The Cytoplasmic Inclusions of the Neurones of Helix aspersa and Limnaea stagnalis

THE purpose of this investigation was to find the form and distribution of the cytoplasmic inclusions in the living neurones of Helix aspersa and Lrmnaea stagnalis, and to reveal the nature of the bodies commonly called ‘Golgi apparatus’, ‘Golgi bodies’, or ‘dictyosomes’, which appear when these cens have been treated in particular ways. Several authors (Brambell, 1923; Boyle, 1937; Thomas, 1947; Moussa, 1950; and Roque, 1954) have addressed themselves to these problems, but have not reached agreement among them-selves.

For this investigation, the cerebral ganglia of Helix aspersa and Limnaea stagnalis were used. The ganglia consist of two groups of neurones which are surrounded by connective tissue; they may easily be separated from this by teasing. The groups of neurones were transferred to 0·7% sodium chloride solution containing 0·2% of 10% anhydrous calcium chloride, slightly flattened under a coverslip, and examined by direct and phase-contrast microscopy.

For supravital staining, neutral red chloride, Nile blue, methylene blue, brilliant cresyl blue, dahlia violet, and Janus green B were used. The dyes were dissolved in distilled water and then diluted with saline to the required concentration. The following method was found to give very satisfactory results:

Prepare a 1% solution of the dye in distilled water for use as a stock solution. Before staining add 2 drops of the dye to 2 ml. of the saline solution; the concentration of the dye is now about 0·04%. Stain a group of neurones in this solution from 10 to 20 minutes, flatten slightly under a coverslip, and examine by direct microscopy.

For intravital staining, 1 ml. of 1% solution of the dye in saline was injected into the haemocoele. The ganglia were removed after 20 or 30 minutes, or sometimes after an hour, and mounted in saline for examination. The animals were always killed by decapitation.

The following methods were used for studying fixed materials:

1. Baker’s standard Sudan black technique (fixation in formaldehydesaline with special postchroming; gelatine sections, coloration with Sudan black B) (Baker, 1944).

2. Sudan black B after fixation in Baker’s ‘Lewitsky-saline’ solution; that is to say, in Lewitsky’s fluid (Flemming-without-acetic) with addition of 0·75% °f sodium chloride (Baker, 1956).

3. Sudan black after fixation in formaldehyde-calcium.

4. Sudan black after simple fixation in formaldehyde-saline.

1. Weigl’s technique (Mann-Kopsch) (Weigl, 1910).

2. Kolatchev’s technique (fixation in Champy’s fluid, washing in running water, post-osmication in 2% osmium tetroxide at 35⁰ C for 3 days; sections were cut at 4μ and mounted unstained in Canada balsam) (Kolatchev, 1916).

3. Aoyama’s silver method (Aoyama, 1929).

4. Fixation in 4% formaldehyde for 24 hours followed by post-osmication in 2% osmium tetroxide for 3 days at 35° C (Baker, 1944).

To show mitochondria, ganglia were fixed in Altmann’s, Helly’s, and Mann’s fluids, and sections were stained by Metzner’s (1928) and Hirschler’s (1927) methods.

The cells of Helix aspersa are unipolar, and about 15 to 70 μ in diameter. Each contains a large nucleus with two or three nucleoli. The nucleus fills about two-thirds of the cell (fig. 1).

With direct microscopy, numerous spherical granules varying in diameter from about 1 to 2/x can be seen in the cytoplasm. Generally they can be divided into two kinds : one kind is yellowish, large, slightly irregular in shape, and mostly aggregated near the axon hillock. Globules of the second kind are colourless, and in the axon a number of them tend to be arranged like a string of beads. Their diameter is about 1 μ, or a little more, but it is never more than 1·5 μ. Both of these kinds of globules are rather highly réfringent. When examined by phase-contrast microscopy, the globules appear darker than the ground cytoplasm, and homogeneous. One or two small granules (satellites’) are usually attached to the larger globules of both kinds. Sometimes a large globule bears three satellites.

In the small neurones the globules are fewer and scattered throughout the cytoplasm with no definite arrangement. The yellowish globules are not present.

The neurones of Limnaea stagnalis (fig. 2) have a similar appearance, but the yellowish globules are of a much deeper yellow and are numerous in both small or large neurones. They are irregular in shape. With phase-contrast microscopy the irregularity of their form is very obvious. The colourless ones, usually spherical, are not numerous.

There is no sign of any crescent- or cap-shaped bodies in the living cell of either Helix or Limnaea.

Nile blue, methylene blue, and brilliant cresyl blue

With Helix neurones, these three dyes all gave exactly similar results. They stained most of the globules, while the ground cytoplasm, the nucleus, and the remainder of the globules were unstained. By the use of these dyes the globules in the cytoplasm of Helix can be divided into three main groups (fig. 1 and fig. 5, p. 54):

These are the same globules that appeared yellowish when examined unstained by direct microscopy. They seem to be formed by aggregation of several small ones. They become green after staining for 10 minutes. The satellites, if present, become blue. They usually gather near the axon hillock, though small ones are also found in other parts of the cytoplasm. Thomas (1947) called them ‘mulberry spheroids’. Cain (1948) found that carotinoids accumulated in them. The green colour results from the mixture of the yellow carotinoid pigment with the blue dye.

These appear homogeneously blue. Their diameter is about i to 2 μ,. Sometimes they have one or two blue satellites. In small neurones (15 μ,) the blue globules are not very numerous. In large neurones (40 to 70 μ) they occur sometimes in the axon hillock among the green globules.

A number of globules do not take up any of these three dyes. Even if the neurones are left in the dye for more than an hour they still remain colourless. They are very easily distinguishable in the axon. These colourless globules are seldom more than 1·5 μ in diameter. They are highly réfringent. Particular attention was paid to them and it was found that the ones in the axon and those in the cytoplasm were indistinguishable.

With Nile blue, but not with other blue dyes, purple globules are occasionally seen in a few of the cells. There is no means of knowing whether these are globules of a special kind, or whether they are ‘blue’ globules that have incorporated a considerable amount of neutral lipid.

Nile blue, methylene blue, and brilliant cresyl blue were also used for intravital staining. The results were exactly the same as with supravital staining.

Most of the globules were stained reddish by this dye, but some of them, especially in the axon, were not coloured. Even after an hour they showed no tendency to be stained. The attached granules (satellites) were always the first to be stained. A similar result was obtained by intravital staining when 1% neutral red was injected into the haemocoele.

This dye was found not to be useful in this investigation. It coloured the ground cytoplasm rather deeply.

Nile blue was used to stain the living neurones of Limnaea (fig. 2). The same kinds of globules were seen as in Helix. The ‘green’ globules are very distinct; most of them bear blue satellites. Blue globules are fewer than Helix-, colourless ones fewer still. Pink globules are seen more often than in Helix.

A group of neurones of Helix aspersa from a very freshly teased cerebral ganglion was transferred, without fixation, to a clean slide with a small amount of saline and attached by slight pressure with a coverslip. They were then put in a saturated solution of Sudan black B in 70% alcohol for 10 minutes, differentiated in 70% alcohol for 5 seconds and in 50% alcohol for one minute, then washed thoroughly in distilled water, and finally mounted in Farrants’ medium. All the globules in the neurones became homogeneously blue-black. Thus all the kinds of globules contain lipid, which is evenly distributed throughout them. There is no differentiation into externum and internum.

Baker’s standard Sudan black method was used. All the lipid globules are coloured blue-black by this method, and seem to be homogeneous. The yellow globules can easily be distinguished from the others, both in form and in distribution. It is not possible to distinguish between the globules that stained blue with Nile blue, methylene blue, or brilliant cresyl blue on one hand and those that do not on the other, for all are equally coloured by Sudan black (fig. 3, A). The cytoplasm shows weak sudanophilia. Occasionally one or two black cap-shaped bodies are seen ; perhaps these are formed by the separation of the lipid constituents from other substances in the globules as a result of the action of the fixative.

When tissue is fixed in Lewitsky-saline and embedded in gelatine, and the sections coloured with Sudan black, the globules are seen in very life-like form (figs. 3, B; 4). The yellow globules still retain their form, but are weakly coloured by Sudan black ; the other kinds of globules are blackened completely.

Many crescent- or cap-shaped bodies appear when Sudan colouring agents are used after fixation in formaldehyde-saline or formaldehyde-calcium without post-chroming.

In general, the results obtained with Limnaea by the use of Sudan black resemble those obtained with Helix. The yellow globules, however, colour more strongly, and the other globules have a tendency to become slightly irregular in shape. Spherical vacuoles, untouched by Sudan black, are sometimes seen; these appear to be artificial, and are not often seen in tissue fixed with Lewitsky-saline.

It will be convenient to describe first the results of the application of the Weigl (Mann-Kopsch) technique to the neurones of Helix (fig. 3, c). This can be divided into two stages: (1) fixation by Mann’s fluid, and (2) postosmication.

Fig. 5 shows the three kinds of globules stained with Nile blue or methyiene blue in the living cell.

The effect of fixation alone is shown in fig. 6. Ganglia were fixed in Mann’s ‘luid and embedded in paraffin without post-osmication. Sections were coloured with Sudan black or Sudan IV and mounted in Farrants’ medium.

The shape is scarcely changed from the living condition. The globule is pale grey, the satellites somewhat reduced in size and darker.

These are much changed in appearance. In optical section one sees a crescent (blue-black with Sudan black, red with Sudan IV) pressed against one side of the globule, the rest being very pale grey like the cytoplasm. The satellites are not so clearly seen as in life.

These are more changed in external form than the other globules, as the surface is no longer smooth. They are blue-black or red, very pale in the centre.

The effect of the application of the whole of the Weigl technique is shown in fig. 7. A comparison of this with fig. 6 shows the result of post-osmication.

The appearance is scarcely changed except that there is a heavy black deposit of osmium round the satellites. When there are two or more satellites, a deposit of osmium may cover and connect them. The whole globule now looks duplex, as though it had an internal and incomplete external part.

The object appearing crescentic in optical section is now black, but the osmium has been deposited in such a way as to thicken the outside of it. Satellites are sometimes enclosed by the deposit of osmium. The external surface of the deposited osmium is somewhat irregular. The rest of the globule is not distinguishable from the cytoplasm.

These are scarcely seen in Weigl preparations, but subsequent colouring with Sudan IV shows them clearly by making them evenly With the Kolatchev technique it is difficult or impossible to distinguish the three kinds of globules. There is a tendency for the osmium to be deposited over the greater part of the globules so that rings as well as crescents are seen in optical section (fig. 3, E). If the sections are bleached and coloured with Sudan black, the yellow globules are easily distinguished by their form; they appear grey.

The technique of Aoyama causes silver to be deposited over nearly the whole surface of the globules, so that many of them appear as rings in optical section. Irregular crescents, not so sharply formed as in Weigl preparations, aie also seen. The silver makes a much thicker and more irregular deposit than the osmium. The yellow globules can be distinguished by their larger size and clear interior (fig. 3, F). When a ganglion is fixed and silvered by Aoyama’s method and frozen sections are coloured with Sudan IV, some of the globules (especially the ‘colourless’ ones in the axon, and the yellow globules) show a reddish internal part and a black silver deposit on the surface.

When tissue was fixed in 4% formaldehyde and post-osmicated, the result was similar to that obtained with Kolatchev’s and Aoyama’s techniques (fig-3, G).

Weigl’s technique was applied to ganglia of Limnaea. The yellow globules can easily be distinguished from the others, because they retain their irregularly globular shape, while the other globules are transformed into ‘crescents’. The osmium is deposited on the satellites of the yellow globules and on the surface of the yellow globules in the vicinity of the satellites, so that a crescentic appearance is given in an optical section. A greater part of the surface of the yellow globules is covered with osmium than in a Helix preparation. The ‘colourless’ globules cannot be distinguished.

With phase-contrast microscopy, the mitochondria in the neurone of Helix aspersa are seen as numerous, fine filaments in the cytoplasm; in the axon they tend to be arranged parallel to its axis (fig. 1). Their length is between 5 and 8 μ,. They are blunt-ended, and often have a granule at one or both ends. These filaments are similar in small and large neurones. They are more easily seen in hypertonic than in isotonic solution, but in the former they disappear in an hour or so.

The mitochondria of Limnaea stagnalis are exceedingly fine, curled filaments. Sometimes the thread is slightly thickened somewhere along its length. There is never a granule at the end, as in Helix.

Janus green B. Satisfactory results were not obtained with this dye in the living cell of either Helix and Limnaea.

Ganglia were fixed in Helly’s, Altmann’s, and Mann’s fluid and stained by Metzner’s (1928) acid fuchsin and Hirschler’s (1927) iron haematoxylin.

The mitochondria of Helix appear as very fine filaments, exactly as seen in the living cell; their granular ends are perhaps even more distinct (fig. 3, H). A number of spherical or cap-shaped fuchsinophil bodies showed after Metzner’s technique. These structures seem to be identical with the ‘blue’ globules in their distribution.

In Limnaea stagnalis the mitochondria appeared as fine, curled filaments. Granular mitochondria were only very rarely seen.

The appearance called ‘Golgi apparatus’, ‘Golgi bodies’, or ‘dictyosomes’ in these cells results from the artificial modification of lipid globules that are easily visible in the living cell. Osmium has a strong tendency to be deposited on the surface of the yellow globules, especially in the vicinity of the satellites. The ‘blue’ globules give a different appearance. The lipid constituent tends to be thrown by the fixative against the edge of the globule on one side, and osmium then accumulates in this artificially produced ‘crescent’ and on its surface. The ‘colourless’ globules do not appear to give rise to any Golgi artifact.

It is not impossible that the cytoplasm in the immediate vicinity of the yellow and ‘blue’ globules may be in some way different from the rest of the cytoplasm at the submicroscopic level, but none of the techniques used in this study has suggested the presence of any kind of special structure round the globules.

It remains to compare the conclusions reached by myself with those reached by other students of the same cell.

My phase-contrast view of the cell agrees well with that of Roque (1954). I differ from him only in classifying the globules by their reactions to vital dyes and not by their sizes. Roque did not investigate the origin of the Golgi artifact. Brambell (1923) made silver preparations resembling my own in general, but did not attempt to identify in the living cell the objects responsible for the reaction of the silver. Boyle (1937) studied the living cell and saw the globules colour with neutral red. He noticed that some of the globules did not colour with neutral red or methylene blue. He considered these to be metamorphosed mitochondria. I have found no support for this view of the origin of the ‘colourless’ globules. He also showed that in frozen sections of material treated by Aoyama’s method, globules became coated by silver on the surface while the interior could be coloured by Sudan IV. He neither stated nor denied there was any relation between the globules seen in the living cell and the crescents and rings seen in the osmium and silver preparations. Thomas’s (1947) ‘mulberry spheroids’ clearly correspond with my yellow globules. He considered that the crescents seen in osmium preparations are in fact over-impregnated mitochondria. He partially bleached Mann-Kopsch preparations and found that the ‘dictyosomes’, now greatly reduced in thickness, could usually be resolved into a row of granules, which he referred to as a mitochondrion. I do not agree with him, because the mitochondria are so numerous that if they were over-impregnated to produce ‘dictyosomes’ one could hardly see the spaces between them. The objects he saw were probably blue’ globules, distorted by the Golgi technique.

Moussa (1950) studied the neurones of Limnaea stagnalis. He noticed that certain globules in the cytoplasm, which he called ‘lipochrome globules’, are naturally coloured golden yellow, and that of the other globules some take up vital dyes while others do not. This is in agreement with my findings. He claimed i o have seen reddish-brown crescents or rims in the golden yellow globules in Hving cells. This is not in conformity with my findings. Moussa considered hat the crescents or rims become separate from the internal part of the globules. The latter constitute the ‘Golgi product’. The crescents and rims now become invisible in life, even by phase-contrast microscopy, but can be silvered or osmicated to form the ‘Golgi dictyosomes’. I have seen nothing that would suggest that this is a true explanation of the origin of the curved black objects seen in a Golgi preparation.

An important difference between Moussa and myself is that he derives the curved black objects (Golgi bodies or dictyosomes) from the yellow globules, whereas I derive them from the ‘blue’ globules. I do not find it necessary to postulate the existence of any totally invisible objects large enough to be seen easily by phase-contrast microscopy.

Young (1932, 1953, ^I950 has remarked that the neutral red globules in the neurones of cephalopods can be swollen or shrunk osmotically, and he concludes that they might be bounded by a semi-permeable membrane. Such a membrane, however, could not be seen by electron microscopy. I have not seen any indication of a membrane in my studies of the lipid globules of Helix and Limnaea.

My observations on Helix entirely agree with those of Roque (1954). Thomas (1947) found that in fixed preparations the threads were often broken up into strings of granules (‘coccoids’). Brambell and Gatenby (1923) and Boyle (1937) described the mitochondria as granules. This is certainly not so in the living cell.

Moussa (1950) described the mitochondria of Limnaea as granular, or sometimes as consisting of rows of granules. This is not true of the living cell.

I wish to express my gratitude to Dr. J. R. Baker for suggesting and supervising this investigation; to Professor A. C. Hardy, F.R.S., for providing me with facilities for working in his Department; to Mr. S. Bradbury and Miss B. M. Jordan for advice on certain matters; and to Mr. P. A. Trotman for supplying me with animals.

The work was done during the author’s tenure of a British Council Scholarship, and study leave from the Department of Zoology, University of Hong Kong.