Communication on the Attempt to Visualize Acetylcholine in Rat Neuromuscular Endplates

Arthur M. Howard - (Scotoni), 8421 Dttlikon/ZH ahoscot@sunrise.ch

file set:http://figshare.com/articles/Neuromuscular_Endplate_Exocytosis/888504
1967 was a time when the issue as to whether or not exocytosis of synaptic vesicles actually takes place was still
questioned. Professor Konrad Akert of the Zrich Brain Research Institute came out with a series of anti-exocytosis
papers, and Professor Heuser of the University Basel published a paper suggesting a pinocytosis/exocytotosis recycling
of synaptic vesicles.
In this same period a fixative was developed by me in the Electron Microscopic Central Laboratory of the University of
Zrich the purpose of which was to visualize acetylcholine in neuromuscular end plates. Unexpectedly, an early stage
of this work unambiguously demonstrated exocytosis of synaptic vesicles, their formation from neurotubules, and the
exocytosis of a neurotubule.
This is a long-delayed report on that work and is followed by an outline strategy for a new approach to the problem
based on an application of the findings of Dudley reported in his paper Observations on Acetylcholine (included on
this disc).
Both the project and myself were terminated on Nov. 4 1968 just as I was preparing to start on the new approach
Note: I am 87 years old and this communication is written from memory. I regret that I am unable to give specific
references as I would have in earlier years I just don't get around as well as formerly.
The Initialization of the Project:
Sometime in mid 1967 Dr. Elvira Nichols, assistant to Professor Peter Waser, head of the Pharmacology
Institute of the University of Zurich came to me in the Electron Microscopic Central Laboratory of the University where
I was working as a technician and asked, ostensibly on orders from Professor Waser, if I thought I could undertake an
attempt to find ein frbung fr Acetylcholine. She offered no information other than a reference to a Jabonero
Frbung.
I was 41 years old and was seemingly at a dead end in my struggle to finally get my PhD and here was a
chance to do a doctoral level study where I would call all the shots with respect to design, strategy and execution of the
study and possibly, if successful, use it as a dissertation paper since, although I had exmatriculated from the university I
had attended all the required courses for the doctorate and then some. I jumped at the chance. (see bio at end).
One glance at the Jabonero paper told me that there was no such thing as the Jabonero Frbung. Jabonero
had employed a fixative consisting of bismuth triiodide/osmium tetroxide which had been developed by Millet (spelling
might be wrong), which in turn was an improvement on a fixative utilizing zinc iodide/osmium tetroxide developed by
Champy in the same laboratory in the Sorbonne. (ref. unavailable - Sorbonne publication).

The Concept
The more complete details of the conceptual approach are laid out in my appeal to the Erziehungsdirektion of
the Kanton of Zrich, submitted just before Christmas 1968, which was denied the following January 1969 on the
grounds that I was only a technician.
On deciding to treat the cell as a particularly delicate test tube in which chemical reactions could be carried
out, I chose the approach of the methodology of inorganic qualitative analysis and a check of the chemistry of
acetylcholine in Beilsteins led me to choose phophotungstic acid as my agent of a general precipitant. The technique
was of necessity reversed- through the looking glass one might say - in that whereas in the chemical approach all
groups of precipitated substances were redisolved and specific groups were reprecipitated, in this case my goal
substance would have to be kept insoluble and the impurities eluted out.
Because of foreseeable solubility difficulties:
a. Phosphotungstic acid precipitates are soluble in alcohol.
b. Phosphotungstic acid precipitates must be kept acid. (The effectiveness of phosphotungstic acid as a
precipitating agent is dependent on aggregation of the molecules, - 3,6,9,12- aggregates of
molecules increase with decreasing pH. The 12 aggregate is the most effective agent.)

It was therefore decided to convert the precipitates into lead sulfide just to see how the fixative would work.
Method
Fixatives:
Phophotungstic acid: A dilution series of phosphotungstic acid was titrated against a standard solution of acetylcholine
and the smallest concentration of phosphotungstic acid which produced the most precipitate was chosen.
To this solution osmium tetroxide or glutaraldehyde was added in the standard concentration.
Trichloroacetic acid: Trichloroacetic acid is highly deliquescent, therefore the anhydrous acid was prepared by heating
a measure in the hood to drive out the water and until dense white clouds of acid were given off. This was then titrated
gravimetrically while warm into distilled water to produce a stock solution which was further diluted as required.
The trichloroacetic acid was added only to the phophotungstic acid/osmium fixatives.
The phosphotungstate fixative had a pH of 2.
Trichloroacetic acid fixative (see below) had a pH of 1.
Diaphragm preparation:
Since the severance of the vagus nerve on decapitation of the rats could be expected
to result in massive impulses down the nerve to the diaphragm with consequent depletion of acetylcholine, speed of
preparation was considered to be of utmost importance.
After decapitation, the torso was cut away on both sides of the diaphragm and the bloody diaphragm, stretched in its rib
cage, was plunged in the fixative. Since rapid fixation after death was the goal, nicety of preparation was ignored. With
practice the time from decapitation to entry into
the fixative was less than one minute.
Conversion of Precipitate: After fixation, the diaphragm was cut away from the rib cage. It was then divided into two
samples. One was immersed in lead nitrate for 15 minutes and the other for 2 hours. They were then treated with
hydrogen sulfide to form lead sulfide, dehydrated and embedded in Araldit (Fluka) epoxy resin and sectioned.
Addition of Trichloacetic Acid
Baeyer (Baeyer. Liebig's Annal. 142. p. 322. 1867) found that of all the solvents he tried for the extraction of
acetylcholine from Capsula bursa pastoris (shepherd's purse, Hirtentaschen) trichloroacetic acid was the most efficient.
Trichloroacetic acid was added to the above fixatives in a dilution series from 1% down to 10-4 %.

Observations
Note: It is difficult to describe micrographs of fixative trials without reference to my laboratory notes. I do have the
complete set of films over 400 7x7 cm Zeiss EM9 Agfa-Gevert Scienta films but since these represent the results of
specific fixation experiments, they are of value only as to the anatomical features one can see in the micrographs.
Unfortunately, my lab. notebook was stolen from the laboratory cloakroom where I had laid it down and forgotten it
when I went home.
What I have done is to go through my film archive, rather blindly looking for those synaptic figures which
seem to be of special interest. For this purpose I have scanned at 3oo dpi for general survey and rescanned at 12000
dpi those films with configurations of special interest. Unfortunately, the EM work was done at magnifications of 6000
and 18000 and much of the illustrations here are disappointing as to resolution, less because of the scan resolution than
of the grain of the film which set an upper limit on magnification.
The scanner was an Epson Perfection 4180.All scans made are available on this disc in JPEG format.
Viewing was done with Photoshop Elements 12. Aside from viewing, the only photoshop features which were
used were lighting and contrast adjustments, enlarging and the cropping tool to extract interesting segments.
All scans can be viewed in their appropriate folders to be found on this disc. All films were of rat diaphragms
fixed with osmium/PTA and substituted with lead sulfide.A few glutaraldehyde-PTA exocytotic films at 38000 EM mag
and 12000dpi scan are also present.
A. Both the osmium and aldehyde fixatives were clean of contamination.
B. Precipitations found in both the osmium and aldehyde fixations were similar, but since this was a fixation
study, and the aldehyde fixations, because of their low contrast were more difficult to read, further work was

concentrated on the osmium/phophotungstic acid fixations. Precipitates were randomly distributed among the synaptic
vesicles. There seemed to be no discernible pattern as to which vesicles had precipitates and which didn't.
C. Vesicle-sized indentations were found in the presynaptic membrane in which precipitate could be seen.
Exocytotic tubules were found.
D. Some neurotubules, on entering the synaptic area showed constrictions which gave them a sausage-string
appearance. Often chains of vesicles could be seen continuing in the line of the tubules. (see:Illustrations folder /film
263 vesicle chains. Barrage and vesicle chains 2 & 3 in Illustrations folder are enlargements from this same plate. )
E. Precipitates formed by a 15 minute immersion in Pb(NO3)2 were punctuate, those from the 2 hour
immersion were course squares.
F. In some clear areas a thin line of precipitate can be seen in the synaptic cleft equidistant from and parallel to
the pre- and postsynaptic membranes.
G. In some areas, clumps of fine black streaks barragecould be observed which stretched completely across
the space between the pre- and postsynaptic membranes. An amorphous cloud of precipitate could be seen in the muscle
at the point where these streaks touched the postsynaptic membrane.(illustrations folder:Barrage). At the end of the
clump of streaks vesicles, broken open, with horseshoe shape and streaks emanating from their interior towards the
presynaptic membrane can be seen. All streaks emanate from vesicles toward the presynaptic membrane and none away
from the membrane.
Note the square shape of the precipitates of cubic crystals of PbS. It is this which gives me hope that the
octahedral form of the acetylcholine-choline dichloroplatinate can be recognized in micrographs.
H. neurotubules were free of precipitate in the phophotungstic acid fixations but were filled with precipitate
when trichloroacetic acid was added. In 10 micron sections, the axons were stained a very dense black.

Discussion
The work described here was done in the 16 months between mid 1967 and November 1968. In the years
which followed I kept up with the field as best I could until 1975 after which I forcibly divorced myself from following
progress in the field for my own mental health since I am, by nature a hands on person and following current research
without being able to do was too painful. Because of this, this communication must be viewed as a period piece almost
half a century old and, since I am unqualified to discuss any of this work in the light of modern knowledge in the field, I
will limit the discussion to the most general knowledge of the field as I knew it then.
Unfortunately, there was hardly a day in the past 45 years when I did not find myself doing thought experiments on the
development of chloroplatinate fixation with the application of Dudley's discoveries. May this communication finally
lay the ghost.
On fixation pH.
EM fixations are usually carried out between pH 7.2 7.4. A general rule in micrology is that acid fixations are
best and indeed, the early workers in ultrastructure micrology did use acid fixation. The trouble in EM work was
embedment. Embedment in early EM work was done in acrylic resins and these tended to explode the tissues. I
believe it was Palade who discovered that it was impossible to rinse the acid out of the tissues and it was this residual
acid which reacted with the acrylics resulting in the explosions. The cure was buffering. With the introduction of
epoxies as embedment agents the buffering was continued. There was no apparent reason to go back to acid fixations.
Phophotungstic acid fixations required acid fixation and I, knowing the history of buffered fixations did not hesitate to
fix at pH 1 and 2. As it turned out, acid fixation revealed synaptic figures which were not demonstrated with buffered
fixatives.
One can ask: Why? What worked, phophotungstic acid or acid fixation? My bet is on pH. I would suggest that
high pH facilitates the breakdown of neurotubules into vesicles.
A. Cleanliness of a fixation is essential for proper assessment of the results. A single fixation with bismuth
triiodide/osmium revealed too much material brought down by the bismuth and I gave up on the fixative after
examining a few grids.
B. The precipitates here were lead sulfide and mean nothing with respect to acetylcholine. Phophotungstic acid
was used as a general precipitating agent and the precipitates represented acetylcholine, choline, K, Ca, Mg, amines,
proteins and anything else.

C & D. The combination of these figures, especially the precipitates within membrane indentations, coupled
with the tubule constrictions present strong evidence for the formation of vesicles from neurotubules and their
subsequent exocytosis. Whether or not these precipitates represented acetylcholine, that was what I hoped to ascertain
with a chloroplatinate fixation.
E. The difference in crystal size with respect to the length of time of immersion provides a useful guide
towards adjusting precipitations of metal salts for viewing. They should be not to small nor so large as to obscure tissue
elements.
F. These figures can been seen in many osmium fixations by many authors and are what one would expect to
see when positively-charged substances are found on an electophoric gel between two positively-charged membranes
as is the case when both the nerve and muscle are in the resting state.
G. The impression is of substance flowing across the inter-membrane space. A valid criticism could be that it is
merely a membrane in planar section. The dense amorphous cloud within the muscle at the other side of the membrane
would lead one to the former conclusion however, and in my missing notebook I referred to them as a barrage. Since
these were studies to determine the effects of various fixations, few high resolution micrographs were made since
magnifications of 6,000 and 18,000 were sufficient.
Does this represent real substance moving across the cleft and if yes, what? ACh? K? The fact that this
substance seems to come solely from both intact and burst vesicles is fully in accord with the observation of quantal
release of neurotransmitter (Fatt & Katz (1952) J. Physiol. 117 109-1239) and strengthens the suspicion that it is indeed
Ach.
Does this negate the theory of transmission by vesicle exocytosis? I think not. To the contrary, this fully
supports the observations of Fatt and Katz. Single vesicle or single microtubule exocytosis would explain their
observation of resting phase mini-potentials as the base quantum of transmitter release and their observation that the
massive release of transmitter is a multiple of the single quantum release could be explained by a non-exocytotic
release of vesicle content through a local destabilization of the both the axon membrane and the membranes of those
vesicles in the vicinity. This we can see as the barrage pattern. Hence, both types of release can be supporting the view
that a single vesicle is the base quantum of synaptic transmission.
All this rests on the assumption that we are dealing with Ach here. While this circumstantial evidence is very
strong and answers most questions, it is still circumstantial. Only positive labeling of the ACh molecule will prove the
case beyond all doubt.
H. I found reference to Baeyer's 1867 paper in Beilsteins. In checking it out I found that one of his interests
here was in recovering as much acetylcholine from his samples of shepherd's purse as possible and trichloroacetic acid
was the best agent of all those which he tested.
In deciding to apply trichloroacetic acid to the tissue I reasoned that if the precipitates I found in the vesicles
represented, at least in part, acetylcholine, and, as it seemed, the vesicles were derived from the neurotubules, then the
lack of precipitate within the tubules meant that the acetylcholine must be bound and trichloroacetic acid, as used by
Baeyer should liberate it. It followed also that the conditions at the synapse were different to those within the
neurotubule such that the acetylcholine was liberated there.
Trichloroacetic acid, in 10% concentration as used by Baeyer is a powerful macerating agent and I dared not
use it in that concentration. I therefore set up a dilution series with the phophotungstic acid/osmium fixative from 1%
down to 10-4 %.
The fixation in all concentrations resulted in a heavy precipitate in the tubules, which, in the light microscope
stained the axons heavy black. This supported the assumption that I might indeed be dealing with acetylcholine. It only
needed to be proven.
If the prospective chloroplatinate fixative proves to be successful, the axons, with the addition of
trichloroacetic acid to the standard fixative, should stain yellow to orange when viewed in the light microscope and, if
the precipitate is not too dense to prevent viewing, octahedral crystals should be discernible. At this point the
experience with the growth of the precipitate of the lead sulfide crystals against immersion time should serve as a
guide. Less and smaller crystals may reveal more.
Orange octahedral crystals, under the proposed conditions of fixation, would be a positive proof of identity and
localization of acetylcholine within the tissues in which they are found.
Note: I could not find the films of PTA-tricloroacetic acid, but I did find a PTA-glut scan of an entire EP with
a barrage hot spot -some 15 films. It is in a separate folder.
A Strategy For The Development of a Specific Fixative For Acetylcholine
Because of the rarity of octahedral crystal forms amongst the substances found in tissues, Dudley's discovery
that acetylcholine chloroplatinate and choline chloroplatinate always form the double compound acetylcholine-choline

dichloroplatinate and that the crystals of this compound are octagonal opens the possibility of designing a fixative
which could specifically label acetylcholine such that it could be followed through the synaptic sequence.
The yields that Dudley obtained, while satisfactory for the chemist whose prime interest is in obtaining
sufficient material for analysis, are unsatisfactory for the micrologist working at the ultra structural level where
maximally efficient labeling and precise localization of the molecule is essential.
Hydrolysis of the acetylcholine, which was a great problem for Dudley is no problem for the micrologist since
any acetylcholine which breaks down to choline would be immediately bound into the less soluble double platinate
compound which is our goal anyway.
On the other hand, we do want to keep these substances nailed down until they have been sufficiently
enmeshed and their location stabilized by the cross-linking component of the fixative.
As cross-linker osmium is out since it not only cross links but stains as well. An aldehyde, formaldehyde out of
paraformadehyde (thermal dissociated, not hydrolyzed dissociated) or glutaraldehyde would be the fixative of choice.
Dudley carried out his extractions with 95% ethanol in which ACh is not soluble. For the ultrastructure
micrologist this not acceptable due to the possible distortions caused by the extreme concentration gradients. An
aqueous fixative would be preferable, but this raises the solubility problem. A fixative out of 30% ethanol would be
preferable and I have gone from aqueous fixative directly into 50% alcohol without harm but this was with already
fixed tissue. As an initial attempt, I would make use of the fact that ACh is insoluble in diethyl ether and make an initial
fixative with 30% ethanol saturated with ether. I know of no instance in the literature where ether has been used in a
fixative but the worst that can happen would be a failed trial. Assuming that ether has no deleterious effects on the
fixation, I would use ether-alcohol throughout the dehydration process. It should mix well with the epoxy resin and,
given its volatility could be easily removed from the blocks by gentle warming should the blocks prove to be too soft.
The trickiest part here is the addition of platinum chloride to our fixative. Initially, I would use the same
method for determining the concentration of platinum chloride as I did with PTA, namely the lowest concentration of
platinum chloride titrated against a standard solution of ACh which yields the most precipitate.
Applying platinum chloride to a tissue should yield chloroplatinates of choline, acetylcholine, potassium,
ammonium, calcium and sundry other unknowns. All of these would have needle shaped precipitates except where
choline and acetylcholine are close enough to each other to react in which case the choline-acetylcholine
dichloroplatinate octahedra would be formed until the smallest amount of reactant has been used up.
But which?
Has all of the Ach been reacted? in which case our job is done and we can proceed to dehydrate and embed.
On the other hand, if, in this micro-locality the ACh is in excess, it will remain as the chloroplatinate and could
be lost. Our EM grid would then have a number of octahedra and a mess of needles of unreacted ACh, choline-cholinedichloroplatinate as well as the chloroplatinates of all of the above-mentioned elements and it becomes obvious that
much preliminary work must be done on the relative solubilities of all of these contaminants so that they can be
removed before the next stage of the fixation can be initiated, namely, the conversion of all residual ACh into the
choline-acetylcholine chloroplatinate.
Assuming: contaminating chloroplatinates have been removed and ALL traces of the original platinum chloride
reactant have been removed, we can then treat the tissue en block or already embedded on the grid with an alcoholic
solution of choline to convert all remaining ACh to the double compound. It is important at this point that none of the
original platinum chloride be present or it would react with the choline to yield a contamination of crystals of the
dicholine-dichlroplatinate.
This was my initial plan of procedure in 1968 when the project was terminated. As with the PTA fixation
where I had to do much donkey work before I would sacrifice my first rat. -Indeed, my first osmium PTA fixation was
on mushroom mycelia just to see how the tissues would look.- I foresee that much preliminary study will be necessary
before initial fixations could be carried out.
In summary: the recipe would be as follows:
Aldehyde in standard fixation concentrations
Platinum chloride-concentration to be determined
30% ether-alcohol
dehydrate in ether-alcohol
additional measures to remove contaminants if necessary
Follow-up treatment after removal of all platinum chloride: choline in 70%
total conversion of ACh.
Rinse to remove unreacted choline and embed..

or higher ether-alcohol to affect

Bio
Born Brooklyn N.Y. In 1926.
Educated in the NY public school system and after service in the US Army Air Force during WWII I used the
GI Bill of Rights and took a Bachelor of Arts degree in 1948 in NYU Washington Square College with a major in
biology, first minor in chemistry and a second minor in psychology.
I entered Bern Medical School in October 1949 and passed everything except physiology with Professor

Alexander von Muralt. Professor von Muralt and I had serious disagreements as to the nature and teaching of
physiology and especially as to what constitutes an examination in the subject. I guess I had been spoiled by 2 semesters
of lectures and laboratory with Professor Alexander Sandow in NYU where I had done quite well in exactly the same
material.
After Bern I entered the University of Zrich in 1953 studying the genetics of arginine dependent mutants of
the yeast Schizosaccharomyces pombe with Pd.Professor Urs Leupold. This was the year Watson and Crick described
the double helix of DNA and molecular biology was an unimaginable dream. During this time of study I attended all
required courses for a PhD including extras such as a summer course in quantative organic microanalysis in the
chemistry institute.
Familial financial difficulties forced me to discontinue my studies and I married my girl friend in July 1958
who died in 2006.
There followed various jobs, salesman in Germany, odd translation work, 5 months praktikant in the
Eidgenossische Versuchsanstalt Wdenswil (study of ester synthesis in the yeast Hansenula minuta), 2 years technician
in Hauser Champignon AG (compost analyses, tracking nematode infections in mushroom beds) and finally as
technician in the EM Central laboratory of the U. Zrich.
From 1969 until 2006 when I retired at age 80 my wife and I owned and operated a boarding kennel for dogs
(Hoscot Hunde Park).
Why do I write this now, after 45 years? I am the type of person who always wants to know, how? what? why?
And this project was discontinued just at the point where I saw the possibility of developing a tool to specifically label a
molecule vital to neurotransmission such that it might make it possible to follow it through the synaptic process.
Unfortunately, what took place in that EM lab was so vile that the love of
my life basic research into
the nature of things, became anathema to me.
Despite that it haunted me, tore me apart and I had to consciously turn off to protect my mental state. And yet,
there was hardly a day in the past 45 years when I didn't find myself doing thought experiments as Einstein put it.
Most recently I came across 2 books: Without Conscience by Robert D. Hare,PhD and The Sociopath Next
Door by Martha Stout PhD and I was able to accept that which, until then, was unthinkable. Namely, I was a pawn on a
Machiavellian chessboard, a chessboard set up and propagated by doctors of medicine, people sworn to do no harm
and, when the pawn threatened to cross the board to the far side it had it be stopped and eliminated. That's o.k. for
chess, but I was a human being.
Understanding what happened to me released a powerful mental block and hence this communication. The
University of Zrich paid for this research and here is the report I refused to write for a pit full of vipers 45 years ago.
-----------------------------------------------------------------------------------------------This communication disc may be freely reproduced and disseminated by any means of communication desired.
Arthur M Howard - (Scotoni) 29 September 2013