Electron microscopic demonstration of hyaluronidase sensible proteoglycans at the presynaptic area in mouse cerebellar cortex

Acta h istochem. Bd. 55, S . 300-316 (1976) Unidad de Investigaciones Bi ologicas, Facultad de Medieina, Universid ad d el Zulia , Maracaibo, Venezuela

Electron microscopic demonstration of hyaluronidase sensible proteoglycans at the presynaptic area in mouse cerebellar cortex By HAYDEE V. CASTEJON and ORLANDO J. CASTEJON With 11 figure;; (R eceived July 26, 1975)

Summary By application of electron cytochemical techniques to cere bella r t issue, the presence of proteo, glycans was demonstrated at th e axoplasmi c matrix of mo ssy fib er en dings. Block s of glut a ra lde h y de (G) fixed mouse cere be llu m wer e processed a ccording t o the following pr ocedures : a) Some pieces of ti ssu e wer e post-fixed in os mi um tet ro x ide, deh ydrated by etha n ol and embedded in araldite . b) Other pieces wer e sec t ioned to 30 IJ.m thick and then imme rsed in Alcian blue solution p H = 2.7 followed b y osm ium t etroxide fixati on, dehydrated and em bedde d in arald it e (GABOUL procedure) . c) Parallel sli ces of (b) previous t o Alcian blue immersion were wa sh ed and in cubated in eit her methanol -Rei , n euraminidase , r ibon uc lease or testi cul ar hyaluronidase with thei r resp ective con trols. d) Other block s of G fixed ti ssu e wit h ou t a n y ot he r trea tment a nd fixat ion wer e dehydrated and e m bed de d in a r a ldite , U lt rathi n sections of n, band c were doubly sta ine d with uranyl acetate and lead cit ra te while ult.r a thin sec t ions of (d) were s ta ine d wit h the osmium coo rdi nat ion co m pou nd Os -DMEDA. The electron mi croscopic study rev ealed at the presynaptic axoplasm of m ossy fiber rosettes, the presence of a GABOUL and Os-DMEDA p ositive electron dense material su rroun d in g synapti c vesicles and co ntin uous with presynapti c dense proj ections. This material wh ich co inc ides with cytonet di stribution wa s resistant t o neuraminid ase and ribonucleas e and sens ible t o h yaluronidase and ca r box y me t hylation . These findings permit 11.S to concl ude that the a x op las m ic mate rial of mossy fiber end ings is co nstit uted by proteoglyeans in wh ich hyaluroni c a cid a nd chond roit in 4and/or 6-s111phato are present. The p rob able im po r t a nce of these prote oglycans in syn a p t ic m echanisms is also discussec.

Introduction Th e existenc e of mucopoly sa cch arides as normal n er ve t iss ue co nstitue nts of se ve ra l mammalian brain has been studied by bi och emical and light mi croscope histochemical analysis (BAIRA'l'I 1953 ; BRANTE 1957, 1959; CASTEJ ON 1969 , 1970a, 1970b; CHOUINARD 1962; CLAUSEN and HANSEN 1963; CLAUSEN a nd RosENKAsT 1962 ; HESS 1953; K URlYAMA and OKADA 1971; MARGOLIS 1967 , 1969 ; MARGOLIS and MARGOLIS 1974 ; ROBINSON and GREEN 1962; SINGH and B ACHHAWAT 1965, 1968 ; SULKIN 1960; SZABO and ROBOZ-EINSTEIN 1962; Vos et al. 1969 ; YOUNG a nd ABOOD 1960) which

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have shown the presence of different fractions of acid mucopolysaccharides such as hyaluronic acid, chondroitin sulphates, heparin, heparan sulphate, dermatan sulphate and other glycoproteins. By light microscope histochemical studies carried out in our laboratory, we have demonstrated hyaluronic acid and chondroitin 4- and/or 6-sulphate in the neuronal cytoplasm of the central nervous system of different vertebrates (CASTEJON 1969, 1970c, 1971) as well as another sulphated polysaccharide resistant to testicular hyaluronidase in the glycocalyx of certain neurons (CASTEJON 1970b, 1970c). Following these studies we have applied some electron cytochemical techniques to nerve tissue using Alcian blue and Os-DMEDA as electron stainings for revealing acid polysaccharides at the mouse cerebellar synaptic region (CASTEJON and CASTEJON 1972, 1972a, 1972b, 1974) and have demonstrated the presence of an electron dense material, presumably a proteoglycan, in the axoplasmic matrix of mossy fiber synaptic endings. PFENNINGER et al. (1969, 1971a, 1971 b) and AKERT et al. (1972) applying the bismuth iodide-uranyl and lead (BIUL) technique to the nerve endings in combination with freeze-etching, have postulated the existence of a presynaptic vesicular grid which seems to be an ideal means of dispensing transmitter towards the receptive element on the postsynaptic surface. In addition, GRAY (1972, 1973, 1974) has described at nerve endings, the presence of a presumed proteinaceous material throughout the presynaptic bag which he has termed "Cytonet". According to the characteristics of the cytonet it would seem to coincide with the axoplasmic dense material described above by us. Since the presynaptic axoplasmic material may be engaged in synaptic transmission mechanism and since proteoglycans or any other negatively charged macromolecule, having a great ability to bind or exchange water and selective ions such as Na" and K+, may be related with ion transport and nerve conduction, we have considered it necessary to follow up the cytochemical studies at nerve endings in order to distinguish the composition of these presumed proteoglycans which are present at the presynaptic area. The present communication primarily deals with the cytochemical demonstration of hyaluronidase labile proteoglycans in the axoplasmic matrix of mouse cerebellum mossy fiber endings. These proteoglycans may partially constitute the chemical network of the presynaptic cytonet. This type of synaptic ending has been chosen because its large dimensions permit such a study,

Material and Methods Adult normal albino mice of both sexes were used. The animals were arranged in 3 groups so that each group received a different preparation procedure for the ultracytochemical study.

First group,- Fixation in glutaraldehyde and OsO 4 Without previous anesthesia, the Ist group was beheaded, the brain was quickly removed and the cerebellum sectioned in small pieces which were fixed by immersion in 3 % glutaraldehyde + 0.1 M phosphate buffer pH = 7.4 for 4 h at 4 °C and postfixed in phosphate buffered 2 % OsO 4 for 2 h. The blocks were then rinsed in a similar buffer, dehydrated in graded ethanol baths and

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embedded in araIdito. Thick sections (0.5 to 1 p,m) were cut and examined by light microscope to determine appropriate areas for thin sectioning. Thick sections were mounted on uncoated copper grids and doubly stained with uranyl acetate and lead citrate at high pH. The thin sections were examined with a Siemens Elmiskop I electron microscope.

Second group; Os-DMEDA technique The 2nd lot of animals was treated according to the SELIGMAN et al. (1968) technique. SmalI pieces of cerebellum were fixed in 5% glutaraldehyde 0.05 M phosphate buffer pH = 7.4 for 2 h at 4°C. Once fixation was completed, the blocks were a1Iowed to rinse for 3 h in a similar buffer to which 0.22 M sucrose was added. Without secondary fixation the pieces were dehydrated by ethanol and embedded in araldite, Ultrathin sections mounted on nickel grids were etched by immersion in dimethylformamide for 20 min and then immersed in 1 % Os-DMEDA in aqueous solution for 16 h at room temperature. After staining, ultrathin sections were washed in several changes of distilled water, dried on filter paper and examined in the electron microscope. Controls consisted of unstained parallel sections which had so little contrast that the electron micrographs were not included here.

Third group;

GABOUL

method

In this group. mouse cerebellum was fixed by immersion in 6.5 % glutaraldehyde (G) phosphate buffer, pH = 7.4 for 2 h. After fixation, tissues were cut into slices of about 30 p,m thick with a SMITH-FARQUHAR tissue sectioner, washed and subsequently immersed in 0.1 % Aloian blue 8 GX (I.C.I. England & Allied Chem. U.S.A.) + acetate buffer pH = 2.7 for 9 to 15 h. After washing, tissues were immersed in 2% Os04 -veronal acctate buffer, pH = 7.4 plus 6.7% sucrose for 2h, dehydrated and embedded in araldite. Ultrathin sections stained with uranyl acetate and lead citrate were examined under the electron microscope.

Methylation Some G fixed and carefully washed 30 p.m slices of tissue were dehydrated by methanol. Methylation took place in 0.1 N HCI in absolute methanol for 15 h at 37 DC foI1owed by rehydration and impregnation in Alcian blue and subsequently in OS04 as described above. Control experiments were made by incubation in 100 % methanol without HCI for the same time and temperature.

Enzymatic digestion Para1Iel slices of same G fixed nerve tissue after washing in phosphate buffer pH = 7.4 were incubated in the following enzymes: a) bovine testicular hyaluronidase (Sigma Type V) 0.05 % in phosphate buffer pH = 5.5 for 3 h at 37 "C. b) neuraminidase from CI perfringes (Sigma Type V) 0.05 % in phosphate buffer pH = 5.5 for 4 hat 37 "C. c) ribonuclease A from bovine pancreas (Sigma Type HA) 1 mg/ml in distilled water for 1 h at 37°C. Control experiments were carried out using the respective buffer solution under identical conditions without enzyme. After incubation, tissues were impregnated in Alcian blue and OS04 and processed as described above. All ultrathin sections were doubly stained with uranyl acetate and lead citrate and examined in the electron microscope.

Results The cerebellar cortex initially fixed with glutaraldehyde, postfixed in OS04 and doubly stained with uranyl and lead salts was only used as a control preparation since its detailed results at the mossy fiber endings have already been published (CASTEJON and CASTEJON 1972a), therefore it is sufficient to say that again we observed the presence of a fine granular, slightly moderate electron dense material de-

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Fig . 1. Glutaraldehydc -osmium t etroxi de f ix ation . U lt rath in section s st a ine d wit h u ranyl ac etate a nd lea d cit ra te . Mossy fiber rosette (l11R) sh owin g a cen t ra l core formed by clustered mitoch ondria (m) and hundreds of clear sphe roi da l synapt ic ve sicles (sv). A sca rce h om ogen eou s f ine gran u lar modera te electron den se m ateri al (at 'r ow ) appears barely v isib le di sp er sed th ro ugho ut t h e a xo plas mic m a trix and surroun d in g th e sy n a p t ic vesi cles. The a rrow p a irs poin t a t t h e asymmetric syna ptic co ntact s with gran u le cell dendri tes (Gr ). X 72 000.

posited throughout t hc mossy axoplas mic rosette and closely sur rounding the synaptic vesicles (Fi g. I). In those regions devoid of synapt ic vesicles or where they appeared fairly apart this material was easily evident . At the level of sy naptic complexes a continuity of such a material with presynaptic dense proj ections was also noted. Os-DMEDA staining The application of the coordination compound Os-Dl\IEDA to the cerebellar tissue distinctively stained the ax oplasmi c matrix of mossy ending as well as its synaptic complexes and dendro-dendritic junctions (Fig . 2). Howev er , the surro unding fine st ruct ural eleme nts such as limiting membranes of mos sy rosettes, granule and GOLGI cell dendrites and those of organelles s uch as synapti c vesicles a nd mitochondria had so little elect ron density tha t it was difficult to make a clea r delineation of them. The remarkable feature noted with this st aining was the select ive elect ron opacity of the axoplasmic matrix which unlike that observed in glut ara lde hyde -OaOj ti ssue , showed a highly cont rasted fine gra nular and fibrillar material sur rounding the syn-

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Fig. 2. Ultrathin section of G-fixed mossy ending etched with dimethylformamide and stained with an aqueous solution of I % Os-DMEDA. The electron micrograph illustrates at the mossy rosette (MR) an increasing amount of a fibrillar and granular axoplasmic dense material which is also seen with a high intensification of its contrast. Note the electron lucent hyaloplasmic matrix of granule cell soma (Gr). X 70000.

aptic vesicles which were seen as clear spheroidal spaces. This dark axoplasmic material of mossy rosettes stood out against the electron lucent hyaloplasmic matrix of granule cell soma and dendrites. A marked contrast of nuclear chromatin and free ribosomes of the neighbouring granule cells was also noted. GABOUL im pregna ted presynaptic endings Because of the poor penetration of Alcian blue into nerve tissue, satisfactory staining was only observed in the periphery of the tissue block. Therefore, ultrathin sections studied have been taken only from the superficial zone of the block. With the GABOUL impregnation method the cerebellar cortex showed an overall staining much better than that seen in the glutaraldehyde-Osfr. fixed tissue. At the mossy fiber rosette a moderate to high electron dense material became evident surrounding synaptic vesicles and spread throughout the ending (Fig. 3). We have already reported similar results in cerebellar tissue initially fixed by vascular perfusion with glutaraldehyde followed by a mixture of glutaraldehyde-Alcian blue, and so as to show this

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Fig. 3. Fixation by immersion in glutaraldehyde-Alcian blue and osmium tetroxide. Ultrathin section stained with uranyl and lead salts (GABOUL technique). The mossy rosette (MR) shows an electron dense homogeneous fine granular material dispersed throughout the axoplasmic matrix and surrounding synaptic vesicles. X 60000.

fact, a reproduction of an electron micrograph of these previous results is included in the present paper (Fig. 4). The GABOULmethod, in contrast to the Os-Dl\lEDA staining, simultaneously offers a good preservation of the fine structural elements including plasma and intracellular membranes. Therefore, it enabled us to establish that certain spheroidal synaptic vesicles were electron lucent while others were moderately stained and that the material surrounding the dense core vesicles appeared of an electron density higher than that covering the clear synaptic vesicles. In addition, synaptic complexes and dendro-dendritic junctions showed a considerable increase in electron density. A continuity between the axoplasmic dense material and the presynaptic dense projections was seen. However, a clear distinction could be made between both, because the presynaptic projections showed an electron density higher than that of the axoplasmic material. The latter, besides, appeared attached to the entire limiting membrane of the mossy fiber ending. Searching for other endings at the molecular layer, positive GABOULreaction was noted at the spine synapses and at

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Fig. 4. Reproduction of previously published micrograph in which cerebellar tissue was primarily fixed by vascular perfusion with glutaraldehydc-Alcian blue mixture followcd by immersion in a fresh similar mixture and postfixcd in osmium tetroxidc. Ultrathin sections stained with uranyl and lead salts. As shown in Fig. 3 an axoplasmic dense substance is scen at the mossy rosette (MR) dispersed in masses and surrounding clear spheroidal synaptic vesicles. X 120000.

some climbing fiber endings. Both endings synapse showed a moderate electron dense axoplasmic matrix.

PURKINJE

cell dendrites and

Effect of Methylation This treatment had a drastic effect on membranous elements which appeared disrupted. However, the general outlines of mossy ending and its organelles as well as its relationship with granule cell dendrites and soma were still recognized. Nuclei, ribosomes, mitochondria and basal membrane of capillary wall appeared moderately

Fig. 5. GABOUL staining technique aftcr carboxymcthylation of G-fixed mossy fiber rosettes. The axoplasmic dense material of the mossy rosette (MR) is only vaguely recognized outlining the synaptic vesicles, indicating thc blocking of carboxyl groups and its unreactivity with Alcian blue. Note also the partial decrease in opacity of the synaptic complex (arrows). Compare with control experiment Fig. 6. X 48000. Fig. 6. Control experiment for carboxymcthylation. Regardless of thc drastic action of methanol on membranous elements, the electron dense axoplasmic material is present at the mossy rosette (MR) surrounding thc synaptic vesicle outlines (triangles). Note also the high electron density of synaptic complexcs (arrows). X 60000.

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a nd O. J .

CASTE JUN

staine d . At mossy endings t he profiles of sy naptic vesicles were almos t undistinguished , the axoplas mie matrix was diminish ed , the extravesicular mat erial appeare d clear and almost unstained (Fig. 5). On the ot her hand, the elect ro n density of synapt ic complexes, dendrodendritic juncti ons a nd presynapti c dense projecti ons was only slight ly diminished. Control experiments (Fig. 6) clearly sho wed t he well st ained synaptic complexes and dendrodend ritic junctions and at the mossy rosettes the profil es of synaptic vesicles surrounded by a gra nula r a nd fib rilla r mat erial. Effe c t o f e n zy m at ic d e gr ad a ti on Slices of cerebellar t issue that had been in cubat ed with either hyaluronidas e, neuraminidase, ribonuclease or in t he correspo nding buffer cont rol solution at times showed its ultrastructure moderately altered giving the impression of a lesser strength of the overa ll staining. H owever , the different organelles could be clearly identified . Hyaluronidase treatment cau sed an almost complete degradation of the GABOUL po sitive axoplasmic material of mossy fiber rosettes (Fig. 7) while the general morphology of these rosettes was not notably altered thus it permitted us to recognize the effect of the enzyme on the a xoplasmic matrix. In the hyaluronidase control ti ssue, regardless of the notable ext ract ive a ction of buffer , the ext ra vesicular mate-

MR MR

7 F ig. 7. The effect of testicular h yaluron idase trea tment on G-fixed mossy endings foll owed by GABO U L t echnique . The en zymatic t reatm ent alm ost com plete ly degra ded the elec t r on den se material present in the m ossy r ose tte {M R} axoplasmi c ma t r ix . Pre - a nd postsynapti c densit ies {arrow } a lso a ppear pa rtially degra ded. x 54000.

Elect ron m icro scopic demonstration

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Fig , 8, This picture may serve as control ex perim en t for hyaluronidase and neuraminidase treatments shown in Figs. 7 and 9. The G-fi xed mossy ending (MR) wa s incubated in the phosphate buffer withou t en zy me . The m oder ately e lect ron de nse material persists in the axop lasmie matrix. X 60000.

rial ofa xoplasm ic matrix was st ill evide nt (Fig. 8). The sy na pt ic densities on the contrary showed a moderate diminution in both hyaluron idase and cont ro l tissue. This effect would be attribut ed t o th e ext ract ive a ction of the buffer. Eventually the GABOUL positive axoplasmic mat erial was obser ved a t contro l t issue , continuous with the sy na pt ic dense pr ojecti ons. In:hyaluronidase treated ti ssue these projecions only a pp eared pa rt iall y a ffected in sp .t.e of the alm ost complete disappearance

MR

9 Fi g . 9. E ffect of neuraminidase in G-f ixed m ossy rosette (i11R) followed by GA B O UL st a ini ng . The elect ro n d ense axoplas mic mater ial is sti ll p resent surrou nd ing sy naptic ves icles. x 48000.

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Fig. 10. Effect of ribonuclease incubation on G-fixed mo~sy ros ette (11'1R) followed by GABOUL staining. N ot e the moderate electron den se material completely filling the mossy ending and surrounding sy na pt ic vesicles and edematous mitochondria. X 48000.

F ig. 11. Th is elect ron mi cr ograph illus t rates the con t ro l experime nt of ri bonuclea se treatmen t . The G-fi xed mossy en d ing (.MR) wa s in cubated in di stilled water on ly withou t en zy me. As is al so sh own in F ig . 10, a n electro n de nse g ran u lar- an d h omogeneous substance appears surroundi ng t he clear sp he ro idal synap t ic v esicles and ede ma to us mi t och ondria. X 60000.

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of the axoplasmic material. At the molecular layer, hyaluronidase digestion also almost completely removed the ext ravesicular GABOUL positive material of spine synapt ic endings . Neuraminidase dig estion affe ct ed t he axoplasmic material (Fig. 9) of mossy fib er rosettes t o a minimal exte nt . This material appeared with a density slight ly lesser than th at of the presynaptic dense proj ections. Th e syna pt ic complexes on the cont rary showed a m oderate weakness in both neuraminidase digest ed and control tissue. In rib onuclease in cubated sections , a n unexpect ed finding was the high preservat ion of t he mo ssy fib er a xpo lasmic material which appeared as a moderate to high electron dens e material complet ely filling t he mossy endi ng a nd surrounding the clear spheroida l synapt ic vesi cles and edematous mitochondria (Fi g. 10). In cont rol tis sue whi ch was only incubated in distill ed water, similar re sults were obtaine d but the a xop lasm ic material appeared mor e elect ron dense (Fig. 11). Presynaptic dense project ions, synaptic complexes and dcndrodendrit ic junct ions were al so seen well stained in both ribonuclease and control ti ssu e.

Discussion In ea rl ier cy toche m ica l st u d ies (CASTEJ6N a nd CAST E J6K 197 2, 1972a ; we h a ve suggeste d tha t the m od era te electron dens e m at eri al presen t in t h e m ossy rosette axop la smi e m a t r ix of m ou se cerebellum m ay be constituted b y p olysa cch arid e. prot ein com p lexes presuma bl y prot eogly ca ns. Prior t o d is cussing our present r esult s , we t hi n k tha t it is n ecessary to briefly con sider t h e presynaptic a rea. Obser va t ion s on n er v e ti ssue processed by various fi x ati on m ethod s h ave con sistently dem on strated th e p resen ce of a presu m ed proteinaceo us m aterial t h ro ug ho u t t he presynapti c b ag (G RAY 1972, 1973 , 1974) wh ich a ppears as a f in e a n d even ly d isp erse d n et work in t he spaces between the vesi cles . Su ch m a terial wh ich h as been refer red to b y G RAY ( 1973, 1974) as t he cytonet, e x te n d s througho ut t h e pre syuupt.ie b a g a n d is a t t ac he d to it s walls at vario us p oints a n d a lso con ve rges onto regul arly spaced points on t he presynapti c mem brane. These special attach me nt p oint s of t he cy to net a re t h e presynapti c d en se p roject ions . The cytone t wou ld a lso seem t o exte n d throu ghout t he who le ne uron a n d a better underst anding of its st ruct ure an d function m ay eluc idate problems of the m ode of action of synapses (GRAY 1973, 1974 ). PFENN IN GE R et a l. (1969) a fter BIUL t re atmen t h av e a lso described the presynapti c grid a s an h exagonal n et wor k in which the GRAY ' S dense projec t ions occupy the nodal po ints and form p olyhedric p ea k s orie nted t oward s the cy t op la sm of t he n erve t erminal. I n th e present st u dy g lutaraldehyd e a n d OS04 f ix a t ion moderately p reserve d an elect ron den se m ateri al in t he axop la smic matrix of m ossy fiber endi n gs. H O P W O O D (197 2) in a r eview on glut arald ehyde fixation , suggest s t hat this fixati ve b esides reacting with prote ins m a y a lso r eact with mueosu bstances , in a way which mi ght be s im ilar t o it s reaction with p olyhydroxyl com p ou n ds ther eby cross-linkin g them. Ther efor e, the fa ct tha t g lu taralde hyde m oder ately preserved the d en se a xo pl as mi c mat eria l, favours the idea t hat t hi s fixative m ay have cro ss-lin ked proteins and mucop olysaccharide -protein com p lexes.

Os-DJ\IEDA staining T he Os-coo rdi nation compound produ ced a hi gh p osit ive react ion in t h e axopla sm ic m a tri x of mossy rosettes , If we tak e into account SELIGMAN 'S (196 8) ex periments in wh ich t he a pplication of this compo und on rat ca rt ilage sho wed an in tensification of contrast of its basop h ilic material which is known to be m ainly constituted b y ch ond ro it in sulphate, we infer that by t he present results, 21

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we are d ealing with a po sitive rea ct ion for a cid polysacch arides p resumably ch ond ro it in sulp ha tes wh ich a re a lso known t o be present at n euron s (CASTEJ6s 1969 , 1970a , 1971 ; MARGOLIS and MARGOLIS 1974 ).

G ABOUL impregnation method The chemical a n d h istoch emical p roper t ies of Alcian bl ue ha ve been widely stud ied by the group of SCOTT et a l , (1964) and QUIl'.'TARELLI eta1. (l964a, b) . This dye is const ituted by a mixture of 4 geo m et rical iso me rs of the cop pe r phthalocyanin which con tains a certain n umber of isothiouron ium groups linked to the phthal ocyanin core by m ethylene b ri dges. T he oni um grou ps im pa rt its cationic c haracter to the d ye whi ch is in tensely att racte d by a ne gative electrost a t ic fi eld and whi ch could be generated in t he ti ssue b y a cidi c gro ups , especia lly those bearing carboxyla ted and sulph ated polyanion s. B EHNKEand ZELANDER (1970) h ave used this d ye in preparative procedure s for electron microscopy a nd m ore rec ently we have a lso a pplied it to nerve tissue (CASTEJ6N 1972; CASTEJ 6N a nd CASTE.J6N 1972a, 1974). The a pp lica t ion of GABOUL method to cere bella r tissue produced a selec ti ve electron opacit y of m ossy fib er axoplasmio matrix. This stain in g technique thereby permitted reproducible identifica tion of the elect r on dense material. Th is f inding suggests the presen ce of ca rb oxy la t ed or sulphated polyanion s t o which Alcian blue is bound. These polyanions may corres po nd to proteoglycans or acidic g lyeo p rote ins such as sialo or su lp hate d glyc opr ote in s. The sensibi lity of these complexes t o m ethyl a ti on an d h yaluronidase digest ion h elped to elu cid ate that they a re m ainly proteoglycans. Methyl ati on is p rincipall y em ployed in h istoch emistry t o bl ock the bas ophilia of polyanion s by esterification of carb oxyl gro ups and the re mo val of O-sulpha te ester . Our resul t s have shown t hat t he GABOUL positi ve e lectro n dense ma t er ial was bloc ke d b y methyl a t ion suggesting that either carbo xyl or su lphate gro u ps of carbohydrate-p rotein com plexes p resen t at m ossy ending were a ffected. In addition , the dense axoplas mic mater ia l wa s sens ible to t esticula r h yaluronidase. This enzyme splits every secon d B -(1 - 4)bond between N- a cet yl glucosamine and gluc uroni c a cid of t he g ly cosami nogly cans y ield ing a tetrasaccharid e as the end product. Since t he glycosam inoglycans h yal uro n ic a cid and ch ondroitin 4- a nd/or 6 -sulphate a re t he m aj or na t ural su bst ra tes of the enzyme, we conclude that t he dense axopl asm ie m at eri al present a t m ossy f iber en d in gs and othe r presynaptic areas, is constituted b y p r ot eogl ycans in wh ich one or several of t hese la t t er polysaccharides are p resen t . These results conf irm and ex te n d t o an ul t ras t ructural level our p re v iou s h ist och emi cal studies a nd arc consistent wit h MARGOLIS and ~1ARGOLIS'S (1974) biochemical f in d in gs in whic h the p resence of t hose polysaccharides wa s d emonstra t ed at n erve cell cy to plasm . Ou r results are al so in agreemen t with those of Vos et al , (196 9) who by su bce llular fra ct ion a t ion of m ou se br a in ob taine d h yaluronic a cid a nd chondroit in sulphate not only from the solu ble cy to plas m but also fro m syna p t oso mes and synaptic vesicles. In addition, K UIlIYAMA a nd OKADA (1971) have d emonstrate d that the major proport ion of 3sS-sul phate was inc orporated into ch on dr oiti n sulpha t e as m uc h in the soluble cytoplasm as in the synaptosomal complex es and cytoplasmic membranes of dev eloping mouse brain. H owever, our results differ from those ob t ai ne d by BONDAREFF and SJ OSTRAND (1969) who studying the effects of t esticular hyaluronidase on the ultrastructure of synaptosomes r eported that ther e was n o a ppare nt alteration after incuba ti on in the en zyme or it s corr espo n d ing buffer. The GA B OU L and Os-DMEDA posit ive material wa s obs erved coextensive with and sim ilar in a ppearance to t he material of p resynap tic dens e pr ojections . Simi la r featu res ha ve been re ported wi th t he E-P TA (GRAY 1973) a nd B IUL methods (PF ENNINGER 1971 a , 1971 b). GRAY (197 2) has a lso cons idered t he dense project ion s as concentrations of cytonet ma te ria l. H owever , in t he p resent study, p resynaptic projecti on s sh owed an electron density h igher t han t hat of t he a x opl asmic material a nd were in addition part ially res istan t to carboxymethylation a nd hyalu ro nidase treatmen t. These fa ct s per mit u s t o suggest that p resynaptic projection s ha ve eithe r a gr eater proteoglycan conden sation or a di fferent che mical com position . Sialic acids occ u r in n ature in gly cosidic linka ge as structural units of oligosaocharides, h om o and

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heteropolysaccharides, glycolipids and glycoproteins (TuPPY and GOTTSCHALK 1972). The presence in sialic acids of a carboxyl group which may eventually be responsible for the positive GABOUL reaction seen at the mossy axoplasm, made us apply neuraminidase as an enzymatic tool in order to cleave the y-ketosidic linkage joining the potential keto group of a terminal N -acylated neuraminic acid to an adjacent sugar residue in a polysaccharide and in this way demonstrate or discard its engagement in the GABOUL positive reaction present at the mossy axoplasmic matrix. After neuraminidase incubation the axoplasmic dense material persisted in spite of the moderate extractive action of buffer in the incubation medium. These findings indicate that carboxyl groups of sialic acids are not the main groups responsible for positive GABOUL reaction. However, sialic acid has been shown to occur in relatively large amounts in synaptosome fractions (BRt:NNGRABER. et al. 1967; WOLFE 1961), bound in part to mucopolysaccharides and in part to gangliosides and at the level of synaptic cleft (BONDAREFF and SJOSTRAND 1969) apparently not associated with mucopolysaccharides. Presumably the configuration of sialic acid into ganglioside or glycoprotein molecule made sialic acid unavailable to the enzyme digestion. In many cases the enzyme is more active against glycopeptides rather than glycoproteins (MARSHALL and NEUBERGER 1972). PFENNINGER (1971 b) has also noted that the neuraminidase-induced loss of acidic groups seems much weaker in synapses in situ than in the synaptosomes. From the above results we conclude that the proteinaceous material presumed by GRAY (1973, 1974) as constituent of cytonet might mostly correspond to proteoglycans in which either hyaluronic acid and chondroitin 4- and/or 6-sulphate or both are present. It must also be said that the granular or fibrillar material observed at the axoplasmic matrix does not reflect the native state of the proteoglycans but probably demonstrate a dehydrated, fixation-precipitated form of the macromolecule. The presence of these proteoglycans surrounding synaptic vesicles may suggest the probable interaction between these complexes and chemical transmitters i. e. acetylcholine. Interaction of acidic glycosaminoglycans with quaternary ammonium compounds has long ago been demonstrated by SCOTT (1955,1961,1962). It has also been confirmed that chondroitin sulphates bind curare and curare-like compounds (CHAGAS et al , 1958; EHRENPREIS and FISHMAN 1960; HASSON and CHAGAS 1959, 1962). Some of these authors (HASSON and CHAGAS 1959, 1962) have suggested that acid glycosaminoglycans may function as receptors for various quaternary compounds or may compete with any other receptor for the quaternary bases. In spite of the fact that up until today the latter suggestion has not been taken into consideration, we feel that it would be of great interest to reevaluate the possible role which proteoglycans play in cellular reaction of quaternary compounds, especially in relation with acetylcholine. Another possibility on the functioning of these protein-polyeaecharide complexes is that they could be involved in surface charges and binding mechanisms, water retention, cation exchange and viscoelastic properties, thus they may be engaged in synaptic transmission mechanism as was referred to in the introduction of the present paper. According to GRAY (1974) the synaptic vesicles lie within the mesh of the cytonet which may therefore also form a framework for guiding and/or moving the vesicles into strategic positions on the presynaptic grid for transmitter release. The findings obtained in cerebellar tissue incubated in ribonuclease have shown that not only was the axoplasmic dense material resistant to ribonuclease but it became more electron dense. Since this effect was also observed in tissue incubated in distilled water without enzyme, we have interpreted this distinct effect in the following way: Incubation media containing phosphate buffer has some extractive effect on tissue; the incubation medium in ribonuclease did not contain salts but only distilled water thus there was no extractive action of salt solution on the stained material of axoplasmic matrix. It must be noted that originally the ribonuclease digestion was only practised in order to study its effect on nerve cell soma. However, the fact that upon casually studying these nerve terminals, we observed a better GABOUL staining at nerve endings incubated with ribonuclease or distilled water only, impelled us to show these findings in the present paper. Nevertheless, it was also noted that mitochondria of these treated tissues were very edematous. This finding in 21*

314

H . V . CASTEJ6N a nd O. J . CASTEJ6N

addi ti on to the ma jor condensation of' a xo p las m io ma trix ca lls our atte ntion t o the possib le con d ens in g a ction of di stilled water on t he axop lasmi c mat erial or the degen era t in g effect (\VESTRUM 1973) that distille d . water may p roduce in a nerv e ti ssue incompletely f ixed .

Acknowledgements The a ut hors a re grea tly indeb t ed to Dr. A . ~f. SELlmIAN of t he S ina i H osp ital of B al t im or eMaryl a n d , U.S.A . for hi s kindness in supplyi ng u s t h e Os -DM E DA coord ina t ion com po u nd use d in t h is s t udy . We fur ther wish to t hank N ELLY MONTIEL, D IONA B OHORQUEZ, NANCY R INCON an d J osE ESPINOZA for va luable t echnical work an d MAVLYN \VHORMS SEMPRUN for skill fu l secretaria l assistance and Iingui st.ic im proveme nts. This project was part ially supported by resea rch gran t N o. 31. 26. S I -0256 from the Consejo N acional de I nvestigaciones Oientificas y Tecno loqicas (CON IC I T ) Venezuela.

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