DEVELOPMENTAL BIOLOGY 84,67-'78 (1981)

Membrane Properties of Cultured Rat Sympathetic Neurons: Morphological Studies of Adrenergic

and Cholinergic Differentiation MARTIN SCHWAB AND STORY LANDIS

Department of Neurobiology, Harvard Medical School, Boston, Massacusetts 02115

Received July 11, 1980; accepted in revised form October 28, 1980

Dissociated neurons from the newborn rat superior cervical ganglion were grown under conditions which lead to either adrenergic or cholinergic differentiation. Lectins and toxins were used to detect differences in the cell membrane associated with transmitter status, age of the neurons, or location on the neurons. These ligands were made visible in the light or electron microscope by coupling to rhodamine or colloidal gold. The density of binding sites for con- canavalin A (Con A), ricin (RCA&, and wheat germ agglutinin (WGA) increased with age in culture on both adrenergic and cholinergic cells. Soybean agglutinin (SBA) binding increased about threefold on adrenergic axons, but failed to increase on neurons induced to become cholinergic by medium conditioned by rat heart cells (CM). The effect of CM on SBA binding paralleled previously described effects of CM on transmitter production; the CM binding pattern developed slowly and was not readily reversible. Mature adrenergic neurons also appeared to bind more WGA than neurons in CM cultures. Tetanus toxin gold binding was uniform, but low, on axons of adrenergic and cholinergic neurons at all ages. In contrast, cholera toxin binding decreased with age on adrenergic axons. Binding sites for SBA and tetanus toxin were found to be less numerous on the cell body surface than on the axonal surface. Thus growth in CM induces fundamental changes in the phenotype of developing sympathetic neurons involving the cell membrane as well as transmitter choice. Differences also appear with maturation and between axonal and somatic cell surface membranes.

INTRODUCTION

The plasma membrane, by carrying receptors for ex- tracellular signal molecules or for contact-mediated in- teraction, plays a key role in the communication of a cell with its environment. The composition of the sur- face membrane can encode specific characteristics of a cell type. Different regions of a neuron could also be identified by characteristic surface membrane compo- nents. During the development of the nervous system, an enormous number of specific connections has to be made, and cell-cell recognition based on specific mem- brane properties is thought to be particularly important (Barondes, 1976; Moscona, 1974). Indeed, when lectins have been used as specific markers for classes of mem- brane glycoproteins and glycolipids, age-related and cell-type specific differences have been found in various parts of the developing nervous system (Denis-Donini et al., 1978; Mintz and Glazer, 1978; Pfenninger and Maylie-Pfenninger, 1978; Sieber-Blum and Cohen, 1978; DeSilva et al., 1979; and Hatten et al., 1979). Most of these studies, however, have involved complex and het- erogeneous tissues, which hamper their interpretation. For this reason we have examined the binding of spe- cific cell surface markers to a single class of neuron, principal neurons from rat sympathetic ganglia, devel- oping in a dissociated cell culture system.

Sympathetic neurons dissociated from the superior cervical ganglia (SCG) of newborn rats can be grown in the virtual absence of nonneuronal cells (Mains and Patterson, 1973a). Under certain growth conditions, the neurons develop many of the properties of mature, ad- renergic sympathetic neurons (Mains and Patterson, 1973a,b; Rees and Bunge, 1974; Burton and Bunge, 1975; Patterson et al., 1975; Landis, 1980). If, however, the neurons are grown in the presence of certain nonneu- ronal cells or medium conditioned by these nonneuronal cells, the cultures develop cholinergic properties (O’Lague et al., 1974, 1975, 1978; Patterson and Chun, 1974, 1977a; Patterson et al., 1975; Johnson et al., 1976; Ko et al., 1976). Conditioned medium appears to induce neurons which have already acquired adrenergic prop- erties to become cholinergic (Johnson et al., 1976; Pat- terson and Chun, 1977b; Landis, 1980); thus the dual function neurons in single-neuron microcultures which release both acetylcholine and norepinephrine are likely to represent neurons in transition from adrenergic to cholinergic function (Furshpan et al., 1976; Landis, 1980; Potter et al., 1980). In viva, a fraction of the principal sympathetic neurons are cholinergic (Sjqvist, 1963a; Aiken and Reit, 1969; Yamauchi et al., 1973). In the cat, these neurons are found mainly in the stellate and lum- bar sympathetic ganglia and they specifically innervate targets such as the eccrine sweat glands of the foot pad

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68 DEVELOPMENTALBIOLOGY VOLUME 84. 1981

(Sjijqvist, 1963a,b). This very specific anatomical and functional relationship raises the possibility that the change in transmitter choice observed with CM in vitro may be only one aspect of a fundamental change in the characteristics of the cell. In fact, some biochemical differences in released proteins and cell surface con- stituents between mature adrenergic and cholinergic SCG cells in vitro have been found (Sweadner and Braun, 1979).

Lectins and toxins have been used in cell biology to investigate differences between the surface constitu- ents of different cell types or regional differences on single cells (Moscona, 1974; Barondes, 1976; Dimpfel et al., 1977; Hansson et al., 1977; Brown and Hunt, 1978; Mirsky et al., 1978). Despite the fact that the lectins are not monospecific, they have been useful markers, mainly for surface membrane glycoproteins. Tetanus toxin binding sites are known to be highly enriched in the neuronal membrane (Dimpfel et al., 1977; Mirsky et al., 1978). Indirect evidence indicates that cholera toxin binds to the ganglioside GM, (Hansson et al., 1977) and tetanus toxin to GDlb and GT, (van Heyningen, 1974; Lee et al., 1979). Sympathetic ganglion cells of the adult rat have receptors for these ligands since in vivo ex- periments have shown that several lectins and toxins specifically bind to and are taken up by their nerve terminals (Dumas et al., 1978; Schwab and Thoenen, 1978; Schwab et al., 1979). In the present morphological study, we have used lectins and toxins as specific mark- ers to examine the surface membranes of cultured SCG cells. We have found age-, region-, and transmitter-re- lated differences in the binding of some but not all of the probes used.

MATERIALSANDMETHODS

Neuron Cd tures

Neurons were mechanically dissociated from the SCG of newborn rats according to the method of Bray (1970) as modified by Mains and Patterson (1973a) and plated onto polystyrene coverslips (Lux Scientific Corp., New- bury Park, Calif.) inserted in Falcon plastic petri dishes (Becton, Dickinson and Co., Oxnard, Calif.) and coated with sterile collagen (Chun, personal communication), The neurons were grown in L-15 COe medium with 20 mMK+ which depolarizes the neurons and strongly sup- ports adrenergic differentiation (Walicke et al., 1977), or in L-15 COz with 50% medium previously conditioned by incubation on heart cells (CM) which induces cho- linergic properties (Patterson and Chun, 1977a). In both cases, the final growth medium contained 5% rat serum and 7s NGF at 1 Fg/ml, but no Methocel. The medium was changed every 2 days. On Days 2 to 4 and 6 to 8,

the cultures were treated with 10 pM cytosine arabi- noside, to suppress the growth and division of nonneu- ronal cells in L-15 COe-based media (Patterson and Chun, 1977a). The neurons were plated at relatively low density in order to obtain sparse cultures and facilitate rinsing and reagent penetration. No differences in neu- ronal health or survival were observed under the dif- ferent growth conditions (Walicke et al., 1977). In sev- eral experiments, the neurons were plated into the central well of a three-chamber culture system as de- scribed by Campenot (1979). Rat serum was included in the growth medium in one side chamber and excluded from the other.

The cultures were routinely examined for lectin or toxin binding at 2, 6, or 21 days of age. In several in- stances, cultures were examined at 6 weeks. In most experiments, one or two sister cultures from each growth condition were fixed with potassium perman- ganate, en bloc stained with uranyl acetate and embed- ded (Landis, 1976, 1980). Thin sections were examined to make sure that the cultures grown in 20 mM K+ contained adrenergic terminals and that the cultures grown in CM contained a majority of terminals which contained no small granular vesicles and appeared cho- linergic.

Fluorescence Microscopg

Con A, RCAGO, SBA, and WGA coupled to rhodamine were purchased from Vector Laboratories, Inc. (Burlin- game, Calif.). The molar ratios of lectin/rhodamine were 2.4 for Con A; 2.3 for SBA, and 0.7 for WGA. All lectins were diluted in PBS, pH 7.4, before use to a final concentration of 50 pg/ml. The same lots of labeled lec- tins were used in a total of six independent experiments. In each separate experiment, comparisons were made between 2-, 6-, and 21-day cultures; 21-day adrenergic and CM cultures were sister cultures.

Cultures were washed three times for 2 min each with phosphate-buffered saline (PBS) containing 0.1% bo- vine serum albumin (BSA) (Sigma Chemical Co., St. Louis, MO.) at room temperature and incubated for 30 min with the fluorescent lectins (20-23°C). To determine nonspecific binding, the lectins were preincubated for 10 min with the specific hapten sugar at 0.2 M. After the lectin incubation, the cultures were washed three times within 1 min with PBS containing BSA and then fixed for 20 min in 1.2% glutaraldehyde in PBS. After a brief rinse, the cultures were mounted in glycerol- PBS (1:l v/v). Rhodamine-specific fluorescence was ex- amined with epiillumination on a Zeiss ICM35 inverted microscope equipped with a HB050 mercury burner, a 580 reflector, and a 510-560 excitation filter. The cul-

SCHWAB AND LANDIS Membranes of Sympathetic Neurons 69

tures were number coded and examined in a double- blind fashion by two independent observers. The inten- sity of the specific fluorescence was rated as very strong, strong, moderate, weak, or very weak. Photomicro- graphs were taken on H & W film (H & W, St. Johns- bury, Vt.) with standardized exposure times. The films were then developed and prints were made under iden- tical conditions in order to facilitate comparisons.

Electron microscopy

Purified SBA and Lens culinaris haemagglutinin B were purchased from Miles-Yeda Ltd. (Rehovot, Israel), phytohemagglutinin from Polysciences (Warrington, Pa.), and cholera toxin from Schwa&Mann (Orange- burg, N. Y.). Highly purified tetanus toxin was a gift from Dr. B. Bizzini, Institut Pasteur, Paris, France. Colloidal gold (particle size 160-200 A) was obtained by reducing gold chloride with 2% sodium citrate (Frens, 1973) and coupled to the lectins or toxins as previously described (Horisberger et al., 1975; Schwab and Thoe- nen, 1978). After two washes by centrifugation (the sec- ond wash taking place immediately before use) these tracer complexes were diluted with PBS. Their concen- tration was adjusted to a standard for each experiment by measuring the light absorption at 420 nm in a Gilson spectrophotometer. The standard concentration con- tained about 10” gold grains/pi. This concentration was determined by counting the grains in the electron mi- croscope in 0.5-~1 aliquots of a serial dilution applied to formvar-coated 1 X 2-mm slot grids. The approxi- mate stoichiometry of one of the complexes was deter- mined using ‘?-labeled SBA and found to be 20-50 SBA molecules per goid grain. ‘%I-SBA with known specific activity was coupled to a known amount of colloidal gold, centrifuged, and diluted to the standard concen- tration. The concentration of SBA molecules was cal- culated from the specific activity and related to the con- centration of gold granules. The same experiment showed that virtually all the lectin (1 mg/50 ml of col- loidal gold) was coupled to the gold and less than 5% was lost during washing and centrifugation.

Cultures were washed three times, 2 min each, with PBS containing 0.1% BSA and were incubated for 30 min at room temperature with the lectin- or toxin-gold complexes. One experiment with tetanus toxin was done on ice and the results obtained were not different from those obtained with incubations at 20-23°C. Nonspecific binding was determined by incubating in the presence of 0.2 M hapten monosaccharide or a 500-fold excess of unlabeled tetanus toxin. The cultures were again rinsed three times with PBS containing BSA, fixed for 3 hr in 3% glutaraldehyde in 0.12 M phosphate buffer, pH 7.4, postfixed in 1.3% Os04, block stained with uranyl ace- tate in acetate buffer, dehydrated, and embedded in

Epon. Thin sections (1000 A) were cut parallel to the culture surface, stained with lead citrate, and viewed with a Phillips 400 electron microscope. Micrographs (15-20) for each culture were taken randomly from freely exposed axonal or cell body membranes on num- ber-coded grids at a magnification of 17,000 and en- larged 2.5 times. Gold grains in contact with the plasma membrane were counted in each sector of freely exposed and perpendicularly cut surface membrane (minimal length about 2 pm), the membrane was measured with a computerized graphic tablet, and the results were expressed as densities. Significant variation between experiments was observed only with cholera toxin; in this case, the amount of binding observed depended on the batch of toxin used. The results from these exper- iments were normalized to the experiment with the highest values. Sister cultures were always used for the comparison of the effect of culture conditions. Students t test was used to test for the significance of the dif- ference of the means for the various experimental sit- uations. In order to reduce variability and facilitate comparisons, in almost every experiment, 2- and 2i-day cultures were incubated with the same lot of lectin or toxin, then fixed and processed at the same time. Each point represents values obtained from two to eight cul- tures.

RESULTS

Light Microscopy

Four lectins, Con A, RCAGO, SBA, and WGA, coupled to rhodamine were used to examine lectin binding sites on axonal and cell body membranes of SCG neurons, after 2, 6, or 21 days in culture, under conditions fa- voring either adrenergic (20 mM K+ in the medium) or cholinergic (rat heart cell CM) differentiation. With all four lectins the nonspecific staining in presence of the hapten sugars (cr-methyl-manno-pyranoside for Con A; galactose for RCA,; N-acetylglucosamine for WGA; N- acetylgalactosamine or galactose for SBA (Brown and Hunt, 1978)) was very faint; this is illustrated for SBA in Figs. lg, h. Significant specific staining of the culture substrate, the collagen, or associated serum- or cell- derived material, was observed with Con A and RCAGO. On neurons and axons, the staining was diffuse and regular; no patching or capping of the fluorescent lectins was seen with the present experimental procedure, i.e., labeling for 30 min at room temperature.

A consistent finding with all four lectins was the marked increase in staining intensity with age. With Con A the staining increased from a low level on both axons and cell bodies after 2 days in culture (Fig. la), to a moderate reaction at 6 days, and bright staining at 3 weeks (Fig. lb). No significant differences were

DEVELOPMENTAL BIOLOGY VOLUME 84, 1981

FIG. 1 . (a) Adrenergic culture at 2 days. Cell bodies and axons of the neurons bind very little rhodamine-Con A. Single ax ons are barely visible ! . {b) Adrenergic cultures at 21 days. Cell bodies and axons are brightly fluorescent when incubated with rhodamine Con A. Single axons al *e clearly visible. The small, brightly fluorescent dots present on some axon bundles are cellular debris that accumul ates with time in cul tu re. (c) Adrenergic culture at 21 days. The somata and axons of adrenergic neurons are brightly fluorescent wher 1 stained with rhodal mi ne-WGA. (d) CM culture at 21 days. The cell bodies and axons of neurons in CM cultures are somewhat less fluoreso ent than those in adr ‘er rergic cultures. This difference is slight but reproducible. (e) Adrenergic culture at 21 days. Axons exhibit distinct staining with rhodal mi ne-SBA. (f) CM culture at 21 days. In contrast to (e) only traces of fluorescence are visible with rhodamine-SBA. (g, h) Incubation in the P resence of the hapten sugar, N-acetylgalactosamine, leads to a very low level of nonspecific binding in adrenergic (9) or CM (h) cu1tur es Cell bodies are barely visible in (g). (a-h) were photographed, developed, and printed under identical conditions. Xl 350.

SCHWABANDLANDIS Membranes of Sympathetic Neurons 71

observed between adrenergic and CM cultures. A sim- ilar result was obtained with RCAso (not illustrated). The level of RCAGO binding at 2 days seemed to be slightly higher than that of Con A. Again, there was no difference between the staining of adrenergic and cholinergic cultures.

WGA yielded distinct and moderate labeling in 2-day- old cultures, increased labeling at Day 6, and prominent labeling at 21 days (Figs. lc, d). A small but reproduc- ible effect of CM was evident at 21 days; CM cultures bound somewhat less WGA than the adrenergic cul- tures grown for 21 days in 20 mM K+ (compare Figs. lc and d). When cultures were grown for 3 weeks in CM or 20 mM Kf and then were switched to 20 mJ4 Kf or CM, respectively, for 2 or for 3 days, the neurons re- tained the intensity of WGA staining appropriate for the original growth conditions.

After staining with SBA, the 2-day-old cultures showed a low level of lectin binding. In the adrenergic cultures, the staining increased to moderate levels at 6 days and was very distinct at Day 21 (Fig. le). In contrast, the SBA binding to neurons grown in CM stayed at a low level (Fig. If). This difference in staining intensity was still pronounced at 6 weeks and was al- ready apparent at 6 days, concomitant with the ap- pearance of detectable levels of ACh synthesis in cul- tures grown under similar conditions (Patterson and Chun, 19’7’7b) and a significant drop in the percentage of small granular synaptic vesicles observed after per- manganate fixation, an indicator of the endogenous pool of norepinephrine (Landis, 1980). If 21-day adrenergic cultures were switched to CM for 3 days, they showed only a slight decrease in fluorescence (not illustrated). No decrease was evident, however, if they were kept in CM for only 2 hr. The slow time course of the effect of CM on SBA binding makes it unlikely that CM com- ponents mask SBA receptors or have a direct enzymatic effect on the binding site. Cultures grown in CM for 3 weeks were not influenced by incubation in 20 mJ4 K+ medium for 2 hr or 3 days.

In addition to this prominent difference in SBA stain- ing intensity between adrenergic and CM cultures, there were indications of regional differences as well: in ad- renergic cultures, at 2 days, cell bodies seemed to bind more SBA than axons, but at 3 weeks, the reverse was true (not illustrated). These differences were confirmed by the electron microscopic analysis described below. In CM cultures at 6- and 21-days dots and short seg- ments of bright fluorescence were present along the faintly fluorescent axon bundles (Fig. If). In order to decide whether these SBA-positive regions were related to bundle formation, we grew neurons in CM in a three- chamber system (Campenot, 1979). Axons grew from cell bodies in the center chamber into two side cham-

bers. One side chamber contained medium with serum, a condition which favors fiber bundling. The other side chamber contained medium without serum, a condition which largely inhibits bundle formation (Campenot, unpublished observations). After incubation with SBA, the streak-like staining was correlated with the occur- rence of fiber bundles (Fig. 2a). In contrast, single axons possessed a uniform and very low density of SBA bind- ing sites (Fig. 2b). This staining pattern was not ob- served with all lectins. For example, reaction with WGA yielded a uniform and strong staining of single axons and bundles.

Electron Microscopg

In a series of pilot experiments, various methods for direct visualization of macromolecular ligands were compared, including horseradish peroxidase, ferritin, and colloidal gold particles. For the present study, the most useful marker proved to be the colloidal gold gran- ules. Preparation of the coupling product minimizes chemical modification since the ligands are electro- statistically adsorbed to the granules and takes several hours. In addition, these highly stable complexes (Schwab and Thoenen, 1978) are easily visible in the EM (160-200 A) and therefore allow rapid sampling of large surface areas. However, these gold complexes are polyvalent; coupling with lzr’I-SBA, for example, indi- cated that approximately 20-50 SBA molecules were adsorbed to each gold particle. This is comparable to previously reported values of 1:lO for several other lec- tins (Horisberger and Vonlanthen, 1979; Maher and Molday, 1979). Because of the polyvalency of the gold complexes, the reported densities of the label do not have a 1:l correspondence with the number of binding sites. Due to their size, the colloidal gold granules have restricted penetration into and washout from axon bun- dles and synaptic clefts. The present analysis has there- fore been confined to freely accessible cell and axon surfaces and does not exclude the possibility that there are sequestered membrane regions, in particular at- tachment sites and synaptic regions, that possess a dif- ferent membrane composition.

In initial screening experiments, phytohemagglutinin and Lens culinaris lectin complexes (hapten sugars N- acetylgalactosamine and mannose, respectively) showed low specific binding to 10 and 21-day-old SCG neurons and no obvious difference between adrenergic and CM cultures. We therefore concentrated on SBA and two ligands reported to be specific for gangliosides, tetanus and cholera toxin.

Soybean agglutinin. A three-fold increase in SBA- gold binding to adrenergic axons, similar to that ob- served with rhodamine-coupled SBA, occurred between

72 DEVELOPMENTAL BIOLOGY VOLUME 84, 1981

FIG. 2. CM chamber culture at 21 days (a, b). Axonal bundling is greatly reduced in the side chambers when serum is excluded from the growth medium. After staining with rhodamine-SBA, single fibers are very faintly and uniformly fluorescent (arrows). Streak- and dot-like fluorescent labeling patterns are characteristic of even very small bundles (asterisk). Dendrites do not extend into the side chambers. X1350.

Day 3 and Day 21 in culture. This increase was absent from cultures grown in CM (Figs. 3a, d; Table 1). Figure 4 shows the densities of SBA-gold binding plotted in a population distribution histogram. There is some overlap between values obtained from adrenergic and CM cultures, and CM cultures clearly contain some ax- ons and cell bodies with SBA binding densities similar to those of adrenergic neurons. This is consistent with evidence that at 3 weeks, CM cultures contain not only cholinergic neurons but also adrenergic and dual-func- tion neurons (Landis, 1980).

Interestingly, the surface membrane of adrenergic cell bodies has different properties than that of axons: the SBA binding of cell bodies was higher than that of axons at 3 days (ratio 1.4), but much lower at 21 days (ratio 0.25, compare Figs. 3a and b). However, the cell bodies of adrenergic and cholinergic neurons differed in SBA binding just as the axonal membranes did (Ta- ble 1; Fig. 4b).

Tetanus Toxin

The density of binding sites for tetanus toxin was low, and in contrast to the lectins studied, it remained unchanged between Day 3 and Day 21 (Table 2). Ac- cordingly, no difference was observed between adren- ergic and cholinergic cultures (Table 2; Figs. 5a, b; Fig. 6, right). On the other hand, a consistent and highly significant difference was seen between tetanus toxin- gold binding to axons and cell bodies; at all ages less toxin bound to cell bodies than to axons (ratio 0.44-0.75, Table 2). Labeling on ice, which prevents lateral mo- bility in membranes, did not influence toxin binding. Excess (500-fold) native toxin, however, displaced most of the label (Table 2).

Cholera Toxin

The binding patterns observed with this toxin dif- fered both from those of the lectins and from tetanus toxin. Cholera toxin binding decreased from Day 3 to Day 21 on axons in adrenergic cultures, but tended to increase on axons in CM cultures (Table 3, Figs. 5c, d). The population distribution histogram of the toxin binding densities showed overlap between adrenergic and CM neurons similar to that seen with SBA binding (Fig. 6). Again this overlap presumably corresponds to the presence of dual function and adrenergic neurons present in the CM cultures. Due to the scatter present in the density values obtained for cell bodies, no con- clusions can be drawn concerning their age differences or regional differences.

DISCUSSION

In the present study, the cell membranes of disso- ciated rat sympathetic neurons developing in culture showed striking differences in the ability to bind cer- tain lectins and toxins. These differences were corre- lated with differences in age, or transmitter choice or cell region. The density of binding sites for Con A, RCAGO, and WGA increased with age (2 to 21 days) un- der culture conditions which lead either to adrenergic or to cholinergic differentiation. In sharp contrast, an increase in SBA binding occurred exclusively on adren- ergic neurons; most neurons in CM cultures retained a low level of SBA binding sites. Cholera toxin binding, on the other hand, decreased with age on adrenergic neurons and slightly increased on cholinergic ones. Tet- anus toxin-gold binding remained at a relatively low level and was unchanged with increasing age under ei- ther adrenergic or cholinergic conditions. From these results it can be concluded that similarities as well as

SCHWAB AND LANDIS Membranes of Sympathetic Neurons 73

quantitative and possibly qualitative differences exist development and the transmitter choice of these cells. in the glycoprotein and/or glycolipid composition of These changes occur during the differentiation and membranes of sympathetic neurons in culture and maturation of neurons which were initially very simi- changes in the membrane composition accompany the lar, if not identical, in their properties. This is an im-

FIG. 3. (a) An axon in a Zl-day-old adrenergic culture binds SBA-colloidal gold complexes heavily. (b) Far fewer SBA-colloidal gold complexes are bound to a cell body in the same culture. (c) Incubation in the presence of a specific hapten sugar, galactose, virtually eliminates the SBA-colloidal gold binding to H-day adrenergic axons. (d) Cholinergic axons in a Zl-day-old CM cultures bind little SBA. The grains accumulated in a patchy distribution may represent trapped SBA-gold complexes or could be the ultrastructural equivalent of the fluorescent SBA patches. (a-d) X51.000.

74 DEVELOPMENTAL BIOLOGY VOLUME 84, 1981

TABLE 1 DENSITY OF LABEL FOR SOYBEAN AGGLUTININ-GOLD ON AXONS AND CELL BODIES OF 3- AND 21-DAY-OLD ADRENERGIC AND CM CULTURES

Grains/~m2, mean + SEM (n)

Axons Cell bodies

Ratio of cell bodies

to axons

3 Days in culture, adrenergic 30.3 f 4.0 (23) 43.6 + 6.2 (14) 1.4

21 Days in culture, adrenergic 107.4 + 7.4 (32) 26.4 + 1.6 (15)b 0.25

21 Days in culture, adrenergic + galactose 4.2 f 1.0 (11) 2.8 + 0.7 (6)

21 Days in CM 20.3 + 1.4 (66pd 15.1 + 2.4 (39) 0.74

21 Days in CM + galactose 8.4 f 1.6 (17) 1.5 k 0.5 (6)

’ Significantly different (P < 0.001) from axons at 3 days.

b Significantly different (P < 0.001) from axons of the same age. ’ Significantly different (P < 0.001) from axons at 3 days and adrenergic axons of the same age. * Range of values considered: 0.1-49.9 grains/pm’ (compare Fig. 5). e Significantly different (P < 0.005) from adrenergic cell bodies and cholinergic axons of the same age.

portant difference from studies on tissue homogenates, where it is difficult to determine whether changes ob- served in the glycoprotein pattern are related to changes in a particular population of cells or to simultaneous changes in more than one population (Mintz and Glazer, 19’78; DeSilva et al., 1979; Engel et cd., 1979).

It seems likely that growth in CM has an effect on the synthesis or probessing of specific membrane con- stituents. We have found that exposure to CM for 2 hr does not change SBA binding. Furthermore, the pattern of SBA binding on neurons grown in CM is not uniform in that CM cultures always contain some axons and cell bodies with SBA binding characteristics typical of ad- renergic neurons. This slow time course and the non-

uniform effect of CM diminish the likelihood that CM components directly mask or enzymatically alter the SBA binding sites. The small number of neurons with adrenergic membrane characteristics in the CM cul- tures at 21 days is consistent with biochemical (Pat- terson and Chun, 1977a), cytochemical (Landis, 1980), and physiological (Furshpan et aZ., 1976; Potter et al., 1980) evidence for the presence of some adrenergic and dual-function neurons among the majority population of cholinergic neurons in these cultures. The fact that some cells with adrenergic lectin binding properties are present in CM cultures also makes it unlikely that the labeling pattern observed in the cultures grown in 20 mM K+ is due to a direct effect of depolarization rather

Soybean Agglutinin

1 t

67

2ld

FIG. 4. The percentage of sampled axons or cell bodies (ordinate) which possessed the indicated binding of SBA-colloidal gold complexes (abscissa) is shown for sister cultures grown for 21 days under adrenergic conditions (L. 15CO2 + 20 mM K+) (n = 32) or under cholinergic conditions (L.COa + CM) (n = 66). Some axons in the CM cultures possess SBA binding characteristic of adrenergic axons. Arrows indicate

the means.

SCHWAB AND LANDIS Membranes of Sympathetic Neurons 75

TABLE 2 DENSITY OF LABEL FOR TETANUS TOXIN-GOLD ON AXONS AND CELL BODIES OF 3- AND 21-DAY-OLD ADRENERGIC AND CM CULTURES

Grains/pm’, mean k SEM (n)

Axons Cell bodies

Ratio of cell bodies to axons

3 Days in culture, adrenergic, labeling at room temperature

3 Days in culture, adrenergic, labeling on ice

3 Days in culture, adrenergic, + excess unlabeled toxin

21 Days in culture, adrenergic 21 Days in culture, adrenergic,

+ excess unlabeled toxin 21 Days in CM 21 Days in CM + excess

unlabeled toxin

18.4 + 1.4 (41) 13.8 k 1.3 (52) 0.75

21.3 + 1.3 (18) 11.7 + 2.2 (lo)* 0.55

2.3 f 0.9 (15) 0.7 + 0.3 (9) 20.3 f 1.3 (68) 9.0 + 1.2 (24)” 0.44

4.5 f 0.7 (35) 2.2 f 0.7 (12) 18.7 f 1.5 (31) 12.9 + 1.8 (5) 0.69

3.1 k 1.0 (11) -

’ Significantly different (P < 0.05) from axons of the same age. *,’ Significantly different (P c 0.01) from axons of the same age.

than to the adrenergic character of the neurons. Fur- ther, the binding properties of SBA on 21-day-old ad- renergic cells can be influenced by CM treatment for 3 subsequent days, but medium containing 20 mM K+ does not reverse the differentiation induced by growth in CM for 21 days. This finding parallels the effects of CM and 20 mM K+ on adrenergic and cholinergic neu- rons with regard to reversibility of their transmitter choice (Walicke et al., 1977). These close correlations of membrane properties and transmitter status in relation to (i) the developmental time course, (ii) the hetero- geneity in CM cultures, and (iii) the reversibility of both these properties strongly suggest that both are a re- flection of fundamental changes in the phenotype of young sympathetic neurons induced by CM.

The molecular significance of the observed binding patterns for these lectins and toxins has to be inter- preted with,caution. Since lectins may bind to several membrane constituents with different affinities (Brown and Hunt, 1978), quantitative or qualitative changes in only one constituent might not be detectable. A further limitation is that fluorescence microscopy of cultured neurons allows only a qualitative estimation of differ- ences. The possibility exists that the SBA and cholera toxin binding sites do not represent the only differences between the cell membranes of adrenergic and cholin- ergic sympathetic neurons. Analysis of one-dimensional gels after surface labeling with borohydrate after ga- lactose oxidase or periodate treatment has indicated, however, that most of the major bands are similar in both types of neurons (Sweadner and Braun, 1979). Reaction of membrane glycoproteins separated on gels or glycolipids separated by thin-layer chromatography with labeled lectins or membrane-directed monoclonal

antibodies will help resolve the question of additional transmitter-specific differences, as well as the nature of the SBA binding site.

Differences were also observed between the mem- branes of axons and cell bodies. Fewer tetanus toxin and SBA binding sites were present on cell bodies than on axons after 21 days in culture. In 3-day-old neurons, tetanus toxin showed a similar difference, but the SBA binding pattern was reversed: axons had fewer sites than cell bodies. These neurons selectively form mor- phologically differentiated synapses with each other (Rees and Bunge, 1974; O’Lague et al., 1975; Landis, 1980) and on themselves (“autapses”) (Landis, 1976). Serial section reconstruction of single neurons in mi- crocultures has shown that the autapses are axosomatic or axodendritic but not axoaxonal (Landis, 1976, 1977, in preparation). This implies that during synaptogen- esis, the growing, highly branched axon is able to dis- tinguish between axonal and dendritic or somatic mem- brane. Whether the particular binding sites identified in the present studies are involved in membrane rec- ognition during synaptogenesis remains to be deter- mined. Biochemical comparisons of axonal and cell body membrane of SCG explant cultures failed to show major differences in the polypeptide (Esteridge and Bunge, 1978) or ganglioside (Cornbrooks et al., 1980) pattern. The quantitative differences described with the present morphological methods are presumably due to the use of highly specific and sensitive methods. Regional dif- ferences in lectin binding have also been described in mouse cerebellum and embryonic chick spinal ganglion cells in vitro (Denis-Donini et al., 1978; Hatten et cd., 1979). Not all lectin binding sites, however, appear to be differentially distributed since Pfenninger and May-

76 DEVELOPMENTAL BIOLOGY VOLUME 84, 1981

FIG. 5. (a) Adrenergic culture at 21 days. Axons bind a few tetanus toxin gold complexes. Varicosities do not label differently than

intervaricose regions. (b) CM culture at 21 days. The binding of tetanus toxin-gold to axons is identical to that observed in adrenergic cultures. One gold granule has been internalized. This was a rare observation. (c) Adrenergic culture at 21 days. Axons bind very little cholera toxin-gold. (d) CM culture at 21 days. More cholera toxin labeling is present on axons.

1% (1978) have reported a uniform distribution of la- linergic cultures. These regions are correlated with fas- beling with WGA and RCA along cultured neurons. ciculation since they are absent from single axons when

In addition to the differences in SBA binding between bundling is prevented. This staining pattern is specific axon and cell body, dot- and streak-like SBA-positive for SBA, and could represent sites where neurites at- regions have been observed in the fiber bundles of cho- tach to each other. Concentration of SBA binding sites

SCHWAB AND LANDIS Membranes of Sympathetic Neurons 77

Cholera toxin .axonr. Zld

Tetanus toxin axons. 2ld

FIG. 6. (Left) The percentage of sampled axons (ordinate) which possessed the indicated binding of cholera toxin-colloidal gold com- plexes (abscissa) is shown for sister cultures grown for 21 days under adrenergicconditions(L.15COz + 20mMK+;n=53)orundercholinergic conditions (L.15COz + CM; n = 85). Some axons in the CM cultures possess cholera toxin binding characteristic of adrenergic axons. Ar- rows indicate the means. (Right) The percentage of sampled axons (ordinate) which possessed the indicated binding of tetanus-toxin gold (abscissa) is shown for sister 21-day adrenergic (n = 68) or CM (n = 31) cultures. There is no significant difference in the histogram. Arrows indicate the means.

at contact points between developing neurons has also been observed for neural crest cells (Sieber-Blum and Cohen, 1978). It was not possible to determine whether adrenergic neurons had such putative SBA binding at- tachment molecules in high number over the entire axonal membrane or whether there was a widely dis- tributed SBA binding component in addition to the pu- tative attachment sites.

TABLE 3 DENSITY OF LABEL FOR CHOLERA TOXIN-GOLD ON AXONS AND CELL

BODIES OF 3- AND 21-DAY-OLD ADRENERGIC AND CM CULTURES

Grains/pm’, mean f SEM (n)

Axons Cell bodies

3 Days in culture, adrenergic

21 Days in culture, adrenergic

21 Days in CM

74.9 + 5.7 (34) (62.8 k 11.8 (12))*

33.6 + 3.4 (53) (12.8 f 3.0 (1l))b 80.0 f 4.5 (85) n.d.d

(r Significantly different (P < 0.001) from axons at 3 days. *Tentative values due to an uncommonly large scatter within and

between the single experiments. ’ Significantly different (P < 0.001) from adrenergic axons of the

same age. d Not determined.

We wish to thank Paul Patterson, Ed Hawrot, Kathleen Sweadner, David Potter, Ed Furshpan, and our other colleagues for their helpful discussions; Doreen McDowell and Claudia Miles for their help with the culturing; Allison Doupe for supplying NGF; Bernard Bizzini (Institut Pasteur, Paris, France) for supplying tetanus toxin; David Freeman for computer assistance; Ed Lamperti and May Hogan for their technical assistance; and Joe Gagliardi and Shirley Wilson for their help with the manuscript. This research was supported by Grants-in-Aid from the Milton Fund and the American Heart As- sociation (78-964) and U. S. Public Health Service Grants (NS 15549, NS 11576, NS 02253) from the National Institute of Neurological and Communicable Diseases and Stroke. M.S. was a Research Fellow of the Swiss National Foundation for Scientific Research (Grant 83-595- 0-78).

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