Cell biological porn

Could there actually be cell biological porn?  Yes indeedy, and hopefully the following is not behind a paywall.  https://www.cell.com/cell/fulltext/S0092-8674(18)31308-4 — [ Cell vol. 175 pp. 1430 – 1442 ’18 ]

For why I find the pictures (and videos) in the article sexy, we have to go back the bad old days of 1962 when I entered medical school and saw my first electron micrograph.  Possessed of an immense ego and a newly minted masters of chemistry, I thought I could look at the pictures and figure out what what going on chemically to produce what was seen, namely Robertson’s unit membrane.  We know what’s going on in cell membranes now, but here’s what I had to deal with back then.

Membranes fixed with osmium tetroxide revealed a characteristic tri-laminar appearance con­sisting of two parallel outer dark (osmiophilic) layers and a central light (osmiophobic) layer.

The osmiophilic layers typically measured 20-25 Å (2.0-2.5nm) in thickness and the osmiophobic layers measured 25-35 Å (2.5-3.5 nm), yielding a total thickness of 65-85 Å (6.5-8.5 nm). This value com­pared favorably with the thickness predicted on the basis of chemical studies.

According to Robertson, the unit membrane consisted of a bimolecular lipid leaflet sandwiched between outer and inner layers of protein organized in the pleated sheet con­figuration. Such an arrangement was presumed to be basically the same in all cell membranes.

Well that was the state of the art back then.  I figured I could do better, particularly since I’d used osmium tetroxide as a chemist to convert olefins to vic-diols.  Little did I know that the osmium was being used because of its high atomic weight (76 protons and over 100 neutrons) making it relatively impenetrable to the electrons of the electron microscope.

But then I looked at what was done to prepare tissue for electron microscopy — fix with glutaraldehyde, then osmium.  Dehydrate the (dead) dissue, and embed it in a monomeric resin which polymerizes to form a solid block of plastic, then cut the block, into a very thin section, place it on a copper grid covered with carbon, pump the air out so the electrons could get through, and take a picture (prayer optional).

As soon as I read this, any hope of chemical analysis disappeared.  It also taught me that it was a very large leap to assume the electron micrographs reflected what was going on in living tissue.

Which is why the above paper is so spectacular.  It uses two types of living cells (COS-7 a fibroblast like cell line from kidney and U2OS, an osteosarcoma cell line). The technique (Grazing Incidence Structured Illumination Microscopy — GI-SIM) is incredibly complicated (but well described in the paper).  It allows you to image events near the part of the cell resting on the microscope stage at 970 Angstrom resolution at rates of ‘up to’ 266 frames/second over thousands of time points.  Recall that the lowest wavelength of visible light is 3,800 Angstroms.

Various dyes are used to differentially stain microtubules, the membranes of the endoplasmic reticulumn (ER), late endosomes (LEs), mitochondria and lysosomes.  To my amazements the pictures look the electron micrographs of yore.

You can watch mitochondria touching the ER and then splitting, ER tubules growing and shrinking and being pulled along LEs riding on microtubules etc. etc. The pictures show the same cell over a period of 4 minutes.

Then to make a neurologist’s day complete, they watch dendritic spines form and unform in cultured hippocampal neurons.

So look at the paper if you can.  You don’t even have to read it th,e pictures are explanatory.

An extraordinarily impressive work, considering where we’ve been.

The bouillabaisse of the synaptic cleft

The synaptic cleft is so small ( under 400 Angstroms — 40 nanoMeters ) that it can’t be seen with the light microscope ( the smallest wavelength of visible light 3,900 Angstroms — 390 nanoMeters).  This led to a bruising battle between Cajal and Golgi a just over a century ago over whether the brain was actually made of cells.  Even though Golgi’s work led to the delineation of single neurons he thought the brain was a continuous network.  They both won the Nobel in 1906.

Semifast forward to the mid 60s when I was in medical school.  We finally had the electron microscope, so we could see synapses. They showed up as a small CLEAR spaces (e.g. electrons passed through it easily leaving it white) between neurons.  Neurotransmitters were being discovered at the same time and the synapse was to be the analogy to vacuum tubes, which could pass electricity in just one direction (yes, the transistor although invented hadn’t been used to make anything resembling a computer — the Intel 4004 wasn’t until the 70s).  Of course now we know that information flows back and forth across the synapse, with endocannabinoids (e. g. natural marihuana) being the major retrograde neurotransmitter.

Since there didn’t seem to be anything in the synaptic cleft, neurotransmitters were thought to freely diffuse across it to being to receptors on the other (postsynaptic) side e.g. a free fly zone.

Fast forward to the present to a marvelous (and grueling to read because of the complexity of the subject not the way it’s written) review of just what is in the synaptic cleft [ Cell vol. 171 pp. 745 – 769 ’17 ] http://www.cell.com/cell/fulltext/S0092-8674(17)31246-1 (It is likely behind a paywall).  There are over 120 references, and rather than being just a catalogue, the single author Thomas Sudhof extensively discusseswhich experimental work is to be believed (not that Sudhof  is saying the work is fraudulent, but that it can’t be used to extrapolate to the living human brain).  The review is a staggering piece of work for one individual.

The stuff in the synaptic cleft is so diverse, and so intimately involved with itself and the membranes on either side what what is needed for comprehension is not a chemist but a sociologist.  Probably most of the molecules to be discussed are present in such small numbers that the law of mass action doesn’t apply, nor do binding constants which rely on large numbers of ligands and receptors. Not only that, but the binding constants haven’t been been determined for many of the players.

Now for some anatomic detail and numbers.  It is remarkably hard to find just how far laterally the synaptic cleft extends.  Molecular Biology of the Cell ed. 5 p. 1149 has a fairly typical picture with a size marker and it looks to be about 2 microns (20,000 Angstroms, 2,000 nanoMeters) — that’s 314,159,265 square Angstroms (3.14 square microns).  So let’s assume each protein takes up a square 50 Angstroms on a side (2,500 square Angstroms).  That’s room for 125,600 proteins on each side assuming extremely dense packing.  However the density of acetyl choline receptors at the neuromuscular junction is 8,700/square micron, a packing also thought to be extremely dense which would give only 26,100 such proteins in a similarly distributed CNS synapse. So the numbers are at least in the right ball park (meaning they’re within an order of magnitude e.g. within a power of 10) of being correct.

What’s the point?

When you see how many different proteins and different varieties of the same protein reside in the cleft, the numbers for  each individual element is likely to be small, meaning that you can’t use statistical mechanics but must use sociology instead.

The review focuses on the neurExins (I capitalize the E  to help me remember that they are prEsynaptic).  Why?  Because they are the best studied of all the players.  What a piece of work they are.  Humans have 3 genes for them. One of the 3 contains 1,477 amino acids, spread over 1,112,187 basepairs (1.1 megaBases) along with 74 exons.  This means that just over 1/10 of a percent of the gene is actually coding for for the amino acids making it up.  I think it takes energy for RNA polymerase II to stitch the ribonucleotides into the 1.1 megabase pre-mRNA, but I couldn’t (quickly) find out how much per ribonucleotide.  It seems quite wasteful of energy, unless there is some other function to the process which we haven’t figured out yet.

Most of the molecule resides in the synaptic cleft.  There are 6 LNS domains with 3 interspersed EGFlike repeats, a cysteine loop domain, a transmembrane region and a cytoplasmic sequence of 55 amino acids. There are 6 sites for alternative splicing, and because there are two promoters for each of the 3 genes, there is a shorter form (beta neurexin) with less extracellular stuff than the long form (alpha-neurexin).  When all is said and done there are over 1,000 possible variants of the 3 genes.

Unlike olfactory neurons which only express one or two of the nearly 1,000 olfactory receptors, neurons express mutiple isoforms of each, increasing the complexity.

The LNS regions of the neurexins are like immunoglobulins and fill at 60 x 60 x 60 Angstrom box.  Since the synaptic cleft is at most 400 Angstroms long, the alpha -neurexins (if extended) reach all the way across.

Here the neurexins bind to the neuroligins which are always postsynaptic — sorry no mnemonic.  They are simpler in structure, but they are the product of 4 genes, and only about 40 isoforms (due to alternative splicing) are possible. Neuroligns 1, 3 and 4 are found at excitatory synapses, neuroligin 2 is found at inhibitory synapses.  The intracleft part of the neuroligins resembles an important enzyme (acetylcholinesterase) but which is catalytically inactive.  This is where the neurexins.

This is complex enough, but Sudhof notes that the neurexins are hubs interacting with multiple classes of post-synaptic molecules, in addition to the neuroligins — dystroglycan, GABA[A] receptors, calsystenins, latrophilins (of which there are 4).   There are at least 50 post-synaptic cell adhesion molecules — “Few are well understood, although many are described.”

The neurexins have 3 major sites where other things bind, and all sites may be occupied at once.  Just to give you a taste of he complexity involved (before I go on to  larger issues).

The second LNS domain (LNS2)is found only in the alpha-neurexins, and binds to neuroexophilin (of which there are 4) and dystroglycan .

The 6th LNS domain (LNS6) binds to neuroligins, LRRTMs, GABA[A] receptors, cerebellins and latrophilins (of which there are 4)_

The juxtamembrane sequence of the neurexins binds to CA10, CA11 and C1ql.

The cerebellins (of which there are 4) bind to all the neurexins (of a particular splice variety) and interestingly to some postsynaptic glutamic acid receptors.  So there is a direct chain across the synapse from neurexin to cerebellin to ion channel (GLuD1, GLuD2).

There is far more to the review. But here is something I didn’t see there.  People have talked about proton wires — sites on proteins that allow protons to jump from one site to another, and move much faster than they would if they had to bump into everything in solution.  Remember that molecules are moving quite rapidly — water is moving at 590 meters a second at room temperature. Since the synaptic cleft is 40 nanoMeters (40 x 10^-9 meters, it should take only 40 * 10^-9 meters/ 590 meters/second   60 trillionths of a second (60 picoSeconds) to cross, assuming the synapse is a free fly zone — but it isn’t as the review exhaustively shows.

It it possible that the various neurotransmitters at the synapse (glutamic acid, gamma amino butyric acid, etc) bind to the various proteins crossing the cleft to get their target in the postsynaptic membrane (e.g. neurotransmitter wires).  I didn’t see any mention of neurotransmitter binding to  the various proteins in the review.  This may actually be an original idea.

I’d like to put more numbers on many of these things, but they are devilishly hard to find.  Both the neuroligins and neurexins are said to have stalks pushing them out from the membrane, but I can’t find how many amino acids they contain.  It can’t find how much energy it takes to copy the 1.1 megabase neurexin gene in to mRNA (or even how much energy it takes to add one ribonucleotide to an existing mRNA chain).

Another point– proteins have a finite lifetime.  How are they replenished?  We know that there is some synaptic protein synthesis — does the cell body send packages of mRNAs to the synapse to be translated there.  There are at least 50 different proteins mentioned in the review, and don’t forget the thousands of possible isoforms, each of which requires a separate mRNA.

Old Chinese saying — the mountains are high and the emperor is far away. Protein synthesis at the synaptic cleft is probably local.  How what gets made and when is an entirely different problem.

A large part of the review concerns mutations in all these proteins associated with neurologic disease (particularly autism).  This whole area has a long and checkered history.  A high degree of cynicism is needed before believing that any of these mutations are causative.  As a neurologist dealing with epilepsy I saw the whole idea of ion channel mutations causing epilepsy crash and burn — here’s a link — https://luysii.wordpress.com/2011/07/17/we’ve-found-the-mutation-causing-your-disease-not-so-fast-says-this-paper/

Once again, hats off to Dr. Sudhof for what must have been a tremendous amount of work

How the brain really works (maybe)

Stare at the picture just below long and hard. It’s where the brain probably does its calculation — no, not the neuron in the center. No, not the astrocyte just above. Enlarge the picture many times. It’s all those tiny little circles and ellipses you see around the apical dendrite. They all represent nerve and glial processes. A few ellipses have very dark borders — this is myelin (which insulates them allowing them to conduct nerve impulses faster, and which also insulates them from being affected by the goings on of nerve processes next to them). Note that most of the nerve processes do NOT have myelin around them.

Now look at the bar at the lower right in the picture which tells you the magnification. 5 um is 5 microns or 50,000 Angstroms or roughly 10 times the wavelength of visible light (4,000 – 8,000 Angstroms). Look at the picture again and notice just how closely the little circles and ellipses are applied to each other (certainly closer than 1/10 of the bar). This is exactly why there was significant debate between two of the founders of neuroHistology — Camillo Golgi and Ramon Santiago y Cajal.

Unlike every other tissue in the body the brain is so tightly packed that it is impossible to see the cells that make it up with the usual stains used by light microscopists. People saw nuclei all right but they thought the brain was a mass of tissue with nuclei embedded in it (like a slime mold). It wasn’t until the late 1800′s that Camillo Golgi developed a stain which would now and then outline a neuron with all its processes. Another anatomist (Ramon Santiago y Cajal) used Golgi’s technique and argued with Golgi that yes the brain was made of cells. Fascinating that Golgi, the man responsible for showing nerve cells, didn’t buy it. This was a very hot issue at the time, and the two received a joint Nobel prize in 1906 (only 5 years after the prizes began).

The paper discussed below gives a possible reason why the brain is built like this — e.g. it’s how it works !!

Pictures are impressive, but could it be all artifact? To see something with an electron microscope (which this picture is) you really have to process the tissue to a fare-thee-well. One example from way back in the day when I started medical school (1962). Electron microscopy was just coming in, and the first thing we were supposed to see was something called the unit membrane surrounding each cell –two dark lines surrounding a light line, the whole mess being about 60 – 80 Angstroms thick. The dark lines were held to be proteins and the light line was supposed to be lipid. Fresh off 2 years of grad school in chemistry, I tried to figure out just what the chemical treatments used to put tissue in a form suitable for electron microscopy would do to proteins and lipids. It was impossible, but I came away impressed with just how vigorous and traumatic what the microscopists were doing actually was.

To make a long story short — the unit membrane was an artifact of fixation. We now know that the cell membrane has a thickness half that of the unit membrane, with all sorts of proteins going through the lipid.

This is something to keep in mind, for you to avoid being snowed by such pictures. Clinical neurologists and neurosurgeons know quite well that a brain lacking oxygen and glucose swells (a huge clinical problem), and dead brain is exactly that.

Even with all these caveats about electron microscopy of the brain, I think the picture above is pretty close to reality. In favor of tight packing is the following work (along with the staining work of over a century ago). [ Proc. Natl. Acad. Sci. vol. 103 pp. 5567 – 5572 ’06 ] injected spheres of different sizes (quantum dots actually) into the rat cerebral cortex, and watched how far they got from the site of injection. Objects ‘as large as’ 350 Angstroms were able to diffuse freely. This was larger than the width seen on electron microscopy (180 Angstroms) but still quite small and too small to be ‘seen’ with visible light.

What’s the point of all this? Simply that the neuropil of the cerebral cortex (all the stuff in the picture which isn’t the cell body of the neuron or the astrocyte) could be where the real computations of the cerebral cortex actually take place. In my opinion, ‘could be’ should be ‘is’ in the previous sentence.

Why? Because of the work described in a previous post — which is repeated in toto below the line of ****

Briefly, the authors of that paper claim to be able to look at the electrical activity of these small processes in the neuropil. How small? A diameter of 5 microns or less. This had never been done before. It was a tremendous technical achievement to do this in a living animal. What they found was that the frequency of spikes in these processes (likely dendrites) during sleep was 7 times greater than the frequency of spikes recorded next to the cell body (soma) which had been done many times before. During wakefulness, the frequency of spikes in the neuropil was 10 fold greater.

I’ve always found it remarkable that most neurons in the cerebral cortex aren’t firing all that rapidly (a few spikes per second — Science vol. 304 pp. 523 – 524, 559 – 563 ’04 ). Neurons (particularly sensory neurons) can fire a lot faster than that — ‘up to’ 500/second.

Perhaps this work explains why — the real calculations are being done in the neuropil by the dendrites.

Even more remarkably, it is possible that the processes of the neuropil are influencing each other without synapses between them because they are so closely packed. The membrane potential shifts the authors measured were much larger than the spikes in the dendrites. So the real computations being performed by the brain might not involve synapses at all ! This would be an explanation of why brain cells and their processes are so squeezed together. So they can talk to each other. No other organ in the body is like this throughout.

This post is already long enough, but the implications are worth exploring further. I’ve written about wiring diagrams of the brain, and how it is at least possible that they wouldn’t tell you how the brain worked — https://luysii.wordpress.com/2011/04/10/would-a-wiring-diagram-of-the-brain-help-you-understand-it/.

There is another possible reason that the wiring diagram wouldn’t be enough to give you an understanding. Here is an imperfect analogy. Suppose you had a complete map of every road and street in the USA, along with the address of every house, building and structure in it. In addition you could also measure the paths of all the vehicles on the roads for one day. Would this tell you how the USA worked? It would tell you nothing about what was going on inside the structures, or how it influenced traffic on the roads.

The paper below is seminal, because for the first time, it allows us to see what brain neurons are doing in all their parts — not just the cell body or the axon (which is all we’ve been able to look at before).

If these speculations are true, the brain is a much more powerful parallel processor than anything we are able to build presently (and possibly in the future). Each pyramidal neuron in the cortex would then be a microprocessor locally influencing all those in its vicinity — and in a cubic millimeter of the cerebral cortex (1,000 x 1,000 x 1,000 microns) there are 20,000 – 100,000 neurons (Science vol. 304 pp. 523 – 524, 559 – 563 ’04).

Fascinating stuff — stay tuned
*****

A staggeringly important paper (if true)

Our conception of how our brain does what it does has just been turned upside down, inside out and from the middle to each end — if the following paper holds up [ Science vol. 355 pp. 1281 eaaj1497 1 –> 10 ’17 ] The authors claim to be able to measure electrical activity in dendrites in a living, behaving animal for days at a time. Dendrites are about the size of the smallest electrodes we have, so impaling them destroys them. The technical details of what they did are crucial, as much of what they report may be artifact due to injury produced by the way they acquired their data.

First a picture of a pyramidal neuron of the cerebral cortex — https://en.wikipedia.org/wiki/Pyramidal_cell — the cell body is only 20 microns in diameter (the giant pyramidal neurons giving rise to the corticospinal tract are much larger with diameters of 100 microns). Look at the picture in the article. If the cell body is (soma) 20 microns the dendrites arising from it (particularly the apical dendrite) are at most 5 microns thick.

Here’s what they did. A tetrode is a bundle of 4 very fine electrodes. Bundle diameter is only 30 – 40 microns with a 5 micron gap between the tips. This allows an intact dendrite to be caught in the gap. The authors note that chronically implanted tetrodes produce an immune response, in which glial cells proliferate and wall off the tetrode, shielding it from the extracellular medium by forming a high impedance sheath. This allows the tetrode to measure the electrical activity of a dendrite caught between the 4 tips (and hopefully little else).

How physiologic is this activity? Remember that epilepsy developing after head trauma is thought to be due to abnormal electrical activity due to glial scars, and a glial scar is exactly what is found around the tetrode. So a lot more work needs to be done replicating this, and studying similar events in neuronal culture (without glia present).

Well those are the caveats. What did they find? The work involved 9 rats and 22 individually adjustable tetrodes. They found that spikes in the dendrites were quite different than the spikes found by a tetrode next to the pyramidal cell body. The dendritic spikes were larger (570 -2,100 microVolts) vs. 80 microVolts recorded extracellularly for spikes arising at the soma. Of course when the soma is impaled by an electrode you get a much larger spike.

More importantly, the dendritic spike rates were 5 times greater than the somatic spike rates during slow wave sleep and 10 times greater during exploration when awake. The authors call these dendritic action potentials (DAPs). Their amplitude was always positive.

They were also able to measure how the membrane potential of the dendrite fluctuated. The membrane potential fluctuations were always larger than the dendritic spikes themselves (by 7 fold). The size of the flucuations correlated with DAP magnitude and rate.

So all the neuronal spikes and axonal action potentials we’ve been measuring over the years (because it was all we could measure), may be irrelevant to what the brain is really doing. Maybe the real computation is occuring within dendrites.

Now we know we can put an electrode in the brain outside of any neuron and record something called a local field potential — which is held to be a weighted sum of transmembrane currents due to synaptic and dendritic activity and arises within 250 microns of the electrode (and probably closer than that).

So fluctuating potentials are out there in the substance of the brain, outside any neuronal structure. Is it possible that the changes in membrane potential in dendrites are felt by other dendrites and if so is this where the brain’s computations are really taking place? Could synapses be irrelevant to this picture, and each pyramidal neuron not be a transistor but a complex analog CPU? Heady stuff. It certainly means goodbye to the McCullouch Pitts model — https://en.wikipedia.org/wiki/Artificial_neuron.