Native and derivatized lectins for in vivo studies of neuronal connectivity and neuronal cell biology

Journal of Neuroscience Methods, 9 (1983) 1 8 5 - 2 0 4 185

E l sev i e r

Review Paper

Native and derivatized lectins for in vivo studies of neuronal connectivity and neuronal cell biology

John Q. Trojanowski Division of Neuropathology, Department of Pathology and Laboratory Medicine, University of Pennsylvania

School of Medicine, Philadelphia, Johnson Pavilion~G2, PA 19104 (U.S.A.)

(Received M a r c h 22nd , 1983)

( A c c e p t e d A u g u s t 23rd, 1983)

Key words: l ec t ins - n e u r o n a l c o n n e c t i v i t y - i n t r a n e u r o n a l t r a n s p o r t - t r a n s c e l l u l a r t r a n s p o r t

Contents

1. In t roduc t ion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185

2. Studies of neuronal connect iv i ty with lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186

2.1. ln t ra-axonal ly t ranspor ted lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 186

2.2. Me thods to vizualize t ransported lectins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 188

2.3. Quan t i t a t ion of lectin sensi t ivi ty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189

2.4. Dete rminants of lectin sensit ivi ty . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190

2.5. L imi ta t ions in the use of lectins in neuroana tomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 191

3. Lectins for in v ivo studies of neuronal cell biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193

3.1. Neuronal membranes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194

3.2. Routes of in t raneuronal t ranspor t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194

3.3. Transcel lular t ranspor t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 196

4. Fur ther modi f ica t ions and uses of lectins in neurobiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198

5. Conclus ions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 198

1. Introduction

Advances in cell biology are critically dependent on the development of new techniques which permit investigators to test their hypotheses. This is exemplified by developments in the neurosciences where methodological innovations are intensely pursued (Heimer and Robards, 1981; Lahue, 1981; Chan-Palay and Palay, 1982; Mesulam, 1982). For example, the elegant work of Hubel (1982) and Wiesel (1982) exploited a powerful array of new neuroanatomical imaging techniques. Among these have been methods for demonstrating neural pathways by using probes which are orthogradely and/or retrogradely transported within neurons and their processes. Tritiated amino acids and the plant enzyme horseradish peroxidase (HRP) have

0 1 6 5 - 0 2 7 0 / 8 3 / $ 0 3 . 0 0 © 1983 E l sev ie r Sc ience P u b l i s h e r s B.V.

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proven to be among the most useful probes for studies of neuronal connectivity and they have also contributed to our understanding of the endocytosis, intraneuronal transport and transcellular transfer of molecules in the nervous system (Lasek et al.. 1968; Kristensson and Olsson, 1971; LaVail and LaVail, 1972: Grafstein and Forman, 1980; Mesulam, 1982). Various methodological innovations have led to significant improvements in the use of HRP (Mesulam, 1982). Recently. attention has focused on the development of more sensitive probes. A category of ligands classed as lectins (Goldstein and Hayes, 1978; Barondes, 1981) appear promising (Schwab et al., 1978; Gonatas et al., 1979). Lectins and related ligands have been used extensively in cell biology as probes for a variety of studies particularly for the investigation of the plasma membrane of cells. Such studies have been reviewed elsewhere (Goldstein and Hayes, 1978; Gonatas and Avrameas, 1977: Gonatas, 1979, 1982). The present review considers recent developments in the use of such probes in neurobiology, especially for studies of neuronal connectivity and for studies of various aspects of the biology of neurons such as intra- and interneuronal transport in vivo.

2. Studies of neuronal connectivity with iectins

A radiolabeled lectin, wheat germ agglutinin (WG), was introduced as a highly sensitive retrogradely transported tracer in 1978 (Schwab et al., 1978). In 1979 conjugates of HRP with various ligands including WG and other lectins were also found to be retrogradely transported (Gonatas et al., 1979; Schwab et al., 1979). In a detailed quantitative study, HRP conjugated with WG (WGHRP) was determined to be superior to native HRP (NHRP) for studies of retrograde axonal transport (Gonatas et al., 1979). The sensitivity of WG was ascribed to the affinity of this lectin for membrane oligosaccharides and its internalization by adsorptive endocytosis (Schwab et al., 1978; Gonatas et al., 1979). N H RP does not bind to membrane moieties and is internalized by fluid phase endocytosis. WG is a lectin derived from wheat with a specific affinity for N-acetylglucosamine and sialic acid moieties which may play a role in defending wheat against pathogens (Mishkin et al., 1982). Despite the uncertainty concerning the function of WG and other lectins, these ligands have been extensively analyzed and exploited as probes in cell biology (Goldstein and Hayes, 1978; Gonatas and Avrameas, 1977: Gonatas, 1979 and 1982; Barondes, 1981).

2.1. Intra-axonally transported lectins The list of currently recognized lectins continues to grow and they appear to be

present in a wide variety of species (Goldstein and Hayes, 1978; Barondes, 1981). The rationale for evaluating a variety of lectins as intra-axonally transported probes is that different lectins bind to different oligosaccharides. Since oligosaccharides on the cell surface of neurons and on the membranes of subcellular organelles appear to be heterogeneously distributed (Hatten et al., 1979; Wood et al., 1981), a number of lectins with different "receptor" specificities have been examined in studies of neuronal connectivity (Table I). Unlike the plant protein HRP, an enzyme that can

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TABLE I

INTRA-AXONALLY TRANSPORTED LECTINS

Each of the lectins listed here has been demonstrated to be intra-axonally transported in native and/or derivatized forms. Cholera toxin and its binding (B) subunit are not considered to be lectins: however, they have some properties in common with lectins and appear to be as sensitive as WGHRP as intra-axonally transported probes. The middle and right hand columns give the molecular weights in kilodaltons (Kd) and binding specificities, respectively, of each of the ligands listed here.

Lectin Mol. wt. Binding specificity (kilodalton)

Wheat germ agglutinin 36 Ricinus communis agglutinin I 120 Ricinus communis agglutinin II 60

Peanut agglutinin 110

Lens culinaris 42-60 Soybean agglutinin 120 Concanavalin A 104 Dolichos bifloris agglutinin 113 Sophora japonica agglutinin 132 Bandieraea simplicifolia lectin 114 Ulex europeaus agglutinin I 43-170

Cholera toxin 84 B subunit of cholera toxin 11.5

N-acetyl-glucosamine and sialic acid lactose and beta-D-galactose lactose, beta-D-galactose and beta-D-galactose-

(l-3)-N-acetyl-galactosamine beta-D-galactose and beta-D-galactose-beta-

(1-3)-N-acetyl-galactosamine alpha-D-mannose and alpha-D-glucose N-acetyl-galactosamine alpha-D-mannose N-acetyl-galactosamine N-acetyl-galactosamine alpha-D-galactose alpha-L-fucose

monosialoganglioside monosialoganglioside

be visual ized in its unmodi f i ed or nat ive form, lectins must be modi f ied or deriva- t ized in some manne r in o rder to be localized or their d i s t r ibu t ion mus t be demons t r a t ed with specific ant i - lect in ant ibodies . In the la t ter case, unmodi f i ed or nat ive lectins may be used. Lect ins may be der ivat ized in a number of ways inc luding label ing with radioisotopes , HRP, f luorochromes, ferri t in etc. The choice of the label one employs to derivat ize and thus visualize a given lectin depends in par t on the exper imenta l object ives of the invest igator . Lect ins have been most c o m m o n l y der iva t ized for studies of neuronal connect iv i ty using radioisotopes , H R P and f luorochromes. The first use of lectins as p robes for neuronal connect iv i ty studies repor ted by Schwab et al. (1978) employed [125I] wheat germ agglutinin. There is l i t t le in fo rmat ion at present concerning the relat ive sensit ivit ies of W G versus other lectins labeled with rad io iso topes for such studies.

Af te r the in t roduc t ion of l ec t in -HRP conjugates as re t rograde ly t r anspor ted markers ( G o n a t a s et al., 1979; Schwab et al., 1979), a large number of H R P conjuga ted lectins were examined and H R P conjugates of W G were de te rmined to be the most sensit ive bo th as o r thograde ly and re t rograde ly t r anspor ted probes (Trojanowski et al., 1981b, 1982). F luo roch rome labeled lectins may also be used as axona l ly t r anspor ted markers. F o r re t rograde t ranspor t studies, W G labeled with f luoroesceine i so th iocyana te (F ITC) was found to be more sensit ive than a large n u m b e r of o ther s imilar ly labeled lectins (Nennesmo and Kris tensson, 1981). A

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recent study suggests that FITC labeled WG (WGFITC) may not be orthogradely transported in sufficient quantities for light microscopic detection (Trojanowski. 1983a). Although HRP conjugates of lectins may prove to be more sensitive than the same lectins labeled with fluorochromes, lectins derivatized with different fluorochromes having different emission spectra provide the possibility for conducting double labeling studies. Such studies are not possible with HRP conjugates of lectins.

The use of native or unmodified lectins offers similar possibilities for conducting double labeling studies by using antibodies raised in different species directed against different lectins. This possibility has not yet been explored. At present there is only a limited experience with a variety of lectins as intra-axonally transported probes visualized with immunohistochemical methods (Lechan et al., 1981: Borges and Sidman, 1982; Ruda and Coulter, 1982; Phillipson and Griffiths, 1983), Once again, WG from among those lectins surveyed, appears to most effectively label neuronal pathways (Borges and Sidman, 1982).

An additional property of certain lectins, which permits them to be used as neuroanatomical probes, is their toxicity. Toxic ricin and other toxic lectins have already been employed in this manner (Harper et al., 1980a; Wiley et al., 1982). Toxic lectins have the potential for use not only as neuroanatomical probes, which identify afferents to a given site by virtue of the cell death of the afferent neurons, but they may also prove to be powerful reagents in behavioral studies. The elimination of specific populations of neurons afferent to a given injection site by toxic lectins could be correlated with behavioral changes.

It is important to recognize that the same lectin in its native and derivative forms are not the same molecules. Even when lectins are conjugated with the same label such as HRP, but different cross-linking reagents are used, the properties of the resulting conjugates may vary (Nygren, 1982', Trojanowski et al., 1981b, 1982). In addition, a given lectin may be comprised of heterogeneous species recognizing different oligosaccharides and having different biological properties. For example, the affinity purification of a given lectin may lead to the production of reagents with different properties when employed as neuroanatomical probes (Margolis et al., 1981). Such factors should be kept in mind when comparing results from different studies and when making decisions regarding which form of a given lectin to use in an experiment.

2.2. Methods to visualize transported lectins The different possibilities for visualizing native and derivatized lectins at an

injection site, in axons and nerve terminals and in retrogradely labeled cell bodies have been alluded to in the previous section. The choice of methods for vizualizing derivatized lectins will depend on the manner in which the lectin is labeled. These methods will, in general, not vary from those used to vizualize the same label under other circumstances. For example rhodamine or FITC labeled lectins are visualized as one would visualize any molecule labeled in the same manner with these fluorochromes. HRP conjugates of lectins are best visualized using tetramethyl benzidine (TMB) as the chromogen for light microscopic studies as described by

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Mesulam (1978, 1982). For EM studies, the choice of the chromogen will depend on the experimental objectives of the investigator. TMB offers enhanced sensitivity but it may polymerize well beyond the sites of the HRP-lectin conjugates. Carson and Mesulam (1982b) have evaluated a variety of chromogens for EM studies and have characterized their advantages and disadvantages. Diaminobenzidine (DAB) with or without the addition of imidazole (Straus, 1982) will likely continue to be highly effective for the ultrastructural demonstration of HRP-lectin conjugates. The possibilities for demonstrating native lectins with immunohistochemical methods are numerous. A number of excellent recent reviews considering the limitations and advantages of various direct and indirect immunohistochemical methods have appeared (Sternberger, 1979; Falini and Taylor, 1983). Studies which have evaluated the relative sensitivities of different chromogens for use in immunohistochemistry are limited but DAB used with imidazole appears more effective than a number of other chromogens in the peroxidase anti-peroxidase technique (Sternberger, 1979; Straus, 1982; Falini and Taylor, 1983; Trojanowski et al., 1983).

2.3. Quantitation of lectin sensitivity By 1981, native or derivatized WG had been examined in only a limited number

of nervous system pathways (Brushart and Mesulam, 1980; Streit, 1980; Lechan et a|., 1981; Margolis and LaVail, 1981; Margolis et al., 1981; Nennesmo et al., 1981; Staines et al., 1981; Trojanowski et al., 1981a and b). Since the "receptors" to which WG binds are heterogeneously distributed on the cell surface of neurons (Hatten et al., 1979) and since other ligands (Streit, 1980; Thoenen and Kreutzberg, 1981) which bind to heterogeneously distributed "receptors" were found to be transported only in selected pathways (for example, retrograde transport of nerve growth factor is restricted to peripheral sympathetic and sensory pathways), it was necessary to rigorously examine the assumption that WGHRP would prove superior to N H RP as a retrogradely and orthogradely transported marker in different central (CNS) and peripheral (PNS) nervous system pathways. In addition, it was reasonable to consider whether or not a number of other HRP conjugated lectins with different oligosaccharide affinities might outperform WG in neuronanatomical studies. Fi- nally, since the method of conjugation may: (a) alter the affinity of the ligand for its "receptors"; (b) alter the biological activity of the ligand; or (c) alter the enzymatic activity of HRP (Nygren, 1982), it was also necessary to critically examine this issue as well. We undertook to systematically examine such factors and included in these studies the endotoxin of Vibrio cholerae, cholera toxin (CT), which binds to monosialoganglioside present on the cell surface of neurons (Joseph et al., 1978; Okada et al., 1982).

In a series of studies (Trojanowski et al., 1981a and b; Trojanowski et al. 1982; Wan et al., 1981a and b) we quantitatively examined the relative sensitivity of N H R P and a large number of HRP conjugates of lectins and CT as orthogradely and retrogradely transported markers in a variety of pathways of the rat PNS and CNS. Conjugates of WG and CT emerged as the most sensitive tracers. These conjugates also revealed the dendritic arborization of retrogradely labeled neurons better than NHRP. Of 3 different cross-linking reagents used to couple WG to HRP,

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no single cross-linking reagent resulted in a conjugate which consistently outper- formed conjugates produced with the other cross-linking reagents. These reagents included glutaraldehyde (Avrameas and Ternynck, 1971), p-benzoquinone (Ternynck and Avrameas, 1976) and periodate (Nakane and Kawoi, 1974). Finally, we demonstrated that the affinity of WG and CT for their specific "receptors" in brain was preserved after conjugation with HRP. In contrast, the biological properties of CT and certain other toxins or lectins are markedly diminished as a result of the conjugation procedure (Schwab et al., 1979; Gonatas et al., 1980: Charalampous et al., 1982). For example, the toxicity of toxic ricin is almost completely lost after conjugation with HRP (Harper et al., 1980a).

It can be concluded from these studies that: (1) HRP conjugates of two ligands, CT and WG, are quantitatively more sensitive retrogradely and orthogradely transported markers than NHRP for neuroanatomical studies; (2) these two conjugates visualize the dendrites of retrogradely labeled neurons in greater detail than NHRP; (3) the affinity of WGHRP and CTHRP for their respective "receptors" is specific and preserved after the conjugation procedure; and (4) no single conjugation method examined yields conjugates which consistently outperform those produced by al- ternative methods. More recent studies reported from other laboratories have provided direct or indirect evidence in support of these observations (Borges and Sidman, 1982; Kistler and Schwartz~ 1982; Robertson and Aldskogius, 1982: Steindler, 1982; Phillipson and Griffiths, 1983).

2.4. Determinants of lectin sensitivity The superior sensitivity of the various forms of WG, other lectins and CTHRP

over NHR P as neuroanatomical probes has not yet been fully explained. Initially it was suggested (Schwab et al., 1978; Gonatas et al., 1979) that this increased sensitivity might be due to the difference in the mode of endocytosis of lectins (adsorptive endocytosis) compared with NHRP (fluid phase endocytosis). However other explanations are worth considering as well. Differences in diffusion at the injection site, uptake along different segments of axons, axon terminals or perikaryal membranes, different rates of orthograde and retrograde transport, different rates of degradation or elimination as well as different rates of endocytosis are but a few of the possibilities which may contribute to differences in the sensitivity of these probes. The elegant work of Gonatas et al. (1980) has indicated that certain lectin-HRP conjugates such as ricin-HRP are indeed internalized by cells in tissue culture at rates that are 100-200 fold greater than the rate at which N H RP is internalized. Similar experimental conditions are impossible to duplicate in vivo but data from studies of the endocytosis of WGHRP by retinal ganglion cells is consistent with the view that the internalization of W G H RP proceeds at a faster rate than the internalization of NHRP (Trojanowski and Gonatas, 1983). It is worth noting that both in vivo and in vitro lectins do not appear to stimulate the fluid phase endocytosis of NHRP (Gonatas et al., 1980; Trojanowski et al., 1982).

Differences in the injection sites produced by NHRP in comparison with CTHRP and WGHRP have been observed (Staines et al,, 1981, Wan et al., 1982a). WGHRP and CTHRP remain at the injection site for a considerably longer time than NHRP

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and they may therefore be available for uptake and transport for longer time periods than NHRP. This difference could contribute to the greater sensitivity W G H R P and CTHRP. The regions of the neuronal plasma membrane which are capable of internalizing W GHRP or other lectins has not been determined. However, two studies have suggested that undamaged axons of passage have only a limited capacity to internalize these probes (Grob et al., 1981; Steindler, 1982). Damaged or transected axons clearly have the capacity to internalize W G H RP (Brushart and Mesulam, 1980) and native WG (Borges and Sidman, 1982). Whether this occurs because of the inward diffusion of W G H R P into transected axons where direct contact with exposed axoplasm is possible or whether internalization takes place across axolemma which has resealed the nerve at a site proximal to the transection has not yet been established. At the same time, it has been observed that axons need not be damaged for the internalization of W G H R P to occur (Trojanowski et al., 1982).

The fastest rate of orthograde intra-axonal transport of radiolabeled WG and W G H R P has been established (Margolis and LaVail, 1981; Trojanowski, 1983). In both the chick (22-33 ram/day) and the rat (about 108 mm/day) this rate is intermediate between the fastest (fast axonal transport) and slowest (slow axonal transport) rates of orthograde intraaxonal transport. That WG may be transported at multiple rates, some of which are slower than what has already been reported, remains to be established. The information presented above indicates that the sensitivity of native and derivatized WG cannot be ascribed to a more rapid arrival of this probe at axon terminals compared with N H R P which is orthogradely transported at a rate of 288-432 m m / d a y (Mesulam and Mufson, 1982).

Differences in the fate of WG versus NHRP after internalization may contribute to the sensitivity of this lectin. For example Wan et al. (1982a) noted that 13 days after a tongue injection of WGHRP or CTHRP, numerous labeled hypoglossal neurons were still present. This is in contrast to studies with NHRP, in the same or other neuroanatomical systems, in which NHRP is no longer detectable in afferent neurons beyond approximately 4-8 days (Heimer and Robards, 1981). In more recent studies with HRP conjugates of the binding (B) subunit of CT (BHRP), labeled cells have been observed in the hypoglossal nucleus as long as 3 weeks after tongue injections of BHRP (Trojanowski, 1983b). It is possible that HRP conjugates of lectins and CT are more resistant to lysosomal degradation or are delivered to a different population of vesicles from those to which N H RP is delivered and thus escape degradation for prolonged periods. Such a mechanism might also explain the enhanced sensitivity observed with certain lectins in comparison with NHRP.

2.5. Limitations in the use of lectins in neuroanatomy

As with any methodology, there are both advantages and limitations to the application of native and derivatized lectins to neuroanatomical studies. The nature of the experiments planned by an investigator will determine which probes and which methods are most appropriate in a given situation. Some of the advantages to be gained from using lectins for studies of neuronal connectivity have been discussed in the foregoing. It is appropriate to consider some of the potential drawbacks of their use.

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Some of the limitations associated with using lectins as neuroanatomical probes are the same as those associated with any of the currently employed tracer techniques (Heimer and Robards, 1981; Mesulam, 1982). The histology of the injection site produced by WGHRP and CTHRP appears similar to that produced by NHRP (Jacobson and Trojanowski, 1974; Trojanowski and Jacobson, 1974; Wan et al., 1982a), and the distinction of the apparent from the effective injection sites is a problem with all tracer methods including those in which lectins are employed. The problem of uptake by fibers of passage appears to be insignificant with WG (Grob et al., 1981; Steindler, 1982), however this potential pitfall should be kept in mind when interpreting results obtained with any neuroanatomical tracer.

A current limitation which may be overcome with increasing use of lectins is the expense of conducting experiments with lectins and the availability of certain types of derivatized lectins compared wi ththe cost and availability of HRP. There is also less experience with lectins than there is with HRP as neuroanatomical tracers. Thus there is a more limited literature concerning lectins with which to compare results obtained from studies in which lectins are used. This may necessitate additional control studies with NHRP or other tracers in order to better interpret such results. This is particularly important in the evaluation of negative results. It should be recalled that initially NHRP was not considered to be orthogradely transported in significant amounts. Subsequently, the failure to consistently observe the orthograde transport of NHRP was found to be due to the relative insensitivity of DAB compared with TMB for the visualization of NHRP (Heimer and Robards, 1981: Mesulam, 1982). The failure to observe lectin transport in a given pathway may similarly be due to technical considerations or it may reflect the absence of lectin binding sites, toxic properties of the lectin, etc. For example, in studies of the turtle and pigeon visual pathways, Schnyder and Kunzle (1983) have convincingly demonstrated both a species specific and pathway specific reduced sensitivity of radiolabeled WG. Injections of [125I] WG into the vitreous body retrogradely labeled neurons in the ishmo-optic nucleus in the pigeon but not in the turtle. They suggested that this may be due to an insufficient number of WG binding sites. We have similarly observed variations in the sensitivity of WGHRP in different neuroanatomical systems of the rat (Trojanowski et al., 1981a, 1982). This is not surprising in view of the heterogenous distribution of lectin binding sites in the nervous system (Gurd, 1977; Hatten et al., 1979). This potential problem may be overcome by the use of a "cocktail" or mixture of lectins with different "receptor" specificities (Trojanowski et al., 1981).

The transcellular transport of WG and possibly other lectins may be a limitation or an advantage depending upon the requirements of the experiment. Tritiated amino acids continue to be used as neuroanatomical tracers despite the fact that they undergo transcellular transport. As is the case with amino acids, the transcellular transport of WG and other ligands appears to be dependent upon the amount injected, survival time and possibly other factors (Grafstein and Forman, 1980; Trojanowski, 1983b). Thus experiments should be conducted in such a way that possible transcellular transport is controlled for if necessary.

There is no "universal" neuroanatomical tracer which will meet every investiga-

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tor's needs. Native and derivatized lectins will probably not replace NHRP, radiolabeled amino acids, fluorochromes etc. in neuroanatomy. Instead, they offer to the investigator an array of probes which differ from these other tracers in specific ways. This may render them particularly advantageous in a given experiment. As they are used with increasing frequency, additional advantages and limitations of their use in neuroanatomy will undoubtably become apparent.

3. Lectins for in vivo studies of neuronal cell biology

The endocytosis and intraneuronal transport of molecules are important physiological aspects of normal neurons which undoubtedly also play a major role in the pathogenesis of disease (Silverstein et al., 1977; Grafstein and Forman, 1980; Pastan and Willingham, 1981; Anderson and Kaplan, 1983; Farquhar, 1983). Endocytosis which occurs on the basis of concentration gradients is termed fluid phase or bulk endocytosis. NHRP is internalized in this manner. Alternatively, endocytosis can be highly selective because it is mediated by binding sites on the cell surface. This mode of internalization is termed adsorptive or receptor mediated endocytosis. Lectins and certain toxins bind to specific components of the cell surface and are internalized by adsorptive endocytosis (Gonatas, 1979 and 1982; Gonatas et. al., 1980). In this discussion, adsorptive endocytosis will be used to designate the mode of internalization of lectins and CT. Ligands with more restricted specificities or binding sites are said to undergo receptor mediated endocytosis which may differ from adsorptive endocytosis (Pastan and Willingham, 1981; Anderson and Kaplan, 1983; Farquhar, 1983).

After internalization by neurons into populations of endocytic vesicles, these vesicles and their contents are translocated from the cell surface to specific sites within the perikaryon and its processes (Grafstein and Forman, 1980; Thoenen and Kreutzberg, 1981). The class of endocytic vesicle receiving the internalized molecule(s) may determine the subsequent fate of the vesicle and its contents. As many as five different rates of orthograde axoplasmic transport have been described (Grafstein and Forman, 1980; Thoenen and Kreutzberg, 1981). They can be generally grouped together as fast (200-400 mm/day), intermediate (4-70 mm/day) and slow (0.25-4 mm/day). Some components traveling at a given rate appear to be distinct. For example, tubulin and neurofilaments only travel in the slowest component (0.25-1.0 mm/day) of slow axonal transport. Retrograde axonal transport has been less well characterized but the rate is approximately 70-290 mm/day. Many of the components carried in the orthograde direction return to the perikaryon via somatopetal transport. The factors which regulate the pathways taken by different internalized molecules in neurons is poorly understood at present.

The endocytosis and intraneuronal transport of ligands are likely of greater biological significance to neurons than the transport of molecules internalized by fluid phase endocytosis in view of the direct role that a variety of ligands are known to play in physiological and pathological events in cells (Grafstein and Forman, 1980; Spencer and Schaumberg, 1980). For example, the ligand tetanus toxin binds

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specifically to polysialogangliosides of the neuronal plasma membrane prior to internalization and transport (Schwab et al., 1979; Yavin et al., 1981). This neuro- toxin is capable of transsynaptic transport and it is postulated that the disinhibitory effect of tetanus toxin is mediated in this manner. Other pathogens such as the chemotherapeutic agent doxorubicin and herpes simplex virus undergo transcellular transfer after retrograde transport and it is speculated that such transfer is an essential step in the pathogenesis of the lesions produced by these agents (Bigotte and Olsson, 1982; Kristensson et al., 1982).

3.1. Neuronal membranes One of the most extensive applications of lectins in cell biology and in neurobiol-

ogy has been in the investigation of cell membranes especially cell surface molecules. Several reviews have appeared on this subject including studies based on work with neuronal cell lines and with primary cultures of neurons (Barondes, 1981; Gonatas, 1979 and 1982; Virtanen et al., 1981). A more limited number of studies have been carried out in the intact nervous system of vertebrates. These have yielded interesting results which suggest that lectins may prove to be effective reagents for characterizing neuronal membranes in vivo.

One conclusion to be drawn from these studies is that the distribution of oligosaccharides of lectin binding sites is heterogeneous both on the cell surface of neurons and on the membranes of subcellular organelles (Gurd, 1977; McLaughlin et al., 1980; Wood et al., 1981; Sanes, et al., 1982). Thus lectins may be useful in mapping the mozaic structure of neuronal membranes as well as in isolating specific molecular components of such membranes. They may also prove effective in determining the fate of internalized or re-cycled cell surface molecules (Dumas et al., 1979; Gonatas, 1982). A potential limitation to their future use in this regard is their lack of specificity for individual macromolecules. Lectins recognize oligosaccharides which may reside on many different macromolecules. Thus, if the experimental objective is to identify or isolate individual macromolecules, monoclonal antibodies specific for epitopes restricted to a given macromolecule may prove to be more effective tools for this purpose (Langley et al., 1982). In addition, conclusions about membrane traffic based upon observations of the movement of lectins may not be valid if the lectin becomes dissociated from its binding site on the membrane subsequent to internalization. Nevertheless, the potential applications of lectins for studies of neuronal membranes warrant further examination. For example, the modulation of neuronal membranes with differentiation can be effectively demonstrated with a battery of lectins at both the light and electron microscopic levels (McLaughlin et al., 1980; Bruckner and Biesold, 1981).

3.2. Routes of intraneuronal transport NHR P has been used extensively as a probe for studies of endocytosis and

intraneuronal transport (Grafstein and Forman, 1980; Rambourg and Droz, 1980; Heimer and Robards, 1981; Mesulam, 1982; Broadwell and Brightman, 1983). It has provided important information concerning the subcellular organdies (coated and uncoated vesicles, multivesicular bodies, cup-shaped organdies, tubules, lysosomes,

195

etc.) available for the translocation of of macromolecules from the cell surface to diverse sites within neurons. Although the organelles involved in intraneuronal transport have been catalogued, there is little information concerning the sequential transfer of macromolecules from one organeUe to another and the routes which they may take within a neuron. Even less is known regarding the factors which determine the routing of vesicular traffic and the extent to which vesicles transporting different macromolecules may differ in their biochemical composition. The disposition of the membranes comprising such organelles after they have transferred their contents to another organelle likewise remains to be determined. The evidence to date suggests that different organelles may be involved in the retrograde and orthograde transport of exogenous fluid phase tracer molecules. For example, the smooth endoplasmic reticulum has been implicated in the orthograde transport of HRP but it does not appear to be involved in the retrograde transport of this protein (LaVail et al., 1980; Rambourg and Droz, 1980).

Recent studies with derivatized and native lectins and CT suggest that N H R P has provided only a limited view of endocytosis and intracellular transport by neurons in vivo. Some of the different ways in which neurons respond to NHRP, compared to native or derivatized lectins and CT, have been described above and are summarized in Table II. These differences can be exploited in order to probe further the cell biology of transport mechanisms in neurons. For example, studies of intraneuronal transport with W G H R P have indicated that the routing of endocytic vesicles is more complex than studies with NHRP would suggest. This is exemplified by the observation of Harper et al. (1980b) that GERL (Golgi Endoplasmic Reticulum Lysosomes) or the Golg i -GERL complex (Novikoff, 1976; Goldfisher, 1982; Gona- tas, 1982) is involved in the intraneuronal transport of conjugates of WG with HRP but not of native HRP. This observation has been confirmed by studies in several types of neurons (Trojanowski et al., 1981; Trojanowski and Gonatas, 1982 and 1983). In addition, the sequential labeling of subcellular organelles with W G H R P appears to be consistent with the hypothesis that this probe is transferred from endocytic vesicles to lysosomes after passage through GERL (Trojanowski and Gonatas, 1983). In contrast, NHRP has not been observed to enter or pass through G ER L or the Golg i -GERL complex of neurons following endocytosis (LaVail and LaVail, 1974; Turner and Harris, 1974; Broadwell and Brightman, 1983). However, there are rare instances in which NHRP has been observed to be translocated to the Golgi apparatus of certain cell types (Oliver, 1982; Broadwell and Oliver, 1983). For instance, of all the different cell types in the adenohypophysis, only somatotrophs appear to permit NHRP to gain access to the Golg i -GERL complex (Broadwell and Oliver, 1983). Although the significance of these differences in the routing of exogenous markers remains to be determined, evidence from both in vivo and in vitro studies suggest that the Golg i -GERL complex plays a pivotal role in routing the traffic of both the membranes and contents of transport vesicles (Gonatas, 1982; Farquhar, 1983).

The differences in the routing of a fluid phase marker such as N H RP compared with an adsorptively internalized marker such as W G H RP may explain differences in the subsequent fate of these tracers. Recent studies have indicated that HRP

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conjugates of WG and CT are not as rapidly eliminated from retrogradety labeled hypoglossal nucleus neurons as N H R P (Wan et al., 1982a). Whether this is due to the fact that W G H R P and CTHRP are transported along different intracellular pathways than those utilized by N H R P has not yet been determined, but this observation could be exploited to elucidate how the intraneuronal transport of proteins is coupled to their ultimate fate in neurons. In other studies of the orthograde transport of radiolabeled WG, it was found that this form of derivatized WG was distributed in an annular region subjacent to the axolemma (LaVail et al.. 1983). This finding suggested that the hypolemmal cisterns, a component of the smooth endoplasmic reticulum, may participate in the orthograde transport of this probe. N H R P is orthogradely transported in part by elements of the smooth endoplasmic reticulum but does not appear to utilize to a significant extent the hypolemmal cisterns (Hanson, 1973; Itaya et al., 1978). It is likely that differences in the mode of endocytosis and in the subcellular organelles involved in the orthograde transport of native and derivatized WG compared with N H R P account for the fact that WG but not N H R P is capable of transcellular transfer following orthograde transport (Itaya et al., 1982; Ruda and Coulter, 1982; Gerfen et al., 1982; LaVail et al., 1983; Trojanowski, 1983b).

3.3. Transcellular Transport The transcellular transport of intra-axonally transported substances was first

reported over 15 years ago (Koor et al., 1967; Grafstein and Forman, 1980). Although a number of macromolecules have been reported to undergo transcellular transfer subsequent to orthograde or retrograde intraneuronal transport, very little information is available on the determining factors which regulate this phenomenon and even less is known of the importance of this phenomenon to neurons and glia (Grastein and Forman, 1980). Transcellular transfer or transport of macromolecules by neurons is to be distinguished from transneuronal or transganglionic transport. The term transneuronal transport is used to refer to the internalization of a marker along a neuronal process (such as a dendrite or axon terminal) followed by somatopetal transport to the neuronal perikaryon and subsequent somatofugal movement into another process. Transganglionic transport refers to the same phenomenon but in addition implies that this process is occurring in a group of neurons such as a ganglion. For example, Brushart and Mesulam (1980) described the transganglionic transport of W G H R P to the central sensory projection sites of afferents from skin and muscle of the rat.

Transsynaptic transport is a specialized case of transcellular transport involving neurons. The term indicates that material is transferred from one neuron to another only across the synaptic cleft. To prove that transcellular transport is transsynaptic it is necessary to demonstrate that exocytosis of the probe occurs at the presynaptic element into the synaptic cleft and that it then undergoes endocytosis by the postsynaptic element. Transcellular transfer following intraneuronal transport would also include the transfer of substances from one neuron to another at sites other than the synapse or transfer to non-neuronal elements such as glia or endothelial cells. The term does not specify the site or sites at which such substances are externalized

197

by a neuron nor whether later internalization involves other neurons or glia or both. Despite the fact that much remains unknown concerning the cell biology of transcellular transfer of neurons, the phenomenon has been successfully exploited in neuroanatomical studies especially in the visual system (Hubel, 1982; Wiesel, 1982).

Although the number and type of substances which are intra-axonally transported is rather extensive, far less is known about the classes of molecules which may undergo transcellular transfer after intra-axonal transport (Grafstein and Forman, 1980). The evidence so far suggests that proteins and protein precursor molecules such as amino acids and sugars may undergo transcellular transfer as well as fluorescent dyes, certain drugs, viral particles and nucleosides (Schwab et al., 1979; Grafstein and Forman, 1980; Bigotte and Olsson, 1982; Kristensson et al., 1982; Davis and McKinnon, 1982). WG in both its native and derivatized forms, has also been recognized to readily undergo transcellular transfer after orthograde transport (Itaya et al., 1982; Ruda and Coulter, 1982; Gerfen et al., 1982; LaVail et al., 1983; Trojanowski, 1983b). This is in contrast with HRP. Transcellular transfer of this enzyme has been demonstrated in some non-mammalian forms (Wilczynski and Zakon, 1982), but at present the consensus is that HRP does not undergo transcellular transfer in mammals (Heimer and Robards, 1981) except under exceptional circumstances such as following direct intraneuronal injections (Triller and Korn, 1981). In one report, Itaya et al. (1978) presented EM photomicrographs suggestive of transcellular transfer of HRP injected with poly-L-ornithine following anterograde transport in the visual system, but this was not evident by light microscopy.

It is interesting to consider whether or not the directionality of intraaxonal transport or the portion of the plasma membrane internalized with a ligand may regulate transcellular transfer by neurons. We have observed the transcellular transfer of WGHRP, CTHRP and BHRP following orthograde transport in the rat visual system but have not been able to detect evidence for the transcellular transport of any of these probes following retrograde transport in the tongue-hypoglossal nucleus system (Trojanowski, 1983b). That transcellular transfer following retrograde transport can occur has been well documented" in the case of some ligands, such as tetanus toxin but not others (Schwab and Thoenen, 1977; Schwab et al., 1979; Streit, 1980) and in the case of certain drugs such as doxorubicin (Bigotte and Olsson, 1982) and certain pathogens such as herpes virus (Kristensson et al., 1982). Other studies have similarly failed to document transcellular transfer of native or derivatized lectins including WG following retrograde transport (Schwab et al., 1978; Schwab et al., 1979; Harper et al., !980b; Lechan et al., 1981; Wan et al., 1982 a and b). Of additional interest in this regard is the fact that in a combined light and electron microscopic study of the transganglionic transport of WGHRP, there was no evidence for the transcellular transfer of this probe (Carson and Mesulam, 1982a).

These observations taken together suggest that, in the case of lectins, endocytic vesicles generated at the perikaryal plasma membrane have the capability of trans- ferring their contents to other neural elements following somatofugal transport. In contrast, endocytic vesicles generated by lectin-membrane interactions from more peripherally located portions of the neuronal plasma membrane, such as that found

198

along dendrites and axons, may not possess a similar capability. This hypothesis, and others which could explain the mechanisms whereby transcellular transfer is governed in neurons can be examined using native and derivatized lectins. A better understanding of this phenomenon is of obvious significance to the cell biology of neuron-neuron and neuron-glia interaction under both normal and pathological conditions.

4. Further modifications and uses of lectins in neurobiology

Many questions regarding the use of native and derivatized ligands as probes in neurobiology remain to be answered. We have begun to address the question of the sensitivity of WG derivatized with different labels. For example, WG tagged with FITC is a less sensitive orthogradely transported probe than HRP conjugates of WG (Trojanowski, 1983a). It is important to verify this preliminary observation in other pathways and to carry out quantitative retrograde labeling studies. Furthermore. native and derivatized forms of the probes described here should be compared with respect to their biological properties and sensitivities as probes for studies of neuronal connectivity. In addition, the toxic property of certain native lectins such as ricin could also be exPloited for combined neuroanatomical and neurobehavioral or neuropathological investigations (Harper et al., 1980a; Wiley et al., 1982). Other lectins without apparent toxic properties by histological examination should be further evaluated for possible metabolic or electrophysiological effects. For example Karpiak et al. (1981) have reported that ligands which bind to monosialoganglio- sides (CT, the B subunit of CT and anti-monosialoganglioside antibodies) induce seizure activity in rodents, lectins recognizing cell surface oligosaccharides may similarly perturb the neuronal plasma membrane and induce abnormal electrical activity. Such ligands could prove to be useful probes for studying epilepsy. The use of lectins to examine various models of neurological diseases has been limited to date but there are a number of reports which indicate that lectins have potential in this regard. A recent study has employed WGHRP to characterize axonal transport abnormalities associated with experimental head trauma (Povlishock et al., 1983). Novel methods for the delivery of lectins could expand the applications or sensitivity of these probes. For example intracellular injections of lectins could be used to characterize the oligosaccharide distribution on the membranes of intracytoplasmic organelles. The use of polyacrylamide gels of WG to provide a slow release delivery of this lectin for neuroanatomical studies has recently been described (Schwanzel- Fukuda et al., 1983). This strategy may further enhance the sensitivity of WG and other lectins as neuroanatomical probes. Perhaps the most exciting applications of lectins in neurobiology will be towards developing a better understanding of both intraneuronal and transcellular transport.

5. Conclusions

The features which distinguish the endocytosis and transport of HRP conjugates of WG and CT from NHRP in the nervous system are presented in Table II. The

TABLE II

SUMMARY OF FEATURES DISTINGUISHING WGHRP & CTHRP FROM NHRP IN VIVO

199

Feature NHRP WGHRP & CTHRP

1. Type of macromolecule. Non-ligand. 2. Affinity for plasma None or at most

membrane, electrostatic affinity.

3. Mode of endocytosis. Fluid phase. 4. Subcellular organelle Vesicles, lyso-

involved in endocytosis somes, tubules and transport, multivesicular

bodies etc.

5. Sensitivity as intra- Extensively used neuronally transported for such studies probe for studies of but less sensitive neuronal connectivity, than WGHRP & CTHRP

6. Rate of orthograde Fast; 288-434 mm/day transport, in rat.

7. Elimination from Cleared by about injection site. 7 days.

8. Elimination from Cleared by about retrogradely labeled 7 days. neurons.

9. Vizualization of dendrites Fair of retrogradely labeled neurons.

10. Transcellular transfer No conclusive after orthograde or evidence for retrograde intraneuronal this in transport, mammals.

Ligands. Specific affinity for defined "receptors".

Adsorptive. Ultrastructurally similar to but biologically different from those associated with NHRP; in addition, transit through GERL & related coated vesicles. Both are about 30-50 times more sensitive than NHRP.

Intermediate for WGHRP; about 108 mm/day in rat. Cleared by about 13 days.

Not cleared even by 21 days.

Superior to NHRP and possibly Golgi method.

Both appear capable of transcellular transfer after orthograde but not retrograde transport.

d a t a r ev iewed he re suggest tha t n a t i v e and de r iva t i zed lec t ins m a y en te r a p o o l of

e n d o c y t i c vesic les wh ich d i f fe r m a r k e d l y in the i r ra te and m o d e of i n t r a n e u r o n a l

t r anspor t , fa te , etc. f r o m the class o f e n d o c y t i c vesic les in to wh ich N H R P is

in te rna l i zed . Thus , it is l ikely tha t lec t ins as wel l as o t h e r re la ted l igands such as C T

wil l p r o v e u s e f u l in p r o v i d i n g n e w ins ights i n to the c o m p l e x and p o o r l y u n d e r s t o o d

p h e n o m e n a of endocy tos i s , i n t r ace l lu la r t r anspo r t a n d t r ansce l lu la r t r ans fe r by

n e u r o n s in vivo. T h e y shou ld also c o n t i n u e to p r o v e useful for s tudies of n e u r o n a l

c o n n e c t i v i t y in the ne rvous sys tem.

Acknowledgements

A p p r e c i a t i o n is exp res sed to Dr . N . K . G o n a t a s for c o n t i n u e d suppor t , encou rage -

m e n t and adv ice as wel l as to Ms. M. O b r o c k a , Mrs. J .O. G o n a t a s , Dr . X.S.T. W a n

21)0

a n d Dr . C .N. L iu for h e l p f u l c o l l a b o r a t i o n . Ms. E l l en S c h w a r t z is t h a n k e d for he lp

in p r e p a r i n g t he m a n u s c r i p t . W o r k f r o m th i s l a b o r a t o r y r e v i e w e d he re was sup-

p o r t e d b y N S - 1 6 7 2 3 , N S - 0 5 5 7 2 a n d K 0 7 N S 0 0 7 6 2 f r o m the N I N C D S .

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Native and derivatized lectins for in vivo studies of neuronal connectivity and neuronal cell biology