Distribution and marketing
In my previous post on the genesis of Crowdsourcing Discovery, I wrote about the neurotransmitter-releasing effects of the class of drugs called amphetamines, in particular amphetamine itself. According to conventional wisdom, amphetamine works by blocking the dopamine transporter protein, DAT, whose normal function is to vacuum up dopamine from synapses after each electrical impulse, thereby resetting the system. So, in the presence of amphetamine, DAT is gummed up, dopamine lingers, and reward centers in the brain go bonkers. Case closed!
Well, not exactly. I also described in detail an alternative mechanism called the weak base model, whereby amphetamines increase neurotransmitter levels by chemically interfering with the sequestration of neurotransmitters into synaptic vesicles. In the years following the debut of the weak base model in 1990, the lab of my Crowdsourcing Discovery collaborator and original weak base proponent, David Sulzer, has observed that the accumulation of amphetamines has other effects on neurons, as well as non-neuronal brain cells, e.g., glia. For example, the toxic effect of unsequestered dopamine, which can be damaging to cellular components in ways that are similar to free radicals.
But Sulzer’s lab observed that amphetamines also have dramatic effects on cell membranes. In 1994, Sulzer’s lab published a paper in which they measured the effects of 24-hr, high-dose methamphetamine treatment on dopamine-expressing neurons grown in the laboratory. Here’s an example of what they saw:
(reproduced from Figure 4 of Cubells et al)
The neuron in A1 was the control, while the neuron in B1 was treated with meth. The meth-treated cells showed evidence of “vacuolation,” or an increase in the number of intracellular compartments (those tiny bubbles). On the right, you can see the same neurons shot through with a laser that illuminated fluorescent microspheres (those white specks) that can only enter the neuron through an ancient process called endocytosis, which involves the transport of cellular cargo in vesicles. What you should take away from these data is the fact that the microspheres reside in the same intracellular compartments induced by meth, suggesting that meth accumulation affects the flow of membranes.
And it wasn’t just Sulzer’s lab that observed membrane-related drug effects. I previously blogged about the seemingly overlooked body of literature from the 1960s-1980s documenting drug distribution across tissues and throughout cells of whole animals treated with psychoactive drugs. In fact, scientists have observed different drug accumulation rates in different tissues, and the cellular adaptations to drug accumulation also vary between tissue types.
Examples of psychoactive drug distribution include the work of Italian researcher Francesco Fornai and colleagues, who published a paper in 2002 on the effects of MDMA (“ecstacy”) on brain cells of mice. They observed curious looking membrane structures inside neurons of the striatum, like the one in the right panel below:
(reproduced from Figure 2E of Fornai et al)
Very similar looking membrane “whorls,” so described because they resemble the patterns on your fingertips, can be seen inside the cells of other organisms treated with chemically related psychoactive drugs, in this case a humble yeast cell exposed to the SSRI antidepressant Zoloft (left panel), unpublished data from the Perlstein lab.
Unfortunately, several lost decades of “high affinity drug target” focus have left pharmacology in a state not too different from swiss cheese, with holes in our basic understanding of how cells respond to drug accumulation, in particular drug accumulation in cell membrane, which is caused by a primordial, in fact prebiotic, chemical attraction between certain psychoactive drugs and phospholipids, the stuff of membranes.
But do these drugs actually get enmeshed in cell membrane, or are these drugs accumulating in some other part of the cell, and we just see ripple effects in the form of aberrant membrane structures?
Autoradiography is the perfect experimental tool to resolve that question. By far the biggest advantage of autoradiography is that you don’t have to alter the structure of the drug being studied. Recall Heisenberg’s uncertainty principle: the act of measurement changes the object you are trying to measure!
A radioactive (“radiolabeled”) version of a drug is infinitesimally heavier than the non-radioactive parent compound. For Crowdsourcing Discovery, we will use amphetamines that contain carbon-14. Perhaps the best way to understand the power of autoradiography is to consider a real world example from the annals of psychopharmacology: lysergic acid diethylamide, or among friends, LSD.
(reproduced from Figure 1 of Yagaloff & Hartig)
What you’re looking at a section of the adult rat brain. The white areas show the distribution of radioactive LSD against a black background. The brightest areas (indicated by the white arrows) are where most of the radioactive LSD wound up. Based on this “map,” one can isolate the regions of high drug accumulation and do further experiments to implicate specific drug targets, also using the radioactive version of the drug.
Now, there have been autoradiography studies using radiolabeled amphetamine before anyone says that we’re claiming to be the first to try this experiment. But those studies were done a long time ago (in the early 1970s), haven’t been reproduced using modern technological advances, and were performed at poor resolution:
(reproduced from Figure 1 of Placidi et al)
You can see swaths of the mouse brain darkening, but each region of radioactive amphetamine accumulation contains too many individual cells to count. If we really want to be able to describe how amphetamines work, we first need to determine precisely where it’s going deep inside cells, down to their membranes.
Believe it or not, that’s not hard to do using a powerful electron microscope that magnifies tiny structures like cell membranes, which are almost 100-times smaller than what can be seen at the limit of light microscopy. Daniel Korostyshevsky (@badomens) has been the Perlstein Lab resident electron microscopist since 2009, and has honed the craft of electron microscopy both in yeast cells and a rat neuronal cell line. The Sulzer Lab has decades of experience working with mice and amphetamines.
Together, we have a team in place that can execute a set of focused autoradiography experiments with amphetamines in lab mice. Could this be funded by traditional sources? In theory, yes. Will that happen any time soon? In practice, probably not. Just consider the age when independent basic research life-scientists get their first stable funding stream (the R01) from the National Institutes of Health: 42. I’m 32. And when I contemplate the 80% rejection rate for R01 grant submissions, crowdfunding starts to look really attractive.
We launch Crowdsourcing Discovery in less than 24 hours! Thanks to everyone who volunteered their time and feedback over the last few weeks. You know who you are, and I’m working on a formal acknowledgement post once we clear the launch pad.
As a reminder to those tuning in for the first time, the following posts cover all the major areas of the project: