Enzymatic Organization of the Subcommissural Organ

Enzymatic Organization of the SubcommissuralOrgan WOLFGANG KOHL

With 9 Figures and I Table

GUSTAV FISCHER VERLAG' STUTTGART· 1975

Dr. WOLFGANG KOHL Institut fUr Histologie und experimentelle Biologie Universitat Miinchen Newadress: Anatomisches Institut Universitat Mainz D-6soo Mainz, Saarstrai3e 19/21

Herrn Professor Dr. med. RUDOLF BACHMANN in herzlicher Dankbarkeit zu seinem 65. Geburtstag gewidmet.

ISBN 3-437-I04IO-I © Gustav Fischer Verlag· Stuttgart· I975 . Aile Rechte vorbehalten Gesamtherstel1ung: Druckerei Mayr, Miesbach

Printed in Germany

Contents Introduction

Discussion

Materials and Methods

I

Results ..

5

1.

2.

Morphological Findings ..

5

a) Structural Organization of the SCO

5

b) Secretory Substance

5

..

Enzymatic Organization of the Ependymal Part of the SCO ..

7

a) Control Reactions .

7

b) Enzymes of Energy-supplying Metabolism ..

8

c) Marker Enzymes of Different Cell Organelles .. 3. Enzymatic Organization of the Hypendymal Part of the SCo .

1.

Methodical Problems

2.

Common Characteristics of the Enzymatic Organization of the SCO Activity of the SCO .

4. Comments Concerning a Species Different Secretory Activity of the SCO Structure-bound Enzyme Activities 6. Remarks on the Hypendymal Part of the SCo .. 7. Concluding Remarks. Summary References

20

22

3. Metabolic Differentiation and Secretory

5. Possible Significance of Some

16

22

Subject Index.

31

Enzymatic Organization of the Sub commissural Organ .

I

Introduction The structural organization of the subcommissural organ (SeO) which is located in the posterior part of the roof of the third ventricle has been described in detail by a number of investigators (for a review see HERRLINGER 1970). Their findings reveal common characteristics of the seo of all species studied so far: the ependymal part consists of a pseudostratified or stratified layer of specialized columnar ependymal cells which cover the ventricular surface of the posterior commissure; they are engaged in the synthesis, transport, and discharge of glycoproteins which released into the third ventricle from the so-called "Reissner's fibre". The small layer of the hypendymal part is situated between the ependymal part and the myelinated fibres of the posterior commissure; this part is a heterogenous composite of secretory hypendymal cells and glial cells and their respective cell processes. A basal secretion into the blood vessels is contested for both of the secretory cell types. The physiological significance of the seo is still not known; the hypotheses about its function are reviewed in detail by STERBA (1972). Only a few investigations have been done so far concerning the enzymatic organization of the seo. However, enzymological analyses yield data which illustrate the metabolic differentiation. The kinds of differentiation of the energysupplying metabolism are the expression of an adjustment to the functional characteristics of tissues, such as the quality and temporal pattern of energy expenditure (see PETTE 1971, lit.). Thus, enzymological data are essential constituents of organ characterization. Biochemical analyses of the seo are difficult because of the small size of this organ and because of the heterogenous composition of the surrounding tissue. Therefore, histochemical and the more difficult microchemical methods are favoured for this purpose. Investigating histochemically glycogenolytic, glycolytic, and oxidative enzymes in the seo of Lampetra planeri (NAUMANN 1968) and Rana temporaria L. (DIEDEREN 1970) the authors con-

eluded an energy supply depending mainly on anaerobic glycolysis. In mammals only a few corresponding data are available concerning mostly single enzymes of the glycolytic and/or oxidative pathway and/or some structure-bound enzymes; these data will be reviewed in the discussion of our results. The present study on the seo of rodents was undertaken in order to investigate the enzymatic organization first of all of the energysupplying metabolism which is the base of organ function; furthermore to investigate the possible significance of some structure-bound enzymes which might be related to the secretory process. Finally species specific variations will be discussed with regard to the secretory activity of the seo which might be different in the four species examined. So our findings may be the base of further experimental studies on this organ and its function. Preliminary results were previously published in abstract form (KOHL 1973).

Materials and Methods We investigated the SCO of 89 male guinea pigs (Pirbright White, 342-930 gm, average weight 501 gm), 95 male rats (Sprague Dawley, 162-394 gm, a. wt. 264 gm), 125 male golden hamsters (not classified, 38-127 gm, a. wt. 66 gm), and 125 male mice (NMRI, 22-41 gm, a. wt. 31,4 gm). All animals were purchased from Firma Baumler, W olfratshausenjObb., and were allowed to be fed upon a standard Altromin® diet and tap water ad libitum for at least one week before killing; they were kept at room temperatnre and natnrallight-dark-rhythm. Most of them were killed between 7.00 and 9.00 a. m. by cervical dissection in Nembutal® anesthesia (50 mgjkg body weight). Some animals were killed at 23.00 and showed no obvious difference of the activity of selected enzymes. There were also no definite seasonal changes. Within 1-2 min a small block of tissue containing the SCO was a) fixed by immersion in formal-calcium (24 hrs), formol-cetylpyridine (48 hrs), Bouin's fluid (24 hrs), Bouin's fluid as modified by BOCK (1967) (7 days), Carnoy's fluid (24 hrs), or tricloracetic acid-ethanol (24 hrs). After embedding in Paraplast® 3-5[J.m sections were sliced for the following reactions: P AS-

2 . WOLFGANG KOHL reaction with and without diastase-digestion (see LILLIE 1965), colloidal iron-method according to Hale as modified by GRAUMANN and CLAUSS (19S8), alcian blue 8 GX (at pH 2,3) (see PEARSE 1968), DNFB-reaction as modified by TRANZER and PEARSE (1964), SH- and SS-groups according to BARRNETT and SELIGMAN (19S2, "DDD-reaction") (see also PEARSE 1968), gallocyanin-chromalum staining for nucleic acids (with and without ribonuclease digestion) according to EINARSON (I9S1) (see also PEARSE 1968). Secretory substance of the SCO was stained by aldehyde-thionin according to PAGET (19S9), chrome alum-hematoxylin-phloxin according to GOMORI (1941) (see also PEARSE 1968), pseudoisocyanin according to STERBA (1964), and chrome alum-gallocyanin according to BOCK (1966). b) For enzyme histochemistry a small tissue block was either fixed in 4% formaldehyde (freshly prepared from paraformaldehyde) in 0,1 M cacodylate buffer, pH = 7,4, containing 0,22 M sucrose (24 hrs), followed by a thorough rinsing in cacodylatesucrose (applied for lysosomal enzymes only), or immediately frozen by CO 2 ; frontal or sagittal sections of 10 [Lm thickness were sliced at _20° C in a cryostat (Dittes-Duspiva model, W. Dittes, Heidelberg); the sections were finger-thawed on clean dry slides and stored in Coplin jars at _20° C, sometimes till the next morning. Just before use they were transferred to room temperature avoiding condensation of water. Within a few minutes they were incubated without fixation or - for hydrolytic enzymes only fixed in 2,S% glutaraldehyde in 0,1 M cacodylate buffer, pH = 7,4, for 30-60 sec; then the sections were rinsed in the corresponding buffer for 2-4 hrs. For each enzyme investigated, tissue blocks of at least two different and frequently of all four species were frozen, stored, and sliced and the sections stored, incubated, and photographed in the same manner.

Reagents: The chemicals used were obtained from the following suppliers: Ladd Research Ind., Burlington, Vt., USA: glutaraldehyde, 70%; Calbiochem, Paesel KG, Frankfurt/M., Germany: naphtol-AS-BI-Nacetyl-~-D-glucosaminide; Fluka, Buchs, Switzerland: p-chloromercuribenzoic acid (p-CMB), NNdimethyl formamide (DMF), NN-dimethyl sulfoxide (DMSO), maleic acid, iso-OMP A, thiamine pyrophosphate tetrahydrate; Serva, Heidelberg, Germany: 8-amino-l, 2, 3, 4-tetrahydrochinoline, acetyl- and butyrylthiocholine jodide, DL-IX-glycerophosphate disodium salt, glucose, glycylglycine, L-lactate sodium salt, naphtol-AS-D-acetate, naphtol-AS-BI~-D-glucuronide, urea, tryptamin HCl; Sigma Chemical Co., St. Louis, Mo., USA: L-glutamic acid sodium salt, ~-glycerophosphate dis odium salt, DLisocitric acid trisodium salt, L-malic acid sodium

salt, naphtol-AS-TR-phosphate sodium salt, nitro blue tetrazolium chloride (NBT), p-nitrocatechol sulfate dipotassium salt, phenazine methosulfate (PMS) , tetranitro blue tetrazolium chloride; Boehringer GmbH, Mannheim, Germany: adenosine-s'diphosphate disodium salt (ADP), adenosine-s'monophosphate disodium salt (AMP), adenosine-s'triphosphate disodium salt (ATP), catalase, cytochrome c, fructose-I,6-diphosphate CHA-salt (fruI,6-P 2), fructose-6-phosphate disodium salt (fru-6-P), gluconate-6-phosphate trisodium salt, glucose-I,6diphosphate CHA-salt (glu-I,6-P 2 ), glucose-I-phosphate disodium salt (glu-I-P), glucose-6-phosphate disodium salt (glu-6-P), D-glyceraldehyde-3-phosphate diethylacetal CHA-salt, glycogen, guanosineS'-diphosphate trilithium salt (GDP), DL-3-hydroxybutyrate sodium salt, inosine-s'-diphosphate trisodium salt (IDP), ~-nicotinamide-adenine dinucleotide grade I (NAD), ~-NADH, nicotinamide-adenine dinucleotide phosphate (NADP), NADPH, pyruvate monosodium salt, succinate disodium salt, uridine-s' -diphosphate dipotassium salt (UDP), uridine-s' -diphosphoglucose disodium salt (UDPglucose), and all the auxiliary enzymes which were freed from (NH4hS04 by dialysis against glycylglycine-buffer, So mM, containing EDTA Na2, S mM, pH 7,6, for several hours at 4°C. Polyvinyl alcohol (PV A) was kindly supplied by WackerChemie GmbH, Miinchen, Germany (Polyviol B OS/ 140). Alcian blue 8 GX was obtained from Imperial Chemical Ind. Ltd., Great Britain. Pseudoisocyanin (N,N' -diethyl 6,6' -dichlorpseudoisocyanin) was the kind gift of Prof. Dr. M. Arnold, Tiibingell, Germany. All other Chemicals were products of E. Merck, Darmstadt, Germany, and were of the highest purity available. All solutions were prepared in double-distilled water. Final concentrations were calculated from the producer's declaration on specific content.

Incubation procedures: For the demonstration of dehydrogenases and dehydrogenase-coupled enzymes the principles introduced by PETTE and BRANDAU (1966) and by SIGEL and PETTE (1969) were applied with some modifications: PVA (see ALTMANN and CHAYEN 1965; ALTMAN 1971) was used instead of agarose-gel (see also JACOBSEN 1969). PVA was used as a 3S% (w/v) stock solution, maintained at 60-70°C. After preliminary trials using the soluble enzymes LDH and G6P-DH as models, the following final concentrations were found to be optimal for the demonstration of dehydrogenases and -coupled enzymes: substrate, coenzyme, and auxiliary enzymes as given below; PMS, 0,3 mM; NaCN, S mM; TNBT, 3,0 mM, dissolved in DMF, 3% (v/v); EDTA Na 2 , 5 mM; glycylglycine buffer, 50 mM; PVA, 18 or preferentially 21% (w/v); pH=7,6. The media

Enzymatic Organization of the Subcommissural Organ . 3 were prepared just before use and then dropped onto the sections. Incubation was carried out at 37°C in the dark using a moist chamber perfused with moistened N 2 • The sections were then rinsed in hand-warm tap water and A. dest., transferred to 10% formol-calcium (IS min or longer) and finally washed in tap water. All the cryostat sections were embedded in glycerol-gelatine. If not noted otherwise, controls were done by either omitting substrate(s) and/or coenzyme and/or PMS and/or cyanide. In the case of hydrolytic enzymes also heat inactivation was applied. Controls were negative if not mentioned.

Composition of the Standard Reaction Media: Formazan substantivity: IS min incubation in a medium lacking substrate and coenzyme which has been reduced by an alkaline ascorbate solution (see below). Tetrazolium substantivity: 30 min incubation without substrate and coenzyme, followed by an alkaline ascorbate reduction (PEARSE and HESS 1961). "Nothing-dehydrogenase": no substrate and/or coenzyme; 10-30 min. Hexokinase (HK; EC 2.7.1.1): glucose,s mM; ATP, 12 mM; Na 2HP0 4 , 5 mM; MgCI 2 , IS mM; G6P-DH, 35 D/ml; NADP, 1,5 mM; 10 min. Phosphoglucomutase (PGluM; EC 2.7.5.1): glu-I-P, 10 mM; gIU-I,6-P 2 , 0,1 mM; MgCI 2 , 1OmM;G6P-DH,35U/ml;NADP, I,5 mM ; 5 min. Uridine-5'-diphosphoglucose pyrophosphorylase (UDPG pyrophosphorylase; EC 2.7.7.9): UDPglucose,s mM; Na 4P 2 0 7 , 10 mM; MgCI 2 , 10 mM; PGluM, 20 U/ml; gIU-I,6-P 2 , 0,1 mM; G6P-DH, 35 U/ml; NADP, 1,5 mM; 10 min; (KOHL, unpublished). Uridine-5' -diphospho-glucose dehydrogenase (UDPG-DH; EC 1.1.1.22): UDP-glucose, 5-30mM; NAD, 1,5 mM; 10-120 min. Glycogen synthetase (EC 2.4.1.II): as modified by SASSE (1966); sections prefixed in abs. ethanol for 10 min; incubation time 180 min; control by p-CMB, I mM. Glycogen: sections prefixed in abs. ethanol for 10 min; P ASreaction; control by diastase-digestion. Glycogen phosphorylase (EC 2,4.1. I): according to TAKEUCHI and KURIAKI (1955); HgCI 2 , 0,1 mM, included; 120 min. Glucose-6-phosphate dehydrogenase (G6PDH; EC 1.1.1.49): glu-6-P, 5 mM; MgCl z or MnCI 2 , 0 and 5 mM; NADP, 1,5 mM; 20-30 min; deoxy glucose-6-P and galactose-6-P were also used as substrates but the enzyme showed only slight activity with these substances. 6-Phosphogluconate dehydrogenase (6-PG-DH; EC 1.1.1.44): gluconate-6phosphate, 5 mM; NADP, 1,5 mM; 20-30 min. Phosphoglucose isomerase (PGI; EC 5.3.1.9): fru-6-P, I and 10 mM; G6P-DH, 35 U/ml; NADP, 1,5 mM; 5-IO min. Fructose-6-phosphate kinase (F6PK; EC 2.7.1.II): fru-6-P, 10 mM; ATP, 2 mM; MgCI 2 , 8 mM; aldolase, 9 U/ml; triosephosphate isomerase, 50 U/ml; GAP-DH, 20 U/ml; Na 2HAs0 4 , 6 mM; NAD, 1,5 mM; pH 8,0; 10 min. Fructose-l,6-diphos-

phatase (FDPase; EC 3.1.3.II): fru-I,6-P 2 , 0,2 and 2,0 mM; MgCI 2 , 10 mM; PGI, 35 U/ml; G6P-DH, 35 U/ml; NADP, 1,5 mM; inhibitor: AMP, 2 and 20mM; 30min; (KOHL, unpublished). Fructose-l,6diphosphate aldolase (ALD; EC 4.1.2.13): fru-I,6-P 2 , 20 mM; triose-phosphate isomerase, 20 U /ml; GAPDH, 50 U/ml; Na 2HAs0 4 , 6 mM; NAD, 1,5 mM; IO min. Glyceraldehyde-3-phosphate dehydrogenase (GAP-DH; EC 1.2.1.12): D-glyceraldehyde-3-phosphate, 1-5 mM (prepared from the diethylacetal); Na 2 HAs0 4 , 6 mM; NAD, 1,5 mM; medium without NaCN; 10-20 min. Lactate dehydrogenase (LDH; EC 1.1.1.27): L-lactate, 100 mM; NAD, 1,5 mM; 10-20 min. LDH subunits: inhibition of M type subunits by addition of urea, 3,5 M, and of H type subunits by pyruvate,s mM (d., McMILLAN 1967; JACOBSEN 1969). Isocitrate dehydrogenase, NAD-dependent (NAD-ICDH; EC 1.1.1.41): DL-isocitrate, 1OomM; ADP, 2mM; MgS0 4 , 8mM; NAD, 1,5 mM; 10-15 min. Isocitrate dehydrogenase, NADPdependent (NADP-ICDH; EC 1.1.1.42): DL-isocitrate, 20 mM; MgS0 4 , 8 mM; NAPD, 1,5 mM; IS min. Succinate dehydrogenase (SDH; EC 1.3.99.1): succinate, 100 mM; 30 min. Malate dehydrogenase, NAD-dependent (NAD-MDH; EC 1.1.1.37): Lmalate, 100 mM; NAD, 1,5 mM; 10 min; preextraction fluid: saccharose, 300 mM; triethanol amine HCl, 10 mM; EDTA Na 2 , 2 mM; pH 7,4; IS min. Malate dehydrogenase, NADP-dependent (NADP-MDH; EC 1.1.1.40): L-malate, 50 mM; MgS0 4 , 8 mM; NADP, 1,5 mM; 30 min. Cytochrome oxidase (CYO; EC 1.9.3.1): according to BURSTONE (1959); sections prefIxed in cold acetone, 5 min; acceptor: 8-amino-I,2,3,4-tetrahydrochinolin; cytochrome c, I mg/ml; 45 min; inhibition by NaCN, 5 mM. 3-Hydroxybutyrate dehydroJ?enase (3HBDH; EC 1. 1. 1.30): DL-3-hydroxybutyrate, 200 mM; NAD, 1,5 mM; IS min. L-Glycerol-3phosphate dehydrogenase (GP-DH; EC 1.1.99.5): DLIX-glycerol-3-phosphate, 200 mM; 20 min. Glycerol3-phosphate dehydrogenase, NAD-dependent (NADGP-DH; EC 1.I.I.8): DL-IX-glycerol-3-phosphate, 200 mM; NAD, 1,5 mM; 20 min. Glutamate dehydrogenase (GIDH; EC 1.4.1.3): L-glutamate, IoomM; ADP, 2 mM; NAD, 1,5 mM; 20 min. Adenosine-5'triphosphatase, Mg++-dependent (ATPase; EC 3.6.1.3): according to WACHSTEIN and MEISEL (1957) but modified: glutaraldehyde-fixed sections; ATP, I mM; MgCI 2 , 10 mM; Pb(N0 3 )2, 2,4 mM; trismaleate-buffer, 80 mM; pH = 7,2; 10-30 min. Monoamine oxidase (MAO; EC 1.4.3,4): according to GLENNER et al. (1957); 30-120 min. NAD(P)HTetrazolium-oxidoreductase (NAD(P)H dehydrogenase; not classified): NAD(P)H 2 , 0,12 and 2,0 mM; 8-20 min. Glucose-6-phosphatase (G6Pase; EC 3.1.3.9): modifiedWACHSTEIN-MEISELmethod(I957): form- and glutaraldehyde-fixed sections; glu-6-P, I and 10 mM; Pb(N0 3)2, 2 mM; cacodylate-buffer,

4 . WOLFGANG KOHL SO mM; pH = 6,6; 90-180 min. The medium was filtered just before use through a Selecta-filter No. 602h to remove precipitate which has been developed during the preparation - perhaps by P-ions contaminating the commercial glu-6-P. The filtered medium remains clearly for several hours. Equimolar concentrations of glu-I-P and ~-glycero-P served as control. Acetyl- and Cholinesterase (AChE and ChE; EC 3.1.1.7 and 3.1.1.8): block-fixed tissue and glutaraldehyde-fixed sections; method according to KARNOVSKyand ROOTS (1964); I-S hrs; both enzymes are inhibited by physostigmine, 0,1 mM; ChE is inhibited by iso-OMPA, 0,1 mM. Nucleoside diphosphatase (NDPase; EC 3.6.1.6): glutaraldehyde-fixed sections; method according to NOVIKOFF and GOLD FISCHER (1961) but modified: !DP, GDP, or UDP, 4 mM; Mg(N0 3)2, 10 mM; Pb(N0 3)2, 2,4 mM; tris-maleate-buffer, 80 mM; pH= 8,0 and 7,2; 30-90 min; complete inhibition by NaF, 10 mM. The media were filtered just before use through a Selecta-filter No. 602h. Thiamine pyrophosphatase (TPPase; EC 3.6.1.9) : thiamine pyrophosphate, 4 mM; other medium as for NDPase; 30-90 min. Acid Phosphatase (AcPase; EC 3.1.3.2): glutaraldehyde-fixed sections; method slightly modified according to BARKA and ANDERSON (1962): ~-glycero-P, 8 mM; Pb(N03lz. 2,4 mM; DMSO, I M; tris-maleate buffer, 40 mM; pH= S,o; instead of ~-glycero-P deoxycytidine monophosphate, 8 mM, was also used; 4S-90 min; complete inhibition by NaF, 10 mM. - The azo-dye-method (BARKA and ANDERSON 1962) was applied to blockfixed tissue using naphtol-AS-TR-phosphate as substrate; 90-180 min. Esterase, E 6oo-resistent (EC 3.1. 1.2): according to THYBUSCH et al. (1966) using naphtol-AS-D-acetate; E 600, 0,01 and I mM; 90-180 min. ~-Glucuronidase (EC 3.2.1.3 I): according to HAYASHI et al. (1964); 60-240 min. Arylsulfatase (EC 3.1.6.1): according to HOPsu-HAVU et al. (1967); 4S-90 min. The azo-dye-method (WOOHSMANN and HARTRODT 1965) was also used with naphtol-AS-BI-sulfate and hexazotized pararosanilin. N-Acetyl-~-D-glucosaminidase (NAGase; EC 3.2.1.29): according to HAYASHI (196S); 60180 min. Alkaline Phosphatase (AIPase; EC 3.1.3.1): according to TRANZER as modified by COLEMAN et al. (1967) using preferentially glutaraldehyde-fixed sections; 10-60 min. Carbonic anhydratase (CAH; EC 4.2.I.I): according to HANSSON (1967); S-7S min; control by acetazolamide, 0,1 mM.

Abbreviations used: AChE, acetylcholinesterase AcPase, acid phosphatase ADP, adenosine-s'-diphosphate ALD, fructose-l,6-diphosphate aldolase AIPase, alkaline phosphatase

AMP, adenosine-s' -monophosphate ATP, adenosine-s' -triphosphate ATPase, adenosine triphosphatase BT, tetrazolium blue CAH, carbonic anhydratase c. ep., ciliated ependyma of the 3rd ventricle ChE, cholinesterase p-CMB, p-chloromercuribenzoic acid CYO, cytochrome oxidase DMF, NN-dimethyl formamide DMSO, NN-dimethyl sulfoxide ep. ch. pI., ependyma of the choriod plexus of the 3rd ventricle ep. SCO, ependymal part of the SCO ER, endoplasmic reticulum FDPase, fructose-l,6-diphosphatase F6PK, fructose-6-phosphate kinase GAP-DH, glyceraldehyde-3-phosphate dehydrogenase GDP, guanosine-s' -diphosphate GIDH, glutamate dehydrogenase G6Pase, glucose-6-phosphatase G6P-DH, glucose-6-phosphate dehydrogenase GP-DH, glycerol-3-phosphate dehydrogenase 3-HBDH, 3-hydroxybutyrate dehydrogenase HK, hexokinase hypo SCO, hypendymal part of the SCO ICDH, isocitrate dehydrogenase !DP, inosine-s' -diphosphate IDPase, inosine diphosphatase LDH, lactate dehydrogenase MAO, monoamine oxidase MDH, malate dehydrogenase N AD, ~-nicotinamide-adenine dinucleotide NADH, reduced NAD N APD, nicotinamide-adenine dinucleotide phosphate NADPH, reduced NAPD NAD(P)H dehydrogenase, NAD(P)H-tetrazoliumoxidoreductase NAGase, N-acetyl-~-D-glucosaminidase NBT, nitro blue tetrazolium chloride NDPase, nucleoside diphosphatase NT, neotetrazolium chloride 6PG-DH, 6-phosphogluconate dehydrogenase PGI, phosphoglucose isomerase PGluM, phosphoglucomutase PMS, phenazine methosulfate PVA, polyvinyl alcohol RNA, ribonucleic acid SCO, subcommissural organ SDH, succinate dehydrogenase TNBT, tetranitro blue tetrazolium chloride TPPase, thiamine pyrophosphatase UDP, uridine-s'-diphosphate UDPG, uridine-s' -diphosphoglucose UDPG-DH, uridine-diphosphoglucose dehydrogenase

Enzymatic Organization of the Subcommissural Organ . 5

Results 1. Morphological Findings

a) Structural Organization of the seo The morphology of the seo of the four investigated rodents is described in detail by PESONEN (1941), WISLOCKI and LEDUC (1952), BOSQUE et al. (1958), OKSCHE (1961), STANK A (1963), PALKOVITS (1965 a), GABRIEL (1970), and HERRLINGER (1970). Though the structuralorganization is essentially alike at the light microscopic level, some species differences have to be mentioned. The ependymal part of the seo is composed of tall, sparsely ciliated columnar cells. The oval-shaped nuclei are located at various levels in the guinea pig and hamster whereas in the rat and mouse they are situated at the very bases of the ependymal cells (see Fig. I). Indentations of the nuclei are observed often in the mouse and hamster, occasionally in the rat but never in the guinea pig. After staining for nucleic

acids, in all species the nuclei appear moderately dark and contain 2-4 intensively stained nucleoli; the latter are sometimes attached to the nuclear membrane. Only in the seo of the hamster some cells contain light nuclei which hold less nucleoli. Some of these faintly stained nuclei seem to be clear of nucleoli (see also Fig. 2). These cells regularly constitute the medial area of the hamster seo. They differ clearly from those of the lateral part where the ep. seo deflects from the posterior commissure to the lateral wall of the third ventricle. The content of cytoplasmic RNA is very low in the seo of all species, relatively the highest in the guinea pig.

b) Secretory Substance Secretory material can be best demonstrated by Bock's chrome alum-gallocyanin stain (d. KOHL and LINDERER 1973). There is no substantial interference by the staining of cytoplasmic RNA. In the guinea pig moderately to

:;IIl&

awe

-

1

~. .

CP

,I'

ha mster

c

rat

b

~.~ ..

-.--- --- - .•...

.... ..... ....

d

Figure 1. Schematic representation of the SCO. a: Guinea pig, b: rat, (: golden hamster, d: mouse (alb sagittal, cld frontal plane). - The pars supracommissuralis consists of a lower ependymal layer in all species. In the pars subcommissuralis the nuclei are located at various levels of the ependymal cells. Only in the guinea pig a well developed "dorsal crest" which is not clearly delimited adjoins the pars retrocommissuralis caudally. The ependyma of the SCO is sharply demarcated from the ciliated ependyma lining the wall of the 3rd ventricle. CP Commissura posterior.

6 . WOLFGANG KOHL

darkly stained secretory granules of small size fill up the cytoplasm of most but not all ependymal cells; these granules are less numerous in the most apical area of the ep. SCQ (Fig. 2a). In contrast, in this area darkly stained granules of larger size a re found. Apical cytoplasmic protrusions bulging into the 3rd ventricle are very often observed and are frequently filled up by the larger-sized granules. In rats the distribution of secretory substance is alike (Fig. 2 b). The granules appear to be of a somewhat larger size; occasionally they are stronger stained by Bock's method. Apically situated granules and protrusions seem to be less in number. In contrast to the foregoing, the ependymal cells of the SCQ of the hamster are largely devoid of small granules. But there are large masses of intensively stained secretory material which are frequently situated near the inferior pole of the nuclei (Fig. 2 c). This accumulation of secretory

a

b

material corresponds ultrastructurally to extremely dilated cisternae of the endoplasmic reticulum which are filled up by secretory substance (KOHL, unpublished; WETZSTEIN 1974). These large secretory sacs occur mainly in cells of the medial part of the SCQ. Cells of the more lateral part of the organ contain granules of small and large size; the lateral cells form also granules-filled apical protrusions as described in the other species. In the ep. SCQ of the mouse secretory substance is most intensely stained in the nuclear region, therefore sometimes hiding the contures of the nuclei. The supranuclear area contains granules of a small size. Because they are only faintly stained they cause a dustlike appearence of this area (Fig. 2 d). Immediately below the surface of the ep. SCQ some intensively stained granules can be observed. Some apical protrusions containing secretory granules bulge into the ventricle, too. In all

c

d

Figure 2. Species different distribution of secretory substance in the sea. a: Guinea pig, b: rat, c: hamster, d: mouse. Chrome alum-gallocyanin stain according to BOCK. x 1060. - In the gu inea pig most of the e pendymal cens are fil1ed up by s mal1 secretory granule~. Only in the most apical part clusters of granules are seen which are intensively stained. These clusters are also observed in apical cytoplasmic protrusions. - In the rat the supranuclear and apical area of the ependymal part contain medium-sized secretory granules. The arrow marks a cytoplasmic protrusion fil1ed up by granules. - In the hamster large masses o f secretory substance are seen most frequently near the inferior pole of the nuclei (arrows). - In the mouse secretory material seems to hide the contures of the nuclei.

Enzymatic Organization of the Sub commissural Organ . 7

species the staining intensity of Reissner's fibre corresponds to the staining of the larger granules. Secretory substance is stained in a corresponding manner by Bock's method as well as by the pseudoisocyanin-, PAS-, DNFB-, and DDDreaction (the latter both for SH- and SS-groups). In all these reactions the small granules show a faint staining, the larger ones a moderate one. Using colloidal iron and alcian blue 8GX there is a moderate taining s of the surface of the c. ep. and a faint staining of the surface of the ep. seQ but secretory substance is not stained.

2. Enzymatic Organization of the Ependymal Part of the seo The various topographical regions of the seQ (for nomenclature see PALKOVITS 1965 a) may differ slightly regarding the activity of individual enzymes but they do not differ in their enzyme pattern. Enzyme activities are generally the lowest in the pars supracommissuralis which consists of a lower ependyma (d. Fig. I). Therefore, we compared only enzyme activities in the most active part, the pars subcommissuralis.

a) Control Reactions There is no staining of any structure by exogenous formazan, i. e. no Jormazan substantivity. Thus, artifacts possibly caused by diffusing formazan can be excluded. The substantivity c>f tetrazolium salts is similar in all species: a dark staining is observed only in nuclei, nucleoli, and myelinated fibres whereas the cytoplasm of the ep. seQ appears merely in a light blue-violet color. This color seems to be somewhat darker in the guinea pig seQ. - Incubation with substrate-free media as well as with media in which additionally coenzyme or PMS or cyanide were omitted leads to a slight or weak staining of the supranuclear region of the c. ep .. The cytoplasm of the ep. seQ is hardly stained. This negligible blind reaction may be due to endogenous substrates and/or coenzymes. It is somewhat stronger in the F6PK-preparations and possibly caused by the higher pH of this

medium. This blind reaction, however, must be separated from non-specific reactions which occur in the demonstration of UDPG pyrophosphorylase for example. Nevertheless, as shown in Figure 4a, in sections incubated without substrate some small contrasting granules are seen in the ep. seQ at higher magnifications. These granules may represent precipitates of TNBTformazan. They are more numerous in the demonstration of cytosolic enzymes, e. g. PGluM, G6P-DH and F6PK (see Figs. 4C, h, i). The formazan precipitates differ clearly in size and distribution from mitochondria. This is obvious when the precipitates of the "nothingdehydrogenase" reaction are compared with the product from the reactions of SDH or NADIeDH (d. Figs. 4a and 4k). NBT-/ormazan precipitates appear as larger granules especially in the seQ. Therefore TNBT was preferred as final electron acceptor. Furthermore, as shown by thin-layer chromatography (Fig. 3), the commercially available TNBT consists of one major blue-violet and two minor nearly negligible components. In contrast, the composition of NBT is a more heterogenous one. - The terms "diffuse" and "granular" will be used in the description of reaction product. The former one implies the diffuse distribution of the reaction product though its appearence is sometimes fmely granular.

Figure 3. Thin-layer chromatogramm of NBT (left) and of TNBT (right) according to TYRER et al. (I969). The different composition of these two tetrazolium salts is obvious.

8 . W OEFGANG KOHL

When using P MS as an intermediate electron carrier incubation times are shortened and reactions become independent of endogenous TNBT-reductases. This fact results in some surprising alterations of staining intensities which are most prominent in the adjoining neuropil but also observed in the seQ. Because TNBT-reductase activities are the lowest in the seQ of the hamster in this species the effect of PMS is most obvious. For this reason the present results correct some of our preliminary ones (KOHL 1973). Cyanide prevents electron transfer from reduced PMS to eYQ by blocking this enzyme. Its effect is particularly conspicuous in regions containing numerous mitochondria, e. g. the neuropil or cerebellum. In these regions the reactions of dehydrogenases and the coupled enzyme assays are enhanced in the presence of cyanide. Some intensification is also seen in the seQ. Inhibition of any reaction was never observed in the presence of cyanide. - An atmosphere of nitrogen is less efficient than cyanide in blocking false electron transport but it seems to decelerate spontaneous reduction of TNBT occuring under normal conditions. Without PVA in the reaction medium glycolytic enzymes for example leak from the sections because they are easily soluble. Nevertheless, diffusion of formazan is not likely to be expected in case of structure-bound mitochondrial enzymes. But we were surprised finding an increase of SDH reaction when adding PVA to the reaction medium. This increase is more distinct using higher concentrations of PVA.

Therefore, this substance was regularly used for the demonstration of dehydrogenases as well as in coupled enzyme assays. A further advantage of PVA is better preservation of the structural integrity of unfixed sections.

b) Enzymes of Energy-Supplying Metabolism Figures 4 b-i illustrate typical staining patterns of enzymes of glucose metabolism in the seQ of the rat. In general, in all species studied the histochemical localization of enzymes related to glucose phosphorylation, glycogen metabolism, pentose phosphate shunt, and glycolysis is similar. Their corresponding reaction product is distributed mainly in the nuclear area and in the supranuclear cytoplasm of the ep. seQ and shows decreasing density towards the apical area of the ependymal cells. In the infranuclear area a similar staining intensity is observed as in the supranuclear region but this part cannot be demarcated clearly from hypendymal cells. In Table 1 relative staining intensities are given which are found in the histochemical demonstration of selected enzymes. The values are only arbitrary and depend on methodical conditions but they may make possible to compare the staining intensities of different brain regions or different species, provided that the conditions are the same. The findings in the seQ are opposed to those in the c. ep. adjoining the seQ and to those in the ep. ch. pI. of the 3rd ventricle. The highest activity level of HK is present in the c. ep. and the ep. ch. pI.; the ep. seQ is

Figure 4. seo of the rat. X 550. - a: "Nothing-dehydrogenase". Only some small granules are observed which may represent coarse formazan precipitates. b: Hexokinase. A diffuse staining of moderate intensity is seen especially in the supranuclear area. c: Phosphoglucomutase. Weak or moderate staining likewise in the supranuclear area. d: UDPG pyrophosphorylase. Weak staining in the same area. e: Glycogen synthetase. Rather strong reaction in the nuclear and supranuclear area.f: Glycogen. Granules decrease in number from the nuclear to the supranuclear area. g: Phosphorylase. There is definitely more reaction product than intrinsic glycogen. h: Glucose-6-phosphate dehydrogenase. Rather strong staining in the supranuclear area. i: Fructose-6-phosphate kinase. Diffuse staining which is only weak or moderate decreases towards the apical area. k: NAD-dependent isocitrate dehydrogenase. Granular distribution of reaction product which is accumulated in the nuclear/supranuclear area as well as in the most apical part. I: NADH dehydrogenase. Granular and diffuse distribution of formazan. m: NADPH dehydrogenase. Only diffuse staining is observed. - In all Figures note also the stronger staining of the basal, respectively hypendymal part of the seo.

Enzymatic Organization of the Subcommissural Organ . 9

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10 • WOLFGANG KOHL

most intensively stained in guinea pigs. In the c. ep. formazan is deposited mainly in the supranuclear area as granules which may correspond to mitochondria. In the seo the reaction product is diffusely distributed in the supranuclear area (Fig. 4b). Only in the hamster the enzyme seems to be more active in the lateral than in the medial part of the seo. - Demonstration of specific G6Pase activity was only possible in the seo. In the different species the staining pattern of this enzyme resembles to that of HK and will be described in detail in connection with other microsomal enzymes. The demonstration of PGluM is made difficult by its high activity which causes diffusion of formazan but a similar localization (Fig. 4c) and the same species differences may be assumed for this enzyme as for HK. - The coupled enzyme assay for UDPG pyrophosphorylase is complicated by non-specific splitting of UDPG which is observed mainly in the seo and which is species different. The reason for this non-specific reaction remained obscure. Nevertheless, specific activity is likewise localized mainly in the supranuclear area of the ep. seo and is higher there than in the c. ep. and ep. ch. pI. (Fig. 4d). - We were not able to demonstrate UDPG-DH activity in the seo whereas parenchymal cells of liver, kidney or cartilage showed moderate activity applying the same method. In all species moderate amounts of glycogen are regularly found in the ep. seo. Glycogen granules are situated mainly in a perinuclear position and - decreasing in number - in the supranuclear area (Fig. 4£). In the seo of the hamster a somewhat lower glycogen content may be supposed (see Table I). In contrast to the ep. seo, the c. ep. contains only a small amount of glycogen granules; their number increases towards the ventral area of the 3rd ventricle though in a somewhat different manner in the four species. In the ep. ch. pI. there is apparently no glycogen. - The demonstration of both glycogen synthetase and phosphorylase activity depends obviously on intrinsic glycogen - at least in the methods applied; consequently high activities of both enzymes are found in the nuclear area and

lower ones in the supranuclear area of the ep. seo (Figs. 4e and g). Both of these enzymes show obvious species differences in the seo (see Table I). Thus, a different ratio of synthetase/ phosphorylase activity might be assumed: a high one in the guinea pig and rat, a lower one in mice, and the lowest in hamsters. Despite of minor differences at the cellular level, in frontal sections both enzymes seem to be equally distributed in all ependymal cells of the seo. Only in the hamster phosphorylase is more active in the lateral part of the organ. In contrast to the high activities found in the seo both enzymes are of low activity in the c. ep., whereas in the ep. ch. pI. they seem to be hardly active (d. Table I). The intracellular location of both NADPdependent enzymes of the pentose phosphate shunt, G6P-DH and 6PG-DH, resembles to the distribution of HK and of the enzymes of glycogen metabolism. The reactions result in a formazan which is diffusely distributed and most dense in the supranuclear area and which decreases in density towards the apical area of the ep. seo (Fig. 4h). Obviously its most apical part is scarcely stained. Comparing the staining pattern of G6P-DH with the distribution of N ADPH-TNBT-oxidoreductase activity (shown in Fig. 4m), their differing location is apparent. In the seo G6P-DH and 6PG-DH are more active in guinea pigs and rats than in hamsters and mice (d. TableI). Figures nand 7 h illustrate the similar staining pattern of G6P-DH activity in the seo of the hamster and mouse. This pattern is only found when PMS is used. The two species are markedly different in their reductase activity (Fig. 7i and k); thus, when not using PMS they differ also in their G6P-DH activity but this activity is more similar in the presence of PMS. Consequently the reductase activity is a clear-cut limiting factor. In presence of PMS the G6P-DH reaction is slightly enhanced by the addition of 5 mM Mg++ or Mn++. Varying concentrations of substrate (0,5-20 mM) and of coenzyme (0,35-3,5 mM) lead to essentially identical results. In raising concentrations of TNBT from 0,5 to 3,0 mM, diffusion of formazan is markedly diminished.

4 4 4

F6PK GAP-DH LDH LDH, with urea LDH, with pyruvate

2-3 3-4

3-4 2

3

4 0-1

2

0

1-2

2

4

1-2 1-2

2-3 2-3

2

2-3 2

3-4

3-4

4 4-5

2 1-2

I

2 2-3

I

I

2-3 0-1 1-2 2

1-2 1-2

3-4

I

4 3 4-5

3

2 1-2 3

2 2-3

mouse

1-2 1-2

2 2 2-3 1-2

I

2-3

0-1 1-2

I

I

2

hamster

4 3

I

1-2

I

3 3

2-3 2 1-2

2-3 3-4 4 2 2-3

4

3 1-2 2

3 3

rat

I

4-5 4-5

3

1-2

4 3

4 4 4 3-4 4-5

4 4-5 3-4

4 4 4 2 1-2

4-5

4 3 3

I

4

guinea pig

4 2

2-3

?

3 3

4 4 1-2 3 3

4 3-4 3

3-4 4 4 2-3 2

4-5

4 0-1

2

?

3 3

4 3 3 3 4

4 3-4 3

3-4 4 3-4 3 2

3-4

3 2-3 2-3

?

4 3 2-3

3-4

?

hamster

3-4

rat

Hypendymal Part

3-4 2

2

?

4 4

4 4 3 3 4

4 3-4 3

4 4 5 2 3-4

4

4 3 4

?

3

mouse

rat

I I

I I

0-1

0

3-4 2-3

4 4 3 3 4

4 4 2-3

I

0-1

0

4 2-3

5 5-6 2 3 1-2

4 4-5 3

I

I

0-1

0

4 2

5 4 3 3-4 5

3-4 5 2-3

I

I

2 2 3

I

2 1-2 1-2

1-2

I

?

0-1 1-2

I

I

0-1

0

3-4 2-3

4 4-5 3-4 3 4

3-4 4 3

I

2 2 2-3

I

I

0

I

3 0

mouse

3-4 0

I

I

2 0-1 0

0

hamster

2 2 2-3 1-2

2

3 0-1 0-1

o

~

guinea pig

Ciliated Ependyma

I

-

-

-

-

5 3-4

6 5 1-2 2-3 3

5 5 4

4 4-5 3-4 3 2

1-2

0-1 0-1 0

5 0

guinea pig

-

-

-

-

-

0

-

-

-

-

0

2 1-2

2 1-2 4 3-4

4-5 3-4 3-4

3 4 2 2 0-1

2

4-5 4 0 2-3 5

4-5 3 3-4

I

4 4 3 2

2-3

0 0

?

0

4 5-6 0-1 3 4-5

6 5 2 3-4 2

5 5 3-4

3 4 4-5 4 2

2

?

0 0

o

? 0

mouse

~

hamster

Neuropil

0-1

5-6 0

rat

Choroid Plexus

In this table an arbitrary estimation was used to indicate the relative amount of reaction product which is formed under conditions described in Methods. Staining is: o=negative; I=justvisible; 2=weak; 3=moderate; 4=ratherstrong; 5=strong; 6=verystrong; ?=l1otdefinite.

AcPase NAGase

TPPase, pH = 7,2

NADH dehydrogenase NADPH dehydrogenase IDPase, pH = 8,0 Diffuse staining

2

I

2 2 2-3

NADP-linked ICDH NADP-linked MDH 3- HBDH GP-DH GlDH

cva

2 3 2

NAD-linked ICDH SDH

3

I

3-4

G6P-DH

I

3 1-2

Glycogen synthetase Glycogen Phosphorylase

guinea pig

Ependymal Part

Sub commissural Organ

Enzyme activity pattern of the SeQ, the ciliated ependyma, the ependyma of the choroid plexus, and the neuropil.

4 3-4

1.

Hexokinase G6Pase

Table

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tJQ

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2.

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When substituting Gal6P or dG6P as substrate the reaction is minimal or negligible. The examined enzymes of the Embdel1Meyerhof pathway are similar regarding their activity and distribution. There is no specific activity of FDPase in the seQ because the reaction is not at all inhibited by AMP. In contrast, in liver and in kidney cortex the inhibitory effect of AMP is definite but incomplete. We were not able to demonstrate the low activity of FDPase known to be present in white muscles (KREBS and WOODFORD 1965) by means of the method applied. Thus, presence of this enzyme in the seQ cannot be strictly excluded. - Because of only minor differences the findings regarding PCl, F6PK, ALD, GAPDH, alJd LDH activity can be summarized as follows: diffusion of formazan is reduced in the PGI reaction by lowering the substrate concentration to 1 mM; diffusion is minimal in the other enzymes; the intracellular location of these enzymes corresponds very well to that of HK and of the enzymes of glycogen metabolism and pentose phosphate shunt; as illustrated in Figure 4i - using F6PK as an examplethe activity of glycolytic enzymes decreases from the supranuclear to the apical region of the ep. SeQ; their activity level is the highest in the guinea pig but the lowest in the hamster (see Table 1); figures 5 a-f show the different activities of F6PK, GAP-DH, and LDH in the seQ of the hamster and mouse. Likewise the enzymes already described, in the hamster seQ the lateral cells are often stronger reactive towards glycolytic enzymes than the medial ones (see also Fig. 5). In the c. ep. the activity of glycolytic enzymes, especially of F6PK and GAP-DH, is lower (Fig. 5 a-d). Because glycolytic enzyme activities are very low in the seQ of the hamster they are apparently equal in the ep. seQ and

the c. ep. of this species. - In the ep. ch. pI. glycolytic enzyme activities resemble to those in the seQ (see also Table I). The histochemical differentiation of LDH isoenzymes or subunits reveals most striking differences. In the c. ep., ep. ch. pl., or cerebellum of all species LDH activity is stronger inhibited by pyruvate (the ratio pyruvate/lactate being 1/20) than by 3,5 M urea. In contrast, this inhibition is inverse in the seQ of the guinea pig, rat, and mouse (Figs. 5 f, h, k), suggesting the predominance of M-type subunits. In the seQ of the hamster, however, LDH activity is not only lower but also unaltered or even slightly enhanced by the addition of urea (see Figs. 5 e, g, i; d. Table 1); it is moderately inhibited by pyruvate - especially in the lateral part of the organ. The distribution of enzymes of the citric acid cycle is illustrated in Figure 4k showing the staining pattern of NAD-dependent IeDH activity in the seQ of the rat. Formazan is deposited as medium-sized granules or clusters of granules which are clearly discernible from so-called formazan precipitates (Fig. 4a). NADIeDH and SDH show an identical distribution of reaction product; they are known to be localized only in mitochondria. The arrangement of reaction product agrees well to that of mitochondria which are accumulated near the apical pole of the nuclei as well as in the most apical part of the ep. SeQ, whereas they are scantily scattered in the intermediate cytoplasm (see STANKA et al. 1964). A further accumulation of mitochondria is observed in the basal part of the ep. seQ but this part is not clearly demarcated from the hypendyma. The staining pattern of different mitochondrial enzymes suggests some species specific variations with regard to number, size, and

Figure 5. Glycolytic enzymes in the SeQ of the hamster (left row) and of the mouse (right row). X 190. - a and b: Fructose-6-phosphate kinase. [ and d: Glyceraldehyde-phosphate dehydrogenase. e and J: Lactate dehydrogenase. g and h: Lactate dehydrogenase in the presence of urea. i and k: Lactate dehydrogenase in the presence of pyruvate. Sections in e-i, respectively f-k are serial ones. - Species different activities are obvious in the SeQ. In the hamster SeQ, note the stronger staining of the apical area of the lateral ependymal cells. The beginning of the ciliated ependyma is marked by small arrows in a and b. Stronger stained basal or hypendymal parts of the SeQ are indicated by large arrows.

Enzymatic Organization of the Sub commissural Organ . 13

I4 . WOLFGANG KOHL

distribution of mitochondria. In the guinea pig mitochondria seem to be more evenly distributed in the ep. SCQ but also enriched in its most apical part. In the hamster they appear to be fewer in number but coarser in size and accumulated especially in the apical cytoplasm of the lateral part of the organ (Figures 6a, i, 7a). In the mouse mitochondria appear as very fine, more evenly distributed granules; they are sometimes concentrated immediately below the surface of the ep. SCQ (Figur 6b). These differences make the comparison of staining intensities more difficult. As shown in Figure 6 a and 6 b, in the SCQ of hamster and mouse the distribution of SD H activity is distinct, indeed, but the activity level does not seem to vary a great deal comparing all ependymal cells of the organ. Despite of this difficulty mitochondrial enzyme activities differ defmitely at least in the SCQ of the guinea pig and hamster (see Table l). Summarizing the findings given in the Table l, only moderate activities of exclusively mitochondrial enzymes, such as NAD-dependent ICDH, SDH, CYQ, 3-HBDH, GP-DH, and GIDH, are found in the SCQ of the guinea pig and rat, and somewhat lower ones in the hamster and mouse. For the last two mentioned species typical examples are illustrated in Figures 6 and 7. - In contrast, the c. ep. and ep. ch. pI. are characterized by relatively high activities of mitochondrial enzymes, the species differences being subtile (see Fig. 6 and Table I). With regard to mitochondrial enzymes the SCQ and c. ep. differ only in quantity and not in quality. Thus, their mitochondrial population might be homogenous. This fact is best demonstrated by means of 3-HBDH activity which corresponds

to the activity levels of NAD-ICDH or SDH only in the SCQ and c. ep. whereas the activity ratio 3-HBDH/SDH is quite distinct in the choroid plexus. In the neuropil there is almost no 3-HBDH activity (see Figs. 6i, k). - It is of interest that the activity level of HK corresponds to some extent to that of mitochondrial enzymes of the citric acid cycle; the distribution of HK, however, appears to be mainly diffuse in the SCQ (d. Figures 4 band k). NAD-ICDH is slightly activated by ADP.The activity of CYQ is low even when cytochrome c is added; its distribution corresponds well to that of NAD-ICDH or SDH (Figures6g, h). - Mitochondrial CP-DH is lacking its extramitochondrial NAD-dependent counterpart which can be clearly demonstrated in liver. - CIDH is intensively activated by the addition of ADP; its distribution is identical to that of the above mentioned enzymes (seeFigures7a, b). - Mg++ -dependent A TPase activity is very low in the SCQ; the fmely granular appearence of its reaction product resembles first of all to the distribution of mitochondria but this location remains unsolved even after prolonged incubation periods. - The activity level of MAQ believed to be located first of all in mitochondria - is very low, too; thus its distribution cannot be clearly demonstrated in the SCQ. All of these enzymes mentioned are thought to be located only in mitochondria. The same localization may be assumed for the SCQ because no diffuse staining of the cytoplasm in the ep. SCQ was observed. Some other enzymes are localized both in and out of mitochondria. This is indicated by an additional diffuse staining which is illnstrated in Figure 41 showing NADH dehydrogenase activity in the rat SCQ. - For

Figure 6. Mitochondrial enzymes in the SCQ of the hamster (left row) and of the mouse (right row). X I90.a and b: Succinate dehydrogenase. In a few but coarse granules are seen especially in the apical area of the lateral cells; in b more numerous fine granules are evenly distributed. c and d: NAD-dependent malate dehydrogenase. e and f: NAD-dependent malate dehydrogenase after extraction of soluble activity. The staining pattern is diffuse before extraction (c and d) but granular after it, thus suggesting localization of this enzyme both in and out of mitochondria. g and h: Cytochrome oxidase. Weak but definite staining in the SCQ, moderate staining in the ciliated ependyma. i and k: 3-Hydroxybutyrate dehydrogenase. The slight staining of the SCQ contrasts with that of the ciliated ependyma. - Arrows as in Fig. 5.

Enzymatic O rganization ofthe Subcommissural Organ . IS

:

..,...,

. ;,j ' ~;

~ .

k

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f' -.

•

~

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I6 . WOLFGANG KOHL

example has NAD-MDH activity which is also related to the tricarboxylic acid cycle a mainly extramitochondrial distribution in the SCQ; that is suggested by the diffuse staining pattern of this enzyme (see Figs. 6c, d). After extraction of soluble MDH activity mitochondrialike particles can be demonstrated in the SCQ (see Figs. 6e, f). Because tissue integrity of brain sections is largely affected by this procedure pre-extraction was not regularly used. Therefore, the intracellular location of the two NADP-dependent enzymes - which are subsidiary to the citric acid cycle - is not definite, either. Judged by the resulting staining pattern NADP-ICDH activity is mainly diffusely distributed in the SCQ but a mitochondrial localization cannot be excluded (Figs. 7C, d). In contrast, in the c. ep. the staining pattern of this enzyme appears mainly granular and the enzyme is more active than in the SCQ (see also Table). - Demonstrating NADP-MDH activity the staining of the ep. SCQ is both diffuse and granular (Figs. 7 e, f); the distribution of these granules resembles to that of exclusively mitochondrial enzymes. The activity of this enzyme varies widely in the SCQ of the different species; in the c. ep., however, its activity is not only higher but also more similar in the different species than in the SCQ (see also Table I).

c) Marker Enzymes of Different Cell Organelles The enzymes so far described are localized partly in the cytosol and partly in mitochondria and are related to the energy-supplying metabolism. Most of the following enzymes are first of all used as markers of different cell organelles such as the endoplasmic reticulum,

-_

.. - - - . _ - - _ . _... _

-._-

---.

Golgi apparatus, and lysosomes; their functional significance is less well defined. Despite of its mitochondrial localization NADH dehydrogel1ase (Fig. 41) might be as well present in the el1doplasmic reticulum of the ep. SCQ: this enzyme is not soluble, consequently structure-bound; its diffuse staining pattern resembles first of all to that of NADPH dehydrogenase (d. Fig. 4m) and to that of G6Pase (d. Fig. 8 a). Both of these enzymes are thought to be specific markers of ER membranes. The activity of NADH dehydrogenase in the ep. SCQ of the different species resembles to the activity of NADPH dehydrogenase and G6Pase, too. In contrast to the rat, in the guinea pig and mouse NADH dehydrogenase is more active (see Table I); furthermore its reaction product seems to be mainly diffusely distributed, even if the incubation period is shortened. In the SCQ of hamsters in which the activity is the lowest the distribution of reaction product is partly diffuse and partly granular. - We did not succeed in differentiating between mitochondrial and microsomal NADH dehydrogenase by means of a different inhibition with amy tal, rotenone, or p-CMB using TNBT or NBT as [mal acceptor. Using NT and BT we were able to demonstrate a partial inhibition of the reaction by p-CMB and amy tal but the formazan of NT and BT is of a very coarse size and therefore unsuitable at a cellular level, i. e. it is not possible to differ between diffuse and granular staining. As shown in Fig. 4m the demonstration of NADPH- TNBT-reductase activity results in a diffuse distribution of formazan in the ep. SCQ; the staining intensity may decrease slightly towards the most apical part of the ependyma. The activity of this enzyme is the highest in the

----

Figure 7. Comparison of various enzyme activities in the SCQ of the hamster (left row) and the mOl4se (right row). x I90. - a and b: Glutamate dehydrogenase. Weak staining and granular localization of reaction product. c and d: NADP-dependent isocitrate dehydrogenase. Formazan seems to be mainly diffusely distributed. e and J: NADP-dependent malate dehydrogenase. Note the obvious species differences. In! staining is diffuse and granular. g and h: Glucose-6-phosphate dehydrogenase. Similar staining intensity in both species. i and k: NADPH-TNBT-oxidoreductase. Most prominent species differences; diffuse distribution of formazan. - Arrows as in Fig. 5.

Enzymatic Organization of the Subcommissural Organ . 17

18 . WOLFGANG KOHL

seQ of the mouse and the lowest in that of the hamster (Figs. 7i, k; d. Table I). - In the seQ G6Pase activity is an amazing finding. The specifity of this reaction is described and discussed elsewhere (KOHL, in preparation). The distribution of this enzyme (see Fig. 8 a) is quite similar to that of NADPH reductase. Its activity level is also species different, in a manner corresponding to that of reductase activity (see Table I; Figs. 7i, k and 8 e, f). There is a nonspecific hydrolysis of glucose-6-phosphate even at pH=6,6; this reaction is probably caused by lysosomal AcPase activity (KOHL, in prep.). In the demonstration of G6Pase activity in the hamster seQ granular deposition of reaction product prevails; that pattern, however, corresponds first of all to the distribution of AcPase activity (d. Figs. 8e and 8i). This fact may rather indicate a non-specific reaction, thus preventing specific demonstration of a supposed low G6Pase activity in the seQ of this species. AChE and ChE were localized in ER membranes of the seQ by means of ultrastructural histochemistry (RECHARDT and LEONIENI 1972, guinea pig and rat). Using the Karnovsky-Roots technique, we obtained positive results for both enzymes only in the guinea pig SeQ; there was not any reaction in the mouse; in the seQ of rats a positive reaction was observed after long incubation periods; in the hamster only ehE activity seemed to be present. When performed at pH= 8,0 IDPase reaction results in a slightly diffuse staining of the ep. seQ of rats (Fig. 8 b). Diffuse distribution of reaction product is more intensively marked in

the guinea pig (see also Table I) and is most intense in the mouse - even at pH = 7,2 (Fig. 8h). In the seQ of the hamster diffuse distribution of reaction product was never observed (Fig. 8 g). - When substituting GDP or UDP as substrate the same species different activity pattern was found but localization of reaction product was not as precise; hence these substrates were not regularly used. - In the c. ep. as well as in glial or nerve cells a comparable diffuse staining was never observed in the NDPase preparations. IDPase activity is also present in thread-like structures which correspond to elements of the Golgi complex (see Fig. 8 b). This staining pattern becomes more distinct when reducing the pH to 7,2. In the seQ of the hamster, only thread-like structures are found at both pH values. - These structures are best demonstrated by the activity of their TPPase, an enzyme which is known to be a Golgi marker. Fig. 8 c illustrates that these structures may be described as small granular configurations connected by straight fine threads. They are arranged parallel to the long axis of the ependymal cells of the seQ and are situated mainly in the upper nuclear and in the supranuclear area. - In the mouse reaction product is distributed in a similar manner as in the rat. In the guinea pig Golgi elements seem to be more numerous and TPPase activity seems somewhat higher (see Table I). Whereas basal cells of the ep. seQ of the guinea pig show typical thread-like structures, in cells of the surface row TPPase activity is localized as a network closely applied to the nuclei. In the seQ of the hamster there is less

Figure 8. Distribution and comparison of hydrolytic enzyme activities. - a-d: SCQ of the rat. x 550. a: Glucose-6-phosphatase. Diffuse distribution of reaction product in the supranuclear and apical area. Non-specific precipitates in the posterior commissure (arrows). b: Nucleoside diphosphatase. A weak diffuse staining in some ependymal cells. Thread-like structures are also observed. c: Thiamine pyrophosphatase. Golgi elements are represented by small granular configurations connected by straight fine threads. d: Acid phosphatase. Medium-sized granules decrease in number from the nuclear towards the apical area. They are also present in apical cytoplasmic protrusions (arrow). e-k: Comparison of hamster (left row) and mouse (right row). X 190. - e andf: Glucose-6-phosphatase. In e reaction product is located preferentially in small granules whereas infit is also diffusely distributed.g and h: Nucleoside diphosphatase. In g only Golgi elements are observed - more distinctly in the lateral part of the SCQ - but in h the diffuse staining predominates. i and k: Acid phosphatase. Incubation time was 45 min in i and 90 min in k. i: Numerous granules especially in the lateral part of the SCQ; k: despite longer incubation staining is less dense. Note apical protrusion (arrow).

Enzymatic Organization of the Sub commissural Organ . 19

reaction product deposited within corresponding incubation periods; thread-like Golgi elements appear more distinct in the lateral than in the medial part of the organ; this distribution

is illustrated in Fig. 8 g by means of the corresponding localization of IDPase activity. Applying both the IDPase and TPPase reaction, in the c. ep. of all species only some granular

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configurations are observed in the supranuclear cytoplasm but there is also a non-specific reaction of the cell surface. Nucleoli are reactive both in the c. ep. and seQ. In the G6Pase, NDPase, and TPPase reactions staining of capillary walls is also observed. Because of the high AIPase activity in capillaries these reactions may not be specific. In the seQ there is no AIPase activity at all, and at pH = 7,2 there is not any hydrolysis of B-glycerophosphate. Typical staining pattern of AcPase activity which is a marker for lysosomes, are illustrated in Figs. 8 d, i and k. Applying a modified Gomori-method, medium-sized and sharply delineated granules are seen and the background staining is negligible. The granules are mainly found in the supranuclear area of the ep. seQ. They decrease in number towards the apical area but clusters of enzyme-marked granules can be observed in apical cytoplasmic protrusions (see Figs. 8 d, k). Species differences exist with regard to the number of granules just as well as to the AcPase activity. The staining intensities given in Table I have to consider species differences in regard to both these findings. As illustrated in Figs. 8 i and 8 k, a higher enzymatic activity in the hamster seQ is suggested by the shorter incubation time needed for the production of precipitate of similar staining intensity compared to the seQ of the mouse. However, in the hamster seQ this higher activity seems to be mainly restricted to the lateral ependymal cells. In the mouse granules are of somewhat smaller size and more evenly distributed in the ep. seQ. Species differences are most pronounced comparing the findings in hamsters and guinea pigs because in the latter species only relatively few granules are found even after prolonged incubation time (d. Table I). However, the species different activities of AcPase in the surrounding neuropil have also to be considered; this fact is illustrated in Figs. 8 i and k, too. - It is remarkable that in AcPase preparations thread-like structures reminding of Golgi elements are occasionally seen.

Using block-fixed tissue and naphtol AS derivates in the demonstration of AcPase, the same species different activities are found but an exclusively granular location cannot be obtained. - In the presence of ro- 5M E608, esterase activity varies in the different species in a manner corresponding but not identical to AcPase activity; within the ependymal cells the distribution of this type of esterase resembles very well to AcPase but a slight diffuse staining is also observed. - ~-Glucuronidase activity is very low in the SeQ; the resulting azo dye is restricted mainly to lysosomes. In the mouse, however, there is no activity at all, not even in the surrounding tissue, and even after very long incubation periods. - The distribution of sulfatase corresponds very well to that of AcPase but only if p-nitrocatechol sulfate is used as substrate. The naphtol AS-BI sulfate is hydrolized only by some cells of the perivascular area. - The activity pattern of N-acetyl glucosaminidase differs from the above mentioned enzymes: its activity is the highest in the seQ of the guinea pig and the lowest in the hamster (Table I), whereas in adjoining brain regions no corresponding species differences are observed. This enzyme is apparently located exclusively in lysosomes, too. Its distribution within the ependymal cells is similar to that of AcPase but a heterogeneity of lysosomes cannot be excluded regarding these two enzymes. - In the c. ep. only low activities of lysosomal enzymes are present in all species. We were not able to demonstrate any activity of CAH in the seQ but this enzyme is highly active in the choroid plexus for example.

3. Enzymatic Organization of the Hypendymal Part of the seQ As described in detail by SCHWINK and WETZSTEIN (I966), PAPACHARALAMPOUS et al. (I968), HERRLINGER (I970), and by KIMBLE and M0LLGARD (I973), the hypo seQ is a heterogenous composite of various glial cells. Basal cell processes of ependymal cells as well as organspecific cells are found which remind of ependymal cells with regard to their content of secre-

Enzymatic Organization of the Subcommissural Organ .

tory material and to many ultrastructural details. They are thought to be secretory hypendymal cells. Astrocytes and oligodendroglia cells are further constituents of the hypo seo. Blood vessels supplying the seo are also found in this part of the organ; occasionally they penetrate into the ependyma; they are also observed between the bundles of the posterior commissure. As described by the authors cited above the basement membrane of the capillaries is bordered by cell processes of astrocytes as well as of secretory ependymal and hypendymal cells. The astrocytic processes are rich in glycogen granules. Secretory cell processes also penetrate deeply between the myelinated fibres of the posterior commissure often accompanied by capillaries. - Secretory cells and their processes constitute most of the hypo seo but a defmite differentiation of the various constituents is difficult even at the level of ultrastructure. The hypo seo is best developed in the guinea pig (see also OKSCHE I961). In tlus species the vascular supply is most dense, too (see Fig. 9a). Therefore, the guinea pig appears to be most suitable for investigating the enzymatic

21

organization of the hypo seo. However, all the fmdings made in this species can be confirmed in the other ones in which the hypendyma and the vascular supply are less developed. At the light microscopic level the morphological characteristics of secretory hypendymal cells resemble to those of the ependymal part. In all species round- or oval-shaped nuclei containing 2-4 darkly stained nucleoli are surrounded partly by small and partly by some larger granules. The former are moderately and the latter intensely stained by Bock's chrome alumgallocyanin. Applying the same method some granules are also found in cell processes penetrating between the fibres of the posterior commissure. - The staining for cytoplasmic RNA is very faint in secretory hypendymal cells. The e nzyme activity pattern of the hyposeo cannot be coordinated to definite cell types. Though most of the histochemical results favour a similar enzymatic organization of both secretory cell types of the seo, it must be emphasized that the relative staining intensities given in the Table I do not only correspond to secretory hypendymal cells but also to all various cells of the hypo seo.

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Figure 9. H ypcnd ymal part of the SCO of the gu inea pig.a x 300,b-1 x 190. - a: Chrome alum-hematoxylinphloxin. N ote the dense vascular suppl y; some capillaries are marked by arrows. b: Glycogen. Granules are concentrated aroun d capillaries (arrows) . c: Glycogen synthetase. High activit y in many hypendymal cells. d: Glucose-6-phosphate dehydrogenase. DiffLlse distribution of formazan in most of the hypendymal cells. e: NAD-dependent isocitrate dehydrogenase. Clusters of larger and more intensively s tained granules around capillaries . .f: NADPH-TNBT-oxidoreductase. The enzyme might be restricted to secretory hypendymal cells.

22 • WOLFGANG KOHL

In the HK reaction most of the hypendymal cells are darkly stained; reaction product is somewhat concentrated around the capillaries. Cell processes penetrating into the commissure react also stongly. - In contrast, reaction product of G6Pase activity is restricted to few hypendymal cells indicating that this enzyme is localized in a distinct cell type. - Many glycogen granules are found especially in the perivascular area (Fig. 9 b). Activities of glycogen synthetase (Fig. 9c) and phosphorylase are very high in the hypo SCQ; as illustrated in Figure 9 c, in both reactions the final product is found in large amounts not only perivascularly but also diffusely distributed in the hypo SCQ, cell processes in the posterior commissure included. In frontal sections the distribution of these two enzymes delimitates the SCQ from the posterior commissure as well as from the adjoining neuropil. The high activities of both G6P-DH and 6PG-DH are unevenly distributed in the hypo SCQ (Fig. 9 d) but diffusion of formazan occurs under the conditions used. The histochemical demonstration of the investigated glycolytic enzymes leads to a stronger staining of most but not all hypendymal cells compared to the ep. SCQ (see Table I). Cell processes in the posterior commissure are also intensely stained. In the remaining part of the hypo SCQ their activity seems to be lower. When differentiating LDH subunits we find their pattern resembling to that of the ep. SCQ but minor differences cannot be excluded (see Figs. 5 e-k). Mitochondrial enzymes (NAD-ICDH, SDH, CYQ, 3-HBDH, GP-DH, GIDH) appear to be evenly distributed as small granules scattered in the hypo SCQ. Around the capillaries, however, clusters of larger-sized granules are present which are also stained more intensely (Fig. ge). Similar clusters are observed in ATPase preparations. Regarding 3-HBDH activity we will emphasize that such a perivascular aggregation of formazan is found only in the SCQ (see also Figs. 6i, k). The perivascular area of the "dorsal crest" which adjoins the SCQ caudally is lacking a similar 3-HBDH activity though its subependymal layer is also densely vascularized (KOHL

1974, guinea pig). - A similar perivascular accumulation of formazan is also noted in the demonstration of enzymes located both in and out of mitochondria (NADP-ICDH, NAD- and NADP-MDH, NADH dehydrogenase). In the intermediate area of the hypo SCQ the distribution of NAD-MDH and NADP-ICDH seems to be mainly diffuse whereas NADPMDH is preferentially located in granules resembling to mitochondria. The fibres which penetrate into the posterior commissure are also characterized by high activities of mitochondrial enzymes. - In the NADH dehydrogenase reaction a similar perivascular accumulation of formazan is observed; in the intermediate area of the hypo SCQ the dye distribution is partly granular and partly diffuse. As listed in the Table 1 the staining intensity of most enzymes which are believed to be located in mitochondria is stronger in the hypendymal than in the ependymal part of the SCQ. This fact is caused mainly by the described perivascular aggregation of reaction product which is regularly found in all species (see also Figs. 6 and 7). In the NADPH dehydrogenase reaction, formazan appears to be diffusely distributed in many but not all hypendymal cells (see Fig. Sf). The distribution of G6Pase activity might correspond to the same cells. Further enzymes which are thought to be located in the endoplasmic reticulum (ChE, AChE, NDPase) are less active in the hypo than in the ep. SCQ; their definite coordination to a distinct cell type is uncertain, too. - In the TPPase reaction the product forms typical networks around the nuclei of many hypendymal cells. -Lysosomal enzymes are located partly in small granules partly in clusters of granules, the latter being often found in the neighbourhood of capillaries. The activity of I ysosomal enzymes seems to be higher in the hypo SCQ, too (d. Table 1).

Discussion 1. Methodical Problems Interpretation of histochemical findings implies first of all discussion of the methods applied and of possible sources of artifacts.

Enzymatic Organization of the Subcommissural Organ . 23

Concerning the demonstration of dehydrogenases and dehydrogenase-linked assay systems, many studies have been performed with regard to the diffusion of soluble enzymes, to PMS as intermediate electron carrier, to cyanide in preventing false electron transport from reduced PMS to CYO, to the concentration of coenzyme and of tetrazolium salt, and with regard to the influence of diffusion of reduced coenzymes and PMS as well as to the substantivity of tetrazolium salts and of their formazan (NACHLAS et al. 1960; PEARSE and HESS 1961; PETTE and BRANDAU 1962; BRODY and ENGEL 1964; Low et al. 1964; FAHIMI and AMARASINGHAM 1964; ALTMANN and CHAYEN 1965; HARDONK 1965; MATHISEN and MELLGREN 1965; FAHIMI and KARNOVSKY 1966; PETTE and BRANDAU 1966; WOHLRAB and FUCHS 1967; McMILLAN 1967; SEIDLER and KUNDE 1969; SIGEL and PETTE 1969; JACOBSEN 1969; DAHL and MELLGREN 1970; ALTMAN 1971; MEIJER 1972 ; WARCHOl, and MARZOTKO 1972; ANDERSON and H0YER 1974; LEEFLANG-DE PIJPER and HULSMANN 1974). The somewhat contrary findings of BROOKE and ENGEL (1966) and of CONKLIN (1966) might be explained either by the omission of gel media or of CYO inhibitors. Summarizing the fmdings of all of the authors cited we state that in the demonstration of dehydrogenases and in coupled reactions: I. the concentration of the enzyme which has to be demonstrated must be deminished by the use of thin sections; 2. the diffusion of soluble enzymes must be prevented by the use of gel media or semipermeable membranes; 3. the concentrations of coenzyme, intermediate electron carrier, CYO inhibitor, and final electron acceptor must be balanced against each other, and they must not be rate-limiting; especially the concentration of the final acceptor has to be as high as practicable; 4. intermediate electron carriers have to be used since endogenous activities of NADHor NADPH-tetrazolium-reductase may be ratelimiting; 5. in the presence of intermediate electron carriers inhibitors of CYO activity have to be used in order to prevent oxidation of reduced carrier by this enzyme; 6. control reactions must be performed on the diffusion of

reduced intermediates as well as on the substantivity of the tetrazolium salt and of its formazan; the "nothing-dehydrogenase" reaction must also be considered. Most of our methods base on the data of PETTE and BRANDAU (1966), of JACOBSEN (1969), and of SIGEL and PETTE (1969) but they were slightly modified. On the requirements mentioned above we may comment as follows: I. Section thickness of IO [Lm may not be the optimum but a definite improvement can be achieved only using at least 5 [Lm sections or using semi-thin sections; that was not practicable for the purpose of this study bue, nevertheless, the results presented are satisfactory because diffusion of formazan - which depends on section thickness to a certain extent (see JACOBSEN 1969) - was only slight or negligible in most cases. 2. Diffusion oIenzymes was diminished by the use of PVA; even in the demonstration of enzymes which are thought to be structurebound the staining intensity was enhanced by the use of PVA; the reason for this effect remains unsolved. PVA containing reaction media are easy to prepare and handle; they make possible the use of auxiliary enzymes which are necessary in coupled reactions. Therefore, PVA was preferred to the technique of semipermeable membranes described by McMILLAN (1967) and by MEIJER (1972). Any pretreatment of sections e. g. with acetone and chloroform - was avoided because this proceeding may result in diminishing or inhibiting enzyme activities. 3. The gel media were tested with regard to their best composition by varying the concentrations of coenzyme, PMS, cyanide, and TNBT; the soluble enzymes LDH and G6P-DH were used as models. The same concentrations were found to be qualified also for the demonstration of all coenzyme dependent reactions (collpled assays included) but we are conscious of the incompleteness of many methodical details which will be the purpose of further studies. 4. The effect oIPMS can be best demonstrated in the neuropil or in the cerebellum both of which show high actIVItIes of mitochondrial oxidative enzymes such as SDH, CYO, or

24 . WOLFGANG KOHL

GP-DH; in these brain regions, however, corresponding activities of mitochondrial coenzymedependent enzymes (e. g. NAD-ICDH or GIDH) as well as of glycolytic enzymes can be demonstrated only in the presence of PMS. When PMS is lacking only low activities of coenzyme-dependent enzymes seem to be present in these brain regions. Such an enzyme activity pattern does certainly not correspond to biochemical results (for this problem see also p. 25). Spontaneous reduction of PMS was largely avoided by performing the histochemical reactions in the dark and in an atmosphere of N2 (see also WARCHOf: and MARZO TKO 1972), immediately after preparing the reaction medium. 5. In the presence of PMS the staining intensity is enhanced up to the maximum in the presence of 5 mM cyanide - a concentration at which CYQ activity is completely blocked. This enhancement can be best demonstrated in the neuropil or cerebellum, too. As similarly stated by JACOBSEN (1969) we did not find any evidence for an enhanced non-enzymatic TNBT reduction in the presence of cyanide. In substrate-free media only insignificant staining occured which did not differ defmitely from that in the absence of everyone substance: substrate, coenzyme, PMS, and cyanide. Positive effectors have to be used in some reactions. For example is GIDH activity enhanced up to the maximum in the presence of ADP; this substance acts in the same way but less effective in the demonstration of NAD-ICDH activity. 6. Diffusion of reduced coenzyme or PMS may occur but this phenomenon does not appear to be important: adjoining cell types which differ morphologically - such as the SCQ, the c. ep., and the neuropil - are characterized in most reactions by quite different enzyme activities which are sharply demarcated; furthermore, quite distinct pattern of cellular localization were observed in the SCQ, depending on the enzyme involved. - The substantivity of TNBT formazan was found to be negligible; the substantivity of TNBT itself causes only a slight staining of the cytoplasm in the SCQ. It may be

discussed that the coarse formazan precipitates of NBT and the fme, granular ones of TNBT might be due to a higher degree of substantivity of tetrazolium salts especially in the SCQ; nevertheless, the resulting problem of precise enzyme localization at the cellular level can be resolved defmitely only by improved methods, e. g. the use of semi-thin sections and new tetrazolium salts. For that reason and for the reason of possible inhibition of enzyme activity pretreatment of sections according to the methods prescribed by W OHLRAB and FUCHS (1967) and by JACOBSEN (1969) in order to reduce TNBT-substantivity of tissues were avoided (see also point 2). - "Nothing-dehydrogenase" reaction which has been recently discussed and reviewed by ANDERSON and H0YER (1974) was tested in gel media omitting substrate and/or coenzyme and/or PMS. As already mentioned staining without substrate was insignificant; staining was also only very slight when substrate, coenzyme, and PMS were omitted. ct.-Hydroxy acid oxidase may interfer with the LDH reaction (see JACOBSEN 1969). This possible artifact was not taken into consideration because this enzyme is thought to be localized in microbodies which have never been described in ultrastructural studies of the SCQ; but this question remains to be answered exactly. Phosphatase activity may convert part of NADP to NAD, thus causing false positive results in the demonstration of NADP-dependent enzymes (LEEFLANG-DE P1]PER and HULSMANN 1974). This interference was not tested, either, but it must be emphasized that at least in the ependymal part of the SCQ AIPase activity is negligible and that ~-glycerophosphate is not hydrolized at pH = 7,2. Furthermore, as shown in the hamster SCQ for example, malate is oxidized in the presence of NAD but not of NADP; so the influence of phosphatase activity - and most probably also of transhydrogenase activity - might be negligible, too. - Finally it may be discussed that in coupled reactions intrinsic activities of auxiliary enzymes such as G6P-DH or GAP-DH may contribute to the reaction and to the final amount of reaction product. Auxiliary enzymes, however, were added in high excess; in the HK

Enzymatic Organization of the Sub commissural Organ . 25

reaction for example the activity and distribution of this enzyme do not parallel to the intrinsic G6P-DH activity. At present only few data are available concerning the relation of enzyme activities in tissue homogenates or fractions to that in tissue sectio11s. ALTMANN (1969a) has found a direct proportion between biochemically measured activity of G6P-DH and the amount of formazan which is formed in tissue sections, provided that PMS is used. Nevertheless, it remains to be verified that in all reactions of our study all the concentrations represent the optimum, and that the reactions are of "zero order" (see also JACOBSEN 1969). As shown by NOLTE and PETTE (1972a, b) by means of comparative photometric determination of the activities of various enzymes, histochemical results "agree well with values derived from measurements of enzyme activity in homogenates". We are, however, conscious of the difficulty or impossibility to define or to judge "enzyme activity" by means of histochemistry: the terms "low" or "high" activity represent only arbitrary and relative quantities in contrast to the well defmed biochemical units. Nevertheless, from bur point of view histochemistry makes possible not only the localization but also the semiquantitative comparison of enzyme activities. Therefore, we compared the staining intensities found in the seo with those of the c. ep. and ep. ch. pI.. We emphasize that our data given in the Table I are only arbitrary and must be completed by quantitative methods. The methods applied in the histochemical demonstration of enzymes of glycogen metabolism have been criticized in detail by ECKNER et al. (1969). Regarding their findings we must emphasize that in the demonstration of both glycogen synthetase and phosphorylase the final amount of glycogen was defmitely higher than the intrinsic glycogen content, i. e. we are dealing with true enzyme activities. However, the activity levels of both enzymes might be absolutely dubious because the histochemical methods disregard all the problems known by biochemical studies. These reactions may be complicated by diffusion because these enzymes

are only partly bound to glycogen; the reactions depend on the primer used; they are influenced by the pH, by the substrate concentration, possibly by a-amylase activity, and by the inhibitory effect of Pi and UDP which are formed in the reaction (see RYMAN and WHELAN 1971). So we may query the observed species different activities first of all for the following reason: the glycogenolytic capacity of most tissues "exceeds their capacitiy for glycogen synthesis by at least an order of magnitude" (SCRUTTON and UTTER 1968). Only in the seo of the mouse our findings are in accordance with this fact. - It is not possible to differentiate histochemically the active and inactive forms of both these enzymes which are thought to be present also in the brain (see BRECKENRIDGE et al. 1962; STALMANS and HERS 1973). Thus, we do n::Jt know the forms we are dealing with. G6Pase activity has been differentiated from non-specific phosphatases (KOHL, in prep.) but further studies have to establish the specific nature of this enzyme on which a comprehensive review has been given by NORD LIE (1971). - Methods for IDPase, TPPase, and AcPase were modified in order to get stable media and precise localization of reaction products at the cellular level and to avoid precipitates and non-specific background staining. For AcPase demonstration the enhancing effect of DMSO (d. GANDER and MOPPERT 1969; SANYAL 1970) was very useful.Our results were less satisfactory about the demonstration of enzymes hydrolysing naphtol substrates and the modifications developed by GOSSRAU (1973) might be useful for further studies. A granular location was achieved at least partly.

2. Common Characteristics of the Enzymatic Organization of the SCO Summarizing the methodical problems we may conclude that the methods applied give reliable information on the activity and distribution of the enzymes investigated in the seo though some details remain to be resolved. In the following, characteristics will be discussed

26 . WOLFGANG KOHL

which are common of the SCO of all species investigated and which differentiate this organ from related ependymal regions such as the c. ep. and ep. ch. pI.. The activity of hexokinase is relatively the lowest in the SCO. The staining pattern of HK resembles to the distribution of mitochondrial enzymes in the c. ep. but differs from their distribution in the SCO. - According to biochemical results brain HK is partitioned into soluble and particulate pools (JOHNSON 1960; BACHELARD 1967; NEWSHOLME et al. 1968). The structurebound enzyme is thought to be mainly associated with the outer mitochondrial membrane (CRAVEN and BASFORD 1969; CRAVEN et al. 1969; COLOWICK 1973, lit.). Both of these parts are very similar in certain characteristics (THOMPSON and BACHELARD 1970) and they are thought to be interconvertible; their ratio varies in different brain regions apparently according to the energy posture, i. e. solubilization increases when the ATP IADP ratio rises; this fact is thought to indicate a regulatory change in the compartmentation of HK during oxidative phosphorylation (WILSON 1968; HOCHMAN 1972; KNULL et al. 1973). - HK is prevailing type I isoenzyme in the brain and only a very low isoenzyme II activity is found (KATZEN and SCHIMKE 1965; WILSON 1967; PURICH et al. 1973). The highest proportion of type II isoenzyme is consistently present in the soluble fractions of various tissues (KATZEN et al. 1970). The two isoenzymes differ in their insulin sensitivity (KATZEN et al. 1970) as well as in their response to various modifiers, especially to G6P, Pi and citrate; that results in a diversity of regulatory properties (Kosow et al. 1973; LUECK and FROMM 1974). When we take these findings into consideration we may presume that in the SCO HK prevails in a soluble state; this possibility might correlate with a relatively high ATP content and consequently a low ATP demand; it may furthermore be suggested that in the SCO the type II isoenzyme predominates which is thought to be inactive during periods of highly active glycogenolysis and glycolysis (LUECK and FROMM 1974). This assumption which will be the purpose of further studies is supported by the

relatively high glycogen content which the SCO may heavily rely on. The reaction catalyzed by the type I isoenzyme of HK is generally regarded as the first committed step of brain glycolysis. On the other hand suggest the presence of G6Pase activity in the SCO that this enzyme may play an important role in the metabolic regulation by its action on glucose-6-phosphate which occupies a central position in carbohydrate metabolism. Though G6Pase is thought to be present in the brain (SACKS and SACKS 1968; PRASANNAN and SUBRAHMANYAN 1968; ROVAINEN et al. 1969) we did not find a comparable specific activity in brain regions adjoining the SCO. The prime function of this enzyme is thought to be the release of free glucose. Thus, this function may be discussed with regard to release of glucose into the cerebrospinal fluid. However, the catalytic activity of G6Pase is a multifunctional one and, therefore, a regulatory role has been suggested (see NORD LIE 1971). This enzyme might be the opposite partner of HK in a glucose-G6P futile cycle which may provide a means for fine regulation of the glucose metabolism (SCRUTTON and UTTER 1968) - also in the SCO. Whereas in liver and kidney G6Pase is involved in gluconeogenesis as one of the key enzymes, our results lack evidence concerning the presence of further gluconeogenetic enzymes. Regarding this route of the EmbdenMeyerhof pathway it has to be considered that the relatively high catalytic capacity of F6PK an enzyme which functions in the direction of glycolysis only - may prevent any net gluconeogenetic flux in the SCO although "brain may have a limited enzymic potential for G6P synthesis from all gluconeogenic precursors" (SCRUTTON and UTTER 1968; see also PRASANNAN and SUBRAHMANYAN 1968; but cf. KREBS and WOODFORD 1965). In the SCO gluconeogenesis might function in the maintenance of the glycogen level because a relatively high amount of glycogen is a common characteristic of this organ in many species (see also SHIMIZU and KUMAMOTO 1952; OKSCHE 1962, 1969; DIEDEREN 1970; M0LLGARD 1972; but cf. SCHAFFNER 1970). The

Enzymatic Organization of the Subcommissural Organ . 27 amount of glycogen is low compared to typical glycogen storing tissues but is significantly higher in the seo than in the c. ep. and ep. ch. pI.; in the latter glycogen is not definitely demonstrable. Since glycogen may represent a simple fuel store, the energy supply of the seo becomes independent from blood glucose levels - at least for short periods of time. This suggestion may correspond to the poor blood supply of the seo of most species (see also WEINDL 1973). Enzymes related to glycogen metabolism, such as PGluM, UDPG pyrophosphorylase, glycogen synthetase and phosphorylase, can be demonstrated clearly in the seo. These enzymes are generally more active in the seo than in the c. ep. and choroid plexus. As already discussed (see p. 25) the divergent findings on phosphorylase activity (reported by SHIMIZU and OKADA 1957; FRIEDE 1959a; NAUMANN 1968; DIEDEREN 1970; our results included) might first of all be a methodical problem; its elucidation is of great interest because glycogenolysis is controlled by this enzyme and by various substances acting on it (LOWRY 1966; VILLAR-P ALAS I and LARNER 1970; RYMAN and WHELAN 1971). Despite of this problem we state that glycogen and the enzymes linked to its metabolism are localized mainly in the nuclear and supranuclear area of the ep. seo - in a similar manner as HK and further enzymes of glucose metabolism. UDPG pyrophosphorylase is not only involved in glycogen metabolism but also in the synthesis of glucuronides and glycosides. - The missing activity of UDPG-DH agrees well with our finding that in rodents the secretory product of the seo is mainly a neutral glycoprotein. Histochemical assays of further enzymes involved in glycoprotein synthesis are not available at present. Compared to both the c. ep. and ep. ch. pI. the maximal catalytic capacity of the pentose phosphate shunt is relatively high. This shunt is probably controlled at the G6P-DH step by substances counteracting the NADPH inhibition of this enzyme (EGGLESTON and KREBS 1974). The metabolic roles proposed for this shunt are

the supply of NADPH for biosyntheses and of pentoses for nucleotide and nucleic acid synthesis. As previously discussed (KOHL and LINDERER 1973), the main function of this shunt in the seo might be the supply of NADPH. NADPH itself might be used partly by the NADPH dehydrogenase of the mono-oxygenase system (seep. 33) partly by the cytoplasmic fatty acid synthetase complex (see also ALTMANN 1969 b). Because the seo does not store lipids it might be presumed that NADPH is required in the synthesis of membrane lipids which are involved in the secretory process. Thus, we may suggest that the activity of both NADP-linked enzymes of the pentose phosphate shunt are related to the secretory activity of the seo. Regarding the activity levels of both enzymes we must emphasize that they are only moderate compared to those in the adrenal cortex, for example. The very strong staining intensity of the G6P-DH reaction reported in the seo of different species (ABE et ai. 1963; DELONG and BALOGH 1965; SCHACHENMAYR 1967; DIEDEREN 1970; SCHihTE 1971) might be explained rather by the fact that these authors did not use an intermediate electron carrier. When PMS is missing in the G6P-DH reaction the seo is the strongest reactive area of the midbrain. The glucose entry system and the reactions catalyzed by HK, F6PK, GAP-DH, and pyruvate kinase are thought to be the rate limiting steps of the glycolytic pathway; amongst them the F6PK reaction is the major control point (LOWRY and PASSONNEAU 1964; LOWRY 1966; SACKTOR et ai. 1966; ROLLESTON and NEWSHOLME 1967 b; SCRUTTON and UTTER 1968; McILWAIN and BACHELARD 1971; NEWSHOLME and START 1973). In the histochemical demonstration of both the F6PK and GAP-DH activity we observed marked staining intensities in the SeQ; the results of the PGI and ALD reaction agree with these fmdings. Thus, we may conclude that the maximal catalytic capacity of the glycolytic pathway is relatively high in the SeQ, is lower in the c. ep., and is similar in the ep. ch. pI. to that of the seo.- To our knowledgeLDH is the sole glycolytic enzyme which has so far been investigated in the seo. Without using

28 • WOLFGANG KOHL

PMS but using relatively long incubation times a moderate to high activity of LDH is reported by DE LONG and BALOGH (1965), NAUMANN (1968), DIEDEREN (1970), SCHAFFNER (1970), and by SCHUTTE (1971). However, this enzyme is regulative only with regard to its different subunits. As shown by McMILLAN and WITTUM (1971) by means of comparative bio- and histochemical studies, the histochemical differentiation of LDH subunits using urea is a reliable method. Consequently we may state that the SCO is characterized by a higher content of M type subunits, whereas in the c. ep., ep. ch. pl., and the neuropil the H type subunits predominate. This finding is supported by our inhibition studies using pyruvate in which the staining pattern is - at least partly - reversed. Based on the findings of KAPLAN and EVERSE (1972, review) on LDH isoenzymes we infer that the SCO relies mainly on glycolysis for its energy supply and that glycolysis might be active even under anaerobic conditions. In contrast, both the c. ep. and ep. ch. pI. depend on aerobic substrate oxidation. The unresolved problem of FDPase activity in the SCO remains of interest because cycling at the F6PK-FDPase level might provide a fine regulation of glycolysis especially with regard to the temporal pattern of energy expenditure, i. e. contino us or discontinous glycolytic flux (see also NEWS HOLME and START 1973, p. 123). Concerning their cellular localization there is growing evidence that the classically "soluble" enzymes of glycolysis may be involved in interactions with particulate cellular components (for example see SCRUTTON and UTTER 1971, p. 293). Because of the partly granular formazan - formed in the demonstration of glycolytic enzymes - we might restrict ourselves to refer to the distinct distribution pattern of glycolytic and mitochondrial enzymes in the SCo. Both the NAD-linked ICDH and the SDH reaction are probable control points of the citric acid cycle (GOLDBERG et al. 1966; LOWRY 1966; but see NEWSHOLME and START 1973). The regulatory role of the NAD-dependent ICDH activity is supported by its activation by ADP.

Judged by their staining intensities the maximal catalytic capacities of both the NAD-ICDH and the SDH reaction are only low in the SCo but are much higher ones both in the c. ep. and ep. ch. pI.. Low or even lacking activities have been reported about SDH (LEDUC and WISLOCKI 1952; FRIEDE 1959b; TALANT! 1959; DELONG and BALOGH 1965; COLMANT 1967; SCHACHENMAYR 1967; LABEDSKY and LIERSE 1968; SCHUTTE 1971), or about both SDH and CYO (SHIMIZU et al. 1957; NAUMANN 1968; SCHAFFNER 1970; DIEDEREN 1970; WENZEL et al. 1970). Though their activities are very low, both enzymes can be clearly demonstrated in the SCo. The lacking evidence of glycerolphosphate cycling supports the idea that glycolytic substrate oxidation is coupled to the function of LDH M-type subunits, i. e. that hydrogen transfer into the extracellular space prevails. On the other hand, hydrogen transfer into mitochondria is made possible by the action of the malate shuttle but this cycling is bidirectional, thus allowing also net transport of NADH into the cytosol. The metabolic signiflCance of both the NADPlinked ICDH and MDH in the brain is unknown. Any interpretation is complicated by the partly cytosolic partly mitochondrial localization of these enzymes (SALGANICOFF and KOEPPE 1968; LOVERDE and LEHRER 1973; FRENKEL and COBOFRENKEL 1973). A dual localization of both enzymes seems to be possible also in the SCO. Probable functions discussed by these authors concern the fixation of CO 2 , the maintenance of intra- and extramitochondrial citric acid cycle intermediates, furthermore a shuttle system for NADP/NADPH, and finally the relation to gluconeogenesis or to synthetic processes. However, as found by PATEL (1974) pyruvate carboxylase plays the major role in the COzfixation of the brain. Because this enzyme was not invcstigated our data lack evidence on this reaction in the SCo and the significance of both the NADP-dependent enzymes remains speculative. According to GOEBELL and PETTE (1967) the ratio intra-/extramitochondrial NADP-linked ICDH activity is related to the functional state of any cell. Because of the widely varying

Enzymatic Organization of the Subcommissural Organ . 29

distribution of this enzyme in the SCO its further differentiation appears to be of interest. Furthermore, in brains of adult rats and mice the NAD-linked ICDH predominates the NADP-dependent enzyme (STEIN et al. 1967; LOVERDE and LEHRER 1973). This ratio seems to be inverse in the SCO, c. ep., and ep. ch. pI. but not in the neuropil. The functional signiflCance of this finding remains also to be elucidated. Cerebral tissues are able to oxidize fatty acids to a very limited extent only (McILWAIN and BACHELARD I971). Thus, glucose represents the main oxidative fuel. In various ketotic states, however, such as during the neonatal period or during starvation or diabetic ketosis, oxidatio11 of ketone bodies can be an important source of metabolic activity (OWEN et al. 1967; ROLLESTON and NEWSHOLME 1967a; KRAUS 1974; RUDERMAN et al. 1974). The controlling factor is the concentration of ketone bodies in plasma and tissues (WILLIAMSON et al. 1971; KREBS et al. I971; SOKOLOFF 1973). Therefore, the presence of 3-HBDH in the SCO, c. ep., and ep. ch. pI. is remarkable, though its activity is very low in the SCo. On the other hand, this enzyme seems to be absent in the neuropil. In this context the studies of MARINETTI et al. (1971) are of interest: they show that the rabbit choroid plexus may use fatty acids as a source of energy. This possible fuel is stored in large lipid inclusion bodies in the ependymal cells of the rabbit choroid plexus (WEINDL et al. 1969) but in our material we did never observe similar inclusion bodies - especially in the SCO. Recapitulating our fmdings regarding the maximal catalytic capacities of enzymes of the energy-supplying metabolism, we state that the ependyma of the choroid plexus is characterized by a low glycogenolytic but both a high glycolytic and a high oxidative capacity, the main fuel being most probably glucose but possibly also fatty acids and ketone bodies. This suggestion is in accordance with in vitro-studies concerning Oz-uptake with corresponding substrates (QUAY 1963,1966; MARINETTI et al. 1971; VOTH et al. 1972). - The ciliated ependyma is characterized by a low glycogenolytic, moderate aerobic glycolytic, and a high oxidative capacity, the

possible fuels being glucose and - under certain conditions - also ketone bodies. To our knowledge in vitro-studies have not been performed on the c. ep. and no data are available concerning a possible oxidation of fatty acids. - The sea, however, is characterized by a relatively high glycogenolytic and high glycolytic but low oxidative capacity; the prevailing fuel is the carbohydrate breakdown, functioning even under anaerobic conditions.

3. Metabolic Differentiation and Secretory Activity of the seQ Both the c. ep. and ep. ch. pI. are favoured by their metabolic differentiation for a high continous activity. This fact is well substantiated for the ep. ch. pI. (see CSERR 1971). In consequence we may infer from our comparative enzymological data that the SCO is only capable either of a high but discontinous or of a contino us but loU! activity. This activity is generally thought to be a secretory one. The synthesis of secretory proteins, their intracellular transport, and their discharge depend on the energy-supplying metabolism (JAMIESON and P ALADE 1968, pancreatic exocrine cells). Therefore, we will discuss our results and conclusions with regard to the various steps of the secretory pathway. As shown in the ependymal part of the rat, the activity of glycogenolytic, glycolytic, and citric acid cycle enzymes are the highest in the nuclear/supranuclear area. The ultrastructural distribution of mitochondria as well as of the cisternae of the RER is essentially alike (see STANKA et aI. 1964). The supranuclear area is the first one which is labelled up to the maximum by 35S-cysteine - a constituent of the secretory material (ERMISCH et aI. 1971). These findings indicate a local correspondance of the energysupplying metabolism with the energy-dependent synthesis of secretory substance. Regarding the metabolic differentiation of the SCO we may suggest that the rate of protein synthesis is relatively lowin this organ. This idea is supported by the following facts: the ATP yield of glycolysis only is low; also the number of mitochondria and the activity of their oxidative

30 • WOLFGANG KOHL

enzymes is low; furthermore, the total RNA content of the ep. SCQ is low and only few ribosomes are attached to the ER membranes; the cisternae of the RER are distended with stored material (see STANKA et al. 1964; HERRLINGER 1970; CHEN et al. 1973); 35S-cysteine is rather slowly incorporated into the sulfhydrylrich secretory glycoproteins which are stored for a long period and discharged intermittently (ERMISCH et al. 1968. 1971; TALANT! 1969; DIEDEREN 1972; STERBA 1972, lit.). The routes of intracellular transport of secretory material are well known in a variety of exocrine and endocrine gland cells but they vary in different cells (see BAINTON and FARQUHAR 1970, discussion). In pancreatic exocrine cells the transport of secretory proteins from transitional elements of the ER to condensing vacuoles is the energy-requiring lock of intracellular transport and depends on oxidative phosphorylation (JAMIESON and PALADE 1968). The same energydependent lock between the ER and the Golgi complex has been described in the cells of the endocrine pancreas (HOWELL 1972; STEINER et al. 1970, 1974) as well as in the somatotrophic cells of the adenohypophysis (HOWELL and WHITFIELD 1973). In contrast, MORRE et al. (1974) in hepatocytes and RAMBOURG et al. (1973) in neurons have claimed the existence of permanent luminal continuity between the RER and the Golgi complex. - Because the metabolic differentiation of the SCQ is quite distinct from that of pancreatic exocrine cells we may infer that in the SCQ synthesis and intracellular transport of secretory material might rely on glycolytic phosphorylation; they might also be controlled by the glycolytic pathway though in aerobic conditions part of the pyruvate will be completely oxidized. This hypothesis is supported by observations which indicate that the route of intracellular transport and the mode of completion of secretory material are different from those of the exocrine pancreas: the ultrastructural findings of STANKA et al. (1964), VIGH et al. (1967), PAPACHARALAMPOUS et al. (1968), HERRLINGER (1970), MURAKAMI et al. (1972), and of CHEN et al. (1973) support the sequestration or fission of secretory vacuoles (or secretory sacs)

from dilated ER cisternae, thus indicating a certain compartmentation of the intracellular transport. These studies, however, lack in evidence that secretory glycoproteins are transported from the ER to the Golgi saccules (CHEN etal. 1973; but cf., WAKAHARA 1974). The role of the Golgi complex in the SCQ is contested (see p. 34). The mode of condensation of secretory material differs from that of salivary glands: condensing vacuoles and secretory granules are missing in the SCQ. The dark or dense granules which originate from the Golgi apparatus and which have been described as secretory granules (PAPACHARALAMPOUS et al. 1968; HERRLINGER 1970; MURAKAMI et al. 1972) are lysosomes (CHEN et al. 1973). Regarding to these findings CHEN et al. (1973) postulated that the secretory material might be completed within the RER cisternae. We may be able to support their idea by the following observation: in histochemical reactions the staining intensity of secretory material is essentially alike in the various parts of the ependymal cells though these reactions base partly on the carbohydrate partly on the protein moiety of the secretory substance. We agree with CHEN et al. (1973) as we cannot find any staining gradient towards either pole of the ep. SCQ. - Nevertheless, we must emphasize that in all species a somewhat different type of secretory material can be observed: granules, which are arranged in the most apical part only, which are larger in diameter, and which are more intensively stained by Bock's method as well as by the P AS- and DDD-reaction. These granules or "large secretory vacuoles" - first described by VIGH et al. (1967) - can be differentiated clearly from lysosomes by means of the diverse distribution of lysosomal marker enzymes; they represent most probably concentrated secretory material (see also PAPACHARALAMPOUS et al. 1968; HERRLINGER 1970). In many secretory systems discharge ofsecretory material is known to depend on oxidative phosphorylation (SCHRAMM 1967; JAMIESON and PALADE 1971, lit.). The accumulation of mitochondria in the most apical part of the ep. SCQwhich can be observed in all species - might support a similar energy demand. This fact,

Enzymatic Organization of the Subcommissural Organ . 3I however, would contradict to a secretory activity of the sea under anaerobic conditions. Thus, discharge of secretory material might also depend on glycolysis. - In contrast to the exocrine pancreatic cells in which exocytosis appears to be the sole way of secretory discharge in the sea exocytosis seems to be paralleled by apocrine secretion, e. g. fission of apical protrusions. These protrusions are often observed on the ventricular surface of the ep. sea; they are filled up by secretory material and by lysosomes - the latter being possibly engaged in the complete discharge of the former one. Apical protrusions are also very numerous during the perinatal period in which the sea appears to be more active (KOHL and LINDERER 1973). A basally situated accumulation of mitochondria which we may confirm by our results has been discussed recently by KIMBLE and M0LLGARD (1973) as one of several evidences for a basal secretion of the ep. sea. We might argue that these mitochondria might also be involved in transport of the reverse direction, e. g. of substances of low molecular weight; furthermore, evidence is lacking for a concentration of secretory material which may compared to that of the most apical area of the ep. sea. Summarizing we discuss that the secretory process in the sea may depend on - and probably is controlled by - the glycolytic pathway. Thus, secretion may be possible even under anaerobic conditions - but during short periods only; in the same way secretion may be independent on blood glucose supply. In normal aerobic conditions glucose is completely oxidized but the low oxidative capacity of the sea suggests that the long-term or contino us activity is also low. Therefore, we may doubt "the very high degree of synthetic activity" proposed by M0LLGARD (1972, human fetus) and by KIMBLE and M0LLGARD (1973, rabbit).

4. Comments Concerning a Species Different Secretory Activity oftheSCO Regarding the obvious species different enzyme activities of both the c. ep. and ep. ch. pI.,

our results agree well with those of BARToNIcEK and LOJDA (1964, 1966). We may follow their suggestion that these differences are not sui generis in character because proportions of several key enzymes of the energy-supplying metabolism appear to be very similar in the different species. - aur results in the sea generally confirm the suggestion of HERRLINGER (1970) concerning a common principle of organization in the sea of all species. Though minor species variations in the molecular activity of individual enzymes may be reflected at the level of enzymatic activity, activities measured at optimum conditions "may, however, be regarded as relative measures of metabolic capacities in comparative analysis" (PETTE 1971). Therefore, some species specific variations ofenzymatic organization are of great interest; they are most obvious when the findings in the guinea pig are compared to those in the hamster. In the sea of the guinea pig the activities of HK, G6Pase, G6P-DH as well as of the glycolytic enzymes are the highest; M type subunits of LDH apparently predominate; the oxidative mitochondrial enzymes seem to be somewhat more active than in the other species. In the sea of the hamster, however, the activities of the enzymes mentioned are the lowest. Though some of them appear to be equally aCtive in the sea of the hamster and the mouse (HK, G6P-DH, oxidative mitochondrial enzymes), some other enzymes - especially the glycogenolytic and glycolytic ones - are less active in the sea of the hamster than in the sea of the mouse; furthermore, in the hamster the relatively low LDH activity seems to be represented mainly by H type subunits; thus, the M type subunits - which are characteristic for the sea of the other species - are apparently lacking. Morphological differences of the sea of both these species concern a) the vascular supply which is most dense in the guinea pig; b) the nuclei which in the guinea pig are darkly stained and contain more numerous nucleoli, whereas in the hamster they are deeply indentated, slightly stained, and contain less numerous nucleoli; c) the total RNA content which seems to be somewhat higher in the guinea pig; d) the

32 . WOLFGANG KOHL

secretory material: in the guinea pig the cells are densely filled up by darkly stained secretory granules. These granules are less numerous in the hamster. In contrast, in the SCO of this species large masses of secretory material are frequently found near the inferior pole of the nuclei; at the level of ultrastructure extremely dilated RER cisternae correspond to these masses and the cisternae contain secretory substance (KoHL unpublished; WETZSTEIN 1974). Similar dilated cisterns have never been observed in the SCO of any other species investigated in this study. Extremely dilated ER cisternae have been reported in the SCO of lower vertebrates and are thought to function in the storage of secretory material (STERBA 1962; STANKA 1967; STERBA et al. 1967; LEATHERLAND and DODD 1968; DIEDEREN 1970). In contrast, in the ep. SCO of the guinea pig the cells contain more numerous narrow cisternae of the RER (d. VIGH et al. 1967; PAPACHARALAMPOUS et al. 1968). The large secretory granules of the most apical part of the ep. SCO and the apical cytoplasmic protrusions are also more numerous in the guinea pig. The variations of the enzymatic organization suggest that the capacity of the energy-supplying metabolism is higher in the SCO of the guinea pig than in the SCO of the hamster. Thus, supported by the morphological differences, we would like to propose the following working hypothesis: the secretory activity of the SCO is higher in the guinea pig than in the hamster and is intermediate in the rat and mouse, i. e., the rate of synthesis, intracellular transport, and discharge of secretory material is the highest and the storage period the shortest in the SCO of the guinea pig. On the other hand, the enzymatic differentiation of the hamster SCO might be adapted to a longer storage period and, consequently, to a lower ratc of discharge of secretory material.

5. Possible Significance of Some Structure-bound Enzymes A species different secretory activity of the SCO may also be supported by species different

activities of enzymes which might be related to the secretory process in a direct or indirect manner. The distinct activity levels of G6Pase might mark a varying content of membranes of the endoplasmic reticulum in the SCO; these membranes might be packed more dense in the guinea pig but less dense in the hamster. This idea is supported by the activity levels of NADPHdehydrogenase which are also species different being high in the guinea pig but low in the hamster. This enzyme is most probably also located in ER membranes. The same might be true of NADH dehydrogenase activity. However, this enzyme is a constituent not only of the microsomal electron transport chains (see DALLNER et al. 1966) but also of the mitochondrial respiratory chain (SINGER and GUTMAN 1971, review). When studying the microsomal NAD(P)H dehydrogenases, tetrazolium salts - especially NT - have been used as artificial electron acceptors just as ferricyanide and cytochrome c. Comparing the different acceptors, a similar activity pattern has been found during the postnatal development of the microsomal system of the liver (DALLNER et al. 1966), or the same solubility of enzyme activities from liver microsomes has been observed (ERNSTER and ORRENIUS 1973). The mitochondrial respiratory chain-linked NADH dehydrogenase and the microsomal enzyme can be differentiated biochemically by rotenone or amy tal (SOTTOCASA et al. 1967) but we failed to differentiate both enzyme activities histochemically. The following reasons might explain this fact: The activity of NADH-NTreductase is very low in the brain. The coarse formazan of NT prevents any differentiation at the cellular level though the reaction is partly inhibited by amy tal or p-CMB. In the demonstration of NADH-TNBT-reductase staining is diffuse and granular but the reaction is not inhibited by amy tal or p-CMB. This might be caused by the higher redox potential of this acceptor (see e. g. KALINA 1966). A similar phenomenon is observed for the coupling between NBT and the respiratory chain (SLATER et al. 1963). For that reason also TNBT may act

Enzymatic Organization of the Sub commissural Organ' 33 closer to the microsomal flavoprotein enzymes than NT. Nevertheless, their quite different distribution pattern may support the localization of NADPH dehydrogenase in the ER membranes and of NADH dehydrogenase in ER membranes as well as in mitochondria. NADPH dehydrogenase - together with cytochrome P-450 - is a component of the microsomal mono-oxygenase system which is found in many organs. In contrast to the liver mono-oxygenases which metabolize many different xenobiotics as well as steroids and fatty acids and which are inducible by drugs (ORRENIUS et al. 1968), the enzyme systems from brain, kidney, intestine, and adrenals are not induced by drugs (FEUER et al. 1971). They show a much more restricted substrate specifity, too. In the steroidogenic tissues only steroids are oxidized, in the kidney cortex predominantly fatty acids, and in the lung mainly drugs (BEND et al. 1973; see also ORRENIUS et al. 1973, review). - The rotenone-insensitive NADH dehydrogenase - together with cytochrome b 5 - is known to constitute a second electron transport system associated with ER membranes and to be involved in mixed-function oxidation (see e. g. HILDEBRANDT and ESTABROOK 1971; OSHINO et al. 1971; HRYCAY and O'BRIEN 1974). Both the microsomal dehydrogenases are thought to be equally distributed between smooth and rough ER membranes (SCHULZE and STAUDINGER 1971). The findings of MELDOLESI et al. (1971) and MORRE et al. (1972, 1974) suggest their presence also in membranes of the Golgi complex. The mono-oxygenase system is thought to be involved in many reactions (see ERNSTER and ORRENIUS 1973) which might also be related to the secretory process of the SCo. In this organ secretory substance might be completed by the ER membranes - as suggested by CHEN et al. (1973). Therefore, the ultrastructural distribution of these enzymes may be of interest. The fact of involvement of both dehydrogenases in the secretory process might be supported by their species different activities which correspond largely to the variations of the energy-supplying metabolism.

Regarding the proposed detoxifying function of the SCo (see STERBA 1972, review), the metabolism of xenobiotics by the mono-oxygenase system is of interest, too; so the question of pharmacological inducibility of this system in the SCO remains to be resolved. However, this induction is thought to be restricted to the liver (FEUER et al. 1971). In the exocrine pancreas cells the increase of this system induced by barbital reflects an increase of the secretory activity but not of drug metabolism (LAVIGNE and MARCHAND 1972). Further inside into the significance of these enzymes in the SCo might be obtained by the determination of their physiological substrates. NDPase activity throughout the ER membranes has been reported in several distinct secretory cells whereas some other glandular cells are lacking this enzyme of which the function is currently unknown (see GOLDFISCHER et al. 1971; PELLETIER and NOVIKOFF 1972; PINSLEY and SCRUTTON 1973; FRUHLING et al. 1974). In the SCo the same localization must be corroborated by ultrastructural histochemistry but this localization is very probable. PINSLEY and SCRUTTON (1973) take into consideration a possible role of ER-bound NDPase activity in the protein synthesis, PELLETIER and NOVIKOFF (1972) speculate about the association of NDPase with glucuronyl transferase. The species different activity of this enzyme in the SCO might be further evidence that NDPase is also related to the secretory process in this organ. Concerning both the AChE and ChE activity in the SCO, the data reported in the literature are very divergent: COLMANT (1967) and SCHUTTE (1971) were not able to demonstrate any activity of both enzymes in the SCo of the rat, whereas RECHARDT and LEONIENI (1972) have demonstrated both enzymes in cytoplasmic, nuclear, and ER membranes of the ep. SCo of the guinea pig and rat. Our own findings disagree with all of them but these differences suggest that this problem might be flfSt of all a methodical one. Because of its localization in the ER which is also observed in nerve cells (see e. g. PANNESE et al. 1974) and

34 . WOLFGANG KOHL because of its proposed role in the secretory process (WELSCH and PEARSE 1969) further work has to be done on AChE activity and distribution in the SCO. The species different activity of TPPase corresponds likewise to the findings so far discussed: the activity is the highest in guinea pigs and the lowest in hamsters. TPPase is thought to be a reliable marker of the Goigi apparatus (see CHEETHAM et aI. 1970, lit.). The enzyme is located in trans-Golgi cisternae and in some closely associated secretory droplets only (CHEETHAM et aI. 1971; BERGERON et aI. 1973; FARQUHAR et aI. 1974; liver). Some further studies suggest that the Golgi-associated TPPase and NDPase activities are likely to be reliable parameters of secretory activity (JONGKIND and SWAAB 1967; JONGKIND 1969, supraoptic nucleus; SMITH and FARQUHAR 1970, adenohypophysis; HAND 1971, salivary gland). Thus, we may suppose that in the SCO the species different TPPase activity reflects also a different secretory activity. The differences in the SCO diverge from the findings in the c. ep. and ep. ch. pI. (see also BARTONICEK and LOJDA 1964). Because of methodical differences the results of BARLOW et aI. (1967, sheep) and of SCHAFFNER (1970, spiny mouse) cannot be compared to our own ones. As already mentioned (see p. 00), the participation of the Golgi apparatus in the elaboration of secretory material in the SCO is contested; the role of the Golgi complex is thought to be a minor one if any (CHEN et aI. 1973). However, tracer studies suggest the completion of glycoproteins within the Golgi apparatus of many tissues (NEUTRA and LEBLOND 1966a, b; WHUR et aI. 1969; ZAGURI et al. 1970; NAKAGAMI et aI. 1971; WEINSTOCK and LEBLOND 1971; BENNETT et aI. 1974). The glycosyl transferases involved in this process have been demonstrated not only in ER membranes but also in Golgi membrane fractions (LAWFORD and SCHACHTER 1966; FLEISCHER et aI. 1969; GINSBURG and NEUFELD 1969; MORRE et aI. 1969; WAGNER and CYNKIN 1969; SCHACHTER et aI. 1970; SPIRO 1970; BERGERON et aI. 1973; MERRITT and MORRE 1973; RONZIO 1973; KEENAN et al. 1974). Though

these facts support evidence for the role of the Golgi complex in the formation of glycoproteins it must be considered that glycoproteins are constituents not only of secretory material but also of lysosomes and of the cell surface coat (see RAMBOURG et aI. 1969). The same idea has been discussed by CHEN et aI. (1973) regarding the intracellular route of secretory material and regarding the role of the Golgi apparatus of the SCO. In addition we may state that P ASstaining of the secretory material is only slight; therefore we may suggest that the glycosyl content of the secretory substance might be relatively low in the SCO of the species investigated. There is some evidence for a cell specific localization of some glycosyl transferases (see MERRITT and MORRE 1973; RONZIO 1973), furthermore for a different distribution of transferases and TPPase within the Golgi complex (FARQUHAR et aI. 1972; BERGERON et aI. 1973), and finally for cell specific routes of the intracellular transport of secretory material (see BAINTON and FARQUHAR 1970, lit.). Thus, the problem of involvement of the Golgi apparatus in the secretory process of the SCO is far from being resolved. The species different TPPase activity which corresponds to the metabolic differences suggests at least an indirect involvement in the process though the secretory glycoproteins might be completed within the ER cisternae. We may speculate wether an experimental stimulation of the SCO might give further insight into this question. Data so far available and concerned with experimental alteration of the seo are very contradictory (see and compare the following results: FARRELL 195 8 ; GILBERT 1958, 1960, 1963; OKSCHE 1962; TAYLOR and FARRELL 1962; VAN DER WAL et aI. 1965; PALKOVITS 1965b, 1968; GILBERT and ARMSTRONG 1966; LEATHERLAND and DODD 1968; BUGNON et al. 1969; M1LIN et al. 1969; SALORINNE et al. 1969; STERBA 1969, 1972; SCHUTTE 1971; DIEDEREN 1972; LEONIENI and RECHARDT 1972). The same problem arises in the lysosomal system for which a regulatory role in the secretory process by absorbing and degrading undis-

Enzymatic Organization of the Subcommissural Organ· 35

charged secretory granules has been demonstrated (SMITH and FARQUHAR 1966; FARQUHAR 1969; SMITH 1969, review). In our studies this system was best demonstrated by its AcPase activity which is known to be localized in lysosomes. Moderate to high activities of this enzyme have been reported in the sea of different species (LEDUC and WISLOCKI 1952; TALANTI 1959; aKSCHE 1962; ALTNER 1968; NAUMANN 1968; KOZLOWSKI 1969; DIEDEREN 1970; SCHAFFNER 1970; WENZEL et al. 1970; SCHUTTE 1971; M0LLcARD 1972). At the level of ultrastructure the enzyme has been found in lysosomes and dense bodies respectively (BARLOW et al. 1967; MURAKAMI et al. 1972) and also in the innermost Golgi saccules (CHEN et al. 1973). AcPase staining of lysosomes as well as their distribution within the sea vary obviously even within the ependymal cells. In the species investigated the staining intensities of lysosomal enzymes differ also in the surrounding neuropil. Therefore we might be reserved in the interpretation of species different results. This reservation may be confirmed by the distribution of NAGase activity which - compared to that of AcPase activity - appears to be inverse in the species investigated. Nevertheless, the different distribution of AcPase activity even in the medial and lateral part of the ep. sea of the hamster corresponds well to the different distribution of several other enzymes which have been already discussed. So it may be suggested that lysosomes might also be involved in the secretory process of the sea - as discussed for the secretory cells of many other glandular structures (SMITH 1969, lit.). Because states of increased secretory activity are paralleled by an increasing AcPase staining (SOBEL 1961 a, b) and by an increasing number of dense bodies (SMITH 1969) we may suppose that in the hamster sea the more numerous lysosomes of the lateral cells indicate a higher secretory activity. As shown by SCHUTTE (1971), the activity of lysosomal enzymes seems to increase in the sea under certain experimental conditions. However - as also evidenced by SMITH and FARQUHAR (1966) and by SMITH

(1969) - it must be considered that suppression of secretion is followed by a progressive increase of AcPase activity and of the number of lysosomes, too. Because both these secretory phases differ in the AcPase activity of the Golgi apparatus, ultrastructural-cytochemical studies will clarify the distribution of AcPase in the sea and the role of lysosomes in the secretory process. - The accumulation of lysosomes in apical cytoplasmic protrusions of the ep. sea together with secretory material might be related to the proposed process of apocrine secretion (d. p. 31).

5. Remarks on the Hypendymal Part oftheSCO Because of the heterogenous composition of the hypo sea our preparations do not allow any defInite coordination of enzyme activities of this part to its different cell types. Though the cells surrounding the hypendymal capillaries an area which is characterized by higher activities of several enzymes - have been identifIed as secretory ependymal and hypendymal cells and as astrocytes (SCHWINK and WETZSTEIN 19 66 ; PAPACHARALAMPOUS et al. 1968; lliRRLINGER 1970), any interpretation of our [mdings must be speculative for the following reasons. The authors cited above have described numerous glycogen granules in the astrocytes of the hypo sea. According to FRIEDE (19 65) astrocytes show a special tendency to contain glycogen; furthermore, they are characterized by a "very little supply of oxidative enzymes, except GIDH" (FRIEDE 1965). Glia cells are also known to contain 3-HBDH (FITZGERALD et al. 1974). Thus, the enzyme pattern of astrocytes has many characteristics in common with the pattern of the secretory cells of the sea. The same is true for oligodendrocytes, at least partly, though they are characterized by "more oxidative enzyme activities than normal astrocytes" (FRIEDE 1965). Therefore we may conclude that the stronger staining intensities of the hypo sea - which are observed in the demonstration of glycogen, of enzymes of glycogen metabolism, of G6P-DH as well as of glycolytic

36 . WOLFGANG KOHL

and of mitochondrial enzymes - are conditioned by the presence of astrocytes and oligodendroglia but not by a different enzymatic organization of secretory hypendymal cells. But no defmite conclusion is allowed concerning the secretory activity of the hypo seQ. G6Pase and - most probably - also NADPH dehydrogenase and AChE seem to be the sole enzymes which are present exclusively in the secretory cells of the seQ - the hypendymal part included. These enzymes might be helpful as markers in further studies on this part of the seQ using serial thin sections.

6. Concluding Remarks The purpose of our study was to investigate the enzymatic organization of the seQ. Enzyme activities may reflect the maximal catalytic capacity of the corresponding metabolic pathways and, therefore, of the energy supply of this organ. Comparing the fmdings in the seQ with the metabolic differentiation of the C. ep. and ep. ch. pl. of the third ventricle we inferred that the functional activity of the seQ - which is thought to be a secretory one - must be rather low. Though many details remain to be resolved our results confirm and extend the findings of NAUMANN (r968) and DIEDEREN (r970) in the seQ of lower vertebrates. We may also confirm the suggestion of HERRLINGER (r970): the species different findings are only quantitative variations of a common principle of organization and function of this organ. The indications for a low secretory activity of the seQ might be helpful in the further discussion of the physiological significance of this organ. Though the seQ - at least in the guinea pig is densely vascularized it may potentially base its energy supply on anaerobic carbohydrate breakdown. We cannot decide if this capability is of biological significance or if this kind of differentiation reflects only the fact that this organ completes its differentiation very early in the phylogenesis as well as in the ontogenesis (see also KOHL and LINDERER 1973). Finally we may propose that the metabolic differentiation

and the mode of secretion characterize this organ as an early but little differentiated region of the brain.

Summary In the subcommissural organ (seQ) of the guinea pig, rat, golden hamster, and mouse the activity and distribution of enzymes related to the energy-supplying metabolism and of some marker enzymes of different cell organelles have been investigated by means of mostly modified histochemical methods. The results were compared with findings in the ciliated ependyma of the ventricular wall and with those in the ependyma of the choroid plexus of the third ventricle. In the ependymal part of the seQ only a moderate activity of hexokinase is observed in its specialized columnar cells whereas a high activity is present both in the ciliated ependyma and the choroid plexus. - The staining pattern of glucose-6-phosphatase is similar to that of hexokinase but this enzyme is found in the seQ only. - Likewise hexokinase, glycogen granules and enzymes related to glycogen metabolism (phosphoglucomutase, uridine-diphosphoglucose pyrophosphorylase, glycogen synthetase and phosphorylase) are regularly found most numerous and active in the nuclear and supranuclear area of the ependymal part. These enzymes are less active in both the other ependymal regions. - Uridine-diphosphoglucose dehydrogenase could not be demonstrated in the

seQ.

The NADP-linked enzymes of the pentose phosphate shunt, glucose-6-phosphate and 6-phosphogluconate dehydrogenase, show a moderate activity which decreases also from the nuclear towards the apical area of the ependymal cells of the seQ. Enzymes of the glycolytic pathway, such as glucosephosphate isomerase, fructose-6-phosphate kinase, fructose-r,6-diphosphate aldolase, glyceraldehyde-3-phosphate and lactate dehydrogenase, are highly active in the seQ and are located mainly in the supranuclear area, too.

Enzymatic Organization of the Sub commissural Organ . 37

Fructose-l,6-diphosphatase could not be demonstrated thus indicating that in the seQ the pathway is most probably only glycolytic but not gluconeogenetic. Compared to the ependyma of the ventricular wall and of the choroid plexus, in the seQ the M type subunits of lactate dehydrogenase predominate. Glycolytic enzymes are also very active in the choroid plexus but less in the ciliated ependyma. Compared to the ciliated ependyma and especially to the ependyma of the choroid plexus, the activities of enzymes which are only present in mitochondria (NAD-linked isocitrate dehydrogenase, succinate dehydrogenase, N ADlinked malate dehydrogenase after preextraction, cytochrome oxidase, 3-hydroxybutyrate and glycerolphosphate and glutamate dehydrogenase) are relatively low. Mitochondria are accumulated near the superior pole of the nuclei as well as in the most apical part of the ependymal cells. - The staining pattern of NADP-linked isocitrate and malate dehydrogenase as well as of NADH dehydrogenase suggests that these enzymes are localized both in and out of mitochondria. The extramitochondrial activity of the first two enzymes might be localized in the cytosol. The extramitochondrial activity of NADH dehydrogenase might be localized in the endoplasmic reticulum. Localization in this organelle is very likely in the case of NADPH dehydrogenase and of glucose-6-phosphatase and is also probable in the case of nucleoside diphosphatase; these enzymes show largely corresponding species different activities. - For acetylcholinesterase and cholinesterase positive results were obtained in the guinea pig only. Thiamine pyrophosphatase appeared to be restricted to the Golgi complex; a species different activity of this enzyme was also observed. The lysosomal system was to be demonstrated in the best way by its acid phosphatase activity. Lysosomes decrease in number towards the apical area of the ependymal cells of the SeQ; clusters of lysosomes are observed in apical cytoplasmic protrusions. A lysosomal localization is likewise very probable for E 60o-resistent esterase, for sulfatase, ~-glucuronidase, and N-

acetyl-glucosaminidase. All of the lysosomal enzymes show species different activities. Alkaline phosphatase and carbonic anhydratase could not be demonstrated in the seQ. In the hypendymal part the enzyme activity pattern resembles to that of the ependymal one. However, glycogen granules appear to be more numerous and glycogen metabolizing enzymes, enzymes of the pentose phosphate shunt as well as glycolytic and mitochondrial enzymes appear to be more active than in the ependymal part. Because of the very heterogenous composition of the hypendyma we failed to coordinate the enzyme activity pattern to the different cell types of the hypendyma. We suppose that the observed differences of staining intensities are conditioned by the presence of astrocytes and oligodendroglia in the hypendyma but not by a diverging enzyme activity pattern of secretory hypendymal cells compared to the ependymal ones. These cells contain apparently glucose-6phosphatase, NADPH dehydrogenase, and acetylcholinesterase whereas astrocytes and oligodendroglia do not. Based on these data the seQ is characterized by a high glycogenolytic and glycolytic but low oxidative capacity. It is concluded that the seQ bases its energy supply predominantly on carbohydrate breakdown which might function even in anaerobic conditions. This kind of metabolic differentiation is in contrast to that of the ciliated ependyma and - more distinct to that of the ependyma of the choroid plexus both of which exhibit a low or lacking glycogenolytic, moderate to high glycolytic but high oxidative capacity. In consequence, it is inferred that this metabolic disposition favours the seQ only for the performance of either a high but discontinous or continous but low secretory activity, the latter being more probable. This idea is discussed regarding the synthesis, intracellular transport, and discharge of secretory substance. The investigated rodents vary in their enzymatic as well as in their morphological organization. In the seQ of the guinea pig the activities of hexokinase, glucose-6-phosphatase, glucose-6phosphate dehydrogenase, glycolytic and mitochondrial enzymes are relatively the highest;

38 . WOLFGANG KOHL

the M type subunits of lactate dehydrogenase predominate. All these activities are relatively the lowest in the seo of the hamster. The seo of the guinea pig is most densely vascularized; it shows apparently a higher RNA content; the membranes of the endoplasmic reticulum are obviously more densely packed; the ependymal cells contain more numerous secretory granules. On the other hand, in the seo of the hamster secretory material is concentrated in the nuclear area; extremely dilated cisternae of the endoplasmic reticulum are observed. From these findings the following hypothesis is deduced: the secretory activity of the seo is higher in the guinea pig than in the hamster and is intermediate in the rat and mouse, i. e., the seo of the guinea pig is favoured for a more continous

or for a higher rate of synthesis, transport, and discharge of secretory material whereas the hamster seo is adapted to a longer storage period and a lower discharge rate of secretory substance. This idea is supported by corresponding species different activities of enzymes which are localized in the endoplasmic reticulum, Golgi apparatus, and lysosomes.

Acknowledgements The author is grateful to Prof. Dr. D. Pette, Konstanz, for critical discussion of this study, furthermore to Mrs. 1. Kohl for technical assistance and to Miss E. Reil for preparing the photographs.

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