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Thiamine pyrophosphatase, acid phosphatase, andalkaline 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)

SummaryAll three phosphatases have been found to be localized mainly in the cortices of bodieswhich have the distribution, size, and shape of the 'blue' and yellow lipid globules.Colouring neurone preparations with the lysochrome Sudan IV, either before or afterincubation for thiamine pyrophosphatase or acid phosphatase activity, shows a sudano-phil reaction in the medullary spaces surrounded by the cortices that hydrolyse bothphosphates. It is concluded that the acid phosphatase and thiamine pyrophosphataseactivities, which in vertebrates are present in the lysosomes and Golgi lamellaerespectively, are mainly found, in these invertebrate neurones, in the phospholipidlamellae which form the externa of certain of the lipid globules present in thecytoplasm.

Introduction

N O V I K O F F and Goldfischer (1961) recently demonstrated the presence ofnucleosidediphosphatase (NDPase) and thiamine pyrophosphatase (TPPase)activity in the lamellae of the Golgi apparatus in certain vertebrate and planttissues. Similarly, Allen and Slater (1961) have shown Golgi-associatedTPPase activity to be present in the epididymis of the mouse. These resultswould suggest that the test for such enzymic activity might be used asa cytochemical technique for depicting the Golgi complex. On the assump-tion that, as these workers have shown, the Golgi lamellae always containsuch an enzyme or enzymes with hydrolytic activity specific for nucleosidedi-phosphates (NDPs) and thiamine pyrophosphate (TPP), then this techniquewould present a marked advance over the classical metal impregnation tech-niques for showing the Golgi apparatus in vertebrate tissues.

The TPPase and NDPase preparations in vertebrates give pictures exactlysimilar to those produced after the standard Golgi methods, and in electronmicrographs show a heavy deposition of lead on the smooth membranes ofthe Golgi lamellae (Novikoff, Essner, Goldfischer, and Heus, 1962). Othercytomembranes 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 incuba-tion in TPP and NDP media, it appears that there is an enzymatic similaritybetween these lamellae in cells of different sorts. This similarity, consideredin 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 bythese investigators, the Golgi apparatus is a homologous structure and a dis-tinct cellular entity. This had formerly been questioned by such investigatorsas Malhotra (1959) and David, Brown, and Mallion (1961), who have shownthat the classical Golgi techniques sometimes darken the same network thatis coloured by basic dyes to reveal the Nissl substance. The Nissl bodiescorrespond to the endoplasmic reticulum with attached ribonucleoproteingranules (Malhotra and Meek, i960).

However, the problem of whether this vertebrate Golgi corresponds to anystructure in the invertebrate cell has remained unsettled. Invertebrate cells,after impregnation with heavy metals, present, not a network as do thevertebrate tissues, but a number of 'dictyosomes' in the shapes of half-moons,crescents, and filaments (Nath, 1944; Baker, 1945; Malhotra, 1961). AlthoughMalhotra, in a histochemical study (1961), has shown that these dictyosomesdiffer chemically from the classical vertebrate Golgi network, many investi-gators still consider these dictyosomes to be homologous with the vertebrateGolgi net.

The invertebrate dictyosomes, for example in the case of the neurones ofthe snail, Helix aspersa (Chou, 1957a), or the neurones of the prawn or cray-fish (Malhotra, i960), have been shown to be due to deposition of silver orosmium on the outer surface of certain of the lipid droplets that are present inthe cytoplasm.

In an electron microscopical investigation of H. aspersa neurones, Chouand Meek (1958) suggested that a structure similar to the typical vertebrateGolgi lamellae was only present in the cells if calcium ions were omitted fromthe fixative. They concluded, therefore, that it represented an artificial dis-tortion, due to the splitting open of the cortex of the phospholipid globules,which are composed of concentric lamellae at the ultrastructural level. Otherworkers (Dalton, i960; McGee-Russell, 1962) who studied the neurones ofH. pomatia, disagreed with this interpretation, believing the lipid globules,and a system of Golgi lamellae similar to the vertebrate Golgi, to be twodistinct and separate cell organelles, both present in Helix.

The present study was therefore undertaken in an attempt to discover thesite of TPPase activity in the neurones of H. aspersa, in the hope of findingout which of the lamellar systems of invertebrate neurones, if any, resemblethe vertebrate Golgi apparatus in their enzyme content.

A study of the distribution of acid phosphatase in this invertebrate cell wasalso undertaken to make a comparison with vertebrate cells, where this enzymeis usually located in the lysosomes (Novikoff, 1961). Sobel (1961) has showna parallel between hormone production and accumulation of acid phosphataseactivity in vertebrate endocrine organs; and it has been shown that neuro-secretory activity (Dalton, i960; Krause, 1961), as well as probable hormonalproduction (Pelluet and Lane, 1961), is present in gastropod neurones. This,as well as the widely accepted theory of the association of the Golgi lamellaewith 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 acidphosphatase activity with that of TPPase activity.

Tests were made for the presence and localization of alkaline phosphataseactivity in order to make a comparison with the results obtained by Chou

Sudan IV was used to colour H. aspersa neurones either before or afterincubation for TPPase and acid phosphatase, as a means of determining thesites of the lipid globules in relation to the phosphatase activity.

ProcedureTest for thiamine pyrophosphatase activity

Neurones from the cerebral ganglion of the snail, H. aspersa, were fixedovernight in formaldehyde-calcium (F/Ca) at 40 C (Baker, 1945). Afterwashing in cold distilled water, the tissue was kept in a sucrose / gum-acaciasolution (Holt, 1959) at 40 C. Frozen sections were cut at 10 /A on a freezingmicrotome and placed in distilled water. The sections were mounted onslides 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 wasremoved by thorough drying of the sections. The sections thus prepared wereeither used at once or kept for a week or two in distilled water at 40 C.Novikoff and Goldfischer (1961) state that such sections may be stored forseveral weeks without loss of enzymic activity. Sometimes neurones wereteased apart after fixation, mounted on slides, dried, and then tested forenzymatic activity.

Sections and teased preparations were incubated for 20 to 45 min at 37° Cin the TPP medium described by Novikoff and Goldfischer (1961). Sincerather weak results were obtained with a 1 M solution of the substrate, a 3 Msolution was used, as Novikoff and Goldfischer found this to be the optimalhigher concentration of substrate. The incubation medium was filtered beforeuse if a precipitate formed during its preparation. After incubation the slideswere rinsed in distilled water, and placed briefly in dilute ammonium sulphidesolution for visualization of the lead reaction-product. The slides weremounted in glycerogel in the usual fashion.

A control for the activity of the TPP incubation medium was carried outon mouse epididymis, where it produced a positive reaction in the Golgi ap-paratus of the epithelial cells, as Novikoff and Goldfischer (1961) had found.

Control sections of H. aspersa neurones were carried through at the sametime as were the experimental tissues. For one form of control, none of theTPP 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 byfixing in cold F/Ca in the same way as described above in the test for TPPase


activity. These cells were then washed and stored in cold sucrose / gum-acacia. Frozen sections were cut at 10^. and mounted on slides as before. Thetissues were incubated at 370 C in o-i M sodium-/?-glycerophosphate mediumat pH 5-0 (Gomori, 1952). The period of incubation was about 1 h; the normalincubation time of 4 h produced too intense a result for the proper resolutionof the elements that hydrolysed the substrate. The final reaction-product wasvisualized by dipping the slides in dilute ammonium sulphide solution.

Control sections were incubated in the same medium to which had beenadded o-oi M sodium fluoride as inhibitor.

Test for alkaline phosphatase activity

Sections were tested for alkaline phosphatase activity by the technique ofGomori (1952). Cerebral neurones were fixed in ethanol/acetone (1:1),embedded in paraffin and sectioned at 6 to 8 p. The slides were incubatedin 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% calciumchloride, 3% cobalt chloride, and dilute ammonium sulphide solutions, asdescribed by Gomori.

Control sections were tested by the same technique, but with the omissionof incubation in the glycerophosphate medium, to check for the presence ofany calcium.

Colouring with Sudan IV to test for the presence of lipids

Novikoff and others (1962), in reference to testing for the activity of NDPaseand TPPase in vertebrate tissues, stated that 'the method permits stainingfor lipids after visualizing [the enzymic] activity in the sections'. Sudan IVwas 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 bycolouring F/Ca material with Sudan IV, and examining for the distributionand coloration of the various lipid globules.

Different sequences of incubation and coloration were used in these experi-ments. First, sections of neurones that had been fixed in F/Ca were incubatedfor TPPase or acid phosphatase activity, as described earlier. After visualiza-tion of the reaction-product with ammonium sulphide, the sections wereexamined, and the enzyme distribution in particular cells noted and drawn.The same section was then treated with a saturated solution of Sudan IV in70% ethanol/acetone (1:1) for 5 min, differentiated with 50% ethanol for1 min, washed with distilled water, and mounted in glycerogel. The sameindividual cells were again examined to observe where uptake of Sudan IVhad occurred.

Secondly, sections of neurones that had been fixed in F/Ca were colouredwith 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 referenceto the lipid globules seen in the first inspection of the same cells.


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 incubatedin the TPPase and acid phosphatase media as usual and examined for en-zymatic activity.

Observations and ResultsThe results were the same regardless of the manner in which the sections

were mounted on the slides. The optimal incubation time for TPPase activitywas found to be about 45 min, for acid phosphatase activity 60 min, and foralkaline phosphatase activity 150 min. Storage of sections for short periodscaused no obvious differences in intensity of reaction.

FIG. 1. Diagram of a section of a cerebral neurone from H. aspersaafter incubation for thiamine pyrophosphatase activity.

The test for TPPase activity produced an intensely positive reaction, local-ized in the form of circular or elliptical bodies, which appeared ring-like insections. These varied from 1 to 2 ̂ in diameter and were dispersed through-out the cytoplasm (fig. 1). This reaction seemed to be on the surface orcortex of globules and was often more intense on one side of the surface thanon the other. Occasionally the enzymic activity seemed to be localized in theform 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 ofirregular bodies which looked as if two or more smaller bodies had coalesced.In the case of the control sections incubated for TPPase activity without anysubstrate, a very slight reaction was sometimes discernible, perhaps due toresidual substrate within the cell. Uranyl nitrate, however, effectively blockedany reaction, giving completely negative results.

4-o6 Lane—Phosphatases in neurones of Helix

Approximately the same localization was observed after incubating for acidphosphatase activity (figs. 2, 3). The cytoplasmic inclusions of these prepara-tions, 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). Thej . control for acid phosphatase, with sodium fluoride inhibition, gave negative',(, , , , - results.

FIG. 2. Diagram of a section of a cerebral neurone from H. aspersaafter incubation for acid phosphatase activity.

There was a great number of positively reacting bodies after incubation foracid phosphatase and TPPase activity; so many that resolution of the separateelements was sometimes impaired. The positively reacting cortices seemedto be evenly distributed, except in some cases where they nearly all occurredon one side of the cell. In these instances the phenomenon was an obviousfixation artifact. In no case were the sites of activity particularly aggregatedin the axon hillock, nor along the length of the axon. The axons that wereobserved contained no element that hydrolysed either TPP or sodium-j8-glycerophosphate.

Alkaline phosphatase activity was localized very intensely in the nuclearand plasma membranes. It was also present in the nucleus, in the chromatinclumps, 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 afterformaldehyde/calcium fixation. Incubated for acid phosphatase activity. (The test forthiamine pyrophosphatase gives the same picture, except that there is no reaction in thenucleus.)

FIG. 3

N. J. LANE


and less violent than the positive acid phosphatase reaction in the nucleus.A fainter positive reaction, indicating some alkaline phosphatase activity, wasseen to be localized on the externa of spheres scattered evenly throughout thecytoplasm. Sometimes this reaction was stronger on one side of a globule thanon the other. Occasionally tiny, apparently homogeneously positive, sphereswere observed. The controls gave negative results. Thus the alkaline phos-phatase activity was in the same site as that observed for the other twophosphatases, but the reaction was very much less intense.

Control sections, coloured only with Sudan IV, contained various sudano-phil globules. The cortices of spheroidal globules (about 1 /x or more indiameter), and of somewhat larger irregular globules, were coloured pinkish-red. Their interna were highly refractile, which made it difficult to ascertainthe colour, although they seemed to be a faint pink or in some cases uncoloured.Heavily coloured, red-orange droplets were scattered about in smaller num-bers. 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 micro-scopy. Larger (1 to2/i) red-orange irregular globules were present, but notin such abundance as the refractile globules. These red-orange bodies weresometimes grouped in the area of the axon hillock.

Post-colouring with Sudan IV, on sections previously incubated for theactivity of both TPPase and acid phosphatase, produced a reddish yellowcolour inside the cortices where the enzyme was localized. There seemed tobe no coloration with the lysochrome within some of the cortices. Tinyscattered 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 colorationwith Sudan IV also indicated that no activity was present in the tiny scatteredsudanophil droplets. A positive reaction was observed around most of therefractile 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 lessintense than that in sections without pretreatment with Sudan IV.

The control sections treated with the ethanol/acetone solution beforeincubation in the TPP and acid sodium-j8-glycerophosphate media, showedthe enzymatic activity in the same sites as described above for cells incubatedand coloured in the normal fashion. However, with the normal incubationtime, the intensity of the reaction was very much decreased.

DiscussionLive preparations and fixed sections of the neurones of H. aspersa were

studied in detail by Chou (1957 a, b). He described three kinds of lipidglobules in these cells. He distinguished the various lipid inclusions by thefollowing names: yellow globules, of irregular shape, which contained caro-tenoid and mixed lipids, with some protein and carbohydrate; spheroidal'blue' globules, which responded to no histochemical tests except those for


phospholipid; and colourless globules, composed of triglyceride. The yellowglobules were aggregated mainly in the axon hillock, the 'blue' spheroids (socalled because they coloured readily with certain blue vital dyes) were scat-tered evenly throughout the cytoplasm, and some of the colourless dropletswere strung out along the basal part of the axon, while others were distributedthroughout the perikaryon. The 'blue' globules measured about i to z /x indiameter, the yellow ones were generally somewhat larger than this, and thecolourless droplets averaged about i JX.

Chou stated (19576) that all three types of globule reacted positively toSudan IV, and that this lysochrome showed the colourless droplets to behomogeneously red. The distribution, size, and shape of the sudanophilglobules that were observed in this study, indicate that they are the 'blue',yellow, and colourless globules described by Chou. The latter's histochemicalobservations (19576) explain the more intense cortical sudanophilia foundhere with the refractile lipid droplets. Both 'blue' and yellow globules arerefractile in fixed preparations, although the 'blue' ones have rather a lowrefractive index during life (Ross and Chou, 1957). Chou noted that the lipidin the yellow globules was more or less restricted to their peripheries; thecentres 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 samesection. Chou stated (1957a) that smaller neurones contained no yellowglobules and fewer 'blue' droplets than did the larger ones. This fact, orsome block to the diffusion of the substrate into these cells, may perhapsaccount for this.

The only enzymatic test performed by Chou was Gomori's test for alkalinephosphatase (19576). This gave a positive result on the surface of the yellowglobules, which Chou noted was in the same position as the phospholipid.

Studies of sections incubated for enzymic activity and subsequentlycoloured with Sudan IV indicated that at least a large proportion of thebodies hydrolysing the phosphates were the cortices of the 'blue' and yellowglobules. Since the intensely sudanophil triglyceride droplets were notevident in the enzyme-incubated preparations before coloration, it is probablethat these colourless lipid globules contained no enzyme activity.

Examinations of neurones coloured with Sudan IV and then incubated forenzyme activity, also suggested that the positive enzymic reaction on thesurface of the lipid globules was limited to the cortices of the 'blue' and yellowones. Further, studies of the axons, where the colourless droplets were oftenstrung out, failed to show evidence of enzymatic activity. There were, in theperikaryon, elements rich in phosphatase which had sudanophobe cores, andit was impossible to state whether these were lipid globules whose sudanophilcortex had been obscured by the enzymatic activity, or whether they weresome other cytoplasmic organelle.


The ultrastructure of the three types of lipid globules is helpful in interpret-ing these results. The lack of enzymatic activity in the colourless droplets isunderstandable if it is assumed that the activity in the 'blue' and yellowglobules is present in the lamellated border of these globules, which Chouand Meek described (1958), for they found that the colourless droplets haveno such lamellar border. They considered that the laminated membranes atthe periphery of the 'blue' globules were probably due to the presence ofphospholipids.

As mentioned earlier, Chou and Meek concluded from their investigationsthat those lamellar membranes in the neurones of H. aspersa which had anappearance similar to the vertebrate Golgi were distortions of the 'blue'phospholipid droplets that had been split open by fixatives not containingcalcium ions. Dalton (i960) and McGee-Russell (1962), in electron-micro-scopical investigations of the neurones of H. pomatia, each described bothlipid globues and Golgi bodies, which were present as distinct and separatecell organelles. The bodies which possess phosphatase activity in H. aspersaand are not definitely lipid globules may perhaps correspond either to split'blue' globules, or to Golgi lamellae. Such a reaction would bear a relation-ship to vertebrate cells, where the Golgi lamellae have high levels of TPPaseactivity (Novikoff and Goldfischer, 1961). The relative sizes of the lipidglobules and the Golgi lamellae are of no use in distinguishing between themin light-microscopical preparations, since the figures are within the samerange; so that no conclusion about this matter can be drawn from the presentevidence. However, it can be stated that the cortices of the 'blue' and yellowlipid globules possess both acid and alkaline phosphatase and TPPaseactivity, 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 assumptionsthat both lipid globules and Golgi lamellar systems are present in Helixneurones, then one would have expected to find the TPPase activity on theGolgi membranes, observable under the light microscope in the form ofdictyosomes or scales. My results indicate that although some activity maybe localized in such a form, most of the enzyme activity which can be seenat the level of light microscopy seems to be on the peripheries of the lipidglobules. These results suggest various hypotheses. It may be that thisconfirms Chou and Meek's interpretation (1958) of the Golgi lamellae inH. aspersa neurones as split phospholipid globules, but possibly the Golgilamellae in invertebrate neurones do not contain the same complex of enzymesas they do in vertebrate cells; or, again, the activity of TPPase in invertebrateneurones may not be restricted to the Golgi lamellae, but may also be presentin the lamellar cortices of such inclusions as lipid globules. The last possi-bility seems the most likely one in consideration of certain facts. The neuronesof H. aspersa examined by myself contain certain bodies which possessTPPase activity and yet which cannot be proved to be lipid droplets. Novikoffand 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 maybe some TPPase in other components of the cell, which hydrolyse the sub-strate more slowly.

In H. aspersa neurones, the activity of all three phosphatases seems to belocalized mainly in the same site, on the cortices of certain lipid globules.In vertebrate cells, however, their sites of activity are usually rather sharplydistinguishable.

From studies on 25 different vertebrate tissues, Novikoff and Goldfischer(1961) concluded that TPPase activity is present on the lamellae of the Golgiapparatus, and hence, that this is an enzyme present on the Golgi membranesin all vertebrate cell types. Novikoff (1961) showed that acid phosphataseactivity was often localized in granules (lysosomes) concentrated in the Golgiregion. The topographical relations between the Golgi membranes and thegranules rich in acid phosphatase that are observed in a number of vertebratetissues, by both light and electron microscopy, suggested a developmental orfunctional 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 befound in the same site, in the Golgi lamellae.

In Helix a relationship between the Golgi membranes and secretory pro-ducts is particularly interesting in connexion with the production of theelementary neurosecretory granules. In H. pomatia, Dalton and McGee-Russell have shown that the neurones contain the typical neurosecretorygranules which are not resolvable at the level of light microscopy, since theyare only 100 to 200 mju, in diameter (Knowles, i960). Strong evidence hasbeen produced to suggest that in both vertebrates and invertebrates these areformed by terminal budding and vesiculation of the Golgi lamellae (Scharrerand Brown, 1961; Bern, Nishioka, and Hagadorn, 1962; von Harnack andLederis, 1962). Novikoff mentioned (1961) that unusually high levels of acidphosphatase activity had been found in some neurosecretory cells andendocrine organs (Sobel, 1961). Also, in the axoplasm of certain cells whichhave been shown to synthesize hormones, much larger (1 p) globules wereobserved which had within them accumulations of neurosecretory vesicles(Knowles, 1962). In certain hypothalamic neurosecretory cells, von Harnackand Lederis observed granules (0-5 to 1-5 /x in diameter) composed of parallelosmiophil lamellae, fine granular material, and vesicles. These, as well as theelementary neurosecretory granules, were usually observed in the vicinity ofGolgi complexes. In Helix neurones, the satellites which are sometimes inassociation with the yellow globules (Chou, 1957a) may perhaps be relatedto the elementary neurosecretory granules. There appears, then to be a pos-sibility that there is a functional relationship of some sort between the Golgilamellae, and globules which may have a lamellar structure (von Harnack andLederis, 1962). Such a relationship may involve the elementary neuro-secretory granules in some way, and perhaps some transfer of enzymaticactivity. A further indication of this is given by the results of Bern and his


colleagues (1962), who, in certain neurones of the gastropod Aplysia, haveobserved transformation of the Golgi complex directly into globules withorange pigmentation.

The enzymatically active lipid globules in H. aspersa show certain similari-ties to granules which have been described in vertebrate tissues. Ogawa andhis colleagues (i960) found that the neutral-red granules of neural cellspossessed acid and alkaline phosphatase activity. These they considered tobe lysosomes. The 'blue' and yellow globules in H. aspersa neurones alsotake up neutral red, as well as displaying phosphatase activity. Koenig (1962)found glycolipoprotein granules with acid phosphatase activity in mammalianneurones, which he believed were identical with lysosomes. He also con-sidered them to correspond to Baker's lipochondria and Chou's phospholipidglobules. Since Chou (19576) found carbohydrate, protein, and mixed lipidsin the yellow globules, and Chou and Meek (1958) suggested that the yellowglobules may originate from the 'blue' phospholipid droplets, it is possiblethat both these lipid globules bear some relationship to Koenig's glycolipo-protein granules.

Although such comparisons indicate that there are certain similaritiesbetween vertebrate and invertebrate neurones in the localization of acidphosphatase, there does seem to be a basic difference between them in thelocalization of a large part of the TPPase activity. However, the resultsrecorded here indicate that these invertebrate neurones possess at least onestructure, the cortical lamellae of the lipid globules, which resembles thevertebrate Golgi apparatus in its enzyme content.

I wish to thank Dr. J. R. Baker, F.R.S., for his invaluable supervisionduring 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 Haywoodfor assistance with photomicrography. This research was carried out duringthe tenure of a Travelling Fellowship from the Canadian Federation of Univer-sity Women, whose financial support is gratefully acknowledged.

ReferencesALLEN, J. M., and SLATER, J. J., 1961. J. Histochem. Cytochem., 9, 418.BAKER, J. R., 1945. Quart. J. micr. Sci., 85, 1.BERN, H. A., NISHIOKA, R. S., and HAGADORN, I. R., 1962. In Neurosecretion, Memoirs of the

Society for Endocrinology, no. 12, edited by Heller and Clark. London (AcademicPress).

CHOU, J. T. Y., 1957a. Quart. J. micr. Sci., 98, 471957&. Ibid., 98, 59.and MEEK, G. A., 1958. Ibid., 99, 279.

DALTON, A. J., i960. In Cell physiology of neoplasia (M. D. Anderson Hospital and TumorInstitute). Austin (University of Texas Press).

DAVID, G. B., BROWN, A. W., and MALLION, K. B., 1961. Quart. J. micr. Sci., 102, 481.GOMORI, G., 1952. Microscopic histochemistry. Chicago (University Press).HARNACK, M. VON, and LEDERIS, K., 1962. From the Conference of European Comparative

Endocrinologists, London. (In press.)HOLT, S., 1959. Exp. Cell Res., suppl. 7, 1.


KNOWLES, F., i960. Nature, Lond., 185, 709.1962. In Neurosecretion, Memoirs of the Society for Endocrinology, no. iz, edited by

Heller and Clark. London (Academic Press).KOENIG, H., 1962. Nature, Lond., 195, 782.KRAUSE, E., i960. Z. Zellforsch., 51, 748.MALHOTRA, S. K., 1959. Quart. J. micr. Sci., 100, 339.

i960. Ibid., IOI, 75.1961. Ibid., 102, 83.and MEEK, G. A., i960. Ibid., 101, 389.

MCGEE-RUSSELL, S. M., 1962. Personal communication.NATH, V., 1944. 31st Indian Science Congress, 6, pp. 1. Delhi (Sri Gouranga Press).NOVIKOFF, A. B., 1961. The cell, vol. 2, edited by Brachet and Mirsky, London (Academic

Press).and GOLDFISCHER, S., 1961. Proc. nat. Acad. Sci., Wash., 47, 802.ESSNER, E., GOLDFISCHER, S., and HEUS, M., 1962. In The interpretation of ultrastructure,Symposium of International Society for Cell Biology, vol. 1, edited by Harris. London(Academic Press).

OCAWA, K., MIZUNO, N., HASHIMOTO, K., FUJII, S., and OKAMOTO, M., i960. Proc. Dept.Anat., Ky6to Univ. School Med., 4, 1. (Quoted from Novikoff (1961).)

PELLUET, D., and LANE, N. J., 1961. Canadian J. Zool., 39, 789.Ross, K. F. A., and CHOU, J. T. Y., 1957. Quart. J. micr. Sci., 98, 341.SCHARRER, E., and BROWN, S., 1961. Z. Zellforsch., 54, 530.SOBEL, H. J., 1961. Endocrinology, 68, 801.

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