Category Archives: Neurology & Psychiatry

Progress has been slow but not for want of trying

Progress in the sense of therapy for Alzheimer’s disease and Glioblastoma multiforme is essentially nonexistent, and we could use better therapy for Parkinsonism. This doesn’t mean that researchers have given up. Far from it. Three papers all in last week’s issue of PNAS came up with new understanding and possibly new therapeutic approaches for all three.

You’ll need some serious molecular biological and cell physiological chops to get through the following.

l. Glioblastoma multiforme — they aren’t living much longer than they were when I started pracice 45 years ago (about 2 years — although of course there are exceptions).

The human ZBTB family of genes consists of 49 members coding for transcription factors. BCL6 is also known as ZBTB27 and is a master regulator of lymph node germinal responses. To execute its transcriptional activity, BCL6 requires homodimerization and formation of a complex with a variety of cofactors including BCL6 corerpressor (BCoR), nuclear receptor corepressor 1 (NCoR) and Silencing Mediator of Retinoic acid and Thyroid hormone receptor (SMRT). BCL6 inhibitors block the interaction between BCL6 and its friends, selectively killing BCL6 addicted cancer cells.

The present paper [ Proc. Natl. Acad. Sci. vol. 114 pp. 3981 – 3986 ’17 ] shows that BCL6 is required for glioblastoma cell viability. One transcriptional target of BCL6 is AXL, a tyrosine kinase. Depletion of AXL also decreases proliferation of glioblastoma cells in vitro and in vivo (in a mouse model of course).

So here are two new lines of attack on a very bad disease.

2. Alzheimer’s disease — the best we can do is slow it down, certainly not improve mental function and not keep mental function from getting worse. ErbB2 is a member of the Epidermal Growth Factor Receptor (EGFR) family. It is tightly associated with neuritic plaques in Alzheimer’s. Ras GTPase activation mediates EGF induced stimulation of gamma secretase to increase the nuclear function of the amyloid precursor protein (APP) intracellular domain (AICD). ErbB2 suppresses the autophagic destruction of AICD, physically dissociating Beclin1 vrom the VPS34/VPS15 complex independently of its kinase activity.

So the following paper [ Proc. Natl. Acad. Sci. vol. 114 pp. E3129 – E3138 ’17 ] Used a compound downregulating ErbB2 function (CL-387,785) in mouse models of Alzheimer’s (which have notoriously NOT led to useful therapy). Levels of AICD declined along with beta amyloid, and the animals appeared smarter (but how smart can a mouse be?).

3.Parkinson’s disease — here we really thought we had a cure back in 1972 when L-DOPA was first released for use in the USA. Some patients looked so good that it was impossible to tell if they had the disease. Unfortunately, the basic problem (death of dopaminergic neurons) continued despite L-DOPA pills supplying what they no longer could.

Nurr1 is a protein which causes the development of dopamine neurons in the embryo. Expression of Nurr1 continues throughout life. Nurr1 appears to be a constitutively active nuclear hormone receptor. Why? Because the place where ligands (such as thyroid hormone, steroid hormones) bind to the protein is closed. A few mutations in the Nurr1 gene have been associated with familial parkinsonism.

Nurr1 functions by forming a heterodimer with the Retinoid X Receptor alpha (RXRalpha), another nuclear hormone receptor, but one which does have an open binding pocket. A compound called BRF110 was shown by the following paper [ Proc. Natl. Acad. Sci. vol. 114 pp. 3795 – 3797, 3999 – 4004 ’17 ] to bind to the ligand pocked of RXRalpha increasing its activity. The net effect is to enhance expression of dopamine neuron specific genes.

More to the point MPP+ is a toxin pretty selective for dopamine neurons (it kills them). BRF110 helps survival against MPP+ (but only if given before toxin administration). This wouldn’t be so bad because something is causing dopamine neurons to die (perhaps its a toxin), so BRF110 may fight the decline in dopamine neuron numbers, rather than treating the symptoms of dopamine deficiency.

So there you have it 3 possible new approaches to therapy for 3 bad disease all in one weeks issue of PNAS. Not easy reading, perhaps, but this is where therapy is going to come from (hopefully soon).

How the brain really works (maybe) – 2

I sent the previous post to a very intelligent friend — a PhD Electrical Engineer who responded as follows

“Correct me if I’m wrong, but it sounds like you are proposing that in addition to direct communication in the nervous system via electrical and chemical synapses, you are proposing that there could also be communication coupling in nerve fibers via local electric fields. But isn’t this a known phenomenon, ephaptic coupling? See
https://en.wikipedia.org/wiki/Ephaptic_coupling”

I didn’t think EE’s knew about such things (but I told you he’s very smart). Here are a few extra points of mine concerning his response and the article in general.

Excellent point. Thanks. What I propose could certainly be called ephaptic transmission. It has been well described between two axons in peripheral nerves (this was the initial description). Ephaptic transmission is fairly well established in muscle (which also has action potentials spreading along the muscle fiber allowing it to contract). Investigation in the brain has primarily been between adjacent neurons or adjacent axons. Questions have arisen as to whether it could be a mechanism of seizure generation.

As far as I can tell, the following ideas are actually original.
(1) Ephaptic transmission could normally occur between dendrites in the cerebral cortex.
(2) The brain and cerebral cortex is built the way it is to allow dendritic ephaptic transmission to occur.
(3) This is the way serious computations are carried out by the cerebral cortex.

Why now? Probably because there was no way of measuring dendritic electric potential changes directly before this paper (prior to this calcium levels in dendrites were used as a surrogate). Another example of new technology driving the science.

I didn’t put it in the original post, but actual paper notes that the potential flucutations across the dendritic membrane were much larger than the fluctuations recorded at the cell body.

People have wondered for years how various electrical activities in the brain could be synchronized over large areas (every electrical wave seen in the electroencephalogram is the activity of hundreds of thousands to millions of neurons). This may be an explanation — previously people had figured that it was coming from neurons lower in the brain (particularly the thalamus) sending axons all over the place stimulating neurons simultaneously. Even this doesn’t really work, because various areas of the brain are separted from each other, axonal speed is thought to be constant, and the impulses have different distances to travel.

One disturbing aspect to the picture in the previous post — If you regard that neuron as embedded in a cube 50 x 50 x 50 microns on a side, you’d get about 8,000 neurons per cubic millimeter (1,000 x 1,000 x 1,000 cubic microns). The literature says over twice that at 20,000 neurons/cubic millimeter.

I doubt that the above constitutes all the implications of these ideas. Any comments? I am quite interested to hear them.

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.

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 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 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 out side 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.

20,000 NanoSensors under the Cell (apologies to Jules Verne)

Too bad Jules Verne isn’t around to read PNAS vol. 114 pp. 1789 – 1794 ’17 where 20,000 fluorescent nanoSensors were placed under a single PC12 cell. PC12 cells are derived from a pheochromocytoma, a tumor which secretes catecholamines like dopamine and norepinephrine. So they’re almost neurons, and they contain vesicles containing dopamine, just like neurons, but they don’t form synapses.

The pictures they show of the cells shows the cell bodies sitting on a slide to be about 100 microns in diameter, with multiple protrusions so how are you going to get 20,000 sensors underneath them. Assuming them to be circular that’s about 3 per square micron. A micron is 10,000 Angstroms. The authors used Single Walled Carbon NanoTubes (SWCNTs) — e.g. rolled up graphene. They have a diameter of from 5 – 20 Angstroms, so there’s plenty of room for many in a square micron.

Here’s what they did. “Previously we found that the corona phase around SWCNTs can be engineered to recognize certain small analytes––a phenomenon we termed Corona Phase Molecular Recognition (CoPhMoRe) (7, 25, 26). Specifically, DNA-wrapped SWCNTs were found to increase their near InfraRed fluorescence in the presence of catecholamines . Here, we synthesized and characterized different DNA/SWCNT com- plexes and identified the best candidates for dopamine detection.

What they found is less remarkable than having the guts to try something like this. They could stimulate the cells to release dopamine using potassium (maddeningly I couldn’t find the concentration anywhere). Then with the density of sensors they could find out where it was released (the edges of the cell) with a time resolution of .1 second. It wasn’t generally released, but in hotspots — what you’d expectd if it were being released due to vesicles containing dopamine fusing with the cell membrane.

Remarkable — hard to see how they’re going to get this sort an array into a living organism, but their use in the study of brain slices can’t be far away.

Norbert Weiner

In the Cambridge Mass of the early 60’s the name Norbert Weiner was spoken in hushed tones. Widely regarded as a genius tutti the assembled genii of Cambridge, that was all I knew about him aside from the fact that he got a bachelor’s degree in math from Tufts at age 14. As a high school student I tried to read Cybernetics, a widely respected book he wrote in 1948, and found it incomprehensible.

Surprisingly, his name never came up again in any undergraduate math courses, graduate chemistry and physics courses, extensive readings on programming and computation (until now).

From PNAS vol. 114 pp. 1281 – 1286 ’17 –“In their seminal theoretical work, Norbert Wiener and Arturo Rosenblueth showed in 1946 that the self-sustained activity in the cardiac muscle can be associated with an excitation wave rotating around an obstacle. This mechanism has since been very successfully applied to the understanding of the generation and control of malignant electrical activity in the heart. It is also well known that self-sustained excitation waves, called spirals, can exist in homogeneous excitable media. It has been demonstrated that spirals rotating within a homogeneous medium or anchored at an obstacle are generically expected for any excitable medium.”

That sounds a lot like atrial fibrillation, a serious risk factor for strokes, and something I dealt with all the time as a neurologist. Any theoretical input about what to do for it would be welcome.

A technique has been developed to cure the arrhythmia. Here it is. “Recently, an atrial defibrillation procedure was clinically introduced that locates the spiral core region by detecting the phase-change point trajectories of the electrophysiological wave field and then, by ablating that region, restores sinus rhythm.” The technique is now widely used, and one university hospital (Ohio State) says that they are doing over 600 per year.

“This is clearly at odds with the Wiener–Rosenblueth mechanism because a further destruction of the tissue near the spiral core should not improve the situation.” It’s worse than that because the summary says “In the case of a functionally determined core, an ablation procedure should even further stabilize the rotating wave”

So theory was happily (for the patients) ignored. Theorists never give up and the paper goes on to propose a mechanism explaining why the Ohio State procedure should work. Here’s what they say.

“Here, we show theoretically that fundamentally in any excitable medium a region with a propagation velocity faster than its surrounding can act as a nucleation center for reentry and can anchor an induced spiral wave. Our findings demonstrate a mechanistic underpinning for the recently developed ablation procedure.”

It certainly has the ring of post hoc propter hoc about it.

The Rorschach test

Despite spending 6 months of a 3 year neurology residency on the psychiatry service (as was typical in those days) the Rorschach test never came up. Of course, it was well known in the wider world, primarily by a joke.

For those who don’t know, the Rorschach test is a series of 10 inkblots and subjects were asked to tell the examiner what they brought to mind.  To learn more about the test see — https://en.wikipedia.org/wiki/Rorschach_test

The joke:  The response to all 10 by one frisky subject was that they reminded him of sex. The examiner asked him why he was so obsessed with sex. The subject asked the examiner why he was showing him dirty pictures.

There is a very interesting review of a book about Dr. Rorschach in the current issue of Science (vol. 355 p.588 ’17). The reviewer is at the Department of Translational Science and Molecular Medicine, Michigan State University, Grand Rapids, MI 49503, USA. Email: erin.mckay@hc.msu.edu

Here is the first part — unfortunately I can’t reproduce it all, as you must be a subscriber to Science —
“We’re all familiar with the inkblots that make up the Rorschach test: black and white, bilaterally symmetrical figures that hover close to familiarity. Or, at least, we think we are. In modern times,the term “Rorschach test” often serves as a metaphor for our divisiveness, as shorthand for an encoded message, or as a warning that appearances

Inkblots were used in psychology to gauge a person’s imagination for nearly two decades before Rorschach developed his version. Rorschach’s contribution was born of his desire to detect the differences in perceptual processes that explained seemingly nonsensical delusions and neuroses. When designing his inkblots, he can be deceiving. But we may not know as much as we think we do about this classic psychological tool or the man behind it, argues Damion Searls in The Inkblots: Hermann Rorschach, His Iconic Test, and The Power of Seeing.

In tracing the story of the inkblots, Searls sets out to restore two vital stipulations of the Rorschach test: that there are good answers and bad answers and that the test is a measure of perception, not of imagination or projection. The book addresses many questions fundamental to understanding the genesis and effectiveness of Rorschach’s eponymous test as well as the life of the man himself.

Hoping to create images that were suggestive of shape and movement, Rorschach hand-painted each of his 10 eponymous inkblots.”

It always seemed incredibly subjective to me (typical of much of psychoanalysis IMHO).

Not so.

I asked two friends long in the field, whose experience and intelligence and hardheadedness isn’t open to question.

The psychiatrist’s response

As a psychiatrist I was never trained in the Rorschach as psychologists are but have generally found them very helpful. In fact, I took one myself back in residency and had the psychologist interpret the results, which at the time left me feeling naked, ie, all my defenses stripped away.

My office mate doesn’t favor it largely for the reasons in the article: the lack of a scientific basis. Since he is a forensic psychiatrist, this drawback is even worse, since one might potentially have to present the results to a jury, which is almost universally likely to view it as hocus pocus even if there was more scientific basis.
There is a technology to interpret the results, but I think an experienced clinician is also key to its results being helpful. It gives a much deeper dimension to the findings s/w similar to other projective tests and relative to more scientifically based tests such as the MMPI.

Interesting article; thanks for sending.

The Psychiatric Nurse’s response

I actually did use the Rorschach test when leading groups on the in patient psychiatric unit at — a prominent Boston Hospiutal (1975-1980). It was always a challenge to get depressed, withdrawn, and psychotic people to express themselves. Trying to be creative and engaging, I would hold up the ink blots and get anywhere from 1-100 word responses……dependent upon their diagnosis! OF COURSE the bipolar manics, with pressured speech, had to be interrupted for the sake of time!

Then, the artist in me would come out. I had people make their own Rorschach’s with paint. It helped engage the withdrawn members in a different format. The response was that those with paucity of speech were able to express themselves in a non-verbal way. There was always more discussion stimulated by their own creations.

Memories are made of this ?

Back in the day when information was fed into computers on punch cards, the data was the holes in the paper not the paper itself. A far out (but similar) theory of how memories are stored in the brain just got a lot more support [ Neuron vol. 93 pp. 6 -8, 132 – 146 ’17 ].

The theory says that memories are stored in the proteins and sugar polymers surrounding neurons rather than the neurons themselves. These go by the name of extracellular matrix, and memories are the holes drilled in it which allow synapses to form.

Here’s some stuff I wrote about the idea when I first ran across it two years ago.

——

An article in Science (vol. 343 pp. 670 – 675 ’14) on some fairly obscure neurophysiology at the end throws out (almost as an afterthought) an interesting idea of just how chemically and where memories are stored in the brain. I find the idea plausible and extremely surprising.

You won’t find the background material to understand everything that follows in this blog. Hopefully you already know some of it. The subject is simply too vast, but plug away. Here a few, seriously flawed in my opinion, theories of how and where memory is stored in the brain of the past half century.

#1 Reverberating circuits. The early computers had memories made of something called delay lines (http://en.wikipedia.org/wiki/Delay_line_memory) where the same impulse would constantly ricochet around a circuit. The idea was used to explain memory as neuron #1 exciting neuron #2 which excited neuron . … which excited neuron #n which excited #1 again. Plausible in that the nerve impulse is basically electrical. Very implausible, because you can practically shut the whole brain down using general anesthesia without erasing memory. However, RAM memory in the computers of the 70s used the localized buildup of charge to store bits and bytes. Since charge would leak away from where it was stored, it had to be refreshed constantly –e.g. at least 12 times a second, or it would be lost. Yet another reason data should always be frequently backed up.

#2 CaMKII — more plausible. There’s lots of it in brain (2% of all proteins in an area of the brain called the hippocampus — an area known to be important in memory). It’s an enzyme which can add phosphate groups to other proteins. To first start doing so calcium levels inside the neuron must rise. The enzyme is complicated, being comprised of 12 identical subunits. Interestingly, CaMKII can add phosphates to itself (phosphorylate itself) — 2 or 3 for each of the 12 subunits. Once a few phosphates have been added, the enzyme no longer needs calcium to phosphorylate itself, so it becomes essentially a molecular switch existing in two states. One problem is that there are other enzymes which remove the phosphate, and reset the switch (actually there must be). Also proteins are inevitably broken down and new ones made, so it’s hard to see the switch persisting for a lifetime (or even a day).

#3 Synaptic membrane proteins. This is where electrical nerve impulses begin. Synapses contain lots of different proteins in their membranes. They can be chemically modified to make the neuron more or less likely to fire to a given stimulus. Recent work has shown that their number and composition can be changed by experience. The problem is that after a while the synaptic membrane has begun to resemble Grand Central Station — lots of proteins coming and going, but always a number present. It’s hard (for me) to see how memory can be maintained for long periods with such flux continually occurring.

This brings us to the Science paper. We know that about 80% of the neurons in the brain are excitatory — in that when excitatory neuron #1 talks to neuron #2, neuron #2 is more likely to fire an impulse. 20% of the rest are inhibitory. Obviously both are important. While there are lots of other neurotransmitters and neuromodulators in the brains (with probably even more we don’t know about — who would have put carbon monoxide on the list 20 years ago), the major inhibitory neurotransmitter of our brains is something called GABA. At least in adult brains this is true, but in the developing brain it’s excitatory.

So the authors of the paper worked on why this should be. GABA opens channels in the brain to the chloride ion. When it flows into a neuron, the neuron is less likely to fire (in the adult). This work shows that this effect depends on the negative ions (proteins mostly) inside the cell and outside the cell (the extracellular matrix). It’s the balance of the two sets of ions on either side of the largely impermeable neuronal membrane that determines whether GABA is excitatory or inhibitory (chloride flows in either event), and just how excitatory or inhibitory it is. The response is graded.

For the chemists: the negative ions outside the neurons are sulfated proteoglycans. These are much more stable than the proteins inside the neuron or on its membranes. Even better, it has been shown that the concentration of chloride varies locally throughout the neuron. The big negative ions (e.g. proteins) inside the neuron move about but slowly, and their concentration varies from point to point.

Here’s what the authors say (in passing) “the variance in extracellular sulfated proteoglycans composes a potential locus of analog information storage” — translation — that’s where memories might be hiding. Fascinating stuff. A lot of work needs to be done on how fast the extracellular matrix in the brain turns over, and what are the local variations in the concentration of its components, and whether sulfate is added or removed from them and if so by what and how quickly.

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So how does the new work support this idea? It involves a structure that I’ve never talked about — the lysosome (for more info see https://en.wikipedia.org/wiki/Lysosome). It’s basically a bag of at least 40 digestive and synthetic enzymes inside the cell, which chops anything brought to it (e.g. bacteria). Mutations in the enzymes cause all sorts of (fortunately rare) neurologic diseases — mucopolysaccharidoses, lipid storage diseases (Gaucher’s, Farber’s) the list goes on and on.

So I’ve always thought of the structure as a Pandora’s box best kept closed. I always thought of them as confined to the cell body, but they’re also found in dendrites according to this paper. Even more interesting, a rather unphysiologic treatment of neurons in culture (depolarization by high potassium) causes the lysosomes to migrate to the neuronal membrane and release its contents outside. One enzyme released is cathepsin B, a proteolytic enzyme which chops up the TIMP1 outside the cell. So what. TIMP1 is an endogenous inhibitor of Matrix MetalloProteinases (MMPs) which break down the extracellular matrix. So what?

Are neurons ever depolarized by natural events? Just by synaptic transmission, action potentials and spontaneously. So here we have a way that neuronal activity can cause holes in the extracellular matrix,the holes in the punch cards if you will.

Speculation? Of course. But that’s the fun of reading this stuff. As Mark Twain said ” There is something fascinating about science. One gets such wholesale returns of conjecture out of such a trifling investment of fact.”

Tidings of great joy

One of the hardest things I had to do as a doc was watch an infant girl waste away and die of infantile spinal muscular atrophy (Werdnig Hoffmann disease) over the course of a year. Something I never thought would happen (a useful treatment) may be at hand. The actual papers are not available yet, but two placebo controlled trials with a significant number of patients (84, 121) in each were stopped early because trial monitors (not in any way involved with the patients) found the treated group was doing much, much better than the placebo. A news report of the trials is available [ Science vol. 354 pp. 1359 – 1360 ’16 (16 December) ].

The drug, a modified RNA molecule, (details not given) binds to another RNA which codes for the missing protein. In what follows a heavy dose of molecular biology will be administered to the reader. Hang in there, this is incredibly rational therapy based on serious molecular biological knowledge. Although daunting, other therapies of this sort for other neurologic diseases (Huntington’s Chorea, FrontoTemporal Dementia) are currently under study.

If you want to start at ground zero, I’ve written a series https://luysii.wordpress.com/category/molecular-biology-survival-guide/ which should tell you enough to get started. Start here — https://luysii.wordpress.com/2010/07/07/molecular-biology-survival-guide-for-chemists-i-dna-and-protein-coding-gene-structure/
and follow the links to the next two.

Here we go if you don’t want to plow through all three

Our genes occur in pieces. Dystrophin is the protein mutated in the commonest form of muscular dystrophy. The gene for it is 2,220,233 nucleotides long but the dystrophin contains ‘only’ 3685 amino acids, not the 770,000+ amino acids the gene could specify. What happens? The whole gene is transcribed into an RNA of this enormous length, then 78 distinct segments of RNA (called introns) are removed by a gigantic multimegadalton machine called the spliceosome, and the 79 segments actually coding for amino acids (these are the exons) are linked together and the RNA sent on its way.

All this was unknown in the 70s and early 80s when I was running a muscular dystrophy clininc and taking care of these kids. Looking back, it’s miraculous that more of us don’t have muscular dystrophy; there is so much that can go wrong with a gene this size, let along transcribing and correctly splicing it to produce a functional protein.

One final complication — alternate splicing. The spliceosome removes introns and splices the exons together. But sometimes exons are skipped or one of several exons is used at a particular point in a protein. So one gene can make more than one protein. The record holder is something called the Dscam gene in the fruitfly which can make over 38,000 different proteins by alternate splicing.

There is nothing worse than watching an infant waste away and die. That’s what Werdnig Hoffmann disease is like, and I saw one or two cases during my years at the clinic. It is also called infantile spinal muscular atrophy. We all have two genes for the same crucial protein (called unimaginatively SMN). Kids who have the disease have mutations in one of the two genes (called SMN1) Why isn’t the other gene protective? It codes for the same sequence of amino acids (but using different synonymous codons). What goes wrong?

[ Proc. Natl. Acad. Sci. vol. 97 pp. 9618 – 9623 ’00 ] Why is SMN2 (the centromeric copy (e.g. the copy closest to the middle of the chromosome) which is normal in most patients) not protective? It has a single translationally silent nucleotide difference from SMN1 in exon 7 (e.g. the difference doesn’t change amino acid coded for). This disrupts an exonic splicing enhancer and causes exon 7 skipping leading to abundant production of a shorter isoform (SMN2delta7). Thus even though both genes code for the same protein, only SMN1 actually makes the full protein.

More background. The molecular machine which removes the introns is called the spliceosome. It’s huge, containing 5 RNAs (called small nuclear RNAs, aka snRNAs), along with 50 or so proteins with a total molecular mass again of around 2,500,000 kiloDaltons. Think about it chemists. Design 50 proteins and 5 RNAs with probably 200,000+ atoms so they all come together forming a machine to operate on other monster molecules — such as the mRNA for Dystrophin alluded to earlier. Hard for me to believe this arose by chance, but current opinion has it that way.

Splicing out introns is a tricky process which is still being worked on. Mistakes are easy to make, and different tissues will splice the same pre-mRNA in different ways. All this happens in the nucleus before the mRNA is shipped outside where the ribosome can get at it.

The papers [ Science vol. 345 pp. 624 – 625, 688 – 693 ’14 ].describe a small molecule which acts on the spliceosome to increase the inclusion of SMN2 exon 7. It does appear to work in patient cells and mouse models of the disease, even reversing weakness.

I was extremely skeptical when I read the papers two years ago. Why? Because just about every protein we make is spliced (except histones), and any molecule altering the splicing machinery seems almost certain to produce effects on many genes, not just SMN2. If it really works, these guys should get a Nobel.

Well, I shouldn’t have been so skeptical. I can’t say much more about the chemistry of the drug (nusinersen) until the papers come out.

Fortunately, the couple (a cop and a nurse) took the 25% risk of another child with the same thing and produced a healthy infant a few years later.

Will flickering light treat Alzheimer’s disease ?

Big pharma has spent zillions trying to rid the brain of senile plaques, to no avail. A recent paper shows that light flickering at 40 cycles/second (40 Hertz) can do it — this is not a misprint [ Nature vol. 540 pp. 207 – 208, 230 – 235 ’16 ]. As most know the main component of the senile plaque of Alzheimer’s disease is a fragment (called the aBeta peptide) of the amyloid precursor protein (APP).

The most interesting part of the paper showed that just an hour or so of light flickering at 40 Hertz temporarily reduced the amount of Abeta peptide in visual cortex of aged mice. Nothing invasive about that.

Should we try this in people? How harmful could it be? Unfortunately the visual cortex is relatively unaffected in Alzheimer’s disease — the disease starts deep inside the head in the medial temporal lobe, particularly the hippocampus — the link shows just how deep it is -https://en.wikipedia.org/wiki/Hippocampus#/media/File:MRI_Location_Hippocampus_up..png

You might be able to do this through the squamous portion of the temporal bone which is just in front of and above the ear. It’s very thin, and ultrasound probes placed here can ‘see’ blood flowing in arteries in this region. Another way to do it might be a light source placed in the mouth.

The technical aspects of the paper are fascinating and will be described later.

First, what could go wrong?

The work shows that the flickering light activates the scavenger cells of the brain (microglia) and then eat the extracellular plaques. However that may not be a good thing as microglia could attack normal cells. In particular they are important in the remodeling of the dendritic tree (notably dendritic spines) that occurs during experience and learning.

Second, why wouldn’t it work? So much has been spent on trying to remove abeta, that serious doubt exists as to whether excessive extracellular Abeta causes Alzheimer’s and even if it does, would removing it be helpful.

Now for some fascinating detail on the paper (for the cognoscenti)

They used a mouse model of Alzheimer’s disease (the 5XFAD mouse). This poor creature has 3 different mutations associated with Alzheimer’s disease in the amyloid precursor protein (APP) — these are the Swedish (K670B), Florida (I716V) and London (V717I). If that wasn’t enough there are two Alzheimer associated mutations in one of the enzymes that processes the APP into Abeta (M146L, L286V) — using the single letter amino acid code –http://www.biochem.ucl.ac.uk/bsm/dbbrowser/c32/aacode.html.hy1. Then the whole mess is put under control of a promoter particularly active in mice (the Thy1 promoter). This results in high expression of the two mutant proteins.

So the poor mice get lots of senile plaques (particularly in the hippocampus) at an early age.

The first experiment was even more complicated, as a way was found to put channelrhodopsin into a set of hippocampal interneurons (this is optogenetics and hardly simple). Exposing the channel to light causes it to open the membrane to depolarize and the neuron to fire. Then fiberoptics were used to stimulate these neurons at 40 Hertz and the effects on the plaques were noted. Clearly a lot of work and the authors (and grad students) deserve our thanks.

Light at 8 Hertz did nothing to the plaques. I couldn’t find what other stimulation frequencies were used (assuming they were tried).

It would be wonderful if something so simple could help these people.

For other ideas about Alzheimer’s using physics rather than chemistry please see — https://luysii.wordpress.com/2014/11/30/could-alzheimers-disease-be-a-problem-in-physics-rather-than-chemistry/