The staggering implications of one axon synapsing on another

It isn’t often that a single paper can change the way we think the brain works.  But such is the case for the paper described in the previous post (full copy below *** ) if the implications I draw from it are correct.

Unfortunately this post requires a deep dive into neuroanatomy, neurophysiology, neuropharmacology and cellular molecular biology.  I hope to put in enough background to make some of it comprehensible, but it is really written for the cognoscenti in these fields.

I’m pretty sure that some of these thoughts are both original and unique

Briefly, the paper provided excellent evidence for one axon causing another to fire an impulse (an action potential).   The fireror was from a neuron using acetyl choline as a neurotransmitter, and the fireree was a dopamine axon going to the striatum.

Dopamine axons are special.  They go all over the brain. The cell body of the parent neuron of the axon to be synapsed on uses dopamine as a neurotransmitter.  It sits in the pars compacta of the substantia nigra a fair piece away from the target they studied (the striatum). “Individual neurons of the pars compacta are calculated to give rise to 4.5 meters of axons once all the branches are summed”  — [ Neuron vol. 96 p. 651 ’17 ].”  These axons release dopamine all over the brain.  There aren’t many dopamine neurons to begin with just 80,000 which is 1 millionth of the current (probably unreliable) estimate of the number of neurons in the brain 80,000,000,000.

Now synapses between neurons are easy to spot using electron microscopy.  The presynaptic terminal contains a bunch of small vesicles and is closely apposed (300 Angstroms — way below anything the our eyes can see) to the post synaptic neuron which also looks different, usually having a density just under the membrane (called, logically enough, post-synaptic density).  Embedded in the postsynaptic membrane are proteins which conduct ions such as Na+, K+, Cl- into the postsynaptic neuron triggering an action potential.

But the dopamine axons going all over the brain have a lot of presynaptic specialization, but in many of the cases the post-synaptic neuron and its postsynaptic density is nowhere to be found (or the receptors for dopamine aren’t near the presynaptic specialization).  This is called volume neurotransmission.

However, in the nuclei studied (the striatum) dopamine synapses on dendrites of the major cell type (the medium spiny neuron) are well described and the 5 receptors for dopamine (called G Protein Coupled Receptors — GPCRs) are found there.  None of the GPCRs conduct ions or trigger action potentials (immediately anyway).  Instead, they produce their effects much more slowly and change the metabolism of the interior of the cell.  This is true for all GPCRs, regardless of the ligand activating them — and humans have 826 GPCR genes.

Note also that volume neurotransmission means that dopamine reaches nonNeuronal tissue — and there is good evidence that dopamine receptors are present on glial cells, pericytes and blood vessels.

The story doesn’t end with dopamine.  There are 3 other similar systems of small numbers of neurons collected into nuclei, using different neurotransmitters, but whose axons branch and branch so they go all over the brain.

These are the locus coeruleus which uses norepinephrine as a neurotransmitter, the dorsal raphe nucleus which uses serotonin and the basal nucleus of Meynert which uses acetyl choline.  There is excellent evidence that the first two (norepinephrine and serotonin) use volume neurotransmission. I’m not sure about those of the basal nucleus of Meynert.

What is so remarkable about the paper, that it allows the receiving neurons to (partially) control what dopamine input it gets.

All norepinephrine receptors are GPCRs, while only one of the 16 or so serotonin receptors conducts ions, the rest being GPCRs.

Acetyl choline does have one class of receptors (nicotinic) which conducts ions, and which the paper shows is what is triggering the axon on axon synapse.  The other class (muscarinic) of acetyl choline receptor is a GPCR.

Addendum 29 September — it goes without saying (although I didn’t say it) that any molecule released by volume neurotransmission doesn’t confine itself to finding targets on neurons.  Especially with norepinephrine, it could bind to receptors for it on the vasculature causing circulatory effects.  They could also bind to GPCRs on pericytes and glia.

Now the paper tested axon to axon firing in one of the four systems (dopamine) in one of the places its axons goes (the striatum).  There is no question that the axons of all 4 systems ramify widely.

Suppose axon to axon firing is general, so a given region can control in someway how much dopamine/serotonin/norepinephrine/acetyl choline it is getting.

Does this remind you of any system you are familiar with?  Perhaps because my wife went to architecture school, it reminds me of an old apartment building, with separate systems to distribute electricity, plumbing, steam heat and water to each apartment, which controls how much of each it gets.

Perhaps these four systems are basically neurological utilities, necessary for  the function of the brain, but possibly irrelevant to the computations it is carrying out, like a mother heating a bottle for her baby in water on a gas stove on a cold winter night.  The nature of steam heat, electricity, water and gas tell you very little about what is going on in her apartment.

The paper is so new (the Neuron issue of 21 September) that more implications are sure to present themselves.

Quibbles are sure to arise.  One is that fact that the gray matter of our brain doesn’t contain much in the way of neurons using acetyl choline as a neurotransmitter.  What it does have is lots of neurons using GABA which we know can act on axons, inhibiting axon potential generation.  This has been well worked out with synapses where the axon emerges from the neuron cell body (the initial segment).  However the different ionic composition of axons in the developing brain results in GABA having an excitatory effect.  Perhaps ionic composition varies in different parts of the neuron.

The work was done in living animals, so the paper contains no electron micrographs.  Such work is sure to be done.  No classical presynaptic apparatus may be present, just two naked axons touching each other and interacting by ephaptic transmission (the term does not appear in the paper).

So a lot of work should be done, the first of which should be replication. As the late Carl Sagan said “extraordinary claims require extraordinary evidence”.

Finally:

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.”

 

The brain gets more complicated the closer you study it

When our tools were blunt, the brain looked a lot simpler than it does now.

Example #1:  Locus coeruleus (LC).  This is a tiny group of neurons deep in the brain.  It looks blue to the naked eye, if you’ve gone to medical school as I have and dissected a brain.  This is held to be due to neuromelanin produced by the neurotransmitter  it uses (norepinephrine).  Neurons using dopamine as a neurotransmitter also produce neuromelanin but it’s brown.  The two differ by just one oxygen atom.

The LC is tiny in primates, only 15,000 – 50,000 neurons depending on species and who you read..  The rat (where most experiments are done) has only 1,500 in a space 1000 microns (1 millimeter) x 200 x 500 microns.

So until now any attempt to stimulate the locus coeruleus with an electrode alerted the animal.

Why? Two reasons.

1. The LC is so small that any electrode stimulating it, stimulated every neuron.

2.  Because that tiny nucleus sends fibers all over the brain, releasing norepinephrine everywhere, and not just at synapses.  This is called volume neurotransmission. Most places on the axons of a LC neuron showing synaptic vesicles (where norepinephrine is found), don’t have a dendrite or any sort synaptic specialization next to them.  So the LC innervates the whole brain, in the same way that our brain innervates our muscles.

Stimulate the LC of a rat and the brain is flooded with norepinephrine and the animal wakes up.

Well that was the case until technology marched on and miniaturization of electrodes allowed us to record from a 10 – 20 neurons at a time in the LC when stimulation was applied.  Those responding to a given stimulus were called ensembles.  Of 285 single LC neurons studied in 15 rats, 115 participated in multiple ensembles, 149 participated only in a single ensemble and 21 didn’t participate in any.  Activity of different ensembles produced different brain states — not all were wide awake.  You can read all about this in Proc. Natl. Acad. Sci. vol. 119 e21116507119 ’22.

Volume neurotransmission is  important because the following neurotransmitters use it — dopamine, serotonin, acetyl choline and norepinephrine. Each has only a small number of cells using them as a transmitter.  The ramification of these neurons is incredible.

For instance, “human serotonergic neurons, which are estimated to extend axons for 350 meters”  [ Science vol. 366 3aaw9997 p. 4 ’19 ], so the fibers are everywhere in the brain.  I couldn’t find a statistic for axons of the locus coeruleus but those of neurons using acetyl choline as a neurotransmitter are estimated to have axons extending for 31 meters.

So now you see why massive release of any of the 4 neurotransmitters mentioned (norepinephrine, serotonin, dopamine, acetyl choline) would have profound effects on brain states.  The four are vitally involved in emotional state and psychiatric disease. The SSRIs treat depression, they prevent reuptake of released serotonin.  Cocaine has similar effects on dopamine.  The list goes on and on and on.

Maybe be we’ll be able to slice and dice these nuclei in the future to produce more subtle effects on brain function.

 

Example #2:  Dendritic diversity  — that’s for next time.  This post is long enough.

How flat can a 100 amino acid protein be?

Alpha-synuclein is of interest to the neurologist because several mutations cause Parkinson’s disease or Lewy Body dementia.  The protein accumulates in the Lewy Bodies of these diseases.  These are concentric hyaline inclusions over 15 microns in diameter found in pigmented brain stem nuclei (substantia nigra, locus coeruleus).

The protein contains 140 amino acids.  It is ‘natively unfolded’ meaning that it has no ordered secondary structure (alpha helix, beta sheet).  No one is sure what it does.  Mouse knockouts are normal, so the mutations must produce something new.

Alpha-synuclein can form amyloid fibrils, which are basically stacks of pancakes made of flattened segments of proteins one on top of the other.

Would you believe that the 100 amino terminal amino acids of alpha-synuclein can form an absolutely flat structure.  Well it does and there are pictures to prove it in PNAS vol. 117 pp. 20305 – 20315 ’20.  Here’s a link if you or your institution has a subscription — https://www.pnas.org/content/pnas/117/33/20305.full.pdf.

This isn’t the usual alpha-synuclein, as it was chemically synthesized with phosphorylated tyrosine at amino acid #39.  Who would have ever predicted that 100 amino acids could form a structure like this?  I wouldn’t. The structure was determined by cryoEM and all the work was done in China.  Very state of the art world class work.  Bravo.

The four hour cure for depression: what is Ketamine doing?

It is a sad state of affairs when you look forward to writing a post on depression.

https://www.fox32chicago.com/news/79-shot-15-fatally-over-fourth-of-july-weekend-in-chicago

https://www.straitstimes.com/asia/south-asia/indian-grooms-wedding-funeral-leave-more-than-100-infected-with-coronavirus

From Nature 2 July — “G4 a type of swine flu virus from China can proliferate in human airway cells.  34/338 pig farm workers in China have antibodies to it.  In ferrets G4 causes lung inflammation and coughing.”

Well that’s enough reason to flee to the solace of the basic neuroscience of depression.

 

 

The drugs we use for depression aren’t great.  They don’t help at least a third of the patients, and they usually take several weeks to work for endogenous depression.  They seemed to work faster in my MS patients who had a relapse and were quite naturally depressed by an exogenous event completely out of their control.

Enter Ketamine which, when given IV, can transiently lift depression within a few hours.  You can find more details and references in an article in  Neuron ( vol. 101 pp. 774 – 778 ’19)  written by the guys at Yale who did some of the original work. However, here’s the gist of the article.  A single dose of ketamine produced antidepressant effects that began within hours peaked in 24 – 72 hours and dissipated within 2 weeks (if ketamine wasn’t repeated).  This occurred in 50 – 75% of people with treatment resistant depression.  Remarkably one third of treated patients went into remission.

This simply has to be telling us something very important about the neurochemistry of depression.

Naturally there has been a lot of work on the neurochemical changes produced by ketamine, none of which I’ve found convincing ( see https://luysii.wordpress.com/2019/10/27/how-does-ketamine-lift-depression/ ) until the following paper [ Neuron  vol. 106 pp. 715 – 726 ’20 ].

In what follows you have to have some basic knowledge of synaptic structure, but here’s a probably inadequate elevator pitch.  Synapses have two sides, pre- and post-.  On the presynaptic side neurotransmitters are enclosed in synaptic vesicles.  Their contents are released into the synaptic cleft when an action potential arrives from elsewhere in the neuron.  The neurotransmitters flow across the very narrow synapse to bind to receptors on the postsynaptic side, triggering (or not) a response of the postsynaptic neuron.  Presynaptic terminals vary in the number vesicles they contain.

Synapses are able to change their strength (how likely an action potential is to produce a postsynaptic response).  Otherwise our brains wouldn’t be able to change and learn anything.  This is called synaptic plasticity.

One way to change the strength of a synapse is to adjust the number of synaptic vesicles found on the presynaptic side.   Presynaptic neurons form synapses with many different neurons.  The average neuron in the cerebral cortex is post-synaptic to thousands of neurons.

We think that synaptic plasticity involves changes at particular synapses but not at all of them.

Not so with ketamine according to the paper.  It changes the number of presynaptic vesicles at all synapses of a given neuron by the same percentage — this is called synaptic scaling.  Given 3 synapses containing 60  50 and 40 vesicles, upward synaptic scaling by 20% would add 12 vesicles to the first 10 to the second and 8 to the third.   The paper states that this is exactly what ketamine does to neurons using glutamic acid (the major excitatory neurotransmitter found in brain).  Even more interesting, is the fact that lithium which treats mania has the opposite effects decreasing the number of vesicles in each synapse by the same percentage.

I found this rather depressing when I first read it, as I realized that there was no chemical process intrinsic to a neuron which could possibly work quickly enough to change all the synapses at once.  To do this you need a drug which goes everywhere at once.

But you don’t. There are certain brain nuclei which send their processes everywhere in the brain.  Not only that but their processes contain varicosities which release their neurotransmitter even where there is no post-synaptic apparatus.  One such nucleus (the pars compacta of the substantia nigra) has extensively ramified processes so much so that “Individual neurons of the pars compact are calculated to give rise to 4.5 meters of axons once all the branches are summed”  — [ Neuron vol. 96 p. 651 ’17 ].  So when that single neuron fires, dopamine is likely to bathe every neuron in the brain.  We think that something similar occurs in the locus coeruleus of the lower brain which has only 15,000 neurons and releases norepinephrine, and also in the raphe nuclei of the brainstem which release serotonin.

It should be less than a surprise that drugs which alter neurotransmission by these neurotransmitters are used to treat various psychiatric diseases.  Some drugs of abuse alter them as well (Cocaine and speed release norepinephrine, LSD binds one of the serotonin receptors etc, etc.)

The substantia nigra contains only 450,000 neurons at birth, so you don’t need a big nucleus to affect our 80 billion neuron brains.

So the question before the house, is have we missed other nuclei in the brain which control volume neurotransmission by glutamic acid?   If they exist, could their malfunction be a cause of mania and/or depression?  There is plenty of room for 10,000 to 100,000 neurons to hide in an 80 billion neuron brain.

Time to think outside the box people. Here is an example:  Since ketamine blocks activation of one receptor for glutamic acid, could there be a system using volume neurotransmission which releases a receptor inhibitor?

Addendum 7 July — I sent a copy of the post to the authors and received this back from one of them. “Thank you very much for your kind words and interest in our work. Your explanation is quite accurate (my only suggestion would be to replace “vesicles” with “receptors”, as the changes we propose are postsynaptic). Reading your blog reassures us that our review article accomplished its main goal of reaching beyond the immediate neuroscience community to a wider audience like yourself.”