Anat. Embryol. 149, 225--239 (1976) 9 by Springer-Verlag 1976

Fluorescence Microscopic Study of the Behaviour of DANS-marked Histidine in the Rat Brain after Intraventricular Injection K a r l Heinz Booz a n d B e r n d Wiesen Fachriehtung Anatomie im Fachbereich Theoretische Medizin der Universit~t des Saarlandes, Homburg/Saar Received March 5, 1976 Summary. Following stereotactic injection of dansyl-histidine into the third ventricle of the rat, uptake and further transport of the amino acid in respect of the ependyma, the choroid plexus and the brain were investigated with fluorescence microscopy, within the framework of a time study. 1. Ependyma. The labelled amino acid, which emits a yellow autofluorescence, is taken up very rapidly by the ependyma and 5 min after injection is demonstrable in the whole of the ventricular lining, the boundary with the subjacent brain being sharply demarcated. In respect of subsequent transport there are regional differences. After 60 min p.i. the histidine has disappeared from some sections of the ependyma. The ependymal lining of the third ventricle is totally depleted 90 min post injeetionem, that of the lateral ventricles 60 and 120 min p.i., that of the aqueduct 40 rain p.i., and that of the fourth ventricle 60 min p.i. 2. Choroid Plexus. At the earliest time of observation permitted by dissecting out etc., all plexuses are found to have taken up histidine. Duration of storage, however, varies. While the plexus of the third ventricle is already depleted in stretches after 60 min and totally depleted after 120 min, the fluorescent amino acid is still discernible in the plexuses of the fourth and lateral ventricles after 180 min. 3. Brain. Penetration of the amino acid into the cerebral tissue commences 10 min p,i. and lasts, taking into account regional temporal differences, 180 rain. The histidine becomes localized foremostly in the following areas: nucl. anterior hypothalami, nuel. ventromedialis hypothalami, nucl. praeopticus, nucl. paraventricularis, stria terminalis, stria medullaris thalami and nucl. septi lateralis. In the cerebellum, DH-storing ceils occur only in the molecular layer. The results of this investigation are compared with transport routes and deposition centres relevant to tryptophan and phenylalanine, which have been studied using the same methodology. Whereas the latter amino acids show definite affinities for serotinergic and dopaminergic nuclear areas, histidine chiefly permeates periventricular structures and hypothalamic nuclear areas. Key-words: Ependyma and plexus - - Brain (Rat) - - Histidine-Intraventrieularinjection-Fluorescence microscopy. Introduetion H i s t a m i n e in inactive, b o u n d form is widely d i s t r i b u t e d in the organism. I t occurs i n t h e skin, lung a n d above all i n the mast cells (Riley, 1963). These cells c o n t a i n a deearboxylase which decarboxylizes histidine to histamine, the latter forming a salt-like b i n d i n g with h e p a r i n (Werle a n d A m a n n , 1955). I n the b r a i n no m a s t cells (Green, 1970) or only a few (Drop, 1972) are found. Although evidence of histaminergie nerve t e r m i n a l s i n the b r a i n is t h u s far lacking, it is nonetheless a s s u m e d t h a t the a m i n e exercises a t r a n s m i t t e r f u n c t i o n (Snyder a n d Taylor, 1975). This is supported b y the results of biochemical studies

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a i m e d a t localizing h i s t a m i n e a n d h i s t a m i n e m e t h y l t r a n s f e r a s e which reveal t h a t b o t h s u b s t a n c e s occur in b r a i n s t r u c t u r e s with p r o p e r t i e s similar to those of t h e p r e s y n a p t i c a x o n s t h a t store s e r o t o n i n a n d n o r a d r e n a l i n e (Kuhar, T a y l o r a n d S n y d e r , 1971 ; Carlini a n d Green, 1963 ; K a t a o k a a n d D e R o b e r t i s , 1967 ; Michaelson a n d Coffmann, 1967). Since h i s t a m i n e , like t h e o t h e r biogenic amines, does n o t pass t h e bloodb r a i n b a r r i e r ( G a r r a t i n i a n d Valze]li, 1965) a n d since W h i t e (1960) was able to show t h a t b y perfusion of t h e ventricles o n l y a b o u t 1% h i s t a m i n e reaches t h e c e r e b r a l tissue, it m u s t be a s s u m e d t h a t a local synthesis t a k e s place a t t h e effective sites. E v i d e n t l y , for h i s t a m i n e t h e r e m u s t exist a C S F - b r a i n b a r r i e r too, t h o u g h this is n o t effective for t h e c o r r e s p o n d i n g a m i n o acid (Seiler, 1966) so t h a t h i s t a m i n e can be s y n t h e s i z e d i n t r a c e r e b r a l l y b y t h e d e c a r b o x y l a t i o n of its precursor. S u p p o r t for this c o n c e p t comes from Schwartz, L a m p a r t a n d Rose (1971) who a f t e r i.p. a p p l i c a t i o n of h i s t i d i n e n o t e d a d i s t i n c t a u g m e n t a t i o n of t h e h i s t a m i n e c o n t e n t of t h e b r a i n which d i d n o t occur after blocMng t h e dec a r b o x y l a s e . T h e e n z y m e h i s t i d i n e d e c a r b o y l a s e is u n e v e n l y d i s t r i b u t e d in t h e brain, e x h i b i t i n g highest a c t i v i t i e s in t h e h y p o t h a l a m u s a n d vermis ccrebelli (White, 1960). I t is p r o b a b l e t h a t h i s t i d i n e reaches t h e b r a i n via either t h e bloods t r e a m or t h e cerebrospinal fluid, a n d o n l y t h e n becomes decarboxylized. T h e p u r p o s e of t h e i n v e s t i g a t i o n r e p o r t e d here has been to as c e r t a i n t h e b e h a v i o r of fluorescence labelled h i s t i d i n e i n j e c t e d into t h e ventricle s y s t e m a n d to c o m p a r e t h e fluorescence-microscopical d a t a concerning resorption, transport, a n d d e p o s i t i o n of t h e h i s t i d i n c in t h e b r a i n with t h e findings of S t a r k a n d F r a n z (1972), F r a n z a n d S t a r k (1972) a n d I~apr/iger (1973) concerning t h e t r a n s p o r t of t r y p t o p h a n , t e t r a c y c l i n e a n d p h e n y l a l a n i n e . Material and Methods From the skull of adult male and female Wistar rats under sodium pentobarbital anaesthesia (50 mg/kg b. w.) a 16 mm 2 piece of bone was trephined just above the superior sagittal sinus, care being taken not to damage the sinus. Through the opening, with the aid of a stereotaetic instrument (cf. Stark and Franz, 1972) 2-4izl dansyl-histidinel& (Serva, HIeidelberg, West Germany), dissolved in approximately 35~ warm Triton (Serva, Heidelberg), corresponding to 0.1 mg/kg b., w., was injected into the third ventricle. The site of injection, located in accordance with the system of coordinates given in the atlas of KSnig and Klippel (1963), was situated 3.180 ~xm before the interauricular frontal plane. This point is favourably placed in the widest part of the ventricle just behind the massa intermedia. The brains were removed 2.5, 5, 10, 15, 20, 30, 40, 60, 90, 120 and 180 rain after injection and frozen as 2-3 frontal sections in isopentane at -79~ This procedure lasts 60 sec. After freeze drying for 3 days (Leybold GT 001) the specimens, under continuous vacuum, were embedded in previously degassed paraffin wax. 7 tzm thick serial sections were cut and stretched on microscopic slides simply by warming to 50~ The specimen were examined with a Leitz fluorescence microscope (Ortholux: I-IBO 200; exciter filter BG 38, BG 12/5 ram; barrier filter K 510). The sections were photographed immediately after having been stretched. Later observations show that there is no decrease of DIt-fluorescence. DH-containing structures are easily distinguishable on account of their yellow fluorescence from the green autofluorescence of the unlabelled brain tissue. 1 abbreviated: DH 2 DH from the firm of Serva contains, besides di-dansylhistidine, also 5% mono-dansylhistidine and 5 % dimethyl-amino-naphthaline sulfonic acid

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Results For presentation of the findings, the nomenclature given in the atlases of KSnig and Klippel (1963) and Zeman and Innes (1963) will be used.

2.5 rain Post Injectionem (cf. Fig. 1 a-f) Already 2.5 min after intraventricular injection of D t t - a shorter interval is precluded by the time required for dissecting out the brains - an irregularuptake of histidine by the ependymal cells and choroid plexuses of all ventricles, except for the ependyma of the fourth ventricle, is observed. Uptake is especially conspicuous in the third ventricle (Fig. 2a) where DtI is to be found in the cytoplasm of all ependymal cells, and less marked in the lateral ventricles, where the rostral ependyma is only moderately labelled and single cells are devoid of D H fluorescence, while the caudal parts fluoresce more strongly. The choroid plexuses of all ventricles fluoresce evenly and strongly except for the connections between the main part of the plexus of the fourth ventricle and its protrusions in the lateral recesses. There is a conspicuously uniform, deep penetration of DH into the tegmen of the fourth ventricle. Here, fluorescing cell processes are also discernible. Fluorescing cells are observable in the nucl. interstitialis striae terminalis praeoptici medialis, paraventricularis, ventromedialis hypothalami and in the striae terminales et medul]ares thalami. 5 rain Post In]ectionem D H can be demonstrated in the ependyma of all ventricles, although the lining of the fourth ventricle as a whole fluoresces less intensely and some cells are devoid of DH. The fluorescing ependymal lining stands out in sharp contrast to the faintly recognizable brain tissue (Fig. 2 c). The fluorescence intensity of DtI has already begun to diminish in the ependymal lining of the aqueduct. But in all plexuses DIt is found evenly distributed. The D H penetrated into the brain tissue is located in cells of the following regions: nuel. interstitialis striae termina]is, nucl. praeopticus medialis, nucl. paraventricularis, nucl. ventromedia]is, stria terminalis, stria medul]aris thalami and the total of stratum moleculare in the cerebellar cortex. 10 rain Post Injectionem While the ependymal lining of the lateral ventricles and that of the fourth ventricle display a uniform yellow fluorescence throughout, decrease of fluorescence is observable in the ependyma of the third ventricle and in that of the aqueduct, some cells containing hardly any DH; hereby the DH is localized at the lateral cell margins but not in the basal parts of the cells. All plexuses (Fig. 2 d) fluoresce with undiminished intensity. The number of anteriorly situated fluorescing cells in the partes latera]is, medialis and centralis of the nucleus ventromedialis has distinctly decreased. On the other hand, the number of fluorescing cells in the nucl. interstitia]is striae terminalis, septi ]ateralis, praeopticus medialis, periventricularis, in the fornix praecommissuralis pars praeoptica et pars hypothalamica, in the stria terminalis and stria medullaris thalami remains constant. Fluorescence is observed for the first time in cells of the nucl. anterior et dorsomedialis hypothalami. The number of fluorescing cells in the molecular layer of the whole cerebellum has clearly augmented.

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Fig. 1 a-f. see text 2.5 min p.i. (a) A 6790 izm, (b) A 6360 ~m, (c) A 5340 Bm, (d) A 4230 Bin, (e) A 1270~m, (f) A6. Abbreviations: CA Commissura anterior; CO Chiasma opticum; CSDD Commissura supraoptica dorsalis, pars dorsalis (Ganser); F Columna fornicis; FLDG Fasciculus ]ongitudinalis dorsalis (Schfitz), pars tegmentalis; F P C P Fornix praccommissuralis, pars praeoptica et hypothalamica; S Subiculum; S M Stria medullaris thalami; S T Stria terminalis; S T C Stria terminalis, pars commissuralis; S T H Stria terminalis, pars hypothalamica; S T I Stria terminalis, pars infracommissuralis; S T P Stria terminalis, pars praecommissuralis; TCC Truncus corporis callosi ; T C M Tractus corticohypothalamicus medialis; cp Nucleus caudatus putamen; ]m Nucleus paraventricu]aris pars magnocellularis; /p Nucleus paraventricularis, pars parvocellularis; ha Nucleus anterior (hypothalami); hd Nucleus dorsomedialis (hypothalami) pars dorsalis; hdv Nucleus dorsomedialis (hypothalami) pars ventralis; hvm Nucleus ventromedialis (hypothalami); pore Nucleus praeopticus medialis; pvr Nucleus periventricularis rotundocellularis; pvs Nucleus periventricularis stellatocellularis; sl Nucleus septi lateralis; st Nucleus interstitialis striae terminalis; ts Nucleus triangularis septi Fig. 2 a~. (a) 2.5 min p.i. Caudal part of the third ventricle. An intensly fluorescing ependyma and which is well marked of the bordering brain tissue can be seen. Magn. 160:1. (b) 2.5 min p.i. Aquaeductus cerebri. D H is stored in the ependyma. A delivery of DH to the brain tissue cannot be recognized. Magn. 160: 1. (c) 5 rain p.i. Third ventricle. The DH-fluorescence is still limited on the ependyma. Magn. 160: 1. (d) 10 rain p.i. Rostral part of the third ventricle. The whole plexus contains DH. Magn. 63 : 1. (e) 15 rain p.i. Lateral ventricle. D H is present in the whole plexus, whereas the ependyma already appears depleted. Magn. 63 : 1. (f) 15 rain p.i. In the nucl. septi lateralis numerous cells fluoresce. Magn. 63:1.

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15 rain Post Injectionem D t t continues to be stored in the plexuses and in the ependymal lining of the lateral ventricles (Fig. 2e) and in t h a t of the fourth ventricle, while the epcndyma of the third ventricle and that of the aqueduct appear depleted. Numerous DI-Lstoring cells are observed to lie in the region of the fornix, from the anterior commissure *o the junction with the corpus eallosum, also beneath the anterior commissure in the area of the fornix praecommissuralis pars praeoptica et hypothalamica. Other areas of storage are the nucl. septi lateralis (Fig. 2f), nucl. ingerstitialis striae terminalis, both parts of the nucl. periventrieularis, the natl. anterior et dorsomedialis hypothalami. Only a few cells fluoresce in the nucl. praeoptieus medialis. Other fluorescing cells are found in all parts of the stria terminalis and stria medullaris thalami as well as in the tractus corgicohypothalamicus medialis. The amount of fluorescing cells in the molecular layer in all parts of the cerebellum is enhanced, while the Purkinje cell layer and the granular layer both display some degree of fluorescence.

20 rain Post In]ectionem D H has largely disappeared from the ependyma; only discrete cells fluoresce. In the plexus of the third ventricle and in stretches in the lateral ventricles the fluorescence intensity has diminished, while the plexus of the fourth ventricle including its protrusions into the recesses fluoresce strongly. There is noticeable change, too, in the situation regarding the periventricular brain areas. I n the region of the nucl. septi lateralis and praeoptieus mcdialis the number of DtIcontaining cells is distinctly less; in the region of the fornix praecommissuralis pars praeoptiea and pars hypoghalamica fluorescing structures are no longer to be seen. A decrease of fluorescence intensity can also be observed in the stria terminalis, where only few cells contain DI-I. I n the region of the nucl. interstitialis striae terminalis DH-congaining cells are no longer seen. By contrast, the number of fluorescing cells in the stria medullaris thalami and in the tractus corticohypothMamieus medialis has augmented. D H fluorescence also occurs in the nuel. paraventrieularis pars magnocellularis and parvocellularis and in some cells of the bundle of Schfitz. In the cerebellum the number of fluorescing cells in the molecular layer remains constant. D H fluorescence is now also observed in cellular processes of the granular layer. 30 min Post lnjectionem (cf. Fig. 3a-/) I n the ependyma, D H is now present only in single cells; however, it is still found in the plexuses (Fig. 4a), mainly in that of the fourth ventricle. An abundance of fluorescing perikarya are observable in the stria terminalis pars commissuralis et praecommissuralis and in some areas of the nucl. interstitialis striae terminMis and of the nucl. sepgi lateralis; these are limited to the vicinity of the anterior commissure and the basal parts of the lateral ventricle. A few cells are discernible in the dorsal parts of the nucl. praeopticus mcdialis (Fig. 4b). I n the nucl. anterior hypothMami some DtLstoring cells occur, their number augmenting caudally (Fig. 4c). Intensively fluorescing cells lie in the stria terminalis hypothalami and in the stria medullaris thalami. Compared to observations made

DANS-marked Histidine in R a t Brain

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Fig. 4 a-f. Magn. d and f 160:1, all others 63: 1. (a)30 min p.i. Lateral ventricle. D H is present in the plexus, the ependyma is vast depleted. (b) 30 min p.i. DH-storing cells are present in the nucleus praeopticus medialis. (c) 30 rain p.i. Fluorescing cells, situated near b y each other, are in the nucleus anterior hypothalami. (d) 40 min p.i. :Numerous cells of the stratum moleculare cerebelli have t a k e n up DH. (e) 60 rain p.i. The plexus chorioideus of the lateral ventricles still contains DH. (f) 180 min p.i. I n the stratum moleculare of the cerebellum fluoresce numerous cells and their processes

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at shorter intervals p.i. the number of these cells is noticeably enhanced. Now the tractus eorticohypothalamicus medialls, too, contains a maximal number of DH-containing cells. For the first time, abundant fluorescing perikaryaappear in the nucl. periventricularis. I n the plane at 6360 ~m a " fluorescing cell p a t h w a y " stretches from the third ventricle over the nuel. anterior hypothalami, the stria terminalis hypothalami, the tractus corticohypothalamicus medialis, the stria medullaris thalami, the stria terminalis and the nucl. periventricularis to the floor of the lateral ventricles. A small number of fluorescing cells are observed also in the nuel. paraventricularis pars magno- et parvoee]lu]aris, in the nucl. dorsomedialis hypothalami pars dorsalis et ventralis and in Schfitz's bundle. I n the molecular layer of the cerebellum a great amount of DH-filled cells are present extending to the border of the Purkinje layer.

40 rain Post Injectionem The situation with regard to ependyma and plexuses is more or less the same as at the previous observational stage. The regions of the stria terminalis and of the nucl. interstitialis striae terminalis, septi lateralis and praeopticus medialis, which at 30 min p.i. were strongly fluorescent, are now depleted of histidine. DItstoring cells lie in the stria medullaris thalami, in the nucl. anterior and dorsomedialls hypothalami, in the nucl. periventricularis, in the commissura supraoptica dorsalis (Ganser) and very sparsely in Schiitz's bundle, while the formerly fluorescing cells in the nucl. paraventricularis, in the tractus corticohypothalamicus media[is, in the stria terminalis and stria terminalis hypothalami are no longer demonstrable. The situation in the cerebellum is unchanged (Fig. 4d).

60 rain Post Injectionem D H is detectable only in the plexuses of the lateral ventricles (Fig. 4e) and in the plexus of the fourth ventricle. The ependymal lining of the third ventricle exhibits no D H content save for a small region in the caudal wall beneath the interthalamic connexus. I n the cerebrum, DH-containing cells are still distinguishable only in the nucl. dorsomedialis hypothalami and in the commissura supraoptica dorsalis. The cells of the stratum moleeularc of the cerebellum and a part of their processes still show strong DH-fluorescence. The midbrain is depleted of DH. 90, 120 and 180 rain Post In]ectionem At all of these intervals p.i., D H cannot be demonstrated in the lateral ventricles except for a moderate yellow fluorescence in the plexuses in the region of telencephalon and diencephalon. The plexus of the fourth ventricle continues to exhibit DH, although its distribution becomes more and more irregular. Solely in the molecular layer of the cerebellum can an abundance of ])H-storing cells still be seen (Fig. 4f). At 180 min p.i. the choroid plexus of the fourth ventricle, too, is virtually depleted of fluorescence. Throughout the whole period of observation the DIt-containing cells in the cerebellar molecular layer remain demonstrable, without a n y noticeable diminishment of fluorescence intensity.

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Discussion The present study is part of an experimental series aimed at elucidating intraccrebral transport pathways. It is a continuation of the studies of Stark and Franz (1972), Franz and Stark(1972) and Raprgger (1973). Since amino acids form fluorescing dansyl compounds the course of their penetration and deposition in tissue can be fluorescence-microscopically traced. Concomitantly it has been tried to clarify the question of whether the transit routes and topographical relationships pertaining to the amino acids tryptophan and phenylalanine pertain also to histidine, a precursor of histamine.

Ependyma Following intravcntrieular injection of DH the fluorescent amino acid is 2.5 rain later, already, found in the epcndymal cells of the third ventricle; the epcndymal lining of the lateral ventricles fluoresces strongly only after 5 rain. Resorption takes place in the ventral rather than in the dorsal parts. In the fourth ventricle an incipient epcndymal fluorescence is first demonstrable, too, after 5 min. Understandably, the ventricle where the injection is made shows the first signs of resorption. Commencement of cpendymal labelling in the ventral parts of the ventricle can be attributed to the pressure of injecting, but it is not clear why the labelling in the ventral parts of the lateral ventricles appears at the same time. Subsequent transport of the amino acid differs both regionally and with regard to time. The lower parts of the third ventricle, beneath the massa intermedia, store DH longer than elsewhere and labelled cellular processes, projecting into the lumen, can be discerned. After 90 rain p.i. DH is no longer demonstrable in the entire ependyma of the third ventricle. D H uptake by the ependyma of the fourth ventricle begins only after 5 min and releasing is concluded after 20 to 30 min p.i. Here there are no observable regional differences other than a rather late and only slight resorption in the canals of the recesses. The lining of the aqueduct participates in resorption of D H to only a slight degree ; although uptake begins immediately, the fluorescence never attains the intensity of that of the rest of the ependyma and depletion of DIt, which begins after 5 min, is concluded 40 min p.i. Jorns (1932) and Bering (1955) interpret intraventricular resorption as diffusional processes, the extent of which is dependent on the subependymal grey or white matter. Studies of Feldberg and Fleisehhaucr (1964), however, showed that with respect to the uptake of intraventricularly applied bromophenol blue by brain tissue, an active transport of the substance is involved which does not take place when the animal is dead. The results of the studies of Stark and Franz (1972) and Rapr~gcr (1973) point also in this direction. The authors assume an active catabolizing function of the epcndyma, since after resorption of tryptophan and phenylalanine by the ependymal ]ining no subcpendymal area of diffusion is demonstrable, despite pronounced differences of concentration as between liquor and cerebral tissue. This concept is further substantiated by the present investigation. Shortly after injection the DtI fluorescence is restricted to the ependyma and subsequently it is localized in nuclei and pathways that arc topographically precisely deter-

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minable. This makes the concept of transport by diffusion seem improbable. Rather, the "enzymic p a t t e r n " of the ependyma comes into consideration. Morphological differences in the ependymal lining of the ventricle system have been evidenced by several studies (fish: JcIorstmann, 1954; Braak, 1963; tortoise and cat : Fleisehhauer, 1957, 1960, 1964 ; homo sapiens: Opalski, 1934 ; Sehimriek, 1966). Schachenmayr (1967) subdivides the ependyma of the third ventricle of the rat into three zones based on histoehemieal differences, in which respect the varying ATPase content, in particular, points to active transport mechanisms. When a comparison is made between the behaviour of intraventrieularly injected Dtt, dansyl-tryptophan and dansyl-phenylManine, chronological and topical ependymal differences in respect of resorption and further transport of the three amino acids become noticeable; these are explicable only if they can be related to differences in substrate-speeifie enzyme equipment. Stark and Franz (1972) noted a uniform distribution in the ependyma and an even release to the brain, whereby it is obscure as to why there should be no uptake of tryptophan by the ependyma of the fourth ventricle. Raprgger (1973) opines t h a t the epend y m a exercises a storage function in respect of dansyl-phenylalanine and infers that a bioehemieally conditioned ependymal resorption pattern must exist for different substances. I-Iistidine, too, is irregularly taken up by the ependyma, and the duration of its deposition in the ependymal cells varies, which might be due to a differing carrier system. In contrast to the findings with regard to tryptophan, both histidine and phenylalanine are taken up by the ependyma of the fourth ventricle. The regional differences in uptake of histidine in the lower part of the third ventricle, beneath the interthalamie eonnexus, are especially conspicuous. Here, resorption b y the apical ependymal sections is of longer duration and the DI-I fluorescence is more intensive than elsewhere. This finding and the fact that in the midbrain aqueduct scarcely any DI-I is taken up b y the ependymal cells, and then over a very brief period, suggest that this is related to the existence of the basement-membrane labyrinths. I n the rat these are more profuse in those parts of the ventricular wall beneath the massa intermedia and virtually absent from the aqueduct (Desaga, 1972; Booz and Desaga, 1973). Choroid Plexus Since all plexuses, even in areas far removed from the site of injection, are seen to have taken up Diet already 2.5 min p.i., an extremely rapid distribution b y the cerebrospinal fluid and an active resorption by the plexus epithelia are to be inferred. Similar observations were made b y Rapr/iger (1973) after intraventricular application of dansyl-phenylManine and by Coben, Coltier, Beaty and Becket (1971), who showed that neutral, acidic and basic amino acids are taken up in vitro by the rabbit ehoroid plexus. The authors hold the view that the ehoroid plexus possesses carrier systems for amino acids and can accumulate and store them, thus playing a major part in regulating the formation and composition of the eerebrospinal fluid. Furthermore, the capacities of secretion, dialysis, ultrafiltration and in addition a combination of filtration and reabsorptiou are all attributed to the epithelia of the ehoroid plexus (Lumsden, 1958). Hence, 8

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transport in two directions is considered to be possible in the cells of the plexus from the blood to the CSF and vice versa. Such two-way processes in the organism are knownly dependent on certain enzymes. Shanta and Manocha (1968) established strong activities for enzymes of the citric acid (T.C.A.) cycle of glycolysis in the plexus epithelia of the rat and a high ATPase activity in the plexus stroma. In contrast to the finding regarding the ependyma, distribution of the oxidative and hydrolytic enzymes in the choroid plexus is equable. This favours the concept that there are, in the cells of the plexus, more complex mechanisms for a twoway transport (Paul, 1968). Voth (1966) saw in the high ATPase activity demonstrable in the plexus, and in the related energy-providing reactions, the preconditions for carrier systems and secretory processes. The observed earlier fluorescence of the extroversions of the choroid plexus of the lateral recessus in comparison with the more centrally lying parts of the fourth ventricle plexus - t~apr/iger (1973) reports a similar finding during tests with dansyl-phenylalanine - could be connected with a jet effect of the lateral apertures causing accelerated flow of CSF (cf. Leonhardt, 1969). When comparing the results of the present study with the findings of Stark and Franz (1972) and Rapr/~ger (1973), considerable agreement is found regarding the behaviour of dansyl-histidine and that of dansyl-phenylalanine. Both substances are rapidly taken up by the entire plexus epithelium and released from different sections at different times, DIt remaining longer in the choroidplexuses of the lateral and fourth ventricles. In contrast, dansyl-tryptophan is not absorbed at all by the choroid plexus of the fourth ventricle. All three amino acids remain stored much longer in the epithelium of the plexus than in the ependymal lining, which points to a storage function of the plexus. Brain

Clearly, uptake of a substance by the ependyma sets the signals for its subsequent liberation. On comparing the behaviour of the three amino acids that provide the most important biogenic amines, tryptophan (Stark and Franz, 1972), phenylalanine (Rapr/iger, 1973) and histidine after their uptake by the ependyma, differences become apparent not only with respect to duration of deposition in the ependyma but also with regard to the temporally phased releasing and affinity to certain nuclear regions. D H is detected early in nuclear areas and pathways adjacent to the ventricles, from whence it disappears after 90 rain, presumably largely catabolized, with exception of the cerebellum. Application of histidine does not reveal an enhancement of fluorescence intensity up to a "maximal fluorescence"as observed by Rapr/~ger (1973) for phenylalanine. While the transport pathways for tryptophan and phenylalanine are clearly directed towards the serotoninergic and monaminergic nuclear areas and there occurs a delineable accumulation in these areas, histidine (under the same experimental conditions) stays predominantly in regions near to the ventricles. Particularly in the midbrain, dansyl-phenylalanine and dansyl-tryptophan can be demonstrated in numerous nuclei and also in cellular processes, whereas D H appaers tobe mainly restricted to Schiitz's bundle. This is probably due to the limited resorption by the ependyma of the aqueduct. In the cerebellum, in the cells of the molecular layer, DH, tryptophan and phenylalanine occur to equal extent. D t t

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probably reaches this region via the ependyma, the choroid plexus and the extracerebral eerebrospinal fluid, but this cannot be the case with t r y p t o p h a n since it is not taken up b y the ependyma of the fourth ventricle (Stark and Franz, 1972). D H shows no affinity for the Purkinje cell layer, these cells taking up exclusively phenylalanine. I t has not been satisfactorily elucidated how the intraventricularly applied amino acids reach certain nuclei and pathways. Stark and Franz (1972) surmise that the slender glial cell processes between ependyma and nerve cells play a role, but in the preparations of the present study they do not show up. Leonhardt (1972), Desaga (1972) and Boozet al. (1972) regard the subependymal basementmembrane labyrinths as a structure of significance for the extraeellular transport of substances. But evidence is lacking as to whether and in what way a selection of substances and transport takes place in the labyrinths. This problem is of relevance since the amino acids used for the investigation, though possessing the same sized molecule, " t r a v e l " with different speeds and to different distances. Further, it can be conjectured whether or not cytopempsis is implicated; Brightman (1965) demonstrated this in the case of ferritin, which after resorption takes the route from the ependyma over the interependymal interstices (where the basement-membrane labyrinths occur) to pcrivascular spaces. (With regard to selective behaviour of the ependyma in respect of other substances, compare Leonhardt, 1974) Several papers (Talor, Gfeller and Snyder, 1972; White, 1959; Snydcr, Glowinski and Axelrod, 1966; Battistin, Grynbaum and Lajtha, 1970) report on uptake and localization of histidine and/or histamine in the brains of various mammals. For comparison, the biochemical studies of Taylor, Gfeller and Snyder (1972) should be consulted. These authors investigated the regional distribution of histamine and histidine in the brain of the rhesus monkey. They found dissimilar conditions to pertain to the two substances. At localities with high histamine content only slight amounts of histidine were present; in regions with little histamine, histidine was found in higher concentrations. By comparing topographical data in respect of sites with the highest concentration of histidine (median eminence, habenula, amygdaloid body and corpus eallosum) and relating these to our findings, it becomes noticeable that dansyl-histidine preferentially accumulates not at these sites but rather in nuclei with high histamine content and correspondingly slight amounts of histidine, foremostly in the nuel. anteriores thalami, periventricu]ares and supraoptici. This suggests a slow decarboxylation of the DH, although the highest activity of histidine decarboxylase is actually found in the hypothalamus (White, 1959, 1960). I n pig brains, histamine occurs in the areas which possess the highest decarboxylase activity (Friede, 1966). The enzyme in question appears to be that which decarboxylizes 5-hydroxytryptophan and dopa (Werle, Schaver and BShler, 1963). References

Battistin, L., Grynbaum, A. : The uptake of various amino acids by the mouse brain in vivo. Brain Res. 29, 85-99 (1971) Booz, K.-H., Desaga, U. : Sub- und interependymale Basalmembranlabyrinthe am Ventrikelsystem und Zentralkanal der weil3en l~atte. Verh. anat. Ges. 67, 607-612 (1973)

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Braak, H. : Das Ependym der Himventrikel von Chimaera monstrosa mit besonderer Berfieksichtigung des Organum vasculosum praeopticum. Z. Zellforsch. 60, 582-608 (1963) Brightman, M. W. : The distribution within the brain of ferritin injected into eerebrospinal fluid compartments. J. Cell Biol. 26, 99-123 (1965) Carlini, E. A., Green, I. P. : The subcellular distribution of histamine, slow reacting substance and 5-hydroxytryptamine in the brain of the rat. Brit. J. Pharmaeol. 20, 264-277 (1963) Coben, L. A., Coltier, E., Beaty, C., Becker, B. : Transport of amino acids by rabbit choroid plexus in vitro. Brain Res. 30, 67-82 (1971) Desaga, U. : Form und Verteilung subependymaler Basalmembranlabyrinthe am Ventrikelsystem der Ratte. Z. Zellforseh. 1112, 553-562 (1972) Draskoei, M., Feldberg, W., Fleischhauer, K., Haranath, P. S. R. : Absorption of histamine into the blood stream on perfusion to the cerebral ventricles and its uptake by brain tissue. J. Physiol. (Lond.) 150, 50-C6 (1960) Drop, I. I. : Mast cells in the central nervous system of several rodents. Anat. Ree. 171, 227-238 (1972) Feldberg, W., Fleischhauer, K. : Penetration of bromphenol blue from the perfused cerebral ventricles into the brain tissue. J. Physiol. (Lond.) 150, 451462 (1960) Fleischhauer, K.: Untersuchungen am Ependym des Zwischen- und Mittelhirns der Landschildkr5te (Testudo graeca). Z. Zellforseh. 46, 729-767 (1957) Fleischhauer, K. : Fluoreszenzmikroskopische Untersuehungen an den Wandungen der Himventrikel der Ii:atze (Seitenventrikel, III. Ventrikel). Z. Zellforsch. 51, 467496 (1960) Franz, H., Stark, M. : Fluoreszenzmikroskopische Untersuchungen fiber die l~esorption und Verteilung yon Tetracyclin im Rattenhirn nach intraventrikul~rer Injektion. Z. Zellforsch. 126,565-579 (1972) Friede, 1%. L. : Topographic brain chemistry. New York-London: Academic Press 1966 Garratini, S., Valzelli, L.: Serotonin. Amsterdam-London-NewYork: Elsevier Publishing Co. 1965 Green, J. P.: Handbook of neurochemistry. Vol. 4, p. 221. London-New York: Plenum press 1970 Horstmann, E. : Die Faserglia des Selaehiergehirns. Z. Zellforsch. 119, 588-617 (1954) Jackson, A., Rose, P. : Observations on the histamine content of the cerebrospinal fluid in man. J. Lab. clin. Med. 34, 250 (1949) Jorns, G.: Experimentelle Untersuchungen fiber die l~esorptionsvorg/~nge in den Hirnkammern. Langenbecks Arch. klin. Chir. 171, 326-360 (1932) Kataoka, K., de l~obertis: Histamine in ;solated small nerve endings and synaptic vesicles of the rat brain. J. Pharmacol. exp. Ther. 1~6, 114-125 (1967) KSnig, J. F. R., Klippel, R. A. : The rat brain. A stereotaxis atlas of the forebrain and lower parts of the brain stem. Baltimore: Williams & Wilkins Co. 1963 Kuhar, M. J., Taylor, K. M., Snyder, S. H.: The subcellular localization of histamine and histamine methyl-transferase in rat brain. J. Neuroehem. 18, 1515-1527 (1971) Leonhardt, H. : Das Ependym. In: Zirkumventrikul~re Organe und Liquor. Bericht fiber das Symposium in SchloB Reinhardsbrunn vom 13. bis 16. Mai 1968. S. 177-186, Hrsg. G. Sterba. Jena: VEB Fischer 1969 Leonhardt, H. : Subependymale Basalmembranlabyrinthe im Hinterhorn des Seitenventrikels des Kaninchengehirns. Z. Zellforseh. 10& 595-604 (1970) Leonhardt, H. : Ober topographisehe Verteilung der subependymalen Basalmembranlabyrinthe im Ventrikelsystem des Kaninchengehirns. Z. Zellforsch. 127, 392-406 (1972) Leonhardt, H.: Ependymstrukturen im Dienst des Stofftransportes zwischen Ventrikelliquor und Hirnsubstanz. In: Ependyma and neurohormonal regulation (A. Nitro ed.), pp. 29-75, Bratislava: Veda, Pupl. House of the Slovak Academy of Sciences 1974 Lumsden, C. E. : Observations on the choroid plexus maintained as an organ in tissue culture. In: The eerebrospinal fluid. Ciba Foundation Symposium, pp. 97-123. London: Churchill 1958 Michaelson, I. A., Coffmann, P. Z. : The subeellular localization of histamine in guinea pig brain. A re-evaluation. Biochem. Pharmacol. 16, 2085-2090 (1967) Oksche, A. : Die Bedeutung des Ependyms ffir den Stoffaustausch zwischen Liquor und Gehim. Anat. Ariz. Erg. 1011, 162-171 (1956)

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OpMski, A.: ?Jber lokMe Unterschiede im Bau der VentrikelwEnde beim Menschen. Z. ges. Neurol. Psychiat. 149, 221-254 (1934) Paul, E. : Histochemisehe Studien an den Plexus chorioidei, an der Paraphyse und am Ependym yon Rana temporaria L. Z. Zellforsch. 91, 519-546 (1968) Rapr~ger, E. J. : Fluoreszenzmikroskopische Untersuchung fiber das VerhMten yon Dansmarkiertem Phenylalanin im Gehirn der Wistarratte nach intraventrikul/~rer Applikation. Z. Zellforseh. 141, 123-144 (1973) Riley, ~I. F. : Functional significance of histamine and heparine in tissue mast cells. Ann. I~.Y. Acad. Sci. 103, 151-163 (1963) Schaehenmayr, W. : tiber die Chemodifferenzierung des Ventrikelependyms der Ratte. Anat. Anz. 120, 361-368 (1967) Schimrick, K. : ~-ber die Wandstruktur der Seitenventrikel und des III. Ventrikels beim Mensehen. Z. Zellforseh. 70, 1-20 (1966) Schwartz, J. C., Lampart, C., Rose, C. : Histamine formation in rat brain in vivo: effects of histidine loads J. Neuroehem. 19, 801-810 (1971) Seiler: Stoffwechsel im ZNS. Stuttgart: Georg Thieme 1966 Shanta, T. R., Manoeha, S. L. : Enzyme histoehemistry of the choroid plexus in rat and squirrel monkey. Histochemie 14, 149-160 (1968) Snyder, S. H., Glowinski, J., Axelrod, A.: The physiologic disposition of h3-histamine in the rat brain. J. Pharmaco]. exp. Ther. 15~, 8-14 (1966) Snyder, S. H., Taylor, K. M. : Histamine in the brain: A neurotransmitter In: S. H. Snyder (ed.): perspectives in neuropharmacology. A tribute to Julius Axelrod. New York: Oxford Univ. Press 1975 Stark, M., Franz, H.: Resorption und Verteilung yon Dansmarkiertem Tryptophan im Rattenhirn nach intraventrikuliirer Injektion. Z. Zellforsch. 126, 536-564 (1972) Taylor, K. M., Gfeller, E., Snyder, S. H. : Regional localization of histamine and histidine in the brain of the rhesus monkey. Brain Res. 41, 171-179 (1972) Taylor, K. M., Snyder, S. H. : Isotopic microassay of histamine, histidinc, histidine decarboxylas~ and histamine methyltransferase in brain tissue. J. Neurochem. 19, 1343-1358 (1972) Vollrath, L. : Ober Bau und Funktion yon Basalmembranen. Dtseh. med. Wschr. 9~, 360-365 (1968) Voth, D. : Untersuchungen fiber die ATPase-Aktivitat im Zytoplasma der Epithelzellen vom Plexus chorioideus des Rindes. Enzym. biol. elin. 7, 203-214 (1966) Werle, E., Amann, R. : Uber die Bindung des Histamins an Heparin. Naturwissenschaften 21, 583 (1955) Werle, E., Schauer, A., Bfihler, H. W. : Klassifizierung histidindecarboxylierender Gewebsund Bakterienenzyme. Arch. int. Pharmacodyn. 145, 198-206 (1963) Withe, T. : Formation and catabolism of histamine in cat brain in vivo. J. Physiol. (Lond.) 152, 299-308 (1960) Zemarm, W., Innes, J. R. 1Y[.: Craigie's neuroanatomy of the rat. New York-London: Academic Press 1963 Prof. Dr. K. H. Booz Fachrichtung Anatomie im Fachbereich Theoretisehe Medizin der Universitiit des Saarlandes D-6650 Homburg/Saar Federal Republic of Germany