Immunohistochemical localization of phosphonoglycosphingolipids in the nervous tissue ofAplysia

Brain Research, 327 (1985) 259-267

259

Elsevier BRE 10536

Immunohistochemical Localization of Phosphonoglycosphingolipids in the Nervous Tissue of Aplysia SACHIKO ABE l, TOSHIRO KUMANISHI2, SHIGEKO ARAKI 1and MEI SATAKE l

t Departments of Neurochemistry and 2Neuropathology, Brain Research Institute, Niigata University, Asahimachi-1, Niigata 951 (Japan) (Accepted May 22nd, 1984)

Key words: new water-soluble glycolipids - - phosphonoglycosphingolipids--Aplysia nervous tissue - tissue localization - - antiserum - - immunohistochemistry

A group of novel phosphonoglycosphingolipidswas isolated from the tissues of Aplysia 1,2 In the present experiment, antiserum was raised against total phosphonoglycosphingolipidsisolated from the ganglia. This antiserum seems specific to the oligosaccharide moiety of the glycolipids. It did not react with gangliosides isolated from mammalian brain. Of the total phosphonoglycosphingolipidsof the ganglion, GGL-V was strongly reactive, but GGL-I was hardly reactive with the antiserum. The indirect immunoperoxidase method in combination with light microscopy revealed staining of fibrous structures in the neuropil of ganglia, connective tracts and peripheral nerves. These fibrous structures often interconnected with supporting cells (glia cells). However, the neuron and its processes were stained not distinctly. Thus our results indicate that some of the major glycolipids isolated from the ganglion are mainly present in extraneuronal components in the nervous tissues of Aplysia.

INTRODUCTION Recently we found a group of novel glycolipids in the nervous and dermal tissues of a g a s t r o p o d , Aplysia kurodai 2,12. These glycolipids were classified chemically as phosphonoglycosphingolipids 1,2, which are similar to gangliosides in having an oligosaccharide chain linked to c e r a m i d e moiety, but differ from gangliosides in having a p h o s p h o n o c o m p o u n d instead of sialic acid. It is interesting that gangliosides are not detectable in the nervous tissue of some invertebrates s,12, including Aplysia, which contain phosphonoglycosphingolipids. Accordingly, it is conceivable that the two kinds of glycolipid exert the same, or a similar biological function in the nervous systems of different species in vivo. Many studies have suggested that the m a j o r sites of location of gangliosides in the nervous tissue are neuronal p l a s m a m e m b r a n e s including synaptic plasma membranes 3.4,7A3-15,18, where the lipid may exert its functions. In the present e x p e r i m e n t , antiserum

was raised against the water-soluble lipid fraction of ganglia, which consists of phosphonoglycosphingolipids, and the cellular location of phosphonoglycosphingolipids in the nervous tissue of Aplysia was studied immunohistochemically to obtain information on the biological functions of this new class of glycolipids. MATERIALS AND METHODS

Materials Phosphatidylcholine was p r e p a r e d from chicken egg yolk by the m e t h o d of Bligh-Dyer 5 using silicic acid (Mallinckrodt Chem. W o r k s ) - H y f l o Super-Cel ( W a k o Pure Chem. Ind. Ltd.), 2:1 (w/w) for column chromatography. Cholesterol ( W a k o Pure Chem. Ind. Ltd.) was recrystallized from absolute ethanol. Globoside was p r e p a r e d from h u m a n erythrocyte ghost as described by M i y a t a k e et al. 19. Bovine serum albumin was a product of Sigma Chem. Comp. and m e t h y l a t e d bovine serum albumin ( M B S A ) was

Correspondence: S. Abe, Department of Neurochemistry, Brain Research Institute, Niigata University, Asahimachi-1, Niigata 951 Japan. 0006-8993/85/$03.30 © 1985 Elsevier Science Publishers B.V. (Biomedical Division)

260 prepared as described by Mandel and Hershey~;. Donkey anti-rabbit Ig,[12-q]F(ab'): fragment, was purchased from Amersham. Plastic TLC plates (Polygram Sil G/UV254) were purchased from Macherey-Nagel.

Preparation of the total phosphonoglycosphingolipid fraction Phosphonoglycosphingolipid fractions of the nerve ganglion and dermal tissues were prepared as described by Araki et al. 1 from Aplysia kurodai, a sea gastropod, caught at Sado Island, Japan. Fresh ganglia or acetone powder of the dermal tissue was homogenized in a Waring blender with 20 vols. of chlor o f o r m - m e t h a n o l - w a t e r (30:60:10, v/v). Chloroform and methanol were added to the extract to give a ratio of chloroform-methanol-water of 8:4:3 by volume. This aqueous upper phase thus separated from the lower phase was washed once with the theoretical lower phase of Folch et al.q dialysed with distilled water, and lyophilized. The lyophilizate was dissolved in a small amount of 10 mM acetic acid and applied to a Sephadex G-50 column. The eluate in the void volume of the column was lyophilized to yield the total phosphonoglycosphingolipid fraction. Glycolipids of the ganglion, denoted as GGL-1V and G G L - V were isolated from the total phosphonoglycosphingolipid fraction by preparative TLC as follows. The lyophilizate, the total phosphonoglycosphingolipid fraction, was dissolved in chloroformmethanol-water (3:3:1, v/v) and applied to a preparative H P T L C plate (Merck). The plate was developed with n - p r o p a n o l - a m m o n i a - w a t e r (75:5:25, v/v). Lipids were located with iodine vapor and extracted with chloroform-methanol-water (3:3:1, v/v).

Dephosphorylation of phosphonoglycosphingolipids The total phosphonoglycosphingolipid fraction of the ganglion was treated with hydrogen fluoride (HF) as described by Itasaka and Hori w.

Preparation of anti-phosphonoglycosphingolipid rum

se-

Rabbits were immunized with glycolipids as described by Inoue and Nojima~ with slight modification. A sample of 2 mg of the total phosphonoglycosphingolipid fraction of the ganglia was mixed with

phosphatidylcholine and cholesterol in a ratio ot: 1:4:30 by weight, and then dissolved in 0.5 ml of chh> roform-methanol (2:1, v/v). This solution was mixed with about 15 ml of physiological saline bv stirring manually. The resulting micelles were collected b~ centrifugation at 20,000 g for 60 min and mixed with l ml of 0 . l % (w/v) MBSA in physiological saline. The mixture was left overnight at 4 °C, and then emulsified with an equal volume of complete Freund's adjuvant (DIFCO Labs.). Then 2 ml of this emulsion was injected into about 50 sites in the skin of the back of a New Zealand rabbit. After 3 weeks, 3 weekly booster injections of 1 mg of the total phosphonoglycosphingolipid fraction of the ganglia in incomplete Freund's adjuvant were given. The antiserum was obtained 10 days after the last injection.

Immunochemical assay (1) Ouchterlony's double diffusion test. Plates made of 1.0%. agarose in physiological saline were used. Undiluted antiserum was placed in the central well and various concentrations of antigens in saline without auxilliary lipids were placed in the outer surrounding wells. (2) Radioimmunodetection of antigens on TLC (RITLC). Antigens on TLC were detected radioimmunologically by the method of Magnani et al.> as described by Mori et al. >. The glycolipids were developed on a plastic TLC plate with n-propanol-ammonia-H_,O (75:5:25, v/v). Then the dried chromatogram was soaked overnight at 4 °C in PBS (8 g of NaC1.0.2 g of KCI, 2.9 g of Na:HPO 4" 12 H20 and/1.2 g of KH_,PO~ per liter, pH 7.5) supplemented with 3% bovine serum albumin, 5% horse serum and 0.1% sodium azide (solution A). Then the chromatogram was immersed in anti-phosphonoglycosphingolipid serum or preimmune rabbit serum diluted 1:30 with solution A and kept at 4 °C. After 6 h the chromatogram was washed with 5 successive changes of PBS and incubated for 12 h at 4 °C with [12sl]-Iabeled donkey anti-rabbit lg,F(ab'): fragment diluted to 1.5 x 11)6 cpm/ml with solution A. It was then washed thoroughly with PBS, air-dried and exposed to Ultrofilm (LKB) for 8 h at -80 °C. (3) Complement fixation test. The microtiter complement fixation test 2~ was used to determine the antigenic titers of glycolipids extracted from tissues of Aplysia. Gelatin veronal buffer, pH 7.5 (145 mM

261 NaCI, 0.15 mM CaC12, 0.5 mM MgCI 2 and 0.1% gelatin in 5 mM veronal buffer pH 7.5) was used as diluent. The glycolipid (1 mg/ml) was diluted 2-fold serially. To each diluted sample (25/A), 4 units of antiserum (25 kd), and 2 units of guinea-pig complement (50/A) were added and kept at 4 °C for 18 h. Then 2.5 × 106 sheep red blood cells sensitized with 3 units of hemolysin (25/~1) were added to the mixtures and incubated at 37 °C for 30 min. The antigenic titers were determined by reading the maximum dilution of the samples which showed 0% hemolysis. The units of antiserum, guinea-pig complement and hemolysin had been determined in the preliminary experiment by reading their maximum dilutions which had shown 100% hemolysis in the absence of glycolipids.

GGL- I SGL-I

GGL-IV

SGL- I I

GGL-V

SGL-I

GGL-IV

SGL-II

GGL-V

Immunohistochemical procedure The procedure 11 used routinely was as follows: the tissues were fixed in 10% formalin in 30 mM phosphate buffer, pH 7.4, and embedded in paraffin. Deparaffinized sections were pretreated with 0.3% H202-methanol to block endogenous peroxidase activity and with normal goat serum to block Fc receptors. Then the sections were incubated with a 1:40 dilution of rabbit anti-phosphonoglycosphingolipid serum in the first reaction and with horseradish peroxidase-labeled goat IgG antibody to rabbit IgG (1:20 dilution, E. Y. Laboratories Inc.) for 1 h in the second reaction. In the control, antiserum diluted 1:40 which had been absorbed with purified phosphonoglycosphingolipids (3 mg per 0.25 ml of the antiserum) or preimmune rabbit serum diluted 1:40 were used in the first reaction. The resulting sections were incubated in D A B - H 2 0 2 solution for 10 min, washed and mounted in balsam. Sections counterstained with hematoxylin-eosin were also prepared. In some experiments, fresh frozen sections were fixed with acetone or 10% formalin and processed as above. RESULTS

Chemical characteristics of the glycolipid fraction used for immunization As described in Materials and Methods, the watersoluble glycolipid fraction isolated from the ganglia of Aplysia was purified by dialysis and gel-chromatography. Several anthrone-positive spots were separated on TLC (Fig. 1 A - G ) . All these spots also gave

Fig. 1. Thin-layer chromatograms of glycolipids isolated from ganglia (G) and dermal tissues (S) ofAplysia kurodai. Materials were separated on HPTLC-plates of silica gel (Merck) in npropanol-ammonia-water (75:5:25, v/v) by the ascending method. Glycolipids were located with anthrone (A) or Dittmer-Lester reagent (B). a positive reaction with Dittmer-Lester reagent for phosphorous (Fig. 1 B - G ) and with ninhydrin, but a negative reaction with resorcinol reagent for sialic acid. The phosphorous compound in the water-soluble lipid fraction was highly resistant to acid hydrolysis, indicating that it had a phosphono structure. As described in the previous paper2, the percentage proportions of these glycolipids which were calculated on their phosphorus contents were: GGL-I, 4%; GGL-III, 14%; GGL-IV, 18%; GGL-V, 60%. The

262 chemical compositions of G G L - V and G G L - I of all above glycolipids are as follows: G G L - V , most probably the same with F G L - V I I and S G L - I I , is ceramide bis[2-aminoethylphosphono ( A E P ) ] pentaoside consisting of 1 mol each of glucose, 3-O-methylgalactose and galactosamine and 2 tool of galactose, and G G L I, p r o b a b l y identical with F G L - I I , is ceramide m o n o ( 2 - A E P ) pentaoside consisting of 1 mol each of glucose, galactosamine and fucose and 2 mol of galactose. Recently, structure of G G L - V has been identified as 3 - O - M e G a l ( 1 - + 3 ) G a l N A c ( I ~ 3 ) [ 6 - O (2'-aminoethylphosphonyl)Gal(1---~2)][2'-aminoethylphosphonyl(--~6)]Gal(1---~4)Glc(1-+l)Cer3.

lmmunological properties of the antiserum In O u c h t e r l o n y ' s double diffusion test, the antiserum gave a single precipitation line with the watersoluble lipid fraction of Aplysia tissues, but not with that of rat brain (Fig. 2). F u r t h e r m o r e , neither bovine brain gangliosides nor globoside from human erythrocytes gave a precipitation line with the antiserum (data not shown). The water-soluble glycolipid

fractions of the 3 tissues of Aplysia that gave precipitation lines in O u c h t e r l o n y ' s test and that of the nerve fiber were d e v e l o p e d on a thin layer plate, and the immunoreactivity of individual lipid were examined by the radioimmunological m e t h o d as described in Materials and M e t h o d s (Fig. 3B). The glycolipid fraction of ganglia treated with H F as described in Materials and Methods was also processed on the same TLC plate. A n o t h e r T L C plate, d e v e l o p e d under the same conditions, was sprayed with anthrone reagent (Fig. 3A). By comparing the densities of the spots of individual glycolipids in the two T L C plates, the glycolipids were divided into two groups: a strongly reactive group, containing G G L - V (SGL-II and F G L - V I I ) and 2 - A E P - f r e e G G L - V ; and non-reactive group, containing G G L - I and F G L - I I . m a r k e d with circles and triangles, respectively in Fig. 3. The oligosaccharide structure of the latter group differed from that of the strongly reactive group 2. In the liver, two anthrone-positive spots of glycolipid were identified: one which co-migrated with S G L - ! and reacted with the antiserum to the same extent as S G L - I : the other, which co-migrated with S G L - I I , reacted only weakly and its a u t o r a d i o g r a p h i c pattern indicated that it was a mixture of glycolipids. The c o m p l e m e n t fixation test was carried out as described in Materials and Methods. The titers of total water-soluble glycolipid of the ganglion, 2 - A E P free total water-soluble glycolipid of the ganglion and G G L - 1 V and G G L - V were 1:8000, 1:4000, 1:1000 and 1:2000, respectively (each original solution con-

s G

s G L r&

free

2-AEP

free

i!~ G G L - I V free

Fig. 2. Ouchterlony's double diffusion test on the reaction of water-soluble lipids of Aplysia tissues and rat brain, with antiserum raised in rabbit against water-soluble glycolipids of Aplysia ganglion. Undiluted antiserum was placed in all the central wells. In all cases, well no. I contained GGL-IV (500 ~ug/ml). Wells no. 2 to no. 6 contained glycolipids of dermal tissue (A), ganglion (B) and liver (C) of Aplysia and those of rat brain (D) at concentrations of 1, 1/2, 1/5, 1/25 and 1/125 mg/ml, respectively.

p

SGL-I

SGL-I

SGL-II

SGL-II

.A

fi5

free

Fig. 3. Radioimmuno-thin-tayer chromatography of glycolipids isolated from tissues of Aplysia. Samples were developed on plastic TLC plates with n-propanol-ammonia-water (75:5:25, v/v). Then plate B was incubated successively with rabbit antiglycolipid serum and the donkey anti-rabbit Ig,[~I] F(ab')2 fragment and exposed to Ultrofilm (LKB). Another plate (A) was sprayed with anthrone reagent. S, dermal tissue; G, ganglion; L, liver: F, nerve fiber; G(HF) glycolipids of ganglion treated with HF. O, GGL-I; I~, FGL-II.

263 tained 1 mg of glycolipid/ml). In another test carried out under the same conditions, the titers of total water-soluble glycolipid of the ganglion, total water-soluble glycolipid of the dermal tissue and total watersoluble glycolipid of the liver were 1:4000, 1:2000 and 1:1000, respectively.

Immunohistochemical findings In the present study, formalin-fixed, paraffin-embedded tissue sections were mainly used. In the ganglia, positive staining was observed in fibrous structures of the neuropil (Fig. 4a, c, d). Staining was seen in the cytoplasm and fine processes of occasional cells with a small oval nucleus, which were probably glial cells (Fig. 4d), thus the staining of fibrous structure in the neuropil was due at least in part to these supporting cells. Generally, there was little, if any, distinct staining of the cytoplasm of nerve cells and their processes (Fig. 4a, c, d). Moreover, strong staining of the plasma membrane of neuronal cell bodies could not be seen (Fig. 4c, d). Staining of the plasma membrane of nerve cell processes, which were occasionally surrounded by intensely stained fibrous structures of the neuropil, was not apparent (Fig. 4c). These findings with immunoperoxidase were identical in all of the ganglia examined, including buccal, abdominal and pedal ones. Proximal portions of peripheral nerves, showed fibrous staining similar to that in the neuropil of ganglia (Fig. 4e). Some of the stained fibrous structures were identified as processes of the supporting cells scattered in the nerve bundles. None of the axons, identified by light-microscopy, stained distinctly. Similar staining was also observed in nerve tracts between ganglia (connective tract, not shown) and in the distal portion of peripheral nerves (Fig. 4g) in subcutaneous and muscle tissues. Nerve cells that existed in small clusters close to subcutaneous or intramuscular nerve bundles showed little or no staining (Fig. 4g). In addition, positive staining was observed in the cytoplasm of small mononuclear cells and foamy cells, both of which were widely distributed, especially in subcutaneous tissues, and were probably of the wandering cell lineage (Fig. 4h). In control sections in which antiserum absorbed with glycolipids isolated from ganglia was used, staining disappeared almost completely in the ganglion

(Fig. 4b), the peripheral nerves (Fig. 4f) and the subcutaneous tissues (not shown). Sections treated with preimmune serum also showed no positive staining. Essentially the same findings were obtained in fresh frozen sections as in formalin-fixed paraffinembedded sections. Positive staining was observed in neuropils of the ganglia, peripheral nerve bundles and widely distributed mononuclear and foamy cells. Fig. 5a shows positively stained neuropils and unstained neurons of the abdominal ganglion in cryostat section fixed with formalin. Fig. 5b shows staining of peripheral nerve bundles encapsulated by unstained perineural sheaths in cryostat section fixed with acetone. DISCUSSION The glycolipids used for the immunization in the present work were isolated from the nervous tissue of a sea gastropod, Aplysia kurodat ~. This is the first time that such glycolipids have been used for immunization. As shown in Fig. 1 and described in the Resuits, the lipid fraction is a mixture of several phosphonoglycosphingolipids. GGL-V, one of the main glycolipids of ganglia has been identified as 3-0MeGal-(1---~ 3)GalNAc(1--~ 3) [6- O- (2'-aminoethylphosphonyl)Gal(1--+2)][2'-aminoethylphosphonyl(--~6)]Gal(1--~4)Glc(1---~l)ceramide using a glycolipid isolated from the dermal tissue of Aplysia 3, SGL-II, which is chemically identical to GGL-V 2. Our data obtained to data strongly suggest that the other glycolipids are similar in structure to GGL-V, and that these glycolipids as a whole constitute a new class of glycolipidz. Gangliosides are not found in Aplysia 8,12. We are trying to determine the cellular and subcellular distribution of this novel group of glycolipid in Aplysia to understand its biological and neurobiological functions. As a first step, in this study we used total phosphonoglycosphingolipids isolated from ganglia for immunization without separating them into individual phosphonoglycosphingolipids. Antiserum was raised effectively when the glycolipids were incorporated into liposomes made of lecithin and cholesterol and injected into rabbit. This antiserum reacted strongly with GGL-V, FGL-VII, SGL-II and 2-AEP free GGL-V on a TLC plate (Fig. 3). These 4 glycolipids

264

265

Fig. 5. a: fresh frozen section, fixed with 10% formalin, of the abdominal ganglion. Positive staining is observed in the neuropil which surrounds unstained nerve cells (N). Indirect immunoperoxidase method. No counterstaining, x 460. b: fresh frozen section, fixed with acetone, of peripheral nerves which extend from the abdominal ganglion. Staining is observed in nerve bundles which are encapsulated with unstained perineural sheath (PS). Indirect immunoperoxidase method. No counterstaining, x 230. s e e m to have the s a m e oligosaccharide chain 2. Rem o v a l of 2 - A E P b y H F did n o t affect the i m m u n o r e activity of G G L - V (Fig. 3B). G G L - I a n d F G L - I I , which h a v e a different oligosaccharide s t r u c t u r e 2 from that of strongly reactive glycolipids, did n o t react with the a n t i s e r u m (Fig. 3B). M o r e o v e r , n o reaction was o b s e r v e d with p r e i m m u n e r a b b i t s e r u m by a u t o r a d i o g r a p h y . T h e facts that the a n t i s e r u m did n o t give a p r e c i p i t a t i o n line in O u c h t e r l o n y ' s test

the a n t i s e r u m does n o t react with c e r a m i d e m o i e t y of lipids. Results of the c o m p l e m e n t fixation test were consistent with those of O u c h t e r l o n y ' s test a n d radioi m m u n o l o g i c a l studies, except that G G L - V , which r e a c t e d most strongly with the a n t i s e r u m on a T L C plate, gave a s o m e w h a t low titer. T h u s , we c o n c l u d e that the a n t i s e r u m reacts with the sugar m o i e t y of the n e w group of glycolipids, especially that of G G L - V , FGL-VII and SGL-II. T h r e e ganglia, n e r v e tracts b e t w e e n ganglia a n d n e r v e fibers a n d the d e r m a l tissue of Aplysia were

with the w a t e r - s o l u b l e lipid fraction of the rat b r a i n (Fig. 2), b o v i n e b r a i n gangliosides, or globoside isolated from h u m a n erythrocytes, a n d did n o t react

stained with the a n t i s e r u m by the indirect i m m u n o -

with G G L - I a n d F G L - I I o n T L C plate, indicate that

peroxidase m e t h o d (Fig. 4). In the ganglia, s t a i n i n g

Fig. 4. a: light micrograph of horizontal section of the abdominal ganglion showing positive staining in the neuropits (NP) of the bilateral hemiganglia. The commissure (C) by which the two neuropils are joined is also similarly stained. Neurons present in the periphery of the ganglion, some of which are indicated by arrowheads, show no distinct staining. Indirect immunoperoxidase method. Paraffin section. Light counterstaining with hematoxylin-eosin, x 46. b: control section. Similar portion to that of a. Section was reacted with antiserum absorbed with glycolipids of ganglia. Staining was almost completely negative. Indirect immunoperoxidase method. Paraffin section. Light counterstaining with hematoxylin-eosin, x 46. c: light micrograph of buccal ganglion showing positive staining in the fibrous structures of the neuropil (NP). Large neuronal processes (P) embedded in stained neuropil show little or no staining. Nerve cell (N) also shows no distinct staining. Indirect immunoperoxidase method. Paraffin section. Light counterstaining with hematoxylineosin. × 230. d: higher magnification of a buccal ganglion showing positive staining in fibrous structures of the neuropil, which are occasionally continuous with processes of small supporting cells (arrows), probably glial in nature. Nerve cell (N) shows no distinct staining. The staining on or near the surface of nerve cell is considered to be of the surrounding fibrous structures. Indirect immunoperoxidase method. Paraffin section. Light counterstaining with hematoxylin-eosin, x 920. e: light micrograph of a cross-section of the proximal portion of a peripheral nerve which extends from the buccal ganglion. Intense staining is observed in fibrous structures of the peripheral nerve tissue, mainly in areas in which axons are small in caliber. Large axons, some of which are indicated by arrows, are unstained. Perineural sheath (PS) is not stained. Indirect immunoperoxidase method. Paraffin section. Light counterstaining with hematoxylin-eosin, x 460. f: control section. Similar portion to that of e. Section was reacted with antiserum absorbed with glycolipids isolated from ganglia. Staining was almost completely negative. Indirect immunoperoxidase method. Paraffin section. Light counterstaining with hematoxylin-eosin, x 460. g: light micrograph of a peripheral nerve in the subcutaneous tissue. The subcutaneous peripheral nerve (arrows) are intensely stained, whereas adjacent subcutaneous nerve cells (N) show no distinct staining. Indirect immunoperoxidase method. Paraffin section. Light counterstaining with hematoxylin-eosin, x 460. h: light micrograph of the subcutaneous tissue showing positive staining in the cytoplasm of mononuclear cells (arrow) and foamy cells (arrowhead). Indirect immunoperoxidase method. Paraffin section. Light counterstaining with hematoxylin-eosin. × 230.

266 was seen mainly in the neuropil, where fibrous structures and supporting cells (glia cells) were stained, and structural continuity between the two was often

colipid in the extra-neural tissues or cells as the mononuclear or the foamy cells as described in the pres-

observed. Similar staining was also observed in the

ent paper was outside the scope of the present work. However, it will deserve extensive studies in future.

nerve tracts between ganglia and the peripheral nerves. In the dermal tissues, the main site of staining

tion of the tissue sections on the immunoperoxidase

was nerve bundles in the subcutis, which showed the same pattern of staining as the proximal portion of the peripheral nerves. The cytoplasm of small mono-

Our preliminary results on the effect of preparastaining (Figs. 4 and 5) indicate that glycolipids in the tissues were stained, but our present data suggest

nuclear cells and foamy cells also stained. It was diffi-

that carbohydrate moieties of the glycolipids will be i m m u n o d e t e r m i n a n t s and cross-reaction of the anti-

cult to recognize stained structures in and along the

bodies with glycoproteins is possible, so the contribu-

neuronal cell bodies and processes by light microsco-

tion of glycoproteins to the staining requires further

py. These histochemical findings suggest that some of the main constituents of the new glycolipid group are

study.

present in the supporting cells, including glia cells in the nervous tissues of Aplysia. But our findings do

ACKNOWLEDGEMENTS

not necessarily prove that phosphonoglycosphingolipids are not present in n e u r o n s or in n e u r o n a l plasma membranes. To reach a final conclusion on this matter, we plan to use the immunohistochemical technique in c o m b i n a t i o n with electron-microscopic

We thank Dr. Y. H o n m a for his valuable suggestions on the histological findings and for collecting Aplysia kurodai, for which we are also thankful to Mr. T. Kitami. Our thanks are also due to Prof. F. Ikuta for his encouragement. This work was support-

studies using antisera that can react with all kinds of

ed by grants for Scientific Research from the Min-

phosphonoglycosphingolipids, including G G L - I . Elucidation of distribution of the new group of gly-

istry of Education of Japan. We also thank Mrs. A. Mitsui for her excellent technical assistance.

REFERENCES

8 Honegger, G. G. and Freyvogel, T. A., Lipide des Zentralnervensystems bei Wirbeltieren und einigen Wirbellosen, Helv. chim. Acta, 46 (1963) 2265-2270. 9 Inoue, K. and Nojima, S., Immunochemical studies of phospholipids. III. Production of antibody to cardiolipin, Biochim. biophys. Acta, 144 (1967) 409-414. 10 Itasaka, O. and Hori, T., Studies on glycosphingolipids of fresh-water bivalves. V. The structure of a novel ceramide octasaccharide containing mannose-6-phosphate found in the bivalve, Corbicula sandai, J. Biochem., 85 (1979) 14691481. 11 Kumanishi, T., The immunoperoxidase method in studies of the nervous system diseases, Advanc. neurol. Sci., 24 (1980) 87-93 (in Japanese). 12 Komai, Y., Matsukawa, S. and Satake, M., Glycolipids in the nervous tissue of invertebrate, J. Biochem., 70 (1971) 367-369. 13 Laev, H., Rapport, M. M.. Mahadik. S. P. and Silverman, A.-J.. Immunohistological localization of ganglioside in rat cerebellum. Brain Research 157 (1978) 136-141. 14 Lapetina. E. G.. Soto. E. F. and Robertis, E. D.. Gangliosides and acetylcholinesterase in isolated membranes of the rat-brain cortex. Biochim. biophys. Acta. 135 (1967) 33-43. 15 Ledeen, R. W. and Yu. R. K.. Structure and enzymic degradation of sphingolipids. In H. G Hers and F. V. Hoof (Eds.), Lysosomes and Storage Diseases. Academic Press. New York and London. 1973. pp. 105-145.

1 Araki, S., Komai, Y. and Satake, M., A novel sphingophosphonoglycolipid containing 3-O-methylgalactose isolated from the skin of the marine gastropod Aplysia kurodai, J. Biochem., 87 (1980) 503-510. 2 Araki, S. and Satake, M., Acidic sphingoglycolipids in invertebrate nervous tissue: presence of several kinds of sphingophosphonogtycolipids in the nervous tissue of Aplysia kurodai, a sea gastropod, Neurosci. Lett., 22 (1981) 179-182. 3 Avrova, N. F., Chenykaeva, E. Y. and Obukhova, E. L., Ganglioside composition and content of rat brain subcellular fractions, J. Neurochem., 20 (1973) 997-1004. 4 Baecque, C. D., Johnson, A. B., Naiki, M., Schwarting, G. and Marcus, D. M., Ganglioside localization in cerebellar cortex: an immunoperoxidase study with antibody to GMI ganglioside, Brain Research, 114 (1976) 117-122. 5 Bligh, E. G. and Dyer, W. J., A rapid method of total lipide extraction and purification, Canad. J. biochem. Physiol., 37 (1959) 911-917. 6 Folch, J., Lees, M. and Sloane Stanley, G. H., A simple method for the isolation and purification of total lipides from animal tissues, J. biol. Chem., 226 (1957) 497-509. 7 Hansson, H.-A., Holmgren, J. and Svennerholm, L., UItrastructural localization of cell membrane GMI ganglioside by cholera toxin, Proc. nat. Acad. Sci. U.S.A., 74 (1977) 3782-3786.

267 16 Magnani, J. L., Smith, D. F. and Ginsburg, V., Detection of gangliosides that bind cholera toxin: direct binding of 12SI-labeled toxin to thin-layer chromatograms, Analyt. Biochern., 109 (1980) 399-402. 17 Mandell, J. D. and Hershey, A. D., A fractionating column for analysis of nucleic acids, Analyt. Biochem., 1 (1960) 6677. 18 Marcus, D. M. and Schwarting, G. A., Immunochemical properties of glycolipids and phospholipids. In H. G. Kunkel and F. J. Dixon (Eds.), Advances in Immunology, VoL 23, Academic Press, New York, San Francisco, London,

1976, pp. 203-240. 19 Miyatake, T., Handa, S. and Yamakawa, T., Chemical structure of the main glycolipid of hog erythrocytes, Jap. J. exp. Med., 38 (1968) 135-138. 20 Mori, E., Mori, T., Sanai, Y. and Nagai, Y., Radioimmuno-thin-layer chromatographic detection of Forssman antigen in human carcinoma cell lines, Biochem. biophys. Res. Commun., 108 (1982) 926-932. 21 Sever, J. L., Application of a microtechnique to viral serological investigations, J. lmmunol., 88 (1962) 320-329.