J. Cell Set. 7, 217-231 (1970) 217

Printed in Great Britain

LANTHANUM STAINING OF NEUROTUBULES

IN AXONS FROM COCKROACH GANGLIA

NANCY J. LANE AND J. E. TREHERNEA.R.C. Unit of Invertebrate Chemistry and Physiology, Department ofZoology, Cambridge University, Cambridge, England

SUMMARY

The axoplasm of the neurons of Penplaneta amencana contains numerous neurotubuleswhich are morphologically similar to the microtubules found in non-nervous tissues aftersectioning or negative staining. In cross-sections of fixed material such tubules usually appearas electron-dense circles containing a less dense core and surrounded by a non-opaque ' clearzone'. However, when cockroach ganglia are fixed and incubated in lanthanum hydroxide,the lanthanum is taken up intracellularly by the axoplasm of certain of the neurons and inthese is found to stain the entire core of the neurotubules, as well as the clear zone. At leastpart of the wall of the tubules remains unstained and appears as a ring of non-opaque subunitsagainst an electron-dense, lanthanum-stained background. Since lanthanum staining, underthe conditions used here, is sometimes considered to demonstrate the presence of acid muco-polysacchandes, its uptake by the neurotubules may indicate that they contain carbohydrateas well as the protein that is generally considered to form part of the microtubular wall. Alter-natively, the lanthanum could indicate the location of other anionic molecules, possibly under-going extra- or intratubular translocation. The extent to which neurotubules could mediatemovements of relatively small water-soluble ions and molecules is considered in relation totheir diffusion through the polyamon matrix represented by the core of the tubules.

INTRODUCTION

The axoplasm of insect neurons, like that of other invertebrate and vertebratenerves (Palay, 1956), contains numerous neurotubules. These organelles resemblemorphologically the 'microtubules' of non-nervous tissues, such as those in mitoticspindles (Roth & Daniels, 1962; Ledbetter & Porter, 1963; Gonatas &Robbins, 1964;Rebhun & Sander, 1967), cilia and flagella (Gibbons & Grimstone, i960; Gibbons,1963) and the cytoplasm of many different cell types (Slautterback, 1963; Ledbetter& Porter, 1964; Sandborn, Koen, McNabb & Moore, 1964; Behnke, 1964). In cross-sections of fixed material, both microtubules and neurotubules are characteristicallycomposed of electron-opaque material in the form of a circle containing a less electron-dense core. In some cases they appear to be surrounded by a non-opaque 'clear zone'(Ledbetter & Porter, 1963; Vivier & Schrevel, 1964; Maser & Philpott, 1964; Behnke,1965; Behnke & Forer, 1966; Silver & McKinstry, 1967; Hamon & Folliot, 1969;Forer, 1969). Certain investigators have suggested that the clear zone surrounding themicrotubules and their non-opaque core may represent additional microtubularcomponents (Behnke & Zelander, 1967; Behnke & Forer, 1967; Silver & McKinstry,1967; Forer, 1969).

Neurotubules both look like microtubules and are about the same size, that is

218 N.jf. Lane and J. E. Trelierne

about 23 nm in diameter. Further, both neurotubules (Kirkpatrick, 1969) andmicrotubules of various kinds (Kiefer, Sakai, Solari & Mazia, 1966; Gall, 1966; Grim-stone & Klug, 1966; Behnke & Zelander, 1967) show a filamentous substructure afternegative staining. Thus neurotubules seem to be, morphologically at any rate, verysimilar to microtubules.

These morphological similarities, however, do not necessarily imply identity orfunctional equivalence, for recent evidence (Behnke & Forer, 1967; Burton, 1968;Rodriguez Echandia & Piezzi, 1968; Behnke, 1970) indicates that not all microtubulesor neurotubules in different systems are equally stable. Further, there are suggestionsthat even within one axon there are variations both in the degree of stability (Forer,Lane & Treherne, in preparation) and in the chemical characteristics of the com-ponent neurotubules which otherwise appear structurally identical (de Lorenzo,Dettbarn & Brzin, 1969).

Chemically the cytoplasmic microtubules of the 9 +2 arrays in cilia and flagella areproteinaceous (Gibbons, 1963), being made up at least in part of a 6s protein dimer(Renaud, Rowe & Gibbons, 1966; Borisy & Taylor, 1967). This 6s protein has thecapacity to bind to colchicine, and since similar colchicine-binding material has beenfound in nervous tissue (Borisy & Taylor, 1967; Weisenberg, Borisy & Taylor, 1968),it can be assumed that at least some of the neurotubules are composed of protein andperhaps contain this 6s component.

The present account reports the demonstration in neurotubules of a substance whichreacts with lanthanum hydroxide, staining the clear zone as well as the neurotubularcore, but leaving at least part of the wall unstained.

MATERIAL AND METHODS

The abdominal nerve cord of the cockroach, Periplaneta americana, was used in this investi-gation. In preparing the tissue for electron-microscopical examination, the ganglia were cut inhalf just prior to fixation to ensure adequate penetration of the various solutions. Both gangliaand connectives were treated with lanthanum according to the procedure of Doggenweiler &Frenk (1965) as modified by Revel & Karnovsky (1967). The nerve cords were fixed for 2 hat room temperature in 3 % glutaraldehyde in o-i M cacodylate buffer (pH 7-4) with sucroseadded to a final concentration of O'2 M and with lanthanum hydroxide added to give a finalconcentration of 1 %. The lanthanum hydroxide was prepared from a 3 % solution of lantha-num nitrate which had been brought to pH 7-8 with 001 N sodium hydroxide. After fixation,the tissues were washed in o 1 M cacodylate buffer, and post-fixed for 2 h at room temperaturei n 1 ' 33% osmium tetroxide in 0-2 M collidine buffer at pH 7-2; lanthanum hydroxide wasadded to both the buffer wash and osmium tetroxide-collidine solution before use, to give afinal concentration of 1 %. Subsequently, the tissues were washed and dehydrated through anascending alcoholic series to 95 % ethanol, to all of which lanthanum was added, followed bytreatment with absolute ethanol, propylene oxide and embedding in Araldite. Ultrathin sec-tions were cut on an LKB Ultratome III, some were stained with lead citrate (Reynolds, 1963),and both stained and unstained sections were observed in a Philips EM 200 or EM 300 elec-tron microscope. Control tissues were treated in the same way as the experimental material,except that lanthanum was omitted from all solutions. Sections of the control tissues werestained with uranyl acetate (Watson, 1958) or lead citrate before examination.

The dimensions of the neurotubules quoted in this report were determined by measure-ments from prints. At least 15 tubules were measured in each case, and from these figuresthe standard deviation was calculated.

Lanthanum staining of neurotubules 219

OBSERVATIONS

In cockroach connectives or ganglia fixed without added lanthanum, neurotubulesare found in varying degrees of abundance in each axon (Fig. 2). They appear to bealigned, for the most part, in arrays roughly parallel to the limiting plasma membrane,so that, although some obliquely cut tubules are evident, cross-sections of axonscontain large numbers of neurotubules which appear in transverse sections as electron-opaque circles 23 ±3 nm in diameter (Figs. 2, 3). The electron-dense material com-prising the wall of the neurotubules measures 5 ± 1 nm in width, and the central corehas a diameter of 12 + 2 nm (see Fig. 1, p. 220). The clear zone (40 ± 5 nm) whichsurrounds them is less evident in these cockroach preparations than in other insecttissues (compare Fig. 3 with the inset) since the background cytoplasm is not veryelectron-dense.

When fixed with lanthanum-containing solutions, some of the axons near the cutedge of the cockroach ganglion took up the lanthanum intracellularly, presumablybecause they had been severed during the act of cutting. This is in contrast to theusual situation where lanthanum acts as an extracellular marker (see Revel & Kar-novsky, 1967; Cancilla, 1968; White & Walther, 1969). Lanthanum was found to beassociated with seemingly all the neurotubules which ran along the axons of thenerve cells which had taken up the lanthanum. Since these sections were unstained,the observed electron opacity could only be presumed to be due to the lanthanumdeposition.

In lanthanum-penetrated axons, the neurotubules were oriented in the same wayas in the controls (Fig. 4). However the appearance of the neurotubules was quitedifferent from the usual situation. The neurotubular central core and clear zone werestained by the lanthanum (Figs. 5, 6), while at least part of the neurotubular wallremained unstained. The effect of lanthanum staining on these sections, then, is toproduce a 'negatively' stained image of the neurotubules (Figs. 4-6). In addition,the dimensions of the tubules differ somewhat from those of control neurotubules, assummarized in Fig. 1. That part of the wall which remains unstained measures3 + 1 nm in width and the diameter of a lanthanum-stained tubule (measured fromthe outer edges of the unstained wall) is 18 ± 1 nm. The diameter of the tubule plusthe lanthanum-stained clear zone is 30 + 3 nm. These are slightly less than thedimensions of the neurotubules in uranyl- and lead-stained control preparations (seeFig. 1). Since the inner core region appears to be the same size whether the tissuehas been treated with lanthanum (12 ± 1 nm) or not (12 + 2 nm), it appears that theunstained circle seen in cross-sections of lanthanum-stained neurotubules must rep-resent only the inner part of the wall. It follows that the outer part of the neurotubularwall may take up lanthanum.

It might be mentioned that, at high magnification, the lanthanum staining appearsto reveal filaments or subunits giving a 'beaded' appearance to part of the wall of theneurotubules (Fig. 6, inset). In addition, non-opaque, 'spoke-like' projections can beseen to extend from the unstained part of the wall into the lanthanum-stained outerzone (Fig. 6, inset); they appear to arise as one per subunit. These are similar in

220 N. J. Lane and J. E. Trelierne

arrangement to the electron-dense projections seen to extend from other microtubules(Bohm & Parker, 1968; Behnke & Forer, 1966).

In the lanthanum-treated preparations, the lanthanum also appears to be associatedwith axoplasmic filaments which take the form of unstained short rods or circularglobules with lanthanum on their periphery (Figs. 5, 6). It is possible that these fila-ments represent neurotubular subunits which have not yet aggregated into completetubular units, although in rat neurons, Wuerker & Palay (1969) consider that directinterconversion between neurofilaments and neurotubules is improbable.

A —

(b)

ABCD

(a)40 ± 5 nm23 ± 3 nm12 ± 2 nm5 ± 1 nm

ABCD

(b)3° ±3i8± 1I2±I3±i

nmnmnmnm

Fig. 1. Diagram summarizing the various dimensions of neurotubules in sectionsstained with (a) uranyl acetate and lead citrate, and (A) lanthanum salts. The blackcircle in (a) and the white circle in (b) represent cross-sections of neurotubules asthey appear after the different preparative methods. The mean diameter and the stan-dard deviation of the mean for the various regions of the tubules are given belowthe diagram.

Lanthanum staining of neurotubules 221

DISCUSSION

It seems to be fairly generally accepted that neurotubules are essentially the sameas microtubules; their name simply distinguishes them from those microtubuleswhich are present in non-nervous tissues (as in Gonatas & Robbins, 1964; Kirk-patrick, 1969). It should be mentioned, however, that there are other interpretationsof neurotubular structure. For example, Sandborn (1966) has suggested that neuro-tubules are continuous with the plasmalemma and other cell membranes. An alter-native arrangement has also been proposed by Rodriguez (1969) who suggested thatneurotubules are formed by 7 nm-thick filaments joined by thin bridges. However,the more widely held view is that, like microtubules, neurotubules are filamentous,with about 6 subfilaments in the side view of each tubule observed in negativelystained preparations; such a substructure has been described in the neurotubules ofmammalian nerve cells (Kirkpatrick, 1969). This suggests that neurotubules maycontain as many as 12 to 13 protein subunits in cross-section, as are present in micro-tubular walls (Andr6 & Thiery, 1963; Gall, 1966; Grimstone & Klug, 1966; Kieferet al. 1966; Behnke & Zelander, 1967). The lanthanum-stained material examinedhere also indicates that the walls of these insect neurotubules are composed of asimilar number of subunits (see Fig. 6, inset).

Certain investigators (Forer, 1969; Silver & McKinstry, 1967) believe it possiblethat microtubules may have additional structural components which are not revealedby conventional methods of fixation, embedding and staining. For example, althoughit has been suggested by some investigators that the clear zone around the peripheryof microtubules and neurotubules might represent an artifact of shrinkage (Maser &Philpott, 1964), it could be a portion of the tubules proper, which merely happensnot to be stained (Silver & McKinstry, 1967). The procedure for staining with lan-thanum hydroxide is one of the methods sometimes used by electron mieroscopists todemonstrate the presence of acid mucosubstances (Overton, 1967; Behnke, 1968). Inthe present experiments the region of the neurotubular clear zone appears to bestained with the lanthanum salts (see Figs. 1 and 4-6), which suggests that there maybe a polysacchande component in the neurotubules which forms an outer ring ofmaterial round the proteinaceous wall. This outer zone of lanthanum-staining materialmeasures approximately 6 nm in width, a value which is fairly similar to the widthof the clear zone in microtubules fixed in a routine fashion (for example, about 6 nmin shrimp nerve fibres (Hama, 1966) or 8-5 nm (Fig. 1)). Of course, as discussedabove, this figure also includes the outer part of the tubule wall normally seen incontrol preparations, in which it is considered to be protein in nature. The less dense,unstained spokes extending from the unstained part of the wall into the dense,lanthanum-staining outer wall may be part of the protein component of the wallsprotruding into the peripheral (muco)substance. Alternatively, it is entirely possiblethat this peripheral region does not form part of the tubules proper, but representssubstances adhering to the tubule wall, or in the process of being moved along it. Asa third possibility, the staining could represent non-specific absorption of the lantha-num to the tubule walls, although, unlike in negatively stained, unfixed preparations,

222 N. J. Lane and J. E. Treherne

this is not to be expected in fixed and sectioned material where the axonal materialsurrounding the neurotubules would remain fixed in position, precluding the non-specific accumulation of substances.

The lanthanum staining of the core of cockroach neurotubules suggests that thetubules contain some sort of material within their lumen. The lanthanum-stainedcores produce an image which resembles that of certain negatively stained micro-tubular preparations where the whole core region has also been stained (Pease, 1963;Andr6 & Thiery, 1963; Kiefer et al. 1966; Gall, 1966; Grimstone & Klug, 1966;Barnicot, 1966). However, Behnke & Zelander (1966, 1967) found that such negativestaining of the core with uranyl acetate or phosphotungstate would occur only atlow pH and not near neutrality. This would appear to indicate that the staining doesnot represent movement of the dye into the tubules, but a pH-specific staining of amaterial already present. Indeed, on the basis of such experiments Behnke & Zelanderrefer to microtubules as 'two component structures'.

Negatively stained preparations of unfixed microtubules, then, reveal staining ofthe whole of the core region of the tubules. However, in sectioned material, densecentral granules located within the non-opaque neurotubular lumen have beenreported to occur in a number of different nervous tissues; these have sometimesbeen described as 'dense-core' neurotubules and have been found in shrimp nervefibres (Hama, 1966), toad nerve cells (Rodriguez Echandia, Piezzi & Rodriguez,1968; Rodriguez, 1969), rat optic nerve (Peters & Vaughn, 1967), sparrow parsnervosa (Bern, Mewaldt & Farner, 1966) and chick embryo retina (Gonatas &Robbins, 1964). Certain investigators (Rodriguez Echandia et al. 1968; Rodriguez,1969) consider that these dense granules represent material undergoing transportrather than structural components of the neurotubules. However, the resultswith lanthanum staining do not provide any further evidence for the existence ornature of central neurotubular granules. Other investigators have similarly suggestedthat neurotubules may have a role in transport (Schmitt, 1968; Kreutzberg, 1969).For example, it has been postulated that they may carry down the axon essential'building stones' which may then be used for the formation of vesicles at theirterminal ends (Blumke & Niedorf, 1965; Pelligrino de Iraldi & de Robertis, 1968;Jarlfors & Smith, 1969). However, whatever its functional significance, the fact thatthe core stains with lanthanum indicates the presence of anionic molecules, possiblymucopolysacchande, within the lumen of the neurotubules.

Recent cytochemical investigations on the localization of acetylcholine esterase(AcChE) in lobster nerves (de Lorenzo et al. 1969) report that reaction product forthis enzyme is to be found in some of the neurotubules of the axons. The materialstaining with lanthanum is unlikely to be AcChE because the cytochemical testsrevealed the enzyme in only a relatively small proportion of the axoplasmic neuro-tubules. Application of lanthanum to the neuromuscular junction, where a higherconcentration of AcChE would be expected, might elucidate this point.

It would appear, then, that the intratubular substance may be in the process oftranslocation, or alternatively may be fixed in position, possibly as part of the neuro-tubular structure. The question of the nature of this substance now arises. Whether

Lanthanum staining of neurotubules 223

it is mucopolysaccharide or not the lanthanum has revealed an apparent matrix ofanionic groups. Even if these were in the process of translocation it would seemreasonable to suppose that movement would be slow relative to that of small, water-soluble ions or molecules. The movement of such diffusible particles along the lumenof a tubule 12 nm in diameter is not likely to be unduly reduced, for there is con-siderable experimental evidence that indicates little restriction to diffusion in a situa-tion in which the size of the ion or molecule is small in relation to the width of thechannel (cf. Pappenheimer, 1953; Nicholls & Kuffler, 1964; Treherne, Lane, More-ton & Pichon, 1970). The presence of an anionic matrix within the neurotubules couldalso be envisaged as allowing relatively rapid movements of small, water-solublecations. This supposition is based on an analogy with cartilage (which for the presentpurposes can be regarded as a network of linear polyanions with fixed negativecharges) where, taking into account the diffusion constants (Maroudas, 1968) anddistribution coefficients (Maroudas, 1969), it can be predicted that the mobility ofsmall cations would not be unduly restricted, although that for small anions wouldprobably be more drastically reduced. It follows, therefore, that neurotubules couldrepresent a series of diffusion channels which would be well adapted for the transportand discharge of small cationic substances at high concentration at their terminations,a characteristic which could conceivably be of physiological significance as an axonaltransporting mechanism.

We would like to express our gratitude to Dr Arthur Forer for helpful discussions concerningthis investigation and for critically reading the manuscript. We are also indebted to Dr D. A.Hayden for valuable criticism and advice. We wish to express our thanks to Mr John Rodfordfor preparing the diagram in Fig. i, and to Miss Yvonne Carter for photographic assistance.

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(Received 18 December 1969)

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226 AT. J. Lane and J. E. Treherne

Fig. 2. Electron micrograph of cockroach central nervous system showing a number ofaxons cut in cross-section. Note the glial cells (g) surrounding them and the distendedextracellular spaces (s). The number of neurotubules (arrows) present in each axonis variable, x 31 600.

Lanthanum staining of neurotubules 227

15-*

228 N. J. Lane andj. E. Treherne

Fig. 3. Portion of an axon from a cockroach ganglion cut in cross-section. Note thespoke-like projections arising from the periphery of some of the neurotubules. Incertain cases a clear zone may be seen round the tubules (arrows) but the low electronopacity of the background cytoplasm often renders this zone imperceptible. (Comparewith inset.) x 107000.

Inset: Cross-section through the ghal cytoplasm surrounding axons in a connectivefrom the central nervous system of the stick insect Caraustus. The clear zones (arrows)around the microtubules and some neurotubules are immediately obvious due tothe moderate electron density of the background cytoplasm, x 70000

Fig. 4. Oblique section through an axon of a cockroach ganglion treated with fixativescontaining lanthanum salts. Note the density due to lanthanum associated with theneurotubules (arrows) in contrast with the other, non-lanthanum-penetrated axonsand surrounding glial cells, x 15400.

Lanthanum staining of neurotubules 229

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230 N. J. Lane and J. E. Treherne

Fig. 5 Longitudinal section through neurotubules in an axon, from the ganglion of acockroach, penetrated by lanthanum. Note the lanthanum deposition on both coreand periphery of the tubules, x 100 500.

Fig. 6. Cross-section through neurotubules in the axon of a cockroach gangliontreated with lanthanum salts. Note the uptake of lanthanum in the cores and on theperiphery of the tubules, especially where indicated by arrows, x 92700.

Inset: Higher magnification showing the unstained, spoke-like projections arisingfrom the unstained neurotubular wall as well as the beaded appearance of the wall,x 292700.

Lanthanum staining of neurotubules 2p: