At one point in time, someone conceptualized the brain as a clock,
another as a telegraph,
somehow, someone else was reminded of a hydraulic pump.
But we’re done with those silly metaphors,
especially that weird pump stuff.
Now we know,
brains are electrical things,
like the bits in computers,
no, like bits in a quantum computer (Lit, 2006),
yes, definitely quantum computers.
Conceptualize it how you want,
but the brain is still an organ,
still a collection of cells.
Inside each of our hundred billion brain cells resides tens of thousands of proteins,
floating in a soup of tens of thousands of smaller molecules,
weaving in and out of cellular organelles,
navigating up and down a meandering axon,
executing the rapid ion fluxes that make the neuron fire,
performing all of the the various tasks of the neuron.
Written & Illustrated by Andrew Neff
15 minute read
Biological treasure chests
primed to revolutionize biomarker research?
Brains are electrical things.
If you want to move your finger,
you could stick an electrode into your motor cortex,
and watch your finger flick.
But voltage isn’t everything.
Molecules underlie all the things cells do,
and variability in their function can impact important biological processes
including regulating the connections a neuron makes,
and the strengths of those connections.
To get a complete picture of the brain,
we can’t ignore molecular biology.
And if we’re going to study molecular neurobiology in 21st century style,
Our goal is to be able to see each individual molecule,
at molecular scale spatial resolution,
and in real time.
All things, in all places, at all times.
Ridiculous, not in our lifetimes, right?
Miraculously, in the past couple decades
some of the most beautifully simple yet sophisticated technologies have been developed,
and are slowly marching us towards all things in all places at all times.
Technologies like next generation sequencing let us simultaneously profile
millions of genes in a single reaction,
rendering the task of knowing complete human genome amateur stuff.
Cooler still are single cell sequencing technologies:
In one technique, individual cells are trapped in a droplet of oil,
elucidating a complete set of data on the DNA or RNA
of each and every cell in a mixed sample (Macosko, 2015).
Or even more mind-blowing and forward thinking are technologies like MERFISH,
a microscopy technique providing researchers with
the sub-cellular location of thousands of RNA molecules in individual cells (Chen, 2015).
Technological progress in molecular biology is proceeding at an unprecedented rate,
and scientists are just itching to apply these technologies to the human brain.
Dashing our hopes, however,
is the sad but inevitable fact that our brains are just not available to do research on.
Cells? sure. Rats? I guess so. But humans? No thanks, science.
But, couldn’t we just take a little teeny tiny chunk of brain tissue?
What if promised that no harm would come to you?
Or, what if I said we were going to take a piece of your brain,
without actually taking a piece of your brain?
Years ago, we discovered that cells secrete small vesicles called exosomes.
Packed with molecular cargo from their parent cell,
exosomes journey into the bloodstream,
where they can be harvested with a simple blood draw.
And if researchers are right,
we might be able to separate these vesicles by their tissue of origin.
Researchers from disparate fields are enthusiastically
adopting the exosome as their window into their favorite organ,
their treasure chest overflowing with molecular delights.
Cancer researchers think that we could study exosomes
as a way of detecting the presence of a tumor.
Neurologists think we could collect exosomes
to monitor the status of neurodegenerative proteins.
Could the contents of exosomes really provide us
with any indication about the way our brains are functioning,
and maybe even deeper questions about who we are?
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Molecular technologies in neuroscience
we’ve already got a few technologies to assess the molecular properties of the brain,
why not just keep going with what we’ve got?
what have we got again?
Murder and Dissection
We could recruit a group of people,
take their brains out, slice em up,
and use any of the wonderful molecular biological technologies
to identify which genes and molecules are present.
One way around the limitation of cold-blooded murder is to take already dead people,
slice up their brains,
and look at the molecules in there.
Famed neuropathologist Will Smith discovered that
at least one person who played professional football
had an abnormal brain pathology characterized by
amyloid plaques and tau tangles,
markers usually present in the brains of people
who have suffered from neurodegenerative disease,
and are usually several decades older (Omalu, 2005).
The biggest issue is that dead people aren’t usually doing anything very interesting.
Without living brains, you can’t track how the molecules change as people do.
This method could be useful to look between individuals at some “lifelong” traits,
to find correlations with psychiatric disease (Shelton, 2011),
or determine whether someone has played professional football,
but not too much else.
PET stands for Positron Emission Tomography.
It’s all in the name, sort of.
Positron Emission is what happens when a chemical isotope decays,
it’s the signal that researchers detect.
Tomography means you can track where this decay occurred
to produce an map, or an image.
The cannabinoid receptor
When you smoke pot, you’re inhaling THC,
through the lungs, into the bloodstream, and up into the brain,
THC find itself a cannabinoid receptor to bind to and the magic starts happening.
Say you want to know where in the brain you can find the cannabinoid receptors.
Using PET imaging,
Your first step is to create a molecule, or “tracer” with the following two properties:
First, it has to contain an isotope that is unstable enough to decay over a reasonable timeframe,
an hour or so should do the trick.
Second, the tracer should have a specific affinity for the cannabinoid receptor.
As the tracer is infused,
it travels throughout the body and eventually into the brain.
Some of the tracers will find a receptor to bind to,
in this case it will stick around a while.
Those that don’t have the good fortune of finding a receptor
will eventually be washed out of your system.
At this point,
you can snap a picture to see where isotope decay is occurring,
and therefore where the cannabinoid receptors are.
PET is an extremely important technology in neuroscience.
It has provided us with detailed molecular information about the living adult brain in unprecedented detail.
But there are serious limitations.
First, the technique can’t localize the signals very precisely.
Analyses are performed on chunks of tissue containing
hundreds of thousands of cells (Moses, 2011).
This would be a bit like trying to understand how to play chess,
except instead of seeing each piece and each square,
you could only see the average of a four square block,
which would be weird, probably uninformative,
and I can’t figure out how to draw it.
Another problem is that the images take a while to acquire.
To locate where the cannabinoid receptors are,
this isn’t really an issue, they probably don’t change their location that quickly.
But many other processes happen far too fast to be observed with PET imaging.
Imagine you are an alien trying to understand how humans swim.
You decide to set up a video camera,
but instead of recording at 20 frames a second, producing a continuous image,
you record at 1 frame a second,
and observe a flickering brown object travelling across the pool.
You see movement,
maybe you can differentiate strokes where the swimmer
is right-side up or upside down,
but there would be no way of understanding arm or leg strokes.
The last problem is that we can’t see all the molecules.
Developing these tracers is extremely difficult and expensive.
But even if we had a tracer to detect every molecule,
the technology still can only be used to detect one molecule at a time,
which is not really the way cells do things.
Instead of designing specialized molecules
to track down and tag human proteins,
we may instead be able to detect a set of naturally occurring molecules
simply based on their magnetic properties.
Packed inside a synaptic vesicle in the terminal bouton of a presynaptic neuron,
glutamate lays in wait of an electrical pulse.
When the pulse arrives, the vesicle docks with the cell membrane,
releasing glutamate into the synapse.
After a short period of diffusion,
some of the glutamates find a receptor on the postsynaptic cell,
triggering the opening of an ion channel,
and an electrical pulse in the post-synaptic neuron,
further propagating the signal.
Glutamate is the most widespread and abundant neurotransmitter in the brain.
To track this molecule in living brains
would be to track most of the things the brain does,
and could provide neuroscience with unprecedented,
non-invasive access to human molecular brain function.
When placed in a magnet,
hydrogen atoms on glutamate align along with the field to minimize their energy state.
Once the atoms have aligned,
infusing energy into these atoms will trigger the atoms to realign against the field
in a higher energy configuration.
Over time the system randomly decays,
realigning hydrogen along with the magnetic field,
back to the low energy state.
As hydrogen releases energy,
each hydrogen atom will do so in a characteristic way,
allowing us to identify which molecules are present.
Some molecular signatures are too similar to distinguish,
and most molecules aren’t abundant enough to detect.
But there is a group that are unique and abundant,
and glutamate happens to be one of them.
Some molecules aren’t really built for PET imaging,
especially smaller ones like glutamate.
MRS allows us to directly assess some of those molecules,
with the added bonus of not having to use radioactive isotopes (Stanley, 2018).
But like PET imaging,
or any non-invasive imaging methods,
a particular set of limitations applies.
Towards the middle of your brain lies a structure called the dorsal anterior cingulate,
which involves itself in processes like attention, and decision making.
Tens of thousands of neurons of variable morphologies,
variable physiological characteristics,
different connection partners,
and most likely differential psychological correlates.
The best resolution MRS can achieve is about the size of a grape,
which happens to be about the size of the dorsal anterior cingulate.
all of that wonderful complexity of the anterior cingulate
is reduced to a single measurement.
In five milliseconds,
ion channels open, allowing sodium and potassium to rush across the membrane,
triggering a voltage change that sweeps through the dendrites,
into the cell body, down the axon and to the synapse,
where glutamate filled vesicles are triggered to fuse with the membrane,
dumping their contents into the synapse,
and inducing an action potential in the next neuron.
The release of glutamate happens in milliseconds,
while image collection happens in seconds.
Thousands of action potentials,
merged into a single reading.
Lastly, the brain functions by precisely coordinating the release of
hundreds of signalling molecules,
with tens of thousands of molecules inside the cell supporting those processes.
Unfortunately, MRS is only sensitive enough to detect five or ten different molecules.
While non-invasive brain imaging technologies have
revolutionized the way neuroscience is done,
and while progress continues to be made,
we are still far from the goal of measuring
all things, and high resolution, in real time.
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a biomarker for the 21st century
If you don’t mind getting just a tiny bit invasive, then we’ve got some options.
You probably wouldn’t want me to scalpel out a chunk of your cerebellum,
but, while you may find it strange and creepy,
you might not mind if I take a nail clipping, or a piece of your hair,
or a urine or stool sample, or even a few drops of blood or cerebrospinal fluid.
Your brain releases all types of molecules into the environment.
Cortisol, the stress hormone,
can be found in hair, nails, blood, everywhere it seems.
So, couldn’t we collect a sample, minimally invasively,
and see whether people with different psychological profiles
exhibit differences in the chemical composition of peripheral fluids and tissues?
We could, and we do,
but there are serious limitations to this approach.
Given that samples like blood interact with every tissue,
perhaps the biggest limitation is knowing where a molecule came from in the first place.
Serotonin based signaling mechanisms are present in the animal kingdom,
because of this it’s thought to have ancient evolutionary origins.
As may be expected of a molecule that’s been with us before we were human,
the distribution of serotonin in the brain is widespread,
and it’s impact on brain function is pervasive.
Of particular relevance to us today
is serotonin’s relationship with mood, sleep, appetite and food intake.
To directly measure neuronal serotonin in humans would be a huge advancement in neuroscience.
And what do you know,
serotonin is in your blood, a whole bunch of it.
But serotonin isn’t just a signalling molecule in the brain,
it’s also a major signalling molecule in the gastrointestinal tract,
and plays a large role in how platelets function in the bloodstream.
Changes in serotonin concentrations in peripheral tissues
could be attributed to changes in any of these original tissues.
But what if there was a way to take a sample,
say from blood,
and partition out the contents.
So we could know, this serotonin came from the intestines,
this serotonin was from the platelets,
and this serotonin came from the brain.
That would be a nice trick wouldn’t it?
Here’s a Neuron
Sometimes, the membrane of the endosome will start budding in,
creating a new, internal, membrane bound structure.
As the internal structure is being formed,
molecular contents from the environment are sucked inside.
Inside your cell are organelles, all of which bounded by a fatty membrane.
Containing your DNA, there is the nucleus,
producing energy is the mitochondria,
collecting and processing newly formed proteins
is the endoplasmic reticulum.
And then you’ve got an organelle called the endosome,
which functions as a reliable molecular sorting center.
When a group of molecules needs to be removed from the cell,
it secretes them.
Another group needs to be degraded,
it ships them off to the lysosome for dismantling.
Sometimes an endosome will fuse with the cell membrane,
releasing its contents,
including what are now called exosomes,
to begin their fantastic voyage.
Exosome may travel nearby,
merging with a neighboring cell membrane and dropping off its contents,
or it might travel great distances,
encountering trials and tribulations,
performing tasks largely unknown.
Like other cells, neurons secrete exosomes (Faure, 2006),
and when stimulated, they tend to release more of them (Lachenal, 2011).
Exosomes can cross the blood brain barrier, at least going into the brain (Alvarez-Erviti, 2011).
Then came the real breakthrough.
The internal molecular contents of exosomes
reflect the molecular contents of the parent cell,
but it’s not just the molecules on the inside.
Some of the membrane-spanning proteins
on the outside are specific to the cells they came from.
If you could find a membrane protein
that’s unique to exosomes derived from particular cell types,
you could isolate your exosomes by that marker,
then break them open, spill out the contents,
and see what sort of brain derived goodies are in there.
From a set of blood samples,
researchers isolated the subset of exosomes studded with the membrane protein L1CAM,
and found the molecular composition of these exosomes reflected
the molecular composition of the subjects brains they were studying (Shi, 2014; Fiandaca, 2015),
supporting their hypothesis that L1CAM-exosomes are brain-exosomes.
Immunoplates for Neural-derived Exosome Isolation
After collecting your blood sample,
start by removing blood cells and cellular debris
by using multiple rounds of centrifugation.
Once you've removed the solids,
you are now ready to isolate exosomes.
You will be supplied with a plate containing wells
that are pre-coated in antibody for neuronal exosomes.
Simply transfer your sample into these wells.
Wash off whatever isn't bound to the plate
and voila, you've got yourself neuronal exosomes.
From here, all you need to do is break open the exosomes
and see what's inside.
To test whether the "neural-derived exosome" plate
actually collects exosomes from neurons,
instead of starting with blood, researchers started with cells,
either neurons, glial cells, colon cells, or blood plasma.
When the starting material was neurons,
the neuron plate yielded quite a bit of material,
but when the starting material was anything else, the neural plate yielded very little.
Where exosomes are going to fit in
Faced with similar obstacles,
researchers from disparate fields are consulting exosomes
to provide them with confidential molecular information on their favorite organ.
It’s exciting when someone recognizes the opportunity to learn new information about human biology.
But before we all leap headfirst,
it’s worthwhile to take a critical perspective on what information exosomes stand to provide us.
Otherwise, if it turns out the info isn’t that useful,
we’re going to have to learn the hard way,
that is, the time and money wasting way.
The human brain, is, well, complicated.
It’s comprised of a spectacularly diverse set of cells,
synthesizing and releasing different neurotransmitters,
generating a unique array of connections,
exhibiting characteristic electrical and physiological properties,
and sometimes relating to distinctive cognitive and behavioral functions.
it’s likely that we can identify molecules that originated in the brain,
but there’s not yet evidence we could use exosomes
to provide us with any better resolution than that.
How about molecules from the hippocampus, or the prefrontal cortex?
Whether exosomes from these cell populations have distinctive membrane proteins is not known.
The brain is incredibly dynamic, and works on a range of timescales.
At extremely fast, millisecond timescales,
neurons perform the operations we’re most familiar with in our lives,
at these timescales we perceive and respond to the world around us.
Exosomes just aren’t produced and secreted fast enough to track anything like this.
However, we’re all familiar with processes that occur over long durations of time,
perhaps experiencing a psychiatric episode, or remission from it.
This is where exosomes may stand to provide interesting and new information.
The last obstacle to exosome research is that
exosomes aren’t completely randomly swallowing up molecules in the cell,
there’s a bit of selectivity there.
A lot of things show up in exosomes,
proteins, RNA, DNA, amino acids,
something from every class of molecule,
but still, not everything finds its way into the exosome (Mathivanan, 2009).
Clinical research in human populations is tremendously challenging,
partly because the range of samples researchers can use is very limited.
In response to these difficulties,
so much of research today is conducted on animals.
With a rat or a mouse,
we can use high risk, high harm, experimental procedures.
We stick electrodes into brains to record neurons,
or dialysis probes to profile molecular environments,
and once the experiment is done,
we can asphyxiate and decapitate the animal, and directly study the tissue itself.
If research continues to support the concept of exosomes as a biomarker of the future,
instead of studying animals whose psychology and behavior is of questionable relevance humans,
we may have an opportunity to apply the spectacular tools of molecular biology to human samples.
Exosomes are not the end,
not even very close,
there’s a ton of information we still won’t have a way of detecting.
Exosomes may, however, provide us with with lots of data that we’ve never seen before.
As a compliment to the existing set of technologies,
depending on what turns out to be true about biology,
we might be able to come to a deeper understanding of the human body,
and maybe even the human brain.
Alvarez-Erviti, Lydia, et al. "Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes." Nature biotechnology 29.4 (2011): 341.
Chen, Kok Hao, et al. "Spatially resolved, highly multiplexed RNA profiling in single cells." Science348.6233 (2015): aaa6090.
Fauré, Julien, et al. "Exosomes are released by cultured cortical neurones." Molecular and Cellular Neuroscience 31.4 (2006): 642-648.
Fiandaca, Massimo S., et al. "Identification of preclinical Alzheimer's disease by a profile of pathogenic proteins in neurally derived blood exosomes: a case-control study." Alzheimer's & dementia: the journal of the Alzheimer's Association 11.6 (2015): 600-607.
Lachenal, Gaelle, et al. "Release of exosomes from differentiated neurons and its regulation by synaptic glutamatergic activity." Molecular and Cellular Neuroscience 46.2 (2011): 409-418.
Litt, Abninder, et al. "Is the brain a quantum computer?." Cognitive Science 30.3 (2006): 593-603.
Macosko, Evan Z., et al. "Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets." Cell 161.5 (2015): 1202-1214.
Mathivanan, Suresh, and Richard J. Simpson. "ExoCarta: a compendium of exosomal proteins and RNA." Proteomics 9.21 (2009): 4997-5000.
Moses, William W. "Fundamental limits of spatial resolution in PET." Nuclear Instruments and Methods in Physics Research Section A: Accelerators, Spectrometers, Detectors and Associated Equipment 648 (2011): S236-S240.
Omalu, Bennet I., et al. "Chronic traumatic encephalopathy in a National Football League player." Neurosurgery 57.1 (2005): 128-134.
Shelton, R. C., et al. "Altered expression of genes involved in inflammation and apoptosis in frontal cortex in major depression." Molecular psychiatry 16.7 (2011): 751.
Shi, Min, et al. "Plasma exosomal α-synuclein is likely CNS-derived and increased in Parkinson’s disease." Acta neuropathologica 128.5 (2014): 639-650.
Stanley, Jeffrey A., and Naftali Raz. "Functional magnetic resonance spectroscopy: the “new” MRS for cognitive neuroscience and psychiatry research." Frontiers in psychiatry 9 (2018): 76.
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