Distribution of filipin-sterol complexes in the myelinated nerve fiber

JOURNAL OF ULTRASIX

UCRJRE RESEARCH 91,

104-l 11 (1985)

Distribution of Filipin-Sterol Complexes in the Myelinated Nerve Fiber G. ALLT,*,t c. E. BLmcr-rAuD,t

M. L. MACKENZIE,t

AND K. SIKRfl

*Department of Anatomy and Biology as Applied to Medicine and tReta Lila Weston Institute of Neurological Studies, Middlesex Hospital Medical School, London WIP 608, United Kingdom Received April 23, 1985, and in revised form September 3, 1985 Using fdipin as a cytochemical probe to reveal the distribution of cholesterol, myelinated peripheral nerve fibers were examined in freeze-fracture replicas. Filipin-sterol complexes were most abundant in the Schwann cell and axonal plasma membranes. In the Schwann cell plasma membrane there was no heterogeneity in complex distribution in relation to the subjacent cytoplasmic network. In myelin lamellae there was a decrease in complexes from outer to inner lamellae and some aggregation of complexes in individual lamellae. The density of complexes in cytoplasmic organelles varied from absent in mitochondria to high in lysosome-like bodies. The results are interpreted in terms of the related biochemical composition and biophysical properties of cell membranes, with particular reference to the myelinated nerve fiber. The influence of diffusion barriers and gradients on the formation of complexes by filipin is considered. Q 1985 Academic

Filipin is a polyene antibiotic which binds specifically to 3-@-hydroxysterols (Norman et al., 1972) the most important of which in biological membranes is cholesterol. The content of cholesterol is a major determinant of such fundamental biophysical properties of membranes as permeability, fluidity, and electrical resistance (Demel and Kruyff, 1976; Sabine, 1977; Finean et al., 1984). Biochemical studies which have determined differences in cholesterol content between membranes (Demel and Kruyff, 1976; Gibbons et al., 1982; Finean et al., 1984) have been complemented in recent years by the use of filipin as a cytochemical marker which has not only confirmed differences between membranes but also revealed a microheterogeneity in the planar distribution of cholesterol (Karnovsky, 1982). While limitations inherent in the use of filipin as a cytochemical probe have rightly been emphasized (Robinson and Karnovsky, 1980; Kamovsky, 1982; Bridgman and Nakajima, 1983; Miller, 1984) much valuable, if tentative, information has accrued from its application. In support of the validity of the technique, Friend and Bearer

(1981) have demonstrated that under optimal conditions the number of filipin-sterol complexes in a membrane is proportional to the amount of sterol experimentally inserted into the membrane. The multimolecular complexes formed between filipin and cholesterol in a 1: 1 ratio produce deformations of biomembranes which can be accurately identified and localized in freeze-fracture replicas as hemispherical protuberances or depressions of -25 nm diameter and in ultrathin sections as characteristic corrugations. Since the technique has apparently not been applied to peripheral nerve, we have undertaken such a study with a particular interest in the myelinated nerve fiber where regional membrane specializations are likely to have especially important physiological correlates. MATERIALS AND METHODS Animals and tissuefiation. Twenty male SpragueDawley rats weighing 300-400 g were used. Animals were anesthetized using intraperitoneal Nembutal supplemented with a mixture of nitrous oxide and oxygen at a rate of 1.0 and 0.3 liter/mm, respectively. Tissue was fixed by vascular perfusion followed by immersion 104

0022/5320/85

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(see below). For perfusion a prewash for 30 set of w 100 ml of filtered 0.15 M sodium cacodylate buffer (pH 7.4) with 0.1% procaine hydrochloride BP at room temperature was immediately followed by 2Oo-400 ml of filtered 4% paraformaldehyde-2% glutaraldehyde fixative in the sodium cacodylate buffer. Heparin (5 units/ml) was added to both solutions prior to use. The perfusion was carried out at a pressure equivalent to 140- 160 mm Hg. The left and right sciatic nerves were exposed, and after additional fixative had been applied locally in situ, were removed for further fixation by immersion (see below). Filipin incubation. To facilitate tissue penetration and labeling by Iilipin four different experimental procedures were investigated : (1) Following vascular perfusion left and right sciatic nerves were removed and cut into small blocks (approximately 1.0 x 0.5 x 0.5 mm) byhandwitharazor blade under a dissecting microscope. Fixation was continued by immersion for a further 8 hr at room temperature on a rotator: for tissue blocks from the left sciatic nerve filipin (100 &ml and 1% dimethyl sulfoxide) was added to the aldehyde fixative while for the right sciatic nerve 8lipin was omitted as a control. (2) Vascular pcrIbsion with lilipin (100 &ml) containing cacodylate-buffered aldehyde fixative ( 100 ml) was achieved via the descending aorta. The sciatic nerves were then excised and cut into small tissue blocks as above. Fixation was continued by immersion in the same fixative with Slipin for 8 hr on a rotator. As a control filipm was omitted from the fixatives. (3) To circumvent the perineurial and blood-nerve barriers intraneural microinjection of fixative, containing filipin, was employed. The left sciatic nerve was microinjected at two adjacent sites with 2 ~1 each. The microinjection technique was as previously described (Ghabriel and Allt, 1982). Following perfusion with fixative alone, immersion fixation in filipin-containing aldehyde fixative was continued for a further 8 hr on a rotator. As a control filipin was omitted from the microinjection and immersion fixatives, tissue blocks being prepared as above. (4) Following fixation by vascular perfusion, the perneurium was removed with line forceps under a binocular dissect& microscope and the nerve fibers were liitly teased using mounted needles (Allt and Gajree, 1980). Subsequent lilipin exposure and continued fixation were as in (1). The most effective technique for the exposure of myelinated nerve fibers to the filipin-fixative mixture was the use of lightly teased fibers which apparently allowed better penetration of filipin resulting in more labeled fibers and was therefore the preferred method. In the other techniques employed, however, regions of maximal Iilipin penetration, for example at the periphery of tissue blocks, produced the same lindings as with teased fibers. Freeze-fructure. For freeze-fracture replication, after thorough rinsing in the buffer, the tissue blocks were

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treated with b&bred glycerol solution: 10% for 30 min followed by 30% for 30 min. The tissue blocks were then mounted on specially designed nerve specimen holders and rapidly frozen in melted Freon 22 precooled .with liquid nitrogen. Fracture and coating of the specimms were paformed at - 1 lO@C(by a snapfracture technique) using a modified Edwards fseezefracture and etching accessory equipped with electron gun evaporators and a quartz crystal thin &lm monitor. The replicas were stmngtbened by coating with a thin layer of 1% parlodian in amyl acetate, which was allowed to dry. This was followed by an overt&M digestion ofthe tissue by treatment with a weak solution of sodium hypochlorite (at a concentration of 0.07% available chlorine). The replicas were collected on 400 mesh copper grids (thin bars), allowed to dry, and were further washed with amyl acetate to remove the parlodian before electron microscopic examination. RESULTS

The Schwann cell plasma membrane of myelinated fibers after filipin treatment was generally heavily labeled with filipin-sterol complexes (Figs. 1 and 2). There was apparently no heterogeneity in the distribution of complexes in the outer Schwann cell plasma membrane in relation to the subjacent cytoplasmic network, either in large fibers where the cytoplasm forms distinct longitudinal and circumferential bands which delimit flat plaque-like areas (Fig. 1) or in small fibers where the cytoplasmic network around the flat areas in less elaborate. Similarly the presence of the two large longitudinal columns of cytoplasm, one on either side of the external mesaxonal furrow, was not correlated with a particular distribution of complexes in the plasma membrane. Also the many plasma membrane pores on the elevated cytoplasmic areas showed no particular association with complexes (Fig. 1). In both the E face and P face of the Schwann cell plasma membrane the complexes were usually discrete and clearly separated from each other (Figs. 1 and 2), while in a minority of fibers the characteristic protuberances were more frequent since they were commonly contiguous with each other. The density of complexes in such fibers was so high as to preclude any accurate quantitative assessment. Furthermore the saponin, tomatin, which is also sterol specific,

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FIG. 1. Schwann cell plasma membrane (P face) overlying a cytoplasmic network of longitudinal columns (PC) and circumferential bands (Pb) and intervening flat plaque-like areas (P). Note the distribution of fdipinsterol complexes over the columns, bands and plaque areas. f, external mesaxon furrow, e, extracellular matrix; arrowheads, plasma membrane pores. x 18 700.

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produces so many characteristic tubular complexes on the Schwann cell plasma membrane that they become confluent (Allt et al., unpublished observation). Saponin is therefore less suitable for revealing variations in the planar distribution of cholesterol than filipin for which most complexes remain discrete. Myelin membranes (Figs. 3 and 4) were less heavily labeled than the Schwann cell plasma membrane. Furthermore there was a consistent decrease in labeling from outer (abaxonal) to inner (adaxonal) myelin lamellae (Fig. 3). Complexes occasionally formed aggregates of varying sizes (Fig. 3) and in some instances these aggregates occurred in particle-free membrane patches (though the extent to which these particular conformations depended on the state of myelin fixation remains to be determined). No differences were apparent between E and P faces of myelin membranes. In some replicas (Figs. 6 and 7) myelin lamellae showed no labeling. The Schwann cell nuclear membranes were consistently heavily and evenly labeled (Fig. 5), though less heavily than the plasma membrane. No consistent differences were observed between inner and outer membranes of the nuclear envelope or between their E and P faces. Membranes of Schwann cell mitochondria (Figs. 6 and 7) contained no complexes while membranes of the endoplasmic reticulum (Fig. 6) and FIG. 2. (mo). Note FIG. 3. complexes x 18000.

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Golgi apparatus were intermediate. By contrast a fairly common, but unidentified, membrane-bound spherical organelle was always medium to heavily labeled (Figs. 6 and 7). The axonal plasma membrane (axolemma) when labeled showed heavy (Fig. 8) or intermediate (Fig. 9) labeling. The distribution of filipin-sterol complexes at nodes of Ranvier and Schmidt-Lamer-man incisures will be discussed in further publications. No E or P faces of membranes in the untreated control fibers showed the protuberances characteristic of filipin labeling. DISCUSSION

The finding in the present study of the heaviest membrane labeling in the plasma membrane (of both the Schwann cell and axon) is consistent with the results of other filipin-sterol freeze-fracture studies (Elias et al., 1979; Gotow and Hashimoto, 1983; Miller, 1984) and with biochemical estimates of a high cholesterol content (Demel and Kruyff, 1976; Gibbons et al., 1982; Finean et al., 1984) for a wide range of animal cells. Thus cholesterol is usually about equimolar with phospholipids in plasma membranes. The functional importance of this characteristically high colesterol content at the surface of the cell remains to be evaluated, but the decreased fluidity and reduced passive permeabilty to ions are the

Schwann cell plasma membrane (E face) and myelin lamellae en face (mf) and obliquely fractured the high density of filipin-sterol complexes at asterisks. e, extracellular matrix; c, collagen. x 28 100. Myelin lamellae obliquely fractured (P face) showing progressive overall decrease in filipin-sterol from the Schwann cell plasma membrane (Sp) through successive layers of myelin lamellae (ml-ma).

FIG. 4. Outer myelin lamellae (upper) show a linear series of membrane corrugations characteristic of a high density of filipin-sterol complexes. x 118 500. FIG. 5. Nuclear membrane of Schwann cell (P face of inner nuclear membrane: many intramembranous particles and nuclear pores as elevations). Membrane is rich in filipin-sterol complexes but less dense than in the plasma membrane (Figs. 1 and 2). m, myelin; SC, Schwann cell cytoplasm; e, extracellular matrix. x 26 300. FIG. 6. Schwann cell cytoplasm showing mitochondria (mi) without complexes and endoplasmic reticulum (rer) and a spherical organelle (1) with a medium density of complexes. m, myelin; Sp, Schwann cell plasma membrane; c, collagen. x 24 500. FIG. 7. Schwann cell cytoplasm in the region of the external mesaxon (arrows). mi, mitrochondrion; 1,spherical organelle; m, myelin; Sp, Schwann cell plasma membrane; arrowhead, fold in outermost myelin lamella characteristic of external mesaxon region. x 46 400.

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FIGS. 8 AND 9. Axolemma showing heavy (Fig. 8, P face) and medium (Fig. 9, E face) complex labeling. a, axon; mf, mo, mt, myelin lamellae en.face, obliquely a nd transversely fractured respectively. Fig. 8 x 56 000, Fig. 9 x 56 000.

most obvious effects (Sabine, 1977; Ostro et al., 1980; Bittman et al., 1984; Finean et al., 1984). The decreasing filipin labeling of myelin membranes from outer to inner lamellae may reflect an in viva heterogeneity in myelin chemical composition which would not be indicated by conventional biochemical fractionation methods (Davison and Peters, 1970; Siegel et al., 1981; Smith, 1983). Alternatively and much more likely, it is related to tight junctional diffusion barriers to filipin along the margins of the myelin sheath (MacKenzie et al., 1984a,b) and diffusion gradients of filipin across the myelin sheath. To distinguish between these two interpretations we are currently making different myelin preparations for filipin labeling which avoid the diffusion barrier and gradient considerations. Cholesterol represents a high proportion of myelin lipids (27% by

dry weight in rat peripheral nerve: Smith, 1983), furthermore myelin has a sterol : lipid ratio similar to that of plasma membranes of other cells (Demel and Kruyff, 1976). It will therefore be important to determine whether the lower filipin labeling of myelin compared with Schwann cell plasma membrane is reproducible when diffision barriers and gradients have been circumvented. The lack of any apparent difference between E and P faces of the myelin membrane in filipin labeling is consistent with biophysical data which indicate that in myelin cholesterol is symmetrically distributed between the two halves of the bilayer membrane (Scott et al., 1980). However, in such assessments of E and P face distribution it has to be borne in mind that sterol molecules have a relatively high rate of transfer across the membrane between bi-

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layers, i.e., “flip flop” (Demel and Kruyff, 1976; Miller, 1984). To take the two extreme examples of filipin labeling of organelles, the absent labeling of mitochondria in the present study is in accord with other filipin-sterol freezefracture studies of various cells (Elias et al., 1979; Gotow and Hashimoto, 1983; Kim and Okada, 1983; McGookey et al., 1983) and with biochemical estimates (Demel and Kruyff, 1976; Finean et al., 1984) of low cholesterol : phospholipid ratios. By contrast, the unidentified organelle with relatively high filipin labeling is likely to be lysosomal or secretory since a variety of filipin-sterol freeze-fracture studies (Orci et al., 1980; Greven and Robenek, 1982; Miller, 1984) and biochemical studies (Finean et al., 1984) have demonstrated that such organelles are intermediate in cholesterol content between plasma membranes and mitochondria. This may be related to the association of these intermediate organelles with the plasma membrane in endocytosis and exocytosis (Greven and Robenek, 1982; McGookey et al., 1983; Finean et al., 1984). We are indebted to The Wellcome support and to Miss B. M. Heyda providing the filipin.

Trust for financial of Upjohn Ltd. for

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