionized water)
-Dip in methyl-peroxide (I forgot this step on my on-my-own … luckily it was tissue already drained of blood, which this step “blocks” for)
-Block (stop the antibody from binding to things you don’t want to see)
-Apply the primary antibody (these proteins bind to what it is you’re looking for (presumably) and then you bind other stuff to them (see below) in order to make the targets visible to the human eye. You “block” these proteins (that will eventually be dyed) from binding to “background”, like blood)
Wait 20 hours…
Apply secondary antibody
Apply Streptavidin Peroxidase
Develop (Apply the color)
Cover slip (imagine shiverring as this is said. Cover-slipping is hard)
Look at ‘em under a microscope!
So that has been my life the last few weeks. It makes for a minimum four day week, with about 4 hours minimum necessary to complete each days task. We do twenty slides at a time. In total, so far, we have stained about 140 slides. We have about that many left to go. We hope to be done sometime in August. (The latest case we are working on, from 2012, has to be stained for a number of other pathological markers…more on that at a latter date).
What am I doing with the rest of my time? Learning to count. If it sounds funny, it should, but really (for those doing it), counting is no laughing matter.
In order to statistically classify numbers of things of interest on very thin sections of very small portions of tissue (brains), one can use a program such as the one we are fortunate enough to have. This processes is called “stereology”, and it is very tedious. The computer, with your help, is able to section a given section into a grid, in which a number of sites for averaging are chosen. The computer is then able to generalize these results as more and more data is fed into it.
Our project is comparing neuronal loss in one hemisphere vs. the other. The expected results, based on the current left-hemisphere language hypothesis (based on studies including almost all aphasias), is that there will be significantly farther degradation in the left hemisphere, especially in parts related (mostly via localization of lesions in aphasics) to language. This will be done during the end of our staining rounds and after (hopefully to be completed by sometime in September??).
Acetylecholinesterase negative stain of basal forebrain in a super-ager. Very clear large (pyramidal?) neuron visible in first, bipolar neuron barely visible in second.
(The first has to be the coolest picture I have ever taken.)
Cresyl violet stains of a 5micron thick hippocampal section of a PPA patient with progranulin mutation. The large neurons with the highly visible axons are white matter: the smaller ones in the gorgeous swirl pattern are grey matter. Grey matter indicates a high load of local connections, and is given its colour by cell bodies. The larger neurons of white matter travel farther, and the insulary material around their axons (out-going processes), myelin, is lipid, or fat, based, and therefore white.
The fairly indistinct and smaller aggregates of violet are cells called glia. They are as numerous as neurons, and play at least a supportive, if not quite active function.
So, I started this week. It was really everything I could have hoped for.
The week opened with a lab meeting, where all of the different projects within the Cognitive Neuroscience and Alzheimer’s Disease Center (CNADC) converge to share their progress.
This was a perfect opportunity to meet everyone in the lab, and find out what was going on. Many of the projects are worked on by small groups, teams, that allow the work to be spread across a couple heads, and sometimes a couple days.
The project I am working on is investigating the effects of a certain genetic mutation (progranulin) on individuals with Primary Progressive Aphasia (PPA).
FTD and PPA
These cases of PPA displayed Fronto-Temporal Dimentia (FTD, a clinical diagnosis) which is associated with Fronto-Temporal Lobular Degeneration (FTLD, a pathological diagnosis). The fine line between clinical and pathological is one I am learning slowly to walk.
Though the cause is largely unknown, the clinical-pathological profile of FTD patients is generally a dementia with an early onset ~(35-55 years of age). The earliest symptoms including loss of inhibition, personality changes, executive dysfunction, social and attentional issues, and language loss.
The disorder can cause severe confusion and emotional distress for those close to the affected. In the course of a few years, family and friends are often thrust from a normal relationship into a very traumatic period, where their loved one undergoes unexplained and jarring changes, and are finally left a caregiver for a relatively young individual.
Primary Progressive Aphasia could be viewed as a type of FTD, though this isn’t quite correct clinically. PPA has its own specific clinical diagnostic criteria, including language loss without amnesic effects, retention of general cognitive abilities (maintenance of routines, etc), all preceding normal dementia symptoms by a few years (these criteria are still being specifically outlined).
Perhaps the best way to delineate PPA is how its founder (a leading member of the CNADC) thought best : (P)rimary - the effects on language are the presenting symptom; (P)rogressive - the effects are seen to grown worse globally; and (A)phasia - the classical clinical diagnosis of loss of language function. PPA could be summarized as a clinical profile of language loss that gets worse in the style of dementia. It is almost always associated with some dementia, the majority of cases being FTD and the remaining either AD or Parkinson’s related.
Progranulin, a gene with a number of distinct and not completely understood functions, fits in to the equation as a heavily reliable genetic indicator of familial FTD and PPA. The project I am currently working on has a goal of quantifying neurons in specific “regions of interest” (ROI’s) and seeing if neuronal loss/atrophy correlates to current models of the brain’s “language network”.
After finding out what everyone else was up to, it was off to the races. I learned how to do my first (of many) immuno-histological stains, using a particular anti-body called NeuN. It is intended to stain certain nucleic proteins in neurons, though there have been some challenges in pathological cases (more to follow). I also learned tons of lab protocol, how to use many different pieces of equipment, on top of getting acquainted with my lab-mates.
It was a whirl-wind, (especially getting up at regular hours!), but it has only queued me up for this week even more.
Aristotle discusses two causes in his Metaphysics (if I do remember correctly); proximate and ultimate. I suppose I want to answer both the how and the why in light of this division.
I woke up one morning this week to an email for which I had waited quite sometime. It was my supervisor finally informing me that all administrative hurdles were cleared, as was I to start this coming week in the laboratory.
I have wanted to work in a neuroscience lab for quite some time now. I was fortunate enough to meet a wonderful professor through a course I took at the Illinois Institute of Technology (IIT or Illinois Tech) who was generous enough to take a liberal arts student on at his lab.
Ultimately, however, I believe it is the training that has been afforded to me via Shimer and IIT that has led me here. While it hasn’t trained me for the specific chemical titrations and stainings I will be doing (maths was my subject at IIT), it has prepared me for something much more important; namely, the ability to pick up a skill as needed.
Many may wonder how a liberal arts program prepares one for such tasks. Its not the “liberal arts” that does so. It is the critical skills garnered through classes set up around a text, with challenging and enriching dialogue created by both faculty and student participants alike.
Shimer has provided me the ability to walk into a 400 level neuro-psychology class, and then a grad level neurology class after, with the confidence that reading the assigned text would be enough to have a serious discussion about it. This showed my professor that, while I was lacking particular technical skills, I would be able to perform both as a technician and participant to dialogue. I think such a skill is invaluable for all thinkers, doers, and citizens.
There are a number of subjects whose domains change the way I would answer this question.
…this particular lab.
The Cognitive Neurology and Alzheimer’s Disease Center (CNADC) provides me with the ability to develop technical skills that deal directly with the interlocking space between anatomy and language while working with dementias, all rapidly growing public health concerns and areas of research. In this sense it provides both materials and space to work with, with an overarching moral and ethical telos.
To be quite honest, because it is here. It offers a range of customization and linking options that will hopefully be as useful and pleasant for readers as for myself. Though I have had to do some major brushing up on html, this has given me a project to keep me busy between beings-in the lab. The Shimer Summer Internship/Mentorship grant asks participants to keep an online record of the internship: this page is such a record. But hopefully it provides some good reading as well. I do not have any other social networking sites (besides professional: See About), and therefore cannot guarantee the kairos of anything I post (See this post).
So, proximately, I can say that it is this internship for which I am writing. But, ultimately, I do have some ulterior motives. Nothing too insidious, as a matter of fact, I hope more helpful than anything.
It seems sometimes that one can tack neuro- or cognitive onto the front of an educated-sounding phrase (e.g. “neuro-plasticity”,”neuro-aesthetics”), and garner an instant, almost mystic, reverence, far outside the technical boundaries it was ever meant to be used within. I hope that by explicating some on work being done in modern neurolinguistics, as well as on some more foundational ideas of modern neuro-psychiatry and cognitive neuroscience, I will not only be able to contextualize some recent advances for a general audience, I will also provide myself with a sandbox in which to play with ideas for my upcoming Shimer senior thesis.
To these ends, near and far, please, enjoy reading.
Peace, love, freedom, and truth,