Thiamine pyrophosphatase, acid phosphatase, and
alkaline phosphatase in the neurones of Helix aspersa
By NANCY J. LANE
(From the Cytological Laboratory, Department of Zoology, University
Museum, Oxford)
With one plate (fig. 3)
Summary
All three phosphatases have been found to be localized mainly in the cortices of bodies
which have the distribution, size, and shape of the 'blue' and yellow lipid globules.
Colouring neurone preparations with the lysochrome Sudan IV, either before or after
incubation for thiamine pyrophosphatase or acid phosphatase activity, shows a sudanophil reaction in the medullary spaces surrounded by the cortices that hydrolyse both
phosphates. It is concluded that the acid phosphatase and thiamine pyrophosphatase
activities, which in vertebrates are present in the lysosomes and Golgi lamellae
respectively, are mainly found, in these invertebrate neurones, in the phospholipid
lamellae which form the externa of certain of the lipid globules present in the
cytoplasm.
Introduction
N O V I K O F F and Goldfischer (1961) recently demonstrated the presence of
nucleosidediphosphatase (NDPase) and thiamine pyrophosphatase (TPPase)
activity in the lamellae of the Golgi apparatus in certain vertebrate and plant
tissues. Similarly, Allen and Slater (1961) have shown Golgi-associated
TPPase activity to be present in the epididymis of the mouse. These results
would suggest that the test for such enzymic activity might be used as
a cytochemical technique for depicting the Golgi complex. On the assumption that, as these workers have shown, the Golgi lamellae always contain
such an enzyme or enzymes with hydrolytic activity specific for nucleosidediphosphates (NDPs) and thiamine pyrophosphate (TPP), then this technique
would present a marked advance over the classical metal impregnation techniques for showing the Golgi apparatus in vertebrate tissues.
The TPPase and NDPase preparations in vertebrates give pictures exactly
similar to those produced after the standard Golgi methods, and in electron
micrographs show a heavy deposition of lead on the smooth membranes of
the Golgi lamellae (Novikoff, Essner, Goldfischer, and Heus, 1962). Other
cytomembranes which hydrolyse NDPs and TPP do so far less rapidly,
producing, during the incubation periods used, a much fainter, or a negligible,
reaction (Novikoff and others, 1962). From the results produced by incubation in TPP and NDP media, it appears that there is an enzymatic similarity
between these lamellae in cells of different sorts. This similarity, considered
in relation to the concordance in structure and dimensions of the Golgi
[Quart. J. micr. Sci., Vol. 104, pt. 3, pp. 401-12, 1963.]
402
Lane—Phosphatases in neurones of Helix
membranes in cells of different sorts, suggests that in the cell types studied by
these investigators, the Golgi apparatus is a homologous structure and a distinct cellular entity. This had formerly been questioned by such investigators
as Malhotra (1959) and David, Brown, and Mallion (1961), who have shown
that the classical Golgi techniques sometimes darken the same network that
is coloured by basic dyes to reveal the Nissl substance. The Nissl bodies
correspond to the endoplasmic reticulum with attached ribonucleoprotein
granules (Malhotra and Meek, i960).
However, the problem of whether this vertebrate Golgi corresponds to any
structure in the invertebrate cell has remained unsettled. Invertebrate cells,
after impregnation with heavy metals, present, not a network as do the
vertebrate tissues, but a number of 'dictyosomes' in the shapes of half-moons,
crescents, and filaments (Nath, 1944; Baker, 1945; Malhotra, 1961). Although
Malhotra, in a histochemical study (1961), has shown that these dictyosomes
differ chemically from the classical vertebrate Golgi network, many investigators still consider these dictyosomes to be homologous with the vertebrate
Golgi net.
The invertebrate dictyosomes, for example in the case of the neurones of
the snail, Helix aspersa (Chou, 1957a), or the neurones of the prawn or crayfish (Malhotra, i960), have been shown to be due to deposition of silver or
osmium on the outer surface of certain of the lipid droplets that are present in
the cytoplasm.
In an electron microscopical investigation of H. aspersa neurones, Chou
and Meek (1958) suggested that a structure similar to the typical vertebrate
Golgi lamellae was only present in the cells if calcium ions were omitted from
the fixative. They concluded, therefore, that it represented an artificial distortion, due to the splitting open of the cortex of the phospholipid globules,
which are composed of concentric lamellae at the ultrastructural level. Other
workers (Dalton, i960; McGee-Russell, 1962) who studied the neurones of
H. pomatia, disagreed with this interpretation, believing the lipid globules,
and a system of Golgi lamellae similar to the vertebrate Golgi, to be two
distinct and separate cell organelles, both present in Helix.
The present study was therefore undertaken in an attempt to discover the
site of TPPase activity in the neurones of H. aspersa, in the hope of finding
out which of the lamellar systems of invertebrate neurones, if any, resemble
the vertebrate Golgi apparatus in their enzyme content.
A study of the distribution of acid phosphatase in this invertebrate cell was
also undertaken to make a comparison with vertebrate cells, where this enzyme
is usually located in the lysosomes (Novikoff, 1961). Sobel (1961) has shown
a parallel between hormone production and accumulation of acid phosphatase
activity in vertebrate endocrine organs; and it has been shown that neurosecretory activity (Dalton, i960; Krause, 1961), as well as probable hormonal
production (Pelluet and Lane, 1961), is present in gastropod neurones. This,
as well as the widely accepted theory of the association of the Golgi lamellae
with secretory products, and Novikoff's suggestion (1961) that lysosomes arise
Lane—Phosphatases in neurones of Helix
403
from the Golgi apparatus, made it also desirable to compare the site of acid
phosphatase activity with that of TPPase activity.
Tests were made for the presence and localization of alkaline phosphatase
activity in order to make a comparison with the results obtained by Chou
Sudan IV was used to colour H. aspersa neurones either before or after
incubation for TPPase and acid phosphatase, as a means of determining the
sites of the lipid globules in relation to the phosphatase activity.
Procedure
Test for thiamine pyrophosphatase activity
Neurones from the cerebral ganglion of the snail, H. aspersa, were fixed
overnight in formaldehyde-calcium (F/Ca) at 4 0 C (Baker, 1945). After
washing in cold distilled water, the tissue was kept in a sucrose / gum-acacia
solution (Holt, 1959) at 4 0 C. Frozen sections were cut at 10 /A on a freezing
microtome and placed in distilled water. The sections were mounted on
slides upon a thin film made from a dilute gelatine solution (Baker, 1945),
or were placed flat on slides in a small amount of distilled water, which was
removed by thorough drying of the sections. The sections thus prepared were
either used at once or kept for a week or two in distilled water at 4 0 C.
Novikoff and Goldfischer (1961) state that such sections may be stored for
several weeks without loss of enzymic activity. Sometimes neurones were
teased apart after fixation, mounted on slides, dried, and then tested for
enzymatic activity.
Sections and teased preparations were incubated for 20 to 45 min at 37° C
in the TPP medium described by Novikoff and Goldfischer (1961). Since
rather weak results were obtained with a 1 M solution of the substrate, a 3 M
solution was used, as Novikoff and Goldfischer found this to be the optimal
higher concentration of substrate. The incubation medium was filtered before
use if a precipitate formed during its preparation. After incubation the slides
were rinsed in distilled water, and placed briefly in dilute ammonium sulphide
solution for visualization of the lead reaction-product. The slides were
mounted in glycerogel in the usual fashion.
A control for the activity of the TPP incubation medium was carried out
on mouse epididymis, where it produced a positive reaction in the Golgi apparatus of the epithelial cells, as Novikoff and Goldfischer (1961) had found.
Control sections of H. aspersa neurones were carried through at the same
time as were the experimental tissues. For one form of control, none of the
TPP substrate was added to the incubation medium; in the other, an inhibitor,
o*oi M uranyl nitrate, was added to the medium (Novikoff and Goldfischer,
1961).
Test for acid phosphatase activity
Cerebral ganglionic tissue was prepared for the acid phosphatase test by
fixing in cold F/Ca in the same way as described above in the test for TPPase
404
Lane—Phosphatases in neurones of Helix
activity. These cells were then washed and stored in cold sucrose / gumacacia. Frozen sections were cut at 10^. and mounted on slides as before. The
tissues were incubated at 370 C in o-i M sodium-/?-glycerophosphate medium
at pH 5-0 (Gomori, 1952). The period of incubation was about 1 h; the normal
incubation time of 4 h produced too intense a result for the proper resolution
of the elements that hydrolysed the substrate. The final reaction-product was
visualized by dipping the slides in dilute ammonium sulphide solution.
Control sections were incubated in the same medium to which had been
added o-oi M sodium fluoride as inhibitor.
Test for alkaline phosphatase activity
Sections were tested for alkaline phosphatase activity by the technique of
Gomori (1952). Cerebral neurones were fixed in ethanol/acetone (1:1),
embedded in paraffin and sectioned at 6 to 8 p. The slides were incubated
in the alkaline 2% sodium-jS-glycerophosphate medium for x\ to 4 h at 370 C.
The reaction-product was visualized by successive periods in 1% calcium
chloride, 3% cobalt chloride, and dilute ammonium sulphide solutions, as
described by Gomori.
Control sections were tested by the same technique, but with the omission
of incubation in the glycerophosphate medium, to check for the presence of
any calcium.
Colouring with Sudan IV to test for the presence of lipids
Novikoff and others (1962), in reference to testing for the activity of NDPase
and TPPase in vertebrate tissues, stated that 'the method permits staining
for lipids after visualizing [the enzymic] activity in the sections'. Sudan IV
was used to determine the localization of the lipid globules described by Chou
(1957 a, b) in the neurones of H. aspersa. A control was first carried out by
colouring F/Ca material with Sudan IV, and examining for the distribution
and coloration of the various lipid globules.
Different sequences of incubation and coloration were used in these experiments. First, sections of neurones that had been fixed in F/Ca were incubated
for TPPase or acid phosphatase activity, as described earlier. After visualization of the reaction-product with ammonium sulphide, the sections were
examined, and the enzyme distribution in particular cells noted and drawn.
The same section was then treated with a saturated solution of Sudan IV in
70% ethanol/acetone (1:1) for 5 min, differentiated with 50% ethanol for
1 min, washed with distilled water, and mounted in glycerogel. The same
individual cells were again examined to observe where uptake of Sudan IV
had occurred.
Secondly, sections of neurones that had been fixed in F/Ca were coloured
with Sudan IV as described above, examined, and a few cells drawn in detail.
The same sections were incubated for acid phosphatase or TPPase activity,
and re-examined to determine the enzymatic activity with particular reference
to the lipid globules seen in the first inspection of the same cells.
Lane—Phosphatases in neurones of Helix
405
A further control was carried out by placing F/Ca material in 70% ethanol/
acetone (1:1) for 5 min, with no Sudan IV added. This was then incubated
in the TPPase and acid phosphatase media as usual and examined for enzymatic activity.
Observations and Results
The results were the same regardless of the manner in which the sections
were mounted on the slides. The optimal incubation time for TPPase activity
was found to be about 45 min, for acid phosphatase activity 60 min, and for
alkaline phosphatase activity 150 min. Storage of sections for short periods
caused no obvious differences in intensity of reaction.
FIG. 1. Diagram of a section of a cerebral neurone from H. aspersa
after incubation for thiamine pyrophosphatase activity.
The test for TPPase activity produced an intensely positive reaction, localized in the form of circular or elliptical bodies, which appeared ring-like in
sections. These varied from 1 to 2 ^ in diameter and were dispersed throughout the cytoplasm (fig. 1). This reaction seemed to be on the surface or
cortex of globules and was often more intense on one side of the surface than
on the other. Occasionally the enzymic activity seemed to be localized in the
form of crescents which sometimes lay on the surface of larger (about 2-5 p)
irregular bodies. A positive reaction was also observed on the surface of
irregular bodies which looked as if two or more smaller bodies had coalesced.
In the case of the control sections incubated for TPPase activity without any
substrate, a very slight reaction was sometimes discernible, perhaps due to
residual substrate within the cell. Uranyl nitrate, however, effectively blocked
any reaction, giving completely negative results.
4-o6
Lane—Phosphatases in neurones of Helix
Approximately the same localization was observed after incubating for acid
phosphatase activity (figs. 2, 3). The cytoplasmic inclusions of these preparations, and those incubated for TPPase activity, could not be distinguished one
,
from the other. However, the clumps of chromatin in the nucleus gave a
'
strongly positive reaction for acid phosphatase activity only (fig. 3). The
j
. control for acid phosphatase, with sodium fluoride inhibition, gave negative
',(,,,, - results.
FIG. 2. Diagram of a section of a cerebral neurone from H. aspersa
after incubation for acid phosphatase activity.
There was a great number of positively reacting bodies after incubation for
acid phosphatase and TPPase activity; so many that resolution of the separate
elements was sometimes impaired. The positively reacting cortices seemed
to be evenly distributed, except in some cases where they nearly all occurred
on one side of the cell. In these instances the phenomenon was an obvious
fixation artifact. In no case were the sites of activity particularly aggregated
in the axon hillock, nor along the length of the axon. The axons that were
observed contained no element that hydrolysed either TPP or sodium-j8glycerophosphate.
Alkaline phosphatase activity was localized very intensely in the nuclear
and plasma membranes. It was also present in the nucleus, in the chromatin
clumps, and in the nucleoli. The reaction in the nucleus was more diffuse
FIG. 3 (plate). Cerebral neurone from H. aspersa. A frozen section cut at 10 n after
formaldehyde/calcium fixation. Incubated for acid phosphatase activity. (The test for
thiamine pyrophosphatase gives the same picture, except that there is no reaction in the
nucleus.)
FIG.
3
N. J. LANE
Lane—Phosphatases in neurones of Helix
407
and less violent than the positive acid phosphatase reaction in the nucleus.
A fainter positive reaction, indicating some alkaline phosphatase activity, was
seen to be localized on the externa of spheres scattered evenly throughout the
cytoplasm. Sometimes this reaction was stronger on one side of a globule than
on the other. Occasionally tiny, apparently homogeneously positive, spheres
were observed. The controls gave negative results. Thus the alkaline phosphatase activity was in the same site as that observed for the other two
phosphatases, but the reaction was very much less intense.
Control sections, coloured only with Sudan IV, contained various sudanophil globules. The cortices of spheroidal globules (about 1 /x or more in
diameter), and of somewhat larger irregular globules, were coloured pinkishred. Their interna were highly refractile, which made it difficult to ascertain
the colour, although they seemed to be a faint pink or in some cases uncoloured.
Heavily coloured, red-orange droplets were scattered about in smaller numbers. These were smaller than the other types of globule, being less that 1 /u,
in diameter and hence beyond the limit of exact measurement by light microscopy. Larger (1 to2/i) red-orange irregular globules were present, but not
in such abundance as the refractile globules. These red-orange bodies were
sometimes grouped in the area of the axon hillock.
Post-colouring with Sudan IV, on sections previously incubated for the
activity of both TPPase and acid phosphatase, produced a reddish yellow
colour inside the cortices where the enzyme was localized. There seemed to
be no coloration with the lysochrome within some of the cortices. Tiny
scattered droplets, measuring less than 1 JX and showing no enzyme activity,
were coloured deep red after the treatment with Sudan IV.
Examinations of sections incubated for enzymatic activity after coloration
with Sudan IV also indicated that no activity was present in the tiny scattered
sudanophil droplets. A positive reaction was observed around most of the
refractile globules, sometimes obscuring the sudanophilia of the cortex.
Sometimes enzymic activity was present only partially around the large,
irregular sudanophil globules. The enzyme activity was in all cases less
intense than that in sections without pretreatment with Sudan IV.
The control sections treated with the ethanol/acetone solution before
incubation in the TPP and acid sodium-j8-glycerophosphate media, showed
the enzymatic activity in the same sites as described above for cells incubated
and coloured in the normal fashion. However, with the normal incubation
time, the intensity of the reaction was very much decreased.
Discussion
Live preparations and fixed sections of the neurones of H. aspersa were
studied in detail by Chou (1957 a, b). He described three kinds of lipid
globules in these cells. He distinguished the various lipid inclusions by the
following names: yellow globules, of irregular shape, which contained carotenoid and mixed lipids, with some protein and carbohydrate; spheroidal
'blue' globules, which responded to no histochemical tests except those for
408
Lane—Phosphatases in neurones of Helix
phospholipid; and colourless globules, composed of triglyceride. The yellow
globules were aggregated mainly in the axon hillock, the 'blue' spheroids (so
called because they coloured readily with certain blue vital dyes) were scattered evenly throughout the cytoplasm, and some of the colourless droplets
were strung out along the basal part of the axon, while others were distributed
throughout the perikaryon. The 'blue' globules measured about i to z /x in
diameter, the yellow ones were generally somewhat larger than this, and the
colourless droplets averaged about i JX.
Chou stated (19576) that all three types of globule reacted positively to
Sudan IV, and that this lysochrome showed the colourless droplets to be
homogeneously red. The distribution, size, and shape of the sudanophil
globules that were observed in this study, indicate that they are the 'blue',
yellow, and colourless globules described by Chou. The latter's histochemical
observations (19576) explain the more intense cortical sudanophilia found
here with the refractile lipid droplets. Both 'blue' and yellow globules are
refractile in fixed preparations, although the 'blue' ones have rather a low
refractive index during life (Ross and Chou, 1957). Chou noted that the lipid
in the yellow globules was more or less restricted to their peripheries; the
centres of the 'blue' globules were diluted with water (Ross and Chou, 1957),
with the phospholipid layers often confined to the cortex (Chou and Meek,
I958)Sometimes the smaller neurones in tissues that had been incubated for
enzymatic activity showed less reactivity than the larger neurones in the same
section. Chou stated (1957a) that smaller neurones contained no yellow
globules and fewer 'blue' droplets than did the larger ones. This fact, or
some block to the diffusion of the substrate into these cells, may perhaps
account for this.
The only enzymatic test performed by Chou was Gomori's test for alkaline
phosphatase (19576). This gave a positive result on the surface of the yellow
globules, which Chou noted was in the same position as the phospholipid.
Studies of sections incubated for enzymic activity and subsequently
coloured with Sudan IV indicated that at least a large proportion of the
bodies hydrolysing the phosphates were the cortices of the 'blue' and yellow
globules. Since the intensely sudanophil triglyceride droplets were not
evident in the enzyme-incubated preparations before coloration, it is probable
that these colourless lipid globules contained no enzyme activity.
Examinations of neurones coloured with Sudan IV and then incubated for
enzyme activity, also suggested that the positive enzymic reaction on the
surface of the lipid globules was limited to the cortices of the 'blue' and yellow
ones. Further, studies of the axons, where the colourless droplets were often
strung out, failed to show evidence of enzymatic activity. There were, in the
perikaryon, elements rich in phosphatase which had sudanophobe cores, and
it was impossible to state whether these were lipid globules whose sudanophil
cortex had been obscured by the enzymatic activity, or whether they were
some other cytoplasmic organelle.
Lane—Phosphatases in neurones of Helix
409
The ultrastructure of the three types of lipid globules is helpful in interpreting these results. The lack of enzymatic activity in the colourless droplets is
understandable if it is assumed that the activity in the 'blue' and yellow
globules is present in the lamellated border of these globules, which Chou
and Meek described (1958), for they found that the colourless droplets have
no such lamellar border. They considered that the laminated membranes at
the periphery of the 'blue' globules were probably due to the presence of
phospholipids.
As mentioned earlier, Chou and Meek concluded from their investigations
that those lamellar membranes in the neurones of H. aspersa which had an
appearance similar to the vertebrate Golgi were distortions of the 'blue'
phospholipid droplets that had been split open by fixatives not containing
calcium ions. Dalton (i960) and McGee-Russell (1962), in electron-microscopical investigations of the neurones of H. pomatia, each described both
lipid globues and Golgi bodies, which were present as distinct and separate
cell organelles. The bodies which possess phosphatase activity in H. aspersa
and are not definitely lipid globules may perhaps correspond either to split
'blue' globules, or to Golgi lamellae. Such a reaction would bear a relationship to vertebrate cells, where the Golgi lamellae have high levels of TPPase
activity (Novikoff and Goldfischer, 1961). The relative sizes of the lipid
globules and the Golgi lamellae are of no use in distinguishing between them
in light-microscopical preparations, since the figures are within the same
range; so that no conclusion about this matter can be drawn from the present
evidence. However, it can be stated that the cortices of the 'blue' and yellow
lipid globules possess both acid and alkaline phosphatase and TPPase
activity, while the colourless droplets, which are without lamellar cortices,
appear to possess no phosphatase activity.
If Dalton (1961) and McGee-Russell (1962) are correct in their assumptions
that both lipid globules and Golgi lamellar systems are present in Helix
neurones, then one would have expected to find the TPPase activity on the
Golgi membranes, observable under the light microscope in the form of
dictyosomes or scales. My results indicate that although some activity may
be localized in such a form, most of the enzyme activity which can be seen
at the level of light microscopy seems to be on the peripheries of the lipid
globules. These results suggest various hypotheses. It may be that this
confirms Chou and Meek's interpretation (1958) of the Golgi lamellae in
H. aspersa neurones as split phospholipid globules, but possibly the Golgi
lamellae in invertebrate neurones do not contain the same complex of enzymes
as they do in vertebrate cells; or, again, the activity of TPPase in invertebrate
neurones may not be restricted to the Golgi lamellae, but may also be present
in the lamellar cortices of such inclusions as lipid globules. The last possibility seems the most likely one in consideration of certain facts. The neurones
of H. aspersa examined by myself contain certain bodies which possess
TPPase activity and yet which cannot be proved to be lipid droplets. Novikoff
and his colleagues (1962) have shown that in the vertebrate cell, although the
4-io
Lane—Phosphatases in neurones of Helix
highest levels of TPPase activity are observed in the Golgi lamellae, there may
be some TPPase in other components of the cell, which hydrolyse the substrate more slowly.
In H. aspersa neurones, the activity of all three phosphatases seems to be
localized mainly in the same site, on the cortices of certain lipid globules.
In vertebrate cells, however, their sites of activity are usually rather sharply
distinguishable.
From studies on 25 different vertebrate tissues, Novikoff and Goldfischer
(1961) concluded that TPPase activity is present on the lamellae of the Golgi
apparatus, and hence, that this is an enzyme present on the Golgi membranes
in all vertebrate cell types. Novikoff (1961) showed that acid phosphatase
activity was often localized in granules (lysosomes) concentrated in the Golgi
region. The topographical relations between the Golgi membranes and the
granules rich in acid phosphatase that are observed in a number of vertebrate
tissues, by both light and electron microscopy, suggested a developmental or
functional interrelationship between the Golgi apparatus and the lysosomes,
and Novikoff and his colleagues mention (1962) that in a few kinds of cells,
including neurones, both TPPase and acid phosphatase activity might be
found in the same site, in the Golgi lamellae.
In Helix a relationship between the Golgi membranes and secretory products is particularly interesting in connexion with the production of the
elementary neurosecretory granules. In H. pomatia, Dalton and McGeeRussell have shown that the neurones contain the typical neurosecretory
granules which are not resolvable at the level of light microscopy, since they
are only 100 to 200 mju, in diameter (Knowles, i960). Strong evidence has
been produced to suggest that in both vertebrates and invertebrates these are
formed by terminal budding and vesiculation of the Golgi lamellae (Scharrer
and Brown, 1961; Bern, Nishioka, and Hagadorn, 1962; von Harnack and
Lederis, 1962). Novikoff mentioned (1961) that unusually high levels of acid
phosphatase activity had been found in some neurosecretory cells and
endocrine organs (Sobel, 1961). Also, in the axoplasm of certain cells which
have been shown to synthesize hormones, much larger (1 p) globules were
observed which had within them accumulations of neurosecretory vesicles
(Knowles, 1962). In certain hypothalamic neurosecretory cells, von Harnack
and Lederis observed granules (0-5 to 1-5 /x in diameter) composed of parallel
osmiophil lamellae, fine granular material, and vesicles. These, as well as the
elementary neurosecretory granules, were usually observed in the vicinity of
Golgi complexes. In Helix neurones, the satellites which are sometimes in
association with the yellow globules (Chou, 1957a) may perhaps be related
to the elementary neurosecretory granules. There appears, then to be a possibility that there is a functional relationship of some sort between the Golgi
lamellae, and globules which may have a lamellar structure (von Harnack and
Lederis, 1962). Such a relationship may involve the elementary neurosecretory granules in some way, and perhaps some transfer of enzymatic
activity. A further indication of this is given by the results of Bern and his
Lane—Phosphatases in neurones of Helix
411
colleagues (1962), who, in certain neurones of the gastropod Aplysia, have
observed transformation of the Golgi complex directly into globules with
orange pigmentation.
The enzymatically active lipid globules in H. aspersa show certain similarities to granules which have been described in vertebrate tissues. Ogawa and
his colleagues (i960) found that the neutral-red granules of neural cells
possessed acid and alkaline phosphatase activity. These they considered to
be lysosomes. The 'blue' and yellow globules in H. aspersa neurones also
take up neutral red, as well as displaying phosphatase activity. Koenig (1962)
found glycolipoprotein granules with acid phosphatase activity in mammalian
neurones, which he believed were identical with lysosomes. He also considered them to correspond to Baker's lipochondria and Chou's phospholipid
globules. Since Chou (19576) found carbohydrate, protein, and mixed lipids
in the yellow globules, and Chou and Meek (1958) suggested that the yellow
globules may originate from the 'blue' phospholipid droplets, it is possible
that both these lipid globules bear some relationship to Koenig's glycolipoprotein granules.
Although such comparisons indicate that there are certain similarities
between vertebrate and invertebrate neurones in the localization of acid
phosphatase, there does seem to be a basic difference between them in the
localization of a large part of the TPPase activity. However, the results
recorded here indicate that these invertebrate neurones possess at least one
structure, the cortical lamellae of the lipid globules, which resembles the
vertebrate Golgi apparatus in its enzyme content.
I wish to thank Dr. J. R. Baker, F.R.S., for his invaluable supervision
during the course of this work. I am grateful to Professor J. W. S. Pringle,
F.R.S., for accommodation in his Department, and to Mr. John Haywood
for assistance with photomicrography. This research was carried out during
the tenure of a Travelling Fellowship from the Canadian Federation of University Women, whose financial support is gratefully acknowledged.
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