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Silver staining of the synapse in the human cerebrum Morrison, George Edward 1951

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SILVER  STAINING  OF THE SYNAPSE  IN THE HUMAN  CEREBRUM  by  GEORGE EDWARD MOR'RI SON, JR.  A THESIS SUBMITTED I N PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF  ARTS  MASTER OF  i n the Department of B i o l o g y and B o t a n y  We a c c e p t t h i s t h e s i s as conforming t o  the  s t a n d a r d r e q u i r e d from c a n d i d a t e s f o r the degree o f MASTER OF  Members o f the Department o f  BIOLOGY a n d BOTANY  THE  UNIVERSITY  OF BRITISH  October,  1951  COLUMBIA.  SILVER STAINING OF THE SYNAPSE IH THE' HUMAN CEREBRUM  ABSTRACT. There has been recent tendency on the part of certain investigators, because of their failure to demonstrate boutons terminaux i n the cerebral cortex by the silver impregnation methods, to suggest that the pericellular fibers i n the cerebral cortex end freely on the cells and that such free terminals are the normal form of synapse i n this part of the central nervous system.  In the present work a histological study was made of  certain areas i n the frontal cerebral and the visual cortex to show the presence of normal boutons terminaux.  It was demon-  strated that boutons occur i n these areas i n sufficient numbers to indicate that these are the normal means of synapse. The direction which further research should take i s suggested.  TABLE OF CONTENTS  Abstract Introduction REVIEW OF LITERATURE (a.)  The Neurone Theorycontact vs . continuity  (b.)  The Synapse 1. The morphology of the synapse . 2. The mechanism of synaptic transmission.  1 11 11 15  EXPERIMENTAL PROCEDURE  19  EXPERIMENTAL RESULTS  22  DISCUSSION  25  SUMMARY  30  ILLUSTRATIONS APPENDIX OF SILVER TECHNIQUES BIBLIOGRAPHY  Acknowledgement i s made to Dr. William C. Gibson, Director of the Crease C l i n i c Research Unit, f o r his generous assistance, advice and many kindnesses during the preparation of t h i s work.  To Dr..A. H. Hutchinson, Head of the Department  of Biology and Botany, sincere gratitude i s extended f o r h i s advice and many considerations. Appreciation i s also due to the members o f the Crease C l i n i c Research Unit who have helped i n many ways  -  and many thanks to my mother, father  and wife f o r t h e i r patience. The work f o r t h i s thesis was c a r r i e d out during the tenure o f a Research Fellowship granted by the Crease Clinic.  INTRODUCTION  The purpose of the present work has been to establish and standardize the simplest reliable and consistent methods for the demonstration of nerve fibers and interneuronal connections i n the cerebral cortex of man i n order to apply these methods to the brains of mental patients coming to autopsy following prefrontal lobotomy. The methods to be studied are reduced silveE and double impregnation silver procedures such as those used by Cajal, Bielschowsky and Del Rio Hortega.  In addition to the main search for dependable methods  of staining for degenerating nerve fibers, there has been an interest i n the " contact vs. continuity " dispute which has so long pointed up the mystery of the interneuronal connection. Some re-examination of the literature pertaining to this controversy, therefore, has been thought advisable, confined largely to a discussion of the histologi c a l evidences. A number of methods have been recorded and discussed i n detail and many others mentioned. Some evidence of the nature of the  1  boutons terminaux ' i n the cerebral cortex of man i s recorded.  - 1 ( a ) The Neurone Theory - Contact vs. Continuity. Knowledge of the structure of the interneuronal connections can only be as accurate as the means used for their demonstration are dependable. Since Golgi i n 1885 developed a technique for staining chrome-hardened nervous tissue with a silver solution many mysteries of the working of the brain have unfolded under the microscope. From observation of spinal cord stained by his new method Golgi became convinced that " i n the grey substance of the spinal cord there exists an exceedingly extensive and delicate network " , which he considered was due to anastomosing of the collateral branches and not as Gerlach, the originator of the reticular hypothesis before supposed, to anastomosing of the dendrites. Retzius, von Kolliker and others ( Retzius, 1908 ) failed to reproduce Golgi's results satisfactorily at that time. Retzius, using Ehrlich's methylene blue method , could find no such network as described by Golgi but i n the ganglia of crawfish he was able to see " not a network of reticulum, but a twistwork ". His and Forel (1883) could not imagine that any protoplasmic connection existed between one nerve c e l l and the next and proposed the theory that the processes of the nerve cells end freely i n the grey matter and are not intertwined i n elaborate network. This theory was not, however, supported by objective evidence and had never been widely accepted. At about this time a young Spanish neurologist and professor of Histology, Santiago Ramon y Cajal, published a series of works on  - 2 -  the minute structure of the brain. Wilhelm His of Leipzig had formed the conviction long before (1883) that nerve cells developed as organs independent of one another. This view was now accepted by Cajal on the basis of his own researches using the Golgi method. Cajal (1891) was unable to see the reticulum described by Golgi and could not see in any place the processes of two different nerve cells anastomosing. " Les fibres s'intrelacaient d'une roaniere fort compliquee, engendrent un plexux enchevetre et serre, mais jamais un reseau ". From his observations on .the cerebellum of the bird " on dirait que chaque element est un canton physiologique absolutment autonome ". Delivering the Croonian Lecture to the Royal Society i n 1894 he said, " Les connexions etablies entre les fibres et les cellules nerveuses ont l i e u au moyen de contact, c'est-a-dire a. laide d'une veritable articulation entre les arborizations variqueuses des cylindresaxes d'une cote, l e corps et les prolongments protoplasmiques de 1'autre. Aussi est on amene a se represente l'axe encephalo-spinal comme un edifice compose d'unites nerveuses superposees, de neurones , suivant 1'expression de Waldeyer."  Waleyer, a erman anatomist, had written G  a resume of Cajal's ideas and popularized the neurone doctrine. In 1891 Held reported his discovery of the " calices " or synapses of the nucleus of the trapezoid body using the methods of Golgi and N i s s l .  In 1897 he published a report of his " Endefusse " which  Auerbach confirmed i n 1899 and called " boutons terminaux  The con-  ception of the continuity between the Endefusse and the reticulum of the neurone was published by Held ( 1904 - 1905 ) and f i n a l l y accepted by Auerbach.  Held believed the Endefusse to be imbedded i n the inter-  - 3s t i c e s of the  p e r i c e l l u l a r nerve net " and saw continuity with the  n  inner reticulum as w e l l .  Auerbach, however, thought that a transparent  c u t i c l e intervened. There existed, then, i n Europe two opposing theories on the i n timate structure of the nervous system.  Golgi, Gerlach and Held  strongly advocated the concept of the Gargantuan spider-web hypothesis of c o n t i n u i t y .  -  the  His, von K o l l i k e r , von Lenhossek, van  Gehuchten and Retzius a l l supported the neurone doctrine of Cajal and Waldeyer  -  the hypothesis of contact and the autonomy of the  neurone• In 1897 an Hungarian zoologist, Stephen Apathy, published a work " Das leitende Element des Nerven-systerns und seine topographischen Beziehungen zu den Z e l l e n " i n which he b u i l t up a theory regarding the minute structure of the nervous system.  In t h i s he  theorized that the n e u r o f i b r i l s form the s p e c i f i c constituent of the nervous system and that they were the independent s t r u c t u r a l parts, the conducting elements.  In the nerve f i b r e s they preserved t h e i r  i n d i v i d u a l i t y , forming r e t i c u l a only i n s p e c i f i c places.  He maintain-  ed that they formed large continuous r e t i c u l a i n the organs of the body and above a l l i n the c e n t r a l grey substance. The evidence f o r these conclusions was obtained with the help of a new gold s t a i n i n g method• Apathy was l a t e r vigorously supported by a German h i s t o l o g i s t , Stephen Bethe, (  ) who became one of the chief opponents of the  neurone theory. The Golgi technique on which these opposing theories are l a r g l y bases was crude compared to the more delicate present day s i l v e r methods •  - u Fresh tissues were placed i n solutions of potassium dichromate beginning with a low concentration, 2 l/2 %» The solution was changed frequently each time increasing the concentration to 3%, L$ or % . The exact degree of hardening of the tissue necessary for optimum silver impregnation had to be attained.  The time required varied  between summer and winter from fifteen days to four months. After hardening the tissues were placed i n a large volume of 0.75$ silver nitrate solution.  The silver bath also was changed several times.  The time required for the impregnation was 24. to 48 hours. Excess of the silver nitrate was washed out with 80$ to 90% alcohol. Thick sections were made freehand, cleared and mounted i n thick xylol-damar, without a cover s l i p .  By this technique the nerve cells and delicate  fib»ps were opaquely stained so that the intracacies of the nerve c e l l processes could be demonstrated but no intracellular detail could be seen. In 1906 Cajal and Bielschowsky simultaneously published a new reduced silver nitrate method for staining nervous tissue. By this new technique  ^ajal was able to demonstrate for the f i r s t time the  intracellular structure and to prove that i n the nerve c e l l processes the neurofibrils always remain within their substance and are not, as Apathy asserted, capable of emerging from i t . Retzius ( 1908 ) summarized the investigations on the subject stating that " Neurone f i b r i l s are to be found i n the nerve cells and their processes, that they farm i n the cells abundant reticula, which are plainly to be seen even i n some of the peripheral terminal organs, and that they do not anastomose outside the particular domains of the c e l l unit or neurons, i e . that they  - 5do not outside these form reticula but plexus•  The several neurones  are connected one with another per contiguitatem. not per continuitatem. Finally, there does not exist any certain proof that the f i b r i l s constitute the sole conducting element After Cajal's severe rebuttal (1908) of Apathy's objections to the neurone theory only weak opposition has been raised to the'neurone doctrine up to the present.  A controversy arose between Bartelmez (193:5)  and Marui (1918) over the vestibular club-endings on the Mauthner's cells of Ameirus. states Marui.  " The contact theory i s a histological impossibility " Bartelmez (1930), with non-metallic stains, settled the  question i n favour of the contact theory and the neurone doctrine. Tiegs, using the reduced silver methods of Cajal and Bielschowsky, revived the controversy by describing the pericellular net which had also been described by Cajal, insisting that i t was the neurofibrillar system which conducted and that there was  tt  no visible gap i n the neuro-  f i b r i l l a r system at the neurone junction ". In 1926 he concluded that a  n  true neurone continuity occurs at the junction of theieurones i n the  spinal cord —  i n which collaterals from various neurones penet-  rate the various dendrites, and , passing into the substance of the nerve cells anastomose, the anastomosis with collaterals from other neurones thus permitting evidently of irfegration and the whole of the integration occurring within the body of the nerve c e l l . n  As late as 1931 Tiegs maintained that " whether there are endbulbs or not there i s protoplasmic continuity between the axone terminals and the intracellular f i b r i l s of the c e l l or dendrite  His histological  preparations demonstrated to Sherrington at that time were not convincing. Ballantyne (1925) explains bouton terminaux and pericellular  networks  as optical illusions due to the use of monocular microscopes I  Windle and Clark (1928) reviewed preparations made by Ranson and found evidence to support the disconuity or contact theory . In answer to Tiegs, they said, " failure  to observe free ends of nerve  fitees i n the young material i s not sufficient evidence on which to assume a nervous syeytium ". They assume that fibres could end on dendrites without end-bulbs - as Cajal assumed with the climbing fibres on the"P8rkinje cells i n the cerebellum. This view has lately been expressed by Glees (194-6). In 1933 Bartelmez and Hoerr gleaned valuable information on returning to a study of the bullhead, Ameirus, i n which the club-shaped endings of certain root fibres of the VIII nerve on the lateral dendrite of Mauthner's cells offer excellent subjects for preparation. They c r i t i c a l l y examined the fixation and staining techniques used i n studies of the synapse.  They suggest that the differences i n opinion concerning  the nature of the synapse are due to differences i n technique. After shrinkage of the tissue i n fixation most synapses are so minute as to be on the verge of the resolving power of the optical microscope. also suggest that the personal factor plays an important role.  They  It i s  not to be wondered at that ''ajal and Held came away from their celebrated conference each certain that his own view was correct, although each had studied the others most convincing preparations. Even scholars like Bielschowsky and Cajal have not discussed the r e l i a b i l i t y of their reduced silver techniques. The need to study living tissue as a control was obvious. Bosler (1927) has been able to see the synapse i n the living tissue of the medusa Rhistoma.  The nervous elements were stained with  a methylene blue leucobase. I t was previously accepted that the coe-  - 7 lenterate nervous system was sycyttoi i n character.  Bosler's prepar-  ations showed only contact at the synapse and no evidence of continuity. He also observed the neurofibrillae i n the living c e l l s .  Their pres-  ence i n the vertebrate systems had not yet been established. Woollard (  ) confirmed Bosler's work. At the junction of the club endings and dendrite of the Mauth-  ner's c e l l i n the Ameirus, Bartelmez and Hoerr could resolve only a single line with no indication of any pericellular network between the two.  They could distinguish clearly that the neurofibrillae were  sharply differentiated but i n no instance could they be seen to pass from the end-feet across the synapse and into the Mauthner cell's dendrite. Bartelmez and Hoerr emphasized the unreliability of a l l reduced silver techniques from the cytological point of view and suggested that they be checked by other methods. From their experiments with the Bielschowsky technique  they concluded that the block method of impreg-  nation i s unreliable and i s inferior to the individual section method. In staining with silver the time allowed for adsorption of the silver solutions i s the most important variable. Ammoniacal alcohol used before mordanting i n silver prepares the tissue for an overlay of silver which may give untrue results.  Bartelmez maintains that formol i s only  to be used as a fixative and must be followed by a mordant to avoid gross shrinkage. Bouton degeneration studies have contributed further convincing evidence i n favour of the neurone theory.  The 'bouton method • also  serves as an invaluable means of tracing fibre tracts to their destination. As early as 1093  Nikolajew investigated the innervation of the  frog's  heart by cutting the vagus nerve to to the heart and studying  microscopically the degeneration of the pericellular endings on the cardiac ganglion c e l l s .  By comparing what he saw with the description  of normal endings given by Smirnow (1890) he could determine which of the endings was affected by the lesion. Since then several investigators have studied neurofibrillar degeneration. Marinesco (1904-1906) studied the degenerating effects of various pathological conditions such as myelitis and hemiplegia on the boutons terminaux.  Lache (1906)  studied post mortem changes i n neurofibrils sixteen hours after death i n humans. Golgi (1908), Achucarro (1909) and Mott (1912) contributed to the information. More recently Lawrentjew (1925) has applied the bouton method to the sympathetic nervous system.  He showed that maximal degeneration  of the boutons terminaux i n the superior cervical ganglion was present five to six days after section of the preganglion fibres.  He confirmed  the work of Nikola jew (1893) and showed that degeneration of the endfeet did not necessarily affect the cells themselves. The work of Hoff and Gibson f i n a l l y proved the nervous nature of the bouton terminaux and indicated a new method for studying fiber tract degeneration. Hoff (1932) published a modification of Cajal's reduced silver method which he used i n his studies of bouton degeneration. Experimental animals were fixed, by perfusion, with 10% chloral hydrate. The central nervous system was then removed to a 10% solution of chloral hydrate for 24 hours and stored i n 96$ alcohol and ammonia. To impregnate, the tissue was placed i n 1.5% smlver nitrate for six days at 37° C. After impregnation the tissue was washed, reduced i n 2% hydroquinone,  - 9 -  dehydrated, imbedded, sectioned at 15 microns and mounted. Studyimg the nature of the normal synapses i n the cat, Hoff found no bouton terminaux at birth.  The f i r s t boutons to appear, 21 days  after birth were boutons de passage. In the adult-t cat he showed boutons in the spinal cord, medulla and on the bodies of nerve cells i n the granular layer of the cerebellar cortex. These normal synapses stained as round or e l l i p t i c a l loops of 2 to 4 microns i n diameter. Around the dendrites Hoff found a meshwork of tortuous fibers some of which ended in bouton terminaux while others passed out of the f£Ld. At no time was he able to demonstrate continuity between boutons and the intracellular neurofibrils.. Hoff published several papers (1933-1934) on his bouton degeneration studies i n the cat, monkey and human. A series of experiments was performed to determine the effect upon boutons of cutting the roots of the afferent fibers to the lumbar and cervical enlargements of the cord. Animals were sacrificed from twenty-four hours to two weeks after the operation and studies made of sections of the cord prepared with the silver technique (1932). After twenty-four hours the boutons were swollen, enlarged and elongated. The swelling had increased after fortyeight hours and the boutons no longer showed the loop-like appearance but were completely solid.  The synapses may reach diameters of four to  seven microns i n degeneration. The seventy-two hour sections showed some boutons almost completely obliterated.  Few abnormal boutons could be  found after four days, and not at a l l after six days. There was s t i l l no evidence to show protoplasmic continuity between the boutons and the intracellular structures. The experiments showed that separation of a nerve fiber from i t s c e l l body i s followed by the degeneration of i t s termination i n the grey matter.  - 10 Foerster, Gagel and Sheehan (1933) confirmed Hoff's work. Sereni and Young (1932) observed a similar course of degeneration in the synapses of cephalopoda. Here the degeneration took place earlier and regeneration of the fibres took place at seven to eighteen microns per hour. Measurement of  1  degeneration time ' showed that i t  varies inversely as the temperature of the water i n which cephalopods are kepji. Lawrentjaw (1934-) considers the boutons to be constant structures i n the synapses of the autonomic nervous system. Gibson (1937) published the results of a carefully correlated series of degeneration experiments as a basis for further work on degeneration, in which i t i s important to differentiate between boutons of longer and those of shorter periods of degeneration.  - 11 THE SYNAPSE The reticular hypothesis was f i n a l l y rejected after experiments on bouton degeneration showed that disintegration of the end-feet had no effect on the effector c e l l .  Boeke (1932) and Stohr (1935)  demonstrated minute threads passing from the terminal nerve fibers to the nerve c e l l .  Ninidez, however, smothered this f i n a l gasp of the  defunct theory by proving that these fibers do not degenerate when the axons leading to the terminals are severed. The way was then open for intensive study of the morphology and the physiology of the synapse i n health and disease. The Morphology of the Synapse The structure of the normal bouton terminaux or endefusse had been variously represented very early by Held, Cajal and Golgi i n an effort to settle the contact vs. continuity controversy. ( see earlier review ) The contribution of Nikolajew, Smirnow, Marinesco, Lache and Lawrentjew as well as the work of Hoff and Gibson have already been discussed.  The contributions of Sereni and Young were also mentioned, and  of Eoerster et. a l . After the introduction of the bouton method i n the tracing of fiber pathways, researchers began to develop measuring sticks for bouton degeneration studies. The f i r s t problem was to set down accurate data as to the size, shape and distribution of the normal end-bulbs i n each area to be investigated.  Bodian (1937) stained the axon endings on the Mauth-  ner's c e l l i n the goldfish using a protargol method. He described them as blackened loops varying i n diameter from 0.5 microns to 7 microns. Phalen and Davenport (1937) demonstrated boutons i n the spinal cords of several veicigbrates using a modified Cajal's stain.  They found a marked  - 12  -  in the size and form of the endings. They also noted that i n Mammals, with the exception of the monkey, the end-bulbs tended to vary i n size with the species. Because of the variation i n size of the boutons, Phalen and Davenport did not consider Regeneration methods of fibre tracing to be satisfactory.  Barr (1930) showed that axon termination  on cells of the lateral groups of the ventral horn are smaller than those on cells of the medial group i n the cat's spinal cord. In his preparations he found that the boutons tended to become smaller on cells undergoing retrograde degeneration. Barnard found no significant difference between normal boutons and those at the end of axones that had been cut, thus emphasizing the unr e l i a b i l i t y of the bouton method of study. An invaluable contribution to the study of normal terminals was made by Jeff Minckler (194-0,4-1,42).  Under his name a series of public-  ations appeared defining five types of terminals i n the human cord ? small loops, large loops, filamented loops, f i b r i l l a t e d loops and opaque or granular loops. He found that each of these might appear as regular, thickened or granular. These endings were usually found i n contact with the c e l l or robust process. They ranged i n size from 0.5 x 1 micron to 3.5 x 5 microns. Varying as a fundtion of the c e l l size, the number of boutons per c e l l ranged from 144- on small sensory c e l l bodies to 1832 on the large c e l l bodies of the posterolateral column. Minckler showed (1941) that the morphological types of endings i n a given area of the human cord remain f a i r l y constant from one individual to the next, regardless of the age, i f the staining technique i s carefully controlled. It was also shown that the different types of terminal occur i n about the same numbers on different parts of the nerve c e l l .  However, gran-  ular forms occurred more frequently i n older persons. It was realized  - 13 that the appearance of the thickened forms may be due to technique such as prolonged fixation i n formaldehyde but Minckler stated that autolytic processes operating up to 24 hours seemed to have l i t t l e effect on the bouton morphology demonstrable with the Cajal technique used.  ( See also Hoff 1931 ). Reports of the demonstration of normal bouton endings i n the  cerebral cortex are remarkable for their scarcity. Hoff, using block silver methods, stained cortical end-feet i n the cerebral cortex of the aat, but with great d i f f i c u l t y .  Cajal (1934) reported boutons i n  the cortex. Poorly rewarded efforts to stain boutons i n the cortex have led some investigators to believe that these are not the normal types of synaptic points i n this part of the nervous system.  Meyer and Meyer (1945)  concluded that " while bouton-like structures around cortical nerve cells can be demonstrated by our present methods under favourable circumstances i t i s obvious that they constitute merely a portion of the terminals . 11  Bielschowsky, (1935) and Cajal (1934) described plexuses formed by the terminal fibres around cortical s t r i a t a l cells and suggest that these may make connection with the dendrites. Degenerating boutons were demonstrated by Greenfield (1939) i n patients with cerebral oedema. King (1942) described briefly pericellular argyrophylic structures i n relation to pyriform cells i n the pyramidal layers which reduced ammoniacal silver solution without further chemical treatment.  In 1946 Glees  stated that " I t seems that the.synapse within the cortex i s mainly represented by free terminals of the pericellular plexus ". However he showed that the pericellular fibres i n the caudate nucleus degenerate after cortical ablation (Glees, 1944) and suggests that these can be  T  -Lo-  used i n degeneration experiments.  The fallacy of this view w i l l be  reviewed i n the discussion.  In spite of i t s apparent potentialities, the  1  bouton  1  method  has not enjoyed the popularity which one might expect. However, i n the hands of a number of investigators i t has yielded many valuable contributions  to the knowledge of the nervous system.  Hoff (1935) used the  bouton method to investigate the terminal distribution of the corticospinal fibers arising i n the premotor area of the monkey. The structure of the lateral geniculate body and the projection of the retina i n the lateral geniculate body were studied, using the bouton method, by Le Gros Clark and Penman (1934) • A series of experiments was carried out (Le Gros Clark, 1941)Glees and e Gros Clark, 1941/ Glees 1941 and L  1942)  to study the termination of the optic nerve" tract fibres i n the lateral geniculate body of the monkey, cat and rabbit. O'Leary (1940) contributed to the knowledge of the lateral geniculate body. The optic centres i n the rat were investigated by Nauta and Van Straaten (1947). Further studies have been carried out by Glees (1944, 1946 and 1947) and by Glees and Meyer (1946) on the cortico-striate connections and the frontal cortex. The reference, by Glees, to the so-called free terminals i n the cortex has already been mentioned. Brodal (1949) used the Glees method i n an experimental study of the spinal afferents to the lateral reticular nucleus of the medulla oblongata i n the cat. Recently (1951) Wall, Glees and Fulton were able to demonstrate direct projections from the orbital surface of the frontal lobe to the ventromedial and paraventricular nuclei of the hypothalamus and the causate nucleus. A report on the use of intravenous methylene blue for studying  - 15 f i b e r degeneration i n the c e n t r a l nervous system has recently been published by W. H. Feindel and A. C. A l l i s o n  (194-8). A c r i t i c a l  discussion of the methods used to study f i b e r degeneration and  suggestions  for the use of methylene blue were published by Feinctel, A l l i s o n and Weddell the same year.  By t h e i r new method they were able to s t a i n en-  larged terminal boutons i n the l a t e r a l geniculate body i n r a b b i t s . The need f o r r e l i a b l e methods of studying f i b e r  degeneration  i n the cerebral cortex became more pressing with the introduction of the s u r g i c a l treatment of c e r t a i n mental disorders by f r o n t a l lobotomy. Most of the knowledge of the projection to and from the f r o n t a l cortex has been derived from experimental observations i n monkeys and apes.  In  a report on the e f f e c t s of p r e f r o n t a l leucotomy, Meyer, Beck and McLardy (194-7) state that " There i s no need, to emphasize the importance of a d e t a i l e d neuro-anatomical i n v e s t i g a t i o n of the brains of patients dying at various i n t e r v a l s a f t e r p r e f r o n t a l leucotomy. "  The methods  which can be used f o r such an i n v e s t i g a t i o n are few, and to date the most promising s t a i n i n g procedures are reduced s i l v e r methods.  The Mechanism of Synaptic  Transmission  A b r i e f discussion of the e a r l i e r l i t e r a t u r e on the mechanism of the synaptic transmission i s thought to be important as giving context to the problem of synaptic s t r u c t u r e . The physiology of nerve a c t i v i t y has received much attention during the past h a l f century and one can say that great strides have been made toward the soluti&ns of many of the problems involved.  However,  the precise nature of the synaptic transmission has not yet been completel y solved.  E a r l y i n the century T. R. E l l i o t  (1905) thought that some  - 16 chemical was responsible for the transmission of the nerve impulse across the synapse; In 1921 Otto Loewi showed that acetylcholine was the substance released by the vagus nerve to the heart and acted directl y on the heart muscle. Dale (1933) tried to extend the  1  neurohumoral '  theory to the neuromuscular junction and the ganglionic synapse. acetylcholine hypothesis ( Dale 1937, Clark 1936  The  ) simp2y stated that  a presynaptic impulse liberated, at the synapse, a minute amount of acetylcholine, which excited the post-synaptic c e l l , thus setting up a synaptic potential. They held that i t was quickly removed by the locally concentrated cholinesterase. ( Brown 1937, Dale 1937, Nachmansohn 194-0 This met with two strong points of opposition.  ).  The neuromusc-  ular transmission occurs much too quickly ( milliseconds ) to be explained by a chemical  reaction. Secondly, the electrical signs of nervous  action do not support the assumption that transmission of the nerve impulse along the axon differs fundamentally from that across the synapse since the excitable properties of the axon and of the c e l l are basically the same. Thus the neurohumoral theory seemed to be unsatisfactory. The electrical theory of nervous transmission, i n i t s early form, was also unsatisfactory. ( Eccles 1936, Erlanger 1939, Lorente de No Monnier 1934)  1939,  In general i t was merely stated that the electrical  currents of the presynaptic impulses set up impulses i n the post-synaptic cell.  The significance of the pre- and post-synaptic responses and of  the rheobase of the post-synaptic c e l l necessitated so much modification of this vague formulation that i t had to be abandoned. Nachmansohn (1945) postulated that the release and removal of acetylcholine was an intracellular process occurring along the neuronal surface and directly connected with the nerve action potenti41.  Thus  the transmitting agent was considered to be the eiwtric current, the  - 17 action potential, but the current was generated by changes i n the mabrane in which a release of acetylcholine i s an essential event. Nachmansohn was able to show that the formation and removal of acetylcholine could take place at a rate consistent with the speed of the nerve impulse. However, Lorente de No (1944) bad shown previously that the transmission of the nerve impulse across the synapse i s unaffected by high concentrations of acetylcholine. A later hypothesis on the synaptic transmission i s the culmination of several years of intensive research by J . C. Eccles (1947). r  Three assumptions are made: f i r s t , that the geometrical situation at the synapse is adsematically represented by the pre-synaptic fiber ending as a cylindrical membrane with a closed and direct apposition to the large plane surface membrane of the post-synaptic c e l l .  Thus he assumes that  a traverse membrane at right angles to the axon exists at the synapse as described by Cajal (1934) and other histologists.  There i s , also,  electrical evidence for a highly resistant transverse membrane. Second, he assumes that, i n general the surface membranes have the electrical properties demonstrated for peripheral nerve and muscle mebranes, such as resistance, electromotive force, capacity and rectification. Third, that the membrane of the immediate post-synaptic region i s specialized, so that large and graduated local responses are set up by polarizing currents. With these basie assumptions the following sequence of events i n synaptic transmission was described. First,the impulse i n the pre-synapt i c nerve fiber generates a current which gives a diphasic effect at the synaptic region of the post-synaptic c e l l with a total duration of not more than 1 millisecond i n mammalian muscle and the spinal cord*, an i n i t i a l anodal focus with cathodal surround; a more intense cathodal  - 18 focus with anodal surround.  Second, this cathodal focus sets up a  brief and intense local response at the synaptic region. Third, from this local response, a catelectrotonus spreads decrementally over the post-synaptic c e l l membrane. Finally a propagated impulse i s set up i n the post-synaptic cells, i f this catelectrotonus i s above a c r i t i c a l value• This summary of two of the more recent theories on the syaaptic transmission gives some indication of the complexity of this problem. Any attempt to discuss the investigations of the past five years would be far beyond the scope of this review. An excellent monograph on the subject has been published by Rosenbleuth (1950). Reviews by Bullock (1951) and Rushton, W. A. H. (1951) indicate the vast and rapidly expand' ing literature.  It can be said that both elecrfcical and humoral trans-  mission are involved i n relay of a nerve impulse but the extent to which each agent i s involved must yet be determined. Rushton concludes his summary  11  * t i s natural to try to explain the two kinds of conduction  upon a common basis, but what has been proved i s that along the nerve fiber the transmission i s electrical*, from the endings of peripheral nerves i t i s pharmacological.  n  - 19 EXPERIMENTAL PROCEDURE The experimental work has been concerned mainly with the s t a i n ing of nerve f i b e r s and the terminal synapses i n the cerebral cortex. Several modifications of the reduced s i l v e r technique have been t r i e d . The stepwise procedure f o r each of these has been included f o r reference purposes i n an appendix.  Certain procedures have been discarded f o r reas-  ons which w i l l be discussed under the heading of experimental  results.  The Cajal's modification f o r frozen sections and the Gros-Bielschowsky method have been used to s t a i n the s p i n a l cord and cortex i n the  dog.  The double impregnation method f o r frozen sections and the Urea-Silver Nitrate method of Ungewitter were used to s t a i n the cerebral cortex i n humans. A l l tissues were f i x e d i n 10$ n e u t r a l formal regardless of the s p e c i a l f i x a t i v e s mentioned i n each method.  In a l l cases frozen sect-  ions were cut at 15 microns.  Cajal's Modification f o r Frozen Sections. ( See Appendix A f o r d e t a i l e d ' " Instructions )  I t was  found advantageous to receive sections from the freezing  microtome i n water containing a few drops of concentrated ammonia. step  2, three to f i v e drops of pyridine per 15 c c . of  proved s u f f i c i e n t .  AgN03  In  solution  Adding greater amounts than t h i s resulted i n a dust-  l i k e p r e c i p i t a t i o n of s i l v e r .  An impregnation time of 6 hours gave the  best r e s u l t s although longer i n the s i l v e r bath seemed to do notharm. S l i d e s of s p i n a l cord, cerebral cortex and cerebellum of dog were stained by t h i s method.  Alternate sections were toned i n gold.  -  The Gros-Bielschiwsky Method  20  -  ( See Appendix B for details )  The block of tissue was soaked i n water containing a few drops of ammonia. Frozen sections were cut at 15 microns and received i n a dish containing a few drops of neutral formalin.  The remaindec of the  procedure was followed as outlined i n McLung's Handbook. (Ferdov's Modif. ) Sections of the cerebral cortex of dog were stained by this method. Alternate sections were toned i n gold. Double Impregnation Method  (See Appendix C )  The procedure outlined i n the Appendix i s Gibson's modification of Rio Hortega's Silver Carbonate Stain for neurofibrils.  Frozen sec-  tions cut at 15 microns were received i n 15 c c . of water plus 10 drops of ammonia. Petri dishes of 96 per cent alcohol and ammonia, 2 per cent silver nitrate plus 5 drops of pyridine, and the silver carbonate solution were maintained at a constant temperature of 45°C. i n a shallow water bath. Five minutes i n the gold chloride were found to be ample when the impregnation was reinforced by heating one minute over a s p i r i t lamp. Sections of human and dog cerebral cortex were stained by this method. Because of the prominence to which the frontal lobes have been raised due to the treatment of certain mental disturbances by frontal lobotomy, i t was decided to determine whether bouton terminaux could be satisfactorily demonstrated i n certain areas of the frontal cortex. For this purpose frozen sections were taken from certain areas of the brain of a mental patient* who died twenty-nine hours after frontal lobotomy,  *  Brain suppled by Dr. Paul Yakovlev, of the Connecticut Lobotomy Project.  - 21 and stained by the double impregnation technique. A transverse slice was removed from the brain immediately anterior to the bilateral lesion. ( F i g . 1 ). Portions were cut from this slice as indicated i n the diagram ( F i g . 2 ), and frozen sections taken from each portion for silver staining. A few sections were also stained which had been taken from the visual cortex, area 18 of Brodmann.  - 22  EXPERIMENTAL RESULTS The silver impregnation of Glees (1%6) and the protargol method of Stotler were found to be unsatisfactory i n this laboratory for our purposes. The method of Glees involved an unnecessarily long procedure which i s a modification of the shorter Gros-Bielschowsky technique. The neurofibrils stain quite black with some tendency for excess precipitation of the silver.  This method was tested on the human cortex  and no bouton terminaux could be found. The protargol procedure gave rather spectacular ' low power ' results from a histological point of view, producing a b r i l l i a n t section i n tones of gold and brown. However, i n our hands the procedure did not show neurofibrils, and moreover, no terminal end-bulbs couldtoefound. Another disadvantage to this method i s the use of protargol which i s d i f f i c u l t to obtain. Cajal s modification for frozen sections 1  proved to be a rather  delicate and exacting method. The amount of pyridine used was  found to  be very important, an excess usually resulted i n the undue precipitation of silver giving • muddy ' preparations.  However this i s a good proced-  ure for demonstration of the larger neurofibrils and boutons i n the cord. The fibers and f i b r i l s i n the cortex were well shorm, but no boutons could be demonstrated.  The background of the untoned sections also  stained f a i r l y heavily so that tracing of the f i b r i l s i s more d i f f i c u l t . The nuclei of the neuroglia pick up the stain f a i r l y heavily. Much of this unnecessary background i s subdued by toning i n gold. The fibers and f i b r i l s then appear more pronounced and are more easily traced. cerebral cortex these conditions also hold true.  In the  - 23 The Gros-Bielschowsky technique produced quite good results i n the dog cortex. ( Figs. 6 and 7 ) The nerve fibers take up the stain much more heavily with this stain so that even some of the larger dendrites appear black. Of several silver nitrate methods used successfully, this, i n my hands, was the most capricious. Using identical times and conditions on consecutive sections and renewing the formalin, silver and ammoniacal silver each time, i t was s t i l l impossible to predict the degree of impregantion. The very high concentration of silver nitrate used might have something to do with this. The Double Impregnation technique was found to be the most dependable and satisfactroy.  By carefully following the specified conditions  and times, excellent preparations could be made almost without f a i l .  In  c e l l preparations of the human cerebral cortex i t was possible to identi f y distinct, ring-like bouton terminaux.  ^he fibers appear to be well  impreganted but not to excess. In a few cases i t was possible to distinguish the neurofibrils leading to the end-feet.  ( Figs. 9, 13 and 14 )•  The Lobotomized Brain. The results here are recorded i n the numerical order of the portions as they are numbered on the diagram of the brain slice used. Portion A.  ( F i g . 1 - 2 ).  This i s from the cingulate gyrus on the medial side  of the cerebral hemisphere. of the medium fibers.  Elongated torpedo-like swellings along many  Distinct ring-like bouton terminaux, ranging i n  size from 1 micron to 3 microns i n diameter, could be found i n every field.  On the average between three and five boutons could be found i n  every f i e l d ,  ^any of these were adjacent to or apparently i n contact  with cells or dendrites. Portion B.  ( Figs, 9 and 10 ).  This i s from the superior frontal gyrus.  Torpedo-  like swellings were again found i n large numbers i n the area. Many of  - 24 -  the fibers seem to have taken up the silver to a greater extent making them appear heavy and irregular.  Boutons could be found i n  almost every f i e l d and often i n contact with c e l l or dendrite. ( Figs. 11 and 12 ). Portions C and D. These are i n the areas 8 and 9 of Brodmann anterior to the premotor area 6. The pattern of the previous section is carried out i n these.  The boutons could be found i n a l l the layers  below layer 1. The fibers show elongated swellings, and an increased take-up of s i l v e r .  Many of the fibers also appear somewhat tortuous.  ( F i g . 13 ). Portion E.  Boutons on dendrites and c e l l bodies.  elongated swellings.  ( F i g . 14 )•  Portion F. Many large pyramidal c e l l s . field.  Fibers show  Boutons i n almost every  Fibers swollen at intervals and sometimes tortuous. ( Figs. 15,  16 and 17 ). Visual Area. Typical visual area showing line of Gennari. Boutons i n this region appeared to be somewhat larger than general, ranging from 1 to 4 microns, i n diameter.  ( F i g . 18 ).  -  25  -  DISCUSSION The multitude of modifications of the reduced silver techniques of Cajal and Bielschowsky which have been published are as an hundred variations on a theme. In spite of the extensive literature on the silver impregnation of nerve fibers and tissues, nearly a l l silver methods can be classified into three basic techniques. A l l involve the adsorption and subsequent reduction of s i l v e r .  In the early procedures  of Cajal and of Bielschowsky, tissues were immersed in solutions of pure silver nitrate i n varying concentrations.  Del Rio Hortega immersed  the already impregnated tissue i n a carefully prepared solution of d i l ute silver carbonate before reduction.  Finally Bodian used, as a source  of the silver ion, a solution of silver albuminose produced by the throp Chemical Company, called ' Protargol . 1  Win-  Each of these techniques  in the laboratories of competent investigators, has produced excellent results. The modification of the basic techniques have usually been introduced into one or more of three important stages in the staining of a piece of tissue. First, much emphasis has been attached to the tissue fixative used. * t would be ridiculous to attempt to l i s t the fixing solutions which have been suggested to prepare nervous tissues for impregnation with s i l v e r .  The most widely used fixative, and that used for a l l  tissues i n this work, i s neutral formalin.  Formalin has the advantages  of universal availability, cheapness, rapid penetration and minimal colour change. Most hospitals and mental institutions use neutral formalin for the preservation of autospy and biopsy material.  It i s therefore  an additional advantage that silver techniques can be successfully applied to formol-fixed tissues.  It i s interesting that Spiegel-Adolf, Henny and  Ashkenaz (1944) have pointed out that even X-ray diffraction studies re-  - 26 veal hardly any changes i n the X-ray diffration pattern of formalinfixed muscles, i n marked contrast to results following the use of other fixatives. The second stage at which modifications may be introduced i s i n the concentration of the silver solution. vary between 1% and 20% silver nitrate. solution.  Concentrations recommended  Most authors suggest a 2%  This seems to be a sufficient concentration to give a  rapid impregnation without resulting i n too great a deposition of s i l ver . Silver solutions of 10$ and above have a tendency to encrust the nerve fibers, and, i n addition , may cause much confusing precipitate. Finally, the reducing solutions may be modified. However, once the silver has been deposited, then any standard reducing solution should be satisfactory provided the correct degree of impregnation has been attained. There are few neurological methods which can be used to show nerve fibers and to trace nerve tracts.  The Marchi method for myelin  degeneration has been widely used and a great part of our anatomical knowledge of the pathways i n the central nervous system has been provided by the ^archi technique. The method depends upon the observation that the products of myelin degeneration can be stained black with osmic acid, while staining of the normal myelin can be prevented by preliminary treatment with a chromic s a l t .  It's failure to demonstrate degen-  erating non-myelinated fibers and degenerating finely myelinated fibers however, i s a serious limitation of this technique. The tracing of such important pathways as the spinothalmic tract, many pathways projecting to and from the hypothalamus and the projection from the cortical suppressor areas to'the basal ganglia are beyond the scope of the Marchi method. Many of the projections to and from the frontal areas are thought  - 27 to be unmyelinated fibers and therefore not demonstrable by the osmium reaction.  The myelin sheaths come to an end some distance from the  precise destination of the degenerated tract and cannot be demonstrated. Thus, ins pite of i t s many uses and advantages, the Marchi method alone cannot satisfy the needs of neuroanatomical investigations i n the frdntal areas. Recent investigations ( Fiendel and Allison, 194-8, Fiendel Allison and Weddell, 1948 ) have shown that intravital methylene blue can give a striking picture of nerve fiber degeneration following experimental lesion of the brain. Feindel, Allison and Weddell have stained bouton ending i n the lateral geniculate body. However, satisfactory results have not yet been obtained using previously fixed experimental material. Therefore the method i s not yet applicable to the investigation of human brains obtained at autopsy, i t should be mentioned here that preliminary experiments i n this laboratory using methylene blue and other non-metallic stains for synapses have so far met with l i t t l e success. It seems therefore that we must return to the silver impregnation methods for the satisfactory demonstration of normal and degenerating nerve fibers and endings. This brings us to a discussion of the stainabilty of the bouton endings i n the cerebral cortex.  They can be shown with varying success  i n most other areas of the central and peripheral nervous system, as has been mentioned previously.  Only with  great difficulty have these  fine endings been seen i n the cortex and then they have been few and far between. The failure of the end-feet to show themselves i n this area has led some investigators to believe that they donot represent  - 28 -  the normal method of nerve fiber termination. The statement of Meyer and Meyer (194-5) that " While boutons-like structures around cortical nerve cells can be demonstrated by our present methods — —  it  i s obvious that they constitute merely a portion of the terminals " may well be reiterated here. But why should the character of the synapse be so different i n the cerebral cortex ?  Glees statement that  " It seems that the synapse within the cortex i s mainly represented by free terminals of the pericellular plexus " i s too easy a rationalization. These pericellular fibers have been clearly demonstrated i n this laboratory. After careful study of many sections, however, no case of contact between these fibers and the cells could be seen except by means of bouton terminaux.  The system of neurofibers and neurofibrils i n  the cerebral cortex i s extremely complex. The processes of some nerve cells, such as those from the foot area of the cortex, have been shown to travias far as three feet to reach their destination.  The fact that  a fiber, or groups of fibers, pass very close to a nerve c e l l en route to their destinations i s no reason to believe that the fiber has anything whatsoever to do with that c e l l .  Most theories on the nature of  synaptic transmission would require the existence of some form of specialized ending. Only with discrete endings such as boutons could one perform purposeful movements. In this laboratory we have been able to see as many as five boutons incontact with one cortical c e l l .  ( Doctor Gibson t e l l s me  that he has seen as many as fifteen boutons i n contact with one cortical c e l l . ) A solution to the problem of staining the endings i n the cortex must be found. Why i s i t so d i f f i c u l t to stain boutons even though i n many cases the nerve fibers right up to the synapse- stain easily ? I t  - 29 -  was thought that acetylcholine might have something to do with the adsorption of silver on nerve fibers and especially in synapses. In this laboratory two dogs were fixed i n an identical manner by perfusion with 10$ formol.  One of the dogs was perfused with a solution of eser-  ine i n physiological saline just before the fixation. Eserine i s an anti-cholinesterase and i t s presence at the synapse would prevent the removal of acetylcholine by the cholinesterase. Thorough histological study of various areas i n the spinal cord, cerebellum and cerebral cortex  showed no difference i n the stainability of the two nervous systems. Perhaps the d i f f i c u l t y i n staining boutons i s a reflection of  the rapid chemical change associated with death, which would be greater i n the synapse because of the blood supply ? I f this i s so, would prevention of this chemical change by some means increase the staining ? A positive approach to the problem must be made. Only after i t s solution can a thorough investigation of the frontal areas i n the human cerebral cortex be made.  SUMMARY  -  A survey o f s e v e r a l reduced S i l v e r foir  techniques  the s t a i n i n g o f nerve f i b e r s and. boutons  termin-  aux i n the c e r e b r a l c o r t e x i s r e p o r t e d . A double impregnation method which i s a m o d i f i c a t i o n o f D e l Rio Hortega's  sliver nitrate - silver  carbonate tech-  nique was found most s a t i s f a c t o r y . The method v/as used the human f r o n t a l the boutons  to study c e r t a i n areas o f  c e r e b r a l c o r t e x and to  demonstrate  terminaux i n these areas.  Evidence i s s u f f i c i e n t to i n d i c a t e that boutons  are the normal  form of i n t e r neuronal  these  synapse  i n t h e c e r e b r a l c o r t e x of the human- and fur ther r e s e a r c h on the s t a i n i n g problems i n me system i s suggested.  nervous  Pig. 1. Diagram of l a t e r a l view of brain to indicate the angle and region of l e s i o n and the area from which the s l i c e was taken.  F i g s . 5 and 4. A few normal, boutons terminaux on l a t e r a l horn c e l l of the cteg spinal cord, (arrows) These two photographs were taken, at slightlyd i f f e r e n t f o c a l l e v e l s , of the same c e l l to indicate the d i f f i c u l t y i n showing the number of b@utons to be found on a c e l l Sy means of photographs. ( mag. 1500 diameters) Stained by Cajal's modification for frozen sections and: toned i n gold.  Figure  4  Fig. 5 C e l l In the cerebral cortex of a  dog.  Stained with Cajal's modification. Note the- d i f f e r e n t i a t i o n of nudlei and. f i b e r s , (mag. X 1500)  Figure 5  Figs. 6 and 7. Cerebral cortex of. dog stalined by the GrosBielschowsky technique. Note the apparently degenerating bouton i n f i g . 7 and the large darklj^ryimpregna&ed one i n f i g . 6. The fibers In general are heavily impregnated (mag. X  1500)  with s i l v e r ,  Figure 6  Figure 7  F i g . 8. Motor area i n cerebral cortex of a dog. Stained by the double impregnation method, and toned i n gold,, (mag. X  1500)  Figure 8  . 9 and 10. Cortex on the cingulate gyrus. Stained by double impregnation method. Toned i n gold. Bcutons terminaux indicated by arrows, (mag. f i g . 9 - X 3000, f i g . 10 - X 1500)  Figure 9  Figure 10  Fig.' 11. Human cortex from portion B (diagram, f i g . 2% Double impregra t i o n . (mag. X 1500) F i g . 12. Human cortex from portion B. Dbl. Impr. (mag. X 1500) F i g . 15. Human cortex from portion C Dbl. Impr. (mag. X 1500)  Figure 11  P i p - t i r e 12  Figure 13  Fig.  14.  Human c o r t e x from p o r t i o n E. Dbl,. Lmpr. (mag.  X  1500)  F i g u r e 14  F i g s .  1 5 ,  16  and.  H u m a n  c o r t e x  ( m a g .  X  1 7 . . f r o m  1 5 0 0 )  p o r t i o n  F.  D b l  I m p r .  Figure 15  gure  16  Figure 17  Fig.  18, Human c o r t e x from the v i s u a l c o r t e x 18 of Brodma=nn. D b l . Impr. (mag.  X  area  1500)  Figure  18  I  A  il  APPENDIX  -  Caial's Modification for Frozen Sections (Gibson,  1950)  (1) Blocks of tissue, fixed for at least one week i n 20 per cent formol, are cut on the freezing microtome at 12-15 microns. (2) Wash the section well i n water and place i n a solution of 12 c c . of 2 per cent silver nitrate, 6 c c . of 96 per cent alcohol, and 5-10 drops of pure pyridine, for six to ten hours at 37° C., or twelve to twenty-four hours i n the cold. The latter i s desirable for the finest detail. Silver nitrate solution up to 5 per cent has been employed successfully i n the cold. (3) Transfer sections to a dish of 98 per cent alcohol where they may remain up to three minutes, depending on the depth of the silver impregnation. (4) Reduce for three minutes i n a solution of 0.3 gm. of hydroquinone, 70 c c of water, 20 c c of formol, and 15 c c . pure acetone• (5) Wash in water and tone i n yellow gold chloride solution, 1 to 500, for five, to ten minutes. (6) Fix i n 5 per cent hypo, wash, mount, dehydrate, balsam, etc B  An Intensified Protargol Method for Paraffin Sections (W.A. Stotler)  (1) Blocks of tissue, fixed i n 15 per cent formalin, Bouin's solution or acetone, are imbedded i n paraffin. Sections cut at 15 . microns, paraffin removed, sections through alcohols to water. (2) Impregnate 18 to 24 hours i n 0 . 1 per cent aqueous solution of protargol. Place 5-10 gm. of copper (shot) i n staining dish and dust 0.25 gm of protargol on 250 c c of water containing the sectidns. The addition of 4 drops of pyridine and 0.25 gm. of sodium glycerophosphate apparently improves the stain. (3) Place sections without washing on a staining rack and flood with a few drops of the reducing solution. Follow the process of reduction and intensification under the microscope and when optimum, wash, dehydrate, and mount. Prepare the unstable reducing solution immediately before use by combining the following solutions in order: Solution A, 6% AgN03, 10 c c , * Solution B, 20 gm. sodium sulphite i n 330 c c . water, 10 c c . Solution C, 35 gm. sodium thiosulphate i n 330 c c . water, 10 c c . ; Solution D, 5 gm. sodium sulphite, 8 gm. Kodak Elon i n 1000 c c . water, 30 c c  3 ^  II  APPENDIX C  -  Gros-Bielschowskv Method (Gibson,  1950)  (1) Fix material i n 12 per cent formalin neutralized with calcium carbonate, from one week to several years. (2) Soak the block of tissue i n water, and cut frfczen sections from 20 to 50 microns into a dish of water containing a few drops of neutral formalin. (3) Place sections i n 20 per cent silver nitrate, one to five minutes• (4) Arrange four capsules of 20 per cent neutral formalin i n a row and l i f t one section omly into the f i r s t . As soon as a white cloud forms at the edge of the section move to the next dish, and similarly into the third and fourth dishes. If clouds s t i l l appear use a f i f t h dish. (5) L i f t the section carefully with a glass needle, and touch i t momentarily on a piece of clean f i l t e r paper to draw off the formalin. Place the section i n a watch glass containing the ammoniacal silver solution (see below). From three to five drops of ammonia should now be added to the watch glass which i s placed under an observation microscope.to control the impregnation. A f a i r l y rapid coloration i s best, and when the nerve trunks i n the section are seen to reach a dark, almost apaque brown-black color the section i s transferred immediately to a solution containing 4 parts of ammonia to 5 of water, for five minutes. (6) Wash u n t i l no odor of ammonia i s detectable. (7) Tone i n a solution of 2 c c . of 1 per cent gold chloride in 20 c c . of water. This suffices for 40 sections i f a drop of gold chloride i s added periodically. (8) Fix i n 5 per cent hypo, dehydrate, and mount i n balsam. Ammoniacal Silver Solution - This must be prepared i n small quantities as required by adding concentrated ammonia to 20 per cent silver nitrate solution u n t i l the precipitate just disappears, leaving a brown tinge throughout the solution. A glass rod should just be visible i n this. A crystal clear i s too ammoniacal. Fresh formalin i n the four reducing capsules, and fresh ammoniacal silver i n the watch glass must be used for each section, regardless of i t s size. The watch glass i s never washed. Its contents are renewed each time. For boutons i t i s best to leave the section only a short time in the 20 per cent silvernitrate dish, and to use the minimum of ammonia i n the watch glass stage.  III  3>  - APPENDIX D Glees' Modification (P. Glees, 194.6) (it) Fix material in 10 per cent formalin or 5 per cent formalin-saline for a period of at least 5 days up to 6 months. (2) Cut frozen sections at a thickness of 15 to 20 microns. (3) Place sections for 12 hours in a dish filled with 50 per cent alcohol to which 6 drops of concentrated ammonia have been added. (Use 6 drops of ammonia for every 50 c c . alcohol and maintain at a constant temperature of 30° throughout.) (4.) Wash sections in distilled water. (5) Place in 10 per cent silver nitrate solution for another 12 hours at room temperature. (6) Wash sections three separate dishes filled with 10 per cent formalin made up with tap water. (7) Transfer sections into the following solution to be left for 30 seconds - 3 parts 20 per cent silver nitrate, 2 parts 96 per cent alcohol, then add concentrated ammonia intil the brown precipitate first formed has redissolved, and then add another 5 drops of ammonia. (8) Transfer into a 10 per cent formalin solution where sections are stained deep brown after 30-60 seconds. (9) Wash briefly in distilled water. (10) Place for 10 seconds in a 10 per cent hypo solution. (11) Wash several times in distilled water and mount via alcohol and creosote. The latter procedure makes the sections soft and transparent. Any excess of creosote is removed by pressing the section firmly under blotting paper.  IV  ^  - APPENDIX E Double Impregnation Method for Neurofibrils (Gibson, 1950) This technique was developed from a method first published by Rio-Hortega in 1921. Is consists essentially of a preliminary imppegnation with a> solution of silver nitrate, followed by treatment with silver carbonate. Fixation t Fix in small blocks or thin slices in 10 per cent formol in distilled water. To this mat be added one drop of pyridine or one drop of ammonia, per cubic centimeter of formol. The optimum fixation period is 10 days at 37° C. or one month in the cold. (1) Cut fixed material on the freezing microtome at 12-15 microns . Wash the sections well in a petri dish of water containing 10 drops of ammonia. Carry through two dishes of pure water. (2) Place in a 12 c c . pyrex glass cup containing a 2 per cent solution of silver nitrate, to which 3-4 drops of pyridine have been added. (3) Heat gently for ten minutes at 45° C., whenthe gray matter will become yellow. (4) Wash very quickly and place in a. similar pyres dish containing a 5 per cent solution of silver carbonate (sosa) with three to four drops of pyridine. (5) Heat gently for ten minutes at 45° C, after which the tissue will take on a tobacco colour. (6) Wash for 15 seconds. (7) Reduce in 10 per cent formol. (8) Tone in yellow gold chloride solution, 1 to 500, in the cold for five minutes. Reinforce by heating the toning bath gently for one minute. Wash quickly. (9) Fix i i a 5 pe$ cent solution of hypo, dehydrate in 96 per cent alcohol, creosote, blot dry, balsam, etc. For material fixed in F.A.B., or for refractory material fixed in formol for several months: (1) Cut sections into water containing ten drops of ammonia. (2) Heat in a pyres cup containing 10 c c . of 96 per cent alcohol and 10 drops of ammonia, for ten minutes at 45 °6. (3) Wash well in three dishes of water* Employ the double impregnation technique as above, reducing in 1 per cent formol without washing after the silver carbonate treatment Five per cent silver carbonate (sosa) Solution of 10 per cent silver nitrate 67 c c Solution of sodium carbonate 5 per cent 267 c c Water tolOOO c c . Add ammonia drop by drop while shaking the solution until the precipitate just dissloves. Filter and sibore in a dark bottle.  -  BIBLIOGRAPHY  -  Auerbach, L.  1099  Das terminale nervennetz i n seinen beziehungen zu dem ganglienzellen der cantralorgane. Monatschr. F . Psychiat. u Neurol. 6, 191  Ballantyne, F.H.  1925  Continuity o f the vertebrate nervous system, Trans. Roy. Soc. (Edinb.) i3_, 663.  Barnard, R.I.  194-0  Experimental changes i n end-feet of Held-Auerbach i n the s p i n a l cord of the c a t , J.Comp. Neurol., 2 2 ,  235-265* Barr, MJL.  1939  Normal and experimentally altered end-bulbs i n the Cat's s p i n a l cord. Anat. R e c , 2 2 , Spppl. 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