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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 IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF ARTS i n the Department o f B i o l o g y and Botany We accept t h i s t h e s i s as conforming to the s tandard r e q u i r e d from candidates 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 COLUMBIA. October , 1951 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 in the cerebral cortex by the silver impregnation methods, to suggest that the pericellular fibers in the cerebral cortex end freely on the cells and that such free terminals are the normal form of synapse in this part of the central nervous system. In the present work a histological study was made of certain areas in the frontal cerebral and the visual cortex to show the presence of normal boutons terminaux. It was demon-strated that boutons occur in these areas in sufficient numbers to indicate that these are the normal means of synapse. The direction which further research should take is suggested. TABLE OF CONTENTS Abstract Introduction REVIEW OF LITERATURE (a.) The Neurone Theory-contact vs . continuity 1 (b.) The Synapse 11 1. The morphology of the synapse . 11 2. The mechanism of synaptic transmission. 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 Clin i c Research Unit, for his generous assistance, advice and many kindnesses during the preparation of this work. To Dr..A. H. Hutchinson, Head of the Department of Biology and Botany, sincere gratitude i s extended for his advice and many considerations. Appreciation i s also due to the members of the Crease Clinic Research Unit who have helped i n many ways - and many thanks to my mother, father and wife for their patience. The work for this thesis was carried out during the tenure of a Research Fellowship granted by the Crease C l i n i c . 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 in the cerebral cortex of man in 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 in 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 histolog-ical evidences. A number of methods have been recorded and discussed in detail and many others mentioned. Some evidence of the nature of the 1 boutons terminaux ' in the cerebral cortex of man is 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 in 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 con-vinced that " in 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 anasto-mosing 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 in 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 cell and the next and proposed the theory that the processes of the nerve cells end freely in the grey matter and are not intertwined in 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 ob-servations 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 in 1894 he said, " Les connexions etablies entre les fibres et les cellules nerveuses ont lieu au moyen de contact, c'est-a-dire a. laide d'une veritable articulation entre les arborizations variqueuses des cylindres-axes d'une cote, le corps et les prolongments protoplasmiques de 1'autre. Aussi est on amene a se represente l'axe encephalo-spinal comme un edi-fice compose d'unites nerveuses superposees, de neurones , suivant 1'expression de Waldeyer." Waleyer, a German anatomist, had written a resume of Cajal's ideas and popularized the neurone doctrine. In 1891 Held reported his discovery of the " calices " or syn-apses of the nucleus of the trapezoid body using the methods of Golgi and Nissl. In 1897 he published a report of his " Endefusse " which Auerbach confirmed in 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 finally accepted by Auerbach. Held believed the Endefusse to be imbedded in the inter-- 3 -stices of the n pericellular nerve net " and saw continuity with the inner reticulum as well. Auerbach, however, thought that a transparent cuticle 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 - the hypothesis of continuity. His, von Kolliker, 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 topograph-ischen Beziehungen zu den Zellen " i n which he b u i l t up a theory regarding the minute structure of the nervous system. In this he theorized that the neurofibrils form the specific constituent of the nervous system and that they were the independent structural parts, the conducting elements. In the nerve fibres they preserved their individuality, forming reticula only i n specific places. He maintain-ed that they formed large continuous reticula i n the organs of the body and above a l l i n the central grey substance. The evidence for these conclusions was obtained with the help of a new gold staining method• Apathy was later vigorously supported by a German histologist, Stephen Bethe, ( ) who became one of the chief opponents of the neurone theory. The Golgi technique on which these opposing theories are largly bases was crude compared to the more delicate present day silve r methods • - u -Fresh tissues were placed in solutions of potassium dichromate be-ginning 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 in 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 in thick xylol-damar, without a cover sl i p . By this technique the nerve cells and delicate fib»ps were opaquely stained so that the intracacies of the nerve cell 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 in the nerve cell processes the neurofibrils always remain within their substance and are not, as Ap-athy asserted, capable of emerging from i t . Retzius ( 1908 ) summarized the investigations on the subject stating that " Neurone fi b r i l s are to be found in the nerve cells and their processes, that they farm in the cells abundant reticula, which are plainly to be seen even in some of the peripheral terminal organs, and that they do not anastomose out-side the particular domains of the cell unit or neurons, i e . that they - 5 -do 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 fibri l s con-stitute 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. " The contact theory is a histological impossibility " states Marui. Bartelmez (1930), with non-metallic stains, settled the question in favour of the contact theory and the neurone doctrine. Tiegs, using the reduced silver methods of Cajal and Bielschow-sky, 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 in the neuro-fi 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 in the spinal cord — in 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 cell n. As late as 1931 Tiegs maintained that " whether there are end-bulbs or not there is protoplasmic continuity between the axone terminals and the intracellular fib r i l s of the cell 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 in the young material is 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 in the cerebellum. This view has lately been express-ed by Glees (194-6). In 1933 Bartelmez and Hoerr gleaned valuable information on returning to a study of the bullhead, Ameirus, in 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 critically examined the fixation and staining techniques used in studies of the synapse. They suggest that the differences in opinion concerning the nature of the synapse are due to differences in technique. After shrinkage of the tissue in fixation most synapses are so minute as to be on the verge of the resolving power of the optical microscope. They also suggest that the personal factor plays an important role. It is 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 reliability 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 in the living tissue of the medusa Rhistoma. The nervous elements were stained with a methylene blue leucobase. It was previously accepted that the coe-- 7 -lenterate nervous system was sycyttoi in character. Bosler's prepar-ations showed only contact at the synapse and no evidence of continuity. He also observed the neurofibrillae in the living cells. Their pres-ence in 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 cell in 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 in no instance could they be seen to pass from the end-feet across the synapse and into the Mauthner cell's den-drite. 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 is unreliable and is inferior to the individual section method. In staining with silver the time allowed for adsorption of the silver solutions is the most important variable. Ammoniacal alcohol used be-fore mordanting in silver prepares the tissue for an overlay of silver which may give untrue results. Bartelmez maintains that formol is 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 in 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 cells. 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 investig-ators 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 in neurofibrils sixteen hours after death in 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 in 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 end-feet did not necessarily affect the cells themselves. The work of Hoff and Gibson finally 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 in 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 in 96$ alcohol and ammonia. To impreg-nate, the tissue was placed in 1.5% smlver nitrate for six days at 37° C. After impregnation the tissue was washed, reduced in 2% hydroquinone, - 9 -dehydrated, imbedded, sectioned at 15 microns and mounted. Studyimg the nature of the normal synapses in 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 in the gran-ular layer of the cerebellar cortex. These normal synapses stained as round or elli p t i c a l loops of 2 to 4 microns in 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 degener-ation studies in 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 forty-eight 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 in 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 its cell body is followed by the degeneration of its termination in 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 in which cephalopods are kepji. Lawrentjaw (1934-) considers the boutons to be constant structures in 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 de-generation, in which i t is important to differentiate between boutons of longer and those of shorter periods of degeneration. - 11 -THE SYNAPSE The reticular hypothesis was finally rejected after experi-ments 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 final 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 in 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 in 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 dis-cussed. The contributions of Sereni and Young were also mentioned, and of Eoerster et. a l . After the introduction of the bouton method in the tracing of fiber pathways, researchers began to develop measuring sticks for bouton degen-eration 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 in each area to be investigated. Bodian (1937) stained the axon endings on the Mauth-ner's cell in the goldfish using a protargol method. He described them as blackened loops varying in diameter from 0.5 microns to 7 microns. Phalen and Davenport (1937) demonstrated boutons in 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 in Mammals, with the exception of the monkey, the end-bulbs tended to vary in size with the species. Because of the variation in 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 in 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 un-reliability 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 in the human cord ? small loops, large loops, filamented loops, fibrillated loops and opaque or granular loops. He found that each of these might appear as regular, thickened or granular. These endings were usually found in contact with the cell or robust process. They ranged in size from 0.5 x 1 micron to 3.5 x 5 microns. Varying as a fundtion of the cell size, the number of boutons per cell ranged from 144- on small sensory cell bodies to 1832 on the large cell bodies of the posterolateral column. Minckler showed (1941) that the morphological types of endings in a given area of the human cord remain fairly constant from one individual to the next, re-gardless of the age, i f the staining technique is carefully controlled. It was also shown that the different types of terminal occur in about the same numbers on different parts of the nerve c e l l . However, gran-ular forms occurred more frequently in older persons. It was realized - 13 -that the appearance of the thickened forms may be due to technique such as prolonged fixation in 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 in the cerebral cortex are remarkable for their scarcity. Hoff, using block silver methods, stained cortical end-feet in the cerebral cortex of the aat, but with great difficulty. Cajal (1934) reported boutons in the cortex. Poorly rewarded efforts to stain boutons in the cortex have led some investigators to believe that these are not the normal types of synaptic points in 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 circumstancesT i t is 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 striatal cells and suggest that these may make connection with the dendrites. Degenerating boutons were dem-onstrated by Greenfield (1939) in patients with cerebral oedema. King (1942) described briefly pericellular argyrophylic structures in relation to pyriform cells in the pyramidal layers which reduced ammoniacal silver solution without further chemical treatment. In 1946 Glees stated that " It seems that the.synapse within the cortex is mainly represented by free terminals of the pericellular plexus ". However he showed that the pericellular fibres in the caudate nucleus degenerate after cortical ablation (Glees, 1944) and suggests that these can be - L o -used in degeneration experiments. The fallacy of this view will be reviewed in the discussion. In spite of its apparent potentialities, the 1 bouton 1 method has not enjoyed the popularity which one might expect. However, in the hands of a number of investigators i t has yielded many valuable contri-butions to the knowledge of the nervous system. Hoff (1935) used the bouton method to investigate the terminal distribution of the cortico-spinal fibers arising in the premotor area of the monkey. The structure of the lateral geniculate body and the projection of the retina in 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 Le Gros Clark, 1941/ Glees 1941 and 1942) to study the termination of the optic nerve" tract fibres in the lateral geniculate body of the monkey, cat and rabbit. O'Leary (1940) contri-buted to the knowledge of the lateral geniculate body. The optic centres in 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 in the cortex has already been mentioned. Brodal (1949) used the Glees method in an experimental study of the spinal afferents to the lateral re-ticular nucleus of the medulla oblongata in 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 paravent-ricular nuclei of the hypothalamus and the causate nucleus. A report on the use of intravenous methylene blue for studying - 15 -fiber degeneration i n the central nervous system has recently been published by W. H. Feindel and A. C. Allison (194-8). A c r i t i c a l discussion of the methods used to study fiber degeneration and suggestions for the use of methylene blue were published by Feinctel, Allison and Weddell the same year. By their new method they were able to stain en-larged terminal boutons i n the l a t e r a l geniculate body in rabbits. The need for reliable methods of studying fiber degeneration i n the cerebral cortex became more pressing with the introduction of the surgical treatment of certain mental disorders by frontal lobotomy. Most of the knowledge of the projection to and from the frontal cortex has been derived from experimental observations i n monkeys and apes. In a report on the effects of prefrontal leucotomy, Meyer, Beck and McLardy (194-7) state that " There i s no need, to emphasize the importance of a detailed neuro-anatomical investigation of the brains of patients dying at various intervals after prefrontal leucotomy. " The methods which can be used for such an investigation are few, and to date the most promising staining procedures are reduced silve r methods. The Mechanism of Synaptic Transmission A brief discussion of the earlier literature on the mechanism of the synaptic transmission i s thought to be important as giving con-text to the problem of synaptic structure. The physiology of nerve activity has received much attention during the past half 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 complete-l y solved. Early 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 direct-ly on the heart muscle. Dale (1933) tried to extend the 1 neurohumoral ' theory to the neuromuscular junction and the ganglionic synapse. The acetylcholine hypothesis ( Dale 1937, Clark 1936 ) simp2y stated that a presynaptic impulse liberated, at the synapse, a minute amount of acetylcholine, which excited the post-synaptic cell, 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 explain-ed 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 cell are basically the same. Thus the neurohumoral theory seemed to be unsatisfactory. The electrical theory of nervous transmission, in its early form, was also unsatisfactory. ( Eccles 1936, Erlanger 1939, Lorente de No 1939, Monnier 1934) In general i t was merely stated that the electrical currents of the presynaptic impulses set up impulses in the post-synaptic c e l l . The significance of the pre- and post-synaptic responses and of the rheobase of the post-synaptic cell 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 in the mabrane in which a release of acetylcholine is 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 is unaffected by high concentra-tions of acetylcholine. A later hypothesis on the synaptic transmission is the culmina-tion of several years of intensive research by J . C. Eccles r (1947). Three assumptions are made: fi 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 is, also, electrical evidence for a highly resistant transverse membrane. Second, he assumes that, in 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 is specialized, so that large and graduated local responses are set up by polarizing currents. With these basie assumptions the following sequence of events in synaptic transmission was described. First,the impulse in the pre-synap-tic nerve fiber generates a current which gives a diphasic effect at the synaptic region of the post-synaptic cell with a total duration of not more than 1 millisecond in 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 cell membrane. Finally a propagated impulse is set up in the post-synaptic cells, i f this catelectrotonus is above a critical 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 in relay of a nerve impulse but the extent to which each agent is involved must yet be determined. Rushton concludes his summary 11 *t is natural to try to explain the two kinds of conduction upon a common basis, but what has been proved is that along the nerve fiber the transmission is electrical*, from the endings of peripheral nerves i t is pharmacological. n - 19 -EXPERIMENTAL PROCEDURE The experimental work has been concerned mainly with the stain-ing of nerve fibers and the terminal synapses i n the cerebral cortex. Several modifications of the reduced silve r technique have been t r i e d . The stepwise procedure for each of these has been included for reference purposes i n an appendix. Certain procedures have been discarded for reas-ons which w i l l be discussed under the heading of experimental results. The Cajal's modification for frozen sections and the Gros-Bielschowsky method have been used to stain the spinal cord and cortex i n the dog. The double impregnation method for frozen sections and the Urea-Silver Nitrate method of Ungewitter were used to stain the cerebral cortex i n humans. A l l tissues were fixed i n 10$ neutral formal regardless of the special fixatives mentioned i n each method. In a l l cases frozen sect-ions were cut at 15 microns. Cajal's Modification for Frozen Sections. ( See Appendix A for detailed ' " Instructions ) It was found advantageous to receive sections from the freezing microtome i n water containing a few drops of concentrated ammonia. In step 2, three to five drops of pyridine per 15 c c . of AgN03 solution proved sufficient. Adding greater amounts than this resulted i n a dust-like precipitation of s i l v e r . An impregnation time of 6 hours gave the best results although longer i n the silver bath seemed to do notharm. Slides of spinal cord, cerebral cortex and cerebellum of dog were stained by this method. Alternate sections were toned i n gold. - 2 0 -The Gros-Bielschiwsky Method ( See Appendix B for details ) The block of tissue was soaked in water containing a few drops of ammonia. Frozen sections were cut at 15 microns and received in a dish containing a few drops of neutral formalin. The remaindec of the procedure was followed as outlined in McLung's Handbook. (Ferdov's Modif. ) Sections of the cerebral cortex of dog were stained by this method. Alternate sections were toned in gold. Double Impregnation Method (See Appendix C ) The procedure outlined in the Appendix is Gibson's modification of Rio Hortega's Silver Carbonate Stain for neurofibrils. Frozen sec-tions cut at 15 microns were received in 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. in a shallow water bath. Five minutes in the gold chloride were found to be ample when the impregnation was reinforced by heating one minute over a spirit 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 in 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. ( Fig. 1 ). Portions were cut from this slice as indicated in the diagram ( Fig. 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 in this laboratory for our purposes. The method of Glees involved an unnecessarily long procedure which is a modification of the shorter Gros-Bielschowsky tech-nique. 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 brilliant section in tones of gold and brown. However, in our hands the procedure did not show neurofibrils, and moreover, no terminal end-bulbs could toe found. Another disadvantage to this method is the use of protargol which is difficult to obtain. Cajal 1s modification for frozen sections proved to be a rather delicate and exacting method. The amount of pyridine used was found to be very important, an excess usually resulted in the undue precipitation of silver giving • muddy ' preparations. However this is a good proced-ure for demonstration of the larger neurofibrils and boutons in the cord. The fibers and fibrils in the cortex were well shorm, but no boutons could be demonstrated. The background of the untoned sections also stained fairly heavily so that tracing of the fibr i l s is more difficult. The nuclei of the neuroglia pick up the stain fairly heavily. Much of this unnecessary background is subdued by toning in gold. The fibers and fib r i l s then appear more pronounced and are more easily traced. In the cerebral cortex these conditions also hold true. - 23 -The Gros-Bielschowsky technique produced quite good results in 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 success-fully, this, in 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 pre-dict 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 depend-able and satisfactroy. By carefully following the specified conditions and times, excellent preparations could be made almost without f a i l . In cell preparations of the human cerebral cortex i t was possible to ident-if 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 dis-tinguish the neurofibrils leading to the end-feet. ( Figs. 9, 13 and 14 )• The Lobotomized Brain. The results here are recorded in the numerical order of the portions as they are numbered on the diagram of the brain slice used. ( Fig. 1 - 2 ). Portion A. This is from the cingulate gyrus on the medial side of the cerebral hemisphere. Elongated torpedo-like swellings along many of the medium fibers. Distinct ring-like bouton terminaux, ranging in size from 1 micron to 3 microns in diameter, could be found in every field. On the average between three and five boutons could be found in every field, ^any of these were adjacent to or apparently in contact with cells or dendrites. ( Figs, 9 and 10 ). Portion B. This is from the superior frontal gyrus. Torpedo-like swellings were again found in large numbers in 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 in almost every field and often in contact with cell or dendrite. ( Figs. 11 and 12 ). Portions C and D. These are in the areas 8 and 9 of Brodmann anterior to the premotor area 6. The pattern of the previous section is carried out in these. The boutons could be found in a l l the layers below layer 1. The fibers show elongated swellings, and an increased take-up of silver. Many of the fibers also appear somewhat tortuous. ( Fig. 13 ). Portion E. Boutons on dendrites and cell bodies. Fibers show elongated swellings. ( Fig. 14 )• Portion F. Many large pyramidal cells. Boutons in almost every fie l d . Fibers swollen at intervals and sometimes tortuous. ( Figs. 15, 16 and 17 ). Visual Area. Typical visual area showing line of Gennari. Boutons in this region appeared to be somewhat larger than general, ranging from 1 to 4 microns, in diameter. ( Fig. 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 silver. In the early procedures of Cajal and of Bielschowsky, tissues were immersed in solutions of pure silver nitrate in varying concentrations. Del Rio Hortega immersed the already impregnated tissue in 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 Win-throp Chemical Company, called ' Protargol 1. Each of these techniques in the laboratories of competent investigators, has produced excellent results. The modification of the basic techniques have usually been intro-duced 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 impreg-nation with silver. The most widely used fixative, and that used for a l l tissues in this work, is neutral formalin. Formalin has the advantages of universal availability, cheapness, rapid penetration and minimal colour change. Most hospitals and mental institutions use neutral form-alin for the preservation of autospy and biopsy material. It is therefore an additional advantage that silver techniques can be successfully applied to formol-fixed tissues. It is interesting that Spiegel-Adolf, Henny and Ashkenaz (1944) have pointed out that even X-ray diffraction studies re-- 26 -veal hardly any changes in the X-ray diffration pattern of formalin-fixed muscles, in marked contrast to results following the use of other fixatives. The second stage at which modifications may be introduced is in the concentration of the silver solution. Concentrations recommended vary between 1% and 20% silver nitrate. Most authors suggest a 2% solution. This seems to be a sufficient concentration to give a rapid impregnation without resulting in too great a deposition of s i l -ver . Silver solutions of 10$ and above have a tendency to encrust the nerve fibers, and, in 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 in the central nervous system has been pro-vided 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 prelimin-ary treatment with a chromic salt. It's failure to demonstrate degen-erating non-myelinated fibers and degenerating finely myelinated fibers however, is 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 its many uses and advantages, the Marchi method alone cannot satisfy the needs of neuroanatomical investigations in 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 ex-perimental lesion of the brain. Feindel, Allison and Weddell have stained bouton ending in the lateral geniculate body. However, satis-factory results have not yet been obtained using previously fixed ex-perimental material. Therefore the method is not yet applicable to the investigation of human brains obtained at autopsy, i t should be mentioned here that preliminary experiments in 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 impreg-nation methods for the satisfactory demonstration of normal and de-generating nerve fibers and endings. This brings us to a discussion of the stainabilty of the bouton endings in the cerebral cortex. They can be shown with varying success in 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 in the cortex and then they have been few and far between. The failure of the end-feet to show themselves in 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 — — i t is 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 in the cerebral cortex ? Glees statement that " It seems that the synapse within the cortex is mainly represented by free terminals of the pericellular plexus " is too easy a rationalization. These pericellular fibers have been clearly demonstrated in this labor-atory. After careful study of many sections, however, no case of con-tact between these fibers and the cells could be seen except by means of bouton terminaux. The system of neurofibers and neurofibrils in the cerebral cortex is 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 cell en route to their destinations is no reason to believe that the fiber has any-thing 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 spec-ialized 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 tells me that he has seen as many as fifteen boutons in contact with one corti-cal c e l l . ) A solution to the problem of staining the endings in the cortex must be found. Why is i t so difficult to stain boutons even though in many cases the nerve fibers right up to the synapse- stain easily ? It - 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 in an identical manner by perfusion with 10$ formol. One of the dogs was perfused with a solution of eser-ine in physiological saline just before the fixation. Eserine is an anti-cholinesterase and its presence at the synapse would prevent the removal of acetylcholine by the cholinesterase. Thorough histological study of various areas in the spinal cord, cerebellum and cerebral cort-ex showed no difference in the stainability of the two nervous systems. Perhaps the difficulty in staining boutons is a reflection of the rapid chemical change associated with death, which would be greater in the synapse because of the blood supply ? If this is so, would prevention of this chemical change by some means increase the staining ? A positive approach to the problem must be made. Only after its solution can a thorough investigation of the frontal areas in the human cerebral cortex be made. SUMMARY -A survey of several reduced S i l v e r techniques fo i r the staining of nerve f i b e r s and. boutons termin-aux i n the cerebral cortex i s reported. A double impregnation method which i s a modification of Del Rio Hortega's s l i v e r n i t r a t e - s i l v e r carbonate tech-nique was found most s a t i s f a c t o r y . The method v/as used to study cert a i n areas of the human fr o n t a l cerebral cortex and to demonstrate the boutons terminaux i n these areas. Evidence i s s u f f i c i e n t to indicate that these boutons are the normal form of inter neuronal synapse i n the cerebral cortex of the human- and fur ther research on the staining problems i n me nervous system i s suggested. Pig. 1. Diagram of lateral view of brain to indicate the angle and region of lesion and the area from which the slice was taken. Figs. 5 and 4. A few normal, boutons terminaux on lateral horn c e l l of the cteg spinal cord, (arrows) These two photographs were taken, at slightly-different focal levels, 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 in gold. F i g u r e 4 Fig. 5 Cell In the cerebral cortex of a dog. Stained with Cajal's modification. Note the- differentiation of nudlei and. fibers, (mag. X 1500) Figure 5 Figs. 6 and 7. Cerebral cortex of. dog stalined by the Gros-Bielschowsky technique. Note the apparently degenerating bouton in f i g . 7 and the large darklj^ryimpregna&ed one in f i g . 6. The fibers In general are heavily impregnated with sil v e r , (mag. X 1500) Figure 6 Figure 7 Fig. 8. Motor area in cerebral cortex of a dog. Stained by the double impregnation method, and toned in gold,, (mag. X 1500) Figure 8 . 9 and 10. Cortex on the cingulate gyrus. Stained by double impregnation method. Toned in 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 tion. (mag. X 1500) Fig. 12. Human cortex from portion B. Dbl. Impr. (mag. X 1500) Fig. 15. Human cortex from portion C Dbl. Impr. (mag. X 1500) Figure 11 P i p - t i r e 12 Figure 13 F i g . 14. Human cortex from portion E. Dbl,. Lmpr. (mag. X 1500) Figure 14 F i g s . 1 5 , 1 6 a n d . 1 7 . H u m a n c o r t e x . f r o m p o r t i o n F . D b l I m p r . ( m a g . X 1 5 0 0 ) Figure 15 g u r e 16 Figure 17 F i g . 18, Human cortex from the v i s u a l cortex - area 18 of Brodma=nn. Dbl. Impr. (mag. X 1500) F i g u r e 18 I il - APPENDIX -A Caial's Modification for Frozen Sections (Gibson, 1950) (1) Blocks of tissue, fixed for at least one week in 20 per cent formol, are cut on the freezing microtome at 12-15 microns. (2) Wash the section well in water and place in 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 in the cold. The latter is desirable for the finest detail. Silver nitrate solution up to 5 per cent has been employed successfully in 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 in 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 in yellow gold chloride solution, 1 to 500, for five, to ten minutes. (6) Fix in 5 per cent hypo, wash, mount, dehydrate, balsam, e t c B An Intensified Protargol Method for Paraffin Sections (W.A. Stotler) (1) Blocks of tissue, fixed in 15 per cent formalin, Bouin's solution or acetone, are imbedded in paraffin. Sections cut at 15 . microns, paraffin removed, sections through alcohols to water. (2) Impregnate 18 to 24 hours in 0 . 1 per cent aqueous sol-ution of protargol. Place 5-10 gm. of copper (shot) in 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 glycero-phosphate 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 op-timum, 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 in 330 c c . water, 10 c c . Solution C, 35 gm. sodium thiosulphate in 330 c c . water, 10 c c . ; Solution D, 5 gm. sodium sulphite, 8 gm. Kodak Elon in 1000 c c . water, 30 c c 3 ^ I I APPENDIX -C Gros-Bielschowskv Method (Gibson, 1950) (1) Fix material in 12 per cent formalin neutralized with calcium carbonate, from one week to several years. (2) Soak the block of tissue in water, and cut frfczen sect-ions from 20 to 50 microns into a dish of water containing a few drops of neutral formalin. (3) Place sections in 20 per cent silver nitrate, one to five minutes• (4) Arrange four capsules of 20 per cent neutral formalin in 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 in 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 is placed under an observation microscope.to control the impregnation. A fairly rapid coloration is best, and when the nerve trunks in the section are seen to reach a dark, almost apaque brown-black color the section is transferred immediately to a solution containing 4 parts of ammonia to 5 of water, for five minutes. (6) Wash until no odor of ammonia is detectable. (7) Tone in 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 is added periodically. (8) Fix in 5 per cent hypo, dehydrate, and mount in balsam. Ammoniacal Silver Solution - This must be prepared in small quantities as required by adding concentrated ammonia to 20 per cent silver nitrate solution until the precipitate just disappears, leaving a brown tinge throughout the solution. A glass rod should just be visible in this. A crystal clear is too ammoniacal. Fresh formalin in the four reducing capsules, and fresh ammoniacal silver in the watch glass must be used for each section, regardless of its size. The watch glass is never washed. Its contents are renewed each time. For boutons i t is best to leave the section only a short time in the 20 per cent silvernitrate dish, and to use the minimum of ammonia in the watch glass stage. 3> III - APPENDIX -D Glees' Modification (P. Glees, 194.6) (it) Fix material in 10 per cent formalin or 5 per cent for-malin-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 cc. alcohol and main-tain 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 precip-itate first formed has redissolved, and then add another 5 drops of ammonia. (8) Transfer into a 10 per cent formalin solution where sec-tions 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 imppeg-nation 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 cc. 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 con-taining 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 cc. 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 treat-ment 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 cc. 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 Ballantyne, F.H. 1925 Barnard, R.I. 194-0 Barr, MJL. 1939 Barr, M.L. 1939 Barr, M.L. 194-0 Bartelmez, G.W. 1915 Bai&elmez, G.W. 1920 Bartelmez, G.W. and 1933 H »L.Hoerr. Bauer, Karl 1932 Beccsrri, N. 1918 Beck, B. 194-9 Beck, E. . 1950 Das terminale nervennetz i n seinen beziehungen zu dem ganglienzellen der cantralorgane. Monatschr. F. Psychiat. u Neurol. 6, 191 Continuity o f the vertebrate nervous system, Trans. Roy. Soc. (Edinb.) i3_, 663. 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