Immunohistochemical localization of galactosyl and sulfogalactosyl ceramide in the brain of the 30-day-old mouse

Brain Research, 211 (1981) 341-354

341

Elsevier/North-Holland Biomedical Press

IMMUNOHISTOCHEMICAL LOCALIZATION OF GALACTOSYL AND SULFOGALACTOSYL CERAMIDE IN THE BRAIN OF THE 30-DAY-OLD MOUSE

B. Z A L C , M. M O N G E , P. D U P O U E Y , J. J. H A U W a n d N. A. B A U M A N N

Laboratoire de Neurochimie, I N S E R M U.134, ( J.J.H. ) Laboratoire de Neuropathologie Charles Foix, Hdpital de la Salpdtridre, 47 Bd de l'HOpital, 75651 Paris, and (P.D.) Laboratoire de Biochimie des Antigdnes, Institut Pasteur, 25 Rue du Dr. Roux, 75015 Paris (France) (Accepted September 18th, 1980)

Key words: i m m u n o h i s t o c h e m i s t r y - - localization - - brain - - galactosylceramide - - sulfatide

SUMMARY

We have used purified antibodies against galactosylceramide (galCb) and sulfogalactosylceramide (sulf) to study the topographical distribution of these two lipid haptens in the brain of the 30-day-old mouse. This study has been conducted, using the indirect immunofluoreseence method, on cerebellum, brain stem and hemispherical tissue sections. Both haptens are present in the myelin sheaths and in the oligodendrocytes within the myelinated bundles. Cortical oligodendrocytes as well as some of the subependymal cells are also galCb-positive but sulf-negative. On the contrary, ciliated ependymal cells and subpial astrocytic processes (especially the Bergmann glia fibers in the cerebellum) are sulf-positive and galCb-negative. Astrocyte cell bodies and other astrocytic cell processes are devoid of both haptens. Lastly, some-sulf positive galCb-negative processes, as yet unidentified, were also found in the periaqueductal gray matter and in the nucleus interpeduncularis.

INTRODUCTION

Galactosylceramide (galCb) and sulfogalactosylceramide (sulf) are mainly concentrated in the brain, and in adult human myelin account for 23 ~ and 4 ~ of the lipids respectively26. These glycosphingolipids are considerably reduced in the hypomyelinated quaking mutant mouseZ and almost absent in the more severely dysmyelinated jimpy mutant2L From these data and others 15, galCb and sulf have both been involved in myelinogenesis and even considered as markers for oligodendrocytes (in central nervous tissue culturesZa,z4). This hypothesis was strengthened by the

342 finding of both galCb and sulf in isolated oligodendroglial cells in a ratio close to the one reported for myelinal. Nevertheless, in addition to its myelin localization, several analytical data were in favor of a neuronal and possibly astroglial localization of sulf. It has been reported that galactolipids were present in bulk isolated neurons and astroglial cells 1,16,2s. De Vries has also reported that both galCb and sulf were present in bovine axons 12. The finding that in these axonal preparations, the sulf/galCb ratio was higher than the one found in myelin lipids, was consistent with previous data showing that the non-myelin portion of white matter may be enriched in sulf 27. As far as synaptosomal plasma membranes are concerned, the data available are contradictory, while some authors could not detect either galCb or sulf in their preparations 6, others reported the presence of either sulf 37 or both galactolipids 16. At last, the implication of sulf in some precise functions also raised the question of its exact localization. Indeed, sulf has been shown to be involved in sodium chloride transportS,18,19,z9, 42 and in the opiate receptors z3 (this latter presumed function being in favor of its neuronal localization). Thus, immunohistochemistry should provide some useful information regarding the localization of these two galactolipids in the central nervous system. When injected into an animal, together with a protein carrier and adjuvants, these glycosphingolipids are antigenic. In 1963, Joffe et al. demonstrated that galCb was the so-called 'lipid hapten' of the CNS 17. Since then, several authors have prepared antisera to galCb (for a more complete review°-,35). We have previously reported the preparation, characterization and purification of specific antibodies of high titer directed respectively against galCb la and sulf4~, using an intravenous procedure of immunization developed by Coulon-Morelec 7. In the present study, we report on the use of both these purified antibodies to localize these two hapten moieties in tissue sections of 30-day-old mouse brains. METHODS

Immunesera and purifieation of antibodies Antisera directed against galCb, sulf and glucosylceramide (gluCb) were prepared in rabbits, as previously describedla, 4x,43. Repeated intravenous injections of a mixture of hapten, auxilliary lipids and a protein carrier were given. The ratios of hapten-cholesterol-human serum albumin used were 0.2:1:4 for the neutral glycolipids, and hapten-lecithin-cholesterol-methylated bovine serum albumin 0.1:0.4:1:1 for the sulf. The antisera thus obtained displayed a high affinity and selectivity for their respective haptens, both in the complement fixation and in the passive agglutination tests. In the indirect immunofluorescence experiments, the use of purified antibodies was required, in order to circumvent non-specific staining. This purification was performed in accordance with Coulon-Morelec s. The immunoadsorbent used consists of cholesterol particles coated with non-covalently bound hapten. When assayed by complement fixation (using 6H50 units of guinea pig complement), the titers of the purified antibodies were 1/256 for the anti-galCb and anti-gluCb, and 1/128 for the anti-sulf. The protein concentration (mg/ml) of each

343 pure antibody preparation used for the immunofluorescence experiments, was 0.15 for the anti-galCb, 0.12 for the anti-gluCb and 0.07 for the anti-sulf. It is noteworthy that whereas, neither the anti-galCb, nor the anti-gluCb were cross-reacting with the other two haptens, the pure anti-sulf had a slight affinity towards galCb, up to a 1/16 dilution, but none whatsoever towards gluCb.

Indirect immunofluorescence Brains of the 30-day-old mice were cut into slabs, embedded in O.C.T. (Miles Laboratories) and quickly frozen in liquid nitrogen-cooled isopentane. Sections 4-8 #m thick were cut at --20 °C in an Ames cryostat and thaw-mounted onto gelatin chrome-alum-coated glass slides. Sections were fixed for 30 min in ice-cold phosphatebuffered 4 ~ formaldehyde, rinsed and then incubated for 5 min at 4 °C in phosphatebuffered (pH 7.4) 0.1 ~o Triton X 100 and washed 4 times for 5 min in phosphatebuffered saline (PBS), pH 7.4. All further incubations were carried out at room temperature. The sections were left in contact with the pure antibody solutions for 60 min. After four 5-min washes in PBS, the sections were exposed for 30 min to antirabbit IgG globulins, conjugated to fluorescein isothiocyanate (Institut Pasteur Production, Paris) diluted 1/100 in PBS (containing 0.1 mg/ml of Evans blue) and then washed 4 times for 5 rnin in PBS before being mounted in glycerol buffered at pH 7.6. This procedure is the one we will refer to as 'standard conditions'. Sections were examined under a Leitz Orthomat fluorescent microscope with a Ploem system for epiillumination (filters BP 450-490, RKP 510, LP 515) and Nomarski's interference contrast optics for transmitted light used in achromatic conditions. In order to be able to examine the sections at a low magnification (objective Achro 4/0.12) we have also used transmitted U.V. light (filters BP 450-490, BG 38, LP 515). Control of the specificity of the staining was performed by incubating adjacent sections with either chromatographically pure normal rabbit IgG (Nordic Lab.) (0.1 mg/ml of PBS) or PBS or myelin-absorbed antibodies solutions in place of the specific antibodies. Antiglycolipid antibodies (or fluorescein conjugated anti-rabbit IgG globulins) were absorbed with myelin by incubating the antibodies solution with lyophilized myelin (7 mg/ml) for 60 min at 37 °C and then for 18 h at 4 °C. Some sections were also fixed in ethanol in place of formaldehyde. As sulf, contrary to galCb, is not susceptible to periodic acid oxidation, we have used this control in order to differentiate between the fluorescence observed with both antibodies. For that purpose, a few sections were incubated, after the Triton X100 treatment, in 1 ~o periodic acid in PBS for 15 min, and then thoroughly washed, prior to the incubation with the different antibodies. Except for the cerebellum, which was cut sagittally or parasagittally, other sections were coronal. Each set of experiments was repeated on at least 3 different brains. The anatomical nomenclature we have used refers to ref. 36. RESULTS

Specificity of the immunofluorescence staining In the standard procedure, galCb and sulf appeared to be localized in myelin.

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345 This staining was not observed unless the sections had been treated with Triton X-100. This was probably due to the high hydrophobicity of the myelinated tract areas. Indeed, in cerebellum sections for instance, the cerebeUar white matter appeared mostly negative in the absence of detergent contrasting with the staining of the deep part of the granule layer. Without preincubating the sections in Triton X-100, cutting the frozen blocks at 4 ffm, instead of 8 #m, slightly improved the staining of the myelin by the anti-galCb or the anti-sulf antibodies. But large areas, especially in the deep white matter of the cerebellum (commissura cerebelli or corpus medulare cerebelli) remained unstained. In preliminary experiments, several treatments, such as methanol or m e t h a n o l - K O H , at various temperatures and concentrations, as well as trypsinization at various pHs and temperatures had been found unsatisfactory. Sections fixed in ethanol at room temperature for 10 min were negative when incubated with either antigalCb or anti-sulf antibodies. Control sections incubated with fluorescent globulins alone were also negative. However, one batch of fluorescent anti-rabbit I g G globulins, was found to bind slightly to myelin. This artefact was overcome by absorbing these globulins on lyophilized myelin prior to use. Brain sections incubated with normal rabbit I g G at a concentration of 0.1 mg/ml also turned out unstained. Nevertheless, a 30-fold higher concentration resulted in a definite increase of the background fluorescence, while some dull fluorescence appeared on the pia arachnoid, the neuronal nuclei and the blood capillaries. No staining was observed on brain sections incubated with anti-gluCb antibodies. After periodic acid treatment, the sections became practically galCbnegative (Fig. lb) whereas the fluorescence observed with the anti-sulf was not altered. On cerebellum sections submitted to periodic acid, the labelling of the granule layer by the anti-sulf was even more intense, suggesting the unmasking of some haptenic sites (Fig. 2b). Myelin-absorbed anti-galCb or anti-sulf antibodies did not react anymore with the brain tissue.

Localization on cerebellar sections (Figs. 1-6) In these sections, all myelinated fiber tracts stained brightly with antibodies to galCb and sulf ~Figs. 1a and 2a). As shown in Fig. 3a, when the nerve fibers were cut longitudinally, the fluorescence had a double-line appearance. But when the fiber tracts were cut transversely, the fluorescence was seen ringing the axons (Fig. 3b). Both

Fig. 1. Sagittai section of two cerebellar folia incubated in standard conditions, with anti-galCb antibodies (1a). Adjacent section (1b) was treated with 1% periodic acid (see Methods) prior to incubations with antibodies. The white matter myelin appeared strongly galCb-positive (la). This contrasts with the absence of fluorescence after periodic acid oxydation (1b). In 1a, the inner part of the granule layer is also seen fluorescent (arrows). W, white matter; G, granule layer; M, molecular layer. Transmitted light. × 40. Fig. 2. Sagittal section of two cerebellar folia, incubated in standard conditions, with anti-sulf antibodies (2a). Adjacent section was treated with 1% periodic acid (see Methods) prior to incubations with antibodies (2b). The white matter appeared brightly fluorescent (2a), but in contrast with Fig. lb, the periodic acid-treated section remained sulf-positive (2b). Note that after periodic acid oxidation, the granule layer is more heavily labelled by the anti-sulf antibodies (arrows). W, white matter; G, granule layer; M, molecular layer. Transmitted light, x 40.

346

Fig. 3. Higher magnification of a cerebellar section stained under standard conditions, with anti-galCb antibodies. In (a) note, the positive myelin sheaths, most of them are cut longitudinally, and the negative looking nuclei of granule cells. Some fluorescence is seen in between the granule cells. Epi-illumination. × 1000. In (b), another field of the same section showing positive myelin sheaths cut transversely. Note the rings of fluorescence around the axons (arrows). Epi-illumination. × 1000. WM, white matter; G, granule cells. Fig. 4. Transverse section of a cerebellar folium showing positive myelin sheaths in the white matter (WM), some fluorescence around the inner granule ceils (G) and negative Purkinje cells (P). Arrow points to one process-like fluorescent structure of the inner granule cell layer. Anti-sulf antibodies. Epi-illumination. x 350. Fig. 5. Cerebellar section stained under standard conditions, with anti-galCb antibodies. Intrafascicular glia is seen galCb-positive. Epi-illumination. × 300. Fig. 6. Cerebellar section stained under standard conditions, with anti-sulf antibodies, showing sulfpositive Bergmann fibers of Golgi epithelial cells. Epi-illumination. x 400. Fig. 7. Coronal section across the brain stem at the level of the aqueductus cerebri, stained in standard conditions, with anti-sulf antibodies. Subpial processes are seen sulf-positive. H, hemisphere; B, brain stem. Epi-illumination. x 750.

347 haptens were also clearly visualized in the intrafascicular glia (Fig. 5). In Figs. la and 2a, one can also see that the deep part of the inner granule layer was also labelled. This fluorescence diminished gradually from the fiber tracts towards the Purkinje cells which at no time were labelled with any of our antibodies. Although less marked for sulf in standard conditions, after exposure to periodic acid, the visualization of sulf mimicked that observed with the anti-galCb in the absence of periodic acid (Fig. 2b). Although at times, the fluorescence seemed to ring the granule cells, the staining more often could not be related to any specific histological feature. Nevertheless, in some field, this fluorescence could well be attributed to myelinated fibers (Fig. 4). The molecular layer was always found to be galCb-negative. On the contrary, the Bergmann fibers were found to be sulf-positive (Fig. 6). Interestingly enough, the epithelial cells of Golgi, which are the cell bodies of these fibers, were sulf-negative. On the same cerebellum sections, the ependymal cells lining the fourth ventricle, as well as some short fiber processes in contact with them, were also rendered brightly fluorescent by the anti-sulf, but not with the anti-galCb (as shown in another area in Fig. 13). Choroid plexus cells were always found galCb- and sulf-negative. Localization across the upper brain stern at the level of the aqueductus cerebri (Figs. 7 and 8) At this level, all the myelinated tracts were stained by anti-galCb, whether they were cut longitudinally as in the commissures (commissura colliculorum anteriorum or decussatio pedunculorum cerebellarium superiorum) or cut transversely as in the ascendant and descendant tracts (such as fibrae longitudinales pontis, lemniscus medialis, tractus corticospinalis and fasciculus medialis). No fluorescence could be detected in non-myelinated areas with the anti-galCb. Sulf was also detected on myelin. Furthermore, only this latter hapten was localized on fiber processes in contact with the pia arachnoid, between the brain stem and the cerebral hemispheres. These sulf-positive fibers were tentatively identified as subpial astrocytic processes (Fig. 7). Sulf was also found in the ependymal cells bordering on the aqueductus cerebri (Fig. 8a). As above for the cerebellum sections, some short processes in contact or in continuity with the ependymal cells were also sulf-positive (Fig. 8a). More unexpected was the finding that some structures in the periaqueductal gray matter (PAG) were sulf-positive. In the PAG, sulf was localized lateroventrally and appeared as thin fiber processes usually in close contact with a neuronal cell body (Fig. 8b). On sections concerning the nucleus interpeduncularis, the same type of processes were found sulf-positive. Localization on sections across the corpus callosum and the chiasma opticum (Figs. 9-13)

Fig. 9 shows a low magnification of such a section stained with anti-galCb. The myelinated tracts can be seen to be highly fluorescent, whether cut longitudinally as the corpus callosum or the commissura anterior, or transversely as in the myelinated bundles of the basal ganglia. At this magnification, one could hardly see any difference when anti-sulf was used instead of anti-galCb. But, here also, each hapten had some selective localization when visualized at higher magnifications. Characteristic ofgalCb

Fig. 8. Coronal section of the brain stem at the level of the periaqueductal grey matter (PAG), stained under standard conditions with anti-sulf antibodies, a: low magnification. Note the positive ependymal cells (E) lining the aqueductus cerebri. Fine arrows indicate sulf-positive processes close to the aqueductus. Arrow heads point to sulf-positive processes located lateroventrally in the PAG. Epi-illumination. × 200. b: a higher magnification of one of those lateroventrally localized sulf-positive processes. Note that this fluorescent process is in contact with a cell body. Epi-illumination. x 1500. Fig. 9. Coronal section showing the corpus callosum (CC) at the level of the chiasma opticum, stained under standard conditions, with anti-galCb antibodies. Note the bright fluorescence of the corpus callosum and related fibers; the myelinated bundles of the striatum (S) are also seen to be fluorescent. The rectangular area is shown at a higher magnification in Fig. 1la. Transmitted light, x 40. Fig. 10. Coronal section at the level of the chiasma opticum, stained under standard conditions, with anti-galCb antibodies. High magnification of a galCb-positive cortical cell of the cingulum. An unfluorescent neighbouring neuron (N) is seen. Epi-illumination. x 900. Fig. 11. Coronal section at the level of the chiasma opticum stained in standard conditions, with antigalC b (a) or anti-sulf (b) antibodies, a : a higher magnification of Fig. 9. Myelinated fibers of the corpus callosum (CC) are brightly fluorescent. GalCb-positive subependymal cells are shown (arrows); arrow heads point to negative adjacent subependymal cells, b: an adjacent section showing the sulf-positive myelinated fibers (CC) and sulf-negative subependymal cells (arrows). Epi-illumination. ~ 350. Fig. 12. Coronal section at the level of the chiasma opticum stained under standard conditions, with anti-sulf antibodies. Ependymal cells lining the ventriculus tertius are seen sulf-positive. Arrows point to sulf-negative tanicytes close to the area of the chiasma opticum (out of field). Epi-illumination. x 250.

349

Fig. 13. Higher magnification of sulf-positive ciliated ependymal cells of ventriculus tertius. Epiillumination (a). In (b), same field as seen with interference contrast optics. Nuclei of the two rows of ependymal cells (E) and collapsed ventricular space (arrow) are seen. All the fluorescent fiber processes could not be traced to their cellular body, even with the interference contrast, x 900.

was the labelling of a few scattered cells in the cingular cortex as well as in the vicinity of the fornix in the nuclei lateralis and fimbrialis septi. From their morphological features (small size, scarcity of cytoplasm) compared to adjacent large cortical neurons, these cells might tentatively be identified as cortical oligodendrocytes (Fig. 10). Part of the layer of subependymal cells, in close contact with the myelinated fibers of the corpus callosum were also observed to fluoresce only with anti-galCb (Fig. 1l a and b). Besides the myelin localization, sulf was found in the ependymal cells of the ventriculi laterali and of the ventriculus tertius. Interestingly enough, in this latter structure, not all ependymal cells were stained; only the ciliated ones were sulfpositive, whereas the tanicytes were sulf-negative (Fig. 12). Around the ventriculi, numerous fiber processes, whether or not in direct contact with ependymal ceils, were

350 heavily stained with anti-sulf as seen in Figs. 12 and 13. Fig. 13 shows a higher magnification of the ciliated ependymal cells with two types of these fibers: some in direct contact with the cell body, and others apparently separated. Other fiber processes in contact with the pia arachnoid of the fissura longitudinalis cerebri were also specifically labelled by the antisulf. DISCUSSION The feasibility of using immunohisto- or cytochemical techniques for the localization of specific glycolipids has already been demonstrated4,11,14,20,25,33,34,as,42-~4. In this study, we present the use of these techniques for the localization of two closely related constituents of the central nervous system: galCb and sulf. As the main antigenic determinant of the studied glycolipids is either galactose or galactose-3sulfate13,41, one might question the possibility of cross-reactivity with some glycoproteins bearing these hexoses as the terminal sugar. However, to our knowledge, galactose-3-sulfate has never been described in glycoproteins but galactose is a common component. Although some glycoproteins may be extracted by organic solvents 21, the absence of staining on tissue sections fixed with ethanol argues well for the lipidic nature of the fluorescence normally observed under standard conditions. Furthermore, if such a cross-reactivity existed, one has then to admit that terminal galactose glycoproteins are localized exclusively in myelin or oligodendrocytes. GluCb is present in the mouse brain at less than 1 ~ of total cerebroside 40 but it could not be detected immunohistochemically. There are various interpretations of this finding: the less convincing possibility would be that this lipid is located in a very specific area which we have not explored. Two other explanations are more probable: either the minute amount of gluCb is below the lowest limit of' sensitivity of our immunohistochemical technique, or these gluCb molecules are not accessible to the antibodies. The slight in vitro cross-reactivity between anti-sulf and galCb does not, as it might seem, interfere in our localization experiments. First of all, for some brain structures, there was no overlapping in the localization of each hapten. Secondly, in the myelinated tracts, which were both galCb and sulf-positive in standard conditions, periodic acid treatment of the brain sections led to a negative outcome for anti-galCb, whereas the anti-sulf remained positive. This is as expected from the fact that sulf is protected from periodic acid oxidation by the sulfate ester in the 3 position of galactose. GalCb as an oligodendrocyte and myelin marker

Firstly, the uniformity of the galCb localizations must be stressed. This hapten was found only in myelin and myelin-related structures. In the fiber tracts, not only the myelin sheaths were found to be fluorescent but also cell bodies in the tracts themselves. These cells appeared either isolated or as forming short chains. These latter were easily identifiable as intrafascicular oligodendroglial cells. The isolated cells of the myelinated tracts have also been tentatively identified as oligodendrocytes, because of their small size, large nucleus and scarce cytoplasm. Their general

351 morphological appearance was very different from that observed for astrocytes using anti-glial fibrillary acidic protein (GFA) antiserumz2. This confirms the data of doublelabelling experiments reported by Raft et al. in dissociated cell cultures of rat CNS33,34. In the hemisphere sections, galCb was found in two extra myelinic localizations: (1) in isolated cortical cells in the cingular cortex or in the nuclei lateralis or fimbrialis septi (Fig. 10); from their morphological appearance, i.e. small size, paucity of the cytoplasm and presence of one or two processes, these cells are thought to be cortical oligodendrocytes; and (2) among the subependymal cells, but only in the layer just in contact with the corpus callosum. This would tend to support Privat and Leblond's hypothesis according to which these subependymal cells are immature oligodendrocytesSL Indeed, according to Privat and Leblond's statement, these subependymal cells in proximity to the myelinated fiber tract (which were found to be galCb-positive) could be considered to be nearly mature oligodendrocytes, ready to migrate in the corpus callosum3°. So, this suggests that accumulation of galCb (but not of sulf) in the oligodendrocyte precedes myelination. In a recent study, Sternberger et al. 3s when comparing the localization in rat CNS of myelin basic protein (MBP) and galCb reported that in the medullar of newborn rats, galCb could not be detected in oligodendroglia while myelinated axons were stained. This is in contradiction with our data, but might be explained by the different methodology they used. More striking was the gradient of fluorescence observed in the cerebellum sections through the granule layer and this for both haptens. Indeed the internal part of the granule layer was always found more stained that the external one. Curiously, an identical gradient of staining has been previously described by De Baecque et al. 9 and confirmed by Laev et al. 16 using anti-GM1 ganglioside antibodies. Whether the staining concerned the granule cells themselves, or is due to nearby myelinated processes between them, is difficult to determine at the light microscope level. The images observed can be interpreted in both ways. If this staining was neuronal, one would then have to explain why only the deep granule cells possess these 3 haptens i.e. GM1, galCb and sulf. On the contrary, if one considers that these 3 glycolipids are myelin components, it is easier to explain this gradient aspect of the staining by an increasing density of myelinated fibers across the granule layer, from the Purkinje cells towards the lamina albae cerebelli. Thus the first major conclusion of our study appears to be the confirmation that galCb can certainly be considered as a myelin and oligodendroglial marker. Can sulf be considered as a myelin marker ?

Concerning sulf, on the other hand, the situation was found to be more complex than for galCb. It is true that the anti-sulf stained (as with anti-galCb) the myelin sheaths and the intrafascicular oligodendroglia. But sulfwas neither present on cortical oligodendrocytes, nor on subependymal cells. Furthermore, sulf was localized in other structures not belonging to the oligodendroglial line (see below). Thus sulf does not appear to be as good an oligodendroglial marker as galCb. Sulf is present in other glial structures

The ependymal cells were indeed highly labelled by the anti-sulf as were some

352 fiber processes in contact with these cells or with the pia arachnoid, especially the Bergmann fibers in the cerebellum. When incubating an adjacent section with an antiG F A antiserum, it was obvious that the labelling of these fibers was identical to that observed with the anti-sulf (except for the absence of staining of the astrocyte cells themselves). This suggests that these sulf-positive fibers are astrocytic processes. The fact that the tanicytes in the ventriculus tertius were devoid of sulf was a surprising finding. It provides an additional specific difference between these two types of ependymal cells. Involvement o f s u l f in the opiate receptors

At last, the finding of some sulf-positive structures in the P A G and the nucleus interpeduncularis has to be stressed for its physiological implication. These two areas of the brain stem are known to be rich in opiate receptors. Furthermore, in the spinal cord, we have also found sulf-positive processes in the substantia gelatinosa of the dorsal horn which also has a high level of opiate receptors. The possibility for sulf as part of the opiate receptor was suggested by H. Loh several years ago 23. Despite numerous papers in support of this hypothesis (reviewed in ref. 24),. it is still controversial 10. Recently, we have shown that, using a pharmacological paradigm, anti-sulf antibodies, when injected stereotaxically in the PAG, inhibit the effects of both morphine and fl-endorphin 9. In another study, we have shown that preincubation of brain stem tissue sections with opiates selectively inhibits the binding of antisulf antibodies on sulf-containing structures in the PAG, but not on other structures such as myelin or ependymal cells 4a. However, without electron microscopic evidence, we cannot determine whether this labelling is neuronal or glial. The selective visualization of sulf in these areas is an additional argument for the involvement of this glycosphingolipid in the opiate receptors. ACKNOWLEDGEMENTS We are greatly indebted to Prof. W. Segal and to Prof. R. Cantrill for reviewing our manuscript. This work was supported in part by Grant 78.7.307 6 from the D G R S T and by Grant C R L 79.5.322.6 B from the I N S E R M . REFERENCES 1 Abe, T. and Norton, W. T., The characterization of sphingolipids from neurons and astroglia of immature rat brain, J. Neurochem., 23 (1974) 1025-1036. 2 Alving, C. R., Immune reactions of lipids and lipid model membranes. In M. Sela (Ed.), The Antigens, Vol. 4, Academic Press, New York, 1977, pp. 1-73. 3 Baumann, N., Jacque, C., Pollet, S. and Harpin, M. L., Fatty acid and lipid composition of the brain of a myelin deficient mutant, the quaking mouse, Europ. J. Biochem., 4 (1968) 340-344. 4 Billecocq, A., Structure des membranes biologiques: localisation des galactosyldiglycerides dans les chloroplastes au moyen des anticorps sp6cifiques, Biochim. biophys. Acta (Amst.), 352 (1974) 245-251. 5 Bolognani, L., Gerzeli, G., De Pilceis Polver, P. and Magnani, P., The sulfatides and some histochemical correlations of the lachrymal glands involved in salt secretion in Chelonia, J. exp. Zool., 195 (1976) 179-190.

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