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A histochemical investigation into the regional distribution of monoamine oxidase in the brain-stem of… Halsey, Nancy M. 1962

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A HISTOCHEMICAL INVESTIGATION INTO THE REGIONAL DISTRIBUTION OF MONOAMINE OXIDASE IN THE BRAIN-STEM OF RABBIT AND CAT WITH ATLAS OF RELATED CONCENTRATIONS by NANCY M. HALSEY B.A. , University of Br i t ish Columbia, I960 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF Master of Science in the Kinsmen Laboratory of Neurological Research Department of Psychiatry We accept this thesis as conforming to the requi red standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1962 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . I t i s understood t h a t copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be allowed without my w r i t t e n permission. Department of R^ JL\dk^  f j^j^^^ ^)n^JTf^ ^ /NTw^p"^ fc^a^L The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, Canada. ABSTRACT In the last decade, considerable interest has been focused on the role of biogenic amines and their function in the central nervous system. Certain of these, norepinephrine and serotonin, have been suggested as neurotransmitters, and evidence has accumulated that r ise in the levels of these amines results in behavioural change. At the same time, i t was found that the enzyme, monoamine oxidase (MAO), u t i l i zed these compounds as substrates, and that inhibit ion of MAO resulted in elevated levels of the catecholamines and serotonin. This knowledge has led to considerable investigation of the areas of brain that might be affected by this inhibi t ion, but beyond the preliminary report of Shimizu et aj_. ( 1 9 5 9 ) l i t t l e had been done to determine the histoehemical local izat ion of MAO by methods of proven speci f ic i ty. A study of the brain-stem has therefore been attempted, to determine the major si tes of MAO ac t iv i ty . Rabbit and cat brain-stem have been used, and the histoehemical method of Glenner, Burtner and Brown ( 1 9 5 9 ) using nitro-blue tetrazolium. Fresh frozen tissue was cut on the cryostat and sections incubated with this solution, a positive result producing a purple-blue formazan precipitate. For identi f icat ion and correlation of brain-stem nucle i , adjac-ent sections were cut and stained with toluidine blue. Controls were run in v i t ro and in vivo with known MAO inhibitors H i as well as by incubation without substrate, application of heat and alteration of pH. F ina l ly , an atlas has been prepared identifying the sites of MAO act iv i ty and suggesting a functional relationship based on these studies. Results indicate that within the brain, the brain-stem i tse l f contains the highest proportion of MAO, which is concentrated within the following regions - choroid plexus, pineal gland, hypothalamus, p i tu i tary , interpeduncular nucleus, habenulo-pedencular t ract , dorsal tegmenta] nucleus, locus coeruleus, area postrema and cranial nerve nucle i , especially the distal portion of the trigeminal nucleus and the dorsal vagal nuclei . The thalamus, infer ior c o l l i c u l i and major f ibre tracts were a l l conspicuously low in MAO. With the exception of the ce l ls of the mesencephalic nucleus of nerve V, act iv i ty did not occur within the body of the neuron, but was present in the neuropil of the neocortex and a l l other posit ive areas of brain-stem. Certain peculiar-i t ies of distr ibut ion were noted for the glandular areas of the pineal , pituitary and choroid plexus. In the anterior pituitary and choroid plexus, MAO was found in intracel lular granules, but within the pineal and posterior pituitary the act iv i ty appeared to l i e in a matrix between eel Is. vi i i ACKNOWLEDGEMENTS I am greatly indebted to Professor William C. Gibson, Or. Harald Scherrer, Dr. Juhn Wada and Drs. Patrick and Edie McGeer, for their stimulating and constructive advice on the subject of this thesis, and I am especially grateful to Miss S . Calthrop, Mrs. I Hemstock, and Mr. Ron Plaster, for their expert technical assistance. INDEX Page INTRODUCTION 1 Part I. CHEMISTRY AND PHARMACOLOGY OF MAO 3 1. History 3 2. Identification and determination of MAO by chemical methods k 3. Distribution of MAO 5 k. MAO in brain development . . 7 5. Substrate location, metabolism and mechanisms of action 7 6. Uptake of substrates in brain 12 7. Inhibition of MAO 13 8. Psychiatric implications 15 Part II. HISTOCHEMICAL INVESTIGATION OF MAO 16 1. Historical development and rationale of histoehemical methods . . 16 2. Comparisons of other work on brain MAO . . . . 18 3. Chemistry of tetrazolium salts 20 k. Chemistry of MAO-tetrazolium reaction . . . . 21 5. Evaluation of reaction mechanisms and speci f ic i ty .-. 22 Part III. EXPERIMENTAL PROCEDURE AND TECHNICAL DATA 23 1. Animal material 23 2. Methods of treatment 23 3. Solutions 2k k. Controls 2k 5. Experimental results 25 6. Photography 32 Part IV. ATLAS OF MAO IN BRAIN-STEM 33 1. Arrangement of atlas 33 2. Key to atlas abbreviations 33 3. Nomenclature 36 k. Atlas ser ia ls 37 5. Description of special areas 64 V INDEX, cont 'd. Page Part V. DISCUSSION 70 1. Review of regional concentrations of MAO . . 70 2 . Comparative histochemistry 7^ 3 . Relationship of regional concentrations to functions of MAO 75 SUMMARY 82 BIBLIOGRAPHY 83 vi INDEX TO TABLES Table Page I Catabol ism of catecholamines showing in f luence of MAO 9 II Tryptophan metabolism in r e l a t i o n s h i p to s e r o t o n i n , melatonin and MAO 10 III Results of i n vi t ro tes ts of var ious drugs on MAO i n h i b i t i o n in rabbi t bra in 25 , v In v ivo tests of MAO i n h i b i t o r s 28 V Substrate tests 29 VI Uptake tests 29 VII E x t i n c t i o n tests 30 VIII F i x a t i o n tests . . . . . 31 IX Regional comparisons of MAO-rich areas of rabbi t bra in -s tem 71 X Diagram showing nucle i of c r a n i a l nerves and concentrat ions of MAO 72 XI O l fac to ry connections showing MAO concentrat ions . . 77 XII Diagram i l l u s t r a t i n g concept of p ineaI -hypothalamic -p i t u i t a r y i n t e r a c t i o n 78 INDEX TO PLATES Plate Page I Dorsal view of rabbit brain-stem showing levels Back of sectioning pocket II Lateral view of rabbit brain-stem " Ml Ventral view of rabbit brain-stem " IV Level of decussation of the pyramids, Sec. 03-8 kO V Through vagal nuclei and area postrema, Sec. 03-15 •• 43 VI Upper levels of vagal nucle i , Sec. 03-16 . . . . . . 45 VII Upper levels of vagal and hypoglossal nucle i , Sec. 03-20 47 VIII Level of isthmus, Sec. 03-36 51 IX Level of superior c o l l l c u l i , Sec. 04-20M 55 X Through mammillary body, Sec. 04-13M 58 XI Hypothalamic region 61 1. INTRODUCTION The purpose of this investigation is to explore the speci f ic areas of brain-stem where monoamine oxidase (MAO) is shown to be most act ive. Only Shimizu et a k (1959) and Smith (unpublished) have attempted any detailed histochem!cal investigation of cerebral MAO, and since a refinement of Shimizu's technique was available here, i t has thus been possible to add to original f indings. For this purpose, histochemical methods using nitro-blue tetrazolium have been used, in conjunction with more common histological techniques, and an atlas of the brain-stem has been prepared, supplemented by hand-drawn maps. Controls were run by in vi tro and in vivo administration of MAO inhibi tors, and an evaluation made of the reaction spec i f i c ! ty . Papez (1929) defines "brain-stem" as that part of the brain which remains after removal of the cerebral hemispheres and cerebellum. In many studies of more recent date, however, the anterior l imits are taken no further than the c o l l l c u l i and may even terminate before them. Thus, in view of the considerable latitude in this respect, i t is proposed in this study to describe "brain-stem" as the area bordered by the optic chiasma anter ior ly, and by the f i r s t cervical nerve posterior ly, excluding the cerebral hemispheres and cerebellum (see plates I, II and III). The brain-stem has been chosen for examination because of i ts phylogenetic and functional importance. The primitive brains of vertebrates were l i t t l e more than brain-stem consisting of cranial reflex mechanisms, which in the mammals became a compact, highly organized structure l inking a l l parts of the old and new brain. In any mixed col lect ion of mammalian 2. brains, the brain-stem shows remarkable similar i ty from species to species, so that, bearing in mind the increase in size and complexity of the cortex and related connections in higher mammals, any wel1-organized brain-stem wil l exemplify the entire mammalian c lass . We know at least that this is so anatomically, but we do not yet know whether this is so chemically. Thus, because the brain-stem is the oldest and most fundamental part of the brain, carrying out reflex arrangements in connection with cranial nerves, regulating conduction systems to the cortex and cerebellum, and engaging in the basic act iv i ty of the reticular formation, i t is of part icular interest to study the chemoarchitecture of this area. Further-more, i t might perhaps be called the "glandular brain" since i t is known that in many parts of the brain-stem, hypothalamus, p i tu i tary , pineal and probably some other areas, neurohumoural mechanisms are at work that exert, and are affected by, internal and external environmental influences of al1 kinds. Above a l l , i t is here that biogenic amines have been found in the highest concentrations, and upon which monoamine oxidase exerts i ts ef fects . It i s , therefore, the purpose of this study to explore the brain-stem in order to determine not only the precise regional locations of this enzyme, but also i ts relative concentrations in the various parts, and to see i f some consistent pattern emerges that would indicate any special l inks be-tween structural units and brain monoamines. 3. PART I. CHEMISTRY AND PHARMACOLOGY OF MAO 1. History. The f i r s t indication that amines could be deaminated in the body by an enzyme was noted by Hare (1928) who showed that mammalian l iver contains a tyramine oxidase. In 1937, Pugh and Quastel observed that melanin-like pigments, firmly bound to t issue, were formed by the oxida-tion of indolethyl amine. They suggested that this reaction might be catalysed by MAO. In the same year, Blaschko et a l . . , Pugh and Quastel, and Kohn, independently concluded that the enzymes oxidizing tyramine, adrenaline and al iphat ic amines were ident ical . Ze l le r (1951) suggested that this enzyme be called "monoamine oxidase" to differentiate i t from diamine oxidase. In 1953, Blaschko and Hellman confirmed that tryptamine and serotonin (5-HT) were oxidized by MAO, converted to an aldehyde, and f ina l l y formed dark brown pigments insoluble in water. They stated that this reaction should also be ut i l i zab le as a histochemical technique. Further work by Blaschko and Phil pot (1953)> Sjoerdsma et a l . . (1955), Udenfriend and Titus (195^), Udenfriend et al.. (1956), and Weissbach ejt a k (1957) has shown conclusively that 5-HT is deaminated by tissue MAO to give 5-hydroxyindoleacetaldehyde, which is then converted to 5-hydroxyindoleacetic ac id . However, in the presence of an electron acceptor such as tetrazolium s a l t s , the conversion to the acid does not take place, and another pathway is made leading to formation of a formazan pigment precipi tate. 4 . 2. I d e n t i f i c a t i o n and Determination o f MAO by Chemical Methods. MAO may now be defined (Davison, 1958) as the enzyme which i s res p o n s i b l e f o r the o x i d a t i v e deamination o f monoamines such as the catecholamines, s e r o t o n i n , tyramine and tryptamine, according to the general equation: R.CH2.NH2 + 0 2 + H 20 • R.CHO + NHj + H 20 2 I s o l a t i o n o f MAO has r e c e n t l y become p o s s i b l e , and i t i s i n t e r e s t -ing to note t h a t i n j e c t i o n o f a p u r i f i e d MAO p r e p a r a t i o n i n t o hypertensive r a t s r e s u l t s In a prolonged lowering o f blood pressure (Davison, 1958). MAO i s destroyed by heating f o r 10 minutes a t 5 0°C, o r by a pH below 6 and above 9-10. I t s optimum pH appears to be around pH 7*3* I t i s i n h i b i t e d by i p r o n i a z i d (1 x \0~l* M) but not by cyanide o r i s o n i a z i d (1 x 10"^). Lagnado and Sourkes (1956) p o s t u l a t e d an amine dehydrogenase system, r e q u i r i n g a h e a t - s t a b l e c o - f a c t o r , present i n l i v e r and r a t b r a i n . Glenner e t a K (1959). however, a f t e r f u r t h e r i n v e s t i g a t i o n using reduc-t i o n by t e t r a z o l i u m s a l t s , could f i n d no evidence o f such a mechanism. Thus, there are no known co-enzymes o r p r o s t h e t i c groups connected w i t h MAO. Several methods have been used f o r determining MAO a c t i v i t y i n t i s s u e s : a) Manometric methods, usi n g tyramine o r tryptamine as s u b s t r a t e , and measuring the oxygen uptake during incubation (Creasey, 1956). The b i o l o g i c a l a c t i v i t y can best be determined on the washed p a r t i c u l a t e f r a c t i o n o f homogenates o r on d i a l y s e d homogenate p r e p a r a t i o n s . This method may then be checked by determining the ammonia o r amine l i b e r a t e d d u r i ng s u b s t r a t e o x i d a t i o n by the Conway technique. 5. b) Measurement of substrate disappearance (Bogdanski et a K , 1956, Sjoerdsma ejt al_., 1955)* The tissue is homogenized with d i s t i l l e d water, and prior to incubation, serotonin is added with phosphate buffer to pH 7.4. This is followed by spectrophotofluorimetric determinations. c) Since these procedures are only satisfactory for studying tissues which contain rather large amounts of enzyme, a new method (Lovenberg .et a l . , 1962) has been worked out for use in organs with low MAO ac t iv i ty , or very small amounts of tissue such as sympathetic ganglia. This highly sensit ive method for measuring MAO in v i t ro , is based on the rate of  indoleacetic acid (IAA) formation from tryptamine. The immediate product of the reaction is indoleacetaldehyde, but i f excess aldehyde dehydrogenase and DPN are added, indole acetic acid can be obtained from the reaction. It is not often possible to quantify the disappearance of small amounts of tryptamine, but corresponding amounts of IAA can easily be detected since the fluorescent properties of the compound permit assay of micro-gram quanti t ies (Lovenberg et a K , 1962). For a l l these biochemical assays, similar controls may be used as in histoehemical methods, v i z . , incubation without substrate, applica-tion of heat, or incubation with an MAO inhibitor of known potency. 3. Distribution of MAO. The occurrence of MAO appears to be fa i r l y widespread in nature. It is known to occur in plants, although this enzyme may not be identical with vertebrate MAO since i t oxidizes not only monoamines but also d i -amines such as cadaverine and spermine. Plant amine oxidase resembles a diamine oxidase in being inhibited by semicarbazide and cyanide (Kenten and Mann, 1952). 6. In the animal world, Blaschko and Hope (1957) have shown that MAO is not confined to vertebrates, but has been found in many invertebrates including molluscs. In invertebrates extensive biochemical studies have been per-formed showing occurrence of the enzyme in tissues such as l i ver , kidney, stomach, intestines, adrenals, lung, pancreas, uterus, placenta, spleen and blood vessels, as well as in the central and peripheral nervous system. It is absent in erythrocytes and plasma, and very low levels are found in muscle (Davison, 1957; Langemann, 19^0 • Nevertheless, although wide variations between species are found, there are consistently high levels in nervous tissue in a l l species. In man, recent assays of MAO have been made by Levine and Sjoerdsma (1962) from post-mortem material obtained 8 hours after death, and from jejunal mucosa by capsule biopsy. They found that the MAO of intestinal mucosa greatly exceeded that of any other t issue, but this was in fresh tissue as against post-mortem t issue, which was l isted in the following order of act iv i ty - l i ve r , kidney, heart, lung, thyroid, adrenal, pancreas, sympathetic ganglion and cerebral cortex. These tests were considered val id in that Davison (1958) considered MAO quite stable even at room temperature for 2k hours. Experiments conducted on brain in this laboratory do not, however, substantiate this finding (see experiment, page 30) • It would appear that s tab i l i ty must vary from tissue to t issue, since pineal , pituitary and placenta tested here do not show the rapid deterioration of MAO that is found in brain post-mortem. This may account for the comparatively low levels found in brain by Levine and Sjoerdsma. Bogdanski et a k (1957) have raised the question of there being 7. more than one MAO, one of which may be more speci f ic for 5-HT, than, for instance, nor-adrenaline. This would be comparable to the relat ively speci f ic and non-specific enzymes involved in acetylcholine metabolism. The exact intracel lular location of MAO has yet to be deter-mined, and histochemical methods are not yet sensitive enough to deter-mine sub-microscopic distr ibution in a l l areas. Hawkins (1952) and Cotzias and Dole (1951) have studied the intracel lu lar local izat ion of MAO in rat l i ve r , and consider i t to be probably exclusively located in  mi tochondria. L i t t l e systematic work has been reported on other t issues, however, although i t is known that MAO is found in the particulate fraction of brain, intestine, lung and kidney. In general, however, i t is believed that MAO is found predominantly in mitochondria. k, MAO in Brain Development. Shimizu (1959a) and Nachmias (I960) have investigated the act iv i ty of MAO in developing rat brain by histochemical and biochemical methods. From their observations i t seems that local differences do not become apparent unti l 3 weeks after b i r th . At a foetal age of 15 days, almost negative act iv i ty was noted, gradually showing conspicuous act iv i ty in the locus coeruleus at the 20th foetal day, with generalized act iv i ty in other regions. Adult concentrations and local izat ion were present after the age of 3 weeks. 5. Substrate Location, Metabolism and Mechanisms of Act ion. Most of the substrates of MAO, the catecholamines, and 5-HT, have been found in granules, many of which, on ultracentrifugation, precipitate below the mitochondrial f ract ion. Blaschko (1958) fe l t that these granules might represent a new type of ce l l organelle, and reports the presence of high ATP wherever catecholamines are high, suggesting 8. that ATP may be involved in holding adrenaline. Hagen (1958) reports that these intracel lular granules containing adrenaline (A) and noradrenaline (NA) measure about 50 mu in diameter and are smaller than histamine granules. ATP is present in the ratio of 4-3:1. Reserpine causes a depletion; iproniazid appears to protect. Serotonin is also found in granules, as well as in platelets and mast ce l ls of rats and mice. Thus, i t is believed that in these s i t es , the substrates of MAO are "bound11 and can only be acted upon by MAO when "free". Precisely what stimulus or mechanism causes the release of these amines is not yet known. Tables I and II show the mechanism of action of MAO on various • substrates. Dixon and Webb (I960) note that monoamines resembling CH3 (CH2)r>NH2> which are only slowly attacked by diamine oxidase, are attacked by MAO at a rate which shows an optimum at a chain length which varies with the preparation used. Primary amines are the best substrates; secondary and tertiary amines are slower, while quaternary salts are negative. The action of MAO is prevented by a methyl group on carbon atom I of the substrate. Other enzymes also appear to be active on some catecholamines and other monoamines. Koelle (1958) reports the act iv i ty of cytochrome oxidase, dopa decarboxylase (tyrosine oxidase), conjugases, and catechol-O-methyl-transferase, as well as MAO. Although Axel rod (1959) has shown that O-methyl-transferase may play a signi f icant part in metabolism, i t is s t i l l held that MAO has a more important biological act ion. Barondes (1962) has noted the action of A, NA and 5-HT in stimulating glucose-1-C , i + oxidation by beef anterior pituitary s l i c e s , a property apparently shared by a number of structurally related amines, TABLE I C a t a b o l i s m of Catecholamines Showing I n f l u e n c e o f MAO Tyramine •DOPA MAO -»*3,4-Dihydroxyphenylpyruvic a c i d Dopamine MAO N-Methyldopamine 3,4-Dihydroxyphenylacetic a c i d m- or p-hydroxy-' 7 p h e n y l a c e t i c a c i d MAO O-Methyldopamine —.<—* »Homovanillic a c i d COMT No r a d r e n a l i n e —?Normetanephrine G l u c u r o n i d 3-Methoxy-4-hydroxyphenyl-e t h a n o l 4/ A d r e n a l i n e ,y 3,4-Dihydroxymandelic a c i d • 3-Methoxy-4-hydroxymandelic a c i d Metanephrine -v 3 - M e t h o x y - 4 - h y d r o x y g l y c o l i c aldehyde < r" S u l f a t e 1 0 . TABLE I I Tryptophan Metabolism i n R e l a t i o n s h i p t o S e r o t o n i n , M e l a t o n i n and MAO Tryptophan 5-Hydroxytryptophan 5-HTP 5-Hydroxytryptamine 5-HT MAO -> Tryptamine MAO I n d o l e a c e t a l d e h y d e 5-Hydroxyindoleacetaldehyde 5-HIA . Pormazon ppt. 5- H y d r o x y i n d o l e a c e t i c A c i d « 5-HIAA N-Acetyl-5-HT N-Acetyl-5-methoxy-t r y p t a m i n e M e l a t o n i n 5-Methoxyindole-a c e t i c a c i d Conjugates 10-Methoxy Harmalan (Mao I n h i b i t o r ) 11 but not by alpha-methylated derivatives. Since alpha-methylated amines are not substrates for MAO, the possib i l i ty that these active amines could stimulate glucose oxidation only after reaction with MAO is suggested. Barondes confirmed this by finding that two different MAO inhibitors could block the effect of the active amines. Further, the possib i l i ty that the aldehydes generated by MAO might be active was tested, since i t is known that the aldehyde metabolites of several of the active amines, and a number of a l iphat ic aldehydes, share the property of stimulating g lucose-1-C^ oxidation, and that the effect of the aldehydes is not blocked by MAO inhibi tors. From the fact that such aldehydes can stimul-ate glucose-l-c'** oxidation, Barondes suggests that the action of MAO may not only be to detoxify and degrade but also to convert neuroamines to an active form. In view of the consistently high levels of MAO found in the pineal in a l l species in this study, and the discovery of the hormone "melatonin" in the pineal , i t is interesting to note that this substance derives from serotonin. The presence of melatonin has been reported by Lerner e t . a l . (1959) in the pineal gland, and Gjarman and Day (1959) have noted the presence of physiologically active amines such as histam-ine, serotonin and catechol amines, as well as enzymes involved in their formation and metabolism. Axel rod and Weissbach (1961) have observed, in addit ion, the presence of hydroxyindole-O-methyl-transferase (found only in pineal) , imidazole-N-methyl-transferase and catechol-O-methyl-transferase. Although N-acetyl-serotonin is found to be the best substrate for hydroxyindole-O-methyl-transferase, other hydroxyindoles are methyl-ated but to a much smaller extent (100;12). The observation that 5-hydroxyindoleacetic a c i d served as s u b s t r a t e f o r the enzyme i s of i n t e r e s t , s i n c e Lerner et a K found 5-methoxyindoleacetic a c i d i n bovine p i n e a l glands (Brown, 1959)* Thus, i t i s becoming apparent that the p i n e a l gland, an organ of which the p h y s i o l o g i c a l f u n c t i o n has so f a r been obscure, possesses c o n s i d e r a b l e biochemical a c t i v i t y . 6. Uptake of Substrates i n B r a i n . Wilson et a K (1962) suggest that a process of a c t i v e t r a n s p o r t may be important i n l o c a l i z i n g the r e l a t i v e l y high concentrations of catecholamines that have been observed (Vogt, 1954) i n c e r t a i n parts of the b r a i n and c e n t r a l nervous system. Although c i r c u l a t i n g c a t e c h o l -amines are excluded from most of the b r a i n , they do enter the p i t u i t a r y (Axelrod e t a K , 1959) as w e l l as c e r t a i n parts of the hypothalamus i n very low c o n c e n t r a t i o n s . The p i t u i t a r y i s one of several s p e c i a l regions w i t h i n the CNS which appear to have no blood-brain b a r r i e r ( W i s l o c k i and Leduc, 1952; Brodie et a_K, I960). I t was f u r t h e r shown by Wilson that i n t r a v e n o u s l y administered e p i n e p h r i n e - ^ was taken up i n the pineal body, area postrema and intercolumnar t u b e r c l e of the c a t , i n concentra-t i o n s c o n s i d e r a b l y i n excess of those i n p a r t s of the b r a i n l y i n g w i t h i n the blood-brain b a r r i e r . A d m i n i s t r a t i o n of reserpine to cats g r e a t l y reduced the c o n c e n t r a t i o n of epinephrine-H^ appearing i n p i t u i t a r y . von E u l e r and L i s h a j k o (1961), studying uptake of catecholamines i n adrenergic nerve granules, have a l s o shown that i s o l a t e d nerve gran-ules can take up the catecholamines, s i m i l a r l y to dopamine uptake i n the adrenal medulla ( B e r t l e r e t aj_., 1961). The NA i s r e t a i n e d i n storage granules when the c o n c e n t r a t i o n of axoplasm i s about lO/ugt/ml, then re-leased a t lower e x t r a - g r a n u l a r c o n c e n t r a t i o n f o l l o w i n g NA f l u x through 13. the axon membrane during nerve stimulation. These facts are of interest in that MAO concentration has been found, in this study, to be high in the choroid plexus and in those other parts outside the blood-brain barrier where epinephrine-H^ was taken up, and i t could be possible, therefore, that MAO acts in some way to regulate in these regions, the levels of active amines. 7. Inhibition of MAO. Work on inhibit ion of MAO was largely pioneered by Zel ler (1951. 1952, 1955) and has since become a major method of experimentation. Mann and Quastel, however, had proposed, in \SkO, that the stimulant action of amphetamine might be related to the inhibit ion of MAO. They believed that amine metabolism proceeded via the formation of intermediate alde-hydes which were depressants of brain ac t iv i ty . It was thought that inhibit ion of MAO decreased the formation of such aldehydes and thereby prevented the onset of mental depression. Later workers (Grana, 1959; Schayer, 1953) showed, however, that stimulant doses of amphetamine and related drugs were not suff ic ient to produce signif icant MAO inhibit ion in the intact animal. When Zel ler showed the MAO inhibitory properties of the anti-tubercular, anti-depressant drug, iproniazid, the possible relationship between enzyme inhibit ion and pharmacologic act iv i ty was seen. A number of other hydrazine compounds have also been shown to be effective MAO inhibitors (Biel et a [ . , 1959; Chessin e t . a k , 1959; Randall et a l . , 1959) and have had therapeutic appl icat ion. Semicarbazide and potassium cyanide, although carbonyl trapping agents, do not inhibit MAO. It is now generally believed (Horita, 1961; Brodie et aj_., 1959; Spector et a]_., 1958) that the inhibit ion of MAO results in a protection and subsequent accumulation of brain serotonin and catecholamines. Thus, the primary cause of the anti-depressant response is due to increase in the levels of these amines. Precisely how the accumulation of amines produces this ef fect , is not yet known. The concentration of stored amines appears to be maintained by a f ine balance between their biosynthesis and d&oxication process. The latter event can occur only after the agents are released from their "bound" to their "free" forms. The administration of a MAO inhibitor results in an upset of the balance between synthesis and degradation, forcing an increase in brain levels of the amine, since synthesis is not affected. Among physiological compounds investigated, only weak MAO inhibitors have been found. The most active is cortisone (Schweppe et a l . 1959) and thyroxine, which depressed enzyme act iv i ty by only 30-50%. The degree of inhibit ion does not influence the amine leve l , according to recent work by Gey and Pletcher (1961). Thus, MAO act iv i ty does not influence MAO content of the brain under physiological conditions. The great excess of MAO suggests a function of rapid inactivation of free monoamines after release from storage depots. In regard to MAO inhibit ion by hydrazines, i t has been shown that these are also effective 0A0 inhibitors as well (Burkard et a l . , I960; Cohn and Shore, i960). Most are also potentiators of various pharmaco-logical agents (Arrigoni-Martel1 i , 1959; Eltherington and Horita, I960; Fouts and Brodie, 1956) and some have sympathomimetic properties, whilst others are weak adrenergic blocking agents (Griesemer et a l . . , 1955). Increase of lact ic acid has also been noted by Gey and Pletcher (I960). In addit ion, many of the anti-MAO hydrazines are effective inhibitors of 5-HTP and dopa decarboxylase (Tabachnick, 1959), thus preventing normal 15. synthesis of amines after MAO blockade. It has also been found that depressants such as barbiturates and anti-convulsants, raise the brain levels of serotonin (Bonnycastle e,t aj_., 1957). New compounds (tranylcypromine, alpha-ethyltryptamine) unrelated to hydrazines, have, however, been found which produce a long last ing, probably irreversible blockade of MAO. These, too, have antidepressant ef fects , thus lending support to the hypothesis that MAO inhibit ion can be responsible for the mechanism of "psychic-energising". Horita (1961) warns that in administering any MAO inhibi tor , "variation in the route of administration may give different patterns of MAO inhibi t ion, in different animals and in different species." 8. Psychiatric Implications. Many theories have been advanced regarding the psychotropic effects of over- or under-supply of MAO substrates in Brain, and i t has thus been postulated that absence or dysfunction of the enzyme could lead to other pathways for the metabolism of physiologically active amines. Adrenaline or serotonin, for example, could be oxidized to hallucinogens. Wool ley and Shaw, in 1954, f i r s t postulated that schizophrenia could be the result of an error in the metabolism of serotonin. Likewise, Hoffer, Osmond and Smythies (1954) postulated the presence of an abnormal adrenal-ine metabolite - adrenochrome - in schizophrenia. Later research has, however, been unable to prove the val id i ty of these theories. It should also be remembered that MAO inhibitors can have more generalized systemic effects such as connective tissue f ibroplas ia , peripheral ganglionic inhibit ion and hypotensives. L i t t l e has been done to correlate abnormal amine metabolism with the pineal , but i t is interesting to note that Giarman et a K (I960) 15a. found the highest pineal gland serotonin in psychotic patients. Further-more, Altschule and Giancola (I960) reported that extracts of pineal cause biochemical changes in schizophrenics. There i s , however, insuff icient knowledge in this area to warrant any conclusions as yet. In spite of the limitations of present knowledge, the fact remains that MAO has extreme biological importance, and that inhibit ion of this enzyme can produce anti-depressant effects with concomitant increase in brain amine levels. Thus, from the many studies on the metabolism and pharmacology of these amines, some investigators (Brodie and Shaw, 1957; Marrazzi and Hart, 1957) have suggested that they might function as neurotransmittors. MAO could thus serve to detoxify the transmitter, in an analogous fashion to AChE. It cannot now be, however, categorically stated that serotonin and the catecholamines are acting as transmitter substances, although there is strong possib i l i ty of such a function. 16. PART II. HISTOCHEMICAL INVESTIGATION OF MAO 1. Historical Development and Rationale of Histoehemical Methods. Since Oster and Schlossman's attempt in 1942 to demonstrate MAO in tissues by means of tyramine incubation, many investigators have explored the poss ib i l i t i es of more speci f ic methods. The method of Oster and Schlossman depended on the ab i l i ty of fresh frozen sections of guinea pig kidney to produce aldehyde when incubated with tyramine and phosphate buffer. Formation of the aldehyde was confirmed by S c h i f f ' s reagent. Gomori (1950) and Pearse (1961) severely c r i t i c i zed the spec i f ic i ty of local izat ion given by this method, since the reaction product was soluble in water and diffused freely in the incubating medium. Pearse therefore suggested that a simultaneous coupling method be used to trap the aldehyde formed at the moment of i ts release, to form an insol -uble precipitate in s i t u , but attempts to do this were unsuccessful. Koelle and Valk (1954) then developed a method using naphthoic acid hydrazide as an aldehyde .capture reagent and 0.01M hydrazine as a blocking agent. In place of tyramine, tryptamine was used as substrate, and 20 per cent sodium sulphate was added to the incubating medium. This method, however, although satisfactory in the majority of mammalian t issues, is not suitable for use on brain because of i ts high content of oxidizable l ip id causing a pseudoplasmal reaction with the hydrazide, and resulting in deeply stained control sections. A method based on pigment formation was attempted by Blaschko and Hellman (1953) by incubating sections with tryptamine hydrochloride at pH 7.4. Arioka and Tanimukai (1957) used the same method, with serotonin 17. as s u b s t r a t e , but found resu l t s i n c o n s i s t e n t . Takamatsu (1958) a l s o developed another method us ing tyramine and potassium t e l l u r i t e , but because of the low redox potent ia l o f the l a t t e r , the method has l im i ted va lue . Pearse (1961) believes that methods r e l y i n g on pigment formation cannot be expected to give accurate l o c a l i z a t i o n of MAO, s ince pigment may be produced from aromatic amines by other enzyme systems in the absence of MAO. The f i r s t method to be based on reduct ion of tet razo l ium s a l t s was reported by F ranc i s (1953) who incubated t i ssues with tyramine and neo-te t razo l ium c h l o r i d e , a method unsuccess fu l l y attempted by other workers. In 1957, Glenner, Burtner and Brown attempted a m o d i f i c a t i o n of the F ranc is technique, and by s u b s t i t u t i n g tryptamine f o r tyramine, and by us ing a more e a s i l y reduc ib le tet razo l ium s a l t , obtained e x c e l l e n t r e s u l t s . Formazan pigment was produced under histochemical cond i t ions from iodo-n i t r o t e t r a z o l i u m (INT) and from n i t r o - b l u e tet razo l ium (NBT), but not from neotetrazol ium o r blue te t razo l ium because of t h e i r lower redox p o t e n t i a l s g i v ing d i f f i c u l t y of reduct ion . The fo l l ow ing tab le shows the ha l f wave p o t e n t i a l s of some of the bet ter known t e t r a z o ltpn s a l t s and t e l l u r i t e (from Pearse, 1961): Oxidant E-^. 2 2 ° . pH 7 . 2 . vo l t s T e l l u r i t e - 0.95 TTC - 0.49 NT - 0.17 BT - 0.16 MTT - 0.11 INT - 0 .09 NBT - 0 .05 Results of the Glenner method f o r MAO l o c a l i z a t i o n corresponded in a l l respects to the K o e l l e - V a l k technique, one method thus lending support to the o t h e r . This method has been used in the present i n v e s t ! g a -18. t ion, and wi l l be fu l ly detailed in the next section. 2. Comparisons of Other Work on Brain MAO. With regard to investigation of MAO in brain by similar methods, Shimlzu, Morikawa and Okada (1959) have used I NT to examine the brain of rodents, Glenner (1957) has made a limited survey using NBT, and Smith (1962) has recently completed a survey of rat brain using NBT. The details of these investigations are tabulated and compared in the following table. Bogdanski's (1955) results, using biochemical methods of evaluation, are included for comparison, but unfortunately do not outl ine the same areas. Regional Comparisons of MAO in Brain by Tetrazolium Methods (Glenner, 1957) Rat, mouse. Rat rabbi t, Rabbit, cat guinea p ig , guinea pig Rat This rabbi t Shimizu Smi th Investiga- Glenner Area et.al i( l959) (1962) tion (1957) Locus coeruleus 4-1 II ++ Habenula +++ M M +++ +++ Midiine nuclear gp. +++ +++ of thalamus Periventricular grey +++ +++ +++ matter Med. nucl . of hypo- T"l"" I- +++ •I 111 thalamus Fasciculus retroflexus +++ t n t * T T T T +++ +++ Interpeduncular nucl . +++ l l l l 11 11 +++ Nucl. of brachium ++ conjunct! vum Dorsal nucl . of vagus n +++ Nucleus ambiguous +++ +++ Inf. ol ivary nucleus +++ +++ Area postrema +++ I1 Oculo-motor nuclei - ++• Neocortex + ++ Hippocampus - neocortex + +++ - dentate gyrus + - Ammon's pyramids - -Amygdala + + 19. Regional Comparisons of MAO in Brain, e t c . , cont 'd. Rat, mouse, Rat rabbit, Rabbit, cat guinea p ig, guinea pig Rat This rabbit Shimizu Smith Investiga- Glenner Area e t a j _ . ( 1 9 5 9 ) ( 1 9 6 2 ) tion ( 1 9 5 7 ) Basal ganglia - caudate ++ ++ - putamen ++ + - globus pal 1idus - -Dorsal tegmental nucl . i + +++ +++ Mesenceph. nucl . of N. V + +++ +++ Other thalamic nuclei + + Mammi1lary body ± + Substantia nigra ++ Red nucleus + + Pineal ± +++-Choroid plexus + Pituitary - anterior ++ (pig) (ox) -posterior +++ +++ Distribution of MAO in Dog Brain by Biochemical Assay (Bogdanski, 1955) (?n^g/5-HT destroyed per g/hr.) Amygdala 968 Hypothalamus 1624 Septal region 1212 Midbrain 8 4 2 Piriform cortex 926 Caudate nucleus 935 Medulla 1117 Thalamus 9 4 0 Hippocampus 1176 Pons 936 Olfactory bulb 573 Cortical grey matter 819 Cerebellum 930 Corpus callosum 4 6 6 + internal capsule Lateral geniculate 8 4 4 Optic tract 701 Fornix 707 20. 3. Chemistry of Tetrazolium Sa l ts . In 1894, von Pechmann and Rune prepared the f i r s t tetrazolium s a l t , triphenyl tetrazolium chloride (TTC), but i t was not unti l 1941 that Kuhn and Jerchel discovered their use In enzyme chemistry and found that a number of colourless tetrazolium salts were reduced to coloured compounds by plant t issues. Synthesis of new compounds was then made and i t is now clear that the ditetrazolturn salts give better histochemical results. The formazan from INT is macrocrystalline, but that of NBT is micro-crysta l l ine . The reaction taking place is as follows: > U f-Hd Ditetrazolium sal t Diformazan (colourless) (blue purple) The above reaction indicates complete reduction, but partial reduction can occur at one end of the molecule only, giving a monoforma-zan which is reddish. Nitro-blue tetrazolium is 2 ,2 ' -d i -p-ni t ro-phenyl -5 ,5 ' -d iphenyl -3 ,3 , - (3»3 ' -d imethoxy-4 ,4 , bipheny1ene) di tetrazolium chloride. With the use of suitable substrate, these compounds can also be used In dehydrogenase chemistry, and are the current choice for local izat ion of 21. succinic dehydrogenase, TPN and DPN^Friede, 1961). k. Chemistry of the MAO-Tetrazolium Reaction. The mechanism by which tetrazolium salts are reduced in the MAO reaction has been investigated by Weissbach et a l . (1957) who demonstrated that in v i t ro . INT was reduced only when an indole amine was oxidized by the MAO of rat l iver mitochondria. Other substrates for MAO did not give rise to products capable of reducing tetrazolium s a l t s . Further elucidation of the mechanism of tetrazole reduction was made possible in 1959 by the synthesis of indole-3-acetaldehyde, the pro-cess being described by Glenner e j taK (1959) as follows: The implication of an aldehyde-intermediate in the formation of this pigment was f i r s t postulated by Blaschko (1953) on the basis of carbonyl reagent inhibit ion of pigment formation. Thus, the product of MAO act iv i ty on tryptamine (IAc) appears to be an intermediate in this particular pigment formation. In the histo-ehemical system, lAc direct ly reduces the tetrazole and forms, not IAA, but an unknown compound which may then go on to pigment formation. This is a somewhat unique mechanism, in that i t stems from a non-enzymatic reduction of an indicator, by the product of an enzyme catalysed reaction. It should be added that no evidence of a diaphorase l ike system in this IAA (oxidation) (aldehyde dehydrogenase) pi gment 22. reduction process has been found (Glenner, 1959)* 5. Evaluation of Reaction Mechanisms and Spec i f i c i t y . It is recognized that the histochemical method has many l imita-t ions, not the least of which is the lack of any completely rel iable method of quantitative determination. Friede (I960), working with succinic dehydrogenase (SDA), attempted a laborious and careful densito-metric evaluation showing reasonable correspondence with microanalytical methods. Others (Farkas et a k , 1962) have recently attempted quantita-tive measurement by chromatography. The reacted tissue section is adhered to chromatographic paper, and chromatography carried out. During this process the formazans provided by SDA shi f t off the section and separate from each other. This method would, however, be of limited value in sections containing several centres of marked ac t iv i ty . By biochemical analytical methods of MAO measurement, dif fusion of enzyme occurs after di f ferent ial centrifugation. Diffusion artefacts are also a hazard of the histochemical method, but these may be minimized by careful handling, and consistent clear-cut results obtained. The general concensus of opinion is that "regional" local izat ion only is poss-ible by the histochemical method for MAO, but in this investigation i t has been possible to obtain repeated patterns of intracel lular detail in identical areas, i . e . , choroid plexus, pituitary and neuropil. Although undoubted weaknesses exist in the method, the actual spec i f ic i ty for the reaction is well established (Weissbach, 1957), and in regions of such complex anatomy as the brain-stem, the advantages of a histochemical approach are s t i l l considerable. 23. PART III. EXPERIMENTAL PROCEDURE AND TECHNICAL DATA 1. Animal Material. A wide in i t i a l survey of brain and spinal cord was made on 34 animals of different ages and sexes, including mouse, rat, rabbit, cat , dog, pig and ox. Tests were performed using different inhibi tors, substrates, methods of k i l l i n g , freezing, f ix ing and durations of enzyme act iv i ty after death. The results of these experiments are summarized at the end of this section. F ina l ly , 6 healthy adult rabbits (mixed sexes) and one adult cat, were selected for brain-stem mapping as outlined in this study. 2. - Methods of Treatment. Animals in the in i t i a l survey were k i l led either by decapitation or by a blow on the head. It was noted, however, that no v is ib le d i f fe r -ences in enzyme local izat ion and concentration could be detected after this method of k i l l i n g , or after administration of ether or pentobarbital. Hence, the f inal animals used for brain mapping were given pentobarbital 30 mg/kilo body weight, followed by perfusion with normal sal ine, of the s t i l l beating heart. This method also c la r i f i ed the picture, in that no confusion could be caused by the presence of blood-borne MAO. MAO has been found in the blood of some species, although generally absent from erythrocytes and plasma. Ini t ial experiments "quick-freezing" tissue with iso-pentane (-140°C) in l iquid nitrogen (-180°C) appeared to cause more cel l disruption and diffusion of enzyme in brain tissue than slower freezing methods. 2k. Hence, the f inal tissues were placed on ice as soon as removed, then mounted on blocks and frozen at -15°C before cutting as soon as possible thereafter, at - 5 °C , cryostat temperature. Friede (1958) found that artefacts, loss of enzyme act iv i ty (SDA) and diffusion of s ta in , are minimized by cutting at a temperature of -3° to -2°C for 60/ii sections. In this case, -5°C was found to be best for these sections cut at 3 0 / i . Cutting was performed in a cryostat, and the sections were mounted on s l ides , then br ief ly dried at room temperature before applying solution. 3 . Solutions (according to Glenner, 1957)• Tryptamine hydrochloride 25 mg (0.005M) Sodium sulfate k g Nitro-blue tetrazolium 5 mg 0.1M phosphate buffer, pH 7.6 5 ml Disti1 led water 15 ml N.N. dimethylformadide 0 .05 ml (solvent for tetrazolium salts) ( o w n i»ocUf«ca.tl.on) Following incubation for 2 hours at 25°C, the sections were washed for 2 minutes before being fixed overnight in formalin vapour and mounted in glycer in-gelat in. k. Controls. Controls used consisted of: a) MAO inhibitors in vivo and in vi tro (See Table Kl and IV, page 25" and 28. b) Destruction by heat (60°C. for 20 minutes). c) Incubation without tryptamine. Controls were run on each area of brain-stem investigated. During The pineal alone, s t i l l gave moderate reaction without substrate. 25. the cutting of the serial sections throughout the brain-stem, adjacent sections were taken for staining with toluidine blue, so that each enzyme-incubated section had i ts equivalent set of stained sections. By a technique of superimposition of the cel l -sta ined s l ide , over the enzyme incubation, i t was thus possible to locate nuclei with precision, using low power opt ics . Sections were cut in a transverse plane. 5. Experimental Results. TABLE III. Results of In Vitro Tests of Various Drugs on MAO Inhibition in Rabbit Brain MAO Inhibition graded v is ib le reaction (1) Hydrazines - MAO inhibitors Iproniazid (Mars!lid) ++ Catron (beta-phenyl-isopropy1- +++ hydrazine) (2) Non-Hydrazines - MAO inhibitors Tranylcypromine (Parnate) ++ Amphetamine (3) Non-MAO Inhibitor Anti-Depressives Imipramine (Tofranil) AmitryptalIne (Elavil ) ++ (4) Muscle Relaxants Flaxedii (gallamine tri-ethiodide) (cat) ++ Tubocurarine + Decamethonium bromide (syncurine) ++ Succinylchol ine (Anectine) +4-Intocostrin (5) Mi seellaneous Aminoguanidine & Pyrogallol Reserpine ++ Dilantin Phenobarbitone 26. Each drug was incubated on fresh frozen tissue sections in O.IM concentrations in standard tryptarnine-tetrazolium solu-tion (Glenner), buffered at pH 7.6, for 2 hours at 25°C. These tests were performed on one set of rabbit brain tissues only, using sections from the same brain as controls. The drug was dissolved in d i s t i l l e d water and added in equal quantities concomitantly with the standard incubating solution. Discussion of Previous Experiment (Table III). Group 1. A l l hydrazines, having either the enclosed or terminal -NH-NH- group, i . e . , Catron -/\r c - c - Ntf- NH^ A l l have been shown to have MAO inhibit ing properties, and re-sults of this experiment, substantiate th is . Group 2. Certain non-hydrazine compounds have also been found to have MAO Inhibiting properties, i . e . , tranylcypromine and amphetamine. Only tranylcypromine was effective here. Group 3. Both groups I and 2 have anti-depressive propert-ies plus MAO-inhibitory ef fects . Group 3 has anti-depressive effects but do not inhibit MAO. Only amitryptaline here showed inhibit ion in vi tro. Group k. This consists of a group of muscle relaxants most of which are quaternary ammonium compounds having muscle end plate ef fects . A l l exhibited MAO inhibit ion except Intocostrin. Group 5. Aminoguanidine is a known DAO inhibitor but not an MAO inhibi tor . Pyrogallol inhibits o-methyl-transferase, and reserpine liberates catecholamines and acts as a t ranqui l l izer . 27. No explanation of these results is possible. Results of this experiment are of interest in the l ight of similar pharmacological work, but have no actual val id i ty in that one set of tissues only was tested and "pre-incubation" as well as "concomitant" incubation with drugs was not done. Glenner (1957) noted that dissimilar results were sometimes obtained by the two methods and believed that inhibit ion by reagents incorporated in the incubation mixture (concomitant) could be caused by: a) an inhibit ion of the enzyme systems per se, b) a reaction of the inhibitor with a substrate ut i l i zed by the enzyme system(s), c) an electron-trap preventing tetrazolium reduction. Semi-carbazide and cyanide, which do not inhibit MAO in the biochemical assays, caused, for instance, a marked concomitant inhibit ion but no inhibit ion by pre-incubation methods. One of the weaknesses of the method of using MAO inhibitors in  vivo or in vi tro, is the d i f f i cu l ty of assessing dosage since some MAO inhibitors are more effective than others and require lower dosage. On the other hand, some species are more susceptible to these drugs than others, and response may vary in duration and magnitude. The following in vivo tests of MAO inhibit ion have perhaps greater va l id i ty than those performed in v i t ro , but the same d i f f i c u l t i e s apply with regard to dosage and species. In this experiment (Table IV) similar dosage was used as in the histoehemical demonstration in rat of MAO inhibit ion by Catron (Mustakallio et al_., 1961), and corroboration of their results was obtained. A dose of 1 x 10-i*M/kg body weight was given, 28. and the rats sacr i f iced 6-18 hours after . No. 1 and 2 are hydrazine MAO inhibitors; 3, 4 and 5 are non-hydrazines, non-MAO inhibitors; and 6 is a non-hydrazine MAO inhibi tor . TABLE IV. In Vivo Tests of MAO Inhibition Species Inhibition 1. Iproniazid rat ++ 2. Catron rat +++ 3. Flaxedi1 cat ++ ( i k. Ami tryptaline cat ++ 5. Reserpine rabbi t -6. Tranylcypromine rabbi t ++ grey matter but reaction in f ibre tractsl) The inhibit ion noted in these in vivo tests with the known MAO inhibitors iproniazid, Catron, and tranylcypromine, is considered to be the mostconclusive and val id control of those used, since true biological inhibit ion correlated with patterns of behaviour observed in the experi-mental animal, especially in rats. In rabbit (No. 6) there was less behavioural agitation but i t was noted that three times the amount of pentobarbital was required to produce anaesthesia. TABLE V. Substrate Test Results of MAO act iv i ty with various substrates (all substrates 0.025 mg) 5-Hydroxytryptamine + positive DL-Normetanephrine ± very s l ight reaction 4-Methoxypheny1 ethyl amine - negative, no reaction 3,4-Dimethoxyphenylethylamine - negative, no reaction TABLE VI. Uptake Test Pre-incubation (25 minutes) with the following was carried out to determine any evidence of uptake Without Tryptamine Wi th Tryptamine DOPA - + 5-Hydroxytryptophan - + Histidine - + TABLE VII. Extinction Test Experiment to test duration of act iv i ty of MAO in hours after death Method: Six adult white rats were sacri f iced by a blow on the head, the bodies then allowed to remain with brain in  s i tu until time of treatment. One hemisphere was then re-moved and quick frozen in iso-pentane in l iquid nitrogen, cut and incubated for MAO. Tine TOST rta*Ten TABLE VIII. Fixation Tests Fixatives used after incubation and washing included: (i) 80% a lcohol, 5 minutes ( i i ) 10% neutral formalin, Ik hours ( i i i ) Formalin vapour (90%) overnight Results: (i) Tissue shrinkage was observed, although demon-stration of f ine detail often improved. ( i i ) Tissue swelling, and some sections lost . ( i i i ) Least tissue changes. No effect on the incubation colour reaction was observ-ed in any of these f ixat ives. 32. 6. Photography. Friede (1961) has noted the d i f f i cu l ty of photographing large, thick sections because of the differences in refractive index of compon-ents of the t issue, and recommended the use of a thin opal glass immediately below the screen. Black and white photography in our case was performed using a ground glass screen with the following equipment: Film: l l ford 35 mm. F.P .3 Camera: Leica with focus s l ide copy attachment and extension bellows Lens: F/3.5 50 mm. Elmar Developer: Kodak, Microdot X 33. PART IV. ATLAS OF MAO IN BRAIN-STEM 1. Arrangement of A t las . The atlas consists of reproductions of transverse sections taken through the brain-stem of rabbit (Plates I, II and II in back pocket) from the region of the f i r s t cervical nerve posteriorly, up to the optic chiasma anter ior ly. These sections were cut at a thickness of 30/u at intervals of approximately 350 fx throughout the brain-stem. Sections were then incubated for MAO, then representative series reproduced as in the following a t las . Adjacent sections were stained with toluidine blue in order to distinguish cel l groups. In addit ion, hand-drawn maps were constructed as keys to the enzyme and cel l -stained sections. Thus, the series consists of: a) Map of each section, as indicated in Plates I, II and 111 of brain-stem (in pocket at back). b) Accompanying legend. c) MAO section photographed in black and white, of representative areas. d) Toluidine blue stained section photographed in black and white, of similar areas. 2. Key to Atlas Abbreviations. o£ ce l l groups of Meesen and Olzewski am ambiguous nucleus ant anterior nucleus of thalamus aq aqueduct ar area postrema arc arcuate or ventrolateral nucleus of thalamus as acoustic s t r ia at acoustic tubercle or dorsal cochlear nucleus 34. be brachium conjunct!vum bon basal optic nucleus (supra-optic) Cj f i r s t cervical nerve ca cornu ammonis cb cerebellum cen central nucleus of dorsal horn cf central tegmental fasciculus eg central grey matter etc commissure of infer ior col Iiculus ci1 ci1iary nucleus cp cerebral peduncle cpl choroid plexus cs cerebrospinal (pyramidal) tracts cvs crossed vestibulospinal tract d ce l l group d of Meesen and Olzewski D dorsal tegmental nucleus (of Gudden) don dorsal ol ivary nucleus dor dorsal nucleus of thalamus dt descending root of trigeminal nerve dur descending vestibular root EW Edinger-Westphal nucleus fc fasciculus cuneatus fg fasciculus grac i l is fn facial nerve fx fornix gf genu of facial nerve hb habenular bodies hp habenulo-peduncular tract hy hypothalamus hyn hypoglossal nucleus ic infer ior col 1iculus int internal capsule ip interpeduncular nucleus i t intercalate nucleus ion infer ior ol ivary nucleus lat lateral nucleus of thalamus 1g lateral geniculate body lc locus coeruleus In lateral nucleus of oblongata mb mammillary body mc Meynert's supraoptic commissure med medial nucleus of thalamus mes mesencephalic nucleus of the trigeminus mg medial geniculate body 35. mi massa intermedia mi medial lemniscus mlf medial longitudinal fasciculus mt mammillo-thalamic tract (Vicq d'Azyr) mrs media] ret icular formation mv dorsal motor nucleus of the vagus n nodulus nbc nucleus of the brachium conjunctivum nc nucleus cuneatus ng nucleus grac i l is n i l nucleus of lateral lemniscus not nucleus of the optic tract (pre-tectal) npc nucleus of the posterior commissure oc opt ic chiasma ocn oculo-motor nerve oh olfacto-habenular tract (ansa peduncularis) om oculomotor nucleus ost olfactory striatum or peduncular nucleus ot optic tract pc posterior commissure peri peri peduncular nucleus (of Jacobsohn) pin pineal body pp pontal protuberance prn pontal raphe nuclei pul pulvinar Q, eel] group Q of Meesen and Olzewski rb restiform body rn red nucleus rs rubro-spinal tract rsx rubro-spinal decussation rt ret icular formation sc superior c o l l i cuius sf sol i tary fasciculus (or descending sensory root of the vagus and glossopharyngeal nerves) sg substantia gelatinosa trigemini sgs superficial grey stratum of the tectum sn substantial nigra sp spinal part of the spinal-accessory nerve sr sensory radiations st spinotectal tract sth spino- and bulbo-thalamic tract including lower trigem-ino-thai amic fibres teg lateral tegmental nucleus of midbrain tn trigeminal nucleus tro trochlear nucleus 36. ts tecto-bulbar and tecto-spinal tract uf uncinate fasciculus va sensory vagal nuclei or ala cinerea vac vagal commissure vc ventral commissure vcn ventral cochlear nucleus ven ventral , or ventromedial nucleus of thalamus vmn ventral medial nucleus von ventral ol ivary nucleus vsc ventral spino-cerebellar tract X decussation of cerebro-spinal tract 3. Nomenclature. Anatomical names of tracts, nuclei and oither structures are according to Papez' "Comparative Neurology", 1929 (Hafner, 1961). Since these did not cover a l l the details required for description of rabbit brain, reference has also been made to Meesen and Olzewski's "Atlas of the Rhombencephalon of the Rabbit" (19^9), Monnier and Gangloff's "Stereotaxic Rabbit Brain Atlas" (1961), Friede's "Histoehemical Atlas of the Cat Brain Stem", and Gi l l Nan's "Atlas of the Mid-Brain of the Rabbit" (19^3). Maps. These are shaded to denote regions of MAO act iv i ty , i . e . , fMl indicates strong act iv i ty K///I indicates moderate act iv i ty Regions of weak act iv i ty are unshaded. Magnification is 10 x. 37. 4. Atlas S e r i a l s . Atlas of MAO Concentrations in Rabbit Brain-Stem • Page Sec. 03-2 Oblongata Below the obex and vagal nuclei 38 I I 03-8 " Level of decussation of the pyramids 39 Plate IV 40 I I 03-9 I I Level of the obex 41 I I 03-15 I I Through vagal nuclei and area postrema 42 Plate V 43 I I 03-16 I I Upper levels of vagal nuclei 44 Plate VI 45 I I 03-20 n Upper levels of vagal and hypoglossal 46 nuclei Plate VI1 47 I I 03-24 I I Level of descending vestibular nucleus 48 I I 03-26 I I Region of facial nerve 49 I I 03-36 n Level of isthmus 50 Plate VIM 51 I I 04-24M Midbrain Level of infer ior c o l l i c u l i 52 I I 04-22M I I Level of c o l l i c u l i 53 I I 04-20M I I Level of superior c o l l i c u l i 54 Plate IX 55 I I 04-17M I I Through mammillary body 56 04-13M Diencephalon Through mammillary body 57 Plate X 58 I I 04-11M n Epithalamic region 59 I I 04-10M I I Hypothalamic region 60 Plate XI 61 it 04-6M it Habenular region 62 ii 04-4M I I Region of optic chiasma 63 38. Sec. 03-2 : Oblongata Below the Obex and Vagal Nuclei In the lower oblongata, the main concentrations of enzyme are seen surrounding the central canal in the region of the dorsal and ventral commissures, and in the greatly enlarged substantia gelatinosa which here becomes the terminal descending nucleus of the trigeminal. The overlying tract of Lissauer can be identif ied lateral ly by oval areas of unreactive ti ssue. This region is unique In the histoehemical brain-stem picture, In that a regular pattern of well-marked act iv i ty can be determined, In which parallel bands of neuropil l i e on the periphery of the trigeminal nucleus, and embrace certain ce l ls with a basket of fine ramifications. Under o i l Immersion, these dendrites and cel l expansions can be seen to possess a double nature, studded with fine granules along their course. Only In the ol ivary nuclei can a rather similar situation be found. 39. Sec* 03-8 : Oblongata Level of Decussation Here there is l i t t l e basic change in regions of MAO act iv i ty from the previous section. The dorsal commissure broadens with strong MAO, and equal concentration continues in the trigeminal nucleus. PfcflT* JZ Sec; 03-9 : Oblongata Level of Obex The vagal commissure contains strong MAO, spreading lateral ly In the ala clnerea of the vagal nucle i . The inferior o l ive has now entered the picture, and shows also strong MAO and marked reaction of the neuropil . The nucleus of the reticular formation shows moderate MAO, so that the MAO reactive areas show up roughly as a figure eight - lateral ly the trigeminal nucle i , medially the vagal commissure, dorsally the nuclei of the fasciculus cuneatus, and ventrally the reticular formation. 42. Sec. 03-15 : Oblongata Through Vagal Nuclei and Area Postrema In "this section, the area postrema appears lateral to the vagal commissure and dorsal to the sensory vagal nucleus. It has approximately the same intensity of reaction as the vagal nuclei and infer ior o l i ve . The trigeminal nucleus, however, is becoming gradually less reactive, so that, in the next section, the nucleus has only mild MAO comparable to the Intervening grey matter of the oblongata. An area of fa i r ly strong MAO appears In the motor nucleus of the vagus, continuing down into the neuropil of the sol i tary fasciculus and ambiguous nucleus. Ventral ly, the lateral nucleus carries moderate MAO. 43. The most prominent reactive area continues to l i e within the neuro-pi l of the vagal nucle i . The hypoglossal nucleus shows only moderate MAO, as do the sol i tary fasciculus, trigeminal nucleus and ret icular formation. Moderate MAO is present In the inferior mri mpnrinr o l i ve . As in the last section, both the central regions of crossed vestibulo-spinal tracts, and the medial lemniscus show minimal ac t iv i ty . In this area of the kth ventr ic le , the choroid plexus is immediately distinguishable under high power by its dense concentration of highly re-<>f ttie m«UII«. active MAO-containing granules. The subependymal tlssue Awithin this region of choroid plexus exhibits an extraordinarily intense granule reaction in the neuropil, almost as though active diffusion were taking place between the choroid plexus and brain stem t issue. ft-ftTE 3ZL 46, Sec. 03-20 : Oblongata Upper Levels of Vagal and Hypoglossal Nuclei The strong MAO in the vagal nuclei continues well defined. In the vagal nucle i , certain small ce l ls appear to have reacted strongly, although the cr iss-cross felt-work of neuropil is the most d ist inct ive pattern. Mild to moderate MAO is shown in the descending vestibular root, ret icular formation and ambiguous nucle i . Ventro-medially, cel l group d of Meesen and Olzewskl shows prominent MAO. Ventral to the strongly reacting subependymal layer, the hypo-glossal and intercalate nuclei show the same moderate reaction throughout the neuropi1. The subependymal layer above the descending vestibular nucleus shows the strongest MAO in this section. Moderate act iv i ty is present in a l l other areas of grey matter, most pronounced In the following nuclei: hypoglossal, vagal, intercalate, descending vestibular, ambiguous, o l ivary , trigeminal, and in cel l group d, and the sol i tary fasciculus. The medial reticulo-splnal tract is weak in MAO. 49. Sec, 03-26 ; Upper Oblongata Region of Facial Nerve From this level of the oblongata, through the midbrain and up to the diencephalon, the most conspicuous feature is the absence of strong MAO. Except for a few scattered areas - locus coeruleus, central grey matter, oculo-motor complex and interpeduncular nucleus, no strongly marked act iv i ty is present. The vagal nuclei and roots have become embedded in the deep surface of the descending vestibular nuclei and are here seen with only moderate MAO, forming a l ine of act iv i ty to the facial nucleus, against the weakly reactive ret icular formation. Along the dorsal surface, below the nodulus, the descending vestibular nuclei show mild to moderate MAO, while along the lateral aspects, the acoustic s t r i a , and moderately reactive dorsal and ventral cochlear nucle i , border the restiform body. 50. Sec. 03-36 : Midbrain Level of Isthmus In the brain-stem at this area, the MAO act iv i ty is confined to the central grey matter, locus coeruleus, mesencephalic root of the trigeminal, nucleus of the lateral leminiscus and of the brachium conjunctivum. This f o l -lows roughly a v-shaped band medio-laterally. In the central grey, the weakly reactive cel l group Q of Meesen and Olzewski is medially located. Lateral to this is the locus coeruleus, d is t inct ly marked, as is the mesencephalic nucleus of N. V. These two areas are strongest in MAO. In the region of the mesencephalic nucleus of N. V, individual ce l ls can be recognised. The MAO is here apparently located only within certain c e l l s , and fa int ly or not at a l l in the neuropil. This is a direct reverse of the usual s i tuat ion, and continues through Into the midbrain areas. A band of moderately MAO reactive tissue occurs in the central pontal raphe nucle i , but the medial reticular formation is almost devoid of MAO. 52. Sec. Oh-24M : Midbrain Level of Inferior Col 1iculI In this section, the most conspicuous feature is the absence of v i r tua l ly any MAO in the inferior c o l l i c u l i . On the other hand, the c o l -l i cu la r commissure and central grey matter are conspicuously reactive, part icular ly in the area immediately ventral to the aqueduct. Ventral again to this are the strongly reactive oculo-motor complex (Nr-~IT1, a**d-the Edlnger-Westphal nucleus), on the ventral borders of which are the pale unreactive tracts of the medial longitudinal fasciculus. The dorsal tegmental nucleus is conspicuously strong in MAO. On the periphery of the ventral part of the central grey matter a few large, oval , deeply stained ce l ls of the mesencephalic trigeminal nucleus l i e in a neuropil of strong MAO. The red nucleus appears to contain a few large reactive c e l l s , as in the substantia nigra. 5 3 . Sec. 04-22M : Midbrain Level of Col 1iculI The inferior c o l l i c u l i continue conspicuously unreactive, but the superior c o l l i c u l i , with moderately strong MAO, come to overl ie them as the tectum of the midbrain. The central grey matter s t i l l contains strong MAO, especially in the dorsal tegmental nucleus, and is carried on into the oculomotor, tmoh1 ear and Edinger-Westphal nuclei . Again, a few ce l ls of the mesencephalic trigeminal nucleus are outlined by the reaction. 54. Sec. 04-20M : Midbrain Level of Superior Col l iculus The superior col l iculus shows a moderate MAO reaction throughout most of i ts layers (N.B., optico-sensory reaction centre), excepting the optic stratum and tecto-spinal tract. Surrounding the aqueduct, the central grey matter continues to show a strong reaction, with the Edinger-Westphal nucleus showing strong MAO between the pale tracts of the medial longitud-inal fasciculus. The lateral tegmental nucleus shows moderate MAO. In this section, however, by far the most predominantly reactive, is the now large interpeduncular nucleus lying Immediately dorsal to the pons. Lateral to this are the pale relat ively unreactlve tracts of the cerebral peduncles, dorsal to which are a few scattered stained cel ls of the substantia nigra. 56* Sec. 0 4 - 1 7 M : Midbrain Through Mammi1lary Body The Interpeduncular nucleus is here the strongest MAO area, with the unreactive mammiIlary body lying on its ventral surface. The central grey and oculomotor regions are diminished in ac t iv i ty , and the tracts of the oculomotor nerve fibres can be seen emerging medial to the cerebral peduncles. Moderate MAO can be seen in the superficial grey substance, in the medial geniculate body and in the peri peduncular nucleus or substantia ni gra. 57 . Sec. 04-13M : Diencephalon Through the MammMIary Body Reference has been made to the unusual reactivity of the habenulo-peduncular tract (fasciculus retroflexus) since, in general, f ibre tracts are unreactive. In this section the two arms of the tract can be seen dorsal to the mammi1lo-thalamic tract. The strongest MAO is to be found in the tuber cinereum, but the fornix and mammi1lo-thalamic tract are unreactive. Moderate act iv i ty is to be found in the central grey, c i l i a r y and oculomotor regions, and in the medial geniculate body. The neuropil of the superior co l l i cus , and peripeduncular nucleus s t i l l contain mild to moderate MAO. 59. Sec. 04-11 : Dfencephalon Epi thalamic Region pin A more complicated pattern of MAO distr ibution is found, in general, in the diencephalon. Starting from the dorsal surface, the superficial grey substance vr (moderate MAO) shelters a strongly MAO reactive p ineal . The unreactive optic tract surrounds the lateral borders of this area, and enters below the superficial grey substance to connect wtth the nucleus of the optic tract above the nucleus of the posterior commissure. Both these nuclei contain moderate MAO. The aqueduct here is surrounded by a narrow zone of moderate MAO, with the reactive habenulo-peduncular tract approaching now its ventral l imit . Very strong MAO is seen in the hypothalamus, and moderate MAO In the medial geniculate and perl peduncular nucle i . 60. Sec. 04-10M : Diencephalon Hypothalamic region In this region of the posterior commissure, the highly reactive pineal organ may s t i l l be seen, but l ies now between the lateral geniculate bodies which contain moderately strong MAO. The pulvinar of the thalamus (weak MAO) enters the picture in this section, and more ventrally the sensory radiations can be distinguished by moderate MAO. The midline nuclei and hypothalamus show conspicuously strong MAO. 61. SEC. ot+.~IO n • \ 62. Sec. Ok-Sn : Dtencephalon Habenular Region The habenulo-peduncular tract can here be seen entering the habenu-lar body, and the olfacto-habenular tract can s t i l l be traced. The medial and central nuclei contain moderate MAO, but l i t t l e or no act iv i ty is apparent in the ventral or ventro-lateral nuclei of the thalamus. The lateral geniculate bodies can no longer be seen in the dorsal region but have come to l i e lateral to the pulvlnar. Thus, the section is almost completely bordered by the pale staining fibres of the optic tract . The whole midline area takes on great signif icance in relationship to MAO, since from the habenular trigones ventromedially, through the midline nuclei of the thalamus to the nuclei of the hypothalamus, Is a region of strong ac t iv i ty , part icularly in the periventricular area. 63. Sec. Ok-kH : Dlencephalon Region of the Optic Chiasma The dorsal nucleus of the thalamus now l ies lateral to the habenu-lar trigone. Weak MAO only is apparent throughout the thalamus, except in the central midline area where moderate MAO is present; higher MAO Is pres-ent in the habenular bodies and hypothalamus. 64. 5. Description of Special Areas. a) Medulla spinal is and spinal cord. In sections taken from the level of the f i r s t cervical nerve, a very similar distr ibution of MAO act iv i ty is seen, as in the rest of the cord at lower levels . Throughout the cord, act iv i ty is usually most marked in the neuropil surrounding the central canal, and in the remaining neuropil of the grey substance. Strong concentrations are often seen in the substantia gelatinosa, and as an exception to neuronal ac t iv i ty , some staining of anterior motor neurons, part icularly at lower levels of the cord. The f ibre tracts appear under low power,to contain l i t t l e or no MAO, but under high power very small reactive granules may be seen. It has not been possible to determine the exact nature of these granules; whether mitochondrial, or associated with other structures such as the gl ia or myelin sheath. b) Blood vessels and meninges. Arteries and veins outside the brain showed sl ight act iv i ty within their wal ls, but no MAO was found in intracerebral cap i l l a r i es . This is in agreement with the findings of Shimizu et a K (1959), but con-trary to those of Arioka and Tanimukai (1957), using a method involving pigment formation on oxidative degradation of tryptamine and serotonin. c) Ventricular walls - ependyma and sub-ependyma. The MAO content of the ependyma ce l ls was variable throughout the brain-stem. In some areas there was almost no evidence of any MAO ac t iv i ty , whereas in others the ependyma and subependymal tissue were intensely strong in MAO. In general, however, the ependyma had only mild to moderate ac t iv i ty , with the subependymal areas showing moderate to strong ac t iv i ty . 65. One o r two p e c u l i a r l y s t r ong MAO areas were noted. One was found i n a t r i a n g u l a r o r wedge-shaped area of subependymal t i s s u e , l ocated between the c e n t r a l grey matter and the medial v e s t i b u l a r nucleus. Aggregations of small round c e l l s could be seen under high power, but no c l e a r - c u t c o r r e l a t i o n s w i t h p a r t i c u l a r c e l l types cound be found. Other s i m i l a r areas were found i n the l a t e r a l w a l l s of the caudal end of the aqueduct, i n the a n t e r i o r medullary velum and near the area postrema. F r i e d e (1961) notes that these areas are a l s o r i c h i n s u c c i n i c dehydro-genase and suggests that they could represent a c e l l type of y e t unknown f u n c t i o n a l s i g n i f i c a n c e , d) Choroid plexus. Shimizu jet aj_. (1959) report that "the choroid plexuses and ependymal c e l l s were weakly p o s i t i v e f o r MAO a c t i v i t y i n each s p e c i e s . " This i s i n c o n t r a s t to the f i n d i n g s i n t h i s i n v e s t i g a t i o n . Under low power, however, Shimizu's statement appears to be t r u e , s i n c e the MAO r e a c t i v e granules are s m a l l , and the cytoplasm i s not r e a c t i v e , i n t h i s study, however, each choroid plexus was shown to c o n t a i n a vast q u a n t i t y of dense, s t r o n g l y r e a c t i v e granules. Large s p e c i f i c c e l l types, unident-i f i e d by r o u t i n e h i s t o l o g i c methods, occurred here and there, somewhat resembling f a t c e l l s , but laden w i t h dense c l u s t e r s o f extremely small r e a c t i v e granules, surrounded by pink cytoplasm. Oddly enough, where d i f f u s i o n o f cytoplasm, o r c e l l - r u p t u r e , had occurred, "pfcnk b a l l o o n s " were formed w i t h i n the plexus. This phenomenon was a l s o observed at the cut ends, o r along the border, of p e r i p h e r a l nerves. In some areas of the 4th v e n t r i c l e choroid plexus, these small MAO granules appear to migrate i n t o the subependymal t i s s u e . The granules 66. are approximately the same size and shape as those observed in l i ve r , by the same reaction, and in lesser quantit ies, in lung. e) Subcommissural organ. This is a high epithelium l ining the ventral surface of the posterior commissure at the rostral end of the aqueduct. Only weak act iv i ty for MAO was noted in rabbit brain. f) Subfornical organ. The subfornical organ, or intercolumnar tubercle, is located under the hlppocampal commissure, and shows strong MAO, confirmed by Shimizu et a l . (1959)* In this connection, i t should perhaps be mentioned that in recent studies on uptake of epinephrine in the brain (Wilson et a l . , 1962) intravenously administered epinephrine-H^ was taken up in the subfornical organ, pineal body and area postrema, in concentrations considerably in excess of those in parts of the brain lying within the blood-brain barr ier . g) Pineal body. In these experiments the pineal body appeared to contain a con-sistent ly higher concentration of enzyme than any other area of the brain. However, Shimizu et al_. (1959) state that "the pineal body . . . showed l i t t l e i f any staining for MAO in animals . . . " (rodents). Comparative studies here have substantiated a strongly positive MAO in human, ox, and pig pineal , as well as in the rabbit. The strongest act iv i ty appears to be in intercel lu lar tissue which runs in rows between the c e l l s . This is part icular ly interesting in that Kappers (I960) demonstrated a very extensive autonomic network in the pineal organ, by means of the Champy technique. In further studies on the pineal , Prop and Kappers (1961) established the presence of me.latonin and serotonin by paper chromatography, 67. although being unable to demonstrate by histological means the presence of aromatic amines or indoles, using acid diazonium, fe r r i c ferricyanide reduction or the chromaffin reaction. This is perhaps understandable in that high concentrations of catecholamines or indoles may be required for these reactions, and are perhaps protected by the high l ip id content of the pineal (Quay, 1957); or i t might be that, unlike other organs, sero-tonin in the pineal is linked to other chemical compounds in such a way that i ts histological demonstration is impossible by the known techniques for indoles. h) Substantia alba. In general the white matter exhibited a weak or negative ac t iv i ty , except for a few f ibre tracts such as the habenulo-peduncular tract men-tioned later . This statement must, however, be made with considerable caution since under low power a completely negative reaction can be assumed which under high power can often be shown to have f ine reactive granules throughout the t issue, their presence masked under low power by the greater quantity of non-reactive t issue. i) Substantia grisea. The MAO reaction in grey matter is almost entirely within the neuropil , and seldom within the soma of any nerve c e l l . This is also substantiated by Shimizu (1959). Friede (1961) defines neuropil as the close juxtaposition of dendritic ramifications, processes of g l ia l ce l ls and, possibly, synapses. These heterogeneous elements form a complex tissue which shows speci f ic histoehemical properties di f fer ing from the properties of the perikarya. That MAO could play a part in synaptic transmission is borne out in this thesis, where, in many regions, wel l -marked c i rcular knobs, many of which exhibit ring shapes resembling 68. boutons terminaux, can be seen in proximity to neurons. These can only be distinguished under high power or oil-immersion, j) Hypophysis. Examination of the pituitary was made in pig and ox, but not in rodents or in cat . It is included here, however, as a matter of general neurological interest in the study of MAO, bearing in mind that reactions may d i f fer in different species. In the pituitary of species studied (approximately one dozen glands) a characterist ic reaction occurs, d is t inct ly different in the anterior from the posterior part. In the pars distal i s , or anterior portion, there is a weak genera} reaction for MAO which under high power, however, appears as a profuse abundance of very f ine , highly reactive granules within the cytoplasm of certain c e l l s . These granules are f iner than those of the choroid plexus, and are located in ce l ls situated on the posterior lateral aspects of the pars distal i s , so that in sections cut as i l lust ra ted , the following pattern of distr ibution can be seen: 69. In the pars nervosa, a moderately strong, diffuse reaction is seen resembling the reaction in cort ical neuropil. There are no dist inguish-able stained c e l l s , but the reactive areas tend to follow the 'sinusoidal pattern of the pineal , and l i e in canals between c e l l s . Three general areas may be distinguished, with the strongest reaction occurring most posteriorly. k) Brain-stem of cat . In general, cat exhibited a much weaker reaction and only poor regional local izat ion was possible. No granules or f ine detail could be picked up under high power by the usual methods used for MAO ident i f ica-t ion. No reference can be found to other histoehemical ident i f icat ion of MAO in this species, so i t is not possible to compare results. Nevertheless, regional local izat ion appeared to be the same as in rabbit, except for the following: (i) No reaction in the habenulo-interpeduncular tract , ( i i ) Very weak reaction in the area postrema and locus coeruleus. ( i i i ) Much weaker general reaction in a l l areas (strongest in choroid plexus and hypothalamus). 70. PART V. DISCUSSION 1. Review of Regional Concentrations of MAO. Table IX on page 71 shows regional comparisons of MAO-rich areas in the brain-stem and from this i t can be seen that the strongest areas of MAO concentration in rabbit brain are in the choroid plexuses, the pineal gland and the interpeduncular nucleus. Not much lower in act iv i ty are the areas of the vagal nucle i , spino-trigeminal nucle i , hypothalamus and periventricular nucle i , habenulae and olivary nucle i . S t i l l rich in MAO, but not so pronounced in intensity of staining, are the dorsal tegmental nucle i , habenulopeduncular tract , pontal raphe nuclei , locus coeruleus and posterior p i tu i tary. The diagram of cranial nerve nuclei (Table X, page 72) shows clearly the importance of MAO in these areas. Most of the brain-stem cranial nerve nuclei appear to contain marked quantities of MAO, so that in sections of the medulla, these areas, and the nuclei of the infer ior o l i v e , stand out as centres of MAO ac t iv i ty . These cranial nerve nuclei are, however, irregular in the amount of MAO they contain, so that the spino-trigeminal, vagal and commissural nuclei stand predominantly high above the rest. A l l other nuclei represented in the diagram are compar-able in intensity. Higher in the brain-stem, other non-cranial nerve nuclei assume importance in MAO act iv i ty . The central grey matter, interpeduncular nucleus, habenular ganglion and hypothalamic areas become prominent, while the thalamic nuclei in general are weak in MAO. 71 TABLE IX R e g i o n a l Comparisons of MAO-Rich Areas i n Rabbit B r a i n Stem Moderate Strong Dorsal and ventral commissure - neuropil M E D U L L A Descending r o o t of t r i g e m i n a l - n e u r o p i l V a g a l n u c l e i - n e u r o p i l Area postrema - n e u r o p i l O l i v a r y n u c l e i - n e u r o p i l R e t i c u l a r f o r m a t i o n - n e u r o p i l - throughout Choroid p l e x u s - g r a n u l e s p 0 N 3 M E S E N C E P H F a c i a l n u c l e i - n e u r o p i l M i d l i n e raphe n u c l e i - n e u r o p i l Locus c o e r u l e u s - n e u r o p i l Mesencephalic n u c l . N.V, - c e l l b o dies * „" J.j V e s t i b u l a r n u c l e i - n e u r o p i l j D o r s a l tegmental n u c l e i - n e u r o p i l ; C e n t r a l g r e y matter i - n e u r o p i l I P i n e a l body ! S u p e r i o r c o l l i c u l i -- n e u r o p i l | Oculo-motor complex - n e u r o p i l i — | M e d i a l g e n i c u l a t e -\ n e u r o p i l 0 ! I n t e r - p e d u n c u l a r nucleus - n e u r o p i l N ' j ; Habenulo-peduncular t r a c t - f i b r e s ^ ^ ^ ^ ^ ^ Habenular b o d i e s - n e u r o p i l P e r i v e n t r i c u l a r n u c l e i - n e u r o p i l Hypothalamus - n e u r o p i l P o s t e r i o r p i t u i t a r y - n e u r o p i l A n t e r i o r p i t u i t a r y - g r a n u l e s ( v e r y f ine).': 72. TABLE Diagram Showing N u c l e i of C r a n i a l Nerve C o n c e n t r a t i o n s of MAO 0 { i ' ^ f - • " N u c l . N. I l l V i s c e r a l " N u c l . N. I l l Somatie - Mesencephalic n u c l . N, V. - tine• W 3. N.V • • W> - N u c l . N. V - Locus Coeruleus Genu o f N. V I I - --ft, M * H - f - N u c l . N. VI I / I /_/_.- N u c l . V I I I ( v e s t i b u l a r ) - N u c l . N- V I I N. IX N. X Area Pestrema T r a c t of S p i n a l V F a s c i c u l u s S o l i t a r i u s N u c l . N. X I I D o r s a l motor n u c l . N. X N u c l . ambiguous Commissural n u c l . N. X N u c l . XI " ' Spin*.! nucleus of N.V. 73. Although not included in the detailed mapping of this study, the whole brain was examined, so that an overall picture could be obtained. It is perhaps suff ic ient to record that diffuse reaction in the cort ical neuropil and hippocampus was consistently present, and that the molecular and granular areas of the cerebellum contained moderate to strong amounts of MAO. At no time was any intracel lular reaction observed in these areas. A special examination was also made of the septal areas of rabbit brain, but no conspicuous act iv i ty was detected. Likewise, the olfactory bulbs were examined with similar results. (See page 18 for act iv i ty In the amygdala, striatum and other regions.) From the general regional concentrations shown in this study, a pattern of act iv i ty emerges, arb i t rar i ly c lass i f ied into the following components: a) Cranio-visceral b) 01factory-l imbic c) Pineal-hypothalamic-pituitary d) Choroid plexus e) Neuropil In order to examine the possible reasons for these regional con-centrations, i t is important to review the main functions of these areas. Why should certain areas possess particular chemical properties, such as have been shown by similar methods for succinic dehydrogenase, DPN and TPN diaphorase, and cytochrome oxidase? Are these areas rich in certain enzymes because of lack of speci f ic inhibitors in those areas, or because of more available substrate? A brief outline of comparative histo-chemistry may help define the functional picture. 74. 2. Comparative Histochemistry and C a p i 1 l a r i z a t i o n . Z e l l e r (1951) has postu lated that cho l ines te rase i s high where MAO is low, and th is has not been cont rad ic ted by the f ind ings of Hebb (1961) or K o e l l e (1954) who have found a c e t y l c h o l i n e s t e r a s e in the supra -o p t i c and paravent r i cu la r nucle i of the hypothalamus. Cavanagh (1961) found that in the hen, in cont rast to mammals, ACh-esterase was present in the cytoplasm of a l l cent ra l neurons examined. In mammals, not a l l neurons conta in ACh -es te rase , where n o n - c h o l i n e r g i c and c h o l i n e r g i c c e l l s are found. Thus, although l o c a l i z e d regions of MAO and ACh-esterase may be found together , i t would seem that MAO is almost always conf ined to the n e u r o p i l . K o e l l e (1954) reports high s p e c i f i c cho l ines te rase a c t i v i t y in the dorsal nucleus of N.X, interpeduncular nucleus (espec-i a l l y pars l a t e r a l i s ) and in the caudate and amygdala. MAO i s a lso high in the f i r s t two a reas , but not in the caudate o r amygdala. K o e l l e fu r the r noted that high ChE a c t i v i t y occurred in near ly a l l motorneurons, and was concentrated at the per ikaryon and along the axons and d e n d r i t e s . Low a c t i v i t y only ex is ted in the neocortex. F r iede (1961) has shown some concordance between c a p i 1 l a r i z a t i o n and s u c c i n i c dehydrogenase (SDA) but th i s i s not always c o n s i s t e n t . For ins tance , in the nucleus dorsal i s vagi which contains low SDA, there i s high c a p i 1 l a r i z a t i o n , high DPN and high MAO. This app l ies a l s o to the area postrema. A g a i n , the i n f e r i o r c o l l i c u l i are almost devoid of MAO, ye t are high in SDA and proport ional in c a p i 1 l a r i z a t i o n , and reverse ly , _ the interpeduncular nucleus shows only moderate SDA compared with i t s high MAO content , and moderate c a p i 1 l a r i z a t i o n . Thus, there does not appear to be any cons is tent c o r r e l a t i o n between the d i s t r i b u t i o n of SDA (or cytochrome oxidase which p a r a l l e l s 75. SDA) and that of MAO, in mammalian b ra in - s tem. 3. Re la t ionsh ip of Regional Concentrat ions of MAO to Functions of  Related A r e a , a) C r a n i o - v i s c e r a l components. Ce r ta in of the c r a n i a l nerve components in the bra in stem are conspicuous f o r t h e i r strong MAO. These are int imate ly connected, p h y l o g e n e t i c a l l y , with the branchial or v i s c e r a l arches , which in the lower aquat ic ver tebrates carry the resp i ra tory organs. They may be c a l l e d "branchiomer ic" nerves, and inc lude the t r igeminal (V) , f a c i a l ( V l l ) , glossopharyngeal (IX) and vagus (X) . Both a f f e r e n t and e f f e r e n t f i b r e s are c a r r i e d , which leave the bra in together and are not separated Into dorsal o r ventra l roo ts . Of the e f f e r e n t branchiomerics , the seventh, n inth and tenth nerves supply many general v i s c e r a l f i b r e s to the glands and v i s c e r a l muscle of the pharynx, larynx and thorac ic and abdominal organs. The a f f e r e n t f i b r e s of these nerves are re lated to v i s c e r a l s e n s i b i l i t y , conveying general impulses from the pharynx, larynx , thorac ic and abdominal v i s c e r a . The sensory root of the tr igeminal nerve mediates touch, pain and temperature from the f a c e , forehead and ectodermal mucous membranes of the mouth and nose, and contains a l s o terminations of soma-t i c a f f e r e n t vagal f i b r e s . Anatomical ly these areas are c l o s e l y connected, and some chemical i n t e r a c t i o n may occur between them, poss ib l y connected with the phenomena of ca rd iac a r r e s t . For ins tance , the area postrema, shown to be an emetic chemoreceptor t r i gge r zone (Wang and Bor ison , 1952) is a f f e c t e d by many medullary c e n t r e s . The vomiting centre i t s e l f i s in c l o s e proximity to the responsive loc i f o r spasmodic r e s p i r a t i o n , i n s p i r a -76. tion and expiration, vaso-motor control , postural tone and the vestibular nucle i . Afferent impulses from the gastrointestinal tract reach this area mainly through the vagus. The vomiting centre is thus influenced, l ike other brain-stem areas, by tonic excitatory and inhibitory nervous and metabolic regulators, in(^h?ch i t would appear that MAO and biogenic amines play a signif icant part. b) 01 factory-1imbic components. In Table XI, page 77, the areas of the olfactory system high in MAO, are shown in conjunction with those of weak MAO. Certain of these brain-stem areas high in MAO, receive connections from the 1imbic system. For instance, the habenular nucleus receives impulses not only from the basal olfactory nuclei of the septal area, but also via the fornix, from the hippocampal cortex. This suggests connections from basic emotional centres, to pathways leading thence to the cranial nuclei of the medulla and to the viscera (See Table XII). In the lamprey, the forebrain lobes send fibres via the cortico-habenular tract , to the habenular body. The habenulo-peduncular tract then passes impulses back to the peduncular region - a system that is common to a l l vertebrates throughout phylogeny. c) Pineal-hypothai amic-pi tui tary components. Because of the consistently high level of MAO demonstrable in the pineal , a new concept of amine interaction, involving these three components, is postulated on the basis of this study. However, with the limited amount of knowledge concerning these mechanisms, i t is possible only to suggest interactions. The following is the evidence on which this concept is based. Although i t is well known that the hypothalamus is phylogenetic-a l ly one of the oldest parts of the vertebrate prosencephalon, forming, TABLE XI O l f a c t o r y Connections Showing MAO C o n c e n t r a t i o n s B a s a l o l f a c t o r y n u c l e i F o r n i x S e p t a l area n u c l e u s 78 TABLE XII Diagram I l l u s t r a t i n g Concept of P i n e a l - H y p o t h a l a m i c - P i t u i t a r y Interaction P i n e a l 7 9 . together with the pi tui tary, a complex neuroglandular mechanism which influences most visceral a c t i v i t i e s , perhaps equally important but neglected in the l i terature of comparative neurology, is the pineal gland and i ts habenular connections. These exist as early as the hypo-thalamic structures. The cyclostomes are the most ancient aquatic forms with a true vertebrate organization of the head region and these possess a well-formed pineal . Between the tectum and habenular nuclei are inserted the stalks of two small pineal "eyes", the fibres of which enter the posterior commissure. They have presumably been termed "eyes" since they occupied a paired frontal position in the brain at this level of phylogency. Thus, some have postulated the existence of a primitive thermo- or 1 ight-receptive mechanism, controll ing endocrine and other functions connected with mating and migration. In addit ion, ;the idea of a chemo-receptor organ suggests i t s e l f , connected with the biosynthesis and catabolism of monoamines. It is we11-known,that melanocytes have their embryonic or igin in nerve t issue, but i t has only recently been discovered that the newly found hormone of the pineal , melatonin, can cause temporary dispersion of the melanocytes in frogs and tadpoles (Lerner, 1 9 6 2 ) . Shortly after the discovery and isolation from the pituitary of the melanocyte-stimulating-hormone (MSH) which causes darkening of frog skin, i t was found that ACTH from the anterior pituitary can also darken frog skin, though not as e f f ic ient ly as MSH. This hormone is effective in man as well as in frogs. Later, a successful attempt by Lerner and his co-workers to isolate the substance from the pineal that lightens frog skin, led to 80. the discovery of the hormone, melatonin. It has now been shown that some tranquilizers potentiate this effect of melanocyte dispersion in frogs, whereas MAO inhibitors tend to prevent i t . It is f e l t that this evidence lends i tse l f to the suggestion of interaction between the pineal , hypothalamus and pi tu i tary , and that MAO could thus be concerned with neurohormones which regulate the endocrine and nervous systems of the body. d) Choroid plexus. Although i t is generally believed that the choroid plexus is concerned with the elaboration of cerebro-spinal f l u i d , i t is also known to be capable of absorbing certain substances from the cerebro-spinal f l u i d . Regarding i ts high content of MAO, however, too l i t t l e is known to warrant any assumptions in this connection. e) Neuropi 1. The fact has been stressed that MAO occurs mainly in the dendrites and ce l l processes surrounding the neurons, rather than within the actual cel l bodies. For lack of a better term, we must designate this area as "neuropil" , and i t is clear that this region is a highly active part of the functioning nervous system. Schade and Baxter (I960) state that dendrites provide about 95% of the receptive surface of the neuron, so that one of the major sources of electrocortical act iv i ty could be a t t r i -buted to summation of post-synaptic potentials (Purpura, 1959). Certain aspects of behaviour may apparently be correlated with the structure and density of the neuropil, so that a depletion in the density of neuropil (and hence the probability of axo-dendritic interaction) is associated with poor learning ab i l i ty (Eayrs, 1961). Flexner et al_. (1950) have shown that the growth of eel I-processes coincides with changes in electro-lyte d istr ibut ion, and Himwich and Petersen ( 1 9 5 9 ) state that similar changes occur in concentrations of glutamic acid and glutamine. In hypothyroidism changes occur in the neuropil connected with anoxia (Eayrs, 1954) and with an apparently irreversible effect on the action of succinic dehydrogenase (Hamburgh and Flexner, 1 9 5 7 ) . That neuropil is also a prominent feature of invertebrates is substantiated by Horridge ( 1 9 6 1 ) who states that a l l invertebrates have nervous systems where a l l the interesting act iv i ty seems to be in the neuropil, and a similar neuropil occurs regionally in vertebrates. These facts are, i t is f e l t , of suff ic ient signif icance to suggest that the concentrations of MAO in the neuropil serve a funda-mentally useful purpose connected with the behaviour of the individual , and that disturbances of MAO and amine metabolism would have related behavioural e f fects . 82. SUMMARY 1. An attempt has been made to give an integrated picture of MAO act iv i ty in rabbit and cat brain-stem. This has been presented in atlas form. 2. A histoehemical method was used which depended on the incubation of fresh frozen tissue sections with tryptamine as substrate and ni tro-blue tetrazolium salts as reduction agents in a pigment production system. Speci f ic i ty for the reaction was established by the use of known MAO inhi bi tors _ij2 vi tro and j_n vivo. 3. Highest concentrations of MAO were found in the visceral auto-nomic centres of the brain-stem, part icularly in the interpeduncular nucleus, hypothalamus and sensory vagal nucle i . k. 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